Wn Journal of Pharmacology and Toxicology ISSN 1816-496X @ Academic Journals Ine. www.academicjournals.comJoumal of Pharmacolegy and Toxicology 3 (2): 102-110, 2008, ISSN 1816-496%, © 2008 Academic Journals Ine. Synthesis and Studies on Some New Fluorine Containing Hydroxypyrazolines and 1H Pyrazoles-as Possible Antiproliferative Agents °B, Sooryanarayana Rao, 'P.M. Akberali, 8. Shivarama Holla and °B.K, Sarojini “Apotex Pharmachem India Pvt, Ltd., PlotNo. 1A, Bommasandra Industrial Area, 4th Phase, 562 158 Bangalore, India "Department of Chemistry, Mangalore University, Mangalagangotri, 74 199-Mangalore, India Department of Chemistry. P.A. College of Engineering, Nadupadavu, Kairangala-574153, Mangalore, India Abstract: A series of twenty four newly synthesized I-aroyl-3-anyl-S-hydrexy- dichloro-s-fluorophenyl) pyrazolines (3) and. 1H-3-aryl-S-hydroxy-5-(2,4-dichloro-5- AluorophenyD-pyrazoles (6) were tested for eytostatc and eytotoxie effets on ina primary three cell line-one dose anticancer assay against NCI-H 460 (Lung), MCF 7(Breast) and SF 268 (CNS), Proliferation of these cancer cell lines was strongly inhibited by eleven compounds, These eleven compounds were then pessed on for evaluation in the full panel of 60 cell fines derived from seven eancer types namely, Lung, Colon, Melanoma, Renal, Ovarian, CNS and Leukemia, These compounds showed antiproliferative activity on the whole cell panel. Compound 1H-pyrazole, 64 [3,4-methylenedioxy at C 3] showed highest activity with Growth Inhibition (Gl) value <10 1M against all tested 60 cell lines except for Leukamia CCRF-CEM, HL-60TB, K-562 call lines. Whereas hy droxypyrazolines 3i, 3k 3m, 30, 3p and 3q showed moderate activity with Gly value <50 wM against all tested 60 cell lines, Compounds 3h, 3¢, 6¢ appear to be less active with Gl Value >100 uM for some of the tested cel lines, Compound 6a appears to be least active with Gl. valle >100 joM for almost all the tested cell lines. The Total Growth Inhibition ¢TGI) and Lethal Concentration (LC,))valtes for the most ative compound [6d] found to be 100 uM. for Leukemia cell lines and for the other cell lines these values remain <20 uM and hence prove to be a cytostatic and eytotoxie for these lines. Hence these newly synthesized pytazole and pyrazoline derivatives showed promising antiproliferative property. Key words: Hydroxypyrazolines, IH-pyrazoles, antiproliferative activity. INTRODUCTION Pyrazolines have been reported to show a broad spectram of biological activities including antibacterial, antifungal, antiinflammatory and antidepressant activities (Elgewro, 1984). The pyrazotine finetion is quite stable and fas inspired chemists to utilize this stable fragment in bioactive moieties to synthesize new compounds possessing biological activities. The presence of fluorine in the ‘molecules at strategic positions alters the activity (Filler and Kabayashi, 1992). Chalcone dibromidss are very usefil synthons in the synthesis of bioactive molecules such as pyrazolines, pyrazoles, isoxazoles, lavones,flavonols, flavanones, aurones, coumarones,tetralones, aziridines etc. The anti- Corresponding Author: BK Saji, Devarinent of Chemisty, PA, College