Metal based drugs: design, synthesis and in-vitro antimicrobial screening of Co(II), Ni(II), Cu(II) and Zn(II) complexes with some new carboxamide derived compounds: crystal structures of N-[ethyl(propan-2-yl)carbamothioyl]thiophene-2-carboxamide and its copper(II) complex.

Abstract A new series of compounds derived from thiophene-2-carboxamide were synthesized and characterized by IR, 1H-NMR and 13C-NMR, mass spectrometry and elemental analysis. These compounds were further used to prepare their Co(II), Ni(II), Cu(II) and Zn(II) metal complexes. All metal(II) complexes were air and moisture stable. Physical, spectral and analytical data have shown the Ni(II) and Cu(II) complexes to exhibit distorted square-planar and Co(II) and Zn(II) complexes tetrahedral geometries. The ligand (L1) and its Cu(II) complex were characterized by the single-crystal X-ray diffraction method. All the ligands and their metal(II) complexes were screened for their in-vitro antimicrobial activity. The antibacterial and antifungal bioactivity data showed that the metal(II) complexes were found to be more potent than the parent ligands against one or more bacterial and fungal strains.


Experimental
All chemicals used were of analytical grade. All metallic salts were used as acetate. Melting points were recorded on Fisher Johns melting point apparatus. Infrared spectra were recorded on Shimadzu FT-IR spectrometer. The C, H and N analysis was carried out using a Perkin Elmer, USA, model. The 1 H-and on JEOL MS Route Instrument. In vitro antibacterial and antifungal properties were studied at HEJ Research Institute of Chemistry, International Centre for Chemical Sciences, University of Karachi, Pakistan and Department of Chemistry, The Islamia University, Bahawalpur, Pakistan.
General procedure for the synthesis of ligands (L 1 -L 3 ) The ligand, N-[ethyl(propan-2-yl)carbamothioyl]thiophene-2-carboxamide (L 1 ) was synthesized by following a reported method 19 , in which a solution of thiophenyl carbonyl chloride (20 mmol, 2.14 mL) in dry acetone (30 mL) was added dropwise to a solution of KSCN (20 mmol, 1.94 g) in acetone (30 mL). The reaction mixture was refluxed for 1 h and then cooled to room temperature. A solution of N-ethyl-isopropyl amine (20 mmol, 2.4 mL) in acetone (20 mL) was added dropwise to the mixture in 20 min and refluxing continued for 2 h. Then KCl was removed by filtration and light yellow filtrate was placed into water (250 mL) and added few drops of conc. HCl under constant agitation. The wax-like material was formed which was separated by filtration, washed several times with cold water and then with diethyl ether. The crude product was purified by recrystallization from a solution mixture of ethanol-dichloromethane (1:1). As a result of recrystallization, fine shiny colorless crystals of (L 1 ) were obtained, which were suitable for X-rays analysis. The same procedure was used for the preparation of other ligands.  , 52 (C 8 ), 46 (C 11 ), 21 (C 9, C 10 ), 15.5 (C 12

General procedure for the synthesis of metal(II) complexes
The solution of Cu(CH 3 COO) 2 .H 2 O (0.5 mmol) in methanol (15 mL) was added dropwise to a stirred solution of ligand N-[ethyl(propan-2-yl)carbamothioyl]thiophene-2-carboxamide (L 1 ) (1 mmol) in methanol (20 mL). The reaction mixture was refluxed for 2 h. The precipitates of dark green color were formed during refluxing. The precipitated product thus formed was filtered, washed with methanol and dried under vacuum. The precipitates were dissolved in a mixture of ethanol and dichloromethane (1:1) and a clear solution was kept in a refrigerator for one week. Suitable dark green color crystals for X-ray studies were obtained for Cu(L 1 ) 2 complex. The same method was used for the preparation of all other complexes.

