Synthesis, anti-inflammatory screening, molecular docking, and COX-1,2/-5-LOX inhibition profile of some novel quinoline derivatives

Article abstract of DOI:10.1016/j.bioorg.2018.03.023

New quinoline compounds comprising pyrazole scaffold through different amide linkages were synthesized. The synthesized compounds were evaluated for their anti-inflammatory activity. Eight compounds (5c, 11b,c, 12c, 14a,b, 20a and 21a) were found to exhib

Full text of DOI:10.1016/j.bioorg.2018.03.023

Accepted Manuscript
Synthesis, anti-Inflammatory screening, molecular docking, and COX-1,2/-5-
LOX inhibition profile of some novel quinoline derivatives
Ibrahim Chaaban, Ola H. Rizk, Tamer M. Ibrahim, Shery S. Henen, El-Sayeda
M. El-Khawass, Aida E Bayad, Ibrahim M. El-Ashmawy, Hisham A. Nematalla
PII:
S0045-2068(18)30056-7
DOI:
https://doi.org/10.1016/j.bioorg.2018.03.023
YBIOO 2313
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
21 January 2018
16 March 2018
18 March 2018
Please cite this article as: I. Chaaban, O.H. Rizk, T.M. Ibrahim, S.S. Henen, E.M. El-Khawass, A.E. Bayad, I.M.
El-Ashmawy, H.A. Nematalla, Synthesis, anti-Inflammatory screening, molecular docking, and COX-1,2/-5-LOX
inhibition profile of some novel quinoline derivatives, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/
j.bioorg.2018.03.023
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Synthesis, anti-Inflammatory screening, molecular docking,
and COX-1,2/-5-LOX inhibition profile of some novel
quinoline derivatives
a
a, b,
c
a
Ibrahim Chaaban , Ola H. Rizk *, Tamer M. Ibrahim , Shery S. Henen , El-
a
d
d,e
Sayeda M. El-Khawass , Aida E .Bayad , Ibrahim M. El-Ashmawy , Hisham A.
f
Nematalla
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
Alexandria 21521, Egypt
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Pharos University in
Alexandria, 21311, Egypt.
c
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh
University, Kafr El-Sheikh 33516, Egypt
d
Pharmacology Department, Faculty of Veterinary Medicine, Alexandria University,
Alexandria, Egypt
e
Department of Veterinary Medicine, Faculty of Agricultural and Veterinary Medicine,
Qassim University, P.O.Box 1482, Buraydah, Al-Qassim, Saudi Arabia
f
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Pharos University
in Alexandria, 21311, Egypt.
ABSTRACT
New quinoline compounds comprising pyrazole scaffold through different amide
linkages were synthesized. The synthesized compounds were evaluated for their anti-
inflammatory activity. Eight compounds (5c, 11b,c, 12c, 14a,b, 20a and 21a) were
found to exhibit promising anti-inflammatory profiles in acute and sub-acute
inflammatory models. They were screened for their ulcerogenic activity and none of them
showed significant ulcerogenic activity comparable to the reference drug celecoxib and
are well tolerated by experimental animals with high safety margin (ALD50> 0.3 g/kg).
Compounds 5c, 11b,c, 12c, 14a,b, 20a and 21a showed significant in vitro LOX
inhibitory activity higher than that of zileuton. In vitro COX-1/COX-2 inhibition study
revealed that compounds 12c, 14a,b and 20a showed higher selectivity towards COX-2
than COX-1. Among the tested compounds, 12c, 14a and 14b showed the highest
inhibitory activity against COX-2 with an IC50 values of 0.1, 0.11 and 0.11μM
respectively. The docking experiments attempted to postulate the binding mode for the
1

most active compounds in the binding site of COX-2 enzymes and confirmed the high
selectivity binding towards COX-2 enzyme over COX-1.
Key words
Quinoline; pyrazole; anti-inflammatory; COX; 5-LOX; docking
1. Introduction
Inflammation is a complicated response of body tissues that arises in response to various
infectious, traumatic or autoimmune diseases, a process that often leads to recovery from
infection and healing. However, if not properly faced, inflammation can result in
persistent tissue damage by infiltrating leukocytes, lymphocytes or damaged collagen [1].
Targeting cyclooxygenase (COX) enzymes is a well-established strategy to treat
inflammation. In this context, inhibiting COX enzyme-associated prostanoid synthesis is
established by using non-steroidal anti-inflammatory drugs (NSAIDs) [2-4].
Nevertheless, long-term clinical usage of NSAIDs is usually associated with significant
side effects including gastro-duodenal erosions, ulceration and nephrotoxicity [5].
Consequently, selective COX-2 inhibitors with lower gastrointestinal side effects
compared with classical NSAIDs were discovered and approved such as celecoxib,
valdecoxib and rofecoxib. Nonetheless, many of them has reported cardiovascular side
effects, and hence, withdrawn from the market [6,7]. Inhibition of COX-1/COX-2 ensues
increased accumulation of leukotrienes through the lipoxygenase (LOX) pathway [8].
LOX catalyzes hydroperoxidation of fatty acids to peroxides [9]. Also, 5-LOX has been
connected to several unwanted effects which are part of the progression of inflammation,
osteoarthritis and asthma [10,11]. Therefore, dual inhibition of 5-LOX/COX would offer
anti-inflammatory effects with lower side effects [12].
Literature survey revealed that substituted quinoline derivatives constitute a promising
group in the search for new more effective anti-inflammatory and analgesic drugs
(
Figure 1) [13-17]. On the other hand, pyrazoles were found to occupy a significant
position in Medicinal Chemistry because of their capability to exhibit a wide range of
bioactivities, particularly as anti-inflammatory [18-20] and analgesic agents [21,22], in
addition to their prominent COX-2 inhibitory activity [23,24]. In specific, the selective
COX-2 inhibitor celecoxib (Figure 1) comprises a substituted diaryl pyrazole as a core
structure [25,26].
Encouraged by these findings, it was found interesting to synthesize and evaluate the
biological activity of some novel structure hybrids encountering the quinoline backbone
bearing a substituted pyrazole moiety aiming to obtain compounds with enhanced anti-
inflammatory activity. The targeted substituted quinolines were designed so as to
2

comprise the 1,3-diaryl pyrazole, pyrazole-3-carbohydrazide, 3,5-dimethylpyrazole and
pyrazolidinedione counterparts through different linkages (Structures A-D, Figure 1).The
substitution pattern of the synthesized compounds was carefully selected so as to confer
different electronic and lipophilic properties to the molecules. All the synthesized
compounds were investigated for their in vivo anti-inflammatory activity and the most
active compounds were further evaluated for their in vitro COX/5-LOX inhibition.A
molecular docking study was also performed for derivatives of highest anti-inflammatory
activity in a trial to investigate the possible mode of action of these compounds.
2. Results and discussion
2.1. Chemistry
The synthetic pathways employed for the synthesis of the intermediate and final
compounds are depicted in schemes 1-3.
In scheme 1, Pfitzinger reaction [13] was adopted to synthesize 6-chloro-2-
methylquinoline-4-carboxylic acid 1 in a very good yield through the condensation of 5-
chloroisatin with acetone in potassium hydroxide solution as described earlier [27].
1
Structure of compound 1 was confirmed by elemental microanalyses, IR, H-NMR in
13
addition to C-NMR spectra. IR spectrum revealed absorption bands corresponding to
-1
-1
1
OH and carboxylic C=O at 3413 cm and 1707 cm respectively, H-NMR spectrum was
characterized by the presence of CH , quinolyl and OH protons at their expected
chemical shifts. while C-NMR showed signals at 25.13 ppm and 167.51 ppm due to
CH and C=O respectively.
3
1
3
3
Esterification of the acid 1 with ethanol in the presence of concentrated sulfuric acid
1
yielded the key intermediate ethyl 6-chloro-2-methylquinoline-4-carboxylate 2. H-NMR
spectrum of compound 2 revealed a triplet at 1.49 ppm and a quartet at 4.52 ppm
assigned to -CH
furnished the corresponding quinoline-4-carbohydrazide derivative 3 [28,29]. H-NMR
showed the disappearance of -CH CH protons and the appearance of D O exchangeable
protons assigned to the NH and NH groups at 4.68 ppm and 9.91 ppm respectively.
2
CH
3
protons. Refluxing 2 with 99% hydrazine hydrate in ethanol
1
2
3
2
2
On the other hand, 1H-pyrazole-4-carbaldehydes 4a-c were adequately prepared utilizing
a previously reported procedure through Vilsmeier-Haack reaction [30].The IR spectra of
-1
the pyrazole 4-carboxaldehydes 4a,c showed a characteristic peak at 1684-1671 cm ,
1
which was attributed to the aldehydic C=O group. Their H-NMR spectra demonstrated
two singlets due to pyrazole-C5-H and the aldehydic proton. Condensing the acid
hydrazide 3 with the appropriate aromatic aldehyde 4a-c in ethanol afforded the target
hydrazones 5a-c [31,32]. The molecular structures of compounds 5a-c were confirmed on
1
the basis of their elemental analyses and spectral data. H-NMR spectra of compounds
5
a-c lacked singlets assigned to NH
2
and aldehydic protons at their previously recorded
3

chemical shifts and showed the presence of a singlet assigned to CH=N at 8.43-8.47 ppm
and a singlet due to NH at 12.04-12.14 ppm. The MS spectrum of 5c showed a molecular
+
•
ion peak (M ) at m/z 530 (15%) which matches its molecular formula C28
H
21Cl
2
N
5
2
O .
1
3
Moreover, C-NMR for compound 5c provided an additional proof for the proposed
structures of compounds 5a-c.
Furthermore, cyclocondensation of the hydrazide 3 with 1,3-dicarbonyl compounds
namely (acetyl acetone and diethyl malonate) aimed at producing quinoline derivatives
bearing pyrazolyl/pyrazolidinyl 1-carbonyl moieties at 4-position. In an attempt to
synthesize the 3,5-dimethylpyrazole derivative 7 by heating with acetyl acetone in
ethanol containing a catalytic amount of acetic acid, [33-35] the hydrazone 6 was
isolated.
Whereas, the target compound 6-chloro-4-[(3,5-dimethyl-1H-pyrazol-1-
yl)carbonyl]2-methylquinoline 7 was prepared by fusing the hydrazide 3 with excess
1
acetyl acetone at 110 °C. H-NMR spectrum of compound 7 was characterized by the
disappearance of singlets previously assigned to NH and NH
of two singlets assigned to pyrazole C-3 and C-5 CH protons at 2.73 ppm and 2.12 ppm
respectively as well as a singlet corresponding to pyrazole C-4 proton in the aromatic
2
protons and the appearance
3
+
•
region. Its mass spectrum showed the molecular ion peak (M ) at m/z 299 (37%)
corresponding to C16 O. An additional confirmation of the proposed structure was
achieved through its C-NMR spectrum which revealed 2 signals at 13.36 ppm and
3.87 ppm corresponding to pyrazole C-3 and C-5 CH . In contrast, fusion of the
H
14ClN
3
1
3
1
3
hydrazide 3 with diethyl malonate at 160˚C [36] afforded the open chain compound 8
while performing the reaction in refluxing ethanol containing sulfuric acid yielded the
pyrazolidine-3,5-dione compound 9. The structure of compound 9 was identified by IR,
1
13
1
H-NMR and C-NMR spectra. H-NMR spectrum was characterized by the appearance
1
3
of a singlet at 3.73 ppm integrated for pyrazolidine C-4 protons. Meanwhile the C-
NMR spectrum showed 3 signals at 159.36, 165.05 and 165.37 ppm attributed to 3 C=O
carbons.
Scheme 2, It is well known that a methyl group positioned α- to an aromatic heterocyclic
nitrogen atom, carries acidic hydrogens. Thus this methyl possesses high tendency to
form a resonance stabilized carbanion. Consequently, such compounds are able to
condense with aldehyde or ketones forming C=C bond. Based on this fact, compound 2
was allowed to condense with 1,3-diaryl pyrazole-4-carbaldehydes 4a-c through its α-
methyl group to afford compounds 11a-c. This reaction was performed in refluxing acetic
anhydride according to reported procedure [37,38]. Similarly, synthesis of compounds
1
2a-c was achieved by heating the ester 2 with morpholine under reflux to afford 10
which was subsequently reacted with 4a-c by heating in acetic anhydride to furnish the
desired compounds. The molecular structures of the prepared compounds 11a-c and 12a-
1
c were confirmed on the basis of their elemental microanalyses, IR, H-NMR spectra for
1
3
all compounds as well as C-NMR and mass spectra for some representative examples.
4

