Design, synthesis and in vitro bactericidal/fungicidal screening of some vanadyl(IV)complexes with mono- and di-substituted ONS donor triazoles

Abstract

A new series of anti-bacterial and anti-fungal mono- and di-substituted triazoles (L¹)–(L⁶) have been synthesized and characterized on the basis of their physical, spectral and analytical data. The ligands (L¹)–(L⁶) on reaction with vanadyl(IV) sulphate led to the formation of vanadyl(IV) metal complexes (1)–(4). The structure of the complexes has been established on the basis of their physical, spectral and elemental analyses data. The synthesized ligands and their vanadyl(IV) complexes have been screened in vitro for anti-bacterial activity against six bacterial species such as, Escherichia coli (ATCC 25922), Shigella flexneri (ATCC 12022), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (ATCC 14028), Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6051) and for in vitro anti-fungal activity against six fungal strains, Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata. The screening results showed the vanadyl complexes to be more bactericidal/fungicidal against one or more bacterial/fungal species. The synthesized compounds were also subjected to brine shrimp bioassay for scrutinizing their cytotoxicity.

• Anti-microbial activity
• mono- and di-substituted triazoles
• vanadyl(IV) complexes

Introduction

Due to emergence of antibiotic resistance to clinically used drugs, it is required that novel, efficient and selective strategies may be designed which could work more aggressively to target the lipoid layer of the microorganisms responsible for drug resistance. Metal complexes have been the best choice so far in designing these candidates with remarkable biological potentials1–3. Variety of triazole derivatives represent an interesting class of compounds showing diversified clinical and pharmacological applications such as anti-bacterial4, anti-fungal5, anti-tumor6, anti-cancer7, anti-inflammatory8, anti-convulsant9, anti-proliferative10 and analgesic properties11.

Advances in medicinal properties of vanadium metal complexes have led to designing and use of its bioactive potential in metal-based drugs12. The most important aspect is; its ability to enhance the activity of insulin mimetic13^,14 in diabetes mellitus of human pathophysiological condition. Vanadyl(IV) complexes with bi-dentate ligands bound to the vanadium metal through oxygen and nitrogen atoms, have been broadly focused in recent years due to their significant efficiency as insulin mimetic compounds15^,16. Orally active medication of these compounds as drug represents an important advancement in the curative approach of human diabetes mellitus. Other potential applications of vanadyl complexes have also been studied in promoting the biological activities such as anti-bacterial17, anti-fungal18, spermicidal19, anti-tumor20, anti-leukemic21 and anti-amoebic22 activity.

Due to increased interest in bioactive potentials of triazoles and vanadium metal, it was thought valuable to combine both the chemistry of triazoles and vanadium metal to form a novel class of vanadium metal based triazoles that could serve as probable bactericidal and fungicidal against resistant bacterial/fungal strains. For this purpose, a new series of vanadyl complexes (1)–(4) with mono-substituted (L¹)–(L³) and di-substituted (L⁴)–(L⁶) (Schemes 1 and 2) triazole-derived compounds have been prepared and screened for their bactericidal/fungicidal activity against a number of bacterial/fungal strains.

Scheme 1. Synthesis of mono-substituted schiff base derived triazoles (L¹)–(L³) and their vanadyl(IV) complexes (1) & (2).

Scheme 2. Synthesis of di-substituted schiff base derived triazoles (L⁴)–(L⁶) and their vanadyl(IV) complexes (3) & (4).

Experimental

Materials and methods

All the chemicals used were of analytical grades. Triazole was purchased from Sigma Aldrich. Fisher Johns melting point apparatus was used for recording melting points. Infrared 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 MSRoute instrument. In vitro bactericidal, fungicidal and cytotoxic properties were studied at HEJ Research Institute of Chemistry, International Centre for Chemical Sciences, University of Karachi, Pakistan.

Synthesis

Procedure for the synthesis of mono-substituted Schiff bases (L¹)–(L³)

An equimolar solution of 3,5-diamino-1,2,4-triazole (0.99 g, 0.01 moles) and 2-acetyl furan (1 mL, 0.01 moles) in 50-mL dry methanol was stirred for 5 h at room temperature, a precipitated product was formed during stirring. It was filtered, washed with methanol (3 × 5 mL), then with diethyl ether (2 × 5 mL) and dried. Recrystallization in a mixture of methanol:dioxane (1:4) afforded TLC pure product (L¹) The same method was applied for the preparation of other ligands (L²)–(L³).

