Accepted Manuscript Synthesis, Characterization, and Biological Evaluation of Schiff Base-Platinum(II) Complexes C. Shiju, D. Arish, N. Bhuvanesh, S. Kumaresan PII: DOI: Reference:

S1386-1425(15)00182-1 http://dx.doi.org/10.1016/j.saa.2015.02.030 SAA 13320

To appear in:

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy

Received Date: Revised Date: Accepted Date:

21 April 2014 6 February 2015 8 February 2015

Please cite this article as: C. Shiju, D. Arish, N. Bhuvanesh, S. Kumaresan, Synthesis, Characterization, and Biological Evaluation of Schiff Base-Platinum(II) Complexes, Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy (2015), doi: http://dx.doi.org/10.1016/j.saa.2015.02.030

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Synthesis, Characterization, and Biological Evaluation of Schiff BasePlatinum(II) Complexes C. Shijua, D. Arisha, N. Bhuvaneshc, and S. Kumaresanb* a

Department of Chemistry, Manonmaniam Sundaranar University, Tirunelveli-627 012, India. b Noorul Islam Center for Higher Education, Kumaracoil, Thuckalay-629 180, TN, India. c Department of Chemistry, Texas A & M University, College Station, Texas-77842, USA

Abstract The platinum complexes of Schiff base ligands derived from 4-aminoantipyrine and a few substituted aldehydes were synthesized and characterized by elemental analysis, mass, 1HNMR, IR, electronic spectra, molar conductance, and powder XRD. The structure of one of the ligands L5 was confirmed by a single crystal XRD analysis. The Schiff base ligand crystallized in the triclinic, space group P-1 with a = 7.032(2) Ǻ, b = 9.479(3) Ǻ, c = 12.425(4) Ǻ, α = 101.636(3)0, β = 99.633(3)0, γ = 94.040(3)0, V = 795.0(4) Ǻ3, Z = 2, F(000) = 352, Dc = 1.405 mg/m3, µ = 0.099 mm-1, R = 0.0378, and wR = 0.0967. The spectral results show that the Schiff base ligand acts as a bidentate donor coordinating through the azomethine nitrogen and the carbonyl oxygen atoms. The geometrical structures of these complexes are found to be square planar. Antimicrobial studies indicate that these complexes exhibit better activity than the ligand. The anticancer activities of the complexes have also been studied towards human cervical cancer cell line (HeLa), Colon Cancer Cells (HCT116) and Epidermoid Carcinoma Cells (A431) and it was found that the [Pt(L3)Cl2] complex is more active. Key words: Schiff base, platinum complexes, Antimicrobial, Antitubercular, Anticancer. * Corresponding author. Tel.: +91 9443182502 E-mail address: [email protected]

1. Introduction It has been well established that the platinum complexes are of biological importance due to their carcinostatic activity and interest in biological chemistry. The carcinostatic action of the platinum complexes is due to their interaction with nuclear DNA [1]. Platinum compounds are among the most important chemotherapeutic agents for treating cancer. Cisplatin (cisdiamminedichloroplatinum(II), has a significant activity in ovarian, testicular, bladder, head and neck, and lung cancer, where it is most commonly used in combination with other drugs [2]. Thus, it has become one of the most successful anticancer drugs used worldwide in almost 50% of solid tumor chemotherapies. Although the initial response rates can be high with cisplatinbased regimen, the clinical utility of the drug is often limited by the onset of acquired or intrinsic resistance [3] and the number of side effects such as kidney damage, vomiting/nausea, and neurotoxicity [4]. The resistance of tumor cells to cisplatin remains a major cause of treatment failure in cancer patients, while the high toxicity of cisplatin limits the dose that can be given to patients. Developing metal complexes as drugs, however, is not an easy task. Schiff base metal complexes have received special attention because of their biological activity. Many Schiff bases are known to be medicinally important and are used to design medicinal compounds [5, 6]. A wide range of Schiff bases have been synthesized and their complexation behaviour studied in recent years. Because of the importance in the area of coordination chemistry, the pyrolozone chemistry of metals is developing very rapidly [7]. 4-Aminoantipyrine derivatives are very important in the field of coordination complexes and also these compounds are reported to exhibit analgesic and anti-inflammatory effects, antiviral, antibacterial, anti-cancer and herbicidal activities. [8-13]. In view of the above applications and importance, in the present

