Synthesis of arylpyrazole linked benzimidazole conjugates as potential microtubule disruptors

Accepted Manuscript Synthesis of Arylpyrazole Linked Benzimidazole Conjugates as Potential Microtubule Disruptors Ahmed Kamal, Anver Basha Shaik, Sowjanya Polepalli, G. Bharath Kumar, Vangala Santhosh Reddy, Rasala Mahesh, Srujana Garimella, Nishant Jain PII: DOI: Reference:

S0968-0896(15)00007-3 http://dx.doi.org/10.1016/j.bmc.2015.01.004 BMC 12007

To appear in:

Bioorganic & Medicinal Chemistry

Received Date: Revised Date: Accepted Date:

31 October 2014 1 January 2015 2 January 2015

Please cite this article as: Kamal, A., Shaik, A.B., Polepalli, S., Bharath Kumar, G., Reddy, V.S., Mahesh, R., Garimella, S., Jain, N., Synthesis of Arylpyrazole Linked Benzimidazole Conjugates as Potential Microtubule Disruptors, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.01.004

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis of Arylpyrazole Linked Benzimidazole Conjugates as Potential Microtubule Disruptors Ahmed Kamal*[a], Anver Basha Shaik[a], Sowjanya Polepalli[b], G. Bharath Kumar[a], Vangala Santhosh Reddy[a], Rasala Mahesh[a], Srujana Garimella[b], Nishant Jain[b] a

Medicinal Chemistry and Pharmacology, bCentre for Chemical Biology (CSIR - Indian Institute

of Chemical Technology, Hyderabad 500007, India. Abstract: In an attempt to develop potent and selective anticancer agents, a series of twenty arylpyrazole linked benzimidazole conjugates (10a-t) were designed and synthesized as microtubule destabilizing agents. The joining of arylpyrazole to the benzimidazole moiety resulted in a four ring (A, B, C and D) molecular scaffold that comprises of polar heterocyclic rings in the middle associated with rotatable single bonds and substituted aryl rings placed in the opposite directions. These conjugates were evaluated for their ability to inhibit the growth of sixty cancer cell line panel of the NCI. Among these some conjugates like 10a, 10b, 10d, 10e, 10p and 10r exhibited significant growth inhibitory activity against most of the cell lines ranging from 0.3-13 µM. Interestingly, the conjugate 10b with methoxy group on D-ring expressed appreciable cytotoxic potential. A549 cells treated with some of the potent conjugates like 10a, 10b and 10d arrested cells at G2/M phase apart from activating cyclin-B1 protein levels and disrupting microtubule network. Moreover, these conjugates effectively inhibited tubulin polymerization with IC50 values of 1.3-3.8 µM. Whereas, the caspase assay revealed that they activate the casepase-3 leading to apoptosis. Particularly 10b having methoxy substituent induced activity almost 3 folds higher than CA-4. Furthermore, a compititve colchicine binding assay and molecular modeling analysis suggests that these conjugates bind to the tubulin successfully at the colchicene binding site. These investigations reveal that such conjugates having pyrazole and benzimidazole moieties have the potential in the development of newer chemotherapeutic agents. Keywords: Anticancer, caspase-3, molecular modeling, pyrazole-benzimidazole conjugates, tubulin polymerization.

Corresponding

authors

Tel.:

+91-40-27193157;

[email protected] (A. Kamal).

1

fax:

+91-40-27193189,

e-mail:

1.Introduction Microtubules are very important structural components of cytoskeleton present in the cell cytoplasm and made up of polymerized α- and β-tubulin heterodimers. Microtubules involved in broad range of cellular functions such as cell division, maintenance of cell structure, cell signaling, cell migration and intracellular transport are critical to the cell survival.1-3 Moreover, these components express dynamic instability where the tubulin dimers polymerize progressively to generate long cylindrical microtubules that subsequently depolymerizes very rapidly resulting individual tubulin units.4-5 This dynamic lengthening and shortening of spindle microtubules decides largely the shape of the mitotic spindle and promotes the appropriate alignment of chromosomes at the spindle centre.6 Therefore, microtubule dynamics is one of the most attractive strategies for the development of cancer therapeutics. A variety of compounds that bind to tubulin-microtubule have been part of the pharmacopoeia of anticancer therapy for decades. In this connection, small molecules that particularly perturb microtubule network and cause mitotic arrest are of immense interest in current cancer chemotherapy.7 Microtubule targeting drugs are well known to interact with tubulin through at least by one of the following binding sites such as laulimalide, taxane/epothilone, vinca alkaloid, and colchicine sites.3 However, colchicine binding site small molecule inhibitors such as colchicine (1), combretastatin A–4 (2) and nocodazole (3) exert their vital biological effects by inhibiting tubulin assembly and perturb dynamic stability (Fig. 1A).8 In addition, some of the natural products and their designed analogues that distinguish colchicine-binding site have received considerable attention towards the discovery of cancer therapeutics. The use of colchicine has been inadequate in the treatment of cancer because of its toxicity, however it can be considered as a significant hallmark for the generation of potent chemotherapeutics.9 Hence, potential attention has risen towards the discovery and development of novel molecules that affect tubulin assembly through impeding multi-drug resistance and acute cytotoxicity. The pyrazole linked benzimidazoles were synthesized keeping in mind both the pyrazole as wellas benzimidazole basic units; benzimidazole containing nocodazole (3) a widely employed antineoplastic agent and a cell cycle synchronizing agent to induce mitotic arrest (Fig. 1A).10-11 Benzimidazole scaffold is the most privileged structure in the field of medicinal chemistry and this residue is a constituent of vitamin B12 that further supports its potential for their development as therapeutic agents (Fig. 1A).12 Another important benzimidazole derivative is 2

(PPTMB) an effective anticancer agent that significantly disrupts microtubule dynamics, leading to mitotic arrest and JNK activation, consequently induces mitochondria-related apoptotic cell death of prostate cancer cells.13 In an endeavor to develop potential anticancer agents, recently we reported terphenyl benzimidazoles as inhibitors of tubulin polymerization that arrest the cells in G2/M phase of the cell cycle.14 Some other heterocyclic compounds, like pyrazoles have received considerable attention owing to their diverse chemotherapeutic properties including versatile antineoplastic activities.15 In addition, some pyrazoles exert their anticancer potential by inhibiting various enzymes and proteins that play a critical role in the cell division.16 Additionally, tri- and tetra-substituted pyrazole derivatives are also known to exhibit potent anticancer efficacy by the inhibition of p38α MAP kinase.17 Structural combinations of heterocyclic rings in certain orders impart potent anticancer activity to the designed molecules.18 Since benzimidazole is a basic building block for various biologically active compounds including tubulin inhibitors, some 2‑aryl-4-benzoyl-imidazole (ABI-III) (4) (Fig. 1A) congeners with basic three ring scaffold (A, B and C) was designed as highly active tubulin polymerization inhibitors.19 We previously reported that a combination of two heterocyclic moieties as a single molecular scaffold could show enhanced cell growth arrest by preventing tubulin polymerization.20-21 Herein, we designed a molecular scaffold that comprises of four rings (A, B, C, D) with a benzimidazole as well as pyrazole basic units and tested for their cytotoxic potential and antitubulin activity. The A (aryl) and D (fused benzene ring of benzimidazole) rings were decorated with carefully selected substituents (-OCH3, OCH2O-, -Cl, -F and –CF3) with a view to generate potent tubulin inhibitors (Fig. 1B). The results of our investigations, which validated the underlying hypothesis is presented in the following sections. 2. Results and Discussion 2.1. Chemistry The synthesis of pyrazole linked benzimidazole conjugates 10a-t described in this study was depicted in Scheme 1. The final compounds were successfully achieved by oxidative cyclization of various o-phenylenediamines with substituted phenyl pyrazole carbaldehydes (9a-d) in the presence of sodium metabisulphite in refluxing ethanol.14 Different acetophenones were utilized in nucleophillic substitution reaction with diethyl oxalate in the presence of sodium ethoxide to 3

afford diketo esters (6a-d) which upon treatment with NH2-NH2.2HCl in ethanol produced the corresponding pyrazole esters (7a-d) in good yields.20 These pyrazole esters underwent reduction by LiAlH4 to yield (3-substitutedaryl-1H-pyrazol-5-yl)methanols (8a-d) and subsequently these alcohols were selectively oxidized by IBX in DMSO to produce

pyrazole carbaldehyde

intermediates (9a-d) in excellent yields. All the target compounds were characterized by IR, 1H NMR, 13C NMR, mass and HRMS spectral data. 2.2. Biological activity 2.2. 1. In vitro cytotoxic activity To understand the structure activity relationship (SAR) we synthesized twenty pyrazole linked benzimidazole conjugates having different substituents on the A and D-rings. A-ring constituted the electron donating group like (OCH3)3, OCH3)2, OCH3), and 3,4-(OCH2O) whereas the Dring possessed both the electron donating as well as withdrawing groups like OCH3, H, F, Cl, Cl2 and CF3. All the synthesized conjugates were evaluated for their anticancer activity against a panel of sixty human cancer cell lines of nine cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast cancer) as per the NCI protocol. Among the twenty conjugates most of them (10a, 10b, 10d, 10e, 10g, 10j, 10k, 10l, 10n, 10o, 10p, 10q, 10r, 10s) were taken up for the single dose (10 µM) screening. The mean growth values (percentage) are summarized in the supporting information (Table S1). Some of these conjugates like 10a, 10b, 10d, 10e, 10p and 10r showed profound inhibitory effect in the primary screening with the mean growth percentage range of 3.04-60.54%. Subsequently, these conjugates were evaluated in five dose screening (0.01, 0.1, 1.0, 10, 100 µM) to examine their potency. These conjugates exhibited a broad spectrum of cytotoxic activity against various cancer cell lines with the GI50 value ranging from 0.34 to 10 µM and their GI50 values (µM) are represented in the supporting information (Table S2). Moreover, these results suggest that conjugates like 10a, 10b, 10d and 10e with trimethoxy phenyl group as A-ring display profound potency than 10p and 10r that possess 3,4-(methylenedioxy) group on the same A-ring. One of the most promising compound 10b that has trimethoxy substituents on the A-ring and monomethoxy on the D-ring manifested potent antiproliferative activity with the GI50 values <1 µM, particularly against T-47D, MDAMB-435, BT-549 (breast) MOLT-4, RPMI-8226, in (leukemia), NCI-H226 (lung) and SK-MEL5 (melanoma) cancer cell lines. Whereas, in case of 10a which is devoid of any substitution on 4

