molecules

Review
Cholesterol-Based Compounds: Recent Advances in
Synthesis and Applications
Hélio M. T. Albuquerque 1, * , Clementina M. M. Santos 1,2 and Artur M. S. Silva 1, *
1 QOPNA & LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, Campus de Santiago,
3810-193 Aveiro, Portugal; clems@ipb.pt
2 Centro de Investigação de Montanha (CIMO) Instituto Politécnico de Bragança, Campus de Santa Apolónia,
5300-253 Bragança, Portugal
* Correspondence: helio.albuquerque@ua.pt (H.M.T.A.); artur.silva@ua.pt (A.M.S.S.);
Tel.: +351-234-370714 (A.M.S.S.)




Received: 2 December 2018; Accepted: 24 December 2018; Published: 29 December 2018


Abstract: This review reports on the latest developments (since 2014) in the chemistry of cholesterol
and its applications in different research fields. These applications range from drug delivery or
bioimaging applications to cholesterol-based liquid crystals and gelators. A brief overview of the
most recent synthetic procedures to obtain new cholesterol derivatives is also provided, as well as
the latest anticancer, antimicrobial, and antioxidant new cholesterol-based derivatives. This review
discusses not only the synthetic details of the preparation of new cholesterol derivatives or conjugates,
but also gives a short summary concerning the specific application of such compounds.

Keywords: cholesterol; drug delivery; bioactive compounds; liquid crystals; gelators;

bioimaging; synthesis

Index
1. Introduction to Cholesterol-Based Compounds ... ... ... ... ... ... ... ... ... ... ... ... ... 1
2. Drug Delivery Applications ... ... ... ... ... ... ... ... ... ... ... ... 3
3. Anticancer, Antimicrobial, and Antioxidant Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
4. Cholesterol-Based Liquid Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
5. Cholesterol-Based Gelators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
6. Bioimaging Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
7. Synthetic Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
8. Miscellaneous . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Conflicts of Interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Abbreviations List . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

1. Introduction to Cholesterol-Based Compounds

Cholesterol (cholest-5-en-3β-ol) is considered to be a lipid-type molecule, being one of the
most important structural components of cell membranes. Chemically, cholesterol is a rigid and
almost planar molecule with a steroid skeleton of four fused rings, three six-membered and one
five-membered, conventionally lettered from A to D (1,2-cyclopentanoperhydrophenanthrene ring
system) (Figure 1A). Therefore, the cholesterol molecule comprises four essential domains (Figure 1B).
In domain I, the polarity of the 3-hydroxy group constitutes an active site for hydrogen bond
interactions with a myriad of biological molecules (e.g., phospholipids in membranes) [1]. In domain

Molecules 2019, 24, 116; doi:10.3390/molecules24010116 www.mdpi.com/journal/molecules

Molecules 2019, 24, 116 2 of 68

Molecules 2018, 23, x 2 of 68

II, the absence of methyl groups at C-4 and C-14 influences directly the planarity of the molecule,
while inindomain
domain III, the
III, natural (R) configuration
the natural at C-20 determines
(R) configuration the “right-handed”
at C-20 determines conformation
the “right-handed”
of the side chain.
conformation of theFinally, in domain
side chain. IV,inthe
Finally, conformation
domain and length of
IV, the conformation thelength
and side chain
of theisside
of prime
chain
relevance
is to intermolecular
of prime contacts [2]. The
relevance to intermolecular presence
contacts [2].ofThe
a hydrophilic
presence 3-hydroxy headgroup
of a hydrophilic on the
3-hydroxy
A-ring,
headgrouptogether
on the with a hydrophobic
A-ring, hydrocarbon
together with body, hydrocarbon
a hydrophobic give the molecule
body,angive
amphiphilic
the moleculenature,
an
which makesnature,
amphiphilic cholesterol
which themakes
most cholesterol
recognized the
sterol.
most recognized sterol.

(A) Numbering
Figure 1. (A) Numbering and
and ring-labeling
ring-labeling convention in cholesterol; (B) four domains of cholesterol.

Cholesterol plays
Cholesterol plays aa vital
vital role
role inin life,
life, particularly
particularly in in cell
cell membranes
membranes and and as as a
a precursor
precursor to to the
the
biosynthesis of several steroid hormones. In cell membranes, which are essentially constituted by aa
biosynthesis of several steroid hormones. In cell membranes, which are essentially constituted by
double layer
double layer of of phospholipids, cholesterol has
phospholipids, cholesterol has great
great influence
influence on on membrane
membrane fluidity,
fluidity, microdomain
microdomain
structure (lipid
structure (lipid rafts),
rafts), and permeability by
and permeability by interacting
interacting withwith both
both thethe hydrophilic
hydrophilic headgroups
headgroups and the
and the
hydrophobic tails of phospholipids. In addition, modifications of the
hydrophobic tails of phospholipids. In addition, modifications of the stereochemistry and oxidation stereochemistry and oxidation
states of
states of the
thefused
fusedrings,
rings,the theside
side chain,
chain, as as
well well as the
as the functional
functional groupsgroups of cholesterol,
of cholesterol, lead
lead to to a
a wide
wide variety
variety of biologically
of biologically important
important molecules,
molecules, suchsuchas as
bilebile acids,
acids, vitaminD,D,and
vitamin and several
several steroid
steroid
hormones [1,2]. Interestingly, 13 Nobel Prizes have been awarded to scientists who studied the
hormones [1,2]. Interestingly, 13 Nobel Prizes have been awarded to scientists who studied the
structure ofofcholesterol,
structure cholesterol, its biosynthetic
its biosynthetic pathway, and metabolic
pathway, regulation.regulation.
and metabolic Unfortunately, cholesterol
Unfortunately,
has gained ahas
cholesterol badgained
reputation a bad because it is increasingly
reputation because itassociated with several
is increasingly cardiovascular
associated and
with several
neurodegenerative diseases, among others [1,3].
cardiovascular and neurodegenerative diseases, among others [1,3].
Over the
Over theyears,
years,cholesterol
cholesterol has risen
has risenas anas attractive starting
an attractive material
starting or a model
material system system
or a model for organic
for
synthesis due to its easily derivatized functional groups, availability,
organic synthesis due to its easily derivatized functional groups, availability, and low cost. Many and low cost. Many useful
chemical
useful and enzymatic
chemical reactions
and enzymatic are noware
reactions widely used forused
now widely multistep steroid transformations,
for multistep leading
steroid transformations,
to products
leading of practical
to products importance.
of practical The chemical
importance. transformations
The chemical transformations range range
from simple ones, ones,
from simple such
such as manipulations of functional groups, to more complex ones, such as C-H activation or C-C
as manipulations of functional groups, to more complex ones, such as C-H activation or C-C
bond formation
bond formation with with organometallic
organometallic reagents. reagents. In In 2014,
2014, aa purely
purely synthetic
synthetic chemistry
chemistry review
review waswas
published,
published, dealing
dealing only
only with
with the the advances
advances in in cholesterol
cholesterol chemistry
chemistry sincesince 2000,
2000, focusing
focusing on on cholesterol
cholesterol
oxidation reactions, substitution of the 3β-hydroxy group, addition
oxidation reactions, substitution of the 3β-hydroxy group, addition to the C5=C6 double bond, to the C5=C6 double bond, C-H
C-H
functionalization, and
functionalization, and C-CC-C bondbond forming
forming reactions. However, this
reactions. However, this review
review paper
paper excluded
excluded simple
simple
derivatization reactions
derivatization reactions of cholesterol such
of cholesterol such as as the
the preparation
preparation of of carboxylic
carboxylic and and inorganic
inorganic acid
acid esters,
esters,
aliphatic and aromatic ethers, simple acetals, or glycosides [4]. From
aliphatic and aromatic ethers, simple acetals, or glycosides [4]. From our perspective, the simpler our perspective, the simpler
chemical transformations
chemical transformations very often lead
very often lead toto thethe preparation
preparation of of new
new cholesterol-based
cholesterol-based molecules
molecules with
with
potential applications in several important research fields. Therefore,
potential applications in several important research fields. Therefore, in this review, we focused ourin this review, we focused our
attention on
attention publications from
on publications from 20142014 to date and
to date and described
described notnot only
only the
the synthesis
synthesis of of cholesterol-based
cholesterol-based
new molecules, but also the application of these molecules
new molecules, but also the application of these molecules in different fields, such asin different fields, such as drug
drug delivery;
delivery;
bioimaging; liquid crystals; gelators; anticancer, antimicrobial, and
bioimaging; liquid crystals; gelators; anticancer, antimicrobial, and antioxidant applications; asantioxidant applications; as well
well
as purely
as purely synthetic
synthetic applications.
applications. However, However, some some interesting
interesting papers
papers published
published beforebefore 2014
2014 were
were
included to
included to fill
fill some
some ofof the
the lacking
lacking papers
papers from from thethe 2014
2014 review
review paper.
paper. Throughout
Throughout the the text,
text, several
several
reaction schemes will be depicted to describe the chemical reaction involved
reaction schemes will be depicted to describe the chemical reaction involved in the preparation of the in the preparation of
the cholesterol-based
cholesterol-based compounds.
compounds. For For simplification
simplification purposes,
purposes, thethestructures
structuresofof cholesterol
cholesterol will
will
consistently be represented using the abbreviations
consistently be represented using the abbreviations depicted in Figure 2. depicted in Figure 2.

Figure 2. Schematic representation of cholesterol-based molecules throughout the manuscript.

bioimaging; liquid crystals; gelators; anticancer, antimicrobial, and antioxidant applications; as well
as purely synthetic applications. However, some interesting papers published before 2014 were
included to fill some of the lacking papers from the 2014 review paper. Throughout the text, several
reaction schemes will be depicted to describe the chemical reaction involved in the preparation of the
cholesterol-based
Molecules 2019, 24, 116 compounds. For simplification purposes, the structures of cholesterol 3 of will
68
consistently be represented using the abbreviations depicted in Figure 2.

Figure 2. Schematic representation of cholesterol-based molecules throughout the

the manuscript.
manuscript.
Molecules 2018,
Molecules 2018, 23,
23, x
x 33 of
of 68
68
2. Drug Delivery Applications
2. Drug
2. Drug Delivery
Delivery Applications
Applications
Drug delivery is a method or process of administering a pharmaceutical compound to achieve a
Drug delivery
Drug
therapeutic delivery ishumans
effect inis aa method
method or
oror processDrug
process
animals. of administering
of administering aa pharmaceutical
delivery systems pharmaceutical compound
compound
can in principle provide toenhanced
to achieve aa
achieve
therapeutic
therapeutic effect
efficacy, reduced in humans
effecttoxicity,
in humans or animals.
or animals.
or both Drug delivery
Drugtypes
for various delivery systems
systems
of drugs. can in principle
can in principle
Liposomes are the mostprovide
provide enhanced
enhanced
common and
efficacy, reduced
efficacy, reduced toxicity,
well-investigated toxicity,
nanocarriers or both
or both for various
for various
for targeted drugtypes
types of drugs.
of
deliverydrugs.
because Liposomes
Liposomes
they havearedemonstrated
are the most
the most common
common and
and
efficiency
well-investigated
well-investigated nanocarriers
nanocarriers for
for targeted
targeted drug
drug delivery
delivery because
because they
they have
have
in several biomedical applications by stabilizing therapeutic compounds, overcoming obstacles to demonstrated
demonstrated efficiency
efficiency
in
in several
several
cellular biomedical
andbiomedical
tissue uptake, applications
applications by stabilizing
by
and improving stabilizing therapeuticofcompounds,
therapeutic
the biodistribution compounds, overcoming
compoundsovercoming obstacles
to target sitesobstacles to
to
in vivo [5].
cellular
cellular and
In and tissue
2014,tissue uptake, and
uptake,
Vabbilisetty and improving
andimproving the biodistribution
the
Sun reported biodistribution of compounds
of
a study of terminal compounds to target
to target sites
triphenylphosphine sites in
incarrying
vivo [5].
vivo [5].
anchorIn 2014,
In 2014, Vabbilisetty
lipidVabbilisetty
effects on and and Sun reported
Sun reported
a liposome a study
a study
surface of terminal triphenylphosphine
of terminal triphenylphosphine
by postchemically carrying anchor
carrying anchor
selective functionalization via
lipid
lipid effects ligation,
effects
Staudinger on aa liposome
on liposome surface by
surface
using lactosyl by postchemically
postchemically
azide selective
as a model selective functionalization
ligand. functionalization
They synthesizedvia via
two Staudinger
Staudinger
different
ligation,
ligation, using lactosyl
using
anchor lipids, lactosyl
one of them azidebased
azide as aa model
as model
on theligand.
ligand. They synthesized
They
cholesterol synthesized two different
two
molecule (Chol-PEG different anchor lipids,
anchor lipids, one
2000 -thiphenylphosphine
one of
of
them
them based
basedwas
3), which on the
on the cholesterol
cholesterol
synthesized molecule
molecule
through (Chol-PEG
an(Chol-PEG 2000-thiphenylphosphine
-thiphenylphosphine
amidation2000reaction 3), which was
3), which 2000
of synthetic Chol-PEG was-NHsynthesized
synthesized
2 1 with
through
through an amidation
an amidation reaction
reaction of
3-diphenylphosphino-4-methoxycarbonylbenzoic of synthetic
synthetic Chol-PEG
Chol-PEG -NH22 11 with
2000-NH
acid N-hydroxysuccinimide
2000 with 3-diphenylphosphino-4-
3-diphenylphosphino-4-
(NHS) active ester 2
methoxycarbonylbenzoic
methoxycarbonylbenzoic
(Scheme 1) [6]. acid N-hydroxysuccinimide
acid N-hydroxysuccinimide (NHS) (NHS) active
active ester
ester 22 (Scheme
(Scheme 1) 1) [6].
[6].

Scheme 1. Synthesis
Scheme Synthesis of
Synthesis ofanchoring
of anchoringlipid
anchoring lipidChol-PEG
lipid Chol-PEG
Chol-PEG 2000
2000
2000 -triphenylphosphine.Reagents
-triphenylphosphine.
-triphenylphosphine. Reagentsand
Reagents and
and conditions:
conditions: a)
conditions:
a)
a) Et
Et3Et N,
N,33N,
CH CH
2 Cl
CH Cl ,, room
roomtemperature
2 , 22room
22Cl temperature
temperature (rt),
(rt), overnight.
overnight.
(rt), overnight.

The authors
The that the
verified that
authors verified the Staudinger ligation could
Staudinger ligation could be be carried out under
carried out under mild
mild reaction
reaction
conditions in aqueous buffers without a catalyst and in high yields.
conditions in aqueous buffers without a catalyst and in high yields. The encapsulation andThe encapsulation and releasing
releasing
capacity ofof
capacity ofthethe
theglycosylated
glycosylated
glycosylated liposome basedbased
liposome
liposome on cholesterol
based were evaluated,
on cholesterol
on cholesterol respectively,
were evaluated,
were evaluated, by entrapping
respectively,
respectively, by
by
5,6-carboxyfluorescein
entrapping (CF)
5,6-carboxyfluoresceindye and(CF)monitoring
dye and the fluorescence
monitoring the
entrapping 5,6-carboxyfluorescein (CF) dye and monitoring the fluorescence leakage. It wasleakage. It
fluorescence was concluded
leakage. It that
was
Chol-PEG2000
concluded
concluded that
that-thiphenylphosphine
Chol-PEG2000
Chol-PEG 3 is particularly 33suitable
-thiphenylphosphine
2000-thiphenylphosphine is for the ligation
is particularly
particularly suitableoffor
suitable water-soluble
for ligationmolecules
the ligation
the of water-
of water-
and can
soluble accommodate
molecules and many
can chemical
accommodate functions,
many being
chemical potentially
functions,
soluble molecules and can accommodate many chemical functions, being potentially useful in useful
being in the coupling
potentially useful of many
in the
the
other
couplingligands
of onto
many liposomes
other ligands for drug
onto delivery
liposomes purposes
for drug [6].
delivery
coupling of many other ligands onto liposomes for drug delivery purposes [6]. purposes [6].
In 2015,
In 2015,aaanew
2015, newmethod
new method
method was reported
was
was reported
reported for the
for deposition
for the deposition
the of a single
deposition lipid bilayer
of aa single
of single onto a hard
lipid bilayer
lipid bilayer ontopolymer
onto aa hard
hard
polymer bead starting from discoidal bicelles and using chemoselective chemistry the
bead
polymerstarting
beadfrom discoidal
starting bicelles
from and using
discoidal chemoselective
bicelles and chemistry
using to hydrophobically
chemoselective anchor
chemistry to
to
lipid assemblies,
hydrophobically anchor
hydrophobically using cholesterol
anchor thethe lipid bearing
lipid assemblies,an oxyamine
assemblies, using linker.
using cholesterol The synthesis
cholesterol bearing
bearing an of oxyamine-terminated
an oxyamine
oxyamine linker.
linker. The The
cholesterolof
synthesis
synthesis of6 involved two steps, starting
oxyamine-terminated
oxyamine-terminated with a66Mitsunobu
cholesterol
cholesterol involved two
involved reaction
two of starting
steps,
steps, compound
starting with
with4, followed
aa Mitsunobu
Mitsunobu by a
reaction of
reaction with hydrazine
of 5compound
compound 4, hydrate
4, followed
followed by (Scheme
by aa reaction
reaction 2)of
[7].
of 55 with
with hydrazine
hydrazine hydrate
hydrate (Scheme
(Scheme 2) 2) [7].
[7].

NH2
NH
HO O Chol 2
HO O
O
O O Chol
O
a)
a) O O
b)
b) O
O O
O Chol
Chol
O N
N O O
O O
O
4
4 O O Chol 6
O O
O
O O Chol
O
6
5
5

Scheme 2.
Scheme Synthesis of
2. Synthesis of oxyamine-terminated
oxyamine-terminated cholesterol
cholesterolconjugate.
conjugate.Reagents
Reagentsand
andconditions:
conditions:(a) PPh33,,
a) PPh
PPh
Scheme 2. Synthesis of oxyamine-terminated cholesterol conjugate. Reagents and conditions: a) 3,
N-hydroxy-phthalimide, diisopropyl
N-hydroxy-phthalimide, diisopropyl azodicarboxylate
azodicarboxylate (DIAD),
(DIAD), tetrahydrofuran
tetrahydrofuran (THF),
(THF), rt,
rt, 16
16 h;
h; (b)
b)
N-hydroxy-phthalimide, diisopropyl azodicarboxylate (DIAD), tetrahydrofuran (THF), rt, 16 h; b)
NH22NH
NH NH2∙H·H2O,
2 O,CH
CH2Cl
2 Cl2rt,, rt,1818h.h.
NH 2NH22∙H 2O, CH 2Cl22,, rt, 18 h.

The
The discoidal
discoidal bicelles
bicelles were
were prepared
prepared in
in water
water media
media upon
upon mixing
mixing
dimyristoylphosphatidylcholine
dimyristoylphosphatidylcholine (DMPC),
(DMPC), dihexanoylphosphatidylcholine
dihexanoylphosphatidylcholine (DHPC),
(DHPC),
dimyristoyltrimethylammonium propane
dimyristoyltrimethylammonium propane (DMTAP),
(DMTAP), and and the
the oxyamine-terminated
oxyamine-terminated cholesterol
cholesterol
derivative 6,
derivative 6, in
in aa specific
specific molar
molar ratio.
ratio. These
These bicelles
bicelles were
were exposed
exposed to to aldehyde-bearing
aldehyde-bearing polystyrene
polystyrene
(PS) beads and readily underwent a change to a stable single lipid bilayer coating at the bead
(PS) beads and readily underwent a change to a stable single lipid bilayer coating at the bead surface.surface.
Molecules 2019, 24, 116 4 of 68

The discoidal bicelles were prepared in water media upon mixing dimyristoylphosphatidylcholine
(DMPC), dihexanoylphosphatidylcholine (DHPC), dimyristoyltrimethylammonium propane
(DMTAP), and the oxyamine-terminated cholesterol derivative 6, in a specific molar ratio.
These bicelles were exposed to aldehyde-bearing polystyrene (PS) beads and readily underwent a
change to a stable single lipid bilayer coating at the bead surface. This approach may be advantageous
in depositing membrane proteins at such surfaces for analytical, diagnostic, or therapeutic applications
(namely drug delivery) [7].
Cholesterol
Molecules
Molecules 2018, 23, xx chloroformate 7 was used as a lipid anchor for hydrophobization of arabinogalactan
2018, 23, 44 of
of 68
68
(AG), a liver-specific high galactose containing a branched polysaccharide, through a two-step reaction
reaction sequence
sequence that yielded
that yielded a novela novel polysaccharide
polysaccharide lipid,lipid, conjugated
conjugated ligand
ligand 9 (Chol-AL-AG),
9 (Chol-AL-AG), withwith a
a bifunctional spacer β-alanine (AL) (Scheme
bifunctional spacer β-alanine (AL) (Scheme 3) [8]. 3) [8].

Synthesis
Scheme 3. Synthesis ofofChol-AL-AG.
Synthesisof Chol-AL-AG. Reaction
Chol-AL-AG.Reaction conditions:
Reactionconditions: a) a)
conditions:
a) β-alanine, soluphor-P,
β-alanine,
β-alanine, NaOH,
soluphor-P,
soluphor-P, THF,
NaOH,
NaOH, rt,
THF,
THF, rt,
2 h;
2rt, h; b)
2 h; arabinogalactan, carbonyldiimidazole
arabinogalactan,carbonyldiimidazole
b)b)arabinogalactan, (CDI),
carbonyldiimidazole(CDI), dimethylaminopyridine
(CDI), dimethylaminopyridine (DMAP),
dimethylaminopyridine (DMAP), dimethyl
(DMAP), dimethyl
sulfoxide (DMSO), 80 ◦°C,
sulfoxide °C, 24 h.
C, 24 h.

Ligand 9
Ligand 9 was
was used
used toto prepare
prepare conventional
conventional liposomes
liposomes (CLs)
(CLs) and
and surface-modified
surface-modified liposomes
liposomes
(SMLs) through
(SMLs) through the
the reverse
reverse phase
phase evaporation
evaporation technique.
technique. These new
These new liposomes
liposomes were
were characterized
characterized
by
by different
different techniques exhibiting the
techniques exhibiting the required
required particle
particle size
size for
for targeting
targeting tumor
tumor and
and infectious
infectious cells.
cells.
In
In vitro
vitrobiological
biologicalstudies
studiesshowed an enhanced
showed binding
an enhanced affinityaffinity
binding and cellular
and uptake
cellularofuptake
SMLs compared
of SMLs
to CLs by HepG2
compared to CLscells, makingcells,
by HepG2 SMLsmaking
an interesting
SMLs an newinteresting
approach fornewtargeted
approachdrugfordelivery
targetedin drug
liver
cancer therapeutics [8].
delivery in liver cancer therapeutics [8].
Crucianelli et
Crucianelli et al. reported in
al. reported in 2014
2014 aa new delivery systemsystem based
based on on liposomes
liposomes containing
containing
dioleoylphosphatidilcholine
dioleoylphosphatidilcholine (DOPC) (DOPC)and andmannose
mannose6-phosphate
6-phosphate (M6P)-functionalized
(M6P)-functionalized cholesterol
cholesterol 14.
ForFor
14. thisthis purpose,
purpose, M6PM6P cholesteryl
cholesteryl conjugate
conjugate (Chol-M6P)was
1414(Chol-M6P) wassynthesized
synthesizedfollowing
following aa three-step
three-step
route starting
route starting from
from cholesterol
cholesterol derivative
derivative 10,
10, as
as depicted
depicted in
in Scheme
Scheme 44 [9].
[9].

Scheme 4.
Scheme Synthesis
4. Synthesis
Synthesis of of
of M6P
M6P M6P cholesteryl
cholesteryl
cholesteryl conjugate
conjugate
conjugate (Chol-M6P).
(Chol-M6P).
(Chol-M6P). Reagents
Reagents
Reagents and conditions:
and conditions:
and conditions: a)
a) N,N′-
a) N,N′-
N,N 0 -dicyclohexylcarbodiimide (DCC), DMAP, CH Cl , rt; b) p-TsOH, MeOH, CHCl ; c) POCl ,
dicyclohexylcarbodiimide (DCC), DMAP, CH Cl , rt; b) p-TsOH, MeOH, CHCl
dicyclohexylcarbodiimide (DCC), DMAP, CH2Cl2, rt;2b) 2p-TsOH, MeOH, CHCl3; c) POCl33, Et3N, dry
2 2 3 ; c) POCl 3 , Et 3N, dry
3
Et ◦ C, rt; d) NaOMe, dry MeOH, then Dowex Na cation exchange resin.
CH
CH 3 N,Cldry
22Cl 22,, 0
CHrt;
0 °C,
°C, Cld)
2rt; 2, 0
d) NaOMe,
NaOMe, dry MeOH,
dry MeOH, then
then Dowex
Dowex Na Na cation
cation exchange
exchange resin.
resin.

This novel
This novel vector
vector system,
system, designed
designed to to target
target lysosomes,
lysosomes, was
was loaded
loaded with
with aa model
model compound
compound
calcein
calcein to
to investigate
investigate intracellular
intracellular trafficking
trafficking in
in aa 3T3-NIH
3T3-NIH cell
cell line
line using
using aa confocal and fluorescence
confocal and fluorescence
microscopy technique. The affinity of the M6P group for the CI-M6PR receptor enabled these
liposomes to carry calcein along the route leading to lysosomes, in opposition to calcein itself, which
did not internalize into cells. These results suggest that liposomes containing Chol-M6P 14 appear to
be promising vectors in the selective targeting of lysosomes for enzyme replacement therapy or
anticancer therapy [9].
Molecules 2019, 24, 116 5 of 68

microscopy technique. The affinity of the M6P group for the CI-M6PR receptor enabled these
liposomes to carry calcein along the route leading to lysosomes, in opposition to calcein itself,
which did not internalize into cells. These results suggest that liposomes containing Chol-M6P
14 appear to be promising vectors in the selective targeting of lysosomes for enzyme replacement
therapy or anticancer therapy [9].
The importance of liposomes in drug delivery applications is well recognized. In this context, Silva
and coworkers reported the synthesis of cholesterol-based neoglycoconjugates of 19 (galactose-Gal
and N-acetylglucosamine-GlcNAc) for further incorporation into liposomes. The glycoconjugates
were synthesized through a copper-catalyzed 1,3-dipolar cycloaddition (CuAAC) reaction of glycosyl
azides (18)
Molecules 2018,with
23, x cholesterol derivative 17 (Scheme 5) [10]. The authors carried out biodistribution
5 of 68
in vivo studies to evaluate the targeting of these carbohydrate-coated liposomes, concluding that they
showed high uptake by the liver, spleen, and kidneys and no significant accumulation into other
organs. Furthermore, it was demonstrated that liposomes with galactose in the surface preferentially
target the liver cells. The
The results
results suggest
suggest that
that this kind of liposome might be a promising delivery
system for therapeutic agents in hepatic diseases [10].

Synthesisofofcholesterol-based
Scheme 5. Synthesis cholesterol-based neoglycoconjugates
neoglycoconjugates derived
derived from
from D -galactose
D-galactose andand
N-
N-acetylglucosamine.
acetylglucosamine. Reagentsand
Reagents andconditions:
conditions: a) 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
hydrochloride (EDAC), DMAP,
hydrochloride DMAP, acetone/THF,
acetone/THF, rt, 4 h; b) glycosyl
h; b) glycosyl azide, CuSO44·∙5H
azide, CuSO 5H22O,
O, AscONa,
AscONa,
THF/H O,rt,
rt,44h;h;c)c)NaOMe,
NaOMe,MeOH, ◦
MeOH,00°C, C,11h.h.
THF/H22O,

To develop
To develop aa drugdrug delivery
delivery system
system for
for potential
potential theranostic applications, Škorpilová
theranostic applications, Škorpilová et et al.
al.
synthesized the
synthesized the fluorescent
fluorescent macrostructure
macrostructure 23 23 containing sesquiterpene lactone
containing sesquiterpene lactone trilobolide
trilobolide (Tb),
(Tb),
cholesterol, and a green-emitting boron dipyrromethene (BODIPY) dye. The
cholesterol, and a green-emitting boron dipyrromethene (BODIPY) dye. The synthesis of compound synthesis of compound
involved aa three-step
23 involved
23 three-step sequence,
sequence, starting
starting from
from the
the CuAAC
CuAAC reaction
reaction ofof propargyl
propargyl cholesterol
cholesterol 20 with
20 with
BODIPY dye 21, followed by functionalization with sesquiterpene lactone trilobolide
BODIPY dye 21, followed by functionalization with sesquiterpene lactone trilobolide (Scheme 6) [11]. (Scheme 6) [11].
This fluorescent cholesterol conjugate 23 was successfully incorporated into liposome
This fluorescent cholesterol conjugate 23 was successfully incorporated into liposome formulations, formulations,
which
which showed
showed promising
promising immunomodulatory
immunomodulatory propertiesproperties in
in primary
primary rat macrophages and
rat macrophages and improved
improved
drug distribution in U-2 OS and HeLa cancer cells. The study of the intracellular
drug distribution in U-2 OS and HeLa cancer cells. The study of the intracellular trafficking trafficking pattern of
pattern
liposomes revealed two populations: One localized on the cell membrane and the
of liposomes revealed two populations: One localized on the cell membrane and the other inside the other inside the cell,
this last
cell, thisone
lastclosely related
one closely to celltodeath.
related This new
cell death. Thisliposomal cholesterol
new liposomal conjugate
cholesterol 23 not only
conjugate retains
23 not only
the biological properties of pure trilobolide, but also enhances bioavailability, and
retains the biological properties of pure trilobolide, but also enhances bioavailability, and thus hasthus has potential
for use in for
potential theranostic applications
use in theranostic [11].
applications [11].
which showed promising immunomodulatory properties in primary rat macrophages and improved
drug distribution in U-2 OS and HeLa cancer cells. The study of the intracellular trafficking pattern
of liposomes revealed two populations: One localized on the cell membrane and the other inside the
cell, this last one closely related to cell death. This new liposomal cholesterol conjugate 23 not only
retains the
Molecules 2019,biological
24, 116 properties of pure trilobolide, but also enhances bioavailability, and thus has
6 of 68
potential for use in theranostic applications [11].

Scheme 6. Synthesis of cholesterol-trilobolide conjugate. Reaction conditions: a) CuSO4∙5H2O, AscONa,

Scheme 6. Synthesis of cholesterol-trilobolide conjugate. Reaction conditions: a) CuSO4 ·5H2 O, AscONa,
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), dimethylformamide (DMF), microwave irradiation
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA), dimethylformamide (DMF), microwave irradiation
(MW), 60 °C,◦ 90 min; b) amino-PEG4-acetylene, N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide
(MW), 60 C, 90 min; b) amino-PEG4 -acetylene, N-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide
hydrochloride (EDCl), DMAP, N-hydroxybenzotriazole (HOBt), DMF, rt, 24 h; c) Tb-N3VA, CuSO4∙5H2O,
hydrochloride (EDCl), DMAP, N-hydroxybenzotriazole (HOBt), DMF, rt, 24 h; c) Tb-N3 VA, CuSO4 ·5H2 O,
AscONa,
Molecules TBTA,
2018,
AscONa, 23, DMF,
x
TBTA, MW,
DMF, MW,60 °C,
60 ◦5C,h.5 h. 6 of 68

Recently,
Recently,LinLinet et
al. reported
al. reported the synthesis of a fluorescent
the synthesis triple-responsive
of a fluorescent block-graft block-graft
triple-responsive copolymer
bearing 27,
copolymer
27, cholesteryl- and pyrenyl-side
bearing cholesteryl- groups, with
and pyrenyl-side a disulfide
groups, with (S-S) bridging
a disulfide point
(S-S) joining
bridging the
point
hydrophilic and hydrophobic
joining the hydrophilic chains. The
and hydrophobic synthesis
chains. of such of
The synthesis polymers relied on
such polymers a typical
relied click
on a typical
click reaction
reaction between between
PNiPAAm PNiPAAm
10 -S-S-P(αN10-S-S-P(αN 3CL)
3 CL)10 26, 10 26, pyrenylmethyl
pyrenylmethyl 4-pentynoate 4-pentynoate 25, and
25, and cholesteryl
cholesteryl 4-pentynoate
4-pentynoate 24, affording 24,PNiPAAm
affording PNiPAAm
10 -S-S-P(αN 103-S-S-P(αN
CL 10 3 CL
-g-PyrePA10 -g-PyrePA
3 /-CholPA 3 /-CholPA
7 ) 27 ) 27
(Scheme
7 (Scheme
7) [12].
7) [12]. Experimental
Experimental resultsresults indicated
indicated that copolymer
that copolymer 27 could
27 could undergo
undergo self-assemblyinto
self-assembly into polymeric
polymeric
performance in aqueous
micelles with excellent fluorescence performance aqueous solution.
solution. The drug-loading capacity of
cholesteryl-grafted copolymer
cholesteryl-grafted copolymer27 27waswasevaluated
evaluatedusingusing doxorubicin
doxorubicin (DOX)
(DOX) as aastemplate
a template
drug, drug,
andandthe
the results
results showed
showed reasonable
reasonable DOX-loading
DOX-loading capacity.
capacity. The authors
The authors also demonstrated
also demonstrated that DOX-
that DOX-loaded
loaded micelles
micelles enter theenter
cells the
at acells at a substantially
substantially faster ratefaster rate than
than their their counterparts,
free-form free-form counterparts,
effectively
effectivelyHeLa
inhibiting inhibiting HeLa cell proliferation
cell proliferation [12]. [12].

Scheme 7. Synthesis of fluorescent triple-responsive block-graft copolymer. Reagents and conditions:

a) CuI, Et33N, THF, 40 ◦°C,
C, 24 h.

In
In 2014,
2014,the
thesynthesis
synthesisofof
a new
a newdual-imaging andand
dual-imaging therapeutic agentagent
therapeutic for improved efficacy
for improved in Boron
efficacy in
Neutron Capture Therapy (BNCT) in cancer treatment was reported [13]. The compound
Boron Neutron Capture Therapy (BNCT) in cancer treatment was reported [13]. The compound consists of
aconsists
carborane
of aunit (ten boron
carborane unitatoms) bearing
(ten boron a cholesterol
atoms) bearing aunit on one side
cholesterol unit(to
onpursue incorporation
one side (to pursue
into
incorporation into the liposome bilayer) and a Gd(III)/1,4,7,10-tetraazacyclododecane complex
the liposome bilayer) and a Gd(III)/1,4,7,10-tetraazacyclododecane monoamide monoamide on
the other side
complex (as an
on the magnetic
other resonance
side (as imaging
an magnetic (MRI) reporter
resonance imagingto attain
(MRI)the quantification
reporter to attainof the
the
quantification of the B/Gd concentration). The synthesis of the target compound Gd-B-AC01 (37)
relied on an eight-step synthetic strategy, which ended with the complexation of 36 with Gd(III) in
aqueous solution at pH 6.5 (Scheme 8). This dual probe 37 was functionalized with a polyethylene
glycol (PEG)ylated phospholipid containing a folic acid residue at the end of the PEG chain. These
liposomes presented interesting features such as the ability to selectively concentrate high amounts
Molecules 2019, 24, 116 7 of 68

B/Gd concentration). The synthesis of the target compound Gd-B-AC01 (37) relied on an eight-step
synthetic strategy, which ended with the complexation of 36 with Gd(III) in aqueous solution at
pH 6.5 (Scheme 8). This dual probe 37 was functionalized with a polyethylene glycol (PEG)ylated
phospholipid containing a folic acid residue at the end of the PEG chain. These liposomes presented
interesting features such as the ability to selectively concentrate high amounts of boron in human
ovarian cancer cells (IGROV-1), enough to perform efficient BNCT treatment with significantly reduced
uptake by healthy cells in the surrounding regions. Furthermore, these liposomes, which can be used
as nanoplatforms to deliver both Gd and B agents, can, in principle, be used for the simultaneous
delivery of antitumor
Molecules 2018, 23, x drugs such as DOX [13]. 7 of 68

Molecules 2018, 23, x 7 of 68

Scheme 8. Synthesis of Gd-B-AC01. Reagents and conditions: ◦ C to rt, 1 h; b)

conditions: a) MsCl, Et3 N, Et22O, O, 00 °C to rt, 1 h;
HO(CH2))33OH, OH,1,4-dioxane,
1,4-dioxane, 120 ◦ C, overnight; c) 3 M CrO in H SO , acetone, rt, overnight; d) DCC,
120 °C, overnight; c) 3 22SO44, acetone, rt, overnight; d) DCC,

CH
CH2Cl Cl22,, rt,
rt, overnight;
overnight; e)e) H
H22,, Pd/C, EtOH/CH2Cl
Pd/C, EtOH/CH 2 Cl (1:1),rt,
2 2(1:1), rt,overnight;
overnight;f)f)33 MM CrO
CrO33 in
in H
H22SO
SO44, ,acetone,
acetone, rt,
rt,
overnight;
overnight; g) g) N-tert-butylDOTAMA-C6-NH
N-tert-butylDOTAMA-C6-NH22,, N-hydroxysuccinimide
N-hydroxysuccinimide (NHS), (NHS),DCC,
DCC,i-PrEt
i-PrEt22N,
N, CH
CH22Cl
Cl2,,
Scheme
rt, 8. Synthesis of Gd-B-AC01.(TFA), Reagents and conditions: a) MsCl, Et3N, Et2O, 0 °C to rt, 1 h; b)
rt, overnight;
overnight; h) h) trifluoroacetic
trifluoroacetic acid
acid (TFA), CH
CH22ClCl2,, rt,
rt, 44 h;
h; i) GdCl
i) GdCl 33,, 1
1MM NaOH,
NaOH, H22O,
H O,rt,
rt,26
26h.h.
HO(CH2)3OH, 1,4-dioxane, 120 °C, overnight; c) 3 M CrO3 in H2SO4, acetone, rt, overnight; d) DCC,
Zhang
CH 2Cl2, and coworkers
rt, overnight; e) Hstudied the behavior
2, Pd/C, EtOH/CH of nanoparticles
2Cl2 (1:1), rt, overnight; f)(NPs) formed
3 M CrO 3 in H2bySOself-assembly
4, acetone, rt, of
amphiphilic
overnight; poly[N-(2-hydroxypropyl)methacrylamide]
g) N-tert-butylDOTAMA-C6-NH2, N-hydroxysuccinimide (pHPMA)(NHS), copolymers bearing
DCC, i-PrEt 2N, CHcholesterol
2Cl2,

side groups (39) as

rt, overnight; h) potential drugacid
trifluoroacetic carriers
(TFA),for
CHsolid tumor
2Cl2, rt, 4 h; i)treatment
GdCl3, 1 M(Figure
NaOH,3) H2[14].
O, rt, 26 h.

Figure 3. Schematic structures of a) cholesterol-free pHPMA-based copolymers (pHPMA-Hyd)

bearing hydrazide groups; b) statistical cholesterol-containing (pHPMA-Chol) groups distributed
along the polymer chain; and c) cholesterol-containing pHPMA-based copolymers carrying the
anticancer drug doxorubicin (DOX) (pHPMA-Chol-DOX).
Figure 3.3.Schematic
Schematicstructures of a)ofcholesterol-free
structures pHPMA-based
a) cholesterol-free copolymers
pHPMA-based (pHPMA-Hyd)
copolymers bearing
(pHPMA-Hyd)
hydrazide
bearing groups; b)
hydrazide statistical
groups; cholesterol-containing (pHPMA-Chol) groups distributed along the
The behavior of such NPsb)instatistical
human serumcholesterol-containing
albumin (HSA)(pHPMA-Chol) groups distributed
protein environment was evaluated
polymer
along thechain; and c)chain;
polymer cholesterol-containing pHPMA-basedpHPMA-based
and c) cholesterol-containing copolymers carrying the anticancer
copolymers carryingdrug
the
using mixed solutions of NPs from polymer conjugates with or without the anticancer drug
doxorubicin
anticancer (DOX)
drug (pHPMA-Chol-DOX).
doxorubicin (DOX) (pHPMA-Chol-DOX).
doxorubicin bounded to them, 39 and 40, respectively. The authors found that in the absence of DOX,
a small
Theamount
behaviorof HSA molecules
of such NPs in bind to the cholesterol
in human groupsprotein
of the NPs by diffusing through the
The behavior of such NPs human serum
serum albumin
albumin (HSA)
(HSA) protein environment
environment was
was evaluated
evaluated
loose
using pHPMA shell or get caught in meshes formed by the pHPMA chains. On the other hand, the
using mixed
mixedsolutions
solutionsof NPs fromfrom
of NPs polymer conjugates
polymer with or without
conjugates with or the anticancer
without the drug doxorubicin
anticancer drug
presence of DOX strongly hinders these interactions, and for that reason the delivery of DOX by these
doxorubicin bounded to them, 39 and 40, respectively. The authors found that in the absence of DOX,
NPs in the human body is not affected by the presence of HSA [14].
a small amount of HSA molecules bind to the cholesterol groups of the NPs by diffusing through the
Recently, Singh and coworkers reported the biofunctionalization of the surface of β-cyclodextrin
loose pHPMA shell or get caught in meshes formed by the pHPMA chains. On the other hand, the
nanosponge 41 (β-CD-NSP) with cholesterol, expecting to improve its cellular binding ability. The β-
presence of DOX strongly hinders these interactions, and for that reason the delivery of DOX by these
CD-NSP was functionalized by grafting cholesterol hydrogen succinate (CHS) through a coupling
NPs in the human body is not affected by the presence of HSA [14].
Molecules 2019, 24, 116 8 of 68

bounded to them, 39 and 40, respectively. The authors found that in the absence of DOX, a small
amount of HSA molecules bind to the cholesterol groups of the NPs by diffusing through the loose
pHPMA shell or get caught in meshes formed by the pHPMA chains. On the other hand, the presence
of DOX strongly hinders these interactions, and for that reason the delivery of DOX by these NPs in
the human body is not affected by the presence of HSA [14].
Recently, Singh and coworkers reported the biofunctionalization of the surface of β-cyclodextrin
nanosponge 41 (β-CD-NSP) with cholesterol, expecting to improve its cellular binding ability.
The β-CD-NSP was functionalized by grafting cholesterol hydrogen succinate (CHS) through a
coupling reaction,
Molecules 2018, 23, x affording β-CD-NSP-CHS 42 (Scheme 9) [15]. 8 of 68

Scheme 9. Synthesis of β-CD-NSP-CHS. Reagents and conditions: a) cholesterol hydrogen succinate

(CHS), EDCl, DMAP,
DMAP,DMF, 45◦°C,
DMF,45 C, 24
24h.
h.

The
The cytotoxicity
cytotoxicity assays
assays showed
showed thatthat β-CD-NSP
β-CD-NSP 41 41 was
was nontoxic
nontoxic and
and that
that the
the surface
surface
biofunctionalized with CHS 42 improved both the therapeutic and drug delivery
biofunctionalized with CHS 42 improved both the therapeutic and drug delivery efficacy of efficacy of DOX.
DOX.
The
The experimental results also
experimental results alsodemonstrated
demonstratedthat thatCHS
CHSgrafting
graftingmay
mayenhance
enhance DOX
DOX adsorption
adsorption duedueto
to the hydrophobic charge on the surface. Therefore, the surface-engineered CD-NSP
the hydrophobic charge on the surface. Therefore, the surface-engineered CD-NSP could be used as could be used
as a carrier
a carrier forfor
lowlow water-soluble
water-soluble small
small drugdrug molecules
molecules to improve
to improve solubility
solubility and and bioavailability
bioavailability in
in site-
site-specific
specific drugdrug delivery
delivery systems
systems [15].[15].
In
In attempting to develop intelligent
attempting to develop an drug delivery
an intelligent for cancer
drug delivery forchemotherapy, Li et al. synthesized
cancer chemotherapy, Li et al.
dual redox/pH-sensitive
synthesized amphiphilic
dual redox/pH-sensitive copolymercopolymer
amphiphilic 44 and 44 cholesterol-modified poly(β-amino
and cholesterol-modified poly(β-
esters)-grafted disulfide poly (ethylene glycol) methyl ether [PAE(-SS-mPEG)-g-Chol].
amino esters)-grafted disulfide poly (ethylene glycol) methyl ether [PAE(-SS-mPEG)-g-Chol]. The The precursor
PAE-SS-mPEG 43 was successfully
precursor PAE-SS-mPEG 43 was synthesized
successfully via Michael-type
synthesized via step polymerization
Michael-type using disulfide
step polymerization
linkage-containing PEG segment. Finally,
using disulfide linkage-containing cholesterol
PEG segment. was incorporated
Finally, into incorporated
cholesterol was the hydroxy-pendant
into the
group trough an esterification reaction, affording the copolymer PAE(-SS-mPEG)-g-Chol
hydroxy-pendant group trough an esterification reaction, affording the copolymer PAE(-SS-mPEG)- 44
(Scheme 10) [16].
g-Chol 44 (Scheme 10) [16].
The authors verified the interesting physicochemical properties of copolymer 44, namely redox
and pH sensitivity. Doxorubicin-loaded hybrid polymer-lipid NPs (DOX-HDPLNPs) were prepared,
and drug-loading capacity, delivery efficacy, and redox- and pH-triggered drug release behavior
in vitro were studied. The results showed that DOX-HDPLNPs enhanced loading capacity and
improved cellular uptake ability, as well as serum stability. The anticancer potential in tumor-bearing
mice was addressed, indicating that the DOX-HDPLNPs prepared with redox- and pH-sensitive
copolymer with disulfides and PEGylated lipid could efficiently enhance therapeutic efficacy with
low cytotoxicity and side effects. Both in vitro and in vivo experiments indicated that DOX-HDPLNPs
enhanced therapeutic efficacy with high cellular uptake and negligible cytotoxicity compared to the
free drug DOX. Therefore, HDPLNPs can be considered to be smart delivery systems for hydrophobic
anticancer drug delivery [16].
Tran et al. developed a copolymer in 2014, constituted of polynorbonene-cholesterol/
poly(ethylene glycol) [P(NBCh9-b-NBPEG)] 45, that undergoes self-assembly to form a long circulating
nanostructure capable of encapsulating the anticancer drug DOX with high drug loading (Figure 4) [17].

Scheme 10. Synthesis of PAE(-ss-mPEG)-g-Chol. Reagents and conditions: a) cholesteryl

chloroformate, DMAP, CH2Cl2, rt, 24 h. * represents only the momomeric structure of the polymer.
synthesized dual redox/pH-sensitive amphiphilic copolymer 44 and cholesterol-modified poly(β-
amino esters)-grafted disulfide poly (ethylene glycol) methyl ether [PAE(-SS-mPEG)-g-Chol]. The
precursor PAE-SS-mPEG 43 was successfully synthesized via Michael-type step polymerization
using disulfide linkage-containing PEG segment. Finally, cholesterol was incorporated into the
hydroxy-pendant group trough an esterification reaction, affording the copolymer PAE(-SS-mPEG)-
Molecules 2019, 24, 116 9 of 68
g-Chol 44 (Scheme 10) [16].

Molecules 2018,
Molecules 2018, 23,
23, xx 99 of
of 68
68

low cytotoxicity
low cytotoxicity and
and side
side effects.
effects. Both
Both in
in vitro
vitro and
and in
in vivo
vivo experiments
experiments indicated
indicated that
that DOX-HDPLNPs
DOX-HDPLNPs
enhanced therapeutic efficacy with high cellular uptake and negligible cytotoxicity
enhanced therapeutic efficacy with high cellular uptake and negligible cytotoxicity compared to compared to the
the
free drug
free drug DOX.
DOX. Therefore,
Therefore, HDPLNPs
HDPLNPs can can be
be considered
considered to to be
be smart
smart delivery
delivery systems
systems for
for hydrophobic
hydrophobic
anticancer drug delivery
anticancer drug delivery [16]. [16].
Tran et
Tran et al.
al. developed
developed aa copolymer
copolymer in in 2014,
2014, constituted
constituted of
of polynorbonene-cholesterol/
polynorbonene-cholesterol/
poly(ethylene glycol) [P(NBCh9-b-NBPEG)] 45, that undergoes
poly(ethylene glycol) [P(NBCh9-b-NBPEG)] 45, that undergoes self-assembly to self-assembly to form
form aa long
long
circulating
circulating nanostructure
nanostructure capable of encapsulating the anticancer
capable of encapsulating the anticancer drug DOX with high drug
drug DOX with highcholesterylloading
drug loading
Scheme
Scheme 10. Synthesis of PAE(-ss-mPEG)-g-Chol.
10. Synthesis PAE(-ss-mPEG)-g-Chol. ReagentsReagents and conditions:
conditions: a) cholesteryl
(Figure 4)
(Figure 4) [17].
[17].
chloroformate,
chloroformate, DMAP,
DMAP,CH
CH22Cl
Cl22, rt, 24 h. * represents only the momomeric
momomeric structure
structure of
of the
the polymer.
polymer.

The authors verified the interesting physicochemical properties of copolymer 44, namely redox
and pH sensitivity. Doxorubicin-loaded hybrid polymer-lipid NPs (DOX-HDPLNPs) were prepared,
and drug-loading capacity, delivery efficacy, and redox- and pH-triggered drug release behavior in
vitro were studied. The results showed that DOX-HDPLNPs enhanced loading capacity and
improved cellular uptake ability, as well as serum stability. The anticancer potential in tumor-bearing
mice was addressed, indicating that the DOX-HDPLNPs prepared with redox- and pH-sensitive
copolymer with disulfides and PEGylated lipid could efficiently enhance therapeutic efficacy with
Figure 4.
Figure 4. Cholesterol-based
4. Cholesterol-based brush
brush block
block copolymer
block copolymer poly(NBCh9)
copolymer poly(NBCh9)xxx-b-(NBMPEG)yy..
poly(NBCh9)

The authors
The authors found
found thatthat the
the doxorubicin-loaded
doxorubicin-loaded nanoparticles
doxorubicin-loaded (DOX-NPs) were
nanoparticles (DOX-NPs) effectively
were effectively
internalized
internalized by
by human
human cervical cancer
cervical cells
cancer (HeLa)
cells and
(HeLa) that they
and showed
that
internalized by human cervical cancer cells (HeLa) and that they showed dose-dependent they dose-dependent
showed cytotoxicity.
dose-dependent
Moreover, theMoreover,
cytotoxicity.
cytotoxicity. DOX-NPs showed
Moreover, the good in vivo
the DOX-NPs
DOX-NPs showed
showed circulation
good in
good time
in and circulation
vivo
vivo preferential time
circulation accumulation
time in tumor
and preferential
and preferential
tissue with reduced
accumulation
accumulation in tumor
in tumoraccumulation
tissue with in
tissue with the heart
reduced
reduced and other vital
accumulation
accumulation theorgans,
in the
in heart and
heart andother
and significantly
other inhibited
vital organs,
vital organs, and
and
tumor growth
significantly in tumor-bearing
inhibited tumor severe
growth in combined
tumor-bearingimmunodeficient
severe combined(SCID)
significantly inhibited tumor growth in tumor-bearing severe combined immunodeficient (SCID) mice. Based
immunodeficient on these
(SCID)
results,
mice. DOX-NPs
mice. Based
Based on can results,
on these
these becomeDOX-NPs
results, useful carriers
DOX-NPs in improving
can become
can become usefultumor
useful delivery
carriers
carriers of hydrophobic
in improving
in improving anticancer
tumor delivery
tumor delivery of
of
drugs [17].
hydrophobic anticancer drugs
hydrophobic anticancer drugs [17]. [17].
A new
A new series
new seriesof
series ofofamphiphilic
amphiphilicdiblock
amphiphilic diblock
diblock terpolymer
terpolymer
terpolymer poly(6-O-methacryloyl-
poly(6-O-methacryloyl-
poly(6-O-methacryloyl- D -galactopyranose)-
-galactopyranose)-
D-galactopyranose)-
D -b-
-b-
-b-poly(methacrylic
poly(methacrylic acid-co-6-cholesteryloxyhexylmethacrylate)
poly(methacrylic acid-co-6-cholesteryloxyhexyl
acid-co-6-cholesteryloxyhexyl methacrylate)bearing
methacrylate) bearing attached
bearing attached galactose
attached galactose and
galactose and
cholesterol grafts [PMAgala-b-P(MAA-co-MAChol)s] 49 were
were prepared
prepared
cholesterol grafts [PMAgala-b-P(MAA-co-MAChol)s] 49 were prepared via Reversible Addition via
via Reversible
Reversible Addition
Fragmentation chain
Fragmentation chainTransfer
chain Transfer(RAFT)
Transfer (RAFT)copolymerization
(RAFT) copolymerization
copolymerization followed
followed
followed byby
by deprotection
deprotection
deprotection of galactose
of galactose
of galactose in the
in in
the
the presence
presence
presence of of trifluoroacetic
of trifluoroacetic
trifluoroacetic acidacid
acid (TFA)
(TFA)
(TFA) (Scheme
(Scheme
(Scheme 11) 11)
11) [18].
[18].
[18].

