Synthesis and Biological Evaluation of 1 , 2 , 4-Triazoles and 1 , 3 , 4-Oxadiazoles Derivatives Linked to 1 , 4-Dihydropyridines Scaffold

A series of diethyl-2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate derivative coupled to 1,3,4-oxadiazole-5-thiones and 1,2,4-triazole-5-thiones moieties at C2,C6 positions of 1,4-dihydropyridine ring system was prepared. This linkage was carried out by the reaction of 1,3,4-oxadiazole-5-thiones and 1,2,4-triazole-5-thiones with 2,6-dibromomethyl-3,5-diethoxycarbonyl-4-phenyl-1,4-dihydropyridine in the presence of potassium carbonate as a weak base and dry acetone as the solvent. The newly synthesized compounds were characterized by FT-IR, 1H NMR, 13C NMR spectral data, elemental analysis and FAB-MS. The synthesized compounds were tested for their antimicrobial and antifungal activity against Escherichia coli and Aspergillus fumigatus in vitro in comparison with Enrofloxacin and Amphotericin as the reference drugs which are normally used for treating such infections. The synthetic compounds showed different inhibition zones against tested bacteria and fungi. Compound 8d showed more antagonistic activity against E. coli and A. fumigatus.


Introduction
Antibiotics are drugs used for treating infection caused by microorganism such as bacteria or fungi and antibiotic resistance is the ability of microorganism to withstand the effect of antibiotics.The resistance of infective bacteria to present antibiotics remains a clinical obstacle in the chemotherapy of many cancers and demands research focused on the discovery of new drugs in the antibiotic drug field.][3][4] Apart from CVS activities (1,4-DHPs), they posses a variety of biological activities such as anti-tubercular, 5 antimicrobial, [6][7] anti-inflammatory, 8 anti-tumor, 9-10 analgesic. 11 substance which has no calcium antagonistic activity but has antibiotic activity would be of value in cancer chemotherapy and has strong ability in overcoming anticancer drug resistance.3] These examples clearly demonstrate the remarkable potential of novel DHP derivatives as a source of valuable drug candidates.

1. Bacterial Strain
The antibacterial and antifungal activity of compounds was assayed according to our perviously published method. 33The antibacterial and antifungal activity of compounds was tested against Escherichia coli and Aspergillus fumigatus.

2 Bacterium and Fungi Culture
The following microorganisms were used in this study to test antimicrobial activity of compounds: E. coli (PTCC 1399) and Aspergillus fumigatus (PTCC 5009).All microorganisms were provided by Persian Culture Collections of Microorganism, Iran.Bacteria were cultured for 24 h at 37 °C in brain heart infusion broth (Merck, Darmstadt, Germany) and A. fumigatus was cultured for 72 h at 5 °C in Saburo Dextrose broth (Merck, Darmstadt, Germany) and were used as inoculums.

3. Susceptibility Tests
The following methods were used to evaluate the activity of the compounds.All tests were repeated three times, using distilled water without compounds as a control to test the inhibitory effect of the solvent.Minimum inhibition concentrations (MIC) of compounds against the tested pathological microorganisms were determined using micro broth dilution method. 34Briefly, serial twofold dilutions of each compounds (10% w/v) were prepared in 96-well micro titer plate ((from 1:2 to 1:8192) containing cation-adjusted Mueller-Hinton broth (Merck, Darmstadt, Germany).Control micro-titer plates containing medium and distilled water at the same dilutions were also made.Bacteria and fungi suspensions were adjusted to the 0.5 McFarland standards (approximately 1 to 2 × 108 CFU/mL).A constant amount of microorganisms were added to all wells and the plates were incubated at 37 and 25 °C for 24-72 hour for E. coli and A. fumigatus, respectively (final inoculates were adjusted to the 10 5 CFU per each well).Each well was examined for growth, comparing each well to the control.The MIC was defined as the lowest concentration of compounds at which there was no visible growth of the organisms.For each test enrofloxacin and amphotericin were used as the control antimicrobial agents.The minimal bactericidal concentration (MBC; the lowest concentration of compounds that resulted in a 99.9% reduction in CFU of the initial inoculums) was determined by plating count the contents of wells that showed no visible growth of bacteria onto Mueller-Hinton agar and Saburo Dextrose agar plates and incubating at 37 and 25 °C for 24-72 h for E. coli and A. fumigatus, respectively.The MBC was considered the lowest concentration of compounds that prevented any colony formation.

