thiadiazoleLinked Oxindoles as Potent Tubulin

CHEMMEDCHEM FULL PAPERS DOI: 10.1002/cmdc.201400069

Synthesis and Biological Evaluation of Imidazo[2,1b][1,3,4]thiadiazole-Linked Oxindoles as Potent Tubulin Polymerization Inhibitors Ahmed 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]

A series of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles composed of an A, B, C and D ring system were synthesized and investigated for anti-proliferative activity in various human cancer cell lines; test compounds were variously substituted at rings C and D. Among them, compounds 7 ((E)-5-fluoro-3-((6p-tolyl-2-(3,4,5-trimethoxyphenyl)-imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one), 11 ((E)-3-((6-ptolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5yl)methylene)indolin-2-one), and 15 ((E)-6-chloro-3-((6-phenyl2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)-

methylene)indolin-2-one) exhibited potent anti-proliferative activity. Treatment with these three compounds resulted in accumulation of cells in G2/M phase, inhibition of tubulin assembly, and increased cyclin-B1 protein levels. Compound 7 displayed potent cytotoxicity, with an IC50 range of 1.1–1.6 mm, and inhibited tubulin polymerization with an IC50 value (0.15 mm) lower than that of combretastatin A-4 (1.16 mm). Docking studies reveal that compounds 7 and 11 bind with aAsn101, bThr179, and bCys241 in the colchicine binding site of tubulin.

Introduction Cancer is a disease in which normal cells dramatically alter their behavior: the multiply uncontrollably, ignore signals to stop, and accumulate to form tumors. Cancer has become a major cause of morbidity throughout the world, accounting for 7.6 million deaths (~ 13 % of all deaths) in 2008. According to the World Health Organization, deaths due to cancer worldwide were projected to continue to rise to over 13.1 million in 2030. There are nearly 200 different types of cancer, each named for the organ or type of cell from which it originates. Cancers of the colon, stomach, lung, liver, and breast cause the greatest number of deaths each year. Levamisole (I) is an immunomodulatory compound toward various cancer cell types including colorectal and breast cancers, melanoma, and leukemia (Figure 1).[1] It was previously shown to affect cell proliferation in different cancers,[2] and to modulate the phosphorylation of proteins involved in cellcycle progression and apoptosis. Levamisole has also been [a] Dr. A. Kamal, M. P. N. Rao, P. Swapna, K. Mullagiri, J. Kovvuri Medicinal Chemistry and Pharmacology CSIR – Indian Institute of Chemical Technology Hyderabad 500 007 (India) E-mail: [email protected] [b] P. Das, S. Polepalli, Dr. N. Jain Chemical Biology, CSIR – Indian Institute of Chemical Technology Hyderabad 500 007 (India) [c] Dr. A. Kamal, V. D. Nimbarte Department of Medicinal Chemistry National Institute of Pharmaceutical Education and Research (NIPER) Hyderabad 500 037 (India) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201400069.

2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

used to treat helminthic infections and in the amelioration of various autoimmune disorders.[3, 4] Furthermore, I has been shown to have anticancer activity in combination with fluorouracil (5-FU) as an adjuvant therapy for tumor-node-metastasis (TNM) stage III (Dukes’ C) colon carcinoma.[5] Imidazo[2,1b]thiazole derivatives of levamisole have been reported as potential antitumor agents.[6] Subsequently, the antitumor activity of 5-formyl-6-arylimidazo[2,1-b]-1,3,4-thiadiazole sulfonamides V were also reported.[7] In 2012 Andreani and co-workers[8] reported that 3-(5-imidazo[2,1-b]thiazolylmethylene)-2-indolinones cause G2/M phase arrest and inhibit tubulin assembly. Tubulin is a heterodimer of two closely related and tightly linked globular polypeptides called a- and b-tubulin, which polymerize to form microtubules. Their function in mitosis and cell division makes them an important target for anticancer drugs. Microtubules and their dynamics are the targets of a chemically diverse group of antimitotic drugs that have been used with great success in the treatment of cancer. There has been considerable interest in the discovery and development of small molecules that affect tubulin polymerization.[9] However, the success of tubulin polymerization inhibitors as anticancer agents has stimulated significant interest in the identification of new compounds that may be more potent or more selective in targeted tissues or tumors. Oxindoles are versatile moieties that display diverse biological activities, including anticancer activity. They exhibit antitumor activity by inhibiting tyrosine kinase receptors such as PDGF-R, VEGF-R, and CDK.[10] One of the most important examples is sunitinib (II), which has been widely used in the treatment of gastrointestinal stromal tumors and metastatic renal ChemMedChem 2014, 9, 1463 – 1475

1463

CHEMMEDCHEM FULL PAPERS

www.chemmedchem.org pared by Vilsmeier–Haack reaction with the corresponding imidazo[2,1-b][1,3,4]thiadiazoles 5 a–e. Conjugates 7–32 were prepared by single-step Knoevenagel reaction between compounds 6 a–e and various substituted oxindoles in ethanol/piperidine. These conjugates were characterized by 1H and 13C NMR spectroscopy, mass spectrometry, HRMS, and IR spectroscopy. All compounds were obtained in pure E isomeric form and confirmed with those previously reported.[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-one conjugates 7–32.

