Synthesis, molecular docking and anti-inflammatory screening of novel quinoline incorporated pyrazole derivatives using the Pfitzinger reaction II

Article abstract of DOI:10.1016/j.bioorg.2014.12.003

In continuation of our study of novel quinolines with anti-inflammatory activity using the Pfitzinger reaction, several new quinoline derivatives were synthesized and tested for their anti-inflammatory and ulcerogenic effect. A docking study on the COX-2

Full text of DOI:10.1016/j.bioorg.2014.12.003

Accepted Manuscript
Synthesis, molecular docking and anti-inflammatory screening of novel quino-
line incorporated pyrazole derivatives using the Pfitzinger reaction II
Said A.H. El-Feky, Zakaria K. Abd El-Samii, Nermine A. Osman, Jasmine
Lashine, Mohamed A. Kamel, Hamdy Kh. Thabet
PII:
S0045-2068(14)00148-5
DOI:
http://dx.doi.org/10.1016/j.bioorg.2014.12.003
YBIOO 1780
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
15 September 2014
Please cite this article as: S.A.H. El-Feky, Z.K. Abd El-Samii, N.A. Osman, J. Lashine, M.A. Kamel, H.K. Thabet,
Synthesis, molecular docking and anti-inflammatory screening of novel quinoline incorporated pyrazole derivatives
using the Pfitzinger reaction II, Bioorganic Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bioorg.2014.12.003
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Synthesis, molecular docking and anti-inflammatory screening
of novel quinoline incorporated pyrazole derivatives using the
Pfitzinger reaction II
Said A. H. El-Fekya,b*, Zakaria K. Abd El-Samiib, Nermine A. Osmanb, Jasmine
Lashineb, Mohamed A. Kamelc and Hamdy Kh. Thabetd,e
aDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Northern Border
University, PO Box 840, Rafha 91911, Saudi Arabia
bDepartment of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig
University, Zagazig, Egypt
cDepartment of Pharmacology, Faculty of Veterinary Medicine, Zagazig University,
Zagazig, Egypt
dDepartment of Chemistry, Faculty of Arts and Science, Northern Border University,
PO Box 840, Rafha 91911, Saudi Arabia
eDepartment of Chemistry, Faculty of Science, Al-Azhar University, Nasr City, Cairo
11284, Egypt
The corresponding author: drsaid.elfeky@yahoo.com
ABSTRACT
In continuation of our study of novel quinolines with anti-inflammatory activity using
the Pfitzinger reaction, several new quinoline derivatives were synthesized and tested
for their anti-inflammatory and ulcerogenic effect. A docking study on the COX-2
binding pocket was carried out for the target compounds to rationalize the possible
selectivity of them against COX-2 enzyme. The most active compounds (5a, 8a and
11a) were found to be superior to celecoxib. Compound 11a demonstrated the highest
anti-inflammatory activity as well as the best binding profiles into the COX-2 binding
site. Moreover, compounds 9c, 9e, 10a and 11a were devoid of ulcerogenic activity.
Keywords: Quinoline derivatives; Pyrazoles; Anti-inflammatory; Ulcerogenic
activity.

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1. Introduction
In our ongoing medicinal chemistry research program [1] we have reported the
synthesis and anti-inflammatory activity of a number of fluorinated quinoline
derivatives. Since, some fluorinated quinolines incorporated benzimidazole
derivatives, such as compound I exhibited more potent anti-inflammatory activity
than celecoxib, it promoted us soon to modify the established structures on the basis
of isosteric replacement [2]. The bioisostere concept is an oversimplification of the
role of scaffolds for activity, unless it plays a pivotal role for function or interaction
such as for β-lactam in penicillins [2]. Moreover, Barreiro et al [3] revealed that
bioisosterism is a useful strategy for the lead optimization process and molecular
modification for rational drug design. On the basis of these results, we surmised that
replacement of fluorine in the 6-position by methoxy group and also replacement of
benzimidazole ring system by pyrazole ring system could give compounds II with
improved anti-inflammatory activity by illustrating the old and well-known isosterism
pyrazole/imidazole having similar geometry and electron features [4] (Figure 1).
Figure 1
Compounds containing N-Aryl-pyrazolone-4-imino and 2,4-imidazolinedione
framework displayed a multitude of biological activities, including antitumor,
antibacterial, antihypertensive, antiplatelet and anti-inflammatory activities [5-8].
Pyrazolines have also been reported to possess antiparasitic [9], antitubercular [10],
insecticidal agents [11], antipyretic [12], anti-inflammatory [13], antidiabetic [14],
tranquillizing [15], muscle relaxant [16], psychoanaleptic [17], anticonvulsant [18],
antihypertensive [19], antidepressant [20] and anticancer [21] activities. They have

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also been found to be Nitric oxide synthase (NOS) inhibitors and had shown
Cannabinoid CB1 receptor antagonist activity [22].
The present work is an extension of our ongoing efforts towards the
development and identification of new molecules; by the bioisostere concept [1,
23,24], we have designed some trisubstituted quinoline derivatives linked to pyrazoles
(5-11). Also, in an attempt to rationalize the possible selective activity of the target
compounds against the COX-2 enzyme, a molecular modeling study was conducted to
check the ability of these new scaffolds to bind to the active site of COX-2 isozyme.
2. Results and discussion
2.1. Chemistry
The reaction sequence used to synthesize the target compounds is utilized in
schemes 1-4. The versatile Pfitzinger reaction was employed to synthesize pertinent
6-methoxy-2-(substitutedphenyl)quinoline-4-carboxylic acids 1a-d in a very good
yield by reacting 5-methoxyisatin with appropriate acetophenones in aqueous ethanol
at reflux temperature [25], Scheme 1. The molecular structure of compounds 1a-d
was confirmed on the basis of their elemental analyses and spectral data. The infrared
spectrum of compounds 1 showed a characteristic band at 3419-3300 cm-1 which is
due to –OH stretching of carboxylic acids in addition to the presence of a stretching
1
band at 1695-1683 cm-1 attributed to C=O functional group. The HNMR spectrum
showed a singlet at δ 13.78 ppm corresponding to the acidic-COOH proton of
quinoline. The corresponding esters 2a-d were obtained by reaction of 1a-d with
ethanol in the presence of concentrated sulfuric acid. These esters were then
hydrazinolyed with hydrazine hydrate in ethanol to give the corresponding
carbohydrazide precursors 3a-d in good yields. The structure of hydrazides 3a-d was
confirmed by elemental analysis and spectral data. Infrared spectrum gave a stretching
1
band at 1712-1709 cm-1 due to the presence of C=O of ester. HNMR spectra gave a
triplet at 1.45-1.41 ppm and a quartet at 4.48-4.46 ppm indicating the 5H of
COOCH2CH3. Infrared spectrum of compounds 3a-d revealed the presence of
absorption band at 3372-3368 due to NH-stretching and 1652-1637 cm-1 due to a
1
carbonyl group. HNMR spectrum of 3a-d showed a singlet at δ 4.78-4.4 indicating
2H of amino group.

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Scheme 1
The hydrazide moiety in compound 3a was exploited to synthesize some
pyrazole derivatives through its reaction with some electrophiles. The
cyclocondensation of hydrazide derivative 3a with chalcones 4a,b [26, 27] in
ethanolic sodium hydroxide afforded pyrazole derivatives 5a,b, Scheme 2. Infrared
spectrum of 5a,b revealed the presence of absorption band at 1674-1651 due to a
1
carbonyl group. HNMR spectrum of 5a showed a doublet at δ 3.58-3.66 indicating
2H of pyrazole.
Scheme 2

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1HNMR spectrum of 5a in DMSO-d6 revealed the presence of a singlet signal
at 3.58-3.66 correponding to methylene miety of pyrazole, a triplet at 4.93-5.04
corresponding to C5-proton of pyrazole, a singlet at 3.97 ppm corresponding to
methoxy group, a singlet at 5.2 ppm corresponding to CH2 benzylic-H in addition to
the presence of aromatic protons at 6.57-7.79 ppm.
Similarly, cyclocondensation of the hydrazides 3a-d with the appropriate β-
dicarbonyl compounds (namely; acetylacetone, benzoylacetone, ethyl acetoacetate &
diethylmalonates) gave the corresponding pyrazolyl derivatives 6a-d, 7a-d, 8a-d, and
9a-h, respectively, Scheme 3. The structures of compounds 6-9 were confirmed by
elemental analyses and spectral data.