N³-[(1E)-1-(furan-2-yl)ethylidene]-1H-1,2,4-triazole-3,5-diamine (L¹)

Yield (1.4g, 73%); m.p. 244 °C; IR (KBr, cm⁻¹): 3350 (NH₂), 3203 (NH, triazole), 1636 (C = N, azomethine), 1605 (C = N, triazole), 1128 (C–O), 1021 (N-N); ¹H NMR (DMSO-d_6, δ, ppm): δ 2.34 (s, CH₃), 6.02 (s, NH₂), 6.95 (dd, J = 4.9, 3.8 Hz, C₃-H), 7.38 (d, J = 4.9 Hz, C₄-H), 7.69 (d, J = 3.8 Hz, C₂-H), 11.88 (s, NH); ¹³C NMR (DMSO-d₆, δ, ppm): δ 15.10 (CH₃), 114.32 (C₄), 122.43 (C₃), 141.81 (C₂), 146.68 (C₅), 152.71 (C₆), 158.48 (C₈), 163.59 (C₇); EIMS (70eV) m/z (%): 191 ([M]⁺, 18), 176 (100), 161 (13), 134 (19), 120 (14), 110 (22), 108 (9), 94 (12), 79 (11), 67 (16), 53 (7); anal. calcd. for C₈H₉N₅O (191.19): C, 50.26; H, 4.74; N, 36.63; Found: C, 50.20; H, 4.71; N, 36.58%.

N³-[(1E)-1-(thiophen-2-yl)ethylidene]-1H-1,2,4-triazole-3,5-diamine (L²)

Yield (1.45 g, 70%); m.p. 252 °C; IR (KBr, cm⁻¹): 3350 (NH₂), 3204 (NH, triazole), 1635 (C = N, azomethine), 1608 (C = N, triazole), 1021 (N-N), 883 (C-S); ¹H NMR (DMSO-d_6, δ, ppm): δ 2.38 (s, CH₃), 5.98 (s, NH₂), 6.90 (d, J = 3.8 Hz, C₃-H), 7.50 (d, J = 3.6 Hz, C₄-H), 7.65 (d, C₂-H), 11.86 (s, NH); ¹³C NMR (DMSO-d_6, δ, ppm): δ 14.90 (CH₃), 125.95 (C₂), 132.30 (C₃), 136.76 (C₅), 138.44 (C₄), 158.17 (C₆), 158.86 (C₈), 163.56 (C₇); EIMS (70eV) m/z (%): 207 ([M]⁺, 17), 192 (100), 151 (14), 136 (9), 123 (13), 110 (10), 109 (19), 84 (15); anal. calcd. for C₈H₉N₅S (207.26): C, 46.36; H, 4.38; N, 33.79; S: 15.47; Found: C, 46.30; H, 4.35; N, 33.74; S: 15.43%.

N³-[(E)-(1-methyl-1H-pyrrol-2-yl)methylidene]-1H-1,2,4-triazole-3,5-diamine (L³)

Yield (1.3 g, 68%); m.p. 231 °C; IR (KBr, cm⁻¹): 3350 (NH₂), 3204 (NH, triazole) 1634 (HC = N, azomethine), 1606 (C = N, triazole), 1020 (N-N); ¹H NMR (DMSO-d_6, δ, ppm): δ 3.45 (s, CH₃), 5.91 (dd, J = 3.8 Hz, C₃-H), 6.0 (s, NH₂), 6.68 (d, J = 3.7 Hz, C₄-H), 6.97 (d, C₂-H), 8.69 (s, HC = N), 11.88 (s, NH); ¹³C NMR (DMSO-d_6, δ, ppm): δ 32.20 (N-CH₃), 113.65 (C₃), 116.96 (C₄), 122.44 (C₂), 130.13 (C₅), 152.73 (C₆), 157.61 (C₈), 162.85 (C₇); EIMS (70eV) m/z (%): 190 ([M]⁺, 19), 175 (100), 159 (13), 147 (11), 133 (14), 110 (11), 107 (16), 83 (9), 80 (11), 66 (8); anal. calcd. for C₈H₁₀N₆ (190.21): C, 50.52; H, 5.30; N, 44.18; Found: C, 50.47; H, 5.27; N, 44.12%.

Procedure for the synthesis of di-substituted schiff bases (L⁴)–(L⁶)

The ligands (L⁴)–(L⁶) have been synthesized by the condensation reaction of 3,5-diamino-1,2,4-triazole with 2-acetyl furan, 2-acetyl thiophene and 1-methylpyrrole-2-carboxaldehyde in (1:2) molar ratio on refluxing. Rest of the procedure was same as for synthesis of (L¹).