cell cancer.[11, 12] Chen and co-workers reported that oxindoles not only inhibit CDK, but also inhibit microtubule polymerization by binding at the colchicine binding site of tubulin.[13] On the other hand, a 3,4,5trimethoxyphenyl ring is present in most of the synthesized and naturally occurring biologically active compounds, including colchicine (IV), combretastatin A-4 (CA-4; III), and its derivatives. Our continued efforts toward the synthesis of a variety of new molecules led to the development of efficient anticancer agents.[14, 15] In view of the biological importance of both imidazothiadiazoles and oxindoles, in this study we designed and synthesized a series of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles. Use of the core imidazothiadiazole scaffold functionalized with 3,4,5-trimethoxyphenyl ring A, an indolinone as ring C, and a phenyl group as ring D resulted in the generation of imidazo[2,1-b][1,3,4]thiadiazolindolin-2-ones. These compounds were evaluated for in vitro anticancer activity, cellcycle effects, and the capacity to inhibit tubulin polymerization.

Results and Discussion Chemistry The imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles were prepared as shown in Scheme 1. Compound 3 was prepared by reaction of 3,4,5-trimethoxybenzoic acid with thiosemicarbazide in POCl3 at reflux. 6-(4Aryl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazoles 5 a–e were obtained in high yield by treating compound 3 with various phenacyl bromides 4 a–e in ethanol with the addition of 3–4 drops of N,N-dimethylformamide. The imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehydes 6 a–e were pre 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Scheme 1. Synthesis of imidazo[2,1-b][1,3,4]thiadiazole–oxindole conjugates 7–32. Reagents 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. (3–4 drops) piperidine, reflux, 3–4 h, 75–85 %.

ChemMedChem 2014, 9, 1463 – 1475

1464


www.chemmedchem.org

Biology Cytotoxicity A sulforhodamine B assay was performed to evaluate the cytotoxic effects of imidazo[2,1-b][1,3,4]thiadiazole-linked oxindoles 7–32 against A549, HeLa, MCF-7, and HCT116 human cancer cell lines, and the IC50 values are listed in Table 1. Compounds 7–15 showed considerable cytotoxicity, with IC50 values ranging from 1.1 to 8.9 mm against A549, HeLa, MCF-7, and HCT116 human cancer cell lines.

Table 1. Cytotoxicity of compounds 7–32 and levamisole on A549, HeLa, MCF-7, and HCT116 human cancer cell lines. Compd A549[b] 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 levamisole

1.1 0.7 5.7 3.1 5.3 0.2 8.5 0.8 2.6 0.9 4.2 0.9 5.0 0.79 6.8 0.8 2.3 1.6 19.5 1.9 18.5 0.2 12 1.1 18.6 0.3 17.4 1.9 3.1 1.3 13.2 0.1 16.4 1.9 31.6 1.5 26.9 1.3 28.4 1.8 29.1 1.9 16.8 0.2 20.7 2.8 18.6 0.7 15.3 2.9 24.5 0.5 > 100

IC50 [mm][a] HeLa[c] MCF-7[d] 1.4 1.7 3.9 0.7 3.8 1.7 2.8 1.2 2.5 0.3 2.5 1.6 3.4 1.3 3.6 2.3 3.2 1.0 12.8 1.2 23.0 0.5 24 0.2 36.9 1.9 33.1 1.8 4.2 1.7 27.8 1.0 20.5 1.2 29.7 2.3 22.4 0.9 15.3 1.5 23.2 1.2 19.7 0.9 22.5 1.7 15.8 3.1 25.9 1.0 28.1 1.1 96

1.2 0.4 2.9 0.8 8.9 1.8 3.0 1.5 2.5 0.9 7.6 0.8 7.4 1.2 7.5 0.9 2.4 1.6 22.9 1.0 21.5 1.6 10.1 0.6 13.1 1.3 12.9 0.3 2.1 1.3 24.6 3.6 21.3 1.1 35.2 2.4 17.7 1.7 25.3 1.2 29.6 2.0 24.9 2.2 21.8 0.7 26.7 1.1 20.0 1.8 21.4 1.4 > 100

HCT116[e] 1.6 0.5 7.3 2.7 8.3 0.7 5.6 2.1 2.9 1.2 5.8 4.2 8.2 1.0 8.4 2.1 3.0 3.0 11.7 0.9 13.5 0.7 13.1 1.1 11.2 1.4 18.6 0.4 15.2 0.3 27.3 4.1 13.5 0.9 20.2 0.4 13.6 0.4 18.9 1.6 26.2 6.2 29.2 1.3 18.2 4.1 14.7 2.7 24.6 5.3 19.5 1.0 > 100

