Synthesis, biological evaluation, docking study and ulcerogenicity profiling of some novel quinoline-2-carboxamides as dual COXs/LOX inhibitors endowed with anti-inflammatory activity

Article abstract of DOI:10.1016/j.ejmech.2016.11.006

A series of novel quinoline-2-carboxamides 15–28 was synthesized and evaluated in?vitro as dual COXs/LOX inhibitors. Compounds 19 and 27 exhibited the highest potency and selectivity for COX-2 inhibitory activity (IC₅₀?=?1.21 and 1.13?μM, respe

Full text of DOI:10.1016/j.ejmech.2016.11.006

Accepted Manuscript
Synthesis, biological evaluation, docking study and ulcerogenicity profiling of some
novel quinoline-2-carboxamides as dual COXs/LOXInhibitors endowed with anti-
inflammatory activity
Mostafa H. Abdelrahman, Bahaa G.M. Youssif, Mohamed A. abdelgawad, Ahmed
H. Abdelazeem, Hussein M. Ibrahim, Abd El Ghany A. Moustafa, Laurent Treamblu,
Syed Nasir Abbas Bukhari
PII:
S0223-5234(16)30944-8
10.1016/j.ejmech.2016.11.006
EJMECH 9039
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 September 2016
Revised Date: 2 November 2016
Accepted Date: 3 November 2016
Please cite this article as: M.H. Abdelrahman, B.G.M. Youssif, M.A. abdelgawad, A.H. Abdelazeem,
H.M. Ibrahim, A.E.G.A. Moustafa, L. Treamblu, S. Nasir Abbas Bukhari, Synthesis, biological evaluation,
docking study and ulcerogenicity profiling of some novel quinoline-2-carboxamides as dual COXs/
LOXInhibitors endowed with anti-inflammatory activity, European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.11.006.
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ACCEPTED MANUSCRIPT
Graphical abstract:
Synthesis, Biological Evaluation, Docking Study and Ulcerogenicity Profiling of
Some Novel Quinoline-2-Carboxamides as Dual COXs/LOXInhibitors Endowed with
Anti-Inflammatory Activity
Mostafa H. Abdelrahman, Bahaa G. M. Youssif, Mohamed A. abdelgawad, Ahmed H.
Abdelazeem, Hussein M. Ibrahim, Abd El Ghany A. Moustafa, Laurent Treamblu, Syed
Nasir Abbas Bukhari

ACCEPTED MANUSCRIPT
Synthesis, Biological Evaluation, Docking Study and Ulcerogenicity
Profiling of Some Novel Quinoline-2-Carboxamides as Dual
COXs/LOXInhibitors Endowed with Anti-Inflammatory Activity
1
2,3*
3,4
Mostafa H. Abdelrahman ,Bahaa G. M. Youssif , Mohamed A. abdelgawad , Ahmed H.
5,6
7,8
8,9
10
Abdelazeem , Hussein M. Ibrahim , Abd El Ghany A. Moustafa , Laurent Treamblu ,
Syed Nasir Abbas Bukhari3*
1
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Al-Azhar University,
2
Assiut 71524, Egypt; Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt; Department of Pharmaceutical Chemistry, College of
Pharmacy, Aljouf University, Aljouf, Sakaka2014, Saudi Arabia; Department ofPharmaceutical
3
4
Organic Chemistry, Faculty of pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt;
5
Department of Medicinal Chemistry, Faculty of pharmacy, Beni-Suef University, Beni-Suef 62514,
6
Egypt; Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Taif
2
University, Cairo, Egypt; Department of Clinical Laboratory Sciences, College of Applied Medical
Sciences, Aljouf University, Aljouf, Sakaka2014, Saudi Arabia; Histology Department, Faculty of
Medicine, Al-Azhar University, Damietta, Egypt; 10School of Natural and Computing Sciences,
University of Aberdeen,Meston Building, Aberdeen, AB243UE, Ireland.
7
1974, Saudi Arabia; Department of Anatomy and Embryology, Faculty of Medicine, Ain Shams
8
9
Short running title:Synthesis,anti-inflammatory activity and docking study of new quinoline-2-
carboxamides
*To whom correspondence should be addressed:
Bahaa G. M. Youssif. Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy,
Assiut University, Assiut 71526, Egypt.
Tel.: (00966)-537240294
E-mail address:bgyoussif@ju.edu.sa
Syed Nasir Abbas Bukhari.Department of Pharmaceutical Chemistry, College of Pharmacy,
Aljouf University, Aljouf, Sakaka2014, Saudi Arabia
Tel.: (00966)-565738896
E-mail address:snab_hussaini@yahoo.com
1

ACCEPTED MANUSCRIPT
Abstract
A series of novelquinoline-2-carboxamides15-28was synthesized and evaluated in vitro as
dual COXs/LOX inhibitors. Compounds 19 and 27 exhibited the highest potency and
selectivity for COX-2 inhibitory activity (IC50 = 1.21 and 1.13
index (COX-1/COX-2) = 6.52 and 7.61, respectively) in comparison to the reference drug
celecoxib (COX-2 IC50 = 0.88 M; selectivity index (COX-1/COX-2) = 8.31). The anti-
µM, respectively; selectivity
µ
inflammatory activity of the newly synthesized compounds was further assessed in vivo using
carrageenan induced paw edema assay. Interestingly, the in vitro results of COXs inhibitory
assay were consistent with that of the in vivo assay where compounds 19 and 27 showed the
highest anti-inflammatory activity with edema inhibition percentages of 59.38% and 65.03%,
respectively compared to celecoxib (71.21%) after 5 h. Moreover, it was found that
compounds 19 and 27 have a superior gastric safety profile comparable to indomethacin. The
molecular docking study of compounds 19 and 27 into COX-2 active site suggested that these
hits assumed binding pattern and interactions similar to that of bromocelecoxib (S-58) as a
cocrystallized ligand explaining their remarkable COX-2 inhibitory activity and
selectivity.Taken together, these results indicated that these derivatives are good leads for
subsequent development into potential anti-inflammatory agents with least gastric damage.
Keywords:Quinoline-2-carboxamides, COXs, LOX, Docking, Anti-inflammatory.
2