of Enginesing, Nadipadav, [S7H1S-Mangalore, India Tel, +918242284701/02003 Fax: +912284705, 102J. Pharmacol. Toxicol., 3 (2): 102-110, 2008 infective and ant-inflammatory properties of chalcone derivatives were reviewed by Nowakowska (Nowakowska, 2607). The literature survey revealed that after 1989, the synthesis of hydroxy Pyruzolines (Stuctie 1) was reported mostly from Holla’s group (Holla etal, 1989; Holla et al, 2006; Holla ef al, 2006; Holla ef al, 2006; Karthikeyan er al, 2007). Bonacorso and others (Bonacorso et al. 2006) reported the regiospecific one step synthesis of heteroaroyl-2-pyrazolines under mild conditions. Prompted by the biological activity of the pyrazole derivatives the present work is undertaken ‘This study presents the synthesis of hydroxypyrazolines, 1H-pyrazoles and to sereen the newly synthesized heterocycles for their antiproliferative activity. MATERIALS AND METHODS, ‘The synthesis ofthe target molecules 3 and 6 which is given inthe Scheme 1 was cartied out by B. Sooryanarayana Rao at the Department of Chemistry, Mangalore University according to the reported procedure (Holla etal, 1989) during his Ph.D. programme. Melting points were taken in open capillary tubes and are uncorrected. IR spectra in KBr pellets were recorded on JASCO FT-IR 5300 Infrared spectrophotometer. 'H NMR spectra were recorded in DMSO-d, on Varian (300 MHZ) spectrometer using TMS as an intemal standard and the mass spectra were recorded on aVG-S-70 micro mas, mass spectrometer oparating at 70eV. The purity ofthe compounds were checked by TLC using ethylacelate:methanal [8:2] solvent system. lodine was used as visualizing agent. The characterization data are given in the Table 1 and 2 and spectral data are given in Table 3. The anticancer studies were carried out at National Institute of Health, Bethesda, Maryland, USA under the Drug Discovery programme Antiproliferative Activity ‘The newly synthesized compounds 3 and 6 were sereened for their antiproliferative activities at NIH, Bethesda, Maryland, USA under the Drug Discovery Programme of NCI according to the procedure suggested by Boyd and Paull (1995) in a primary three cell line-one dose anticancer assay against NCI-H 460 (Lung), MCF 7(Breast) and SF 268 (CNS), Inthe current protocol each cell line isinoculated on an incubated micro titer plate. The test agents were added at a single concentration and the culture was incubated for 48 h, Endpoint determinations were made with Sulforhodamine B, a protein binding dye. Results for each test agents were reported as the percent grovith of the treated cells when compared with the untreated control cells. Compounds which reduce the growth of any one of the cell lines to 32% or less (negative numbers indicate cell kill) in primary three cel line-one dose anticancer assay were considered as active and these compounds were then passed on to 60-cel line sereening studies, 103J. Pharmacol. Toxicol., 3 (2): 102-110, 2008 co Br HAC 2A-C1; 34(0CH)5 34-0080 R= Cg 4-CIC; Py Scheme 1: Synthesis of I-aroyl-3-aryl-S-tyydroxy-5-2,4-dichloto-S-fluorophenyl) pyrazolines (3) and 1H-pyrazoles (6) “able 1: Charactervation dita of 1sroy-3-ay Sy