Scheme 11. Synthesis

Scheme 11.
11. Synthesisofof
Synthesis ofPMAgala-b-P(MAA-co-MAChol)s.
PMAgala-b-P(MAA-co-MAChol)s.
PMAgala-b-P(MAA-co-MAChol)s. Reaction
Reaction
Reaction conditions:
conditions:
conditions: a) 02,2′-azobis(2-
a) 2,2
a) -azob
2,2′-azobis(2-
is(2-methylpropionitrile) (AIBN), toluene, 80 ◦ C, 8 h; b) TFA/CH Cl (1:2), rt, 32 h.
methylpropionitrile) (AIBN),
methylpropionitrile) (AIBN), toluene,
toluene, 80
80 °C,
°C, 88 h;
h; b)
b) TFA/CH Cl22 (1:2),
TFA/CH22Cl (1:2), rt, 32
2 2 rt, 32 h.
h.

The new
The new terpolymers
terpolymers (49)
(49) were
were studied
studied for
for in
in vitro
vitro DOX
DOX release,
release, and
and the
the results
results revealed
revealed high
high
stability of the DOX-loaded terpolymer micelles under neutral conditions and significantly
stability of the DOX-loaded terpolymer micelles under neutral conditions and significantly fast fast
responsive DOX
responsive DOX release.
release. In
In addition,
addition, the
the results
results of
of fluorescence
fluorescence microscopy
microscopy revealed
revealed thatthat the
the DOX
DOX
encapsulated in the synthesized diblock terpolymer PMAgala -b-P(MAA
encapsulated in the synthesized diblock terpolymer PMAgala18-b-P(MAA26-co-MAChol9)/DOX
18 26 -co-MAChol 9)/DOX
Molecules 2019, 24, 116 10 of 68

The new terpolymers (49) were studied for in vitro DOX release, and the results revealed
high stability of the DOX-loaded terpolymer micelles under neutral conditions and significantly
fast responsive DOX release. In addition, the results of fluorescence microscopy revealed that the
DOX encapsulated in the synthesized diblock terpolymer PMAgala18 -b-P(MAA26 -co-MAChol9 )/DOX
micelles could be uptaken and delivered into cell nuclei in an efficient way, and their intracellular
trafficking pathway could be altered compared to the free DOX control. The new terpolymers (49)
could therefore
Molecules 2018, 23, xbe strongly considered for future smart nanoplatforms toward efficient antitumor10drug
of 68
delivery [18].
In 2014,
2014, aareduction-responsive
reduction-responsive polymersome
polymersome based on the
based on amphiphilic blockblock
the amphiphilic copolymer PEG-
copolymer
SS-PAChol 52 was
PEG-SS-PAChol developed.
52 was TheThe
developed. synthesis of 52
synthesis was
of 52 achieved
was achievedusing
usingPEG-SS-Br
PEG-SS-Br50,
50, aa versatile
atom transfer radical polymerization (ATRP) macroinitiator, and a cholesterol-containing acrylate 51, 51,
using CuBr as as aa catalyst
catalystand
andN,N,N 0 ,N”,N”-pentamethyldiethylenetriamine (PMDETA)
N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA) as a ligand
[19].
(Scheme 12) [19].

Scheme 12. Synthesis of reduction-sensitive block copolymer

12. Synthesis PEG-SS-PAChol. Reagents and
copolymer PEG-SS-PAChol.
conditions: a) CuBr, N,N,N 0 ,N”,N”-pentamethyldiethylenetriamine (PMDETA), ◦ C,
(PMDETA), toluene, 80 °C,
N,N,N′,N′′,N′′-pentamethyldiethylenetriamine 18 h.

The
The polymersome
polymersome5252was wasstudied
studied to come
to comeup with robust
up with nanocarriers
robust able toable
nanocarriers release their content
to release their
inside the cells upon contact with the intracellular reducing environment. The physical
content inside the cells upon contact with the intracellular reducing environment. The physical crosslinking by
acrosslinking
smectic phase of 52 in the hydrophobic sublayer, as well as the introduction of a
by a smectic phase of 52 in the hydrophobic sublayer, as well as the introduction of adisulfide bridge
that links bridge
disulfide the hydrophilic
that linksPEG and hydrophobic
the hydrophilic PEG andblocks present in 52,
hydrophobic werepresent
blocks key features
in 52, that
weregavekey
stability, robustness, and reduction sensitivity to the polymersome. The results showed
features that gave stability, robustness, and reduction sensitivity to the polymersome. The results sensitivity of
the block copolymer 52 to reduction, and the fluorescence dequenching of calcein
showed sensitivity of the block copolymer 52 to reduction, and the fluorescence dequenching of both in glutathione
(GSH)
calceinsolution
both in and in vitro (GSH)
glutathione with the mouse and
solution macrophage cells pretreated
in vitro with the mouse with GSH-OEtcells
macrophage demonstrated
pretreated
the breakdown of polymersome under reduction conditions. To achieve significant
with GSH-OEt demonstrated the breakdown of polymersome under reduction conditions. To achieve calcein release,
high concentrations
significant of GSHhigh
calcein release, and concentrations
long incubationoftimes GSHwere necessary.
and long Thesetimes
incubation reduction-responsive
were necessary.
polymersomes
These reduction-responsive polymersomes (52) could be used as drug carriers withslow
(52) could be used as drug carriers with very long circulation profiles and very release
long
kinetics [19].
circulation profiles and slow release kinetics [19].
Recently,
Recently, twotwo new
new sterol-anchored
sterol-anchored polyethylene
polyethylene glycols, 55 and
glycols, 55 58, were
and 58, were reported
reported as as potential
potential
alternatives to conventional phosphatidylethanolamine-PEGs. Their
alternatives to conventional phosphatidylethanolamine-PEGs. Their synthesis relied on synthesis relied on thethe
esterification reaction of cholesterol derivatives 53 and 56 with PEGs 54 and 57,
esterification reaction of cholesterol derivatives 53 and 56 with PEGs 54 and 57, as depicted in Scheme as depicted in
Scheme
13 [20]. 13 [20].
The authors studied the biophysical properties of liposomes containing these two sterol-anchored
PEGs, 55 and 58, which exhibited an array of canonical PEGgylated-liposome behaviors including
retention of encapsulated small molecules, low serum protein adsorption, and reduced cellular uptake,
yet they did not exhibit long circulation [20].
Polymeric micelles are known for their variety of therapeutic applications. In this field,
two amphiphilic polymers were successfully synthesized using hyaluronic acid (HA), cholesterol,
and octadecanoic acid as hydrophobic groups. Only the synthesis of cholesterol containing
polymer HA-SA-CYS-Chol 60 is depicted in Scheme 14, since the other hydrophobic groups do
not fit in the scope of this paper. Nevertheless, the authors concluded that different properties of
hydrophobic groups of the amphiphilic carrier are closely implicated in the stability and drug-loading
capacity of the amphiphilic carrier and micelles. HA-SA-CYS-Chol 60 presented a lower critical

Scheme 13. Synthesis of sterol-anchored polyethylene glycols (PEGs). Reagents and conditions: a)
EDCl, DMAP, CHCl3, rt, 24 h.

The authors studied the biophysical properties of liposomes containing these two sterol-
anchored PEGs, 55 and 58, which exhibited an array of canonical PEGgylated-liposome behaviors
disulfide bridge that links the hydrophilic PEG and hydrophobic blocks present in 52, were key
features that gave stability, robustness, and reduction sensitivity to the polymersome. The results
showed sensitivity of the block copolymer 52 to reduction, and the fluorescence dequenching of
calcein both in glutathione (GSH) solution and in vitro with the mouse macrophage cells pretreated
with GSH-OEt demonstrated the breakdown of polymersome under reduction conditions. To achieve
Molecules 2019, 24, 116 11 of 68
significant calcein release, high concentrations of GSH and long incubation times were necessary.
These reduction-responsive polymersomes (52) could be used as drug carriers with very long
circulation
micellar profiles and producing
concentration, slow release kinetics (DTX)-loaded
docetaxel [19]. micelles of a smaller particle size, higher
Recently, two
encapsulation new sterol-anchored
efficiency, polyethylene
and drug loading, glycols, 55toand
when compared the 58, were
other reported as tails
hydrophobic potential
[21].
alternatives toin vivo
Furthermore, conventional phosphatidylethanolamine-PEGs.
animal studies Their synthesis
revealed very good tumor-targeting propertiesrelied on the
and efficient
esterification
antitumor reaction
effects of cholesterol
at very derivativeswith
low concentrations, 53 and 56 systemic
low with PEGs 54 and of
toxicity 57,HA–SA–CYS–Chol
as depicted in Scheme 60
13 [20]. [21].
micelles

Molecules 2018, 23, x 11 of 68

octadecanoic acid as hydrophobic groups. Only the synthesis of cholesterol containing polymer HA-
SA-CYS-Chol 60 is depicted in Scheme 14, since the other hydrophobic groups do not fit in the scope
of this paper. Nevertheless, the authors concluded that different properties of hydrophobic groups
of the amphiphilic carrier are closely implicated in the stability and drug-loading capacity of the
amphiphilic carrier and micelles. HA-SA-CYS-Chol 60 presented a lower critical micellar
concentration, producing docetaxel (DTX)-loaded micelles of a smaller particle size, higher
encapsulation efficiency, and drug loading, when compared to the other hydrophobic tails [21].
Furthermore, in vivo animal studies revealed very good tumor-targeting properties and efficient
antitumor effects at very low concentrations, with low systemic toxicity of HA–SA–CYS–Chol 60
Scheme 13. Synthesis
Scheme 13. Synthesis of
of sterol-anchored
sterol-anchored polyethylene
polyethylene glycols
glycols (PEGs).
(PEGs). Reagents and conditions:
Reagents and a)
conditions: a)
micelles [21].
EDCl, DMAP, CHCl , rt, 24 h.
EDCl, DMAP, CHCl33, rt, 24 h.

The authors studied the biophysical properties of liposomes containing these two sterol-
anchored PEGs, 55 and 58, which exhibited an array of canonical PEGgylated-liposome behaviors
including retention of encapsulated small molecules, low serum protein adsorption, and reduced
cellular uptake, yet they did not exhibit long circulation [20].
Polymeric micelles are known for their variety of therapeutic applications. In this field, two
amphiphilic polymers were successfully synthesized using hyaluronic acid (HA), cholesterol, and

Scheme
Scheme 14. Synthesis of
14. Synthesis of HA-SA-CYS-Chol. Reagents and
HA-SA-CYS-Chol. Reagents and conditions:
conditions: a) Cystamine dihydrochloride,
a) Cystamine dihydrochloride,
NaOH, ◦
NaOH, CHCl
CHCl33,, 50
50 °C,
C, 22 h;
h; b) HA-SA, PyBOP,
b) HA-SA, PyBOP, DMAP,
DMAP, DMSO/FM (4:1), rt,
DMSO/FM (4:1), rt, 22 days.
days.

A new liposomal
A new liposomalformulation
formulationfor fordrug
drugdelivery
delivery purposes
purposes waswas recently
recently developed,
developed, based
based on
on the
the N-terminal
N-terminal cholesterol
cholesterol conjugation
conjugation with
with a amitochondria-penetrating
mitochondria-penetratingpeptide peptide (MPP)
(MPP) sequence,
sequence,
consisting of four amino acids [phenylalanine-arginine-phenylalanine-lysine (FRFK)].
consisting of four amino acids [phenylalanine-arginine-phenylalanine-lysine (FRFK)]. More More specifically,
the synthesis
specifically, theofsynthesis
cholesterol-phenylalanine-arginine-phenylalanine-lysine
of cholesterol-phenylalanine-arginine-phenylalanine-lysine (Chol-FRFK) 64 was
(Chol-FRFK)
achieved by coupling
64 was achieved cholesteryl
by coupling chloroformate
cholesteryl 7 with amino
chloroformate 7 withacid-bound resins (62),
amino acid-bound followed
resins by resin
(62), followed
cleavage using TFA and the removal of protecting groups (Scheme 15) [22]. The authors
by resin cleavage using TFA and the removal of protecting groups (Scheme 15) [22]. The authors developed the
liposomes
developedusing dioleoyl-sn-glycero-3-phosphoethanolamine
the liposomes (DOPE) and Chol-FRFK
using dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and for delivery
64 Chol-FRFK
of
64 for delivery of the hydrophobic drug antimycin A specifically targeted toward mitochondriaA549
the hydrophobic drug antimycin A specifically targeted toward mitochondria and lung cancer and
cells.
lung The results
cancer A549 indicated thatresults
cells. The this formulation
indicated can
thateffectively deliver the
this formulation canencapsulated drug to the
effectively deliver the
mitochondria because of the small size and moderately cationic charge of the liposomes,
encapsulated drug to the mitochondria because of the small size and moderately cationic charge of enabling
cellular uptakeenabling
the liposomes, with lowcellular
toxicity. The liposomes
uptake were found
with low toxicity. to be stablewere
The liposomes for long
foundperiods at room
to be stable for
temperature, and they acted synergistically with antimycin A, leading to the complete
long periods at room temperature, and they acted synergistically with antimycin A, leading to the disruption of
inner membrane
complete potential
disruption [22].
of inner membrane potential [22].
In 2016, six new cholesterol-derived cationic lipids, 68–73, were synthesized via ether or ester
linkages with different head groups (Scheme 16), which were used to create cationic liposomes for
nonviral gene delivery vectors [23]. The authors studied the relationship between the structure of the
synthesized lipids and the transfection efficiency and optimized gene transfection conditions of the
liposomes. They found that the chemical structure of head groups and the linkage between
cholesterol and head groups play important roles in gene delivery efficiency. Furthermore, lipids 69
transfection-efficient. The authors found that redox activities of co-liposomes and their lipoplexes
could be regulated using the alkyl ferrocene moiety. The vesicles possessing ferrocene in the reduced
state induced an efficient gene transfection capability using pEGFP-C3 plasmid DNA in three cell
lines, even better than the commercial lipofectamine 2000 (Lipo 2000). This evidence suggests that
these redox-driven systems could be used in gene delivery applications where transfection needs
Molecules 2019, 24, 116
to
12 of 68
be performed spatially or temporally [24].
Molecules 2018, 23, x 12 of 68

cholesterols 76 and 77, as well as 79 and 80, were incorporated into co-liposomes and shown to be
transfection-efficient. The authors found that redox activities of co-liposomes and their lipoplexes
could be regulated using the alkyl ferrocene moiety. The vesicles possessing ferrocene in the reduced
state induced an efficient gene transfection capability using pEGFP-C3 plasmid DNA in three cell
lines, even better than the commercial lipofectamine 2000 (Lipo 2000). This evidence suggests that
these redox-driven systems could be used in gene delivery applications where transfection needs to
be performed spatially or temporally [24].

Scheme 15. Synthesis of cholesterol-phenylalanine-arginine-phenylalanine-lysine

15. Synthesis (Chol-FRFK).
cholesterol-phenylalanine-arginine-phenylalanine-lysine (Chol-FRFK).
Reagents and
and conditions: a)N,N-diisopropylethylamine
conditions: a) N,N-diisopropylethylamine(DIPEA),
(DIPEA),CH
CH2 Cl22,,rt,
2Cl rt,16
16h;h;b)b)TFA/TIS/H
TFA/TIS/H22O
(95:2.5:2.5), 28 ◦°C,
C, 16 h.

In 2016, six new cholesterol-derived cationic lipids, 68–73, were synthesized via ether or ester
linkages with different head groups (Scheme 16), which were used to create cationic liposomes for
nonviral gene delivery vectors [23]. The authors studied the relationship between the structure of
the synthesized lipids and the transfection efficiency and optimized gene transfection conditions
of the liposomes. They found that the chemical structure of head groups and the linkage between
cholesterol
Schemeand 15.head groups
Synthesis ofplay important roles in gene delivery efficiency. Furthermore,
cholesterol-phenylalanine-arginine-phenylalanine-lysine lipids 69
(Chol-FRFK).
and 73 exhibited
Reagents andhigher transfection
conditions: efficiency and lower(DIPEA),
a) N,N-diisopropylethylamine toxicity than
CH2Clthose of h;
2, rt, 16 theb)tested commercial
TFA/TIS/H 2O

liposomes DC-Chol
(95:2.5:2.5), 28 °C,and
16 h.lipofectamine 2000, even in the presence of serum [23].

Scheme 16. Synthesis of cholesterol-based cationic lipids. Reagents and conditions: a) p-TsCl,
pyridine, CHCl3, rt, 23 h; b) hexane-1,6-diol, anhydrous 1,4-dioxane, reflux, 7 h; c) CBr4, PPh3, CH2Cl2,
rt, 6 h; d) Et3N, MeOH/EtOAc (1:1), 90 °C in screw-top pressure tube, 48 h; e) HN3, PPh3, DIAD, 3
days; f) Me2N∙HCl, NaOH, toluene, 90 °C in screw-top pressure tube, 72 h; g) Et2N, K2CO3,
MeOH/EtOAc (1:1), 90 °C in screw-top pressure tube, 48 h; h) 2-chloroethanol, acetonitrile, 90 °C in
screw-top pressure tube, 24 h; i) Boc-aminohexanoic acid, DCC/NHS, THF, 0 °C, 7 h; j) TFA, CH2Cl2,
0 °C, 4 h.

Scheme 16. 16.Synthesis

Synthesisof of
cholesterol-based
cholesterol-based cationic lipids.
cationic Reagents
lipids. and conditions:
Reagents a) p-TsCl,a)pyridine,
and conditions: p-TsCl,
CHCl3 , rt,CHCl
pyridine, 23 h;3b)
, rt,hexane-1,6-diol, anhydrous
23 h; b) hexane-1,6-diol, 1,4-dioxane,
anhydrous reflux, 7 reflux,
1,4-dioxane, h; c) CBr 4 , c)
7 h; PPh 3 , 4CH
CBr 2 Cl
, PPh , rt,2Cl
3,2CH 6 h;
2,

d) Et ◦ C in screw-top pressure tube, 48 h; e) HN , PPh , DIAD, 3 days; f)

rt, 3 N,
6 h; d)MeOH/EtOAc
Et3N, MeOH/EtOAc (1:1), 90(1:1), 90 °C in screw-top pressure tube, 48 h;3 e) HN 3 3, PPh3, DIAD, 3
Me2 N·f) HCl, ◦ C in screw-top pressure tube, 72 h; g) Et N, K CO , MeOH/EtOAc
days; MeNaOH,
2N∙HCl,toluene,
NaOH,90toluene, 90 °C in screw-top pressure tube, 2 722 h; 3g) Et2N, K2CO3,
(1:1), ◦
90 C in screw-top pressure tube, 48pressure
h; h) 2-chloroethanol, 90 ◦ C in
MeOH/EtOAc (1:1), 90 °C in screw-top tube, 48 h; h)acetonitrile,
2-chloroethanol, screw-top pressure
acetonitrile, 90 °C in
tube, 24 h; i) Boc-aminohexanoic acid, DCC/NHS, THF, 0 ◦ C, 7 h; j) TFA, CH Cl , 0 ◦ C, 4 h.
screw-top pressure tube, 24 h; i) Boc-aminohexanoic acid, DCC/NHS, THF, 02°C,2 7 h; j) TFA, CH2Cl2,
0 °C, 4 h.
In 2015, Vulugundam and coworkers reported the design and synthesis of new redox-active
monomeric 76 and 77, and dimeric (gemini) 79 and 80, cationic lipids based on ferrocenylated
cholesterol derivatives for the development of gene delivery systems (Scheme 17). The cationic
Molecules 2019, 24, 116 13 of 68

cholesterols 76 and 77, as well as 79 and 80, were incorporated into co-liposomes and shown to be
transfection-efficient. The authors found that redox activities of co-liposomes and their lipoplexes
could be regulated using the alkyl ferrocene moiety. The vesicles possessing ferrocene in the reduced
state induced an efficient gene transfection capability using pEGFP-C3 plasmid DNA in three cell
lines, even better than the commercial lipofectamine 2000 (Lipo 2000). This evidence suggests that
these redox-driven systems could be used in gene delivery applications where transfection needs to be
Molecules 2018,spatially
performed 23, x or temporally [24]. 13 of 68
Molecules 2018, 23, x 13 of 68

Scheme
Scheme 17.
Scheme 17.Synthesis
17. Synthesis
Synthesis of
of redox-active
of
redox-active ferrocene
redox-active containing
ferrocene
ferrocene cationic
containing
containing monomeric
cationic
cationic (CHM-C6F
monomeric
monomeric and
and CHM-
(CHM-C6F
(CHM-C6F and
CHM-
C11F)
C11F) and gemini (CHD-C6F and CHD-C11F) cholesteryl lipids. Reagents and conditions: a)
and
CHM-C11F) gemini
and (CHD-C6F
gemini and
(CHD-C6F CHD-C11F)
and cholesteryl
CHD-C11F) lipids.
cholesteryl Reagents
lipids. and
Reagents conditions:
and conditions:
a)
MeOH/EtOAc
a) MeOH/EtOAc
MeOH/EtOAc (1:1),
(1:1), reflux,
(1:1), 4–6
reflux,
reflux, days.
4–64–6 days.
days.

A series
A series of
of macrocycle
macrocycle polyamine
macrocycle polyamine (cyclen
polyamine (cyclen and
and 1,4,7-triazacyclononane
1,4,7-triazacyclononane (TACN))-based
1,4,7-triazacyclononane (TACN))-based cationic
cationic
lipids 85 and 88, bearing cholesterol as a hydrophobic
hydrophobic tail, were
were synthesized
synthesized through
lipids 85 and 88, bearing cholesterol as a hydrophobic tail, were synthesized through ring-opening ring-opening
reactions (Scheme18).
reactions (Scheme
(Scheme 18).
18). These
These These cationic
cationic
cationic lipids,lipids,
lipids, 85 88, and
and 85
85 and 88, were 88,
were usedwere
used in used in combination
in combination
combination with with
with 1,2-dioleoyl-
1,2-dioleoyl-
1,2-dioleoyl-sn-glycero-
sn-glycero- -3-phosphoethanolamine
-3-phosphoethanolamine (DOPE) to (DOPE)
prepare to prepare
lipoplexes, whichlipoplexes, which
efficiently
sn-glycero- -3-phosphoethanolamine (DOPE) to prepare lipoplexes, which efficiently condense DNA efficiently
condense DNA
condense
into DNA into
into nanoparticles
nanoparticles nanoparticles
with
with aa proper with
proper size
size and
anda proper
zeta size and[25].
zeta potential
potential zeta potential [25].
[25].

Scheme 18. Synthesis of macrocyclic polyamine (cyclen and 1,4,7-triazacyclononane

Scheme 18. Synthesis of macrocyclic polyamine (cyclen and 1,4,7-triazacyclononane (TACN))-based
(TACN))-based
cationic lipids bearing a cholesterol tail.
tail. Reagents and conditions:
conditions: a) tetrabutylammonium
tetrabutylammonium bromide
cationic lipids bearing a ◦cholesterol tail. Reagents and conditions: a) tetrabutylammonium bromide
(TBAB), NaOH, H2O, 40 °C, C, 24 h;
h; b) EtOH,
EtOH, reflux, 60 h; c)c) CH Cl22,,rt,
rt, 6 h.
(TBAB), NaOH, H2O, 40 °C, 24 24 h; b)
b) EtOH, reflux,
reflux, 60 h; c) TFA,
TFA, CH22Cl
Cl2, rt, 66 h.
h.
Lipid 85,
Lipid 85, containing cyclencyclen as aa headgroup,
headgroup, demonstrated lower lower toxicity and and better transfection
transfection
Lipid 85, containing
containing cyclen as as a headgroup, demonstrated
demonstrated lower toxicitytoxicity and better
better transfection
efficiency (TE)
efficiency in vitro, when compared to the commercial reference lipofectamine 2000 in both 7402
efficiency (TE)
(TE) in in vitro,
vitro, when
when compared
compared to to the
the commercial
commercial reference
reference lipofectamine
lipofectamine 2000 2000 in
in both
both 7402
7402
and
and A549 cancer cells. Furthermore, the authors rationalized the good serum tolerance of 85 due to the
and A549
A549 cancer
cancer cells.
cells. Furthermore,
Furthermore, the the authors
authors rationalized
rationalized thethe good
good serum
serum tolerance
tolerance of of 85
85 due
due toto
presence
the of a hydroxy group in its structure. These promising results indicated that cationic-lipid 85
the presence
presence of of aa hydroxy
hydroxy group
group in in its
its structure.
structure. These
These promising
promising results
results indicated
indicated thatthat cationic-lipid
cationic-lipid
should
85 be considered for nonviral gene vectors in in vivo applications [25].
85 should
should bebe considered
considered for for nonviral
nonviral genegene vectors
vectors inin in
in vivo
vivo applications
applications [25].[25].
Aiming
Aiming to extend the existent library of polycationic amphiphiles, Puchkoval. et al. designed
Aiming to extend the existent library of polycationic amphiphiles, Puchkov
to extend the existent library of polycationic amphiphiles, Puchkov et et al. designed
designed andand
and synthesized
synthesized amolecule,
new molecule, based
92, on on triethylenetetramine and cholesterol (a spermine
synthesized a new molecule, 92, based on triethylenetetramine and cholesterol (a spermine analogue
a new 92, based triethylenetetramine and cholesterol (a spermine analogue
analogue containing
containing the same number of amino groups but differing in the number of methylene
containing thethe same
same number
number of of amino
amino groups
groups but
but differing
differing in
in the
the number
number of of methylene
methylene units).
units). The
The
units). The
synthesis synthesis of the polycationic amphiphile 92 was based on the selective transformation of
synthesis of the polycationic amphiphile 92 was based on the selective transformation of primary
of the polycationic amphiphile 92 was based on the selective transformation of primary
primary
amines amines into secondary ones via nitrobenzenesulfonamides, and the molecule of cholesterol
amines into
into secondary
secondary ones ones via
via nitrobenzenesulfonamides,
nitrobenzenesulfonamides, and and the
the molecule
molecule of of cholesterol
cholesterol waswas
was incorporated
incorporated through alkylation of bis(sulfonamide) 89 with bromo derivative of cholesterol
incorporated through
through alkylation
alkylation of of bis(sulfonamide)
bis(sulfonamide) 89 89 with
with bromo
bromo derivative
derivative of of cholesterol
cholesterol 90 90
90 (Scheme
(Scheme 19) [26]. The authors the used the triethylenetetramine-based amphiphile 92 to prepare
(Scheme 19)19) [26].
[26]. The
The authors
authors used
used the triethylenetetramine-based
triethylenetetramine-based amphiphile
amphiphile 92 92 to
to prepare
prepare cationic
cationic
liposomes
liposomes andand concluded
concluded that that the
the transfection
transfection properties
properties ofof delivery
delivery nucleic
nucleic acids
acids inin eukaryotic
eukaryotic cells
cells
were
were inferior to those with amphiphiles based on spermine. Despite the polyamines
inferior to those with amphiphiles based on spermine. Despite the polyamines
(triethylenetetramine
(triethylenetetramine and and spermine)
spermine) having
having thethe same
same number
number of of amino
amino groups,
groups, their
their distribution
distribution was
was
significantly different, which may have resulted in the difference in their transfection
significantly different, which may have resulted in the difference in their transfection activity [26]. activity [26].
Molecules 2019, 24, 116 14 of 68

cationic liposomes and concluded that the transfection properties of delivery nucleic acids in
eukaryotic cells were inferior to those with amphiphiles based on spermine. Despite the polyamines
(triethylenetetramine and spermine) having the same number of amino groups, their distribution was
Molecules
Molecules 2018,
2018, 23,
significantly xx
different,
23, 14
14 of
of 68
which may have resulted in the difference in their transfection activity [26]. 68

Scheme 19.
19. Synthesis
Scheme 19. Synthesis of
Synthesis of polycationic amphiphile
polycationic amphiphile
amphiphile based based on
based on triethylenetetramine.
triethylenetetramine. Reagents
on triethylenetetramine. Reagents and
and
◦
conditions: a)
conditions: Cs22CO
a)a) Cs
Cs 2 CO
CO 3, ,DMF,
DMF,80
3, 3 DMF, 8080°C,
°C,C,55 5h;
h; h;b) C
C66H
b) b) C565SH,
H K
K22CO
H5 SH,
SH, ,, DMF,
K233CO
CO rt,
rt, 1.5
3 , DMF,
DMF, rt, h;
1.5 1.5c)
h; c)h;44 c)
MM 4HCl,
HCl, 1,4-
M HCl,
1,4-
dioxane/CH 2Cl2 (1:5),
1,4-dioxane/CH Cl rt,
(1:5),
dioxane/CH2Cl2 (1:5), rt, 1.5 h.
2 2 1.5 h.
rt, 1.5 h.

A newly designed
A newly
newly designedarginine-conjugated
designed arginine-conjugatedcholesterol
arginine-conjugated cholesterolderivative,
cholesterol derivative,
derivative, 94,94,
94, was
waswas recently
recently
recently reported
reported
reported for for
for the
the
the preparation
preparation of of cationic
cationic liposomes
liposomes and
and their
their interaction
interaction with
with paclitaxel
paclitaxel (PTX),
(PTX),
preparation of cationic liposomes and their interaction with paclitaxel (PTX), a widely usedaa widely
widely used
anticancer
anticancer drug. The synthesis
drug. The
The synthesis of
synthesis of cholesterol-arginine
of cholesterol-arginine ester
cholesterol-arginine ester (CAE)
ester (CAE) conjugate
(CAE) conjugate 94
conjugate was carried
94 was
94 carried out
out
through N-amidation
through N-amidation of L-arginine ethyl ester 93 with cholesteryl chloroformate 7 (Scheme 20)
of L-arginine ethyl ester 93 with cholesteryl chloroformate
chloroformate 7
7 (Scheme
(Scheme 20) [27].
20) [27].
[27].

Scheme 20. Synthesis of cholesterol-arginine conjugate. Reagents

Reagents and conditions: THF, 10–15 ◦ C,
conditions: a) THF,
Scheme 20. Synthesis of cholesterol-arginine conjugate. Reagents and
and conditions: a)
a) THF, 10–15 °C,
°C,
6–7 h.
6–7 h.

The
The authors conductedmolecular molecular dynamic simulations well as wellinasvitro
in vitro studies withPTX-
the
The authors
authors conducted
conducted molecular dynamic dynamic simulations
simulations as as well asas in vitro studies
studies with
with the
the PTX-
PTX-loaded
loaded liposomes.resultsThe results showed that these cationic liposomes enhanced loading efficiency
loaded liposomes.
liposomes. The The results showed
showed that that these
these cationic
cationic liposomes
liposomes enhanced
enhanced loading
loading efficiency
efficiency and
and
and stability
stability over over
the the conventional
conventional liposomes,
liposomes, which which
can be can be rationalized
rationalized based onbased
the on the
hydrogen hydrogen
bonding
stability over the conventional liposomes, which can be rationalized based on the hydrogen bonding
bonding
between between CAE and PTX and the deeper penetration of PTX in the bilayer. Moreover, these
between CAE CAE and and PTX PTX and
and the
the deeper
deeper penetration
penetration of of PTX
PTX in in the
the bilayer.
bilayer. Moreover,
Moreover, these these novel
novel
novel liposomes demonstrated
liposomes improved cytotoxicity in three different cell lines (MDA MB 231, H5V,
liposomes demonstrated
demonstrated improvedimproved cytotoxicity
cytotoxicity in in three
three different
different cell
cell lines
lines (MDA
(MDA MB MB 231,
231, H5V,
H5V, and
and
and HDMEC)
HDMEC) and and enhanced
enhanced endothelial
endothelial cell cell migration
migration inhibition
inhibition compared
compared toto conventional
conventional liposomes.
liposomes.
HDMEC) and enhanced endothelial cell migration inhibition compared to conventional liposomes.
The
The absence of genotoxicity makesmakes cholesterol-arginine ester an interesting biocompatible
94 an biocompatible cationic
The absence
absence of of genotoxicity
genotoxicity makes cholesterol-arginine
cholesterol-arginine ester ester 9494 an interesting
interesting biocompatible cationic cationic
ligand
ligand in
in drug
drug delivery
delivery applications
applications [27].
[27].
ligand in drug delivery applications [27].
The
The design and synthesis of thermosensitive polymers of N-(2-hydroxypropyl)methacrylamide
The design
design andand synthesis
synthesis of of thermosensitive
thermosensitive polymers
polymers of of N-(2-hydroxypropyl)methacrylamide
N-(2-hydroxypropyl)methacrylamide
mono/dilactate
mono/dilactate of different
different molecular
molecular weights weights andand composition
composition bearing
bearing a cholesterol anchor anchor 98
mono/dilactate of different molecular weights and composition bearing aa cholesterol
of cholesterol anchor 98 98
(Chol-pHPMAlac)
(Chol-pHPMAlac) was
was reported
reported in
in 2014
2014 (Scheme
(Scheme 21).
21). These
These new
new cholesterol-based
cholesterol-based polymers
polymers were
98 were
98
(Chol-pHPMAlac) was reported in 2014 (Scheme 21). These new cholesterol-based polymers 98 were
incorporated
incorporated onto liposome formulations loaded loaded with DOX. DOX. The authors concluded that that the release
incorporated onto onto liposome
liposome formulations
formulations loaded with with DOX. The The authors
authors concluded
concluded that the the release
release
of
of DOX from such liposome formulations was effective at low temperatures and could be adjusted
of DOX
DOX from from such
such liposome
liposome formulations
formulations was was effective
effective at at low
low temperatures
temperatures and and could
could be be adjusted
adjusted
according
according to the
to the grafting density
density of Chol-pHPMAlac Chol-pHPMAlac 98 with
Chol-pHPMAlac 98. Chol-pHPMAlac a cloud point of
according
◦ to the grafting
grafting density of of Chol-pHPMAlac 98. 98. Chol-pHPMAlac 98 98 with
with aa cloud
cloud point
point of
of
19.0
19.0 C and aa M n of 10.0
10.0 kDa showed interesting releasing
releasing features becausebecause it was was stable at body
19.0 °C
°C and
and a M Mnn of
of 10.0 kDakDa showed
showed interesting
interesting releasing features
features because it it was stable
stable atat body
body
temperature,
temperature, releasing itsitscontent only under hyperthermia conditions. These releasing features make
temperature, releasing
releasing its content
content only only under
under hyperthermia
hyperthermia conditions.
conditions. These
These releasing
releasing features
features
these
make liposomes interesting for local drug delivery using hyperthermia [28].
make these
these liposomes
liposomes interesting
interesting for for local
local drug
drug delivery
delivery using
using hyperthermia
hyperthermia [28]. [28].
Molecules 2019,
Molecules 2018, 23, 116
x 15 of 68
Molecules 2018, 24,
23, x 15
15 of
of 68
68
Molecules 2018, 23, x 15 of 68

Scheme 21. Synthesis of poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate with a cholesterol

Scheme 21.
Scheme 21. Synthesis of poly(N-(2-hydroxypropyl)methacrylamide
Synthesis of
of poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate with
mono/dilactate with aa cholesterol
cholesterol
Scheme 21. Synthesis
anchor (Chol-pHPMAlac). poly(N-(2-hydroxypropyl)methacrylamide mono/dilactate
Reagents and conditions: a) AIBN, 1,4-dioxane, 70 °C, 24with
h. a cholesterol
anchor (Chol-pHPMAlac).
anchor (Chol-pHPMAlac). Reagents and
Reagents and conditions:
and conditions: a) AIBN, 1,4-dioxane,
AIBN, 1,4-dioxane,
a) AIBN, 70
1,4-dioxane, 70 °C,
◦
70 °C, 24
C, 24 h.
24 h.
h.
anchor (Chol-pHPMAlac). Reagents conditions: a)
Recently, Asayama and coworkers reported a byproduct-free PEGylation method for the
Recently, Asayama
Recently, Asayama and coworkers
coworkers reported
reported aa byproduct-free
byproduct-free PEGylation
PEGylation method for the the
modification ofAsayama
Recently, and strategy
insulin. The coworkers reported
involves theareaction
byproduct-free PEGylation
of cholesterol method for
chloroformate the
7 with
insulin. The
modification of insulin. The strategy
strategy involves
involves the
the reaction of cholesterol chloroformate 7 with with
modification
aminopropyl of insulin.
mPEG The
in the strategyof involves
presence the reaction
triethylamine to affordofthe
cholesterol
conjugatechloroformate
Chol-U-Pr-mPEG7 with
99
aminopropyl mPEG in the presence of triethylamine to afford the conjugate Chol-U-Pr-mPEG 99
aminopropyl mPEG in the with
(Scheme 22), complexation presence of in
insulin triethylamine to afford
aqueous solution, andthe conjugatefreeze-drying
subsequent Chol-U-Pr-mPEG
[29]. 99
(Scheme 22),
(Scheme 22), complexation
complexation with
with insulin
insulin in
in aqueous
aqueous solution,
solution,and
andsubsequent
subsequentfreeze-drying
freeze-drying[29].
[29].
(Scheme 22), complexation with insulin in aqueous solution, and subsequent freeze-drying [29].

Scheme 22. Synthesis of Chol-U-Pr-mPEG and complexation with insulin. Reagents and conditions:
Scheme 22. Synthesis of Chol-U-Pr-mPEG
Scheme 22. Chol-U-Pr-mPEG and and complexation
complexation with
with insulin.
insulin. Reagents and conditions:
Scheme 22. Synthesis
a) aminopropyl mPEG,ofEtChol-U-Pr-mPEG ◦ C,and
3N, CHCl3, 40 °C, 24 h.complexation with insulin. Reagents and conditions:
a) aminopropyl
aminopropyl mPEG,
mPEG, EtEt33N, CHCl3,, 40 °C, 24 h.
24 h.
a) aminopropyl mPEG, Et3N, CHCl3, 40 °C, 24 h.
The Chol-U-Pr-mPEG/insulin
The Chol-U-Pr-mPEG/insulin complex complex notnot only
only preserved
preserved the insulin conformation,
conformation, but also
The Chol-U-Pr-mPEG/insulin complex not only preserved the insulin
insulin conformation, but also also
The Chol-U-Pr-mPEG/insulin
was shown
shown to
to bebe effective
effective in in
itsits complex
protection
protection fromnot
from only
hydrolysispreserved
hydrolysisby by the insulin
protease
protease and and
in conformation,
the insuppression but
the suppression
of also
of
blood
was shown to be effective in its protection from hydrolysis by protease and in the suppression of
was shown
blood glucose
glucose to
levels in be effective
levels
micein in
mice [29].its protection from hydrolysis by protease and in the suppression of
blood glucose levels in[29].
mice [29].
blood glucose levels in mice [29].
3. Anticancer, Antimicrobial, and Antioxidant Compounds Compounds
3. Anticancer, Antimicrobial, and Antioxidant Compounds
3. Anticancer, Antimicrobial, and Antioxidant Compounds
Many new newcholesterol
cholesterol derivatives
derivativesbearing a wide
bearing a range
wide of bioactive
range scaffolds scaffolds
of bioactive have beenhave
developed
been
Many new cholesterol derivatives bearing a wide range of bioactive scaffolds have been
in theMany
search
developed new
infor cholesterol
thenewsearch for derivatives
anticancer, bearing
newantimicrobial,
anticancer, a wide range
or antioxidant
antimicrobial, of bioactive
agents
or with improved
antioxidant scaffolds
agents have
efficacy.
with Inbeen
this
improved
developed in the search for new anticancer, antimicrobial, or antioxidant agents with improved
developed
context, in the
Rodríguez search
et al. for new
described anticancer,
an antimicrobial,
efficient synthesis of or antioxidant agents
(6E)-hydroximinosteroid
efficacy. In this context, Rodríguez et al. described an efficient synthesis of (6E)-hydroximinosteroid with improved
homodimers
efficacy. In this context, Rodríguez et al. described an efficient synthesis of (6E)-hydroximinosteroid
efficacy.
(105)
homodimers In this
linking two context,
(105) steroidal
linking Rodríguez
monomers
two et al.
steroidal atdescribed an
position 3at
monomers efficient
ofposition 3synthesis
the steroid of via
scaffold
of the steroid(6E)-hydroximinosteroid
ruthenium-catalyzed
scaffold via ruthenium-
homodimers (105) linking two steroidal monomers at position 3 of the steroid scaffold via ruthenium-
homodimers
cross-metathesis (105)reaction
linking two
catalyzed cross-metathesis steroidal
(Scheme
reaction 23). monomers23). at
The synthesis
(Scheme position
The the 3precursor
ofsynthesisof the
of steroid scaffoldwas
themonomers
precursor via ruthenium-
carried was
monomers out
catalyzed cross-metathesis reaction (Scheme 23). The synthesis of the precursor monomers was
catalyzed
through
carried out cross-metathesis
a five-step
throughreaction
a five-step reaction
sequence (Scheme
reactionstarting 23).
from
sequence The synthesis
cholesterol
starting from28 of the precursor
(Scheme 23)
cholesterol [30].
28 (Scheme monomers
23) [30]. was
carried out through a five-step reaction sequence starting from cholesterol 28 (Scheme 23) [30].
carried out through a five-step reaction sequence starting from cholesterol 28 (Scheme 23) [30].

Scheme 23. Synthesis of (6E)-hydroximinosteroid

23. Synthesis (6E)-hydroximinosteroid homodimers. Reagents and
homodimers. Reagents and conditions: a)
Scheme 23. Synthesis of (6E)-hydroximinosteroid homodimers. Reagents and conditions: a)
Scheme 23. Synthesis
montmorillonite
montmorillonite K-10,
K-10,CHCl
CHCl 55◦°C,
of 3(6E)-hydroximinosteroid
3,, 55 C, 77days;
days;b) homodimers. Reagents
b)m-chloroperoxybenzoic
m-chloroperoxybenzoic acid
acid and CHCl
(m-CPBA),
(m-CPBA),conditions:
CHCl 3 , rt, a)
2020
3, rt, h;
montmorillonite K-10, CHCl3, 55 °C, 7 days; b) m-chloroperoxybenzoic ◦ C, 45 min; e) Hacid (m-CPBA), CHCl3, rt, 20
montmorillonite
h; CrO
c) c) CrO
3 , H
3 , H
2 O,
2 O, K-10, CHCl
butan-2-one,
butan-2-one,rt,
3 , 55
2
rt, h;
2 °C,
d)
h; d)7
SOdays;
SOCl
2 2
2 Cl,b)
2, m-chloroperoxybenzoic
pyridine,
pyridine, 00 °C, 45 min; acid
2
2 , (m-CPBA),
Lindlar
Lindlar CHCl
catalyst,
catalyst, , rt,
pyridine,
pyridine,
3 20
h; c) CrO3, H2O, butan-2-one, rt, 2 h; d) SO2Cl2, pyridine, 0 °C, 45 min; e) H2, Lindlar catalyst, pyridine,
◦ C, 0
rt, c)
h; 5 h; f) 3Grubbs
CrO 1st generation
, H2O, butan-2-one, rt, catalyst,
2 h; d) SOCH
2Cl222Cl 22,, 45
45 °C,
, pyridine, overnight;
°C, 45 min;
overnight; g)e)
g) NH
NH ,OH ·HCl,catalyst,
H222OH∙HCl,
Lindlar pyridine,
pyridine, rt,11h.h.
pyridine,
rt,
rt, 5 h; f) Grubbs 1st generation catalyst, CH2Cl2, 45 °C, overnight; g) NH2OH∙HCl, pyridine, rt, 1 h.
rt, 5 h; f) Grubbs 1st generation catalyst, CH2Cl2, 45 °C, overnight; g) NH2OH∙HCl, pyridine, rt, 1 h.
Molecules 2019, 24, 116 16 of 68
Molecules
Molecules 2018,
2018, 23,
23, xx 16
16 of
of 68
68

The
The cytotoxic
cytotoxic activity
activity ofof (6E)-hydroximinosteroid
(6E)-hydroximinosteroid homodimers
homodimers (105)
homodimers (105) was
(105) was evaluated
was evaluated in
evaluated in vitro
in vitro
using human lung carcinoma A549,
using human lung carcinoma A549, colon colon adenocarcinoma
adenocarcinoma HCT-116, human Caucasian glioblastoma
HCT-116, human Caucasian glioblastoma
multiform
multiform T98G,
T98G, andand human
human pancreatic
pancreatic adenocarcinoma
adenocarcinoma PSN1 PSN1 cells.
cells. Only
Only homodimer
homodimer 105
homodimer 105 (n
(n == 2)
2)
showed
showed selective
selective cytotoxicity
cytotoxicity against
against HCT-116
HCT-116 cells:
cells: However,
However, it it presented
presented no no activity
activity against
against thethe
remaining
remaining cell lines. Nevertheless,
cell lines. Nevertheless, the the monomer
monomer counterparts
counterparts 106106 and
and 107
107 showed
showed better
better cytotoxic
cytotoxic
activity against all cell lines when compared to
activity against all cell lines when compared to homodimer homodimer
homodimer 105 105 [30].
105 [30].
[30].
Richmond
Richmond et et al.
al. reported
reported the
the synthesis
synthesis of
of four
four new
new (6E)-hydroximinosteroids
(6E)-hydroximinosteroids (109),
(6E)-hydroximinosteroids (109), starting
starting from
from
the corresponding
the corresponding ketones
correspondingketones (108)
ketones(108) derived
(108)derived from
derivedfrom cholesterol.
fromcholesterol.
cholesterol. The
The
The authors
authors
authors evaluated
evaluated
evaluated the
thethe cytotoxicity
cytotoxicity
cytotoxicity of
of all
of
all
all the
the prepared
prepared
the compounds
compounds
prepared compounds (109)(109) and
and compared
and compared
(109) the
the results
the results
compared to thoseto
results toofthose of
of five
five polyhydroxylated
five polyhydroxylated
those sulfated
polyhydroxylated
sulfated
analogs analogs
sulfated(110) (110)
(110) (Scheme
(Scheme
analogs 24) [31]. 24)
(Scheme 24) [31].
[31].

Scheme 24.
24. Synthesis
Scheme 24. Synthesis
Synthesis of (6E)-hydroximinosteroids.
(6E)-hydroximinosteroids. Reagents
of (6E)-hydroximinosteroids. Reagents and
Reagents conditions: a)
and conditions: NH ·HCl,
NH222OH∙HCl,
a) NH OH
OH∙HCl,
NaOAc ·
NaOAc∙3H3H 22O,
O, EtOH,
EtOH,
NaOAc∙3H2O, EtOH, rt. rt.
rt.

Upon evaluation
Upon evaluationofof
evaluation thethe
of cytotoxic
the activity
cytotoxic
cytotoxic of theof
activity
activity ofsteroidal
the oxime 109
the steroidal
steroidal against
oxime
oxime 109two
109 prostate
against
against twocarcinoma
two prostate
prostate
cell lines (PC-3 and LNCaP), the authors concluded that oxime 109 (R 1 = R4 = OH, R2 4= R3 = H)2 was3
carcinoma
carcinoma cell cell lines
lines (PC-3
(PC-3 and
and LNCaP),
LNCaP), the the authors
authors concluded
concluded that
that oxime
oxime 109
109 (R
(R1 == R
1
R4 == OH,
OH, R R2 == R
R3
5 = H, R6 = OSO
=the
H) most
was active
the compound
most active for PC-3,
compound while
for for
PC-3, LNCaP
while the
for trisulfated
LNCaP the analog 110
trisulfated
= H) was the most active compound for PC-3, while for LNCaP the trisulfated analog 110 (R = H, R6(R
analog 110 (R 55 = H,
3 Na)
R 6

was
== OSO
OSO the most
33Na)
Na) was
wasactive
the one [31].
the most
most active
active one
one [31].
[31].
A
A new greener methodology involving steroidal
new greener methodology involving steroidal epoxides
epoxides as as intermediates
intermediates forfor the
the synthesis
synthesis of of
steroidal β-aminoalcohols
steroidal β-aminoalcohols
β-aminoalcohols was was recently
recently reported.
reported. The synthesis of
The synthesis of β-aminoalcohol
β-aminoalcohol
β-aminoalcohol 112 112 involved
involved two two
steps:
steps: i) Epoxidation
Epoxidation of
i) Epoxidation cholesterol
cholesterol 28
of cholesterol 28 conducted
conducted by m-chloroperoxybenzoic
by m-chloroperoxybenzoic
m-chloroperoxybenzoic acid acid (m-CPBA);
acid (m-CPBA);
(m-CPBA); and and ii)
and ii)
ii)
solvent-free aminolysis
solvent-free aminolysis ofof epoxide 111 mediated
epoxide 111 mediated by by sulfated
sulfated zirconia
sulfated zirconia(Scheme
zirconia (Scheme25)
(Scheme 25)[32].
25) [32].
[32].

H
H
H H
H
H
a)
a) b)
b)
HO
HO
HO
HO HN
HO :: (5:1)
(5:1) HN
HO
HO HO O
28 O 112
28 111
111 112

Scheme 25.
Scheme Synthesis of
25. Synthesis of 6β-phenylaminocholestan-3β,5α-diol.
6β-phenylaminocholestan-3β,5α-diol. Reagents and
6β-phenylaminocholestan-3β,5α-diol. Reagents
Reagents and conditions:
and conditions: a)
conditions: a) m-CPBA,
a) m-CPBA,
m-CPBA,
CH Cl , 30 min; b) ZrO /SO 2− (50% w/w), aniline, 120 ◦ C, 6 h.
CH222Cl
Cl222,, 30
30 min;
min; b)
b) ZrO
ZrO222/SO
/SO442−4 (50%
(50% w/w),
w/w), aniline,
aniline, 120
120 °C,
°C, 66 h.
2−
h.

The antiproliferative
The activity of the cholesterol-based β-aminoalcohol 112 112 was evaluated using
The antiproliferative
antiproliferative activity
activity of
of the
the cholesterol-based
cholesterol-based β-aminoalcohol
β-aminoalcohol 112 was was evaluated
evaluated using
using
MCF-7 cells,
MCF-7 cells, andthe the results showed better cytotoxic effects than cholesterol 28 itself, byeither by
MCF-7 cells, and
and the results
results showed
showed better
better cytotoxic
cytotoxic effects
effects than
than cholesterol
cholesterol 28 28 itself,
itself, either
either by crystal
crystal
crystalstaining
violet violet stainingor (CVS) or 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromideassays.
(MTT)
violet staining (CVS)
(CVS) or 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium
3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide bromide (MTT)(MTT) assays.
assays.
Furthermore,Furthermore, cell images obtained by Harris’ hematoxylin and eosin staining protocol
Furthermore, cell
cell images
images obtained
obtained byby Harris’
Harris’ hematoxylin
hematoxylin and and eosin
eosin staining
staining protocol
protocol evidenced
evidenced
evidenced of
formation formation of bodies
apoptotic bodiesofbecause of the presence of cholesterol β-aminoalcohol 112 in
formation of apoptotic bodies because of the presence of cholesterol β-aminoalcohol 112
apoptotic because the presence of cholesterol β-aminoalcohol 112 in
in aa dose-
dose-
a dose-dependent
dependent fashion [32].
dependent fashion
fashion [32].
[32].
The synthesis
The of new steroidal 5α,8α-endoperoxides starting from
from different steroids, including
The synthesis
synthesis of of new
new steroidal
steroidal 5α,8α-endoperoxides
5α,8α-endoperoxides starting
starting from different
different steroids,
steroids, including
including
cholesterol, was
cholesterol, was reported, involving
involving a four-step synthetic protocol. It It involved thethe introduction of of a
cholesterol, was reported,
reported, involving aa four-step
four-step synthetic
synthetic protocol.
protocol. It involved
involved the introduction
introduction of aa
diene
diene in the cholesterol 28 structure through allylic bromination followed by elimination, and finally a
diene in
in the
the cholesterol
cholesterol 28 28 structure
structure through
through allylic
allylic bromination
bromination followed
followed by by elimination,
elimination, andand finally
finally
photoinduced
aa photoinduced formation ofof the cholesterol-based 5α,8α-endoperoxide 115 (Scheme 26)26) [33].
photoinduced formation
formation of the the cholesterol-based
cholesterol-based 5α,8α-endoperoxide
5α,8α-endoperoxide 115 115 (Scheme
(Scheme 26) [33].
[33].
Molecules 2018, 23, x 17 of 68
Molecules 2019, 24, 116 17 of 68
Molecules 2018, 23, x 17 of 68

Scheme 26. Synthesis of cholesterol-based 5α,8α-endoperoxide. Reagents and conditions: a) Ac2O,

CH2Cl2, pyridine,
Scheme
Scheme 26. rt, 6 h;
26. Synthesis
Synthesis ofb)
of 1. N-bromosuccinimide
cholesterol-based
cholesterol-based (NBS), NaHCOReagents
5α,8α-endoperoxide.
5α,8α-endoperoxide. 3, cyclohexane,
Reagents reflux, 1 h; 2.
and conditions:
and conditions: a) TBAB,
a) Ac
Ac2O,O,
tetrabutylammonium
CH Cl , pyridine, rt, fluoride
6 h; b) (TBAF),
1. THF, 0 °C,
N-bromosuccinimide 4 h; c)
(NBS),NaOMe,
NaHCO MeOH,
CH22Cl22, pyridine, rt, 6 h; b) 1. N-bromosuccinimide (NBS), NaHCO3, cyclohexane, 3 , rt, 12 h;
cyclohexane, d) O 2, eosin
reflux, 1
reflux, 1 h; 2. TBAB,h; Y,
2.
pyridine,
TBAB, hv, 0 °C, 30
tetrabutylammonium min. fluoride (TBAF), ◦
tetrabutylammonium fluoride (TBAF), THF,THF,
0 °C,0 4 C,
h; 4c)h;NaOMe,
c) NaOMe, MeOH,
MeOH, rt,rt,
1212h;h;d)d)OO2,2 ,eosin
eosinY,
Y,
pyridine, hv, 0 ◦ C, 30 min.
pyridine, hv, 0 °C, 30 min.
The authors evaluated the in vitro antiproliferative activities of the 5α,8α-endoperoxides against
human The authors
Thecancer
authors evaluated
cell the in
lines derived
evaluated the in from
vitro antiproliferative
vitro antiproliferative
various human cancer activities
activities of the
types,
of the 5α,8α-endoperoxides
such as human hepatocellular
5α,8α-endoperoxides against
against
human
cancer cancer
cell cell
lines lines
(HepG2, derived
SK-Hep1)from various
and human human
breast cancer
cancer types,
cell such
lines
human cancer cell lines derived from various human cancer types, such as human hepatocellular as human
(MDA-MB-231, hepatocellular
MCF-7). cancer
It was
cell
cancerlines
found cell (HepG2,
thatlines
some SK-Hep1)
compounds
(HepG2, and
SK-Hep1) human
exhibited breast cancer
potentbreast
and human cell
anticancer lines (MDA-MB-231,
cancer activities MCF-7).
through inducing
cell lines (MDA-MB-231, It
MCF-7). was
cancer found
cell
It was
that some
apoptosis compounds
against the exhibited
four potent
tested anticancer
cancer cell activities
lines, through
particularly
found that some compounds exhibited potent anticancer activities through inducing cancer cell inducing
the cancer cell
cholesterol-based apoptosis
5α,8α-
against
apoptosis theagainst
endoperoxide four tested
115, the cancer
which cell
the lines,
wastested
four most particularly
promising
cancer the cholesterol-based
derivative,
cell lines, presenting
particularly 5α,8α-endoperoxide
IC 50 values ranging
the cholesterol-based 115,
from
5α,8α-
which
8.07 to was
12.25the
endoperoxide μMmost
[33].
115, promising
which was thederivative, presenting
most promising IC50 values
derivative, ranging from
presenting IC50 8.07
valuesto 12.25
ranging µMfrom[33].
A six-step synthetic
8.07 to 12.25 μM [33]. route based on cholesterol 28 as a starting material was designed to prepare
two newAnew steroidal
steroidal
six-step thiadiazole
routederivatives,
thiadiazole
synthetic 121 (R
derivatives,
based 121= H,
on cholesterol (R28Me),
= asH,awith
Me),anwith
startingA-homo an lactam
material A-homo andlactam
was designeda B-norsteroidal
toand a B-
prepare
skeleton
norsteroidal(Scheme 27)
skeleton [34]. The
(Scheme antiproliferative
27) [34]. The activity of
antiproliferative compounds
activity
two new steroidal thiadiazole derivatives, 121 (R = H, Me), with an A-homo lactam and a B- 118–121
of against
compounds various
118–121 cancer
against
cell linescancer
various
norsteroidal wasskeleton
evaluated,
cell lines and
was
(Scheme the results
evaluated,
27) [34]. and
Theshowed
the thatshowed
results
antiproliferativecompounds 120 (R = Ph)
that compounds
activity of compounds 120and
(R Ph)(Rand
=121
118–121 = Me)
121
against
displayed
(R = Me)cancer
various excellent
displayed selective
excellent
cell lines inhibition to the
selective inhibition
was evaluated, A-549 (human
to the showed
and the results lung
A-549 (human carcinoma) cell line,
lung carcinoma)
that compounds with
120 (R cell IC
= Ph)line, values
50andwith
121
of
IC 7.8 and
values 8.0
of µM,
7.8 respectively
and 8.0 μM, [34].
respectively [34].
(R = Me) displayed excellent selective inhibition to the A-549 (human lung carcinoma) cell line, with
50

IC50 values of 7.8 and 8.0 μM, respectively [34].

