Spectral and crystallographic study of pyridinic analogues of nimesulide: Determination of the active form of methanesulfonamides as COX-2 selective inhibitors

Article abstract of DOI:10.1021/jm020920n

Compound 7, N-(3-phenoxy-4-pyridinyl)trifluoromethanesulfonamide, showed in vitro (whole blood assay) a strong inhibitory activity on the two cyclooxygenase (COX) enzymes (IC₅₀(COX1) = 2.2 μM and IC₅₀(COX-2) = 0.4 μM), being more act

Full text of DOI:10.1021/jm020920n

5182
J . Med. Chem. 2002, 45, 5182-5185
Brief Articles
Sp ectr a l a n d Cr ysta llogr a p h ic Stu d y of P yr id in ic An a logu es of Nim esu lid e:
Deter m in a tion of th e Active F or m of Meth a n esu lfon a m id es a s COX-2 Selective
In h ibitor s
Fabien J ule´mont,*,† Xavier de Leval,† Catherine Michaux,‡ J acques Damas,§ Caroline Charlier,‡
Franc¸ois Durant,‡ Bernard Pirotte,† and J ean-Michel Dogne´†
Natural and Synthetic Drugs Research Center, Department of Medicinal Chemistry, Universite´ de Lie`ge, 1, Avenue de
l’Hoˆpital, Tour 4(+5) Sart-Tilman, B-4000 Lie`ge, Belgium, Department of Molecular and Structural Chemistry, Universite´ de
Namur, 61 Rue de Bruxelles, B-5000 Namur, Belgium, and Department of Human Physiology, Universite´ de Lie`ge,
17 Place Delcour, 4020 Lie`ge, Belgium
Received April 25, 2002
Compound 7, N-(3-phenoxy-4-pyridinyl)trifluoromethanesulfonamide, showed in vitro (whole
blood assay) a strong inhibitory activity on the two cyclooxygenase (COX) enzymes (IC50(COX-
1) ) 2.2 µM and IC50(COX-2) ) 0.4 µM), being more active but less COX-2-selective than
nimesulide. Physicochemical studies and structural analyses indicated that the anionic
sulfonamidate species seemed to be the active form of methanesulfonamides, which optimally
interacted with the COX enzymes’ active sites.
Sch em e 1a
In tr od u ction
Prostaglandins (PGs) are key mediators involved in
the inflammation processes. They are synthesized by
cyclooxygenases (COXs) from arachidonic acid.1 COX-1
is a constitutive enzyme responsible for the physiological
production of PGs. This enzyme is involved in several
homeostatic processes and is thus considered as a
“housekeeping” enzyme. In contrast, COX-2 is an induc-
ible enzyme, which is mainly produced during inflam-
mation processes. The inhibition of COX-1 is thought
to be responsible for the side effects of the nonsteroidal
antiinflammatory drugs (NSAID).2 Antiinflammatory
drugs inhibit the synthesis of PGs by inhibiting these
enzymes. Following the discovery of the second isoform
of cyclooxygenase,3 the search for new inhibitors of the
COX-2 isoform has led to the development of selective
drugs such as celecoxib.4
At the present time, nimesulide is marketed as a
NSAID and is reported as a COX-2-selective methane-
sulfonamide.5 The methanesulfonamide class has been
widely developed in the literature.6,7 All compounds of
this class present the same chemical skeleton: a ben-
zene ring substituted by an electron-withdrawing group
at the 4-position.
To determine the ionic state of drugs from the pyri-
dinic methanesulfonamide class expected to interact
with the active site of the COX enzymes, four deriva-
tives have been synthesized. Their chemical structures
have been determined from X-ray, IR, and NMR experi-
ments. Their inhibitory effects on the COXs and their
a
(i) H2O2, CH3COOH, ∆; (ii) HNO3, H2SO4, ∆; (iii) C6H4O-Na+,
∆; (iv) Fe, H2O/CH3COOH, ∆; (v) R-SO2Cl, K2CO3/CH3CN; (vi)
KOH, ∆; (vii) CH3I, K2CO3/CH3CN.
selectivity have been evaluated by a whole blood assay.