paper we report the synthesis, characterization, and biological activity of platinum complexes with the Schiff bases derived from 4-aminoantipyrene and substituted benzaldehydes. 2. Experimental 2.1. Materials 4-Aminoantipyrene, N,N’-dimethylaminobenzaldehyde, 4-isopropylbenzaldehyde, 4bromobenzaldehyde, 3-nitrobenzaldehyde, and 4-nitrobenzaldehyde were purchased from Merck. The human cervical cancer cell line (HeLa) and Colon Cancer Cells (HCT116) were obtained from National Centre for Cell Science (NCCS), Pune. K2PtCl4 was purchased from Sigma-Aldrich. All other reagents and solvents were purchased from commercial sources and were of analytical grade. 2.2. Physical Measurements Elemental analysis was done using a Perkin-Elmer elemental analyzer. Molar conductance of the complexes was measured in DMSO (10-3 M) solutions using a Coronation Digital Conductivity Meter. The electrospray (ESI) mass spectra were recorded on a THERMO Finnigan LCQ Advantage max ion trap mass spectrometer. Samples (10 µL) (dissolved in solvent such as methanol/ chloroform/ dichloromethane) were introduced into the ESI source through Finnigan surveyor autosampler. The 1H NMR spectra were obtained on a JEOL GSX 400 FT–NMR spectrometer. IR(KBr) spectra were recorded on a JASCO FT/IR-410 spectrometer in the 4000-400 cm-1 region. The electronic spectra were recorded on a Perkin Elmer Lambda-25 UV-VIS spectrometer. Powder XRD was recorded on a Rigaku Dmax X-ray diffractometer with Cu-Kα radiation. 2.3. Synthesis of Schiff Base Ligands 4-Aminoantipyrene (1mmol) in MeOH (20 mL) was taken in a 100 mL RB (Round Bottomed) flask. A solution of substituted benzaldehyde (1 mmol) in absolute MeOH (20 mL)

was then added slowly to the flask. The reaction mixture was vigorously stirred for about 5 h. The volume of the mixture was reduced to half of the initial volume under reduced pressure and an excess of anhydrous ether was added. A yellow precipitate was formed, which was collected by vacuum filtration and washed several times with anhydrous ether and then dried in vacuo over anhydrous CaCl2. The purity of the Schiff base ligand was checked by TLC. The yield of the isolated ligands was found to be 65-72%. The synthetic route for Schiff base ligands is shown in Scheme 1. {(E)-4- [4-N,N’dimethylbenzylideneamino]-1, 5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one}(L1). Yellow crystals, yield 68%. 1H NMR (CDCl3) δ: 9.64 (s CH=N), 2.45 (s, C-CH3), 3.24 (s, N-CH3), 6.69-7.75 (m, ArH), 3.08 (s, N-(CH3)2). IR (KBr, cm-1) ν: 1647 (C=O), 1610 (C=N), 1371 (C-N), 1455 (N-CH3), 3047 (Ar CH). Anal. calcd for C20H22N4O: C 71.83, H 6.63, N 16.75; found: C 72.15, H 6.21, N 16.52. UV-vis (CH2Cl2): λ/nm 362, 215. {(E)-4- [4-isopropylbenzylideneamino]-1, 5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one}(L2). Pale yellow crystals, yield 65%. 1H NMR (CDCl3) δ: 9.55 (s CH=N), 3.16 (s, C-CH3), 3.34 (s,-CH3), 7.30-7.80 (m, ArH), 2.50 (s, C-(CH3)2). IR (KBr, cm-1) ν: 1650 (C=O), 1595 (C=N), 1366 (C-N), 1455 (N-CH3), 3048 (Ar CH). Anal. calcd for C21H23N3O: C 75.65, H 6.95, N 12.60; found: C 76.02, H 7.18, N 12.21. UV-vis (CH2Cl2): λ/nm 329, 244, 215. {(E)-4- [4-bromobenzylideneamino]-1, 5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one}(L3). Yellow crystals, yield 72%. 1H NMR (CDCl3) δ: 9.70 (s CH=N), 2.43 (s, C-CH3), 3.16 (s, N-CH3), 7.26-7.73 (m, ArH). IR (KBr, cm-1) ν: 1649 (C=O), 1591 (C=N), 1376 (C-N), 1428 (NCH3), 3056 (Ar CH). Anal. calcd for C18H16BrN3O: C 58.39, H 4.36, N 11.35; found: C 59.08,H 4.89, N 11.76. UV-vis (CH2Cl2): λ/nm 340, 255, 210. {(E)-4- [3-nitrobenzylideneamino]-1, 5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one}(L4).

