Metal-based ethanolamine-derived compounds: a note on their synthesis, characterization and bioactivity

Abstract

Metal-based ethanolamines, (L¹)–(L⁴) coordinated with Co(II), Cu(II), Ni(II) and Zn(II) metals in 1:2 (metal:ligand) molar ratio to produce new compounds have been reported. These compounds were screened for their bactericidal/fungicidal activity against a number of bacterial (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Bacillus subtilis) and fungal strains (Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata) alongside against a shrimp species known as Artemia salina. The screening results indicated that metal complexes have significantly higher activity than uncomplexed ligands against one or more bacterial/fungal species due to chelation. The ligand (L⁴) displayed good bacterial and fungal activity as compared to other ligands. The antibacterial results revealed that the Zn(II) complex (16) of (L⁴) was found to be the most active complex and Co(II) complex (14) of the same ligand (L⁴), demonstrated the highest antifungal activity.

Keywords:

• Antibacterial/antifungal
• ethanolamine-derived compounds
• metal(II) complexes

Introduction

Bioactive transition metal complexes have gained increasing interest in metal-based drug chemistry especially as therapeutic agents. Since their discovery in 1864, Schiff’s bases have contributed immensely to the development of co-ordination chemistry and acting as an inspiration for the development of novel compounds1. The Schiff bases are characterized by an imine group and are formed as a condensation product2 of primary amine with an aldehyde or ketone in the presence of a dehydrating agent such as MgSO₄. A number of Schiff’s base metal complexes have displayed bioactivity as antibacterial3,4, antifungal5, anticancer6,7,8, anti-inflammatory9, anticonvulsant10, antiviral11 and analgesic12 agents. Although metal-based ethanolamines have historically been subjected of many investigations13,14 due to their use as buffers15, catalysts16, inhibitors17, ion exchangers18, electroplating and dyes19, now they are garnering attention for their biological20 role in plant growth21 regulation, bacterial and fungal metabolism and as a cytotoxic agent22. Metal-based compounds have also been reported to improve electron transfer and efficiency in protein bioelectrochemistry23,24.

One of the advantages of using Schiff’s bases is the ability to control and tune the electronic and steric properties of the ligand attached to the metal. One of the strategies is an appropriate choice of precursor, because that plays a pivotal role by allowing control over nature of donor atoms, number of chelating moieties and the extent of denticity. The new series of ethanolamine-derived compounds (L¹)–(L⁴), (E)-2-(((2-hydroxyethyl)imino)methyl)phenol (L¹), (E)-2-(((2-hydroxyethyl)imino)methyl)naphthalen-1-ol (L²), (E)-2-((4-nitrobenzylidene)amino)ethanol (L³) and (E)-5-bromo-2-(((2-hydroxyethyl) imino)methyl)phenol (L⁴) were synthesized from the reaction of 2-hydroxy-1-naphthaldehyde, 2-hydroxybezaldehyde, 5-bromo-2-hydroxybezaldehyde and 4-nitrobenzaldehyde, respectively, with ethanolamine in equimolar ratio (Scheme 1). The synthesized ligands were further coordinated with Co(II), Cu(II), Ni(II), and Zn(II) metals in 1:2 (metal:ligand) molar ratio to produce new metal complexes (Scheme 2).

Scheme 1. Preparation of ligands.

Scheme 2. Metal(II) complexes.

The synthesized ligands and their metal(II) complexes have been screened in vitro for antibacterial activity against six bacterial species (Escherichia coli, Shigella flexneri, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Bacillus subtilis) and for in vitro antifungal activity against six fungal strains (Trichophyton longifusus, Candida albicans, Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata). Cytotoxicity of the compounds was determined through the help of brine shrimp bioassay.

Materials and methods

All the chemicals used were of analytical grade and were purchased from Sigma Aldrich. Infrared spectra of solid compounds (as KBr disc) were recorded on a Nicolet FT-IR Impact 400D infrared spectrometer. Elemental analysis was carried out on Perkin Elmer and 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. UV-Visible spectra were recorded on ultraviolet spectrometer-1700 (Shimadzu, Japan) in the frequency range of 250–800 nm. Molar conductance of metal complexes was measured using an Inolab Cond 720 Conductivity Bridge at room temperature using 0.001 molar solutions in DMF. A Stanton SM12/S Gouy balance was used to measure the magnetic susceptibility of the metal complexes at room temperature using mercury acetate ligand as a standard. In vitro antibacterial, antifungal and cytotoxic properties were studied in collaboration with Research Institute of Chemistry, International Centre for Chemical Sciences, University of Karachi, Pakistan and School of Science and Engineering, Teesside University, UK.

Synthesis of ligands

Different aldehydes such as 2-hydroxy-1-naphthaldehyde, 2-hydroxybezaldehyde, 5-bromo-2-hydroxybezaldehyde and 4-nitrobenzaldehyde were individually dissolved in ethanol (25 mL) and added respectively to a refluxed solution of ethanolamine in ethanol (10 mL) in an equimolar ratio. The reaction mixture was refluxed for 3 h and consequently precipitated while being monitored. The solid product thus obtained was filtered, washed with ethanol and dried. It was re-crystallized in hot ethanol/methanol (1:1). The same method was used for the preparation of all other ligands (L¹)–(L⁴).

