Various Routes to Synthesise 3-Thioxo-1,2,4-Triazine-5-One Derivatives as Antimicrobial Agents

Reda M. Abdel-Rahman, Wafa A. Bawazir^*

Department of Chemistry, Faculty of Science, KAU, Jeddah, Saudi Arabia.

DOI: 10.4236/ijoc.2018.82014                

Abstract

A simple condensation afforded some new 3-thioxo-1,2,4-triazin-5-one derivatives (4, 6 and 8). Utilizing a facile condensation of (E)-4-(4’-bromo styryl)-2-oxo-3-buteneoic acid with thiosemicarbazide, dithioic formic acid hydrazide, and thiocarbahydrazide in different conditions. Structures of these compounds were confirmed by elemental and spectral analysis. The preliminary biocidal activity of these products were evaluated against some microbial and compared to Mycostatine and piperacillin as antibiotics were most of derivatives exhibited good activity.

Keywords

Synthesis, Antimicrobial, 3-Thioxo-1, 2, 4-Triazin-5-Ones

1. Introduction

Nitrogen containing heterocyclic compounds have a huge interests by synthetic chemists, due to their various pharmacological activities [1] [2] . Triazine derivatives expose a wide pharmaceutical activity such as antiviral [3] [4] , antiproliferative and antitumor [5] . Moreover, their anti-HIV [6] [7] , anti-cancer [8] , anti-microbial [9] , antileukemic and anti-convulsant activity were in vitro supported [10] [11] .

For the significant impact of 1,2,4-triazine moieties at the synthesis area of biologically active compounds 3-thioxo-1,2,4-triazin-5-one derivatives were targeted. It has a great attention mainly due to their medicinal properties, as well as biocidal molluscicidal agent against some snails [12] . In addition, most of functionally 1,2,4-triazones form stable metal-complexes with metal ions [13] . Also, the reactivity of 3-thioxo-1,2,4-triazin-5-ones depends onto the active groups, type of activation nature of substitution, leaving moities, character of nucleophile engaged, site selectivity and polarity of solvent used [14] [15] [16] .

Based upon these observations, the present work tends to use various routes to synthesise 6-substituted-3-thioxo-1,2,4-triazin-5-one derivatives as Sencore analogue (Figure 1) [14] , and evaluate their antimicrobial activity in compare with Mycostatine and pipiricillin as standard antibiotic.

2. Results and Discussion

2.1. Chemistry

In order to synthesise targeted compounds, (E)-4-(4’-Bromophenyl)-2-oxo-3-buteneoic acid (1) used as starting material, which was prepared via treatment of 4-bromobenzaldehyde with sodium pyruvate in aq NaOH then ice bath [17] . Also dithioic formic hydrazide (2) synthesized by addition of CS₂ to hydrazine hydrate in dioxan at room temperature with continuous stirring (m.p 130˚C) [18] . Thiocarbohydrazide produced from reflux CS₂ with hydrazine hydrate in aqueous ethanol (m.p 170˚C) [19] .

Condensation of compound 1 with thiosemicarbazide by reflux in EtOH-NaOAc produced thiosemicarbazone 3 which upon boiling with K₂CO₃-EtOH afforded 6-(4-bromostyryl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)one 4, (Scheme 1).

On the other hand, condensation of compound 1 with dithioic formic acid hydrazide (2) in reflux EtOH-AcOH yielded the hydrazine 5. A successful ring closure reaction of 5 occurred after reflux with sulfanilamide in DMF lead to the direct formation of 4-aryl-6-(4’-bromostyryl) -3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)one (6) (Scheme 1). Formation of 6 might be take place via a nucleophilic attack of amino-group of sulfa to SH of compound 5 followed by heterocyclisation (Scheme 2).

Most of 6-alkyl-4-amino-3-thioxo-1,2,4-triazin-5-ones and their alkylated systems are used as herbicides Sencore [14] . Thus, in hope to obtaining new biocidal compounds having high affects towards microbes, 4-amino-6-(4’-bromostyryl)-3-thioxo-1,2,4-triazin-5-one (8) synthesised via simple condensation of compound 1 with thiocarbohydrazide (1:1 by mole) in warm EtOH-AcOH to give the hydrazine 7 which subjected to ring closure reaction by reflux in absolute EtOH containing a few drops of Conc. HCl (1%, 1 mL) afforded the target 8 (Scheme 1). Both the obtained compounds 4, 6 & 8 have stable tautomeric structural formula, thione $\rightleftarrows$ thiol (C=S $\rightleftarrows$ C-SH).

