Structural elucidation and biological significance of 2-hydroxy-1-naphthaldehyde derived sulfonamides and their first row d-transition metal chelates

Abstract

2-Hydroxy-1-naphthaldehyde derived sulfonamides and their first row d-transition metal chelates [cobalt (II), copper (II), nickel (II) and zinc (II)] have been synthesized and characterized. The nature of bonding and structure of all the compounds have been deduced from elemental analyses, infrared, ¹H NMR, ¹³C NMR, mass spectrometry, electronic spectra, magnetic susceptibility and conductivity measurements. An octahedral geometry has been suggested for all the complexes. The metal complexes were screened for their antibacterial and antifungal activities on different species of pathogenic bacteria and fungi and their biopotency has been discussed. The results of these studies revealed that all compounds showed moderate to significant antibacterial activity against all bacterial strains and good antifungal activity against various fungal strains. In-vitro cytotoxic properties of all the compounds against Artemia salina was also studies by brine shrimp bioassay.

Keywords::

• Sulfonamides
• Metal (II) Complexes
• Antibacterial
• Antifungal
• Cytotoxicity

Introduction

Sulfonamides are well-renowned for their antibacterial 1-3, antitumor [4], diuretic [5], anti-carbonic anhydrase [6,7], hypoglycaemic [8], anti-thyroid [9] and protease inhibitor [10] activities. Many drugs possess modified pharmacological and toxicological potentials when administered in the form of metallic compounds. The most widely studied metallic ions in this respect are cobalt (II), copper (II), nickel (II) and zinc (II) because they form low molecular weight complexes which are proved to be more beneficial against several diseases 11-14. Sulfonamides have been known to attract much attention into this promising area of metal based sulfa drugs. It was primarily inspired by the successful introduction of metal based compounds of sulfadiazine to stop and/or heal bacterial infections 15-17. Biological properties of the metal complexes exclusively depend on the ease of cleaving the bond between the metal ion and the ligand. It is therefore, vital to understand coordination behaviour and relationship of the metals and the ligands in biological systems. In view of the versatile biological chemistry of sulfonamides and to identify the coordination properties we have consequently, activated a program 18-28, in synthesizing and designing various metal based sulphonamides and look into their structural and biological behaviour. In the same continuation, we herein describe the preparation, and characterization of Co(II), Cu(II) Ni(II) and Zn (II) complexes with 2-hydroxy-1-naphthaldehyde derived sulfonamides, 4-[(2-hydroxynaphthalen-1-yl)methyleneamino] benzenesulfonamide, 4-[{(2-hydroxynaphthalen-1-yl)methyleneamino}methyl]benzenesulfonamide, and 4-[2-{(2-hydroxynaphthalen-1-yl)methyleneamino}ethyl] benzenesulfonamide. Also, in-vitro antibacterial, antifungal and cytotoxic properties of these compounds have been evaluated and reported in the present paper.

Materials and methods

All reagents and solvents used were of analytical grades; Elemental analyses were carried out with a LECO-CHNS-9320 model. ¹H and ¹³C-NMR spectra of compounds were recorded with a Bruker Spectrospin Avance DPX-400 using TMS as internal standard and DMSO d₆ as solvent. Infrared spectra of the compounds were recorded on a Philips Analytical PU 9800 FTIR spectrophotometer. The melting points were determined with a Gallenkamp melting point apparatus. In vitro antibacterial, antifungal and cytotoxic properties were studied at HEJ Research Institute of Chemistry, International Center for Chemical Sciences, University of Karachi, Pakistan.

Synthesis of ligands

Synthesis of 4-[(2-hydroxynaphthalen-1-yl)methyleneamino] benzenesulfonamide (L₁)

To an ethanolic (30 ml) solution of sulfanilamide (1.21 g, 0.007 moles), 2-hydroxy-1-naphthaldehyde (1.21 g, 0.007 moles) in ethanol (15 ml) was added with stirring. The solution was refluxed for 2 h. The precipitates thus formed during refluxing, were cooled to room temperature and collected by suction filtration. Washing thoroughly with ethanol (2 × 10 ml), afforded TLC pure product (1.90 g, 83% yield).

The same method was applied to prepare ligands (L₂) and (L₃).

