Article

Design, Synthesis, and Molecular Docking Study of

Novel Heterocycles Incorporating 1,3,4-Thiadiazole
Moiety as Potential Antimicrobial and Anticancer
Agents
Mohamed El-Naggar 1, Hanan A. Sallam 2, Safaa S. Shaban 2, Salwa S. Abdel-Wahab 3,
Abd El-Galil E. Amr 4,5,*, Mohammad E. Azab 2, Eman S. Nossier 6 and Mohamed A. Al-Omar 4
1 Chemistry Department, Faculty of Sciences, University of Sharjah, Sharjah 27272, UAE;
m5elnaggar@yahoo.com
2 Synthetic Organic Laboratory, Chemistry Department, Faculty of Science, Ain Shams University,

Cairo 11566, Egypt, dr.salam.h.a@gmail.com (H.A.S.); safashaban@ymail.com (S.S.S.);

meazabali2015@yahoo.com (M.E.A.)
3 Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt,

New Cairo 11835, Egypt; salwa_elsayed@ymail.com

4 Pharmaceutical Chemistry Department, Drug Exploration and Development Chair (DEDC),

College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia; malomar1@ksu.edu.sa
5 Applied Organic Chemistry Department, National Research Centre, Cairo 12622, Egypt

6 Pharmaceutical Medicinal Chemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University,

Cairo 11754, Egypt; dr.emannossier@gmail.com

* Correspondence: aamr@ksu.edu.sa; Tel.: +966-543-074-312

Received: 22 February 2019; Accepted: 13 March 2019; Published: 18 March 2019

Abstract: A new series of 5-(3,5-dinitrophenyl)-1,3,4-thiadiazole derivatives were prepared and

evaluated for their in vitro antimicrobial, antitumor, and DHFR inhibition activity. Compounds 9,
10, 13, and 16 showed strong and broad-spectrum antimicrobial activity comparable to Amoxicillin
and Fluconazole as positive antibiotic and antifungal controls, respectively. Compounds 6, 14, and
15 exhibited antitumor activity against four human cancer cell lines, CCRF-CEM leukemia, HCT-15
colon, PC-3 prostate, and UACC-257 melanoma cell lines using Doxorubicin as a reference drug.
Compounds 10, 13, 14, and 15 proved to be the most active DHFR inhibitors with an IC50 range of
0.04 ± 0.82–1.00 ± 0.85 µM, in comparison with Methotrexate (IC50 = 0.14 ± 1.38 µM). The highly
potent DHFR inhibitors shared a similar molecular docking mode and made a critical hydrogen
bond and arene‒arene interactions via Ser59 and Phe31 amino acid residues, respectively.

Keywords: 1,3,4-thiadiazole; antimicrobial; anticancer; dihydrofolate reductase; molecular docking

1. Introduction
Dihydrofolate reductase (DHFR) is a prevalent enzyme that is present in all prokaryotic and
eukaryotic cells. It has a key role in folate metabolism and subsequently DNA and RNA synthesis
[1,2]. Inhibition of DHFR revealed importance in the development of therapeutic agents against
anticancer drugs as well as bacterial and parasitic infections [3–7]. DHFR inhibitors exhibit a vital
role in clinical medicine, like the use of Methotrexate in neoplastic diseases, inflammatory bowel
diseases, rheumatoid arthritis, psoriasis, and asthma [8].
1,3,4-Thiadiazole is a privileged five-membered ring system that has gained prominence by
exploring broad biological activity spectrum due to the presence of the N=C‒S moiety. From the
literature survey, it was noticed that 1,3,4-thiadiazole derivatives possess many pharmacological
activities, such as antimicrobial, anti-hepatitis B viral, antitubercular, antileishmanial, analgesic,

Molecules 2019, 24, 1066; doi:10.3390/molecules24061066 www.mdpi.com/journal/molecules

Molecules 2019, 24, 1066 2 of 21

anti-inflammatory, anticancer, anticonvulsant, central nervous system (CNS) depressant,

antioxidant, molluscicidal, antidiabetic, diuretic, and antihypertensive activities [9–18].
Furthermore, many drugs containing a 1,3,4-thiadiazole nucleus have been approved, such as
methazolamide, which is used as a carbonic anhydrase inhibitor, and acetazolamide, which is a
modulator of anticancer therapies in combination with different cytotoxic agents [19,20]. Other
thiadiazole-containing drugs include Cefazedone and Cefazolin sodium, which have
broad-spectrum antibiotic action [21] (Figure 1).
H
N O O N N O Cl N S
O N N
S NH2 S NH2
N S N S O N S N
O H O O O N
Cl CO2H S
Methazolamide Acetazolamide Cefazedone

H
N S
N
N N O N S N
N O N
S
O O Na

Cefazolin sodium

Figure 1. Some drugs on the market possessing a 1,3,4-thiadiazole scaffold.

Recently, some pharmacophores containing 1,3,4-thiadiazoles have been explored for their
potential antitumor and antimicrobial activities via inhibition of DHFR. For example, 2-hydrazono-
1,3,4-thiadiazole I was reported to possess promising antitumor and antimicrobial activities [22].
Moreover, compound II belongs to the 3-(4-nitrophenyl)-5-(thiophen-2-yl)-2,3-dihydro-1,3,4-
thiadiazole skeleton, has high anticancer potency comparable to Cisplatin, and was allocated with
DHFR inhibition [23].
Considering the findings above and as a continuation of our efforts towards the development of
biologically active heterocyclic compounds [24–34], we undertook the design and synthesis of some
novel 1,3,4-thiadiazole prototypes that possess the advantages of pharmacophores (I, II), as outlined
in Figure 2. All the newly hybrid compounds comprising the 1,3,4-thiadiazole motif were evaluated
for their antimicrobial and anticancer activities through a study of their in vitro inhibitory activity
enzyme against DHFR enzyme, followed by molecular docking studies to get insight into the
interactions and binding modes in the active site of this enzyme.
Molecules 2019, 24, 1066 3 of 21

CH3

H
N
S
O O
N N
N
S N
CH3
H3CO
I

DHFR inhibitor, anticancer, antimicrobial

NO2

N N
N
S N Cl
S
II

DHFR inhibitor, anticancer

Structural modification

O 2N N N

O2N
2-20

Figure 2. Structures of previously reported 1,3,4-thiadiazoles I, II with DHFR inhibitory activity and
the strategy employed for designing the lead compounds 2–20.

2. Results and Discussion

2.1. Chemistry
In this study, compound 5-(3,5-dinitrophenyl)-1,3,4-thiadiazol-2-amine (1) was used as the key
starting material to synthesize different heterocyclic compounds.
Thus, when compound 1 was treated with carbon disulfide and potassium hydroxide, followed
by stirring with hydrazine hydrate, thiosemicarbazide derivative 2 was produced.
Compound 2 was used as a precursor to construct different heterocyclic ring systems such as
thiadiazole, thiazole, and pyrimidine through the reaction with different reagents.
Consequently, compound 2 was treated with phenylalanine/pyridine, benzoic acid/POCl3,
and/or CS2/NaOH to give 1,3,4-thiadiazol-2-yl-1,3,4-thiadiazole derivatives 3–5 in a good yield.
Reacting compound 5 with phenacyl bromide in the presence of potassium carbonate produced the
acetophenone derivative 6 (Scheme 1).
Molecules 2019, 24, 1066 4 of 21

Scheme 1. Synthesis route for compounds 2–6.

Compound 3 was formed via a nucleophilic attack of the NH2 group of thiosemicarbazide on
the carbonyl group of phenylalanine through tetrahedral mechanism, followed by elimination of the
water molecule; then ring closure takes place via the elimination of another water molecule from the
thiol and OH of the carboxylic group. However, in the case of compound 4 the carboxylic group is
converted into acid chloride and as a result a thiadiazole ring is formed by the elimination of water
and HCl molecules. In compound 5, a nucleophilic attack of NH2 group of thiosemicarbazide on the
C=S group was followed by ring closure through the elimination of the H2S molecule.
The structures of the products were in accordance with their elemental analysis and spectral
data, where the IR spectrum of compound 3 showed bands at 3415-3365, 3257, 2958, and 2924, for the
NH2, NH, CH2, and CH groups, respectively. Its 1H-NMR spectrum exhibited peaks at δ 3.17, 3.31
(m, 2H, CH2-Ph), 4.08 (t, 1H, CH), 6.45 (s, 2H, NH2), and 11.21 (s, 1H, NH).
As proof of the proposed structure of compound 4, its IR spectrum displayed a stretching
frequency of 3265 (NH) and the 1H-NMR spectrum had peaks at δ 7.33–7.66 (m, 5H, Ph-H) and 10.22
(s, 1H, NH). For compound 5, the IR spectrum showed two characteristic absorption bands at 3261
(NH) and 2561 (SH), which indicates the presence of the compound in the thiol form, besides the
1H-NMR peaks at δ 10.13 and 12.44 corresponding to the NH and SH groups. The IR spectrum of
compound 6 lacked the band of the SH group and showed bands at 3269 (NH) and 2921 (CHaliph.),
and the 1H-NMR spectrum displayed peaks at δ 4.96 (s, 2H, S‒CH2‒CO), 7.37–7.69 (m, 5H, Ph-H) and
10.29 (s, 1H, NH).
Furthermore, thiosemicarbazide 2 reacted with bis-methylthiomethylene-barbituric acid (7),
chloroacetic acid/sodium acetate, and/or malonic acid in the presence of acetyl chloride to give
barbituric, thiazolidin-4-one, and thiobarbituric derivatives 8–10, respectively (Scheme 2).
Molecules 2019, 24, 1066 5 of 21

Scheme 2. Synthesis route for compounds 8–10.