1
H-NMR spectra of compounds 11a-c were characterized by triplet and quartet assigned
to -CH CH protons in addition to aryl, quinolyl, -CH=CH- and pyrazolyl C-5 protons at
their expected chemical shifts. The mass spectrum of compound 11a showed a molecular
2
3
+
•
1
ion peak (M ) at m/z 548 (41%) corresponding to C29
H
20Cl
3
N
3
2
O . While the H-NMR
spectra of compounds 12a-c revealed multiplets assigned to morpholinyl protons in
addition to aryl, quinolyl, -CH=CH- and pyrazolyl C-5 protons at their expected chemical
1
3
shifts. Investigating the C-NMR spectra of compound 11a and 12c provided a
substantial affirmation for the assigned structure of these compounds. The chalcones
1
4a,b were prepared through the reaction of 10 with aryl glyoxal 13a,b in acetic
1
anhydride[37,38]. The H-NMR spectra of chalcones 14a,b showed the presence of two
doublets, each assigned to a vinylic proton with a coupling constant 15.6-15.8 Hz
indicating their existence as E isomers [39]. Attempts to cyclize the chalcones 14a,b into
the corresponding N-arylpyrazole derivatives 15a-dusing phenyl hydrazine and aryl
hydrazines utilizing reaction conditions previously reported for the synthesis of
analogous compounds [40] went in vain.
Scheme 3, Knorr pyrazole synthesis [41] was adopted for the synthesis of the pyrazole-3-
carboxylate esters 16a-c which were then reacted with hydrazine hydrate in ethanol to
produce the respective hydrazides 17a-c [42]. IR of compound 17a-c exhibited strong
absorption bands attributed to NH , NH, C=O and C=N. Moreover, oxidation of 2-methyl
2
quinoline derivatives 2 and 10 using selenium dioxide in dioxane afforded the
corresponding aldehydes 18 and 19 [43]. The latter compounds were then stirred at room
temperature with the appropriate 1,5-diaryl-1H-pyrazole-3-carbohydrazide 17a-c in
glacial acetic acid to furnish the target hydrazones 20a-c and 21a-c respectively [44]. The
structures of the target compounds 20a-c and 21a-c were determined by elemental
1
13
microanalyses and by studying the IR, H-NMR spectra for all compounds as well as C-
NMR and mass spectra for some representative examples. IR spectra of compounds 20a-
c and 21a-c were characterized by absorption bands assigned to NH, C=O, C=N and C-
1
O-C functional groups. H-NMR spectra of compounds 20a-c and 21a-c revealed the
disappearance of the aldehydic and NH
to CH=N, D O exchangeable NH andpyrazole-C4 protons, which therefore indicates the
2
protons and the appearance of singlets assigned
2
1
3
successful combination of the two heterocyclic moieties. Finally C-NMR spectra of 20a
and 21b provided a further confirmation for their intended structures.
2
2
.2. Biological evaluation
.2.1. Anti-inflammatory activity
The anti-inflammatory (AI) activity of the synthesized compounds was evaluated by
utilizing the formalin-induced paw edema bioassay [45, 46] using celecoxib (20 mg/kg)
as a reference standard anti-inflammatory agent. The paw edema was employed as a
5

model for acute and sub-acute inflammation. The data obtained were presented in
Tables 1 and 2) and expressed as means ± SE. Statistical differences of control and test
(
groups was carried out using the Analysis of Variance (ANOVA) followed by ‘Student–
Newman–Keuls Multiple Comparison Test. They were performed using computer
package of the Statistical Analysis System (SAS, 1987), SAS Incorporation Institute. The
difference in results was considered significant when p < 0.05.
2
.2.1.1. Formalin-induced paw edema bioassay (acute inflammatory model).
In this acute inflammatory model [45,46], each test compound was dosed orally (20
mg/kg body weight) 1 h prior to induction of inflammation by formalin injection.
Celecoxib was utilized as a reference drug at a dose of 20 mg/kg, po. The anti-
inflammatory activity was then calculated 1–4 h after induction and presented in (Table
1
) as the mean paw volume (mL) in addition to the percentage anti-inflammatory activity
(
AI %).
A comparative study of the anti-inflammatory activity of the test compounds relative to
the reference drug celecoxib at different time intervals revealed the following:
After one hour, twelve compounds showed distinctive pharmacokinetic profiles as shown
from their rapid onset of action which was higher than celecoxib at a dose of 20 mg/kg,
po. A remarkable AI% was recorded for compounds 12c, 14a,b, 20a (78%); 5c, 11b
(
75%); 7, 11c, 12b, 21c (71%) and 21a (67%) when compared to celecoxib (60%). In
addition, compound 21b showed good AI% (60%) which was equivalent to the reference
drug. After two hours interval, the data indicated that eight compounds namely; 5c, 14a
(
75%); 11b (72%); 11c, 12c (70%); 20a (67%) and 21a,b (64%) showed significant anti-
inflammatory activity superior to celecoxib (59%). Whereas, compound 12b (59%)
displayed equipotent activity to the standard drug. The other tested compounds were less
potent than the reference drug. Regarding the AI activity after four hours’ time interval as
a criterion for comparison, it was found that four compounds; 11c (86%), 11b (74%), 12c
(
70%) and 14a (66%) showed remarkable effect in inhibiting the paw edema when
compared to celecoxib (66%). Whereas, compounds 5c, 14b (64%), 20a (62%) and 21a
60%) showed moderate activity. The most active compounds 5c, 11b,c, 12c, 14a,b, 20a
(
and 21a were further tested at 5, 10, 20, 40 and 50 mg/kg body weight in order to
determine their ED50 values after two hours. Compound 11c was found to be more potent
(
ED50 = 10.70 mg/kg, Table 1) than celecoxib (ED50 = 15.11 mg/kg), whereas the rest of
the compounds were found to be nearly equipotent to the reference drug (ED50 = 13.15-
7.12 mg/kg).
1
6

2
.2.1.2. Formalin-induced paw edema bioassay (sub-acute inflammatory model).
For this sub-acute inflammatory model [45,46], inflammation was induced by formalin
injection in the first and third days, and test compounds were administered once orally (at
2
0 mg/kg daily) for 7 days. Again, celecoxib was used as a reference AI agent. In this
st
th
assay, the anti-inflammatory activity was calculated at 1 and 8 days after induction and
presented in (Table 2) as the mean paw volume and the percentage anti-inflammatory
activity (AI %).
The results obtained revealed that at the first day, six compounds namely; 5c, 14a, 20a,
2
5
1
1c, 21a and 11b displayed significant anti-inflammatory activity (74, 62, 62, 62, 60 and
7% respectively) which was higher than celecoxib (51%). Meanwhile, compounds 7 and
4b were nearly equivalent to the reference celecoxib with AI 48%. The data obtained at
day eight revealed that compound 14b (62%); 5c, 11c (58%) and 20a (56%) were found
to be nearly as effective as the reference celecoxib (60%). The other compounds
exhibited moderate to weak activity.
Further interpretation of the anti-inflammatory activity of the test compounds in pre-
mentioned screens (Tables 1, 2) revealed that 5c, 11b,c, 14a,b, 20a and 21a showed
remarkable activity in both the acute and subacute inflammatory models. On the other
hand, compounds 12b, 12c and 21b displayed pronounced activity in the acute
inflammatory model nevertheless they proved to be less active in the subacute
inflammatory model. This fact would suggest that such type of compounds might be
effective in managing acute inflammation, while they would be less effective in
controlling chronic inflammatory conditions.
2
.2.1.3. Ulcerogenic activity
Eight compounds namely; 5c, 11b,c, 12c, 14a,b, 20a and 21a that exhibited promising
anti-inflammatory profiles in the pre-mentioned animal models were further evaluated for
their ulcerogenic potential in rats [47]. Gross observation of the isolated rat stomach
showed a normal stomach texture for compounds 12c and 14b with no observable
hyperemia indicating a superior GI safety profile (no ulceration) in the population of the
test animals at an oral dose of 300 mg/kg, when administered twice daily in fasted rats.
Whereas, compounds 5c, 11b, c, 14a, 20a and 21a showed weak ulceration effect (10-
2
0%) compared to the reference drug celecoxib which was found to cause no ulceration
under the same experimental conditions. It is worth to mention that, indomethacin; the
reference standard AI drug was found to cause 100% ulceration.
2
.2.1.4. Acute toxicity
The same selected compounds 5c, 11b,c, 12c, 14a,b, 20a and 21a were further evaluated
for their approximate acute lethal dose ALD50 in male rats using a literature method [48].
7

The results indicated that all of the tested compounds proved to be non-toxic and are well
tolerated by the experimental animals. The compounds showed a high safety margin
when screened at graded doses (0.1–0.4 g/kg, po), where their ALD50 values were found
to be > 0.4 g/kg.
2
.2.2. In vitro COX-1/COX-2 inhibition assay
The same selected compounds 5c, 11b,c, 12c, 14a,b, 20a and 21a were further evaluated
for their ability to inhibit COX-1 and COX-2 isoenzymes using the in vitro colorimetric
COX (ovine) inhibitor assay method [49], which utilizes the peroxidase component of
cyclooxygenase. The peroxidase activity is assayed colorimetrically by monitoring the
appearance of oxidized N,N,N’,N’-tetramethyl-p-phenylenediamine (TMPD), which is
produced during the reduction of PGG2 to PGH2, at 590 nm. The efficacies of the tested
compounds were determined as the concentration causing 50% enzyme inhibition (IC50
)
and recorded in Table 3.
The data revealed that the compounds 12c, 14a,b and 20a showed higher selectivity
towards COX-2 than COX-1. Among the tested compounds, 12c, 14a and 14b showed
the highest inhibitory activity against COX-2 with an IC50 values of 0.1, 0.11 and 0.11μM
respectively whereas 11b was the least selective towards COX-2 inhibition with an IC50
value of 0.63 μM. These results indicate that the most active compounds possess selective
inhibitory activity towards COX-2 enzyme.
2
.2.3. In vitro LOX inhibition assay
The same selected compounds 5c, 11b,c, 12c, 14a,b, 20a and 21a were evaluated for
their in vitro ability to inhibit 5-lipoxygenase enzyme, using Abnova 5-lipoxygenase
inhibitor screening assay kit (catalog no. KA1329, Cayman Chemicals), according to the
manufacturer’s instructions. The results were recorded in Table 3 as the compound’s
concentration causing 50% enzyme inhibition (IC50) and they are the means of three
determinations. All compounds showed comparable range of 5-LOX inhibitory activity
with IC50 values range 1.76 - 2.24 µM. Such activities are comparable to the reference
diclofenac sodium. However, comparing their activity to zileuton as a reference drug
appears to show zileuton possessing the least inhibitory activity (4.51 µM), nevertheless,
with the same range of low micromolar range of activity. In conclusion, these results
highlight promising 5-LOX inhibitory activity of the tested compounds.
2
.2.4. Molecular docking
The aim of the modeling pat in our study is to rationalize the in vitro biological results
observed for the most active compounds towards COX-2 inhibition.
8