any-S-(2.4-dchloro:S-fuophers) gyrazotnes (3) ‘Analysis 6) found (alelated) Yiew Rt ey H N or 8 SLSIGLS 34084) 64652) cH, 78 602106013) 3.64370) 5.98.10) ca, 8 S84758.99) 3.8889 64610) Cae MH $822158.35) 3.16817) 5.84592) GH 76 565218696) 3.09302) 5.961601) 3 cH, TB S3269580 26161) 5.45.62) 4 4-C1CH, PR SSSHSS3) 3.1882 160) sh ACI: 16 S4BKSSOL) 34684) 5.72.67) 3 4.C1Ca 68 SHINS39) 271A 7H 3.31838) 31 ACI, 8 S32KSBS1) 32830 5.44552) * 4c, 6 S3185301) 25061) 55x50) 31 OIG, PR 491649.) 22125 5.55.62) 38 pyri) 9 $8258.60) 3.28321) 9.679.77) ae 40cH, yd Jo 870218739) 330847) 9.02013) 30 S40CH, pyr 68 $0856.33) 36186) 8488.57) 3p B4s0CEO) pric T 38345860) 28295) TABS) 4 “cl pyri! Tl S443(S12) 27379 —_R91B.0H, = 24.1, py oF 8027(5050) 248240) 8371842) 104J. Pharmacol. Toxicol., 3 (2): 102-110, 2008 ‘able 2 Characterization data of 3 4ev.S-(44 DeAD-purwoles (6) ‘analysis (99) found (caleuned Compounds No. R © H N 6a H 5841863) 2882.93) 9.07(9.13) & 40cH, 5648(50.97) 3216.26) 8361830) 6 34OCH) '55.36(85.58) 3490350) 751768) 6 34(0CHL0) 4375470) 2521256) 7189798) fe 4cl n 523465270) 2230.34) 812819) a 24-01 20 2759.89 170.89), 7.400745) “able 5: Spectral dita of some of the newly synthesized compounds ComposndNo. Spectral data a ‘3a! IR (KB ise) Yoga (6m): 3242(0-F), 3099 (CH), 1GENC=O) 1372 (C—O), 1081 (CF), TB (CCH Se: IR OBE tise) Ynye (am): $2520.40, 3014 (CAH), 1646(C=0) 1597 (CN), 1514 (C-0),1086 (C-), 731 (C-Ci) 3-8 (KBE dis) Yu (Im): 3048 (CHD, 1GHLC-O), 1608 (CN), 1580 (C=C), 1068 (C-F), 146 (C.C).3a: “FINMIC 300 MHZ, (CDCL*DMSO-,): 5 7.86 (, 1H. AME, Jutogo™ 78 H2) 8.0104, 18, APH J°10.6 H2), 806 (1H, APH, fap 6.3 2), 18. 7-72, OFL ArH). $43 (, IH,-OF, 8.62(dd, 2H,CH, J=18.3H2), se 'ENMR 300 MHZ. (CDCI): 67.86 IH, APH, Jyeqg7 7.8H2), 801G, 1H, Ar-HJM10.6 Hz), 8.06 (1H, AM-H, ayn 63 H2),7.43-7.96 (on, 9H ArH, $39 (, 1H-OF), 3.6244, 2H,CH, T= 1828), Pa "ag NMR 300 OZ (CDCI): 8 739 (4, IH, APH, Jyyage= 718 He), 7.71 IH, ArH I= 10.6 Hz) 7.94, 1, APH, Jp ya = 63 Ha), 7397.43 (my THE APD, 5.41 (, TH-OF), 3784, 24, CH, J = 18682). 31H NMIR 400 MHZ (CDCI) 8 6 854d, IH, ArH, 3= 8 1Hz), 7.086, 1H, ArHJ= 10642), 71, 1H, ArH, J= 18 Hz), 12864, 1H, ArH, J= 6982), 7.93, 1H, [APH,J = 6982) 7.43 mn, 3H, Ar-H), 603 (6, 2H-OCH,O-), 539 6, 1-0), 3.59 (dd, 2H, CH, 3 16H) sk: HNMR 400 MHZ (CCL): 7.2166, IH, APH, 736 , 7H, Ar-#D,7.5704, 1H, ArH J = 10.6 Hy), 786 (1H, AvH, J~ 8542), $42 @ IHOH), 365 (dh, 2H.CH, 8 6H2)3n: "HNMR 400 MHZ (CDCI): 8 6 95(4, 2H, Ar-H, J= 8 THz), 745 (1H, ACH GHz), 7.674, 2H, ArH, J= 88 Hz), 182 (4, IH, Ar-H, J= 10.182), 826 (1H, ArH, (liz) 285 (5, SHOCH,) 534 (6, IH-OH), 3.61(dd, 2H, CH, J=18. 6812 3e miz 402/404 IM’ M12), 44/446 (10%, MELO), 425/427 19%, A84-Mucrne radical), M042 Mv-berzoie acid) 191 (10% 2claichiro:S-fluerobenzontileradiel cation 1371586 phenyl cyanide radical cation), 108(100% berzoyl cation), 77 (45% phenyl cation)Si mz 22/824 (20% Mi M'#2), $0806 (25%, MP-H,0), 485487 (15% SOF-lucrne radial), 366/368 (106 Mt ehlorabenzoie acid) 191 (10%, 2yt-dichloro-Stluoroberzonitile radial cation), 681% 3 methoxy phenyl cyanide radical cation), 139(100% benzoyl cation), LLL (48% 3.4-dimethosy benzyl cation) Sr: v2 497/490 (208 MY