New 7-piperazinylquinolones containing (benzo[d]imidazol-2-yl)methyl moiety as potent antibacterial agents

Article abstract of DOI:10.1007/s11030-018-9834-3

A series of 7-piperazinylquinolones containing a (benzo[d]imidazol-2-yl)methyl moiety were designed and synthesized as new antibacterial agents. The antibacterial activity of title compounds was evaluated against Gram-positive (Staphylococcus aureus, Staphylococcus epidermidis and Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumonia) microorganisms. Among the tested compounds, the N1-cyclopropyl derivative 4a showed the highest activity against S. aureus, S. epidermidis, B. subtilis and E. coli (MIC = 0.097 μ g/mL), being 2–4 times more potent than reference drug norfloxacin. A structure-activity relationship study demonstrated that the effect of the nitro group on the benzimidazole ring depends on the pattern of substitutions on the piperazinylquinolone.

Full text of DOI:10.1007/s11030-018-9834-3

Molecular Diversity
https://doi.org/10.1007/s11030-018-9834-3
ORIGINAL ARTICLE
New 7-piperazinylquinolones containing
(benzo[d]imidazol-2-yl)methyl moiety as potent antibacterial agents
Hojat-Allah Arab1 · Mohammad Ali Faramarzi2 · Nasrin Samadi3 · Hamid Irannejad4 · Alireza Foroumadi5 ·
Saeed Emami4
Received: 25 September 2017 / Accepted: 14 May 2018
© Springer International Publishing AG, part of Springer Nature 2018
Abstract
A series of 7-piperazinylquinolones containing a (benzo[d]imidazol-2-yl)methyl moiety were designed and synthesized as
new antibacterial agents. The antibacterial activity of title compounds was evaluated against Gram-positive (Staphylococcus
aureus, Staphylococcus epidermidis and Bacillus subtilis) and Gram-negative (Escherichia coli, Pseudomonas aeruginosa
and Klebsiella pneumonia) microorganisms. Among the tested compounds, the N1-cyclopropyl derivative 4a showed the
highest activity against S. aureus, S. epidermidis, B. subtilis and E. coli (MIC = 0.097 μg/mL), being 2–4 times more potent
than reference drug norfloxacin. A structure-activity relationship study demonstrated that the effect of the nitro group on the
benzimidazole ring depends on the pattern of substitutions on the piperazinylquinolone.
Keywords Antibacterial activity · 1H-benzo[d]imidazole, 7-piperazinylquinolones · Docking study
Introduction
essential cellular enzymes that catalyze the double-strand
breakage of DNA and are responsible for the maintenance
of DNA topology and conformation during the DNA tran-
scription and replication. Thus, the quinolones block the
transcription and the DNA replication, which results in
bacterial cell death [2–4]. The success of quinolones in clin-
ical practice and their widespread use originate from their
selective action on the bacterial topoisomerases over their
eukaryotic equivalents [5].
Structurally, thequinoloneantibacterialsrelatetonalidixic
acid (Fig. 1), a naphthyridone analog with poor pharmacoki-
netic properties and limited activity against Gram-negative
bacteria [6]. Extensive SAR studies to increase the effi-
cacy against bacteria have led to discovery of norfloxacin
(Fig. 1) with potent and broad spectrum activity [7]. The
distinct structural features of this agent are the presence of
fluorine at 6-position and piperazinyl group at 7-position
of the quinolone nucleus. Subsequently, several new mem-
bers of 6-fluoro-7-piperazinylquinolones such as enoxacin,
lomefloxacin, and ciprofloxacin (Fig. 1) have emerged with
enhanced activity against Gram-negatives and improved
pharmacokinetic profile. Generally, these quinolones had
good activity against Gram-negative bacteria, but their activ-
ities against clinically important Gram-positive pathogens
includingStaphylococcusaureusandStreptococcuspneumo-
niae were moderate [8]. Furthermore, the emerging resistant
Quinolone antibacterials are one of the most important
classes of chemotherapeutic agents frequently used for the
treatment of community- or hospital-acquired infectious
diseases [1]. Quinolones exert their antibacterial effect by
inhibiting two type-II bacterial topoisomerase enzymes,
DNA-gyrase and/or topoisomerase IV. These enzymes are
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11030-018-9834-3) contains supplementary
material, which is available to authorized users.