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3
3
3
3
3
3
3
3
3
3
3
1
1
3
1
1
1
3
1
3
3
3
1
3
1
3
3
3
1
3
1
3
Scheme 3

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Finally, cyclocondensation of the hydrazides 3a-d with ethoxy ethylidene-
malononitriles gave the corresponding pyrazolyl derivatives 10a-d. On the other hand,
the reaction of compounds 3a-d with ethyl 2-cyano-3-ethoxyacrylate afforded the
pyrazolyl derivatives 11a-h, and the other possible structure 12 was excluded on the
basis of elemental analysis and spectral data, Scheme 4.

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Scheme 4

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2.2. Molecular docking results
Considering the promising biological results and for the reason of tackling the
interaction mode of the synthesized compounds with the COX-2 enzyme. The
protein structure used for docking was prepared using COX2-
celecoxib complex (PDB ID: 6COX) as a template by protein
modification function of Molecular operating environment (MOE)
[28].
The molecular docking studies suggested that all the tested compounds show
different bindings modes with the active site amino acids through hydrogen bonding
and hydrophobic interactions. Among all the tested compounds, compound 9a, 10a,
10b and 11a form a common binding mode as indicated from their molecular docking
score (-10.24, -10.25, -10.27, -10.26, respectively). The binding modes of compound
11a and COX-2 were depicted in Figure 2. All the interactions of compound 11a with
COX-2 were exhibited in Figure 3. In the binding mode, compound 11a is potently
bound to the binding site of COX-2 via hydrophobic interactions and binding is
stabilized by hydrogen bonds. The oxygen atom of the methoxy group on compound
11a formed a hydrogen bond with the Ser530 while, the interaction between Arg120
and p-chlorophenyl group is a cation-pi interaction. Also, the amino group formed a
hydrogen bond with Val523 and there is a hydrogen bond interaction between ligand
quinoline ring and Arg120. In addition, there are two other hydrogen bond
interactions between carbonyl group and Tyr355, and oxygen atom of the methoxy
group and Arg120. From this modeling results, it was suggested that compound 11a
have a promising activity on this target (COX-2) enzyme.

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Figure 2: Docking of compound 11a into COX-2 active site.
Figure 3: Interactions of compound 11a with COX-2.

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2.3. Biological studies
2.3.1. In vitro cyclcooxygenese (COX) inhibition assay:
The compounds synthesized in this work were evaluated for their ability to
inhibit COX-1 and COX-2 using an ovine COX-1/COX-2 assay kit (Catalog No.
560101, Cayman chemical Inc. Ann Arbor. MI (USA) according to the protocol
recommended by the supplier. IC50 (µM) are determined as the means of two
determination acquired and the deviation from the mean is <100% of the mean value.
The selectivity index value (SI values) was defined as IC50 % (COX-1)/IC50 (COX-2).
In the assay system, the IC50 values of celecoxib on COX-1 and COX-2 were
determined to be > 50 and 0.2 µM, respectively indicating that celecoxib is a selective
COX-2 inhibitor. None of the tested compounds showed inhibitory activity on COX-1
down to 50 µM. Compounds 9e, 11c and 11e show almost 100-times lower potency
(IC50: 40-49µm) compared with compound 9a, 10a, 10b and 11a (IC50: 0.26-0.43µm).
Some of the tested compounds (5a, 5b, 9a, 10a and 11a) were found to be potent and
selective similar to celecoxib against COX-2. IC50 values in µM (table 1) acquired by
the determination of the in vitro ability of the tested compounds to inhibit COX-2
showed that compound 11a with chlorine substituents of quinolone scaffold on the
phenyl ring was more potent and selective COX-2 inhibitor (IC50 = 0.26 µM, SI = >
192.33) compared with the reference drug celecoxib (IC50 = 0.28 µM, SI = > 178.57).
this is in accordance to our previous reported data [1] that the lead compound I which
show specially for COX-2 receptor that produce the potent anti-inflammatory activity,
compound 11a is a structural analogue of the lead compound I with similar electronic
and steric characteristics.
Compounds 6a, 6c, 7a, 7e, 8a, 8c and 8g have less potency and selectivity
compared with compound 11a due to the presence of bulky methyl group in the
position 3 of pyrazole moiety. In addition, the presence of methyl group at C-4 of
phenyl ring at 2-position of quinolone scaffold decrease also the potency and
selectivity as in compound 9g and 11c. this is an evidence that electron withdrawing
substituents at C-4 of phenyl group is very important for selective inhibition of COX-
2 [30].

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2.3.2. In vivo anti-inflammatory studies
Compounds 5a, 5b, 6a, 6c, 7a, 7e, 8a, 8c, 8g, 9a, 9e, 9g, 10a, 10e, 11a, 11c,
11e and 11g were evaluated for their anti-inflammatory activity using carragenin
induced edema bioassay method in rats [29]. Celecoxib was used as a reference
standard representing selective COX-2 inhibitor non-steroidal anti-inflammatory
agent. Compounds 5a, 7a, 8a, 8c, 8g, 10a and 10e, exhibited good anti-inflammatory
activity comparable to or slightly better than celecoxib (table 2). This is consistent
with the reported data [30] that COX-2 activity is sensitive to the lipophilic nature of
C-7 and C-8 quinoline substituents. Also, this adds further evidence that these
compounds may be functioning as COX-2 inhibitors. Moreover, replacement of
fluorine in 6-position in quinoline scaffold by methoxy substituent has nearly the
same lipophilic nature, thereby enhancing the rates of absorption and transportation in
vivo. As all tested compounds exhibited a promising anti-inflammatory activity, this
indicates that replacement of fluorine at the p-position by another electron
withdrawing group like chlorine (compound 11a) gave a good evidence that
designation of COX-2 inhibition is sensitive to the p-position of C-2 of phenyl ring
[30]. Moreover, the substituents on the pyrazole ring have a great effect on activity;
compound 5a which has 4-bromophenyl at position 3 has more anti-inflammatory
activity than compound 5b which has 4-chlorophenyl substitution at the same side.
This may be due to the higher lipophilicty of bromo drugs than that of chlorodrug
derivatives [31].
2.3.3. Analgesic activity
All compounds tested for their anti-inflammatory activities and celecoxib
reference drug were screened for their analgesic activity using the hot plate method of
Jacob and Bsovski [32]. The results presented in table 3 showed that all tested
compounds except celecoxib elicited a non-significant change when compared with
control group except compound 11a which show a significant increase in the reaction
time compared with control group.
2.3.4. Ulcerogenic activity
The target compounds were evaluated for their ulcerogenic potential in rats
using indomethacein as a reference drug. The incidence of ulcer score was calculated

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according to 1 to 5 scoring system of Wilhelmi and Menasse-Gdynia [33]. The ulcer
index was calculated according to the method of Pauls et al [34]. All drug doses were
calculated according to Paget and Barnes [35]. As illustrated in table in table 4, the
most active compounds 9c, 9e, 10a and 11a were devoid of any ulcerogenic activity.
These data can be added to our previous published data [1] in which, incorporation
quinoline skeleton with different azoles may provide lead compounds devoid of any
ulcerogenic activity.
3. Conclusion
The importance of the potent quinoline class of compounds has been
established in the search for effective anti-inflammatory agents [36]. In the current
investigation, a new scaffold of quinoline linked to pyrazoles were synthesized and
evaluated as anti-inflammatory agents. Also, fluorine at 6-position of quinoline
scaffold has been replaced by methoxy group and also fluorine at p-position of 2-
phenyl group has been replaced by electron withdrawing and electron donating
substituents. The results of this study revealed that, lipophilic substituents at 6-
position and electron withdrawing substituents at p-position of phenyl group are very
important to act as selective COX-2 inhibitor [1]. Compound 11a was found to have
the best anti-inflammatory activity as well as the best binding profiles with COX-2
isozyme. Moreover, some of the synthesized and evaluated compounds were devoid
of any ulcerogenic activity. Therefore, these derivatives may represent good lead
compounds for the development of potent and selective anti-inflammatory agents.
4. Experimental
4.1. General
Melting points were determined on a Gallenkamp melting point apparatus and are
uncorrected. Infrared spectra were recorded on a Pye Unicam SP 1000 infrared
spectrophotometer and SHIMADZU infrared spectrophotometer using potassium
1
bromide discs and expressed in wave number (cm-1). H NMR spectra were recorded
on a Varian-Mercury 200 MHz or 300 MHz spectrometer in DMSO-d6 and BRUKER
400 MHz spectrometer in DMSO-d6. Chemical shifts were expressed in parts per

