Design, synthesis and molecular modeling of pyrazole-quinoline-pyridine hybrids as a new class of antimicrobial and anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2014.01.018

A new series of pyrazole-quinoline-pyridine hybrids were designed based on molecular hybridization technique and synthesized by a base-catalyzed cyclocondensation reaction through one-pot multicomponent reaction. All compounds were tested for in vitro ant

Full text of DOI:10.1016/j.ejmech.2014.01.018

European Journal of Medicinal Chemistry 76 (2014) 549e557
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and molecular modeling of pyrazoleequinolinee
pyridine hybrids as a new class of antimicrobial and anticancer agents
Chetan B. Sangani, Jigar A. Makawana, Xin Zhang, Shashikant B. Teraiya, Lin Lin,
*
Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of pyrazoleequinolineepyridine hybrids were designed based on molecular hybridization
technique and synthesized by a base-catalyzed cyclocondensation reaction through one-pot multicom-
ponent reaction. All compounds were tested for in vitro antibacterial and anticancer activities. Enzyme
inhibitory activities of all compounds were carried out against FabH and EGFR. Of the compounds
studied, majority of the compounds showed effective antibacterial as well as anticancer activity against
Received 3 December 2013
Received in revised form
7 January 2014
Accepted 13 January 2014
Available online 25 January 2014
used strains and cancer cell lines respectively. Compound 7k (IC50 ¼ 0.51 ꢀ 0.05
displayed the most potent inhibitory activity with IC50 of 3.1 M against FabH as compared to other
member of the series. In the molecular modeling study, compound 7k was bound in to the active pocket
of EGFR with three hydrogen bond and one ecation interaction with minimum binding energy Gb
ꢁ54.6913 kcal/mol, as well as compound 7b was bound in to the active site of FabH with hydrogen bond
and esigma interactions with minimum binding energy
mM) against EGFR and 7b
m
Keywords:
4H-Quinoline
p
D
¼
1H-Pyrazole-4-carbaldehyde
Pyridine
One-pot reaction
Antibacterial activity
Anticancer activity
Molecular modeling
p
D
Gb ¼ ꢁ45.9125 kcal/mol.
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
always been observed in these tumors, approximately in 60% of all
tumors [6]. EGFR and ErbB-2 are the hottest targets in current
research and their over expression or abnormal activation often
cause cell malignant transformation. Also they have relationship
with postoperative adverse, radiotherapy and chemotherapy
resistance and tumor angiogenesis [9].
Along with the living habits and environment changes, cancer
has become the major cause of death in both developing and
developed countries [1]. Until now one significant way to induce
the occurrence of cancers is still by mutation or mis-regulation of
cell cycle regulatory genes and proteins to guide an abnormal
control of cell proliferation [2]. Epidermal growth factor receptor
(EGFR) is a kind of tyrosine kinase firstly reported in the literature
[3,4]. It has become one of the targets of anticancer drug research
and development because of it’s widely distribution in the cell and
important role in cell life. EGFRs are distributed in mammalian
epithelial cell membranes and have relationships with cell prolif-
eration, death, and differentiation. They are junctions to deliver
extracellular growth signals intracellular. EGFR family comprise
four members, including: EGFR (HER1/ErbB-1), ErbB-2 (HER2/neu),
ErbB-3 (HER3), and ErbB-4 (HER4) [5]. EGFR tyrosine kinase-
mediated cell growth signaling pathway plays an important role
in the formation and development of many types of solid tumors,
such as nonsmall cell lung cancer [6], head and neck cancer [7] and
glioblastomas [8]. Overexpression of EGFR family receptors have
Besides, bacteria resistant to known therapies are a growing
threat across the globe. An increasing fraction of bacterial isolates
shows reduced susceptibility to our most trusted antibiotics. In
order to prevent this serious medical problem, the discovery of new
types of antibacterial agents or the expansion of bioactivity of the
previous drugs is a very important task [10]. Therefore, in recent
years, the research has been focused on the development of new
antibacterial agents, which may act through structure design and
novel targets, overcoming the problem of acquired resistance. One
of the most attractive biochemical pathways that could be targeted
for new antibacterial agents is the fatty acid biosynthesis (FAS). This
pathway has been demonstrated to be essential for the bacteria cell
survival [11] and differs considerably from human FAS pathway.
While in humans fatty acid synthesis occurs in a homodimeric
multifunctional enzyme [12,13] in bacteria the pathway is
composed of various discrete enzymes and each one can be
considered a putative molecular target. Those features make the
type II FAS pathway a potential target for new antimicrobial agents.
* Corresponding author. Tel.: þ86 25 83592572.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.018

550
C.B. Sangani et al. / European Journal of Medicinal Chemistry 76 (2014) 549e557
A key enzyme in this pathway is the
b
-ketoacyacyl carrier
pyrazole, quinoline and pyridine a modification on the 1 and 4-
position on 4H-quinoline by pyridine and 1H-pyrazole-4-
carbaldehydes respectively may bring significant changes in
pharmacological activities and may provide new classes of thera-
peutically active compounds, with this hope and as a part of our
current studies in developing new therapeutically active agents via
combination of two therapeutically active moieties [16e21], we
report herein the preparation of N-pyridinyl-4-pyrazolyl-4H-
quinoline 7ael derivatives via MCR approach i.e. one pot base-
catalyzed cyclocondensation reaction of 1H-pyrazole-4-
protein synthase III (FabH), which is the responsible enzyme of the
first pathway reaction and play an important regulatory role. FabH
has also been demonstrated to be essential for organism survival
and it is presented in a wide number of important human patho-
gens. Furthermore, some chemical compounds have shown to
inhibit FabH from diverse microl-organisms, including multi-drug
resistant strains [14,15] These facts support the idea that FabH can
be used as an effective molecular target for the development of
new antimicrobial agents. Over the past few years, we have been
principally engrossed in the synthesis of pyrazole incorporating
structures for biological evaluations [16e21] on the premise that
the several 4-functionally substituted N-arylpyrazole derivatives
identified as antimicrobial [22e24], anti-inflammatory (COX-2
inhibitor and ulcerogenic activity) [23], antitubercular [24], anti-
tumor [25,26], anticancer [27e30] as well as inhibitory activity
against FabH [31]. Furthermore, quinoline moiety is found in a
large variety of naturally occurring compounds and also chemi-
cally useful synthons bearing diverse bioactivities [32e42]
including EGFR inhibitory activity [43]. Also, no one can ignore
the role of pyridine as an appreciable pharmacophore for antimi-
crobial and anticancer activity [44,45] including EGFR inhibitory
activity [46]. Moreover, multicomponent reactions were employed
as a powerful tool to synthesize diverse and complex heterocyclic
compounds due to their advantages of the intrinsic atom economy,
simpler procedures, structural diversity, energy savings, and
reduced waste [47e49]. In view of biological significance of
carbaldehydes, malononitrile/ethylcyanoacetate and
naminone.
b-pyridinyle
2. Chemistry
The synthetic approach adopted to obtain the target compounds
is depicted in Scheme 1. The starting material 1-aryl-5-chloro-3-
methyl-1H-pyrazole-4-carbaldehydes 2aec was prepared accord-
ing to literature procedure [16] by VilsmeiereHaack reaction of 1-
aryl-3-methyl-1H-pyrazol-5(4H)-one 1aec. The required 3-(pyr-
idin-3-ylamino)cyclohex-2-enones 5a,
b were synthesized by
nucleophilic addition reaction of 1,3-cyclohexanedione/dimedone
3a, b and 3-aminopyridine 4 at 120 ꢂC for 30 min under solvent free
condition [32]. The title compounds 7ael were prepared via one-
pot three component cyclocondensation reaction between 2aec,
5a, b and malononitrile/ethylcyanoacetate 6a, b in ethanol con-
taining a catalytic amount of piperidine in good to excellent yields.