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Graphical abstract:
3

ACCEPTED MANUSCRIPT
1
- Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are considered the most frequently used
remedies to alleviate inflammation, fever and pain. It was reported that NSAIDs exert their
action mainly through inhibiting the activity of cyclooxygenase enzymes (COXs) which are
responsible for the biosynthesis of prostaglandins (PGs), prostacyclin (PGI2) and
thromboxanes (TXA2) [1, 2]. Only one form of the COX enzymes, COX-1, was known until
1
990 and it was thought to be the main cause of inflammation and its inhibition resulted in
undesired side effect [3]. To date, three forms of cyclooxygenase isozymes are known;
constitutive COX-1, induced COX-2 and COX-3 that remain under investigation [1, 4]. On
the other hand, lipoxygenases (LOXs) are group of enzymes that are the main factors in the
leukotrienes (LTs) production process and catalyze the oxidation of lipoproteins (LDL, HDL)
to atherogenic forms. Thus, COXs and LOXs are two metabolic inflammatory mediators
produced from biotransformation of arachidonic acid (AA)[1, 2]. It was assumed that the
inhibition of the COX-1 enzyme causes some of the undesired side effects of NSAIDssuch as
ulceration, decrease renal function and gastrointestinal haemorrhage. However, the inhibition
of COX-2 was found to reduce pain, inflammation and fever but with increasing the risk of
cardiovascular diseases [3, 5-8].
Until recently, it was thought that the ideal NSAID should inhibit only COX-2 enzyme
activity without a ecting COX-1 enzyme activity. Accordingly, many selective COX-2
ff
compounds were introduced in the past decade and marketed as NSAIDs with less gastric
damage[9]. Unfortunately, some of these drugs such as VIOXX* (rofecoxib) were withdrawn
from the market due to the reported increase in the cardiovascular diseases risk[9].Several
strategies were conducted to find novel NSAIDs devoid of both cardiovascular risk and
gastrointestinal side effects. Of these strategies that have been emerged are antagonism of
leukotrienes receptor[10], the dual inhibition of COXs/LOX enzymes[1, 11], the dual
inhibition of COXs/iNOS enzymes [12] and nitric oxide (NO) donating NSAIDs [13, 14].
4

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Obviously, it is well-documented that PGs and LTs have a complementary role in the
occurrence and development of the inflammatory process. Moreover, the dual inhibition of
COXs and LOX may participate in counteracting the vascular changes appeared during
inflammation and leucocyte induced GI damage [15]. It has been suggested that blocking
both LT and PG production might have synergistic effects and achieve optimal anti-
inflammatory activity with less gastric liability [16].
Two drugs, tepoxalin I and licofelone (ML3000)II, were reported as dual inhibitors of COX
and LOX with potent anti-inflammatory/analgesic potentials and better gastric tolerance
profiles even at higher doses, Fig. 1[16-19]. In addition, Reddy and co-workers designed and
synthesized a racemic indolylpyrazoline-based compound III as dual COX/LOX inhibitor
with IC values of COX-2 = 3.9
ꢀM, 5-LOX = 94
ꢀM, 12-LOX = 3 ꢀM and 15-LOX = 36
5
0
ꢀM[11]. Furthermore, quinoline nucleus is an important building block and privileged
structure that is widely used in drug design and discovery to develop several bioactive
molecules with various pharmacological activities. Among these reported bioactive
quinoline-based compounds, quinoline-4-carboxylic acid (IV) which showed remarkable
potency and higher COX-2 selectivity (IC = 0.043 µM; selectivity index (COX-1/COX-2)>
5
0
5
13) than celecoxib (IC50 = 0.06 µM and SI = 405) [20]. One more COX-2 selective
quinoline-based compound, 2-(4-(methylsulfonyl)phenyl)-3-phenylquinoline-4-carboxylic
acid ( ), was introduced by the same researchers’ group and it exhibited high potency and
V
COX-2 selectivity (IC50 = 0.07 µM and SI = 687.1) more than celecoxib [21]. Recently, a
series of 2-chloro-quinoline-based imidazopyridines and imidazothiazoles was indicated as
soybean 15-LOX inhibitors where imidazo[2,1-b]thiazole derivative (VI) displayed the
highest inhibitory activity against 15-LOX with IC50 value of 11.5 µM [22], Fig. 1
.
5

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Cl
Cl
OH
O
F C
3
N OH
O
N
N
N
N
N
SO NH2
2
N
H
H CO
3
Cl
Tepoxalin, I
Licofelone, II
III
S
COOH
N
COOH
N
HN
Cl
N
N
N
SO Me
2
SO Me
2
VI
IV
V
Fig. 1.Chemical structures of some reported dual COX/LOX compounds (I-III) and quinoline-based
COX or LOX bioactive compounds (IV-VI)
Encouraged by these findings, we continued our work in this area, and in the current study we
describe herein the design and synthesis of novel set ofquinoline-2-carboxamide-
basedderivatives as dual COX and LOX inhibitors with anti-inflammatory potential promoted
with the significant reported COX and LOX activity of quinoline-privileged structure. A new
pharmacophore model, Fig. 2, was constructed and it consists of three regions; the quinoline
template, carboxamide linker and hydrophobic area with or without an amino tail. In order to
study the iterative structure-activity relationship, various amines with variable polarity,
basicity and size were used and the influence of these changes on the activity was
investigated. In addition, our focus was to examine the effect of these new compounds on the
gastric mucosa using histopathological and scanning electron microscopic studies.
Fig. 2. The pharmacophoric model of the newly designed quinoline-2-carboxamide scaffold.
6

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2.
Results and discussion
2.1. Chemistry
Phenethylamine derivatives 4-6and
outlined in scheme 1 as previously described[23].
alkylation using dibromoalkanes in refluxing toluene and in the presence of diisopropylamine
DIPEA) to yield benzyl cyanide intermediates1-3. These cyanide derivatives were reduced
to the corresponding amines 4-6using LiAlH . On the other hand, compound was prepared
by bis-methylation of -aminobenzylcyanide using a reductive amination method followed by
8
were synthesized following the general pathway
p
-Aminobenzyl cyanide was subjected to
(
4
8
p
4
reduction by LiAlH .
a
b
H N
2
R
R
C N
C N
: R = piperidyl
NH2
1
2
3
4
5
6
: R = piperidyl
: R = pyrrolidinyl
:R = 2-methylpyrrolidinyl
: R = pyrrolidinyl
:R = 2-methylpyrrolidinyl
c
d
N
N
C N
NH2
7
8
Scheme 1: Synthesis of phenethylamine derivatives4-6and8. Reagents and reactionconditions:
(
a) 1,5-Dibromopentane, 1,4-dibromobutane or 1,4-dibromopentane DIPEA, toluene, reflux, 20 h; (b)
o
LiAlH
rt, 3h; (d) LiAlH
4
, Et
2
O, 0 C to rt, overnight; (c) 37% aqueous formaldehyde, NaBH
3
CN, acetic acid, CH
3
CN,
o
4
, Et
2
O, 0 C to rt, overnight.
The preparation of 1-benzyl-3-aminopiperidine was also accomplished using a three-step
procedure, Scheme 2 [24]. Treatment of ethyl nipecotate with benzyl bromide in the presence
of Na
monohydrate to give a quantitative yield of acyl hydrazide10. Curtius rearrangement of
using the combination of NaNO and TFAprovided the desired product11 in 86% yields.
2 3
CO afforded compound 9, whichwas subsequently treated with hydrazine
10
2
1
13
The structures of compounds 9-11were confirmed by H NMRand C NMR spectroscopic
analyses as well as HRESI-MS spectrometry, which were in agreement withthe data reported
in the literature[24].
7