The mechanism for the formation of compound 8 could be as follows: first, an Michael addition
reaction between the NH2 of the thiosemicarbazide 2 and the bis-methyl-thiomethylene barbituric
acid (7), resulting in the elimination of the CH3SH group to give intermediate (A), which underwent
intramolecular cyclization through the elimination of the second CH3SH group to furnish the
pyrimidine-2,4–6-trione derivative 8.
The structure of compound 8 was elucidated from its elemental analysis and spectral data. The
IR spectrum revealed the following bands: 3322–3189 (NH groups) and 1744, 1678, and 1655 (C=O
groups). The 1H-NMR spectrum exhibited four peaks at δ 9.34, 11.19, 12.67, and 12.78 (4s, 4H, 4NH).
Compound 9 could be formed via nucleophilic attack of the nitrogen atom of thiosemicarbazide
on the carbonyl group of chloroacetic acid with elimination of the H2O molecule; then ring closure
takes place through nucleophilic attack of the sulfur atom (in the thiol form) on the carbon atom
(attached to Cl), synchronized with elimination of the HCl molecule.
The spectral data were in accordance with the proposed structure of compound 9, where the IR
spectrum showed characteristic stretching frequencies at 3378, 3254 (NH2), and 1681 (C=O), while
the 1HNMR spectra displayed peaks at δ 3.86 (s, 2H, C5 thiazolidine) and 6.41 (s, 2H, NH2).
The attack of two NH groups of thiosemicarbazide on the two carbonyl groups of malonic acid
resulted in ring closure with elimination of two water molecules to produce compound 10,
accompanied by acylation of the NH2 group.
Meanwhile, compound 1 was allowed to react with phenylisothiocyante in dioxane to produce
the thiadiazole derivative 11, which may be formed as a result of the nucleophilic attack of the NH2
on the carbon atom of the isothiocyant derivative (Scheme 3).
Molecules 2019, 24, 1066 6 of 21

Ph Ph
N N N PhCOCH2Br/TEA
O2N
S N S

(12)
NO2

N N S
N N
PhNCS/dioxane O2N Ph
O2N S NH NH
S NH2

(1) NO2 (11)

NO2

CH2(CO2H)2/
AcCl

N N S
N N S
O2N Ph O2N Ph
PhN2Cl/AcONa S N N
S N N

O O O O

NO2 N NO2
(14) (13)
NHPh

N N S
O2N Ph PhCHO/piperidine
S N N

O O
NO2
(15) Ph

Scheme 3. Synthesis route for compounds 11–15.

Compound 11 was supported by its spectral data: its IR spectrum displayed stretching
absorption bands at 3329 and 3256, corresponding to two NH groups. At the same time, 1H-NMR
revealed peaks at δ 10.49, 11.33 (2s, 2H, 2NH), and δ 7.45–7.89 (m, 5H, Ar-H).
The reactivity of compound 11 was explored through the reaction with phenacyl bromide in the
presence of triethylamine as a catalyst, and/or malonic acid/acetyl chloride to give thiazole and
thiobarbituric derivatives 12 and 13, respectively (Scheme 3).
The formation of compound 12 could be via a nucleophilic attack of NH on the carbonyl group,
resulting in the elimination of a water molecule; then, the sulfur in the thiol form attacks the
methylene group, leading to ring closure, accompanied by elimination of HBr molecule to produce
the thiazole derivative 12.
The spectral data of compound 13 showed the following bands in the IR spectrum: 2961
(CHaliph), and 1702, 1688 (C=O), and 1H-NMR displayed peaks at δ 3.31 (s, 2H, C5 pyrimidine) and
7.32–7.88 (m, 5H, Ar‒H).
Compound 13 was coupled with benzene diazonium chloride in the presence of sodium acetate
and/or condensation with benzaldehyde in the presence of piperidine to produce phenyl hydrazone
and thiadiazole derivatives 14 and 15, respectively (Scheme 3).
The IR spectrum of 14 exhibited a stretching frequency band at 3276, corresponding to the NH
group, and its 1H-NMR spectrum revealed peaks at δ 7.25–7.69 (m, 10H, Ar-H) and 12.55 (s, 1H, NH),
while the 1H-NMR of 15 showed peaks at δ 6.84 (s,1H, olefinic proton) and 7.41–7.77 (m, 10H, Ar-H).
The present investigation was extended to demonstrate the reactivity of dimethyl
carbon-imidodithioate derivative 16 towards some amine derivatives in order to synthesize different
heterocyclic systems. Compound 16 was prepared through the treatment of the 2-aminothiadiazole
derivative 1 with carbon disulfide in a basic medium followed by S-methylation using methyl
iodide.
When compound 16 was allowed to react with amines, namely o-aminophenol,
o-amino-thiophenol, o-phenylenediamine, and/or ethylene diamine in dimethylformamide,
Molecules 2019, 24, 1066 7 of 21

benzo-oxazole, benzothiazole, benzoimidazole, and dihydroimidazole derivatives 17–20 were

produced, respectively (Scheme 4).

Scheme 4. Synthesis route for compounds 16–20.

The formation of compounds 17–20 was proposed to take place via the nucleophilic attack of
the amino and HX groups on the carbon atom of thioacetal followed by elimination of two MeSH
molecules. This can be shown as a speculated mechanistic pathway in Scheme 5.

N N H N N N
S Me -MeSH NH
O2N H O2N
S N S N
X SMe
SMe
16 H (A) X
NO2 NO2
X = O , S, NH H
-MeSH

N N N N
N HN
O2N O 2N
S N S N
H X X

NO2 17-20 NO2

Scheme 5. Speculated mechanism for the formation of compounds 17–20.

The elemental analyses and spectral data were in accordance with the proposed structures of
compounds 17–20. The IR spectra showed stretching absorption bands at 3389–3272 for NH groups.
Also, the 1H-NMR spectra of compounds 17–19 showed peaks at δ 7.17–7.51 (m, 4H, Ar-H), 9.78 (s,
1H, NH), 7.36–7.67 (m, 4H, Ar-H), 11.06 (s, 1H, NH), 7.11–7.43 (m, 4H, Ar-H), 9.96 (s, 1H, NH) and
11.78 (s, 1H, NH), respectively, while the 1H-NMR spectrum of compound 20 exhibited peaks at δ
2.81 (t, 2H, CH2-imidazole), 2.69 (t, 2H, CH2-imidazole), 9.83 (s, 1H, NH), and 10.66 (s, 1H, NH),
respectively.
Molecules 2019, 24, 1066 8 of 21

2.2. Biological Evaluation

2.2.1. Antimicrobial Sensitivity Assay

All the synthesized compounds 1–20 were screened for their antimicrobial activity against
Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis), Gram-negative bacteria
(Pseudomonas aeruginosa and Escherichia coli), and yeast-like pathogenic fungi (Aspergillus niger and
Candida albicans). The antimicrobial screening was carried out using a standard well agar diffusion
assay according to Cheesbrough et al. [35]. The broad-spectrum antibiotic Amoxicillin and the
antifungal Fluconazole were used at a concentration of 100 µg/mL as positive controls. The obtained
results (Table 1) revealed that the tested compounds 9, 10, 13, and 16 showed the highest
antimicrobial activity. Compound 13 is the most potent one since it demonstrated antimicrobial
activity higher than that of the standard drugs (Figures 3 and 4). The rest of the tested compounds
had antimicrobial activity that ranged from moderate to weak.

Table 1. In vitro antimicrobial activities of the tested compounds 1–20 at 100 µg/mL and expressed as
inhibition zone diameter (mm).

Diameter of Inhibition Zone (mm)

Gram Positive Bacteria Gram Negative Bacteria Fungi
Compd. No. Staphylococcus Bacillus Pseudomonas Escherichia Aspergillus Candida
aureus subtilis aeruginosa coli niger albicans
1 12 10 14 8 6 9
2 25 24 22 23 17 18
3 0 0 0 2 0 0
4 0 0 2 3 0 0
5 20 19 20 21 16 18
6 10 11 13 12 9 8
8 9 7 8 10 8 9
9 27 28 26 26 21 23
10 28 29 27 27 22 24
11 13 14 16 15 11 13
12 0 2 0 2 0 0
13 30 29 28 27 23 25
14 18 13 11 9 8 12
15 16 19 13 10 6 15
16 28 26 25 27 22 22
17 8 8 6 9 7 6
18 4 3 0 3 2 3
19 5 0 6 4 3 4
20 21 18 20 22 16 18
Amoxicillin 28 27 24 25 - -
Fluconazole - - - - 20 22
Molecules 2019, 24, 1066 9 of 21

Figure 3. Antimicrobial activity of the most potent compounds against different bacterial strains.

Figure 4. Antifungal activity of the most potent compounds against different fungal strains.