Scheme 1. Synthetic pathway for the synthesis of title derivatives 7ael.

C.B. Sangani et al. / European Journal of Medicinal Chemistry 76 (2014) 549e557
551
The reaction occurs via an in situ initial formation of the heter-
ylidenenitrile, containing the electron-poor C]C double bond,
from the Knoevenagel condensation between pyrazole-4-
carbaldehyde and malononitrile/ethylcyanoacetate by loss of wa-
ter molecules. Finally, Michael addition of 5a, b to the initially
formed unsaturated nitrile, i.e. nucleophilic attack of enaminone to
the cyano moiety affords cyclized 4H-quinoline derivatives 7ael
(Scheme 2).
the 50 upstream to the EGFR sequences. Sf-9 cells were infected for
3 days for protein expression. Sf-9 cell pellets were solubilized at
0 ꢂC in a buffer at pH 7.4 containing 50 mM HEPES, 10 mM NaCl, 1%
Triton, 10
m
M ammonium molybdate, 100
g/mL aprotinin, 10 g/mL leupeptin, 10
g/mL benzamidine HCl for 20 min followed by 20 min centri-
m
M sodium vanadate,
10
16
m
m
m
mg/mL pepstatin, and
fugation. Crude extract supernatant was passed through an equil-
ibrated Ni-NTA superflow packed column and washed with 10 mM
and then 100 mM imidazole to remove nonspecifically bound
material. Histidinetagged proteins were eluted with 250 and
500 mM imidazole and dialyzed against 50 mM NaCl, 20 mM
3. Biological assay
HEPES, 10% glycerol, and 1 mg/mL each of aprotinin, leupeptin, and
3.1. Experimental method for cell proliferation assay
pepstatin for 2 h. The entire purification procedure was performed
at 4 ꢂC or on ice [50].
The antiproliferative activities of 4H-quinoline derivatives 7ael
were determined using a standard (MTT)-based colorimetric assay
(Sigma). Briefly, cell lines were seeded at a density of 7 ꢃ 103 cells/
well in 96-well microtiter plates (Costar). After 24 h, exponentially
growing cells were exposed to the indicated compounds at final
concentrations ranging from 0.1 to 40 mg/mL. After 48 h, cell sur-
The EGFR kinase assay was set up to assess the level of auto-
phosphorylation based on DELFIA/Time-Resolved Fluorometry.
Compounds 7ael was dissolved in 100% DMSO and diluted to the
appropriate concentrations with 25 mM HEPES at pH 7.4. In each
well, 10
recombinant enzyme (1:80 dilution in 100 mM HEPES) for 10 min
at room temperature. Then, 10
L of 5ꢃ buffer (containing 20 mM
HEPES, 2 mM MnCl2, 100 M Na3VO4, and 1 mM DTT) and 20 L of
mL compound was incubated with 10 mL (5 ng for EGFR)
vival was determined by the addition of an MTT solution (20 mL of
m
5 mg/mL MTT in PBS). After 6 h, 100 mL of 10% SDS in 0.01 N HCl
was added, and the plates were incubated at 37 ꢂC for a further 4 h;
optical absorbance was measured at 570 nm on an LX300 Epson
Diagnostic microplate reader. Survival ratios are expressed in per-
centages with respect to untreated cells. IC50 values were deter-
mined from replicates of 6 wells from at least two independent
experiments.
m
m
0.1 mM ATPe50 mM MgCl2 were added for 1 h. Positive and
negative controls were included in each plate by incubation of
enzyme with or without ATPeMgCl2. At the end of incubation,
liquid was aspirated, and plates were washed three times with
wash buffer. A 75
mL (400 ng) sample of europiumlabeled anti-
phosphotyrosine antibody was added to each well for another 1 h
of incubation. After washing, enhancement solution was added and
the signal was detected by Victor (Wallac Inc.) with excitation at
340 nm and emission at 615 nm. The percentage of autophos-
phorylation inhibition by the compounds was calculated using
3.2. General procedure for preparation, purification of EGFR
inhibitory assay
A 1.6 kb cDNA encoded for the EGFR cytoplasmic domain (EGFR-
CD, amino acids 645-1186) were cloned into baculoviral expression
vectors pBlueBacHis2B and pFASTBacHTc (Huakang Company
China), separately. A sequence that encodes (His)6 was located at
the following equation: 100%
control ꢁ negative control)]. The IC50 was obtained from curves of
ꢁ
[(negative control)/(positive
percentage inhibition with eight concentrations of compound. As
Scheme 2. Plausible mechanistic pathway for title derivatives 7ael.

552
C.B. Sangani et al. / European Journal of Medicinal Chemistry 76 (2014) 549e557
Table 2
the contaminants in the enzyme preparation are fairly low, the
majority of the signal detected by the anti-phosphotyrosine anti-
body is from EGFR.
Antibacterial activity of compounds 7ael.
Compounds
Minimum inhibitory concentrations (mg/mL)
Gram positive
Gram negative
3.3. Experimental method for antibacterial activity
B. subtilis
S. aureus
E. coli
P. aeruginosa
ATCC6633
ATCC6538
ATCC35218
ATCC13525
The antibacterial activity of the synthesized compounds 7ael
was tested against Bacillus subtilis, Staphylococcus aureus, Escher-
ichia coli and Pseudomonas aeruginosa and using MH medium
(MuellereHinton medium: casein hydrolyzate 17.5 g, soluble starch
1.5 g, beef extract 1000 mL). The MICs (minimum inhibitory con-
centrations) of the test compounds were determined by a colori-
metric method using the dye MTT (3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyl tetrazoliumbromide) [51]. A stock solution of the
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
6.25
3.13
12.5
50
25
25
12.5
6.25
6.25
3.13
12.5
>50
0.39
1.56
50
6.25
25
25
25
12.5
>50
50
3.13
12.5
>50
50
25
1.56
50
25
25
6.25
50
50
3.13
25
6.25
6.25
1.56
3.13
50
50
12.5
50
6.25
25
25
6.25
>50
12.5
>50
25
synthesized compound (100 mg/mL) in DMSO was prepared and
graded quantities of the test compounds were incorporated in
specified quantity of sterilized liquid MH medium. A specified
quantity of the medium containing the compound was poured into
microtitration plates. Suspension of the microorganism was pre-
pared to contain approximately 105 cfu/mL and applied to micro-
titration plates with serially diluted compounds in DMSO to be
tested and incubated at 37 ꢂC for 24 h. After the MICs were visually
Kanamycin B
Penicillin G
1.56
6.25
3.13
6.25
kanamycin. The culture was shaken for 4 h at 37 ꢂC, and then
induced with 0.5 mM isopropyl -thiogalactopyranoside (IPTG).
