Design, synthesis, and biological evaluation of ketoprofen analogs as potent cyclooxygenase-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.06.094

A new series of ketoprofen analogs were synthesized to evaluate their biological activities as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1 and COX-2 inhibition studies showed that all compounds were potent and selective inhibitors of the COX-2 isozyme with IC₅₀ values in the highly potent 0.057-0.085 μM range, and COX-2 selectivity indexes in the 115 to >1298.7 range. Compounds possessing azido pharmacophore group (8a and 8b) exhibited highly COX-2 inhibitory selectivity and potency even more than reference drug celecoxib. Molecular modeling studies indicated that the azido substituent can be inserted deeply into the secondary pocket of COX-2 active site for interactions with Arg⁵¹³.

Full text of DOI:10.1016/j.bmc.2010.06.094

Bioorganic & Medicinal Chemistry 18 (2010) 5855–5860
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis, and biological evaluation of ketoprofen analogs as
potent cyclooxygenase-2 inhibitors
*
Afshin Zarghi , Razieh Ghodsi
Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of ketoprofen analogs were synthesized to evaluate their biological activities as selective
cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1 and COX-2 inhibition studies showed that all com-
pounds were potent and selective inhibitors of the COX-2 isozyme with IC50 values in the highly potent
0.057–0.085 lM range, and COX-2 selectivity indexes in the 115 to >1298.7 range. Compounds possess-
ing azido pharmacophore group (8a and 8b) exhibited highly COX-2 inhibitory selectivity and potency
Received 18 May 2010
Revised 28 June 2010
Accepted 29 June 2010
Available online 3 July 2010
even more than reference drug celecoxib. Molecular modeling studies indicated that the azido substitu-
Keywords:
ent can be inserted deeply into the secondary pocket of COX-2 active site for interactions with Arg513
.
Cyclooxygenase-2 inhibition
Ketoprofen analogs
Quinoline-4-carboxylic acid
Molecular modeling
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
IDs and our quinoline-4-carboxylic acid scaffold. As part of our re-
search program, aimed at discovering new selective COX-2
inhibitors, we focused our attention on the synthesis, COX inhibi-
tory and some molecular modeling studies of 2-(4-(substituted)
phenyl)quinoline-4-carboxylic acid derivatives possessing benzoyl
moiety at C-6 or C-8 of quinoline ring. The rational for the design of
these compounds was based on ketoprofen structure as a part of 2-
aryl-quinoline-4-carboxylic acid scaffold (Fig. 1) in our previously
reported COX-2 inhibitors.
Non-stroidal anti-inflammatory drugs (NSAIDs) are widely used
for the treatment of inflammation, fever, and pain. However, be-
cause NSAIDs inhibit both isoforms of cyclooxygenase (COX) (con-
stitutive COX-1, responsible for cytoprotective effects; inducible
COX-2, responsible for inflammatory effects), they are associated
with well-known side effects such as gastrointestinal side effects
and renal function suppression.1,2 It is known that selective COX-
2 inhibitors can provide anti-inflammatory agents devoid of the
undesirable effects associated with classical non-selective NSA-
IDs.3 In addition to the role of COX-2 in inflammatory disorders
such as rheumatoid arthritis and osteoarthritis, it is also implicated
in cancer and angiogenesis. In this regard, several epidemiologic
studies have been reported that inhibitors of COX-2 enzyme reduce
the risk of colorectal, breast, and lung cancer, and COX-2 is ex-
pressed in these cancers.4,5 As a consequence, increasing interest
has been devoted to the synthesis of selective inhibitors of COX-
