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Biology-Oriented Syntheses (BIOS) of Novel Santonic-1,3,4-oxadiazole Derivatives under Microwave-Irradiation and their Antimicrobial Activity.

Byline: Uzma Salar, Ghulam Abbas Miana, Khalid Mohammed Khan, Farzana Naz, Nida Iqbal Siddiqui, Muhammad Taha, Saima Tauseef, Saifullah Khan and Shahnaz Perveen

Summary: Novel 2-thio substituted 1,3,4-oxadiazole derivatives of santonic acid (13-18) were synthesized. The synthesis of these derivatives was comprised of six steps which start from the basic hydrolysis of a-santonin 1 to the santoninic acid 2 followed by in situ rearrangement of 2 into santonic acid 3. Santonic acid 3 was then converted into its acyl imidazole derivative 4 followed by hydrazinolysis to give santonic carbohydrazide 5 which was further converted into santonic-1,3,4- oxadiazole-2-thiol 6. Santonic-1,3,4-oxadiazole-2-thiol 6 was alkylated to afford 2-thio substituted 1,3,4-oxadiazole derivatives of santonic acid (13-18).

All the synthetic steps were carried out under microwave irradiation in controlled parameters. Compounds 13-18 along with intervening intermediates 5 and 6 were evaluated for their antimicrobial potential. Compound 14 showed appreciably good activity against Staphylococcus epidermidis. On the other hand compounds 6 and 17 demonstrated good activity against Escherichia coli and Shigella flexeneri, respectively. Compound 6 showed good antifungal activity. The synthesized compounds were characterized by different spectroscopy techniques.

Keywords: a-Santonin; Santonic acid; Antibacterial activity; Antifungal activity.

Introduction

a-Santonin, isolated from various species of Artemisia [1,2], is a naturally occurring plant sesquiterpene lactone which is well known for a variety of biological activities such as antimicrobial, antiprotozoal, phytotoxic [3-7], antiinflammatory [8], antipyretic activity [9]. Different compounds derived from santonin also exhibit cytotoxicity against different cancer cell lines [10-14]. Santonin also serves as an intermediate for various asymmetric syntheses leading to the important natural products and related compound [15].

1,3,4-oxadiazole-2(3H)-thione and its structural analogues possesses a variety of biological activities [16] including bactericidal [17], fungicidal [18] and also known to have strong inhibitory activity against cyclooxygenase, lipoxygenase [19], monoamine oxidase, succinate dehydrogenase [20], and carbonic anhydrase enzymes [21-23]. The search for nontoxic and selective antibacterial agents has now turn out to be a main area of interest in medicinal chemistry [24,25].

There is diverse range of antibacterial agents available, but still resistance of pathogenic bacteria to drugs is persistently raising. A clear-cut way of identifying new drugs and treatment of various infectious diseases is problematic due to the chemical diversity and diverse mechanisms of actions of drugs which result in the reappearance of various diseases such as malaria which were thought to be controlled [26]. So, the sighting of new antibacterial agents is a crucial area of investigation and it leads to the increasing number of current research in favor of the development of novel antibacterial agents in order to triumph over this serious medical issue [27].

Our aim was to synthesize the new chemical entities derived from santonin with oxadiazole moiety and to evaluate them for the antimicrobial activities. Furthermore, it was thought to carry out most of the transformation under microwave irradiation which is an efficient means of synthesis [28], and it also offers various benefits for performing syntheses, including yields, most importantly enhancement in reaction rate and clean chemistry [29].

Compounds 13-18 along with intervening intermediates 5 and 6 were evaluated for their antimicrobial potential and showed encouraging results.

Experimental

General Information

Thin layer chromatography (TLC) was performed on pre-coated silica gel aluminum plates (Kieselgel 60 F-254, 0.20 mm, Merck, Darmstadt, Germany). Chromatograms were visualized by using a handhold UV lamp at (254 and 365 nm) or iodine vapors. Electron impact mass spectra (EI MS) were recorded on a Finnigan MAT-311A, Germany (70eV) spectrometers and the data are tabulated as m/z. 1HNMR spectroscopic analysis was performed on Avance Bruker AM spectrometers 300, 400 and 500 MHz machine.

Splitting patterns for NMR spectra were as follows s, singlet; d, doublet; t, triplet; m, multiplet. Chemical shifts are reported in d (ppm) and coupling constants are given in Hz. CHN analysis was performed on a Carlo Erba Strumentazione- Mod-1106, Italy. Commercially available (Flulka Aldrich) phenacyl bromides were purchased and used as received. All the synthetic steps were performed on CEM Matthews, NC 28105 microwave synthesizer.

Bioassay

Antibacterial Assay

The disc diffusion method [30] was adopted to check out the antibacterial potential of compounds 326. Bacterial cultures mixed with pre sterile physiological (0.85%) saline solution were subjected for 24 h incubation. Turbidity of all samples was agreed with the standard inoculum of 0.5 Mac- Farland scale [~106 CFU/mL]. The Mueller Hinton agar (Oxoid) plates were seeded with all bacterial cultures grown in Mueller Hinton broth (Oxoid). All prepared discs containing test comounds were positioned on to the surfaces and plates were incubated for 24 h at 37 C. Results were notified by measuring the zone of inhibitions in mm. DMSO was utilized as negative control. Gentamicin was utilized as positive control.