The culture was grown for 4 h, and harvested by centrifugation
(30 min at 15,000 rpm).
b-D
determined on each of the microtitration plates, 50
mL of PBS
(phosphate buffered saline 0.01 mol/L, pH 7.4, Na2HPO4$12H2O
2.9 g, KH2PO4 0.2 g, NaCl 8.0 g, KCl 0.2 g, distilled water 1000 mL)
containing 2 mg of MTT/mL was added to each well. Incubation was
continued at room temperature for 4e5 h. The content of each well
Harvested cells containing His-tagged ACP, ACPS and FabHs
were lysed by sonication in 20 mM Tris, pH 7.6, 5 mM imidazole,
0.5 M NaCl and centrifuged at 20,000 rpm for 30 min. The super-
natant was applied to a Ni-NTA agarose column, washed and eluted
using a 5e500 mM imidazole gradient over 20 column volumes.
Eluted protein was dialyzed against 20 mM Tris, pH 7.6, 1 mM DTT
and 100 mM NaCl. Purified FabHs were concentrated up to 2 mg/mL
and stored at ꢁ80 ꢂC in 20 mM Tris, pH 7.6, 100 mM NaCl,1 mM DTT
and 20% glycerol for enzymatic assays. Purified ACP contains the
apo-form that needs to be converted into the holo-form. The con-
version reaction is catalyzed by ACP synthase (ACPS). In the final
volume of 50 mL, 50 mg ACP, 50 mM Tris, 2 mM DTT, 10 mM MgCl2,
was removed, and 100 mL of isopropanol containing 5% 1 mol/L HCl
was added to extract the dye. After 12 h of incubation at room
temperature, the optical density (OD) was measured with a
microplate reader at 550 nm. The observed MICs are presented in
Table 2.
3.4. E. coli FabH purification and activity assay
Full-length E. coli acyl carrier protein (ACP), acyl carrier protein
synthase (ACPS) and b-ketoacyl-ACP synthase III (FabH) were
individually cloned into pET expression vectors with an N-terminal
His-tag (ACP, ACPS in pET19; FabH in pET28).
All proteins were expressed in E. coli strain BL21(DE3). Trans-
formed cells were grown on LuriaeBertani (LB) agar plates sup-
plemented with kanamycin (30 mg/mL). Sodium dodecyl sulfatee
polyacrylamide gel electrophoresis (SDSePAGE) analysis was used
to screen colonies for overexpression of proteins. One such positive
colony was used to inoculate 10 mL of LB medium with 30 mg/mL of
kanamycin and grown over night at 37 ꢂC, 1 mL of which was used
600 mM CoA and 0.2 m
M ACPS was incubated for 1 h at 37 ꢂC. The pH
of the reaction was then adjusted to approximately 7.0 using 1 M
potassium phosphate. Holo-ACP was purified by fractionation of
the reaction mixture by Source Q-15 ion exchange chromatography
using a 0e500 mM NaCl gradient over 2 column volumes.
In a final 20
0.5 mM DTT, 0.25 mM MgCl2 and 2.5 mM holo-ACP were mixed
with 1 nM FabH, and H2O was added to 15 L. After 1 min incu-
bation, a 2 L mixture of 25 M acetyl-CoA and 0.75
Ci [3H] acetyl-
CoA was added for FabH reaction for 25 min. The reaction was
stopped by adding 20 L of ice-cold 50% TCA, incubating for 5 min
on ice, and centrifuging to pellet the protein. The pellet was washed
mL reaction, 20 mM Na2HPO4/NaH2PO4, pH 7.0,
m
m
m
m
m
to inoculate 100 mL LB medium supplemented with 30 mg/mL of
with 10% ice-cold TCA and resuspended with 5 mL of 0.5 M NaOH.
The incorporation of the 3H signal in the final product was read by
liquid scintillation. When determining the inhibition constant
(IC50), inhibitors were added from a concentrated DMSO stock such
that the final concentration of DMSO did not exceed 2%.
Table 1
Inhibition of EGFR kinase and antiproliferative activity IC50
7ael.
(
m
M) of compounds
Hep G2
Compound
EGFR
A549
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
18.02 ꢀ 0.12
10.12 ꢀ 0.05
4.15 ꢀ 0.30
32.13 ꢀ 0.42
14.70 ꢀ 0.09
24.05 ꢀ 0.10
2.09 ꢀ 0.05
0.91 ꢀ 0.02
6.15 ꢀ 0.07
11.18 ꢀ 0.10
0.51 ꢀ 0.05
1.03 ꢀ 0.02
0.032 ꢀ 0.002
21.05 ꢀ 0.01
15.10 ꢀ 0.06
2.15 ꢀ 0.03
12.18 ꢀ 0.19
10.67 ꢀ 0.04
19.10 ꢀ 0.05
3.18 ꢀ 0.01
0.25 ꢀ 0.13
2.10 ꢀ 0.09
20.08 ꢀ 0.19
0.18 ꢀ 0.09
0.21 ꢀ 0.16
0.13 ꢀ 0.01
10.20 ꢀ 0.02
29.18 ꢀ 0.11
9.13 ꢀ 0.06
15.17 ꢀ 0.21
4.83 ꢀ 0.15
1.02 ꢀ 0.08
7.15 ꢀ 0.03
1.30 ꢀ 0.05
5.84 ꢀ 0.12
24.40 ꢀ 0.04
4.04 ꢀ 0.05
6.21 ꢀ 0.16
0.12
3.5. Docking simulations
Molecular docking of all compounds into the three-dimensional
EGFR complexstructure (PDB code: 1M17) http://www.rcsb.org/
pdb/explore/explore.do?structureId¼1M17 as well as into the
three-dimensional X-ray structure of E. coli FabH (PDB code: 1HNJ)
http://www.rcsb.org/pdb/explore/explore.do?structureId¼1HNJ
were carried out using the AutoDock software package (version 4.0)
as implemented through the graphical user interface AutoDock
Tool Kit (ADT 1.4.6) [52].
