Copper(II) and cobalt(II) complexes of 5-methyl pyrazole-3-carboxylic acid: synthesis, X-ray crystallography, thermal analysis and in vitro antimicrobial activity

Article abstract of DOI:10.1080/00958972.2018.1520984

The coordination behavior of 5-methylpyrazole-3-carboxylic acid (Hmpca) has been demonstrated by the solid state isolation and characterization of [Cu(mpca)₂(H₂O)]·3H₂O (1) [Cu(mpca)₂]·H₂O (2) and [Co(mpca)₂(H₂O)₂] (3). The new compounds are characterized by X-ray crystallography, thermogravimetric analysis and DFT study. The redox properties of the complexes are examined by cyclic voltammetric analysis. The antibacterial and antifungal activities of the compounds against eight bacteria (Escherichia coli, Enterococcus faecalis, Bacillus subtilis, Klebsiella pneumoniae, Proteus vulgaris, Staphylococcus aureus, Pseudomonas aeruginosa and Salmonella typhi) and two fungi (Aspergillus flavus and Candida albicans) are screened using modified agar well diffusion method. The metal complexes demonstrate better inhibition on all bacteria and fungi than the ligand. The high lipophilicity of the complexes accounts for good inhibitory action toward microbes. Among the reported complexes, 3 emerges as an excellent antifungal agent and a better antibiotic than standard fluconazole. The structure and activity relationship indicate that complexes having sufficient Jahn–Teller distortion with high logP values, cross the cell membrane of the microbes creating intercellular damage.

Full text of DOI:10.1080/00958972.2018.1520984

Journal of Coordination Chemistry
ISSN: 0095-8972 (Print) 1029-0389 (Online) Journal homepage: http://www.tandfonline.com/loi/gcoo20
Copper(II) and cobalt(II) complexes of 5-methyl
pyrazole-3-carboxylic acid: Synthesis, X−ray
crystallography, thermal analysis and in vitro
antimicrobial activity
Ananyakumari Santra, Paula Brandao, Harekrishna Jana, Gopinath Mondal,
Pradip Bera, Abhimanyu Jana & Pulakesh Bera
To cite this article: Ananyakumari Santra, Paula Brandao, Harekrishna Jana, Gopinath Mondal,
Pradip Bera, Abhimanyu Jana & Pulakesh Bera (2018): Copper(II) and cobalt(II) complexes of 5-
methyl pyrazole-3-carboxylic acid: Synthesis, X−ray crystallography, thermal analysis and inꢀvitro
antimicrobial activity, Journal of Coordination Chemistry, DOI: 10.1080/00958972.2018.1520984
To link to this article: https://doi.org/10.1080/00958972.2018.1520984
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Copper(II) and cobalt(II) complexes of 5-methyl pyrazole-3-carboxylic acid:
Synthesis, X−ray crystallography, thermal analysis and in vitro
antimicrobial activity
ANANYAKUMARI SANTRA†, PAULA BRANDAO‡, HAREKRISHNA JANA¶, GOPINATH MONDAL†,
PRADIP BERA†, ABHIMANYU JANA† and PULAKESH BERA1†
†Post Graduate Department of Chemistry, Panskura Banamali College (Vidyasagar University),
Panskura R.S, Midnapore (East), West Bengal, India – 721152
‡Department of Chemistry, CICECO, University of Aveiro, 3810−193 Aveiro, Portugal
¶Department of Microbiology, Panskura Banamali College, Panskura R.S, Midnapore (East),
West Bengal, India – 721152
The coordination behavior of 5-methylpyrazole-3-carboxylic acid (Hmpca) has been
demonstrated by the solid state isolation and characterization of [Cu(mpca)2(H2O)]·3H2O (1)
[Cu(mpca)2]·H2O (2) and [Co(mpca)2(H2O)2] (3). The new compounds are characterized by
X-ray crystallography, thermogravimetric analysis and DFT study. The redox properties of the
complexes are examined by cyclic voltammetric analysis. The antibacterial and antifungal
activities of the compounds against eight bacteria (E. coli, S. fecalis, B. subtilis, K. pneumonia,
P. vulgaris, S. aureus, P. aeruginosa and S. typhi) and two fungi (A. flavus and C. albicans) are
screened using modified agar well diffusion method. The metal complexes demonstrate better
inhibition on all bacteria and fungi than the ligand. The high lipophilicity of the complexes
accounts for good inhibitory action toward microbes. Among the reported complexes, 3 emerges
as an excellent antifungal agent and a better antibiotic than standard fluconazole. The structure
and activity relationship indicates that complexes having sufficient Jahn-Teller distortion with
high logP values, cross the cell membrane of the microbes creating intercellular damage.
Keywords:
Pyrazolyl;
Inner-metallic;
Thermogravimetry;
Antibacterial-antifungal;
Mono/polymeric
1. Introduction
Heterocyclic compounds containing nitrogen, sulfur and oxygen and their metal complexes are
biologically active materials towards bacteria, fungi and viruses [1-5]. Heterocyclic compounds
1Corresponding author. Email: pbera.pbc.chem@gmail.com

