Indian Journal of Chemistry Vol. 38B, June 1999, pp. 684 - 695

Synthesis and pharmacological evaluation of some novel 5-aryl-6-arylamino-l-phenylpyrazolo[3,4-dlpyrimidin-4(5H)-ones as analgesic

and anti-inflammatory agents

Chamanlal J Shishoo*, Urvish S Pathak, Ishwarsinh S Rathod, Kishore S Jain, Laxmivenkatesh G Nargund, Ashok D Taranallit , Hiren Patel, Vivek Kumar & Vikas S Shirsath.

Department of Pharmaceutical Chemistry, L.M.College of Pharmacy, Navrangpura, Ahmedabad 380 009 t H.Q.K.L.E.'s College of Pharmacy, J.N.Medical College Campus, Belgaum 590 OW, India.

Received 23 February 1998; accepted (revised) 24 November 1998

A series of novel title compounds 4a-o has been synthesized through two different synthetic routes. While the first involves the condensation of pyrazole o-aminoester 1 with aryl isothiocyanates 2, the second involves cyciocondensation of alkyl isothiourea ethers of sym-diarylthioureas 5 with 1. The compounds have been evaluated for analgesic, as well as, anti-inflammatory activities in rodents. The lead compound, 4c (LM-22835), exhibits analgesic activity comparable to morphine and aspirin at the dose levels of 10 mg/kg p.o. and 100 mg/kg p.o. In the acetic acid induced writhing test, in mice, it has been found to be superior to aspirin in the rat caudal immersion test. Efforts towards optimization of lead compound 4c have resulted in identifying more active compounds. Compounds 4b, 4c, 4e, 4h, 4i and 40 exhibit anti­inflammatory activity superior to aspirin at 100 mg/kg, p.o. in the colton pellet induced granuloma test(rats); compound 40, is also found to be superior to aspirin when evaluated by the carageenan induced rat paw edema test. Acute toxicity studies reveal that these compounds are non-toxic upto 4.0 g/kg, p.o. in mice. The lead compound, 4.:, has been found to be safe and without any untoward effects in the 30, as well as, 60 days chronic toxicity studies in mice.

Non-narcotic analgesic and anti-inflammatory drugs represent one of the most widely prescribed categories of drugs'. NSAIDs are used extensively not only in rheumatology but also in other fields of medicine like treatment of gout, gynaecology and paediatrics2

•

Most of the drugs in therapeutic use have a drawback of bearing an acidic functionality and therefore the side effects associated with it. More or less, all the NSAIDs and OTe analgesics suffer from adverse side effects like allergic reactions, GIT disturbances, irritation, nausea, vertigo, drowsiness. dizziness etc. In order to circumvent these problems, extensive search for an ideal NSAID still continues.

The pyrazolo[3,4-d]pyrimidines have been described as biologically active agents.305 The most

X

~NH ~ I, )l ~I ;:-(

'N N R

I,R=H.X=O

II, R = H, X = S HO

III , R = OH. X = 0 IV

H

OHOH

widely used pyrazolo[3,4-d]pydmidines, allopurinol I, thiopurinol nand oxyallopurinol III are established inhibitors of xanthine oxidase and thus interfere in the biosynthesis of uric acid, the causative agent of gout. This group of compounds also exhibits antineoplastic activitl. Antitumour and antiviral compounds have been synthesized based on the antibiotic formycin IV, a nucleoside of pyrazolo[3,4-d]pyrimidine7

.

Potent anti-inflammatory aDd analgesic activities have been reported in a number of 5-arylpyrazolo[3,4-d]pyrimidines V and VI8

.9

•

Herein, we report the synthesis, analgesic and anti­inflammatory activities and results of our toxicity studies of novel 5-aryl-6-arylamino-I-phenylpyra-

v VI

SHlSHOO et. al.: SYNTHESIS OF PYRAZOLO[3,4-djPYRIMIDINES 685

zolo[3,4-d]pyrimidin-4(5H)-ones VI(Table I). synthetic routes were developed.

Results The title compounds were synthesized through the

annealation of a pyrimidine ring on the suitably substituted pyrazole ring substrate. Two different

Method I. This involved the reaction of pyrazole 0-

aminoester 1 with a variety of aryl isothiocyanates 2 in a weakly basic solvent like pyridine at 135°C. The reaction presumably proceeds through the formation of an intermediate 6-mercaptopyrazolo[3,4-d]pyri-

Compd R

4a H

4b H

4c H

4d H

4e H

4f H

4g H

4h

4i

4j

4k

41

40

Table I - 5-Aryl-6-arylamino-l-phneylpyrazolo[3,4-djpyrimidin-4(5H)-ones 4a-v.

Ar mpoc Yield Mol. formulae UV(MeOH)' lR (KBr) ~MR (Solvent)" (%) (M+)f (nm) (0, ppm)

198-200 (E)

180-82 (E)

228-30 (E)

194-96 (E-C)

218-20 (E-C)

240-42 (E-C)

248-50 (E-C)

250-52 (E-C)

240-42 (E-C)

270-72 (E)

216-18 (E-C)

238-40 (E-C)

280-82 (E-C)

29b C2jH2INsO 76c

62d

3 1b C2sH21Ns03 81 c

60d

C23HISChNsO (449)

C23HISBr2NsO (537)

30b

C24HI9NsOS 71 c (425) 66d

82c C26H23NsOS 63d

78c C26H23NsOS 68d (453)

207.4

210.6

209.6

256.6

261.9

3413(NH); 1700 (CO)

