DOI: 10.1002/cmdc.201400069

Synthesis and Biological Evaluation of Imidazo[2,1-b][1,3,4]thiadiazole-Linked Oxindoles as Potent TubulinPolymerization InhibitorsAhmed Kamal,*[a, c] M. P. Narasimha Rao,[a] Pompi Das,[b] P. Swapna,[a] Sowjanya Polepalli,[b]

Vijaykumar D. Nimbarte,[c] Kishore Mullagiri,[a] Jeshma Kovvuri,[a] and Nishant Jain[b]

Introduction

Cancer is a disease in which normal cells dramatically altertheir behavior: the multiply uncontrollably, ignore signals tostop, and accumulate to form tumors. Cancer has becomea major cause of morbidity throughout the world, accountingfor 7.6 million deaths (~13 % of all deaths) in 2008. Accordingto the World Health Organization, deaths due to cancer world-wide were projected to continue to rise to over 13.1 million in2030. There are nearly 200 different types of cancer, eachnamed for the organ or type of cell from which it originates.Cancers of the colon, stomach, lung, liver, and breast cause thegreatest number of deaths each year.

Levamisole (I) is an immunomodulatory compound towardvarious cancer cell types including colorectal and breast can-cers, melanoma, and leukemia (Figure 1).[1] It was previouslyshown to affect cell proliferation in different cancers,[2] and tomodulate the phosphorylation of proteins involved in cell-cycle progression and apoptosis. Levamisole has also been

used to treat helminthic infections and in the amelioration ofvarious autoimmune disorders.[3, 4] Furthermore, I has beenshown to have anticancer activity in combination with fluo-rouracil (5-FU) as an adjuvant therapy for tumor-node-metasta-sis (TNM) stage III (Dukes’ C) colon carcinoma.[5] Imidazo[2,1-b]thiazole derivatives of levamisole have been reported as po-tential antitumor agents.[6] Subsequently, the antitumor activityof 5-formyl-6-arylimidazo[2,1-b]-1,3,4-thiadiazole sulfonamidesV were also reported.[7] In 2012 Andreani and co-workers[8] re-ported that 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indoli-nones cause G2/M phase arrest and inhibit tubulin assembly.Tubulin is a heterodimer of two closely related and tightlylinked globular polypeptides called a- and b-tubulin, whichpolymerize to form microtubules. Their function in mitosis andcell division makes them an important target for anticancerdrugs. Microtubules and their dynamics are the targets ofa chemically diverse group of antimitotic drugs that have beenused with great success in the treatment of cancer. There hasbeen considerable interest in the discovery and developmentof small molecules that affect tubulin polymerization.[9] Howev-er, the success of tubulin polymerization inhibitors as anticanc-er agents has stimulated significant interest in the identifica-tion of new compounds that may be more potent or more se-lective in targeted tissues or tumors.

Oxindoles are versatile moieties that display diverse biologi-cal activities, including anticancer activity. They exhibit antitu-mor activity by inhibiting tyrosine kinase receptors such asPDGF-R, VEGF-R, and CDK.[10] One of the most important exam-ples is sunitinib (II), which has been widely used in the treat-ment of gastrointestinal stromal tumors and metastatic renal

A series of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindolescomposed of an A, B, C and D ring system were synthesizedand investigated for anti-proliferative activity in various humancancer cell lines; test compounds were variously substituted atrings C and D. Among them, compounds 7 ((E)-5-fluoro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)-imidazo[2,1-b]-[1,3,4]thiadiazol-5-yl)methylene)indolin-2-one), 11 ((E)-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one), and 15 ((E)-6-chloro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-

methylene)indolin-2-one) exhibited potent anti-proliferative ac-tivity. Treatment with these three compounds resulted in accu-mulation of cells in G2/M phase, inhibition of tubulin assembly,and increased cyclin-B1 protein levels. Compound 7 displayedpotent cytotoxicity, with an IC50 range of 1.1–1.6 mm, and inhib-ited tubulin polymerization with an IC50 value (0.15 mm) lowerthan that of combretastatin A-4 (1.16 mm). Docking studiesreveal that compounds 7 and 11 bind with aAsn101, bThr179,and bCys241 in the colchicine binding site of tubulin.

[a] Dr. A. Kamal, M. P. N. Rao, P. Swapna, K. Mullagiri, J. KovvuriMedicinal Chemistry and PharmacologyCSIR – Indian Institute of Chemical TechnologyHyderabad 500 007 (India)E-mail : ahmedkamal@iict.res.in

[b] P. Das, S. Polepalli, Dr. N. JainChemical Biology, CSIR – Indian Institute of Chemical TechnologyHyderabad 500 007 (India)

[c] Dr. A. Kamal, V. D. NimbarteDepartment of Medicinal ChemistryNational Institute of Pharmaceutical Education and Research (NIPER)Hyderabad 500 037 (India)

Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cmdc.201400069.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &1&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS

cell cancer.[11, 12] Chen and co-workers reported thatoxindoles not only inhibit CDK, but also inhibit micro-tubule polymerization by binding at the colchicinebinding site of tubulin.[13] On the other hand, a 3,4,5-trimethoxyphenyl ring is present in most of the syn-thesized and naturally occurring biologically activecompounds, including colchicine (IV), combretastatinA-4 (CA-4; III), and its derivatives.

Our continued efforts toward the synthesis of a va-riety of new molecules led to the development of ef-ficient anticancer agents.[14, 15] In view of the biologi-cal importance of both imidazothiadiazoles and oxin-doles, in this study we designed and synthesizeda series of imidazo[2,1-b][1,3,4]thiadiazole-linked oxin-doles. Use of the core imidazothiadiazole scaffoldfunctionalized with 3,4,5-trimethoxyphenyl ring A, anindolinone as ring C, and a phenyl group as ring D re-sulted in the generation of imidazo[2,1-b]-[1,3,4]thiadiazolindolin-2-ones. These compoundswere evaluated for in vitro anticancer activity, cell-cycle effects, and the capacity to inhibit tubulin poly-merization.

Results and Discussion

Chemistry

The imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoleswere prepared as shown in Scheme 1. Compound 3was prepared by reaction of 3,4,5-trimethoxybenzoicacid with thiosemicarbazide in POCl3 at reflux. 6-(4-Aryl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazoles 5 a–e were obtained in high yieldby treating compound 3 with various phenacyl bro-mides 4 a–e in ethanol with the addition of 3–4drops of N,N-dimethylformamide. The imidazo[2,1-b]-[1,3,4]thiadiazole-5-carbaldehydes 6 a–e were pre-

pared by Vilsmeier–Haack reac-tion with the correspondingimidazo[2,1-b][1,3,4]thiadiazoles5 a–e. Conjugates 7–32 wereprepared by single-step Knoeve-nagel reaction between com-pounds 6 a–e and various substi-tuted oxindoles in ethanol/piper-idine. These conjugates werecharacterized by 1H and 13C NMRspectroscopy, mass spectrome-try, HRMS, and IR spectroscopy.All compounds were obtained inpure E isomeric form and con-firmed with those previously re-ported.[16, 17]

Figure 1. Structures of levamisole (I), sunitinib (II), combretastatin A-4 (III), colchicine (IV), 5-formyl-6-arylimidazo-[2,1-b][1,3,4]thiadiazolesulfonamide derivatives V, and synthesized imidazo[2,1-b][1,3,4]thiadiazole-indolin-2-oneconjugates 7–32.

Scheme 1. Synthesis of imidazo[2,1-b][1,3,4]thiadiazole–oxindole conjugates 7–32. Re-agents and conditions : a) POCl3, reflux, 4 h, 90 %; b) EtOH, reflux, 12 h, 80–85 %;c) POCl3 + DMF, reflux, 5 h, 75–80 %; d) 5- or 6-substituted oxindoles, EtOH, cat. (3–4drops) piperidine, reflux, 3–4 h, 75–85 %.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &2&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

Biology

Cytotoxicity

A sulforhodamine B assay was performed to evaluate the cyto-toxic effects of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles7–32 against A549, HeLa, MCF-7, and HCT116 human cancercell lines, and the IC50 values are listed in Table 1. Compounds7–15 showed considerable cytotoxicity, with IC50 values rang-ing from 1.1 to 8.9 mm against A549, HeLa, MCF-7, and HCT116human cancer cell lines.

Compound 7, which is fluorinated on ring C and hasa methyl group on the ring D, exhibited potent cytotoxicitywith IC50 values in the range 1.1–1.6 mm. In comparison, conju-gate 11, devoid of any substitution on ring C, also displayedpotent anti-proliferative activity, with IC50 values of 2.5–2.9 mm.Moreover, compound 15, chlorinated at the 6-position onring C and lacking ring D substitution, and 21, with a 5-fluorogroup on ring C, and a methoxy group on ring D, both exhibit-ed potent cytotoxicity.

However, compounds with 5-methoxy (8), 5-chloro (9), and6-chloro (10) substituents on ring C showed moderate cytotox-icity (IC50 values 2–8 mm). Overall, activity was decreased incells treated with compounds that contain chloro, fluoro, or

methoxy groups and that are devoid of substitution on indoli-none (ring C) and that have a fluoro group on ring D. Based onthe results of sulforhodamine B assays, compounds either me-thylated or that lack substitution on ring D showed the mostpotent activities in the series; therefore, the activities of themost potent compounds can be ranked in the order of 7 > 11> 15 > 21. To further determine their potency in other celltypes, conjugates 7, 11, 15, and 21 were evaluated in thesixty-cell-line panel of the US National Cancer Institute (NCI),which includes leukemia, melanoma, renal, lung, colon, ovary,prostate, breast, and central nervous system cancer cell lines;compounds 11 and 21 were selected for the five-dose assay(Table 2). These compounds displayed significant cytotoxicityin most of the NCI panel cell lines, with GI50 values rangingfrom 0.30 to 5.8 mm. In particular, compound 11 showed prom-ising cytotoxicity, with GI50 values of 0.30 and 0.42 mm againstOVCAR-4 (ovarian cancer) and HOP-92 (lung cancer) cell lines.Taken together, results from the cytotoxicity assays agreed re-markably with the NCI 60-cell-line screen. We then elucidatedwhether the anti-proliferative properties of 7, 11, and 15 weredue to inhibition of tubulin polymerization. To investigate thispossibility, 7, 11, and 15 were incubated at varying concentra-tions with tubulin, and polymerization assays were performed.Compound 7, which potently inhibited cell growth, also signifi-cantly decreased tubulin assembly, with an IC50 value of0.15 mm. Compounds 11 and 15 were only slightly less potent,with IC50 values of 1.23 and 2.11 mm, respectively.

Effect of the compounds on cell cycle

Because the compounds inhibit tubulin polymerization, weevaluated their ability to stall cells at the G2/M phase. A549cells were treated with test compounds at 5 mm for 24 h. Cellswere then harvested and analyzed by flow cytometry for DNAcontent (Figure 2). The control cells treated with DMSOshowed 26.5 % arrested in G2/M phase. In contrast, compounds7 and 11 increased the cell population in G2/M phase by 67.3and 64.8 %, respectively. Moreover, cells treated with 15 mani-fested 66.7 % of cells in G2/M phase.

