Accepted Manuscript Original article Synthesis, spectroscopic characterization and antimicrobial activity evaluation of new tridentate Schiff bases and their Co(II) complexes Ganesh More, Darshana Raut, K. Aruna, Sakina Bootwala PII: DOI: Reference:
S1319-6103(17)30060-1 http://dx.doi.org/10.1016/j.jscs.2017.05.002 JSCS 876
To appear in:
Journal of Saudi Chemical Society
Received Date: Revised Date: Accepted Date:
13 February 2017 1 May 2017 2 May 2017
Please cite this article as: G. More, D. Raut, K. Aruna, S. Bootwala, Synthesis, spectroscopic characterization and antimicrobial activity evaluation of new tridentate Schiff bases and their Co(II) complexes, Journal of Saudi Chemical Society (2017), doi: http://dx.doi.org/10.1016/j.jscs.2017.05.002
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Synthesis, spectroscopic characterization and antimicrobial activity evaluation of new tridentate Schiff bases and their Co(II) complexes Ganesh More1, Darshana Raut2, K. Aruna2 and Sakina Bootwala1* 1
Department of Chemistry, 2 Department of Microbiology,
Wilson College, Mumbai, India-400007. *
Corresponding Author: Email:
[email protected]
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INTRODUCTION Schiff bases are the compounds with the general structure R2C=NR' (R' ≠ H), either secondary ketimines or secondary aldimines first reported by Hugo Schiff in 1864. Azomethine are secondary aldimines with a general formula RHC=N-R’ where R and R’ are alkyl, aryl, cyclo alkyl or heterocyclic groups. The presence of C=N linkage is essential for biological properties. Wide applications of Schiff base in food, dye, polymer, catalysis, agrochemical and optical material industries makes them extensively studied class of compounds in inorganic chemistry [1,2]. 2-Aminothiophene derivatives are one of the important heterocyclic compounds found in several biologically active and natural compounds. This class of compounds has demonstrated a broad spectrum of activities and several applications in agrochemicals, pharmaceuticals and dyes industries as well as in biodiagnostics, electronic and optoelectronic devices [3]. The Schiff base ligands with sulphur, oxygen and nitrogen donor atoms act as good chelating agents forming stable chelates with transition metal ions. Such metal complex on coordination shown to exhibit increased potential biological activity [4] has prompted us to prepare a Schiff base by condensing aromatic o-hydroxyl aldehyde with 2-amino thiophene derivative.
Cobalt is essential ultra trace element for all animals. It is present in the form of coenzyme adenosylcobalamine in human body and plays important role in generation of erythrocytes in bone marrow in the conduction of nerve impulse and overall growth. The primary biological reservoir of cobalt is cobalamine also known as vitamin B12 [5,6]. Cobalt complexes are widely studied for their antiviral, antibacterial activities and as chaperones in prodrugs design [7]. Co(III) complexes are potential prodrugs capable of undergoing bioreduction, producing a bioactive agent . Some of the cobalt complexes derived from Hexacarbonyl dicobalt and Schiff base imidazole have shown to posses potential antitumor (breast cancer) as well as antiviral (HIV-1) properties respectively. Cobalt possesses diverse bioactive properties that can be tailored to design well-controlled selective drugs [8-10].
Tuberculosis (TB) is an airborne contagious disease usually caused by M. tuberculosis (MTB) [11], has been present in humans since ancient times and is most widespread public health hazard of 21st century. It ranks alongside HIV as a leading cause of death globally [12]. India is world’s
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third highest TB laden country, accounting for 23% global incidences of TB [13] and is consistently growing with recent discovery of TDR-TB (Totally Drug Resistant Tuberculosis) [14]. Urinary Tract Infections (UTIs) is also most common infectious disease, accounting for over 150 million cases annually [15,16]. UTIs are caused by gram negative uropathogens viz. E.coli, K.pneumoniae, Citrobacter spp, P.aeruginosa and Proteus spp [17]. Of late, treatment of these common infectious diseases has become more difficult due to the emergence of antibiotic resistant strains like MBL (Metallo- β-lactamase) and ESBL (Extended spectrum β-lactamase) producers. Extended spectrum β-lactamases (ESBL) are functionally defined as β-lactamases that are able to hydrolyze extended-spectrum cephalosporins (cefotaxime or ceftazidime) and monobactams (aztreonam) and these are inhibited by β-lactamase inhibitors, such as clavulanic acid, whereas they cannot hydrolyze cephamycins (cefoxitin or cefotetan) or carbapenems (imipenem or meropenem) efficiently [18-22]. MBLs are bacterial zinc enzymes that are able to hydrolyze most β-lactam antibiotics [23]. This multiple drug resistance is developed due to the indiscriminate use of commercial antimicrobial drugs frequently used in the treatment of infectious diseases. The emergence of ever-increasing Multiple Drug Resistant (MDR) microbial strains has become a severe health hazard to human-kind and one of the biggest challenges to global drug discovery programs [24-27]. Latest study revealed that thiophene containing compounds are more potent anti-tubercular drugs against MTB and in some cases comparable or even exceeding standard lead compounds [2830]. Herein we report the synthesis and spectroscopic characterization of a series of Co(II) complexes and their antimicrobial activity and antitubercular activity.
