# Synthesis, Spectral Characterization, and Biological Activities of Some Metal Complexes Bearing an Unsymmetrical Salen-Type Ligand, (Z)-1-(((2-((E)-(2-Hydroxy-6-methoxybenzylidene)amino)phenyl)amino) methylene) Naphthalen-2(1H)-one

Authors: Quang Trung Nguyen; Phuong Nam Pham Thi; Quang Do Bui; Van Tuyen Nguyen
Journal: Heteroatom Chemistry
 (2023)

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## Abstract

An unsymmetrical salen-type Schiff base ligand, (Z)-1-(((2-((E)-(2-hydroxy-6-methoxybenzylidene)amino)phenyl)amino)methylene)naphthalen-2(1H)-one, and its Zn(II), Cu(II), Co(II), Mn(II), and Fe(III) complexes were synthesized and characterized by mass (MS), nuclear magnetic resonance (NMR), infrared (IR), ultraviolet-visible (UV-Vis) spectra, and effective magnetic moments. The thermal analyses of the obtained ligand and metal complexes were conducted by thermogravimetric analysis (TGA). Antimicrobial activity of the unsymmetrical Schiff base ligand and its metal complexes were examined forStaphylococcus aureus as Gram-positive bacteria and Escherichia coli as Gram-negative bacteria. In vitro anticancer property of synthetic compounds was estimated against human cancer cell lines, a subline of Hela tumor cell line (KB), and a human liver cancer cell line (HepG-2) as well.


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## Body

## 1. Introduction

From the discovery of cisplatin in the mid-1960s, metal-based anticancer candidates have received increasing attention nowadays [1]. Therefore, the development of novel metal-based compounds with potential biological activity has played an important role in bioinorganic chemistry. The nature of the metal and the structure of the organic ligands could be the main factors that can affect the therapeutic activity of such compounds [2].Salen-type Schiff base ligands with particularly attractive features in their structure and nature are one of the most useful and popular Schiff base ligands [3]. The popularity of tetradentate N2O2 bis-Schiff base ligands has come from their possibility of synthesis and ability to coordinate a high diversity of transition metals in various oxidation states and geometries [4, 5]. The bioactivity of metal complexes containing salen-type Schiff base ligands has been considerably studied. They can show rich biological applications as antibacterial, antifungal, and antitumor activities [6–8]. As usual symmetrical Schiff bases of 1,2‐ and 1,3‐diamines, where the same type of simple aldehydes or ketones are condensed on both nitrogen atoms, and their complexes are extensively reported [9–13]. However, unsymmetrical Schiff bases of diamines where two different carbonyl compounds are conducted on the nitrogen atoms have been paid attention recently [14–16]. Besides the electronic properties of unsymmetrical Schiff bases using different electron donating and/or withdrawing groups at the two imine units [17–19], various ions of transition metals coordinated with these unsymmetrical salen ligands were interestingly needed to study as well. In this research, we continue to report the synthesis and characterization of a new asymmetric salen-type Schiff base (Z)-1-(((2-((E)-(2-hydroxy-6-methoxybenzylidene)amino)phenyl)amino)methylene)naphthalen-2(1H)-one and its Zn(II), Cu(II), Co(II), Mn(II) and Fe(III) complexes. The antimicrobial activity of a Gram-positive bacterium (Staphylococcus aureus) and a Gram-negative bacterium (Escherichia coli), as well as in vitro cytotoxicity of synthetic compounds against human cancer cell lines, KB and HepG-2, were examined.


## 2. Materials and Methods

All Chemicals used in this study includingo-phenylenediamine (98%), 2-hydroxy-1-naphthaldehyde (tech.), Co(OAc)2.4H2O, and Mn(OAc)2.4H2O have been obtained from Across Organics. The other metal salts such as Zn(OAc)2.2H2O, CuCl2.2H2O 98.0%, FeCl3.6H2O 99.0%, and anhydrous Na2CO3 98.5% were purchased from Xilong Scientific Co., Ltd., China. The solvents were purified by following laboratory procedures. 2-Hydroxy-6-methoxybenzaldehyde was synthesized from 3-methoxyphenol by Reimer–Tiemann reaction [20]: 1H-NMR (CDCl3, 500 MHz, δ (ppm), J (Hz)): 11.96 (s, 1H-CHO), 10.34 (s, 1H-OH), 7.41 (t, J = 8.5, 1H-Ar), 6.52 (d, J = 8.5, 1H-Ar), 6.37 (d, J = 8.5, 1H-Ar), 3.89 (s, 3H-MeO).


### 2.1. Instrumentation

A Bruker Advance 500 MHz NMR spectrometer was used to record NMR spectra using d6-DMSO as the solvent. The high-resolution mass spectrometry (m/z) was measured on a Sciex X500 QTOF spectrometer as +IDA-TOF-MS. On Agilent 6310 Ion Trap spectrometer, mass spectra (m/z) were determined in electrospray ionization (ESI) mode. FT-IR (KBr pellet, 400–4000 cm−1) spectra were taken with a Perkin Elmer Spectrum Two spectrophotometer. UV-Visible absorption spectra of the synthetic compounds were measured on A Perkin Elmer Lambda UV-35 spectrophotometer in methanol solution (3.0 × 10−5 M). Magnetic susceptibility measurements of obtained complexes were determined at room temperature using a magnetic susceptibility balance, Mark 1 with serial No. 25179, of Sherwood Scientific Ltd. Thermogravimetric analysis was carried out on Setaram thermal analyzer, Labsys TG/DSC 1600, under a dynamic flow of air (100 µl/min) and a heating rate of 10°C/min from ambient temperature to about 870°C.


### 2.2. Synthesis of (Z)-1-(((2-((E)-(2-Hydroxy-6-methoxybenzylidene)amino)phenyl)amino)methylene) Naphthalen-2(1H)-One

This unsymmetrical salen-type ligand was prepared by one-pot method including two-step reaction similarly according to the known procedure [18, 19, 21].(Z)-1-(((2-((E)-(2-hydroxy-6-methoxybenzylidene)amino)phenyl)amino)methylene)naphthalen-2(1H)-one (H2L): yellow solid, 72.5%; +IDA-TOF-MS (m/z) (Figure S1a): 397.1535 [M+] (Cal. 397.4459); FT-IR (KBr, cm−1) (Figure S4a): 2924 (ν, C-H), 2615 (ν, O-H), 1607 (ν, C=N), 1562 (ν, C=C), 1470, 1316, and 1250 (ν, C-N), 1185 (ν, C-O), 1089, 824, 738 (δ, C-H), 469; 1H-NMR (DMSO-d6, 500 MHz, δ (ppm) (Figures S2a and S2b), J (Hz)): δ 15.55 (d, J = 6.0, 1H, NH), 13.33 (s, 1H, OH), 9.62 (d, J = 6.0, 1H, HC-N), 9.12 (s, 1H, HC=N), 8.46 (d, J = 8.0, 1H, Naph), 7.91 (t, J = 8.0, 2H, Naph), 7.76 (d, J = 7.0, 1H, 1H-Naph), 7.50 (t, J = 7.0, 1H, Sal), 7.42 (t, J = 8.0, 1H, 1H-Ph), 7.42-7.37 (m, 3H, 1H-Naph, 2H-Ph), 7.33 (t, J = 7.0, 1H, Ph), 6.96 (d, J = 9.0, 1H, Sal), 6.58 (m, 2H, 1H-Naph, 1H-Sal), 3.87 (s, 3H, OCH3); 13C-NMR (DMSO-d6, 125 MHz, δ (ppm)) (Figures S2c and S2d): δ 171.78 (1C, C=O), 161.85 (1C, C=N), 160.39 (1C, C-OH), 160.17 (1C, C-OCH3), 154.73 (1C, HC-NH), 141.46 (1C, N-CAr), 137.40 (1C, HN-CAr), 137.17 (1C, Naph), 135.18 (1C, Naph), 133.16 (1C, Sal), 128.91 (1C, Naph), 128.04 (1C, Naph), 127.68 (1C, Naph), 127.23 (1C, Naph), 126.48 (1C, Ph), 123.43 (1C, Naph), 122.54 (1C, Ph), 120.23 (1C, Ph), 120.08 (1C, Naph), 118.93 (1C, Naph), 109.38 (1C, Ph), 108.73 (1C, Sal), 108.48 (1C, Sal), 101.28 (1C, Sal), 56.01 (1C, OCH3); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1 M−1) (Figure S5a): 231 (30,666), 273 (14,667), 336 (14,000), 450 (7,333), 470 (6,667).