Biological activity
Antibacterial studies (in-vitro) The synthesized ligands (L 1 -L 3 ) and their respective metal(II) complexes were tested against four Gram-negative (E. coli, S. sonnei, P. aeruginosa and S. typhi) and two Gram-positive (St. aureus and B. subtilis) bacterial strains by the disc diffusion method 20,21 . The test compounds (ligand/complex) were dissolved (10 mg/mL) in DMSO. A known volume (10 mL) of the solution was applied with the help of a micropipette onto the sterilized filter paper discs. The discs were dried at room temperature over night and stored in sterilized dry containers. Discs soaked with 10 mL of DMSO and dried in air at room temperature were used as the negative control. The standard antibiotic discs used as positive control were prepared as mention above in the laboratory DOI: 10.3109/14756366.2015.1050011 by applying a known concentration of the standard antibiotic solution. Ampicillin was used as a standard antibiotic. Bacterial culture was grown in nutrient broth medium at 37 o C overnight and spread on to solidified nutrient agar medium in Petri plates using sterilized cotton swabs. Test and control disks were then applied to the medium surface with the help of sterilized forceps. The plates were incubated at 37 o C for 24-48 h. The results were recorded by measuring the zone of inhibition in mm against each compound 21 . The experiments were carried out in triplicate and the values obtained were statistically analyzed.

Antifungal activity (in-vitro)
Antifungal activity of all the compounds was studied against six fungal strains (T. longifusus, C. albicans, A. flavus, M. canis, F. solani and C. glabrata) according to literature protocol 21,22 . Sabouraud dextrose agar (Oxoid, Hampshire, England) was seeded with 10 5 (cfu) mL À1 fungal spore suspensions and transferred to petri dishes. Discs soaked in 20 mL (200 mg/mL in DMSO) of test compounds were placed at different positions on the agar surface. The plates were incubated at 32 o C for 7 days. The results were recorded as percentage of inhibition and compared with the standard drugs miconazole and amphotericin B.

Chemistry
The ligands (L 1 -L 3 ) were synthesized by the reaction of potassium thiocyanate with thiophene-2-carbonyl chloride, 5-methyl-thiophene-2-carbonyl chloride and 5-bromo-furan-2-carbonyl chloride, respectively, in dry acetone followed by condensation of the resulting product with N-ethyl-isopropyl amine (Scheme 1). The ligands (L 1 -L 3 ) thus formed were soluble in ethanol, ethyl acetate, DMF and DMSO; however, slightly soluble in tetrahydrofuran, diethyl ether and insoluble in aliphatic and aromatic hydrocarbons. The metal(II) complexes were obtained by stoichiometric reaction of the corresponding ligands with metals [Co(II), Ni(II), Cu(II) and Zn(II)] as acetate in a molar ratio of metal:ligand (M:L) as 1:2 (Scheme 1). All metal(II) complexes were air and moisture stable. They were soluble in mixture of ethanol and dichloromethane (1:1), DMF and DMSO. The ligands and their metal(II) complexes were characterized by their physical, spectral and analytical data. The structure of ligand, N-[ethyl(propan-2-yl)carbamothioyl]thiophene-2-carboxamide (L 1 ) and its Cu(L 1 ) 2 complex was determined from single crystal X-ray diffraction data. Physical measurements and analytical data of the metal(II) complexes are given in Supplementary Tables 1 and 2.
Spectral characterization of ligands (L 1 -L 3 ) and their metal(II) complexes