EXPERIMENTAL Materials and methods: All chemical used were of AR grade. Solvents were purified and distilled for synthesis and physical measurements. The IR spectra were recorded in KBr disc on a Perkin Elmer Model 1600 FTIR Spectrophotometer. The 1H and
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C NMR spectra were recorded in CDCl3 and
DMSO-d6 solvent respectively on Mercury plus 300 MHz NMR spectrometer. Melting points were determined in open capillary tubes and are uncorrected. Electronic spectra were recorded on a UV-Vis Jasco Spectrophotometer Model V-630. Molar conductance was measured in DMF
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(10-3 M solution) on an ELICO Digital conductivity meter Model CM-180 at room temperature. Mass spectra were recorded on Varian Inc, USA made Liquid Chromatograph Mass Spectrometer. The elemental analysis was carried out by Thermo finnigan CHNS(O) Analyzer, while metal content was determined by ARCOS, ICP-Atomic Emission Spectrometer and by standard methods [31]. Magnetic susceptibility measurements were carried out using Hg[Co(SCN)4] as calibrant by Gouy balance. Thermo gravimetric study was carried out on a Perkin Elmer diamond TGA instrument. Powder XRD analysis was carried out on PANanalytical X-ray diffractometer.
Synthesis of ligand: The starting material, ethyl 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate (1) was prepared according to Gewald synthesis [32]. To a solution of this 2-amino thiophene derivative (0.01 mol) in ethanol (20 ml) was added to o-hydroxyl aldehyde derivative (0.01 mol) (2a-2d) dissolved in ethanol (20 ml) in small portion with constant stirring. The resulting mixture was refluxed on water bath for 2-3 hours. On cooling the solution, the Schiff base (HL1-HL4) crystallized. It was filtered, washed and dried. Further purified and recrystallized from ethanol [33]. The reaction scheme is represented in Figure-1.
Ethyl 2-amino-5,6-dihydro-4H-cyclopenta[b]thiophene-3-carboxylate - (1) [C10H13NO2S] Yield: 72%; M.P.: 900C; Colour: Brown; 1H NMR (CDCl3, 300 MHz) δ: 1.291.34 (t, 3H, -CH3), 2.25-2.84 (m, 6H, cylopentane ring), 4.20-4.27 (q, 2H, -CH2-), 5.84 (s, 2H, NH2); FTIR (KBr, υ, cm-1): 3415 (-NH), 3297, 3174 (-NH2), 1644 (C=O), 1263 (C-N), 1575, 1421, 1371(thiophene ring).
Ethyl 2-{[(E)-(2-hydroxyphenyl)methylidene]amino}-5,6-dihydro-4H-cyclopenta[b] thiophene-3-carboxylate - (HL1) [C17H17NO3S] Yield: 80%; M.P.: 1280C; Colour: Golden Yellow; 1H NMR (CDCl3, 300 MHz) δ: 1.36-1.41 (t, 3H, -CH3), 2.34-2.43 (m, 2H, cylopentane ring ), 2.88-3.00 (m, 4H, cylopentane ring), 4.35-4.42 (q, 2H, -CH2-), 6.88-7.39 (m, 4H, salicyladehyde ring), 8.54 (s, 1H, -CH=N- ), 12.94 (s, 1H,-OH);
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C NMR (DMSO-d6, 75 MHz) δ: 159.49 (-CH=N-), 160.11 (-C-OH),
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162.11 (-C=O-), 157.95, 145.41, 136.22, 133.82, 132.88, 120.36, 119.34, 119.03, 116.84, 60.27, 30.18, 29.36, 26.74, 14.10; FTIR (KBr, υ, cm-1): 3140 (-OH), 1682 (C=O), 1597 (C=N), 1310 (C-O), 616 (C=S); LCMS m/z: [M]+ 315.9; Elemental analysis, found (calc.): C 64.45% (64.68%), H 5.45% (5.39%), N 4.20% (4.44%), S 9.96% (10.15%).
Ethyl 2-{[(E)-(5-chloro-2-hydroxyphenyl)methylidene]amino}-5,6-dihydro-4Hcyclopenta[b]thiophene-3-carboxylate - (HL2) [C17H16NO3SCl] Yield: 87%; M.P.: 1660C; Colour: Yellow orange; 1H NMR (CDCl3, 300 MHz) δ: 1.36-1.40 (t, 3H, -CH3), 2.34-2.44 (m, 2H, cylopentane ring), 2.89-3.00 (m, 4H, cylopentane ring), 4.35-4.42 (q, 2H, -CH2-), 6.93-7.33 (m, 3H, salicyladehyde ring), 8.45 (s, 1H, -CH=N-), 12.93 (s, 1H,-OH);
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C NMR (DMSO-d6, 75 MHz) δ: 158.12 (-CH=N-), 158.70 (-C-OH),
162.08 (-C=O-), 157.39, 145.56, 137.09, 133.13, 131.40, 122.66, 120.94, 120.29, 118.95, 60.37, 30.15, 29.36, 26.77, 14.10; FTIR (KBr, υ, cm-1): 3073 (-OH),
1704 (C=O), 1598 (C=N),
1311(C-O), 619 (C=S); LCMS m/z: [M]+ 349.9; Elemental analysis, found (calc.): C 58.64% (58.31%), H 4.59% (4.57%), N 3.89% (4.00%), S 8.94% (9.15%).