2 by means of modification of well-known non-selective agents
such as indomethacine,6–8 zomepirac,9 flurbiprofen,10 meclofena-
mic acid,11 or ketoprofen.12–14 On the other hand, we recently re-
ported several investigations describing the design, synthesis,
and a molecular modeling study for a group of 2-phenyl-4-car-
boxyl quinolines (Fig. 1, A) possessing a methyl sulfonyl COX-2
pharmacophore at the para position C-2 phenyl ring.15 Our results
showed that quinoline ring is a very suitable scaffold for COX-2
inhibitory activity. Therefore, it is interesting to synthesize and
evaluate the hybrid structure of ketoprofen as a well-known NSA-
2. Chemistry
The target 6- or 8-benzoyl-2-(4-(methylthio)phenyl)quinoline-
4-carboxylic acid derivatives and 6- or 8-benzoyl-2-(4-(acetam-
ido)phenyl)quinoline-4-carboxylic acid derivatives were obtained
employing a Doebner reaction.16 As illustrated in Scheme 1, appro-
priate benzaldehyde (1), pyrrovic acid (2), and appropriate amine
(3) were heated in acetic acid to provide 4-carboxyl quinolines
(4 and 5, 15–31%) and then oxidation of methylthio substituent
was carried out using oxone by previously reported method17 to give
6a and 6b (67–70%). Hydrolysis of acetamido derivatives 5a and 5b
under acidic condition gave the corresponding amines 7a and 7b
(47%). Diazotization of 7a and 7b with sodium nitrite followed by
treatment with sodium azide afforded the azide derivatives 8a and
8b (20–35%).18
3. Results and discussion
A new group of 2-(4-(substituted)phenyl)quinoline-4-carbox-
ylic acid possessing a benzoyl moiety at C-6 or C-8 position of
* Corresponding author. Tel.: +98 21 8820096; fax: +98 21 88665341.
E-mail address: azarghi@yahoo.com (A. Zarghi).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.06.094

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A. Zarghi, R. Ghodsi / Bioorg. Med. Chem. 18 (2010) 5855–5860
COOH
O
COOH
N
SO 2Me
A
Ketoprofen
COOH
N
O
COOH
N
O
R
R
Designed compounds
Figure 1. Chemical structures of ketoprofen and 4-carboxyl quinoline (A) lead compounds and our designed scaffolds.
COOH
CHO
R3
O
R3
+
+
a
R1
COOH
NH2
R2
N
R2
1
2
3
R1
4a, R1 = SMe, R2 = PhCO, R3 = H
4b, R1 = SMe, R2 = H, R3 = PhCO
5a, R1 = NHCOMe, R2 = PhCO, R3 = H
5b, R1 = NHCOMe, R2 = H, R3 = PhCO
COOH
N
COOH
N
R3
R3
b
R2
R2
SCH3
SO2CH3
4a, 4b
6a, R2 = PhCO, R3 = H
6b, R2 = H, R3 = PhCO
COOH
N
COOH
N
COOH
R3
R3
R3
c
d
N
R2
R2
R2
NHCOCH3
N3
NH2
5a, 5b
7a
7b
,
R2 = PhCO, R3 = H
,
R2 = H, R3 = PhCO
8a, R2 = PhCO, R3 = H
8b, R2 = H, R3 = PhCO
Scheme 1. Reagents and conditions: (a) acetic acid, reflux, 20 h; (b) oxone/THF, 3–5 h; (c) HCl, reflux, 5 h; (d) NaNO2/HCl and then NaN3, 0–5 °C, 2 h.
quinoline ring was designed based on ketoprofen structure. SAR
data (IC50 values) acquired by determination of the in vitro ability
of the title compounds to inhibit the COX-1 and COX-2 isozymes
showed that all compounds were potent and selective inhibitors
of the COX-2 isozyme with IC50 values in the highly potent 0.057–
0.085 lM range, and COX-2 selectivity indexes in the 115 to
>1298.7 range (Table 1). Our results showed that the COX inhibition
was sensitive to the position of benzoyl group on the quinoline ring.
Accordingly, compounds possessing benzoyl on the C-6 (5b, 6b, and
8b) were more selective COX-2 inhibitors compared with those

A. Zarghi, R. Ghodsi / Bioorg. Med. Chem. 18 (2010) 5855–5860
5857
Table 1
compounds possessing N3 substituent (8a and 8b) exhibited high
potency and selectivity on COX-2 inhibition which may be due to
the insertion of N3 deeply into the secondary pocket of COX-2 bind-
In vitro COX-1 and COX-2 enzyme inhibition assay data
COOH
ing site where it undergoes electrostatic interaction with Arg513
.