3403(NH); 1700 (CO)

3416 (NH); 1705 (CO)

3392(NH); 1710 (CO)

3406(NH); 1705 (CO)

3400(NH); 1690 (CO)

341O(NH); 1690 (CO)

3450(NH); 1700 (CO)

3450(NH); 1700 (CO)

6.1-6.3(s,1 H, Ar-NH, 0 20 exchangable), 7.1-8.3 (m, 16H, Ar-H & proton at 3)

2.2-2.5(d, 6H, Ar-CHl at 5 & 6, 6.8(s, tH, Ar­NH, 0 20 exchangable), 7.0-8.2 (m, 14H, Ar-H & proton at 3)

3.7-3.9(d,6H,Ar-OCH3 at 5 & 6, 6.4 (s, IH, Ar­NH, 0 20 exchangable), 6.6-7.6(m, I3H, Ar-H), 8.3(s, tH proton at 3)

6.2 (s, IH, Ar-NH, 0 20 exchangable),8.2-8.7 (m, 14H, Ar-H & proton at 3)

-Contd

686 INDIAN J. CHEM, SEC. B, JUNE 1999

Table I - 5-Aryl-6-arylamino-l-phneylpyrazolo[3,4-dJpyrimidin-4(5H)-ones 4a-v.( --Contd).

40 SCH3 4-Brt::JI4 298-300 27b C24H17Br2N50S (CH-EA) 65d

4p H 4-CH(CH3hC6H4 179-80 76d C~29N50 207.8 3415(NH);

(E-C) (463) 1700 (CO)

4q H 4-C2HsOC6H4 169-71 29b C27H2SN50 209.2 3399(NH); 1.25- 1.6 (t of t, 6H,

(E-C) 75c 1700 (CO) ArOCH2CH3 at 5 &. 6, 66<1 3.8-4.3 (q of q, 4H,

ArOCH2CH3 at 5 &. 6, 7.8-8.1 (m, \3H, Ar-H), 8.3(s, \H, proton at 3)

4r H 4-FC6H4 2\0-12 73c C23H16F2N50 208.6 3406(NH); 6.8-6.9(broad, I H, Ar-

(E-C) (415) 1720 (CO) NH), 7.0-7.8 (m,13H, Ar-H), 7.9-8.1 (5, I H, proton at 3)

45 H 2,4-diCH3C6H3 189-91 62c C27H25N50 211.6 3403, 1.8-2.3(m,12H,Ar-CH3

(E-C) (435) 3291(NH); at 5 &. 6), 6.1 (5, \H,Ar-1700 (CO) NH).6.9-7.5(m, 11H,

Ar-H), 8.1(s,lH, proton at 3)

4t H 2,3-diCH3C6H3 241-42 65c C27H25N50 2\0.2 3402,3258

(E-C) (435) (NH); 1700 (CO.)

4u H 2,5-diCH3C6H3 177-79 70c C27H2SN5O 212.0 3399(NH); (E-C) 1710 (CO)

4v H 2,6-diCH3C6H3 261-63 76c C27H2SNsO 243.8 3476,3381 (E-C) (NH);

1700 (CO)

(')C=Cloroform, CH=Cyclohexane, E=Ethanol, EA=Ethyl Acetate. (b)Prepared by method - I, (C)Prepared by method - II, (d)Prepared by method - III, (e)Satisfactory elemental analysis (± 0.4 %) for percentage of carbon and hydroen obtained., (f)Mole.Wt. determination by mass spectra, (g)A.max in methanol.

midin-4-one 3, which on further reaction with an additional mole of aryl isothiocyanate 2 leads to the target compound, 4. This unusual direct formation of the 6-arylamino-5-arylpyrazolo[3,4-d]pyrirnidinA-one 4 in the above condensation of 5-amino-4-ethoxycarbonyl-l-phenylpyrazole 1 with aryl isothiocyanate 2 has been presumed to proceed through the intermediate 6-mercaptopyrazolopyrimi­dinA-one 3. However, all attempts to isolate the presumed intermediate 3 under the reaction conditions met with failure (Scheme la, Table I). Though the isolation of 3 was not successful, its unequivocal synthesis under carefully controlled conditions and subsequent conversion to the target compounds has been achieved successfully ( Scheme Ib, Table II). However, the longer reaction time (30-35 hr) with lower overall yields (-25-35%) of the target compounds makes this route less attractive. Therefore,

an alternative one step, facile and single pot route was developed.

The unusual direct formation of 2-substituted­aminopyrazolopyrimidine-4-one by the condensation of 5-arnino-4-ethoxycarbonyl-l-phenylpyrazole (4) with arylisothiocyanate has been presumed to proceed via the 2-mercaptopyrazolopyrimidin-4-one inter­mediate, 3. This essentially a one-pot reaction has been extended to synthesize a series of novel 3-aryl-2-arylamino-pyrazolopyrimidine-4-ones, The formation of 4, can be explained by the mechanism1o,11 depicted in Scheme lb.