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Scheme 2: Synthesis of 1-benzyl-3-aminopiperidine 11
Benzyl bromide, Na CO , DCM /H O (2:1), reflux, 3 h; (b) NH
O, 0 C for 2 h then 80 C for 2 h.
.
Reagents and reaction conditions: a)
2
3
2
2
NH , ethanol, reflux, 3 h; (c) NaNO
2
2
,
o
o
TFA, H
2
The synthetic route for preparation of quinoline-2-carboxamides 15-28was shown in Scheme
. Condensation of -chloroaniline with diethyl oxalpropionate afforded the corresponding
3
p
imine 12, which was then cyclized in polyphosphoric acid to give quinoline-2-carboxylic acid
1
ethyl ester13 in excellent yield over the two steps. The significant feature of HNMR
spectrum of compound 13 is the appearance of different sets of protons corresponding to the
protons in the quinoline ring, a singlet signal of one proton integration at
δ
11.86ppm assigned
1.36 ppm corresponding to OCH CH
group as well as aromatic protons of
to hydroxyl group, quartet at
δ
δ
4.43 ppm and triplet at
δ
2
3
moiety, and singlet signal at
2.19ppmassigned to CH
3
13
quinoline ring. C NMR and HRESI-MS also confirmed the structure of compound 13. The
quinoline-2-carboxylate 13 was hydrolyzed under basic conditions to its carboxylic acid 14
which was then coupled with the appropriate primary or secondary amine using BOP as the
,
coupling reagent in the presence of
N,
N-diisopropylethylamine (DIPEA) to give quinoline-2-
1
carboxamides 15-28. All the structures of quinoline-2-carboxamides were confirmed by H
13
NMR and C NMR spectroscopic analyses as well as HRESI-MS spectrometry. Compound
as an example of this series was identified by the appearance of extra peaks, which were
15
1
13
not presented in the carboxylic acid starting material 14in both the HNMR and CNMR
1
spectra as well as via HRESI-MS. The HNMR spectrum of 15revealed the appearance of a
signal at
δ 8.83 ppm assigned to amide NH, two sets of doublets at δ 7.08 and δ6.84 ppm with
coupling constant of 8.1 Hz each assigned to the phenyl protons which is indicative of
aromatic para-disubstitution, two signals of two protons integration each at
δ 3.47 (q) and
8

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δ
2.75 (t) ppm attributed to NHCH CH group, and piperidinyl protons. The structure of
2
2
+
1
5was further confirmed by HRESI-MS which gave a molecular ion m/z 424.1787 [M+H] ,
consistent with the molecular formula C24
3
H27ClN O
2
of the desired product.
O
N
O
Cl
O
O
a
Cl
NH2
O
O
O
O
O
1
2
b
OH
OH
N
OH
N
Cl
c
Cl
Cl
d
O
OH
R
N
O
O
O
1
5-28
14
13
R see Table 1
Scheme 3.Synthesis of the target quinoline-2-carboxamides 15-28
.
Reagents and
o
reactionconditions: (a) Acetic acid, ethanol, reflux, 48 h; (b) PPA, 120 C, 2 h; (c) 5% NaOH, ethanol,
o
8
0 C, 4 h; (d) Appropriate amine, BOP, DIPEA, DCM, overnight, rt.
2
2
.2. Anti-inflammatory activity
.2.1. In vitro cyclooxygenase (COX) inhibition assay
The in vitro biological activity assay was operated to investigate the ability of the
newlysynthesizedcompounds to inhibit both bovine COX-1 and COX-2 subtypes. A
colorimetric enzyme immunoassay (EIA) kit was used to screen the isozyme-specific
inhibition [25]. The potency of the testing compounds was determined as the concentration
causing 50% enzyme inhibition (IC ). Also, the COX-2 selectivity indexes (SI values)
5
0
werecalculated, is defined as IC50 (COX-1) / IC50 (COX-2) and compared with that of the
standard drug celecoxib. All compounds were tested and the data obtained were listed in
Table 1
.
9

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As shown in Table 1, all compounds except 17, 20,22,23and 25 (IC >2.5 µM) displayed
50
good inhibitory activities against COX-2 and were more potent inhibitors of COX-2 (IC =
5
0
1
.13- 2.11 µM range) than COX-1 (IC50 = 7.50-10.40 µM range) with COX-2 selectivity
indexes (SI) inthe range of 4.43-7.61. However, in all cases, the measured activities were
lower than that of celecoxib.The effects of substituents introduced into quinoline-2-
carboxamide core of compounds15-28 demonstrated that benzimidazole and
phenylpiperazine groups were the most appropriate substitutions and the corresponding
compounds19 and 27, respectively were the most potent COX-2 inhibitors in this series (19
,
IC50 = 1.21µM, SI = 6.52; 27, IC50 = 1.13µM, SI = 7.61). Compound 22(with a phenyl
piperidine substituent in quinoline-2-carboxamide core) was the least potent compound as
COX-2 inhibitor (IC = 3.54µM, SI = 3.70).
50
2
.2.2. In vitro lipoxygenase (LOX) inhibition assay
Compounds15 17 19 20 23 24 and 26-28were evaluated for their ability to inhibit
,
,
,
,
,
lipoxygenase (LOX) enzyme using the LOX enzyme assay kit [25]. The obtained data is
listed in Table 1. Regarding 15-LOX inhibitory activity, compounds 19and27 were the most
active as 15-LOX inhibitors with (IC50 = 6.18 and 5.91 µM, respectively) compared that of
meclofenamate sodium (IC50 = 5.64 µM). Compounds 15
, 24, 26 and 28 showedmoderate
inhibitory activity against 15-LOX enzyme in the range of (IC = 6.54-7.14 µM) while 17
5
0
and 20 were the lowest as lipoxygenase inhibitors (IC50 = 9.87 and10.41 µM, respectively).
10

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Table 1
:
In vitro COX-1, COX-2 inhibition and 15-LOXof compounds15-28and the
reference drug celecoxib for COX and meclofenamte sodium for LOX.
a
a
COX-1
(IC50
µM)
COX-2
(IC50
µM)
c
Comp.
No
Selectivity
ratio (SI)
LOX
R
b
IC50 µM
1
5
10.4
2.11
4.92
5.10
7.32
1
6
9.8
1.91
ND
1
7
11.4
2.54
4.48
9.87
1
1
8
9
8.3
7.9
1.74
1.21
4.77
6.52
ND
6.18
2
2
0
1
12.4
8.3
3.14
1.67
3.94
4.97
10.41
ND
2
2
13.1
3.54
3.70
ND
2
2
3
4
10.7
7.8
2.81
1.41
3.80
5.53
8.43
6.54
2
2
5
6
11.4
9.6
2.78
1.91
4.10
5.02
ND
7.14
2
2
7
8
8.6
7.5
7.32
-
1.13
1.69
0.88
-
7.61
4.43
8.31
-
5.91
6.74
-
Celecoxib
Meclofenamate
Sodium
5.64
11