Scheme 27. 27. Synthesis

Synthesis ofofsteroidal
steroidalthiadiazoles.
thiadiazoles.Reaction
Reactionconditions:
conditions:a)a)O3O/(CH
3/(CH33))22S; b)Al
S; b) Al22O
O33/Ph;
/Ph; c)
Jones reagent, acetone,
acetone, rt;
rt;d) NH22NHCSNHR,
d)NH NHCSNHR,EtOH, EtOH,AcOH,
AcOH,8080 ◦ C; e) NH OH·HCl, NaOAc, EtOH,
Scheme 27. Synthesis of steroidal thiadiazoles. Reaction conditions: a) O3/(CH3)2S; b) Al2O3/Ph; 60
°C; e) NH 2OH∙HCl,
2 NaOAc, EtOH, c)
60 ◦ 0 ◦ C.
°C; f)C;reagent,
Jones
f) SOCl
SOCl 2 , THF,
2, THF, 0 °C.
acetone, rt; d) NH2NHCSNHR, EtOH, AcOH, 80 °C; e) NH2OH∙HCl, NaOAc, EtOH, 60
°C; f) SOCl2, THF, 0 °C.
In 2017,Martínez-Pascual
In 2017, Martínez-Pascualet etal. al. reported
reported a newa three-step
new three-step
methodmethod for the synthesis
for the synthesis of 6a-aza-ofB-
6a-aza- B -homo
homoInsteroidal steroidal
lactam lactam
124 using 124 using cholesterol
cholesterol 28 as a 28 as
starting a starting
material. material.
This new This new
methodologymethodology
involved
2017, Martínez-Pascual et al. reported a new three-step method for the synthesis of 6a-aza-B-
involved the formation
the formation of a hydroximino intermediate 123 obtained in a two-step sequence from the
homo steroidaloflactam
a hydroximino intermediate
124 using cholesterol 28 123
as a obtained in a two-step
starting material. sequence
This new from theinvolved
methodology starting
starting
cholesterolcholesterol 28
28 (Scheme (Scheme 28) [35].
28) [35]. intermediate 123 obtained in a two-step sequence from the starting
the formation of a hydroximino
cholesterol 28 (Scheme 28) [35].
Molecules 2018, 23, x 18 of 68

Molecules 2019, 24, 116 18 of 68

Molecules 2018, 23, x 18 of 68

Scheme 28. Synthesis of 6a-aza-B-homo lactams. Reagents and conditions: a) NaNO2, Ac2O, BF3OEt2,
AcOH, rt, 1 h; b) Na2CO3, CH2Cl2/MeOH (1:1), reflux, 2.5 h; c) SOCl2, dioxane, rt, 20 min.

The new compound 124 was evaluated as an antiproliferative agent against six human solid
tumor cell lines, displaying only moderate activity against the screened cell lines [35].
D’yakonov
Scheme et al. synthesized
28. Synthesis of 6a-aza-B-homo two lactams.
new hybrid
lactams. Reagents
Reagentscompounds
and
and conditions: based
conditions: on cholesterol
a)NaNO
a) NaNO Ac22O, BF33and
22,, Ac OEt22,,1,14-
tetradeca-(5Z,9Z)-dienedicarboxylic
AcOH, rt, 1 h; b) Na 2CO33,, CH CH22Cl acid, (1:1),
Cl2/MeOH
2 /MeOH
127 and
(1:1), 129,
reflux,
reflux, which
2.5
2.5 h;h;c)c)SOClwere
SOCl synthetic
, dioxane,
2, 2dioxane, 20analogues
rt,rt, 20min.min. of natural
(5Z,9Z)-dienoic acids. The synthetic methodology relied on the preparation of cholesterol-based
The126
oximes
The new
new andcompound
128 and their
compound 124 was
124 evaluated
further
was as
as an
esterification
evaluated an antiproliferative agent
agent against
using 1,14-tetradeca-(5Z,9Z)-dienedicarboxylic
antiproliferative against six six human
human solidsolid
tumor
tumor cell
acid (Scheme lines, displaying
29)displaying
cell lines, [36]. Theonlyonly moderate
authors activity
evaluated
moderate activity against
theagainst
in vitro the screened
thecytotoxic cell
screenedactivities lines
cell lines [35].[35].
of the synthesized
D’yakonov
compounds
D’yakonov 126–129et al.
et al.
against synthesized
Jurkat two
synthesized twonewnew
(leukemia), hybrid
K562
hybrid compounds
(myelogenous
compounds based
leukemia),
based on cholesterol
U937
on cholesterol (lung),
and HeLa and
1,14-
1,14-tetradeca-(5Z,9Z)-dienedicarboxylic
(cervical), and Hek293 (kidney) human
tetradeca-(5Z,9Z)-dienedicarboxylic acid,acid,
cell
127 127
lines. and
andThe 129, which
129,results
whichshowed were synthetic
that the hybrid
were synthetic analogues
analogues of
moleculesnatural
of natural127
(5Z,9Z)-dienoic
and 129 efficiently
(5Z,9Z)-dienoic acids.
acids. The apoptosis
induced
The synthetic methodology
synthetic methodology
of the studied reliedrelied
cell onand
lines
on the were
the preparation
substantially
preparation of cholesterol-based
of more cytotoxic
cholesterol-based
oximes
oximes 126
than their and
and128 128and
126cholesterol their
oxime
and further
precursors
their further esterification
126 and 128
esterification using
[36].1,14-tetradeca-(5Z,9Z)-dienedicarboxylic
using 1,14-tetradeca-(5Z,9Z)-dienedicarboxylic acid
(Scheme 29) [36].
acid Cholesterol The
(Scheme 29)28[36]. authors
was The evaluated
usedauthors
as a template the in vitro
for thethe
evaluated cytotoxic
synthesis activities
in vitroofcytotoxic of the
a series ofactivities synthesized
2-methoxybenzoate compounds
analogs,
of the synthesized
126–129
compounds against
bearing function Jurkat
groups
126–129 (leukemia),
such Jurkat
against K562
as carbonyl (myelogenous
131, hydroxyl
(leukemia), leukemia),
132, U937 (lung), HeLa
and thiosemicarbazones
K562 (myelogenous leukemia), U937 (cervical),
133, whichHeLa
(lung), and
were
Hek293
evaluated(kidney)
as human
potential cell
new lines. The
anticancer results
agents. showed
The that
synthetic
(cervical), and Hek293 (kidney) human cell lines. The results showed that the hybrid
route molecules
involved the 127
reaction and 129
of efficiently
cholesterol
hybrid molecules 127
induced
28 with
and apoptosis
129 efficiently of
2-methoxybenzoyl the studied
induced cell lines
chloride
apoptosis and and
of the were substantially
thestudied
subsequent more
functionalization
cell lines and were cytotoxic
of thethan7 their
substantially morecholesterol
position of the
cytotoxic
oxime
steroid precursors
core with 126 and
several 128 [36].
functional groups
than their cholesterol oxime precursors 126 and 128 [36]. (Scheme 30) [37].
Cholesterol 28 was used as a template for the synthesis of a series of 2-methoxybenzoate analogs,
bearing function groups such as carbonyl 131, hydroxyl 132, and thiosemicarbazones 133, which were
evaluated as potential new anticancer agents. The synthetic route involved the reaction of cholesterol
28 with 2-methoxybenzoyl chloride and the subsequent functionalization of the 7 position of the
steroid core with several functional groups (Scheme 30) [37].

Scheme 29. 29. Synthesis

Synthesisofofcholesterol-based
cholesterol-based (5Z,9Z)-dienoic acids.
(5Z,9Z)-dienoic Reagents
acids. and conditions:
Reagents a) m-CPBA,
and conditions: a) m-
CH 2 Cl2 ;CH
CPBA, b) 2CrO , HCrO
Cl2; 3b) 2 O; c)
3, SOCl , pyridine;
H2O; 2c) d) NH2 OH
SOCl2, pyridine; d)·HCl,
NH2pyridine;
OH∙HCl, e) celite, pyridinium
pyridine; dichromate
e) celite, pyridinium
(PDC), t-BuOH,
dichromate (PDC),benzene;
t-BuOH, f) (5Z,9Z)-tetradeca-5,9-dienedioic acid, DMAP,acid,
benzene; f) (5Z,9Z)-tetradeca-5,9-dienedioic EDCl, CH2 ClEDCl,
DMAP, 2 , rt, 12
CHh.2Cl2,
rt, 12 h.
Cholesterol 28 was used as a template for the synthesis of a series of 2-methoxybenzoate analogs,
bearing function groups such as carbonyl 131, hydroxyl 132, and thiosemicarbazones 133, which were
Scheme 29. Synthesis of cholesterol-based (5Z,9Z)-dienoic acids. Reagents and conditions: a) m-
evaluated as potential new anticancer agents. The synthetic route involved the reaction of cholesterol
CPBA, CH2Cl2; b) CrO3, H2O; c) SOCl2, pyridine; d) NH2OH∙HCl, pyridine; e) celite, pyridinium
28 with 2-methoxybenzoyl chloride and the subsequent functionalization of the 7 position of the
dichromate (PDC), t-BuOH, benzene; f) (5Z,9Z)-tetradeca-5,9-dienedioic acid, DMAP, EDCl, CH2Cl2,
steroid core with several functional groups (Scheme 30) [37].
rt, 12 h.
Molecules 2018, 23, x 19 of 68
Molecules 2019, 24, 116 19 of 68
Molecules 2018, 23, x 19 of 68

Scheme 30. Synthesis of cholesteryl 2-methoxybenzoates. Reagents and conditions: a) 2-

methoxybenzoyl
Scheme 30.
Scheme chloride,ofof
Synthesis
30. Synthesis pyridine,
cholesteryl
cholesteryl h; b) CrO3, pyridine, Reagents
rt, 122-methoxybenzoates.
2-methoxybenzoates. CHReagents
2Cl2, rt, 24and
and h;conditions:
c)conditions:
CeCl2, NaBH 4,
a)
a) 2-
EtOH/CH 2 Cl 2
methoxybenzoyl ,
2-methoxybenzoyl rt, 30 min; d)
chloride,
chloride, EtOH, AcOH,
pyridine, 80 °C.
pyridine,rt,rt,1212h;h;b) CrO33, ,pyridine,
b) CrO pyridine, CH
CH22Cl
Cl2,2 ,rt,
rt,24
24 h;
h; c)
c) CeCl
CeCl2,, NaBH
NaBH4,,
EtOH/CH22Cl
Cl2,2rt,
, rt,3030min;
min;d)d)EtOH,
EtOH,AcOH,
AcOH,80 ◦ C.
80°C.
EtOH/CH
All of the synthesized cholesterol derivatives were evaluated for their in vitro antiproliferative
All of
activities
All the
the synthesized
ofagainst cholesterol
cholesterol derivatives
CNE-2 (nasopharyngeal),
synthesized BEL-7402
derivatives were(liver),
were evaluated
evaluated HepG2for their
for their in vitro
(liver),
in and antiproliferative
vitro Skov3 (ovarian)
antiproliferative
activities
human against
cancer CNE-2
cells, as (nasopharyngeal),
well as HEK-293T BEL-7402
human (liver),
kidney HepG2
epithelial
activities against CNE-2 (nasopharyngeal), BEL-7402 (liver), HepG2 (liver), and Skov3 (liver),
cells. and
The Skov3
results(ovarian) human
demonstrated
(ovarian)
cancer
that
human thecells, as cells,
presence
cancer well as
as HEK-293T
of the 7-hydroxy
well human kidney
humanepithelial
group (compound
as HEK-293T cells. The
132) doubled
kidney epithelial the resultsThedemonstrated
antiproliferative
cells. thatover
activity
results demonstrated the
presence
the of the
nonhydroxylated 7-hydroxy group
compound (compound
130. 132)
Furthermore, doubled
none ofthe antiproliferative
the evaluated
that the presence of the 7-hydroxy group (compound 132) doubled the antiproliferative activity over activity
compounds over
showedthe
nonhydroxylated
inhibitory compound
activity on HEK-293T
the nonhydroxylated 130.
compoundnormal Furthermore, none of
cells, makingnone
130. Furthermore, the evaluated
themofgood compounds
the candidates showed inhibitory
for cancer treatment
evaluated compounds showed
activity
[37]. on HEK-293T normal cells, making them good candidates for
inhibitory activity on HEK-293T normal cells, making them good candidates for cancer treatment cancer treatment [37].
[37]. The synthesis of a bis(cyclam)capped cholesterol lipid (139) was recently reported by Peters and
coworkers,
coworkers, who
whoalso
The synthesis ofevaluated
also aevaluated itsits
bioactivity
bis(cyclam)capped using
bioactivity using
cholesterolprimary
primary chronic
lipid (139) lymphocytic
chronic
was lymphocytic
recently leukemia
reported by (CLL)
leukemia
Peters cells.
(CLL)
and
The synthesis
cells. The of
synthesisthe bis(cyclam)capped
of the bis(cyclam)capped cholesterol lipid
cholesterol relied
lipidon a four-step
relied
coworkers, who also evaluated its bioactivity using primary chronic lymphocytic leukemia (CLL) on a methodology,
four-step as depicted
methodology, as
in Scheme
depicted in31 [38].
Scheme It was
31 found
[38]. It was that the
found bis(cyclam)capped
that the bis(cyclam)capped cholesterol lipid
cholesterol
cells. The synthesis of the bis(cyclam)capped cholesterol lipid relied on a four-step methodology, as 139 was
lipid water-soluble
139 was water-
and self-assembled
soluble
depicted and
in Scheme into
self-assembled micellar
31 [38]. into and
micellar
It was nonmicellar
found and the aggregates
thatnonmicellar in water.in
aggregates
bis(cyclam)capped The authors
water.
cholesterol The also139
found
authors
lipid alsothat
was the
found
water-
bis(cyclam)capped
that the cholesterol
bis(cyclam)capped lipid
cholesterol139 was
lipidas
139effective
was asas the commercial
effective as the
soluble and self-assembled into micellar and nonmicellar aggregates in water. The authors also found drug AMD3100
commercial drug in reducing
AMD3100
chemotaxis
in reducing
that along CXCL12
chemotaxis
the bis(cyclam)capped gradients,
along CXCL12
cholesterol showing thatwas
gradients,
lipid 139 139as
showingmay be effective
that
effective139asmaytheinbe disrupting
effective in
commercial the migration
disrupting
drug AMD3100 of
the
CLL cells into
migration
in reducing protective
ofchemotaxis
CLL cells intoniches
along such asniches
protective
CXCL12 the bone
suchmarrow
gradients, and
as the bone
showing thatlymphoid
marrow
139 mayand organs
be [38].inorgans
lymphoid
effective [38].the
disrupting
migration of CLL cells into protective niches such as the bone marrow and lymphoid organs [38].

Scheme 31. Synthesis of bis(cyclam)-capped cholesterol lipid. Reagents Reagents and conditions:
conditions: a) DIPEA,
a) DIPEA,
sonication for 30 min, then DMF,
DMF, rt,
rt, 30
30 min;
min; b) (Boc)
(Boc)33cyclam
cyclam
b)cholesterol or
or cyclam,
cyclam, NaHCO
NaHCO 33, MeCN, reflux,
reflux, 48 h;
48 h;
Scheme 31. Synthesis of bis(cyclam)-capped lipid. Reagents and conditions: a) DIPEA,
c) trifluoroacetic anhydride
c) TFA, trifluoroacetic anhydride(TFAA),
(TFAA),CH CH2Cl
2 Cl , rt,
2,2rt, 4 4 h;
h; d) d) HCl,
HCl, MeCN,
MeCN, rt, rt, min;
25 25 min;
e) e) cholesteryl
cholesteryl 3β-
sonication for 30 min, then DMF, rt, 30 min; b) (Boc)3cyclam or◦cyclam, NaHCO3, MeCN, reflux, 48 h;
3β-(N-hydroxysuccinimidyl)
(N-hydroxysuccinimidyl) carbamate,
carbamate, NaHCO
NaHCO 3 , MeCN,
, MeCN, 75 overnight.
75 °C, C, overnight.
c) TFA, trifluoroacetic anhydride (TFAA), CH23Cl 2, rt, 4 h; d) HCl, MeCN, rt, 25 min; e) cholesteryl 3β-

(N-hydroxysuccinimidyl) carbamate, NaHCO3, MeCN, 75 °C, overnight.

Molecules 2018, 23,
Molecules 23, xx 20 of 68
68
Molecules 2018,
2019, 24, 116 20
20 of
of 68

In 2015,
In 2015, aa paper
paper describing
describing the the synthesis,
synthesis, asas well
well as
as the
the antimicrobial
antimicrobial andand cytotoxic
cytotoxic activities,
activities, of
of
ten pharmacophoric
ten pharmacophoric motifs through
In 2015, a paper describing
motifs through CuAAC of
the synthesis,
CuAAC of chloroquinoline
as chloroquinoline
well and glucose
as the antimicrobial
and glucose azide substrates
and cytotoxic
azide substrates with
activities, of
with
propargyl
ten compounds
pharmacophoric such
motifs as chalcones,
through CuAAC theophylline,
of and
chloroquinoline cholesterol
and was
glucose
propargyl compounds such as chalcones, theophylline, and cholesterol was published. Within the published.
azide Within
substrates the
with
scope ofof this
propargyl
scope this review, only
compounds
review, only as
such thechalcones,
the synthesistheophylline,
synthesis of cholesterol-based
of cholesterol-based derivatives
and cholesterol 141 and
and 143
was published.
derivatives 141 143 is presented
Within
is presented
the scope
(Scheme
of 32). Interestingly,
this review,
(Scheme 32). Interestingly, the results
only the synthesis
the results from the
the antimicrobial
antimicrobial
of cholesterol-based
from derivativesevaluation
141 and 143
evaluation showed that among
is presented
showed that among
(Schemethe ten
the ten
32).
synthesized
Interestingly, conjugates,
the results triazole
from the 143 exhibited
antimicrobial the highest
evaluation antibacterial
showed activity
that among against
the
synthesized conjugates, triazole 143 exhibited the highest antibacterial activity against E. coli and S. ten E. coli and
synthesized S.
aureus, and
and triazole
conjugates,
aureus, moderate
moderate antifungal activity
exhibitedactivity
143antifungal against
the highest A. flavus
flavus and
antibacterial
against A. and C.
C. albicans.
activity albicans. Furthermore,
against E. the sugar-
coli and S. aureus,
Furthermore, the sugar-
and
cholesterol
moderate conjugate
antifungal 143 displayed
activity against the
A. best in
flavus vitro
and cytotoxic
C. activity
albicans. against
Furthermore, the
theprostate
cholesterol conjugate 143 displayed the best in vitro cytotoxic activity against the prostate cancer PC3 cancer
sugar-cholesterolPC3
cell line
line [39].
conjugate
cell [39].
143 displayed the best in vitro cytotoxic activity against the prostate cancer PC3 cell line [39].

Scheme 32.
Scheme 32. Synthesis
32. Synthesis of
Synthesis of pharmacophoric
of pharmacophoric motifs
pharmacophoric motifs by copper-catalyzed
motifs by copper-catalyzed 1,3-dipolar
1,3-dipolar cycloaddition
cycloaddition
(CuAAC). Reaction conditions: a) CuSO 4 ∙5H O,
·5H22O,
(CuAAC). Reaction conditions: a) CuSO44∙5H 2 L-AA,
O,LL-AA, THF/H
-AA,THF/H
THF/H2O
2 O
2O
(4:1), reflux.
(4:1),
(4:1), reflux.
reflux.

Two cholesterol
Two
Two derivatives
cholesterol derivatives (3β-azidocholest-5-ene
derivatives (3β-azidocholest-5-ene (144)
(3β-azidocholest-5-ene(144) and
(144)and (3β)-3-(prop-2-yn-1-yloxy)-
and(3β)-3-(prop-2-yn-1-yloxy)-
(3β)-3-(prop-2-yn-1-yloxy)- --
cholest-5-ene
-cholest-5-ene (20))
(20)) were
were used as
as starting
starting materials
materials for the preparation
preparation of three-motif
cholest-5-ene (20)) were used as starting materials for the preparation of three-motif pharmacophoricpharmacophoric
including cholesterol,
conjugates including
conjugates cholesterol, 1,2,3-triazole,
cholesterol, 1,2,3-triazole, and
1,2,3-triazole, and either
eitheraa achalcone,
andeither chalcone,
chalcone,aa alipophilic
lipophilic residue,
lipophilicresidue, oror
residue,or aa
acarbohydrate
carbohydrate
carbohydrate tag tag
tag [40].[40].
[40]. The first
The first
Theset first
set of cholesterol
of cholesterol conjugates was
set of cholesterol
conjugates was prepared
prepared
conjugates through
was the reaction
prepared
through the reaction
through of 3β-
of 3β-
the
azidocholest-5-ene
reaction of 144 with
3β-azidocholest-5-ene propargylated
144 with chalcones
propargylated or lactose
chalcones or
azidocholest-5-ene 144 with propargylated chalcones or lactose derivatives under CuAAC derivatives
lactose under
derivatives CuAAC
under
conditions,
CuAAC
conditions, affordingaffording
conditions,
affording chalcone chalcone
chalcone conjugates
conjugates 145 and
and 146
conjugates
145 146 and lactose
and lactose
145 and conjugates
146 andconjugates 147 and
lactose conjugates
147 and 148
148 (Scheme
and 148
147 (Scheme
33) [40].
[40]. 33) [40].
(Scheme
33)

Scheme 33. Synthesis

Scheme 33.
33. Synthesis of
Synthesis ofsugar
of sugaror
sugar orchalcone-triazole
or chalcone-triazolecholesterol
chalcone-triazole cholesterolconjugates.
cholesterol conjugates.
conjugates. Reagents and
Reagents
Reagents conditions:
and
and a)
conditions:
conditions:
propargyl
a) chalcone
a) propargyl
propargyl or or
chalcone
chalcone sugar, CuSO
or sugar,
sugar, 4 ·5H
CuSO
CuSO 2 O,
∙5H
44∙5H
L-AA,
O,
22O, L THF/H
-AA,
L-AA, THF/H
THF/H 2O O(5:1),
22O (5:1),reflux,
(5:1), reflux,333h.h.
reflux, h.

A secondsetset
A second
second of cholesterol
of cholesterol
cholesterol conjugates
conjugates was prepared
was prepared
prepared once again
againonce againCuAAC
through through CuAAC of
of (3β)-3-(prop-
(3β)-3-(prop-
A set of conjugates was once through CuAAC of
(3β)-3-(prop-2-yn-1-yloxy)cholest-5-ene
2-yn-1-yloxy)cholest-5-ene (20)(20) with
with azido(20) alkanols
azido with azido alkanols
(149) (149) and 3β-azidocholest-5-ene
and 3β-azidocholest-5-ene
3β-azidocholest-5-ene (144),
(144), affording
affording
2-yn-1-yloxy)cholest-5-ene alkanols (149) and (144),
affording cholesterol-triazole
cholesterol-triazole alkanols alkanols
alkanols (150)
(150) and (150) and a triazole-linked
and aa triazole-linked
triazole-linked cholesterol cholesterol
cholesterol dimer dimer
dimer (152), (152), respectively
(152), respectively
respectively (Scheme
(Scheme
cholesterol-triazole
(Scheme 34) [40]. Furthermore, compound 150 was converted in the respective bromo alkane 151
34) [40]. Furthermore, compound 150 was converted in the respective bromo alkane 151 through aa
34) [40]. Furthermore, compound 150 was converted in the respective bromo alkane 151 through
through a substitution
substitution reaction
reaction in reaction
in the in
the presencethe
presence of presence
of CBr of CBr
CBr44 (Scheme
(Scheme 34)
4 (Scheme
34) [40].34)
[40]. A [40]. A carbohydrate-tagged
A carbohydrate-tagged
carbohydrate-tagged set set of
of
substitution
set of cholesterol
cholesterol compounds
compounds was was prepared
prepared by the by the CuAAC
CuAAC reaction reaction
of of (3β)-3-(prop-2-yn-1-yloxy)-
(3β)-3-(prop-2-yn-1-yloxy)- -cholest-
cholesterol compounds was prepared by the CuAAC reaction of (3β)-3-(prop-2-yn-1-yloxy)- -cholest-
-cholest-5-ene
5-ene (20)
5-ene (20) with
with(20)
thewith
the the appropriate
appropriate
appropriate glycosyl
glycosyl glycosyl
azides azides
azides 153 and
153 and andaffording
153155,
155, 155, affording
affording compounds
compounds
compounds 154 and
154 and and
154 156,
156,
156, respectively,
respectively, upon upon cleavage
upon cleavage
cleavage of of
of the the
the acetylacetyl protecting
acetyl protecting
protecting groupsgroups
groups (Scheme (Scheme
(Scheme 34) 34)
34) [40].
[40].[40].
respectively,
Molecules 2018, 23, x 21 of 68
Molecules 2019, 24, 116 21 of 68
Molecules 2018, 23, x 21 of 68

Scheme 34. Synthesis of cholesterol-triazole dimer, cholesterol-triazole alkanes, and cholesterol-

triazole
Schemecarbohydrates.
Scheme SynthesisofReagents
34.Synthesis
34. and conditions:
cholesterol-triazole
of a)cholesterol-triazole
dimer,
cholesterol-triazole dimer, CuSO 4∙5H2O, L-AA, THF/H
alkanes,
cholesterol-triazole 2O cholesterol-triazole
and
alkanes,(5:1),
andreflux, 3 h; b)
cholesterol-
CBr 4, PPh3, CH2Reagents
carbohydrates. Cl 2, rt, overnight;
and c) NaOMe,
conditions: a)
triazole carbohydrates. Reagents and conditions: a)MeOH,
CuSO · rt,
5H
4 CuSO 2
O,h.
L -AA, THF/H
2 4∙5H2O, L-AA, THF/H
2 O (5:1),
2O (5:1), reflux, 3CBr
reflux, 3 h; b) 4,
h; b)
PPh
CBr43,, PPh
CH23,Cl2 , rt,
CH 2Clovernight; c) NaOMe,
2, rt, overnight; MeOH,
c) NaOMe, rt, 2 h.
MeOH, rt, 2 h.
Another carbohydrate-tagged compound, 159, was synthesized through the reaction of
Another
cholesterol
Another 28carbohydrate-tagged
with an appropriate compound,
carbohydrate-tagged glycosyl donor
compound, wasin
159, 159,
157 synthesized
a three-step
was through
protocol,
synthesized theasreaction
through theofreaction
depicted cholesterol
in Scheme of
28
35 with
[40]. an
The appropriate
authors glycosyl
screened donor
all the 157 in a three-step
cholesterol conjugatesprotocol,
for as
their depicted
in vitro
cholesterol 28 with an appropriate glycosyl donor 157 in a three-step protocol, as depicted in Scheme in Scheme
antimicrobial 35 [40].
and
The authors
anticancer
35 [40]. The screened
activities. all the cholesterol
authors Among
screened allallcompounds,conjugates for their inforvitro antimicrobial
the chalcone-triazole-cholesterol
the cholesterol conjugates their and anticancer
in vitroderivative 145 (R
antimicrobial and=
activities.
NMe2) wasAmong
anticancer the oneallwith
activities. compounds,
Among the most the chalcone-triazole-cholesterol
promising
all compounds, antimicrobial activity,derivative 145
being as active
the chalcone-triazole-cholesterol (Ras= the
NMe
derivative 2 ) (R
was=
controls
145
the one
against with
E. the
coli, S. most
aureus promising
and C. antimicrobial
albicans. activity,
Concerning thebeing as
cytotoxic active as
potential
NMe2) was the one with the most promising antimicrobial activity, being as active as the controls theofcontrols
the against
cholesterol
E. coli, S.
conjugates,
against E. aureus
coli, S.and C. albicans.
aureus and C. Concerning
the cholesterol-triazole-lactoside the cytotoxic
congener
albicans. Concerning 147the potential
displayed
cytotoxic ofpotential
the the in
best cholesterol
vitro conjugates,
cytotoxic
of the effect
cholesterol
the cholesterol-triazole-lactoside
against the prostate cancer PC3 congener
cell line, 147
with displayed
similar the best
cytotoxicity in
to vitro
that cytotoxic
of DOX,
conjugates, the cholesterol-triazole-lactoside congener 147 displayed the best in vitro cytotoxic effect effect
used against
as a controlthe
prostate
[40]. cancer PC3 cell line, with similar cytotoxicity to that of DOX, used as a
against the prostate cancer PC3 cell line, with similar cytotoxicity to that of DOX, used as a control control [40].
[40].

Scheme
Scheme 35. Synthesis of
35. Synthesis of carbohydrate-tagged
carbohydrate-tagged cholesterol
cholesterol conjugates. Reagents and
conjugates. Reagents and conditions:
conditions:
a)TMSOTf,
a)TMSOTf, MeCN,
MeCN, rt,
rt, 15
15 min;
min; b)
b) NaOMe,
NaOMe, MeOH,
MeOH, rt,rt,7575min;
min;c)c)1. 1.
NaOH,
NaOH, 1,4-dioxane/MeOH/H
1,4-dioxane/MeOH/H 22O
O
Scheme 35. Synthesis of carbohydrate-tagged cholesterol conjugates. Reagents and conditions:
(6:3:1),
(6:3:1), rt, 8 h
rt, 8 h then HCl
then HCl (pH
(pH 5), rt,
5), rt, overnight; 2. Ac O/pyridine (1:2), rt, overnight; 3. NaOMe, MeOH,
a)TMSOTf, MeCN, rt, 15 min; b) overnight; 2. Ac2O/pyridine
NaOMe, MeOH, 2
rt, 75 min;(1:2),
c) 1. rt, overnight;
NaOH, 3. NaOMe, MeOH,
1,4-dioxane/MeOH/H 2O
rt,
rt, 22 h.
h.
(6:3:1), rt, 8 h then HCl (pH 5), rt, overnight; 2. Ac2O/pyridine (1:2), rt, overnight; 3. NaOMe, MeOH,
rt, 2 h.
AA new
new methodology
methodology for for the
the synthesis
synthesis of of steroidal
steroidal pyrazolines (162) through
pyrazolines (162) through the
the reaction
reaction of
of
cholest-5-en-7-ones
cholest-5-en-7-ones (160)
(160) with
with 2,4-dinitrophenylhydrazine
2,4-dinitrophenylhydrazine (161)
(161) was
was reported
reported by
by Shamsuzzaman
Shamsuzzaman and
and
A new methodology for the synthesis of steroidal pyrazolines (162) through the reaction of
coworkers
coworkers inin2016 (Scheme
2016 36) [41].
(Scheme 36)The reaction
[41]. The proceeded by
reaction (161)a well-known
proceeded by a1,4-/1,2-addition/dehyd
well-known 1,4-/1,2-
cholest-5-en-7-ones (160) with 2,4-dinitrophenylhydrazine was reported by Shamsuzzaman and
ration mechanism
addition/dehydration to an α,β-unsaturated
mechanism to an carbonyl
α,β-unsaturated compound.
carbonyl The new
compound.steroid-based
The new pyrazolines
steroid-based
coworkers in 2016 (Scheme 36) [41]. The reaction proceeded by a well-known 1,4-/1,2-
(162) were evaluated
pyrazolines for their infor
(162) weremechanism
evaluated vitro antibacterial
their activity against
in vitro antibacterial activitythree different
against three strains (E.
different coli,
strains
addition/dehydration to an α,β-unsaturated carbonyl compound. The new steroid-based
Corynebacterium
(E. xerosis, and
coli, Corynebacterium S. epidermidis),
xerosis, and in which compound
S. epidermidis), in which 162 (R = H) was
compound 162 the
(R =most
H) active
was against
the most
pyrazolines (162) were evaluated for their in vitro antibacterial activity against three different strains
(E. coli, Corynebacterium xerosis, and S. epidermidis), in which compound 162 (R = H) was the most
Molecules 2019, 24, 116 22 of 68
Molecules 2018, 23, x 22 of 68

C. xerosis
active and S.
against C.epidermidis,
xerosis andwith minimum with
S. epidermidis, inhibitory
minimum concentrations
inhibitory similar to the positive
concentrations similarcontrol
to the
gentamicin.
Scheme Compound
36. Synthesis 162
of (R =
steroidalH) also demonstrated
pyrazolines. Reagents moderate
and activity
conditions:
positive control gentamicin. Compound 162 (R = H) also demonstrated moderate activity against a) against
DMSO, fungal
AcOH, strains
reflux, Mucor
21–
azygosporus,
fungal35strains
h. Claviceps purpurea, and
Mucor azygosporus, A. niger,
Claviceps being the
purpurea, andmost effective
A. niger, beingcompound tested. The
the most effective in vitro
compound
anticancer
tested. Theactivity
in vitroagainst
anticancerfive human
activitycancer
against cell lines
five (SW480
human (colon),
cancer cell HeLa
lines (cervical),
(SW480 (colon), A549 (lung),
HeLa
HepG2 The same
(hepatic),research
HL-60 group reported
(leukemia)) of a green simple
pyrazolines synthesis
(162) was of
also
(cervical), A549 (lung), HepG2 (hepatic), HL-60 (leukemia)) of pyrazolines (162) was also screened, steroidal
screened, 2H-pyran-2-ones
with the chlorinated(163),
starting
compound
with from 3-substituted
162 (R = Cl)
the chlorinated cholest-5-en-7-ones
the most162
compound active
(R =[41]. (160) and
Cl) the most active [41]. ethyl acetoacetate in the presence of
chitosan as an ecofriendly heterogeneous catalyst (Scheme 37) [42]. The synthesized steroidal 2H-
pyran-2-ones (163) were tested in vitro against two cancer cell lines (HeLa (cervical) and Jurkat
(leukemia)) and one normal cell line (PBMC: Peripheral blood mononuclear cell). All the tested
compounds (163) exhibited moderate-to-good activity against the two human cancer cell lines and
were less toxic against the noncancer cell line. Furthermore, the antioxidant potential of these new
compounds (163) was also evaluated, exhibiting lower 2,2-diphenyl-1-picrylhydrazyl radical (DPPH)
radical scavenging activity than the positive control, ascorbic acid [42].
A series of new steroidal pyrimidine derivatives (167) was prepared through the
multicomponent
Scheme 36. reaction
Synthesis
36. Synthesis ofofof cholestan-6-ones
steroidal
steroidal pyrazolines.
pyrazolines. (164)
Reagents with
Reagents and urea
and (166) and
conditions:
conditions: benzaldehyde
a) DMSO,
a) DMSO,
AcOH, AcOH, (165)
reflux, 21–in the
reflux,
presence
21–35
35 of
h. h. trimethylsilyl chloride (TMSCl) as catalyst (Scheme 38) [43]. The antitumor activity of
these steroidal pyrimidine-functionalized scaffolds (167) was screened against three human cancer
The same
cell lines,
The same research group
MDA-MB231
research group
(breast),reported aa green
green simple
HeLa (cervical),
reported simple synthesis
and HepG2
synthesis of steroidal
steroidal
(hepatic),
of 2H-pyran-2-ones
and 2H-pyran-2-ones
one noncancer normal (163),
(163),
starting
cell line,from
starting PBMC,
from 3-substituted
by MTT assay.
3-substituted cholest-5-en-7-ones
All tested compounds
cholest-5-en-7-ones (160) and
(160) ethylethyl
showed
and acetoacetate
cytotoxicities in against
acetoacetate the presence
in the of chitosan
the presence
three cancer of
as an
chitosanecofriendly heterogeneous
cell lines.asParticularly,
an ecofriendly compound catalyst (Scheme
167 (R =catalyst
heterogeneous 37)
H) exhibited[42].
(Scheme The
the37)synthesized
highest
[42]. The steroidal
cytotoxicity
synthesized 2H-pyran-2-ones
against the three
steroidal 2H-
(163)
cancerwerecell tested
pyran-2-ones lines. in vitro
(163) were against
However, all cases
tested two cancer
in were
vitro cellthan
lower
against lines
two (HeLa
DOX, used
cancer (cervical)
as alines
cell and
positive Jurkat
(HeLa control(leukemia))
[43]. The
(cervical) andandauthorsone
Jurkat
normal
also cell
addressed line (PBMC:
the Peripheral
antioxidant blood
activity mononuclear
of the pyrimidine cell). All the
compounds
(leukemia)) and one normal cell line (PBMC: Peripheral blood mononuclear cell). All the tested tested compounds
(167), concluding (163) exhibited
that these
moderate-to-good
new compounds
compounds activity against
(163) presented
exhibited reduced the
DPPH
moderate-to-good two human
radical,
activitycancer
hydroxyl cellradical,
against linestwo
the and were
nitric
human less
oxide toxic cellagainst
radical,
cancer and
linesHandthe
2O2

noncancer
scavenging
were cell
less toxic line.
potential Furthermore,
against than -ascorbic
the Lnoncancer the acid,
antioxidant aspotential
a control.ofthe
usedFurthermore,
cell line. these
Moreover, newthe
antioxidant compounds
IC 50 values
potential (163) was new
ofpointed
these also
out
evaluated,
compounds exhibiting
that the scavenging lower
(163) wasactivity 2,2-diphenyl-1-picrylhydrazyl
of the tested
also evaluated, compounds
exhibiting radical
were (DPPH)
in the orderradical
lower 2,2-diphenyl-1-picrylhydrazyl scavenging
of nitric oxide activity
radicalradical
(DPPH) <
than
radicalthe
hydrogen positive
scavenging control,
peroxideactivity ascorbic
< DPPHthan radical acid [42].
the <positive
hydroxyl radical
control, [43]. acid [42].
ascorbic
A series of new steroidal pyrimidine derivatives (167) was prepared through the
multicomponent reaction of cholestan-6-ones (164) with urea (166) and benzaldehyde (165) in the
presence of trimethylsilyl chloride (TMSCl) as catalyst (Scheme 38) [43]. The antitumor activity of
these steroidal pyrimidine-functionalized scaffolds (167) was screened against three human cancer
cell lines, MDA-MB231 (breast), HeLa (cervical), and HepG2 (hepatic), and one noncancer normal
cell line, PBMC, by MTT assay. All tested compounds showed cytotoxicities against the three cancer
cell lines. Particularly, compound 167 (R = H) exhibited the highest cytotoxicity against the three
Scheme 37. 37.Synthesis
Synthesis of of
steroidal 2H-pyran-2-ones.
steroidal 2H-pyran-2-ones. Reaction conditions:
Reaction a) ethyla)acetoacetate,
conditions: chitosan,
ethyl acetoacetate,
cancer cell lines. However, all cases were lower than DOX, used as a positive control [43]. The authors
MeOH,
chitosan,reflux,
MeOH, 13–16 h. 13–16 h.
reflux,
also addressed the antioxidant activity of the pyrimidine compounds (167), concluding that these
new A compounds
series of new presented
steroidalreduced
pyrimidine DPPH radical, hydroxyl
derivatives (167) was radical,
preparednitricthroughoxidetheradical, and H2O2
multicomponent
scavenging potential than
reaction of cholestan-6-ones (164) with urea (166) and benzaldehyde (165) in the presenceout
L -ascorbic acid, used as a control. Moreover, the IC 50 values pointed of
that the scavenging
trimethylsilyl chloride activity
(TMSCl) of the as tested
catalyst compounds
(Scheme 38) were[43].in the Theorder of nitricactivity
antitumor oxide radical
of these <
hydrogen peroxide < DPPH radical < hydroxyl radical [43].
steroidal pyrimidine-functionalized scaffolds (167) was screened against three human cancer cell
lines, MDA-MB231 (breast), HeLa (cervical), and HepG2 (hepatic), and one noncancer normal cell line,
PBMC, by MTT assay. All tested compounds showed cytotoxicities against the three cancer cell lines.
Particularly, compound 167 (R = H) exhibited the highest cytotoxicity against the three cancer cell lines.
However, all cases were lower than DOX, used as a positive control [43]. The authors also addressed
the antioxidant activity of the pyrimidine compounds (167), concluding that these new compounds
presented reduced DPPH radical, hydroxyl radical, nitric oxide radical, and H2 O2 scavenging potential
than L-ascorbic acid, used as a control. Moreover, the IC50 values pointed out that the scavenging
Scheme 37. Synthesis of steroidal 2H-pyran-2-ones. Reaction conditions: a) ethyl acetoacetate,
activity of the tested compounds were in the order of nitric oxide radical < hydrogen peroxide < DPPH
chitosan, MeOH, reflux, 13–16 h.
radical < hydroxyl radical [43].
Molecules 2018, 23, x 23 of 68
Molecules 2019, 24, 116 23 of 68
Molecules 2018, 23, x 23 of 68

Scheme 38. Synthesis of steroidal pyrimidines. Reagents and conditions: a) trimethylsilyl chloride
(TMSCl), DMF/MeCN
Scheme 38. (1:2),
Synthesis of 90 °C, pyrimidines.
steroidal
steroidal 13–14.5 h.
pyrimidines. conditions: a) trimethylsilyl chloride
Reagents and conditions:
◦
(TMSCl), DMF/MeCN (1:2),90
DMF/MeCN (1:2), 90°C,C,13–14.5
13–14.5h.
h.
The cases in which the attachment of a heterocycle in the steroid backbone changes the biological
properties of the
The cases steroid
in which themolecule
attachment areofnot heterocycle
so rare, and
a heterocycle in the steroid
often are backbone
steroid an interesting platform
changes for the
the biological
properties
development of the
of steroid
new molecule
pharmacophores. are Innot so
this rare,
context, and often
Saikia et are
al. an interesting
reported the platform
synthesis
properties of the steroid molecule are not so rare, and often are an interesting platform for the of for the
steroidal
development of new
new pharmacophores.
heterocyclic compounds
development of pharmacophores.
(170) throughIn Inthe
thissolvent-free
this context, Saikia
context, Saikia et al.
al. reported
reported the
microwave-assisted
et theepoxide
synthesis
synthesis of steroidal
ring
of steroidal
opening
heterocyclic
with nitrogencompounds
nucleophiles
heterocyclic compounds [44].
(170) through
The firstthe solvent-free
series of microwave-assisted
N-heterocycles was epoxide
synthesized by
through the solvent-free microwave-assisted epoxide ring opening ring
the opening
reaction of
nitrogen
with nucleophiles
nitrogen with
nucleophiles the
[44]. epoxide
The first169, which
series of N-heterocycles
was prepared in
was a three-step
synthesized synthetic
by
with nitrogen nucleophiles [44]. The first series of N-heterocycles was synthesized by the reaction of the route
reaction
starting
of
nitrogen from
nitrogen cholesterol
nucleophiles
nucleophiles acetate
with
with 125
thethe (Scheme
epoxide
epoxide 39)
169,
169, [44].was
which
which wasprepared
preparedininaa three-step
three-step synthetic
synthetic route
route
starting from cholesterol acetate
starting from cholesterol acetate 125 125 (Scheme
(Scheme 39)
39) [44].
[44].

Scheme 39. Synthesis cholesterol-based N-heterocycles. Reagents and conditions: a) t-BuOOH, RuCl3,
cyclohexane,
Scheme rt, 4 h; b)cholesterol-based
Synthesis
39. Synthesis HCl, MeOH, reflux, 1 h; c) m-CPBA,
N-heterocycles.
N-heterocycles. Reagents ,and
CHCl3and rt, 5conditions:
h; d) N-heterocycle,
conditions: neat,RuCl
a) t-BuOOH, MW, 3,,
33
140 °C, 6–16
cyclohexane, min.
rt, 4 h; b) HCl, MeOH, reflux, 1 h; c) m-CPBA, CHCl , rt, 5 h; d) N-heterocycle, neat,
cyclohexane, rt, 4 h; b) HCl, MeOH, reflux, 1 h; c) m-CPBA, CHCl333, rt, 5 h; d) N-heterocycle, neat, MW, MW,
140 ◦ C, 6–16 min.
140 °C, 6–16 min.
The synthesis of another set of N-heterocycles, 173, was accomplished using a mixture of
The synthesis
The
epoxides (171 (α) of
synthesis ofanother
and 172 (β)
another of N-heterocycles,
set(4:1))
set 173, was
ofasN-heterocycles,
starting materials, accomplished
173,which using ausing
were obtained
was accomplished mixture
a of
through epoxides
the direct
mixture of
(171 (α) and
epoxidation
epoxides (171172
of(α)(β)and
(4:1))
172as
cholesterol starting
acetate
(β) 125materials,
(4:1)) startingwhich
as(Scheme 40) were
[44].
materials, obtained
It iswhich
worthwerethrough
noticing thecompound
that
obtained direct epoxidation
through 173
the was
direct
of cholesterol
obtained
epoxidation acetate 125 (Scheme
as aofdiastereomeric
cholesterol acetate 40) [44].
mixture,
125 which It upon
(Scheme is worth noticing that compound
40) recrystallization
[44]. It is worth in ethanol
noticing 173 was obtained
provided
that pure173
compound as a
alcohol
was
diastereomeric
173.
obtained mixture, which
as a diastereomeric upon recrystallization
mixture, in ethanol provided
which upon recrystallization pureprovided
in ethanol alcohol 173.
pure alcohol
173.

Scheme 40. Synthesis cholesterol-based N-heterocycles. conditions: a) m-CPBA, CHCl3,

N-heterocycles. Reagents and conditions:
rt, 5 h; b) imidazole or benzimidazole, neat, MW, 140 ◦ C, 10 min, recrystallization.
benzimidazole, neat, MW, 140 °C, 10 min, recrystallization.
Scheme 40. Synthesis cholesterol-based N-heterocycles. Reagents and conditions: a) m-CPBA, CHCl33,
rt, 5 h; b) imidazole or benzimidazole, neat, MW, 140 °C, 10 min, recrystallization.
Molecules 2018, 23, x 24 of 68
Molecules 2019, 24, 116 24 of 68
Molecules 2018, 23, x 24 of 68
The authors also considered the dehydration of the obtained cholesterol-based N-heterocycles
(170 and
The 173), which
authors alsowas successfully
considered accomplishedofusing
the dehydration a catalytic
the obtained
obtained amount of sulfuric
cholesterol-based
cholesterol-based acid in acetic
N-heterocycles
acid,and
(170 affording compounds
173), which 174 and 175,
was successfully respectivelyusing
accomplished (Scheme 41) [44].
a catalytic amount of sulfuric acid in acetic
acid, affording compounds 174 and 175, respectively
respectively (Scheme
(Scheme 41)
41) [44].
[44].

Scheme 41. Dehydration of cholesterol-based N-heterocycles. a) H2SO4 (cat.), AcOH, 80 °C, 4–6 h.
◦ C, 4–6 h.
Scheme 41. Dehydration
Scheme 41. Dehydration of cholesterol-based N-heterocycles.
of cholesterol-based a) H
N-heterocycles. a) (cat.), AcOH,
H22SO44 (cat.), AcOH, 80
80 °C, 4–6 h.
Finally, the in vitro antibacterial activity of all compounds was evaluated, and the N-
Finally, the
heterocycles
Finally, the inin
170 vitro antibacterial
(Het
vitro activity
= 4-nitroimidazole,
antibacterial of all compounds
activity of allpiperidine,was evaluated,
compounds morpholine,
was evaluated, N-heterocycles
and thethiomorpholine,
and the N-
170 (Het = 4-nitroimidazole,
tetrahydroisoquinoline) and piperidine,
dehydrated morpholine,
N-heterocycles thiomorpholine,
174
heterocycles 170 (Het = 4-nitroimidazole, piperidine, morpholine, thiomorpholine, (Het = tetrahydroisoquinoline)
4-nitroimidazole, and
morpholine)
dehydrated N-heterocycles
demonstrated moderate and
tetrahydroisoquinoline) 174
effects (Het = 4-nitroimidazole,
against the
dehydrated morpholine)
tested microorganisms
N-heterocycles demonstrated
174 (Het =(E.4-nitroimidazole, moderate
coli, P. syringae, B. effects
subtilis, P.
morpholine)
against
vulgaris the tested
and S. microorganisms
aureus). (E.
Specifically,coli, P. syringae,
compound B. subtilis,
170 (Het P. vulgaris
= and
demonstrated moderate effects against the tested microorganisms (E. coli, P. syringae, B. subtilis,and
piperidine,S. aureus). Specifically,
morpholine, P.
compound
vulgaris and170S.(Het
thiomorpholine) = piperidine,
inhibited
aureus). all morpholine,
the tested
Specifically, strains,
compound andthe
and thiomorpholine)
170 (Het
170 inhibited allmorpholine,
(Het= tetrahydroisoquinoline)
= piperidine, the tested strains,
derivative
and
and the 170
showed
thiomorpholine)(Het inhibited
inhibition = tetrahydroisoquinoline)
againstall three gram-negative
the tested derivative
strains, theshowed
bacterial
and inhibition
170strains,
(Het E. coli, against threeand
P. syringae,
= tetrahydroisoquinoline) gram-negative
P. vulgaris.
derivative
bacterial
The strains,
authors also E. coli, P.
concluded syringae,
that and
the P. vulgaris.
removal of The
the authors
hydroxyl also concluded
group that
decreased
showed inhibition against three gram-negative bacterial strains, E. coli, P. syringae, and P. vulgaris. the
theremoval of the
antimicrobial
hydroxyl
activity
The group
of thealso
authors decreased
tested the
compounds
concluded antimicrobial
that[44]. activity of the tested compounds [44].
the removal of the hydroxyl group decreased the antimicrobial
Recently,
Recently,
activity Morake
Morake
of the tested and coworkers
compounds [44]. synthesized a series of artemisinin-cholesterol conjugates, 177, 177,
179, 180, 182, 184,
184, 186,
186, and
and 188,
188, expecting
expecting that the putative cholesterol transporters
Recently, Morake and coworkers synthesized a series of artemisinin-cholesterol conjugates, 177, may enhance the
activity of the parent
179, 180, 182, 184, 186,drugdrug (artemisinin)
(artemisinin)
and 188, against malaria and tuberculosis [45]. The
expecting that the putative cholesterol transporters may enhance the conjugates were
designed to have different O- or
activity of the parent drug (artemisinin) N-linkers, such as
against ester,
ether, and
malaria ester, and
and carbamate,
tuberculosis [45]. varying the length
The conjugates wereof
each linker as well. The first set of conjugates, 177, 179–180, was synthesized
designed to have different O- or N-linkers, such as ether, ester, and carbamate, varying the length of from cholesterol 28 or
cholesteryl
each linker chloroformate
as well. The first 7 with
set ofdihydroartemisinin
dihydroartemisinin
conjugates, 177, 179–180, 178 or
178 or artesunate
artesunate 176 (Scheme
176
was synthesized from 42)
(Scheme 42) [45].
[45]. 28 or
cholesterol
cholesteryl chloroformate 7 with dihydroartemisinin 178 or artesunate 176 (Scheme 42) [45].

