Journal Pre-proofs

Design, synthesis, and molecular docking study of new piperazine derivative as

potential antimicrobial agents

Mahadev Patil, Anurag Noonikara Poyil, Shrinivas D. Joshi, Shivaputra A. Patil,

Siddappa A. Patil, Alejandro Bugarin

PII: S0045-2068(19)31140-X
DOI: https://doi.org/10.1016/j.bioorg.2019.103217
Reference: YBIOO 103217

To appear in: Bioorganic Chemistry

Received Date: 17 July 2019

Revised Date: 19 August 2019
Accepted Date: 22 August 2019

Please cite this article as: M. Patil, A.N. Poyil, S.D. Joshi, S.A. Patil, S.A. Patil, A. Bugarin, Design, synthesis, and
molecular docking study of new piperazine derivative as potential antimicrobial agents, Bioorganic Chemistry
(2019), doi: https://doi.org/10.1016/j.bioorg.2019.103217

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Design, synthesis, and molecular docking Leave this area blank for abstract info.
study of new piperazine derivatives as
potential antimicrobial agents
Mahadev Patila, Anurag Noonikara Poyilb, Shrinivas D. Joshic, Shivaputra A. Patild, Siddappa A.
Patila*, and Alejandro Bugarine*
aCentre for Nano & Material Sciences, Jain University, Jain Global Campus, Bangalore 562112, Karnataka,

India.
bDepartment of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX 76019, USA
cNovel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry, S. E. T’s College of

Pharmacy, Sangolli Rayanna Nagar, Dharwad 580 002, Karnataka, India.

dPharmaceutical Sciences Department, College of Pharmacy, Rosalind Franklin University of Medicine and

Science, 3333 Green Bay Road, North Chicago, IL 60064, USA

eDepartment of Chemistry and Physics, Florida Gulf Coast University, Fort Myers, FL 33965, USA.

R4 R4
R1 NCO
N R3 N R3
toluene H
HN R1 N N
R2 40-45 ºC, 1 h
* Metal-free reaction conditions O
R2
* Intermolecular amidation 17 examples
* Easy access to piperazines derivatives Fluorinated adducts
 1

Bioorganic Chemistry
journal homepage: www.elsevier.com

Design, synthesis, and molecular docking study of new piperazine derivative as

potential antimicrobial agents
Mahadev Patila, Anurag Noonikara Poyilb, Shrinivas D. Joshic, Shivaputra A. Patild, Siddappa A. Patila*,
Alejandro Bugarine
aCentre for Nano & Material Sciences, Jain University, Jain Global Campus, Bangalore 562112, Karnataka, India.
bDepartment of Chemistry & Biochemistry, University of Texas at Arlington, Arlington, TX 76019, USA.
cNovel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry, S. E. T’s College of Pharmacy, Sangolly Rayanna Nagar, Dharwad

580 002, Karnataka, India.

dPharmaceutical Sciences Department, College of Pharmacy, Rosalind Franklin University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL

60064, USA.
eDepartment of Chemistry and Physics, Florida Gulf Coast University, Fort Myers, FL 33965, USA.

ARTICLE INFO ABSTRACT

Article history: Herein, we describe the successful design and synthesis of seventeen new 1,4-diazinanes,
Received compounds commonly known as piperazines. This group of piperazine derivatives (3a-q) were fully
Received in revised form characterized by 1H NMR, 13C NMR, FT-IR, and LCMS spectral techniques. The molecular
Accepted structure of piperazine derivative (3h) was further established by single crystal X-ray diffraction
Available online analysis. All reported compounds were evaluated for their antibacterial and antifungal potential
against five bacterial (Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae,
,,Keywords: Acinetobacter baumannii, and Pseudomonas aeruginosa) and two fungal strains (Candida albicans
Piperazine derivatives and Cryptococcus neoformans). The complete bacterial screening results are provided. As
Synthesis documented, piperazine derivative 3e performed the best against these bacteria. Additionally, data
Characterization obtained during molecular docking studies are very encouraging with respect to potential utilization
Molecular docking of these compounds to help overcome microbe resistance to pharmaceutical drugs, as explicitly
Antimicrobial activity noted in this manuscript.
2009 Elsevier Ltd. All rights reserved.

———
 Corresponding authors. Tel.: +1-239-745-4464: e-mail address: abugarin@fgcu.edu (A. B.) e-mail address: p.siddappa@jainuniversity.ac.in (S. P.)
2 Bioorganic Chemistry
1. Introduction
Bacterial and fungal infections are posing serious health threats globally in large part due to their increasing resistance to a large
number of known antimicrobial drugs [1, 2]. It is well known that microbes can develop resistance to pharmaceuticals by altering their
target site, enzymatic resistance, and expression of efflux pumps. Drug efflux is particularly significant as it decreases drug uptake in the
cells [3]. The ever-escalating drug resistance problem has encouraged medicinal chemists to design and develop novel drug candidates to
help mitigate this problem. In this context, we have examined a variety of relatively small bioactive molecules for use as pre-clinical drug
candidates. For example, we have previously examined potential pharmacophores such as chalcones [4], ureas [5], and N-heterocyclic
carbene-metal complexes [6-13]. The present manuscript significantly extends our studies on this important research topic.
Nitrogen heterocycles remain an attractive topic for small molecular drug design and discovery. Among all heterocycles, piperazines,
the six-membered nitrogen-containing heterocyclic ring, are certainly an established important pharmacophore in medicinal chemistry
[14, 15]. For example, the piperazine moiety is present in the core structure of many important commercial fluoroquinoline antibiotics
such as: Norfloxacin, Ciprofloxacin, Gatifloxacin, Grepafloxacin, Sparfloxacin, and Levofloxacin [14] (Fig. 1). Of particular significance
is the fact that combining the piperazine moiety with other heterocyclic ring systems, such as tetrazole, has resulted in new antifungal
agents [15]. Such prior studies have clearly identified the potential use of piperazine derivatives as important pharmacophores.
Fig. 1. Antibiotics containing piperazine moiety.
Therefore, in continuation of our research efforts to identify novel new effective antimicrobial agents, we herein describe the design,
synthesis, antimicrobial evaluation, and molecular docking of a wide range of new 1,4-di(hetero)aryl substituted piperazine analogs.
2. Result and Discussion
2.1. Chemistry
The general synthetic approach employed to prepare these piperazine derivatives (3a-q) is outlined in Scheme 1 (for reaction
optimization see the ESI). The starting materials for the synthesis of piperazine derivatives, namely: 1-benzhydrylpiperazine, 1-((4-
fluorophenyl)(phenyl)methyl) piperazine, 1-((2-fluorophenyl)(4-fluorophenyl)methyl) piperazine and 11-(piperazin-1-
R4
R4
N R3
R1 NCO N R3 H
toluene R1 N N
HN
40-45 °C, 1 h
R2 O
1a-d 2a-e R2 3a-q

F N
S

N N N N
H H H H
N N N N N N N N
F F
3a, 78% 3b, 74% 3c, 70% 3d, 76%
O O O O
Me Me Me Me