(E)-2-(((2-hydroxyethyl)imino)methyl)naphthalen-1-ol (L¹)

Yield: (1.64 g, 76%); mp 151 °C; color (dark yellow); IR (KBr, cm⁻¹): 3438 (aryl-OH), 3385(alc-OH), 1627 (–HC=N), 1603 (C–H) and 1584 (C=C); ¹H NMR (ppm d₆-DMSO): δ 3.65 (s, 1H, alc-OH), 4.50 (t, 2H, C₁₂–H), 5.85 (t, 2H, C₁₁–H), 7.15 (d, 1H, J = 9.0 Hz, C₃–H), 7.41 (t, 1H, J = 7.4 Hz, C₆–H), 7.53 (t, 1H, J = 7.4 Hz, C₇–H), 7.80 (d, 1H, J = 7.8 Hz, C₄–H), 7.95 (d, 1H, J = 9.0 Hz, C₅–H), 8.22 (d, 1H, J = 8.5 Hz, C₈–H), 8.53 (s, 1H, C₁₃–H), 9.88 (s, 1H, aryl-OH); 13C NMR (DMSO-d₆): δ 59.3 (C₁₁), 69.5 (C₁₂), 116.4 (C₁), 122.1 (C₆), 125.5 (C₃), 126.2 (C₈), 126.9 (C₁₀), 127.7 (C₇), 128.9 (C₅), 134.3 (C₉), 136.4 (C₄), 159.6 (C₂), 162.8 (C₁₃); Mass Spectrum (ESI): [M]⁺= 215.25. UV-Visible: 27 335 and 31 284 cm⁻¹. Anal. Calc. for C₁₃H₁₃NO₂ (215.25) C, 72.54; H, 6.09; N, 6.51; O, 14.87 and found C, 72.48; H 6.04; O, 14.83.

(E)-2-(((2-hydroxyethyl)imino)methyl)phenol25 (L²)

Yield (1.22 g, 74%); mp 92 °C; color (yellow); IR (KBr, cm⁻¹): 3450 (aryl-OH), 3380(alc-OH),1625 (–HC=N) and 1596 (C=C); ¹H NMR (ppm d₆-DMSO): δ 3.63 (s, 1H, alc-OH),4.50 (t, 2H, C₁₂-H), 5.85 (t, 2H, C₁₁–H), 7.0 (t, 1H, J = 8.5 Hz, C₄–H), 7.11 (d, 1H, J = 7.9 Hz, C₆–H), 7.45 (t, 1H, J = 7.5 Hz, C₅–H), 7.69 (d, 1H, J = 7.8 Hz, C₃–H), 8.50 (s, 1H, C₁₃–H), 9.91(s, 1H, aryl-OH); 13C NMR (DMSO-d₆): δ 59.4 (C₁₁), 69.7 (C₁₂), 117.5 (C₃), 119.3 (C₁), 121.7(C₅), 131.1 (C₆), 133.6 (C₄), 159.9 (C₂), 162.5 (C₁₃); Mass Spectrum (ESI): [M]⁺= 165.19. UV-Visible: 27 233 and 31 050 cm⁻¹. Anal. calcd. for C₉H₁₁NO₂ (465.19): C, 65.44; H, 6.71; N, 8.48; O, 19.37. Found: C, 65.38; H, 6.67; N, 19.33.

(E)-4-Bromo-2-(((2-hydroxyethyl)imino)methyl)phenol (L³)

Yield (1.51 g, 78%); mp: 99 °C, color (orange yellow); IR (KBr, cm⁻¹): 3453 (aryl-OH), 3382(alc-OH), 1622 (–HC=N) and 1590 (C=C); ¹H NMR (ppm d₆-DMSO): δ 3.67 (s, 1H, alc-OH),4.50 (t, 2H, C₁₂–H), 5.85 (t, 2H, C₁₁–H), 7.12 (d, 1H, J = 8.7 Hz, C₃–H), 7.50 (dd, 1H, J = 8.7, 2.5 Hz, C₅–H), 8.10 (d, 1H, J = 2.5 Hz, C₆–H), 8.57 (s, 1H, C₁₃–H), 9.93 (s, 1H, aryl-OH); ¹³C NMR (DMSO-d₆): δ 58.9 (C₁₁), 68.1 (C₁₂), 118.7 (C₃), 122.5 (C₁), 125.2 (C₅), 127.7 (C₄), 133.1(C₆), 159.2 (C₂), 161.9 (C₁₃); mass spectrum (ESI): [M]⁺= 165.19. UV-Visible: 27 461 and 31 338 cm⁻¹. Anal. calcd. forC₉H₁₀BrNO₂ (244.1): C, 44.29; H, 4.13; Br, 32.74; N, 5.74. Found: C, 44.23; H, 4.11; Br,32.68; N, 5.70.

(E)-2-((4-nitrobenzylidene)amino)ethanol25 (L⁴)

Yield (1.55 g, 80%); mp: 88 °C, color (reddish brown); IR (KBr, cm⁻¹): 3460 (aryl-OH), 3390(alc-OH), 1632 (–HC=N), 1595 (C=C) and 1424 (NO₂); ¹H NMR (ppm d₆-DMSO): δ 3.69 (s, 1H, alc-OH), 4.50 (t, 2H, C₁₂–H), 5.85 (t, 2H, C₁₁–H), 8.10 (dd, 2H, J = 8.7, 2.5 Hz, C₂–H and C₆–H), 8.33 (dd, 2H, J = 8.7, 2.5 Hz, C₃–H and C₅–H), 8.65 (s, 1H, C₁₃–H); 13C NMR (DMSO-d₆): δ 59.3 (C₁₁), 68.6 (C₁₂), 120.6 (C₃), 122.4 (C₅), 130.5 (C₁), 131.0 (C₆), 152.3

(C₄), 158.8(C₂), 162.2 (C₁₃); mass spectrum (ESI): [M]⁺= 194.19. UV-Visible: 27 590 and 31 410 cm⁻¹. Anal. calcd. for C₉H₁₀N₂O₃ (194.19): C, 55.67; H, 5.19; N, 14.44. Found: C, 55.62; H, 5.16; N, 14.11.

