2.1 General information

The melting points of the all the synthesized compounds were measured on a liquid paraffin bath in open capillary tubes and are uncorrected. The progress of the chemical reaction as well as the purity of the target compounds was checked by using TLC plates, pre-coated with silica gel G in solvent systems of toluene: ethyl acetate: formic acid (5:4:1, v/v/v) and benzene: acetone (9:1, v/v). The spots on TLC plates were visualized after exposing to iodine vapors or under UV-light. Chemical synthesis was carried out in a scientific microwave synthesizer (model No. CATA-R, Catalyst systems, India). Flash chromatography technique was used to purify the target compounds using hexane and ethyl acetate mixture as an eluent mixture. ¹H- and ¹³C-Nuclear magnetic resonance (NMR) spectra of the pure compounds in DMSO-d₆/CDCl₃ were recorded on Bruker spectrospin DPX-300 MHz instrument. Tetramethylsilane was used as an internal reference and the exchangeable protons of OH and NH were confirmed by the D₂O shaking test. Mass spectra were recorded on LCMS/MS (Perkin–Elmer and LABINDIA, Applied Biosystem) model no. API 3000 and is presented as m/z. IR spectra of the compounds were recorded on FT/IR (Jasco, Japan), model no. 410. An elemental analysis was done on a Perkin–Elmer 240 analyzer and was found in the range of ±0.5% for each element analyzed (C, H and N).

2.2 Synthesis

2.3 In vitro anticancer methodology

The medium RPMI 1640 having a 5% fetal bovine serum and 2 mM l-glutamine was used to grow the human tumor cell lines. The Microtiter plates were inoculated with the cancer cells followed by incubation at ideal conditions for growth, such as 37 °C temperature, 5% CO₂, 95% air and 100% relative humidity for a period of 24 h before treatment with the tested compounds. After the incubation for 24 h, cell population of each cell line at the time of sample addition (Tz) was measured by fixing two plates of each cell line with TCA in situ. The sample was dissolved in DMSO at 400-fold of the desired final maximum test concentration and stored in frozen conditions until its further use in experiments. An aliquot of frozen concentrate of test sample was shaken to liquefy the content at the time of sample addition to cell lines and diluted to twofold with the complete medium containing 50 μg/mL gentamicin of the desired final maximum concentration. In addition to this, another four, 10-fold or ½ log serial dilutions were prepared to have five different concentrations and a control. The different dilutions of sample in aliquots of 100 μL were added to the appropriate Microtiter wells having 100 μL of medium to obtain the desired final sample concentrations. After the addition of sample, the plates were again incubated for an additional 48 h at the standard temperature, air and humidity conditions. An accurately measured 50 μL of cold 50% (w/v) TCA (final concentration, 10% TCA) was gently added to fix the cells in situ and incubated at 4 °C for a duration of 60 min. The supernatant was rejected and the plates were thoroughly washed five times with tap water and finally dried in air. A 4% (w/v) solution of Sulforhodamine B (SRB) (100 μL) prepared in 1% acetic acid was added to each Microtiter well and the plates were incubated at room temperature for another 10 min. A 10 mM Trizma base was subsequently added to solubilize the bound stain and the absorption of resulting mixture was recorded at 515 nm using an automated micro plate reader (Grever et al., 1992; Monks et al., 1991). A total of seven absorbance (optical density) readings were recorded at [time zero, (Tz), control growth (C) and test growth in the presence of sample at five concentration levels (Ti)]. The Percentage Growth (PG) i.e. the effect of the compound on growth of a cell line was calculated as by using the following formula: $$\begin{array}{c}\text{If}(\text{Mean OD test}-\text{Mean ODtzero})>0.\text{then}\hfill \\ \text{PG}=100\times (\text{Mean ODtest-Mean ODtzero})/(\text{Mean ODcrtl}-\text{Mean ODtzero})\hfill \\ \text{If}(\text{Mean ODtest}-\text{Mean ODtzero})<0.\text{then}\hfill \\ \text{PG}=100\times (\text{Mean ODtest}-\text{Mean ODtzero})/\text{Mean ODtzero}\hfill \end{array}$$ where Mean OD_tzero represents an average of optical density (OD) measurements of SRB-derived color just before exposure of cells to the screened sample. Mean OD_test is the average of optical OD measurements of SRB-derived color after 48 h exposure of cells to the screened sample. Mean OD_ctrl stands for an average of OD measurements of SRB-derived color after 48 h with no exposure of cells to the test compound. Each concentration was expressed as the log₁₀ (molar or μg/mL) and the response parameters values of GI₅₀, TGI and LC₅₀ were interpolated which indicate the concentrations at which the PG is +50, 0 and −50 respectively.

