Preparation of CF31,3 Diones

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Letters in Drug Design & Discovery, 2005,2, 329-340 329

Synthesis and Cyclooxygenase-2 (COX-2) Inhibiting Properties of 1,5-Diarylpyrazoles Possessing N-Substitution on the Sulfonamide (SO2NH2)Moiety

Manojit Pala,*, Venugopal Rao Veeramanenia, Sanjeev Kumara, Akhila Vangoorib,Ramesh Mullangib, Parimal Misrab, Shaikh Abdul Rajjaka, Vidya B. Lohraya,Seshagiri Rao Casturiband Koteswar Rao Yeleswarapua

aChemistry, bBiology, Discovery Research, Dr. Reddys Laboratories Ltd., Bollaram Road, Miyapur, Hyderabad500049, India

Recei ved D ecember 2 9, 2004: Accep ted Apri l 05, 2005

Abstract: A number of novel 1,5-diarylpyrazoles possessing N-substitution on the sulfonamide (SO2NH2)moiety were synthesized and tested for COX-1/COX-2 inhibition in vitro. Many of these 1,1-dioxo-2,3-dihydrobenzo[d]isothiazolyl substituted 1,5-diarylpyrazoles, where the SO2NH2group was a part of the fusedring, showed COX inhibitory activity. Few of them were identified as selective COX-2 inhibitors. StructureActivity Relationship study within the series are discussed.

Keywords:1,5-Diarylpyrazoles, COX-1 & COX-2 inhibition, Structure Activity Relationship (SAR) study.

INTRODUCTION osteoarthritis and rheumatoid arthritis is presently in phaseIII clinical development. Apart from its role in rheumatoidarthritis and osteoarthritis, COX-2 is also implicated incolon cancer and angiogenesis. [6a-c]Since the progressionof Alzheimers disease has shown to be reduced among someusers of NSAIDs, the chronic treatment with COX-2inhibitors therefore may be effective for the treatment ofinflammatory neurodegenerative disorders, without causingGI damage [6d].

1,5-Diarylpyrazoles are the focus of many recent reports[1] because of their widespread use in the development ofcyclooxygenase-2 (COX-2) inhibitors such as celecoxib.Selective COX-2 inhibitors currently provide effectivetreatment against pain and inflammation with reducedgastrointestinal side effects associated with traditional non-steroidal anti-inflammatory drugs (NSAIDs). NSAIDs reducethe pain and swelling of joints by inhibiting prostaglandinsynthesis via blocking arachidonic acid (AA) oxygenation to

prostaglandin G2 (PGG2). PGG2 is the precursor tonumerous prostaglandins including those that provide

protection for the gastric mucosal. Until the discovery ofsecond and inducible isozyme (COX-2) recently, a singlecyclooxygenase (COX) enzyme was thought to beresponsible for all of the catalysis of AA to PGG2. After thediscovery of COX-2, the methanesulfonanilide (NS-398) andthe diarylheterocycle (DUP-697) were first identified as non-ulcerogenic anti-inflammatory agents (Fig. 1) [2].Subsequent research and rational drug design resulted in anumber of potent and selective COX-2 inhibitors, whichvalidated the initial concept that a selective COX-2 inhibitorwould elicit effective anti-inflammatory activity without theadverse ulcerogenic effect associated with the use of NSAIDsthat inhibit both COX-1 and COX-2. Accordingly, celecoxib[3] and rofecoxib [4], followed by valdecoxib [5a] and

etoricoxib [5b] became the first and second-generationselective COX-2 inhibitors (Fig.1) to enter the market. Veryrecently, parecoxib sodium [5c], a water-soluble prodrug ofvaldecoxib has also been marketed for the parenteraltreatment of postoperative pain. In addition, a new COX-2inhibitor lumiracoxib [5d] developed for the treatment of

Several strategies have been reported on the modificationof the known and non-selective inhibitors for the design anddevelopment of novel COX-2 inhibitors. These include

lengthening or derivatization of the carboxylic side chain othe indomethacin [7a,b], development of 5-methylsulfonyderivatives of indole-2-carboxylic acids (structurally relatedto indomethacin) [6c] and modification of the basicframework of zomepirac or flurbiprofen [7d,e]. The chemicalstructures of these COX-2 inhibitors including lumiracoxibclearly indicate that the basic framework of diarylheterocyclesis not the exclusive prerequisite for COX-2 inhibition. Thiswas supported by the recent report on the development ofnovel tetrahydro-2H-isoindoles as COX-2 inhibitor, where1,2-disubstitution by two aryl groups on a central core wasmissing [7f]. In spite of several reports on deviation from thecommon structural features of diarylheterocycles i.e. 1,2substitution and/ or the sulfonyl moiety on the aromatic

ring, the main effort has been devoted to thediarylheterocycle class [7g-l] (as is exemplified by the recentreport on the development of 1,5-diarylimidazoles [7i], 2,3-diarylindoles [7j], deracoxib [7k], 3,4-diarylpyran-2-ones[7l], DRF4848 [9f] etc. as COX-2 inhibitors), perhaps due tothe early success in the discovery of two selective COX-2inhibitors e.g. celecoxib and rofecoxib (while rofecoxib has

been withdrawn from the market recently, its recall notnecessarily indicates that all COX-2 inhibitors are equivalenand risky for human patients [4c] ). Celecoxib belongs to adiaryl heterocyclic class where two aryl moieties are attached

*Address correspondence to this author at the Discovery Research, Dr.Reddys Laboratories Ltd., Bollaram Road, Miyapur, Hyderabad 500049,India; Fax: 91-40-23045438 /23045007; E-mail: [email protected] Publication No. 340A.

1570-1808/05 $50.00+.00 2005 Bentham Science Publishers Ltd.


