INTRODUCTION:

Recently much interest of the use of microwave oven for the synthesis of heterocyclic compounds has been shown and that is due to substantial reduction in reaction time and better yield of the product¹. Coupling of two techniques, that is, organic reaction using microwave irradiation has been field, which had shown excellent results leading to development of many reaction procedures, which are environmental friendly falling in the domain of ‘Green Chemistry’.

Benzothiazole derivatives are reported to possess antibacterial², anti-inflammatory and anticonvulsant activities. Therefore it was considered worthwhile to explore the synthesis of compounds built upon benzothiazole skeleton incorporating pyrazolyl moiety with the hope of potentiating the activity of two such units in the same compound.

For the present investigation we have prepared 2-{[(4-substituted phenyl azomethylene)-3-substituted phenyl] pyrazol-1-yl} benzothiazoles (5a-o) starting from 2-amino benzothiazole (1) which was converted to corresponding benzotiazol-2-yl hydrazine (2) using hydrazine hydrate. The available hydrazino group was exploited to give benzotiazol-2-yl hydrazones analogues (3) on reacting with different methyl phenyl ketones. Vilsmeier Haack reaction³ was applied to benzothiazole hydrazones to obtain corresponding formylated pyrazolyl benzothiazoles (4). Addition of substituted anilines to (4) resulted the corresponding products (5a-o) (Scheme I).

Anti-bacterial activity:

A total of 15 compounds were subjected to antibacterial screening against Staphylococcus aureus ATCC 3750, Salmonella typhi NCTC 786 using tube dilution technique⁴. Ampicillin and Trimethoprim and were used as the standard drugs against S. aureus and S. typhi, respectively.

Anti-fungal activity:

The SAR of antifungal agents has revealed that the basic nitrogen-carbon linkage is an essential feature for the activity⁵. All the 15 synthesized compounds were screened for antifungal activity against standard strain of Candida albicans ATCC 10231 by Sabouraud-Dextrose broth by tube dilution method using Miconazole as standard drug.

EXPERIMENTAL:

All raw materials used in the synthesis have been obtained from Fluka AG (Buchs, Switzerland) and Aldrich Chemical and Co. Inc. (Milwaukee, WI, USA). All the commercial solvents used in the experimental work were redistilled and dried before use. Melting points of the synthesized compounds was recorded on Thermonik Melting Point Apparatus (Campbell Electronics, Mumbai, India) and are uncorrected. TLC was performed on silica gel coated plates using iodine vapour as visualizing agent. IR spectra were recorded on Hartmann-Braun, MB series (Bomem, Quebec, Canada), ¹H NMR spectra were recorded on 300 MHz instrument (Jeol Ltd., Tokyo, Japan), using deuterated solvent DMSO, at Institute of Science, Mumbai. Elemental analyses were carried out on at SAIF, IIT Powai, Mumbai.

General method for preparation of Benzothiazol-2-yl hydrazine (2)

Concentrated HCl (6ml) was added dropwise with stirring to hydrazine hydrate (6ml) at 5-10°c, to it ethylene glycol

Table-1: Physical properties and antimicrobial activity of compounds 5a-o.

Comp	Substituent		Yield (%)	M.P. (C)	Antimicrobial activity (µg/ml) (MIC)
	Substituent				*S. aureus* ATCC 3750	S. typhi NCTC 786	*C. albicans* ATCC 10231	*A.. niger* ATCC 16404
	(R)	(R’)			*S. aureus* ATCC 3750	S. typhi NCTC 786	*C. albicans* ATCC 10231	*A.. niger* ATCC 16404
5a	3,4-dichloro	2,5-dimethoxy	68	196	50	100	200	100
5b	3,4-dichloro	4-fluoro	71	148	100	100	100	100
5c	3,4-dichloro	3,4-dichloro	72	205	50	100	N.A	200
5d	3,4-dichloro	4-chloro	68	214	100	50	100	100
5e	3,4-dichloro	4-methoxy-2-nitro	73	143	100	100	200	200
5f	3,4-dichloro	4,5-dichloro-2-nitro	72	282	200	100	100	200
5g	3,4-dichloro	3,4-dimethyl	69	261	100	50	100	100
5h	2,4-dihydroxy	2,5-dimethoxy	79	263	200	N.A	200	100
5i	2,4-dihydroxy	2,4-difluoro	76	269	200	100	200	100
5j	2,4-dihydroxy	4-fluoro	70	150	N.A	200	100	50
5k	4-fluoro	3-chloro-4-fluoro	75	155	100	100	100	100
5l	4-fluoro	4-fluoro	80	99	50	50	50	50
5m	4-fluoro	2,4-difluoro	69	120	40	100	50	40
5n	4-fluoro	3,4-dichloro	71	274	100	50	100	100
5o	4-fluoro	2,5-dimethoxy	78	189	40	100	40	50

 Elemental analysis (C, H, N) of the compounds was in the range of µ 0.4%.

 Ampicillin (MIC-0.04 µ g/ml) used as standard against S. aureus.

 Trimethoprim (MIC 0.01 µ g/ml) used as standard against S. typhi.

 Miconazole (MIC 6.25 µ g/ml) as standard against C. albicans and A. niger.

 N.A.- Inactive at 200 µ g/ml.