containing pyrazole rings have interest due to their applications in the pharmaceutical and
agrochemical industries [6]. Pyrazole derivatives have wide range of applications like anti-
hyperglycemic [7], analgesic [8], anti-inflammatory [9], antipyretic [10], antibacterial [11],
hypoglycaemic [12] and sedative−hypnotic activities [13]. Pyrazole derivatives such as
celecoxib, rimonabant, fomepizole and sildenafil were established as selective drugs [14].
Celecoxib demonstrated anti-inflammatory effect and inhibited cox-2 [15]. Rimonabant is a
cannabixiod receptor and is used for obesity treatment. Fomepizole and bindenafil inhibit alcohol
dehydrogenase and phosphodiesterase, respectively [16]. Some pyrazole derivatives also have
non nucleoside HIV−1 reverse transcriptase inhibitory activities [17-19]. Their transition metal
complexes are also active metallobiomolecules. The acid and amido derivatives of pyrazole
compounds are important heterocycles for synthesis of metallobiomolecules [20]. Copper and
cobalt complexes of pyrazolyl derivatives showed excellent antibacterial and antifungal activities
for generation of several coordination and non−covalent interactions [20-23]. For these reasons,
large numbers of copper complexes were synthesized to screen the biological activities and some
of them were active both in vivo and in vitro [15, 23]. Fascinated by the significant biological
applications of transition metal complexes of pyrazolyl compounds, comprehensive work has
been taken up to synthesize and screen the antimicrobial activity of pyrazole derivatives. Herein
we report the synthesis, characterization and antimicrobial activities of three new complexes of
copper(II) and cobalt(II) derived from 5–methylpyrazole–3–carboxylic acid. The structure-
activity relationship has been discussed.
2. Experimental
2.1. Materials
Reagent grade CuCl2∙2H2O and Co(NO3)2∙6H2O were purchased from Merck India Chemical
Company and used without purification. The salt Cu(ClO4)2∙6H2O was prepared by laboratory
process from CuCO3 (Merck, India) and perchloric acid (Merck, India). Solvent ethanol
(Changshu Yangyuan Chemical, China), methanol (Merck, India) and dichloromethane (Merck,
India) were dried and distilled before use. 1-Octanol, hydrazine hydrate, ethyl acetate, pyruvic
acid and hydrazine sulphate were purchased from Himedia, India. Caution! Perchlorate salts are
explosive. Care should be taken in handling the perchlorate salts.

2.2. Characterization
Elemental analyses (C, H, N, and S) of the ligand and complexes were performed using a
FISONS EA−1108 CHN analyzer. The molar conductance of 10–3 (M) solutions of 1, 2 and 3 in
DMF:MeOH (1:1) were measured at 30 C using a Thermo Orion model 550A conductivity
meter and a dip-type cell with platinized electrode. The FTIR analyses were performed by using
a Perkin Elmer Spectrum Two Spectrophotometer (400–4000 cm–1). The samples were prepared
as KBr pellets using anhydrous KBr salt. The magnetic susceptibility measurements were done
using a Sherwood Scientific Cambridge (UK) magnetometer. UV–Visible absorption spectra of
samples were recorded on a Perkin Elmer Lambda 35 spectrophotometer from 200–800 nm at
room temperature. Single crystal X–ray diffractions (XRD) of 1-3 were carried out on a Bruker
SMART APEX II X–ray diffractometer equipped with graphite–monochromatic Mo−Kα
radiation (λ = 0.71073 Å) and 16 CCD area detector. The intensity data were collected in the π
and ω scan mode, operating at 50 kV, 30 mA at 296 K [24]. The data reductions were performed
using SAINT and SADABS programs [25]. All calculations in the structural solution and
refinement were performed using the Bruker SHELXTL program [26]. The structures were
solved by the heavy atom method and refined by full-matrix least-squares methods. All non–
hydrogen atoms were refined anisotropically; hydrogens were geometrically positioned and fixed
with isotropic thermal parameters. The final electron density maps showed no significant
difference. Cyclic voltammetry studies were performed at room temperature in methanol using
tetrabutylammonium perchlorate as a supporting electrolyte on a CH Instrument electrochemical
workstation model no. CHI630E. The conventional three-electrode assembly is comprised of a
platinum working electrode, a platinum wire auxiliary electrode and a Ag/AgCl reference
electrode. Thermogravimetric analysis was performed using a Perkin Elmer Thermal Analyzer
TGA4000 instrument at a heating rate 20 °C/min under nitrogen.
2.3. Synthesis of ligand and metal complexes
2.3.1. Synthesis of 5–methylpyrazole–3–carboxylic acid (Hmpca). 5–Methylpyrazole–3–
carboxylic acid (Hmpca) was prepared from the hydrolysis of 5–methylpyrazole–3–
carbohydrazide (mpch) [27]. Aqueous solution of sodium ethyl acetopyruvate (A) (10 g,
0.05 mol) is taken in a 250 mL round bottom flask and allowed to react with hydrazine sulphate
(7 g, 0.05 mol) in the presence of anhydrous Na2CO3 (scheme 1). A light yellow product ethyl 5–

