264 Chapter - 5

CHAPTER 5

Synthesis characterization and

pharmacological activity of Nimesulide

based sulphonamides

265 Chapter - 5

5. Synthesis, Characterization and pharmacological

activity of Nimesulide based sulphonamides

5.1 Introduction

Sulfonamides play a versatile role in many medicinal chemistry

analogue programs, these are a very important class of compounds in

the pharmaceutical industry, being widely used anticancer, anti-

inflammatory and antiviral agents. Organic sulfur compounds are

widespread in numerous natural products and widely used as various

artificial chemicals. Not only the medicinal importance the sulfonamides

like Naphtholic naphthalene sulfonate containing sulfonamide group and

its derivatives can be used to synthesize universal dyes, acid dyes, and

cationic dyes.

There is an urgent need for processes involved in structural

modifications of lead compounds that enhance pharmacological activity,

improve pharmacokinetic properties, or reduce unwanted side effects in

the field of drug discovery. Epidemiological and animal model studies

have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) can

act as chemopreventive agents. A growing body of experimental and

epidemiological evidence suggests that the use of NSAIDs (non-steroidal

anti-inflammatory drugs) may decrease the incidence of mammary

cancer, tumor burden, and tumor volume.

266 Chapter - 5

Celecoxib, a COX-2 (cyclooxygenase 2) selective inhibitor, shows

strong chemopreventive activity against mammary carcinoma in rats in

some studies.

1

In addition to COX inhibition, these small molecules could target

other molecular pathways. The COX-2 inhibitor nimesulide is able to

suppress the development of 2-amino-1-methyl-6-phenylimidazo [4,5-

b]pyridine (PhIP)-induced mammary gland carcinogenesis in rats .

Researchers proved that nimesulide also suppresses aromatase

activity and expression in several breast cancer cell lines In brief, COX-

2 inhibitors benefit breast cancer patients in several ways. Firstly, they

decrease PGE2 production which subsequently promotes tumor

invasiveness, angiogenesis, and progression. Secondly, they inhibit some

kinases which are related with tumor growth. Thirdly, they

decrease aromatase activity which is the key enzyme for

the biosynthesis of estrogen.

The cyclooxygenase-2 (COX-2) inhibitor nimesulide shows

anticancer effects in several cancer cell lines via COX-2-dependent and -

267 Chapter - 5

independent mechanisms. The molecular structure of nimesulide was

used as a starting scaffold to design novel sulfonanilide analogs and

examine the structural features that contribute to this anticancer effect.

2

Other research demonstrated that nimesulide also suppressed

aromatase activity and expression in several breast cancer cell lines.

Nimesulide derivatives which do not have COX-2 inhibitory activity were

more active than nimesulide to target aromatase. Further study reveals

that several nimesulide analogs were able to selectively inhibit Her2

overexpressing breast cancer cell proliferation, which suggests that they

are potentially able to overcome AI resistant breast cancer cell growth.

Because of the unique character of nimesulide derivatives, we propose

that the modification of the structure might change the drug from a

COX-2 inhibitor to an anti-cancer agent.

Garcinol (camboginol) from the fruit rind of Guttiferae species

shows anti-carcinogenic and anti-inflammatory properties.

268 Chapter - 5

Garcinol potently interferes with 5-lipoxygenase and microsomal

prostaglandin (PG) E2synthase enzymes that play pivotal roles

in inflammation and tumorigenesis.

In cell-free assays, garcinol inhibited the activity of purified 5-

lipoxygenase and blocked the mPGES-1-mediated conversion of PGH2 to

PGE2 with IC50 values of 0.1 and 0.3 μM respectively. Garcinol

suppressed 5-lipoxygenase product formation also in intact human

neutrophils and reduced PGE2 formation in interleukin-1β-stimulated

A549 human lung carcinoma cells as well as in human whole blood

stimulated by lipopolysaccharide. In contrast, neither Ca2+-ionophore

(A23187)-induced arachidonic acid release in neutrophils nor COX-2

activity in A549 cells or whole blood measured as formation of 6-keto

PGF1α, or isolated human recombinant COX-2 were significantly affected

by garcinol (≤30 μM). Together the high potency of garcinol to selectively

suppress PGE2 synthesis and 5-lipoxygenase product formation provides

a molecular basis for the anti-inflammatory and anti-carcinogenic effects

of garcinol and rationalizes its therapeutic use (Figure 5.1).

