Synthesis of formazans from Mannich base of 5-(4-chlorophenyl amino)-2-mercapto-1,3,4-thiadiazole as antimicrobial agents Academic research paper on "Chemical sciences"

Arabian Journal of Chemistry (2014) 7, 181-187

King Saud University Arabian Journal of Chemistry

www.ksu.edu.sa www.sciencedirect.com

ORIGINAL ARTICLE

Synthesis of formazans from Mannich base of 5-(4-chlorophenyl amino)-2-mercapto-1,3,4-thiadiazole as antimicrobial agents

Pramilla Sah *, Pratibha Bidawat, Manu Seth, Chandra Prakash Gharu

Department of Chemistry, J.N.V. University, Jodhpur 342 005, Rajasthan, India

Received 3 September 2010; accepted 23 October 2010 Available online 29 October 2010

KEYWORDS

Mannich base; 1,3,4-Thiadiazole; Formazans; Antimicrobial

Abstract 5-(4-Chlorophenyl amino)-2-mercapto-1,3,4-thiadiazole (I) was refluxed with formaldehyde and ammonium chloride in ethanol yielding the Mannich base 5-(4-chloro phenyl amino)-3-aminomethyl-2-mercapto-1,3,4-thiadiazole (II). Esterification with 4-chloro-(2,6-dinitro phenoxy)-ethyl acetate (III) under anhydrous conditions gave the intermediate (IV). Subsequent hydrazinolysis with hydrazine hydrate gave the corresponding hydrazide 3-amino methyl-5-(4-chloro phenyl amino)-2-mercapto-4'-(2',6'-dinitro phenoxy)-acetyl hydrazide (V). The hydrazide was converted into the Schiff bases (VIa b) by reacting with 2-chlorobenzaldehyde and 3-meth-oxy-4-hydroxy benzaldehyde in presence of methanol containing 2-3 drops of acetic acid. Diazotisation with aromatic amines, sulphanilic acid and sulphur drugs gave the formazans (VIIa g) respectively. Chemical structures have been established by elemental analysis and the spectral techniques of FTIR, 1H NMR and mass. Antimicrobial activity (in vitro) was evaluated against the two pathogenic bacterial strains. Escherichia coli and Salmonella typhi, three fungal strains Aspergillus niger, Penicillium species and Candida albicans. The compounds have shown moderate activity.

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1. Introduction

* Corresponding author. Tel.: +91 0291 2642340. E-mail address: pramilla_s@yahoo.co.uk (P. Sah). Peer review under responsibility of King Saud University.

Thiadiazole system is a cyclic analogue of thiosemicarbazide having the toxophoric N=C=S linkage. This heterocyclic system is responsible for a broad spectrum of biological activities, i.e. antimicrobial (Akhtar et al., 2007), antidepressant (Bahadur and Singh, 1980), antitubercular (Bauer et al., 1966), antioxidant (British Pharmacopeia, 1953), antidiabetic (Giri and Singh, 1967), antileishmanial (Haydon et al., 2009), anti-HIV (Jamode et al., 2008), anti-inflammatory (Joshi et al., 2008), etc.

Formazan derivatives have been synthesized by blocking the reactive hydrogen of a Schiff base or a hydrogen with diazotised

1878-5352 © 2010 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.Org/10.1016/j.arabjc.2010.10.023

solution of aromatic amines and sulpha drugs. These coloured compounds have been reported to possess antibacterial and antiviral activities against Gomphrena Mossaic Virus (GMV), Sunhemp Rosette Virus (SRV) in vitro and in vivo (Kumar et al., 2008, 2007; Manoj Kumar et al., 2008; Mishra et al., 1978; Mukherjee and Shukla, 1981). The Sulphon-amides were the first effective chemotherapeutic agents to be employed systematically for the prevention and cure of the bacterial infection in the human beings and also used to treat some urinary tract

infections, leprosy and in combination with other drugs in fungal diseases such as toxoplasmosis (Paria et al., 1982; Pattan et al., 2008). It is also reported that benzenesulphonamide substituted on the nitrogen of sulphonamido group such as sul-phathiazole, sulphadiazine, and sulphaguanidine are known to possess antimicrobial activities (Pattanayak et al., 2009; Rustagi et al., 2003; Sah and Sinha, 2006).

Insecticidal activity has also been reported by quinazolin substituted formazans (Sah et al., 2010).

