Investigation of Ugi-4CC derived 1H-tetrazol-5-yl-(aryl) methyl piperazinyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid: Synthesis, Biology and 3D-QSAR analysis

Article abstract of DOI:10.1016/j.ejmech.2014.03.069

Novel series of 7-piperazinylquinolones with tetrazole derivatives were synthesized and evaluated for their antibacterial activity against various strains of Staphylococcus aureus. All the synthesized compounds showed significant in vitro antibacterial ac

Full text of DOI:10.1016/j.ejmech.2014.03.069

Accepted Manuscript
Investigation of Ugi-4CC derived 1H-tetrazol- 5-yl-(aryl) methyl piperazinyl-6-fluoro-4-
oxo-1, 4-dihydroquinoline-3-carboxylic acid: Synthesis, Biology and 3D-QSAR
Analysis
Kuldeep Chauhan, Pratiksha Singh, Vikash Kumar, Praveen K. Shukla, Mohammad
Imran Siddiqi, Prem.M.S. Chauhan
PII:
S0223-5234(14)00283-9
10.1016/j.ejmech.2014.03.069
EJMECH 6853
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 10 September 2013
Revised Date: 20 March 2014
Accepted Date: 22 March 2014
Please cite this article as: K. Chauhan, P. Singh, V. Kumar, P.K. Shukla, M.I. Siddiqi, P.M.S. Chauhan,
Investigation of Ugi-4CC derived 1H-tetrazol- 5-yl-(aryl) methyl piperazinyl-6-fluoro-4-oxo-1, 4-
dihydroquinoline-3-carboxylic acid: Synthesis, Biology and 3D-QSAR Analysis, European Journal of
Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.03.069.
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Graphical abstract
Investigation of Ugi-4CC derived 1H-tetrazol- 5-yl-(aryl) methyl
piperazinyl-6-fluoro-4-oxo-1,
4-dihydroquinoline-3-carboxylic
acid:
Synthesis, Biology and 3D-QSAR Analysis
a
b
c
b
Kuldeep Chauhan, Pratiksha Singh, Vikash Kumar, Praveen K. Shukla, Mohammad Imran
c
a
Siddiqi and Prem. M. S. Chauhan *
a
Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031, U.P., India
b
Microbiology Division, CSIR-Central Drug Research Institute, Lucknow 226031, U.P., India
c
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, U.P.,
India
A new class of 7-piperazinyl quinolone-tetrazole derivatives were synthesized via using Ugi-
4
CC reaction. Most of the synthesized compounds exhibited potent antibacterial activity
against various strains of Staphylococcus aureus.

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Highlights:
ꢀ
ꢀ
ꢀ
ꢀ
Synthesis of 7-piperazinylquinolones-tetrazole derivatives via using Ugi-4CC.
Some of synthesized derivatives showed excellent antibacterial activity.
These compounds displayed no toxicity against mammalian cell line L-929.
It is a successful approach to overcome the problem of resistance.

ACCEPTED MANUSCRIPT
Investigation of Ugi-4CC derived 1H-tetrazol- 5-yl-(aryl) methyl
piperazinyl-6-fluoro-4-oxo-1,
4-dihydroquinoline-3-carboxylic
acid:
Synthesis, Biology and 3D-QSAR Analysis
a
b
c
b
Kuldeep Chauhan, Pratiksha Singh, Vikash Kumar, Praveen K. Shukla, Mohammad Imran
c
a
Siddiqi and Prem. M. S. Chauhan *
a
Medicinal & Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow 226031, U.P., India
b
Microbiology Division, CSIR-Central Drug Research Institute, Lucknow 226031, U.P., India
c
Molecular and Structural Biology Division, CSIR-Central Drug Research Institute, Lucknow 226031, U.P.,
India
Abstract: Novel series of 7-piperazinylquinolones with tetrazole derivatives were
synthesized and evaluated for their antibacterial activity against various strains of
Staphylococcus aureus. All the synthesized compounds showed significant in vitro
antibacterial activity against Gram-positive bacteria whereas some compounds displayed
moderate activity against Gram-negative bacteria. Among all the synthesized compounds,
compounds (6a-c, 6e-g, 6i-k, 6m, 6’f and 6’m) were found to be more effective with MIC
ranging from (0.78-3.12 µg/mL) against Staphylococcus aureus (ATCC-29213) than the
control; ciprofloxacin (MIC = 25 µg/mL). Moreover, these analogues displayed no toxicity
up to MIC = 0.39 µg/mL against mammalian cell line L-929. Furthermore, to correlate the
biological activities of synthesized compounds with their 3D conformation, we attempted 3D-
QSAR study.
Keywords: Antibacterial activity; Cytotoxicity; 3D-QSAR Analysis; Fluoroquinolones;
Tetrazole.
*
Corresponding author mailing address: Medicinal and Process Chemistry Division, CSIR-
Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow-
26031, U.P, India, Phone: +91 522 2771940, 2771942; Fax: +91 522 2771941. Email:
prem_chauhan_2000@yahoo.com; premsc58@hotmail.com
2
1

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1
. Introduction
Despite increasing focus and research in medicinal chemistry, Antibiotic resistance to
presently available antibacterial agents is a growing health problem in the community and
hospitals [1]. On a worldwide basis, nosocomial multidrug-resistant Gram-positive
staphylococcal pathogen isolates and found to display resistance to frontline antibacterials
such as methicillin, vancomycin and gentamycin, and the urgent need for safe and affordable
antibacterial agents capable to overcome the resistance is required [2-4]. The numbers of
solutions to remove the problem of bacterial resistance are possible and the most prevalent
approach is to continue modifying the existing classes of antibacterial agents to provide new
analogues [5, 6].
An initial study reported that quinolones are the significant class of antibacterial,
antitubercular [7] and antimalarial agents [8], which are extensively approved for the
treatment of human infections [9]. The beginning of nalidixic acid in 1962 has shown the
new avenue for the patients with the bacterial infections and also the dramatic impact in
changing morbidity and mortality rate compared with the chemotherapy during 1940s [8].
But in the period of 1980s, the first fluoroquinolone norfloxacin [10] and other quinolone
derivatives like ciprofloxacin [11], sparfloxacin [12] and trovafloxacin [13] (Figure 1) have
launched which changed the landscape of antibacterial chemotherapy and displayed activity
against both Gram-negative and Gram-positive bacterial pathogens. The structure activity
relationship (SAR) studies showed that, the fluorine atom and the 1-alkyl, 1,4-dihydro-4-
oxoquinoline-3-carboxylic acid skeleton of fluoroquinolones is responsible for potency
represented in binding with type-II topoisomerase enzymes, DNA gyrase and topoisomerase
IV [14, 15]. Moreover, it is believed that the 6-fluoro and 7-piperazinyl groups are
responsible for the broad spectrum and antipseudomonal activities of fluoroquinolones.
Furthermore, it is clear that chemical modifications at C-7 is suitable for controlling of the
2

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pharmacokinetic properties and hence changes in the cell permeability of these antibiotics
[
16]. Recently Emami et. al synthesized mannich bases of 7-piperazinylquinolones with kojic
acid and chlorokojic acid as potent antibacterials [17].
In this regards, tetrazole is a class of heterocycles with wide range of applications that
is receiving considerable attention [18]. Tetrazole and their derivatives exhibited various
biological applications such as anti-allergic, antibiotic, antihypertensive, antibacterial and
anticonvulsant agents [19-22]. Previously we have synthesized 8-fluoro Norfloxacin
derivatives and their hybrid with triazines and pyrimidine which showed potent activity
against methicillin and vancomycin resistant strains of S. aureus [23]. In our effort and
continuation of our ongoing anti-infective research programme [24-28], considering our
previous work. We envisaged the incorporation of tetrazole scaffolds with 7-
piperazinylquinolones.
In order to identify molecular properties that have the largest impact on antibacterial
activity of the synthesized compounds, we performed the QSAR (Quantitative structure
activity relationship) analysis [29]. QSAR have been applied for decades in the improvement
of relationships between physicochemical properties of chemical substances and their
biological activities to achieve a reliable statistical model for prediction of the activities of
new chemical entities [30]. In this contest 3D-QSAR has emerged as a natural extension to
the classical Hansch and Free-Wilson approaches, which develops the three-dimensional
properties of the ligands to predict their biological activities using robust chemometric
techniques such as PLS, G/PLS, ANN etc. Several success stories of QSAR have attracted
the medicinal chemists to investigate the relationship of structural properties with biological
activity [31]
3

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2
. Chemistry Overview
The realization of a facile access towards 1H-tetrazol-5-yl-(aryl) methyl piperazinyl-
6
-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid from readily available substrates, a
small library of 32 compounds were synthesized for the structure activity relationship (SAR).
The synthesis of norfloxacin analogues possessing Ugi derived substructure (6a-p and 6’a-p)
is outlined in Scheme 1. The intermediate ethyl 6, 7-difluoro-4-oxo-1, 4-dihydroquinoline-3-
carboxylate 3 was achieved via cyclization of diethyl 2-(3, 4-difluorophenylamino)
methylene) malonate 2 in diphenyl ether under refluxing condition (~250 °C), which was
obtained from 3, 4-difluoro aniline 1. The treatment of 3 with ethyliodide and K CO in dry
2
3
DMF at 90 °C produced ethyl 1-ethyl-6,7-difluoro-4-oxo-1,4-dihydroquinoline-3-carboxylate
in good yield and with 1-(bromomethyl)-4-(trifluoromethyl)benzene NaH mediated N-
4
alkylation in DMF at 90 °C gave ethyl 6,7-difluoro-4-oxo-1-(4-(trifluoromethyl)benzyl)-1,4-
dihydroquinoline-3-carboxylate 4’ in quantitative yield. Further, the nucleophilic substitution
th
at 7 position of 4 and 4’ with piperazino in the presence of K CO using acetonitrile as
2
3
solvent under refluxing condition offered their respective derivatives 5 and 5’ in 84 and 80%
yield respectively. In this context, compound 5 and 5’ was allowed to react with different
commercially available aldehydes, isocyanides and azidotrimethylsilane (TMSN ) in
3
anhydrous methanol to form the desired quinolone-tetrazole ester derivatives [32]. Esters
were hydrolyzed to their respective carboxylic acids 6a-p and 6’a-p by treating with aqueous
NaOH in methanol (1:1) quantitatively.
3
. In vitro antibacterial assay
All the synthesized compounds (6a-p and 6’a-p) were screened for antibacterial
activity against Gram-positive bacteria Staphylococcus aureus (ATCC-25923), S. aureus
(ATCC-700699), S. aureus (ATCC-29213) and S. aureus (ATCC-33592) and Gram-negative
4

