Indian Journal of Chemistry Vol. 38B, September 1999, pp. 1075-1085

Synthesis of novel 1,2-(un) substituted-3-amino-5-aryl-6-arylaminopyrazolo [3,4-d]pyrimidin-4(5H)-ones and their biological activities

C J Shishooa*, T Ravikumar at , K S Jaina, T S Rathod", T P Gandhib & M C Satiab

"Department of Pharmaceutical Chemistry, L. M. College of Pharmacy, Navrangpura, Ahmedabad 380009, India. bDepartment of Pharmacology, Cadil a Pharmaceuticals, Maninagar, Ahmedabad 380008, India

Received 5 December 1998; accepted (revised) 3 May 1999

A series of novel 1,2-(un)substituted-3-amino-5-aryl-6-arylaminopyrazolo[3A-dl pyrimidin-4(5H)-ones 5-7 has been synthesised th rough the cyclocondensation of the corresponding, hitherto unreported 3-aryl-2-arylamino-5-cyano-6-methylsulfonylpyrimidin-4-ones 3 or 4 with hydrazine hydrate or substituted hydrazines. Compounds 6a and 7a exhibited analgesi c activity superior to that of the standards, aspirin & pentazocine. These compounds also exhibited signifi cant anti­inf1ammatory activ ity comparable to that of dicl ofenac sodium. The free radical scavenging activity, as well as, the ulcerogenic potential of some of the acti ve compounds in the series was also determined.

Most of the widely used non-steroidal analgesic and anti-inflammatory drugs (NSAIDs) are acidic in nature. However, their therapeutic potential is limited due to the frequently observed gastro-intestinal side effects assoc iated with them. There is a need to develop a non acidic, non steroidal analgesic and anti­inflammatory drug. The side effects of the acidic NS AIDs are the result, at least in part, of a direc t contact mechanism, whi ch is the combination of the local irritation produced by the acidic group in the molecule and the local inh ibi tion of the cytoprotective ac tions of prostaglandi ns on the gastric mucosa I .

Whi le acidic NSAIDs have higher tendency to accummulate in the stomach ' wall soon after oral absorption2

, certain basic NSAIDs have been shown to accumulate in inflammatory exudates3

. Thus, basic NSAID compounds are gaining attention due to their potentially favourable physico-chemical properties.

Among various NSAIDs and non-narcotic analgesics, notable are the pyrazolone de rivatives, namely antipyrine, aminopyrine, pheny lbutazone, oxyphenbutazone and analgin . Thus the pyrazole ring system seems to be a potential pharmacophore for analgesic and anti-inflam matory actlv ltles. Pyrazolopyrimidine, al\opuriniol and its congeners oxypurinol and th iopurinol , inhibit the enzyme, xanthine oxidase and interfere in the biosynthesis of uric acid , the causative agent of gout4

. Of the various

t Present address: K.B.lnstilute of Pharmaceutical Education & Research, Gandhinagar 382023 ,lndia

pyrazolopyrimidines , the pyrazolo[3 ,4-d]pyri mi dines are of special interest, as a wide range of biological actIVIties, particularly, analgesic and anti ­inflammatory activities have been exhibi ted by them5

-11

Earlier, we have reported the synthesis of condensed pyri midin-4-ones 1 for potent analgesic and anti-inflammatory ac tivities l2

.13 Recently a series

of these dervatives has been synthesized and evaluated for the ir biological activities 14. The lead compounds in this series, exhibited analges ic activity superior and comparable to aspi rin and an ti­inflammatory activ ity comparable La dic lofenac sodium, when evaluated in rodents. In recent years a few reports, mainly by Japanese workers have appeared in the literature on the synthesis and analgesic and/or anti-inflammatory activities of some basic, 3-substituted-aminopyrazolo[3 ,4-dJpyrimidin-4-ones 25

-7

• We now report the synthesis and evaluati on of some novel 1,2-(un) substi tuted-3-amino-5-ary l-6-arylaminopyrazolo-[3,4-d]pyrimidin-4(5H)-ones (5, 6 and 7) fo r analges ic and anti­inflammatory acti vities.

1076 INDIAN J. CHEM. SEC. B, SEPTEMBER 1999

Reactive oxygen species are implicated in the induction and prolongation of the inflammatory process l5

. A number of therapeutically useful NSATDs have been shown to act by virtue of their free radical scavenging properties 19-23. Free radicals are implicated in a variety of degenerative processes like arthritis inflammation, liver damage, as well as cardiovascular effects 16-21. The very fact that superoxide dismutase has been observed to suppress symptomatically many inflammation pathologies, demonstrates the involvement of free radicals in inflammation. Nowadays, free radical scavenger activity is used to assess the potential of many anti-

inflammatory compounds. 1,1-Dipheny 1-2-picry 1-hydrazine (DPPH*) is used as a free radical producing agent24. Free radical scavengers if added to the solution of DPPH*, exert their antioxidant effect and reduce DPPH*, which results in the reduction of the opticaf density of the DPPH* solution at Amax 516 nm. In the present study we have also investigated the free radical scavenging properties of the title compounds.

