Abstract: Objective: The emergence of resistance to the major classes of antibacterial drugs is recognized as a serious health concern. Chemotherapy of cervical cancer, a devastating cancer with increasing worldwide incidence and mortality rates is largely ineffective. The discovery and development of effective antibacterial/anticancer agents is urgently needed. The present study reports on the evaluation of antibacterial and anticancer properties of some azo compounds containing thiophene backbone. Methodology: The antibacterial activity of the synthesized compounds was assessed by performing Minimum Inhibitory Concentration (MIC), Minimum Bactericidal Concentration (MBC) and the time-kill kinetic study, while the cytotoxic activity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay using HeLa (cancer cell lines) and Vero (normal) cells. Results: All of the compounds tested here showed significant antibacterial activity against the strains of Gram-positive bacteria, Staphylococcus aureus and Gram-negative multi-drug resistance bacteria, Vibrio cholerae (causative agent of cholera) and Shigella flexneri (causative agent of shigellosis), as well as the significant cytotoxic activity (LC50, 1.03-106.73 μg mL–1) against HeLa cells. Among these, compounds 3, 6 and 7 showed the most antibacterial (MIC, 8-64 μg mL–1) and cytotoxic (LC50, 1.03-2.37 μg mL–1) activities with selectivity index values ranged from 71.19-144.80, which were much higher than that of the reference drug, paclitaxol. The presence of the nitro functional group in compounds 3 and 6 could explain their good antibacterial and cytotoxic activities. Conclusion: These results indicate that the synthesized compounds have both antibacterial and anticancer properties with no toxicity to normal cells.
INTRODUCTION
Drug resistance refers to a situation in which the drugs that usually destroy the bacteria no longer do so. In different organisms, multi-drug resistance mechanisms are established differently. Microbial resistance to antibiotics can be classified as natural or acquired resistance. In the former, the bacteria are "Intrinsically" resistant, while the latter refers to bacteria that are susceptible to develop resistance to antibiotics despite the fact that they are usually sensitive. Mutations in chromosomal genes, acquisition of mobile genetic elements like plasmids and transposons, which are carriers of antibiotic resistance genes are frequent causes of acquired resistance. The development of microbial resistance to chemotherapeutic agents usually results from their wide-spread exploitation in the treatment of infectious diseases. The emergence of resistance to the major classes of antibacterial drugs is recognized as a serious health concern. The study for new antibacterial agents with different action modes has always remained paramount1. Azo compounds, with wide range of biological properties are very promising to this effect. The specific pathway of metabolism generally attributes this activity to azo colorants. In vivo, there is an enzyme-catalyzed reduction of the azo bond2. The azoreductase activity was found in liver3, in digestive tract bacteria4,5 of mammals, as well as in the skin of bacteria such as Staphylococcus aureus6. This reaction results in the azo-bond cleavage and the release of the corresponding aromatic amines originating from the azo dye7. The products can be more or less toxic than the parent molecules and thus this process can decrease or increase any toxic or carcinogenic effects of the dyes8,9.
Substituted thiophene and their biheterocycles have received considerable attention during last two decades as they are endowed with wide range of therapeutic properties such as analgesic10, antibacterial11, antioxidant and anti-inflammatory12, antifungal13, anticancer14 and local anaesthetic activity15. New compounds with enhanced biological activities can be obtained by the fusion of thiophene with heterocyclic nuclei. Thienopyrimidines have some special characteristics among these compounds. Many of these derivatives exhibit antiallergic16, antibacterial17, antidepressant18, antidiabetic19, analgesic and anti-inflammatory20 activities. Diverse and useful biological activities are exhibited by coumarin and its derivatives, which are ubiquitously distributed in nature21,22. These compounds have numerous medical applications including antitumor and anti-HIV therapy23,24, Central Nervous System (CNS) stimulation25, antibacterial26,27, anti-inflammatory28,29 and anti-coagulant properties30. Hydroxycoumarins are well noted for their antioxidants potentials, which protect injury by scavenging reactive oxygen species31. Coumarin derivatives have been reported for their cytostatic and cytotoxic activities32. The above observations prompted to synthesize the title compounds with the presumption that the association of azo group with coumarin and thiophene skeleton would produce new compounds with significant antibacterial and cytotoxic activities. Recently, a series of arylazothiophenes dyes in the form of their ammonium sulfate salts from diazonium salts of 3-aminothieno (3,4-c) coumarins and aromatic amines was synthesized33. Reports have also shown that 3-aminothieno (3,4-c) thienocoumarins and azo dyes derived from these compounds are of great potential interest34,35. The present study reports on the antibacterial activity of the synthesized azo compounds against the strains of Gram-positive bacteria such as Staphylococcus aureus and Gram-negative multi-drug resistance bacteria such as Vibrio cholerae and Shigella flexneri, as well as the cytotoxic activity against HeLa cells.