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million (ppm) with tetramethylsilane (TMS) as an internal standard. As for the proton
13
magnetic resonance, D2O was carried out for NH and OH protons. C-NMR spectra
were obtained in DMSO-d6 on Avance 125 MHz spectrometers; the chemical shifts
are expressed in δunits. MS spectra were measured with an HP 5995 instrument and
Hewlett Packard 5988 spectrometer Micro Analytical Center, Egypt. Elemental
analyses (C, H, and N) were performed on VARIO El ELEMENTER apparatus at the
Micro Analytical Centre, Cairo University, Giza, Egypt. All compounds were
routinely checked by thin-layer chromatography (TLC) on aluminum-backed silica
gel plates. All solvents were dried by standard methods.
4.1.1. General method for the synthesis of compounds (1a-d)
A mixture of 5-methoxyisatin, (10 mmol), 33% potassium hydroxide (10 ml)
in ethanol (20 ml), and 4-substituted acetophenone (10 mmol) was heated under
reflux for 9-18h. The reaction mixture was concentrated under reduced pressure and
the residue was diluted with water (50 ml) and extracted with ether (3 × 50 ml). The
aqueous layer was neutralized with 1M hydrochloric acid. The precipitated solid was
filtered, washed with water, dried and crystallized from ethanol.
4.1.1.1. 2-(4-Chlorophenyl)-6-methoxyquinoline-4-carboxylic acid (1a)
Yield 70%; m.p. 264–266 oC ; IR (KBr, cm-1): 3300-2700 (br, OH), 3064-3003 (CH
1
aromatic), 2968, 2837 (CH aliphatic), 1689 (C=O), 1618 (C=N), 1587 (C=C). H-
NMR (200 MHz DMSO-d6, δppm): 3.97 (s, 3H, OCH3) 7.6-8.4 (m, 8H, aromatic H),
13.78 (s, 1H, OH, D2O exchangeable); Anal. Calcd. For C17H12ClNO3 (313.5): C,
65.08; H, 3.86; N, 4.46. Found: C, 65.19; H, 4.07; N, 4.15.
4.1.1.2. 2-(4-Bromophenyl)-6-methoxyquinoline-4-carboxylic acid (1b)
Yield 71%; m.p. 279-280 oC; IR (KBr, cm-1): 3400 (OH), 3007 (CH aromatic), 2964-
3835 (CH, aliphatic), 1689 (C=O), 1616 (C=N), 1589 (C=C).1H NMR (200 MHz,
DMSO-d6, δppm): 3.97 (s, 3H, OCH3), 7.55-8.47 (m, 8H, aromatic H), 13.62 (s, 1H,
OH, D2O exchangeable); Anal. Calcd. For C17H12BrNO3 (358): C, 57.00; H, 3.38; N,
3.91, Found: C, 56.90; H, 3.32; N, 3.84.

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4.1.1.3. 6-Methoxy-2-p-tolylquinoline-4-carboxylic acid (1c)
Yield 65%; m.p. 243-245oC; IR (KBr, cm-1): 3400 (OH), 3064, 3007 (CH aromatic),
2993, 2841 (CH aliphatic), 1695 (C=O), 1618 (C=N), 1587 (C=C). 1H NMR (200
MHz, DMSO-d6, δppm): 2.45 (s, 3H, CH3), 3.98 (s, 3H, OCH3), 7.42-8.49 (m, 8H,
aromatic H), 13.64 (s, 1H, OH, D2O exchangeable); Anal. Calcd. For C18H15NO3
(293.32): C, 73.71; H, 5.15; N, 4.78, Found: C, 73.50; H, 5.37; N, 4.47.
4.1.1.4.
6-Methoxy-2-(4-methoxyphenyl)quinoline-4-carboxylic
acid
(1d)
Yield 60%; m.p. 243.7-244oC; IR (KBr, cm1): 3419 (OH), 3032, 3005 (CH aromatic),
2968, 2841 (CH aliphatic), 1683 (C=O), 1616 (C=N), 1581 (C=C).1H NMR (200
MHz, DMSO-d6, δppm): 3.91 (s, 3H, OCH3), 3.97 (s, 3H, OCH3), 7.15-8.47 (m, 8H,
aromatic H), 13.6 (s, 1H, OH, D2O exchangeable); Anal. Calcd. For C18H15NO4
(309.32): C, 69.89; H, 4.89; N, 4.53, Found: C, 69.77; H, 4.77; N, 4.48.
4.1.2. General method for the synthesis of compounds (2a-d)
A mixture of the appropriate 4-carboxylic acids (1a-d) (10 mmol), absolute
ethanol (20 ml) and concentrated sulfuric acid (2 ml) was refluxed for 10 h. Excess
ethanol was distilled under reduced pressure; the mixture was allowed to cool, diluted
with water then rendered alkaline with sodium bicarbonate solution. The precipitate
was filtered, washed with water and crystallized from ethanol.
4.1.2.1. Ethyl 2-(4-chlorophenyl)-6-methoxyquinoline-4-carboxylate (2a).
Yield 65%; m.p. 128-129 oC; IR (KBr, cm-1): 3032-3007 (CH aromatic), 2981, 2902
1
(CH aliphatic), 1712 (C=O), 1624 (C=N). H NMR (300 MHz, DMSO-d6, δ ppm):
1.44 (t, 3H, CH3), 3.92 (s, 3H, OCH3), 4.5 (q, 2H, CH2), 7.51-8.45 (m, 8H, aromatic
H). Anal. Calcd. For C19H16ClNO3 (341.5): C, 66.77; H, 4.72; N, 4.10; Found: C,
66.46; H, 4.65; N, 3.84.
4.1.2.2. Ethyl 2-(4-Bromophenyl)-6-methoxyquinoline-4-carboxylate (2b)
o
Yield 75%; m.p. 134-136 C; IR (KBr, cm-1): 3010 (CH aromatic), 2981, 2900 (CH
aliphatic), 1712 (C=O), 1622 (C=N).1H NMR (300 MHz, DMSO-d6, δppm): 1.44 (t,

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3H, CH3), 3.93 (s, 3H, OCH3), 4.5 (q, 2H, CH2), 7.5-8.46 (m, 8H, aromatic H). Anal.
Calcd. For C19H16BrNO3 (386.24): C, 59.08; H, 4.18; N, 3.63; Found: C, 58.81; H,
4.36; H, 3.97.
4.1.2.3. Ethyl 6-methoxy-2-p-tolylquinoline-4-carboxylate (2c)
Yield 60%; m.p. 115-117oC; IR (KBr, cm-1): 3007(CH aromatic), 2984, 2842 (CH
aliphatic), 1712 (C=O), 1614 (C=N).1H NMR (400 MHz, DMSO-d6, δppm): 1.45 (t,
3H, CH3), 2.41 (s, 3H, CH3), 3.92 (s, 3H, OCH3), 4.5 (q, 2H, CH2), 7.52-8.48 (m, 8H,
aromatic H). Anal. Calcd. For C20H19NO3 (321.37): C, 74.75; H, 5.96; N, 4.36;
Found: C, 74.92; H, 6.18; N, 4.18.
4.1.2.4. Ethyl 6-methoxy-2-(4-methoxyphenyl)quinoline-4-carboxylate (2d)
o
Yield 59%; m.p. 96-98 C; IR (KBr, cm-1): 3112 (CH aromatic), 2948, 2829 (CH
aliphatic), 1708 (C=O), 1609 (C=N). 1H NMR (400 MHz, DMSO-d6, δppm): 1.45 (t,
3H, CH3), 3.90 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 4.48 (q, 2H, CH2), 7.52-8.48 (m,
8H, aromatic H). Anal. Calcd. For C20H19NO4 (337.37): C, 71.20; H, 5.68; N, 4.15;
Found: C, 70.93; H, 5.70; N, 3.84.
4.1.3. General method for the synthesis of compounds (3a-d)
To a solution of 2a-d (10 mmol) in ethanol (20 ml), hydrazine hydrate (97%, 3
ml) was added and refluxed for 3-7 h. After cooling, the precipitate was filtered,
washed with water dried and crystallized from ethanol.
4.1.3.1. 2-(4-Chlorophenyl)-6-methoxyquinoline-4-carbohydrazide (3a)
Yield 75%; m.p. 233-234 oC. IR (KBr, cm-1): 3269 (NHs), 3050 (CH aromatic), 2950,
2800 (CH aliphatic), 1637 (C=O), 1618 (C=N).1H NMR (200 MHz, DMSO-d6, δ
ppm): 3.94 (s, 3H, OCH3), 4.78 (s, 2H, NH2, D2O exchangeable), 7.57-8.37 (m, 8H,
aromatic H), 10.08 (s, 1H, NH, D2O exchangeable). Anal. Calcd. For C17H14ClN3O2
(327.5): C, 62.30; H, 4.31; N, 12.82; Found: C, 62.49; H, 4.73; N, 12.69.
4.1.3.2. 2-(4-Bromophenyl)-6-methoxyquinoline-4-carbohydrazide (3b)
o
Yield 82%; m.p. 242-244 C; IR (KBr, cm-1): 3313, 3271 (NH, NH2), 3050 (CH
aromatic), 2956, 2860 (CH aliphatic), 1640 (C=O). 1H NMR (200 MHz, DMSO-d6, δ

