Indian Journal of Chemistry Vol. 42 B. August 2003 . pp. 1950- I 957

Synthesis of novel heterocyclic compounds: Routes to pyrazolyl 1,2,3-triazoles and their biological activity evaluation

Ajay Kumara.h

, Mofazzal Husa ina.c

, Ashok K Prasad", lshwar Singh", Archana Vats", Nawal K Sharma,,·b, Sunil K Sharma", Raj inder K GuptaC

, Carl E Olsend, Marc E Brackec

, Richard A Grossb & Virinder S Parmara.b.-

"Bi oorganic Laboratory. Department of Chem istry, Uni vers ity of Delhi. De lhi- 110 007. Indi a; bDepartmel1l o r C hem ist ry and C hemi cal Eng ineering, Polytechnic Uni versity, Si x Metrotech Center, Brooklyn. Ne w York 11 20 I, US A: "Schoo l o f

Biotechnology , Guru Gobind Si ngh Indrapras tha Uni versity. Kashmere Gate, De lhi 11 0006. India: dChemistry Department. Royal Veterinary and Agri cultural Uni versity, Frederiksberg C , DK - 187 1 Cope nhagen. Denmark; CLaboratory of Exper i­mental Cancero logy, Department of Radiotherapy and Nuclear Medicine. University Hospita l. De Pinte laan 185. B-9000

Ge nt , Be lg ium

Received 3 October 2002; accepted (revised) 6 March 2003

A series of 5-aryl-3-cyanomethy lpyrazoles have been sy nthesized by re Ouxing 6-aryl-3-cyano-4-methy lt 10-2H-pyran -2-ones wi th hydrazi ne. T he acti ve methyle ne moiety o f these pyrazoles has further been ex pl o ited to build 1,4-d isubstituted 5-amino- I ,2.3-triazolcs via the ir base-cata lyzed condensation with 3/4-nitrophenyl azidcs. All these compou nds have bee n characteri zed by detail ed spectral ana lys is and chemi cal transformat ions LO confirm the ir struc tures unambiguously. whi ch were proposed inconc lusive ly five decades ago. Furthe r. these pyrazolyltriazoles have been tes ted as anti invasi ve agents aga inst so lid tumors and as antimycobacLeri a l agents.

Living organisms find diffi culty in constructing N-N bonds that limit the natural abundance of compounds hav ing such bonds. Py razol es and their de rivatives,1 a c lass of compounds conta ining the N-N bond ex hibit a wide range of bi ological act ivi ti es, e.g. antipyre ti c,2.3 antiinfl ammatory,4 gastric secretion stimulatory,S anridepressent,6 antihypercho lesterolemic ,7 anti­bacteria l,s antifa lari a l,9 antioxidants lO and against rheumatoid arthritis. 11.I 2 The recent success of a pyrazo le COX-lJ inhibitor has further hi ghl ighted the importance of these heterocycles in medicina l chemistry.1 3 Pyrazo le de ri vati ves a lso find applica­tions as herbicides,14.15 fungicides,15 pesti c ides and

insecticides. 16 Triazo les and the ir derivatives are another class of hete rocycles, which have been widely studi ed for their medicina l and industri a l applications. 17 We envi saged that incorporation of both the triazole and pyrazole moieties in one molecule might enhance the biolog ical activities of the resulting compounds. Based on our earli er experience of synthes izi ng different classes of heterocycl ic compounds,1 8-23 we have desig ned and developed a strategy to bring together the pyrazole and tri azo le moieti es in a sing le structure.

1,4- Disubstituted 1,2,3 -tri azo les have, earlier been synthes ized by Dimorth24 in 1909 using base­catalyzed condensation of aryl azides with phenyl

acetonitri les. He also reported that I ,4-di substituted 5-amino- l ,2,3-tri azo les undergo a rather fac ile and ap­parently revers ible isomerization to 5-(substituted)­amino-4-substituted-l ,2,3-tri azo les (Figure 1). Li eber and coworkers25.26 lale r (in 1957) made more ex haus­tive examination of the effects of substituents on the position of the equi.librium between these two iso­meric triazo les; kinetics and energetics o f the iso meri­zation were a lso studi ed . However, thei r hypothesis of isomeri zation was based on the melti ng po in t and ti­tration data of the crude products on ly . They were unable to separate the two isomers of uch triazo les, a lso the compounds were not fully charac teri zed from their spectral data. [n this investi gation, we report a simi lar strategy involving the base-cata lyzed condensation of 3/4-nitrophenyl azides w ith 5-ary l-3-cyanomethy lpyrazoles leading to the formatio n of a series of 5-amino-4-(5-ary lpyrazol-3-y l)- I- (3l4-nitro­phenyl)-1 ,2,3-triazoles, all of which have been unam­biguously characte ri zed from the ir Y, JR , IH NM R,

H2N 5 4 R"

F==T N / N3

R' / 1 ' N/ 2

1,4-Disubstituted-5-amino-1 ,2,3-triazole

R'HN R"

f==( H/ N' N~N

4-Substituted-5-substituted­amino-1,2,3-triazole

Figure I - Isomeriza tion of I A-di substituled-5-amino- 1 ,2.3-triazole

KUMAR el 01.: SYNTHES IS OF NOVEL HETEROCYCLIC COMPOUNDS

SCH3 CN (iv) (i) , (ii) CH3S~SCH3 (iii)