The a-amino ester reacts with aryl isothiocyanates to yield a 2-mercapto-3-aryl-5,6-disubstituted pyrirni­dinA-one. This 2-mercapto intermediate adds one more molecule of aryl isothiocyanate to give a four membered ring, 1,3-thazetidin-2-thione, by internal

SHISHOO et. al.: SYNTHESIS OF PYRAZOLO[3,4-djPYRIMIDINES 687

0 0

"r { OC,H, ~ r R OC2H5

NHAr N / +

~ 1350 C N, N~ ' N NH2 • cb NHA, © c pyridine

II S

1. :R",H 2

1b : R '" HSCH3 I , r~ A, 0

R N )-- ,Ar

}--NHAr R N ft- }--SH N, N ArNCS

N .-- N, N

©. N

© 3

Scheme Ia u 0 II "r--t n OC2HS

ArNCS NaH I DMF

I OC2HS

N ' N~NH + • N ' N~N- C- NWAr RT, stirring I 2

I H II C6HS 2 C6HS S

tfik

• ArNCS

~ N SH

C6 HS

3

1a , R = H, 1b, R = SCH3 ,

4 Scheme Ib

cycJization, which then loses one molecule of carbon disulfide to yield 3-aryl-2-arylaminopyrazolopyrimi­dine-4-ones, 4 (Scheme Ib). The identity of the

compounds has been separately established by their synthesis by an un-ambiguous route involving the 0-

ami noesters 1 and sym-diarylthiourea 5.

688 INDIAN 1. CHEM, SEC. 8, JUNE 1999

Table 11- 5-Aryl-6-mercapto-l-phenylpyrazolo[3,4-dJpyrimidin-4(5H)-one 3a-j.

R:-lN/N N' \ ~SH

'N N

© Compd R Ar mp (0C), Yield Mol.formulab (M+)" IR(KBr)

(Solvent)" (%) 3a H -C6HS 193-94 69 C 17HI2N40S (320) 3300,1720

(E-C) 3b H 2-CH3C6tLl 180-82 70 CIsHI4N40S 3310,1710

(E-C) 3c H 4-CH3C6l-LJ 199-201 72 CIsHI4N40S 3300,1710

(E-C) 3d H 2-OCH3C6~ 220-22 70 CIsHI4N402S 3380,1720

(E-C) 3e H 4-OCH3C6~ 286-87 64 CIsHI4N402S (350) 3400,1720

(E-C) 3f H 4-CIC6H4 221-22 69 C 17HIICIN40S (354) 3360,1730

(E-C) 3g H 4-8rC6~ 230-32 66 C 17H I1 8rN4OS 3360,1730

(E-C) 3h H 4-0C2HsC6H4 218-20 69 CI9HI6N402S 3320,1710

(E-C) 3i H 4-CH(CH3hC6H4 210-13 61 C2oHISN40S 3450,1740

(E-C) 3j H 4-FC6H4 211-13 67 C 17H 11 FN4OS 3360,1730

(E-C)

(a)C : Chloroform, E : Ethanol, (b)Satisfactoty Elemental Analysis(± 0.4%) for percentage of carbon & hydrogen obtained. (C)MoI.Wt. determination by mass spectra.

Method II. This involves the condensation of equimolar quantities of the pyrazole o-aminoester 1 with the methyl isothiourea ether of sym-disubstituted arylthiourea 5 in DMF at reflux temperature in the presence of catalytic amounts of anhydrous K2CO). The reaction can be envisaged to be proceeding via the guanidine intermediate 6 arising out of the nucleophilic attack of the amino group of 1, on 5 with the concomitant loss of Methyl mercaptan (MeSH). The intermediate, 6 undergoes subsequent intramole­cular cyclisation to the target compound 4 (Scheme II,

5

Table I). All the new synthesized compounds 4a-o have

been characterized on the basis of their correct elemental analysis and spectral data. Analgesic activity

Acetic acid induced writhing test (mice). The analgesic effect of the test compounds on the acetic acid induced writhing in ntice was compared with standard analgesic drugs, morphine (10 mg/kg, p.o. and 100 mg/kg, p.o.) and aspirin (100 mg/kg, p.o.). The test compounds showed significant inhibition of

6

Scheme II

SHISHOO et. al.: SYNTHESIS OF PYRAZOLO[3,4-d]PYRIMlDINES 689

Table III - Effect of test compounds on acetic acid induced writhing at the dose level of 10 mg/kg.

Animal Testg Compound Number Mean no. of writhes ',' value % Reduction in no. of wriths (% analgesia) group No. (Dose mg/kg, p.o.) tS.E.

Control (0.5mL of 2% acacia 47.0 t 1.61 suspension, p.o.)

II Morphine (5.) 21.8 t 0.84* 13.816 53.16

III Aspirin (10 ) 26.3 t 0.70* 11.734 44.04 IV 4b (10) 24.4 t 0.68* 12.848 44.08 V 4c (10) 23.0tO.71* 13.559 51.06 VI 4e (10) 26.0 t 0.63* 12.083 44.68 VII 4h (10) 28.3 t 1.03* 9.737 39.78 VIII 4i (10) 24.6 t 0.90* 13.813 47.65 IX 40 (10) 27.3 t 0.73* 11 .089 41.91

n = 10 animals, * = Significantly different from control (p < 0.01).

Table IV - Effect of test compounds on acetic acid induced writhing at the dose level of 100 mg/kg, p.o.

Animal Test Compound Number (Dose) Mean no. of wriths t S.E. 't'value % Reduction in no. of wriths group No. (% analgesia)

X Aspirin (100 mg/kg, p.o.) 16.0 ± 0.90* 16.71 65.59 XI 4b 20.7 t 0.77 * 14.64 55.95 XII 4c 19.2 t 0.66 * 15.89 59.14 XIII 4e 23.0 ± 0.57 * 13.96 51.06 XIV 4h 25.1 ± 0.90 * 11 .82 46.59 XV 4i 20.5 ± 0.76* 14.80 56.38 XVI 40 24.7 to.68* 12.69 47.44

n = 10 animals, * = Significantly different from control (p < 0.01).

writhing in mice when compared to the control, i.e., 53.16%, 44.04% and 65.59%, respectively. Similarly, all the test compounds (4b, 4c, 4e, 4h, 4i and 40) produced significant reduction in the number of writhes at dose levels of 10 mg/kg, p.o. and 100 mg/kg, p.o. The lead compound, 4c exhibited 5l.06% and 59.14% reduction in the number of writhes at the dose levels of 10 mg/kg and 100 mg/kg, p.o. (Tables III and IV).

