Synthesis of 5-arylidine amino-1,3,4-thiadiazol-2-[(N-substituted benzyol)]sulphonamides endowed with potent antioxidants and anticancer activity induces growth inhibition in HEK293, BT474 and NCI-H226 cells

Abstract A series of imines 5-amino-1,3,4-thiadiazol-2-[(N-substituted benzyol)]sulphonamide derivatives were synthesized from various aromatic aldehydes and substituted with benzoyl acetazolamides under different reaction conditions and were evaluated for their antioxidant and free radical scavenging, antimitotic activity by Allium cepa meristem root model and cytotoxicity activity against HEK 293 (human epidermal kidney cell line), BT474 (breast cancer cell line) and NCI-H226 (lung cancer cell line) by MTT assay. Some of the synthesized compounds showed moderately potent cytotoxicity compared to indisulam. Graphical abstract A series of imines 5-amino-1,3,4-thiadiazol-2-[(N-substituted benzyol)]sulphonamide derivatives (9a–j); 5-amino-1,3,4-thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (4a–g); 5-(4-acetamido phenyl sulphonamido)-1,3,4-thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (6a–g); and 5-(4-amino phenyl sulphonamido)-1,3,4-thiadiazol-2-[N-(substituted benzoyl)]sulphonamide (7a–g) were synthesized from acetazolamide and were investigated for the in vitro anticancer by MTT assay, free radical scavenging and antimitotic activity by Allium cepa root meristem model. Experimental observations indicate that synthesized compounds were moderately potent anticancer agents.


Introduction
The rapid spread of cancer has sparked an intense worldwide search for new compounds, which may be used in designing anticancer drugs. The search of more effective anticancer agent has focused to a large extent on the design of molecules capable of recognizing and binding to target DNA base sequences. Development of anticancer drugs with fewer or no side effects is important for the treatment for cancer. The search for such potential anticancer drugs has led to the discovery of synthetic small molecules with anti-carcinogenic activity and limited harmful side effects particularly with respect to the immune system. Research in this area is expanding rapidly, and some promising leads have emerged. Heterocyclic moieties can be found in a large number of compounds, which display biological activity. The biological activity of the compounds is mainly dependent on their molecular structures (Salimon et al., 2010). A vast number of 1,3,4-thiadiazoles have been reported as potential pharmacologically active compounds with antimicrobial (Patil and Biradar, 2001;Zamani et al., 2004;Sharma et al., 2006), antiviral (Pandey et al., 2004), antitubercular (Oruc et al., 2004;Desai et al., 1984), anticonvulsant (Shrivastava et al., 1999;Kumar et al., 2003;Gupta et al., 2008;Stillings et al., 1986;Jatav et al., 2008), CNS depressant , hypoglycaemic (Hanna et al., 1995;Pattan et al., 2009), anti-inflammatory (Sharma et al., 2008;Varandas et al., 2005) and anticancer (Noolvi et al., 2011;Kumar et al., 2010) properties. At the same time, the 1,3,4-thiadiazole fragment appears in a number of clinically used drugs such as acetazolamide; methazolamide; butazolamide (diuretic); sulfamethiazole (antibacterial); cefazolin, cefazedone (antibiotic); atibeprone (anti-depressant); glybuthiazole, glybuzole (antidiabetic); and tebuthiuron (insecticide) (Wilson and Gisvold, 1991;Abrahum, 2003;Supran et al., 2003).
Schiff bases, contain an azomethine group, derived from aromatic aldehydes and aromatic amines, have potential for both chemical and biological activities (Dhar and Taploo, 1982;Pacheco et al., 1970). This is due to the presence of carbon-nitrogen double bond having potential receptorbinding ability. Schiff bases are also one of the intensively investigated classes of aromatic and heteroaromatic compounds. This class of compounds showed a variety of applications ranging from anticancer (Sharma et al., 1998;Kuzmin et al., 2005), antibacterial (More et al., 2002;Vaghasiya et al., 2004), diuretic (Supran et al., 1996), antifungal (Manrao et al., 1982(Manrao et al., , 1995(Manrao et al., , 2001 and antiparasitic activity (Rathelot et al., 2002). They have also medicinal importance and are used in drug design due to their activity against a wide range of organisms (Khan et al., 2002;Verma et al., 2004). Schiff bases are used as substrates in the preparation of a number of industrially and biologically active compounds via closure, cycloaddition and replacement reactions (Taggi et al., 2002).