N,N′-bis[(1E)-1-(furan-2-yl)ethylidene]-1H-1,2,4-triazole-3,5-diamine (L⁴)

Yield (2.1g, 74%); m.p. 229–231 °C; IR (KBr, cm⁻¹): 3205 (NH, triazole), 1637 (C = N, azomethine), 1607 (C = N, triazole), 1130 (C–O), 1020 (N-N); ¹H NMR (DMSO-d_6, δ, ppm): δ 2.34 (s, 6H, CH₃), 6.95 (dd, J = 4.9, 3.8 Hz, C₃-H, -H), 7.38 (d, 2H, J = 4.9 Hz, C₄-H, -H), 7.69 (d, 2H, J = 3.8 Hz, C₂-H, -H), 11.88 (s, 1H, NH); ¹³C NMR (DMSO-d₆, δ, ppm): δ 15.10 (2×CH₃), 114.34 (C₄, ), 122.45 (C₃, ), 141.80 (C₂, ), 146.69 (C₅, ), 152.72 (C₆, ), 164.44 (C₇, C₈); EIMS (70eV) m/z (%): 283 ([M]⁺, 24), 268 (35), 254 (11), 190 (100), 176 (23), 120 (20), 108 (11), 94 (18), 78 (13); anal. calcd. for C₁₄H₁₃N₅O₂ (283.28): C, 59.36; H, 4.63; N, 24.72; Found: C, 59.48; H, 4.59; N, 24.68%.

N,N′-bis[(1E)-1-(thiophen-2-yl)ethylidene]-1H-1,2,4-triazole-3,5-diamine (L⁵)

Yield (2.35g, 75%); m.p. 226–227 °C; IR (KBr, cm⁻¹): 3206 (NH, triazole), 1635 (C = N, azomethine), 1609 (C = N, triazole), 1021 (N-N), 885 (C-S); ¹H NMR (DMSO-d_6, δ, ppm): δ 2.38 (s, 6H, CH₃), 6.90 (d, 2H, J = 3.8 Hz, C₃-H, -H), 7.50 (d, 2H, J = 3.6 Hz, C₄-H, -H), 7.65 (d, 2H, C₂-H, -H), 11.86 (s, 1H, NH); ¹³C NMR (DMSO-d_6, δ, ppm): δ 14.90 (2×CH₃), 125.95 (C₂, ), 132.30 (C₃, ), 136.76 (C₅, ), 138.44 (C₄, ), 158.17 (C₆, ), 163.36 (C₇, C₈); EIMS (70eV) m/z (%): 315 ([M]⁺, 23), 300 (100), 286 (17), 206 (21), 192 (14), 136 (26), 124 (9), 110 (8), 83 (9), 78 (7); anal. calcd. for C₁₄H₁₃N₅S₂ (315.42): C, 53.31; H, 4.15; N, 22.20; S: 20.33; Found: C, 53.27; H, 4.19; N, 22.17; S: 20.35%.

N,N′-bis[(1E)-(1-methyl-1H-pyrrol-2-yl)methylidene]-1H-1,2,4-triazole-3,5-diamine (L⁶)

Yield (2.2g, 78%); m.p. 218–219 °C; IR (KBr, cm⁻¹): 3204 (NH, triazole) 1634 (HC = N, azomethine), 1606 (C = N, triazole), 1020 (N-N); ¹H NMR (DMSO-d_6, δ, ppm): δ 3.45 (s, 6H, CH₃), 5.91 (dd, 2H, J = 3.8 Hz, C₃-H, -H), 6.68 (d, 2H, J = 3.7 Hz, C₄-H, -H), 6.97 (d, 2H, C₂-H, -H), 8.69 (s, 2H, HC = N), 11.88 (s, 1H, NH); ¹³C NMR (DMSO-d_6, δ, ppm): δ 32.20 (N-CH₃), 113.65 (C₃, ), 116.96 (C₄, ), 122.44 (C₂, ), 130.13 (C₅, ), 152.73 (C₆, ), 160.15 (C₇, C₈); EIMS (70eV) m/z (%): 315 ([M]⁺, 12), 300 (4), 207 (93), 192 (100), 174 (4), 151 (20), 136 (11), 124 (11), 110 (16), 97 (8), 84 (8); anal. calcd. for C₁₄H₁₅N₇ (281.32): C, 59.77; H, 5.37; N, 34.85; Found: C, 59.72; H, 5.34; N, 34.81%.

Synthesis of vanadyl(IV) complexes (1)

To a hot magnetically stirred dioxane (20 mL) solution of N³-[(1E)-1-(furan-2-yl)ethylidene]-1H-1,2,4-triazole-3,5-diamine (0.382 g, 0.002 moles), a methanol solution (20 mL) of VOSO₄.5H₂O (0.163g, 0.001 moles) 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. It was recrystallized in a mixture of water:dioxane (1:3) to obtain TLC pure complex (1) (Schemes 1 and 2). All other complexes (2)–(4) were prepared following the same method.

Pharmacology

In vitro bactericidal, fungicidal, minimum inhibitory concentration (MIC) and cytotoxicity were studied according to following procedures.