Antifungal Assay

Antifungal activity of synthesized compounds 326 was also checked by employing disc diffusion method [30]. Concisely, a small fraction of fungal culture was shifted to normal saline 2-3 ml in a screw capped tube with few glass beads (diameter = 1 mm) and vortexes for about 10 min to formulate a homogeneous suspension fungal cultures. Sabouraud dextrose agar (SDA) plates were seeded with these fungal suspensions. Sterile filter discs containing stock solution 10 l were positioned on to the surfaces. Plates were incubated for one week at room temperature. Results were notified by measuring the zone of inhibitions in mm. Ketoconazole was utilized as positive control for antifungal activity.

Experimental Procedure for the Microwave Assisted Synthesis of Santonic Acid (3)

Commercially available a-santonin 1 (1 mmol) was added to concentrated aqueous solution of potassium hydroxide (2 ml) in microwave vial which was equipped with a magnetic stirrer and irradiated for 30 min with each pulse of 10 sec at a set temperature, pressure and power control (Temperature = 50 C, Power = 30 Watt and Pressure = 30 psi).

The progress of reaction was monitored by TLC analysis. Santoninic acid 2 was formed first which was analyzed by taking a small fraction from the reaction mixture after 5 min. The small aliquot was extracted with the ethyl acetate and right after drying characterized by the spectroscopic analysis EIMS, HNMR and CHN etc. The reaction mixture was further irradiated for 25 min. Santoninic acid was then rearranged in situ to santonic acid 3. After completion of the reaction, it was extracted with ethyl acetate (3 x 10 ml), organic layer was dried over anhydrous Na2SO4, evaporated on a rotary evaporator to afford viscous gummy product.

Experimental Procedure for the Microwave-Assisted Synthesis of Imidazolyl derivative of Santonic Acid (4)

A mixture of santonic acid (1 mmol), 1,1- carbonyl diimidazole (1 mmol) in acetonitrile (2 ml) and triethylamine (1 mmol) as a base were taken in microwave vial (2-5 ml). It was irradiated for 5 min with each pulse of 10 second at a set temperature, pressure and power control as mentioned earlier. The progress of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was evaporated on a rotary evaporator and washed with 10 ml hexane. A viscous gummy product was obtained.

Experimental Procedure for the Microwave-Assisted Synthesis of Santonic Carbohydrazide (5)

Imidazolyl derivative of santonic acid 4 (1 mmol) was taken in hydrazine hydrate (2 ml) into a microwave vial (2-5 ml) and irradiated for 20 min with each pulse of 10 sec after keeping into the microwave cavity at a set temperature, pressure and power control. The reaction mixture was turned into the dark yellow color. The progress of the reaction was monitored by TLC analysis. After completion of the reaction, the reaction mixture was poured into the cooled distilled water. Yellow precipitates were formed which were filtered and crystallized from ethanol.

Experimental Procedure for the Microwave-Assisted Synthesis of Santonic-1,3,4-oxadiazole-2-thiol (6)

Santonic carbohydrazide (1 mmol), 95% ethanol (2 ml), NaOH (1 g) and CS2 (1 ml) was taken in a microwave vial, equipped as previously described, irradiated for 20 minutes with each pulse of 10 sec. Reaction progress was monitored via TLC analysis. After completion, the reaction mixture was poured in 10% HCl while keeping the flask on an ice bath. Stirred the reaction mixture for 15 minutes, light yellow precipitates were appeared which were then filtered, washed with distilled water (50 ml) and dried in air. The crude product was crystallized by using ethanol.

General Procedure for the Microwave-Assisted Syntheses of 2-Thio-substituted derivatives of Santonic-1,3,4-oxadiazole (13-18).

Santonic-1,3,4-oxadiazole-2-thiol (1 mmol), different derivatives of phenacyl bromide (1 mmol) in ethanol (3 ml) and triethylamine (1 mmol) as base were taken into a microwave vial, equipped as described before and irradiated in 10 second pulses for 30 min. Progress of the reaction was monitored by comparative TLC. After completion of reaction, mixture was allowed to cool and poured into the beaker containing crushed ice. Precipitates were formed, filtered and washed with distilled water. The solid product so obtained was crystallized from ethanol.

Spectroscopic Data

(2R)-2-[(1S,4aS)-1-Hydroxy-4a,8-dimethyl-7-oxo1,2,3,4,4a,7-hexahydro-2-naphthal-enyl] propanoic acid (2)

Yield: 60%; 1HNMR (500 MHz, DMSO-d6): 11.97 (s, 1H, COOH), 6.86 (d, J = 9.9 Hz, 1H, H 6), 6.11 (d, J = 9.6 Hz, 1H, H-5), 5.34 (br.s, 1H, OH), 4.38 (d, J = 11.1 Hz, 1H, H-1), 2.99 (m, 1H, H-1'), 2.12 (s, 3H, CH3-10), 2.01 (m, 1H, H-2), 1.84-1.53 (m, 2H, CH2-3), 1.43-1.38 (m, 2H, CH2-4), 1.19 (s, 3H, CH3-9), 1.14 (d, J = 4.5 Hz, 3H, CH3-11); 13C NMR (75 MHz, DMSO-d6): 182.3 (C=O, C-3), 176.5 (C=O, C-12), 155.3 (CH, C-1), 151.3 (C, C-5), 128.6 (C, C-4), 126.4 (CH, C-2), 81.6 (CH, C-6), 52.1 (CH, C-7), 41.3 (C, C-10), 41.1 (CH, C-11), 38.1 (CH2, C-9), 32.1 (CH2, C-8), 25.4 (CH3, C), 12.8 (CH3, C), 10.5 (CH3, C); EI MS m/z (% rel. abund.): 246 (M+, 51.6), 231 (38.2), 173 (100.0), 135 (47.8), 91 (37.0); Anal. Calcd for C15H20O4, C = 68.16, H = 7.63, Found C = 68.18, H = 7.65; IR (KBr, cm-1): 3245 (OH), 3155 (O-H), 1731 (C=O), 1684 (C=O), 1645 (C=C), 1469 (C-H), 1174 (C-H), 723 (C-H);