Chemistry The title compounds 5-7 have been synthesised

through the cyc1ocondensation of the corresponding 3-aryl-2-arylamino-5-cyanopyrimidin-4-ones (3 and

R-©lOO CN

~I rAYN SCH3

R~H

R-fA 0 ~~CN

~~~SO,cH, 3

lNH~H2> H:zO DMF, anhyd. K2C03

5

R, NHNH2> DMF anhyd. K~03

Scheme I

2a - 9

Scheme II

4

1

3a - 9

....

SHISHOO el al.: PYRAZOLO[3,4-djPYRIMlDINES & THEIR BIOLOGICAL ACTIVITIES 1077

4) with hydrazine and substituted hydrazines Scheme I.

The intermediates, 3-aryl-2-arylamino-5-cyano-6-methylthiopyrimidin-4-ones 3 (Scheme II) were synthesized by the condensation of equimolar quantities of a-cyanoketene S,N-acetal 1 and the corresponding S-methyl diarylisothioureas 2a-gin dimethylformamide in the presence of catalytic amount of anhydrous potassium carbonate under reflux. The starting materials a-cyanoketene S,N­acetal 1 and S-methyl diarylisothioureas 2a-g were prepared by the reported literature procedures25

•26

.

While the 6-methylthio group readily undergoes the nucleophilic displacement with hydrazine, it failed to do so with substituted hydrazines, namely, methyl and phenylhydrazine. Therefore, the 6-methylthio group was oxidised with H20 2 / acetic acid to the corresponding 6-methylsulfonyl group, a better leaving group that could be readily displaced with these substituted hydrazines (Scheme III).

The structural assignments for the target compounds S-7 and their intermediates 3 and 4 are based on spectral (JR, mass and PMR) and analytical data. The physical and spectral properties are shown in the Tables I-V.

Analgesic activity

(a) Writhing method27• Writhing was induced through

intraperitoneal administration of acetic acid into swiss albino mice. Two of the compounds 6a and 7a, when given by oral route, exhibited better protection to the animals from .the writhes, compared to aspirin (reference standard), while compound 6c was found to be equipotent to aspirin Table VI. (b) Hot Plate Method28

• The title compounds were also evaluated for the analgesic activity by hot plate method. Many of the compounds exhibited considerable analgesic activity comparable to the centrally acting analgesic drug, pentazocine (reference standard). Again, compounds, 6a and 7a, along with the compounds 6c, 7e and 7f, were found to be the most potent of all (Table VII).

Anti-inflammatory activity The title compounds were also screened for anti­

inflammatory actIvItIes in albino wistar rats, employing the formalin induced rat paw edema test model. Percentage reduction in the inflammation (i.e., reduction in the hind paw edema volume of the animals) after 3 hrs of administration of formalin and the test compounds was compared with that of the

o ~A 0 ~A :iN tWiAcOH 'YN ",CS ~"UA ~)l~N'OA

3a-9 4a-9

R = a,-H; b. 2-CH3; C, 4-CH3; d. 4-OCH3; e. 4-0C2H5; f. 4-CI; g. 2,4-di CH3

Scheme III

animals administered with formalin and the reference standard, diclofenac sodium. The compounds, 6a, 6f, Sc, 7a and Sf, show significant anti-inflammatory activity in the same rank order and is comparable to that of the reference standard, diclofenac sodium (Table VIII).

Ulcerogenic potential2~ The test compounds, 6a and 7a, showed lower

ulcer indices (0.0 at dose of 400 mg, p.o.) compared to the reference stancfard, aspirin (0.35 at same dose). Also two other compounds, . 5a and Se, have lower ulcer indices than aspirin, viz., 0.2 and 0.25 respectively.

Free radical scavenging activity

All the title compounds synthesized revealed some free radical scavenging activi ty30, as seen through their ability to reduce the methanolic coloured solution of 1,I-diphenyl-2-picryl hydrazide (DPPH*) leading to the reduction in its O.D. at "'max 516 nm. Compounds 7e and 7f, possess the most significant activity of all the compounds tested ( Table IX).

Discussion

The analgesic and in vivo anti-inflammatory activities of the synthesized title compounds were evaluated by using the functional models of acetic acid induced writhings in mice and by hot-plate method and formalin induced inflammation in the rat. These models represent the responses of clinically observed pain and inflammation diseases and are reliable predictors of the analgesic and anti­inflammatory efficacy of the NSAIDs .

