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Il Farmaco 55 (2000) 331–337

Synthesis and antimicrobial activity of1-aryl-2-amino-3-(4-arylthiazol-2-yl)/(benzothiazol-2-yl)guanidines

Ram Lakhan a,*, Bimal Prasad Sharma a, Bishwa Nath Shukla b

a Department of Chemistry, Banaras Hindu Uni6ersity, Varanasi 221 005, Indiab Department of Microbiology, Institute of Medical Sciences, Banaras Hindu Uni6ersity, Varanasi 221 005, India

Received 10 January 1999; accepted 16 February 2000

Abstract

The synthesis of fifteen new 1-aryl-2-amino-3-(4-arylthiazol-2-yl)/(benzothiazol-2-yl)guanidines is described. They were screenedfor their antimicrobial susceptibility by the standard disc diffusion method of the World Health Organization (WHO) and theactivities compared with that of standard strain of Escherichia coli NCTC 10418. The sensitive aminoguanidines were furthersubjected to the minimum inhibitory concentration (MIC) test. © 2000 Elsevier Science S.A. All rights reserved.

Keywords: Antimicrobial activity; Minimum inhibitory concentration; Guanidines

1. Introduction

A number of thiazolyl and benzothiazolyl guanidineshave been reported to exhibit antitubercular, antimalar-ial, CNS depressant, analgesic [1–8] and antimicrobialactivity against both Gram-positive and Gram-negativebacteria [9]. The related N-aminoguanidines are veryscarcely reported in the literature; an exception beingtheir report as tuberculostatic agents [10]. Therefore, weconsidered it interesting to synthesize and evaluate theantibacterial activity of some new 1-aryl-2-amino-3-(4-arylthiazol-2-yl)/(benzothiazol-2-yl)guanidines.

The synthetic approach to these compounds [2,4] isoutlined in Schemes 1 and 2. The starting materials,2-amino-4-arylthiazoles and 2-aminobenzothiazole wererefluxed with substituted phenyl isothiocyanates in drybenzene. Subsequent desulfurization of the resultingthioureas 1 and 3 with yellow lead oxide in ethanol inthe presence of hydrazine hydrate gave 2 and 4, respec-tively, in 45–75% yields. The structure of the com-pounds 1–4 were confirmed by elemental analyses, IRand 1H NMR spectral data.

2. Experimental

All melting points (m.p.) were determined in opencapillaries on a Gallenkamp apparatus and are uncor-rected. The purity of the compounds was routinelychecked by thin layer chromatography (TLC) using

Scheme 1.* Corresponding author. Tel.: +91-542-21 1979; fax: +91-542-31

7074.

0014-827X/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.PII: S 0 0 1 4 -827X(00 )00032 -X

R. Lakhan et al. / Il Farmaco 55 (2000) 331–337332

Scheme 2.

(broad, 1H, NH, D2O exchangeable), 6.90–8.00 (m,10H, ar), 11.25–11.50 (broad, 1H, NH, D2Oexchangeable).

2.2. 1-p-Bromophenyl-3-(4-p-totylthiazol-2-yl)-2-thiourea (1g)

The compound was prepared by condensing 2-amino-4-p-tolylthiazole with p-bromophenyl isothio-cyanate in dry benzene as above; yield 58%, m.p.225°C. Analysis: C17H14BrN3S2 (C, H, N, S). IR (KBr,cm−1): 3450 m, 3350 m (N�H str), 1660 m, 1600 m,1540 s (aromatic ring), 1230 s (C�S str). NMR (DMSO-d6): d 2.20 (s, 3H, CH3), 7.13–7.90 (m, 10H, 9 ar andone NH, D2O exchange), 9.04 (s, 1H, NH, D2O ex-change). Other thioureas 1 were prepared similarly in46–73% yields. Their m.p. and characteristic IR data(Nujol) are reported in Table 1.