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a
The concentrationof the test compound required to produce 50% inhibitionof COX-1 or COX-2. The
results (IC , µM) is themean of twodeterminations acquired using an ovine COX-1/ COX-2 assay
5
0
Kits(Cayman Chemicals Inc., Ann Arbor, MI, USA) and the deviation from the mean is <10% of the
b
c
mean value. In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50). Soya bean 15-LOX assay kit.
ND = Not Determined
2
.2.3. In vivo anti-inflammatory activity
The newly synthesized compounds 15-28 were evaluated for their in vivo anti-inflammatory
potential using carrageenan induced paw edema bioassay in rats where celecoxib was used as
a reference drug[26, 27]. The results were presented as percentage of edema inhibition at time
interval 1, 3 and 5 h,Table 2.The anti-inflammatory results revealed that the tested
compounds showed a gradual increase of the anti-inflammatory activity up to its maximum
after 5 h. Compounds 19 and 27 were the most active compounds showing 83% and 91%
anti-inflammatory activitycompared to that of celecoxib. In addition, results showed that22
with benzyl piperidine moiety was found to be the least active one with 53% activity
comparable to celecoxib after 5 h.
12

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Table 2: The percentages of edema inhibition of compounds 15-28and celecoxib
%
Edema inhibition
3h
Compound no.
1
h
5h
1
1
1
1
1
2
2
2
2
2
2
2
2
2
5
6
7
8
9
0
1
2
3
4
5
6
7
8
39.80±0.14
41.03±0.15
35.77±0.24
38.86±0.65
45.24±0.74
30.06±0.54
39.14±0.68
31.41±0.14
31.10±0.27
43.47±0.22
34.16±0.22
40.48±0.26
49.54±0.24
41.05±0.34
57.08±0.22
46.93±0.45
47.97±0.65
40.87±0.25
45.60±0.65
50.08±0.46
35.42±0.15
46.90±0.35
35.90±0.95
37.54±0.76
50.08±0.81
39.47±0.73
47.31±0.84
61.02±0.15
47.98±0.12
68.39±0.14
48.16±0.14
49.70±0.72
43.39±0.84
47.02±0.28
59.38±0.29
36.76±0.14
48.57±0.29
37.82±0.29
37.98±1.20
51.99±0.25
41.26±0.67
49.02±0.61
65.03±0.36
49.86±0.46
71.21±0.22
Celecoxib
The anti-inflammatory activity (the percentage of edema inhibition)was calculated according to the
following equation [27]:
%
Edema = [(V
R
-V
L
) control - (V
R
-V
L
) treated / (V
R L R
-V ) control] × 100 Where, V : Average right paw
thickness, V
L
: Average left paw thickness.
2
.2.4. In vivo ulcer liability activity
The occurrence of gastrointestinal (GI) damage (bleeding and ulceration) is probably
amongst the most prevalent and serious side effects associated with the use of non-steroidal
anti-inflammatory drugs (NSAIDs)[28, 29]. Observation of the gastrointestinal mucosa for
the presence of lesion following oral administration of 50 mmol/kg of the tested compounds
13

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as well as the reference drug has been taken as an indication for the ulcerogenic effects[28].
Compounds 19and 27were selected for this test. The results of ulcerogenic effect of these
compounds revealed that both of them showed superior safety profile when compared to
indomethacin since they showed ulcer index =2.66 and 2.5, respectively and weresafer than
reference indomethacin (UI = 17.23)as shown in Table 3.These data represented the potential
medicinal valueof these compounds as anti-inflammatory agents, since they have better safety
margin than indomethacinon gastric mucosa.
Table 3.Ulcerogenic effects of compounds 19,27 compared to indomethacin
1
9
27
(n = 5)
Indomethacin
Observations
(
n = 5)
(n = 5)
No. of rats with
ulcer
+
+
+++++
Mild
Mild
2.5
Severe
17.23
Severity of ulcer
Ulcer index
2.66
The ulcer index was calculated by the following formula
∶
U
I
= U
N S A I
+ U +U /10 where U is
the ulcer index, U
each stomach.
N
is ulcer number, U
S
is the ulcer score and U is the ulcer surface area for
A
2
.2.5. Histopathological studies (Light microscopic study)
The stomachs of rat used in ulcerogenicity test were subjected to histopathological study to
visualize the deep effects of indomethacin and the tested compounds 19 and 27 on the
stomach mucosa, submucosa and mucosal glands [30]. It was found that the transverses
sections in the stomach wall of the rats of control group showed no histopathological effect
with normal mucosa.The normal mucosa contains the gastric glands, which are formed of
isthmus, neck, and base (Fig. 3), while the transverses sections in the stomach of the rats
treatedwith indomethacin exhibited ulcer formation with marked maceration of gastric
14

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mucosa, necrosis and lymphocytic infiltration.In addition, in some animals, there was
evidence of submucosal widening, indicating edema (Fig. 4). Higher safety profiles were
observed with compounds 19and 27, where the transverses sections showed normal mucosal
glands with no histopathological effects apart from mild congestion with compound 19
(Fig.5)and focal lymphocytic infiltration with compound 27 (Fig. 6).
Fig. 3: Light microscopic pictures of the gastric mucosa of control rats: (A) showing the
gastric glands [isthmus (I), neck (N) and base (B)], muscularis mucosa (MM), submucosa (S)
and musculosa(M)(H&E 100×). (B) Higher magnification of the upper part of mucosa
showing the surface mucous cells extending into gastric pits(arrow) (H&E 200×).
Fig.4: Light microscopic pictures of the gastric mucosa of indomethacin treated rats showing:
(A) ulcer with marked maceration of gastric mucosa(U), dilated congested bloodvessels(C)
and inflammatory infiltrate (arrow),submucosal edema(E) (H&E 100×). (B)Higher
magnification of the previous section showing exfoliation of superficial epithelial cells
(Thick arrow) and necrotic cells (Thin arrow)(H&E 200×).
15

ACCEPTED MANUSCRIPT
Fig.5: Light microscopic pictures of the gastric mucosa of compound 19treated rats showing:
(A) nearly normal architecture (H&E 100×). (B) Higher magnification of the previous section
showing mild congestion of mucosal blood vessels(arrow) (H&E 200×).
16