Effect of compounds on microtubules

Pharmacological inhibition of microtubule formation results ina profound disruption of the cellular microtubule network, andas such, spindle fibers composed of microtubules are routinelydisrupted from treatments with anti-tubulin agents.[18] Theimidazo[2,1-b][1,3,4]thiadiazole–oxindole conjugates inhibit cell

Table 1. Cytotoxicity of compounds 7–32 and levamisole on A549, HeLa,MCF-7, and HCT116 human cancer cell lines.

Compd IC50 [mm][a]

A549[b] HeLa[c] MCF-7[d] HCT116[e]

7 1.1�0.7 1.4�1.7 1.2�0.4 1.6�0.58 5.7�3.1 3.9�0.7 2.9�0.8 7.3�2.79 5.3�0.2 3.8�1.7 8.9�1.8 8.3�0.710 8.5�0.8 2.8�1.2 3.0�1.5 5.6�2.111 2.6�0.9 2.5�0.3 2.5�0.9 2.9�1.212 4.2�0.9 2.5�1.6 7.6�0.8 5.8�4.213 5.0�0.79 3.4�1.3 7.4�1.2 8.2�1.014 6.8�0.8 3.6�2.3 7.5�0.9 8.4�2.115 2.3�1.6 3.2�1.0 2.4�1.6 3.0�3.016 19.5�1.9 12.8�1.2 22.9�1.0 11.7�0.917 18.5�0.2 23.0�0.5 21.5�1.6 13.5�0.718 12�1.1 24�0.2 10.1�0.6 13.1�1.119 18.6�0.3 36.9�1.9 13.1�1.3 11.2�1.420 17.4�1.9 33.1�1.8 12.9�0.3 18.6�0.421 3.1�1.3 4.2�1.7 2.1�1.3 15.2�0.322 13.2�0.1 27.8�1.0 24.6�3.6 27.3�4.123 16.4�1.9 20.5�1.2 21.3�1.1 13.5�0.924 31.6�1.5 29.7�2.3 35.2�2.4 20.2�0.425 26.9�1.3 22.4�0.9 17.7�1.7 13.6�0.426 28.4�1.8 15.3�1.5 25.3�1.2 18.9�1.627 29.1�1.9 23.2�1.2 29.6�2.0 26.2�6.228 16.8�0.2 19.7�0.9 24.9�2.2 29.2�1.329 20.7�2.8 22.5�1.7 21.8�0.7 18.2�4.130 18.6�0.7 15.8�3.1 26.7�1.1 14.7�2.731 15.3�2.9 25.9�1.0 20.0�1.8 24.6�5.332 24.5�0.5 28.1�1.1 21.4�1.4 19.5�1.0levamisole >100 96 >100 >100

[a] 50 % inhibitory concentration; values are the mean �SD of three indi-vidual experiments determined after 48 h treatment. [b] NSC lung cancer.[c] Human epithelial cervical cancer. [d] Human breast adenocarcinoma.[e] Colon cancer.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &3&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

proliferation, tubulin assembly, and arrest cells at the G2/Mphase (Table 3). To further validate their effect on microtubules,we treated A549 cells with compounds 7, 11, and 15 for 24 h.Immunofluorescence analyses revealed that the conjugatesprofoundly alter microtubule the network. Treated cells also

exhibit a typical rounded mitoticphenotype, and DAPI was usedto counter-stain the nuclei(Figure 3).

Effect of compounds on endoge-nous tubulin in A549 cells

Microtubules are present in a dy-namic equilibrium with free tu-bulin monomers. Anti-tubulincompounds disrupt this dynamicequilibrium. Microtubule-depoly-merizing agents increase theamount of free tubulin mono-mer in cells, thereby preventingmicrotubule polymerization.[19]

As compounds 7, 11, and 15 effi-ciently arrest cells at the G2/Mphase and inhibit tubulin assem-bly, we elucidated their ability toblock endogenous tubulin poly-merization. A549 cells weretreated with compounds 7, 11,and 15 at 5 mm for 24 h. Wethen permeabilized cells withdetergent-containing buffers(details in the Experimental Sec-tion below). The insoluble andsoluble fractions were analyzedfor tubulin (Figure 4). DMSO-treated cells demonstrated equalamounts of tubulin across thesoluble and insoluble fractions.In contrast, 7 and 11 effecteda marked increase in the solublefraction similar to CA-4-treatedcells. In comparison, paclitaxel-treated cells possessed increasedlevels of tubulin in the insoluble

fraction. Next, we analyzed whether the cells arrested at theG2/M phase were indeed mitotic in nature. We confirmed theirstate by assaying for the mitotic marker cyclin-B1. As expected,treatments with compound 7 induced increased levels ofcyclin-B1, when compared with 11 and 15. b-actin was used asa loading control.

Molecular docking studies

Among the synthesized compounds, 7 and 11 displayed signif-icant cytotoxicity, tubulin polymerization inhibition with IC50

values less than or equal to that of CA-4, and arrested cells atthe G2/M phase of the cell cycle. Molecular docking studieswere performed to determine whether the active conjugates 7and 11 exert their cytotoxicity and capacity to block tubulinpolymerization by binding to tubulin at the colchicine bindingsite. Docking analyses suggest that both compounds occupy

Table 2. In vitro cytotoxicity of compounds 11 and 21 against the NCI panel of 60 human cancer cell lines.

Cancer panel/cell line GI50 [mm][a] Cancer panel/cell line GI50 [mm][a]

11[b] 21[c] 11[b] 21[c]

Leukemia MelanomaCCRF-CEM – 5.49 MDA-MB-435 – 3.39HL-60 (TB) >100 2.17 SK-MEL-2 16.3 5.58K-562 – 13.1 SK-MEL-28 – 9.58MOLT-4 – 4.54 SK-MEL-5 0.59 1.64RPMI-8226 – 11.3 UACC-257 – 3.35SR 2.35 23.5 UACC-62 5.47 13.3

Non-Small Cell Lung Cancer Ovarian CancerA549/ATCC 2.12 2.13 IGROV 1 4.75 3.39HOP-62 4.14 1.94 OVCAR-3 2.90 2.32HOP-92 0.42 2.02 OVCAR-4 0.30 1.77NCI-H226 22.5 16.8 OVCAR-5 >100 6.14NCI-H23 4.54 4.25 OVCAR-8 3.71 5.34NCI-H322M 10.3 2.74 NCI/ADR-RES 3.05 21.9NCI-H460 1.98 3.42 SK-OV-3 15.2 2.84NCI-H522 10.2 5.44

Renal CancerColon Cancer 786-0 5.80 16.0COLO 205 – 1.61 A498 7.28 12.2HCC-2998 >100 5.25 ACHN 4.97 3.29HCT116 2.59 14.2 CAKI-1 15.4 4.48HCT15 2.68 2.50 RXF 393 2.24 3.96HT29 – 2.18 SN 12C 5.25 14.2KM12 >100 4.04 TK-10 1.69 2.26SW-620 – 3.21 UO-31 8.44 15.0

CNC Cancer Prostate CancerSF-268 3.63 4.40 PC-3 81.1 24.7SF-295 5.04 3.00 DU-145 2.55 1.87SF-539 12.9 56.0SNB-19 7.89 15.1 Breast CancerSNB-75 0.55 10.3 MCF-7 – 1.66U251 1.59 1.77 MDA-MB-231/ATCC 3.51 5.25

HS 578T 0.50 16.2Melanoma BT-549 4.69 14.9LOX IMVI – 6.88 T-47D 3.13 2.01MALME-3M 0.76 9.15 MDA-MB-468 4.84 2.53M14 – 5.51

[a] Compound concentration required to decrease cell growth to half that of untreated cells. [b] 11(NSC775003). [c] 21 (NSC774982).

Table 3. Anti-tubulin polymerization activity of compounds 7, 11, 15 andCA-4.

Compd IC50 [mm][a]

7 0.15�0.311 1.23�1.015 2.11�0.7CA4 1.16�0.1

[a] Concentration of compounds required to inhibit tubulin assembly by50 %; values are the mean �SD of two independent experiments per-formed in triplicate.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &4&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

the colchicine binding site of a/b-tubulin (Figure 5). The 3,4,5-tri-methoxyphenyl group of theconjugates anchors the moleculein the a/b interface of tubulin,similar to the binding mode ofcolchicines.

This binding pattern is sup-ported by the hydrogen bond-ing interactions between the3,4,5-trimethoxyphenyl ring andaGln11 and bAsn258, the distan-ces from which are in the rangeof 1.8–2.2 �. Moreover, rings B ofcompounds 7 and 11 are in-volved in hydrophobic interac-tions with aThr179. Some hydro-gen bonding interactions werealso observed between ring Cand bThr353, bCys241, andaAsn101 of a/b-tubulin. The p-tolyl groups of 7 and 11 are in-volved in hydrogen bonding in-teractions with aSer178. Basedon the analysis of docking re-sults, we conclude that com-

pounds 7 and 11 occupy the colchicine binding site at the a/b interface of tubulin (Table 4).

Overall, some determinant hydrophobic interactions wereobserved between ring C and aAsn101 and bCys241, and be-

Figure 2. Antimitotic effects of b) 7, c) 11, and d) 15 by FACS analysis. Induction of cell-cycle G2/M arrest by com-pounds 7, 11, and 15. A549 cells were harvested after treatment at 5 mm for 24 h. Untreated cells and a) DMSO-treated cells served as controls. Flow cytometry was used to quantify the percentage of cells in each phase of thecell cycle.

Figure 3. Effect of 7, 11, and 15 on microtubule dynamics. A549 cells wereindependently treated with 7, 11, and 15 at 5 mm for 24 h. Cells were thenfixed and stained for tubulin antibody (green), and DAPI was used as coun-ter-stain (blue). The merged images of cells stained for tubulin and DAPI areshown at right.

Figure 4. Distribution of tubulin in polymerized versus soluble fractions:A549 cells were treated with compounds 7 and 11 at 5 mm for 24 h. Tubulinwas detected by immunoblot analysis. Cyclin-B1 protein levels were measurewith specific antibodies, and b-actin was used as a loading control.

Table 4. Comparisons of docking scores of synthesized compounds 7and 11 with colchicine (IV).

IV �8.84 �8.84 �8.84 �8.84 �8.84 �8.84

7 �10.22 �10.21 �9.91 �9.67 �9.57 �9.5711 �9.19 �9.16 �9.10 �9.08 �9.08 �7.45

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &5&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

tween ring B and bAla180 and bThr179. Hydrophobic interac-tions between ring A and bLeu255, bLeu248, aGln11, andbAsn249 were also observed (Figure 6). Some hydrophobic in-teractions are similar to thoseobserved for colchicine (IV) ;however, a few additional inter-actions were also observed withaGln11, bAsn258, aThr179,bThr353, aAsn101, and aSer178.

Conclusions

We synthesized a series ofimidazo[2,1-b][1,3,4]thiadiazole–oxindoles composed of A, B, C,and D ring systems. Two ofthese conjugates were selectedfor the NCI 60-cell-line panel,five-dose screen. SAR analysis re-veals that amongst the conju-gates synthesized, those witha methyl group and devoid ofany substitution on ring D exhib-it significant activity. Compounds7, 11, and 15 displayed potentcytotoxicity, with IC50 values of1.1–3.2 mm. These three com-pounds exert their cytotoxic ac-tivity by inhibition of tubulinpolymerization, with IC50 valuesof 0.15, 1.23, and 2.11 mm, re-spectively. They also induce cell-

cycle arrest in the G2/M phase, disruption of microtubule net-works, and increase levels of cyclin-B1. Tubulin disassembly bycompounds 7 and 11 was further supported by western blot

Figure 5. Superposition of the docked conformations of 11 (green sticks) and 7 (blue sticks) over the X-ray crystal structure of the colchicine binding site oftubulin (PDB ID: 3E22).

