Design, synthesis and biological evaluation of pyridine acyl sulfonamide derivatives as novel COX-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.09.034

A series of pyridine acyl sulfonamide derivatives (1-24) have been designed and synthesized and their biological activities were also evaluated as potential cyclooxygenase-2 (COX-2) inhibitors. Among all the compounds, compound 23 displayed the most poten

Full text of DOI:10.1016/j.bmc.2011.09.034

Bioorganic & Medicinal Chemistry 19 (2011) 6827–6832
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and biological evaluation of pyridine acyl sulfonamide
derivatives as novel COX-2 inhibitors
Xiang Lu , Hui Zhang , Xi Li, Guo Chen, Qing-Shan Li, Yin Luo, Ban-Feng Ruan, Xian-Wei Chen,
⇑
Hai-Liang Zhu
State Key Laboratory of Pharmaceutical Biotechnology, Nanjing University, Nanjing 210093, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyridine acyl sulfonamide derivatives (1–24) have been designed and synthesized and their
biological activities were also evaluated as potential cyclooxygenase-2 (COX-2) inhibitors. Among all
the compounds, compound 23 displayed the most potent COX-2 inhibitory activity with an IC50 of
0.8 lM. Antitumor and anti-inflammatory assays indicated that compound 23 owned high antiprolifera-
tive activity against B16-F10, HepG2 and MCF-7 cancer cell lines as well as COX-2-derived prostaglandin
Received 11 August 2011
Revised 18 September 2011
Accepted 19 September 2011
Available online 25 September 2011
E2 (PGE2) inhibitory activity of murine macrophage RAW 264.7 cell line with IC50 values of 2.8, 1.2, 1.8
Keywords:
and 0.15
lM, respectively. Docking simulation was performed to position compound 23 into the
Pyridine acyl sulfonamide derivatives
Cyclooxygenase-2 inhibition
Structure–activity relationship
Molecular docking
COX-2 active site to determine the probable binding model.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
mize the risk of unwanted side effects.8 On the other hand, synthesis
of the pyridine ring system and its derivatives occupy an important
place in the realm of synthetic pharmaceutical chemistry, due to
their therapeutic and pharmacological properties. They have
emerged as integral backbones of over 7000 existing drugs. The pyr-
idine ring is also an integral part of anticancer and anti-inflamma-
tory agents. Among them, some compounds showed their
remarkable effects on COX-2 inhibitor potency in vitro, according
to previous researches.9,10 It encouraged us to continuously screen
new pyridine acyl sulfonamide derivatives as potential COX-2 inhib-
itory agents. The two combined substructures, pyridine ring and sul-
fonamide, might exhibit synergistic effect in antitumor and anti-
inflammatory activities. The objectives of present work are (1) to
synthesize new pyridine acyl sulfonamide derivatives; (2) to evalu-
ate their anticancer, anti-inflammatory and anticyclooxygenases
activities; (3) to explore the binding modes of these compounds at
the active site of COX-2 by docking simulations.
Since the early 1990s, it has been reported that cyclooxygenases
(COXs) are responsible for the production of prostaglandins H2,
which is a precursor for the biosynthesis of prostaglandins, throm-
boxanes and prostacyclins.1 COXs exist in two distinct isoforms, a
constitutive form cyclooxygenase-1 (COX-1) and an inducible form
cyclooxygenase-2(COX-2). TheCOX-1isahousekeepingenzymeex-
pressed in resting cells of most tissues involved in protection of gas-
tric mucosa, platelet aggregation and renal blood flow. Whereas the
COX-2 is rapidly induced in response to a variety of pro-inflamma-
tory stimuli such as tumor necrosis factor-a (TNF-a), interleukines
and growth factors, and plays an important role in pain, oncogenesis,
various acute and chronic inflammatory symptoms.2–6 Therefore,
molecules that selective inhibition of COX-2 enzymatic activity
would be of significant therapeutic value.1
Classical nonsteroidal anti-inflammatory drugs (NSAIDs) have
been widely used in the treatment of acute and chronic inflamma-
tion states. However, all these drugs cause untoward side effects re-
lated to COX-1 inhibition, among which gastrointestinal irritation
leading to ulcers and bleeding is the most common.7 This promising
concept of COX-2 selective inhibition led to the development of a
large number of new molecules such as celecoxib, valdecoxib and
nimesulide. Among these structures, sulfonamide is a promising
skeleton that has shown the COX-2 selective inhibition, but mini-
2. Results and discussion
2.1. Chemistry
The synthesis route of compounds 1–24 followed the general
pathway outlined in Scheme 1. Coupling pyridinecarboxylic acid,
substituted benzenesulfonamide amides, 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethyl-
aminopyridine (DMAP) were dissolved in CH2Cl2 and refluxed to
give the desired compounds 1–24. Among these compounds,
6–10, 12–14, 16–18 and 20–24 are reported for the first time.