ACCEPTED MANUSCRIPT
Fig. 6: Light microscopic pictures of the gastric mucosa of compound 27treated rats showing:
(A) nearly normal architecture (H&E 100×). (B) Higher magnification of the previous section
showing focal lymphocytic infiltration(arrow) (H&E 200×).
2
.2.6. Scanning electron microscopic study
Scanning electron microscopic study[31] showed thatthe surface of the stomach of control
rats showed gastric pits surrounded and lined by surface mucous cells. These cells appeared
dome-shaped with well-demarcated boundaries. The mucus was seen as small globular
masses over the cells (Fig. 7-A). The scanning results of the gastric mucosa in the group
exposed to indomethacin showed loss of the normal architecture, compared to the control
group. There was shedding of surface mucous cells leaving the underlying gastric pit walls
and openings with honeycomb shape. The cells lining the gastric pit walls were necrotic and
filling the pits (Fig. 7-B). The scanning of the groups treated with compounds19and27
revealed preservation of the normal architecture of the gastric mucosal surface, which
appeared similar to the control group with slight loos of demarcation between surface mucus
cells in some areas (Fig. 8-A and8-B).
Fig. 7: SEM photomicrograph of gastric mucosal surface of rats. (A) Control group: showing dome-
shaped surface mucous cells (thick arrow) that surround the gastric pits (arrowhead) with well-
demarcated boundaries. Mucus appears as globules over the cells (Thin arrow). X I000.(B)
Indomethacin group: showing complete shedding of the surface mucous cells leaving the walls and
openings ofthe gastric pits with a shape of honeycomb. The gastric pits show a necrotic tissue inside
(
arrow). X 500 and X 1000.
17

ACCEPTED MANUSCRIPT
Fig. 8: SEM photomicrograph of gastric mucosal surface of rats treated with compound 19 (A) and
compound 27 (B), respectively showing the gastric pits (arrow) surrounded by surface mucous cells
with loss of demarcation between the cells (arrow head). X 500 and X 1000.
2
.3. Molecular docking study
In an attempt to explain the biological activity and the difference in selectivity profiles of the
newly synthesized quinoline-2-carboxamides toward COX subtypes based on their
orientation and binding patterns, three representative compounds 19, 27 (active) and
22
(inactive) were docked into the active sites of COX-1 and COX-2, respectively, using
LIGANDFIT embedded in Discovery Studio software[32]. The 3D crystal structures of both
enzymes COX-1 and COX-2 complexes with their cocrystallized ligands (PDB codes: 1PGF
and 1CX2, respectively) were used for this study. The presence of an additional side pocket is
one of the well-known characteristics of COX-2 binding site. This side pocket was
createdbecause of the replacement of the amino acid residue Ile523 in COX-1 with the less
bulky Val523 in COX-2, which increases the volume of the COX-2 active site [33].
Moreover, it makes the COX-2 protein accommodate bulkier structures and allows other
interactions with amino acid residues such as Arg513, substituted by a His513 in COX-1 [34,
3
5]. It was reported that the traditional COX-2 inhibitors assume binding with Arg513 in the
COX-2 additional pocket, often via sulfone or sulfonamide groups, to achieve their activity
and selectivity over COX-1. In doing so, we evaluated the docking results of two of the most
active compounds in our novel quinoline-2-carboxamide derivatives, 19 and 27, into COX-2
18

ACCEPTED MANUSCRIPT
active sites. Obviously, these two hits exhibited similar binding pattern and interactions to
that of the cocrystallizedbromocelecoxib, S-58 ligand, Fig.9. It was clear from Fig.9(A&C)
that the orientation of the benzimidazole and phenylpiperazine moieties in 19 and
27,respectively within the additional side pocket of COX-2 was similar to fitting of
sulfonamide moiety of S-58 into the same side pocket. In addition, compound 19 formed two
H-bonds with Leu352 and Ser353 residues, Fig.9(C). Interestingly and because of the larger
volume of COX-2 active site, it could accommodate the bulky moieties, benzimidazole and
phenylpiperazine, in compounds 19 and 27 with almost the same binding pattern within the
entire COX-2 active site. Furthermore, positioning the quinoline nucleus in both derivatives,
19 and 27, within the main active site and forming hydrophobic interactions with Ile325,
Leu525 and Ala527 residues, correlates well with the position and hydrophobic interactions
of the trifluoromethylpyrazole fragment of S-58 with the same residues, Fig.9. Overall, these
interactions and binding pattern of 19 and 27 into COX-2 active site may contribute to the
remarkable COX-2 inhibitory activity and selectivity of these compounds.
Fig.9. (A) Docking and binding pattern of compound 27 into COX-2 active site (PDB code:
1
CX2). (B) The superimposition of the docked pose 27 (red) and the co-crystallized S-58
cyan) within active site of COX-2. (C) Docking and binding pattern of compound 19 into the
same COX-2 binding pocket. (D) The superimposition of the docked pose 19 (red) and the
(
19

ACCEPTED MANUSCRIPT
co-crystallized S-58 (cyan) within active site of COX-2. Dashed green lines represent
hydrogen bonds. All hydrogens were removed for the purposes of clarity.
On the contrary, docking of the inactive compound 22 into COX-2 active site revealed that
this compound adopted different binding pattern and disposition compared to the
cocrystallized ligand, S-58, Fig.10(B), where the quinoline-2-carboxamide nucleus was
deeply embedded inside the side corridor instead of the benzylpiperidine moiety as in
compound 27,Fig.10(A).
Fig.10. (A) Docking and binding pattern of compound 22 into COX-2 active site (PDB code:
CX2). (B) The superimposition of the docked pose 22 (green) and the co-crystallized S-58
cyan) within active site of COX-2. Dashed green lines represent hydrogen bonds. All
1
(
hydrogens were removed for the purposes of clarity.
On the other hand, the results of the docking simulation of 22 into COX-1 active site showed
that this compound was forced to adopt a longitudinal binding pattern due to presence of
Ile523, which closed the side pocket and pushed the compound to the bottom of the active
site, Fig.11.It could be noticed that the benzylpiperidine moiety in 22 seems to extremely
protrude outside the COX-1 active site, compared to cocrystallized ligand IMM owing to the
low affinity of 22 against COX-1 subtype. Apparently, this difference in binding modes and
disposition of 22 into both active sites of COX-1 and COX-2 compared with the
cocrystallized ligands could be the reason behind the inferior activity, which reflected its low
in vivo anti-inflammatory potential.
20

ACCEPTED MANUSCRIPT
Fig.11. (A) Docking and binding pattern of compound 22 into COX-1 active site (PDB code:
1
PGF). (B) Docking and binding pattern of the co-crystallized ligand IMM,1-(4-
iodobenzoyl)-5-methoxy-2-methyl indole-3-acetic acid, into COX-1 active site (PDB code:
PGF). (C) The superimposition of the docked pose 22 (red) and the co-crystallized
1
IMM(cyan) within the active site of COX-1. Dashed green lines represent hydrogen bonds.
All hydrogens were removed for the purposes of clarity.
Finally, to validate our docking protocol settings, we have re-docked the co-crystallized
ligand S-58 after being extracted from the used 3D protein structure of COX-2 (pdb code:
1
CX2) using the same protocol settings utilized for docking simulation of our compounds 19
,
22
and 27. It was clear that the used docking protocol closely reproduced the bound structure
with RMSD value of 0.83 A° confirming the confidence in our docking study. The
comparison between the docked pose of S-58 and the experimental co-crystallized structure
has been shown in Fig.12
.
Fig. 12. Comparison between the docked pose of S-58 (red) as produced by
dockingexperiment and the co-crystallized ligand of this inhibitor within COX-2 (green, PDB
code: 1CX2).
21

