2-Aminobenzothiazole containing novel Schiff bases derivatives: Search for new Antibacterial agents

. The benzothiazole and Schiff base moieties are crucial functionalities due to their wide variety of biological activities and have a wide range of therapeutic properties. Keeping in view the importance of these organic moieties, a new series of 2-Aminobenzothiazole containing novel Schiff bases derivatives were synthesized by sequential reaction. The structures of the synthesized compounds were confirmed by their analytical and spectral data. The synthesized compounds were evaluated for their in vitro antibacterial activity against gram positive and gram negative bacteria. Synthesized compounds showed significant activity against microorganisms, which can be correlated with the privileged heterocyclic structural scaffolds.


INTRODUCTION
Today, we are facing one of the major problems in the context of infectious diseases is the persistent increase and spread of antimicrobial resistance. Thus, studies for the identification of new targets and drugs for the action of infectious diseases are at the forefront. Many heterocyclic nuclei, benzimidazole, triazine, benzothiazole have been lately reviewed as antimicrobial agents. [1,2] Our attention was focused to the benzothiazole nucleus. [3] Benzothiazole can serve as unique and versatile scaffolds in research area, especially in synthetic as well as in pharmaceutical chemistry because of its potent and significant pharmacological activities. In fact, benzothiazole derivatives possess a wide spectrum of biological applications such as antimicrobial, [4] anticancer, [5] Anthelmintic, [6] Anti-diabetic, [7] Anti-tuberculosis, [8] antitumor, [9] Antitrypanosomal, [10] antiviral, Antibacterial, antioxidant, Anti-glutamate and Anti-parkinsonism, analgesic, Anti-inflammatory, Antifungal, Anti-leishmanial, Anticonvulsant, [11] etc. Schiff bases, named after Hugo Schiff, [12] are designed when any primary amine treated with an aldehyde or a ketone under particular conditions. A Schiff base is known as imine or azomethine, is a nitrogen analogue of an aldehyde or ketone in which the carbonyl group has been replaced by an imine or azomethine group. They are some of the most widely used organic compounds. They are used as catalysts, pigments and dyes, intermediates in organic synthesis, and as polymer stabilizers. [13] Schiff bases have also been shown to exhibit a broad range of biological activities such as antiviral, antibacterial, anti-inflammatory, antimalarial, antifungal, anti-proliferative and antipyretic properties. [14,15] Imine or azomethine groups are present in various natural, naturally-derived, non-natural compounds and some compound which shows various biological activities. [16,17]. There are very scarce recent literature data on antimicrobial potentials of benzothiazole containing Schiff base moiety that should combine favorable structural properties of both Schiff bases moiety and benzothiazole moiety. Therefore, in the present paper, we have prepared a set of new N-(4-(1-(benzo[d]thiazol-2-ylimino)ethyl)phenyl)-2-(benzo[d]thiazol-2-ylthio) acetamide derivatives and evaluated for their in-vitro antibacterial activities against Gram-positive and Gram-negative bacteria.

EXPERIMENT SECTION
All starting materials and other reagents were purchased from commercial suppliers. Thin layer chromatography (TLC) which was performed on silica gel G 60 F 254 (Merck) plates and eluted with the appropriate solvent ratios (v/v). The melting points were recorded on optimelt automated melting point system and were uncorrected. For column chromatography 60-120 mesh LR (Merck) silica gel was used. IR spectra were recorded on a Perkin-Elmer 377 spectrophotometer in KBr with absorption in cm -1 . 1 H NMR spectra were recorded on Bruker AV 400 MHz using DMSO-d 6 as solvent and TMS as an internal standard. Chemical shifts are reported in parts per million (δ in ppm). Mass spectra were recorded at Advion Expression CMS, USA. Elemental analysis was performed on the Vario MICRO cube, elementar CHN analyzer serial no.: 15084053.

General procedure for the synthesis of N-(4-acetylphenyl)-2-chloroacetamide (2):
A mixture of 4-amino acetophenone 1 (0.01 mol) with chloroacetylchloride (0.015 mol) and Triethylamine (4-5 drops) in 25 ml RBF using DMF as solvent. The reaction mixture was stirred for 4 hours at Room temperature. The completion of the reaction was checked by TLC with Mobile phase Toluene: Acetone (30 %). The solution was poured into ice water. The product obtained was filtered by using a vacuum pump and crystalline it in Ethanol. [18]

General procedure for the Synthesis of N-(4-Acetylphenyl)-2-(benzo[d]thiazole-2-ylthio) acetamide (4):
Above synthesized compound N-(4-acetyl phenyl) -2-chlroacetamide 2 (0.01 mol) was further reacted with 2-mercatobenzothiazole 3 (0.01 mol). The reaction was stirred at room temperature for 4 hrs in the presence of K 2 CO 3 (0.02 mol) and acetone as a solvent. The completion of the reaction was monitored by TLC using Toluene: Acetone (20 %) as a mobile phase, the product was poured into water and stirred for 1 hour. The obtained precipitate was collected and dried. The product was crystallized into methanol.

Determination of Antibacterial Activity
Antibacterial activities of 6a-6g were carried out in Nutrient-agar plates by well diffusion assay. Cultures were activated in Nutrient broth. Isolates were inoculated in Nutrient broth and incubated at 37 o C for 24 hours for activation of cultures and then centrifuged at 3000 rpm for 15 min and the supernatant was collected to study antibacterial activity.

ILCPA Volume 53
Using in-vitro agar well diffusion method, antimicrobial activity experiments were carried out. ) was inoculated in molten agar and poured into sterile plates than allowed to solidify. Wells with 5mm diameter were prepared at equal distance in solidified agar plates using cup-borer. Various derivatives 6a-6g with 1000 µg/ml concentration were inoculated in the wells of nutrient agar whereas test microorganisms were inoculated by pour plate technique. The plates were incubated at 37 o C for 24 hours. The inhibition zones were measured at the end of the incubation period.

Chemistry
The synthetic pathway for preparation of different derivative of N-(4-(1-(benzo[d]thiazol-2ylimino)ethyl)phenyl)-2-(benzo[d]thiazol-2-ylthio) acetamide (6a-6g) is shown in Scheme 1. To explore the scope and limitations of this reaction further, we extended our studies to the use of various substituted 2-aminobenzothiazole and chloroacetylchloride in the presence of diethyl amine. It was gratifying to observe that most of the tested substrates exhibited satisfactory reactivity profiles, in all cases leading to a link sequence such as hydrazine hydrate and 2-aminobenzothiazole that readily afforded the target product (Table 1).  6. The appearance of a molecular ion peak at 384.8 mass unit supports the structure of compound 6a.

Antibacterial Activity
The antibacterial activity of the synthesized compound (6a-6g) was carried out on Nutrient-agar plates by well-diffusion assay against test culture. Cultures were triggered in Nutrient broth. Isolates inhibits the above mentioned organisms or not were studied and zone of inhibition was measured in terms of zone diameter and with the help of that zone index was calculated where streptomycin was used as standard.

Determination of activity index
The activity index of the probiotic culture was calculated as:  Table 2, revealed that all compounds tested showed some degree of antibacterial activity. The antibacterial activity of all compounds except 6c showed more than 83.3 % inhibition than the standard against Enterobacter aerogens bacteria. When compound 6b, 6c, 6d, 6f, and 6g also shows good activity to standard drug against Micrococcus luteus gram positive bacteria. Compounds 6c-6g more pronounced activity compared to streptomycin against Escherichia coli bacteria. Compound 6b, 6c and 6g have showed very close activity to standard drug against Bacillus cereus gram positive bacteria.