The IC₅₀ value has been renamed by NCI into special concentration parameters (GI₅₀, TGI and LC₅₀). Percentage growth inhibition was calculated as follows: $$\begin{array}{c}[(\text{Ti}-\text{Tz})/(\text{C}-\text{Tz})]\times 100\text{for concentrations for which Ti}>/=\text{Tz}\hfill \\ [(\text{Ti}-\text{Tz})/\text{Tz}]\times 100\text{for concentrations for which Ti}<\text{Tz}\hfill \end{array}$$

Three dose response parameters viz. GI₅₀, TGI and LC₅₀ were calculated for each cell line subpanel. GI₅₀ or Growth inhibition of 50% was deduced from [(Ti − Tz)/(C − Tz)] × 100 = 50 (It is the drug concentration which results in a 50% decrease in the net protein increase). Total growth inhibition (TGI) was calculated from the Ti = Tz (i.e. a concentration at which the total growth inhibition is 100%) while LC₅₀ was calculated from [(Ti − Tz)/Tz] × 100 = −50 (concentration of the drug which results in a 50% reduction in the measured protein at the end of the drug exposure as compared to that in the beginning) indicating a net loss of the cells (Holbeck et al., 2010; Boyd and Paull, 1995). The dose response curve is plotted to get a fair idea about the growth percentage inhibition of cell lines at a particular concentration of the tested sample. The points at which the curve crosses the horizontal grid lines correspond to parameters; GI₅₀ (crosses at +50 line), TGI (crosses at 0 line) and LC₅₀ (crosses at −50 line), respectively.

2.4 Molecular docking studies

The molecular docking studies were performed with the help of a Maestro 9.0 docking software (Schrodinger Inc. USA) on the 3D structure of DNA topoisomerase complex enzyme. A windows 7 based 64 bit operating systems using an HCl computer [Intel (R) Core (TM) i5-2400 CPU @ 3.10 GHz, 8 GB memory] was used to carry out the docking studies. The 3D structure of DNA topoisomerase enzyme for the study was downloaded from the Protein Data Bank (PDB ID: 1SC7). It has 96% similarity with the human cell enzyme and all active site residues in the vicinity of cofactor have exact counterparts. The downloaded structure was further refined for ideal docking results (Staker et al., 2005). The PDB enzyme structure was thoroughly analyzed for missing atoms, bonds and/or contacts. All the residues and water molecules except ligand from the enzyme structure were removed manually. A builder molecule was used to construct the ligand molecules and then to obtain a stable structure, the energy of the molecule was also minimized. With the help of a grid box, the active sites were generated on the molecule. The conformation corresponding to the lowest energy was selected and subjected to an energy minimization.