Orange yellow crystals, yield 70%. 1H NMR (CDCl3) δ: 9.80 (s CH=N), 2.52 (s, C-CH3), 3.21 (s, N-CH3), 7.25-8.75 (m, ArH). IR (KBr, cm-1) ν: 1647 (C=O), 1592 (C=N), 1382 (C-N), 1456 (N-CH3), 3059 (Ar CH). Anal. calcd for C18H16N4O3: C 64.28, H 4.79, N 16.66; found: C 65.11, H 5.04, N 16.14. UV-vis (CH2Cl2): λ/nm 347, 254, 214. {(E)-4- [4-nitrobenzylideneamino]-1, 5-dimethyl-2-phenyl-1H-pyrazol-3(2H)-one}(L5). Yellow crystals, yield 68%. 1H NMR (CDCl3) δ: 9.78 (s CH=N), 2.51 (s, C-CH3), 3.22 (s, N-CH3), 7.25-8.25 (m, ArH). IR (KBr, cm-1) ν: 1649 (C=O), 1597 (C=N), 1381 (C-N), 1430 (NCH3), 3041 (Ar CH). Anal. calcd for C18H16N4O3: C 64.28, H 4.79, N 16.66; found: C 65.02, H 4.65, N 16.42. UV-vis (CH2Cl2): λ/nm 390, 260, 214. 2.4. Synthesis of the Schiff Base Metal Complexes The Schiff base (1mmol) in MeOH (25 mL) and a little dichloromethane (for better solubility) was taken in a 100 mL RB flask. K2PtCl4 (1mmol) in MeOH (15 mL) and water (10 mL) mixture was then added slowly to the flask. The reaction mixture was vigorously stirred for about 8 h. The volume of the reaction mixture was concentrated to half by removing the solvent under reduced pressure and kept at room temperature. The complexes were obtained as solids, washed with water followed by a little cold methanol, and dried in vacuo over anhydrous CaCl2. The synthetic route of platinum complexes is given in Scheme 2. The yield was found to be 5563%. [Pt(L1)Cl2] Brown solid, yield 58%. ESI-MS: m/z 601 [M+]. 1H NMR (CDCl3) δ: 9.74 (s CH=N), 2.34 (s, C-CH3), 3.08 (s, N-CH3), 6.68-7.74 (m, ArH), 2.99 (s N-(CH3)2). IR (KBr, cm-1) ν: 1621(C=O), 1588 (C=N), 1370 (C-N), 1455 (N-CH3), 3047 (Ar CH), 434 (M-O), 501 (M-N).

Ʌc(Ohm-1 cm2 mol-1) 2.3. Anal. calcd for C20H22Cl2N4OPt: C 40.01, H 3.69, N 9.33; found: C39.78, H 3.98, N 9.83. UV-vis (CH2Cl2): λ/nm 521, 415, 366, 256. [Pt(L2)Cl2] Brown solid, yield 60%. ESI-MS: m/z 600 [M+]. 1H NMR (CDCl3) δ: 9.80 (s CH=N), 3.12 (s, C-CH3), 3.30 (s, N-CH3), 7.29-7.82 (m, ArH), 2.48 (s C-(CH3)2). IR (KBr, cm-1) ν: 1620(C=O), 1587 (C=N), 1366 (C-N), 1455 (N-CH3), 3049 (Ar CH), 436 (M-O), 506 (M-N). Ʌc(Ohm-1 cm2 mol-1) 2.1. Anal. calcd for C21H23Cl2N3OPt: C 42.08, H 3.87, N 7.01; found: C41.81, H 4.06, N 7.43. UV-vis (CH2Cl2): λ/nm 512, 380, 262. [Pt(L3)Cl2] Brown solid, yield 55%. ESI-MS: m/z 634 [M+]. 1H NMR (CDCl3) δ: 9.88 (s CH=N), 2.35 (s, C-CH3), 3.09 (s, N-CH3), 7.03-7.76 (m, ArH). IR (KBr, cm-1) ν: 1633 (C=O), 1586(C=N), 1430 (N-CH3), 3054 (Ar CH), 431 (M-O), 501 (M-N). Ʌc (Ohm-1cm2 mol-1) 3.6. Anal. calcd for C18H16BrCl2N3OPt: C 33.98, H 2.53, N 6.60; found: C 34.27, H 2.84, N 6.91. UV-vis(CH2Cl2): λ/nm 525, 415, 317, 260. [Pt(L4)Cl2] Brown solid, yield 57%. ESI-MS: m/z 602 [M+]. 1H NMR (CDCl3) δ: 10.09 (s CH=N), 2.34 (s, C-CH3), 3.08 (s, N-CH3), 7.25-8.86 (m, ArH). IR (KBr, cm-1) ν: 1635 (C=O), 1587(C=N), 1381 (C-N), 1455 (N-CH3), 3062 (Ar CH), 434 (M-O), 502 (M-N). Ʌc (Ohm-1cm2 mol-1) 1.9. Anal. calcd for C18H16Cl2N4O3Pt: C 35.89, H 2.68, N 9.30; found: C 36.19, H 2.79, N 9.11. UV-vis (CH2Cl2): λ/nm 518, 415, 360, 258, 214. [Pt(L5)Cl2] Brown solid, yield 63%. ESI-MS: m/z 602 [M+]. 1H NMR (CDCl3) δ: 9.88 (s CH=N), 2.52 (s, C-CH3), 3.23 (s, N-CH3), 7.25-8.41 (m, ArH). IR (KBr, cm-1) ν: 1620 (C=O),