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

Compound 7, which is fluorinated on ring C and has a methyl group on the ring D, exhibited potent cytotoxicity with IC50 values in the range 1.1–1.6 mm. In comparison, conjugate 11, devoid of any substitution on ring C, also displayed potent anti-proliferative activity, with IC50 values of 2.5–2.9 mm. Moreover, compound 15, chlorinated at the 6-position on ring C and lacking ring D substitution, and 21, with a 5-fluoro group on ring C, and a methoxy group on ring D, both exhibited potent cytotoxicity. However, compounds with 5-methoxy (8), 5-chloro (9), and 6-chloro (10) substituents on ring C showed moderate cytotoxicity (IC50 values 2–8 mm). Overall, activity was decreased in cells treated with compounds that contain chloro, fluoro, or 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

methoxy groups and that are devoid of substitution on indolinone (ring C) and that have a fluoro group on ring D. Based on the results of sulforhodamine B assays, compounds either methylated or that lack substitution on ring D showed the most potent activities in the series; therefore, the activities of the most potent compounds can be ranked in the order of 7 > 11 > 15 > 21. To further determine their potency in other cell types, conjugates 7, 11, 15, and 21 were evaluated in the sixty-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 cytotoxicity in most of the NCI panel cell lines, with GI50 values ranging from 0.30 to 5.8 mm. In particular, compound 11 showed promising cytotoxicity, with GI50 values of 0.30 and 0.42 mm against OVCAR-4 (ovarian cancer) and HOP-92 (lung cancer) cell lines. Taken together, results from the cytotoxicity assays agreed remarkably with the NCI 60-cell-line screen. We then elucidated whether the anti-proliferative properties of 7, 11, and 15 were due to inhibition of tubulin polymerization. To investigate this possibility, 7, 11, and 15 were incubated at varying concentrations with tubulin, and polymerization assays were performed. Compound 7, which potently inhibited cell growth, also significantly decreased tubulin assembly, with an IC50 value of 0.15 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, we evaluated their ability to stall cells at the G2/M phase. A549 cells were treated with test compounds at 5 mm for 24 h. Cells were then harvested and analyzed by flow cytometry for DNA content (Figure 2). The control cells treated with DMSO showed 26.5 % arrested in G2/M phase. In contrast, compounds 7 and 11 increased the cell population in G2/M phase by 67.3 and 64.8 %, respectively. Moreover, cells treated with 15 manifested 66.7 % of cells in G2/M phase. Effect of compounds on microtubules Pharmacological inhibition of microtubule formation results in a profound disruption of the cellular microtubule network, and as such, spindle fibers composed of microtubules are routinely disrupted from treatments with anti-tubulin agents.[18] The imidazo[2,1-b][1,3,4]thiadiazole–oxindole conjugates inhibit cell ChemMedChem 2014, 9, 1463 – 1475

1465


www.chemmedchem.org

Table 2. In vitro cytotoxicity of compounds 11 and 21 against the NCI panel of 60 human cancer cell lines. Cancer panel/cell line Leukemia CCRF-CEM HL-60 (TB) K-562 MOLT-4 RPMI-8226 SR

GI50 [mm][a] 11[b] 21[c] – > 100 – – – 2.35

5.49 2.17 13.1 4.54 11.3 23.5

Cancer panel/cell line Melanoma MDA-MB-435 SK-MEL-2 SK-MEL-28 SK-MEL-5 UACC-257 UACC-62

GI50 [mm][a] 11[b] 21[c] – 16.3 – 0.59 – 5.47

3.39 5.58 9.58 1.64 3.35 13.3

exhibit a typical rounded mitotic phenotype, and DAPI was used to counter-stain the nuclei (Figure 3). Effect of compounds on endogenous tubulin in A549 cells

Microtubules are present in a dynamic equilibrium with free tubulin monomers. Anti-tubulin Non-Small Cell Lung Cancer Ovarian Cancer compounds disrupt this dynamic A549/ATCC 2.12 2.13 IGROV 1 4.75 3.39 equilibrium. Microtubule-depolyHOP-62 4.14 1.94 OVCAR-3 2.90 2.32 HOP-92 0.42 2.02 OVCAR-4 0.30 1.77 merizing agents increase the NCI-H226 22.5 16.8 OVCAR-5 > 100 6.14 amount of free tubulin monoNCI-H23 4.54 4.25 OVCAR-8 3.71 5.34 mer in cells, thereby preventing NCI-H322M 10.3 2.74 NCI/ADR-RES 3.05 21.9 microtubule polymerization.[19] NCI-H460 1.98 3.42 SK-OV-3 15.2 2.84 NCI-H522 10.2 5.44 As compounds 7, 11, and 15 effiRenal Cancer ciently arrest cells at the G2/M Colon Cancer 786-0 5.80 16.0 phase and inhibit tubulin assemCOLO 205 – 1.61 A498 7.28 12.2 bly, we elucidated their ability to HCC-2998 > 100 5.25 ACHN 4.97 3.29 HCT116 2.59 14.2 CAKI-1 15.4 4.48 block endogenous tubulin polyHCT15 2.68 2.50 RXF 393 2.24 3.96 merization. A549 cells were HT29 – 2.18 SN 12C 5.25 14.2 treated with compounds 7, 11, KM12 > 100 4.04 TK-10 1.69 2.26 and 15 at 5 mm for 24 h. We SW-620 – 3.21 UO-31 8.44 15.0 then permeabilized cells with CNC Cancer Prostate Cancer detergent-containing buffers SF-268 3.63 4.40 PC-3 81.1 24.7 (details in the Experimental SecSF-295 5.04 3.00 DU-145 2.55 1.87 tion below). The insoluble and SF-539 12.9 56.0 SNB-19 7.89 15.1 Breast Cancer soluble fractions were analyzed SNB-75 0.55 10.3 MCF-7 – 1.66 for tubulin (Figure 4). DMSOU251 1.59 1.77 MDA-MB-231/ATCC 3.51 5.25 treated cells demonstrated equal HS 578T 0.50 16.2 amounts of tubulin across the Melanoma BT-549 4.69 14.9 LOX IMVI – 6.88 T-47D 3.13 2.01 soluble and insoluble fractions. MALME-3M 0.76 9.15 MDA-MB-468 4.84 2.53 In contrast, 7 and 11 effected M14 – 5.51 a marked increase in the soluble [a] Compound concentration required to decrease cell growth to half that of untreated cells. [b] 11 fraction similar to CA-4-treated (NSC775003). [c] 21 (NSC774982). cells. In comparison, paclitaxeltreated cells possessed increased levels of tubulin in the insoluble fraction. Next, we analyzed whether the cells arrested at the proliferation, tubulin assembly, and arrest cells at the G2/M G2/M phase were indeed mitotic in nature. We confirmed their phase (Table 3). To further validate their effect on microtubules, state by assaying for the mitotic marker cyclin-B1. As expected, we treated A549 cells with compounds 7, 11, and 15 for 24 h. treatments with compound 7 induced increased levels of Immunofluorescence analyses revealed that the conjugates cyclin-B1, when compared with 11 and 15. b-actin was used as profoundly alter microtubule the network. Treated cells also a loading control. Table 3. Anti-tubulin polymerization activity of compounds 7, 11, 15 and CA-4. Compd

IC50 [mm][a]

7 11 15 CA4

0.15 0.3 1.23 1.0 2.11 0.7 1.16 0.1

[a] Concentration of compounds required to inhibit tubulin assembly by 50 %; values are the mean SD of two independent experiments performed in triplicate.


Molecular docking studies Among the synthesized compounds, 7 and 11 displayed significant cytotoxicity, tubulin polymerization inhibition with IC50 values less than or equal to that of CA-4, and arrested cells at the G2/M phase of the cell cycle. Molecular docking studies were performed to determine whether the active conjugates 7 and 11 exert their cytotoxicity and capacity to block tubulin polymerization by binding to tubulin at the colchicine binding site. Docking analyses suggest that both compounds occupy ChemMedChem 2014, 9, 1463 – 1475

1466


www.chemmedchem.org

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

the colchicine binding site of a/ b-tubulin (Figure 5). The 3,4,5-trimethoxyphenyl group of the conjugates anchors the molecule in the a/b interface of tubulin, similar to the binding mode of colchicines. This binding pattern is supported by the hydrogen bonding interactions between the 3,4,5-trimethoxyphenyl ring and aGln11 and bAsn258, the distances from which are in the range of 1.8–2.2 . Moreover, rings B of compounds 7 and 11 are involved in hydrophobic interactions with aThr179. Some hydrogen bonding interactions were also observed between ring C and bThr353, bCys241, and aAsn101 of a/b-tubulin. The ptolyl groups of 7 and 11 are involved in hydrogen bonding interactions with aSer178. Based on the analysis of docking results, we conclude that com-

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. Tubulin was detected by immunoblot analysis. Cyclin-B1 protein levels were measure with specific antibodies, and b-actin was used as a loading control.

pounds 7 and 11 occupy the colchicine binding site at the a/ b interface of tubulin (Table 4). Overall, some determinant hydrophobic interactions were observed between ring C and aAsn101 and bCys241, and be-

Table 4. Comparisons of docking scores of synthesized compounds 7 and 11 with colchicine (IV). Figure 3. Effect of 7, 11, and 15 on microtubule dynamics. A549 cells were independently treated with 7, 11, and 15 at 5 mm for 24 h. Cells were then fixed and stained for tubulin antibody (green), and DAPI was used as counter-stain (blue). The merged images of cells stained for tubulin and DAPI are shown at right.