### 2.3. Preparation of Unsymmetrical Salen-Type Complexes

Unsymmetrical salen-type Schiff base complexes of Zn(II), Cu(II), Co(II), Mn(II), and Fe(III) were prepared from the obtained ligand with each metal salt of Zn(OAc)2.2H2O, CuCl2.2H2O, Co(OAc)2.4H2O, Mn(OAc)2.4H2O, and FeCl3.6H2O in 1 : 1 molecular ratio following the published procedure similarly [22–24] in the presence of Na2CO3 and ethanol as solvent.


#### 2.3.1. [Zn(L).H2O]

Yellow solid, yield 85%.ESI-MS (m/z) (Figure S1b): 458.9 [M-H2O]+ (Cal. 460.8); FT-IR (KBr, cm−1) (Figure S4b): 3207 (ν, O-H, H2O), 3029 (ν, C-H), 1610 (ν, C=N), 1538 (ν, C=C), 1433, 1380, 1248 (ν, C-N), 1184 (ν, C-O), 1098, 829, 740 (δ, C-H), 551 (Zn-N), 451 (Zn-O); 1H-NMR (DMSO-d6, 500 MHz, δ (ppm), J (Hz)) (Figures S3a and S3b): δ 9.76 (s, 1H, HC-N), 9.40 (s, 1H, HC=N), 8.41 (d, J = 7.0, 1H, Naph), 8.09 (dd, J = 7.0; 1.0, 1H, Naph), 7.78 (d, J = 7.5, 1H, Naph), 7.68 (m, 2H, Naph), 7.46 (dt, J = 6.0; 1.0, 1H, Sal), 7.38 (m, 2H, Ph), 7.23-7.18 (m, 2H, Ph), 6.95 (d, J = 8.0, 1H, Sal), 6.34 (d, J = 7.0, 1H-Sal), 6.07 (d, J = 6.5, 1H-Naph), 3.85 (s, 3H, OCH3); 13C-NMR (DMSO-d6, 125 MHz, δ (ppm)) (Figures S3c and S3d): δ 173.72 (1C, C=O), 173.14 (1C, C=N), 161.61 (1C, C-OH), 156.26 (1C, C-OCH3), 156.03 (1C, HC-N), 140.57 (1C, CAr-N), 139.60 (1C, CAr-N), 135.78 (1C, Naph), 135.55 (1C, Naph), 135.07 (1C, Sal), 128.74 (1C, Naph), 127.46 (1C, Naph), 127.14 (1C, Naph), 126.96 (1C, Naph), 126.69 (1C, Ph), 125.61 (1C, Naph), 121.67 (1C, Ph), 119.53 (1C, Ph), 116.93 (1C, Naph), 116.27 (1C, Naph), 115.74 (1C, Ph), 109.73 (1C, Sal), 108.96 (1C, Sal), 94.49 (1C, Sal), 55.72 (1C, OCH3); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5b): 244 (24,667), 268 (12,000), 335 (13,333), 403 (10,667), 445 (8,000); diamagnetic.


#### 2.3.2. [Cu(L)]

Reddish brown solid, yield 89%.ESI-MS (m/z) (Figure S1c): 457.9 [M]+ (Cal. 458.9); FT-IR (KBr, cm−1) (Figure S4c): 3064 (ν, C-H), 1602 (ν, C=N), 1532 (ν, C=C), 1433, 1361, 1250 (ν, C-N), 1191 (ν, C-O), 1095, 830, 737 (δ, C-H), 536 (Cu-N), 471 (Cu-O); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5c): 245 (32,667), 270 (22,000), 345 (17,333), 362 (16,667), 425 (13,667); µeff = 2.03 BM.


#### 2.3.3. [Co(L)]

Brown solid, yield 91%.ESI-MS (m/z) (Figure S1d): 453.8 [M]+ (Cal. 454.3); FT-IR (KBr, cm−1) (Figure S4d): 3033 (ν, C-H), 1603 (ν, C=N), 1527 (ν, C=C), 1455, 1354, 1253 (ν, C-N), 1197 (ν, C-O), 1095, 833, 738 (δ, C-H), 563 (Co-N), 504 (Co-O); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5d): 258 (64,000), 374 (26,333), 470 (13,333); µeff = 4.34 BM.


#### 2.3.4. [Mn(L)]

Brown solid, yield 83%.ESI-MS (m/z) (Figure S1e): 448.9 [M] + (Cal. 450.3); FT-IR (KBr, cm−1) (Figure S4e): 3030 (ν, C-H), 1602 (ν, C=N), 1532 (ν, C=C), 1426, 1366, 1255 (ν, C-N), 1196 (ν, C-O), 1092, 823, 740 (δ, C-H), 557 (Mn-N), 503 (Mn-O); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5e): 247 (43,667), 375 (23,333), 453 (10,333); µeff = 5.85 BM.


#### 2.3.5. [Fe(L) (Cl)]

Brown solid, yield 91%.ESI-MS (m/z) (Figure S1f): 449.9 [M-Cl]+ (Cal. 450.2); FT-IR (KBr, cm−1) (Figure S4f): 2924 (ν, C-H), 1597 (ν, C=N), 1533 (ν, C=C), 1456, 1362, 1256 (ν, C-N), 1194 (ν, C-O), 1092, 837, 742 (δ, C-H), 563 (Fe-N), 481 (Fe-O), 411 (Fe-Cl); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5f): 225 (29,000), 272 (15,433), 301 (16,333), 350 (16,667), 433 (8,000); µeff = 6.08 BM.


## 2.1. Instrumentation

A Bruker Advance 500 MHz NMR spectrometer was used to record NMR spectra using d6-DMSO as the solvent. The high-resolution mass spectrometry (m/z) was measured on a Sciex X500 QTOF spectrometer as +IDA-TOF-MS. On Agilent 6310 Ion Trap spectrometer, mass spectra (m/z) were determined in electrospray ionization (ESI) mode. FT-IR (KBr pellet, 400–4000 cm−1) spectra were taken with a Perkin Elmer Spectrum Two spectrophotometer. UV-Visible absorption spectra of the synthetic compounds were measured on A Perkin Elmer Lambda UV-35 spectrophotometer in methanol solution (3.0 × 10−5 M). Magnetic susceptibility measurements of obtained complexes were determined at room temperature using a magnetic susceptibility balance, Mark 1 with serial No. 25179, of Sherwood Scientific Ltd. Thermogravimetric analysis was carried out on Setaram thermal analyzer, Labsys TG/DSC 1600, under a dynamic flow of air (100 µl/min) and a heating rate of 10°C/min from ambient temperature to about 870°C.


## 2.2. Synthesis of (Z)-1-(((2-((E)-(2-Hydroxy-6-methoxybenzylidene)amino)phenyl)amino)methylene) Naphthalen-2(1H)-One

This unsymmetrical salen-type ligand was prepared by one-pot method including two-step reaction similarly according to the known procedure [18, 19, 21].(Z)-1-(((2-((E)-(2-hydroxy-6-methoxybenzylidene)amino)phenyl)amino)methylene)naphthalen-2(1H)-one (H2L): yellow solid, 72.5%; +IDA-TOF-MS (m/z) (Figure S1a): 397.1535 [M+] (Cal. 397.4459); FT-IR (KBr, cm−1) (Figure S4a): 2924 (ν, C-H), 2615 (ν, O-H), 1607 (ν, C=N), 1562 (ν, C=C), 1470, 1316, and 1250 (ν, C-N), 1185 (ν, C-O), 1089, 824, 738 (δ, C-H), 469; 1H-NMR (DMSO-d6, 500 MHz, δ (ppm) (Figures S2a and S2b), J (Hz)): δ 15.55 (d, J = 6.0, 1H, NH), 13.33 (s, 1H, OH), 9.62 (d, J = 6.0, 1H, HC-N), 9.12 (s, 1H, HC=N), 8.46 (d, J = 8.0, 1H, Naph), 7.91 (t, J = 8.0, 2H, Naph), 7.76 (d, J = 7.0, 1H, 1H-Naph), 7.50 (t, J = 7.0, 1H, Sal), 7.42 (t, J = 8.0, 1H, 1H-Ph), 7.42-7.37 (m, 3H, 1H-Naph, 2H-Ph), 7.33 (t, J = 7.0, 1H, Ph), 6.96 (d, J = 9.0, 1H, Sal), 6.58 (m, 2H, 1H-Naph, 1H-Sal), 3.87 (s, 3H, OCH3); 13C-NMR (DMSO-d6, 125 MHz, δ (ppm)) (Figures S2c and S2d): δ 171.78 (1C, C=O), 161.85 (1C, C=N), 160.39 (1C, C-OH), 160.17 (1C, C-OCH3), 154.73 (1C, HC-NH), 141.46 (1C, N-CAr), 137.40 (1C, HN-CAr), 137.17 (1C, Naph), 135.18 (1C, Naph), 133.16 (1C, Sal), 128.91 (1C, Naph), 128.04 (1C, Naph), 127.68 (1C, Naph), 127.23 (1C, Naph), 126.48 (1C, Ph), 123.43 (1C, Naph), 122.54 (1C, Ph), 120.23 (1C, Ph), 120.08 (1C, Naph), 118.93 (1C, Naph), 109.38 (1C, Ph), 108.73 (1C, Sal), 108.48 (1C, Sal), 101.28 (1C, Sal), 56.01 (1C, OCH3); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1 M−1) (Figure S5a): 231 (30,666), 273 (14,667), 336 (14,000), 450 (7,333), 470 (6,667).


## 2.3. Preparation of Unsymmetrical Salen-Type Complexes

Unsymmetrical salen-type Schiff base complexes of Zn(II), Cu(II), Co(II), Mn(II), and Fe(III) were prepared from the obtained ligand with each metal salt of Zn(OAc)2.2H2O, CuCl2.2H2O, Co(OAc)2.4H2O, Mn(OAc)2.4H2O, and FeCl3.6H2O in 1 : 1 molecular ratio following the published procedure similarly [22–24] in the presence of Na2CO3 and ethanol as solvent.


### 2.3.1. [Zn(L).H2O]

Yellow solid, yield 85%.ESI-MS (m/z) (Figure S1b): 458.9 [M-H2O]+ (Cal. 460.8); FT-IR (KBr, cm−1) (Figure S4b): 3207 (ν, O-H, H2O), 3029 (ν, C-H), 1610 (ν, C=N), 1538 (ν, C=C), 1433, 1380, 1248 (ν, C-N), 1184 (ν, C-O), 1098, 829, 740 (δ, C-H), 551 (Zn-N), 451 (Zn-O); 1H-NMR (DMSO-d6, 500 MHz, δ (ppm), J (Hz)) (Figures S3a and S3b): δ 9.76 (s, 1H, HC-N), 9.40 (s, 1H, HC=N), 8.41 (d, J = 7.0, 1H, Naph), 8.09 (dd, J = 7.0; 1.0, 1H, Naph), 7.78 (d, J = 7.5, 1H, Naph), 7.68 (m, 2H, Naph), 7.46 (dt, J = 6.0; 1.0, 1H, Sal), 7.38 (m, 2H, Ph), 7.23-7.18 (m, 2H, Ph), 6.95 (d, J = 8.0, 1H, Sal), 6.34 (d, J = 7.0, 1H-Sal), 6.07 (d, J = 6.5, 1H-Naph), 3.85 (s, 3H, OCH3); 13C-NMR (DMSO-d6, 125 MHz, δ (ppm)) (Figures S3c and S3d): δ 173.72 (1C, C=O), 173.14 (1C, C=N), 161.61 (1C, C-OH), 156.26 (1C, C-OCH3), 156.03 (1C, HC-N), 140.57 (1C, CAr-N), 139.60 (1C, CAr-N), 135.78 (1C, Naph), 135.55 (1C, Naph), 135.07 (1C, Sal), 128.74 (1C, Naph), 127.46 (1C, Naph), 127.14 (1C, Naph), 126.96 (1C, Naph), 126.69 (1C, Ph), 125.61 (1C, Naph), 121.67 (1C, Ph), 119.53 (1C, Ph), 116.93 (1C, Naph), 116.27 (1C, Naph), 115.74 (1C, Ph), 109.73 (1C, Sal), 108.96 (1C, Sal), 94.49 (1C, Sal), 55.72 (1C, OCH3); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5b): 244 (24,667), 268 (12,000), 335 (13,333), 403 (10,667), 445 (8,000); diamagnetic.


### 2.3.2. [Cu(L)]

Reddish brown solid, yield 89%.ESI-MS (m/z) (Figure S1c): 457.9 [M]+ (Cal. 458.9); FT-IR (KBr, cm−1) (Figure S4c): 3064 (ν, C-H), 1602 (ν, C=N), 1532 (ν, C=C), 1433, 1361, 1250 (ν, C-N), 1191 (ν, C-O), 1095, 830, 737 (δ, C-H), 536 (Cu-N), 471 (Cu-O); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5c): 245 (32,667), 270 (22,000), 345 (17,333), 362 (16,667), 425 (13,667); µeff = 2.03 BM.


### 2.3.3. [Co(L)]

Brown solid, yield 91%.ESI-MS (m/z) (Figure S1d): 453.8 [M]+ (Cal. 454.3); FT-IR (KBr, cm−1) (Figure S4d): 3033 (ν, C-H), 1603 (ν, C=N), 1527 (ν, C=C), 1455, 1354, 1253 (ν, C-N), 1197 (ν, C-O), 1095, 833, 738 (δ, C-H), 563 (Co-N), 504 (Co-O); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5d): 258 (64,000), 374 (26,333), 470 (13,333); µeff = 4.34 BM.