The 1,2-substituted (methyl/phenyl) compounds showed significant reduction in writhings as well as significan t delay in response to temperature (Table VI and VII), suggesting significant analgesic activity. However, 1,2-unsubstituted ti tle compounds showed lesser analgesic activity as compared to 1,2-substituted compounds. .

1078 INDIAN J. CHEM . SEC. B, SEPTEMBER 1999

Table I- Physical and spectra l characte ristics of 3-ary l-2-ary lamino-5 -cyano-6-methyl thiopyri midin-4-ones 3a-g

Compd R Mol. fo rmula* mpoC Spectra l data (Mol Wt) (%Yield)b

3a -H CIsHI4N40S 248-51 UV(CH2C12) :235.6n m (334.39) (95) IH NMR(OMSO-d6+OzO):8 .9-9.I(s, IH ,NH, 0 20 exchangeable) 7.3 -7.8

(m , IOH, Ar-H), 2.5(s,3H,S-CH3)' MS: m/z 334(M+):; 238,2 18, 182,147,105,77,67,55.

3b 2-CH) CZOHI8N40S 270-72 UV(CHzClz) : 235 .6nm (362.434) (90)

3c 4-CH) C2oHI SN40S 258-60 UV (CH 2C11) : 234.8 nm (362.434) (92) I H NMR(DMSO-d6+OZO) : 8.8-9.0(5,1 H,NH,020 exchangeable), 7.2-7 .5

(m,8H,Ar-H)2.4-2.6(d,6H,ArCH3),2.3(s,3 H,S-CH3)·

MS: (m/z) : 362(M+); 357,332,246,175,136, 128,118,106,91 ,65.

3d 4-CH)0 C"ol-I l sN4O)S 302-05 UV(CH2Ci2) : 236.2n1l1 (394.446) (9-+) IH NMR(OMSO-do+020): 8.8-S.9(s,IH,NI,', 0 20 exchangeJble),6.9-

7.7(m,8H, Ar-H ),3 .8-4.1 (d ,6H,Ar-OCHJ),2.4(s,3H,S-CHJ) .

3e 4-Czl-IsO Cn H2"N4O.lS 236-40 UV(C I-I2CI2) : 236.6nm (422.494) (86) IH NMR(COCi3+OMSO-cl6) : 7.8-7.9(s, I H,NH),6 .7-7 .3(m,S H, A r-H ),3 .8-

4.2(1I1,4H,-CH2-), 2.3(s,3H,S-Clh), 1.2-1 .6(m,6I-I ,-CHz-CHJ). MS: (m/z) : 422(M+);393,2S3,258, 189, 136,258.1 89, 136, IOS,75.

3f · ·CI C ,sHI2C1 2N4OS 271-74 UV(CH2CI2) : 238.3 nll1 (403 .:>8) (90)

3g 2,4-t1iCH3 C22H2:N j OS 252-56 UV(CI 11C12) : 236.8 n111 (422.4(/ ) (83) 'Ii NMR(OMSO-d6): 6.8-7 .S(m,7H, Ar-H & NH),2.6 (s,3 1-I ,S-CH3) .. 2.1-

2.5(m, 12H,A r-CHJ)' MS: (m/z) : 390(. t); 375,270,25 1,195 ,1 30, 121, 111,9 1,77.

\atisf:lctory micro anJ lysis ~±n.4% of C,I I) was ohtJinetl fo r at! compounds b All compounds were recrystall isetl from ethanol-ch lorofo rm

The compounds 6a and 7a exhibited signi fican tly higher activity than the aspi rin at p< 0.05 aild 6b,6c,7c,7d and 7f are compar;1tive to aspirin. Thus our data indicates that th substit ution at 1 or 2 posit ion of the pyrazole ring increases the analgesic activi ty.

Similarly, substitution on ,he 5-aryl or 6-ary lamillo rings showed less significant effect on the analgesic activity.

Most of the title compounds showed good analgesic ac tivity by hot plate method and are comparable to the centrally act ing analgesic drug like pentazocine.

O ne of the major dra wback of NSAIDs is gastro­intestinal side effects, particul arly gastrointestinal ulcers. Interest ingly, the lead compounds, 6a and 7a, showed '0 ' u lcer index at dose of 400 mg/kg co mpared to the reference. aspirin (ulcer index 0.35 at same dose).

In rar paw oedema model for ant i-inflammatory 3ctivity the compounds 6a and (if showed significantiy higher activity than the reference and Se, Sf and 7a are showed comparable activities to reference diclofenac sodium.

Free rad icals playa dual role in the promotion of inflammation, through the activation of phagocytes as well as activation of cyclooxygenase. Therefore, the

..

SHISHOO el at.: PYRAZOLO[3,4-djPYRIMIDINES & THEIR BIOLOGICAL ACTIVITIES 1079

TabIe-U-Physical and Spectral characteristics of 3 -aryl-2-arylamino-5-cyano-6-mcthylsulfonyipyrimidin-4-ones 4a-g

-Compd R Mol. formulaa mpoC Spectral data (Mol Wt) (%Yield)b

4a -H ClsHI4N40)S 262-64 UV(CH2Cl;) : 248.2nm (366.38) (70) IH NMR(DMSO-d6 +D20 )6.7-7.7(m,IIH, Ar-H & NH),2 .8(s,3H,S02CH)).

MS: (mlz) : 366(M+):; 334,304, 195,182, \68,147,105,92,77.

4b 2-CH) C20H 18N40 )S 236·42 UV(CH2CI2) : 24S.8nm (394.43) (68)

4c 4-CH) C20H 18N40 3S 248·52 UV(CH2Cl2) : 249.4 nm (394.43) (68) IH NMR(DMS O-d6 +D20 ) : 8.5-S.7(s, I H,NH, D20 exchangeable),6.9-7.5

(m,SH,A r- H) 3. I (s,3H,S02CH) ,2.3-2.5(d,6H ,Ar·CH) . MS: (mJz) : 394(M+ ); 362,223,182, 165,147,119,105,91 ,77,65.

4d 4-CH)O C2oHISN"OsS 248-50 UV(CH~CI 2) : 248.5nm (426.43) (65) IH NM R(DMSO-d6 +D20 ) : 9.2-9.3(s,lH,NH, D20 exchangeab1e),7.0-