4.2. 2-[(1R,5S)-1,5-Dimethyl-4,7-dioxotri- cyclo[4.4.0.02,8]dec-8-yl]propanoic acid (3)

Yield: 50%; 1HNMR (500 MHz, DMSO-d6): 11.64 (s, 1H, COOH), 2.76-2.73 (m, 1H, H-11), 2.58 (dd, J = 13, 17.5 Hz, 1H, CH2-2a), 2.51 (br.d, J = 14.5 Hz, 1H, CH-5), 2.42 (q, J = 7.0, 14.0 Hz, 1H, CH2-2b), 2.15-2.09 (m, 1H, CH-4), 2.06 (br.t, J = 3.5 Hz,1H, CH-1), 1.97 (br.s, 1H, CH2-8a), 1.68 (dt, J = 4.0, 12.5 Hz, 1H, CH2-8b), 1.56-1.50 (m, 1H, CH29a), 1.33 (dd, J = 4.0, 10.0 Hz, 1H, CH2-9b), 1.29 (s, 3H, CH3-14), 1.17 (d, 3H, CH3-13), 0.94 (d, 3H, CH3-15); 13C NMR (75 MHz, DMSO-d6): 174.2 (C=O, C-12), 170.4 (C=O, C-6), 165.0 (C=O, C-3), 64.3 (CH, C-1), 63.4 (CH, C-5), 60.2 (C, C-7), 41.1 (CH, C-4), 40.3 (CH, C-11), 39.3 (CH2, C-2), 36.0 (CH2, C-9), 29.2 (C, C-10), 26.5 (CH2, C-8), 15.9 (CH3, C), 13.5 (CH3, C), 13.2 (CH3, C); EI MS m/z (% rel. abund.): 264 (M+, 45.0), 218 (34.0), 246 (23.5); 203 (54.0) 191 (43.0), 162 (52.2); Anal. Calcd for C15H20O4, C = 68.16, H = 7.63, Found C = 68.18, H = 7.65; IR (KBr, cm-1): 3285 (O-H), 1731 (C=O), 1684 (C=O), 1461 (C-H), 1182 (C-H), 734 (C-H);

4.3. (1R,5S)-8-[2-(1H-Imidazol-1-yl)-1-methyl-2- oxoethyl]-1,5-dimethyltricyclo[4.4.0.02,8] decane-4,7- dione (4)

Yield: 65%; 1H-NMR (300 MHz, CD3OD): 8.43 (s, 1H, H-2'), 7.91 (d, J(4',5') = J(5',4') = 7.8 Hz, 2H, H-4', 5'), 2.74-2.60 (m, 1H, H-11), 2.58 (dd, J = 13, 17.5 Hz, 1H, CH2-2a), 2.52 (br.d, J = 14.5 Hz, 1H, CH-5), 2.40 (q, J = 7.0, 14.0 Hz, 1H, CH2-2b), 2.15-2.10 (m, 1H, CH-4), 2.07 (br.t, J = 3.5 Hz,1H, CH-1), 1.98 (br.s, 1H, CH2-8a), 1.68 (dt, J = 4.0, 12.5 Hz, 1H, CH2-8b), 1.56-1.50 (m, 1H, CH2-9a), 1.35 (dd, J = 4.0, 10.0 Hz, 1H, CH2-9b), 1.28 (s, 3H, CH314), 1.18 (d, 3H, CH -13), 0.97 (d, 3H, CH -15); 13C NMR (75 MHz, DMSO-d6): 174.3 (C=O, C-12), 170.4 (C=O, C-6), 165.0 (C=O, C-3), 136.5 (CH, C2'), 130.6 (CH, C-4'), 117.3 (CH, C-5'), 64.2 (CH, C1), 63.3 (CH, C-5), 60.0 (C, C-7), 41.2 (CH, C-4), 40.2 (CH, C-11), 39.4 (CH2, C-2), 36.1 (CH2, C-9), 29.7 (C, C-10), 26.5 (CH2, C-8), 15.9 (CH3, C), 13.0 (CH3, C), 12.7 (CH3, C); EI MS m/z (% rel. abund.): 314 (M+, 8.0), 299 (34.0), 233 (23.5); 191 (62.2), 177 (79.0), 149 (36); Anal.