IR spectra
The main IR vibrational bands of the synthesized ligands (L 1 -L 3 ) and their metal(II) complexes were found in their expected region. The characteristic IR bands, are given in ''Experimental'' section and Table 1. The IR spectra of newly synthesized ligands showed 23 the strong peak at 3265-3280 cm À1 due to N-H vibrations and medium peaks at 1675-1680 and 1235-1250 cm À1 , respectively, due to carbonyl (C¼O) and thiocarbonyl (C¼S) vibrations, which strongly support the preparation of desired compounds. The ligands (L 1 ) and (L 2 ) showed bands at 880-885 due to (C-S) vibrations assigned 24 to thiophene and (L 3 ) displayed band at 1160 cm À1 due to (C-O) vibrations assigned to furane moiety. A weaker peak at 690 cm À1 was also observed by ligand (L 3 ), which was due 25 to v(C-Br) vibrations. The IR spectra of all the metal(II) complexes exhibited major changes in comparison to the spectra of the subsequent ligands. The most prominent change observed was, the carbonyl (C¼O) and thiocarbonyl (C¼S) bands originally appearing at 1675-1680 and 1235-1250 cm À1 in the spectra of the ligand, shifted to lower frequency by 13-15 cm À1 at 1660-1670 and 1220-1237 cm À1 , respectively, in the spectra of metal(II) complexes indicating 1,26 the involvement in coordination with the metal(II) ions. The decrease in frequency is due to delocalization of electrons 27 . The N-H vibrations appearing in the ligands at 3265-3280 cm À1 were also disappeared in the metal complexes giving a clue of deprotonation, which may undergo through tautomerism. However, keto form was identified as a stable product. Coordination of carbonyl-O and thiocarbonyl-S is further justified Scheme 1. Preparation of ligands (L 1 -L 3 ) and their metal(II) complexes. by the appearance of new bands at 450-460 and 525-536 cm À1 corresponding to M-S and M-O linkages, respectively. This linkage is also supported by X-ray structure of the Cu(L 1 ) 2 complex as shown in Figures 3 and 4.

H-NMR spectra
The spectra of all the ligands (L 1 -L 3 ) showed a broad singlet peak at 11.53-11.62 ppm due to N-H proton 1,28 and the protons of ethyl and isopropyl groups (C 8 -H, C 9 -H, C 10 -H, C 11 -H and C 12 -H) were observed as doublet to multiplet at 0.93-2.60 ppm. The methyl (CH 3 ) protons of (L 2 ) and C 3 -H to C 5 -H protons of all ligands were observed at 2.45 and 7.10-8.19 ppm as a singlet and doublet, respectively. On comparison of the spectra of ligand with those of Zn(II) complexes, the N-H protons disappeared which is also supported by the IR spectra and X-rays technique and all other remaining protons underwent downfield shift by 0.05-0.15 ppm due to coordination and increased conjugation 30 .

Electronic spectra
The electronic spectral values of Co(II), Ni(II), Cu(II) and Zn(II) complexes in DMF are recorded in Table 1. The spectra of Co(II) complexes showed only one absorption band at 17 995-18 380 cm À1 assigned to the transition 4 A 2 (F) ! 4 T 1 (F) 31 . This in turn, propose tetrahedral geometry for the Co(II) complexes. This is also supported by magnetic moment values (3.78-3.94 B.M) of Co(II) complexes 31 . The Ni(II) complexes exhibited two absorption bands at 13 390-13 515 cm À1 and at 18 770-19 230 cm À1 assigned to the transitions 1 A 1g ! 1 B 2g and 1 A 1g ! 1 A 2g , respectively, which give clue for square-planar geometry 32 . The electronic spectra of Cu(II) complexes showed low-energy absorption bands at 15 190-15 660 cm À1 assigned to the transitions, 2 B 1g ! 2 E 1g . The high-energy bands at 21 160-21 640 cm À1 was assigned to the transition, 2 B 1g ! 2 A 1g . These transitions as well as the measured magnetic moment values (1.83-190 B.M) propose a square-planar geometry for Cu(II) complex 33 . The diamagnetic Zn(II) complexes did not show any d-d transitions and their spectra were dominated 32 only by the charge transfer band at 27 190-27 360 cm À1 proposing a tetrahedral geometry for the Zn(II) complexes.