MP-+2), 481/482 (25%, MT-H0), 462464 (13%, 481- fMuorineraiical, 376378 (10% MT-nicoinic acid) 191 70%, 24 ‘Dvorcberznitrile radial cation), 163.3% 2,4ichloro-Sfhoro phenyl ralicaleaticn), 166(100% pyrideyl cation, 7916 pyri cation), & "HNMR 300 MHZ (CDCI): 6 385 (6 3H, OCH,) 697m, 3H, Ard), 7256, 1H, CAH praca, 7-52(d, IH, APH, J= 69H), 7.6l(m, 2H, Ar-H, 4.34 1H-NE). of 'H_ NMR. 300 MHZ. (CDC) 8 7.17, IH, CLH pyrazole), 7.38 (84, 1H, Ar-H, J= 2 Hi, 7.5200, 1H, APH, J= 682), 7.6200, 1H, ADH, J=9.5 Hz), 16S, 1H, APHL, J= 85H, 4.34 LN RESULTS AND DISCUSSION Im the present anticancer sereening program of L-arayl-3aryl-S-hydroxy-5-(2,4-dichloro-5- flurophenyl) pyrazolines, I-benzoyl-3-phenyl-5-hydroxy-5-(2,4-dichloro-S-flurophenyl) pyrazoline 3e, I-t-chlorobenzoyl-3+4-methoxyphenyl)-S-hydroxy-S-(2,4-dichloro-S-fluropheny!) pyrazoline 3h, 1-(4-chlorobenzoyl)-3-C-chlorophenyl)-S-hydroxy-5-(2,4-dicloro-S-urophenyl) pyrazoline 3k, 1-(4-chloroberzoyl)-3-(34-dimethoxyphenyl)-5-hydroxy-5.(2,4-dichloro-5-flurophenyl) pyrazoline 3i, I-pyridoyl-3-phenyl-S-hydroxy-5-(2,4-lichloro--furopheny) pyrazoline 3m, 1-pyridoyl-3- I-tiehloro-S-flurophenyl) pyrazoline 30, I-pyridoyl-3-44 iydroxy-5-(2,4-dichloro-S-flurophenyl) pyrazoline 34, L-pyridayl-3434- ‘methylinedioxypheny!)-5-hydroxy-5-(,4-dichloro-5-fhurophenyl) pyrazoline 3p and 1H pyrazoles 3ephenyl-5.(2,4-dichloro-S-flurophenyl) pyrazole 6a, 3-(3.4-dimethoxyphenyl)-5-(24-dichloro-5- flurophenyl) pyrazole 6e, (3,4-methytinedioxypheny!)-5.(2,4-dichloro-S-flurophenyl) pyrazole 6d, 105J. Pharmacol. Toxicol., 3 (2): 102-110, 2008 ‘Table: Prelrinay in vivo nicancer serena 2 data of Active |-aroyl-3-aryl-S dy dry-524-ichloeo-Shurephes)) tazolines (3) and Sary-S 424 chloro S-fhrop hen - MET gyrazole (6), Growth percentage nse Compounds _No. Sample eone.*10"*0M0 * * 713822 sh 13824 xe 713825 3i am Breast MCF? 713326 Tisees 30 713085 ia 715686 5p 715687 fa 6 68 71688 7153680 a 6 713500 a 2 ‘Fined cancetaton assay (000 pM, sandard NCI protoca, Percent cell owt redaction flfowing 48H incubation with test compounds (optical density, sulforhodamine procedure), = Active when grow percentage i =32%6 for any of the thre cll ines “Table 5 Sixty ell line in vitro anticancer serening of (3) and (6) (Gt, uM) Gly, uM Panetiell line 3k shea Sq__p_ awe Lenkemin CORFCEM «188502 199 Ud 133 Bo 6 380 Hoe) 2 SLs 049 ne isl 22 136 100 K-50 73s 407 583 itd 166 379 461 508 MOLT-+ 935 128 43. 897 645133 us >100 4122272 m2 350 728 8817 HI 968 216 204 287100 40.7293 218 196 aos 61s 2 200 162 190 Iso ots 399236 me Md 28 >10 206 207 is6 128 162 S078 bss 2024 280 100300226, 322 238 27 ml 28928 186152 410 3213 2618 10 102312 Ml 123 997 151 >100 19.2.8 BS M8 Ral 163 m0 87 262 M8 136 180-1022 GBF 196 123 M8 17 Ho 21 469 257 191 28 269 7100 6d 438 MS 180 22 BE M10 6 S38 306 182 199 100 292-240 163 1s 191 2100 183264 Isa 2 167 30 159 Lao 400 413-26 10S 319 164 ST 182 71033380 164 M3 167 SIS 269190 ‘Melanoma LOXIME = 182,79 TTD S739 16S 992184290 MALMESM 306 100 S19, 28.3 DG Bd 100 30d 49 Mu 27 ua 736 189 S18 210 758.0 az SKMEL-2 170 27-290 aus m7 >10 796 = 170 SKMB.28 M3 5100498 21198 ass 100 4873.28J. Pharmacol. Toxicol., 3 (2): 102-110, 2008 ‘Table $: Cone Gln, BE Panetlell, line SK MELS VACC-257 WACC-62 ‘Ovarian cancer IGRVOL OVCAR-S OVCAR- OVCAR'S OVCARS SK-OV-3 oa >100 261 a8 100 194 Renal cancer 780-0 189 >100 we ml mi 186 100 138 A498 136716 se _ : ‘ACHN 213 >100 Me 232 213 178 635 193 CAKE be o74 186333241 287 >100 5358, RXF-98 239 >100 615 167 185 7 5100 199) sNIzc Ms 391 2) 2 iss 185 =100 248 TK: 527 >100 B68 4.0 308 391 >100 222 vost 90s 169 308 286 174 173 100) 292 Prostate cancer Pes od 20 4 GA 2S 126 19 2S m0 2 292 Di-ids 273-100 30 851 321-279 169-209 >100, S83 Breast cancer MCF Ts 90 sk ask NCUADRRES 184 483 G2 2s 18s 100251363 MDAMB: M2100 422 187 3 S84 308188 2sUATCC BSs78r 390 >100 mo 20 3R0 100437 as MDAMB35 167 186 269 2 S10 462491 MDAN 6 24 S76 26 23 S10 «28 610 Br-s9 22 2100 2100 = 203. 210 100302399 TD 995 577508 627123, 359100285 220, possessed growth percentage to less than 32% against all the tested 3 cancer cell ines and were regarded as active compounds, Presereen results are given in Table 4. These 6 compounds were then passed on for evaluation inthe fll panel of 60 call lines derived from seven cancer types namely, Lung, Colon, Melanoma, Renal, Ovarian, CNS and Leukemia. These compounds showed antiproliferative activity on the whole cell panel. The screening data is presented in Table 5. Compound 1#-pyrazole 6d, (3,4-methylinedioxyphenyl)-5-(2,4-dichloro-S-flurophenyl) pyrazole, showed highest activity with Growth Inhibition (Glo) value <10 tM against all tested 60 cell lines except for Leukatnia CCRF-CEM, HL-60TB, K-562 cell lines. Whereas hydroxypyrazolines 3i, 3k, 3m, 3o, 3p and 3q showed moderate activity with Gly value <50 uM against all tested 60 cal lines ‘Compounds 3h, 3, 6¢ appear to be less active with Gl., value >100 jiM for some of the tested ell lines, Compound 6a appears to be least active with Gl. value >100 1M for almost all the tested ell lines, ‘The most active compound éd emerged as most effective against Non-small cell Lung cancer cells A549/ATCC = 2.93 1M, EKVX = 2.09 uM, HOP-62 = 2.36 uM, HOP-92= 2.07 uM, NCI- SSM, NCI-H23 = 2.26 pM, NCIH322M = 2.73 M, NCI-H460 = 3.12 uM, NCI- 3.12 AM among the tested cel lines ‘The Total Growth Inhibition (TGD and Lethal Concentration (LC.) values for the most active ‘compound [64] is given in Table 6, For leukemia cel lines both parameters are more than 100 Manel for the other cell lines these values remain less than 20 1M and hence proves to be a cytostatic and cytotoxic for these lines. With this information it is immature to comment on structure activity relationship. However, it appears that the presence of 3.4-methylenedioxy and pyridyl moieties may also contribute to their enhanced activity. 107J. Pharmacol. Toxicol., 3 (2): 102-110, 2008 ‘oble 6 Total Growth Inhibition (TGI) and Lishal Concerration (LCS0) of 3-3.4-Methylenedioxy phen) SQ dichloro-S-urophen DD gyrazole (6d) Pantie _ line Lc80 GN) Leukemia CCRF-CEM S100 HL-60 (TB) 100 KS02 100 MOLT-+ S100 RPMI-8226, 100 SR P10 ‘Non-smal el Tung cancer AMB/ATCC EKVX HOP-62 HoP-92 NeHHI26 NOLES NCTH20M N60 NOTH Colon cancer sat 2s 500 iss 6.02 >100 196 es 100 P00 340 P00 626 S100 1.63 MA 297 S91 ns HS 568 753 3M 638 Melanoma LOXIMVI 833 