ACCEPTED MANUSCRIPT
3
. Conclusion
In the present study, a series of quinoline-2-carboxamide derivatives was synthesized and
evaluated in vivo as potential anti-inflammatory agents suggesting the dual COXs/LOX
inhibition as a molecular mechanism of their action. The in vitro assays results revealed that
compounds 19 and 27 possessing benzimidazole and phenyl piperazine groups were the most
potent COX-2 inhibitors with IC50 values of 1.21 and 1.13 µM, respectively while the same
compounds showed remarkable activity against 15-LOX with IC50 values of 6.18 and
5.91µM, respectively. Conversely, compound 22 bearing phenyl piperidine moiety was the
least potent compound as COX-2 inhibitor with IC50 value of 3.54µM. Meanwhile, the in vivo
carrageenan induced rat paw edema assay results showed that the tested compounds showed a
gradual increase of the anti-inflammatory activity and interestingly compounds 19 and 27
with highest in vitro activity were the most active compounds so far. Furthermore,
compounds 19 and 27were subjected to ulcerogenicity study and the results indicated that
these two compounds have higher gastric safety profiles than indomethacin. The transverses
sections showed normal mucosal glands with no histopathological effects. Also, the electron
scanning microscopic study of the groups treated with compounds 19 and 27 revealed
preservation of the normal architecture of the gastric mucosal surface. The docking study
supported and explained the results obtained from the in vitro COXs assays based on the
structural features and binding patterns of the compounds inside the active sites. Obviously,
the two active hits 19 and 27 adopted similar binding patterns and interactions to that of the
cocrystallized bromocelecoxib, S-58 ligand where the benzimidazole and phenyl piperazine
moieties in 19 and 27, respectively assumed the same positioning of sulfonamide moiety of
S-58 within the additional side pocket of COX-2. On the contrary, compound 22 exhibited
different binding mode and disposition compared to the cocrystallized ligand, S-58 reflecting
the inactivity of this ligand against COX-2. Combining the in vitro
, in vivo and ulcerogenicity
results, this study led to discovery of two promising compounds with high potency against
22

ACCEPTED MANUSCRIPT
COX-2 and LOX that warranted continued preclinical and further development as potential
anti-inflammatory agents with much better gastric safety profiles.
4
. Experimental
4
.1. Chemistry
All the chemicals used were of analytical grade and purified by standard methods prior to
use. Silica gel column chromatography was carried out using kieselgel 60 (merck). TLC
analysis was performed on aluminium-backed plates coated with silica gel 60 F254 (Merck).
Melting points (mp) were determined using a GalenKampmelting point apparatus andare
uncorrected. Components were visualized using potassium permanganate solution and UV
light. NMR Spectra were taken using a Varian Unity INOVA 400 MHz and Bruker AC250
MHz spectrometers for proton and carbon at university of Aberdeen. All numbers referring to
NMR data obtained are in parts per million (ppm). High resolution mass spectrometric data
were obtained using Thermo Instruments MS system (LTQ XL/LTQ Orbitrap Discovery)
coupled to a Thermo Instruments HPLC system (Accela PDA detector, Accela PDA
autosampler and Pump) at university of Aberdeen.Compounds
1, 4, 7 and 8[23],9 and
10[24]were prepared according to reported procedures.
4.1.1. General procedure for synthesis of benzylcyanides(1-3).
To a stirred solution of p-aminobenzylcyanide (3.00 g, 22.7 mmol, 1.0 eq.) in toluene (75
mL), 1,5-dibromopentane, 1,4-dibromobutane or 1,4-dibromopentane (24.97 mmol, 1.1 eq.)
and DIPEA (7.9 mL, 2 eq.) were added. The reaction mixture was refluxed for 20 h in an oil
bath. After cooling, excess toluene was added and the red brown precipitate (Br-salt) was
collected by filtration. The filtrate was washed with water, dried over anhydrous MgSO
evaporated under reduced pressure to afford the target products.
4
, and
23

ACCEPTED MANUSCRIPT
4
.1.1.1. 2-(4-(Pyrrolidin-1-yl)phenyl)acetonitrile (2).Compound
to the synthetic procedure given above as pale brown solid in yield of 92%, mp 82-83 C.; H
NMR (400 MHz, CDCl 7.13 (d, = 8.8 Hz, 2H, Ar-H), 6.52 (d, = 8.0 Hz, 2H, Ar-H),
.62 (s, 2H, CH CN), 3.26 (t, = 6.0 Hz, 4H, pyrrolidin-H), 2.10-1.95 (m, 4H, pyrrolidin-
H); C NMR (62.5 MHz, CDCl
2 was synthesized according
o
1
3
)
δ
J
J
3
2
J
1
3
3
)
δ
147.6, 128.8, 118.9, 115.8, 112.0, 47.6, 25.5, 22.8
+
(
CH CN);HRESI-MSm/z calcd.for [M+H] C H N : 187.1230, found: 187.1232.
2
12 15
2
4.1.1.2. (±)2-(4-(2-Methylpyrrolidin-1-yl)phenyl)acetonitrile
(3
).Compound
3
was
synthesized according to the synthetic procedure given above asreddish brown oilin yield of
1
9
2%; H NMR (250 MHz, CDCl )
δ
7.13 (d,
= 6.0 Hz, 1H, pyrrolidin-H), 3.62 (s, 2H, CH
pyrrolidin-H), 3.15 (q,
J
= 8.8 Hz, 2H, Ar-H), 6.55 (d,
J
J
= 8.5 Hz, 2H,
= 7.3 Hz, 1H,
3
Ar-H), 3.86 (t,
J
2
CN), 3.40 (t,
J
= 7.8 Hz, 1H, pyrrolidin-H), 2.22-1.88 (m, 3H, pyrrolidin-H), 1.70
13
(
3 3
m, 1H, pyrrolidin-H), 1.16 (d, J = 6.5 Hz, 3H, CH ); C NMR (62.5 MHz, CDCl ) δ 146.8,
1
28.8, 118.9, 115.7, 112.1, 53.7, 48.2, 33.1, 23.3, 22.7, 19.2;HRESI-MS m/z calcd.for
+
[
M+H] C H N : 201.1386, found: 201.1384.
13
17
2
4
.1.2. General procedure for the synthesis of phenethylamine derivatives (4-6).
(3.8 g, 99.9 mmol, 10 eq.) in dry Et O (30 mL) at 0 C in a two-
necked rounded bottom flask maintaining anhydrous conditions under nitrogen atmosphere,
benzylcyanide derivatives (1-3) (9.99 mmol, 1 eq.) dissolved in dry Et O (50 mL) was added.
o
To a suspension of LiAlH
4
2
2
The reaction mixture was stirred overnight at rt. A saturated solution of Na
2
SO
4
was slowly
O. The
O. The organic
layer was washed with brine, dried over anhydrous MgSO and evaporated under reduced
o
added at 0 C with stirring for an additional 30 min followed by addition of H
2
reaction mixture was filtered and the residue was washed three times with Et
2
4
pressure to give the correspondingphenethylamine derivatives (4-6).
24