2
.2.4.1. COX-2 docking
Selecting a definite X-ray structure for COX-2 as a docking model does not emerge to be
significant according to literature [38, 50]. We selected a celecoxib co-crystallized model
(
PDB code: 3NL1) since it is used as a reference compounds for our in vitro biological
evaluation. Also, benchmarking of the widely used docking tools (e.g., GOLD
ChemPLP], Glide [SP] and AutoDock Vina) employing DEKOIS 2.0 benchmark sets
[
showed a good screening performance with all three docking tools [51]. This guided us to
employ GOLD (ChemPLP) as scoring function.
Based on the docking fitness distribution of the docking compounds and reference
standards, celecoxib appeared to be among the high scored binders while indomethacin
and diclofenac showed the lowest docking fitness compared to other compounds (Table
4
). Compounds 20a and 21a demonstrate the highest fitness among the most active
compounds. This observation is attributable to the fact that 20a, 21a and 12c show the
highest topological and size features (e.g. molecular weight, molecular volume and
molecular surface area) compared to other biologically active compounds. Such fact
would usually reward the docking score based on superior molecular size. [52-54]
However, compound 12c do not benefit from such topological features since its
postulated binding mode is dissimilar (data not shown).
Pose prediction employing GOLD (ChemPLP) has been validated by successful pose-
retrieval experiment of the co-crystallized ligand (celecoxib) in the binding site of COX-2
(
RMSD < 1 Å), as shown in Figure 2.
We focus on the most active compounds (14a, 14b and 20a) for elucidating the binding
modes of the docking outcome.
Rationalizing the selectivity of the most active compounds towards COX-2 over COX-1
by docking experiments, we find that these compounds cannot afford a specific binding
mode in the binding site of COX-1 (data not shown). This observation is attributable to
the fact that the topological features and the general size of the binding site of COX-1 is
much smaller than COX-2.[55-57] Besides, the size features (e.g., molecular weight) of
1
4a, 14b, 12c and 20a (Table 4) are generally above the average range of the diverse and
representative COX-1 and COX-2 ligands reported in DEKOIS 2.0 benchmark sets (the
average molecular weight of COX-1 and COX-2 in DEKOIS 2.0 benchmark set are 333.8
(
±62.97) and 373.6 (±50.65), respectively).
The docking poses of the 14a, 14b and 20a compounds showed to access more residues
in the binding site compared to the co-crystallized ligand due to scaffold differences and
substitution pattern (Figure 2B). Since 14a and 14b are closely related congeneric
9

analogues while 20a represents a different pyrazole hybrid, we show in Figure 2B only
docking poses of 14a and 20a.
Binding interactions are inspected to be mainly favorable hydrophobic contacts. For
instance in 14a, the quinolinyl core is packed between Val509 and Ser339 residues,
Figure 3. The chloro group of the quinolinyl moiety is packed between the backbone of
Phe504, Ile503 and Ala502. Also, the benzoyl group shows hydrophobic contacts with
the side chains of Met99, Leu345, Ile331, Leu517 and Leu103. However, the
morpholinyl group established a H-bonding interaction with the side chain of Ser516.
Compound 14b resembles the binding mode of its congener 14a elucidated by their
optimum overlay of their docking poses, Figure 3. Only exception is that the p-
cholobenzoyl group of 14b showed a flip to avoid a possible clash with Met99 and
Ile331. As a result, the p-cholobenzoyl group is directed towards the Tyr101. Overall,
this binding mode produced a comparable docking fitness to 14a.
Compound 20a interacts with the essential residues of the binding site of COX-2
similarly as celecoxib (Figure 2B), however, with extended binding cleft towards the
solvent exposed area via the quinolinyl moiety. The nitrogen atom of quinolinyl moiety
shows H-bonding interaction with His75, as seen in Figure 4. Likewise, the NH group of
the hydrazonocarbonyl group demonstrates H-bonding interaction with Ser339. The N-
cholorophenyl of the pyrazolyl moiety is packed between Val102, Met99 and Leu517
while the 5-chlorophenyl group is packed between Trp373, Tyr371, Phe367 and Leu370.
Unlike celecoxib, the pyrazolyl moiety of 20a is packed between Val509 and Ser339.
Based on our analysis of the docking fitness and postulated binding modes of different
conformers and geometrical isomers we conclude that E isomers appeared to be
distinctively favored for the docked compounds. This highlights the importance of
geometrical isomers for high affinity binding to COX-2.
2
.3. Conclusion
The aim of the present study was to synthesize and investigate the anti-inflammatory
activity of some quinoline derivatives bearing pyrazole moiety through different
linkage with the hope of discovering new lead structures devoid of the GI side effects
associated with conventional NSAIDs. The obtained results clearly revealed that
compounds 5c, 11b,c, 12c, 14a,b, 20a and 21a showed anti-inflammatory activity similar
to or even higher than that of celecoxib. In vitro COX-1/COX-2 inhibition study revealed
that among the synthesized compounds, compounds 12c, 14a and 14b showed the highest
inhibitory activity against COX-2 with an IC50 values of 0.1, 0.11 and 0.11μM
respectively. Additionally,
showed significant in vitro LOX
inhibitory activity higher than that of zileuton and
fi
The
10

docking experiments attempted to postulate the binding mode for the most active
compounds in the binding site of COX-2 enzymes and confirmed the high selectivity
binding towards COX-2 enzyme over COX-1. Consequently, such type of compounds
would represent a fruitful matrix that deserves further investigation and derivatization.
3. Experimental
3
.1. Chemistry
All reagents and solvents were purchased from commercial suppliers and were dried and
purified when necessary by standard techniques. Melting points were determined in open
glass capillaries using Stuart capillary melting point apparatus (Stuart Scientific Stone,
Staffordshire, UK) and are uncorrected. Infrared (IR) spectra were recorded on Perkin-
Elmer 1430 infrared spectrophotometer (Perkin Elmer, Beaconsfield, UK) and measured
-
1
13
by Ú cm 1 scale using KBr cell. H NMR spectra and C-NMR were scanned on
Mercury 300 MHz spectrophotometer or on Jeol-NMR 500 MHz spectrophotometer in
CDCl
3
or DMSO-d
6
and are reported as δ values (ppm) relative to tetramethylsilane
(
TMS) as an internal standard.
3
.1.1. 6-Chloro-2-methylquinoline-4-carboxylic acid (1)
Acetone (24 ml, 330 mmol) was gradually added with stirring and cooling to a solution of
-chloroisatin (4.00 g, 22 mmol) in 33% (w/v) aqueous potassium hydroxide (19 mL).
5
The reaction mixture was heated under reflux with stirring for 16 hours. It was then
concentrated to half its original volume, left to attain room temperature, treated with ice
(
100g) and filtered. The filtrate was acidified with glacial acetic acid whereupon a
precipitate was formed. The product was filtered, washed with water, dried and
-1
crystallized from ethanol. Yield: 90%; Mp: 265-7 ˚C. IR (KBr, cm ): broad band at 3413
1
(
OH); 1707 (C=O); 1603 (C=N). H-NMR (500 MHz, DMSO-d6, δ ppm): 2.67 (s, 3H,
CH ); 7.74 (d, 1H, J= 8.6 Hz, quinolyl-C -H); 7.88 (s, 1H, quinolyl-C -H); 7.97 (d, 1H,
J= 8.6 Hz, quinolyl-C -H); 8.70 (s, 1H, quinolyl-C -H); 13.97 (s, 1H, OH, D
exchangeable). C-NMR (75.44 MHz, DMSO-d , δ ppm): 25.13 (quinoline-C2-CH
24.10, 124.66, 124.71, 130.52, 131.43, 132.07, 135.14, 147.13, 159.94 (quinoline C-5,
C-4a, C-6, C-3, C-8, C-7, C-4, C-8a and C-2 respectively); 167.51 (C=O). Anal. Calcd
ClNO (221.64): C, 59.61; H, 3.64; N, 6.32. Found: C, 59.79; H, 3.70; N, 6.41.
3
7
3
8
5
2
O
1
3
6
3
);
1
for C11
H
8
2
3
.1.2. Ethyl 6-chloro-2-methylquinoline-4-carboxylate (2)
A solution of 1 (8.00 g, 36 mmol) in absolute ethanol (60 mL) containing concentrated
sulfuric acid (7 mL) was heated under reflux for 15 hours. The reaction mixture was
concentrated to a smaller volume, allowed to attain room temperature and then carefully
poured with stirring onto excess 10% sodium hydrogen carbonate solution. The ester
obtained was filtered, washed with water, dried and crystallized from ethanol. Yield:
-1
8
5%; Mp: 94-5˚C. Yield 7.62 g (85%), m.p. 94-5˚C. IR (KBr, cm ):1715 (C=O); 1589
11

1
(
-
C=N); 1249, 1168 (C-O-C). H-NMR (300 MHz, CDCl
CH CH ); 2.80 (s, 3H, CH ); 4.52 (q, 2H, J= 6.9 Hz, -CH
-H); 7.85 (s, 1H, quinolyl-C -H); 8.03 (d, 1H, J= 9 Hz, quinolyl-C
s, 1H, quinolyl-C -H). Anal. Calcd for C13 (249.69): C, 62.53; H, 4.84; N,
.61. Found: C, 62.70; H, 4.90; N, 5.68.
3
, δ ppm): 1.49 (t, 3H, J= 6.9 Hz,
CH ); 7.68 (d, 1H, J= 9 Hz,
-H); 8.79
2
3
3
2
3
quinolyl-C
(
5
7
3
8
5
H
12ClNO
2
3
.1.3. 6-Chloro-2-methylquinoline-4-carbohydrazide (3)
A solution of ethyl 6-chloro-2-methylcinchoninate 2 (2.50 g, 10 mmol) in absolute
ethanol (20 mL) was added with stirring to 99% hydrazine hydrate (2 ml, 40 mmol). The
reaction mixture was heated under reflux for 2 hours whereupon white crystals started to
separate out. After attaining room temperature, the crystalline product was filtered,
washed thoroughly with water, dried and recrystallized from ethanol. Yield: 98 %; Mp:
-
1
1
2
45-6 ˚C. IR (KBr, cm ): 3334, 3257, 3180 (NH
NMR (300 MHz, DMSO-d , δ ppm): 2.68 (s, 3H, CH
exchangeable); 7.49 (s, 1H, quinolyl-C -H); 7.77 (d, 1H, J= 9 Hz, quinolyl -C
d, 1H, J= 9 Hz, quinolyl-C -H); 8.19 (s, 1H, quinolyl-C -H); 9.91 (s, 1H, NH, D
exchangeable). C-NMR (75.44 MHz, DMSO-d , δ ppm): 25.09 (quinoline C-2 CH
21.54, 124.03, 124.57, 130.62, 131.23, 131.30, 140.34, 146.54, 159.67 (quinoline C-5,
C-4a, C-6, C-3, C-8, C-7, C-4, C-8a and C-2 respectively); 165.93 (C=O). Anal. Calcd
O (235.67): C, 56.06; H, 4.28; N, 17.83. Found: C, 56.13; H, 4.26; N,
2
, NH); 1642 (C=O); 1588 (C=N). H-
); 4.68 (s, 2H, NH , D
-H); 8.00
6
3
2
2
O
3
7
(
8
5
2
O
1
3
6
3
);
1
for C11
H10ClN
3
1
8.02.
3
.1.4. 1,3-Diaryl-1H-pyrazole-4-carbaldehydes (4a-c)
To a solution of phenylhydrazine hydrochloride or 4-chlorophenylhydrazine
hydrochloride (70 mmol) in ethanol containing anhydrous sodium acetate (70 mmol), was
added an equimolar amount of p-substituted acetophenone (70 mmol). The reaction
mixture was heated under reflux for 2 hours, filtered while hot to remove the sodium
chloride formed and set aside to attain room temperature. The reaction mixture was then
cooled in an ice bath whereupon a precipitate started to separate. The obtained
hydrazones were filtered, washed with ether and allowed to dry. Under anhydrous
conditions, phosphorus oxychloride (13.80 g, 8.40 mL, 90 mmol) was dropwise added
over a period of 30 minutes to a cold well stirred dry dimethylformamide (38.00 g, 40 ml,
5
20 mmol). Cooling and stirring were continued for further 45 minutes. The appropriate
previously prepared hydrazone (40 mmol) was portionwise added to the well stirred cold
mixture. After attaining room temperature, the reaction mixture was heated for 3 hours at
7
5 °C, allowed to attain room temperature and then poured onto crushed ice (55 g) and
water (100 mL). The reaction mixture was further boiled whereupon a white precipitate
separated out. After attaining room temperature, the crude product was filtered, washed
with water, dried and crystallized from ethanol.
12