VIa : R = 2-chloro, V'Ib : R = 3-methoxy, 4-hydroxy

VII.a-b : R = 2-chloro, Ri = 4-nitro, 2-methyl-4-nitro

VII,., : R = 2-chloro, Ri = hydroxyl, amino, guanidino

VIIf.g : R = 3-methoxy, 4-hydroxy, Rr = pyrimidinyl, 4,6-dimethyl pyrimidinyl

Scheme 1

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Table 2 Antibacterial activity.a

Compound E. coli S. typhi

VIIa ± +

VIIb + ±

VIIc ± -

VIId ± -

VIIe ± ±

VIIf ± -

VIIg - -

Disc size: 5 mm Duration: 24 h. Control: DMSO.

Standard: ampicillin, streptomycin (20-25 mm). + +: Highly active (20-25 mm). + : Active (15-19 mm). ±: Moderately active (8-12 mm). -: No activity. a Concentration: 500 ig/disc.

In view of these observations, we have synthesized thiadia-zole substituted formazans in a single molecular framework as possible antimicrobial agents.

2. Materials and methods

Melting points were determined in open capillary tubes in a 'Neolab' electrical apparatus and are uncorrected. FTIR was carried out on Schimadzu 8101 spectrophotometer in KBr pellets. 1H NMR was recorded on a DPX 300 MHz Brucker spectrophotometer in DMSO and Mass spectra on JEOL SX 102/ DA 6000 Mass spectrophotometer data system using Argon/ Xenon (6 kV, 10 MA) as the FAB gas.

5-(4-Chlorophenyl amino)-2-mercapto-1,3,4-thiadiazole (I) was synthesized by the known procedure (Shashikant et al., 2008), while 4-chloro-2,6-dinitrophenoxy ethyl acetate (III) was prepared by our earlier method (Srivastava et al., 1983). The sulpha drugs obtained from the local market were purified by reported methods (Srivastava and Mishra, 1988).

2.1. 5-(4-Chlorophenyl amino)-3-amino methyl-2-mercapto-1 ,3,4-thiadiazole (II)

Equimolar ratio of 5-(4-chlorophenyl amino)-2-mercapto-1,3,4-thiadiazole, ammonium chloride and formaldehyde were refluxed in ethanol (10 ml) for 5-6 h. On cooling, a solid separated out which was filtered, dried and recrystallized from eth-anol. Yield 60%; M.P. 125 0C; Molecular Formula C9H9N4S2Cl;% Calcd. (Found); C% = 39.63 (39.70); H% = 3.30 (3.34); N% = 20.55 (20.58); S% = 23.48 (23.45).

FTIR (KBr, cm-1): 3415 (-NH2), 3219 (-NH), 1380 (N=C=S), 1223 (N-N=C), 1170 (C-S-C), 739 (-Cl); *H NMR (DMSO, d ppm, 300 MHz), 3.39 (s, 2H, CH2), 4.11 (s, 2H, NH2), 7.52-7.80 (m, 4H, ArH), 9.22 (s, 1H, NH).

2.2. 3-Aminomethyl-[5-(4-chlorophenyl amino)-2-mercapto-1 ,3 ,4-thiadiazol-5-yl]-4' -(2" ,6" -dinitrophenoxy)-ethyl acetate (IV)

Mannich base (0.01 mol) (II) and 0.001 mol of the ester (III) were refluxed in dry acetone (10 ml) and 0.002 mol of anhy-

drous potassium carbonate for 5-6 h. The unreacted potassium carbonate was removed by decantation. Excess acetone was distilled off and the solution on cooling gave a solid which was filtered, dried and recrystallized from methanol.

Yield 70%; M.P. 120 0C; Molecular Formula C19H17N6O7-S2Cl; % Calcd. (Found); C% = 42.18 (42.22); H% = 3.14 (3.17); N% = 15.54 (15.50); S% = 11.84 (11.92).

FTIR (KBr, cm-1): 3310 (-NH), 1710 (>C=O), 1530 (-NO2), 1380 (N=C=S), 1223 (N-N=C), 740 (-C-Cl).

2.3. 3-Aminomethyl-[5-(4-chlorophenyl amino)-2-mercapto-1,3,4-thiadiazol-5-yl]-4'-(2" ,6"-dinitrophenoxy)-acetyl hydrazide (V)

0.01 mol of (IV) and 0.07 mol (5 ml) of hydrazine hydrate (80%) were refluxed in absolute ethanol (8-10 ml) for about 4-5 h. Excess ethanol was distilled off. The solution was cooled and left overnight. A solid separated out which was filtered, dried and recrystallized from ethanol.

