Synthesis and antimicrobial screening of novel 1,3-dioxolanes linked to N-5 of 5H-1,2,4-triazino[5,6-b]indole-3-thiol

1-[(2,2-Dimethyl-1,3-dioxolan-4-yl)methyl]-1H-indole-2,3-dione (10). Yield: 69 %; red syrup; Anal. Calcd for C14H15NO4 (FW: 262.27): C, 64.36; H, 5.79; N, 5.36 %. Found: C, 64.25; H, 5.59; N, 5.33 %; IR (KBr, cm–1): 2996.67, 1760.87, 1710.99, 1614.64, 1467.38, 1446.22, 1384.49, 1270.17, 1208.96, 1052.74; 1H-NMR (200 MHz, CDCl3, δ / ppm): 1.26, 1.31 (6H, 2s, 2 × CH3), 3.74–3.85 (3H, m, N–CH2 & H-3′a), 4.04 (1H, dd, J1 = 6.4 Hz, J2 = 8.8 Hz, H-3′b), 4.36–4.42 (1H, m, H-2′), 7.03–7.10 (2H, m, Ar-H), 7.51–7.59 (2H, m, Ar-H). 2-[1,2-Dihydro-{(2,2-dimethyl-1,3-dioxolan-4-yl)methyl}-2-oxo-3H-indol-3-ylidene]hydrazinecarbothioamide (17). Yield: 84 %; orange crystals; m.p.: 168–170 °C; IR (KBr, cm–1): 3359.77, 3245.97, 3151.47, 2968.24, 1691.46, 1610.45, 1473.51, 1361.65, 1228.57, 1066.56; 1H-NMR (300 MHz, DMSO-d6, δ / ppm): 1.21 & 1.27 (6H, 2s, 2 × CH3), 3.74 (1H, dd, J1 = 5.6 Hz, J2 = 8.5 Hz, H-3′a), 3.87 (2H, d, J = 5.5 Hz, N–CH2), 4.04 (1H, dd, J1 = 6.4 Hz, J2 = 8.5 Hz, H-3′b), 4.33–4.39 (1H, m, H-2′), 7.14 (1H, t, J = 7.5 Hz, Ar-H), 7.24 (1H, d, J = 7.9 Hz, Ar-H), 7.41 (1H, t, J = 7.6 Hz, Ar-H), 7.70 (1H, d, J = 7.6 Hz, Ar-H), 8.73 (1H, s, D2O-exchangeable, NHc), 9.08 (1H, s, D2O-exchangeable, NHb), 12.35 (1H, s, D2O-exchangeable, NHa); 13C-NMR (125 MHz, DMSO-d6, δ / ppm): 25.21 (CH3), 26.52 (CH3), 42.31 (N–CH2), 66.46 (CH2–O), 72.93 (CH–O), 108.92, 110.76, 119.25, 120.60, 122.91, 130.89, 131.00, 143.18 (CMe2 & Ar-C), 161.06 (C=O), 178.66 (C=S); MS (70 eV, m/z (%)): 334.1 (M+, 16.83), 221.0 (37.84), 205.0 (53.62), 188.0 (42.23), 145.0 (15.67), 144.0 (22.40), 131.1 (28.37), 130.0 (28.33), 118.0 (16.80), 117.0 (46.27), 116.0 (26.50), 101.0 (42.68), 89.0 (24.34), 77.1 (21.28), 76.1 (21.04), 60.0 (100.00); FABMS (m/z): Calcd. for [C15H18N4O3S]+: 334.1100. Found: 334.1041.


General
The starting chemicals and reagents used in this study were purchased from Sigma-Aldrich.Melting points were determined with a Mel-Temp apparatus and are uncorrected.Thin--layer chromatography (TLC) was performed on Baker-Flex silica gel 1B-F plates using ethyl acetate/petroleum ether (4:1 volume ratio, b.p: 60-80 °C) as eluent and the compounds were detected by UV light absorption.FT-IR spectra were measured on a Shimadzu 8400 S spectrometer. 1 H-NMR spectra were recorded on a Bruker Avance AV 300 spectrometer (300 MHz) and Varian Gemini spectrometer (200 MHz). 13C-NMR spectra were measured on a JEOL ECA-500 spectrometer at 125 MHz.The chemical shifts (δ) are given in parts per million (ppm) relative to TMS as an internal standard.The coupling constant values (J) are reported in Hz.Mass spectrometry was realized using electron ionization (EI) on a Finnigan MAT 312 spectrometer and fast atom bombardment (FAB) on a Karatos MS 50 spectrometer.Microanalysis was performed on a Vario elementar EL III analyzer.
Analytical and spectral data of the synthesized compounds are given in Supplementary material to this paper.
The antimicrobial tests were performed in the Pharmaceutical Microbiology Department, Faculty of Pharmacy, Alexandria University.Column chromatography was performed using silica gel (200-400 mesh, Merck) and anhydrous sodium sulfate was employed as a drying agent.
2 (17).To a solution of compound 10 (1.30 g, 5 mmol) in water (20 mL) was added a solution of hydrazinecarbothioamide (0.455 g, 5 mmol) in water (5 mL) and two drops of glacial acetic acid.The reaction mixture was heated under reflux for 2 h.The product that separated out on cooling was filtered and recrystallized from ethanol.Yield: 84 %.
Synthesis of 1,3-dioxolane analogues 20 and 21.Method a: compound 19 (1.58 g, 5 mmol) in a solution of sodium hydroxide (0.20 g, 5 mmol) in water (10 mL) was stirred for 10 min until dissolution.The required allyl/benzyl halide (5 mmol) was then added dropwise and stirring was continued for a further 30 min.The separated solid was filtered, washed with water and recrystallized from ethanol.Method b: a stirred solution of compounds 23 or 24 (5  mmol) in dry DMF (10 mL) was treated with NaH (0.28 g, 5 mmol, 60 % in mineral oil) in a 50 mL closed Erlenmeyer flask and compound 15 (1.43 g, 5 mmol) was added under stirring for 24 h.The reaction mixture was processed as before to give products that were found to be identical with those obtained from method a.

