Synthesis, Characterization and Antimicrobial Evaluation of some Schiff Bases and their Thiazolidinone Products

Introduction

Schiff’s bases, characterized by the presence of imine or azomethine (-C=N-) group, constitute an important class of organic synthetic compounds. Imines owing to –C=N- linkage are useful as precursors in diverse organic synthesis and exhibit wide spectrum of biological properties, viz. pesticidal¹, fungicidal^2-4, bactericidal^5,6, bacteriostatic⁷, anticancer^8,9, antiviral^10,11 etc. They have been reported as novel ligands informing complexes of unusual coordination numbers and isomeric structures with metals¹², as dyes¹³ and analytical reagents ^{14, 15}. Vanillin containing Schiff’s bases act as a weak inhibitor of tyrosinase and display both antimutagenic and comutagenic properties in Escherichia coli¹⁶ in addition to antimicrobial activities against various microbes and several pharmacological properties¹.

Thiazolidinone derivatives were reported to display antitumor¹⁷, antituberculor¹⁸, anti-HIV¹⁹, analgesic²⁰, anti-inflammatory²⁰, ulcerogenic²¹, and antibacterial²² and antifungal²² activities. Therefore it was envisaged that compounds containing vanillin and thiazolidinone moieties would result in compounds of interesting biological properties. In the present study vanillin was treated with isomeric nitro-and methoxy anilines to produce Schiff’s bases. The Schiff’s bases were subjected to cyclocondensation reactions with thioglycolic acid to produce 4-thiazolidinones. The chemical structures of the synthesized compounds were confirmed by means of elemental analyses, molecular weight determination, and IR, ¹H, & ¹³C and DEPT-135 NMR. All the compounds were screened against Gram-positive and Gram-negative bacteria and fungi.

Experimental

Synthesis Scheme

Synthesis of 1, 4-thiazolidinones involved two steps

Reagent And conditions

FIGURE 1:(a)Respective primary amines, dry ethanol, reflux 3h;(b) Thioglycolic acid, dry acetone, anhydrous CH3COONa, aqueous Na2CO3,  reflux 3h Click here to View figure

 

Step I

All the Schiff’s bases were prepared by mixing equimolar (0.5 mol) saturated solutions of each of substituted anilines with vanillin in dry ethanol and reaction mixtures were refluxed for 3 h on water bath. The solids precipitated or obtained on evaporation of the solvent as residues were washed with water and ether to remove unreacted vanillin and amine, if any, successively. Products were crystallized from acetone or methanol and dried in oven at 60-70 ⁰C.

 Step II

For the synthesis of 1, 4-thiazolidinones, saturated solution of Schiff’s bases (0.25 mol) in acetone was  mixed with thioglycolic acid in 1:3 molar ratio and 5 gram anhydrous sodium acetate was added to the reaction  mixture and refluxed for h. Hot reaction mixtures were filtered, cooled to room temperature and neutralized with aqueous Na₂CO₃ solution to remove unreacted acid; precipitates of products were filtered and washed with water repeatedly to ensure complete removal of sodium salt(s) and dried in air. All the compounds were purified by crystallization from ethanol or ether.

The purity of the synthesized compounds was checked by thin-layer chromatography and the impure samples were purified either by column chromatography or by washing with the solvent as identified by TLC.

Physico-Chemical And Microbial Analysis

Microanalaytical analyses were performed on a Vario-el III, elemental-R analyzer. Melting points were determined in open glass capillaries using MP-D Mitaruma Rikero Kiygo electro-thermal melting point apparatus. Molecular weights of the compounds were determined by Rast`s method with camphor as solvent. IR spectra in 500 cm^-1 – 4000 cm^-1 range were recorded in KBr medium on FT-IR Shimadzu spectrometer. ¹H spectra, ¹³C and DEPT-135 NMR spectra were recorded in CDCl₃ on Bruker 400 MHz Advanced spectrometer.

Antimicrobial (antibacterial and antifungal) activities of the azomethines and thiazolidinones, were tested in vitro using disc diffusion method against Staphylococcus aureus and Escherichia coli bacteria in Mueller Hinton Agar (MHA) medium and against Aspergillus niger and Rhizoctoia bataticola fungi in Potato Dextrose Agar (PDA) medium. The degree of bactericidal and fungicidal activities was determined by measuring diameter of inhibition zone and compared with the standard drugs ampicillin and bavistin, respectively.

