Indian Journal of Chemistry Vol. 438, February 2004, pp. 385-388

Bisheterocyclic synthesis and antimicrobial studies on some biologically significant 2-[N- (3' -chloro-4' -substituted azetidinone-2)] amino-4-hydroxypurines

Pratibha Sharma*, Ashok Kumar & Shikha Sharma

School of Chemical Sciences, Devi Ahilya University, Indore 52 017, India

Received 5 February 2003; accepled (revised) 22 July 2003

Present communication describes the synthesis of a series o f 2-[N- (3'-chloro-4'-substituted azetidinone-2)]amino-4-hydroxypurines 4a-h. 2-Amino-4-hydroxypurine 2 formed by the [4+ I] cycloaddition of 2, 5, 6-triamino-4-hydroxypyrimidine with formic acid is subjected to diazotisation followed by coupling with an appropriate active methylene compound under alkaline conditions. The product is treated with chloroacetyl chloride in the presence of triethyl amine and I , 4-dioxane to yield 2-[N- (3'-chloro-4'-substituted azetidinone-2)]amino-4-hydroxypurines 4a-h . The st ructures of the compounds have been establi shed on the basis of spectroscopic data. All the compounds have been tested for their antim­icrobial act ivity against 8 .sublilis, E.coli, P.dill1il1ula, S.aureus and B.pwllilus. They show significant antimicrobial activity.

IPC: Int.CL7 C 07 D 473/00 II A61 P 31104

The chemistry of purine or imidazo[4, 5-d] pyri­midine ring system has been one of the most widely investigated. During the last few years, many purines l

-7 have been claimed to have a multitude of

useful biological and pharmacological activities, but the main area of activity apart from antitu­mour/antileukemia field include their use as immuno­suppressive, antibiotic, antiallergics and cardiac stimulants. Similarly, azetidine8

-lo and its derivatives

have also been found to be associated with significant antibiotic activity. Keeping thi s view in consideration, synthesis of some substituted azetidinone purines has been carried out by refluxing together ethyl cynoace­tate and guanidine in equimolar quantities for 3 hr in the presence of freshly prepared sodium ethoxide so­lution. The resultant compound on nitration followed by successive reduction and strong heating with for­mic acid provides fine crystals of 2-amino-4-hydroxypurines 2. In order to ex tend the sy nthesis to obtain desi red azetidinone purines, 2 was diazotised

and coupled with an appropriate <1, B-unsaturated compound in an cquimolar ratio under alkaline condi ­tions and then refluxing with chloroacetyl chloride in the presence of triethylamine and 1, 4-dioxane for 4

hr. After successful work-up the crystals of 2-[N- (3'­chloro-4'-substituted azetidinone-2)] amino-4-hydro­xypurines 4a-h were obtained as yellow to brown crystals (Scheme I). Compounds 1 and 2 had been synthesized as per the procedure ll

. 12 reported and modified by us. Structures of all the synthesized compounds were established by their consistent JR,

NMR and mass spectral data and elemental analysis and purity was ascertained by TLC (methanol and toluene, 4:6).

Experimental Section

The 1 H NMR spectra were recorded at 300 MH z with a Bruker Advance DPX 300 instrument using TMS as an internal standard. Mass spectra were re­corded at 70 eV ionising voltage on FB2603X. LRP and are presented as mlz (% reI. int.). IR spectra were run on a Perkin-Elmer model 377 spectrophotometer in KBr pellets. Analytical thin layer chromatography was performed using E. Merck silica gel 0.50 mm plate (Merck No. 5700) . Melting points were deter­mined in open capillary tubes using an electric melt­ing point apparatus and are uncorrected.

Synthesis of 2, 5, 6-triamino-4-hydroxypyrimi­dine 1. A mixture of ethyl cyanoacetate (2.23mL, 0.02 mole) and guanidine hydrochloride (1.91 g, 0.02 mole) was refl uxed in sodium ethoxide solution (pre­pared from 6 g of sodium metal and 50 mL absolute alcohol) . The solid product obtained was filtered and dried. The product was nitrated and then reduced by the standard method 13 to yield 2, 5, 6-triamino-4-hydroxypyrimidine I , m.p. 16S-6'r'C, yield 68%.