Erlotinib

C.B. Sangani et al. / European Journal of Medicinal Chemistry 76 (2014) 549e557
553
Table 3
4. Results and discussion
E. coli FabH inhibitory activity of synthetic compounds 7ael.
Hemolysis LC30a (mg/ml)
4.1. Analytical results
Compounds
E. coli FabH IC50
(m
M)
7a
7b
7c
7d
7e
7f
7g
7h
7i
31.8
3.1
12.7
22.1
10.8
5.6
10.1
8.3
6.8
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
The structural elucidation of the synthesized compounds 7ael
was carried out by FT-IR, 1H NMR, 13C NMR, elemental analysis and
mass spectrometry. In FT-IR spectra, compounds 7ael exhibited an
absorption band around 1635e1645 and 1660e1680 cmꢁ1 for (C]
O) stretching and 765e740 cme1 for CeCl group. The characteristic
absorption bands for all the compounds were observed in the
range of 3445e3350 and 3190e3150 cmꢁ1 corresponding to
asymmetrical and symmetrical stretching of NH2 group and
2205e2185 cmꢁ1 for C^N group. In 1H NMR spectra of com-
pounds 7ael, a sharp singlet peak of methine proton (H4) was
7j
7k
7l
4.5
5.7
9.8
a
Lytic concentration 30%.
appeared around
d 5.12e5.38 ppm. A most characteristic positive
signal observed around
d
34.58e35.28 ppm was indicated
methane carbon (C4) in 13C NMR spectra of compounds 7ael
which confirm the formation of quinoline ring. Also the signal at
Most of the tested compounds showed potent E. coli FabH inhibi-
tory activity. Among them, compound 7b showed the most potent
around
carbonitrile and
with ester group. In 13C NMR spectra, the negative signals around
169.32e169.48 and 194.87e195.62 were arise for the carbonyl
carbon (C]O) of ester and cyclohexenone ring, respectively. The
obtained elemental analysis values are in consonance with theo-
retical data. Mass spectra of title compounds showed expected
molecular ion peak Mþ corresponding with proposed molecular
mass.
d
59.52e59.64 ppm is assigned to carbon attached with
inhibitory with IC50 of 3.1
antibacterial activity of 7b. Among other compounds, compound 7f
(IC50 ¼ 5.6 M), 7j (IC50 ¼ 4.5 M) and 7k (IC50 ¼ 5.7 M) have
found to possess effective E. coli FabH inhibitory activity.
mM. This result supported the potent
d
77.95e78.05 ppm is assigned to carbon attached
m
m
m
d
Structure Activity Relationship (SAR) was carried out from
antibacterial and anticancer activities. According to the activity
data, it has been observed that the change in R1, R2 and R3
substitutions may lead to change in the activity against employed
strains and cancer cells. In the case of both activities, R1 ¼ CH3
and Cl may play an important role for the better activity, while
R1 ¼ H may decrease the activity. Besides, in the case of anti-
bacterial activity, R2 ¼ H is more effective than R2 ¼ CH3 against
Gram negative bacteria, while R2 ¼ H and R3 ¼ COOEt against
Gram positive bacteria may play an important role for the better
activity. Moreover, reviewing and comparing the activity data, it
is worthy to mention that the antibacterial and anticancer ac-
tivities of the target compounds depend not only on the bicyclic
heteroaromatic pharmacophore, but also on the nature of the
substituents.
Generally, in the molecular modeling, the most stable pro-
teineligand complex structure can be obtained by binding of
compound into the active site of protein with lowest binding
energy. Due this stability, particular compounds lead to the
efficient activity. In the case of antibacterial activity, our
modeling results revealed that the compound 7b (R1 ¼ CH3,
R2 ¼ H, R3 ¼ CN) showed lowest binding energy by interaction
with FabH leads to stable protein-ligand complex. The lowest
binding energy may lead to the improvement in the activity,
while increasing of the binding energy and poor activity was
observed in compound 7d (R1 ¼ H, R2 ¼ CH3, R3 ¼ CN).
In the case of anticancer activity, eCH3 at R1 and R2 position, and
eCOOEt at R3 position (7k) may responsible for most effective
activity by binding into the active pocket of EGFR with lowest
binding energy. This also creates the most stable proteineligand
complex structure than other compounds of the series. In brief, it
has been observed that compounds having R1 ¼ CH3 were found
to have lower binding energies and efficient antibacterial as well
as anticancer activity.
4.2. Biological evaluation
4.2.1. Antiproliferation and EGFR inhibitory activity
All compounds were tested against EGFR kinase as well as
against cancer cell A549 (adenocarcinomic human alveolar basal
epithelial cell line) and Hep G2 (liver cancer cell line). Upon
investigation of antiproliferative activity of compounds 7ael, it has
been observed that compounds 7h (IC50 ¼ 0.25 ꢀ 0.13
(IC50 ¼ 0.18 ꢀ 0.09 M) and 7l (IC50 ¼ 0.21 ꢀ 0.16 M) against A549
as well as compounds 7f (IC50 ¼ 1.02 ꢀ 0.08 M) and 7h
M) against Hep G2 showed most effective
mM), 7k
m
m
m
(IC50 ¼ 1.30 ꢀ 0.05
m
activity as compared to other compounds.
As shown in Table 1, compounds 7h (IC50 ¼ 0.91 ꢀ 0.02
mM), 7k
mM) displayed the
(IC50 ¼ 0.51 ꢀ 0.05
m
M) and 7l (IC50 ¼ 1.03 ꢀ 0.02
most potent inhibitory activity against EGFR as compared to other
compounds and less comparable to the positive control erlotinib
(IC50 ¼ 0.032 ꢀ 0.02
mM).
4.2.2. Antibacterial and E. coli FabH inhibitory activity
Upon investigation of antibacterial activity (Table 2), it has been
observed that majority of the compounds have shown effective
activity against used strains. Against Gram negative bacteria E. coli,
compound 7b (MIC ¼ 1.56
which is more effective than penicillin G (MIC ¼ 3.13
comparable to kanamycin B (MIC ¼ 1.56 g/mL), while compound
g/mL) showed comparable activity to penicillin G
g/mL) and 7h
mg/mL) showed most effective activity
mg/mL) and
m
7i (MIC ¼ 3.13
m
(MIC ¼ 3.13
(MIC ¼ 6.25
(MIC ¼ 6.25
m
g/mL). Compounds 7e (MIC ¼ 6.25
m
m
g/mL) showed comparable activity to penicillin G
mg/mL) against Gram negative bacteria P. aeruginosa.
Against Gram positive bacterial S. aureus, compounds 7i
(MIC ¼ 3.13
(MIC ¼ 6.25
penicillin G (MIC ¼ 6.25
g/mL) showed effective activity as compared to
other compounds against B. subtilis.
