In vitro antibacterial, antifungal and cytotoxic activities of some triazole Schiff bases and their oxovanadium(IV) complexes

Abstract

The condensation reaction of 3,5-diamino-1,2,4-triazole with methoxy-, chloro-, bromo-, iodo- and nitro-substituted 2-hydroxybenzaldehydes formed triazole Schiff bases (L¹)–(L⁶). The synthesized ligands have been characterized through physical, spectral and analytical data. Furthermore, the reaction of synthesized Schiff bases with the oxovanadium(IV) sulphate in (1:2) (metal:ligand) molar ratio afforded the oxovanadium(IV) complexes (1)–(6). All the complexes were non-electrolytic and showed a square-pyramidal geometry. The synthesized compounds have been screened for in-vitro antibacterial, antifungal and brine shrimp bioassay. The bioactivity data showed the complexes to be more active than the original Schiff bases.

Keywords::

• Triazole Schiff bases
• oxovanadium(IV) complexes
• antibacterial
• antifungal
• cytotoxic activities

Introduction

Triazole derivatives have attracted a wide spread attention¹ due to their diverse coordination behaviour and biological activities. 1,2,4-Triazoles have been known for a potential use in a wide variety of therapeutically interesting drugs including CNS stimulant, anti-anxiety² and also antimycotic compounds such as fluconazole, intraconazole and voriconazole³. In view of the significant therapeutic uses, the chemistry of 1,2,4-triazoles represents an important class of compounds owing to their synthetic and effective biological properties like antibacterial⁴, antifungal⁵, anticonvulsant⁶, antiproliferative⁷, antitumor⁸, antitubercular⁹, anticancer¹⁰, anti HIV¹¹ and antiviral¹².

Vanadium chemistry has established its strong role as antimicrobial¹³, anti-tumor¹⁴, anti-leukemic¹⁵, spermicidal¹⁶, anti-amoebic¹⁷, antioxidant¹⁸ and osteogenic¹⁹ activity in bioinorganic chemistry. Oxovanadium(IV) complexes are known as potential inhibitors of various enzymes. Recent advances in catalytic and medicinal properties of vanadium complexes have motivated their designing, synthesis and potential bioactive use in drug chemistry. Another important thrust of vanadium chemistry in the context of medicinal application has arisen from their ability to promote the insulin mimetic²⁰ activity in patho-physiological state of diabetes mellitus in humans. This biological and catalytic relevance of vanadium has promoted the synthesis of model vanadium compounds containing O, N donor ligands.

In view of all the above facts and as a continuation of our research work on the synthesis and biological properties of triazole derived Schiff base ligands, we wish to report a series of oxovanadium(IV) complexes with newly synthesized biologically active ligands prepared by the condensation reaction of 3,5-diamino-1,2,4-triazole with methoxy-, chloro-, bromo-, iodo- and nitro-substituted 2-hydroxybenzaldehydes. These triazole ligands and their oxovanadium(IV) complexes have been subjected to in vitro antibacterial activity against bacterial strains such as, Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Bacillus subtilis and for antifungal activity against fungal strains, Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata. These compounds were also studied for in vitro cytotoxic activity. The reported series of compounds could be an appreciable extension in the chemistry of vanadium as vanadium based new class of antibacterial and antifungal agents.

Experimental

All the chemicals used were of analytical grade. 1,2,4-Triazole was purchased from Sigma Aldrich. Fisher Johns melting point apparatus was used for recording melting points. IR spectra were recorded on SHIMADZU FT-IR spectrophotometer. Elemental analysis was carried out on Perkin Elmer (USA model). ¹H and ¹³C NMR spectra were recorded on a Bruker Spectrospin Avance DPX-400 spectrometer using TMS as internal standard and d₆-DMSO as a solvent. Electron impact mass spectra (EIMS) were recorded on JEOL MS Route instrument. In vitro antibacterial, antifungal and cytotoxic properties were studied at HEJ Research Institute of Chemistry, International Centre for Chemical Sciences, University of Karachi, Pakistan.

Synthesis of (2-{(E)-[(5-amino-1H-1,2,4-triazol-3-yl)imino]methyl}phenol (L¹)

An equimolar solution of 3,5-diamino-1,2,4-triazole (0.99g, 10 mmol) and 2-hydroxybenzaldehyde (1.07 mL, 10 mmol) in methanol (50 mL) was vigorously stirred for 3 h. A precipitated product was formed during stirring which was cooled to room temperature, filtered, washed with methanol (3 × 5 mL), then with diethyl ether (2 × 5 mL) and dried under vacuum. Crystallization was carried out in a mixture of methanol:dioxane (1:4) to obtain TLC pure product (L¹) in 75% yield. The same method was applied for the preparation of all other ligands (L²)–(L⁶) (Scheme 1).

Scheme 1.  Preparation of triazole Schiff base ligands (L¹)–(L⁶) and their oxovanadium(IV) complexes (1)–(6).