There are many connections between carbonic anhydrase (CA) and cancer (Supuran, 2008;Supuran and Scozzafava, 2000;Pastorek et al., 1994;Pastorekova et al., 1997;Chegwidden et al., 2001). It is well known that some CA isozymes are predominantly found in cancer cells and are lacking from their normal counterparts (Pastorek et al., 1994;Pastorekova et al., 1997;Chegwidden et al., 2001), and these are two transmembrane isozymes CA IX and CA XII. Isozyme CA XIV was the last one to be discovered among the 15 CA isoforms of this widespread metalloprotein known up to now in human . Kaunisto et al. (2002) and Parkkila et al., (2001Parkkila et al., ( , 2002 revealed CA XIV distribution in the human body as well as potential physiological/pathological roles. It has been observed that hCA XIV is highly abundant in the brain, kidney, colon, small intestine, urinary bladder, liver and spinal cord Parkkila et al., 2001Parkkila et al., , 2002Fujikawa-Adachi et al., 1999;Ashida et al., 2002). Similar to isozymes CA IX and CA XII, CA XIV is a transmembrane protein with the active site oriented extracellularly, but unlike the first two proteins, isozyme XIV is not associated with tumour cells (Pastorek et al., 1994;Kaunisto et al., 2002;Parkkila et al., 2001Parkkila et al., , 2002Ashida et al., 2002). Membrane-associated human carbonic anhydrase (hCAs) isozymes IX, XII and XIV (Fujikawa-Adachi et al., 1999;Tureci et al., 1998) like other hCAs regulate pH and carbon dioxide (CO 2 )-bicarbonate anion (HCO 3 ) homoeostasis, through the catalysis of the reversible hydration of CO 2 to give HCO 3 and proton (Hþ). The expression level of isozymes hCA IX and XII is elevated in response to hypoxia, and research on the involvement of these isozymes in cancer has progressed considerably in recent years, particularly for hCA IX (Tureci et al., 1998;Wykoff et al., 2000;Parkkila et al., 2000;Svastova et al., 2004;Cecchi et al., 2005). It has been confirmed that hCA IX is a high-activity CA isozyme responsible for the extracellular acidification (pHe) of the tumour microenvironment. Multiple downstream effects of this reduced pHe are associated with tumour progression and poor prognosis (Parkkila et al., 2000;Svastova et al., 2004). Aromatic sulphonamide compounds have been shown to reverse the effect of tumour acidification, to inhibit the growth of cancer cells and to suppress tumour invasion mediated by these CAs (Tureci et al., 1998;Wykoff et al., 2000;Parkkila et al., 2000;Svastova et al., 2004;Cecchi et al., 2005;Brzozowski et al., 2010).
Thus, the data from these many physiological studies appear to have identified a CA-mediated, hypoxic tumourspecific pathway. This provides firm grounds for exploring the effects of this class of compounds as a novel approach to discriminate between healthy cells and cancerous cells, specifically targeting hypoxic tissues, an attractive attribute that is lacking in many existing cancer therapies (Minchinton and Tannock 2006;Kamb, 2005).

Pharmacological evaluation
Antioxidant and free radical scavenging activity ABTS Á1 radical, lipid peroxidation, DPPH radical, superoxide anion and nitric oxide anion radical scavenging activity has been used as a quick and reliable parameter to assess the in vitro antioxidant activity. Each method relates to the generation of a different radical, acting through a variety of mechanisms and the measurement of a range of end points at a fixed time point or over a range Rice-Evans, 1994, 1996). The different concentrations of the synthesized compounds showed antioxidant activities in a dose-dependent manner. Comparative IC 50 (nM/mL) inhibitory concentrations of synthesized compounds against different free radicals are reported in Table 1. All the tested compounds showed statistically significant (P \ 0.05) IC 50 values. Among the tested compounds, (9c) is the most potent compound and had lowest IC 50 (nM/mL) value against DPPH radical, nitric oxide anion and lipid peroxidation, while (9e) and (9f) showed maximum potency against ABTS Á1 radical and superoxide anion radical, respectively. The study also indicates that the compounds (9c), (9d) and (9f) showed the smaller IC 50 (nM/mL) values even than respective standards, indicating that these compounds are more potent than the standard, and reveals that the electron-donating functional group like -OCH 3 (9c and 9d) or the functional group like -OH having the ability to bind with free radical (9f) is responsible for the potency.