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330 Letters in Drug Design & Discovery, 2005, Vol. 2, No. 4 Pal et a

S

F

H3CO2S

Br NN

H2NO2S

H3C

CF3

O

H3CO2S

O

O

N

S

H2N

OO

CH3

O

NO2

HNSO2CH3

NS-398

N

N

H3CO2S

H3C

Cl

DuP 697

Rofecoxib (11)

Celecoxib (10)

Valdecoxib Etoricoxib

Fig. (1). Some selective COX-2 inhibitors.

to the adjacent positions of the central pyrazole ring. The 4-benzenesulfonamide group attached to the nitrogen atom ofthe pyrazole ring is thought to be responsible for its efficacyand COX-2 selectivity in different models of inflammation.Recent study revealed that this class of compounds could beuseful as dual COX-2 / 5-LO (5-lipooxygenase) inhibitors forthe effective management of inflammatory diseases [8a].Moreover, unlike rofecoxib, this class of compounds is notsusceptible to aerial oxidation under physiologicalconditions. Due to their interesting pharmacological and

pharmacokinetic properties, 1,5-diarylpyrazoles have been

utilized for the development of selective COX-2 inhibitorsby many research groups [8b-I]. In most of the these cases,structure activity relationship (SAR) studies were carried outwith various modifications on the central pyrazole ring, 1-(4-

benzenesulfonamide) or 5-phenyl group, individuallykeeping the sulfonamide (-SO2NH2) moiety intact (Fig. 2).While the replacement of SO2NH2 by an azido (N3)

bioisostere retained COX-2 inhibitory activity [8j], a nitro(NO2) or methanesulfonamide (NHSO2Me) group, which candispose a pair of oxygen atoms such as SO2, abolishedCOX-2 inhibitory activity [3b]. Alkylation i.e. N-methylation or N,N-dimethylation of the sulfonamide moietyalso resulted in compounds that did not show COX-2inhibitory activity [3b, 8k]. However, linking of thesulfonamide moiety with the adjacent benzene ring through amethylene bridge and its subsequent effect on COX activityhas not been studied extensively earlier. In connection withour effort on the development of cyclooxygenase inhibitors[9], we have recently reported the synthesis and COX-2inhibiting properties of a new class of 1,5-diarylpyrazolescontaining substituted benzensulfonamide moiety as

pharmacophore [10a]. In further pursuance of our study onCOX inhibitors, we became interested in the synthesis of1,5-diarylpyrazole derivatives containing fused 1,1-dioxoisothiazolyl group. To the best of our knowledge, onlyone example of such compound has been reported earlier

[10b]. Herein, we describe the synthesis, COX inhibitingproperties and molecular modeling studies for a novel classof 1,5-diarylpyrazoles that possess 1,1-dioxo-2,3dihydrobenzo[d]isothiazol-5-yl group as a pharmacophore in

place of 4-benzenesulfonamide moiety.

NN

R4

YX

R1S

R3

OO

R1= NH2, CH3X; Y = CH, N

R2= F, Me, CH2OH

R3= CF3, CHF2, CN, COOH etc.

R4= aryl, OiPr

R2

Fig. (2). Some 1,5-diarylpyrazoles as COX-2 inhibitors.

RESULTS AND DISCUSSION

Chemistry

The majority of the diarylpyrazoles listed in the Table 1were synthesized according to Schemes 1-3. 1,1-Dioxo-2,3dihydrobenzo[d]isothiazole-5-yl hydrazine 18; the keystarting material for most of the compounds was synthesizedfrom 3-fluorotoluene 13(Scheme 1) [11a]. Chlorosulfonationof 13 followed by ammonolysis, led to the formation of 4-fluoro-2-methyl benzenesulfonamide 15 in good yieldOxidative cyclization of 15 in the presence of alkaline

potassium permanganate solution followed by the treatmenwith dilute hydrochloric acid afforded benzo[d]isothiazinederivative 16. The amidic carbonyl group of 16 was thenreduced to a methylene group to afford 17, which ontreatment with anhydrous hydrazine provided 18. This wasthen converted to a hydrochloride salt 19 whichwhen reactedwith the dicarbonyl derivatives 20 [11b-d] (Scheme 2)yielded 1,5-diarylpyrazoles (1-4, 8) as major product [11e-f]The methylsulfone derivatives (5-7, 9) were prepared(Scheme 3) by controlled oxidation of appropriatemethylsulfanyl derivatives (e.g.4, 8 etc.).


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Synthesis and Cyclooxygenase-2 (COX-2) Inhibiting Properties Letters in Drug Design & Discovery, 2005, Vol. 2, No. 4 33

NH

S

OO

F

NH

S

OO

O

FMeF

SO2NH2

HN

S

O

O

NHNH2

MeF

SO2 Cl

HN

S

O

O

NHNH2.HCl

MeF a bc

d

ef

13 14 15 16

19 18 17

Scheme 1. Reagents and conditions: (a) ClSO3H, 0 C, 4h., 74% yield; (b) NH3, dioxane, 0 C, 6h., 79% yield; (c) NaOH, H2O,KMnO4, 6h., dilute HCl, 46% yield; (d) EtOH, Conc. HCl, Zn powder, 0-25 C, 2h, 33% yield; (e) NH2NH2, CH3CN, reflux, 6h., yield68%; (f) HCl, EtOH, quantitative yield.

All the compounds synthesized were tested initially forselectivity and potency against human COX-2 (expressed insf9 insect cells using baculovirus) and COX-1 (RamSeminal vesicles) enzyme [12a]. In vivo efficacy wasevaluated using carrageenan-induced rat paw edema model

[12b] and percentage of inhibition was calculated at the doseindicated in the text.

replacement of 4-fluorophenyl group by 3-fluoro-4-methylphenyl moiety (2, Table 1) did not improve the

potency or selectivity. Based on the available experimentadata [3b] and our experience [10], we anticipated that the

presence of a stronger electron donating group e.g. methoxy

or methylsulfanyl at the C-4 of the 5-aryl moiety mightimprove the potency as well as selectivity. Accordingly, therelated derivatives 3 and 4 were synthesized and tested invitro. Both the compounds (3 and 4, Table 1) showedimproved potency in COX inhibition but were identified asnon-selective inhibitors of COX-1 and COX-2. Both 3and 4showed significant inhibition (~50-60 %) of COX-1 even atlower concentration i.e. at 10 M. (Compound 3synthesized viaother route inhibited COX-2 insignificantlyat 100 M) [10b]. We therefore focused on electronwithdrawing groups specifically those, which are known toinfluence the binding of the diaryl heterocyclic class ofcompounds with COX-2 pocket e.g. SO2Me and SO2NH2[13]. Since methylsulfonyl group is known to impart betterCOX-2 selectivity than sulfonamide, we therefore preferredthis group at the C-4 position of the 5-aryl moiety of 1According to our expectation, the methylsulfonyl moiety atthe same position suppressed the COX-1 inhibition with