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bacteria Escherichia coli and Klebsiella pneumoniae using standard techniques and
compared to the standard drugs ciprofloxacin and gentamycin. The bacterial strains were
grown on nutrient agar at 37 °C. After 24 h of incubation, bacterial cells were suspended in
normal saline containing Tween 20 at 0.05% at a concentration of approximately 1.0-2.0 ×
7
1
0 cells/mL by matching with 0.5 McFarland standards. The activity of compounds was
determined as per NCCLS protocol using Mueller Hinton broth (Becton Dickinson, USA) in
9
6-well tissue culture plates [33].
4
. Structure activity relationship
The results of compounds 6a-p and 6’a-p were presented in Table 1. At a glance,
compounds having N-ethyl substitution (6a-p) were found to be more potent in assessment
with the compounds having 4-(trifluoromethyl) benzyl group as N-substituent (6’a-p) of
quinolone ring. Compounds (6a-h) having t-butyl substituent at R , Compound 6a which
1
contained the benzylidene at R position displayed significant MIC = 0.39 µg/mL against S.
2
aureus (ATCC-25923), and MIC = 25, 0.78 and 0.39 µg/mL against M-R S. aureus (ATCC-
7
00699), S. aureus (ATCC-29213), and G-R S. aureus (ATCC-33592), respectively which is
more potent than that of ciprofloxacin and gentamycin against S. aureus (ATCC-25923) and
S. aureus (ATCC-29213). Compound 6b (R = 4-pyridine) exhibited potent MIC values
2
(
MIC = 1.56, >50, 1.56 and 1.56 µg/mL) against S. aureus (ATCC-25923), M-R S. aureus
ATCC-700699), S. aureus (ATCC-29213), and G-R S. aureus (ATCC-33592). While
(
compound 6c having R = 3, 4, 5-trimethoxy benzylidene showed MIC values (MIC = 1.56,
2
5
0, 3.12 and 3.12 µg/mL) against S. aureus (ATCC-25923), M-R S. aureus (ATCC-700699),
S. aureus (ATCC-29213), and G-R S. aureus (ATCC-33592). Surprisingly, in case of 4-
isopropylbenzylidene compound 6d activity diminished. Furthermore, compound 6e with 2-
furfuralehyde substituent found to be more potent than gentamycin and display equal potency
with ciprofloxacin. Compound 6f, having 4-fluorobenzyledene also showed good activity
5

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against S. aureus (ATCC-25923), M-R S. aureus (ATCC-700699), S. aureus (ATCC-
2
9213), and G-R S. aureus (ATCC-33592) than controls; ciprofloxacin and gentamycin.
Compounds 6g exhibited potent activity against all the strains of S. aureus while Compounds
h showed remarkable activity. Similarly, in compounds (6i-p) having cyclohexyl substituent
at R compound 6i R = benzylidene showed potent activity MIC = 1.56 µg/mL against S.
6
1
,
2
aureus (ATCC-25923) and MIC = 12.5, 3.12 and 1.56 µg/mL against M-R S. aureus ATCC
00699, S. aureus (ATCC-29213), and G-R S. aureus (ATCC-33592) respectively which is
7
more potent than ciprofloxacin and gentamycin. Compound 6j having R = 4-pyridine
2
showed MIC = 1.56 µg/mL against S. aureus (ATCC-25923) and MIC = >50, 3.12 and 3.12
µg/mL M-R S. aureus (ATCC-700699), S. aureus (ATCC-29213), and G-R S. aureus
(
ATCC-33592) respectively. While compound 6k = 2, 3, 4 trimethoxy benzylidene activity
enhance against S. aureus (ATCC-29213), and similar MIC values against G-R S. aureus
ATCC-33592). Surprisingly, the substitution with R = 4-isopropylbenzylidene compound 6l
(
2
activity diminished in all strains of S. aureus. Furthermore, compound 6m R = 2-
2
furfuraldehyde found to be more potent than gentamycin with MIC values 0.78, 25, 1.56 and
0
.78 µg/mL against S. aureus (ATCC-25923), M-R S. aureus (ATCC-700699), S. aureus
(ATCC-29213), and G-R S. aureus (ATCC-33592). Compound 6n, having 4-
fluorobenzyledene at R displayed good activity against S. aureus (ATCC-25923) while
2
activity reduced in resistant strains of S. aureus. In case of compounds 6o and 6p R =
2
naphthyledene and 4-methoxynaphthyledene, showed good activity against S. aureus
(ATCC-25923), M-R S. aureus (ATCC-700699), S. aureus (ATCC-29213), and G-R S.
aureus (ATCC-33592). Compounds (6’a-p) were having 4-(trifluoromethyl) benzyl group as
N-substituent and in this sequence compounds (6’a-h) were having t-butyl substituent at R1.
Compounds 6’a, 6’b and 6’c which contained the R = benzylidene, 4-pyridine and 3, 4, 5-
2
trimethoxy benzylidene respectively exhibited less potent activity against S. aureus (ATCC-
6

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2
5923), M-R S. aureus (ATCC-700699), S. aureus (ATCC-29213), and G-R S. aureus
(
ATCC-33592). In case of compound 6’d R = 4-isopropylbenzylidene showed potent
2
activity with MIC values 3.12, 3.12, 6.25 and 3.12 µg/mL against S. aureus (ATCC-25923),
M-R S. aureus (ATCC-700699), S. aureus (ATCC-29213), and G-R S. aureus (ATCC-
3
3592). In case of compounds 6’e, 6’g and 6’h having R = 2-furfuraldehyde, naphthyledene
2
and 4-methoxynaphthyledene, showed nil activity against all the strains of S. aureus.
Interestingly compound 6’f having R = 4-fluorobenzyledene, displayed potent MIC values
2
(
(
(
MIC = 6.25, 25, 3.12 and 6.25 µg/mL) against S. aureus (ATCC-25923), M-R S. aureus
ATCC-700699), S. aureus (ATCC-29213), and G-R S. aureus (ATCC-33592). Compounds
6’i-p) were having cyclohexyl substituent at R position showed no potent activity against all
1
the strains of S. aureus except compound 6’m, which showed good activity (MIC = 0.78, 50,
1
.56 and 0.78 µg/mL) against (MIC = 6.25, 25, 3.12 and 6.25 µg/mL) .
Some compounds of this series showed moderate to good activity against Gram
negative bacteria Escherichia coli and Klebsiella pneumoniae. Compounds 6e, 6j and 6’m
exhibited potent activity against Escherichia coli and compounds 6j, 6m and 6’m showed
significant activity against Klebsiella pneumoniae which are presented in Table 2.
5
. Cytotoxicity evaluation
The cytotoxicity effect of the selected compounds of 7-piperazinylquinolones-
tetrazole derivatives were evaluated against mammalian cells, mouse fibroblast cell line L-
9
29. Compounds 6e did not exhibit any toxicity up to MIC 0.78 ꢀg/mL also showed in Figure
. However, a decline in cell survival of compounds 6e, 6m and 6k was observed at 50
2
ꢀ
g/mL, 25 ꢀg/mL and 12.5 ꢀg/mL concentration for the compounds as shown in the Figure 3.
7