Synthesis of 2-amino-4-hydroxypurine 2. Com­pound 1 (0.02 mole) was strongly heated (l80°C) for 6-8 hr with formic acid (6 g). On cooling white col­oured crystals of the compound were formed . The product was recrystallized from a mixture of ethanol -DMF (l : 1, v/v) and the purity of the compound was

386 INDIAN 1. CHEM., SEC B, FEB RUARY 2004

where,

:X0H N o N ~

II H I I CH R'-C I~ /

'C=N/N IN N R"-C" H 8 3 (vii)

OH

N:XN~ COR' I CH I ;,... /

R"OC-C-N-HNAN N I I

Ci-C-C=O H I H 4a-h

R' R" R' R" a CH) C H) c OC2HS OC2HS b C H) OC2HS f C6HS CoHs c CH) NHC6HS g CN OC2HS d OCH) OCH) h CH2Br OC2HS

Reagents and Conditions: (i) C2HsONa refl ux 3 hr, (i i) HI ·O/H2S04, (ii i) Sn/HCI, (iv) HCOOH /';., 6-8 hr, (v) H 0 2/HC1 , O-SoC, (vi) RCOC H2COR", CH)COONa + NaOH, EtOH, (v ii ) Et)N I I A-dioxane, C ICOCH2CI, reflux , 4 Ilr.

Scheme I

ascertained on the bas is of TLC (methanol: toluene; 4:6), m.p.I78-80°C, yield 65%.

Synthesis of 2-[N- (3'-chloro-4'-substituted azet­idinone-2)]amino-4-hydroxypurines 4a-h. General procedure. Diazotised solution of compound 2 was

coupled with an equimolar quantity of an active me­thylene compound under alkaline conditions [sodium acetate (1 .82 g, 0.01 mole) and sodium hydroxide (0.40 g, 0.01 mole)] to yield crystals of .3a-h. The de­rivatives 3a-h were added to a stirred solution of chloroacetyl chloride (2.90 mL, 0.02 mole) and triethylamine (2.30 mL, 0.02 mole) in 1, 4-dioxane (50 mL). After 2 hr the reaction vessel was kept at room temperature for 30 min and then refluxed for 4 hr. On cooling the compound obtained was filtered, thoroughly washed with water and dried.

4a: m.p. 178-80°C, yield 70%. Anal. Calcd for C12H1I04N6CI: C, 42.54; H, 3.25; N, 24 .8 1. Found: C, 42.44; H, 3.11; N, 24.79%; IR (v, cm-I): 3600 (-OH), 3450 (-NH), 3250 (-NH ring) , 3060 (C-H, sp\ 2870 (C-H, sp\ 1750 (C=O, four membered) , 1720 (­COCH3), 1660 (C=C/C=N), 550 (C-C1); IHNMR (8, ppm): 2.0 (s, 6H, 2 x CH3), 4.1 (s, CH-CI), 8.1 (s, CH ring) , 9.5 (s, NH-N), 9.8 (s, NH ring) , 12.5 (bs, -OH); MS: (mlz, % abundance) 338 [M+] (45), 340 [M+2] (15) , 79 (100), 203 (40) .

4b: m.p. 175-79°C, yield 75%. Anal. Calcd for C I3 HI30sN6CI: C, 42.33; H, 3.52; N, 22.79. Found: C, 42.28; H, 3.34; N, 22.50%; IR (v, cm· I): 3600 (-OH), 3480 (-NH), 3280 (-NH ring), 3060 (C-H, Sp2), 2870 (C-H, Sp3), 1740 (C=O, four membered), 1730 (­COCH3) , 1660 (C=C/C=N), 722 (-CH2' rocking) , 548 (C-C1); 'HNMR (8, ppm): 1.1 (t, 3H, CH3), 1.9 (s, 3H, CH3), 4.0 (q, 2H, CH2), 4 .5 (s, CH-CI), 7.9 (s, CH ring) , 9.1 (s, NH-N), 9.7 (s, NH ring), 14.1 (bs, OH); MS: (rn/z, % abundance) 368 [Nt] (30), 370 [M+2] (10), 79 (100), 281 (55).

4c: m.p. 172-74°C, yield 72%. Anal. Calcd for CI7HI404N7CI : C, 49.09; H, 3.36; N, 3.58. Found: C, 48 .9; H, 3.30; N, 3.42%; IR (v, cm·I): 3600 (-OH), 3448 (-NH), 3251 (-NH ring), 3058 (C-H, Sp2), 2872 (C-H, Sp3), 1751 (C=O, four membered) , 1720 (-COCH3), 1660 (C=C/C=N), 1600, 1500 (C=C ring str), 552 (C-C1) ; IHNMR (8, ppm): 2.1 (s, 3H, CH3),

4.3 (s, CH-CI), 7.5 (s, 5H, C6Hs), 8.0 (s , CH ring), 9.0 (s, NH-N), 9.6 (s, NH ring), 14.0 (bs, OH); MS: (rn/z, % abundance) 415 [M+] (60), 417 [M+2] (20), 79 (100), 290 (25).