Caudal Immersion test (rats). Aspirin and the test compounds at the dose levels of 100 mg/kg, p.o. significantly prolonged the tail withdrawal reflux time at half an hour, 1 hr, 3 hr, and 6 hr, after their admjni stration , compounds 4c and 4h being the most active. Compound 4c exhibited 64.33 % and 77.57 % analgesic effect after 0.5 hr and 1.0 hr of administra­tion, respectively. Compound 4h showed 65.86% inhibition of tail withdrawal reflux after 3.0 hour (Table V).

Anti-inflammatory activity Carageenan induced rat paw edema test.

Aspirin at the dose level of 100 mg/kg showed significant inhibition of edema (62.01 % at the third hour). The test compounds 4b, 4c, 4e, 4h, 4i & 40 at the dose level of 100 mg/kg exhibi ted significant anti-

inflammatory action against the carageen an induced rat paw edema (45.91, 45.91, 60.09, 60.09, 56.00 and 66.10, respectively at the third hour) (Table VI).

Cotton pellet induced granuloma test (rats). Aspirin significantly inhibited the granuloma formation with mean dry weight of 47.55 mg ± 2.013 compared to the control (68.67 ± 2.965). All the test compounds were found to be more active than standard aspirin with mean granuloma dry weights of 39.70 ± l.88 (4b), 36.71 ± 1.20 (4c), 37.94 ± 1.96 (4e), 47.55 ± 2.01 (4h), 26.37 ± 0.88 (4i), and 34.33 ± l.77 (41) (Table VII).

Ulcerogenic potential. The mean ulcerogenic potential of aspirin (33.33 ± 4.94) and the compounds 4b, 4c and 4e (21.66 ± 4.77,23.33 ± 4.14, and 25 .00 ± 4.28, respectively) is statistically significant when compared with control (only 10.00 ± l.825). However, the compounds 4h, 4i and 41 did not increase the mean gastric ulcerogenic potential and are not significantly ulcerogenic as compared to the control (Table VIII).

Toxicological study Acute toxicity (mice). In an experiment carried

out to determine the acute toxici ty of the test

690 INDIAN 1. CHEM, SEC. B, JUNE 1999

Table V - Analgesic effect of aspirin and test compounds on caudal immersion test in rats.

Timc(hr) 30 min. I hr 3 hr 6 hr

Tesl XiSE ' 1' ' p' % inhi- XiSE ' 1' 'p' % inhi- XiSE . (' ' p' % inhi Xi SE .(' 'p' % inhi Compds (sec.) bilion (sec.) bilion (sec.) bilion (sec.) bilion

Conlrol 1.57 i 1.65± 1.67 i 1.63 ct 0.5 mL 0.033 0.061 0.0334 0.0333 2% gum acacia' Aspirin 1.77 i 3.34 <0.001 42.73 2.35 i 10.63 <0.001 42.42 2.11 i 4.70 <0.001 26.34 1.88 ± 2.86 < 0.02 15.33

0.049 0.022 0.087 0.079 4b 2.13 i 6.74 <0.001 35.66 2.6Oi 7.24 <0.001 57.57 2.53 i 9. 11 <0.001 5 1.49 1.62± 0.1 72 N.S. 0.61

0.076 0.115 0.088 0.047 4c 2.58 i 8.13 < 0.00 1 64.33 2.93± 11.87 <0.001 77.57 2.25 i 5.23 <0.001 34.73 1.57 ± 0.280 N.S. 5.68

0.119 0.088 0. 105 0.221 4e 2.33 i 8.61 <0.001 48.40 2.55 i 9. 15 <0.001 54.54 2.01 i 3.63 <0.01 20.35 1.53 t 0.672 N.S. 6. 13

0.088 0.076 0.087 0.494 4h 2.05 i 5.75 <0.001 30.57 2.82 i 12.38 < 0.001 70.90 2.77i 11.1 <0.001 65.86 1.83 ± 4.24 <0.0 1 12.26

0 .076 0.070 0.049 8 0.033 4i 1.83 i 3.99 < 0.01 16.56 2.50 i 10.54 <0.001 51.51 2.00i 3.74 <0.01 19.76 1.72 :< 1.54 N.S. 5.52

0.055 0.516 0 .081 0 .047 40 1.88 i 4.50 < 0.01 19.74 2.55 i 8.35 <0.001 54.54 2. 13 i 7.07 <0.001 27.54 1.68 :r 0.72 N.S. 3.06

0.060 0 .088 0.055 0.060

*all the test compounds and aspirin were administered at the dose level of 100 mg/kg, p.o.; n = six animals/group. #0.5 mL per 100 gm body weight.

compounds 4b, 4c, 4e, 4h, 4i and 40, by both oral as well as intraperitonial routes neither toxicity nor mortality was revealed for any compounds at dose levels as high as 3999.39 mg/kg (4 gmlkg), indicating the compounds to be reasonably safe by these routes.

Chronic toxicity of compound 4c (rats). No pathological changes observed in the kidneys. Also, the effect on liver and changes observed revealed not much of significant chronic toxicity in both 30 days and 60 days studies.

