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 Ahily a University, Indore 52 017, India

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

Present communication describes the synthesis of a seri es o f 2-[N- (3'-chloro-4'-substituted azetidinone-2)]amino-4hyd roxypurines 4a-h. 2-Amino-4-hydroxypurine 2 formed by the [4+ I] cycloaddition of 2, 5, 6-triamino-4hydroxypyrimidine 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 tri ethy l 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 compou nds have been tested for their antimicrobial act ivity aga inst 8 .sublilis, E.coli, P.dill1il1ula, S.aureus and B.pwllilus. They show significant antimicrobial activit y.

IPC: Int.CL 7 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 yea rs, many
7
purines l - 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, azetidine 8- lo and its derivatives
have also been found to be associated with significant
antibiotic activity. Keeping thi s view in consideration,
sy nthesis of so me 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-4hydroxypurines 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)] a mino-4-hydroxypurines 4a-h were obtained as yellow to brown
crystals (Scheme I). Compounds 1 and 2 had been
sy nthesized as per the procedure ll . 12 reported and
modifi ed 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 1H 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 FB 2603X . 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 electri c 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 so luti on (prepared from 6 g of sodium metal and 50 mL absolute
alcohol). The so lid product obtained was filtered and
dried. The product was nitrated and then red uced by
the standard method 13 to yield 2, 5, 6-triamino-4hydroxypyrimidine I , m.p. 16S-6'r'C, yield 68 %.
Synthesis of 2-amino-4-hydroxypurine 2. Compound 1 (0.02 mole) was strongly heated (l80C) 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 ethano l DMF (l : 1, v/v) and the purity of the compound was

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

386

0H

oII

H I

:X

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

CH

OH

N:XN~
I CH

COR'
I
;,...
R"OC-C-N-HNAN
I

Ci-C-C=O
I

N
H

4a-h

where,
R'
R"
R'
R"
CH)
C H)
c OC2HS OC2HS
C H)
OC2HS
f
C6HS
CoHs
C H)
NH C6HS g
CN
OC2HS
OCH)
OCH)
h CH 2Br OC2HS
Reagents and Conditions: (i) C 2HsONa refl ux 3 hr, (i i)
HI O/H 2S0 4 , (ii i) Sn/HCI, (iv) HCOO H /';., 6-8 hr, (v)
H 0 2/HC1 , O-SoC, (v i) RCOC H2COR" , CH)COONa + NaOH,
EtOH, (v ii ) Et)N I I A-dioxane, C ICOCH 2C I, reflux , 4 Ilr.
a
b
c
d

Scheme I
ascertained on the bas is of TLC (methanol: toluene;
4:6), m.p.I78-80C, yield 65%.
Synthesis of 2-[N- (3'-chloro-4'-substituted azetidinone-2)]amino-4-hydroxypurines 4a-h. General
procedure. Diazotised so lution of compound 2 was

coup led 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. 178-80C, 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 (COCH 3), 1660 (C=C/C=N), 550 (C-C1 ); IHNMR (8,
ppm): 2.0 (s, 6H, 2 x CH 3), 4.1 (s, CH-C I), 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. 175-79C, yield 75 %. Anal. Calcd for
C I3 HI30sN6C I: 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 (COCH 3) , 1660 (C=C/C=N), 722 (-CH2' rocking) , 548
(C-C1); 'HNMR (8, ppm) : 1.1 (t, 3H, CH 3), 1.9 (s, 3H,
CH3 ), 4.0 (q, 2H, CH 2), 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. 172-74C, 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
(-COCH 3), 1660 (C=C/C=N), 1600, 1500 (C=C ring
str), 552 (C-C1) ; IHNMR (8, ppm): 2.1 (s, 3H, CH 3 ),
4.3 (s, CH-CI), 7.5 (s, 5H , C6H s), 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. 172-73C, 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 (COCH 3 ), 1740 (-C=O, four membered), 1590 (C=C/C=N), 650 (C-C1); IHNMR (8,
ppm): 3.8 (s, 6H, 2 x CH 3), 4.3 (s, CH-CI), 8.1 (s, CH