ACCEPTED MANUSCRIPT
4.1.2.1. 2-(4-(Pyrrolidin-1-yl)phenyl)ethanamine (5). Compound
5
was synthesized
1
according to the synthetic procedure given above as reddish brown oil in yield of 78%; H
NMR (250 MHz, CDCl
.26 (t, = 6.5 Hz, 4H, pyrrolidin-H), 2.90 (t,
Hz, 2H, CH CH NH ), 2.10-1.95 (m, 4H, pyrrolidin-H), 1.29 (s, 2H, NH
146.6, 129.6, 126.4, 111.8, 47.8, 44.0, 39.9, 25.5;HRESI-MS m/z calcd.for
3
)
δ
7.05 (d,
J
= 8.3 Hz, 2H, Ar-H), 6.52 (d,
J
= 8. 8 Hz, 2H, Ar-H),
NH ), 2.64 (t, = 6. 8
); C NMR (62.5
3
J
J
= 7.0 Hz, 2H, CH CH
2
2
2
J
13
2
2
2
2
MHz, CDCl3)
δ
+
[
M+H] C H N : 191.1543, found: 191.1540.
12
19
2
4
.1.2.2. (±)2-(4-(2-Methylpyrrolidin-1-yl)phenyl)ethanamine (6)
.
Compound
6
was
synthesized according to the synthetic procedure given above as reddish brown oil in yield of
1
9
1%; H NMR (250 MHz, CDCl
3
)
δ
7.05 (d,
= 6.0 Hz, 1H, pyrrolidin-H), 3.35 (t,
7. 8 Hz, 1H, pyrrolidin-H), 2.91 (t, = 6.3 Hz, 2H, CH CH NH ), 2.85-2.50 (m, 4H,
CH CH NH ), 2.20-1.82 (m, 3H, pyrrolidin-H), 1.70 (m, 1H, pyrrolidin-H), 1.16 (d,
Hz, 3H, CH
J
= 8.3 Hz, 2H, Ar-H), 6.53 (d,
J = 8.3 Hz, 2H,
Ar-H), 3.83 (t,
J
J
= 7.3 Hz, 1H, pyrrolidin-H), 3.15 (q, J
=
J
2
2
2
2
2
2
J = 6.25
13
3
); C NMR (62.5 MHz, CDCl
3
)
δ
145.9, 129.6, 125.9, 111.9, 53.7, 48.4, 43.6,
+
3
8.5, 33.2, 23.4, 19.6;HRESI-MS m/z calcd.for [M+H]
05.1701.
.1.3. Procedure for synthesis of compound9 ((±) Ethyl 1-benzylpiperidine-3-carboxylate).
13 21 2
C H N : 205.1699, found:
2
4
To a solution of ethyl nipecotate (5.94 mL, 38.24 mmol, 1 eq.) in a mixture of DCM (80 mL)
and H O (40 mL), benzyl bromide (4.77 mL, 1.05 eq.) and Na CO (5.06 g, 1.25 eq.) were
2
2
3
added and the resulting reaction mixture was refluxed for 3h. The organic layer was separated
from aqueous layer, washed with brine, dried over MgSO , and evaporated under reduced
4
pressure. The residue was purified by flash chromatography on silica gel using a mixture of
1
ethylactate, hexanes (1:6) as eluent to give
MHz, CDCl 7.29-7.18 (m, 5H, Ar-H), 4.11 (q,
3.6 Hz, 1H, CHa-Ph), 3.44 (d, = 13.2 Hz, 1H, CHb-Ph), 2.90 (m, 1H, piperidin-H), 2.68
m, 1H, piperidin-H), 2.54 (m, 1H, piperidin-H), 2.20 (m, 1H, piperidin-H), 2.01 (m, 1H,
9
as colorless oil in yield of 87%; H NMR (400
3
)
δ
2 3
J = 7.2 Hz, 2H, OCH CH ), 3.51 (d, J =
1
J
(
25

ACCEPTED MANUSCRIPT
piperidin-H), 1.90 (m, 1H, piperidin-H), 1.69 (m, 1H, piperidin-H), 1.52 (m, 2H, piperidin-
13
H), 1.19 (t,
J
= 7.2 Hz, 3H, OCH CH ); C NMR (62.5MHz, CDCl ):
δ
174.4, 138.4, 129.1,
2
3
3
+
1
28.2, 127.0, 63.3, 60.3, 55.5, 53.7, 42.0, 27.0, 24.6, 14.2;HRESI-MS m/z calcd.for [M+H]
: 248.1645, found: 248.1646.
15 2
C H22NO
4.1.4. Procedure for synthesis of compound10 ((±) 1-Benzylpiperidine-3-carbohydrazide).
A mixture of ethyl 1-benzylpiperidine-3-carboxylate( )(5.1 g, 20.62 mmol, 1 eq.) and
9
hydrazine monohydrate (10 mL, 10 eq.) in ethanol (10 mL) was stirred overnight under
reflux. After cooling, the solvent and excess of hydrazine were removed under reduced
1
pressure to afford compound 10as colorless oil in yield of 99%; H NMR (400 MHz, CDCl
3
)
δ
9.00 (s br, 1H, NH), 7.32-7.17 (m, 5H, Ar-H), 3.79 (s br, 2H, NH
2
), 3.47(d, J = 12.4 Hz,
1
H, CHa-Ph), 3.38 (d, = 12.4 Hz, 1H, CHb-Ph), 2.62 (m, 2H, piperidin-H), 2.45 (m, 1H,
J
piperidin-H), 2.23 (m, 2H, piperidin-H), 1.87 (m, 1H, piperidin-H), 1.72-1.48 (m, 3H,
13
piperidin-H); C NMR (100 MHz, CDCl )
δ
175.4, 137.3, 129.2, 128.4, 127.4, 63.4, 54.4,
3
+
5
3.7, 40.8, 26.7, 22.7; HRESI-MS m/z calcd.for [M+H] C13
H
20
N
3
O: 234.1601, found:
2
34.1604.
.1.5. Procedurefor synthesis of compound11 ((±) 1-Benzyl-3-aminopiperidine).
4
To a solution of (10) (5 g, 21.43 mmol, 1 eq.) in H
2
O (30 mL), trifluoroacetic acid (4.95 mL,
o
3
eq.) and NaNO (2.22 g, 1.5 eq.) were successively added with stirring for 2 h at 0 C then
2
o
heated at 80 C for additional 2 h . 6 N NaOH (11 mL) was added and the reaction mixture
was extracted twice with DCM, then with EtOAc. The combined organic layers were dried
over MgSO
4
and concentrated under reduced pressure to afford 11as reddish brown oil in
1
yield of 86%; H NMR (400 MHz, CDCl
3
)
δ
7.27-7.16 (m, 5H, Ar-H); 3.46 (d,
= 10.8 Hz, 1H, CHb-Ph); 2.83 (m, 1H, piperidin-H); 2.68 (m, 1H,
piperidin-H); 2.53 (m, 1H, piperidin-H); 2.27 (s br, 2H, NH ); 2.02 (m, 1H, piperidin-H);
.84-1.72 (m, 2H, piperidin-H); 1.63 (m, 1H, piperidin-H); 1.49 (m, 1H, piperidin-H); 1.09
J = 13.2 Hz,
1
H, CHa-Ph); 3.41 (d, J
2
1
26