### 2.3.4. [Mn(L)]

Brown solid, yield 83%.ESI-MS (m/z) (Figure S1e): 448.9 [M] + (Cal. 450.3); FT-IR (KBr, cm−1) (Figure S4e): 3030 (ν, C-H), 1602 (ν, C=N), 1532 (ν, C=C), 1426, 1366, 1255 (ν, C-N), 1196 (ν, C-O), 1092, 823, 740 (δ, C-H), 557 (Mn-N), 503 (Mn-O); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5e): 247 (43,667), 375 (23,333), 453 (10,333); µeff = 5.85 BM.


### 2.3.5. [Fe(L) (Cl)]

Brown solid, yield 91%.ESI-MS (m/z) (Figure S1f): 449.9 [M-Cl]+ (Cal. 450.2); FT-IR (KBr, cm−1) (Figure S4f): 2924 (ν, C-H), 1597 (ν, C=N), 1533 (ν, C=C), 1456, 1362, 1256 (ν, C-N), 1194 (ν, C-O), 1092, 837, 742 (δ, C-H), 563 (Fe-N), 481 (Fe-O), 411 (Fe-Cl); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5f): 225 (29,000), 272 (15,433), 301 (16,333), 350 (16,667), 433 (8,000); µeff = 6.08 BM.


## 2.3.1. [Zn(L).H2O]

Yellow solid, yield 85%.ESI-MS (m/z) (Figure S1b): 458.9 [M-H2O]+ (Cal. 460.8); FT-IR (KBr, cm−1) (Figure S4b): 3207 (ν, O-H, H2O), 3029 (ν, C-H), 1610 (ν, C=N), 1538 (ν, C=C), 1433, 1380, 1248 (ν, C-N), 1184 (ν, C-O), 1098, 829, 740 (δ, C-H), 551 (Zn-N), 451 (Zn-O); 1H-NMR (DMSO-d6, 500 MHz, δ (ppm), J (Hz)) (Figures S3a and S3b): δ 9.76 (s, 1H, HC-N), 9.40 (s, 1H, HC=N), 8.41 (d, J = 7.0, 1H, Naph), 8.09 (dd, J = 7.0; 1.0, 1H, Naph), 7.78 (d, J = 7.5, 1H, Naph), 7.68 (m, 2H, Naph), 7.46 (dt, J = 6.0; 1.0, 1H, Sal), 7.38 (m, 2H, Ph), 7.23-7.18 (m, 2H, Ph), 6.95 (d, J = 8.0, 1H, Sal), 6.34 (d, J = 7.0, 1H-Sal), 6.07 (d, J = 6.5, 1H-Naph), 3.85 (s, 3H, OCH3); 13C-NMR (DMSO-d6, 125 MHz, δ (ppm)) (Figures S3c and S3d): δ 173.72 (1C, C=O), 173.14 (1C, C=N), 161.61 (1C, C-OH), 156.26 (1C, C-OCH3), 156.03 (1C, HC-N), 140.57 (1C, CAr-N), 139.60 (1C, CAr-N), 135.78 (1C, Naph), 135.55 (1C, Naph), 135.07 (1C, Sal), 128.74 (1C, Naph), 127.46 (1C, Naph), 127.14 (1C, Naph), 126.96 (1C, Naph), 126.69 (1C, Ph), 125.61 (1C, Naph), 121.67 (1C, Ph), 119.53 (1C, Ph), 116.93 (1C, Naph), 116.27 (1C, Naph), 115.74 (1C, Ph), 109.73 (1C, Sal), 108.96 (1C, Sal), 94.49 (1C, Sal), 55.72 (1C, OCH3); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5b): 244 (24,667), 268 (12,000), 335 (13,333), 403 (10,667), 445 (8,000); diamagnetic.


## 2.3.2. [Cu(L)]

Reddish brown solid, yield 89%.ESI-MS (m/z) (Figure S1c): 457.9 [M]+ (Cal. 458.9); FT-IR (KBr, cm−1) (Figure S4c): 3064 (ν, C-H), 1602 (ν, C=N), 1532 (ν, C=C), 1433, 1361, 1250 (ν, C-N), 1191 (ν, C-O), 1095, 830, 737 (δ, C-H), 536 (Cu-N), 471 (Cu-O); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5c): 245 (32,667), 270 (22,000), 345 (17,333), 362 (16,667), 425 (13,667); µeff = 2.03 BM.


## 2.3.3. [Co(L)]

Brown solid, yield 91%.ESI-MS (m/z) (Figure S1d): 453.8 [M]+ (Cal. 454.3); FT-IR (KBr, cm−1) (Figure S4d): 3033 (ν, C-H), 1603 (ν, C=N), 1527 (ν, C=C), 1455, 1354, 1253 (ν, C-N), 1197 (ν, C-O), 1095, 833, 738 (δ, C-H), 563 (Co-N), 504 (Co-O); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5d): 258 (64,000), 374 (26,333), 470 (13,333); µeff = 4.34 BM.


## 2.3.4. [Mn(L)]

Brown solid, yield 83%.ESI-MS (m/z) (Figure S1e): 448.9 [M] + (Cal. 450.3); FT-IR (KBr, cm−1) (Figure S4e): 3030 (ν, C-H), 1602 (ν, C=N), 1532 (ν, C=C), 1426, 1366, 1255 (ν, C-N), 1196 (ν, C-O), 1092, 823, 740 (δ, C-H), 557 (Mn-N), 503 (Mn-O); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5e): 247 (43,667), 375 (23,333), 453 (10,333); µeff = 5.85 BM.


## 2.3.5. [Fe(L) (Cl)]

Brown solid, yield 91%.ESI-MS (m/z) (Figure S1f): 449.9 [M-Cl]+ (Cal. 450.2); FT-IR (KBr, cm−1) (Figure S4f): 2924 (ν, C-H), 1597 (ν, C=N), 1533 (ν, C=C), 1456, 1362, 1256 (ν, C-N), 1194 (ν, C-O), 1092, 837, 742 (δ, C-H), 563 (Fe-N), 481 (Fe-O), 411 (Fe-Cl); UV-Vis (MeOH, 3 × 10−5 M, λ/nm, ε/cm−1·M−1) (Figure S5f): 225 (29,000), 272 (15,433), 301 (16,333), 350 (16,667), 433 (8,000); µeff = 6.08 BM.


## 3. Biological Properties

### 3.1. Antimicrobial Activity

In vitro antimicrobial activity of H2L and its metal complexes were evaluated using the disk diffusion method [15] against a Gram-positive bacterium, Staphylococcus aureus (ATCC 13709), and a Gram-negative bacterium, Escherichia coli (ATCC 25922), which were previously grown in Mueller–Hinton Agar (MHA) medium. The tested solutions of 1 mg/mL in DMSO were prepared for all the unsymmetrical salen-type Schiff base ligands and their metal complexes. The MHA (15 mL) kept at ca. 45°C was poured into the 90 mm Petri dishes which were inoculated with the bacterial strains of 100 µL from their culture media (ca. 106 CFU/mL) and allowed to solidify. Then, holes of 6 mm diameter were punched carefully. These holes were filled with 50 µL of the tested solutions. Petri dishes were incubated for 24 hrs at 37°C. The diameter of the inhibition zone in millimeters (mm) of each sample was determined and used to evaluate the antimicrobial activity of tested compounds which was performed in Table 1 and Supplementary File S7. Ampicillin was used as the standard reference drug. DMSO alone was used as the negative control under the same condition for each microorganism.Table 1
Antibacterial activities of the unsymmetrical salen-type ligand and its metal complexes.
CompoundDiameter of inhibition zone (mm)S. aureusE. coliH2L9.00[Zn(L).H2O]00[Cu(L)]00[Co(L)]00[Mn(L)]16.50[Fe(L)Cl]12.00Ampicillin27.011.0DMSO00