Biological Results

Results of in vitro assays of the compounds synthesizedhave been summarized in Table 1 [12c]. Compound 1, thefirst compound synthesized in this series, has a central

pyrazole ring of which one nitrogen atom was attached with1,1-dioxo-2,3-dihydrobenzo[ d]isothiazol-5-yl group. An arylgroup such as 4-fluorophenyl moiety occupied the C-5

position of the pyrazole ring. At a concentration of 100 M,1 showed selectivity in COX-2 inhibition over COX-1 withmoderate potency. Since celecoxib (10, Figure 1) possesses a4-methylphenyl moiety at the C-5 position of the pyrazolering, we anticipated that the presence of a mild electrondonating methyl group at the para position of the 5-arylmoiety of 1 might improve the potency. However, the

Ar

O

Ar

O

R

OH

NN

Ar

NHS

R

O O

a b

1 - 4, 8 20 21

Ar = R =

1 4-fluorophenyl CF3

2 3-fluoro-4-methylphenyl CF3

3 4-methoxyphenyl CF3

4 4-methylsulfanylphenyl CF3

8 2-flouro-4-methylsulfanylphenyl CHF2

Scheme 2. Reagents and conditions: (a) NaH, DMF, 0 C-room temp., RCO2Et, 24h., 12-97% yield; (b) 19, EtOH, reflux, 32-65%yield.


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NN

Ar

NH

S

R

OO

NN

Ar'

NH

S

R

OO

a

5 - 7, 9

Ar' = R =

5 4-methylsulfonylphenyl CF3

6 3-methyl-4-methylsulfonylphenyl CF3

7 2-flouro-4-methylsulfonylphenyl CF3

9 2-flouro-4-methylsulfonylphenyl CHF2

Scheme 3. Reagents and conditions: (a) oxone, acetone H2O (4: 1), room temp., 3h., 15-35% yield.

little effect on COX-2 potency (compound 5-7, Table 1).Compound 6for example did not inhibit COX-1, whereas 5and 7showed insignificant inhibition of COX-1 at 100 M.

Although the role of methylsulfonyl moiety in theenhancement of COX-2 selectivity of 5, 6 & 7 is not clearlyunderstood, presumably this polar group, which can formhydrogen bond through the oxygen atom, helps thesecompounds in binding with COX-2 more effectively.

Nevertheless, the methylsulfonyl moiety at the C-4 positionof the 5-aryl ring appeared to be promising for theenhancement of COX-2 selectivity and prompted us forfurther investigation. While all these compounds (5-7)showed good inhibition of COX-2 at the concentration of100 M, their potency was found to be reduced except in thecase of 7 when measured at a lower concentration i.e. at 10M. Presence of a fluorine at the C-3 position of the 5-(4-methylsulfonylphenyl) group of 7, perhaps reinforced its

binding with COX-2 when compared with 5and 6.

clear at the moment, this however, was not unexpected as asimilar observation was noted earlier [3b] that led to theidentification of deracoxib [7k] during the SAR studies on

1,5-diarylpyrazole class.The selectivity of celecoxib has been shown to result

from the benzenesulfonamide moiety [13b]. This groupbinds in a COX-2 pocket, which is relatively polar and isless restricted in contrast to COX-1 enzyme. This could also

be the reason for COX-2 selectivity of 5, 6 and 7, whichperhaps possess necessary structural requirements for theselective inhibition for COX-2 over COX-1. Analysis of themost energetically favored conformation of 7, in the COX-2complex, revealed that this 1,5-diarylpyrazole binds wellwith the COX-2 pocket [14]. The orientation and hydrogen-

bonding interactions of compound 7 within the COX-2binding site is shown in Fig. 3. Cyclic sulfonamide i.e. 1,1dioxo-2,3-dihydrobenzo[d]isothiazol-5-yl group o

compound 7binds in a polar region surrounded by residuesHis90, Gln192 and Arg513. Different sets of hydrogen

bonding interactions with residues His90 (N-H....O=S, 2.70; all distances are for dX-X), Leu352 (C=O....H-N, 3.07), Tyr385 (O-H...O=S, 3.00 ), Arg513 (N-H....O=S3.08 ) and Arg120 (NH....N2, 3.47 ) are observedAdditional methylene group in cyclic sulfonamide moietyforms favorable hydrophobic interactions with Leu352 andPhe518. The trifluromethyl group is bound in a pockeformed by Val116, Arg120, Val349, Tyr355, Leu531 andLeu359. The F atom in the trifluoromethyl (-CF3) acts as anacceptor to form a hydrogen bond with NH group ofArg120 side chain. Methylsulfonyl group binds in ahydrophobic pocket formed by Met522, Phe381, Tyr385 and

Trp387, where sulfone oxygen (SO2) forms strong hydrogenbond with Tyr385. It is noteworthy that 7extends its acidicsulfonamide moiety rather than methylsulfonyl group to the

polar region of the COX-2 pocket, perhaps due to the morepolar nature of -SO2NH- group [15a]. The binding pocket fothe 5-aryl moiety is long and narrow as the side of the

binding site bordered by Tyr385 is sterically restricted[15b]. This clearly disfavors the insertion of the bulky 1,1-dioxo-2,3-dihydrobenzo[d]isothiazol-5-yl group in this

pocket. However, to gain further evidence on the role of 1,1dioxo-2,3-dihydrobenzo[d]isothiazol-5-yl group an analogue