2-{(E)-[(5-Amino-1H-1,2,4-triazol-3-yl)imino]methyl}phenol (L¹)

Yield: 75% (1.52 g); colour (yellow); m.p. 181–183°C; ¹H NMR (DMSO-d₆, 400 MHz): δ 6.01 (s, 2H, NH₂), 6.98 (t, 1H, J = 8.4 Hz, C₄-H), 7.12 (d, 1H, J = 7.8 Hz, C₆-H), 7.41 (t, 1H, J = 7.4 Hz, C₅-H), 7.63 (d, 1H, J = 7.9 Hz, C₃-H), 8.8 (s, 1H, C₇-H), 10.31 (s, 1H, OH), 12.22 (s, 1H, NH); ¹³C NMR (DMSO-d₆): δ 117.67 (C₆), 118.97 (C₂), 121.23 (C₄), 130.35 (C₃), 133.18 (C₅), 158.12 (C₁), 159.75 (C₉), 160.63 (C₇), 163.28 (C₈); IR (KBr, cm⁻¹): 3420 (OH), 3350 (NH₂), 3190 (NH), 1631 (HC=N), 1594 (C=N, triazole), 1025 (N-N); EIMS (70eV) m/z (%): 203 ([M]⁺, 100), 186 (85), 172 (48), 161 (36), 147 (81), 132 (72), 120 (47), 104 (70), 91 (31), 77 (84); Anal. Calcd. for C₉H₉N₅O (203.20): C 53.20, H 4.46, N 34.47; Found C 53.16, H 4.43, N 34.45.

2-{(E)-[(5-Amino-1H-1,2,4-triazol-3-yl)imino]methyl}-3-methoxyphenol (L²)

Yield: 83% (1.93 g); colour (light yellow); m.p. 252–253°C; ¹H NMR (DMSO-d₆, 400 MHz): δ 2.86 (s, 3H, OCH₃), 6.02 (s, 2H, NH₂), 6.98 (dd, 1H, J = 8.8, 8.9 Hz, C₄-H), 7.12 (d, 1H, J = 8.8 Hz, C₅-H), 7.53 (d, 1H, J = 8.9 Hz, C₃-H), 8.78 (s, 1H, C₇-H), 10.22 (s, 1H, OH), 12.18 (s, 1H, NH); ¹³C NMR (DMSO-d₆): δ 55.39 (OCH₃), 119.72 (C₂), 121.86 (C₄), 124.23 (C₅), 129.55 (C₃), 149.95 (C₁), 151.2 (C₆), 156.65 (C₉), 157.19 (C₇), 162.28 (C₈); IR (KBr, cm⁻¹): 3420 (OH), 3346 (NH₂), 3187 (NH), 2910 (OCH₃), 1628 (HC=N), 1595 (C=N, triazole), 1022 (N-N); EIMS (70eV) m/z (%): 233 ([M]⁺, 13), 218 (100), 202 (32), 177 (9), 171 (19), 164 (7), 150 (12), 134 (27), 123 (14), 104 (20), 77 (22); Anal. Calcd. for C₁₀H₁₁N₅O₂ (233.23): C 51.50, H 4.75, N 30.03, O 13.72; Found C 51.47, H 4.72, N, 29.98.

2-{(E)-[(5-Amino-1H-1,2,4-triazol-3-yl)imino]methyl}-5-chlorophenol (L³)

Yield: 73% (1.74 g); colour (light yellow); m.p. 222–224°C; ¹H NMR (DMSO-d₆, 400 MHz): δ 6.04 (s, 2H, NH₂), 6.97 (d, 1H, J = 7.8 Hz, C₆-H), 7.48 (dd, 1H, J = 7.8, 2.4 Hz, C₅-H), 7.84 (d, 1H, J = 2.4 Hz, C₃-H), 8.87 (s, 1H, C₇-H), 10.26 (s, 1H, OH), 12.27 (s, 1H, NH);¹³C NMR (DMSO-d₆): δ 119.05 (C₆), 120.27 (C₂), 125.52 (C₄), 131.72 (C₃), 133.13 (C₅), 156.12 (C₉), 159.63 (C₇), 160.33 (C₁), 163.35 (C₈); IR (KBr, cm⁻¹): 3428 (OH), 3345 (NH₂), 3192 (NH), 1636 (HC=N), 1606 (C=N, triazole), 1032 (N-N), 819 (C-Cl); EIMS (70eV) m/z (%): 237 ([M]⁺, 100), 221 (5), 205 (5), 181 (18), 166 (6), 154 (11), 131 (14), 127 (10), 111 (8), 75 (7); Anal. Calcd. for C₉H₈ClN₅O (237.65): C 45.49, H 3.39, N 29.47; Found C 45.46, H 3.36, N 29.45.

2-{(E)-[(5-Amino-1H-1,2,4-triazol-3-yl)imino]methyl}-5-bromophenol (L⁴)

Yield: 77% (2.17 g); colour (light yellow); m.p. 227–229°C; ¹H NMR (DMSO-d₆, 400 MHz): δ 6.03 (s, 2H, NH₂), 6.97 (d, 1H, J = 8.8 Hz, C₆-H), 7.58 (dd, 1H, J = 8.8, 2.4 Hz, C₅-H), 8.04 (d, 1H, J = 2.4 Hz, C₃-H), 8.83 (s, 1H, C₇-H), 10.24 (s, 1H, OH), 12.25 (s, 1H, NH); ¹³C NMR (DMSO-d₆): δ 116.02 (C₄), 119.78 (C₆), 121.34 (C₂), 133.09 (C₃), 136.53 (C₅), 156.12 (C₉), 159.92 (C₁), 158.78 (C₇), 164.89 (C₈); IR (KBr, cm⁻¹): 3424 (OH), 3343 (NH₂), 3192 (NH), 1634 (HC=N), 1603 (C=N, triazole), 1027 (N-N), 564 (C-Br); EIMS (70eV) m/z (%): 282 ([M]⁺, 100), 266 (13), 250 (7), 225 (20), 212 (8), 199 (26), 171 (5), 157 (9), 103 (14), 76 (11); Anal. Calcd. for C₉H₈BrN₅O (282.09): C 38.32, H 2.86, N 24.83; Found C 38.28, H 2.84, N 24.81.

2-{(E)-[(5-Amino-1H-1,2,4-triazol-3-yl)imino]methyl}-5-iodophenol (L⁵)

Yield: 80% (2.63 g); colour (deep yellow); m.p. 231–232°C; ¹H NMR (DMSO-d₆, 400 MHz): δ 6.03 (s, 2H, NH₂), 6.85 (d, 2H, J = 8.6 Hz, C₆-H), 7.72 (dd, 2H, J = 8.6, 2.2 Hz, C₅-H), 8.19 (d, 1H, J = 2.2 Hz, C₃-H), 8.80 (s, 1H, C₇-H), 10.21 (s, 1H, OH), 12.21 (s, 1H, NH); ¹³C NMR (DMSO-d₆): δ 114.97 (C₄), 118.65 (C₆), 120.76 (C₂), 140.17 (C₃), 142.24 (C₅), 156.12 (C₉), 158.69 (C₇), 159.42 (C₁), 163.26 (C₈); IR (KBr, cm⁻¹): 3426 (OH), 3344 (NH₂), 3188 (NH), 1630 (HC=N), 1599 (C=N, triazole), 1027 (N-N); EIMS (70eV) m/z (%): 329 ([M]⁺, 100), 312 (36), 298 (5), 273 (15), 245 (9), 203 (10), 146 (14), 131 (16), 119 (5), 103 (5), 76 (12); Anal. Calcd. for C₉H₈IN₅O (329.09): C 32.85, H 2.54, N 21.28; Found C 32.81, H 2.51, N 21.25.

2-{(E)-[(5-Amino-1H-1,2,4-triazol-3-yl)imino]methyl}-5-nitrophenol (L⁶)

Yield: 67% (1.66 g); colour (dark yellow); m.p. 242–244°C; ¹H NMR (DMSO-d₆, 400 MHz): δ 6.07 (s, 2H, NH₂), 7.19 (d, 1H, J = 8.7 Hz, C₆-H), 8.24 (dd, 1H, J = 8.7, 2.3 Hz, C₅-H), 8.75 (d, 1H, J = 2.3 Hz, C₃-H), 8.93 (s, 1H, C₇-H), 10.31 (s, 1H, OH), 12.30 (s, 1H, NH); ¹³C NMR (DMSO-d₆): δ 118.17 (C₆), 120.85 (C₂), 126.93 (C₃), 135.62 (C₅), 140.75 (C₄), 156.12 (C₉), 160.16 (C₇), 162.52 (C₁), 166.05 (C₈); IR (KBr, cm⁻¹): 3431 (OH), 3347 (NH₂), 3199 (NH), 1638 (HC=N), 1607 (C=N, triazole), 1350 (NO₂), 1034 (N-N); EIMS (70eV) m/z (%): 248 ([M]⁺, 22), 232 (100), 216 (20), 192 (31), 172 (5), 166 (19), 149 (22), 138 (11), 122 (14), 76 (10); Anal. Calcd. for C₉H₈N₆O₃ (248.19): C 43.55, H 3.25, N 33.86; Found C 43.52, H 3.22, N 33.83.

Synthesis of oxovanadium(IV) complex (1)

To a hot magnetically stirred dioxane (50 mL) solution of 2-{(E)-[(5-amino-1H-1,2,4-triazol-3-yl)imino]methyl}phenol (0.41g, 2 mmol), a methanol solution (30 mL) of VOSO₄.5H₂O (0.163g, 1 mmol) was added. The mixture was refluxed for 3 h during which a precipitated product was formed. It was then cooled to room temperature. The precipitates thus formed were filtered, washed with methanol, dioxane and then with diethyl ether and dried under vacuum. It was crystallized in a mixture of water:dioxane (1:3) to obtain TLC pure complex (1). All other complexes (2)–(6) were prepared following the same method (Scheme 1).

Estimation of vanadium metal

The vanadium content of complex (1) was determined volumetrically by using 0.1N KMnO₄ solution as an oxidizing agent in the presence of dil. H₂SO₄. A 0.1 g sample of the vanadyl complex (1) was placed in a silica crucible, decomposed by gentle heating and then by adding 1–2 mL of conc. HNO₃, two to three times. An orange coloured mass (V₂O₅) was obtained after decomposition and complete drying. It was dissolved in minimum amount of dil. H₂SO₄ and the solution so obtained was diluted with distilled water to 100 mL in a measuring flask. The amount of vanadium in the tested sample was calculated²¹ by using the standard relationship of 1mL of 0.1N KMnO₄ considered as 0.005094 g of vanadium. All other vanadyl complexes (2)–(6) were estimated following the same method.

Pharmacology

The test procedures for in-vitro antibacterial, antifungal, minimum inhibitory concentration (MIC) and cytotoxicity bioassay have already been reported²².

Results and discussion

The triazole Schiff base ligands (L¹)–(L⁶) were prepared by the condensation reaction of 3,5-diamino-1,2,4 triazole with a series of methoxy-, chloro-, bromo-, iodo- and nitro-substituted 2-hydroxybenzaldehydes under stirring (Scheme 1). All the synthesized ligands were air, moisture stable. These were all amorphous solids which melted at 181–252°C. All the ligands were only soluble in dioxane, DMF and DMSO. The oxovanadium(IV) complexes (1–6) of the prepared ligands were synthesized in a molar ratio 1:2 (metal:ligand). All the oxovanadium(IV) complexes were light green to dark green coloured amorphous solids. All the complexes decomposed on heating rather than melting. They were all only soluble in DMSO and DMF. The elemental analysis and solubility data strongly recommended that these complexes were monomers and showed 1:2 stoichiometry of type ML₂ where M is metal and L is ligand. The analytical data given in (Table 1), also agreed well with the proposed structure of the complexes (Scheme 1).

Table 1.  Physical and analytical data of the oxovanadium(IV) complexes.

No.	Structure	MW (g/mole)	M.P (dec.)	Yield (%)	Found (calculated) %
		Formula	(°C)	[Colour]	C	H	N	V
(1)	[VO(L^1-H)2]	[471.33]	229–232	81	45.84	3.41	29.67	10.77
		C18H16N10O3V		[Sea green]	(45.87)	(3.42)	(29.72)	(10.81)
(2)	[VO(L^2-H)2]	[531.38]	252–255	78	45.19	3.76	26.32	9.56
		C20H20N10O5V		[Deep green]	(45.25)	(3.79)	(26.39)	(9.59)
(3)	[VO(L^3-H)2]	[540.22]	251–253	80	39.98	2.58	25.90	9.41
		C18H14Cl2N10O3V		[Light green]	(39.94)	(2.61)	(25.93)	(9.43)
(4)	[VO(L^4-H)2]	[629.12]	243–245	82	34.32	2.23	22.23	8.07
		C18H14Br2N10O3V		[Sea green]	(34.36)	(2.24)	(22.26)	(8.10)
(5)	[VO(L^5-H)2]	[723.12]	236–239	79	29.86	1.93	19.35	7.02
		C18H14I2N10O3V		[Deep green]	(29.90)	(1.95)	(19.37)	(7.04)
(6)	[VO(L^6-H)2]	[561.32]	272–275	72	38.47	2.53	29.90	9.05
		C18H14N12O7V		[Green]	(38.51)	(2.51)	(19.95)	(9.08)

IR spectra

Some of the distinctive IR spectral bands of the triazole ligands and their oxovanadium (IV) complexes are reported in experimental part and (Table 2). The triazole ligands have potentially active donor sites for coordination such as, azomethine-N), hydroxyl-O which have the capability of coordinating with the vanadium metal atom. All the ligands showed bands at 3420–3431, 3343–3350, 3187–3199, 1594–1607 and 1022–1034 cm⁻¹ respectively due to v(OH), v(NH₂), v(N-H), v(C=N) and v(N-N) vibrations. Two bands²³ at 3310 and 3350 cm⁻¹ were observed due to the presence of two amino groups of the original 3,5-diamino-1,2,4-triazole moiety. All triazole ligands showed an absence of one of the amino group band at 3310 cm⁻¹ emerging into a new band of azomethine v(HC=N) linkage²⁴ at 1628–1638 cm⁻¹ giving thus a clue for condensation of one amino group of the triazole moiety; however, another band of amino group at 3350 cm⁻¹ remained unchanged giving a clue of its non-participation in condensation reaction. The ligand (L²) exhibited band at 2910 cm⁻¹ due to v(OCH₃) group. Moreover, the ligands (L³) and (L⁶) showed vibrations at 810 and 1355 cm⁻¹ due to Cl and NO₂ groups, respectively. A weaker band at 564 cm⁻¹ was observed by the ligand (L⁴) which was assigned to v(C-Br) vibration.

Table 2.  Physical and spectral data of oxovanadium(IV) complexes.

