Design, synthesis, and biological properties of triazole derived compounds and their transition metal complexes

Abstract

Triazole derived Schiff bases and their metal complexes (cobalt(II), copper(II), nickel(II), and zinc(II)) have been prepared and characterized using IR, ¹H and ¹³C NMR, mass spectrometry, magnetic susceptibility and conductivity measurements, and CHN analysis data. The structure of L², N-[(5-methylthiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine, has also been determined by the X-ray diffraction method. All the metal(II) complexes showed octahedral geometry except the copper(II) complexes, which showed distorted octahedral geometry. The triazole ligands and their metal complexes have been screened for their in vitro antibacterial, antifungal, and cytotoxic activity. All the synthesized compounds showed moderate to significant antibacterial activity against one or more bacterial strains. It is revealed that all the synthesized complexes showed better activity than the ligands, due to coordination.

Keywords::

• Triazole
• metal complexes
• antibacterial
• antifungal
• cytotoxic

Introduction

Triazoles are widely known to possess broad-spectrum biological activities such as antibacterial^1–4, antifungal^5–8, anticonvulsant^9–11, antitumor^12–14, antitubercular^15–19, antimicrobial^20–24, anticancer^25–27, analgesic²⁸, cytotoxic²⁹, insecticidal, herbicidal, plant growth regulatory^30–32, and antiproliferative activities^33,34. These are extensively used as prospective ligands in a variety of bioinorganic syntheses. Due to their significant biological applications they have gained much attention in bioinorganic and metal-based drug chemistry. In view of its structural and biological importance, we have synthesized a series of triazole derived Schiff bases, N-(thiophen-2-ylmethylidene)-1H-1,2,4-triazol-3-amine (L¹), N-[(5-methylthiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L²), N-[(3-methylthiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L³), N-[(5-chlorothiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L⁴), and N-[(5-nitrothiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L⁵), from the reaction of 3-amino-1,2,4-triazole and methyl-, chloro-, and nitro-substituted thiophene-2-carboxaldehydes. It coordinates to metal ions such as Cu(II), Co(II), Ni(II), and Zn(II) in different ways depending upon the donor sites of the ligand^35–40. These compounds have been investigated for their in vitro antibacterial activity against four Gram-negative (Escherichia coli, Shigella sonnei, Pseudomonas aeruginosa, Salmonella typhi) and two Gram-positive (Staphylococcus aureus, Bacillus subtilis) bacterial strains and for antifungal activity against Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani, and C. glabrata fungal strains. These studies indicate that all the compounds show moderate to significant activity that increases upon coordination/chelation. These compounds were also checked in the in vitro brine shrimp bioassay.

Materials and methods

All reagents and solvents used were of Analar grade. All metals were used as the chloride salts. Melting points were recorded on a Fisher Johns melting point apparatus. Infrared (IR) spectra were recorded on a Shimadzu FT-IR spectrometer. C, H, and N analysis was carried out using a PerkinElmer model. ¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recorded in dimethylsulfoxide (DMSO)-d₆ using tetramethylsilane (TMS) as internal standard on a Bruker Spectrospin Avance DPX-500 spectrometer. Electron impact mass spectra (EIMS) were recorded on a Jeol MS Route instrument. In vitro antibacterial, antifungal, and cytotoxic properties were studied at the HEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Pakistan and the Department of Chemistry, The Islamia University of Bahawalpur, Pakistan.

Synthesis of ligands

N-(Thiophen-2-ylmethylidene)-1H-1,2,4-triazol-3-amine (L1)

A mixture of thiophene-2-carboxaldehyde (1.12 g, 0.93 mL, 10 mmol) and 3-amino-1,2,4-triazole (0.84 g, 10 mmol) in methanol (40 mL) was refluxed for 5 h with monitoring by thin layer chromatography (TLC). The reaction mixture was cooled to room temperature and filtered; within 1 h a fine off-white solid product separated from the clear solution. It was filtered, washed with methanol, dried, and recrystallized from hot ethanol. The same procedure was used for the synthesis of all other ligands.

Physical, analytical, and spectral data of the ligands (L1–L5)

N-(Thiophen-2-ylmethylidene)-1H-1,2,4-triazol-3-amine (L1)

Yield (1.26 g, 71%); m.p. 172°C; IR (KBr, cm⁻¹): 3175 (NH), 1628 (HC=N), 1611 (C=N, triazole), 1570, 1540 (C=C), 1020 (N-N), 960 (C-S); ¹H NMR (DMSO-d₆): δ 7.24 (dd, 1H, J = 4.6, 4.0 Hz, C₄-H), 7.76 (d, 1H, J = 4.0 Hz, C₃-H), 7.83 (d, 1H, J = 4.6 Hz, C₅-H), 8.25 (s, 1H, C₆-H), 9.30 (s, 1H, triazole), 13.98 (s, 1H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 126.7 (C₅), 129.5 (C₄), 132.6 (C₃), 143.6 (C₂), 152.7 (C₈), 156.2 (C₆), 157.9 (C₇); EIMS (70 eV) m/z (%): 178 ([M]⁺, 77), 177 (100), 151 (6), 145 (11), 137 (15), 122 (12), 110 (20), 96 (11), 69 (16); Anal. Calcd. for C₇H₆N₄S (178.21): C, 47.18; H, 3.39; N, 31.44. Found: C, 47.30; H, 3.41; N, 31.35%.

N-[(5-Methylthiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L2)

Yield (1.40g, 73%); m.p. 168°C; IR (KBr, cm⁻¹): 3185 (NH), 965 (C-S), 1632 (HC=N), 1612 (C=N, triazole), 1575, 1545 (C=C), 1020 (N-N); ¹H NMR (DMSO-d₆): δ 2.5 (s, 3H, C₅-CH₃), 6.96 (d, 1H, J = 3.0 Hz, C₄-H), 7.60 (d, 1H, J = 3.0 Hz, C₃-H), 8.20 (s, 1H, C₆-H), 9.20 (s, 1H, triazole), 13.95 (s, 1H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 15.4 (CH₃), 128.2 (C₄), 130.9 (C₃), 137.6 (C₅), 142.9 (C₂), 153.5 (C₈), 156.5 (C₆), 158.2 (C₇); EIMS (70 eV) m/z (%): 192 (M⁺, 81%), 191 (100), 177 (9), 159 (22), 124 (26), 122 (15), 109 (10), 97 (10), 69 (15); Anal. Calcd. for C₈H₈N₄S (192.24): C, 49.98; H, 4.19; N, 29.14. Found: C, 50.10; H, 4.10; N, 29.20%

N-[(3-Methylthiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L3)

Yield (1.42 g, 74%); m.p. 172°C; IR (KBr, cm⁻¹): 3180 (NH), 970 (C-S), 1626 (HC=N), 1615 (C=N, triazole), 1568, 1540 (C=C), 1020 (N-N); ¹H NMR (DMSO-d₆): δ 2.40 (s, 3H, C₃-CH₃), 7.06 (d, 1H, J = 4.5 Hz, C₄-H), 7.75 (d, 1H, J = 4.5 Hz, C₅-H), 8.20 (s, 1H, C₆-H), 9.25 (s, 1H, triazole), 13.90 (s, 1H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 14.4 (CH₃), 128.4 (C₂), 129.3 (C₄), 132.9 (C₅), 140.6 (C₃), 153.1 (C₈), 156.8 (C₆), 158.2 (C₇); EIMS (70 eV) m/z (%): 192 (M⁺, 68 %), 191 (40), 177 (100), 150 (13), 134 (15), 124 (31), 123 (40), 109 (21), 97 (22), 80 (11), 70 (14), 65 (13); Anal. Calcd. for C₈H₈N₄S (192.24): C, 49.98; H, 4.19; N, 29.14. Found: C, 49.80; H, 4.00; N, 29.3%.

N-[(5-Chlorothiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L4)

Yield (1.47 g, 69%); m.p. 178°C; IR (KBr, cm⁻¹): 3190 (NH), 970 (C-S), 1627 (HC=N), 1610 (C=N, triazole), 1570, 1540 (C=C), 1020 (N-N), 820 (C-Cl); ¹H NMR (DMSO-d₆): δ 7.39 (d, 1H, J = 3.5 Hz, C₄-H), 7.70 (d, 1H, J = 3.5 Hz, C₃-H), 8.5 (s, 1H, C₆-H), 9.24 (s, 1H, triazole), 13.99 (s, 1H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 128.9 (C₃), 132.5 (C₄), 135.6 (C₅), 146.2 (C₂), 153.3 (C₈), 156.9 (C₆), 158.6 (C₇); EIMS (70 eV) m/z (%): 212 (M⁺, 34), 211 (60), 196 (44), 177 (46), 162 (27), 161 (100), 155 (13), 149 (13), 134 (17), 112 (15), 76 (17), 69 (15), 51 (11); Anal. Calcd. for C₇H₅ClN₄S (212.66): C, 39.54; H, 2.37; N, 26.35. Found: C, 39.70; H, 2.34; N, 26.26%.

N-[(5-Nitrothiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L5)

Yield (1.74 g, 79%); m.p. 203°C; IR (KBr, cm⁻¹): 3175 (NH), 980 (C-S), 1624 (HC=N), 1612 (C=N, triazole), 1560, 1540 (C=C), 1370 (NO₂), 1020 (N-N); ¹H NMR (DMSO-d₆): δ 7.72 (d, 1H, J = 4.1 Hz, C₃-H), 7.87 (d, 1H, J = 4.1 Hz, C₄-H), 8.30 (s, 1H, C₆-H), 9.27 (s, 1H, triazole), 14.00 (s, 1H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 129.9 (C₃), 133.8 (C₄), 141.2 (C₅), 147.0 (C₂), 153.6 (C₈), 157.7 (C₆), 159.1 (C₇); EIMS (70 eV) m/z (%): 223 (M⁺, 75), 182 (10), 177 (100), 150 (30), 136 (17), 123 (15), 109 (27), 95 (33), 82 (10), 69 (23); Anal. Calcd. for C₇H₅N₅S (223.21): C, 37.67; H, 2.26; N, 31.38. Found: C, 37.78; H, 2.34; N, 31.26%.

X-ray structure of N-[(5-methylthiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L2)

The X-ray structure of one of the ligands, N-[(5-methylthiophen-2-yl)methylidene]-1H-1,2,4-triazol-3-amine (L²), was determined and published⁴¹ by us elsewhere, and is presented here as Figures 1 and 2 for authentication.

Figure 1.  ORTEP diagram of a single molecule in asymmetric unit of L².

Figure 2.  The unit cell packing in L².

General procedure for the preparation of metal(II) complexes (1–20)

Cobalt(II) complex with N-(thiophen-2-ylmethylidene)-1H-1,2,4-triazol-3-amine (L1)

A warm ethanol (20 mL) solution of Co(II)Cl₂.6H₂O (0.238 g, 1 mmol) was added drop-wise to a magnetically stirred solution of N-(thiophen-2-ylmethylidene)-1H-1,2,4-triazol-3-amine (L¹) (0.356 g, 2 mmol) in ethanol (25 mL). The mixture was refluxed for 2 h and cooled to room temperature. On cooling, a colored precipitate product was formed which was filtered, washed with ethanol and then ether, and dried. Crystallization from aqueous ethanol (30:70) gave the desired metal complex. Physical, analytical, and spectral data of Co(II), Ni(II), Cu(II), and Zn(II) complexes are given in Tables 1 and 2. The same method was used for the preparation of all other complexes.

Table 1.  Physical measurements and analytical data of the metal(II) complexes.

