Synthesis and antibacterial, antimycobacterial and docking studies of novel N-piperazinyl fluoroquinolones

Article abstract of DOI:10.1007/s00044-012-0074-2

The present study deals with the synthesis of some novel fluoroquinolone derivatives as antibacterial and antitubercular agents. The titled compounds 7a-g and 8a-g were found to possess comparable or more potent activity than the reference compounds ciprofloxacin, norfloxacin, isoniazid and rifampicin. The synthesized compounds showed activity against S. aureus and C. bacterium, whereas poor activity was observed against P. aeruginosa and E. coli. These compounds were subjected to in vitro cytotoxicity study by MTT assay, and their selectivity index was calculated. Compound 7d was found to be the most efficient antimycobacterial agent amongst the series. Molecular docking revealed that synthesized derivatives and target proteins were actively involved in a binding pattern and had significant correlation with biological activity. Graphical abstract: Novel N-piperazinyl fluoroquinolone derivatives were synthesized and evaluated for their in vitro antibacterial, antimycobacterial and cytotoxic properties. Activity results were compared with the docking results. [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00044-012-0074-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:818–831
DOI 10.1007/s00044-012-0074-2
ORIGINAL RESEARCH
Synthesis and antibacterial, antimycobacterial and docking
studies of novel N-piperazinyl fluoroquinolones
•
Kapil M. Agrawal Gokul S. Talele
Received: 16 June 2011 / Accepted: 18 April 2012 / Published online: 11 May 2012
Ó Springer Science+Business Media, LLC 2012
Abstract The present study deals with the synthesis of
some novel fluoroquinolone derivatives as antibacterial and
antitubercular agents. The titled compounds 7a–g and
8a–g were found to possess comparable or more potent
activity than the reference compounds ciprofloxacin, nor-
floxacin, isoniazid and rifampicin. The synthesized com-
pounds showed activity against S. aureus and C. bacterium,
whereas poor activity was observed against P. aeruginosa
and E. coli. These compounds were subjected to in vitro
cytotoxicity study by MTT assay, and their selectivity
index was calculated. Compound 7d was found to be the
most efficient antimycobacterial agent amongst the series.
Molecular docking revealed that synthesized derivatives
and target proteins were actively involved in a binding
pattern and had significant correlation with biological
activity.
manifestations caused by Mycobacterium tuberculosis,
which has plagued humans throughout the recorded and
archaeological history (Dutt and Stead, 1999). The number
of individuals succumbing to this disease has been
increasing vastly because of the HIV/AIDS pandemic
(Cambau et al., 1994; Grosset, 1992; Tsukamura et al.,
1985). Treatment outcomes of MDR-TB are poor with low
cure rates of around 60 % and high recurrence rates close
to 30 % after standard short-course TB treatment (Becerra
et al., 2000; Espinal et al., 2000; Migliori et al., 2002).
Besides being increasingly popular in treatment of TB,
complicated by intolerance or relative contraindication for
first-line drugs, fluoroquinolones (FQs) are important for
improving treatment outcomes of MDR-TB as second-line
drugs (Yew et al., 2000; Migliori et al., 2008a, b). Fluo-
roquinolones, such as ciprofloxacin, gatifloxacin, spar-
floxacin, moxifloxacin, etc. have demonstrated potential for
shortening treatment duration, (Rustomjee et al., 2008;
Conde et al., 2009; Dorman et al., 2009) and have been
recommended in the treatment of XDR-TB (WHO/HTM/
TB/, 2008). During the past few decades, several fluoro-
quinolone (FQ) antibacterial drugs have been used to treat
M. tuberculosis and bacterial infections because of their
favourable pharmacokinetic profiles.
Keywords Fluoroquinolone (FQ) ꢀ Antibacterial ꢀ
Antimycobacterial ꢀ Cytotoxicity ꢀ Docking study
Introduction
Tuberculosis (TB) is the oldest documented, chronic
necrotizing bacterial infection with a wide variety of
In the present scenario, much of the quinolone anti-
bacterial research indicates that functionality at C-7 posi-
tion of 6-fluoroquinolone can be enhanced with the entry of
bulkier substituents. Moreover, C-7 substitution is the most
adaptable site for chemical change and is an area that
determines potency and target preferences (Foroumadi
et al., 2005; Domagala et al., 1988; Chu et al., 1985; Shen
et al., 1989). Hence, there is the need for the development
of novel antimycobacterial agents to combat mycobacterial
and bacterial infections, with reduced toxicity profile and
resistance (Rattan et al., 1998).
K. M. Agrawal (&)
Department of Pharmaceutical Chemistry, R. C. Patel Institute of
Pharmaceutical Education and Research, Shirpur, Dhule 425405,
Maharashtra, India
e-mail: contactkap@yahoo.co.in
G. S. Talele
Department of Pharmaceutical Chemistry, Nasik Gramin
Shikshan Prasarak Mandal College of Pharmacy, Triambak
Road, Anjaneri, Nasik, Maharashtra 422213, India
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Med Chem Res (2013) 22:818–831
819
At present, we synthesized the substituted C-5 ben-
zoylmethylenethio-1,3,4-thiadiazole which was fused with
C-7 and N-1 piperazinyl FQs. They could be considered as
hybrid compounds (Gadad and Talath, 2006; Manzo et al.,
1992). The titled compounds were subjected to in vitro
antibacterial, antimycobacterial and cytotoxicity studies to
examine their relationship between structural modifications
and biological activities with reduced toxicity profile.
Finally, these biological activity findings and dock score
have helped in predicting a potent/efficient agent against
bacteria and M. tuberculosis from the synthesized deriva-
tives; these objectives of our study were really achieved
with the help of molecular docking study in which the
binding patterns of the synthesized derivatives with tar-
geted protein, DNA gyrase (subunit A), was significantly
compared (Ghosh and Bagchi, 2010).
mechanism involving substituted 2-chloro-5-benzoyl-
methylenethio-1,3,4-thiadiazole 6a–g and piperazinyl fluo-
roquinolones with weak base sodium-bi-carbonate in the
presence of dimethylformamide, which seemed to be a
convenient route to fulfil our objectives. The structure of
novel synthesized derivatives 7a–g and 8a–g were eluci-
dated on the basis of physical constants, spectral data (FTIR,
1H-NMR, 13C-NMR and FAB MASS) and elemental
analysis.
In general, FTIR spectra of compounds 7a–g and 8a–
g gave representative broad absorption bands ranging around
3,457–3,400 cm-1 for –OH str., indicating carboxylic acid
group in the final structure; and 3,120–2,910 cm-1 for aro-
matic C–H str., indicating the presence of 2-oxo-2-p(substi-
tuted)phenylethylthio moiety. Aliphatic CH2-stretching
vibrations appeared between 2,850 and 2,822 cm-1 for
methylene bridge, which confirmed the 2-oxo-2-p(substi-
tuted)phenylethylthio-1,3,4-thiadiazole linker with N-piper-
azinyl FQ derivatives. 1750–1710, 1694–1660 and 1645–
1620 cm-1 indicated the presence of three C=O str. groups in
titled compounds (The C=O as carboxylic, mono aryl and
4-oxo quinolonic ketonic group, respectively). Absorption
peaks appeared around 705–610 cm-1 for aliphatic C–S str.
vibration, justifying the presence of 1,3,4-thiadiazole group.