R2
Accordingly, the binding interactions of the most selective COX-2
inhibitor (8b) within the COX-2 binding site were investigated.
The most stable enzyme–ligand complex of 2-(4-(azido)phenyl)-
6-benzoyl-quinoline-4-carboxylic acid possessing a N3 COX-2 phar-
macophore group within the COX-2 binding site (Fig. 2) shows that
the para-N3-phenyl moiety is oriented towards the COX-2 second-
ary pocket (Arg513, Phe518, and Val523). All the nitrogen atoms of
N3 can form remarkable binding interactions with NH groups of
Arg513 (distances <4 Å). The azido substituent has the potential to
undergo electrostatic binding interactions with amino acid resi-
dues, particularly Arg513, lining the secondary pocket of COX-2.18
In addition, our docking studies showed that one of the O-atoms
of carboxylic acid group can form hydrogen binding interaction
with amino group of Arg120 (distance = 2.0 Å) whereas the other
O-atom is about 2.8 Å away from OH of Tyr355. Also, the distance be-
tween the O-atom of C@O group attached to C-6 quinoline ring and
OH of Ser530 is 3.4 Å which can provide a hydrogen binding interac-
tion. On the other hand, the nitrogen atom of quinoline ring is close
to NH group of Ala527 (distance = 4.7 Å) and the phenyl ring of ben-
zoyl moiety is close to hydrophobic side chain of Leu117. These data
together with experimental results can explain the high potency
and selectivity of compound 8b. Similarly, molecular modeling
study of compound 8a shows that it binds in the primary binding
site such that the para-N3 substituent is inserted into the secondary
N
R1
R3
a
Compound R1
R2
H
R3
IC50
(l
M)
Selectivity
index (SI)b
COX-1
COX-
2
5a
PhCO
NHCOCH3
9.2
21.8
15.9
24.4
0.080
0.085
0.069
0.077
115.0
256.5
231.1
316.8
1150
5b
H
PhCO NHCOCH3
SO2Me
PhCO SO2Me
N3
PhCO N3
SO2Me
6a
6b
PhCO
H
H
8a
8b
PhCO
H
H
65.55 0.057
>100
13.4
24.3
0.077 >1298.7
A
H
H
0.091
0.06
147.2c
405
Celecoxib
a
Values are means of two determinations acquired using an ovine COX-1/COX-2
assay kit and the deviation from the mean is <10% of the mean value.
b
In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
c
Ref. 15.
having benzoyl on the C-8 (5a, 6a, and 8a) of quinoline ring. This ef-
fect may be explained by steric parameter for interaction with COX-
1 active binding site. However, the isomers having C-8 benzoyl moi-
ety showed more potency for COX-2 inhibitory activity. SAR data
also indicated the COX inhibition was very sensitive to the type of
substituent at the para position of C-2 phenyl ring so that it can af-
fect both potency and selectivity on COX-2 inhibitory activity. The
relative COX-2 potency and COX-2 selectivity profiles for ketopro-
fen derivatives with respect to the substituents at the para position
of C-2 phenyl ring was N3 ꢀ SO2Me > NHCOMe. Interestingly,
pocket present in COX-2 and forms hydrogen bonding interaction
0
with NH groups of Arg513 (distances <4 ÅA). Also, the carboxylic acid
substituent of compound 8a is close to NH groups of Arg120 and can
bind to this amino acid in the same manner as compound 8b. How-
ever, the orientation of benzoyl moiety of 8a is different from that of
8b into the active site of COX-2, which may explain the selectivity
and potency differences of these isomers. The binding interactions
Figure 2. Docking 2-(4-(azido)phenyl)-8-phenylcarbonylquinoline-4-carboxylic acid (8a, in green), 2-(4-(azido)phenyl)-6-phenylcarbonylquinoline-4-carboxylic acid (8b, in
pink) and ketoprofen (in yellow) in the active site of murine COX-2.

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A. Zarghi, R. Ghodsi / Bioorg. Med. Chem. 18 (2010) 5855–5860
of ketoprofen molecule within the COX-2 binding site were also
investigated and interestingly the binding interactions of ketopro-
fen are similar to compound 8b into the COX-2 binding site
(Fig. 2). These results showed that when the benzoyl group is at
C-6 position of quinoline ring, the molecule is more like ketoprofen,
hence showing better selectivity than the isomer possessing C-8
benzoyl group.