-17-
ppm): 3.97 (s, 3H, OCH3), 4.4 (br. s, 2H, NH2, D2O exchangeable), 7.65-8.28 (m, 8H,
aromatic H), 11.85 (s, 1H, NH, D2O exchangeable). Anal. Calcd. For C17H14BrN3O2
(372.22): C, 54.86; H, 3.79; N, 11.29; Found: C, 54.72; H, 3.53; N, 11.12.
4.1.3.3. 6-Methoxy-2-p-tolylquinoline-4-carbohydrazide (3c)
o
Yield 78%; m.p. 206-208. C; IR (KBr, cm-1): 3372, 3287 (NH2), 3175 (NH), 3000
(CH aromatic), 2975, 2838 (CH aliphatic), 1652 (C=O). 1H NMR (400 MHz, DMSO-
d6, δ ppm): 2.5 (s, 3H, CH3), 3.85 (s, 3H, OCH3), 4.70 (s, 2H, NH2, D2O
exchangeable), 7.09-8.23 (m, 8H, aromatic H), 10.01 (s, 1H, NH, D2O
exchangeable). Anal. Calcd. For C18H17N3O2 (307.35): C, 70.34; H, 5.58; N, 13.67;
Found: C, 70.19; H, 5.38; N, 13.52.
4.1.3.4. 6-Methoxy-2-(4-methoxyphenyl)quinoline-4-carbohydrazide (3d)
o
Yield 70%; m.p. 159-161 C; IR (KBr, cm-1): 3368, 3286 (NH2), 3177 (NH), 3010
(CH aromatic), 2978, 2838 (CH aliphatic) 1650 (C=O). 1H NMR (300 MHz, DMSO-
d6, δ ppm): 3.84 (s, 3H, OCH3) 3.87 (S, 3H, OCH3), 4.71 (S, 2H, NH2, D2O
exchangeable), 7.08-8.23 (m, 8H, aromatic H), 10.0 (s, 1H, NH, D2O exchangeable).
Anal. Calcd. For C18H17N3O3 (323.35): C, 66.86; H, 5.30; N, 13.00; Found: C, 66.54;
H, 5.12; N, 12.71.
4.1.4. General procedure for the synthesis of compounds (5a,b)
A mixture of the acid hydrazide (3b) (10 mmol) and the corresponding
chalcones (4a-b) (10 mmol) was refluxed in ethanol (30 ml) containing NaOH (10
mmol) for 6 hours. The mixture was cooled and the precipitated product was filtered,
washed and crystallized from aqueous ethanol.
4.1.4.1. (5-(4-(Benzyloxy)phenyl)-3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazol-1-yl) (2-
(4-bromophenyl)-6-methoxyquinolin-4-yl)methanone (5a)
o
Yield 60%; m.p. 189-191 C; IR (KBr, cm-1): 3062, 3032 (CH aromatic), 2916, 2850
(CH aliphatic) 1651(C=O), 1600 (C=N), 1589 (C=C). 1H NMR (200 MHz, DMSO-d6,
δppm): 3.33 – 3.44 (d, 2H, C4-H of pyrazole), 3.92 (s, 3H, OCH3), 4.27 (t, 1H, C5-H
of pyrazole), 5.20 (s, 2H, benzylic-H), 6.88-7.78 (m, 21H, aromatic H). Anal. Calcd.
For C39H29BrClN3O3 (703.02): C, 66.63; H, 4.16; N, 5.98; Found: C, 66.41; H, 4.40;
N, 5.79.

-18-
4.1.4.2. (5-(4-(Benzyloxy)phenyl)-3-(4-bromophenyl)-4,5-dihydro-1H-pyrazol-1-yl) (2-
(4-bromophenyl)-6-methoxnyquinolin-4-yl)methanone (5b)
o
Yield 65%; m.p. 180-182 C; IR (KBr, cm-1): 3062, 3032 (CH aromatic), 2920, 2862
1
(CH aliphatic), 1674 (C=O), 1604 (C=N), 1581 (C=C). H NMR (200 MHz, DMSO-
d6, δppm): 3.58-3.66 (d, 2H, C4-H of pyrazole), 3.97 (s, 3H, OCH3), 4.93-5.04 (t, 1H,
C5-H of pyrazole), 5.2 (s, 2H, benzylic-H), 6.57-7.79 (m, 21H, aromatic H); Anal.
Calcd. For C39H29Br2N3O3 (745.06): C, 62.67; H, 3.91; N, 5.62; Found: C, 62.60; H,
4.12; N, 5.31.
4.1.5. General procedure for the synthesis of compounds (6a-d)
A solution of acid hydrazide (3a-d) (10 mmol) and acetylacetone (10 mmol) in
dry DMF (5 ml) was heated for 6h. The mixture was cooled and poured on ice water
(20 ml). The separated solid was filtered, washed with water and crystallized from
ethanol.
4.1.5.1. (2-(4-Chlorophenyl)-6-methoxyquinolin-4-yl)(3,5-dimethyl-1H-pyrazol-1-
yl)-methanone (6a)
o
Yield 83%; m.p. 280-282 C; IR (KBr, cm-1): 3062 (CH aromatic), 2924, 2843 (CH
1
aliphatic), 1700 (C=O), 1620 (C=N), 1588 (C=C). H NMR (300 MHz, DMSO-d6, δ
ppm): 2.50 (s, 3H, CH3), 2.72 (s, 3H, CH3), 3.81 (s, 3H, OCH3), 6.3 (s, 1H, pyrazole
H), 7.00-8.29 (m, 8H, aromatic H). Anal. Calcd. For C22H18ClN3O2 (391.11): C,
67.43; H, 4.63; N, 10.72; Found: C, 67.09; H, 4.74; N, 10.46.
4.1.5.2. (2-(4-Bromophenyl)-6-methoxyquinolin-4-yl)(3,5-dimethyl-1H-pyrazol-1-
yl)-methanone (6b)
o
Yield 82%; m.p. 295-297 C; IR (KBr, cm-1): 3019 (CH aromatic), 2929, 2842 (CH
aliphatic), 1692 (C=O), 1617 (C=N), 1588 (C=C). 1H NMR (400 MHz, DMSO-d6, δ
ppm): 2.80 (s, 3H, CH3), 3.16 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 6.96 (s, 1H, Pyrazole
H), 7.50-8.45 (m, 8H, aromatic H). Anal. Calcd. For C22H18BrN3O2 (435.06): C,
60.56; H, 4.16; N, 9.63; Found: C, 60.50; H, 4.14; N, 9.85.
4.1.5.3. (3,5-dimethyl-1H-pyrazol-1-yl)(6-methoxy-2-p-tolylquinolin-4-
yl)methanone (6c)