269 Chapter - 5

Figure 5.1 Mechanism of anti-inflammatory and anti-carcenogenic

activity of gercinol

Arachidonic Acid

Gercinol LTA4

PGH2

Leukotrienes

PGI2 PGE2

PGI2 Synthase

mPGES-1

6-Keto PGF1

5-LipoxygenaseCOX-1COX-2

Nimesulide, a preferential COX-2 inhibitor is a non-carboxylic acid

nonsteroidal anti-inflammatory drug (NSAID) that has been in patient’s

use for the treatment of pain for more than 20 years.1 Derivatives of

nimesulide have shown antiviral, anticancer2 and COX-2 inhibiting3

properties.

We anticipated that combination of structural features of these

NSAIDs nimesulide (2) with substituted sulfonyl chlorides in a single

molecule would provide novel agents possessing potent pharmacological

activities. Herein we report the synthesis, structure analysis and in vitro

pharmacological evaluation of a series of hybrid molecules based on

sulfonamides.

270 Chapter - 5

Figure 5.2 Design of novel molecules based on nimesulide and sulfonyl

Chlorides

O

NHSO2CH3

NO2

O

NHSO2CH3

NHSO2Ar

ArSO2Cl

2 A

Pharmacologically active derivatives of Nimesulide

Renard and his team reported4 the synthesis and the

pharmacological evaluation of pyridine analogues of nimesulide, a COX-2

preferential inhibitor. The cyclooxygenases inhibitory activities were

evaluated in vitro using a human whole blood mode. According to the in

vitro results, a selection of compounds exhibiting moderate to high COX-

2/COX-1 selectivity ratio were further evaluated in vivo in a moderate of

λ carrageenan-induced pleurisy in rats. Some of the selected compounds

(3) displayed similar or improved anti-inflammatory properties when

compared to nimesulide and celecoxib.

N

X

NHS

O

O

R

R = CH3, CF3X = O, S, NH

3

271 Chapter - 5

Kavitha and his team have described a simple and rapid synthesis5

of novel cyclic substituted imides (4) from nimesulide. The aromatic

amine prepared from nimesulide was reacted with a variety of cyclic

anhydrides in the presence of sodium acetate to afford the desired

products. Some of the compounds synthesized showed anti-inflammatory

activity when tested in rats.

O

NHSO2CH3

N OO

4

Pyridinic analogues6 of nimesulide were given by Julemont and his

team. Compound 4, N-(3-phenoxy-4-pyridinyl)trifluoromethane

sulfonamide, showed in vitro a strong inhibitory activity on the two

cyclooxygenase enzymes, being more active but less COX-2 selective than

nimesulide. Physicochemical studies and structural analyses indicated

that the anionic sulfonamide species seemed to be the active form of

methanesulfonamides, which optimally interacted with COX enzymes

active sites.

272 Chapter - 5

N

O

NHSO2CF3

5

A series of COX-2 selective inhibitor nimesulide derivatives were

synthesized.7 Their anti-cell proliferation activities were evaluated with a

long term estrogen deprived MCF-7aro (LTEDaro) breast cancer cell line,

which is the biological model of aromatase inhibitor resistance for

hormone dependent breast cancer. Compared to nimesulide which

inhibited LTEDaro cell proliferation with an IC50 at 170.30μM, several

new compounds showed IC50 close to 1.0 μM. The results suggest that A

position as 2, 5 dimethyl or dichloro benzyl is the best fit.

HNO

R2

R1

N

S OO

6

Julemont and his team has synthesized the pyridinic analogues of

nimesulide8 based on their major ionic state at the physiological pH of

7.4 the alkane sulfonamides 7 and the anionic trifluoromethane

sulfonamides 8 compounds. Pharmacological evaluation in vitro showed

273 Chapter - 5

that pyridinium compounds are generally more selective toward COX-2

when compared to their pyridinic analogues. Compound (8) displayed a

better COX-2 selectivity when compared to nimesulide. Compound ()

showed a similar in vivo profile as nimesulide when tested in the rat aw

odema.