Synthesis of the transition metal(II) complexes

All complexes were prepared according to the standard procedure in which a methanol solution (20 mL) of the respective metal(II) as a chloride (5 mmol) was added to a refluxed methanol solution (30 mL) of the ligand (10 mmol). The mixture was further refluxed for 3 h leading to a precipitated product. It was then cooled to room temperature, filtered, washed with methanol and finally with diethyl ether. The precipitated product thus obtained was dried and recrystallized in a mixture of hot aqueous methanol:ethanol (1:2) to obtain TLC pure product.

NMR data of the Zn(II) complexes

[Zn(L¹)₂] (4): ¹H NMR (DMSO-d₆, δ, ppm): 3.78 (s, 1H, alc-OH), 4.61 (t, 2H, C₁₂–H), 5.91 (t, 2H, C₁₁–H), 7.26 (d, 1H, C₃–H), 7.46 (t, 1H, C₆–H), 7.59 (t, 1H, C₇–H), 7.90 (d, 1H, C₄–H), 7.99(d, 1H, C₅–H), 8.26 (d, 1H, C₈–H), 8.64 (s, 1H, C₁₃–H); 13C NMR (DMSO-d₆, δ, ppm): 60.1(C₁₁), 70.3 (C₁₂), 117.5 (C₁), 129.1 (C₆), 125.5 (C₃), 126.7 (C₈), 127.5 (C₁₀), 128.2 (C₇), 129.6(C₅), 135.0 (C₉), 136.4 (C₄), 160.6 (C₂), 163.9 (C₁₃).

[Zn(L²)₂] (8): ¹H NMR (DMSO-d₆, δ, ppm): 3.79 (s, 1H, alc-OH), 4.62 (t, 2H, C₁₂–H),5.94 (t, 2H, C₁₁–H), 7.07 (t, 1H, C₄–H), 7.18 (d, 1H, C₆–H), 7.51 (t, 1H, C₅–H), 7.79 (d, 1H, C₃–H), 8.63 (s, 1H, C₁₃–H); 13C NMR (DMSO-d₆, δ, ppm): δ 60.4 (C₁₁), 70.7 (C₁₂), 117.5 (C₃), 119.3 (C₁), 123.3 (C₅), 131.1 (C₆), 134.2 (C₄), 161.0 (C₂), 163.6 (C₁₃).

[Zn(L³)₂] (12): ¹H NMR (DMSO-d₆, δ, ppm): 3.70 (s, 1H, alc-OH), 4.60 (t, 2H, C₁₂–H), 5.95 (t, 2H, C₁₁–H), 7.23 (d, 1H, C₃–H), 7.55 (dd, 1H, C₅–H), 8.19 (d, 1H, C₆–H), 8.61 (s, 1H, C₁₃–H);¹³C NMR (DMSO-d₆, δ, ppm): δ 59.8 (C₁₁), 69.1 (C₁₂), 118.7 (C₃), 123.5 (C₁), 125.7 (C₅), 128.3(C₄), 133.8 (C₆), 160.2 (C₂), 163.0 (C₁₃).

[Zn(L⁴)₂] (16): ¹H NMR (DMSO-d₆, δ, ppm): 4.59 (t, 2H, C₁₂–H), 5.93 (t, 2H, C₁₁–H), 8.18(dd, 2H, C_2–-H and C₆–H), 8.39 (dd, 2H, C₃–H and C₅–H), 8.62 (s, 1H, C₁₃–H), 10.3 (s, 4H, H₂O); 13C NMR (DMSO-d₆, δ, ppm): δ 60.2 (C₁₁), 69.7 (C₁₂), 121.4 (C₃), 122.9 (C₅), 131.5 (C₁), 131.8 (C₆), 152.9 (C₄), 159.7 (C₂), 163.3 (C₁₃).

Biological activity

Antibacterial activity (in vitro)

All the newly synthesized ethanolamine-derived compounds (L¹)–(L⁴) and their metal(II) complexes (1)–(16) were screened in vitro for their antibacterial activity against four Gram-negative (E. coli, S. flexneri, P. aeruginosa, S. typhi) and two Gram-positive (S. aureus, B. subtilis) bacterial strains by agar-well diffusion method26. The wells (6 mm in diameter) were dug in the media with the help of a sterile metallic borer with centers of 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 a negative and positive control, respectively. The plates were incubated at 37 °C for 24 h. Activity was determined by measuring the diameter of clear 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.

Antifungal activity (in vitro)

Antifungal activity 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 days. The results were recorded as percentage of inhibition and compared with the standard drugs miconazole and amphotericin B.

Minimum inhibitory concentration (MIC)

Compounds containing promising antibacterial activity were selected for minimum inhibitory concentration (MIC) studies. The MIC was determined using the disc diffusion technique27 by preparing discs containing diluted samples at 10, 25, 50, and 100 g mL⁻¹ concentrations of the compounds along with standards at the same concentrations.

Cytotoxicity (in vitro)

Brine shrimp assay was done using the protocol of Meyer et al.28, the details of which are reproduced here. Brine shrimp (Artemia salina leach) eggs were hatched in a shallow rectangular plastic dish (22 × 32 cm), filled with artificial seawater prepared from commercial salt mixture and double-distilled water. An unequal partition was made in the plastic dish with the help of a perforated device where approximately 50 mg of eggs were sprinkled into the darkened larger compartment while smaller matter compartment was exposed to the ordinary light. After two days, nauplii were collected from the unsheltered 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 5, 50 and 500 μg/mL were transferred to nine vials (three for each dilutions were used for each test sample and LD₅₀ was calculated as the mean of these 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 seawater and 10 shrimps were added to each vial (30 shrimps/dilution) and the volume was adjusted with seawater to 5 mL per vial28. After 24 h the number of survivors was counted. Data were analyzed by a computer program to determine the LD₅₀ values29.