Scheme 42. 42. Synthesis of O-linked

O-linked artemisinin-cholesterol
artemisinin-cholesterol conjugates. Reagents and conditions: a)
conjugates. Reagents
BF · OEt , CH Cl , 0 ◦ C to rt, overnight; b) DCC, DMAP, CH Cl , rt, 4 h; c) DMAP, Et N, CH Cl , rt,
3 ∙OEt
Scheme
3 2 , CH 2 Cl 2
242. Synthesis
2 2 , 0 °C to rt, overnight; b) DCC, DMAP, CH 2Cl 2 , rt,
of O-linked artemisinin-cholesterol conjugates.
2 2 4 h; c) DMAP, Et
Reagents 3N,
and CH2Cl22, rt,
3 conditions:2 18
a)
18 h.
h. 3∙OEt2, CH2Cl2, 0 °C to rt, overnight; b) DCC, DMAP, CH2Cl2, rt, 4 h; c) DMAP, Et3N, CH2Cl2, rt, 18
BF
h.
Molecules 2019, 24, 116 25 of 68
Molecules 2018,
Molecules 2018, 23,
23, xx 25 of
25 of 68
68

second set
A second set of
of conjugates,
conjugates, 182,
182, 184,
184, and
and 186,
186, was synthesized
was synthesized
186, was starting
synthesized starting from aa specific
starting from specific artemisinin
specific artemisinin
appropriately substituted with a piperazine group at C-10, through reaction
derivative, 181 or 183, appropriately
with the
with the appropriate
appropriate cholesterol
appropriate cholesterol derivative,
cholesterol derivative, 777 or
derivative, or 185
or 185 (Scheme
185 (Scheme43)
(Scheme 43)[45].
43) [45].
[45].

Scheme 43. Synthesis

Scheme 43.
43. Synthesis of N-linked artemisinin-cholesterol
of N-linked
N-linked artemisinin-cholesterol conjugates.
artemisinin-cholesterol conjugates. Reagents
conjugates. Reagents and
and conditions: a)
conditions: a)
Et 3N, CH2Cl
Cl2, ,rt,
rt,overnight;
overnight;b)
b)Et
Et3
Et3N, CH22Cl22, rt, overnight; b) Et3N, N,
N,THF,
THF, reflux,
reflux,24
24
3 THF, reflux, 24 h.h.
h.

Furthermore,
Furthermore, thetheauthors
the authorsdesigned
authors designedaa afinal
designed final
final setset
set of of
of compounds
compounds
compounds bearing
bearing
bearing a carbamate
aa carbamate
carbamate linker,
linker,
linker, 188.
188. The
188. The
The synthesis
synthesis of
synthesis of
of this this
this set set
set of of compounds
of compounds
compounds was was carried
was carried
carried out out through
out through
through an an amidation
an amidation reaction
amidation reaction of cholesteryl
reaction of cholesteryl
chloroformate
chloroformate 7 with the appropriate amine derivative, 187, bearing different lengths
7 with the appropriate amine derivative, 187, bearing different lengths of
of alkyl
alkyl chains
chains
(Scheme 44) [45].
(Scheme 44) [45].

Scheme 44. Synthesis

Scheme 44.
44. Synthesis of of O-linked
O-linked artemisinin
O-linked artemisinin cholesteryl
artemisinin cholesteryl carbamates:
cholesteryl Reagents and
carbamates: Reagents
carbamates: and conditions: a)
conditions: a)
Et3 N, CH22Cl ◦ C to rt, overnight.
Cl22, ,00°C
Et3N, CH2Cl2, 0 °C to to rt,
rt, overnight.
overnight.

The
The antimalarial activity of
antimalarial activity
activity of the
the novel
novel artemisinin-cholesterol
artemisinin-cholesterol conjugates
conjugates 177,
177, 179,
179, 182,
182, 184,
184, 186,
186,
The antimalarial of the novel artemisinin-cholesterol conjugates 177, 179, 182, 184, 186,
and
and 188 were
and 188
188 were evaluated
were evaluated against Plasmodium
evaluated against
against Plasmodium falciparum
Plasmodium falciparum (Pf
falciparum (Pf)) NF54,
(Pf)
NF54, K1,
NF54, K1, and
and W2
K1, and W2 strains,
W2
in
in which
strains, in
strains, which the
which the
the
conjugates
conjugates ofof 186
of 186 (N-linked
186 (N-linked artemisinin-cholesterol
(N-linked artemisinin-cholesterol conjugates)
artemisinin-cholesterol conjugates)
conjugates) were were
were thethe most
the most active
most active derivatives.
active derivatives.
derivatives.
conjugates
However,
However, the
the potency
potency of
of these
these compounds was
compounds was lower
lower than
than the
the precursors
precursors artemether
artemether andand artesunate.
artesunate.
However, the potency of these compounds was lower than the precursors artemether and artesunate.
The
The authors
authors rationalized these results
rationalized these
these results based
based onon the
the low
low solubility
solubility in in
in the
the culture
culture medium
medium givengiven by
by
The authors rationalized results based on the low solubility the culture medium given by
cholesterol
cholesterol moiety,
moiety, which
which may
may have
haveaffected
affectedthe efficacies
the of
efficacies the
of artemisinin-cholesterol
the artemisinin-cholesterol conjugates. On
conjugates.
cholesterol moiety, which may have affected the efficacies of the artemisinin-cholesterol conjugates.
the
On other hand,hand,
the other
other concerning the activities
concerning the activities
activities Mycobacterium
against against tuberculosis
Mycobacterium (Mtb) H37Rv,
tuberculosis theH37Rv,
(Mtb) conjugates
the
On the hand, concerning the against Mycobacterium tuberculosis (Mtb) H37Rv, the
displayed enhanced
conjugates displayed efficacy
displayed enhanced over the
enhanced efficacy parent
efficacy over drug
over the artemisinin
the parent
parent drug [45].
drug artemisinin
artemisinin [45].
[45].
conjugates
The
The synthesis
synthesis of
of three
three new
new cholesterol
cholesterol conjugates,
conjugates, 190, 193,
190, 193, and
and 194,
194, via
via CuAAC
CuAAC reaction
reaction was
was
The synthesis of three new cholesterol conjugates, 190, 193, and 194, via CuAAC reaction was
recently
recently reported
reported [46].
[46]. These
These conjugates
conjugates were
were prepared
prepared either to have
either to a ferrocene-chalcone
have a ferrocene-chalcone moiety 190
moiety
recently reported [46]. These conjugates were prepared either to have a ferrocene-chalcone moiety
or sugar
190 or moieties
or sugar 193
sugar moieties and
moieties 193 194
193 and as
and 194 well,
194 as both
as well, linked
well, both
both linkedby a
linked by triazole group
by aa triazole
triazole group(Scheme
group (Scheme 45) [46].
(Scheme 45)45) [46].
[46].
190
Molecules 2018, 23, x 26 of 68
Molecules 2019, 24, 116 26 of 68
Molecules 2018, 23, x 26 of 68

Scheme 45. Synthesis of cholesterol-triazole conjugates bearing ferrocene-chalcone and sugar

moieties.
Scheme 45.Reagents andofconditions:
45.Synthesis
Synthesis a) L-AA, conjugates
cholesterol-triazole CuSO
of cholesterol-triazole 4·5H2bearing
O, THF/H
conjugates 2O (6:1), 60–80 °C,
ferrocene-chalcone
bearing 4 h.sugar
and
ferrocene-chalcone andmoieties.
sugar
◦
Reagents and conditions:
moieties. Reagents a) L-AA, CuSO
and conditions: 4 ·5H
a) L-AA, 2 O, THF/H
CuSO O (6:1),2O
4·5H2O,2THF/H 60–80
(6:1), C, 4 h. °C, 4 h.
60–80
The antimicrobial activities of these cholesterol conjugates were evaluated in vitro against E. coli,
The
S. aureus, antimicrobial
A. flavus, and
The antimicrobial activities of
C. albicans.
activities these cholesterolthe
Surprisingly,
of these cholesterol conjugates were
authors were
conjugates evaluated
foundevaluated
that the in in vitro
vitro against
against
cholesterol E. coli,
conjugate
E. coli,
S. aureus,
bearing A. flavus, and
ferrocene-chalcone C. albicans.
moiety Surprisingly,
190 was the
completely authors
inactive found that
against all the
the cholesterol
tested
S. aureus, A. flavus, and C. albicans. Surprisingly, the authors found that the cholesterol conjugate conjugate
bacteria. On the
bearing
other
bearing ferrocene-chalcone
hand, sugar conjugates
ferrocene-chalcone moiety
moiety 190 was
193 190
and 194 completely
was inactive
showed moderate
completely against
against all
inactiveinhibitory the
the tested
all activity
tested bacteria.
against On
On the
E. coli,
bacteria. A.
the
other
flavus,hand,
and sugar
C. conjugates
albicans, being 193
even and
less 194 showed
potent than moderate
control inhibitory
compounds activity
ampicillinagainst
other hand, sugar conjugates 193 and 194 showed moderate inhibitory activity against E. coli, A. and E. coli, A.
amphotericin flavus,
B
and
[46]. C. albicans, being even less potent than control compounds ampicillin and amphotericin
flavus, and C. albicans, being even less potent than control compounds ampicillin and amphotericin B B [46].
Employing a one-pot multicomponent reaction procedure using (thio)semicarbazide
[46]. Employing
hydrochloride
Employing 196aand ethyl 2-chloroacetoacetate
one-pot multicomponent 195 allowed
reaction the preparation
procedure using of(thio)semicarbazide
a series of steroidal
oxazole and thiazole derivatives (197) (Scheme 46) [47].
hydrochloride 196 and ethyl 2-chloroacetoacetate 195 allowed the preparation of a series of steroidal
oxazole and thiazole derivatives (197) (Scheme 46) [47].

Scheme 46. Synthesis of steroidal oxazole/thiazole

oxazole/thiazole derivatives.
derivatives. Reagents
Reagents and
and conditions:
conditions: a)
a) NaOAc,
NaOAc,
EtOH, reflux, 6–10 h.
Scheme 46. Synthesis of steroidal oxazole/thiazole derivatives. Reagents and conditions: a) NaOAc,
EtOH, reflux, 6–10 h.
antimicrobialactivity
The antimicrobial activityofof thethe
new new steroidal
steroidal compound
compound was evaluated
197 evaluated
197 was againstagainst
two gram-two
gram-negative
negative (E. coli and
(E. coli and activity
The antimicrobial P. aeruginosa)
P. aeruginosa)
of the new and
andsteroidaltwo gram-positive
two gram-positive
compound 197 bacterial
bacterial strains
strains (S.
was evaluated (S. aureus
aureus
against and
twoand
gram- L.
L.
monocytogenes).
negative (E. coli Additionally,
Additionally, the bioactivity
and P. aeruginosa) and two against
against pathogenic bacterial
pathogenic
gram-positive fungi (C.
fungi albicans
(C. albicans and
strains and C. neoformans)
C. neoformans)
(S. aureus and L.
was also addressed. The authors found that most of the compounds exhibited good
monocytogenes). Additionally, the bioactivity against pathogenic fungi (C. albicans and C. neoformans) antibacterial and
antifungal
antifungal activity against the tested
was also addressed. The authors found that most strains. In addition, the compounds also showed
In of the compounds exhibited good antibacterial and interesting
antibiofilm activity against S. aureus biofilm. Molecular
antifungal activity against the tested strains. In addition, docking studies showed
the compounds also effective binding of
showed interesting
the steroidal compound 197 with amino acid residues of DNA gyrase and glucosamine-6-phosphate
antibiofilm activity against S. aureus biofilm. Molecular docking studies showed effective binding of
synthase through
the steroidal hydrogen
compound 197 bonding
with amino interactions [47]. of DNA gyrase and glucosamine-6-phosphate
acid residues
Given
synthase increasingbonding
the hydrogen
through importance of steryl
interactions [47]. ferulates [3-O-(trans-4-feruloyl)sterols] in
pharmaceutical
pharmaceutical applications,
Given theapplications, Begum
increasing Begum and
importance coworkers
and coworkers
of sterylreported
reportedthe microwave-assisted
ferulates synthesis
the [3-O-(trans-4-feruloyl)sterols]
microwave-assisted of steryl
synthesis of
in
ferulates
steryl from
ferulates several
from steroids
several [48].
steroidsThe synthesis
[48]. The of cholesterol-based
synthesis of steryl
cholesterol-based
pharmaceutical applications, Begum and coworkers reported the microwave-assisted synthesis of ferulate 199
steryl is exemplified
ferulate 199 is
in Scheme
exemplified 47, in
in which
Scheme microwave
47, in (MW)
which irradiation
microwave played
(MW) a crucial
irradiationrole in the
played esterification
steryl ferulates from several steroids [48]. The synthesis of cholesterol-based steryl ferulate 199 isa crucial step
role inwith
the
trans-4-O-acetylferulic
esterification
exemplified in step withacid
Scheme in [48].
47,198
trans-4-O-acetylferulic
which microwave acid 198
(MW)[48].irradiation played a crucial role in the
esterification step with trans-4-O-acetylferulic acid 198 [48].
Molecules
Molecules 2018,
2019, 23,
24, x116 2727of
of 68
68
Molecules 2018, 23, x 27 of 68

Scheme 47. Synthesis of cholesterol-based steryl ferulate. Reagents and conditions: a) DCC, DMAP,
CH 2Cl2, MW,
Scheme 100 °C, 15ofmin;
47. Synthesis b) K2CO3, CHCl
cholesterol-based 3/MeOH
steryl (2:1),Reagents
ferulate. reflux, 6 and
Reagents h.
and conditions:
conditions: a) DCC, DMAP,
a) DCC, DMAP,
CH2Cl
Cl22,,MW,
MW, 100 ◦ C, 15 min; b) K CO , CHCl /MeOH (2:1), reflux, 6 h.
100 °C, 15 min; b) K22CO3,3 CHCl3/MeOH
3 (2:1), reflux, 6 h.
The authors evaluated the antioxidant capacity (DPPH radical scavenging, total antioxidant
The
capacity, authors
The and reducing
authors evaluated
power)
evaluated the
theof antioxidant
all synthesized
antioxidant capacity (DPPH
(DPPH radical
steryl ferulates
capacity scavenging,
in comparison
radical scavenging, total
total antioxidant
to equimolar mixtures
antioxidant
capacity,
of and
steryl ferulates
capacity, reducing
and reducing power)
and power)
γ-oryzanol of all
of all(a synthesized
natural mixture
synthesized steryl ferulates in comparison
of sterylinferulates,
steryl ferulates comparison to
abundant equimolar
to equimolar mixtures
in cereal bran
mixtures
of
of steryl
layers). ferulates
sterylThe results
ferulates and
and γ-oryzanol
showed that the
γ-oryzanol (a natural
(amixture
natural mixture
ofmixtureof steryl
steryl ferulatesferulates,
of steryl and abundant
γ-oryzanol
ferulates, in
abundantwascereal
ain bran
better
cereallayers).
radical
bran
The results
scavenger showed
than most that the
individual mixture of
ferulates, steryl ferulates
including the and γ-oryzanol
cholesterol-based
layers). The results showed that the mixture of steryl ferulates and γ-oryzanol was a better radical was a
one,better
199 radical
[48]. scavenger
than most individual
scavenger ferulates, including
than most individual ferulates,the cholesterol-based
including one, 199 [48].
the cholesterol-based one, 199 [48].
4. Cholesterol-Based Liquid Crystals
4. Cholesterol-Based Liquid
4. Cholesterol-Based Liquid Crystals
Crystals
A liquid crystal is basically a state of matter that has properties between those of conventional
liquids A liquid
and thosecrystal of is basically
solid crystals. a state
Theof matter that has properties between those of conventional
A liquid crystal is basically a state ofclassification
matter that has of liquid
propertiescrystals was proposed
between in the 19th
those of conventional
liquids
century and
and those
is of
based solid
on crystals.
molecular The classification
arrangement. of
Since liquid
then,
liquids and those of solid crystals. The classification of liquid crystals was proposed in the crystals
liquid was proposed
crystals have in
been the 19th
dividedcentury
into
19th
and
smecticis based
century(from on
and isthe molecular
Greek
based onword arrangement.
molecular “smegma”, Since then,
meaningSince
arrangement. liquid
soap)then, crystals
and nematic have been
(from the
liquid crystals have divided
Greekbeen into
word smectic
“nema”,
divided into
(from
meaning
smectic the(from
Greek
thread)theword
Greek “smegma”,
crystals. In smectic
word meaning
“smegma”, soap)
liquidmeaning andsoap)
crystals, nematicand (from
molecules the(from
are arranged
nematic Greektheword
so that
Greek “nema”,
their
word meaning
major axes
“nema”,
thread)
are
meaning crystals.
parallel, andIncrystals.
thread) smectic
their centersliquid
In of crystals,
smectic mass liemolecules
liquid in one plane.
crystals, are arranged
moleculesThereare soarranged
are that
many their somajor
that axes
different are
smectic
their parallel,
majorphases
axes
and their centers
characterized
are parallel, and of mass
by different
their lie
types
centers in oneand
of plane.
degrees
mass lieThere
of one
in areplane.
manyand
positional different
There are smectic
orientational phases
many order.
different The characterized
most common
smectic phases by
different
ones
characterizedtypes
are the smectic and degrees
A phase,
by different of
typesinandpositional
which degrees and orientational
the molecules
of positional areand order.
oriented The most
along the
orientational common
layer
order. The ones
normal, are
and the
most common the
smectic
smectic A
ones areCthe phase,
phase, in
smectic which
in which
A phase, the molecules
the in molecules
which the are oriented
aremolecules
tilted awayalong the
are from layer
oriented normal,
it. Nematic
along the and
phases the
layer are smectic C phase,
the simplest
normal, and the
in which
liquid the
smecticcrystalline molecules
C phase, in phases are
whichformed, tilted away
the molecules from
since they it. Nematic
only away
are tilted have fromphases
long-range are the simplest
orientational
it. Nematic phases are liquid
order crystalline
the (of, e.g.,
simplest
phases
molecules, formed,
liquid crystalline since
columns) theyno
and
phases only
degree
formed, haveof long-range
long-range
since they onlyorientational
translational order
order(of,
have long-range [49].e.g., molecules,
There a columns)
is also order
orientational chiral(of, and
variant
e.g.,
no
of degree of
nematic
molecules, or long-range
smecticand
columns) translational
phases,
no degree whenof the order [49]. translational
molecules
long-range There
of theisliquid
alsoorder
acrystalline
chiral
[49].variant of
substance
There nematic
is also are or smectic
chiral,
a chiral with
variant
phases,
these when the
phasesordenoted
of nematic molecules
smectic N* of
or Sm(A/B)*
phases, the
when theliquid crystalline
(anmolecules
asterisk denotes substancea chiral
of the liquid are chiral, with these
phase), respectively.
crystalline substance are phases
These denoted
phases
chiral, with
N*
are or
these Sm(A/B)*
often called
phases (an asterisk
the cholesteric
denoted N* or Sm(A/B)*denotes
phases,(an a chiral
because phase),
asterisk they respectively.
wereafirst
denotes chiral These
observed phases
phase), for are often
cholesterolThese
respectively. called
derivatives
phasesthe
cholesteric
[49]. phases, because they were first observed for cholesterol
are often called the cholesteric phases, because they were first observed for cholesterol derivatives derivatives [49].
In 2014,
[49]. In 2014, Hiremath
Hiremathreported reportedthethe synthesis
synthesis of two
of two newnew seriesseries of cholesterol-biphen-4-yl
of cholesterol-biphen-4-yl 4-(n-
4-(n-alkoxy)benzoate
alkoxy)benzoate
In 2014, Hiremath conjugates
conjugates (203), the
reported (203),
linked linked through
throughofeither
synthesis two new either
odd-parity odd-parity
series or or
of even-parity even-parity spacers
spacers (Scheme
cholesterol-biphen-4-yl 4-(n-
(Scheme
48) [50]. The48) compounds
alkoxy)benzoate [50].conjugates
The compounds
in 203(203), in 203through
arelinked
optically areactive,
opticallyand active,
either both and of
series
odd-parity both series ofshow
or conjugates
even-parity conjugates show
a frustrated
spacers (Scheme
a48)
frustrated
liquid[50]. Theliquid
crystalline crystalline
state,
compounds with
in 203 astate,
arewith a thermodynamically
thermodynamically
optically active,stable
and bothtwiststable
graintwist
series grain boundary
ofboundary
conjugates phase
showwith aphase with
a chiral
frustrated
aliquid
chiralcrystalline
smectic smectic
C structureC state,
structure
with(TGBC*)
(TGBC*) over an over an exceedingly
exceedingly
a thermodynamically wide wide
twistthermal
thermal
stable range
grain [50].range [50].phase with a chiral
boundary
smectic C structure (TGBC*) over an exceedingly wide thermal range [50].

Scheme
Scheme 48. Synthesis
Synthesis of cholesterol-biphen-4-yl 4-(n-alkoxy)benzoate conjugates. Reagents Reagents and
and
conditions:
Scheme 48.a)
conditions: bisphenol,
a)Synthesis K22CO
K
bisphenol,of CO33, ,DMF,
DMF,85 ◦
85°C,C,24
24h;h;b)
cholesterol-biphen-4-yl DCC,
DCC,DMAP,
DMAP, CH
CH22Cl
4-(n-alkoxy)benzoate
b) ,2 ,40 ◦
40°C,C,2424h.h.
Cl2conjugates. Reagents and
conditions: a) bisphenol, K2CO3, DMF, 85 °C, 24 h; b) DCC, DMAP, CH2Cl2, 40 °C, 24 h.
The author
The author explained
explained such such behavior
behavior based
based on on the
the combined
combined effect
effect of extended
extended geometry
geometry
(conformation),
(conformation),
The authorstrongstrong chirality,
chirality,
explained andthe
such and theenantiomeric
behavior enantiomeric
based on the excess
excess of of
combined thethe molecules.
molecules.
effect Furthermore,
Furthermore,
of extended geometrythe
the conjugates
conjugates
(conformation), of 203
of 203strong with
with an an odd-parity
odd-parity
chirality, spacer
and spacer show an additional
show an additional
the enantiomeric phase,
excess ofphase, the blue one.
the blue one.
the molecules. The clearing
clearing
Furthermore, the
transition
transition
conjugatestemperatures
of 203 with and associated
the associated
an odd-parity enthalpies
enthalpies
spacer show an alternate
alternate where
where
additional odd
the odd
phase, members
the members exhibit
blue one.exhibit lower
lower
The clearing
values
values compared
compared
transition totothose
temperatures those
andofof even
even
the members.
members.
associated These
These results
results
enthalpies clearly
clearly
alternate demonstrate
demonstrate
where the oddthat thatgeometry
the
members the geometry
exhibit (rod-
lower
(rod-like
like
values andconformation)
andcompared
bent bentto conformation)
and the
those of even and the These
thermal
members. thermal
behavior behavior
of the
results ofdemonstrate
the conjugates
conjugates
clearly of 203that of 203
are the
greatly are greatly
influenced
geometry (rod-
influenced
by
likethe
and byparity
spacer
bent the spacer
[50]. parity
conformation) and[50].
the thermal behavior of the conjugates of 203 are greatly influenced
by theA spacer
A series of
of similar
similar
parity conjugates
[50].conjugatesofof 206, containing
206, containing cholesterol, triazole,
cholesterol, and and
triazole, biphenylene units,units,
biphenylene were
synthesized
were A seriesvia
of CuAAC
synthesized via CuAAC
similar chemistry
conjugates (Scheme
chemistry
of 49). 49).
(Scheme
206, Different
containing flexible
Different spacers
flexible
cholesterol, werewere
spacers
triazole, and introduced
introduced
biphenylene in the
in
units,
were synthesized via CuAAC chemistry (Scheme 49). Different flexible spacers were introduced in
Molecules 2019, 24, 116 28 of 68
Molecules 2018, 23, x 28 of 68
Molecules 2018, 23, x 28 of 68

the system
the system to to evaluate
evaluate the
the effect
effect on
on the
the mesophase
mesophase formation
formation as
as well
well as
as the
the influence
influence of
of the
the presence
presence
system to evaluate the effect on the mesophase formation as well as the influence of the presence of a
of a triazole
of a triazole linker
linker [51].
triazole linker [51].[51].

Scheme 49. Synthesis of

Synthesisof cholesterol-biphenylene
ofcholesterol-biphenylene dimers.
cholesterol-biphenylenedimers. Reagents
dimers.Reagents
Reagentsand conditions:
and a) NaN
conditions: 3, DMF, 70
a) NaN
Scheme
Scheme 49.
49. Synthesis and conditions: a) NaN 3 , DMF,
3, DMF, 70
°C, overnight;
70 ◦ C, b) CuI,
overnight; b) THF,
CuI, reflux,
THF, 6 h.6 h.
reflux,
°C, overnight; b) CuI, THF, reflux, 6 h.

The authors
authors concluded
concluded that
that short
short (n(n === 55 and 6) and medium
medium (n = 7, 8, and 9) alkyl spacers exhibit
The authors concluded that short (n and 6) and medium (n = 7, 8, and 9) alkyl spacers exhibit
enantiotropic
enantiotropic SmA*
SmA* and
andmonotropic
monotropic SmC*SmC* phases,
phases,whereas
whereasthe conjugate
the possessing
conjugate the longest
possessing spacer
the longest
longest
enantiotropic SmA* and monotropic SmC* phases, whereas the conjugate possessing the
(n = 10)(nfavors
spacer the formation of enantiotropic SmA SmA and N* phases. A close correlation between the
spacer (n == 10)
10) favors
favors the formation
the formation of enantiotropic
of enantiotropic SmA and and N* phases.
N* phases. A close
A close correlation
correlation between
between
transition
the temperatures
transition temperatures andandthethe
increase
increase in in
thethe length
length ofofthe
themethylene
methylene spacer
spacer was
was also
also observed,
the transition temperatures and the increase in the length of the methylene spacer was also observed,
and a higher
higher clearing
clearingpoint
pointwas
wasobserved
observedfor forthethe even
even spacers.
spacers. Further
Further comparison
comparison studies
studies withwith
(S)-
and a higher clearing point was observed for the even spacers. Further comparison studies with (S)-
(S)-2MBbip-n-Chol
2MBbip-n-Chol 207 207207 (Scheme
(Scheme 49)49) demonstrated
demonstrated that
that thetriazole
the triazolering
ringplays
plays a crucial
crucial rolerole in the
2MBbip-n-Chol (Scheme 49) demonstrated that the triazole ring plays aa crucial role in the
mesophase
mesophase formation,
formation, wherein
wherein apart
apart from
from thethe
molecular
molecular dipole,
dipole,thethe
subtle electrostatic
subtle interaction
electrostatic and
interaction
mesophase formation, wherein apart from the molecular dipole, the subtle electrostatic interaction
van
and der
vanWaals forcesforces
der Waals
Waals enhance the SmC*
enhance phasephase
the SmC*
SmC* [51]. [51].
and van der forces enhance the phase [51].
A study involving
involving thethe design,
design,synthesis,
synthesis,and andmesomorphic
mesomorphicproperties
properties ofofthethe first
first examples
examples of
A study involving the design, synthesis, and mesomorphic properties of the first examples of
of cholesterol-based
cholesterol-based calixarene
calixarene liquid
liquid crystals
crystals was was reported
reported in in by
2015 2015
GuobyandGuocoworkers
and coworkers [52]. [52].
Novel
cholesterol-based calixarene liquid crystals was reported in 2015 by Guo and coworkers [52]. Novel
Novel cholesterol-1,3-bis-substituted
cholesterol-1,3-bis-substituted calix[4]arene
calix[4]arene 209 andand and cholesterol-tetra-substituted
209cholesterol-tetra-substituted calix[4]arene
calix[4]arene 210
cholesterol-1,3-bis-substituted calix[4]arene 209 cholesterol-tetra-substituted calix[4]arene 210
210 derivatives
derivatives were were synthesized
synthesized by by cholesterol-chlorinated
reacting reacting cholesterol-chlorinated derivatives derivatives
(208) with (208) with
calix[4]arene,
derivatives were synthesized by reacting cholesterol-chlorinated derivatives (208) with calix[4]arene,
calix[4]arene,
as depicted
depicted in inas depicted
Scheme 50.in Scheme 50.
as Scheme 50.

Scheme
Scheme 50. 50.Synthesis of calix[4]arene-cholesterol
Synthesis derivatives.
of calix[4]arene-cholesterol ReagentsReagents
derivatives. and conditions:
and a) calix[4]arene,
conditions: a)
Scheme 50. Synthesis of calix[4]arene-cholesterol derivatives. Reagents and conditions: a)
K 2 CO3 , KI, MeCN,
calix[4]arene, K2COreflux, 24 h; b)reflux,
3, KI, MeCN, 208, K24
2 CO , KI,
h; 3b) MeCN,
208, K2CO3reflux, 36 h. reflux, 36 h.
, KI, MeCN,
calix[4]arene, K2CO3, KI, MeCN, reflux, 24 h; b) 208, K2CO3, KI, MeCN, reflux, 36 h.
The liquid crystalline behaviors of cholesterol-calix[4]arene compounds 209 and 210 were studied,
The liquid
The liquid crystalline
crystalline behaviors of of cholesterol-calix[4]arene compounds
compounds 209 209 and
and 210
210 were
were
and both showed excellent behaviors
mesomorphiccholesterol-calix[4]arene
properties of the columnar molecular arrangement of
studied,
studied, and both showed
and bothbowlic excellent
showedcolumn, mesomorphic
excellentwith
mesomorphic properties of the columnar molecular arrangement
the calix[4]arene cholesterolproperties of the columnar
units as ancillary molecularFurthermore,
lateral columns. arrangement
of the
of the calix[4]arene
calix[4]arene bowlic
bowlic column,
column, with
with cholesterol
cholesterol units
units as
as ancillary
ancillary lateral
lateral columns.
columns. Furthermore,
Furthermore,
the authors demonstrated that compounds with longer spacers and more cholesterol units, such as
the authors
the authors demonstrated
demonstrated that compounds
that compounds with longer
with[52]. spacers and more cholesterol units,
longer spacers and more cholesterol units, such such as
as
210, are better for good mesomorphic properties
210, are
210, are better for
better for good
good mesomorphic
mesomorphic properties [52].
properties [52].
Following this study, similar calix[4]arene-cholesterol derivatives with Schiff-base bridges (213)
Following this
Following this study,
study, similar
similar calix[4]arene-cholesterol
calix[4]arene-cholesterol derivatives
derivatives with
with Schiff-base
Schiff-base bridges
bridges (213)
(213)
were synthesized (Scheme 51), and the influence of complexation behaviors on their mesomorphic
were synthesized
were synthesized (Scheme
(Scheme 51),51), and the influence of complexation behaviors on their mesomorphic
properties was investigated [53].and
Likethe
theinfluence
previousofcholesterol-calix[4]arene
complexation behaviorscompounds
on their mesomorphic
(210), these
properties
properties waswas investigated
investigated [53].
[53]. Like
Like the previous
the previous cholesterol-calix[4]arene
cholesterol-calix[4]arene compounds (210), these
these
Schiff-base bridged compounds (213) presented mesomorphic properties with acompounds (210),
molecular arrangement
Schiff-base bridged
Schiff-base bridged compounds
compounds (213) (213) presented
presented mesomorphic
mesomorphic properties
properties with
with a molecular
molecular
of the calixarene bowlic column and Schiff-base cholesterol units as ancillary lateralacolumns as
arrangement of the calixarene bowlic column and Schiff-base cholesterol units as
arrangement of the calixarene bowlic column and Schiff-base cholesterol units as ancillary lateral ancillary lateral
columns as
columns as well.
well. However,
However, upon upon complexation
complexation with with AgClO
AgClO44,, no
no mesophase
mesophase was was observed,
observed,
Molecules 2019, 24, 116 29 of 68
Molecules 2018, 23, x 29 of 68

well. However,
suggesting that upon complexation
the mesomorphic with AgClO
properties 4 , no mesophase
of compound wasbe
213 could observed,
tuned bysuggesting that the
ion-complexation
mesomorphic
behavior [53]. properties of compound 213 could be tuned by ion-complexation behavior [53].

Scheme 51. Synthesis of calix[4]arene-cholesterol derivatives with Schiff-base bridges. Reagents and
MeOH/CHCl333(1:1),
conditions: a) AcOH, MeOH/CHCl (1:1),reflux,
reflux,88h.h.

Recently, novel
novel columnar
columnar liquid
liquid crystals
crystals (LCs)
(LCs) based
based onon symmetric
symmetric hairpin-shaped
hairpin-shaped cholesterol
cholesterol
tetramers
tetramers with Schiff-base spacers were prepared, and their mesomorphic were
with Schiff-base spacers were prepared, and their mesomorphic behaviors investigated
behaviors were
by different by
investigated techniques. The new molecules
different techniques. The new were synthesized
molecules through thethrough
were synthesized reactionthe
between
reactiona
cholesterol
between dimer, 214, dimer,
a cholesterol and phenylenediamines or bis-hydrazides
214, and phenylenediamines working asworking
or bis-hydrazides spacers containing
as spacers
hydrogen bonds,
containing affording
hydrogen bonds,compounds 215 and 216215
affording compounds (Scheme 52)(Scheme
and 216 [54]. 52) [54].

Synthesisofofcholesterol
52. Synthesis
Scheme 52. cholesterol tetramers
tetramers with
with Schiff-base
Schiff-base bridges.
bridges. Reagents
Reagents and conditions:
and conditions: a) p-
a) p-phenylenediamine
phenylenediamine or o-phenylenediamine,
or o-phenylenediamine, AcOH, AcOH,
CHCl CHCl
33/EtOH
3 /EtOH
(1:4), (1:4), reflux,
reflux, 12 h; 12
b)h; b) malonic
malonic bis-
bis-hydrazide
hydrazide or adipic
or adipic bis-hydrazide,
bis-hydrazide, AcOH,
AcOH, CHCl
CHCl 3 /EtOH
33/EtOH (1:4),(1:4), reflux,
reflux, 12 h.12 h.

The results
The resultsindicated
indicated good hexagonal
good columnar
hexagonal liquid liquid
columnar crystalline behaviors,
crystalline with threewith
behaviors, molecules
three
arranged asarranged
molecules a disc of the
as acolumnar hexagonal
disc of the columnarstate. In addition,
hexagonal state.the
In symmetric cholesterol
addition, the symmetrictetramers with
cholesterol
rigid cores or hydrogen-bonding cores strongly favored the formation of a columnar mesophase
tetramers with rigid cores or hydrogen-bonding cores strongly favored the formation of a columnar [54].
The preparation
mesophase [54]. of a series of tetramers (218), based on azobenzene decorated with cholesterol
units,The
was also recently
preparation of a reported. These oligomeric
series of tetramers (218), basedcompounds bearing
on azobenzene different
decorated withalkyl spacers
cholesterol
were synthesized by reacting azobenzene tetracarboxylic acid (217) with cholesteryl derivatives
units, was also recently reported. These oligomeric compounds bearing different alkyl spacers were (200)
(Scheme 53) [55].
synthesized by reacting azobenzene tetracarboxylic acid (217) with cholesteryl derivatives (200)
(Scheme 53) [55].
Molecules 2019, 24, 116 30 of 68
Molecules 2018, 23, x 30 of 68

Scheme 53. Synthesis of

of azobenzene-centered
azobenzene-centeredcholesterol-based
cholesterol-basedtetramers.
tetramers.Reagents
Reagents and
and conditions:
conditions: a)
a) KOH,
KOH, H2HO,2O, tetraoctylammonium
tetraoctylammonium bromide
bromide (TOAB),
(TOAB), reflux,
reflux, 5 h.5 h.

Among the
Among the synthesized compounds, it
synthesized compounds, it was
was found
found that
that oligomers
oligomers with
with nn= 1, 5,
= 1, 5, and
and 88 exhibited
exhibited
an enantiotropic
an enantiotropic N* N* phase,
phase, while
while the
the other
other oligomers showed aa monotropic
oligomers showed monotropic N* N* phase,
phase, upon
upon cooling
cooling
from an
from an isotropic
isotropic state. Interestingly, oligomers
state. Interestingly, oligomerswithwithnn == 11 and
and 88 formed
formed spherulites
spherulites inin their
their crystalline
crystalline
state, dispersed
state, dispersed forfor hundreds
hundreds of of micrometers
micrometers in in the
the case
case ofof the
the oligomer with n
oligomer with n= 1. Moreover,
= 1. Moreover, both
both
oligomers (n = = 1 and 8)8) had
had photoisomerization
photoisomerization in in dilute
dilute solutions
solutions and
and Langmuir
Langmuir monolayers,
monolayers, in
opposition
opposition to to the
the liquid
liquid crystalline
crystalline state,
state, in
in which
which nono photoisomerization
photoisomerization was was observed
observed [55].
[55].
Cholesterol-based nonconventional liquid crystals have been
Cholesterol-based nonconventional liquid crystals have been studied by Guptastudied by Gupta andand coworkers.
coworkers.
They reported
reported the thesynthesis
synthesis of novel
of novel functional
functional discotic
discotic oligomeric
oligomeric materials
materials based onbased on
3,4,9,10-
3,4,9,10-tetrasubstituted perylene, one of which bore the cholesterol units of
tetrasubstituted perylene, one of which bore the cholesterol units of 220 (Scheme 54) [56]. 220 (Scheme 54) [56].

Scheme 54.
Scheme Synthesis of
54. Synthesis of cholesterol-perylene-based
cholesterol-perylene-based liquid
liquid crystals
crystals (LCs).
(LCs). Reagents
Reagents and
and conditions:
conditions: a)
a)
KOH, H O, 70 ◦ C, 2 h, then HCl (aq); b) TOAB, reflux, 3 h.
KOH, H22O, 70 °C, 2 h, then HCl (aq); b) TOAB, reflux, 3 h.

The cholesterol
The cholesterol derivative
derivative 220 was found
220 was found to
to be
be aa nonconventional
nonconventional LC LC at
at room
room temperature:
temperature:
However, a monotropic nematic (N*) phase on cooling was achieved. The authors
However, a monotropic nematic (N*) phase on cooling was achieved. The authors also demonstrated also demonstrated
that the
that the combination
combination of of rod
rod and
and disc-like
disc-like moieties
moieties sufficiently
sufficiently perturbed
perturbed the molecular shape
the molecular to yield
shape to yield
calamitic mesophases. Additionally, this hybrid material showed interesting fluorescence
calamitic mesophases. Additionally, this hybrid material showed interesting fluorescence emission emission
properties,
properties, making
making itit suitable
suitable for
for aa range
range of
of optoelectronic
optoelectronic applications
applications [56].
[56].
Recently, the
Recently, the synthesis
synthesis of ofperylene
perylenederivatives
derivativeswith
withtwotwo(223)
(223)oror four
four cholesterol
cholesterol units
units (225)
(225) at
at bay-position or both in bay-position and imide position, respectively, was reported
bay-position or both in bay-position and imide position, respectively, was reported (Scheme 55). The (Scheme 55).
The authors
authors addressed
addressed the influence
the influence of theof the number
number as wellasaswell
the as the position
position of the cholesterol
of the cholesterol units
units on the
on the mesomorphic and photophysical properties of these new liquid crystals
mesomorphic and photophysical properties of these new liquid crystals [57]. The authors concluded [57]. The authors
concluded
that that more cholesterol
more cholesterol units significantly
units significantly lowered thelowered the mesophase
mesophase temperature,
temperature, created created wider
wider scopes
scopes
of phaseof transfer
phase transfer temperatures,
temperatures, and increased
and increased the fluorescence.
the fluorescence. Furthermore,
Furthermore, it was
it was found
found that
that a
a longer spacer between perylene and cholesterol units was ideal for mesomorphic
longer spacer between perylene and cholesterol units was ideal for mesomorphic properties as well properties as well
as to
as to enhance
enhance the
the fluorescence
fluorescence of of the
the compounds
compounds [57].
[57].
A year later, Chen et al. reported the synthesis of three different perylene-based liquid crystals
bearing different bay-rigid spacers (228). These new liquid crystals were synthesized starting from
a perylene derivative (227) with six alkyl chains on the imides positions by coupling two phenyl
(biphenyl or naphthyl)-bridging cholesterol units (226) at bay positions (Scheme 56) [58]. Investigations
addressing the mesomorphic properties of these perylene-based compounds (228) demonstrated that
Molecules 2019, 24, 116 31 of 68

all derivatives ordered hexagonal columnar liquid crystalline behaviors, despite the functionalization
of the bay positions with aromatic spacers. Derivatives with larger and rigid aromatic spacers
presented higher phase transition temperatures as well as smaller scopes of mesophase temperatures.
The authors also concluded that rigid and larger aromatic groups showed stronger emission and
higher fluorescence quantum yield. These results suggested that by adjusting the structures of spacers
on the bay position, both mesomorphic and photophysical properties are likely to be tuned depending
on the purpose
Molecules 2018, 23, xof the liquid crystal [58]. 31 of 68

Scheme 55. 55. Synthesis

Synthesisofofcholesterol-perylene
cholesterol-perylene liquid
liquid crystals.
crystals. Reagents
Reagents and conditions:
and conditions: a) 3K, 2DMF,
a) K2 CO CO3,
95 ◦ C, 95
DMF, 10 °C,
h; b)
10Kh;
2 CO
b) 3K, 2DMF, 105 ◦ C,
CO3, DMF, 10520°C,
h. 20 h.

Aiming
A to explore
year later, Chen et the potentially
al. reported interesting
the synthesis of mesomorphic properties of liquid
three different perylene-based liquid crystals,
crystals
Champagne
bearing and bay-rigid
different coworkers reported
spacers the
(228). synthesis
These of a synthetic
new liquid liquid
crystals were crystal dimer
synthesized (233)
starting fromanda
two of its monomer analogues (231) based on cholesterol mesogens [59].
perylene derivative (227) with six alkyl chains on the imides positions by coupling two phenyl The synthesis relied
on the CuAAC
(biphenyl reaction of a cholesteryl
or naphthyl)-bridging cholesterolazide (229)
units withatα,ω-di-O-propargyl-TEG
(226) bay positions (Scheme (232) and
56) [58].
O-monopropargylated-TEG (230) linkers, as depicted in Scheme 57. Several
Investigations addressing the mesomorphic properties of these perylene-based compounds (228) experimental studies were
carried out, showing
demonstrated that allthat both monomers
derivatives ordered (231) as well
hexagonal as the dimer
columnar (233)
liquid formed behaviors,
crystalline a smectic Adespite
liquid
crystalline
the phase withof
functionalization comparable layer spacing.
the bay positions The authors
with aromatic explained
spacers. this feature
Derivatives with by the formation
larger and rigid
of a bilayer structure in the case of the monomers (231) and a monolayer
aromatic spacers presented higher phase transition temperatures as well as smaller scopes structure for the dimer (233).
of
Concerningtemperatures.
mesophase the thermal stability of the
The authors self-assembled
also concluded that phases, the clearing
rigid and temperature
larger aromatic groupsincreased
showed
around 10 ◦ C from 231 (R = Ac) to 231 (R = H). Molecular modeling studies rationalized the features
stronger emission and higher fluorescence quantum yield. These results suggested that by adjusting
of the liquid crystalline
the structures of spacers phases
on the based
bay on the different
position, chemical functional
both mesomorphic groups present
and photophysical in each class
properties are
of materials,
likely allowing
to be tuned different
depending onkinds of intermolecular
the purpose interactions,
of the liquid such as dipole-dipole interaction,
crystal [58].
hydrogen-bonding,
Aiming to explore as well
theaspotentially
London dispersion
interestingforces, which greatly
mesomorphic affectedofthe
properties self-assembly
liquid crystals,
behavior of the
Champagne andthree cholesterol
coworkers derivatives
reported [59]. of a synthetic liquid crystal dimer (233) and two
the synthesis
of its monomer analogues (231) based on cholesterol mesogens [59]. The synthesis relied on the
CuAAC reaction of a cholesteryl azide (229) with α,ω-di-O-propargyl-TEG (232) and O-
monopropargylated-TEG (230) linkers, as depicted in Scheme 57. Several experimental studies were
carried out, showing that both monomers (231) as well as the dimer (233) formed a smectic A liquid
crystalline phase with comparable layer spacing. The authors explained this feature by the formation
of a bilayer structure in the case of the monomers (231) and a monolayer structure for the dimer (233).
Concerning the thermal stability of the self-assembled phases, the clearing temperature increased
around 10 °C from 231 (R = Ac) to 231 (R = H). Molecular modeling studies rationalized the features
Molecules 2018, 23, x 32 of 68
Molecules 2018,
Molecules 2019, 24,
23, 116
x 32 of
32 of 68
68
Molecules 2018, 23, x 32 of 68

Scheme 56. Synthesis of perylene-based liquid crystals bearing different bay-rigid spacers. Reagents
and conditions:
Scheme a) K2CO
56. Synthesis , DMF, 100 °C, 24
of 3perylene-based h. crystals bearing different bay-rigid spacers. Reagents
liquid
Scheme 56. Synthesis of perylene-based liquid crystals bearing different bay-rigid spacers. Reagents
and conditions: a) K2CO3, DMF, 100 °C,◦ C,24 h.
and conditions: a) K22CO
CO33, ,DMF,
DMF, 100
100 °C, 24h.
24 h.

Scheme 57. Synthesis of cholesteryl dimer and two cholesteryl monomers. Reagents and conditions:
a) CuI, DIPEA,
Scheme acetone,
57. Synthesis of 55
of °C, overnight.
cholesteryl
cholesteryl dimerand
dimer andtwo
twocholesteryl
cholesterylmonomers.
monomers.Reagents
Reagents and
and conditions:
conditions: a)
Scheme 57. Synthesis of ◦cholesteryl dimer and two cholesteryl monomers. Reagents and conditions:
a) CuI,
CuI, DIPEA,
DIPEA, acetone,
acetone, 55 55C,°C, overnight.
overnight.
a)
TheCuI, DIPEA, acetone,
synthesis 55 °C,aliphatic
of four new overnight.
polycarbonate copolymers (mPEG43-b-P(MCC-Cn)51 (236) (n
= 1–4)
Thecontaining
The synthesis
synthesis of cholesteryl
of four
fournew groups polycarbonate
newaliphatic
aliphatic as side chain copolymers
polycarbonate mesogenic
copolymers units
(mPEG
(mPEGwas achieved nthrough
43 -b-P(MCC-C
43-b-P(MCC-C )51 (236)
n)51
the
(n
(236) =
(n
The synthesis of four new aliphatic polycarbonate copolymers (mPEG 43-b-P(MCC-Cn)51 (236) (n
=coupling
1–4)
1–4) reaction
containing
containing between
cholesteryl mPEG
groups
cholesteryl groups-b-PMCC
as43side chain 51 (235) with
mesogenic
as side a side
chain units wascarboxyl
mesogenic units group
achieved through
was and
thechiral
achieved cholesteryl
coupling
through reaction
the
=derivatives
1–4) containing
between mPEG(234) cholesteryl
with
-b-PMCC different groups
(235)numbers
with a as
sideside
of chaingroup
methylene
carboxyl mesogenic
groups,
and units
bearing
chiral was
a achieved
terminal
cholesteryl through
hydroxyl
derivatives (234) the
group
with
coupling reaction43 between51mPEG43-b-PMCC51 (235) with a side carboxyl group and chiral cholesteryl
coupling reaction
(Scheme numbers
different 58) between
[60]. with
of mPEG43-b-PMCC
methylene 51 (235) with a side carboxyl group and chiral cholesteryl
derivatives (234) differentgroups,
numbers bearing a terminal groups,
of methylene hydroxylbearing
group (Scheme
a terminal58) [60].
hydroxyl group
derivatives (234) with different numbers of methylene groups, bearing a terminal hydroxyl group
(Scheme 58) [60].
(Scheme 58) [60].

Scheme
Scheme 58. Synthesisofofblock
58. Synthesis block copolymers
copolymers mPEG
mPEG 43 -b-P(MCC-C
43-b-P(MCC-C )51 . Reagents
n)51.nReagents and conditions:
and conditions: a)
a) DCC,
DCC,
DMAP,DMAP,
CH ClCH
2. 2 Cl2 .
Scheme 58. 2Synthesis of block copolymers mPEG43-b-P(MCC-Cn)51. Reagents and conditions: a) DCC,
Scheme 58. Synthesis of block copolymers mPEG43-b-P(MCC-Cn)51. Reagents and conditions: a) DCC,
DMAP,
The CH 2Cl2.
The authors
DMAP, CH2Clstudied
authors 2.
studied the
the liquid
liquid crystal
crystal behavior
behavior of
of both
both chiral
chiral cholesteryl
cholesteryl compounds
compounds (234)
(234) and
and
the
the block
block copolymers based
copolymers based on cholesterol
cholesterol (236). The
The results demonstrated
demonstrated that the chiral
chiral compounds
compounds
The authors studied theon
liquid crystal(236).
behavior results that compounds
of both chiral cholesteryl the (234) and
(234)
(234) exhibited
The
exhibited anstudied
authors
an enantiotropic
enantiotropic mesophase
the liquid
mesophase ofan
of anSmA
crystal behaviorSmA phase
of phase
both andcholesteric
chiral
and cholesteric
cholesteryl phase except
compounds
phase except for
(234)
for 234
and
234 (n
the block copolymers based on cholesterol (236). The results demonstrated that the chiral compounds
the block copolymers based on cholesterol (236). The results demonstrated that the chiral compounds
(234) exhibited an enantiotropic mesophase of an SmA phase and cholesteric phase except for 234 (n
(234) exhibited an enantiotropic mesophase of an SmA phase and cholesteric phase except for 234 (n
Molecules 2019, 24, 116 33 of 68
Molecules 2018, 23, x 33 of 68

(n = 1),
= 1), which
which only
only showed
showed anan SmA
SmA phase.The
phase. Theblock
blockcopolymers
copolymersshowed
showedan anenantiotropic
enantiotropic mesophase
mesophase
of an SmA phase except for mPEG
SmA phase except for mPEG4343 -b-P(MCC-C ) (236) (n
51 (236) (n =
11 51 = 1), with the mesophase temperature
range of the copolymers (236) being greater than those of the the corresponding
corresponding chiral
chiral compounds
compounds (234).
(234).
It was
wasalso
alsoconcluded
concludedthat
thata longer
a longer spacer
spacer tended
tended to stabilize
to stabilize thethe mesophase
mesophase moremore
thanthan a shorter
a shorter one
one and showed a wide mesophase range. These new polycarbonate copolymers
and showed a wide mesophase range. These new polycarbonate copolymers with longer with longer spacers
based on cholesterol exhibited mesophase states below body temperature, which makes them good
candidates for drug delivery applications [60].[60].
The synthesis of the cholesterol-triazine-BODIPY
cholesterol-triazine-BODIPY trimers 239 and
trimers 239 and 240 with one or two cholesterol
cholesterol
units involved the reaction of cyanuric chloride-substituted BODIPY derivative 238 with an esterified
cholesterol derivative (237), using different
different reaction
reaction conditions
conditions (Scheme
(Scheme 59)
59) [61].
[61].

59. Synthesis
Scheme 59. Synthesis ofof cholesterol-triazine-BODIPY
cholesterol-triazine-BODIPY trimers.
trimers. Reagents and
and conditions: a) Na
conditions: a) Na22CO33,,
acetone, rt, 6 h; b) 237, Na22CO33,, THF,
THF, reflux,
reflux, 10 h. BODIPY:
BODIPY: Boron dipyrromethene.