Physical measurements, analytical estimations and spectral properties of the ligands and zinc (II) complexes

4-[(2-hydroxynaphthalen-1-yl)methyleneamino] benzenesulfonamide (L₁)

Yield 83%; m.p. 280–82°C; IR (KBr, cm^{− 1}): 3392 (NH₂), 3315 (OH), 1597 (HC = N), 1345, 1110 (S = O), 956 (S–N), 841 (C–S); ¹H NMR (DMSO-d₆, δ, ppm): 7.20 (s, 2H, SO₂NH₂), 7.50–7.85 (m, 4H, N-Ph), 7.90–8.25 (m, 6H, naphthalene), 9.15 (s, 1H, azomethine), 10.85 (s, 1H, OH); Anal. Calcd. for C₁₇H₁₄N₂O₃S (326.38): C, 62.56; H, 4.32; N, 8.58; Found: C, 62.75; H, 4.42; N, 8.53%; Mass spectrum (ESI) [M]⁺ = 325.9. ¹³C NMR (δ, ppm): 138.2 (C₁-phenyl), 128.6 (C₂,C₆-phenyl), 122.6 (C₃,C₅-phenyl), 156.4 (C₄-phenyl), 160.0 (C = N, azomethine), 108.5 (C₁-naphthalene), 158.8 (C₂-naphthalene), 118.4 (C₃-naphthalene), 132.4 (C₄-naphthalene), 126.8–135.1 (C₅,C₆,C₇,C₈,C₉,C₁₀-naphthalene); ¹H NMR of Zn (II) complex (DMSO-d6, δ, ppm): 7.55 (s, 2H, SO₂NH₂), 7.90–8.15 (m, 4H, N-Ph), 8.20–8.55 (m, 6H, naphthalene), 9.45 (s, 1H, azomethine); ¹³C NMR of Zn (II) complex (δ, ppm): 138.2 (C₁-phenyl), 128.6 (C₂,C₆-phenyl), 122.6 (C₃,C₅-phenyl), 165.2 (C₄-phenyl), 172.3 (C = N, azomethine), 114.6 (C₁-naphthalene), 170.1 (C₂-naphthalene), 122.3 (C₃-naphthalene), 126.8–135.1 (C₄,C₅,C₆,C₇,C₈,C₉,C₁₀-naphthalene).

4-[{(2-hydroxynaphthalen-1-yl)methyleneamino}methyl]benzenesulfonamide (L₂)

Yield 81%; m.p. 240–42°C; IR (KBr, cm^{− 1}): 3392 (NH₂), 3315 (OH), 1592 (HC = N), 1345, 1110 (S = O), 956 (S–N), 841 (C–S); ¹H NMR (DMSO-d₆, δ, ppm): 4.63 (s, 2H, CH₂), 7.20 (s, 2H, SO₂NH₂), 7.50–7.85 (m, 4H, N-Ph), 7.90–8.25 (m, 6H, naphthalene), 9.00 (s, 1H, azomethine), 10.85 (s, 1H, OH); ¹³C NMR (δ, ppm): 136.7 (C₁-phenyl), 127.2 (C₂,C₆-phenyl), 129.3 (C₃,C₅-phenyl), 142.1 (C₄-phenyl), 64.4 (CH₂), 160.8 (C = N, azomethine), 114.6 (C₁-naphthalene), 158.8 (C₂-naphthalene), 118.4 (C₃-naphthalene), 132.4 (C₄-naphthalene), 126.8–135.1 (C₅,C₆,C₇,C₈,C₉,C₁₀-naphthalene); Anal. Calcd. for C₁₈H₁₆N₂O₃S (340.40): C, 63.51; H, 4.74; N, 8.23; Found: C, 63.55; H, 4.65; N, 8.20%. Mass spectrum (ESI) [M]⁺ = 341.15. ¹H NMR of Zn (II) complex (DMSO-d6, δ, ppm): 4.95 (s, 2H, CH₂), 7.55 (s, 2H, SO₂NH₂), 7.90–8.15 (m, 4H, N-Ph), 8.20–8.55 (m, 6H, naphthalene), 9.40 (s, 1H, azomethine); ¹³C NMR of Zn (II) complex (δ, ppm): 136.7 (C₁-phenyl), 127.2 (C₂,C₆-phenyl), 129.3 (C₃,C₅-phenyl), 142.1 (C₄-phenyl), 70.1 (CH₂), 171.3 (C = N, azomethine), 119.8 (C₁-naphthalene), 169.2 (C₂-naphthalene), 121.2 (C₃-naphthalene), 126.8-135.1 (C₄,C₅,C₆,C₇,C₈,C₉,C₁₀-naphthalene).