1HNMR spectra for 7a–g and 8a–g showed broad singlet
around d 14.49–15.09 ppm, confirming the presence of acid
proton of –COOH. The characteristic singlet was arrested in
the final compounds around d 4.44–4.82 ppm for –CH2
methylene proton, which confirmed the reaction between a-
bromoketones 4a–g and 2-amino-5-mercapto-1,3,4-thiadia-
zole 2. Quinolone protons splitted and appeared as singlet for
2nd proton of quinolone at d 8.46–8.68 ppm, and doublets
appeared between the ranges at d 7.80–7.98 and 7.51–7.68 for
5th and 8th quinolone proton, respectively. Aryl protons
appeared at d 7.22–7.54 as multiplets for 5H (7a and 8a)
protons, but when the substitutions (–Cl, –Br, –NO2, –CH3,
–OCH3 and –F) occured at para position in aryl group
(7b–g and 8b–g), the remaining fourarylprotons (4H)splitted
into two protons each and appeared as multiplets at different
d positions which seemed to be at d 8.10–8.78 and
6.82–8.18 ppm for four protons. Eight piperazinyl protons for
7a–g were characterized as multiplets and splitted into set of
four protons at d 3.67–3.73 and 3.50–3.61 ppm and at d
3.41–3.58 and 3.18–3.32 ppm for compounds 8a–g, respec-
tively. Compounds 7a–g for four cyclopropylic protons
appeared as multiplets at d 1.22–1.43 ppm, and remaining
one proton of cyclopropyl appeared as multiplets at d
3.41–3.58 ppm along with piperazinyl protons. Compounds
8a–g for N-bonded five ethylic (CH2CH3) protons splitted as
quartet (2H, d 4.20–4.54) and triplet (3H, d 1.18–1.42 ppm).
N-bonded both cyclopropylic and ethylic protons 7a–g and
8a–g showed characteristic peaks indicating that both the FQs
are distinguishably synthesized. The final derivatives 7e and
Result and discussion
Chemistry
In the present study, we synthesized few reported substituted
phenacyl bromides and 2-amino-5-mercapto-1,3,4-thiadia-
zole which in turn were used to prepare the newly synthe-
sized N-piperazinyl fluoroquinolonic derivatives, preceded
by chlorobenzoylmethylenethio-1,3,4-thiadiazole 6a–g.
The synthesis of 2-amino-5-mercapto-1,3,4-thiadiazole 2
involved the method of direct cyclization through thiosem-
icarbazide 1 in the presence of carbon-di-sulphide and dry
ethanol (Foroumadi et al., 1999; Freund and Imgart, 1895).
The synthesis of phenacyl bromide/a-bromoketones 4a–
g was based on free radical reaction mechanism followed by
side-chain halogenation of alkylbenzene via homolytic fis-
sion of bromide ion in the presence of lewis acid (anhydrous
aluminium chloride) that easily abstracted the benzoylic
hydrogen ion from the substituted acetophenones 3a–g, with
hydrogen bromide getting released and the substituted
phenacyl bromides 4a–g being formed (Morrison and Boyd,
2004). The synthesis of 2-amino-5-benzoylmethylenethio-
1,3,4-thiadiazoles 5a–g was carried out by reacting a-bro-
moketones 4a–g and 2-amino-5-mercapto-1,3,4-thiadiazole
2 via dehydrobromination mechanism, in the presence of
ethanolic potassium hydroxide which abstracted the hydro-
gen bromide. The synthesized compounds 5a–g were
directly converted to 2-chloro-5-benzoylmethylenethio-
1,3,4-thiadiazole 6a–g by diazotization of amines followed
by chlorination with concentrated hydrochloric acid in
the presence of NaNO2 and copper powder. The synthe-
sis of 1-cyclopropyl/ethyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-
oxo-2-p(substituted)-phenylethylthio)-1,3,4-thiadiazol-2-yl}
piperazin-1-yl]-4-oxoquinoline-3-carboxylic acid 7a–g and
8a–g was based on aromatic nucleophilic substitution
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Med Chem Res (2013) 22:818–831
7f were also confirmed by characteristic peaks as singlets
around d 2.35 for methylic and d 3.33 ppm for methoxyl
proton and d 2.38 and d 3.82 for (8e and 8f), respectively.
In 13C-NMR, the higher d values appeared at around
194.1–195.2, 177.5–178.5 and 165–167 for three carbonyl
carbons which are present in compounds 7a–g and 8a–
g. The characteristic peak as singlet for C–S was observed
at 164–165 d ppm, confirming the formation of 2-oxo-2-
p(substituted)phenylethylthio linker in final derivatives.
Nitro-substituted 7d and 8d appeared at higher d values
152.8 ppm as compared to other substitutions because of
electron-withdrawing nature of nitro group. On the con-
trary, the electron-releasing nature of methyl and methoxyl
group 7e, 7f, 8e, and 8f shifted the d values at upfield
position at 24.3, 24.4, 55.8 and 56.1 ppm, respectively.
Aliphatic CH2 methylene carbon showed the characteristic
peak at d 37.5–38.5 ppm in the final derivatives. Four
piperazinyl carbons of FQs appeared at around d 49.6 and
49.3 ppm. Cyclopropyl and ethylic group in 7a–g and 8a–
g represented characteristic peaks at around at d 36, 5.6, 49
and 13 ppm, respectively. The FAB Mass spectra showed
molecular ion peak data for the selected compounds. All
the compounds produced satisfactory elemental analysis
( 0.4 %).
in case of 7e, 7f, 8e and 8f. On the contrary 7b–7d, 8b–8d
resulted in encouraging MIC values from 0.5 to 2.5 lg/mL.
Compounds 7d, 8d, 7b and 8b (MIC = 0.5, 1.0, 1.0,
1.5 lg/mL respectively) exhibited a prominent activity as
compared to first-line anti-TB drugs isoniazid (INH,
MIC = 1.5 lg/mL) and rifampicin (RIP, MIC = 1.0
lg/mL). The results are depicted in Table 1.
Cytotoxicity study
Selected compounds 7b–7d, 8b–8d were examined for in
vitro cytotoxicity study (IC50) by MTT assay in mamma-
lian vero cell lines from the kidney of African green
monkey, organism Cercopethicus aethiops, followed by
determination of selectivity index (SI), (IC50/MIC). The
results were expressed in lM, but for determination of SI,
these values were converted into lg/mL. Compounds
having high SI are categorized as non-toxic agent (Sriram
et al., 2006). Compound 7d was found to be least toxic
SI [ 12.2 as compared to reference RIP, (SI = 10) INH,
(SI = 8), compounds 8d, 7b, 7c, 8b, and 8c were near to
non-toxic, as per the SI i.e. [10.2, 3.0, 3.0, 2.67 and 2.0
respectively. The presence of domain as 2-oxo-2-p-phe-
nylethylthio-1,3,4-thiadiazole with nitro as p-substituted in
ciprofloxacin/norfloxacin analogue, makes it the most
efficient antimycobacterial agent. The results of cytotox-
icity studies are depicted in Table 1.
Antibacterial activity
Antibacterial activity of 7a–g and 8a–g was performed by
broth microdilution method. Ciprofloxacin and norfloxacin
were used as reference. Amongst all the synthesized
compounds, 7d (MIC = 0.5) was found to be more potent
than reference drugs, whereas, compounds 7f, 8d and 8f,
(MIC = 1–2) possessed activity comparable to the refer-
ence, against Gram-positive bacterial strains, but all these
compounds were not able to produced enhanced antibac-
terial activity against Gram-negative organisms. These
observed antibacterial activity suggests that; all these
compounds were not able to exert their effects against
comparatively tough (double layered and excessive lipids
containing) cell wall of Gram-negative organisms; how-
ever, these compounds were effective against single cell
wall layer containing Gram-positive organisms. Besides
the possibility of resistance development against Gram-
negative organisms cannot be ruled out. The results of
antibacterial studies are presented in Table 1.
SAR and docking study
1- Introduction of 1,3,4-thiadiazole carrying benzoylme-
thylenethio moiety at N-4 position on the piperazine
ring was well tolerated in terms of Gram-positive
activity, exemplified by the potency of ciprofloxacin
and norfloxacin analogues. The absence of substitu-
tions at R position in FQ derivatives 7a and 8a did not
produce enhanced activity.
2- The presence of chloro, bromo, or nitro substituents at
2-oxo-2-p-phenylethylthio-1,3,4-thiadiazole linked to
N-4 piperazinyl quinolone 7b–7d, 8b–8d, resulted in
prominent activity with MIC = 0.5–2.5 lg/mL.
3- The presence of methyl and methoxyl substituent 7e,
7f, 8e and 8f, showed moderate activity while
compound 7a and 8a which were unsubstituted did
not show enhanced activity.