Lead Optimization. An attempt was made to undertake the lead optimization studies of this series by synthesizing additional derivatives of the lead compound. Five analogues of 4c namely, 4f, 4p, 4q, 4r and 4s were synthesized with an increased lipophilicity at the aromatic rings and tested for their analgesic activity in mice by hot-plate method. All the newly synthesized derivatives exhibited analgesic activity superior to 4c. The EC50 of 4c is 5.4 mg whereas these analogs have an EC50 in the range of 3.9-5.0 mg.

Discussion

The pyrazole ring seems to be the potential pharmacophore for analgesic and anti-inflammatory activity. The present study involves the synthesis and screening of newer derivatives of pyrazolopyrimi­dines, namely, pyrazolo[3,4-d]pyrimidin-4(5H)-ones, appropriately substituted at the 5 and 6 position, for the analgesic and anti-inflammatory activity, and also having variations at position 3. This novel series of non-acidic 5-aryl-6-arylamino-l-phenylpyrazolo[3,4-d]pyrimidin-4(5H)-ones exhibited significant anal-

gesic and anti-inflammatory activities in rodents (mice and rats) at the dose levels of 10 rngfkg and 100 mg/kg, p.o. However, these compounds lack activity at lower dose levels of 1 mg/kg, p.o. When evaluated by acetic acid induced writhing in mice, analgesic activity comparable to that of morphine ( 5 mg/kg, p.o.) and aspirin (10 mg/kg and 100 mg/kg, p.o.) was exhibited. The lead compounds 5-(4-methyl)-phenyl-6-( 4-methyl)phenylamino-l-pheny Ipyrazolo[3,4-d]pyrimidin-4(5H)-one 4c, exhibited 51.06 % and 59.14 % reduction in number of acetic acid induced writhes at dose levels of 10 mg/kg, 100 mg/kg, p.o. , respectively. The activity is dose dependent. In the caudal immersion test in rats (thermal stimulus), the lead compound 4c, exhibited 64.33% and 77.57% analgesic effect after 0.5 and l.0 hrs of administration (100 mg/kg, p.o.). The analgesic activity is signi­ficantly more than aspirin (42.73% and 42.42% respectively).

This novel series of pyrazolo[3,4-d]pyrimidin-4-one 4, also exhibits significant anti--inflammatory activity by two different models, namely, the carageenan induced rat paw edema model and the cotton pellet induced granuloma in rat model. In both the models, the 3-methylthio derivatives are more potent than the 3-unsubstituted analogs. Thus compounds 4h, 4i and 41 exhibited significant anti­inflammatory action against the carageenan induced edema in rat paw (60.09, 56.00 and 66.00 % respectively at the third hour), which is comparable to aspirin (62.01 %) at the same dose level of 100 mg/kg, p.o. In the cotton pellet induced granuloma (rat) model , all the compounds were found to exhibit

..

SHISHOO et. af.: SYNTHESIS OF PYRAZOLO[3,4-djPYRIMIDINES 691

Table VI- Anti-inflammatory effect of aspirin and test compounds on carageen an induced rat paw edema.

Time in I hr 3 hr 6 hr Hour

Test X±SE ' I' 'p' % inhi- X±SE 't' 'p' % inhi- X±SE ' t' 'p' % Compd (mL) bition (mL) bition (mL) inhi-

bition

Control 0.183 ± 0.416± 0.633± ImL 0.0105 0.047 0.061 2% gum acacia Aspirin 0.252 ± 2.77 < 0.02 27.32 0.158± 5.14 <0.001 62.01 0.366± 3.88 <0.01 42.18

0.049 0.015 0.030 4b 0.150± 1.56 <N.S. 18.03 0.225± 3.89 <0.01 45 .91 0.533± 1.19 N.S. 15.79

0.018 0.011 0.057 4c 0.158 ± 1.86 <N.S. 13.66 0.225± 3.89 <0.01 45.91 0.533± 1.19 N.S. 15.79

0.008 0.011 0.057 4e 0.150± 1.98 <N.S. 18.03 0. 166± 4.94 <0.001 60.09 0.525± 1.65 N.S . 17.06

0.012 3 0.016 0.021 4h 0.158 ± 1.69 <N.S. 13.66 0.166± 5.11 < 0.001 60.09 0.550± 1.24 <0.01 13.11

0.010 0.010 8 0.025 4i 0.150± 1.98 <N.S. 18.03 0.183± 4.71 < 0.001 56.00 0.523± 1.70 N.S. 17.37

0.011 0.210 0.019 40 0.158 ± 1.38 <N.S. 13.66 0.141± 5.31 <0.001 66.10 0.575± 6.89 N.S . 9.16

0.015 0.020 0.021

* All the test compounds and aspirin were administered at the dose level of 100 mglkg, p.o.; n = six animals/group.

activity far superior to aspirin at the dose level of 100 mg/kg, p.o . Interestingly, the methylthio analogs, 4h, 4i, and 41 were superior (46.54%,61.59% and 50.00% respectively) to aspirin (30.75 %) and 4b, 4c and 4e (42.18%, 46.50% and 44.75% respectively). This indicates the positive influence of the 3-methylthio moiety on the anti-inflammatory activity of the compounds. Compounds 4h, 4i and 41 also have more prominent effect on causing decrease in the weight of the adrenal glands of the animals, compared to aspirin and other test compounds. Of interest is that the ulcerogenic potential of these 3-methylthio deri­vatives, 4h, 4i and 41 is lower than the 3-unsub­stituted analogues, as well as, aspirin.