2.2.2. Structure–Activity Relationship for Antimicrobial Activity

By analysis of the previous results and concerning the structural modifications that occurred
only at position-2 (p-2) of the parent 1,3,4-thiadiazole scaffold, it was found that: a 1,3,4-Thiadiazole
derivative 1 having free amino group at p-2 showed moderate inhibitory activity against all tested
strains. Substitution of NH2 group with thiocarbohydrazide in compound 2 produced excellent
activity, while its phenyl congener 11 displayed moderate activity. Furthermore, substitution of the
amino group with dimethyl carbonimidodithioate in compound 16 exhibited high inhibitory activity
and was approximately equipotent with the reference.
The existence of a new 1,3,4-thiadiazole ring via the NH linker revealed various levels of
antimicrobial activity:
 a drop in the potency, illustrated in substitution at p-2 of the new 1,3,4-thiadiazole moiety with
1-amino-2-phenylethyl in compound 3 or with phenyl in compound 4;
 elevated potency if the same position in the new 1,3,4-thiadiazole moiety is substituted with a
free thioxo group in compound 5; and
 moderate activity but only about half that of compound 5 if the thioxo group is substituted with
phenylethane-1-one in compound 6, or p-2 is linked to pyrimidine-2,4,6-trione scaffold in
compound 8.
Direct attachment of the thiazole moiety achieved excellent potency in compound 9, while
attachment via an imine group led to a drop in activity in compound 12.
Moreover, direct attachment of a thioxopyrimidine-4,6-dione scaffold led to excellent activity
with substitution at N-1 either with phenyl as in 13 or with acetyl acetamide as in 10. It was noted
that insertion of 2-phenyl hydrazone in compound 14 or benzylidine in compound 15, to p-5 of the
newly formed 2-thioxopyrimidine-4,6-dione of 10 and 13, decreased the activity to a moderate level.
Upon attachment of an imidazole moiety via a NH linker there was significant antimicrobial
activity in compound 20, followed by a drastic drop due to cyclization forming benzo[d]imidazole
derivative 19 and its congeners benzo[d]oxazole and benzo[d]thiazole derivatives 17, 18,
respectively.

2.2.3. In Vitro Anticancer Activity

All synthesized compounds 1–20 were investigated for their in vitro cytotoxic activity against
four human carcinoma cell lines, CCRF-CEM, HCT-15, PC-3, and UACC-257, using Doxorubicin as a
standard anticancer drug. The anticancer activity was expressed as IC50 values (the concentration of
test compounds required to kill 50% of the cell population) in (µM) ± SEM from three replicates.
The results depicted in Table 2 revealed that the most potent compounds are 15 > 14 > 6, in
descending order, against all tested cell lines. Compounds 8 and 12 exhibited moderate activity
Molecules 2019, 24, 1066 10 of 21

about half that of the reference drug. The rest of the tested compounds showed weak cytotoxic
activity.

Table 2. In vitro cytotoxic activity of the tested compounds 1–20 against different human cancer cell
lines.

Compound IC50 (Mean ± SEM) (µM) a

Number CCRF-CEM HCT-15 PC-3 UACC-257 MCF-10A
DOX b 6.78 ± 0.7 5.17 ± 0.2 4.56 ± 0.4 7.34 ± 0.5 377.33 ± 2.55
1 90.7 ± 1.2 79.8 ± 2.6 88.2 ± 1.6 81.6 ± 1.1 497.54 ± 5.44
2 74.56 ± 3.8 67.20 ± 2.9 70.18 ± 3.2 71.20 ± 3.9 586.43 ± 4.53
3 51.88 ± 1.6 30.12 ± 1.8 27.24 ± 1.2 46.44 ± 1.1 464.34 ± 3.47
4 39.28 ± 2.6 32.75 ± 2.2 29.52 ± 1.5 36.33 ± 2.4 383.63 ± 2.56
5 77.74 ± 2.6 65.66 ± 3.3 82.55 ± 1.9 80.66 ± 2.2 252.74 ± 2.45
6 10.99 ± 0.3 9.87 ± 0.5 9.92 ± 1.2 12.67 ± 0.6 461.85 ± 5.58
8 19.19 ± 0.5 17.24 ± 0.9 15.44 ± 1.4 22.66 ± 1.3 373.96 ± 6.49
9 76.22 ± 2.6 69.65 ± 2.7 81.59 ± 1.3 83.33 ± 2.9 262.07 ± 4.50
10 40.44 ± 1.9 36.76 ± 1.8 33.88 ± 1.1 38.55 ± 2.1 351.89 ± 6.68
11 71.26 ± 3.2 63.27 ± 2.3 66.14 ± 3.6 73.34 ± 3.5 380.68 ± 5.57
12 15.78 ± 0.8 15.01 ± 0.5 12.14 ± 1.1 18.38 ± 1.6 373.47 ± 6.45
13 49.36 ± 1.2 52.72 ± 1.2 48.88 ± 2.1 55.55 ± 0.9 460.56 ± 8.64
14 9.66 ± 0.6 8.59 ± 0.7 6.99 ± 1.2 9.20 ± 0.8 474.44 ± 8.72
15 6.99 ± 0.4 5.28 ± 0.5 4.67 ± 0.3 7.41 ± 0.5 352.35 ± 7.53
16 79.32 ± 3.1 68.33 ± 4.3 80.59 ± 1.7 76.61 ± 1.7 473.26 ± 8.44
17 67.45 ± 3.4 61.27 ± 2.3 62.56 ± 2.6 70.77 ± 1.5 382.35 ± 7.35
18 58.58 ± 4.4 55.67 ± 1.6 61.21 ± 1.1 65.34 ± 2.2 491.24 ± 9.53
19 84.44 ± 2.2 69.87 ± 3.3 86.95 ± 1.5 83.54 ± 2.9 382.32 ± 8.72
20 83.34 ± 4.2 71.64 ± 4.1 92.34 ± 4.2 88.64 ± 4.1 473.23 ± 9.83
aIC50, compound concentration required to inhibit tumor cell proliferation by 50%, b positive control,
SEM = standard error mean; each value is the mean of three values.

Comparing the IC50 values obtained for the synthesized derivatives against CCRF-CEM,
HCT-15, PC-3, and UACC-257 with those obtained against non-tumorigenic MCF-10A cells, we can
conclude that the synthesized derivatives have much less toxicity against normal cells.

2.2.4. Structure–Activity Relationship for Anticancer Activity

Regarding the chemical structural modifications at p-2 of the parent 1,3,4-thiadiazole moiety, it
was observed that: incorporation of 2-thioxopyrimidine-4,6-dione having benzylidene group at p-5
in compound 15 gave the highest cytotoxic activity (IC50 = 6.99 ± 0.4, 5.28 ± 0.5, 4.67 ± 0.3 and 7.41 ±
0.5µM, respectively), which is equipotent to the reference drug (IC50 = 6.78 ± 0.7, 5.17 ± 0.2, 4.56 ± 0.4
and 7.34 ± 0.5 µM, respectively). Alteration of benzylidene with 2-phenyl hydrazone in compound
14 maintained high potency but slightly less than compound 15 (IC50 = 9.66 ± 0.6, 8.59 ± 0.7, 6.99 ± 1.2
and 9.20 ± 0.8 µM, respectively).
Also, increased potency was obtained upon addition of a new 1,3,4-thiadiazole ring substituted
with thio-1-phenylethan-1-one group via a NH linker in compound 6 (IC50 = 10.99 ± 0.3, 9.87 ± 0.5,
9.92 ± 1.2 and 12.67 ± 0.6 µM, respectively).
On the other hand, moderate activity was caused by the attachment of new 1,3,4-thiadiazole
ring bearing pyrimidine-2,4,6-trione group via a NH linker in compound 8 (IC50 = 19.19 ± 0.5, 17.24 ±
0.9, 15.44 ± 1.4 and 22.66 ± 1.3 µM, respectively) or by insertion of a 3,4-diphenylthiazole scaffold via
an imine linker in compound 12 (IC50 = 15.78 ± 0.8, 15.01 ± 0.5, 12.14 ± 1.1 and 18.38 ± 1.6 µM,
respectively).

2.2.5. Dihydrofolate Reductase (DHFR) Inhibition

The synthesized compounds 1–20 were evaluated as inhibitors of bovine liver DHFR using a
reported procedure [36]. Results were summarized as IC50 values in Table 3 and Methotrexate was
used as a positive control. As illustrated in Table 3, compounds 10, 13, 14, and 15 proved to be the
most active inhibitors with an IC50 range from 0.04 ± 0.82 to 1.00 ± 0.85 µM, in comparison with
Molecules 2019, 24, 1066 11 of 21

Methotrexate (IC50 = 0.14 ± 1.38 µM). However, the rest of the tested compounds showed moderate
to weak inhibitory activity, with an IC50 range of 8.46 ± 0.13–36.48 ± 0.72 µM.

Table 3. In vitro inhibitory activities of the tested compounds 1–20 against DHFR enzyme.

Compound IC50 (Mean ± SEM) (µM)

Number DHFR
1 17.26 ± 0.43
2 14.33 ± 0.81
3 28.54 ± 0.22
4 31.57 ± 0.30
5 15.38 ± 0.12
6 13.45 ± 0.23
8 20.54 ± 0.14
9 8.46 ± 0.13
10 1.00 ± 0.85
11 17.13 ± 0.90
12 36.48 ± 0.72
13 0.09 ± 0.91
14 0.08± 0.37
15 0.04 ± 0.82
16 10.24 ± 0.97
17 27.42 ± 0.35
18 24.71 ± 1.26
19 29.00 ± 1.25
20 11.80 ± 0.79
Methotrexate 0.14± 1.38
IC50: Compound concentration required to inhibit DHFR enzyme activity by 50%, SEM = standard
error mean; each value is the mean of three values.

The inhibitory activity of the tested derivatives could be correlated to structure variation and
modifications. By investigating the variation in selectivity of the highly potent compounds 10, 13, 14,
and 15 over the DHFR enzyme, it was revealed that the existence of thioxopyrimidine-4,6-dione
moiety at p-2 of the parent 1,3,4-thiadiazole scaffold led to enhanced activity, and the potency order
was 15 > 14 > 13 > 10. Structure–activity relationships in these compounds demonstrated that
compounds with substitution at p-5 of the newly formed 2-thioxopyrimidine-4,6-dione (14, 15, with
excellent anticancer activity) showed more potent inhibitory activity against the DHFR enzyme than
those having no substituents (10, 13, with excellent antimicrobial activity).