This indicates that electron withdrawing group increases
activity while electron releasing group slightly raise in
activity while unsubstituted derivatives lacks the activity.
To explore further, all the synthesized compounds were
subjected to Grid based molecular docking studies. DNA
gyrase-A is one of the few thoroughly characterized and
well validated targets in anti TB and antibacterial inhibitors
Antimycobacterial activity
The activity was performed against M. tuberculosis H37Rv
strain by broth dilution assay using Mueller–Hinton agar
medium. Results revealed that 7a and 8a did not produce
significant results, whereas moderate activity was observed
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Med Chem Res (2013) 22:818–831
821
Table 1 Antibacterial, antimycobacterial, cytotoxicity and SI data for 7a–g and 8a–g
R
F
O
O
S
S
N
N
O
OH
N
N
N
R'
Compounds
R
R0
Gram-positive organismsa
Gram-negative organismsb
M. TB
MICc
ICd50
SIe
Sa
16
Cb
16
Pa
Ec
32
7a
7b
7c
7d
7e
7f
H
[64
Resist
–
–
–Cl
4
8
1
8
2
8
2
4
[64
[64
[64
[64
[64
32
16
16
32
16
32
16
1
3
3
–Br
2
6
3
–NO2
–CH3
–OCH3
–F
0.5
0.5
Resist
10
[6.1
[12.2
4
1
4
–
–
–
–
–
–
7g
5
8a
H
–C2H5
–C2H5
–C2H5
–C2H5
–C2H5
–C2H5
–C2H5
32
4
8
2
8
2
4
2
2
–
–
16
2
4
1
4
1
2
1
2
–
–
[64
[64
[64
[64
[64
[64
32
[64
32
Resist
1.5
2.5
1
–
–
8b
–Cl
4
2.67
8c
–Br
32
5
2
8d
–NO2
–CH3
–OCH3
–F
16
[10.2
[10.2
8e
[64
[64
[64
2
Resist
10
–
–
–
8f
–
8g
5
–
–
Ciprofloxacin*
Norfloxacin*
Rifampicin*
Isoniazid*
4
–
–
–
4
2
–
–
–
–
–
1
[10
[12
[10
8
–
–
1.5
* Reference drugs
a
Values expressed in lg/mL, Corynebacterium (Cb) ATCC 29355, Staphylococcus aureus (Sa) ATCC25365
Values expressed in lg/mL, Pseudomonas aeruginosa (Pa) ATCC 25619, Escherichia coli (Ec) MTCC1089
Mycobacterium tuberculosis H37Rv strain: (values expressed in lg/mL)
b
c
d
e
IC50 inhibition concentration (inhibited 50 % of total cells in lM and converted to lg/mL for SI calculation)
SI selectivity index (ratio between IC50 and M. tuberculosis MIC value)
123

822
Med Chem Res (2013) 22:818–831
like fluoroquinolones (Ghosh and Bagchi, 2010). The
docking results revealed that some of the synthesized
fluoroquinolone derivatives had good affinity to the active
site residue with respect to reference drugs ciprofloxacin,
norfloxacin and isoniazid (Table 2). The compounds
7a–7d, 7f, 7g, 8d–8f and 8g showed better Glide score
(G-score) as compared to reference ciprofloxacin and
norfloxacin with respect to crystal structure of Staphylo-
coccus aureus. Similarly compounds 7e, 7g and 8g showed
better G-score than reference isoniazid with respect to
crystal structure of M. tuberculosis (Fig. 1).
group showed the hydrophobic interaction with Gly, Val
and Ala residues which impart the effective binding affinity
to the synthesized fluoroquinolone derivatives. Hence,
compound 7d showed better biological activities despite
low G-score.
Highly active compounds
Compound 7b produced significant G-score of -9.37
indicating high binding affinity of the ligand towards DNA
gyrase for S. aureus. The chloro group held at C-5 ben-
zoylmethylenethio-1,3,4-thiadiazole of 7b, produced
strong hydrophobic interactions with Ala1118, Glu1088,
Gly1117, the carboxyl group at C-3 and C-4 position in
quinolone ring of 7b, showed hydrogen bonding interaction
with Gly459 and Asp437 residue, whereas Phe172, Ala173
and Gln168 participated in electrostatic interactions, which
are essential characteristics for drug receptor interaction.
However, 7b showed significant antimycobacterial activity
despite of moderate G-score (-2.43) as compared to
standard isoniazid (-3.2).
In case of M. tuberculosis, the carboxyl group at 3rd
position in quinolone ring interacts mainly with Arg120
and Gly161 residue, which participated in hydrogen
bonding, whereas Phe172, Ala173 and Gln168 participated
in electrostatic interactions. It was also found to interact on
the same position, with Gly459, Asp437, Arg458 and DG9
residues of S. aureus, which participated in hydrogen
bonding and electrostatic interaction, which are essential
characteristics for drug receptor binding. Amongst the
series, compound 7d possessed higher H-bonding and
electrostatic score i.e. -1.49 and -0.79 than isoniazid.
Besides H-bonding and electrostatic interaction, hydro-
phobic interactions are also one of the major interactive
forces in ligand recognition and binding pattern (Ghosh
and Bagchi, 2010). As per Fig. 2, substituted derivatives at
N-4 position of benzoylmethylenethio-1,3,4-thiadiazole
Moderately active compounds
Compounds 7e, 7f, 8e and 8f produced moderate activity
with moderate G-scores of -5.95, -8.97, -7.53 and -8.53
Table 2 Docking results of synthesized compounds 7a–g and 8a–g
S. no. Ligands
PDB: 2XCT (S. aureus)
PDB: 2BM7 (M. tuberculosis)
G-score Lipophilic EvdW HBond Electrostatic score G-score Lipophilic EvdW HBond Electrostatic score
1
7a
7b
7c
7d
7e
7f
-9.24
29.37
-9.24
-7.96
-5.95
-8.97
-9.1
-6.7
-0.91 -1.62
-0.92 21.65
-0.81 -1.64
-0.71 -1.30
-0.54 -0.35
-0.92 -1.53
-0.96 -1.59
-0.96 -0.39
-0.91 -1.33
-1.34 -0.62
-0.96 -1.52
-1.85 -0.76
-0.99 -0.64
-0.63 -0.84
-1.02 -0.43
-1.67 -0.41
-2.93
-2.43
-3.11
-2.57
-3.65
-2.22
-3.74
-2.54
-3.05
-2.5
-3.84
-3.98
-2.86
-1.72
-3.14
-2.7
0
0
0
-0.01
-0.05
0.02
2
-6.8
3
-6.78
-5.96
-5.06
-6.53
-6.55
-6.83
-6.91
-4.96
-6.28
-6.08
-6.89
-6.73
-5.95
-5.12
–
4
21.49 20.79
5
0
0
-0.54
-0.46
0.14
6
7
7g
8a
8b
8c
8d
8e
8f
-3.82
-3.04
-3.01
-3.13
-4.06
-3.22
-4.06
-3.79
-0.52
–
0
8
-7.17
-5.15
-2.85
-8.76
-7.53
-8.53
-8.2
0
-0.25
-0.58
9
-0.7
10
11
12
13
14
15
16
17
-0.22 -0.37
-2.22
-3.06
-2.22
-3.66
-2.12
–
0
-0.7
0
0.28
-0.44
-0.49
-0.33
8g
0
Ciprofloxacin -7.58
22.12 -0.41
Norfloxacin
Isoniazid
-7.49
–
–
–
–
-3.2
-1.2
-0.7
-0.56
Bold value indicates high G-score amongst the series
G-score total GlideScore, Lipophilic EvdW lipophilic term derived from hydrophobic grid potential and fraction of the total protein ligand vdW
energy, HBond ChemScore H-bond pair term, Electrostatic score electrostatic reward
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Med Chem Res (2013) 22:818–831
823
Fig. 1 Binding interaction of synthesize ligands with S. aureus DNA
gyrase complex (PDB: 2XCT) domain. a Binding pose of highest
scoring compound 7b (green stick). b Binding pose of the most active
compound 7d (green stick). Dotted yellow bond showing H-bond
interaction with binding site residue (Color figure online)
Fig. 2 Binding interaction of synthesize ligands with M. tuberculosis
DNA complex (PDB: 2BM7) domain. a Binding pose of highest
scoring compound 7g (green stick). (b) Binding pose of the most
active compound 7d (green stick). Dotted yellow bond showing
H-bond interaction with binding site residue. Grey surface showing
lipophilic area and brown colour showing electrostatic area (Color
figure online)
against DNA gyrase from S. aureus and -3.65, -2.22,
-3.06 and -2.22 against M. tuberculosis DNA gyrase
respectively. However, 7f and 8f produced higher G-scores
than 7e and 8e. Here once again our assumption proved
that electron withdrawing group has significant impact over
the biological activity, as compared to electron releasing
substituents (the presence of O with –CH3 in 7f and 8f
compared with –CH3 group in 7e and 8e) as shown in
Tables 1 and 2.