3.1 Chemistry

The target compounds were prepared as per the synthetic route outlined in Schemes 1–3. The starting material 4-(1H-benzo[d]imidazol-2-yl)-4-oxobutanoic acid (1) was synthesized by oxidative cyclization of 1,2 diaminobenzene with α-ketoglutaric acid in an acidified solution (4NHCl). The compound (1) was converted to an ethyl ester (2) by simple Fischer esterification reaction followed by treatment with hydrazine hydrate to obtain 4-(1H-benzo[d]imidazol-2-yl)-4-oxobutane hydrazide (3). The hydrazide (3) upon reaction with chloroacetic acid in the presence of cyclizing agent, (POCl₃) under microwave irradiation yielded a compound (4). The chloro group at the 5th position of the oxadiazole ring of compound 1-(1H-benzo[d]imidazol-2-yl)-3-(5-(chloromethyl)-1,3,4-oxadiazol-2-yl) propan-1-one (4) was further substituted with eight heterocyclic secondary amines. The reaction of compound (4) with substituted secondary amines was carried out in the presence of NaOAc by employing microwave radiations to accomplish the synthesis of 1-(1H-benzo[d]imidazol-2-yl)-3-(5-(methylsubstituted)-1,3,4-oxadiazol-2-yl)propan-1-ones (4a–g) (Scheme 1). The compounds of the series (5a–f); (1-(1H-benzo[d]imidazol-2-yl)-3-(5-mercaptosubstituted-1,3,4-thiadiazol-2-yl) propan-1-one) were synthesized by reacting compound 1-(1H-benzo[d]imidazol-2-yl)-3-(5-mercapto-1,3,4-thiadiazol-2-yl)propan-1-one (5) with six different chloro compounds (Scheme 2). Compound (3) was also cyclized with CS₂/KOH in ethanol to produce, 1-(1H-benzo[d]imidazol-2-yl)-3-(5-mercapto-1,3,4-oxadiazol-2-yl) propan-1-one (6) which on further treatment with hydrazine hydrate gave a compound 7, (3-(4-amino-5-mercapto-4H-1,2,4-triazol-3-yl)-1-(1H-benzo[d]imidazol-2-yl)propan-1-one). Benzimidazole clubbed triazolo compounds (7) were then condensed with two substituted methyl chloride and two carbonyl chloride compounds to get benzimidazole bearing substituted triazolo-thiadiazine and triazolo-thiadiazole derivatives i.e. 1-(1H-benzo[d]imidazol-2-yl)-3-(6-substituted-7H-[1,2,4]triazolo[3,4-b] [1,3,4]thiadiazin-3-yl)propan-1-one (8a–b) and 1-(1H-benzo[d]imidazol-2-yl)-3-(6-(substituted)-[1,2,4]triazolo [3,4-b][1,3,4]thiadiazol-3-yl)propan-1-one (9a–b), respectively (Scheme 3).

The structures of newly prepared compounds were elucidated using modern, sophisticated analytical techniques viz. FT-IR, ¹H & ¹³C NMR and mass spectral data. The results of C, H, N analysis were consistent with the proposed structures and found within ±0.5% as compared with the theoretical values. In general, for all the synthesized compounds, the absorption bands for carbonyl (C⚌O) and secondary amino (N—H) were observed in the range 1666–1728 and 3317–3429 cm⁻¹, respectively. The absorption peaks around 2599 and 1660 cm⁻¹ were accounted for S-H and C⚌N. In ¹H NMR spectra, the presence of a singlet around δ 12.3 is indicative of ring N—H and another singlet further downfield, at δ 13.4 is assigned to S—H, both these signals were disappeared upon D₂O shaking, which confirm the presence of these groups in the synthesized molecules. The appearance of peaks at δ 173.1 and 154.9 in ¹³C NMR spectra could be related to C⚌O and C⚌N. The distinct bands observed in IR spectra nearly at 1384, 1060 and 1028 cm⁻¹ were accounted for N—N⚌C, C—S—C and C—O—C which indicates the incorporation of oxadiazole and thiadiazole ring in the benzimidazole analogs. ¹H NMR spectra of all the compounds exhibited two triplets at appropriate position around δ 2.8 (J = 6.9 Hz) and 3.2 (J = 7.2 Hz) and also in ¹³C NMR spectra two signals were obtained around δ 30.2 and 27.9, which could be assigned to two methylene groups (—CH₂—CH₂—) spacer, linker or bridge groups between the benzimidazole ring and the other heterocyclic rings such as oxadiazole, thiadiazole, triazolo-thiadiazole, and triazolo-thiadiazine. The signals for benzimidazole hydrogens in proton NMR appeared as doublet, triplet at around δ 7.6, 7.4 and 7.2 (7.8 Hz, 7.5 Hz, 7.5 Hz). The characteristic signals in ¹³C NMR spectra which appeared at around δ 161.1, 159.1 are related to oxadiazole carbon ring and other signals at δ 162.3, 160.7 are indicative of the thiadiazole carbon ring. The 3D, optimization and viewer of few designed molecules such as 4f, 5e, 9a and clinically used anticancer drug, bendamustine have been shown with space fill model (Fig. 3).