methylpyrazole–3–carboxylate (B) is obtained. The ester (B) separated from the solution is
filtered off, washed with water and dried in an oven at 100 C. Recrystallization from aqueous
ethanol gave the pure ester (B) in colorless form melting at 82 C. The dried ester (0.04 mol,
6.2 g) is allowed to reflux with hydrazine hydrate (100%) (0.06 mol, 3.0 g) at water bath
temperature for 1 h. A white crystalline precipitate of 5–methylpyrazole–3–carbohydrazide
(mpch) is obtained which is filtered off, washed with aqueous ethanol and dried in a vacuum
desiccator. The dried mpch melts at 153 C. The yield is 4.2 g (75%). The dry mpch (15 mmol,
2.1 g) is taken in excess hydrazine hydrate (30 mL) and refluxed for 5 h. The desired ligand
(Hmpca) is obtained by removing excess hydrazine by vacuum distillation. The product thus
obtained is recrystallized from dry ethanol. Purity of the product was checked by the
determination of melting point (M.P. 239 C).
2.3.2. Synthesis of [Cu(mpca)2(H2O)]·3H2O (1) and [Cu(mpca)2]·H2O (2). Hmpca (5 mmol,
0.63 g) dissolved in 15 mL in dry methanol is allowed to mix with a methanolic solution of
CuCl2·2H2O (2.25 mmol, 0.38 g) (20 mL) in a 100 mL round bottom flask. The resulting blue
solution is refluxed for 6 h whereupon a blue crystalline product separates. The product is
filtered off, washed with methanol and dried over silica gel. The yield is 0.86 (72%) for
[Cu(mpca)2(H2O)]·3H2O (1). Elemental analysis for 1 (C10H14CuN4O6): Calculated: C, 31.10; H,
4.66; N, 14.53 and Found: C, 31.05; H, 4.54; N, 14.85. IR assignments (in cm–1): ν(C=N) (1425),
ν(C-N) (1299), ν(N-N) (1041) and ν(C-O) (1627).
In similar way, Cu(ClO4)2∙6H2O (2.25 mmol, 0.83 g) in 20 mL dry methanol is mixed
with a dry methanolic solution (15 mL) of Hmpca (5 mmol, 0.63 g). The resulting solution is
refluxed for 4 h; slow evaporation of the solution at room temperature furnished blue crystalline
compound which is filtered off, washed with methanol and dried over silica gel. The yield is
0.51 g (68%) for [Cu(mpca)2]·H2O (2). Elemental analysis for 2 (when n=1) (C10H12CuN4O5):
Calculated: C, 36.16; H, 3.61; N, 16.87 and Found: C, 36.29; H, 3.42; N, 16.91. IR assignments
(in cm–1): ν(C=N) (1369), ν(C-N) (1285), ν(N-N) (1034) and ν(C-O) (1550).
2.3.3. Synthesis of [Co(mpca)2(H2O)2] (3). Hmpca (5 mmol, 0.63 g) dissolved in 15 mL
hydrazine hydrate (100%) is mixed with a hot methanolic solution of Co(NO3)2·6H2O (20 mL)
(2.25 mmol, 0.65 g) with constant stirring. The resulting solution is refluxed for 4 h and the solid

thus obtained is filtered, washed with dry methanol, and dried in vacuum. Yield is 0.54 g (71%)
for C10H14CoN4O6 (3). Elemental analysis: Calculated: C, 34.76; H, 4.05; N, 16.22 and Found:
C, 34.56; H, 3.89; N, 16.57. IR assignments (in cm–1): ν(C=N) (1476), ν(C-N) (1271), ν(N-N) (1041)
and ν(C-O) (1697).
Complexes 1, 2 and 3 are soluble in common polar solvents like methanol, ethanol, DMF,
and DMSO. The molar conductivity values of 20, 15 and 23 Ω–1cm2mol–1 for 1, 2 and 3,
respectively, in DMF:MeOH (1:1) with concentration 10–3 (M) are indicative of nonelectrolyte
nature of the complexes. The observed spin only magnetic moment values are 2.32, 2.22 and
3.58 BM for 1, 2 and 3, respectively. The magnetic moment measurements indicate high spin
cobalt(II) center in 3.
Scheme 1. Synthesis of Hmpca ligand and 1-3.
2.4. Determination of the logP by using the shake−flask method
Lipophilicity of the ligands and complexes are determined by calculating logP values according
to the shake-flask method [6]. Compounds under investigation are weighted out accurately and
partitioned between equal volumes of water and 1-octanol (5 mL each) by shaking for 30 h at
room temperature to assure full partitioning of the analyzed compounds between water and
1-octanol phases. Then, the solution is centrifuged at 3500 rpm for 10 minutes to separate into
two layers. The concentration of the compounds in different layers is estimated by the optical