Calcd for C18H22N2O3, C = 68.77, H = 7.05, N = 8.91, Found C = 68.79, H = 7.07, N = 8.93; IR (KBr, cm-1): 1712 (C=O), 1665 (C=O),1645 (C=N), 1640 (C=C), 1450 (C-H), 1150 (C-H), 735 (C-H);

4.4. 2-[(1R,5S)-1,5-Dimethyl-4,7- dioxotricyclo[4.4.0.02,8]dec-8-yl]propanohydrazide (5)

Yield: 52%; 1H-NMR (300 MHz, CD3OD): 7.92 (s, 1H, NH), 5.67 (br.s, 1H, NH2), 2.72-2.58 (m, 1H, H-11), 2.56 (dd, J = 13, 17.5 Hz, 1H, CH22a), 2.49 (br.d, J = 14.5 Hz, 1H, CH-5), 2.39 (q, J = 7.0, 14.0 Hz, 1H, CH2-2b), 2.10-2.05 (m, 1H, CH-4), 2.03 (br.t, J = 3.5 Hz,1H, CH-1), 1.95 (br.s, 1H, CH28a), 1.66 (dt, J = 4.0, 12.5 Hz, 1H, CH2-8b), 1.521.49 (m, 1H, CH2-9a), 1.30 (dd, J = 4.0, 10.0 Hz, 1H, CH2-9b), 1.26 (s, 3H, CH3-14), 1.15 (d, 3H, CH3-13), 0.93 (d, 3H, CH3-15); 13C NMR (75 MHz, DMSOd6): 174.2 (C=O, C-12), 170.3 (C=O, C-6), 165.5 (C=O, C-3), 64.6 (CH, C-1), 63.2 (CH, C-5), 60.3 (C, C-7), 41.2 (CH, C-4), 40.2 (CH, C-11), 39.3 (CH2, C2), 36.1 (CH2, C-9), 29.7 (C, C-10), 26.5 (CH2, C-8), 15.6 (CH3, C), 13.5 (CH3, C), 13.2 (CH3, C); EI MS m/z (% rel. abund.): 278 (M+, 5.0), 260 (44.0), 250 (43.5), 203 (82.2), 163 (60.0), 136 (23.0); Anal.

Calcd for C13H13Cl2N3O3, C = 64.73, H = 7.97, N = 10.06, Found C = 64.76, H = 8.00, N = 10.09; IR (KBr, cm-1): 3354 (NH), 3267 (NH), 2860 (C-H), 1730 (C=O), 1695 (C=O), 1686 (C=O), 1455 (C- H),1365 (C-H), 730 (C-H);

(1R,5S)-1,5-Dimethyl-8-[(1R)-1-(5-sulfanyl-1,3,4- oxadiazol-2-yl)ethyl]tricycle [4.4.0.02,8] dec ane-4,7- dione. (6)

Yield: 47%; 1H-NMR (300 MHz, DMSOd6): 10.55 (s, 1H, SH), 2.78-2.74 (m, 1H, CH-11), 2.52 (br.s, 2H, CH2-2), 2.49-2.45 (m, 1H, CH-5), 2.39-2.35 (m, 1H, CH-4), 2.13-2.07 (m, 1H, CH-1), 1.97 (br.s, 1H, CH2-8a), 1.53 (br.s, 1H, CH2-8b), 1.42-1.37 (m, 2H, CH2-9), 1.23 (s, 3H, CH3-14), 1.18 (d, 3H, CH3-13), 0.99 (d, 3H, CH3-15); 13C NMR (75 MHz, DMSO-d6): 170.4 (C=O, C-6), 167.6 (C, C5'), 165.1 (C=O, C-3), 155.3 (C, C-2'), 64.6 (CH, C1), 63.2 (CH, C-5), 60.3 (C, C-7), 41.4 (CH, C-4), 40.2 (CH, C-11), 39.3 (CH2, C-2), 36.6 (CH2, C-9), 29.3 (C, C-10), 26.5 (CH2, C-8), 15.6 (CH3, C), 13.5 (CH3, C), 13.2 (CH3, C); EI MS m/z (% rel. abund.): 316 (1.4), 288 (1.6), 261 (1.2), 191 (2.9), 177 (3.1), 149 (38.1); Anal. Calcd for C16H20 N2O3S, C = 59.98, H = 6.29, N= 8.74; Found: C = 60.00, H = 6.31, N= 8.76; IR (KBr, cm-1): 1675 (C=O), 1657 (C=O), 1645 (C=N), 1465 (C-H), 1376 (C-H), 1300 (C-O), 742 (C-H);

(1R,5S)-8-[(1R)-1-(5-{[2-(3,4-Dichlorophenyl)-2- oxoethyl]sulfanyl}-1,3,4-oxadiazol-2-yl)ethyl]-1,5- dimethyltricyclo[4.4.0.02,8]decane-4,7-dione (13)

Yield: 42%; 1H-NMR (400 MHz, DMSO- d6): 7.96-7.71 (m, 3H, H-2', 5', 6'), 5.74 (s, 2H, - SCH2), 3.11-3.09 (m, 1H, CH-11), 3.08-3.06 (m, 2H, CH2-2), 2.49 (br.s, 2H, CH-5), 2.48 (br.s, 1H, CH-4), 2.13 (m, 1H, CH-1), 1.58 (br.s, 2H, CH2-8), 1.491.37 (m, 2H, CH2-9), 1.17 (s, 3H, CH3-14), 1.15 (d, 3H, CH3-13), 0.99 (d, 3H, CH3-15); 13C NMR (75 MHz, DMSO-d6): 189.2 (C=O, C-1'''), 170.4 (C=O, C-6), 167.6 (C, C-5'), 165.1 (C=O, C-3), 155.3 (C, C-2'), 137.5 (C, C-4''), 136.3 (C, C-1''), 133.4 (C, C-3''), 130.1 (CH, C-2''), 129.8 (CH, C-5''), 128.2 (CH, C-6''), 64.1 (CH, C-1), 63.0 (CH, C-5), 60.3 (C, C-7), 41.3 (CH, C-4), 40.2 (CH, C-11), 39.4 (CH2, C2), 38.0 (CH2, C-2'''), 36.1 (CH2, C-9), 29.7 (C, C10), 26.9 (CH2, C-8), 15.6 (CH3, C), 13.4 (CH3, C), 13.2 (CH3, C); EI MS m/z (% rel. abund.): 510 (M+2, 1.1), 508 (M+1, 1.7), 392.5 (1.1), 173 (47.2), 145 (20.7), 44 (100.0); Anal.

