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Spectroscopic and Biological Investigations of 4-[(1E)-N-(2-

aminophenyl) ethanimidoyl]-3-methyl-1-phenyl-1H-pyrazol-5-ol and

its Copper (II) Complex

Ogbuagu, O. E.

, Ezenweke, L. O.

, Ojiako, E. N.

, Achonye, C.C.

, Okolo, A. J.

, Ndupu, R. O.

Silas,

C.U.

Department of Chemistry, Alvan Ikoku Federal University of Education Owerri, Imo State, Nigeria.

Department of Pure & Industrial Chemistry, Chukwuemeka Odumegwu Ojukwu University Uli,

Anambra State, Nigeria.

Department of Science Laboratory Technology, Anambra State Polytechnic, Mgbakwu, Anambra State

Nigeria.

Department of Chemistry and Biochemistry, Miami University, Oxford, OH 45056 USA.

Department of Chemistry, Kingsley Ozumba Mbadiwe University, Ideato, Imo State, Nigeria

DOI: https://dx.doi.org/10.51584/IJRIAS.2025.101100009

Received: 28 October 2025; Accepted: 03 November 2025; Published: 29 November 2025

ABSTRACT

This study reports the synthesis, characterization, and antimicrobial evaluation of a novel Schiff base ligand (4-

[(1E)-N-(2-aminophenyl) ethanimidoyl]-3-methyl-1-phenyl-1H-pyrazol-5-ol) derived from 4-acyl pyrazolone

and its copper (II) complex. The work aims to correlate the structural modifications induced by metal

coordination with variations in biological activity. The experimental procedure involved the condensation of 4-

acyl pyrazolone with 1,2-diaminobenzene to form the Schiff base ligand, followed by complexation with

copper (II) chloride dihydrate (CuCl₂·2H₂O) to yield the corresponding Cu (II) complex. Structural elucidation

was achieved using elemental analysis, molar conductivity, infrared (IR), ultraviolet–visible (UV–Vis), proton

nuclear magnetic resonance (¹H NMR), and gas chromatography–mass spectrometry (GC–MS) techniques.

Spectroscopic analyses confirmed the formation of the Schiff base through the characteristic azomethine (C=N)

absorption at 1636 cm⁻¹ and coordination to Cu(II) via N,N,O donor sites, as evidenced by metal–ligand (M–L)

bands at 667.2 cm⁻¹. The Cu(II) complex displayed a higher melting point and molar conductivity than the free

ligand, indicating greater thermal stability and a non-electrolytic nature. Antimicrobial activities were assessed

against Salmonella typhi, Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, and Candida

albicans using the agar well diffusion method following CLSI standards. The Cu(II) complex exhibited

enhanced antimicrobial efficacy compared to the free ligand, except against S.aureus. Minimum inhibitory

concentration (MIC) and minimum bactericidal/fungicidal concentration (MBC/MFC) values further confirmed

improved potency upon complexation. The increased activity of the Cu (II) complex is attributed to enhanced

lipophilicity and cell membrane permeability in accordance with Tweedy’s chelation theory. Overall, the

synthesized Cu (II) complex demonstrates promising potential for development as a broad-spectrum

antibacterial and antifungal agent.

Keywords: Schiff base ligand, Copper(II) complex, Antimicrobial activity, Spectroscopic characterization.

INTRODUCTION

Schiff bases are an important family of organic compounds formed by the condensation of primary amines

with aldehydes or ketones to form an imine/azomethine group (C=N). These compounds have attracted interest

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because of their structural flexibility, ease of preparation, and diverse coordination behaviour. When

coordinated to transition-metal ions, Schiff bases often exhibit improved biological and catalytic activities due

to changes in their electronic and steric environments (Ceramella et al., 2022; Manohar et al., 2022). Over the

years, Schiff-base metal complexes have found applications as antibacterial, antifungal, anticancer, and

antioxidant agents, demonstrating that coordination can significantly modulate the pharmacological potential of

the parent ligands (Silas et al., 2025)

Within this broad class, derivatives of 4-acylpyrazolone occupy a special position because of the chemical

versatility of the pyrazolone moiety (Okolo et al., 2025). The 4-acyl-5-pyrazolone system behaves as a β-

dicarbonyl framework that undergoes keto–enol tautomerism and coordinates metals through oxygen and

nitrogen donor atoms. These properties make 4-acylpyrazolones excellent building blocks for new Schiff-base

ligands and their metal complexes (Idemudia et al,, 2016). Studies have shown that transition metals such as

copper, nickel, and zinc readily form stable chelates with acylpyrazolone derivatives, producing geometrically

diverse complexes with interesting optical and biological properties (Shaikh et al., 2022).

Spectroscopic characterization provides essential insight into the formation and bonding patterns of such

compounds. In the infrared region, the appearance of a strong azomethine (C=N) stretching vibration around

1600–1650 cm⁻¹ confirms the condensation reaction, whereas coordination to a metal center is usually

accompanied by shifts in characteristic bands and the emergence of new metal–ligand vibrations below 700

cm⁻¹ (Ahmed et al., 2022). Complementary data from ultraviolet–visible, nuclear magnetic resonance,

elemental analysis, and molar-conductivity measurements aid in deducing the coordination environment,

stoichiometry, and geometry of the complexes (Xi et al., 2020). Together, these techniques allow reliable

correlation between structural features and physicochemical behavior.

The enhancement of antimicrobial activity observed after metal complexation of Schiff bases has often been

explained through chelation and lipophilicity concepts. According to Tweedy’s chelation theory, the partial

sharing of metal ion positive charge with the donor atoms of the ligand reduces metal polarity, delocalizes

electron density over the chelate ring, and increases overall lipophilicity. This facilitates diffusion of the

complex across microbial cell membranes and improves interaction with intracellular targets (Alezzy et al.,

2022). Overtone’s principle similarly proposes that lipid-soluble compounds pass more easily through cell

envelopes, accounting for the higher activity of coordinated species (Nandini & Amutha Selvi, 2025).