(24ml) and 2-amino benzothiazole (1) (0.03 mol), were added and refluxed for 3 hrs. On cooling solid separated out, which was filtered, washed with water and recrystallized form ethanol. M.P.=195°C IR (KBr); 3321, 3202, 1651, 1460 (C=N), 1195, 1159, 816. ¹H NMR (DMSO); d5.04, (2H, s, NH₂), d7.43 (1H, t, Ar-H), d7.60 (1H, q, Ar-H), d7.72 (1H, d, Ar-H), d9.34 (1H, s, NH).

Synthesis of Benzothiazol-2-yl hydrazones (3a-c):

Compound 2 (1.5mmol), appropriate acetophenone (3,4-dichloro, 2,4-dihydroxy and 4-fluoro) (2.2mmol) and glacial acetic acid (2-3drops) were taken in absolute alcohol (20ml) and refluxed on a water bath for 5-13 hrs. On cooling solid separated out, which was filtered, washed with little water and crystallized from ethanol.

3a: M.P.= 216°C. IR (KBr); 3148, 3049, 2908, 1568, 1459 (NHNH=C), 1341, 1126, 918, 937. ¹H NMR (DMSO); d2.45 (3H, s, CH₃), d7.22 (1H, m, Ar-H), d7.47 (1H, d, Ar-H), d7.76 (1H, m, Ar-H), d11.63 (1H, s, NH).

Synthesis of Formylated pyrazole benzothiazole (4a-c)

· Method A (conventional)

Compound 3 (5mmol) was dissolved in Vilsmeier Haack reagent (DMF) (6ml) in POCl₃ (1.5ml) and stirred on room temperature for 4 hrs, then the contents were poured over crushed ice (previously neutralized with NaHCO₃), solid separated out, which was filtered washed with water, dried and crystallized from ethanol.

· Method B (MWI)

The hydrazone (3a-c) (0.004M) was added in mixture of Vilsmeier-Haack reagent in an open Erlenmeyer flask and irradiated in microwave oven for 5 sec. After completion of the reaction, product was worked up as described in method A.

4a: M.P.=200-202°C IR (KBr); 3131, 3062, 2923 (CHO), 2360, 2342, 1695, 1610, 1548, 1463(C=N),1356, 1207, 898. ¹H NMR (DMSO); d7.49, (1H, m, Ar-H), d7.63 (3H, s, Ar-H), d8.03 (2H, s, Ar-H), d9.75 (1H, s, >C=CH), 10.14 (1H, s, CHO).

Synthesis of 2-{[(4-substituted phenyl azomethylene)-3-substituted phenyl] pyrazol-1-yl} benzothiazoles (5a-o)

Compound 4 (0.001M) was dissolved in 10-15ml benzene. Add (0.001M) of substituted aniline followed by addition of 1-2 drops of glacial acetic acid and reflux on water bath for 5-10hrs. The H₂O formed is removed using Dean-Stark apparatus. On cooling solid separates out, which was filtered, washed with little water and recrystallised from ethanol/DMF. The physical characteristics are recorded in Table 1. Spectral data of the selected compounds are given in Table 2. The yields of the compounds were moderate to good (55-80%) ( Table 1). All other reactions were facile and afforded almost quantitative yield of the products. All the synthesized compounds were stable at room temperature.

Table 2: Spectral data of selected synthesized compounds.

Compd.

IR spectral data cm^-1

NMR spectral data

d= ppm

3156 (NH-stretching)

1619 (C=N)

1566 (C=C)

7.9 (s, 1H, 5-CH)

6.9-7.4 (m, 11H, ArH)

2.17 (s, 1H, N=CH)

1601 (C=N)

3566 (NH-stretching)

3300 (-OH)

8.06 (s, 1H, 5-CH)

7.1-7.8 (m, 11H, ArH)

2.21 (s, 1H, N=CH)

3288 (NH-stretching)

1621 (C=N)

1523 (C=C)

8.36 (s, 1H, 5-CH)

7.5-8.12 (m, 12H, ArH)

2.25 (s, N=CH)

Biological screening:

The results of the biological screening are given in Table 3. Ampicillin (MIC-0.04 mg/ml) and Trimethoprim (MIC-0.01 mg/ml) were used as the standard drugs for S. aureus and S. typhi resp. Also, Miconazole (MIC 0.01 mg/ml) was used as a standard drug against fungal species. Two compounds 5m and 5o-exhibited activity at 40mg/ml against S.aureus. Rest of the compounds exhibited activity in the range of 50-200mg/ml.

Compounds 5l, 5m and 5o exhibited activity upto 40mg/ml while rest of the compounds exhibited moderate activity against the fungal species.

ACKNOWLEDGEMENT:

The authors are thankful to SAIF (Sophisticated Analytical Instrument Facility), IIT (Indian Institute of Technology), Powai, Mumbai for carrying out the elemental analysis (CHN) and also thankful to Institute of Science, Fort, Mumbai for recording the NMR spectra.

REFERENCES:

1. Caddick S., Tetrahedron, 51, (1995), 10403.

2. P. Gopkumar, B. Shivakumar, E. Jaychandran, A.N. Nagappa, Indian J. Heterocyclic Chem., 11 (2001), 39.

3. S. Salvi, P.T. Perumal, Indian J. Chem., 39B (2000), 163.

4. E. Joan Stokes, Clinical Bacteriology, 4^th Edition (1995), 226.

5. Wilson Giswold’s “Textbook of Organic Medicinal Chemistry and Pharmaceutical Chemistry”, 11^th Edition.

Received on 03.11.2009 Modified on 25.03.2010

Research J. Pharm. and Tech.3 (4): Oct.-Dec.2010; Page 1044-1046

S. typhi

Scheme I:

ACKNOWLEDGEMENT: