Design, synthesis and anti-bacterial studies of piperazine derivatives against drug resistant bacteria

Article abstract of DOI:10.1016/j.ejmech.2019.01.062

In current research, five series of mono- and di-substituted piperazine derivatives have been synthesized. For di-substituted derivatives, ciprofloxacin was selected and hybrids were synthesized via substitution at piperazinyl-N⁴. In vitro anti

Full text of DOI:10.1016/j.ejmech.2019.01.062

Accepted Manuscript
Design, synthesis and anti-bacterial studies of piperazine derivatives against drug
resistant bacteria
Saba Tahir, Tariq Mahmood, Faiqa Dastgir, Ihsan ul-Haq, Amir Waseem, Umer
Rashid
PII:
S0223-5234(19)30081-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.062
EJMECH 11069
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 2 October 2018
Revised Date: 24 January 2019
Accepted Date: 24 January 2019
Please cite this article as: S. Tahir, T. Mahmood, F. Dastgir, I. ul-Haq, A. Waseem, U. Rashid, Design,
synthesis and anti-bacterial studies of piperazine derivatives against drug resistant bacteria, European
Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.01.062.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, Synthesis and Anti-bacterial Studies of Piperazine
Derivatives against Drug Resistant Bacteria
Saba Tahir 1, Tariq Mahmood 1, Faiqa Dastgir2, Ihsan-ul-Haq2,
Amir Waseem 3, Umer Rashid 1, *
1Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus,
Abbottabad-22060, Pakistan
2 Department of Pharmacy, Faculty of Biological Sciences, Quaid-i-Azam University,
Islamabad 45320, Pakistan
3 Department of Chemistry, Quaid-i-Azam University, Islamabad 45320, Pakistan
Corresponding author. *E-mail: umerrashid@cuiatd.edu.pk; umer_rashid39@hotmail.com
(Umer Rashid);
Tel. Off.: +92 (992) 383591-6
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Abstract
In current research, five series of mono- and di-substituted piperazine derivatives have been
synthesized. For di-substituted derivatives, ciprofloxacin was selected and hybrids were
synthesized via substitution at piperazinyl-N4. In vitro antibacterial studies of all synthesized
compound were carried out against American Type Culture Collection (ATCC) strains; E. coli
(ATCC 25922), P. aeruginosa (ATCC 15442), K. pneumoniae (ATCC 1705), B. subtilis (ATCC
6633) and S. aureus (ATCC 6538). The potent series of compounds were further evaluated for
their potential against clinically isolated resistant strains of E. coli, P. aeruginosa, S. aureus, and
S. hemolytic. The reaction of piperazinyl-NH of ciprofloxacin with selected drugs resulted in
pronounced growth inhibition of standard as well as resistant bacterial strains. Hybrid
compounds 14b, 16a, 16d and CGS-20 showed excellent bacterial growth inhibition against
standard and resistant strains. In vitro results were further correlated by using in silico tools.
Molecular docking studies were carried out using MOE (Molecular Operating Environment)
software. DNA gyrase used as a target and all compounds were docked against this specific
target.
Key words: Piperazine; Ciprofloxacin hybrids; Isoniazid; Gabapentin; Resistant strains
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1. Introduction
In human history, the discovery of antibiotics was a turning point that has revolutionized medical
science for the treatment of chronic parasitic, fungal and bacterial infections [1,2]. Infectious
diseases represent a significant health threat and have affected significant portions of the human
population globally [3,4]. However, due to rapid outbreak of resistance, the efficacy of these
wonder drugs is diminishing against their targeted bacteria [5,6]. Drug resistance incidences are
increasing every year throughout the world and majority of Hospital acquired infections are
caused by ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae,
Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species) pathogenic
bacteria [7-9]. World Health Organization (WHO) pays utmost attention toward urgent discovery
of novel antibiotics against resistant strains [10,11]. New therapeutic agents are not able to show
their antimicrobial functions to intracellular targets due to outer bacterial-envelope, which is
efficient in restricting the passage of these drugs [12,13].
In recent years, drug design researchers followed the hybrid drug strategy to discover synthetic
antimicrobial scaffolds that may overcome drug resistance. Different chemical fragments fused
together with cleavable or non-cleavable tether through covalent bond to form biologically active
heteromeric molecules. Cleavable linker constitutes pro-drug approaches as compared to non-
cleavable linker approach. Prototypical drug hybrids may utilize either one of the pharmacophore
or both pharmacophoric domains simultaneously by performing its antibacterial action [14-16].
Among other hybrids reported, ciprofloxacin drug hybrids (Figure 1) possess broad
antimicrobial spectrum against Gram-negative bacilli with different biological active fragments.
Trimethoprim-ciprofloxacin hybrid BP-4Q-002 (1) exhibits potent activity against Gram-positive
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(S. epidermidis and S. aureus) and Gram-negative E. coli with MIC50 = 0.5 µg/mL and 1 µg/mL
[17]. Wang et al. synthesized tyrosyl-tRNA synthetase (TyrRS) inhibitor 2 by linking aryl furan
with ciprofloxacin pharmacophore and it exhibited better antibacterial activity (MIC50 = 0.11
µg/mL) than the parent ciprofloxacin [18]. Naringenin-ciprofloxacin drug hybrid (3) exhibits
activity against methicillin resistant S. aureus (MRSA) with MIC50 = 0.29 µg/mL and multidrug
resistant E. coli with MIC50 = 0.71 µg/mL [19]. A number of fluoroquinolone dimers linked with
various linkers at C-7 position (4, Figure 1) have also been reported as antibacterial agents.
These dicarboxylic acids containing dimers have shown promising in vitro antibacterial activities
against the tested organisms [17, 20].
O
N
O
O
N
O
F
F
NH2
N
OH
OH
O
O
O
N
N
O
N
N
N
H2N
O
O
F
1
2
O
N
O
O
O
O
O
F
O
O
OH
OH
F
F
OH
HO
N
N
N
N
N
N
O
N
R
R
N
Linker
HO
4
3
Figure 1: Structures of various reported ciprofloxacin drug hybrids
We have previously described trimethoprim derivatives and drug-amino acid conjugates as
potent antibacterial agents [21,22]. With the aim to further expand our antibacterial research in
the battle against bacterial resistance, herein we report the design, synthesis and biological
evaluation of mono- and di-substituted piperazine derivatives. Piperazine derivatives have played
important role in improving the potency of antibacterial drugs [23]. Ciprofloxacin (CPX) is a
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piperazine containing fluoroquinolone antibiotic and exerts its action by the inhibition of
bacterial DNA gyrase. Extensive structure activity relationship (SAR) has shown that carboxylic
acid and carbonyl groups at C-3 and C-4 are essential for DNA gyrase inhibiting activity (Figure
2a). Linking of piperazinyl-N4 (7-position of CPX) with a different drug via non-cleavable
linkage resulted in a variety of novel dual action antibacterial agents [24-26]. Based on these
studies, we have planned to synthesize hybrids linked to piperazine moiety through diverse
linkers. The overall strategy for the synthesis of piperazine derivatives is shown in Figure 2b.
We envisioned that hybrids containing two different drugs (CPX and drugs 5-8) may be able to
show improvement of efficacy than parent drug or even impart a new mechanism of action by
acting on dual or multiple bacterial targets.
2. Results and discussion
2.1. Design strategy
To explore the further chemical space for antibacterial drug design, some commercially available
drugs were introduced. We have selected four different marketed drugs; p-amino benzoic acid
(PABA, 5), sulfanilamide (SNA, 6) gabapentin (GP, 7) and isoniazid (INH, 8). PABA (5) is a
cyclic amino acid and is used for the synthesis of folic acid. Structurally related compound of
PABA is p-aminobenzene sulfonamide (sulfanilamide, 6). It is used as short acting anti-infective
drug for the treatment of trauma infection due to hemolytic streptococcus. Moreover, it is also
used as intermediate for the synthesis of sulfa-drugs. SNA blocks folic acid synthesis by
inhibiting dihydropteroate synthase and inhibits the synthesis of nucleic acid. Gabapentin (GP) is
a gamma-amino acid drug originally synthesized as a lipophilic analogue of gamma-
aminobutyric acid (GABA). Lipophilic domain of GP (i.e. cyclohexane ring) could facilitate
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bacterial cell wall permeability. Moreover, the idea to use GP and PABA was to synthesize
dicarboxylic acid derivatives like 4 (Figure 1). In strategy 1, we pursued the synthesis of mono-
substituted piperazine derivatives with selected drugs (5-8, Figure 2) separated by a methylene
linker. While in strategy 2, we planned to synthesize hybrids of ciprofloxacin with above
mentioned drugs (5-8) tethered through methylene, ethylene and gabapentin linkers.