In majority of the compounds synthesized,trifluoromethyl (CF3) group occupied the C-3 position of the

pyrazole ring. The effect of replacement of this moiety withdifluoromethyl group was also investigated (compd 8 & 9,Table 1). Results of in vitro assay of compounds 8 & 9 atthe concentration of 10 M suggested that trifluoromethylgroup was superior to its difluoro counterpart (compd 7vs 9,Table 1), which was in agreement with the results observedearlier [3b]. Recent theoretical studies on inhibitionmechanism of COX-2 revealed that the CF3group increasesthe magnitude of drug-protein interactions due to thefavorable contribution from electrostatic and van der Waalsinteraction [13a]. Elimination of this group in the case of

celecoxib decreases the activity due to a change indesolvation energy and to a decrease of the drug-protein vander Waals interaction. The drug-protein interactions appearedto be less efficient in the case of 9when the trifluoromethylgroup of 7was replaced by a difluoromethyl group and wasreflected by the in vitro activity of these compounds.Interestingly, unlike a similar 3-trifluoromethyl analog e.g.4, compound 8 showed COX-2 selectivity in spite of

possessing a strong electron donating methylsulfanyl groupat C-4 position of its 5-aryl ring and was identified as a more

potent COX-2 inhibitor than its methylsulfone derivative 9at 10 M. Although the reason for such observation is not


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Table 1. InVitroData for 1,5-diaryl pyrazoles

CompdStructure % of inhibition @ 100Ma

COX-1 COX-2

1NN

NHS

F3 C

O O

F

6 45

2NN

NHS

F3C

O O

Me

F

13 49

3

NN

NHS

F3 C

O O

OMe

98

57 (10)

93

60 (10)

4

NN

NHS

F3C

O O

SMe

8751 (10)

9745 (10)

5

NN

NHS

F3C

O O

SO2Me

10 84

32 (10)

6

NN

NHS

F3C

O O

SO2 Me

Me

0 99

49 (10)


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(Table 1). contd.....

CompdStructure % of inhibition @ 100Ma

COX-1 COX-2

7

NN

NHS

F3C

O O

SO2MeF

23 86

85 (10)

8 NN

NHS

F2HC

O O

F SMe

12 (10) 63 (10)

9 NN

NHS

F2HC

O O

F SO2Me

0 (10) 38 (10)

10 Celecoxiba 0 100

11 Rofecoxib 0 100

12 Indomethacin 100 97aHuman COX-2 (expressed in sf9 insect cells using baculovirus) and COX-1 (Ram Seminal vesicles) enzyme. Figures in the brackets indicate concentration in M. Theresult is the average of at least three determinations, and the deviation from the mean is 100. This was then selected for further study in vivo. Anti-inflammatory activity of compound 7was tested in standardrat model of inflammation. In carrageenan-induced rat pawedema assay, compound 7 showed ~30 % and ~20 %inhibition when dosed orally at 30 and 10 mg/kgrespectively. Notably, unlike celecoxib, compound 7

possesses a 3-fluoro-4-methanesulfonylphenyl group insteadof 4-methylphenyl moiety at the C-5 position of the pyrazolering. Celecoxib is known to undergo metabolism viahydroxylation of the aromatic methyl group (i.e. 4-methylphenyl group) to generate a hydroxymethylmetabolite, followed by additional oxidation to a carboxylicacid metabolite [16b]. Because of its structural dissimilaritywith celecoxib, a similar metabolic pathway was unlikely inthe case of 7. Thus, compound 7 was expected to showdifferent pharmacokinetics than celecoxib that was implicated

by their stability in the presence of rat liver microsome


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Fig. (3).Molecule 7 in the binding pocket of COX-2. The hydrogen bonding interactions are shown as red colored broken lines. Theligand is shown in orange (ball and stick). All protein hydrogens are removed for clarity.

(RLM) in vitro. As expected, compound 7was found to bemore stable than celecoxib in the presence of RLM after 2h.

However, detailed pharmacokinetic studies are in progress toconfirm its superior pharmacokinetics over celecoxib in vivo.

3-trifluoromethylpyrazoles, a strong electron-donating groupsuch as methoxy or methylsulfanyl group at the C-4 position

of the 5-aryl ring resulted in compounds that inhibited bothCOX-1 & COX-2, whereas a mild electron donating groupe.g. fluoro and methyl induced COX-2 selectivity. Anelectron-withdrawing group such as methylsulfone at thesame position increased the COX-2 selectivity as well as

potency. Thus, our study indicates that 1,1-dioxo-2,3dihydrobenzo[d]isothiazolyl group could be utilized as a

pharmacophore for the inhibition of cyclooxygenase. Themolecular modeling study revealed that the favorablehydrophobic interactions between the bridged methylene

CONCLUSION AND PERSPECTIVES

We have described efficient synthesis of a novel class of1,5-diarylpyrazole derivatives that possess N-substitution onthe sulfonamide moiety. These compounds are characterizedas moderately potent inhibitors of cyclooxygenase enzymes.Structure-activity relationship (SAR) study revealed that for

Table 2. In Vitro (Enzyme Assay) and In Vivo (Rat Paw Edema Assay) Data for 1,5-Diarylpyrazoles

Compound IC50 (M)a Selectivity index (COX-1/ COX-2) In Vivo Rat paw edemab

(30mg/kg)

COX-1 COX-2

5 >150c 120.55 > 12 n.d.

6 >150c 9.00.9 > 16 n.d.

7 >200c 1.870.15 > 100 304

Celecoxib 15.330.03 0.070.005 ~219 492

Indomethacin 0.0670.001 7.80.11 ~0.0085 n.d.

aThe result is the mean value of two determinations, and the deviation from the mean is


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group and the COX-2 pocket enabled the 1,1-dioxo-2,3-dihydrobenzo[d]isothiazolyl moiety to function as a new

pharmacophore. Moreover, a larger group like methylsulfonecould be accommodated in the hydrophobic pocket formed

by residues Met352, Phe381, Tyr358 and Trp387. Insummary, these results clearly encourage further design andSAR studies around the cyclic sulfonamide moiety,especially by replacing the central pyrazole ring with other

heterocycles. Compound 7 showed selective inhibition inCOX-2 over COX-1, and therefore represents a new class ofCOX-2 inhibitor. Anti-inflammatory activity of compound 7has also been demonstrated in vivo model.