⇑
Corresponding author. Tel.: +86 25 8359 2572; fax: +86 25 8359 2672.
E-mail address: zhuhl@nju.edu.cn (H.-L. Zhu).
These two authors equally contributed to this paper.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.034

6828
X. Lu et al. / Bioorg. Med. Chem. 19 (2011) 6827–6832
R
O
COOH
O
S
1 eq. RPhSO2NH2
1 eq. EDCI/DMAP
N
O
H
N
N
Synthesis of compounds 1-5
R
R
HOOC
COOH
O
O
2 eq. RPhSO2NH2
O
O
S
S
2 eq. EDCI/DMAP
N
H
N
N
O
O
H
N
Synthesis of compounds 6-10
R
O
O
COOH
COOH
S
1 eq. RPhSO2NH2
N
H
O
N
1 eq. EDCI/DMAP
N
Synthesis of compounds 11-14
R
O
O
S
1 eq. RPhSO2NH2
N
H
O
N
N
1 eq. EDCI/DMAP
Synthesis of compounds 15-19
H
N
H
O
S
O
S
N
2 eq. RPhSO2NH2
N
O
O
O
O
2 eq.E DCI/DMAP
HOOC
N
COOH
R
R
Synthesis of compounds 20-24
R= H, F, Cl, Br or Me
Scheme 1. Synthesis route of compounds 1–24. Reagents and conditions: EDCI/DMAP, CH2Cl2, reflux, 8–10 h.
Table 1
These compounds gave satisfactory elementary analytical and
spectroscopic data. 1H NMR and ESI MS spectra were consistent
with the assigned structures. Furthermore, the crystal structure
of compound 7 was determined by single crystal X-ray diffraction
analysis and the crystal data are presented in Table 1 and
Figure 1.
Crystallographic data and structure refinements for compound 7
Compound
Empirical formula
Formula weight
Crystal system
Space group
a (Å)
7
C
19H13F2N3O8S2
513.46
Monoclinic
C2/c
22.700(5)
8.4010(17)
10.970(2)
90
91.36(3)
90
2091.4(7)
4
1.631
1048.0
2609/2002
0.328
0.0777/0.2292
0.0634/0.2094
1.099
2.2. Biological evaluation
b (Å)
c (Å)
a
(°)
The data of the anti-inflammatory activities of the pyridine acyl
sulfonamide derivatives (1–24) were summarized in Table 2. It can
be seen that most of the synthesized compounds exhibited
remarkable and selective inhibitory activities against the COX-2
isozyme, but weak to COX-1. Among these compounds, 23 showed
the most potent COX-2 inhibitory activity (IC50 = 0.8
sults of western blotting assay further confirmed that compound
23 with the concentration above 0.75 M could obviously inhibit
b (°)
c
(°)
V (Å3)
Z
Dc/g cmꢀ3
F (000)
lM). The re-
Reflections collected/unique
Absorption coefficient (mmꢀ1
)
l
R1; wR2 [I > 2
R1; wR2 (all data)
GF
r
(I)]
lipopolysaccharide (LPS)-induced COX-2 expression in murine
macrophage RAW 264.7 cell line (Fig. 3).

X. Lu et al. / Bioorg. Med. Chem. 19 (2011) 6827–6832
6829
Table 3
Inhibition (IC50) of B16-F10, MCF-7 and HepG2 cells proliferation
Compound
R1
IC50 (lM)
B16-F10
MCF-7
HepG2
1
2
3
4
5
6
7
H
F
Cl
Br
Me
H
14.5
13.8
11.9
9.7
18.5
11.3
8.8
11.5
10.6
10.1
8.3
15.5
10.6
7.9
10.4
9.0
8.6
6.7
12.3
9.2
F
6.2
8
9
Cl
Br
Me
F
Cl
Br
Me
H
10.6
7.2
9.2
13.8
12.4
8.8
16.2
14.2
10.9
9.4
4.2
4.1
5.4
3.3
10.0
12.4
7.9
6.3
20.2
9.9
8.5
8.1
5.0
12.1
5.3
3.7
3.8
1.2
4.2
95.5
Figure 1. Crystal structure diagrams of compound 7. H atoms are shown as small
spheres of arbitrary radii.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Celecoxib
10.8
13.9
10.1
8.7
14.1
10.6
11.0
9.8
6.8
11.8
9.3
4.6
4.2
Table 2
Inhibition (IC50) of COX-1, PGE2 and COX-2 in lipopolysaccharide (LPS)-activated
murine macrophage RAW 264.7 cells
F
Cl
Br
Me
H
8.5
Compound
R1
IC50 (lM)
16.7
10.7
10.0
6.2
2.8
12.2
85.6
COX-1
PGE2
COX-2
F
1
2
3
4
5
6
7
8
H
F
Cl
Br
Me
H
>50
>50
9.5
8.6
3.2
2.8
8.8
2.6
1.9
1.7
1.2
2.2
9.2
8.9
5.5
9.8
4.5
3.3
2.8
1.9
5.9
1.6
1.1
0.87
0.15
2.1
0.10
15.8
11.7
6.6
4.9
18.2
7.9
5.6
3.4
2.8
8.5
12.1
12.6
7.8
19.4
8.8
6.3
6.0
3.6
8.0
Cl
Br
Me
1.8
6.2
40.8
35.8
40.5
>50
36.9
40.2
29.8
42.5
>50
F
Cl
Br
Me
F
Cl
Br
Me
H
9
20.2
inhibitory activity (IC50 = 1.2
and 1.8 M for MCF-7) better than that of the positive control
celecoxib.
lM. Among them, compound 23 displayed the most potent
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Celecoxib
l
M for HepG2, 2.8 M for B16-F10,
l
>50
l
41.2
38.0
>50
Subsequently SAR studies were performed to determine how
the substituents of the subunits affected the COX-2 inhibitory
activities. Compounds 1–24 with para substituted on phenyl ring
exhibited significant COX-2 inhibitory activities in order of
Br > Cl > F > Me. The results demonstrated that an electron-with-
drawing halogen group may be helpful to improve COX-2 inhibi-
tory activities while electron donating methyl substituted may
show some relatively negative effects. The results also indicated
that in the case of constant phenyl ring substituents, change of var-
ious kinds of pyridine carboxylic acids skeletons could also affect
the activities of these compounds. Compounds with 2-pyridine-
carboxylic acid skeleton (15–19) showed more potent activities
than those with nicotinic acid (1–5) and isonicotinic acid (11–14)
skeletons. Moreover, several symmetrically substituted derivatives
with 2,6-pyridinedicarboxylic acid and pyridine-3,5-dicarboxylic
acid skeleton (6–10, 20–24) displayed more potent than the corre-
sponding nonsymmetrically substituted derivatives (1–5, 15–19).
Molecular symmetry, as a broad structural property, is frequently
present in many drugs.18 The biological activities of the designed
compounds suggested that molecular symmetry might be a valid
approach to obtain potent new COX-2 inhibitory agents.
>50
F
43.8
39.7
>50
Cl
Br
Me
H
>50
35.8
40.3
41.5
35.6
39.8
27.5
3.7
2.6
1.9
0.8
5.6
0.12
F
Cl
Br
Me
Prostaglandin E2 (PGE2) is one of the principal mediators of
inflammation, which is biosynthesised via COX-2 enzymes.11 As
shown in Table 2, compounds 23 was found to be promising inhibit
PGE2 with an IC50 value of 0.15
lM, which was comparable to that
of celecoxib (IC50 = 0.10 M). These data suggested that pyridine
l
acyl sulfonamide derivatives inhibited the inflammatory activity
via the COX-2 pathway and were devoid of toxicity due to the ab-
sence of COX-1 inhibition which is maintenance of normal physio-
logical functions.12–15
Molecular docking was performed to study the biding model of
compound 23 with COX-2 based on the crystal structure of COX-2
protein (PDB code: 1cx2). The binding model of compounds 23 and
catalytic site of COX-2 are depicted in Figure 2. It was showed that
the amide group and the oxygen of carboxyl group of compound 23
exhibited hydrogen bond with Leu 352 and Arg 513 in COX-2 (dis-
tance = 2.76 and 2.30 Å, respectively). In addition, the oxygen
atoms of the sulfonamide system formed two hydrogen bonds with
the amino hydrogen of Phe518 and Arg530 (length: Arg530 N–
Hꢁ ꢁ ꢁO = 1.88 Å and Phe518 N–Hꢁ ꢁ ꢁO = 2.33 Å). Overall, these results
can provide a good explanation for the high potency of compound
23 towards COX-2 protein.
Furthermore, being associated with inflammation, it is well doc-
umented that COX-2 is especially overexpressed in many human
cancer entities such as melanoma, hepatic carcinoma, breast can-
cer and colorectal cancer, which is assumed to play an important
role in carcinogenesis by stimulating angiogenesis, tissue invasion,
metastasis and apoptosis inhibition.6,16,17 Herein, compounds 1–
24 were evaluated for their anticancer activities against the three
cancer cell lines including melanoma cell line (B16-F10), human
breast cancer cell line (MCF-7) and liver cancer cell line (HepG2).
The results were summarized in Table 3. It was observed that pyr-
idine acyl sulfonamide derivatives (1–24) have been found to show
good inhibitory activities displaying IC50 values between 1.2 and

6830
X. Lu et al. / Bioorg. Med. Chem. 19 (2011) 6827–6832
8.00–8.02 (m, 2H), 8.23–8.25 (m, 1H), 8.76–8.78 (m, 1H), 9.00
(m, 1H). ESI-MS: 263.0 (C12H11N2O3S, [M+H]+). Anal. Calcd for
12H10N2O3S: C, 54.95; H, 3.84; N, 10.68. Found: C, 54.74; H,
C
3.85; N, 10.72.