MATERIALS AND METHODS
Experimental: All melting points are uncorrected and were determined with a Reichert Thermovar Microscope and a Buchi 530 melting point apparatus. The IR spectra were measured with a SHIMADZU FTIR-8400S and Perkin Elmer FT-IR 2000 spectrometers. The UV spectra were recorded with a Beckman U-640 spectrophotometer. Combustion analysis were carried with Yanaco CHN corder MT-6 (Yanaco Analytical Instruments Corp., Kyoto, Japan). The HREIMS and EIMS (direct inlet 70 eV) were measured on Jeol JMS AX-500 and AX-700 spectrometers. The 1H and 13C-NMR spectra were recorded in DMSO-d6 with a Jeol JNM ECA-600 and AL-400 spectrometers with TMS and/or the residual solvent signals as internal references. Coupling constants J in brackets are reported in hertz. Simulated 1H and 13C (1H)-NMR spectra were performed with an ACD-NMR spectra simulation programme and with Chemdraw software.
Reagents and starting materials: All the reagents mentioned in this study were purchased from Aldrich and Fluka and were used without further purification. Starting 2-aminothiopenes (1 and 2) have been prepared according to literature procedures as published earlier36-38.
Preparation of diazonium salt solution: Dry sodium nitrite (2.07 g, 3 mmol) was slowly added over a period of 30 min to concentrated sulphuric acid (10 mL) with occasional stirring. The solution was cooled at 0-5°C. The 2-aminothiophene substrates 1 and 2 were dissolved in DMSO (10 mL) and cooled at 0-5°C. The nitrosyl sulphuric acid solution was added to the 2-aminothiophene’s solutions, the temperature was maintained between 0-5°C. The clear diazonium salt solution thus obtained was used immediately in the coupling reactions.
4H-2-(p-amino-5-nitrophenylazo)thieno[3,4-c]chromen-4- one hydrogen sulphate (3): Para nitroaniline (414 mg, 3 mmol) was dissolved in DMSO (10 mL) and then cooled in an ice-bath at 0-5°C. The diazonium solution of 1 previously prepared was added dropwise over 1 h and then 15 mL sodium acetate solution (10%) was added to the mixture. The solid material that formed was crystallized from the hot water to give the title compound 3 (601 mg, 46%) as a red powder. Crystallisation of the solid residue from aqueous ethanol gave the title compound 3 (465 mg, 33%) as orange powder, mp 233-234°C; IR (potassium bromide) 3552, 3533, 3012, 2825, 2802, 2717, 2538, 1726, 1679 and 1498 cm–1; λmax (THF) (log ε) 203 (4.45), 213 (4.44), 235 (4.80), 273 (4.83), 282 (4.84), 325 (4.38), 382.0 (4.13), 387 (4.11) and 471 nm (3.44); 1H NMR (DMSO-d6, 300 MHz) δ 14.32 (broad s, 1H, HSO4‾, deuterium oxide-exchangeable), 8.73 (d, 1H, 4’-H, J = 8.5 Hz), 8.70 (s, 1H, 6’H), 7.95 (d, 1H, 3’-H, J = 8.5 Hz), 7.84 (broad s, 3H, NH3+, deuterium oxide-exchangeable), 7.80 (ddd, 1H, 7-H, J = 2.0, 6.8 and 7.5 Hz), 7.82 (dd, 1H, 9-H, J = 2.0 and 8.0 Hz), 7.80 (dd, 1H, 6-H, J = 2.0 and 6.8 Hz), 7.54 (s, 1H, 1-H), 7.53 (ddd, 1H, 8-H, J = 2.0, 6.8 and 8.0 Hz); 13C (1H) NMR (DMSO-d6, 300 MHz) δ 163.6 (C-4), 156.7 (C-9b), 155.0 (C-3a), 152.7 (C-5a), 147.9 (C-5’), 135.5 (C-3), 135.3 (C-2’), 128.9 (C-7), 128.9 (C-9a), 128.9 (C-3’), 127.1 (C-9), 127.1 (C-1’), 125.5 (C-8), 125.3 (C-4’), 125.3 (C-6’), 118.1 (C-6), 117.0 (C-1); ms: (EI) m/z (%) 530 (1), 477 (1), 449 (5), 402 (1), 367 (1), 321 (1), 291 (2), 255 (1). Anal. Calcd. for C17H12N4O8S2: C, 43.96; H, 2.60; N, 12.06. Found C, 44.07; H, 2.58; N, 12.19.