N

O

NHO2S

CH3

Cl

N

HN

NHO2S

CF3

7 8

Synthesis of a series of compounds9 structurally related to the

anti-inflammatory agent nimesulide has been accomplished via Pd-

catalyzed C–C bond forming reactions. Thus 4-iodo derivative, prepared

from nimesulide, participated in Sonogashira (9), Heck (10) and Suzuki

(11) coupling reactions to afford the corresponding alkynyl, alkenyl and

aryl substituted products. Some of the compounds synthesized were

tested for anti-inflammatory activities in vivo.

O

NHCOCH3

R

R = C(CH3)2OH, CH2(CH2)4CH3 C(CH3)3

O

NHCOCH3

R'

R' = CO2CH3 CO2C2H5 CO2nBu C2H5 CN

O

NHCOCH3

R'' = OCH3 CH2OH F

R''

9 10 11

274 Chapter - 5

5.2 Present work

We report the synthesis of benzene sulfonamides as hybrid

molecules derived from nimesulide by straight forward sulfonylation of

the key intermediate nimesulide based N-(4-Amino-2-phenoxy-phenyl)-

methane sulfonamide with an appropriate and commercially available

sulfonyl chlorides. Thus reduction of 2 with Sn/HCl10 followed by the

treatment of 12 with sulfonyl chlorides 13 in the presence of chloroform

gave the compound 14a-d (Scheme 1).

Scheme 1

O

NHSO2CH3

NO2

O

NHSO2CH3

NH2

O

NHSO2CH3

HN

Sn/HCl ArSO2Cl (13)

SO2Ar

2 1214a-d

275 Chapter - 5

Table 5.1 Comparison of Time and Yield of products 14a-d

Sl. No. ArSO2Cl

(13)

Product

(14)

Time & Yield

(%)

1.

S

O

O

Cl

CH3

13a

O

NHSO2CH3

HNS

O

O

CH3

14a

9 h / 48

2.

S

O

O

Cl

NHCOCH3

13b

O

NHSO2CH3

HNS

O

O

NHCOCH3

14b

8 h / 51

3.

S

O

O

Cl

Cl

13c

O

NHSO2CH3

HNS

O

O

Cl

14c

14 h / 31

4.

S

O

O

Cl

CH3H3C

13d

O

NHSO2CH3

HNS

O

O

CH3H3C

14d

14 h / 32

276 Chapter - 5

All the compounds were characterized by spectral analysis IR,

MASS, and 1HNMR. Structure of the compound was characterized by X-

ray analysis and was found to be in the form of a monomer.

Figure 5.3 ORTEP view of compound N-[4-(4-methanesulfonylamino-3-

phenoxy phenylsulfamoyl)-phenyl]-acetamide (14b)

277 Chapter - 5

5.3 Conclusion

In conclusion, reduction of nimesulide (2) followed by treating the

N-(4-Amino-2-phenoxy-phenyl)-methane sulfonamide (12) with different

sulfonyl chlorides (13a-d) provided novel benzene sulfonamide

derivatives. But unfortunately we were able to synthesize very few

compounds 14a-d, because of various drawbacks such as long reaction

time, tedious manipulations in the isolation of the pure products, and

very less yield. Many trials were made by changing the solvent, reaction

conditions, and the reagent, but we were not successful in preparing

these sulfonamides in good yield.

5.4 Experimental section

General procedure for the preparation of benzene sulfonamides

N-(4-amino-2-phenoxy phenyl)methanesulfonamide (12, 1 g, 3.56 mmol)

was dissolved in dry chloroform and triethylamine (0.6 mL) was added to

it. The solution was cooled to 0ºC and benzene sulfonyl chloride (13,

3.56 mmol) was added drop wise with stirring. The reaction mixture was

then stirred at room temperature for the time given in Table 5.1, poured

into water (20 mL) and extracted with chloroform (3 × 25 mL). Organic

layers were collected, combined, washed with 10% HCl (10 mL) followed

by water (2 × 10 mL), dried over anhydrous Na2SO4 and concentrated.

