INTRODUCTION:

1, 3, 5 Triazine derivatives have been shown to possess remarkable antitubercular activity¹.2-chloro-s-triazine herbicides induce aromatase (CYP19) activity in H295R human adrenocortical carcinoma cells². Several derivatives of s-triazines show antibacterial activity³ and 2-hydrazino-α-naptha-[2, 3]-thiazole⁴ have significant antifungal property. Hetrocyclic hydrazino systems when condensed with aromatic aldehydes to afford Schiff bases are effective against pathogenic fungi⁵ and associated with antimicrobial and herbicide activity⁶ considering the biological potential of the Schiff bases of the triazines it was thought pertintent to synthesize the compounds. According to scheme 1

MATERIALS AND METHODS:

Melting points of synthesized compounds were determined in open capillary tubes and are therefore uncorrected. The structures of compounds were established on the basis of elemental analysis and spectral data. The IR spectra were recorded in range of 4000-450cm^-1 using KBr pellets on a FTIR RX1 Perking-Elmer spectro-photometer. H NMR spectra were recorded on a Bruker DRX300Mhz spectrophoto-meter using CDCI₃/DMSO-d₆ as solvent. The FAB mass spectra were recorded on Jeol SX-102/DA-6000 spectrometer data system using argon/xenon (6KV, 10mA) as FAB gas. Purity of synthesized compounds was checked by silica gel-G plates of 2mm thickness using benzene and ethyl acetate (9:1 and 1:9) as solvent system and iodine chamber as developer.

Experimental:

Preparation of alkyl thiourea:

Methyl (m.p.119^oC) and butyl thiourea (m.p.80^oC) were prepared by addition of ammonia to corresponding alkyl isothiocyanates. Cyclohexylthiourea (m.p.172^oC) was synthesized by reaction of benzoyl isothiocyanate and cyclohexylamine.

General methods of Preparation of S-benzyl-N-alkyl isothiourea:

Alkylthiourea (9.0g, 0.1mol.) was dissolved in alcohol (100ml). Then benzylchloride (12.7g, 0.1mol.) was added to it. .The reaction mixture was refluxed for about two and half hours. Excess of solvent was removed on flash evaporator. The residue was cooled in freezing mixture and neutralized with very dilute and cold solution of sodium bicarbonate. The free S-benzyl-N-alkyl isothiourea was extracted with ether. The ethereal layer was dried over anhydrous sodium sulphate. Removal of ether gave the desired free base in quite good yield. By adopting similar procedure other S-benzyl_N-alkyl isothiourea was prepared.

Preparation of 2-benzylmercapto-1-methyl-4-phenyl-1, 6-dihydro-1, 3, 5-triazine-6-thione:

A solution of benzoylisothiocyanate (6.5g, 0.04mol.) was added to a solution S-benzyl-N-methyl isothiourea (7.2g, 0.04mol.) in acetone over a period of 15 minutes. An exothermic reaction took place and a golden yellow solid separated after 30 minutes. It was recrystallised with benzene.

Yield : 9.0g (69%); mp146^oC

IR (KBr) : 1624(C=N), 1109(C=S), 659(C-S-C) cm^-1

NMR (CDCl₃) : δ 3.65(s, 3H, CH₃), 4.52(s, 2H, CH₂), 7.1-8.2(m,10H, ArH)

Elemental Analysis: Calcd. For C₁₇H₁₅N₃S₂, C, 62.76; H, 4.61; N, 12.92; S, 19.69%

Found, C, 62.58; H, 4.64; N, 12.97; S, 19.62%

Following similar procedure different 1-alkyl-2-benzylmercapto-4-(phenyl/4-chlorophenyl)-1,6- di-hydro-1, 3, 5-triazine-6-thiones were prepared and their details are given in Table 1.

Preparation of 2-hydrazino-1-alkyl-4-phenyl-1, 6-dihydro-1, 3, 5-triazine-6-thione:

2-Benzylmercapto-1-methyl-4-phenyl-1, 6-dihydro-1,3,5-triazine-6-thione(6.5g,0.02mol.) was dissolved in excess of benzene(300ml),hydrazinehydrate (100%,1.0g,0.02mol.) was added and the reaction mixture was stirred at 30^oC for 10h. A greenish yellow precipitate thus formed was filtered and washed with excess of petroleum ether and benzene to remove benzyl mercaptan and starting material if left unreacted. The resulting hydrazino compound was recrystallised with ethanol/DMF (4:1) mixture.