### 3.2. Cytotoxicity Assay

Colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays were used to estimatein vitro cytotoxicity of the unsymmetrical salen-type Schiff base ligand H2L and their complexes against human cancer cell lines, KB and HepG-2, according to Mosmann’s modified method [25, 26]. The IC50 (the 50% inhibition concentration was defined as the compound concentration causing 50% inhibition of cell growth) values were estimated and presented in Table 2.Table 2
Cytotoxicity ofH2L and its metal complexes.
CompoundIC50 (µM)KBHep-G2H2L46.14 ± 3.7843.29 ± 3.57[Zn(L).H2O]>250>250[Cu(L)]>250>250[Co(L)]4.45 ± 0.267.40 ± 0.62[Mn(L)]4.68 ± 0.653.67 ± 0.27[Fe(L)Cl]2.64 ± 0.116.07 ± 0.44Ellipticine1.22 ± 0.201.34 ± 0.20


## 3.1. Antimicrobial Activity

In vitro antimicrobial activity of H2L and its metal complexes were evaluated using the disk diffusion method [15] against a Gram-positive bacterium, Staphylococcus aureus (ATCC 13709), and a Gram-negative bacterium, Escherichia coli (ATCC 25922), which were previously grown in Mueller–Hinton Agar (MHA) medium. The tested solutions of 1 mg/mL in DMSO were prepared for all the unsymmetrical salen-type Schiff base ligands and their metal complexes. The MHA (15 mL) kept at ca. 45°C was poured into the 90 mm Petri dishes which were inoculated with the bacterial strains of 100 µL from their culture media (ca. 106 CFU/mL) and allowed to solidify. Then, holes of 6 mm diameter were punched carefully. These holes were filled with 50 µL of the tested solutions. Petri dishes were incubated for 24 hrs at 37°C. The diameter of the inhibition zone in millimeters (mm) of each sample was determined and used to evaluate the antimicrobial activity of tested compounds which was performed in Table 1 and Supplementary File S7. Ampicillin was used as the standard reference drug. DMSO alone was used as the negative control under the same condition for each microorganism.Table 1
Antibacterial activities of the unsymmetrical salen-type ligand and its metal complexes.
CompoundDiameter of inhibition zone (mm)S. aureusE. coliH2L9.00[Zn(L).H2O]00[Cu(L)]00[Co(L)]00[Mn(L)]16.50[Fe(L)Cl]12.00Ampicillin27.011.0DMSO00


## 3.2. Cytotoxicity Assay

Colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assays were used to estimatein vitro cytotoxicity of the unsymmetrical salen-type Schiff base ligand H2L and their complexes against human cancer cell lines, KB and HepG-2, according to Mosmann’s modified method [25, 26]. The IC50 (the 50% inhibition concentration was defined as the compound concentration causing 50% inhibition of cell growth) values were estimated and presented in Table 2.Table 2
Cytotoxicity ofH2L and its metal complexes.
CompoundIC50 (µM)KBHep-G2H2L46.14 ± 3.7843.29 ± 3.57[Zn(L).H2O]>250>250[Cu(L)]>250>250[Co(L)]4.45 ± 0.267.40 ± 0.62[Mn(L)]4.68 ± 0.653.67 ± 0.27[Fe(L)Cl]2.64 ± 0.116.07 ± 0.44Ellipticine1.22 ± 0.201.34 ± 0.20


## 4. Results and Discussion

### 4.1. Synthesis and Spectral Characterization

The ligand and complexes were obtained in high yields (72.5 and 83–91%), and they are stable to air and moisture. The synthetic compounds can be dissolved in dichloromethane as well as in methanol, acetonitrile, and dimethylsulfoxide. The ligand was characterized by +IDA-TOF-MS, FT-IR, NMR, and UV-Vis spectra. Its complexes were analysed by ESI-MS, IR, UV-Vis spectroscopies, and magnetic susceptibility. On mass spectra, the pseudomolecular ion signals of the ligand and complexes are found as [M]+, [M-H2O]+ or [M-Cl]+ which are quite suitable to the calculated masses for suggested formulae.NMR spectra of the ligandH2L were recorded in DMSO-d6. On 1H-NMR, there were typical signals of one double peak at 15.55 ppm for 1 NH proton, a single peak at 13.33 ppm for 1 OH proton, a double peak at 9.62 ppm for 1 HC-N proton, and a single peak at 9.12 ppm for HC=N proton. This is proved by tautomerism between enol-imine and keto-amine forms when 1 proton of NH and 1 proton of OH were found (Scheme 1). The protons of aromatic rings were found at 8.46-6.57 ppm and the protons of the CH3O group at 3.87 ppm as a single peak. On 13C-NMR of H2L, there were characteristic signals of C=O carbon at 171.78 ppm, C=N carbon at 161.85 ppm, C-OH carbon at 160.37 ppm, and C-NH carbon at 154.73 ppm. The other aromatic carbon peaks were observed at 141.46-101.28 ppm. The methoxy carbons were found at 56.01 ppm as a single signal. Difference from paramagnetic complexes, the NMR spectra of diamagnetic Zn(II) complex can be recorded by NMR spectrometer. On the 1H-NMR spectrum, the absence of NH and OH protons for the Zn(II) complex ensured the deprotonation of NH and OH protons upon complexation with Zn(II). The protons of HC-N and HC=N groups at 9.76 and 9.40 ppm, respectively, were shifted downfield compared to the ones of H2L when the coordination was observed. The aromatic protons appeared at 8.41-6.07 ppm, and methoxy protons were found at 3.85 ppm as a single peak reasonably. On the 13C-NMR spectrum, the typical signals of C=O at 173.72 ppm, C=N at 173.14 ppm, C-OH at 161.61 ppm, and C-NH at 156.03 ppm were shifted to downfield compared to the free ligand which confirmed the coordination between H2L with Zn(II) at nitrogen and oxygen atoms. The observed peaks of aromatic carbons were at 140.57-94.49 ppm, and the methoxy carbon was found at 55.72 ppm.Scheme 1
Synthesis of the unsymmetrical salen-type ligand and its metal complexes.On the IR spectrum ofH2L, the weak broad signal at 2615 cm−1 can be attributed to the stretching vibration of the o-O-H group [27]. There are the characteristic signals at 1607 cm−1 for the stretching vibration of C=N, at 1250 cm−1 for νC-N, and at 1185 cm−1 for νC-O. The disappearance of νO-H and the presence of new bands at 563-536 and 504-451 cm−1 for M-N and M-O bondings, respectively, on the IR spectra of the obtained complexes proved the coordination of metals and the ligand [17, 21, 28]. The characteristic bands for νC=N, νC-N, and νC-O in the complexes are lightly moved to the higher or lower field from the bands in the free ligand. The IR spectra data assured that the coordination of the ligand to the studied metals at nitrogen and oxygen atoms. On the IR spectrum of [Zn(L).H2O], there was a typical signal at 3207 cm−1 which may be assigned to the stretching vibration of O-H of coordinated H2O [29–32].UV-Visible spectra ofH2L and its metal complexes were recorded in methanol solution and presented in Figure 1. The UV-Vis spectrum of H2L showed main bands at 231–273 nm assigned to the π-π∗ electronic transition of aromatic rings, at 336 nm relative to n-π∗ transition of C=N and C-O groups and at 450 nm attributed to the n-π∗ transition within C=O [21, 33]. During the coordination of the ligand to metals, the electronic absorption spectra of synthetic complexes show new MLCT bands at 403 nm for Zn(II) complex, at 425 nm for Cu(II) complex, at 470 nm for Co(II) complex [34–36], at 453 nm for Mn(II) complex, and at 433 nm for Fe(III) complex. The d-d absorptions seem to have no appearance at this complexes’ concentration in methanol solution (3 × 10−5 M). The effective magnetic moments were calculated to be diamagnetic for Zn(II) complex [36, 37]. The effective magnetic moments measured experimentally were 2.03 BM corresponding to one unpaired electron supporting the distorted square-planar geometry for Cu(II) complex [37, 38]; 4.34 BM relative to the presence of three unpaired electrons in the octahedral structure for Co(II) complex [33, 37]; 5.85 BM indicated the octahedral geometry for Mn(II) complex [37, 39]; and 6.08 BM due to the presence of five unpaired electrons attributed the octahedral structure for Fe(III) complex [37, 40] probably.Figure 1
Electronic absorption spectra of obtained compounds.