No.	ΩM (Ω^−1 cm^2 mol^−1)	B.M (µeff)	λmax (cm^−1)	IR (cm^−1)
(1)	35.9	1.69	13435 (744), 18239 (548), 26226 (381), 37272 (268)	1618 (C=N), 1377 (C-O), 960 (V=O), 525 (V-O), 423 (V-N)
(2)	42.3	1.71	13542 (738), 18723 (534), 26189 (382), 37155 (269)	1616 (C=N), 1376 (C-O), 958 (V=O), 519 (V-O), 425 (V-N)
(3)	39.6	1.77	13536 (738), 18641 (536), 26355 (379), 37346 (268)	1619 (C=N), 1378 (C-O), 967 (V=O), 529 (V-O), 433 (V-N)
(4)	42.9	1.70	13478 (742), 18504 (540), 26312 (378), 37272 (268)	1620 (C=N), 1382 (C-O), 959 (V=O), 523 (V-O),432 (V-N)
(5)	41.3	1.67	13458 (743), 18519 (540), 26378 (379), 37189 (269)	1622 (C=N), 1383 (C-O), 961 (V=O), 523 (V-O), 429 (V-N)
(6)	37.6	1.67	13567 (737), 18728 (534), 26316 (380), 37409 (267)	1626 (C=N), 1386 (C-O), 970 (V=O), 529 (V-O), 438 (V-N)

On comparison of IR spectra of oxovanadium (IV) complexes with the spectra of ligands indicated that the triazole ligands were principally coordinated with the vanadium(IV) metal atom bidentately. The coordination modes of bonding are discussed as under:

• (i) All the ligands showing IR spectral band at 1628–1638 cm⁻¹ due to azomethine, ν(HC=N) shifted²⁵ to lower frequency (12–15 cm⁻¹) at 1616–1626 cm⁻¹ indicating the coordination of the azomethine-N with the vanadium(IV) metal atom.

• (ii) Absence of band at 3420–3431 cm⁻¹ due to ν(OH) and in turn, appearance of a new band at 1376–1386 cm⁻¹ assigned²⁶ to ν(C-O) in the oxovanadium(IV) complexes, confirmed deprotonation and coordination of hydroxyl-O to the oxovanadium(IV) metal.

• (iii) Te appearance of a new lower frequency bands at 519–529 and 423–438 cm⁻¹ were assigned to ν(V-O) and ν(V-N) respectively indicated the coordination of these ligands with the oxovanadium(IV) metal ion through hydroxyl-O and azomethine-N.

• (iv) A new band which was not observed in the spectra of the ligands but appeared in the spectra of complexes at 959–970 cm⁻¹ was assigned to the presence (V=O)²⁷.

• (v) The bands at 3343–3350, 3187–3199, 1594–1607 and 1022–1034 cm⁻¹ respectively due v(NH₂), v(N-H), v(C=N) and v(N-N) vibrations in the spectra of triazole moiety remained unchanged indicating their non-involvement in the coordination.

All the above mentioned evidences supported the coordination of the ligands with the vanadium (IV) metal via the azomethine-N and deprotonation of hyroxyl-OH groups.

¹H NMR spectra

All the ligands (L¹)–(L⁶) displayed²⁸ distinguishing protons of azomethine (CH=N) and NH group at 8.78–8.93 and 12.18–12.3 ppm, respectively as a singlet. Also, distinctive OH proton of all the ligands was observed at 10.20–10.31 ppm as a singlet, which disappeared on exchange with D₂O. The ligand (L¹) showed peaks of protons C₄-H and C₅-H at 6.98 and 7.41 ppm, respectively as a triplet and, C₃-H and C₆-H protons were observed at 7.12 and 7.63 ppm, respectively as a doublet. The methoxy (OCH₃) protons of ligand (L²) were observed at 3.86 ppm as a singlet. The C₅-H proton was observed at 6.98 ppm as a double doublet. Remaining protons, C₄-H and C₆-H appeared at 7.12 and 7.53 ppm, correspondingly as doublet. The ligands, (L³-L⁵) showed C₃-H protons as a doublet at 6.97, 6.97 and 6.85 ppm, and C₄-H protons appeared as double doublet at 7.48, 7.58 and 7.72 ppm, respectively. Due to the inductive effect of azomethine (CH=N) functional group, the C₆-H protons were deshielded at 7.84, 8.04 and 8.19 ppm, as a doublet. However, the ligand (L⁶) exhibited C₃-H proton at 7.19 ppm as a doublet. It was found that due to electron withdrawing effect of the NO₂ group, C₄-H proton appeared as double doublet at 8.24 ppm. All the ligands demonstrated³¹ characteristic amino (NH₂) proton at 6.01–6.07 ppm as a singlet which provided an evidence for condensation of only one amino group of triazole moiety. The conclusions drawn from this study provided further support to the modes of bonding discussed earlier in IR spectra. All the ligands having protons due to heteroaromatic/aromatic groups were found as to be in their expected region. However, the number of protons calculated from the¹H NMR integration curves and those obtained from the values of the expected analytical data (CHN) results confirmed the proposed structures of the Schiff base ligands.