Yield : 3.0g (64%); mp160^oC

IR (KBr) : 3327, 3273, 3063(NHNH₂), 1630(C=N), 1064(C=S), cm^-1

NMR (DMDSO-d₆) : δ 2.9(s, 3H, CH₃), 7.3-8.3(m, 5H, ArH)

Elemental Analysis : Calcd. for C₁₀H₁₁N₅S,C,51.50; H,4.72; N,30.04; S,13.73%

Found, C, 51.48; H, 4.75N, 30.08; S, 13.60%

Similarly other derivatives were prepared.

Preparation of Schiff bases:

Preparation of N-(1-Alkyl-4-phenyl-6-thioxo-1, 6-dihydro-1, 3, 5-triazine-2-yl)-N’-(4-chloro-benzylidine) hydrazine:

2-Hydrazino-1-alkyl-4-phenyl-1, 6-dihydro-1,3,5-triazine-6-thione(2.33g,0.01mol) was dissolved in absolute alcohol (50ml) and 4-chlorobenzaldehyde (1.4g,0.01mol) and 1-2 drops of glacial acetic acid was added to it, and the reaction mixture was refluxed for about 8h, then it was filtered and the filtrate was allowed to cool overnight and the title compound was separated out as fine yellow needles, which was recrystallised from ethanol.

Yield : 2.7g (76%); mp166^oC

IR (KBr) : 3439(NH), 1624 (C=N), 1089 (C=S) cm^-1

NMR (DMDSO-d₆) : δ 2.5(s, 3H, CH₃₎, 7.4-8.1(m, 9H, ArH 8.5)

(s, 1 H,N=CH), 10.5 (s,1H,NH)

Elemental Analysis : Calcd. for C₁₇H₁₄ClN₅S,C,57.38; H,3.93; N,19.69; S,9.00%

Found, C, 57.42; H, 3.89N, 19.73; S, 8.97%

Similarly other derivatives were prepared and their physical constrants are recorded in Table-I.

In-vitro Antibacterial Study:

The Invitro antibacterial activity of the series of synthesized compounds was evaluated against five pathogenic bacteria. The bacterial strains were procured from the bacterial repository of department of microbiology I. M. S. B. H. U. Varanasi.

N, N-dimethyl formamide (Ranbaxy) N, N-dimethyl sulpoxide (CDH) were used to prepare stock solution of standard and synthesized drugs. Subsequent dimension of the stock were also made in above solvent. Mueller Hinton media (Hi-media) was used to subculture various strains of micro-organism as well for diversing the MIC of

synthesized compounds by agar diffusion method. Normal saline was used to prepare in column of the bacteria to be used for in vitro antimicrobial study.

Graded amounts of synthesized compounds were incorporated into measured amount of M.H agar media. The media was subsequently inoculated and insulated the lowest concentration of the test compound that completely inhibited growth agar plate, disregarding a single colony or faint haze caused by the inoculums was considered as minimum inhibitory concentration (MIC) of that compound.

Stock solution of test compound was prepared in dimethyl formamide, dimethyl sulphoxide with a concentration of 10mg/2ml. The stock solution was then double diluted and used for study. The sterilized petri dishes were marked with 1-10 column on the bottom for the respective micro organism. The double diluted tests (1ml) were added to the petri dishes, diluted further by addition of 19ml of MHA media (previously cold to 60^oC). Such that plates containing 500mg/ml of test drug to lower concentration was prepared. Under expected condition, the dilute bacterial suspension was inoculated as spots using sterilized swabs. After inoculation, the plates were incubated at 37^oC for 24hrs, MIC was noted. Reference compounds trimethoprim and sulphamethoxazole were used. The results are given in Table II.

Table-I N-(1-Alkyl-4-phenyl-6-thioxo-1, 6-dihydro-1, 3, 5-triazine-2-yl)-N’-arylidine hydrazine