-19-
o
Yield 70%; m.p. 242-244 C; IR (KBr, cm-1): 3062 (CH aromatic), 2930, 2839 (CH
1
aliphatic), 1688 (C=O), 1626 (C=N), 1544 (C=C). H NMR (300 MHz, DMSO-d6, δ
ppm): 2.50 (s, 3H, CH3), 2.51 (s, 3H, CH3), 2.81 (s, 3H, CH3) 3.92 (s, 3H, OCH3),
6.97 (s, 1H, pyrazole H), 7.48-8.46 (m, 8H, aromatic H). Anal. Calcd. For
C23H21N3O2 (371.43): C, 74.37; H, 5.70; N, 11.31; Found: C, 74.66; H, 5.98; N,
11.20.
4.1.5.4. (3,5-dimethyl-1H-pyrazol-1-yl)(6-methoxy-2-(4-methoxyphenyl)quinolin-4-
yl)methanone (6d)
o
Yield 73%; m.p. 260-262 C; IR (KBr, cm-1): 3090 (CH aromatic), 2968, 2925, 2831
1
(CH aliphatic), 1627 (C=O), 1547 (C=C). H NMR (400 MHz, DMSO-d6, δ ppm):
2.50 (s, 3H, CH3), 2.51 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 3.84 (s, 3H, OCH3), 6.93 (s,
1H, pyrazole H), 7.07-8.22 (m, 8H, aromatic H). Anal. Calcd. For C23H21N3O3
(387.43): C, 71.30; H, 5.46; N, 10.85; Found: C, 71.36; H, 5.81; N, 10.71.
4.1.6. General procedure for the synthesis of compounds (7a-d)
To a solution of compound (3a-d) (10 mmol) and potassium hydroxide (20 mmol) in
ethanol (20 ml), benzoylacetone (10 mmol) was added. The mixture was refluxed for
15h. The reaction mixture was cooled, diluted with water and neutralized with
hydrochloric acid. The separated solid was filtered, dried and crystallized from
ethanol.
4.1.6.1. (2-(4-Chlorophenyl)-6-methoxyquinolin-4-yl)(5-methyl-3-phenyl-1H-
pyrazol-1-yl)-methanone (7a)
o
Yield 88%; m.p. 352-354 C; IR (KBr, cm-1): 3062 (CH aromatic), 2928, 2839 (CH
1
aliphatic), 1680 (C=O), 1620 (C=N), 1544 (C=C). H NMR (400 MHz, DMSO-d6, δ
ppm): 2.48 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 7.48-8.47 (m, 13H aromatic H +
pyrazole H). Anal. Calcd. For C27H20ClN3O2 (453.92): C, 71.44; H, 4.44; N, 9.26;
Found: C, 71.36; H, 4.56; N, 9.36.
4.1.6.2. (2-(4-Bromophenyl)-6-methoxyquinolin-4-yl)(5-methyl-3-phenyl-1H-pyrazol-
1-yl)-methanone (7b)

-20-
o
Yield 82%; m.p.
360 C; IR (KBr, cm-1): 3089 (CH aromatic), 2925, 2830 (CH
1
aliphatic), 1691 (C=O), 1616 (C=N), 1500 (C=C). H NMR (300 MHz, DMSO-d6, δ
ppm): 2.50 (s, 3H, CH3), 3.92 (s, 3H, OCH3), 6.23 (s, 1H, pyrazole H), 7.47-8.23 (m,
13H, aromatic H). Anal. Calcd. For C27H20BrN3O2 (498.37): C, 65.07; H, 4.04; N,
8.43; Found: C, 64.79; H, 3.90; N, 8.12.
4.1.6.3. (6-Methoxy-2-p-tolylquinolin-4-yl)(5-methyl-3-phenyl-1H-pyrazol-1-yl)
methanone (7c).
o
Yield 60%; m.p. 320-322 C; IR (KBr, cm-1): 3066 (CH aromatic), 2920, 2830 (CH
aliphatic), 1685 (C=O), 1620 (C=N), 1540 (C=C). 1H NMR (DMSO-d6, 400 MHz): δ
2.03 (s, 3H, CH3), 2.38 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 6.87 (s, 1H, pyrazole H),
7.34-8.44 (m, 13H, aromatic H). Anal. Calcd. For C28H23N3O2 (433.50): C, 77.58; H,
5.35; N, 9.69; Found: C, 77.50; H, 5.29; N, 9.60.
4.1.6.4. (6-Methoxy-2-(4-methoxyphenyl)quinolin-4-yl)(5-methyl-3-phenyl-1H-
pyrazol-1-yl) methanone (7d)
o
Yield 63%; m.p. 332-334 C; IR (KBr, cm-1): 3063 (CH aromatic), 2935, 2830 (CH
1
aliphatic), 1690 (C=O), 1635 (C=N), 1560 (C=C). H NMR (400 MHz, DMSO-d6, δ
ppm): 2.00 (s, 3H, CH3), 3.86 (s, 3H, OCH3), 3.95 (s, 3H, OCH3) 6.85 (s, 1H,
pyrazole H), 7.11-8.28 (m, 13H, aromatic H). Anal. Calcd. For C28H23N3O3 (449.50):
C, 74.82; H, 5.16; N, 9.35; Found: C, 74.78; H, 5.40; N, 9.24.
4.1.7. General procedure for the synthesis of compounds (8a-d)
Ethyl acetoacetate (3 mmol) was added to a solution of the requisite acid hydrazide
3a-d (2 mmol) in glacial acetic acid (15 ml). The reaction mixture was refluxed for
18h. The reaction mixture was then cooled and the precipitated solid was filtered,
washed with water and crystallized from aqueous ethanol.
4.1.7.1. 1-(2-(4-Chlorophenyl)-6-methoxyquinoline-4-carbonyl)-3-methyl-1H-pyrazol
-5(4H)-one (8a).

-21-
o
Yield 86%; m.p. 277-279 C; IR (KBr, cm-1): 3174 (CH aromatic), 3005 (CH
1
aromatic), 2974 (CH aliphatic), 1696 (C=O), 1658 (C=O). H NMR (DMSO-d6, 300
MHz): δ1.99 (s, 3H, CH3), 3.44 (s, 2H, CH2), 3.92 (s, 3H, OCH3), 7.48-8.31 (m, 8H,
aromatic H). Anal. Calcd. For C21H16ClN3O3 (393.82): C, 64.05; H, 4.09; N, 10.67;
Found: C, 64.20; H, 4.43; N, 10.82.
4.1.7.2. 1-(2-(4-Bromophenyl)-6-methoxyquinoline-4-carbonyl)-3-methyl-1H-pyrazol
-5(4H)-one (8b)
o
Yield 82 %; m.p. 284-286 C; IR (KBr, cm-1): 3086 (CH aromatic), 2974, 2846 (CH
1
aliphatic), 1696 (C=O), 1657 (C=O). H NMR (DMSO-d6, 300 MHz): δ1.99 (s, 3H,
CH3), 3.40 (s, 2H, CH2), 3.92 (s, 3H, OCH3), 7.48-8.24 (m, 8H, aromatic H). Anal.
Calcd. For C21H16BrN3O3 (438.27): C, 57.55; H, 3.68; N, 9.59; Found: C, 57.09; H,
3.93; N, 9.00.
4.1.7.3. 1-(6-Methoxy-2-p-tolylquinoline-4-carbonyl)-3-methyl-1H-pyrazol-5(4H)-
one (8c)
o
Yield 72 %; m.p. 264-266 C; IR (KBr, cm-1): 3005 (CH aromatic), 2927, 2837 (CH
1
aliphatic), 1695 (C=O), 1661 (C=O). H NMR (DMSO-d6, 400 MHz): δ1.99 (s, 3H,
CH3), 2.38 (s, 3H, CH3), 3.38 (s, 2H, CH2), 3.91 (s, 3H, OCH3), 7.35-8.22 (m, 8H,
aromatic H). Anal. Calcd. For C22H19N3O3 (373.40): C, 70.76; H, 5.13; N, 11.25;
Found: C, 71.11; H, 5.73; N, 11.46.
4.1.7.4. 1-(6-Methoxy-2-(4-methoxyphenyl)quinoline-4-carbonyl)-3-methyl-1H-
pyrazol-5(4H)-one (8d)
o
Yield 70 %; m.p. 271-272 C; IR (KBr, cm-1): 3000 (CH aromatic), 2964, 2833 (CH
1
aliphatic), H NMR (DMSO-d6, 400 MHz): δ1.99 (s, 3H, CH3), 3.40 (s, 2H, CH2),
3.90 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 7.49-8.28 (m, 8H, aromatic H). Anal. Calcd.
For C22H19N3O4 (389.40): C, 67.86; H, 4.92; N, 10.79; Found: C, 67.90; H, 5.11; N,
10.79.
4.1.8. General procedure for the synthesis of compounds (9a-h)