Conclusions
In brief, a series of N-piperazinyl fluoroquinolone deriva-
tives 7a–g and 8a–g in which N-4 hydrogen of piperazinyl
group of ciprofloxacin and norfloxacin is substituted with
various chlorobenzoylmethylenethio-1,3,4-thiadiazol moi-
eties has been synthesized. These compounds showed
prominent in vitro antibacterial activity against Gram-
positive organisms, whereas poor activity was observed
against Gram-negative organisms than ciprofloxacin and
norfloxacin. The characteristics of the substituted func-
tional groups at C-7 position of quinolone ring system have
strong influence on the spectrum and extent of antimyco-
bacterial activity. Electron withdrawing groups such as
–Cl, –Br, –NO2 substituents, produced greater/comparable
antimycobacterial activity than the electron releasing
groups as substituents (R in Scheme 1). Especially, nitro
Least active compound
Unsubstituted analogues 7a and 8a produced good G-score
[-9.24 and -7.17 (DNA gyrase from S. aureus)] and
[-2.93 and -2.54 (DNA gyrase from M. tuberculosis)] but
minimal anti-bacterial and anti-mycobacterial activity,
because of -0.91 and -0.96 and 0.00 hydrogen bonding
energy for both 7a and 8a with the respective receptors.
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Med Chem Res (2013) 22:818–831
R
Scheme 1 R: (a) = H, (b) =
–Cl, (c) = –Br, (d) = –NO2,
(e) = –CH3, (f) = –OCH3 and
(g) = –F. (i) EtOH, KOH/4 h
stirring, rt; (ii) NaNO2, HCl,
0–5 °C/stirring; and (iii) DMF,
NaHCO3,/18–20 h reflux/
120 °C. Note: (4a–g). 1
HS
S
2
NH2
+
O
N
N
4(a -g)
CH2Br
R
(i)
Reaction should be carried out
in fuming hood with strong
alkali trap and the reaction
assembly to be surrounded by
strong ammonia vapour which
masks the bromine fumes. 2
Improved yield may be obtained
by exposing the flask to the light
of two 300-watt tungsten lamps
during the bromination
N
S
N
O
S
5(a -g)
NH2
R
(ii)
N
N
O
S
(Coleman and Honeywell,
1943)
S
CH3
Cl
6 (a -g)
OH
N
OH
N
O
HN
N
O
HN
(iii)
O
N
O
O
F
F
O
N
R
(iii)
F
OH
O
N
O
R
N
F
OH
N
S
N
O
S
N
N
N
S
7 (a -g)
O
S
H3C
8 (a -g)
N
N
substituted analogues 7d and 8d were more active than
other compounds. Interestingly, fluoro substitution 7g and
8g were not able to produce enhanced antimycobacterial
activity than chloro and bromo substituted analogues. On
the contrary, the presence of electron releasing groups like
methyl, methoxyl 7e, 7f, 8e and 8f resulted in declined
activity. Encouraging antimycobacterial agents were
selected for in vitro cytotoxicity studies, in which 7d and
8d were found to be the most efficient (potent with least
cytotoxicity) antimycobacterial agents. As per docking
studies, compounds did not show any clear correlation
between G-score and biological activities; however, com-
pound 7d showed strong (MIC = 0.5 lg/mL) biological
activity and low G-score as compared to reference. This
might be due to high score of H-bonding and electrostatic
force, which is key factor for drug receptor binding pattern.
However, the role of substitutions in the titled compounds
is unclear to explain the exact mechanism of action, for
giving variations in results, as all the substitutions (R),
were held at the same para position. Therefore, our studies
could provide tremendous space for chemists and phar-
macists to further explore in the same region and search for
efficient antimycobacterial agents.
Experimental
Materials and methods
The pure FQ drugs used in the synthesis were procured as
gift samples from Mcleods Pharmaceuticals Ltd., Mumbai
and IPCA laboratories Ratlam, India. Reagents, starting
materials and solvents were purchased from common
commercial suppliers (S.D. Fine chemicals, Rankem,
E.merck, Himedia, Spectrochem). Melting points were
determined using Thiels tube (paraffin bath) and are
uncorrected. The purification of synthesized compounds
was achieved by passage through column chromatography
on silica gel (mesh 230–400) with the indicated solvent
system. Thin layer chromatography was performed on
precoated silica gel 60 F254 plates (E. Merck Co.
Darmstadt, Germany). Infra red spectra was recorded using
1
KBr disc on a Nicolet MX-1 FTIR spectrophotometer, H
NMR spectra were recorded at 400 MHz and 13C NMR
spectra were recorded at 100 MHz on a Bruker AM
spectrometer, IISc Bangalore, India and their chemical
shifts are reported in d ppm units with respect to TMS as
internal standard. Microanalysis for C, H, N was performed
123

Med Chem Res (2013) 22:818–831
825
in a Heraeus CHN Rapid Analyzer. The FAB Mass spectra
were recorded on Autospec Mass spectrometer, IICT,
Hyderabad, India data System using Argon/Xenon (6 kV,
10 mA) as the FAB gas. The accelerating voltage was
10 kV and the spectra were recorded at room temperature.
All new compounds yielded spectral data consistent with
the proposed structure and microanalysis within 0.4 % of
the theoretical values.
The solid was removed by filtration, washed with water and
ether. 5a–g was obtained, m.p. 88–91 °C (60–65 %).
General procedure for 2-chloro-5-benzoylmethylenethio-
1,3,4-thiadiazoles 6a–g
5-Benzoylmethylenethio-2-amino-1,3,4-thiadiazole 5a–g
(10 mmol) was ground with an excess of sodium nitrite
(30 mmol) and the mixture was introduced in small portions
with continuous stirring, into an ice cooled solution of
30 mL conc. hydrochloric acid and 15 mL water, main-
tained at 0–5 °C, containing copper powder (0.1 times). The
reaction mixture was allowed to cool at room temperature,
and heated to 75 °C for 1 h. The reaction mixture was cooled
again and extracted with chloroform (50 mL 9 3). The
combined extracts were washed with sodium bicarbonate
solution, dried over anhydrous sodium sulphate for over-
night and chloroform was evaporated under reduced pres-
sure and recrystallized from ethanol to yield 6a–g. The
reaction was monitored through TLC, purification was
achieved by column chromatography with chloroform:
methanol 9:1 as mobile phase, m.p. 90–110 °C (55–60 %).
Synthesis of 2-amino-5-mercapto-1,3,4-thiadiazole 2
(Foroumadi et al., 1999; Freund and Imgart, 1895)
A mixture of thiosemicarbazide 1 0.1 mol and carbon-di-
sulphide 0.1 mol were refluxed gently for half an hour.
After cooling, 90 mL water was added and the mixture was
refluxed for 4 h and filtered. The solution was neutralized
with potassium hydroxide. The precipitate was filtered and
crystallized from ethanol and recrystallized from aqueous
ethanol to get white crystalline mass 2. M.p. 230–232 °C
dec. (75–80 %).