the D-ring showed notable activity against MDA-MB-435 cells with GI50 value of 0.90 µM. The conjugates 10e-dichloro, 10p-unsubstitution and 10r-flouro substitution on the D-ring and a common 3,4-(methylenedioxy) on their A-ring also demonstrated significant cytotoxic effect against most of the cell lines in sub-micromolar levels, however in lesser magnitude when compared to 10a, 10b and 10d. Further all the conjugates (10a-t) were also evaluated for their antiproliferative activity against five representative human cancer cell lines namely, HepG2 (liver carcinoma), A549 (lung cancer), MCF 7 (breast cancer) Hela (cervical cancer) and DU145 (prostate cancer) as shown in Table 2. The nocodazole and CA-4 were employed as standard references. Most of these compounds shown appreciable cytotoxic effect against all the cell lines tested by MTT assay. In this study as well the 10b inhibits significantly the growth of A549 cells (IC50 = 0.7 µM) and Hela cells (IC50 = 0.9 µM) with a best average IC50 value of 1.44 µM (the IC50value is average of five celllines). Similarly, 10a which is devoid of substitution on the Dring also manifested superior growth inhibitory effect on the lung cancer cells with an IC50 value of 1.2 µM and the average IC50 value is 2.48 µM. However, the conjugate 10f, 10o and 10t (average IC50=22.2, 38.4 and 25.0 µM) with an electron withdrawing trifluoromethane group on the D-ring showed diminished cytotoxic effect, specifically on the A549 cells with IC50 value of 24.0 µM. The conjugates with a fluorine substituent such as 10c (15.8 µM), 10h (16.4 µM), 10l (30.7 µM) and 10r (10.3 µM) also shown least efficacy but not lesser than trifluoromethane bearing conjugates. Interestingly, replacement of donating groups in case of 10b, 10k and 10q to withdrawing groups (10f, 10o and 10t) on the D-ring decreases the cytotoxic activity. Therefore, the optimal activity order of substitutions on the D-ring is OCH3 > H > Cl > (Cl)2 > F > CF3. Based on these results, the conjugates anchorage with trimethoxy group on A-ring (10a-f) exert profound activity when compared to the conjugates with 3,4-(methylenedioxy) group on the same ring (10a-f) (Fig. 1B). In contrast, the conjugates with dimethoxy and monomethoxy substituents on the A ring (10g-o) display reduced cytotoxicity. Thus, an optimal activity order of substitution on the A-ring is (OCH3)3> -OCH2O- > (OCH3)2 > OCH3. Moreover, these conjugates (10a, 10b and 10d) that exhibit significant growth inhibitory effect in the NCI 60 cell line study also shows similar cytotoxic effect against MTT assay against five cancer cell lines.

5

2.2. 2. Inhibition of cellular tubulin polymerization Since conjugates of pyrazole linked benzimidazole elicit significant antiproliferative activity, we hypothesized that these conjugates can inhibit tubulin assembly. To investigate whether the cytotoxic effect of these compounds were related to an interaction with microtubule network, the most active conjugates 10a, 10b and 10d were examined for their effect on the inhibition of tubulin assembly. Thus, we incubated tubulin with varying concentrations of the potential conjugates to determine the IC50 values for antitubulin activity. The results of tubulin polymerization inhibition assay are summarized in Table 2. All the lead conjugates exhibit remarkable activity comparable to combretastatin-A4. Interestingly the conjugate 10b that showed substantial increase in antiproliferative activity also inhibits tubulin polymerization significantly with an IC50 value of 1.3 µM. However, the other two promising conjugates 10a and 10d inhibited tubulin assembly with IC50 values of 2.5 µM and 3.8 µM respectively (Table 2).

2.2. 3. Analysis of cell cycle distribution Flow cytometry is routinely employed to distinguish the population of cells in different phases of cell cycle based on the DNA content of the cells.22 The antiproliferative activity of the pyrazole linked benzimidazole conjugates correlates to the cell cycle arrest as analyzing by the cell cycle distribution. Since conjugates 10a, 10b and 10d inhibit tubulin polymerization apart from promising cytotoxicity, A549 cells were treated with 10a, 10b and 10d at 5 µM for 24 h and observed that the cells accumulated significantly in G2/M phase of the cell cycle. Cells treated with potent conjugate 10b showed 79% arrest of cells in G2/M phase. Whereas, 10a and 10d results in increase of mitotic arrest by 77% and 70% respectively, however the lower population of cells were observed in the S phase. In contrast, DMSO treated cells showed a majority of the cell content in G1 phase (67.7%) of the cell cycle (Fig. 2).

2.2. 4. Effect on cellular cyclin-B1 levels by immunoblot analysis To obtain further insight into the results of tubulin inhibitory activity, we investigated the effect of these conjugates on cyclin-B1. This protein is one of the important regulatory proteins of 6

mitosis and accumulation of cyclin-B1 is an indication for G2/M arrest.23 Thus, we treated A549 cells with lead conjugates at 5 µM concentrations for 24 h and performed immunoblot analysis for cyclin-B1. For comparision, combretastatin A-4 and colchicine were included as positive controls and tubulin as loading control. Interestingly, compounds like 10a and 10b that show potent antitubulin activity, considerably induced cyclin-B1 protein levels like colchicine as well as CA-4 treated cells. However, 10d exhibited negligible effect on the cyclin-B1 protein. Therefore, the accumulation of cyclin-B1 levels suggests that these conjugates demonstrate cytotoxic effect through cell cycle arrest at mitosis (Fig. 3).

2.2. 5. Effect of 10a, 10b and 10d on microtubule network The collection of spindle microtubules, microtubule-associated proteins (MAPs) and the microtubule organizing centres (MTOC) is called as mitotic spindle. These are key components of the cell that play a critical role in the segregation of chromosomes during cell division.24 Occurrence of irregular spindle fibres due to disrupted microtubule network is a hallmark of cells treated with antitubulin agents. The inhibitors of tubulin assembly cause severe perturbation in the microtubule dynamics leading to irregular morphology. Since some of the the pyrazole linked benzimidazoles significantly inhibit the growth of cells, tubulin polymerization and accumulate the cells in G2/M phase of the cell cycle. Therefore, it was considered of interest to understand the effect of these conjugates on the microtubule network. In order to determine if the cell cycle arrest is due to spindle abnormality, A549 cells were treated with 5 µM concentrations of 10a, 10b and 10d were and stained with tubulin antibody. Immunofluorescence analysis reveals that the cells treated with these conjugates exhibited a rounded or irregular morphology that is typical of mitotic arrest. Moreover, the chromatin was also condensed in the nuclei, suggesting that it is metaphase cell arrest. Whereas, cells treated with DMSO demonstrated a normal and intact tubulin organization. The A549 cells were also counterstained with DAPI to visualize the morphology of nuclei (Fig. 4).

2.2. 6. Distribution of soluble versus polymerized tubulin in cells

7

Since inhibition of tubulin polymerization disturbs the microtubule dynamics, we evaluated the levels of soluble versus polymerized forms of tubulin in A549 cells following treatment with 5 µM of 10a and 10b for 24 h. In addition, cells were treated with combretastatin A-4 (1µM), as positive and DMSO as negative controls in parallel experiments. Western blot analysis reveals that the amount of tubulin protein in both soluble and polymerized fractions was approximately the same in DMSO treated cells. Combretastatin A-4 treated cells exhibited a shift of tubulin from the polymerized fraction into the soluble fraction. As expected the cells treated with 10a and 10b had exhibited enhanced tubulin content in the soluble fraction. Specifically, cells treated with 10b showed a more distinct shift in tubulin balance, with most of the tubulin present in the soluble fraction similar to that of positive control. Therefore, increased tubulin in soluble fraction of cells treated with these conjugates corroborated with the inhibition of tubulin assembly and arrest of cells in G2/M phase (Fig. 5).

2.2. 7. Competitive tubulin-binding studies Based on the literature survey, it is well established that the colchicine exerts enhanced fluorescence when complexed with tubulin.25 Many reports suggest that tubulin polymerization inhibitors competitively displace the colchicine form tubulin-colchicine complex leading to a remarkable quenching of fluorescence.26 Since, the conjugates 10a, 10b and 10d exhibited significant inhibitory effects on tubulin polymerization compared to that of combretastatin A-4, it was immense of interest to evaluate their target site of binding on tubulin. In this view, a fluorescence based competitive tubulin-binding assay was performed to determine whether these conjugates bind at colchicene binding site of tubulin.27 The tubulin-colchicine complex emits fluorescence at 435 nm when excited at 380 nm. A series of experiments were performed with tubulin-colchicine complexes to record the fluorescence in the presence and absence of the test compounds 10a, 10b and 10d, whereas CA-4 and paclitaxel were employed as a positive control and negative control respectively. The results revealed that a significant decrease in fluorescence when tubulin-colchicine complexes were incubated with compounds of interest (10a, 10b and 10d) at 37 oC for 60 min. The affinity of the test conjugates towards the colchicine site is increased with concentration as evidenced by decrease in fluorescence emitted by tubulin bound colchicine complex. Similarly CA-4, a known colchicine site competitor also shown reduced 8

fluorescence in a concentration dependent manner. Further, no significant change in the fluoroscene was observed with paclitaxel, which does not affect the binding of colchicine to tubulin, clearly demonstrates that the test compounds are binding at colchicine site (Fig. 6). 2.2.8. Effect on caspase-3 The term apoptosis refers to a programmed cell death exemplify by nuclear damage, cell shrinkage and fragmentation of cellular DNA. It is well established that molecules inhibit microtubule polymerization cause mitotic arrest and eventually lead to apoptosis.28, 30 Caspase-3 is one of the essential proteins of cysteine-aspartic acid protease family that play a vital role in cell apoptosis. Therefore, the effect of these conjugates in the induction of successful apoptosis in A549 cells was investigated. Thus, cells were treated with 10a, 10b and 10d at 5 µM concentrations for 48 h and were examined for the activation of caspase-3 activity, wherein colchicine as well as combretastatin A-4 were employed as reference standards. Notably the conjugates 10a, 10b and 10d significantly activate caspase-3, thereby causing caspase-3 induced apoptosis. The optimalorder of these conjugates on caspase-3 activity is shown as 10b > 10a > 10d (Fig. 7). 3. Molecular modeling To rationalize the experimental results obtained, molecular modeling studies were performed on these promising conjugates (10a, 10b and 10d). Autodock4 was employed and these compounds were docked in the colchicine binding site of the tubulin (PDB code: 3E22).29 The colchicine binding site is generally located at the interface of α,β-tubulin heterodimers.30 The α,β-subunits are shown as light green and salmon colored ribbons respectively (Fig. 8). The amino acid residues that interacting with these conjugates are depicted as green sticks while other residues are shown as lines and the hydrogen bonds are represented as red dashes. All the promising conjugates docked with tubulin in a similar manner at the colchicine site of tubulin. The trymethoxyphenyl A-ring is buried in the α-subunit of unsubstituted is placed in the β-subunit. Although, trimethoxy phenyl group is a hallmark for the tubulin binding at colchicine site, these conjugates are built with trimethoxy substituent on A-ring. The most potent conjugate 10b with a methoxy substitution on the D-ring occupies the β-subunit, an important hydrogen bonding interactions were observed between O atom of methoxy group and OH of βtyr202 (O---H). 9

Moreover, trimethoxyphenyl group present as A-ring of 10b show polar interactions with αAsn101 and βLys254 (O---HN). Additionally, the pyrazole NH of the same conjugate formed a hydrogen bond with NH of βLeu255 (NH---NH). Whereas, benzimidazole NH develops a weak electrostatic interactions with βCys241. It is indicated that the presence of methoxy group on the D-ring of 10b favored to occupy the trimethoxy substituent of A-ring at the interface of α,β−subunits. On the other hand the binding pose of 10a also exhibited similar type of interactions comparable to 10b and sandwiched at the interface of α, β−subunits of tubulin. A strong hydrogen bond was found between O of trimethoxy group of A-ring and NH of βLys254 (O---HN). Interestingly, the O atom of the same residue forms a strong hydrogen bond with NH of the pyrazole (NH---O). In addition to these interactions a weak hydrogen bond was observed between methoxy O of the A-ring and OH of the tyr224 (O---OH). Moreover, some weak electrostatic interactions were identified with amino acid residues βval181, β met259 and

βcys241. Among the three potent conjugates 10d with a chlorine atom on the D-ring showed diminished antitubulin activity, which might be unsuccessful docking. Only a few hydrogen bonding interactions were observed with tyr224 of α-subunit and Asn258 β-subunit whereas, some hydrophobic interactions were also identified with βlys254, met259, leu255 and βcys241 of β−subunit explaining that loss in activity of compound 10d when compared to 10a and 10b. However, B and C-rings are buried at the interface of the two subunits of the tubulin. Specifically, 10b is a promising tubulin polymerization inhibitor is sandwiched between the

βLeu248, βLys254 and βcys241 of β-subunit and αAsn101 and αSer178 of α-subunit, exhibited successful molecular interactions with tubulin. A pose of cocrystalized structure colchicine superimposed with 10a also visualized to gain further insight of our findings (Fig. 8).

4. Conclusion In summary, a series of arylpyrazole linked benzimidazole conjugates (10a-t) comprising of A, B, C and D ring system were synthesized and evaluated for their cytotoxic activity against different human cancer cell lines. Among them some representative conjugates like 10a, 10b, 10d, 10e, 10p and 10r were tested through NCI five dose screen. The most potent conjugates 10a, 10b and 10d that possess trimethoxy phenyl group as A-ring exhibited profound 10

cytotoxicity in most of the cancer cell lines at submicromolar concentrations. All the synthesized compounds (10a-t) also showed significant antiproliferative activity against five cancer cell lines such as HepG2 (liver carcinoma), A549 (lung cancer), MCF 7 (breast cancer) Hela (cervical cancer) and DU-145 (prostate cancer) with the IC50 values ranging from 0.7-42 µM. A549 cells treated with some of the potent conjugates like 10a, 10b and 10d caused accumulation of the cells at G2/M phase of the cell cycle, activate cyclin-B1 protein levels and disrupt microtubule system. As hypothesized, the lead compounds 10a, 10b and 10d elicit their cytotoxic activity by inhibition of tubulin polymerization with the IC50 values 1.3 µM, 2.5 µM and 3.8 µM respectively. These representative conjugates also activate the caspase-3 leading to induction of apoptotic cell death. Furthermore, molecular modeling analysis revealed that these conjugates considerably occupied colchicine binding site of the tubulin. Finally, it is established such conjugates containing pyrazole and benzimidazole moieties could be effective inhibitors of tubiulin polymerization that are further amenable for the development of related newer molecules as potential chemotherapeutic drugs. 5. Experimental Section 5.1. General All the chemicals and reagents were purchased from Aldrich (Sigma–Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Company, Ward Hill, MA, USA), or Spectrochem Pvt. Ltd. (Mumbai, India). Progresses of all the reactions were monitored by TLC performed on silica gel glass plates containing 60 GF-254, and visualizations were achieved by UV light or iodine indicator. Purification of the compounds has done by the application of Column chromatography with Merck 60–120 mesh silica gel. 1H NMR spectra were recorded on Bruker UXNMR/XWIN-NMR (300 MHz) or Inova Varian-VXR-unity (400, 500 MHz) instruments.

13

C NMR spectra were recorded on Bruker UXNMR/XWIN-NMR (75 MHz)

instrument. Chemical shifts (δ) are reported in ppm downfield from an internal TMS standard. ESI spectra were recorded on a Micro mass Quattro LC using ESI+ software with capillary voltage 3.98 kV and ESI mode positive ion trap detector. High-resolution mass spectra (HRMS) were recorded on a QSTAR XL Hybrid MS–MS mass spectrometer. Melting points were determined with an Electro thermal melting point apparatus and are uncorrected. 5.2. General procedures 5.2.1. Synthesis of ethyl 2,4-dioxo-4-(substituted phenyl)butanoates 6(a-d) 11

To the freshly prepared sodium ethoxide added an appropriate amount of diethyl oxalate (1.0 mol) at 0 °C. After proper dissolution, different acetophenones 5(a-d) (1.0 mol) were charged slowly in small portions by maintaining the temperature at 0 °C. The stirring was continued at room temperature for 4 h and completion of reaction was confirmed by TLC. The reaction mixture was neutralized with diluted H2SO4 and extracted with ethyl acetate to afford diketo esters 6(a-d) in good yields (85-90%). These compounds were taken as such in the next step for the preparation of phenyl pyrazole esters. 5.2.2. Synthesis of ethyl 3-substituted phenyl-1H-pyrazole-5-carboxylates 7(a-d) To each of the di keto esters 6(a-d) (1.0 mol) obtained in the earlier step was added NH2NH2.2HCl (1.5 mol) in ethanol and the reaction mixture heated to reflux for 3 h. Reactions were monitored by TLC using ethyl acetate and hexane as mobile phase. The solvent was removed under vacuum then added water to the residue followed by extracted with ethyl acetate (50 ml X 4). The organic layer so obtained was dried on anhydrous NaSO4 and evaporated the solvent to yield crude products. Subsequently these compounds were purified by column chromatography using ethyl acetate and hexane. The pure compounds 7(a-d) were eluted at 30-40% of ethyl acetate with good yields (70-80%). 5.2.2.1. Ethyl 3-(3,4,5-trimethoxyphenyl)-1H-pyrazole-5-carboxylate (7a) This compound was prepared employing the method described above using trimethoxy acetophenone as starting material. Yellow solid; (yield 75.0%): Rf = 0.5 (50% ethyl acetate/hexane); 1H NMR (400MHz, CDCl3); δ 1.33-1.40 (t, 3H, J1=6.7 Hz, J2=7.5Hz, -CH3), 3.88 (s, 3H, -OCH3), 3.95 (s, 6H, -OCH3) 4.33-4.45 (q, 2H, J1=6.7 Hz, J2=7.5Hz, CH2), 7.01 (s, 2H, ArH), 7.05 (s, 1H, ArH) ppm; MS (ESI) m/z 307[M+H]. 5.2.2.2. 2 ethyl 3-(3,4-dimethoxyphenyl)-1H-pyrazole-5-carboxylate (7b) This compound was prepared employing the method described above using 3,4-dimethoxy acetophenone as starting material. Pale yellow solid; (yield 78.0%): Rf = 0.3 (30% ethyl acetate/hexane); 1H NMR (300MHz, CDCl3); δ 1.23-1.31 (t, 3H, J1=7.1 Hz, -CH3), 3.85 (s, 3H, OCH3), 3.90 (s, 3H, -OCH3) 4.19-4.32 (q, 2H, J1=7.1 Hz, -CH2), 6.88 (d, 1H, J1=7.1 Hz, -ArH), 6.94 (s, 1H, ArH), 7.21-7.28 (m, 1H, ArH) 7.29-7.34 (m, 1H, ArH) 9.67 (brs, 1H, -NH) ppm; MS (ESI) m/z 277[M+H]. 5.2.2.3. Ethyl 3-(4-methoxyphenyl)-1H-pyrazole-5-carboxylate (7c)

12

This compound was prepared employing the method described above using trimethoxy acetophenone as starting material. Yellow solid; yellow solid; (yield 80.0%): Rf = 0.3 (30% ethyl acetate/hexane); 1H NMR (300MHz, CDCl3); δ 1.23-1.37 (t, 3H, J1=6.7 Hz, J2=7.5Hz, -CH3), 3.81 (s, 3H, -OCH3), 4.17-4.36 (q, 2H, J1=6.7 Hz, J2=7.5Hz, CH2), 6.8 (s, 1H, ArH), 6.90 (d, 2H, J = 2.2Hz, ArH), 7.62 (d, 2H, J = 9.0Hz, ArH) ppm; MS (ESI) m/z 247[M+H]. 5.2.2.4. Ethyl 3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carboxylate (7d) This compound was prepared employing the method described above using trimethoxy acetophenone as starting material. Pale yellow colored solid; (yield 75.0%): Rf = 0.3 (40% ethyl acetate/hexane); 1H NMR (500MHz, CDCl3); δ 1.33-1.40 (t, 3H, J1=6.7 Hz, J2=7.5Hz, -CH3), 4.33-4.45 (q, 2H, J1=6.7 Hz, J2=7.5Hz, CH2), 6.0 (s, 2H, OCH2O), 6.86 (d, 1H, J = 8.3Hz, ArH)7.05 (s, 1H, ArH), 7.21-7.28 (m, 2H, ArH) ppm; MS (ESI) m/z 261[M+H]. 5.2.3. Synthesis of (3-substitutedphenyl-1H-pyrazol-5-yl)methanols 8(a-d) To the each pyrazole carboxylates 7(a-d) produced in the previous step was added LiAH4 (0.5 mol) in dry THF at 0 °C and continued the stirred for 1h at room temperature. After completion the reaction, added saturated NH4Cl solution drop wise to quench the LiAlH4. The solvent THF was removed under vacuum then extracted with ethyl acetate (100 ml X 4). The organic layers were dried on anhydrous Na2SO4 and evaporated ethyl acetate to yield color less solid products of (3-substitutedphenyl-1H-pyrazol-5-yl)methanols 8(a-d) (yield 70-80%). The pyrazole alcohols so produced in this step were pure, and taken as such for the oxidation. 5.2.4. Synthesis of 3-subtitutedphenyl-1H-pyrazole-5-carbaldehydes 9(a-d) To each (3-substitutedphenyl-1H-pyrazol-5-yl)methanols 9(a-d) produced in the earlier step was added IBX (1.2 mol) in DMSO and stirred for 1 h at room temperature. After completion of the reaction added ice cold water to the reaction mixture and extracted with ethyl acetate (50 ml X 4). The organic layers were dried on anhydrous Na2SO4 and evaporated the ethyl acetate to obtain pure compounds of 3-subtitutedphenyl-1H-pyrazole-5-carbaldehydes 9(a-d) in good yields (80-85%). These pyrazole carbaldehydes were as such taken in the next step for the preparation of pyrazole linked benzimidazole conjugates (10a-t). 5.2.4. 1. 3-(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde (9a) 3-(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 9a was prepared using above method by the addition of (3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)methanol 8a (2.64g. 10 mmol) IBX (3.36g 1.2 mmol). Yellow colored solid; (2.09g. yield 80%): Rf = 0.3 (40% ethyl 13

acetate/hexane); 1H NMR (400MHz, CDCl3); δ 3.82 (s, 3H, -OCH3), 3.92 (s, 6H, -OCH3), 6.857.25 (m, 3H, ArH), 9.96 (s, 1H, CHO) ppm; MS (ESI) m/z 263[M+H]. 5.2.4. 2. 3-(3, 4-dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde (9b) 3-(3, 4-dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 9b was prepared using above method by the addition of (3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)methanol 8b (2.34g. 10 mmol) IBX (3.36g 1.2 mmol). Yellow colored solid; (1.85g. yield 82%): Rf = 0.4 (40% ethyl acetate/hexane); 1H NMR (300 MHz,CDCl3); δ 3.87 (s, 3H, -OCH3), 3.91 (s, 3H, -OCH3), 6.937.04 (m, 1H, ArH), 7.27-7.47 (m, 2H, ArH), 7.86-8.10 (m, 1H, ArH) 9.93 (s, 1H, CHO) ppm; MS (ESI) m/z 233[M+H]. 5.2.4.3. 3-(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde (9c) 3-(4-methoxyphenyl)-1H-pyrazole-5-carbaldehyde 9c was prepared using above method by the addition of (3-(4-methoxyphenyl)-1H-pyrazol-5-yl)methanol 8c (2.04g. 10 mmol) IBX (3.36g 1.2 mmol). Yellow colored solid; (1.71g. yield 85%): Rf = 0.3 (40% ethyl acetate/hexane); 1H NMR (400MHz, CDCl3); δ 3.85 (s, 3H, -OCH3), 7.78-7.82 (m, 1H, ArH), 7.86-7.93 (m, 2H, ArH), 7.95-8.03 (m, 2H, ArH) 9.95 (s, 1H, CHO) ppm; MS (ESI) m/z 203[M+H]. 5.2.4.4. 3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde (9d) 3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 9d was prepared using above method by the addition of (3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)methanol 8d (2.18g. 10 mmol) IBX (3.36g 1.2 mmol). Yellow colored solid; (1.83g. yield 80%): Rf = 0.3 (40% ethyl acetate/hexane); 1H NMR (300MHz,CDCl3); δ δ 6.03 (s, 2H, -OCH2O), 7.35-7.45 (m, 1H, ArH), 7.87-7.94 (m, 2H, ArH), 8.10-8.17 (m, 1H, ArH) 10.2 (s, 1H, CHO ppm; MS (ESI) m/z 217[M+H]. 5.2.5. General procedure for synthesis of pyrazole linked benzimidazole conjugates 10(a-t) To the each 3-subtitutedphenyl-1H-pyrazole-5-carbaldehydes (9a-d) (1.0 mmol) obtained in the above step was added various substituted o-phenylenediamines (1.0 mmol) and catalytic amount of Na2S2O5 (5 mg). The reaction mixture was stirred at a temperature of 85 °C for 3-4 h and the completion of reaction was confirmed by TLC. The reaction mixture was cooled to room temperature and extracted with ethyl acetate (50 X 4 mL). The combined organic layer was washed with water, brine solution and dried over anhydrous Na2SO4. Evaporation of the solvent under vacuum yielded crude product and further this was purified by column chromatography

14

using ethyl acetate/hexane to afford pure compounds of pyrazole linked benzimidazole conjugates 10(a-t) in good yields (75-85%). 5.2.5.1. 2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10a) This compound was synthesized employing the same procedure described above by the addition of 3-(3, 4, 5-trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 9a (262 mg, 1.0 mmol) and ophenylenediamine (108 mg, 1.0 mmol). The compound was obtained as yellow solid. Yield: (285 mg, 81.4%); mp: 184-185 °C; 1H NMR (400 MHz, CDCl3); δ 3.76 (s, 6H, -OCH3), 3.84 (s, 3H, OCH3), 6.83 (s, 2H, ArH), 7.17-7.29 (m, 3H, ArH), 7.59 (s, 2H, ArH);

13

C NMR (75 MHz,

CDCl3 +DMSO-d6): δ 54.8, 59.3, 100.3, 101.6, 120.9, 124.3, 136.6, 145.4, 152.2 ppm; IR (KBr) (νmax/cm-1): ν = 3471, 3063, 1691, 1588, 1506, 1427, 1309, 1272, 1243, 1309, 1272, 1243, 1187, 999 cm-1; MS (ESI) m/z 351[M+H]; HR-MS (ESI) m/z for C19H19N4O3 calculated m/z: 351.1451, found m/z: 351.1451. 5.2.5.2.

5-methoxy-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole

(10b) This compound was prepared using the procedure described above by the addition of 3-(3,4,5trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 9a (262 mg, 1.0 mmol) and 4-methoxybenzene1,2-diamine (138 mg, 1.0 mmol). The compound obtained as yellow colored solid. Yield: (305 mg, 80.1%); mp: 190-192 °C; 1H NMR (400 MHz, CDCl3); δ 3.87 (s, 6H, -OCH3), 3.93 (s, 6H, OCH3), 6.87 (d, 1H, J = 8.8 Hz, ArH), 7.08 (s, 2H, ArH), 7.23 (s, 1H, ArH), 7.47-7.54 (m, 2H, ArH);

13

C NMR (75 MHz, CDCl3+DMSO-d6): δ 55.1, 55.6, 59.8, 100.8, 102.5, 111.1, 125.0,

137.4, 145.7, 152.9, 155.6 ppm; IR (KBr) (νmax/cm-1): ν = 3069, 2936, 2832, 1737, 1587, 1505, 1464, 1420, 1271, 1126, 1017, 994 cm-1 ; MS (ESI) m/z 381 [M+H]; HR-MS (ESI) m/z for C20H21N4O4 calculated m/z: 381.1557, found m/z: 381.1559. 5.2.5.3.5-fluoro-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10c) This compound was prepared using the procedure described above by the addition of 3-(3,4,5trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 9a (262 mg, 1.0 mmol) and 4-fluorobenzene1,2-diamine (126 mg 1.0 mmol). The compound obtained as brown solid. Yield: (285 mg, 77.4%); mp: 180-183 °C; 1H NMR (400 MHz, CDCl3); δ 3.86 (s, 3H, -OCH3), 3.93 (s, 6H, OCH3), 6.90-7.01 (m, 1H, ArH), 7.08 (m, 2H, ArH), 7.22 (s, 1H, ArH), 7.59 (s, 2H, ArH); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 54.8, 59.3, 100.2, 101.7, 108.8, 109.1, 131.6, 136.7,

15

146.7, 152.2, 156.3, 159.4 ppm; IR (KBr) (νmax/cm-1): ν = cm-1 ; MS (ESI) m/z 369 [M+H]; HRMS (ESI) m/z for C19H18N4O3F calculated m/z: 369.1357, found m/z: 369.1357. 5.2.5.4.5-chloro-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10d) This compound was prepared using the procedure described above by the addition of 3-(3,4,5trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 9a (262 mg, 1.0 mmol) and 4-chlorobenzene1,2-diamine (142 mg 1.0 mmol). The compound obtained as yellow solid Yield: 310 mg (80.5%); mp: 204-206 °C; 1H NMR (300 MHz, CDCl3); δ 3.86 (s, 3H, -OCH3), 3.94 (s, 6H, OCH3), 7.08 (s, 2H, ArH), 7.18 (d, 1H, J = 8.4 Hz, ArH), 7.24 (s, 1H, ArH), 7.51-7.62 (m, 1H, ArH), 7.64 (s, 1H, ArH);

13

C NMR (75 MHz, CDCl3+DMSO-d6): δ 54.2, 58.3, 100.0, 101.1,

120.3, 124.7, 126.1, 128.3, 135.9, 138.7, 151.5 ppm; IR (KBr) (νmax/cm-1): ν = 3280, 3170, 3165, 2956, 1740, 1596, 1508, 1483, 1440, 1326, 1248, 1136, 998 cm-1; MS (ESI) m/z 385 [M+H]; HR-MS (ESI) m/z for C19H18N4O3Cl calculated m/z: 385.1061, found m/z: 385.1062. 5.2.5.5. 5,6-dichloro-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10e) This compound was prepared using the procedure described above by the addition of 3-(3,4,5trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde

9a

(262

mg,

1.0

mmol)

and

4,5-

dichlorobenzene-1,2-diamine (175 mg 1.0 mmol). The compound obtained as yellow colored solid Yield: 310 mg (74%); mp: 218-220 °C; mp: 190-192 °C; 1H NMR (300 MHz, CDCl3); δ 3.85 (s, 3H, -OCH3), 3.93 (s, 6H, -OCH3), 7.07 (s, 2H, ArH), 7.22 (s, 1H, ArH), 7.69 (s, 2H, ArH), 12.7 (brs, 1H, -NH), 13.3 (brs, 1H, -NH); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 55.0, 59.5, 100.7, 101.9, 124.3, 137.0, 152.4 ppm; IR (KBr) (νmax/cm-1): ν = 3227, 3068, 2933, 1708, 1630, 1591, 1509, 1477, 1454, 1412, 1303, 1238, 1125, 996 cm-1; MS (ESI) m/z 419 [M+H]; HR-MS (ESI) m/z for C19H17N4O3Cl2 calculated m/z: 419.0672, found m/z: 419.0666. 5.2.5.6.

5-(trifluoromethyl)-2-(3-(3,4,5-trimethoxyphenyl)-1H-pyrazol-5-yl)-1H-

benzo[d]imidazole (10f) This compound was prepared using the procedure described above by the addition of 3-(3,4,5trimethoxyphenyl)-1H-pyrazole-5-carbaldehyde

9a

(262

mg,

1.0

mmol)

and

4-

(trifluoromethyl)benzene-1,2-diamine (176 mg 1.0 mmol). The compound obtained as light brown colored crystal Yield: 320 mg (76.5%); mp: 188-190 °C; 1H NMR (300 MHz, CDCl3); δ 3.70 (s, 6H, -OCH3), 3.83 (s, 3H, -OCH3), 6.78 (s, 2H, ArH), 7.17 (s, 1H, ArH), 7.41 (d, 1H, J = 16

8.3 Hz, ArH), 7.50-7.61 (m, 1H, ArH), 7.82 (s, 1H. ArH); 13C NMR (75 MHz, CDCl3+DMSOd6): δ 54.9, 59.4, 100.7, 101.8, 117.7, 122.0, 122.4, 122.8, 124.03, 125.6, 136.8, 144.4, 148.1, 152.3 ppm; IR (KBr) (νmax/cm-1): ν = 3147, 2942, 1591, 1505, 1469, 1418, 1332, 1247, 1164, 1126, 1051, 1002, 938 cm-1; MS (ESI) m/z 419 [M+H]; HR-MS (ESI) m/z for C20H18N4O3F3 calculated m/z: 419.1325, found m/z: 419.1319. 5.2.5.7. 2-(3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10g) This compound was prepared using the procedure described above by the addition of 3-(3,4dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 9b (232 mg 1.0 mmol) and o-phenylenediamine (108 mg, 1.0 mmol). The compound obtained as yellow colored solid Yield: 240 mg (74.9%); mp: 196-198 °C; 180-182 °C; 1H NMR (300 MHz, CDCl3); δ 3.91 (s, 3H, -OCH3), 3.94 (s, 3H, OCH3), 6.97 (d, 1H, J = 8.4 Hz, ArH), 7.19-7.24 (m, 3H, ArH), 7.33-7.36 (m, 1H, ArH), 7.40 (s, 1H, ArH), 7.58-7.68 (m, 2H, ArH); 13C NMR (75 MHz, CDCl3 +DMSO-d6): δ 54.5, 99.6, 107.8, 110.5, 113.6, 116.8, 120.8, 137.4, 145.0, 147.7 ppm; IR (KBr) (νmax/cm-1): ν = 3450, 3128, 2957, 1753, 1622, 1588, 1562, 1491, 1445, 1329, 1295, 1245, 1126, 1041, 937 cm-1 ; MS (ESI) m/z 321 [M+H]; HR-MS (ESI) m/z for C18H17N4O2 calculated m/z: 321.1346, found m/z: 321.1343. 5.2.5.8. 2-(3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)-5-fluoro-1H-benzo[d]imidazole (10h) This compound was prepared using the procedure described above by the addition of 3-(3,4dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 9b (232 mg 1.0 mmol) and 4-fluorobenzene-1,2diamine (126 mg 1.0 mmol). The compound obtained as brown colored solid Yield: (265 mg, 78.3%); mp: 179-181 °C; 1H NMR (300 MHz, CDCl3); δ 3.84 (s, 3H, -OCH3), 3.89 (s, 3H, OCH3), 6.95-7.07 (m, 2H, ArH), 7.22 (s, 1H, ArH), 7.25-7.34 (m, 1H, ArH), 7.38 (d, 1H, J = 8.9 Hz, ArH), 7.44 (s, 1H, ArH), 7.54 (s, 1H, ArH), 8.21 (brs, 1H, -NH);

13

C NMR (75 MHz,

CDCl3 +DMSO-d6): δ 54.9, 100.1, 108.2, 110.9, 114.1, 117.2, 121.3, 122.0, 137.8, 144.6, 145.7, 148.2 ppm; IR (KBr) (νmax/cm-1): ν = 3421, 3108, 2956, 2836, 1691, 1576, 1545, 1496, 1414, 1286, 1190, 1169, 1076, 947 cm-1; MS (ESI) m/z 339 [M+H]; HR-MS (ESI) m/z for C18H17N4O3F calculated m/z: 339.1257, found m/z: 339.1256. 5.2.5.9.

5,6-dichloro-2-(3-(3,4-dimethoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole

(10i) This compound was prepared using the procedure described above by the addition of 3-(3,4dimethoxyphenyl)-1H-pyrazole-5-carbaldehyde 9b (232 mg 1.0 mmol) and 4,5-dichlorobenzene1,2-diamine (175 mg 1.0 mmol). The compound obtained as brown colored solid Yield: (295 mg, 17

75.8%); mp: 192-194 °C; 1H NMR (400 MHz, CDCl3); δ 3.91 (s, 3H, -OCH3), 3.94 (s, 3H, OCH3), 6.96 (d, 1H, J = 8.0 Hz, ArH), 7.18 (s, 1H, ArH), 7.34 (d, 1H, J = 8.0 Hz, ArH), 7.38 (s, 1H, ArH), 7.62 (s, 1H, ArH), 7.71 (s, 1H, ArH), 13.25 (brs, 1H, NH);

13

C NMR (75 MHz,

CDCl3 +DMSO-d6): δ 54.4, 54.5, 99.8, 107.7, 110.3, 116.8, 123.7, 147.7 ppm; IR (KBr) (νmax/cm-1): ν = 3422, 3258, 2924, 2859, 1615, 1589, 1508, 1441, 1409, 1257, 1171, 1021, 969 cm-1; MS (ESI) m/z 390 [M+H]; HR-MS (ESI) m/z for C19H19N4O3 calculated m/z: 389.0572, found m/z: 390.0568. 5.2.5.10. 2-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10j) This compound was prepared using the procedure described above by the addition of 3-(4methoxyphenyl)-1H-pyrazole-5-carbaldehyde (9c) (202 mg, 1.0 mmol) and o-phenylenediamine (108 mg, 1.0 mmol). The compound obtained as yellow colored solid. Yield: (266 mg, 78% yield); mp: 174-176 °C; 1H NMR (300 MHz, CDCl3); δ 3.87 (s, 3H, -OCH3), 6.99 (d, 1H, J = 8.4 Hz, ArH), 7.18-7.28 (m, 4H, ArH), 7.53 (s, 1H, ArH), 7.64 (s, 1H, ArH), 7.73 (d, 2H, J = 8.1 Hz, ArH);

13

C NMR (75 MHz, CDCl3+DMSO-d6): δ 54.6, 100.1, 113.7, 121.4, 126.2, 159.0

ppm; IR (KBr) (νmax/cm-1): ν = 3326, 3162, 3010, 1725, 1655, 1610, 1580, 1525, 1476, 1468, 1270, 1215, 1162, 1115, 1080, 1040, 975 cm-1 ; MS (ESI) m/z 291[M+H]; HR-MS (ESI) m/z for C17H15N4O calculated m/z: 291.1240, found m/z: 291.1238. 5.2.5.11. 5-methoxy-2-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10k) This compound was prepared using the procedure described above by the addition of 3-(4methoxyphenyl)-1H-pyrazole-5-carbaldehyde (9c) (202 mg, 1.0 mmol) and 4-methoxybenzene1,2-diamine (138 mg, 1.0 mmol). The compound obtained as yellow colored solid Yield: (265 mg, 82.8%); mp: 188-189 °C; 1H NMR (400 MHz, CDCl3); δ 3.83 (s, 6H, -OCH3), 6.82 (s, 1H, ArH), 6.96-7.07 (m, 2H, ArH), 7.09-7.16 (m, 1H, ArH), 6.41-7.51 (m, 1H, ArH), 7.71-7.81 (m, 2H, ArH), 7.13-8.21 (m, 1H, ArH); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 53.7, 54.1, 98.9, 110.1, 112.8, 125.3, 126.4, 129.0, 130.7, 139.3, 154.6, 158.0 ppm; IR (KBr) (νmax/cm-1): ν = 3613, 3238, 2951, 2833, 1617, 1537, 1504, 1458, 1251, 1199, 1157, 1027 cm-1 ; MS (ESI) m/z 321 [M+H]; HR-MS (ESI) m/z for C18H17N4O2 calculated m/z: 321.1346, found m/z: 321.1343. 5.2.5.12. 5-fluoro-2-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10l) This compound was prepared using the procedure described above by the addition of 3-(4methoxyphenyl)-1H-pyrazole-5-carbaldehyde (9c) (202 mg, 1.0 mmol) and 4-fluorobenzene-1,2diamine (126 mg 1.0 mmol). The compound obtained as yellow colored solid Yield: (225 mg, 18

72.9%); mp: 196-198 °C; 1H NMR (400 MHz, CDCl3); δ 3.87 (s, 3H, -OCH3), 6.96-7.01 (m, 2H, ArH), 7.16 (s, 1H, ArH), 7.49-7.55 (m, 3H, ArH), 7.71 (d, 2H, J = 8.3 Hz, ArH); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 53.3, 98.8, 112.5, 124.9, 155.4, 157.6, 158.5 ppm; IR (KBr) (νmax/cm-1): ν = 3618, 3242, 2961, 1615, 1534, 1497, 1459, 1347, 1302, 1259, 1179, 1137, 1107, 1024, 960 cm-1; MS (ESI) m/z 309 [M+H]; HR-MS (ESI) m/z for C17H14FN4O calculated m/z: 309.1151, found m/z: 309.1158. 5.2.5.13. 5-chloro-2-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10m) This compound was prepared using the procedure described above by the addition of 3-(4methoxyphenyl)-1H-pyrazole-5-carbaldehyde (9c) (202 mg, 1.0 mmol) and 4-fluorobenzene-1,2diamine (126 mg 1.0 mmol) and 4-chlorobenzene-1,2-diamine (142 mg 1.0 mmol). The compound obtained as yellow colored solid Yield: (250 mg, 76.9%); mp: 184-186 °C; 1H NMR (400 MHz, CDCl3); δ 3.85 (s, 3H, -OCH3), 6.99 (d, 1H, J = 8.6 Hz, ArH), 7.11-7.20 (m, 2H, ArH), 7.49-7.69 (m, 2H, ArH), 7.72 (d, 2H, J = 8.4 Hz, ArH), 7.81 (s, 1H, ArH); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 53.5, 99.0, 112.6, 120.4, 125.0, 157.8 ppm; IR (KBr) (νmax/cm-1): ν = 3614, 3230, 3060, 2923, 2853, 1615, 1533, 1502, 1458, 1413, 1254, 1176, 1026 cm-1 ; MS (ESI) m/z 325[M+H]; HR-MS (ESI) m/z for C17H18N4OCl calculated m/z: 325.0856, found m/z: 325.0841. 5.2.5.14.

5,6-dichloro-2-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole

(10n) This compound was prepared using the procedure described above by the addition of 3-(4methoxyphenyl)-1H-pyrazole-5-carbaldehyde (9c) (202 mg, 1.0 mmol) and and 4,5dichlorobenzene-1,2-diamine (175 mg 1.0 mmol). The compound obtained as yellow colored solid Yield: (270 mg, 75.2%); mp: 189-191 °C; 1H NMR (500 MHz, CDCl3); δ 3.87 (s, 3H, OCH3), 6.99 (d, 1H, J = 8.3 Hz, ArH), 7.15 (s, 1H, ArH), 7.53 (s, 3H, ArH), 7.67-7.76 (m, 2H, ArH);

13

C NMR (75 MHz, CDCl3+DMSO-d6): δ 53.5, 99.2, 112.6, 123.1, 125.1, 132.5, 137.9,

148.0, 157.8 ppm; IR (KBr) (νmax/cm-1): ν = 3620, 3236, 3121, 2926, 2857, 1685, 1620, 1582, 1536, 1476, 1448, 1323, 1265, 1150, 1120, 1100, 1092, 1025, 966 cm-1 ; MS (ESI) m/z 360 [M+H]; HR-MS (ESI) m/z for C17H13N2OCl2 calculated m/z: 360.0466, found m/z: 360.0463. 5.2.5.15.

2-(3-(4-methoxyphenyl)-1H-pyrazol-5-yl)-5-(trifluoromethyl)-1H-

benzo[d]imidazole (10o)

19

This compound was prepared using the procedure described above by the addition of 3-(4methoxyphenyl)-1H-pyrazole-5-carbaldehyde

(9c)

(202

mg,

1.0

mmol)

and

4-

(trifluoromethyl)benzene-1,2-diamine (176 mg 1.0 mmol). The compound obtained as brown colored solid Yield: 265 mg (74.0%); mp: 203-205 °C; 1H NMR (500 MHz, CDCl3); δ 3.86 (s, 3H, -OCH3), 7.00 (d, 2H, J = 8.6 Hz, ArH), 7.17 (s, 1H, ArH), 7.45 (d, 1H, J = 8.3 Hz, ArH), 7.73 (d, 1H, J = 8.3 Hz, ArH), 7.89 (s, 3H, ArH);

13

C NMR (75 MHz, CDCl3+DMSO-d6): δ

54.7, 100.1, 109.3, 109.6, 113.8, 126.3, 156.8, 159.0, 160.0 ppm; IR (KBr) (νmax/cm-1): ν = 3619, 3233, 2968, 2847, 1617, 1535, 1507, 1463, 1442, 1332, 1265, 1161, 1122, 1027, 963 cm-1 ; MS (ESI) m/z 359 [M+H]; HR-MS (ESI) m/z for C18H14N4OF3 calculated m/z: 359.1114, found m/z: 359.1114. 5.2.5.16. 2-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-1H-benzo[d]imidazole (10p) This compound was prepared using the procedure described above by the addition of 3(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 9d (216 mg

1.0 mmol) and o-

phenylenediamine (108 mg, 1.0 mmol). The compound obtained as light yellow colored solid Yield: 195 mg (75%); mp: 206-208 °C; 1H NMR (400 MHz, CDCl3); δ 6.04 (s, 2H, -OCH2O-), 6.89 (d, 1H, J = 8.6 Hz, ArH), 7.16 (s, 1H, ArH), 7.19-7.24 (m, 1H, ArH), 7.30 (s, 1H, ArH), 7.58-7.68 (m, 4H, ArH); IR (KBr) (νmax/cm-1): ν = 3142, 2916, 1660, 1602, 1575, 1482, 1436, 1383, 1341, 1280, 1215, 1145, 1090, 960 cm-1 ; MS (ESI) m/z 305 [M+H]; HR-MS (ESI) m/z for C17H13N4O2 calculated m/z: 305.1033, found m/z: 305.1030. 5.2.5.17. 2-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-5-methoxy-1H-benzo[d]imidazole (10q) This compound was prepared using the procedure described above by the addition of 3(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 9d (216 mg

1.0 mmol)

and 4-

methoxybenzene-1,2-diamine (138 mg, 1.0 mmol). The compound obtained as light yellow colored solid Yield: (260 mg, 77.8%); mp: 186-188 °C; 1H NMR (300 MHz, CDCl3); δ 3.87 (s, 3H, -OCH3), 6.01 (s, 2H, -OCH2O-), 6.89 (d, 1H, J = 8.4 Hz, ArH), 7.13 (s, 1H, ArH), 7.26-7.31 (m, 1H, ArH), 7.49 (s, 4H, ArH);

13

C NMR (75 MHz, CDCl3+DMSO-d6): δ 53.8, 98.2, 98.9,

99.5, 103.9, 104.1, 106.9, 109.7, 117.0, 117.5, 130.4, 145.7, 146.2, 154.2 ppm; IR (KBr) (νmax/cm-1): ν = 3420, 3261, 3101, 1690, 1665, 1630, 1545, 1412, 1326, 1300, 1248, 1205, 1185, 1165, 1025, 992 cm-1; MS (ESI) m/z 335 [M+H]; HR-MS (ESI) m/z for C18H15N4O3 calculated m/z: 335.1138, found m/z: 335.1136. 20

5.2.5.18.

2-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-5-fluoro-1H-benzo[d]imidazole

(10r) This compound was prepared using the procedure described above by the addition of 3(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d (150 mg

0.0694 mmol) and 4-

fluorobenzene-1,2-diamine (126 mg 1.0 mmol). The compound obtained as light brown colored solid Yield: (240 mg, 74.5%); mp: 190-192 °C; 1H NMR (500 MHz, CDCl3); 6.03 (s, 2H, OCH2O-), 6.89 (d, 1H, J = 8.6 Hz, ArH), 6.92-6.98 (m, 1H, ArH), 7.12 (s, 1H, ArH), 7.24-7.34 (m, 2H, ArH), 7.59 (s, 1H, ArH), 7.63 (s, 1H, ArH); 13C NMR (75 MHz, CDCl3+DMSO-d6): δ 100.4, 100.7, 105.4, 108.1, 118.8, 147.0, 147.5 ppm; IR (KBr) (νmax/cm-1): ν = 3132, 3016, 2920, 1612, 1586, 1540, 1425, 1412, 1365, 1280, 1251, 1140, 1026, 975, 940 cm-1 ; MS (ESI) m/z 323 [M+H]; HR-MS (ESI) m/z for C17H12FN4O2 calculated m/z: 323.09443, found m/z: 323.09399. 5.2.5.19.

2-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-5,6-dichloro-1H-

benzo[d]imidazole (10s) This compound was prepared using the procedure described above by the addition of 3(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d (150 mg 0.0694 mmol) and 4,5dichlorobenzene-1,2-diamine (175 mg 1.0 mmol). The compound as brown colored solid Yield: (290 mg, 77.7%); mp: 180-182 °C; 1H NMR (400 MHz, CDCl3); δ 6.04 (s, 2H, -OCH2O-), 6.90 (d, 1H, J = 8.4 Hz, ArH), 7.12 (s, 1H, ArH), 7.24-7.32 (m, 1H, ArH), 7.70 (s, 1H, ArH), 7.75 (s, 2H, ArH);

13

C NMR (75 MHz, CDCl3+DMSO-d6): δ 99.8, 104.4, 104.5, 107.2, 111.2, 117.9,

121.6, 123.5, 132.5, 134.0, 139.7, 141.8, 146.2, 146.6 ppm; IR (KBr) (νmax/cm-1): ν = 3129, 2909, 1609, 1575, 1499, 1467, 1422, 1480, 1416, 1251, 1100, 1041, 971, 938 cm-1 ; MS (ESI) m/z 373 [M+H]; HR-MS (ESI) m/z for C17H11N4O2Cl2calculated m/z: 373.0253, found m/z: 373.0256. 5.2.5.20.

2-(3-(benzo[d][1,3]dioxol-5-yl)-1H-pyrazol-5-yl)-5-(trifluoromethyl)-1H-

benzo[d]imidazole (10t) This compound was prepared using the procedure described above by the addition of 3(benzo[d][1,3]dioxol-5-yl)-1H-pyrazole-5-carbaldehyde 11d (150 mg

0.0694 mmol) and 4-

(trifluoromethyl)benzene-1,2-diamine (176 mg 1.0 mmol). The compound obtained as yellow colored solid Yield: (265 mg, 71.2%); mp: 178-180 °C; 1H NMR (300 MHz, CDCl3); δ 6.04 (s, 2H, -OCH2O-), 6.90 (d, 1H, J = 8.3 Hz, ArH), 7.18 (s, 1H, ArH), 7.30 (s, 1H, ArH), 7.51 (s, 4H, ArH); IR (KBr) (νmax/cm-1): ν = 3220, 3120, 2936, 1665, 1620, 1572, 1566, 1470, 1428, 1383, 21

1245, 1160, 1125, 1104, 1092, 1010, 956 cm-1 ; MS (ESI) m/z 373 [M+H]; HR-MS (ESI) m/z for C18H12N4O2F3 calculated m/z: 373.0259, found m/z: 373.0256. 5.3. Cell Cultures, maintenance and antiproliferative evaluation All cell lines used in this study were purchased from the American Type Culture Collection (ATCC, United States). A549, MCF-7, and HeLa were grown in Dulbecco's modified Eagle's medium (containing 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C). HepG2 and DU145 cells were cultured in Eagle's minimal essential medium (MEM) containing non-essential amino acids, 1 mM sodium pyruvate, 10 mg/mL bovine insulin, and 10% FBS. Cells were trypsinized when sub-confluent from T25 flasks/60 mm dishes and seeded in 96-wel plates. The synthesized test compounds were evaluated for their in vitro antiproliferative in four different human cancer cell lines. A protocol of 48 h continuous drug exposure was used, and a MTT cell proliferation assay was used to estimate cell viability or growth. The cell lines were grown in their respective media containing 10% fetal bovine serum and were seeded into 96-well microtiter plates in 200 µL aliquots at plating densities depending on the doubling time of individual cell lines. The microtiter plates were incubated at 37 °C, 5% CO2, 95% air, and 100% relative humidity for 24 h prior to addition of experimental drugs. Aliquots of 2 µL of the test compounds were added to the wells already containing 198 µL of cells, resulting in the required final drug concentrations. For each compound, four concentrations (0.01, 0.1, 1, 10, and 100 µM) were evaluated, and each was done in triplicate wells. Plates were incubated further for 48 h, and the assay was terminated by the addition of 10 µL of 5% MTT and incubated for 60 min at 37 °C. Later, the plates were air-dried. Bound stain was subsequently eluted with 100 µL of DMSO, and the absorbance was read on an multimode plate reader (Tecan M200) at a wavelength of 560 nm. Percent growth was calculated on a plate by plate basis for test wells relative to control wells. The above determinations were repeated thrice. The growth inhibitory effects of the compounds were analyzed by generating dose response curves as a plot of the percentage surviving cells versus compound concentration. The sensitivity of the cancer cells to the test compound was expressed in terms of IC50, a value defined as the concentration of compound that produced 50% reduction as compared to the control absorbance. IC50 values are indicated as means ± SD of three independent experiments.31 5.4. Dot-blot assay

22

Cells were trypsinized when sub-confluent from T25 flasks/60 mm dishes and seeded in 6-well plates. The pyrazole-oxadiazole conjugates were evaluated for their activity against Cyclin B1. A549 cells were treated with 5 µM concentrations of (10a, 10b and 10d) for 24 h. Susbequently, cells were harvested and proteins were quantified using Amido Black followed by densitomerty analysis. Equal amount of protein were blotted on nitrocellulose membrane using Bio-Dot SF microfiltration apparatus (Bio-Rad). Briefly, nitrocellulose membrane and 3 filters papers (Whatmann 3) were soaked in IX TBS solution for 10 min. Later, the filter papers, membrane were arranged in the apparatus and connected to vacuum pump (Millipore). The membranes were rehydrated using 100 µl of 1X TBS by vacuum filtration. Subsequently, 50 µl volumes of equal protien samples were blotted on the membrane and washed with 200 µl of 1X TBS through application of vacuum. The blot was blocked with 5% blotto for 1 h at room temperature. Immunoblot analysis was performed as described previously using UVP, biospectrum 810 imaging system.32 5.5. Analysis of cell cycle A549 cells were grown in 60 mm dishes and were incubated for 24 h in the presence or absence of test compounds 10a, 10b and 10d at 5 µM 10 µM concentrations. Cells were harvested using Trypsin-EDTA, fixed with ice-cold 70% ethanol at 4 oC for 30 min, ethanol was removed by centrifugation and cells were stained with 1 mL of DNA staining solution [0.2 mg of Propidium Iodide (PI), and 2 mg RNase A] for 30 min in dark at 37°C as described earlier. The DNA contents of 20,000 events were measured by flow cytometer (BD FACSCanto II). Histograms were analyzed using FCS express 4 plus.32 5.6. Tubulin polymerization assay An in vitro assay for monitoring the time-dependent polymerization of tubulin to microtubules was performed employing a fluorescence-based tubulin polymerization assay kit (BK011, Cytoskeleton, Inc.) according to the manufacturer’s protocol. The reaction mixture in a final volume of 50 µl in PEM buffer (80 mM PIPES, 0.5 mM EGTA, 2 mM MgCl2, pH 6.9) in 384 well plates contained 2 mg/mL bovine brain tubulin, 10 µM fluorescent reporter, 1 mM GTP in the presence or absence of test compounds at 37 oC. Tubulin polymerization was followed by monitoring the fluorescence enhancement due to the incorporation of a fluorescence reporter into microtubules as polymerization proceeds. Fluorescence emission at 420 nm (excitation wavelength is 360 nm) was measured for 1 h at 1-min intervals in a multimode plate reader 23