(10 g, yield 79%). mp 171-172 C (lit17b 172-173 C); 1HNMR (200 MHz, DMSO-d6) 7.9 (m, 1H), 7.4 (s, 2Hexchangeable with D2O), 7.2 (m, 2H), 2.59 (s, 3H).

Preparation of 5-Fluoro-1,1,3-Trioxo-2,3-Dihydrobenzo[d]isothiazine (16) [17c]

To a mixture of 15(10g, 67 mmol) and aqueous sodiumhydroxide (28g, 70.3 mmol) solution (100 mL) was added

potassium permanganate (18g, 114 mmol) in small portionsover a period of 5 hours with vigorous stirring. The mixturewas then stirred for 6 hours and filtered through celite. Thefiltrate was collected and neutralized with cold dilutehydrochloric acid and the separated solid was filtered off. Thefiltrate was collected again and treated with concentratedhydrochloric acid until the pH was 2.0. The separated solidwas filtered and dried to afford the expected compound (4.3g46%) as a white solid. mp > 200 C (lit [17c] 218-220 C)1H NMR (200 MHz, DMSO-d6) 8.9 (bs, 1Hexchangeable with D2O), 8.2 (m, 1H), 7.8 (m, 2H).

EXPERIMENTAL

Chemical Methods

All the solvents used were commercially available anddistilled before use. Reactions were monitored by thin-layerchromatography (TLC) on silica gel plates (60 F254;Merck), visualizing with ultraviolet light or iodine spray.Flash chromatography was performed on silica gel (SRL230-400 mesh) using distilled petroleum ether, ethylacetate,dichloromethane, chloroform and methanol. 1H NMR spectrawere determined in CDCl3, DMSO-d6 or MeOH-d4 solutionon Varian Gemini 200 MHz spectrometers. Proton chemicalshifts ( ) are relative to tetramethylsilane (TMS, = 0.00)as internal standard and expressed in ppm. Spinmultiplicities are given as s (singlet), d (doublet), t (triplet),and m (multiplet) as well as b (broad). Coupling constants(J) are given in hertz. Infrared spectra were recorded on aPerkin-Elmer 1650 FT-IR spectrometer. UV spectra wererecorded on a Shimadzu UV 2100S UV-vis recordingspectrophotometer. Melting points were determined using aBuchi melting point B-540 apparatus and are uncorrected.MS spectra were obtained on a HP-5989A mass

spectrometer. Microanalyses were performed using a Perkin-Elmer 2400 C H N S/O analyzer. All yields reported areunoptimized. Celecoxib was prepared according to theliterature [3b] procedure. Rofecoxib was prepared accordingto the procedure described in WO 9500501. Acetophenoneswere either purchased or prepared according to the proceduredescribed in the literature.

Preparation of 1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazol-5-yl Fluoride (17)

To a solution of 16 (2g, 10 mmol) in ethanol (5 mL)was added concentrated hydrochloric acid (15 mL) at 0 oCfollowed by the portion wise addition of zinc powder. Thereaction mixture was stirred for 2 hours at 25 C and thentreated with cold sodium bicarbonate solution until the pHof the mixture became 9. The mixture was filtered throughcelite pad and the filtrate was extracted with EtOAc (3 x 50mL). Combined organic layer was collected, washed withwater (2 x 50 mL), dried over anhydrous Na2SO4 andconcentrated under reduced pressure to give the requiredcompound (0.6g, yield 33%). 1H NMR (200 MHz, DMSO-d6) 7.90 (m, 2H, ArH & NH), 7.40 (m, 2H), 4.40 (d, J =4.0 Hz, 2H, CH2).

Preparation of 1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazol-5-

ylhydrazine (18)

A solution of 17 (0.55g, 2.94 mmol) in acetonitrile (10mL) was treated with anhydrous hydrazine (0.6 mL) and themixture was refluxed for 6 hours. The solvent was thenremoved under reduced pressure and the residue was treatedwith water (10 mL). The separated solid was filtered andwashed with cold water (2 x 50 mL) to give the required

product (0.4g, yield 68 %). 1H NMR (200 MHz, DMSO-d6)7.60 (s, 1H), 7.40 (m, 2H), 6.70 (m, 3H, NH), 4.20 (d, J= 5.0 Hz, 2H, CH2).

Preparation of 4-Fluoro-2-Methyl-1-BenzenesulfonylChloride (14) [17a]

To a cooled solution (0 C) of chlorosulfonic acid (57.0g, 450 mmol) was added 3-fluorotoluene (10 g, 90 mmol)slowly at 0 C. The mixture was then stirred for 4 hours at 0C and allowed to stand for overnight at same temperature.

The reaction mixture was poured into crushed ice. Oily layerseparated was collected and washed with water to yield thedesired compound as a liquid (14g, yield 75%). 1H NMR(200 MHz, CDCl3) 8.10 (m, 1H), 7.10 (m, 2H), 2.79 (s,3H, CH3).

Preparation of 1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazol-5-ylhydrazine Hydrochloride (19)

To a solution of 18(0.4g) in ethanol (5 mL) was addedi-propanol (3 mL) saturated with dry hydrochloric acid at 25C and the mixture was stirred for 1.5 hours at the sametemperature. The solvent was removed under low pressure toyield the required compound in quantitative yield and theresidue was used directly for the next reaction without further

purification.Preparation of 4-Fluoro-2-Methyl-1-Benzenesulfonamide(15) [17b]

Step 1. General Method for the Preparation of 1,3-Diketones (21)

To a cooled solution of 14 (14g, 67.0 mmol) in dioxane(30 mL) was added 25% aqueous solution of ammonia (140mL) with vigorous stirring. The stirring continued for 6hours at 0 C and separated solid was filtered, washed withwater (2x 50 mL), to give the title compound as white solid

To a solution of acetophenone (20, 2.7 mmol) indimethylformamide was added 60% oil suspension ofsodium hydride (3.24 mmol) at 10 C under nitrogen


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atmosphere and the mixture was stirred for 10 minutes. Tothis mixture was added appropriate ethylacetate (3.24 mmol)at the same temperature and stirring was continued for 12hours. This mixture was then poured into ice-coldhydrochloric acid solution (100 mL) and was stirred for 10minutes. Solid separated was extracted with ethyl acetate (2x 50 mL). Combined organic layer was collected, washedwith water (2 x 50 mL), dried over anhydrous Na2SO4 and

concentrated under vacuum. The residue isolated waspurified by column chromatography using 5% EtOAC-petroleum ether to give the required compound 21.