No.	M.P (dec.) (°C)	Yield (%)	Found (Calc.) (%)
			C	H	N
1 [Co(L^1)2]Cl2 [486.26] C14H12N8S2Cl2Co	230–232	58	34.62 (34.58)	2.42 (2.49)	22.97 (23.04)
2 [Ni(L^1)2]Cl2 [486.03] C14H12N8S2Cl2Ni	237–239	57	34.66 (34.60)	2.58 (2.49)	23.10 (23.06)
3 [Cu(L^1)2]Cl2 [490.88] C14H12N8S2Cl2Cu	234–236	60	34.42 (34.25)	2.40 (2.46)	22.95 (22.83)
4 [Zn(L^1)2]Cl2 [492.74] C14H12N8S2Cl2Zn	244–246	58	34.22 (34.13)	2.50 (2.45)	22.67 (22.74)
5 [Co(L^2)2]Cl2 [514.32] C16H16N8S2Cl2Co	223–225	59	37.43 (37.36)	3.20 (3.14)	21.66 (21.79)
6 [Ni(L^2)2]Cl2 [514.08] C16H16N8S2Cl2Ni	231–23	61	37.30 (37.38)	3.06 (3.14)	21.89 (21.80)
7 [Cu(L^2)2]Cl2 [518.93] C16H16N8S2Cl2Cu	238–240	58	37.14 (37.03)	3.18 (3.11)	21.70 (21.59)
8 [Zn(L^2)2]Cl2 [520.77] C16H16N8S2Cl2Zn	235–237	58	37.0 (36.90)	3.01 (3.10)	21.60 (21.52)
9 [Co(L^3)2]Cl2 [514.32] C16H16N8S2Cl2Co	215–217	62	37.31 (37.36)	3.22 (3.14)	21.71 (21.79)
10 [Ni(L^3)2]Cl2 [514.08] C16H16N8S2Cl2Ni	220–222	59	37.27 (37.38)	3.20 (3.14)	21.86 (21.80)
11 [Cu(L^3)2]Cl2 [518.93] C16H16N8S2Cl2Cu	228–230	58	36.95 (37.03)	3.02 (3.11)	21.48 (21.59)
12 [Zn(L^3)2]Cl2 [520.77] C16H16N8S2Cl2Zn	234–236	57	37.05 (36.90)	3.04 (3.10)	21.66 (21.52)
13 [Co(L^4)2]Cl2 [555.16] C14H10N8S2Cl4Co	222–224	62	30.12 (30.29)	1.86 (1.82)	20.30 (20.18)
14 [Ni(L^4)2]Cl2 [554.91] C16H16N8S2Cl4Ni	225–227	60	30.05 (30.30)	1.89 (1.82)	20.39 (20.19)
15 [Cu(L^4)2]Cl2 [559.77] C16H16N8S2Cl4Cu	232–234	58	29.89 (30.04)	1.74 (1.80)	20.30 (20.02)
16 [Zn(L^4)2]Cl2 [561.61] C16H16N8S2Cl4Zn	229–231	59	30.20 (29.94)	1.89 (1.79)	20.10 (19.95)
17 [Co(L^5)2]Cl2 [576.26] C14H10N10S2O4Cl2Co	258–260	63	29.04 (29.18)	1.68 (1.75)	24.39 (24.31)
18 [Ni(L^5)2]Cl2 [576.02] C14H10N10S2O4Cl2Ni	265–267	58	29.37 (29.19)	1.70 (1.75)	24.54 (24.32)
19 [Cu(L^5)2]Cl2 [580.87] C14H10N10S2O4Cl2Cu	269–271	61	29.12 (28.95)	1.79 (1.74)	24.28 (24.11)
20 [Zn(L^5)2]Cl2 [582.71] C14H10N10S2O4Cl2Zn	278–280	57	29.01 (28.86)	1.68 (1.73)	23.91 (24.04)

Table 2.  Conductivity, magnetic, and spectral data of metal(II) complexes.

No.	ΩM (Ω^−1 cm^2 mol^−1)	B.M. (μeff)	λmax (cm^−1)	IR (cm^−1)
1	84.6	4.32	8565, 17,545, 29,990	3175 (NH), 1613 (HC=N), 1600 (C=N), 1565, 1540 (C=C), 1020 (N-N), 960 (C-S), 520 (M-N), 365 (M-S)
2	82.8	3.42	9895, 16,105, 29,272	3175 (NH), 1612 (HC=N), 1601 (C=N), 1565, 1540 (C=C), 1020 (N-N), 960 (C-S), 525 (M-N), 370 (M-S)
3	89.5	1.49	14,725, 25,350	3175 (NH), 1610 (HC=N), 1600 (C=N), 1565, 1540 (C=C), 1020 (N-N), 960 (C-S), 523 (M-N), 367 (M-S)
4	85.9	Dia	28,530	3175 (NH), 1610 (HC=N), 1600 (C=N), 1565, 1540 (C=C), 1020 (N-N), 960 (C-S), 520 (M-N), 364 (M-S)
5	87.8	4.4	8610, 17,580, 29,910	3175 (NH), 1617 (HC=N), 1602 (C=N), 1565, 1540 (C=C), 1020 (N-N), 965 (C-S), 520 (M-N), 364 (M-S)
6	83.0	3.28	10,130, 16,210, 29,355	3175 (NH), 1616 (HC=N), 1601 (C=N), 1565, 1540 (C=C), 1020 (N-N), 965 (C-S), 522 (M-N), 368 (M-S)
7	88.6	1.43	14,670, 25,427	3175 (NH), 1613 (HC=N), 1599 (C=N), 1565, 1540 (C=C), 1020 (N-N), 965 (C-S), 526 (M-N), 365 (M-S)
8	88.0	Dia	28,628	3175 (NH), 1616 (HC=N), 1600 (C=N), 1565, 1540 (C=C), 1020 (N-N), 965 (C-S), 528 (M-N), 363 (M-S)
9	78.6	4.55	8590, 17,520, 30,150	3180 (NH), 1611 (HC=N), 1602 (C=N), 1565, 1540 (C=C), 1020 (N-N), 970 (C-S), 529 (M-N), 369 (M-S)
10	82.5	3.37	10,105, 16,185, 29,491	3180 (NH), 1610 (HC=N), 1603 (C=N), 1565, 1540 (C=C), 1020 (N-N), 965 (C-S), 522 (M-N), 364 (M-S)
11	87.00	1.60	14,670, 25,540	3180 (NH), 1609 (HC=N), 1604 (C=N), 1565, 1540 (C=C), 1020 (N-N), 965 (C-S), 525 (M-N), 366 (M-S)
12	86.00	Dia	28,680	3180 (NH), 1611 (HC=N), 1601 (C=N), 1565, 1540 (C=C), 1020 (N-N), 965 (C-S), 527(M- N), 360 (M-S)
13	79.3	4.35	8675, 17,610, 30,180	3190(NH), 1608(HC=N), 1599 (C=N), 1560, 1540 (C=C), 1020 (N-N), 970 (C-S), 820 (C-Cl), 531 (M- N), 367 (M-S)
14	81.0	3.5	9960, 15,988, 29,375	3190 (NH), 1609 (HC=N), 1598 (C=N), 1560, 1540 (C=C), 1020 (N-N), 970 (C-S), 825 (C-Cl), 529 (M-N), 369 (M-S)
15	78.7	1.64	14,910, 25,350	3190 (NH), 1611 (HC=N), 1599 (C=N), 1560 1540 (C=C), 1020 (N-N), 970 (C-S), 824 (C-Cl), 527 (M -N), 360 (M-S)
16	81.2	Dia	28,465	3175 (NH), 1612 (HC=N), 1597 (C=N), 1560, 1540 (C=C), 1020 (N-N), 970 (C-S), 830 (C-Cl), 523 (M-N), 364 (M-S)
17	82.6	4.55	8502, 17,710, 29,970	3175 (NH), 1610 (HC=N), 1602 (C=N), 1560, 1540 (C=C), 1370 (C-NO2), 1020 (N-N), 980 (C-S), 520 (M-N), 362 (M-S)
18	78.8	3.48	9980, 15,875, 29,472	3175(NH), 1609(HC=N), 1596 (C=N), 1560, 1540 (C=C), 1375 (C-NO2), 1020 (N-N), 980 (C-S), 525(M- N), 370 (M-S)
19	83.00	1.59	14,815, 25,445	3175 (NH), 1605 (HC=N), 1599 (C=N), 1560, 1540 (C=C), 1372 (C-NO2), 1020 (N-N), 980(C-S), 521 (M-N), 360 (M-S)
20	89.5	Dia	28,538	3175 (NH), 1608 (HC=N), 1600 (C=N), 1560, 1540 (C=C), 1372 (C-NO2), 1020 (N-N), 980 (C-S), 529 (M-N), 366 (M-S)

NMR data of Zn(II) complexes

[Zn(L1)2Cl2] (4)

¹H NMR of Zn(II) complex (DMSO-d₆): δ 7.30 (dd, 2H, J = 4.6, 4.0 Hz, C₄-H), 7.80 (d, 2H, J = 4.0 Hz, C₃-H), 7.95 (d, 2H, J = 4.6 Hz, C₅-H), 8.50 (s, 1H, C₆-H), 9.46 (s, 2H, triazole), 14.0 (s, 2H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 127.5 (C₅), 129.8 (C₄), 132.8 (C₃), 144.5(C₂), 153.9 (C₈), 158.3, (C₆), 159.4 (C₇).

[Zn(L2)2Cl2] (8)

¹H NMR of Zn(II) complex (DMSO-d₆): δ 2.6 (s, 6H, C₅-CH₃), 7.0 (d, 2H, J = 3.0 Hz, C₄-H), 7.69 (d, 2H, J = 3.0 Hz, C₃-H), 8.43 (s, 2H, C₆-H), 9.38 (s, 2H, triazole), 14.05 (s, 2H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 15.6 (CH₃), 128.4 (C₄), 131.1 (C₃), 139.1 (C₅), 143.6 (C₂), 154.9 (C₈), 158.7 (C₆), 159.7 (C₇).

[Zn(L3)2Cl2] (12)

¹H NMR of Zn(II) complex (DMSO-d₆): δ 2.5 (s, 6H, C₃-CH₃), 7.12 (d, 2H, J = 4.5 Hz, C₄-H), 7.95 (d, 2H, J = 4.5 Hz, C₅-H), 8.40 (s, 2H, C₆-H), 9.45 (s, 2H, triazole), 14.00 (s, 2H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 14.5 (CH₃), 129.0 (C₂), 129.4 (C₄), 134.6 (C₅), 140.7 (C₃), 154.2 (C₈), 158.7 (C₆), 159.3 (C₇).

[Zn(L4)2Cl2] (16)

¹H NMR of Zn(II) complex (DMSO-d₆): δ 7.44 (d, 2H, J = 3.5 Hz, C₄-H), 7.75 (d, 2H, J = 3.5 Hz, C₃-H), 8.7 (s, 2H, C₆-H), 9.40 (s, 2H, triazole), 14.05 (s, 2H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 129.0 (C₃), 132.6 (C₄), 136.7 (C₅), 147.0 (C₂), 154.7 (C₈), 159.2 (C₆), 160.0 (C₇).

[Zn(L5)2Cl2] (20)

¹H NMR of Zn(II) complex (DMSO-d₆): δ 7.79 (d, 2H, J = 4.1 Hz, C₃-H), 7.95 (d, 2H, J = 4.1 Hz, C₄-H), 8.59 (s, 2H, C₆-H), 9.45 (s, 2H, triazole), 14.07 (s, 2H, triazole, NH); ¹³C NMR (DMSO-d₆): δ 130.0 (C₃), 133.9 (C₄), 142.7 (C₅), 147.8 (C₂), 154.4 (C₈), 159.9(C₆) 160.6 (C₇).

