Acta Pharm. 63 (2013) 19–30 Original research paper DOI: 10.2478/acph-2013-0001 Synthesis and biological evaluation of some novel pyrazolopyrimidines incorporating a benzothiazole ring system MOHAMMED AFZAL AZAM* In the present study, a series of benzothiazol derivatives 3a-l LOGANATHAN DHARANYA containing pyrazolo[3,4-d]pyrimidine moiety at the second CHARU CHANDRAKANT MEHTA SUMIT SACHDEVA position were synthesized and characterized by analytical and spectral data. The compounds were tested for their in vitro Department of Pharmaceutical antimicrobial activity. Compounds 1-(1,3-benzothiazol-2- Chemistry, J. S. S. College of Pharmacy yl)-3-methyl-4-phenyl-1H-pyrazolo[3,4-d]pyrimidine (3a), 1- Ootacamund, India (1,3-benzothiazol-2-yl)-4-(4-chlorophenyl)-3-methyl-1H-py- razolo[3,4-d]pyrimidine (3d) and 1-(1,3-benzothiazol-2-yl)- 3-methyl-4-substituted phenyl-1H-pyrazolo[3,4-d]pyrimidi- nes (3h-j) showed significant inhibitory activity against P. aeruginosa whereas compounds 1-(1,3-benzothiazol-2-yl)-4- (2-chlorophenyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidine (3b), 2-[1-(1,3-benzothiazol-2-yl)-3-methyl-1H-pyrazolo[3,4-d]pyri- midin-4-yl]phenol (3e), 1-(1,3-benzothiazol-2-yl)-4-(3,4-di- methoxyphenyl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidine (3h), 4-[1-(1,3-benzothiazol-2-yl)-3-methyl-1H-pyrazolo[3,4-d]pyri midin-4-yl]-N,N-dimethylaniline (3j) and 1-(1,3-benzothia- zol-2-yl)-3-methyl-4-[2-phenylvinyl]-1H-pyrazolo[3,4-d]pyri- midine (3k) were found to be active against C. albicans. Some of these synthesized compounds were evaluated for their in vivo acute toxicity, analgesic, anti-inflammatory, and ulcerogenic actions. The tested compound 4-[1-(1,3-benzo- thiazol-2-yl)-3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl]-N, N-dimethylaniline (3j) exhibited maximum analgesic and anti-inflammatory activities. Compounds 1-(1,3-benzothiazol- -2-yl)-3-methyl-4-(3-nitrophenyl)-1H-pyrazolo[3,4-d]pyrimi- dine (3i)and3j showed a significant gastrointestinal protec- tion compared to the standard drug diclofenac sodium. Keywords: benzothiazoles, pyrazolo[3,4-d]pyrimidine, anti- Accepted September 28, 2012 microbial activity, anti-inflammatory activity A major mechanism of action of nonsteroidal anti-inflammatory drugs (NSAIDs) is the lowering of prostaglandin (PG) production through inhibition of the cyclooxygenase (COX) enzyme, which catalyzes the conversion of arachidonic acid into PG (1). Since PG * Correspondence; e-mail: [email protected] 19 M. A. Azam et al.: Synthesis and biological evaluation of some novel pyrazolopyrimidines incorporating a benzothiazole ring system, Acta Pharm. 63 (2013) 19–30. has a dual function, mediation of inflammation (2) and cytoprotection (3) in the stomach and intestine, long-term usage of NSAIDs to relieve the symptoms of inflammation and pain always results in gastrointestinal (GI) disorders and renal toxicity (4). It is known that bacterial infections often produce pain and inflammation. In normal practice, chemotherapeutic, analgesic, and anti-inflammatory drugs are prescribed simulta- neously, which increases the risk of developing NSAIDs-related complications, especial- ly in the elderly, patients