Figure 6. Binding poses of a, b) 7 and c, d) 11 within the a/b interface of tubulin. Shown are the trimethoxyphenylgroup and N atom (blue) of the oxindole ring involved in hydrophobic and hydrogen bonding interactions.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &6&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

analysis. Moreover, docking studies provided some molecularinsight into the binding mode of compounds 7 and 11, whichbind at the colchicine binding site in a/b interfaces of poly-merized tubulin. Based on these results, it is evident that com-pounds of this structural class are acquiescent to further modi-fications and function as a suitable template for the design ofa new class of tubulin polymerization inhibitors for the treat-ment of cancer.

Experimental Section

Chemistry

All chemicals and reagents were obtained from Aldrich (Sigma–Al-drich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson MattheyCo., Ward Hill, MA, USA), or Spectrochem Pvt. Ltd. (Mumbai, India),and were used without further purification. TLC was performed onglass plates containing silica gel 60 GF254, and visualization wasachieved by UV light or iodine indicator-monitored reactions.Column chromatography was performed with Merck 60–120 meshsilica gel. 1H and 13C NMR spectra were recorded on BrukerUXNMR/XWIN-NMR (300 MHz) or Inova Varian VXR Unity (400,500 MHz) instruments. Chemical shifts (d) are reported in ppmdownfield from an internal TMS standard. ESIMS spectra were re-corded on a Micro-mass Quattro LC instrument equipped with anESI mode positive ion trap detector and running ESI + software;capillary voltage was 3.98 kV. High-resolution mass spectra (HRMS)were recorded on a QSTAR XL Hybrid MS–MS mass spectrometer.Melting points were determined with an electrothermal meltingpoint apparatus, and are uncorrected. 1H and 13C NMR, IR, andHRMS spectra of final compounds 7–32 are provided in the Sup-porting information.

Preparation of 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-amine (3): POCl3 (21 mL) was added to a mixture of 3,4,5-trime-thoxybenzoic acid (1, 10 g, 47 mmol) and thiosemicarbazide (2,4.27 g, 47 mmol), and the mixture was heated at 75 8C for 30 min.After cooling to room temperature, water was added slowly. Thereaction mixture was further heated at 110 8C for 4 h. After cooling,the mixture was adjusted to pH 8 by dropwise addition of a 50 %NaOH solution under stirring. The precipitate obtained was filteredand washed with water (2 � 100 mL). The pale-yellow solid ob-tained (11.3 g, 90 %) was dried and used directly for the next step.

General procedure for the preparation of 6-(4-substitutedphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazoles 5 a–e : A mixture of 3 (1 equiv) and 4 a–e (1 equiv) along with a few drops of DMF was held at reflux inethanol (30 mL) for ~20–24 h. The solvent was evaporated, andthe remainder was dissolved in EtOAc (100 mL), washed with a sa-turated sodium bicarbonate solution (2 � 100 mL), and the separat-ed organic layer was dried over anhydrous Na2SO4. The organiclayer was concentrated under reduced pressure, and the crudeproduct was purified by column chromatography with EtOAc/hexane (2:8) as the eluent to afford compounds 5 a–e with highpurity.

6-p-Tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole (5 a): Compound 5 a was prepared according tothe general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-p-tolylethanone4 a (3.19 g, 14.98 mmol) to obtain pure product 5 a as pale-yellowsolid (4.56 g, 80 % yield). 1H NMR (500 MHz, CDCl3): d= 7.99 (s, 1 H),

7.73 (d, J = 7.9 Hz, 2 H), 7.22 (d, J = 7.9 Hz, 2 H), 7.08 (s, 2 H), 3.96 (s,6 H), 3.92 (s, 3 H), 2.38 ppm (s, 3 H); MS (ESI, m/z): 382 [M + H]+ .

6-Phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole (5 b): Compound 5 b was prepared according tothe general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-phenylethanone4 b (3.92 g, 14.98 mmol) to obtain pure product 5 b as pale-yellowsolid (4.45 g, 81 % yield). 1H NMR (500 MHz, CDCl3): d= 8.02 (s, 1 H),7.84–7.82 (m, 2 H), 7.43–7.40 (m, 2 H), 7.32–7.28 (s, 1 H), 7.08 (s, 2 H),3.96 (s, 6 H), 3.92 ppm (s, 3 H); MS (ESI, m/z): 368 [M + H]+ .

6-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole (5 c): Compound 5 c was prepared according tothe general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-(4-methoxyphe-nyl)ethanone 4 c (3.43 g, 14.98 mmol) to obtain pure product 5 c aspale-yellow solid (4.75 g, 80 % yield). 1H NMR (300 MHz, CDCl3): d=7.93 (s, 1 H), 7.74 (d, J = 8.6 Hz, 2 H), 7.07 (s, 2 H), 6.94 (d, J = 8.6 Hz,2 H), 3.96 (s, 6 H), 3.92 (s, 3 H), 3.84 ppm (s, 3 H); MS (ESI, m/z): 398[M + H]+ .

6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole (5 d): Compound 5 d was prepared according tothe general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-(4-chloropheny-l)ethanone 4 d (3.46 g, 14.98 mmol) to obtain pure product 5 d aspale-yellow solid (5.10 g, 85 % yield). 1H NMR (500 MHz, CDCl3): d=8.00 (s, 1 H), 7.75 (d, J = 8.5 Hz, 2 H), 7.39–7.37 (m, 2 H), 7.08 (s, 2 H),3.96 (s, 6 H), 3.93 ppm (s, 3 H); MS (ESI, m/z): 402 [M + H]+ .

6-(4-Fluorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole (5 e): Compound 5 e was prepared according tothe general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thia-diazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-(4-fluoropheny-l)ethanone 4 e (3.22 g, 14.98 mmol) to obtain pure product 5 e aspale-yellow solid (4.72 g, 82 % yield). 1H NMR (300 MHz, CDCl3): d=8.40 (s, 1 H), 8.01 (d, J = 8.5 Hz, 2 H), 7.24–7.27 (m, 2 H), 7.14 (s, 2 H),3.98 (s, 6 H), 3.94 ppm (s, 3 H); MS (ESI, m/z): 386 [M + H]+ .

General procedure for the preparation of 6-(4-substitutedphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole-5-carbaldehydes 6 a–e : The Vilsmeier reagentwas prepared at 0–5 8C by dropping POCl3 (5.4 equiv) into a stirredsolution of dry DMF (6.5 equiv) in CHCl3 (10 mL). Compounds 5 a–e (1 equiv) in CHCl3 (100 mL) was added dropwise to the Vilsmeierreagent while maintaining stirring and cooling. The reaction mix-ture was kept for 3 h at room temperature and under reflux for24 h. Chloroform was removed under reduced pressure, and theresulting oil was poured into crushed ice. The mixture was extract-ed with EtOAc (2 � 200 mL), and the separated organic layer wasdried over anhydrous Na2SO4. The organic layer was concentratedunder reduced pressure, and the crude product was purified bycolumn chromatography using EtOAc/hexane (3:7) as the eluent toafford compounds 6 a–e with high purity.

6-p-Tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole-5-carbaldehyde (6 a): Compound 6 a was pre-pared according to the general procedure, using compound 5 a(3 g, 7.8 mmol), POCl3 (3.96 mL, 42.4 mmol), and DMF (3.95 mL,50.7 mmol) to obtain pure product 6 a as a yellow solid (2.41 g,55 % yield). 1H NMR (300 MHz, CDCl3): d= 10.09 (s, 1 H), 7.78 (d, J =7.9 Hz, 2 H), 7.32 (d, J = 7.9 Hz, 2 H), 7.16 (s, 2 H), 3.98 (s, 6 H), 3.94(s, 3 H), 2.44 ppm (s, 3 H); MS (ESI, m/z): 410 [M + H]+ .

6-Phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole-5-carbaldehyde (6 b): Compound 6 b was pre-

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &7&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

pared according to the general procedure, using compound 5 b(3 g, 8.17 mmol), POCl3 (4.12 mL, 44.1 mmol), and DMF (4.12 mL,53.1 mmol) to obtain pure product 6 b as a pale-yellow solid(2.45 g, 76 % yield). 1H NMR (300 MHz, CDCl3): d= 10.10 (s, 1 H),7.85–7.82 (m, 2 H), 7.41–7.43 (m, 2 H), 7.34–7.30 (m, 1 H), 7.09 (s,2 H), 3.98 (s, 6 H), 3.94 ppm (s, 3 H); MS (ESI, m/z): 396 [M + H]+ .

6-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole-5-carbaldehyde (6 c): Compound 6 c was pre-pared according to the general procedure, using compound 5 c(3 g, 7.55 mmol), POCl3 (3.81 mL, 40.8 mmol), and DMF (3.80 mL,49.0 mmol) to obtain pure product 6 c as a pale-yellow solid(2.51 g, 78 % yield). 1H NMR (300 MHz, CDCl3): d= 10.10 (s, 1 H),7.89 (d, J = 8.8 Hz, 2 H), 7.41–7.43 (m, 2 H), 7.15 (s, 2 H), 7.03 (d, J =8.8 Hz, 2 H), 3.98 (s, 6 H), 3.94 (s, 3 H), 3.88 ppm (s, 3 H); MS (ESI, m/z): 426 [M + H]+ .

6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole-5-carbaldehyde (6 d): Compound 6 d was pre-pared according to the general procedure, using compound 5 d(3 g, 7.4 mmol), POCl3 (3.76 mL, 40.3 mmol) and DMF (3.77 mL,48.6 mmol) to obtain pure product 6 d as a pale-yellow solid(2.43 g, 76 % yield). 1H NMR (300 MHz, CDCl3): d= 10.15 (s, 1 H),7.92 (d, J = 8.4 Hz, 2 H), 7.47 (d, J = 8.4 Hz, 2 H), 7.15 (s, 2 H), 3.98 (s,6 H), 3.94 ppm (s, 3 H); MS (ESI, m/z): 430 [M + H]+ .

6-(4-Fluorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazole-5-carbaldehyde (6 e): Compound 6 e was pre-pared according to the general procedure, using compound 5 e(3 g, 7.7 mmol), POCl3 (3.92 mL, 42.0 mmol), and DMF (3.93 mL,50.6 mmol) to obtain pure product 6 e as a pale-yellow solid(2.56 g, 80 % yield). 1H NMR (500 MHz, CDCl3): d= 10.14 (s, 1 H),8.00–7.96 (m, 2 H), 7.20 (t, J = 8.6 Hz, 2 H), 7.14 (s, 2 H), 3.98 (s, 6 H),3.94 ppm (s, 3 H); MS (ESI, m/z): 414 [M + H]+ .

