Synthesis of Acetylenic Derivatives of a Substituted 1, 3, 4-Thiadiazole as Antibacterial Agents

124 Synthesis of Acetylenic Derivatives of a Substituted 1, 3, 4-Thiadiazole as Antibacterial Agents Anwar A. Tamer and Ahlam J. Qassir Department of Pharmaceutical Chemistry, College of Pharmacy, University of Baghdad, Baghdad, Iraq Abstract Thiadiazole is a heterocyclic compound that exhibits a wide variety of pharmacological activities such as anticancer, antibacterial, antifungal, antimicrobial, anti‐inflammatory, analgesic and anticonvulsant. 2, 5 disubstituted 1, 3, 4-thiadiazole constitutes an important class of compounds for new drug development because it acts as "hydrogen binding domain" and "two-electron donor system." It also serves as a constrained pharmacophore. This research highlights the recently synthesized Schiff base and mannich base derivatives and investigation of their chemical and biological behavior. Depending on this information’s new derivatives of 1, 3, 4-thiadiazole were synthesized and in the hope of having some activities as antibacterial and antifungal. These are: 1. N-(5-((4-(piperidin-1-yl) but-2-yn-1-yl) thio)-1, 3, 4-thiadiazol-2-yl) acetamide compound (4). 2. 1-(4-chlorophenyl)-N-(5-((4-(piperidin-1-yl) but-2-yn-1-yl) thio)-1, 3, 4 thiadiazol-2-yl) methanimine compound (6). 3. 1-(4-chlorophenyl)-N-(5-(prop-2-yn-1-ylthio)-1, 3,4thiadiazol-2-yl)methanimine compound(7). The characterization of mentioned compounds was performed by FTIR spectroscopy, HNMR, measurements of their physical properties, and studying of biological activity of the synthesized compounds by well diffusion method.


Introduction
Heterocyclic compounds are the cyclic organic compounds which contain at least one heteroatom, the most common heteroatoms are the nitrogen, oxygen, and sulfur but heterocyclic rings containing other heteroatoms are also widely known (1) .
Heterocyclic compounds are considered as one of the principal classes of organic compounds, which are used in many biological fields, due to their activities. Biological molecules such as DNA and RNA, chlorophyll, hemoglobin, vitamins and many more contain the heterocyclic ring in the significant skeleton (2) . Heterocycles are used in the development of several pharmaceutically essential compounds in a wide manner. The nitrogen and sulfur heterocyclic systems are important because of their physicochemical properties like lipophilicity with relevance to the design of new drugs (3) . Thiadiazole nuclei have antimicrobial, antiinflammatory,  anticancer,  anticonvulsant,  antidepressant, antioxidant, radio protective, antileishmanial activities , antimicrobial, antitubercular,  antifungal, analgesic, oxidative inhibitors, anti Hpylori, herbicides, dyes, lubricants and analytical reagents (4).. (6) .
The Mannich reaction is a crucial C-C bond forming reaction that is widely used in the synthesis of many biologically active natural compounds (7) .
Mannich reactions are three component condensation reactions involving carbonyl compounds, which exist as enol-keto tautomeric forms, formaldehyde and a primary or secondary amine (8) .
Schiff bases are an essential class of compounds due to their flexibility, structural similarities with natural biological substances and the presence of imine (-N=CH-) which is involved in the mechanism of transformation and racemization reaction in the biological system. These novel compounds could also act as valuable ligands whose biological activity has been shown to increase on complexation (12) . Also, they have a wide range of biological activities especially anti-bacterial (13) , anti-inflammatory (14) , anti-fungal (15) , anti-tumor (16) anti-oxidant (17) , antimicrobial, anthelmintic, anti-inflammatory, analgesic .antipyretic, antitubercular, diuretic, hypoglycemic, anticonvulsant, anti-HIV, cytotoxic (18) Recently, the severe infectious diseases caused by gram positive and gram negative pathogenic bacteria have inflated to threat level around the world. This increases, as well as the emergence of bacteria immune to ordinarily used antibiotics, has resulted in the need to develop new categories of antibacterial agents to conflict infections (19) .

Material and Methods
Chemicals used during the synthesis were supplied by hyper-chem (China). Completion of reactions and the purity of compounds were monitored by thin-layer chromatography (TLC), using Silica gel GF254 (type 60) pre-coated aluminum sheets, Merck (Germany) exposed to UV-254 nm light. Two solvent systems were used ethyl acetate: hexane (3:7) and methanol: hexane (8:2). Melting points were detected by using Stuart SMP3 melting point apparatus in open capillary tubes, and are uncorrected. The infrared spectra were performed in KBr disc, (υ, cm -1 ), using FTIR Spectrophotometer (Shimadzu, WQF-520, Japan).