1572(C=N), 1380 (C-N), 1431 (N-CH3), 3042 (Ar CH), 438 (M-O), 507 (M-N). Ʌc (Ohm-1cm2 mol-1) 2.8. Anal. calcd for C18H16Cl2N4O3Pt: C 35.89, H 2.68, N 9.30; found: C 36.21, H 2.93, N 9.55. UV-vis (CH2Cl2): λ/nm 517, 371, 306, 257. 2.5. Crystal Structure Determination A Leica MZ 75 microscope was used to identify a suitable red block with very well defined faces with dimensions (max, intermediate, and min) 0.38mm x 0.26 mm x 0.09 mm from a representative sample of crystals of the same habit. The crystal mounted on a nylon loop was then placed in a cold nitrogen stream (Oxford) maintained at 110 K. A BRUKER APEX 2 X-ray (three-circle) diffractometer was employed for crystal screening, unit cell determination, and data collection. The goniometer was controlled using the APEX2 software suite, v2008-6.0. The sample was optically centered with the aid of a video camera such that no translations were observed as the crystal was rotated through all positions. The detector was set at 6.0 cm from the crystal sample (APEX2, 512x512 pixel). The X-ray radiation employed was generated from a Mo sealed X-ray tube (Kα= 0.70173Å with a potential of 40 kV and a current of 40 mA) fitted with a graphite monochromator in the parallel mode (175 mm collimator with 0.5 mm pinholes). Sixty data frames were taken at widths of 0.5°. These reflections were used in the auto-indexing procedure to determine the unit cell. A suitable cell was found and refined by nonlinear least squares and Bravais lattice procedures. The unit cell was verified by examination of the h k l overlays on several frames of data by comparing with both the orientation matrices. No supercell or erroneous reflections were observed. After careful examination of the unit cell, a standard data collection procedure was initiated using omega scans. Integrated intensity information for each reflection was obtained by reduction of the data frames with the program APEX2 [14]. The integration method employed a three dimensional profiling algorithm and all data were corrected

for Lorentz and polarization factors, as well as for crystal decay effects. Finally the data was merged and scaled to produce a suitable data set. The absorption correction program SADABS [15] was employed to correct the data for absorption effects. Systematic reflection conditions and statistical tests of the data suggested the space group P-1. A solution was obtained readily using SHELXTL (XS) [16]. Hydrogen atoms were placed in idealized positions and were set riding on the respective parent atoms. All non-hydrogen atoms were refined with anisotropic thermal parameters. The structure was refined (weighted least squares refinement on F2) to convergence [16, 17]. Olex2 was employed for the final data presentation and structural plots [17]. 2.6. Antimicrobial Activities Antibacterial and antifungal properties of the ligand and its complexes were tested in vitro against the bacterial species Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus ; fungal species, Aspergillus niger, Aspergillus flavus, and Candida albicans by the disc diffusion method. Amikacin, ofloxacin, and ciprofloxacin were used as standards for antibacterial activity and nystatin was used as a standard for antifungal activity. The test organisms were grown on nutrient agar medium in petri plates. The compounds were prepared in DMSO and soaked in filter paper disc of 5 mm diameter and 1 mm thickness. The discs were placed on the previously seeded plates and incubated at 37oC and the diameter of inhibition zone around each disc was measured after 24 h for antibacterial and 72 h for antifungal activities. The minimum inhibitory concentration (MIC) was determined by serial dilution technique [18]. 2.7. Antimycobacterial Activity Screening by Resazurin Microplate Assay (REMA). The anti-TB activity of the compounds was tested by resazurin microplate assay (REMA) as per Martin et al (2003) [19] with slight modification. Resazurin, a redox dye, is blue in its

oxidized state. In the presence of viable cells it is reduced into resorufin, which is pink in color. M. tuberculosis H37Rv was grown in Middlebrook 7H9 broth (Difco BBL, Sparks, MD, USA) supplemented with 10 % OADC (Becton Dickinson, Sparks, MD, USA) and 0.5% glycerol. The optical density of the bacterial culture was adjusted to McFarland 1.0 unit and 50 µ L from this suspension was used as the inoculum. Stock solutions of the test compounds were prepared in dimethylformamide (DMF) and were added to fresh medium in the wells of a 96-well microplate to which 50 µL inoculum was added making the total assay volume 200 µL. The final concentrations of the test molecules were 1, 10 and 100 µg/mL. Growth control wells contained the medium and M. tuberculosis H37Rv alone. Rifampicin (1.0 µg/mL) served as positive control for inhibition of growth. Negative control wells contained the highest volume of DMF used in test wells without any compound. After incubation at 37 ºC for 7 days, 15 µL of 0.01% resazurin (Sigma, St. Louis. MO, USA) solution in sterile water was added to the first growth control wells and incubated for 24 hours. Once the first set of growth controls turned pink, the dye solution was added to the second set of growth controls and the test wells, and incubated for 24 hours at 37 ºC. Blue color in the wells containing the test compounds would indicate inhibition of growth and pink would indicate lack of inhibition of growth of M. tuberculosis [20]. 2.8. In vitro Anti Cancer Activity The human cervical cancer cell line (HeLa) and Colon Cancer Cells (HCT116) were grown in Eagles Minimum Essential Medium (EMEM) containing 10% fetal bovine serum (FBS). The cells were maintained at 37 0C, 5% CO2, 95% air, and 100% relative humidity. Maintenance cultures were passaged weekly, and the culture medium was changed twice a week. The monolayer cells were detached with trypsin-ethylenediaminetetraacetic acid (EDTA) to make single cell suspension and viable cells were counted using a hemocytometer and diluted