4-[2-{(2-hydroxynaphthalen-1-yl)methyleneamino}ethyl] benzenesulfonamide (L₃)

Yield 80%; m.p. 197–98°C; IR (KBr, cm^{− 1}): 3392 (NH₂), 3315 (OH), 1591 (HC = N), 1345, 1110 (S = O), 956 (S–N), 841 (C–S); ¹H NMR (DMSO-d₆, δ, ppm): 2.91 (t, 2H, CH₂), 3.88 (t, 2H, CH₂), 7.20 (s, 2H, SO₂NH₂), 7.50–7.85 (m, 4H, N-Ph), 7.90–8.25 (m, 6H, naphthalene), 9.00 (s, 1H, azomethine), 10.85 (s, 1H, OH); ¹³C NMR (δ, ppm): 136.9 (C₁-phenyl), 127.2 (C₂,C₆-phenyl), 128.0 (C₃,C₅-phenyl), 142.6 (C₄-phenyl), 37.5 (CH₂-C₄), 61.3 (CH₂–N = ) 160.8 (C = N, azomethine), 114.6 (C₁-naphthalene), 158.8 (C₂-naphthalene), 118.4 (C₃-naphthalene), 132.4 (C₄-naphthalene), 126.8–135.1 (C₅,C₆,C₇,C₈,C₉,C₁₀-naphthalene); Anal. Calcd. for C₁₉H₁₈N₂O₃S (354.43): C, 64.39; H, 5.12; N, 7.90; Found: C, 64.45; H, 5.22; N, 7.83%. Mass spectrum (ESI) [M]⁺ = 354.8. ¹H NMR of Zn (II) complex (DMSO-d6, δ, ppm): 3.15 (t, 2H, CH₂), 4.20 (t, 2H, CH₂), 7.55 (s, 2H, SO₂NH₂), 7.90–8.15 (m, 4H, N-Ph), 8.20–8.55 (m, 6H, naphthalene), 9.40 (s, 1H, azomethine); ¹³C NMR of Zn (II) complex (δ, ppm): 136.9 (C₁-phenyl), 127.2 (C₂,C₆-phenyl), 128.0 (C₃,C₅-phenyl), 142.6 (C₄-phenyl), 37.5 (CH₂-C₄), 65.5 (CH₂–N = ), 171.3 (C = N, azomethine), 119.8 (C₁-naphthalene), 169.2 (C₂-naphthalene), 121.2 (C₃-naphthalene), 126.8–135.1 (C₄,C₅,C₆,C₇,C₈,C₉,C₁₀-naphthalene).

Synthesis of metal (II) complexes

Synthesis of Co(II) complex with 4-[(2-hydroxynaphthalen-1-yl)methyleneamino] benzenesulfonamide [Co(L₁-H)₂(H₂O)₂] (1)

To a hot magnetically stirred dioxane (10 ml) solution of 4-[(2-hydroxynaphthalen-1-yl)methyleneamino] benzenesulfonamide (L1) (0.65 g, 0.002 moles), an aqueous solution (15 ml) of Co (II) Cl₂.6H₂O (0.24 g, 0.001 moles) was added. Then buffer solution (pH = 10, 2 ml) was added in it to maintain the pH of the reaction mixture. The mixture was then refluxed for 1 h. The precipitates formed during refluxing, were cooled to room temperature, collected by suction filtration and washed with small amount of dioxane (1 × 5 ml), ether (2 × 10 ml) and dried. Unfortunately, only microcrystalline powder could be obtained, which was impossible to be used for X-ray structural determinations.

The same method was used for the preparation of all other complexes (2)-(12).

Biological activity

Antibacterial bioassay (in-vitro)

All the synthesized compounds (L₁)-(L₃) and metal (II) complexes (1)-(12) were screened in-vitro for their antibacterial activity against four Gram-negative (E. coli, S. flexenari, P. aeruginosa, S. typhi) and two Gram-positive (S. aureus, B. subtilis) bacterial strains by the agar-well diffusion method [29,30]. 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 (1 mg/ml in DMSO) was introduced in the respective wells. Other wells supplemented with DMSO and reference antibacterial drug, imipenum, served as negative and positive controls, respectively. The plates were incubated immediately at 37°C for 24 h. Activity was determined by measuring the diameter of zones showing complete inhibition (mm). In order to clarify any participating role of DMSO in the biological screening, separate studies were carried out with the solutions alone of DMSO and they showed no activity against any bacterial strains.