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6
. Development of 3D-QSAR models
In order to correlate the biological activities of synthesized compounds with their 3D
conformation, we attempted 3D-QSAR study Figure 4. CoMFA and CoMSIA studies on
thirty
compounds
1H-tetrazol-5-yl-(aryl)methyl
piperazinyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid are resulted in acceptable statistical model. Initially total
thirty two molecules were selected for 3D-QSAR study, however during model development
we found compound 6e and 6’e as outliers, and they were not included in final 3D-QSAR
2
2
models. Partial least square (PLS) analysis in case of CoMFA showed cross validated r (r )
cv
2
2
of 0.501 at 3 component. Non cross validated r (r ncv) and standard error of estimate (SEE)
showed value of 0.831 and 0.315 respectively. Field contribution depicted that steric
contribution and electrostatic contribution explains 76.6 % and 23.4 % of variance in activity.
2
2
We also obtained predictive r (r pred) greater than 0.5, which validates CoMFA model. For
2
predictive r , we used 7 compounds belonging to test set (Figure 5 and Figure 6). Similarly
2
2
PLS analysis in case of CoMSIA showed r of 0.542, r
of 0.821 and SEE of 0.324 at 3
ncv
cv
2
components. Field contribution details are given in Table 3. Predictive r value (0.710) was
better than CoMFA, indicating that the model was very good in predicting activities of test
set compounds.
Contour maps obtained after 3D-QSAR analysis were mapped on compounds under
study. In CoMFA green and yellow contours represents sterically favoured and disfavoured
region respectively (Figure 7). Figure 8 and Figure 9) Electrostatic favourable regions are
represented by red contour and disfavoured region by blue contour. Large green contour near
piperazinyl attached carbon showed that bulky group is favourable in compound 6’c (MIC =
6
6.37 µM) and 6’g (MIC = 35.05 µM) when compare to compound 6’a (MIC = 75.38 µM).
1
Similarly at position-N presence of green contour indicates that bulkier is favourable near
this region, that’s why 7’d (MIC = 70.89 µM) is more active than 7d (MIC = 86.91 µM).
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During analysis of electrostatic contour map we found blue contour near para position of
piperazinyl attached benzene ring ,which suggests greater activity of 6c (MIC = 2.50 µM)
1
than 6f (MIC = 2.83 µM). At N -position red contour map display that electronegative group
is favourable near this region, which explain why 7’d (MIC = 70.89 µM) is better active than
7
d (MIC = 86.91 µM). Similarly we also analyzed CoMSIA contour maps for steric,
electrostatic, hydrophobic, H-bond acceptor and H-bond donor. Steric and electrostatic
contour maps were almost similar to CoMFA. Favourable hydrophobic contour maps
1
(
magenta) explained that at N -position substitution by more hydrophobic group will increase
the activity of compounds, for example compound 6’d (MIC = 70.89 µM) is more active than
compound 6d (MIC = 86.9 µM). In case of H-bond acceptor map, we see a large green
contour near para position of piperazinyl attached benzene ring , which indicates that an
acceptor group is favourable here and explains the higher activity of compound 6’c (MIC =
6
6.37 µM) than compound 6’a (MIC = 75.38 µM).
7
. Conclusion
In conclusion, the concept of developing new effective antibacterials by the introduction of
new substituents on quinolone pharmacophore is a successful approach to conquer the
problem of resistance. In this observation, we have chosen to explore norfloxacin moiety by
incorporated tetrazole scaffold at the N-4 position of the C-7 piperazin-1-yl group of newly
developed norfloxacin entity. Among all, compounds (6a-c, 6e-g, 6i-k, 6m, 6’f and 6’m)
were found to be more effective with MIC ranging from (0.78-3.12) µg/mL against
Staphylococcus aureus (ATCC-29213) than the control; ciprofloxacin (MIC = 25 µg/mL).
Moreover, these analogues displayed no toxicity up to MIC = 0.39 µg/mL against
mammalian cell line L-929. Furthermore thirty two compounds were scrutinized by CoMFA
2
and CoMSIA techniques of 3D-QSAR. Good predictive rpred values for CoMFA and
9

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CoMSIA models reflected the robustness of the predictive ability. Through CoMFA and
CoMSIA studies, we were able to explain the effect of 3D conformation on biological
activities of compounds. These promising and safe results of 7-piperazinylquinolones-
tetrazole derivatives against various strains of S. aureus make them possible drug candidate
for the treatment of bacterial infection.
8
. Experimental Section
8
.1 General information
All reagents and solvents were commercially available and were used without further
1
13
purification. H NMR and C NMR spectra were recorded on Bruker Supercon Magnet
Avance DRX-300 or DPX-200 FT spectrometers using TMS as an internal reference and the
samples were dissolved in suitable deuterated solvents (Chemical shifts (δ) are given in ppm
relative to TMS and coupling constants (J) in Hz). IR spectra were recorded on a FTIR
spectrophotometer Shimadzu 8201 PC and are reported in terms of frequency of absorption
-1
(
cm ). Electro Spray Ionisation Mass spectra (ESI-MS) were recorded by micromass quattro
II instrument. HR-DART MS were recorded on JEOL, JMS T100LC Accu TOF. Column
chromatography purifications were performed in flash using 60-120 or 100-200 Mesh silica
gel. Thin-layer chromatography (TLC) was carried out with silica gel plates (silica gel 60
F254), that were visualized by exposure to ultraviolet light. Purity of all tested compounds
was ascertained on the basis of their elemental analysis and was carried out on Carlo-Erba-
1
108 instrument. The melting points were recorded on an electrically heated melting point
apparatus and are uncorrected.
.2 Representative Procedure for the Synthesis of 7-(4-((1-tert-butyl-1H-tetrazol-5-yl)
phenyl) methyl) piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic
acid (6a)
8
(
10

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A solution of compound 5 (300 mg, 0.864 mmol), benzaldehyde (0.08 mL, 0.864 mmol) and
tert-butyl isocyanide (0.06 mL, 0.864 mmol) were stirred in anhydrous methanol (5 mL) at
room temperature for 10 min. Subsequently, trimethylsilyl azide (0.13 mL, 1.03 mmol) was
added and the resulting mixture was further stirred for 8~12 h. On completion of the reaction
(checked by TLC analysis), the methanol was removed in vacuo and further these reaction
mixture was reflux in NaOH solution in 50% methanol/water after completion the reaction
the methanol was removed in vacuo and the crude product was purified by chromatography
on silica gel (eluting with 2% methanol in chloroform) to afford the target product
6a as
White solid, Yield = 82%, mp = 228-230 °C. IR (KBr): 3439, 2989, 1703, 1629, 1218, 1141
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.08 (s, 1H), 8.65 (s, 1H), 8.05 (d, J = 13.0 Hz, 1H),
3
H
7
2
1
6
5
.38-7.36 (m, 5H), 6.81 (d, J = 5.7 Hz, 1H), 5.33 (s, 1H), 4.30 (bs, 2H), 3.32 (s, 4H), 3.00-
13
.96 (m, 2H), 2.69-2.65 (m, 2H), 1.69 (s, 9H), 1.55 (s, 3H). C NMR (75 MHz, CDCl ): δ
3
C
76.9, 167.2, 154.0, 147.1, 137.0, 134.7, 129.7, 128.8, 120.6, 112.8, 112.5, 108.3, 103.9,
+
5.1, 61.5, 50.1, 41.0, 30.3, 14.4. ESI-MS C H FN O (m/z): 534.0 (M + H) ; HRMS: calc.:
2
8
32
7
3
+
+
34.2623 (MH ); Found: 534.2653 (MH ).
The following compounds 6b-p and 6’a-p were prepared using a procedure similar to that
described for compound 6a.
8
.3 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (pyridin-4-yl) methyl) piperazin-1-yl)-1-ethyl-6-
fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6b)
White solid, Yield = 85%, mp = 220-222 °C. IR (KBr): 3426, 2996, 1721, 1630, 1262, 1133
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.02 (s, 1H), 8.66 (d, J = 4.8 Hz, 3H), 8.04 (d, J =
3
H
1
2.9 Hz, 1H), 7.41 (d, J = 5.7 Hz, 2H), 6.80 (d, J = 6.7 Hz, 1H), 5.40 (s, 1H), 4.30 (bs, 2H),
1
3
3
.31 (s, 4H), 3.03-2.99 (m, 2H), 2.69-2.65 (m, 2H), 1.74 (s, 9H), 1.57 (t, J = 6.2 Hz, 3H). C
NMR (75 MHz, CDCl + DMSO-d ): δ 176.5, 166.6, 159.6, 158.1, 153.8, 151.6, 151.4,
3
6
C
1
48.6, 145.5, 145.4, 142.5, 137.4, 128.3, 121.1, 120.0, 117.3, 113.5, 111.7, 111.3, 109.6,
11

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1
07.6, 106.2, 62.8, 61.3, 49.9, 49.4, 49.1, 29.7, 14.3. ESI-MS C H FN O (m/z): 535.0 (M
2
7
31
8
3
+
+
+
+
H) ; HRMS: calc.: 535.2575 (MH ); Found: 535.2578 (MH ).
8
.4 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (3, 4, 5-trimethoxyphenyl) methyl) piperazin-1-yl)-1-
ethyl-6-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6c)
White solid, Yield = 88%, mp = 232-234 °C. IR (KBr): 3406, 3015, 1714, 1624, 1218, 1125
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.06 (s, 1H), 8.65 (s, 1H), 8.04 (d, J = 13.0 Hz, 1H),
3
H
7
3
.41 (d, J = 5.7 Hz, 2H), 6.81 (d, J = 6.7 Hz, 1H), 5.19 (s, 1H), 4.30 (bs, 2H), 3.85 (s, 9H),
1
3
.33 (s, 2H), 2.94 (s, 2H), 2.94 (s, 2H), 2.67 (s, 2H), 1.74 (s, 9H), 1.58 (t, J = 6.8 Hz, 3H). C
NMR (75 MHz, CDCl ): δ 177.0, 167.2, 154.2, 153.4, 147.1, 138.4, 130.2, 112.9, 107.0,
3
C
1
03.9, 65.5, 61.5, 60.8, 56.3, 50.4, 50.0, 30.3, 14.4. ESI-MS C H FN O (m/z): 624.0 (M +
31 38 7 6
+
+
+
H) ; HRMS: calc.: 624.2940 (MH ); Found: 624.2947 (MH ).
8
.5 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (4-isopropylphenyl) methyl) piperazin-1-yl)-1-ethyl-
6
-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6d)
Cream solid, Yield = 91%, mp = 218-220 °C. IR (KBr): 3436, 2962, 1724, 1628, 1218, 1138
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.07 (s, 1H), 8.64 (s, 1H), 8.03 (d, J = 9.7 Hz, 1H),
3
H
7
4
1
1
5
.36 (d, J = 5.9 Hz, 2H), 7.22 (d, J = 5.9 Hz, 2H), 6.80 (d, J = 4.9 Hz, 1H), 5.28 (s, 1H), 4.29-
.26 (m, 2H), 4.09 (bs, 1H) 3.32 (s, 4H), 2.98-2.95 (m, 4H), 2.91-2.88 (m, 3H), 1.81(s, 3H),
13
.70 (s, 9H), 1.56 (t, J = 5.1 Hz, 3H). C NMR (75 MHz, CDCl ): δ 176.9, 167.2, 154.3,
3
C
49.6, 147.1, 146.0, 145.9, 137.0, 131.9, 129.6, 126.8, 120.6, 112.8, 108.3, 103.9, 65.0, 61.5,
+
0.1, 49.7, 33.7, 30.3, 23.8, 23.8, 14.4. ESI-MS C H FN O (m/z): 576.0 (M + H) ; HRMS:
3
1
38
7
3
+
+
calc.: 576.3093 (MH ); Found: 576.3085 (MH ).
8
.6 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (furan-2-yl) methyl) piperazin-1-yl)-1-ethyl-6-
fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6e)
Light brown solid, Yield = 89%, mp = 196-198 °C. IR (KBr): 3440, 2987, 1717, 1625, 1220,
-1 1
1
131 cm . H NMR (300 MHz, CDCl ): δ 15.02 (s, 1H), 8.65 (s, 1H), 8.06 (d, J = 13.0 Hz,
3 H
12