Results and discussion

The condensation reaction of ethanolamine with 2-hydroxy-1-naphthaldehyde/2-hydroxy benzaldehyde/5-bromo-2-hydroxybenzaldehyde/4-nitrobenzaldehyde in an equimolar ratio afforded four ligands (L¹)–(L⁴) (Scheme 1). These colored ligands were stable in both air and moisture. These microcrystalline solids melted at 88–151 °C and were soluble in DMSO and DMF at room temperature and in methanol and ethanol on heating. The ligands (L¹)–(L³) were found to be tridentate and the ligand (L⁴) to be bidentate which reacted readily with Co(II), Cu(II), Ni(II) and Zn(II) metals as their chlorides in methanol to form their metal(II) complexes (Scheme 2).

All the synthesized metal(II) complexes were microcrystalline and had an intense color except Zn(II) complexes, which were off-white. The metal(II) complexes decomposed (Table 1) without melting and were insoluble in common organic solvents such as ethanol, methanol, dichloromethane and acetone but soluble in DMSO and DMF. The spectral data and elemental analysis of the prepared ligands and their metal(II) complexes were in a good agreement with their proposed structures, indicating the high purity of all the compounds. The analytical data of the complexes indicated a 1:2 metal: ligand stoichiometry.

Table 1. Physical measurements and analytical data of metal (II) complexes (1)–(16).

					Elemental analysis (%)Calc (Found)
No	Structure	Yield (%)	MW/Formula	M.P (°C)	C	H	N	M
1	[Co(L^1–H)2]	70	[487.41]C26H24N2O4Co	166–168	64.07(64.01)	4.96 (4.92)	5.75 (5.72)	12.09 (12.04)
2	[Ni(L^1–H)2]	70	[487.17] C26H24N2O4Ni	220–222	64.10 (64.01)	4.97 (4.92)	5.75 (5.71)	12.05 (12.01)
3	[Cu(L^1–H)2]	75	[492.02]C26H24N2O4Cu	185–187	64.77 (64.71)	4.92 (4.89)	5.69 (5.65)	12.92 (12.88)
4	[Zn(L^1–H)2]	83	[493.89]C26H24N2O4Zn	197–199	63.23 (63.18)	4.90 (4.86)	5.67 (5.62)	13.24 (13.20)
5	[Co(L^2–H)2]	71	[387.30]C18H20N2O4Co	175–176	55.82 (55.77)	5.21 (5.18)	7.23 (7.20)	15.22 (15.18)
6	[Ni(L^2–H)2]	74	[387.06]C18H20N2O4Ni	230–232	55.86 (55.80)	5.21 (5.17)	7.24 (7.20)	15.16 (15.12)
7	[Cu(L^2–H)2]	81	[391.91]C18H20N2O4Cu	194–196	55.16 (55.11)	5.14 (5.10)	7.15 (7.12)	16.21 (16.17)
8	[Zn(L^2–H)2]	82	[393.77]C18H20N2O4Zn	270–272	54.90 (54.85)	5.12 (5.09)	7.11 (7.06)	16.61 (16.56)
9	[Co(L^3–H)2]	85	[545.09]C18H18Br2N2O4Co	225–227	39.66 (39.61)	3.33 (3.29)	5.14 (5.11)	10.81 (10.77)
10	[Ni(L^3–H)2]	80	[544.85]C18H18Br2N2O4Ni	194–196	39.68 (39.64)	3.33 (3.29)	5.14 (5.10)	10.77 (10.73)
11	[Cu(L^3–H)2]	84	[549.70]C18H18Br2N2O4Cu	214–216	39.33 (39.29)	3.30 (3.27)	5.10 (5.07)	11.56 (11.52)
12	[Zn(L^3–H)2]	80	[551.56] C18H18Br2N2O4Zn	211–212	39.20 (39.15)	3.29 (3.26)	5.08 (5.04)	11.86 (11.82)
13	[Co(L^4)2]	74	[481.32]C18H22N4O8Co	176–177	44.92 (44.87)	4.61 (4.57)	11.64 (11.60)	12.24 (12.20)
14	[Ni(L^4)2]	82	[481.07]C18H22N4O8Ni	206–208	44.94 (44.89)	4.61 (4.58)	11.65 (11.60)	12.20 (12.16)
15	[Cu(L^4)2]	77	[485.84]C18H22N4O8Cu	190–191	44.49 (44.44)	4.56 (4.53)	11.53 (11.50)	13.08 (13.05)
16	[Zn(L^4)2]	78	[487.80] C18H22N4O8Zn	278–280	44.32 (44.27)	4.55 (4.51)	11.49 (11.45)	13.41 (13.37)