Figure 2: The overall strategies to synthesize target compounds of current research
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2.2. Chemistry
The synthesis of piperazine derivatives (10a-d) is shown in Scheme 1. A one-pot three
component reaction of piperazine 9, amines (5-8) and formalin (40 %) resulted in target Mannich
base derivates 10a-d [27].
a
HN R
HN
NH
HN
N
10a-d
9
O
H
O
N
OH
O
O
S
R=
O NH2
OH
N
10a
10d
10c
10b
Scheme 1: Synthesis of Mannich bases (10a-d), Reagents and condition: (a) CH2O (37-40%), R-NH2 (5-
8), MeOH/H2O (9:1), 25°C, Overnight stirring
In our previous study, trimethoprim-amino acid hybrids with triazine linker showed promising
antibacterial activity [25]. In current study, we tried here again triazine linker and synthesized
target compound 12 by the reaction of INH (8) and triazine. The intermediate obtained was then
reacted with piperazine (Scheme 2).
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CH3
O
H3C
N
O
N
N
N
H
O
N
H
NH2
N
Cl
O
N
H
N
N
O
N
a
b
H
N
N
H
NH
N
N
11
12
N
8
Scheme 2: Synthesis of piperazine antibacterial derivative (12), Reagents and condition: (a) K2CO3, THF,
2,4-dichloro-6-methoxy-1,3,5-triazine, Reflux 5-6h (b), THF, K2CO3, 9, Reflux 4-5 h.
The synthesis of target compounds 14a-d is shown in Scheme 3. Here again, a single step
synthetic route was followed by using ciprofloxacin (CPX, 13), amine containing drugs (5-8),
formalin (37%) to obtain Mannich base derivatives 14a-d. The reaction yield was ranged from
73-89%.
O
N
O
O
O
F
F
OH
OH
a
N
N
N
H
N
N
HN
R
13
14a-d
O
H
O
N
OH
O
S
O
R =
O NH2
OH
N
14a
14b
14d
14c
Scheme 3: Synthesis of ciprofloxacin derivatives (14a-d), Reagents and condition: (a) CH2O (37-40%),
Appropriate amines R-NH2 (5-8), EtOH, 25°C.
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In the synthesis of the target compounds 16a-d (Scheme 4), a two-steps protocol was followed.
In first step, amine compounds (5-8) were reacted with 1-bromo-2-chloro ethane in N,N-
dimethylformamide (DMF) to obtain 15a-d. The reactions were carried out under alkaline
conditions and resulting products were obtained in 10-12 hours in 60.6 % to 78.0% yield. The
target molecules 16a-d were obtained by the reaction of ciprofloxacin and compounds 15a-d
using acetonitrile as solvent. Reactions were carried out by stirring overnight at 60 oC.
O
N
O
F
OH
a
b
R
Cl
N
R NH2
N
H
R
N
N
H
5-8
15a-d
16a-d
O
H
O
N
OH
O
S
O
R=
O NH2
OH
N
16a
16b
16c
16d
Scheme 4: Synthesis of ciprofloxacin derivatives 16a-d. (a) Appropriate amines (5-8), 1-bromo-2-chloro
ethane, K2CO3, DMF, 40-50 oC; (b) Compounds 15a-d, CPX (13), K2CO3 and acetonitrile, stir overnight
at 60 oC
.