SHARMA

e/

01. : BISHETEROCYCLIC SYNTH ES IS & A NTIMICROB IAL STUDIES ON PURINES

ring), 9.1 (s, NH-N ), 9.7 (5, NH rin g), 13.9 (bs, 01-1 );
MS : (mlz, % abundance) 370 rM +l (36), 372 [M+2]
( 12), 235 (40), 79 ( 100).
4e: m.p. I77-79C, yield 80%. Ana l.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 (COOC2 Hs), 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 . 170-75C, 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 rin g), 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 rin g), 9.4 (5, NH-N), 9.7 (5 , NH ring), 13.8 (bs,
OH) ; MS: (mlz, % ab und ance) 462 rM +l (45), 464
rM+21 ( 15), 327 (29), 79 ( 100).
4g: m.p. I79-82C, 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, CH 3) , 4.4 (q, 2H , CH 2), 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. 170-75C, yie ld 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 rin g), 3050 (C-H, sp\ 2860
Table I -

(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, CH 2), 4.6 (s, CH - CI ), 8.2 (s, CH rin g) ,
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 takin g th e
weighed qu antity vi z., peptone 5 g, bee f ex tract I g.
agar-aga r 20 g and di stilled water 1000 mL, and th e
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/cm 2 . All th e 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
100e. Medium was coo led to 37C and homogeneOLl S suspension was prepared by transferrin g aseptically a loopful of all the corresponding mi croorga nism 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 th e 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 meth od
of Rapperl 4. Petridi shes were incubated at 3T'C for 24
hr. The inhibiti on zone for each test solution was
measured in mm .
All the sy nthesised compounds were screened fo r
their antimicrobial activity against P.dil7lillllla ,
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

Antibacterial activity of 4a-h (zo ne of inhibiti on in %)

E. coli

387

P.dilllinll/a

. S.allreus

B.plllllillls

Compd

MR

B. s/lb/ilis

4a
4b
4c
4d
4e
4f
4g
4h

59.73

- 12.42

-4.11

-5 . 11

5.86

3.95

63.57

-25 . 15

- 10.9 1

-18.16

-7.43

-8.80
-23. 12

71.82

-30.56

-25.05

-28.42

-14.40

62.80

-24.24

-9.87

-16. 8 1

3.76

1.55

69.42

-27.02

-19.8 1

-21.59

-11.10

- 15.1 8
-25.68

79.37

-35.86

-29 .50

-32.06

- 15.60

60.90

- 18.68

-5 .86

-6. 12

4.81

2. 19

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 antimicrobi al acti vity

against B. subfilis, while least against S.aureus. A systemati c perusal of the data presented in Table I, reveal
th at compound 4f was found to be more active in the
seri es. The data in Table I reveal that the acti vity
show n by sy nthesized purines follow the pattern :
B. su bf i I is> P.dim i Ilil fa>. col b B.pUll I i IlIs>5. aII reus.

To stud y qu antitati ve structure-act ivity relationship

(QSA R), molecul ar refrac ti ve index (M R) by the

method of Dreisbac h 16 has bee n calcul ated and reported in Table I. A peru sal of data revea l th at th e
dru g acti vity increases with th e increase of molec ul ar
refractive index (MR)' Molec ul ar refracti ve index
(MR) is correlated linea rl y to th e drug acti vity. It has
also been concluded th at alk ylati on of carboxy li c
gro up, introduction of phenyl group on ni trogen atom
and electron attracting group (-8 r) increase the antimicrobi al acti vity.
The sy nthesized co mpounds have bee n subj ected to
ac ute tox icity stu dy to find out LDso va lues . These
compounds do not show any tox icity upto dose of
9.85 mg/kg body we ight in rats.
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