4.2.2. N-(4-Fluorophenylsulfonyl)nicotinamide (2)
White powders, mp: 245–246 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.46–7.49 (m, 2H), 7.56–7.58 (m, 1H), 8.06–8.09 (m, 2H), 8.26–
8.28 (m, 1H), 8.77–8.79 (m, 1H), 9.01 (d, J = 1.6 Hz, 1H). ESI-MS:
281.0 (C12H10FN2O3S, [M+H]+). Anal. Calcd for C12H9FN2O3S: C,
51.42; H, 3.24; N, 9.99. Found: C, 51.24; H, 3.25; N, 9.95.
Figure 2. Binding mode of compound 23 with COX-2; ligand is represented as wire,
the interacting residues are depicted as stick and ball. The H-bond (green) is
displayed as line.
4.2.3. N-(4-Chlorophenylsulfonyl)nicotinamide (3)
White powders, mp: 241–243 °C. 1H NMR (300 MHz, DMSO-d6)
d: 7.58–7.62 (m, 1H), 7.84 (d, J = 8.6 Hz, 2H), 7.93 (d, J = 9.1 Hz, 2H),
8.29–8.33 (m, 1H), 8.78–8.81 (m, 1H), 9.02 (d, J = 2.0 Hz, 1H). ESI-
MS: 298.1 (C12H10ClN2O3S, [M+H]+). Anal. Calcd for C12H9ClN2O3S:
C, 48.57; H, 3.06; N, 9.44. Found: C, 48.31; H, 3.31; N, 9.22.
4.2.4. N-(4-Bromophenylsulfonyl)nicotinamide (4)
White powders, mp: 255–356 °C. 1H NMR (300 MHz, DMSO-d6)
d: 7.51–7.61 (m, 1H), 7.82–7.85 (m, 2H), 7.90–7.93 (m, 2H), 8.28–
8.32 (m, 1H), 8.77–8.79 (m, 1H), 9.01 (d, J = 2.2 Hz, 1H). ESI-MS:
343.0 (C12H10BrN2O3S, [M+H]+). Anal. Calcd for C12H9BrN2O3S: C,
42.24; H, 2.66; N, 8.21. Found: C, 42.37; H, 2.67; N, 8.23.
Figure 3. Inhibitory effect of compound 23 on COX-2 protein expression. RAW
264.7 cells (5 ꢂ 105 cells) were incubated in 12 well culture plate for 24 h, and then
treated with compounds and LPS (1 lg/ml) for 18 h. After incubation, cells were
lysed, and protein was applied on SDS–polyacrylamide gel. The level of COX-2
protein expression was examined by western blotting analysis.
4.2.5. N-Tosylnicotinamide (5)
White powders, mp: 225–227 °C. 1H NMR (300 MHz, DMSO-d6)
d: 2.40 (s, 3H), 7.44 (d, J = 8.0 Hz, 2H), 7.52–7.57 (m, 1H), 7.90 (d,
J = 8.2 Hz, 2H), 8.23 (d, J = 8.0 Hz, 1H), 8.77–8.79 (m, 1H), 8.99 (d,
J = 1.8 Hz, 1H). ESI-MS: 277.1 (C13H13N2O3S, [M+H]+). Anal. Calcd
for C13H12N2O3S: C, 56.51; H, 4.38; N, 10.14. Found: C, 56.38; H,
4.39; N, 10.10.
3. Conclusions
In summary, we have prepared a series of pyridine acyl sulfon-
amide derivatives (1–24) and evaluated their biological activities.
All of these compounds can inhibit the growth of three cancer cell
lines. Compound 23 were most exciting, as it inhibited the cancer
cell lines more potently than standard anticancer agents (cele-
coxib). Moreover promising anti-inflammatory activities were also
obtained by those compounds. Bioavailability and toxicity test re-
vealed the compounds to be nontoxic with drug properties. The re-
sults of this study may find a leading compound (23) toward the
development of new therapeutic agent to fight against cancer
and inflammation.
4.2.6. N3,N5-Bis(phenylsulfonyl)pyridine-3,5-dicarboxamide (6)
White powders, mp: 290–292 °C. 1H NMR (300 MHz, DMSO-d6)
d: 7.62–7.67 (t, J = 7.5 Hz, 4H), 7.70–7.75 (t, J = 7.1 Hz, 2H), 8.00 (d,
J = 7.2 Hz, 4H), 8.68 (t, J = 2.1 Hz, 1H), 9.12 (d, J = 2.0 Hz, 2H). ESI-
MS: 446.0 (C19H16N3O6S2, [M+H]+). Anal. Calcd for C19H15N3O6S2:
C, 51.23; H, 3.39; N, 9.43. Found: C, 51.29; H, 3.47; N, 9.40.