Bactericidal activity (in vitro)

All the newly synthesized triazole Schiff bases (L¹)–(L⁶) and their oxovanadium(IV) complexes (1)–(4) were screened in vitro for their bactericidal activity against four Gram-negative (Escherichia coli (ATCC 25922), Shigella flexneri (ATCC 12022), Pseudomonas aeruginosa (ATCC 27853), Salmonella typhi (ATCC 14028), and two Gram-positive Staphylococcus aureus (ATCC 25923) and Bacillus subtilis (ATCC 6051) bacterial strains by the agar-well diffusion method23. The wells (6 mm in diameter) were dug in the media with the help of a sterile metallic borer with centers at least 24-mm apart. Two to eight hours old bacterial inocula containing approximately 10⁴–10⁶ colony-forming units (CFU/mL) were spread on the surface of the nutrient agar with the help of a sterile cotton swab. The recommended concentration of the test sample (1mg/mL in DMSO) was introduced in the respective wells. Other wells supplemented with DMSO and reference anti-bacterial drug, imipenum, served as negative and positive controls, respectively. The plates were incubated at 37 °C for 24 h. Activity was determined by measuring the diameter of zones showing complete inhibition (mm). In order to confirm the effect of DMSO in the biological screening, alternate studies on DMSO solution showed no activity against any bacterial strains.

Fungicidal activity (in vitro)

Fungicidal activity of all compounds was studied against six fungal strains (T. longifusus, C. albican, A. flavus, M. canis, F. solani and C. glabrata). Sabouraud dextrose agar (Oxoid, Hampshire, England) was seeded with 10⁵ (cfu) mL⁻¹ fungal spore suspensions and transferred to petri plates. Discs soaked in 20 mL (200 µg/mL in DMSO) of the compounds were placed at different positions on the agar surface. The plates were incubated at 32 °C for 7 d. The results were recorded as percentage of inhibition and compared with standard drugs miconazole and amphotericin B.

Minimum inhibitory concentration

Compounds containing promising anti-bacterial activity were selected for MIC studies24. The MIC was determined using the disc diffusion technique by preparing discs containing 10, 25, 50 and 100 μg mL⁻¹ concentrations of the compounds along with standards at the same concentrations.

Cytotoxicity (in vitro)

Brine shrimp (Artemia salina leach) eggs were hatched in a shallow rectangular plastic dish (22 × 32 cm), filled with artificial seawater, which was prepared with commercial salt mixture and double distilled water. An unequal partition was made in the plastic dish with the help of a perforated device. Approximately 50 mg of eggs were sprinkled into the large compartment, which was darkened while the matter compartment was opened to ordinary light. After 2 d a pipette collected nauplii from the lighted side. A sample of the test compound was prepared by dissolving 20 mg of each compound in 2 mL of DMF. From this stock solutions 500, 50 and 5 µg/mL were transferred to 9 vials (three for each dilutions were used for each test sample and LD₅₀ is the mean of three values) and one vial was kept as control having 2 mL of DMF only. The solvent was allowed to evaporate overnight. After 2 d, when shrimp larvae were ready, 1 mL of seawater and 10 shrimps were added to each vial (30 shrimps/ dilution) and the volume was adjusted with seawater to 5 mL per vial25. After 24 h the number of survivors was counted. Data were analyzed by Finney, computer program to determine the LD₅₀ values26.

Statistical analysis

Standard deviation27 is a generally used measure of variability or diversity in statistics and probability theory. It indicates how much variation or dispersion is there from the average or mean values. A low standard deviation shows that the data points tend to be very close to the average, while high standard deviation indicates that the data are distributed over a wide range of values. Standard deviation was used as a statistical tool to check the variation of triplicate anti-bacterial and anti-fungal results.

Results and discussion

Chemistry

New series of triazole derived Schiff bases (L¹)–(L⁶) have been synthesized by the condensation reaction of 3,5-diamino-1,2,4-triazole with 2-acetyl furan, 2-acetyl thiophene and 1-methylpyrrole-2-carboxaldehyde in (1:1) molar ratio on stirring (Scheme 1) and (1:2) molar ratio on refluxing (Scheme 2). The most stable structures of the synthesized ligands have been established on the basis of their physical, spectral (IR, ¹H and ¹³C NMR and mass spectrometry) and analytical (CHN analysis) data. All the prepared ligands were stable at room temperature, non-hygroscopic and colored amorphous solid. The vanadyl(IV) complexes (1)–(4) of these ligands were prepared in a molar ratio of 1:2 (metal:ligand). The ligands were soluble in dioxane at room temperature, soluble in methanol on heating and the vanadyl(IV) complexes were only soluble in DMF and DMSO. Physical and analytical data of the complexes (Table 1) showed these complexes were monomers having stoichiometry of the type ML₂ (metal:ligand).

Table 1. Physical and analytical data of the vanadyl(IV) complexes (1)–(4).