Copper(II) complexes garners significant attention because Cu(II) possesses variable coordination geometries,

moderate redox potential, and intrinsic antimicrobial capability. Schiff bases derived from 4-acylpyrazolone

and coordinated to Cu(II) have been reported to exhibit square-planar or distorted octahedral structures and it

tends to display stronger biological effects than their free ligands (Barad et al., 2023; Wang et al., 2019)

This investigation focuses on the synthesis of a Schiff base formed by condensing a 4-acylpyrazolone

derivative with an aromatic diamine and its subsequent complexation with Cu(II) ions. Comprehensive

spectroscopic analyses: FT-IR, UV–Vis,

H NMR, elemental analysis, molar-conductivity, and GC–MS were

employed to elucidate the structural features of both the ligand and the complex. Their antimicrobial efficacy

was then examined against selected bacterial and fungal strains to assess how coordination influences

biological activity. This study therefore bridges structural chemistry and bioactivity, aiming to clarify and

analyse how metal–ligand interactions in acyl pyrazolone based Schiff bases contribute to enhanced

antimicrobial performance.

MATERIALS AND METHODS

All reagents and solvents utilized in this study were of analytical reagent grade and were used without further

purification. Ethyl acetoacetate (C₆H₁₀O₃), phenylhydrazine (C₆H₈N₂), diethyl ether, ethanol, 1,4-dioxane,

acetyl chloride, and hydrochloric acid were procured from Sigma-Aldrich. Additional reagents and metal salts

were obtained from Loba Chemie. Distilled water of high purity was also used as supplied. Culture media such

as nutrient agar, Sabouraud’s dextrose agar, and culture broth were obtained from the Department of

Microbiology, Federal Polytechnic Nekede, Imo State, Nigeria.

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Instrumentation and Measurements analyses (C, H, N, and O) were performed on a Perkin Elmer 2400 Series II

elemental analyzer. Molar conductivity measurements were recorded using a Philips PW 9506 conductivity

meter. The solubility of the synthesized Schiff base ligand and its copper (II) complex was determined in

various solvents including water, ethanol, methanol, DMSO, petroleum ether, and n-hexane. Infrared (IR)

spectra were obtained in KBr discs using an Agilent Cary 630 FTIR spectrophotometer, with absorption

frequencies reported in the range of 4000–650 cm⁻¹. The proton nuclear magnetic resonance (¹H NMR) spectra

were recorded on a Nanalysis X685 spectrometer operating at 60 MHz, using deuterated DMSO (d₆) as the

solvent. Gas chromatography–mass spectrometry (GC–MS) analyses were conducted on a Varian 3800/4000

GC–MS system with a run time of 45 minutes and a mass range up to 500/1000 m/z. Ultraviolet–visible (UV–

Vis) spectra were recorded using an Agilent Cary 630 UV spectrophotometer within the wavelength range of

200–800 nm.

METHODS

Synthesis of Tridentate Schiff Base Ligand (4-[(1E)-N-(2-aminophenyl) ethanimidoyl]-3-methyl-1-

phenyl-1H-pyrazol-5-ol)

Step 1: Synthesis of 3-Methyl-1-Phenyl -Pyrazol-5-one {Precursor-P}

The precursor ligand, designated as P, was synthesized following the method previously described by Furniss

et al. (1989), with slight modifications (Figure 1). Ethyl acetoacetate (49 ml, 0.384 mol) was mixed with

phenyl hydrazine (36.5ml, 0.370 mol) in a large evaporating dish. The reaction mixture was heated over a

boiling water bath inside a fume cupboard and continuously stirred with a stirrer for approximately two hours.

As the reaction proceeded, a reddish syrup-like mass was formed. After heating for two hours, the mixture was

removed from the water bath and allowed to cool for about five minutes. Diethyl ether (100 ml) was then

added, and the mixture was stirred vigorously, leading to gradual solidification of the syrup. The resulting

solid, which was insoluble in ether, was collected and washed thoroughly with diethyl ether (100 mL) to

remove impurities. The washing was repeated, the crude product was then recrystallized from ethanol (100

mL) to yield pale yellow crystals. Yield: 78%; M.p.: 127°C; Molecular formula: C₁₀H₁₀N₂O; IR (KBr, cm⁻¹):

3125.4 (N–H stretch), 2924.1 (C–H stretch), 1697.2 (C=O), 1496.5 (C=C stretch), 1343.7 (C–N stretch), 1198

(C–N cyclic); GC–MS (m/z): Calc., 174; Found, 174.0.

Figure 1: synthesis of 3 methyl-1-phenyl-pyrazol-5-one (P)

STEP 2: Synthesis of Acyl Pyrazolone (4-acetyl-3-methyl-1-phenyl-5-pyrazolone)

The compound 4-acetyl-3-methyl-1-phenyl-5-pyrazolone was synthesized following a slightly modified

procedure of Jensen (1959), (Figure 2). 3-methyl-1-phenyl-5-pyrazolone (17.4 g, 0.1 mol) was dissolved in 80

mL of 1,4-dioxane contained in a three-necked round-bottom flask equipped with a reflux condenser and a

magnetic stirrer. The solution was gently warmed and stirred until complete dissolution was achieved.

Anhydrous calcium hydroxide (12 g) was then added to the reaction mixture, and heating was continued.

