Synthesis , Crystal Structure and Antimicrobial Properties of an Anhydrous Copper ( II ) Complex of Pyridine-2-Carboxylic Acid

A copper(II) complex with picolinic acid as ligand has been synthesised and characterised by elemental analysis, magnetic susceptibility, Fourier transform infrared spectroscopy (FTIR) and ultraviolet-visible spectroscopic techniques. The crystal structure of the complex has been determined by single crystal X-ray diffraction technique and the ligand was found to coordinate through Nand O-atoms. The ligand and the complex were screened for their activity against resistant strains of fungi (Candida albicans ATCC P37039, Candida albicans 194 B, Candida glabrata 44B, Cryptococcus neoformans) and bacteria (Staphylococcus aureus CIP 7625, Pseudomonas aeruginosa CIP 76110, Salmonella typhi and Escherichia coli ATCC 25922) isolated from humans in Cameroon.


Introduction
Pyridine-2-carboxylic acid (picolinic acid) which is a naturally occurring product of the degradation of tryptophan is a biologically important ligand found in some enzymes.It has metal ion-chelating activity and is an active agent in some drugs (Masui 2001, Beuerle & Pichersky 2002, Kalinowska et al. 2007, Kukovec et al. 2013).Picolinic acid and its metal complexes has been the subject of intense research activity recently due to their broad spectrum of physiological activity as well as for the design of new metallopharmaceuticals (Yasui et al. 2002, Chaudhary et al. 2005, Kalinowska et al. 2007, Mautner & Massoud 2007, Kukovec et al. 2013).It has been shown to bind different metal ions including alkali metals, transition metals and lanthanides (Goher et al. 1992, Goher et al. 1993, Goher & Mautner 1994, Goher & Mautner 1995, Goher et al. 1996, Mautner et al. 1997, Barandika et al. 1999, Chattopadhyay et al. 2003, Mautner & Massoud 2007, Casas et al. 2008, Siddiqui 2012, Wang et al. 2012).This may be due to the versatile bonding nature of the picolinate ligand.A literature survey of the reported crystal structures of picolinic acid with diverse metal ions indicates picolinic acid exhibits different bonding modes which include monodentate through oxygen atom of the carboxyl group or through its pyridine nitrogen atom and bidentate through nitrogen and oxygen atoms (Yasui et al. 2002, Kalinowska et al. 2007).The ligand can also form polynuclear complexes in which it acts as a bridge between metal ions (Wu et al. 2005, Girginova et al. 2007).The N, O-chelating bonding mode and H-bonding between the carboxylate moiety and water molecules are predominant (Kalinowska et al. 2007, Kukovec et al. 2013).
The increasing resistance to antibiotics necessitates the search for new and more potent antimicrobial agents.Metal complexes of biologically active ligands are a target for the development of new drugs.We have reported the antimicrobial activity of some metal complexes of the ligands 1,10-phenanthroline and 2,2'-bipyridine (Agwara et al. 2010, Ndosiri et al. 2013), hexamethylenetetramine (Agwara et al. 2012) and 4,4'-bipyridine (Akum et al. 2014), where a general increase in antimicrobial activity was observed upon complexation.
In this paper we report the synthesis, X-ray crystal structure of an anhydrous Cu(II) picolinate complex as well as its antimicrobial properties against some resistant fungal and bacterial strains isolated from humans in Cameroon.

Materials
All chemicals were of reagent grade and they were used as supplied without further purification.

Characterization Techniques
Elemental analyses (C,H,N) was carried out on a Perkin-Elmer automated model 2400 series II CHNS/O analyser at the University of Zululand (South Africa).The melting point/decomposition temperatures were recorded using the LEIC VMHB Kofler system.Conductivity measurement was carried out in distilled water using a HANNA multimeter H19811-5; pH/•C/EC/TDS meter at room temperature.The FTIR spectrum was recorded on a Bruker FT-IR tensor 27 spectrophotometer directly on a small sample of the complex in the range 200 -4000 cm -1 while the electronic spectrum was recorded on a Varian, Cary 50 UV-Visible spectrophotometer.
The magnetic susceptibility value of the complex was determined by the Gouy method at room temperature.The calibrant employed was Hg[Co(SCN) 4 ] for which a magnetic susceptibility of 1.644×10 -5 cm 3 g -1 was taken.Correction for diamagnetism of the constituent atoms was calculated using Pascal's constants (König 1966).The effective magnetic moment value, μ eff , was calculated from the equation: (1) where χ M is the magnetic susceptibility and T is the absolute temperature.