IV

8.84

8.84

8.84

8.84

8.84

8.84

7 11

10.22 9.19

10.21 9.16

9.91 9.10

9.67 9.08

9.57 9.08

9.57 7.45

ChemMedChem 2014, 9, 1463 – 1475

1467


www.chemmedchem.org

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 of tubulin (PDB ID: 3E22).

tween ring B and bAla180 and bThr179. Hydrophobic interactions between ring A and bLeu255, bLeu248, aGln11, and bAsn249 were also observed (Figure 6). Some hydrophobic interactions are similar to those observed for colchicine (IV); however, a few additional interactions were also observed with aGln11, bAsn258, aThr179, bThr353, aAsn101, and aSer178.

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

Conclusions We synthesized a series of imidazo[2,1-b][1,3,4]thiadiazole– oxindoles composed of A, B, C, and D ring systems. Two of these conjugates were selected for the NCI 60-cell-line panel, five-dose screen. SAR analysis reveals that amongst the conjugates synthesized, those with a methyl group and devoid of any substitution on ring D exhibit significant activity. Compounds 7, 11, and 15 displayed potent cytotoxicity, with IC50 values of 1.1–3.2 mm. These three compounds exert their cytotoxic activity by inhibition of tubulin polymerization, with IC50 values of 0.15, 1.23, and 2.11 mm, respectively. They also induce cell-

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


ChemMedChem 2014, 9, 1463 – 1475

1468

CHEMMEDCHEM FULL PAPERS analysis. Moreover, docking studies provided some molecular insight into the binding mode of compounds 7 and 11, which bind at the colchicine binding site in a/b interfaces of polymerized tubulin. Based on these results, it is evident that compounds of this structural class are acquiescent to further modifications and function as a suitable template for the design of a new class of tubulin polymerization inhibitors for the treatment of cancer.

Experimental Section Chemistry All chemicals and reagents were obtained from Aldrich (Sigma–Aldrich, St. Louis, MO, USA), Lancaster (Alfa Aesar, Johnson Matthey Co., Ward Hill, MA, USA), or Spectrochem Pvt. Ltd. (Mumbai, India), and were used without further purification. TLC was performed on glass plates containing silica gel 60 GF254, and visualization was achieved by UV light or iodine indicator-monitored reactions. Column chromatography was performed with Merck 60–120 mesh silica gel. 1H and 13C NMR spectra were recorded on Bruker UXNMR/XWIN-NMR (300 MHz) or Inova Varian VXR Unity (400, 500 MHz) instruments. Chemical shifts (d) are reported in ppm downfield from an internal TMS standard. ESIMS spectra were recorded on a Micro-mass Quattro LC instrument equipped with an ESI 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 melting point apparatus, and are uncorrected. 1H and 13C NMR, IR, and HRMS spectra of final compounds 7–32 are provided in the Supporting information. Preparation of 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2amine (3): POCl3 (21 mL) was added to a mixture of 3,4,5-trimethoxybenzoic 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. The reaction 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 filtered and washed with water (2 100 mL). The pale-yellow solid obtained (11.3 g, 90 %) was dried and used directly for the next step. General procedure for the preparation of 6-(4-substituted phenyl)-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 in ethanol (30 mL) for ~ 20–24 h. The solvent was evaporated, and the remainder was dissolved in EtOAc (100 mL), washed with a saturated sodium bicarbonate solution (2 100 mL), and the separated organic layer was dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the crude product was purified by column chromatography with EtOAc/ hexane (2:8) as the eluent to afford compounds 5 a–e with high purity. 6-p-Tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole (5 a): Compound 5 a was prepared according to the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-p-tolylethanone 4 a (3.19 g, 14.98 mmol) to obtain pure product 5 a as pale-yellow solid (4.56 g, 80 % yield). 1H NMR (500 MHz, CDCl3): d = 7.99 (s, 1 H), 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org 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 to the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-phenylethanone 4 b (3.92 g, 14.98 mmol) to obtain pure product 5 b as pale-yellow solid (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 to the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-(4-methoxyphenyl)ethanone 4 c (3.43 g, 14.98 mmol) to obtain pure product 5 c as pale-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 to the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-(4-chlorophenyl)ethanone 4 d (3.46 g, 14.98 mmol) to obtain pure product 5 d as pale-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 to the general procedure, using 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-amine 3 (4 g, 14.98 mmol) and 2-bromo-1-(4-fluorophenyl)ethanone 4 e (3.22 g, 14.98 mmol) to obtain pure product 5 e as pale-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-substituted phenyl)-2-(3,4,5-trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazole-5-carbaldehydes 6 a–e: The Vilsmeier reagent was prepared at 0–5 8C by dropping POCl3 (5.4 equiv) into a stirred solution 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 Vilsmeier reagent while maintaining stirring and cooling. The reaction mixture was kept for 3 h at room temperature and under reflux for 24 h. Chloroform was removed under reduced pressure, and the resulting oil was poured into crushed ice. The mixture was extracted with EtOAc (2 200 mL), and the separated organic layer was dried over anhydrous Na2SO4. The organic layer was concentrated under reduced pressure, and the crude product was purified by column chromatography using EtOAc/hexane (3:7) as the eluent to afford 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 prepared 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 preChemMedChem 2014, 9, 1463 – 1475

1469

CHEMMEDCHEM FULL PAPERS 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 prepared 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 prepared 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 prepared 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[2,1b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (7): Compound 7 was 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 (s 2H), 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 (ESI m/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): Compound 8 was prepared according to the method described for compound 7, employing 6 a (200 mg, 0.48 mmol) and 5-methoxyindolin-2-one (79 mg, 0.48 mmol) to obtain pure product 8 as a 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 for C30H27O5N4S: 555.16967, found: 555.16907 [M + 1] + . 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org (E)-5-Chloro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (9): Compound 9 was 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) calcd for C29H24O4N4ClS: 559.12013, found: 559.11907 [M + 1] + . (E)-6-Chloro-3-((6-p-tolyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (10): Compound 10 was prepared according to the method described for compound 7, employing 6 a (200 mg, 0.48 mmol) and 6-chloroindolin2-one (81 mg, 0.48 mmol) to obtain pure product 10 as a yellow solid (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 of compound 6 a (200 mg, 0.48 mmol) and indolin-2-one (64 mg, 0.48 mmol) was dissolved in ethanol (6 mL) and piperidine (3–4 drops) was added. The reaction mixture was heated at reflux for 3 h. Progress of the reaction was monitored by TLC, and the precipitate formed on cooling was collected by filtration, washed twice a small amount of ethanol to obtain the pure product 11 as a 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; 1 H 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); 13 C 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 (ESI m/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): Compound 12 was prepared according to the method described for compound 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 ChemMedChem 2014, 9, 1463 – 1475

1470

CHEMMEDCHEM FULL PAPERS [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): Compound 13 was prepared according to the method described for compound 7, employing 6 b (200 mg, 0.5 mmol) and 5-methoxyindolin-2-one (82 mg, 0.5 mmol) to obtain pure product 13 as a 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 for C29H25O5N4S: 541.15402, found: 541.15342 [M + 1] + . (E)-5-Chloro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (14): Compound 14 was prepared according to the method described for compound 7, employing 6 b (200 mg, 0.5 mmol) and 5-chloroindolin-2one (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): calcd for C28H22O4N4ClS: 545.10448, found: 545.10397 [M + 1] + . (E)-6-Chloro-3-((6-phenyl-2-(3,4,5-trimethoxyphenyl)imidazo[2,1b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (15): Compound 15 was prepared according to the method described for compound 7, employing 6 b (200 mg, 0.5 mmol) and 6-chloroindolin-2one (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[2,1b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (16): Compound 16 was prepared according to the method described for compound 7, employing 6 b (200 mg, 0.5 mmol) and 5-fluoroindolin-2one (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 = 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org 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): Compound 17 was prepared according to the method described for compound 7, employing 6 c (200 mg, 0.47 mmol) and indolin-2one (62 mg, 0.47 mmol) to obtain pure product 17 as a yellow solid (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 for C29H25O5N4S: 541.15402, found: 541.15329 [M + 1] + . (E)-5-Methoxy-3-((6-(4-methoxyphenyl)-2-(3,4,5trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (18): Compound 18 was prepared according to the method described for compound 7, employing 6 c (200 mg, 0.47 mmol) and 5-methoxyindolin-2-one (76 mg, 0.47 mmol) to obtain 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-(3,4,5trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (19): Compound 19 was prepared according to the method described for compound 7, employing 6 c (200 mg, 0.47 mmol) and 5-chloroindolin-2-one (78 mg, 0.47 mmol) to obtain 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-(3,4,5trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (20): Compound 20 was prepared according to the method described for compound 7, employing 6 c (200 mg, 0.47 mmol) and 6-chloroindolin-2-one (78 mg, 0.47 mmol) to ChemMedChem 2014, 9, 1463 – 1475

1471

CHEMMEDCHEM FULL PAPERS 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 for C29H24O5N4ClS: 575.11504, found: 575.11586 [M + 1] + . (E)-5-Fluoro-3-((6-(4-methoxyphenyl)-2-(3,4,5trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (21): Compound 21 was prepared according to the method described for compound 7, employing 6 c (200 mg, 0.47 mmol) and 5-fluoroindolin-2-one (70 mg, 0.47 mmol) to obtain pure 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 described for compound 7, employing 6 c (200 mg, 0.47 mmol) and 5-nitroindolin-2-one (83 mg, 0.47 mmol) to obtain pure product 22 as a 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); 13 C 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): Compound 23 was prepared according to the method described for compound 7, employing 6 d (200 mg, 0.46 mmol) and indolin-2one (62 mg, 0.46 mmol) to obtain pure product 23 as a yellow solid (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): calcd for 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 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org (24): Compound 24 was prepared according to the method described for compound 7, employing 6 d (200 mg, 0.46 mmol) and 5-methoxyindolin-2-one (75 mg, 0.46 mmol) to obtain pure product 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 for C29H24O5N4ClS: 575.11502, found: 575.11529 [M + 1] + . (E)-5-Chloro-3-((6-(4-chlorophenyl)-2-(3,4,5trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (25): Compound 25 was prepared according to the method described for compound 7, employing 6 d (200 mg, 0.46 mmol) and 5-chloroindolin-2-one (77 mg, 0.46 mmol) to obtain 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; 1 H 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-(3,4,5trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (26): Compound 26 was prepared according to the method described for compound 7, employing 6 d (200 mg, 0.46 mmol) and 6-chloroindolin-2-one (77 mg, 0.46 mmol) to obtain 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 described for compound 7, employing 6 d (200 mg, 0.46 mmol) and 5-fluoroindolin-2-one (70 mg, 0.46 mmol) to obtain pure product 27 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, ChemMedChem 2014, 9, 1463 – 1475