¹³C NMR spectra

The ligands (L¹)–(L⁶) showed the triazole carbons C₈ and C₉ at 156.12–166.05 ppm. In addition, the characteristic azomethine carbons C₇ of the ligands were observed in the region at 157.19–160.63 ppm. The downfield shifting of C₁ was observed at 158.12–160.12 ppm due to the attachment of hydroxyl (OH) group at C₁ position except the ligand (L²) which possessed same peak at 149.95 ppm due to electron donating effect of methoxy-C attached to adjacent carbon at C₆ position showing its peak at 151.2 ppm. Similarly, the carbons C₂ of all the triazole ligands were found in the region at 118.97–121.34 ppm due to the inductive effect of azomethine carbon. However, the peaks for the carbons C₃-C₆ were observed at 114.97–142.26 ppm. Also, the peaks for C₄ carbon of ligands (L³) and (L⁶) appeared downfield at 125.52 and 140.75 ppm, respectively due to the electronegative nature of chloro and nitro groups at this position. The same peaks for C₄ carbon of (L⁴) and (L⁵) were observed upfield at 114.97 and 116.02 ppm respectively, due to lesser electronegative nature of bromo and iodo groups. Moreover, the methoxy-C in (L²) appeared in the region at 55.39 ppm. Furthermore, all these studies indicated that the expected values of carbon atoms compromised well with the number of carbon atoms present in the proposed structures of the compounds.

Conductance and magnetic susceptibility measurements

The molar conductance values of the oxovanadium(IV) complexes (1)–(6) were taken in DMF and showed their conductance values in the range 35.9–42.9 Ω⁻¹ cm² mol⁻¹ (Table 2), which is an indicative of non-electrolytic²⁹ nature. The effective magnetic moments of the oxovanadium(IV) complexes at room temperature were found in the range at 1.70–1.82 B.M. indicative of presence of a single electron consistent with half-spin (S = 1/2) orbital contribution. The oxovanadium(IV) complexes possessed +4 oxidation state for vanadium which is an indicative of square pyramidal geometry. These values were compatible to the reported values for their square-pyramidal geometry³⁰. Moreover, the data suggested that the prepared complexes were all mononuclear.

UV/visible spectra

The UV/visible spectra of all the oxovanadium(IV) complexes in DMF displayed three (Table 2) distinctive low to high intensity bands (v₁, v₂ and v₃) which were assigned to b₂ (d_xy) → e_π(d_xz, d_yz), b₂ (d_xy) → b₁ (d_x² _{− y}²) and b₂ (d_xy) → a₁ (d_z²) transitions, respectively. The first band observed at 13435–13567 cm⁻¹ was assigned to b₂ → e_π d-d transitions. The second band was observed at 18239–18728 cm⁻¹ which can be attributed to b₂→ b₁ and the presence of third band at 26189–26378 cm⁻¹ can be assigned to the transitions b₂→ a₁. The fourth band of high intensity observed at 37155–37409 cm⁻¹ was due to metal → ligand charge transfer (MLCT). All these observations provided a strong evidence for the complexes to show a square-pyramidal geometry³⁰.

Biological screening

Antibacterial bioassay (in vitro)

The results obtained from in-vitro antibacterial studies are summarised in Table 3. The antibacterial studies revealed that all the Schiff base ligands and their oxovanadium(IV) complexes contributed significantly towards enhancing the biological activity. It is evident that coordination makes the ligands strongly antibacterial and inhibits the growth of bacteria more than the parent ligand. All compounds were tested against four Gram-negative (E. coli, S. flexneri, P. aeruginosa, S. typhi) and two Gram-positive (S. aureus, B. subtilis) bacterial strains. The results obtained were compared with that of the standard drug imipenum. The percentage of activity was compared with the activity of the standard drug considering its activity as 100% (Figures as Supplementary data). All ligands and their oxovanadium(IV) complexes possessed wide-ranging biological activity against all Gram-negative and Gram-positive bacterial strains. The obtained results showed that the ligand (L¹) exhibited a significant (57–61%) activity against (c) and (f) strain and rest of all the strains showed moderate (35–52%) activity. The ligand (L²) displayed a significant (39–50%) activity against all the strains except (c) strain which demonstrated weaker (30%) activity. Similarly, the ligand (L³) showed significant (58–69%) activity against (a), (b), (e) and (f) bacterial strains and moderate (41–50%) activity was observed against (c) and (d). However, the ligand (L⁴) exhibited significant (55–57%) activity against (a), (c) and (d) strain and moderate (39–46%) was observed against (b), (e) and (f). Moreover, the ligands (L⁵) and (L⁶) showed overall significant (57–71%) activity against all the bacterial strains. In addition, all the oxovanadium(IV) complexes (1)–(6) possessed overall significant (55–84%) activity against all the bacterial strains. It is confirmed that antibacterial activity was overall enhanced³¹ upon coordination.