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Figure 3 The 3D, optimization and 3D, viewer with space fill model of designed molecules 4f, 5e, 9b and marketed anticancer drug (Bendamustine). The different groups of structure showed with different colors as such carbon with white color, hydrogen with cyan color, nitrogen with red color, oxygen with lime color, sulfur with yellow color and chlorine with magenta color.

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3.2 In vitro anticancer screening

A total of fifteen compounds were submitted to NCI for in vitro anticancer screening against 60 human cell lines obtained from nine clinically isolated cancer types and were granted NCS-codes (Table 1). The prepared compounds were tested at a single dose and added at a concentration (1 × 10⁻⁵ M) followed by incubation of culture for the duration of 48 h. A protein binding dye “Sulforhodamine B” was used for the end point determinations (Shoemaker, 2006; Grever et al., 1992). The result of each tested compound on the growth of cells is expressed in terms of percent growth of treated cells in comparison with the untreated control cells.

Those compounds which diminished or inhibited the cell line growth to 32% or less were regarded (a negative value is suggestive of cell kills) as in vitro active (Table 1) (Corona et al., 2009). Among all the tested compounds, 4b, 4f, 4g, 5e, 8b and 9a were observed to be active against CCRF-CEM (leukemia), MDA-MB-435 (melanoma), MLT-4 (leukemia), CCRF-CEM (leukemia) and K-562 (leukemia) cell lines respectively. Except compound 4f, all other agents exhibited low antiproliferative activity. The compound 4f (NSC: 761982/1) was the most active anticancer agent which met the pre – determined criteria of growth inhibition and thus was further chosen for the NCI full panel of five dose assay method at 10-fold dilutions of five different concentrations (0.01, 0.1, 1, 10 and 100 μM) (Table 2). Compound 4f showed remarkable antiproliferative activity at all the five dosage levels and therefore, further referred to Biological Evaluation Committee of NCI for advanced study (Monks et al., 1991).