2.3. 1-p-Bromophenyl-3-(benzothiazol-2-yl)-2-thiourea(3a)

The compound was prepared by refluxing 2-aminobenzothiazole with p-bromophenyl isothio-cyanate in dry benzene as above; yield 95%, m.p.231°C. Analysis: C14H10BrN3S2 (C, H, N, S). IR (KBr,cm−1): 3480 m, 3340 w (N�H str), 1660 w, 1640 s, 1550s (aromatic ring), 1240 (C�S str). NMR (DMSO-d6): d

6.50–8.45 (m, 8H, ar), 9.20 (broad, 1H, NH, D2Oexchange), 11.92 (broad, 1H, NH, D2O exchange). Byfollowing this procedure, thioureas 3b–d were alsoprepared. Their yields, m.p. and characteristic IR data(Nujol) are given in Table 2.

2.4. 1-p-Bromophenyl-2-amino-3-(4-phenylthiazol-2-yl)guanidine (2a)

A mixture of 1-p-bromophenyl-3-(4-phenylthiazol-2-yl)-2-thiourea (4.0 g, 0.01 mol), yellow lead oxide (2.2 g,

silica gel G (E. Merck). A Jeol FX-90Q Fourier trans-form spectrometer was used for recording 1H NMRspectra at 27°C with TMS as an internal standard. AJasco FT-IR 5300 spectrophotometer was used for IRspectra and a Perkin–Elmer CHN Analyzer 240C forelemental analyses. The analytical values for all the newcompounds were within 90.3% of the theoreticalvalues.

2.1. 1-p-Bromophenyl-3-(4-phenylthiazol-2-yl)-2-thiourea (1a)

An equimolecular mixture of 2-amino-4-phenylthia-zole (4.0 g, 23 mmol) and p-bromophenyl isothio-cyanate (5.3 g, 25 mmol) in 30 ml of dry benzene wasrefluxed in a water-bath for 10 h. After cooling, thesolid product was filtered and washed with ether fol-lowed by dilute HCl. The product was finally washedwith water and crystallized from ethanol to give color-less crystals; yield 73%, m.p. 255°C. Analysis:C16H12BrN3S2 (C, H, N, S). IR (Nujol, cm−1): 3400 m,3345 s (N�H str), 1640 s, 1590 m, 1460 m (aromaticring), 1200 s (C�S str). NMR (DMSO-d6): d 3.50–3.89

Table 1Characterization data of 1-aryl-3-(4-arylthiazol-2-yl)-2-thioureas (1)

Comp. Ar X m.p. (°C) Molecular formula Characteristic IR bands (cm−1)

nC�SnNH

4-BrC6H4 H 255 C16H12BrN3S2 3400 m, 3345 s 1200 s1aC6H5CH2 H 1781b C17H15N3S2 3350–3250 br 1210 m

H 148 C16H12ClN3S21c 3250 s, 3240 s2-ClC6H4 1205 s3-ClC6H41d H 210 C16H12ClN3S2 3400 m, 3330 m 1205 s

H 222 C17H17N3OS21e 3460–3200 br4-C2H5OC6H4 1200 s1f 1205 m3420 m, 3280 mC16H12N4O2S2120H2-NO2C6H4

3450 m, 3350 mC17H14BrN3S2225 1230 sCH34-BrC6H41g1h C6H5CH2 CH3 237 C18H17N3S2 3440–3350 br 1220 m

2-ClC6H4 CH3 1941i C17H14ClN3S2 3480 s, 3320 m 1205 s1210 s3400 m, 3200 mC17H14ClN3S22283-ClC6H4 CH31j

4-C2H5OC6H4 CH3 205 C19H19NOS2 3400–3250 br1k 1210 s

R. Lakhan et al. / Il Farmaco 55 (2000) 331–337 333

Table 2Characterization data of 1-aryl-3-(benzothiazol-2-yl)-2-thioureas (3)