with a prior history of peptic ulcer disease and patients with impaired kidney functions. Hence, there is a pressing need for drugs having both anti- microbial and analgesic/anti-inflammatory activities with minimum adverse effects. Pyrazolo[3,4-d]pyrimidine and related fused heterocycles are of interest as potential bioactive molecules and have received a great deal of attention due to their useful anti- bacterial (5), antifungal (6) and anti-inflammatory (7) properties. In addition, many ben- zothiazole derivatives exhibit a wide variety of biological activities [antimicrobial (8, 9) and anti-inflammatory (10, 11)]. Based on the above observations and with the aim of developing new dual acting antimicrobial/anti-inflammatory agents with minimum ul- cerogenic effects, it appeared of interest to link the benzothiazole nucleus at the second position to some pyrazolo[3,4-d]pyrimidine moiety. This is an attempt to investigate the influence of such hybridization and structure variation on the anticipated biological ac- tivities, hoping to add some synergistic biological significance to the target molecules. In the present investigation, we aimed to synthesize novel benzothiazole incorporated py- razolo[3,4-d]pyrimidine derivatives and evaluate their antimicrobial, analgesic, anti-in- flammatory and ulcerogenic activities. EXPERIMENTAL Requisite chemicals were of reagent grade and were purified when necessary. Melt- ing points were determined in open glass capillaries and are uncorrected. The reaction progress was routinely monitored by thin layer chromatography (TLC) on silica gel pla- tes (Merck 60 F254, Germany). The IR spectra were recorded on a Shimadzu 8400S FT-IR spectrometer (Japan) in KBr pellets and band positions are given in reciprocal centime- ters (cm–1). The 1HNMRand13C NMR spectra were recorded using a Bruker AV-III 400 spectrometer (Germany) with dimethyl sulfoxide (DMSO)-d6 as solvent. Chemical shifts were reported in ppm using the solvent as internal standard. Mass spectra were mea- sured on a Shimadzu LC-MS 2010A mass spectrometer (Japan) equipped with a manual injector, a photodiode array detector, an electrospray ionization source and a single-qua- drupole mass analyzer in both positive and negative ion modes. Elemental analyses were carriedoutonaFlashEA1112seriesinstrument (Thermo Finnigan, Italy). 2-Hydrazino-1,3-benzothiazole (1) and 1-(1,3-benzothiazol-2-yl)-3-methyl-1,4-dihy- dro-5H-pyrazol-5-one (2) were prepared by the procedure described in the literature (12, 13). Synthesis of 1-(1,3-benzothiazol-2-yl)-3-methyl-4-substituted phenyl-1H-pyrazolo[3,4-d]pyri- midine derivatives (3a-l). General procedure A mixture of 1-(1,3-benzothiazol-2-yl)-3-methyl-1,4-dihydro-5H-pyrazol-5-one (2) (10 mmol, 2.31 g), urea (10 mmol, 0.60 g) and appropriate araldehyde (10 mmol, for 20 M. A. Azam et al.: Synthesis and biological evaluation of some novel pyrazolopyrimidines incorporating a benzothiazole ring system, Acta Pharm. 