Chemical synthesis
The target compound were synthesized by multistep reaction as shown in scheme 1 .

Antimicrobial activity
The antimicrobial activity of the final compounds was evaluated in the University of Baghdad / College of Education for Pure Sciences-Ibn Al-Haitham by the Advisory Office of the Central Service Laboratory.A preliminary antibacterial have been carried using the well diffusion method. The synthesized compounds were evaluated for their antimicrobial activity in vitro against three types of tested microorganisms (Staph. aureus., and Bacillus subtilis as a Gram-positive bacteria) and (klebsiella pneomonae, and E. coli) as a Gram-negative bacteria) were clinically activated and maintained on nutrient agar for examining antibacterial activity. Ampicillin was used as a standard drug for antibacterial activity.

Synthesis of compound (2)
Compound (2) was prepared by alkylating the potassium salt of compound (1) with propargyl bromide. It is logical to assume that the alkylation step followed an SN2 mechanism. The reaction is started by nucleophilic attack of the sulfide anion on the propargyl bromide affording the desired alkylated thiadiazole derivative. No allylic rearrangement was observed (26) .

Synthesis of compound (3)
In the acetylation of compound (2), where this step includes the synthesis of amide; it was done by treatment of an amine with acetic anhydride in the presence of a few drops of sulphuric acid as catalyst. Switching of the amino group into the acetamido group by acetylation modifies the interaction of the nitrogen lone pair with the π-electron system of the aromatic ring so that the ring is less powerfully activated toward electrophilic attack (27) .

Synthesis of compound (4)
Mannich reaction is a nucleophilic addition reaction that involves the condensation of a compound with active hydrogen(s), with an amine (primary or secondary) and formaldehyde (any aldehyde) (9) .

Synthesis of compound (5)
Acid hydrolysis reaction occurs by nucleophilic addition of water to the protonated amide, followed by transfer of a proton from oxygen to nitrogen to make the nitrogen a better leaving group and for subsequent elimination. The steps are reversible with the equilibrium shifted towards the product by the protonation of the NH2 in the final step (28) .

Synthesis of compound (6) and (7)
The mechanism of Schiff base formation is a reversible, acid catalyzed process, begins with nucleophilic addition of the primary amine to a carbonyl group (aldehyde or ketone) followed by a transfer of a proton from nitrogen to oxygen to yield neutral amino alcohol or carbinolamine. Protonation of the carbinolamine oxygen by an acid catalyst then converting the (-OH) group into a better leaving group (-OH2), and E1-like loss of water produces an iminium ion which after the loss of a proton from nitrogen gives the Schiff base and regenerate the acid catalyst to afford compounds (6) and (7) (29) (30) .  The recorded data in Table (1) lead to the following conclusions: All the synthesized compounds showed antimicrobial activity against G ( + ve) and G (ve) bacteria, but some of them showed no activity against (staphylococcus aureas) (compound 4 and compound 6) at concentration (0.01) mg/ml and even in concentration (0.02 mg/ml). Compound (7) showed activity against previously mentioned G ( + ve) (staphylococcus aureas) but at higher conc. showed higher antibacterial activity and it is Schiff base derivative, its benzene ring containing an ewithdrawing group (Cl) at the para position For compounds (6) which are a combination of Mannich base and Schiff base derivative showed moderate to higher antibacterial activity at both concentrations against tested bacteria.

Conclusions
New derivatives of 2-amino 5-mercapto1, 3, 4 thiadiazole were successfully synthesized using the conventional method. The synthesis of these proposed compounds was successfully performed by the stated procedures as previously described. The results obtained from this investigation were achieved according to the data shown by the physical and chemical analysis including (TLC, melting point, FTIR and 1HNMR analysis). Compound 4, 6 & 7 exhibit good antimicrobial activity comparable with marketable compounds. The antimicrobial evaluation indicated that the newly synthesized compounds showed moderate to high antimicrobial activity in comparison to Ampicillin for gram-positive bacteria and also have highest anti-microbial activity for gram negative bacteria. The compound (7) showed an excellent antimicrobial activity, and highest activity against staphylococcus areus compared to ampicillin.