with a medium containing 5% FBS to give a final density of 1x105 cells/mL. One hundred microlitres per well of cell suspension were seeded into 96-well plates at a plating density of 10,000 cells/well and incubated to allow for cell attachment at 370C, 5% CO2, 95% air, and 100% relative humidity. After 24 h, the cells were treated with serial concentrations of the test samples. They were initially dissolved in neat dimethylsulfoxide (DMSO) to prepare the stock (200 mM) and stored frozen prior to use. At the time of sample addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with serum free medium. Additional three, 2 fold serial dilutions were made to provide a total of five sample concentrations. Aliquots of 100 µL of these different sample dilutions were added to the appropriate wells already containing 100 µL of medium, resulting in the required final sample concentrations. Following sample addition, the plates were incubated for an additional 48 h at 370C, 5% CO2, 95% air, and 100% relative humidity. The medium without samples served as control and a triplicate was maintained for all concentrations [21]. MTT Assay MTT is a yellow water soluble tetrazolium salt [(3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide)]. Succinate-dehydrogenase, a mitochondrial enzyme in living cells cleaves the tetrazolium ring, converting the MTT to an insoluble purple formazan. Thus, the amount of formazan produced is directly proportional to the number of viable cells. After 48h of incubation, 15µL of MTT (5mg/mL) in phosphate buffered saline (PBS) was added to each well and incubated at 370C for 4h. The medium with MTT was then flicked off and formazan crystals obtained were solubilized in 100µL of DMSO. The absorbance at 570 nm was measured using a micro plate reader [22]. The % cell inhibition was determined using the following formula. % cell inhibition = 100 - Abs (sample)/Abs (control) x100

Nonlinear regression graph was plotted between % cell inhibition and log10 concentration and IC50 was determined using GraphPad Prism software. 3. Results and Discussion 3.1. Crystallographic Study X-ray diffraction data and structure refinement for the ligand L5 are shown in Table 1. The Schiff base ligand crystallized in the triclinic, space group P-1 with a = 7.032(2) Ǻ, b = 9.479(3) Ǻ, c = 12.425(4) Ǻ, α = 101.636(3)0, β = 99.633(3)0, γ = 94.040(3)0, V = 795.0(4) Ǻ3, Z = 2, F(000) = 352, Dc = 1.405 mg/m3, µ = 0.099 mm-1, R = 0.0378, and wR = 0.0967. Important bond lengths and bond angles are shown in the Tables 2a and 2b. All the bond lengths and bond angles were within the normal range [23]. The ORTEP diagram of the molecule is shown in Figure 1. From the crystal data, the pyrazolone ring (C9/C8/C10/N3/O3) was found to be almost planar. The phenyl ring attached to the pyrazolone ring has a deviation with torsion angles of 33.7(2)0 (N3-N4-C13-C14) and -147.5(1)0 (N3-N4-C13-C18). The C11 and C12 atoms are located on the opposite side of the pyrazolone plane with a torsion angle of -20.5(2)0 (C11-C9N3-C12). The atom O3 is slightly deviated from the pyrazolone mean plane, with torsion angles of 175.6(1)0 (C9-C8-C10-O3) and -171.4(1)0 (O3-C10-N4-N3). Because of the conjugation through the imino double bond C7=N2, the pyrazolone and the C1-C6 phenyls are approximately coplanar with the torsion angles value of 0.1(2)0 (C10-C8-N2-C7), -176.9(1)0 (C9-C8-N2-C7), -179.5(1)0 (C4-C7-N2-C8), -174.1(1)0 (C5-C4-C7-N2), 6.9(2)0 (C3-C4-C7-N2). The packing arrangement of the Schiff base ligand in the crystal lattice is shown in Figure 2. 3.2. Characterization of Schiff Base Ligand The schiff base ligands (Ln) were synthesized in the present work all yellowish in color and soluble in common organic solvents. The elemental analysis data of the ligands was in good