408 ‘MALME-3M 48 S100 ML 29 100 SK MEL: 390 97 SK MEL-28 10 484 SK MEL-S 355 uACC257 282 00 VACC-62 : : ‘Ovarian cancer IGRVvOL aus OvcAR-S 825 OVCAR- 277 OVCAR'S Si OVCARS 698 SK-OV-3 1s. ‘Renal cancer 386 796 an 735 262 P10 44s 4 6.96 5.07 108J. Pharmacol. Toxicol., 3 (2): 102-110, 2008 ‘Table 6: Contre a Panetiel — line organ 1Lc80 GN Prostate cancer POS a 23 Di-ids 130 eu Breast cancer MCE? : . NCUADR-RES 163100 s 783 100 P00 MDAMB-435 343 P00 MDAN 331 S100 Br-si9 168 S40 TD 1. 733 CONCLUSIONS With the history of pyrazole derivatives as potential bioactive moieties and in @ hope to find potential molecules with antiproliferaive activity we synthesized fluorine containing hydroxypyrazolines and pyrazoles. The newly synthesized compounds were characterized by analytical and spectral studies. In the present anticancer screening program of I-aroyl-3-aryl-5- hydrexy-5.(2.4-dichloro-S-fluropheny!) pyrazolines, compounds 3c, 3h, 3k, 3i, 3m, 30, 3q, 3p and 1H pyrazoles 6a, 6c, 6d were emerged a5 active compounds, IH-pyrazole 6d [3,4-methylenedioxy atC 3] showed highest activity with Growth Inhibition (Gly) value <10 j1M against ll tested 60 ell lines except for Leukamia CCRF-CEM, HL-60TB,K-562 cell lines. Whereas hydroxy pyrazoline compounds 31 [3,4-methylenedioxyphenyl at C3 and 4-chloro phenyl at C | substitution), 3k [4-chloro phenyl at C 3 and 4-chloro phenyl at C 1 substitution), 3mfphenyl at C3 and pyridyl at C1 substitution], 30 [3,4-dimethoxyphenyl at C3 and pyridyl at Cl substation), 3p [34- ‘methylenedicxyphenyl at C3 and pyridyl at C1 substitution] and 3q [4-chloro phenyl at C3 and Pytidyl at C1 substitution) showed moderate activity with Glyy value 100 jtM for some of the tested cell ines. Compound 6a [phenyl at © 3 substitution] appears to be least active with Glzp value >100 pM for almost all the tested cell Lines, The Total growth inhibition (TGI) and Lethal Concentration (LC. values for the ‘most active compound [6d] is given. For leukemia cell lines both parameters are >100 4M and for the ‘other cel lines these values remain <20 iM and hence proves to bea cytostatic and eytotoxie for these lines. It appears that the presence of 3,4-methylenedioxy and pyridyl moieties may contribute to their enhanced activity. However, it is hoped that these pyrazole derivatives may emerge as potential ‘compounds for antiproliferative activity ACKNOWLEDGMENTS: ‘The authors are grateful to Head, RSC, CDRI, Lucknow and The Director, RSLC, Punjab University, Chandigarh, for providing microanalysis, IR, 'H NMR and mass spectral data, The authors ae grateful to Dr. V.L. Narayanan, National Institutes of Health (NIE), Bethesda, Maryland, USA, for the antitumor activity screening studies reported in this research, 109J. Pharmacol. Toxicol., 3 (2): 102-110, 2008 REFERENCES Bonacorso, H.G., AP. Wentz, R-V. Lourega, C.A. Cechinel, T'S. Moraes, H.S. Coelho, N. Zanatta, MAP. Martins, M. Hoetner and SH. Alves, 2006, Trifluoromethyl-containing, pyrazolinyl (p-tolyl) sulphpones: The synthesis and structure of promising antimicrobial agents, J. Fluor Chem, 127: 1066-1072, Boyd, MR, and K.D. Paull, 1995. 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