Ethyl 2-{[(E)-(5-bromo-2-hydroxyphenyl)methylidene]amino}-5,6-dihydro-4Hcyclopenta[b]thiophene-3-carboxylate - (HL3) [C17H16NO3SBr] Yield: 81%; M.P.: 1640C; Colour: Orange; 1H NMR (CDCl3, 300 MHz) δ: 1.361.40 (t, 3H, -CH3), 2.34-2.44 (m, 2H, cylopentane ring), 2.89-3.00 (m, 4H, cylopentane ring), 4.35-4.42 (q, 2H, -CH2-), 6.92-7.48 (m, 3H, salicyladehyde ring), 8.46 (s, 1H, -CH=N-), 12.95 (s, 1H,-OH);
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C NMR (DMSO-d6, 75 MHz) δ: 158.03 (-CH=N-), 159.09 (-C-OH), 162.06 (-
C=O-), 157.41, 145.54, 137.08, 135.87, 134.36, 120.92, 119.23, 118.36, 110.02, 60.36, 30.14, 29.04, 26.75, 14.16; FTIR (KBr, υ, cm-1): 3060 (-OH), 1708 (C=O), 1598 (C=N), 1311(C-O), 626 (C=S); LCMS m/z: [M]+ 393.9; Elemental analysis, found (calc.): C 51.90% (51.74%), H 4.15% (4.06%), N 3.39% (3.55%), S 8.07% (8.12%).
Ethyl 2-{[(E)-(2-hydroxynaphthalen-1-yl)methylidene]amino}-5,6-dihydro-4Hcyclopenta[b]thiophene-3-carboxylate - (HL4) [C21H19NO3S] Yield: 76 %; M.P.: 1600C; Colour: Red; 1H NMR (CDCl3, 300 MHz) δ: 1.38-1.43 (t, 3H, -CH3), 2.35-2.45 (m, 2H, cylopentane ring), 2.90-3.01 (m, 4H, cylopentane ring), 4.38-
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4.45 (q, 2H, -CH2-), 7.19-8.16 (m, 6H, napthaldehyde ring), 9.40 (s, 1H, -CH=N-), 14.85 (s, 1H,-OH); 13C NMR (DMSO-d6, 75 MHz) δ: 162.24 (-CH=N-), 158.35 (-C-OH), 162.81 (-C=O), 155.01, 145.32, 135.79, 132.14, 129.11, 128.22, 127.51, 124.00, 120.91, 119.65, 119.28, 109.52, 109.31, 60.27, 30.27, 29.37, 26.81, 14.23; FTIR (KBr, υ, cm-1): 3046 (-OH), 1704 (C=O), 1598 (C=N), 1307 (C-O), 617 (C=S); LCMS m/z: [M]+ 365.9; Elemental analysis, found (calc.): C 68.52% (68.96%), H 5.32% (5.20%), N 3.91% (3.83%), S 8.62% (8.76%).
Synthesis of metal complexes: To a magnetically stirred hot ethanolic solution of ligand 3a-3d (20 ml, 0.01 mol), a hot ethanolic solution of Cobalt(II) chloride (10 ml, 0.005 mol) was added in small parts. After complete addition of the metal salt solution, the pH was adjusted to 6.5 by adding ethanolic ammonia. It was then refluxed for 6-8 hours in a water bath and the solution was reduced to half and kept overnight. The complex [Co(L)2] formed was filtered, washed sequentially with aqueous ethanol and ether. Finally the complex was dried in vacuum over P4O10.
[Co(L1)2] Brown solid of CoC34H32N2O6S2 (687.69); FTIR (KBr, υ, cm-1): 1653 (C=O), 1567 (C=N), 1337 (C-O), 617 (C=S), 518 (M←O), 424(M←N); Elemental analysis, found (calc.): C 60.22% (59.33%), H 4.52% (4.65%), N 4.16% (4.07%), S 9.16% (9.30%), Co 8.12% (8.57%); Molar conductivity (DMF): 5.90 (Ω-1cm2 mol-1).
[Co(L2)2] Maroon solid of CoC34H30N2O6S2Cl2 (756.58); FTIR (KBr, υ, cm-1): 1651 (C=O), 1575 (C=N), 1343 (C-O), 618 (C=S), 518 (M←O), 422(M←N); Elemental analysis, found (calc.): C 54.22% (53.93%), H 4.02% (3.97%), N 3.63% (3.70%), S 8.25% (8.46%), Co 7.67% (7.79%); Molar conductivity (DMF): 1.20 (Ω-1cm2 mol-1).
[Co(L3)2] Red coloured solid of CoC34H30N2O6S2Br2 (845.48); FTIR (KBr, υ, cm-1): 1651 (C=O), 1573 (C=N), 1333 (C-O), 627 (C=S), 516 (M←O), 414 (M←N); Elemental analysis, found (calc.): C 6
48.96% (48.26%), H 3.66% (3.55%), N 3.40% (3.31%), S 7.25% (7.57%), Co 7.01% (6.97%); Molar conductivity (DMF): 2.00 (Ω-1cm2 mol-1).