Yield 75%; M.P. 125 0C; Molecular Formula C17H15N8O6-S2Cl; % Calcd. (Found); C% = 38.74 (38.80); H% = 2.84 (2.92); N% = 21.27 (21.31); S% = 12.15 (12.09).

FTIR (KBr, cm-1): 3325, 3420 (NHNH2), 3310 (-NH), 1645 (>C=O), 1528 (-NO2), 1382 (N=C=S), 1221 (N-N=C), 742 (-C-Cl).

2.4. 3-[Aminomethyl-5-(4-chlorophenyl amino)-2-mercapto-1,3,4-thiadiazol-5-yl]-4'-[(2'' ,6''-dinitrophenoxy)]-methyl amido-2''' -chloro-benzylidene mine (VIa)

Equimolar ratio of the hydrazide (V) and 2-chloro benzaldehyde were refluxed for 6 h in presence of methanol (10 ml) containing 2-3 drops of acetic acid. Excess solvent was distilled off and the solution was left overnight in a refrigerator. A solid separated out which was filtered, dried and recrystallized by repeated washing with petroleum ether (60-800) (Scheme 1).

Yield 70%; M.P. 110 0C; Molecular Formula C24H18N8O6-S2Cl2; % Calcd. (Found); C% = 44.37 (44.41); H% = 2.77 (2.80); N% = 17.25 (17.19); S% = 9.86 (9.79).

FTIR (KBr, cm-1): 3310 (-NH), 1648 (>C=O), 1625 (-N=C), 1525 (-NO2), 1385 (N=C=S), 1220 (N-N=C), 742 (-C-Cl). 1H NMR (DMSO, d ppm, 300 MHz), 3.38 (s, 2H, CH2), 4.26 (s, 2H, OCH2), 5.18 (s, 1H, N=CH), 7.517.94 (m, 10H, ArH), 8.11 (s, 1H, CONH), 8.46 (s, 1H, CH2-NH), 9.12 (s, 1H, C6H4NH).

VIb: Yield 65%; M.P. 120 0C; Molecular Formula C25H21-N8O8S2Cl; % Calcd. (Found); C% = 45.42 (45.39); H% = 3.71 (3.11); N% = 16.95 (16.89); S% = 9.68 (9.71).

FTIR (KBr, cm-1): 3320 (-NH), 3242 (OHbonded), 1642(>C=O), 1625 (-N=C), 1525 (-NO2), 1385 (N=C=S), 1220 (N-N=C), 742 (-C-Cl).

2.5. 1-(4'''-nitro phenyl)-3-(chloro phenyl)-5-[(3'-amino methyl)-(2'',6"-dinitro phenoxy acetyl)-5' -(4-chlorophenyl amino)-2' -mercapto-1' ,3' ,4' -thiadiazol-5' -yl]-formazan ( VIIa)

4-Nitro aniline (0.015 mol) was dissolved in 3 ml glacial acetic acid and stirred with 2 ml con. HCl at 0 0C. A solution of 1 gm sodium nitrite in 4 ml H2O was then added drop wise with constant shaking, maintaining the temperature below 0-5 0C. The diazotised product was then added gradually with stirring to a

cold solution of 0.01 mol of compound (VIa) initially dissolved in 0.01 mol sodium acetate in minimum quantity of ethanol. The reaction product was allowed to stand at room temperature overnight and decomposed by pouring in ice cold water. The solid obtained was filtered, repeatedly washed with cold water, dried and recrystallized with petroleum ether (6080 0C).

The same procedure was followed for compound VIIb.

2.6. 1-(N-(sulphanilamido)-3-(2-chloro phenyl)-5-[(3' -aminomethyl)-(2'',6''-dinitro phenoxy acetyl)-5'-(4-chlorophenyl amino)-2'-mercapto-1',3',4'-thiadiazol-5' -yl]-formazan (VIIc)

Sulphanilic acid (0.01 mol) was initially dissolved in con. HCl (3 ml) and cooled in an ice bath. An aqueous solution of sodium nitrite (0.01 mol) was added gradually with constant shaking, maintaining the temperature below 0-5 0C. The diazotised product was further treated with an equimolar ratio of the Schiff base (VIa) initially dissolved in an ethanolic solution of sodium acetate (0.01 mol). The contents were left for 24 h at room temperature and then poured into crushed ice. A solid separated out which was filtered, repeatedly washed with cold water dried and recrystallized with petroleum ether (60-80 0Q.