Saeed Emami
sd_emami@yahoo.com
B
1
Student Research Committee, Faculty of Pharmacy,
Mazandaran University of Medical Sciences, Sari, Iran
2
Department of Pharmaceutical Biotechnology, Faculty of
Pharmacy and Biotechnology Research Center, Tehran
University of Medical Sciences, Tehran, Iran
3
Department of Drug and Food Control, Faculty of Pharmacy,
Tehran University of Medical Sciences, Tehran, Iran
4
Department of Medicinal Chemistry and Pharmaceutical
Sciences Research Center, Faculty of Pharmacy, Mazandaran
University of Medical Sciences, Sari, Iran
5
Department of Medicinal Chemistry, Faculty of Pharmacy
and The Institute of Pharmaceutical Sciences (TIPS), Tehran
University of Medical Sciences, Tehran, Iran
123

Molecular Diversity
O
4
5
3
COOH
6
7
2
H3C
N
N1
8
CH3
Nalidixic acid
O
N
O
N
O
F
COOH
CH3
F
COOH
F
COOH
H3C
HN
N
N
N
N
N
F
HN
HN
CH3
CH3
1b, Enoxacin
1a, Norfloxacin
1c, Lomefloxacin
O
N
O
N
F
COOH
F
COOH
H3C
HN
N
N
OCH3
HN
1d, Ciprofloxacin
1e, Gatifloxacin
Fig. 1 Examples of 7-piperazinylquinolones derived from nalidixic acid
bacteria especially in strains of MRSA have reduced the
efficiency of these agents due to their excessive and inap-
propriate use for the past few years [9–12]. Thus, recent
efforts have focused on the design of new quinolones with
improved antibacterial activity against Gram-positives, while
retaining the good Gram-negative activity of early agents
such as norfloxacin and ciprofloxacin [8,11,13]. Extensive
SAR studies demonstrated that the substituent at position 7
greatlyinfluencesthepotency, spectrumandsafetyprofilesof
quinolones. Furthermore, this position of the quinolone core
is more adaptable for structural modification and is suitable
for attachment of bulky substituents [8].
O
OH
O
N
NH
N
H
F
(A)
Fig. 2 Structure of benzimidazole derivative A targeting DNA-gyrase
B enzyme
Besides quinolones, reported studies have shown that var-
ious benzimidazole derivatives exhibit remarkable antibac-
terial activity. Recently, Janupally et al. identified benzim-
idazole A (Fig. 2) targeting DNA-gyrase B enzyme of S.
aureus with low micromolar inhibitory activity. Seenaiah
et al. described 2-(thiomethyl)benzimidazoles B (Fig. 3) as
antimicrobial agents with good activity against S. aureus
[14]. A series of nitro-substituted benzimidazoles C contain-
ing phenoxymethyl moiety was synthesized by Hosamani
123

Molecular Diversity
Y
R
N
N
O
S
N
O
N
F
COOH
N
H
N
H
O2N
N
X
N
R
(B)
(C)
HN
CF3
CF3
R1
1a-e
HN
N
N
H
(D)
Piperazinylquinolone
antibacterials
Benzimidazole-based
antibacterials
O
F
COOH
R2
N
X
N
R
N
N
N
H
R1
4
Designed antibacterials
Fig. 3 Examples of benzimidazole-based antibacterial agents B–D, piperazinylquinolones and designed benzimidazole-based quinolone antibac-
terials 4
and co-workers and screened for in vitro antibacterial activ-
ity. The results showed that the introduction of nitro group
on the aromatic ring enhances the activity against most of
the microorganisms [15]. In a study by Zhang et al., some
2-substituted benzimidazoles were synthesized and evalu-
ated for in vitro antimicrobial activity. Compound D with
MIC values of 2–16 μg/mL was the most potent derivative
[16].
incorporated a benzimidazol-2-ylmethyl moiety on the piper-
azine ring of representative piperazinylquinolones (Fig. 3).
Thus, we report here the synthesis, antibacterial activity
and docking studies of N-[(benzimidazol-2-ylmethyl]-7-
piperazinylquinolones 4.
Experimental
With these in mind and attending to the fact that the C-7
heterocyclic substituents such as 1,3,4-thiadiazole [17–19],
thiophene[20–24], furan[25], benzopyran[26,27], andpyran
[28] in quinolones could greatly influence the inhibition
of the target enzyme, cell permeability, and bioactivity, we
Chemistry
Thepiperazinylquinolones1a–ewerepurchasedfromSigma-
Aldrich Company. Other starting materials, reagents and
123

Molecular Diversity
solvents were purchased from Merck Company and used
without further purification. The progress of reactions was
checked by thin-layer chromatography (TLC) using pre-
coated silica gel 60 F254 plastic sheets (Merck). Melting
points were determined in open glass capillaries using Bibby
Stuart Scientific SMP3 apparatus (Stuart Scientific, Stone,
UK) and are uncorrected. The IR spectra were recorded on a
PerkinElmer FT-IR spectrophotometer using KBr disks. The
NMR spectra were recorded using Bruker 400 or 500 spec-
trometers and chemical shifts are expressed as δ (ppm) using
tetramethylsilane (TMS) as internal standard. Signal mul-
tiplicities are presented as s (singlet), br s (broad singlet),
d (doublet), t (triplet), q (quartet), and m (multiplet). Also,
coupling constants are reported in Hertz (Hz). Mass spec-
tra were obtained using a HP 5937 Mass Selective Detector
(Agilent technologies). Elemental analyses were carried out
on a CHN-O-rapid elemental analyzer (GmbH-Germany) for
C, H and N, and the results are within 0.4% of the theoret-
ical values.