Antifungal activity (in-vitro)

Antifungal activities of all compounds were studied against six fungal cultures. 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 all compounds were placed at different positions on the agar surface. The plates were incubated at 32°C for seven days. The results were recorded [31] as % of inhibition and compared with standard drugs miconazole and amphotericin B.

Minimum inhibitory concentration (MIC)

Compounds containing high antibacterial activity (over 80%) were selected for minimum inhibitory concentration (MIC) studies. The minimum inhibitory concentration was determined using the disc diffusion technique by preparing discs containing 10, 25, 50 and 100 μg/ml of the compounds and applying the protocol [32].

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 two days nauplii were collected by a pipette 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 two days, when 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 by Finney computer program to determine the LD₅₀ values [33,34].

Result and discussion

Chemistry, composition and characterization of the ligands

The sulfonamide derived ligands (L₁)-(L₃) were prepared as shown in Scheme 1. All ligands were only soluble in Dioxane, DMF and DMSO. The composition of the ligands is consistent with their microanalytical data.

Scheme 1 Preparation of ligands.

Chemistry, composition and characterization of the metal (II) complexes

The metal (II) complexes (1)-(12) of the ligands (L₁)-(L₃) were prepared according to the following equations:

Physical measurements and Analytical data for complexes (1)-(12) is given in Table I.

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

		M.P (dec.)	Yield	Calc. (Found) %
	No.	(^oC)	(%)	C	H	N
1	[Co(L1-H)2(H2O)2] [745.70]	271–275	85	54.76 (54.62)	4.06 (4.33)	7.51 (7.42)
	C34H30N4O8S2 Co
2	[Cu(L1-H)2(H2O)2] [750.31]	266–271	81	54.43 (54.37)	4.03 (3.87)	7.47 (7.44)
	C34H30N4O8S2 Cu
3	[Ni(L1-H)2(H2O)2] [745.45]	256–260	84	54.78 (54.66)	4.06 (4.21)	7.52 (7.42)
	C34H30N4O8S2 Ni
4	[Zn(L1-H)2(H2O)2] [752.15]	273–278	80	54.29 (54.39)	4.02 (4.00)	7.45 (7.53)
	C34H30N4O8S2 Zn
5	[Co(L2-H)2(H2O)2] [773.75]	290–295	72	55.88 (55.78)	4.43 (4.28)	7.24 (7.15)
	C36H34N4O8S2 Co
6	[Cu(L2-H)2(H2O)2] [778.36]	288–293	75	55.55 (55.43)	4.40 (4.69)	7.20 (7.13)
	C36H34N4O8S2 Cu
7	[Ni(L2-H)2(H2O)2] [773.51]	270–275	77	55.90 (55.87)	4.43 (4.56)	7.24 (7.16)
	C36H34N4O8S2 Ni
8	[Zn(L2-H)2(H2O)2] [780.21]	274–279	72	55.42 (55.51)	4.39 (4.47)	7.18 (7.08)
	C36H34N4O8S2 Zn
9	[Co(L3-H)2(H2O)2] [801.80]	262–266	83	56.92 (56.86)	4.78 (4.68)	6.99 (6.94)
	C38H38N4O8S2 Co
10	Cu(L3-H)2(H2O)2] [806.42]	270–275	81	56.60 (56.42)	4.75 (4.87)	6.95 (6.68)
	C38H38N4O8S2 Cu
11	[Ni(L3-H)2(H2O)2] [801.56]	260–265	85	56.94 (56.87)	4.78 (4.72)	6.99 (6.95)
	C38H38N4O8S2 Ni
12	[Zn(L3-H)2(H2O)2] [808.26]	253–258	80	56.47 (56.56)	4.74 (4.82)	6.93 (6.82)
	C38H38N4O8S2 Zn

Conductance and magnetic susceptibility measurements

The molar conductance values (in DMF) for complexes (1)-(12) fall within the range 82–91 Ω^{− 1} cm² mol^{− 1}, showing their non-electrolytic [35] nature. The room temperature magnetic moment values of the complexes are given in Table II. The observed magnetic moment (4.82–4.85 B.M.) is consistent with half-spin octahedral cobalt (II) complexes. The magnetic moment values (1.79–1.83 B.M.) measured for the copper (II) complexes lie in the range expected for a d⁹- system, which contain one unpaired electron with octahedral geometry [36]. The measured values (3.30–3.32 B.M.) for the nickel (II) complexes suggest [37] octahedral geometry for these complexes (Scheme 2). The zinc (II) complexes were found to be diamagnetic as expected.