agreement with those calculated for the suggested formulae. The IR spectra of the Schiff base ligands exhibit bands in the range 1591-1610 cm-1 due to the azomethine group ν(C=N). Strong bands present in the region 1647-1650 cm-1 can be assigned to the carbonyl stretching frequency. The Schiff base ligands also display bands at 1366-1382 cm-1, due to the stretching vibration of the C-N. N-CH3 stretching frequencies were shown in the range 1428-1456 cm-1 [13]. In the UV spectra of the ligands, absorption bands were observed in the region 203-390 nm, that could be attributed to the π-π* and n-π* transitions in the benzene ring or -C=N or C=O groups. The 1HNMR spectrum of the Schiff bases were recorded in CDCl3. The spectra display two sharp signals at 2.43–2.52 and 3.16–3.24 ppm with an integration equivalent to three protons corresponding to the N-CH3 and C-CH3 groups. The aromatic ring protons were appeared at 6.69-8.75 ppm range. The azomethine proton for the Schiff bases was resonated at 9.55–9.80 ppm as a sharp singlet. The 1H-NMR spectral data are provided in Table 3. 3.3. Characterization of Platinum-Schiff Base Complexes The platinum complexes are air stable. All the complexes were colored, soluble in DMSO, DMF, THF and CHCl3. The analytical and physical characterizations of the complexes are given in Table 6. The analytical data show that the metal to ligand ratio was 1:1 in all the complex systems. The composition of the complexes is [Pt(Ln)Cl2], where Ln is the Schiff base ligand. The mass spectra of the Platinum complexes [Pt(L1)Cl2], [Pt(L2)Cl2], [Pt(L3)Cl2], [Pt(L4)Cl2], [Pt(L5)Cl2] show molecular ion peaks at m/z 601 (M+, 31%), 600 (M+, 43%), 634 (M+, 37%), 602 (M+, 28%)], and 602 (M+, 33%) respectively, which coincide with the formula weights of the Schiff base complexes. The low molar conductance values of the metal complexes reveal their non-electrolytic nature. 3.4. 1H NMR Spectra

The 1H NMR spectra of the platinum complexes were recorded at room temperature in CDCl3 solution and is shown in the Table 4. Compared to the Schiff base ligands, the platinum complexes show a slight upfield shift of 0.01–0.23 ppm in the resonance peaks of the aromatic ring. The azomethine proton in the complexes shows a downfield shift of 0.10-0.29 ppm. The shielding observed indicates the coordination of nitrogen atom to Pt(II) ion. 3.5. IR Spectra The IR spectra provide valuable information about the coordinating sites of ligands. The changes that might have taken place during the complexation can be determined by comparing the IR spectra of the complexes with those of the free ligands. Only important peaks, which have been either shifted or newly appeared, are discussed. The bands at 1591-1610 cm-1 for the azomethine group of free ligands were shifted to lower frequency in the range ~1572-1588 cm-1 in the complexes is indicative of the coordination of the azomethine nitrogen atom to the metal ion. The peaks in the region 1647-1650 cm-1 for the carbonyl stretching frequency of the Schiff base ligands were shifted to lower frequency in the range ~1620-1635 cm-1. This indicates the coordination of carbonyl oxygen to the metal ion. Coordination of the Schiff bases ligand to the metal through the azomethine nitrogen or carbonyl oxygen atom was expected to reduce the electron density in the azomethine or carbonyl link and to lower the vibration [13]. The spectrum of all the metal complexes show new bands in the 415–480 cm-1 and 405-430 cm-1 regions, which may probably be due to the formation of M-N and M-O bonds respectively [24]. 3.6. Electronic Spectra Based on the positions and number of d-d transition peaks, the electronic spectra are used for assigning the steriochemistries of metal ions in the complexes. From the data, it was found that all the complexes were of square planar geometry. Electronic spectra of the ligands and their

complexes were recorded in CH2Cl2 solution. The wide range bands were observed due to either the π-π* and n-π* of azomethine and carbonyl chromophores or charge transfer transition arising from π electron interactions between the metal and ligands, which involved either ligand to metal or metal to ligand electron transfer. Substitution on the phenyl ring in the Schiff base can influence the position of the charge transfer band. The electronic spectra of the free ligand showed strong absorption band in the region 203-390 nm. It could be due to the π-π* and n-π* transitions in the benzene ring or azomethine and carbonyl groups [13]. The absorption bands slightly changed and some additional bands also were present in the spectra of the metal complexes (203-525 nm). This is due to the metal to ligand charge transfer. The absorption shift and intensity change in the spectra of the metal complexes most likely originated from the metallation, increased the conjugation and delocalization of the whole electronic system resulting in the energy change of the π-π* and n-π* transitions of the conjugated chromophore [25]. 3.7. Powder XRD Powder XRD patterns of the platinum complexes were recorded over 2θ = 0-80 range. From the data, it was observed that [Pt(L2)Cl2], [Pt(L3)Cl2], and [Pt(L4)Cl2] complexes did not exhibit well defined crystalline peaks indicating their amorphous nature. Only [Pt(L1)Cl2] complex showed sharp peaks indicating its crystalline nature. The average crystalline sizes of the complexes were calculated using Scherrer’s formula [26]. The complex had an average crystallite size of 29 nm, suggesting that to be nanocrystalline. 4. Biological Studies 4.1. Antimicrobial Activity The results of the antimicrobial activities are summarized in Table 5. The standard error for the experiment is ± 0.001 cm-1 and the experiment was repeated three times under similar