The precipitated solid was collected by filtration, washed
with water and recrystallized from methanol to give desired
product 4a–j.
7-(4-((1H-Benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)-1-
cyclopropyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxy
lic acid (4a)
White solid; yield 86%; m.p. 248–251 ◦C; IR (KBr, cm−1):
1
3414, 1627, 1579, 1487, 1456, 1390, 1306, 1259, 750; H
NMR (500 MHz, DMSO-d6): 0.81–1.39 (m, 4H, cyclo-
propyl), 2.64 (br s, 4H, piperazine), 3.27 (br s, 4H, piper-
azine), 3.54–3.69 (m, 1H, cyclopropyl), 3.83 (s, 2H, CH2),
7.05–7.23 (m, 2H, H-5 and H-8 quinolone), 7.35–7.53 (m,
2H, benzimidazole), 7.53–7.66 (m, 2H, benzimidazole), 8.66
(s, 1H, H-2, quinolone), 12.40 (s, 1H, NH). MS (m/z, %):
461 (M+, <1), 368 (6), 235 (8), 183 (9), 125 (100), 105
(21), 91 (61), 77 (30), 57 (48), 43 (83). Anal. Calcd for
C25H24FN5O3: C, 65.07; H, 5.24; N, 15.18; Found: C, 64.85;
H, 5.30; N, 15.32.
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-(4-((5-nitro-1H-
benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)-4-oxoquino
line-3-carboxylic acid (4b)
General procedure for the preparation of 2-(chloromethyl)
benzimidazole derivatives 3a or 3b
A mixture of o-phenylenediamine derivative 2a or 2b
(10 mmol) and ethyl chloroacetate (13 mmol) in diluted
hydrochloric acid (4 M, 10 mL) was stirred at room temper-
ature for 3–5 h. Then, the reaction mixture was heated under
reflux for another 3–5 h. After cooling the reaction, water
was added and the pH was adjusted to 9 by adding ammonia
solution. The precipitated solid was collected by filtration
and washed with water to give corresponding compounds 3a
or 3b.
Pale yellow solid; yield 72%; m.p. 229–232 ◦C; IR (KBr,
cm−1): 3385, 1627, 1579, 1515, 1486, 1455, 1338, 1310,
1260, 832, 741; 1H NMR (400 MHz, DMSO-d6): 0.80–1.60
(m, 4H, cyclopropyl), 2.73 (br s, 4H, piperazine), 3.21–3.70
(m, 4H, piperazine, and 1H cyclopropyl), 3.92 (s, 2H, CH2),
7.10–7.89 (m, 2H, H-5 and H-8 quinolone), 7.91–8.50 (m,
3H, benzimidazole), 8.64 (s, 1H, H-2, quinolone), 13.14 (s,
1H, NH). MS (m/z, %): 506 (M+, < 1), 413 (5), 264 (6),
252 (5), 236 (5), 222 (5), 134 (6), 118 (32), 105 (9), 92
(29), 81 (24), 69 (71), 57 (63), 43 (100). Anal. Calcd for
C25H23FN6O5: C, 59.28; H, 4.58; N, 16.59; Found: C, 59.36;
H, 4.45; N, 16.60.
2-(Chloromethyl)-1H-benzo[d]imidazole (3a)
Pale yellow solid; yield 80%; m.p. 142–145 ◦C; IR (KBr,
cm−1): 3419, 2852, 2687, 1621, 1434, 1442, 1316, 1279,
1142, 1033, 736, 744; 1H NMR (400 MHz, DMSO-d6): 4.93
(s, 2H, CH2), 7.17–7.27 (m, 2H, benzimidazole), 7.53–7.59
(m, 2H, benzimidazole).
7-(4-((1H-Benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)-1-
ethyl-6-fluoro-1,4-dihydro-4-oxoquinoline-3-carboxylicacid
(4c)
White solid; yield 77%; m.p. 89–92 ◦C; IR (KBr, cm−1):
1
2-(Chloromethyl)-5-nitro-1H-benzo[d]imidazole (3b)
Pale yellow solid; yield 67%; m.p. 109–112 ◦C; IR (KBr,
cm−1): 3105, 2921, 1630, 1517, 1474, 1341, 1260, 1066,
1015, 889, 834, 738; 1H NMR (400 MHz, DMSO-d6): 5.00
(s, 2H, CH2), 7.60–8.70 (m, 3H, benzimidazole), 13.43 (br
s, 1H, NH).
3402, 1623, 1579, 1494, 1455, 1403, 1340, 1260, 748; H
NMR (500 MHz, DMSO-d6): 1.39 (t, 3H, CH3), 2.58–2.84
(m, 4H, piperazine), 3.20–3.39 (m, 4H, piperazine), 3.83 (s,
2H, CH2), 4.44 (q, 2H, CH2-Me), 7.04–7.95 (m, 2H, H-5
and H-8 quinolone, and 4H, benzimidazole), 8.42 (br s, 1H,
benzimidazole), 8.94 (s, 1H, H-2 quinolone), 12.44 (br s, 1H,
NH). MS (m/z, %): 449 (M+, < 1), 196 (14), 139 (9), 118
(14), 106 (50), 91 (100), 77 (57), 65 (78), 51 (57). Anal. Calcd
for C24H24FN5O3: C, 64.13; H, 5.38; N, 15.58; Found: C,
63.94; H, 5.40; N, 15.85.