				Calc. (Found) %
No.	Structure formula	MW in g/mole [M.P in °C]	Yield % [color]	C	H	N	V
1	[VO(L^1)2]SO4 C16H18N10O7SV	[545.32]	70	35.21	3.33	25.69	9.34
		252–254	[Dark green]	(35.17)	(3.29)	(25.64)	(9.30)
		2	[VO(L^2)2]SO4 C16H18N10S3O5V	[577.45]	68	33.28	3.14	24.24	8.82
260–262	[Green]			(33.22)	(3.11)	(24.20)	(8.78)
3	[VO(L^3)2]SO4 C28H26N10O9SV			729.51	74	46.05	3.56	19.19	6.98
		[260–262]	[Green]	(46.22)	(3.67)	(19.27)	(6.96)
		4	[VO(L^4)2]SO4 C28H26N10S5O5V	793.77	77	42.33	3.27	17.63	6.41
[273–275]	[Green]			(42.39)	(3.35)	(17.68)	(6.48)

IR spectra

The most important and distinguishing IR spectral bands are reported in experimental part and Table 2. All ligands possessed potentially active donor sites such as, azomethine nitrogen (–HC = N), triazole nitrogens, acetylfuran-O and acetylthiophene-S which have ability to coordinate with the vanadium metal atom. Originally, 3,5-diamino-1,2,4-triazole had two bands at 3310 and 3350 cm⁻¹ due to two amino groups. Ligands of Scheme 1 showed an absence of a band at 3310 cm⁻¹ emerging into a new band due to the azomethine linkage at 1634–1636 cm⁻¹ however, a band at 3350 cm⁻¹ remained unchanged, thus giving evidence for condensation of only one amino group of the triazole moiety to produce mono-Schiff bases28. However, the ligands of Scheme 2 showed the absence of the bands at 1735, 3310 and 3350 cm⁻¹ originally assigned to carbonyl v(C = O) of aldehydes and two v(NH₂) groups of triazole moiety. The absence of these bands and instead, appearance of a strong new band of azomethine v(HC = N) linkage at 1634–1637 cm⁻¹ gave a clue of condensation of carbonyl v(C = O) group with both amino groups of triazole moiety, thus giving bi-Schiff bases29. The bands appearing in the ligands at 883–885 and 1128–1130 cm⁻¹ were assigned to (C–S) and (C–O) vibrations. All these ligands displayed bands at 3203–3206, 1605–1608 and 1020–1021 cm⁻¹ respectively due to the presence of N–H, C = N and N–N vibrations of triazole moiety30.

Table 2. Physical, spectral and IR data of the vanadyl(IV) complexes.

No.	ΩM (Ω^−1cm^−1 mol^−1)	B.M (μeff)	λmax (cm^−1)	IR (cm^−1)
1	95	1.71	13 290, 18 502, 26 879, 36 104	1621 (C = N), 1086 (SO4), 978 (V = O), 868 (C–S), 495 (V–O), 422 (V–N).
2	98	1.73	13 334, 18 468, 26 846, 36 154	1620 (C = N), 1084 (SO4), 977 (V = O), 1115 (C–O), 476 (V–S), 417 (V–N)
3	92	1.74	13 275, 18 528, 26 892, 36 094	1622 (C = N), 1083 (SO4), 976 (V = O), 868 (C–S), 497 (V–O), 422 (V–N).
4	95	1.73	13 341, 18 438, 26 869, 36 142	1618 (C = N), 1082 (SO4), 980 (V = O), 1115 (C-O), 475 (V–S), 417 (V–N).

On comparison of IR spectral data of all the vanadyl(IV) complexes with the data of Schiff base ligands, the absorption modes indicated that the triazole Schiff bases were principally coordinated to the vanadium(IV) metal ion bidentately. The coordination modes of bonding are discussed below:

1. all the ligands showing IR spectral bands at 1634–1637 cm⁻¹ due to azomethine, ν(HC = N) shifted31 to lower frequency (15–17 cm⁻¹) at 1618–1622 cm⁻¹thus indicating the coordination of the azomethine-N with the vanadium(IV) metal atom.

2. the bands at 883–885 and 1128–1130 cm⁻¹ assigned to ν(C–S) and ν(C–O) of thiophene and furan moiety shifted to lower frequency side (15 cm⁻¹) at 868 and 1115 cm⁻¹ suggesting the coordination of thiophene-S and furan-O with the vanadium(IV) metal atom.

3. the appearance of weaker low-frequency new bands at 417–422, 475–476 and 495–497 cm⁻¹ were attributed to and v(vanadium-N), v(vanadium-S) and v(vanadium-O) confirming32 the coordination of vanadium metal atom with the ligands via azomethine-N, thiophene-S and furan-O.

4. two new bands which were not observed in the spectra of the ligands but appeared in the spectra of complexes at 1082–1086 and 976–980 cm⁻¹ were assigned to the presence of (SO₄) group33 outside the coordination sphere and (V = O) of vanadyl34, respectively.