F N
S

N N N N
H H H H
N N N N N N N N
F F

O O O O
Cl 3e, 75% Cl 3f, 71% Cl 3g, 70% Cl 3h, 72%

O O
O O F
F F OH N
OH S
N N
N N
N HN N
N N HN
H H H CH3 H
N N N N N N N N
F Ciprofloxacin
F Norfloxacin CH3
F N
O O O O 3l, 71%
3i, 77% 3j, 74% F 3k, 70% F N
F F O O
R5 O
F H O
OH N
H3C HO N
N N
HN O N O CH3 N
H
Levofloxacin
S
H3C R6
Piperazine
N N Gatifloxacin N
H H N Cl NH2 O H
NO N
Cl N N Cl N N H F
F Cl N N3 O
CH O OH
F
OH HF3C O
O 3m, 74% O 3n, 76% N N 3q, 73%
Cl Cl H3C O 3o, 72%, R 5=H, R 6=F Cl
N N 75%, R =F, R =H HN F
Cl 3p, 5 6
HN CH3
Grepafloxacin Sparfloxacin

yl)dibenzo[b,f][1,4]thiazepine were synthesized following published procedure [16, 17], ( also see the supporting information). All
compounds (3a-q) were prepared in a simple, one-step reaction of previously prepared aryl isocyanates and mono-substituted piperazines
in toluene at 40-45 ºC. This method has the advantages of fast reactions (1 h), easier work-up, mild reaction conditions, and good yields
(70 to 78%).
 3
Scheme 1. Direct synthesis of disubstituted-piperazine derivatives

2.2. Spectroscopic characterization

The newly synthesized piperazine derivatives containing aryl moieties (3a-q) were characterized using IR, 1H NMR,13C NMR, and
mass spectrometry. The spectral data of the newly synthesized compounds (3a-q) are detailed in the experimental section. The FT-IR
spectra, for all derivatives, were recorded in the region from 4000 to 400 cm-1, wherein the observed bands at 3356-3251 cm-1 were
assigned to the (NH) groups of the urea moiety. The IR spectra of all derivatives show stretching frequencies around 1628-1707 cm-1,
which correspond to the (C=O) groups. Weak to medium absorptions, observed around 2814–2956 cm-1, are assigned to the =C–H
stretch of the aromatic ring. Structural information of the compounds was further established by using 1H NMR and 13C NMR spectra.
The 13C NMR spectra of these derivatives exhibit a signal characteristic of the (C=O) functional groups between 154.9–152.2 ppm.
2.3. X-ray crystallography
X‐ray quality single crystals were obtained from recrystallization, in chloroform-d solution, of the N-(4-chlorophenyl)-4-
(dibenzo[b,f][1,4]thiazepin-11-yl)piperazine-1-carboxamide (3h). The crystals provided a good diffraction pattern and thus an
unequivocal molecular structure for this compound. The compound (3h) was found to crystallize in a monoclinic unit cell and P21 space
group. The crystal structure of compound (3h) is shown in Fig. 2. The crystal data and refinement details are found in Table 1, whereas
selected bond lengths and bond angles are provided in Table 2. The asymmetric unit of the N-(4-chlorophenyl)-4-
(dibenzo[b,f][1,4]thiazepin-11-yl)piperazine-1-carboxamide (3h) contains two separate molecular components, with different
conformations. X-ray structure reveals that both these components are nonplanar. There are no lattice-held water molecules or organic
solvent molecules in the unit cells of the determined structures. The Cl groups are coplanar with the attached benzene rings which are

N S
N
H
N N

O 3h
Cl

nearly antiparallel to the other benzene rings. The C-C-C bond angles in the aromatic ring are close to 120°, thus suggesting that the
carbon atoms are sp2 hybridized. The C-C bond distances in the aromatic rings are in the normal range of 1.34–1.51 Å, which is
characteristic of delocalized aromatic rings. The molecular packing diagram shows four layers of molecules, which are independently
arrange in the unit cell. Molecules forming each layer are connected through the intermolecular hydrogen bonding formed between NH
and C=O. In each layer, the molecules are alternatively parallel.
Fig. 2. X-ray diffraction structure of 3h; molecule; thermal ellipsoids are drawn on the 50% probability level. Additional data check CCDC 1939260.

Table 1.
Crystal data and structure refinement for 3h.
Identification code 3h
Empirical formula C24H21ClN4OS
Molecular formula C24H21ClN4OS
Formula weight 449.0
Temperature 293K
Crystal system Monoclinic
Space group P21
Unit cell dimensions a = 6.1263(5) Å
b = 37.4701(9) Å
c = 9.5021(6)Å
α = 90°
 =102.626(12)°
γ = 90°
Volume 2128.49(7) Å3
Z 4
4 Bioorganic Chemistry
Radiation type Mo-K Software: APEX2 and SAINT (Bruker, 2014) [18], SHELXS97 and SHELXL2013
Density (calculated) 1.4011Mg/m3 (Sheldrick, 2008) [19], and JANA2006 [20].
Absorption coefficient 0.303mm–1
F(000) 936 Table 2.
Crystal size 0.32 x 0.28 x 0.05mm3 Selected bond lengths [Å] and angles (°) for compound 3h.
Theta range for data collection 2.17 to 28.44°. Bond lengths [Å] 3h Bond lengths [Å] 3h
Index ranges –8<=h<=8, –49<=k<=49, –
S(1)-C(13) 1.773 (3) N(4)-C(12) 1.408 (4)
12<=l<=12
Diffractometer Bruker SMART Absorption correction Multi- S(1)-C(20) 1.779 (4) N(5)-C(33) 1.470 (4)
APEXII scan (SADABS; Bruker, 2014) S(2)-C(40) 1.781 (4) N(5)-C(34) 1.378 (5)
Reflections collected 10550 S(2)-C(41) 1.779 (4) N(5)-C(47) 1.469 (5)
Independent reflections 10550 [R(int) = 0.0408] O(1)-C(8) 1.230 (4) O(2)-C(31) 1.237 (4)
Completeness to θmax 99 % N(1)-C(34) 1.286 (5) N(6)-C(11) 1.368 (5)
Max. and min. Transmission 0.908 and 0.985 N(1)-C(35) 1.390 (4) N(6)-C(18) 1.453 (4)
N(2)-C(7) 1.431 (5) C(30)-N(7) 1.431 (5)
Goodness-of-fit on F2 1.30
N(2)-C(8) 1.363 (4) N(7)-C(31) 1.340 (5)
Final R indices [I>2sigma(I)] R1 = 0.1321, wR2 = 0.0408
N(3)-C(8) 1.367 (5) C(31)-N(8) 1.357 (5)
R indices (all data) R1 = 0.0440, wR2 = 0.0509
N(3)-C(9) 1.469 (4) N(8)-C(32) 1.460 (4)
ρmax,ρ min (e-/ Å3) 0.27(e-/ Å3), -0.25(e-/ Å3)
N(3)-C(19) 1.457 (4) N(8)-C(48) 1.479 (5)
N(4)-C(11) 1.280 (5)
Bond angles [°] 3h Bond angles [°] 3h
C(13)-S(1)-C(20) 95.04 (18) S(1)-C(13)-C(12) 120.4 (3)
C(40)-S(2)-C(41) 96.40 (18) S(1)-C(13)-C(14) 119.8 (3)
C(34)-N(1)-C(35) 123.5 (3) N(6)-C(18)-C(19) 110.7 (3)
C(7)-N(2)-C(8) 119.8 (3) N(3)-C(19)-C(18) 110.8 (3)
C(8)-N(3)-C(9) 124.2 (3) C(29)-C(30)-N(7) 120.0 (3)
C(8)-N(3)-C(19) 116.4 (3) N(7)-C(30)-C(39) 120.6 (3)
C(9)-N(3)-C(19) 114.0 (3) C(30)-N(7)-C(31) 120.7 (3)
C(11)-N(4)-C(12) 122.4 (3) O(2)-C(31)-N(7) 120.5 (3)
C(33)-N(5)-C(34) 117.6 (3) O(2)-C(31)-N(8) 122.0 (3)
C(33)-N(5)-C(47) 109.5 (3) N(7)-C(31)-N(8) 117.5 (3)
C(34)-N(5)-C(47) 118.3 (3) C(31)-N(8)-C(32) 126.2 (3)
N(2)-C(7)-C(6) 119.3 (3) C(31)-N(8)-C(48) 118.1 (3)
N(2)-C(7)-C(17) 122.5 (4) C(32)-N(8)-C(48) 111.9 (3)
O(1)-C(8)-N(2) 120.3 (3) N(8)-C(32)-C(33) 110.0 (3)
O(1)-C(8)-N(3) 121.5 (3) N(1)-C(34)-N(5) 119.5 (3)
N(2)-C(8)-N(3) 118.2 (3) N(1)-C(34)-C(46) 125.8 (3)
N(3)-C(9)-C(10) 112.0 (3) N(5)-C(34)-C(46) 114.5 (3)
C(10)-N(6)-C(11) 121.4 (3) N(1)-C(35)-C(36) 118.6 (3)
C(10)-N(6)-C(18) 111.4 (3) N(1)-C(35)-C(40) 123.5 (3)
C(11)-N(6)-C(18) 122.8 (3) S(1)-C(40)-C(3) 119.8 (3)
N(4)-C(11)-N(6) 119.6 (3) S(1)-C(40)-C(35) 120.1 (3)
N(4)-C(11)-C(25) 125.6 (3) S(2)-C(41)-C(42) 120.8 (3)
N(6)-C(11)-C(25) 114.2 (3) S(2)-C(41)-C(46) 119.3 (3)
N(4)-C(12)-C(13) 124.2 (3) N(5)-C(47)-C(48) 110.8 (3)
N(4)-C(12)-C(27) 117.7 (3)