General procedure for the synthesis of compounds 4a–j
Amixtureofpiperazinylquinolone1(0.3mmol), 2-(chlorome
thyl)benzimidazole derivative 2 (0.33 mmol) and sodium
bicarbonate (0.3 mmol) in N,N-dimethylformamide (4 mL)
was stirred at room temperature for 72 h. After completion
of the reaction (monitored by TLC), water (10–15 mL) was
added and the mixture was left in the refrigerator overnight.
1-Ethyl-6-fluoro-1,4-dihydro-7-(4-((5-nitro-1H-benzo[d]
imidazol-2-yl)methyl)piperazin-1-yl)-4-oxoquinoline-3-car
boxylic acid (4d)
123

Molecular Diversity
Pale yellow solid; yield 98%; m.p. 265–268 ◦C; IR (KBr,
cm−1): 3424, 3191, 1732, 1630, 1525, 1508, 1466, 1339,
1309, 1259, 1200, 1130, 1026, 994, 802, 755; 1H NMR (400
MHz, DMSO-d6): 1.39 (br s, 3H, CH3), 2.60–2.80 (m, 4H,
piperazine), 3.92 (s, 2H, CH2), 4.33–4.80 (m, 2H, CH2-Me),
7.17 (br s, 1H, H-8 quinolone), 7.60–8.20 (m, 3H, benzimi-
dazole and H-5 quinolone), 8.42 (br s, 1H, benzimidazole),
8.93 (s, 1H, H-2 quinolone), 13.43 (br s, 1H, NH). MS (m/z,
%): 494 (M+, < 1), 368 (5), 265 (5), 239 (6), 193 (7), 177
(8), 163 (8), 125 (37), 106 (17), 91 (65), 77 (27), 57 (51), 43
(100). Anal. Calcd for C24H23FN6O5: C, 58.30; H, 4.69; N,
17.00; Found: C, 58.41; H, 4.53; N, 16.79.
57 (60), 43 (100). Anal. Calcd for C27H27FN6O6: C, 58.90;
H, 4.94; N, 15.27; Found: C, 59.23; H, 4.88; N, 15.29.
7-(4-((1H-Benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)-1-
ethyl-6-fluoro-1,4-dihydro-4-oxo-1,8-naphthyridine-3-car
boxylic acid (4g)
White solid; yield 27%; m.p. 186–189 ◦C; IR (KBr, cm−1):
1
3564, 1717, 1634, 1476, 1445, 1340, 1260, 807, 744; H
NMR (500 MHz, DMSO-d6): 1.36 (t, 3H, J = 7.05 Hz,
CH3), 2.62–2.74 (m, 4H, piperazine), 3.80 (s, 2H, CH2),
3.84–3.93 (m, 4H, piperazine), 4.46 (q, 2H, J = 7.05 Hz,
CH2Me), 7.09–7.18 (m, 2H, H-5 and H-6 benzimidazole),
7.45 (d, 1H, J = 7.5 Hz, H-4 benzimidazole), 7.55 (d, 1H,
J= 7.70 Hz, H-7 benzimidazole), 8.06 (d, 1H, J= 13.55, H-
5 naphthyridine), 8.96 (s, 1H, H-2 naphthyridine), 12.37 (s,
1H, NH), 15.31 (s,1H, COOH). MS (m/z, %): 450 (M+, 2),
304 (48), 276 (8), 234 (8), 219 (10), 204 (11), 191 (13), 164
(11), 145 (11), 131 (32), 91 (33), 71 (33), 57 (64), 43 (100).
Anal. Calcd for C23H23FN6O3: C, 61.32; H, 5.15; N, 18.66;
Found: C, 61.30; H, 5.25; N, 18.58.