5. all the ligands showed bands at 3205–3208, 1606–1609 and 1020–1021 cm⁻¹ respectively due to the presence of N–H, C = N and N–N vibrations of triazole moiety that remained unchanged in all the complexes indicating that these groups did not participate in the coordination phenomenon.

All the above mentioned evidences supported the coordination of the triazole ligands with the vanadium(IV) metal via the azomethine-N, thiophene-S and/or furan-O groups.

¹H NMR spectra

The ¹H NMR spectra of mono-substituted Schiff bases demonstrated35 characteristic amino (NH₂) protons at 5.98–6.02 ppm as a singlet, which provided evidence for condensation of one amino group of the triazole moiety. The ligands (L¹)-(L⁶) showed methyl (CH₃) and NH protons at 2.42–3.45and 11.86–11.88 ppm, respectively as a singlet. However, the C₃–H & –H protons of (L¹) and (L⁴) were observed at 6.95 ppm as a double doublet. In addition, the C₄–H & –H and C₂–H & –H protons were found at 7.38 and 7.69 ppm, respectively as a doublet. Similarly, C₃–H and –H protons found in ligand (L²) and (L⁵) were observed at 6.90 ppm as a double doublet and C₄–H & –H and C₂–H & –H protons at 7.50 and 7.65 ppm as a doublet. But in case of ligand (L³) & (L⁶), C₃–H and –H protons appeared at 5.91 ppm as a double doublet, C₄–H & –H and C₂–H & –H protons at 6.68 and 6.97 ppm as a doublet and were shielded due to methyl group and shifted upfield. Azomethine protons were observed at 8.69 ppm as a singlet in ligand (L³) and (L⁶). All the protons due to heteroaromatic groups were found in their expected region36. However, the number of proton calculated from the ¹HNMR integration curves and those obtained from the values of the expected microanalysis (CHN) confirmed the proposed structures of the Schiff bases. The conclusions drawn from this study provided further support to the modes of bonding discussed earlier in IR spectra.

¹³C NMR spectra

The ¹³C NMR spectra of the Schiff bases showed triazole carbons (C₇) and (C₈) in the region at 157.61–164.44 ppm, respectively. The azomethine carbons (C₆) and () of the ligands appeared in the region at 152.71–158.17 ppm. The furanyl and thienyl carbons were found in the region at 113.65–149.86 ppm. The methyl groups of the ligands (L¹)–(L⁶) appeared in the region at 14.9–32.20 ppm. All the protons due to heteroaromatic groups were found in their expected region. The present studies are well supported by their IR and ¹H NMR spectral data. Furthermore, all these studies indicated that expected values of carbon atoms compromised well with the number of carbon atoms present in the proposed structures of the compounds.

Mass spectra

The mass spectral data and fragmentation pattern of the triazole ligands strongly confirmed the formation of the ligands possessing proposed structures and also, their bonding pattern. The molecular masses of ligands (L¹)–(L⁶), were found at m/z 191 (calcd. 191.19) for fragment [C₈H₉N₅O]⁺, m/z 207 (calcd. 207.26) for fragment [C₈H₉N₅S]⁺, m/z 190 (calcd. 190.21) for fragment [C₈H₁₀N₆]⁺, m/z 283 (calcd. 283.28) for fragment [C₁₄H₁₃N₅O₂]⁺, m/z 315 (calcd. 315.42) for fragment [C₁₄H₁₃N₅S₂]⁺ and m/z 281 (calcd. 281.32) for fragment [C₁₄H₁₅N₇]⁺, respectively. The most stable base peak fragment [C₇H₆N₅O]⁺ of ligand (L¹) was observed at m/z 176. Moreover, ligand (L²) displayed the base peak stable fragment [C₇H₆N₅S]⁺ at m/z 192 and (L³) showed the stable base peak having fragment [C₇H₇N₆]⁺ at m/z 175. Similarly, the most stable base peak fragment [C₈H₈N₅O]⁺ of ligand (L⁴) was observed at m/z 190. Moreover, ligand (L⁵) displayed the base peak stable fragment [C₈H₈N₅S]⁺ at m/z 207 and (L⁶) showed the stable base peak having fragment [C₈H₁₀N₆]⁺ at m/z 192, as these were the most expected stable fragments. The fragmentation pattern followed the cleavage of C = N (exocyclic as well as endocyclic), C = C, C–C, C–O and C–S bonds. Fragmentation pattern of one ligand (L¹) is shown as Figure 1.

Figure 1. Proposed mass fragmentation pattern of triazole ligand (L¹).

Molar conductance and magnetic susceptibility

The molar conductance values of the complexes (1)–(4) were taken in DMF which fall in the range 92–98 Ω⁻¹cm² mol⁻¹ (Table 2) indicating electrolytic nature22 of the vanadyl(IV) complexes. The magnetic moment (1.71–1.73 BM) values of the complexes were carried out at room temperature (Table 2) and supported the square-pyramidal geometry37.