-22-
A mixture of the requisite acid hydrazide 3a-d (2 mmol) and diethyl malonate or
diethyl 2-bromomalonate (4 mmol) and sodium methoxide (0.22 g, 4 mmol) was
refluxed in methanol (20 ml) for 18h. Excess solvent was removed under reduced
pressure; the remaining residue was dissolved in water (20 ml). The aqueous solution
was acidified with 2N hydrochloric acid to pH 3-4. The precipitated solid was filtered,
washed with water, dried and crystallized from ethanol.
4.1.8.1. 1-(2-(4-Chlorophenyl)-6-methoxyquinoline-4-carbonyl)pyrazolidine-3,5-
dione (9a).
Yield 86 %; m.p. 236-238oC; IR (KBr, cm-1): 3171 (NH), 3018 (CH aromatic), 2932,
1
2839 (CH aliphatic), 1693 (3 C=O), 1614 (C=N). H NMR (DMSO-d6, 300 MHz): δ
3.92 (s, 3H, OCH3), 4.03 (s, 2H, CH2), 7.53-8.46 (m, 8H, aromatic H), 10.43 (s, 1H,
NH, D2O-exchangeable). 13C NMR (CDCl3, 125 MHz): δ55.42 (CO-CH2-CO), 55.59
(CH3-O-), 103.42 (C=C-OCH3), 116.96 (C=C), 122.63-131.87 (aromatic carbons),
134.42 (C-Cl), 136.87 (C-C of phenyl), 144.60 (C-CO), 152 (C-phenyl), 158.43 (C-
OCH3), 165.29 (HN-CO-CH2), 165.87 (N-CO-CH2), 167.66 (C-CO-N). Anal. Calcd.
For C20H14ClN3O4 (395.80): C, 60.69; H, 3.57; N, 10.62; Found: C, 60.64; H, 4.04; N,
10.36.
4.1.8.2. 1-(2-(4-Bromophenyl)-6-methoxyquinoline-4-carbonyl)pyrazolidine-3,5-
dione (9b).
Yield 73 %; m.p. 242-244 oC; IR (KBr, cm-1): 3163 (NH), 3008 (CH aromatic), 2924,
1
2840 (CH aliphatic), 1656 (3 C=O), 1614 (C=N). H NMR (DMSO-d6, 300 MHz): δ
3.92 (s, 3H, OCH3), 4.02 (s, 2H, CH2), 7.49-8.25 (m, 8H, aromatic H), 10.49 (s, 1H,
NH, D2O exchangeable). Anal. Calcd. For C20H14BrN3O4 (440.25): C, 54.56; H,
3.21; N, 9.54; Found: C, 55.00; H, 3.36; N, 9.53.
4.1.8.3. 1-(6-Methoxy-2-p-tolylquinoline-4-carbonyl)pyrazolidine-3,5-dione (9c).
Yield 65 %; m.p. 224-226 oC; IR (KBr, cm-1): 3250 (NH), 3050 (CH aromatic), 2930
(CH aliphatic), 1689 (3 C=O), 1615 (C=N). 1H NMR (DMSO-d6, 400 MHz): δ2.37
(s, 3H, CH3), 3.90 (s, 3H, OCH3), 3.98 (s, 2H, CH2), 7.49-8.25 (m, 8H, aromatic H),
8.41 (s, 1H, NH, D2O exchangeable). Anal. Calcd. For C21H17N3O4 (375.38): C,
67.19; H, 4.56; N, 11.19; Found: C, 67.58; H, 4.70; N, 11.05.

-23-
4.1.8.4. 1-(6-Methoxy-2-(4-methoxyphenyl)quinoline-4-carbonyl)pyrazolidine-3,5-
dione (9d).
Yield 63 %; m.p. 230-232 oC; IR (KBr, cm-1): 3183 (NH), 3025 (CH aromatic), 2999,
2838 (CH aliphatic), 1674 (3 C=O), 1617 (C=N). 1H NMR (DMSO-d6, 400 MHz): δ
3.84 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.94 (s, 2H, CH2), 7.08-8.27 (m, 8H,
aromatic H), 10.43 (s, 1H, NH, D2O exchangeable). Anal. Calcd. For C21H17N3O5
(391.38): C, 64.45; H, 4.38; N, 10.74; Found: C, 64.33; H, 4.13; N, 10.72.
4.1.8.5. 4-Bromo-1-(2-(4-chlorophenyl)-6-methoxyquinoline-4-carbonyl)
pyrazolidine-3,5-dione (9e).
Yield 89 %; m.p. 260-262 oC; IR (KBr, cm-1): 3377 (NH), 3025 (CH aromatic), 2932
(CH aliphatic), 1654 (3 C=O), 1621(C=N). 1H NMR (DMSO-d6, 400 MHz): δ3.88 (s,
3H, OCH3), 4.21 (s, 1H, pyrazole-C4-H), 7.47-8.31 (m, 9H, 8 aromatic H + 1H, NH
D2O exchangeable). Anal. Calcd. For C20H13BrClN3O4 (474.69): C, 50.60; H, 2.76; N,
8.85; Found: C, 50.68; H, 2.63; N, 8.85.
4.1.8.6. 4-Bromo-1-(2-(4-bromophenyl)-6-methoxyquinoline-4-carbonyl)-pyrazo-
lidine-3,5-dione (9f).
Yield 82 %; m.p. 282-284 oC; IR (KBr, cm-1): 3385 (NH), 2928 (CH aliphatic), 1652
(3 C=O), 1621 (C=N). 1H NMR (DMSO-d6, 300 MHz): δ3.91 (s, 3H, OCH3), 3.96 (s,
1H, pyrazole-C4-H), 7.47-8.33 (m, 9H, 8 aromatic H + 1H, NH). Anal. Calcd. For
C20H13Br2N3O4 (519.14): C, 46.27; H, 2.52; N, 8.09; Found: C, 46.77; H, 2.75; N,
7.90.
4.1.8.7. 4-Bromo-1-(6-methoxy-2-p-tolylquinoline-4-carbonyl)pyrazolidine-3,5-dione
(9g).
Yield 74 %; m.p. 250-252 oC; IR (KBr, cm-1): 3363 (NH), 3022 (CH aromatic), 2925
(CH aliphatic), 1649 (3 C=O), 1619 (C=N). 1H NMR (DMSO-d6, 300 MHz): δ2.38 (s,
3H, CH3), 3.92 (s, 1H, OCH3), 4.02 (s, 1H, pyrazole-C4-H), 7.34-8.32 (m, 8H,
aromatic H), 10.00 (s, 1H, NH) Anal. Calcd. For C21H16BrN3O4 (454.27): C, 55.52;
H, 3.55; N, 9.25; Found: C, 55.96; H, 4.15; N, 9.53.

-24-
4.1.8.8. 4-Bromo-1-(6-methoxy-2-(4-methoxyphenyl)quinoline-4-carbonyl)pyrazo-
lidine-3,5-dione (9h)
Yield 76 %; m.p. 257-259 oC; IR (KBr, cm-1): 3384 (NH), 3000 (CH aromatic), 2927,
1
2837 (CH aliphatic), 1659 (3 C=O), 1614 (C=N). H NMR (DMSO-d6, 300 MHz): δ
3.85 (s, 3H, OCH3), 3.95 (s, 1H, OCH3), 4.00 (s, 1H, pyrazole-C4-H), 7.06-8.39 (m,
8H, aromatic H), 10.19 (s, 1H, NH, D2O exchangeable). Anal. Calcd. For
C21H16BrN3O5 (470.27): C, 53.63; H, 3.43; N, 8.94; Found: C, 53.42; H, 3.91; N,
8.91.
4.1.9. General procedure for the synthesis of compounds (10a-h)
To a solution of the requisite 3a-d (10 mmol) in dry DMF (10 ml), an
equimolar amount of 2-(ethoxymethylene)malononitrile or 2-(1-ethoxyethylidene)-
malononitrile (10 mmol) was added. The mixture was refluxed for 8h. The reaction
mixture was then cooled and poured on ice water (20 ml). The separated solid was
filtered and crystallized from ethanol.
4.1.9.1. 5-Amino-1-(2-(4-chlorophenyl)-6-methoxyquinoline-4-carbonyl)-1H-
pyrazole -4-carbonitrile (10a)
Yield 79%; m.p. 300-302oC; IR (KBr, cm-1): 3418, 3255 (NH2), 3020 (CH aromatic),
1
2928, 2833 (CH aliphatic), 2218 (CN), 1665 (C=O), 1620 (C=N). H NMR (400
MHz, DMSO-d6, δppm): 3.92 (s, 3H, OCH3), 4.79 (s, 2H, NH2 D2O- exchangeable ),
7.46 - 8.29 (m, 9H, 8H aromatic H + 1H, pyrazole H). Anal. Calcd. For C21H14ClN5O2
(403.82): C, 62.46; H, 3.49; N, 17.34; Found: C, 62.56; H, 3.47; N, 17.24.
4.1.9.2. 5-Amino-1-(2-(4-bromophenyl)-6-methoxyquinoline-4-carbonyl)-1H-
pyrazole -4-carbonitrile (10b)
o
Yield 73%; m.p. 325-327 C; IR (KBr, cm-1): 3424, 3292 (NH2), 3006 (CH
aromatic), 2958, 2832 (CH aliphatic), 2222 (CN), 1679 (C=O) 1617 (C=N), 1578
(C=C). 1H NMR (400 MHz, DMSO-d6, δppm): 3.95(s, 3H, OCH3), 5.03 (s, 2H, NH2
D2O exchangeable ), 7.12 (s,1H, pyrazole H), 7.46-8.39 (m, 8H, aromatic H). Anal.
Calcd. For C21H14BrN5O2 (448.27): C, 56.27; H, 3.15; N, 15.62; Found: C, 56.57; H,
3.05; N, 15.28.