7-(4-((1H-Benzo[d]imidazol-2-yl)methyl)-3-methylpiper
azin-1-yl)-1-cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-4-
oxoquinoline-3-carboxylic acid (4e)
White solid; yield 84%; m.p. 200–203 ◦C; IR (KBr, cm−1):
1
3391, 1725, 1619, 1511, 1457, 1319, 1274, 1063, 746; H
NMR (400 MHz, DMSO-d6): 0.90–1.07 (m, 2H, cyclo-
propyl), 1.08–1.12 (m, 2H, cyclopropyl), 1.18 (d, 3H, J =
6.0 Hz, CH3-piperazine), 2.52–2.60 (m, 1H, piperazine),
2.62–2.72 (m, 1H, piperazine), 2.80–2.89 (m, 1H, piper-
azine), 3.02–3.10 (m, 1H, piperazine), 3.21–3.32 (m, 1H,
piperazine), 3.72 (s, 3H, OCH3), 3.76 (d, 1H, J = 14.8
Hz, piperazine), 4.10 (d, 1H, J = 14.4 Hz, piperazine),
7.00–7.20 (m, 2H, benzimidazole), 7.40–7.60 (m, 2H, benz-
imidazole), 7.72 (d, 1H, J = 12.0 Hz, H-5 quinolone), 8.68
(s, 1H, H-2 quinolone), 12.30 (s, 1H, NH). 13C NMR (100
MHz, DMSO-d6): 9.36, 9.44, 16.21, 41.24, 50.92, 51.78,
52.44, 55.53, 57.45, 63.40, 107.00 (d, JC,F = 22.3 Hz),
111.66, 118.86, 121.42, 122.22, 134.66 (d, JC,F = 14.8
Hz), 139.44, 143.68, 146.30, 150.97, 152.55, 155.98 (d,
JC,F = 250.9 Hz), 166.18, 176.73. MS (m/z, %): 505 (M+,
1), 273 (8), 259 (9), 245 (8), 217 (8), 188 (22), 174 (15), 160
(8), 145 (92), 131 (100), 118 (17), 104 (24), 91 (50), 77 (50),
63 (25), 41 (56). Anal. Calcd for C27H28FN5O4: C, 64.15;
H, 5.58; N, 13.85; Found: C, 64.19; H, 5.41; N, 13.50.
1-Ethyl-6-fluoro-1,4-dihydro-7-(4-((5-nitro-1H-benzo[d]
imidazol-2-yl)methyl)piperazin-1-yl)-4-oxo-1,8-naphthyrid
ine-3-carboxylic acid (4h)
Pale yellow solid; yield 69%; m.p. 191–194 ◦C; IR (KBr,
1
cm−1): 3385, 1632, 1575, 1478, 1338, 1257, 823, 742; H
NMR (500 MHz, DMSO-d6): 1.38 (t, 3H, J= 7.0 Hz, CH3),
2.70 (br s, 4H, piperazine), 3.83–3.93 (m, 4H piperazine,
and 2H CH2), 4.38 (q, 2H, CH2Me), 7.41–8.40 (m, 4H, H-5
naphthyridine and benzimidazole), 8.91 (s, 1H, H-2 naph-
thyridine). MS (m/z, %): 495 (M+, < 1), 369 (5), 321 (5),
285 (7), 197 (15), 183 (7), 169 (10), 154 (13), 140 (10), 125
(16), 109 (5), 97 (11), 83 (32), 69 (37), 55 (80), 43 (100).
Anal. Calcd for C23H22FN7O5: C, 55.76; H, 4.48; N, 19.79;
Found: C, 56.00; H, 4.65; N, 19.92.
7-(4-((1H-Benzo[d]imidazol-2-yl)methyl)-3-methylpiper
azin-1-yl)-1-ethyl-6,8-difluoro-1,4-dihydro-4-oxoquinoline-
3-carboxylic acid (4i)
1-Cyclopropyl-6-fluoro-1,4-dihydro-8-methoxy-7-(3-met
hyl-4-((5-nitro-1H-benzo[d]imidazol-2-yl)methyl)piperazin-
1-yl)-4-oxoquinoline-3-carboxylic acid (4f)
White solid; yield 97%; m.p. 169–172 ◦C; IR (KBr, cm−1):
3424, 1717, 1622, 1542, 1525, 1457, 1330, 1274, 1249, 1053,
1
1018, 745; H NMR (500 MHz, DMSO-d6): 1.17 (d, 3H,
Pale yellow solid; yield 92%; m.p. 198–201 ◦C; IR (KBr,
cm−1): 3382, 1717, 1618, 1579, 1519, 1448, 1339, 1285,
J = 5.6 Hz, CH3-piperazine), 1.41 (t, 3H, J = 7.0 Hz,
CH3), 2.60–2.72 (m, 1H, piperazine), 2.80–2.91 (m, 1H,
piperazine), 2.94–3.12 (m, 1H, piperazine), 3.20–3.36 (m,
2H, piperazine), 3.79 (d, 1H, J = 14.9 Hz, piperazine),
4.06 (d, 1H, J = 14.9 Hz, piperazine), 4.46–4.63 (m, 2H,
CH2), 7.09–7.51 (m, 2H, benzimidazole), 7.46 (d, 1H, J=
7.2 Hz, benzimidazole), 7.56 (d, 1H, J = 7.2 Hz, benzimi-
dazole), 7.77–7.90 (m, 1H, H-5 quinolone), 8.91 (s, 1H, H-2
quinolone), 12.28 (s, 1H, NH). MS (m/z, %): 481 (M+, < 1),
335 (7), 251 (8), 236 (8), 221 (8), 208 (7), 193 (7), 179 (10),
165 (13), 145 (12), 125 (100), 104 (23), 91 (72), 77 (51), 55
1
1065, 830, 740; H NMR (400 MHz, DMSO-d6): 0.60–
1.30 (m, 4H, cyclopropyl, 3H, CH3-piperazine), 2.52–2.60
(m, 1H, piperazine), 2.60–2.70 (m, 1H, piperazine), 2.80–
3.32 (m, 3H, piperazine), 3.72 (s, 3H, OCH3), 3.73–4.40 (m,
2H, piperazine), 7.50–7.89 (m, 2H, benzimidazole and H-
5 quinolone), 7.80–8.20 (m, 1H, benzimidazole), 8.30–8.51
(m, 1H, benzimidazole), 8.67 (s, 1H, H-2 quinolone), 12.88
(s, 1H, NH). MS (m/z, %): 550 (M+, 1), 319 (6), 255 (7),
177 (9), 145 (8), 131 (22), 111 (8), 97 (15), 83 (24), 70 (20),
123

Molecular Diversity
(54), 43 (92). Anal. Calcd for C25H25F2N5O3: C, 62.36; H,
5.23; N, 14.55; Found: C, 62.35; H, 5.11; N, 14.81.