Yield (%) m.p. (°C) Molecular formula Characteristic IR bands (cm−1)Comp. Ar

nNH nC�S

95 2213a C14H10BrN3S24-BrC6H4 3480 m, 3340 w 1240 m55 181 C15H13N3S2C6H5CH2 3300 s, 3280 m3b 1200 m

2-ClC6H43c 51 155 C14H10ClN3S2 3240 s, 3180 m 1210 m3d 494-C2H5OC6H4 198 C16H15N3OS2 3440 s, 3280 m 1230 s

0.01 mol) and hydrazine hydrate (1.5 ml, 0.03 mol) in30 ml of ethanol was heated in a sealed glass tube on awater-bath for 8 h. Lead sulfide was filtered while hotand the residue extracted with hot ethanol. The filtrateswere concentrated to get the required guanidine, whichwas crystallized from ethanol to form light yellowcrystals; yield 45%, m.p. 240°C. Analysis: C16H14BrN5S(C, H, N, S). IR (KBr, cm−1): 3420 m, 3350 w (NH2

and NH str), 1640 s, 1600 s, 1540 s, 760 m. NMR(DMSO-d6): d 5.27 (s, 2H, NH2, D2O exchange),6.90–8.00 (m, 10H, ar), 8.85 (s, 1H, NH, D2Oexchange), 10.79–11.66 (broad, 1H, NH, D2O ex-change). By the above procedure, a series of 1-aryl-2-amino-3-(4-phenylthiazol-2-yl)guanidines (2b– f) wereprepared and crystallized from ethanol. Their yields,m.p., characteristic IR and NMR data are recorded inTable 3.

2.5. 1-p-Bromophenyl-2-amino-3-(4-p-tolylthiazol-2-yl)guanidine (2g)

The guanidine was prepared from 1-p-bromophenyl-3-(4-p-tolylthiazol-2-yl)-2-thiourea by heating for 7 h;yield 48%, m.p. 114°C. Analysis: C17H16BrN5S (C, H,N, S). IR (KBr, cm−1): 3450 m, 3350 m (NH2 andNH str), 1640 m, 1600 m, 1540 s (aromatic ring),760 s. NMR (DMSO-d6): d 2.35 (s, 3H, CH3), 6.00(s, 2H, NH2, D2O exchange), 7.12–7.90 (m, 10H, 9 arand one NH, D2O exchange), 9.00 (s, 1H, NH,D2O exchange). Similarly, a series of 1-aryl-2-amino-3-(4-p-tolylthiazol-2-yl)guanidines (2h–k) were preparedand crystallized from ethanol. Their yields, m.p., char-acteristic IR and NMR (DMSO-d6) data are given inTable 3.

2.6. 1-p-Bromophenyl-2-amino-3-(benzothiazol-2-yl)guanidine (4a)

The compound was prepared from 1-p-bromophenyl-3-(benzothiazol-2-yl)-2-thiourea as above. The heatingperiod was 6 h; yield 75%, m.p. 190°C. Analysis:C14H12BrN5S (C, H, N, S). IR (KBr, cm−1): 3600–3200 broad (NH2 and NH str), 1620 m, 1600 m, 1560 s(aromatic ring), 740 m. NMR (DMSO-d6): d 6.83–8.39

(m, 10H, 8 ar and NH2, D2O exchange), 9.35 (s, 1H,NH, D2O exchange), 11.35–11.88 (broad, 1H, NH,D2O exchange).

Similarly, a series of 1-aryl-2-amino-3-(benzothiazol-2-yl)guanidines (4b–d) were prepared. Their yields,m.p. and characteristic IR and NMR data are recordedin Table 4.