conditions. DMSO was used as a negative control and amikacin, ofloxacin, and ciprofloxacin were used as positive standards for antibacterial studies. Nystatin was used as a reference for antifungal studies. These compounds exhibit moderate to strong antimicrobial activity. Comparatively a better activity was found for the bacteria rather than the fungi. The platinum complexes exhibit a higher activity than the free ligand towards bacteria as well as fungal species. Again the platinum complexes show equal or better activity compared to the negative controls such as amikacin, ofloxacine, and ciprofloxacin. The platinum complex with bromo substituted Schiff base shows better activity compared to the other platinum complexes. The antimicrobial activity of the Schiff base ligand was very poor but that of the platinum complexes was greater than those of the free ligand, this indicates that the complexation to metal enhances the activity of the ligand. This is explained on the basis of Overtone’s concept and chelation theory [27]. Chelation tends to make the ligand a more powerful and potent bacterial agent. In a chelated complex, the positive charge of the metal is partially shared with donor atoms present in the ligands and there is an electron delocalization over the whole chelated ring. This, in turn, increases the lipoid layers of the bacterial membranes. Generally, it is suggested that the chelated complexes deactivate various cellular enzymes, which play a vital role in various metabolic pathways of these microorganisms. Other factors such as solubility, conductivity, and dipole moment are also being the possible reasons for increasing the biological activity of the metal complexes. Platinum complexes have an ability to bind the DNA of the cell; this may also be a reason for the better activity towards the organisms. In our case, the platinum complex [Pt(L3)Cl2] shows a strong activity, especially against the Gram-negative bacteria such as E. coli. and B. subtilis. There are certain Gram-negative organisms that proved difficult to be treated [28]. It is therefore believed that all the complexes which are biologically active against the

Gram-negative strains may have something to do with the barrier function of the envelope of these Gram negative strains [29,30]. Thus the compounds reported by us may possess a possible antitumour effect [31]. 4.2. In vitro Anti Cancer Activity The reliable criteria for judging the efficacy of any anticancer drug are prolongation of life span, improving the clinical, haematological, biochemical profile, and reduction in viable tumour cell count in the host [32]. In order to evaluate the biological effects of the Schiff base ligands and their platinum complexes on cancer cells, we used the compounds to treat HeLa (Human Cervical Cancer Cells), HCT116 (Colon Cancer Cells), and A431(Epidermoid Carcinoma Cells) at the concentrations of 6.25, 12.5, 25, 50, and 100 µM for 48 h. The untreated cells were used as a control. Cell growth inhibition was analyzed by MTT assay and the results showed that the complexes and the ligands exhibited an inhibitory effect on the proliferation of HeLa, HCT116 and A431 cells in a dose-dependent manner (Fig. 3a, 3b, and 3c). The Schiff base ligands have very low activity against all the three type of cancer cells, the IC50 (Table 6) value is above 100 µM. Platinum complexes shows better activity compared to the free ligand. Among them, [Pt(L2)Cl2] and [Pt(L3)Cl2] complexes show the most potent inhibitory effect on the growth of all the cells compared to the remaining platinum complexes and the free ligand. The IC50 values in the platinum complexes for HeLa cancer cells are better than the reported values (82-209 µM) exhibited by some Ru(II) complexes with benzo[i]dipyrido[3,2-a:2’,3’-c] phenazine and a few more neutral ligands [33]. The IC50 values for our metal complexes and the free ligand against HCT116 cancer cells show moderate activity compared to the IC50 value of the clinicaly used drug such as etoposide (29.6 µM) [34]. The activity of the metal complexes and the free ligand towards HeLa cancer cells are not much significant, compared to the known metal-free

anticancer agents such as estramustine (IC50 ∼1.5 to 3.0 µM ), [35] noscapine (IC50 ∼22 µM) [36] as well as metal-bound anticancer reagents such as cisplatin (IC50 ∼8 µM) [37]. The activity of the metal complexes and the free ligand towards A431 cancer cells are also not much significant compared to the clinically used drugs. From the IC50 values of platinum complexes on both the cancer cells, it is understood that these complexes are more active on HCT116 cancer cells than on the HeLa cancer cells. 4.3. Anti-tubercular activity Resazurin microtiter assay (REMA) was used for the determination of antimycobacterial activity. The results indicate that all these Schiff base ligands have no inhibition against M. tuberculosis H37Rv growth upto 100 µM concentration. The reported Schiff base and platinum complexes have no significant activity compared to the commercial drugs. At 100 µM all the platinum complexes inhibit the growth of tubercular bacteria. The complex [Pt(L3)Cl2] show better activity compared to the other complexes and the free ligand. This complex starts to inhibit the growth of the organism at 50 µM concentration. The results are tabulated in Table 7. 5. Conclusion Platinum complexes with the Schiff base ligands derived from 4-aminoantipyrine and substituted aldehydes were synthesized and characterized by spectral analysis. The structure of the Schiff base ligand L5 has been verified by its single crystal XRD. The coordination of the Schiff base to the metal atom was found to be through the azomethine nitrogen and the carbonyl oxygen. The geometry of the complexes is assigned as square planar. The antimicrobial studies reveal that the complexes show higher activity than the ligand. The complex [Pt(L3)Cl2] shows better activity towards gram negative bacteria such as E-coli and B. subtilis compared to the other complexes. The complex [Pt(L3)Cl2] show better activity compared to the other complexes

and the free ligand towards M. tuberculosis H37Rv. In vitro anticancer activity is not significant compared to the known anticancer agents such as estramustine, noscapine, and cisplatin [38]. The [Pt(L3)Cl2] complex is more active than the other two complexes and the free ligand on all the three cancer cells (HeLa, HCT116, and A431). Acknowledgements One of the authors (C. Shiju) thanks Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, for the biological studies.