The compound 4f (NSC: 761982/1) displayed remarkable significant cytotoxic potential against all the investigated cell lines which represent diverse sub-panels with GI₅₀ values obtained between 0.09 to 16.2 μM falling within the sensitive range and exhibiting an outstanding antiproliferative activity (Table 3). The compound was also found to be sensitive against some individual cell lines and demonstrated the highest activity against CNS cancer cell lines, such as SNB-75 (GI₅₀ 0.09, TGI 1.39, LC₅₀ > 100 and log₁₀GI₅₀ −7.0, log₁₀TGI −5.86, log₁₀LC₅₀ >−4.00). The analyzed date also indicated an evident sensitivity profile against colon cancer subpanel (GI₅₀ value vary from 0.23 to 15.20 μM), least for HT29 and highest for HCT-15 cell lines. The tested compound 4f was also noted to be quite sensitive against other cell lines like in leukemia, melanoma, CNS, prostate, breast cancer, etc., and in each case the concentration required by the compound to exhibit the activity was observed to be under 2 μM. All the screened melanoma cancer cell lines were sensitive against the tested compound and GI₅₀ value was observed to be less than 1.56 μM. The maximum inhibition of growth of cell lines was noted against the SNB-75 CNS cancer cell line (GI₅₀ value 0.09 μM) and the least growth inhibitory activity against ovarian cancer, NCI/ADR-RES ovarian cancer cell line (GI₅₀ value 16.2 μM). The rest of all other subpanel cell lines exhibited maximum sensitivity against the tested compound with not more than 16.2 μM concentrations (Table 3). LC₅₀ values for the majority of the cell lines were greater than 100 μM with exception to COLO 205 and DU-145, where LC₅₀ was observed to be very low (20.2 μM & 24.3 μM, respectively) (Table 3). The log molar concentration (logGI₅₀) values of compound 4f against various cell lines ranged from −7.00 to −4.79. The minimum concentration (−7.00) was observed against CNS cancer subpanel of SNB-75 cell line, while for NCI/ADR-RES cell line of ovarian cancer subpanel, logGI₅₀ was the highest (−4.79). The majority of the cell lines of subpanel showed logTGI and logLC₅₀ values to be more than >−4.00 and except COLO 205 (colon cancer) and DU-145 (prostate cancer) cell lines for which logLC₅₀ were noted to be −4.69 and −4.61, respectively. Furthermore, a mean graph midpoint (MG-MID) value of 4f was also calculated for logGI₅₀, logTGI and logLC₅₀ parameters. MG-MID value is the averaged activity parameter of GI₅₀, TGI, or LC₅₀ values of all cell lines in the subpanel or the full panel toward the tested compound. The values were found on the lower side, logGI₅₀ (−6.04), logTGI (−4.38) and logLC₅₀ (−4.02) which indicates efficacy of the screened compound (Table 4). The selective index, which is a ratio of average sensitivity of all cell lines to the average sensitivity of all cell lines of a particular subpanel toward the tested compound, was also calculated to measure the compound selectivity toward cell lines (Rostom, 2006). The selective index values between 3 and 6 indicate moderate selectivity; ratios >6 is considered to have high selectivity toward the corresponding cell line, while compounds not meeting either of these criteria are referred to as nonselective i.e. mild selectivity toward the corresponding subpanel. Compound 4f, in the study exhibited moderate selectivity toward prostate cancer cell lines with a selective index 3.66 and observed to be mild selective against breast cancer, melanoma, leukemia and CNS cancer with a selective index of 2.78, 2.71, 2.55 and 2.03, respectively (Table 3). A dose response curve of synthetic compound 4f after exposure to various cancer cell lines in NCI60 panel was plotted between log₁₀ of respective molar concentration of the sample versus percentage growth (PGs). The horizontal grid lines for comparison purpose are drawn across the plot at percentage growth values of +50, 0 and −50. The curved lines were coded with different colors as per origin of tissues such as blue color for lung cancer; red color for leukemia cell line; the gray color for central nervous system cancer; green color for colon cancer; purple color for ovarian cancer; pink color for breast cancer cell line; coral color for melanoma; golden color for renal cancer; turquoise color for prostate cancer cell lines (Fig. 4). The plots of percentage growth in cancer cell lines vs sample concentration at five different dose levels (1 log dilutions from 10⁻⁴ mol/L to 10⁻⁸ mol/L) after treatment with compound 4f are shown in Fig. 5.

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Figure 4 Dose response curves for all cell lines in the NCI 60 panel after exposed with compound 4f (NSC: 761982) of different subpanels with originated colors and shapes.

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Figure 5 Dose response curves at NCI fixed protocol, (μM) for all cell lines with different subpanels in the NCI 60 panel after treatment with compound 4f (NCS: 761982/1). The curves of tested compound obtained from the NCI’s in vitro disease-oriented human tumor cells line on nine cancer diseases at five concentrations (1 log dilutions from 10⁻⁴ mol/L to 10⁻⁸ mol/L). The tissue originated color and shapes of NCI, subpanel cell lines indicative of growth percentage inhibition with concentration of tested sample.