3. Antimicrobial screening results

The 1-aryl-2-amino-3-(4-arylthiazol-2-yl)guanidines(2) and 1-aryl-2-amino-3-(benzothiazol-2-yl)guanidines(4) were screened for their antimicrobial activity againstseventeen different pathogenic strains of microorgan-isms such as Staphylococcus aureus, Staphylococcus epi-dermidis, Escherichia coli, Klebsiella pneumoniae,Pseudomonas aeruginosa, Citrobacter freundii, Proteus6ulgaris, Pro6idencia rettgeri, Edwardsiela tarda,Salmonella typhi, Salmonella typhimurium, Strigelladysenteriae, Vibrio cholerae 01 classical, Vibrio choleraenon 01, Vibrio parahaemolyticus, Aeromonas hydrophilaand Plesimonas shigelloides. The antimicrobial suscepti-bility was determined by the Stokes disc diffusion tech-nique [11] whereas minimum inhibitory concentration(MIC) was determined by the Stokes and Ridgewayplate dilution method [11]. Zones of inhibition of thetest microorganisms were compared with that of thecontrol NCTC strain of E. coli 10418.

The antimicrobial susceptibility was measured at aconcentration of 40 mg/disc. The analysis of variouspatterns of susceptibility is shown in Table 5. Theresults reveal that there are a wide variety of combina-tions of the synthesized compounds to which strainswere sensitive. The synthesized N-aminoguanidines aremost active against S. epidermidis, K. pneumoniae, S.dysenteriae, V. cholerae 01 classical, V. cholerae non 01and A. hydrophila. In general, those aminoguanidineswhich have para or meta halogen substituent in thephenyl ring show remarkable antibacterial activity.

The sensitive aminoguanidines from antimicrobialsusceptibility test were further subjected to the MIC testas follows. Freshly prepared solutions of synthesizedcompounds diluted to different concentrations were

R. Lakhan et al. / Il Farmaco 55 (2000) 331–337334

Tab

le3

Cha

ract

eriz

atio

nda

taof

1-ar

yl-2

-am

ino-

3-(4

-ary

lthi

azol

-2-y

l)gu

anid

ines

(2)

Mol

ecul

arm

.p.

Ar

Cha

ract

eris

tic

IRba

nds

1H

NM

Rin

d(D

MSO

-d6)

Com

p.X

Yie

ld(%

)(c

m−

1)

form

ula

(°C

)

nN

H2,

NH

nC�N

3420

m,

3350

w16

40s

5.27

(s,

2H,

NH

2,

D2O

exch

ange

),6.

90–8

.00

(m,

10H

,ar

),8.

85(s

,1H

,N

H,

C1

6H

14B

rN5S

240

45H

2a4-

BrC

6H

4

D2O

exch

ange

),10

.79–

11.6

6(b

r,1H

,N

H,

D2O

exch

ange

)34

40s,

3260

m16

20s

2.83

(s,

1H,

NH

,D

2O

exch

ange

),4.

52(d

,2H

,C

H2),

4.53

–5.2

2(b

r,2H

,N

H2,

C6H

5C

H2

2bH

6014

2C

17H

17N

5S

D2O

exch

ange

),7.

00–8

.00

(m,

11H

,ar

),10

.13

(br,

1H,

NH

,D

2O

exch

ange

)34

10s,

3260

m16

10m

3.40

(s,

2H,

NH

2,

D2O

exch

ange

),6.

00(s

,1H

,N

H,

D2O

exch

ange

),6.

66–8

.32

C1

6H

14C

lN5S

182

2c62

H2-

ClC

6H

4

(m,

11H

,10

aran

dN

H,

D2O

exch

ange

)34

50s,

3225

s16

40m

2d5.

98(s

,2H

,N

H2,

D2O

exch

ange

),6.

71–7

.85

(m,

10H

,ar

),9.

00(s

,1H

,N

H,

3-C

lC6H

4H

6010

2C

16H

14C

lN5S

D2O

exch

ange

),11

.00–

11.6

6(b

r,1H

,N

H,

D2O

exch

ange

)34

80–3

250

br16

30s

–14

52e

C1

8H

19N

5O

S63

H4-

C2H

5O

C6H

4

2-N

O2C

6H

4C

16H

14N

6O

2S

3440

s,32

50s

1610

s–

H46

2f15

02g

3450

m,

3350

m4-

BrC

6H

416

40m

2.35

(s,

3H,

CH

3),

6.00

(s,

2H,

NH

2,

D2O

exch

ange

),7.