4d: m.p. 172-73°C, yield 78%. Anal. Calcd for C1 2H1106N6CI: C, 38.86; H, 2.96; N, 22.67. Found: C, 38.75; H, 2.68; N, 22.54%; IR (v, cm· I): 3650 (-OH), 3340 (-NH), 3280 (-NH ring), 3070 (C-H, Sp2), 2880 (C-H, Sp3), 1750 (COCH3), 1740 (-C=O, four mem­bered), 1590 (C=C/C=N), 650 (C-C1); IHNMR (8, ppm): 3.8 (s, 6H, 2 x CH3), 4.3 (s, CH-CI), 8.1 (s, CH

SHARMA e / 01. : BISHETEROCYCLIC SYNTH ESIS & ANTIMICROB IAL STUDIES ON PURINES 387

ring), 9.1 (s , NH-N), 9.7 (5, NH ring), 13.9 (bs, 01-1 ); MS : (mlz, % abundance) 370 rM+l (36), 372 [M+2] ( 12), 235 (40), 79 (100).

4e: m.p. I 77-79°C, yield 80%. Anal.Calcd for CI4 1-1 1S0 6N6CI: C, 42.15 ; H, 3.76; N, 21.07 . Found: C, 42. 10; H, 3.61 ; N, 20.9 1%; IR (v, cm' \ 3660 (-01-1 ), 3348 (-NH), 3260 (-NH ring), 3060 (C-H Sp2), 2875 (C-I-I , sp\ 1755 (COOC2Hs), 1730 (-C=O, four membered), 1600 (C=C/C=N), 722 (-CI-I 2), 660 (C-Cl); IHNMR (8, ppm): 1.1 (t, 6H , 2 x CH,), 3.8 (q, 4H , 2 x CH 2) . 4.5 (s, CH-Cl), 7.9 (s, CI-I ring), 9.0 (s,

H-N ), 9.5 (s, NI-I ring) , 14.1 (bs, OH); MS : (mlz, % abundance) 398 rM +] (75) , 400 [M+2] (25), 79 ( 100), 263 (60).

4f: m.p . 170-75°C, yield 78%. Anal. Calcd for C22 l-1l s0 4N6CI: C, 57.08; H, 3.24; N, 18. 16. Found : C, 56.91 ; H, 3.20;N, 18. 10%; IR (v, cm'I ): 3640 (-OH), 3400 (-NH), 3260 (-NH ring), 3050 (C-H, sp\ 2860 (C- I-I , sp\ 1740 (C=O, four membered), 1725 (­COC(\ Hs), 1600 (C=C/C=N), 600 (C-Cl ); IHNMR (8, ppm): 4.2 (s, CH-Cl), 7.4 (s, 10H, 2 x C(\ Hs), 8.3 (5 , CH ring), 9.4 (5, NH-N), 9.7 (5 , NH ring), 13.8 (bs, OH) ; MS: (mlz, % abundance) 462 rM +l (45), 464 rM+21 ( 15), 327 (29), 79 ( 100).

4g: m.p. I 79-82°C, yield 75%. Anal.Calcd for C13 I-1 IOOsN7CI: C, 4 1. 10; H, 2.63 ; N, 25.82. FOLlnd :C, 40.9 1: 1-1 ,2.60; N, 25.38%; IR (v, cm' I) : 3640 (-01-1), 3400 (-NH), 3270 (-NH ring), 3050 (C-H , Sp2), 2860 (C-I-I , sp\ 1740 (C=O, fOLlr membered), 1590 (C=C/C=N), 1425 (C-N), 530 (C-Cl); IHNMR (8, ppm): 1.1 (t, 31-1, CH3) , 4.4 (q, 2H , CH2), 4.6 (5, CH­CI), 7.9 (5, CH ring), 9. 1 (5, NH-N), 9.5 (s, NI-I ring), 13.8 (bs, 01-1 ); MS: (mlz, % abundance) 379 [M+] (45), 38 1 [M+2] ( 15), 244 (28), 79 (100).