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The dose response curve of compound 4f (NSC: 761982) plotted for seven subpanels of colon cancer (Fig. 6), illustrates the endpoint calculations for GI_50, TGI and LC₅₀ at five dose concentrations. A value equal to ‘0’ growth percent suggests no net growth or multiplication during the whole assay and the number of cells at remain equals to quantity at time zero. The calculated endpoints for the cell line COLO 205 of colon subpanel (red open circle) were found as GI₅₀ = 0.35, TGI = 1.36 and LC₅₀ = 20.2. All other cell lines of the colon cancer subpanel such as HCC-2998 (red open diamond), HCT-116 (red open triangle), HCT-15 (red open square), HT29 (solid blue circle), KM12 (solid blue diamond), and SW-620 (solid blue triangle) were found to be less sensitive than COLO 205 against the screened compound (Fig. 6). The in vitro anticancer activity of compound 4f, the most potent and active compound among the entire library of synthesized compounds based on rational design was also compared with the biological data obtained from the NCI web site for the clinically used anticancer drugs (Bendamustine and Chlorambucil) in terms of potency (μmol/L) by three response parameters and the results are presented in Table 5. The tabulated results clearly indicate that the compound 4f has lower mean values of log molar concentration for response parameters viz. GI₅₀ and TGI₅₀ and slightly higher mean value of LC₅₀ as compared to clinically used anticancer agents, bendamustine and chlorambucil. Also, the mean graph midpoint GI₅₀ value (arithmetical mean value of treated cancer cell lines) of the most potent compound 4f was observed to be only 2.09 μM, which is much lower than the reference anticancer agents (60 and 52 μM, respectively) suggesting that the benzimidazole endowed 1,3,4 oxadiazole compound holds promise as a potential anticancer agent. It has been reported in the literature that benzimidazole derivatives act by inhibiting DNA topoisomerase complex (Selcen et al., 2009; Singh and Tandon, 2011) and their binding mode to DNA varies from intercalation to groove binding based on the number of benzimidazole rings (Kubota et al., 1999). Therefore, there is a high probability that antiproliferative effects of compound 4f, a benzimidazole derivative which is attached to pyrimidine and 1,3,4 oxadiazole rings could be due to DNA intercalation. However, the studies focusing on the mechanism of action of these derivatives are currently under progress in our laboratory.

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Figure 6 Dose response curve of Colon cancer subpanel of compound 4f (NSC: 761982) showing end point calculations for COLO 205 cell line at five dose concentrations (1 log dilution from 10⁻⁴ mol/L to 10⁻⁸ mol/L).

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3.3 Structural activity relationship (SAR)

On the basis of the obtained results, it can be concluded that, benzimidazole analogues endowed with oxadiazole possess excellent antiproliferative activities as compared to the other benzimidazole clubbed thiadiazole, triazolo-thiadiazines and triazolo-thiadiazoles derivatives. It was observed that the presence of electron withdrawing groups such as oxygen at ortho (2nd position), meta (3rd position) or para (4th position) position on aromatic ring of target compounds influences the antiproliferative activity. Compound 4g having free oxygen group at ortho (2-one) position of phenyl ring, chemically as, 1-((5-(3-(1H-benzo [d] imidazol-2-yl)-3-oxopropyl)-1,3,4-oxadiazol-2-yl)methyl)-4-amino pyrimidin-2(1H)-one, demonstrated high sensitivity (78.75%) against NCI cancer cell lines panel and likewise when the same group is disubstituted on ortho and para (2,4-dione) positions of phenyl ring, an augment in the sensitivity (20.03%) was observed for the compound, 3-(5-(3-(1H-benzo [d] imidazol-2-yl)-3-oxopropyl)-1,3,4-oxadiazol-2-yl)methyl)-5-methylpyrimidine-2,4(1H, 3H)-dione (4f). Contrary to this, the electron donating groups such as methoxy (—OCH₃) attached at para (4th position) position of the phenyl ring, a decrease in the sensitivity (93.97%) was observed for compound 8b, namely, 1-(1H-benzo[d]imidazol-2-yl)-3-(6-(4-methoxy phenyl)-7H-[1,2,4] triazolo [3,4-b] [1, 3] thiazin-3-yl) propan-1-one. Similarly, the presence of methyl group also decreased the sensitivity (94.39%) as observed for the compound 5e, namely, 1-(1H-benzo[d]imidazol-2-yl)-3-(5-(benzylthio)-1,3,4-thiadiazol-2-yl) propan-1-one. However, the sensitivity of unsubstituted aromatic ring such as in compound 9a noted to be increased to (92.86%) in comparison with the compounds bearing electron donating groups such as methoxy and methyl as in compounds 8b and 5e.