-25-
4.1.9.3. 5-Amino-1-(6-methoxy-2-p-tolylquinoline-4-carbonyl)-1H-pyrazole-4-
carbonitrile (10c)
o
Yield 68%; m.p. 290-292 C; IR (KBr, cm-1): 3232 (NH2), 3018 (CH aromatic),
2930, 2832 (CH aliphatic), 2200 (CN), 1668 (C=O). 1H NMR (300 MHz, DMSO-d6, δ
ppm): 2.38 (s, 3H, CH3 ), 3.92 (s, 3H, OCH3), 4.8 (s, 2H, NH2 D2O-exchangeable),
7.34 - 8.17 (m, 9H, 8H, aromatic H + 1H, pyrazole H). Anal. Calcd. For C22H17N5O2
(383.40): C, 68.92; H, 4.47; N, 18.27; Found: C, 69.17; H, 4.80; N, 18.90.
4.1.9.4. 5-Amino-1-(6-methoxy-2-(4-methoxyphenyl)quinoline-4-carbonyl)-1H-
pyrazole-4-carbonitrile (10d)
Yield 70%; m.p. 286-288oC; IR (KBr, cm-1): 3170 (NH2), 3020 (CH aromatic), 2928,
1
2836 (CH aliphatic), 2216 (CN), 1671 (C=O). H NMR (400 MHz, DMSO-d6, δ
ppm): 3.88 (s, 3H, OCH3), 3.95 (s, 3H, OCH3), 6.00 (s, 2H, NH2 D2O-exchangeable),
7.09 (s, 1H, pyrazole H), 7.47 - 8.26 (m, 8H, aromatic H). Anal. Calcd. For
C22H17N5O3 (399.40): C, 66.16; H, 4.29; N, 17.53; Found: C, 66.12; H, 4.07; N,
17.62.
4.1.9.5. 5-Amino-1-(2-(4-chlorophenyl)-6-methoxyquinoline-4-carbonyl)-3-methyl-
1H-pyrazole-4-carbonitrile (10e)
Yield 83%; m.p. 232-234oC; IR (KBr, cm-1): 3432, 3300 (NH2), 3099 (CH aromatic),
1
2933, 2834 (CH aliphatic), 2198 (CN), 1695 (C=O) 1616 (C=N), 1572 (C=C). H
NMR (400 MHz, DMSO-d6, δ ppm): 2.00 (s, 3H, CH3), 3.95 (s, 3H, OCH3), 7.50-
8.43 (m, 8H, aromatic H), 11.12 (s, 2H, NH2 D2O-exchangeable ). Anal. Calcd. For
C22H16ClN5O2 (417.85): C, 63.24; H, 3.86; N, 16.76; Found: C, 62.92; H, 3.96; N,
16.78.
4.1.9.6. 5-Amino-1-(2-(4-bromophenyl)-6-methoxyquinoline-4-carbonyl)-3-methyl-
1H-pyrazole-4-carbonitrile (10f)
o
Yield 77%; m.p. 246-248 C; IR (KBr, cm-1): 3430, 3295 (NH2), 3099 (CH
aromatic), 2932, 2834 (CH aliphatic), 2223 (CN), 1694 (C=O), 1617 (C=N), 1572
1
(C=C). H NMR (300 MHz, DMSO-d6, δ ppm): 2.05 (s, 3H, CH3), 3.95 (s, 3H,
OCH3), 6.43 (s, 2H, NH2, D2O- exchangeable), 7.55-8.50 (m, 8H, aromatic H). Anal.

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Calcd. For C22H16BrN5O2 (462.30): C, 57.16; H, 3.49; N, 15.15; Found: C, 57.20; H,
3.60; N, 15.25.
4.1.9.7. 5-Amino-1-(6-methoxy-2-p-tolylquinoline-4-carbonyl)-3-methyl-1H-
pyrazole-4-carbonitrile (10g)
o
Yield 74 %; m.p. 221-223 C; IR (KBr, cm-1): 3414, 3299 (NH2), 3097 (CH
aromatic), 2921, 2840 (CH aliphatic), 2224 (CN), 1696 (C=O), 1617 (C=N), 1571
(C=C). 1H NMR (400 MHz, DMSO-d6, δppm): 1.99 (s, 3H, CH3), 2.10 (s, 3H, CH3)
3.94 (s, 3H, OCH3), 6.85 (s, 2H, NH2, D2O-exchangeable), 7.36-8.46 (m, 8H,
aromatic H). Anal. Calcd. For C23H19N5O2 (397.43): C, 69.51; H, 4.82; N, 17.62;
Found: C, 69.80; H, 5.05; N, 17.64.
4.1.9.8. 5-Amino-1-(6-methoxy-2-(4-methoxyphenyl)quinoline-4-carbonyl)-3-methyl-
1H-pyrazole-4-carbonitrile (10h)
Yield 76%; m.p. 226-228 oC; IR (KBr, cm-1): 3423, 3297 (NH2), 3020 (CH aromatic),
2931, 2841 (CH, aliphatic), 2216 (CN), 1693 (C=O), 1626 (C=N), 1562 (C=C). 1H
NMR (400 MHz, DMSO-d6, δppm): 2.00 (s, 3H, CH3) , 3.92 (s, 3H, OCH3), 3.94 (s,
3H, OCH3), 7.45-8.44 (m, 10H, aromatic H + NH2, D2O- exchangeable). Anal. Calcd.
For C23H19N5O3 (413.43): C, 66.82; H, 4.63; N, 16.94; Found: C, 66.91; H, 4.82; N,
16.62.
4.1.10. General procedure for the synthesis of compounds (11a-h)
To a solution of 3a-d (1 mmol) in dry DMF (4 ml), an equimolar amount of ethyl 2-
cyano-3-ethoxyacrylate or ethyl 2-cyano-3-ethoxybut-2-enoate (1 mmol) was added.
The mixture was refluxed for 8h. The reaction mixture was then cooled and poured on
ice water (20 ml). The separated solid was filtered and crystallized from ethanol.
4.1.10.1. Ethyl 5-amino-1-(2-(4-chlorophenyl)-6-methoxyquinoline-4-carbonyl)-1H-
pyrazole-4-carboxylate (11a)
Yield 88%; m.p. 229-231 oC; IR (KBr, cm-1): 3452, 3345 (NH2), 3020 (CH aromatic),
1
2983, 2936 (CH aliphatic), 1700 (2 C=O), 1614 (C=N), 1551 (C=C). H NMR
(DMSO-d6, 300 MHz): δ 1.27 (t, 3H, CH3), 3.84 (s, 3H, OCH3) 4.26 (q, 2H, CH2),
7.13 (s, 1H, pyrazole H), 7.50 - 8.41 (m, 8H, aromatic H), 9.51 (s, 2H, NH2, D2O