(E)-5-Fluoro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (7): Compound 7was prepared according to the method described for compound 7,employing 6 a (200 mg, 0.48 mmol) and 5-fluoroindolin-2-one(73 mg, 0.48 mmol) to obtain pure product 7 as a yellow solid(206 mg, 78 % yield); mp: 308–310 8C; IR (KBr): n= 3427, 3173,2936, 1709, 1613, 1520, 1474, 1432, 1414, 1362, 1337, 1292, 1241,1131, 1069, 1000, 802, 771, 759, 732, 652, 620, 589 cm�1; 1H NMR(300 MHz, CDCl3 + [D6]TFA): d= 7.89, (bs, 1 H), 7.57 (s, 2 H), 7.35 (s2H), 7.10 (s, 2 H), 7.10–6.92 (m, 2 H), 6.59 (d, J = 7.5 Hz, 1 H), 7.36 (s,2 H), 3.97 (s, 3 H), 3.90 (s, 6 H), 2.40 ppm (s, 3 H); 13C NMR(75.47 MHz, CDCl3 + [D6]TFA): d= 168.1, 157.9, 153.7, 145.2, 143.2,141.7, 140.7, 137.0, 127.7, 122.9, 118.5, 118.0, 117.8, 113.5, 111.9,104.8, 61.3, 56.3, 21.5 ppm; MS (ESI, m/z): 543 [M + 1]+ ; HRMS (ESIm/z) calcd for C29H24O4N4FS: 543.14968, found: 543.14855 [M + 1]+ .

(E)-5-Methoxy-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo-[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (8): Com-pound 8 was prepared according to the method described forcompound 7, employing 6 a (200 mg, 0.48 mmol) and 5-methox-yindolin-2-one (79 mg, 0.48 mmol) to obtain pure product 8 asa red solid (210 mg, 78 % yield); mp: 286–288 8C; IR (KBr): n= 3432,2935, 1699, 1628, 1482, 1463, 1411, 1316, 1249, 1129, 1100, 1002,822, 749, 645 cm�1; 1H NMR (300 MHz, CDCl3): d= 8.13, (bs, 1 H),7.95 (s, 1 H), 7.70 (d, J = 7.9 Hz, 2 H), 7.21 (d, J = 7.7 Hz, 2 H), 6.95 (s,2 H), 6.78–6.70 (m,2 H), 6.49 (s, 1 H), 3.91 (s, 3 H), 3.86 (s, 6 H), 3.43(s, 3 H), 2.37 (s, 3 H); 13C NMR (75 MHz, CDCl3 + [D6]TFA): d= 167.9,155.6, 153.8, 145.1, 142.8, 141.9, 140.2, 134.9, 130.6, 127.6, 123.1,122.8, 121.1, 118.3, 117.5, 117.2, 112.5, 111.7, 104.7, 61.3, 56.3, 56.0,21.3 ppm; MS (ESI, m/z): 555 [M + 1]+ ; HRMS (ESI m/z) calcd forC30H27O5N4S: 555.16967, found: 555.16907 [M + 1]+ .

(E)-5-Chloro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (9): Compound 9was prepared according to the method described for compound 7,employing 6 a (200 mg, 0.48 mmol) and 5-chloroindolin-2-one(81 mg, 0.48 mmol) to obtain pure product 9 as a yellow solid(215 mg, 79 % yield); mp: 292–294 8C; IR (KBr): n= 3432, 2935,1699, 1628, 1482, 1463, 1411, 1316, 1249, 1129, 1100, 1002, 822,749, 645 cm�1; 1H NMR (300 MHz, CDCl3): d= 8.13, (bs, 1 H), 7.95 (s,1 H), 7.70 (d, J = 7.9 Hz, 2 H), 7.21 (d, J = 7.7 Hz, 2 H), 6.95 (s, 2 H),6.78–6.70 (m, 2 H), 6.49 (s, 1 H), 3.91 (s, 3 H), 3.86 (s, 6 H), 3.43 (s,3 H), 2.37 ppm (s, 3 H); 13C NMR (75.47 MHz, CDCl3 + [D6]TFA): d=167.9, 155.6, 153.8, 145.1, 142.8, 141.9, 140.2, 134.9, 130.6, 127.6,123.1, 122.8, 121.1, 118.3, 117.5, 117.2, 112.5, 111.7, 104.7, 61.3, 56.3,56.0, 21.3 ppm; MS (ESI, m/z): 559 [M + 1]+ ; HRMS (ESI m/z) calcdfor C29H24O4N4ClS: 559.12013, found: 559.11907 [M + 1]+ .

(E)-6-Chloro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (10): Compound10 was prepared according to the method described for com-pound 7, employing 6 a (200 mg, 0.48 mmol) and 6-chloroindolin-2-one (81 mg, 0.48 mmol) to obtain pure product 10 as a yellowsolid (217 mg, 80 % yield); mp: 346–348 8C; IR (KBr): n= 3435, 3135,2937, 1708, 1612, 1522, 1482, 1456, 1346, 1240, 1213, 1131, 1072,1000, 808, 699, 575, 529, 488 cm�1; 1H NMR (300 MHz, CDCl3 +[D6]TFA): d= 7.83, (bs, 1 H), 7.58, (d, J = 8.1 Hz, 1 H), 7.36 (s, 2 H),7.03 (s, 3 H), 6.88 (d, J = 7.1 Hz, 1 H), 6.80 (d, J = 7.7 Hz, 1 H), 3.96 (s,6 H), 3.88 (s, 6 H), 2.42 ppm (s, 3 H); 13C NMR (75.47 MHz, CDCl3 +[D6]TFA): d= 167.8, 153.8, 142.9, 142.0, 137.4, 130.6, 127.8, 126.8,123.0, 122.8, 118.9, 117.4, 111.7, 104.7, 61.3, 56.3, 21.3 ppm; MS(ESI, m/z): 559 [M + 1]+ ; HRMS (ESI m/z) calcd for C29H24O4N4ClS:559.12013, found: 559.11971 [M + 1]+ .

(E)-3-((6-p-Tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (11): A mixture ofcompound 6 a (200 mg, 0.48 mmol) and indolin-2-one (64 mg,0.48 mmol) was dissolved in ethanol (6 mL) and piperidine (3–4drops) was added. The reaction mixture was heated at reflux for3 h. Progress of the reaction was monitored by TLC, and the pre-cipitate formed on cooling was collected by filtration, washedtwice a small amount of ethanol to obtain the pure product 11 asa yellow solid (207 mg, 80 % yield); mp: 294–296 8C; IR (KBr): n=3427, 3169, 2936, 2832, 2358, 1697, 1616, 1488, 1465, 1368, 1348,1315, 1291, 1241, 1002, 844, 820, 779, 742, 733, 623, 524, 489 cm�1;1H NMR (300 MHz, CDCl3 + [D6]TFA): d= 9.40 (bs, 1 H), 7.80 (s, 1 H),7.68–7.50 (m, 1 H), 7.40–7.20 (m, 4 H), 7.00 (s, 2 H), 6.98 (s, 1 H),6.91–6.79 (m, 2 H), 3.93 (s, 3 H), 3.84 (s, 6 H), 2.40 ppm (s, 3 H);13C NMR (75 MHz, CDCl3 + [D6]TFA): d= 170.7, 165.5, 153.8, 141.9,141.2, 140.8, 130.3, 130.1, 127.8, 125.9, 123.5, 122.2, 121.0, 110.4,104.3, 61.0, 56.3, 21.3 ppm; MS (ESI, m/z): 525 [M + 1]+ ; HRMS (ESIm/z) calcd for C29H25O4N4S: 525.15910, found: 525.15948 [M + 1]+ .

(E)-3-((6-Phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b]-[1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (12): Compound12 was prepared according to the method described for com-pound 7, employing 6 b (200 mg, 0.5 mmol) and indolin-2-one(67 mg, 0.5 mmol) to obtain pure product 12 as a yellow solid(206 mg, 80 % yield); mp: 292–294 8C; IR (KBr): n= 3431, 3017,3077, 3017, 2938, 2832, 1709, 1611, 1585, 1522, 1486, 1464, 1431,1414, 1349, 1297, 1238, 1129, 997, 779, 734, 692, 625 cm�1; 1H NMR(300 MHz, CDCl3): d= 8.80, (bs, 1 H), 7.94 (s, 1 H), 7.83 (d, J = 7.1 Hz,2H), 7.52–7.33 (m, 3 H), 7.15 (t, J = 7.5, 7.3 Hz, 1 H), 6.94–6.85 (m,4 H), 6.79 (t, J = 7.5 Hz, 1 H), 3.90 (s, 3 H), 3.82 ppm (s, 6 H); 13C NMR(75.47 MHz, CDCl3 + [D6]TFA): d= 168.1, 157.9, 153.7, 145.3, 143.2,141.7, 140.7, 137.0, 130.7, 127.7, 122.9, 121.3, 118.5, 118.0, 117.8,113.5, 113.3, 111.9, 104.8, 61.3, 56.3, 21.2 ppm; MS (ESI, m/z): 511

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &8&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

[M + 1]+ ; HRMS (ESI, m/z) calcd for C28H23O4N4S: 511.14345, found:511.14215 [M + 1]+ .

(E)-5-Methoxy-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo-[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (13): Com-pound 13 was prepared according to the method described forcompound 7, employing 6 b (200 mg, 0.5 mmol) and 5-methoxyin-dolin-2-one (82 mg, 0.5 mmol) to obtain pure product 13 asa pale-red solid (229 mg, 84 % yield); mp: 289–291 8C; IR (KBr): n=3418, 3159, 2998, 2937, 2833, 1710, 1628, 1583, 1515, 1483, 1460,1358, 1327, 1250, 1197, 1128, 1107, 1093, 1070, 1035, 1000, 845,805, 787, 703, 651, 610 cm�1; 1H NMR (300 MHz, CDCl3): d= 8.39 (s,1 H), 7.98 (s, 1 H), 7.82 (d, J = 7.3 Hz, 2 H), 7.41 (t, J = 7.3, 7.6 Hz, 2 H),7.35 (t, J = 7.3 Hz, 1 H), 6.96 (s, 2 H), 6.77 (d, J = 8.3 Hz, 1 H), 6.73 (dd,J = 8.5, 2.4 Hz, 1 H), 6.47 (d, J = 2.2 Hz, 1 H), 3.91 (s, 3 H), 3.87 (s, 6 H),3.42 ppm (s, 3 H); 13C NMR (75 MHz, CDCl3 + [D6]TFA): d= 154.9,147.7, 147.4, 128.5, 126.0, 125.2, 122.4, 111.5, 109.9, 56.0, 55.8, 32.7,28.4 ppm; MS (ESI, m/z): 541 [M + 1]+ ; HRMS (ESI, m/z): calcd forC29H25O5N4S: 541.15402, found: 541.15342 [M + 1]+ .

(E)-5-Chloro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (14): Compound14 was prepared according to the method described for com-pound 7, employing 6 b (200 mg, 0.5 mmol) and 5-chloroindolin-2-one (84 mg, 0.5 mmol) to obtain pure product 14 as a yellow solid(223 mg, 81 % yield); mp: 303–305 8C; IR (KBr): n= 3418, 3078,2999, 2938, 2834, 1714, 1623, 1605, 1581, 1514, 1481, 1463, 1410,1359, 1329, 1282, 1234, 1152, 1128, 1107, 1002, 927, 855, 845, 802,780, 717, 703, 593, 581, 550, 471 cm�1; 1H NMR (300 MHz, CDCl3):d= 9.45 (s, 1 H), 7.93 (s,1 H), 7.72–7.61 (m, 2 H), 7.57–7.51 (m, 3 H),7.21 (d, J = 8.1 Hz, 1 H), 7.11 (s, 2 H), 6.90 (d, J = 8.3 Hz, 1 H), 6.78 (s,1 H), 3.96 (s, 3 H), 3.91 ppm (s, 6 H); 13C NMR (75 MHz, CDCl3 +[D6]TFA): d= 170.8, 168.2, 153.8, 145.7, 142.0, 140.9, 139.3, 131.8,130.9, 128.5, 127.8, 126.4, 126.0, 122.8, 121.4, 118.7, 112.0, 104.8,61.3, 56.3 ppm; MS (ESI, m/z): 545 [M + 1]+ ; HRMS (ESI, m/z): calcdfor C28H22O4N4ClS: 545.10448, found: 545.10397 [M + 1]+ .