Acetyl chloride (7.1 mL, 0.1 mol) was introduced dropwise over a period of two minutes, resulting in the

formation of a thick paste. The reaction mixture was subsequently refluxed for three hours. After completion of

reflux, the mixture was allowed to cool for about 20 minutes and then poured into 200 mL of cold dilute

hydrochloric acid (2 M) with continuous stirring. Acidification decomposed the calcium complex, leading to

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the precipitation of cream-colored crystals of 4-acetyl-3-methyl-1-phenyl-5-pyrazolone. The product was

filtered, washed with cold water, and dried. Yield: 50%; M.p.: 242 °C.

COCl

Ca(OH)

Dioxane/80ml/3hrs

` R = CH

Figure 2: synthesis of 4-acetyl-3-methyl-1 -phenyl-5-pyrazolone

STEP 3: Procedure for the Synthesis of Tridentate Schiff Base Ligand (4-[(1E)-N-(2-

aminophenyl)ethanimidoyl]-3-methyl-1-phenyl-1H-pyrazol-5-ol).

The Schiff base ligand, 4-[(1E)-N-(2-aminophenyl)ethanimidoyl]-3-methyl-1-phenyl-1H-pyrazol-5-ol (L), was

synthesized according to the procedure described by Nasridas et al. (2015), with slight modifications (Scheme

3). An ethanolic hot solution (50 mL) of 1,2-diaminobenzene (1.08 g, 0.01 mol) was mixed with an equimolar

ethanolic solution (50 mL) of 4-acetyl-3-methyl-1-phenyl-5-pyrazolone (2.16 g, 0.01 mol). The resulting

mixture was refluxed for three hours in the presence of a few drops of glacial acetic acid as a catalyst. After

refluxing, the reaction mixture was allowed to cool to room temperature and left to stand overnight. Cream-

colored crystalline precipitates were obtained, filtered, washed with cold ethanol, and dried to yield the Schiff

base ligand of excellent purity. Yield: 73% , M.P: 180  C C

O (L), Anal. Found(%) : C, 66.99; H, 6.20;

N, 18.29;O, 5.53; Calc(%): C, 70.11; H, 6.54; N, 18.17; O,5.18. IR (KBr, cm-1-): 3496.2 (N-H Stretch),

3236.3 (O-H), 2961 (C-H Stretch), 1636.3 (azomethine C=N), 1561.8 (C=C Stretch), 1323,2 (C-N Stretch),

1073 (C-O):. UV-Vis.(DMSO) λmax(nm);298, ε (8.16 x 104), H NMR (DMSO-d6, 400 MHz) δ (ppm): 6.85–

6.50 (m, 1H, Ar–H), 5.10 (s, 2H, OH or NH2), 1.23 (s, 1.5H, CH3).. GC-MS (m/z): cal., 308; found, 308.2.

N NH

Ethanol

Reflux

R = CH

Figure 3: Synthesis of Tridentate Schiff Base Ligand (4-[(1E)-N-(2aminophenyl) ethanimidoyl] -3-methyl-1-

phenyl-1H-pyrazol-5-ol).

Synthesis of the Copper Complex 4-[(1E)-N-(2aminophenyl) ethanimidoyl] -3-methyl-1-phenyl-1H-

pyrazol-5-ol (CuL

)

The copper (II) complex of the Schiff base ligand was synthesized by reflux condensation. The Schiff base

ligand, 4-[(1E)-N-(2-aminophenyl) ethanimidoyl]-3-methyl-1-phenyl-1H-pyrazol-5-ol (1.54 g, 5 mmol), was

dissolved in 100 ml of ethanol and stirred for approximately five minutes to obtain a homogeneous solution.

Separately, copper (II) chloride dihydrate (0.85 g, 5 mmol) was dissolved in 20 ml of ethanol. Both solutions

were gently warmed for a few seconds to ensure complete dissolution while preventing solvent evaporation,

after which they were mixed together. The resulting mixture was refluxed for six hours under continuous

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stirring. During reflux, the solution was gradually concentrated to about half of its original volume. Upon

cooling, the solid product formed was filtered, washed repeatedly with hot water and then with hot ethanol to

remove any unreacted materials or impurities. The Black-coloured complex obtained was dried under vacuum

and recrystallized from dimethylformamide (DMF) to yield a pure copper (II) complex suitable for further

characterization. ). Yield: 75%, M.p.: >250  C, C

CuN

(CoL

), Anal. Found (%) : C, 63.61; H, 5.63; N,

16.57;O, 4.75; Cu, 9.44. Calc (%): C, 63.56; H, 5.93; N, 16.47; O,4.70; Cu, 9.34. IR (KBr, cm-1-): 3339.4 (N-

H Stretch), 3179.4 (O-H), 1617.1 (azomethine C=N), 1524.5 (C=C Stretch), 1233.7(C-N Stretch), 1103.3 (C-

O), 667.2 (M-L): UV-Vis.(DMSO) λmax (nm);286, ε (3.58 x 104). GC-MS (m/z): cal., 679.5; found, 6. Below

(Figure 4) is a schematic equation of the synthesis of the Metal complex (Copper Complex of 4-[(1E)-N-

(2aminophenyl)ethanimidoyl] -3-methyl-1-phenyl-1H-pyrazol-5-ol).

Reflux

CuCl

.2H

Figure 4: Equation of the synthesis of the Metal complex (Copper Complex 4-[(1E)-N-(2aminophenyl)

ethanimidoyl] -3-methyl-1-phenyl-1H-pyrazol-5-ol).