+ - I OCHCH ~ .. NC~OCH2CH3 NC 2 3 R '=inCH,

-

R

o 0 0

1 2 R

CN

(v)

R'~N3 R,}-!I

5. R' = H; R" = N02

5b R' = N02; Roo = H

.. ¢PR'Roo

5"' I ~ 3'" .0 (vi)

H2N 1 '" 6d.6f 5',/ ~ and 6g

/N N' 3'

6

o

3

2-4 R

a b c d e f

Compd

6a 6b 6c 6d 6e 6f 69 6h 6i 6j

6k

H CH3

OCH3 F CI Br

R R'

H N02

CH3 N02

OCH3 N02

F N02

CI N02

Br N02

H H CH3 H OCH3 H CI H Br H

ROO

H H H H H R

H N02

N02

N02

N02 N02

R

II ROO

R'~~ ~ '/ \

Isomer of6 (not formed)

/ N N'

R 7d, 7f and 79

ROO

~COCH3

R' r; ~ ~ - N-COCH

'/ \ 3 .................. _ ...• / N

N'

R

'/ \ / N N I COCH3

Isomeric acetate (not formed)

Scheme I. Reagents and Conditions: (i) EtOH, Na, DoC, stirring 5-6h; (ii) CH31 , MeOH,

~oC, stirring 2-3h; (iii) DMF, appropriately substituted acetophenone, KOH, 30°C, stirring ' 6h; (iv) MeOH, NH2NH2 , refluxing 5-6h; (v) EtOH, sodium t-butoxide , 0-5°C, stirring 24-96h;

(vi) acetic anhydride, cat. amount of DMAP, 25-28°C.

1951

l.1C MR and mass spectral data. Surpri singly , we did not observe the formation of the isomeric products, i.e. the 4-(5-arylpyrazol-3-y l)-5-(3/4-nitrophenyl)­amino- I ,2,3-tri azo les (Scheme I).

Results and Discussion Eleven pyrazoly ltri azo les 6a-6k have been

synthes ized by the condensatio n of 3-cyano­methylpyrazoles 4a-4f with 3-/4-nitrophenyl azide SalSb in the presence of sod ium te rt-butoxide in

ethano l at 0-5°C in 70 to 95% yield (Scheme I). 3-Cyanomethylpyrazolcs 4a-4f, in turn were synthe­sized in four steps start ing with the condensat ion of ethy l cyanoacetate with carbon disulfide in the presence of sodium ethox ide, fo llowed by methylation of the resulted disodio salt with methy l iodide to afford ethyl 2-cyano-3,3-bis-methy lthioacry late (1 ), The reaction of ac rylate 1 with corresponding acetophenones 2a-2f resulted in the fo rmat ion of pyranones 3a-3f, which on condensation with

1952 INDI AN J. CHEM ., SEC B, AUGUST 2003

hydrazine in methano l affo rded 3-cyano methyl­pyrazo les 4a-4f in 50-60% yie lds ' 8.22 (Scheme I). It has been observed that the rate of tri azo le format ion with 4-nitropheny l azide 5b was faste r than that with 3-nitropheny l azide Sa. We were able to get more than 85% isolated y ields o f the pyrazo ly ltri azo les 6a-6f prepared from 4-nitropheny l az ide 5b in 24-48 hr, whereas the y ie lds were less than 40% in the case of corresponding reactions with 3-nitrophenyl azide Sa. The y ie ld of pyrazo ly ltriazo le fo rmati on with 3-nitropheny l azide Sa was inc reased by filtering of the precipitated product afte r 48 hr and by further continuing the stirring o f the filtrate at 50-55°C for another 40-48 hr. The y ie lds were inc reased from 40 to nearl y 70% under the modi fied condition o f the reacti on. It has been observed that the contro l of initi al condensation temperature at 0-5°C is necessary as at higher temperature , the y ie lds decreased signi ficantly. T hi s decrease in y ie lds may be due to the instability of the nitropheny l az ide or due to the occurrence of side reactio ns. Hi gher y ie lds in the case of reactions with 4-nitro pheny l azide 5b o ver those with 3-nitrophenyl az ide Sa may be due to the g reater charge delocali zatio n on the fo rmer making it more stable than the latter.

All the pyrazoly ltriazo les 6a-6k are new com­pounds and the ir structures have unambiguously been establi shed o n the bas is of the ir spectral data (IR, UV, ' H NMR , '3C NMR and ElMS). Due to the poor solu­bility of pyrazo ly ltri azo les in organic sol vents, CDCl3 contai ni ng 5-10% deuterated tri fl uoroaceti c acid was used LO carry out the ir NMR spectra l record ing . The ' H NMR spectra of pyrazo ly ltri azo les 6a-6k exhibited resonances fo r a ll the proto ns covalentl y linked to carbo n (ef Experimenta l). The peaks in the 'H NMR spectra of 6a-6k were ass igned o n the bas is of com­parison of 'H NMR spectra of the ir precursors 4a-4f and 5aJ5b. The di sappearance of the acti ve methy lene proton signal resonating at around b 4.00 ppm in the ' H N M R spectra of 4a-4f and that of the CN band at around 2200 cm-' present in the ir IR spectra indicated the linkage of the azide to the acti ve methylene group of the pyrazole to form pyrazo ly ltri azoles 6a-6k. The IR spectra of a ll the pyrazo lyltri azoles 6a-6k exhib­ited two absorption bands between 3202-3443 cm-' (ef Experimenta l), very characteri stic of a primary amino group. Thus, the presence of the primary amino