The antiinflammatory effects of these drugs have also
been determined in vivo by a carrageenan-induced rat
paw oedema test.
* To whom correspondence should be addressed. Phone: 32-4-
3664366. Fax: 32-4-3664362. E-mail: f.julemont@ulg.ac.be.
† Department of Medicinal Chemistry, Universite´ de Lie`ge.
‡ Universite´ de Namur.
§ Department of Human Physiology, Universite´ de Lie`ge.
10.1021/jm020920n CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/03/2002

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5183
Ta ble 1. Estimated IC50 Values from Whole Blood Assay and Effect on Rat Paw Oedema for Compounds 6, 7, 12, and 13 and
Nimesulidea
whole blood assay
antiinflammatory effect
on rat paw oedema
IC50(COX-1)
IC50(COX-2)
IC50(COX-1)/
IC50(COX-2)
compd
(µM)
(µM)
10 mg/kg dose
30 mg/kg dose
control
6
7
12
13
4.9
2.2
ni
ni
22.0
37.5
0.4
ni
ni
1.3
0.13
5.28
NT
74.7 ( 7.2
NT
84.6 ( 4.7
58.0 ( 6.0
toxic
54.1 ( 17.5
NT
65.6 ( 16.8
54.0 ( 7.0
96.0 ( 8.7
97.3 ( 13.7
97.0 ( 3.0
nimesulide
16.92
a
ni: no inhibition at 100 µM. NT: not tested. Results on rat paw oedema are expressed as percentage of growth of the paw after
injection of carrageneen (mean ( standard deviation; n ) 6).
Ch em istr y
found to be toxic after an intraperitoneal injection at a
dose of 30 mg/kg (proconvulsant). Compound 7 pre-
sented a dose-dependent antiinflammatory effect. At 30
mg/kg, the antiinflammatory effect of compound 7 was
similar to that of 10 mg/kg nimesulide. Compound 13,
which was inactive in vitro, presented an antiinflam-
matory effect at a dose of 30 mg/kg. This activity could
be explained by a metabolization of the product to
generate nimesulide. It is hypothesized that compound
13 acts as a prodrug of nimesulide.
The synthesis of compound 6 has been previously
described8 but has been optimized in order to easily
obtain compounds 6 and 7. Those two compounds were
synthesized in five steps. The first four steps of Scheme
1A led to the aminopyridine 5. The synthesis started
with oxidation of 3-bromopyridine 1 by hydrogen per-
oxide in the presence of acetic acid. This oxidation was
followed by nitration at the 4-position of the pyridine
N-oxide 2 by a mixture of nitric acid and sulfuric acid
at 90 °C. After that, the bromine atom was substituted
with sodium phenolate to give compound 4. The nitro
and the N-oxide groups were then reduced by iron in
the presence of acetic acid and water to afford the
aminopyridine 5. Compounds 6 and 7 were obtained by
reaction of 5 with the appropriate sulfonyl chloride.
The synthesis of compound 12, a geometric isomer of
compound 6, was achieved in four steps (Scheme 1B).
This scheme presents an alternative pathway to the
previous process used for the synthesis of 10.9 First, the
chlorine atom of 2-chloro-3-nitropyridine was substi-
tuted with sodium phenolate. In the second step, the
nitro group of compound 9 was reduced by iron in the
presence of acetic acid and water to produce the ami-
nopyridine 10. Compound 10 reacted with an excess of
mesyl chloride to form the sulfonimide 11. This sulfon-
imide was hydrolyzed by KOH in aqueous medium
under reflux to afford compound 12.
Str u ctu r a l An a lysis
To determine the ionic state of the compounds in
solution at the physiological pH of 7.4, we have evalu-
ated the pKa values of compounds 6, 7, and 12 by
spectrophotometry. Compound 6 has two pKa values of
2.98 and 8.13. Compounds 7 and 12 were found to have
a first pKa value under 1 and a second of 6.1 and 7.85,
respectively. Nimesulide has a pKa value of 6.56.10 By
this method, the pKa cannot be associated with the
species in equilibrium.