Figure 1. SENCORE®.

Scheme 1. Formation of 3-thioxo-1,2,4-triazin-5-one derivatives (4, 6 and 8).

UV absorption spectra gives us a good indication about their electronic distributions. λ_max of both compounds 6 & 8 showed higher than that open structures 5 & 7. λ_max of 6 (408), 5 (320), and 8 (425), 7 (420)nm. Also absorption bands of both 6 and 8 are slightly higher which might be due to extended conjugation of hetero-systems. IR spectra of all the obtained acyclic systems 3, 5 & 7 showed significant ν at 3500, 3200, 3100, 1610, 1200 and 750 cm⁻¹ attribute the presence of OH, NH₂, NH, HC=CH, C=S and C-Br fundamental groups, while that of cyclic compounds 4, 6 & 8 reduced a lack of OH groups, which confirm that heterocyclization reaction. ¹HNMR spectrum of compound 4 recorded a resonated signals at δ 13.3, 11.5 for NH & NH of 1,2,4-triazine. In addition δ 8.0 - 7.3 ppm assigned aromatic and CH=CH protons with disappearance of both

Scheme 2. A plausible suggested mechanism via a nucleophilic attack of amino-group of sulfa to SH of compound 5 followed by heterocyclization.

OH and SH protons, while that of compound 8 showed at δ 7.8 - 7.7, 7.6 - 7.5 ppm (each double, 2H, aryl protons), 7.3-7.2 (coupling CH=CH-) 4.3 - 4.1 ppm for CH=CH- and 2.8, 2.3 ppm assigned NH₂(N⁴) & NH(N²).

¹³C spectrum of compound showed a resonated signals at δ 180, 165, 150 & 142 ppm attribute C=S, C=O, HC=CH & C=N of 1,2,4-triazine nucleus, in addition δ at 130 - 120 ppm for aromatic carbons.

Mass fragmentation pattern of functionalized 1,2,4-triazines obtained recorded a lower intensity of that molecular ion with a base peak at 183, featured that 4-bromophenylethylene radical (Figure 2).

A higher stability of compound 8 as Sencore analogues, may be due to a various tautomeric and H-bonding via internal effects (Figure 3).

2.2. The Antimicrobial Evaluation

The newly prepared compounds 3 - 8 have been evaluated as antimicrobial agents against Gram-negative and Gram-positive bacteria as Escherichia Coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Staphylococcus Aureus,

Figure 2. A predicted Mass fragmentation pattern.

Figure 3. The tautomeric effects and H-bonding of 8.

were recovered on Nutrient agar and MacConkey agar. Two fungi, Candida albicans and Aspergillus fumigatus were isolated on Sabouraud Dextrose Agar (Oxide). The in vitro tested use agar diffusion disk method [20] . DMSO used as solvent and the antimicrobial potentialities of the tested compounds were estimated by placing pastoralized filter paper disks (11 mm diameter) impregnated with 50 mg/disk, and showed inhibition zones (IZ) measured after 24 - 28 h incubation at 37˚C for bacteria and after 5 days incubation at 28˚C for fungi. References antibiotic disks (11 mm in diameter) supplied by (BBL), piperacillin (PIP) and fungicide Mycostatine were used for comparison [21] . The results obtained outline in Table 1.

From the results obtained in Table 1. We can conclude that: Generally, the compounds 4, 6 and 8 exhibit a growth inhibition activity more than open derivatives. The compound numbered 8 recorded the highest inhibition activity more than other compounds including the standard antibiotic used. The Structure Activity Relationship (SAR) showed that presence of a sulfonamide, mercapto and amino groups led to enhance the activity. Minimum inhibitory concentration (MIC) for the active synthesised compounds as 4, 6 and 8 displayed a very good antimicrobial activity in compared with the standard antibiotic used.