Antimicrobial/ Antifungal Analyses of the Schiff base Ligand (L) and its Cu (II) Complex (CuL

)

The antibacterial and antifungal activities of the Schiff base ligand (L) and its Copper(II) complex (CuL₂) were

evaluated using the disc diffusion method (Jayarajan et al., 2010). Test organisms included Staphylococcus

aureus, Streptococcus pyogenes, Escherichia coli, Salmonella typhi, and Candida albicans. The compounds

(200 µg/mL in DMSO) were tested on nutrient or Sabouraud agar plates, with Levofloxacin and Nystatin

serving as positive controls, and DMSO as the negative control. Plates were incubated at 37 ± 1°C for bacteria

and 28 ± 1°C for fungi, and activity was determined by measuring inhibition zones (mm).

Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal /Fungicidal (MFC/MBC)

Concentrations (MBC/MFC) were determined by the broth dilution method. Serial dilutions of the test

compounds were prepared in suitable broth media and inoculated with standardized microbial suspensions.

After incubation, the MIC was recorded as the lowest concentration showing no visible growth, while the

MBC/MFC was the lowest concentration showing no microbial recovery on agar plates (Weigand et al., 2008).

RESULTS AND DISCUSSION

The precursor compound, 3-methyl-1-phenyl-pyrazol-5-one, underwent acylation with acetyl chloride to

produce an acyl pyrazolone derivative. This intermediate was subsequently condensed with 1,2 diamino

benzene to yield the Schiff base, 4-[(1E)-N-(2-aminophenyl)ethanimidoyl]-3-methyl-1-phenyl-1H-pyrazol-5-

ol. The resulting ligand was then reacted with copper (II) chloride dihydrate to form the corresponding copper

(II) complex. The precursor, Schiff base ligand, and its copper complex were characterized using various

analytical and spectroscopic techniques.

Physicochemical Properties of the Pyrazolone (3-Methyl-1-Phenyl -Pyrazol-5-one) Schiff base Ligands

(L) and its Cu (II) Complex (CuL

)

The synthesized compounds exhibited distinct physical properties that support their structural transformation.

Table 1 shows the formation of the Schiff base ligand (L) resulted in a cream-colored compound with a melting

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point (180 °C) and moderate conductivity (10.8 μS/cm), upon coordination with Cu(II) ions, the resulting

complex (CuL₂) appeared as a black solid with a higher melting point (>250 °C) and slightly higher

conductivity (15.9 μS/cm). These changes are consistent with metal–ligand chelation, which typically enhances

thermal stability and slightly alters the electronic environment, producing intense coloration due to d–d

transitions. The progressive increase in melting point and conductivity values confirms successful Schiff base

formation and complexation with copper (II). The data align with previous findings that Schiff base metal

complexes are thermally stable, non-electrolytic, and exhibit strong metal–ligand interactions through

azomethine nitrogen, enolic oxygen and primary amine nitrogen atoms (Ogbuagu et al., 2025).

Table 1 Physicochemical Properties of the Schiff base Ligands (4-[(1E)-N-(2-aminophenyl) ethanimidoyl]-3-

methyl-1-phenyl-1H-pyrazol-5-ol) and its Cu (II) Complex (CuL

)

Compounds

Molecular Formula

% Yield

Colour

Melting Point (∘C)

Conductivity (μs/cm)

Cream

180

10.8

CuL

CuN

Black

>250

15.9

L- 4-[(1E)-N-(2aminophenyl) ethanimidoyl] -3-methyl-1-phenyl-1H-pyrazol-5-ol

CuL

- Cu (II) Complex 4-[(1E)-N-(2aminophenyl) ethanimidoyl] -3-methyl-1-phenyl-1H-pyrazol-5-ol

Solubility Profile of L and CuL

in various solvents

Table 2 shows solubility profile of Schiff base, and copper complex. Schiff base ligand (L) showed slight

solubility in DMSO, ethanol, and methanol but was insoluble in petroleum ether, n-hexane, and water,

indicating a moderately polar nature. In contrast, the Cu (II) complex (CuL₂) was insoluble in polar

solvents (DMSO, ethanol, and methanol) and nonpolar solvents, reflecting decreased polarity due to metal–

ligand coordination. Coordination induces reduction in polarity and increased lattice stability. These

observations further support the formation of a stable Cu(II) complex.

Table 2 Solubility Profile of Schiff base Ligand (L) and its Cu (II) Complexes (CuL

) in various solvents

Compounds

Solvents

DMSO

Petroleum Ether

N-Hexane

Ethanol

Methanol

Water

CuL

Infrared Spectral Analysis

The IR spectral data of the Schiff base ligand (L), and its copper(II) complex (CuL₂) are presented in Table 3.

The emergence of an azomethine (C=N) stretch at 1636.3 cm⁻¹, indicating successful Schiff base formation

through condensation of the carbonyl group with an amine (Fig 5). In the spectrum of the Cu(II) complex, the

C=N stretching frequency shifted to a lower value (1617.1 cm⁻¹), suggesting coordination of the

azomethine nitrogen to the metal ion. The O–H stretching vibration observed at 3236.3 cm⁻¹ in the ligand

also shifted to 3179.4 cm⁻¹ in the complex, implying involvement of the enolic oxygen in chelation and a

downward shift of the C-N stretching frequency(Fig 6). Additionally, new absorption bands appearing around

667 cm⁻¹ correspond to M–O/M–N vibrations, confirming the formation of metal–ligand bonds. The observed

shifts in the C=N, O–H, C-N and C–O frequencies, along with the presence of new metal-sensitive bands,

collectively support a tridentate coordination mode of the ligand through azomethine nitrogen, enol oxygen

and diamino nitrogen atoms. These findings align with literature reports on pyrazolone-based Schiff base metal

complexes (Achonye et al,. 2024).