3
1
.1.4.1. 1,3-bis(4-chlorophenyl)-1H-pyrazole-4-carbaldehyde (4a). Yield: 96%; Mp: 169-
-1
1
70 °C. IR (KBr, cm ): 2947, 2842 (CH aldehyde), 1684 (C=O); 1607 (C=N). H-NMR
, δ ppm): 7.5 (dd, 4H, J1= 8.4, J2 = 4.2 Hz, pyrazolyl-C3,4-
(
300 MHz, CDCl
3
chlorophenyl-C3,5-H and pyrazolyl-N1-4-chlorophenyl-C3,5-H); 7.75 (d, 2H, J= 8.7 Hz,
pyrazolyl-C3-4-chlorophenyl-C2,6-H); 7.83 (d, 2H, J= 8.4 Hz, pyrazolyl-N1-4-
chlorophenyl-C2,6-H); 8.51 (s, 1H, pyrazolyl-C
5
-H); 10.05 (s, 1H, CHO). Anal. Calcd for
C
l6
H
10Cl
2
N
2
O )713ꢀ13(: C, 60.59; H, 3.18; N, 8.83. Found: C, 60.71; H, 3.21; N, 9.04.
3
.1.4.2. 3-(4-methoxyphenyl)-1-phenyl-1H-pyrazole-4-carbaldehyde (4b). Yield: 96%;
Mp: 169-170 °C [58].
3
.1.4.3. 1-(4-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazole-4-carbaldehyde (4c).
-1
Yield: 88%; Mp: 136-7 °C. IR (KBr, cm ):2921, 2815 (CH aldehyde), 1671 (C=O); 1605
1
(
7
C=N); 1219, 1176 (C-O-C). H-NMR (300 MHz, CDCl
.04 (d, 2H, J= 8.1 Hz, 4-methoxyphenyl-C3,5-H); 7.48 (d, 2H, J= 8.1 Hz, 4-
methoxyphenyl-C2,6-H); 7.73-7.80 (m, 4H, 4-chlorophenyl-H); 8.49 ( s, 1H, pyrazolyl-
-H); 10.05 (s, 1H, CHO). Anal. Calcd for C17 )71.ꢀ3ꢁ(: C, 65.29; H, 4.19;
N, 8.96. Found: C, 65.37; H, 4.24; N, 9.08.
3
, δ ppm): 3.88 (s, 3H, OCH );
3
C
5
H13ClN
2
O
2
3
.1.5. N'-[(1,3-Diaryl-1H-pyrazol-4-yl)methylene]6-chloro-2-methylquinoline-4-
carbohydrazides (5a-c)
A solution of the appropriate 1,3-diaryl-1H-pyrazole-4-carbaldehyde 4a-c(2 mmol) in
absolute ethanol (30 ml) was gradually added to a solution containing an equimolar
amount of the acid hydrazide 3 (0.47 g) in absolute ethanol (30 ml). The reaction mixture
was heated under reflux for 2 hours and then set aside for an overnight whereupon white
crystals separated out. The products obtained were filtered, washed with water, dried and
recrystallized from ethanol.
3
.1.5.1.
N'-[(1,3-bis(4-chlorophenyl)-1H-pyrazol-4-yl)methylene]6-chloro-2-
-1
methylquinoline-4-carbohydrazide (5a). Yield: 71%; Mp: 298-300 °C. IR (KBr, cm ):
1
3
3
2
182 (NH); 1652 (C=O); 1595 (C=N). H-NMR (300 MHz, DMSO-d
H, CH ); 7.39-7.85 (m, 9H, the two chlorophenyl-H and quinolyl-C
H, quinolyl-C7,8-H); 8.17 (s, 1H, quinolyl-C -H); 8.43 (s, 1H, CH=N); 9.12 (s, 1H,
-H); 12.11 (s, 1H, NH, D O exchangeable). Anal. Calcd for C27
534.82): C, 60.63; H, 3.39; N, 13.09. Found: C, 60.72; H, 3.43; N, 13.32.
6
, δ ppm): 2.70 (s,
3
3
-H); 8.00-8.07 (m,
5
pyrazolyl-C
(
5
2
H
18Cl
3
5
N O
3
.1.5.2.
6-Chloro-N'-[(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)methylene]-2-
-1
methylquinoline-4-carbohydrazide (5b). Yield: 94%; Mp: 275-6 °C. IR (KBr, cm ):
3
d
1
190 (NH); 1652 (C=O); 1599 (C=N); 1250-1175 (C-O-C). H-NMR (300 MHz, DMSO-
, δ ppm): 2.73 (s, 3H, CH ); 3.82 (s, 3H, OCH ); 7.07 (d, 2H, J= 9 Hz, 4-
6
3
3
methoxyphenyl-C3,5-H); 7.29-7.86 (m, 8H, 4-methoxyphenyl-C2,6-H, phenyl-H and
13

quinolyl-C
.47(s, 1H, CH=N); 9.04 (s, 1H, pyrazolyl-C
Anal. Calcd for C28 (69ꢁꢀ94): C, 67.81; H, 4.47; N, 14.12. Found: C, 67.87; H,
3
-H); 8.03 (d, 2H, J= 8.7 Hz, quinolyl-C7,8-H); 8.20 (s, 1H, quinolyl-C
5
-H);
8
5
-H). 12.04 (s, 1H, NH, D O exchangeable).
2
H22ClN
5
O
2
4
.49; N, 14.19.
3
.1.5.3.
6-Chloro-N'-[(1-(4-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-
yl)methylene]-2-methylquinoline-4-carbohydrazide (5c).(Yield: 94%; Mp: 270-2 °C. IR
-
1
1
(
KBr, cm ): 3187 (NH); 1653 (C=O); 1596 (C=N); 1249-1175 (C-O-C). H-NMR (300
MHz, DMSO-d , δ ppm): 2.72 (s, 3H, CH ); 3.82 (S, 3H, OCH ); 7.08 (d, 2H, J= 8.7 Hz,
-methoxyphenyl-C3,5-H) ; 7.60 (d, 2H, J= 8.4 Hz, 4-chlorophenyl-C3,5-H); 7.67 (d, 2H,
J= 8.4 Hz, 4-chlorophenyl-C2,6-H); 7.71 (s, 1H, quinolyl-C -H); 7.78-8.11 (m, 4H, 4-
methoxyphenyl-C2,6-H and quinolyl-C7,8-H); 8.20 (s, 1H, quinolyl-C -H); 8.46 (s, 1H,
CH=N); 9.09 (s, 1H, pyrazolyl-C -H); 12.05 (s, 1H, NH, D O exchangeable). C-NMR
75.44 MHz, DMSO-d , δ ppm): 24.71 (CH ); 55.22 (OCH ); 114.14 (4-methoxyphenyl
6
3
3
4
3
5
1
3
5
2
(
6
3
3
C-3/5); 120.17 (4-chlorophenyl C-2/6); 120.42 (pyrazole C-4); 121.29 (4-methoxyphenyl
C-1); 123.37, 123.80 and 124.02 (quinoline C-5, C-3 and C-7 respectively); 127.51 (4-
chlorophenyl C-3/5); 129.44 (4-methoxyphenyl C-2/6); 129.70 (pyrazole C-5); 130.30,
1
(
1
30.87 (quinoline C-8 and C-6 respectively); 131.02 (4-chlorophenyl C-4); 131.10
quinoline C-4a); 137.83 (4-chlorophenyl C-1); 139.13 (quinoline C-4); 142.34 (CH=N);
46.12 (quinoline C-8a); 152.12 (quinoline C-2); 159.23 (pyrazole C-3); 159.69 ( 4-
+
•
+•
methoxyphenyl C-4); 161.81 (C=O). MS (m/z, %): 532 (M +2) (5); 531(M +1) (13);
5
1
+
•
30 (M )(15) 176 (100) Anal. Calcd for C28
3.20. Found: C, 63.48; H, 3.97; N, 13.31).
H
21Cl
2
N
5
2
O (ꢁ75ꢀ65): C, 63.40; H, 3.99; N,
3
.1.6. 6-Chloro-2-methyl-N'-(1-methyl-3-oxobutylidene)quinoline-4-carbohydrazide (6).
Acetylacetone (0.15 mL, 1.5 mmol) was added to a solution containing an equimolar
amount of acid hydrazide 3 (0.35g) in ethanol (30 mL). The reaction mixture was heated
under reflux for 4 hours and then set aside to attain room temperature whereupon a
precipitate was formed. The precipitate obtained was filtered, washed with 50% cold
-1
ethanol, dried and crystallized from ethanol. Yield: 89 %; Mp: 160-1˚C. IR (KBr, cm ):
1
3
3
232 (NH); 1633 (C=O); 1598 (C=N). H-NMR (300 MHz, DMSO-d
H, CH -C=N-NH); 1.99 (s, 3H, CH -C=O); 2.67 (s, 3H, quinolyl-C -CH
); 3.05 (d, 1H, J= 18.9 Hz, CH ); 6.97 (s, 1H, NH, D O exchangeable);
.38 (s, 1H, quinolyl-C -H) ; 7.76 (d, 1H, J= 9 Hz, quinolyl-
6
, δ ppm): 1.78 (s,
3
3
2
3
); 2.90 (d, 1H,
J= 18.9 Hz, CH
7
C
ppm): 15.64(CH
9
2
2
2
3
-H); 7.71 (s, 1H, quinolyl-C
5
3
1
7
-H); 7.99 (d, 1H, J= 9.3 Hz, quinolyl-C
8
-H). C-NMR (75.44 MHz, DMSO-d
6
, δ
-C=O); 52.21 (CH );
1.02 (quinoline C-5), 119.54, 122.91, 123.38, 130.06, 130.57, 130.74 and 142.92 (C-4a,
C-3, C-7, C-8, C-6, C-4 and C-8a respectively); 145.69 (CH -C=N); 156.86 (quinoline C-
); 159.19 (NH-C=O); 164.07 (CH -C=O). Anal. Calcd for C16 (317.77):
C,60.47; H, 5.08; N, 13.22. Found: C, 60.56; H, 5.13; N, 13.34.
3
-C=N-NH); 24.70 (quinoline C-2 CH
3
); 25.82 (CH
3
2
3
2
3
H
16ClN
3
O
2
14

3
.1.7. 6-Chloro-4-[(3,5-dimethyl-1H-pyrazol-1-yl)carbonyl]2-methylquinoline (7)
A mixture of acid hydrazide 3 (0.47 g, 2 mmol) and excess acetylacetone (3 mL, 30
mmol) was heated at 110 °C for 6 hours. The reaction mixture was set aside to attain
room temperature whereupon a precipitate was formed. The product obtained was
triturated with diethyl ether to break the lumps formed, filtered, washed with diethyl
-1
ether, dried and crystallized from ethanol. Yield: 68 %; Mp: 199-200˚C. IR (KBr, cm ):
1
1
710 (C=O); 1598 (C=N). H-NMR (500 MHz, CDCl
CH ); 2.73 (s, 3H, pyrazolyl-C -CH ); 2.78 (s, 3H, quinolyl-C
pyrazolyl-C -H); 7.42 (s, 1H, quinolyl-C -H); 7.63 (d, 1H, J= 8.4 Hz, quinolyl-C
.71 (s, 1H, quinolyl-C -H); 8.02 (d, 1H, J= 8.4 Hz, quinolyl-C
MHz, DMSO-d , δ ppm): 13.36 and 13.87 (pyrazole C-3 and C-5 CH
C-2 CH ); 112.28 (pyrazole C-4); 121.37, 122.86, 123.44, 130.43, 130.86, 131.18 and
40.23 (quinoline C-5, C-4a, C-3, C-7, C-8, C-6 and C-4 respectively); 144.70 (pyrazole
C-5); 145.67 (quinoline C-8a); 153.34 (pyrazole C-3); 158.95 (quinoline C-2); 166.48
3
, δ ppm): 2.12 (s, 3H, pyrazolyl-C
-CH ); 6.11 (s, 1H,
-H);
-H). C-NMR (75.44
); 24.76 (quinoline
5
-
3
3
3
2
3
4
3
7
13
7
5
8
6
3
3
1
+
•
+•
+•
(
C
C=O). MS (m/z, %):301 (M +2) (13); 300 (M‏ + 1) (8); 299 (M‏ ) (37).Anal. Calcd for
O (299.75): C, 64.11; H, 4.71; N, 14.02. Found: C, 64.23; H, 4.68; N, 14.13.
16
H
14ClN
3
3
.1.8.. Ethyl 3-[2-(6-chloro-2-methylquinolin-4-ylcarbonyl)hydrazino]-3-oxopropanoate
(8)
A mixture of acid hydrazide 3 (0.35 g, 1.5 mmol) and excess diethyl malonate (2 mL,
3.2 mmol) was heated at 160˚C for half an hour. The reaction mixture was set aside to
1
attain room temperature whereupon a precipitate was formed. The product obtained was
triturated with diethyl ether, filtered, washed with diethyl ether, air dried and crystallized
-1
from ethanol. Yield: 90 %; Mp: 200-2˚C. IR (KBr, cm ): 3184 (NH); broad band
1
centered at 1744 (C=O); 1268, 1164 (C-O-C). H-NMR (300 MHz, DMSO-d
ppm):1.22 (t, 3H, J= 7.2 Hz, -CH CH ); 2.70 (s, 3H, CH ); 3.42 (s, 2H, CH ); 4.14 (q,
H, J= 7.2 Hz, -CH CH ); 7.57 (s, 1H, quinolyl-C -H); 7.80 (d, 1H, J= 9 Hz, quinolyl-C
H); 8.02 (d, 1H, J= 9 Hz, quinolyl-C -H); 8.31 (s, 1H, quinolyl-C -H); 10.39, 10.73 (2s,
O exchangeable). Anal. Calcd for C16 (349.77): C, 54.94; H,
.61; N, 12.01. Found: C, 55.13; H, 4.67; N, 12.14.
6
, δ
2
3
3
2
2
2
3
3
7
-
8
5
2
4
H, 2NH, D
2
H
16ClN
3
O
4
3
.1.9. 1-(6-Chloro-2-methylquinolin-4-ylcarbonyl) pyrazolidine-3,5-dione (9)
Diethyl malonate (0.5 mL, 3.28 mmol) was added to a solution containing an equimolar
amount of acid hydrazide 3 (0.77 g, 3.28 mmol) in absolute ethanol (40 mL) containing
one drop of concentrated sulphuric acid. The reaction mixture was heated under reflux for
3
hours whereupon a white precipitate started to form. The reaction mixture was set aside
to attain room temperature for complete precipitation and the precipitate formed was
filtered, washed with cold ethanol, dried and crystallized from ethanol. Yield: 70 %; Mp:
-1
1
1
68-170˚C. IR (KBr, cm ): 3251 (NH); 1702, 1641 (C=O). H-NMR (300 MHz, DMSO-
15