2.3. Molecular Modeling Studies

To gain a better understanding of the potency of the studied compounds and guide further SAR
studies, we proceeded to examine the interaction of compounds 10, 13, 14, and 15 via the X-ray
crystallographic structure of DHFR (PDB ID: 1DLS) [37]. The co-crystallized ligand Methotrexate
was redocked into the pocket sites of DHFR and revealed docking score energies of −11.4 kcal/mol at
a root mean square deviation (RMDS) value of 9.1. The molecular docking was performed by
inserting compounds 10, 13, 14, and 15 into the ATP binding site of DHFR. All docking runs applied
the LigandFit Dock protocol of Molecular Operating Environment (MOE, 10.2008) software [38,39].
The docking scores for compounds 10, 13, 14, and 15 were all in the range −15.6 to −12.3 kcal/mol.
Representations of the docking results of these compounds and DHFR are given in Figures 5 and 6.
Inspection of the binding modes demonstrated that all compounds were potently bound to the
ATP binding site of DHFR via arene‒arene interaction between the centroids of Phe31 and
3,5-dinitrophenyl moiety, and a hydrogen bond acceptor between the sidechain of Ser59 and one
oxygen of thioxopyrimidine-4,6-dione.
Furthermore, compound 10 was stabilized by another hydrogen bond acceptor between the
oxygen of acetamide moiety and the side chain of Tyr121 (distance: 2.95 Å). In compound 14, a dual
Molecules 2019, 24, 1066 12 of 21

hydrogen bonding network was involved between the nitrogen atom and the NH proton of the
phenylhydrazone moiety, and the side chains of Ser59 and Thr56, respectively (distance: 2.39 and
2.42 Å, respectively). The other oxygen of thioxopyrimidine-4,6-dione in compound 15 mediated a
strong hydrogen bond acceptor with the side chains of Thr56 (distance: 2.49 Å) (Figure 5).

A B

C D

Figure 5. Two-dimensional representation of the interactions of compounds 10 (A), 13 (B), 14 (C),

and 15 (D) with the DHFR enzyme pocket amino acids.

From the docking results, it is evident that the thioxopyrimidine-4,6-dione moiety linked to the
1,3,4-thiadiazole scaffold contributes to the activity of compounds 10, 13, 14, and 15 through
H-bonds to Ser59, which considerably strengthens the binding interaction (Figure 6).

A B
Molecules 2019, 24, 1066 13 of 21

C D

Figure 6. Three-dimensional representation of the interactions of compounds 10 (A), 13 (B), 14 (C),

and 15 (D) with the DHFR enzyme pocket amino acids.

3. Materials and Methods

3.1. Chemistry
All solvents, reagents, and chemicals were obtained from Alfa Aesar (Ward Hill, MA, USA) and
Sigma-Aldrich (St. Louis, MO, USA). All melting points are not corrected and were measured on a
Stuart SMP 30 advanced digital electric melting point apparatus (Cole-Parmer, Staffordshire, UK).
Infrared spectra were recorded on a Shimadzu FT-IR 8300 E (Shimadzu Corporation, Kyoto, Japan),
using KBr discs and are reported as ν cm−1. A Bruker Avance III spectrometer (Bruker Corporation,
Rheinstetten, Germany) was used to record 1H-NMR spectra at 400 MHz using TMS as an internal
standard and DMSO-d6 as a solvent. 13C-NMR spectra were recorded on the same spectrometer at
100 MHz using the same solvent. MS spectra were measured on a Shimadzu GC-MS-QP-1000 EX
mass spectrometer instrument operating at 70 eV. The elemental analyses of the new compounds
were recorded on a Perkin-Elmer CHN-2400 analyzer (Waltham, MA, USA) and carried out at the
Microanalytical Centre, Cairo University, Cairo, Egypt. The microanalysis showed that the observed
values were within ±0.4% of theoretical values. The homogeneity of the compounds and the progress
of the chemical reactions were monitored by TLC silica gel plates (60F254, Merck, Munchen,
Germany). The biological evaluation was conducted at the Department of Pharmacology, Faculty of
Pharmacy, Mansoura University, Egypt. Compound 1 [40] was prepared using a previously
reported method [41] (m.p. 217–219 °C, Let. 215 °C).

3.1.1. N-(5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl)hydrazinecarbothioamide (2)

To a solution of compound 1 (2.67 g, 0.01 mol), in THF (10 mL), potassium hydroxide (2.5 g) in
tetrahydrofuran (10 mL) was added portion-wise with shaking. To the reaction mixture, carbon
disulfide (1.2 mL, 0.02 mol) was added with stirring for 1 h in an ice bath at 5 °C, followed by the
addition of hydrazine hydrate (0.98 mL, 0.02 mol) with stirring for 1 h at 60 °C. The reaction mixture
was concentrated under a vacuum, and the obtained solid was filtered off and crystalized from
dioxane to produce the thiosemicarbazide derivative 2. Yield: 87%, m.p: 260–262 °C, IR (KBr, cm−1): ν =
3430–3273 (NH2, NH), 3083 (C‒Harom), 1629 (C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ = 5.33 (s, 2H,
NH2, D2O exchangeable), 8.79, 9.13, 9.31 (3s, 3H, Ar-H), 11.11, 11.67 (2s, 2H, 2 NH, D2O exchangeable).
13C-NMR (100 MHz, ppm, DMSO-d6): δ = 125.3, 127.6, 133.4, 142.5, 147.9, 150.6, 184.2 (9 C). MS (EI, 70

eV): m/z (%) = 341 [M+, 13]. Analysis for C9H7N7O4S2 (341.32): Calcd. C, 31.67; H, 2.07; N, 28.73. Found:
C, 32.01; H, 1.89; N, 29.08.
Molecules 2019, 24, 1066 14 of 21

3.1.2. 5-(1-Amino-2-phenylethyl)-N-(5-(3,5-dinitrophenyl)-1,3,4-thiadiazol-2-yl)-1,3,4-thiadiazol-2-
amine (3)
A mixture of the thiosemicarbazide derivative 2 (3.41 g, 0.01 mol) and phenylalanine (1.65 g,
0.01 mol) in pyridine (15 mL) was heated in an oil bath at 180–200 °C for 6 h. After cooling, the
reaction mixture was treated with a cold Na2CO3 solution (10%). The formed solid was filtered off,
washed with H2O, and crystallized from ethanol to give 3. Yield: 51%, m.p.: 238–240 °C, IR (KBr,
cm−1): ν = 3415–3257 (NH2, NH), 3058 (C‒Harom), 2958 (C‒Haliph), 1630 (C=N). 1H-NMR (400 MHz,
ppm, DMSO-d6): δ = 3.17 (d, 2H, CH2-Ph), 4.08 (t, 1H, J = 6.8 Hz, CH-N), 6.45 (s, 2H, NH2, D2O
exchangeable), 7.48–7.71 (m, 5H, Ph-H), 8.68, 9.01, 9.26 (3s, 3H, Ph-H), 11.21 (s, 1H, NH, D2O
exchangeable). 13C-NMR (100 MHz, ppm, DMSO-d6): δ = 37.9, 55.1, 125.4, 126.1, 128.2, 128.8, 129.9,
130.2, 132.7, 136.3, 143.2, 147.1, 150.3, 156.9 (18 C). MS (EI, 70 eV): m/z (%) = 470 [M+, 9]. Analysis for
C18H14N8O4S2 (470.48): Calcd. C, 45.95; H, 3.00; N, 23.82. Found: C, 46.23; H, 3.29; N, 24.19.

3.1.3. 5-(3,5-Dinitrophenyl)-N-(5-phenyl-1,3,4-thiadiazol-2-yl)-1,3,4-thiadiazol-2-amine (4)

To a mixture of 2 (3.41 g, 0.01 mol), benzoic acid (1.22 g, 0.01 mol) and phosphorous oxychloride
(5 mL) were added and refluxed in a water bath for 45 min, then cooled and quenched with cold
water (10 mL). The resulting solution was refluxed for an additional 4 h and filtered while hot. The
filtrate was cooled and triturated with an aqueous potassium hydroxide solution. The obtained solid
was separated by filtration, dried, and crystallized from dioxin to produce compound 4. Yield 70%,
m.p. 277–279 °C, IR (KBr, cm−1): ν = 3265 (NH), 3033 (CHarom), 1616 (C=N). 1H-NMR (400 MHz, ppm,
DMSO-d6): δ = 7.33–7.66 (m, 5H, Ph-H), 8.64, 9.07, 9.22 (3s, 3H, Ph-H), 10.22 (s, 1H, NH, D2O
exchangeable). 13C-NMR (100 MHz, ppm, DMSO-d6): δ = 124.5, 126.8, 127.5, 128.4, 129.8, 130.4, 131.3,
137.2, 143.2, 147.7, 151.1, 157.8 (16 C). MS (EI, 70 eV): m/z (%) = 427 [M+, 24]. Analysis for C16H9N7O4S2
(427.41): Calcd. C, 44.96; H, 2.12; N, 22.94. Found: C, 45.29; H, 2.00; N, 23.28.

