INTRODUCTION:

Epilepsy is one of the most common neurological diseases. It is commonly accepted that a significant proportion of patients suffering from seizures are resistant to drug treatment. The ratio of intractable patients has not been reduced despite the marketing of several new anticonvulsant agents in recent years. The search for anticonvulsant agents with a more selective activity and lower toxicity continues to be an area of investigation in medicinal chemistry [1].

Thiazine derivatives are an important class of heterocyclic compounds reported to possess a wide spectrum of biological properties such as anticonvulsant [2], antimicrobial [10-15], anti-inflammatory [16-19], anticancer [20, 21], antidiabetic [22], analgesic [23], immunotropic [24], antitubercular [25], antianxiety [26], insecticidal, anesthetic, ulcerogenic, etc. Thiazines exerted anticonvulsantactivity in models of generalized seizure which are well documented in the literature. These observations have prompted us to synthesize more number of new derivatives having thiazine nucleus. Hence the main objectives of the present work is to synthesize, characterize and evaluate some of the thiazines like 2-iminothiazines prepared from a compound having chalcone nucleus for their anticonvulsant and antimicrobial activity. In the earlier days, the Chinese used mouldy soybean curd to treat carbuncles, boils and other infections. Greek physicians used wine, myrrh and inorganic salts. In the middle ages, certain types of honey were used to prevent infections following arrow wounds, of course, in those days, there was no way of knowing that bacteria were the cause of these infections. Methods of vaccination were studied and research was carried out to try and find effective antibacterial agents or antibiotics¹. Sir Alexander Fleming’s accidental discovery of the antibacterial properties of penicillin in 1929 is largely credited with initiating the modern antibiotic era. Penicillin became available in quantities sufficient in 1941. Several antibiotics such as streptomycin, chloramphenicol, chlortetracycline and other antibacterial agents such as sulphonamides were identified sooner or towards the end of world war II. Since then, many novel drugs including antibiotics were discovered entirely by chemical synthesis. An ever increasing understanding of the nature of disease, how cells work, and how drugs influence these processes led to the development of numerous classes of antimicrobial agents, which are most commonly used of all drugs. For example, 30% or more of all hospitalized patients are treated with one or more courses of antimicrobial therapy. Death from an incurable bacterial infection came to be considered a thing of past. The remarkable success of antimicrobial drugs generated a misconception in the late 1960s and early 1970s that infectious diseases were conquered. However, four decades later, infectious diseases remain the second leading cause of death in the world. This is mainly due to the emergence of chemotherapeutic agent-resistant microorganisms [5]. The extensive use of antibiotics has provided powerful forces for the selection of microbes that either carried mutations conferring resistance or had the enhanced ability to mutate to resistance in the face of the antibiotic. The mutated bacteria have acquired new genes, producing novel machinery to overcome the action of many antibiotics. As a result, now with every possible bacterial infection, resistance to antibiotic treatment is a common phenomenon⁶. Furthermore, the emergence of multi-drug resistant bacteria has created a situation in which there are very few options for infections with certain microorganisms. Chalcones, on the other hand, are α, β-unsaturated enones with a broad range of biological activities and also acts as key synthon in the chemical synthesis of heterocyclic compounds [7].

Fig. 1

METHODOLOGY:

General procedure for synthesis of 2,6-disubstitutedarylidene-4-methylcyclohexanones^3,4:

Substituted aromatic aldehyde (0.02mol) was added to a mixture of 30 ml of 10% sodium hydroxide and 4-methl cyclohexanone (0.01mol) in 50 ml of ethyl alcohol, and stirred at 20-25⁰C for 2 hrs. Later, reaction mixture was kept in an ice chest over night. The product obtained was filtered, washed with ice cold water followed by ice-cold ethanol, dried and recrystalized from dimethylformamide.). The physical data was given in Table-I. Physical and spectral data of the synthesized compounds, the yellow color solid, mp.154-155 ⁰C, Yield: 79 %, 3b) UV 3b): 319, IR (KBr) (V_max,cm^-1): 3d) 1664(C=O), 1601, 1575, 1464(Ar), 1136(C-Cl), 829(C=C). 3a) ¹H NMR (CDCl₃): δ=1.2 (d, 3H, CH₃), 1.7(m, 1H, CH), 2.5 (s, 6H, 2 X CH₃), 2.6-3.2 (d, 4H, 2XCH₂), 7.3-7.5(m, 8H, ArH), 7.8(s, 2, methine), Mass 3a: calculated 426.17, observed 427 (M+1). Elemental analysis found: Carbon 59.19 (59.17), Hydrogen 3.78 (3.75).