Significantly, these compounds are non-toxic even at dose levels as high as, 4.0 gm/kg, p.o. The lead compound 4c, in this series exhibited no toxic effects or side effects in the 30 and 60 days chronic toxicity study. 'Thus, thi s series is an interesting and potential series for finding a non-acidic, less ulcerogenic analgesic and anti-inflammatory drug with a dose level as low as 100 mg/kg.

With a view to optimizing the activity of the lead compound 4c, additional analogs of 4c were synthesized with various lipophilllic groups on the aromatic rings. By the lead optimization techniques,

the additional derivatives of compound 4c were synthesized (4f, 4p, 4q, 4r and 4s) and evaluated for the analgesic activity by hot-plate method in mice. All the newly synthesized compounds exhibited activity superior to 4c and are under further studies.

It is generally observed that the electron releasing substituents (-CH3) on ortho or para position of the aromatic rings at 5 and 6 positions leads to increased activity probably by increasing the aromaticity of the pyrimidine ring whereas electron withdrawing substituents (-Cl, F) reduce the activity . The bulk at para position however is not favourable since the poi Pr derivative shows reduced activity.

Experimental Section

Melting points were determined in open capillaries and are uncorrected. The UV spectra were recorded in methanol on Beckman spectrophotometer model (Beckman, USA); IR spectra recorded in KBr on a Perkin Elmer 841, grating spectrophotometer, (Perkin­Elmer, USA) and mass spectra on a Varian Atlas CH-7 spectrometer at 70 eV ionizing beam, using direct insertion probe. Satisfactory microanalysis (± 0.4% of the calculated values) was obtained for all the compounds.

The pyrazole o-aminoesters ' 2.t3 la, lb and the N­

arylisothiocyanates l4 2 and the methyl isothioureas of

692 INDIAN 1. CHEM, SEC. B, JUNE 1999

Table VII - Anti-inflammatory effect of test compounds and aspirin on cotton pellet induced granuloma dry weight ( expressed in mg/lOO gm body weight) and effect of aspirin and test compounds on gastric mucosa (ulcerogenic effect).

Compd Anti-inflammatory activity Ulcerogenic activity (dose)

Mean ± S.E p Percentage Mean ± S.E p

Control 68.67±2.96 IO.OO± 1.82 (I mL 2% acasia) Aspirin 47.55±2.01 5.88 <0.001 30.75 33.33±4 .. 94 4.42 < 0.01 (l00 mg/kg) 4b 39.70± 1.88 8.24 <0.001 42 .18 23.33±4.94 2.52 < 0.05 (100 mg/kg) 4c 36.71 ± 1.96 9.98 < 0.001 46.54 21.66±4.77 2.28 <0.05 (100 mg/kg) 4e 37.94± 1.96 8.60 <0.001 48.54 25.00±4.28 3.22 <0.01 (100 mg/kg) 4h 36.81 ± 1.96 9.86 <0.001 46.64 18.33±4.77 1.63 N.S. (100 mg/kg) 4i 26.37±O.88 13.66 <0.001 61.59 20.00±55.16 1.82 N.S. (100 mg/kg) 41 34.33± 1.77 9.93 < 0.001 50.00 16.66±3.33 1.75 N.S. ( 100 mg/kg)

Ulcer Scoring: I: Denuded epithelium = 10,2: Petechial and frank haemorrhages = 20, 3: one or two ukers = 30, 4: multiple ulcers = 40., 5: perforated ulcers = 50.

sy m.-diary!thioureas I5 5 were prepared by literature methods. All the other reagents were of reagent grade.

General procedure for the synthesis of 5-aryl-amino- 1 -phenylpyrazolo[3, 4-d]pyrimidin-4(sH)-ones, 4a-v. Method I - A mjxture of 1 ( 0.01 mole) and the appropriate N-aryl isothiocyanate (0.04 mole) in dry pyridine (10-15 mL) was refluxed at 135-40°C for 30 hr. The reaction mixture was allowed to cool to room temperature and poured into dilute hydrochloric acid (10% viv, ag. 300 mL). The solid separated was filtered, washed with water and dried. Recrystallization from appropriate solvent yielded a crystalline product. (Table I).

Method II - An eguimolar mixture of pyrazole 0-

aminoester (1, 0.02 mole) and the appropriate S­methylisothiourea(s, 0.02 mole) in the presence of catalytic amounts of anhydrous K2CO) in DMF (30 mL) was refluxed until the evolution of methyl mercaptan gas ceased, completely. The reaction mixture was aUowed to cool to room temperature and thereafter poured into ice-water (250 mL). The solid separated out was filtered, washed with water, dried :llld recrystalli sed from a suitable solvent (Table I).

Method III- General procedure. An aqueous mixture of 3 Jnd the appropriate aryl isothiocyanate (2,0.01 mole) in dry pyridine (15 mL) was refluxed at 135-40 °c fo r 30 hr. The reaction mixture was allowed to cool to room temperature and poured into

dilute hydrochloric acid (10% viv, ag. 300 mL). The solid separated out was filtered, washed with water and dried. Recrystallization from appropriate solvent yielded a crystalline product. (Scheme Ib, Table I).