Calcd for C24H24N2O4Cl2S, C = 56.81, H = 4.77, N= 5.52; Found: C = 56.83, H = 4.79, N= 5.54; IR (KBr, cm-1): 1672 (C=O), 1660 (C=O), 1640 (C=N), 1657 (C=C), 1458 (C-H), 1370 (C-H), 1315 (C-O), 725 (C-H);

(1R,5S)-1,5-Dimethyl-8-[(1S)-1-(5-{[2-(3- nitrophenyl)-2-oxoethyl]sulfanyl}-4H-pyrazol-3-yl) ethyl]tricyclo[4.4.0.02,8]decane-4,7-dione (14)

Yield: 45%; 1H-NMR (400 MHz, DMSOd6): 8.65 (br.s, 1H, H-2'), 8.51-8.47 (m, 1H, H-4'), 8.39-8.35 (m, 2H, H-5',6'), 5.79 (s, 2H, -SCH2), 3.32 (br.s, 1H, CH-11), 3.10-3.07 (m, 2H, CH2-2), 2.49 (br.s, 2H, CH-5), 2.40 (br.s, 1H, CH-4), 2.15 (m, 1H, CH-1), 1.55 (br.s, 2H, CH2-8), 1.45-1.33 (m, 2H, CH2-9), 1.17 (s, 3H, CH3-14), 1.14 (d, 3H, CH3-13), 0.80 (d, 3H, CH3-15); 13C NMR (75 MHz, DMSO- d6): 189.6 (C=O, C1'''), 170.7 (C=O, C-6), 167.9 (C, C-5'), 165.3 (C=O, C-3), 155.3 (C, C-2'), 147.6 (C, C-3''), 137.7 (C, C-1''), 134.7 (CH, C-6''), 129.3 (CH, C-5''), 28.1 (CH, C-4''), 122.2 (CH, C-2''), 64.6 (CH, C-1), 63.3 (CH, C-5), 60.2 (C, C-7), 41.2 (CH, C-4), 40.2 (CH, C-11), 39.2 (CH2, C-2), 38.0 (CH2, C-2'''), 36.1 (CH2, C-9), 29.7 (C, C-10), 26.6 (CH2, C-8), 15.9 (CH3, C), 13.5 (CH3, C), 13.2 (CH3, C); EI MS m/z (% rel. abund.): 510 (M+2, 1.1), 508 (M+1, 1.7), 392.5 (1.1), 173 (47.2), 145 (20.7), 44 (100.0); Anal.

Calcd for C24H25N3O6S, C = 59.61, H = 5.21, N= 8.69; Found: C = 59.64, H = 5.24, N= 8.73; IR (KBr, cm-1): 1685 (C=O), 1667 (C=O), 1645 (C=N), 1650 (C=C), 1545 (N-O), 1490 (N-O), 1462 (C-H), 1375 (C-H), 1355 (N-O), 1320 (C-O), 732 (C-H);

(1R,5S)-1,5-Dimethyl-8-[(1S)-1-(5-{[2-(4- methylphenyl)-2-oxoethyl]sulfanyl}-4H-pyrazol-3- yl)ethyl]tricyclo[4.4.0.02,8]decane-4,7-dione (15)

Yield: 40%; 1H-NMR (500 MHz, DMSOd6): 7.77-7.59 (m, 4H, H-2', 3', 5', 6'), 5.12 (s, 2H, -SCH2), 3.61 (br.s, 1H, CH-11), 3.10-3.07 (m, 2H, CH2-2), 2.49 (br.s, 2H, CH-5), 2.40 (br.s, 1H, CH-4), 2.28 (br.s, 1H, CH-1), 1.57 (br.s, 2H, CH2-8), 1.481.32 (m, 2H, CH2-9), 1.22 (s, 3H, Ar-CH3), 1.19 (s, 3H, CH3-14), 1.15 (d, 3H, CH3-13), 0.99 (d, 3H, CH3-15); 13C NMR (75 MHz, DMSO-d6): 189.2 (C=O, C-1'''), 170.8 (C=O, C-6), 167.7 (C, C-5'), 165.4 (C=O, C-3), 155.2 (C, C-2'), 132.6 (C, C-1''), 128.7 (CH, C-3''), 128.7 (CH, C-5''), 128.3 (CH, C2''), 128.3 (CH, C-6''), 127.6 (C, C-4''), 64.6 (CH, C1), 63.2 (CH, C-5), 60.0 (C, C-7), 41.2 (CH, C-4), 40.1 (CH, C-11), 39.3 (CH2, C-2), 38.0 (CH2, C-2'''), 36.1 (CH2, C-9), 29.7 (C, C-10), 26.6 (CH2, C-8), 21.6 (CH3, C), 15.6 (CH3, C), 13.0 (CH3, C), 12.8 (CH3, C); EI MS m/z (% rel. abund.): 450 (3.2), 373 (7.5), 119 (94.6), 91 (43.0), 44 (100.0); Anal.