To elucidate the ionic state of the molecules in the
solid state, we have studied the compounds by IR
spectrometry and crystallography. The IR spectra of
compounds 6 and 7 revealed that the two drugs appear
to be pyridinium ions in the solid state. Those com-
pounds present three broad absorption bands between
2800 and 2650 cm-1 expected for the presence of a
N+-H bond. In contrast, compound 12 presents a strong
absorption band at 3226 cm-1 that can be associated
with a N-H bond. This strong absorption band is also
Nimesulide reacted with iodomethane in the pres-
ence of potassium carbonate in acetonitrile to produce
compound 13 (N-methyl-N-(4-nitro-2-phenoxyphenyl)-
methanesulfonamide, N-methylnimesulide) in good yield.
present in the spectrum of nimesulide at 3284 cm-1
Finally, compound 13 is devoid of any absorption band
near 3200 cm-1 as well as between 2800 and 2650 cm-1
.
P h a r m a cologica l Eva lu a tion
.
Compounds 6, 7, 12, and 13 and nimesulide have been
evaluated as COX inhibitors in vitro and as NSAID in
vivo. For the in vitro evaluation (whole blood assay),
each drug was studied in triplicate at drug concentra-
tions ranging from 100 to 0.01 µM and the IC50 values
were calculated. In this assay, the COX-1 activity was
measured as TXB2 production after blood coagulation,
and the COX-2 activity was measured as PGE2 produc-
tion after stimulation by lipopolysaccharide (LPS). Table
1 reports the results obtained with compounds 6, 7, 12,
and 13 and nimesulide. As observed, compound 6
inhibited both COX-1 and COX-2 but was a COX-1
preferential inhibitor. In contrast, the results with
compound 7 and nimesulide showed that the two drugs
were COX-2 preferential inhibitors, compound 7 being
more potent on COX-2.
A previous crystallographic study of 6 confirmed that
this drug was a zwitterionic pyridinium compound
substituted in the 4-position by an anionic sulfonami-
date group.11 Figure 1 reports an ORTEP view of the
crystallographic structures of compounds 7 and 12.
Compound 7 was also found to be in a comparable
zwitterionic pyridinium form in the solid state. In
contrast, the X-ray structure of 12 revealed that the
drug was present in the crystal as an un-ionized
pyridine compound substituted with a nonanionic meth-
anesulfonamido group. Furthermore, this analysis re-
vealed that in 7 the C9-N10-C11 angle was 121.1° and
that in 12 the C8-N9-C10 angle was 116.4°. This
information also led to the conclusion that 7 was
crystallized as a pyridinium compound and 12 as an un-
ionized pyridine compound. Finally the structure of
compound 13 has been confirmed by a recently pub-
lished crystallographic study.12
Those compounds, except 12, were also evaluated in
a rat paw oedema study. In this test, compound 6 was

5184 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23
Brief Articles
F igu r e 2. Hypotheses for the attribution of the pKa values
for compounds 6 and 7.
the pyridinium/pyridine equilibrium. For compound 7,
the pKa value of the equilibrium sulfonamide/sulfona-
midate could be estimated as being less than 1 and the
pKa value of 6.81 could be associated with the equilib-
rium pyridinium/pyridine. For compound 12, in con-
trast, the spectral and structural analyses show that
in the solid state and in solution this compound is
present as an un-ionized pyridine compound substituted
by an un-ionized sulfonamide group. The protonated
pyridinium nitrogen atom of compound 12 in acidic
medium is expected to exhibit a pKa value less than 1,
and the sulfonamide function has a pKa value of 7.85.
F igu r e 1. ORTEP view of compounds 7 (top) and 12 (bottom).
Con clu sion
At the physiological pH of 7.4, compound 6 is expected
to be mainly present as a positively charged pyridinium
species substituted with an anionic sulfonamidate moi-
ety (84.9%), compound 7 a nonprotonated pyridinic
compound substituted by an anionic sulfonamidate
moiety (79.5%), compound 12 a nonprotonated pyridinic
compound substituted by a nonionic sulfonamide moiety
(73.8%), and nimesulide an anionic sulfonamidate
(87.0%). By comparison of the activity of nimesulide and
compound 13, we can conclude that inhibition of the
COX enzymes probably requires the deprotonation of
the sulfonamide function. Furthermore, the weak activ-
ity of compound 6 compared to 7 leads to the conclusion
that the molecules should not bear a positively charged
nitrogen atom on the pyridine ring. By comparison of
the activity of compounds 6 and 12, we can also conclude
that the substitution at the 2- and 3-position of the
pyridine ring is unfavorable for obtaining a COX inhibi-
tor. Thus, optimal binding site interactions are obtained
with anionic sulfonamidate compounds devoid of a
positively charged nitrogen atom on the aromatic ring.