General procedure fol' the synthesis of s-aryl-6-mercapto-l pheny Ipyrazolo[3,4 -d]pyrimidin -4( sR)­ones, 3a-j. To a suspension of ' odium hydride (50%, 1.0 g, 0.02 mole) in DMF (30 mL) was added the pyrazolo o-aminoester (1, 0.02 moles). The reaction mixture was cooled and treated dropwise, under stirring, with the solution of the appropriate aryl isothiocyanate (2, 0.02 mole) in DMF (10 mL). The reaction mixture was stirred for 8 hr. at room temperature, poured into ice-water and filtered. The clear filtrate was acidified (10% wiv of aq. HCI) to pH 5-6 and the solid filtered, washed with water, dried and recrystallised from an appropriate sol vent (Table II).

Biological methods

General conditions of experimental animals. Experiments were carried out using laboratory animals namely, albino rats (Wistar strain) and albino mice (Swiss strain) of either sex. The animals wcre housed at temperature 24 ± 1°C and humidity 50-70% fo r 14 hr and 10 hr dark cycles. The animals were given food and water ad libitum, unless specified otherwise. For the studies the ani.mal s were selected at random.

SHISHOO et. al.: SYNTHESIS OF PYRAZOLO[3,4-djPYRIMlDINES 693

Table VIII - Analgesic activity of 5-aryl-6arylamino-l-phenylpyrazolo[3,4-djpyrimidin-4(5H)-ones by Hot plate method (lead optimisation).

H 0 ~R

uCN

N'N N..J-N-@-R

@" Compd R Mean increase in latency period

t SEM (sec)

2.5 mg 5.0mg 7.5 mg

4a -H 0.7tO.06 2.7tO.18 6.4tO.5 4c -CH) 1.4tO.12 5.8tO.4l 8.5±O.72 4p -CH(CH)h 1.8tO.16 8.5tO.65 13tO.93 4e -OCH) 0.5tO.04 1.3 to. 10 3.4tO.21 4q -OC2HS 1.6tO.12 7.ltO.80 9.ltO.83 4r -F 1.7tO.18 7.2tO.52 8. ltO.54 4f -CI I .0tO.1 I 7.8tO.63 9.3tO.82 4g -Br 0.06tO.08 1.8±O.13 4.6tO.50 4t -2,3-diCH) 1.0tO.10 1.8tO.09 4.3tO.38 4s -2,4-diCH) 2.5tO.20 8.6tO.54 11 .8tO.6 4u -2,5-diCH) 0.8tO.09 1.5tO.11 3.4tO.23 4v -2,6-diCH) 1.3tO.24 2.9tO.21 9.6tO.75

2.5 mg

6.7 12.03 15.9 5.0 14.4

15.04 9.0 7.0 8.84 20.0 7.0 11.5

% Analgesic activity

5.0mg

27.1 51.0 75.0 11.5 63.0 64.0 67.0 16.0 15.9 78.0 13.2 25.6

7.5 mg

56.5 75.0 116.8 30.0 80.6 72.0 83.0 41.0 38.9 118.0 30.0 85.0

ECso (mg)

7.2 5.4

4.10 12.0 4.9 5.0 4.8 8.6 9.7 3.9 12.3 5.6

Std.: Pentazocin at 10 mg, 11.3 t 0.99 secs.(Mean increase in latency period). Five new compounds, 4p, 4q, 4r, 4f & 4s were found to be more active than 4c (LM - 22835).

Initially, all the newly synthesized compounds 4a­o were roughly evaluated for analgesic activity by tail flick method in rats, using morphine as a standard (dose 10 mg/kg, b.wt., i.p. for both test and standard). Six compounds revealed good activity. Thus, of the fifteen compounds synthesized, only six compounds (4b, 4c, 4e, 4h, 4i, and 40) were selected for their detailed pharmacological investigation (analgesic and anti-inflammatory activities, ulcerogenic potential, effects on adrenal galnds, investigation into mechanism of analgesic action and toxicity studies, i.e., chronic and acute toxicity studies) . All the test drugs were suspended in 2% gum acacia and used for the studies. Control in all the experiments was treated with 0.5 mL / 100gm of 2% gum acacia suspension.

Analgesic activity

Acetic acid induced writhing test in mice (Chemical stimulus)16. Albino mice of either sex (20-25 gms, body wt.) were divided into total 16 groups of 10 animals each. The first group was the control group and received 0.5 mL/100 gm b.wt of aqueous gum acacia (2% w/v) suspension. The second and third groups received morphine (5 mg/kg,b.wt,

p.o.) and aspirin (10 mg/kg,b.wt,p.o.), respectively. The other groups viz; IV to IX received the corres­ponding test compounds (4b, 4c, 4e, 4h, 4i and 40) orally at the same dose level.

The remaining groups X to XVI received aspirin and the test compounds in same order at the dose levels of 100 mg/kg, p.o. Nearly 30 minutes after the drug was administered, acetic acid (1 mL of 0.6% v/vIlOOgmb.wt) was given intraperitonially to the animals. The writhing movements of the animals were recorded for 15 min. The mean of the writhing in different groups was compared statistically using students '1' test. (Table III and IV).

Caudal Immersion test in albino wistar rats (Thermal stimulus)17. Wi star albino rats of ei ther sex (150-200 gm) were divided into eight groups of six animals each. The animals were deprived of food 12 hr prior to the experiment and only water, ad libitum was allowed. The first group served as the control group and received 0.5 mL/100 mL of 2% w/v aq. gum acacia suspension, orally. Of the remaining seven groups, six groups (I to VI) received 100 mg/kg, body wt. of the test compounds 4b, 4c, 4e, 4h, 4i, and

694 INDIAN 1. CHEM, SEC. B, JUNE 1999

40, respectively, and the last group (VII) received aspirin at the same dose. All the drugs were administered 1 hr prior to the commencement of estimation of reaction time. The temperature of water in the organ bath was set at 55 ± 0.5 DC. The reaction time was determined by immersing the tail of the animal upto caudal portion into hot water and time taken by the animal to withdraw it clearly out of the water, was recorded. Observations were repeated thereafter at an interval of 30 min, 1 hr, 3 hr, and 6 hr (Table V). The percent analgesic activity was calcul­ated according to followi ng formula,

Xt - Xc % Analgesia = x 100

Xc

Where, Xt = Response time for test compound treated group Xc = Response time for control group.