The other formazans VIId-VIIg were synthesized by the same method.

The physical and analytical data of the synthesized formaz-ans are given in Table 1.

VIIa: FTIR (KBr, cm-1): 3340 (-NH), 1637 (>C=O), 1620 (C=N), 1600 (N=N), 1532 (NO2), 741 (-C-Cl). 1H NMR (DMSO, d ppm, 300 MHz), 3.38 (s, 2H, CH2), 4.09 (s, 2H, OCH2), 7.49-7.84 (m, 14H, ArH), 8.20 (s, 1H, CONH), 8.41 (s, 1H, CH2NH), 9.23 (s, 1H, C6H4NH). Mass spectra: M + . 798; 800 (M+2); 802 (M+4).

VIIb: FTIR (KBr, cm-1): 3338 (-NH), 1641 (>C=O), 1620 (C=N), 1615 (N=N), 1535 (-NO2), 740 (-C-Cl).

VIIc: FTIR (KBr, cm-1): 3424 (-OH), 1642 (>C=O), 1600 (N=N), 1540 (NO2), 1375, 1125 (SO2, pasym, psym), 730 (Cl). 1H NMR (DMSO, d ppm, 300 MHz), 3.27 (s, 2H, CH2), 4.11 (s, 2H, OCH2), 7.25-7.73 (m, 14H, ArH), 8.14 (s, 1H, CONH), 8.32 (s, 1H, CH2NH), 9.13 (s, 1H, C6H4NH), 10.19 (s, 1H, OH), Mass spectra: M+. 833, 837 (M+2), 842 (M+4).

Table 3 Antifungal activity.a

Compound Aspergillus niger Penicillium sp. Candida albicans

Duration: 72 h. Control: DMSO.

Standard: griseofulvin, gentamycin. Medium: PDA (potato dextrose agar). + +: No growth, no fungal colony (highly active). + : 0-20% Growth, 1-2 fungal colony (active). ±: 20-40 Growth, 2-4 fungal colony (moderately active). -: >40% Growth, more than four fungal colonies (no activity). a Concentration: 500 ig/disc.

VIId: FTIR (KBr, cm-1): 3445 (NH2, broad), 1639 (>C=O), 1590 (N=N), 1530 (-NO2), 1370, 1115 (SO2, pasym, psym), 740 (-Cl).

VIIe: FTIR (KBr, cm-1): 3441 (NH2, broad), 1638 (>C=O), 1595 (N=N), 1525 (-NO2), 730 (-Cl), 1375, 1125 (SO2, -^/asym, -y/sym).

VIIf: FTIR (KBr, cm-1): 3350 (-NH), 3242 (-OH), 1641 (>C=O), 1600 (N=N), 1535 (-NO2), 1370,1120 (SO2, pasym, psym), 735 (-Cl). 1H NMR (DMSO, d ppm, 300 MHz), 2.5 (s, 1H, SO2NH), 3.25 (s, 2H, CH2), 4.18 (s, 2H, OCH2), 4.56 (s, 3H, OCH3), 7.42-7.97 (m, 16H, ArH), 8.20 (s, 1H, CONH), 8.48 (s, 1H, CH2NH), 9.15 (s, 1H, C6H4NH), 10.11 (s, 1H, OH), Mass spectra: M + . 921.5, 924 (M + 2).

VIIg: FTIR (KBr, cm-1): 3355 (-NH), 3242 (-OH), 1645 (>C=O), 1590 (N=N), 1540 (-NO2), 1379, 1125 (SO2, •y/asym, -y/sym), 732 (-Cl).

3. Antimicrobial screening

Antimicrobial screening of the formazans was done following the disc diffusion technique (Tripathi and Mishra, 2007). All the compounds (VIIa-g) were screened for their in vitro antibacterial activity against Escherichia coli and Salmonella typhi at 500 ig/disc with Streptomycin and Ampicillin as the standard drugs. Antifungal activity was conducted against Asper-gillus niger, Penicillium sp. and Candida albicans at the same concentration (500 ig/disc) using Griseofulvin and Gentamy-cin as the standard drugs. The zone of inhibition was recorded in mm after incubation of plates for 24 h (antibacterial) and 72 h (antifungal) at 37 0C (see Tables 2 and 3).