3. Experimental

The melting point recorded on Stuart scientific SMP3 (Bibby, UK) melting point apparatus and reported as uncorrected. A Perkin Elmer (Lambda EZ-2101) double beam spectrophotometer (190 - 1100 nm) used for recording the electronic spectra. A Perkin Elmer model RXI-FT-IR 55,529 cm⁻¹ used for recording the IR spectra. A Brucker advance DPX 400 MHz using TMS as an internal standard for recording the ¹H NMR ¹³C nmr spectra in deuterated DMSO (δ in ppm). AGC-MS-QP 1000 Ex model used for recording the mass spectra. Elemental analysis performed on Micro Analytical Center of National Reaches Center-Dokki, Cairo, Egypt.

Table 1. The Antimicrobial evaluation of the new compounds 3 - 8.

A: E. coli, B: K. pneumonia, C: P. aeruginosa, D: S. coccus, E: C. albicans, F: A. fumigates, P: Pipericillin, M: Mycostatine.

・ (2Z,3E)-4-(4-bromophenyl)-2-(2-carbamothioylhydrazineylidene)but-3-enoic acid (3)

Thiocarbohydrazide obtained from reflux hydrazine hydrate with CS₂ in aq EtOH for 30 min. then cooled and filtered to give the solid (m.p 170˚C). A mixture of 1 (0.01 mol) in AcOH (50.0 ml) and thiosemicarbazide (0.01 mol) in hot water (10.0 ml) reflux for 1 h, cooled then powered onto ice. The solid obtained was filtered off and crystallized from MeOH to give 3. Yield 80%, m.p 209˚C - 210˚C. IR ν_max cm⁻¹: 3500 - 3300 (OH, NH₂), 3120 (NH), 1700 (C=O), 1600 (CH=CH), 1190 (C-S), 750 (C-Br). ¹HNMR (400 MHz, DMSO-d₆) δ_H ppm: 8 - 7.8 (d, 1H, Ar H), 7.56 (d, 1H, ArH), 7.35 (s, 1H, ArH), 7.25 (s, 1H, ArH), 7.2 - 6.9 (d, d, CH=CH), 5.49 (s, 1H, OH), 4.2 (s, 1H, NHCS), 3.3 (s, 2H, NH₂CS). Analytical data for C₁₁H₁₀N₃SO₂Br (328); Calculated C 40.24; H 3.04; N, 12.80; S 9.75%, Found: C 40.01; H, 3.00; N 12.55; S 9.49%.

・ 6-(4-bromostyryl)-3-thioxo-3,4-dihydro-1,2,4-triazin-5(2H)-one (4)

Compound 3 (1.0 gm) in ethanol (100.0 ml) with K₂CO₃ (5.0 gm) reflux for 4 h, cooled then powered onto ice-HCl. The resultant solid filtered off and recrystallized from ethanol to give 4. Yield 65%, m.p. 190˚C - 192˚C. UV (λ) nm: 375. IR (ν)cm⁻¹: 3180, 3150 (2NH), 1670 (C=O), 1620 (CH=CH-), 1580 (C=N), 1350 (cyclic NCSN), 1180 (C-S). ¹HNMR (400 MHz, DMSO-d₆) δ_H ppm: 13.3, 11.5 (each s, NH, NH), 8.0 - 7.3 (d, 1H, ArH), 7.56 (d, 1H, Ar-H), 7.35 (s, 1H, ArH), 7.2 - 6.9 (d,d, CH=CH), coupling CH=CH), 7.1 - 6.9 (d, d, 4-substituted phenyl), 4.3 - 4.1 (m, CH=CH),. Analytical data, for C₁₁H₈N₃SOBr (310); Calculated C 42.58; H 2.58; N 13.54; S 10.32%, Found: C 42.19; H 2.30; N 13.11; S 10.00%.