2.4. Antimicrobial activity

All newly synthesized compounds (3a-q) were screened against standard one Gram-positive i.e. S. aureus and four Gram-negative i.e.
E. coli, P. aeruginosa, K. pneumonia and A. baumannii bacterial strains. In addition, all these compounds were also screened against two
fungal strains of C. albicans and C. neoformans. The results of the in-vitro antimicrobial activity are summarized in Table 3. In regard to
P. aeruginosa and A. baumannii, the in vitro assay results, from all piperazine derivatives (3a-q), showed poor or even no growth
inhibition (Table 3) indicating that cell impermeability could be the cause.
On the other hand, it was observed that all piperazine derivatives (3a-q) showed medium to good growth inhibition (Table 3) against
Gram-negative bacteria E. coli. The increased growth inhibition is presumably due to easy penetration of compounds into the lipid
membranes of these organism. Regarding Gram positive bacteria S. aureus, compounds (3a, 3d-l, and 3o-q) exhibited medium growth
inhibition. Likewise, compounds (3a, 3e, 3f, 3i, 3k, and 3p) showed medium growth inhibition against K. pneumonia. Also, compound
(3g) exhibited very good growth inhibition (30.15%) towards C. albicans fungal strain, whereas (3a-f) and (3h-3q) showed moderate
growth inhibition. Regarding fungal strain C. neoformans, piperazine derivatives 3c, 3e, 3m, 3n, and 3o displayed medium to good growth
inhibition while remaining adducts; 3a, 3b, 3d, 3f-l, and 3p-q did not show any growth inhibition.

Table 3.
Antimicrobial activity of compounds (3a–q) with the concentration set at 32 g/mL in DMSO.
Percentage of inhibition of antibacterial and antifungal growth[a]
Antibacterial activity Antifungal activity
Compound Gram-positive Gram-negative bacteria
(#) Staphylococcus Escherichia Pseudomonas Klebsiella Acinetobacter Candida Cryptococcus
aureus coli aeruginosa pneumoniae baumannii albicans neoformans
 5
3a 12 ±4 18±2 3±4 14±8 8±18 5±4 -18±1
3b -4 ±5 12±2 -5±2 -1±8 13±3 12±1 -7±4
3c -1832 191 66 -812 -3529 44 2312
3d 116 178 -84 5.40.1 -215 44 -13
3e 53 26 3 9.00.1 156 46 43 1210
3f 81 203 -55 112 93 61 0 1
3g 74 62 -30.4 -93 -142 308 -82
3h 74 92 -6 1 -34 -1913 73 -3113
3i 78 182 3.70.1 121 -28 37 -227
3j 146 192 -32 1010 33 1 1 -48
3k 102 17.10.2 -11 17 2 99 -0.80.1 -123
3l 136 132 -32 42 -312 52 -2616
3m -24 173 -2.10.2 102 6 17 26 95
3n -12 28 142 -1 2 -69 -4231 56 246
3o 74 252 31 48 416 54 9 10
3p 82 182 25 114 78 6 7 -82
3q 144 113 -1 6 -25 -289 43 -26

[a] Highest percentile of antibacterial/antifungal growth inhibition are highlighted in bold. Data are expressed as the mean ±SD. SD = Standard Deviation

2.5. Molecular docking studies

To understand the mechanism of anti-microbial activity of the synthesized compounds, molecular modelling and docking studies were
performed on the X-ray crystal structure of E. coli 24 kDa domain in complex with clorobiocin (PDB code: 1KZN; resolution 2.30 Å)
using Surflex-Dock programme of Sybyl-X software. All 17 inhibitors were docked into the active site of the enzyme as shown in Fig.
3(A & B). The predicted binding energies of the compounds are listed in Table 4. The docking study revealed that all the compounds
exhibited very good docking scores against E. coli.
Table 4.
Surflex docking score (kcal/mol) of the piperazine derivatives for E. coli (PDB ID: 1KZN)

Mol. # C Scorea Crash Scoreb Polar Scorec D Scored PMF Scoree G Scoref Chem Scoreg

Clorobiocin ligand 12.14 -1.21 5.33 -189.513 -76.407 -135.711 -34.514

3a 2.93 -0.87 0.00 -66.065 -20.007 -115.193 -16.162
3b 5.48 -1.69 1.46 -114.895 -23.772 -236.789 -25.490
3c 3.60 -4.44 1.32 -122.691 -18.028 -243.061 -24.762
3d 5.38 -0.62 0.96 -103.551 -31.084 -173.693 -27.155
3e 5.41 -1.77 0.64 -128.063 -12.168 -225.387 -24.369
3f 5.54 -1.99 0.92 -131.171 -12.123 -213.098 -24.211
3g 3.64 -0.82 0.05 -95.843 -8.012 -131.015 -19.207
3h 4.99 -0.61 1.04 -109.661 -35.048 -170.947 -27.589
3i 5.55 -2.28 1.33 -131.189 1.405 -231.666 -30.644
3j 2.72 -1.19 0.00 -93.647 8.001 -149.443 -15.933
3k 5.20 -1.39 0.00 -122.741 -1.386 -230.423 -21.916
3l 5.30 -0.76 1.11 -102.217 -28.486 -168.898 -26.264
3m 3.94 -3.38 0.04 -142.042 -6.368 -245.636 -26.355
3n 3.97 -1.02 1.46 -95.522 -7.291 -154.593 -21.745
3o 2.77 -2.56 0.00 -108.918 3.070 -145.135 -20.229
3p 2.41 -0.92 1.82 -77.155 -18.425 -101.856 -15.749
3q 5.24 -1.74 1.81 -123.589 -36.455 -205.531 -30.026

aCScore (Consensus Score) integrates a number of popular scoring functions for ranking the affinity of ligands bound to the active site of a receptor and reports
the output of total score.
b Crash-score revealing the inappropriate penetration into the binding site. Crash scores close to 0 are favorable. Negative numbers indicate penetration.
c Polar indicating the contribution of the polar interactions to the total score. The polar score may be useful for excluding docking results that make no hydrogen

bonds.
d D-score for charge and van der Waals interactions between the protein and the ligand.
6 Bioorganic Chemistry
e PMF-score indicating the Helmholtz free energies of interactions for protein-ligand atom pairs (Potential of Mean Force, PMF).
f G-score showing hydrogen bonding, complex (ligand-protein), and internal (ligand-ligand) energies.
g Chem-score points for H-bonding, lipophilic contact, and rotational entropy, along with an intercept term.