Synthesis of a-bromoketones/phenacyl bromide 4a–
g (Cowper and Davidson, 1943; Naidir and Khim, 1982)
General procedure for N-piperazinyl quinolone derivatives
7a–g and 8a–g
Equimolar quantity of substituted acetophenones 3a–g and
appropriate anhydrous solvent such as ether, acetone, car-
bon-di-sulphide, methanol, chloroform or glacial acetic
acid were taken into two necked flask with magnetic bead,
and fitted with bromine dropping funnel and reflux con-
denser. The reaction condition was maintained either in
cold or at room temperature, anhydrous aluminium chlo-
ride 0.1 g was introduced, bromine (0.084 mol) was added
gradually to the flask with stirring. After addition of bro-
mine, appropriate solvent was added to the reaction mix-
ture. Compounds 4a–g were obtained as brownish yellow
to colourless crystalline mass, washed twice with solvents,
and recrystallised from methanol to get colourless shining
lachrymatory crystals.
A mixture of equimolar quantities of 2-chloro-5-benzoyl-
methylenethio-1,3,4-thiadiazole 6a–g, and substituted pip-
erazinyl quinolone, along with sodium-bi-carbonate in
5 mL dimethylformamide was heated on oil bath and
refluxed at 120 °C for 18–20 h. After cooling 10 mL of
cold water was added to the reaction mixture and the
precipitate was filtered, washed with water twice and re-
crystallized from dimethylformamide–water to yield the
titled compounds. For physical constants please refer
Table 3.
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
phenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-yl]-4-
oxoquinoline-3-carboxylic acid (7a)
Melting point ranges of 4a–g; R = H, Cl, Br, NO2, CH3,
OCH3, F; 50–52°, 90–92°, 103–105°, 94–96°, 52–54°,
68–70°, 48–50° respectively (56–61 %, Scheme 1).
IR (KBr) cm-1: 3420 (O–H str.), 3020 (Ar. C–H str.), 2830
(Ali. C–H str.), 1730 (Carboxylic C=O str.), 1680 (Ar.,
C=O str.), 1620 (4-Oxo quinolone, C=O str.), 742 (out of
plane, Ar. C–H def.), 680 (Ali. C–S str.) 1H NMR (DMSO-
d6), d ppm: 15.03 (s, 1H, COOH), 8.52 (s, 1H, H2-quino-
line), 7.94 (d, 1H, H5-quinoline, J = 13.04 Hz), 7.63 (d,
1H, H8-quinoline, J = 7.44 Hz), 7.54 (m, 5H, Ar.), 4.80 (s,
2H, Ali. CH2), 3.81–3.87 (m, 4H, piperazine), 3.35–3.77
(m, 4H, piperazine and 1H, CH, cyclopropyl), 1.21–1.42
(m, 4H, cyclopropyl). 13C NMR (DMSO-d6) d ppm: 194.1,
177.3, 166.2, 163.8, 148, 144.4, 143.5, 136.7, 133.2, 128.5,
116.4, 110.2, 100.5, 49.4, 48.9, 36.4, 5.4. MS (FAB) m/z:
General procedure for 2-amino-5-benzoylmethylenethio-
1,3,4-thiadiazole 5a–g
Addition of 0.1 mol of 85 % potassium hydroxide to a
slurry of 0.1 mol of 2-amino-1,3,4-thiadiazole-5-mercapto
2 in 10 mL of water produced a brownish solution. Solu-
tion was clarified with activated charcoal and diluted with
28.5 mL of ethanol. 0.1 mol of various substituted a-bro-
moketones 4a–g were added rapidly with stirring. Thick
reaction mixture was formed, stirred vigorously and cooled
for 30 min, and then diluted with 200 mL of cold water.
123

826
Med Chem Res (2013) 22:818–831
Table 3 Physical constants for 7a–g and 8a–g
R
F
O
O
S
S
N
N
O
OH
N
N
N
R'
Compounds
R
H
R0
Yield
63
Colour
Brown
M.P.
(°C)
Rf
7a
248–250
242–246
241–244
244–46
0.42
7b
7c
7d
7e
7f
–Cl
53
58
60
62
54
50
Brown
Brown
0.44
0.62
0.32
0.40
0.34
0.52
–Br
–NO2
–CH3
–OCH3
–F
Brownish yellow
Yellowish brown
Brown
244–247
240–242
168–170
7g
Colourless
8a
8b
8c
8d
8e
8f
H
–C2H5
–C2H5
–C2H5
–C2H5
–C2H5
–C2H5
–C2H5
62
52
53
54
57
62
50
Brownish yellow
Light brown
Brown
234–238
236–240
238–241
231–232
239–242
229–232
162–165
0.46
0.37
0.56
0.48
0.32
0.36
0.52
–Cl
–Br
–NO2
–CH3
–OCH3
–F
Yellow
Brown
Brown
8g
Off-white
M? 584. Anal. Calcd. for C27H24FN5O4S2: C 57.33, H
4.28, N 12.38. Found: C 57.31, H 4.30, N 12.36.
744 (out of plane, Ar. C–H def.), 684 (Ali. C–S str.) 1H NMR
(DMSO-d6) d ppm: 15.04 (s, 1H, OH), 8.54 (s, 1H, H2-
quinoline), 8.12 (m, 2H, Ar.), 7.92 (d, 1H, H5-quinoline,
J = 13.0 Hz), 7.60 (d, 1H, H8-quinoline, J = 7.42 Hz), 7.24
(m, 2H, Ar.), 4.54(s, 2H, CH2),3.78–3.84(m,4H, piperazine),
3.54–3.60 (m, 4H, piperazine and 1H, CH, cyclopropyl),
1.22–1.34 (m, CH2, 4H, cyclopropyl) ppm.13C NMR
(DMSO-d6) d ppm:194.5, 177.3, 166.5, 164.5, 149.2, 144.8,
143.9, 139.2, 135.2, 130.4, 128.7, 117.7, 111.5, 101.4, 49.7,
49.6, 36.2, 5.3 ppm. Anal. Calcd. for C27H23ClFN5O4S2: C
54.04, H 3.86, N 11.67. Found: C 54.02, H 3.85, N 11.69.
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
chlorophenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-
yl]-4-oxoquinoline-3-carboxylic acid (7b)
IR (KBr) cm-1: 3448 (O–H str.), 3120 (Ar.C–H str.), 2822
(Ali. C–H str.) 1720 (Carboxylic C=O str.), 1690 (Ar. C=O
str.), 1622 (4-Oxo quinolone C=O str.), 780 (Ar. C–Cl str.),
123

Med Chem Res (2013) 22:818–831
827
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
bromophenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-
yl]-4-oxoquinoline-3-carboxylic acid (7c)
cyclopropyl), 2.35 (s, 3H, Ar.CH3, methyl), 1.21–1.44 (m,
CH2, 4H, cyclopropyl) ppm. 13C NMR (DMSO-d6) d ppm:
194.5, 176.4, 166.4, 164.5, 149.6, 144.3, 141.7, 133.7,
129.5, 128.4, 116.5, 109.4, 100.5, 49.2, 51.4, 36.2, 24.4,
5.2 ppm. Anal. Calcd. for C28 H26FN5O4S2: C58.02, H 4.52,
N12.08. Found: C 58.00, H 4.50, N 12.10.
IR (KBr) cm-1: 3408 (O–H str.), 2990 (Ar. C–H str.), 2845
(Ali. C–H str.) 1710 (Carboxylic C=O str.), 1686 (Aryl
C=O str.), 1628 (4-Oxo quinolone C=O str.), 750 (out of
plane, Ar. C–H def.), 686 (Ali. C–S str.) 1H NMR (DMSO-
d6) d ppm: 14.90 (s, 1H, OH), 8.50 (s, 1H, H2-quinoline),
8.10 (m, 2H, Ar.), 7.80 (d, 1H, H5-quinoline, J =
13.02 Hz), 7.68 (d, 1H, H8-quinoline, J = 7.44 Hz), 7.22
(m, 2H, Ar.), 4.65 (s, 2H, CH2), 3.76–3.82 (m, 4H, piper-
azine), 3.58–3.64 (m, 4H, CH2, piperazine and 1H, CH,
cyclopropyl), 1.22–1.35 (m, CH2, 4H, cyclopropyl) ppm.