4. Results and discussion

The FTIR spectrum of compound II gave a distinct vibration at 3415 cm-1 which was characterised as the amino group vibrations. The 1H NMR spectrum gave two singlets at d 3.39 and another slightly downfield at d 4.11 integrating for two protons each. These were identified as the methylene and the amino group protons. Conversion of this Mannich base into the ester (IV) gave two new stretching vibrations at 1710 cm-1 and 1530 cm-1 in its IR spectra which indicated the presence of a carbonyl and nitro group supporting the process of esterification.

Hydrazinolysis led to the formation of the hydrazide (V). Stretching vibrations at a slightly lower field around 1645 cm-1 were visualized which were the carbonyl group vibrations part of an amido group. Two new vibrations at 3325 and 3420 cm-1 indicated the presence of an NHNH2 group.

The hydrazide on reacting 2-chloro benzaldehyde gave the substituted benzylidene imine (VIa). A new vibrational mode at 1625 cm-1 was characterised for the C=N group. The 1H NMR gave two singlets for two protons each at d 3.38 and d 4.26 while another singlet for one proton at d 5.18, respectively. The signal for the single proton was that of a methine proton supporting and confirming the formation of the Schiff base.

The Schiff base when treated with the aromatic amine gave the formazan (VIIa). The FTIR spectra gave an extra vibration at 1600 cm-1 for the azo group. The 1H NMR showed the absence of signal at d 5.18 while a multiplet integrating for fourteen aromatic protons was visible between d 7.49 and 7.84. When diazotised solution of sulphanilic acid was taken the

Figure 2 Activity shown by A. niger.

FTIR spectrum gave two vibrations at 1125 cm-1 and 1375 cm-1 which were the symmetric and asymmetric vibrations of the -SO2 group. Band at 3424 cm-1 was also observed identified as that of a free hydroxyl group. Presence of the hydroxy group was also indicated by the appearance of a very downfield signal at d 10.19 in its 1H NMR spectrum.

The mass spectra of compound VIIa gave the molecular ion peak at M + 798, with two isotopic peaks at 800 (M + 2) and 802 (M+4) respectively indicating the presence of two halogen atoms. The mass spectra of two other formazans, i.e. VIIc and VIIf were taken. The molecular ion peaks were visible at M + 833 with isotopic peaks at 837 and 842 in compound VIIc while the molecular ion peak in VIIf was visible at M+ 921.5 and the isotopic peak at 924 (M + 2), respectively. These mass fragments were in concordance with the molecular weight of the formaz-ans, hence supporting their chemical structures (see Fig. 3).

4.1. Antibacterial activity

Compound (VIIb) showed maximum activity (15-19 mm) zone size against E. coli while all other compounds showed moderate activity (see Fig. 1).

MASS FRAGMENTATION OF 1 -(¿"'-NITROPHENYL )-3—(2-CHLOPOPHENYl> - 5- [(3-AMIN OMET HYL)-(2". 6"-DI NITROPHENOXYACETYL ) - 5-A-C HLORO -PHENYL AMINO-2-MERCAPTO-i;3>'-THIADIAZOL-5-YL-] -FORMAZAN (Vila)

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Against S. typhi, only one derivative (VIIa) showed the It shows that Penicillium species was the most inhibited by

maximum inhibition while two formazans compound (VIIb, - the formazans among the three fungal strains. VIIe) showed moderate activity (see Fig. 2).

4.2. Antifungal activity

Among the substituted formazans, compound (VIIa) was active against all two fungal strains, i.e. A. niger and Penicillium species with the development of 1-2 fungal colonies after 72 h incubation followed by VIIb which was active only against Penicillium.

Against C. albicans only three derivatives VIIa, VIIb and VIIf were moderately active.

Acknowledgements

The authors are thankful to SAIF (Sophisticated Analytical Instrument Facility), CDRI (Central Drug Research Institute), Lucknow for FTIR, *H NMR and Mass spectral analysis. Thanks are also to the Head, Department of Microbiology, M.D.M. Hospital, Jodhpur for the microbial screening and the Head, Department of Chemistry, J.N.V. University, Jodh-pur for providing the laboratory facilities.

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