7.5(m,8H, Ar-H),3.1-3.5(d,6H,Ar-OCH), 2.8 (s,3H, S02CH]) . MS: (mJz) : 42(i(M+):394,379,300,272,255,239, 197, 175, 147, 122, 108,77,75.

4e 4-C2HsO C22H22N40 SS 236-40 UV(CH2CI2) : 249.6nm (454.49) (55)

4f 4-CI ClsH 12C12N. 0 3S 255-58 UV(CH 2Cl2) : 251.2nm (435 .24) (62)

4g 2.4-diCH) Cn Hn N4O)S 205- 10 UV(CH2CI2): 247.lnm (408.3) (56)

'Satisfactory micro analysis (±O,4% C,H) was obtained for all com pounds . bAil compounds were recrys tall ised frolll ethanol·C'hbroform

scavengers of free radical s have a protecti ve effec t against inflammation. The tit le compounds when tested :'or scavenging activity o f free rad ical (DPPH*) revealed some free rarlical scavenging activ ity .

In conclusion the title series of compounds has a pot ntial of offering a nonacidic analges ic NSAID candidate. Compou nds, Sc, Sf, 6a, 6f and 7a have significant anti-infbmmatory activi ty and 6a and 7a, exhibit better activities than the res t in the series . 7e and 7f have high free rad ical activity also.

Experimental Section

Melting points were determi ned in open capi llaries and are uncorrected. The IR spectra were recorded in potassium bromide on a Perkin-Elmer 837, grating spectrophotometer, 'H NMR spectra on a Varian A-60 spectrometer using TMS as an internal standard and the mass spectra on a Varian Atlas CH-7 spectrometer at 70e V ionising beam using direct insertion probe.

Satisfactory microanalysi s (± 0.4% of the calculated val ues) was obtained for all the compounds.

Synthesis of 3-aryl-2arylamino-S-cyano-6-thiomethylpyrimidin-4(J-l)-ones 3a-g. General procedure. Ar. equimolar mi xture of 2-carbethoxy-2-cyano-l-amino- l-methylth ioacrylonitrile (lS.6g, 0.1 mole) and an appropri ate S-methyl diarylisothiourea 2a-g in dimethylformamide containing catalytic amount of potassiu m carbonate was retluxed for 4-5 hr. The reaction mixture was then cooled to room temperature and poured on ice- water mixture (50 mL). The solid obtai ned was fil tered, washed, and dried and crystnll ised from a suitable sol vent (Table I) .

Hydrogen peroxide oxida tion of 3-aryl-2-arylamino-5-cyano-6-methylthiopyrimidin-4-oncs to 3-aryl-2-arylamino-5-cyano-6-methylsulphonyl­pyrimidin-4-oncs 4a-g. General procedure. To a suspension of the appropriate 3-aryl-2-arylamino-5-

1080 INDIAN J. CHEM. SEC. B, SEPTEMBER 1999

Table 111- Physical and spectral characteristics of 3-amino-5-aryl-6-arylaminopyrazolo[3,4-dJpyrimidin-4(5H)-ones Sa-g

Compd R Mol. formula' (Mol Wt)

Sa -H C 17H'4N60 (318.33)

Sb 2-CH3 C'9H,sN6O (346.38)

Sc 4-CH3 C'9H,sN6O (346.38)

Sd 4-0CH3 C'9H,sN60 3

(378.38)

Se 4-0C2HS C2,H22N60 3 (406.44)

Sf 4-CI C17H12CI2N60 (387.23)

Sg 2,4-diCHJ C2,H22N60 (374.44)

mpoCb

(% Yield)

273-75 (62)

170-72 (61)

170-73 (58)

162-65 (62)

240-42 (56)

180-82 (60)

208-12 (53)

Spectral data

UV(CH2CI2) 238.4nm 'H NMR(DMSO-d6 +020) : 6.8-7.7 (m,ll H, Ar-H & N-NH),6.1-6.5 (s,IH,NH, 0 20 exchangeable),4.5-5.0(s,2H,NH2, 0 20 exchangeable). MS: (mlz): 318(M+); 239,226,194,182,147,132,105,77,67,55.

UV(CH2CI2) : 239.2nm 'H NMR(DMSO-d6): 7.2-7.6(m,9H,Ar-H & N-NH), 6.8-6.9(s,IH,NH), 5.1-5.4(s,2H,NH2),2.3-2.7(d,6I-i,Ar-CH3)

MS (mlz) : 346(M+); 240,222,173,131,107,91,65,46.

UV(CH2CI2) : 240nm 'H NMR(DMSO-d6 +D20) : 7.0-7.7(m,9H,Ar-H & N-NH), 6.7-6.8(s,lH,NH, 0 20 exchangeable), 5.0-5.3(s, 2H,NH2, D20 exchangeable),2.3-2.7(d,6H,Ar-CHJ) MS (mlz): 346(M+); 240,222,173,131,124,117,106,91,65

UV(CH2Ch) : 239.4nm 'H NMR(DMSO-d6 +D20): 7.0-7.6(m,9H,Ar-H &N-NH), 6.9-7.0(s,lH,NH, 0 20 exchangeable),5.1-5.4(s,2H,NH2, 0 20 exchangeable), 3.8-4.0(d,6H,Ar-OCHJ)' MS (mlz): 378(M+); 256,195,147,135,121,108,91,78.

UV(CH2CI2) : 240.2nm 'H NMR(DMSO-d6+D20): 6.6-7.3(9H,Ar-H & N-NH), 4.9-5. I (s,2H,NH2,

0 20 exchangeable), 3.3-4.2 (m,4H,-CHr ), I.I-I.5(m,6H,-CH2-CHJ) MS (mlz): 406(M+); 377,270,242,186,137,109,77,65.

UV(CH2Ci2) : 242.3nm 'H NMR(DMSO-~+ 0 20) : 7.3-8.4(m,9H,Ar-H & NH), 5.0-5.6(s,2H,NH2 0 20 exchangeable)

UV(CH2Ci2) : 239.4nm

, Satisfactory micro analysis (±0.4% of C,H) was obtained for all compounds. b All compounds were recrystallised from ethanol-chloroform.

cyano-6-methylthiopyrimidin-4-one 3a-g (0.01 mole) in 20-30 mL glacial acetic acid, hydrogen peroxide (15-20 mL 30% v/v) was added dropwise with stirring at 60°C for 30 min. The reaction mixture was further stirred at 60°C for an additional one hour. After allowing to stand at room temperature for 15-20 minutes, the clear solution obtained was poured into ice-water mixture (30 mL). The solid obtained was filtered, washed with water, dried and crystallised from a suitable solvent (Table II).

1,2-Unsubstituted-3-amino-5-aryl-6-arylamino­pyrazolo[3,4-d]pyrimidin-4(5H)-ones Sa-g. General

procedure. To a solution of the appropriate 3-aryl-2-arylamino-5-cyano-6-methylthiopyrimidin-4-ones(3a­g, 0.01 mole) in dimethylformamide, hydrazine hydrate(0.03 mole) . was added and the reaction mixture was refluxed in the prescence of catalytic amount of anhydrolls potassium carbonate for 7--8hrs. After the reaction was complete(t1c), reaction mixture was poured on ice-water (50mL). The solid obtained was filtered, washed with water, dried and recrystallised from a suitable sol vet (Table III).

I-Methyl and 2-phenyl-3-amino-5-aryl-6-amino­pyrazolo[3,4-d]pyr imidin-4(5H)-oQes 6a-g and 7a -

SHISHOO et al.: PYRAZOLO[3,4-dlPYRIMIDINES & THEIR BIOLOGICAL ACTIVITtES 1081

Table IV-Physical and spectral data of I-methyl -3-amino-5-aryl-6-arylaminopyrazolo[3,4-dJpyrimidin-4(5H)-ones 6a-g

Compd R

6a -H

6b 2-CHJ

6c 4-CH3

6d 4-CHJO

6e 4-C2H50

6f 4-CI

6g 2,4-diCHJ

Mol. formulaa (Mol wt)

CI S HI6N60 (332.358)

C2oH20N60 (360.41)

C2oH20N60 (360.41 )

C2oH20N60 J (391.40)

C22H24N60 3 (420.46)

C IsH I4 CI2N6O (401.39)

C22H24N60 (388.46)

mpoC (%Yield,b

238-40 (72)

264-68 (71)

245-47 (74)

298-302 (69)

230-34 (63)

198-202 (65)

257-60 (55)

Spectral data

UV(CH2CI2) : 257nm IH NMR(DMSO-d6): 6.9-7.7 (m,IIH, Ar-H & NH),5 .0-5.3, (s,2H,NH2), 3.3-3.7(s,3H,N-CHJ)' MS: (mJz): 332(M+):;331,24I,240,166,138,119,103,9I,77,65.

UV(CH2CI2) : 258.2nm MS: (mJz) : 360(M+); 254,138,116,51,77, 65.

UV(CH2Cl2) : 257.4nm IH NMR(DMSO-d6): 6.7-7.3(m,9H,Ar-H & NH), 4.8-5. I (s,2H,NH2), 3.4-4.0(s, 3H,N-CHJ), 1.8-2.3(d,6H,Ar-CHJ) MS: (mJz): 360(M+); 345, 254,180,155,138,131,117,106,91 , 77,65.

UV(CH2CI2) : 257.8nm IH NMR(DMSO-~) : 7.0-7 .7(m,8H.Ar-H), 6.8-6.90(s,1 H,NH ),5.0-5.3(s,2H,NH2), 3.8-4.0(d,6H,Ar-OCHJ)' 3.4-3.7(s,3H,N-CHJ)' MS: (mJz): 391(M+); 270,255,149,105,91,77,57.

UV(CH2CI2) : 257.4nm MS: (mJz): 420(M+); 391,284,256,210,186,149.

UV(CH2Cl2) : 259.5nm : IH NMR(DMSO-d6) : 6.8-7.8(m,9H,Ar-H & NH), 5.0-5.2(s,2H,NH2)3.0-3.2(s, 3H,N-CHJ)'

UV(CH2Ch) : 257.4nm

a Satisfactory micro analysis (±O.4% of C,H) was obtained for all compounds b All compounds were recrystallised from ethanol-chloroform

g. G~neral procedure. To a solution of the appropriate 3-aryl - 2 - arylamino - 5-cyano-6-methyl-sulphonyl-pyrimidin-4-one (4a-g, 0.01 mol} in dimethylformamide; methylhydrazine/phenylhydra­zine (0.02 mole/O.03 mole, respectively) was added and the reaction mixture was refluxed in the prescence of catalytic amount of anhydrous potassium carbonate for 10-12 hrs (TLC). The reaction mixture was thereafter poured into an ice-water mixture (50mL). The solid thus obtained was filtered, washed with dil.HCI (5 % v/v) solution and with water, dried and crystallised from a suitable solvent (Table IV and V).

Pharmacological methods

General conditions of experimental animals. The experiments were carried out using laboratory animals

such as albino mice (swiss strain) and albino rats (wi star strain). Inhouse breeding of these animals was carried out at the department of pharmacology,Cadila Pharmaceuticals Ltd.,Ahmedabad (India). The animals were hOllsed at 24,+ 1°C and humidity of 50-70 % with 14h light and lOh dark cycles. The animals were given food (The feed was prepared instantly by mixing wheat 80%, gram 15%, mineral oil 2%, milk powder 2.5%, salt 0.5% and boiled to cook) and tap water ad libitum, unless specified in any particular method.

Analgesic activity

(a) Writhing Method27 .• Five groups of swiss albino

wistar mice were adminstered orally the test compound or the aspirin (300mg/Kg). Sixty minutes later 0.3mL of 0.6% (v/v) of acetic acid was

1082 INDIAN J. CHEM. SEC. B, SEPTEMBER 1999

TauJc V -- Physical and spectral data of 2-phenyl -3-amino-5-aryl-6-arylaminopyrazolo[3 ,4-dJpyrimidin-4(5H)-ones 7a-g

~~~ N)"N "N,,©

R~H Compd R Mol. formulaa mp oC Spectral data

(Mol wt) (%Yield)b

7a -H C2)H 1SN6O 232-36 UV(CH2CI2) : 264.6nm (394.22) (70) IH NMR(DMSO-J6+D20): 7.3-7.8 (111 , !5H, Ar-H), 7.2-7.3(s, IH,NH,

D20 exchangeable),6.4-6.6(s,2H, Nfh D20 exchangeable). MS: (rnJz): 394(W): ; 303,182, 14 9, 119, 105,91 , 77,65 ,57.

7b 2-CH3 C25H22N~0 266-68 UV(CI-I l Cl2) : 265.8nm (422.48) (75)

7c 4-CH) C25H22N60 258-60 UV(CH2Cl 2) ~66.1 nm (422.48) (74) IH NMR(DMSO-d6): 6.8-7.8(m,14H,Ar-H & NH), 5.7-5.8(s, 2H,NHz),

3.4-3.6(d,6H,Ar-CH)

7d 4-CH)0 C25H22N60 3 310-13 UV(CH2CI2) : 266.5nm (454.48) (71 ) IH NMR(DMSO-d6): 6.6-7.3(m,14H,Ar-H & NH), 5.0-5.2(s,2H,NHz),

3.6-3 .9(d,6H,Ar-OCH3)· MS: (mlz) : 454(M+); 440,348, 333,325,226, 202, 123, 108,93,77

7e 4-C2H50 C27 H26N6O) 240-43 lJV(CH2CI2) : 267.3nl11 (482 .53) (56) MS : (mlz) : 482(M+); 462,354,348, 154, 106,95,77.