4H-2-(p-N-phenylaminophenylazo)thieno[3,4-c]chromen-4-one hydrogen sulphate (4): Diphenylamine (507 mg, 3 mmol) was dissolved in DMSO (10 mL) and then cooled in an ice-bath at 0-5°C. The diazonium solution of 1 previously prepared was added dropwise over 1 h and then 15 mL sodium acetate solution (10%) was added to the mixture. Crystallisation from methanol gave the title compound 4 (564 mg, 37%) as a yellow powder, mp 236-238°C; IR (potassium bromide): 3371, 3259, 3205, 3176, 2731, 2135, 1965, 1755, 1733, 1672, 1494, 1446 and 1406 cm–1; λmax (THF) (log ε) 204 (4.24), 213 (4.26), 235 (4.61), 272 (4.65), 282 (4.65), 325 (4.16) and 389 nm (3.88); 1H NMR (DMSO-d6, 300 MHz) δ 14.54 (broad s, 1H, HSO4‾, deuterium oxide-exchangeable), 8.81 (d, 2H, 2’-H and 6’-H, J = 7.0 Hz), 8.72 (d, 2H, 2’’-H and 6’’-H, J = 8.0 Hz), 8.20 (broad s, 1H, NH2+, deuterium oxide-exchangeable), 7.86 (d, 2H, 3’-H and 5’-H, J = 7.0 Hz), 7.57 (d, 1H, 9-H, J = 8.0 Hz), 7.32 (dd, 2H, 3’’-H and 5’’-H, J = 7.0 and 7.5 Hz), 7.27 (dd, 1H, 4’’-H, J = 7.5 and 7.0 Hz), 7.20 (dd, 1H, 8-H, J = 9.1 and 9.2 Hz), 7.00 (m, 7-H, 1H), 6.85 (d, 1H, 6-H, J = 8.5 Hz), 6.70 (s, 1-H, 1H); ms: (EI) m/z (%) 356 (11), 337 (22), 296 (7), 240 (7), 205 (1), 183 (100). Anal. Calcd. for C23H17N3O6S2: C, 55.75; H, 3.46; N, 8.48. Found C, 55.81; H, 3.53; N, 8.41.
4H-4-imino-2-(p-aminophenylazo)thieno[3,4-c]chromen hydrogen sulphate (5): The reaction mixture of the diazonium sulphate of 2 with aniline was worked up as above to afford compound 5 (349 mg, 27%) as a red powder, mp>230°C; IR (potassium bromide): 3652, 3203, 3178 (NH), 3047 (arom. C-H), 2918, 1448 and 1404 cm–1; λmax (THF) (log ε) 204 (4.23), 217 (4.26), 236 (4.64), 269 (4.71), 283 (4.66) and 474 nm (3.76); 1H NMR (DMSO-d6, 300 MHz) δ 9.30 (broad s, 1H, = NH, deuterium oxide-exchangeable), 8.96 (broad s, 1H, HSO4‾, deuterium oxide-exchangeable), 7.75 (s, 1H, thiophenic proton), 7.62 (m, 2H, aromatic protons), 7.45 (m, 2H, aromatic protons), 7.40 (m, 2H, aromatic protons), 7.25 (m, 2H, aromatic protons), 7.17 (broad s, 3H, NH3+, deuterium oxide-exchangeable); ms: (EI) m/z (%) 512 (1), 418 (M+, 1), 402 (7), 367 (7), 330 (11), 301 (2), 243 (4), 169 (1). Anal. Calcd. for C17H14N4O5S2: C, 48.80; H, 3.35; N, 13.40. Found C, 49.02; H, 3.45; N, 13.54.