3.1.4. 5-{[5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl]amino}-1,3,4-thiadiazole-2-thiol (5)

To a solution of thiosemicarbazide 2 (3.41 g, 0.01 mol) in ethanolic sodium hydroxide (20 mL,
2%), carbon disulfide (0.9 mL, 0.015 mol) was added with stirring for 30 min. The reaction mixture
was refluxed for 12 h, and, after cooling, acidified with hydrochloric acid. The separated solid was
filtered off, washed with water, and crystallized from dioxane to give compound 5. Yield 62%, m.p.
286–288 °C, IR (KBr, cm−1): ν = 3261 (NH), 3044 (C‒Harom), 1622 (C=N). 1H-NMR (400 MHz, ppm,
DMSO-d6): δ = 8.71, 9.11, 9.29 (3s, 3H, Ar-H), 10.13 (s, 1H, NH, D2O exchangeable), 12.23 (s, 1H, NH,
D2O exchangeable). 13C-NMR (100 MHz, ppm, DMSO-d6): δ = 126.8, 128.4, 129.8, 131.3, 147.7, 151.1,
157.8, 181.5 (10 C). MS (EI, 70 eV): m/z (%) = 383 [M+, 20]. Analysis for C10H5N7O4S3 (383.38): Calcd.
C, 31.33; H, 1.31; N, 25.58. Found: C, 30.98; H, 1.52; N, 25.95.

3.1.5. 2-((5-((5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl)amino)-1,3,4-thiadiazol-2-yl)thio)-1-
phenylethan-1-one (6)
To equimolar amount of 5 (3.83 g, 0.01 mol) and phenacyl bromide (1.99 g, 0.01 mol) were
dissolved in dry acetone (30 mL), potassium carbonate anhydrous (1.38 g, 0.01 mol) was added,
followed by refluxing on a water bath for 10 h. The reaction mixture was filtered and the filtrate was
poured over cooled water; the obtained solid was filtered off and crystallized from benzene to
produce compound 6. Yield 62%, m.p. 230–232 °C, IR (KBr, cm−1): ν = 3269 (NH), 3057 (C‒Harom), 2921
(C‒Haliph), 1618 (C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ = 4.96 (s, 2H, S-CH2-CO), 7.37–7.69 (m,
5H, Ph-H), 8.75, 9.17, 9.24 (3s, 3H, Ar-H), 10.29 (s, 1H, NH, D2O exchangeable). 13C-NMR (100 MHz,
ppm, DMSO-d6): δ = 40.5, 126.2, 127.5, 128.7, 129.1, 129.7, 130.7, 131.5, 132.3, 146.9, 151.16, 152.2,
156.9, 179.6 (18 C). MS (EI, 70 eV): m/z (%) = 501 [M+, 32]. Analysis for C18H11N7O5S3 (501.51): Calcd. C,
43.11; H, 2.21; N, 19.55. Found: C, 42.80; H, 2.00; N, 19.21.
Molecules 2019, 24, 1066 15 of 21

3.1.6. 5-(5-((5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl)amino)-1,3,4-thiadiazol-2(3H)-ylidene)
pyrimidine-2,4,6(1H,3H,5H)-trione (8)
To a solution of compound 2 (3.41 g, 0.01 mol) in methanol (30 mL), 5-[bis(methylthio)-
methylene] barbituric acid (7) (2.32 g, 0.01 mol) was added with stirring and refluxed for 5 h. The
reaction mixture was left to cool and the separated precipitate was filtered off and recrystallized
from dioxane to give compound 8. Yield 67%, m.p. 293–295 °C, IR (KBr, cm−1): ν = 3322–3189 (NH),
1744, 1678, 1655 (3 C=O). 1H-NMR (400 MHz, ppm, DMSO-d6): δ = 8.70, 9.14, 9.30 (3s, 3H, Ph-H), 9.34,
11.19, 12.67, 12.78 (4s, 4H, 4NH, D2O exchangeable). 13C-NMR (100 MHz, ppm, DMSO-d6): δ = 113.7,
127.5, 128.2, 130.7, 132.3, 146.4, 148.1, 154.8, 157.2, 162.6, 167.2, 167.8 (14 C). MS (EI, 70 eV): m/z (%) =
477 [M+, 23]. Analysis for C14H7N9O7S2 (477.39): Calcd. C, 35.22; H, 1.48; N, 26.41. Found: C, 34.88; H,
1.70; N, 26.81.

3.1.7. 3-[5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl]-2-hydrazonothiazolidin-4-one (9)

A mixture of thiosemicarbazide 2 (3.41 g, 0.01 mol) and chloroacetic acid (1 mL, 0.01 mol),
sodium acetate (2.46 g, 0.03 mol) in acetic acid (20 mL) was refluxed for 6 h, cooled, and poured on
water/ice. The obtained solid was filtered off, washed with water, and recrystallized from acetic acid
to produce thiazolidin-4-one derivative 9. Yield 78%, m.p. 246–248 °C, IR (KBr, cm−1): ν = 3378, 3254
(NH2), 2909 (C‒Haliph), 1681 (C=O), 1623 (C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ = 3.86 (s, 2H,
CH2, thiazolidinone), 6.41 (s, 2H, NH2, D2O exchangeable), 8.77, 9.17, 9.28 (3s, 3H, Ph-H). 13C-NMR
(100 MHz, ppm, DMSO-d6): δ = 31.2, 126.9, 129.6, 131.5, 133.8, 147.5, 150.4, 155.2, 177.7 (11 C). MS (EI,
70 eV): m/z (%) = 381 [M+, 20]. Analysis for C11H7N7O5S2 (381.34): Calcd. C, 34.65; H, 1.85; N, 25.71.
Found: C, 35.01; H, 2.12; N, 26.03.

3.1.8. N-Acetyl-N-[3-(5-(3,5-dinitrophenyl)-1,3,4-thiadiazol-2-yl]-4,6-dioxo-2-thioxotetrahydro-
pyrimidin-1(2H)-yl)acetamide (10)
A mixture of 2 (3.41 g, 0.01 mol), malonic acid (1.04 g, 0.01 mol) and acetyl chloride (8 mL) was
refluxed for 10 h. The mixture was poured into cold water with stirring; the precipitated solid was
filtered off and crystallized from dioxane to give compound 10. Yield 59%, m.p. 271–273 °C, IR (KBr,
cm−1): ν = 2944 (C‒Haliph), 1710, 1691, 1661 (3 C=O), 1616 (C=N). 1H-NMR (400 MHz, ppm, DMSO-d6):
δ = 2.72 (s, 6H, 2CH3), 3.23 (s, 2H, CH2, pyrimidine), 8.73, 9.14, 9.32 (3s, 3H, Ph-H). 13C-NMR (100
MHz, ppm, DMSO-d6): δ = 24.1, 41.9, 127.5, 129.9, 132.5, 137.6, 148.1, 151.3, 163.5, 164.8, 168.5, 175.9
(16 C). MS (EI, 70 eV): m/z (%) = 493 [M+, 18]. Analysis for C16H11N7O8S2 (493.43): Calcd. C, 38.95; H,
2.25; N, 19.87. Found: C, 39.31; H, 2.00; N, 20.20.

3.1.9. 1-[5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl]-3-phenylthiourea (11)

To aminothiazole derivative 1 (2.67 g, 0.01 mol) in dry dioxane (30 mL) and phenylisothio-
cyanate (0.01 mol, 1.35 g), anhydrous potassium carbonate (1.38 g, 0.01 mol) was added with stirring
and refluxed for 12 h, cold to room temperature then poured over ice/water. The obtained solid was
filtered off, dried, and crystallized from dioxane to produce 11. Yield 81%, m.p. 264–266 °C, IR (KBr,
cm−1): ν = 3329, 3256 (2NH), 3080 (C‒Harom.), 1626 (C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ =
7.45–7.89 (m, 5H, Ph-H), 8.68, 9.18, 9.29 (3s, 3H, Ph-H), 10.49, 11.33 (2s, 2H, 2NH, D2O exchangeable).
13C-NMR (100 MHz, ppm, DMSO-d6): δ = 127.3, 129.5, 131.6, 132.5, 134.1, 137.8, 147.7, 150.9, 154.5,

159.2, 182.9 (15 C). MS (EI, 70 eV): m/z (%) = 402 [M+, 16]. Analysis for C15H10N6O4S2 (402.40): Calcd. C,
44.77; H, 2.50; N, 20.89 Found: C, 45.08; H, 2.77; N, 21.21.

3.1.10. N-[5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl]-3,4-diphenylthiazol-2(3H)-imine (12)

A mixture of 11 (4.02 g, 0.01 mol) and phenacyl bromide (1.99 g, 0.01 mol) in EtOH (50 mL), in
the presence of TEA (0.5 mL), was refluxed for 10 h, then cooled to r.t. The formed solid was filtered
off, washed with water, dried, and crystallized from ethanol to produce imine derivative 12. Yield
51%, m.p. 251–253 °C, IR (KBr, cm−1): ν = 3061 (C‒Harom.), 1629 (C=N). 1H-NMR (400 MHz, ppm,
Molecules 2019, 24, 1066 16 of 21

DMSO-d6): δ = 6.65 (s, 1H, CH, thiazole), 7.41–7.91 (m, 10H, 2 Ph-H), 8.74, 9.12, 9.35 (3s, 3H, Ph-H).
C-NMR (100 MHz, ppm, DMSO-d6): δ = 115.4, 122.4, 125.7, 126.8, 127.7, 129.9, 130.8, 131.4, 133.2,
13

133.9, 136.4, 137.6, 143.2, 147.9, 152.6, 155.2, 159.5 (23 C). MS (EI, 70 eV): m/z (%) = 502 [M+, 25].
Analysis for C23H14N6O4S2 (502.52): Calcd. C, 54.97; H, 2.81; N, 16.72. Found: C, 55.30; H, 3.00; N,
17.04.