Antimitotic activity
The levels of the physicochemical parameters of Allium cepa (root number and root length) were recorded after treatment with various drugs at 0, 48 and 72 h and found to cause significant inhibition in the growth of roots in comparison with negative control and positive control. From the observations, it has been revealed that average root length in (9f) treatment group was decreased significantly (1.06 cm) compared with that of the negative control (3.93 cm) after 72 h of treatment. The root morphology was nearly normal during the negative control treatment, but at positive control and synthesized compound groups, the roots morphology showed an obvious difference in its appearance in that it turned to slightly yellowish to brownish in colour. Its cytotoxic effect was evident in the form of shortening and decaying of roots, while progressive increases in root length and root numbers were observed in control group. The cytotoxic effect of tested compounds inhibits root growth and mitosis to a significant extent. The compound 9f showed lowest mitotic index (0.41 %) with highest activity among all the treatment groups, and it was also observed that the number of nondividing cells increased in all treatment groups other than negative control. As there is no antimitotic principle in water, it was considered as negative control. Ethyl methanesulphonate (EMS) was treated as positive control treatment group and induces DNA damage by a direct mechanism, acting at various sites as a monofunctional ethylating agent of nucleotides (Budavari, 1989;Sega, 1984).

Cytogenetic analysis
With the objective of investigating the possible mechanism involved in root growth inhibition, cytogenetic analysis was performed (Angayarkanni et al., 2007;Auti et al., 2010;Pavlica et al., 2000). All the tested compounds provoked strong inhibition of the mitotic index, where a statistically significant difference in relation to the control, and the decrease in the mitotic index was positively correlated with the electron-releasing group (Table 2). Changes in chromosome and cellular morphology were observed with increasing time. Partial c-mitosis (colchicine-like mitosis) and full c-mitosis, with partially functional spindles and completely normal mitotic phases, were seen in the various cells of the same root tip between 6-and 72-h time period. Cytogenetic alterations were investigated, and the results are depicted in Table 2. All the tested compounds induced chromosome and cytological alterations in treatment groups. An analysis of chromosome aberrations showed that most of the fragments detected in the different treatments were of chromosome type. The observation of chromosome breaks showed the clastogenic effect of tested compounds. The occurrence of chromosome fragments allows observation of statistically significant differences at tested synthesized compounds. In addition to the chromosome fragments, sticky metaphase and polar deviations (wrong directions of chromosome movement) were also observed. In general, it is possible to observe an increase in different abnormalities as the nucleophilic functional group concentration increased. In Allium test, a strong toxic effect of tested compounds was observed, supported by great occurrence of sticky metaphases, leading to cellular death (mitotic index decrease). All the tested compounds produced a significant decrease in mitotic index were time dependent at the treatment of 1 mg/mL. There was a statistically significant increase in total aberrant cells (P \ 0.05) (aberrant cells include chromosome breaks, thickness and polar deviation) as compared with the negative control (Table 2); however, the highest value of aberrant cells is shown by the positive control. Statistical analysis showed that the genotoxic activities of the tested compounds induced micronuclei in the root tip meristem cells of A. cepa. Micronucleus formation in 1,000 cells per slide (%MNC value) was also increased in tested compounds and in positive control EMS compared with negative control, which is statistically significant (P \ 0.05).
In the light of the results obtained in the present study, these observations above may be due to the genotoxic and nucleotoxic action of the compounds or the disturbance of the formation of spindle fibres during cell division, which leads to chromosomal aberrations. Stickiness and clumping of the chromosomes were some of the most common effects of these tested compounds on the treated root tips. Stickiness usually leads to the formation of anaphase and telophase bridges, and this ends up inhibiting post-telophase, metaphase and cytokinesis, respectively, and thus hampering cell division.

In vitro cytotoxicity activity by MTT assay method
All the synthesized compounds prepared by Scheme I and previously reported (Chhajed et al., 2007(Chhajed et al., , 2013 compounds were subjected to anticancer activity. CTC 50 (cytotoxic concentration at which 50 % of the cells are dead after drug exposure) determined for test and standard compound with the help of MTT assay HEK 293 (epidermal kidney cell line), BT474 (breast cancer cell line) and NCI-H226 (lung cancer) cell lines by MTT method (Freshney, 2000;Edmondson et al., 1988;Prasad et al., 2005;Chiruvella et al., 2008). The viability of control cells was designated as 100 %, and the others were expressed as percentage compared to the control. The results were compared with standard drug indisulam (ISL). The results demonstrated a strong dose-dependent growth inhibition in treated cell lines. It showed that different cells had a  compounds showed that the presence of NH linker between aryl moiety which is substituted by electron-withdrawing group and 1,3,4-thiadiazole ring has been recognized as potent anticancer agent. Substitution on phenyl ring with chloro, methoxy and nitro group gives better anticancer activity. The order of cytotoxic activity was electron-withdrawing group on phenyl [ electron-donating group on phenyl [ phenyl.