ACCEPTED MANUSCRIPT
1
4
1
1
H), 7.45 (s, 1H), 6.80 (d, J = 6.3 Hz, 1H), 6.50 (s, 2H), 5.53 (s, 1H), 4.30 (bs, 2H), 3.32 (s,
1
3
H), 3.10 (s, 2H), 2.76 (s, 2H), 1.75 (s, 9H), 1.62 (s, 3H). C NMR (75 MHz, CDCl ): δ
3
C
76.9, 167.2, 152.0, 148.0, 147.1, 145.9, 143.0, 137.1, 120.6, 112.8, 112.5, 110.8, 108.3,
+
04.0, 61.8, 58.0, 50.0, 49.8, 30.0, 14.4. ESI-MS C H FN O (m/z): 524.0 (M + H) ;
26
30
7
4
+
+
HRMS: calc.: 524.2416 (MH ); Found: 524.2446 (MH ).
.7 7(4-((1-Tert-butyl-1H-tetrazol-5-yl) (4-fluorophenyl) methyl) piperazin-1-yl)-1-ethyl-6-
8
fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6f)
White solid, Yield = 82%, mp = 217-219 °C. IR (KBr): 3421, 3020, 1722, 1626, 1217, 1137
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.06 (s, 1H), 8.63 (s, 1H), 8.01 (d, J = 12.8 Hz, 1H),
3
H
7
.45 (s, 2H), 7.09 (t, J = 8.1 Hz, 2H), 6.79 (s, 1H), 5.32 (s, 1H), 4.29 (s, 2H), 3.31 (s, 4H),
1
3
2
.93 (s, 2H), 2.65 (s, 2H), 1.70 (s, 9H), 1.54 (s, 3H). C NMR (75 MHz, CDCl + DMSO-
3
d ): δ 176.6, 166.5, 165.4, 159.6, 159.1, 158.6, 158.1, 154.8, 151.5, 150.6, 150.6, 149.0,
6
C
1
1
44.2, 137.5, 133.4, 125.8, 121.2, 120.5, 117.4, 117.2, 116.9, 113.6, 111.7, 109.7, 107.7,
+
06.7, 63.3, 63.0, 51.1, 29.5, 14.6. ESI-MS C H F N O (m/z): 552.0 (M+H) ; HRMS:
2
8
31
2
7
3
+
+
calc.: 552.2529 (MH ); Found: 552.2550 (MH ).
8
.8 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (naphthalen-1-yl) methyl) piperazin-1-yl)-1-ethyl-6-
fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6g)
White solid, Yield = 90%, mp = 231-233 °C. IR (KBr): 3431, 2987, 1709, 1625, 1218, 1133
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.1 (s, 1H), 8.63 (s, 1H), 8.45 (d, J = 8.7 Hz, 1H),
3
H
8
1
1
1
5
5
.03 (d, J = 12.9 Hz, 1H), 7.94-7.84 ( m, 3H), 7.38 (t, J = 7.6 Hz, 1H), 7.05 (d, J = 7.1 Hz,
H), 6.75 (d, J = 6.2 Hz, 1H), 6.39 (s, 2H), 4.25 (bs, 2H), 3.26 (s, 4H), 2.85-2.83 (m, 4H),
1
3
.90 (s, 3H), 1.54 (s, 9H). C NMR (75 MHz, CDCl ): δ 176.2, 168.1, 153.3, 147.6, 145.4,
3
C
37.5, 131.9, 128.0, 127.7, 126.6, 125.9, 124.1, 111.9, 113.4, 107.2, 105.0, 102.8, 61.9, 60.2,
+
1.2, 49.3, 48.1, 31.0, 14.2. ESI-MS C H FN O (m/z): 584.0 (M + H) ; HRMS: calc.:
3
2
34
7
3
+
+
84.2779 (MH ); Found: 584.2796 (MH ).
13

ACCEPTED MANUSCRIPT
8
.9 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (4-methoxynaphthalen-1-yl) methyl) piperazin-1-yl)-
1
-ethyl-6-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6h)
Off white solid, Yield = 85%, mp = 198-200 °C IR (KBr): 3428, 2982, 1705, 1634, 1208,
-
1 1
1
2
8
3
1
1
141 cm . H NMR (300 MHz, CDCl ): δ 15.09 (s, 1H), 8.62 (s, 1H), 8.38 (t, J = 6.8 Hz,
3 H
H), 8.03 (d, J = 13.1 Hz, 1H), 7.68 (t, J = 6.4 Hz, 1H), 7.59 (t, J = 7.7 Hz, 1H), 6.94 (d, J =
.0 Hz, 1H), 6.74 (d, J = 6.3 Hz, 1H), 6.67 (d, J = 8.2 Hz, 1H), 4.24 (bs, 2H), 3.99 (s, 3H),
1
3
.24 (s, 6H), 2.85 (s, 4H), 2.69-2.65 (m, 2H), 1.55 (s, 9 H). C NMR (75 MHz, CDCl ): δ
3
C
76.9, 167.3, 156.2, 153.7, 147.0, 146.1, 146.0, 137.1, 132.5, 128.6, 127.4, 126.3, 125.5,
23.3, 123.0, 112.7, 112.4, 108.2, 103.9, 102.6, 61.4, 60.6, 55.6, 50.5, 49.7, 48.6, 30.0, 14.4.
+
+
ESI-MS C H FN O (m/z): 614.0 (M + H) ; HRMS: calc.: 614.2885 (MH ); Found:
3
3
36
7
4
+
6
9
14.2898 (MH ).
.0 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (phenyl) methyl) piperazin-1-yl)-1-ethyl-6-fluoro-4-
oxo-1, 4-dihydroquinoline-3-carboxylic acid (6i)
White solid, Yield = 74%, mp = 233-235 °C. IR (KBr): 3442, 2951, 1716, 1668, 1231, 1142
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.07 (s, 1H), 8.65 (s, 1H), 8.05 (d, J = 12.8 Hz, 1H),
3
H
7
2
1
4
5
9
.41-7.36 (m, 5H), 6.83 (s, 1H), 4.99 (s, 1H), 4.31 (s, 2H), 3.38 (s, 4H), 2.90 (s, 2H), 2.68 (s,
1
3
H), 2.08 (s, 4H), 1.92 (s, 3H), 1.73 (s, 7H). C NMR (75 MHz, CDCl ): δ 177.0, 166.8,
3
C
53.6, 153.2, 148.5, 138.4, 137.6, 130.9, 128.8, 126.5, 105.7, 65.3, 60.9, 58.2, 56.3, 50.9,
+
9.4, 32.8, 30.5, 25.4, 13.7. ESI-MS C H FN O (m/z): 560.0 (M +H); HRMS: calc.:
3
0
34
7
3
+
+
60.2779 (MH ); Found: 560.2786 (MH );
.1 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (pyridin-4-yl) methyl) piperazin-1-yl)-1-ethyl-6-
fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6j)
White solid, Yield = 88%, mp = 213-215 °C. IR (KBr): 3436, 3019, 1716, 1628, 1218, 1136
-1 1
cm . H NMR (300 MHz, CDCl + CD OD): δ 8.69 (d, J = 17.0 Hz, 3H), 8.03 (d, J = 12.2
3
3
H
Hz, 1H), 7.50 (s, 2H), 7.35 (s, 1H), 6.89 (s, 1H), 5.13 (s, 1H), 4.43-4.31 (m, 4H), 3.48-3.38
14