7f 4-Cl C2J H16 CI2N6O 182-84 UV(CH2Clz) : 273.3nm (463 .32) (68)

7g 2,4-diCH) C27 i-lz6N6O 265-68 UV(C1-l2CI2) : 268 .4nm (436.49) (58) MS: (mlz) : 436(M+); 368,346,1 89,165,108,95,78 ,68.

3 Sati sfac to ry micro analysis(±O.4% C,H) was obtained for all compounds b Ail cumpou nds were recrystail ised from ethanol-chl oroform

adrninstered intraperitoneally and after 5 min the number of writhings were counted for each animal for the next 10 min . The results were then expressed as percen t inhibition in number of wri things from the control g roup (Table VI).

(b) H OI Plate Method28. The test compounds or

pentazoci ne prepared in 0.1 % (w/v) CMC was adminslered to swiss albino wister mice. One hour later the animals were placed on a hot plate mai n! ai ned at :''is.O+O.5(\C and time for paw licking were recorded. Each group served as its own control.

Calc ulation of activity at each individual dose was done with respect to the standard, us ing the follow ing formula (Table VII).

% Activ ity =

Mean latency peri od (drug treated)

Mean latency period (std. treated)

Mean latency period (before administering drug)

Mean latency period (before administering std.)

where, drug=test compou nd, std=pentazocine.

lJlcerogenic potential

The acute ulcerogen ic potent ial of the compounds Sa, Se, 6a and 7a were measurt:d by the method of Ganguly el a1. 29

• Aspirin (400 mg/kg) and the compounds (400mg/k g) were suspended in 1 % aqueous carbethoxymethy l cellulose sol ution by keeping the adminstrating volume constant i.e 0.5 mL and were adminstered oral ly to valious groups of albino wistar mice (24 hr fasted) each containing 5 ani mals of either sex . Four hours later the animals were sacrified. The stomachs were removed and opened along the greater curvature to determine the ulcer index. The ulcer index was calculated by using the equation:

10 Ulcer Index = -;

X

SHISHOO et al.: PYRAZOLO[3,4-d]PYRIMIDINES & THEIR BIOLOGICAL ACTIVITIES 1083

Table VI- Analgesic activity (writhing method)

Mean No.of % Reduction in No.of Writhings + SEM Writhings from control

Sa 18.00+01.27" 19.64

Sb 18.00+01.10" 19.65

Sc 11.75+00.90" 47.55*

Sd 13.00+01.20" 41.96*

Se 14.3 +02.40 36.20*

Sf 19.40+ 13.40" 13.40

Sg 15.28+01.20" 32. 15*

Aspirin 05.94 ± 0.83 73.50* *

Control 22.40±0.87

6a 00.75+00.70" 97.02 c **

6b 12.20+00.70 51.59*

6c 09.80+01.60 61.11*

6d 20.20+02.40" 19.84

6e 20.11 +02.10" 20.25

6f 13.50+01.40 46.40*

6g 13.76+00.95" 45.40*

Aspirin 8.60±0.67 65.67**

Control 25.20±1.04

7a 05.80+00.50 73.15**

7b 13.20+00.50" 38.89*

7c 09.40+00.50 56.48*

7d 10.00+01.50 53.70*

7e 11.77+01.21 45.50*

7f 10.80+00.80 50.00*

7g 13.28+01.12" 38.50*

Aspirin 7.60±0.73 64.80*

Control 21.60±1.28

a Compounds were dosed at dose levels of 300mg/kg,p.o. n = 5 albino wistar mice bNo Mortality was observed upto 1.5g/kg

c ED50 = (144.54) mg * Significantly different from control at p < 0.05. ** Significantly different from control at p < 0.00 I. " Significantly different from aspirin at p < 0.05.

h X Total Stomach Area (in .mm)

were = - --- -------'-----'-Total Lesion Area (in. mm)

Anti-inflammatory activity

Anti-inflammatory effect of each compound was measured simultaneously against run controls using the method of Turner3o. Groups of four albino wistar rats weighing between 200-250 gms fasted for 24hr,

Table VII- Analgesic activity (by hot plate method)

Compd ED50 in mg/Kg

Sa 05.76

Sb 05.75

Sc 01.58

Sd 02.60

Se 09.70

Sf 04.49

Sg 04.83

6a 01.20

6b 09.90

6c 00.51

6d 18.95

6e 10.33

6f 06.56

6g 14.72

7a 00.63

7b 15.76

7c 01.07

7d 19.30

7e 00.66

7f 00.72

7g 07.59

Compounds were dosed at dose levels of 10 mg/kg, p.o. ; n = 5 albino wistar mice

were dosed lOmg/kg orally with the test title compounds and reference dicIofenac sodium lh before injection of O.lrnL of 3.5% v/v formalin into the subplantar region of the hind paw. All the suspensions for oral dose were prepared in 1 rnL propyleneglycol containing O.lrnL of Tween-80 and adminstered in constant volume, i.e., 0.5 rnL/rat. Paw volume measured immediately and for each hour upto 3h by plethysomograph. The change in the paw volume was compared with that in vehicle (l rnL propylene glycol contain ing O. l rnL of Tween 80, 0.5mLlrat) treated control animals and was expressed as % odema inhibition using the formula (Table VIII).