Clorobiocin [Fig. 4(A-C)] was found to have hydrogen bonding interactions with ASP73 (2.04Å; 3.72Å), ASN46 (1.72 Å; 2.29Å;
2.67Å) and ARG136 (2.49Å; 1.91Å; 2.52Å). As depicted in Fig. 5(A-C), compound 3e makes two hydrogen bonding interactions at the
active site of the enzyme (PDB ID: 1KZN), one interaction was the oxygen atom of carbonyl group with hydrogen of THR165 (-C=O-
-----H-THR165, 2.31 Å) and remaining another hydrogen bonding interaction raised from the hydrogen atom of CONH group with
oxygen of ASP73 (NH-----O-ASP73, 2.00 Å). As depicted in Fig. 6(A-C), compound 3c, makes three hydrogen bonding interactions at
the active site of the enzyme (PDB ID: 1KZN). Among those, two interactions were of oxygen atom of carbonyl group with hydrogen
atoms of THR165 and GLY77 (C=O------H-THR165, 2.66 Å; C=O------H-GLY77, 2.59 Å) and the remaining another hydrogen bonding
interaction raised from the hydrogen atom of CONH group with oxygen of ASP73 (NH-----O-ASP73, 2.06 Å). Fig. 7(A & B) represents
the hydrophobic and hydrophilic amino acids surrounding compounds 3c and 3e.
All the compounds showed consensus score in the range 5.55-2.41, indicating the summary of all forces of interaction between ligands
and the protein. These scores indicate that molecules preferentially bind to protein in comparison to the reference clorobiocin (Table 4).
It was found that hydrogen bond formation with ASP73 amino acid residue may be responsible for the antibacterial activity compared
to that observed for clorobiocin.

Fig. 3. Docked view of all compounds at the active site of the enzyme PDB ID: 1KZN.
 13

Fig. 4. Docked view of Clorobiocin at the active site of the enzyme PDB ID: 1KZN.

Fig. 5. Interaction of compound 3e at the binding site of enzyme (PDB ID: 1KZN).
8 Bioorganic Chemistry

Fig. 6. Interaction of compound 3c at the binding site of enzyme (PDB ID: 1KZ).

Fig. 7. A) Hydrophobic amino acids surrounded to compounds 3c (cyan color) and 3e (green color)). B) Hydrophilic amino acids surrounded 3c and 3e.

3. Conclusion B

In summation, we have designed and synthesized a new series of piperazine derivatives containing bisaryl/thiazepine moieties. The
structures of all molecules were confirmed through IR, 1H NMR, 13C NMR, and mass spectroscopy. In addition, the chemical structure
of compound 3h was clearly confirmed by single crystal X-ray diffraction analysis. All piperazine derivatives were screened for anti-
microbial properties. In general, based on the biological evaluation results, compound 3e showed the best inhibition of antimicrobial
growth towards the bacteria and fungi strains evaluated. Additionally, gram-negative bacteria were also most inhibited with 3e. Finally,
the molecular docking studies of all prepared molecules were carried out and the results revealed that all the piperazine derivatives showed
very good docking score against E coli.