[Co(L4)2] Brown coloured solid of CoC42H36N2O6S2 (787.81); FTIR (KBr, υ, cm-1): 1668 (C=O), 1578 (C=N), 1333 (C-O), 618 (C=S), 513 (M←O), 415 (M←N); Elemental analysis, found (calc.): C 64.45% (63.98%), H 4.33% (4.57%), N 3.31% (3.55%), S 8.00% (8.12%), Co 7.67% (7.48%); Molar conductivity (DMF): 6.20 (Ω-1cm2 mol-1). Biological studies: Test organisms: Gram-negative uropathogens were collected from local hospitals and pathological laboratories situated in Mumbai and characterized for ESBL and MBL production in our laboratory in our previous studies [34,35]. Five ESBL and 5 MBL producing uropathogens that included 5 representative isolates of each of the following genera, i.e., Klebsiella, Escherichia, Pseudomonas, Proteus and Citrobacter were used in the current study (Table 1). These isolates were maintained on Luria-Bertani (LB) agar slants supplemented with 100µg/ml of ampicillin and stored at refrigerated condition.
Antimicrobial susceptibility of uropathogens: Antibiotic sensitivity profile of the pathogens was studied using Kirby Bauer method [36]. Antibacterial activity screening: The antibacterial activity of Schiff base ligands and their respective Co(II) complexes was assayed by agar well diffusion method [37-40] The metal complexes were dissolved in HPLC grade DMSO to obtain final concentration of 25 µg/ml. A loopfull of each of the test isolates were inoculated in 10 ml of Brain Heart infusion (BHI) broth and incubated at 37°C for 24h in order to obtain actively growing log phase cultures. Sterile 20 ml of molten Mueller and Hinton agar butt was cooled to around 40°C and then seeded with 0.4 ml test culture (0.1 O.D. at 540nm) and poured into a 9cm diameter aneubra Petri plate. Using a sterile cork borer (8mm diameter), wells was punched in each plate after solidification of the medium. About 50µl of the
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test sample (metal complex) was then added to the wells and incubated at temperature of 37°C for 24h to observe the zones of inhibition against the metal complex. Control wells were also set up using 50µl of DMSO as a solvent for each isolate. The experiment was carried out in triplicates, while the results were reported as mean ± Standard Deviation (SD).
Anti-tubercular activity screening: All the prepared Schiff base ligands and their Co(II) complexes were screened for their in vitro antimycobacterial activity against M. tuberculosis (H37 RV strain) using Microplate Alamar Blue Assay (MABA). A stock solution of each compound (1 mg/ml) was prepared in sterile deionized water to test the final drug concentration in the range 0.8–100 µg/ml. The 96-well microtitre plates were added with 200 µl of sterile de-ionized water in all outer perimeter wells to minimize evaporation of medium in the test wells during incubation, while 100 µl of the Middlebrook 7H9 broth with serial dilution of compounds were added directly on the plate. Plates were covered and sealed with paraffin and incubated at 37ºC for five days. On sixth day, 25µl of freshly prepared 1:1 mixture of Almar Blue reagent and Tween 10% and Tween 80% was added to the plate and incubated at 37ºC for another 24 hours. On the following day, plates were read and MIC (Minimum inhibitory concentration) was determined as the lowest concentration of compound that prevented colour change from blue to pink [41].
RESULTS AND DISCUSSION Structure of Schiff base ligands: Analytical data suggested that o-hydroxyl aldehyde derivative (2a-2d) condensed with Ethyl 2amino-4, 5, 6, 7 tetrahydrobenzo(b)thiophene 3-carboxylate (1) in 1:1 molar ratio to form Schiff base ligand (HL1, HL2, HL3 and HL4).
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H-NMR Spectra:
The 1H-NMR spectra of the Schiff base ligands were recorded in CDCl3 solvent using TMS as internal reference over the range of 0–16 ppm. The disappearance of the singlet at 5.84 ppm of
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aminothiophene (1) and appearance of new singlet peak in the range of 8.45–9.40 ppm can be assigned to the azomethine protons which confirmed the condensation of aminothiophene and ohydroxyl aldehyde derivative. The singlet observed in the range of 12.93-14.85 ppm is attributable to the phenolic -OH of aldehyde derivative (2a-2d). Also a set of multiplets observed in the range 6.88–8.16 ppm can be ascribed to the aromatic protons. The important 1H NMR signals are listed in Table 2.
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C NMR Spectra:
The
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C NMR spectra of the Schiff base ligands were recorded in DMSO-d6 solvent. In the
spectra, ester carbonyl carbon appeared most downfield in the range 162.06-162.81 ppm followed by phenolic carbon in the range of 158.35- 160.11 ppm and azomethine carbon in the range 158.03- 162.24 ppm. The
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C spectral data confirmed the formation of the Schiff bases.
The important 13C NMR signals are summarized in Table 2.