density values obtained from UV-Vis spectroscopy. Concentration of organic, aqueous phase and
logP values are in table 1.
2.5. DFT calculations
The ground state electronic structure calculations have been carried out using the X-ray
coordinates of the complex with the help of DFT [28] methods with the Orca 2.7 program [29].
Becke’s hybrid function [29] with the Lee–Yang–Parr (LYP) correlation function [30] was used
throughout the study. LANL2DZ valence and effective core potential functions were used. All
energy calculations were performed using the self-consistent field “tight” option of the Orca 2.7
program to ensure sufficiently well converged values for the state energies.
2.6. Determination of antibacterial and antifungal activity
Antibacterial sensitivity is tested by the agar well diffusion method using Mueller–Hilton agar
media. The said diffusion method is employed for the determination of antibacterial activities of
the compounds following the method described by Vanden Berghe and Vlietinck in 1991 [31].
The compounds are dissolved with required amount of methanol to make up a concentration
5000 μg/mL. From this stock solution different concentrations of working solution (ranges from
1000 to 25 μg/mL) was prepared. Eight species of pathogenic bacteria, Escherichia coli,
Salmonella typhi, Klebsiella pneumoniae, Staphylococcus aureus, Proteus vulgaris,
Pseudomonas aeruginosa, Bacillus subtilis and Streptococcus faecalis, are used to screen the
antibacterial activities of the compounds [32]. Pathogenic bacterial strains are incubated at 37 °C
for 24 h in sterile nutrient broth. The inocula size of swabbed pathogen was adjusted to deliver
106 CFU/mL on the surface of Mueller−Hilton agar media. 25 mL of Mueller–Hinton Agar
media and 100 μL inoculums of bacterial strain are allowed to solidify in petridishes. Wells are
made into solidified agar media with the help of sterilized cup−borer. 100 μL of prepared sample
solution is poured into the respective wells and the plates are incubated overnight at 37 °C.
Methanol is used as a control. The experiment is repeated three times under strict aseptic
condition and the antibacterial activity of each compound was expressed in terms of the mean
diameter of zone of inhibition (in cm) produced by the respective compound.
Similarly, antifungal sensitivity is tested by the agar well diffusion method using PD agar
media [32]. Two species of pathogenic fungi, Aspergillus flavus and Candida albicans, are used

to screen the antifungal activity of the pyrazolyl compounds. Pathogenic fungal strain are
inoculated in sterile potato dextrose broth and incubated at 25 °C for 48 to 72 h. Petri dishes
containing 25 mL of potato dextrose agar with 100 μL inoculums of fungal strain and media is
allowed to solidify. The remaining work up process is similar to that of antibacterial sensitivity
test. The stock solution (5000 µg/mL) was made by dissolving the antibiotic in sterile distilled
water. From this stock solution 1000 µg/mL was prepared for the determination of the sensitivity
of fungi.
3. Results and discussion
Hydrolysis of 5–methyl pyrazole–3–carbohydrazide (Hmpch) occurs in the presence of
hydrazine hydrate. The title complexes are obtained by reaction of the ligand with the
corresponding metal salt. The alkaline hydrolysis of mpch gives high yield of the ligand in the
present procedure. The anion in the solution influences the crystallization as evidenced by the
formation of different copper(II) complexes. Monomeric and polymeric copper(II) complexes
are isolated in the presence of chloride and perchlorate ions, respectively. This is an instance of
anion dependent crystallization of copper(II) complexes. On the other hand, cobalt(II) salt in the
solution always furnish monomeric complex of Hmpca irrespective of the anion present in the
reaction medium. The reducing environment prevents the oxidation of cobalt(II) species during
reaction. The use of hydrazine as solvent has the advantage to prepare complexes of cobalt(II).
The spin only magnetic moment values of the complexes reveal the presence of one and three
unpaired electrons in copper(II) and cobalt(II) complexes.
3.1. FTIR analysis
The important IR assignments (in cm−1) of the complexes are shown in figure 1. The stretching
frequencies of the C=N, C−N and N−N(pyrazole) bonds are obtained in the range 1425-1467,
1271-1299 and 1034-1041 cm−1, respectively, in the complexes. Similar IR frequencies are also
observed in several pyrazole derivatives [33]. All peaks are obtained in lower frequency range in
2 than those observed in 1 and 3. This proves the polymeric nature of 2 where polymerization
effectively reduced the respective bond orders. The peak at 1550-1697 cm−1 in the complexes
can be assigned for the stretching vibration of C−O of carboxylate. The observed lower

frequency range of ν(CO) in the complexes with respect to free carboxylic acid (1690-
1710 cm−1) revealed that the coordination occurred through oxygen of the acid group [33, 34].
3.2. Single crystal X-ray crystallography
Single crystal X-ray analyses of the complexes provide the molecular arrangement and
coordination environment of the transition metal chelates. Figure 2 shows the ORTEP diagram of
1 and crystal refinement parameters are given in table 2. Selected bond lengths and angles of the
complexes are given in tables 3 and 4, respectively. The 5–methylpyrazole–3–carboxylic acid
(Hmpca) acts as a uninegative bidentate ligand, coordinating through N and O. Complex 1 is a
monomer and crystallizes with space group Pī. The role of water molecules in the crystal packing
of 1 is very important. The 1D propagation of molecule competes with the parallel propagation
of molecules of water of crystallization making a cylindrical channel of water. The D…A bond
distance (2.03 Å) indicates considerable H−bonding in the molecule. The H bonds are shown in
figure S1 and the corresponding values are given in table S1. Copper is square pyramidal with
the base formed by two nitrogen and two oxygen atoms of mpca–. The fifth coordination site is
occupied by a water molecule (figure 2). Copper(II) is coordinated by pyrazolyl N and anionic O
of the acid group of mpca–. The bond lengths Cu−N1 and Cu−N2 are 1.941 Å and 1.954 Å,
respectively, and the bond lengths Cu−O2 and Cu−O4 are equal (1.963 Å), whereas the Cu–O1
length is 2.466 Å, which indicates the axial position of the square pyramidal structure is
occupied by H2O (table 3).
The same ligand furnishes a polymeric copper(II) complex in the presence of
Cu(ClO4)2·6H2O designated as 2. Figure 3 represents the ORTEP diagram of 2, where the copper
ion completed five coordination sites to form a distorted square pyramidal complex with the
pyrazolyl N and carboxylic O of mpca–. Four basal positions are occupied by N and O atoms of
two mpca– and the fifth position is occupied by the oxygen of the mpca– of a neighboring
molecule. A polymeric structure is formed through Cu–O–Cu bonding (figure 4). The
propagation of polymeric chains in the structure is facilitated by several non-covalent
interactions. The π-π interactions were found with centroid to centroid distance 3.739 Å in 1
(figure S2). Contribution of CH-π interaction is also interpreted with centroid to HC 3.15 Å. The
H-bond interactions of 2 are shown in figure S3.