278 Chapter - 5

The residue was purified by re-crystallization from chloroform ethyl

acetate mixture.

Preparation of N-(4-methanesulfonylamino-3-phenoxy-phenyl)-4-

methyl-benzenesulfonamide (14a):

O

NHSO2CH3

HNS

O

O

CH3

This compound was prepared according to the general procedure

using N-(4-amino-2-phenoxy phenyl)methanesulfonamide (12, 1 g, 0.003

mol), benzene sulfonyl chloride (13a, 0.52 g, 0.003 mol), triethylamine

(1.0 mL, 0.007 mol) and solvent (15 mL) as described in general

procedure to give 48% of the product as white solid; mp 142 ºC; IR (KBr

cm-1): 3331, 3230, 1659, 1590; MS (ES): m/z 433 (M+, 100%); 1H NMR

(200 MHz, DMSO-d6) δ 10. 2(s, 1H), 9.2 (s, 1H), 7.5-6.5 (m, 12H), 2.9 (s,

3H), 2.3 (s, 3H); Molecular formula: C20H20N2O5S2.

279 Chapter - 5

Synthesis of N-[4-(4-methanesulfonylamino-3-phenoxy

phenylsulfamoyl)-phenyl]-acetamide (14b):

O

NHSO2CH3

HNS

O

O

NHCOCH3

This compound was prepared according to the general procedure

using N-(4-amino-2-phenoxy phenyl)methanesulfonamide (12, 1 g, 0.003

mol), 4-Acetylamino-benzene sulfonyl chloride (13b, 0.696 g, 0.003 mol),

triethylamine (1.0 mL, 0.007 mol) and solvent (15 mL) as described in

general procedure to give 51% of the product as white solid; mp 186 ºC;

IR (KBr cm-1): 3231, 3315, 1658, 1604; MS (ES): m/z 476 (M+, 100%);

1HNMR (200 MHz, DMSO-d6): δ 9.87 (s, 1H), 9.77 (s, 1H), 8.17 (s, 1H),

6.74-7.78 (m, 12H), 2.16 (s, 3H), 2.89 (s, 3H), 9.77 (s, 1H), Molecular

formula: C21H21N3O6S2.

Synthesis of 4-Chloro-N-(4-methanesulfonylamino-3-phenoxy-

phenyl)-benzenesulfonamide (14c):

O

NHSO2CH3

HNS

O

O

Cl

280 Chapter - 5

This compound was prepared according to the general procedure

using N-(4-amino-2-phenoxy phenyl)methanesulfonamide (12, 1 g, 0.003

mol), 4-chloro-benzene sulfonyl chloride (13c, 0.62 g, 0.003 mol),

triethylamine (1.0 mL, 0.007 mol) and solvent (15 mL) as described in

general procedure to give 31% of the product as off white solid; mp:

1580C; IR (KBr cm-1): 3386, 3318, 1692, 1590, 1090; MS (ES) m/z: 453

(M+, 100%), 1HNMR (400 MHz, DMSO-d6): δ 9.71 (bs, 1H, NH, D2O

exchangeable), 7.78 (m, 6H, ArH), 7.55 (d, 1H, J=12Hz), 7.44 (t, 2H,

J=12Hz), 7.23 (t, 1H, J=12Hz), 6.97 (d, 2H, J=12Hz), 6.12 (bs, 1H, NH,

D2O exchangeable), 2.01 (s, 3H); Molecular formula: C19H17ClN2O5S2.