### 4.2. Thermogravimetric Analysis

The thermal analysis of compounds was based on the thermogravimetric analysis (TGA) method at the temperature of 40–870°C. The results of thermal analysis of the ligand and metal complexes in this study based on the degradation steps were used to evaluate the water molecules, the chemical compositions, and the residual amount of the tested compounds (Table3) (Figures S6a–S6f). The ligand decomposition behaviour comprises two steps in which the first step, decomposition at 140–320°C, is assigned to the loss of methoxy and hydroxy groups (observed about 16.3%, calculated about 16.4%). The second step of thermal decomposition, which occurs at 320–870°C, is assigned to the loss of all most organic compounds (observed at about 76.8%, calculated at 77.5%). Above 870°C, two carbon atoms are still remaining.Table 3
TGA data of the ligand and its metal complexes.
CompoundTemperature range (°C)Weight loss (%)AssignmentsFoundCalc.H2L150–320320–870>87016.376.86.916.477.56.1Loss of methoxy and hydroxyl groupsLoss of all most organic residue2C atoms[Zn(L).H2O]150–250250–375375–870>8703.86.567.821.93.86.567.722.0Loss of a coordinated H2OLoss of methoxy groupLoss of 2C4H4 groups and C14H7N2OZnO and 2C atom[Cu(L)]45–150150–410410–870>870∼17.757.924.4∼18.159.222.7Loss of uncoordinated H2OLoss of methoxy and C4H4 groupsLoss of C4H4 group and C14H7N2OCuO and 2C atom[Co(L)]40–150150–440440–850>850∼18.864.416.8∼18.365.116.6Loss of uncoordinated H2OLoss of methoxy and C4H4 groupsLoss of C4H4 group and C16H7N2OCoO[Mn(L)]40–150150–500500–865>865∼34.050.016.0∼30.154.115.8Loss of uncoordinated H2OLoss of methoxy and 2C4H4 groupsLoss of the remain organic C16H7N2OMnO[Fe(L)Cl]40–150150–375375–630630–870>870∼7.729.040.822.5∼7.527.743.421.4Loss of uncoordinated H2Oloss of HCl gasLoss of methoxy and 2C4H4 groupsLoss of the remaining organic C14H7N2O0.51/2 Fe2O3 and 2C atomThe Zn(II), Cu(II), and Mn(II) complexes were decomposed through three steps. The TGA curve of[Zn(L).H2O] shows the loss of coordinated water (3.8%) in the range of 150–250°C [32]. Then, the weight reduced by 6.5% can be assigned to the loss of the methoxy group (calc. 6.5%) in the range 250–375°C. The third step of the thermal decomposition, which occurs in the range 375–870°C, could be assigned to the loss of 2C4H4 and C14H7N2O groups (67.8%, calc. 67.7%). Finally, the stable compound ZnO is formed above 870°C. The TGAs of other complexes show similar results. The loss of uncoordinated water of [Cu(L)] occurs at 45–150°C, and then the loss of methoxy and a C4H4 group (17.7%, calc. 18.1%) is observed in the range 150–410°C. The loss of remaining C4H4 and C14H7N2O groups takes place within the range 410–870°C (57.9, calc. 59.2%), and finally, the stable compound CuO is formed above 870°C [41, 42]. The TGA of [Co(L)] shows the loss of moisture in the range of 40–150°C. Then, the loss of the methoxy and a C4H4 occurs in the range of 150–440°C (18.8%, calc. 18.3%). The weight reduced in the range of 440–850°C (64.4%, calc. 65.1%) can be assigned to the loss of C4H4 and C16H7N2O groups. CoO could be found above 850°C [20, 43]. The TGA of [Mn(L)] performs the loss of uncoordinated water in the range 40–150°C. Then, the loss of methoxy and two C4H4 groups occurs in the range of 150–500°C (34.0%, calc. 30.1%). In the range of 500–865°C, the loss of the C16H7N2O group could be recorded (50.0%, calc. 54.1%), and MnO is finally formed above 865°C [24]. The TGA of [Fe(L)Cl] showed four decomposition steps. The loss of uncoordinated water takes place within the range of 40–150°C. The loss of HCl gas occurs at 150–375°C (7.7%, calc. 7.5%). Then the loss of methoxy and two C4H4 is at 375–630°C, and the loss of the remaining organic group is at 630–870°C. Finally, the stable compound Fe2O3 could be obtained above 870°C [44].


### 4.3. Bioactive Studies

#### 4.3.1. Antimicrobial Activity

In the antibacterial activity study, the unsymmetrical salen-type ligand and its metal complexes were tested against Gram-positive and Gram-negative bacteria,S. aureus and E. coli, respectively. The antimicrobial assay was carried out by the disk diffusion method in Muller Hinton–Agar medium using DMSO as the negative control and ampicillin as the positive reference standard. The inhibition efficiencies of the tested compounds shown in Table 1 indicate that the Zn(II), Cu(II), and Co(II) complexes have no activity for both Gram-positive and negative bacteria, S. aureus and E. coli, respectively. Unsymmetrical Mn(II) and Fe(III) complexes possess the antibacterial potential for only the Gram-positive bacteria, S. aureus, better than the ligand H2L with the inhibition zone diameters of 16.5 and 12.0 mm compared with 9.0 mm, respectively. However, their antimicrobial activity is still far from the standard drug, ampicillin, with the inhibition zone diameter of 27.0 mm. The Gram-positive bacteria and Gram-negative bacteria have different cell walls which can induce the different interactions between the tested compounds and bacteria, and some compounds can inhibit only Gram-positive bacteria [37]. Besides the structure and nature of the ligand, the electronic property of central metals seems to be the main reason which indicates the antibacterial activities of the studied metal complexes, and so some metal complexes are good and effective, but some are infective.


#### 4.3.2.In Vitro Cytotoxicity of H2L and Its Metal Complexes

The cytotoxic activity of the metal complexes was tested for human cancer cells, KB and Hep-G2. The obtained results showed that the cytotoxic activity of H2L is moderate for both human cancer cells with IC50 = 46.14 µM for KB and 43.29 µM for Hep-G2 (Table 2). The complexes [Zn(L).H2O] and [Cu(L)] are not active for KB and Hep-G2 (IC50 > 250 µM) when the remaining metal complexes are good active (IC50 < 10 µM) and are more active than the ligand. [Mn(L)] and [Fe(L)Cl] exhibited the best cytotoxicity for Hep-G2 and KB with IC50 = 3.67 and 2.64 µM, respectively.