Table II.  Analytical conductivity, magnetic and spectral data of metal (II) complexes.

No.	ΩM	B.M	λmax	IR
	(Ω^1 cm^2 mol^1)	(μeff)	(cm^1)	(cm^1)
1.	82.8	4.82	7405,17445	1572 (C = N),1395 (C–O),1345, 1110 (SO2),
			20585,29315	956 (S–N), 841 (C–S), 441(M–N), 525 (M–O)
2.	87.5	1.81	14995,19160,	1567 (C = N), 1395 (C–O), 1345, 1110 (SO2),
			30375	956(S–N), 841 (C–S), 442(M–N), 530 (M–O)
3.	85.6	3.30	10395,15705,	1562 (C = N), 1395 (C–O), 1345, 1110 (SO2),
			26455,29870	956 (S–N), 841 (C–S), 439(M–N), 540 (M–O)
4.	82.0	Dia	28935	1568 (C = N), 1395 (C–O),1345, 1110 (SO2),
				956(S–N), 841 (C–S), 440(M–N), 528 (M–O)
5.	89.7	4.85	7365,17510,	1569 (C = N), 1395 (C–O), 1345, 1110 (SO2),
			20640,29355	956(S–N), 841 (C–S), 444(M–N), 535 (M–O)
6.	83.2	1.83	15155,19205,	1571 (C = N), 1395 (C–O),1345, 1110 (SO2),
			30355	956(S–N), 841 (C–S), 442(M–N), 525 (M–O)
7.	88.0	3.31	10440,15785,	1570 (C = N), 1395 (C–O),1345, 1110 (SO2),
			26495,30955	956(S–N), 841 (C–S), 438(M–N), 533 (M–O)
8.	82.9	Dia	29125	1568 (C = N), 1395 (C–O),1345, 1110 (SO2),
				956 (S–N), 841 (C–S), 439(M–N), 538 (M–O)
9.	91.0	4.84	7295,17495,	1569 (C = N), 1395 (C–O),1345, 1110 (SO2),
			20505,29370	956(S–N), 841 (C–S), 444(M–N), 530 (M–O)
10.	88.2	1.79	14985,19180,	1567(C = N), 1395 (C–O),1345, 1110 (SO2),
			30385	956(S–N), 841 (C–S), 443(M–N), 535 (M–O)
11.	83.1	3.32	10405,15690,	1570 (C = N), 1395 (C–O), 1345, 1110 (SO2),
			26535,29995	956(S–N), 841 (C–S), 438(M–N), 537 (M–O)
12.	82.7	Dia	28980	1568 (C = N), 1395 (C–O), 1345, 1110 (SO2),
				956(S–N), 841 (C–S), 439(M–N), 528 (M–O)

Scheme 2 Proposed structure of the metal complex.

IR spectra

The important IR spectral bands of the ligands and its metal complexes are given in experimental and in Table II. All ligands contain various potential electron pair donor sites. In the IR spectra of the ligands a broad band observed at 3315 cm^{− 1} and a sharp band at 1591–1597 cm^{− 1} are assigned [38] to the ν(OH) and (C = N) modes respectively. Evidence of the nitrogen bonding of the azomethine (C = N) group to the central metal atom stems from the shift of the ν(C = N) frequency to lower frequency by 25–35 cm^{− 1} (1562–1572 cm^{− 1}) in all of its metal complexes. This is further supported by the appearance of the new bands at 438–444 cm^{− 1} due to the ν(M-N) band [39].

The coordination through the hydroxyl oxygen is revealed by disappearance of the mode at 3315 cm^{− 1} and appearance of a new band at 1395 cm^{− 1} due to the C–O mode. This is further confirmed by the appearance of the new band at 525–540 cm^{− 1} due to ν(M-O) in the metal complexes. The bands in the ligand due to ν_asymm(SO₂) and ν_symm(SO₂) appear at 1345 and 1110 cm^{− 1}, respectively [40]. These bands remain almost unchanged in the complexes, indicating that this group is not participating in coordination. This is supported by the unchanged ν(S–N) and ν(C–S) modes appearing at 956 and 841 cm^{− 1}, respectively [41,42], in the ligands after complexation. All the other potential electron pair donor sites of the ligands do not participate in coordination as their IR frequencies remain almost unchanged after complexation.