The
The cholesterol-triazine-BODIPY
cholesterol-triazine-BODIPY trimers
trimers239 and
239 240 andexhibited distinct mesomorphic
240 exhibited properties,
distinct mesomorphic
dependent on the number of cholesterol units. The one-cholesterol unit
properties, dependent on the number of cholesterol units. The one-cholesterol unit derivative derivative 239 showed nematic
239
liquid
showed crystal behavior,
nematic liquidwhile
crystalthe behavior,
two-cholesterolwhile unitthe 240 was a hexagonal
two-cholesterol unitcolumnar
240 was liquid crystal.
a hexagonal
The photophysical
columnar liquid crystal.properties of both compounds
The photophysical properties were ofalso
bothaddressed,
compounds and thealso
were authors concluded
addressed, and
that both derivatives presented good fluorescence intensities with
the authors concluded that both derivatives presented good fluorescence intensities with higher quantum yields andhigher
larger
Stokes
quantum shifts when
yields and compared to their
larger Stokes precursors.
shifts The authors
when compared claimed
to their that this
precursors. The study reported
authors the that
claimed first
examples of cholesterol-BODIPY liquid crystals, in which the introduction
this study reported the first examples of cholesterol-BODIPY liquid crystals, in which the of a cholesterol unit was
favorable
introductionfor of
both liquid crystalline
a cholesterol unit wasbehavior
favorableand forimproved fluorescence
both liquid [61].
crystalline behavior and improved
The synthesis
fluorescence [61]. of two series of λ-shaped dicholesteryl-based conjugates, 242 and 245, containing
a Schiff
Thebase core linking
synthesis two cholesteryl
of two series of λ-shapedester units was reported.
dicholesteryl-based The first
conjugates, 242series of compounds
and 245, containing
was prepared
a Schiff base corebased
linkingon atwo Williamson
cholesteryletherification
ester units was between
reported.theThe
Schiff-base
first series(241) and cholesteryl
of compounds was
bromo-alkanoates
prepared based on(200) to afford XSB-n-Chol
a Williamson etherification (n =between
4–10) derivatives (242) (Scheme
the Schiff-base (241) and 60)cholesteryl
[62]. The synthesis
bromo-
of SB-10-Chol
alkanoates (200)(244) was slightly
to afford different
XSB-n-Chol (n =and4–10)involved the alkylation
derivatives of 2,4-dihydroxybenzadehyde
(242) (Scheme 60) [62]. The synthesis of
(243) by cholesteryl
SB-10-Chol (244) was bromo-decanoate
slightly different (200)
andfollowed
involvedbythe condensation
alkylation of with 4-aminophenol to afford
2,4-dihydroxybenzadehyde
OHSB-10-Chol (245) (Scheme 60) [62]. The study of the liquid
(243) by cholesteryl bromo-decanoate (200) followed by condensation with 4-aminophenol crystal properties of the conjugates 242
to afford
and 245 indicated that the compounds had enantiotropic chiral nematic behavior,
OHSB-10-Chol (245) (Scheme 60) [62]. The study of the liquid crystal properties of the conjugates 242 with an exception for
short
and 245conjugates,
indicated which formed
that the an additional
compounds SmA phase along
had enantiotropic with
chiral the narrow
nematic intermediary
behavior, with anTGB phase.
exception
All compounds showed mesogenic properties, as they could form oily
for short conjugates, which formed an additional SmA phase along with the narrow intermediary streaks, fan-shaped filaments, and
Grandjean
TGB phase.textures in the liquid
All compounds crystalline
showed state. The
mesogenic authors also
properties, found
as they that long
could formspacer compounds
oily streaks, fan-
vitrified to form stable
shaped filaments, andcholesteric
Grandjeanglassy states
textures instead
in the liquid of crystallization.
crystalline state. Furthermore,
The authorsthe mesomorphic
also found that
temperature
long spacer range increased
compounds alongside
vitrified to the
form length
stableof the spacer (from
cholesteric n =states
glassy 4 to n =instead
10), showing an odd-even
of crystallization.
alternation
Furthermore, on the
the mesomorphic
clearing and transition
temperaturetemperatures [62]. alongside the length of the spacer (from
range increased
n = 4 to n = 10), showing an odd-even alternation on the clearing and transition temperatures [62].
Molecules 2019, 24, 116 34 of 68
Molecules 2018,
Molecules 2018, 23,
23, xx 34 of
34 of 68
68

Scheme 60.
Scheme 60. Synthesis of λ-shaped
Synthesis of λ-shaped chiral liquid
λ-shaped chiral
chiral liquid crystal
liquid crystal trimers.
crystal trimers. Reagents
trimers. Reagents and
Reagents and conditions:
and conditions:a)
conditions: a) K
a) K222CO
K CO33,,, KI,
KI,
KI,
DMF,
DMF, 90 ◦°C, 24 h; b) K CO , acetone, reflux, 8 h; c) 4-aminophenol, EtOH, AcOH, reflux, 5 h.
DMF, 90 C, 24 h; b) K2CO33,, acetone,
90 °C, 2 3 acetone, reflux,
reflux, 88 h;
h; c) 4-aminophenol, EtOH, AcOH, reflux, 5 h.
c) 4-aminophenol,

In 2015, Frizon
In 2015, Frizon and
and coworkers
coworkers described
described the synthesis of
the synthesis preliminary studies
and preliminary
of and studies on the thermal
on the thermal
and
and photophysical properties of
photophysical properties of selenium liquid crystals
selenium liquid crystals containing cholesterols 247,
containing cholesterols 247, 249, 251, and
249, 251, and
253. synthesis
synthesis of
of three
three new
new series
series of
of selenide
selenide 247/251
247/251 and
and diselenide
diselenide compounds
253. The synthesis of three new series of selenide 247/251 and diselenide compounds 249/253 was
The compounds 249/253
249/253 was
accomplished via
accomplished via esterification
esterification ofof cholesterol
of cholesterol 28
cholesterol 28 with
28 with the
with theappropriate
the appropriateselenide
appropriate selenide246/250
selenide 246/250 or
246/250 or diselenide
diselenide
acid 248/252 (Scheme
acid 248/252
248/252 (Scheme 61)
(Scheme61) [63].
61)[63].
[63].

61. Synthesis
Scheme 61.
Scheme 61.
of cholesterol-based
cholesterol-based selenides
Synthesis of cholesterol-based selenides and
selenides and diselenides.
and
Reagents and conditions: (a)
diselenides. Reagents
diselenides. and conditions: (a)
DMAP,
DMAP, DCC,
DCC, CH
CH 22Cl
Cl22,, rt,
rt, overnight.
overnight.
DMAP, DCC, CH2Cl2, rt, overnight.

All synthesized
synthesized compounds
All synthesized compounds presented
presentedgood good thermal
thermal stability.
stability. Six
Six of them
of them showed liquid
them showed
showed liquid crystal
crystal
All compounds presented good thermal stability. Six of liquid crystal
properties,
properties, inin which
in which selenide
which selenide 251
selenide 251 and
251 and alkyl
and alkyl diselenides
alkyl diselenides
diselenides 249249 (n
249 (n= 2)
(n == 2) and
2) and 249
and 249
249 (n(n = 3)
(n == 3) exhibited
3) exhibited
exhibited anan SmC*
an SmC*
SmC*
properties,
mesophase,
mesophase, whereaswhereasaryl diselenide
aryl diselenide253, with
253, higher
with structural rigidity, showed
higher structural
structural rigidity, a chiral
showedenantiotropic
mesophase, whereas aryl diselenide 253, with higher rigidity, showed aa chiral
chiral
smectic A (SmA*)
enantiotropic smectic mesophase.
smectic A Furthermore,
A (SmA*)
(SmA*) mesophase. all these new
mesophase. Furthermore, selenide-cholesterol
Furthermore, all all these
these newcompounds showed
new selenide-cholesterol
selenide-cholesterolhigher
enantiotropic
glutathione
compounds peroxidase-like
showed activity
higher than the standard
glutathione ebselen, activity
peroxidase-like with selenide
than 249
the (nstandard
= 2) the most activewith
ebselen, [63].
compounds showed higher glutathione peroxidase-like activity than the standard ebselen, with
A series
selenide 249 (n of=glycosteroids
(n 2) the
the most (256) [63].
most active
active constituted by cholesterol and distinct glycosidic moieties were
selenide 249 = 2) [63].
synthesized
A series by
series of coupling
of glycosteroidspropargyl
glycosteroids (256) 1-S-propargyl
(256) constituted
constituted D -glucose, and
by cholesterol
cholesterol D -galactose, or L-rhamnose
distinct glycosidic
glycosidic moieties (255)
wereto
A by and distinct moieties were
cholesterol
synthesized by scaffold 254
by coupling through
coupling propargyl a CuAAC
propargyl 1-S-propargyl reaction
1-S-propargyl D (Scheme 62)
-glucose, D
D-glucose, [64]. This
-galactose, or
D-galactose, study aimed
or LL-rhamnose to analyze
-rhamnose (255) (255) to if
to
synthesized
the sugar structure
cholesterol scaffold as 254well as theaheteroatom
through CuAAC reaction linked(Scheme
reaction to the anomeric
62) [64]. position
[64]. This
This study
study had an impact
aimed on the
to analyze
analyze if
cholesterol scaffold 254 through a CuAAC (Scheme 62) aimed to if
liquid-crystalline
the sugar structure properties
as well asof the
the glycosteroids
heteroatom (256).
linked to The
the mesomorphic
anomeric temperature
position had an range
impact found
on the
the sugar structure as well as the heteroatom linked to the anomeric position had an impact on the
for the glycosteroids
liquid-crystalline (256) was
properties higher
of the
the than that generally
glycosteroids Thereported
(256). The mesomorphic in thetemperature
literature, but similar
range found to
liquid-crystalline properties of glycosteroids (256). mesomorphic temperature range found
that reported
for the for
the glycosteroids other
glycosteroids (256) glycosteroids.
(256) was
was higher All
higher than the
than that studied
that generallyglycosteroids
generally reported
reported in (256)
in the showed
the literature, great
literature, but phase
but similar stability
similar to to that
that
for
compared
reported to those
for other already studied,
glycosteroids. Allandthe interestingly,
studied glycosteroids
glycosteroids (256)(256) (sugargreat
showed = D-glucose;
phase X = S)
stability
reported for other glycosteroids. All the ◦ studied glycosteroids (256) showed great phase stability
showed
compared notodecomposition
alreadyeven
those already at 200
studied, andC.interestingly,
These resultsglycosteroids
interestingly, offer new possibilities
(256) (sugar in the
(sugar Ddevelopment X == of
compared to those studied, and glycosteroids (256) == D -glucose; X
-glucose; S)
S)
new
showedhigh-temperature
no decomposition captors
even oratdetectors
200 °C. [64].
These results offer new possibilities in the development of
showed no decomposition even at 200 °C. These results offer new possibilities in the development of
new high-temperature
new high-temperature captorscaptors oror detectors
detectors [64].
[64].
Molecules 2019, 24, 116 35 of 68
Molecules 2018,
Molecules 2018, 23,
23, xx 35 of
35 of 68
68

Scheme 62.
Scheme
Scheme 62. Synthesis
Synthesis of
Synthesis of O-
of O- and
and S-glyco-triazole-cholesterol
S-glyco-triazole-cholesterol derivatives.
S-glyco-triazole-cholesterol derivatives. Reagents
derivatives. Reagents and
Reagents and conditions:
and conditions: a)
conditions: a)
a)
CuSO
CuSO 4∙5H2O, AscONa, 1,4-dioxane/H2O (4:1), 80 °C,
·5H22O,
CuSO44∙5H O,AscONa,
AscONa,1,4-dioxane/H
1,4-dioxane/H2O O (4:1),
(4:1), 8080 ◦
°C, 24
C,
24 h.
24
h. h.
2

5. Cholesterol-Based
5. Cholesterol-Based GelatorsGelators
Low molecular
Low molecular
molecular weight
weightorganic
weight organicgelators
organic gelators(LMOGs)
gelators (LMOGs)are
(LMOGs) aresmall
are smallorganic
small organic
organic molecules
molecules
molecules that self-assemble
that
that self-assemble
self-assemble in
water
in or organic
in water
water or solvents,
or organic
organic forming
solvents,
solvents, a 3D network
forming
forming aa 3D
3D network that entraps
network the liquid
that entraps
that entraps thephase,
the liquid
liquid resulting in gel formation.
phase, resulting
phase, resulting in gel
in gel
In recent
formation. years,
In these
recent classes
years, of
these compounds
classes of have
compounds attracted
have much
attracted attention
formation. In recent years, these classes of compounds have attracted much attention because of their much because
attention of their
because range
of of
their
applications,
range of
range for example
of applications,
applications, for as
for examplealternative
example as biomaterials
as alternative
alternative biomaterials for drug
biomaterials for delivery
for drug or
drug deliverytissue
delivery or engineering
or tissue [65,66].
tissue engineering
engineering
New generations
[65,66]. New of steroidal
generations of low molecular
steroidal low mass
molecular gelators
mass
[65,66]. New generations of steroidal low molecular mass gelators (LMGs) are usually (LMGs)
gelators are usually
(LMGs) designed
are usuallythrough
designed
designed the
assembly
through of
the various
assembly building
of units
various such
buildingas a steroid
units derivative
such as a (S),
steroid a linker unit
derivative
through the assembly of various building units such as a steroid derivative (S), a linker unit (L), and (L),
(S), and
a often
linker an
unit aromatic
(L), and
platform
often an
often (A) around
an aromatic
aromatic which the
platform
platform (A)steroid
(A) aroundunits
around which
which canthebesteroid
the positioned
steroid units
units through
can be
can be linkers.
positioned
positioned Thethrough
good gelation
through linkers.
linkers.ability
The
The
of the
good steroidal
gelation LMGs
ability led
of to
thethe development
steroidal LMGs of a
ledseries
to of
the steroid-based
development
good gelation ability of the steroidal LMGs led to the development of a series of steroid-based gelators
of a commonly
series of classified
steroid-based as
ALS, arranged
gelators in
commonly A(LS)
gelators commonly classified 2 , A(LS)
classified as
as , LS,
ALS, or LS
arranged
3 ALS, arranged 2 molecular
in types
A(LS) 2 , [65].
A(LS) 3 , LS, or LS 2 molecular
in A(LS)2, A(LS)3, LS, or LS2 molecular types [65]. types [65].
In 2014, an interesting study was
In 2014, an interesting study was reported involving reported involving the the design
design of of an
an uncommon
uncommon class class of
of
cholesteryl-based
cholesteryl-based triangular
triangular A(LS) -type
33-type low
low molecular
molecular mass
mass gelators
gelators
cholesteryl-based triangular A(LS)3-type low molecular mass gelators and the exploration of their and
and the exploration of their
gelation and
gelation and anion-sensing
anion-sensing applications.
applications. The The design
design strategy
strategy was was basedbased on on placing
placing three
three cholesteryl
cholesteryl
derivatives using
derivatives using linker
linker units
units around
around melamine
melamine
melamine or or
or benzene-1,3,5-tricarbonyl
benzene-1,3,5-tricarbonyl chloride
benzene-1,3,5-tricarbonyl chloride as as aromatic
aromatic
platform precursors. The
The synthesis
synthesis of compounds
compounds 257 and 259
platform precursors. The synthesis of compounds 257 and 259 involved the reaction of cholesterylinvolved the reaction of cholesteryl
cholesteryl
chloroformate 77 with
chloroformate with different
different
different amines
amines in
amines in one-
in one- or
one- or two-step
or two-step procedures
two-step procedures(Scheme
procedures (Scheme63)
(Scheme 63)[67].
63) [67].
[67].

Scheme 63.
Scheme 63. Synthesis of
63. Synthesis
Synthesis of triangular
of triangular A(LS)
triangular -type cholesteryl
A(LS)333-type
-type cholesteryl derivatives
cholesteryl derivatives with
derivatives with different
with aromatic
different aromatic
platforms and
platforms and linkers.
and linkers. Reagents
linkers. Reagents and
Reagents and conditions:
and conditions:a) melamine,
conditions: a) Et
a) melamine,
melamine,
3 N, MeCN, reflux,
N, MeCN,
Et33N, 9 days;
MeCN, reflux, b) hydrazine
reflux, 99 days;
days; b)b)
platforms Et
hydrate (n hydrate
hydrazine = 0) or ethylenediamine
(n = 0) or (n = 2) or 1,3-diaminopropane
ethylenediamine (n = 2) or (n = 3), Et3 N,
1,3-diaminopropane (n CH
= 3),2 Cl
Et 23,N,
rt, CH
overnight;
2Cl2, rt,
hydrazine hydrate (n = 0) or ethylenediamine (n = 2) or 1,3-diaminopropane (n = 3), Et3N, CH2Cl2, rt,
c) benzene-1,3,5-tricarbonyl
overnight; chloride, Etchloride,
c) benzene-1,3,5-tricarbonyl
benzene-1,3,5-tricarbonyl 3 N, THF,Et rt,3N,
Et overnight.
THF, rt, overnight.
overnight; c) chloride, 3N, THF, rt, overnight.

This study
This study also involved
study also
also involved the evaluation of
the evaluation
evaluation gelation and
of gelation
gelation and self-assembly
self-assembly properties of
properties of this
of this new class
this new
new class
This involved the of and self-assembly properties class
of compounds
of compounds
compounds by by comparing
by comparing
comparing them them
them to to the
to the existing
the existing cholesteryl-based
existing cholesteryl-based
cholesteryl-based LMGs. LMGs.
LMGs. The The results
The results indicated
results indicated
indicated thatthat
of
that
the the gelation
gelation and and self-assembly
self-assembly properties
properties of of compounds
compounds 257257 and259
and couldbe
259could be controlled
controlled by by
the gelation and self-assembly properties of compounds 257 and 259 could be controlled by
modification
modification ofof the
of the structural
the structural features
structural features of
features of the
of the A(LS)
the A(LS) -type
A(LS)33-type
3 molecule.
-type molecule. Increasing
molecule. Increasing the
Increasing the length
the length
length ofof the
of the linker
the linker
linker
modification
units, the fibrous xerogel networks assembled into more porous fiber networks.
units, the fibrous xerogel networks assembled into more porous fiber networks. Moreover, the Moreover, the
Molecules 2019, 24, 116 36 of 68
Molecules
Molecules 2018,
2018, 23,
23, xx 36
36 of
of 68
68

authors
units, thefound that
fibrous the compounds
xerogel 257 and 259
networks assembled intocould
more be usedfiber
porous as selective
networks.sensors for F−−the
Moreover, , and their
authors
selectivity
found that could be enhanced
the compounds 257by
andincreasing
259 couldthe
bechain
used aslength of their
selective linkerfor
sensors −
units
F , [67].
and their selectivity
couldTwo new cholesterol-based
be enhanced by increasing thecompounds
chain length(261) were
of their also
linker reported
units [67]. as fluoride-responsive
organogels.
Two new Their design was based
cholesterol-based on the (261)
compounds coupling
wereof compounds
also reported asinfluoride-responsive
260, bearing azo units as the
organogels.
chromophore
Their design was andbased
a pyrazole group asofthe
on the coupling anion acceptor,
compounds in 260, with theazo
bearing cholesteryl chloroformate
units as the chromophore7
(Scheme
and 64) [68].
a pyrazole group as the anion acceptor, with the cholesteryl chloroformate 7 (Scheme 64) [68].

Scheme 64. Synthesis of cholesterol-azobenzyl organogels. Reagents

cholesterol-azobenzyl organogels. Reagents and
and conditions:
conditions: a)
a) Et
Et333N, CHCl333,,
rt, 24 h.

The
The authors
authors observed
observed that
that structural
structural modifications
modifications on on thethe benzyl
benzyl core
core of
of compound
compound 261 261 (R
(R == H
H
or
or NO222), hydrogen bonding, hydrophobic interactions, as well as π-π stacking interactions, had
NO ), hydrogen bonding, hydrophobic interactions, as well as π-π stacking interactions, had
considerable
considerable influence
influence on
on the
the gel-sol
gel-sol transition
transition properties.
properties. Moreover,
Moreover, they they also
also found
found that
that the
the gel
gel was
selectively fluoride-responsive among the tested anions, expressing gel-sol
selectively fluoride-responsive among the tested anions, expressing gel-sol transition transition and red-purple
color
color changes
changes easily
easily detected
detected by by the
the naked
naked eye
eye [68].
[68].
Following the purposes
Following the purposes of
of the
the selective
selective detection
detection ofFF−−−,, a new coumarin-based
of coumarin-based supramolecular
supramolecular
gelator
gelator (267)
(267) was
was designed
designed [69].
[69]. The
The reported
reported compound
compound 267
267 follows
followsaasimple
simplearchitecture
architecturethatthatbears
bearsa
coumarin-appended
a coumarin-appended 1,2,3-triazole coupled
1,2,3-triazole with with
coupled cholesterol, synthesized
cholesterol, in a six-step
synthesized route as depicted
in a six-step route as
in Schemein65.
depicted The coumarin
Scheme moiety acts
65. The coumarin as a fluorescence
moiety signaling unit,
acts as a fluorescence the 1,2,3-triazole
signaling as a linker
unit, the 1,2,3-triazole
and
as a as an anion
linker and asbinding
an anionsite, and cholesterol
binding as a hydrophobic
site, and cholesterol surface.
as a hydrophobic surface.

Scheme
Scheme 65. 65.Synthesis of triazole-linked
Synthesis cholesterol-coumarin.
of triazole-linked cholesterol-coumarin.ReagentsReagents
and conditions: a) chloroacetyl
and conditions: a)
chloride,
chloroacetylpyridine, drypyridine,
chloride, CH2 Cl2 , dry
rt, 10CHh;22b)
Cl22MeCN,
, rt, 10 h;NaN 3 , reflux,
b) MeCN, 5 h;33,c)
NaN propargyl
reflux, 5 h; c)alcohol,
propargyl CuSO 4 , Cu
alcohol,
turning,
CuSO44, Cu EtOH, reflux,
turning, 90 ◦ C,
EtOH, 6 h; d)
reflux, 90methanesulfonyl chloride, CH
°C, 6 h; d) methanesulfonyl 2 Cl2 , Et3CH
chloride, N, 22rt,
Cl30 min;
22, Et e) LiBr,
33N, rt, THF,
30 min; e)
rt, 8 h; f) 6,7-dihydroxycoumarin, MeCN,
LiBr, THF, rt, 8 h; f) 6,7-dihydroxycoumarin,2MeCN, Cs CO 3 , reflux, 36 h.
Cs22CO33, reflux, 36 h.

The authors concluded

The authors concludedthat thatcooperative
cooperative hydrogen
hydrogen bonding
bonding between
between phenolic
phenolic OH and OH aand a
1,2,3-
1,2,3-triazole
triazole ring asring
wellasaswell as hydrophobic-hydrophobic
hydrophobic-hydrophobic interactions
interactions of the cholesteryl
of the cholesteryl groups in
groups in compound
compound 267 played a crucial role in the formation of an organogel. Furthermore,
267 played a crucial role in the formation of an organogel. Furthermore, it was demonstrated that it was demonstrated
that compound organogel − and HP O 3− detection by means of gel phase
compound 267 267
organogel waswas sensitive
sensitive forforF−−F and HP22O2 773−
3−7 detection by means of gel phase
transformation
transformation as as well as fluorimetrically,
well as fluorimetrically, showing
showing considerable
considerable changes
changes inin emission
emission properties
properties [69].
[69].
A novel cholesterol-based organogelator containing D-A (donor-acceptor)
A novel cholesterol-based organogelator containing D-A (donor-acceptor) pairs pairs (salicylaldehyde
and naphthalimide
(salicylaldehyde andunits) (272) was synthesized
naphthalimide units) (272)[70].wasThe synthetic [70].
synthesized strategy
Therelied on the
synthetic introduction
strategy relied
of the electron-rich salicylaldehyde group into a naphthalimide-based
on the introduction of the electron-rich salicylaldehyde group into a naphthalimide-based organogelator through a
Schiff-base reaction (Scheme 66). This cholesterol-based organogelator (272)
organogelator through a Schiff-base reaction (Scheme 66). This cholesterol-based organogelator (272) was found to form
stable and chiral
was found to formgels with
stable anddifferent optical
chiral gels withproperties and morphologies
different optical properties and in several organicinsolvents.
morphologies several
An interesting feature of compound 272 was the changing of the color and
organic solvents. An interesting feature of compound 272 was the changing of the color and emissionemission color of the
organogel in benzene, which varied from yellow-green to red during
color of the organogel in benzene, which varied from yellow-green to red during the the thermoreversible sol-gel
transformation,
thermoreversible demonstrating for the firstdemonstrating
sol-gel transformation, time solvent-controlled
for the firstmultiple color emission achieved
time solvent-controlled multiple in
color emission achieved in a monocomponent gel system. This feature makes the organogel 272 quite
suitable for applications in optical switches, sensors, and smart materials [70].
Molecules 2019, 24, 116 37 of 68

a monocomponent gel system. This feature makes the organogel 272 quite suitable for applications in
optical
Moleculesswitches,
Molecules 2018, 23, xx sensors, and smart materials [70].
2018, 23, 37 of
37 of 68
68

Scheme 66.
Scheme 66. Synthesis
66. Synthesis of
of cholesterol
cholesterol naphthalimide-based
cholesterol naphthalimide-based gelators.
naphthalimide-based gelators. Reagents
gelators. Reagents and conditions:
Reagents and conditions: a) a)
EDAC/BtOH; b)
EDAC/BtOH;
EDAC/BtOH; b) NH
b)NH NH
NH222NH ∙H
2 ·H
NH22∙H O, CuCl,
2 O,
22O, EtOH,
CuCl,EtOH,
CuCl, reflux,
reflux,33 3days;c)
EtOH,reflux, days;c) salicylaldehyde,
days;c)salicylaldehyde, EtOH,
salicylaldehyde,EtOH, reflux,
EtOH,reflux, 2424h.
reflux,24 h.h.

To develop new
To develop newsupramolecular
supramolecular gelators,
gelators, Panja
Panja and and coworkers
coworkers synthesized
synthesized pyrrole pyrrole and
and furan-
furan-based pyridine/pyridinium
based pyridine/pyridinium bisamides
bisamides containing
containing cholesteryl
cholesteryl units
units in in their
their architecture
architecture [71]. [71].
The
The synthesis
synthesis of cholesterol-based
of cholesterol-based bisamides
bisamides (274)
(274) waswas achievedthrough
achieved throughthe thecoupling
coupling reaction
reaction of
of
cholesteryl
cholesteryl chloroacetate
chloroacetate derivate
derivate 262
262 with
with the
the pyridine
pyridine ring
ring nitrogens
nitrogens in
in bisamide
bisamide 273
273 (Scheme
(Scheme 67).
67).

Scheme 67. Synthesis

Scheme 67. Synthesis ofof cholesteryl-bisamides. Reagents and
cholesteryl-bisamides. Reagents and conditions:
conditions: a)
a) 1.
1. DMF, MeCN, reflux,
DMF, MeCN, reflux, 33
days; 2. NH PF , DMF/MeOH
PF666,, DMF/MeOH
days; 2. NH444PF (1:10,
DMF/MeOH (1:10, v/v),
(1:10, v/v),
v/v), H H
H22O,
O,O, hot,
hot, 30
2 hot, 30 min.
30 min.
min.

The gelation properties

The gelation properties of of both bisamide 273
both bisamide and bisamides
273 and bisamides with
with aa cholesteryl
cholesteryl unitunit attached
attached (274)
(274)
were
were evaluated.
evaluated. In In aqueous
aqueous DMSO,DMSO, compound
compound 274 274 (X
(X == O)
O) exhibited
exhibited nongelation
nongelation properties,
properties, while
while
compound
compound 274 (X = NH) produced a light yellow colored gel. This suggests that the heteroatomthe
274 (X = NH) produced a light yellow colored gel. This suggests that the heteroatom of of
aromatic linker
the aromatic played
linker a crucial
played role role
a crucial in gelation. The organogel
in gelation. The organogelformed by compound
formed by compound 274 (X 274= NH)
(X =
revealed itselfitself
NH) revealed to betoa be
good anion
a good sensor,
anion sincesince
sensor, the gel
the state waswas
gel state selectively
selectivelyruptured
ruptured intointo
solution
solutionin
the presence of F − and
− AcO − anions.
− Interestingly, the gel rupture induced by F − was−recovered upon
in the presence of F− and AcO− anions. Interestingly, the gel rupture induced by F− was recovered
the
uponaddition of Fe3+of
the addition . This3+.feature
Fe3+ is veryisuseful
This feature in the in
very useful visual distinction
the visual of F− from
distinction AcO−AcO
of F−− from anions [71].
−− anions

[71]. A different kind of fluorescent organogelator based on cholesterol containing benzothiadiazole

fluorophores 276 and
A different kind278 was designed
of fluorescent and synthesized
organogelator by Sun
based and coworkers
on cholesterol (Schemebenzothiadiazole
containing 68). The authors
aimed to understand
fluorophores 276 andthe 278role
was of designed
hydrogenand bonding and π–πbyinteractions
synthesized and to study
Sun and coworkers the changes
(Scheme 68). Theof
fluorescent properties in the process of gelation of cholesterol-based π-conjugated
authors aimed to understand the role of hydrogen bonding and π–π interactions and to study the organogels [72].
The of
changes authors studied
fluorescent three methods
properties in the of gel preparation
process of gelation(heating-cooling
of cholesterol-based process, ultrasonic
π-conjugated
treatment,
organogelsand [72].mixed solvents, at room temperature) and found that π–π and H-bonding interactions
should be the key contributors in forming gels of 276, while in gel formations of 278, only π–π
interactions seemed to matter. The obtained results suggest that these two multiple-stimuli responsive
luminescent gels, 276 and 278, can be used as smart soft materials sensitive to temperature, solvent,
ultrasound, and Hg2+ [72].
Recently, Panja and Ghosh reported three related works involving cholesterol conjugates bearing
three different moieties (dithioacetal 280, diaminomalononitrile 281, and diazine 282 functional
groups) for sensing a series of cations such as Hg2+ , Cu2+ , Ag2+ , and Fe2+ [73–75]. The three
cholesterol conjugates were synthesized using the same three-step methodology, except for the

Scheme 68.
Scheme 68. Synthesis
Synthesis of
of cholesterol-benzothiadiazole
cholesterol-benzothiadiazole based based compounds.
compounds. Reagents
Reagents and
and conditions:
conditions: a)
a)
4-iodoaniline, Et N, CH 2 Cl 2, rt, 12 h; b) 4-iodophenol, K2CO 3 , acetone, reflux, 24 h; c) 4,7-diethynyl-
4-iodoaniline, Et3N, CH2Cl2, rt, 12 h; b) 4-iodophenol, K2CO3, acetone, reflux, 24 h; c) 4,7-diethynyl-
3

2,1,3-benzothiadiazole, CuI,
2,1,3-benzothiadiazole, Pd(PPh33))44,, di-isopropylamine,
CuI, Pd(PPh di-isopropylamine, THF,
THF, reflux,
reflux, 24
24 h.
h.
 The gelation properties of both bisamide 273 and bisamides with a cholesteryl unit attached (274)
were evaluated. In aqueous DMSO, compound 274 (X = O) exhibited nongelation properties, while
compound 274 (X = NH) produced a light yellow colored gel. This suggests that the heteroatom of
the aromatic linker played a crucial role in gelation. The organogel formed by compound 274 (X =
NH) revealed itself to be a good anion sensor, since the gel state was selectively ruptured into solution
Molecules 2019, 24, 116 38 of 68
in the presence of F− and AcO− anions. Interestingly, the gel rupture induced by F− was recovered
upon the addition of Fe3+. This feature is very useful in the visual distinction of F− from AcO− anions
Molecules 2018, 23, x 38 of 68
[71]. step, which involved the reaction of the intermediate benzaldehyde 279 with 1-dodecanethiol,
final
A different kind ofand
diaminomalononitrile, fluorescent
hydrazine organogelator based onconjugates
to afford cholesterol cholesterol280, containing
281, andbenzothiadiazole
282, respectively
The authors studied three methods of gel preparation (heating-cooling process, ultrasonic 2+ and
fluorophores
(Scheme 69). 276
Theand 278 was designed and
cholesterol-dithioacetal synthesized
conjugate by Sun
280 was used and forcoworkers (Scheme
the detection of Hg 68). The
treatment, and mixed solvents, at room temperature) and found that π–π and H-bonding interactions
authors aimed
incorporated two todistinct
understand the rolei)ofAhydrogen
components: cholesterolbonding
motif to and
assistπ–π interactions and
the self-assembly to molecules
of the study the
should be the key contributors in forming gels of 276, while in gel formations of 278, only π–π
changes hydrophobic
through of fluorescent propertiesand
interaction; in ii)the process
a thiol part of
thatgelation
was used of ascholesterol-based
the reaction-based π-conjugated
recognition
interactions seemed to matter. The obtained results suggest that these two multiple-stimuli
organogels
unit [72].
of the molecule [73].
responsive luminescent gels, 276 and 278, can be used as smart soft materials sensitive to temperature,
solvent, ultrasound, and Hg2+ [72].
Recently, Panja and Ghosh reported three related works involving cholesterol conjugates
bearing three different moieties (dithioacetal 280, diaminomalononitrile 281, and diazine 282
functional groups) for sensing a series of cations such as Hg2+, Cu2+, Ag2+, and Fe2+ [73–75]. The three
cholesterol conjugates were synthesized using the same three-step methodology, except for the final
step, which involved the reaction of the intermediate benzaldehyde 279 with 1-dodecanethiol,
diaminomalononitrile, and hydrazine to afford cholesterol conjugates 280, 281, and 282, respectively
(Scheme 69). The cholesterol-dithioacetal conjugate 280 was used for the detection of Hg2+ and
incorporated two distinct components: i) A cholesterol motif to assist the self-assembly of the
Scheme
Scheme 68. Synthesis of
68. Synthesis ofcholesterol-benzothiadiazole
cholesterol-benzothiadiazolebasedbasedcompounds.
compounds.Reagents
Reagents and
and conditions:
conditions: a)
molecules through hydrophobic interaction; and ii) a thiol part that was used as the reaction-based
a) 4-iodoaniline, Et
4-iodoaniline, Et3N, CH N, CH Cl
3 2Cl2, rt,
2 12, rt, 12 h; b) 4-iodophenol, K
2 h; b) 4-iodophenol, K2CO3, acetone, 2 CO , acetone, reflux, 24 h; c)
3reflux, 24 h; c) 4,7-diethynyl-
recognition unit of the molecule [73]. CuI, Pd(PPh ) , di-isopropylamine, THF, reflux, 24 h.
4,7-diethynyl-2,1,3-benzothiadiazole,
2,1,3-benzothiadiazole, CuI, Pd(PPh3)4, di-isopropylamine,
3 4 THF, reflux, 24 h.

69. Synthesis of cholesterol-based

Scheme 69. cholesterol-based dithioacetal. Reagents and conditions: a) chloroacetyl
dithioacetal. Reagents
chloride, pyridine,
pyridine, dry dry CHClCHCl33, , rt,rt, 1010h;h;b)b)4-hydroxybenzaldehyde,
4-hydroxybenzaldehyde,K2K 2 CO
CO 3, 3 , MeCN,
MeCN, 5 h;5 h; c)
c) 1-
1-dodecanethiol, BF · OEt , dry CH Cl , 0 ◦ C, 30 min; d) diaminomalenonitrile, dry benzene, reflux, 3
dodecanethiol, BF 3∙OEt
3 2 , dry
2 CH 2 Cl
2 22 , 0 °C, 30 min; d) diaminomalenonitrile, dry benzene, reflux,
days; e) NH2NH ·H22O,
NH22∙H O,dry
drybenzene,
benzene,reflux,reflux,33days.
days.

The 2+ of the cholesterol-dithioacetal conjugate,

The authors
authors studied
studied the the sensing
sensing mechanism
mechanism for for Hg
Hg2+ of the cholesterol-dithioacetal conjugate,
realizing that the
realizing that the specific
specific Hg Hg2+2+-induced
-induceddeprotection
deprotectionofofthe the thioacetal
thioacetal functionality
functionality of of
280280 resulted
resulted in
in sol-to-gel transition in DMF/H O (1:1, v/v) through the formation
sol-to-gel transition in DMF/H2O (1:1, v/v) through the formation of precursor aldehyde 279. The
2 of precursor aldehyde 279.
The
authorsauthors also claimed
also claimed that thisthat
wasthis was chemodosimeter
the first the first chemodosimeter
that functionsthatasfunctions
a selectiveas“naked-eye”
a selective
“naked-eye” 2+
Hg2+-detectorHg -detectorinbysitu
by showing showing in situ
sol-to-gel sol-to-gel
conversion conversion [73].
[73].
The cholesterol-diaminomalononitrile
The cholesterol-diaminomalononitrile conjugate 281 was conjugate 281 wasfound
foundtoto form
form supramolecular
supramolecular gels
gels in
in dimethylformamide
dimethylformamide (DMF)/H2O and (DMF)/H 2 O and 1,2-dichlorobenzene, as confirmed by rheological
1,2-dichlorobenzene, as confirmed by rheological studies. In studies.
In addition,
addition, thethe authors
authors verified
verified thatthatthethe
gelgel formed
formed in DMF/H
in DMF/H 2 O was
2O was more more stable
stable andand robust
robust thanthan
the
the one obtained from 1,2-dichlorobenzene, due to strong intermolecular
one obtained from 1,2-dichlorobenzene, due to strong intermolecular forces among the gelators forces among the gelators
in
in DMF/H
DMF/H O. Furthermore,
2O. 2Furthermore, it was
it was alsoalso established
established that that cholesterol-diaminomalononitrile
cholesterol-diaminomalononitrile 281 gel gel
281was
was selective for visual recognition 2+ and Cu2+ ions, and for sensing hydrazine based on the
selective for visual recognition of ofHgHg
2+ and Cu2+ ions, and for sensing hydrazine based on the
dosimetric
dosimetric interaction of the malononitrile motif with
interaction of the malononitrile motif with hydrazine
hydrazine [74].[74].
Concerning
Concerning the the cholesterol-diazine
cholesterol-diazine conjugateconjugate 282, the authors
282, the authors demonstrated
demonstrated that that it
it could
could form
form
nice gels with Ag + and Fe3+ ions in a CHCl /CH OH mixture solvent, using the diazine moiety
nice gels with Ag and Fe ions in a CHCl3/CH
+ 3+ 3 3OH 3 mixture solvent, using the diazine moiety as a
as a metal + and Fe3+ with the aid of
metal ion ion binding
binding site.site.
TheThe gelator
gelator 282282was was abletotodistinguish
able distinguishAg Ag+ and Fe3+ with the aid of
tetrabutylammonium chloride, tetrabutylammonium bromide
tetrabutylammonium chloride, tetrabutylammonium bromide or fluoride, and ammonium or fluoride, and ammonium thiocyanate.
Furthermore, the authors proved that there was no interference of Fe 2+ ions in the detection of Fe3+
thiocyanate. Furthermore, the authors proved that there was no interference of Fe ions in the 2+

ions, as inof
detection the
Fecase of most
3+ ions, as in chemosensors
the case of most and gelators [75].and gelators [75].
chemosensors
The effect of different spacer lengths containing two, three, five, six, ten, or twelve carbon atoms
on cholesterol-based azobenzene organogels 285 and 286 was investigated [76]. For this purpose, a
series of seven azobenzene-cholesterol compounds was synthesized through esterification reactions
Molecules 2019, 24, 116 39 of 68

The effect of different spacer lengths containing two, three, five, six, ten, or twelve carbon atoms on
cholesterol-based
Molecules 2018,
Molecules 23, xx azobenzene organogels 285 and 286 was investigated [76]. For this purpose, a 39
2018, 23, series
39 of 68
of 68
of seven azobenzene-cholesterol compounds was synthesized through esterification reactions of
cholesterol
of derivatives
cholesterol of 283 (bearing
derivatives of 283different spacer
(bearing with 40 -carboxy-4-methoxyazobenzene
lengths)spacer
different lengths) with 4′-carboxy-4-
methoxyazobenzene
284 carried out in the 284 carried
presence out 0in
of N,N the presence of N,N′-dicyclohexylcarbodiimide
-dicyclohexylcarbodiimide (DCC) and
(DCC) and dimethylaminopyridine
(DMAP) in dichloromethane,
dimethylaminopyridine (DMAP)as depicted in Scheme 70.as depicted in Scheme 70.
in dichloromethane,

Scheme 70.
Scheme 70. Synthesis
Synthesis of azobenzene-cholesterol
azobenzene-cholesterol compounds with different different spacers.
spacers. Reagents
spacers. Reagents and and
a) TsCl,
TsCl, DMAP,
DMAP, Et N, CH
Et333N, CH222Cl
Cl222,,, 48
48 ◦ C, 12 h; b) HO(CH
°C,
conditions: a)
conditions: DMAP, Et CH 48 °C, 12 h; b) HO(CH222))nnnOH,
OH, 1,4-dioxane,
OH,1,4-dioxane, reflux,
reflux, 444 h;
1,4-dioxane,reflux, h; c)
h; c)
DCC, DMAP,
DCC, DMAP, CH
DMAP, CH222Cl
CH Cl222,, 24
24 h.
h.

Typical reversible trans-cis

Typical reversible and cis-trans
trans-cis and cis-trans isomerization
isomerization of of the
the azobenzene
azobenzene units
units was
was observed
observed
upon UV-Vis irradiation, giving the compounds 285 and 286 recoverable photoresponsive
upon UV-Vis irradiation, giving the compounds 285 and 286 recoverable photoresponsive properties. properties.
Differential scanning
Differential scanning calorimetry studiesstudies revealed that the spacer length plays a crucial role in in the
the
gelation
gelation phenomenon.
phenomenon. Interestingly,
Interestingly, among
among the
the tested
tested compounds,
compounds, only
only 285285
(n (n
= =
6) 6)
couldcould
form form a
a gel,
gel, and
and in specific
in specific solvents
solvents such
such as as ethanol,isopropanol,
ethanol, isopropanol,and andbutan-1-ol.
butan-1-ol.Furthermore,
Furthermore, the the authors
authors
concluded that the solvents, intermolecular
concluded that the solvents, intermolecular H-bonding, and van der Waals interactions affected
interactions affected the the
aggregation mode and morphology of the
aggregation mode and morphology of the gels [76]. gels [76].
In 2016,
In 2016, aa study
studywaswasreported
reportedon onliquid
liquidcrystal
crystal(LC)
(LC) and
and gelation-based
gelation-based self-assembly,
self-assembly, as as well
well as
as
thethe photoresponsive
photoresponsive behavior
behavior of aofnew
a new unsymmetrical
unsymmetrical azobenzene-cholesterol
azobenzene-cholesterol basedbased dimesogen,
dimesogen, 288
288 [77].
[77]. ThisThis molecule
molecule assembles
assembles a CN a CN
groupgroup at one
at one endend
andand a cholesterol
a cholesterol carbonate,
carbonate, fixed
fixed through
through an
an oxyethylene
oxyethylene spacer,
spacer, to the
to the opposite
opposite endend of the
of the azobenzene
azobenzene unit
unit (Scheme
(Scheme 71).71).

Scheme 71.
Scheme 71. Synthesis of
71. Synthesis of azobenzene-cholesterol-based
azobenzene-cholesterol-based dimesogen.
azobenzene-cholesterol-based Reagents and
dimesogen. Reagents
dimesogen. Reagents and conditions: a)
conditions: a)
pyridine, toluene, 100 ◦ C, 9 h.
°C,
pyridine, toluene, 100 °C, 9 h.

Compound 288
Compound 288 presented
presentedthe thecapacity
capacityofofacting
actingas as
a chiral mesogenic
a chiral mesogenicdyedye
dopant to induce
dopant a high
to induce a
helical-twisting chiral phase in the common nematic phase of 5CB. In addition, the gels
high helical-twisting chiral phase in the common nematic phase of 5CB. In addition, the gels of 288 of 288 formed in
organic solvents
formed exhibited
in organic multiple
solvents stimuli-responsive
exhibited behaviors upon exposure
multiple stimuli-responsive to environmental
behaviors upon exposure stimuli
to
such as temperature, light, and shear forces. The photoresponsive character was also
environmental stimuli such as temperature, light, and shear forces. The photoresponsive characterproven in solution,
in
wasLCalso
andproven
gel states. These properties
in solution, in LC and givegel
to states.
compoundThese288 potential give
properties applications in displays,
to compound as chiral
288 potential
mesogenic dye dopants, photochemical molecular switches, and new versatile LMGs
applications in displays, as chiral mesogenic dye dopants, photochemical molecular switches, and [77].
A new series
new versatile LMGsof[77].
liquid crystal gelators (290) with photoresponsive and aggregation-induced
emission
A new (AIE)
seriesproperties was synthesized
of liquid crystal gelators (290)bywith
connecting cholesterol
photoresponsive andderivatives 200 and
aggregation-induced
tetraphenylethylene (an important AIEgen) to a central azobenzene moiety
emission (AIE) properties was synthesized by connecting cholesterol derivatives 200 and through esterification
tetraphenylethylene (an important AIEgen) to a central azobenzene moiety through esterification
reaction (Scheme 72) [78]. The authors included variations in the alkyl chain spacer (n = 0, 1, 3, 5) to
adjust the distance between cholesterol and azobenzene, while a fixed alkyl chain was placed
between azobenzene and tetraphenylethylene (Scheme 72). The liquid crystal properties of
compounds in 290 were assessed, and the results showed that all compounds exhibited, in pure state,
Molecules 2019, 24, 116 40 of 68

reaction (Scheme 72) [78]. The authors included variations in the alkyl chain spacer (n = 0, 1, 3, 5) to
adjust the distance between cholesterol and azobenzene, while a fixed alkyl chain was placed between
azobenzene and tetraphenylethylene (Scheme 72). The liquid crystal properties of compounds in
Molecules 2018, 23, x 40 of 68
290 were assessed, and the results showed that all compounds exhibited, in pure state, smectic A LC
phases,
smecticenantiotropic
A LC phases, forenantiotropic
290 (n = 0) and
for (n
290= (n
3),=but monotropic
0) and (n = 3), butformonotropic
290 (n = 1) and (n =(n5).= The
for 290 1) andgelation
(n =
properties of compound 290 demonstrated that 290 (n = 3) and (n = 5) form stable
5). The gelation properties of compound 290 demonstrated that 290 (n = 3) and (n = 5) form stable gels gels in appropriate
solvents or solvent
in appropriate mixtures,
solvents whilemixtures,
or solvent 290 (n =while
0) and (n(n
290 = =1)0)cannot
and (n form gels inform
= 1) cannot a range
gels in of asolvents.
range
Anof interesting
solvents. An feature of both
interesting 290 (n
feature of =both
3) and
290 (n
(n == 3)
5)and
LMOGs
(n = 5)isLMOGs
that they havethey
is that significantly enhanced
have significantly
emissions
enhancedinduced
emissions by induced
molecularby self-assembly into fibril into
molecular self-assembly or ribbon-like nanostructures
fibril or ribbon-like [78].
nanostructures [78].

Scheme
72.72. Synthesisofoftrans-C
Synthesis trans-C -Chol. Reagents
Reagents and conditions:
conditions:a)
a)KK2CO 3, 3DMF,
, DMF,5050 ◦ C,
°C, 2424
h. h.
Scheme n n-Chol. 2 CO

Three
Three new cholesteryl-based
new cholesteryl-basedA(LS) 2 - and
A(LS) A(LS)A(LS)
2- and 3 -type LMGs,
3-type 292, 294,
LMGs, 292,and 296,
294, andwithout hydrogen
296, without
bond linkers,
hydrogen were
bond reported
linkers, wereinreported
the literature, synthesized
in the literature, throughthrough
synthesized esterification reactions
esterification of acid
reactions
of acid chlorides
chlorides 291, 293,291,
and293,
295 and
with295 with cholesterol
cholesterol 28 in the28presence
in the presence
of DMAP of DMAP (Scheme
(Scheme 73) [79].
73) [79]. The
The study
study of the gelation properties in various organic solvents indicated that the
of the gelation properties in various organic solvents indicated that the number and position of the number and position
of the substituents
substituents in the cholesteryl
in the cholesteryl moietiesmoieties
attachedattached
to a benzeneto a benzene
ring had ring had ainfluence
a great great influence
on the on the
gelation
asgelation
well as inas well as in the aggregation
the aggregation behaviors behaviors of the2 -A(LS)
of the A(LS) 2- and A(LS)
and A(LS) 3 -type 3-type
LMOGs. LMOGs.
Among Among
these these
three
three gelators,
gelators, 294 and 294
296and 296 showed
showed efficient
efficient gelation
gelation abilities
abilities even even without
without hydrogen
hydrogen bondlinkers,
bond linkers,in
in contrast with the meta-substituted 292, which did not gelate in any
contrast with the meta-substituted 292, which did not gelate in any tested solvent [79]. tested solvent [79].
Recently, the synthesis of a new pillar[6]arene-functionalized cholesterol derivative (298), acting
as an LMG, was reported in the literature [80]. In this new compound, the host–guest pillar[6]arene 300
was linked to a cholesterol unit by the long alkyl chain, as well as amide groups (Scheme 74). This new
pillar[6]arene-cholesterol 298 was found to form an organogel in cyclohexane/hexan-1-ol (10:1, v/v),
which was reversibly responsive to temperature, share stress, and partially host–guest interaction
introduced by ferrocenyl iminium derivative 299. In the case of the addition of ferrocenyl iminium
derivative 299, the organogel could be tuned into a solution and tuned back into the organogel
upon addition of per-butylated pillar[6]arene 300. This interesting feature could be explained
on the basis of host–guest interactions of individual 300 with cationic guest 299 that bound with
pillar[6]arene-cholesterol gelator 298 [80].
In 2015, the development of a new kind of self-healing, degradable, and biocompatible
polypeptide hydrogel based on self-assembly between cholesterol-modified triblock poly(L-glutamic
acid)-block-PEG-block-poly(L-glutamic acid) [(PLGA-b-PEG-b-PLGA)-g-Chol] 302 and β-cyclodextrin
(β-CD)-modified poly(L-glutamic acid) (PLGA-g-β-CD) 303 (Figure 5) was reported in the literature [81].
The authors observed that the hydrogel formation was based on the host and guest linkage
Scheme 73. Synthesis of cholesterol-based A(LS)n-type gelators. Reagents and conditions: a) DMAP,
between β-cyclodextrin (β-CD) and cholesterol, and that their viscoelastic behavior depended on polymer
Et3N, toluene, 90 °C, 2 days; b) DMAP, Et3N, toluene, 90 °C, 5 days.
concentration as well as the β-CD/Chol molar ratio. Those hydrogels showed very interesting self-healing
capabilities, goodthe
Recently, cytocompatibility,
synthesis of a newexcellent flexibility, and quick colorant
pillar[6]arene-functionalized diffusion.
cholesterol With all(298),
derivative these acting
features,
it is anticipated that these self-healable hydrogels may have important applications in tissue engineering
as an LMG, was reported in the literature [80]. In this new compound, the host–guest pillar[6]arene [81].
300 was linked to a cholesterol unit by the long alkyl chain, as well as amide groups (Scheme 74). This
new pillar[6]arene-cholesterol 298 was found to form an organogel in cyclohexane/hexan-1-ol (10:1,
v/v), which was reversibly responsive to temperature, share stress, and partially host–guest
interaction introduced by ferrocenyl iminium derivative 299. In the case of the addition of ferrocenyl
 of acid chlorides 291, 293, and 295 with cholesterol 28 in the presence of DMAP (Scheme 73) [79]. The
study of the gelation properties in various organic solvents indicated that the number and position
of the substituents in the cholesteryl moieties attached to a benzene ring had a great influence on the
gelation as well as in the aggregation behaviors of the A(LS)2- and A(LS)3-type LMOGs. Among these
three 2019,
Molecules gelators, 294 and 296 showed efficient gelation abilities even without hydrogen bond linkers,
24, 116 41 of 68
in contrast with the meta-substituted 292, which did not gelate in any tested solvent [79].

Molecules 2018, 23, x 41 of 68

iminium derivative 299, the organogel could be tuned into a solution and tuned back into the
organogel upon addition of per-butylated pillar[6]arene 300. This interesting feature could be
explained on the basis of host–guest interactions of individual 300 with cationic guest 299 that bound
with pillar[6]arene-cholesterol gelator 298 [80].
Molecules 2018, 23, x 41 of 68

iminium derivative 299, the organogel could be tuned into a solution and tuned back into the
organogel upon addition of per-butylated pillar[6]arene 300. This interesting feature could be
explained
Scheme
Scheme on the
73.73. basis ofofhost–guest
Synthesis
Synthesis interactions
ofcholesterol-based
cholesterol-based A(LS)of
A(LS) individual
-type
nn-type gelators.
gelators.300 with cationic
Reagents
Reagents guest 299
andconditions:
and conditions: that
a) a) bound
DMAP,
DMAP,
with pillar[6]arene-cholesterol
Et Et
N,3N,
toluene, ◦
toluene,9090 C,
°C,22days;
days;b)gelator
b)DMAP, 298
DMAP,Et [80].
Et3N,
N, toluene, 90 ◦
90 °C,
C,55days.
days.
3 3

Recently, the synthesis of a new pillar[6]arene-functionalized cholesterol derivative (298), acting

as an LMG, was reported in the literature [80]. In this new compound, the host–guest pillar[6]arene
300 was linked to a cholesterol unit by the long alkyl chain, as well as amide groups (Scheme 74). This
new pillar[6]arene-cholesterol 298 was found to form an organogel in cyclohexane/hexan-1-ol (10:1,
v/v), which was reversibly responsive to temperature, share stress, and partially host–guest
interaction
Schemeintroduced byof
74. Synthesis ferrocenyl iminium derivative
pillar[6]arene-functionalized 299. In theReagents
cholesterol. case of the
andaddition of a)
conditions: ferrocenyl
11-
bromoundecanoyl chloride, DIPEA, CHCl3, rt, 12 h; b) Mono-OH-P[6], Cs2CO3, DMF, rt, 24 h.