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Table 3: Selected Infra-red absorption bands of Schiff base Ligand (L) and its Cu (II) Complexes (CuL

)

S/N

Compounds

(N-H)

(O-H)

(C-H)

(C=N)

(C=C)

(C-N)

(C-O)

M-L

O [L]

3496.2

3236.3

2961

1636.3

1561.8

1323.2

1073.5

CuN

(CuL

3339.4

3179.4

1617.1

1524.5

1233.7

1103.3

667

Fig 5: IR Spectrum for Schiff base Ligand -L (4-[(1E)-N-(2aminophenyl) ethanimidoyl] -3-methyl-1-phenyl-

1H-pyrazol-5-ol)

Fig 6: IR Spectrum for Cu (II) Complex

Percentage Elemental Analysis

Table 4 shows elemental analysis of the synthesized Schiff base ligand (L) and its copper (II) complex (CuL

)

showed good agreement between the experimental and calculated values, confirming the purity and proposed

molecular formula of the compounds. For the ligand, the experimental values correspond well with the

calculated values. In the copper (II) complex (CuL

), the experimental values closely match the theoretical

data. The slight reduction in the percentages of carbon, hydrogen, and nitrogen upon complexation indicates

successful coordination of the ligand to the copper (II) ion

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Table 4: Percentage Elemental Analysis of L and CuL

Compounds

Molar Mass (g/mol)

L(C

306

69.99

(70.11)

6.20

(6.54)

18.29

(18.17)

5.53

(5.18)

CuL

CuN

)

679.5

63.61

(63.56)

5.63

(5.93)

16.57

(16.47)

4.75

(4.70)

9.44

(9.34)

Gas Chromatography-Mass Spectroscopy Analysis of Schiff base Ligand (L)and its Cu(II) complex

(CuL

)

The mass spectral analysis (Table 5) supports the formation of the Schiff base ligand and its copper (II)

complex. The Schiff base ligand (L) displayed a molecular ion peak at m/z 308.2 (Fig 7), which agrees with its

calculated mass (308 g/mol). Fragment ions at m/z 293, 275, 232, 136, 125, and 58 correspond to the loss of

methyl, amine, and aromatic groups, indicating cleavage around the azomethine and pyrazolone moieties. The

copper(II) complex (CuL

) recorded a molecular ion peak at m/z 680.8 (Fig 8), which is in close agreement

with the calculated molecular weight (679.5 g/mol). The major fragment peaks at m/z 662.2, 603.1, and 510.0

represent stepwise loss of ligand fragments and small neutral species, suggesting a stable metal-ligand

coordination structure.

Fig 7 GC-MS spectrum of Schiff base Ligand (L) with a few fragment structure

The presence of a characteristic isotopic pattern for copper around m/z 680.8 further confirms metal the

coordination number of six (6) was deduced from the molecular ion value of the copper(II) complex (CuL

which indicates two (2) tridentate ligands coordinate to the metal ion which is indicative of an octahedral

geometry around the Cu(II)centre. Overall, the close correspondence between the experimental and theoretical

data from both elemental and mass spectral analyses confirms the successful synthesis, purity, and proposed

structural formulations of the Schiff base ligand and its Cu(II) complex.

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Fig 8: GC-MS spectrum of Cu(II) metal complex (CuL

) with a few fragment structure

Table 5: Gas Chromatography-Mass Spectroscopy Analysis of Schiff base Ligand (L)and its Cu(II) complex

(CuL).

Compounds

Calculated

Molecular

Mass (g/mol)

Observed

Molecular ion

)

Observed

Fragment ions

Coordination

Number

L (C

308

308.2

58.0, 125.0, 136.0, 232.2, 275.0, 293.0

CuL

CuN

)

679.5

680.8

662.2, 603.1, 510.0

L- 4-[(1E)-N-(2aminophenyl) ethanimidoyl] -3-methyl-1-phenyl-1H-pyrazol-5-ol

CuL

- Cu (II) Complex 4-[(1E)-N-(2aminophenyl) ethanimidoyl] -3-methyl-1-phenyl-1H-pyrazol-5-ol

Ultra Violet -Visible Spectra Analysis of L and CuL

Table 6 shows the UV–visible spectra of the Schiff base ligand (L) and its copper(II) complex (CuL

), the UV-

Vis Spectra of L exhibit strong absorption peaks at 298 nm (ε = 8.16 × 10⁴ L·mol⁻¹·cm⁻¹) and that of CuL

peak of 286 nm (ε = 3.58 × 10⁴ L·mol⁻¹·cm⁻¹). These absorptions are associated with electronic transitions

within the ligand’s conjugated system (Fig 9), primarily of the π→π* and n→π* types. A slight shift of the

absorption band toward a shorter wavelength in the complex, along with a reduction in intensity, indicates that

metal coordination affects the electronic environment of the ligand, leading to minor adjustments in orbital

energy levels and transition probabilities. The absence of any observable d–d band in the spectrum of the

copper (II) complex is attributed to the transitions are both spin- and Laporte-forbidden, making them

intrinsically weak (ε values are low). These low-intensity bands were overshadowed by the much stronger

ligand-based transitions occurring in the UV region. Consequently, the spectral features observed are

dominated by ligand-centred excitations.

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(a) (b)

FIG 9: UV-VIS Spectrum (a) Schiff Base Ligand L (b) Cu(II) Complex (CuL

)

TABLE 6: Ultra Violet -Visible Spectral Data of L and CuL

Compounds

Maximum wavelength

max

(nm)

Molar Absorptivity

ε (L mol

-1

)

Electronic transitions

O (L)

298

8.16 x 10

π – π*

n - π*

CuN

(CuL

)

286

3.58 x 10

π – π*

n - π*

HNMR Spectral Analysis of 4-[(1E)-N-(2aminophenyl) ethanimidoyl] -3-methyl-1-phenyl-1H-pyrazol-5-

ol (L)

Table 7 shows the

HNMR analysis in DMSO of the Schiff base Ligand. The data support the formation of a

Schiff-base-type compound containing aromatic and aliphatic regions, along with a hydrogen-bonded hydroxyl

and amine functionality. The presence of aromatic resonances between 6.5–6.9 ppm and a downfield

exchangeable proton at 5.1 ppm is consistent with a conjugated enol-imine system typical of 4-

acetylpyrazolone derivatives. The small methyl singlet at 1.23 ppm further confirms substitution on the ligand

backbone.