d
6
, δ ppm):2.69 (s, 3H, quinolyl-C
quinolyl-C -H); 7.85 (d, 1H, J= 8.7 Hz, quinolyl-C
-H); 8.18 (s, 1H, quinolyl-C -H); 11.05 (s, 1H, NH, D
75.44 MHz, DMSO-d , δ ppm): 24.41 (quinoline C-2 CH
2
-CH
3
); 3.73 (s, 2H, pyrazolidinyl-C
4
-H); 7.65 (s, 1H,
3
7
-H); 8.05 (d, 1H, J= 8.7 Hz, quinolyl-
1
3
C
(
8
5
2
3
O exchangeable). C-NMR
); 40.32 (pyrazolidine C-4,
6
under DMSO); 121.47, 121.83, 122.96, 123.61, 130.38, 130.89, 131.68, 137.28 and
45.33 (quinoline C-); 159.36 ,165.05 , 165.37 (3 C=O). Anal. Calcd for C14
303.70): C, 55.37; H, 3.32; N, 13.84. Found: C, 55.48; H, 3.35; N, 13.92.
1
H10ClN
3
O
3
(
3
.1.10. 6-Chloro-2-methyl-4-(morpholin-4-yl) carbonylquinoline (10)
A mixture of ethyl 6-chloro-2-methylquinoline-4-carboxylate 2 (3.25 g, 13 mmol) and
excess morpholine (6.10 g, 6.00 mL, 70 mmol) was heated under reflux for 24 hours, the
reaction mixture was left to attain room temperature and then poured onto cold water (50
mL). The product separated was filtered, washed several times with water, dried and
-1
crystallized from ethanol. Yield: 70 %; Mp: 144-5˚C. IR (KBr, cm ): 1637(C=O); 1594
1
(
3
(
C
C=N); 1108, 1066 (C-O-C). H-NMR (300 MHz, CDCl
.17-3.26 (m, 2H, morpholin-C3a,5a-H); 3.54-3.62 (m, 2H, morpholin-C3e,5e-H); 3.79-4.06
m, 4H, morpholin-C2,6-H); 7.25 (s, 1H, quinolyl-C -H); 7.69 (d, 1H, J= 9 Hz, quinolyl-
-H); 8.06 (d, 1H, J= 9 Hz, quinolyl-C -H). Anal. Calcd
(290.74): C, 61.97; H, 5.20; N, 9.64. Found: C, 62.08; H, 5.25; N, 9.79.
3
, δ ppm): 2.78 (s, 3H, CH );
3
3
7
-H); 7.77 (s, 1H, quinolyl-C
5
8
for C15
H15ClN
2
O
2
3
.1.11. Ethyl 2-[2-(1,3-diaryl-1H-pyrazol-4-yl)ethenyl]-6-chloroquinoline-4-carboxylates
(11a-c)
To a solution of (1.00 g, 4 mmol) of ethyl 6-chloro-2-methylcinchoninate 2 in acetic
anhydride (20 mL), was added an equimolar amount of the appropriate 1,3-diaryl-1H-
pyrazole-4-carbaldehyde 4a-c.The reaction mixture was heated under reflux for 20 hours
and was then set aside to attain room temperature. The precipitates formed were filtered,
washed with ethanol, dried and crystallized from glacial acetic acid.
3
.1.11.1.
Ethyl
2-[2-(1,3-bis(4-chlorophenyl)-1H-pyrazol-4-yl)ethenyl]-6-
-1
chloroquinoline-4-carboxylate (11a). Yield: 71%; Mp: 180-2 °C. IR (KBr, cm ): 1722
1
(
3
C=O); 1590 (C=N); 1239-1172 (C-O-C). H-NMR (500 MHz, CDCl
H, J= 6.9 Hz, CH CH ); 4.49 (q, 2H, J= 6.9 Hz, CH CH ); 7.15 (d, 1H, J= 16.00 Hz, -
CH=CH-); 7.41-7.47 (m, 4H, pyrazolyl-N1-4-chlorophenyl-C3,5-H and pyrazolyl-C3-4-
chlorophenyl-C3,5-H); 7.62-7.67 (m, 6H, quinolyl-C -H, -CH=CH-, pyrazolyl-N1-4-
chlorophenyl-C2,6-H and pyrazolyl-C3-4-chlorophenyl-C2,6-H); 7.94-8.03 (m, 2H,
3
, δ ppm): 1.47 (t,
2
3
2
3
3
1
3
quinolyl-C-7,8-H); 8.21 (s, 1H, quinolyl-C
75.44 MHz, CDCl , δ ppm):14.54 (-CH CH
); 120.50 (pyrazole C-4); 121.82 (pyrazole-N1-4-chlorophenyl C-2/6); 124.85, 124.96
5
-H); 8.71 (s, 1H, pyrazolyl-C
5
-H). C-NMR
(
3
3
2
3
); 62.41 (-CH CH ); 119.95 (quinoline C-
2
3
and 125.44 (quinoline C-5, C-4a and C-6 respectively); 125.69 (pyrazole-C3-4-
chlorophenyl C-2/6); 128.41 (pyrazole-N1-4-chlorophenyl C-3/5 and pyrazole-C3-4-
16

chlorophenyl C-3/5); 129.26 (pyrazole C-5); 129.94 (quinoline C-8 and pyrazole-C3-4-
chlorophenyl C-1); 130.17 (pyrazole-N1-4-chlorophenyl C-4); 131.27 (quinoline C-7);
1
1
3
32.84 (-CH=CH-); 133.96 (quinoline C-4); 134.93 (pyrazole-C3-4-chlorophenyl C-4);
38.30 (pyrazole-N1-4-chlorophenyl C-1); 147.72 (quinoline C-8a); 151.96 (pyrazole C-
+
•
+•
); 155.56 (quinoline C-2); 165.98 (C=O). MS (m/z, %): 550(M +2) (31); 549(M +1)
+
•
(
39); 548(M ) (41). Anal. Calcd for C29
H
20Cl
3
N
3
O (548.85): C, 63.46; H, 3.67; N, 7.66.
2
Found: C, 63.62; H, 3.74; N, 7.82.
3
.1.11.2.
Ethyl
2-[2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)ethenyl]-6-
-1
chloroquinoline-4-carboxylate (11b). Yield: 64%; Mp: 210-2 °C. IR (KBr, cm ): 1725
1
(
(
4
(
C=O); 1595 (C=N); 1242-1172 (C-O-C, ester); 1092-1027 (C-O-C, OCH
300 MHz, DMSO-d , δ ppm): 1.41 (t, 3H, J= 6.9 Hz, CH CH ); 3.85 (s, 3H, OCH
.47 (q, 2H, J= 6.9 Hz, CH CH ); 7.14 (d, 2H, J= 6.9 Hz, 4-methoxyphenyl-C3,5-H); 7.37
t, 1H, J= 7.8 Hz, phenyl-C -H); 7.46 (d, 1H, J= 16.20 Hz, -CH=CH-); 7.57 (t, 2H, J= 7.8
Hz, phenyl-C3&5-H ); 7.67 (d, 2H, J= 9 Hz, 4-methoxyphenyl-C2,6-H); 7.74 (s, 1H,
quinolyl-C -H); 7.75-8.15 (m, 5H, phenyl-C2,6-H, quinolyl-C7,8-H and -CH=CH-); 8.61
s, 1H, quinolyl-C -H); 9.17 (s, 1H, pyrazolyl-C -H). Anal. Calcd for C30
509.98): C, 70.65; H, 4.74; N, 8.24. Found: C, 70.73; H, 4.75; N, 8.31.
3
). H-NMR
6
2
3
3
);
2
3
4
3
(
(
5
5
H24ClN
3
O
3
3
6
.1.11.3. Ethyl 2-[2-(1-(4-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-yl)ethenyl]-
-1
-chloroquinoline-4-carboxylate (11c). Yield: 60%; Mp: 208-9 °C. IR (KBr, cm ): 1726
1
(
(
(
1
(
C=O); 1594 (C=N); 1242-1175 (C-O-C, ester); 1088-1027 (C-O-C, OCH
300 MHz, CDCl , δ ppm): 1.51 (t, 3H, J= 7.2 Hz, CH CH ); 3.91 (s, 3H, OCH
q, 2H, J= 7.2 Hz, CH CH ); 7.08 (d, 2H, J= 8.7 Hz, 4-methoxyphenyl-C3,5-H); 7.32 (d,
H, J= 16.50 Hz, -CH=CH-); 7.48 (d, 2H, J= 8.7 Hz, 4-chlorophenyl-C3,5-H); 7.69-7.79
m, 6H, 4-methoxyphenyl-C2,6-H, 4-chlorophenyl-C2,6-H, -CH=CH- and quinolyl-C -H);
-H); 8.76 (s, 1H, pyrazolyl-
(544.43): C, 66.18; H, 4.26; N, 7.72. Found: C,
3
). H-NMR
3
2
3
3
); 4.55
2
3
3
8
C
.10-8.14 (m, 2H, quinolyl-C7,8-H); 8.32 (s, 1H, quinolyl-C
-H). Anal. Calcd for C30
6.24; H, 4.28; N, 7.88.
5
5
H
23Cl
2
N
3
O
3
6
3
.1.12. 6-Chloro-2-[2-(1,3-diaryl-1H-pyrazol-4-yl)ethenyl]-4-(morpholin-4-
ylcarbonyl)quinolines (12a-c)
The appropriate 1,3-diaryl-1H-pyrazole-4-carbaldehyde 4a-c (3.5 mmol) was added to a
solution
containing
an
equimolar
amount
of
6-chloro-2-methyl-4-
morpholinocarbonylquinoline 10 (1.02 g) in acetic anhydride (20 mL). The reaction
mixture was heated under reflux for 20 hours and was then set aside to attain room
temperature. The precipitates obtained were filtered, washed with ethanol, dried and
crystallized from glacial acetic acid.
3
.1.12.1.
6-Chloro-2-[2-(1,3-bis(4-chlorophenyl)-1H-pyrazol-4-yl)ethenyl]-4-
-1
(morpholin-4-ylcarbonyl) quinoline (12a).Yield: 50%; Mp: 235-7 °C. IR (KBr, cm ):
17