(E)-6-Chloro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (15): Compound15 was prepared according to the method described for com-pound 7, employing 6 b (200 mg, 0.5 mmol) and 6-chloroindolin-2-one (84 mg, 0.5 mmol) to obtain pure product 15 as a yellow solid(223 mg, 81 % yield); mp: 322–324 8C; IR (KBr): n= 3428, 3127,2939, 2836, 1711, 1658, 1632, 1585, 1481, 1443, 1456, 1432, 1414,1298, 1279, 1171, 1155, 1128, 1105, 1072, 996, 918, 840, 804, 761,733, 695, 669, 652, 626, 599, 534 cm�1; 1H NMR (300 MHz, CDCl3 +[D6]TFA): d= 7.82 (s, 1 H), 7.75–7.66 (m, 3 H), 7.56 (s, 2 H), 7.29 (s,1 H), 7.09 (d, J = 8.8 Hz, 2 H), 6.87 (d, J = 7.9 Hz, 1 H), 6.76 (d, J =8.3 Hz,1 H), 3.96 (s, 3 H), 3.91 ppm (s, 6 H); 13C NMR (75 MHz,CDCl3 + [D6]TFA): d= 171.0, 167.8, 153.8, 142.1, 137.4, 132.0, 131.7,129.9, 129.7, 129.3, 127.8, 126.6, 123.3, 122.9, 122.8, 118.7, 117.3,111.7, 104.8, 104.7, 61.2, 56.4, 56.3 ppm; MS (ESI, m/z): 545 [M + 1]+

; HRMS (ESI, m/z): calcd for C28H22O4N4ClS: 545.10448, found:545.10416 [M + 1]+ .

(E)-5-Fluoro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (16): Compound16 was prepared according to the method described for com-pound 7, employing 6 b (200 mg, 0.5 mmol) and 5-fluoroindolin-2-one (76 mg, 0.5 mmol) to obtain pure product 16 as a yellow solid(218 mg, 82 % yield); mp: 276–278 8C; IR (KBr): n= 3417, 3174,2938, 2835, 1710, 1658, 1639, 1620, 1566, 1519, 1443, 1475, 1415,1393, 1338, 1239, 1180, 1128, 997, 840, 733, 708, 694, 651,632 cm�1; 1H NMR (300 MHz, CDCl3 + [D6]TFA): d= 7.90 (s, 1 H),7.68–7.54 (m, 5 H), 7.29 (s, 1 H), 7.13 (s, 2 H), 6.99 (s, 1 H), 6.59 (d, J =

8.3 Hz, 1 H), 4.01 (s, 6 H), 3.92 ppm (s, 3 H); 13C NMR (75 MHz,CDCl3 + [D6]TFA): d= 168.6, 168.2, 153.8, 142.0, 137.0, 132.3, 131.9,130.0, 129.8, 129.3, 127.8, 123.2, 122.8, 118.4, 118.1, 117.7, 113.5,111.8, 104.9, 104.8, 61.3, 56.5, 56.3 ppm; MS (ESI, m/z): 529 [M + 1]+

; HRMS (ESI, m/z): calcd for C28H22O4N4FS: 529.13403, found:529.13312 [M + 1]+ .

(E)-3-((6-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (17): Com-pound 17 was prepared according to the method described forcompound 7, employing 6 c (200 mg, 0.47 mmol) and indolin-2-one (62 mg, 0.47 mmol) to obtain pure product 17 as a yellowsolid (215 mg, 85 % yield); mp: 304–306 8C; IR (KBr): n= 3419, 3181,2936, 2832, 1711, 1613, 1588, 1519, 1504, 1487, 1465, 1414, 1432,1368, 1346, 1302, 1252, 1213, 1175, 1131, 1095, 1032, 1004, 829,807, 778, 757, 749, 735, 640, 622, 590, 524 cm�1; 1H NMR (300 MHz,CDCl3 + [D6]TFA): d= 7.78 (s, 1 H), 7.62 (d, J = 8.6 Hz, 2 H), 7.28 (d,J = 7.5 Hz, 1 H), 7.04–6.98 (m, 5 H), 6.92–6.82 (m, 2 H), 3.93 (s, 3 H),3.84 ppm (s, 6 H); 13C NMR (75 MHz, CDCl3 + [D6]TFA): d= 171.1,167.1, 162.3, 153.7, 144.7, 141.6, 141.0, 139.8, 131.5, 129.5, 125.9,123.2, 122.9, 120.2, 118.1, 116.9, 115.4, 111.3, 104.8, 61.3, 56.3,55.5 ppm; MS (ESI, m/z): 541 [M + 1]+ ; HRMS (ESI, m/z): calcd forC29H25O5N4S: 541.15402, found: 541.15329 [M + 1]+ .

(E)-5-Methoxy-3-((6-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-ne)indolin-2-one (18): Compound 18 was prepared according tothe method described for compound 7, employing 6 c (200 mg,0.47 mmol) and 5-methoxyindolin-2-one (76 mg, 0.47 mmol) toobtain pure product 18 as a yellow solid (214 mg, 80 % yield); mp:292–294 8C; IR (KBr): n= 3428, 2935, 2836, 1701, 1633, 1607, 1586,1485, 1464, 1414, 1358, 1302, 1251, 1205, 1175, 1130, 1069, 1035,835, 819, 780, 755, 734, 646, 588 cm�1; 1H NMR (300 MHz, CDCl3 +[D6]TFA): d= 7.82 (s, 1 H), 7.61 (d, J = 8.4 Hz, 2 H), 7.10 (s, 2 H), 7.05–6.94 (m, 3 H), 6.88 (dd, J = 8.4, 2.0 Hz, 1H), 6.49 (s, 1 H), 3.99 (s, 3 H),3.90 (d, J = 7.1 Hz, 9 H), 3.58 ppm (s, 3 H); 13C NMR (75 MHz, CDCl3 +[D6]TFA): d= 171.1, 167.7, 155.5, 153.7, 144.8, 141.6, 139.8, 135.1,129.4, 123.0, 121.1, 118.1, 117.9, 117.7, 117.1, 115.5, 112.9, 111.9,104.8, 61.4, 56.3, 56.2, 55.5 ppm; MS (ESI, m/z): 571 [M + 1]+ ; HRMS(ESI, m/z): calcd for C30H27O6N4S: 571.16458, found: 571.16467 [M +1]+ .

(E)-5-Chloro-3-((6-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-ne)indolin-2-one (19): Compound 19 was prepared according tothe method described for compound 7, employing 6 c (200 mg,0.47 mmol) and 5-chloroindolin-2-one (78 mg, 0.47 mmol) toobtain pure product 19 as a yellow solid (205 mg, 76 % yield); mp:297–299 8C; IR (KBr): n= 3418, 3165, 2939, 2836, 1701, 1625, 1598,1482, 1465, 1414, 1364, 1336, 1252, 1305, 1252, 1222, 1174, 1149,1129, 1106, 1066, 1034, 1005, 915, 837, 805, 730, 704, 651, 586,545, 525 cm�1; 1H NMR (300 MHz, CDCl3 + [D6]TFA): d= 7.89 (s, 1 H),7.56 (d, J = 7.9 Hz, 2 H), 7.24 (s, 1 H), 7.12 (s, 2 H), 7.04 (d, J = 7.7 Hz,2 H), 6.95 (d, J = 8.3 Hz, 1 H), 6.80 (s, 1 H), 3.98 (s, 3 H), 3.92 (s, 6 H),3.87 ppm (s, 3 H); 13C NMR (75 MHz, CDCl3 + [D6]TFA): d= 168.0,162.5, 153.8, 141.7, 139.3, 131.1, 129.5, 128.7, 126.3, 122.9, 121.5,118.4, 118.2, 115.7, 112.2, 104.9, 61.4, 56.3, 55.5 ppm; MS (ESI, m/z):575 [M + 1]+ ; HRMS (ESI, m/z): calcd for C29H24O5N4ClS: 575.11504,found: 575.11527 [M + 1]+ .

(E)-6-Chloro-3-((6-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-ne)indolin-2-one (20): Compound 20 was prepared according tothe method described for compound 7, employing 6 c (200 mg,0.47 mmol) and 6-chloroindolin-2-one (78 mg, 0.47 mmol) to

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &9&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

obtain pure product 19 as a yellow solid (205 mg, 76 % yield); mp:352–354 8C; IR (KBr): n= 3408, 3120, 2940, 2836, 1711, 1611, 1487,1461, 1416, 1303, 1283, 1256, 1216, 1175, 1154, 1133, 1105, 1072,995, 918, 828, 804, 728, 706, 650, 620, 599, 523, 504 cm�1; 1H NMR(300 MHz, CDCl3 + [D6]TFA): d= 7.78 (s, 1 H), 7.61 (d, J = 8.5 Hz, 2 H),7.06–7.04 (m, 5 H), 6.91 (d, J = 8.2 Hz, 1 H), 6.81 (d, J = 8.2 Hz,1 H),3.97 (s, 3 H), 3.88 ppm (s, 9 H); 13C NMR (75 MHz, CDCl3 + [D6]TFA):d= 171.2, 167.6, 162.4, 153.8, 145.1, 142.0, 137.3, 129.6, 126.8,122.9, 122.7, 118.9, 118.3, 117.5, 115.5, 111.7, 104.7, 61.2, 56.3,55.5 ppm; MS (ESI, m/z): 575 [M + 1]+ ; HRMS (ESI, m/z): calcd forC29H24O5N4ClS: 575.11504, found: 575.11586 [M + 1]+ .

(E)-5-Fluoro-3-((6-(4-methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-ne)indolin-2-one (21): Compound 21 was prepared according tothe method described for compound 7, employing 6 c (200 mg,0.47 mmol) and 5-fluoroindolin-2-one (70 mg, 0.47 mmol) to obtainpure product 21 as a yellow solid (204 mg, 78 % yield); mp: 297–299 8C; IR (KBr): n= 3408, 2937, 2835, 1713, 1608, 1585, 1519, 1475,1414, 1361, 1249, 1175, 1127, 1005, 861, 840, 757, 735, 705, 590,565, 547 cm�1; 1H NMR (300 MHz, CDCl3 + [D6]TFA): d= 7.87 (s, 1 H),7.59 (d, J = 7.9 Hz, 2 H), 7.10 (s, 2 H), 7.06–6.92 (m, 4 H), 6.60 (d, J =7.4 Hz, 1 H), 3.97 (s, 3H), 3.88 ppm (s, 9H); 13C NMR (75 MHz,CDCl3 + [D6]TFA): d= 171.2, 167.6, 162.4, 153.8, 145.1, 142.0, 137.3,129.6, 126.8, 122.9, 122.7, 118.9, 118.3, 117.5, 115.5, 111.7, 104.7,61.2, 56.3, 55.5 ppm; MS (ESI, m/z): 559 [M + 1]+ ; HRMS (ESI, m/z):calcd for C29H24O5N4FS: 559.14460, found: 559.14456 [M + 1]+ .