In 2015, the development of a new kind of self-healing, degradable, and biocompatible

polypeptide hydrogel based on self-assembly between cholesterol-modified triblock poly(L-glutamic
acid)-block-PEG-block-poly(L-glutamic acid) [(PLGA-b-PEG-b-PLGA)-g-Chol] 302 and β-cyclodextrin
(β-CD)-modified poly(L-glutamic acid) (PLGA-g-β-CD) 303 (Figure 5) was reported in the literature
Scheme 74. Synthesis
Scheme74. of pillar[6]arene-functionalized
Synthesis of pillar[6]arene-functionalized cholesterol.
cholesterol. Reagents
Reagents and and conditions:
conditions: a) 11- a)
[81].
11-bromoundecanoyl chloride,
bromoundecanoyl chloride, DIPEA,
DIPEA, CHCl
CHCl 3 , 12
3, rt, rt, h;
12b)
h;Mono-OH-P[6],
b) Mono-OH-P[6], Cs32,CO
Cs2CO 3 , DMF,
DMF, rt, 24rt,
h. 24 h.

In 2015, the development of a new kind of self-healing, degradable, and biocompatible

polypeptide hydrogel based on self-assembly between cholesterol-modified triblock poly(L-glutamic
acid)-block-PEG-block-poly(L-glutamic acid) [(PLGA-b-PEG-b-PLGA)-g-Chol] 302 and β-cyclodextrin
(β-CD)-modified poly(L-glutamic acid) (PLGA-g-β-CD) 303 (Figure 5) was reported in the literature
[81].

Figure 5.5.Chemical
Figure Chemicalstructures
structuresof
of(PLGA-b-PEG-b-PLGA)-g-Chol andPLGA-g-β-CD.
(PLGA-b-PEG-b-PLGA)-g-Chol and PLGA-g-β-CD.

6. Bioimaging Applications
The authors observed that the hydrogel formation was based on the host and guest linkage
between β-cyclodextrin (β-CD) andfluorescence
Imaging techniques, particularly cholesterol, imaging
and that techniques,
their viscoelastic behavior
have become depended
powerful toolsonfor
polymer concentration as well as the β-CD/Chol molar ratio. Those hydrogels showed
noninvasive visualization of biological processes in real time with high spatial resolution. Methods veryto
interesting self-healing capabilities, good cytocompatibility, excellent flexibility, and quick
“see into the body” or “see into cells” are essential for the diagnosis and treatment of a disease, as colorant
diffusion.
well With allinto
as for research these
thefeatures,
basic it is anticipated
processes that these bioimaging
of life. Therefore, self-healabletechniques
hydrogels may have
Figure 5. Chemical structures of (PLGA-b-PEG-b-PLGA)-g-Chol and PLGA-g-β-CD. to visualize
important applications in tissue engineering [81].
The authors observed that the hydrogel formation was based on the host and guest linkage
6. Bioimaging Applications
between β-cyclodextrin (β-CD) and cholesterol, and that their viscoelastic behavior depended on
polymer concentration
Imaging techniques,asparticularly
well as the β-CD/Chol
fluorescence molar techniques,
imaging ratio. Those hydrogels
have showed tools
become powerful very
interesting self-healing
for noninvasive capabilities,
visualization good cytocompatibility,
of biological excellent
processes in real flexibility,
time with high and quick
spatial colorant
resolution.
Molecules 2018, 23, x 42 of 68
Molecules 2019, 24, 116 42 of 68

disease, as well as for research into the basic processes of life. Therefore, bioimaging techniques to
visualize physiological
physiological or pathophysiological
or pathophysiological changes inchanges
the body inand
the body and cells
cells have have
become become increasingly
increasingly important
important in biomedical
in biomedical sciences [82].
sciences [82].
The synthesis of a series
series of
of BODIPY-based
BODIPY-based fluorogenic dyes was reported, involving the CuAAC
reaction of a nonfluorescent BODIPY-azide, 304, with a series of nonfluorescent alkyne alkyne molecules,
molecules,
including
including O-propargylated
O-propargylated cholesterol
cholesterol 20 (Scheme 75) [83]. The most interesting molecule molecule was the
cholesterol-linked
cholesterol-linked dyedye 305, which presented red-shifted absorption and emission wavelengths and
displayed
displayed its
its preferential
preferentialaccumulation
accumulationatatthetheintracellular
intracellularmembranes
membranes over thethe
over plasma membrane
plasma membraneof
HeLa cells. This result offers potential applications of cholesterol-BODIPY conjugate
of HeLa cells. This result offers potential applications of cholesterol-BODIPY conjugate 305 in 305 in the
bioimaging of cholesterol trafficking
trafficking in
in living
living cells
cells and organisms [83].
and organisms [83].

Scheme
Scheme 75. 75. Synthesis
Synthesis of BODIPY-triazole-cholesterol fluorescent
of BODIPY-triazole-cholesterol fluorescent dye.
dye. Reagents
Reagents and
and conditions:
conditions: a)
a)
CuSO444,, AscONa,
AscONa, CHCH222Cl
Cl222/H22O O
/H 2 (2:1), rt.rt.
(2:1),

Byrd and
andcoworkers
coworkers reported the synthesis
reported of a crosslinker
the synthesis containing
of a crosslinker two independent
containing cholesterol
two independent
units, with units,
cholesterol or without
with ora photoaffinity label, guided
without a photoaffinity byguided
label, computational methods methods
by computational based onbased
a model
on
afor the transfer
model for the of a cholesterol
transfer molecule molecule
of a cholesterol between two proteins,
between two NPC1 andNPC1
proteins, NPC2and[84]. The synthesis
NPC2 [84]. The
synthesis of crosslinker
of crosslinker 314 (without
314 (without a photoaffinity
a photoaffinity label) label) involved
involved several
several steps,
steps, especially
especially because
because of
of
thethe demanding
demanding six-step
six-step syntheticroute
synthetic routeofofone
oneofofthe
theportions
portionsthat
that constitutes
constitutes the
the crosslinker
crosslinker 314
(Scheme 76) [84].

Scheme 76. cholesterol-based crosslinker. Reagents

76. Synthesis of cholesterol-based Reagents andand conditions:
conditions: a) p-TsOH, MeOH,
a) p-TsOH, MeOH,
◦ C, TsCl, pyridine, 25 ◦ C, 4 h; c) KOAc, DMF/H O, reflux, 12 h; d) TBDMSCl, imidazole,
25 °C, 24 h; b)
b) TsCl, pyridine, 25 °C, 4 h; c) KOAc, DMF/H 2
22O, reflux, 12 h; d) TBDMSCl, imidazole,
pyridine/DMF, 25 ◦ C, 1.5 h; e) LiAlH , THF, 0 ◦ C, 1.5 h; f) (PhO) P(O)N , DPPA, PPh , DIAD, THF, 0
25 °C,
pyridine/DMF, 1.5 h; e) LiAlH444, THF, 0 °C, 1.5 h; f) (PhO)222P(O)N333, DPPA, PPh333, DIAD, THF, 0
◦ C, 22 h; g) Ph P, H O, THF, reflux, 3 h; h) DCC, DMAP, Cl222,,00to
to25 ◦ C, 1.5 h; i) TBAF, THF,
25°C,
to 25 °C, 22 h; g) Ph333P, H222O, THF, reflux, 3 h; h) DCC, DMAP, CH222Cl 1.5 h; i) TBAF, THF,
◦ C, 24 h.
25 °C, 24 h.

Another cholesterol-based crosslinker (322) with a photoaffinity label was also synthesized
(Scheme 78) [84]. The synthesis of such a compound involved two stages: i) The preparation of an
Molecules 2019, 24, 116 43 of 68

Another cholesterol-based crosslinker (322) with a photoaffinity label was also synthesized
(Scheme 78) 77.
Scheme [84]. The synthesis
Synthesis of such
of carboxylic acidacholesterol
compound involvedReagents
derivative. two stages: i) The preparation
and conditions: a) t-BuOK,of an
appropriate carboxylic acid
t-butyl bromoacetate, cholesterol
toluene, 25 °C, 17moiety
h; b) 70%(318) (Scheme PDC,
aq t-BuOOH, 77) [84]; and
celite, ii) the 25
benzene, linkage between
°C, 24 h; c)
compounds andPd/C,
31810%
H2 (1 atm), 312 (previously
MeOH/CH2Clsynthesized) (Scheme
2, 25 °C, 14 h; d) HCO2H,76)Et2[84].
O, 65 °C, 4 h.

Molecules 2018, 23, x 43 of 68

Scheme 77.78.Synthesis
Synthesisof of
carboxylic acid cholesterol
cholesterol-based derivative.
crosslinker with Reagents and conditions:
photoaffinity label. Reagentsa) t-BuOK,
and
t-butyl bromoacetate,
conditions: toluene,
a) PPh3, THF/H 2O,25
◦ C, 173h;
reflux, b)70%
h;b) 318,aq t-BuOOH,
DCC, DMAP,PDC, CH2celite, °C, 1.5 h;25c)◦ C,
Cl2, 25benzene, 24 h; c) H2
HF.pyridine,
appropriate carboxylic acid cholesterol moiety (318) (Scheme 77) [84]; and ii) the linkage between
(1 atm),
THF, 25 10% Pd/C,
°C, 48 h; d) MeOH/CH
NH3, H2NOSO , 25I2◦, C,
2 Cl23H, 14 h; d) HCO
MeOH/THF, 2 H,
0 to 25Et 1565h.◦ C, 4 h.
2 O,
°C,
compounds 318 and 312 (previously synthesized) (Scheme 76) [84].
The authors claimed that with the appropriate connection of the two cholesterol molecules 314
and 322, both proteins (NPC1 and NPC2) are simultaneously occupied in a manner that stabilizes the
protein–protein interaction, allowing detailed structural analysis of the resulting complex.
Furthermore, the introduction of a photoaffinity label in one of the cholesterol moieties, 322, should
allow the covalent attachment of one of the units into its respective protein-binding pocket. The
compounds synthesized in this work may be interesting tools for studying the transfer of cholesterol
between cholesterol-binding proteins [84].
Two cholesterol-based fluorescent lipids, 326 and 329, were synthesized using
nitrobenzoxadiazole (NBD) or rhodamine B, respectively, linked by an ether alkyl chain (Scheme 79).
Compounds 326 and 329 were incorporated into liposome formulations, aiming to create and validate
their use as fluorescent probes for lipoplex tracking, without interfering with green fluorescent
protein (GFP) [85]. The authors concluded that both compounds 326 and 329 did not interfere with
the expression of GFP plasmid, obtaining live cell images without any interference. Furthermore,

77. Synthesis
Scheme 78.
Scheme Synthesis of
of carboxylic
carboxylic acid
acid cholesterol
cholesterol derivative. Reagents and
derivative. Reagents and conditions:
conditions: a) t-BuOK,
a) t-BuOK,
t-butyl bromoacetate, ◦ C, 17 h; b) 70% aq t-BuOOH, PDC, celite, benzene, 25 ◦ C, 24 h; c) H
t-butyl bromoacetate,toluene,
toluene,25
25 °C, 17 h; b) 70% aq t-BuOOH, PDC, celite, benzene, 25 °C, 24 h; c)2
(1 ◦ ◦ C, 4 h.
H2atm), 10%10%
(1 atm), Pd/C, MeOH/CH
Pd/C, MeOH/CH 2 Cl 2 , 225
2Cl , 25 C,
°C,1414h;h;d)d)HCO
HCO2 H,
2H,Et
Et2 2O,
O,65
65 °C, 4 h.

The authors claimed that with the appropriate connection of the two cholesterol molecules 314
and 322, both proteins (NPC1 and NPC2) are simultaneously occupied in a manner that stabilizes the
protein–protein interaction, allowing detailed structural analysis of the resulting complex. Furthermore,
the introduction of a photoaffinity label in one of the cholesterol moieties, 322, should allow the
covalent attachment of one of the units into its respective protein-binding pocket. The compounds
synthesized in this work may be interesting tools for studying the transfer of cholesterol between
cholesterol-binding proteins [84].
Two cholesterol-based fluorescent lipids, 326 and 329, were synthesized using nitrobenzoxadiazole
(NBD) or rhodamine B, respectively, linked by an ether alkyl chain (Scheme 79). Compounds 326
Molecules 2019, 24, 116 44 of 68

and 329 were incorporated into liposome formulations, aiming to create and validate their use as
fluorescent probes for lipoplex tracking, without interfering with green fluorescent protein (GFP) [85].
The authors
Molecules 2018, concluded
23, x that both compounds 326 and 329 did not interfere with the expression of 44GFP
of 68
plasmid, obtaining live cell images without any interference. Furthermore, microscopic observations
clearly showed
microscopic that these fluorescent
observations lipidsthat
clearly showed hadthese
minimal self-quenching
fluorescent andminimal
lipids had photobleaching effects.
self-quenching
The results indicated
and photobleaching that The
effects. the results
synthesized compounds
indicated 326 and 329compounds
that the synthesized may be considered
326 and 329 formay
the
development
be consideredoffor fluorescent probes to trace
the development the intracellular
of fluorescent probestrafficking of cholesterol-derived
to trace the cationic
intracellular trafficking of
liposomes [85].
cholesterol-derived cationic liposomes [85].

Synthesisof
Scheme 79. Synthesis offluorescent
fluorescentlipids.
lipids.Reagents
Reagents
andand conditions:
conditions: a) acrylonitrile,
a) acrylonitrile, 18-crown-6,
18-crown-6, aq
aq KOH/CH Cl ; b)
KOH/CH2Cl2;2b) 2NiCl2∙6H2O, NiCl · 6H
2 Boc O, Boc
2 2O, NaBH
2 O, NaBH
4/MeOH;/MeOH; c) TFA/CH Cl
4 c) TFA/CH2Cl2; d) NBD-Cl,
2 2 ; d) NBD-Cl, CHCl 3 rt,
,
CHCl3, rt, overnight;
overnight;
e) e) TsCl, CHCl
TsCl, pyridine, pyridine, CHCl3 ; f) propane-1,3-diol,
3; f) propane-1,3-diol, anhydrous
anhydrous 1,4-dioxane;
1,4-dioxane; g) rhodamine
g) rhodamine B, DCC, B, DCC,
DMAP,
DMAP,
CH 2Cl2, CH
rt, 22 Cl
h.2 , rt, 2 h.

Reibel et 18 polymer compounds based on linear PEG 332 and novel

Reibel et al.
al. prepared
prepared radiolabeled-
radiolabeled-18FF polymer compounds based on linear PEG 332 and novel
linear-hyperbranched amphiphilic polyglycerol
linear-hyperbranched amphiphilic polyglycerol (hbPG) 334, (hbPG) using
334,using cholesterol
cholesterol as aaslipid
28 28 a lipid anchor
anchor via
via CuAAC chemistry of propargylated compounds 330 and 333 with radiolabeled- 18 F azide 331
CuAAC chemistry of propargylated compounds 330 and 333 with radiolabeled-18F azide 331 (Scheme
(Scheme
80) [86]. 80) [86].
The authors also carried out direct labeling of cholesterol 28 with 18 F (Scheme 80)
and performed in vivo positronCh-PEG emission tomography
18
27-CH2-triazole-TEG- F
(PET)
18
F studies as well as ex vivo
biodistribution studies in mice with both polymers (Ch-PEG 18
Chol O O 27 -CH 2 -triazole-TEG- F 332 and
O
Ch-PEG30 -hbPG 27
330 24 -CH2 -triazole-TEG- F 334) and
18 18OF-cholesteryl fluoride 336. These three new

+ a)
derivatives were incorporated into liposome formulations. N The results showed that both polymers
332 18
N F
Owere quickly 18
F Chol O 18 F-cholesteryl fluoride 336 showed
332 and
N3 334 O excreted by O renal function,
N whereas
27
some retention 331in the lung, liver, and spleen. Liposome formulations with the new polymers showed
O
different physical properties from HO those of the conventional liposomes with 18 F-cholesteryl fluoride
HO
336, as well as fast uptake by theO liver, OH
spleen, and lung. Furthermore, the novel hbPG-polymer O
HO
liposomes of 334 showed OH O
OH similar behavior O to the PEG-shielded vesicles, enhancing HO multifunctionality
OH O
OH
without
Chol
the loss of pharmacokinetic
O
O O properties. This approach opens new possibilitiesOH
O OH in theN Nfield of
O OH O
O O O O OH OH O N
polymer trackingO in vivo and liposome O
O
O
tracing inb)mice via PET [86]. OH OH
30 O O
O OH
In 2015, Palakollu and Kanvah OH O O
OH designed and synthesized cholesterol-conjugated chromophores
O Chol
O O OH O O O O O OH
HO OH OH O O
of α-cyanostilbene/diene
HO 338O and OH 340 exhibiting intramolecular 30 O charge transfer
O O (ICT) and
333 OH OH
aggregation-induced +enhanced emission (AIEE). Compounds 338 and
O O OH were
340 OH easilyO prepared
OH HO OH O OH
from the reaction of cholesterol chloroformate 7 with either a stilbene334 HO 337 or diene OH
derivative 339
18 OH
O F
N3
(Scheme 81) [87]. O OH
331 Ch-PEG30-hbPG24-CH2-triazole-TEG-18F

O
Chol Chol Chol
OH c) S d) 18
O F
28 335 O 336

Scheme 80. Synthesis of radioactive polymers Ch-PEG27-CH2-triazole-TEG-18F, Ch-PEG30-hbPG24-

CH2-triazole-TEG-18F, and 3-[18F]Fluoro-cholest-5-ene. Reagents and conditions: a) CuSO4, AscONa,
PBS, DMSO, 70 °C, 15 min; b) CuSO4, AscONa, PBS, EtOH, 70 °C, 15 min; c) methanesulfonyl chloride,
Et3N, CH2Cl2, rt, 16 h; d) tetrabutylammonium hydroxide (TBAH), [18F]Fluoride, MeCN, 120 °C, 20
min.

The authors also carried out direct labeling of cholesterol 28 with 18F (Scheme 80) and performed
in vivo positron emission tomography (PET) studies as well as ex vivo biodistribution studies in mice
 CH2Cl2, rt, 2 h.

Reibel et al. prepared radiolabeled-18F polymer compounds based on linear PEG 332 and novel
linear-hyperbranched amphiphilic polyglycerol (hbPG) 334, using cholesterol 28 as a lipid anchor via
CuAAC chemistry of propargylated compounds 330 and 333 with radiolabeled-18F azide 331 (Scheme
Molecules 2019, 24, 116 45 of 68
80) [86].

18
F
Ch-PEG27-CH2-triazole-TEG-18F
Chol O O
O
27 O
330
+ a) N 332 18
N F
O 18
F Chol O
N3 O O N
27
331
O
HO
HO O
O
Molecules 2018, 23, x OH HO 45 of 68
OH O
OH O HO O
OH OH
O 27-CH O 2-triazole-TEG-18F 332 and Ch-PEG30-hbPG24-CH2-triazole-TEG- OH N
with both
Chol polymers (Ch-PEG O O OH OH O N
O O O O O OH OH O
18F 334) and 18F-cholesteryl O N
O
30 O
fluoride O 336. O These three b) new derivatives were incorporated
OH OH into liposome
O O
OH Chol and 334 were O O OH
formulations. The results O O OH showed OH thatO both polymers 332 O O Oquickly
O Oexcreted OHby renal
HO OH OH O O
O O liver, O and spleen.
function, whereasHO18F-cholesteryl fluoride OH 336 showed some retention 30 Oin the lung,
OH
333 OH OH OH O
Liposome formulations O O
+ with OH the new polymers showed different HO physical properties
OH O from
OH those of
the conventional liposomes with 18F-cholesteryl fluoride 336, asHO well334 as fast uptake OH
by the liver,
18 OH
O F
N3 O OH
spleen, and lung. Furthermore,
331
the novel hbPG-polymer liposomes of 334 showed similar behavior
Ch-PEG30-hbPG24-CH2-triazole-TEG-18F
to the PEG-shielded vesicles, enhancing multifunctionality without the loss of pharmacokinetic
properties. This approach opens new possibilities O in the field of polymer tracking in vivo and
Chol
Chol Chol S
OH c) d) 18
F
liposome tracing in mice via PET [86]. O
O
28 335 336
In 2015, Palakollu and Kanvah designed and synthesized cholesterol-conjugated chromophores
18 F,
Scheme
Scheme 80.80. SynthesisSynthesis
of α-cyanostilbene/diene of 338 andof 340
radioactive radioactive
polymers Ch-PEG
exhibiting polymers Ch-PEGcharge
27-CH2-triazole-TEG-
intramolecular 27 -CH -triazole-TEG-
18F, 2Ch-PEG
transfer 30-hbPG
(ICT)24- and

Ch-PEG -hbPG24 -CH -triazole-TEG- 18 18

F, and 3-[ F]Fluoro-cholest-5-ene. Reagents and 4conditions:
CH -triazole-TEG-
30 18 F, 2and 3-[ 18 F]Fluoro-cholest-5-ene. Reagents
aggregation-induced enhanced emission◦ (AIEE). Compounds 338 and 340 were easily◦prepared from
2 and conditions: a) CuSO , AscONa,
a)
PBS,CuSO 4 , AscONa,
DMSO, 70 °C, 15PBS,
min; DMSO,
b) CuSO70 C, 15 min;
4, AscONa, b) CuSO , AscONa, PBS, EtOH, 70 C,chloride,
15 min;
the reaction of cholesterol chloroformate 7 withPBS, eitherEtOH, 704°C,
a stilbene 15337
min;orc)diene
methanesulfonyl
derivative 339 (Scheme
c) methanesulfonyl chloride, Et N, CH Cl , rt, 16 h; d) tetrabutylammonium
Et3N, CH2Cl2, rt, 16 h; d) tetrabutylammonium hydroxide (TBAH), [ F]Fluoride, MeCN, 120 °C, 20
3 2 2 18 hydroxide (TBAH),
81) [87].
18 ◦
[min.
F]Fluoride, MeCN, 120 C, 20 min.

The authors also carried out direct labeling of cholesterol 28 with 18F (Scheme 80) and performed
in vivo positron emission tomography (PET) studies as well as ex vivo biodistribution studies in mice

Scheme 81. Synthesis of

81. Synthesis of cholesterol-tethered
cholesterol-tethered aggregation-induced
aggregation-induced enhanced
enhanced emission
emission (AIEE)
fluorogens. Reagents and conditions: a) pyridine, benzene, reflux, 12 h.

The
The authors
authors carefully
carefully studied
studied thethe absorption
absorption andand emission
emission properties
properties of of both
both cholesterol
cholesterol
conjugates
conjugates 338 and 340 and their parent chromophores 337 and 339. An ICT behavior was
338 and 340 and their parent chromophores 337 and 339. An ICT behavior was observed
observed
for
for diene
diene compounds
compounds 339 339 and 340, whereas
and 340, whereas forfor stilbene
stilbene compounds
compounds 337 337 and 338 aa remarkable
and 338 remarkable AIEEAIEE
behavior was detected. The lack of AIEE characteristics in dienes may be explained
behavior was detected. The lack of AIEE characteristics in dienes may be explained by the competing by the competing
nonradiative
nonradiative losses
lossesdue
duetotodouble
doublebondbondflexibility. Nevertheless,
flexibility. Nevertheless, thethe
most
mostinteresting conclusion
interesting of the
conclusion of
optical properties study was that the random aggregates formed by stilbene 337
the optical properties study was that the random aggregates formed by stilbene 337 in aqueous media in aqueous media
became
became highly
highly ordered
ordered upon
upon cholesterol
cholesterol conjugation 338. Furthermore,
conjugation 338. Furthermore, the the interaction
interaction with
with sodium
sodium
cholate
cholate stimulated the formation of self-assembled structures in nanoscale dimensions, making these
stimulated the formation of self-assembled structures in nanoscale dimensions, making these
conjugates the starting point for the development of several bioimaging
conjugates the starting point for the development of several bioimaging probes [87]. probes [87].
In
In 2016,
2016, Wercholuk
Wercholuk and and coworkers
coworkers synthesized
synthesized aa fluorescent-labeled
fluorescent-labeled cholesterol
cholesterol molecule
molecule (342)
(342)
by
by treating cholesteryl chloroformate 7 with 4-amino-1,8-naphthalimides (341) (Scheme 82) [88]. [88].
treating cholesteryl chloroformate 7 with 4-amino-1,8-naphthalimides (341) (Scheme 82) The
The authors expected that such conjugates might serve one of two roles,
authors expected that such conjugates might serve one of two roles, depending on whether the depending on whether
the toxicity
toxicity of fluorophore
of the the fluorophore was retained
was retained in theinconjugates:
the conjugates: As reporters
As reporters for following
for following in vivo in vivo
uptake
uptake or catabolism of cholesterol, or as “Trojan horse” antibiotics. The results
or catabolism of cholesterol, or as “Trojan horse” antibiotics. The results pointed out that the new pointed out
that the new(342)
compounds compounds (342)light
emitted blue emitted blue light
in nonpolar in nonpolar
solvents, and its solvents, and incorporated
lipid portion its lipid portion
into
liposomal membrane bilayers quickly, leaving the fluorophore exposed to the external aqueous
environment. Compounds in 342 were incubated with Mycobacterium smegmatis mc2 155, which
displayed stable integration of the fluorescent-labeled cholesterols into bacterial membranes in vivo.
Although fluorophores are toxic to prokaryotic cells, the new cholesterol conjugates (342) are not,
and therefore they could be considered for the evaluation of cholesterol uptake in prokaryotic
Molecules 2019, 24, 116 46 of 68

incorporated into liposomal membrane bilayers quickly, leaving the fluorophore exposed to the
external aqueous environment. Compounds in 342 were incubated with Mycobacterium smegmatis mc2
155, which displayed stable integration of the fluorescent-labeled cholesterols into bacterial membranes
in vivo. Although fluorophores are toxic to prokaryotic cells, the new cholesterol conjugates (342) are
not, and therefore they could be considered for the evaluation of cholesterol uptake in prokaryotic
organisms
Molecules [88].
Molecules 2018,
2018, 23,
23, xx 46
46 of
of 68
68

82. Synthesis
Scheme 82. Synthesis of
of cholesterol-naphthalimide
cholesterol-naphthalimide conjugates. Reagents
Reagents and conditions: a) DIPEA,
EtOAc, 55 ◦°C,
C, 5 days.

In
In the
the same
same year,
year, Bernhard
Bernhard et et al. reported an
al. reported an interesting
interesting paper
paper inin which
which they
they studied
studied two
two
strategies the bioconjugation
strategies for the bioconjugationof ofbombesin
bombesin(BBN),
(BBN),aawell-known
well-knownpeptide,
peptide,thethe receptor
receptor ofof which
which is
is overexpressed
overexpressed at at
thethe surfaceofoftumor
surface tumorcells
cellsand
andwhich
whichhas hasbeen
beenconjugated
conjugatedin inseveral
several probes
probes [89].
[89].
They
They used
used subphthalocyanines
subphthalocyanines (SubPcs),
(SubPcs), which
which are
are interesting
interesting probes
probes for
for optical
optical imaging.
imaging. One
One of
of
these
these strategies
strategies involved
involved the
the entrapping
entrapping of
of SubPc
SubPc into
into a a liposome
liposome and
and subsequently
subsequently grafting
grafting BBN
BBN to
to
thethe SubPc-containing
SubPc-containing liposome
liposome totoafford
affordaabiovectorized
biovectorizedliposome.
liposome. TheThe synthesis
synthesis of
of cholesterol
cholesterol
derivatives 346 and 347 used in their work was achieved by the reaction of dimethylaminopropyne
derivatives 346 and 347 used in their work was achieved by the reaction of dimethylaminopropyne 344
or
3443-azidodimethylpropylamine
or 3-azidodimethylpropylamine 345 with345cholesterol bromo ester
with cholesterol bromo to afford
343 ester 343cholesteryl-ammonium
to afford cholesteryl-
species 346 (alkynyl) and 347 (azide), respectively (Scheme 83) [89].
ammonium species 346 (alkynyl) and 347 (azide), respectively (Scheme 83) [89].

Scheme
Scheme 83.83. Synthesis
Synthesis of
of cholesteryl-ammonium
cholesteryl-ammonium compounds. Reagents and
compounds. Reagents and conditions:
conditions: a) acetone,
a) acetone,
reflux, 3 h.
reflux, 3 h.

Once
Once the
the cholesteryl-ammonium
cholesteryl-ammonium species species 346346 and 347 were
and 347 were prepared,
prepared, thethe pre-bioconjugation
pre-bioconjugation
strategy
strategy started from grafting the biomolecule to one liposome’s component (i.e.,
started from grafting the biomolecule to one liposome’s component (i.e., cholesterol
cholesterol
additive) prior to the preparation of the liposome, to afford BBN-cholesterol
additive) prior to the preparation of the liposome, to afford BBN-cholesterol conjugates 348 and 349. conjugates 348
and 349. The conjugation of BBN-azide with cholesteryl-alkyne 346 (i.e.,
The conjugation of BBN-azide with cholesteryl-alkyne 346 (i.e., pre-functionalization by copper- pre-functionalization
by copper-catalyzed
catalyzed click chemistry)clickwas
chemistry)
carried out was carried
in the out of
presence incopper
the presence of copper
sulfate and sulfate and
sodium ascorbate as
sodium ascorbate as the reducing agents (Scheme 84) [89]. Alternatively, BBN-bicyclononyne
the reducing agents (Scheme 84) [89]. Alternatively, BBN-bicyclononyne and cholesteryl-azide 347 and
cholesteryl-azide
were reacted without 347 were
the Cureacted without
catalyst the Cu
to afford catalyst 349
conjugate to afford conjugate
(Scheme 84) [89]. (Scheme
349This 84) [89].
strategy was
This strategy was employed using liposomes containing graftable cholesterol derivatives,
employed using liposomes containing graftable cholesterol derivatives, revealed itself as a more revealed itself
as a moreapproach
suitable suitable approach in addressing
in addressing the stabilitythe stability
of SubPcs,of SubPcs,
and was and was achieved
achieved by copper-free
by copper-free click-
click-chemistry on the outer face of the liposome. This study demonstrated
chemistry on the outer face of the liposome. This study demonstrated that both azido- and that both azido- and
alkynyl-
alkynyl-liposomes
liposomes are goodare good
entry entry
points forpoints for a bioconjugation
a bioconjugation or biovectorization
or biovectorization approach approach
(on the outer (onface
the
outer face of the liposome), which offers a second chance for fluorophores with
of the liposome), which offers a second chance for fluorophores with no reactive functional group no reactive functional
group available
available on theironbackbone,
their backbone,
a way of a way of imitating
imitating bioconjugation
bioconjugation with a biomolecule
with a biomolecule (i.e., an
(i.e., an indirect
indirect
approach approach
offered tooffered to achieve
achieve future site-specific
future site-specific targetingtargeting of tumors)
of tumors) [89]. [89].
Molecules 2019, 24, 116 47 of 68
Molecules 2018,
Molecules 2018, 23,
23, xx 47of
47 of 68
68

R
R
O N
N
O
O O
Chol N N
N
N
N Chol
Chol a)
a) Chol N N
O O
O N
O
346 348
348 O
346 O
R
R
N
N
O
O H
H
O O
O
O
N Chol b) Chol
Chol N
N
N Chol b) O N
N33
N O
O O N
347 N N
N
347 349
349 N

HN
HN S
S
NH
NH N
N
O
O OO O
O O
O
H
H H
H H
H H
H
R ==
R O
O N
N N
N N
N N
N NH22
NH
O
O N
N N
N N
N N
N
H
H H
H H
H H
H
O
O O
O O
O O
O O
O

H22N
H N O
O

Scheme 84.
Scheme Synthesis
84.Synthesis
84. Synthesis of of bombesin
of bombesin
bombesin (BBN)-cholesterol
(BBN)-cholesterol
(BBN)-cholesterol conjugates.
conjugates.
conjugates. ReagentsReagents
Reagents and conditions:
and conditions:
and conditions: a) N-2-2-
a) N-2-2-
a) N-2-2-[2-(N-azidoethoxy)ethoxy]acetyl-bombesin,
[2-(N-azidoethoxy)ethoxy]acetyl-bombesin, CuSO ∙5H CuSO
O,
[2-(N-azidoethoxy)ethoxy]acetyl-bombesin, CuSO4∙5H2O, AscONa,
4 2 ·
AscONa,
4 5H O, AscONa,
DMF, rt, 8 h;DMF,
b) rt, 8 h; b)
N-2-[2-(2-(N-4-
2 DMF, rt, 8 h; b) N-2-[2-(2-(N-4-
N-2-[2-(2-(N-4-(((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)amino)ethoxy)ethoxy]acetyl-bombesin,
(((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)amino)ethoxy)ethoxy]acetyl-bombesin, DMF,
(((bicyclo[6.1.0]non-4-yn-9-ylmethoxy)carbonyl)amino)ethoxy)ethoxy]acetyl-bombesin, DMF, rt,rt, 15
15
DMF, rt, 15 h.
h.
h.

A series
A of
series of new
of new hybrid
new hybrid compounds
hybrid compounds (Ch-DAINs), 355,
compounds (Ch-DAINs),
(Ch-DAINs), 355, 356, and 360,
356, and 360, bearing
bearing aa green
a green fluorescent
green fluorescent
fluorescent
A series 355, 356, and 360, bearing
protein-chromophore
protein-chromophore analogue,
analogue, 4-(diarylmethylene)imidazolinone (DAIN),
4-(diarylmethylene)imidazolinone (DAIN), and and aa cholesten
cholesten oror
protein-chromophore analogue, 4-(diarylmethylene)imidazolinone (DAIN), and a cholesten or
cholestane, was
cholestane, was recently
was recently reported
recently reported as
reported as a candidate
as aa candidate for
candidate for viscosity-dependent
for viscosity-dependent
viscosity-dependent and and cholesterol-responsive
and cholesterol-responsive
cholesterol-responsive
cholestane,
fluorescent
fluorescent molecules [90].
molecules [90]. The
Thesynthesis
synthesisofofCh(en)-DAINs
Ch(en)-DAINs355 and356
355and wascarried
356was carriedout
outthrough
through
fluorescent molecules [90]. The synthesis of Ch(en)-DAINs 355 and 356 was carried out through aa
a condensation
condensation reaction
reaction of methyl
of methyl
methyl imidatesimidatesor 352
352 or or 358 (obtained
358 (obtained
(obtained from cholestenone
from cholestenone
cholestenone through
through Beckmann
Beckmann
condensation reaction of imidates 352 358 from through
Beckmann rearrangement
rearrangement followed
followed by followed by
by methylation) methylation)
methylation) with with N-(diarylmethylene)
with N-(diarylmethylene)
N-(diarylmethylene) glycinates
glycinates 353glycinates
353 or
or 354 353
354 (Scheme
(Schemeor 354
85)
rearrangement 85)
(Scheme
[90]. 85) [90].
Likewise, Likewise, Ch(an)-DAINs
Ch(an)-DAINs 359 and 360 359 and
were 360 were
obtained obtained
following the following
same the same
synthetic synthetic
strategy with
[90]. Likewise, Ch(an)-DAINs 359 and 360 were obtained following the same synthetic strategy with
strategy with an
an additional
additional additional hydrogenation
double-bond double-bond hydrogenation
(Scheme step
step (Scheme (Scheme 85) [90].
85) [90].
[90].
an double-bond hydrogenation step 85)

Scheme 85.
Scheme 85. Synthesis
Synthesis of
Synthesis of Ch-DAINs.
of Ch-DAINs. Reagents
Ch-DAINs. Reagents
Reagents andand conditions: a)
and conditions: a) NH
a) NH OH∙HCl, NaOAc,
OH·HCl,NaOAc,
NH222OH∙HCl, EtOH,
NaOAc,EtOH, reflux;
EtOH,reflux;
reflux;
b) phenylpropanolamine (PPA),
b) phenylpropanolamine (PPA), 130 °C,130 ◦
°C, 2 steps; c) MeOTf, CH Cl
C, 2 steps; c) MeOTf, CH22Cl22,, rt,
2 2 , rt, 24
rt, 24 h;
24 h; d)
h; d) AcOH,
d) AcOH, toluene,
AcOH, toluene, 50
toluene, 50 ◦ C,
°C,
50 °C,
48 h;
48 h; e)
e) Pd/C
Pd/C (10%),
Pd/C (10%), MeOH,
(10%),MeOH,
MeOH,rt,rt,
rt,777days;
days;
days;f)f) AcOH,
f)AcOH, toluene,
AcOH,toluene, reflux,
toluene,reflux, overnight.
reflux,overnight.
overnight.

Among the tested compounds,

Among compounds, cholesten
cholesten DAINs
DAINs 355 and 356
355 and 356 increased their fluorescence
intensity
intensity in
inviscous solvents
viscous such
solvents as
such triglycerides.
as Besides,
triglycerides. compound
Besides, 355 showed
compound good
355 cholesterol-
showed good
cholesterol-responsive
responsive emission,
emission, which which
increased increased
linearly withlinearly with of
the amount thecholesterol
amount ofincholesterol in the lipid
the lipid bilayer. The
responsiveness displayed by cholesten DAIN 355 to cholesterol was improved relatively to the
known viscosity probes, 9-(2,2-dicyanovinyl)julolidine (DCVJ) and Laurdan [90].
Molecules 2019, 24, 116 48 of 68

bilayer. The responsiveness displayed by cholesten DAIN 355 to cholesterol was improved relatively
Molecules
to 2018, 23,
the known
Molecules 2018, 23, xx
viscosity probes, 9-(2,2-dicyanovinyl)julolidine (DCVJ) and Laurdan [90]. 48 of
48 of 68
68

Synthetic Applications
7. Synthetic Applications
The regio- and and stereoselective
stereoselective formation of O-glycosidic
stereoselective O-glycosidic bonds between
O-glycosidic between carbohydrates
carbohydrates and
steroids is still a demanding process, despite the considerable progress in carbohydrate chemistry in
The direct electrochemical glycosylation
the last years. The glycosylation of
of steroids
steroids is
is an alternative: However,
However, it it has
several drawbacks. In In attempting
attempting toto solve
solve the
the problem,
problem, Tomkiel
Tomkiel et al. screened several derivatives of
cholesterol as
cholesterol as sterol
sterol donors
donors in
in electrochemical
electrochemical reactions
reactions with
with sugar
sugar alcohols
alcohols [91].
[91]. The
The authors
authors tested
tested
substrates in
sixteen cholesterol substrates in the
in the presence
the presence of
presence of1,2:3,4-di-O-isopropylidene-α-
of 1,2:3,4-di-O-isopropylidene-α-DD
1,2:3,4-di-O-isopropylidene-α- -galactopyranose
D-galactopyranose
-galactopyranose
diphenylphosphate 361
(362), concluding that cholesteryl diphenylphosphate
diphenylphosphate 361 was
was the
the best
best compound
compound forfor the
the purpose,
purpose,
affording 3β-O-(1 0 0 0 0
3β-O-(1′,2′:3′,4′-di-O-isopropylidene-α-
,2 :3 ,4 -di-O-isopropylidene-α-D 0
-galactopyranos-6′-yl)-cholest-5-ene (363)
-galactopyranos-6 -yl)-cholest-5-ene(363)
DD-galactopyranos-6′-yl)-cholest-5-ene in
(363) in 54%
in 54%
3β-O-(1′,2′:3′,4′-di-O-isopropylidene-α-
yield (Scheme 86) [91].

Scheme 86.86. Electrochemical

86.Electrochemical oxidation
Electrochemical of cholesteryl
oxidation
oxidation of cholesteryl diphenylphosphate
of cholesteryl in the
the presence
diphenylphosphate
diphenylphosphate in presence of 1,2:3,4-di-
in the of 1,2:3,4-di-
presence of
O- isopropylidene-α- D -galactopyranose.
1,2:3,4-di-O-isopropylidene-α- -galactopyranose.
O-isopropylidene-α-D-galactopyranose.
D

Following this work,

Following this work, the
the same
same authors
authors reported
authors reported in
reported in 2015
in 2015 the
2015 the use
the use of
use of3α,5α-cyclocholestan-6β-yl
of 3α,5α-cyclocholestan-6β-yl
3α,5α-cyclocholestan-6β-yl
alkyl
alkyl and
and aryl
aryl ethers
ethers (364)
(364) as
as aa cholesteryl
cholesteryl donor
donor in
in the
the electrochemical
electrochemical synthesis
synthesis of
of glycoconjugates
glycoconjugates
(363)
(363) (Scheme 87) [92]. The reaction worked well for all the tested compounds, but the
(Scheme
(Scheme 87) [92].
[92]. The reaction worked well for all the tested compounds, but the best
best yields
yields
were achieved for ethyl, benzyl, phenyl, and tert-butyldimethylsilyl (TBDMS) ethers
were achieved for ethyl, benzyl, phenyl, and tert-butyldimethylsilyl (TBDMS) ethers (51%, 50%,
were (51%, 50%, 58%,
58%,
and
and 52%,
52%, respectively).
52%, respectively).Unfortunately,
respectively). Unfortunately,
Unfortunately, an an
isomerization
an isomerization
isomerizationside side
reaction
side was observed
reaction
reaction for the less
was observed
was observed reactive
for the
for the less
less
cholesteryl
reactive esters,
cholesterylaffording
esters, the compounds
affording the in 365
compounds (Scheme
in 365 87) [92].
(Scheme
reactive cholesteryl esters, affording the compounds in 365 (Scheme 87) [92]. 87) [92].

Scheme
Scheme 87. Anodic oxidation
87. Anodic oxidation of
of 3α,5α-cyclocholestan-6β-yl alkyl and
3α,5α-cyclocholestan-6β-yl alkyl aryl ethers.
and aryl ethers.

To
To develop
To step-economy
develop step-economy
develop syntheses of
step-economy syntheses
syntheses ofcholesteryl
of cholesterylglycosides,
cholesteryl glycosides,Davis
glycosides, Davisand
Davis andcoworkers
and coworkersreported
coworkers reportedaa
reported
a methodology
methodology for
methodology for
for the the synthesis
the synthesis
synthesis of of
of α-
α-D α- D -cholesteryl glycosides
-cholesteryl glycosides
D-cholesteryl glycosides 369
369 and369 and
and 372,
372, using372, using
one-potone-pot
using aa one-pot a per-O-
per-O-
per-O-trimethylsilyl
trimethylsilyl glycosyl glycosyl
iodide iodide glycosylation
glycosylation (Scheme (Scheme
88) 88)
[93]. [93].
The The methodology
methodology
trimethylsilyl glycosyl iodide glycosylation (Scheme 88) [93]. The methodology relied first on relied relied
first onfirst
the
the
on the generation
generation of of
glucosyl glucosyl
or or
galactosylgalactosyl
iodide iodide
through through
the the
reactionreaction
of of
per-O-TMSper-O-TMS
generation of glucosyl or galactosyl iodide through the reaction of per-O-TMS glucoside 366 or 370 glucoside glucoside
366 or 366
370
or
with with iodotrimethylsilane
370iodotrimethylsilane
with iodotrimethylsilane (TMSI),
(TMSI), (TMSI),
which
which which
was was
was directly
directly
directly cannulated
cannulated
cannulated into
into
into aa asolution
solutionof
solution of cholesterol,
of cholesterol,
cholesterol,
tetrabutylammonium
tetrabutylammonium iodide
tetrabutylammonium iodide
iodide (TBAI),
(TBAI), and N,N-diisopropylethylamine
(TBAI), and
and N,N-diisopropylethylamine (DIPEA),
N,N-diisopropylethylamine (DIPEA),
(DIPEA), and and the
the mixture
and the mixture was
mixture was
was
stirred for
stirred for
stirred 2 days
for 22 days
days at at room
at room temperature.
room temperature.
temperature. After After
After that, that,
that, the the
the productproduct
product was was
was treated treated
treated with with
with methanol methanol
methanol and and Dowex- and
Dowex-
Dowex-50WX8-200
50WX8-200 acidic
50WX8-200 acidicto
acidic resin
resin toresin
removeto remove
remove the theprotecting
the silyl
silyl silyl protecting
protecting groups,
groups,groups, affording
affording
affording compounds
compounds
compounds 367 and
367 and
367 and 371
371
371 (Scheme
(Scheme 88) 88) [93].
[93]. These
These glycosides
glycosides were
were subsequently
subsequently esterified
esterified using
using
(Scheme 88) [93]. These glycosides were subsequently esterified using regioselective enzymatic regioselective
regioselective enzymatic
enzymatic
acylation
acylation of
acylation of the
of the 6-hydroxy
the 6-hydroxy group
6-hydroxy group with
group with tetradecanoyl
with tetradecanoyl vinyl
tetradecanoyl vinyl ester
vinyl ester 368 (Scheme
ester 368
368 (Scheme 88)
(Scheme 88) [93].
88) [93].
[93].
Molecules 2019, 24, 116 49 of 68
Molecules 2018,
Molecules 2018, 23,
23, xx 49 of
49 of 68
68

Scheme 88.
Scheme
Scheme 88. Synthesis of cholesteryl α-
88. α-D
α- D -glucopyranoside
-glucopyranoside and
D-glucopyranoside and its
and its enzymatic
its enzymatic regioselective
enzymatic regioselective acylation.
regioselective acylation.
acylation.
Reagents and
and conditions:
conditions: a) a) iodotrimethylsilane
iodotrimethylsilane (TMSI),(TMSI), OH-Chol,
OH-Chol, tetrabutylammonium
Reagents and conditions: a) iodotrimethylsilane (TMSI), OH-Chol, tetrabutylammonium (TBAI),
tetrabutylammonium iodide iodide
iodide
(TBAI), DIPEA,
DIPEA, 4DIPEA,
(TBAI), 4
Å molecular Å molecular sieves,
sieves, CHsieves,
4 Å molecular CH
2 Cl2 , rt,CH Cl
48 2h;
2
Clb)
2 , rt, 48 h; b) Dowex-50WX8-200,
Dowex-50WX8-200,
2, rt, MeOH, 2 h, rt;
48 h; b) Dowex-50WX8-200, MeOH, 2
c) Novozym
MeOH, h, rt;435,
2 h, rt; c)
c)
Novozym
acetone, ◦
435,
40435, acetone,
C, acetone, 40 °C,
°C, 24
24 h; d) Novozym 24 h;
h; d) Novozym
435, Novozym
THF/pyridine 435, (4:1), ◦
THF/pyridine (4:1),
40 C, 96 (4:1),
h. 40 40 °C,
°C, 96
96 h.
h.
Novozym 40 d) 435, THF/pyridine

This methodology
This methodology
methodology involving
involving
involving the glycosylation
the of cholesterol
the glycosylation
glycosylation followed byfollowed
of cholesterol
of cholesterol enzymaticby
followed byregioselective
enzymatic
enzymatic
acylation allowed
regioselective expansion
acylation of
allowed the acylated
expansion α-cholesteryl
of the acylated glycoside inventory
α-cholesteryl
regioselective acylation allowed expansion of the acylated α-cholesteryl glycoside inventory to include
glycoside galactose
inventory to
to
analogues. The
include galactose
include glycosylation
galactose analogues.
analogues. The of per-O-silylated
The glycosylation glucose
glycosylation of provided
of per-O-silylated better
per-O-silylated glucose α-selectivity
glucose provided (39:1)
provided betterthan past
better α-
α-
syntheses
selectivity (8:1 α-selectivity)
(39:1) than past and higher
syntheses glycosylation
(8:1 α-selectivity) yields
and due
higherto the armed
glycosylation
selectivity (39:1) than past syntheses (8:1 α-selectivity) and higher glycosylation yields due to the nature
yieldsof per-O-silyl
due to the
donors
armed [93]. of
armed nature
nature of per-O-silyl
per-O-silyl donors
donors [93].
[93].
Mao and
Mao and coworkers
coworkers developed
developed a a novel
novel glycosyl
glycosyl coupling
coupling reaction,
reaction, involving
involving aa photoinduced
photoinduced
photoinduced
direct activation
activation mechanism
mechanism of
of thioglycosides
thioglycosides (373)
(373) andandsubsequent
subsequent
direct activation mechanism of thioglycosides (373) and subsequent O-glycosylation in O-glycosylation
O-glycosylation in thethe
in absence
the of
absence
absence
photosensitizer
of photosensitizer [94]. In
[94]. Intheir
theirstudies,
studies, the
theauthors
authors used
used several
several sugars,
sugars, amino
amino acids,
of photosensitizer [94]. In their studies, the authors used several sugars, amino acids, and cholesterolacids, and
and cholesterol
28 (75%)
28 (75%) as
as substrates
substrates (Scheme
(Scheme 89). The authors
89). The authors showed
showed that that the
the activation
activation of of thioglycosides
thioglycosides upon upon
UV irradiation followed
followed by
by the oxidation
oxidation of
of Cu(OTf)
Cu(OTf) 2 led to the in situ formation of species that could
2
UV irradiation followed by the oxidation of Cu(OTf)2 led to the in situ formation of species that could
undergo glycosylation
undergo glycosylation to to afford
afford glycosides
glycosides without
without the the need
need for
need for aaa photosensitizer.
for photosensitizer. The
photosensitizer. The proposed
proposed
mechanism
mechanism involved
involved i) homolytic
i) homolytic cleavage
cleavageof a C-S
of a bond
C-S to
bond generate
to
mechanism involved i) homolytic cleavage of a C-S bond to generate a glycosyl radical a glycosyl
generate a radical
glycosyl and ii)
radical oxidation
and ii)
and ii)
to an oxacarbenium
oxidation to an ion
oxacarbeniumpromoted ion
oxidation to an oxacarbenium ion promoted by by Cu(OTf)
promoted by, and sequential
Cu(OTf) 2 , and O-glycosylation
sequential [94].
O-glycosylation
2 Cu(OTf)2, and sequential O-glycosylation [94]. [94].

Scheme
Scheme 89.Glycosylation
Scheme 89.
89. Glycosylation
Glycosylation
of cholesterol.
of
Reagents
of cholesterol.
cholesterol. and conditions:
Reagents
Reagents
a) Cu(OTf)
and conditions:
and conditions: 2 , activated
a) Cu(OTf)
a) Cu(OTf) 4 Å molecular
2, activated 4 Å
2, activated 4 Å
sieves, CH2sieves,
molecular Cl2 , rt,CH
5 days.
2Cl2, rt, 5 days.
molecular sieves, CH2Cl2, rt, 5 days.
In
In 2015,
2015, Davis
Davis and
and coworkers
coworkers reported
reported the
the synthesis
synthesis ofof cholesteryl-α-
cholesteryl-α-D D -lactoside 378 via
-lactoside 378
D-lactoside 378 via
via
In 2015, Davis and coworkers reported the synthesis of cholesteryl-α-
generation
generation and
and trapping
trapping of
of stable
stable β-lactosyl
β-lactosyl iodide
iodide 376.
376. The
The iodide
iodide derivative
derivative 376 was prepared
376 was prepared
generation and trapping of stable β-lactosyl iodide 376. The iodide derivative 376 was prepared
quantitatively under non-in
quantitatively under
under non-in situ
situ anomerization
anomerization and and metal-free
and metal-free conditions
conditions by by reacting
by reacting commercially
commercially
quantitatively non-in situ anomerization metal-free conditions reacting commercially
available β-per-O-acetylated
available β-per-O-acetylated lactose
β-per-O-acetylated lactose 375
lactose 375with trimethylsilyl
375 with iodide
with trimethylsilyl [95].
trimethylsilyl iodide The introduction
iodide [95].
[95]. The of cholesterol
The introduction
introduction of
available of
occurred under
cholesterol microwave
occurred under conditions
microwave to afford
conditions the
to corresponding
afford the glycoconjugate
corresponding 377 in 59%377
glycoconjugate yield
in
cholesterol occurred under microwave conditions to afford the corresponding glycoconjugate 377 in
(Scheme
59% yield 90). Cholesterol
yield (Scheme
(Scheme 90). glycoconjugate
90). Cholesterol 377
Cholesterol glycoconjugatewas further
glycoconjugate 377 deacetylated
377 was
was further using sodium
further deacetylated methoxide
deacetylated using
using sodium
sodiumto
59%
afford cholesteryl
methoxide to afford -lactoside 378
α-Dcholesteryl α- D
in 88% yield
-lactoside 378 (Scheme
in 88% 90).
yieldThis glycosylation
(Scheme 90). This method can be
glycosylation
methoxide to afford cholesteryl α-D-lactoside 378 in 88% yield (Scheme 90). This glycosylation
employed
method can on
can be sterically
be employed demanding
employed on nucleophiles
on sterically
sterically demanding such as cholesterol
demanding nucleophiles
nucleophiles such and
such as has potential
as cholesterol
cholesterol and applications
and has in
has potential
potential
method
accessing structurally
applications diverse
in accessing
accessing cholesteryl
structurally glycoside
diverse analogs
cholesteryl [95]. analogs [95].
glycoside
applications in structurally diverse cholesteryl glycoside analogs [95].
Molecules 2019, 24, 116 50 of 68
Molecules 2018, 23, x 50 of 68
Molecules 2018, 23, x 50 of 68

Scheme 90. Glycosylation

Scheme of
Glycosylation of cholesterol. Reagents
of cholesterol. and
Reagents and conditions:
and conditions:
conditions: a) a) TMSI, CHCl
CHCl333,,, 10
TMSI, CHCl 10 min;
10 min; b)
min; b)
Scheme 90.
90. Glycosylation cholesterol. Reagents a) TMSI, b)
cholesterol,
cholesterol, CHCl
CHCl 3, MW, 110 ◦
, MW, 110 °C,
C, 2
2 h;
h; c)
c) NaOMe,
NaOMe, MeOH/CH
MeOH/CH 2Cl2 (10:1), rt, 2 h.
Cl (10:1), rt, 2 h.
cholesterol, CHCl3, MW, 110 °C, 2 h; c) NaOMe, MeOH/CH22Cl2 2(10:1), rt, 2 h.