group at C-5' pos iti on was indicated, thereby showing the formation of only one set of isomeric compounds, i.e. the 5-amino-4-(5-arylpyrazo l-3-yl )-I-(3/4-nitro­pheny l)- 1,2,3-tri azo les in the present study . In order

to further confirm the structures of compounds 6a-6k, three representati ve pyrazoly ltriazo les 6d, 6f and 6g were acety lated with aceti c anhydride and d imethy l­aminopyridine (DMAP) to the tri aceta tes 7d, 71' and 7g, respecti vely (Scheme I ). The ' H NMR spectral anal ys is o f the tri acetates 7d, 7f and 7g revealed the presence o f two sets o f N-acety l proto ns resonating

between 8 2 .34-2.40 and 2.59-2 .67 for 6H and 3H, respec ti ve ly which c lea rl y ind icate the forma ti o n of 5-(3/4- nitrophenyl)am ino-4-(5-ary Ipyrazo l-3-y 1)- 1-(3/4- nitropheny l)- 1,2,3-tri azo les 6a-6k and not the o ther isomers, i. e. 4-(5-arylpyrazo l-3-yl)-5-(3/4-nitropheny l)amino- I ,2,3-tri azo les in the condensat ion step (v) (Scheme I ). If the isomeri c py razo ly ltri azo les had formed , the th ree N-acety I moi eti es in the ' H NMR spectra of the ir tri acetates (sh wn a lte rnati ve ly in Scheme I ) would have been seen as three d istinct peaks. Al so the fo rmati o n of isomeri c pyrazo ly ltri a­zoles hav ing secondary amino group would have ex­hibited onl y one absorpti on band in the reg ion 3200-3500 cm-' in the ir IR spectra for the am ino group. As no such peaks or absorptio ns were observed in the 'H NMR spectra of the triacetates 7d, 7f and 7g or the IR spectra of the pyrazo lyltriazo les 6a-6k, it is proved that the structures o f the py razolyltri azo les obta ined under the present reacti on conditions a re the S-amino­I ,4-di ary l- 1 ,2,3-triazo les 6a-6k, th s resolving the ambi guity in lite rature on such compounds since last fi ve decades.

Antiinvasive activity evaluation of 6a-6k Invasion is the hallmark of malignant tumors, and

is responsibl e for the bad prog nosis of the un treated cancer pati ent. Oncologists nowadays di spose of sur­gery, radiotherapy, chemotherapy and immunotherapy as effi cient tools to tackle tumor growth. In vas ion however is a more res istant problem than growth , and anti-invas ive agents are mainly lack ing in c lini cal practice. Progress in this fi e ld can be expected fro m a bette r knowledge of the mo lecular mechanisms of invasion, from the deve lopment of re levant in vas ion models in vitro and from the synthes is and iso lation of new compounds as candidates for anti -in vas ive drugs. As a part of our continued search for new antiin vas ive agents, we tested the pyrazoly ltri azoles 6a-6k in an organotypic assay for invasion . The assay of antiinva­sive activity was based on confrontation of invas ive human MCF-7/6 mammary carc inoma cell lines on embryonic chick heart fragments . Briefly , nine day old embryonic chick heart fragments were precultured and confronted with aggregates of MCF-7/6 cells.

KUMAR el al. : SYNTHESIS OF NOVEL HETEROCYCLIC COMPOUNDS 1953

After an overnight incubation on the top of semi-solid agar, the confronting pairs were cultured for another 8 days in a suitable medium. The interaction between MCF-7/6 carcinoma cells and embryonic chick heart fragments was evaluated histologically . In such con­fronting cultures, effect on the tumor cells can be di s­cerned from effects on the normal host ti ssue. Human MCF-7/6 mammary carcinoma cell s were used in thi s assay, because they can in vade the normal ti ssue fragment within one week. Although compounds con­taining pyrazol yl moiety exhibited a strong antiin va­sive acti vity in our earlier studies,19.27 surpri singly, the pyrazolyltriazoles 6a-6k showed no inhibition of in­vasion, and histologically their cultures were similar to control.

Antimycobacterial activity evaluation of 6a-6k Tuberculosi s is one of the world's most infectious

diseases which kills one person every fifteen seconds across the globe. 28 It is estimated that almost one bil­lion new infections will occur from Mycobacterium tuberculosis , the causative agent of tuberculosi s, in the next twenty years. One of the important reasons of increase in the incidence of tuberculosis is the recent explosion of multi-drug resistant strains of Mycobac­terium tuberculosis. The severity of the situation has led the World Health Organi zation to declare tubercu­losis as a global emergency.29 Tuberculosis has not faced any new class of drugs with new mechanism of action for more than 30 years. Encouraged by the an­timicrobial activity of azoles,3o e.g. fluconazole, keto­conazole and miconazole we tested the antimycobac­terial activity of all the eleven pyrazolyltriazoles 6a-6k against Mycobacterium tuberculosis H37 R v (cf Experimental); none of them exhibited antituberculor activity at 6.25 J.lg/mL concentration.