We can conclude that electron-releasing group on phenyl ring is responsible for less activity.

Conclusion
Thiadiazoles are mesoionic system, a poly-heteroatomic system containing a five-membered heterocycle associated with a conjugation of p and p electrons and distinct regions of positive and negative charges leading to highly polarizable derivatives. This distinctive characteristic allows mesoionic compounds to effectively cross-cellular membranes and interact with biological molecules in unique ways. The good liposolubility of the sulphur atom in the heterocycle might also have a positive effect on the biological activity and pharmacokinetic properties of thiadiazole-containing compounds. The thiadiazole ring possesses similar chemical properties to the pyrimidine ring and can be considered a bioisostere. Given that the pyrimidine structure is found in nucleobases, components of nucleotides and the building blocks of DNA and RNA, it seems likely that thiadiazole could readily interact with DNA and RNA, potentially explaining the broad and often potent activity. Furthermore, this activity against DNA suggests that thiadiazoles derivatives could potentially be used for chemical intervention at the gene level. Compounds containing thiadiazole with high potency have been reported here, and some of them displayed excellent activities against a range of tumour cells. The ability of thiadiazoles to target DNA could explain their potential anticancer activity as uncontrolled DNA replication/cell division is a hallmark of neoplastic diseases. Furthermore, the heteroatoms of the thiadiazole are able to form interactions, such as hydrogen bonds, with biological targets that include key kinases that participate in tumorigenesis, such as CA IX and XII. The sulfonyl group of sulphonamides is similar to the carbonate ion and can competitively inhibit CAs. Compounds containing a thiadiazole, a benzene bioisostere, should also possess high inhibitory activity when bonded with a sulphamide group. From lead compound, acetazolamide, some of the most potent compounds were synthesized and evaluated several sulphonamides as inhibitors of in vitro cancer cell growth compared with selective hCA IX inhibitor, indisulam. The affinity of 1,3,4-thiadiazole for hCA increases significantly when substituted with sulphonamides connected with Schiff base. These results indicate that the thiadiazole ring has receptor-binding ability in the context of hCA IX inhibition and in the prevention of cancer associated with CA.

Synthetic study
Melting points were determined in one-end-open capillary tubes on a Thermonik Precision melting point apparatus (C-PMP-2, Mumbai, India) and presented without any corrections. The IR spectra (m, cm -1 ) were recorded in KBr tablets using Shimadzu FT-IR 8400s spectrophotometer. 1 H nuclear magnetic resonance ( 1 H-NMR) spectra were recorded for the compounds on Varian EM-390 apparatus by using TMS as an internal standard. 13 C-NMR spectra were recorded for the compounds on Bruker Avance II 400 NMR Spectrometer apparatus using TMS as an internal standard, and chemical shifts are reported in ppm (d-scale).
Elemental analysis of the obtained compounds was performed for C, H, N, S using Elemental Vario EL III Carlo Erba 1106 analyzer. The maximum percentage differences between calculated and found values for each element were within the error and amounted to ±0.4 %. The completion of reaction and the purity of the obtained compounds were checked by TLC on aluminium oxide 60 F254 plates (Merck Co., Whitehouse Station, NJ, USA), in a CHCl3/C2H5OH (3:1, v/v) solvent system. The spots were developed in iodine chamber and visualized under ultra violet lamp (k = 254 nm).