3.1.11. 1-[5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl]-3-phenyl-2-thioxodihydropyrimidine-4,6
(1H,5H)-dione (13)
To a mixture of equimolar amounts of phenylthiourea derivative 11 (4.02 g, 0.01 mol) and
malonic acid (1.04 g, 0.01 mol), acetyl chloride (5 mL) was added. The reaction mixture was heated in
an oil bath at 140–150 °C for 4 h, then poured onto crushed ice/water. The reaction mixture was
alkaloid with NaOH (pH= 9–10). The resulting solid was filtered off, washed with water, and
crystallized from dioxane to give compound 13. Yield 66%, m.p. 282–284 °C, IR (KBr, cm−1): ν = 3037
(C‒Harom.), 2961 (C‒Haliph), 1702, 1688 (C=O), 1624 (C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ =
3.31 (s, 2H, CH2, pyrimidine) and 7.32–7.88 (m, 5H, Ph-H), 8.78, 9.10, 9.30 (3s, 3H, Ph-H). 13C-NMR
(100 MHz, ppm, DMSO-d6): δ = 42.4, 126.3, 127.8, 129.5, 130.3, 131.4, 136.7, 143.2, 147.3, 153.1, 155.7,
169.2, 169.9, 187.2 (18 C). MS (EI, 70 eV): m/z (%) = 470 [M+, 20]. Analysis for C18H10N6O6S2 (470.43):
Calcd. C, 45.96; H, 2.14; N, 17.86. Found: C, 45.61; H, 1.98; N, 18.20.

3.1.12. 1-[5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl]-3-phenyl-5-(2-phenylhydrazono)-2-thioxo-
dihydro-pyrimidine-4,6(1H,5H)-dione (14)
Benzene diazonium chloride (0.01 mol) [prepared by the addition of NaNO2 (0.01 mol) to
distilled aniline (0.01 mol) in concentrated HCl (4 mL) at 0–5 °C under stirring] was added dropwise
to a solution of thiobarbituric derivative 13 (4.7 g, 0.01 mol) in ethanol (40 mL) containing sodium
acetate (2 g), with stirring at 0–5 °C for 3 h. The reaction mixture was left at room temperature for 2
h; the solid product was filtrated off, washed with water and crystallized from dioxane to give
hydrazone derivative 14. Yield 58%, m.p. 267–269 °C, IR (KBr, cm−1): ν = 3276 (NH), 3029 (C‒Harom.),
1700, 1690 (C=O), 1620 (C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ = 7.25–7.69 (m, 10H, 2Ph-H),
8.73, 9.14, 9.22 (3s, 3H, Ph-H), 12.55 (s, 1H, NH, D2O exchangeable). 13C-NMR (100 MHz, ppm,
DMSO-d6): δ = 124.2, 126.5, 127.8, 129.9, 130.7, 131.1,131.8, 132.3, 133.4, 136.1, 139.3, 144.2, 146.8, 152.8,
154.8, 167.3, 168.5, 180.4 (24 C). MS (EI, 70 eV): m/z (%) = 558 [M+, 26]. Analysis for C24H14N8O6S2
(574.55): Calcd. C, 50.17; H, 2.46; N, 19.50. Found: C, 49.80; H, 2.64; N, 19.18.

3.1.13. 5-Benzylidene-1-[5-(3,5-dinitrophenyl)-1,3,4-thiadiazol-2-yl]-3-phenyl-2-thioxodihydro-
pyrimidine-4,6(1H,5H)-dione (15)
A mixture of 13 (4.7 g, 0.01 mol) and benzaldehyde (1.06 g, 0.01 mol) in EtOH (40 mL),
containing piperidine (0.5 mL) as a catalyst, was heated under reflux for 8 h. The obtained solid was
collected by filtration and crystallized from dioxane to give 15. Yield 58%, m.p. 257–259 °C, IR (KBr,
cm−1): ν = 3055 (C‒Harom.), 1698, 1682 (2 C=O), 1627 (C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ =
6.84 (s, 1H, CH), 7.41–7.77 (m, 10H, 2Ph-H), 8.75, 9.08, 9.28 (3s, 3H, Ph-H). 13C-NMR (100 MHz, ppm,
DMSO-d6): δ = 121.1, 125.3, 126.2, 127.6, 129.3, 130.1, 131.5, 132.8, 134.2, 137.3, 140.7, 144.4, 146.2,
150.6, 153.2, 155.4, 168.1, 169.2, 181.5 (25 C). MS (EI, 70 eV): m/z (%) = 574 [M+, 27]. Analysis for
C25H14N6O6S2 (558.54): Calcd. C, 53.76; H, 2.53; N, 15.05. Found: C, 53.40; H, 2.79; N, 14.71.

3.1.14. Dimethyl (5-(3,5-dinitrophenyl)-1,3,4-thiadiazol-2-yl)carbonimidodithioate (16)

Aminothiadiazole derivative 1 (2.67 g, 0.01 mol) was stirred in DMF (30 mL), then sodium
hydroxide (5 mL, 20 M), carbon disulfide (1.52 g, 0.02 mol), and methyl iodide (1.43 g, 0.01 mol) were
added in sequence at intervals of 30 min and stirring continued for 6 h. The mixture was then poured
into an ice/water mixture with vigorous stirring. The formed precipitate was filtered off, washed
with water, dried, and recrystallized from dioxane to give 16. Yield 79%, m.p. 211–213 °C, IR (KBr,
cm−1): ν = 3026 (C‒Harom.), 2887 (C‒Haliph), 1620 (C=N), 1334 (C‒S). 1H-NMR (400 MHz, ppm,
Molecules 2019, 24, 1066 17 of 21

DMSO-d6): δ = 2.56 (s, 6H, 2 CH3), 8.66, 9.13, 9.26 (3s, 3H, Ph-H). 13C-NMR (100 MHz, ppm,
DMSO-d6): δ = 13.3, 126.5, 129.6, 132.8, 151.3, 153.8, 157.4, 168.1 (11 C). MS (EI, 70 eV): m/z (%) = 371
[M+, 14]. Analysis for C11H9N5O4S3 (371.40): Calcd. C, 35.57; H, 2.44; N, 18.86. Found: C, 35.21; H,
2.71; N, 19.22.

3.1.15. Synthesis of N-[5-(3,5-dinitrophenyl)-1,3,4-thiadiazol-2-yl]benzo[d]azole derivatives 17–19

A solution of 16 (3.71 g, 0.01 mol) in 15 mL N,N-dimethyl formamide was added to a solution of
amino derivatives (namely, o-aminophenol, o-aminothiophenol, o-phenylenediamine, 0.01 mol) in
15 mL N,N-dimethyl formamide with stirring at room temperature for 30 min. The reaction mixture
was refluxed for 8–10 h, left to cool, then poured on a water/ice mixture with stirring. The resulting
solid was filtered and recrystallized from a solvent to produce compounds 17–19, respectively.

3.1.16. N-[5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl]benzo[d]oxazol-2-amine (17)

Yield 61%, m.p. 254–256 °C (EtOH), IR (KBr, cm−1): ν = 3389–3272 (NH), 3044 (C‒Harom.) and 1627
(C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ = 7.17–7.51 (m, 4H, Ph-H), 8.62, 9.18, 9.32 (3s, 3H,
Ph-H), 9.78 (s, 1H, NH, D2O exchangeable). 13C-NMR (100 MHz, ppm, DMSO-d6): δ = 120.2, 122.3,
125.6, 127.9, 130.5, 132.8, 138.1, 144.9, 146.6, 150.6, 154.1, 155.9, 157.4 (15 C). MS (EI, 70 eV): m/z (%) =
384 [M+, 16]. Analysis for C15H8N6O5S (384.33): Calcd. C, 46.88; H, 2.10; N, 21.87. Found: C, 46.55; H,
2.32; N, 22.20.

3.1.17. N-[5-(3,5-Dinitrophenyl)-1,3,4-thiadiazol-2-yl]benzo[d]thiazol-2-amine (18)

Yield 68%, m.p. 281–283 °C (Dioxane), IR (KBr, cm−1): ν = 3364 (NH), 3039 (C‒Harom.), 1631
(C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ = 7.36–7.67 (m, 4H, Ph-H), 8.69, 9.22, 9.33 (3s, 3H,
Ph-H), 11.06 (s, 1H, NH, D2O exchangeable). 13C-NMR (100 MHz, ppm, DMSO-d6): δ = 119.6, 122.9,
126.1, 127.5, 131.7, 132.8, 137.3, 139.9, 145.8, 151.1, 154.5, 156.3, 158.8 (15 C). MS (EI, 70 eV): m/z (%) =
400 [M+, 16]. Analysis for C15H8N6O4S2 (400.39): Calcd. C, 45.00; H, 2.01; N, 20.99. Found: C, 44.68; H,
2.29; N, 21.34.

3.1.18. N-(1H-Benzo[d]imidazol-2-yl)-5-(3,5-dinitrophenyl)-1,3,4-thiadiazol-2-amine (19)

Yield 63%, m.p. 274–276 °C (DMF). IR (KBr, cm−1): ν = 3355, 3282 (NH), 3052 (C‒Harom.), 1628
(C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ = 7.11–7.43 (m, 4H, Ph-H), 8.74, 9.14, 9.27 (3s, 3H,
Ph-H), 9.96 (s, 1H, NH, D2O exchangeable), 11.78 (s, 1H, NH, D2O exchangeable). 13C-NMR (100
MHz, ppm, DMSO-d6): δ = 120.9, 121.7, 122.4, 127.1, 128.1, 131.2, 138.1, 138.9, 146.3, 151.1, 155.2,
157.5, 159.4 (15 C). MS (EI, 70 eV): m/z (%) = 383 [M+, 19]. Analysis for C15H9N7O4S (383.34): Calcd. C,
47.00; H, 2.37; N, 25.58. Found: C, 46.70; H, 2.18; N, 25.25.