A new efficient method for the synthesis of cholesteryl glucosides starting from sucrose 379 was
A new efficient method for the synthesis of cholesteryl glucosides starting from sucrose 379 was
recently developed [96]. This method lays down a five-step synthetic route that involves the initial
recently developed [96]. This method lays down a five-step synthetic route that involves the initial
protection of
of disaccharide
disaccharide379379hydroxy
hydroxygroups,
groups,and
andupon acidic
upon hydrolysis
acidic at its
hydrolysis at anomeric center,
its anomeric the
center,
protection of disaccharide 379 hydroxy groups, and upon acidic hydrolysis at its anomeric center, the
pyranosyl
the moiety
pyranosyl 381 is
moiety 381converted into into
is converted trichloroacetimidate derivative
trichloroacetimidate 383 383
derivative (Scheme 91). 91).
(Scheme
pyranosyl moiety 381 is converted into trichloroacetimidate derivative 383 (Scheme 91).

Scheme Synthesis of
91. Synthesis
Scheme 91. of cholesteryl
cholesteryl glucoside
glucoside starting
starting from
from sucrose.
sucrose. Reagents
Reagents and
and conditions:
conditions: a) BnBr,
a) BnBr,
Scheme 91. Synthesis of cholesteryl glucoside starting from sucrose. Reagents and conditions: a) BnBr,
NaH,
NaH, DMF,
DMF, rt,
rt, 4.5
4.5 h; b) conc.
h; b) conc. HCl,
HCl, acetone,
acetone, reflux,
reflux, 1.5
1.5 h; c) trichloroacetonitrile,
h; c) trichloroacetonitrile, NaH,
NaH, CH
CH22ClCl22,, rt,
rt, 44 h;
h;
NaH, DMF, rt, 4.5 h; b) conc. HCl, acetone, reflux, 1.5 h; c) trichloroacetonitrile, NaH, CH2Cl2, rt, 4 h;
d) TMSOTf, 4 Å molecular sieves, CH 2 Cl2 , rt, 1.5 h; e) Pd(OH) , EtOH/cyclohexene
d) TMSOTf, 4 Å molecular sieves, CH2Cl2, rt, 1.5 h; e) Pd(OH)2, EtOH/cyclohexene (2:1), reflux.
2 (2:1), reflux.
d) TMSOTf, 4 Å molecular sieves, CH2Cl2, rt, 1.5 h; e) Pd(OH)2, EtOH/cyclohexene (2:1), reflux.
The final two steps rely on the formation of the glycosidic bond to cholesterol 28 followed by
The final two steps rely on the formation of the glycosidic bond to cholesterol 28 followed by the
The finaloftwo
the removal thesteps rely ongroups,
protecting the formation of the
affording theglycosidic bond to cholesterol
desired cholesteryl glucoside28384 followed
(Scheme by 91).
the
removal of the protecting groups, affording the desired cholesteryl glucoside 384 (Scheme 91). The
removal
The of the
authors protecting
claimed that thegroups,
majoraffording
advantagethe desired
of this cholesteryl
strategy was theglucoside
use of the384 (Scheme
readily 91). The
available and
authors claimed that the major advantage of this strategy was the use of the readily available and
authors claimed that the major advantage of this strategy was the use of
cheap sucrose 379 as starting material. In addition, the methodology proved to be fast, cost-saving, the readily available and
cheap sucrose 379 as starting material. In addition, the methodology proved to be fast, cost-saving,
cheap
and sucrose 379 as
high-yielding, starting material.
representing In addition,
a competitive the methodology
preparation method for proved to be fast,
these natural cost-saving,
compounds [96].
and high-yielding, representing a competitive preparation method for these natural compounds [96].
and high-yielding,
In 2014, Algay representing
and coworkers a competitive
extensivelypreparation
explored method for these natural
the versatility of nitrilecompounds
oxide alkyne [96].
In 2014, Algay and coworkers extensively explored the versatility of nitrile oxide alkyne
In 2014, Algay
cycloaddition (NOAC) and coworkers
chemistry for extensively
the formation explored the versatility
of cholesterol conjugates of anchored
nitrile oxide alkyne
by way of
cycloaddition (NOAC) chemistry for the formation of cholesterol conjugates anchored by way of a
cycloaddition
a polar, aromatic, (NOAC) chemistry
metabolically for the
stable formation
isoxazole nucleusof cholesterol
[97]. The firstconjugates
series of anchored
compounds byproduced
way of a
polar, aromatic, metabolically stable isoxazole nucleus [97]. The first series of compounds produced
polar,
in this aromatic, metabolically
paper involved stable isoxazole nucleus
i) the microwave-assisted formation [97].ofThe first series
propargyl ethers of (386)
compounds
in 62%–70% produced
yield
in this paper involved i) the microwave-assisted formation of propargyl ethers (386) in 62%–70%
in this paper involved i) the microwave-assisted formation of propargyl ethers
(Scheme 92a), and ii) the reaction of cholesterol propargyl ethers (386) with phenyl nitrile oxide (generated (386) in 62%–70%
yield (Scheme 92a), and ii) the reaction of cholesterol propargyl ethers (386) with phenyl nitrile oxide
yield
in situ(Scheme 92a), and ii)oxime
from benzaldehyde the reaction of cholesterol
upon exposure propargylsolution
to an ethanolic ethers (386) with phenyl(Scheme
of chloramine-T) nitrile oxide
92b).
(generated in situ from benzaldehyde oxime upon exposure to an ethanolic solution of chloramine-
(generated in situ from benzaldehyde oxime upon exposure to an ethanolic
This last reaction was carried out at room temperature or under microwave heating depending on the solution of chloramine-
T) (Scheme 92b). This last reaction was carried out at room temperature or under microwave heating
T) (Scheme
length of the92b). Thisbetween
spacing last reaction was lipid
the bulky carried
andouttheatreacting
room temperature
alkyne, affordingor under microwave
isoxazoles (387) in heating
fair to
depending on the length of the spacing between the bulky lipid and the reacting alkyne, affording
depending
excellent on the
yields length of[97].
(35%–91%) the The
spacing
authorsbetween
extendedthe abulky lipid and
bit further the reacting
this reaction alkyne,
to prepare affording
biologically
isoxazoles (387) in fair to excellent yields (35%–91%) [97]. The authors extended a bit further this
isoxazoles
relevant (387) in fair
cholesterol to excellent
fluorescent probes yields
such(35%–91%) [97]. The (391)
as steroid–coumarin authors(75%)extended a bit further this
and steroid–azobenzene
reaction to prepare biologically relevant cholesterol fluorescent probes such as steroid–coumarin
reaction to prepare biologically relevant cholesterol fluorescent probes such
conjugates (389) (56%) (Scheme 92). It is known that long-chain hydrophilic linkers are very attractive as steroid–coumarin
(391) (75%) and steroid–azobenzene conjugates (389) (56%) (Scheme 92). It is known that long-chain
(391)
for (75%) and steroid–azobenzene
bioconjugation and therefore, in this conjugates (389)authors
paper, the (56%) (Scheme 92). It is three
also synthesized known thatether-linked
new long-chain
hydrophilic linkers are very attractive for bioconjugation and therefore, in this paper, the authors also
hydrophilic linkers are very attractive for bioconjugation
isoxazole-cholesterol conjugates (396) in 29%–58% yield (Scheme 92) [97]. and therefore, in this paper, the authors also
synthesized three new ether-linked isoxazole-cholesterol conjugates (396) in 29%–58% yield (Scheme
synthesized three new ether-linked isoxazole-cholesterol conjugates (396) in 29%–58% yield (Scheme
92) [97].
92) [97].
Molecules 2019, 24, 116 51 of 68
Molecules 2018, 23, x 51 of 68
Molecules 2018, 23, x 51 of 68

Scheme 92.
Scheme
Scheme 92. Synthesis of
Synthesis of
92. Synthesis 3,5-disubstituted
of 3,5-disubstituted isoxazoles.
3,5-disubstituted isoxazoles. Reagents
isoxazoles. Reagents and
Reagents and conditions:
and conditions: a)
conditions: a) MK-10,
MK-10, CHCl
a) MK-10, CHCl
CHCl333,,,MW,
MW,
MW,
90 ◦
°C,
90 °C, 17
C, 17 h;
17 h; b)
h; b) Ch-T, EtOH,
Ch-T, EtOH,
b) Ch-T, n = 1 MW
n == 11 MW at
MW at 60–100
at 60–100
60–100 °C ◦
°C for
C for 30
for 30 min
30 min to
min to 1 h and n = 2 or
and nn ==22or
to 11 hh and 4 rt
or44 rt for
rt for 1 h;
for 11 h; c)
h; c) Ch-T,
Ch-T,
c) Ch-T,
90 EtOH, n
EtOH, rt,
EtOH, rt, 11 h;
h; d)
d) MK-10, CHCl333,, MW,
MK-10, CHCl 90 ◦°C,
MW, 90 °C,
C, 88 h;
h; e)
e) NH
NH222OH∙HCl,
OH∙HCl, pyridine,
OH·HCl, pyridine, EtOH,
pyridine, EtOH, MW,
EtOH, MW,
MW, 125 ◦ C, 1 h; f)
125 °C,
°C, 1 h; f)
(prop-2-yn-1-yloxy)benzene, Ch-T,
(prop-2-yn-1-yloxy)benzene,
(prop-2-yn-1-yloxy)benzene, Ch-T, EtOH,
Ch-T, EtOH,rt,
EtOH, rt,17
rt, 17h.
17 h.
h.

Another series
Another series of
of isoxazole-cholesterol conjugates (401)
isoxazole-cholesterol conjugates was also
(401) was also prepared,
prepared, starting
starting from
from
cholesterol chloroformate 7 and bearing an amidocarbamate linker following the four-step synthetic
cholesterol chloroformate 7 and bearing an amidocarbamate linker following the four-step synthetic
route depicted
route depicted in
in Scheme
Scheme 93
93 [97].
[97].

Scheme 93. Synthesis of amidocarbamate

93. Synthesis
Synthesis amidocarbamate cholesterol
cholesterol conjugates. Reagents and conditions:
conjugates. Reagents
Reagents conditions: a)a)
Scheme 93. of amidocarbamate cholesterol conjugates. and conditions:
ethylenediamine,
ethylenediamine, toluene,
toluene, rt, 17rt,h; b)
17 4-(dimethoxymethyl)benzoic
h; b) acid, DCC, DMAP,
4-(dimethoxymethyl)benzoic acid, CH 2 Cl2 /toluene,
DCC, DMAP,
ethylenediamine, toluene, rt, 17 h;◦ b) 4-(dimethoxymethyl)benzoic acid, DCC, DMAP,
rt,
CH17
CH
h;2/toluene,
2Cl c) NH2 OH rt,·17
HCl, EtOH,
h; c) MW, 125 EtOH,
NH2OH∙HCl, C, 1 h;MW,
d) Ch-T, propargyl
125 °C, alcohol,
1 h; d) Ch-T, or phenyl
propargyl propargyl
alcohol,
2Cl2/toluene, rt, 17 h; c) NH2OH∙HCl, EtOH, MW, 125 °C, 1 h; d) Ch-T, propargyl alcohol, or phenyl
ether,
or phenyl
EtOH, rt, 17
propargyl ether,h.
ether, EtOH,
EtOH, rt,rt, 17
17 h.
h.
propargyl
The
The nontrivial synthesis
nontrivial synthesis of aryl
synthesis of
of ethers
aryl ethers of
ethers of natural
of natural alcohols
natural alcohols drove
alcohols drove the
drove the authors
the authors to
to test
authors to test the
the NOAC
NOAC
The nontrivial aryl test the NOAC
chemistry
chemistry in
in the
the assembly
assembly of
of aryl
aryl ether
ether cholesterol
cholesterol conjugates
conjugates [97].
[97]. Therefore,
Therefore, isoxazole-linked
isoxazole-linked aryl
aryl
chemistry in the assembly of aryl ether cholesterol conjugates [97]. Therefore, isoxazole-linked aryl
cholesterol ether
cholesterol ether 404
ether 404was prepared
404 was
was prepared from
prepared from the aldehyde-functionalized
from the aryl
the aldehyde-functionalized ether
aldehyde-functionalized aryl 402 through
aryl ether
ether 402 subsequent
402 through
through
cholesterol
oximation
subsequent and cycloaddition
oximation and reactions, asreactions,
cycloaddition depicted in
as Scheme
depicted94.
in Scheme 94.
subsequent oximation and cycloaddition reactions, as depicted in Scheme 94.
Finally, the authors used the potential of NOAC chemistry to prepare a steroidal glycoconjugate,
407, and the selective tethering of one or two cholesterol units, 409 and 410, respectively, to a thymidine
skeleton was demonstrated by trapping of the same dipole by 50 -protected mono- or bis-propargylated
thymidines (Scheme 95) [97].

Scheme 94.
Scheme 94. Synthesis
Synthesis of
of isoxazole-linked
isoxazole-linked aryl
aryl cholesterol
cholesterol ether.
ether. Reagents
Reagents and
and conditions:
conditions: a)a)
NH OH∙HCl, pyridine,
NH22OH∙HCl, pyridine, EtOH,
EtOH, MW,
MW, 125
125 °C,
°C, 11 h;
h; b)
b) (prop-2-yn-1-yloxy)benzene,
(prop-2-yn-1-yloxy)benzene, Ch-T,
Ch-T, EtOH,
EtOH, rt,
rt, 17
17 h.
h.
Molecules 2019, 24, 116 52 of 68

Scheme 93. Synthesis of amidocarbamate cholesterol conjugates. Reagents and conditions: a)

In 2016, Alarcón-Manjarrez
ethylenediamine, toluene, rt, and 17 coworkers reported the synthesis of
h; b) 4-(dimethoxymethyl)benzoic twoDCC,
acid, dimeric steroidal
DMAP,
terephthalates, 415 and 416, from epimeric 4,5-seco-cholest-3-yn-5-ols 413 and 414, using
CH2Cl2/toluene, rt, 17 h; c) NH2OH∙HCl, EtOH, MW, 125 °C, 1 h; d) Ch-T, propargyl alcohol, or phenyl a five-step
synthetic routeether,
propargyl withEtOH,
cholesterol
rt, 17 h.28 as a starting material [98]. The synthetic route first involved the
Oppenauer oxidation of cholesterol 28, followed by epoxidation, to afford a mixture of epoxides (411)
(α:β The
= 1:4) (Scheme synthesis
nontrivial 96). Then,ofan Eschenmoser-Tanabe
aryl fragmentation
ethers of natural alcohols drove thefollowed
authorsby to carbonyl group
test the NOAC
reduction
chemistryprovided the epimeric
in the assembly of arylalkynols 413 and 414
ether cholesterol in a 1:2 ratio
conjugates (Scheme
[97]. 96). isoxazole-linked
Therefore, Finally, the treatment
aryl
of each one of
cholesterol the epimeric
ether 404 was alkynols and 414
prepared413from the with terephthaloyl chloride
aldehyde-functionalized led ether
aryl to the 402
symmetrical
through
axial and equatorial
subsequent oximationdimers 415 and 416, respectively
and cycloaddition reactions, as(Scheme
depicted96)
in [98].
Scheme 94.

Molecules 2018, 23, x 52 of 68

Finally, the authors used the potential of NOAC chemistry to prepare a steroidal glycoconjugate,
407, Scheme
and the selective tethering
Synthesis of
of one or two cholesterol
of isoxazole-linked
units, 409 and 410, conditions:
respectively, to a
Scheme 94.94. Synthesis isoxazole-linked aryl
aryl cholesterol
cholesterol ether. Reagents and
ether. Reagents and conditions: a)a)
thymidine
NH skeleton
OH was demonstrated
·HCl,pyridine,
pyridine, EtOH,MW,
MW, by trapping
125 ◦ of (prop-2-yn-1-yloxy)benzene,
the same dipole by 5′-protected mono- or bis-
NH22OH∙HCl, EtOH, 125 °C, C,
1 h;1b)
h;(prop-2-yn-1-yloxy)benzene,
b) Ch-T,Ch-T,
EtOH,EtOH, rt,
rt, 17 h.
propargylated
17 h. thymidines (Scheme 95) [97].

95. Synthesis
Scheme 95. Synthesis of
of glycol-
glycol- and
and thymidine-cholesterol
thymidine-cholesterol conjugates.
conjugates. Reagents and conditions: a)
Ch-T, EtOH,
Ch-T, EtOH, rt,
rt, 17
17 h.
h.

The authors
In 2016, proceeded to crystallographic
Alarcón-Manjarrez and coworkersanalysis of the compounds
reported the synthesis andofconcluded that the
two dimeric facial
steroidal
hydrophobicity of the steroidal skeletons had crucial influence on the crystal packing in which
terephthalates, 415 and 416, from epimeric 4,5-seco-cholest-3-yn-5-ols 413 and 414, using a five-step the dimeric
molecules were forced
synthetic route to accommodate
with cholesterol 28 as athese fragments
starting only[98].
material withThe
a few hydrogen-bonding
synthetic interactions.
route first involved the
This feature originated a cisoid conformation for 415 and a linear conformation for 416 [98].
Oppenauer oxidation of cholesterol 28, followed by epoxidation, to afford a mixture of epoxides (411)
(α:β Shibuya et al. reported
= 1:4) (Scheme 96). Then,in 2016 the synthesis of (24S)-hydroxycholesterol
an Eschenmoser-Tanabe fragmentation followed (24S-OHChol)
by carbonyl esters,
group
which are involved
reduction providedintheneuronal
epimericcell death, through
alkynols 413 andcatalysis with
414 in a 1:2 acyl-CoA:cholesterol
ratio (Scheme 96). Finally,acyltransferase-1
the treatment
(ACAT-1)
of each one [99]. Theepimeric
of the authorsalkynols
studied the
413 esterification
and 414 with of (24S)-OHChol
terephthaloyl 417 with
chloride cis-oleoyl
led to chloride
the symmetrical
under basic
axial and conditions
equatorial and 415
dimers obtained mono-oleates
and 416, respectively418 and 419
(Scheme 96)and bis-oleate 420 in 39%, 9%, and
[98].
20% yields, respectively (Scheme 97). The protection of (24S)-OH with a trifluoroacetyl group was
also attempted, affording mono-trifluoroacetates 421 and 422 in 33% and 14% yields, respectively,
and the bis-trifluoroacetate 423 in 21% yield (Scheme 97) [99]. The authors took advantage of the
mono-trifluoroacetate 421 to prepare the stearoyl and palmitoyl esters 427 and 428 in 68% and 75%
yields, respectively, as depicted in Scheme 98 [99]. Finally, the authors also reported the use of esters
of unsaturated long-chain fatty acids, such as linoleic (LA), arachidonic (AA), and docosahexaenoic
(DHA), to react with cholesterol derivative 422 in order to prepare linoleate 430, arachidonoate 431,
 In 2016, Alarcón-Manjarrez and coworkers reported the synthesis of two dimeric steroidal
terephthalates, 415 and 416, from epimeric 4,5-seco-cholest-3-yn-5-ols 413 and 414, using a five-step
synthetic route with cholesterol 28 as a starting material [98]. The synthetic route first involved the
Oppenauer
Molecules 2019,oxidation
24, 116 of cholesterol 28, followed by epoxidation, to afford a mixture of epoxides53(411)
of 68
(α:β = 1:4) (Scheme 96). Then, an Eschenmoser-Tanabe fragmentation followed by carbonyl group
reduction provided the epimeric alkynols 413 and 414 in a 1:2 ratio (Scheme 96). Finally, the treatment
and
of docosahexaenoate
each one of the epimeric432 esters,
alkynolsin 413
52%,and
74%,
414and 66%
with yields, respectively,
terephthaloyl chloride in
leda to
two-step synthetic
the symmetrical
route, as depicted in Scheme 99 [99].
axial and equatorial dimers 415 and 416, respectively (Scheme 96) [98].

Molecules 2018, 23, x 53 of 68

The authors proceeded to crystallographic analysis of the compounds and concluded that the
facial hydrophobicity of the steroidal skeletons had crucial influence on the crystal packing in which
the dimeric molecules were forced to accommodate these fragments only with a few hydrogen-
bonding interactions. This feature originated a cisoid conformation for 415 and a linear conformation
for 416 [98].
Shibuya et al. reported in 2016 the synthesis of (24S)-hydroxycholesterol (24S-OHChol) esters,
which are involved in neuronal cell death, through catalysis with acyl-CoA:cholesterol
acyltransferase-1 (ACAT-1) [99]. The authors studied the esterification of (24S)-OHChol 417 with cis-
oleoyl chloride under basic conditions and obtained mono-oleates 418 and 419 and bis-oleate 420 in
39%, 9%, and 20% yields, respectively (Scheme 97). The protection of (24S)-OH with a trifluoroacetyl
group was also attempted, affording mono-trifluoroacetates 421 and 422 in 33% and 14% yields,
respectively, and the bis-trifluoroacetate 423 in 21% yield (Scheme 97) [99]. The authors took
advantage of the mono-trifluoroacetate 421 to prepare the stearoyl and palmitoyl esters 427 and 428
in 68% and 75% yields, respectively, as depicted in Scheme 98 [99]. Finally, the authors also reported
the use of esters of unsaturated long-chain fatty acids, such as linoleic (LA), arachidonic (AA), and
Scheme 96. Synthesis of dimeric
dimeric steroidal
steroidal terephthalates.
terephthalates. Reagents and conditions:
conditions: a) Al(O-i-Pr)33,,
docosahexaenoic (DHA), to react with cholesterol derivative 422 in order to prepare linoleate 430,
cyclohexanone, toluene, reflux, 1.5 h;
cyclohexanone, b) NaOH/MeOH
h; b) NaOH/MeOH (10%), H22O22 (30%),(30%), CH22Cl
Cl22, , rt,
rt, 72
72 h;
h; c)
c)
arachidonoate 431, and docosahexaenoate 432 esters, in 52%, 74%, and 66% yields, respectively, in a
TsNHNH2,, CH22ClCl22/AcOH
/AcOH(1:1),
(1:1),rt,rt,2.5
2.5h;h;d) NaBH4,4 ,MeOH,
d) NaBH MeOH,rt,
rt, 30
30 min;
min; e) terephthaloyl chloride,
e) terephthaloyl chloride,
two-step synthetic route, as depicted in Scheme 99 [99].
DMAP, Et N, toluene, reflux, 5 h.
DMAP, Et33N, toluene, reflux, 5 h.

Synthesisofof(24S)-OHChol
Scheme 97. Synthesis (24S)-OHChololeates
oleatesandand trifluoroacetates.
trifluoroacetates. Reagents
Reagents andand conditions:
conditions: a)
a) cis-
cis-oleoyl chloride, Et N, DMAP, THF, 0 ◦ C to rt, 16 h; b) 2-trifluoroacetoxypyridine, CH Cl , rt, 20 h.
oleoyl chloride, Et3N,3DMAP, THF, 0 °C to rt, 16 h; b) 2-trifluoroacetoxypyridine, CH2Cl2, rt,2 20 h.

Recently, Sarkar et al. reported interesting work dealing with the preparation of diverse ring-A
or ring-B oxo-functionalized steroids in a green fashion involving solvent-free solid supports [100].
The authors used cholesterol derivatives such as 4β-hydroxycholesterol 433, which was functionalized
into three different keto-steroids, 434, 435, and 436, in 55%, 10%, and 10% yields, respectively,
employing p-toluenesulfonic acid and SiO2 (silica 60–120 mesh) as solid support (Scheme 100) [100].
Interestingly, if the reaction was attempted in the solution phase at room temperature using either
dichloromethane or ethanol as solvents, cholest-4-en-3-one 434 was obtained exclusively in 64% and
60% from dichloromethane and ethanol, respectively. The procedure on solid silica was applied to
the other cholesterol derivative, namely 4β,7α-dihydroxycholesterol 437, which was converted into
four distinct keto-steroids: (i) cholest-5-en-7-one (438, 8%), (ii) cholesta-3,5-dien-7-one (439, 13%), (iii)
cholesta-4,6-dien-3-one (440, 17%), and (iv) 5a-cholestane-4,7-dione (441, 7%) (Scheme 100) [100]. It is
worthScheme
noticing98.that if the reaction
Synthesis of 4β,7α-dihydroxycholesterol
of (24S)-OHChol stearoyl and palmitoyl437 wasReagents
esters. carried outandinconditions:
dichloromethane
a)
as solvent,
TBDMSCl,cholesta-4,6-dien-3-one (440,
imidazole, DMF, rt, 16 54%)
h; b) 7 Nwas
NHfound to bert,
3 in MeOH, the only
3 h; productchloride
c) stearoyl formed.orThis was found
palmitoyl
to bechloride,
a facile procedure
Et3N, DMAP, forTHF,
the synthesis
0 °C to rt,of
16dienone 440 from AcOH,
h; d) HF.pyridine, cholesterol
50 °Cvia triol
to rt, h. [100].
16 437
Cholesterol derivatives can also be used as starting materials for the synthesis of fused nitrogen
heterocycles. This was the case for 4-cholesten-3-one 350, which was involved in the preparation of
A-ring dehydropiperazine 443 (90% yield) through a microwave-assisted annulation reaction with
ethylenediamine 442 in the presence of basic alumina (Scheme 101) [101].

Scheme 99. Synthesis of (24S)-OHChol linoleoyl, arachidonoyl and docosahexaenoyl esters. Reagents
and conditions: a) linoleoyl chloride, arachidonoyl chloride or docosahexaenoyl chloride, Et3N,
arachidonoate 431, and docosahexaenoate 432 esters, in 52%, 74%, and 66% yields, respectively, in a
two-step synthetic route, as depicted in Scheme 99 [99].
two-step synthetic route, as depicted in Scheme 99 [99].

Molecules 2019, 24, 116 54 of 68

The proposed mechanism should encompass the initial oxidation of the allylic protons of the
conjugated ketone via enolate intermediate to afford a diketo intermediate. Then, the condensation
Scheme
with Scheme 97. Synthesis of (24S)-OHChol
ethylenediamine oleates and trifluoroacetates. Reagents and conditions: a) cis-
97. Synthesisfollowed by a Michael
of (24S)-OHChol oleates andaddition and autoxidation
trifluoroacetates. reactions
Reagents and afforded
conditions: a) cis- the
oleoyl chloride, Et3N, DMAP, THF, 0 °C to rt, 16 h; b) 2-trifluoroacetoxypyridine, CH2Cl2, rt, 20 h.
dehydropiperazine derivatives
oleoyl chloride, Et3N, DMAP, [101].
THF, 0 °C to rt, 16 h; b) 2-trifluoroacetoxypyridine, CH2Cl2, rt, 20 h.

Molecules 2018, 23, x 54 of 68

Recently, Sarkar et al. reported interesting work dealing with the preparation of diverse ring-A
or ring-B oxo-functionalized steroids in a green fashion involving solvent-free solid supports [100].
The Scheme
Scheme
98.used
authors98. Synthesis
Synthesis
Synthesis
of (24S)-OHChol
cholesterol derivatives
of (24S)-OHChol
stearoyl and palmitoyl
such
stearoyl
esters. Reagents and 433,
as 4β-hydroxycholesterol
and palmitoyl esters. Reagents
conditions:
Reagents and conditions:
a) was
whicha)
a)
TBDMSCl,
functionalized imidazole,
into three DMF, rt,
different 16 h; b) 7 N
keto-steroids,NH 3 in MeOH, rt, 3 h; c) stearoyl chloride or palmitoyl
434, 435, and 436, in 55%, 10%, and 10% yields,
imidazole, DMF,
TBDMSCl, imidazole, DMF, rt, 16 h; b) 7 N NH3 in MeOH, rt, 3 h; c) stearoyl chloride or palmitoyl
chloride, Et
respectively, 3N, DMAP, THF, 0 ◦
employing °C to rt,
p-toluenesulfonicrt, 16
16 h;
h; acid
d) HF.pyridine,
HF.pyridine,
and SiO2AcOH,AcOH, 50 ◦°C
(silica50 to rt,
rt, 16
16 h. as solid support
chloride, Et N, DMAP,
33N, DMAP, THF, 0 °CC to d) HF.pyridine, AcOH, 5060–120
C to
°C rt,mesh)
16 h.
h.
(Scheme 100) [100]. Interestingly, if the reaction was attempted in the solution phase at room
temperature using either dichloromethane or ethanol as solvents, cholest-4-en-3-one 434 was
obtained exclusively in 64% and 60% from dichloromethane and ethanol, respectively. The procedure
on solid silica was applied to the other cholesterol derivative, namely 4β,7α-dihydroxycholesterol
437, which was converted into four distinct keto-steroids: i) cholest-5-en-7-one (438, 8%), (ii) cholesta-
3,5-dien-7-one (439, 13%), (iii) cholesta-4,6-dien-3-one (440, 17%), and (iv) 5a-cholestane-4,7-dione
(441, 7%) (Scheme 100) [100]. It is worth noticing that if the reaction of 4β,7α-dihydroxycholesterol
437 was carried out in dichloromethane as solvent, cholesta-4,6-dien-3-one (440, 54%) was found to
Synthesis of
Scheme 99. Synthesis of (24S)-OHChol
(24S)-OHChol linoleoyl,
linoleoyl, arachidonoyl and docosahexaenoyl esters. Reagents
Scheme
be the 99. Synthesis
only product of (24S)-OHChol
formed. This linoleoyl,
was arachidonoyl
found to bearachidonoyl
a facile or and docosahexaenoyl
procedure for the chloride,esters.
synthesis ofReagents
dienone 440
and conditions:
conditions:a)a)linoleoyl
linoleoyl chloride,
chloride, arachidonoylchloride
chloridedocosahexaenoyl
or docosahexaenoyl Et3 N, DMAP,
chloride, Et3N,
fromand conditions:
cholesterol
◦
THF, 0 THF,
DMAP,
via
C to rt,
a)
16 h;
0 °C
linoleoyl
triol 437
rt, 716Nh;NH
to b)
chloride, arachidonoyl
[100].
b) 7inN MeOH, ◦ chloride or docosahexaenoyl chloride, Et3N,
0 C, 3 h.0 °C, 3 h.
NH3 in MeOH,
3
DMAP, THF, 0 °C to rt, 16 h; b) 7 N NH3 in MeOH, 0 °C, 3 h.

Scheme 100.
Scheme 100.Oxo-functionalization of 4β-hydroxycholesterol.
Oxo-functionalization Reagents
of 4β-hydroxycholesterol. and conditions:
Reagents a) p-TSA/SiO
and conditions: 2,
a) p-
120 ◦ C, 5 or 10 min.
TSA/SiO2, 120 °C, 5 or 10 min.

Recently, Ansari
Cholesterol and coworkers
derivatives can also be reported an efficient
used as starting andfor
materials green syntheticofmethod
the synthesis for the
fused nitrogen
preparation
heterocycles. This was the case for 4-cholesten-3-one 350, which was involved in the preparationasofa
of steroidal pyridines [102]. Such methodology relied on the utilization of MgO NPs
heterogeneous,
A-ring mild, and reusable
dehydropiperazine 443 (90%catalyst,
yield)inthrough
a multicomponent one-pot protocol,
a microwave-assisted takingreaction
annulation advantage of
with
the usefulness of the microwave irradiation as an alternative heating
ethylenediamine 442 in the presence of basic alumina (Scheme 101) [101]. source. The series of substituted
fused pyridines (444) were obtained in 80%–89% yield from the reaction of steroidal ketones (164) with
malononitrile/methylcyanoacetate, benzaldehyde, and ammonium acetate in ethanol using MgO NPs
as a catalyst (Scheme 102) [102].
One of the key mechanistic steps in this kind of multicomponent reaction is the standard
Knoevenagel condensation of benzaldehyde and malononitrile/methyl cyanoacetate. The effect

Scheme 101. Synthesis of 4′-dehydrocholest-4-eno[3,4-e]piperazin-6-one. Reagents and conditions: a)

basic Al2O3, MW, 120 °C, 5 min.
 Scheme 100. Oxo-functionalization of 4β-hydroxycholesterol. Reagents and conditions: a) p-
TSA/SiO
Molecules 2, 120
2019, 24, 116 °C, 5 or 10 min. 55 of 68

Cholesterol derivatives can also be used as starting materials for the synthesis of fused nitrogen
of MgO NPs can
heterocycles. Thisbewas
rationalized on this
the case for basis since they350,
4-cholesten-3-one are which
knownwas
as a involved
highly effective heterogeneous
in the preparation of
base catalyst for Michael addition and Knoevenagel condensation reactions with Mg 2+ (Lewis acid)
A-ring dehydropiperazine 443 (90% yield) through a microwave-assisted annulation reaction with
and O2− (Lewis base)
ethylenediamine 442 insites
thealong withofvarious
presence cationic(Scheme
basic alumina and anionic
101)vacancies
[101]. in the lattice [102].

Molecules 2018, 23, x 55 of 68

Scheme 101.
Scheme Synthesis of
101. Synthesis 40 -dehydrocholest-4-eno[3,4-e]piperazin-6-one. Reagents
of 4′-dehydrocholest-4-eno[3,4-e]piperazin-6-one. Reagents and
and conditions:
conditions: a)
a)
Molecules
basic2018, O x, MW, 120 ◦ C, 5 min.
Al 23,
basic Al22O33, MW, 120 °C, 5 min.
55 of 68

The proposed mechanism should encompass the initial oxidation of the allylic protons of the
Scheme 102. Synthesis of steroidal fused pyridines. Reagents and conditions: a) benzaldehyde,
conjugated ketone via enolate intermediate to afford a diketo intermediate. Then, the condensation
malononitrile or methylcyanoacetate, MgO nanoparticles (NPs), EtOH, MW, 70 °C, 20–25 min.
with ethylenediamine followed by a Michael addition and autoxidation reactions afforded the
dehydropiperazine derivatives [101].
One of the key mechanistic steps in this kind of multicomponent reaction is the standard
Recently, Ansari and coworkers reported an efficient and green synthetic method for the
Knoevenagel condensation of benzaldehyde and malononitrile/methyl cyanoacetate. The effect of
preparation of steroidal pyridines [102]. Such methodology relied on the utilization of MgO NPs as
MgOScheme
NPs can be rationalized on this basis
Synthesis of steroidal since they are knownand as aconditions:
highly effective heterogeneous
102.
102.
a heterogeneous, mild, and reusablefused
Synthesis fused pyridines.
pyridines.
catalyst, Reagents
in aReagents
multicomponent one-pota) benzaldehyde,
a) benzaldehyde,
protocol, taking
base malononitrile
catalyst for Michael addition and Knoevenagel condensation
or methylcyanoacetate, MgO nanoparticles (NPs),(NPs), EtOH, reactions
EtOH, MW,
MW, 70 ◦ with Mg
C, 20–25
70 °C,
2+ (Lewis acid)
20–25 min.
min.
advantage of the usefulness of the microwave irradiation as an alternative heating source. The series
and O2− (Lewis base) sites along with various cationic and anionic vacancies in the lattice [102].
of substituted
A-ringfused pyridines
of cholesterol
cholesterol (444)
was were
also obtained in
functionalized80%–89%
by yield
fusing from the
pyrimidines reaction
at of
the steroidal
steroidal
The
One A-ring
of the ofkey mechanistic was also
steps functionalized
in this kind of by fusing pyrimidines
multicomponent at the
reaction is steroidal
the standard2,3-
ketones (164) These
2,3-position. with malononitrile/methylcyanoacetate,
new steroidal compounds were benzaldehyde,
synthesized and ammonium
through a acetate in
microwave-assisted
position. These new steroidal compounds were synthesized through a microwave-assisted
Knoevenagel condensation of benzaldehyde and malononitrile/methyl cyanoacetate. The effect of three-
ethanol using MgOreaction
three-component NPs as aofcatalyst (Scheme 102) [102].
2-hydroxymethylene-3-ketosteroid
component
MgO NPs canreaction of 2-hydroxymethylene-3-ketosteroid
be rationalized on this basis since they are known(445), as(445), benzaldehydes
benzaldehydes
a highly (446), and
(446),
effective heterogeneous
ammonium acetate,
base catalystacetate,
for Michaelaffording
affording cholesterol-fused
cholesterol-fused
addition and Knoevenagel pyrimidines
pyrimidines (447) (447)
condensation inyields
inreactions
good good
with yields
(78%–88%) (78%–88%)
Mg2+ (Lewis (Scheme
acid)
(Scheme
103) 103)
[103]. [103].
and O (Lewis base) sites along with various cationic and anionic vacancies in the lattice [102].
2−

The A-ring of cholesterol was also functionalized by fusing pyrimidines at the steroidal 2,3-
position. These new steroidal compounds were synthesized through a microwave-assisted three-
component reaction of 2-hydroxymethylene-3-ketosteroid (445), benzaldehydes (446), and
ammonium acetate, affording cholesterol-fused pyrimidines (447) in good yields (78%–88%) (Scheme
103) [103].

Scheme 103. Synthesis of cholesterol-fused pyrimidines. Reagents and conditions:

conditions: a) NH44OAc, silica
gel (60–120 mesh), MW, 120 ◦°C,
MW, 120 C, 6 min.

The authors’
The authors’mechanism
mechanismwas was based
based on:on:
(i) microwave-assisted
i) microwave-assisted reaction of ammonia
reaction of ammonia (released from
(released
decomposition of ammonium acetate) with 2-hydroxymethylene-3-ketosteroid
from decomposition of ammonium acetate) with 2-hydroxymethylene-3-ketosteroid to afford a β- to afford a β-aminoketoimine
intermediate; (ii) their
aminoketoimine condensation
intermediate; reaction
ii) their with benzaldehydes
condensation reaction to afford
with a diamine intermediate;
benzaldehydes and (iii)
Scheme 103. Synthesis of cholesterol-fused pyrimidines. Reagents and conditions: a) toNH afford a diamine
4OAc, silica
cyclization
intermediate; and subsequent auto-oxidation to give the cholesterol-fused pyrimidines [103].
gel (60–120and iii)MW,
mesh), cyclization and subsequent auto-oxidation to give the cholesterol-fused
120 °C, 6 min.
A two-step
pyrimidines [103].method for the preparation of steroid-fused 4,6-diaryl substituted pyridines has
beenA reported
Thetwo-step
authors’ [104].
method Theforsynthetic
mechanism thewas protocol
preparation
based of relied
on: on the Michael
steroid-fused 4,6-diaryladdition
i) microwave-assisted substituted
reaction of
of 5α-cholestan-3-one
pyridines (released
ammonia has been
448 with
reported chalcones (generated
[104]. The synthetic
from decomposition of ammonium in situ
protocolacetate) by
relied on the base-catalyzed reaction
the2-hydroxymethylene-3-ketosteroid
with of acetophenones
Michael addition of 5α-cholestan-3-one 448 (449)
to afford with
a β-
and benzaldehydes
chalcones (generated (446)),
in affording
situ by 3,5-diaryl-1,5-dicarbonyl
the base-catalyzed 5α-cholestan-3-one
reaction of acetophenones
aminoketoimine intermediate; ii) their condensation reaction with benzaldehydes to afford a diamine derivatives
(449) (450)
and
(88%–94%)
benzaldehydes (Scheme
intermediate; and 104).
(446)), Then,
iii)affording
cyclizationthe intermediates (450)
3,5-diaryl-1,5-dicarbonyl were used as substrates
5α-cholestan-3-one
and subsequent auto-oxidation in a microwave-assisted
to givederivatives (450) (88%–
the cholesterol-fused
solid
94%) phase
(Scheme
pyrimidines reaction
104). with
[103]. Then,urea thein the presence of
intermediates (450) ·OEt2used
BF3were to giveas 4,6-diaryl
substratessubstituted pyridines (451)
in a microwave-assisted
in good
solid yields
Aphase
two-step (81%–93%)
reaction with
method for(Scheme
urea in the104)presence
the preparation [104]. of BF3∙OEt2 4,6-diaryl
of steroid-fused to give 4,6-diaryl
substituted substituted
pyridinespyridines
has been
(451) The authors
in good
reported [104].yieldsproposed
(81%–93%)
The synthetic a mechanism
(Scheme
protocol for the
104)on
relied formation
[104]. of a pyridine ring, which
the Michael addition of 5α-cholestan-3-one may start
448 with
with
the
chalcones (generated in situ by the base-catalyzed reaction of acetophenones (449) with
release of ammonia by urea under microwave heating, which forms an imine by reaction and
one carbonyl group. Next, the BF · OEt -promoted nucleophilic attack of the
benzaldehydes (446)), affording 3,5-diaryl-1,5-dicarbonyl 5α-cholestan-3-one derivatives (450) (88%–
3 2 imine NH-group on the
activated carbonyl functionality facilitated an aza-cyclization reaction, affording
94%) (Scheme 104). Then, the intermediates (450) were used as substrates in a microwave-assisted a 1,4-dihydropyrinine
intermediate upon which
solid phase reaction witharomatization gave theofdesired
urea in the presence BF3∙OEt4,6-diarylpyridines [104].
2 to give 4,6-diaryl substituted pyridines

(451) in good yields (81%–93%) (Scheme 104) [104].

 Scheme 103. Synthesis of cholesterol-fused pyrimidines. Reagents and conditions: a) NH4OAc, silica
gel (60–120 mesh), MW, 120 °C, 6 min.

The authors’ mechanism was based on: i) microwave-assisted reaction of ammonia (released
from decomposition
Molecules 2019, 24, 116 of ammonium acetate) with 2-hydroxymethylene-3-ketosteroid to afford 56 aof β-
68
aminoketoimine intermediate; ii) their condensation reaction with benzaldehydes to afford a diamine
intermediate; and iii) cyclization and subsequent auto-oxidation to give the cholesterol-fused
In 2015, [103].
pyrimidines Schulze and coworkers developed a new method for the synthesis of model asphaltene
compounds. The
A two-step method reportedformethodology
the preparationwasofbased on a multicomponent
steroid-fused cyclocondensation
4,6-diaryl substituted reaction
pyridines has been
of 2-aminoanthracene
reported 452 with
[104]. The synthetic aromatic
protocol aldehydes
relied on the and 5-α-cholestan-3-one
Michael 448 (Scheme 105)
addition of 5α-cholestan-3-one 448[105].
with
The authors found that the actual catalyst for this reaction was indeed
chalcones (generated in situ by the base-catalyzed reaction of acetophenones (449) and hydriodic acid, which is
formed in situ from the reaction of iodine with water. Carrying the reaction
benzaldehydes (446)), affording 3,5-diaryl-1,5-dicarbonyl 5α-cholestan-3-one derivatives (450) (88%–under anhydrous
conditions,
94%) (Scheme it was
104).proven
Then, the thatintermediates
iodine itself (450)
did notwerepromote
used asthe reaction,inas
substrates generally assumed.
a microwave-assisted
Using this methodology, the authors prepared a library of optically active
solid phase reaction with urea in the presence of BF3∙OEt2 to give 4,6-diaryl substitutedsteroidal naphthoquinolines
pyridines
(453)
(451) in
in acceptable
good yieldsyields (40%–53%)
(81%–93%) [105].
(Scheme 104) [104].

Molecules 2018, 23, x 56 of 68

The authors proposed a mechanism for the formation of a pyridine ring, which may start with
the release of ammonia by urea under microwave heating, which forms an imine by reaction with
one carbonyl group. Next, the BF3∙OEt2-promoted nucleophilic attack of the imine NH-group on the
activated carbonyl functionality facilitated an aza-cyclization reaction, affording a 1,4-
dihydropyrinine intermediate upon which aromatization gave the desired 4,6-diarylpyridines [104].
In 2015, Schulze and coworkers developed a new method for the synthesis of model asphaltene
compounds. The Synthesis
Scheme 104. reported ofmethodology was based
cholesterol-fused on a multicomponent
4,6-diarylpyridines. cyclocondensation
Reagents and
Reagents and conditions: a)
conditions: reaction
KOH,
a) KOH,
of 2-aminoanthracene 452 with aromatic
·OEt2, ,MW,
toluene, rt, 6 h; b) urea, BF3∙OEt aldehydes
MW, 140 ◦
140 °C,C,88min.
min.and 5-α-cholestan-3-one 448 (Scheme 105) [105].
3 2

105. Synthesis
Scheme 105. Synthesis of
of model
model asphaltene
asphaltene compounds.
compounds. Reagents
Reagents and
and conditions: a)II22,, H
conditions: a) H22O/THF,
O/THF,
reflux, 5 days.

8. Miscellaneous
The authors found that the actual catalyst for this reaction was indeed hydriodic acid, which is
formed
Thein situ of
design from the reaction of
(supra)molecular iodine and
switches with water. Carrying
machines the reaction
has a key feature underto anhydrous
that relates the control
conditions,
of it was
mechanical proven
motions at that iodine itself
the molecular did not
level. promote
In this field, the reaction,
rotaxanes as generally
have attracted assumed. Using
much attention
this methodology,
because authors prepared
they offer the possibility a library
of restricting theoffreedom
optically
ofactive
motionsteroidal
to somenaphthoquinolines (453)
well-defined pathways,
in acceptable yields (40%–53%) [105].
such as the translational motion of a rotaxane’s ring along its axis in a shuttling manner. The synthesis
of a novel nonsymmetrical bistable pH-sensitive rotaxane with a cholesterol stopper at one end and
8. tetraphenylmethane
a Miscellaneous group at the other end (457), has been reported [106]. The synthesis of both
terminal
The ends wasofchallenging,
design and therefore
(supra)molecular switchesweand
onlymachines
describe here
has athe
key final step, which
feature consisted
that relates of
to the
joining both axes of the nonsymmetrical rotaxane, the alkyne 454, and the azide 455 through
control of mechanical motions at the molecular level. In this field, rotaxanes have attracted much CuAAC
chemistry, affording
attention because compound
they 456 (Scheme
offer the possibility of 106). The formation
restricting the freedomof aofpH-sensitive
motion to somebistable rotaxane
well-defined
457 was achieved by methylation of the triazole ring using methyl iodide (Scheme 106).
pathways, such as the translational motion of a rotaxane’s ring along its axis in a shuttling manner.The authors
verified that the crown ether part changed its preferred position on the axis because of the
The synthesis of a novel nonsymmetrical bistable pH-sensitive rotaxane with a cholesterol stopper at protonation
one end and a tetraphenylmethane group at the other end (457), has been reported [106]. The
synthesis of both terminal ends was challenging, and therefore we only describe here the final step,
which consisted of joining both axes of the nonsymmetrical rotaxane, the alkyne 454, and the azide
455 through CuAAC chemistry, affording compound 456 (Scheme 106). The formation of a pH-
sensitive bistable rotaxane 457 was achieved by methylation of the triazole ring using methyl iodide
Molecules
Molecules 2018, 23, 116
2019, 24, x 57
57 of
of 68
68

polyethylene glycol monomethyl ether (mPEG-OH) 460 (Scheme 107). The authors evaluated the
state of aof
behavior secondary amine. More
these copolymers specifically,
in aqueous media,the crown ether
concluding that was
they located aroundto
self-assembled the secondary
form unique
ammonium ion as the best binding site in the protonated form. On the other hand,
nanostructures, including disk-like micelles. The experimental results also suggested that the NMR analysis
showed that
prepared upon deprotonation
copolymers can be usedof the
as ammonium
inexpensiveion, the stabilizers
steric triazolium for
ion became the better
liposomes, makingbinding
them
site, which
suitable forcaused
severalthe ring to shuttle
biomedical along the
applications axis toward this position (Scheme 106) [106].
[107].

Scheme 106. 106. Synthesis

Synthesis ofof cholesterol-based
cholesterol-based rotaxane.
rotaxane. Reagents
Reagents and andconditions: a)Cu(CH
conditions: a) Cu(CH33CN)
CN)44BF44,,
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, CH
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, CH2Cl
2 Cl
2,2 ,rt,
rt,4848 h;h; b) MeI, rt,
b) MeI, rt, 72
72 h; c) NaBF44,,
CH22ClCl22/acetone/H
/acetone/H2O2 O (1:1:2),
(1:1:2), rt,rt,
1818
h. h.

Venkataraman and coworkers reported the two-step synthesis of cholesterol-functionalized

aliphatic N-substituted 8-membered cyclic carbonate monomer 459 (Scheme 107) [107].
Cholesterol-based monomer 459 was employed in organocatalytic ring-opening polymerization to
produce PEGylated amphiphilic diblock copolymers (using a commercially available macroinitiator),
polyethylene glycol monomethyl ether (mPEG-OH) 460 (Scheme 107). The authors evaluated the
behavior of these copolymers in aqueous media, concluding that they self-assembled to form unique
nanostructures, including disk-like micelles. The experimental results also suggested that the prepared
copolymers can be used as inexpensive steric stabilizers for liposomes, making them suitable for
several biomedical applications [107].
Recently, a cholesterol-modified poly(L-cysteine) copolymer, 466, that can undergo unusual
Scheme 107. Synthesis of PEGylated amphiphilic diblock copolymers. Reagents and conditions: a)
micelle-to-vesicle transformation of polypeptides triggered by oxidation, was synthesized following
diethanolamine, Na2CO3, THF/H2O (2:1), 0 °C to rt, 16 h; b) ethyl chloroformate, Et3N, THF, 0 °C to rt,
a three-step protocol starting from cholesteryl
16 h; c) 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU),3-bromopropylcarbamate
CH2Cl2, rt, 2 h. 462 (Scheme 108) [108].
The thioether groups in the side chains of 466 were further oxidized to the corresponding sulfone
derivative 467 (Scheme
Recently, 108). The authors
a cholesterol-modified demonstrated
poly( that oxidation
L-cysteine) copolymer, 466,ofthat
the thioether groups
can undergo in the
unusual
side chains could change the packing characteristics of cholesterol groups and the peptide
micelle-to-vesicle transformation of polypeptides triggered by oxidation, was synthesized following backbone,
aresulting in the
three-step transformation
protocol of a cholesteryl
starting from β-sheet to an α-helix conformation, combined
3-bromopropylcarbamate with108)
462 (Scheme an interesting
[108]. The
morphological transition from micelle-like structures to vesicles. Moreover, changing
thioether groups in the side chains of 466 were further oxidized to the corresponding sulfone the secondary
structure as
derivative 467well as the108).
(Scheme morphology
The authorsendowed the polymer
demonstrated assemblies
that oxidation of thewith excellent
thioether specificity
groups in the
side chains could change the packing characteristics of cholesterol groups and the peptide backbone,
Molecules 2019, 24, 116 58 of 68

Scheme 106. Synthesis of cholesterol-based rotaxane. Reagents and conditions: a) Cu(CH3CN)4BF4,

for controlled payload release and improved cell interaction
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, in response
CH2Cl2, rt, 48 to rt,
h; b) MeI, ROS.72 These interesting
h; c) NaBF 4,

formulations had excellent

CH2Cl2/acetone/H anticancer
2O (1:1:2), rt, 18 h. properties both in vitro and in vivo [108].

Molecules 2018, 23, x 58 of 68

resulting in the transformation of a β-sheet to an α-helix conformation, combined with an interesting

morphological transition from micelle-like structures to vesicles. Moreover, changing the secondary
structure as well as the morphology endowed the polymer assemblies with excellent specificity for
Scheme
Scheme 107.107. Synthesis
Synthesis of
of PEGylated
PEGylated amphiphilic
amphiphilic diblock
diblock copolymers. Reagents and
copolymers. Reagents and conditions: a)
conditions: a)
controlled payload release and improved cell interaction in response to ROS. These interesting
◦ C to ◦ C to
diethanolamine, Na CO , THF/H
diethanolamine, Na2CO33, THF/H2O O (2:1), 0 rt, 16 h; b) ethyl chloroformate, Et
2 (2:1), 0 °C to rt, 16 h; b) ethyl chloroformate, Et3N, N, THF, 0
3 THF, 0 °C to rt,
formulations had excellent anticancer properties both in vitro and in vivo [108].
rt, 16 h; c) 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), CH Cl , rt, 2 h.
16 h; c) 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), CH2Cl22, rt,
2 2 h.