Calcd for C25H28N2O4S, C = 66.35, H = 6.24, N= 6.19; Found: C = 66.38, H = 6.27, N= 6.22; IR (KBr, cm-1): 1675 (C=O), 1668 (C=O),1649 (C=N), 1650 (C=C), 1454 (C-H), 1374 (C-H), 1319 (C-O), 735 (C-H);

(1R,5S)-1,5-Dimethyl-8-{(1S)-1-[5-({2-[4- (methylsulfonyl)phenyl]-2-oxoethyl}sulfanyl)-4H- pyrazol-3-yl]ethyl}tricyclo[4.4.0.02,8]decane-4,7- dione (16)

Yield: 30%; 1H-NMR (500 MHz, DMSOd6): 7.77-7.59 (m, 4H, H-2', 3', 5', 6'), 5.15 (s, 2H, -SCH2), 3.31 (br.s, 1H, CH-11), 3.19-3.13 (m, 2H, CH2-2), 2.49 (br.s, 2H, CH-5), 2.48 (br.s, 1H, CH-4), 2.26 (br.s, 1H, CH-1), 1.59 (br.s, 2H, CH2-8), 1.481.35 (m, 1H, CH2-9), 1.29 (s, 3H, -SO2-CH3), 1.22 (s, 3H, CH3-14), 1.16 (d, 3H, CH3-13), 1.14 (d, 3H, CH3-15); 13C NMR (75 MHz, DMSO-d6): 189.7 (C=O, C-1'''), 170.5 (C=O, C-6), 167.6 (C, C-5'), 165.4 (C=O, C-3), 155.3 (C, C-2'), 145.1 (C, C-4''), 140.2 (C, C-1''), 129.7 (CH, C-2''), 129.7 (CH, C-6''), 128.0 (CH, C-3''), 128.0 (CH, C-5''), 64.2 (CH, C-1), 63.0 (CH, C-5), 60.0 (C, C-7), 47.9 (S-CH3, C), 41.2 (CH, C-4), 40.1 (CH, C-11), 39.3 (CH2, C-2), 38.0 (CH2, C-2'''), 36.5 (CH2, C-9), 29.7 (C, C-10), 26.6 (CH2, C-8), 15.9 (CH3, C), 13.1 (CH3, C), 12.9 (CH3, C); EI MS m/z (% rel. abund.): 475 (12.4), 447 (11.6), 183 (60.9), 121 (34.3), 44 (100.0); Anal.

Calcd for C25H28N2O6S2, C = 58.12, H = 5.46, N= 5.42; Found: C = 58.16, H = 5.50, N= 5.46; IR (KBr, cm-1): 1670 (C=O), 1665 (C=O),1654 (C=N), 1658 (C=C), 1450 (C-H), 1371 (C-H), 1340 (S=O), 1323 (C-O), 1150 (S=O), 730 (C-H);

(1R,5S)-1,5-Dimethyl-8-((1S)-1-{5-[(2-oxo-2- phenylethyl)sulfanyl]-4H-pyrazol-3-yl}ethyl)tri cyclo [4.4.0.02,8]decane-4,7-dione (17)

Yield: 38%; 1H-NMR (500 MHz, DMSOd6): 8.02-7.88 (m, 1H, H-2'), 7.68-7.50 (m, 3H, H3', 4', 5'), 7.34-7.20 (m, 1H, H-6'), 5.06 (s, 2H, SCH2), 2.75-2.70 (m, 1H, CH-11), 2.50 (br.s, 2H, CH2-2), 2.45-2.40 (m, 1H, CH-5), 2.35-2.30 (m, 1H, CH-4), 2.10-2.05 (m, 1H, CH-1), 1.92 (br.s, 1H, CH2-8a), 1.50 (br.s, 1H, CH2-8b), 1.40-1.35 (m, 2H, CH2-9), 1.20 (s, 3H, CH3-14), 1.15 (d, 3H, CH3-13), 0.98 (d, 3H, CH3-15); 13C NMR (75 MHz, DMSOd6): 189.5 (C=O, C-1'''), 170.4 (C=O, C-6), 167.5 (C, C-5'), 165.3 (C=O, C-3), 155.2 (C, C-2'), 135.1 (C, C-1''), 133.3 (CH, C-4''), 128.6 (CH, C-2''), 128.6 (CH, C-6''), 128.5 (CH, C-3''), 128.5 (CH, C-5''), 64.2 (CH, C-1), 63.4 (CH, C-5), 60.2 (C, C-7), 41.2 (CH, C-4), 40.1 (CH, C-11), 39.0 (CH2, C-2), 38.5 (CH2, C-2'''), 36.5 (CH2, C-9), 29.6 (C, C-10), 26.8 (CH2, C-8), 15.6 (CH3, C), 13.4 (CH3, C), 13.2 (CH3, C); EI MS m/z (% rel. abund.): 347 (12.4), 288 (11.6), 191 (60.9), 177 (34.3), 44 (100.0); Anal.

Calcd for C24H26N2O4S, C = 65.73, H = 5.98, N= 6.39; Found: C = 65.75, H = 6.00, N= 6.41; IR (KBr, cm-1): 1665 (C=O), 1659 (C=O), 1640 (C=N), 1655 (C=C), 1450 (C-H), 1371 (C-H), 1323 (C-O), 737 (C- H);

(1R,5S)-8-[(1S)-1-(5-{[2-(4-Bromophenyl)-2- oxoethyl]sulfanyl}-4H-pyrazol-3-yl)ethyl]-1,5- dimethyltricyclo[4.4.0.02,8]decane-4,7-dione (18)