Finally, in the in vitro study, compound 7 was found
to be more potent than nimesulide but less COX-2-
selective. Furthermore, this compound presented an
interesting in vivo antiinflammatory activity. Thus, this
compound will serve as a lead structure in the design
of new potent and selective nonsteroidal antiinflamma-
tory drugs.
Compounds 6, 7, and 12 were also studied by 1H NMR
in deuterated DMSO. All compounds presented an
exchangeable proton. For nimesulide, the proton of the
sulfonamide function was found at a chemical shift of
10.13 ppm. For compounds 6, 7, and 12 in the same
solvent, the chemical shift of the exchangeable proton
appeared at 11.87, 13.90, and 9.64 ppm, respectively.
By comparison of the respective chemical shifts of the
N-H proton, it can be hypothesized that nimesulide and
compound 12 present a protonated sulfonamide function
(-SO2NH-; expected chemical shift around 10 ppm)
and that compounds 6 and 7 are zwitterions with a
pyridinium N+-H proton found in a more deshielded
position (around 12-14 ppm), and in the para position,
a sulfonamidate (SO2N--) function is present. Such a
structural feature is confirmed by the chemical shift of
the CH3 group, which was found at 2.88, 3.15, and 3.17
for 6, 12, and nimesulide, respectively. Because of the
proximity of the negative charge in 6, the methyl group
appeared to be shielded in compound 6 compared to 12
and nimesulide.
For compounds 6 and 7, two hypotheses can be
proposed for the attribution of the pKa values to the
equilibrium involved (Figure 2). In the first hypothesis,
the lowest pKa value should be associated with the
equilibrium pyridinium/pyridine and the highest with
the equilibrium sulfonamide/sulfonamidate. In the sec-
ond hypothesis, in contrast, the more acidic pKa value
should be associated with the equilibrium sulfonamide/
sulfonamidate and the second with the equilibrium
pyridinium/pyridine. The spectral and X-ray analyses
of compounds 6 and 7 show that in the solid state and
in solution in DMSO-d6 those compounds are in the
zwitterionic pyridinium sulfonamidate form. We can
then conclude that for 6 the pKa value of 2.98 could be
attributed to the sulfonamide/sulfonamidate equilibrium
and that the pKa value of 8.13 could be attributed to
Exp er im en ta l Section
Hu m a n Wh ole Blood Assa y. The compounds have been
evaluated by a whole blood assay performed on human blood
as described by de Leval.13
Ca r r a geen a n -In d u ced Ra t P a w Oed em a . Wistar rats
(250 g) were treated with an intraperitoneal injection of the
drug at the appropriate concentration (solution at 10 mg/mL
in DMSO). Lambda carrageenan (0.1 mL, 1%) was injected 1
h later in the plantar region of the right-hand paw. Three
hours thereafter, the rats were euthanized by injection of

Brief Articles
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 23 5185
Inhibition and Progress to Date. Med. Res. Rev. 1996, 16, 181-
200.
(6) Talley, J . Selective inhibitors of Cyclooxygenase-2 (COX-2). Prog.
Med. Chem. 1999, 36, 201-234.
(7) Inaba, T.; Tanaka, K.; Takeno, R.; Nagaki, H.; Yoshida, C.;
Takano, S.; Synthesis and Anti-inflammatory Activity of
7-Methanesulfonylamino-6-phenoxychromones. Antiarthritic
Effect of the 3-Formylamino Compound (T-614) in Chronic
Inflammatory Disease Models. Chem. Pharm. Bull. 2000, 48,
131-139.
(8) Cignarella, G.; Viaello, P.; Berti, F.; Rossoni, G. Synthesis and
Pharmacological Evaluation of Derivatives Structurally Related
to Nimesulide. Eur. J . Med. Chem. 1996, 31, 359-364.