Conclusion A facile synthetic procedure has been developed

for the sy nthesis of a new class of heterocyclic com­pounds, i.e. 5-amino-4-(5-arylpyrazol-3-yl)-I-(3/4-nitrophenyl)-1 ,2,3-triazoles. The initial condensation temperature between nitrophenyl azides and 3-cyano­methylpyrazoles is very important and has to be main­tained at 0-5°C to obtain high yields. Higher yields in the case of 4-nitrophenyl az ide over the 3-nitrophenyl azide have been obtained probably due to greater charge delocali zation in the former. The pyrazolyltri­azoles sy nthesi zed showed very little potential as anti­invas ive agents against solid tumors or antimycobac­terial agents against Mycobacterium tuberculosis .

Experimental Section General: All the melting points were determined

either in a sulphuric ac id bath or on a Mettler FP62 instrument and are uncorrected. The UV and IR spec­tra were recorded on a Beckmann DU-2 spectropho­tometer and Shimadzu model 435 spectrophotometer, respecti vel y. The proton (I H) and carbon (DC) N M R spectra were recorded on a Bruker DPX300 A vance specrometer at 300 & 75.13 MHz, respec ti vely. The chemical shifts in parts per million (ppm) for IH and I3C NMR spectra were referenced relative to tetra me­thylsilane (TM S) as an internal reference at 0.00 ppm. Electron impact mass spectra were recorded either on a Varian MAT 31lA or Jeol AX 505W mass spec­trometer at 70 eV. Ethyl cyanoacetate, carbon di sul­phide, methyl iodide, substituted acetophenones, hy­drazine, meta/para nitroanilines, sodium azide, tert­butanol and anhydrous ethanol were purchased from Aldrich Chemical Co.

General method of preparation of 6-aryl-3-cyano-4-methylthio-2H-pyran-2-ones 3a-3f. To a solution of ethyl 2-cyano-3,3-bis(methylthio)pro­penoate l8 (1, 4.34 g, 0.02 mole) in DMF (40 mL), KOH (2.24 g, 0.04 mole) was added and the solution stirred for 10 min under N2-atmosphere, followed by the addition of corresponding acetophenone (2, 0.02 mole). The reaction mixture was stirred fo r 6 hr at 28-30°C, allowed to settle and the reddish-brown coloured supernatant was poured onto crushed ice (200 g) and the contents were stirred for 4 hr when a yellow solid precipitated out. It was filtered, dried and purified by column chromatography over sili ca gel using ethyl acetate-petroleum ether as eluent to afford pyranones, which were crystallized from acetone to yellow needles in 44 to 50% yield and identified on the basis of spectral data and by their compari son with h d · h I' 1831 31 t ose reporte III t e (terature. . .-

General method of preparation of 5-aryl-3-cyanomethylpyrazoles 4a-4f. To a solution of 6-aryl-3-cyano-4-methylthio-2H-pyran-2-ones (3a-3f, 6 mmole) in meth anol (30 mL), hydrazine monohydrate (9 mmole) was added and the reac tion mixture was refluxed for 5-6 hr. The progress of the reacti on was monitored by TLC; on completion, the reaction mi x­ture was concentrated under reduced pressure, poured over crushed ice (200 g) and stirred vigorously until a brownish-yellow solid precipitated out. It was filtered , washed with water (2x25 mL), dried, and column chromatographed over silica gel using ethyl acetate­petroleum ether as eluent to afford 4a-4f as white sol­ids, which were crystallized from ethyl acetate to

1954 INDIAN 1. CHEM., SEC B, AUGUST 2003

white shining needles in 50 to 60% yields. All the pyrazoles 4a-4f were identified on the basis of spec­tra l data and by their comparison with those reported in the literature.22

General method of preparation of pyrazolyltria­zoles 6a-6k. To a solution of 3-cyanomethylpyrazoles (4a-4f, 2 mmole) in anhydrous ethanol (15 mL), so­dium terl-butoxide (3 mmole) was added and the mix­ture st irred for 30 min at O°e. 3/4-Nitropheny l azide 5a15b (2 mmole) was added to the above reaction mixture in one lot and the contents stirred for 2 hr at O°C and then stirring continued at 0-5°C for 24-48 hr. Yellow precipitate started appearing in the reaction after 2-3 hr of stirring, which was filtered on comple­tion of the reaction, washed with ethanol-water (1: 1), followed by pure ethanol and dried under reduced pressure to afford the pure pyrazolyltriazoles 6a-6f in 70-95% yields and 6g-6k in 35-40% yields. The fil­trates of the reaction mixtures between pyrazoles 4a, 4b, 4c, 4e and 4f and 3-nitrophenyl azide Sa were fur­ther stirred at 50-55°C for 40-48 hr to afford pyra­zolyltriazoles 6g-6k in a combined yield of 65-70%. All the pyrazolyltriazoles 6a-6k were found to be novel compounds and they were unambiguously iden­tified on the bas is of their spectral analysis and acety­lation studies.