The E. coli FabH inhibitory potency of the synthetic 7ael de-
rivatives was examined and the results are summarized in Table 3.
m
g/mL) showed more effective activity and 7b
g/mL) showed comparable activity as compared to
g/mL). Compounds 7b (MIC ¼ 3.13 g/mL)
4.2.3. Molecular docking study with EGFR
m
To gain better understanding on the potency of all compounds
and guide further SAR studies, we proceeded to examine the
interaction of those with EGFR (PDB code: 1M17) by molecular
docking, which was performed by simulation of compounds into
the ATP binding site in EGFR. The binding energy of all the com-
pounds is mentioned in Table 4. Of the compounds studied,
m
m
and 7j (MIC ¼ 3.13
m

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C.B. Sangani et al. / European Journal of Medicinal Chemistry 76 (2014) 549e557
Table 4
Binding energy of compounds 7ael with EGFR.
compound 7k was nicely bound into the active site of EGFR with
minimum binding energy
DGb ¼ ꢁ54.6913 kcal/mol. The binding
model of compound 7k and EGFR was depicted in Fig. 1a and b.
The amino acid residues which had interaction with EGFR were
labeled. In the binding mode, compound 7k was nicely bound to
the ATP binding site of EGFR through hydrophobic interaction
and the binding was stabilized by three hydrogen bonds and one
Compounds
Binding energy DGb
7a
7b
7c
7d
7e
7f
7g
7h
7i
ꢁ47.5404
ꢁ44.2319
ꢁ48.4589
ꢁ38.3829
ꢁ47.2833
ꢁ43.7073
ꢁ49.4853
ꢁ52.0321
ꢁ48.6366
ꢁ47.0647
L54.6913
ꢁ51.9798
p
ecation interaction. Among them one hydrogen bond forms H
ꢀ
atom of eNH2 group and ARG817 (distance: 2.45462 A, DHA
angle: 172.9ꢂ, HAY angle: 147.4ꢂ), second one between O atom of
ꢀ
carbonyl group and THR830 (distance: 2.23299 A, DHA angle:
7j
7k
7l
138.3ꢂ, HAY angle: 139.3ꢂ), and third one between H atom ofꢂeNH2
ꢀ
group and ASP831 (distance: 2.32111 A, DHA angle: 103.1 , HAY
angle: 163.8ꢂ). One
pecation bond forms between N atom of
Bold number indicate the lower binding energy.
ꢀ
pyridine ring and PHE699 with distance 5.87883 A. From this
binding model, it could be concluded that three hydrogen bond
and
pecation interaction are responsible for the effective EGFR
inhibitory of compound 7k.
4.2.4. Molecular docking study with FabH
Similarly, to gain better understanding on the potency of all
compounds and guide further SAR studies, molecular docking of
compounds and E. coli FabH was performed on the binding model
based on the E. coli FabHeCoA complex structure (PDB code:
1HNJ). The FabH active site generally contains a catalytic triad
tunnel consisting of Cys-His-Asn, which is conserved in various
bacteria. This catalytic triad plays an important role in the regu-
lation of chain elongation and substrate binding. Since the alkyl
chain of CoA is broken by Cys of the catalytic triad of FabH, in-
teractions between Cys and substrate appear to play an important
role in substrate binding. Of the compounds studied, compound
7b was nicely bound to active site of the FabH with hydrogen
bonds and
pesigma interactions with minimum binding energy
D
Gb ¼ ꢁ45.9125 kcal/mol. The binding energy of all the com-
pounds is mentioned in Table 5. The binding model of compound
7b and FabH was depicted in Fig. 2a and b. Among them hydrogen
bonds formed with between oxygen atom of cyclohexanꢂedione
ꢀ
ring and ASN210 (distance: 2.37469 A, DHA angle: 161.5 , HAY
angle: 104.1ꢂ) and
pesigma interaction formed between pyrazole
ꢀ
ring and GLY209 with distance: 2.63465 A. From this binding
model, it could be concluded that hydrogen and esigma
p
interaction are responsible for the effective FabH inhibitory of
compound 7b.
5. Conclusion
In conclusion, the aim of the present investigation was to
design a new series of 4H-quinoline derivatives by introduction
Table 5
Binding energy of compounds 7ael with FabH.
Compounds
Binding energy DGb
7a
7b
7c
7d
7e
7f
7g
7h
7i
ꢁ38.9723
L45.9125
ꢁ37.9588
ꢁ35.1931
ꢁ38.3578
ꢁ39.7733
ꢁ37.8427
ꢁ38.6283
ꢁ39.8193
ꢁ40.1617
ꢁ39.7644
ꢁ39.7296
7j
7k
7l
Fig. 1. (a) 3D binding model of compound 7k into the active pocket of EGFR. (b) 2D
binding model of compound 7k into the active pocket of EGFR.
Bold number indicate the lower binding energy.

C.B. Sangani et al. / European Journal of Medicinal Chemistry 76 (2014) 549e557
555
6. Experimental
6.1. Chemistry
All chemicals and reagents used in the current study were of
analytical grade. Melting points (uncorrected) were determined on
an XT4 MP apparatus (Taike Corp., Beijing, China). All the 1H NMR
and 13C NMR spectra were recorded on a Bruker DPX300 model
Spectrometer in DMSO-d6 and chemical shifts were reported in
ppm (d). FTIR spectra (KBr) were run on a Nexus 870 FT-IR spec-
trophotometer. ESI-MS spectra were recorded on a Mariner System
5304 mass spectrometer. Elemental analyses were performed on a
CHN-O-Rapid instrument. TLC was performed on the glass backed
silica gel sheets (Silica Gel 60 GF254) and visualized in UV light
(254 nm).
6.1.1. General procedure for the synthesis of compounds 7ael
A 100 mL round bottomed flask, fitted with a reflux condenser,
was charged with a mixture of 5-chloro-3-methyl-1-phenyl-1H-
pyrazole-4-carbaldehyde 2aec (1 mmol),
b-enaminone 5a, b
(1 mmol), malononitrile/ethylcyanoacetate 6a, b (1 mmol) and a
catalytic amount of piperidine (0.2 mmol) in ethanol (10 mL). The
reaction mixture was heated under reflux for 2.5e3 h and the
progress of the reaction was monitored by TLC. After the completion
of reaction (as evidenced by TLC), the reaction mixture was cooled to
room temperature and stirred magnetically for further 20 min, the
solid mass separated was collected by filtration, washed well with
ethanol (15 mL) and purified by leaching in equal volume ratio of
chloroform and methanol (15 mL) to obtain pure solid sample 7ael.