4.2.7. N3,N5-Bis(4-fluorophenylsulfonyl)pyridine-3,5-
dicarboxamide (7)
White powders, mp: 302–304 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.48 (t, J = 8.72 Hz, 4H), 7.95 (s, 2H), 8.07–8.10 (m, 4H), 8.68 (s,
1H), 9.13 (d, J = 1.8 Hz, 2H). ESI-MS: 482.0 (C19H14F2N3O6S2,
[M+H]+). Anal. Calcd for C19H13F2N3O6S2: C, 47.40; H, 2.72; N,
8.73. Found: C, 47.35; H, 2.73; N, 8.75.
4. Experimental
4.1. Chemistry
The synthesis of pyridine acyl sulfonamide derivatives was fol-
lowed the general reaction pathway outlined in Scheme 1. Com-
pounds 1–24 were synthesized by coupling substituted
benzenesulfonamide with nicotinic acid, isonicotinic acid, picolinic
acid, pyridine-2,6-dicarboxylic acid or pyridine-3,5-dicarboxylic
acid, using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
hydrochloride (EDCI), 4-dimethylaminopyridine (DMAP) as con-
densing agent. The mixture was refluxed in anhydrous CH2Cl2 for
8–10 h. The products were extracted with ethyl acetate. The extract
was washed successively with HCl, NaHCO3 and water, dried over
Na2SO4, filtered and evaporated. The residue was purified by column
chromatography using petroleum ether and ethyl acetate (1:1).
4.2.8. N3, N5-Bis(4-chlorophenylsulfonyl)pyridine-3,5-
dicarboxamide (8)
White powders, mp: 305–307 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.69–7.70(dd, J1 = 2.0, J2 = 6.9, 4H), 7.97–7.99 (dd, J1 = 2.0,
J2 = 6.9, 4H), 8.66 (t, J = 2.1 Hz, 1H), 9.10 (d, J = 2.1 Hz, 2H). ESI-
MS:
515.9(C19H14Cl2N3O6S2,
[M+H]+).
Anal.
Calcd
for
C19H13Cl2N3O6S2: C, 47.37; H, 2.55; N, 8.17. Found: C, 47.31; H,
2.56; N, 8.15.
4.2.9. N3,N5-Bis(4-bromophenylsulfonyl)pyridine-3,5-
dicarboxamide (9)
4.2. Spectral properties of intermediate compounds
White powders, mp: 309–311 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.87 (d, J = 8.7 Hz, 4H), 7.93 (d, J = 8.7 Hz, 4H), 8.69 (t, J = 2.1 Hz,
1H), 9.13 (d, J = 2.1 Hz, 2H). ESI-MS: 604.8 (C19H14Br2N3O6S2,
[M+H]+). Anal. Calcd for C19H13Br2N3O6S2: C, 37.83; H, 2.17; N,
6.97. Found: C, 37.72; H, 2.16; N, 6.95.
4.2.1. N-(Phenylsulfonyl)nicotinamide (1)
White powders, mp: 242–244 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.53– 7.56 (m, 1H), 7.63–7.66 (m, 2H), 7.72 (t, J = 7.5 Hz, 1H),

X. Lu et al. / Bioorg. Med. Chem. 19 (2011) 6827–6832
6831
4.2.10. N3,N5-Ditosylpyridine-3,5-dicarboxamide (10)
4.2.19. N-Tosylpicolinamide (19)
White powders, mp: 303–305 °C. 1H NMR (300 MHz, DMSO-d6)
d: 2.40–2.50(m, 6H), 7.44 (d, J = 8.2 Hz, 4H), 7.89 (d, J = 8.4 Hz, 4H),
8.66 (t, J = 2.0 Hz, 1H), 9.10 (t, J = 2.0 Hz, 2H), 9.12 (d, J = 2.0 Hz,
2H). ESI-MS: 474.0 (C21H20Br2N3O6S2, [M+H]+). Anal. Calcd for
White powders, mp: 220–222 °C. 1H NMR (500 MHz, DMSO-d6)
d: 2.50 (s, 3H), 7.43 (d, J = 8.0 Hz, 2H), 7.68–7.71 (m, 1H), 7.91–7.93
(d, J = 8.3 Hz, 1H), 8.00–8.04 (m, 1H), 8.69–8.71 (m, 1H), 12.18 (s,
1H). ESI-MS: 278.5 (C13H13N2O3S, [M+H]+). Anal. Calcd for
C21H19Br2N3O6S2: C, 53.27; H, 4.04; N, 8.87. Found: C, 53.24; H,
C13H12N2O3S: C, 56.51; H, 4.38; N, 10.14. Found: C, 56.30; H,
4.03; N, 8.85.
4.40; N, 10.12.