(E)-3-((6-(4-Methoxyphenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-[2,1-b][1,3,4]thiadiazol-5-yl)methylene)-5-nitroindolin-2-one (22):Compound 22 was prepared according to the method describedfor compound 7, employing 6 c (200 mg, 0.47 mmol) and 5-nitroin-dolin-2-one (83 mg, 0.47 mmol) to obtain pure product 22 asa yellow solid (211 mg, 77 % yield); mp: 298–300 8C; IR (KBr): n=3412, 3088, 2937, 2837, 1716, 1607, 1519, 1485, 1465, 1412, 1368,1335, 1313, 1254, 1193, 1149, 1128, 1107, 1074, 1003, 904, 843, 777,750, 738, 708, 686, 650, 599, 584, 547 cm�1; 1H NMR (300 MHz,CDCl3 + [D6]TFA): d= 8.20 (dd, J = 8.6, 2.0 Hz, 1 H), 8.09 (s, 1), 7.67(d, J = 1.8 Hz, 1 H), 7.59 (d, J = 8.6 Hz, 2 H), 7.16 (s, 1 H), 7.13 (s, 2 H),6.94 (d, J = 8.4 Hz, 2 H), 3.98 (s, 3 H), 3.92 (s, 6 H), 3.82 ppm (s, 3 H);13C NMR (75 MHz, CDCl3 + [D6]TFA): d= 171.1, 168.3, 162.4, 153.8,145.8, 143.2, 142.0, 129.5, 127.0, 123.1, 122.8, 121.3, 120.5, 118.4,117.7, 115.4, 110.8, 104.8, 61.3, 56.4, 55.5 ppm; MS (ESI, m/z): 586[M + 1]+ ; HRMS (ESI, m/z): calcd for C29H24O7N5S: 586.13910, found:586.13965 [M + 1]+ .

(E)-3-((6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (23): Com-pound 23 was prepared according to the method described forcompound 7, employing 6 d (200 mg, 0.46 mmol) and indolin-2-one (62 mg, 0.46 mmol) to obtain pure product 23 as a yellowsolid (203 mg, 80 % yield); mp: 318–320 8C; IR (KBr): n= 3152, 3077,2937, 2833, 1709, 1612, 1586, 1520, 1483, 1464, 1432, 1414, 1349,1307, 1286, 1238, 1173, 1130, 1093, 1071, 996, 829, 779, 758, 737,718, 635, 621 cm�1; 1H NMR (300 MHz, CDCl3 + [D6]TFA): d= 7.76 (s,1 H), 7.59 (d, J = 8.3 Hz, 2 H), 7.49 (d, J = 8.3 Hz, 2 H), 7.34 (t, J =7.5 Hz, 1 H), 7.06 (s, 3 H), 6.92 (t, J = 7.5 Hz, 1 H), 6.81 (d, J = 7.7 Hz,1 H), 3.98 (s, 3 H), 3.87 ppm (s, 6 H); 13C NMR (75 MHz, CDCl3 +

[D6]TFA): d= 171.0, 167.9, 153.7, 145.3, 141.8, 141.2, 138.4, 131.9,130.2, 129.0, 125.8, 124.5, 123.2, 122.9, 120.1, 116.3, 111.4, 104.8,61.3, 56.3 ppm; MS (ESI, m/z): 545 [M + 1]+ ; HRMS (ESI, m/z): calcdfor C28H22O4N4ClS: 545.10448, found: 545.10440 [M + 1]+ .

(E)-3-((6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-[2,1-b][1,3,4]thiadiazol-5-yl)methylene)-5-methoxyindolin-2-one

(24): Compound 24 was prepared according to the method de-scribed for compound 7, employing 6 d (200 mg, 0.46 mmol) and5-methoxyindolin-2-one (75 mg, 0.46 mmol) to obtain pure prod-uct 24 as red solid (217 mg, 81 % yield); mp: 284–286 8C; IR (KBr):n= 3418, 3164, 2941, 2830, 1700, 1631, 1519, 1483, 1464, 1438,1408, 1359, 1289, 1255, 1171, 1128, 1091, 1036, 1012, 921, 893,832, 788, 746, 634, 623, 565 cm�1; 1H NMR (300 MHz, CDCl3 +[D6]TFA): d= 7.80 (s, 1H), 7.59 (d, J = 8.4 Hz, 2 H), 7.48 (d, J = 8.4 Hz,2 H), 7.11 (s, 2 H), 6.98 (d, J = 8.4 Hz, 1 H), 6.87 (dd, J = 8.6, 2.2 Hz,1 H), 6.44 (d, J = 2.0 Hz, 1H), 3.99 (s, 3H), 3.90 (s, 6H), 3.58 ppm (s,3H); 13C NMR (75 MHz, CDCl3 + [D6]TFA): d= 170.9, 167.3, 155.6,153.8, 146.1, 142.1, 140.4, 137.6, 134.8, 131.0, 130.0, 128.9, 126.0,122.9, 121.1, 118.9, 117.6, 116.7, 112.9, 112.4, 111.5, 104.6, 61.1, 56.3,55.8 ppm; MS (ESI, m/z): 575 [M + 1]+ ; HRMS (ESI, m/z): calcd forC29H24O5N4ClS: 575.11502, found: 575.11529 [M + 1]+ .

(E)-5-Chloro-3-((6-(4-chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-ne)indolin-2-one (25): Compound 25 was prepared according tothe method described for compound 7, employing 6 d (200 mg,0.46 mmol) and 5-chloroindolin-2-one (77 mg, 0.46 mmol) toobtain pure product 25 as a yellow solid (210 mg, 78 % yield); mp:286–288 8C; IR (KBr): n= 3427, 3160, 2939, 2835, 1706, 1629, 1606,1585, 1514, 1480, 1464, 1431, 1414, 1340, 1297, 1238, 1171, 1128,1090, 1066, 1002, 914, 895, 835, 759, 719, 647, 588, 577, 552 cm�1;1H NMR (300 MHz, CDCl3 + [D6]TFA): d= 7.86 (s, 1 H), 7.60 (d, J =8.4 Hz, 2 H), 7.49 (d, J = 8.1 Hz, 2 H), 7.29 (d, J = 8.4 Hz, 1 H), 7.13 (s,2 H), 7.00 (d, J = 8.3 Hz, 1 H), 6.87 (s,1 H), 3.99 (s, 3 H), 3.92 ppm (s,6 H); 13C NMR (75 MHz, CDCl3 + [D6]TFA): d= 170.7, 168.1, 153.8,141.9, 139.4, 138.3, 131.2, 130.3, 129.0, 128.6, 126.0, 122.9, 121.4,118.2, 112.1, 104.8, 61.3, 56.3 ppm; MS (ESI, m/z): 579 [M + 1]+ ;HRMS (ESI, m/z): calcd for C28H21O4N4 Cl2S: 579.06551, found:579.06610 [M + 1]+ .

(E)-6-Chloro-3-((6-(4-chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-ne)indolin-2-one (26): Compound 26 was prepared according tothe method described for compound 7, employing 6 d (200 mg,0.46 mmol) and 6-chloroindolin-2-one (77 mg, 0.46 mmol) toobtain pure product 26 as a yellow solid (210 mg, 78 % yield); mp:336–338 8C; IR (KBr): n= 3428, 3138, 2939, 2836, 1713, 1613, 1585,1519, 1479, 1459, 1431, 1414, 1346, 1307, 1284, 1239, 1214, 1172,1154, 1129, 1071, 994, 920, 829, 761, 739, 721, 671, 642, 599,547 cm�1; 1H NMR (300 MHz, CDCl3 + [D6]TFA): d= 7.55 (s, 1 H),7.61–7.52 (m, 4 H), 7.13 (s, 1 H), 7.05 (s, 2H), 6.92 (d, J = 7.5 Hz, 1 H),6.79 (d, J = 8.1 Hz, 1 H), 4.00 (s, 3 H), 3.90 ppm (s, 6 H); 13C NMR(75 MHz, CDCl3 + [D6]TFA): d= 171.0, 168.0, 153.8, 145.6, 142.2,141.8, 138.6, 137.9, 130.3, 129.1, 126.7, 124.4, 123.2, 122.9, 119.2,118.6, 116.7, 112.1, 104.8, 61.4, 56.3 ppm; MS (ESI, m/z): 579 [M +1]+ ; HRMS (ESI, m/z): calcd for C28H21O4N4 Cl2S: 579.06551, found:579.06673 [M + 1]+ .

(E)-3-((6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-[2,1-b][1,3,4]thiadiazol-5-yl)methylene)-5-fluoroindolin-2-one(27): Compound 27 was prepared according to the method de-scribed for compound 7, employing 6 d (200 mg, 0.46 mmol) and5-fluoroindolin-2-one (70 mg, 0.46 mmol) to obtain pure product27 as a yellow solid (209 mg, 80 % yield); mp: 315–317 8C; IR (KBr):n= 3433, 3174, 3077, 2939, 2836, 1709, 1637, 1619, 1587, 1519,1475, 1432, 1414, 1363, 1339, 1304, 1286, 1239, 1182, 1129, 1093,1070, 1000, 830, 763, 713 cm�1; 1H NMR (300 MHz, CDCl3 + [D6]TFA):d= 7.83 (s, 1 H), 7.64–7.48 (m, 4 H), 7.11 (s, 2 H), 7.03 (d, J = 4.9 Hz,2 H), 6.59 (d, J = 8.3 Hz, 1 H), 4.00 (s, 3 H), 3.91 ppm (s, 6 H); 13C NMR(75 MHz, CDCl3 + [D6]TFA): d= 170.9, 168.3, 157.3, 153.8, 145.7,141.8, 139.3, 138.7, 137.2, 130.3, 129.0, 124.4, 122.9, 121.0, 118.9,

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &10&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

118.5, 118.1, 117.8, 113.4, 113.0, 112.2, 112.1, 104.9, 61.4, 56.3 ppm;MS (ESI, m/z): 563 [M + 1]+ ; HRMS (ESI, m/z): calcd for C28H21O4N4 ClFS: 563.09506, found: 563.09582 [M + 1]+ .