General procedure for the preparation of 4-aryl-8-arylidene-2-imino-5,6- dihydro-4H,7H-6-methyl-(3,1)benzothiazines^5,6:

A mixture of 2,6-disubstitutedarylidene-4-methyl cyclohexanones(0.01mol) and thiourea (0.015mol) was refluxed in isopropyl alcohol for 16 hrs in the presence of aqueous potassium hydroxide (0.01mol). Later, solvent was removed by distillation and residue obtained was treated with ice cold water, filtered, dried and recrystalized from ethanol. The purity of the synthesized compounds was confirmed by the thin layer chromatography (TLC). The physical data was given in table-I. Physical & spectral data of the synthesized compounds, the yield of product, mp.230-231 ⁰C, Yield: 73 %. [8a] UV 288, 8b) IR (KBr) (V_max,cm^-1), 3418 (NH), 3209 (Cyclic NH), 1564 (C=N), 1482 (CH₃), 1306 (C-O-C), 1063 (C-N), 8a) ¹H NMR (Amx– 100 MHz, CDCl₃₎ Chemical shift: δ= 0.9 (d, Me, 3H), 1.5-2.6(m, CH₂CHCH_2,5H) 5.4 (s, S-CH, 1H), 6.5 (s, Imine NH, 1H), 6.7 (s, Cyclic NH, 1H), 7.1 - 7.4 (m, ArH, 6H), 7.9 (s, Methine H, 1H); 8a) Mass spectral data Calculated 484.28 observed 485(M⁺1); 8a) Elemental analysis calculated values: Carbon 54.56, Hydrogen 3.75, Nitrogen 5.78, Observed values: Carbon 54.59, Hydrogen 3.73, Nitrogen 5.95.

Table I: Physical data of the synthesized compounds

Compound No.	Ar	Mol. formula	Yield (%)	M.P(⁰c)	R_f
[4a]	3,4-Cl₂C₆H₃	C₂₂H₁₈N₂SCl₄	74	230-232	0.52
[4b]	2,4,5-(OMe)₃C₆H₂	C₂₈H₃₂N₂O₆S	64	168-170	0.71
[4c]	2,4-F₂C₆H₃	C₂₂H₁₈N₂SF₄	68	231-233	0.69
[4d]	2,4-Cl₂C₆H₃	C₂₂H₁₈N₂SCl₄	72	225-227	0.56
[4e]	p-FC₆H₄	C₂₁H₁₈N₂SF₂	76	209-212	0.66

(x:y)* = Ethyl acetate: Cyclohexanone

Table II: Anticonvulsant Activity by PTZ Induced Model

Groups	Latency of Convulsion (in min)	*Time of Death^(in min)**
Control (Normal saline)	1.071 ± 0.048	5.72±0.3116
Standard (Diazepam)	0.00 ± 0.00***	30.00 ± 0.00***
[4a]	1.606 ± 0.1887	21.62 ± 2.668**
[4b]	11.36 ± 0.3411**	25.48 ± 2.857**
[4c]	1.675 ± 0.1661	27.77 ± 1.058**
[4d]	1.506 ± 0.1284	8.987 ± 0.506
[4e]	3.359 ± 0.1426**	13.97± 0.593**

Values are expressed as mean ± SEM of each group (n=6) and are significant when done ONE-WAY ANOVA with followed by Dunnett’s multiple comparison test. *** p <0.001 when compared with disease control.

Anticonvulsant Activity:

Healthy Swiss Albino mice of male sex, weighing 18-22 g at the start of the experiment were used as experimental animals in the present study. Mice are useful experimental animals precisely because they are so adaptable to the laboratory environment. They are easy to maintain and handle. Moreover, one can have the best laboratory animals by breeding them locally. Besides preventing environmental variation, inbreeding provides a homogeneous population of animals for experiments. Room temperature was 25 °C ± 2 °C, humidity was 45-55 % with a light period of 12 hrs (06:00 to 18:00).

Maximum Tolerated Dose (MTD):

MTD study was carried out as per the OECD/425 guidelines and different doses checked were 55 mg / kg, 175 mg / kg, and 550 mg / kg. Mice of male sex with a body weight between 18 and 22 g were used. The experimental protocol for the pharmacological screening was done in accordance with the guidelines prescribed by an Institutional Animal Ethics Committee (CPCSEA No: 1292/ac/09/CPCSEA). The test compounds and the standard drug, diazepam (30mg/kg) were given orally to group of 6 mice. Another group of 6 mice served as control. 60 min after oral administration, 80 mg/kg PTZ was injected through intra-peritoneal route. Each animal was placed into an individual plastic cage for observation lasting 30 min. Latency of convulsion, % of convulsion, time of death, % of survival were recorded.