Lipid peroxidation inhibitory activity Egg lecithin (3 mg/ mL phosphate buffer, pH 7.4) was sonicated in an ultrasonic sonicator for 10 min to ensure proper liposome formation. Test samples or standard, ascorbic acid (100 lL) of different concentrations (10, 20, 30, 40 50 and 100 lg/ mL) was added to liposome mixture (1 mL); the control was without test sample. Lipid peroxidation was induced by adding ferric chloride (10 lL, 400 mM) and L-ascorbic acid (10 lL, 200 mM). After incubation for 1 h at 37°C, the reaction was stopped by adding hydrochloric acid (2 mL, 0.25 N) containing trichloroacetic acid (150 mg/ mL), thiobarbituric acid (3.75 mg/mL) and butylated hydroxy anisole (0.50 mg/mL). The reaction mixture was subsequently boiled for 15 min, cooled and centrifuged at 1,000 rpm for 15 min, and the absorbance of the supernatant was measured at 532 nm (Duh and Yen, 1997). The IC 50 values of all tested compounds are reported in Table 1. The % inhibition at different concentrations was calculated by the following formula where V t = mean absorption of test compound, V c = mean absorption of control.
The IC 50 (nM/mL) value was derived from the % inhibition at different concentrations.
DPPH radical scavenging activity Compounds of SC series were evaluated for their in vitro free radical scavenging activities by 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay method (Blois, 1958;Shishoo et al., 1999;Chhajed et al., 2007). To determine the free radical scavenging activity, a method based on the reduction of a methanolic solution of the coloured DPPH radical was used. To a set of test tubes containing methanol (3 mL), DPPH reagent (2 mg/mL) (50 lL) was added. The initial absorbance was measured. To these test tubes, methanolic solution of different test solutions (1 mg/mL) were added (10-50 lL). Ascorbic acid (0.5 mg/mL) was also added in the concentration of 10, 20, 30, 40, 50 and 100 lL. After 20 min, absorbance was recorded at 516 nm. The experiment was performed in triplicate. The percentage reduction in absorbance was calculated from the initial and final absorbance of each solution (Dhar and Taploo, 1982). The IC 50 (nM/mL) values are shown in Table 1.
Superoxide anion radical scavenging effect Measurement of superoxide anion scavenging activity of the synthesized compound was taken based on the method described by Nishimiki et al. (1972) and slightly modified. About 1 mL of nitroblue tetrazolium (NBT) solution (156 lM NBT in 100 mM phosphate buffer, pH 7.4), NADH solution (1 mL) (reduced form of b-nicotinamide adenine dinucleotide) (468 lM in 100 mM phosphate buffer, pH 7.4) and sample solution (0.1 mL) of compounds (10,20,30,40,50 and 100 lg) in distilled water were mixed and the reaction started by adding phenazine methosulphate (PMS) solution (100 lL) (60 lM PMS in 100 mM phosphate buffer, pH 7. 4). The reaction mixture was incubated at 25°C for 5 min, and the absorbance at 560 nm was measured against blank samples. Catechin was used as reference compound. All the experiments were performed in triplicate, and the results were averaged. The percentage of inhibition was determined by comparing the results of control and test samples. The IC 50 (nM/mL) value are depicted in Table 1.
Nitric oxide radical scavenging effect Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions, which were measured by the Griess reaction (Marcocci et al., 1994;Green et al., 1982). Scavenger of nitric oxide competes with oxygen leading to reduced production of nitric oxide (Mondal et al., 2006). The reaction mixture (3 mL) containing sodium nitroprusside (10 mM) in phosphatebuffered saline (PBS) and the compounds in different concentrations (10, 20, 30, 40, 50 and 100 lg) were incubated at 25°C for 150 min. At every 30-min interval, the incubated sample (0.5 mL) was removed and Griess reagent (1 % sulphanilamide, 0.1 % naphthylethylene diamine dihydrochloride in 2 % H 3 PO 4 ) (0.5 mL) was added. The absorbance of the chromophore formed was measured at 546 nm. All the analyses were performed in triplicate, and the results were averaged. The percentage inhibition of nitric oxide generated was measured by comparing the absorbance values of control and test. Curcumin was used as a reference compound. The IC 50 (nM/mL) values are reported in Table 1.
In vitro antimitotic activity by Allium cepa (onion) meristem root model Small bulbs (1.5-2.0 cm in diameter) of the common onion, A. cepa (2n = 16), were purchased from vendor at a local market. Prior to initiating the test, the outer scales of the bulbs and the dry bottom plate were removed without destroying the root primordia. The roots of A. cepa were grown in distilled water in Erlenmeyer flasks (200 mL capacity) under laboratory conditions (dark 24°C). For each synthesized compound sample, after reaching a length of 3 cm (±0.5 cm), a series of six bulbs were placed in distilled water (pH 7.3) for 48 h and then onion roots were treated with the synthesized compound at 1 mg/mL concentrations of each tested compound. The test tubes were kept in an incubator at 22 ± 1°C, and the test samples were changed daily at the same time. Several of the newly formed root tips were then cut from each bulb and examined for any visible morphological abnormalities. The bulbs with satisfactory root lengths (2-2.5 cm) were used in the study, while those with exceptionally long or short roots were discarded (on average 2-3 bulbs). Therefore, individual sets of five bulbs were used for each extract sample.