12–7

.90

(m,

10H

,ar

and

CH

348

114

C1

7H

16B

rN5S

NH

,D

2O

exch

ange

),9.

00(s

,1H

,N

H,

D2O

exch

ange

)34

00s,

3300

s16

40m

2.30

(s,

3H,

CH

3),

4.52

(d,

2H,

CH

2),

4.79

(s,

2H,

NH

2,

D2O

exch

ange

),C

6H

5C

H2

C1

8H

19N

5S

178

2h49

CH

3

7.00

–8.0

0(m

,11

H,

aran

dN

H,

D2O

exch

ange

),10

.12

(s,

1H,

NH

,D

2O

exch

ange

)84

C1

7H

16C

lN5S

3450

–310

0br

1630

s1.

28(s

,3H

,C

H3),

3.39

(s,

2H,

NH

2,

D2O

exch

ange

),6.

70(s

,1H

,N

H,

D2O

2i2-

ClC

6H

4C

H3

58ex

chan

ge),

6.70

–8.4

4(m

,10

H,

aran

dN

H,

D2O

exch

ange

)34

00m

,33

25m

1635

m–

136

(dec

.)68

C1

7H

16C

lN5S

2j3-

ClC

6H

4C

H3

2k34

00–3

310

br4-

C2H

5O

C6H

416

20s

1.33

(t,

3H,

CH

3),

2.21

(s,

3H,

CH

3),

3.90

(q,

2H,

CH

2),

4.52

–5.3

1(b

r,2H

,C

H3

4565

C1

9H

21N

5O

SN

H2,

D2O

exch

ange

),5.

66(s

,1H

,N

H,

D2O

exch

ange

),6.

50–8

.00

(m,

9H,

ar),

8.26

(s,

1H,

NH

,D

2O

exch

ange

)

R. Lakhan et al. / Il Farmaco 55 (2000) 331–337 335

Tab

le4

Cha

ract

eriz

atio

nda

taof

1-ar

yl-2

-am

ino-

3-(b

enzo

thia

zol-

2-yl

)gua

nidi

nes

(4)

Mol

ecul

arY

ield

Cha

ract

eris

tic

IRba

nds

Ar

1H

NM

Rin

d(D

MSO

-d6)

Com

p.m

.p.

(cm

−1)

(°C

)fo

rmul

a(%

)

nN

H2,

NH

nC�N

C1

4H

12B

rN5S

3600

–320

0br

1620

m6.

83–8

.39

(m,

10H

,ar

and

NH

2,

D2O

exch

ange

),9.

35(s

,1H

,N

H,

D2O

exch

ange

),4-

BrC

6H

44a

7519

011

.35–

11.8

8(b

r,1H

,N

H,

D2O

exch

ange

)16

40m

1.31

(s,

1H,

NH

,D

2O

exch

ange

),3.

11–3

.79

(br,

2H,

NH

2,

D2O

exch

ange

),4.

44(d

,34

00–3

100

brC

15H

15N

5S

85–8

8(d

ec.)

70C

6H

5C

H2

4b2H

,C

H2),

6.53

–8.3

5(m

,10

H,

aran

dN

H,

D2O

exch

ange

)2-

ClC

6H

434

00br

,32

00br

1640

s–

7021

04c

C1

4H

12C

lN5S

C1

6H

17N

5O

S36

00–3

100

br16

40s

–4d

4-C

2H

5O

C6H

459

115

R. Lakhan et al. / Il Farmaco 55 (2000) 331–337336

added to Mueller–Hinton agar (Hi-Media), at 45–50°C. It was mixed well and poured into 100-mmpetridishes; 20 ml gave a depth of about 4 mm. Plateswere dried prior to experiment and each plate wasdivided into equal sections. Overnight broth cultures ofthe test strains and control of E. coli NCTC 10418 werediluted to 1:1000 in phosphate buffer solution PBS (pH7.4) to a concentration of about 104–105 viable cells/ml.