6.1.1.1. 2-Amino-4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-5-
oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-hexahydroquinoline-3-carbonitrile
(7a). Yield: 89%; mp 250e252 ꢂC; IR (KBr, nmax, cmꢁ1): 3435 & 3155
(asym. & sym. stretching of NH2), 2195 (C^N stretching), 1670 (C]
O stretching), 750 (CeCl stretching); 1H NMR (DMSO-d6)
d ppm:
1.66e2.24 (m, 6H, 3ꢃ CH2), 2.41 (s, 3H, CH3), 5.17 (s, 1H, quinoline
H4), 5.60 (s, 2H, NH2), 7.62e8.68 (m, 9H, AreH); 13C NMR (DMSO-
d6) d ppm: 12.98 (AreCH3), 21.23, 28.73 (2C, CH2), 34.60 (C4), 36.34
(CH2eCO), 59.58 (CeCN), 112.19, 119.22, 121.80, 125.53, 127.44,
130.27, 132.12, 136.19, 138.23, 142.18, 144.17, 148.45, 150.20, 151.47,
153.59, 159.41 (16C, AreC), 195.54 (C]O); Anal. Calcd. for
C
25H21ClN6O (456.93 g/mol): C, 65.71; H, 4.63; N, 18.39 (%); Found:
C, 65.48; H, 4.87; N, 18.17 (%). MS (m/z): 456.1 (Mþ), 458.1 [M þ 2].
6.1.1.2. 2-Amino-4-(5-chloro-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-
5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-hexahydroquinoline-3-carbonitrile
(7b). Yield: 84%; mp 242e244 ꢂC; IR (KBr, nmax, cmꢁ1): 3430 & 3160
(asym. & sym. stretching of NH2), 2185 (C^N stretching), 1675 (C]
Fig. 2. (a) 3D binding model of compound 7b into the active site of FabH. (b) 2D
binding model of compound 7b into the active site of FabH.
O stretching), 760 (CeCl stretching); 1H NMR (DMSO-d6)
d ppm:
1.72e2.32 (m, 6H, 3ꢃ CH2), 2.36, 2.44 (s, 6H, 2ꢃ CH3), 5.15 (s, 1H,
of substituted 1H-pyrazole at the C-4 position and pyridine ring
at N-1 position for aiming their potent antimicrobial and anti-
cancer activities. Some magnificent biological results have been
obtained with the pyrazoleequinoloneepyridine hybridized
scaffold and it has been concluded that majority of the com-
pounds showed effective antibacterial as well as anticancer
activities. In the case of inhibitory activity, compounds 7h, 7k
and 7l against EGFR as well as compounds 7b, 7f, 7j and 7k
against FabH have found to be most effective members of the
series. According to this, it is worthy to mention that the pyr-
quinoline H4), 5.59 (s, 2H, NH2), 7.55e8.59 (m, 8H, AreH); 13C NMR
(DMSO-d6)
d ppm: 12.90, 19.40 (AreCH3), 21.28, 28.66 (2C, CH2),
34.66 (C4), 36.30 (CH2eCO), 59.64 (CeCN), 111.90, 120.17, 122.01,
125.45, 127.68, 130.88, 132.21, 136.11, 138.47, 142.36, 144.45, 148.98,
150.17, 151.40, 153.47, 159.69 (16C, AreC), 195.50 (C]O); Anal.
Calcd. for C26H23ClN6O (470.95 g/mol): C, 66.31; H, 4.92; N, 17.84
(%); Found: C, 66.52; H, 5.13; N, 17.63 (%). MS (m/z): 470.2 (Mþ),
472.2 [M þ 2].
6.1.1.3. 2-Amino-4-(5-chloro-1-(4-chlorophenyl)-3-methyl-1H-pyr-
azol-4-yl)-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-hexahydroquinoline-3-
carbonitrile (7c). Yield: 75%; mp 218e220 ꢂC; IR (KBr, nmax, cmꢁ1):
3440 & 3150 (asym. & sym. stretching of NH2), 2200 (C^N
stretching), 1660 (C]O stretching), 745 (CeCl stretching); 1H NMR
azoleequinolineepyridine hybrids as
a useful template for
further development of more antimicrobial and anticancer
compounds that deserve further investigation and derivatization
in order to discover the scope and limitation of its biological
activities.
(DMSO-d6)
d
ppm: 1.69e2.21 (m, 6H, 3ꢃ CH2), 2.50 (s, 3H, CH3), 5.19

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C.B. Sangani et al. / European Journal of Medicinal Chemistry 76 (2014) 549e557
(s, 1H, quinoline H4), 5.67 (s, 2H, NH2), 7.62e8.73 (m, 8H, AreH);
13C NMR (DMSO-d6)
ppm: 13.09 (AreCH3), 21.20, 28.75 (2C, CH2),
36.48 (CH2eCO), 58.99 (OCH2), 77.98 (CeCOOEt), 112.24, 120.29,
125.31, 126.61, 127.12, 130.80, 139.79, 142.58, 145.76, 147.16, 151.52,
152.33, 152.47, 155.25, 159.69 (15C, AreC), 169.40 (COO), 195.49
(C]O); Anal. Calcd. for C27H26ClN5O3 (503.98 g/mol): C, 64.35; H,
5.20; N, 13.90 (%); Found: C, 64.62; H, 5.47; N, 14.15 (%). MS (m/z):
503.2 (Mþ), 505.2 [M þ 2].
d
34.58 (C4), 36.39 (CH2eCO), 59.55 (CeCN), 112.32, 119.87, 121.91,
125.43, 127.65, 130.42, 132.17, 136.19, 138.47, 142.36, 144.58, 148.32,
150.21, 151.45, 154.54, 159.53 (16C, AreC), 195.47 (C]O); Anal.
Calcd. for C25H20Cl2N6O (491.37 g/mol): C, 61.11; H, 4.10; N, 17.10
(%); Found: C, 61.39; H, 4.32; N, 17.32 (%). MS (m/z): 490.1 (Mþ),
492.1 [M þ 2].
6.1.1.8. Ethyl 2-amino-4-(5-chloro-3-methyl-1-p-tolyl-1H-pyrazol-4-
yl)-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (7h). Yield: 83%; mp 235e237 ꢂC; IR (KBr, nmax, cmꢁ1):
3360 & 3175 (asym. & sym. stretching of NH2), 1665 (C]O
stretching), 1645 (C]O stretching), 750 (CeCl stretching); 1H NMR
6.1.1.4. 2-Amino-4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-yl)-
7,7-dimethyl-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-hexahydroquinoline-
3-carbonitrile (7d). Yield: 87%; mp 267e269 ꢂC; IR (KBr, nmax
,
cmꢁ1): 3435 & 3155 (asym. & sym. stretching of NH2), 2205 (C^N
(DMSO-d6)
d
ppm: 0.95 (t, 3H, CH3), 1.76e2.30 (m, 6H, 3ꢃ CH2),
stretching), 1680 (C]O stretching), 740 (CeCl stretching); 1H NMR
2.37, 2.43 (s, 6H, 2ꢃ CH3), 3.96 (q, 2H, OCH2), 5.29 (s, 1H, quinoline
(DMSO-d6)
CH2), 2.46 (s, 3H, CH3), 5.12 (s, 1H, quinoline H4), 5.63 (s, 2H, NH2),
7.51e8.55 (m, 9H, AreH); 13C NMR (DMSO-d6)
ppm: 13.14 (Are
d
ppm: 0.84, 1.00 (s, 6H, 2ꢃ CH3), 1.85e2.72 (m, 4H, 2ꢃ
H4), 7.08 (s, 2H, NH2), 7.57e8.69 (m, 8H, AreH); 13C NMR (DMSO-
d6) d ppm: 13.16, 19.47 (AreCH3), 14.70 (CH3), 21.18, 28.64 (2C, CH2),
d
35.19 (C4), 36.48 (CH2eCO), 59.00 (OCH2), 78.02 (CeCOOEt), 112.45,
119.39, 125.20, 126.55, 126.98, 130.35, 139.27, 142.19, 145.95, 147.18,
151.70, 152.17, 152.30, 155.11, 160.05 (15C, AreC), 169.48 (COO),
195.62 (C]O); Anal. Calcd. for C28H28ClN5O3 (518.01 g/mol): C,
64.92; H, 5.45; N,13.52 (%); Found: C, 65.19; H, 5.29; N,13.67 (%). MS
(m/z): 517.2 (Mþ), 519.2 [M þ 2].