Subsequently, we further investigated the effect of linker type and synthesized CPX-SAN hybrid
named CGS-20. Here, we selected gabapentin linker. The synthetic process is outlined in
Scheme 5. Boc-protected gabapentin (17) was synthesized by reaction of GP (7) with di-tert
-
butylcarbonate ( -BuOCO)2O in a mixture of H2O/THF (10:1) as reported by Lebedyeva et al
t
[28]. In the next step, the coupling of Boc-protected gabapentin (17) and sulfanilamide was
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carried out using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). 1-Hydroxy-
benzotriazole (HOBt) was used to avoid racemization and triethylamine (Et3N) was used to
maintain the pH. Finally, the deprotected compound 19 was reacted with CPX in a Mannich type
reaction to obtain final product CGS-20. All the synthesized compounds were purified and
characterized using spectroscopic technique (See Supporting Information). The structures of
all the synthesized compounds are shown in Figure S-1 (See Supporting Information).
O
H2N
HO
OH
Boc
N
a
H
O
17
7
H
N
H2N
H
N
Boc
b
NH
c
17
6
O
S
O
S
O
NH2
O
NH2
O
O
19
18
O
N
O
O
N
O
O
S
O
F
OH
d
F
OH
H2N
O
N
19
N
N
HN
H
N
N
H
13
CGS-20
Scheme 5: Synthesis of potent antibacterial drug CGS-20, Reagents and condition: (a) (BOC)2O,
H2O/THF, Na2CO3, rt, stirring, overnight (b) EDC, HOBt, Et3N, CHCl3 (c) CF3COOH, N2,
DCM; (d) CH2O (37-40%), MeOH/H2O (9:1), 25°C, overnight stirring.
1
13
The structures of all the synthesized compounds were verified by H NMR, C NMR, LC-MS
and elemental analysis. Purity of the series 14a-d, 16a-d and CGS-20 was analyzed by using
reversed phase HPLC. The elemental analysis results were found within +0.4% of the theoretical
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values. In 1H-NMR spectrum, the evident signals of methylene linker protons of the
monosubstituted piperazine derivatives 10a-d appeared at δ≈4.3 ppm except for 10c (δ=4.11
ppm). All the -NH protons of the Mannich base derivatives (10a-d and 14-d) appeared as broad
1
singlets. Unsubstituted piperazine-NH in 10a-d appeared at
≈
1.85 ppm as broad singlet. The H
NMR signals for compounds 14a-d were verified by a singlet in the range of 3.74-4.09 ppm
corresponding to -NCH2N- group. For compounds 16a-d, two methylene protons (-NCH2CH2N-)
showed triplets as the evident signals. In DMSO-d6, the characteristic peaks of carboxylic acid
proton (-OH) of ciprofloxacin, GP and PABA were observed in deshielded region. The -OH
proton of CPX appeared in the range of 12.8 ppm. While -OH of GP and PABA appeared in the
range of 14.3-14.5 ppm.
2.3 In vitro antibacterial studies
All the synthesized compounds 10a-d, 12, 14a-d, 16a-d and CGS-20 were screened against
standard two Gram-positive i.e. S. aureus (ATCC-6538), B. subtilis (ATCC-6633) and three
Gram-negative i.e. E. coli (ATCC-25922), P. aeruginosa (ATCC-15442) and K. pneumonia
(ATCC-1705) strains of bacteria (Table 1). Moreover, all these compounds were also screened
against four clinically isolated resistant strains of S. aureus (MRSA), S. hemolytic, E. coli and P.
aeruginosa. The in vitro activities against standard and resistant strains, expressed as minimum
inhibitory concentration (MIC) in µg/mL and micromolar (µM) in brackets, are tabulated in
Table 1 and Table 2 respectively. Ciprofloxacin, cefixime and roxithromycin were used as
standard drugs (Tables 1-2). Table 1 enlists the in vitro MIC values of the synthesized
compounds against standard strains.
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The in vitro assay results showed that piperazine analogues 10a, 10b and 10d were not able to
achieve the desired results and showed poor or even no activity against all the tested strains
(Table1) suggesting that cell impermeability may be the cause. Compounds 10c and 12 have
shown some modest activity against Gram-positive tested strains (MIC range 16-32 µg/mL).