・ (2Z,3E)-4-(4-bromophenyl)-2-(2-dithiocarboxyhydrazineylidene)but-3-enoic acid (5)

A mixture of 1 (0.01 mol) and dithioic formic acid hydrazide 2 (0.01 mol) in AcOH-EtOH (1:1, 50 ml) reflux for 1h, cooled then poured onto ice. The resultant solid filtered off and crystallized from EtOH to give 5. Yield 70%, m.p. 193˚C - 195˚C. IR (ν)cm⁻¹: 3480 (OH), 3180 (NH), 1610 (CH=CH-), 1710 (C=O), 1570 (C=N), 1180 (C-S), 710 (C-Br).¹HNMR (400 MHz, DMSO-d₆) δ_H ppm: 7.75 (d, 1H, ArH), 7.40 (d, 1H, ArH), 7.25 (s, 1H, ArH), 7.25 - 6.8 (d, d, CH=CH), 5.78 (s, 1H, SH), 5.41 (s, 1H, OH), 4.1 (s, 1H, NHCS). Analytical data, for C₁₁H₉N₂S₂O₂Br (345); Calculated C 38.26; H 2.60; N 8.11; S 18.55%, Found: C 38.15; H 2.35; N 8.01; S 18.30%.

・ 4-(6-(4-bromostyryl)-5-oxo-3-thioxo-2,5-dihydro-1,2,4-triazin-4(3H)-yl)benzenesulfonamide (6)

Equimolar amounts of 5 and sulfanilamide in DMF (100.0 mL), reflux for 6h, cooled then poured onto ice. The yielded solid filtered off and crystallized from Methanol to give 6. Yield 66%, m.p. 178˚C - 180˚C. UV (λ) nm: 408. IR (ν) cm⁻¹: 3080 (NH), 1690 (C=O), 1610 (CH=CH-), 1350, 1280 (SO₂-NH₂) 1188 (C-S), 910, 830 (p-substituted phenyl). ¹HNMR (400 MHz, DMSO-d₆) δ_H ppm: 11.8 (s, 1H, NH), 8.0 - 7.8 (d, d, p-substituted phenyl), 7.2 - 7.0 (m, 2H, coupling CH=CH) 6.9 - 6.6 (m, 4H, aromatic), 3.4 (s, 2H, NH₂). Analytical data, for C₁₆H₁₃N₄S₂O₃Br (451); Calculated. C 43.77; H 3.00; N 12.01; S 13.73%, Found: C 43.55; H 2.88; N 11.70; S 13.51%.

・ (2Z,3E)-4-(4-bromophenyl)-2-(2-(hydrazinecarbonothioyl)hydrazineylidene)but-3-enoic acid (7)

A mixture of 1 (0.01 mol) and thiocarbohydrazide (0.01 mol) in AcOH-EtOH (1:1, 100 mL) reflux for h, cooled then poured onto ice. The solid that obtained filtered off and crystallised from EtOH to give 7. Yield 65%, m.p. 155˚C - 157˚C. UV (λ) nm: 420. IR (ν) cm⁻¹: 3500 - 3400 (OH, NH₂), 3300, 3180 (NH, NH), 1700 (C=O), 1610(CH=CH-), 1190 (C=S), 880 (p-substituted phenyl) 710 (C-Br). ¹HNMR (400 MHz, DMSO-d₆) δ_H ppm: 7.81 (d, 1H, ArH), 7.55 (d, 1H, ArH), 7.35 (s, 1H, ArH), 7.2 - 6.9 (d,d, CH=CH), 4.11 (s, 1H, NHCS), 3.8 (s,1H, NH-NH₂), 3.30 (s, 2H, NH₂). Analytical data, for C₁₁H₉N₄SOBr (325); Calculated C 40.61; H 2.76; N 17.23; S 9.84%, Found: C 38.02; H 3.01; N 16.12; S 9.25%.