5-A mino-l-( 4-nitrophenyl)-4-(5-phenylpyrazol-3-yl)-1,2,3-triazole 6a. It was obtained as a yellow­ish-white solid, m.p. >255°e. UV (MeOH): 256 nm; IR (KBr): 3428,3364, 3282, 1633, 1596, 1519, 1339, 1270, 1106, 964, 949, 855 and 824 cm· l

; IH NMR (CDCI,-TFA): 87.37 (1H, s, C-4H), 7.38-7.55(3H, m, C-3" H, C-4"H and C-5"H), 7.76-7 .80(2H, m, C-2"H and C-6"H), 7.85(2H, d, J = 9.0 Hz, C-2"'H and C-6"'H) and 8.52(2H, d, J = 9.0 Hz, C-3"'H and C-5"'H); 13C NMR (CDClrTFA): 8 102.31(C-4), 112.12(C-2 '" and C-6"'), 121.18(C-I"), 125.60, 126.04, 126.78 and 129.93(C-2", C-4", C-3''', C-5'" and C-6"), 132.62(C-3" and C-5 "), and 138.07, 138.30, 141.45, 148.79 and 149.32(C-l "', C-3, C-4''', C-4', C-5, C-5'); ElMS (% reI. int.) : mlz 347([Mt, 4),346(25),3 18(22),301(12), 272(47),262(12),244(11),208(30),194(21),178(80), 151(45), 143(16), 128(18), 104(15), 97(100) and 57(90).

5-Amino-4-[5-( 4-methylphenyl)pyrazol-3-yIJ-l­(4-nitrophenyl)-1,2,3-triazole 6b. It was obtained as a yellowish-white solid, m.p. >255°e. UV (MeOH): 26 1 nm; IR (KBr) : 3426, 3366, 3285, 2925, 1634, 1597, 1519, 1337, 1274, 1108,965 and 814 cm· l

; IH NMR (CDClrTFA): 82.44 (3H, s, CH3), 7.34(1H, s,

C-4H), 7.35(2H, d, J = 7.9 Hz, H-3", H-5"), 7.65(2H, d, J = 7.9 Hz, C-2"H and C-6"H), 7.85(2H, d, J = 9.1 Hz, C-2"'H and C-6"'H) and 8.50(2H, d, J = 9.1 Hz, C-3'''H and C-5'''H); 13C NMR (CDCb-TFA): 8 21.43(CH3), 10 1.64(C-4), 117 .38 and 121.36(C- l" and C-4"), 125.38, 125.91 and 126.62(C-2", C-2"', C-3", C-5", C-6" and C-6'''), 130.49(C-3" ', C-5" '), 138.23, 138.54, 141.03 and 143.33(C-3, C-4', C-4'" and C-5), and 148.59 and 149.08(C- I '" and C-5'); ElMS (% reI. int. ): mlz 361([Mt. 15), 360(47), 258(21), 223(42), 214(100), 197(67), 185(52), 172(77), 151(18), 142(21), 138(36),136(65),1 18(45), 97(50), 83(70), 71 (68), 63(95) and 57(75).

5-Amino-4-[ 5-( 4-methoxyphenyl)pyrazol-3-yl]-l­(4-nitrophenyl)-1,2,3-triazole 6c. It was obtained as a yellow solid, m.p. >255°e. UV (MeOH): 270 nm; IR (KBr) : 3422,3366,3283, 1633, 1596, 1519, 1495 , 1338, 1306, 1287, 1267, 1183, 1105 , 1031 , 964 and 814 cm· l

; IH NMR (CDCh-TFAj.: 8 3.92(3H, s, OCH3), 7.08(2H, d, J = 8.9 Hz, C-3"H and C-5"H), 7.26(1H, s, C-4H), 7.73(2H, d, J = 8.9 Hz, C-2"H and C-6"H), 7.87(2H, d, J = 9.1 Hz, C-2'''H and C-6"'H) and 8.52(2H, d, J = 9. 1 Hz, C-3'''H and C-5'''H); J3C NMR (CDCI)-TFA): 8 55.67(OCH3), 101.37(C-4), 11 2. 12(C-3" and C-5"), 115.43(C-2 '" and C-6"'), 121.17(C-l "), 125.57, 126.03(C-2", C-3"', C-5 '" and C-6"), 138.06, 140.50, 141.46, 148.79 and 149.40(C­I''', C-3, C-4''', C-4' , C-5 and C-5') and 162.80(C-4"); ElMS (% reI. int.): mlz 377([Mt , 15), 376(26), 364(50),346(86) , 301 (15),290(20),272(18),262(12), 243(14), 219( 16), 208(26), 187(3 1), 178(100), 136(20), 125(25), 111(49),97(37),83(46), 71(53) and 57(58) .

5-Amino-4-[ 5-( 4-fluorophenyl)pyrazol-3-ylJ-l-( 4-nitrophenyl)-1 ,2,3-triazole 6d. It was obtained as a yellowish-white solid, m.p. 226°e. UV (MeOH): 255 nm; IR (KEr) : 3430, 3380, 3232, 1634, 1598, 1521 , 1417,1342,1268,1224,1107,965 and 857 cm· l

;

IH NMR (CDCI3-TFA): 8 7.23-7.42(3H, m, C-3"H, C-4H and C-5"H), 7.67-7.82(2H, m, C-2"H and C-6"H), 7.87(2H, d, J = 9.0 Hz, C-2'''H and C-6"'H) and 8.53(2H, d, J = 9.0 Hz, C-3"'H and C-5"'); J3C NMR (CDCb-TFA): 8 101.93(C-4), 107.63(C-4"), 112.16 (C-2'" and C-6"'), 116.68(C-3" and C-5"), 121.21 (C-l "), 125.66, 126.07 and 129.09(C-3"', C-5"', C-2", C-6", C-5), 138.04, 138.19, 141.74, 145.93 and 148.83(C-l "', C-3 , C-4', C-4'" and C-5'); ElMS (% reI. int.): mlz 365([Mt, 16), 364(67), 346(69), 336(28), 319(23), 306(15), 290(52), 289(29), 272(24), 26 1 (18), 220(13), 208(38), 187(50), 178(100), 153(26),

KUMAR el at.: SYNTHESIS OF NOVEL HETEROCYCLIC COMPOUNDS 1955

146(14), 122(35), 111(43), 97(40), 95(32), 83(37), 70(50) and 57(51).