Promising activity was observed for ciprofloxacin hybrids against all the tested strains. Mannich
base hybrid derivatives (14a-d) of ciprofloxacin and drugs (5-8) showed interesting activity
profile against all the tested strains (MIC range 0.5-32
conjugate 14b emerged as most potent compound of this series and showed activity against both
type of tested strains (MIC = 0.5 g/mL for Gram-positive and 1 g/mL for Gram-negative
strains). Ciprofloxacin-gabapentin hybrid (14c) showed only the modest activity against Gram-
negative strains with MIC value of 32 g/mL. However, it has shown good inhibition data
against Gram negative strains (MIC = 8 g/mL). The lengthening of spacer between
ciprofloxacin and drugs (5-8) resulted in target hybrids 16a-d. The best activity against Gram-
positive strains was shown by INH-CPX hybrid 16d (MIC = 0.5 g/mL). It is interesting to note
µg/mL). Ciprofloxacin-sulfanilamide
µ
µ
µ
µ
µ
here that hybrid of pyrazinamide (another first-line antituberculotic drug like INH) with
ciprofloxacin reported to have shown moderate to poor inhibition against tested bacterial strains
[29]. The most interested compound synthesized in current research is CPX-sulfanilamide hybrid
(CGS-20) with gabapentin as spacer. The target compound CGS-20 (logP = 3.12, Table 1) not
only demonstrates promising potency against Gram-negative strains (MIC = 0.5 µg/mL) but also
augments anti-Gram-positive bacterial potency. In vitro activity of the four drugs (5-8) used to
synthesize hybrids with ciprofloxacin was also carried out. Only SNA (6) have shown some
activity against S. aureus (MIC = 64
µ
g/mL). Other three compounds have shown no inhibition
in tested concentration.
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Based on obtained results, the compounds exhibiting promising activities (14a-d, 16a-d and
CGS-20) were subsequently tested against four clinically isolated resistant strains of S. aureus
(MRSA), S. hemolytic, E. coli and P. aeruginosa. The results are enlisted in Table 2.
Compounds 14a and 14b showed noticeable difference in activity between S. aureus (8
µg/mL
and 0.5 g/mL respectively, Table 1) and MRSA (32 g/mL and 4 g/mL respectively, Table
µ
µ
µ
2). Another interesting result was shown by CPX-sulfanilamide hybrids 14b and 16b. For
standard ATCC strains, MIC value of 14b>16b. In contrast to methylene linker containing 14b,
ethylene linker containing compound 16b showed a twofold increase in activity against MRSA
and S. hemolytic. Hybrid 16c showed similar activity for both S. aureus standard strain and
MRSA. However, 16c showed improved activity against Gram-negative resistant strains (Table
2). Hybrid compound CGS-20 have shown improved anti-Gram-positive inhibition (MIC for
MRSA = 0.5
µg/mL), while its potency against resistant E. coli and P. aeruginosa remains same.
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Table 1: Partition coefficient (LogP) and minimum inhibitory concentration (MIC) of different series of compounds against the standard Gram-
positive and Gram-negative strains.
Minimum inhibitory concentration µg/mL (µM)b
R
Gram-positive
Gram-negative
LogPa
P.
K.