hydrogen atoms were merged with ADT. The Lamarckian
genetic algorithm (LGA) was used as a search engine. The
active site was defined using AutoGrid 4.2. For docking on
2XCT, the grid size was set to 46 × 50 × 60 points with a grid
spacing of 0.375 on the mass of the X-ray ligand in the crystal
structure complex (X = 2.582, Y = 43.778, Z = 69.248)
and for docking on 3TTZ, the grid size was set to 40 × 44
× 44 points with the default setting centered on X = 0.922,
Y = 4.822, Z = 24.285. Amaximumnumberofenergyeval-
uations were set to 2,500,000. For each of the 10 independent
runs, a maximum number of 27,000 LGA operations were
generated on a single population of 150 individuals. Operator
weights for crossover, mutation, and elitism were set to 0.80,
0.02, and 1, respectively [28,30]. Also, for evaluation of our
docking protocol, the native ligand was re-docked into the
active site of gyrase A and gyrase B and the resulting poses
were compared to their co-crystallized conformations. The
resulting conformations of the native ligand was shown to
be the same as co-crystallized one with the RMSD values of
0.315 and 2.065 Å (based on the heteroatoms), respectively.
1-Ethyl-6,8-difluoro-1,4-dihydro-7-(3-methyl-4-((5-nitro-1H-
benzo[d]imidazol-2-yl)methyl)piperazin-1-yl)-4-oxoqu
inoline-3-carboxylic acid (4j)
Pale yellow solid; yield 96%; m.p. 199–202 ◦C; IR (KBr,
cm−1): 3386, 1618, 1523, 1474, 1340, 1249, 1067, 831,
1
739; H NMR (500 MHz, DMSO-d6): 1.12–1.21 (m, 3H,
CH3-piperazine), 1.30–1.50 (m, 3H, CH3), 2.67–2.79 (m,
1H, piperazine), 2.80–2.94 (m, 1H, piperazine), 2.95–3.15
(m, 1H, piperazine), 3.17–3.34 (m, 2H, piperazine), 3.86 (d,
1H, J = 14.9 Hz, piperazine), 4.15 (d, 1H, J = 14.9 Hz,
piperazine), 4.40 (br s, 2H, CH2), 4.56 (br s, 2H, CH2), 7.71
(br s, 1H, benzimidazole), 7.77–7.93 (m, 1H, H-5 quinolone),
8.08 (br s, 1H, benzimidazole), 8.43 (br s, 1H, benzimida-
zole), 8.90 (s, 1H, H-2 quinolone). MS (m/z, %): 526 (M+,
< 1), 368 (7), 313 (5), 265 (6), 239 (7), 131 (7), 111 (8),
97 (17), 83 (28), 70 (14), 57 (64), 43 (100). Anal. Calcd
for C25H24F2N6O5: C, 57.03; H, 4.59; N, 15.96; Found: C,
56.86; H, 4.60; N, 16.01.
Agar dilution method for MICs determination
Results and discussion
The minimum inhibitory concentrations (MICs) of target
compounds 4a–j were determined by the conventional agar
dilution method [29]. The compounds (10.0 mg) were dis-
solved in DMSO (1 mL) and then diluted with distilled
water (9 mL). Further progressive twofold serial dilution with
molten sterile Mueller–Hinton agar was performed to obtain
the required concentrations of 100, 50, 25, 12.5, 6.25, 3.125,
1.56, 0.78, 0.39, 0.19, 0.097, 0.049, and 0.025 μg/mL. The
medium containing the test compounds was dispensed into
a sterile Petri dish and allowed to solidify. Petri dishes were
inoculated with 1–5 × 104 CFU and incubated at 37 ◦C for
18 h. The MIC was defined as the lowest concentration of
the test compound, which resulted in no visible growth.
Chemistry
The general route for the synthesis of compounds 4a–j is
outlined in Scheme 1. The o-phenylenediamine derivative
(2a or 2b) was reacted with ethyl chloroacetate in diluted
hydrochloric acid under reflux to give corresponding 2-
(chloromethyl)benzimidazole (3a or 3b, respectively) [31].