S-Amino-4-[S-( 4-chlorophenyl)pyrazol-3-yl]-1-(4-nitrophenyl)-1,2,3-triazole 6e. It was obtained as a yellowish-white solid, m.p. >255°C. UV (MeOH): 268 nm; IR (KBr): 3428, 3367, 3282, 1633, 1597, 1521 ,1483, 1339, 1273, 1099,965, 855 and 820 em-i; 'H NMR (CDCI3-TFA): 8 7.33(1H, s, C-4H), 7.55(2H, d, J = 8.6 Hz, C-2"H and C-6"H), 7.72(2H, d, J = 8.6 Hz, C-3"H and C-5"H), 7.87(2H, d, J = 9.0 Hz, C-2'''H and C-6"'H) and 8.52(2H, d, J = 9.0 Hz, C-3'''H and C-5'''H) ; '3C NMR (CDClr TFA): 8 102.09(C-4), 112.14(C-2'" and C-6''') , 121.20 and 122.71(C-l" and C-4") : 125.6 1, 126.06 and 128.02(C-2" , C-3", C-3''', C-5''' , C-5" and C-6"), 138.02, 138.3 1, 139.17 and 141.63(C-3, C-4''', C-4' and C-5), and 148.28 and 148.8(C- I'" and C-5'); ElMS (% reI. int.): mlz 382([M+lt, 25), 381([Mt, 17), 380(90), 364(95), 346( 1 00), 306( 17), 203(23), 179(9), 170(32), 149(13), 138(13), 111(21),97(32) and 57(31).

5-Amino-4-[ 5-( 4-bromophenyl)pyrazol-3-yl]-1-( 4-nitrophenyl)-1,2,3-triazole 6f. It was obtained as a yellowish-white solid, m.p. >255°C. UV (MeOH): 270 nm; IR (KBr): 3413, 3380, 3283, 1635, 1596, 1517, 1481,1333,1275, 1111,1077,1048,966 and 857 em·'; 'H NMR (CDCh-TFA): 8 7.39(1H, s, C-4H) , 7.66(2H, d, J = 8.6 Hz, C-3"H and C-5"H), 7.74(2H, d, J = 8.6 Hz, C-2"H and C-6"H), 7.88(2H, d, J = 8.9 Hz, C-2"'H and C-6"'H) and 8.54(2H, d, J = 8.9 Hz, C-3'''H and C-5'''H); '3C NMR (CDCI3-TFA): 8 102.54(C-4) , 112.09(C-2'" and C-6'''), 116.99 and 121.14(C-1" and C-4"), 125.62, 126.09 and 128.18(C-2", C-3", C-3''', C-5''', C-5" and C-6"), and 138.04, 138.55, 141.40, 148.18 and 148.84(C- l''' , C-3 , C-4', C-4''', C-5, C-5'); ElMS (% reI. int. ): mlz 426([Mt, 4), 424(6), 376(100), 364(31), 318(8), 301(6), 272(15), 245( 10), 228(9), 208(8), 199(11), 182(11 ), 170(26) and 57(27).

5-Amino-l-(3-nitrophenyl)-4-( 5-phenylpyrazol-3-yl)-1,2,3-triazole 6g. It was obtained as a yellow­ish-white solid, m.p. 215°C. UV (MeOH): 255 nm; IR (KBr): 3430, 3318, 3211, 1633, 1589, 1526, 1488, 1351, 1264, 1109, 1073, 963, 899 and 799 em-i; 'H NMR (CDCh-TFA): 8 7.35(1H, s, C-4H), 7.53-7.63(3H, m, C-3"H, C-4"H and C-5"H), 7.76-7.79(2H, m, C-2"H and C-6"H), 7.87-8.02(2H, m, C-5'''H and C-6'''H) and 8.48-8.53(2H, m, C-2'''H and C-4'''H); I3C NMR (CDCI3+TFA): 8 102.10(C-4), 112. 12(C-2'''), 121.18 and 124.22(C-l", C-4" and C-6'''), 126.76(C-3" and C-5"), 129.91 (C-2" and C-6"),

130.72, 131.95 , 132.56 and 133.98(C-4', C-4''', C-5 and C-5'''), and 138.35, 141 .50, 149.09 and 149.27(C­I ''', C-3, C-3'" and C-5'); ElMS (% reI. int.): mlz 347([Mt, 25), 346(100), 301(28), 260(26), 170(48), 169(22), 155(21), 111(25),97(43),83(42),69(47) and 57(40).