In 3, the cobalt is octahedrally surrounded by the ligand as shown in figure 5. The
asymmetric unit contains half of the whole molecule where a C2 axis passes through cobalt and
symmetric operation C2 generates the whole molecule. The cis form is predominant in the solid
state. This may be due to the process of crystallization of the compound in the polar solvent [35].
The typical higher values of Co−N(pz) and Co−O(carboxylate) than reported CoIII−N and
CoIII−O bonds in octahedral complexes clearly suggest +2 oxidation state of cobalt in 3 [35]. In
the structure of 3, the H-bonding aspects are very important. Strong H-bonding occurs in each H
attached with pyrazole N and coordinated water with oxygen. H-bonds are extended three
dimensionally as shown in figure S4. No CH-π interaction was involved in the proposed
structure. Ligand containing N–N donor sites are involved for coordination and two water
molecules are attached to the cobalt making it six-coordinate octahedral (figure 5). The cobalt(II)
octahedral complex is stabilized by strong chelation through the donor pyrazolyl nitrogen and
carboxylate oxygen.
3.3. Thermogravimetric analysis
Thermal decomposition of all three complexes is carried out. As shown in figure 6, TG curve of
1 shows three significant mass losses at 100-135 °C (18.3%), 135-350 °C (5.6%) and 340-700 °C
(47.1%). The first step corresponds to the loss of three moles of water of crystallization followed
by the second step which accounts for the loss of one mole of coordinated water from the
coordination site. About 47.1% mass loss in the third stage of decomposition may be loss of two
moles of methyl pyrazole. There is similarity in the decomposition of 2 with the isostructural 1
but the decomposition steps are not clearly observed in 2. The reason may be the polymeric
nature of 2. Unlike 1, the decomposition of 2 starts at RT which reveals that the sample may
contain moisture. A mass loss up to 300 °C is about 18% (calc. 18.68%) which may be the
combined loss of one unit CO2 and one mole of water of crystallization. The certain mass losses
at 290-390 °C is the loss of remaining organics i.e., methyl pyrazole unit giving CuO residue
(23.96%). Further heating of the compound beyond 500 °C metallic copper is obtained with
residual percentage 19.1 [36]. As shown in figure 6 (c), the decomposition of 3 starts at 150 °C
giving a weight loss about 86%, which can be attributed to the combined lose of two moles of
coordinated water molecules (calc. 10.42%) preceding the loss of two moles of 5-methyl
pyrazole-3-carboxylic acid (calc. 71.84%) giving a residue 17% of metallic cobalt [33, 37].

3.4. Cyclovoltammetry study
The electrochemical behaviors of the present compounds have been studied as these categories
of compounds have wide applications in biology and chemistry. The redox properties give
insight into their metabolic facts and biological activities. Copper(II) complexes 1 and 2 in
methanol solution (10−3 M) in the presence of tertiary butyl ammonium perchlorate as a
reference electrolyte versus Ag/AgCl, saturated KCl, at a scan rate 100 mVs–1, display
irreversible reductive responses at −0.561V (C1) in 1 and −0.346V (C1) in 2 which correspond to
the reduction Cu(II)→Cu(I) as shown in figure 7. The other peak on cathodic scan at +0.559V
(C2) in 1 and +0.738V (C2) in 2 can be assigned for the reduction of Cu(I) to Cu(0). In the
subsequent anodic scan, the anodic counterparts (A1, A2) are not prominent. A broad peak at
+0.979V (A2) in 1 and +1.153V (A2) in 2 are assigned for Cu(0)→Cu(I). The difference in the
values of reduction potential in the copper complexes may be due to the different coordination
environment around copper(II) e.g., monomeric structure of 1 and polymeric structure of 2,
respectively [38]. The CV of 3 also shows irreversible redox process. Irreversible reductive
responses at −0.253V (C1) and +0.262V (C2) correspond to Co(III)→Co(II) and Co(II)→Co(I),
respectively. The only one observable anodic peak at +0.458 (A2) can be attributed to the
oxidation of Co(II)→Co(III).
3.5. DFT study
Density functional theory is used to give insight about the structural aspects of the prepared
complexes. Representative complexes 1 and 3 are investigated by DFT study. DFT study of 1
shows that HOMO is completely localized on carboxylic acid group of the ligand molecule;
similar features are also observed for HOMO(n-1) and LUMO(n+1). However, LUMO in 1 is
mainly localized both on pyrazole ring and carboxylic acid group (figure 8). Coordinated water
molecule does not contribute much to the HOMO(n-1), HOMO, LUMO and LUMO(n-1). In 3,
HOMO(n-1) and HOMO are fully localized on carboxylic acid group and coordinated water
molecules. Pyrazole moiety does not contribute to the HOMO(n-1) and HOMO. LUMO and
LUMO(n+1) of 3 are mainly composed with pyrazole unit, carboxylic acid group and
coordinated water molecules. The theoretical studies register that the coordinated water