3.4 Molecular docking studies

A number of research studies conducted elsewhere have reported that benzimidazole derivatives act by inhibiting DNA topoisomerase complex (Selcen et al., 2009; Singh and Tandon, 2011). Therefore, the molecular interactions of the compound 4f with the target protein, topoisomerase enzyme complex were studied with the help of Maestro 9.0 molecular docking software. The most fitting binding modes of compound 4f in the active site of topoisomerase enzyme (1SC7) are presented in Figs. 7 and 8.

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Figure 7 Binding mode of compound 4f into the binding sites of topoisomerase enzyme (PDB code: 1SC7) showing hydrogen bond (yellow dotted lines) with Arg 364 and pi interaction with Arg 364 and DG 12.

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Figure 8 Lig plot of compound 4f showing interaction into the binding sites of topoisomerase enzyme (PDB code: 1SC7), hydrogen bond (pink dotted line) with Arg 364 (1.73 Å and 2.39 Å) and pi interaction (green solid line) with Arg 364 (4.83 Å) and DG 12 (4.29 Å).

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Docking of the most active synthesized compound 4f into the enzyme active site yielded a number of molecular interactions showing hydrogen bond, π interaction and hydrophobic interactions between the drug and enzyme and are considered to be accountable for the noted affinity of the compound. Though, compound 4f lacks Zwitter ion but it is able to form hydrogen bonds with the enzyme through its secondary amino group of benzimidazole/pyrimidine rings and carbonyl group with the Arg 364 residue which is the same residue where the natural inhibitor binds (Staker et al., 2005). In the hydrogen bond interaction between the nitrogen (—N—) of the imidazole ring of the compound 4f and the carboxyl group (C⚌O) of the side chain residue of Arg 364 (1.73 Å), the former acts as a hydrogen bond donor while the later behaves as a hydrogen bond acceptor. Further, in the second Hydrogen bond interaction, the carbonyl group (C⚌O) of the compound 4f acts as the hydrogen bond acceptor and an amino group (N—H) of the side chain residue of Arg 364 (2.39 Å) is a hydrogen bond donor (Fig. 7). The amide group of compound 4f seems to have an important role in strong hydrogen bonding because the lone pair electrons on nitrogen atom of the amide delocalized into the carbonyl group of compound. Pi–π interactions were also observed between the compound and the binding site of enzyme, which are considered to play a significant role in the inhibitory activity. As it can be seen in the Lig plot (Fig. 8) of compound which shows the interactions with binding site, it appears that oxadiazole and phenyl ring of benzimidazole are properly oriented toward the more lipophilic area of 1SC7 binding site and form CH-π interaction with Arg 364 (4.83 Å) and DNA pointed DG 12 (4.29 Å) resides. In addition to this, several hydrophobic interactions were also observed between the phenyl ring, oxadiazole and pyrimidine ring of compound 4f with the amino acid residues of the enzyme topoisomerase including DA 14, DA 13, Tgp 11, Lys 532, Thr 718, Ile 535 and Asp 533 and shown in Figs. 7 and 8. The compound 4f was found to have a glide score value of −5.39, indicating a high affinity and better interaction between the compound and the enzyme.