1
1
3
643 (C=O); 1535 (C=N); 1249-1113 (C-O-C). H-NMR (300 MHz, DMSO-d
6
, δ ppm):
.13-3.25 (m, 2H, morpholin-C3a,5a-H); 3.39-3.64 (m, 2H, morpholin-C3e,5e-H); 3.70-3.88
(
m, 4H, morpholin-C2,6-H); 7.40 (d, 1H, J= 16.20 Hz, -CH=CH-); 7.59-7.63 (m, 4H,
pyrazolyl-N1-4-chlorophenyl-C3,5-H and pyrazolyl-C3-4-chlorophenyl-C3,5-H); 7.71-7.81
m, 5H, pyrazolyl-N1-4-chlorophenyl-C2,6-H, pyrazolyl-C3-4-chlorophenyl-C2,6-H and -
CH=CH); 7.88 (s, 1H, quinolyl-C -H); 8.00 (d, 2H, J= 8.7 Hz, quinolyl-C7,8-H); 8.04 (s,
H, quinolyl-C -H); 9.22 (s, 1H, pyrazolyl-C -H). Anal. Calcd for C31
589.90): C, 63.12; H, 3.93; N, 9.50. Found: C, 63.18; H, 3.97; N, 9.58.
(
3
1
(
5
5
H
23Cl
3
N
4
O
2
3
.1.12.2.6-Chloro-2-[2-(3-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-4-yl)ethenyl]-4-
-
1
(morpholin-4-ylcarbonyl)quinoline (12b). Yield: 60%; Mp: 251-2 °C. IR (KBr, cm ):
1
1
(
639 (C=O); 1533 (C=N); 1247-1115 (C-O-C); 1176-1056 (C-O-C, OCH
300 MHz, DMSO-d , δ ppm): 3.14-3.25 (m, 2H, morpholin-C3a,5a-H); 3.40-3.60 (m, 2H,
morpholin-C3e,5e-H); 3.72-3.80 (m, 4H, morpholin-C2,6-H); 3.85 (s, 3H, OCH ); 7.12 (d,
H, J= 8.85 Hz, 4-methoxyphenyl-C3,5-H); 7.32-7.43 (m, 2H, -CH=CH- and phenyl-C
H); 7.55 (t, 2H, J= 8.4 Hz, phenyl-C3,5-H); 7.68 (d, 2H, J= 8.85 Hz, 4-methoxyphenyl-
2,6-H); 7.73-7.81 (m, 3H, -CH=CH- and phenyl-C2,6-H); 7.82 (s, 1H, quinolyl-C -H);
.93-8.03 (m, 3H, quinolyl-C5,7,8-H); 9.14 (s, 1H, pyrazolyl-C -H). Anal. Calcd for
3
). H-NMR
6
3
2
4
-
C
7
3
5
C
32
H27ClN
4
O
3
(551.03): C, 69.75; H, 4.94; N, 10.17. Found: C, 69.83; H, 4.96; N, 10.32.
3
.1.12.3.
6-Chloro-2-[2-(1-(4-chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-
yl)ethenyl]-4-(morpholin-4-ylcarbonyl)quinoline (12c). Yield: 65%; Mp: 240-1 °C. IR
-
1
(
KBr, cm ): 1639 (C=O); 1533 (C=N); 1249-1114 (C-O-C); 1177-1087 (C-O-C, OCH
3
).
, δ ppm): 3.15-3.26 (m, 2H, morpholin-C3a,5a-H); 3.56-
.58 (m, 2H, morpholin-C3e,5e-H); 3.73-3.82 (m, 4H, morpholin-C2,6-H); 3.85 (s, 3H,
OCH ); 7.11 (d, 2H, J= 9 Hz, 4-methoxyphenyl-C3,5-H); 7.38 (d, 1H, J= 16.20 Hz, -
1
H-NMR (300 MHz, DMSO-d
6
3
3
CH=CH-); 7.61 (d, 2H, J= 9 Hz, 4-chlorophenyl-C3,5-H); 7.67 (d, 2H, J= 9 Hz, 4-
methoxyphenyl-C2,6-H); 7.72-7.80 (m, 3H, -CH=CH- and 4-chlorophenyl-C2,6-H); 7.83
(
s, 1H, quinolyl-C
3
-H); 7.97-8.02 (m, 3H, quinolyl-C5,7,8-H); 9.17 (s, 1H, pyrazolyl-C
H). C-NMR (75.44 MHz, DMSO-d , δ ppm): 47.01 (morpholin C-3/5); 55.20 (OCH
6.14 (morpholin C-2/6); 114.28 (quinoline C-3); 117.36 (4-methoxyphenyl C-3/5);
19.00 (pyrazole C-4); 119.95 (4-chlorophenyl C-2/6); 123.30 (quinoline C-4a); 123.51
5
-
);
1
3
6
3
6
1
(
4-methoxyphenyl C-1); 124.63, 125.46 (quinoline C-5 and C-6); 127.27 (4-
methoxyphenyl C-2/6); 128.20 (4-chlorophenyl C-3/5); 129.39 (pyrazole C-5); 129.49
quinoline C-8); 129.57 (4-chlorophenyl C-4); 130.66 (quinoline C-7); 130.83 (CH=CH);
(
1
8
1
31.29 (CH=CH); 137.97 (4-chlorophenyl C-1); 141.41, 146.30 (quinoline C-4 and C-
a); 151.56 (pyrazole C-3); 155.99 (quinoline C-2); 159.54 (4-methoxyphenyl C-4);
65.25 (C=O). Anal. Calcd for C32
H
26Cl
2
N
4
3
O (585.48): C, 65.65; H, 4.48; N, 9.57.
Found: C, 65.72; H, 4.52; N, 9.65.
18

3
.1.13. Phenyl glyoxal or 4-chlorophenyl glyoxal (13a,b).
Compound13a (R = H), yield: 91%; Mp: 75-6 ˚C [59].Compound13b (R = Cl), yield:
8
2%; Mp:115-6 ˚C [60]
3
.1.14. 3-[6-Chloro-4-(morpholin-4-ylcarbonyl)quinolin-2-yl]-1-arylprop-2-en-1-ones
(
14a,b)
Phenyl glyoxal or 4-chlorophenyl glyoxal 13a,b (3.75 mmol) was added to a solution of
-chloro-2-methyl-4-morpholinocarbonylquinoline 10 (1.00 g, 3.5 mmol) in acetic
6
anhydride (20 mL).The reaction mixture was heated under reflux for 5 hours, set aside to
attain room temperature and then poured onto ice. The precipitates formed were filtered,
washed with water and crystallized from 70% aqueous ethanol.
3
.1.14.1. 3-[6-Chloro-4-(morpholin-4-ylcarbonyl) quinolin-2-yl]-1-phenylprop-2-en-1-
-
1
one (14a). Yield: 91%; Mp: 143-5 °C. IR (KBr, cm ): 1709 (C=C-C=O); 1634 (N-C=O);
1
2
1
546 (C=N); 1111-1100 (C-O-C). H-NMR (300 MHz, DMSO-d
H, morpholin-C3a,5a-H); 3.40-3.65 (m, 2H, morpholin-C3e,5e-H); 3.70-3.95 (m, 4H,
-H and phenyl-H); 8.15-8.23
-H); 8.37 (d, 1H, J= 15.6 Hz, -CH=CH-
(406.86): C, 67.90; H, 4.71; N, 6.89. Found: C, 67.98;
6
, δ ppm): 3.10-3.35 (m,
morpholin-C2,6-H); 7.46-7.91 (m, 7H, CH=CH, quinolyl-C
3
(
)
m, 2H, quinolyl-C7,8-H); 8.31 (s, 1H, quinolyl-C
. Anal. Calcd for C23
5
H
19Cl
1
N
2
O
3
H, 4.68; N, 6.96.
3
2
.1.14.2. 3-[6-Chloro-4-(morpholin-4-ylcarbonyl)quinolin-2-yl]-1-(4-chlorophenyl)prop-
-1
-en-1-one (14b). Yield: 91%; Mp: 259-260 °C. IR (KBr, cm ): 1714 (C=C-C=O); 1641
1
(
N-C=O); 1592 (C=N); 1248-1239 (C-O-C). H-NMR (300 MHz, DMSO-d
6
, δ ppm):
3
.17-3.26 (m, 2H, morpholin-C3a,5a-H); 3.40-3.64 (m, 2H, morpholin-C3e,5e-H); 3.72-3.90
(
m, 4H, morpholin-C2,6-H); 7.68 (d, 2H, J= 8.4 Hz, 4-chlorophenyl-C3,5-H); 7.80 (d, 1H,
J= 15.75, -CH=CH-); 7.85-7.90 (m, 2H, quinolyl-C3,7-H); 8.16 (d, 1H, J= 9 Hz, quinolyl-
-H); 8.20 (d, 2H, J= 8.4 Hz, 4-chlorophenyl-C2,6-H); 8.31 (s, 1H, quinolyl-C -H); 8.35
d, 1H, J= 15.75 Hz, -CH=CH-). Anal. Calcd for C23 (441.31): C, 62.60; H,
C
(
8
5
H
18Cl
2
N
2
O
3
4
.11; N, 6.35. Found: C, 62.67; H, 4.13; N, 6.49.
3
.1.15. Ethyl 1,5-diaryl-1H-pyrazole-3-carboxylates (16a-c).
1
Compound16a (R = R = 4-Cl-C
6
H
4
), yield: 57%; Mp: 102-2 ˚C [61]. Compound16b (R
), yield: 87%; Mp: 67-9˚C [62]. Compound16c (R = 4-Cl-
), yield: 64%; Mp: 116-7˚C [62].
1
=
C
6
H
5
, R = 4-OCH
3
-C
6
H
4
1
C
6
H
4
, R = 4-OCH
3
-C
6
H
4
3
.1.16. 1,5-Diaryl-1H-pyrazole-3-carbohydrazides (17a-c)
A solution of the appropriate ethyl 1,5-diaryl-1H-pyrazole-3-carboxylate 16a-c (10
mmol) in ethanol (30 mL) was gradually added with stirring to 99% hydrazine hydrate (5
mL, 100 mmol). The reaction mixture was heated under reflux for 5 hours, concentrated
19

under diminished pressure to (15 mL) and allowed to attain room temperature. The
products separated were filtered, washed with cold 50% aqueous ethanol, dried and
crystallized from ethanol.
3
.1.16.1. 1,5-Bis(4-chlorophenyl)-1H-pyrazole-3-carbohydrazide (17a). Yield: 91%; Mp:
-1
1
1
10-2 °C. IR (KBr, cm ): 3345, 3274, 3131 (NH
, δ ppm): 4.50 (s, 2H, NH
-H); 7.29 (d, 2H, J= 8.4 Hz, pyrazolyl-N1-4-chlorophenyl-C3,5-H); 7.36 (d,
H, J= 8.7 Hz, pyrazolyl-C5-4-chlorophenyl-C3,5-H); 7.46 (d, 2H, J= 8.7 Hz, pyrazolyl-
C5-4-chlorophenyl-C2,6-H); 7.53 (d, 2H, J= 8.4 Hz, pyrazolyl-N1-4-chlorophenyl-C2,6
H); 9.59 (s, 1H, NH, D O exchangeable). Anal. Calcd for C16 O (347.20): C,
5.35; H, 3.48; N, 16.14. Found: C, 55.43; H, 3.50; N, 16.22.
2
, NH); 1676 (C=O); 1618 (C=N). H-
NMR (300 MHz, DMSO-d
pyrazolyl-C
6
2
, D O exchangeable); 7.04 (s, 1H,
2
4
2
-
2
H
12Cl
2
N
4
5
3
.1.16.2. 5-(4-Methoxyphenyl)-1-phenyl- 1H-pyrazole-3-carbohydrazide (17b).Yield:
-1
6
6%; Mp: 162-3 °C. IR (KBr, cm ): 3325, 3271, 3100 (NH
2
, NH); 1649 (C=O); 1592
, δ ppm): 3.75 (s, 3H,
O exchangeable); 6.92-6.95 (m, 3H, pyrazolyl-C -H and 4-
methoxyphenyl-C3,5-H); 7.18 (d, 2H, J= 8.7 Hz, 4-chlorophenyl-C3,5-H); 7.35 (d, 2H, J=
1
(
C=N); 1245, 1171 (C-O-C). H-NMR (300 MHz, DMSO-d
6
OCH ); 4.49 (s, 2H, NH , D
3
2
2
4
8
1
1
.7 Hz, 4-methoxyphenyl-C2,6-H); 7.50 (d, 2H, J= 9 Hz, 4-chlorophenyl-C2,6-H); 9.55 (s,
H, NH, D O exchangeable). Anal. Calcd for C17 (308.33): C, 66.22; H, 5.23; N,
8.17. Found: C, 66.35; H, 5.21; N, 18.34.
2
H
16
N
4
O
2
3
.1.16.3. 1-(4-chlorophenyl)-5-(4-methoxyphenyl)-1H-pyrazole-3-carbohydrazide (17c).
-1
Yield: 82%; Mp: 140-2 °C. IR (KBr, cm ): 3325, 3271, 3100 (NH
2
, NH); 1649 (C=O);
, δ ppm): 3.75 (s, 3H,
1
1
592 (C=N); 1245, 1171 (C-O-C). H-NMR (300 MHz, DMSO-d
OCH ); 4.49 (s, 2H, NH , D
methoxyphenyl-C3,5-H); 7.18 (d, 2H, J= 8.7 Hz, 4-chlorophenyl-C3,5-H); 7.35 (d, 2H, J=
6
3
2
2
O exchangeable); 6.92-6.95 (m, 3H, pyrazolyl-C -H and 4-
4
8
1
.7 Hz, 4-methoxyphenyl-C2,6-H); 7.50 (d, 2H, J= 9 Hz, 4-chlorophenyl-C2,6-H); 9.55 (s,
H, NH, D O exchangeable). Anal. Calcd for C17 (342.78): C, 59.57; H, 4.41;
2
H15ClN
4
O
2
N, 16.34. Found: C, 59.65; H, 4.44; N, 16.51.
3
.1.17. Ethyl 6-chloro-2-formylquinoline-4-carboxylate (18)
A mixture of equimolar amounts of ethyl 6-chloro-2-methylcinchoninate 2 (1.12 g, 4.5
mmol) and selenium dioxide (0.50 g) in dioxane (40 mL) was heated under reflux with
stirring for 4 hours. The reaction mixture was then carefully filtered while hot to get rid
of the precipitated selenium metal. The filtrate was concentrated to a smaller volume and
left to attain room temperature. The product separated was filtered, washed with cold
-
ethanol, dried and crystallized from ethanol. Yield: 56%; Mp: 160-2 °C. IR (KBr, cm
1
)
:2988, 2863 (CH aldehyde); broad band centered at 1707 for two (C=O); 1599 (C=N);
1
1
242, 1164 (C-O-C). H-NMR (300 MHz, CDCl
3
, δ ppm): 1.50 (t, 3H, J= 7.2 Hz, -
-H); 8.25
CH CH ); 4.55 (q, 2H, J= 7.2 Hz, -CH CH ); 7.82 (d, 1H, J= 9 Hz, quinolyl-C
2
3
2
3
7
20