Yield: 34%; 1H-NMR (500 MHz, DMSOd6): 7.92-7.56 (m, 4H, H-2', 3', 5', 6'), 5.12 (s, 2H, -SCH2), 2.77-2.73 (m, 1H, CH-11), 2.55 (br.s, 2H, CH2-2), 2.47-2.43 (m, 1H, CH-5), 2.37-2.33 (m, 1H, CH-4), 2.12-2.07 (m, 1H, CH-1), 1.95 (br.s, 1H, CH2-8a), 1.55 (br.s, 1H, CH2-8b), 1.42-1.37 (m, 2H, CH2-9), 1.22 (s, 3H, CH3-14), 1.15 (d, 3H, CH313), 0.99 (d, 3H, CH3-15); 13C NMR (75 MHz, DMSO- d6): 189.1 (C=O, C-1'''), 170.4 (C=O, C-6), 167.9 (C, C-5'), 165.3 (C=O, C-3), 155.2 (C, C-2'), 134.6 (C, C-1''), 131.4 (CH, C-3''), 131.4 (CH, C-5''), 129.6 (CH, C-2''), 129.6 (CH, C-6''), 127.3 (C, C-4''), 64.2 (CH, C-1), 63.3 (CH, C-5), 60.0 (C, C-7), 41.2 (CH, C-4), 40.1 (CH, C-11), 39.3 (CH2, C-2), 38.4 (CH2, C-2'''), 36.1 (CH2, C-9), 29.6 (C, C-10), 26.5 (CH2, C-8), 15.9 (CH3, C), 13.4 (CH3, C), 13.1 (CH3, C); EI MS m/z (% rel. abund.): 459 (26.8), 276 (88.2), 275(100), 183 (64.7); Anal.

Calcd for C24H25N2O4BrS, C = 55.71, H = 4.87, N = 5.41; Found: C = 55.73, H = 4.89, N= 5.43; IR (KBr, cm-1): 1672 (C=O), 1660 (C=O),1647 (C=N), 1658 (C=C), 1455 (C-H), 1378 (C-H), 1328 (C-O), 727 (C-H);

Results and Discussion

Chemistry

Santoninic acid 2 was synthesized via basic hydrolysis of a-santonin as per literature protocol [22]. Santoninic acid is quite unstable and relactonized back to santonin due to its kinetic nature. Santoninic acid was detected between the reaction first and second step by monitoring the TLC and characterized after a mini workup as described in the experimental procedure, however, on long it was rearranges in situ into the more stable, thermodynamic product santonic acid 3 which is a isomer of santoninic acid. Both, the isomers were synthesized and their structures were elucidated using all available spectroscopic techniques.

Furthermore, santonic acid was treated with 1,1-carbonyl diimidazole (CDI) in order to convert into its acyl imidazole derivative 4. CDI is very reactive, have high affinity towards the carboxylic functional group and reacts immediately with the COOH functional group followed by the removal of carbon dioxide and one imidazole ring and converted into its acyl imidazole derivative 4. The products were fully characterized using different spectroscopic techniques EIMS, NMR, CHN and IR.

Hydrazinolysis of acyl imidazole derivative 4 was carried out to furnish santonic carbohydrazide 5 by treating the acyl imidazole derivative 4 with hydrazine hydrate. Due to good leaving group character, acyl imidazole is much prone for nucleophilic attack of hydrazine hydrate. So hydrazine attacks on electrophilic carbon of carbonyl group followed by the departure of imidazole ring and converted into santonic carbohydrazide 5.

Santonic-1,3,4-oxadiazole-2-thiol 6 was successfully synthesized by cyclization of santonic carbohydrazide 5 with carbon disulphide in the presence NaOH and ethanol as a solvent. Furthermore, 2-mercapto substitution on oxadiazole ring was achieved by treating 6 with the different phenacyl halides 7-12 to afford products 13-18.

Table-1: Structures of 2-Thio substituted derivatives of Santonic-1,3,4-oxadiazole.

###Compound No.###R###(% Yield)

###13###42

###14###45

###15###40

###16###30

###17###38

###18###34

All the six chemical transformation were performed under microwave irradiations at a set temperature, pressure and power control (Temperature = 50 C, Power = 30 Watt and Pressure = 30 psi) with each pulse of 10 sec. The reaction conditions (Temperature = 50 C, Power = 30 Watt and Pressure = 30 psi) was pre-optimized and it was found that increase in temperature, pressure and power leads to the decomposition of the products and decrease in all parameters required longer time for the transformations. The set conditions (Temperature = 50 C, Power = 30 Watt and Pressure = 30 psi) has given the best results. As far as the pulse time concerned, all the reactions was performed in closed vessel by giving each pulse of 10 sec and after each pulse the reaction mixture was allowed to cool for 10 sec. The continuous heating would increase the pressure inside the vessel and can cause the bursting of vessel.

Antimicrobial Activity

Compounds 5, 6, and 13-18 were evaluated to check their potential against Gram-positive as well as Gram-negative bacterial strains by utilize standard disc diffusion method. DMSO was employed as a control [30]. Gram-Positive bacterial strains used in this study include Bacillus subtilis, Corynebacterium xerosis, Corynebacterium diphtheria, Staphylococcus aureus (MRSA), Staphylococcus aureus, Streptococcus faecalis, Staphylococcus epidermidis, Staphylococcus saprophyticus and Streptococcus pyogenes.

Results listed in Table-2 display zone diameter of growth inhibition (mm) that showed all synthetic derivatives demonstrated weak to good potential against Gram-positive bacterial strains. A key role in antibacterial potential is the lipophilicity of the drugs in the sense that cell membranes only allow the channel to the lipid-soluble materials [31, 32]. Therefore, it is worth-mentioning that all the synthetic compounds were much lipophilic mainly due to hydrocarbon skeleton and also they have not the substituent like hydroxyl which decreases the lipophilicity of the compounds.