% Protection = (lOO-(lOO/C X D)

where C = increase in paw volume in control group; D = increase in paw volume in treated group.

Free radical scavenging activity31

To determine the reducing activity of the stable radical 1, l-dipheny l-2-picrylhydrazyl (DPPH*), a

1084 INDIAN 1. CHEM. SEC. B, SEPTEMBER 1999

Table VIn - Anti-inflammatory activity

Compda In itial Mean Paw Final Mean Paw % Reduction in Volume ±SEM Volume ±SEM (after 3hr) Inflammation

(after 3 hr)

Sa 16.138+0.715 19.762+0.656 20.15

Sb 18.725 +0.545 21.700+ 0.789 15.15

Sc 16.125+ 1.560 18.200+ 1.575 51.84

Sd 16.625 + 1.463 20.450+ 2.180 11 .30

Se 16.950+0.940 19.250+0.904 14.80

Sf 15.833+0.722 19.580+ 1.176 46.88

Sg 15 .330+0.869 17.950+0.494 36.50

6a 16.666+ 1.084 18.580+ 1.050 60.97*

6b 15.900+0.330 20.500+0.590 40.90

6c 13.900+0.720 17.500+0.990 40.81

6d 14.000+0.690 22.000+0.610 30.63

6e 17.1 63+0.899 18.430+ 1.338 28.15

6f 16.766+ 1.640 19.670+2.362 59.82*

6g 14.230+ 1.260 19.350+ 1.077 28.90

7a 16.766+0.539 19.580+0.949 48.04

7b 14.875 + 1.270 22.500+0.957 04.54

7c 14.880+0.54 1 22.250+.0893 10.60

7d 18.875+1.120 20.280+ 1.018 15.50

7e 16.875+1.197 20.000+0.840 17.80

7f 18.225+0.752 20.448+0.616 40.52

7g 19.600+0.92 1 21.710+1.015 25.12

Diclofenac sodium 15.00+1.160 19.200+ 1.27 47.83

*Significantly higher acti vity than diclofcnac sodi um at p<0.05. a Compounds were adminstered at dose 10mglkg, n = 5 albino rats

Table IX-Free radical (DPPH' ) scavenging property

Compd Initi al Final %Activity Compd Initial Final %Activity

Sa 0.976 + 0.012 0.790 + 0.006 19.06 6e 1.005 + 0.012 0.882 + 0.007 12.24

Sb 0.968 + 0.005 0.715 + 0.004 26.14 6f 0.980 +0.005 0.944 + 0.005 3.68

Sc 0.906 + 0.003 0.553 + 0.012 38.96 6g 0.977 + 0.006 0.899 + 0.013 7.98

Sd 0.817 + 0.014 0.641 + 0.014 21.54 7a 0.954 + 0.003 0.835 +0.004 12.47

Se 1.172 + 0.007 0.719 + 0.006 38.65 7b 0.945 + 0.012 0.845 + 0.009 10.58

Sf 0.946 +0.013 0.63 1 + 0.004 33.30 7c 1.110 + 0.004 0.988 + 0.016 10.99

Sg 1.035 + 0.003 0.749 + 0.015 27.63 7d 0.957 + 0.005 0.609 + 0.019 36.47

6a 0.987 +0.016 0.912 + 0.004 7.30 7e 0.901 + 0.014 0.118+0.004 86.90

6b 0.997 +0.008 0.881 + 0.008 11.64 7f 0.894 + 0.013 0.148 + 0.002 83.45

6c 0.956 +0.013 0.808 + 0.009 15.48 7g 0.998 + 0.009 0. 843 + 0.004 15.53

6d 0.930 + 0.009 0.760 + 0.015 18.28

Conc . of compounds = IOmg/DMSO:Methanol, 0.5:0.5mL (100~L for test) Conc. of DPPH = 2.6mg/2mL methanol (75~L for test)

SHISHOO et al .: PYRAZOLO[3,4-d]PYRIMIDINES & THEIR BIOLOGICAL ACTIVITIES 1085

mixture of methanol and dimethylsulfoxide was chosen in order to solubilize and test all the derivatives, under same conditions. In a test tube containing 3 mL methanol, was added 75)..lL DPPH (2 .6 mg/2mL methanol) and initial optical density of the solution was recorded at A 516 nm. The test compounds 100 )..ll each (10 mg in DMSO:methanol, 0.5 : 0.5 mL) were added to the above 3mL mixture and was allowed to stand for 20,min at room temperature and thereafter its optical density recorded. The antiradical activity was measured by the decrease in optical density of methanolic solution of coloured DPPH at A 516 nm. The activity was expressed as % reduction of DPPH* (Table IX).

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