Biological activity

Antibacterial studies

All the newly synthesized compounds (L¹–L⁵) and their respective metal(II) chelates (1–20) were tested against four Gram-negative (E. coli, S. sonnei, P. aeruginosa, S. typhi) and two Gram-positive (S. aureus, B. subtilis) bacterial strains by the disk diffusion method⁴². The test compounds (ligand/complex) were dissolved in DMSO to obtain 10 mg/mL solutions. A known volume (10 µL) of solution was applied with the help of a micropipette onto sterilized filter paper disks. The disks were dried at room temperature overnight and stored in sterile dry containers. Disks soaked with 10 µL of DMSO and dried in air at room temperature were used as the negative control. The standard antibiotic disks used as the positive control were either purchased from the manufacturer or prepared as above in the laboratory by applying a known concentration of standard antibiotic solution. Bacterial cultures were grown in nutrient broth medium at 37°C overnight and spread onto solidified nutrient agar medium in Petri plates using sterilized cotton swabs in a standard microbiological working environment⁴². Test and control disks were then applied to the solidified medium surface with the help of sterilized forceps. The plates were incubated at 37°C for 12–15 h. The results were recorded by measuring the zone of inhibition in mm against each compound⁴². Ampicillin was used as the reference compound. Experiments were carried out in triplicate and the values obtained were statistically analyzed.

Antifungal activity (in vitro)

Antifungal activities of all compounds were studied⁴³ against six fungal strains (T. longifusus, C. albicans, A. flavus, M. canis, F. solani, and C. glabrata). Sabouraud dextrose agar (Oxoid, Hampshire, England) was seeded with 10⁵ cfu/mL fungal spore suspensions and transferred to Petri plates. Disks soaked in 20 mL (200 µg/mL in DMSO) of the compounds were placed at different positions on the agar surface⁴⁴. The plates were incubated at 32°C for 7 days. The results were recorded as percentage of inhibition and compared with standard drugs miconazole and amphotericin B.

Minimum inhibitory concentration

Compounds containing significant antibacterial activity (over 80%) were selected for minimum inhibitory concentration (MIC) studies. The minimum inhibitory concentration was determined using the disk diffusion technique by preparing disks containing 10, 25, 50, and 100 µg/mL of the compounds and applying the protocol⁴⁴.

In vitro cytotoxicity

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 a commercial salt mixture and double distilled water⁴⁵. 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 smaller compartment was opened to ordinary light. After 2 days, nauplii were collected by a pipette from the light side. A sample of test compound was prepared by dissolving 20 mg of each compound in 2 mL of DMSO. From this stock solution, 500, 50, and 5 µg/mL were transferred to nine vials (three for each dilution 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 DMSO only. The solvent was allowed to evaporate overnight. After 2 days, when the shrimp larvae were ready, 1 mL of sea-water and 10 shrimps were added to each vial (30 shrimps/dilution) and the volume was adjusted with sea-water to 5 mL per vial. After 24 h the number of survivors was counted. Data were analyzed using Finney’s computer program to determine the LD₅₀ values⁴⁶.

Results and discussion

Chemistry

The Schiff base derivatives of triazole (L¹–L⁵) were prepared by refluxing an appropriate amount of 3-amino-1,2,4-triazole with a series of methyl-, chloro-, and nitro-substituted thiophene-2-carboxaldehydes, as shown in Scheme 1. All triazole derivatives were only soluble in methanol, ethanol, dioxane, dimethylformamide, and dimethylsulfoxide. The compositions were consistent with their microanalytical and mass spectral data. The metal(II) complexes (1–20) were prepared in a stoichiometric (metal:ligand, 1: 2) molar ratio. Cobalt, copper, nickel, and zinc were used as the chlorides. Physical measurements and analytical data of complexes 1–20 are given in Tables 1 and 2.

Scheme 1.  Preparation of ligands.

Conductance and magnetic susceptibility measurements

The electrolytic nature of the metal complexes (1–20) was indicated by molar conductance values (in dimethylformamide, DMF) (Table 2), which fell in the range 78.6–89.5 Ω⁻¹ cm² mol⁻¹, showing an electrolytic nature⁴⁷. The magnetic moment values of the solid metal complexes obtained at room temperature are also given in Table 2. The magnetic moment values for Co(II) and Ni(II) complexes were found to be in the ranges of 4.32–4.55 B.M. and 3.28–3.50 B.M., respectively, indicative of three and two unpaired electrons for Co(II) and Ni(II) ions in an octahedral environment⁴⁸. The magnetic moment values 1.43–1.64 B.M. for Cu(II) complexes are indicative of one unpaired electron per Cu(II) ion, which suggests that the structures of copper(II) complexes had spin-free distorted octahedral geometry⁴⁹. All the Zn(II) complexes were found to be diamagnetic.

IR spectra

The characteristic bands of IR spectra of the ligands (L¹–L⁵) and their metal complexes are given in “Materials and methods” above and in Table 2. The presence of a strong new band in the ligands at 1624–1632 cm⁻¹ gave a clue of condensation of the carbonyl v(C=O) group of thiophene-2-carboxaldehyde with the amino (NH₂) group of triazole to develop an azomethine (HC=N) linkage⁵⁰, which is also supported by the absence of spectral bands at 1715 and 3325 cm⁻¹ originally assigned to carbonyl v(C=O) and amine v(NH₂) stretching vibrations. The IR spectral bands of the ligands at 1610–1615 cm⁻¹ and 1540–1575 cm⁻¹ were assigned to triazole (C=N) and thiophene (C=C), respectively. The IR bands of the ligands at 1624–1632 and 1610–1615 cm⁻¹ assigned to the azomethine (HC=N) vibration and triazole (C=N), respectively, were shifted to lower frequencies at 1603–1616 and 1596–1604 cm⁻¹, by 15–20 cm^{−1 51}, indicating the coordination of azomethine (HC=N) and (C=N) of triazole with the metal(II) ions. In addition to this, new weak bands appeared in all complexes at lower frequency regions 360–370 cm⁻¹ and 520–532 cm⁻¹, which indicated the coordination of metal–sulfur (M-S) and metal–nitrogen (M-N), respectively. The IR spectra of the ligands (L¹–L⁵) and their metal(II) complexes confirmed coordination of the ligands with the metal(II) ions tridentately through the sulfur of thienyl and nitrogens of azomethine (HC=N) and triazole (C=N)⁵².