molecules in 3 have higher involvement with the metal center than 1. Thus it can be concluded
that 1 is more labile than 3 with respect to water substitution.
3.6. Antimicrobial activity
The diploid nature and longer generation time of fungi compared to bacteria make them different
with respect to interaction with antimicrobial agents [39-41]. Most of the antibacterial agents
inhibit the formation of peptidoglycan of cell wall resulting in cell death. Other prominent ways
to destroy bacteria are protein synthesis inhibitors, topoisomerase inhibitor and metabolic
pathway inhibitor. Whereas most antifungal agents target the formation and function of
ergosterol which is an important component of fungal cell membrane. The ability of the ligands
and complexes to inhibit the growth of several bacteria and fungi are assessed using disc
diffusion, agar well diffusion, and minimum inhibitory assays. The in vitro antimicrobial
activities are given in table 5 and MIC values of the compounds are presented in table 6. Camera
views of the agar experiments are provided in the Supplementary Data (figure S5). Results show
that the antimicrobial activities of the complexes are greater than the ancillary ligands i.e.,
Hmpca and mpch. This can be explained on the basis of lipophilic character of the compounds.
The lipophilicity of a compound is the measure of cell membrane permeation. Mostly disposition
ability of a drug depends on the capacity to cross the cell membrane [42, 43]. The general trend
of the logP (where P is the partition coefficient) values is 0.645 (3) > 0.396 (1) > 0.253 (Hmpca)
> 0.174 (mpch) > 0.101 (2) (table 1). The antibacterial effect of the tested compounds follows
the above logP order. Complex 3 has the highest logP (0.645) and exhibits the highest
antimicrobial property. The lowest logP value is attesting the lowest activity of 2. The polymeric
nature of 2 restricts the distribution of molecule in the organic (1-octanol) phase compared to
monomeric complexes 1 and 3. The logP order among the ligands can be explained considering
the increased polarity of the ligands. 5-Methyl pyrazole carbohydrazide (mpch) and/or 5-methyl
pyrazole 3-carboxylic acid (Hmpca) is expected to have higher polarity than the metal
complexes. Both the ligands, mpch and Hmpca, which differ from each other solely by their
functional group, exhibit low antibacterial activity (MIC values 500−600 µg/mL) against
K. pneumonia and C. albicans compared to metal complexes. The complexes obtained from the
reaction of ligand and metal ions are where the primary charge(s) of the metal ion is satisfied by
the ligand molecule. Absence of any free anion(s) in the complexes makes them lipophilic in

nature. Lipophilicity of compound favors permeation through the lipid layer of the cell
membrane. The present study concludes that complexes with high logP show better
antimicrobial activity than the original chelator. Among the complexes, cobalt(II) complex (3)
exhibits the greatest antimicrobial activity with MIC values 25−100 µg/mL and the lowest
activity is exhibited by polymeric 2 against different bacteria and fungi. These findings are
graphically represented in figure 9.
3.7. Structure-activity relationship
Sensitivity study and the MIC values reveal that 1 and 3 show best results (figure 7). Copper(II)
(d9) and high spin cobalt(II) (d7) have electron arrangement (d 2)2(d 2)1 and (d 2)1, respectively.
2
x -y
z
z
The asymmetric arrangement registers a strong tetragonal distortion which allows weak
coordination with H2O in the axial position. The distortion in 1 and 3 makes them labile with
respect to H2O substitution. DFT results also suggest higher lability of the water molecule in 1
than 3. The lability of the complexes favors oxygen binding to the metal center, which further
leads to generation of different reactive oxygen species (ROS). The generated ROS are
responsible for microbial cell destruction. The lesser activity of 2 than 1 and 3 can be explained
on the basis of non availability of coordinated water molecule in the coordination site and
polymeric structure making it inert with respect to ligand substitution. Additionally, the
compounds have high value of lipophilicity (logP) (P refers partition coefficient) (table 1). The
high lipophilicity shows high permeation to the cell wall of the microbes inhibiting metabolism
and protein synthesis.
4. Conclusion
Complexes of copper(II) and cobalt(II) with 5-methylpyrazole–3–carboxylic acid (Hmpca) are
synthesized and characterized by X-ray crystallography, TGA, DFT and cyclic voltammetry
study. Both monomeric and polymeric copper(II) complexes are obtained from the influence of
different counter ions in the reaction mixture. Monomeric 1 adopts a square pyramidal structure
where pyrazolyl N and O (carboxylate) of ligand occupy the corners of the square plane, and the
axial position is occupied by a water molecule. The polymeric structure of 2 is obtained where
the square planes are attached with other square planes through an axial O (carboxylate).
Cobalt(II) complex (3) has distortion in its structure due to Jahn-Teller effect which holds two