2-(2-amino-5-nitrophenylazo)-4-(2-hydroxyphenyl)-thiophene-3-carboxylic acid hydrogen sulphate (6): Para-nitroaniline (414 mg, 3 mmol) was dissolved in DMSO (10 mL) and then cooled in an ice-bath at 0-5°C. The diazonium solution of 2 previously prepared was added dropwise over 1 h and then 15 mL sodium acetate solution (10%) was added to the mixture. Crystallisation of the resulted solid material from aqueous ethanol gave compound 6 (144 mg, 10%) as a red powder, mp 190-192°C; IR (potassium bromide): 3290, 3269, 3236, 2914, 2804, 2705, 2667, 1733, 1676, 1602 and 1444 cm–1; λmax (THF) (log ε) 203 (4.53), 213 (4.52), 239 (5.04), 268 (4.99), 286 (4.98), 325 (5.18), 448 (4.16) and 553 nm (3.48); 1H NMR (DMSO-d6, 300 MHz) δ 8.90 (dd, 1H, 4’’-H, J = 1.4 and 1.3 Hz), 7.67 (s, 1H, 6’’-H), 7.62 (broad s, 3H, NH3+, deuterium oxide-exchangeable), 7.34 (d, 1H, 6’-H, J = 2.0 Hz), 7.30 (dd, 1H, 4’-H, J = 2.0 and 7.0 Hz), 7.27 (dd, 1H, 5’-H, J = 2.0 and 7.0 Hz), 7.23 (d, 1H, 3’-H, J = 1.8 Hz), 7.20 (d, 1H, 3’’-H, J = 1.8 Hz), 7.19 (s, 1H, 5-H); 13C (1H) NMR (DMSO-d6, 300 MHz) δ 184.5 (COOH), 155.0 (C-2’), 153.6 (C-3), 153.4 (C-2’’), 147.8 (C-4), 135.4 (C-5’’), 135.2 (C-2), 130.0 (C-4’), 128.9 (C-6’), 125.4 (C-5), 125.2 (C-4’’), 125.2 (C-6’’), 125.3 (C-1’), 119.9 (C-1’’), 117.5 (C-5’), 117.0 (C-3’’), 114.9 (C-3’); ms: (FAB+) m/z (%) 384 (1), 383 (1), 307 (60), 429 (11), 248 (100), 154 (91). Anal. Calcd. for C17H14N4O9S2: C, 42.32; H, 2.92; N, 11.61. Found: C, 42.25; H, 3.01; N, 11,69.
Azo bis[4-(2-hydroxy-phenyl)-thiophene-3-carboxylic acid -2-yl] (7): Compound 2 (648 mg, 3 mmol) was dissolved in DMSO (10 mL) and then cooled in an ice-bath at 0-5°C. The diazonium solution of 2 previously prepared was added dropwise over 1 h and then 15 mL of sodium acetate solution (10%) was added in the mixture. The solid precipitate was filtered and crystallized from methanol to give compound 7 (29 mg, 35%) as yellow powder mp>230°C; IR (potassium bromide): 3807, 3484, 2299, 1750, 1726 and 1444; λmax (THF) (log ε) 203 (4.60), 215 (4.59), 234 (4.92), 276 (4.94), 281 (4.96), 325 (4.61) and 385 nm (4.34); 1H NMR (DMSO-d6, 300 MHz) δ 14.32 (broad s, 1H, COOH, deuterium oxide-exchangeable); 8.66 (dd, 1H, 3’-H, J = 1.0 and 6.0 Hz); 8.02 (broad s, 1H, NH, deuterium oxide-exchangeable); 7.84 (ddd, 1H, 4’-H, J = 1.0, 6.1 and 6.8 Hz); 7.60 (s, 2H, 5-H), 7.56 (ddd, 2H, 5’-H, J = 1.0, 6.4 and 8.8 Hz), 7.31 (d, 2H, 6’-H, J = 6.4 Hz), 6.98 (broad s, 2H, OH, deuterium oxide-exchangeable); 13C (1H) NMR (DMSO-d6, 300 MHz) δ 184.4 (COOH), 155.0 (C-2’), 154.0 (C-3), 153.7 (C-1’), 147.6 (C-4), 135.5 (C-2), 128.8 (C-6’), 125.4 (C-5), 120.4 (C-5’), 117.3 (C-3’), 114.8 (C-4’); ms: (EI) m/z (%) 364 (2), 402 (1), 248 (12), 247 (100), 231 (40), 214 (12), 203 (14), 189 (24), 144 (28), 77 (10). Anal. Calcd. for C22H16N2O10S3: C, 46.80; H, 2.86; N, 4.96. Found C, 46.65; H, 2.88; N, 4.80.