Aliquots of 0.01 ml of the diluted cultures werespotted on the different sectors on the agar platescontaining the serial dilutions 2.0, 1.0, 0.5, 0.25 and

0.125 mg/ml of the synthesized compounds. Inoculawere allowed to dry and the plates were incubated at30°C overnight. All the test strains were also inoculatedon a control plate containing no synthesized com-pound. The MIC of the synthesized compound was thelowest concentration of the compound per milliliter ofmedium that completely inhibited visible growth of thecorresponding test strain.

The MIC of the various antimicrobial agents forspecies tested is shown in Table 6. The MIC values forthe compounds showed a range from 0.125 to 2 mg/ml.

Table 5Antimicrobial screening results of 1-aryl-2-amino-3-(4-arylthiazol-2-yl)guanidines (2) and 1-aryl-2-amino-3-(benzothiazol-2-yl)guanidines (4)a

Microorganisms Comp. no.

2g2f2e2d2c 4c4a2k2j 4d2i2b2a 4b2h

S R R R R S SSR R RS. aureus S R R SS R S R S S S S SR R S R RS. epidermidis SS R R R R S S S RR R S R RE. coli S

S S R S R S SRK. pneumoniae SRRSRRSR S R S R R RP. aeruginosa R R R S R R R RR R S R S R SRR SC. freundii R R R S R

RS R S S R S R RR R S R R SP. 6ulgarisRS R S R S S S RR R S R R SP. rettgeri

R R S R R R SRE. tarda SRRRRRSS R R S S R R R RS. typhi SS R S R R

R S R R S S RRRS. typhimurium SRRSRSRS R S S S S S SR R S R R SS. dysenteriaeRS S R R S S S SR R S R R SV. cholerae 01 classical

S R S S R R SRV. cholerae non 01 SRRSSRSR R S S S R R S SV. parahaemolyticus RS R S R R

S R S S R S SRRA. hydrophila S R SRRSR SP. shigelloides SS R R R SR R S R R S R

a S=sensitive; R=resistant. .

Table 6MIC of 1-aryl-2-amino-3-(4-arylthiazol-2-yl)guanidines (2) and 1-aryl-2-amino-3-(benzothiazol-2-yl)guanidines (4)a

Microorganisms Comp. no.

2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 4a 4b 4c 4d

2 2S. aureus 1 10.125 1 0.125S. epidermidis 0.125 2 0.125 1 0.125 12 2 1E. coli 2 11 1K. pneumoniae 2 1 2

P. aeruginosa 0.5 2 1C. freundii 2 1 2P. 6ulgaris 1P. rettgeri 2 2E. tardaS. typhi 2S. typhimurium 0.5

1 1S. dysenteriae 2 2 1 0.5V. cholerae 01 classical 2 2 2 2 2 1

1 0.5V. cholerae non 01 2 1 2 0.52 2V. parahaemolyticus 2 2 12 1A. hydrophila 2 1 2 2 1

1P. shigelloides 2 2 1 1

a MIC=minimum inhibitory concentration. The figures are in mg/ml.

R. Lakhan et al. / Il Farmaco 55 (2000) 331–337 337

In the present study, all seventeen isolates were resistantto one or more antimicrobial agents while the MICvalues did not exceed 0.125 mg/ml for any bacterialpathogens tested.

Acknowledgements

The authors wish to thank Professor R. Balaji Rao,Head of the Department of Chemistry and to the Headof the Department of Microbiology, IMS, BanarasHindu University for providing the necessary facilities.The financial assistance by the Tribhuvan University,Kathmandu, Nepal, for the award of a Teacher Re-search Fellowship under the Faculty Development pro-gram to B.P.S. is also gratefully acknowledged.

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[4] P.N. Bhargava, M.R. Chaurasia, Some new potential antituber-culars: benzothiazolyl guanidines, Curr. Sci. 37 (1968) 347.

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