13C NMR (DMSO-d6) d ppm: 194.3, 177.2, 167.2, 164.1,
148.7, 144.6, 144.9, 135.7, 131.6, 127.5, 116.8, 109.2,
100.7, 49.3, 49.4, 36.4, 5.3 ppm. MS (FAB) m/z: M? 654.
Anal. Calcd. for C27H23BrFN5O4S2: C 50.31, H 3.60, N
10.87. Found: C 50.33, H 3.62, N 10.89.
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
methoxyphenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-
yl]-4-oxoquinoline-3-carboxylic acid (7f)
IR (KBr) cm-1: 3457 (O–H str.), 2920 (Ar. C–H str.), 2838
(Ali. C–H str.), 1740 (Carboxylic C=O str.), 1674 (Ar.
C=O str.), 1636 (4-Oxo quinolone C=O str.), 1029 (C–O–C
str.) 747 (out of plane, Ar. C–H def.), 650 (Ali. C–S str.) 1H
NMR (DMSO-d6) d ppm: 15.07 (s, 1H, OH), 8.58 (s, 1H,
H2-quinoline), 8.14 (m, 2H, Ar.), 7.93 (d, 1H, H5-quino-
line, J = 13.03 Hz), 7.58 (d, 1H, H8-quinoline,
J = 7.48 Hz), 6.84 (m, 2H, Ar.), 4.54 (s, 2H, CH2),
3.71–3.77 (m, 4H, CH2, piperazine), 3.51–3.58 (m, 4H,
CH2, piperazine and 1H, CH, cyclopropyl), 3.33 (s, 3H, Ar.
–OCH3, methoxyl), 0.92–1.42 (m, 4H, CH2, cyclopropyl),
13C NMR (DMSO-d6) d ppm:194.6, 176.7, 166.3, 165.2,
149.2, 144.3, 141.2, 133.4, 129.8, 114.2, 116.2, 108.6,
100.1, 56.1, 49.3, 36.2, 5.2 ppm. MS (FAB) m/z: M?
595.6. Anal. Calcd. for C28 H26FN5 O5 S2: C 56.46, H 4.40,
N 11.76. Found: C 56.44, H 4.42, N 11.74.
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
nitrophenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-yl]-
4-oxoquinoline-3-carboxylic acid (7d)
IR (KBr) cm-1: 3400.53 (O–H str.), 2910.00 (Ar.C–H str.),
2848 (Ali. C–H str.) 1750 (Carboxylic C=O str.), 1690 (Aryl
C=O str.), 1635 (4-Oxo quinolone C=O str.), 1348 (Ar.
–N=O str.), 748 (out of plane, Ar. C–H def.), 705 (Ali. C–S
str.) 1H NMR (DMSO-d6) d ppm: 15.09 (s, H, OH), 8.68 (m,
2H, Ar.), 8.51 (s, 1H, H2-quinoline), 8.11 (m, 2H, Ar.), 7.81
(d, 1H, H5-quinoline, J = 13.04 Hz), 7.59 (d, 1H, H8-
quinoline, J = 7.48 Hz), 4.53 (s, 2H, CH2), 3.74–3.80 (m,
4H, piperazine), 3.56–3.64 (m, 4H, CH2 piperazine and 1H,
CH, cyclopropyl), 1.58–1.62 (m, 4H, CH2, cyclopro-
pyl) ppm. 13C NMR (DMSO-d6) d ppm: 195.2, 177.7,
167.9, 165.2, 152.8, 149.4, 145.3, 142.9, 136.5, 130.5,
121.5, 117.8, 110.7, 101.4, 49.7, 50.3, 37.3, 5.6 ppm. MS
(FAB) m/z: M? 611.08. Anal. Calcd. for C27 H23FN6O6 S2:
C 53.11, H 3.80, N 13.76. Found: C 53.10, H 3.82, N 13.78.
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
fluorophenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-
yl]-4-oxoquinoline-3-carboxylic acid (7g)
IR (KBr) cm-1: 3446 (O–H str.), 2912 (Ar. C–H str.), 2840
(Ali. C–H str.) 1745 (Carboxylic C=O str.), 1688 (Ar. C=O
str.), 1642 (4-Oxo quinolone C=O str.), 740 (out of plane,
1
Ar. C–H def.), 610 (Ali. C–S str.) H NMR (DMSO-d6)
d ppm: 15.07 (s, 1H, OH), 8.55 (s, 1H, H2-quinoline), 8.12
(m, 2H, Ar.), 7.90 (d, 1H, H5-quinoline, J = 13.01 Hz),
7.54 (d, 1H, H8-quinoline, J = 7.40 Hz), 7.37 (m, 2H, Ar.),
4.44 (s, 2H, CH2), 3.68–3.72 (m, 4H, CH2, piperazine),
3.48–3.54 (m, 4H, CH2, piperazine and 1H, CH, cyclopro-
pyl), 1.22–1.42 (m, 4H, CH2, cyclopropyl) ppm. 13C NMR
(DMSO-d6) d ppm: 195.1, 177.7, 167.5, 166.6, 165.4, 148.4,
144.6, 141.5, 132.7, 130.4, 115.5, 109.5, 100.5, 56.3, 49.4,
36.3, 5.6 ppm. Anal. Calcd. for C27H23F2N5O4S2: C 56.56,
H 3.97, N 12.00. Found: C 56.54, H 3.99, N 12.02.
1-Cyclopropyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
tolylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-yl]-4-
oxoquinoline-3-carboxylic acid (7e)
IR (KBr) cm-1: 3440 (O–H str.), 2911 (Ar. C–H str.), 2840
(Ali. C–H str.), 1740 (Carboxylic C=O str.), 1665 (Aryl
C=O str.), 1626 (4-Oxo quinolone C=O str.), 744 (out of
plane, Ar. C–H def.), 660 (Ali. C–S str.) 1H NMR (DMSO-
d6) d ppm: 15.06 (s, 1H, OH), 8.56 (s, 1H, H2-quinoline),
8.10 (m, 2H, Ar.), 7.98 (d, 1H, H5-quinoline,
J = 13.08 Hz), 7.62 (d, 1H, H8-quinoline, J = 7.50 Hz),
7.02 (m, 2H, Ar.), 4.48 (s, 2H, CH2), 3.78–3.84 (m, 4H, CH2,
piperazine), 3.48–3.56 (m, 4H, CH2, piperazine and 1H, CH,
1-Ethyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-
phenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-yl]-4-
oxoquinoline-3-carboxylic acid (8a)
IR (KBr) cm-1: 3435 (O–H str.), 3122 (Ar. C–H str.), 2825
(Ali. C–H str.) 1742 (Carboxylic C=O str.), 1667 (Ar. C=O
123

828
Med Chem Res (2013) 22:818–831
1-Ethyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
nitrophenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-yl]-
4-oxoquinoline-3-carboxylic acid (8d)
str.), 1622 (4-Oxo quinolone C=O str.), 752 (out of plane,
1
Ar. C–H def.), 640 (Ali. C–S str.) H NMR (DMSO-d6)
d ppm: 14.70 (s, 1H, OH), 8.48 (s, 1H, H2-quinoline), 7.88
(d, 1H, H5-quinoline, J = 13.04 Hz), 7.56 (d, 1H, H8-
quinoline, J = 7.42 Hz), 7.22 (m, 5H, Ar.), 4.54 (q, 2H,
NCH2CH3, J = 6.94 Hz), 4.42 (s, 2H, CH2), 3.58 (m, 4H,
piperazine), 3.10–3.30 (m, 4H, piperazine), 1.42 (t, 3H,
NCH2CH3, J = 6.92 Hz) ppm. 13C NMR (DMSO-d6)
d ppm: 194.2, 177.7, 166.5, 164.2, 148.2, 144.5, 143.7,
140.5, 136.7, 133.2, 128.9, 118.4, 116.5, 109.4, 100.1,
49.8, 49.3, 38.5, 13.1 ppm. MS (FAB) m/z: M? 553. Anal.
Calcd. for C26H24FN5O4S2: C 56.41, H 4.37, N 12.63.
Found: C 56.44, H 4.38, N 12.65.
IR (KBr) cm-1: 3438 (O–H str.), 2932 (Ar. C–H str.), 2837
(Ali. C–H str.) 1742 (Carboxylic C=O str.), 1672 (Ar. C=O
str.), 1629 (4-Oxo quinolone C=O str.), 1354 (Ar. –N=O
str.), 760 (out of plane, Ar. C–H def.), 672 (Ali. C–S str.)
1H NMR (DMSO-d6) d ppm: 15.07 (s, 1H, OH), 8.78 (m,
2H, Ar.), 8.46 (s, 1H, H2-quinoline), 8.14 (m, 2H, Ar.),
7.82 (d, 1H, H5-quinoline, J = 13.09 Hz), 7.60 (d, 1H, H8-
quinoline, J = 7.44 Hz), 4.55 (s, 2H, CH2), 4.25 (q, 2H,
NCH2CH3, J = 6.97 Hz), 3.41 (m, 4H, piperazine),
3.14–3.24 (m, 4H, piperazine), 1.20 (t, 3H, NCH2CH3,
J = 6.90 Hz) ppm. 13C NMR (DMSO-d6) d ppm: 194.7,
178.6, 168.7, 165.2, 152.8, 148.1, 144.3, 143.7, 142.9,
140.1, 134.9, 130.2, 129.7, 121.0, 109.5, 100.7, 50.9, 50.1,
38.7, 13.5 ppm. MS (FAB) m/z: M? 598.12. Anal. Calcd.
for C26H23FN6O6S2: C 52.17, H 3.87, N 14.04. Found: C
52.20, H 3.89, N 14.06.
1-Ethyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
chlorophenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-
yl]-4-oxoquinoline-3-carboxylic acid (8b)
IR (KBr) cm-1: 3436 (O–H str.), 2916 (Ar. C–H str.), 2850
(Ali. C–H str.) 1738 (Carboxylic C=O str.), 1690 (Ar. C=O
str.), 1624 (4-Oxo quinolone C=O str.), 864 (Ar.C–Cl str.),
1
1-Ethyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
tolylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-yl]-4-
oxoquinoline-3-carboxylic acid (8e)
737 (out of plane, Ar. C–H def.), 690 (Ali. C–S str.) H
NMR (DMSO-d6) d ppm: 14.52 (br, s, 1H, OH), 8.46 (s,
1H, H2-quinoline), 8.30 (m, 2H, Ar.), 7.84 (d, 1H, H5-
quinoline, J = 13.00 Hz), 7.52 (d, 1H, H8-quinoline,
J = 7.40 Hz), 7.24 (m, 2H, Ar.), 4.64 (s, 2H, CH2), 4.52
(q, 2H, NCH2CH3, J = 6.94 Hz), 3.55 (m, 4H, piperazine),
3.15–3.24 (m, 4H, piperazine), 1.40 (t, 3H, NCH2CH3,
J = 6.92 Hz) ppm. 13C NMR (DMSO-d6) d ppm: 194.3,
178.3, 167.5, 164.5, 148.3, 144.7, 144.2, 140.5, 138.7,
136.5, 134.9, 130.2, 128.8, 116.7, 109.7, 100.5, 49.7, 49.8,
38.6, 13.3 ppm. MS (FAB) m/z: [M?1]? 588.42. Anal.
Calcd. for C26 H23ClFN5O4S2: C 53.10, H 3.94, N 11.91.
Found: C 53.12, H 3.96, N11.89.
IR (KBr) cm-1: 3434 (O–H str.), 3011 (Ar. C–H str.), 2908
(Ar. C–CH3 str.), 2842 (Ali. C–H str.) 1740 (Carboxylic
C=O str.), 1694 (Ar. C=O str.), 1638 (4-Oxo quinolone
C=O str.), 744 (out of plane, Ar. C–H def.), 647 (Ali. C–S
1
str.) H NMR (DMSO-d6) d ppm: 14.78 (br, s, 1H, OH),
8.57 (s, 1H, H2-quinoline), 8.24 (m, 2H, Ar.), 7.98 (d, 1H,
H5-quinoline, J = 13.07 Hz), 7.60 (d, 1H, H8-quinoline,
J = 7.44 Hz), 6.98 (m, 2H, Ar.), 4.50 (s, 2H, CH2), 4.22
(q, 2H, NCH2CH3, J = 6.98 Hz), 3.47 (m, 4H, piperazine),
3.12–3.22 (m, 4H, piperazine), 2.38 (s, 3H, Ar.CH3) 1.28
(t, 3H, NCH2CH3, J = 6.94 Hz) ppm. 13C NMR (DMSO-
d6) d ppm: 193.8, 177.1, 167.2, 164.6, 147.3, 144.1, 142.8,
142.2, 139.1, 133.8, 129.5, 129.0, 128.7, 108.7, 100.5,
49.5, 50.1, 38.3, 24.3, 13.1 ppm. Anal. Calcd. For
C27H26FN5O4S2 : C 57.13, H 4.62, N 12.34. Found: C
57.15, H 4.61, N 12.32.
1-Ethyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
bromophenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-
yl]-4-oxoquinoline-3-carboxylic acid (8c)
IR (KBr) cm-1: 3437 (O–H str.), 2975 (Ar. C–H str.), 2828
(Ali. C–H str.) 1747 (Carboxylic C=O str.), 1660 (Ar. C=O
str.), 1628 (4-Oxo quinolone C=O str.), 729 (out of plane, Ar.
C–H def.), 670 (Ali. C–S str.) 1H NMR (DMSO-d6) d ppm:
15.09 (s, 1H, OH), 8.50 (s, 1H, H2-quinoline), 8.12 (m, 2H,
Ar.), 7.81 (d, 1H, H5-quinoline, J = 13.08 Hz), 7.57 (d, 1H,
H8-quinoline, J = 7.41 Hz), 7.20 (m, 2H, Ar.), 4.50 (s, 2H,
CH2), 4.22 (q, 2H, NCH2CH3, J = 6.90 Hz), 3.45 (m, 4H,
piperazine), 3.10–3.20 (m, 4H, piperazine), 1.38 (t, 3H,
NCH2CH3, J = 6.92 Hz) ppm. 13C NMR (DMSO-d6)
d ppm: 194.2, 178.4, 167.7, 164.5, 147.8, 143.8, 143.2, 139.9,
135.8, 134.7, 131.6, 131.0, 129.8, 127.4, 109.2, 100.2, 50.4,
49.6, 38.2, 13.1 ppm. Anal. Calcd. for C27H23BrFN5O4S2 : C
50.31, H 3.60, N 10.87. Found: C 50.33, H 3.62, N 10.84.
1-Ethyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
methoxyphenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-
yl]-4-oxoquinoline-3-carboxylic acid (8f)
IR (KBr) cm-1: 3433 (O–H str.), 2934 (Ar. C–H str.), 2830
(Ali. C–H str.) 1735 (Carboxylic C=O str.), 1690 (Ar. C=O
str.), 1645 (4-Oxo quinolone C=O str.), 1031 (Ar.C–O–C
str.), 740 (out of plane, Ar. C–H def.), 674 (Ali. C–S str.)
1H NMR (DMSO-d6) d ppm: 15.05 (s, 1H, OH), 8.58 (s,
1H, H2-quinoline), 8.18 (m, 2H, Ar.), 7.94 (d, 1H, H5-
quinoline, J = 12.98 Hz), 7.54 (d, 1H, H8-quinoline,
123

Med Chem Res (2013) 22:818–831
829
J = 7.38 Hz), 6.82 (m, 2H, Ar.), 4.82 (s, 2H, CH2), 4.20
(q, 2H, NCH2CH3, J = 6.94 Hz), 3.82 (s, 3H, Ar. –OCH3,
methoxyl), 3.45 (m, 4H, piperazine), 3.10–3.20 (m, 4H,
piperazine), 1.30 (t, 3H, NCH2CH3, J = 6.96 Hz) ppm.