Antimicrobial testing
The synthesized 1,3-dioxolanes were screened for their antibacterial activity against Gram-positive bacteria Staphylococcus aureus (ATCC 6538P) and Gram-negative bacteria Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 9027) and their antifungal activity against Candida albicans (ATCC 2091) by the agar well-diffusion technique, 20 using a 1 mg per 1 mL solution in ethanol.Each tested organism was cultured in 3 mL of sterile nutrient broth and incubated for 18 h at 37 °C.Aseptically, 0.4 mL was taken with a glass pipette from resultant microbial growth and transferred into 40 mL of warm agar in one sterile flask for each organism.The seeded agar was poured into sterile Petri dishes (≈15 cm in diameter) onto a level surface to obtain a layer of about 4 mm thickness and the plates were then left to solidify.Cups of 8 mm in diameter were made using a cork borer, the sample size for all the compounds was fixed at 65 µL and the plates were then incubated at 37 °C for 24 h.The diameter of each resultant growth inhibition zone (IZ) in mm was accurately measured in three different directions and its mean was calculated.The minimal inhibitory concentration (MIC) was evaluated in μg mL -1 by a broth dilution method. 21Clotrimazole (0.01 g mL -1 ) and imipenem (10 mcg per disc) were used as standard drugs.

RESULTS AND DISCUSSION
The reactions required for the synthesis of the target compounds are outlined in Schemes 1 and 2. Thus, (R)-( 2  (17) in 84 % yield.The IR spectrum of 17 showed bands at 3359.77, 3245.97 and 3151.47 cm -1 , characteristics for the NH and NH 2 groups, and two bands at 1691.46 and 1228.57cm -1 due to C=O and C=S stretching vibrations, respectively.No SH band was observed at 2500-2600 cm -1 , the range in which the SH stretching vibrations are most likely to appear.This clearly shows that no thione-thiol tautomerism occurred in this compound in the solid state.Its 1 H-NMR spectrum in DMSO-d 6 showed also the presence of three downfield singlet peaks at δ 12.35, 9.08, and 8.73 ppm corresponding to NH a , NH b and NH c , respectively (Scheme 2).The configuration about imine linkage (C=N) in 17 was assigned as Z due to the existence of intramolecular hydrogen bonding between the ketonic oxygen at C-2 of the indole ring and H a of thiosemicarbazone moiety. 24 green synthesis of the cyclized compound 5-[(2,2-dimethyl-1,3-dioxolan--4-yl)methyl]-2,5-dihydro-3H-1,2,4]-triazino[5,6-b]indole-3-thione (18) was achieved by the dehydrative cyclization of 17 in aqueous potassium carbonate.The reaction was completed in 7 h at reflux and the yield reached 75 %.However, it was obtained in one pot by refluxing 10 with hydrazinecarbothioamide in aqueous potassium carbonate for 10 h, but the yield was improved to 82 %.The 1 H-NMR spectrum of compound 18 in DMSO-d 6 showed a D 2 O-exchangeable singlet at δ 14.62 ppm due to SH proton, evidencing its existence in solution in the thiol form 19 in which the aromatic ring character (14-π electrons) is preserved.The ethylenic proton geminal to the terminal methyl group resonated as a multiplet at δ 5.94-6.14ppm, while two doublet peaks for the ethylenic proton neighbors to sulfur appeared at δ 6.84 ppm (J = 15.0Hz, E-isomer) and 7.08 ppm (J = 9.6 Hz, Z-isomer) with an E:Z ratio of 2:3, respectively.An allylic rearrangement of compound 20 was not detected in deuterochloroform (Supplementary material).

Antimicrobial results
The results of the antimicrobial studies presented in Table I revealed that the synthesized compounds showed moderate antimicrobial activity against the fungus C. albicans with growth inhibition zones (IZ) in the range of 12-15 mm relative to clotrimazole as antifungal medication (IZ = 17 mm).However, they were inactive against the Gram-positive bacteria S. aureus and Gram-negative strains E. coli and P. aeruginosa, except 5-[(2,2-dimethyl-1,3-dioxolan-4-yl)methyl]-________________________________________________________________________________________________________________________ (CC) 2019 SCS.

TABLE I .
In vitro antimicrobial activity of the synthesized compounds against C. albicans; IZ: mean growth inhibition zones; errors: ±5 %; MIC: minimal inhibitory concentration; inhibition level, % = 100(IZ of compound/IZ of reference drug)