Preparation of 8 and 9: Compound 1 (0.651 g, 3 mmol) was dissolved in DMSO (10 mL) and then cooled in an ice-bath at 0-5°C. The diazonium solution of aniline previously prepared was added dropwise over 1 h and then 15 mL of sodium acetate solution (10%) was added to the mixture. The solid material that was formed was filtered and crystallized from hot water to afford compound 9 (370 mg, 26%) as a red powder. The solid residue was crystallized from methanol to yield the title compound 8 (388 mg, 40%) as a red powder.
4H-3-amino-1-phenylazo-1-thieno[3,4-c]chromen-4-one (8): Melting point 258-259°C [lit.39 232-234°C from DMF/ethanol (5:3)]; IR (potassium bromide): 3207, 3107, 3062, 2896, 2875, 2845, 2580, 1874, 1731, 1683, 1488 and 1404 cm–1; λmax (THF) (log ε) 211 (3.67), 253 (4.11), 291 (3.89), 336 (3.66) and 478 nm (4.12); 1H NMR (DMSO-d6, 300 MHz) δ 8.82 (dd,1H, H-9, J = 8.0 and 1.8 Hz), 7.77 (d, 1H, H-2’ and H-6’, J = 7.5 Hz), 7.60 (ddd, 1H, H-7, J = 1.8, 8.0 and 8.0 Hz), 7.55 (dd, 1H, H-3’ and H-5’, J = 7.9 and 8.0 Hz), 7.44 (d, 1H, H-4’, J = 7.7 Hz), 7.37 (ddd, 1H, H-8, J = 1.0, 7.7 and 7.8 Hz), 7.35 (dd, 1H, H-6, J = 7.4 and 8.6 Hz); 13C (1H) NMR (DMSO-d6, 300 MHz) δ 168.5 (C-3), 158.4 (C-4a), 153.0 (C-1’ and C-8a), 152.0 (C-2), 135.0 (C-9), 133.0 (C-8b), 131.8 (C-7), 129.6 (C-3’ and C-5’), 129.0 (C-6), 128.8 (C-8), 125.1 (C-4’), 122.1 (C-2’ and C-6’), 117.5 (C-5), 117.0 (C-2a); ms: (EI) m/z (%) 321 (6), 280 (22), 247 (22), 208 (100), 190 (24).
2-amino-4-(2-hydroxyphenyl)-5-phenylazothiophene-3-carboxylic acid (9): Melting point 150°C; IR (potassium bromide): 3425, 3315, 3143, 3056, 3028, 1741, 1676, 1483 and 1460 cm–1; λmax (THF) (log ε) 203 (3.87), 212 (3.78), 220 (3.91), 247 (4.47), 258 (4.48), 288 (4.33), 345 (4.02), 461 (4.52) and 471 nm (4.54). Anal. Calcd. for C17H13N3O3S: C, 60.18; H, 3.83; N, 12.39. Found: C, 59.50; H, 3.61; N, 12.46.
Antibacterial assays
Microbial growth conditions: A total of six bacterial strains were tested for their susceptibility to compounds and these strains were taken from a laboratory collection (Kindly provided by Dr. T. Ramamurthy, NICED, Kolkata). Among the clinical strains of Vibrio cholerae used in this study, strains NB2, CO6 and SG24 belonged to O1 and O139 serotypes, respectively. All these strains were able to produce cholera toxin and hemolysin and are multi-drug resistant. The other strains used in this study were V. cholerae non-O1, non-O139 (strain PC2, positive for hemolysin production) and Shigella flexneri 2a. The American Type Culture Collection (ATCC) strain, Staphylococcus aureus ATCC 25923 was also used for quality control. The bacterial strains were maintained on agar slant at 4°C and subcultured on a fresh appropriate agar plates 24 h prior to any antibacterial test. Mueller Hinton Agar (MHA) was used for the activation of bacteria. Mueller Hinton Broth (MHB) and nutrient agar (Hi-Media) were used for the MIC and MBC determinations, respectively.