(
d, 1H, J= 9 Hz, quinolyl-C
8
-H); 8.55 (s, 1H, quinolyl-C
3
-H); 8.97 (s, 1H, quinolyl-C -
5
H); 10.24 (s, 1H, CHO). Anal. Calcd for C13
H
10ClNO
3
(263.68): C, 59.22; H, 3.82; N,
5
.31. Found: C, 59.35; H, 3.85; N, 5.39.
3
.1.18. 6-Chloro-4-(morpholin-4-ylcarbonyl)quinoline-2-carbaldehyde (19)
A mixture of equimolar amounts of 6-chloro-2-methyl-4-morpholinocarbonylquinoline
0 (2.04 g, 7 mmol) and selenium dioxide (0.78 g)in dioxane (40 mL) was heated under
1
reflux with stirring for 4 hours. The reaction mixture was carefully filtered while hot to
get rid of the precipitated selenium metal. The filtrate was concentrated to a smaller
volume, left to attain room temperature and then allowed to cool in an ice bath. The
product separated was filtered, washed with ethanol, dried and crystallized from dioxane.
-1
Yield: 67%; Mp: 224-5 °C. IR (KBr, cm ): 2913, 2851 (CH aldehyde); 1708 (HC=O);
1
1
3
(
635 (C=O); 1554 (C=N); 1271-1115 (C-O-C). H-NMR (300 MHz, CDCl
.15-3.33 (m, 2H, morpholin-C3a,5a-H); 3.52-3.73 (m, 2H, morpholin-C3e,5e-H); 3.81-4.07
-H); 7.92 (s, 1H, quinolyl-
-H); 8.26 (d, 1H, J= 9 Hz, quinolyl-C -H); 10.21 (s, 1H,
(304.73): C, 59.12; H, 4.30; N, 9.19. Found: C,
3
, δ ppm):
m, 4H, morpholin-C2,6-H); 7.83 (d, 1H, J= 9 Hz, quinolyl-C
7
C
3
-H); 7.94 (s, 1H, quinolyl-C
5
8
CHO). Anal. Calcd for C15
H
13ClN
2
O
3
5
9.19; H, 4.37; N, 9.34.
3
.1.19. Ethyl 6-chloro-2-{[(1,5-diaryl-1H-pyrazol-3-ylcarbonyl)hydrazono] methyl}
quinoline-4-carboxylates (20a-c)
The appropriate 1,5-diaryl-1H-pyrazole-3-carbohydrazide 17a-c (1 mmol) was gradually
added to a solution containing an equimolar amount of the aldehyde 18 (0.26 g) in glacial
acetic acid (20 mL). The reaction mixture was stirred at 25˚C until a precipitate started to
form and was then set aside for an overnight to complete precipitation. The precipitates
formed were filtered, washed with ethanol, dried and crystallized from glacial acetic acid.
3
.1.19.1.
Ethyl
6-chloro-2-{[(1,5-bis(4-chlorophenyl)-1H-pyrazol-3-
ylcarbonyl)hydrazono]methyl}quinoline-4-carboxylate (20a). Yield: 82%; Mp: 240-2 °C.
-1
IR (KBr, cm ): 3186 (NH); 1718 (C=O); 1665 (NH-C=O); 1592 (C=N); 1236-1087 (C-
1
O-C). H-NMR (500 MHz, CDCl
H, J= 6.9 Hz, -CH CH ); 7.14-7.52 (m, 9H, CH=N, two chlorophenyl-H); 7.76 (s, 1H,
quinolyl-C -H); 7.96 (d, 1H, J= 8.4 Hz, quinolyl-C -H); 8.10 (s, 1H, quinolyl-C -H);
.85-8.88 (m, 2H, quinolyl-C -H and pyrazolyl-C -H); 10.58 (s, 1H, D O exchangeable,
NH). C-NMR (75.44 MHz, DMSO-d , δ ppm): 13.94 (-CH CH ); 62.20 (-CH CH );
07.98 (pyrazole C-4); 119.44 (quinoline C-3); 123.96 (pyrazole-N1-4-chlorophenyl C-
/6); 126.10, 126.25 (quinoline C-5 and C-6 respectively); 128.55 (pyrazole-C3-4-
3
, δ ppm): 1.50 (t, 3H, J= 6.9 Hz, -CH
2
CH ); 4.53 (q,
3
2
2
3
3
7
5
8
8
4
2
1
3
6
2
3
2
3
1
2
chlorophenyl C-2/6); 128.62 (pyrazole-N1-4-chlorophenyl C-3/5); 128.86 (pyrazole-C3-
-chlorophenyl C-3/5); 129.19 (pyrazole-C3-4-chlorophenyl C-1); 129.47, 130.59
quinoline C-4a and C-8 respectively); 130.89 (pyrazole-N1-4-chlorophenyl C-4);
4
(
21

1
33.40, 134.25 (quinoline C-7 and C-4 respectively); 135.21 (pyrazole-C3-4-
chlorophenyl C-4); 137.20 (pyrazole-N1-4-chlorophenyl C-1); 139.32 (pyrazole C-3);
44.58 (pyrazole C-5); 145.16 (CH=N); 145.76, 151.83 (quinoline C-8a and C-2
respectively); 158.16 (HN-C=O); 164.42 (C=O-OC ). Anal. Calcd for C29
592.86): C, 58.75; H, 3.40; N, 11.81. Found: C, 58.88; H, 3.47; N, 11.88.
1
2
H
5
H
20Cl
3
N
5
O
3
(
3
.1.19.2.
Ethyl
6-chloro-2-{[(5-(4-methoxyphenyl)-1-phenyl-1H-pyrazol-3-
ylcarbonyl)hydrazono]methyl}quinoline-4-carboxylate (20b). Yield: 74%; Mp: 200-2 °C.
-1
IR (KBr, cm ): 3210 (NH); 1723 (C=O); 1705 (NH-C=O); 1609 (C=N); 1296-1081 (C-
1
O-C); 1198-1032 (C-O-C, OCH
.2 Hz, -CH CH ); 3.83 (s, 3H, OCH
J= 7.8 Hz, 4-methoxyphenyl-C3,5-H); 7.12-7.60 (m, 8H, CH=N, 4-methoxyphenyl-C2,6-H
and phenyl-H); 7.76 (s, 1H, quinolyl-C -H); 8.04 (d, 1H, J= 7.5 Hz, quinolyl-C -H); 8.10
s, 1H, quinolyl-C -H); 8.81-8.87 (m, 2H, quinolyl-C -H and pyrazolyl-C -H); 10.60 (s,
H, D O exchangeable, NH). Anal. Calcd for C30 (554.00): C, 65.04; H, 3.37;
N, 12.64. Found: C, 65.16; H, 4.41; N, 12.78.
3
). H-NMR (300 MHz, CDCl
3
, δ ppm): 1.51 (t, 3H, J=
7
2
3
3
); 4.54 (q, 2H, J= 7.2 Hz, -CH
2
CH ); 6.87 (d, 2H,
3
3
7
(
1
5
8
4
2
H24ClN
5
O
4
3
.1.19.3. Ethyl 6-chloro-2-{[(1-(4-chlorophenyl)-5-(4-methoxyphenyl)-1H-pyrazol-3-
ylcarbonyl)hydrazono]methyl}quinoline-4-carboxylate (20c). Yield: 78%; Mp: 220-2 °C.
-1
IR (KBr, cm ): 3157 (NH); 1723 (C=O); 1664 (NH-C=O); 1612 (C=N); 1202-1083 (C-
1
O-C); 1172-1033 (C-O-C, OCH
3
). H-NMR (500 MHz, CDCl
); 3.82 (s, 3H, OCH ); 4.53 (q, 2H, J= 6.9 Hz, -CH
H, CH=N, 4-methoxyphenyl-H and4-chlorophenyl-H); 7.72 (s, 1H, quinolyl-C
.94 (d, 1H, J= 8.4 Hz, quinolyl-C -H); 8.07 (s, 1H, quinolyl-C -H); 8.82-8.86 (m, 2H,
-H and pyrazolyl-C -H); 10.62 (s, 1H, D O exchangeable, NH). Anal. Calcd
(588.44): C, 61.23; H, 3.94; N, 11.90. Found: C, 61.39; H, 4.01; N,
3
, δ ppm): 1.49 (t, 3H, J=
CH ); 6.85-7.49 (m,
-H);
6
9
7
.9 Hz, -CH
2
CH
3
3
2
3
3
7
5
quinolyl-C
8
4
2
for C30
H
23Cl
2
N
5
O
4
1
2.04.
3
.1.20. N'-[6-Chloro-4-(morpholin-4-ylcarbonyl)quinolin-2-ylmethylene]-1,5-diaryl-1H-
pyrazole-3-carbohydrazides (21a-c)
The appropriate 1,5-diaryl-1H-pyrazole-3-carbohydrazide 17a-c(1 mmol) was gradually
added to a solution containing an equimolar amount of 6-chloro-4-(morpholin-4-
ylcarbonyl)quinoline-2-carbaldehyde 19 (0.30g)in glacial acetic acid (25 mL).The
reaction mixture was stirred at 25˚C until a precipitate started to form and was left for an
overnight to complete precipitation. The precipitates formed were filtered, washed with
ethanol, air dried and crystallized from glacial acetic acid.
3
.1.20.1.
N'-[6-Chloro-4-(morpholin-4-ylcarbonyl)quinolin-2-ylmethylene]-1,5-bis(4-
chlorophenyl)-1H-pyrazole-3-carbohydrazide (21a).Yield: 50%; Mp: 190-2 °C. IR (KBr,
22