(9) Eatough, J .; Fuller, L.; Good, R.; Smalley, R. Synthesis of
Polynuclear Heterocycles. Part II. Cyclisations of 2- and 4-sub-
stituted 3-Amino and 3-Nitro-pyridines. J . Chem. Soc. C 1970,
1874-1878.
(10) Singh, S.; Shardra, N.; Mahajan, L. Spectrophotometric Deter-
mination of pKa of Nimesulide. Int. J . Pharm. 1999, 176, 261-
264.
(11) Michaux, C.; Charlier, C.; J ulemont, F.; Dogne, J .-M.; de Leval,
X.; Norberg, B.; Pirotte, B.; Durant, F. The N-(3-phenoxy-4-
pyridinyl)methanesulfonamide, Strict Pyridinic Analogue of
Nimesulide, a Selective Inhibitor of Cyclooxygenase-2. Acta
Crystallogr., in press.
(12) Michaux, C.; Charlier, C.; J ulemont, F.; Norberg, B.; Dogne, J .-
M.; Pirotte, B.; Durant, F. FJ 6, N-methyl-(4-nitro-2-phenox-
yphenyl)methanesulfonamide. Acta Crystallogr. 2001, E57,
o1012-o1013.
nembutal (100 mg/kg) and the paws were cut at the ankle.
The swelling was calculated as a percentage increase in the
weight of the control paw.
p Ka Deter m in a tion . The procedure for the determination
and calculation of the pKa value was essentially the same as
that described by Albert and Serjeant.14
Ack n ow led gm en t. This study was supported by
grants from the “Communaute´ Franc¸aise de Belgique”
and from the National Fund for Scientific Research
(F.N.R.S., Belgium), of which B. Pirotte is Research
Associate.
Su p p or tin g In for m a tion Ava ila ble: Procedure for the
synthesis of compounds 6, 7, 12, and 13, crystallographic data,
and elemental analyses. This material is available free of
charge via the Internet at http://pubs.acs.org.
Refer en ces
(1) Dannhardt, G.; Kiefer, W. Cyclooxygenase InhibitorssCurrent
Status and Future Prospects. Eur. J . Med. Chem. 2001, 36, 109-
126.
(2) Crofford, L.; Lipsky, P.; Brooks, P.; Abramson, S.; Simon, L.;
van de Putte, L. Basic Biology and Clinical Application of Specific
Cyclooxygenase-2 Inhibitors. Arthritis Rheum. 2000, 43, 4-13.
(3) Xie, W.; Chipman, J .; Robertson, D.; Erikson, R. L.; Simmons,
D. Expression of a Mitogen-Responsive Gene Encoding Prostag-
landin Synthase Is Regulated by mRNA Splicing. Proc. Natl.
Acad. Sci. U.S.A. 1991, 2692-2696.
(4) Amgad, G.; Praveen Rao, P. N.; Knaus, E. E. Design and
Synthesis of 4,5-Diphenyl-4-isoxazolines: Novel Inhibitors of
Cyclooxygenase-2 with Analgesic and Antiinflammatory Activity.
J . Med. Chem. 2001, 44, 2921-2927.
(5) Griswold, D. E.; Adams, J . L. Constitutive Cyclooxygenase (COX-
1) and Inducible Cyclooxygenase (COX-2): Rational for Selective
(13) de Leval, X.; Delarge, J .; Devel, P.; Neven, P.; Michaux, C.;
Masereel, B.; Pirotte, B.; David, J .-L.; Henrotin, Y.; Dogne, J .-
M. Evaluation of Classical NSAID and COX-2 Selective Inhibi-
tors on Purified Ovine Enzymes and Whole Blood. Prostaglan-
dins, Leukotrienes Essent. Fatty Acids 2001, 64, 211-216.
(14) Albert, A.; Serjeant, E. P. Refinements of Potentiometric Titra-
tion: Apparatus and Calculation. In The Determination of
Ionization Constants: A Laboratory Manual; Ed Chapman &
Hall Ltd.: London, 1971; pp 342-369.
J M020920N