Table 3.  Antibacterial activity (concentration used 1 mg/mL of DMSO) of triazole Schiff bases and oxovanadium(IV) complexes.

Bacteria	Compounds [zone of inhibition (mm)]
	(L^1)	(L^2)	(L^3)	(L^4)	(L^5)	(L^6)	(1)	(2)	(3)	(4)	(5)	(6)	(A)	SD
Gram-negative
(a)	15	14	17	16	17	18	20	18	24	19	26	24	10	29
(b)	11	12	18	13	18	19	17	17	25	16	18	22	12	31
(c)	17	09	15	17	17	18	19	16	18	26	19	25	11	30
(d)	11	13	12	16	18	19	16	21	19	22	20	24	10	29
Gram-positive
(e)	11	12	18	12	17	16	16	19	22	16	21	22	08	26
(f)	17	14	16	11	20	19	21	18	20	17	16	23	09	28
SA	3.01	1.86	2.28	2.48	1.17	1.17	2.14	1.72	2.80	3.98	3.41	1.21	1.41	1.72
Average of bacterial strains
	13.3	11.7	16	13	16.7	18.2	17.5	17.8	21.5	19.3	20	23.3	10	28.8

(a) = E. coli, (b) = S. flexneri, (c) = P. aeruginosa, (d) = S. typhi (e) = S. aureus, (f) = B. Subtilis; <10 = weak; 11–15 = moderate; 16 and above = significant; (A), simple triazole; SD, standard drug = Imipenum; SA, Statistical analysis.

Antifungal bioassay (in vitro)

In vitro antifungal studies of all the compounds were carried out against T. longifusus, C. albican, A. flavus, M. canis, F. solani and C. glabrata fungal strains (Table 4). All ligands and their oxovanadium(IV) complexes possessed good antifungal activity against one or more fungal strains. The results of inhibition obtained from these studies were compared with those of the standard drugs, miconazole and amphotericin B (Figures as Supplementary data). From the antifungal results, it was shown that the ligand (L¹) exhibited significant (58%) activity against (b), moderate (37–48%) against (a), (c), (e) and (f) but showed no activity against (d) fungal strain. However, (L²) observed significant (56%) activity against (b), moderate (35–46%) against (a) and (d)–(f) but inactive against (c) fungal strain. Similarly, the ligand (L³) showed significant (55–62%) activity against (a), (c) and (f), and moderate (37–46%) against (b), (d) and (e) fungal strains. Also, significant (54–56%) activity was observed by the ligand (L⁴) against (b) and (d), and moderate (42–44%) against (a), (c), (e) and (f) fungal strains. Beside this, (L⁵) showed significant (55%) activity against (d), moderate (38–42%) against (b), (c) and (f). Also, weaker (22–26%) activity was shown against (a) and (e) fungal strains. Ligand (L⁶) showed significant (54–61%) activity against (a-d) and moderate (45–48%) was observed against (e) and (f) fungal strains. The oxovanadium(IV) complex (1) showed significant (54–67%) activity against (a), (b), (e) and moderate (36–40%) against (c), (d) and (f) strains. In addition, the complex (2) possessed significant (55–70%) activity against (b), (d) and (f) but moderate (34–49%) activity was displayed against (a), (c) and (e) strains. The complex (4) possessed significant (59–77%) activity against (a), (b) and (f) but moderate (38–43%) against (c), (e) strains. The complex (5) possessed moderate (38–45%) activity against (a) and (e) strains and rest of all strains showed significant (54–69%) activity. The complexes (3) and (6) showed overall significant (54–81%) activity against all strains.

Table 4.  Antifungal activity (concentration used 200 µg/mL) of triazole Schiff bases and oxovanadium(IV) complexes.

Organism	Compounds [% Inhibition]
	(L^1)	(L^2)	(L^3)	(L^4)	(L^5)	(L^6)	(1)	(2)	(3)	(4)	(5)	(6)	SD
(a)	46	35	58	42	26	61	56	43	75	59	45	81	A
(b)	58	41	46	56	38	54	67	55	62	77	56	70	B
(c)	43	00	62	23	42	56	38	34	80	38	63	63	C
(d)	00	56	43	54	55	60	40	70	57	43	69	78	D
(e)	37	34	37	35	22	48	54	49	54	43	38	62	E
(f)	49	46	55	48	39	45	36	62	73	64	54	57	F
SA	22	18	19	18	16	19	17	16	19	16	70	70
Average of fungal strains
	38.8	34.7	50.2	43	37	53.2	48.5	52.2	66.8	54	47	68.5

(a) = T. longifusus (b) = C. albicans (c) = A. flavus (d) = M. canis (e) = F. solani (f) = C. glabrata SD = Standard Drugs MIC µg/mL; A = Miconazole (70 µg/mL:1.6822 × 10⁻⁷ M/mL), B = Miconazole (110.8 µg/mL:2.6626 × 10⁻⁷ M/mL), C = Amphotericin B (20 µg/mL:2.1642 × 10⁻⁸ M/mL), D = Miconazole (98.4 µg/mL:2.3647 × 10⁻⁷ M/mL), E = Miconazole (73.25 µg/mL: 1.7603 × 10⁻⁷ M/mL), F = Miconazole (110.8 µg/mL: 2.66266 × 10⁻⁷ M/mL). SA= Statistical analysis.