(Tecan M200). To determine the IC50 values of the compounds against tubulin polymerization, the compounds were pre-incubated with tubulin at varying concentrations (0.01, 0.1, 1, 10 and 20 µM). Assays performed under similar conditions as employed for polymerization assays as described above.33 5.8. Immunohistochemistry of tubulin and analysis of nuclear morphology A549 cells were seeded on glass cover slip, incubated for 24 h in the presence or absence of test compounds 10a, 10b and 10d at a concentration of 5 µM. Cells grown on coverslips were fixed in 3.5% formaldehyde in phosphate-buffered saline (PBS) pH 7.4 for 10 minutes at room temperature. Cells were permeablized for 6 minutes in PBS containing 0.5% Triton X-100 (Sigma) and 0.05% Tween-20 (Sigma). The permeablized cells were blocked with 2% BSA (Sigma) in PBS for 1h. Later, the cells were incubated with primary antibody for tubulin from (sigma) at (1:200) diluted in blocking solution for 4h at room temperature. Subsequently the antibodies were removed and the cells were washed thrice with PBS. Cells were then incubated with FITC labeled anti-mouse secondary antibody (1:500) for 1h at room temperature. Cells were washed thrice with PBS and mounted in medium containing DAPI. Images were captured using the Olympus confocal microscope FLOW VIEW FV 1000 series and analyzed with FV10ASW 1.7 series software. 5.9. Western blot analysis of soluble versus polymerized tubulin A549 cells were seeded in 12-well plates at 1×105 cells per well in complete growth medium. Following treatment of cells with respective compounds 10a, 10b and 10d for duration of 24 h, cells were washed with PBS and subsequently soluble and insoluble tubulin fractions were collected. To collect the soluble tubulin fractions, cells were permeablized with 200 µL of prewarmed lysis buffer [80 mM Pipes-KOH (pH 6.8), 1 mM MgCl2, 1 mM EGTA, 0.2% Triton X100, 10% glycerol, 0.1% protease inhibitor cocktail (Sigma-Aldrich)] and incubated for 3 min at 30 oC. Lysis buffer was gently removed, and mixed with 100 µL of 3×Laemmli’s sample buffer (180 mM Tris-Cl pH 6.8, 6% SDS, 15% glycerol, 7.5% β-mercaptoethanol and 0.01% bromophenol blue). Samples were immediately heated to 95 oC for 3 min. To collect the insoluble tubulin fraction, 300 µL of 1×Laemmli’s sample buffer was added to the remaining cells in each well, and the samples were heated to 95 °C for 3 min. Equal volumes of samples were run on an SDS-10 % polyacrylamide gel and were transferred to a nitrocellulose membrane employing semidry transfer at 50 mA for 1h. Blots were probed with mouse anti-human α24

tubulin diluted 1:2,000 ml (Sigma) and stained with rabbit anti-mouse secondary antibody coupled with horseradish peroxidase, diluted 1:5000 ml (Sigma). Bands were visualized using an enhanced Chemiluminescence protocol (Pierce) and radiographic film (Kodak).34 5.10. Competitive tubulin binding assay The various concentrations (5 µM, 10µM, 15 µM and 20 µM) of conjugates 10a, 10b and 10d were coincubated with 3 µM colchicines in 30 mM Tris buffer containing 3 µM tubulin at 37oC for 60 min. The standard CA-4 was employed as a positive control whereas peclitaxel was used as the negative control. After incubation, the fluorescence of tubulin-colchicine complex was measured by using Tecan multimode reader at excitation wavelength of 380 nm and emission wavelength of 435 nm; whereas 30 mM Tris buffer was used as a blank. The raw fluorescence values were normalized first by subtracting the fluorescence of the buffer and then setting the fluorescence of 3 µM tubulin with 3 µM colchicine to 100%. Values represented were ± SD of at least three independent experiments. 5.11. Caspase-3 Assay A549 cells were seeded in 12 well plates as mentioned above and were treated with different compounds 10a, 10b and 10d at 5µM concentration. After 24 h of incubation with the compound, cells were lysed using lysis buffer and incubated at 4 oC for 10 mins which then collected and spun at 4 oC, 10000 rpm for 10 mins. The supernatant was collected which contains the protein. The protein was quantified employing Bradford Assay. Equal amount of protiens were added with assay buffer and caspase substrate which gives fluorescence units thereby calculated the activity against caspase.34 6. Molecular Modeling AutoDock4 was employed to dock lead pyrazole linked benzimidazoles in colchicine binding site of tubulin.[35-36] Initial Cartesian coordinates for the protein-ligand complex structure were derived from crystal structure of tubulin (PDB ID: 3E22). The protein targets were prepared for molecular docking simulation by removing water molecules, bound ligands. Hydrogen atoms and Kollman charges were added to each protein atom. Auto-Dock Tools-1.5.6 (ADT) was used to prepare and analyze the docking simulations for the AutoDock4. Coordinates of each compound were generated using Chemdraw11.0 followed by MM2 energy minimization. The interaction of protein and ligands in binding pocket for Autodock4 was defined by a Grid box. The grid box was created with 60 points equally in each direction of x, y, and z. AutoGrid4 was 25

used to produce grid maps for AutoDock4 calculations where the search space size utilized grid points of 0.375 Å. The Lamarckian genetic algorithm was opted to search for the best conformers. Each docking experiment was performed 100 times, yielding 100 docked conformations. Parameters used for the docking were as follows: population size of 150; random starting position and conformation; maximal mutation of 2 Å in translation and 50 degrees in rotations; elitism of 1; mutation rate of 0.02 and crossover rate of 0.8; and local search rate of 0.06. Simulations were performed with a maximum of 1.5 million energy evaluations and a maximum of 50000 generations. Final docked conformations were clustered using a tolerance of 1.0 Å root mean square deviation. The best model was picked based on the best stabilization energy. Final figures for molecular modeling were visualized by using PyMol.37 Supporting information: Mean growth percentage for inhibition (Table S1), GI50 values for NCI selected compounds (Table S2), five dose results of selected compounds, 1H NMR and 13C NMR spectra of final compounds are provided. Acknowledgements A.B.S, G.B.K thanks CSIR, New Delhi for the award of senior research fellowships. We also thank CSIR for financial support under the 12th Five Year plan project “Affordable Cancer Therapeutics” (CSC0301) and “Small Molecules in Lead Exploration (SMiLE)” (CSC0111). Notes and References 1.

Perez, E.A. Mol Cancer Ther. 2009, 8, 2086.

2.

Dumontet, C.; Jordan, M.A. Nat Rev Drug Discov. 2010 10, 790-803. Review. Erratum in: Nat Rev Drug Discov. 2010, 9, 897.

3.

Jordan, A.; Wilson, L. Nat. Rev. Cancer. 2004, 4, 253.

4.

Lu, Y.; Chen, J.; Xiao, M.; Li, W.; Miller, D.D. Pharm. Res. 2012, 29, 2943.

5.

Jordan, A.; Kamath, K. Curr. Cancer. Drug. Targets. 2007, 7, 730.

6.

Mitchison, T.; Kirschner, M. Nature.1984, 312, 5991, 237.

7.

Bacher, G.; Beckers, T.; Emig, P.; Klenner, T.; Kutscher, B. Pure Appl. Chem. 2001, 73, 1459.

8.

Bhattacharyya, B.; Panda, D.; Gupta, S.; Banerjee, M. Med Res Rev. 2008, 28, 155.

9.

Jain, N.; Yada, D.; Shaik, T.B.; Vasantha, G.; Reddy, P.S.; Kalivendi, S.V.; Sreedhar, B. Chem. Med. Chem. 2011, 6, 859.

10.

Attia, S.M. J Appl Toxicol.2013, 33, 426. 26

11.

Samson.; Donoso, J.A.; Heller-Bettinger, I.; Watson, D.; Himes, R.H. J Pharmacol Exp Ther. 1979, 208, 411.

12.

Hörig, J.A.; Renz, P. Eur J Biochem. 1980, 105, 587.

13.

Chang, L.; Chang, CS.; Chiang, PC.; Ho, YF.; Liu, JF.; Chang, KW.; Guh, JH. Br J Pharmacol. 2010, 160, 1677.

14.

Kamal, A.; Reddy, M.K.; Shaik, T.B.; Rajender.; Srikanth, Y.V.; Reddy, V S.; Kumar, G.B.; Kalivendi, S.V. Eur. J. Med. Chem, 2012, 50, 9.

15.

Shen, L.; Zhu, J.; Li, M.; Zhao, B.X.; Miao, J.Y. Eur J Med Chem. 2012, 54, 287.

16.

El-Dakdouki, H.; Adamski, N.; Foster, L.; Hacker, M.P.; Erhardt, P.W. J. Med. Chem. 2011, 54, 8224.

17.

Abu Thaher, B.; Arnsmann, M.; Totzke, F.; Ehlert, JE.; Kubbutat, MH.; Schächtele, C.; Zimmermann, MO.; Koch, P.; Boeckler, FM.; Laufer, SA. J. Med. Chem. 2012, 55, 961.

18.

Li, C.M.; Wang, Z.; Lu, Y.; Ahn, S.; Narayanan, R.; Kearbey, J.D.; Parke, D.N.; Li, W.; Miller, D.D.; Dalton. Cancer. Res. 2011, 71, 216.

19.

Chen, J.; Wang, Z.; Li, CM.; Lu, Y.; Vaddady, PK.; Meibohm, B.; Dalton, JT.; Miller, DD.; Li, W. J Med Chem. 2010, 53, 7414.

20.

Kamal, A.; Shaik, A.B.; Jain, N.; Kishor, C.; Nagabhushana, A.; Supriya, B.; Kumar, G.B.; Chourasiya, S.S.; Suresh, Y.; Mishra, R.K.; Addlagatta, A. Eur. J. Med.Chem. 2013. DOI: 10.1016/j.ejmech.2013.10.077.

21.

Kamal, A.; Reddy, V.S.; Karnewar, S.; Chourasiya, S.S. Shaik, A.B.; Nagabhushana, A.; Kumar, G.B.; Kishor, C.; Addlagatta, A.; Kotamraju, S. Chem. Med. Chem. 2013, 8, 2015.

22.

Frisa, S.; Jacobberger, J.W. PLoS One. 2009, 4, e7064.

23.

Hwang, A.; Maity, A.; McKenna, W.G.; Muschel, R.J. J Biol Chem. 1995, 270, 28419.

24.

Walczak, E.; Heald, R. Int Rev Cytol. 2008, 265, 111.

25.

Bhattacharyya.; Wolff, J. Proc Natl Acad Sci U S A. 1974, 71, 2627.

26.

Bhattacharyya.; Howard, R.; Maity, SN.; Brossi, A.; Sharma, PN.; Wolff, J. Proc Natl Acad Sci U S A. 1986, 83, 2052.

27.

Risinger, L.; Westbrook, CD.; Encinas, A.; Mülbaier, M.; Schultes, CM.; Wawro, S.; Lewis, JD.; Janssen, B.; Giles, FJ.; Mooberry. SL. J Pharmacol Exp Ther. 2011, 336, 652.

27

28.

Beale, M.; Allwood, D.M.; Bender, A.; Bond, P.J.; Brenton, J.D.; Charnock-Jones, D.S.; Ley, S.V.; Myers, R.M.; Shearman, J.W.; Temple, J.; Unger, J.; Watts, C.A.; Xian, J.; ACS Med Chem Lett. 2012, 19, 177.