Inoculum preparation: Suspensions of bacteria were prepared in MHB from cells arrested during their logarithmic phase growth (4 h) on MHB at 37°C. The turbidity of the microbial suspension was read spectrophotometrically at 600 nm and adjusted to an OD of 1.0 with MHB, which is equivalent to 2×108 CFU mL–1. From this prepared solution, other dilutions were made with MHB to yield 1×106 CFU mL–1.
Determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC): The MIC and MBC of compounds 3-9 were assessed using the broth microdilution method recommended by the National Committee for Clinical Laboratory Standards40,41 with slight modifications. The 96-well round bottom sterile plates were prepared by dispensing 180 μL of the inoculated broth (1×106 CFU mL–1) into each well. Twenty microliters aliquot of the compounds were added. The concentrations of sample tested were 0.125, 0.25, 0.50, 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024 and 2048 μg mL–1. Dilutions of ampicillin and chloramphenicol served as positive controls, while broth with 20 μL of DMSO was used as negative control. The ATCC strain Staphylococcus aureus ATCC 25923 was included for quality assurance purposes. Plates were covered and incubated for 24 h in ambient air at 37°C. After incubation, Minimum Inhibitory Concentrations (MIC) were read visually, all wells were plated to nutrient agar (Hi-Media) and incubated. Minimal Bactericidal Concentration (MBC) was defined as a 99.9% reduction in CFU from the starting inoculums after 24 h incubation interval.
Time-kill kinetic study (for antimicrobial drugs) against Staphylococcus aureus: Time-kill kinetic assay was performed as previously described42 with minor modifications. Cultures of bacteria in MHB (1×106 CFU mL–1) were incubated separately at 37°C for 0, 2, 4, 6, 10 and 24 h in the absence (control) and in the presence of the reference drug/test compounds at MIC and MBC of each sample. Compounds 3, 6 and 7 and ampicillin were used in the time-kill kinetic experiment. The final concentration of DMSO was 1%. A control sample was made using DMSO 1% and the inoculum. At each incubation time point, liquids (50 μL) were removed from the test solution for ten-fold serial dilution. Thereafter, a 100 μL liquid from each dilution was spread on the surface of the MHA plates and incubated at 37°C for 24 h and the number of CFU mL–1 was counted. Experiments were carried out in triplicate. Time-kill curves were constructed by plotting the surviving log10 of number of CFU mL–1 against time (h).
Cytotoxicity assays: HeLa (Human cervical cancer cell line) and Vero cells (African green monkey kidney cells, normal non-cancer cells), obtained from the American Type Culture Collection (ATCC) were used in this study. Cytotoxic activity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, Sigma, USA) assay43 against HeLa cells and Vero cells. This cell viability assay is based on living cell’s property to transform the MTT dye tetrazolium ring into a purple-colored formazan structure due to the action of mitochondrial and other dehydrogenases inside the cell. The color intensity yielded by the cell population is directly proportional to the number of viable cells and one can quantify the absorbance measurements using mathematical parameters. Compounds 3-9 were prepared from the stock solutions by serial dilution in RPMI 1640 to give a volume of 100 μL in each well of a microtiter plate (96-well). Each well was filled with 100 μL of cells at 2×105 cells mL–1. The assay for each concentration of extract was performed in triplicates and the culture plates were kept at 37°C with 5% (v/v) CO2 for 24 h. After removing the supernatant of each well and washing twice by PBS, 20 μL of MTT solution (5 mg mL–1 in PBS) and 100 μL of medium were then introduced. After 4 h of incubation, 100 μL of DMSO was added to each well to dissolve the formazan crystals and the absorbance values at 490 nm were measured with a microplate reader (Bio-RAD 680, USA). The relative cell viability (%) was expressed as a percentage relative of treated cells to the untreated control cells (TC/UC×100). The rate of cell inhibition was calculated using the following equation:
The LC50 values were calculated as the concentration of test sample resulting in a 50% reduction of absorbance compared to untreated cells. Paclitaxal served as positive control.
