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Acta Pharm. 62 (2012) 221236 DOI: 10.2478/v10007-012-0017-y Original research paper BEATRIZ C. C. SOUZA1 TIAGO B. DE OLIVEIRA1 THIAGO M. AQUINO1 MARIA C. A. DE LIMA1 IVAN R. PITTA1 SUELY L. GALDINO1 EDELTRUDES O. LIMA2 TERESINHA GONÇALVES-SILVA1 GARDÊNIA C. G. MILITÃO1 LUCIANA SCOTTI2 MARCUS T. SCOTTI3 FRANCISCO J. B. MENDONÇA Jr.4 1 Departamento de Antibióticos Universidade Federal de Pernambuco Recife-PE 50670-910,Brazil 2 Departamento de Ciências Farmacêuticas Universidade Federal da Paraíba João Pessoa-PB, 58051-970, Brazil 3 Departamento de Engenharia e Meio Ambiente, Universidade Federal da Paraíba, Campus IV; Rio Tinto-PB 58297-000, Brazil A series of 2-[(arylidene)amino]-cycloalkyl[b]thiophene-3-carbonitriles (2a-x) was synthesized by incorporation of substituted aromatic aldehydes in Gewald adducts (1a-c). The title compounds were screened for their antifungal activity against Candida krusei and Criptococcus neoformans and for their antiproliferative activity against a panel of 3 human cancer cell lines (HT29, NCI H-292 and HEP). For antiproliferative activity, the partial least squares (PLS) methodology was applied. Some of the prepared compounds exhibited promising antifungal and proliferative properties. The most active compounds for antifungal activity were cyclohexyl[b]thiophene derivatives, and for antiproliferative activity cycloheptyl[b]thiophene derivatives, especially 2-[(1H-indol-2-yl-methylidene)amino]-5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carbonitrile (2r), which inhibited more than 97 % growth of the three cell lines. The PLS discriminant analysis (PLS-DA) applied generated good exploratory and predictive results and showed that the descriptors having shape characteristics were strongly correlated with the biological data. Keywords: cycloalkyl[b]thiophene derivatives, antifungal activity, antiproliferative activity, PLS-DA, Pentacle program 4 Departamento de Ciências Biológicas, Universidade Estadual da Paraíba CCBSA, João Pessoa-PB 58070-450, Brazil Accepted April 27, 2012 2-Aminothiophene derivatives are an important class of heterocycles found in several biologically active and natural compounds. This class of compounds has demonstrated a broad spectrum of activities and applications as pharmaceuticals and agrochemicals, dyes, biodiagnostics, and electronic and optoelectronic devices (1). * Correspondence; e-mail: email@example.com B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. Substituted 2-aminothiophenes are a class of heterocycles that have attracted a great deal of research interest due their great usefulness as precursors of molecules with pharmacological properties. They have been reported to exert antitubercular (2), anti-inflammatory (3), antimicrobial (4) and antianxiety (5) properties. A survey of the literature also reveals that substituted 2-aminothiophenes are potent and selective inhibitors of human leukocyte elastase (6), kinesin spindle protein (KPS) (7) and adenosine A1 receptor allosteric enhancers (8). Antifungal (9) and antitumor (10) properties have also been extensively described, resulting in marketed antifungal agents such as sertaconazole. The above prompted us to synthesize a new series of 2-[(arylidene)amino]-cycloalkyl[b]thiophene-3-carbonitrile derivatives by incorporating different substituted aromatic aldehydes at the 2-amino position of the thiophene ring, thereby affording Schiff bases. These were screened for their in vitro antifungal and antiproliferative activities. Partial Least Squares discriminant analysis (PLS-DA) was applied, using Pentacle, to the antiproliferative activity of cycloalkyl[b]thiophene derivatives. EXPERIMENTAL All melting points were measured on a Quimis-340.27 apparatus (Quimis, Brazil) and are uncorrected. IR spectra were recorded using potassium bromide pellets on a Bruker IFS-66 IR spectrophotometer (Bruker, USA). NMR were recorded on a Unity Plus-300 MHz-Varian spectrometer (Varian, USA) using tetramethylsilane as internal standard. Chemical shifts are reported in ppm (d), and coupling constants (J) are reported in Hz. HRMS were recorded on a Delsi-Nermag R1010C mass spectrometer (Delsi-Nermag, France) with 70 eV electron impact. Elemental analyses were performed using an EA 1110 CHNS-O elemental analyzer (CE instruments, UK). The results were found to be in accord (± 0.4 %) with the calculated values. All reactions were monitored by TLC on 0.25 mm silica gel plates (60F254, Merck, Germany) using binary mixtures of hexane/ethyl acetate in different proportions (from 9:1 to 1:1, V/V). Spectral, physical and analytical data of all newly synthesized compounds are listed in Tables IIII. General synthesis procedure 2-[(Arylidene)amino]-cycloalkyl[b]thiophene-3-carbonitriles (2a-x). An equimolar mixture of 1a-c and substituted aromatic aldehyde in ethanol with 0.5 mL of acetic acid was stirred under reflux for 2 h and then cooled to room temperature. Water was added and the solid that precipitated out was filtered under vacuum, washed with water, dried and recrystallized from absolute ethanol (Scheme 1). Antifungal activity The in vitro antifungal activity of synthesized compounds 2a-o, 2r-x was investigatedfor two species of pathogenic fungi: Candida krusei LM08 and Criptococcus neoformans ICB59. These strains were supplied by the URM Culture Collection of the Department of Mycology, Department of Pharmaceutical Sciences of the Federal University of Paraíba, Brazil. Minimum inhibitory concentration (MIC) values were determined by the micro222 B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. Scheme 1. dilution broth method using microdilution plates according to the guidelines of the National Committee for Clinical and Laboratory Standards (NCCLS). The MIC for all strains was set at the lowest concentration of the antifungal agent that completely inhibited the growth of the organism, as detected by the naked eye when compared to the control group. All the strains were stored in mineral oil at 18 °C. The viability test and subsequent taxonomic confirmation were carried out according to Barnett et al. (12). In order to obtain an inoculum of 2.5 ´ 103 cells mL1, each strain was cultured in a tube containing 20 mL of Sabouraud dextrose agar plus yeast extract at 35 °C for two days. After this time, suspensions were prepared in a sterile physiological solution (0.85 %) and mixed in a shaker. The inoculum was adjusted to 90 % transmittance at 530 nm, as measured on a spectrophotometer. Stock solutions of tested compounds were freshly prepared in dimethyl sulfoxide (DMSO), which had no effect on the organism at the concentration studied, aliquoted, and stored at 20 °C in stock solution at a final concentration of 1024 mg mL1. Decimal dilutions of thiophene stock solutions were prepared in free RPMI 1640 cell cul- B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. ture medium (Sigma, USA) and buffered to pH 7.0 with 0.165 mol L1 of morpholinopropanesulphonic acid (MOPS, Sigma, USA). Miconazole and 5-fluorocitosine were used as reference drugs for anti-Candida and anti-Cryptococcus activity, respectively, and were tested under similar conditions. Microdilution plates containing serial dilutions (from 1024 to 1 µg mL1) of each compound were inoculated with each organism. Each plate included a positive control (fungi without any compound), negative control (medium only) and reference drugs. The microdilution plates were incubated at 35 °C and were read visually after 24 and 72 h of incubation. All tests were performed in duplicate and the results were expressed as the arithmetic mean of MIC values obtained in the two trials (13). The bands established for antifungal activity were: excellent activity (50 to 500 mg mL1); moderate activity (600 to 1000 mg mL1); inactive or