X-Ray Crystallography
A suitable single blue crystal with regular trigonal prismatic shape (0.33×0.31×0.28mm 3 ) was selected and mounted on a glass fiber and fixed on a goniometer head.Data collection was performed at room temperature (293 K) on an Xcalibur3 diffractometer equipped with a CCD camera using Mo-Kα radiation (λ = 0.71073 Å) from an enhanced optic X-ray tube operating at 50 kV and 40 mA, and a detector-to-crystal distance of 50 mm, by means of omega scan of width 1° and an exposure time of 20 sec.Data integration and numerical absorption corrections were carried out with the CrysAlis software package yielding 2560 independent reflections out of the 4844 collected reflections.The final cell parameters were obtained using all measured reflections.The structure was solved by direct methods and full-matrix least-squares techniques based on  2 values against all reflections and they converge to final R 1 = 0.063 for observed reflections (I > 2(I)) and wR 2 = 0.162 for all.The relatively high residuals are due to the difficulties encountered during absorption correction and racemic twin refinements.The thermal displacement of all non-hydrogen atoms was refined anisotropically.The H atoms bonded to C atoms were positioned geometrically and refined using a riding model with distance restraints of C-H = 0.93 Å, and with U iso (H) = 1.2U eq (C).

Antimicrobial Tests
The antimicrobial tests were carried out in the Laboratory of Phytobiochemical and Medicinal Plant Study, University of Yaounde I.The tests were done on eight microorganisms, 4 yeasts, Candida albicans ATCC P37039, Candida albicans 194 B, Candida glabrata 44B, Cryptococcus neoformans and 4 bacterial strains Staphylococcus aureus CIP 7625, Pseudomonas aeruginosa CIP 76110, Salmonella typhi and Escherichia coli ATCC 25922 obtained from Centre Pasteur Yaoundé , Cameroon.Preliminary screening was carried out using Muller Hinton Agar.The disk diffusion method from the protocol described by the National Committee for Clinical Laboratory Standard (NCCLS, 2004) was used for preliminary screening.Mueller-Hinton agar was prepared from a commercially available dehydrated base according to the manufacturer's instructions.Several colonies of each microorganism was collected and suspended in saline (0.9% NaCl).Then, the turbidity of the test suspension was standardized to match that of a 0.5 McFarland standard (approximately 1.5 x 10 8 CFU/ml for bacteria or 1 x 10 6 -5 x 10 6 cells/mL for yeast).Each compound or reference was accurately weighed and dissolved in the appropriate diluents (DMSO at 10%, Methanol at 10% or distilled water) to yield the required concentration (2 mg/mL for the compound or 1 mg/mL for the reference drug), using sterile glassware.
Whatman filter paper No. 1 was used to prepare disks approximately 6 mm in diameter, which were wrapped with aluminum paper and sterilized by autoclaving.Then, 25 µl of stock solutions of compound or positive control were delivered to each disk, leading to 50 µg of compound or 25 µg of reference drug.
The dried surface of a Müeller-Hinton agar plate was inoculated by flooding over the entire sterile agar surface with 500 µl of inoculum suspensions.The lid was left ajar for 3 to 5 minutes, but no more than 15 minutes, to allow for any excess surface moisture to be absorbed before applying the drug impregnated disks.Disks containing the complex or reference drugs were applied within 15 minutes of inoculating the MHA plate.Six disks per petri dish were plated.The plates were inverted and placed in an incubator set to 35°C.After 18 hours (for bacteria) or 24 hours (for yeasts) of incubation, each plate was examined.The diameters of the zones of complete inhibition (as judged by the unaided eye) were measured, including the diameter of the disk.Zones were measured to the nearest whole millimeter, using sliding calipers or a ruler, which was held on the back of the inverted petri plate.All experiments were carried out in duplicate.The compound was considered active against a microbe if the diameter of the inhibition zone was greater than 6 mm.

Synthesis of the Complex
The reaction of picolinic acid with CuCl 2 .2H 2 O in a water/ethanol mixture (3:1 v/v) yielded the complex of formula Cu(Pic) 2 which has the same molecular formula like that of the complexes Cu(Pic) 2 (Zurowska et al. 2007) and Cu(Pic) 2 (Zurowska et al. 2004) previously reported.Table 1 represents the physical data of the complex.The complex, obtained in moderate yield (64 %) has a sharp melting point of 116 º C and it undergoes a colour change from blue to brown around this temperature.The molar conductivity value of 60 Ω -1 cm 2 mol -1 for the Cu(Pic) 2 complex corresponds to a non-electrolyte, suggesting the molecular nature of the complex.The complex has a room temperature magnetic moment of 1.63 BM, which is less than the spin-only value of 1.73 BM.This is consistent with the antiferromagnetic exchange coupling between the copper(II) ions (Zurowska et al. 2004)