Results And Discussion

Theoretically proposed molecular formulae of the compounds are in conformity of experimental data of molecular weights and elemental analyses (Table-1).

Table 1: Structure, melting point, yield and analyses data of compounds

Compound	Colour	Yield(%)	M.P.(oc)	Molecular weight: Calcd.(Found)	Analyses: Calcd.(Found) %
					C	H	N	S
1a	Smoky white	51.9	78±1	272 (264.7)	61.76 (62.20)	4.44 (4.90)	10.29 (10.20)	–
1b	Light yellow	96.2	132±1	272 (273.8)	61.76 (61.21)	4.44 (4.62)	10.29 (10.22)	–
1c	White	94.2	103±1	272 (283.6)	61.76 (61.65)	4.44 (4.68)	10.29 (10.17)	–
1d	Yellow	94.0	119±1	257 (248.1)	70.02 (69.76)	5.88 (6.12)	5.44 (5.33)	–
1e	Light yellow	58.4	126±1	257 (256.1)	70.02 (69.26)	5.88 (6.54)	5.44 (5.82)	–
1f	White	99.2	154±1	257 (259.5)	70.02 (69.80)	5.88 (5.46)	5.44 (5.40)	–
2a	Yellow	51.3	116±1	346 (345.2)	55.48 (55.00)	4.07 (4.24)	8.09 (7.73)	9.26 (9.05)
2b	Orange	59.4	113±1	346 (360.9)	55.48 (54.92)	4.07 (4.18)	8.09 (8.16)	9.26 (9.36)
2c	Light yellow	65	166±1	346 (336.4)	55.48 (55.26)	4.07 (4.41)	8.09 (7.84)	9.26 (9.10)
2d	Orange	62.3	119±1	331 (317.6)	61.61 (60.98)	5.17 (5.31)	4.23 (4.28)	9.68 (9.35)
2e	Yellow orange	62.1	134±1	331 (330.8)	61.61 (59.87)	5.17 (5.13)	4.23 (3.98)	9.68 (9.90)
2f	Pale orange	81.5	141±1	331 (354.5)	61.61 (60.69)	5.17 (4.86)	4.23 (3.99)	9.68 (9.29)

 

Where

C₇H₇O₂-CH=N-ArX

C₇H₇O₂-C₃H₃NOS-ArX

o-NO2

m-NO2

o-OCH3

m-OCH3

p-OCH3

The common characteristic groups of 1a-f and 2a-f of azomethines and thiazolidinones, display ν C-OH & δ C-OH (phenolic), ν C-O-C, aromatic ν C=C & ν C-H, aliphatic ν C-H, ν C-NO₂ (symm.) & ν C-NO₂ (asymm.)  and ortho, meta and para substitution vibrations in their infrared spectra in 3215-3482 cm^-1 & 1029-1154 cm^-1, 1265-1378 cm^-1, 1452-1595 cm^-1 &  2954-3106 cm^-1, 2921-3105 cm^-1, 1265-1377 cm^-1 & 1515-1666 cm^-1, and ca.747-727 cm^-1, ca.777-789 cm^-1 and ca.843-836 cm^-1 regions respectively. A strong band observed in 1a-f at 1592-1668 cm^-1 range assigned to CH=N clearly indicates formation of the new azomethines owing to condensation of CHO group of vanillin with NH₂ group of primary amine and absence of any peak of CHO group supports the proposed structure of 1a-f. Cyclocondensation of azomethine compounds with thioglycolic acid leads to the formation of thiazolidinones containing C=O, C-S-C, C-N and CH₂ groups of heterocyclic ring with elimination of water. IR spectra of these compounds exhibit ν C=O (cyclic), ν C-S-C( ring) and  ν C-N( ring) vibrations in 1608-1667 cm^-1, 650-722 cm^-1 and 1300-1367 cm^-1 regions respectively whereas heterocyclic ring methylene group (CH₂) displayed its ν C-H symmetric, ν C-H asymmetric and δ C-H bands in 2852-2855 cm^-1, 2921-2955 cm^-1 and 1447-1463 cm^-1 ranges respectively. The absence of ν CH=N peak and presence of these characteristic groups of heterocyclic ring obviously conform the cyclization of the azomethines leading to the formation of thiazolidinones (2a-f)²³.