4. Experimental section: Materials and Methods

4.1. General Considerations
All chemicals, including isocyanates, were procured commercially from Sigma-Aldrich chemical company and were used without
further purification. All solvents purchased were of analytical grade and were used without further purification. All reactions were carried
out under aerobic conditions, in oven-dried glassware, with magnetic stirring. Heating was accomplished by either a heating mantle or
silicone oil bath. Reactions were monitored by thin-layer chromatography (TLC) performed on 0.25 mm Merck TLC silica gel plates,
using UV light as a visualizing agent. Purification of reaction products was carried out by flash column chromatography using silica gel
60 (230-400 mesh). Yields refer to chromatographically pure material. Concentration in vacuo refers to the removal of volatile solvent,
using a rotary evaporator attached to a dry diaphragm pump (10-15 mm Hg), followed by pumping to a constant weight with an oil pump
(<300 mTorr). 1H NMR spectra were recorded on a JEOL Eclipse Plus 500 (500 MHz), and are reported relative to CDCl3 (δ 7.26) or
DMSO-d6 (δ 2.50). 1H NMR coupling constants (J) are reported in Hertz (Hz) and multiplicities are indicated as follows: s (singlet), d
(doublet), t (triplet), quint (quintet), m (multiplet). Proton-decoupled 13C NMR spectra were recorded on JEOL Eclipse Plus 500 (125
MHz) and reported relative to CDCl3 (δ 77.00) or DMSO-d6 (δ 39.52). X-ray diffraction data for compound (3h) was collected using Mo-
 9
Kα radiation and a Bruker SMART APEXII diffractometer [15]. The structure was solved by direct method using SHELXS-97 and
refined by full-matrix least-squares on F2 for all data using SHELXL-97 at 100 K [21]. An analytical absorption correction, based on the
shape of the crystal, was performed. All hydrogen atoms were added at calculated positions and refined using a riding model. Anisotropic
thermal displacement parameters were used for all non-hydrogen atoms. Additional details involving data collection and reliability factors
are listed in Table 1. CCDC 1939260 (for 3h), contain the supplementary crystallographic data for this paper. These data can be obtained
free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif.
4.2. Synthesis
4.2.1. General experimental procedure for synthesis of piperazine derivatives
To a solution of isocyanate (1.877 mmol) in toluene (2.5 mL) was added a solution of a monosubstituted piperazine (1.877 mmol) in
toluene (1.0 mL). The reaction mixture was heated at 40-45 °C for 30 to 60 minutes. The reaction mixture was then cooled down to room
temperature (22-25 °C) and the resulting solids were filtered and washed with more toluene (2.0 mL). The wet solids were then placed in
2.0 mL of toluene, stirred at room temperature for about 30 minutes, filtered and washed with toluene (1.0 mL) to obtain the crude
disubstituted piperazine derivative. Finally, all crude derivatives were purified by silica-gel column chromatography using a mixture of
dichloromethane/methanol (9:1) to afford pure piperazines products.
4.2.1.1. Synthesis of 4-benzhydryl-N-(p-tolyl)piperazine-1-carboxamide (3a)
Compound (3a) was synthesized from 4-methyl phenyl isocyanate (0.25g, 1.877 mmol) and 1-benzhydrylpiperazine (0.473 g, 1.877
mmol) according to the general procedure. White solid. Yield: 78% (0.56 g).1H NMR (CDCl3, 500 MHz) δ 7.43 (d, J = 7.5 Hz, 4H), 7.30
(t, J = 7.5 Hz, 4H), 7.22-7.19 (m, 4H), 7.06 (d, J = 8.0 Hz, 2H), 6.33(s, 1H), 4.25 (s, 1H), 3.47 (t, J = 4.5 Hz, 4H), 2.42 (t, J = 4.5 Hz,
4H), 2.29 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ 155.2, 142.2, 136.3, 132.6, 129.3, 128.6, 127.8, 127.1, 75.9, 51.5, 44.2, 20.7.IR (KBr):
ῡ =3257.05, 2952.2, 2810.92, 1637.37, 1598.01, 1287.71, 809.10. LC-MS for C25H27N3O: m/z = 386 [M+H]+.
4.2.1.2. Synthesis of 4-((4-fluorophenyl)(phenyl)methyl)-N-(p-tolyl)piperazine-1-carboxamide (3b)
Compound (3b) was synthesized from 4-methylphenyl isocyanate (0.25g, 1.877 mmol) and 1-((4-
fluorophenyl)(phenyl)methyl)piperazine (0.50 g, 1.877 mmol) according to the general procedure. White solid. Yield: 74%, (0.56 g).1H
NMR (CDCl3, 500 MHz) δ 7.40-7.38 (m, 4H), 7.30 (t, J = 7.0 Hz, 2H), 7.23-7.19 (m, 3H), 7.06 (d, J = 8.0 Hz, 2H), 6.99 (t, J = 8.5 Hz,
2H), 6.39 (s, 1H), 4.24 (s, 1H), 3,46 (t, J = 4.5 Hz, 4H), 2.41-2.39 (m, 4H), 2.29 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ 161.8 (d, 1JC,F =
244.4 Hz), 155.2, 141.9, 138.0, 136.3, 132.6, 129.3, 129.2, 127.7, 127.2, 120.2, 115.5, 115.3, 75.1, 51.4, 44.1, 20.7. IR (KBr): ῡ = 3313.58,
2790.2, 2948.15, 1637.09, 1515.01, 1237.92, 813.40. LC-MS for C25H26FN3O: m/z = 404 [M+H]+.
4.2.1.3. Synthesis of 4-((2-fluorophenyl)(4-fluorophenyl)methyl)-N-(p-tolyl)piperazine-1-carboxamide (3c)
Compound (3c) was synthesized from 4-methylphenyl isocyanate (0.25 g, 1.877 mmol) and 1-((2-fluorophenyl)(4-
fluorophenyl)methyl)piperazine (0.54 g, 1.877 mmol) according to the general procedure. White solid. Yield: 70% (0.55 g). 1H NMR
(CDCl3, 500 MHz) δ 7.58 (t, J = 7.0 Hz, 1H), 7.42-7.39 (m, 2H), 7.20 (d, J = 8.5 Hz, 3H), 7.13 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.0 Hz,
2H), 6.99 (t, J = 8.5 Hz, 3H), 6.41 (s, 1H), 4.70 (s, 1H), 3.46 (t, J = 4.5 Hz, 4H), 2.45-2.38 (m, 4H), 2.29 (s, 3H). 13C NMR (CDCl3, 125
MHz) δ 161.9 (d, 1JC,F = 244.4 Hz), 160.5 (d, 1JC,F = 244.4 Hz), 155.2, 136.9, 136.3, 132.7, 129.5 (d, 3JC,F = 8.4 Hz), 129.3, 128.8, 128.7,
128.5 (d, 3JC,F = 8.4 Hz), 128.4, 124.4, 120.3, 115.7, 115.4 (d, 2JC,F = 21.4 Hz),, 65.9, 51.3, 44.1, 20.7. IR (KBr): ῡ = 3265.25, 2810.19,
2952.8, 1634.57, 1508.80, 1249.81, 811.38. LC-MS for C25H25F2N3O: m/z = 422 [M+H]+.
4.2.1.4. Synthesis of 4-(dibenzo[b,f][1,4]thiazepin-11-yl)-N-(p-tolyl)piperazine-1-carboxamide (3d)
Compound (3d) was synthesized from 4-methylphenyl isocyanate (0.25 g, 1.877 mmol) and 11-piperazin-1-yl-dibenzo
[b,f][1,4]thiazepine (0.55 g, 1.877 mmol) according to the general procedure. White solid. Yield = 76% (0.61 g). 1H NMR (DMSO-d6,
500 MHz) δ 8.50 (s, 1H), 7.57-7.56 (m, 1H), 7.49-7.43 (m, 3H), 7.40-7.38 (m, 1H), 7.34 (d, J = 8.5 Hz, 2H), 7.22-7.16 (m, 1H), 7.05-
7.03 (m, 3H), 6.91 (td, J = 7.5, 1.0 Hz, 1H), 3.60-3.47 (m, 8H), 2.23 (s, 3H). 13C NMR (DMSO-d6, 125 MHz) δ 160.1, 155.2, 148.5,
138.7, 137.8, 133.5, 132.1, 132.0, 131.4, 130.7, 129.3, 129.1, 129.0, 128.9, 128.8, 127.2, 125.1, 122.7, 119.9, 43.4, 20.4. IR (KBr): ῡ =
3251.4, 2849.73, 2916.4, 1638.69, 1513.45, 1250.15, 810.81. LC-MS for C25H24N4OS: m/z = 429 [M+H]+.
4.2.1.5. Synthesis of 4-benzhydryl-N-(4-chlorophenyl) piperazine-1-carboxamide (3e)
Compound (3e) was synthesized from 4-chloro phenyl isocyanate (0.25 g, 1.627 mmol) and 1-benzhydrylpiperazine (0.41 g, 1.627
mmol) according to the general procedure. White solid. Yield = 75% (0.495 g). 1H NMR (DMSO-d6, 500 MHz) δ 8.60 (s, 1H), 7.48 (dt,
J = 9.0, 2.5 Hz, 2H), 7.44 (d, J = 7.5 Hz, 4H), 7.30 (t, J = 7.5 Hz, 4H), 7.25 (dt, J = 9.5, 2.5 Hz, 2H), 7.19 (t, J = 7.5 Hz, 2H), 4.34 (s,
1H), 3.46 (t, J = 4.5 Hz, 4H), 2.32 (t, J = 4.0 Hz, 4H). 13C NMR (DMSO-d6, 125 MHz) δ 154.7, 142.5, 139.5, 128.6, 128.1, 127.6, 127.0,
125.3, 121.0, 74.8, 51.4, 43.8. IR (KBr): ῡ = 3298.40, 2793.08, 2954.76, 1635.53, 1529.81, 1240.67, 821.77. LC-MS for C24H24ClN3O:
m/z = 406 [M+H]+.
4.2.1.6. Synthesis of N-(4-chlorophenyl)-4-((4-fluorophenyl)(phenyl)methyl)piperazine-1-carboxamide (3f)
Compound (3f) was synthesized from 4-chloro phenyl isocyanate (0.25 g, 1.627 mmol) and 1-((4-fluorophenyl)
(phenyl)methyl)piperazine (0.44 g, 1.627 mmol) according to the general procedure. White solid. Yield = 71% (0.49 g). 1H NMR (DMSO-
d6, 500 MHz) δ 8.67 (s, 1H), 7.50-7.41 (m, 6H), 7.31 (t, J = 7.0 Hz, 2H), 7.25 (d, J = 9.0 Hz, 2H), 7.20 (t, J = 7.5 Hz, 1H), 7.13 (t, J = 8.0
Hz, 2H), 4.38 (s, 1H), 3.47 (s, 4H), 2.30 (s, 4H). 13C NMR (DMSO-d6, 125 MHz) δ 161.07 (d, 1JC,F= 240.9 Hz), 154.7, 142.3, 139.6,
138.7, 129.5 (d, 3JC,F = 8.4 Hz), 128.6, 128.1, 127.6, 127.0, 125.2, 121.0, 115.3 (d, 2JC,F = 20.3 Hz), 73.7, 51.3, 43.8. IR (KBr): ῡ = 3305.20,
2791.24, 2955.9, 1634.24, 1504.97, 1282.91, 829.67. LC-MS for C24H23ClFN3O: m/z = 424 [M+H]+.
4.2.1.7. Synthesis of N-(4-chlorophenyl)-4-((2-fluorophenyl)(4-fluorophenyl)methyl)piperazine-1-
carboxamide (3g)
10 Bioorganic Chemistry
Compound (3g) was synthesized from 4-chlorophenyl isocyanate (0.25 g, 1.627 mmol) and 1-((2-fluorophenyl)(4-
fluorophenyl)methyl)piperazine (0.469 g, 1.627 mmol) according to the general procedure. White solid. Yield = 70% (0.50 g). 1H NMR
(CDCl3, 500 MHz) δ 7.57 (t, J = 7.0 Hz, 1H), 7.40 (dd, J = 8.0, 6.0 Hz, 2H), 7.27-7.25 (m, 2H), 7.21-7.18 (m, 3H), 7.13 (t, J = 7.5 Hz,
1H), 6.99 (t, J = 8.5 Hz, 3H), 6.50 (s, 1H), 4.70 (s, 1H), 3.47 (t, J = 4.5 Hz, 4H), 2.46-2.39 (m, 4H). 13C NMR (CDCl3, 125 MHz) δ 161.1
(d, 1JC,F= 244.4 Hz), 160.6 (d, 1JC,F = 245.6 Hz), 154.7, 137.5, 136.8, 129.5 (d, 3JC,F = 7.1 Hz), 128.8, 128.6 (d, 3JC,F = 8.4 Hz), 128.4,
128.0, 124.4, 121.2, 115.8, 115.6, 115.4, 65.9, 51.2, 44.1. IR (KBr): ῡ = 3264.76, 2818.08, 2951.4, 1640.27, 1508.64, 1280.07, 825.60.
LC-MS for C24H22ClF2N3O: m/z = 442 [M+H]+.
4.2.1.8. Synthesis of N-(4-chlorophenyl)-4-(dibenzo[b,f][1,4]thiazepin-11-yl)piperazine-1-carboxamide (3h)
Compound (3h) was synthesized from 4-chloro phenyl isocyanate (0.25 g, 1.627 mmol) and 11-Piperazin-1-yl-dibenzo [b,f][1,4]
thiazepine (0.48 g, 1.627 mmol) according to the general procedure. White solid. Yield = 72% (0.526 g). 1H NMR (CDCl3, 500 MHz) δ
7.54 (d, J = 7.5 Hz, 1H), 7.41 (dd, J = 7.3, 1.0 Hz, 1H), 7.39-7.35 (m, 1H), 7.34-7.30 (m, 4H), 7.24-7.18 (m, 3H), 7.11 (dd, J = 8.0, 1.0
Hz, 1H), 6.93 (td, J = 7.5, 1.5 Hz, 1H), 6.65 (s, 1H), 3.73-3.48 (m, 8H). 13C NMR (CDCl3, 125 MHz) δ 160.7, 154.9, 140.0, 137.5, 133.6,
132.3, 132.2, 131.2, 129.2, 129.0, 128.9,128.8, 128.5, 128.2, 125.3, 123.5, 121.3, 43.6. IR (KBr): ῡ = 3356, 2840.53, 2891.01, 1626.31,
1518.95, 1239.22, 803.49. LC-MS for C24H21ClN4OS: m/z = 449 [M+H]+.
4.2.1.9. Synthesis of 4-benzhydryl-N-(4-fluorophenyl) piperazine-1-carboxamide (3i)
Compound (3i) was synthesized from 4-fluorophenyl isocyanate (0.25 g, 1.823 mmol) and 1-benzhydrylpiperazine (0.46 g, 1.823
mmol) according to the general procedure. White solid. Yield = 77% (0.546 g). 1H NMR (CDCl3, 500 MHz) δ 7.42 (d, J = 7.0 Hz, 4H),
7.31-7.28 (m, 6H), 7.22-7.17 (m, 2H), 6.98-6.93 (m, 2H), 6.31 (s, 1H), 4.26 (s, 1H), 3.47 (s, 4H), 2.43 (s, 4H). 13C NMR (CDCl3, 125
MHz) δ 158.9 (d, 1JC,F = 240.8 Hz), 155.1, 142.1, 134.8, 128.6, 127.9, 127.2, 121.9 (d, 3JC,F = 8.4 Hz), 115.8 (d, 2JC,F = 22.6 Hz), 75.9,
51.5, 44.2. IR (KBr): ῡ = 3302.39, 2794.53, 2951.98, 1630.84, 1510.68, 1287.08, 834.38. LC-MS for C24H24FN3O: m/z = 390 [M+H]+.
4.2.1.10. Synthesis of N-(4-fluorophenyl)-4-((4-fluorophenyl)(phenyl)methyl)piperazine-1-carboxamide (3j)
Compound (3j) was synthesized from 4-fluorophenyl isocyanate (0.25 g, 1.823 mmol) and 1-((4-fluorophenyl)
(phenyl)methyl)piperazine (0.49 g, 1.823 mmol) according to the general procedure. White solid. Yield = 74% (0.549g). 1H NMR (CDCl3,
500 MHz) δ 7.40-7.37 (m, 4H), 7.31-7.27 (m, 3H), 7.25-7.20 (m, 2H), 7.00-6.94 (m, 4H), 6.31 (s, 1H), 4.25 (s, 1H), 3.47 (t, J = 4.5 Hz,
4H), 2.42 (t, J = 3.0 Hz, 4H). 13C NMR (CDCl3, 125 MHz) δ 160.89, 158.9 (d, 1JC,F = 240.8 Hz), 155.1, 141.9, 137.9, 134.8, 129.3 (d,
3J
C,F = 7.3 Hz), 128.7, 127.3, 122.0, 121.9, 115.5 (d, JC,F = 21.4 Hz), 115.4 (d, JC,F = 22.6 Hz), 75.1, 51.4, 44.2. IR (KBr): ῡ = 3311.8,
2 2