S. aureus
B. subtilis
E. coli
aeruginosa
pneumoniae
R N
NH
O
256
(1087.9)
0.74
64 (272.1)
NIc
64 (272.1)
NI
256 (1087.9)
NI
256 (1087.9)
NI
10a
NH
HO
O
-0.07
NI
10b
10c
S
NH
H2N O
O
HO
1.05
32 (118.7)
32 (118.7)
64 (237.5)
64 (237.5)
64 (237.5)
N
H
-0.75
NI
NI
NI
NI
NI
10d
12
O
H
N
N
O
N
H
0.29
16 (49.2)
16 (49.2)
32 (98.4)
32 (98.4)
64 (196.8)
N
N
N
CH3
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H
N
2.82
2.01
8 (16.6)
8 (16.6)
4 (8.3)
1 (1.9)
4 (8.3)
8 (16.6)
14a
14b
HO
O
H
N
0.5d (0.9)
0.5 (0.9)
1 (1.9)
1 (1.9)
O
S
H2N
O
NH
3.13
32 (62.1)
32 (62.1)
8 (15.5)
8 (15.5)
8 (15.5)
14c
HO
O
O
H
N
1.32
2.54
2 (4.1)
1 (2.0)
4 (7.7)
1 (2.0)
4 (7.7)
4 (7.7)
4 (7.7)
1 (2.0)
14d
16a
N
H
N
OH
O
0.5 (1.0)
0.5 (1.0)
N
H
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O
S
H2N
1.73
2.85
2 (3.7)
2 (3.7)
4 (7.5)
4 (7.5)
4 (7.5)
4 (7.5)
16b
16c
O
N
H
O
HO
16 (30.2)
16 (30.2)
4 (7.50)
8 (15.1)
N
H
H
N
O
N
H
1.00
3.12
0.5 (1.0)
1 (1.4)
0.5 (1.0)
1 (1.4)
2 (4.0)
2 (4.0)
2 (4.0)
16d
N
O
S
H2N
O
O
0.5 (0.7)
0.5 (0.7)
0.5 (0.7)
CGS-20
N
H
N
H
Ciprofloxacine
Cefiximee
8
8
-
8
8
8
-
0.1(0.125)
NI
64 (378)
NI
0.1 (0.06)
0.5 (0.06)
0.1 (0.06)
Roxithreomycine
0.04(0.039)
-
-
-
p-Amino benzoic acid (PABA)
Sulfanilamide (SNA)
Gabapentin (GP)
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
NI
Isoniazid (INH)
NI
aLogP value was calculated by using ChemDraw; bThe reported MIC values in µg/mL. MIC value in µM are shown in parenthesis. These MIC values are an
c
d
average of at least three individual measurements; No inhibition was found in tested concentrations; MIC values in µg/mL of most active compounds are
highlighted (bold values); eStandard drugs.
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Table 2: Minimum inhibitory concentration (MIC) of ciprofloxacin-drug hybrids against the clinically
isolated Gram-positive and Gram-negative bacterial strains.
Compounds
Minimum inhibitory concentration (MIC), µg/mL( µM)
Gram-positive
MRSAa
32 (66.5)b
4 (7.7)
Gram-negative
S. hemolytic
4 (8.3)
4 (7.7)
32 (62.1)
4 (8.3)
NT
E. coli
4 (8.3)
4 (7.7)
4 (7.7)
16 (33.2)
0.5 (1.0)
4 (7.5)
2 (3.7)
4 (8.0)
0.5 (0.74)
4
P. aeruginosa
4 (8.3)
4 (7.7)
4 (7.7)
16 (33.2)
0.5 (1.0)
4 (7.5)
2 (3.7)
4 (8.0)
0.5 (0.74)
4
14a
14b
14c
14d
16a
16b
16c
16d
32 (62.1)
1c (2.0)
8 (16.1)
2 (3.7)
16 (30.2)
2 (4.0)
0.5 (0.74)
32
2 (3.7)
NTd
4 (8.0)
NT
4
0.25
CGS-20
Ciprofloxacine
Cefiximee
-
0.75
0.5
b
aMethicillin resistant S. aureus; The reported MIC values in µg/mL.cMIC value in µM are shown in parenthesis.
c
These MIC values are an average of at least three individual measurements; MIC values in µg/mL of most active
compounds are highlighted (bold values); d Not tested; e Standard drugs.