¹H NMR spectra

¹H NMR spectra of the free ligands and their diamagnetic zinc (II) complexes were recorded in DMSO-d₆. The ¹H NMR spectral data along with the possible assignments is recorded in the experimental part. All the protons due to heteroaromatic/aromatic groups were found as to be in their expected region [43]. The conclusions drawn from these studies lend further support to the mode of bonding discussed in their IR spectra. The coordination of the azomethine nitrogen is inferred by the downfield shifting of the –CH = N– proton signal from 9.00–9.15 ppm in the ligand to 9.40–9.45 ppm in the complexes. Hydroxyl proton at 10.85 ppm in the spectra of Zn (II) complexes of ligands (L₁)-(L₃) disappeared indicating deprotonation and coordination of the oxygen with the metal ion All other protons underwent downfield shifting by 0.25–0.35 ppm due to the increased conjugation [44] and coordination with the metal atoms. Furthermore, the number of protons calculated from the integration curves, and those obtained from the values of the expected CHN analyses agree well with each other.

¹³C NMR spectra

¹³C NMR spectra of the free ligands and their diamagnetic zinc (II) complexes were also recorded in DMSO-d₆. The ¹³C NMR spectral data along with the possible assignments is recorded in the experimental part. The carbons atoms due to heteroaromatic/aromatic groups were found as to be in their expected region [43]. The conclusions drawn from these studies present further support to the mode of bonding discussed in their IR and ¹H NMR spectra. Downfield shifting of the –CH = N– signal from 160.0–160.8 ppm in the ligands to 171.3–172.3 ppm in its metal (II) complexes revealed coordination of the azomethine nitrogen to the metal atom. All other carbons underwent downfield shifting by 0.35–11.0 ppm due to the increased conjugation and coordination with the metal atoms [44]. Furthermore, the presences of the number of carbons agree well with the expected values.

Mass spectra

The mass spectral data is consistent with the formulations: C₁₇H₁₄N₂O₃S, 325.9 (calcd., 326.38): C₁₈H₁₆N₂O₃S, 341.15 (calcd., 340.40): C₁₉H₁₈N₂O₃S, 354.8 (calcd., 354.43) of the ligands. The base peak for (L₁) was observed at m/e 245.9 for fragment [C₁₇H₁₂NO]⁺ and for (L₂) and (L₃) at 169.82 for fragment [C₁₁H₈NO]⁺ as these are expected to be the most stable fragments.

Electronic spectra

The Co(II) complexes exhibited well-resolved, low-energy bands at 7,295–7,405 cm^{− 1}, 17,445–17,510 cm^{− 1} and a strong high-energy band at 20,505–20,640 cm^{− 1} (Table I) which are assigned [45] to the transitions ⁴T_1g(F) → ⁴T_2g(F), ⁴T_1g(F) → ⁴A_2g(F) and ⁴T_1g(F) → ⁴T_2g(P) in an octahedral geometry [46]. A high intensity band at 29,315–29,370 cm^{− 1} was assigned to the metal to ligand charge transfer. The magnetic susceptibility measurements for the solid Co (II) complexes are also indicative of three unpaired electrons per Co (II) ion suggesting [47] consistency with their octahedral environment.

The electronic spectra of the Cu (II) complexes (Table I) showed two low-energy weak bands at 14,985–15,155 cm^{− 1} and 19,160–19,205 cm^{− 1} and a strong high-energy band at 30,355–30,385 cm^{− 1} and may be assigned to ²B_1g → ²A_1g and ²B_1g → ²E_g transitions, respectively [48]. The strong high-energy band, in turn, is assigned to metal → ligand charge transfer. Also, the magnetic moment values for the copper (II) are indicative of anti-ferromagnetic spin-spin interaction through molecular association indicative of their octahedral geometry [49].