7.35 (d,J= 8.8 Hz, 1H), 7.26 7.11 (m, 4H), 6.79 (s, 1H)4.80 (bs, 1H, NH), 4.53 (d, J= 4.9 Hz, 2H, CH2), 2.50 (s3H, SCH3); IR (Nujol) cm.-1 1604, 1465; MS (CI, iButane) 426 (M+1, 100); found C, 50.77; H, 3.19; N, 9.95C18H14F3N3O2S2requires C, 50.82; H, 3.32; N, 9.88 %.

5-[3-Difluoromethyl-5-(2-Fluoro-4-Methylsulfanylphenyl)-1H-1-Pyrazolyl]-1,1-Dioxo-2,3-Dihudrobenzo[d]isothiazole(8)

The title compound was synthesized from 2-fluoro-4-methylsulfanylacetophenone using the two step procedure asdescribed above: yield 65 %; mp 178-180 oC; 1H NMR(200 MHz, CDCl3) 7.71 (d, J = 8.3 Hz, 1H), 7. 53 (s1H), 7.35-6.91 (m, 4H), 6.79 (s, 1H), 6.78 (t, J = 55 Hz1H, CHF2), 4.83 (bs, 1H, NH), 4.52 (d, J = 4.5 Hz, 2HCH2), 2.52 (s, 3H, SCH3); MS (CI, i-Butane) 425 (M+

100), 391 (10), 360 (30); found C, 50.80; H, 3.22; N, 9.91C18H14F3N3O2S2requires C, 50.82; H, 3.32; N, 9.88 %.

Step 2. General Method for the Preparation of 1,5-Diarylpyrazoles (1-4, 8)

A mixture of hydrazine hydrochloride 19 and 1,3-diketone 21 in ethanol was heated to reflux with vigorousstirring under nitrogen atmosphere for 12 hours. Ethanol wasremoved under low vacuum and the residue isolated was

purified by column chromatography using EtOAc-petroleumether (1:2) to give the expected product.

Step 3. General Method for the Preparation of 1,5-Diarylpyrazoles (5-7, 9)

5-[5-(4-Fluorophenyl)-3-Trifluoromethyl-1H-1-Pyrazolyl]-1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazole (1)

To a solution of methylsulfanylsubstituted1,5

diarylpyrazole (0.36 mmol) in acetone-water (4:1, 40 mL)was added oxone (potassium monopersulphate triple salt0.73 mmol) at 25 oC and the mixture was stirred for 3 hoursat the same temperature. Acetone was removed under lowvacuum and the residue was diluted with water (20 ml).Solid separated was filtered, washed with water (5 ml)followed by petroleum ether (5 ml) to give the required

product.

The title compound was synthesized from 4/-fluoroacetophenone using the two step procedure as describedabove: yield 20 %; mp 194197 oC; 1H NMR (200 MHz,CDCl3) 7.74 (d,J = 8.0 Hz, 1H), 7.53 (s, 1H), 7.20 (m,5H), 6.70 (s, 1H), 4.90 (bs, 1H, NH), 4.50 (d, J = 4.0 Hz,2H); IR (Nujol) cm.-1 1605, 1455; MS (CI, i-Butane) 398(M+1, 100); found C, 51.37; H, 3.09; N, 10.55;C17H11F4N3O2S requires C, 51.39; H, 2.79; N, 10.58 %. 5-[5-(4-Methylsulfonylphenyl)-3-Trifluoromethyl-1H-1-

Pyrazolyl]-1,1-Dioxo-2,3-Dihydro Benzo[d]isothiazole (5)5-[5-(3-Fluoro-4-Methylphenyl)-3-Trifluoromethyl-1H-1-Pyrazolyl]-1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazole (2) The title compound was synthesized from 4 using the

procedure as described above. Yield 67 %; mp 228 230 C1H NMR (200 MHz, CDCl3) 8.0 (d, J = 8.3 Hz, 2H)

7.78 (d,J= 8.3 Hz, 1H), 7.55 (s, 1H), 7.47 (d, J = 8.3 Hz2H), 7.33 (d,J = 9.7 Hz, 1H), 6.90 (s, 1H), 4.78 (bs, 1HNH), 4.54 (s, 2H, CH2), 3.12 (s, 3H, SO2CH3); IR (Nujol)cm.-11607,1496; MS (CI, i-Butane) 458 (M+1, 100); foundC, 47.37; H, 3.09; N, 9.05; C18H14F3N3O4S2 requires C47.26; H, 3.08; N, 9.19 %.

The title compound was synthesized from 3-fluoro-4-methylacetophenone using the two step procedure as

described above: yield 26 %; mp 175 176o

C,1

H NMR(200 MHz, CDCl3) 7.70 (d, J = 8.0 Hz, 1H), 7.53 (s,1H), 7.20 (m, 1H), 7.0 (s, 3H), 6.70 (s, 1H), 4.90 (bs, 1H,

NH), 4.50 (d,J= 4.0 Hz, 2H, CH2), 2.27 (s, 3H, CH3); IR(Nujol) cm.-1 1610; MS (CI, i-Butane) 412 (M+1, 100);found C, 52.37; H, 3.29; N, 10.15; C18H13F4N3O2Srequires C, 52.55; H, 3.19; N, 10.21 %.

5-[5-(3-Methyl-4-Methylsulfonylphenyl)-3-Trifluoromethyl-1H-1-Pyrazolyl]-1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazole(6)

5-[5-(4-Methoxyphenyl)-3-Trifluoromethyl-1H-1-Pyrazolyl]-1,1-Dioxo-2,3-Dihydro Benzo[d]isothiazole (3)

The title compound was synthesized from 4/-methoxyacetophenone using the two step procedure asdescribed above: yield 60 %; mp 75 77 oC; 1H NMR (200MHz, CDCl3) 7.70 (d, J = 8.0 Hz, 1H), 7.52 (s, 1H),

7.33 (d,J= 8.0 Hz, 1H), 7.14 (d, J= 9.0 Hz, 2H), 6.89 (d,J= 9.0 Hz, 2H), 6.7 (s, 1H), 4.98 (bs, 1H, NH), 4.49 (d, J= 5.0 Hz, 2H, CH2), 3.8 (s, 3H, OCH3); IR (Nujol) cm.-1

1610, 1470; MS (CI, i-Butane) 410 (M+1, 100); found C,52.67; H, 3.39; N, 10.35; C18H14F3N3O3S requires C,52.81; H, 3.45; N, 10.26 %.

The title compound was synthesized from 5-[5-(3-methyl-4-methylsulfanylphenyl)-3-trifluoromethyl-1H-1-

pyrazolyl]-1,1-dioxo-2,3-dihydrobenzo[d]isothiazole usingthe procedure as described above: yield 28 %; mp 134 136o

C,1

H NMR (200 MHz, CDCl3) 8.03 (d, J = 8.3 Hz1H), 7.76 (d,J = 8.3 Hz, 1H), 7.55 (s, 1H), 7.327.15 (m

3H), 6.86 (s, 1H), 4.92 (bs, 1H, NH), 4.54 (d, J = 4.8 Hz2H, CH2), 3.13 (s, 3H, SO2CH3), 2.72 (s, 3H, CH3); IR(Nujol) cm.-1 1608, 1481; MS (CI, i-Butane) 472 (M+1100), 407 (10); found C, 48.37; H, 3.37; N, 8.99C19H16F3N3O4S2requires C, 48.40; H, 3.42; N, 8.91 %.5-[5-(4-Methylsulfanylphenyl)-3-Trifluoromethyl-1H-1-

Pyrazolyl]-1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazole (4) 5-[5-(3-Methyl-4-Methylsulfanylphenyl)-3-Trifluoromethyl-1H-1-Pyrazolyl]-1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazoleThe title compound was synthesized from 4-

methylsulfanylacetophenone using the two step procedure asdescribed above: yield 62 %; mp 177-179 C, 1H NMR (200MHz, CDCl3) 7.74 (d, J = 8.3 Hz, 1H), 7.55 (s, 1H),

5-[5-(3-methyl-4-methylsulfanylphenyl)-3-trifluoromethyl1H-1-pyrazolyl]-1,1-dioxo-2,3-dihydrobenzo[d]isothiazolewas prepared from 3-methyl-4-methylsulfanylacetophenone


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according to the procedure described in step 1 & 2. Yield 50%; mp 159 160 oC, 1H NMR (200 MHz, CDCl3) 7.70(d,J= 8.3 Hz, 1H), 7.55 (s, 1H), 7.33 (d, J = 8.3 Hz, 1H),7.08 7.04 (m, 2H), 6.90 (d,J= 7.8 Hz, 1H), 6.73 (s, 1H),4.82 4.80 (m, 1H), 4.51 (d, J = 4.8 Hz, 2H, CH2), 2.47(s, 3H, SCH3), 2.29 (s, 3H, CH3); IR (Nujol) cm.-1 1606,1464; MS (CI, i-Butane) 440 (M+1, 100); found C, 51.89;H, 3.65; N, 9.81; C19H16F3N3O2S2 requires C, 51.93; H,

3.67; N, 9.56 %..

initiation of enzymatic reaction in the presence ocompound/vehicle. The reaction was initiated by theaddition of 100 M arachidonic acid and 120 M TMPDThe enzyme activity was measured by estimation of theinitial velocity of TMPD oxidation over the first 25 s of thereaction, followed by tracking the increase in absorbance at603 nM. The IC50 values were calculated using nonlinearregression analysis.

In Vivo Screening Methods. Carrageenan-Induced RatPaw Edema5-[5-(2-Fluoro-4-Methylsulfonylphenyl)-3-

Trifluoromethyl-1H-1-Pyrazolyl]-1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazole (7)

Male Wistar rats (120-140 g) were fasted for 16 h beforethe experiment. Compounds were suspended in 0.25%carboxymethylcellulose and administered orally in a volumeof 10 mL/kg 2 h before carrageenan injection. Paw edemawas induced in rats by intradermal injection of 50 L of 1%

-carrageenan in saline into the plantar surface of the righthind paw. Paw volume was measured before and 3 h aftercarrageenan injection by plethysmometer (Ugo-Basile, Italy).Paw edema was compared with the vehicle control group and

percent inhibition was calculated in comparison to thevehicle group.

The title compound was synthesized from 5-[5-(2-fluoro-4-methylsulfanylphenyl)-3-trifluoromethyl-1H-1-pyrazolyl]-1,1-dioxo-2,3-dihydrobenzo[ d]isothiazole using the

procedure as described above: yield 69 %; mp 230 232 oC,1H NMR (200 MHz, CDCl3): 7.86 7.59 (m, 5H), 7.34(d,J = 8.3 Hz, 1H), 7.0 (s, 1H), 4.45 (d, J = 3.9 Hz, 2H,CH2), 3.18 (s, 3H, SO2CH3); MS (CI, i-Butane) 476 (M+1,100); found C, 45.49; H, 2.75; N, 8.81; C18H13F4N3O4S2requires C, 45.47; H, 2.76; N, 8.84 %.

5-[5-(2-Fluoro-4-Methylsulfanylphenyl)-3-Trifluoromethyl-1H-1-Pyrazolyl]-1,1-Dioxo-2,3-

Dihydrobenzo[d]isothiazole

Molecular Modeling

Three-dimensional structure building and all modelingwere performed using the SYBYL Program package, version6.9 [14] on Silicon Graphics Octane 2 workstations with theIRIX 6.5 operating system. Energy minimizations were

performed using the Tripos force field with a distancedependent dielectric and the Powell conjugate gradientalgorithm with a convergence criterion of 0.05 kcal/ (mol )Partial atomic charges were calculated using GasteigerMarsili method. The co-crystal structure of murine apo-COX-2 (6COX) with SC-558 [13b] was used. A manualdocking procedure was applied and the obtained receptor-ligand complexes were optimized using above procedure

Different conformations and orientations of each ligandwithin the binding pocket were explored, and each time theligand-receptor complex was re-minimized. On the basis ofinteraction energy, one complex structure for each ligand wasselected and further analysis was carried out.

5-[5-(2-fluoro-4-methylsulfanylphenyl)-3-trifluoromethyl-1H-1-pyrazolyl]-1,1-dioxo-2,3-dihydrobenzo[d]isothiazolewas prepared from 2-fluoro-4-methylsulfanylacetophenoneaccording to the procedure described in step 1 & 2. yield 58%; mp 159 161oC, NMR (200 MHz, CDCl3): 7.71 (d, J= 8.3 Hz, 1H), 7.56 (s, 1H), 7.326.89 (m, 4H), 6.81 (s,1H), 4.94 (bs, 1H, NH), 4.52 (d, J = 3.4 Hz, 2H, CH2),2.50 (s, 3H, SCH3); MS (CI, i-Butane) 444 (M+1, 100);found C, 48.89; H, 2.95; N, 9.21; C18H13F4N3O2S2requires C, 48.75; H, 2.95; N, 9.48 %.

5-[3-Difluoromethyl-5-(2-Fluoro-4-Methylsulfonylphenyl)-1H-1-Pyrazolyl]-1,1-Dioxo-2,3-Dihydrobenzo[d]isothiazole(9)

The title compound was synthesized from 8 using theprocedure as described above: yield 35 %; mp >200 oC; 1HNMR (200 MHz, CDCl3) 7.78-7.34 (m, 6H), 6.93 (s,1H), 6.81 (t, J = 54.7 Hz, 1H, CHF2), 4.31 (s, 2H, CH2),3.09 (s, 3H, SO2CH3); MS (CI, i-Butane) 458 (M+1, 11),393 (100); found C, 47.29; H, 3.19; N, 8.95;C18H14F3N3O4S2requires C, 47.26; H, 3.08; N, 9.19 %.

ACKNOWLEDGEMENTS

The authors sincerely thank Dr. A. Venkateswarlu, DrR. Rajagopalan and Prof. J. Iqbal for their constantencouragement and the Analytical Department for spectralsupport.

Biological Assays. In Vitro Biochemical Assays

REFERENCESSpectrophotometric Assay of COX-1 and COX-2

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[8] (a) Barbey, S.; Goossens, L.; Taverne, T.; Cornet, J.; Choesmel,V.; Rouaud, C.; Gimeno, G.; Yannic-Arnoult, S.; Michaux, C.;Charlier, C.; Houssin, R.; Henichart, J.-P. Bioorg. Med. Chem.

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[13] (a) Soliva, R.; Almansa, C.; Kalko, S. G.; Luque, F. J.; Orozco, M.J. Med. Chem. 2003, 46, 1372-1382; (b) Kurumbail, R. G.;Stevens, A. M.; Gierse, J. K.; McDonald, J. J.; Stegeman, R. A.;Pak, J. Y.; Gildehaus, D.; Miyashiro, J. M.; Penning, T. D.; Seibert,K.; Isakson, P. C.; Stallings, W. C.Nature1996, 384, 644-648.

trifluoromethyl-1H-5-pyrazolyl]-1-benzenesulfonamide and 4-[1(4-methoxyphenyl)-3-trifluoromethyl-1H-5-pyrazolyl]-1-benzen-esulfonamide were identified as potent inhibitors of both COX-1and COX-2. See ref 3b; (b) Paulson, S. K.; Zhang, J. Y.; Breau, A.P.; Hribar, J. D.; Liu, N. W. K.; Jessen, S. M.; Lawal, Y. M.Cogburn, J. N.; Gresk, C. J.; Markos, C. S.; Maziasz, T. J.Schoenhard, G. L.; Burton, E. G. Drug Metabolism and

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[14] Molecular modeling was performed using the SYBYL prorogrampackage version 6.9. SYBYL, version 6.9 Molecular ModellingSoftware; Tripos Associates Inc.: 1699 South Hanley Road, St.Louis, MO 6314. The molecule 7 was sketched and minimizedusing the Tripos force field. A co-crystal structure of murine apo-

COX-2 (6COX) with SC-55812was used for docking.

[17] (a) Giri, S.; Nizamuddin, Gupta, H. C.J. Indian Chem. Soc.198259, 365-366; (b) Huntress, E. H.; Carten, F. H. J. Am. Chem. Soc

1940, 62,511-514; (c) Lombardino, J. G. J. Org. Chem.1971, 361843-1845.[15] (a) A recent study has shown that the recognition site of COX-2 isvery flexible and can adopt its structure to very subtle structuralchanges in drug. See ref 13a. This perhaps offer an alternativeexplanation for the insertion of a relatively large and bicyclic 1,1-dioxo-2,3-dihydrobenzo[d]isothiazolyl group into the COX-2

pocket near Val523; (b) Price, M. L. P.; Jorgensen, W. L. J. Am.Chem. Soc. 2000, 122,9455-9466.

[18] (a) Hemler, M.; Lands, W. E. J. Biol. Chem. 1976, 251, 5575-5579; (b) Cromlish, W. A.; Payette, P.; Culp, S. A.; Ouellet, M.Percival, M. D.; Kennedy, B. P. Arch. Biochem. Biophys. 1994314, 193-199; (c) Copeland, R. A.; Williams, J. M.; Giannaras, J.

Nurnberg, S.; Covington, M.; Pinto, D.; Pick, S.; Trzakos, J. MProc. Natl. Acad. Sci. USA 1994,91,11202-11206.

[16] (a) A similar observation was noted during SAR studies on 1,5-diaryl pyrazoles earlier. For example 4-[1-(4-fluorophenyl)-3-


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Preparation of CF31,3 Diones

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