Hemolytic assay: Whole blood (10 mL) from a healthy man was collected into a conical tube containing heparin as an anticoagulant (blood group O was used). Authorization for the collection of blood was obtained from the Medical and Ethical Committee. The written informed consent for participation in the study was obtained from a parent of 40 years old. Erythrocytes were harvested by centrifugation for 10 min at 1,000x g and room temperature and washed three times in PBS solution. The top layer (plasma) and the next, milky layer (buffy coat with a layer of platelets on top of it) were then carefully aspirated and discarded. The cell pellet was resuspended in 10 mL of PBS solution and mixed by gentle aspiration with a pasteur pipette. This cell suspension was used immediately.
For the normal human red blood cells, which are in suspension, the cytotoxicity was evaluated as previously described44. Compounds 3-9 at concentrations ranging from 32-512 μg mL–1 were incubated with an equal volume of 1% human red blood cells in phosphate buffered saline (10 mM PBS, pH 7.4) at 37°C for 1 h. Ampicillin and chloramphenicol were tested simultaneously. Non-hemolytic and 100% hemolytic controls were buffer alone and buffer containing 1% triton X-100, respectively. Cell lysis was monitored by measuring the release of hemoglobin at 540 nm. Percent hemolysis was calculated as follows: [(A595 of sample treated with compound-A595 of sample treated with buffer)/(A595 of sample treated with triton X-100-A595 of sample treated with buffer)]×100.
Statistical analysis: Statistical analysis was carried out using Statistical Package for Social Science (SPSS, version 12.0). The experimental results were expressed as the Mean±Standard Deviation (SD). Group comparisons were performed using one way ANOVA followed by Waller-duncan Post hoc test. A p-value of 0.05 was considered statistically significant.
RESULTS AND DISCUSSION
Chemical analysis: The azo compounds 3-9 (Fig. 1) used in this study were synthesised according to previous experimental procedures33. The yields, the melting points and all the spectroscopic data for these compounds described in the present study are in full agreement with those originally reported.
Antibacterial activity: The azo compounds 3-9 were examined in vitro against bacterial species and the results are depicted in Table 1. All the compounds showed different degrees of antibacterial activities against the tested bacterial pathogens. Staphylococcus aureus was the most sensitive microorganisms, while V. cholerae CO6 and V. cholerae 2 were the most resistant. The lowest MIC value for these tested compounds (8 μg mL–1) were obtained with compounds 3 and 7 on S. aureus and with compound 3 on S. flexneri, while the lowest MBC value of 16 μg mL–1 were obtained on S. aureus and S. flexneri with compounds 3 and 7. However, the highest MIC value of 256 μg mL–1 was recorded on V. cholerae CO6, while the highest MMC value of 256 μg mL–1 were obtained on V. cholerae CO6 with compounds 5c and 10 and on V. cholerae SG24 (1) with compounds 4 and 5. A lower MBC/MIC (≤4) value signifies that a minimum amount of the tested compounds are used to kill the bacterial species whereas, a higher value (MBC/MIC>4) signifies the use of comparatively higher dose of the compounds are needed for the control of the microorganism45.
No activity was noted for ampicillin against V. cholerae NB2, V. cholerae PC2 and S. flexneri at concentrations up to 512 μg mL–1.
However, these bacterial strains were found to be sensitive to most of the tested compounds. In addition, the antibacterial activities of the tested compounds (MIC, 8-256 μg mL–1) were in some cases equal or stronger than those of the reference antibiotics such as ampicillin and tetracycline, highlighting their effective antibacterial potency.
Collectively, these studies showed that the tested compounds have broad-spectrum antibacterial activity and is effective against multi-drug resistant (MDR) bacteria. Taking into account the medical importance of the tested bacteria, this result can be considered as promising in the perspective of developing new antibacterial drugs from azo compounds of 3-aminothieno (3,4-c) coumarins. The present study demonstrated the antibacterial activity of azo dyes against the bacterial species such as V. cholerae, the causative agent of dreadful disease cholera and S. flexneri, the causative agent of shigellosis. Although, azo compounds have been reported to possess antimicrobial activity10,12,16,25,27, no study has been reported on the activity of these compounds against these types of MDR pathogenic strains. The strains of V. cholerae and S. flexneri included in the present study were MDR clinical isolates and these were resistant to commonly used drugs such as ampicillin, streptomycin, tetracycline, nalidixic acid, furazolidone and co-trimoxazole etc. Although no definite structure-activity relationship could be determined from this study, some structural features that might have influenced the antibacterial activity of these azo compounds can be drawn from the comparison of the chemical structures of compounds with different activities. Compound 3 was the most active azo compound, followed by 7, 6, 4, 9, 5 and 8. Together, it appears that, 2-hydroxyl, 2-methoxy and 10-carboxyl groups play a greater role in increasing the antibacterial activity based on the substitution patterns of the aromatic rings. Compound 9 may be generated by hydrolysis of compound 8. The later (64 - 256 μg mL–1) is less active than compound 9 (32 - 128 μg mL–1). The presence of the coumarin function in compound 8 and the presence of hydroxyl and carboxylic acid groups in compound 9 could be responsible for the difference in the activity of these two compounds.