5-Amino-4-[S-( 4-methylphenyl)pyrazol-3-yl]-1-(3-nitrophenyl)-1,2,3-triazole 6h. It was obtained as a yellowish-white solid, m.p. >255°C. UV (MeOH): 285 nm; IR (KBr): 3415, 3296, 2908, 1636, 1532, 1461, 1377, 1352, 1270, 1119 and 1103; 'H NMR (CDCh-TFA): 8 2.45(3H, s, CH3), 7.33(IH, s, C-4H), 7.38(2H, d, J = 8.0 Hz, C-3"H and C-5"H), 7.67(2H, d, J = 8.0 Hz, C-2"H and C-6"H), 7.92-8.03(2H , m, C-5'''H and C-6'''H) and 8.49-8.53(2H, m, C-2'''H and C-4'''H); '3C NMR (CDCI3+TFA): 8 21.39(CH3), 101.84(C-4), 112.IO(C-2'''), 120.26 and 121.21(C-l" and C-4'''), I 25.92(C-4"), 126.64(C-3" and C-5 "), 130.61(C-2" and C-6"), 130.76 and 131.94(C-5'" and C-6'''), and 133.94, 138.03, 141.67, 143.74, 149.08 and 149.53 (C-1''', C-3, C-3''', C-4' , C-C-5 and C-5'); ElMS (% reI. int.): mlz 361([Mt, 25), 360(100), 346(17), 183(30),95(17), 83(27) and 57(33).

5-Amino-4-[ 5-( 4-methoxyphenyl)pyrazol-3-yl]-1-(3-nitrophenyl)-1,2,3-triazole 6i. It was obtained as a yellowish-white solid, m.p. 241-244°C. UV (MeOH): 263 nm; IR (KBr): 3415, 3296, 3208, 1631, 1589, 1541, 1527, 1504, 1420, 1352, 1248, 1177, 1110, 1029,965,899 and 837 em·'; 'H NMR (CDClr TFA): 8 3.92(3H, s, OCH3), 7.07(2H, d, J = 8.6 Hz, C-3"H and C-5"H), 7.32(lH, s, C-4H), 7.74(2H, d, J = 8.6 Hz, C-2"H and C-6"H), 7.91(1H, t, J = 8.1 Hz, C-6"'H), 8.01(1H, d, J = 8.0 Hz, C-5"'H) and 8.52(2H, br s C-2'''H and C-4'''H); '3C NMR (CDCl3-TFA): 8 55.95(OCH3), 101.57(C-4), 115.60(C-3" and C-5" ), 120.48(C-2'" and C-6'''), 125.57(C-l "), 128.83(C-2" and C-6"), 130.28, 130.87(C-3'" and C-5''') , 132.12 (C-4"'), 134.63(C- l"') , 139.05(C-3), 141.42(C-4'), 149.12 and 149.48(C-5 and C-5') and 162.96(C-4").

5-Amino-4-[S-( 4-chlorophenyl)pyrazol-3-yl]-1-(3-nitrophcnyl)-1,2,3-triazolc 6j. It was obtained as a yellowish-white solid, m.p. >255°C. UV (MeOH): 260 nm; IR (KBr): 3443, 3325, 3202, 1632, 1590, 1541 , 1527, 1489, 1354, 1267, 1107, 1090,966,901 and 829 em·'; 'H NMR (CDCI3-TFA): 8 7.36(1H, s, C-4H), 7.56(2H, d, J = 8.7 Hz, C-3"H and C-5"H) , 7.74(2H, d, J = 8.7 Hz, C-2"H and C-6"H), 7.87-8.02(2H, m, C-5'''H and C-6'''H) and 8.49-8.52(2H, m, C-2'''H and C-4'''H); '3C NMR (CDCb+TFA): 8 102.31(C-4), I 12.61(C-2'''), 120.16, 121.20 and

1956 INDIAN J. CHEM ., SEC B, AUGUST 2003

122.9 1(C- l" , CA'" and C-6'''), 128.06(C-3" and C-5"), 130.67, 131.92 and 134.04(C-2", C-4", CA', C-5'" and C-6"), and 138.69. 138.76 and 141.28(C-3 , C-3"', C-5 ), and 147.88 and 149 .JO(C-I''', C-5'); ElMS (% re I. int. ): mlz 382(fM+ Ir, 45), 38 1(fMr, 25), 380( I 00), 346(55), 2 16(47), 203(22), 172(25) , 170(25) , 97(55) , 9 1 (25),69(47) and 60(55) .

5-Amino-4-[5-( 4-bromophenyl)pyrazol-3-yl]-1-(3-nitrophcnyl)-1,2,3-triazole 6k . It was obtained as a

ye llowish-white so lid, m.p. 220°e. UY (MeOH) : 264 nm; IR (KBr): 3405,3308,3210, 1635, 1538, 1482, 1352, 1285, 11 6 1, 1075 , 968,894 and 828 cm· l

; IH

NMR (COCb-TFA): 8 7.33(IH, s, CAH), 7.64(2H, d, J = 8.6 Hz, C-3"H and C-5 "H), 7 .73 (2H, d, J = 8.6 Hz, C-2" H and C-6"H), 7.91(lH, m, C-S'''H), 8.0 I ( I H, d, J = 8.5 Hz, H-6''') . 8.50-8 .53(2H, m, C-2'''H and C-4'''H); 13C NMR (CDCh-TFA):

8 102.06(C-4), 11 2. 12(C-2''') , 120.17, 123 . 10 and 126. 11 (C-l" , C-4", CA'" and C-6'''), 128. 10(C-2" and C-6") , 132.08 and 133.32(C-3", C-S''' and C-5"), and 133 .82, 138. 19, 14 1.90, 148.46 and 149.05(C- I ''', C-3, C-3"', CA', C-5 and C-5 '); ElMS (% re I. int .): mlz 426(1"Mr, 20), 375(28), 345(60), 288(12), 252(100), 250(92) , 2 14( 18), 208(8) , 193(6), 173(22), 155(9), 136(14), 127(20) , 97( 19),90(38) and 57(32).