Recently, a cholesterol-modified poly(L-cysteine) copolymer, 466, that can undergo unusual

micelle-to-vesicle transformation of polypeptides triggered by oxidation, was synthesized following
a three-step protocol starting from cholesteryl 3-bromopropylcarbamate 462 (Scheme 108) [108]. The
thioether groups in the side chains of 466 were further oxidized to the corresponding sulfone
derivative 467 (Scheme 108). The authors demonstrated that oxidation of the thioether groups in the
side chains could change the packing characteristics of cholesterol groups and the peptide backbone,

Scheme 108.
Scheme Synthesis of copolymers
108. Synthesis copolymers PEG-PCys-Chol
PEG-PCys-Chol and and PEG-PCys-Chol-O
PEG-PCys-Chol-O22.. Reagents
Reagents and
conditions: a)a)22NNNaOH,
NaOH,TBAI,TBAI,CHCl
CHCl ◦
conditions: 3 /EtOH
3/EtOH (7:26),
(7:26), rt,h;48b)h;triphosgene,
rt, 48 b) triphosgene,
THF,THF,
50 °C,504 h;C,c)4PEG-
h; c)
PEG-NH , THF, 35 ◦ C, 72 h; d) 10% H O , 5% AcOH, 37 ◦ C, 16 h, dialysis.
NH2, THF, 35 °C, 72 h; d) 10% H2O2, 5% AcOH, 37 °C, 16 h, dialysis.
2 2 2

Gramine [N-(1H-indol-3-ylmethyl)-N,N-dimethylamine]
Gramine [N-(1H-indol-3-ylmethyl)-N,N-dimethylamine] is is aa well-known
well-known indole
indole derivative
derivative and
and
is often used as synthon for the preparation of a large variety of substituted indoles
is often used as synthon for the preparation of a large variety of substituted indoles with importantwith important
biological activities.
biological activities. InInthis
thiscontext,
context,Kozanecka
Kozaneckaand and coworkers
coworkers reported
reported thethe
useuse
of of gramine
gramine (470)
(470) to
to synthesize
synthesize cholesterol
cholesterol (471)
(471) andandcholestanol
cholestanoldimers
dimers(472)
(472)consisting
consistingofoftwo
two molecules
molecules of
of sterols
sterols
connected by
connected by an
an N(CH
N(CH33))22 group
group (Scheme
(Scheme 109)
109) [109].
[109].
These new steroid dimers (471 and 472) were shown to interact in vitro with the human erythrocyte
membrane, changing the discoid erythrocyte shape, which resulted in induced stomatocytosis or
echinocytosis. The authors also demonstrated that these new dimers were capable of interfering with
membrane phospholipid asymmetry and loosening the molecular packing of phospholipids in the bilayer
at sublytic concentrations. Moreover, the dimers 471 and 472 possessed a higher capacity for changing
the erythrocyte membrane structure and its permeability than steroids alone did [109].
A new multifunctional pyridine derivative was synthesized and studied as an efficient initiator for
the polymerization of diethylvinylphosphonate (DEVP). The authors used a new pyridine compound
(473) in the thiol-ene click reaction (a well-established coupling method) to link together poly-DEVP
and thiocholesterol 95 (Scheme 110) [110].
Compound 474 exhibited good thermal response and low cytotoxicity against human embryonic
Scheme 109. Synthesis of cholesterol and cholestanol dimers. Reagents and conditions: a) MeCN,
renal cell lines (HEK-293) and immortalized human microvascular endothelial cells (HMEC-1). It was
reflux, 5 h.
concluded that the introduction of the thiocholesterol anchor unit was advantageous regarding toxicity
whenThese
comparednew tosteroid
polymers without
dimers (471functionalization.
and 472) were The shownthiocholesterol
to interact conjugate 474 isthe
in vitro with interesting
human
for many applications, since it is water-soluble, thermo-responsive, and
erythrocyte membrane, changing the discoid erythrocyte shape, which resulted in inducedbiocompatible [110].
stomatocytosis or echinocytosis. The authors also demonstrated that these new dimers were capable
of interfering with membrane phospholipid asymmetry and loosening the molecular packing of
phospholipids in the bilayer at sublytic concentrations. Moreover, the dimers 471 and 472 possessed
a higher capacity for changing the erythrocyte membrane structure and its permeability than steroids
alone did [109].
 Gramine [N-(1H-indol-3-ylmethyl)-N,N-dimethylamine] is a well-known indole derivative and
is often used as synthon for the preparation of a large variety of substituted indoles with important
biological activities. In this context, Kozanecka and coworkers reported the use of gramine (470) to
synthesize
Molecules 2019,cholesterol
24, 116 (471) and cholestanol dimers (472) consisting of two molecules of sterols
59 of 68
connected by an N(CH3)2 group (Scheme 109) [109].

Molecules 2018, 23, x 59 of 68

Scheme 109.
Scheme Synthesis of
109. Synthesis of cholesterol
cholesterol and
and cholestanol
cholestanol dimers. Reagents and
dimers. Reagents and conditions:
conditions: a) MeCN,
a) MeCN,
reflux, 5 h.
reflux, 5 h.
Molecules 2018, 23, x 59 of 68

These new steroid dimers (471 and 472) were shown to interact in vitro with the human
erythrocyte membrane, changing the discoid erythrocyte shape, which resulted in induced
stomatocytosis or echinocytosis. The authors also demonstrated that these new dimers were capable
of interfering with membrane phospholipid asymmetry and loosening the molecular packing of
Scheme 110. Synthesis of poly-DEVP thiocholesterol. Reagents and conditions: a) AIBN, 70 °C, 24 h.
phospholipids in the bilayer at sublytic concentrations. Moreover, the dimers 471 and 472 possessed
DEVP: Diethylvinylphosphonate.
a higher capacity for changing the erythrocyte membrane structure and its permeability than steroids
◦ C, 24 h.
Scheme
aloneScheme
did [109].110. Synthesis of poly-DEVP thiocholesterol. Reagents
110. Reagents and
and conditions:
conditions: a) AIBN, 70
a) AIBN, 70 °C, 24 h.
Compound
DEVP:
DEVP:
474 exhibited good thermal response and low cytotoxicity against human embryonic
Diethylvinylphosphonate.
A newDiethylvinylphosphonate.
multifunctional pyridine derivative was synthesized and studied as an efficient initiator
renal cell lines (HEK-293) and immortalized human microvascular endothelial cells (HMEC-1). It was
for the polymerization of diethylvinylphosphonate (DEVP). The authors used a new pyridine
To takethat
concluded
Compound advantage of the important
the exhibited
introduction the biological
ofthermal properties
thiocholesterolandanchor of cholesterol and glutathione
unit wasagainst
advantageous for the
regarding
compound (473) 474
in the thiol-ene good
click response
reaction low cytotoxicity
(a well-established coupling method) human embryonic
to link together
cells,
renal a cholesterol-glutathione
toxicity when
cell lines compared
(HEK-293) to
and (Chol-GSH)
polymers without
immortalized bioconjugate
human (478) was
functionalization.
microvascular The designed and(HMEC-1).
thiocholesterol
endothelial cells used as a model
conjugate It474
wasis
poly-DEVP and thiocholesterol 95 (Scheme 110) [110].
amphiphilic
interesting biomolecule
for many to make
applications,a co-assembly
since it is with lysozyme
water-soluble, using a dialysis-assisted
thermo-responsive,
concluded that the introduction of the thiocholesterol anchor unit was advantageous regarding and approach
biocompatible[111].
The synthetic
[110].
toxicity when route
comparedtoward the Chol-GSH
to polymers withoutbioconjugate 478 involved
functionalization. a five-step reaction
The thiocholesterol conjugatesequence,
474 is
including
To esterification,
take advantage 1,3-dipolar
of the cycloaddition,
important biologicaland thiol-disulfide
properties of exchange
cholesterol reactions
and
interesting for many applications, since it is water-soluble, thermo-responsive, and biocompatible (Scheme
glutathione for 111).
the
cells,
[110].The
a authors applied
cholesterol-glutathione a dialysis-assisted
(Chol-GSH) method of
bioconjugate Ch-GSH
(478) and
was lysozyme
designed to
and prepare
used asbioactive
a model
self-assembled
amphiphilic structures,
biomolecule
To take advantage ofto which
themake showed that hydrophobic
a co-assembly
important biological with lysozyme
properties cholesterol located in the walls,
using a dialysis-assisted
of cholesterol and glutathione forand
approach the
hydrophilic
[111]. The GSH
synthetic and lysozyme
route toward on the
the inner
Chol-GSHand outer surfaces.
bioconjugate This
478 result
involved
cells, a cholesterol-glutathione (Chol-GSH) bioconjugate (478) was designed and used as a model was
a explained
five-step based
reaction
on the electrostatic
sequence,
amphiphilic including interaction
biomolecule to makebetween
esterification, GSH and
a1,3-dipolar
co-assembly lysozyme,
cycloaddition,
with which provided
and using
lysozyme a driving
thiol‒disulfide exchange
a dialysis-assisted force for the
reactions
approach
self-assembly,
(Scheme
[111]. The111). maintaining
synthetic route the bioactivity
toward of lysozyme
the Chol-GSH in the self-assembly
bioconjugate process
478 involved [111].
a five-step reaction
sequence, including esterification, 1,3-dipolar cycloaddition, and thiol‒disulfide exchange reactions
(Scheme 111).

111. Synthesis
Scheme 111. Synthesis of ofcholesterol-glutathione
cholesterol-glutathione(Ch-GSH)(Ch-GSH)bioconjugate.
bioconjugate.Reagents
Reagents and conditions:
and a)
conditions:
2-bromoacetyl bromide, Et3Et
N,3N,
CH Cl , rt, overnight; b) NaN , THF/H O (4:1), 50 ◦ C, overnight;
a) 2-bromoacetyl bromide, CH
2 22 Cl 2, rt, overnight; b) NaN 3 3 , THF/H 22 °C, c)
4-pentynoic acid, CuBr, PMDETA, THF/DMF (4:1), 35 ◦ C, 48 h; d) 2-(pyridine-2-yldisulfanyl)ethanol,
CuBr,of
Scheme 111. Synthesis PMDETA, THF/DMF
cholesterol-glutathione °C, 48 h;
(Ch-GSH) d) 2-(pyridine-2-yldisulfanyl)ethanol,
bioconjugate. Reagents and conditions:
EDCl, DMAP,
DMAP, CH
CH 22Cl22, rt, 24 h; (e) glutathione, THF/H
THF/H O
22 O(3:1),
(3:1),rt,rt,66h.h.
a) 2-bromoacetyl bromide, Et3N, CH2Cl2, rt, overnight; b) NaN3, THF/H 2O (4:1), 50 °C, overnight; c)

4-pentynoic acid, CuBr, PMDETA, THF/DMF (4:1), 35 °C, 48 h; d) 2-(pyridine-2-yldisulfanyl)ethanol,

9. Conclusions
The authors
EDCl, DMAP, applied
CH2Cl2, rt, a 24
dialysis-assisted
h; (e) glutathione, method
THF/H2of O Ch-GSH
(3:1), rt, 6 h. and lysozyme to prepare bioactive
self-assembled structures,
In this review, the role which showed that compounds
of cholesterol-based hydrophobicincholesterol locatedareas
different research in the walls,
such and
as drug
hydrophilic
delivery, GSH
biologicaland lysozyme
activities, on
liquid the inner
crystals, and outer
gelators, surfaces.
bioimaging, This
andresult
purely was explained
synthetic
The authors applied a dialysis-assisted method of Ch-GSH and lysozyme to prepare bioactive based on
applications
the
was electrostatic
highlighted.
self-assembled interaction between
In the drug
structures, GSH
whichdelivery and
showed field, lysozyme, which
several examples
that hydrophobic provided a driving
of cholesterol
cholesterol force for
derivatives
located in the self-
the walls,were
and
assembly, maintaining
hydrophilic the bioactivity
GSH and lysozyme on theofinner
lysozyme in thesurfaces.
and outer self-assembly process
This result was[111].
explained based on
the electrostatic interaction between GSH and lysozyme, which provided a driving force for the self-
9. Conclusions
assembly, maintaining the bioactivity of lysozyme in the self-assembly process [111].
In this review, the role of cholesterol-based compounds in different research areas such as drug
9. Conclusions
delivery, biological activities, liquid crystals, gelators, bioimaging, and purely synthetic applications
Molecules 2019, 24, 116 60 of 68

highlighted due to their applications in preclinical and clinical liposomal drug formulations to decrease
membrane fluidity and provide favorable drug retention properties. Furthermore, in the last few years,
some series of new cholesterol derivatives have also been developed for pharmacological applications
as anticancer, antimicrobial, or antioxidant agents. In the bioimaging field, cholesterol has been used as
a lipid anchor attached to fluorophores to study cellular membrane trafficking, imaging of cholesterol
density, and liposome tracing, among many other bioimaging applications. The fact that cholesterol
conjugates have much scientific interest in the field of materials science due to their liquid crystal
phase behavior, as well as the ability to promote self-organization and hydrophobic interactions in
aqueous media (gelation properties), was also demonstrated in this review. In this review, a general
perspective was given of the main applications of cholesterol derivatives in several research fields,
but also a concise perspective of the advances in their synthetic chemistry. Therefore, we described
the synthetic pathway for different cholesterol derivatives alongside the corresponding application of
the new compounds to furnish a general view from the synthetic and biological aspects of the most
recently reported cholesterol-based compounds.

Funding: Thanks are due to the University of Aveiro, Instituto Politécnico de Bragança, FCT/MEC for financial
support of the QOPNA (FCT UID/QUI/00062/2013) and CIMO (UID/AGR/00690/2013) research units, through
national funds, and where applicable cofinanced by the FEDER, within the PT2020 Partnership Agreement; and
also to the Portuguese NMR Network. This work was also supported by the Integrated Programme of SR&TD
“pAGE–Protein aggregation Across the Lifespan” (reference CENTRO-01-0145-FEDER-000003), co-funded by
the Centro 2020 program, Portugal 2020, European Union, through the European Regional Development Fund.
H. M. T. Albuquerque thanks the pAGE project for his Post-Doc grant (BPD/UI98/4861/2017).
Conflicts of Interest: The authors declare no conflict of interest.

Abbreviations List
Ac acetyl
Ac2 O acetic anhydride
AcOH acetic acid
AG arabinogalactan
AIBN 2,20 -azobis(2-methylpropionitrile)
AIEE aggregation induced enhanced emission
AL β-alanine
AscONa sodium ascorbate
ATRP atom transfer radical polymerization
BBN bombesin
Bn benzyl
Boc2 O di-tert-butyl decarbonate
BODIPY boron dipyrromethene
BtOH N-hydroxybenzotriazole
Bz benzoyl
CAE cholesterol-arginine ester
β-CD β-cyclodextrin
β-CD-NSP β-cyclodextrin nanosponge
CDI carbonyldiimidazole
CF 5,6-carboxyfluorescein
Chol cholesterol
Chol-OA oxyamine-terminated cholesterol
CHS cholesterol hydrogen succinate
Ch-T chloramine-T
CL conventional liposomes
CuAAC copper(I)-catalyzed azide-alkyne cycloaddition
CVS crystal violet staining
Cyclen 1,4,7,10-tetraazacyclododecane
CYS cystamine
Molecules 2019, 24, 116 61 of 68

DAIN 4-(diarylmethylene)imidazolinone
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene
DCC N,N 0 -dicyclohexylcarbodiimide
DCVJ 9-(2,2-dicyanovinyl)julolidine
DHPC dihexanoylphosphatidylcholine
DIAD diisopropyl azodicarboxylate
DIPEA N,N-diisopropylethylamine
DMAP dimethylaminopyridine
DMF dimethylformamide
DMPC dimyristoylphosphatidylcholine
DMSO dimethyl sulfoxide
DMTAP dimyristoyltrimethylammonium propane
DNA deoxyribonucleic acid
DOPC dioleoylphosphatidilcholine
DOPE 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
DOX doxorubicin
DOX-NPs doxorubicin loaded nanoparticles
DPPA diphenylphosphoryl azide
DPPH 2,2-diphenyl-1-picrylhydrazyl radical
DTX docetaxel
EDAC 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride
EDCl N-(3-dimethylaminopropyl)-N 0 -ethylcarbodiimide hydrochloride
Et2 N diethylamine
Et3 N triethylamine
Et2 O diethyl ether
EtOAc ethyl acetate
EtOH ethanol
GFP green fluorescent protein
GSH glutathione
HA hyaluronic acid
hbPG linear-hyperbranched amphiphilic polyglycerol
HOBt N-hydroxybenzotriazole
HSA human serum albumin
IC50 half-maximal inhibitory concentration
ICT intramolecular charge transfer
L -AA L -ascorbic acid
LC liquid crystal
LMGs low-molecular-weight gelators
MeCN acetonitrile
MeOTf methyl triflate
m-CPBA m-chloroperoxybenzoic acid
min minutes
MPP mitochondria-penetrating peptide
MTT 3-(4,5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide
MW microwave irradiation
NaOAc sodium acetate
NaOMe sodium methoxide
NBD nitrobenzoxadiazole
NBS N-bromosuccinimide
NHS N-hydroxysuccinimide
NOAC nitrile oxide alkyne cycloaddition
NPs nanoparticles
PBS phosphate-buffered saline
PDC pyridinium dichromate
PEG polyethylene glycol
Molecules 2019, 24, 116 62 of 68

PET positron emission tomography

pHPMA poly[N-(2-hydroxypropyl)-methacrylamide]
PMDETA N,N,N 0 ,N”,N”-pentamethyldiethylenetriamine
PPA phenylpropanolamine
PPh3 triphenylphosphine
p-TsCl p-toluenesulfonyl chloride
PTX paclitaxel
PyBOP benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate
RAFT reversible addition fragmentation chain transfer
rt room temperature
SA succinic anhydride
SCID severe combined immunodeficient
SML surface-modified liposomes
SubPcs subphthalocyanines
TACN 1,4,7-triazacyclononane
Tb trilobolide
TBAB tetrabutylammonium bromide
TBAF tetrabutylammonium fluoride
TBAH tetrabutylammonium hydroxide
TBAITBDMS tetrabutylammonium iodidetert-butyldimethylsilyl
TBDMSCl tert-butyldimethylsilyl chloride
Tb-N3 VA trilobolide 8-O-azidovalerate
TBTA tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
t-BuOH tert-butanol
t-BuOK potassium tert-butoxide
t-BuOOH tert-butyl hydroperoxide
TE transfection efficiency
TEG tetraethylene glycol
TFA trifluoroacetic acid
TFAA trifluoroacetic anhydride
THF tetrahydrofuran
TIS triisopropylsilane
TMSCl trimethylsilyl chloride
TMSITMSOTf iodotrimethylsilanetrimethylsilyl trifluoromethanesulfonate
TOAB tetraoctylammonium bromide
TsCl p-toluenesulfonyl chloride

References
1. Cerqueira, N.M.F.S.A.; Oliveira, E.F.; Gesto, D.S.; Santos-Martins, D.; Moreira, C.; Moorthy, H.N.; Ramos, M.J.;
Fernandes, P.A. Cholesterol Biosynthesis: A Mechanistic Overview. Biochemistry 2016, 55, 5483–5506.
[CrossRef] [PubMed]
2. Nes, W.D. Biosynthesis of Cholesterol and Other Sterols. Chem. Rev. 2011, 111, 6423–6451. [CrossRef]
[PubMed]
3. Habchi, J.; Chia, S.; Galvagnion, C.; Michaels, T.C.T.; Bellaiche, M.M.J.; Ruggeri, F.S.; Sanguanini, M.; Idini, I.;
Kumita, J.R.; Sparr, E.; et al. Cholesterol catalyses Aβ42 aggregation through a heterogeneous nucleation
pathway in the presence of lipid membranes. Nat. Chem. 2018, 10, 673–683. [CrossRef] [PubMed]
4. Morzycki, J.W. Recent advances in cholesterol chemistry. Steroids 2014, 83, 62–79. [CrossRef] [PubMed]
5. Sercombe, L.; Veerati, T.; Moheimani, F.; Wu, S.Y.; Sood, A.K.; Hua, S. Advances and Challenges of Liposome
Assisted Drug Delivery. Front. Pharmacol. 2015, 6, 286. [CrossRef] [PubMed]
6. Vabbilisetty, P.; Sun, X.-L. Liposome surface functionalization based on different anchoring lipids via
Staudinger ligation. Org. Biomol. Chem. 2014, 12, 1237–1244. [CrossRef] [PubMed]
7. Saleem, Q.; Zhang, Z.; Petretic, A.; Gradinaru, C.C.; Macdonald, P.M. Single Lipid Bilayer Deposition on
Polymer Surfaces Using Bicelles. Biomacromolecules 2015, 16, 1032–1039. [CrossRef] [PubMed]
Molecules 2019, 24, 116 63 of 68

8. Pathak, P.O.; Nagarsenker, M.S.; Barhate, C.R.; Padhye, S.G.; Dhawan, V.V.; Bhattacharyya, D.;
Viswanathan, C.L.; Steiniger, F.; Fahr, A. Cholesterol anchored arabinogalactan for asialoglycoprotein receptor
targeting: Synthesis, characterization, and proof of concept of hepatospecific delivery. Carbohydr. Res. 2015,
408, 33–43. [CrossRef]
9. Crucianelli, E.; Bruni, P.; Frontini, A.; Massaccesi, L.; Pisani, M.; Smorlesi, A.; Mobbili, G. Liposomes
containing mannose-6-phosphate-cholesteryl conjugates for lysosome-specific delivery. RSC Adv. 2014, 4,
58204–58207. [CrossRef]
10. Silva, A.T.M.; Maia, A.L.C.; de Oliveira Silva, J.; de Barros, A.L.B.; Soares, D.C.F.; de Magalhaes, M.T.Q.; Jose
Alves, R.; Ramaldes, G.A. Synthesis of cholesterol-based neoglycoconjugates and their use in the preparation
of liposomes for active liver targeting. Carbohydr. Res. 2018, 465, 52–57. [CrossRef]
11. Škorpilová, L.; Rimpelová, S.; Jurášek, M.; Buděšínský, M.; Lokajová, J.; Effenberg, R.; Slepička, P.; Ruml, T.;
Kmoníčková, E.; Drašar, P.B.; Wimmer, Z. BODIPY-based fluorescent liposomes with sesquiterpene lactone
trilobolide. Beilstein J. Org. Chem. 2017, 13, 1316–1324. [CrossRef] [PubMed]
12. Lin, Y.-K.; Fang, J.-Y.; Wang, S.-W.; Lee, R.-S. Synthesis and characterization of triple-responsive
PNiPAAm-S-S-P(αN3CL-g-alkyne) copolymers bearing cholesterol and fluorescence monitor.
React. Funct. Polym. 2018, 130, 29–42. [CrossRef]
13. Alberti, D.; Toppino, A.; Geninatti Crich, S.; Meraldi, C.; Prandi, C.; Protti, N.; Bortolussi, S.; Altieri, S.;
Aime, S.; Deagostino, A. Synthesis of a carborane-containing cholesterol derivative and evaluation as a
potential dual agent for MRI/BNCT applications. Org. Biomol. Chem. 2014, 12, 2457–2467. [CrossRef]
[PubMed]
14. Zhang, X.; Niebuur, B.-J.; Chytil, P.; Etrych, T.; Filippov, S.K.; Kikhney, A.; Wieland, D.C.F.; Svergun, D.I.;
Papadakis, C.M. Macromolecular pHPMA-Based Nanoparticles with Cholesterol for Solid Tumor Targeting:
Behavior in HSA Protein Environment. Biomacromolecules 2018, 19, 470–480. [CrossRef] [PubMed]
15. Singh, P.; Ren, X.; Guo, T.; Wu, L.; Shakya, S.; He, Y.; Wang, C.; Maharjan, A.; Singh, V.; Zhang, J.
Biofunctionalization of β-cyclodextrin nanosponges using cholesterol. Carbohydr. Pol. 2018, 190, 23–30.
[CrossRef] [PubMed]
16. Li, J.; Ma, Y.J.; Wang, Y.; Chen, B.Z.; Guo, X.D.; Zhang, C.Y. Dual redox/pH-responsive hybrid polymer-lipid
composites: Synthesis, preparation, characterization and application in drug delivery with enhanced
therapeutic efficacy. Chem. Eng. J. 2018, 341, 450–461. [CrossRef]
17. Tran, T.-H.; Nguyen, C.T.; Gonzalez-Fajardo, L.; Hargrove, D.; Song, D.; Deshmukh, P.; Mahajan, L.;
Ndaya, D.; Lai, L.; Kasi, R.M.; et al. Long Circulating Self-Assembled Nanoparticles from
Cholesterol-Containing Brush-Like Block Copolymers for Improved Drug Delivery to Tumors.
Biomacromolecules 2014, 15, 4363–4375. [CrossRef] [PubMed]
18. Wang, Z.; Luo, T.; Sheng, R.; Li, H.; Sun, J.; Cao, A. Amphiphilic Diblock Terpolymer
PMAgala-b-P(MAA-co-MAChol)s with Attached Galactose and Cholesterol Grafts and Their Intracellular
pH-Responsive Doxorubicin Delivery. Biomacromolecules 2016, 17, 98–110. [CrossRef] [PubMed]
19. Jia, L.; Cui, D.; Bignon, J.; Di Cicco, A.; Wdzieczak-Bakala, J.; Liu, J.; Li, M.-H. Reduction-Responsive
Cholesterol-Based Block Copolymer Vesicles for Drug Delivery. Biomacromolecules 2014, 15, 2206–2217.
[CrossRef] [PubMed]
20. Dolor, A.; Kierstead, P.; Dai, Z.; Szoka, F.C. Sterol-modified PEG lipids: Alteration of the bilayer anchoring
moiety has an unexpected effect on liposome circulation. Chem. Commun. 2018, 54, 11949–11952. [CrossRef]
[PubMed]
21. Zhu, Z.; Li, D.; Li, Y.; Yang, X.; Pan, W. In vitro–in vivo evaluation of hyaluronic acid-based amphiphilic
copolymers for tumour targeted delivery: The role of hydrophobic groups. RSC Adv. 2017, 7, 23942–23953.
[CrossRef]
22. Mallick, S.; Thuy, L.T.; Lee, S.; Park, J.I.; Choi, J.S. Liposomes containing cholesterol and
mitochondria-penetrating peptide (MPP) for targeted delivery of antimycin A to A549 cells. Colloids Surf. B
2018, 161, 356–364. [CrossRef] [PubMed]
23. Ju, J.; Huan, M.-L.; Wan, N.; Hou, Y.-L.; Ma, X.-X.; Jia, Y.-Y.; Li, C.; Zhou, S.-Y.; Zhang, B.-L. Cholesterol
derived cationic lipids as potential non-viral gene delivery vectors and their serum compatibility. Bioorg. Med.
Chem. Lett. 2016, 26, 2401–2407. [CrossRef] [PubMed]
Molecules 2019, 24, 116 64 of 68

24. Vulugundam, G.; Kumar, K.; Kondaiah, P.; Bhattacharya, S. Efficacious redox-responsive gene delivery
in serum by ferrocenylated monomeric and dimeric cationic cholesterols. Org. Biomol. Chem. 2015, 13,
4310–4320. [CrossRef] [PubMed]
25. Wang, H.-J.; He, X.; Zhang, Y.; Zhang, J.; Liu, Y.-H.; Yu, X.-Q. Hydroxyl-containing non-viral lipidic gene
vectors with macrocyclic polyamine headgroups. RSC Adv. 2015, 5, 59417–59427. [CrossRef]
26. Puchkov, P.A.; Perevoshchikova, K.A.; Kartashova, I.A.; Luneva, A.S.; Kabilova, T.O.; Morozova, N.G.;
Zenkova, M.A.; Maslov, M.A. Polycationic amphiphiles based on triethylenetetramine and their transfection
efficacy. Russ. J. Bioorg. Chem. 2017, 43, 561–569. [CrossRef]
27. Monpara, J.; Kanthou, C.; Tozer, G.M.; Vavia, P.R. Rational Design of Cholesterol Derivative for Improved
Stability of Paclitaxel Cationic Liposomes. Pharm. Res. 2018, 35, 90–107. [CrossRef]
28. van Elk, M.; Deckers, R.; Oerlemans, C.; Shi, Y.; Storm, G.; Vermonden, T.; Hennink, W.E. Triggered Release
of Doxorubicin from Temperature-Sensitive Poly(N-(2-hydroxypropyl)-methacrylamide mono/dilactate)
Grafted Liposomes. Biomacromolecules 2014, 15, 1002–1009. [CrossRef]
29. Asayama, S.; Nagashima, K.; Negishi, Y.; Kawakami, H. Byproduct-Free Intact Modification of Insulin by
Cholesterol End-Modified Poly(ethylene glycol) for in Vivo Protein Delivery. Bioconj. Chem. 2018, 29, 67–73.
[CrossRef]
30. Rega, M.; Jiménez, C.; Rodríguez, J. 6E-Hydroximinosteroid homodimerization by cross-metathesis processes.
Steroids 2007, 72, 729–735. [CrossRef]
31. Richmond, V.; Careaga, V.P.; Sacca, P.; Calvo, J.C.; Maier, M.S. Synthesis and cytotoxic evaluation of four new
6E-hydroximinosteroids. Steroids 2014, 84, 7–10. [CrossRef] [PubMed]
32. Soto-Castro, D.; Lara Contreras, R.C.; Pina-Canseco, M.D.S.; Santillan, R.; Hernandez-Huerta, M.T.; Negron
Silva, G.E.; Perez-Campos, E.; Rincon, S. Solvent-free synthesis of 6β-phenylamino-cholestan-3β,5α-diol and
(25R)-6β-phenylaminospirostan-3β,5α-diol as potential antiproliferative agents. Steroids 2017, 126, 92–100.
[CrossRef] [PubMed]
33. Bu, M.; Cao, T.; Li, H.; Guo, M.; Yang, B.B.; Zhou, Y.; Zhang, N.; Zeng, C.; Hu, L. Synthesis and biological
evaluation of novel steroidal 5α,8α-endoperoxide derivatives with aliphatic side-chain as potential anticancer
agents. Steroids 2017, 124, 46–53. [CrossRef] [PubMed]
34. Huang, Y.; Yang, C.; Zhan, J.; Gan, C.; Liu, Z.; Pang, C.; Chen, H.; Cui, J. Synthesis and antiproliferative
activity of novel A-homo-B-norsteroid thiadiazole derivatives. Tetrahedron Lett. 2017, 58, 2952–2954.
[CrossRef]
35. Martínez-Pascual, R.; Meza-Reyes, S.; Vega-Baez, J.L.; Merino-Montiel, P.; Padrón, J.M.; Mendoza, Á.;
Montiel-Smith, S. Novel synthesis of steroidal oximes and lactams and their biological evaluation as
antiproliferative agents. Steroids 2017, 122, 24–33. [CrossRef]
36. D’Yakonov, V.A.; Tuktarova, R.A.; Dzhemileva, L.U.; Ishmukhametova, S.R.; Yunusbaeva, M.M.;
Dzhemilev, U.M. Catalytic cyclometallation in steroid chemistry V: Synthesis of hybrid molecules based on
steroid oximes and (5Z,9Z)-tetradeca-5,9-dienedioic acid as potential anticancer agents. Steroids 2018, 138,
14–20. [CrossRef]
37. Huang, Y.; Wen, H.; Zheng, J.; Gan, C.; Pang, L.; Pang, C.; Liu, X.; Zhan, J.; Cui, J. Synthesis, characterization,
and biological evaluations of some steryl 2-methoxybenzoates as anticancer agents. Nat. Prod. Res. 2018, 1–5.
[CrossRef]
38. Peters, A.D.; McCallion, C.; Booth, A.; Adams, J.A.; Rees-Unwin, K.; Pluen, A.; Burthem, J.; Webb, S.J.
Synthesis and biological activity of a CXCR4-targeting bis(cyclam) lipid. Org. Biomol. Chem. 2018, 16,
6479–6490. [CrossRef]
39. Aly, M.R.E.S.; Saad, H.A.; Mohamed, M.A.M. Click reaction based synthesis, antimicrobial, and cytotoxic
activities of new 1,2,3-triazoles. Bioorg. Med. Chem. Lett. 2015, 25, 2824–2830. [CrossRef]
40. Aly, M.R.E.S.; Saad, H.A.; Abdel-Hafez, S.H. Synthesis, antimicrobial and cytotoxicity evaluation of new
cholesterol congeners. Beilstein J. Org. Chem. 2015, 11, 1922–1932. [CrossRef]
41. Shamsuzzaman; Khanam, H.; Dar, A.M.; Siddiqui, N.; Rehman, S. Synthesis, characterization, antimicrobial
and anticancer studies of new steroidal pyrazolines. J. Saudi Chem. Soc. 2016, 20, 7–12. [CrossRef]
42. Shamsuzzaman; Mashrai, A.; Khanam, H.; Asif, M.; Ali, A.; Sherwani, A.; Owais, M. Green synthesis and
biological evaluation of steroidal 2H-pyrans as anticancer and antioxidant agents. J King Saud Univ. Sci.
2015, 27, 1–6. [CrossRef]
Molecules 2019, 24, 116 65 of 68

43. Ali, A.; Asif, M.; Alam, P.; Jane Alam, M.; Asif Sherwani, M.; Hasan Khan, R.; Ahmad, S.; Shamsuzzaman.
DFT/B3LYP calculations, in vitro cytotoxicity and antioxidant activities of steroidal pyrimidines and their
interaction with HSA using molecular docking and multispectroscopic techniques. Bioorg. Chem. 2017, 73,
83–99. [CrossRef] [PubMed]
44. Saikia, P.; Kaishap, P.P.; Goswami, J.; Singh, A.K.; Deka Boruah, H.P.; Gogoi, S.; Boruah, R.C. Synthesis of
steroidal and nonsteroidal vicinal heterocyclic alcohols, N-(1-cycloalkenyl)heterocycles and their antibacterial
studies. Steroids 2014, 84, 36–45. [CrossRef] [PubMed]
45. Morake, M.; Coertzen, D.; Ngwane, A.; Wentzel, J.F.; Wong, H.N.; Smit, F.J.; Birkholtz, L.M.; Pietersen, R.D.;
Baker, B.; Wiid, I.; et al. Preliminary Evaluation of Artemisinin-Cholesterol Conjugates as Potential Drugs for
the Treatment of Intractable Forms of Malaria and Tuberculosis. ChemMedChem 2018, 13, 67–77. [CrossRef]
[PubMed]
46. Aly, M.R.E.S.; El Azab, I.H.; Gobouri, A.A. Synthesis, antimicrobial and photoelectric potency of new
ferrocene-based congeners. Mon. Chem. 2018, 149, 505–517. [CrossRef]
47. Ansari, A.; Ali, A.; Asif, M.; Rauf, M.A.; Owais, M.; Shamsuzzaman. Facile one-pot multicomponent synthesis
and molecular docking studies of steroidal oxazole/thiazole derivatives with effective antimicrobial,
antibiofilm and hemolytic properties. Steroids 2018, 134, 22–36. [CrossRef]
48. Begum, A.; Borah, P.; Chowdhury, P. Microwave (MW) promoted high yield expedient synthesis of steryl
ferulates—A class of novel biologically active compounds: A comparative study of their antioxidant activity
with that of naturally occurring γ-oryzanol. Steroids 2016, 107, 37–44. [CrossRef]
49. Wöhrle, T.; Wurzbach, I.; Kirres, J.; Kostidou, A.; Kapernaum, N.; Litterscheidt, J.; Haenle, J.C.; Staffeld, P.;
Baro, A.; Giesselmann, F.; Laschat, S. Discotic Liquid Crystals. Chem. Rev. 2016, 116, 1139–1241. [CrossRef]
50. Hiremath, U.S. Synthesis and characterization of novel chiral dimers exhibiting highly frustrated liquid
crystal phases. Tetrahedron 2014, 70, 4745–4753. [CrossRef]
51. Yeap, G.-Y.; Alshargabi, A.; Mahmood, W.A.K.; Han, C.-C.; Lin, H.-C.; Santo, M.; Ito, M.M. Synthesis,
characterization and molecular organization for induced smectic phase of triazole ring in non-symmetric
liquid crystalline dimer. Tetrahedron 2015, 71, 3939–3945. [CrossRef]
52. Guo, H.; Yang, F.; Liu, W.; Lai, J. Novel supramolecular liquid crystals: Synthesis and mesomorphic properties
of calix[4]arene-cholesterol derivatives. Tetrahedron Lett. 2015, 56, 866–870. [CrossRef]
53. Zhang, X.; Guo, H.; Yang, F.; Yuan, J. Ion complexation-controlled columnar mesophase of
calix[4]arene–cholesterol derivatives with Schiff-base bridges. Tetrahedron Lett. 2016, 57, 905–909. [CrossRef]
54. Xiong, J.; Lin, X.; Guo, H.; Yang, F.; Lai, J. Liquid crystalline oligomers derived from cholesterol: Synthesis
and columnar mesomorphism. Liq. Cryst. 2018, 45, 362–369. [CrossRef]
55. Gupta, M.; Pal, V.; Pal, S.K. Photo-responsive liquid crystals derived from azobenzene centered
cholesterol-based tetramers. New J. Chem. 2018, 42, 8765–8772. [CrossRef]
56. Gupta, S.K.; Setia, S.; Sidiq, S.; Gupta, M.; Kumar, S.; Pal, S.K. New perylene-based non-conventional discotic
liquid crystals. RSC Adv. 2013, 3, 12060–12065. [CrossRef]
57. Zhu, M.; Guo, H.; Yang, F.; Wang, Z. Synthesis, mesomorphic and photophysical properties of novel triads
and pentads of perylene liquid crystals with cholesterol units at the bay-position. RSC Adv. 2017, 7, 4320–4328.
[CrossRef]
58. Chen, S.; Hong, B.; Guo, H.; Yang, F. The mesomorphic and photophysical properties of perylene liquid
crystals with different bay-rigid spacers. Liq. Cryst. 2018, 45, 793–800. [CrossRef]
59. Champagne, P.-L.; Ester, D.; Aldosari, S.; Williams, V.E.; Ling, C.-C. Synthesis and comparison of
mesomorphic behaviour of a cholesterol-based liquid crystal dimer and analogous monomers. Liq. Cryst.
2018, 45, 1164–1176. [CrossRef]
60. Liu, X.; Guo, Z.; Xie, Y.; Chen, Z.; Hu, J.; Yang, L. Synthesis and liquid crystal behavior of new side chain
aliphatic polycarbonates based on cholesterol. J. Mol. Liq. 2018, 259, 350–358. [CrossRef]
61. Xiong, Y.; Zheng, S.; Zhu, L.; Guo, H.; Yang, F. Novel liquid crystals with high fluorescence: Synthesis,
mesomorphic and photophysical properties of cholesterol-triazine-BODIPY trimers. J. Mol. Struct. 2018,
1164, 311–316. [CrossRef]
62. Ooi, Y.-H.; Yeap, G.-Y. λ-Shaped liquid crystal trimers with dual terminal cholesteryl moieties: Synthesis and
concomitant of N*, SmA and cholesteric glassy phases. Liq. Cryst. 2018, 45, 204–218. [CrossRef]
Molecules 2019, 24, 116 66 of 68

63. Frizon, T.E.; Jamal, R.; Sumbal, S.; Bechtold, I.H.; Gallardo, H.; Braga, A.L. Synthesis of Functionalized
Organoselenium Materials: Selenides and Diselenides Containing Cholesterol. Eur. J. Org. Chem. 2015, 2015,
3470–3476. [CrossRef]
64. Beaulieu, R.; Gottis, S.; Meyer, C.; Grand, E.; Deveaux, V.; Kovensky, J.; Stasik, I. Cholesteryl and diosgenyl
glycosteroids: Synthesis and characterization of new smectic liquid crystals. Carbohydr. Res. 2015, 404, 70–78.
[CrossRef]
65. Skilling, K.J.; Citossi, F.; Bradshaw, T.D.; Ashford, M.; Kellam, B.; Marlow, M. Insights into low molecular
mass organic gelators: A focus on drug delivery and tissue engineering applications. Soft Matter 2014, 10,
237–256. [CrossRef] [PubMed]
66. Draper, E.R.; Adams, D.J. Low-Molecular-Weight Gels: The State of the Art. Chem 2017, 3, 390–410. [CrossRef]
67. Devi, M.; Dhir, A.; Dhir, A.; Pooja; Pradeep, C.P. New triangular steroid-based A(LS)3 type gelators for
selective fluoride sensing application. RSC Adv. 2014, 4, 27098–27105. [CrossRef]
68. Geng, L.; Feng, G.; Wang, S.; Yu, X.; Xu, Z.; Zhen, X.; Wang, T. Fluoride-responsive organogel containing
azobenzyl and cholesterol units. J. Fluorine Chem. 2015, 170, 24–28. [CrossRef]
69. Ghosh, K.; Panja, S. Coumarin-based supramolecular gelator: A case of selective detection of F− and
HP2 O7 3− . RSC Adv. 2015, 5, 12094–12099. [CrossRef]
70. Pang, X.; Yu, X.; Xie, D.; Li, Y.; Geng, L.; Ren, J.; Zhen, X. Tunable multicolor emissions in a monocomponent
gel system by varying the solvent, temperature and fluoride anion. Org. Biomol. Chem. 2016, 14, 11176–11182.
[CrossRef]
71. Panja, S.; Ghosh, S.; Ghosh, K. Pyridine/pyridinium symmetrical bisamides as functional materials:
Aggregation, selective sensing and drug release. New J. Chem. 2018, 42, 6488–6497. [CrossRef]
72. Huibin, S.; Shujuan, L.; Qiang, Z.; Wei, H. Multiple-Stimuli Responsive Luminescent Gels Based on
Cholesterol Containing Benzothiadiazole Fluorophores. Chin. J. Chem. 2015, 33, 1140–1144.
73. Panja, A.; Ghosh, K. Selective sensing of Hg2+ via sol–gel transformation of a cholesterol-based compound.
Supramol. Chem. 2018, 30, 722–729. [CrossRef]
74. Panja, A.; Ghosh, K. Diaminomalenonitrile-decorated cholesterol-based supramolecular gelator:
Aggregation, multiple analyte (hydrazine, Hg2+ and Cu2+ ) detection and dye adsorption. New J. Chem. 2018,
42, 13718–13725. [CrossRef]
75. Panja, A.; Ghosh, K. Cholesterol-based diazine derivative: Selective sensing of Ag+ and Fe3+ ions through
gelation and the performance of metallogels in dye and picric acid adsorption from water. Mater. Chem. Front.
2018. [CrossRef]
76. Ren, Y.; Wang, B.; Zhang, X. Synthesis of photoresponsive cholesterol-based azobenzene organogels:
Dependence on different spacer lengths. Beilstein J. Org. Chem. 2015, 11, 1089–1095. [CrossRef]
77. Tan, X.; Li, Z.; Xia, M.; Cheng, X. Reversible photoresponsive chiral liquid crystal and multistimuli responsive
organogels based on a cholesterol-azobenzene dimesogen. RSC Adv. 2016, 6, 20021–20026. [CrossRef]
78. Yu, X.; Chen, H.; Shi, X.; Albouy, P.-A.; Guo, J.; Hu, J.; Li, M.-H. Liquid crystal gelators with photo-responsive
and AIE properties. Mater. Chem. Front. 2018. [CrossRef]
79. Shimasaki, T.; Okamiya, Y.; Sato, R.; Hara, K.; Nakamura, T.; Teramoto, N.; Shibata, M. Synthesis, structure
and properties of cholesterol-based A(LS)2- and A(LS)3-type gelators without hydrogen bond linkers.
Tetrahedron 2016, 72, 1517–1523. [CrossRef]
80. Yao, C.; Sun, Q.; Xia, W.; Zhang, J.; Lin, C.; Wang, L. Ferrocenyl-guest tunable organogel constructed from a
Pillar[6]arene-functionalized cholesterol derivative. J. Organomet. Chem. 2017, 847, 68–73. [CrossRef]
81. Li, G.; Wu, J.; Wang, B.; Yan, S.; Zhang, K.; Ding, J.; Yin, J. Self-Healing Supramolecular Self-Assembled
Hydrogels Based on Poly(L-glutamic acid). Biomacromolecules 2015, 16, 3508–3518. [CrossRef] [PubMed]
82. Guo, Z.; Park, S.; Yoon, J.; Shin, I. Recent progress in the development of near-infrared fluorescent probes for
bioimaging applications. Chem. Soc. Rev. 2014, 43, 16–29. [CrossRef] [PubMed]
83. Chauhan, D.P.; Saha, T.; Lahiri, M.; Talukdar, P. BODIPY based ‘click on’ fluorogenic dyes: Application in
live cell imaging. Tetrahedron Lett. 2014, 55, 244–247. [CrossRef]
84. Byrd, K.M.; Arieno, M.D.; Kennelly, M.E.; Estiu, G.; Wiest, O.; Helquist, P. Design and synthesis of a
crosslinker for studying intracellular steroid trafficking pathways. Bioorg. Med. Chem. 2015, 23, 3843–3851.
[CrossRef] [PubMed]
Molecules 2019, 24, 116 67 of 68

85. Kim, B.-K.; Seu, Y.-B.; Choi, J.-S.; Park, J.-W.; Doh, K.-O. Synthesis and validation of novel cholesterol-based
fluorescent lipids designed to observe the cellular trafficking of cationic liposomes. Bioorg. Med. Chem. Lett.
2015, 25, 3893–3896. [CrossRef] [PubMed]
86. Reibel, A.T.; Müller, S.S.; Pektor, S.; Bausbacher, N.; Miederer, M.; Frey, H.; Rösch, F. Fate of Linear and
Branched Polyether-Lipids In Vivo in Comparison to Their Liposomal Formulations by 18 F-Radiolabeling
and Positron Emission Tomography. Biomacromolecules 2015, 16, 842–851. [CrossRef] [PubMed]
87. Palakollu, V.; Kanvah, S. Cholesterol-tethered AIEE fluorogens: Formation of self-assembled nanostructures.
RSC Adv. 2015, 5, 33049–33057. [CrossRef]
88. Wercholuk, A.N.; Thuman, J.M.; Stanley, J.L.; Sargent, A.L.; Anderson, E.S.; Allen, W.E. Incorporation of
fluorophore-cholesterol conjugates into liposomal and mycobacterial membranes. Bioorg. Med. Chem. 2016,
24, 1045–1049. [CrossRef] [PubMed]
89. Bernhard, Y.; Gigot, E.; Goncalves, V.; Moreau, M.; Sok, N.; Richard, P.; Decréau, R.A. Direct
subphthalocyanine conjugation to bombesin vs. indirect conjugation to its lipidic nanocarrier.
Org. Biomol. Chem. 2016, 14, 4511–4518. [CrossRef]
90. Ikejiri, M.; Mori, K.; Miyagi, R.; Konishi, R.; Chihara, Y.; Miyashita, K. A hybrid molecule of a GFP
chromophore analogue and cholestene as a viscosity-dependent and cholesterol-responsive fluorescent
sensor. Org. Biomol. Chem. 2017, 15, 6948–6958. [CrossRef]
91. Tomkiel, A.M.; Kowalski, J.; Płoszyńska, J.; Siergiejczyk, L.; Łotowski, Z.; Sobkowiak, A.; Morzycki, J.W.
Electrochemical synthesis of glycoconjugates from activated sterol derivatives. Steroids 2014, 82, 60–67.
[CrossRef] [PubMed]
92. Tomkiel, A.M.; Biedrzycki, A.; Ploszynska, J.; Narog, D.; Sobkowiak, A.; Morzycki, J.W.
3α,5α-Cyclocholestan-6β-yl ethers as donors of the cholesterol moiety for the electrochemical synthesis of
cholesterol glycoconjugates. Beilstein J. Org. Chem. 2015, 11, 162–168. [CrossRef] [PubMed]
93. Davis, R.A.; Fettinger, J.C.; Gervay-Hague, J. Tandem Glycosyl Iodide Glycosylation and Regioselective
Enzymatic Acylation Affords 6-O-Tetradecanoyl-α-D-cholesterylglycosides. J. Org. Chem. 2014, 79, 8447–8452.
[CrossRef] [PubMed]
94. Mao, R.-Z.; Guo, F.; Xiong, D.-C.; Li, Q.; Duan, J.; Ye, X.-S. Photoinduced C-S Bond Cleavage of Thioglycosides
and Glycosylation. Org. Lett. 2015, 17, 5606–5609. [CrossRef] [PubMed]
95. Davis, R.A.; Fettinger, J.C.; Gervay-Hague, J. Synthesis of cholesteryl-α-D-lactoside via generation and
trapping of a stable β-lactosyl iodide. Tetrahedron Lett. 2015, 56, 3690–3694. [CrossRef] [PubMed]
96. Weiss, S.; Neu, P.M.; Ludwig, C.; Schober, S.; Mittelbach, M. Novel Method for the Synthesis of Cholesteryl
Glucosides starting from Disaccharides. Eur. J. Lipid Sci. Technol. 2018, 120, 1700389. [CrossRef]
97. Algay, V.; O’Sullivan, J.; Heaney, F. C-3β-Tethered Functional Cholesterol Conjugates by Nitrile Oxide Alkyne
Cycloaddition (NOAC). Eur. J. Org. Chem. 2014, 2014, 2522–2532. [CrossRef]
98. Alarcon-Manjarrez, C.; Arcos-Ramos, R.; Alamo, M.F.; Iglesias-Arteaga, M.A. Synthesis, NMR and crystal
characterization of dimeric terephthalates derived from epimeric 4,5-seco-cholest-3-yn-5-ols. Steroids 2016,
109, 66–72. [CrossRef] [PubMed]
99. Shibuya, K.; Watanabe, T.; Urano, Y.; Takabe, W.; Noguchi, N.; Kitagishi, H. Synthesis of
24(S)-hydroxycholesterol esters responsible for the induction of neuronal cell death. Bioorg. Med. Chem. 2016,
24, 2559–2566. [CrossRef]
100. Sarkar, A.; Das, J.; Ghosh, P. p-TsOH-Catalyzed one-pot transformation of di- and trihydroxy steroids towards
diverse A/B-ring oxo-functionalization. New J. Chem. 2017, 41, 9051–9060. [CrossRef]
101. Borthakur, M.; Barthakur, M.G.; Boruah, R.C. Microwave promoted one-pot synthesis of novel A-ring fused
steroidal dehydropiperazines. Steroids 2008, 73, 539–542. [CrossRef] [PubMed]
102. Ansari, A.; Ali, A.; Asif, M. Microwave-assisted MgO NP catalyzed one-pot multicomponent synthesis of
polysubstituted steroidal pyridines. New J. Chem. 2018, 42, 184–197. [CrossRef]
103. Barthakur, M.G.; Gogoi, S.; Dutta, M.; Boruah, R.C. A facile three-component solid phase synthesis of
steroidal A-ring fused pyrimidines under microwave irradiation. Steroids 2009, 74, 730–734. [CrossRef]
[PubMed]
104. Dutta, M.; Saikia, P.; Gogoi, S.; Boruah, R.C. Microwave-promoted and Lewis acid catalysed synthesis of
steroidal A- and D-ring fused 4,6-diarylpyridines. Steroids 2013, 78, 387–395. [CrossRef] [PubMed]
Molecules 2019, 24, 116 68 of 68

105. Schulze, M.; Scott, D.E.; Scherer, A.; Hampel, F.; Hamilton, R.J.; Gray, M.R.; Tykwinski, R.R.; Stryker, J.M.
Steroid-Derived Naphthoquinoline Asphaltene Model Compounds: Hydriodic Acid Is the Active Catalyst
in I2 -Promoted Multicomponent Cyclocondensation Reactions. Org. Lett. 2015, 17, 5930–5933. [CrossRef]
[PubMed]
106. Berg, M.; Nozinovic, S.; Engeser, M.; Lützen, A. A Cholesterol Containing pH-Sensitive Bistable [2]Rotaxane.
Eur. J. Org. Chem. 2015, 2015, 5966–5978. [CrossRef]
107. Venkataraman, S.; Mineart, K.P.; Prabhu, V.M.; Hedrick, J.L.; Yang, Y.Y. Cholesterol functionalized aliphatic
N-substituted 8-membered cyclic carbonate. Polym. Chem. 2018, 9, 2434–2437. [CrossRef]
108. Liu, H.; Wang, R.; Wei, J.; Cheng, C.; Zheng, Y.; Pan, Y.; He, X.; Ding, M.; Tan, H.; Fu, Q.
Conformation-Directed Micelle-to-Vesicle Transition of Cholesterol-Decorated Polypeptide Triggered by
Oxidation. J. Am. Chem. Soc. 2018, 140, 6604–6610. [CrossRef] [PubMed]
109. Kozanecka, W.; Mrowczynska, L.; Pospieszny, T.; Jasiewicz, B.; Gierszewski, M. Synthesis, spectroscopy,
theoretical and biological studies of new gramine-steroids salts and conjugates. Steroids 2015, 98, 92–99.
[CrossRef] [PubMed]
110. Schwarzenböck, C.; Schaffer, A.; Pahl, P.; Nelson, P.J.; Huss, R.; Rieger, B. Precise synthesis of
thermoresponsive polyvinylphosphonate-biomolecule conjugates via thiol–ene click chemistry. Polym. Chem.
2018, 9, 284–290. [CrossRef]
111. Tao, Y.; Ma, X.; Cai, Y.; Liu, L.; Zhao, H. Coassembly of Lysozyme and Amphiphilic Biomolecules Driven by
Unimer–Aggregate Equilibrium. J. Phys. Chem. B 2018, 122, 3900–3907. [CrossRef] [PubMed]

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).