4.2.20. N2,N6-Bis(phenylsulfonyl)pyridine-2,6-dicarboxam
ide(20)
4.2.11. N-(4-Fluorophenylsulfonyl)isonicotinamide (11)
White powders, mp: 221–222 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.42–7.46 (t, J = 8.8 Hz, 2H), 7.86 (d, J = 6.1 Hz, 2H), 8.03–8.06 (m,
2H), 8.77–8.78 (d, J = 4.6, 2H). ESI-MS: 281.0 (C12H10FN2O3S,
[M+H]+). Anal. Calcd for C12H9FN2O3S: C, 51.42; H, 3.24; N, 9.99.
Found: C, 51.48; H, 3.27; N, 9.96.
White powders, mp: 313–315 °C. 1H NMR (300 MHz, DMSO-d6)
d: 7.66–7.70 (t, J = 7.4 Hz, 4H), 7.75–7.79 (m, 2H), 8.08–8.11 (d,
J = 7.1 Hz, 4H), 8.17–8.25(m, 3H), 12.99 (s, 2H). ESI-MS: 446.9
(C19H16N3O6S2, [M+H]+). Anal. Calcd for C19H15N3O6S2: C, 51.23;
H, 3.39; N,9.43. Found: C,51.43; H,3.40; N, 9.45.
4.2.21. N2,N6-Bis(4-fluorophenylsulfonyl)pyridine-2,6-dicarb
oxamide (21)
4.2.12. N-(4-Chlorophenylsulfonyl)isonicotinamide (12)
White powders, mp: 235–238 °C. 1H NMR (300 MHz, DMSO-d6)
d: 7.65–7.67 (d, J = 8.6 Hz, 2H), 7.91–7.98 (m, 4H), 8.79 (d,
J = 6.0 Hz, 2H). ESI-MS: 297.0 (C12H10ClN2O3S, [M+H]+). Anal. Calcd
for C12H9ClN2O3S: C, 48.57; H, 3.06; N, 9.44. Found: C, 48.47; H,
3.07; N, 9.41.
White powders, mp: 315–317 °C. 1H NMR (300 MHz, DMSO-d6)
d: 7.49–7.55 (t, J = 8.8 Hz, 4H), 8.15–8.19 (m, 4H), 8.21–8.26(m,
3H), 12.97 (s, 2H). ESI-MS: 482.0 (C19H14F2N3O6S2, [M+H]+). Anal.
Calcd for C19H13F2N3O6S2: C, 47.40; H, 2.72; N, 8.73. Found: C,
47.32; H, 2.71; N, 8.75.
4.2.13. N-(4-Bromophenylsulfonyl)isonicotinamide (13)
White powders, mp: 246–248 °C. 1H NMR (300 MHz, DMSO-d6)
d: 7.78–7.81 (d, J = 8.6 Hz, 2H), 7.87–7.92 (t, J = 8.1 Hz, 4H), 8.79 (d,
J = 6.2 Hz, 2H). ESI-MS: 340.9 (C12H10BrN2O3S, [M+H]+). Anal. Calcd
for C12H9BrN2O3S: C, 42.24; H, 2.66; N, 8.21. Found: C, 42.42; H,
2.67; N, 8.23.
4.2.22. N2,N6-Bis(4-chlorophenylsulfonyl)pyridine-2,6-dicarbox
amide (22)
White powders, mp: 325–326 °C. 1H NMR (300 MHz, DMSO-d6)
d: 7.88–7.91 (d, J = 8.6 Hz, 4H), 7.99–8.03 (d, J = 8.6 Hz 4H), 8.22–
8.27 (m, 3H), 12.99 (s, 2H). ESI-MS: 515.0(C19H14Cl2N3O6S2,
[M+H]+). Anal. Calcd for C19H13Cl2N3O6S2: C, 44.37; H, 2.55; N,
8.17. Found: C, 44.32; H, 2.54; N, 8.19.
4.2.14. N-Tosylisonicotinamide (14)
White powders, mp: 230–232 °C. 1H NMR (300 MHz, DMSO-d6)
d: 2.50 (s, 3H), 7.42–7.45 (d, J = 8.4 Hz, 2H), 7.77–7.79 (m, 2H),
7.87–7.90 (d, J = 8.4 Hz, 2H), 8.74–8.76 (m, 2H). ESI-MS: 277.9
(C13H13N2O3S, [M+H]+). Anal. Calcd for C13H12N2O3S: C, 56.51; H,
4.38; N, 10.14. Found: C, 56.70; H, 4.36; N, 10.18.