ACCEPTED MANUSCRIPT
13
(
m, 1H, piperidin-H); C NMR (62.5 MHz, CDCl ):
δ
138.3, 129.2, 128.2, 127.0, 63.2, 62.0,
3
+
5
1
4
3.5, 47.9, 33.5, 23.5; HRESI-MS m/z calcd.for [M+H] C H N : 191.1543, found:
1
2
19
2
91.1537.
.1.6. Procedure for synthesis of compound12 (Diethyl 3-((4-chlorophenyl)imino)-2-methyl
pentanedioate).
To a solution of 4-chloroaniline (2 g, 15.68 mmol) and diethyloxalpropionate (3.8 g, 18.81
mmol) in ethanol (30 mL), acetic acid (10 drops) was added and the reaction mixture heated
under reflux under for 48 h. After completion of the reaction, ethanol was removed by vacuo
and the residue was extracted with ethyl acetate and washed with saturated sodium
4
bicarbonate solution and brine. Organic layer was dried over MgSO and evaporated to give
crude product which purified by column chromatography on silica using EtOAc/hexanes
1
(
1:10) to give pure imine 12as an oil in yield of 84%; H NMR (250 MHz, CDCl )
δ
7.19 (d,
J
3
=
8.50 Hz, 2H, Ar-H), 6.88 (d,
.35-4.00 (m, 4H, 2 x CH CH ), 1.82 (d,
= 7.25 Hz, 3H, CH
J
= 8.50 Hz, 2H, Ar-H), 4.75 (q,
J
= 7.2 Hz, 1H, CHCH
3
),
4
2
3
J
= 7.2 Hz, 3H, CHCH
3
), 1.29 (t,
J
= 7.0 Hz, 3H,
170.63,
13
CH
2
CH
3
), 1.12 (t,
J
2
CH
3
); C NMR (62.5 MHz, CDCl
3
)
δ
164.73, 146.56, 139.20, 129.22, 129.05, 122.04, 97.17, 61.95, 60.34, 14.37, 13.78, 13.17.
4.1.7. Procedurefor synthesis of compound13 (Ethyl 6-chloro-4-hydroxy-3-methylquinoline-
2-carboxylate).
To the compound (12) (12.28 mmol), polyphosphoric acid (15 g) was added and the reaction
mixture was stirred at 120 °C for 2 h to form a dark brown solid then water (50 mL) was
slowly added to quench the excess acid and stirring with reflux was continued for 0.5 h. The
formed precipitate was filtered and washed with water then Et O, and dried to provide pure
2
o
1
quinoline-2-carboxylate 13as white solid in yield of 92%, mp 82-83 C; H NMR (400 MHz,
DMSO- 11.86 (s, 1H, OH), 7.98 (t, = 2.8 Hz, 1H, Ar-H), 7.89 – 7.79 (m, 1H, Ar-H),
= 8.8, 2.5 Hz, 1H, Ar-H), 4.43 (q, = 7.1 Hz, 2H, CH CH ), 2.19 (s, 3H, CH ),
= 7.1 Hz, 3H, CH CH ); C NMR (101 MHz, DMSO-d6 165.25, 138.07, 132.80,
d
6
)
δ
J
7
.67 (td,
.36 (t,
J
J
2
3
3
1
3
1
J
) δ
2
3
27

ACCEPTED MANUSCRIPT
1
32.60, 128.28, 124.37, 124.20, 121.86, 121.79, 119.31, 63.06, 14.39, 11.79; HRESI-MS m/z
-
calcd.for [M-H] C H ClNO : 264.0433, found: 264.0430.
13
11
3
4.1.8. Procedurefor synthesis of compound14 (6-Chloro-4-hydroxy-3-methylquinoline-2-
carboxylic acid).
To a solution of quinoline ester (13) (1 g, 3.76 mmol) in ethanol (25 mL), 5% NaOH (20 mL)
o
was added.The reaction mixture was kept at 80 C with stirring for 4 h. The residue after
removal of ethanol under reduced pressure was then taken into water and acidified with 5%
HCl. The formed precipitate was filtered, washed with water, and dried under vacuum to
o
1
give14as white solid in yield of 96%, mp243-245 C; H NMR (400 MHz, DMSO-
d
6
)
δ
11.84
= 9.0,
(s, 1H, OH), 7.98 (d,
J
= 2.5 Hz, 1H, Ar-H), 7.86 (d,
J
= 9.0 Hz, 1H, Ar-H), 7.66 (dd,
J
13
2
.5 Hz, 1H, Ar-H), 2.22 (s, 3H, CH ); C NMR (62.5 MHz, DMSO-d6) 176.56, 164.86,
3
1
37.64, 136.77, 132.24, 127.90, 123.96, 123.80, 121.46, 118.98, 11.39; HRESI-MS m/z
-
calcd.for [M-H] C11
H
7
ClNO
3
: 236.0120, found: 236.0116.
4.1.9. General procedure for preparation of quinoline-2-carboxamides (15-28).
A mixture of 6-chloro-4-hydroxy-3-methylquinoline-2-carboxylic acid (14) (1.05 mmol),
BOP (0.6 g, 1.37 mmol), and DIPEA (0.37 mL, 2.10 mmol) in DCM (30 mL) was stirred for
1
0 min at rt before addition of an appropriate primary or secondary amine(1.26 mmol)
dissolved in DCM (5 mL) and the resulting reaction mixture was stirred overnight at rt. The
formed precipitate was filtered, washed with Et O and dried to afford pure desired quinoline-
-carboxamides (15-28).
2
2
28