Acetylation of pyrazolyltriazoles 6d, 6f and 6g. Pyrazolyltri azo les 6d, 6f and 6g (80-95 mg) were stirred with acetic an hydride and catalytic amount of d imethylaminopyridine (O MAP) for 5-8 hr at 25-

28°C. The reacti on was monitored on TLC and on completion, the reacti on mixture was poured into ice­cold water (20 mL) and the compound ex tracted with ethyl acetate (2x25 mL) and purified by column chromatography to afford the acetates 7d , 7f and 7g, respectively in 80-85 % yie lds. Compound 7d : I H NMR (CDCl3): 8 2.40(6H, s, C-5'N(COCH3h ), 2.59(3H, s, N I COCH3), 7 . 1 0-7.20(2H, m, C-3"H and C-S" H), 7 .30( JH, s, C-4H), 7.40-7.50(2H, m, C-2"H and C-6"H), 7.77(2H, d, J = 9.0 Hz, C-2'''H and C-6'''H) and 8.45(2H, d, J = 9.0 Hz, C-3'''H and C-S'''H); Compound 7f: IH NMR (COCb): 8 2.35(6H, s, C-5'N(COCH3h), 2.65(3H, s, N I COCH3), 7.23(1 H, s, CAH), 7.32(2H, d, J = 8.6 Hz, C-3"H and C-5" H), 7.56(2H, d, J = 8.6 Hz, C-2" H and C-6"H), 7 .77(2H, d, J = 8.9 Hz, C-2'''H and C-6'''H) and 8.46(2H, d, J = 8.9 Hz, C-3'''H and C-S'''H); Compound 7g: 'H NMR (COCl, ): 8 2.34(6H, S, C-5'N(COCH3)2), 2.67(3H, S,

N I COCH3) , 7.29(1 H, s, CAH), 7.45(3H, m, C-3"H, CA" H and C-5"H), 7.85(2 H, m, C-2"H and C-6"H),

7 .87-7.99(2H, m, C-5"'H and C-6'''H) and 8.48-8.53(2H, m, C-2"'H and CA'''H).

General method of biological activity evaluation In vitro assay for invasion. The assay consists of

three-dimensional confrontations between tumor cell s and normal ti ssue. 33 MCF-7 cell s. used as tumor cell s were ori ginally established from a pleural effusion of a breast adenocarcinoma patient. 34 These cells, whose identity was confirmed in our laboratory,3) are in va­sive both in vilro36 and in vivo.J7 Fragments of nine­day-old embryonic chick heart were precultured and selected for a diameter of 0.4 mm. These precultured heart fragments (PHF) were confronted individually with an aggregate of MCF-7/6 cells (diameter 0.2 mm), first on top of semi-solid agar overn ight to al­low attachment, and subsequentl y in liquid medium for eight days in suspension cultu re . The cultures were then fixed and embedded indi viduall y in paraf­fin , and seri a ll y secti oned for histolog ical ana lys is . In order to evaluate the interaction between the MCF-7/6 cell s and PHF, the secti ons were stained with haema­toxy lin-eosin o r with an immunohi stochemical tech­nique to reveal MCF-7 anti gens with the 50 I 0 mono­clonal antibody. 38 Occupation and destruction of the PHF was considered as typ ical for invas ion, while growth around the PHF was scored as absence of in­vasIOn.

Treatments. All compounds were d issolved in di­methyl sulfox ide (OMSO) to g ive a stock soluti on of 10.1 M from which further dilutions in culture me­dium were prepared. For each compound at least two

confro ntati ons were treated in suspension with 10 11M fo r eight days. If this concentration appeared to be anti-in vasive or' cy totoxic, lower concentrati ons (I or 0. 1 11M ) were app li ed . Control cultures were treated with solvent alone (OMSO at corresponding concen­trations) .

In vitro evaluation of antimycobacterial activity. Pre liminary screening against Mycobacterium tuber­culosis H37R v (A TCC 27294) has been conducted at a concentration of 6 .25 )lg/ml in BACTEC J 2B me­dium using a broth microdiluti on assay, the Mi­croplate Alamar Blue Assay (MABA). 39 Compounds exhibiting fluorescence were tested in the BACTEC 460 radiometric system.39

Acknowledgement Financial support from Council of Scientific and

Industrial Research (CS IR, India) and Dani sh Interna­tional Development Agency (DANIDA, Den mark) is

KUMAR et al.: SYNTHES IS OF NOV EL HETEROCYCLIC CO MPOUNDS 1957

highly acknowledged. We thank TAACF, Southern Research Institute, Birmingham, Alabama, USA for the evaluation of the anti mycobacterial activit ies of our compounds.

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