CH3), 26.99, 28.25 (2C, CH3), 32.05 (C(CH3)2), 34.65 (C4), 38.03
(CH2), 49.88 (CH2eCO), 59.52 (CeCN), 112.22, 119.34, 121.67, 125.73,
127.58, 130.45, 132.34, 136.37, 138.61, 142.45, 144.71, 148.80, 150.20,
151.47, 153.59, 159.60 (16C, AreC), 195.07 (C]O); Anal. Calcd. for
C
27H25ClN6O (484.98 g/mol): C, 66.87; H, 5.20; N, 17.33 (%); Found:
C, 66.58; H, 4.94; N, 17.57 (%). MS (m/z): 484.1 (Mþ), 486.1 [M þ 2].
6.1.1.9. Ethyl 2-amino-4-(5-chloro-1-(4-chlorophenyl)-3-methyl-1H-
pyrazol-4-yl)-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-hexahydroquinoline-
3-carboxylate (7i). Yield: 71%; mp 272e274 ꢂC; IR (KBr, nmax, cmꢁ1):
3370 & 3190 (asym. & sym. stretching of NH2), 1665 (C]O
stretching), 1645 (C]O stretching), 755 (CeCl stretching); 1H NMR
6.1.1.5. 2-Amino-4-(5-chloro-3-methyl-1-p-tolyl-1H-pyrazol-4-yl)-
7,7-dimethyl-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-hexahydroquinoline-
3-carbonitrile (7e). Yield: 79%; mp 229e231 ꢂC; IR (KBr, nmax
,
cmꢁ1): 3445 & 3160 (asym. & sym. stretching of NH2), 2190 (C^N
stretching), 1675 (C]O stretching), 755 (CeCl stretching); 1H NMR
(DMSO-d6)
(s, 3H, CH3), 3.94 (q, 2H, OCH2), 5.38 (s, 1H, quinoline H4), 7.11 (s,
2H, NH2), 7.61e8.76 (m, 8H, AreH); 13C NMR (DMSO-d6)
ppm:
d
ppm: 0.98 (t, 3H, CH3), 1.65e2.18 (m, 6H, 3ꢃ CH2), 2.48
(DMSO-d6)
CH2), 2.35, 2.39 (s, 6H, 2ꢃ CH3), 5.14 (s, 1H, quinoline H4), 5.64 (s,
2H, NH2), 7.62e8.68 (m, 8H, AreH); 13C NMR (DMSO-d6)
ppm:
d
ppm: 0.82, 0.98 (s, 6H, 2ꢃ CH3), 1.86e2.77 (m, 4H, 2ꢃ
d
d
12.93 (AreCH3), 14.74 (CH3), 21.26, 28.72 (2C, CH2), 35.22 (C4),
36.44 (CH2eCO), 58.95 (OCH2), 78.05 (CeCOOEt), 112.48, 119.65,
125.63, 126.94, 127.20, 130.67, 139.82, 143.03, 146.13, 147.23, 151.80,
152.18, 152.40, 156.11, 159.11 (15C, AreC), 169.35 (COO), 195.57 (C]
O); Anal. Calcd. for C27H25Cl2N5O3 (538.43 g/mol): C, 60.23; H, 4.68;
N, 13.01(%); Found: C, 60.01; H, 4.90; N, 13.26 (%). MS (m/z): 537.1
(Mþ), 539.1 [M þ 2].
13.20,19.60 (AreCH3), 27.05, 28.28 (2C, CH3), 32.00 (C(CH3)2), 34.63
(C4), 37.95 (CH2), 49.85 (CH2eCO), 59.60 (CeCN), 111.89, 120.02,
122.15, 125.45, 127.64, 130.45, 132.17, 136.30, 138.64, 142.43, 144.70,
148.24, 119.15, 151.40, 154.15, 160.19 (16C, AreC), 194.99 (C]O);
Anal. Calcd. for C28H27ClN6O (499.01 g/mol): C, 67.39; H, 5.45; N,
16.84 (%); Found: C, 67.54; H, 5.19; N, 17.05 (%). MS (m/z): 498.2
(Mþ), 500.2 [M þ 2].
6.1.1.10. Ethyl 2-amino-4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-
4-yl)-7,7-dimethyl-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (7j). Yield: 82%; mp 223e
225 ꢂC; IR (KBr, nmax, cmꢁ1): 3355 & 3185 (asym. & sym. stretching
of NH2), 1670 (C]O stretching), 1640 (C]O stretching), 760 (CeCl
6.1.1.6. 2-Amino-4-(5-chloro-1-(4-chlorophenyl)-3-methyl-1H-pyr-
azol-4-yl)-7,7-dimethyl-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-
hexahydroquinoline-3-carbonitrile (7f). Yield: 76%; mp 257e259 ꢂC;
IR (KBr, nmax, cmꢁ1): 3440 & 3165 (asym. & sym. stretching of NH2),
2195 (C^N stretching), 1665 (C]O stretching), 765 (CeCl stretch-
stretching); 1H NMR (DMSO-d6)
d ppm: 0.83 (s, 3H, CH3), 0.96 (t,
ing); 1H NMR (DMSO-d6)
d
ppm: 0.85, 1.01 (s, 6H, 2ꢃ CH3), 1.88e
3H, CH3), 0.99 (s, 3H, CH3), 1.84e2.74 (m, 4H, 2ꢃ CH2), 2.38 (s, 3H,
2.75 (m, 4H, 2ꢃ CH2), 2.40 (s, 3H, CH3), 5.16 (s, 1H, quinoline H4),
CH3), 3.95 (q, 2H, OCH2), 5.25 (s,1H, quinoline H4), 7.09 (s, 2H, NH2),
5.69 (s, 2H, NH2), 7.60e8.71 (m, 8H, AreH); 13C NMR (DMSO-d6)
7.53e8.68 (m, 9H, AreH); 13C NMR (DMSO-d6)
d ppm: 13.01 (Are
d
ppm: 13.05 (AreCH3), 27.01, 28.20 (2C, CH3), 32.03 (C(CH3)2),
CH3), 14.79 (CH3), 26.95, 28.19 (2C, CH3), 32.02 (C(CH3)2), 34.23
(C4), 37.99 (CH2), 49.90 (CH2eCO), 59.03 (OCH2), 78.00 (CeCOOEt),
111.96, 119.28, 124.94, 126.73, 127.26, 131.12, 140.13, 143.17, 145.54,
147.09, 152.07, 152.38, 152.61, 155.96, 160.16 (15C, AreC), 169.32
(COO), 195.15 (C]O); Anal. Calcd. for C29H30ClN5O3 (532.03 g/mol):
C, 65.47; H, 5.68; N, 13.16 (%); Found: C, 65.78; H, 5.96; N, 12.86 (%).