IR Spectra: IR spectra of Schiff base ligands shows band in the region 3000-3200 cm−1 corresponding to phenolic hydroxyl group (-OH) and strong peak in the regions of 1597-1598 cm−1 corresponding to the azomethine group (C=N). A strong band appearing in the region 1682-1704 cm−1 was assigned to ester carbonyl band. Substituted thiophene ring vibrations were observed at 1540, 1440 and 1360 cm−1 [42]. Important IR peaks are listed in Table 3.
Electronic Spectra: The electronic absorption spectra of the Schiff base ligands in DMF showed two strong bands in the range 41666-38461 cm−1 and 33333-31250 cm−1 for the phenolimine form, assignable to π→π* and n→π* transitions respectively. Also weaker bands at 35714 cm−1 and 25000 cm−1 for quinoneamine form are observed, confirming phenolimine form of Schiff base ligands under consideration [42].
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Mass Spectra: From the Spectral evaluation the desired molecular weights of Schiff base ligands were observed. Intense molecular ion peaks in the mass spectra established the formation of the Schiff base ligands. The proposed molecular formula of these Schiff base ligands were confirmed by comparing their molecular formula weights with m/z values. Schiff base ligands exhibited parent peaks due to molecular ions [M]+ at m/z 315.9 (calculated m/z 315.3), 349.9 (calculated m/z 349.8), 393.9 (calculated m/z 394.3) and 365.9 (calculated m/z 365.4) for HL1, HL2, HL3 and HL4 respectively. Based on 1H and
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C NMR Spectra, electronic spectra, mass spectra and FTIR, the proposed
structures of the Schiff base ligands (HL1, HL2, HL3 and HL4) are given in Figure-2.
Structure of the metal complexes: Schiff base ligands formed well defined complexes with the CoCl2 salt. Physico-chemical data recommended 1:2 (metal: ligand) stoichiometry of the complexes. Negligible molar conductance values indicated non electrolytic nature of the metal complexes. All the Co(II) complexes were colored, stable, non hygroscopic and decomposed over 170°C. The complexes are insoluble in common organic solvents except DMF and DMSO. Purity of the ligand and its complexes has been checked by TLC.
FTIR Spectra: IR spectra of the Schiff base ligands and its complexes are compared in order to determine the coordination sites on complexation. The important IR bands and their assignments are listed in Tables 3. The broad band appearing around 3000-3200 cm−1 is assigned to phenolic –OH in Schiff base ligands (HL1-HL4), which is absent in IR spectra of the metal complexes and υ(C-O) band appearing in the region 1307-1311 cm-1 in Schiff base ligand is increased by 22-32 cm-1 in the respective Co(II) complex spectra, suggesting complexation of Schiff base ligand with central metal atom through phenolic -OH. Ester carbonyl frequency υ(C=O) in the region 16821708 cm−1 is decreased by 29-57 cm-1 on chelation, indicating coordination by ester carbonyl
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group to the Co(II) ion. The azomethine υ(C=N) peak at 1597-1598 cm-1 is shifted to lower frequency by 20-30 cm-1 in metal complexes confirming involvement of azomethine nitrogen in coordination with metal ion. The medium intensity band observed in the region 616-627 cm−1 is assigned to υ(C-S) vibrations, which remained unaltered, representing non involvement of thiophene sulphur in the coordination. The IR spectra of all the metal complexes exhibited prominent non-ligand band around 513-518 cm−1 range which can be assigned to υ(M←O) and 415-424 cm−1 to υ(M←N) stretching vibrations respectively.
Electronic Spectra and Magnetic moments: Electronic spectra of Co(II) complexes were recorded in DMF at room temperature (Figure-4). All Co(II) complexes showed three absorption bands in the range 10000-10200 cm−1, 19400– 20000 cm−1 and 23000-25000 cm−1. These bands can be assigned to the three spin allowed transitions, 4T1g (F) → 4T2g (F) (ν1), 4T1g (F) → 4A2g (F) (ν2) and 4T1g (F) → 4T1g (P) (ν3) respectively. Using Konig equqtions [43], the covalency factor (β) and transition ratio (ν2/ν1) were observed in the range 0.87-0.96 and 1.91-1.98 respectively, while LFSE values were obtained between -79.35 to -74.94 kJ/mol. Lower B values of the complexes than the free ion, indicates orbital overlap and delocalization of d-orbitals [44]. Magnetic moment values of Co(II) complexes were observed in the range 4-5 BM which confirmed the octahedral geometry of the complexes. The electronic spectral data and magnetic moments of the complexes are illustrated in Table 4.
Thermogravimetric Studies: The thermal decomposition analysis of the [Co(L3)2] metal complex was carried out under nitrogen atmosphere from room temperature to 1000°C. The metal complex was stable up to 180°C, indicating absence of coordination water. The thermogram of [Co(L3)2] complex is given in Figure-5, which shows two stage decomposition pattern. First TG loss corresponds to the aminothiophene moiety in the temperature range 200-400°C (found 46.08 %, calc. 46.19 %). Second TG loss occurred in the temperature range 450-700°C with a loss of salicylaldehyde moiety (found 45.02 %, calc. 44.95 %) leaving behind cobalt oxide as residue (found 8.90 %,
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calc. 8.86 %). Observed TG data is in good agreement with the proposed molecular formula of the cobalt complex.