S. No.	Ar	Mol. Formula	Yield (%)	M.P. (^oC)	Elemental Analysis
					Calculated (Found)%
					C	H	N	S
R=CH₃ Ar=C₂H₅
1.	4-ClC₆H₄	C₁₇H₁₄ClN₅S	76	166	57.38 (57.42)	3.93 (3.89)	19.69 (19.73)	9.00 (8.97)
2.	4-CH₃OC₆H₄	C₁₈H₁₇N₅OS	66	165	61.53 (61.55)	4.84 (4.88)	19.94 (19.97)	9.11 (9.08)
3.	3-CH₃C₆H₄	C₁₈H₁₇N₅S	72	150	64.47 (64.45)	5.07 (5.11)	20.89 (20.92)	9.55 (9.51)
4.	4-NO₂C₆H₄	C₁₇H₁₄N₆O₂S	69	170	55.73 (55.69)	3.82 (3.78)	22.95 (22.93)	8.74 (8.69)
R=n-C₄H₉; Ar=4-ClC₆H₄
5.	4-ClC₆H₄	C₂₀H₁₉Cl₂N₅S	69	188	55.55 (55.14)	4.39 (4.45)	16.20 (16.24)	7.40 (7.39)
6.	4-CH₃OC₆H₄	C₂₁H₁₂ClN₅OS	61	156	58.94 (58.95)	5.14 (5.19)	16.37 (16.42)	7.48 (7.53)
7.	3-CH₃C₆H₄	C₂₁H₂₂ClN₅S	69	159	61.23 (61.25)	5.34 (5.28)	17.21 (16.97)	7.77 (7.82)
8.	4-NO₂C₆H₄	C₂₀H₁₉ClN₆O₂S	68	151	54.23 (54.19)	4.29 (4.32)	18.98 (19.02)	7.23 (7.18)
R=C₆H₁₁ Ar=C₆H₅
9.	4-ClC₆H₄	C₂₂H₂₂ClN₅S	56	238	62.33 (62.02)	5.19 (5.39)	16.52 (16.59)	7.55 (7.35)
10.	4-CH₃OC₆H₄	C₂₃H₂₅N₅OS	49	173	85.87 (65.80)	5.96 (6.05)	16.70 (16.77)	7.63 (7.51)
11.	3-CH₃C₆H₄	C₂₃H₂₅N₅S	55	265	68.48 (68.42)	6.20 (6.25)	17.361(17.30)	7.94 (7.80)
12.	4-NO₂C₆H₄	C₂₂H₂₂N₆O₂S	58	255	60.82 (60.71)	5.06 (4.90)	19.38 (19.31)	7.37 (7.17)

Table-II Invitro Antibacterial activities of N-(1-Alkyl-4-phenyl-6-thioxo-1, 6-dihydro-1, 3, 5- triazine-2-yl)-N’arylidine hydrazine, 2 (MIC in mg/ml)

Compound No.	Microorganism
Compound No.	*E. coi*	*S. aureus*	*Bacillus subtilis*	*Salmonella typhi*	*Shigella dysentrae*
1	0.152	1.22	625	9.76	156.26
2	1250	625	2500	2500	325
3	1250	2500	625	1250	156.26
4	9.76	4.88	9.76	78.12	19.53
5	9.76	78.12	156.25	312.5	39.06
6	1250	2500	1250	625	2500
7	2500	1250	625	78.12	156.12
8	4.88	39.06	78.12	4.88	2.44
9	0.61	0.152	1.22	0.152	19.53
10	9.76	78.12	625	312.5	325
11	312.5	2500	1250	2500	2500
12	78.12	36.06	19.53	39.06	19.53
Trimethoprim	19.53	75000	1250	9.76	-
Sulphamethoxazole	2500	5000	1250	2500	2500

RESULTS AND DISCUSSION:

The triazines inhibited growth of various bacteria. The different series of compound with two major variations one at the size of the alkyl substituent and other in the phenyl ring. From the results it is apparent as the alkyl chain is increased from methyl to butyl to cyclo hexane there is increase in the spectrum of antibacterial activity. Further substitution in the phenyl ring at affected the antibacterial activity. The chloro substituent derivatives are active followed by nitro substituent derivatives. The compound no 9 with cyclo hexane and 4-chloro substitution was found to be most active in the series against different organism. This may be perhaps due to greater lipophilicity of the cyclohexyl ring as compared to methyl or butyl. This property may help the compound to disrupt the lipoidal membrane of the micro-organism. Further the introduction of a chloro group in phenyl ring increases the metabolic stability of the drug. Most of the compounds are superior in activity as compared to sulphamethoxazole and some are more active than trimethoprim. The compound no 9 has emerged as a lead molecule from these studies and further molecular modifications can be attempted to increase the antibacterial activity of the compounds.

AKNOWLEDGEMENTS:

U.R.T.BHU and C.D.R.I. Lucknow

REFERENCES:

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3. Megen, M.A. Aldema, I.Rabbani, and Alearez, F.E., J.Hetero.Chem.,17(1),77(1980)

4. Leymarie, Martine, Pays, M.B. and Richer J.C; J.Hetero.Chem, 17, 1175 (1980)

5. Ferro, W.H.,Hanna C., and Schueler, F.W; J.Am.Pharm. Assoc, 43, 370, 1954.

6. Brown, F.C.; Chem.Rev.61, 463, 1961.

Received on 01.08.2009 Modified on 05.10.2009

Asian J. Research Chem. 3(1): Jan.-Mar. 2010; Page 36-39