Compound 14 was found to be the most active compound as it showed significantly good inhibitory activity against Staphylococcus epidermidis and remaining compounds showed moderate to good activity for the same bacterial strains. All the compounds revealed moderate to good inhibitory potential against Bacillus subtilis, Staphylococcus aureus, Staphylococcus saprophyticus and weak to moderate activity against Corynebacterium xerosis, Corynebacterium diphtheria, Staphylococcus aureus (MRSA), Streptococcus pyogenes, and Streptococcus faecalis.

Results for the potential of compounds 5, 6, and 13-18 against Gram-negative bacteria are listed in Table-3, Gram-negative bacterial strains utilized in this study include Escherichia coli, Escherichia coli(MDR), Enterobacter aerogene, Pseudomonas aeruginosa, Salmonella typhi, Salmonella paratyphi A, Salmonella paratyphi B, Shigella flexeneri, Shigella dysenteriae, Vibrio choleriae, and Aeromonas. Two compounds 6 and 17 showed good activities. Compounds 6 gave good activity against Escherichia coli and 17 showed activity against Shigella flexeneri. However, rest of the compounds showed weak to moderate potential against all Gram- negative bacterial strains.

Inhibitory potential of compounds 5, 6, and 13-18 were checked in vitro for their antifungal potential against eight fungal cultures by employing agar plate technique [33]. The linear growth of the all fungus used Aspergillus niger, Aspergillus flavus, Rhizopus sp., Penicillium spp., Candida albican, Candida tropicalis, Mucor and Saccharomyces cerevisiae were acquired by measuring the diameter of the fungal colony after one week. Results are collected in Table-4 as zone diameter of growth inhibition (mm). All the compounds showed weak to moderate activities against Aspergillus flavus, Candida albican, Candida tropicalis. Compound 6 showed good however, compounds 15-18 demonstrated the weak activities against Aspergillus niger. All the compounds are completely inactive aginst Penicillium spp., Rhizopus sp., Mucor, and Saccharomyces cerevisiae.

Table 2: In vitro antibacterial activity against Gram-positive bacteria (Inhibition zones in mm using the disc diffusion method.

###Gram-positive bacteria###Zone Inhibition (mm) of Compounds

###5###6###13###14###15###16###17###18###Gentamicin

###Bacillus subtilis###15###15###15###15###15###14###15###15###22

###Corynebacterium xerosis###11###12###11###-###9###-###9###13###22

###Corynebacterium diphtheriae###15###14###13###13###12###15###14###15###25

###Staphylococcus aureus (MRSA)###10###11###10###10###10###10###10###10###20

###Staphylococcus aureus###14###14###15###15###15###14###14###15###25

###Streptococcus faecalis###10###11###9###11###10###10###11###10###25

###Staphylococcus epidermidis###14###15###16###18###16###16###14###14###28

###Streptococcus pyogenes###11###10###10###12###12###11###11###10###25

###Staphylococcus saprophyticus###15###16###15###15###15###15###12###15###09

Table-3: In vitro antibacterial activity against Gram negative bacteria (Inhibition zones in mm using the disc diffusion method.

###Gram negative bacteria###Zone Inhibition (mm) of Compounds

###5###6###13###14###15###16###17###18###Gentamicin

###Escherichia coli###12###16###14###12###15###12###12###12###29

###Escherichia coli(MDR)###10###14###12###12###14###11###10###10###20

###Enterobacter aerogene###11###12###12###13###12###11###11###11###22

###Pseudomonas aeruginosa###10###12###11###13###12###12###11###11###22

###Salmonella typhi###13###14###15###15###15###15###14###15###25

###Salmonella paratyphi A###14###14###14###14###14###14###14###14###25

###Salmonella paratyphi B###11###11###11###11###9###11###11###11###25

###Shigella flexeneri###13###14###12###14###12###12###16###12###28

###Shigella dysenteriae###11###14###13###12###12###13###12###11###23

###Vibrio choleriae###12###15###14###15###14###13###13###12###25

###Aeromonas###10###14###12###12###14###11###10###10###27

Table-4: In vitro antifungal activity (Inhibition zones in mm using the disc diffusion method.

###Zone Inhibition (mm) of Compounds

###Names of Fungus###5###6###13###14###15###16###17###18###Ketoconazole

###Aspergillus niger###-###14###-###-###8###8###11###11###24

###Aspergillus flavus###9###14###9###11###10###12###12###11###24

###Rhizopus sp.###-###-###-###-###-###-###-###-###22

###Penicillium spp.###-###-###-###-###-###-###-###-###22

###Candida albican###10###12###12###10###12###9###8###10###22

###Candida tropicalis###9###10###10###11###10###9###11###9###22

###Mucor###-###-###-###-###-###-###-###-###24

###Saccharomyces cerevisiae###-###-###-###-###-###-###-###-###22mm

Conclusions

Novel 2-thio substituted 1,3,4-oxadiazole derivatives of santonic acid (13-18) were synthesized in six steps in microwave irradiation under controlled parameters. Eight newly synthesized compounds (5, 6, 13, 14, 15, 16, 17, and 18) were evaluated for their antimicrobial inhibitory potential which showed weak to significantly good activity.

Acknowledgment

The authors are thankful to Pakistan Academy of Sciences, Pakistan, Project No. 59/PAS/1335.

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