X-ray diffraction Studies: The powder sample of [Co(L3)2] complex was scanned for 2θ ranging from 10° to 90° at the wavelength of 1.5406 A° on PANanalytical X-ray diffractometer. The maximum reflection was observed at 2θ=19.6538° corresponding to interplanar distance of d=4.5133 A°. The single crystals of the complexes were difficult to isolate from the solvent, hence powder diffraction data were acquired [42]. The diffractogram presented in Figure 6, indicated high degree of crystallinity with sharp crystalline peaks of the complex. Using Scherer’s formula [45,46], the average crystallite size of the complex was calculated, the [Co(L3)2] complex has an average crystallite size of 53 nm. On the basis of FTIR, elemental analysis, molar conductance, electronic spectra, magnetic susceptibility and thermogravimetric analysis, the proposed structure of the complex is shown in Figure-3.
Antimicrobial Studies: Table 5 represents the Antimicrobial resistance profile of gram negative uropathogens which was carried out using Kirby Bauer method. All isolates used in the study were Multiple Drug Resistant (i.e resistant to more than 3 antibiotics) including resistance to 3rd generation cephalosporins (viz. ceftazidime, cefotaxime and ceftriaxone). The effect of Schiff base ligands and Co(II) complexes against these test isolates are indicated in Table 6. DMSO (solvent) did not show any zone of inhibition against the test organisms. All the Schiff base ligands and their Cobalt complexes showed considerable zones of inhibition against all the test isolates except for ESBL producing Klebsiella pneumoniae (kp), which showed no zone of inhibition against any compound and MBL producing Proteus mirabilis (isolate 607), which showed no zone of inhibition against Schiff base ligand HL3 and HL4 and their respective cobalt complex. Schiff bases and their respective Co(II) complexes have shown similar microbial activity against Escherichia coli-10 (Ec-10) and Klebsiella pneumoniae-7 (Kp-7) ESBL producers and
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Pseudomonas aeruginosa (isolate-85), and Citrobacteramalonaticus (isolate-135) MBL producers. Schiff base ligands were found resistant against Citrobacterdiversus (Citro-2), Proteus mirabilis-7 (Pro-7) ESBL uropathogens and Proteus mirabilis (isolate-607), Klebsiella pneumonia (isolate-618) and Escherichia coli (isolate-220) MBL uropathogens, while their respective Co(II) complexes were found sensitive to these uropathogens on chelation. Maximum activity was observed against MBL producing C. amalonaticus (isolate 135) with zones of inhibition ranging between 13-22 mm against cobalt complexes. The enhanced antimicrobial activity of cobalt complexes against the test isolates can be attributed to the concept of chelation. As the positive charges of the metal are partially shared with the donor atoms present in the ligands and there is possible π-electron delocalization over the metal complex formed, the lipophilic character of the metal complex increases and favors its permeation more efficiently through the lipid layer of the micro-organisms. This allows easy binding and penetration of the complex in the cellular structure of the pathogens [47].
A recent study reported antimicrobial activity of cobalt complexes against both gram-positive as well as gram-negative bacteria, with zones of inhibition in the range of 15-17mm [48]. Tharmaraj and co-workers also reported zones of inhibition of cobalt complexes in the range of 14-19mm. Few authors have reported antibacterial as well as antifungal activities of cobalt complexes [49]. High susceptibility of cobalt complexes was indicated by saha et al, 2009. In his study, the zones of inhibition were found to be in the range of 22-31mm against bacterial cultures and 21-45mm against fungal cultures [50]. On the contrary, Aiyelabola and co-workers have reported moderate susceptibility of cobalt complexes (zone of inhibition 6-20mm) against bacterial and fungal isolates [51].
Anti-Tubercular Studies: Mycolic acids which are hydroxyl fatty acids present in the mycobacterial cell wall are important key structure for survival and growth of M. tuberculosis bacteria. Majority of first line drugs against M. tuberculosis works by inhibiting biosynthesis of mycolic acids leading to mycobacterial cell death [52,53]. Recent advancements revealed aminothiophene carboxylate such as methyl 2-amino-5-benzylthiazole-4-carboxylate is more potent compound against M.
13
tuberculosis H37 RV strain with MIC lower than thiolactomycin and isoniazid [54]. Enticed by these results, Schiff base ligands and their Co(II) complex were evaluated against Mycobacterium tuberculosis (H37 RV strain ATCC No- 27294) by Microplate Alamar Blue Assay (MABA). All compounds exhibited moderate antitubercular activity with 100% inhibitory activity against Mycobacterium tuberculosis at 25 µg/ml concentration. Schiff base ligands and Co(II) complexes exhibited same antitubercular activity indicating non involvement of Co ion in the inhibition process. It shows that the activity of the complexes cannot be merely associated to the presence of the metal ion [55, 56]. Test results are indicated in Table 7.