1H NMR spectra

¹H NMR spectral data of the free ligands (L¹–L⁵) and their Zn(II) complexes are recorded in “Materials and methods.” The exhibited signals of all protons of the Schiff bases due to heteroaromatic/aromatic groups were found to be in their expected regions⁵³. ¹H NMR spectra of the Schiff bases displayed protons due to azomethine (C₆-H) and triazole (C₈-H) at δ 8.20–8.50 and δ 9.20–9.30 respectively, as singlets. The ¹H NMR spectrum of L¹ exhibited thienyl C₃-H and C₅-H as a doublet at 7.76 ppm and 7.83 ppm, respectively. However, the thienyl C₄-H of L¹ appeared as a double doublet at 7.24 ppm. The peaks appearing at 6.96 ppm and 7.60 ppm were assigned to the protons C₄-H and C₃-H, respectively, of the thienyl ring as a doublet of L^{2 54}. The spectrum of L³ showed thienyl C₄-H and C₅-H as a doublet at 7.06 ppm and 7.75 ppm, respectively. ¹H NMR spectra of ligands L₂ and L₃ exhibited methyl protons C₃-CH₃ and C₅-CH₃ as singlets at δ 2.4–2.5. In the case of compounds L₂ and L₃, as C₄-H was shielded from the electron-donating effect of methyl (CH₃) at the 3- and 5-positions, these appeared upfield at 6.96 ppm and 7.06 ppm as compared to L¹ at 7.24 ppm. The ¹H NMR spectrum of L⁴ displayed C₃-H and C₄-H as a doublet at 7.70 ppm and 7.39 ppm, respectively. The thienyl proton of C₃-H and C₄-H appeared as a doublet at 7.72 ppm and 7.87 ppm of compound L⁵, respectively. Due to the electron-withdrawing effect of the NO₂ group in compound L⁵, thienyl C₄-H appeared downfield at 7.87 ppm as compared to the thienyl C₄-H at 7.24 ppm in ligand L^{1 55}. A broad singlet at δ 13.90–14.0 displayed the NH proton of triazole in all the ligands, which disappeared on exchangement with D₂O. By comparing ¹H NMR spectra of diamagnetic Zn(II) complexes with the free ligands, the proton signals of the triazole ring (C₈-H) and azomethine (CH=N) were assigned a downfield shift by 0.20–0.40 ppm upon coordination. All other protons underwent a downfield shift by 0.11–0.24 ppm due to the increased conjugation⁵⁶.

13C NMR spectra

¹³C NMR spectra of the free Schiff bases and their diamagnetic zinc(II) complexes were recorded in DMSO-d₆. The ¹³C NMR spectral data of the free Schiff bases and their zinc(II) complexes along with possible assignments are reported in “Materials and methods.” The carbons were found to be in their expected regions⁵³. These studies were well supported by the IR and ¹H NMR spectral data. The azomethine carbon (C₆) of all the Schiff bases appeared in the region of δ 156.2–157.7 ppm⁵⁵. The ¹³C NMR spectrum of L¹ showed that the thienyl carbons C₂, C₃, C₄, and C₅ were found at 143.6, 132.6, 129.5, and 126.7 ppm, respectively. All thienyl carbons in all ligands were present in the region of δ 126.7–147.0. All the ligands showed triazole carbons at δ 152.7–159.1. The methyl groups of ligands L² and L³ appeared in the region of δ 14.4–15.5. Downfield shifting of the azomethine carbon (C₆) from 156.2–157.7 ppm in the triazole Schiff bases to 158.3–159.9 ppm in the metal(II) complexes revealed coordination of the azomethine to the metal atom. Similarly, the carbon atom attached to the N of the triazole ring that participated in the coordination of carbon also showed a downfield shift by 0.24–2.40 ppm⁵⁴.

Mass spectra

The electron impact mass spectra (EIMS) gave compositions: C₇H₆N₄S, 178.0 (calcd. 178.21); C₈H₈N₄S, 192.0 (192.24); C₈H₈N₄S, 192.2 (192.24); C₇H₅ClN₄S, 212.2 (212.6); and C₇H₅N₅O₂S, 223.2 (223.21). L¹ showed a base peak at 177 of fragment [C₇H₅N₄S]⁺; for L² this was observed at 191 of fragment [C₈H₇N₄S]⁺; for L³ at 177.2 of fragment [C₇H₅N₄S]⁺; for L⁴ at 177.2 of fragment [C₇H₅N₄S]⁺; and for L⁵ at 177.2 of fragment [C₇H₅N₄S]⁺; these are the most expected stable fragments. The most probable fragmentation pattern was the cleavage of C=N (exocyclic as well as endocyclic), C=C, C-C, and C-S bonds.

Electronic spectra

The electronic spectral values of Co(II), Ni(II), Cu(II), and Zn(II) complexes are recorded in Table 2. The electronic spectra of Co(II) complexes generally showed three absorption bands in the regions 8502–8675, 17,520–17,710, and 29,910–30,180 cm⁻¹, which may be assigned to ⁴T_1g → ⁴T_2g(F), ⁴T_1g → ⁴A_2g(F), and ⁴T_1g → ⁴T_g(P) transitions, respectively, and are suggestive of octahedral geometry^49,55 around the Co(II) ion. The electronic spectral data of Ni(II) complexes showed d–d bands in the regions 9960–10,130, 15,875–16,210, and 29,272–29,491 cm⁻¹, assignable respectively to the transitions ³A_2g(F) → ³T_2g(F), ³A_2g(F) → ³T_1g(F), and ³A_2g(F) → ³T_2g(F), which are characteristic of Ni(II) in octahedral geometry^48,56. The electronic spectra of Cu(II) complexes showed an absorption band in the region 14,670–14,910 cm⁻¹, which may be assigned to the transition ²E_g → ²T_2g. The high-energy band at 25,350–25,540 cm⁻¹ is due to forbidden ligand → metal charge transfer. On the basis of the electronic spectra, a distorted octahedral geometry around the Cu(II) ion is suggested^49,57. The Zn(II) complexes were diamagnetic; they did not show any d–d transition and their spectra were dominated⁵⁸ only by the charge transfer band at 28,465–28,728 cm⁻¹.