Synthesis of N-(4-methanesulfonylamino-3-phenoxy-phenyl)-2,4-

dimethyl-benzenesulfonamide (14d):

O

NHSO2CH3

HNS

O

O

CH3H3C

This compound was prepared according to the general procedure

using N-(4-amino-2-phenoxy phenyl)methanesulfonamide (12, 1 g, 0.003

mol), 3,5-Dimethyl-benzene sulfonyl chloride (13d, 0.61 g, 0.003 mol),

triethylamine (1.0 mL, 0.007 mol) and solvent (15 mL) as described in

general procedure to give 32% of the product as white solid; mp: 164-

281 Chapter - 5

1660C; IR (KBr cm-1): 3378, 3326, 1653, 1590; MS (ES) m/z: 447 (M+,

100%), 1HNMR (400 MHz, DMSO-d6): δ 10.58 (bs, 1H, NH, D2O

exchangeable), 9.21 (bs, 1H, NH, D2O exchangeable), 8.19 (s, 1H), 7.42

(m, 3H), 7.24-7.12 (M, 3H), 7.01-6.90 (m, 2H), 6.54 (s, 1H), 6.52 (m, 1H),

2.91 (s, 3H), 2.45 (s, 6H); Molecular formula: C21H22N2O5S2.

282 Chapter - 5

5.5 References

1. L. J. Roberts, J. D. Morrow, J. G. Hardman, L. E. eds. Limbird.

2001, 687. McGraw-Hill, New York.

2. S. Karakuş, S. G. Küçükgüzel, I. Küçükgüzel, E. D. Clercq, C.

Pannecouque, G Andrei, R. Snoeck, F. Şahin, O. F. Bayrak, Eur.

J. Med. Chem. 2009, 44, 3591.

3. C. Michaux, C. Charlier, F. Julémont, Leval, X. de Leval, JM.

Dogné, B. Pirotte, F. Durant, Eur. J. Med. Chem. 2005, 40, 1316.

4. R. Jean-Francois, A. Deniz, G. Nancy, B. Pirotte, X. de Levalt. J.

Med. Chem. 2009, 52, 5864–5871.

5. K.Kavitha, V. R. Reddy, K. MukkantI, S. Pal. J. Braz. Chem. Soc.

2010, 21, 6, 1060-1064.

6. F. Julemont, X. de Leval, C. Michaux, J. Damas, C. Charlier, F.

Durant, B. Pirotte, J. M. Dogne. J. Med. Chem. 2002, 45, 5182-

5185.

7. Bin Su, Shiuan Chen. Bioorg. Med. Chem. Lett. 2009, 19(23):

6733–6735

8. F. Julemont, X. de Leval, C. Michaux, R. Jean-Francüois, W. Jean-

Yves, M. Jean-Louis, J. Damas, D. Jean-Michel, B. Pirotte. J. Med.

Chem. 2004, 47, 6749-6759.

283 Chapter - 5

9. D. Shylaprasad, C. Vijay kumar, K. Mukkanti, S. Pal. Appl.

Organometal. Chem. 2010, 24, 680–684.

10. S. Pericherla, J. Mareddy, R .D. P. Geetha, P. V. Gollapudi, S.

Pal, J. Braz. Chem Soc. 2007, 18, 384.

284 Chapter - 5

5.6 Some important spectra of the compounds

Figure 5.4 1HNMR spectrum of N-(4-methanesulfonylamino-3-phenoxy-

phenyl)-4-methyl-benzenesulfonamide (14a)

Figure 5.5 13CNMR spectrum of N-(4-methanesulfonylamino-3-phenoxy-

phenyl)-4-methyl-benzenesulfonamide (14a)

O

NHSO2CH3

HNS

O

O

CH3

O

NHSO2CH3

HNS

O

O

CH3

285 Chapter - 5

Figure 5.6 Mass spectrum of N-(4-methanesulfonylamino-3-phenoxy-phenyl)-

4-methyl-benzenesulfonamide (14a)

Figure 5.7 IR spectrum of N-(4-methanesulfonylamino-3-phenoxy-phenyl)-4-

methyl-benzenesulfonamide (14a)

O

NHSO2CH3

HNS

O

O

CH3

O

NHSO2CH3

HNS

O

O

CH3

286 Chapter - 5

Figure 5.8 1HNMR spectrum of N-[4-(4-methanesulfonylamino-3-phenoxy

phenylsulfamoyl) -phenyl]-acetamide (14b)

O

NHSO2CH3

HNS

O

O

NHCOCH3