As received grade chemicals used during this study were procured from Sigma; the solvents were dried as reported [14]. C, H, and N analyses were carried out on a Yanaco high-speed CHN analyzer; antipyrene was used as a reference, while antimony was estimated according to the reported procedure [15]; melting points were recorded on Gallenkmp capillary melting point apparatus and are uncorrected. FTIR spectra of all the compounds were taken on Bruker FTIR spectrophotometer TENSOR27 using OPUS software in the range of 5000-400 cm-1 (ZnSe). 1H and 13C NMR spectra in DMSO were recorded on a multinuclear Avance 300 and 75 MHz FT NMR spectrometer operating at room temperature, i.e., 25 C. Thermoanalytical measurements were carried out using a Perkin Elmer Thermogravimetric/differential thermal analyzer (YRIS Diamond TG-DTA High Temp. Vacu.) consuming variable heating rates between 0.5°C/min and 50°C/min. HR FAB-MS spectra were obtained from a double-focusing mass spectrometer Finnigan (MAT 112).
Synthesis of ligands
Phthalic anhydride (5 g, 33.77 mM) was dissolved in acetic acid (100 mL), and a cold solution of amino acid (33.77 mM, i.e. 2.53 g, 3 g, 5.58 g, and 4.63 g of glycine, β-alanine, L-phenylalanine, and 4-aminobenzoic acid, respectively) in acetic acid (75 mL) was added to it. This mixture was stirred at room temperature for 3 hours resulting in white precipitate. The white precipitate was washed several times with cold water and recrystallized from water.
Synthesis of I
Yield: 72%. C10H9NO5: Calcd. (%): C 53.82, H 4.06, and N 6.28; Found (%): C 53.27, H 3.86, N 6.01; FAB-MS (m/z) 224 (M + 1); IR ν 3293 (N-H), 1684 (C-N), 1592 (CO2)as, and 1353 (CO2)s, Δν (CO2): 239 cm-1. 1H NMR (DMSO-d6, 300 MHz) 12.8 (s, COOH), 12.1 (s, COOH), 8.31 (s, NH), 7.02-7.61 (Ar), and 3.62 (s, CH2). 13C NMR (DMSO-d6, 75 MHz) 174.7 (COOH), 170.2 (CONH), 168.9 (COOH), 107-138 (Ar), and 44.6 (CH2).
Synthesis of II
Yield: 67%. C11H11NO5: Calcd. (%): C 55.70, H 4.67, N 5.90; Found (%): C 55.24, H 4.03, N 5.42. FAB-MS (m/z) 238 (M + 1). IR ν 3372 (N-H), 1670 (C-N), 1581 (CO2)as, 1345 (CO2)s, Δν (CO2): 236 cm-1. 1H NMR (DMSO-d6, 300 MHz) 12.7 (s, COOH), 12.5 (s, COOH), 8.39 (s, NH), 7.07-7.59, (m, Ar) 3.51 (t, CH2 J: 3.42), 2.33 (t, CH2 J: 4.1). 13C NMR (DMSO-d6, 75 MHz) 173.2 (COOH), 168.8 (COOH), 142.4 (CONH), 109-140 (Ar), 40.4 (CH2), 35.2 (CH2).
Synthesis of III
Yield: 80%. C17H15NO5: Calcd. (%): C 65.17, H 4.83, N 4.47; Found (%): C 64.86, H 4.32, N 4.11. FAB-MS (m/z) 314 (M + 1). IR ν 3380 (N-H), 1686 (C-N), 1577 (CO2)as, 1361 (CO2)s, Δν (CO2): 216 cm-1. 1H NMR (DMSO-d6, 300 MHz) 12.6 (s, COOH), 12.2 (s, COOH), 8.43 (s, NH), 7.02-7.51 (m, Ar), 5.06 (q, CH, J: 8.8), 3.4 (d, CH2, J: 10.1). 13C NMR (DMSO-d6, 75 MHz) 171.4 (COOH), 170.0 (COOH), 144.1 (CONH), 111-138 (Ar), 61.2 (CH), 36.1 (CH2).
Synthesis of IV
Yield: 70%. C15H11NO5: Calcd. (%): C 63.16, H 3.89, N 4.91; Found (%): C 63.02, H 3.43, N 4.60. FAB-MS (m/z) 286 (M + 1). IR ν 3388 (N-H), 1672 (C-N), 1566 (CO2)as, 1371 (CO2)s, Δν (CO2): 195 cm-1. 1H NMR (DMSO-d6, 300 MHz) 12.3 (s, COOH), 11.8 (s, COOH), 8.52 (s, NH), 7.13-8.33 (m, Ar). 13C NMR (DMSO-d6, 75 MHz) 176.7 (COOH), 165.4 (COOH), 148.2 (CONH), 120-136 (Ar).
Synthesis of antimony complexes
Aqueous solution of SbCl3 was made by dissolving 0.5 g (2.19 mM) in 10 mL, and a few drops of dil. HCl were added; to this solution, equimolar amount of ligand 2.19 mM, i.e. 0.48 g, 0.52 g, 0.69 g, and 0.62 g, respectively, for I-IV dissolved in ethanol (20 mL). The mixture was stirred at room temperature for 15 min, for adjustment of pH, and one drop of ammonia was added which resulted in the formation of a precipitate. The precipitate was filtered and washed with warm 70% ethanol and recrystallized from water.