Biological activity

Antibacterial bioassay (in vitro)

Antibacterial activity of the title Schiff bases and their metal chelates was determined against four Gram-negative (E. coli, S. sonnei, P. aeruginosa, S. typhi) and two Gram-positive (S. aureus, B. subtilis) bacterial strains (Table 3) according to the literature protocol⁴². The results for all the synthesized compounds were compared with those of the standard drug ampicillin (Figures 3 and 4). The ligands showed varying degrees of inhibitory effect: low, moderate, and significant, on the growth of the different tested strains, while their metal complexes had only moderate to significant inhibitory effects on the growth of the different tested strains (Table 3). The Schiff base ligand L¹ possessed significant activity (53–64%) against (c), (d), and (e), moderate activity (45–46%) against (a) and (b), and weaker activity (24%) against (f). The antibacterial activity of compound L² was found to be significant (56–69%) against (a), (c), and (f), moderate (48–50%) against (b) and (e), and weaker (24%) against (d). Significant activity (64–66%) was observed for ligand L³ against (b) and (d), moderate (44–48%) against (c), (e), and (f), and weaker (30%) against (b). The ligand L⁴ showed good antibacterial activity (53–66%) against (d), (e), and (f), moderate (46%) activity against (a) and (c), and weaker (33%) against (b). Ligand L⁵ also showed significant activity (53–66%) against (c), (e), and (f), moderate activity (50%) against (a) and (d), and weaker (25%) against (b). Compounds 1–20 showed overall significant activity (53–85%) against (a), (b), (c), (d), (e), and (f). However, moderate activity (37–50%) was observed for compounds 1, 2, 9, 10, 11, and 12 against (a), 1, 6, 8, 13, 14, 17, and 19 against (b), 9, 10, 11, and 16 against (c), 5 and 8 against (d), 2, 9, and 11 against (e), and 1–6 and 20 against (f). The antibacterial results (Table 3) evidently show that the activity of the Schiff base compounds was enhanced on coordination with the metal ion. Enhancement in activity of the Schiff bases upon coordination can be explained on the basis of chelation theory. Chelation reduces the polarity of the metal ion to a significant extent due to overlapping with donor groups. Further, the delocalization of π-electrons over the whole chelate ring is increased, which in turn enhances the lipophilicity of the complexes⁵⁹. The increased lipophilicity in turn enhances the penetration of complexes into lipid membranes, thus killing more bacteria.

Table 3.  Antibacterial bioassay (concentration used 1 mg/mL of DMSO) of ligands and metal(II) complexes.

Compound	Zone of inhibition (mm)
	Gram-negative				Gram-positive		SA
	(a)	(b)	(c)	(d)	(e)	(f)
L^1	12	11	17	18	16	07	3.50
L^2	18	12	18	09	14	17	3.33
L^3	08	16	15	18	12	14	2.56
L^4	12	08	15	17	16	17	2.67
L^5	13	06	17	14	18	16	3.00
1	12	12	19	21	17	12	3.50
2	13	17	18	19	13	14	2.33
3	17	16	23	20	17	13	2.56
4	18	17	17	22	16	11	2.22
5	20	16	19	12	17	13	2.50
6	21	12	18	18	18	15	2.33
7	19	16	25	17	16	21	2.67
8	21	12	18	13	17	24	3.50
9	13	19	14	20	13	18	2.83
10	12	20	13	23	16	20	3.67
11	12	18	15	21	14	19	2.83
12	13	17	18	20	19	20	1.89
13	17	12	19	19	17	21	2.17
14	18	11	19	20	19	19	2.22
15	16	16	18	22	20	21	2.17
16	20	16	15	17	18	24	2.44
17	16	12	19	18	19	20	2.22
18	16	16	15	21	23	17	2.67
19	19	11	22	17	21	19	2.78
20	17	16	23	18	20	15	2.22
SD	26	24	32	28	27	29	2.00

Note. (a), E. coli; (b), S. sonnei; (c), P. aeruginosa; (d), S. typhi; (e), S. aureus; (f), B. subtilis. <10, weak; >10, moderate; >16, significant. SD, standard drug (ampicillin); SA, statistical analysis.

Figure 3.  Comparison of antibacterial activity.

Figure 4.  Average antibacterial activity.

In vitro antifungal bioassay

The antifungal screening of all compounds was carried out against T. longifusus, C. albicans, A. flavus, M. canis, F. solani, and C. glabrata fungal strains (Table 4) according to the literature protocol⁴³. Some of the Schiff base derivatives of triazole showed moderate to significant degrees of inhibitory effect on the growth of the tested strains, whereas some showed either low or no inhibitory effect. However, the Schiff base L¹ showed significant activity (53–54%) against (c) and (e), L² showed significant activity (54%) against (f), L³ showed significant activity (57%) against (e), L⁴ showed significant activity (62%) against (d), and L⁵ showed significant activity (59–66%) against (d) and (e). The results given in Table 4 show that compounds 6 and 8 possessed significant activity (53–54%) against (a), 11 and 14 showed significant activity (54–57%) against (b), 1, 3, 4, 6, and 17 had significant activity (54–59%) against (c), 9, 11, and 13–20 possessed significant activity (54–72%) against (d), 1, 2, 4, 8, and 17-20 had significant activity (53–74%) against (e), and 5, 7, 10, 11, 12, and 13 showed significant activity (53–67%) against (f). Most of the other compounds showed moderate and only a few possessed weaker activity against T. longifusus, C. albicans, A. flavus, M. canis, F. solani, and C. glabrata fungal strains. The results of inhibition were compared with the results for the standard drugs, miconazole and amphotericin B⁴⁴ (Figures 5 and 6).