CONCLUSION In summary, we have synthesized, Schiff bases by condensation of ethyl-2-amino-5,6-dihydro4H-cyclopenta[b]thiophene-3-carboxylate and O-hydroxyl aldehyde derivative and their Co(II) complexes. Physico-chemical and various spectral studies revealed, monobasic trident nature of ligands with ONO donor atoms towards central Co(II) ion and octahedral geometry of complexes with 1:2 (metal: ligand) stoichiometry. All compounds exhibited moderate antitubercular activity against M. tuberculosis (H37Rv) while most of the synthesized compounds have shown to exhibit promising antimicrobial activity against multidrug resistant gram negative uropathogens.
Conflict of Interests: The authors declare that there is no conflict of interests regarding the publication of this paper.
Acknowledgment: The authors thank Dr. V. J. Sirwaya, Principal Wilson College, Mumbai, for providing research facility and Dr. Kishore Bhat of Governmental Dental College, Belgaum, for providing anti-TB activity. The authors are thankful to Dr. P. L. Paulose and Dr. Sujata Patil for XRD analysis. The authors also wish to thank Mr. Mustapha Mandewale, Dr. Vijay Veer and Dr. Chandrakanth Gadipelly for Spectral analysis. The authors acknowledge IIT SAIF, Mumbai for analytical support. This research did not receive any specific grant from funding agencies in the public, commercial or not-for-profit sectors. 14
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20
List of Figures: Fig. No.
Title
Page No.
1
Synthesis of ligand
2
2
Structures of Schiff base ligands (HL1-HL4)
3
3
Proposed structure of Co(II) complexes
4
4
Electronic spectra of Co(II) complexes
5
5
Thermogram of [Co(L3)2] complex
6
6
XRD diffractogram of [Co(L3)2] complex
7
21
Figure 1: Synthesis of ligand
22
Figure 2: Structures of Schiff base ligands (HL1-HL4)
23
Figure 3: Proposed structure of Co(II) complexes
24
Figure 4: Electronic spectra of Co(II) complexes
25
Figure 5: Thermogram of [Co(L3)2] complex
26
Figure 6: XRD diffractogram of [Co(L3)2] complex
27
List of Tables: Table No.
Title
Page No.
1
Culture name and their type used in study
2
2
13
3
3
IR spectral data of Schiff base ligands and Co(II) complexes
4
4
Electronic and magnetic field parameters of Co(II) complexes
5
5
Antibiotic resistance profile of ESBL and MBL producers
6
6
Antibacterial activity of Schiff base ligands and Co(II) complexes
7
7
Anti-TB activity of Schiff base ligands and Co(II) complexes
8
C & 1H NMR signals for Schiff base ligand
Table 1: Culture name and their type used in study Culture Type Cultures code name ESBL
Citro-2
Full form Citrobacter diversus-2
28
Ec- 10
Escherichia coli- 10
Kp
Klebsiella pneumoniae
Kp-7
Klebsiella pneumoniae- 7
Pro- 7
Proteus mirabilis- 7
85
Pseudomonas aeruginosa
135
Citrobacter amalonaticus
220
Escherichia coli
607
Proteus mirabilis
618
Klebsiella pneumoniae
MBL
Table 2: 13C & 1H NMR signals for Schiff base ligands Schiff base ligand
C=O Ester
-C-OH Phenolic
-CH=N- Azomethine
HL1
162.11
160.11
159.49
NMR
HL2
162.08
158.70
158.12
signals
HL3
162.06
159.09
158.03
(ppm)
HL4
162.81
158.35
162.24
-NH2 Amine
-C-OH Phenolic
-CH=N- Azomethine
13
C
Schiff base ligand 1
H
(1)
5.84
-
-
NMR
HL1
-
12.94
8.54
29
signals
HL2
-
12.93
8.45
(ppm)
HL3
-
12.95
8.46
HL4
-
14.85
9.40
Table 3: IR spectral data of Schiff base ligands and Co(II) complexes υ(O-H)
υ(C=O)
phenolic
ester
HL1
3200-3000
1682
1597
1310
616
-
-
HL2
3200-3000
1704
1598
1311
619
-
-
HL3
3200-3000
1708
1598
1311
626
-
-
HL4
3200-3000
1704
1598
1307
617
-
-
[Co(L1)2]
-
1653
1567
1337
617
518
424