(E)-3-((6-(4-Chlorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo-[2,1-b][1,3,4]thiadiazol-5-yl)methylene)-5-nitroindolin-2-one (28):Compound 28 was prepared according to the method describedfor compound 7, employing 6 d (200 mg, 0.46 mmol) and 5-nitroin-dolin-2-one (82 mg, 0.46 mmol) to obtain pure product 28 asa yellow solid (209 mg, 76 % yield); mp: 287–289 8C; IR (KBr): n=3418, 3129, 3088, 2939, 2837, 1708, 1627, 1604, 1522, 1481, 1465,1431, 1414, 1338, 1242, 1224, 1193, 1146, 1130, 1091, 1075, 1000,833, 749, 735, 649 cm�1; 1H NMR (300 MHz, CDCl3 + [D6]TFA): d=8.23 (dd, J = 8.6, 1.8 Hz, 1 H), 8.07 (s, 1 H), 7.70 (d, J = 1.7 Hz, 1 H),7.58 (d, J = 8.3 Hz, 2 H), 7.44 (d, J = 8.1 Hz, 2 H), 7.17 (d, J = 8.8 Hz,1 H), 7.12 (s, 2 H), 3.99 (s, 3 H), 3.91 ppm (s, 6 H); 13C NMR (75 MHz,CDCl3 + [D6]TFA): d= 170.9, 168.4, 153.7, 146.1, 143.1, 141.8, 138.5,130.3, 129.0, 127.7, 127.4 125.1, 122.9, 121.1, 120.3, 117.4, 111.4,111.1, 104.8, 61.3, 56.3 ppm; MS (ESI, m/z): 590 [M + 1]+ ; HRMS (ESI,m/z): calcd for C28H21O6N5ClS: 590.08956, found: 590.09038 [M + 1]+

.

(E)-5-Chloro-3-((6-(4-fluorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-ne)indolin-2-one (29): Compound 29 was prepared according tothe method described for compound 7, employing 6 e (200 mg,0.48 mmol) and 5-chloroindolin-2-one (80 mg, 0.46 mmol) toobtain pure product 29 as a yellow solid (218 mg, 80 % yield); mp:322–324 8C; IR (KBr): n= 3407, 3139, 3005, 2941, 2837, 1714, 1624,1605, 1536, 1500, 1483, 1458, 1432, 1357, 1234, 1221, 1206, 1175,1156, 1128, 1107, 1064, 999, 907, 871, 802, 754, 728, 636, 622, 583,572, 538 cm�1; 1H NMR (300 MHz, CDCl3 + [D6]TFA): d= 7.88 (s, 1 H),7.65–7.64 (m, 2 H), 7.29–7.20 (m, 3 H), 7.14 (s, 2 H), 6.96 (d, J =8.4 Hz, 1 H), 6.79 (s, 1 H), 3.99 (s, 3 H), 3.93 ppm (s, 6 H); 13C NMR(75 MHz, CDCl3 + [D6]TFA): d= 170.7, 167.9, 165.4, 163.4, 153.8,142.0, 139.4, 130.9, 130.0, 128.4, 126.0, 122.9, 121.4, 118.5, 117.3,117.2, 111.9, 104.7, 61.2, 56.3 ppm; MS (ESI, m/z): 563 [M + 1]+ ;HRMS (ESI, m/z): calcd for C28H21O4N4ClFS: 563.09506, found:563.09539 [M + 1]+ .

(E)-3-((6-(4-Fluorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)-5-methoxyindolin-2-one (30):Compound 30 was prepared according to the method describedfor compound 7, employing 6 e (200 mg, 0.48 mmol) and 5-me-thoxyindolin-2-one (78 mg, 0.46 mmol) to obtain pure product 30as a yellow solid (218 mg, 81 % yield); mp: 288–290 8C; IR (KBr): n=3400, 3158, 2943, 2834, 1711, 1631, 1596, 1503, 1484, 1461, 1433,1357, 1299, 1217, 1197, 1169, 1129, 1107, 1092, 1067, 1035, 998,841, 806, 756, 721, 706, 694, 641 cm�1; 1H NMR (300 MHz, CDCl3 +[D6]TFA): d= 7.81 (s, 1 H), 7.70–7.66 (m, 2 H), 7.21 (t, J = 8.3 Hz, 2 H),7.11 (s, 2 H), 6.93 (d, J = 8.4 Hz, 1 H), 6.85 (dd, J = 8.6, 2.2 Hz, 1 H),6.41 (d, J = 2.0 Hz, 1H), 3.98 (s, 3 H), 3.90 (s, 6H), 3.56 ppm (s, 3 H);13C NMR (75 MHz, CDCl3 + [D6]TFA): d= 170.9, 167.6, 166.0, 162.6,155.6, 153.8, 145.7, 142.0, 134.9, 134.8, 129.9, 123.2, 122.8, 121.0,117.3, 117.0, 116.7, 112.9, 112.4, 111.9, 111.6, 104.6, 61.3, 56.3, 56.2,55.8 ppm; MS (ESI, m/z): 559 [M + 1]+ ; HRMS (ESI, m/z): calcd forC29H24O5N4FS: 559.14460, found: 559.14501 [M + 1]+ .

(E)-5-Fluoro-3-((6-(4-fluorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-ne)indolin-2-one (31): Compound 31 was prepared according tothe method described for compound 7, employing 6 e (200 mg,0.48 mmol) and 5-fluoroindolin-2-one (73 mg, 0.46 mmol) to obtainpure product 31 as a yellow solid (216 mg, 82 % yield); mp: 330–332 8C; IR (KBr): n= 3429, 3177, 2940, 2846, 1710, 1619, 1524, 1475,

1413, 1432, 1363, 1339, 1173, 1157, 1128, 1090, 1070, 1000, 924,903, 863, 837, 806, 761, 735, 655, 617, 587 cm�1; 1H NMR (300 MHz,CDCl3 + [D6]TFA): d= 7.85 (s, 1 H), 7.65–7.59 (m, 2 H), 7.20 (d, J =7.9 Hz, 2 H), 7.12 (s, 2 H), 7.04–6.98 (m, 2 H), 6.58 (d, J = 8.4 Hz, 1 H),4.00 (s, 3 H), 3.92 ppm (s, 6 H); 13C NMR (75 MHz, CDCl3 + [D6]TFA):d= 171.0, 167.7, 163.3, 157.8, 153.8, 142.0, 137.1, 130.0, 122.9,121.2, 118.8, 117.8, 117.6, 117.3, 117.1, 113.3, 113.1, 111.7, 104.6,61.2, 56.3 ppm; MS (ESI, m/z): 547 [M + 1]+ ; HRMS (ESI, m/z): calcdfor C28H21O4N4F2S: 547.12461, found: 547.12525 [M + 1]+ .

(E)-6-Chloro-3-((6-(4-fluorophenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methyle-ne)indolin-2-one (32): Compound 32 was prepared according tothe method described for compound 7, employing 6 e (200 mg,0.48 mmol) and 5-fluoroindolin-2-one (73 mg, 0.46 mmol) to obtainpure product 32 as a yellow solid (218 mg, 80 % yield); mp: 338–340 8C; IR (KBr): n= 3409, 3139, 2944, 2839, 1712, 1633, 1613, 1503,1484, 1459, 1431, 1414, 1345, 1307, 1230, 1155, 1128, 1103, 1072,994, 903, 842, 803, 725, 705, 694, 599, 576, 522 cm�1; 1H NMR(300 MHz, CDCl3 + [D6]TFA): d= 7.76 (s, 1 H), 7.66–7.61 (m, 2 H),7.28–7.23 (m, 2 H), 7.14 (s, 1 H), 7.06 (s, 2 H), 6.92 (d, J = 8.3 Hz, 1 H),6.83 (d, J = 8.3 Hz, 1 h), 4.01 (s, 3 H), 3.91 ppm (s, 6 H); 13C NMR(75 MHz, CDCl3 + [D6]TFA): d= 171.2, 168.0, 166.5, 163.1, 153.8,142.2, 141.7, 138.0, 130.3, 130.1, 126.7, 123.3, 122.9, 118.6, 117.6,117.3, 116.7, 112.1, 104.9, 61.4, 56.3 ppm; MS (ESI, m/z): 563 [M +1]+ ; HRMS (ESI, m/z): calcd for C28H21O4N4ClFS: 563.09506, found:563.09601 [M + 1]+ .

Biology

Cell culture, maintenance, and anti-proliferative evaluation : All celllines used in this study were purchased from the American TypeCulture Collection (ATCC, USA). A549, MCF-7, HCT116 and HeLacells were grown in Dulbecco’s modified Eagle’s medium (DMEM,containing 10 % fetal bovine serum (FBS) in a humidified atmos-phere of 5 % CO2 at 37 8C). Cells were trypsinized when sub-conflu-ent from T25 flasks/60 mm dishes and seeded in 96-well plates.The test compounds were evaluated for their in vitro anti-prolifera-tive activity against four different human cancer cell lines, usinga protocol of 48 h continuous drug exposure; a sulforhodamine Bcell proliferation assay was used to estimate cell viability orgrowth. The cell lines were grown in their respective media con-taining 10 % FBS and were seeded into 96-well microtiter plates in200 mL aliquots at plating densities according to the doubling timeof the given cell line. The microtiter plates were incubated at 37 8C,5 % CO2, 95 % air, and 100 % relative humidity for 24 h prior to theaddition of test compounds. Aliquots (2 mL) of the test compoundswere added to the wells already containing cells in 198 mL, result-ing in the required final drug concentrations. For each compound,five concentrations (0.01, 0.1, 1, 10, and 100 mm) were evaluated,and each was done in triplicate wells. Plates were incubated fur-ther for 48 h, and the assay was terminated by the addition of10 % TCA and incubated for 60 min at 4 8C. Later, the plates wereair-dried and washed thrice with water. The cells were then incu-bated with 0.57 % sulforhodamine B dye for 1 h at 37 8C, andplates were washed thrice with 1 % acetic acid. The air-dried plateswere reconstituted with 50 mL 10 mm Tris buffer (pH 8.0), and theabsorbance was read at 510 nm in EnSpire, multimode reader.Before the start of treatments, a plate was terminated at 24 h,which served as time zero (t0). The plates were then terminatedafter 48 h, containing DMSO-treated control (tc) and five-dose-treat-ed cells (ti). The IC50 values were calculated by linear interpolationfrom plots of percent growth versus log concentrations of com-

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &11&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

pounds in mm. IC50 values are indicated as means �SD of three in-dependent experiments.[18]

Cell-cycle analysis : A549 cells in 60 mm dishes were incubated for24 h in the presence or absence of test compounds 7, 11, and 15at 5 or 10 mm. Cells were harvested with Trypsin-EDTA, and thenfixed with ice-cold 70 % ethanol at 4 8C for 30 min. Ethanol was re-moved by centrifugation (400 g, 10 min), and cells were stainedwith 1 mL DNA staining solution [0.2 mg propidium iodide (PI),2 mg RNase A] for 30 min as described earlier.[18] The DNA contentsof 20 000 events were measured by flow cytometry (BD FACSCan-to II). Histograms were analyzed using FCS express 4 plus.[18]

Tubulin polymerization assays : An in vitro assay for monitoring thetime-dependent polymerization of tubulin to microtubules wasperformed by using a fluorescence-based tubulin polymerizationassay kit (BK011, Cytoskeleton Inc.) according to the manufacturer’sprotocol. The reaction mixture in a final volume of 10 mL in PEMbuffer (80 mm PIPES, 0.5 mm EGTA, 2 mm MgCl2, pH 6.9) in 384-well plates contained 2 mg mL�1 bovine brain tubulin, 10 mm fluo-rescent reporter, and 1 mm GTP in the presence or absence of testcompounds at 37 8C. Tubulin polymerization was followed by mon-itoring the fluorescence enhancement due to the incorporation ofa fluorescence reporter into microtubules as polymerization pro-ceeds. Fluorescence emission at 420 nm (excitation wavelength360 nm) was measured for 1 h at 1-min intervals in a multimodeplate reader (Tecan M200). To determine the IC50 values of the com-pounds against tubulin polymerization, the compounds were pre-incubated with tubulin at varying concentrations (1, 5, 10 and20 mm). Assays were performed under similar conditions as usedfor the polymerization assays described above.[18]