The reaction of compounds 3a or 3b with appropriate 7-
piperazinylquinolones 1a–e in the presence of NaHCO3 in
DMF afforded final compounds 4a–j.
Antibacterial activity
The antibacterial activity of synthesized compounds 4a–j
was evaluated against Gram-positive (Staphylococcus aureus
ATCC 6538, Staphylococcus epidermidis ATCC 12228 and
Bacillus subtilis ATCC 6633) and Gram-negative (E. coli
ATCC 8739, Pseudomonas aeruginosa ATCC 9027 and
Klebsiella pneumonia ATCC 10031) microorganisms. The
minimum inhibitory concentrations (MICs) of all com-
pounds were determined compared to norfloxacin as standard
quinolone antibacterial. The MIC values of compounds 4a–
j are listed in Table 1. At a glance, all compounds with
the exception of 4h showed significant antibacterial activ-
ity. Compound 4a with a MIC value of 0.097 μg/mL was
the most active compound against Gram-positive bacteria,
S. aureus, S. epidermidis and B. subtilis. It was 2–4 times
more potent than norfloxacin against these microorganisms.
In addition, compounds 4b and 4i exhibiting MIC values of
Computational method
Crystal structures of topoisomerase II DNA-gyrase, subunits
A (PDB code: 2XCT) and subunit B (PDB code: 3TTZ)
were retrieved from Protein Data Bank and used for docking
studies. Discovery Studio v.4.0 (Accelrys Inc., San Diego,
CA, USA) was used for preparing the chemical structures of
ligands and proteins and to compute the ligand-protein inter-
actions. The amino groups in both proteins and ligands were
protonated at physiological pH, and carboxylic acid groups
were ionized to carboxylate anions. Docking study was per-
formed with AutoDock 4.2 software (La Jolla, CA, USA),
employing AutoDock Tools (ADT) graphical interface in
MGLTools 1.5.6. For the proteins and the ligands, Kollman
and Gasteiger charges were computed, respectively, and all
123

Molecular Diversity
O
O
F
COOH
Cl
R2
N
Cl
EtO
HCl
NH2
R2
N
X
N
R
N
H
NH2
HN
=
R2
3a:
3b:
R2
H
NO2
2a:
2b:
R2= H
R1
=
1a-e
R
2= NO2
DMF
NaHCO3
F
O
COOH
R2
X=CH; CF; COMe; N
R = cyclopropyl; ethyl
R1 = H; Me
N
X
N
R
N
N
N
H
R2 = H; NO
2
R1
4a-j
Scheme 1 Synthesis of compounds 4a–j
Table 1 MIC values (μg/mL) of compounds 4a–j against different microorganisms
O
F
COOH
R2
N
X
N
R
N
N
N
H
R1
Compounds
X
R
R1
R2
S.a.
S.e.
B.s.
E.c.
P.a.
K.p.
4a
C–H
C–H
C–H
C–H
C–OMe
C–OMe
N
c-Pr
c-Pr
Et
H
H
0.097
0.78
3.125
3.125
12.5
6.25
1.56
100
0.097
0.39
6.25
25
0.097
1.56
6.25
6.25
12.5
6.25
6.25
12.5
1.56
0.39
0.195
0.097
0.78
1.56
0.78
1.56
0.78
25
25
3.125
3.125
12.5
6.25
50
4b
H
NO2
H
25
4c
H
50
4d
Et
H
NO2
H
25
4e
c-Pr
c-Pr
Et
Me
Me
H
100
25
100
6.25
25
4f
NO2
H
3.125
25
4g
6.25
50
4h
N
Et
H
NO2
H
> 100
50
> 100
100
100
1.56
100
25
4i
C–F
C–F
Et
Me
Me
0.78
6.25
0.39
1.56
25
4j
Et
NO2
50
25
Norfloxacin
0.39
0.195
0.39
S.a., Staphylococcus aureus ATCC 6538; S.e., Staphylococcus epidermidis ATCC 12228; B.s., Bacillus subtilis ATCC 6633; E.c., Escherichia
coli ATCC 8739; P.a., Pseudomonas aeruginosa ATCC 9027; K.p., Klebsiella pneumonia ATCC 10031
123

Molecular Diversity
Table 2 Binding free energies
(kcal/mol) for selected
compounds on the DNA-gyrase
A and B
Compound
Gyrase A (ꢀG binding)
Gyrase B (ꢀG binding)
4a
−11.87
−10.72
−7.8
Norfloxacin
−8.39
Fig. 4 Docking pose of compound 4a in the active site of topoisomerase II DNA-gyrase subunit A. Double-stranded DNA is shown in thin lines
and light color. (Color figure online)
0.78 μg/mL were slightly less potent than norfloxacin against
S. aureus. Theactivityofcompound4bagainstS. epidermidis
was comparable to that of norfloxacin (MIC = 0.39 μg/mL).
Against B. subtilis, the MIC of compound 4j was close to that
of norfloxacin.