IR spectra

The significant and distinctive IR spectral bands are reported in the experimental part and in Table 2. All Schiff’s base ligands possessed potentially active donor sites capable of coordinating with metal atoms such as azomethine nitrogen (–HC=N), aliphatic and aromatic hydroxyl (–OH) groups. The Schiff bases (L¹)–(L⁴) possessed the characteristic azomethine (–HC=N) stretching30 at 1622–1632 cm⁻¹ giving a clue of condensation product. All the ligands (L¹)–(L⁴) displayed aliphatic hydroxyl (–OH) stretching at 3380–3390 cm⁻¹, however, ligands (L¹)–(L³) additionally showed aromatic hydroxyl (–OH) stretching31 at 3438–3390 cm⁻¹. The ligand (L⁴) showed a band at 1424 cm⁻¹ resulting from NO₂ vibrations. The comparison of the IR spectra of the Schiff’s bases (L¹)–(L⁴) with their metal(II) complexes (1)–(16) indicated that the Schiff bases were principally coordinated to the metal(II) atoms in a bi- and tridentate fashion. The vibrations of azomethine-N group present in all the metal complexes (1)–(16) and aliphatic-O appearing in the spectra of metal(II) complexes (1)–(12) shifted to lower frequency (07–15 cm⁻¹) at 1608–1623 and 3365–3377 cm⁻¹, respectively, representing the mode of coordination of the azomethine nitrogen32 and hydroxyl-O with the metal(II) atoms. Absence of bands at 3438–3490 and 3380–3390 cm⁻¹ due to aliphatic and aromatic ν(OH) and consequent appearance of a new band33 at 1370–1377 cm⁻¹ attributed to ν (C–O) in the metal(II) complexes (1)–(16), confirmed the deprotonation and coordination of aromatic and aliphatic ν (OH) to the metal(II) atoms. The following observations further support the mode of chelation:

• Appearance of the new bands at 520–543 and 440–460 cm⁻¹ could be assigned to v(M–O) and v(M–N) vibrations34 in the metal complexes which are absent in ligands.

• The metal(II) complexes (13)–(16) demonstrated35 new broad bands at 3468–3478 cm⁻¹ which can likely be attributed to water molecules coordinated to the metal atoms. These bands were only observed in the spectra of the metal complexes.

These clues supported the evidence of the participation of azomethine-N and deprotonation/coordination of hydroxyl-O with the metal(II) ions.

Table 2. Conductivity, magnetic and spectral data of metal(II) complexes (1)–(16).

No	ΩM (Ω^−1 cm^2 mol^−1)	B.M μeff	λm (cm^−1)	IR (cm^−1)
1	15.9	4.35	8723, 17 690, 29 820	3370 (OH), 1616 (HC = N), 1370 (C–O), 520 (M–N), 442 (M–O)
2	15.7	3.56	8635, 17 717, 25 817, 29 781	3372 (OH), 1615 (HC = N), 1372 (C–O), 535 (M–N), 450 (M–O)
3	14.6	1.66	8897, 17 783, 29 816	3371 (OH), 1620 (HC = N), 1373 (C–O), 529 (M–N), 448 (M–O)
4	14.9	Dia	28 915	3373 (OH), 1618 (HC = N), 1371 (C–O), 533 (M–N), 450 (M–O)
5	16.2	4.53	8650, 17 729, 29 841	3366 (OH), 1612 (HC = N), 1372 (C–O), 522 (M–N), 446 (M–O)
6	15.8	3.41	8712, 17 761, 25 778, 29 738	3365 (OH), 1610 (HC = N), 1370 (C–O), 536 (M–N), 445 (M–O)
7	15.6	1.71	8937, 17 892, 29 812	3365 (OH), 1615 (HC = N), 1371 (C–O), 526 (M–N), 443 (M–O)
8	15.1	Dia	28 928	3368 (OH), 1613 (HC = N), 1373 (C–O), 532 (M–N), 440 (M–O)
9	16.2	4.38	8680, 17 523, 29 831	3368 (OH), 1611 (HC = N), 1377 (C–O), 540 (M–N), 449 (M–-O)
10	15.2	3.48	8786, 17 777, 25 719, 29 781	3367 (OH), 1610 (HC = N), 1376 (C–O), 524 (M–N), 453 (M–O)
11	16.0	1.74	8727, 17 765, 29 788	3370 (OH), 1612 (HC = N), 1375 (C–O), 536 (M–N), 448 (M–O)
12	15.0	Dia	28 841	3377 (OH), 1608 (HC = N), 1377 (C–O), 528 (M–N), 451 (M–O)
13	14.6	4.45	8790, 17 800, 29 805	3470 (H2O), 1620 (HC = N), 540 (M–N), 455 (M–O)
14	16.5	3.51	8696, 17 871, 25 719, 29 778	3471 (H2O), 1622 (HC = N), 544 (M–N), 459 (M–O)
15	15.4	1.70	8866, 17 857, 29 858	3470 (H2O), 1623 (HC = N), 541 (M–N), 460 (M–O)
16	15.1	Dia	28 932	3473 (H2O), 1619 (HC = N), 543 (M–N), 463 (M–O)

¹H NMR spectra

¹H NMR spectra of the ligands (L¹)–(L⁴) and their diamagnetic Zn(II) complexes were recorded in DMSO-d₆ and the details are provided in the experimental section. ¹H NMR spectradisplayed36 characteristic azomethine (–HC=N) protons at 8.53–8.65 ppm as a singlet providing a strong evidence for the condensation of amino group of the ethanolamine with aldehydes. All of the ligands (L¹)–(L⁴) displayed aliphatic (–OH) proton at 5.63–5.99 ppm as a singlet and C₁₁–H and C₁₂–H protons at 4.50 and 5.85 ppm as triplet, respectively. The ligands (L¹)–(L³) possessed aromatic (–OH) proton at 9.88–9.93 ppm. Ligand (L¹) displayed other C_3–5–H and C₈–H protons as a double of doublet at 7.15–8.22 ppm, respectively, but C₆–H and C₇–H protons appeared as a triplet at 7.41 and 7.53 ppm, respectively. Similarly, the ligand (L²) exhibited C₄–H and C₅–H protons at 7.0 and 7.45 ppm as triplet and, C₃–H and C₆–H protons as a doublet at 7.11 and 7.69 ppm, respectively. The C₃–H and C₆–H protons of ligand (L³) were found at 7.12 and 8.10 ppm as a doublet and C₅–H proton observed at 7.50 ppm as a double of doublet. ¹H NMR spectra of the ligand (L⁴) showed the C₂–H, C₆–H and, C₃–H and C₅–H protons at 8.10 and 8.33 ppm as double of doublet. The disappearance of hydroxyl proton at 9.88–9.93 ppm in the spectra of Zn(II) complexes confirmed deprotonation and coordination of the hydroxyl-O with the metal atom. The coordination of the azomethine-N was evident by downfield shifting of all the proton signals in their Zn(II) complexes. A strong singlet was observed at 10.3 ppm in the Zn(II) complex (16) due to the coordination of water molecule with the Zn(II) metal atom. All protons underwent downfield shift by 0.05–0.12 ppm due to the increased conjugation on coordination with the zinc metal atom. The number of protons calculated from the integration curves34 and obtained values of the expected CHN analysis agreed well with each other.