1472

CHEMMEDCHEM FULL PAPERS 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 Cl FS: 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 described for compound 7, employing 6 d (200 mg, 0.46 mmol) and 5-nitroindolin-2-one (82 mg, 0.46 mmol) to obtain pure product 28 as a 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-(3,4,5trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (29): Compound 29 was prepared according to the method described for compound 7, employing 6 e (200 mg, 0.48 mmol) and 5-chloroindolin-2-one (80 mg, 0.46 mmol) to obtain 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[2,1b][1,3,4]thiadiazol-5-yl)methylene)-5-methoxyindolin-2-one (30): Compound 30 was prepared according to the method described for compound 7, employing 6 e (200 mg, 0.48 mmol) and 5-methoxyindolin-2-one (78 mg, 0.46 mmol) to obtain pure product 30 as 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); 13 C 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 for C29H24O5N4FS: 559.14460, found: 559.14501 [M + 1] + . (E)-5-Fluoro-3-((6-(4-fluorophenyl)-2-(3,4,5trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (31): Compound 31 was prepared according to the method described for compound 7, employing 6 e (200 mg, 0.48 mmol) and 5-fluoroindolin-2-one (73 mg, 0.46 mmol) to obtain pure 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, 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org 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): calcd for C28H21O4N4F2S: 547.12461, found: 547.12525 [M + 1] + . (E)-6-Chloro-3-((6-(4-fluorophenyl)-2-(3,4,5trimethoxyphenyl)imidazo[2,1-b][1,3,4]thiadiazol-5-yl)methylene)indolin-2-one (32): Compound 32 was prepared according to the method described for compound 7, employing 6 e (200 mg, 0.48 mmol) and 5-fluoroindolin-2-one (73 mg, 0.46 mmol) to obtain pure 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 cell lines used in this study were purchased from the American Type Culture Collection (ATCC, USA). A549, MCF-7, HCT116 and HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, containing 10 % fetal bovine serum (FBS) in a humidified atmosphere of 5 % CO2 at 37 8C). Cells were trypsinized when sub-confluent from T25 flasks/60 mm dishes and seeded in 96-well plates. The test compounds were evaluated for their in vitro anti-proliferative activity against four different human cancer cell lines, using a protocol of 48 h continuous drug exposure; a sulforhodamine B cell proliferation assay was used to estimate cell viability or growth. The cell lines were grown in their respective media containing 10 % FBS and were seeded into 96-well microtiter plates in 200 mL aliquots at plating densities according to the doubling time of 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 the addition of test compounds. Aliquots (2 mL) of the test compounds were added to the wells already containing cells in 198 mL, resulting 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 further for 48 h, and the assay was terminated by the addition of 10 % TCA and incubated for 60 min at 4 8C. Later, the plates were air-dried and washed thrice with water. The cells were then incubated with 0.57 % sulforhodamine B dye for 1 h at 37 8C, and plates were washed thrice with 1 % acetic acid. The air-dried plates were reconstituted with 50 mL 10 mm Tris buffer (pH 8.0), and the absorbance 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 terminated after 48 h, containing DMSO-treated control (tc) and five-dose-treated cells (ti). The IC50 values were calculated by linear interpolation from plots of percent growth versus log concentrations of comChemMedChem 2014, 9, 1463 – 1475