Fig 10: H’NMR Spectrum of Schiff base Ligand (L)

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Table 7- ¹H NMR Spectral Data (in DMSO-d₆) for a Schiff base Ligand(L)

Chemical Shift

(ppm)

Multiplicity

Integration

Tentative Assignment

Proton Type

6.8491, 6.6809,

6.5013

Multiplet (appears as

three distinct peaks)

0.8223

Aromatic protons

Ar-H

5.1035

Singlet

1.7828

-OH or N-H

Hydroxyl or

Amine proton

2.5000

Singlet

Not integrated (solvent

peak)

DMSO-d6

Solvent

1.2318

Singlet

Appears as a small peak

CH3

Methyl proton

Antimicrobial Susceptibility Analysis of Schiff base Ligand (L) and its Cu(II) complex (CuL

)

The antimicrobial activity of the Schiff base ligand (L) and its copper (II) complex (CuL₂) was evaluated

against selected Gram-positive, Gram-negative, and fungal strains, namely Staphylococcus aureus,

Streptococcus pyogenes, Escherichia coli, Salmonella typhi, and Candida albicans. The results were compared

with standard antibiotics: Levofloxacin (LEV) for bacterial strains and Nystatin (NY) for fungal strain (Table

8). The Schiff base ligand (L) exhibited moderate antibacterial and antifungal activity, with the highest

inhibition zone recorded against S. aureus, indicating significant potency against Gram-positive bacteria.

However, its activity against E. coli and S. pyogenes were comparatively lower, reflecting limited efficacy

against some Gram-negative and Gram-positive organisms. Upon complexation with copper (II) ion, there was

a notable enhancement in antimicrobial activity across almost all tested microorganisms(Fig 11). The Cu(II)

complex (CuL₂) showed the highest zone of inhibition against C. albicans and S. typhi , followed by S.

pyogenes and E. coli . This increase in activity upon chelation supports the chelation theory. This enhances

lipophilicity and facilitates penetration through microbial cell membranes, thereby improving biological

efficacy (Zoubi, 2013). Comparatively, the CuL₂ complex demonstrated comparable or superior activity to the

reference drugs in certain cases. For instance, against S. typhi and C. albicans, CuL₂ (26 mm and 32 mm,

respectively) surpassed LEV (20 mm) and NY (28 mm). However, LEV remained more effective against E.

coli and S. aureus (32 mm and 36 mm, respectively). Overall, the data indicate that metal complexation

significantly improves the antimicrobial potential of the parent Schiff base, making CuL₂ a promising candidate

for further biological evaluation (Ogbuagu et al 2025).

Table 8: Antimicrobial Susceptibility data of Schiff base ligand (L) and its Cu(II) complex (CuL

)

Test Organisms Formulation/Zone of Inhibition (mm)

Compounds

S. typhi

E. Coli

S. Aureus

S.Pyogenes

C. albicans

CuL

LEV

Key:

mm = Millimeter

Clinical Laboratory Standard Institute guideline for antimicrobial agents (CLSI)

Resistant (0 – 12 mm) Susceptible (16 mm and above) LEV = Levofloxacin

NY = Nystatin - = Not determined µg/ml = Microgram per millilitre ND = Not detected

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Fig 11: Antimicrobial Susceptibility Bar chart of Schiff base ligand (L) and its Cu(II) complex (CuL

)

The MIC (Minimum inhibitory concentration) data (Table 9) reveals the Schiff base ligand (L) and its copper

(II) complex (CuL₂) exhibit broad-spectrum antimicrobial activity against the tested bacterial and fungal

strains. However, a distinct improvement in activity is observed upon complexation with Cu (II), as

evidenced by the generally lower MIC values of CuL₂ compared with the free ligand. The Schiff base ligand

(L) displayed the highest sensitivity against Staphylococcus aureus (MIC = 62.5 µg/mL), indicating strong

inhibition toward Gram-positive bacteria. In contrast, higher MIC values (500 µg/mL) were recorded for E.

coli and S. pyogenes, suggesting lower susceptibility of these organisms to the free ligand (Fig 12a). The

moderate inhibition against S. typhi and C. albicans (250 µg/mL each) indicates partial effectiveness against

Gram-negative and fungal species. Upon complexation with copper(II), there was a twofold reduction in MIC

values for most organisms, signifying enhanced inhibitory potency. The CuL₂ complex exhibited MIC values

of 125 µg/mL against S. typhi and C. albicans, and 250 µg/mL against E. coli and S. pyogenes, while

maintaining the same MIC (62.5 µg/mL) against S. aureus. This demonstrates that chelation substantially

increases the biological efficacy of the ligand, likely by improving its lipophilicity and cell membrane

permeability (Jayarajan et al, 2010).

Table 9: Minimum Inhibitory Concentration analysis data for L and CuL

Test organisms Formulations/Concentrations (µg/ml)

Compounds

S. typhi

E. Coli

S. Aureus

S.Pyogenes

C. albicans

250

500

62.5

500

250

CuL

125

250

62.5

250

125

This observation aligns with the chelation theory (Lever, 1984; Mohamed et al,, 2006), which explains that

complex formation reduces the polarity of the metal ion and enhances its ability to penetrate microbial cell

membranes. Consequently, the Cu(II) complex becomes more effective at interacting with intra-cellular bio-

molecules, leading to greater antimicrobial activity. Overall, the results confirm that metal complexation

significantly enhances the antimicrobial potential of the Schiff base ligand, with CuL₂ showing promising

activity comparable to standard antimicrobial agents in similar studies.