2793.8, 2951.8, 1633, 1509, 1209.9, 835.8. LC-MS for C24H23F2N3O: m/z = 408 [M+H]+.
4.2.1.11. Synthesis of N-(4-fluorophenyl)-4-((2-fluorophenyl)(4-fluorophenyl)methyl)piperazine-1-
carboxamide (3k)
Compound (3k) was synthesized from 4-fluorophenyl isocyanate (0.25 g, 1.823 mmol) and 1-((2-fluorophenyl)(4-
fluorophenyl)methyl)piperazine (0.525 g, 1.823 mmol) according to the general procedure. White solid. Yield = 70% (0.543g). 1H NMR
(DMSO-d6, 500 MHz) δ 8.55 (s, 1H), 7.64 (t, J = 7.0 Hz, 1H), 7.46-7.43 (m, 4H), 7.29-7.21 (m, 2H), 7.17-7.11 (m, 3H), 7.05 (t, J = 9.0
Hz, 2H), 4.71 (s, 1H), 3.47 (s, 4H), 2.35-2.28 (m, 4H). 13C NMR (DMSO-d6, 125 MHz) δ 160.2, 160.0 (d, 1JC,F= 242.0 Hz), 158.3,
156.4, 155.0, 137.2, 136.8, 129.7, 128.9, 128.7, 124.8, 121.4, 121.3, 115.7, 115.4 (d, 2JC,F = 21.5 Hz), 114.8 (d, 2JC,F = 22.6 Hz), 65.7,
51.2, 43.8. IR (KBr): ῡ = 3307, 2814.18, 1628.47, 1506.23, 1223.83, 816.77. LC-MS for C24H22F3N3O: m/z = 426 [M+H]+.
4.2.1.12. Synthesis of 4-(dibenzo[b,f][1,4] thiazepin-11-yl)-N-(4-fluorophenyl)piperazine-1-carboxamide
(3l)
Compound (3l) was synthesized from 4-fluorophenyl isocyanate (0.25 g, 1.823 mmol) and 11-piperazin-1-yl-dibenzo
[b,f][1,4]thiazepine (0.538 g, 1.823 mmol) according to the general procedure. White solid. Yield = 71% (0.56 g). 1H NMR (DMSO-d6,
500 MHz) δ 8.64 (s, 1H), 7.58-7.55 (m, 1H), 7.49-7.45 (m, 5H), 7.39 (dd, , J = 7.5, 2.0 Hz, 1H), 7.20 (td, J = 7.5, 1.0 Hz, 1H), 7.11-7.05
(m, 2H), 7.03 (dd, J = 7.8, 1.5 Hz, 1H), 6.91 (td, J = 7.5, 1.0 Hz, 1H), 3.61-3.39 (m, 8H). 13C NMR (DMSO-d6, 125 MHz) δ 160.1, 158.4,
156.5, 155.2, 148.5, 138.7, 136.7, 133.4, 132.1, 132.0, 131.4, 129.3, 129.1, 129.0, 128.2, 127.3, 125.1, 122.8, 121.5 (d, 3JC,F = 8.3 Hz),
114.8 (d, 2JC,F = 22.6 Hz), 43.4. IR (KBr): ῡ = 3342.7, 2859.21, 2892.1, 1641.98, 1508.33, 1240.54, 812.81. LC-MS for C24H21FN4OS:
m/z = 433 [M+H]+.
4.2.1.13. Synthesis of 4-benzhydryl-N-(3,4-dichlorophenyl)piperazine-1-carboxamide (3m)
Compound (3m) was synthesized from 3,4-dichlorophenyl isocyanate (0.25 g, 1.329 mmol) and 1-benzhydrylpiperazine (0.335 g,
1.329 mmol) according to the general procedure. White solid. Yield = 74% (0.433 g). 1H NMR (DMSO-d6, 500 MHz) δ 8.76 (s, 1H),
7.82 (d, J = 2.0 Hz, 1H), 7.46-7.41 (m, 6H), 7.3 (t, J = 8.0 Hz, 4H), 7.25-7.14 (m, 2H), 4.34 (s, 1H), 3.47 (t, J = 4.5 Hz, 4H), 2.33-2.29
(m, 4H). 13C NMR (DMSO-d6, 125 MHz) δ 154.4, 142.5, 140, 130.6, 130.1, 128.6, 127.6, 127.0, 122.9, 120.4, 119.2, 74.7, 51.3, 43.8. IR
(KBr): ῡ = 3258.54, 2814.75, 2953, 1642.66, 1505.68, 1245.13, 823.55. LC-MS for C24H23Cl2N3O: m/z = 441 [M+H]+.
4.2.1.14. Synthesis of N-(3,4-dichlorophenyl)-4-((4-fluorophenyl)(phenyl)methyl)piperazine-1-carboxamide
(3n)
Compound (3n) was synthesized from 3,4-dichlorophenyl isocyanate (0.25 g, 1.329 mmol) and 1-((4-fluorophenyl)
(phenyl)methyl)piperazine (0.36 g, 1.329 mmol) according to the general procedure. White solid. Yield = 76% (0.46 g). 1H NMR (DMSO-
d6, 500 MHz) δ 8.83 (s, 1H), 7.83 (s, 1H), 7.47-7.41 (m, 6H), 7.31 (t, J = 7.5 Hz, 2H), 7.20 (t, J = 7.0 Hz, 1H), 7.13 (t, J = 8.5 Hz, 2H),
4.38 (s, 1H), 3.47 (s, 4H), 2.30 (s, 4H). 13C NMR (DMSO-d6, 125 MHz) δ 161.1 (d, 1JC,F = 240.9 Hz), 154.4, 142.2, 140.9, 138.6, 130.5,
130.1, 129.5 (d, 3JC,F = 8.4 Hz), 128.6, 127.6, 127.0, 122.8, 120.3, 119.2, 115.3 (d, 2JC,F = 21.4 Hz), 73.7, 51.2, 43.8. IR (KBr): ῡ = 3301.2,
2791.11, 2952.46, 1637.27, 1505, 1239.68, 823.96. LC-MS for C24H22Cl2FN3O: m/z = 459 [M+H]+.
 11
4.2.1.15. Synthesis of N-(3,4-dichlorophenyl)-4-((2-fluorophenyl)(4-fluorophenyl)methyl)piperazine-1-
carboxamide (3o)
Compound (3o) was synthesized from 3,4-dichlorophenyl isocyanate (0.25 g, 1.329 mmol) and 1-((2-fluorophenyl) (4-fluorophenyl)
methyl) piperazine (0.383g, 1.329 mmol) according to the general procedure. White solid. Yield = 72% (0.456 g). 1H NMR (DMSO-d6,
500 MHz) δ 8.79 (s, 1H). 7.81 (d, J = 2.0 Hz, 1H), 7.65-7.62 (m, 1H), 7.46-7.41 (m, 4H), 7.29-7.21 (m, 2H), 7.17-7.11 (m, 3H), 4.71 (s,
1H), 3.47 (t, J = 4.5 Hz, 4H), 2.39-2.29 (m, 4H). 13C NMR (DMSO-d6, 125 MHz) δ 161.7 (d, 1JC,F = 240.9 Hz), 160.5 (d, 1JC,F = 243.3
Hz), 154.9, 141.4, 137.7, 131.1, 130.7, 130.2 (d, 3JC,F = 8.4 Hz), 129.5 (d, 3JC,F= 8.4 Hz), 129.2, 129.1, 125.3, 123.4, 120.9, 119.8, 116.3,
116.0 (d, 2JC,F = 20.3 Hz), 66.1, 51.6, 44.3. IR (KBr): ῡ = 3267.2, 2815.5, 2953, 1640.51, 1507.84, 1244.97, 821.59. LC-MS for
C24H21Cl2F2N3O: m/z = 477 [M+H]+.
4.2.1.16. Synthesis of 4-(bis(4-fluorophenyl) methyl)-N-(3,4-dichlorophenyl)piperazine-1-carboxamide (3p)
Compound (3p) was synthesized from 3,4-dichlorophenyl isocyanate (0.25 g, 1.329 mmol) and 1-(bis(4-fluorophenyl)
methyl)piperazine (0.383 g, 1.329 mmol) according to the general procedure. White solid. Yield = 75% (0.475 g). 1H NMR (DMSO-d6,
500 MHz) δ 8.77 (s, 1H), 7.81 (d, J = 2.5 Hz, 1H), 7.46-7.41 (m, 6H), 7.15-7.12 (m, 4H), 4.43 (s, 1H), 3.46 (t, J = 4.5 Hz, 4H), 2.29 (t, J
= 4.5 Hz, 4H). 13C NMR (DMSO-d6, 125 MHz) δ 162.1, 160.2, 154.4, 140.9, 138.3, 130.6, 130.1, 129.5, 129.4, 122.9, 120.4, 119.2,
115.5, 115.3, 72.7, 51.1, 43.8. IR (KBr): ῡ = 3305.63, 2789.71, 2952.67, 1637.30, 1505.19, 1235.69, 818.05. LC-MS for C24H21Cl2F2N3O:
m/z = 477 [M+H]+.