H3 & H5, J = 8.4 Hz), 7.57 (t, 2H, phenyl H3 & H5, J = 7.6 Hz), 7.69
(t, 1H, phenyl H4, J = 7.4 Hz), 7.80 (d, 2H, phenyl H2 & H6,
J = 8.1 Hz), 8.10 (d, 1H, quinoline H8, J = 9.4 Hz), 8.22 (d, 1H, quin-
oline H7, J = 8.4 Hz), 8.26 (d, 2H, 4-methylthiophenyl H2 & H6,
J = 8.4 Hz), 8.51 (s, 1H, quinoline H3), 9.06 (s, 1H, quinoline H5),
14.2 (s, 1H, COOH), LCMS (ESI): 400.6 (M+1)+ 100.
5.1.3. 2-(4-(Acetamido)phenyl)-8-benzoyl-quinoline-4-
carboxylic acid (5a)
4. Conclusions
Yield: 31%; yellow crystalline powder; mp: 310–311 °C; IR
(KBr):
m
(cmꢁ1) 3347 (NH) 2973–2263 (OH), 1708, 1657, 1646
This study indicates that (i) 2-aryl-4-carboxyl quinoline is a
suitable scaffold (template) to design COX-1/COX-2 inhibitors, (ii)
COX-1/COX-2 inhibition is very sensitive to the substituent of the
para position of C-2 phenyl ring, (iii) COX-1/COX-2 inhibition is af-
fected by the position of benzoyl moiety on the quinoline ring, and
(iv) 2-(4-(azido)phenyl)-6 or 8-benzoyl-quinoline-4-carboxylic
acid (8a or 8b) exhibited highly COX-2 inhibitory potency and
selectivity even more than celecoxib.
(C@O), 1H NMR (DMSO-d6): d 2.01 (s, 3H, Me), 7.46 (t, 2H, phenyl
H3 & H5, J = 7.70 Hz), 7.53 (d, 2H, 4-acetamidophenyl H3 & H5,
J = 8.7 Hz), 7.58–7.69 (m, 5H, phenyl H2, H4 & H6 & 4-acetamid-
ophenyl H2 & H6), 7.77 (t, 1H, quinoline H6, J = 7.8 Hz), 7.91 (d,
1H, quinoline H7, J = 7.0 Hz), 8.38 (s, 1H, quinoline H3), 8.75 (d,
1H, quinoline H5, J = 9.4 Hz), 10.04 (s, 1H, NH), 14.01 (s, 1H, COOH),
13C NMR (DMSO-d6): d 24.9, 119.5, 119.7, 123.8, 127.9, 128.4,
128.5, 129.3, 130.0, 130.2, 132.5, 133.7, 138.5, 139.5, 139.7,
142.1, 146.8, 155.7, 168.2, 169.4, 198.5, LCMS (ESI): 411.6 (M+1)+
100. Anal. Calcd for C25H18N2O4: C, 73.16; H, 4.42; N, 6.83. Found:
C, 73.38; H, 4.20; N, 6.81.
5. Experimental section
All chemicals and solvents used in this study were purchased
from Merck AG and Aldrich Chemical. Melting points were deter-
mined with a Thomas-Hoover capillary apparatus. Infrared spectra
were acquired using a Perkin Elmer Model 1420 spectrometer. A
Bruker FT-500 MHz instrument (Brucker Biosciences, USA) was
used to acquire 1H NMR spectra with TMS as internal standard.
Chloroform-D and DMSO-d6 were used as solvents. Coupling con-
stant (J) values are estimated in hertz (Hz) and spin multiples are
given as s (singlet), d (double), t (triplet), q (quartet), m (multiplet),
and br (broad). The mass spectral measurements were performed
on an 6410Agilent LCMS triple quadrupole mass spectrometer
(LCMS) with an electrospray ionization (ESI) interface. Microanaly-
ses, determined for C and H, were within 0.4% of theoretical values.