-1
1
cm ): 3247 (NH); 1687 (C=O); 1634 (NH-C=O); 1534 (C=N); 1208-1113 (C-O-C). H-
NMR (500 MHz, CDCl , δ ppm): 3.12-3.34 (m, 2H, morpholin-C3a,5a-H); 3.47-3.52 (m,
H, morpholin-C3e,5e-H); 3.81-4.11 (m, 4H, morpholin-C2,6-H); 7.11-7.68 (m, 9H, CH=N
and two chlorophenyl-H); 7.78 (s, 1H, quinolyl-C -H); 7.90 (d, 1H, J= 9.2 Hz, quinolyl-
-H); 8.12 (d, 1H, J= 9.2 Hz, quinolyl-C -H); 8.30 (s, 1H, quinolyl-C -H); 8.58 (s, 1H,
pyrazolyl-C -H); 10.86 (s, 1H, D O exchangeable, NH). Anal. Calcd for C31
633.91) C, 58.74; H, 3.66; N, 13. 26. Found: C, 58.88; H, 3.62; N, 13.44.
3
2
3
C
7
8
5
4
2
H
23Cl
3
N
6
O
3
(
:
3
.1.20.2.
N'-[6-Chloro-4-(morpholin-4-ylcarbonyl)quinolin-2-ylmethylene]-5-(4-
methoxyphenyl)-1-phenyl-1H-pyrazole-3-carbohydrazide (21b). Yield: 63%; Mp: 287-9
-
1
°
(
2
3
7
C. IR (KBr, cm ): 3279 (NH); 1702 (C=O); 1644 (NH-C=O); 1603 (C=N); 1244-1103
1
C-O-C); 1193-1077 (C-O-C, OCH
3
). H-NMR (500 MHz, CDCl
H, morpholin-C3a,5a-H); 3.47-3.65 (m, 2H, morpholin-C3e,5e-H); 3.80 (s, 3H, OCH
.82-4.04 (m, 4H, morpholin-C2,6-H); 6.82-6.85 (m, 2H, 4-methoxyphenyl-C3,5-H); 7.13-
.54 (m, 8H, CH=N, phenyl-H and 4-methoxyphenyl-C2,6-H); 7.67 (s, 1H, quinolyl-C
-H); 7.84 (s, 1H, quinolyl-C -H); 8.00 (d, 1H, J=
-H); 8.36 (s, 1H, pyrazolyl-C -H); 10.60 (s, 1H, D O exchangeable,
NH). C-NMR (125.75 MHz, CDCl , δ ppm): 42.40 (morpholin C-3/5); 47.64 (OCH );
6.92 (morpholin C-2/6); 109.02 (pyrazole C-4); 116.59 (quinoline C-3); 119.89 (4-
methoxyphenyl C-3/5); 123.37 (4-methoxyphenyl C-1); 123.83, 124.10 (phenyl C-2/6);
25.47 (phenyl C-4); 126.15, 128.76 and 128.86 (quinoline C-4a, C-5 and C-6
respectively); 129.24 (4-methoxyphenyl C-2/6); 131.63, 131.96 (phenyl C-3/5); 135.33,
36.74 (quinoline C-8 and C-7 respectively); 139.33 (phenyl C-1); 140.01, 142.59
pyrazole C-3 and C-5 respectively); 145.00, 145.61 (quinoline C-4 and C-8a
respectively); 146.43 (CH=N); 151.74 (quinoline C-2); 158.17 (NH-C=O); 159.86 (4-
3
, δ ppm): 3.12-3.38 (m,
3
);
3
-
H); 7.76 (d, 1H, J= 8.4 Hz, quinolyl-C
.4 Hz, quinolyl-C
7
5
8
8
4
2
1
3
3
3
6
1
1
(
methoxyphenyl C-4); 165.58 (C=O). Anal. Calcd for C32
H, 4.57; N, 14.12. Found: C, 64.68; H, 4.61; N, 14.21.
H27ClN
6
O
4
(595.05) C, 64.59;
:
3
.1.20.3.
N'-[6-Chloro-4-(morpholin-4-ylcarbonyl)quinolin-2-ylmethylene]-1-(4-
chlorophenyl)-5-(4-methoxyphenyl)-1H-pyrazole-3-carbohydrazide (21c).Yield: 64%;
-1
Mp: 185-6 °C. IR (KBr, cm ): 3257 (NH); 1688 (C=O); 1623 (NH-C=O); 1531 (C=N);
1
1
3
3
207-1112 (C-O-C); 1175-1085 (C-O-C, OCH
.14-3.40 (m, 2H, morpholin-C3a,5a-H); 3.50-3.75 (m, 2H, morpholin-C3e,5e-H); 3.80 (s,
H, OCH ); 3.81-4.08 (m, 4H, morpholin-C2,6-H); 6.82-7.48 (m, 9H, CH=N, 4-
-H); 7.84 (s,
-H); 8.40 (s, 1H, quinolyl-C
O exchangeable, NH). Anal. Calcd for
(629.49): C, 61.06; H, 4.16; N, 13. 35. Found: C, 61.13; H, 4.19; N,
3
). H-NMR (500 MHz, CDCl
3
, δ ppm):
3
methoxyphenyl-H and 4-chlorophenyl-H); 7.77 (d, 1H, J= 9.15, quinolyl-C
H, quinolyl-C -H); 7.94 (d, 1H, J= 9.15 Hz, quinolyl-C
H); 8.81 (s, 1H, pyrazolyl-C -H); 11.03 (s, 1H, D
7
1
3
8
5
-
4
2
C
32
H
26Cl
2
N
6
O
4
1
3.51.
23

3
3
.2. Anti-inflammatory (AI) activity
.2.1. Formalin-induced paw edema bioassay (acute inflammatory model)
Male albino rats weighing 180–200 g were used throughout the assay [45,46]. They were
kept in the animal house under standard condition of light and temperature with free
access to food and water. The animals were randomly divided into groups each of five
rats. One group of five rats was kept as a control and another group received the standard
drug celecoxib [at a dose of 20 mg/kg body weight per os (po)]. A solution of formalin
(
2%, 0.1 mL) was injected into the subplanter region of the left hind paw under light
ether anesthesia 1h after oral administration (po) of the test compound (at a dose level of
0 mg/kg body weight). The paw volume (mL) was measured by means of water
2
plethysmometer and re-measured again 1, 2 and 4 h after administration of formalin. The
edema was expressed as an increase in the volume of paw, (Table 1) and the percentage
of edema inhibition for each rat and each group was obtained as follows:
%
Inhibition = (Vt–Vo) control–(Vt–Vo) tested compound / (Vt–Vo) control ×100
Where Vo = volume of edema at zero time interval.
Vt = volume of edema at specific time interval.
3
.2.2. Formalin-induced paw edema bioassay (sub-acute inflammatory model)
Rats in the first experiment were given the same test compounds at a dose level of 20
mg/kg body weight daily for 7 consecutive days. A solution of formalin (2%, 0.1 mL)
was injected into the subplanter region of the left hind paw under light ether anesthesia 1
h after oral administration (po) of the test compound. A second injection of formalin (2%,
0
.1 mL) was given on the third day. The changes in the volume of paw were measured
plethymographically at the first and eighth days [45,46]. (Table 2)
3
.2.3. Ulcerogenic activity
Male albino rats (180–200 g) were divided into groups each of five animals and were
fasted for 12 h prior to the administration of the test compounds. Water was given ad
libitum. Control group received 1% gum acacia orally. Other groups received celecoxib
or the test compounds orally in two equal doses at 0 and 12 h for three successive days at
a dose of 300 mg/kg per day. Animals were sacrificed by diethyl ether 6 h after the last
dose and their stomachs were removed. An opening at the greater curvature was made
and the stomach was cleaned by washing with cold saline and inspected with a
3
×magnifying lens for any evidence of hyperemia, hemorrhage, definite hemorrhagic
erosion or ulcer [47].
24

3
.2.4. Acute toxicity
Twelve groups of rats (180–200 g) each consists of five animals, were used in this test
48]. The animals were fasted for 24 h prior to administration of the test compounds. The
[
compounds were given orally in graded doses of 0.1–0.4 g/kg body weight, po. The
compounds were screened at graded doses for their acute lethal doses (ALD50) and the
mortalities were recorded at each dose level after 24 h.
3
.2.5. Determination of effective dose 50 (ED50)
The selected compounds were further tested at 5, 10, 20, 40, and 50 mg/Kg body weight
and the ED50 was determined by measuring the inhibition of the edema volume 2 h after
formalin injection. (Table 1)
3
.2.6. Statistical analysis of data
The data obtained are presented as means  SE of the mean. The concentration-dependent
effects of various drugs were evaluated statistically by the randomized block design
analysis of Variance (ANOVA) followed by Student–Newman–Keuls Multiple
Comparison Test. The difference in results was considered significant when P < 0.05.
3
.2.7. In vitro COX-1/COX-2 inhibition assay
The inhibitory COX activity of the tested compounds and the reference was assayed
using Cayman colorimetric COX (ovine) inhibitor screening assay kit (Catalog No.
7
60111, Cayman Chemicals, Ann Arbor, MI) according to the manufacturer’s
instructions [49]. Aliquots (170 mL) of the assay buffer (0.1N Tris- HCl, pH 8.0), heme,
and enzyme ovine (COX-1 or COX-2) were placed in a 96-well plate. The test
compounds were added to the aliquots. The plate was carefully shaken for a few seconds
and then incubated for 5 min at 25 °C. The colorimetric substrate N,N,N’,N’-tetramethyl-
p-phenylenediamine (20 mL, TMPD) and arachidonic acid (20 mL) were added to the
aliquots. The plate was carefully shaken for a few seconds and then incubated for 5 min
at 25 °C. The absorbance was measured at 590 nm using a 96-well Tecan Safire plate
reader. The mixture of 160 mL of assay buffer and 10 mL of heme served as a
background control. The mixture of 150 mL of assay buffer, 10 mL of heme, and 10 mL
of ovine COX-1 or COX-2 showed 100% initial activity as a control experiment in the
absence of inhibitor. Diclofenac, celecoxib and indomethacin were used as reference
standards in the study. The assays were performed in triplicate and the IC50 values were
calculated from the concentration curves by means of GraphPad software PRISM.
3
.2.8. In vitro 5-LOX inhibitory assay
The inhibitory activity of the test compounds against soya bean 5-LOX was evaluated by
using Abnova lipoxygenase assay kit (catalog no. KA1329, Cayman Chemicals). IC50
25

values of the tested compounds were carried out according to procedures and instructions
given with the assay kit and in accordance to previously reported methods [63].
3
.3. Docking Studies
Preparation of COX crystal structures:
Coordinates for COX-1 and COX-2 crystal structures was retrieved from the Protein Data
Bank (PDB code: 1EQG and 3LN1 respectively) and handled consequently with
Molecular Operating Environment program (MOE) [64]. Redundant chains, non-essential
ions, water molecules and ligands were discarded. The crystal structures were treated
with “QuickPrep” module at default settings. This includes additions of bond orders,
formal charges and explicit hydrogen atoms to the complex structure. Subsequently, the
most appropriate protonation states and optimization of the H-bond network were
performed employing “Amber: 10EHT” force field. Prepared protein structures were
saved as PDB files which were further used for GOLD docking. The ligand-protein in-
place minimization was also performed.
Preparation of the selected compounds for docking:
The molecules were built and prepared by MOE. ‘Molecule wash’ function was used to
generate meaningful protonation states by deprotonating strong acids and protonating
strong bases (if required). Energy minimization of all molecules was performed using the
Amber: 10EHT force field at a gradient of 0.01 RMSD (i.e. if the gradient falls below
RMSD, the minimization terminates). Conformational search was conducted to sample
maximum number of minimized conformations including different stereoisomers and
ring conformations (if necessary) using “Stochastic” method, with option “Allow
unconstrained double bond rotation” enabled to sample different geometrical isomers of
the double bond-containing compounds. All other options were set at default levels. This
ended up to total of 4254 conformations of the compounds under investigation. The
resulting conformers were saved as SD file for the docking experiments.
Docking experiments:
GOLD (version 5.2) [65-70] was used for docking experiments against COX-1 and COX-
2
crystal structures. Residues of the binding site were defined by specifying the
coordinates of the co-crystallized ligands and using a cutoff radius of 8 Å, with the
detect cavity’ option enabled. The scoring function used for GOLD docking experiments
was ChemPLP. The search efficiency of the genetic algorithm was at 50% to handle the
254 conformations in a meaningful time period. The best docking solution per
‘
4
conformation were saved consequently. This docking approach was validated by
successful pose-retrieval of the co-crystallized ligands of COX-2 crystal structures. All
graphical representations in Figures 2-4 were rendered using MOE.
26

Dedication
This work is dedicated to the memory of El-Sayeda M. El-Khawass.
Acknowledgement
Authors thank Prof. Dr. Frank Boeckler, Lab for Molecular Design & Pharmaceutical
Biophysics, Eberhard Karls University Tuebingen, Germany, for granting access to some
computational tools.
Declaration of interest
The authors report no conflicts of interest. The authors alone are responsible for the
contents and writing the paper.
Ethical conduct of research
The protocols used in the present study followed the guidelines set in ‘The Guide for the
Care and Use of Laboratory Animals’, and got approval By ACUC, Faculty of Pharmacy,
Alexandria University, Project No. 82 at 23/11/2016. Copy of approval is available upon
request.
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