4.2.1.17. Synthesis of 4-(dibenzo[b,f][1,4]-thiazepin-11-yl)-N-(3,4-dichlorophenyl)piperazine-1-

carboxamide (3q)
Compound (3q) was synthesized from 3,4-dichlorophenyl isocyanate (0.25 g, 1.329 mmol) and 11-piperazin-1-yl-dibenzo
[b,f][1,4]thiazepine (0.392 g, 1.329 mmol) according to the general procedure. White solid. Yield = 73% (0.469 g). 1H NMR (CDCl3, 500
MHz) δ 7.58 (d, J = 2.0 Hz, 1H), 7.54 (d, J = 7.5 Hz, 1H), 7.41 (dd, J = 7.8, 1.0 Hz, 1H), 7.39-7.30 (m, 4H), 7.21-7.18 (m, 2H), 7.11 (dd,
J = 8.0, 1.5 Hz, 1H), 6.93 (td, J = 7.8, 1.0 Hz, 1H), 6.70 (s, 1H), 3.63-3.49 (m, 8H). 13C NMR (CDCl3, 125 MHz) δ 160.7, 154.5, 140.0,
138.5, 133.6, 132.5, 132.4, 132.3, 131.3, 129.2, 128.9, 128.5, 126.2, 125.3, 123.5, 121.6, 119.2, 43.6. IR (KBr): ῡ = 3239.58, 2870.6,
2898.6, 1632.92, 1503.66, 1243.45, 807.94. LC-MS for C24H20Cl2N4OS: m/z = 484 [M+H]+.
4.3. Antimicrobial studies
Samples were prepared in DMSO and water to a final testing concentration of 32 μg/mL or 20 μM (unless otherwise indicated in the
data sheet), in a 384-well, non-binding surface plate (NBS) for each bacterial/fungal strain, and in duplicate (n = 2), keeping the final
DMSO concentration to a maximum of 1% DMSO [22-26]. All the sample preparations for antimicrobial studies where done using liquid
handling robots.
Antimicrobial Assay
Primary antimicrobial screening study via whole cell growth inhibition assays were carried out using compounds (3a-q) at a single
concentration, in duplicate (n = 2). The inhibition of growth was measured against five bacteria: Escherichia coli (E. coli) ATCC 25922,
Klebsiella pneumonia (K. pneumoniae) ATCC 700603, Acinetobacter baumannii (A. Baumannii) ATCC 19606, Pseudomonas aeruginosa
(P. Aeruginosa) ATCC 27853) and Staphylococcus aureus (S. aureus) ATCC 43300, and two fungi: Candida albicans (C. albicans) ATCC
90028 and Cryptococcus neoformans (C. Neoformans) ATCC 208821 [27].
Procedure
All bacteria were cultured in Cation-adjusted Mueller Hinton broth (CAMHB) at 37 °C overnight. A sample of each culture was then
diluted 40-fold in fresh broth and incubated at 37 °C for 1.5-3 h. The resultant mid-log phase cultures were diluted (CFU/mL measured
by OD600), then added to each well of the compound containing plates, giving a cell density of 5x105 CFU/mL and a total volume of 50
μL. All plates were covered and incubated at 37 °C for 18 h without shaking.
Analysis
Inhibition of bacterial growth was determined measuring absorbance at 600 nm (OD600), using a Tecan M1000 Pro monochromator
plate reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control
(bacteria without inhibitors) on the same plate as references. The significance of the inhibition values was determined by modified Z-
scores, calculated using the median and MAD of the samples (no controls) on the same plate. Samples with inhibition value above 80%
and Z-Score above 2.5 for either replicate (n = 2 on different plates) were classed as actives. Samples with inhibition values between 50
- 80% and Z-Score above 2.5 for either replicate (n = 2 on different plates) were classed as partial actives.
Antifungal Assay
Procedure
Fungi strains were cultured for 3 days on Yeast Extract-Peptone Dextrose (YPD) agar at 30 °C. A yeast suspension of 1 x 106 to 5 x
106 CFU/mL (as determined by OD530) was prepared from five colonies. The suspension was subsequently diluted and added to each
well of the compound-containing plates giving a final cell density of fungi suspension of 2.5 x 103 CFU/mL and a total volume of 50 μL.
All plates were covered and incubated at 35 °C for 24 h without shaking.
Analysis
12 Bioorganic Chemistry
Growth inhibition of C. albicans was determined measuring absorbance at 530 nm (OD530), while the growth inhibition of C.
neoformans was determined measuring the difference in absorbance between 600 and 570 nm (OD600-570), after the addition of resazurin
(0.001% final concentration) and incubation at 35 °C for additional 2 h. The absorbance was measured using a Biotek Synergy HTX plate
reader. The percentage of growth inhibition was calculated for each well, using the negative control (media only) and positive control
(fungi without inhibitors) on the same plate. The significance of the inhibition values was determined by modified Z-scores, calculated
using the median and MAD of the samples (no controls) on the same plate. Samples with inhibition value above 80% and Z-Score above
2.5 for either replicate (n = 2 on different plates) were classed as actives. Samples with inhibition values between 50-80% and Z-Score
above 2.5 for either replicate (n = 2 on different plates) were classed as partial actives.
4.4. Docking simulations
For the docking of ligands to protein active sites, and for estimating the binding affinities of docked compounds, Surflex-Dock module,
a fully automatic docking tool available on Sybyl X-2.0version (Tripos Inc.), was used in this study [28]. Docking simulations: The X-
ray Crystal Structure of E. coli 24 kDa Domain in Complex with clorobiocin (PDB code: 1KZN; resolution 2.30A; http://www. rcsb.org)
was obtained from protein data bank in PDB format as starting point. Protein structure with all water molecules deleted was used for
docking simulations. Mislabeled atom types from the pdb file were corrected, subsequently, proline F angles were fixed at 700 side chain
amides were checked to maximize potential hydrogen bonding, side chains were checked for close van der Waals contacts, and essential
hydrogens were added. The model was checked for conformational problems using the module ProTable from Sybyl. Ramachandran plot
of the backbone torsion angles phi and psi, local geometry and the location of buried polar residues/exposed non-polar residues were
examined. The protein was subjected to energy minimization following the gradient termination of the Powell method for 3000 iterations
using Kollman united force field, with non-bonding cut-off set at 9.0 and the dielectric constant set at 4.0. Energy minimization for
synthesized compounds, including clorobiocin, were carried out by the Powell method for 3000 iterations using Tripos force field and
Gasteiger charge with nonbonding cut-off set at 9.0 and the dielectric constant set at 4.0. The synthesized compounds were docked to
DNA gyrase subunit A (PDB code: 1KZN) using Surflex-Dock programme in Sybyl software by incremental construction approach of
building the structure in the active site so as to favor the binding affinity. Finally, the docked ligands were ranked based on a variety of
scoring functions that have been compiled into the single consensus score (C-score).

Acknowledgments

Florida Gulf Coast University and the University of Texas at Arlington partially supported this work. The ACS Petroleum Research
Fund supported this research (grant # 58269-ND1). The authors also acknowledge Dr. Delphine Gout for X-ray data collection and UTA
for additional instrumentation. The authors thank DST-Nanomission, India (SR/NM/NS-20/2014), DST-SERB, India
[SERB/F/1423/2017–18 (File No. YSS/2015/000010)] and Jain University, India for financial support.
Supplementary Material
Supplementary data [1H and13C NMR data of all the compounds (3a-q)] associated with this article can be found, in the online version,
at http://

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Design, Synthesis, and Molecular Docking Study, of New Piperazine Derivatives

as Potential Antimicrobial Agents
Mahadev Patila, Anurag Noonikara Poyilb, Shrinivas D. Joshic, Shivaputra A. Patild, Siddappa A. Patila*,
Alejandro Bugarinb

HIGHLIGHTS:

1. A simple method for the direct access to piperazines is documented

2. Good functional group tolerance, including fluorinated compounds
3. Excellent docking scores were observed
4. Extensive biological screening for both bacteria and fungi are reported
5. Comprehensive adducts’ characterization was performed

———
 Corresponding authors. Tel.: +1-817-272-9399: e-mail address: bugarin@uta.edu (A. B.) e-mail address: p.siddappa@Jainuniversity.ac.in