ACCEPTED MANUSCRIPT
1
3
(
m, 10H), 2.83 (s, 2H), 2.67 (s, 2H), 2.00 (s, 5H) . C NMR (75 MHz, CDCl + DMSO-d ):
3 6
δC 176.5, 166.6, 153.7, 150.8, 148.5, 145.5, 142.4, 137.4, 128.0, 121.1, 117.3, 113.4, 109.6,
1
5
9
1
07.6, 106.0, 60.1, 57.9, 49.9, 49.4, 49.0, 33.2, 25.0, 24.7, 14.2. ESI-MS C H FN O (m/z):
29 33 8 3
+
+
+
61.0 (M + H) ; HRMS: calc.: 561.2732 (MH ); Found: 561.2749 (MH ).
.2 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (3, 4, 5-trimethoxyphenyl) methyl) piperazin-1-yl)-
-ethyl-6-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6k)
White solid, Yield = 89%, mp = 186-188 °C. IR (KBr): 3442, 2986, 1709, 1632, 1241, 1123
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.03 (s, 1H), 8.64 (s, 1H), 8.03 (d, J = 12.9 Hz, 1H),
3
H
6
2
1
5
.82 (s, 1H), 6.66 (s, 2H), 4.85 (s, 1H), 4.36 (s, 2H), 3.84 (s, 9H), 3.39 (s, 4H), 2.84 (s, 2H),
1
3
.69 (s, 2H), 2.03-1.88 (m, 9H), 1.75-1.56 (m, 5H). C NMR (75 MHz, CDCl ): δ 160.5,
3
C
59.9, 154.3, 140.3, 138.1, 121.5, 116.7, 112.9, 107.6, 105.4, 63.7, 61.0, 59.7, 56.3, 51.9,
+
0.9, 46.6, 32.9, 31.9, 24.9, 24.6, 24.3, 13.8. ESI-MS C H FN O (m/z): 650.0 (M +H);
3
3
40
7
6
+
+
HRMS: calc.: 650.3096 (MH ); Found: 650.3119 (MH );
.3 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (4-isopropylphenyl) methyl) piperazin-1-yl)-1-
9
ethyl-6-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6l)
Light yellow solid, Yield = 93%, mp = 214-216 °C. IR (KBr): 3437, 2939, 1719, 1623, 1218,
-
1 1
1
1
4
2
1
5
6
9
127 cm . H NMR (300 MHz, CDCl ): δ 15.04 (s, 1H), 8.66 (s, 1H), 8.06 (d, J = 13.0 Hz,
3 H
H), 7.33 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 7.8 Hz, 2H), 6.83 (d, J = 6.6 Hz, 1H), 5.28 (s, 1H),
.95 (s, 2H), 4.38 (s, 2H), 4.29 (s, 1H), 3.37 (s, 4H), 2.91-2.87 (m, 3H), 2.68 (s, 2H), 2.08-
1
3
.00 (m, 4H), 1.92 (t, J = 8.7 Hz, 6H), 1.73 (s, 7H). C NMR (75 MHz, CDCl ): δ 176.3,
3
C
67.1, 155.4, 153.0, 148.2, 139.8, 131.6, 129.5, 112.9, 108.2, 105.1, 66.0, 61.5, 60.8, 56.8,
+
1.5, 50.2, 31.8, 23.8, 14.5. ESI-MS C H FN O (m/z): 602.0 (M +H); HRMS: calc.:
3
3
40
7
3
+
+
02.3249 (MH ); Found: 602.3274 (MH ).
.4 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (furan-2-yl) methyl) piperazin-1-yl)-1-ethyl-6-
fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6m)
15

ACCEPTED MANUSCRIPT
White solid, Yield = 84%, mp = 196-198 °C IR (KBr): 3430, 2967, 1712, 1632, 1241, 1132
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.04 (s, 1H), 8.66 (s, 1H), 8.06 (d, J = 13.0 Hz, 1H),
3
H
7
2
.47 (s, 1H), 6.82 (s, 1H), 6.58 (s, 1H), 6.44 (s, 1H), 5.27 (s, 1H), 4.33 (s, 2H), 3.35 (s, 4H),
1
3
.84 (s, 2H), 2.67 (s, 2H), 2.05-1.99 (m, 4H), 1.88-1.81 (m, 3H), 1.58-1.41 (m, 7H). C
NMR (75 MHz, CDCl ): δ 176.9, 167.1, 151.3, 147.3, 143.3, 137.0, 120.6, 112.8, 112.5,
3
C
1
11.9, 108.2, 103.9, 58.6, 58.2, 50.3, 49.8, 33.1, 33.0, 25.5, 24.8, 14.4. ESI-MS C H FN O
28 32 7 4
+
+
+
(
m/z): 550.0 (M + H) ; HRMS: calc.: 550.2572 (MH ); Found: 550.2596 (MH ).
9
.5 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (4-fluorophenyl) methyl) piperazin-1-yl)-1-ethyl-6-
fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6n)
White solid, Yield = 90%, mp = 205-207 °C. IR (KBr): 3430, 2988, 1718, 1646, 1231, 1143
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.02 (s, 1H), 8.06 (d, J = 13.4 Hz, 2H), 7.44 (s, 2H),
3
H
7
.11 (t, J = 7.8 Hz, 2H), 6.82 (s, 1H), 5.20 (s, 1H), 4.30 (s, 4H), 3.30 (s, 2H), 2.83 (s, 2H),
1
3
2
.65 (s, 2H), 2.06-1.97 (m, 4H), 1.88-1.79 (m, 3H), 1.61-1.53 (m, 7H). C NMR (75 MHz,
CDCl ): δ 178.1, 167.5, 160.2, 154.3, 153.4, 147.6, 137.8, 130.1, 113.0, 107.2, 103.8, 66.2,
3
C
+
6
1.7, 60.2, 56.1, 50.8, 50.2, 31.0, 14.5. ESI-MS C H F N O (m/z): 578.0 (M + H) ;
30 33 2 7 3
+
+
HRMS: calc.: 578.2685 (MH ); Found: 578.2694 (MH ).
9
.6 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (naphthalen-1-yl) methyl) piperazin-1-yl)-1-ethyl-
-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6o)
6
White solid, Yield = 81%, mp = 208-210 °C. IR (KBr): 3436, 3019, 1720, 1627, 1218, 930
-1 1
cm . H NMR (300 MHz, CDCl ): δ 14.8 (s, 1H), 8.54 (s, 1H), 8.42 (d, J = 8.6 Hz, 1H),
3
H
8
7
4
.00 (d, J = 13.0 Hz, 1H), 7.92-7.81 ( m, 2H), 7.71-7.62 (m, 2H), 7.41 ( t, J = 7.7 Hz, 1H),
.04 ( d, J = 7.3 Hz, 1H), 6.78 (d, J = 6.4 Hz, 1H), 6.42 (s, 1H), 4.26-4.22 (m, 2H), 3.38 (s,
13
H), 2.86 (s, 2H), 2.66 (s, 2H), 2.03-1.96 (m, 4H), 1.86-1.78 (m, 3H), 1.60-1.52 (m, 7H). C
NMR (75 MHz, CDCl ): δ 176.0, 168.3, 153.2, 147.9, 145.8, 137.9, 131.4, 128.2, 127.8,
3
C
1
26.1, 125.7, 124.5, 111.2, 113.7, 107.0, 105.4, 102.6, 61.8, 61.2, 51.3, 49.6, 48.3, 31.0, 30.6,
16

ACCEPTED MANUSCRIPT
+
2
5.8, 24.6, 14.3. ESI-MS C H FN O (m/z): 610.0 (M + H) ; HRMS: calc.: 610.2936
34
36
7
3
+
+
(
MH ); Found: 610.2961 (MH ).
9
.7 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (4-methoxynaphthalen-1-yl) methyl) piperazin-1-
yl)-1-ethyl-6-fluoro-4-oxo-1, 4-dihydroquinoline-3-carboxylic acid (6p)
-
White solid, Yield = 86%, mp = > 250 °C IR (KBr): 3438, 2982, 1713, 1634, 1232, 1131 cm
1
1
.
H NMR (300 MHz, CDCl ): δ 15.0 (s, 1H), 8.63 (s, 1H), 8.37 (t, J = 6.9 Hz, 2H), 8.11 (d,
3
H
J = 13.0 Hz, 1H), 7.71 (t, J = 6.5 Hz, 2H), 7.62 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 8.2 Hz, 1H),
6
.64 (d, J = 8.2 Hz, 1H), 6.31 (s, 1H), 4.94 (s, 1H), 4.32 (s, 3H), 3.34 (s, 4H), 3.01-2.95 (m,
H), 2.72 (s, 2H), 2.65-2.61 (m, 2H), 1.83-1.64 (m, 7H), 1.58 (t, J = 6.5 Hz, 3H), 1.28-1.17
2
1
3
(
m, 4H). C NMR (75 MHz, CDCl ): δ 176.3, 167.4, 156.6, 152.6, 148.2, 147.0, 137.0,
3 C
1
5
33.6, 129.0, 127.1, 126.8, 125.3, 121.0, 113.0, 112.6, 108.4, 103.4, 102.8, 61.9, 60.7, 55.9,
1.9, 49.5, 48.8, 32.7, 31.0, 26.9, 25.5, 24.0, 14.6. ESI-MS C H FN O (m/z): 640.0 (M +
3
5
38
7
4
+
+
+
H) ; HRMS: calc.: 640.3042 (MH ); Found: 640.3056 (MH ).
.8 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (phenyl) methyl) piperazin-1-yl)-6-fluoro-4-oxo-1-
4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’a)
White solid, Yield = 86%, mp = 198-200 °C. IR (KBr): 3430, 2978, 1709, 1641, 1220, 1131
9
(
-1 1
cm . H NMR (300 MHz, CDCl ): δ 14.9 (s, 1H), 8.82 (s, 1H), 8.00 (s, 2H), 7.64 (s, 4H),
3
H
7
.48 (s, 5H), 5.26 (s, 1H), 4.41 (s, 2H), 3.10 (s, 3H), 2.86 (s, 2H), 2.56 (s, 3H), 1.66 (s, 9H).
1
3
C NMR (75 MHz, CDCl ): δ 176.2, 168.1, 155.1, 154.0, 149.2, 138.8, 131.3, 128.3, 113.2,
3
C
1
08.6, 104.2, 66.1, 62.0, 60.9, 57.2, 51.6, 51.2, 31.0. ESI-MS C H F N O (m/z): 664.0
34 33 4 7 3
+
+
+
(
M +H); HRMS: calc.: 664.2653 (MH ); Found: 664.2682 (MH ).
9
.9 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (pyridin-4-yl) methyl) piperazin-1-yl)-6-fluoro-4-
oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’b)
Yellow solid, Yield = 90%, mp = 227-229 °C IR (KBr): 3439, 2989, 1732, 1641, 1209, 1142
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.0 (s, 1H), 8.77 (s, 1H), 8.64 (s, 2H), 8.00 (d, J = 9.6
3
H
17