4.2.23. N2,N6-Bis(4-bromophenylsulfonyl)pyridine-2,6-dicarb
oxamide (23)
White powders, mp: 330–332 °C. 1H NMR (300 MHz, DMSO-d6)
d: 7.75–7.78 (d, J = 8.6 Hz, 4H), 8.08–8.11 (d, J = 8.6 Hz 4H), 8.22–
8.26 (m, 3H), 12.99 (s, 2H). ESI-MS: 604.8(C19H14Br2N3O6S2,
[M+H]+). Anal. Calcd for C19H13Br2N3O6S2: C, 37.83; H, 2.17; N,
6.97. Found: C, 37.88; H, 2.16; N, 6.99.
4.2.15. N-(Phenylsulfonyl)picolinamide (15)
White powders, mp: 221–213 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.62- 7.65 (t, J = 7.8 Hz, 2H), 7.70–7.72 (m, 2H), 7.97 (d,
J = 7.8 Hz, 1H), 8.02–8.05 (t, J = 7.9 Hz, 3H), 8.71 (d, J = 4.7 Hz,
1H), 12.34 (s, 1H). ESI-MS: 263.0 (C12H11N2O3S, [M+H]+). Anal.
Calcd for C12H10N2O3S: C, 54.95; H, 3.84; N, 10.68. Found: C,
54.75; H, 3.86; N, 10.65.
4.2.24. N2,N6-Ditosylpyridine-2,6-dicarboxamide (24)
White powders, mp: 310–312 °C. 1H NMR (300 MHz, DMSO-d6)
d: 2.41 (s, 6H), 7.46–7.49 (d, J = 8.4 Hz, 4H), 7.96 (d, J = 8.4 Hz, 4H),
8.20–8.24(m, 3H), 12.89 (s, 2H). ESI-MS: 474.0 (C21H20N3O6S2,
[M+H]+). Anal. Calcd for C21H19N3O6S2: C, 53.27; H, 4.04; N, 8.87.
Found: C, 53.18; H, 4.03; N, 8.89.
4.2.16. N-(4-Fluorophenylsulfonyl)picolinamide (16)
White powders, mp: 225–228 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.46–7.49 (m, 2H), 7.71–7.74 (m, 1H), 8.00 (d, J = 7.8 HZ, 1H),
8.04–8.07 (m, 1H), 8.09–8.12 (m, 2H), 8.71 (d, J = 4.1,1H). ESI-MS:
280.6 (C12H10FN2O3S, [M+H]+). Anal. Calcd for C12H9FN2O3S: C,
51.42; H, 3.24; N, 9.99. Found: C, 51.46; H, 3.23; N, 9.96.
4.3. Crystal structure determination
Crystal structure of compound 7 were determined on a Nonius
CAD4 diffractometer equipped with graphite-monochromated Mo
0
K
(k = 0.71073 ÅA) radiation. The structure was solved by direct
a
methods and refined on F2 by full-matrix least-squares methods
using SHELX-97. All the nonhydrogen atoms were refined anisotrop-
ically. All the hydrogen atoms were placed in calculated positions
and were assigned fixed isotropic thermal parameters at 1.2 times
the equivalent isotropic U of the atoms to which they are attached
and allowed to ride on their respective parent atoms. The contribu-
tions of these hydrogen atoms were included in the structure-fac-
tors calculations.
4.2.17. N-(4-Chlorophenylsulfonyl)picolinamide (17)
White powders, mp: 245–247 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.70–7.75 (m, 3H), 8.00–8.04 (m, 3H), 8.04–8.09 (m, 1H), 8.71 (d,
J = 4.7 Hz, 1H). ESI-MS: 297.0 (C12H10ClN2O3S, [M+H]+). Anal. Calcd
for C12H9ClN2O3S: C, 48.57; H, 3.06; N, 9.44. Found: C, 48.54; H,
3.07; N, 9.47.
4.2.18. N-(4-Bromophenylsulfonyl)picolinamide (18)
White powders, mp: 255–256 °C. 1H NMR (500 MHz, DMSO-d6)
d: 7.72–7.75 (m, 1H), 7.84–7.86 (m, 2H), 7.94–7.96 (m, 2H), 8.01 (d,
J = 7.7 Hz, 1H), 8.06–8.09 (m, 1H), 8.71 (d, J = 4.2 Hz, 1H). ESI-MS:
340.9 (C12H10BrN2O3S, [M+H]+). Anal. Calcd for C12H9BrN2O3S: C,
42.24; H, 2.66; N, 8.21. Found: C, 42.28; H, 2.65; N, 8.19.
4.4. Anticancer assay
The antiproliferative activity of the prepared compounds was
determined using a standard (MTT)-based colorimetric assay.19
Briefly, HepG2, B16-F10 and MCF-7 cell lines were seeded at a

6832
X. Lu et al. / Bioorg. Med. Chem. 19 (2011) 6827–6832
density of 2 ꢂ 104 cells/ml in 96-well microtiter plates. After 24 h,
Acknowledgements
exponentially growing cells were exposed to the compounds
(1–24) at final concentrations ranging from 0.1 to 100 lM. After
This work was supported by Jiangsu National Science Founda-
tion (No. BK2009239) and the Fundamental Research Funds for
the Central Universities (Nos. 1092020804 & 1106020824).