1473

CHEMMEDCHEM FULL PAPERS pounds in mm. IC50 values are indicated as means SD of three independent experiments.[18] Cell-cycle analysis: A549 cells in 60 mm dishes were incubated for 24 h in the presence or absence of test compounds 7, 11, and 15 at 5 or 10 mm. Cells were harvested with Trypsin-EDTA, and then fixed with ice-cold 70 % ethanol at 4 8C for 30 min. Ethanol was removed by centrifugation (400 g, 10 min), and cells were stained with 1 mL DNA staining solution [0.2 mg propidium iodide (PI), 2 mg RNase A] for 30 min as described earlier.[18] The DNA contents of 20 000 events were measured by flow cytometry (BD FACSCanto II). Histograms were analyzed using FCS express 4 plus.[18] Tubulin polymerization assays: An in vitro assay for monitoring the time-dependent polymerization of tubulin to microtubules was performed by using a fluorescence-based tubulin polymerization assay kit (BK011, Cytoskeleton Inc.) according to the manufacturer’s protocol. The reaction mixture in a final volume of 10 mL in PEM buffer (80 mm PIPES, 0.5 mm EGTA, 2 mm MgCl2, pH 6.9) in 384well plates contained 2 mg mL 1 bovine brain tubulin, 10 mm fluorescent reporter, and 1 mm GTP in the presence or absence of test compounds at 37 8C. 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 360 nm) was measured for 1 h at 1-min intervals in a multimode plate reader (Tecan M200). To determine the IC50 values of the compounds against tubulin polymerization, the compounds were preincubated with tubulin at varying concentrations (1, 5, 10 and 20 mm). Assays were performed under similar conditions as used for the polymerization assays described above.[18] Western blot analysis of soluble versus polymerized tubulin and cyclin-B1: Cells were seeded in 12-well plates at 1 105 cells per well in complete growth medium. Cells were treated with compounds 7 or 11 for 24 h. Subsequently, cells were washed with phosphate-buffered saline (PBS), and soluble and insoluble tubulin fractions 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 % Triton X-100, 10 % glycerol, AND 0.1 % protease inhibitor cocktail (Sigma– Aldrich)] and incubated for 3 min at 30 8C. The lysis buffer was gently removed, and mixed with 100 mL 3 Laemmli sample buffer (180 mm Tris·Cl pH 6.8, 6 % SDS, 15 % glycerol, 7.5 % b-mercaptoethanol, and 0.01 % bromophenol blue). Samples were immediately heated at 95 8C for 3 min. To collect the insoluble tubulin fraction, 300 mL 1 Laemmli sample buffer was added to the remaining cells in each well, and the samples were heated at 95 8C for 3 min. Equal volumes of samples were separated by 10 % SDS-PAGE and then transferred to a nitrocellulose membrane using semidry transfer at 50 mA for 1 h. Blots were probed with mouse anti-human atubulin diluted 1:2000 mL (Sigma) and stained with rabbit antimouse secondary antibody coupled with horseradish peroxidase, diluted 1:5000 mL (Sigma). Bands were visualized using an enhanced Chemiluminescence protocol (Pierce) and radiographic film (Kodak). For cyclin-B1 immunoblots, cells were seeded in 12-well plates at 1 105 cells per well in complete medium and treated with various concentrations of 7, 11, and 15 for 24 h. After treatment, cells were washed twice with PBS and lysed in 1 SDS sample buffer. Proteins were separated, transferred, probed, and analyzed similarly to tubulin. The primary anti-cyclin-B1 antibody was employed at 1:1500 and b-actin (Sigma) and horseradish peroxidase coupled goat anti-rabbit secondary antibody diluted 1:5000 (Sigma).[20] 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

www.chemmedchem.org Immunohistochemistry and nuclear morphology analysis: A549 cells were seeded on a glass cover slip, incubated for 24 h in the presence or absence of test compounds 7, 11, and 15 at 5 mm. Cells grown on cover slips were fixed in 3.5 % formaldehyde in PBS pH 7.4 for 10 min at room temperature. Cells were permeabilized for 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 primary antibody for tubulin (Sigma) at 1:200 diluted in blocking solution for 4 h at room temperature. The antibodies were then removed, and the cells were washed thrice with PBS. Cells were then incubated with FITC-labeled anti-mouse secondary antibody (1:500) for 1 h at room temperature. Cells were washed thrice with PBS and mounted in medium containing DAPI. Images were captured using an Olympus confocal microscope and analyzed with Provision software (instrument: FLOW VIEW-FV 1000 Series; software: FV 10 ASW 1.7 Series).[19] Docking: All geometries were optimized in Gaussian 09 using PM3 semi-empirical method.[20] The protein structure was downloaded from RCSB Protein Data Bank (PDB ID: 3E22).[21] Docking studies were performed using AutoDock 4.2 software.[22] The analysis of intermolecular 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 fellowships. The authors also acknowledge the Council of Scientific & Industrial Research (CSIR), India, for financial support under the 12th Five-Year Plan project “Affordable Cancer Therapeutics (ACT)” (CSC0301) and Small Molecule in Lead Exploration (SMiLECSC-0111). Keywords: antitumor agents · docking · imidazothiadiazolelinked 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.

ChemMedChem 2014, 9, 1463 – 1475

1474

CHEMMEDCHEM FULL PAPERS [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. Nimbarte, V. Srinivasulu, M. V. P. S. Vishnuvardhan, C. S. Reddy, ChemMedChem 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. Anthony, 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-


www.chemmedchem.org 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. Kobayashi, 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. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador, J. J. Dannenberg, S. Dapprich, 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, EMBO Rep. 2008, 9, 1101 – 1106. [22] G. M. Morris, R. Huey, W. Lindstrom, M. F. Sanner, R. K. Belew, D. S. Goodsell, 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 April 8, 2014

ChemMedChem 2014, 9, 1463 – 1475

1475