poor activity (1000 mg mL1) (14). Antiproliferative activity All synthesized compounds were selected by the National Cancer Institute (NCI) Developmental Therapeutic Program for in vitro cell line screening. Antiproliferative assays were performed according the US NCI protocol as described elsewhere (15). The compounds were evaluated in one primary dose (25 mL mL1) for three human cancer cell lines: colon carcinoma (HT29), lung cancer (NCI H-292) and laryngeal carcinoma (HEP) cells. The cells lines were supplied by the Rio de Janeiro Cell Bank of the Federal University of Rio de Janeiro (UFRJ), Brazil. The cytotoxicity/survival of cells in the presence or absence of the experimental agent was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) method, as described previously (16). Cells harvested in the log phase of growth were counted and seeded in triplicate (105 cells per 100 mL per well) in 96-well microculture plates in a complete free Dulbecco's modified eagle's medium (DMEM). After 24 h of incubation at 37 °C and 5 % CO2, the cultures were treated with the tested compounds at a single concentration of 25 mg mL1 in DMSO (100 mL per well). Each plate also included a positive control (cells without any compound) and a negative control (medium only). After 72 h of exposure to the compounds, 25 mL of MTT was added to each well. After 4 h at 37 °C, the reaction was stopped and the formazan crystals formed by MTT metabolism were solubilized by additing 50 mL of DMSO to each well. The cellular metabolism of MTT was quantified by reading the absorbance of the solubilized product at 595 nm with a 96-well plate reader attached to a spectrophotometer. Results for each tested compound were reported as the percentage growth of the treated cells compared to the untreated control cells. The mean ± SD of three independent experiments for each compound was calculated. The compounds were classified as possessing no activity (0 % growth inhibition), low activity (up to 30.0 % growth inhibition), moderate activity (between 31.0 % and 70.0 % growth inhibition) and high activity (> 70.0 % growth inhibition) for each cell line tested. Computational methods All compounds were drawn (2D structures) using ChemDraw 8.0. They were imported by the Spartan program for Windows 8.0 and were converted into 3D models. B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. These models were minimized using the MM+ method (17) and atomic partial charges were assigned using the AM1 semiempirical method (18). These methods employed 1000 interactions, 100 cycles of optimization, and 10 conformers of lowest minimum energy. The selected dihedrals were evaluated by rotation in accordance with the standard (default) conditions of the program, in which the number of simultaneous variations was 1 to 8. Acyclic chains were submitted to rotations from 60 to 180o and torsion rings were in the range of 30 to 120o. Conformational search was applied and the EMIN conformer was selected and saved in sdf format (19). The compounds were imported using the Pentacle program and the PLS methodology was applied. The Pentacle software, produced by Molecular Discovery (Italy), is a computational tool for computing alignment-free molecular descriptors, also called GRid-INDependent descriptors or GRIND (20). The software is based on Molecular Interaction Fields and describes the ability of the molecules to interact with other molecules and does not require superimposition of compounds. The Pentacle program uses the GRID force field to characterize potential polar and hydrophobic interaction sites around target molecules by water (H2O), hydrophobic (DRY) and carbonyl oxygen (O) and amide nitrogen (N1) probe. We calculated a binary free PLS-DA for the three cell lines investigated: HT29, NCI H-292 and HEP. The PLS-DA is a partial least squares regression of a set (Y) of binary variables describing the categories of a