ACCEPTED MANUSCRIPT
Hz, 1H), 7.67 (s, 4H), 7.34 (s, 2H), 6.52 (s, 1H), 5.63 (s, 1H), 4.96 (s, 2H), 3.35 (s, 4H), 2.78
1
3
(
s, 2H), 2.56 (s, 2H), 1.69 (s, 9H). C NMR (75 MHz, CDCl + DMSO-d ): δ 171.1, 157.9,
3 6 C
1
54.9, 148.9, 142.6, 132.9, 131.0, 129.8, 67.1, 66.5, 61.1, 56.3, 50.4, 50.0, 34.7. ESI-MS
+
+
C H F N O (m/z): 665.0 (M +H); HRMS: calc.: 665.2606 (MH ); Found: 665.2637
33
32
4
8
3
+
(
MH ).
1
0.1 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (3, 4, 5-trimethoxyphenyl) methyl) piperazin-1-yl)-
-fluoro-4-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’c)
6
Off white solid, Yield = 78%, mp = 225-227 °C. IR (KBr): 3439, 3013, 1719, 1624, 1219,
-
1 1
1
2
4
1
5
126 cm . H NMR (300 MHz, CDCl ): δ 15.02 (s, 1H), 8.80 (s, 1H), 7.96 (d, J = 12.6 Hz,
3 H
H), 7.63 (s, 2H), 7.26 (s, 2H), 6.64 (s, 2H), 5.53 (s, 2H), 5.12 (s, 1H), 3.81 (s, 9H), 3.11 (s,
1
3
H), 2.81 (s, 2H), 2.55 (s, 2H), 1.69 (s, 9H). C NMR (75 MHz, CDCl ): δ 177.2, 166.8,
3
C
54.2, 153.3, 148.4, 138.4, 137.6, 130.1, 126.5, 112.5, 108.5, 107.0, 104.7, 65.5, 61.5, 60.8,
+
6.3, 50.3, 49.6, 30.3. ESI-MS C H F N O (m/z): 754.0 (M +H); HRMS: calc.: 754.2970
3
7
39
4
7
6
+
+
(
MH ); Found: 754.2962 (MH ).
1
0.2 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (4-isopropylphenyl) methyl) piperazin-1-yl)-6-
fluoro-4-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’d)
White solid, Yield = 93%, mp = 242-244 °C. IR (KBr): 3446, 2988, 1729, 1632, 1221, 1143
-1 1
cm . H NMR (300 MHz, DMSO-d ): δ 15.1 (s, 1H), 9.23 (s, 1H), 7.86 (d, J = 13.0 Hz,
6
H
1
1
1
1
H), 7.73 (s, 2H), 7.52 (s, 2H), 7.44 (d, J = 7.8 Hz, 2H), 7.29 (d, J = 7.8 Hz, 2H), 7.04 (s,
H), 5.96 (s, 1H), 5.41 (s, 2H), 4.68 (s, 2H), 3.12 (s, 4H), 2.89 (s, 2H), 2.87-2.82 (m, 1H),
1
3
.87 (m, 6H), 1.72 (s, 9H). C NMR (75 MHz, DMSO-d ): δ 177.0, 166.4, 160.2, 152.8,
6
C
48.0, 131.8, 129.8, 128.6, 126.5, 126.0, 64.1, 62.0, 60.6, 57.1, 49.9, 30.3, 24.0. ESI-MS
+
+
C H F N O (m/z): 706.0 (M +H); HRMS: calc.: 706.3123 (MH ); Found: 706.3146
37
39
4
7
3
+
(
MH ).
18

ACCEPTED MANUSCRIPT
1
0.3 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (furan-2-yl) methyl) piperazin-1-yl)-6-fluoro-4-
oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’e)
-1
White solid, Yield = 89%, mp = 242-244 °C. IR (KBr): 3436, 3019, 1626, 1218, 1128 cm .
1
H NMR (300 MHz, CDCl ): δ 14.9 (s, 1H), 8.78 (s, 1H), 8.06 (d, J = 13.0 Hz, 1H), 7.65 (d,
3
H
J = 7.3 Hz, 2H), 7.42 (s, 1H), 7.28 (s, 3H), 6.59-6.53 (m, 1H), 6.43 (s, 1H), 5.53 (s, 2H), 5.16
1
3
(
s, 1H), 3.12 (s, 4H), 2.76 (s, 2H), 2.58 (s, 2H), 1.75 (s, 9H). C NMR (75 MHz, CDCl ): δ
3 C
1
5
76.2, 167.1, 155.2, 154.0, 148.2, 138.4, 130.2, 110.9, 107.0, 103.9, 68.3, 61.5, 60.8, 52.3,
+
0.4, 50.0, 30.1. ESI-MS C H F N O (m/z): 654.0 (M +H); HRMS: calc.: 654.2446
32
31
4
7
4
+
+
(
MH ); Found: 654.2472 (MH ).
1
0.4 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (4-fluorophenyl) methyl) piperazin-1-yl)-6-fluoro-
-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’f)
4
-
White solid, Yield = 86%, mp = >250 °C. IR (KBr): 3451, 2998, 1729, 1685, 1231, 1153 cm
1
1
.
H NMR (300 MHz, CDCl ): δ 15.0 (s, 1H), 8.74 (s, 1H), 8.04 (d, J = 13.0 Hz, 2H), 7.53
3
H
(d, J = 6.7 Hz, 2H), 6.83- 6.76 (m, 4H), 6.54 (s, 2H), 5.18 (s, 1H), 4.31 (s, 2H), 3.27 (s, 4H),
1
3
2
1
.95 (s, 2H), 2.83 (s, 2H), 1.78 (s, 9H). C NMR (75 MHz, CDCl ): δ 176.4, 167.3, 154.4,
3 C
53.6, 147.3, 138.2, 130.1, 113.0, 107.2, 105.2, 65.4, 61.8, 59.3, 56.1, 52.0, 51.3, 30.2. ESI-
+
+
MS C H F N O (m/z): 682.0 (M +H); HRMS: calc.: 682.2559 (MH ); Found: 682.2566
34
32
5
7
3
+
(
MH ).
1
0.5 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (naphthalen-1-yl) methyl) piperazin-1-yl)-6-fluoro-
-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’g)
4
White solid, Yield = 91%, mp = 196-198 °C. IR (KBr): 3423, 2981, 1702, 1626, 1219, 1134
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.2 (s, 1H), 8.56 (s, 3H), 8.43 (d, J = 8.6 Hz, 1H),
3
H
8
7
2
.02 (d, J = 13.0 Hz, 1H), 7.93-7.88 ( m, 3H), 7.72-7.63 (m, 3H), 7.42 ( t, J = 7.6 Hz, 1H),
.02 ( d, J = 7.2 Hz, 1H), 6.76 (d, J = 6.2 Hz, 1H), 5.19 (s, 1H), 4.30 (s, 2H), 3.35 (s, 4H),
1
3
.96 (s, 2H), 2.66 (s, 2H), 1.78 (s, 9H). C NMR (75 MHz, CDCl ): δ 177.2, 166.3, 155.4,
3
C
19

ACCEPTED MANUSCRIPT
1
54.7, 147.4, 138.5, 130.2, 114.0, 108.2, 104.3, 66.0, 62.5, 61.6, 52.5, 51.6, 30.5. ESI-MS
+
+
C H F N O (m/z): 714.0 (M +H); HRMS: calc.: 714.2810 (MH ); Found: 714.2826
38
35
4
7
3
+
(
MH ).
1
0.6 7-(4-((1-Tert-butyl-1H-tetrazol-5-yl) (4-methoxynaphthalen-1-yl) methyl) piperazin-1-
yl)-6-fluoro-4-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid
6’h)
White solid, Yield = 79%, mp = 210-212 °C. IR (KBr): 3436, 2898, 1719, 1628, 1218, 1153
(
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.1 (s, 1H), 8.68 (s, 3H), 8.03 (d, J = 12.8 Hz, 1H),
3
H
7
1
9
1
.74-7.65 (m, 3H), 7.42 (d, J = 5.8 Hz, 2H), 6.81 (d, J = 6.7 Hz, 2H), 6.69 (s, 2H), 5.19 (s,
H), 4.36 (s, 2H), 3.83 (s, 3H), 3.31 (s, 2H), 2.98 (s, 2H), 2.93 (s, 2H), 2.68 (s, 2H), 1.73 (s,
1
3
H). C NMR (75 MHz, CDCl ): δ 177.3, 166.8, 155.0, 154.5, 147.3, 138.8, 131.0, 113.2,
3
C
07.6, 103.8, 66.4, 62.8, 61.9, 56.2, 52.4, 51.3, 30.3. ESI-MS C H F N O (m/z): 744.0
3
9
37
4
7
4
+
+
+
(
M +H); HRMS: calc.: 744.2915 (MH ); Found: 744.2936 (MH ).
1
0.7 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (phenyl) methyl) piperazin-1-yl)-6-fluoro-4-oxo-1-
(
4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’i)
White solid, Yield = 90%, mp = 186-188 °C. IR (KBr): 3451, 2989, 1703, 1635, 1221, 1142
-1 1
cm . H NMR (300 MHz, CDCl ): δ 14.91 (s, 1H), 8.80 (s, 1H), 8.04 (d, J = 12.8 Hz, 1H),
3
H
7
.66 (d, J = 7.4 Hz, 2H), 7.37 (s, 5H), 7.25 (s, 2H), 6.59 (d, J = 5.3 Hz, 1H), 5.49 (s, 2H),
1
3
4
.90 (s, 1H), 3.16 (s, 4H), 2.76 (s, 2H), 2.59 (s, 2H), 1.92-1.78 (m, 11H). C NMR (75 MHz,
CDCl ): δ 177.1, 166.9, 154.6, 153.1, 152.1, 148.5, 145.6, 145.5, 137.7, 137.3, 129.0, 128.7,
3
C
1
26.5, 112.7, 112.5, 108.4, 104.8, 64.9, 58.1, 57.8, 50.6, 49.4, 32.8, 32.6, 25.3, 24.6. ESI-MS
+
+
C H F N O (m/z): 690.0 (M +H); HRMS: calc.: 690.2810 (MH ); Found: 690.2842
36
35
4
7
3
+
(
MH ).
1
0.8 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (pyridin-4-yl) methyl) piperazin-1-yl)-6-fluoro-4-
oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’j)
20