Result and Discussion
The main synthetic route for substituted 1,3,4-oxadiazole-5-thiones 2a-d involves an initial reaction between carboxylic acid hydrazides 1a-d and carbon disulfide in basic ethanol solution, followed by acidification with di-Scheme 1. Synthesis of 1,3,4-oxadiazole-5-thione and 1,2,4-triazole-5-thione derivatives lute hydrochloric acid which resulted in the precipitation of oxadiazole.6] 1-Substituted-4-phenylthiosemicarbazides 3a-d were prepared by the condensation of carboxylic acid hydrazides 1a-d with phenylisothiocyanate under reflux in absolute ethanol.8][39] The existence of thiol-thione tautomerism is known for the compounds 2a-d and 4a-d and generally one form is predominant.In the present study, the thione structure was dominated in the solid state.These constitutional isomers were distinguished by IR and 1 H NMR. The appearance of a C=S absorption peak in the region 1248-1278 cm -1 indicated that the oxadiazoles and triazoles are in their thione form.][37][38][39] 4-Phenyl-1,4-dihydro-2,6-dimethyl-3,5-pyridine dicarboxylic acid ester (diethyl 2,6-dimethyl-4-phenyl-1,4-dihydropyridine-3,5-dicarboxylate)derivative (compound 5) was synthesized by condensation of benzaldehyde with two equivalents of β-ketoester in the presence of a nitrogen donor such as ammonia or ammonium acetate according to the procedure reported in the literature. 40he 1 H NMR spectrum of compound 5 shows a characteristic singlet in the range of δ H 5-7 ppm which was due to the NH proton of the 1,4-DHP ring and another important singlet at 5 ppm which was attributed to the CH at C4 of the 1,4-DHP ring.Allylic bromination is the replacement of a hydrogen on a carbon adjacent to a double bond and N-bromosuccinimide (NBS) is a brominating agent that is used as the source of bromine in radical reactions that are used for allylic bromination.The synthesis of 2,6-dibromomethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine (compound 6) was carried out by bromination of corresponding 2,6-dimethyl-1,4-dihydropyridine (compound 5) with NBS in methanol according to the procedure reported in the literature. 41Replacement of the bromines of compound 6 with 1,2,4-triazole-5-thiones 4a-d or 1,3,4oxadiazole-5-thiones 2a-d was carried out in the presence of potassium carbonate as a weak base in dry acetone to afford the corresponding coupled 1,4-dihydropyridines (7a-d and 8a-d) (Scheme 2).The structures identification for compounds 7a-d and 8a-d was based on spectroscopic methods.In the IR spectra the disappearance of the C=S absorption peak in the region 1248-1278 cm -1 and furthermore the absence of NH peak at 12-14 ppm support the connection of oxadiazole and triazoles to 1,4-DHP ring.
The CH 2 X protons at positions C2 and C6 of symmetrically substituted 1,4-dihydropyridine ring become diastereotopic and provide an AB system in the corresponding 1 H NMR spectra.The extent of the observed anisochrony of the methylene protons should be influenced by the spatial conformation of ester groups and the formation of a CH•••O=C intramolecular hydrogen bonding. 41he in vitro antibacterial and antifungal activities of the synthesized compounds against E. coli and A. fumigatus are shown in Tables 1 and 2. The minimal inhibition concentration for enrofloxacin as the reference antibacterial drug is 36 µg/µL concentration and for amphotericin was 28 µg/µL of drug in the same test conditions.From the data presented in Tables 1 and 2 it is clear that compounds 8a-d showed better antifungal activities than compounds 7a-d and the compounds 7c,7d,8d exhibited better and equipotent activity against E. coli.The compound 8d is highly active against E. coli and A. fumigatus.

1. General
The melting points of all compounds were recorded on a Philip Harris C4954718 apparatus without calibration.IR spectra were recorded on Thermo Nicolet Nexus 670 FT-IR spectrometer and 1 H and 13 C NMR spectra measured with Bruker Avance 300 MHz spectrometer.Mass spectra were recorded on a JEOL-JMS 600 (FAB MS) instrument.Thin layer chromatography (TLC) analyses were carried out on silica gel plates.All chemicals were purchased from Merck (Tehran, Iran) and used as received by standard procedures.All of the instruments, chemicals and solvents were dried according to standard methods.Freshly distilled solvents were used throughout, and anhydrous solvents were dried according to the method reported by Perrin and Armarego.Microanalyses were performed on a Leco Analyzer 932.

2. General Procedure for the Synthesis of 2a-d
Compounds 2a-d were synthesized by the reaction of CS 2 (6.7 mL, 0.1 mol) with a suspension of carboxylic acid hydrazides 1a-d (0.1 mol) in chloroform (100 mL) in the presence of Et 3 N (15 mL, 0.1 mol).After refluxing the reaction mixture for 3 h, the resulting solution was filtered and acidified with acetic acid (20 mL, 20% v/v).

3. General Procedure for the Synthesis of 4a-d
Equimolar quantities of carboxylic acid hydrazides 1a-d (0.01 mol) and phenylisothiocyanate (1.35 g, 0.01 mol) in appropriate amount of absolute ethanol were refluxed for 6-8 h.The formed precipitate of 1-substituted-4-phenylthiosemicarbazides 3a-d was filtered, and then a suspension of 0.04 g thiosemicarbazides 3a-d in sodium hydroxide 0.4 g (0.01 mol, as a 2N solution) was refluxed for 6 h.

General Procedure for the Synthesis of Compounds 7a-d and 8a-d
A mixture of 2a-d or 4a-d (2 mmol), K 2 CO 3 (0.276 g, 2 mmol) and acetone (20 mL) were stirred at room temperature for 30 minute, and then 0.487 g (1 mmol) com-

Conclusion
In this study we demonstrated the synthesis, antibacterial and antifungal activity of new derivatives of 1,4-dihydropyridines bearing 1,2,4-triazole and 1,3,4-oxadiazole moieties at C2 and C6 of 1,4-DHP ring system.It should be noted that compound 8d exhibited the most potent activity against E. coli and A. fumigates.The structure-activity relationship of the compounds showed that substitution at the position 2, 6 of the pyridine ring enhances biological activity.Our results will have an impact on further investigation in this field in search of 1,4-dihydropyridine compounds connected with 1,2,4-triazole and 1,3,4-oxadiazole moieties as antibacterial and antifungal agents.

Table 1 .
In vitro antibacterial activity of synthetic compounds against E. coli.