Synthesis of V
Yield: 58%. C10H7ClNO5Sb: Calcd. (%): C 31.74, H 1.86, N 3.70, Sb 32.18; Found (%): C 31.21, H 1.45, N 3.39, Sb 31.80. FAB-MS (m/z) 377, 379 (M + 2). IR ν 3231 (N-H), 1655 (C-N), 1561 (CO2)as, 1320 (CO2)s, Δν (CO2): 241, 450 (N → Sb), 574 (O-Sb) cm-1. 1H NMR (DMSO-d6, 300 MHz) 8.24 (s, NH), 7.11-7.61 (m, Ar), 3.87 (s, CH2). 13C NMR (DMSO-d6, 75 MHz) 177.4 (CONH), 174.7 (COO), 170.2 (COO), 107-138 (Ar), 40.2 (CH2).
Synthesis of VI
Yield: 58%. C11H9ClNO5Sb: Calcd. (%): C 33.67, H 2.31, N 3.57, Sb: 31.03; Found (%): C 33.28, H 2.08, N 3.19, Sb: 30.67. FAB-MS (m/z) 391, 393 (M + 2). IR ν 3265 (N-H), 1643 (C-N), 1551 (CO2)as, 1302 (CO2)s, Δν (CO2): 249, 442 (N → Sb), 582 (O-Sb) cm-1. 1H NMR (DMSO-d6, 300 MHz) 12.7 (s, COOH), 12.5 (s, COOH), 8.39 (s, NH), 7.07-7.59, (m, Ar) 3.51 (t, CH2 J: 3.42), 2.33 (t, CH2 J: 4.1). 13C NMR (DMSO-d6, 75 MHz) 181.4 (CONH), 170.0 (COO), 160.1 (COO), 122-142 (Ar), 33.3 (CH2NH), 27.1 (CH2).
Synthesis of VII
Yield: 51%. C17H13ClNO5Sb: Calcd. (%): C 43.58, H 2.80, N 2.99, Sb 25.99; Found (%): C 43.20, H 2.50, N 2.67, Sb 25.34. FAB-MS (m/z) 467, 469 (M + 2). IR ν 3276 (N-H), 1666 (C-N), 1540 (CO2)as, 1311 (CO2)s, Δν (CO2): 229, 425 (N → Sb), 580 (O-Sb) cm-1. 1H NMR (DMSO-d6, 300 MHz) 8.24 (s, NH), 7.10-8.1 (m, Ar), 5.06 (t, CH, J: 9.7), 3.42 (d, CH2, J: 9.3). 13C NMR (DMSO-d6, 75 MHz) 180.6 (CONH), 174.0 (COO), 169.5 (COO), 125-136 (Ar), 66.8 (CH), 30.6 (CH2).
Synthesis of VIII
Yield: 58%. C15H9ClNO5Sb: Calcd.(%): C 40.90, H 2.06, N 3.18, Sb 27.64; Found (%):C 40.71, H 1.89, N 2.91, Sb 27.22. FAB-MS (m/z) 439, 441 (M + 2). IR ν 3266 (N-H), 1678 (C-N), 1541 (CO2)as, 1336 (CO2)s, Δν (CO2): 137, 446 (N → Sb), 568 (O-Sb) cm-1. 1H NMR (DMSO-d6, 300 MHz) 8.16 (s, NH), 7.06-8.55 (m, Ar). 13C NMR (DMSO-d6, 75 MHz) 183.2 (CONH), 170.4 (COO), 172.8 (COO), 123-137 (Ar).
Anti-leishmanial activity
Anti-leishmanial activity of the compound was carried out on the pre-established cultures of L. major (JISH118), L. major (MHOM/PK/88/DESTO), L. tropica (K27), L. infantum (LEM3437), L. mex mex (LV4) and L. donovani (H43). Parasites were cultured in medium M199 with 10% foetal bovine serum; 25 mM of HEPES, and 0.22 μg of penicillin and streptomycin, respectively at 24°C in an incubator. 1 mg of each test compound (I-VIII) was dissolved in 1 mL of water, ethanol, methanol and DMSO according to their solubilities. 1 mg of Amphotercin B was also dissolved in 1 mL of DMSO as reference drug. Parasites at log phase were centrifuged at 3000 rpm for 3 min. Parasites were diluted in fresh culture medium to a final density of 2 × 106 cells/mL. In 96-well plates, 180 μL of medium was added in different wells. 20 μL of the extracts was added in medium and serially diluted. 100 μL of parasite culture was added in all the wells. Four rows left for negative and positive controls: water, ethanol, methanol and DMSO, respectively, serially diluted in medium whereas positive control contained varying concentrations of standard antileishmanial compound, i.e. AmphotericinB. The plates were incubated for 72 h at 24°C. Results were analyzed through dose versus response by using nonlinear regression curve fit with Graphad Prims5.
Anti-fungal activity
Agar tube dilution method was used for screening antifungal activities against Aspergillus Flavus, Aspergillus Fumigants, Aspergillus Niger, and Fusarium Solani. A sample of Media supplemented with DMSO and reference antifungal drugs was used as negative and positive control, respectively. Tubes were then incubated at 27°C for 4-7 days and examined twice weekly during incubation. Standard drug, Miconazole, used for the above stated fungi, growth in media containing sample under test were determined by linear growth (mm) measuring, and percent inhibition of growth was calculated with reference to negative control using formula.