## 4.1. Synthesis and Spectral Characterization

The ligand and complexes were obtained in high yields (72.5 and 83–91%), and they are stable to air and moisture. The synthetic compounds can be dissolved in dichloromethane as well as in methanol, acetonitrile, and dimethylsulfoxide. The ligand was characterized by +IDA-TOF-MS, FT-IR, NMR, and UV-Vis spectra. Its complexes were analysed by ESI-MS, IR, UV-Vis spectroscopies, and magnetic susceptibility. On mass spectra, the pseudomolecular ion signals of the ligand and complexes are found as [M]+, [M-H2O]+ or [M-Cl]+ which are quite suitable to the calculated masses for suggested formulae.NMR spectra of the ligandH2L were recorded in DMSO-d6. On 1H-NMR, there were typical signals of one double peak at 15.55 ppm for 1 NH proton, a single peak at 13.33 ppm for 1 OH proton, a double peak at 9.62 ppm for 1 HC-N proton, and a single peak at 9.12 ppm for HC=N proton. This is proved by tautomerism between enol-imine and keto-amine forms when 1 proton of NH and 1 proton of OH were found (Scheme 1). The protons of aromatic rings were found at 8.46-6.57 ppm and the protons of the CH3O group at 3.87 ppm as a single peak. On 13C-NMR of H2L, there were characteristic signals of C=O carbon at 171.78 ppm, C=N carbon at 161.85 ppm, C-OH carbon at 160.37 ppm, and C-NH carbon at 154.73 ppm. The other aromatic carbon peaks were observed at 141.46-101.28 ppm. The methoxy carbons were found at 56.01 ppm as a single signal. Difference from paramagnetic complexes, the NMR spectra of diamagnetic Zn(II) complex can be recorded by NMR spectrometer. On the 1H-NMR spectrum, the absence of NH and OH protons for the Zn(II) complex ensured the deprotonation of NH and OH protons upon complexation with Zn(II). The protons of HC-N and HC=N groups at 9.76 and 9.40 ppm, respectively, were shifted downfield compared to the ones of H2L when the coordination was observed. The aromatic protons appeared at 8.41-6.07 ppm, and methoxy protons were found at 3.85 ppm as a single peak reasonably. On the 13C-NMR spectrum, the typical signals of C=O at 173.72 ppm, C=N at 173.14 ppm, C-OH at 161.61 ppm, and C-NH at 156.03 ppm were shifted to downfield compared to the free ligand which confirmed the coordination between H2L with Zn(II) at nitrogen and oxygen atoms. The observed peaks of aromatic carbons were at 140.57-94.49 ppm, and the methoxy carbon was found at 55.72 ppm.Scheme 1
Synthesis of the unsymmetrical salen-type ligand and its metal complexes.On the IR spectrum ofH2L, the weak broad signal at 2615 cm−1 can be attributed to the stretching vibration of the o-O-H group [27]. There are the characteristic signals at 1607 cm−1 for the stretching vibration of C=N, at 1250 cm−1 for νC-N, and at 1185 cm−1 for νC-O. The disappearance of νO-H and the presence of new bands at 563-536 and 504-451 cm−1 for M-N and M-O bondings, respectively, on the IR spectra of the obtained complexes proved the coordination of metals and the ligand [17, 21, 28]. The characteristic bands for νC=N, νC-N, and νC-O in the complexes are lightly moved to the higher or lower field from the bands in the free ligand. The IR spectra data assured that the coordination of the ligand to the studied metals at nitrogen and oxygen atoms. On the IR spectrum of [Zn(L).H2O], there was a typical signal at 3207 cm−1 which may be assigned to the stretching vibration of O-H of coordinated H2O [29–32].UV-Visible spectra ofH2L and its metal complexes were recorded in methanol solution and presented in Figure 1. The UV-Vis spectrum of H2L showed main bands at 231–273 nm assigned to the π-π∗ electronic transition of aromatic rings, at 336 nm relative to n-π∗ transition of C=N and C-O groups and at 450 nm attributed to the n-π∗ transition within C=O [21, 33]. During the coordination of the ligand to metals, the electronic absorption spectra of synthetic complexes show new MLCT bands at 403 nm for Zn(II) complex, at 425 nm for Cu(II) complex, at 470 nm for Co(II) complex [34–36], at 453 nm for Mn(II) complex, and at 433 nm for Fe(III) complex. The d-d absorptions seem to have no appearance at this complexes’ concentration in methanol solution (3 × 10−5 M). The effective magnetic moments were calculated to be diamagnetic for Zn(II) complex [36, 37]. The effective magnetic moments measured experimentally were 2.03 BM corresponding to one unpaired electron supporting the distorted square-planar geometry for Cu(II) complex [37, 38]; 4.34 BM relative to the presence of three unpaired electrons in the octahedral structure for Co(II) complex [33, 37]; 5.85 BM indicated the octahedral geometry for Mn(II) complex [37, 39]; and 6.08 BM due to the presence of five unpaired electrons attributed the octahedral structure for Fe(III) complex [37, 40] probably.Figure 1
Electronic absorption spectra of obtained compounds.


## 4.2. Thermogravimetric Analysis

The thermal analysis of compounds was based on the thermogravimetric analysis (TGA) method at the temperature of 40–870°C. The results of thermal analysis of the ligand and metal complexes in this study based on the degradation steps were used to evaluate the water molecules, the chemical compositions, and the residual amount of the tested compounds (Table3) (Figures S6a–S6f). The ligand decomposition behaviour comprises two steps in which the first step, decomposition at 140–320°C, is assigned to the loss of methoxy and hydroxy groups (observed about 16.3%, calculated about 16.4%). The second step of thermal decomposition, which occurs at 320–870°C, is assigned to the loss of all most organic compounds (observed at about 76.8%, calculated at 77.5%). Above 870°C, two carbon atoms are still remaining.Table 3
TGA data of the ligand and its metal complexes.
CompoundTemperature range (°C)Weight loss (%)AssignmentsFoundCalc.H2L150–320320–870>87016.376.86.916.477.56.1Loss of methoxy and hydroxyl groupsLoss of all most organic residue2C atoms[Zn(L).H2O]150–250250–375375–870>8703.86.567.821.93.86.567.722.0Loss of a coordinated H2OLoss of methoxy groupLoss of 2C4H4 groups and C14H7N2OZnO and 2C atom[Cu(L)]45–150150–410410–870>870∼17.757.924.4∼18.159.222.7Loss of uncoordinated H2OLoss of methoxy and C4H4 groupsLoss of C4H4 group and C14H7N2OCuO and 2C atom[Co(L)]40–150150–440440–850>850∼18.864.416.8∼18.365.116.6Loss of uncoordinated H2OLoss of methoxy and C4H4 groupsLoss of C4H4 group and C16H7N2OCoO[Mn(L)]40–150150–500500–865>865∼34.050.016.0∼30.154.115.8Loss of uncoordinated H2OLoss of methoxy and 2C4H4 groupsLoss of the remain organic C16H7N2OMnO[Fe(L)Cl]40–150150–375375–630630–870>870∼7.729.040.822.5∼7.527.743.421.4Loss of uncoordinated H2Oloss of HCl gasLoss of methoxy and 2C4H4 groupsLoss of the remaining organic C14H7N2O0.51/2 Fe2O3 and 2C atomThe Zn(II), Cu(II), and Mn(II) complexes were decomposed through three steps. The TGA curve of[Zn(L).H2O] shows the loss of coordinated water (3.8%) in the range of 150–250°C [32]. Then, the weight reduced by 6.5% can be assigned to the loss of the methoxy group (calc. 6.5%) in the range 250–375°C. The third step of the thermal decomposition, which occurs in the range 375–870°C, could be assigned to the loss of 2C4H4 and C14H7N2O groups (67.8%, calc. 67.7%). Finally, the stable compound ZnO is formed above 870°C. The TGAs of other complexes show similar results. The loss of uncoordinated water of [Cu(L)] occurs at 45–150°C, and then the loss of methoxy and a C4H4 group (17.7%, calc. 18.1%) is observed in the range 150–410°C. The loss of remaining C4H4 and C14H7N2O groups takes place within the range 410–870°C (57.9, calc. 59.2%), and finally, the stable compound CuO is formed above 870°C [41, 42]. The TGA of [Co(L)] shows the loss of moisture in the range of 40–150°C. Then, the loss of the methoxy and a C4H4 occurs in the range of 150–440°C (18.8%, calc. 18.3%). The weight reduced in the range of 440–850°C (64.4%, calc. 65.1%) can be assigned to the loss of C4H4 and C16H7N2O groups. CoO could be found above 850°C [20, 43]. The TGA of [Mn(L)] performs the loss of uncoordinated water in the range 40–150°C. Then, the loss of methoxy and two C4H4 groups occurs in the range of 150–500°C (34.0%, calc. 30.1%). In the range of 500–865°C, the loss of the C16H7N2O group could be recorded (50.0%, calc. 54.1%), and MnO is finally formed above 865°C [24]. The TGA of [Fe(L)Cl] showed four decomposition steps. The loss of uncoordinated water takes place within the range of 40–150°C. The loss of HCl gas occurs at 150–375°C (7.7%, calc. 7.5%). Then the loss of methoxy and two C4H4 is at 375–630°C, and the loss of the remaining organic group is at 630–870°C. Finally, the stable compound Fe2O3 could be obtained above 870°C [44].