water molecules weakly. The loosely bound water molecules are labile with respect to ligand
substitution. The lability of the present complexes favors formation of reactive oxygen species
(ROS) for the cell destruction of microbes under investigation. Moreover, the present complexes
have high lipophilicity showing better permeation to the cell wall leading to cell death. These
combining effects of lability and lipophilicity need to be explored to search for new drugs.
Supplementary data
CCDC 1826509, 1826510 and 1826511 for 1, 2 and 3, respectively. Crystallographic data can be
obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EZ, UK;
Fax: (+44) 1223 336033; E-mail: deposit@ccdc.cam.ac.uk.
Acknowledgement
We gratefully acknowledge the grant from Council for Scientific and Industrial Research
(CSIR), Government of India through the project [No. 1(2858)/16/EMR−II]. Panskura Banamali
College acknowledges the grants received from Department of Science and Technology (DST),
Govt. of India through FIST program (Sanction no. SR/FST/College−295 dated 18/11/2015).
Conflict of interest
The authors declare no conflict of interest.
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Figure 1. FTIR analysis of 1-3.
Figure 2. ORTEP of 1.

Figure 3. ORTEP of 2. *Symmetry equivalent for O2i (i = x, 1/2-y, 1/2+z).
Figure 4. Polymeric structure of 2 viewed along the b axis (hydrogens are omitted for clarity).

Figure 5. ORTEP of 3.
Figure 6. Thermogravimetric analysis of 1-3.

Figure 7. Cyclic voltammetry study of 1-3 in the presence of tertiary butyl ammonium
perchlorate in methanol.
Figure 8. Surface plots of 1 and 3.

Figure 9. Graphical presentation of sensitivity of the compounds against different bacteria and
fungi.
Table 1. Lipophilicity of the compounds determine by shake−flask method using UV-Vis
spectroscopy.
1-Octanol
Water
Concentration
Sample
logP
Concentration
Absorbance
Absorbance
(g/L)
2.40
2.51
2.07
1.83
2.08
(g/L)
1.39
1.68
0.83
1.45
0.47
Hmpca
3.7923
3.3218
3.2156
2.5292
3.4714
2.116
2.2590
1.4972
2.014
0.2371
0.1743
0.3968
0.1010
0.6459
mpch
1
2
3
0.7934

Table 2. Crystal refinement parameters of 1-3.
Parameters
1
2
3
CCDC
1826509
385.83
1826511
331.79
1826510
345.18
Mr
Moiety formula
a (Å)
C10H12CuN4O5·3(H2O)
6.9787(6)
9.0648(7)
12.8706(10)
97.289(2)
91.744(3)
112.178(2)
Triclinic
150K
C10H10CuN4O4·H2O
8.2401(2)
15.6756(5)
9.9014(3)
90
C10H14CoN4O6
14.7463(13)
14.7463(13)
32.114(3)
90
b (Å)
c (Å)
α (°)
β (°)
104.88
90
γ (°)
90
120
Crystal system
Temperature
Volume
Space group
μ (mm−1)
Z
Monoclinic
150K
Trigonal
150 K
745.15(10)
Pī
1236.06(6)
P 21/c
6047.7(14)
R −3 c
1.515
1.794
1.312
2
4
18
F (000)
394.0
676.0
3150.0
F (000ˊ)
Dcalc (g cm−3)
R (reflections)
wR2 (reflections)
394.84
677.55
3157.38
1.686
1.711
1.783
0.0337 (3269)
0.0858 (4037)
0.0279 (2835)
0.0705 (3348)
0.0964 (1337)
0.2252 (1499)

Table 3. Selected bond lengths (Å) of 1-3.
1
2
3
Atoms
Length (Å)
Atoms
Length (Å)
Atoms
Length (Å)
Cu−O1
Cu−O2
Cu−O4
Cu−N1
Cu−N3
O2−C1
O3−C1
O4−C6
O5−C6
N1−N2
N1−C2
N2−C4
N3−N4
N3−C7
2.466(2)
1.963(1)
1.963(1)
1.941(2)
1.954(1)
1.290(2)
1.236(2)
1.291(3)
1.234(2)
1.342(2)
1.340(3)
1.348(3)
1.343(2)
1.338(3)
Cu−O1
Cu−O3
Cu−N1
Cu−N3
Cu−O2
O1−C1
O2−C1
O2−Cu
O3−C6
O4−C6
N1−N2
N1−C2
N3−N4
N2−C4
1.988(1)
1.987(1)
1.955(2)
1.970(2)
2.209(2)
1.278(2)
1.245(3)
2.209(2)
1.271(2)
1.252(2)
1.342(2)
1.336(3)
1.344(2)
1.354(3)
Co–O1
Co–O2
Co–N1
Co–O1
Co–O2
Co–N1
O2–C1
O3–C1
N1–N2
N1–C2
N2–C4
C1–C2
C2–C3
C3–C4
2.079
2.107
2.126
2.079
2.107
2.126
1.26(1)
1.25(1)
1.346(8)
1.32(1)
1.34(1)
1.487(9)
1.41(1)
1.37(1)