category variable on a set (X) of predictor variables. PLS-DA looks for components correlated with the unfolded class while describing a large number of variations in X, i.e., directions able to discriminate between the classes. It is a compromise between the usual discriminant analysis and a discriminant analysis of the significant principal components of predictor variables. For the construction of binary PLS matrices the, compounds which showed cytotoxicity greater than 20 % were considered active = 1, and the compounds that did not reach 20 % inactive = 0. RESULTS AND DISCUSSION Chemistry 2-Amino-cycloalkyl[b]thiophene-3-carbonitriles (1a-c) were first synthesized by the reaction of malononitrile with a cyclic ketone and elemental sulphur in the presence of morpholine following the Gewald procedure (9, 10). Treatment of 1a-c with substituted aromatic aldehydes afforded 2-[(arylidene)amino]-cycloalkyl[b]thiophene-3-carbonitrile (2a-x). The Schiff bases were prepared in satisfactory yield (except 2q). Compounds 2a-g and 2j were also previously described in the literature (9). Chemical structures of all newly synthesized compounds (2h, 2i and 2k-x) were characterized on the basis of their physical, analytical and spectral data, and were in full agreement with the proposed structures (Tables IIII). The main vibrational bands of the title compounds are given in Table III. IR spectra showed absorption bands at about 29602911 and 22232212 cm1, characteristic of C-H and CºN stretching vibrations, respectively. 1H NMR and 13C NMR data of the compounds obtained are given in Table II and are consistent with the proposed structures. 1H NMR spectra did not display signs of 2225 B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. Table I. Physical and analytical data of 2h, i, k-x Molecular formula (Mr) C17H15BrN2S C16H13FN2S C23H20N2OS C17H15N3O2S C18H18N2S C19H20N2S C20H22N2S C20H21N3OS C20H21N3S C19H17N3S C18H13N3S C19H15N3S C20H17N3S C13H10N2S2 C14H12N2S2 C15H14N2S2 Analysis (calcd./found) (%) C 56.83/56.83 67.58/67.55 74.16/74.14 62.75/62.71 73.43/73.44 73.99/73.98 74.49/74.48 68.35/68.34 71.61/71.61 71.44/71.40 71.26/71.25 71.90/71.87 72.48/72.44 60.43/60.41 61.73/61.70 62.90/62.89 N 7.80/7.77 9.85/9.85 7.52/7.50 12.91/12.88 9.51/9.48 9.08/9.07 8.69/8.65 11.96/11.95 12.53/12.51 13.15/13.12 13.85/13.85 13.24/13.21 12.68/12.67 10.84/10.80 10.28/10.25 9.78/9.77 H 4.21/4.23 4.61/4.62 5.41/5.43 4.65/4.66 6.16/6.19 6.54/6.54 6.88/6.91 6.02/6.03 6.31/6.34 5.36/5.40 4.32/4.33 4.76/4.80 5.17/5.19 3.90/3.92 4.44/4.46 4.93/4.95 Compd. 2h 2i 2k 2l 2m 2n 2o 2p 2q 2r 2s 2t 2u 2v 2w 2x M. p. (°C) 144 9395 140142 126 8890 9495 9395 159160 211 180182 200202 175177 204206 174175 123125 115117 Yield (%) 87 65 92 90 92 84 70 74 18 90 67 53 79 81 88 76 -aminothiophene protons (NH2), but rather CH=N protons, highlighting the success of the final synthetic step in obtaining the Schiff bases. Signals of the CH=N protons were observed as a singlet in the 8.028.95 ppm region, almost always as the stronger deshielding signal. The strongest shielding signals and peaks in 1H NMR and 13C NMR were attributed to cycloalkyl protons and carbons. These appeared at 1.622.99 ppm in 1H NMR, and at 21.832.0 ppm in 13C NMR. Further support was obtained from the 13C NMR spectra, which exhibited resonance at 114.0115.9 ppm attributed to the CºN group. The HRMS of all synthesized compounds exhibited (M+H)+ molecular ion peaks, which is in agreement with the molecular formulas. For compounds 2v and 2x, the molecular ion peaks found were [M+Na]+. Antifungal activity All synthesized compounds (except 2p and 2q) were evaluated for their in vitro antifungal activity against Criptococcus neoformans and Candida krusei. The data presented in Table IV show that the tested compounds more efficiently inhibited the growth of C. neoformans than the C. krusei strain. The MIC values ranged from excellent (50500 mg mL1) to moderate (6001500 mg mL1) for half of the tested compounds, although none exhibited greater antifungal activity than the reference drugs 5-fluorocytosine and miconazole. B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. Table II. NMR data for 2h, i, k-x Compd. 