Compounds 4a–f showed good inhibitory activity against
E. coli (MIC values ≤ 1.56 μg/mL). Compound 4a showed
the highest activity toward E. coli (MIC = 0.097 μg/mL),
being two times superior to norfloxacin. The remaining com-
pounds were less active than reference drug against this
Gram-negative strain. Most compounds exhibited low or no
activity against P. aeruginosa. However, compound 4f had
moderate activity (MIC = 6.25 μg/mL), being fourfold less
potent than norfloxacin. In terms of activity against K. pneu-
monia, compounds 4a, 4b and 4f with MIC values of 3.125
μg/mL were the most active compounds, although weaker
than norfloxacin.
For example, the insertion of a nitro group in compound 4a
resulted in compound 4b with 4–16 times decreasing activ-
ity against Gram-positives. In contrast, nitro compound 4f
was found to be 2- to 16-fold more potent than its analog
4e against tested strains. The comparison of naphthyridine
derivatives 4g and 4h with their quinolone congeners 4c and
4d revealed that the replacement of CH at 8-position with N
did not improve the antibacterial profile.
The obtained results with this limited series of benzimi-
dazole compounds demonstrated that the most potent analog
4a belongs to the non-nitro compounds. This finding is in
contrast to our previous studies with nitrofuran, nitrothio-
phene and nitroimidazole analogs of piperazinylquinolones
[32–35].
Docking study
The obtained results against Gram-positive and Gram-
negative bacteria revealed that cyclopropyl is more favorable
than ethyl as N–1 substituent. The cyclopropyl analog 4a was
2–64 times more potent than the corresponding ethyl deriva-
tive 4c against different strains. The introduction of a nitro
group on the benzimidazole ring had different effect depend-
ing on the type of substituent on the quinolone nucleus.
Molecular docking is a valuable tool for studying the inter-
actions between ligand and target at a molecular level and to
rationalize experimental data for designing novel drugs. With
the aim of rationalizing the obtained antibacterial activity
data, the most potent compound 4a along with the reference
drug norfloxacin were chosen for docking into the active sites
of DNA-gyrase A and B.
123

Molecular Diversity
Fig. 5 2D map of compound 4a within the active site of topoisomerase II DNA-gyrase subunit A
The calculated binding free energies of compound 4a and
norfloxacin on the two DNA-gyrase subunits A and B are
summarized in Table 2. As shown in Table 2, docking on
gyrase A resulted in significantly higher free energy of bind-
ing compared to gyrase B. These results showed that the
possible mechanism of antibacterial action is mainly through
the inhibition of gyrase A.
The best docking pose of compound 4a in the active site
of gyrase A is illustrated in Fig. 4. The quinolone ring of
compound 4a was intercalated between base pairs in double-
Conclusion
A series of benzimidazole-based 7-piperazinylquinolones
were designed and synthesized as new antibacterial agents.
The biological results revealed that most of the com-
pounds had significant activity against Gram-positive bac-
teria including S. aureus, S. epidermidis and B. subtilis.
Although, some compounds showed remarkable activity
against E. coli and K. pneumonia, the activity of these com-
pounds against P. aeruginosa was marginal. Among the
tested compounds, the N1-cyclopropyl derivative 4a bearing
no substituent at R1 and R2 and C-8 of quinolone showed
the highest activity against S. aureus, S. epidermidis, B. sub-
tilis and E. coli (MIC = 0.097 μg/mL), being 2–4 times more
potentthannorfloxacin. ThelimitedSARstudydemonstrated
that the effect of nitro- substituent on the benzimidazole
ring depends on the pattern of substitutions on the piper-
azinylquinolone. The docking study of compound 4a into
the active site of topoisomerase II DNA-gyrase revealed that
the benzimidazole part of the prototype molecule 4a partici-
pates in additional interactions with the target active site.
π
π
stacking. Furthermore, the
stranded DNA and forms
−
β-keto acid system of the quinolone scaffold was coordi-
nated to the manganese ion (Mn2+). The chelation with
manganese ion was further confirmed by measuring the dis-
tance between the two oxygen atoms and Mn2+ ion (3.0 and
1.6 Å). Extra interactions were found between carboxylate
π
-
oxygen and Ser1084 in the form of hydrogen bond, and
cation interaction between phenyl ring of quinolone moiety
and amino group of adenine and guanine nucleotides. The
protonated piperazine-N4 atom of compound 4a interacts
with the carboxylate ion of Asp437 and the benzimidazole-
N1 has hydrogen bond with oxygen atom of ribose ring in
double-strandedDNA. Finally, thebenzimidazolemoietyhas
Acknowledgements This work was supported by a Grant (No. 833)
from the Research Council of Mazandaran University of Medical Sci-
ences, Sari, Iran.
π
-cation interactions with adenosine nucleotide as depicted
in Fig. 5. As mentioned above, the free energy of binding in
the active site of gyrase A for compound 4a was estimated to
be − 11.87 kcal/mol, which is higher than that of norfloxacin.
Therefore, the promising antibacterial activity of compound
4a may be attributed to the additional interactions of the ben-
zimidazole part with the target site.
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