Minimum inhibitory concentration

All the synthesized triazole ligands and their oxovanadium(IV) complexes possessing promising antibacterial activity (above 80%) were selected for minimum inhibitory concentration (MIC) studies and obtained results are reported in (Table 5). The antibacterial results indicated that only compounds (3)–(6) were found to display activity greater than 80%, therefore these compounds were further selected for their MIC screening. The compound (3) possessed activity against E. coli, S. flexneri and S. aureus and no activity was observed against P. aeruginosa, S. typhi and B. subtilis. The compound (4) only showed activity against P. aeruginosa and compound (5) against E. coli and S. aureus. Similarly, the compound (6) possessed activity against all bacterial strains except S. flexneri. The MIC values of these compounds fall in the range 2.199 × 10⁻⁷ to 2.832 × 10⁻³ M. The compound (6), however, was found to be the most active showing maximum inhibition 2.199 × 10⁻⁷ M against S. aureus.

Table 5.  Minimum inhibitory concentration (M/mL) of the selected compounds (3), (4), (5) and (6) against selected bacteria.

Microorganisms	MIC (M/mL)^a
	(3)	(4)	(5)	(6)
Gram-negative
E. coli	3.210 × 10^−4	ND	2.125 × 10^−5	5.413 × 10^−4
S. flexneri	4.713 × 10^−3	ND	ND	ND
P. aeruginosa	ND	4.543 × 10^−4	ND	2.832 × 10^−3
S. typhi	ND	ND	ND	4.653 × 10^−6
Gram-positive
S. aureus	4.339 × 10^−5	ND	7.129 × 10^−4	2.199 × 10^−7
B. subtilis	ND	ND	ND	5.715 × 10^−5

^aEach compound was measured three times, with a standard deviation (SD) less than 10% in all the cases. ND is not determined at tested concentrations.

Cytotoxic bioassay (in vitro)

All the synthesized Schiff base ligands (L¹)–(L⁶) and their oxovanadium(IV) complexes (1)–(6) were subjected to cytotoxic activity (brine shrimp bioassay). It is evident from the data reported in (Table 6), that all ligands were found to be inactive for their bioassay showing LD₅₀ values in the range of 2.835 × 10⁻⁴ to 3.926 × 10⁻² M/mL. Only, the compounds (3) and (6) showed activity at (LD₅₀ = 4.116 × 10⁻⁵) and (LD₅₀ = 5.615 × 10⁻⁴), respectively. The data clearly showed that only the oxovanadium complexes exhibited effective cytotoxic nature rather than the parent uncomplexed ligands (L¹)–(L⁶). It was interesting to note that only bromo- and nitro-substituted complexes possessed potent cytotoxic activity against Artemia salina. This activity relationship may help development of certain cytotoxic agents for clinical application.

Table 6.  Brine shrimp bioassay data of triazole Schiff bases (L¹)–(L⁶) and oxovanadium(IV) complexes (1)–(6).

Ligands/complexes	LD50 (M/mL)
(L^1)	>4.315 × 10^− 4
(L^2)	>3.926 × 10 ^− 2
(L^3)	>2.181 × 10^− 3
(L^4)	>4.116 × 10^− 2
(L^5)	>2.835 × 10^− 4
(L^6)	>3.825 × 10 ^− 3
(1)	>3.456 × 10^− 3
(2)	>2.632 × 10^− 3
(3)	4.116 × 10^− 5
(4)	>2.236 × 10^− 2
(5)	>3.356 × 10^− 4
(6)	5.615 × 10^− 4

Conclusion

The prepared triazole ligands act as bidentate ligands for coordination with the vanadium metal via azomethine-N and benzilidene-O. Antimicrobial results indicated that all the oxovanadium (IV) complexes possessed enhanced biological activity against one or more bacterial/fungal strains as compared to their uncomplexed parent triazole ligands. In general, it is believed that the functional groups present in the compounds such as azomethine-N and other heteroatoms like oxygen and nitrogen are responsible for improved biological activity. Our present studies are reported with the conclusion that those compounds which were biologically inactive became active and less biologically active became more upon complexation/coordination with the vanadium metal atom. It has been suggested that the chelation process reduces the polarity of the metal ion by coordinating with ligands, which in turn increases the lipophilic nature of the vanadium metal. This lipophilic nature of metal thus enhances its penetration through the lipoid layer of cell membrane of the microorganism and thus potentially killing more of them.

Declaration of interest

The authors declare no conflicts of interest.

Acknowledgments

The authors are thankful to Higher Education Commission (HEC), Government of Pakistan for the award to carry out this research work. We are also indebted to HEJ research Institute of Chemistry, University of Karachi, Pakistan, for providing their help in taking NMR and mass spectra and, for the help in carrying out antibacterial, antifungal and brine shrimp bioassays.