¹³C NMR spectra

The ¹³C-NMR spectra of the Schiff bases and their diamagnetic Zn(II) complexes were recorded in DMSO-d₆ and are reported along with their possible assignments in the experimental section and all the carbon atoms were found in the expected region. The ¹³C-NMR spectra of the Schiff base ligands (L¹)–(L⁴) displayed distinctive and representative (C₁₁, C₁₂) carbons of ethylene group and (C₁₃) carbons of azomethine (–HC=N) at 58.9–69.7 and 161.9–162.8 ppm, respectively. The remaining other (C₁)–(C₁₀) carbons of aromatic rings were observed at 116.4–159.9 ppm. Downfield shifting of the azomethine carbons found in the uncomplexed Schiff bases from161.9–162.8 ppm to 163.0–163.9 ppm in their Zn(II) complexes was due to shifting of electronic density toward the Zn(II) ion. Similarly, all carbons of methylene groups and aromatic rings being near to the coordination sites also showed downfield shifting by 0.5–1.1 ppm due to the increased conjugation and coordination with the metal atoms. The downfield shifting also confirmed the coordination of the azomethine to the zinc metal atom. Moreover, the presence of the number of carbons is well in agreement with the expected values37. Furthermore, the conclusions drawn from these studies present further support to the modes of bonding discussed in their IR and ¹H NMR spectra.

Mass spectra

The mass fragmentation pattern of the ligands (L¹)–(L³) followed the cleavage of C=N (exocyclic) and C=C bonds. The mass spectral data and the stable fragmentation values of the ligands have already been detailed in the experimental section. All the ligands showed pronounced molecular ion peaks. The data of the Schiff bases shown by mass spectra strongly confirmed the formation of the ligands possessing proposed structures and also, their bonding pattern.

Molar conductances and magnetic measurements

Molar conductance studies of the metal(II) complexes (1)–(16) were carried out in DMF and their molar conductance data (14.6–16.9 ohm⁻¹ cm² mol⁻¹) showed (Table 1) that these complexes were non electrolytic38 in nature. The magnetic moment (B.M) values of all the metal(II) complexes (1)–(16) were measured at room temperature. The observed magnetic moment value of Co(II)complexes were found in the range of 4.35–4.53 B.M indicating that Co(II) complexes as a high-spin with potentially three unpaired electrons in an octahedral environment39. The Ni(II) complexes displayed magnetic moment values in the range of 3.41–3.56 B.M indicative of two unpaired electrons per Ni(II) ion suggesting these complexes to have an octahedral40 geometry. The measured magnetic moment values 1.66–1.74 B.M for Cu(II) complexes are indicative of one unpaired electron per Cu(II) ion for d⁹-system suggesting octahedral41 geometry. All the Zn(II) complexes were found to be diamagnetic42 as expected.

Electronic spectra

The UV-Visible spectra of all the ligands demonstrated two bands at 27 233–27 590 cm⁻¹ and 31 050–31 410 cm⁻¹, respectively. The first band appeared due to n–π^∗ transition of –C=N group, while the second band would be assigned to π–π^∗ transition of the phenyl group. After complexation, n–π^∗ transition of ligand shifted to a higher wavelength which revealed the coordination of ligand with metallic ions43,44. The electronic spectra of Co(II) complexes generally showed45 three absorption bands in the region 8650–8790, 17 523–17 800 and 29 805–29 841 cm⁻¹ which may be assigned to 4T₁g→4T₂g(F), 4T₁g→4A₂g(F) and 4T₁g→4Tg(P) transitions respectively, and are suggestive of octahedral geometry around the Co(II) ion. The electronic spectral data of Ni(II) complexes showed46 the bands in the region 8635–8786, 17 717–17 871 and 25 719–25 717 cm⁻¹ assigned, respectively, to the d–d transitions of 3A₂g(F)→3T₂g(F) and 3A₂g(F)→3T₁g(F). Also, a strong band due to metal to ligand charge transfer was appeared at 29 738–29 781 cm⁻¹. The electronic spectra of all the Cu(II) complexes exhibited47 absorption bands in the region at 8727–8937 and 17 765–17 892 cm⁻¹ which may be assigned to the transitions 2Eg→2T₂g. The high energy band at 29 788–29 858 cm⁻¹ was due to forbidden ligand to metal charge transfer. On the basis of electronic spectra, octahedral geometry around the Cu(II) ion was suggested. The Zn(II) complexes did not show any d–d transition thus showing diamagnetic nature and their spectra were dominated only by a charge transfer band48 at 28 841–28 932 cm⁻¹.