The electronic spectra of the Ni (II) complexes showed d-d bands in the region 10,395–10,440, 15,690–15,785 and 26,455–26,535 cm^{− 1}. These are assigned [50] to the transitions ³A_2g(F) → ³T_2g(F), ³A_2g(F) → ³T_1g(F) and ³A_2g(F) → ³T_2g(P), respectively, consistent with their well-defined octahedral configuration. The band at 29,870–29,995 cm^{− 1} was assigned to metal → ligand charge transfer. The magnetic measurements showed two unpaired electrons per Ni (II) ion suggesting [51] also an octahedral geometry for the Ni (II) complexes. The electronic spectra of the Zn (II) complexes exhibited only a high-intensity band at 28,935–29,125 cm^{− 1} and are assigned [52] to a ligand-metal charge transfer.

Biological activity

Antibacterial bioassay (in-vitro)

All compounds were tested against four Gram-negative (E. coli, S. flexenari, P. aeruginosa, S. typhi) and two Gram-positive (S. aureus, B. subtilis) bacterial strains (Table III) according to literature protocol [29,30]. The results were compared with those of the standard drug imipenum (Figure 1). All ligands showed moderate to significant activity against all Gram-negative and Gram-positive bacterial strains except the activity of all compounds against strain (b) where no moderate to significant activity was observed. Compounds (1)–(12) exhibited overall a significant activity against E. coli, P. aeruginosa, S. typhi, S. aureus and B. subtilis. However a moderate activity was observed by compound (3), (6) against (a), (L₁), (L₂), (L₃) against (c) and (d), (L₁) against (f). Antibacterial activity is overall enhanced after complexation of the ligands (Figure 2). However the Zinc (II) complexes of all the ligands were observed to be the most active against all species. It was interesting to note that methyl and ethyl carbon chain in the ligands and their respective metal chelates had an impact on the bactericidal activity. As the carbon chain increased in compounds (5)-(12) the bactericidal activity was increased as compared to the other compounds of the series (1)-(4) where there was no methyl or ethyl carbon chain present.

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

	Compound [zone of inhibition (mm)]
Bacteria	L1	L2	L3	1	2	3	4	5	6	7	8	9	10	11	12	SD
Gram-negative
(a)	18	16	17	19	16	15	22	17	15	21	24	19	20	19	26	30
(b)	7	8	6	11	12	13	13	11	11	10	12	11	11	10	14	27
(c)	14	13	15	19	18	16	23	18	19	20	22	18	19	20	23	26
(d)	12	13	13	24	18	18	24	20	20	19	23	20	17	18	25	27
Gram-positive
(e)	17	17	16	21	18	17	24	19	22	19	24	19	20	18	26	30
(f)	15	16	19	22	17	18	25	19	20	18	23	20	19	22	25	28

(a) = E. coli (b) = S. flexenari (c) = P. aeruginosa (d) = S. typhi (e) = S. aureus (f) = B. subtilis 10 < :weak; >10:moderate; >16:Significant SD = Standard Drug (Imipenum).

Figure 1 Comparison of antibacterial activity.

Figure 2 Average antibacterial activity of ligands versus metal (II) complexes.

Antifungal bioassay (in-vitro)

The antifungal screening of all compounds was carried out against T. longifusus, C. albican, A. flavus, M. canis, F. solani and C. glaberate fungal strains (Table IV) according to the literature protocol [31]. All synthesized compounds showed good antifungal activity against different fungal strains. Compound (10) and (11) showed good antifungal activity against all the fungal strains. The results of inhibition were compared with the results of inhibition of standard drugs miconazole and amphotericin B and individual synthesized compounds were also compared (Figure 3). Effect of metal complexation on antifungal activity of the ligands can be seen (Figure 4).

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

	Compound (% inhibition)
Organism	L1	L2	L3	1	2	3	4	5	6	7	8	9	10	11	12	SD
(a)	80	75	0	35	40	20	45	65	60	0	78	30	45	25	70	A
(b)	0	0	45	45	35	60	40	0	0	75	65	60	65	60	55	B
(c)	30	70	80	0	55	55	35	40	40	40	45	45	60	50	0	C
(d)	80	60	0	60	0	0	90	35	85	85	70	0	35	55	85	D
(e)	36	80	0	90	65	70	0	50	30	45	0	20	80	85	80	E
(f)	0	0	30	35	0	80	60	0	75	0	70	0	40	40	45	F

(a) = T. longifucus (b) = C. Albicans (c) = A. flavus (d) = M. canis (e) = F. Solani (f) = C. glaberata.