The MBC/MFC activities of the Schiff base ligand (L) and its copper(II) complex (CuL₂). The ligand (L)

exhibited limited bactericidal activity(Fig 12b), showing an MBC value of 250 µg/mL only against S. aureus,

while no lethal effect was observed against S. typhi, E. coli, S. pyogenes, and C. albicans up to the highest

S. typhi E. Coli S. Aureus S.Pyogenes C. albicans

Antimicrobial Susceptibility Testing

L CuL2 LEV NY

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tested concentration (>500 µg/mL). Complexation with copper(II) significantly broadened the antimicrobial

spectrum of the ligand. The Cu(II) complex (CuL₂) demonstrated bactericidal/fungicidal activity against S.

aureus (MBC = 250 µg/mL), S. typhi (MBC = 500 µg/mL), S. pyogenes (MBC = 500 µg/mL), and C. albicans

(MFC = 500 µg/mL), whereas E. coli remained resistant (MBC > 500 µg/mL). The enhanced activity of the

complex compared to the free ligand is attributed to the chelation effect, which increases lipophilicity and

facilitates penetration through the microbial cell membrane, leading to greater disruption of essential biological

processes (Table 10). Among all the tested organisms, S. aureus was the most susceptible, with both the ligand

and its Cu(II) complex exhibiting bactericidal activity at 250 µg/mL. The broader activity of CuL₂ against both

Gram-positive and Gram-negative bacteria, as well as C. albicans, indicates that metal coordination enhances

the antimicrobial potential of the ligand.

Table 10: Minimum Bactericidal/Fungicidal Concentration data for L and CuL

Test organisms Formulations/Concentrations (µg/ml)

Compounds

S. typhi

E. Coli

S. Aureus

S.Pyogenes

C. albicans

250

CuL

500

250

500

(a) (b)

Fig 12; (a)Minimum Inhibitory Concentration MIC (b) Minimum bactericidal /Fungicidal concentration

(MBC/MFC)

CONCLUSION

A novel Schiff base ligand derived from 4-acyl pyrazolone and its Cu(II) complex were successfully

synthesized and thoroughly characterized using spectroscopic and analytical methods. The results from IR,

UV–Vis, ^1H NMR, and GC–MS analyses confirmed the formation of the azomethine linkage and

coordination through the nitrogen and oxygen donor atoms, while elemental and conductivity measurements

verified the proposed stoichiometry and non-electrolytic nature of the Cu(II) complex. Observed spectral shifts,

enhanced thermal stability, and the emergence of M–O and M–N bands collectively evidenced strong metal–

ligand bonding. Biological evaluation revealed that coordination with copper(II) markedly enhanced the

antimicrobial properties of the ligand. As supported by chelation theory, complexation reduces the metal ion’s

polarity, increases lipophilicity, and facilitates membrane permeability—thereby strengthening interactions

with microbial biomolecules. The free ligand displayed limited activity, while the Cu(II) complex exhibited

broader antimicrobial and antifungal efficacy, particularly against Staphylococcus aureus.

100

200

300

400

500

600

Minimum Inhibitory

concentration (MIC)

L CuL2

100

200

300

400

500

600

Minimum

Bactericidal/fungicidal

concentrarion (MBC/MFC)

L CuL2

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Overall, these findings demonstrate that metal complexation not only stabilizes the Schiff base framework but

also significantly enhances its bioactivity. The synthesized Cu(II) complex shows promising broad-spectrum

antimicrobial potential, indicating that pyrazolone-derived Schiff base metal complexes could serve as valuable

scaffolds for the design of new antimicrobial agents

REFERENCES

1. Achonye, C.C., Ezenweke, L.O., Ojiako, E.N., and Ogbuagu, O.E. (2024). “Synthesis,

Characterization and Antimicrobial Activities of Pyrrolidin-2-Ylidene-2,(4-Chlorophenyl)

Semicarbazone and Its Cd(II) Complex”. Journal of Materials Science Research and Reviews 7

(4):649–662. https://doi.org/10.9734/jmsrr/2024/v7i4357

2. Ahmed, A. A., Kurama, U. M., Buba, U. S., & Wakil, I. M. (2022). Synthesis and evaluation of

antimicrobial activity of two Schiff bases derived from cyclohexylamine. Algerian Journal of

Chemical Engineering (AJCE), 1(1), 49–54.

3. Alezzy, A. A. M., Alnahari, H., & Al-horibi, S. A. (2022). Short review on metal complexes of Schiff

bases containing antibiotic, and bioactivity applications. Journal of Chemistry and Nutritional

Biochemistry, 3(2), 44–57. https://doi.org/10.48185/jcnb.v3i2.671

4. Barad, S. V., Chaudhari, K., Jadeja, R. N., Roy, H., & Butcher, R. J. (2023). Cytotoxicity assay and

gene expression studies of acylpyrazolone-based square planar Cu(II) complexes: Synthesis,

characterization and computations. Journal of Coordination Chemistry, 76(11–12), 1955–1983.

5. Ceramella, J., Iacopetta, D., Catalano, A., Cirillo, F., Lappano, R., & Sinicropi, M. S. (2022). A review

on the antimicrobial activity of Schiff bases: Data collection and recent studies. Antibiotics, 11(2), 191.

https://doi.org/10.3390/antibiotics11020191

6. Clinical and Laboratory Standards Institute (CLSI). (2020). Performance standards for antimicrobial

susceptibility testing (30th ed.). CLSI document M100.