Biological screening

Antibacterial bioassay (in vitro)

The newly produced Schiff’s bases (L¹)–(L⁴) and their metal(II) complexes (1)–(16) were screened for their in vitro antibacterial activity against E. coli, S. flexneri, P. aeruginosa, S. typhi, S. aureus and B. subtilis bacterial strains according to the standard procedures26 and results are reported in Table 3. The obtained results were compared with those of the standardized drug sample of imipenum (Figure 1). The synthesized ligand (L¹) displayed a weaker (07–08 mm clearance zone (cz) or zone of inhibition) activity against E. coli and B. subtilis and the remaining strains displayed moderate (10–13 mm) activity. Ligand (L²) presented a weaker (8 mm) activity against S. typhi and the remaining strains showed moderate (9–13 mm) activity. Ligand (L³) demonstrated moderate (11–12 mm) activity against S. flexneri, P. aeruginosa and B. subtilis and, other remaining strains observed significant (16–17 mm) activity. The ligand (L⁴) experienced overall significant (15–17 mm) activity. The metal complex (1) showed moderate (14 mm) activity against E. coli and B. subtilis and other remaining strains experienced significant (15–19 mm) activity. Complex (2) showed moderate (12–14 mm) activity against P. aeruginosa and B. subtilis and left behind strains practiced significant (17–18 mm) activity. Compound (3) exhibited moderate (12–13 mm) activity against S. typhi and B. subtilis and, left over demonstrated significant (16–18 mm) activity. Beside this, the compound (4) revealed significant (15–20 mm) activity against all bacterial strains except E. coli which observed moderate (13 mm) activity. Also, the complexes (5) and (6) displayed moderate (12–14 mm) activity against Salmonella typhi and Staphylococcus aureus and, remaining strains revealed significant (17–19 mm) activity. However, the complexes (7)–(16) displayed overall significant (15–25 mm) activity against all bacterial strains. The data reported in Table 3 clearly indicates that (L⁴) exhibited overall good bacterial activity as compared to other three ligands. The Zn(II) complex (16) of (L⁴) were found to be the most active complexes. The metal(II) complexes showed40 higher activity results upon complexation rather than their uncomplexed Schiff’s bases.

Figure 1. Comparison of antibacterial activity of Schiff’s bases versus metal(II) complexes.

Table 3. Antibacterial bioassay of ligands (L¹)–(L⁴) and metal(II) complexes (1)–(16).

[Zone of inhibition (mm)]
	Gram-negative				Gram-positive
Compounds	(a)	(b)	(c)	(d)	(e)	(f)	(SA)	Average
L^1	08	13	11	10	13	07	2.50	10.33
L^2	13	11	12	08	09	11	1.86	10.67
L^3	16	12	11	17	16	12	2.61	14.00
L^4	16	17	15	17	16	15	0.89	16.00
1	14	19	17	15	19	14	2.59	16.50
2	17	18	12	17	18	14	2.45	16.00
3	16	17	18	13	16	12	2.34	15.33
4	13	16	19	16	20	15	2.59	16.50
5	18	17	19	14	12	18	2.73	16.33
6	16	18	16	13	14	16	1.76	15.50
7	17	19	15	16	16	16	1.38	16.50
8	20	17	19	15	16	17	2.50	17.67
9	20	21	17	22	22	18	2.10	20.00
10	22	16	19	19	23	21	2.53	20.00
11	20	17	16	21	19	20	1.94	18.83
12	18	24	19	23	20	21	2.32	20.83
13	21	23	16	20	19	18	2.58	19.67
14	20	17	21	22	24	22	2.37	21.00
15	19	22	17	19	25	20	2.80	20.33
16	22	21	22	24	21	21	1.17	21.83
SD	25	26	25	27	27	28	1.17	25.83

Average activity of ligand (L¹)–(L⁴) = 12.75 mm; Average activity of complexes (1)–(16) = 18.74 mm; (a) E. coli (b) S. flexneri (c) P. aeruginosa (d) S. typhi (e) S. aureus (f) B. subtilis; SD: standard drug (Imipenum); weaker = 0–08 mm, moderate = 09–14 mm, above 14 mm = significant, SA: statistical analysis.

Antifungal bioassay (in-vitro)

The antifungal screening of all the synthesized 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 protocol23. The results of inhibition were compared with the results of standard drugs, miconazole and amphotericin B (Figure 2). Ligand (L¹) exhibited significant (55%) activity against A. flavus fungal strain, moderate (37–45%) activity against T. longifusus, M. canis and C. glabrata and weaker (17%) activity against C. albicans and but inactive against F. solani begging further investigation why that may be? Similarly, the ligand (L²) possessed significant (56%) activity against C. albicans and moderate (38–49%) activity against T. longifusus, A. flavus, F. solani, weaker (30%) activity against C. glabrata but no activity was observed in A. flavus. However, the compound (L³) and (L⁴) displayed overall significant (55–63%) activity against all fungal strains. The complex (1) displayed significant (61–63%) activity against T. longifusus, A. flavus and C. glabrata fungal strains, moderate (37–50%) activity against C. albicans and M. canis, and weaker (16%) activity against F. solani. Likewise, complex (2) had significant (55–77%) activity against T. longifusus, A. flavus, M. canis, and C. glabrata, and displayed weaker (25–26%) activity against remaining two strains. Similarly, the compound 3 demonstrated significant (55–69%) activity against T. longifusus, A. flavus, M. canis, and C. glabrata, moderate (42%) against C. albicans and weaker (23%) activity against F. solani. The complex (4) comparably possessed significant (55–65%) activity against T. longifusus, A. flavus, M. canis, and C. glabrata and moderate (36–37%) activity was observed with rest of the fungal strains. On the other hand, the compound (5)–(8) showed overall significant (55–75%) activity against all strains except M. canis which displayed weaker (17–30%) activity. The compounds (9)–(16) displayed overall significant (55–78%) activity against fungal strains due to Br and NO₂ groups attached with the aromatic rings. (L⁴) showed overall good fungal activity as compared to other three ligands due to nitro substitution. The Co(II) complex (14) of (L⁴) was found to be the most active complex. The metal (II) complexes showed enhanced31 activity results rather than their uncomplexed Schiff’s bases upon complexation.