Table 4.  Antifungal bioassay (concentration used 200 µg/mL) of ligands and metal(II) complexes.

Compound	% Inhibition
	(a)	(b)	(c)	(d)	(e)	(f)	SA
L^1	29	08	53	42	54	21	18.07
L^2	46	22	29	10	39	54	13.00
L^3	33	46	00	50	57	48	15.00
L^4	24	42	08	62	39	47	14.00
L^5	32	24	48	59	66	18	16.50
1	38	12	59	34	59	29	13.67
2	41	22	38	48	61	36	09.00
3	24	26	57	51	44	39	10.50
4	35	16	56	44	55	33	11.83
5	39	38	43	16	49	67	11.00
6	54	40	58	12	43	46	10.78
7	44	27	37	24	45	56	09.50
8	53	35	39	14	53	45	10.50
9	20	42	39	57	46	50	08.67
10	14	46	44	49	25	67	14.22
11	41	54	10	54	23	54	15.22
12	24	49	25	50	52	53	11.78
13	33	43	20	72	45	43	10.78
14	38	57	09	63	41	29	14.17
15	29	34	14	56	43	52	10.67
16	39	43	28	59	38	53	08.44
17	35	29	53	59	74	20	17.17
18	39	21	37	65	59	31	13.33
19	41	29	44	67	69	14	16.00
20	34	15	36	70	68	23	18.67
SD	A	B	C	D	E	F

Note. (a), T. longifusus; (b), C. albicans; (c), A. flavus; (d), M. canis; (e), F. solani; (f), C. glabrata. SD, standard drug; MIC: 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, 2.6626 × 10⁻⁷ M/mL). SA, statistical analysis.

Figure 5.  Comparison of antifungal activity.

Figure 6.  Average antifungal activity.

Minimum inhibitory concentration (MIC)

The antibacterial results for all the synthesized compounds obtained after preliminary screening showed that compounds 6, 8, 10, 16, and 18 were the most active (above 80%). These five compounds were therefore selected for minimum inhibitory concentration (MIC) studies (Table 5). The MIC of these compounds was in the range 35.48–70.66 µg/mL. The MIC results in Table 5 show that compound 18 was the most active. It inhibited the growth of S. aureus at 35.48 µg/mL.

Table 5.  Minimum inhibitory concentration (µg/mL) of selected compounds 6, 8, 10, 16, and 18 against selected bacteria.

	6	8	10	16	18
Gram-negative
E. coli	60.66	44.37	—	—	—
S. sonnei	—	—	50.12	—	—
S. typhi	—	—	59.63	—	—
Gram-positive
S. aureus	—	—	—	—	35.48
B. subtilis	—	56.76	—	70.66	—

In vitro cytotoxic bioassay

The synthesized ligands (L¹–L⁵) and their metal(II) complexes (1–20) were screened for their cytotoxicity (brine shrimp bioassay) using the protocol of Meyer et al.⁴⁵. The cytotoxic data recorded in Table 6 reveal that only six compounds, 3, 4, 7, 14, 15, and 20, displayed potent cytotoxic activity, LD₅₀ = 4.47 × 10⁻⁵ to 2.52 × 10⁻⁴ M, against Artenia salina, while all other compounds can be considered as almost inactive in this assay. It was interesting to note that the metal complexes showed potent cytotoxicity as compared to the ligands. The values of LD₅₀ ⁴⁶ of the synthesized compounds from Table 6 show that the cytotoxic activity of the copper complexes is better than of the other metal complexes. This activity relationship may help to serve as a basis for future direction toward the development of certain cytotoxic agents for clinical application.

Table 6.  Brine shrimp bioassay data of the ligands (L¹–L⁵) and their metal(II) complexes (1–20).

Compound	LD50 (M/mL)
L^1	>8.15 × 10^−4
L^2	>8.66 × 10^−4
L^3	>3.64 × 10^−4
L^4	>4.51 × 10^−4
L^5	>4.46 × 10^−4
1	>7.54 × 10^−4
2	>9.39 × 10^−4
3	4.47 × 10^−5
4	5.80 × 10^−5
5	5.48 × 10^−4
6	>4.48 × 10^−4
7	1.92 × 10^−4
8	9.44 × 10^−4
9	>6.32 × 10^−4
10	>4.86 × 10^−4
11	>8.16 × 10^−4
12	>5.78 × 10^−4
13	>9.08 × 10^−4
14	2.52 × 10^−4
15	4.61 × 10^−5
16	>4.51 × 10^−4
17	>4.94 × 10^−4
18	>3.72 × 10^−4
19	>4.59 × 10^−4
20	9.60 × 10^−5

Conclusions

The target compounds were achieved and characterized successfully (Figure 7). Their screening results revealed that the antibacterial and antifungal activity increases upon chelation/coordination. Chelation reduces the polarity of the metal ion, which in turn increases the lipophilic nature of the metal. This lipophilic character experienced by the metal ions further enhances effective penetration through the lipid layer of the cell membrane of the microorganism by killing the bacteria more efficiently. Further, it has been suggested that some functional groups such as azomethine (HC=N), or heteroatoms present in the compounds, may play an important role in increasing the biological activity of the synthesized compounds.

Figure 7.  Proposed structure of the metal complexes.

Acknowledgements

We also thankful to the HEJ Research Institute of Chemistry, University of Karachi, Pakistan, for providing help in recording NMR and mass spectra and also the antibacterial and antifungal assays.

Declaration of interest

One of the authors (M.H.) is grateful to the Higher Education Commission (HEC), Government of Pakistan, for an award to carry out this research.