UV/visible spectra

The UV/Visible spectra of the vanadyl(IV) complexes in DMF showed three (Table 3) distinct 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 () and B₂ (d_xy) → A₁ () transitions, respectively. The first band found at 13 275–13 341 cm⁻¹was assigned to B₂→ E_π d-d transitions. The second band was observed at 18 438–18 528 cm⁻¹ which can be attributed to B₂→ B₁ and the presence of third band at 26 869–26 892 cm⁻¹ can be assigned to the transitions B₂→ A₁. The fourth band of high intensity shown at 36 094–36 142 cm⁻¹ was assigned to metal → ligand charge transfer (MLCT). All these findings provided a strong evidence for the complexes to have a square–pyramidal geometry38.

Table 3. Bactericidal activity (concentration used 1 mg/mL of DMSO) of Triazole schiff bases and their vanadyl(IV) complexes [Zone of Inhibition (mm)].

Bacteria	Compounds [zone of Inhibition (mm)]
	(L^1)	(L^2)	(L^3)	(L^4)	(L^5)	(L^6)	(1)	(2)	(3)	(4)	(A)	SD
Gram-negative
(a)	16	13	14	18	14	16	22	19	24	19	10	29
(b)	14	12	12	16	13	17	19	18	21	17	12	30
(c)	19	15	14	19	17	18	25	24	25	24	11	30
(d)	12	14	14	14	17	18	18	20	17	20	10	29
Gram-positive
(e)	17	16	16	17	18	20	22	23	22	24	08	27
(f)	16	11	17	16	14	19	21	19	23	18	09	28
SA =	1.83	1.51	1.90	2.32	2.48	3.20	2.48	2.43	2.83	3.01	1.41	1.72

(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) = 3,5-Diamino-1,2,4-triazole; SD, standard drug = Imipenum; SA, statistical analysis.

Pharmacology

Bactericidal activity (in vitro)

The results obtained from in vitro bactericidal activity are summarized in (Table 3) and as (Figure 2). All the synthesized 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 according to literature protocol. The results obtained were compared with that of the standard drug imipenum. The percentage activity was compared with the activity of the standard drug considering its activity as 100%. All these ligands and their vanadyl(IV) complexes showed bactericidal activity against all Gram-negative and Gram-positive bacterial strains. The ligand (L¹) showed a significant (16–19 mm, 55–63%) activity against all bacterial strains except S. Flexneri and S. Typhi which possessed moderate (12–14 mm, 41–47%) activity. The ligand (L²) exhibited significant (16 mm, 59%) activity against S. Aureus and remaining strains possessed moderate (11–15 mm, 39–50%) activity. The ligand (L³) possessed significant (16–17 mm, 59–61%) activity against S. Aureus and B. Subtilis bacterial strains and rest of strains showed moderate (12–14 mm, 40–48%) activity. The ligand (L⁴) possessed overall significant (16–19 mm, 57–63%) activity against all bacterial strain except S. Typhi which displayed moderate (14 mm, 48%) activity. Similarly, the ligand (L⁵) exhibited significant (17–18 mm, 59-67%) activity against P. aeruginosa, S. typhi and S. aureus, and rest of the strains E. coli, S. flexneri and B. Subtilis showed moderate (13–14 mm, 43–50%) activity. However, the ligand (L⁶) displayed overall significant (16–20 mm, 55–74%) activity against all bacterial strains. Moreover, all the vanadyl(IV) complex (1)–(4) showed overall significant (17–25 mm, 57–89%) activity against all bacterial strains. The results revealed39 that all the ligands and their vanadyl(IV) complexes contributed significantly towards enhancing the bactericidal activity. It is evident from the reported data that coordination makes the ligands to inhibit the growth of bacteria more than the parent ligand.

Figure 2. Comparison of bactericidal activity of triazole schiff bases versus vanadyl(IV) complexes.

Fungicidal activity (in vitro)

In vitro fungicidal activity of all the compounds were carried out against six fungal strains; T. Longifusus, C. Albican, A. Flavus, M. Canis, F. Solani and C. Glabrata (Table 4) according to the literature protocol. The results of inhibition obtained were compared with those of the standard drugs, miconazole and amphotericin B (Figure 3). The activity was measured in percentage inhibition. The ligand (L¹) had shown significant (55–65%) activity against all fungal strain except A. Flavus and C. Glabrata which showed moderate (45–51%) activity. The (L²) possessed significant (54–57%) activity against A. Flavus and M. Canis, and rest of strains displayed moderate (35–52%) activity. On the other hand, (L³) displayed overall significant (56–70%) activity against all fungal strains. The ligand (L⁴) exhibited significant (57%) activity against C. Albican and remaining strains showed moderate (44–52%) activity. The (L⁵) displayed overall significant (56–67%) activity against all strains. However, (L⁶) exhibited weaker (35–40%) activity against T. Longifusus and C. Albican, and remaining strains displayed significant (55–70%) activity, respectively. On comparison with the vanadyl(IV) complexes, all the complexes (1)–(6) possessed overall significant (54–82%) activity against all fungal strains except C. Albicans fungal strain of (2) which possessed moderate (48%) activity. It was also concluded that the heterocyclic aldehyde ring system and imine (–CH = N) group play a key role in enhancing the activity. On the other hand, the comparison of average values of the complexes versus ligands, a conclusion can be drawn that the fungicidal activity is overall enhanced upon chelation with the vanadyl(IV) metal atom40.