4h: m.p. 170-75°C, yield 78%. Anal.Calcd for CI ,HI20 sN6CIBr: C, 34.86; 1-1 ,2 .68 ; N, 18.77. Found : C, 34.71; H, 2.6 1; N, 18.69; IR (v, cm' I): 3620 (-OH), 3430 (-NH), 3270 (-NH ring), 3050 (C-H, sp\ 2860

(C-H , sp\ 1750 (-COCH 3), 1730 (C=O, four mem­bered), 1640 (C=C/C=N), 610 (C-Br), 560 (C-Cl ); IHNMR (8, ppm): 1.1 (t , 3H, CH 3), 3.7 (s, 2H, CI-I 2),

4.2 (q, 2H, CH2), 4.6 (s, CH - CI ), 8.2 (s, CH ring) , 9.4 (s, NH-N ), 9.9 (s, NH -N), 13.6 (bs, OH); MS : (mlz, % abundance) 447 [M+] (24), 449 [M+21 (8), 45 I [M+4] (6), 3 11 (30), 79 ( 100).

Antimicrobial activity Nutrient agar media was prepared by taking the

weighed quantity vi z., peptone 5 g, beef ex tract I g. agar-agar 20 g and di stilled water 1000 mL, and the pH of the medi a was adjusted to 6.5. The mi xture was autoclaved for half an hour at a pressure of 1.5 kg/cm2

. All the glass apparatus were sterili zed fo l­lowed by washing with di still ed water, dried and rinsed with spirit then wrapped with paper and ti ed with thread and kept in oven for half an hour at 100°e. Medium was coo led to 37°C and homogene­OLl S suspension was prepared by transferring asepti­cally a loopful of all the corresponding mi croorgan­ism from fresh subcu lture into agar medium foll owed by vigorous shaking, 20 mL of this medium was poured into each sterilized petridishes under asept ic conditions and allowed to set. After preparing the me­dium, the paper di sc (6 mm) was immersed in seeded agar containing petridishes. The solution (0.1 mL) was dropped into the filter paper di sc by using method of Rapperl 4. Petridi shes were incubated at 3T'C for 24 hr. The inhibition zone for each test solution was measured in mm.

All the synthesised compounds were screened fo r their antimicrobial activity against P.dil7lillll la , B. sublilis, E.coli, s'aureus and B.pumilus using stan­dard antibiotic drug purinthol as control. Inhibition (%) = [ (a-~)/a] x 100 was calculated ls and presented in Table I, where a and ~ stand for inhibition zone of control drug and inhibition of the synthesized COIll­pound, respectively. The screening results indicate that

Table I - Antibacterial activity of 4a-h (zone of inhibition in %)

Compd MR B. s/lb/ilis E. coli P.dilllinll/a . S.allreus B.plllllillls

4a 59.73 - 12.42 -4.11 -5 . 11 5.86 3.95

4b 63.57 -25. 15 - 10.9 1 -18.16 -7.43 -8.80

4c 71.82 -30.56 -25.05 -28.42 -14.40 -23. 12

4d 62.80 -24.24 -9.87 -16.8 1 3.76 1.55

4e 69.42 -27.02 -19.8 1 -21.59 -11.10 -15.1 8

4f 79.37 -35.86 -29.50 -32.06 - 15.60 -25.68

4g 60.90 -18.68 -5 .86 -6. 12 4.81 2. 19

4h 70.56 -28.50 -20.49 -22.10 12.23 - 18.25

388 INDI AN J. CH EM., SEC B, FEBRUARY 2004

compounds exhibit excellent antimicrobial acti vity against B. subfilis, while least against S.aureus. A sys­tematic perusal of the data presented in Table I, reveal that compound 4f was found to be more active in the series. The data in Table I reveal that the acti vity shown by synthesized purines follow the pattern : B. su bf i I is> P. dim i Ilil fa>£. col b B. pUll I i IlIs>5. a II reus.

To study quantitati ve structure-act ivity relationship (QSA R), molecul ar refrac ti ve index (MR) by the method of Dreisbach 16 has been calcul ated and re­ported in Table I. A peru sal of data revea l that the drug acti vity increases with the increase of molecul ar refractive index (MR)' Molecul ar refracti ve index (MR) is correlated linearly to the drug acti vity. It has also been concluded that alkylation of carboxy li c group, introduction of phenyl group on ni trogen atom and electron attracting group (-8 r) increase the antim­icrobial acti vity.

The synthesized compounds have been subj ected to acute tox icity study to find out LDso va lues . These compounds do not show any tox icity upto dose of 9.85 mg/kg body weight in rats.

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