3.1.20. N-(4,5-Dihydro-1H-imidazol-2-yl)-5-(3,5-dinitrophenyl)-1,3,4-thiadiazol-2-amine (20)

To a solution of dimethyl carbonimidodithioate derivative 16 (3.71 g, 0.01 mol) in dimethyl-
formamide (15 mL), ethylene diamine (1.2 g, 0.02 mol), in DMF (10 mL) was added drop wise with
stirring at room temperature. The reaction mixture was refluxed for 8 h, left to cool, then poured
onto water/ice with stirring. The obtained solid was filtered off, washed with water, dried, and
recrystallized from ethanol to produce compound 20. Yield 55%, m.p. 227–229 °C, IR (KBr, cm−1): ν =
3371, 3272 (NH), 3049 (C‒Harom.), 1622 (C=N). 1H-NMR (400 MHz, ppm, DMSO-d6): δ = 2.69 (t, 2H, J =
8.2 Hz, CH2-imidazole), 2.81 (t, 2H, J = 8.2 Hz, CH2-imidazole), 8.70, 9.10, 9.25 (3s, 3H, Ph-H), 9.83 (s,
1H, NH, D2O exchangeable), 10.66 (s, 1H, NH, D2O exchangeable). 13C-NMR (100 MHz, ppm,
DMSO-d6): δ = 46.9, 47.5, 121.7, 127.5, 131.8, 146.9, 153.8, 154.6, 159.4 (11 C). MS (EI, 70 eV): m/z (%) =
335 [M+, 12]. Analysis for C11H9N7O4S (335.30): Calcd. C, 39.40; H, 2.71; N, 29.24. Found: C, 39.74; H,
3.00; N, 24.95.
Molecules 2019, 24, 1066 18 of 21

3.2. Biological Evaluation

3.2.1. Antimicrobial Sensitivity Assay

The antimicrobial activities of the synthesized compounds were evaluated against four bacterial
strains, Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, and Escherichia coli, and two
fungal strains, Aspergillus niger and Candida albicans, using a standard well agar diffusion assay
according to Cheesbrough et al. [35]. Plates containing nutrient agar medium and sabouraud
dextrose agar medium (for bacteria and fungi, respectively) were surface-inoculated with 106
CFU/mL of freshly prepared microorganisms. Using a 6-mm sterile cork borer, wells were punched
in the agar and filled separately with 100 µL of the tested compounds (100 µg/mL in DMSO). The
plates were left in a refrigerator for 2 h to allow diffusion of the tested compounds. After that, the
plates were incubated for 24 h at 37 °C for bacteria and for 72 h at 28 °C for fungi, then the inhibition
zones surrounding the wells were measured in millimeters. Amoxicillin and Fluconazole were used
as the standard against bacteria and fungi, respectively, using the same concentration (100 µg/mL).

3.2.2. In Vitro Anticancer Activity

The newly synthesized heterocyclic compounds were evaluated for their in vitro cytotoxicity
against four cancer cell lines, CCRF-CEM (leukemia), HCT-15 (human colon carcinoma), PC-3
(prostate cancer) and UACC-257 (melanoma, skin cancer) cell lines, which were obtained from
Sigma-Aldrich Chemical Company, USA. DOX (Doxorubicin) was utilized as a reference drug
according to a previously reported MTT method [38,39].

3.2.3. Dihydrofolate Reductase (DHFR) Inhibition

The in vitro DHFR enzyme inhibition assessment was carried out in a confirmatory diagnostic
unit, Vacsera, Egypt. All synthesized derivatives 1–20 were screened against DHFR using
Methotrexate as a reference according to a previously reported method [36]. The results are reported
as IC50 values of enzymatic activity in Table 3.

3.3. Molecular Modeling Studies

The docking study was performed using Molecular Operating Environment (MOE®) 2008.10
software [38,39]. The X-ray crystal structure of the dihydro–folate reductase enzyme was
downloaded from the Protein Data Bank website (PDB ID: 1DLS) [37]. Regularization and
optimization for ligand and protein were done. The performance of the docking method was
evaluated by re-docking the crystal ligand into the assigned active DHFR enzyme to evaluate a
root-mean-square deviation value. Then, the molecular docking was applied for compounds 10, 13,
14, and 15 into ATP binding site of DHFR according to the reported method [38,39].

4. Conclusions
In summary, a series of 1,3,4-thiadiazole derivatives 2–20 incorporating with different
heterocyclic systems was designed and synthesized. All synthesized compounds were examined for
their in vitro antimicrobial, antitumor, and DHFR inhibition activity. The antimicrobial results
exhibited the ability of the compounds 9, 10, 13, and 16 to inhibit the growth of a panel of six strains
with higher inhibition zones in comparison with the reference drugs. In addition, the cytotoxic
activity against four cell lines illustrated that compounds 6, 14, and 15 have mostly prevented cell
growth with lower IC50 values. Based on the data obtained from the DHFR inhibition study,
compounds 10, 13, 14, and 15, containing a thioxopyrimidine-4,6-dione moiety at p-2 of the parent
1,3,4-thiadiazole ring, were the most potent derivatives in comparison with Methotrexate. Moreover,
the docking study indicated that compounds 10, 13, 14, and 15 showed good fitting and caused
favorable contacts in the binding site of DHFR enzyme. Therefore, the obtained mark points have
been proposed as an explanation for the unique activity of such derivatives and could be used as a
Molecules 2019, 24, 1066 19 of 21

template for further development and future optimization of new antimicrobial and anticancer
agents via DHFR inhibition.

Author Contributions: M.E,N, H.A.S., S.S.S., and S.S.A.-W. performed most of the experiments; A.E.-G.E.A.
and M.E.A. analyzed the data; E.S.N. contributed to the modeling studies; M.A.A.-O. contributed to the
anticancer activity assays; all authors read and approved the final manuscript.

Funding: This research was funded by the Vice Deanship of Scientific Research Chairs, Deanship of Scientific
Research, King Saud University.

Acknowledgments: The authors are grateful to the Deanship of Scientific Research, King Saud University for
funding through the Vice Deanship of Scientific Research Chairs.

Conflicts of Interest: The authors declare no conflict of interest.

References
1. Schnell, J.R.; Dyson, H.J.; Wright, P.E. Structure, dynamics, and catalytic function of dihydrofolate
reductase. Annu. Rev. Biophys. Biomol. Struct. 2004, 33, 119–140.
2. Huang, H.; Lu, W.; Li, X.; Cong, X.; Ma, H.; Liu, X.; Zhang, Y.; Che, P.; Ma, R.; Li, H.; et al. Design and
synthesis of small molecular dual inhibitor of falcipain-2 and dihydrofolate reductase as antimalarial
agent. Bioorg. Med. Chem. Lett. 2012, 22, 958–962.
3. Borst, P.; Quellette, M. New mechanisms of drug resistance in parasitic protozoa. Annu. Rev. Microbiol.
1995, 49, 427–460.
4. Foye, W.O.; Lemke, T.L.; Williams, D.A. Principles of Medicinal Chemistry, 4th ed.; Williams and
Wilkins: Media, PA, USA, 2005; pp. 442–456.
5. Champe, P.C.; Harvey, R.A. Lippincott’s Illustrated Reviews: Biochemistry, 2nd ed.; Lippincott Williams
and Wilkins: Philadelphia, PA, USA, 1994; pp. 350–356.
6. Berman, E.M.; Werbel, L.M. The renewed potential for folate antagonists in contemporary cancer
chemotherapy. J. Med. Chem. 1991, 34, 479–485.
7. El-Gazzar, Y.I.; Georgey, H.H.; El-Messery, S.M.; Ewida, H.A.; Hassan, G.S.; Raafat, M.M.; Ewida,
M.A.; El-Subbagh, H.I. Synthesis, biological evaluation and molecular modeling study of new
(1,2,4-triazole or 1,3,4-thiadiazole)-methylthio-derivatives of quinazolin-4(3H)-one as DHFR
inhibitors. Bioorg. Chem. 2017, 72, 282–292.
8. Al-Rashood, S.T.; Aboldahab, I.A.; Nagi, M.N.; Abouzeid, L.A.; Abdel-Aziz, A.A.M.; Abdel-hamide,
S.G.; Youssef, K.M.; Al-Obaida, A.M.; El-Subbagha, H.I. Synthesis, dihydrofolate reductase inhibition,
antitumor testing, and molecular modeling study of some new 4(3H)-quinazolinone analogs. Bioorg.
Med. Chem. 2006, 14, 8608–8621.
9. Jain, K.; Sharma, S.; Vaidya, A.; Ravichandran, V.; Agrawal, R.K. 1,3,4-Thiadiazole and its derivatives:
A review on recent progress in biological activities. Chem. Biol. Drug Des. 2013, 81, 557–576.
10. Kushwaha, N.; Kushwaha, S.K.S.; Rai, A.K. Biological activities of thiadi¬azole derivatives: A review.
Int. J. Chem. Res. 2012, 4, 517–531.
11. Gomha, S.M.; Salah, T.A.; Abdelhamid, A.O. Synthesis, characterization, and pharmacological
evaluation of some novel thiadiazoles and thiazoles incorporating pyrazole moiety as anticancer
agents. Monatsh. Chem. 2015, 146, 149–158.
12. Gomha, S.M.; Abdel-aziz, H.M. Synthesis and antitumor activity of 1,3,4-thiadiazole derivatives
bearing coumarine ring. Heterocycles 2015, 91, 583–592.
13. Siddiqui, N.; Ahuja, P.; Ahsan, W.; Pandeya, S.N.; Alam, M.S. Thiadiazoles: Progress report on
biological activities. J. Chem. Pharm. Res. 2009, 1, 19–30.
14. Bhattacharya, P.; Leonard, J.T.; Roy, K. Exploring QSAR of thiazole and thiadiazole derivatives as
potent and selective human adenosine A3 receptor antagonists using FA and GFA techniques. Bioorg.
Med. Chem. 2005, 15, 1159–1165.
15. Foroumadi, A.; Kargar, Z.; Sakhteman, A.; Sharifzadeh, Z.; Feyzmohammadi, R.; Kazemi, M.; Shafiee,
A. Synthesis and antimyco¬bacterial activity of some alkyl [5-(nitroaryl)-1,3,4-thiadiazol-2-
ylthio]propionates. Bioorg. Med. Chem. Lett. 2006, 16, 1164–1167.
16. Kumar, D.; Kumar, N.M.; Chang, K.H.; Shah, K. Synthesis and anticancer activity of
5-(3-indolyl)-1,3,4-thiadiazoles. Eur. J. Med. Chem. 2010, 45, 4664–4668.
Molecules 2019, 24, 1066 20 of 21