7. Furniss, B. S., Hannaford, A. J., Smith, P. W. G., & Tatchell, A. R. (1989). Vogel’s textbook of

practical organic chemistry (5th ed., p. 1150),

8. Idemudia, O. G., Sadimenko, A. P., & Hosten, E. C. (2016). Metal complexes of new bioactive

pyrazolone phenylhydrazones; crystal structure of 4-acetyl-3-methyl-1-phenyl-2-pyrazoline-5-one

phenylhydrazone Ampp-Ph. International Journal of Molecular Sciences, 17(5), 687.

https://doi.org/10.3390/ijms17050687

9. Jayarajan, R., Vasuki, G., & Rao, P. S. (2010). Synthesis and antimicrobial studies of tridentate Schiff

base ligands with pyrazolone moiety and their metal complexes. Organic Chemistry International,

2010, Article 648589. https://doi.org/10.1155/2010/648589

10. Jensen, B. S. (1959). The synthesis of 1-phenyl-3-methyl-4-acyl pyrazol-5-ones. Acta Chemica

Scandinavica, 13(8), 1668–1670.

11. Lever, A. B. P. (1984). Inorganic electronic spectroscopy (2nd ed.). Elsevier.

12. Manohar, V., Sridhar, M., Jyothi, P., & Battini, S. (2022). Bioactive metal-Schiff base complexes: A

review on antimicrobial and anticancer potential. Revista Electronica de Veterinaria, 25(2), Article

1886. https://doi.org/10.69980/redvet.v25i2.1886

13. Mohamed, G. G., Omar, M. M., & Hindy, A. M. (2006). Metal complexes of Schiff bases: Preparation,

characterization, and biological activity. Turkish Journal of Chemistry, 30, 361–382

14. Nandini, B., & Amutha Selvi, M. (2025). Metal complexes with Schiff bases – A review of their

antimicrobial activities. Current Bioactive Compounds, 21(5), e15734072305971.

https://doi.org/10.2174/0115734072305971240816114116

15. Narsidas, P., Shashikant, T., Rikin, P., Hitesh, B., Harshur, J., & Vasudev, T. (2015). Synthesis,

antimicrobial and antioxidant activities of some 5-pyrazolone based Schiff bases. Journal of Saudi

Chemical Society, 19, 36–41.

16. National Center for Biotechnology Information (NCBI). (2024). PubChem Compound Summary for

CID 24749, 3-methyl-1-phenyl-pyrazol-5-one. https://pubchem.ncbi.nlm.nih.gov/compound/24749

17. Okolo, A. J., Ezenweke, L. O., Ojiako, E. N., Oragwu, I. P., Okwuego, P. O., Ogbuagu, O. E., Silas, C.

U., & Chukwuemeka, N. U. (2025). Synthesis and antimicrobial study of Fe(II) complex of Schiff base

derived from 4-acyl antipyrine and substituted aniline. International Journal of Research and

INTERNATIONAL JOURNAL OF RESEARCH AND INNOVATION IN APPLIED SCIENCE (IJRIAS)

ISSN No. 2454-6194 | DOI: 10.51584/IJRIAS |Volume X Issue XI November 2025

Page 98

www.rsisinternational.org

Innovation in Applied Science, 10(10), Article 0031.

https://doi.org/10.51584/IJRIAS.2025.1010000031

18. Ogbuagu, O. E., Ezenweke, L. O., Ojiako, E. N., Achonye, C. C., Okolo, A. J., Silas, C. U., & Ndupu,

R. O. (2025). Synthesis, spectroscopic characterization and antimicrobial assessment of 4-acetyl

pyrazolone Schiff base and its cobalt(II) complex. Journal of Materials Science Research and Reviews,

8(4), 845–866. https://doi.org/10.9734/jmsrr/2025/v8i4445

19. Shaikh, I., Travadi, M., Jadeja, R. N., Butcher, R. J., & Pandya, J. H. (2022). Crystal feature and

spectral characterization of Zn(II) complexes containing Schiff base of acylpyrazolone ligand with

antimalarial action. Journal of the Indian Chemical Society, 99(5), 100428.

https://doi.org/10.1016/j.jics.2022.100428

20. Silas, C. U., Ezenweke, L. O., Enedoh, M. C., Ogbuagu, O. E., & Okolo, A. J. (2025). Synthesis,

characterization, and antimicrobial activities of (E)-2-(2-aminothiazol-4-yl)-N'-(2-

nitrobenzylidene)acetohydrazide ligand and its Co(II) complex. Journal of Materials Science Research

, 8(4), 821–833. https://doi.org/10.9734/jmsrr/2025/v8i4443

21. Wang, J., Xu, G., Zhang, Y., Luo, H., Li, J., Zhang, L., & Jia, D. (2019). Copper(II) complexes with 4-

acyl pyrazolone derivatives and diimine coligands: Synthesis, structural characterization, DNA binding

and antitumor activity. New Journal of Chemistry, 43, 2529–2539.

https://doi.org/10.1039/C8NJ02695E

22. Wiegand, I., Hilpert, K., & Hancock, R. E. W. (2008). Agar and broth dilution methods to determine

the minimal inhibitory concentration (MIC) of antimicrobial substances. Nature Protocols, 3(2), 163–

175.

23. Xi, W., Song, F., Xia, X., & Song, X. (2020). Tuned structure and DNA binding properties of metal

complexes based on a new 4-acylpyrazolone derivative. New Journal of Chemistry, 44, 2281–2290.

https://doi.org/10.1039/C9NJ05948B

24. Zoubi, W. (2013). Biological activities of Schiff bases and their complexes: A review of recent works.

International Journal of Organic Chemistry, 3, 73–95. https://doi.org/10.4236/ijoc.2013.33A008