[Co(L2)2]
-
1651
1575
1343
618
518
422
[Co(L3)2]
-
1651
1573
1333
627
516
414
[Co(L4)2]
-
1668
1578
1333
618
513
415
Compound
υ(C=N)
υ(C-O)
υ(C=S) υ(M←O) υ(M←N)
azomethine phenolic thiophene
Table 4: Electronic and magnetic field parameters of Co(II) complexes Complex Absorption
Tentative
Magnetic
(ν2/ν1)
Dq
B
β
LFSE
30
band
assignment
(cm−1)
[Co(L1)2]
[Co(L2)2]
[Co(L3)2]
[Co(L4)2]
moment
(cm−1)
(cm−1)
(kJ/mol)
(BM)
10080
4
T1g (F) → 4T2g (F)
20000
4
23866
4
10204
4
19455
4
25000
4
10040
4
19685
4
23095
4
10050
4
19417
4
24691
4
T1g (F) → 4A2g (F)
4.76
1.98
991.94
908
0.94
-79.35
4.78
1.91
925.12
923
0.95
-74.01
4.79
1.96
964.49
844
0.87
-77.16
4.82
1.93
936.72
931
0.96
-74.94
T1g (F) → 4T1g (P)
T1g (F) → 4T2g (F)
T1g (F) → 4A2g (F) T1g (F) → 4T1g (P)
T1g (F) → 4T2g (F)
T1g (F) → 4A2g (F) T1g (F) → 4T1g (P)
T1g (F) → 4T2g (F)
T1g (F) → 4A2g (F) T1g (F) → 4T1g (P)
Table 5: Antibiotic resistance profile of ESBL and MBL producers Cultures code name
Full form
Citro-2
Citrobacter diversus-2
Ec- 10
Escherichia coli- 10
Kp
Klebsiella pneumoniae
Kp-7
Klebsiella pneumoniae- 7
Antibiotic sensitivity test Sensitive Intermediate Resistant ESBL PRODUCERS AS, BA, CF, PC, ZN, GM, AK, GF, CH, RC, CI, CF TT, OX, RP, ZX, CB, NA, TE NX, AG, CU, CP, FG, PB BA, CF, PC, RC, CI, TE, ZN, CH, GM, PB, AS TT, OX, RP, ZX, CB, NA, AK, GF NX, AG, CU, CP, FG AS, BA, CF, PC, CH,RC, CI, TE, ZN, GF, TT, OX, RP, GM, AK ZX, CB, NA, NX, AG, CU, CP, FG, PB AS, BA, CF, PC, CH,RC, CI, OX, TE ZN, GM, AK, GF, TT, RP, ZX, CB, NA, NX, AG, CU,
31
CP, AS, RP, NA, CF, PC, AK RC, GM, GF MBL PRODUCERS
Pro- 7
Proteus mirabilis- 7
85
Pseudomonas aeruginosa
AK, GF, PB
RC, CF, PC
135
Citrobacter amalonaticus
CH, PB
AK
220
Escherichia coli
CH, AK, GF
AS, ZN
607
Proteus mirabilis
-
CH, GM
618
Klebsiella pneumoniae
-
-
CP, FG, PB BA, CH, CI, TE, ZN, TT, OX, ZX, CB, NX, AG, CU, FG, PB AS, CI, TE, ZN, GM, TT, OX, RP, ZX, CB, BA, CH, NA, NX, AG, CU, CP, FG, AS, BA, CF, PC, RC, CI, TE, ZN, GM, GF, TT, OX, RP, ZX, CB, NA, NX, AG, CU, CP, FG, CB, AS BA, CF, PC, RC, CI, TE, GM, TT, OX, RP, ZX, CB, NA, NX, AG, CU, CP, FG, PB AS, BA, CF, PC, RC, CI, TE, ZN, AK, GF, TT, OX, RP, ZX, CB, NA, NX, AG, CU, CP, FG, PB AS, BA, CF, PC, CH,RC, CI, TE, ZN, GM, AK, GF, TT, OX, RP, ZX, CB, NA, NX, AG, CU, CP, FG, PB
Key: TT -Ticarcillin/clavulanic acid, OX- Oxytetracycline, RP – Ceftriaxone, ZX – Cefepime, CB – Cefuroxime, NA - Naladixic acid, NX- Norfloxacin, AG - Amoxycillin/clavulanic acid, CU – Cefadroxil,CP - Cefoperazone, FG- Ceftazidime, PB - Polymixin B, AS – Ampicillin, BA - Co-trimaxazole, CF – Cefotaxime, PC- Pipperacillin, CH – Chloramphenicol, RC – Ciprofloxacin, CI – Ceftizoxime, TE – Tetracycline, ZN – Ofloxacin, GM – Gentamicin, AK –Amikacin, GF – Gatifoxacin
Table 6: Antibacterial activity of Schiff base ligands and their Co(II) complexes Zone of Inhibition in mm Sr. No.
Cultures code name
1
Citro-2
2
Ec- 10 ESB L
3
[Co(L3)2 ]
HL4
[Co(L4)2 ]
DMSO (solvent)
13
0
12
0
12
0
11
12
10
12
10
12
0
0
0
0
0
0
0
0
0
HL1
[Co(L1)2]
HL2
[Co(L2)2]
0
13
0
11
12
0
HL
3
Kp
4
Kp-7
11
12
12
12.5
10
12
12
11
0
5
Pro- 7
0
12
0
12
0
12
0
13
0
32
6
85
7
135 MB L
12
15
15
14
15
12.5
15
12
0
16
22
17
17
15
13
15
16
0
0
16
11
12
11
13
0
11
0
8
220
9
607
0
13
0
10
0
0
0
0
0
10
618
0
12
0
11
10
10
0
12
0
Table 7: Anti-TB activity of Schiff base ligands and their Co(II) complexes Compound
Ligand
MIC (µg/ml)
HL1
25.0
HL2
25.0
HL3
25.0
33
Complex
HL4
25.0
[Co(L1)2]
25.0
[Co(L2)2]
25.0
[Co(L3)2]
25.0
[Co(L4)2]
25.0
Pyrazinamid e Standard
Streptomycin Ciprofloxaci n
3.125 6.250 3.125
34