## 4.3. Bioactive Studies

### 4.3.1. Antimicrobial Activity

In the antibacterial activity study, the unsymmetrical salen-type ligand and its metal complexes were tested against Gram-positive and Gram-negative bacteria,S. aureus and E. coli, respectively. The antimicrobial assay was carried out by the disk diffusion method in Muller Hinton–Agar medium using DMSO as the negative control and ampicillin as the positive reference standard. The inhibition efficiencies of the tested compounds shown in Table 1 indicate that the Zn(II), Cu(II), and Co(II) complexes have no activity for both Gram-positive and negative bacteria, S. aureus and E. coli, respectively. Unsymmetrical Mn(II) and Fe(III) complexes possess the antibacterial potential for only the Gram-positive bacteria, S. aureus, better than the ligand H2L with the inhibition zone diameters of 16.5 and 12.0 mm compared with 9.0 mm, respectively. However, their antimicrobial activity is still far from the standard drug, ampicillin, with the inhibition zone diameter of 27.0 mm. The Gram-positive bacteria and Gram-negative bacteria have different cell walls which can induce the different interactions between the tested compounds and bacteria, and some compounds can inhibit only Gram-positive bacteria [37]. Besides the structure and nature of the ligand, the electronic property of central metals seems to be the main reason which indicates the antibacterial activities of the studied metal complexes, and so some metal complexes are good and effective, but some are infective.


### 4.3.2.In Vitro Cytotoxicity of H2L and Its Metal Complexes

The cytotoxic activity of the metal complexes was tested for human cancer cells, KB and Hep-G2. The obtained results showed that the cytotoxic activity of H2L is moderate for both human cancer cells with IC50 = 46.14 µM for KB and 43.29 µM for Hep-G2 (Table 2). The complexes [Zn(L).H2O] and [Cu(L)] are not active for KB and Hep-G2 (IC50 > 250 µM) when the remaining metal complexes are good active (IC50 < 10 µM) and are more active than the ligand. [Mn(L)] and [Fe(L)Cl] exhibited the best cytotoxicity for Hep-G2 and KB with IC50 = 3.67 and 2.64 µM, respectively.


## 4.3.1. Antimicrobial Activity

In the antibacterial activity study, the unsymmetrical salen-type ligand and its metal complexes were tested against Gram-positive and Gram-negative bacteria,S. aureus and E. coli, respectively. The antimicrobial assay was carried out by the disk diffusion method in Muller Hinton–Agar medium using DMSO as the negative control and ampicillin as the positive reference standard. The inhibition efficiencies of the tested compounds shown in Table 1 indicate that the Zn(II), Cu(II), and Co(II) complexes have no activity for both Gram-positive and negative bacteria, S. aureus and E. coli, respectively. Unsymmetrical Mn(II) and Fe(III) complexes possess the antibacterial potential for only the Gram-positive bacteria, S. aureus, better than the ligand H2L with the inhibition zone diameters of 16.5 and 12.0 mm compared with 9.0 mm, respectively. However, their antimicrobial activity is still far from the standard drug, ampicillin, with the inhibition zone diameter of 27.0 mm. The Gram-positive bacteria and Gram-negative bacteria have different cell walls which can induce the different interactions between the tested compounds and bacteria, and some compounds can inhibit only Gram-positive bacteria [37]. Besides the structure and nature of the ligand, the electronic property of central metals seems to be the main reason which indicates the antibacterial activities of the studied metal complexes, and so some metal complexes are good and effective, but some are infective.


## 4.3.2.In Vitro Cytotoxicity of H2L and Its Metal Complexes

The cytotoxic activity of the metal complexes was tested for human cancer cells, KB and Hep-G2. The obtained results showed that the cytotoxic activity of H2L is moderate for both human cancer cells with IC50 = 46.14 µM for KB and 43.29 µM for Hep-G2 (Table 2). The complexes [Zn(L).H2O] and [Cu(L)] are not active for KB and Hep-G2 (IC50 > 250 µM) when the remaining metal complexes are good active (IC50 < 10 µM) and are more active than the ligand. [Mn(L)] and [Fe(L)Cl] exhibited the best cytotoxicity for Hep-G2 and KB with IC50 = 3.67 and 2.64 µM, respectively.


## 5. Conclusion

In this report, (Z)-1-(((2-((E)-(2-hydroxy-6-methoxybenzylidene)amino)phenyl)amino)methylene) naphthalen-2(1H)-one and its Zn(II), Cu(II), Co(II), Mn(II), and Fe(III) complexes were synthesized and characterized by spectrometries. The obtained spectral data supported the tautomerism of the ligand, and it must have one arm in keto-amine and the other in enol-imine form. The synthetic metal complexes have different geometries such as square-planar structure for Cu(II) complex and octahedral geometry for Co(II), Mn(II), and Fe(III) complexes. The thermogravimetric analysis data exhibited uncoordinated and coordinated H2O in the obtained complexes. The ligand was decomposed almost completely, while metal complexes were decomposed to form the metal oxides. Mn(II) and Fe(III) complexes showed the antibacterial potential for Gram-positive bacteria, S-aureus, better than the ligand H2L. The in vitro cytotoxicity for KB and Hep-G2 cancer cell lines indicated that while Zn(II) and Cu(II) complexes have no activity, the metal complexes such as Co(II), Mn(II), and Fe(III) complexes exhibited good cytotoxic activity, much better than the ligand. [Mn(L)] and [Fe(L)Cl] showed the best cytotoxicity for Hep-G2 and KB with IC50 = 3.67 and 2.64 µM, respectively.



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*Source: 4563958-2023-02-04.xml*