・ 4-Amino-6-(4-bromostyryl)-3-thioxo-3,4-dihydro-1,2,4-triazine-5(2H)-one (8)

Compound 7 (1.0 gm) in aq. HCl (1%, 100 mL), ethanol (100.0 mL), reflux for 2 h, cooled then neutralized with NaHCO₃. The produced solid filtered off and crystallized from MeOH, to give 8. Yield 50%, m.p. 176˚C - 178˚C. UV (λ) nm: 425. IR (ν) cm⁻¹: 3300 - 3180 (NH₂, NH), 1700 (C=O), 1640 (deformation NH₂), 1610 (HC=CH), 1480 (CH=CH), 1188 (C=S), 890 (p-substituted phenyl) 720 (C-Br).). ¹HNMR (400 MHz, DMSO-d₆) δ_H ppm: 12.0 (s, 1H, NH), 7.8 - 7.7, 7.6 - 7.5 (m, 2H, aromatic protons), 7.3 - 7.2 (coupling -CH=CH-), 4.3 - 4.1 (m, -CH=CH-), 2.88 (s, 2H, NH₂). ¹³C nmr spectrum (DMSO-d₆) δ_C ppm: 180 (C=S), 165 (C=O), 150 (C=C), 142 (C=N), 130 - 120 (aromatic carbons). Analytical data, for C₁₁H₉N₄SOBr (325); Calculated C 40.61; H 2.76; N 17.23; S 9.84%, Found: C 40.15; H 2.55; N 17.01; S 9.59%,