For the verification of IR results, ¹H and ¹³C NMR spectra of the compounds have been examined. ¹H NMR spectra of azomethines (1a-f) display singlet in δ 3.80- 4.00, δ 7.70-8.40 and δ 8.40- 9.85 regions characteristics  of –OCH₃, -OH and –CH=N groups, respectively and multiplet bands of benzene rings in δ 6.27- 8.06 range. ¹H NMR spectra of thiazolidinones  however displayed signals in δ 3.85- 4.40, δ 8.10- 9.85, δ 8.10- 8.75 and δ 5.55- 6.20 regions for  their OCH₃, OH(phenolic), CH-N (ring) and -S-CH₂ (ring) groups respectively; benzene protons exhibit multiplets in δ 6.10-7.60 ppm region²⁴. ¹³C NMR spectra of 1a-f of carbon containing groups-OCH_3, C-OH, CH=N and C-NO₂ exhibit their characteristic signals in δ 56,  δ 147-153, δ 159-191 and δ 136-147, respectively along with benzene ring carbons in δ 107-161 range.¹³C NMR spectra of 2a-f display signals of carbon containing groups (Ar)O-CH₃, C-OH (phenolic),  C-N (ring), C=O (ring), C-S (ring) and CH₂ (ring) in δ 56-66,  δ 139-152, δ 55-56,  δ 166-191, δ 55-56 and δ 30-34 ranges  respectively and aromatic carbon signals in δ 109-158 region. In NO₂ substituted products C-NO₂ signal is exhibited in δ 136-148 range. Signals corresponding to C-N (ring), C-S (ring) and O-CH₃ group have been seemed to be overlapped. All the NMR inferences are totally in conformity of IR results²⁵.DEPT-135 spectra of azomethines also displaying O-CH₃ and CH=N signals at the same ppm as observed in ¹³C NMR spectra and seven aromatic hydrogen bonded carbons exhibit seven peaks in upward direction as expected. Thiazolidinones also display peaks of OCH_3, CH-N and aromatic hydrogen bonded carbons exhibit peaks at the same ppm as observed in¹³C NMR spectra in upward direction as expected²⁵.

The results of antimicrobial studies reveal less significant antibacterial activity of both series of compounds, against both test bacteria; m- methoxy thiazolidinone however exhibits better results against E.coli among all others. All the compounds show better antifungal activities than bactericidal properties against both fungi tested. Although several azomethines and thazolidinones exhibit highly significant results, p-nitro thiazolidinone has highest antifungal action against R. bataticola in both the concentrations used, better than standard drug bavistin (Table 2)²⁶.

Table 2: Inhibitory zone diameters (%) of azomethines and thiazolidinones against tested bacteria and fungi strains by paper disc diffusion method

Compound	Antibacterial activity				Antifungal activity
	S. aureus		E.coli		A.niger		R.bataticola
	10 μL/ disc	20 μL/ disc	10 μL/ disc	20 μL/ disc	10 μL/ disc	20 μL/ disc	10 μL/ disc	20 μL/ disc
1a	32	32	27	27	30	36	39	39
1b	32	36	27	31	65	59	58	65
1c	32	36	23	27	50	73	58	65
1d	32	32	23	27	30	36	65	62
1e	32	36	23	31	40	41	54	65
1f	36	36	23	27	30	32	58	73
2a	32	41	27	31	40	50	50	62
2b	36	45	31	35	45	55	54	50
2c	32	41	27	35	70	64	100	115
2d	32	41	27	31	30	36	23	35
2e	36	45	39	50	45	41	27	35
2f	36	41	31	42	30	27	31	35
DMSO	–	–	–	–	–	–	–	–
Ampicillin	100	100	100	100	–	–	–	–
Bavistin	–	–	–	–	100	100	100	100

 

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