5.1.4. 2-(4-(Acetamido)phenyl)-6-benzoyl-quinoline-4-
carboxylic acid (5b)
Yield: 15%; yellow crystalline powder; mp: 286–288 °C; IR
(KBr):
m
(cmꢁ1) 3348 (NH), 3094–1995 (OH), 1721, 1663, 1633
(C@O), 1311, 1150 (SO2), 1H NMR (DMSO-d6): d 2.04 (s, 3H, Me),
7.57 (t, 2H, phenyl H3 & H5, J = 7.7 Hz), 7.68 (t, 1H, phenyl H4,
J = 7.4 Hz), 7.77 (d, 2H, 4-acetamidophenyl H3 & H5, J = 8.7 Hz),
7.80 (d, 2H, phenyl H2 & H6, J = 7.2 Hz), 8.10 (d, 1H, quinoline H8,
J = 10.5 Hz), 8.20 (d, 1H, quinoline H7, J = 8.7 Hz), 8.27 (d, 2H, 4-
acetamidophenyl H2 & H6, J = 8.7 Hz), 8.49 (s, 1H, quinoline H3),
9.06 (s, 1H, quinoline H5), 10.18 (s, 1H, NH), 14.2 (s, 1H, COOH),
13C NMR (DMSO-d6): d 25.0, 119.9, 120.7, 123.2, 129.0, 129.5,
129.9, 130.4, 130.6, 130.9, 132.6, 133.7, 135.7, 137.8, 139.2,
142.5, 150.8, 158.4, 168, 169.5, 196.1, LCMS (ESI): 411.6 (M+1)+
100. Anal. Calcd for C25H18N2O4: C, 73.16; H, 4.42; N, 6.83. Found:
C, 73.34; H, 4.24; N, 6.89.
5.1. General procedure for preparation of 6- or 8-benzoyl-2-(4-
(substituted)phenyl)quinoline-4-carboxylic acid (4 and 5)
To a 50 ml round-bottom flask was added appropriate aldehyde
(1, 4.73 mmol) and pyrrovic acid (2, 0.82 g, 9.35 mmol), immedi-
ately a white precipitate produced, then 20 ml acetic acid was
added and the mixture was heated at 100 °C to solve the precipi-
tate then refluxed for 30 min. After that 2- or 4-aminobenzophe-
none (3, 1.86 g, 9.45 mmol) was added and refluxed for 3–6 h.
After cooling, the produced precipitate was filtered and washed
with acetic acid, ethanol, ether, and hexane and crystallized in eth-
anol (yields: 15–31%).
5.2. General procedure for preparation of 6- or 8-benzoyl-2-(4-
(methylsulfonyl)phenyl)quinoline-4-carboxylic acid (6)
One gram of 2-(4-(methylthio)phenyl)-6- or 8-benzoyl-quino-
line-4-carboxylic acid was dissolved in 10 ml THF and 5 g oxone
in THF/water was added. The mixture was stirred at room temper-
ature for 3–5 h, after evaporation of THF, the residue was extracted
with ethyl acetate and dried with sodium sulfate and then evapo-
rated, the product was recrystallized in ethanol (yields: 67–70%).
5.1.1. 8-Benzoyl-2-(4-(methylthio)phenyl)quinoline-4-
carboxylic acid (4a)
Yield: 25%; pale brown crystalline powder; mp: 261–263 °C; IR
(KBr):
5.2.1. 8-Benzoyl-2-(4-(methylsulfonyl)phenyl)quinoline-4-
carboxylic acid (6a)
m
(cmꢁ1) 3295–2335 (OH), 1682, 1669 (C@O), 1H NMR
(DMSO-d6): d 2.50 (s, 3H, Me), 7.18 (d, 2H, 4-methylthiophenyl
H3 & H5, J = 8.4 Hz), 7.45 (t, 2H, phenyl H3 & H5, J = 7.7 Hz), 7.59
(t, 1H, phenyl H4, J = 7.4 Hz), 7.65–7.67 (m, 4H, 4-methylthiophenyl
H2 & H6 & phenyl H2 & H6), 7.77 (t, 1H, quinoline H6, J = 7.8 Hz),
7.91 (d, 1H, quinoline H7, J = 6.8 Hz), 8.40 (s, 1H, quinoline H3),
8.75 (d, 1H, quinoline H5, J = 8.5 Hz), 14.09 (s, 1H, COOH), LCMS
(ESI): 400.6 (M+1)+ 100.
Yield: 67%; cream crystalline powder; mp: 228–230 °C; IR
(KBr):
m
(cmꢁ1) 3419–2678 (OH), 1739, 1695 (C@O), 1300, 1140
(SO2), 1H NMR (DMSO-d6): d 3.24 (s, 3H, Me), 7.52 (t, 2H, phenyl
H3 & H5, J = 7.7 Hz), 7.67 (t, 1H, phenyl H4), 7.71 (d, 2H, phenyl
H2 & H6, J = 8.3 Hz), 7.89–7.93 (m, 3H, quinoline H6 & 4-meth-
ylsulfonylphenyl H2 & H6), 7.97 (d, 2H, 4-methylsulfonylphenyl
H3 & H5, J = 8.4 Hz), 8.03 (d, 1H, quinoline H7, J = 7.1 Hz), 8.57 (s,
1H, quinoline H3), 8.86 (d, 1H, quinoline H5, J = 9.4 Hz), 14.2 (s,
1H, COOH), 13C NMR (DMSO-d6): d 44.2, 120.5, 124.4, 128.2,
128.6, 128.7, 129.0, 129.5, 130.0, 130.5, 133.9, 139.2, 139.4,
140.0, 142.7, 146.6, 154.5, 168.1, 198.1, LCMS (ESI): 432.5 (M+1)+
100. Anal. Calcd for C24H17NO5S: C, 66.81; H, 3.97; N, 3.25. Found:
C, 66.99; H, 3.80; N, 3.22.
5.1.2. 6-Benzoyl-2-(4-(methylthio)phenyl)quinoline-4-
carboxylic acid (4b)
Yield: 16%; orange crystalline powder; mp: 221–223 °C; IR
(KBr):
m
(cmꢁ1) 3490–2500 (OH), 1696, 1652 (C@O), 1H NMR
(DMSO-d6): d 2.53 (s, 3H, Me), 7.41 (d, 2H, 4-methylthiophenyl

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5859
5.2.2. 6-Benzoyl-2-(4-(methylsulfonyl)phenyl)quinoline-4-
carboxylic acid (6b)
(C@O), 1H NMR (DMSO-d6): d 7.05 (d, 2H, 4-azidophenyl H2 & H6,
J = 8.6 Hz), 7.46 (t, 2H, phenyl H3 & H5, J = 7.7 Hz), 7.60 (t, 1H, phe-
nyl H4, J = 7.4 Hz), 7.65 (d, 2H, phenyl H2 & H6, J = 7.3 Hz), 7.77 (d,
2H, 4-azidophenyl H3 & H5, J = 8.6 Hz), 7.80 (t, 1H, quinoline H6,
J = 8.4 Hz), 7.92 (d, 1H, quinoline H7, J = 7.0 Hz), 8.42 (s, 1H, quino-
line H3), 8.75 (d, 1H, quinoline H5, J = 8.5 Hz), 14.2 (s, 1H, COOH),
13C NMR (DMSO-d6): d 119.8, 120.3, 124.0, 128.2, 128.4, 129.4,
129.4, 130, 130.1, 133.9, 134.8, 138.8, 139.3, 139.8, 142.2, 146.7,
155.2, 168.2, 198.3, LCMS (ESI): 417.5 (M+23)+ 100. Anal. Calcd
for C23H14N4O3: C, 70.05; H, 3.58; N, 14.21. Found: C, 69.88; H,
3.70; N, 14.02.
Yield: 70%; pale yellow crystalline powder; mp: 246–248 °C; IR
(KBr):
m
(cmꢁ1) 3542–2650 (OH), 1696, 1661 (C@O), 1311, 1150
(SO2), 1H NMR (DMSO-d6): d 3.21 (s, 3H, Me), 7.57 (t, 2H, phenyl
H3 & H5, J = 7.6 Hz), 7.69 (t, 1H, phenyl H4, J = 7.4 Hz), 7.81 (d, 2H,
phenyl H2 & H6, J = 7.4 Hz), 8.09 (d, 2H, 4-methylsulfonylphenyl
H2 & H6, J = 8.4 Hz), 8.13 (d, 1H, quinoline H8, J = 10.1 Hz), 8.27
(d, 1H, quinoline H7, J = 8.7 Hz), 8.55 (d, 2H, 4-methylsulfonylphe-
nyl H3 & H5, J = 8.4 Hz), 8.6 (s, 1H, quinoline H3), 9.08 (s, 1H, quin-
oline H5), 14.2 (s, 1H, COOH), 13C NMR (DMSO-d6): d 44.3, 121.5,
123.9, 128.5, 129.2, 129.5, 129.8, 130.7, 131.3, 133.8, 136.6,
137.6, 139.7, 142.8, 142.9, 150.5, 157.1, 167.7, 196.0, LCMS (ESI):
432.5 (M+1)+ 100. Anal. Calcd for C24H17NO5S: C, 66.81; H, 3.97;
N, 3.25. Found: C, 70.03; H, 3.79; N, 3.23.
5.4.2. 2-(4-(Azido)phenyl)-6-benzoyl-quinoline-4-carboxylic
acid (8b)
Yield: 35%; orange crystalline powder; mp: 207–209 °C; IR (KBr):
m
(cmꢁ1) 3602–2415 (OH), 2132, 2105 (N3), 1700, 1659 (C@O), 1H
5.3. General procedure for preparation of 2-(4-(amino)phenyl)-
6- or 8-benzoyl-quinoline-4-carboxylic acid (7)
NMR (DMSO-d6): d 7.25 (d, 2H, 4-azidophenyl H2 & H6, J = 8.5 Hz),
7.57 (t, 2H, phenyl H3 & H5, J = 7.6 Hz), 7.68 (t, 1H, phenyl H4,
J = 7.4 Hz), 7.80 (d, 2H, phenyl H2 & H6, J = 7.4 Hz), 8.10(d, 1H, quin-
oline H8, J = 10.1 Hz), 8.20 (d, 1H, quinoline H7, J = 8.7 Hz), 8.33 (d,
2H, 4-azidophenyl H3 & H5, J = 8.5 Hz), 8.5 (s, 1H, quinoline H3),
9.05 (s, 1H, quinoline H5), 14.2 (s, 1H, COOH), 13C NMR (DMSO-d6):
d 120.5, 120.8, 123.4, 129.5, 129.9, 130.0, 130.4, 130.6, 130.9,
133.7, 135, 136.0, 137.7, 139.4, 142.5, 150.6, 157.8, 167.9, 196.0,
LCMS (ESI): 417.5 (M+23)+ 100. Anal. Calcd for C23H14N4O3: C,
70.05; H, 3.58; N, 14.21. Found: C, 70.24; H, 3.73; N, 14.43.
To
a 50 ml round-bottom flask was added 2-(4-(acetam-
ido)phenyl)-6- or 8-benzoyl-quinoline-4-carboxylic acid (1 mmol),
then 1 ml HCl (1 N) and 3 ml HCl (12 N) were added and refluxed
for 5 h then the solution was neutralized with NaOH (10%), the
pH was adjusted to 5–6, the dark red precipitate was formed.
The precipitate was filtered and washed with water and used with-
out further purification (yields: 47%).