MS (m/z): 531.2 (Mþ), 533.2 [M þ 2].
34.61 (C4), 38.05 (CH2), 50.00 (CH2eCO), 59.62 (CeCN), 112.07,
119.84, 120.98, 125.28, 127.23, 130.40, 132.17, 136.30, 138.68, 142.46,
144.78, 148.80, 150.22, 151.40, 154.64, 160.10 (16C, AreC), 195.87
(C]O); Anal. Calcd. for C27H24Cl2N6O (519.43 g/mol): C, 62.43; H,
4.66; N, 16.18 (%); Found: C, 62.72; H, 4.88; N, 15.91 (%). MS (m/z):
518.1 (Mþ), 520.1 [M þ 2].
6.1.1.7. Ethyl 2-amino-4-(5-chloro-3-methyl-1-phenyl-1H-pyrazol-4-
yl)-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (7g). Yield: 88%; mp 276e278 ꢂC; IR (KBr, nmax, cmꢁ1):
3365 & 3180 (asym. & sym. stretching of NH2), 1660 (C]O
stretching), 1635 (C]O stretching), 760 (CeCl stretching); 1H NMR
6.1.1.11. Ethyl 2-amino-4-(5-chloro-3-methyl-1-p-tolyl-1H-pyrazol-
4-yl)-7,7-dimethyl-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (7k). Yield: 79%; mp 280e
282 ꢂC; IR (KBr, nmax, cmꢁ1): 3350 & 3180 (asym. & sym. stretching
of NH2), 1675 (C]O stretching), 1635 (C]O stretching), 770 (CeCl
(DMSO-d6)
(s, 3H, CH3), 3.93 (q, 2H, OCH2), 5.30 (s, 1H, quinoline H4), 7.10 (s,
2H, NH2), 7.54e8.70 (m, 9H, AreH); 13C NMR (DMSO-d6)
ppm:
13.07 (AreCH3), 14.73 (CH3), 21.22, 28.70 (2C, CH2), 36.42 (C4),
d
ppm: 0.97 (t, 3H, CH3),1.68e2.26 (m, 6H, 3ꢃ CH2), 2.45
stretching); 1H NMR (DMSO-d6)
d ppm: 0.86 (s, 3H, CH3), 0.97 (t,
3H, CH3), 0.98 (s, 3H, CH3), 1.88e2.73 (m, 4H, 2ꢃ CH2), 2.36, 2.47 (s,
d
6H, 2ꢃ CH3), 3.92 (q, 2H, OCH2), 5.32 (s, 1H, quinoline H4), 7.13 (s,
2H, NH2), 7.54e8.69 (m, 8H, AreH); 13C NMR (DMSO-d6)
d ppm:

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557
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13.04, 19.52 (AreCH3), 14.77 (CH3), 27.03, 28.17 (2C, CH3), 32.09
(C(CH3)2), 34.28 (C4), 38.10 (CH2), 50.02 (CH2eCO), 58.97 (OCH2),
77.95 (CeCOOEt), 112.16, 119.58, 125.47, 126.78, 127.23, 130.45,
139.65, 143.14, 145.93, 148.20, 151.84, 151.98, 152.40, 155.17, 159.79
(15C, AreC), 169.44 (COO), 195.02 (C]O); Anal. Calcd. for
C
30H32ClN5O3 (546.06 g/mol): C, 65.99; H, 5.91; N, 12.83 (%);
Found: C, 65.65; H, 6.14; N, 13.15 (%). MS (m/z): 545.2 (Mþ), 547.2
[M þ 2].
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6.1.1.12. Ethyl
2-amino-4-(5-chloro-1-(4-chlorophenyl)-3-methyl-
1H-pyrazol-4-yl)-7,7-dimethyl-5-oxo-1-(pyridin-3-yl)-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (7l). Yield: 73%; mp 262e264 ꢂC;
IR (KBr, nmax, cmꢁ1): 3365 & 3185 (asym. & sym. stretching of NH2),
1665 (C]O stretching), 1640 (C]O stretching), 765 (CeCl
stretching); 1H NMR (DMSO-d6)
d ppm: 0.84 (s, 3H, CH3), 0.98 (t,
3H, CH3), 0.99 (s, 3H, CH3), 1.87e2.74 (m, 4H, 2ꢃ CH2), 2.43 (s, 3H,
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2427e2432.
CH3), 3.94 (q, 2H, OCH2), 5.28 (s, 1H, quinoline H4), 7.12 (s, 2H, NH2),
7.63e8.74 (m, 8H, AreH); 13C NMR (DMSO-d6)
d ppm: 13.10 (Are
CH3), 14.72 (CH3), 26.97, 28.23 (2C, CH3), 32.01 (C(CH3)2), 34.26
(C4), 37.93 (CH2), 49.96 (CH2eCO), 58.93 (OCH2), 77.97 (CeCOOEt),
112.25, 120.20, 125.43, 126.72, 127.21, 131.04, 139.84, 142.60, 146.11,
147.22, 151.82, 152.19, 153.16, 156.01, 160.14 (15C, AreC), 169.38
(COO), 195.10 (C]O); Anal. Calcd. for C29H29Cl2N5O3 (566.48 g/
mol): C, 61.49; H, 5.16; N,12.36 (%); Found: C, 61.74; H, 5.41; N,12.73
(%). MS (m/z): 565.2 (Mþ), 567.2 [M þ 2].
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Acknowledgment
This work was supported by the National Forestry Commonweal
Project (200904001), by the project (No. BY2012136) from the
Science & Technology Agency of Jiangsu Province and by the pro-
jects (Nos. CXY1213 & CXY1222) from the Science & Technology
Bureau of Lianyuangang City of Jiangsu Province.
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Appendix A. Supplementary data
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Supplementary data related to this article can be found at http://
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