SD = Standard Drugs MIC μg/ml; A = Miconazole (70 μg/ml:1.6822 × 10^{− 7} moles/ml),B = Miconazole (110.8 μg/ml:2.6626 × 10^{− 7} moles/ml), C = Amphotericin B (20 μg/ml:2.1642 × 10^{− 8} moles/ml), D = Miconazole (98.4 μg/ml:2.3647 × 10^{− 7} moles/ml), E = Miconazole (73.25 μg/ml: 1.7603 × 10^{− 7} moles/ml), F = Miconazole (110.8 μg/ml: 2.66266 × 10^{− 7} moles/ml).

Figure 3 Comparison of antifungal activity.

Figure 4 Average antifungal activity in ligands versus metal (II) complexes.

Minimum inhibitory concentration (MIC) for antibacterial activity

The preliminary antibacterial screening showed that compounds (1), (4), (8) and (12) were the most active ones (above 80%). These compounds were therefore, selected for antibacterial minimum inhibitory concentration (MIC) studies (Table V).

Table V.  Minimum inhibitory concentration (moles/ml of the selected compounds (1), (4), (8) and (12) against selected bacteria.

No.	1	4	8	12
Gram-negative
E. coli	–	–	1.282 × 10^− 7	6.186 × 10^− 8
P. aeruginosa	–	6.647 × 10^− 8	3.204 × 10^− 8	3.093 × 10^− 8
S. typhi	1.341 × 10^− 7	3.324 × 10^− 8	3.834 × 10^− 8	1.237 × 10^− 8
Gram-positive
S. aureus	–	1.330 × 10^− 7	6.408 × 10^− 8	1.237 × 10^− 7
B. subtilis	–	6.647 × 10^− 8	3.204 × 10^− 8	6.186 × 10^− 8

Cytotoxic bioassay (in-vitro)

All the synthesized compounds were screened for their cytotoxicity (brine shrimp bioassay) using the protocol of Meyer et al. [33]. From the data recorded in Table VI, it is evident that three compounds, (2), (6) and (10) displayed potent cytotoxic activity against Artemia salina, while the other compounds were almost inactive for this assay. The compound (2) showed activity (LD₅₀ = 5.358 × 10^{− 4} moles/ml), compound (6) showed activity (LD₅₀ = 6.745 × 10^{− 4} moles/ml), compound (10) showed activity (LD₅₀ = 4.898 × 10^{− 4} moles/ml) in the present series of compounds. It was interesting to note that only copper complexes showed potent cytotoxicity whereas the other metal complexes did not. This activity relationship may help to serve as a basis for future direction towards the development of certain cytotoxic agents for clinical applications.

Table VI.  Brine shrimp bioassay data of the ligands (L₁)-(L₃) and their metal (II) complexes (1)-(12).

Compound	LD50 (moles/ml)
L1	>3.064 × 10^− 3
L2	>2.938 × 10^− 3
L3	>2.821 × 10^− 3
1	>1.341 × 10^− 3
2	5.358 × 10^− 4
3	>1.341 × 10^− 3
4	>1.330 × 10^− 3
5	>1.292 × 10^− 3
6	6.745 × 10^− 4
7	> 1.293 × 10^− 3
8	> 1.282 × 10^− 3
9	> 1.247 × 10^− 3
10	4.898 × 10^-4
11	> 1.248 × 10^-3
12	> 1.237 × 10^-3

Conclusion

The enhancement of antibacterial/antifungal activity in ligands (L₁)-(L₃) upon chelation is rationalized on the basis of their structures and the mode of coordination/chelation. It has been suggested that chelation reduces the polarity of the metal ion 53-56 on partial sharing of its positive charge with the donor groups. The process of chelation increases the lipophilic nature of the metal atom, which in turn favors 57-59 its permeation through the lipoid layer of cell membrane of the micro-organism. It has also been suggested that some functional groups such as azomethine or heteroaromatics present in these compounds display [60,61] extensive biological activities that may be responsible for the increase of hydrophobic character and liposolubility of the molecules. It ultimately enhances activity of the compounds and the biological utilization ratio.

Acknowledgements

One of us (HAS) is grateful to Higher Education Commission (HEC), Government of Pakistan for the award to carry out this research. We are also indebted to HEJ research Institute of Chemistry, University of Karachi, Pakistan, for help in providing NMR and mass spectra and also the antibacterial and antifungal assays.