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Figure caption’s

Scheme 1

Synthetic route for Schiff base ligands

Scheme 2

Synthetic route for Schiff base-platinum complexes

Fig. 1

ORTEP Diagram of (E)-4- [4-Nitrobenzylideneamino]-1,5-dimethyl-2-phenyl-1H pyrazol-3(2H)-one

Fig. 2

Packing Structure of (E)-4- [4-Nitrobenzylideneamino]-1,5-dimethyl-2-phenyl1H-pyrazol-3(2H)-one

Fig. 3a

Growth inhibition based on concentration (HeLa)

Fig. 3b

Growth inhibition based on concentration (HCT116)

Fig. 3c

Growth inhibition based on concentration (A431)

Synthetic route for Schiff base ligands

Synthetic route for Schiff base-platinum complexes

Fig. 1. ORTEP Diagram of (E)-4- [4-Nitrobenzylideneamino]-1,5-dimethyl-2-phenyl-1Hpyrazol-3(2H)-one

Fig. 2. Packing Structure of (E)-4- [4-Nitrobenzylideneamino]-1,5-dimethyl-2-phenyl-1Hpyrazol-3(2H)-one

Fig. 3a. Growth inhibition based on concentration (HeLa)

Fig. 3b. Growth inhibition based on concentration (HCT116)

Fig. 3c. Growth inhibition based on concentration (A431)

Table 1. Crystal data and structure refinement for L5 Compound CCDC Empirical formula Formula weight Temperature Wavelength Crystal system Space group Unit cell dimensions

Volume Z Density (calculated) Absorption coefficient F(000) Crystal size Theta range for data collection Index ranges Reflections collected Independent reflections Completeness to theta = 27.50° Absorption correction Max. and min. transmission Refinement method Data / restraints / parameters Goodness-of-fit on F2 Final R indices [I>2sigma(I)] R indices (all data) Largest diff. peak and hole

L5 917904 C18 H16 N4 O3 336.35 110(2) K 0.71073 Å Triclinic P-1 a = 7.032(2) Å b = 9.479(3) Å c = 12.425(4) Å 795.0(4) Å3

α= 101.636(3)°. β= 99.633(3)°. γ = 94.040(3)°.

2 1.405 Mg/m3 0.099 mm-1 352 0.38 x 0.26 x 0.09 mm3 2.21 to 27.50°. -9100

96

>100

>100

79

75

69

>100

92

98

>100

>100

>100

84

88

>100

>100

>100

L5

>100

91

>100

76

87

>100

>100

[Pt(L1)Cl2]

11

9

10

7

14

18

12

[Pt(L2)Cl2]

9

10

15

9

10

13

10

[Pt(L3)Cl2]

4

5

7

4

8

8

11

[Pt(L4)Cl2]

12

13

15

9

12

14

17

[Pt(L5)Cl2]

10

9

12

10

10

12

13

Amikacina

05

05

04

05

-

-

-

Ciprofloxacinb

05

05

05

05

-

-

-

Ofloxacinc

10

2.5

2.5

05

-

-

-

Nystatind

-

-

-

-

06

06

05

a, b, c, d

Standard

Table 6. IC50 values of the compounds on the cancer cells Compound

IC50 (µM) HCT116

HeLa

A431

L1

>100

>100

>100

L2

>100

>100

>100

L3

>100

>100

>100

L4

>100

>100

>100

L5

>100

>100

>100

[Pt(L1)Cl2]

29.4

34.8

31.89

[Pt(L2)Cl2]

28.2

35.7

26.03

[Pt(L3)Cl2]

21.5

24.8

22.87

[Pt(L4)Cl2]

32.8

36.3

34.94

[Pt(L5)Cl2]

30.5

32.4

30.77

Table 7. Antimycobacterial activity of the compounds Compound L1 L2 L3 L4 L5 [Pt(L1)Cl2] [Pt(L2)Cl2] [Pt(L3)Cl2] [Pt(L4)Cl2] [Pt(L5)Cl2]

100µg/mL N N N N N P P P N P

N = No inhibition P = Inhibition

50µg/mL N N N N N N N P N N

25µg/mL N N N N N N N N N N

12.5µg/mL N N N N N N N N N N

6.25µg/mL N N N N N N N N N N

Highlights
The platinum complexes of Schiff base ligands were synthesized and characterized
The structure of one of the ligands was confirmed by a single crystal XRD analysis
The geometrical structures of these complexes are found to be square planar
Antimicrobial studies indicate that these complexes exhibit better activity than the ligands
[Pt(L3)Cl2] shows better activity towards M. tuberculosis H37Rv