-27-
exchangeable). 13C NMR (CDCl3, 125 MHz): δ 14.40 (CH2CH3), 55.70 (CH3-O),
59.38 (CH2CH3), 93.64 (C=C-OCH3), 102.78 (C=C-NH2) 118.14 (C=C), 123-139.07
(aromatic carbons), 136.75 (C-Cl) 139.07 (C-C of phenyl), 143.74 (pyrazole carbon),
144.08 (C-CO), 151.46 (C-NH2), 153.77 (C-Phenyl), 158.27 (C-OCH3), 162.85 (C=O
ester), 169.11 (C-CO-N). Anal. Calcd. For C23H19ClN4O4 (450.87): C, 61.27; H, 4.25;
N, 12.43; Found: C, 61.16; H, 4.30; N, 12.61.
4.1.10.2. Ethyl 5-amino-1-(2-(4-bromophenyl)-6-methoxyquinoline-4-carbonyl)-1H-
pyrazole-4-carboxylate (11b).
Yield 89%; m.p. 238-239 oC; IR (KBr, cm-1): 3459, 3348 (NH2), 3020 (CH aromatic),
1
2943, 2838 (CH aliphatic), 1692 (2 C=O), 1616 (C=N), 1552 (C=C). H NMR
(DMSO-d6, 300 MHz): δ1.28 (t, 3H, CH3), 3.91 (s, 3H, OCH3) 4.26 (q, 2H, CH2),
7.11 (s, 1H, pyrazole H), 7.50 - 8.39 (m, 10H, aromatic H + NH2, D2O
exchangeable). Anal. Calcd. For C23H19BrN4O4 (495.33): C, 55.77; H, 3.87; N, 11.31;
Found: C, 55.49; H, 3.73; N, 11.40.
4.1.10.3. Ethyl 5-amino-1-(6-methoxy-2-p-tolylquinoline-4-carbonyl)-1H-pyrazole-4-
carboxylate (11c).
Yield 65%; m.p. 223-225 oC; IR (KBr, cm-1): 3493, 3224 (NH2), 3005 (CH aromatic),
1
2926, 2837 (CH aliphatic) 1689 (2 C=O), 1617 (C=N), 1544 (C=C). H NMR
(DMSO-d6, 400 MHz): δ1.43 (t, 3H, CH3), 2.34 (s, 3H, CH3), 3.91 (s, 3H, OCH3)
4.49 (q, 2H, CH2), 7.34 - 8.42 (m, 9H, aromatic H, 1H, pyrazole H), 9.61 (s, 2H, NH2,
D2O exchangeable). Anal. Calcd. For C24H22N4O4 (430.46): C, 66.97; H, 5.15; N,
13.02; Found: C, 67.03; H, 5.37; N, 13.31.
4.1.10.4. Ethyl
5-amino-1-(6-methoxy-2-(4-methoxyphenyl)quinoline-4-carbonyl)-
1H-pyrazole-4-carboxylate (11d).
Yield 62%; m.p. 224-226 oC; IR (KBr, cm-1): 3466, 3347 (NH2), 3084 (CH aromatic),
1
2942, 2834 (CH aliphatic), 1712 (2 C=O), 1616 (C=N), 1554 (C=C). H NMR
(DMSO-d6, 400 MHz): δ1.26 (t, 3H, CH3), 3.91 (s, 3H, OCH3), 3.94 (s, 3H, OCH3),
4.26 (q, 2H, CH2), 7.07 (s, 1H, pyrazole H), 7.46-8.31 (m, 8H, aromatic H), 9.31 (s,
2H, NH2, D2O exchangeable). Anal. Calcd. For C24H22N4O5 (446.46): C, 64.57; H,
4.97; N, 12.55; Found: C, 64.39; H, 4.81; N, 12.50.

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4.1.10.5. Ethyl 5-amino-1-(2-(4-chlorophenyl)-6-methoxyquinoline-4-carbonyl)-3-
methyl-1H-pyrazole-4-carboxylate (11e).
Yield 86%; m.p. 230-232oC; IR (KBr, cm-1) 3464, 3345 (NH2), 3062 (CH aromatic),
1
2933, 2836 (CH aliphatic), 1703 (2 C=O), 1616 (C=N), 1550 (C=C). H NMR
(DMSO-d6, 400 MHz): δ1.28 (t, 3H, CH3), 2.08 (s, 3H, CH3) 3.83 (s, 3H, OCH3),
4.27 (q, 2H, CH2), 7.09–8.38 (m, 8H, aromatic H), 9.56 (s, 2H, NH2, D2O
exchangeable). Anal. Calcd. For C24H21ClN4O4 (464.90): C, 62.00; H, 4.55; N, 12.05;
Found: C, 61.80; H, 4.47; N, 11.85.
4.1.10.6. Ethyl 5-amino-1-(2-(4-bromophenyl)-6-methoxyquinoline-4-carbonyl)-3-
methyl-1H-pyrazole-4-carboxylate (11f).
o
Yield 81%; m.p. 232-234 C; IR (KBr, cm-1): 3463, 3344 (NH2), 3063 (aromatic H),
1
2932, 2838 (CH aliphatic), 1701 (2 C=O), 1615 (C=N), 1550 (C=C). H NMR
(DMSO-d6, 300 MHz): δ1.28 (t, 3H, CH3), 2.09 (s, 3H, CH3) 3.83 (s, 3H, OCH3),
4.25 (q, 2H, CH2), 7.50 – 8.39 (m, 8H, aromatic H), 9.22 (s, 2H, NH2, D2O
exchangeable). Anal. Calcd. For C24H21BrN4O4 (509.35): C, 56.59; H, 4.16; N, 11.00;
Found: C, 56.37; H, 4.00; N, 10.83.
4.1.10.7. Ethyl 5-amino-1-(6-methoxy-2-p-tolylquinoline-4-carbonyl)-3-methyl-1H-
pyrazole-4-carboxylate (11g).
Yield 64%; m.p. 228-229 oC; IR (KBr, cm-1): 3466, 3344 (NH2), 3021 (CH aromatic),
1
2917, 2840 (CH aliphatic), 1692 (2 C=O), 1614 (C=N), 1550 (C=C). H NMR
(DMSO-d6, 400 MHz): δ1.28 (t, 3H, CH3), 2.08 (s, 3H, CH3), 2.38 (s, 3H, CH3) 3.82
(s, 3H, OCH3), 4.25 (q, 2H, CH2), 7.34–8.31 (m, 8H, aromatic H), 9.15 (s, 2H, NH2,
D2O-exchangeable). Anal. Calcd. For C25H24N4O4 (444.48): C, 67.55; H, 5.44; N,
12.60; Found: C, 67.32; H, 5.33; N, 12.76.
4.1.10.8. Ethyl 5-amino-1-(6-methoxy-2-(4-methoxyphenyl)quinoline-4-carbonyl)-3-
methyl-1H- pyrazole-4-carboxylate (11h).
Yield 66%; m.p. 220-221 oC; IR (KBr, cm-1): 3444, 3327 (NH2), 3066 (CH aromatic),
1
2926, 2835 (CH aliphatic), 1698 (2 C=O), 1610 (C=N), 1551 (C=C). H NMR
(DMSO-d6, 300 MHz): δ1.28 (t, 3H, CH3), 2.09 (s, 3H, CH3), 3.82 (s, 3H, OCH3),
3.84 (s, 3H, OCH3), 4.28 (q, 2H, CH2), 7.11-8.30 (m, 8H, aromatic H), 9.44 (s, 2H,

-29-
NH2, D2O exchangeable). Anal. Calcd. For C25H24N4O5 (460.48): C, 65.21; H, 5.25;
N, 12.17; Found: C, 65.05; H, 5.39; N, 12.30.
4.2. Molecular docking methodology
All the molecular modeling studies were carried out on Intel Pentium 1.6GHZ
processor, 512 MB memory with Windows XP operating system using Molecular
Operating Environment (MOE 2007-09 Chemical Computing Group, Canada)24 as
the computational software.
Target compounds optimization
The compounds were constructed using builder interface of MOE program, and then
were subjected to energy minimization with MMFF94X force and the partial charges
were automatically calculated. Systematic conformational import of the MOE is used
to produce different conformers for each compound. The obtained database was then
saved as mdb files be used in the docking calculations.
Preparation of the enzyme structure
The 3D coordinations of the inhibitor celecoxib of murine COX-2 enzyme (PDB
code: 6cox) were obtained from the freely accessible Protein data bank and used for
molecule docking studies. In this structure, three ligands are bound to the enzyme:
HEM (protoporphyrin IX containing FE), NAG (N-acetyl-D-glucosamine) and
celecoxib. The non-relevant compounds HEM and NAG were removed for the study.
Docking of the target molecules to the COX-2 active site
Docking of the conformational database of the compounds was done using
MOE-dock software through the following methodology: (i) The enzyme active site
was loaded and the dock tool was initiated where the program parameters were
adjusted as follow: Ligand atoms as the docking site, triangle matcher as the
placement methodology, London dG as scoring methodology, and Forcefield as
refinement (default values used) (ii) The mdb file of the ligands to be docked was
loaded and Dock calculations were run. The obtained ligand-enzyme complex model