Molar conductivity and magnetic properties of the metal(II) complexes
Molar conductance was carried out in DMF solution and the results reported in Table 1 indicate the values in a lower range (10.9-18.2 À1 cm 2 mol À1 ), thus showing their non-electrolytic nature 34 . The magnetic moment values of Co(II) complexes were found in the range of 3.78-3.94 B.M, expected for three unpaired electrons and are in agreement with their tetrahedral geometry 35 . The Cu(II) and Ni(II) complexes showed eff values compatible for their square-planer geometry. The Zn(II) complexes exhibited diamagnetic nature 36 .  X-Ray crystallographic studies of ligand (L 1 ) and Cu(L 1 ) 2 complex Single crystal X-ray structure of ligand (L 1 ) The molecular structure of N-[ethyl(propan-2-yl)carbamothioyl]thiophene-2-carboxamide (L 1 ) (Figure 1) showed their expected bond lengths and angles 37,38 . Data collection and refinement of (L 1 ) are listed in Supplementary  Table 4). This information indicated partial electron delocalization within the C5-N1-C6-N2 fragment. The C6-N2 bond, adjacent to the alkyl group, was slightly shorter than C6-N1. These bond distances were in good agreement with those observed in literature, as reported in the Cambridge Structural Database 37,38 .
Molecular structure of Cu(L 1 ) 2 complex The molecular structure of Cu(L 1 ) 2 complex (Figure 2) was obtained from X-ray single crystal studies and, the data collection and crystal refinement parameters are given in Supplementary  Table 3 and selected bond lengths and bond angles are given in  Supplementary Tables 7 and 8. The structure of Cu(L 1 ) 2 complex showed the bond lengths and angles 15 as expected. The Cu(L 1 ) 2 complex existed as a monomer unit in which each copper Cu1 atom was coordinated with each oxygen and each sulfur of two ligands ( Figure 2) in a cis-fashion, with slightly distorted square planar geometry 15 . In both chelate rings, the distances of N1-C6 (1.333(6) Å ) and N3-C17 (1.337(6) Å ) in the thiourea fragment were almost same, but the distances of N1-C5 (1.310(6) Å ) and N3-C16 (1.325(6) Å ) (Supplementary Table 8) were slightly different, which supported the distorted square planar geometry of the complex. The bond lengths of the carbonyl O1-C5 ¼ 1.273(5) Å ; O2-C16 ¼ 1.267(5) Å and thiocarbonyl S1-C6 ¼ 1.746(4) Å ; S3-C17 ¼ 1.716(5) Å groups were in between those for double and single bonds (Supplementary Table 7). The investigated

Impact of metal/ligand coordination on bioactivity
In vitro antibacterial bioassay Antibacterial activity of newly synthesized ligands (L 1 -L 3 ) and their metal(II) complexes was determined against four Gramnegative (E. coli, S. sonnei, P. aeruginosa and S. typhi) and two Gram-positive (S. aureus and B. subtilis) bacterial strains and obtained data are recorded in Table 2. The antibacterial activity of prepared compounds was compared with the activity of standard drug (ampicillin). The synthesized compounds showed varying degree of inhibitory effects: low (up to 10 mm), moderate (up to 11-15 mm) and significant (above 15 mm). The obtained data indicated that all the ligands (L 1 -L 3 ) showed moderate (11-15 mm) (16-20 mm) activity against all the remaining tested bacterial strains. Conclusively, the ligands (L 1 -L 3 ) possessed smaller average (11.5 mm) activity against different strains than the average (14.3 mm) activity of the metal(II) complexes, which showed that the activity is enhanced 39,40 upon coordination. The comparative and average activity data of the ligands and their metal(II) complexes is reproduced in Figure 3.

Conclusions
All the newly synthesized ligands (L 1 -L 3 ) act as bidentate and coordinate through carbonyl-O and thiocarbonyl-S to the metal(II) ions. The bonding of ligands to the metal(II) ion is supported by their physical, analytical and spectral data. These observations are further supported by the X-ray crystallographic data of Cu(L 1 ) 2 complex. In vitro antibacterial and antifungal studies of the ligands and their metal(II) complexes against representative bacterial and fungal strains showed that the ligands (L 1 -L 3 ) and their Co(II), Ni(II), Cu(II) and Zn(II) complexes were found to have moderate to significant activity. However, against one or more bacterial strains the bioactivity of the ligands enhanced upon coordination with the metal ions.