2.3 Docking studies
Due to the important role in bacterial survival, DNA gyrase was selected as target for the
docking studies of the synthesized compounds. Docking studies were carried out using
Molecular Operating Environment (MOE 2016.08, Chemical Computing Group, Canada)
software. Three-dimensional structure of DNA gyrase from S. aureus (PDB code 5CDQ) co-
crystalized with moxifloxacin was retrieved from protein data bank (PDB). The docking
algorithm was validated by re-docking of native ligand moxifloxacin. The computed root means
square deviation (RMSD) between the experimental and re-docked poses was found 0.874 Å. In
general, the following features of the docked compounds were observed; (1) Almost all the
active compounds have shown binding pose similar to that of moxifloxacin; (2) Hydrophobic
interactions were observed with DNA bases (adenine and guanine); (3) hydrogen bonding
interactions were also found between compounds and Ser84/Glu88; (4) Most of the ciprofloxacin
derivatives coordinate with Mg2+ through -C=O of carboxylic acid and quinolone ring. Three-
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dimensional ribbon diagram of the binding-pose of CGS-20 into the binding site of 5CDQ is
shown in Figure 3 a-c. The Figure 3a showed that CGS-20 binds between two base-pairs and
mode of binding is similar to that of moxifloxacin. Three-dimensional and two-dimensional
interaction plot is shown in Figure 3b and 3c respectively. CGS-20 forms
π-π stacking
interactions with base pairs DA2013 and DG2009. Piperazine nitrogen also interacts with Ser84
forming hydrogen bond interactions. The two-dimensional docking poses of some important
compounds are shown in Supporting Information.
Figure 3: (a) Three-dimensional (3D) ribbon diagram of the binding pose of CGS-20 into the binding
site of DNA gyrase from S. aureus (PDB code 5CDQ); (b)3D interaction plot generated via discovery
studio visualizer showing π-π stacking interactions with base pairs and Ser84 (representing with stick
model); (c) 3D interaction plot generated via MOE software.
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2.4 Preliminary in silico pharmacokinetic studies
We have predicted human intestinal absorption and carcinogenicity of the synthesized
compounds by using online AdmetSAR server (http://lmmd.ecust.edu.cn/admetsar1). All the
synthesized compounds are predicted to be absorbed in the human intestine (HIA) and are found
non-carcinogenic (Table S-1 in Supporting Information).
3. Conclusions
To summarize, a set of five new series of piperazine derivatives were synthesized and evaluated
for their antibacterial potential against standard as well as resistant bacterial strains.
Monosubstituted piperazine derivatives with methylene linker were almost ineffective against all
the tested strains. The only test-derivative of isoniazid and piperazine synthesized with triazine
linker has shown some activity. In current research, we continued with one and two-carbon
linkers and synthesized ciprofloxacin-drug hybrids. The reaction of piperazinyl-NH with selected
drugs resulted in pronounced growth inhibition of standard as well as resistant bacterial strains.
The CPX-drug hybrids with methylene linker (14a-d) showed MIC range of 0.5-32
hybrids with ethylene linker (16a-d) exhibited antibacterial potential in the range of 0.5-16
g/mL for Gram-positive strains and 0.5-4 g/mL for Gram-negative strains. Apart from
µg/mL. The
µ
µ
methylene and ethylene linker, introduction of cyclohexyl ring enhanced the hydrophobicity. The
resulted hybrid of ciprofloxacin and sulfanilamide (CGS-20) has shown pronounced inhibition
for both type of tested standard strains. However, it showed improved anti-Gram-positive
inhibition (resistant strain, MIC for MRSA = 0.5
µg/mL by compound CGS-20), while the
potency against resistant E. coli and P. aeruginosa remains same. Most of the other compounds
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exhibited same activity profile for resistant strains except for methicillin resistant S. aureus
(MRSA).
DNA gyrase was selected as target for the docking studies of the synthesized compounds.
Besides the hydrophobic interactions with DNA bases and hydrogen bonding interactions with
amino acid residues of the enzyme, coordination with Mg2+ is essential for strengthening of
ligand-enzyme complex.
To develop new antibacterials, we intend to extend our investigations to include other quinolone-
drug hybrids, hydrophobic linkers and to establish more extensive SAR. Moreover, a future
detailed in vitro study on the target enzyme will be taken into consideration as an extension of
current research.
Acknowledgement
Dr. Umer Rashid is thankful to Higher Education Commission for financial support for the
purchase of MOE license under HEC-NRPU project 5291/Federal/NRPU/R&D/HEC/2016.
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Highlights
•
•
•
•
•
Mono- and di-substituted piperazine derivatives were synthesized
In di-substituted derivatives, ciprofloxacin-drug hybrids were synthesized
Compounds were evaluated against normal and resistant bacterial strains
Excellent in vitro bacterial strain inhibition by some compounds
Molecular docking studies on DNA gyrase as target