Western blot analysis of soluble versus polymerized tubulin andcyclin-B1: Cells were seeded in 12-well plates at 1 � 105 cells perwell in complete growth medium. Cells were treated with com-pounds 7 or 11 for 24 h. Subsequently, cells were washed withphosphate-buffered saline (PBS), and soluble and insoluble tubulinfractions were collected. To collect the soluble tubulin fractions,cells were permeabilized with 200 mL pre-warmed lysis buffer[80 mm PIPES-KOH (pH 6.8), 1 mm MgCl2, 1 mm EGTA, 0.2 % TritonX-100, 10 % glycerol, AND 0.1 % protease inhibitor cocktail (Sigma–Aldrich)] and incubated for 3 min at 30 8C. The lysis buffer wasgently removed, and mixed with 100 mL 3 � Laemmli sample buffer(180 mm Tris·Cl pH 6.8, 6 % SDS, 15 % glycerol, 7.5 % b-mercaptoe-thanol, and 0.01 % bromophenol blue). Samples were immediatelyheated at 95 8C for 3 min. To collect the insoluble tubulin fraction,300 mL 1 � Laemmli sample buffer was added to the remainingcells in each well, and the samples were heated at 95 8C for 3 min.Equal volumes of samples were separated by 10 % SDS-PAGE andthen transferred to a nitrocellulose membrane using semidry trans-fer at 50 mA for 1 h. Blots were probed with mouse anti-human a-tubulin diluted 1:2000 mL (Sigma) and stained with rabbit anti-mouse secondary antibody coupled with horseradish peroxidase,diluted 1:5000 mL (Sigma). Bands were visualized using an en-hanced Chemiluminescence protocol (Pierce) and radiographic film(Kodak). For cyclin-B1 immunoblots, cells were seeded in 12-wellplates at 1 � 105 cells per well in complete medium and treatedwith various concentrations of 7, 11, and 15 for 24 h. After treat-ment, cells were washed twice with PBS and lysed in 1 � SDSsample buffer. Proteins were separated, transferred, probed, andanalyzed similarly to tubulin. The primary anti-cyclin-B1 antibodywas employed at 1:1500 and b-actin (Sigma) and horseradish per-oxidase coupled goat anti-rabbit secondary antibody diluted1:5000 (Sigma).[20]

Immunohistochemistry and nuclear morphology analysis : A549 cellswere seeded on a glass cover slip, incubated for 24 h in the pres-ence or absence of test compounds 7, 11, and 15 at 5 mm. Cellsgrown on cover slips were fixed in 3.5 % formaldehyde in PBSpH 7.4 for 10 min at room temperature. Cells were permeabilizedfor 6 min in PBS containing 0.5 % Triton X-100 (Sigma) and 0.05 %Tween-20 (Sigma). The permeabilized cells were blocked with 2 %BSA (Sigma) in PBS for 1 h. Later, the cells were incubated with pri-mary antibody for tubulin (Sigma) at 1:200 diluted in blocking solu-tion for 4 h at room temperature. The antibodies were then re-moved, and the cells were washed thrice with PBS. Cells were thenincubated with FITC-labeled anti-mouse secondary antibody(1:500) for 1 h at room temperature. Cells were washed thrice withPBS and mounted in medium containing DAPI. Images were cap-tured using an Olympus confocal microscope and analyzed withProvision software (instrument: FLOW VIEW-FV 1000 Series; soft-ware: FV 10 ASW 1.7 Series).[19]

Docking : All geometries were optimized in Gaussian 09 using PM3semi-empirical method.[20] The protein structure was downloadedfrom RCSB Protein Data Bank (PDB ID: 3E22).[21] Docking studieswere performed using AutoDock 4.2 software.[22] The analysis of in-termolecular interactions was performed with PyMOL v. 0.99.[23]

Acknowledgements

M.P.N.R. , P.S. , K.M., and J.K. acknowledge the Council of Scientific& Industrial Research/University Grants Commission (CSIR-UGC),New Delhi (India), for the award of senior research fellowships;they are thankful to DST (India) for the award of Inspire fellow-ships. The authors also acknowledge the Council of Scientific &Industrial Research (CSIR), India, for financial support under the12th Five-Year Plan project “Affordable Cancer Therapeutics(ACT)” (CSC0301) and Small Molecule in Lead Exploration (SMiLE-CSC-0111).

Keywords: antitumor agents · docking · imidazothiadiazole-linked oxindoles · tubulin

[1] H. C. Stevenson, I. Green, J. M. Hamilton, B. A. Calabro, D. R. Parkinson,J. Clin. Oncol. 1991, 9, 2052 – 2066.

[2] J. S. Kovach, P. A. Svingen, D. J. Schaid, J. Natl. Cancer Inst. 1992, 84,515 – 519.

[3] T. Friis, A. M. Engel, B. M. Klein, J. Rygaard, G. Houen, Angiogenesis 2005,8, 25 – 34.

[4] M. Guminska, T. Kedryna, E. Marchut, Biochem. Pharmacol. 1986, 35,4369 – 4374.

[5] M. Artwohl, T. Holzenbein, L. Wagner, A. Freudenthaler, W. Waldhausl,Br. J. Pharmacol. 2000, 131, 1577 – 1583.

[6] A. Andreani, D. Bonazzi, M. Rambaldi, Arch. Pharm. 1982, 315, 451 – 456.[7] A. K. Gadad, S. S. Karki, V. G. Rajurkar, B. A. Bhongade, Arzneim.-Forsch.

1999, 49, 858 – 863.[8] A. Andreani, M. Granaiola, A. Locatelli, R. Morigi, M. Rambaldi, L. Varoli,

N. Calonghi, C. Cappadone, G. Farruggia, C. Stefanelli, L. Masotti, T. L.Nguyen, E. Hamel, R. H. Shoemaker, J. Med. Chem. 2012, 55, 2078 – 2088.

[9] a) F. Pellegrini, D. R. Budman, Cancer Invest. 2005, 23, 264 – 273; b) S.Honore, E. Pasquier, D. Braguer, Cell. Mol. Life Sci. 2005, 62, 3039 – 3065.

[10] a) H. N. Bramson, J. Corona, S. T. Davis, S. H. Dickerson, M. Edelstein, S. V.Frye, R. T. Gampe, P. A. Harris, A. Hassell, W. D. Holmes, R. N. Hunter, K. E.Lackey, B. Lovejoy, M. J. Luzzio, V. Montana, W. J. Rocque, D. Rusnak, L.Shewchuk, J. M. Veal, D. H. Walker, L. F. Kuyper, J. Med. Chem. 2001, 44,4339; b) M. A. Bogoyevitch, D. P. Fairlie, Drug Discovery Today 2007, 12,622; c) G. Cerchiaro, A. M. C. Ferreira, J. Braz. Chem. Soc. 2006, 17, 1473.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &12&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

[11] L. Sun, C. Liang, S. Shirazian, Y. Zhou, T. Miller, J. Cui, J. Y. Fukuda, J. Y.Chu, A. Nematalla, X. Wang, H. Chen, A. Sistla, T. C. Luu, F. Tang, J. Wei,C. Tang, J. Med. Chem. 2003, 46, 1116.

[12] R. Roskoski, Jr. , Biochem. Biophys. Res. Commun. 2007, 356, 323.[13] Z. Chen, P. J. Merta, N. H. Lin, S. K. Tahir, P. Kovar, H. L. Sham, H. Zhang,

Mol. Cancer Ther. 2005, 4, 562 – 568.[14] A. Kamal, N. V. S. Reddy, V. L. Nayak, V. S. Reddy, B. Prasad, V. D. Nim-

barte, V. Srinivasulu, M. V. P. S. Vishnuvardhan, C. S. Reddy, ChemMed-Chem 2014, 9, 117 – 128.

[15] A. Kamal, V. S. Reddy, K. Santosh, S. S. Chourasiya, A. B. Shaik, G. B.Kumar, C. Kishor, M. K. Reddy, M. P. N. Rao, N. Ananthamurthy, K. V. S.Ramakrishna, A. Anthony, K. Srigiridhar, ChemMedChem 2013, 8, 2015 –2025.

[16] A. Andreani, A. Locatelli, A. Leoni, M. Rambaldi, R. Morigi, R. Bossa, M.Chiericozzi, A. Fraccari, I. Galatulas, Eur. J. Med. Chem. 1997, 32, 919 –924.

[17] A. Andreani, M. Granaiola, A. Leoni, A. Locatelli, R. Morigi, M. Rambaldi,G. Giorgi, L. Salvini, V. Garaliene, Anti-Cancer Drug Des. 2001, 16, 167 –174.

[18] M. A. Reddy, N. Jain, D. Yada, C. Kishore, V. J. Reddy, P. S. Reddy, A. An-thony, S. V. Kalivendi, B. Sreedhar, J. Med. Chem. 2011, 54, 6751 – 6760.

[19] N. Jain, D. Yada, T. B. Shaik, G. Vasantha, P. S. Reddy, S. V. Kalivendi, B.Sreedhar, ChemMedChem 2011, 6, 859 – 868.

[20] Gaussian 09 (Revision B.1), M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E.Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, B. Men-

nucci, G. A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian,A. F. Izmaylov, J. Bloino, G. Zheng, J. L. Sonnenberg, M. Hada, M. Ehara,K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O.Kitao, H. Nakai, T. Vreven, J. A. Montgomery, J. E. Peralta, F. Ogliaro, M.Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin, V. N. Staroverov, R. Kobaya-shi, J. Normand, K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar, J.Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene, J. E. Knox, J. B. Cross, V.Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev,A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin, K. Moro-kuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dap-prich, A. D. Daniels, Farkas, J. B. Foresman, J. V. Ortiz, J. Cioslowski, D. J.Fox, Gaussian Inc. , Wallingford, CT (USA), 2010.

[21] A. Cormier, M. Marchand, R. B. G. Ravelli, M. Knossow, B. Gigant, EMBORep. 2008, 9, 1101 – 1106.

[22] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Good-sell, A. J. Olson, J. Comput. Chem. 2009, 30, 2785 – 2791;http://autodock.scripps.edu/.

[23] The PyMOL Molecular Graphics System, Version 0.99, Schrçdinger LLC,New York, NY (USA); http://www.pymol.org/.

Received: January 29, 2014

Published online on && &&, 0000

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &13&

These are not the final page numbers! ��

CHEMMEDCHEMFULL PAPERS www.chemmedchem.org

FULL PAPERS

A. Kamal,* M. P. N. Rao, P. Das,P. Swapna, S. Polepalli, V. D. Nimbarte,K. Mullagiri, J. Kovvuri, N. Jain

&& –&&

Synthesis and Biological Evaluation ofImidazo[2,1-b][1,3,4]thiadiazole-LinkedOxindoles as Potent TubulinPolymerization Inhibitors

Imidazzling microtubule blockers! Aseries of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindole conjugates were synthe-sized and evaluated for anticancer po-tential. Conjugate 7 displayed promisingcytotoxicity, arrested cells at the G2/Mphase, and showed potent tubulin poly-merization inhibition with an IC50 valueof 0.15 mm.

� 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 0000, 00, 1 – 14 &14&

These are not the final page numbers! ��