Figure 2. Comparison of antifungal activity of Schiff’s bases versus metal(II) complexes.

Table 4. Antifungal bioassay of ligands (L¹)–(L⁴) and metal(II) complexes (1)–(16).

[% Inhibition (mm)]
Compounds	(a)	(b)	(c)	(d)	(e)	(f)	(SA)	Average
L^1	45	17	55	37	00	40	20.18	32.33
L^2	38	56	49	00	43	30	19.77	36.00
L^3	56	60	61	56	63	59	2.79	59.17
L^4	62	58	59	63	61	55	2.94	59.67
1	62	37	63	49	16	61	18.63	48.00
2	55	25	75	77	26	58	22.81	52.67
3	59	42	62	55	23	69	16.66	51.67
4	65	36	64	56	37	57	12.91	52.50
5	57	68	66	17	75	66	20.97	58.17
6	61	69	72	28	69	71	16.94	61.67
7	55	73	67	30	66	75	16.72	61.00
8	56	64	68	25	68	69	17.03	58.33
9	71	75	70	67	72	74	2.88	71.50
10	75	69	57	73	70	67	6.32	68.50
11	69	74	70	65	69	65	3.39	68.67
12	70	78	68	74	73	60	6.19	70.50
13	72	75	70	61	72	64	5.37	69.00
14	78	65	57	73	70	67	7.20	68.33
15	69	58	70	65	72	65	5.01	66.50
16	75	68	72	77	79	68	4.62	73.17
SD	A	B	C	D	E	F	–	–

Average activity of ligands (L¹)–(L⁴) = 46.79%; Average activity of complexes (1)–(16) = 62.51%; (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); weaker = 0–33%, moderate = 34–54%, 55–100% = significant; SA = statistical analysis.

Minimum inhibitory concentration (MIC)

The synthesized ligands and their transition metal(II) complexes showing promising antibacterial activity (> 80%) were selected for MIC studies i.e. the compounds (9), (10) and (12)–(16) and the results are reported in Table 5. The MIC values of these compounds fall in the range 23.11–46.62 g/mL. Among these, the compound (14) was found to be the most active possessing maximum inhibition of 16.11 μg/mL against the bacterial strain S. Aureus.

Table 5. MIC (μg/mL) of the selected compounds (9), (10) and (12)–(16) against selected bacteria.

Compounds	E. coli	S. flexneri	P. aeruginosa	S. typhi	S. aureus
9	–	46.21	–	37.16	25.17
10	30.16	–	–	–	39.24
12	–	36.35	–	43.22	–
13	23.71	28.67	–	–	–
14	–	–	32.11	46.62	16.11
15	–	25.00	–	–	26.35
16	39.10	18.17	42.65	25.31	–

Cytotoxicity (in vitro)

The Schiff bases and their metal(II) complexes were screened for their cytotoxicity (brine shrimp bioassay) by using Meyer protocol28. The data recorded in Table 6 indicated that none of the compounds either ligands or complexes showed cytotoxicity (1.64 × 10⁻³ to 4.71 × 10⁻³) against Artemia salina. The cytotoxic data recorded in Table 6 revealed that almost all compounds, ligands as well as metal complexes were inactive but it was interesting to note that the metal complexes displayed better potent cytotoxicity as compared to their parent ligands. This activity relationship may help to serve as a basis for future direction toward the development of certain cytotoxic agents for clinical applications.

Table 6. Cytotoxicity (in vitro) of ligands (L¹)–(L⁴) and their metal(II) complexes (1)–(16).

Compounds	LD50 (M/mL)	Compounds	LD50 (M/mL)	Compounds	LD50 (M/mL)
L^1	>2.19 × 10^−3	4	>1.64 × 10^−3	11	>4.71 × 10^−3
L^2	>1.81 × 10^−3	5	>3.19 × 10^−3	12	>2.68 × 10^−3
L^3	>3.11 × 10^−3	6	>4.04 × 10^−3	13	>2.51 × 10^−3
L^4	>2.19 × 10^−3	7	>4.59 × 10^−4	14	>2.11 × 10^−3
1	>3.81 × 10^−3	8	>2.81 × 10^−3	15	>4.19 × 10^−4
2	>2.11 × 10^−3	9	>3.15 × 10^−3	16	>3.81 × 10^−3
3	>4.10 × 10^−3	10	>1.98 × 10^−3

Conclusions

The synthesized ethanol-derived Schiff’s bases act as bi and tridentate ligands for coordination with the Co(II), Cu(II), Ni(II) and Zn(II) metal atoms. Physical (magnetic and molar conductance), spectral (IR, NMR, electronic) and analytical (C, H, N and metalloelements percentage) data confirmed that the Schiff base ligands are coordinated with the Co(II), Cu(II), Ni(II) and Zn(II) metal atoms via azomethine-N, naphthalene-O, salicylidene-O and ethanol-O showing an octahedral geometry. The obtained results of antibacterial and antifungal activities indicated that the metal complexes possessed better biological activity against one or more bacterial and/or fungal strains as compared to their parent uncomplexed ligands. It can be asserted that azomethine-N, nitro, bromo and oxygen were the functional groups in the compounds potentially responsible for the enhancement of bacterial and fungal activities. However, brine shrimp assays revealed that these compounds were limited in their cytotoxicity hinting at some divergent apoptotic mechanisms.

Declaration of interest

The authors declare no conflict of interest.

Acknowledgements

The authors are grateful 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 antibacterial, antifungal and brine shrimp bioassays.