29.

Nitesh, S.; Vaibhav, J.; Ranjan, P.; Somnath, K.; Sarita, D.; Neha, T.; Purusottam, M.; Garima, P.; Maneesh, K.; Dipon, D.; Shakti, R.; Satapathy, Sumit, S.; Sankar, K.; Guchhait, N. K.; Chanakya and Bharatam, P.V. Med. Chem. Commun., 2014, 5, 766.

30.

Kamal, A.; Sultana, F.; Ramaiah, M.J.; Srikanth, Y.V.; Viswanath, A.; Kishor, C.; Sharma, P.; Pushpavalli, S.N.; Addlagatta, A.; Pal-Bhadra, M.; ChemMedChem.2012, 6, 292.

31.

Kamal, A.; Rao, M.P.; Swapna, P.; Srinivasulu, V.; Bagul, C.; Shaik, A.B.; Mullagiri, K.; Kovvuri, J.; Reddy, V.S.; Vidyasagar, K.; Nagesh, N. Org Biomol Chem. 2014, 12, 2370.

32.

Kamal, A.; Shaik, AB.; Polepalli, S.; Reddy, VS.; Kumar, GB.; Gupta, S.; Krishna, KV.; Nagabhushana, A.; Mishra, RK.; Jain, N. Org Biomol Chem. 2014, 12, 7993.

33.

Reddy, M.A.; Jain, N.; Yada, D.; Kishore, C.; Vangala, J.R.; Surendra, R. P.; Addlagatta, A.; Kalivendi, S.V.; Sreedhar, B. J. Med. Chem. 2011, 54, 6751.

34.

Vangala, R.; Dudem, S.; Jain, N.; Kalivendi, S.V. J. Biol. Chem. 2014, 289, 12612.

35.

AutoDock,version4.0;http://www.scripps.edu/mb/olson/doc/autodock/.

36.

DeLano, W. L. Scientific, San Carlos, CA, USA, http://www.pymol.org., 2002.

37.

Ravelli, R.B.; Gigant, B.; Curmi, P.A.; Jourdain, I.; Lachkar, S.; Sobel, A.; Knossow, M. Nature. 2004, 428,198.

28

Tables Table 1 Structures of the compounds 10a-t and invitro antiproliferative activity (IC50+SD (µM) against selected human cancer cell lines.

IC50+S.D (µM)a HepG2b A549c MCF 7d HeLae DU-145f Avg IC50 3,4,5(OCH3)3 H 2.6±0.3 1.2±0.1 3.0±0.2 1.3±0.6 4.3±0.6 2.48 10a 3,4,5(OCH3)3 5-OCH3 1.8±0.5 0.7±0.1 1.6±0.7 0.9±0.2 2.2±0.2 1.44 10b 3,4,5(OCH3)3 5-F 12.9±0.4 3.5±0.3 22.2±1.7 18.4±1.8 22.1±1.5 15.8 10c 3,4,5(OCH3)3 5-Cl 4.1±0.5 3.0±0.4 2.8±0.3 2.2±0.3 5.0±0.4 3.42 10d 3,4,5(OCH3)3 5,6(Cl)2 8.2±0.5 13.7±1.0 2.4±1.5 6.7±0.9 2.6±0.9 6.72 10e 3,4,5(OCH3)3 5-CF3 15.8±1.0 24.0±1.3 27.±0.9 16.6±0.9 28.0±1.4 22.2 10f 3,4,(OCH3)2 H 10±0.2 6.1±0.9 6.3±0.8 8.3±1.2 13.3±1.7 8.8 10g 3,4,(OCH3)2 5-F 15.8±0.4 12.3±0.3 18.8±0.6 11.2±0.7 24.3±1.1 16.4 10h 3,4,(OCH3)2 5,6(Cl)2 19.7±1.4 27.1±0.8 26.6±0.9 23.5±1.7 41.0±0.8 27.5 10i 4-OCH3 H 22.8±1.5 30.0±2.2 25.7±0.7 28.8±1.9 32±0.12 27.8 10j 4-OCH3 5-OCH3 31.0±1.7 17.0±0.8 13.0±1.0 29.0±0.9 27.5±1.2 23.5 10k 4-OCH3 5-F 29.1±1.4 38.0±1.9 28.5±0.8 21.3±0.5 37.0±0.5 30.7 10l 4-OCH3 5-Cl 30.4±1.0 36.4±1.4 25.0±1.9 22.7±1.3 39.3±2.1 30.7 10m 4-OCH3 5,6(Cl)2 35.5±1.6 40.7±1.2 31.0±1.2 33.1±0.7 42.0±1.6 36.4 10n 4-OCH3 5-CF3 42.0±2.2 36.8±1.4 39.5±1.9 37±1.6 39±2.1 38.4 10o 3,4-(OCH2O)- H 6.1±0.5 2.5±0.3 10.1±0.8 12.5±1.0 3.8±0.5 7.0 10p 3,4-(OCH2O)- 5-OCH3 13±1.2 10.8±0.5 14.9±1.1 19.9±1.4 23.4±1.7 16.4 10q 3,4-(OCH2O)- 5-F 3.6±1.2 12.1±0.3 4.7±0.2 13.2±0.1 18.3±0.5 10.3 10r 3,4-(OCH2O)- 5,6(Cl)2 27.4±1.2 45.6±1.7 34.1±1.6 12.9±1.6 37.6±1.7 31.5 10s 3,4-(OCH2O)- 5-CF3 23.0±0.9 38.4±1.3 15.7±1.6 14.9±0.8 33.4±2.4 25.0 10t 1.1+ 0.5 0.8 + 0.1 1.7+ 0.6 3.2 + 0.3 1.7+ 0.4 1.7 Nocodazole 0.10±0.02 0.13±0.01 0.17±0.01 0.11+0.01 0.15±0.02 0.13 CA-4 Cell lines were treated with different concentrations of pyrazole-benzimidazole conjugates. Cell viability was measured employing MTT assay. aConcentration required to inhibition 50% cell growth and the values represent mean ± S.D. from three different experiments performed in triplicates. bHepG2: liver carcinoma cells cA549: lung adenocarcinoma epithelial cells. dMCF-7: breast adenocarcinoma cell line. eHeLa: cervix cancer cell line. fDU-145 prostate cancer cell line. R1

R2

29

Table 2 Inhibition of tubulin polymerization of compounds 10a, 10b and 10d

a

Compound

IC50 in µM

10a

2.5 ± 0.2

10b

1.3 ± 0.3

10d

3.8 ± 0.6

CA-4a

0.96±0.01

Combretastatin-A (CA-4) was employed as the reference standard. Figures

30

Fig. 1A Structures of some anticancer molecules: Colchicine (1), combretastatin (2), nocodazole (3), 2-aryl-4-benzoyl-imidazole series (ABI) (4), arylpyrazole inked benzimidazole conjugates (10a-t) and lead conjugate (10b)

Fig. 1B Structure and activity relationship of arylpyrazole linked benzimidazole conjugates: The compounds that possessing 3,4,5-trimethoxyphenyl as A-ring and various substituents on the Dring showed significant anticancer activity. 3,4,5-trimethoxyphenyl ring is a hall mark for tubulin binding. Whereas, the substituents on the D-ring that alter their cytotoxic activity is represented as R group.

31

Fig. 2 Anti-mitotic effects of 10a, 10b and 10d by FACS analysis: Induction of cell cycle G2/M arrest by compounds 10a, 10b and 10d. A549 cells were harvested after treatment at 5µM for 24h. Flow cytometric analysis of DNA content demonstrates that 10a, 10b and 10d arrest A549 cancer cells in G2/M phase and 10b (79.04%) is the most effective in the assay, followed by 10a (77.32%) and then 10d (70.23%). DMSO treated cells served as control. The percentage of cells in each phase of cell cycle was quantitatively measured.

32

Fig. 3. Western blot analysis of Cyclin-B1: Treatment of A549 cells with 5 µM concentrations of 10a, 10b and 10d for 24 h resulted an increase in Cyclin B1. The potent compound 10b show an expression of cyclin B1 levels significantly. Tubulin was employed as loading control. CA-4, colchicine (col.) were used as positive control and DMSO as negetive control.

Fig. 4 Effect of 10a, 10b and 10d on microtubule network: Immunofluorescence images of A549 cells stained with anti-β-tubulin antibody FITC-conjugated and then observed by confocal microscopy, magnification at 60x. Cells were exposed to 5µM of representative compounds for 24 h and then fixed and analyzed by fluorescence microscopy. The potent inhibitors (10a, 10b and 10d) of tubulin assembly show an irregular or rounded morphology. Cells were also counterstained with DAPI to visualize the nuclei.

33

Fig. 5 Distribution of tubulin in polymerized vs soluble fractions: A549 cells were treated with 5 µM of 10a and 10b for 24 h. This image represents soluble and polymerized fractions of tubulin for the most potent compounds 10a and 10b. In the case of 10b more soluble fraction is observed when compared to 10a. Combretastatin A-4 was used as positive control. The amount of tubulin was detected by western blot analysis.

Fig 6. Fluorimetric analysis of competitive colchicine binding of conjugates 10a, 10b and 10d: All the test conjugates indicated concentrations were incubated with 3 µM of tubulin and 3 µM colchicine for 60 min at 37 °C. Combretastatin A-4 was employed as a positive control whereas taxol was used as negative control. Fluorescence values are normalized to tubulin-colchicine complex (control).

34

Flouresence Units

12

Caspase-3 Assay

10 8 6 4 2 0

Fig. 7 Effect of compounds 10, 10b and 10d on caspase-3 activity: A549 cells were treated with compounds 10, 10b and 10d at 5 µM concentrations for 24 h. CA4 as well as colchicine were treated as positive controls. The most potent compound 10a activated the caspase-3 three times better than CA4. The other two potent compounds showed significant effect on the caspase-3 but, less magnitude when compare to 10b.

35

Fig. 8 Molecular modeling poses of the lead conjugates 10a, 10b, 10d and colchicine with 10a in colchicine binding site of the tubulin: The panel of images render the docking simulations of proposed ligands at the interface of α, β-tubulin. All the ligands are visualized in stick models (yellow color). The pale green and salmon color ribbons represent α- and β-tubulin subunits respectively. The significant residues are shown in green stick model and potential inter molecular hydrogen bonding interactions were indicted in red dashes. While the residues involved in other molecular interactions are shown as lines. The last image (10a+col) represents 10a along with colchicine occupy the same site in the tubulin. Images were generated with PyMol programme.

36

Scheme 1. Synthesis of pyrazole linked benzimidazole conjugate. Reagents and conditions : (i) NaOEt, Diethyl oxalate, EtOH, 0°C-rt, 4 h; (ii) NH2-NH2.2HCl, EtOH, reflux 3 h; (iii) LiAlH4, THF, 0°C-rt, 1-2 h; (iv) IBX, dry DMSO, rt, 1 h; (v) Na2S2O5, EtOH, o-phenylenediamines, rt, 34 h.

37

Graphical abstract

Synthesis of Arylpyrazole Linked Benzimidazole Conjugates as Potential Microtubule Disruptors

38