Figure 3. Comparison of fungicidal activity of triazole schiff bases versus vanadyl(IV) complexes.

Table 4. Fungicidal activity (concentration used 200 µg/mL) of triazole schiff bases and their vanadyl(IV) complexes [% inhibition].

	Compounds
Organism	(L^1)	(L^2)	(L^3)	(L^4)	(L^5)	(L^6)	(1)	(2)	(3)	(4)	SD
(a)	55	43	56	46	59	40	71	54	59	76	100
(b)	59	35	60	57	62	61	74	48	64	78	100
(c)	51	54	58	44	64	70	59	68	65	77	100
(d)	56	57	61	45	67	57	67	70	72	80	100
(e)	65	52	56	52	56	35	75	74	76	73	100
(f)	45	48	70	48	64	55	57	57	63	82	100
SA =	20.58	4.86	7.72	8.86	20.15	13.16	4.32	10.58	8.80	7.37

(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 (MIC)

The synthesized ligands and their vanadyl(IV) complexes showing promising bactericidal activity (above 80%) were selected for MIC studies and obtained results are reported in Table 5. The obtained results indicated that all the vanadyl(IV) complexes (1)–(4) were found to display activity more than 80%, therefore, these complexes were selected for their MIC screening. The MIC values of these compounds (1)–(4) fall in the range 4.54 × 10⁻⁸ to 1.64 × 10⁻⁷ M. Among these, the compound (1) was found to be the most active possessing maximum inhibition 4.54 × 10⁻⁸ M against bacterial strain P. Aeruginosa.

Table 5. Minimum inhibitory concentration (M/mL) of the selected compounds (1)–(4) against selected bacteria and in vitro cytotoxic bioassay of ligands, (L¹)–(L⁶) and vanadyl(IV) complexes (1)–(4).

	Minimum inhibitory concentration (M/mL)				Cytotoxic bioassay
No.	E. Coli	P. Aeruginosa	S. Aureus	B. Subtilis	LD50 (M/mL)
(L^1)	–	–	–	–	>7.19 × 10^−4
(L^2)	–	–	–	–	>5.81 × 10^−3
(L^3)	–	–	–	–	>6.11 × 10^−4
(L^4)	–	–	–	–	>7.19 × 10^−2
(L^5)	–	–	–	–	>5.81 × 10^−4
(L^6)	–	–	–	–	>6.11 × 10^−4
(1)	–	2.62 × 10^−7	3.54 × 10^−7	–	>6.11 × 10^−4
(2)	–	1.64 × 10^−7	1.67 × 10^−7	–	>4.14 × 10^−4
(3)	1.68 × 10^−8	4.54 × 10^−8	5.42 × 10^−7	1.98 × 10^−8	>3.69 × 10^−4
(4)	–	1.76 × 10^−8	2.16 × 10^−8	–	>4.14 × 10^−4

Cytotoxicity (in vitro)

The Schiff bases and their vanadyl(IV) complexes were screened for their cytotoxicity (brine shrimp bioassay) by using Meyer protocol25. The data recorded in Table 5 clearly indicated that no compound either ligands or complexes showed potent cytotoxic activity against Artemia salina. This activity relationship may help to serve as a basis for the future research pursuits in designing and development of cytotoxic agents used for clinical purposes.

Conclusion

The synthesized triazole Schiff bases act as bidentate ligands for coordination with the vanadium metal atom. Physical (magnetic and molar conductance), spectral (IR, NMR, electronic) and analytical (C, H, N and V percentage) data revealed that the Schiff base ligands are coordinated with the vanadium metal atom via azomethine-N, furanyl-O and/or thienyl-S showing a square-pyramidal geometry. The obtained results of bactericidal and fungicidal activities indicated that the vanadyl(IV) complexes possessed more biological activity against one or more bacterial and/or fungal strains as compared to their parent uncomplexed ligands. Generally, it is claimed that functional groups found in the compounds like azomethine-N and other heteroatoms (N, O and S) are responsible for the enhancement of bactericidal and fungicidal activities. Our present studies are reported with the conclusion that those compounds which were biologically active, became more active and less biologically active became more upon complexation/coordination with the vanadium metal atom.

Acknowledgements

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

Declaration of interest

The authors report no conflict of interest and are responsible for the contents and writing of the article.