ANTIMICROBIAL SCREENING:

Antibacterial Activity: In vitro antibacterial activity of the synthesized compounds were tested against the standard cultures of gram-positive Staphylococcus aureus and Bacillus subtilis and gram-negative Escherichia coli and Pseudomonas aeruginosa by using cup plate agar diffusion method. Bacteria were cultured in nutrient agar medium and the solutions of the compounds were dissolved in dimethyl sulfoxide (DMSO 1ml) at 100 µg/mL concentration and the compounds incubated at 37 ⁰C for 24 hrs. After incubation period, the inhibition zones were measured in mm, and the known antibiotics like ciprofloxacin as standard drug were used for the comparison at the same concentration [9]. The results are tabulated in the Table III.

Antifungal Activity:

The antifungal activity of the compounds were evaluated against two fungal organisms’ Candida albicans and Asperigillus niger by using cup plate agar dilution diffusion method and inoculated at 28±1 ⁰C for seven days. After seven days, inhibition in fungal growth was determined as a difference in growth between control plates and those treated with test compound, and compared with the standard drug (fluconazole) and the results were tabulated in the Table III. All the tested compounds showed optimum antimicrobial activity with minor differences in their zone of inhibitions when compared with the standard drugs.

Table III: Antimicrobial Activity

Compounds	Diameter of zone of inhibition in mm (MIC µg/ml)
Compounds	*S.aureus*	*Bacillus subtilis*	*E.coli*	*P.aeruginosa*	*C. albicans*	*A. niger*
4a	22	21	19	20	24	23
4b	21	20	17	18	23	22
4c	21	20	18	17	22	23
4d	20	19	20	16	23	21
4e	23	17	18	17	24	20
Standard	25	26	25	26	26	26

Standard Drugs used (ciprofloxacin and fluconazole)

Fig.2: Graphical representation of antimicrobial activity

EXPERIMENTAL:

GENERAL:

Melting points of the synthesized compound were determined by open capillary tubes and were uncorrected. The purity of the final synthesized compounds was checked by precoated silica gel G, using ethyl acetate and cyclohexanone as a solvent system. The chemicals and reagents used in the present were of AR and LR grade, procured from Aldrich, Sigma, Hi-media, Merck, NR chem., S.D. Fine Chemical Ltd. The Infrared spectra of the synthesized compounds were recorded on a Fourier Transform IR spectrometer (Model Shimadzu 8700) in the range of 400-4000 (cm^-1) using KBr disks. ¹H NMR (100 MHz) spectra were measured on Amx – 100 MHz NMR spectrometer using CDCl₃as a solvent and chemical shifts (δ) are reported in parts per million downfield from internal standard Tetramethylsilane (TMS). Mass spectra were recorded on a Mass spectrophotometer at 70eV (Model Shimadzu) by LC-MS. All the chemicals used were reagent grade and were used as such without further purification.

RESULTS AND DISCUSSION:

The structures of new compounds prepared during the present investigation have been authentically established by their UV, IR, NMR, Mass spectral studies and elemental analysis. In the following section the spectral studies of some selected compounds have been dealt. The compounds 2, 6-disubstitutedarylidene-4-methylcyclohexanones [3a-e] have been prepared by reaction of 4-methylcyclohexanone [1] with aromatic aldehydes [2a-e which is an example for Claisen-Schmidt condensation. The compounds α,α'-bis(substitutedarylidene)-4-methylcyclohexanones [3a-e] have been prepared by reaction of 4-methylcyclohexanone [1] with aromatic aldehydes [2a-e] which is another example for Claisen-Schmidt condensation. The formation of α,α'-bis(2,4,5-trimethoxybenzylidene)-4-methylcyclohexanone [3b] from [1] has been indicated by its UV spectrum. The substrate [1] exhibited l_max at 287 nm. The compound [3b] exhibited l_max at 319 nm. This change in l_maxclearly indicate that the bathochromic shift is attributed because of C=CH Ar chromophore. The formation of α,α'-bis(2,4-dichlorobenzylidene)-4-methylcyclohexanone [3d] from [1] has also been indicated by its IR spectrum. The substrate [1 exhibited n_max at 1715 cm^-1 which is due to carbonyl group (C=O)¹¹⁹. The compound [3d] exhibited n_max at 1664cm^-1 due to carbonyl group (C=O). The appearance of a band at 1664 cm^-1 is mainly due to the presence of two C=CH-Ar chromophore. This clearly indicates the formation of [3d]. The formation of α,α'-bis(3,4-dichlorobenzylidene)-4-methylcyclohexanone [3a] has been confirmed by its mass spectrum. The molecular ion peak of [3a] is observed at 427, which is in good agreement with the proposed molecular weight. The compound [3a] also has shown an additional (M+3) peak at 430, (M+6) peak at 433 which proves the isotopic nature of four chlorine atoms. The compounds 4-substitutedaryl-8-substitutedarylidene-2-imino-6-methyl-5,6-dihydro-4H,7H-(3,1)benzothiazines [4a-e] have been prepared by cyclocondensation of α,α'-bis (substitutedarylidene)-4-methylcyclohexanones [3a-e] with thiourea which is an example for Michael addition. The formation of 4-(2,4,5-trimethoxyphenyl)-8-(2,4,5-trimethoxybenzylidene)-2-imino-6-methyl-5,6-dihydro-4H,7H-(3,1)benzothiazine [4b] from α,α'-bis(2,4,5-trimethoxybenzylidene)-4-methylcyclohexanone [3b] has been indicated by its UV spectrum. The compound [3b] exhibited l_max at 319 nm. The compound [4b] exhibited l_max at 247 nm. These observations indicate that the hypsochromic shift is attributed because of cyclocondensation. The formation of 4-(2,4-dichlorophenyl)-8-(2,4-dichlorobenzylidene)-2-imino-6-methyl-5,6-dihydro-4H,7H-(3,1)benzothiazine [4d] from α,α'-bis(2,4-dichlorobenzylidene)-4-methylcyclohexanone [3d] has also been indicated by its IR spectrum. The compound [3d] exhibited n_max at 1664 cm^-1 due to carbonyl group (C=O). The compound [4d] exhibited n_max at 3180 cm^-1 and 2962 cm^-1 due to stretching vibrations of imine NH and cyclic NH respectively. The absence of 1664 cm^-1 and presence of at 3180 cm^-1 and 2962 cm^-1 in [4d]clearly indicate its formation. Similarly, the formations of other thiazines have been revealed by their IR spectra. The formation of [4a] has also been elucidated by its ¹H NMR spectrum. The presence of signals d1.5-2.7 (m, (CH₂)₃, 6H), 4.9 (s, CH-S, 1H), 6.5 (s, imine H, 1H), 7.1-7.5 (m, ArH + cyclic NH, 4H+1H), 7.1-7.5 (m, ArH, 6H), 7.7 (s, methine H, 1H) clearly indicates the formation of [4a]. The formation of 4-(3,4-dichlorophenyl)-8-(2,4-dichlorobenzylidene)-2-imino-6-methyl-5,6-dihydro-4H,7H-(3,1)benzothiazine [4a] has also been confirmed by its Mass spectrum. The molecular ion peak of [4a] has been observed at 485, which is in good agreement with the calculated molecular weight. The compound [4a] also has shown an additional (M+4) peak at 489, (M+6) peak at 491 which proves the isotopic nature of four chlorine atoms. Similarly other compounds [4c] and [4e] have also been confirmed by their Mass spectra finally the structures of compounds are confirmed by their elemental analyses.

CONCLUSION:

We have reported for synthesis and biological screening of some of novel substituted thiazine derivatives. All the synthesized compounds were screened for their antimicrobial activity against, human pathogenic gram-positive and gram-negative microorganisms and fungal organisms using ciprofloxacin and fluconazole as standard reference, along with anticonvulsant activity by PTZ induced model. Modern accessible anticonvulsant drugs are capable to efficiently control epileptic seizures in about 50 % of the patients; another 25 % may show improvement whereas the leftovers does not benefit significantly, furthermore, undesirable side effects from the drugs used clinically often render treatment difficult; so that a demand for new types of anticonvulsants exists, From pentylenetetrazole (PTZ) induced model it can be clearly notified that 1,3-thiazines possess significant anticonvulsant activity but less than the standard. The observations of biological results of all the new synthesized thiazines reveal that the % of protection increases with various substitutions in the following order, Cl< F < OMe. Further substitution of the 4-substitutedaryl-8-substitutedarylidene-2-imino-5,6-dihydro-4H,7H-1, 3-benzothiazines with electron donating groups and o/ p directors could probably lead to better anticonvulsant activity.

ACKNOWLEDMENT:

The authors express their sincere gratitude to the management for encouragement and for providing the essential requirements to carry out this research work. We would also like to thank Mr. Kadiri Sunil Kumar, Associate Professor, Department of Pharmacology, Vijaya College of Pharmacy for help in pharmacological screening.

CONFLICTS OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 24.11.2016 Modified on 20.12.2016

Res. J. Pharmacology & Pharmacodynamics.2017; 9(1): 13-18.

DOI: 10.5958/2321-5836.2017.00003.9