ACCEPTED MANUSCRIPT
White solid, Yield = 85%, mp = 244-246 °C. IR (KBr): 3422, 2992, 1710, 1628, 1219, 1132
-1 1
cm . H NMR (300 MHz, CDCl ): δ 14.8 (s, 1H), 8.78 (s, 1H), 8.65 (s, 3H), 8.02 (d, J = 9.6
3
H
Hz, 2H), 7.65 (s, 2H), 7.37 (s, 2H), 6.57 (s, 1H), 5.51 (s, 1H), 4.98 (s, 2H), 3.14 (s, 4H), 2.74
1
3
(
s, 2H), 2.55 (s, 2H), 1.99-1.92 (m, 5H), 1.75-1.68 (m, 6H). C NMR (75 MHz, CDCl +
3
DMSO-d ): δ 176.9, 166.4, 154.4, 152.4, 151.9, 150.2, 145.4, 143.3, 140.5, 137.8, 128.1,
6
C
1
26.2, 125.8, 124.7, 120.0, 111.8, 107.9, 106.9, 60.8, 57.4, 56.4, 33.1, 25.1. ESI-MS
+
+
C H F N O (m/z): 691.0 (M +H); HRMS: calc.: 691.2762 (MH ); Found: 691.2789
35
34
4
8
3
+
(
MH ).
1
0.9 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (3, 4, 5-trimethoxyphenyl) methyl) piperazin-1-
yl)-6-fluoro-4-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid
6’k)
White solid, Yield = 79%, mp = >250 °C. IR (KBr): 3440, 2978, 1702, 1622, 1217, 1133 cm
(
-
1
1
.
H NMR (300 MHz, CDCl ): δ 14.92 (s, 1H), 8.82 (s, 1H), 8.07 (d, J = 12.8 Hz, 1H), 7.69
3
H
(d, J = 5.8 Hz, 3H), 6.63 (s, 4H), 5.49 (s, 1H), 4.75 (s, 2H), 4.40-4.28 (m, 2H), 3.82 (s, 9H),
1
3
3
.17 (s, 4H), 2.73 (s, 4H), 1.93-1.73 (m, 5H), 1.70-1.65 (m, 6H). C NMR (75 MHz, CDCl ):
3
δC 177.2, 167.1, 155.8, 152.6, 147.3, 138.2, 131.0, 112.6, 108.0, 103.7, 66.1, 62.0, 61.6, 55.8,
+
5
2.6, 51.2, 31.2. ESI-MS C H F N O (m/z): 780.0 (M +H); HRMS: calc.: 780.3127
39
41
4
7
6
+
+
(
MH ); Found: 780.3141 (MH ).
1
1.0 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (4-isopropylphenyl) methyl) piperazin-1-yl)-6-
fluoro-4-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’l)
White solid, Yield = 92%, mp = 193-195 °C. IR (KBr): 3440, 2988, 1721, 1639, 1238, 1141
-1 1
cm . H NMR (300 MHz, DMSO-d ): δ 15.1 (s, 1H), 9.21 (s, 1H), 7.88 (d, J = 12.7 Hz,
6
H
1
1
1
H), 7.71 (s, 2H), 7.51 (s, 2H), 7.41 (d, J = 7.7 Hz, 2H), 7.28 (d, J = 7.7 Hz, 2H), 7.03 (s,
H), 5.96 (s, 1H), 5.41 (s, 2H), 4.68 (s, 2H), 4.42-4.29 (m, 2H), 3.12 (s, 4H), 2.89-2.85 (m,
1
3
H), 1.92-1.71 (m, 5H), 1.87-1.66 (m, 6H), 1.21-1.16 (m, 6H). C NMR (75 MHz, DMSO-
21

ACCEPTED MANUSCRIPT
d ): δ 178.0, 167.1, 153.9, 150.3, 149.0, 132.5, 129.6, 128.1, 126.8, 126.2, 106.2, 103.8,
6
C
+
6
2.2, 57.2, 49.8, 33.5, 33.0, 30.3, 25.1. ESI-MS C H F N O (m/z): 732.0 (M +H); HRMS:
39 41 4 7 3
+
+
calc.: 732.3279 (MH ); Found: 732.3315 (MH ).
1
1.1 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (furan-2-yl) methyl) piperazin-1-yl)-6-fluoro-4-
oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’m)
White solid, Yield = 87%, mp = 216-218 °C. IR (KBr): 3445, 2937, 1719, 1625, 1219, 1138
-1 1
cm . H NMR (300 MHz, CDCl ): δ 8.79 (s, 1H), 8.04 (d, J = 12.8 Hz, 1H), 7.65 (d, J = 7.2
3
H
Hz, 2H), 7.43 (s, 1H), 7.29 (s, 3H), 6.58-6.52 (m, 2H), 6.40 (s, 1H), 5.50 (s, 2H), 5.18 (s, 1H),
1
3
3
.11 (s, 5H), 2.74 (s, 2H), 2.55 (s, 2H), 1.98 (s, 6H), 1.76 (s, 4H). C NMR (75 MHz,
CDCl ): δ 177.1, 166.8, 154.5, 152.0, 151.3, 148.5, 147.2, 145.5, 145.4, 143.3, 143.2, 137.6,
3
C
1
2
37.3, 126.5, 112.8, 112.6, 111.8, 110.8, 108.5, 104.8, 58.6, 58.1, 57.8, 50.1, 49.5, 33.0, 29.6,
+
5.4, 25.3, 24.7. ESI-MS C H F N O (m/z): 680.0 (M +H); HRMS: calc.: 680.2602
34
33
4
7
4
+
+
(
MH ); Found: 680.2616 (MH ).
1
1.2 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (4-fluorophenyl) methyl) piperazin-1-yl)-6-fluoro-
-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’n)
4
White solid, Yield = 83%, mp = 228-230 °C. IR (KBr): 3452, 2991, 1718, 1631, 1208, 1152
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.1 (s, 1H), 8.73 (s, 1H), 8.04 (d, J = 12.8 Hz, 2H),
3
H
7
4
1
1
.52 (d, J = 6.7 Hz, 2H), 6.81-6.73 (m, 4H), 6.52 (s, 2H), 5.17 (s, 1H), 4.32 (s, 2H), 3.24 (s,
1
3
H), 2.95 (s, 2H), 2.82 (s, 2H), 1.93 (s, 6H), 1.76 (s, 5H). C NMR (75 MHz, CDCl ): δ
3
C
76.5, 167.2, 154.6, 152.3, 151.0, 148.6, 147.3, 145.6, 143.2, 137.0, 126.7, 112.9, 111.4,
10.1, 108.6, 105.2, 58.7, 58.2, 57.6, 52.0, 50.1, 33.2, 29.8, 25.6. ESI-MS C H F N O
36
34
5
7
3
+
+
+
(
m/z): 708.0 (M +H); HRMS: calc.: 708.2716 (MH ); Found: 708.2751 (MH ).
1
1.3 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (naphthalen-1-yl) methyl) piperazin-1-yl)-6-
fluoro-4-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylic acid (6’o)
22

ACCEPTED MANUSCRIPT
White solid, Yield = 78%, mp = 219-221 °C. IR (KBr): 3442, 2988, 1710, 1648, 1228, 1141
-1 1
cm . H NMR (300 MHz, CDCl ): δ 14.9 (s, 1H), 8.79 (s, 1H), 8.49 (d, J = 7.8 Hz, 2H),
3
H
7
1
4
1
2
7
1
.97 (d, J = 13.0 Hz, 2H), 7.89 (t, J = 7.5 Hz, 2H), 7.60-7.43 (m, 4H), 6.61 (s, 1H), 5.72 (s,
H), 5.53 (s, 2H), 3.17 (s, 4H), 2.86 (s, 2H), 2.72 (s, 2H), 1.82-1.62 (m, 7H), 1.24-1.17 (m,
1
3
H). C NMR (75 MHz, CDCl ): δ 176.8, 166.9, 152.8, 148.4, 134.1, 131.3, 130.6, 129.3,
3
C
29.0, 127.0, 126.5, 124.9, 123.2, 112.9, 112.5, 108.5, 61.4, 58.1, 50.6, 49.7, 32.7, 32.4, 25.1,
+
+
4.5. ESI-MS C H F N O (m/z): 740.0 (M +H). HRMS: calc.: 740.2966 (MH ); Found:
4
0
37
4
7
3
+
40.3002 (MH ).
1.4 7-(4-((1-Cyclohexyl-1H-tetrazol-5-yl) (4-methoxynaphthalen-1-yl) methyl) piperazin-1-
yl)-6-fluoro-4-oxo-1-(4-(trifluoromethyl) benzyl)-1, 4-dihydroquinoline-3-carboxylicacid
6’p)
White solid, Yield = 89%, mp = 223-225 °C. IR (KBr): 3441, 3011, 1721, 1642, 1209, 1132
(
-1 1
cm . H NMR (300 MHz, CDCl ): δ 15.2 (s, 1H), 8.92 (s, 1H), 8.48 (d, J = 7.7 Hz, 2H),
3
H
8
5
1
1
5
.01 (d, J = 12.6 Hz, 2H), 7.88-7.83 (m, 2H), 7.62-7.52 (m, 4H), 6.70 (s, 2H), 5.85 (s, 2H),
.74 (s, 1H), 4.26 (s, 3H), 3.21 (s, 4H), 2.88 (s, 2H), 2.76 (s, 2H), 1.82-1.73 (m, 7H), 1.22-
13
.15 (m, 4H). C NMR (75 MHz, CDCl + DMSO-d ): δ 177.0, 166.8, 156.1, 153.2, 148.6,
3
6
C
35.0, 131.6, 130.9, 128.8, 128.2, 127.0, 126.6, 125.0, 124.2, 112.7, 112.3, 109.5, 62.5, 58.0,
+
6.4, 51.3, 50.1, 32.6, 32.2, 25.0, 24.7. ESI-MS C H F N O (m/z): 770.0 (M +H); HRMS:
4
1
39
4
7
4
+
+
calc.: 770.3072 (MH ); Found: 770.3087 (MH ).
1
1
1.5 Biological methods
1.5.1 In vitro antibacterial assay:
The targeted 7-piperazinylquinolone-tetrazole derivatives (6a-p and 6’a-p) were
screened against Gram-positive bacteria S. aureus (ATCC-25923), S. aureus (ATCC-
00699), S. aureus (ATCC-29213) and S. aureus (ATCC-33592) and Gram-negative bacteria
Escherichia coli and Klebsiella pneumoniae where Ciprofloxacin and Gentamycin were used
7
23