4. Conclusion

Various routes to synthetic polyfunctional 1,2,4-triazine derivatives have been used (E)4-aryl-2-oxo-3-buteneoic acid (1), dithioic formic acid hydrazide, thiosemicarbazide and thiocarbohydrazide in different media. Most of these targets exhibit a very good antibacterial and antifungal activity in compared with the reference antibiotics used.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Eicher, T., Hauptmann, S. and Speicher, A. (2013) The Chemistry of Heterocycles: Structures, Reactions, Synthesis, and Applications. John Wiley & Sons.
[2]	Gautam, N., Ajmera, N., Gupta, S. and Gautam, D.C. (2012) Synthesis, Spectral Characterization and Biological Evaluation of 4H-1,4-Benzothiazines, Their Sulfones and Ribofuranosides. European Journal of Chemistry, 3, 106-111. https://doi.org/10.5155/eurjchem.3.1.106-111.489
[3]	Pandey, V.K., Yadav, S., Joshi, M.N. and Bajpai, S.K. (2000) Antiviral Activity of s-Triazines. Biological Memoirs, 26, 48-51.
[4]	Bishnoi, A. and Saxena, R. (2001) Synthesis of Hexahydro-1, 3, 5-Tri (8-Aryl-Aminomethyl-7-Hydroxy-4-Methyl-Quinolinyl)-s-Triazines as Possible Antiviral Agents. Indian Journal of Heterocyclic Chemistry, 11, 47-50.
[5]	Krauth, F., Dahse, H.M., Rüttinger, H.H. and Frohberg, P. (2010) Synthesis and Characterization of Novel 1,2,4-Triazine Derivatives with Antiproliferative Activity. Bioorganic & Medicinal Chemistry, 18, 1816-1821. https://doi.org/10.1016/j.bmc.2010.01.053
[6]	Abdel-Rahman, R.M., Makki, M.S.T. and Al-Romaizan, A.N. (2014) Synthesis of Novel Fluorine Substituted Isolated and Fused Heterobicyclic Nitrogen 1, 2, 4-Triazin-5-One Moiety as Potential Inhibitor towards HIV-1 Activity. 247-268.
[7]	Abdel-Rahman, R.M. (2001) Role of Uncondensed 1, 2, 4-Triazine Compounds and Related Heterobicyclic Systems as Therapeutic Agents—A Review. Pharmazie, 56, 18.
[8]	Makki, M.S.I., Abdel-Rahman, R.M. and Khan, K.A. (2014) Fluorine Substituted 1, 2, 4-Triazinones as Potential Anti-HIV-1 and CDK2 Inhibitors. Journal of Chemistry. https://doi.org/10.1155/2014/430573
[9]	Makki, M.S.T., Rahman, R.M.A. and Ali, O.A.A. (2016) Synthetic Approach for Novel Fluorine Substituted Thiadiazaphosphole Fused 1, 2, 4-Triazinopyridiazine Moieties as Bactericidal Agents. Asian Journal of Chemistry, 28, 917. https://doi.org/10.14233/ajchem.2016.19555
[10]	Kumar, A., Mukerjee, S.K. and Bhattacharya, S.K. (1983) Synthesis of N3-4-Substituted-aryl-N1-(alkyl/aryl-Substituted Aryl) Triazene-N1-Oxides as Potential Anticonvulsant Agents. Pharmazie, 38, 66.
[11]	Katsoulas, A., Rachid, Z., Brahimi, F., McNamee, J. and Jean-Claude, B.J. (2005) Engineering 3-Alkyltriazenes to Block bcr-abl Kinase: A Novel Strategy for the Therapy of Advanced bcr-abl Expressing Leukemias. Leukemia Research, 29, 693-700. https://doi.org/10.1016/j.leukres.2004.11.012
[12]	TawfekMakki, M.S., Rahman, R.A. and AbuAli, O.A. (2015) Synthesis of Some More New Fluorinated 1, 2, 4-triazino [3, 4-b][1, 3, 4] Thiadiazolonesand Their Molluscicidal Against Selective Snails-Part I. Journal of Chemistry, 9, 162-175.
[13]	Asiri, A.M., Baghlaf, A.O., Abdel-Rahman, R.M., Khan, S.A. and Ishaq, M. (2013) Synthesis and Spectroscopic Studies of Ru (II) Complexes of 1, 2, 4-Triazoles, 1, 2, 4-Triazines and Pyrimidine Derivatives. Asian Journal of Chemistry, 25, 7779. https://doi.org/10.14233/ajchem.2013.14604
[14]	Abdel-Rahman, R.M. (2000) Chemistry of Uncondensed 1,2,4-Triazines: Part Ii-Sulfur Containing 5-Oxo-1,2,4-Triazin-3-Yl Moiety: An Overview. Phosphorus, Sulfur, and Silicon and the Related Elements, 166, 315-357. https://doi.org/10.1080/10426500008076552
[15]	Cascioferro, S., Parrino, B., Spanò, V., Carbone, A., Montalbano, A., Barraja, P., Diana, P. and Cirrincione, G. (2017) An Overview on the Recent Developments of 1,2,4-Triazine Derivatives as Anticancer Compounds. European Journal of Medicinal Chemistry, 142, 328-375. https://doi.org/10.1016/j.ejmech.2017.08.009
[16]	Abdel-Rahman, M., R., ST Makki, M., E Ali, T., and A Ibrahim, M. (2013) 1,2,4-Triazine Chemistry Part III: Synthetic Strategies to Functionalized Bridgehead Nitrogen Heteroannulated 1,2,4-Triazine Systems and Their Regiospecific and Pharmacological Properties. Current Organic Chemistry, 10, 136-160.
[17]	Zaher, H.A., Jahine, H., Sherif, O. and Mohammady, R. (1979) Uncondensed 1,2,4-Triazines. 1. Behavior of 3-Hydrazino-5,6-Diphenyl-1,2,4-Triazine towards Acylating Agents, Activated Alkenes and Carbonyl-Compounds. Indian Journal of Chemistry—Section B, 18, 316-319.
[18]	Makki, M.S.T., Al-Romaizan, A.N. and Abdel-Rahman, R.M. (2010) Synthesis of Some More New Sulfur Compounds Bearing Heterocyclic Systems as Biocidal Agents-Part Ⅰ: Synthetic of 2-Thioxo Thiadiazinone Derivatives. Journal of Chemistry and Chemical Engineering, 4, 41-49.
[19]	Zamani, H.A., Rajabzadeh, G., Ganjali, M.R. and Norouzi, P. (2007) Determination of Gadolinium (III) Ions in Soil and Sediment Samples by a Novel Gadolinium Membrane Sensor Based on 6-methyl-4-{[1-(2-thienyl) methylidene] amino} 3-thioxo-3,4-dihydro-1,2,4-triazin-5-(2H)-one. Analytica Chimica Acta, 598, 51-57. https://doi.org/10.1016/j.aca.2007.07.028
[20]	Barry, A.L. (1980) Susceptibility Testing: Diffusion Test Procedures, Manual of Clinical Microbiology. American Society for Microbiology, Washington DC.
[21]	Weinstein, M.J. and Nagman, G.H. (1978) Antibiotics. Elsevier Sci. Publ. Company, Amsterdam, 464.