2h 1H NMR (d ppm)a 13C NMR (d ppm)a 1.641.74 (m, 4H, CH2), 1.841.88 (m, 2H, CH2), 2.762.81 (m, 4H, CH2), 7.58 (d, 2H, J = 8.7 Hz, Ar-H), 7.78 (d, 2H, J = 8.7 Hz, Ar-H), 8.34 (s, 1H, CH=N) 1.841.90 (m, 4H, CH2), 2.632.70 (m, 4H, CH2), 7.12 (d, 2H, J = 8.7 Hz, Ar-H), 7.91 (d, 2H, J = 8.7 Hz, Ar-H), 8.36 (s, 1H, CH=N) 1.731.89 (m, 4H, CH2), 2.622.69 (m, 4H, CH2), 5.12 (s, 2H, OCH2), 7.03 (d, 2H, J = 8.7 Hz, Ar-H), 7.347.46 (m, 5H, Ar-H), 7.87 (d, 2H, J = 8.7 Hz, Ar-H), 8.32 (s, 1H, CH=N) 1.661.75 (m, 4H, CH2), 1.851.89 (m, 2H, CH2), 2.792.82 (m, 4H, CH2), 8.06 (d, 2H, J = 8.7 Hz, Ar-H), 8.28 (d, 2H, J = 8.7 Hz, Ar-H), 8.43 (s, 1H, CH=N) 1.69 (m, 4H, CH2), 1.86 (m, 2H, CH2), 2.41 (s, 3H, CH3), 2.78 (m, 4H, CH2), 7.25 (d, 2H, J = 8.1 Hz, Ar-H), 7.81 (d, 2H, J = 8.1 Hz, Ar-H), 8.38 (s, 1H, CH=N) 1.26 (t, 3H, J = 7.5 Hz, CH3), 1.641.74 (m, 4H, CH2), 1.841.88 (m, 2H, CH2), 2.73 (q, 2H, J = 7.5 Hz, CH2), 2.752.81 (m, 4H, CH2), 7.28 (d, 2H, J = 8.1 Hz, Ar-H), 7.84 (d, 2H, J = 8.1 Hz, Ar-H), 8.39 (s, 1H, CH=N) 1.72 (d, 6H, J = 6.9 Hz, CH3), 1.631.73 (m, 4H, CH2), 1.831.91 (m, 2H, CH2), 2.752.81 (m, 4H, CH2), 2.96 (d, 1H, J = 6.9 Hz, CH), 7.31 (d, 2H, J = 8.1 Hz, Ar-H), 7.84 (d, 2H, J = 8.1 Hz, Ar-H), 8.38 (s, 1H, CH=N) 1.801.88 (m, 4H, CH2), 2.612.67 (m, 4H, CH2), 3.30 (t, 4H, J = 4.8 Hz, NCH2), 3.84 (t, 4H, J = 4.8 Hz, OCH2), 6.88 (d, 2H, J = 9 Hz, Ar-H), 7.80 (d, 2H, J = 9 Hz, Ar-H), 8.26 (s, 1H, CH=N) 1.85 (m, 4H, CH2), 2.07 (m, 4H, CH2), 2.66 (m, 4H, CH2), 3.43 (m, 4H, CH2), 6.62 (d, 2H, J = 8.4 Hz, Ar-H), 7.94 (d, 2H, J = 8.4 Hz, Ar-H), 8.30 (s, 1H, CH=N) 1.651.72 (m, 4H, CH2), 1.841.88 (m, 2H, CH2), 2.752.82 (m, 4H, CH2), 7.297.42 (m, 4H, Ar-H), 7.67 (d, 1H, J = 3 Hz, Ar-H), 8.60 (s, 1H, CH=N) 157.1, 140.3, 136.7, 133.9, 132.1, 130.5, 126.8, 114.9, 109.7, 31.9, 30.6, 29.1, 27.7, 27.0 166.8, 163.5, 159.4, 157.3, 135.1, 132.5, 131.5, 131.4, 116.2, 115.9, 114.4, 107.0, 25.1, 24.2, 23.0, 21.9 162.1, 160.4, 158.2, 136.0, 134.7, 131.5, 131.3, 128.6, 128.1, 128.1, 127.4, 115.1, 114.6, 105.8, 70.0, 25.0, 24.2, 23.0, 21.9 155.8, 155.0, 149.3, 140.9, 140.4, 138.6, 129.6, 123.9, 114.5, 111.4, 31.8, 30.7, 29.0, 27.6, 26.9 158.8, 158.1, 143.0, 139.9, 135.7, 132.5, 129.5, 129.3, 115.0, 108.7, 31.9, 30.5, 29.1, 27.7, 27.1, 21.7 158.8, 158.1, 149.3, 139.9, 135.7, 132.7, 129.5, 128.4, 115.0, 108.7, 31.9, 30.5, 29.1, 29.0, 27.8, 27.1, 15.2 158.7, 158.1, 153.8, 139.9, 135.6, 132.8, 129.4, 126.9, 115.0, 108.7, 34.2, 31.9, 30.5, 29.0, 27.7, 27.0, 23.6 161.1, 158.3, 153.7, 134.5, 131.1, 130.8, 125.6, 114.9, 113.9, 105.0, 66.4, 47.4, 25.0, 24.2, 23.0, 21.9 2i 2k 2l 2m 2n 2o 2p 2q 198.3, 190.2, 158.3, 151.6, 134.5, 133.1, 115.0, 112.3, 111.2, 102.8, 48.0, 47.6, 25.4, 25.3, 24.9, 24.3, 23.1, 22.0 160.5, 153.4, 139.3, 136.9, 133.5, 132.1, 124.8, 124.2, 123.0, 122.7, 116.0, 115.7, 111.3, 106.7, 32.0, 30.5, 29.1, 27.9, 27.2 2r B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. Table II. continued 2s 2.48 (q, 2H, J = 7.6 Hz, CH2), 2.91 (t, 2H, J = 7.6 Hz, CH2), 2.99 (t, 2H, J = 7.6 Hz, CH2), 7.79 (t, 1H, J = 7.6 Hz, Ar-H), 7.87 (t, 1H, J = 7.6 Hz, Ar-H), 8.08 (d, 1H, J = 4.8 Hz, Ar-H), 8.40 (d, 1H, J = 8.4 Hz, Ar-H), 8.95 (d, 1H, J = 8.4 Hz, Ar-H), 8.02 (s, 1H, CH=N), 9.03 (d, 1H, J = 4.8 Hz, Ar-H) 1.821.88 (m, 4H, CH2), 2.652.74 (m, 4H, CH2), 7.91 (d, 2H, J = 6 Hz, Ar-H), 8.90 (t, 2H, J = 11.2 Hz, Ar-H), 8.95 (s, 1H, CH=N), 9.02 (d, 2H, J = 6 Hz, Ar-H) 1.691.72 (m, 4H, CH2), 1.88 (m, 2H, CH2), 2.792.82 (m, 4H, CH2), 7.667.79 (m, 2H, Ar-H), 7.84 (d, 1H, J = 8.4 Hz, Ar-H), 8.17 (d, 1H, J = 4.5 Hz, Ar-H), 8.90 (d, 1H, J = 8.4 Hz, Ar-H), 8.93 (s, 1H, CH=N), 9.00 (d, 1H, J = 4.5 Hz, Ar-H) 2.362.45 (m, 2H, CH2), 2.802.92 (m, 4H, CH2), 6.81 (dd, 1H, J = 1.5, 3.6 Hz, Ar-H), 7.11 (d, 1H, J = 3.6 Hz, Ar-H), 7.64 (d, 1H, J = 1.5 Hz, Ar-H), 8.25 (s, 1H, CH=N) 1.821.91 (m, 4H, CH2), 2.632.69 (m, 4H, CH2), 6.58 (dd, 1H, J = 1.5, 3.3 Hz, Ar-H), 7.11 (d, 1H, J = 3.3 Hz, Ar-H), 7.65 (d, 1H, J = 1.5 Hz, Ar-H), 8.23 (s, 1H, CH=N) 163.5, 154.3, 150.0, 149.0, 145.3, 140.1, 137.1, 130.2, 129.7, 128.3, 125.2, 123.9, 122.1, 114.3, 104.7, 30.2, 28.0, 27.3 2t 158.4, 155.5, 150.0, 149.0, 137.1, 135.8, 134.8, 130.2, 129.7, 128.2, 125.2, 123.9, 122.1, 114.0, 109.2, 25.3, 24.2, 22.9, 21.8 156.3, 155.1, 149.8, 148.8, 140.8, 138.7, 137.2, 130.0, 129.7, 128.2, 125.2, 123.9, 122.0, 114.5, 111.4, 31.8, 30.7, 29.0, 27.6, 26.9 164.9, 151.2, 146.9, 145.2, 144.8, 137.4, 118.0, 114.7, 112.9, 101.6, 30.1, 28.1, 27.2 159.7, 151.3, 146.9, 146.3, 135.3, 132.3, 117.8, 114.3, 112.9, 106.1, 25.1, 24.2, 22.9, 21.9 2u 2v 2w 2x 157.7, 151.2, 146.8, 146.1, 140.3, 1.621.72 (m, 4H, CH2), 1.821.86 (m, 2H, CH2), 2.742.79 (m, 4H, CH2), 6.57 (dd, 1H, J = 1.5, 3.6 Hz, 136.0, 117.7, 114.9, 112.8, 108.4, Ar-H), 7.10 (d, 1H, J = 3.6 Hz, Ar-H), 7.64 (d, 1H, J 31.9, 30.4, 29.1, 27.7, 27.0 = 1.5 Hz, Ar-H), 8.23 (s, 1H, CH=N) CDCl3 Compounds 2t and 2u, with MIC values of 64 mg mL1, and compounds 2d, 2g, 2j, 2k, 2m, 2o and 2v with MIC values of 128 mg mL1, exhibited excellent antifungal activity against Criptococcus neoformans. Other tested compounds were inactive, or showed moderate activity (2a). The most active compounds for anti-Cryptococcus activity were 2t and 2u, which have in common the presence of a quinoline moiety linked to the 2-amino position of the thiophene ring. This result corroborates the data of Boateng and collaborators (21) for a series of benzothieno[3,2-b]quinoline derivatives. The compounds showed moderate activity against C. krusei. Compounds 2i, 2k and 2t were the most active (MIC = 512 mg mL1), followed by compounds 2a, 2g, 2j, 2r and 2s (MIC values at 1024 mg mL1). The present study also confirmed that the presence of the cyclohexyl ring linked to the C-4 and C-5 position of the thiophene ring, as found in compounds 2d, 2g, 2i-k and 2t, increases antifungal activity. The cyclopentyl (2a, 2s and 2v) and cycloheptyl (2l-o and 2r) derivatives displayed similar antifungal activity profiles and were, generally, two to four times less active than the cyclohexyl derivatives (9). B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. Table III. HRMS and IR data for 2h, i, k-x IR (KBr) (nmax, cm1) CH CN CH CN CH CN CH CN CH CN CH CN CH CN CH CN CH CN CH CN CH CN CH CN CH CN CH CN CH CN CH CN (2916) (2223) (2940) (2222) (2938) (2218) (2929) (2220) (2912) (2222) (2928) (2216) (2927) (2216) (2945) (2213) (2928) (2212) (2911) (2220) (2924) (2220) (2937) (2217) (2930) (2219) (2960) (2220) (2936) (2213) (2929) (2215) HRMS m/z (ES+) Calculated 358.0139 284.0783 372.1296 325.0884 294.1190 308.1347 322.1503 351.1405 335.1456 319.1143 303.0830 317.0986 331.1143 258.0285 272.0441 286.0598 Found 359.0940 285.0903 373.1119 326.0718 295.1067 309.1455 323.1641 352.1217 336.1291 320.1003 304.0767 318.1099 332.1270 281.0148a 273.0399 309.0227a Compd. 2h 2i 2k 2l 2m 2n 2o 2p 2q 2r 2s 2t 2u 2v 2w 2x [M+Na]+ Antiproliferative activity Table V summarizes the in vitro antiproliferative effects of cycloalkyl[b]thiophene derivatives 2a-x against the three human cancer cell lines. Compounds 2a, 2c, 2f-g, 2k-l, 2p-q and 2s-v exhibited antiproliferative activity below the concentration of 25 mg mL1. The results indicate that the size of the cycloalkyl ring coupled at the C-4 and C-5 position of the thiophene ring is essential for antiproliferative activity. Increasing the si229 B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. Table IV. Antifungal activity of the synthesized compounds, miconazole and 5-fluorocitosine MIC (mg mL1) C. neoformans 2a 2b 2c 2d 2e 2f 2g 2h 2i 2j 2k 2l 2m 2n 2o 2r 2s 2t 2u 2v 2w 2x 5-Fluorocytosine Miconazole No inhibition NT Not tested