17. Sharma, B.; Verma, A.; Prajapati, S.; Sharma, U.K. Synthetic methods, chemistry, and the
anticonvulsant activity of thiadiazoles. Int. J. Med. Chem. 2013, 2013, 348948.
18. Mathew, V.; Keshavayya, J.; Vaidya, V.P.; Giles, D. Studies on synthesis and pharmacological
activities of 3,6-disubstituted-1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles and their dihydro analogues. Eur.
J. Med. Chem. 2007, 42, 823–840.
19. Masereel, B.; Rolin, S.; Abbate, F.; Scozzafava, A.; Supuran, C.T. Carbonic anhydrase inhibitors:
Anticonvulsant sulfonamides incorporating valproyl and other lipophilic moieties. J. Med. Chem. 2002,
45, 312–320.
20. Said, H.M.; Hagemann, C.; Carta, F.; Katzer, A.; Polat, B.; Staab, A.; Scozzafava, A.; Anacker, J.; Vince,
G.H.; Flentje, M.; et al. Hypoxia induced CA9 inhibitory targeting by two different sulfonamide
derivatives including Acetazolamide in human Glioblastoma. Bioorg. Med. Chem. 2013, 21, 3949–3957,
doi.:10.1016/j.bmc.2013.03.068.
21. Li, Y.; Geng, J.; Liu, Y.; Yu, S.; Zhao, G. Thiadiazole—A Promising Structure in Medicinal Chemistry.
Chem. Med. Chem. 2013, 8, 27–41.
22. Riyadh, S.M.; El-Motairi, S.A.; Ahmed, H.E.A.; Khalil, K.D.; Habibe, E.E. Synthesis, biological
evaluation, and molecular docking of novel thiazoles and [1,3,4]thiadiazoles incorporating
sulfonamide group as DHFR Inhibitors. Chem. Biodivers. 2018, 15, doi: 10.1002/cbdv.201800231.
23. Gomha, S.M.; Edrees, M.M.; Muhammad, Z.A.; El-Reedy, A.A.M. 5-(Thiophen-2-yl)-1,3,4-thiadiazole
derivatives: Synthesis, molecular docking and in vitro cytotoxicity evaluation as potential anticancer
agents. Drug Des. Dev. Ther. 2018, 12, 1511–1523.
24. El-Hashash, M.A.; Azab, M.E.; Faty, R.A.; Amr, A.-G. Synthesis, antimicrobial and anti-inflammatory
activity of some new benzoxazinone and quinazolinone candidates. Chem. Pharm. Bull. 2016, 64,
263–271.
25. Azab, M.E.; Rizk, S.A.; Mahmoud, N.F. Facile Synthesis, characterization and antimicrobial
evaluation of novel heterocycles, schiff bases and N-nucleosides bearing phthalazine moiety. Chem.
Pharm. Bull. 2016, 64, 349–350.
26. Sayed, G.H.; Azab, M.E.; Anwer, K.E.; Raouf, M.A.; Negm, N.A. Pyrazole, pyrazolone and
enaminonitrile pyrazole derivatives: Synthesis, characterization and potential in corrosion inhibition
and antimicrobial applications. J. Mol. Liq. 2018, 252, 329–338. doi:10.1016/j.molliq.2017.12.156.
27. Sayed, G.H.; Azab, M.E.; Anwer, K.E.; Negm, N.A. Antimicrobial and cytotoxic activities of some
novel heterocycles bearing pyrazole moiety. J. Heterocycl. Chem. 2018, 55, 1615–1625.
28. Amr, A.E.; Abo-Ghalia, M.H.; Abdalah, M.M. Synthesis of new (Nα-dipicolinoyl)-bis-L-valyl-L-
phenylalanyl linear and macrocyclic bridged peptides as anti-inflammatory agents. Arch. Pharm. 2007,
340, 304–309, doi:10.1002/ardp.200600187.
29. Al-Omar, M.A.; Amr, A.E. Synthesis of some new pyridine-2,6-carboxamide-derived schiff bases as
potential antimicrobial agents. Molecules 2010, 15, 4711–4721, doi:10.3390/molecules15074711.
30. Amr, A.E.; Abdel-Latif, N.A.; Abdalla, M.M. Synthesis of some new testosterone derivatives fused
with substituted pyrazoline ring as promising 5α-reductase inhibitors. Acta Pharm. 2006, 56, 203–218.
31. Abdel-Wahab, B.F.; Mohamed, S.F.; Amr, A.E.; Abdalla, M.M. Synthesis and reactions of
thiosemicarbazides, triazoles, and Schiff bases as antihypertensive α-blocking agents. Monatshefte fur
Chemie 2008, 139, 1083–1090, doi:10.1007/s00706-008-0896-2.
32. Amr, A.E.; Abdulla, M.M. Synthesis and anti-inflammatory activities of new cyanopyrane derivatives
fused with steroidal nuclei. Arch. Pharm. 2006, 339, 88–95, doi:10.1002/ardp.200500209.
33. Khalifa, N.M.; Al-Omar, M.A.; Amr, A.E.; Haiba, M.E. HIV-1 and HSV-1 virus activities of some new
polycyclic nucleoside pyrene candidates. Int. J. Biol. Macromol. 2013, 54, 51–56,
doi:10.1016/j.ijbiomac.2012.11.015.
34. Abdalla, M.M.; Al-Omar, M.A.; Bhat, M.A.; Amr, A.E.; Al-Mohizea, A.M. Steroidal pyrazolines
evaluated as aromatase and quinone reductase-2 inhibitors for chemoprevention of cancer. Int. J. Biol.
Macromol. 2012, 50, 1127–1132, doi:10.1016/j.ijbiomac.2012.02.006.
35. Cheesbrough, M. District Laboratory Practice in Tropical Countries; Cambridge University Press:
Cambridge, UK, 2000; Part 2.
36. Pignatello, R.; Sapmpinato, G.; Sorrenti, V.; Vicari, L.; Di-Giacomo, C.; Vanella, A.; Puglisi, G.
Aliphatic α-γ-bis(amides) of Methotrexate. Influence of chain length on in-vitro activity against
sensitive and resistant tumour cells. Pharm. Pharmacol. Commun. 1999, 5, 299–305.
Molecules 2019, 24, 1066 21 of 21

37. Lewis, W.S.; Cody, V.; Galitsky, N.; Luft, J.R.; Pangborn, W.; Chunduru, S.K.; Spencer, H.T.;
Appleman, J.R.; Blakley, R.L. Methotrexate-resistant variants of human dihydrofolate reductase with
substitutions of leucine 22. Kinetics, crystallography, and potential as selectable markers. J. Biol. Chem.
1995, 270, 5057–5064.
38. Amr, A.E.; Abo-Ghalia, M.H.; Moustafa, G.; Al-Omar, M.A.; Nossier, E.S.; Elsayed, E.A. Design,
synthesis and docking studies of novel macrocyclic pentapeptides as anticancer multi-targeted kinase
inhibitors. Molecules 2018, 23, 2416, doi:10.3390/molecules 23102416.
39. Elzahabi, H.S.A.; Nossier, E.S.; Khalifa, N.M.; Alasfoury, R.A.; El-Manawat, M.A. Anticancer
evaluation and molecular modeling of multi-targeted kinase inhibitors based pyrido[2,3-d]-
pyrimidine scaffold. J. Enzym. Inhib. Med. Chem. 2018, 33, 546–557, doi:10.1080/14756366.2018.1437729.
40. Vachala, S.D.; Bhargavi, B. Design, synthesis and screening of novel 5-substituted-1,3,4-thiadiazol-2-
amines and their Schiff bases. J. Chem. Pharm. Res. 2014, 6, 377–389.
41. Gür, M.; Şener, N.; Muğlu, H.; Çavuş, M.S.; Özkan, O.E.; Kandemirli, F.; Şener, İ. New
1,3,4-thiadiazole compounds including pyrazine moiety: Synthesis, structural properties and
antimicrobial features. J. Mol. Struct. 2017, 1139, 111–118.

Sample Availability: Samples of the compounds are available from the authors.

© 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/).