48 h, cell survival was determined by the addition of an MTT solu-
tion (0.5 mg/mL MTT in DMEM cell culture medium). After 4 h
incubation, the optical absorbance was measured at 570 nm on
an ELISA microplate reader. In all experiments three replicate wells
were used for each drug concentration. Each assay was carried out
at least three times.
References and notes
1. Ramana-Reddy, M. V.; Billa, V. K.; Pallela, V. R.; Mallireddigari, M. R.;
Boominathan, R.; Gabriel, J. L. Bioorg. Med. Chem. 2008, 16, 3907.
2. Ghodsi, R.; Zarghi, A.; Daraei, B.; Hedayati, M. Bioorg. Med. Chem. 2010, 18,
1029.
3. Herschman, H. R. Biochim. Biophys. Acta 1996, 1299, 125.
4. Xu, X. C. Anticancer Drugs. 2002, 13, 127.
5. Jiménez, P.; García, A.; Santander, S.; Piazuelo, E. Curr. Pharm. Des. 2007, 13,
2261.
6. Wuest, F.; Kniess, T.; Bergmann, R.; Pietzsch, J. Bioorg. Med. Chem. 2008, 16,
4.5. Molecular modeling (docking) studies
Molecular docking of compounds into the three dimensional
COX-2 complex structure (PDB code: 1cx2) was carried out using
the Molsoft ICM-Pro software package (version 3.5-0a).
7662.
7. Jule´mont, F.; Leval, X. D.; Michaux, C.; Renard, J. F.; Winum, J. Y. J. Med. Chem.
2004, 47, 6749.
4.6. In vitro cyclooxygenase (COX) inhibition assays
8. Renard, J. F.; Arslan, D.; Garbacki, N.; Pirotte, B.; Leval, X. D. J. Med. Chem. 2004,
52, 5864.
9. Ouimet, N.; Chan, C. C.; Charleson, S.; Claveau, D.; Gordon, R. Bioorg. Med. Chem.
Lett. 1999, 9, 151.
10. Hamdy, N. A.; Gamal-Eldee, A. M. Eur. J. Med. Chem. 2009, 44, 4547.
11. Cardile, V.; Lombardo, L.; Granata, G.; Perdicaro, A.; Balazy, M.; Santagati, A.
Bioorg. Med. Chem. 2009, 17, 1991.
The ability of the test compounds 1–24 to inhibit COX-1 and
COX-2 was determined using chemiluminescent enzyme assays
kit (Cayman Chemical, Ann Arbor, MI, USA) according to previously
reported method.20
12. Eren, G.; Unlu, S.; Nunez, M. T.; Labeaga, L.; Ledo, F. Bioorg. Med. Chem. 2010, 18,
6367.
13. Zarghi, A.; Zebardast, T.; Daraie, B.; Hedayati, M. Bioorg. Med. Chem. 2009, 17,
5369.
14. Abouzid, K.; Bekhit, S. A. Bioorg. Med. Chem. 2008, 16, 5547.
15. Laneuville, O.; Breuer, D. K.; DeWitt, D. L.; Hla, T.; Funk, C. D.; Smith, W. L. J.
Pharmacol. Exp. Ther. 1994, 271, 927.
16. Piva, R.; Belardo, G.; Santoro, M. Antioxid. Redox Signal. 2006, 8, 478.
17. Giri, R. S.; Thaker, H. M.; Giordano, T.; Williams, J.; Rogers, D. Bioorg. Med. Chem.
2010, 18, 2796.
4.7. Anti-inflammatory assay
The inhibitory effects of samples on PGE2 expression were eval-
uated in lipopolysaccharide (LPS)-activated murine macrophage
RAW 264.7 cells, using a method modified from that previously
reported.21
18. Ibáñez, E.; Plano, D.; Font, M.; Calvo, A.; Prior, C.; Palop, J. A.; Sanmartín, C. Eur.
J. Med. Chem. 2011, 46, 265.
4.8. Western immunoblot analysis
19. Lv, P. C.; Li, H. Q.; Sun, J.; Zhou, Y.; Zhu, H. L. Bioorg. Med. Chem. 2010, 18, 4606.
20. Amr, A. G.; Abdulla, M. M. Bioorg. Med. Chem. 2006, 14, 4341.
21. Li, W. F.; Zhou, P.; Zhang, Y. M.; He, L. C. J. Ethnopharmacol. 2009, 17, 6348.
22. Cheenpracha, S.; Park, E. J.; Yoshida, W. Y.; Barit, C.; Wall, M. Bioorg. Med. Chem.
2010, 18, 6598.
RAW 264.7 cells were pretreated with compounds 23 at the
concentrations of 0.75, 1 and 1.5
lM for 15 min before treatment
with 1 g/mL LPS for 18 h and examining the expression of COX-
l
2 protein. Cells were lysed with lysis buffer. Western immunoblot
analysis was performed using a method described by Cheenpracha,
et al.22