63 (2013) 19–30. benzaldehyde 1.06 g, 2,3 or 4-chlorobenzaldehyde 1.40 g, 2 or 4-hydroxybenzaldehyde 1.22 g, 4-methoxybenzaldehyde 1.36 g, 3,4-dimethoxybenzaldehyde 1.66 g, 3-nitrobenz- aldehyde 1.51 g, 4-dimethylaminobenzaldehyde 1.49 g, cinnamaldehyde 1.32 g, 2-furfur- aldehyde 0.96 g) in absolute ethanol (30 mL) was heated under reflux on a water bath for 7–8 h. The mixture was concentrated in vacuo and cooled to room temperature. The sepa- rated solid was filtered, washed several times with water, dried and recrystallized from appropriate solvent to give the title compounds 3a-l. Physicochemical and spectral data for the synthesized compounds are given in Ta- bles I and II. Synthetic route is represented in Scheme 1. Solvent for crystallization: for 3a, 3c-f, 3h, 3i acetone, for 3b, 3j chloroform and me- thanol 1:1, for 3g, 3l methanol, for 3k chloroform and petroleum ether 1:1 Table I. Physicochemical properties of the synthesized compounds 3a-l N N CH3 N S Ar N N Formula Yield M.p. Analysis (calcd./found, %) Compd. Ar (Mr) (%) (°C) C H N C H N S 3a C H 19 13 5 66 165 66.47/66.50 3.79/3.82 20.40/20.46 6 5 (343.40) C19H12ClN5S 3b 2-Cl-C6H4 67 228 60.31/60.29 3.17/3.20 18.51/18.56 (377.85) C19H12ClN5S 3c 3-Cl-C6H4 61 203 60.31/60.36 3.17/3.13 18.51/18.49 (377.85) C19H12ClN5S 3d 4-Cl-C6H4 70 185 60.31/60.28 3.17/3.13 18.51/18.56 (377.85) C19H13N5OS 3e 2-HO-C6H4 66 225 63.50/63.46 3.62/3.68 19.49/19.54 (359.40) C19H13N5OS 3f 4-HO-C6H4 61 280 63.50/63.53 3.62/3.58 19.49/19.45 (359.40) C20H15N5OS 3g 4-CH3O-C6H4 69 165 62.53/62.49 4.21/4.19 18.77/18.81 (373.43) C21H17N5O2S 3h 3,4-di-CH3O-C6H3 62 198 62.53/62.49 4.21/4.19 17.36/17.31 (403.45) C19H12N6O2S 3i 3-NO2-C6H4 72 171 58.76/58.80 3.09/3.13 21.64/21.69 (388.40) C21H18N6S 3j 4-N(CH3)2-C6H4 67 240 65.28/65.32 4.66/4.69 21.76/21.81 (386.47) C21H15N5S 3k C6H5-CH=CH 61 195 68.29/68.33 4.06/4.11 18.97/18.92 (369.44) C H N OS 3l 2-furyl 17 11 5 62 180 61.26/61.31 3.30/3.36 21.02/21.08 (333.36) 21 22 Acta Pharm. M. A. Azam Table II. Spectral characterization of the new compounds Compd. IR (KBr) (n,cm–1) 1HNMR(d, ppm) 13CNMR(d, ppm) MS 63 (m/z) et al. (2013) 19–30. 3a 3057 (Ar-CH), 2972 (CH3), 1633 (C=N), 8.21 (s, 1H, N-CH=N), 8.03–7.69 (m, 4H, ArH), 343 : Synthesis and biological evaluation o + 1600 (Ar-C=C), 756 (C-S-C) 7.54–7.21 (m, 5H, ArH), 2.48 (s, 3H, CH3) (M ) 3b 3048 (Ar-CH), 2970 (CH3), 1627 (C=N), 8.28 (s, 1H, N-CH=N), 7.99–7.81 (m, 4H, ArH), 376 + 1599 (Ar-C=C), 752 (C-S-C), 727 (C-Cl) 7.79–7.26 (m, 4H, ArH), 2.45 (s, 3H, CH3) (M –2) 3c 3057 (Ar-CH), 2979 (CH3), 1631 (C=N), 8.31 (s, 1H, N-CH=N), 8.17–7.86 (m, 4H, ArH), 378 + 1595 (Ar-C=C), 755 (C-S-C), 729 (C-Cl) 7.76–7.38 (m, 4H, ArH), 2.39 (s, 3H, CH3) (M ) 3d 3070 (Ar-CH), 2976 (CH3), 1626 (C=N), 8.34 (s, 1H, N-CH=N), 8.11–7.83 (m, 4H, ArH), 380 + 1610 (Ar-C=C), 756 (C-S-C), 727 (C-Cl) 7.72–7.31 (m, 4H, ArH), 2.15 (s, 3H, CH3) (M +2) 3e 3064 (OH), 3034 (Ar-CH), 2971(CH3), 1610 8.44 (s, 1H, N-CH=N), 8.18–7.76 (m, 4H, ArH), 359 + (C=N), 1595 (Ar-C=C), 758 (C-S-C) 7.69–7.38 (m, 4H, ArH), 5.23 (brs, 1H, OH, D2O (M ) exchangeable), 2.35 (s, 3H, CH3) f some novel pyrazolopyrimidines incorp 3f 3066 (OH), 3030 (Ar-CH), 2981(CH3), 1630 8.28 (s, 1H, N-CH=N), 7.88–7.78 (m, 4H, ArH), 358 + (C=N), 1579 (Ar-C=C), 763 (C-S-C) 7.64–7.27 (m, 4H, ArH), 5.02 (brs, 1H, OH, D2O (M –1) exchangeable), 2.29 (s, 3H, CH3) 3g 3010 (ArH), 2966 (CH3), 1622 (C=N), 1580 8.41 (s, 1H, N-CH=N), 8.11–7.70 (m,