a Solvent: DMSO Compd.a 512 128 128 128 128 128 128 256 64 64 128 10 NT C. krusei 1024 1024 512 1024 512 512 1024 1024 512 NT 50 ze of the cycloalkyl ring helps increase the capacity to inhibit cell growth. Cycloheptyl derivatives 2r, 2n, 2o and 2x were thus the most active compounds, while all cyclopenthyl derivatives were inactive compounds. The antiproliferative activity of the compounds was greater against the HEP cells compared to the other two cell lines, as can be seen in compounds 2i, 2n, 2o and 2x with inhibition of 95.4, 57.3, 71.0 and 52.6 %, respectively. The most active cycloalkyl[b]thiophene compound identified in this study was 2r, which inhibited the growth of HT29, NCI H-292 and HEP cancer cell lines by 100.0, 100.0 and 97.5 %, respectively. B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. Cyclohepthyl[b]thiophene derivatives also demonstrated that the size of the alkyl group in the para position of the benzylidene moiety seemed to be important for antiproliferative activity. In this context, a comparison of substituent effects revealed that the replacement of i-propyl (2o) by an ethyl (2n) group resulted in a slight reduction in antitumor activity (reduction of 71.0 to 57.3 % for the HEP cell, and of 45.7 to 42.5 % for the NCI H-292 cell), and the replacement of ethyl (2n) by a methyl (2m) group resulted in a substantial loss of activity (reduction of 57.3 to 22.1 % for the HEP cell, 42.5 to 25.8 % for the NCI H-292 cell and 61.6 to 11.0 % for the HT29 cell). Another structural feature that can be associated with complete loss of activity in these derivatives is the presence of bulky or large arylidenes linked to the 2-amino position, as can be seen in compounds which have a quinoline moiety (2s, 2t and 2u), and in compounds 2k, 2p and 2q, which represent 4-benzyloxy-benzylidene, 4-morpholinyl-benzylidene and 4-pyrrolidinyl-benzylidene, respectively. Computational methods The goal of PLS regression is to provide a dimension reduction strategy in a situation where we want to relate a set of response variables Y to a set of predictor variables X. The PLS-DA is a PLS regression where Y is a set of binary variables describing the effects of a category variable on X; i.e., the number of dependent, or response, variables is equal to the number of categories. The best way to extract information from the PLS-DA is graphically, by plotting the obtained matrices. Table V. Antiproliferative activity of the synthesized compounds Inhibition (%)b,c HT29 2b 2d 2e 2h 2i 2j 2m 2n 2o 2r 2w 2x a b c Compd.a 11.1 ± 2.7 24.9 ± 3.8 0.0 ± 0.0 11.3 ± 4.8 53.7 ± 1.9 4.9 ± 0.6 11.0 ± 0.6 61.6 ± 2.0 28.9 ± 1,5 100.0 ± 0.5 34.7 ± 0.7 25.0 ± 2.0 NCI H-292 30.4 ± 1.1 24.2 ± 5.5 29.5 ± 3.4 23.5 ± 1.2 50.9 ± 4.0 18.4 ± 0.2 25.8 ± 1.1 42.5 ± 2.7 45.7 ± 3.3 100.0 ± 0.0 33.9 ± 3.8 27.9 ± 2.7 HEP 20.8 ± 6.4 1.6 ± 1.0 0.0 ± 3.1 11.3 ± 0.4 95.4 ± 0.0 3.7 ± 4.8 22.1 ± 0.5 57.3 ± 5.0 71.0 ± 3.9 97.5 ± 2.5 24.1 ± 2.7 52.6 ± 2.9 Solvent: DMSO 25 mg mL1 Mean ± SD, n = 3. B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. PLS-DA was applied in this study to improve the results obtained by the classic image analysis and to identify the significant spots responsible for the differences in antiproliferative activity against the three cell lines, which were similar to each other. The models presented the following statistical indices: R2 ~ 0.80 coefficient of determination, the squared multiple correlation coefficient, which is the total variance of the response explained by a regression model; SDEP ~ 0.70 standard deviation error of prediction, also known as standard error in prediction SEP or PSE, which is the function of the predictive residual sum of squares; Q2 ~ 0.60 predictive ability of the model. A large difference was observed between active (triangle up) and inactive (triangle down) compounds for the three cell lines (Fig. 1). This graph plots X-scores (T) against Y-scores (U). It provides a clear idea of the correlation between the X's and the Y's obtained in the model for each of the LVs. LVs are linear combinations of the original X-variables. The variables highlighted in the studies with the three cell lines differ only by Angstroms but showed that hydrogen bond acceptor regions were characteristic of the active compounds. The plot weight, obtained in the study with the NCI H-292 cell line, reflects the behavior observed in all investigations (Fig. 2). Active compounds showed greater interactions between the probes DRY-DRY, N1-N1 and DRY-N1. The most active compound (2r) was the only one that exhibited an O-N1 interaction (represented by the variable VAR246) (triangle up). In Fig. 3 this variable corresponds to an interaction between 4.40 and 4.80 Å, and characterizes a hydrogen bond acceptor region. Fig. 1. Plot scores obtained from PLS-DA, distribution and separation of the set by the best model. Active (triangle up) and inactive (triangle down) compounds against the investigated cell lines: a) HT 29 cell line, b) NCI H-292 cell line, and c) HEP cell line. 232 B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. Fig. 2. Plot weight obtained with the NCI H-292 cell line, selecting the active compounds. Greater intensity and number of positive influence of the variables are generated on the interaction of probes DRY-DRY, N1-N1 and DRY-N1 in active compounds. Fig. 3. Disclosure of O-N1 interaction, VAR 246, in the most active compound (2r) (triangle up). CONCLUSIONS In short, novel thiophene derivatives substituted with an arylidene moiety were synthesized, characterized on the basis of their physical, analytical, and spectral data, and preliminarily evaluated for their in vitro antifungal and antiproliferative properties. Antifungal studies revealed that the results for the antifungal activity of cycloalkyl[b]thiophene derivatives ranged from excellent to moderate and that the most promising compounds were those that had a cyclohexyl ring linked to the thiophene ring (2d, 2g, 2i-k and 2t). The results for antiproliferative activity indicate that the most active compounds are cycloheptyl[b]thiophene derivatives, especially 2-[(1H-indol-2-yl-methylidene)amino]5,6,7,8-tetrahydro-4H-cyclohepta[b]thiophene-3-carbonitrile (2r). B. C. C. Souza et al.: Preliminary antifungal and cytotoxic evaluation of synthetic cycloalkyl[b]thiophene derivatives with PLS-DA analysis, Acta Pharm. 62 (2012) 221236. The chemometric tool (PLS-DA) applied in this study generated good exploratory and predictive results. The behavior of the binary free PLS-DAwas similar for the three human cancer cell lines studied. The descriptors having shape characteristics were strongly correlated with the biological data. Higher weight variables highlight the interactions between the DRY-DRY N1-N1, DRY-N1 probes. Hydrogen bond acceptor regions are characteristic of active compounds and the presence of O-N1 interaction, as found in 2r, increased the antitumor activity. Finally, it can be concluded that cycloalkyl[b]thiophene derivatives could be considered promising compounds for the discovery of new antitumor agents. Further investigation of the mode of action at molecular level needs to be carried out, and more extensive studies are needed to determine additional physicochemical and biological parameters in order to provide a deeper insight into the SAR and to optimize the efficacy and safety of this series of compounds. Acknowledgements. We are indebted to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and UEPB, through the Programa de Incentivo à Pós-Graduação e Pesquisa/ PRPGP for financial support. The authors thank the CNPQ and the Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco (FACEPE) for scholarships of Luciana Scottiand Beatriz C. C. Souza.
Acta Pharmaceutica – de Gruyter
Published: Jun 1, 2012
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