Description of the Crystal Structure
Crystal structural data for Cu(pic) 2 are summarized in Table 2.The ORTEP view of the crystal structure together with the atom numbering scheme are shown in Fig. 1, the axial Cu-O interactions are shown in Fig. 2, while the packing diagram of the complex seen along the crystallographic a-axis is shown in Fig. 3.The complex crystallizes in the triclinic crystal system with space group P1 (Nr 1).This is different from the crystal system (monoclinic) and space group (P2 1 /c) for Cu(pic) 2 complexes reported in the literature with similar molecular formulae (Zurowska et al. 2004, Zurowska et al. 2007).In the crystal structure of the complex, each Cu atom is covalently bonded to two picolinates through N-and O-atoms.The Cu-O (Cu-O1 = 1.953(9)Å, Cu-O2 = 1.954(10)Å) bond lengths are identical while the Cu-N (Cu-N1 = 1.970(14)Å, Cu-N2 = 1.966(14)Å) bond lengths are also similar but slightly longer than the Cu-O distance.These values are similar to those reported in the literature for Cu(II)-picolinate complexes (Zurowska et al. 2004, Zurowska et al. 2007, Dutta et al. 2008).The Cu(II) ion is in a distorted octahedral environment in which the basal plane is occupied by N 2 O 2 from two picolinates and elongated in the axial direction by weak Cu-O (Cu-O3 = 2.777 Å; Cu-O5 = 2.822 Å) axial interactions (Fig. 2) between Cu atoms of one plane and carboxylate O-atoms of an adjacent plane.This type of axial interaction (Cu-O = 2.745 and 2.770 Å) has been reported for a Cu(pic) 2 polymorph (Zurowska et al. 2007).The axial interaction leads to an infinite 1-D layered structure viewed along the crystallographic a-axis (Fig. 3).Selected crystal data of two identical complexes found in the literature (Zurowska et al. 2004, Zurowska et al. 2007) and which are polymorphic to each other are compared with crystal data of the title complex in Table 3.The title complex differs structurally from those in the literature (Zurowska et al. 2004, Zurowska et al. 2007) in terms of crystal system, space group and unit cell parameters.The methods of syntheses of these complexes also differ.Bond lengths and angles for the complex are presented in Table 4. FTIR analysis on a powdered sample of the title compound shows that the characteristic absorption bands of C=O and C-O for the ligand at 1715 cm -1 and 1347 cm -1 , respectively have disappeared in the complex (Parajón-Costa et al. 2004).The asymmetric υ as (COO -) and symmetric ν s (COO-) stretching vibrations of the carboxylate group have appeared in the 1629 cm -1 and 1374 cm -1 regions, respectively (Parajón-Costa et al. 2004, Zurowska et al. 2004, Zurowska et al. 2007, Kukovec et al. 2008).The difference (Δν = ν as -ν sym ) of 256 cm -1 indicates monodentate carboxylate coordination (Zurowska et al. 2004, Zurowska et al. 2007).This high value of Δν (> 200 cm -1 ) suggests a highly asymmetrical bridging ("pseudo-monodentate" coordination) (Zurowska et al. 2007).This observation is consistent with the crystal structure of the complex.The ʋ C=N band has shifted from 1657 cm -1 in the ligand to 1626 cm -1 in the complex.Absorption bands at 445 cm -1 and 293 cm -1 have been assigned to ν(Cu-O) and ν(Cu-N) vibrations, respectively (Parajón-Costa et al. 2004).The absence of a broad band in the 3100-3500 cm -1 region indicates the absence of water in the complex.This observation is consistent with the crystal structure.

Visible Spectroscopy
The electronic absorption spectrum of the complex shows a single broad band centred at 19880 cm -1 (503 nm).This single band is explained by a d-d transition in the Cu(II) ion which has been assigned to 2 E g → 2 T 2g transition.The observed band is consistent with an octahedral geometry for Cu(II) complexes (Lee 2003).

Antimicrobial Tests
The assay compares the inhibition of fungal and bacterial cell growth with known antifungal and antibacterial agents, respectively.The ligand, metal salt and the complex were tested against resistant fungal and bacteria strains isolated from humans in Cameroon.The diameter of inhibition zones are presented in Table 5 and a histogram shown in Fig. 4. The higher the diameter of the inhibition zone (IZ) the more active the compound.The ligand showed considerable activity only against the fungus C. albicans 194B while the metal complex showed increased activity against most of the pathogens.This indicates that the interaction of metal ions with the ligand plays an important role in enhancing its antimicrobial activity.The complex showed greater activity than the ligand and the metal salt.Generally, the Cu(II) complex exhibited its strongest antifungal effect against the fungi C. neoformans and C. albicans 194B, while an enhanced antibacterial activity was observed against the bacteria species P. aeruginosa.This observation is consistent with results found in the literature (Oladipo et al. 2013).This increase in activity could be due to the reduction of the polarity of the metal ion by partial sharing of the positive charge with the ligand's donor atoms so that there is electron delocalization within the metal complex.This may increase the hydrophobic and lipophilic character of the metal complex, enabling it to permeate the lipid layer of the organism killing them more effectively (Chohan et al. 2001, Nfor et al. 2013).

Conclusion
The compound Cu(Pic) 2 has been synthesised and characterised by elemental analysis, FTIR and XRD spectroscopic techniques.The coordination geometry around the copper(II) ion can be best described as a ''Jahn Teller'' distorted octahedron with two axial bonds elongated.The complex was screened for its antimicrobial activity against four resistant strains each of fungi and bacteria isolated from humans in Cameroon.The complex showed a greater activity than that of the ligand towards these pathogens.

Figure 1 .
Figure 1.ORTEP view of the complex with atom numbering scheme

Table 2 .
Crystal data and structure refinement for [Cu(Pic) 2 ]

Table 3 .
Comparative crystal data of the complexes

Table 5 .
Diameter of inhibition zones