5.3.1. 2-(4-(Amino)phenyl)-8-benzoyl-quinoline-4-carboxylic
acid (7a)
6. Molecular modeling and biological evaluation
Yield: 47%; red crystalline powder; mp: 348–350 °C (decom-
Docking studies were performed using Autodock software
Version 3.0. The coordinates of the X-ray crystal structure of the
selective COX-2 inhibitor SC-558 bound to the murine COX-2 en-
zyme was obtained from the RCSB Protein Data Bank (1cx2) and
hydrogens were added. The ligand molecules were constructed
using the Builder module and were energy minimized for 1000
iterations reaching a convergence of 0.01 kcal/mol Å. The energy
minimized ligands were superimposed on SC-558 in the PDB file
1cx2 after which SC-558 was deleted. The purpose of docking is
to search for favorable binding configuration between the small
flexible ligands and the rigid protein. Protein residues with atoms
greater than 7.5 Å from the docking box were removed for effi-
ciency. These docked structures were very similar to the mini-
mized structures obtained initially. The quality of the docked
structures was evaluated by measuring the intermolecular energy
of the ligand–enzyme assembly.19,20
posed); IR (KBr):
m
(cmꢁ1) 3467, 3352 (NH2), 3232 (OH), 1657,
1645 (C@O), 1H NMR (DMSO-d6): d 6.42 (s, 2H, NH2), 7.39–7.43
(m, 5H, 4-aminophenyl H3 & H5 & phenyl H3–H5), 7.62–7.78 (m,
5H, 4-aminophenyl H2 & H6 & phenyl H2 & H6 & quinoline H6),
7.88 (s, 1H, quinoline H3), 8.13 (s, 1H, quinoline H7), 8.78 (s, 1H,
quinoline H5), LCMS (ESI): 369.7 (M+1)+ 100.
5.3.2. 2-(4-(Amino)phenyl)-6-benzoyl-quinoline-4-carboxylic
acid (7b)
Yield: 47%; red crystalline powder; mp: 267–270 °C; IR (KBr):
m
(cmꢁ1) 3420, 3351 (NH2), 3239 (OH), 1662, 1644 (C@O), 1H NMR
(DMSO-d6): d 6.67 (s, 2H, NH2), 7.56–7.78 (m, 5H, 4-aminophenyl
H3 & H5 & phenyl H3–H5), 7.9–8.2 (m, 6H, 4-aminophenyl H2
&
H6 & phenyl H2 & H6 & quinoline H7 & H8), 8.35 (s, 1H, quinoline
H3), 9.01 (s, 1H, quinoline H5), LCMS (ESI): 369.7 (M+1)+ 100.
5.4. General procedure for preparation of 2-(4-(azido)phenyl)-6-
or 8-benzoyl-quinoline-4-carboxylic acid (8)
7. In vitro cyclooxygenase (COX) inhibition assays
The assay was performed using an enzyme chemiluminescent
kit (Cayman Chemical, MI, USA) according to our previously re-
ported method.21 The Cayman chemical chemiluminescent COX
(ovine) inhibitor screening assay utilizes the heme-catalyzed
hydroperoxidase activity of ovine cyclooxygenases to generate
luminescence in the presence of a cyclic naphthalene hydrazide
and the substrate arachidonic acid. Arachidonate-induced lumines-
cence was shown to be an index of real-time catalytic activity and
demonstrated the turnover inactivation of the enzyme. Inhibition
of COX activity, measured by luminescence, by a variety of selective
and non-selective inhibitors showed potencies similar to those ob-
served with other in vitro and whole cell methods.
2-(4-(Amino)phenyl)-6- or 8-benzoyl-quinoline-4-carboxylic
acid (0.5 mmol) was suspended in 10 ml water in a 50 ml flask.
The flask was placed in ice bath so that the temperature of the mix-
ture reached to 0 and 3 ml of HCl (12 N) was added. Then 0.5 g
(7.2 mmol) NaNO2 was dissolved in water and added to the mixture
drop wisely. Then 5 ml THF was added, the temperature was kept
between 0 and 5, the solution was stirred for 1.5 h. After that NaN3
(0.5 g, 7.6 mmol) was dissolved in water and slowly added to the
solution and the mixture was stirred for 2 h, THF was evaporated
and the precipitate was filtered and washed with water, the solid
was added to a hot solution of HCl (3 N), after cooling, the solid
was filtered and washed with water and CHCl3 (yields: 20–35%).
Supplementary data
5.4.1. 2-(4-(Azido)phenyl)-8-benzoyl-quinoline-4-carboxylic
acid (8a)
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.06.094. These data
Yield: 20%; orange crystalline powder; mp: 202–204 °C; IR
(KBr):
m
(cmꢁ1) 3336–2517 (OH), 2136, 2103 (N3), 1693, 1670

5860
A. Zarghi, R. Ghodsi / Bioorg. Med. Chem. 18 (2010) 5855–5860
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