Time-kill kinetic study: The time-kill kinetic study for compounds 3, 6 and 7 against Staphylococcus aureus (as a function of incubation time) is shown in Fig. 2. It can be noted that significant reduction of the bacterial population is observed with the tested samples and ampicillin at a concentration corresponding to their MBC values. At this concentration, all the bacterial population was completely killed after 6 h of incubation with compounds 3 and 7 and 10 h with ampicillin and compound 6.
Hemolytic activity: To investigate the potential use of compounds, the cellular toxicity also has to be determined. In this study, none of the tested compounds showed hemolytic activities against human red blood cells at concentrations up to 512 μg mL–1 (results not shown) indicating that it is non-toxic to normal cells.
Cytotoxicity activity: Compounds 3-9 were evaluated for their cytotoxicity against human cancer cells (HeLa cells) and normal non-cancer cells (Vero cells) and the results are presented in Table 2. The tested compounds showed significant cytotoxicity to HeLa cells (LC50 = 1.03-106.73 μg mL–1) when compared with Vero cells (LC50 = 34.38-257.76 μg mL–1). The positive control paclitaxol values of 47.63 and 593.69 nM against HeLa and Vero cells, respectively. Interestingly, the cytotoxicity of compounds 3, 6 and 7 can be considered more important when taking into consideration the criterion of the American National Cancer Institute (NCI) regarding the cytotoxicity of pure compounds (LC50<4 μg mL–1) l)46.
The presence of the nitro function in compounds 3 and 6 could explain their good cytotoxic activity. The ring opening of coumarin function in compound 3 into hydroxyl and carboxylic acid groups has no effect in the cytotoxity of the resultant compound 6.
Selectivity is important because most anticancer drugs presently in use induce serious adverse effects. In the present study, Selectivity Index (SI) of active compounds were determined in order to investigate, whether the cytotoxic activity was specific to cancer cells. The SI of the samples is defined as the ratio of cytotoxicity (LC50 values) on normal cells (Vero cells) to cancer cells (HeLa cells): SI = LC50 on Vero cells/LC50 on HeLa cells. Selectivity Index (SI) values of the compounds 5 and 8 against HeLa cells are 4.11 and 8.30, respectively and could be considered as poor when taking in consideration that the ratio for a good therapeutic index for a remedy or drug should be ten47. However, the Selectivity Index (SI) values of the compounds 3, 6, 7 and 8 against the HeLa cells ranged from 71.19-144.80 and could be considered significant to be used as anticancer agent. This is the first report on the cytotoxicity of compounds 3-9 against HeLa cells. These results are consistent with the use of compounds 3, 6 and 7 for treating breast cancer.
CONCLUSION
In short, known azo compounds containing thiophene backbone were synthesized, characterized on the basis of their physical, analytical and spectral data and preliminarily evaluated for their in vitro antibacterial and cytotoxic properties. The results indicate that compounds 3, 6 and 7 have good antibacterial and anticancer activities with no toxicity to human red blood cells and normal Vero cells. The presence of the nitro function in compounds 3 and 6 could explain their good antibacterial and anticancer activities. Finally, it can be concluded that these azo compounds could be considered promising compounds for the discovery of new antibacterial and antitumor agents. Further investigations 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.
ACKNOWLEDGMENTS
This study was supported by the Indian Ministry of Education and Research through their CV Raman fellowship grant to Dr. Jean-de-Dieu Tamokou for his Post Doctoral fellowship studies. The authors also acknowledge financial support from the research grant committees of both the University of Dschang and the Cameroonian Ministry of Higher Education.