Analgesic activity by hot-plate method (mice)18,19. Albino mice of either sex (15-20 g) were di vided into different groups of 6 animals each. The paw li cking response of each animal was recorded by pl ac ing the animal on a hot plate maintained at the temperature of 55 ± 0.5 DC, before the administration of compounds. Three dose levels of each compound was administered to three different groups/ compound. These levels were, 2.5 , 5.0 , and 7.5 mg/kg, i.p .. The standard group received pentazocin at dose of 10 mg/kg, i.p . After the interval of 60 minutes, the paw licking responses were recorded. Calculation of the activity at each individual dose was done with respect to the standard , usi ng followin g formula,

Mean latency (drug treated)

Mean latency (before admn . of drug)

% Act ivity = --------------Mean latency - Mean latency (s td treated) before adm n. of drug)

x 100

The results of the analgesic activity at the three di ffere nt doses were correlated and ECso values were calcu lated for each compound (see Table VIII).

Anti-inflammatory activity

Carageenan induced rat paw edema teseo. Albino rats of either sex (150-200 gm wt) were div ided into eight groups of 6 animals each. Animals were deprived of food 12 hI' prior to experiment and only water was allowed ad libitum. While the first group was controL and rece ived 1 mL of 2% w/v gum

acacia suspension in water/kg b.wt, p.o., the second group received aspirin (l00 mg/kg b.wt. ), orally. The remaining six groups received the test compounds at the same dose, p.o.

One hour after the administration of the compounds, carageen an suspension (0.1 mL of 1 % w/v suspension in gum acacia) was injected into the subplantar region of the left hind paw of the animals. Immediately, the paw volume was measured using a plethismometer (initial paw volume). Thereafter, the paw volume was measured everyone hour till 6 hours after the carageen an administration. The difference between initial and subsequent readings gave the edema volume for the corresponding time.

Edema volume control (Vc) and treated(Vt) were calculated and percentage (%) inhibition was calcul­ated by the formula,

Percentage (%) inhibition = 100 x (I-VtNe)

Results were analyzed by students ' t' test. (Table V).

Cotton wool pellet granuloma method (ratsi l•

The method of D' Arcy et at., with slight modification was adopted. Albino rats of either sex (150-200 gm wt.) were divided into eight groups of 6 animals each. Under light ether anaesthesia, the gro in area and axill a of each animal were shaved and cleaned with alcohol. Under aseptic conditions, small incisions were made on both the sides of groin and axi\[a and four subcutaneous pockets were made by separating skin and the underlying tissues . Sterilized cotton pellets(10 mg) were pushed into three pockets and in the fourth pocket, 2 em sterilized grass pith was implanted. Wounds were closed with sterilized si lk threads. The first group was the control group and received 0.5 mL of 2% w/v /kg gum acacia suspension in water, p. o. while the blind group received aspmn (100 mg/kg,b.wt. ,p.o.), the remaining six groups recei ved the corresponding test compounds at same dose, orally, once a day for 8 days.

On the 9th day, the animals were sacrificed and the pelletes and grass pith covered with granuloma tissue were dissected out. Each pellet was removed of fat and extraneous tissue and dried overnight in hot air oven at 60DC and weighed. The dissected grass piths were immedi ately kept in 10% formal in solution and further subjected to histological studies. At the sa me time adrenal glands were careful ly removed and weighed.

The difference in weight of cotton pellets before implantation and the weight of the dried pell et after di ssection was calculated to obta in the granuloma

SHISHOO et. al.: SYNTHESIS OF PYRAZOLO[3.4-d]PYRIMIDINES 695

weight. The mean dry cotton pellet granuloma was calculated for the treated groups and compared with that of the control. The percent inhibition of the granuloma was calculated as follows,

percent (%) inhibition = 100 ( 1 - WtlWc) ;

Wt = Wt. of pellets in treated groups,

Wc = Wt of pellets in control group (Table VII) .

Ulcerogenic potential22• The compounds were

evaluated for their ulcerogenic potential in order to examine their gastrointestinal side effects. The stomach of the sacrificed animals of the cotton pellet granuloma experiment were taken out. The stomach was opened along the greater curvature and examined for hemorrhages and ulcers with the help of hand lens and compared with that of aspirin treated groups. An arbitrary scoring system was followed as follows,

I, denuded epithelium - 10 II, petechal and frank hemorrhage = 20 III, one or two ulcers = 30 IV, mUltiple ulcers = 40 V, perforated ulcers = 50

Ulcer index was calculated as mean score of ulcer severity in various treated and control groups (Table VIII).

Toxicity studies on compounds23

Acute toxicity studies (mice). Albino mice (6-8 weeks old, 20-25 g) were divided into different group of 10 animals each, and different doses of the compounds were admini stered oralIy, as well as, intraperitonially . Each group consisted of 5 males and 5 females. The animals were kept under observation for 7 days and LDso was calculated with 95% confidence limits for 50% mortality, by the method of Litchfield and Wilcoxon.

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