13C NMR (DMSO-d6) d ppm: 194.2, 177.5, 168.5, 165.1,
164.9, 148.2, 144.5, 142.5, 139.5, 130.2, 129.8, 129.1,
114.2, 109.3, 100.7, 55.9, 50.2, 49.7, 38.5, 13.2 ppm. MS
(FAB) m/z: M? 583.49. Anal. Calcd. for C27H26FN5O5S2:
C 55.56, H 4.49, N 12.00. Found: C 55.54, H 4.47, N 12.12.
supplemented with DMSO at the same dilutions as used in
the experiments (Goto et al., 1982).
In vitro antitubercular activity
In vitro antitubercular screening was performed using
M. tuberculosis virulent H37Rv strain. The MIC of each drug
was determined by broth dilution assay. A frozen culture of
Middlebrook 7H9 broth supplemented with 10 % ADC
(albumin dextrose catalase) and 0.2 % glycerol was thawed
and diluted in broth to 2 9 105 cfu/mL and used as inocu-
lum. In the assay, U-tubes were used to accommodate
compounds in 0.1–25 lg/mL dilutions i.e. (25, 20, 15, 10,
05, 2.5, 1.5, 1.0, 0.5 and 0.1). Each test compound was dis-
solved in DMSO and then diluted in broth at twice the desired
concentration. The final concentration of DMSO in the assay
medium was 1.3 %. Each U-tube was then inoculated with
0.05 mL of standardized culture and then incubated at 37 °C
for 21 days. The growth in U-tubes was compared with
visibility against positive control (without drug), negative
control (without drug and inoculum) and standard isoniazid
and rifampicin (Suling et al., 2000; Yajko et al., 1995).
1-Ethyl-6-fluoro-1,4-dihydro-7-[4-{5-(2-oxo-2-p-
fluorophenylethylthio)-1,3,4-thiadiazol-2-yl}piperazin-1-
yl]-4-oxoquinoline-3-carboxylic acid (8g)
IR (KBr) cm-1: 3440 (O–H str.), 2889 (Ar. C–H str.), 2847
(Ali. C–H str.) 1742 (Carboxylic C=O str.), 1688 (Ar. C=O
str.), 1652 (4-Oxo quinolone C=O str.), 762 (out of plane,
1
Ar. C–H def.), 665 (Ali. C–S str.) H NMR (DMSO-d6)
d ppm: 14.49 (s, 1H, OH), 8.56 (s, 1H, H2-quinoline), 8.22
(m, 2H, Ar.), 7.91 (d, 1H, H5-quinoline, J = 13.04 Hz),
7.51 (d, 1H, H8-quinoline, J = 7.35 Hz), 7.35 (m, 2H,
Ar.), 4.74 (s, 2H, CH2), 4.24 (q, 2H, NCH2CH3,
J = 6.92 Hz), 3.42 (m, 4H, piperazine), 3.14–3.32 (m, 4H,
piperazine), 1.18 (t, 3H, NCH2CH3, J = 6.98 Hz) ppm.
13C NMR (DMSO-d6) d ppm: 194.7, 177.2, 168.9, 167.3,
165.7, 148.2, 144.7, 143.2, 139.7, 132.1, 130.4, 115.4,
109.3, 100.2, 50.5, 49.6, 38.2, 13.0 ppm. Anal. Calcd. for
C26H23F2N5O4S2: C 54.63, H 4.06, N 12.25. Found: C
54.65, H 4.10, N 12.26.
In vitro cytotoxic activity
MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra
sodium bromide)] is a pale yellow substrate that is cleaved
by living cells to yield a dark blue formazan product. This
process requires active mitochondria, and even freshly
dead cells do not cleave significant amount of MTT. Thus,
the amount of MTT cleaved is directly proportional to the
number of viable cells present, which is quantified by
colorimetric methods. This assay was performed at Desh-
pande Laboratories, Bhopal, (MP) India using the standard
operating procedures. The test compounds were dissolved
in DMSO and serially diluted with complete medium to get
the range of test concentration. DMSO concentration was
kept \0.1 % in all the test compounds. Cell lines main-
tained in appropriate conditions were seeded in 96-well
plates and treated with different concentrations of the test
samples and incubated at 37 °C, 5 % CO2 for 96 h. MTT
reagent was added to the wells and incubated for 4 h; the
dark blue formazan product formed by the cells was dis-
solved in DMSO in a safety cabinet and read at 550 nm.
Percentage inhibitions were calculated and plotted with the
concentrations and used to calculate the IC50 values in
micromolar (Sriram et al., 2006).
Biological assay
In vitro antibacterial activity
Broth microdilution method was employed for preliminary
in vitro antibacterial activity. Activity was investigated
against Gram-positive organisms, S. aureus and Coryne-
bacterium, (ATCC-25365 and ATCC-29355 strain); and
Gram-negative organisms, Pseudomonas aeruginosa and
Escherichia coli, (ATCC 25619 and MTCC-1089 strain).
The activity of the test compounds was compared with
ciprofloxacin and norfloxacin as standards.
Twofold serial dilutions of the test compounds and
reference drugs were prepared in Mueller–Hinton agar
medium. Test compounds and standard drugs (6.4 mg)
were dissolved in dimethylsulphoxide (DMSO, 1 mL) and
diluted with distilled water (9 mL). Progressive double
dilutions with melted Mueller–Hinton agar were performed
to obtain the required concentrations of 64, 32, 16, 8, 4, 2, 1
and 0.5 lg/mL. Petri dishes were inoculated with
1–5 9 104 colony-forming units (cfu/mL) and incubated at
37 °C for 18 h. A control was performed with the medium
Docking study
Degree of affection between the synthesized novel
molecules and target in terms of structural and chemical
complementation was explored by advanced scientific
123

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Cowper RM and Davidson LH (1943) Phenacyi bromide. In: Blatt AH
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In the present study, X-ray crystal structure of the
˚
twinned 3.35 A structure of S. aureus gyrase complexed
with ciprofloxacin and DNA gyrase (PDB entry: 2XCT) for
antibacterial activity and structure of M. tuberculosis
complexed with isoniazid and DNA gyrase (PDB entry:
2BM7) was taken from PDB (www.rscb.org). Before
docking, the proteins were prepared by means of the pro-
¨
tein preparation wizard (Maestro-v8.0 Schrodinger, 2007)
by removing the water molecule and cofactors from the
proteins, optimizing hydrogen bonding and deleting the
ligand present in crystal structure. Solvent molecules were
deleted, and bond order for crystal ligands and proteins
˚
were adjusted. The structures were minimized up to 0.30 A
RMSD. The ligands were built using Maestro v-8.0 (Lig-
¨
Prep-v2.1 Schrodinger, 2007) build panel and prepared by
LigPrep-v2.1 (Metropolis et al., 1953) by means of the
OPLS-2005 force field using extra precision (XP) mode of
Glide v-4.5. All the molecules were docked into active site
of target molecule, and final scoring was carried out in
terms of G-score multi-ligand scoring function.
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activities of some 7-[4-(5-amino-[1,3,4] thiadiazole-2-sulfonyl)-
piperazin-1-yl] fluoroquinolone derivatives. Eur J Med Chem
41:918–924
Acknowledgments The authors express heartfelt thanks to Dr. S.J.
Surana, Principal, R.C. Patel Institute of Pharmaceutical Education &
Research Shirpur, North Maharashtra University, Jalgaon (Maha-
rashtra), India for providing the necessary facilities to carry out this
research. The authors also thank Dr. S.B. Bari and Mr. Shailesh Jain
for providing the necessary guidance for the docking studies. The
authors offer their sincere gratitude to Dr. Amit Deshpande, Chair-
man, Deshpande Laboratories, Bhopal (MP), India for his help in
carrying out cytotoxicity studies, The authors are also thankful to
Mcleods pharmaceutical Ltd. Mumbai and Ipca laboratories Ratlam,
India for providing free samples as pure drug of fluoroquinolones.The
authors are extremely grateful to the departments of Physical, Inor-
ganic and Organic Chemistry and NMR research centre, IISc Ban-
galore, India for facilitating elemental and spectral analyses.
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structure based screening of combinatorial libraries. J Mol
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