Distilled water (pH 7.3) was used as a negative control, and EMS (2 9 10 -2 M) used as a positive control mutagen (Fiskesjo, 1993(Fiskesjo, , 1997. After 24 h of exposure, several root tips were removed from the bulbs, fixed in 3:1 (v/v) ethanol (90 %)/glacial acetic acid (45 %) and stored overnight at 4°C. The next day, they were placed in 70 % (v/v) aqueous alcohol and refrigerated until used. Allium roots were softened by digesting with HCl and rinsed the roots in water. After removing the water from the third rinse, the roots were covered with the orcein acetate stain. The roots were incubated in the stain for 12 min. During this time, the very tip of the root begins to turn red as the DNA stains the numerous small actively dividing cells at the tip. A root was transferred to the centre of a clean microscope slide, and a drop of water was added. Using a razor blade most of the unstained part of the root was cut off and discarded. The root tip was covered with a cover slip and then carefully pushed down on the cover slide with the wooden end of a dissecting probe. Care should be taken to push hard, but do not twist or push the cover slide sideways. The root tip should spread out to a diameter about 0.5-1 cm. Five slides were prepared per bulb.
Determination of cytotoxicity and genotoxicity The following parameters were used for the determination of cytotoxicity and genotoxicity: (i) the mitotic index (MI) was calculated as the ratio between the number of mitotic cells and the total number of cells scored and expressed as percentage using following formula as per standard procedures.

Mitotic index ¼ Number of dividing cells Total number of cells Â 100
(ii) Chromatin aberrations (stickiness, breaks and polar deviation) were used as end points for the determination of cytogenetic effects, and micronuclei (MNC) were scored in interphase cells per 1,000 cells (% MNC) (Freshney, 2000). (iii) The most frequent abnormalities are shown in microphotographs. After 72 h of exposure to the test samples, the root lengths were measured and used as an index of general toxicity. The results for mitotic index and root length are expressed as percentage of the negative and positive controls. Visible morphological modifications, such as changes in root consistency and colour as well as the presence of swelling (c-tumours), hooks or twists in the roots, were also observed.

In vitro cytotoxicity activity by MTT assay method
Cell line and culture medium The cancer cell line cultures of HEK 293 (epidermal kidney cell line), BT474 (breast cancer cell line) and NCI-H226 (lung cancer) were obtained from Pasteur Institute of India, Coonoor, India, and were cultured in RPMI-1640 and 10 % heat-activated New born calf serum with antibiotics [penicillin (1,000 I. U./mL), streptomycin (100 lg/mL) and amphotericin B (25 lg/mL)]. The cells were maintained at 37°C in a humidified atmosphere with 5 % CO 2 and were subcultured twice a week.
Determination of cytotoxicity by microculture tetrazolium (MTT) assay The monolayer cell culture (100 lL) was trypsinized, and the cell count was adjusted to 3. 0 9 10 5 cells/mL using medium containing 10 % new born calf serum. To each well of the 96-well microtitre plate, the diluted cell suspension (approximately 10,000 cells) (0.1 mL) was added and kept for 24 h in incubator at 37°C in 5 % CO 2 atmosphere for cell monolayer formation. After 24 h, when a partial monolayer was formed at the bottom of the well, the supernatant was flicked off, the monolayer was washed once, and different drugs, i.e. synthesized compounds (100 lL), were added to the cells in microtitre plates. The plates were then incubated at 37°C for 3 days in 5 % CO 2 atmosphere, and microscopic examination was carried out and observations recorded every 24 h. After 72 h, the sample solution in the wells was flicked off; MTT dye (50 mL) was added to each well; plates were gently shaken and incubated for 4 h at 37°C in 5 % CO 2 incubator. The supernatant was removed and propanol (50 lL) was added; the plates were gently shaken to solubilize the formed formazan. The absorbance was measured using a microplate reader at a wavelength of 490 nm (Edmondson et al., 1988;Prasad et al., 2005;Chiruvella et al., 2008;Chang et al., 2007).