ACCEPTED MANUSCRIPT
as a reference drugs. The bacterial strains were grown on nutrient agar at 37 °C. After 24 h of
incubation, bacterial cells were suspended in normal saline containing Tween 20 at 0.05 % at
7
a concentration of approximately 1.0-2.0 × 10 cells/mL by matching with 0.5 McFarland
standards. The activity of compounds was determined as per NCCLS protocol using Mueller
Hinton broth (Becton Dickinson, USA) in 96-well tissue culture plates. Proper growth
control, drug control and the negative control were adjusted onto the plate. Compounds were
dissolved in DMSO at a concentration of 1 mg/mL and 20 µL of this was added to each well
of 96-well tissue culture plate having 180 µL Mueller Hinton broth. From here the solution
was serially diluted resulting in two fold dilution of the test compounds in subsequent wells.
1
00 µL of Mc Farland matched bacterial suspension was diluted in 10 mL of media and then
00 µL of it was added in each well and kept for incubation. The maximum concentration of
1
compounds tested was 50 µg/mL. The micro-titer plates were incubated at 35 °C in a moist,
dark chamber and MICs were recorded spectrophotometrically after 24h using SOFT max
Pro 4.3 Software (Molecular Devices, Sunnyvale, USA).
1
1.5.2 In vitro cytotoxicity evaluation assay
The toxicity evaluation of lead compounds 6e, 6m and 6k against murine fibroblast
cell line L-929 was used. All test compounds were solubilised in DMSO (1mg/mL). The cell
line L-929 was grown in RPMI 1640 medium supplemented with 10% FBS and 1 X
o
antimycotic and antibacterial solution (sigma, USA) at 37 C in incubator having 5% CO .
2
5
The confluent fibroblast cells are collected from tissue culture flask and maintained 1 x 10
cells per milliliter in RPMI serum free medium. Hundred microliters of cell suspension
prepared in RPMI serum free medium was dispensed in 96 well tissue culture plates and
incubated for 5h to adhere the cells properly. In the other replica plate test compounds were
diluted in 2 fold manner (25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, 0.09 ꢀg/mL) were added
to the growing cells along with control (with no compounds) and incubated for 24h in
24

ACCEPTED MANUSCRIPT
o
incubator at 37 C. After incubation wells were washed with PBS and 200 µL of MTT
solution (0.5 mg of MTT in RPMI 1640 medium) was added to each well and incubated for
4
h at 37 °C. After incubation 100 ꢀL DMSO was added to each well and incubated for 30
minute at room temprature O. D was determined spectrophotometrically at 570 nm and 630
nm. The morphology of the cells was observed using Giemsa stain under Phase contrast
microscope. After fixation of the cells in 96 well tissue culture plates, Giemsa stain was
added to each well and incubated for 30 minute at room temprature. The excess stain was
removed by thorough washing with phosphate buffer saline and the culture plates were air
dried and observed under a phase contrast microscope [34, 35].
1
1.6 Material and methods of 3D-QSAR Analysis
Thirty two molecules belonging 1H-tetrazol-5-yl-(aryl) methyl piperazinyl-6-fluoro-
4
-oxo-1, 4-dihydroquinoline-3-carboxylic acid were selected for CoMFA
[36] and CoMSIA
[
37
]
studies. MIC values of reported molecules were converted into respective PIC (-Log
5
0
IC ). All molecules were drawn using sketch option in Sybyl 7.1
[
38]. These compounds
5
0
were subjected to energy minimization by Powell gradient method after assigning Gasteiger
Huckel charges. Minimization was performed till convergence. Data set was randomly
divided into training set of twenty five molecules and test set of seven molecules. Alignment
is considered crucial step in developing 3D-QSAR model [25]. Molecules in data set were
aligned by automatic database alignment on common substructure.
CoMFA (comparative molecular field alignment) method uses previously aligned
molecules to generate steric and electrostatic fields. For this purpose all molecules were
placed automatically in defined cubic GRID with grid spacing 2Aº. Steric (Lennard-jones 6-
1
2 potential) and electrostatic field (columbic potential) for each molecule were calculated
3
using +1.0 charged SP carbon atom. Minimum Column filtering was set to 2.0 kcal/mol to
improve the signal to noise ratio by omitting those lattice points, whose energy variation was
25

ACCEPTED MANUSCRIPT
below this threshold. Steric and electrostatic fields generated were scaled by the CoMFA-
Standard method in SYBYL with default cut-off energy (30 kcal/mol). The calculated
descriptors were used to develop CoMFA model. CoMSIA method has advantage over
CoMFA in way that it calculates also field created by Hydrogen acceptor, Hydrogen donor
3
and Hydrophobic groups. SP C atom with +1 charge was used to calculate steric and
electrostatic field. Hydrogen and hydrophobic fields were calculated using.
Partial least square is a statistical method to calculate linear relationship between a
dependent variable and set of predictor variable. CoMFA and CoMSIA models were
developed by considering PIC values as dependent variable and field values as independent
5
0
variables. Both models were subjected to LOO (Leave one out) cross-validation. After getting
2
2
optimal no of components, no validation was performed to get r .Predictive r was
ncv
calculated using test set molecules in order to further verify the models.
Acknowledgments
The author K.C is grateful to Council of Scientific and Industrial Research for the financial
support in the form of fellowship and also thankful to S.A.I.F. Division of CDRI, Lucknow,
for providing the spectroscopic data.
References
[
[
[
1] G. V. Doern, K. P. Heilmann, H. K. Huynh, P. R. Rhomberg, S. L. Coffman, A. B.
Brueggemann, Antimicrob. Agents Chemother. 45 (2001) 1721–1729.
2] C. G. Giske, D. L. Monnet, O. Cars, and Y. Carmeli, Antimicrob. Agents Chemother.
5
2 (2008) 813–821.
3] H. Y. Kim, J. A. Wiles, Q. Wang, G. C. G. Pais, E. Lucien, A. Hashimoto, D. M.
Nelson, J. A. Thanassi, S. D. Podos, M. Deshpande, M. J. Pucci, and B. J. Bradbury
J. Med. Chem. 54 (2011) 3268-3282.
,
26

ACCEPTED MANUSCRIPT
[
4] T. L. Smith, M. L. Pearson, K. R. Wilcox, C. Cruz, M. V. Lancaster, B. R. Dunn, F.
C. Tenover, M. J. Zervos, J. D. Band, E. White, W. R. Jarvis, N. Engl. J. Med. 340
(1999) 493–501.
[
[
5] V. T. Andriole, Drugs 58 (1999) 1-5.
6] H. W. Boucher, G. H. Talbot, J. S. Bradley, J. E. Jr. Edwards, D. Gilbert, L. B. Rice,
M. Scheld, B. Spellberg, J. Bartlett, Clin. Infect. Dis. 48 (2009) 1–12.
7] H. G. Kathrotiya, M. P. Patel, Eur. J. Med. Chem. 63 (2013) 675-684.
8] T. Rodrigues, A. S. Ressurreicao, F. P. d. Cruz, I. S. Albuquerque, J. Gut, M. P.
Carrasco, D. Gonçalves, R. C. Guedes, D. J.V.A. dos Santos, M. M. Mota, P. J.
Rosenthal, R. Moreira, M. Prudêncio, F. Lopes, Eur. J. Med. Chem. 69 (2013) 872-
[
[
8
80.
[
[
9] C. M. Oliphant, G. M. Green, Am. Fam. Physican, 65 (2002) 455-462.
10]
11]
H. Koga, A. Itoh, S. Murayama, S. Suzue, T. Irikura, J. Med. Chem. 23
1980) 1358-1363.
R. Wise, J. M. Andrews, L. J. Edwards, Antimicrob. Agents Chemother. 23
1983) 559-564. (b) K. Grohe, H. Heitzer, Liebigs Ann. Chem. (1987) 29-37.
(
[
(
[
[
12]
13]
J. J. Schentag, Clin. Ther. 22 (2000) 372-387.
T. D. Gootz, K. E. Brighty, M. R. Anderson, S. L. Haskell, J. A. Sutcliffe, M.
J. Castaldi, S. A. Miller, Presented at the 32th Meeting of the ICAAC,
Anaheim, CA, 1992; Abstract 751.
[
[
[
14]
15]
16]
N. Harnett, S. Brown, C. Krishnan, Antimicrob. Agents Chemother. 35 (1991)
1
911- 1913.
G. Cheng, H. Hao, M. Dai, Z. Liu, Z. Yuan, Eur. J. Med. Chem. 66 (2013)
55-562.
S. Emami, A. Shafiee, and A. Foroumadi, Iran. J. Pharm. Res. 3 (2005) 123-
5
27