Table 4. Selected bond angles (°) of 1-3.
1
2
3
Atoms
Angle (°)
Atoms
Angle (°)
Atoms
Angle (°)
O1–Cu–O2
O1–Cu–O4
O2–Cu–O4
O1–Cu–N1
O1–Cu–N3
O2–Cu–N1
O2–Cu–N3
O4–Cu–N1
O4–Cu–N3
N1–Cu–N3
Cu–O2–C1
Cu–O4–C6
Cu–N1–N2
Cu–N1–C2
N2–N1–C2
N1–N2–H2
N1–N2–C4
H2–N2–C4
Cu–N3–N4
Cu–N3–C7
N4–N3–C7
88.28(6)
99.74(6)
171.62(6)
89.22(7)
93.30(6)
82.12(7)
99.73(6)
95.48(7)
82.33(6)
176.91(7)
114.3(1)
115.4(1)
139.1(1)
113.0(1)
106.1(2)
122(2)
O1−Cu−O2
O1−Cu−O3
O3−Cu−O2
O1−Cu−N1
O3−Cu−N1
O3−Cu−N3
O1−Cu−N3
N1−Cu−N3
N1−Cu−O2
N3−Cu−O2
Cu−O1−C1
C1−O2−Cu
Cu−O3−C6
Cu−N1−N2
Cu−N1−C2
N2−N1−C2
N1−N2−C4
H2−N2−C4
Cu−N3−N4
Cu−N3−C7
N4−N3−C7
99.54(6)
160.53(5)
99.90(6)
81.55(6)
94.93(6)
81.91(6)
96.99(6)
166.36(6)
97.10(6)
96.51(6)
115.0(1)
126.4(1)
113.8(1)
139.4(1)
114.1(1)
106.4(1)
111.1(2)
129(1)
O1–Co–O2
O1–Co–O1
O1–Co–O2
O1–Co–N1
O1–Co–N1
O2–Co–N1
O2–Co–O1
O2–Co–O2
O2–Co–N1
N1–Co–O1
N1–Co–O2
N1–Co–N1
O1–Co–O2
O1–Co–N1
O2–Co–N1
Co–O2–C1
Co–N1–N2
Co–N1–C2
N2–N1–C2
N1–N2–C4
O2–C1–O3
173.5
88.1
88.9
96.7
95.2
77.7
88.9
94.6
90.9
95.2
90.9
163.3
173.5
96.7
77.7
116.2
139.5
111.6
105.9(7)
111.6(7)
124.2(8)
111.7(2)
126(2)
140.1(1)
113.4(1)
106.1(2)
140.9(1)
112.9(1)
106.1(1)

Table 5. Antimicrobial activity of specific concentration (1000 µg/mL) of different synthetic metal complex
compared with control by agar well diffusion method.
Zone of inhibition (in cm) against (Mean±SD)
Bacteria
Fungi
Gram negative
Gram positive
Sample
P.
C.
albicans
E. coli
S. typhi
B. subtilis K. pneumoniae P. vulgaris S. aureus
S. fecalis
A. flavus
aeruginosa
Hmpca
1.1 ±0.03 0.9 ±0.01 1.2 ±0.05
1.2 ±0.05 1.1± 0.05 0.9 ±0.05
0.9 ±0.05 1.4 ±0.06 2.2 ±0.01
1.0 ±0.05 1.0 ±0.06 1.1 ±0.01
2.2 ±0.03 0.9 ±0.06 1.9 ±0.05
1.5 ±0.05
0.9 ±0.01
1.8 ±0.05
1.6 ±0.05
1.9 ±0.01
-
1.1± 0.05 1.2±0.05 1.3 ±0.01
1.1 ±0.03
0.9 ±0.01 1.6±0.01
0.9 ±0.01 1.5±0.05
1.2 ±0.01 1.5 ±0.01
0.9 ±0.01 0.9 ±0.01
1.0 ±0.01 2.9±0.01
1.1± 0.17 1.2±0.05
mpch
0.9± 0.05 0.9±0.05 1.9 ± 0.05 0.9 ±0.05
1.6±0.05
1.2±0.05
1.9±0.01
-
1.8±0.01 1.6 ±0.06
1.5±0.01 0.9 ±0.06
2.1±0.01 2.2 ±0.06
2.3 ±0.05
1.2 ±0.05
2.4 ±0.03
-
1
2
3
Flucanazole
-
-
-
-
-
Control
MeOH
0.9±0.05
0.9±0.05
0.9±0.05
0.9±0.050.9±0.05
0.9±0.05 0.9±0.05
0.9±0.05
0.9±0.05
0.9±0.05
Table 6. MIC (minimum inhibitory concentration) value of different synthetic compound against
bacteria and fungi.
MIC values (µg/ml) of samples
Bacteria
Fungi
Sample
Gram negative
Gram positive
K.
P.
P.
S.
A.
C.
E. coli
S. typhi B. subtilis
S. aureus
pneumoniae vulgaris
aeruginosa fecalis
flavus albicans
Hmpca
−
−
−
−
−
−
−
−
−
600
500
100
300
−
−
−
−
−
−
−
−
−
−
500
500
100
−
mpch
−
25
−
−
−
50
500
−
300
400
300
−
3
2
1
−
400
−
500
300
−
−
100
−
200
200

Graphical abstract