Imidazopyridine linked triazoles as tubulin inhibitors, effectively triggering apoptosis in lung cancer cell line.

A library of new imidazopyridine linked triazole hybrid conjugates (8a-r) were designed, synthesized and evaluated for their cytotoxicity against four cancer cell lines namely, human lung (A549), human prostate (DU-145), human colon (HCT-116) and breast (MDA-MB 231) cancer. These conjugates exhibited good to moderate activity against the tested human cancer cell lines. Two of the conjugates (8g and 8j) showed significant antitumor activity against human lung cancer cell line (A549) with IC50 values of 0.51 µM and 0.63 µM respectively. Flow cytometry analysis revealed that these conjugates arrested the cell cycle at G2/M phase in human lung cancer cell line (A549). Immune-histochemistry and tubulin polymerization assay suggest inhibition of tubulin. Hoechst staining, annexin V and DNA fragmentation by tunnel assay suggested that these compounds induce cell death by apoptosis. Overall, the current study demonstrates that the synthesis of imidazopyridine linked triazole conjugates as promising anticancer agents causing G2/M arrest and apoptotic-inducing ability.

Synthesis of imidazo-thiadiazole linked indolinone conjugates and evaluated their microtubule network disrupting and apoptosis inducing ability.

A series of imidazo[2,1-b][1,3,4]thiadiazole linked indolinone conjugates were synthesized and investigated for antiproliferative activity in different human cancer cell lines by changing various substitutions at indolinone and phenyl ring systems. Among them conjugates 7, 14 and 15 were exhibited potent antiproliferative activity with GI50 values from 0.13 to 3.8 µΜ and evaluated for cell cycle analysis, tubulin polymerization assay and apoptosis. Treatment with 7, 14 and 15 were resulted in accumulation of cells in G2/M phase, inhibition of tubulin assembly, disruption of microtubule network. Inhibition of tubulin polymerization was further supported by Western blot analysis. In addition, the conjugates (7, 14 and 15) also showed apoptosis in HeLa cell line, detailed biological studies such as Hoechst 33,258 staining, DNA fragmentation and caspase-3 assays suggested that these compounds induce cell death by apoptosis. Docking studies revealed that these compounds (7, 14 and 15) bind with αAsn101, αThr179, αSer178, ßCys241, ßLys254 and ßLys352 in the colchicine-binding site of the tubulin.

Design and synthesis of 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates as tubulin polymerization inhibitors.

1,2,3-Triazolo linked benzo[d]imidazo[2,1-b]thiazole conjugates (5a-v) were designed, synthesized and evaluated for their cytotoxic potency against some human cancer cell lines like DU-145 (prostate), HeLa (cervical), MCF-7 (breast) HepG2 (liver) and A549 (lung). Preliminary results revealed that some of these conjugates like 5f and 5k exhibited significant antiproliferative effect against human breast cancer cells (MCF-7) with IC50 values of 0.60 and 0.78µM respectively. Flow cytometric analysis of the cell cycle demonstrated an increase in the percentage of cells in the G2/M phase which was further authenticated by elevation of cyclin B1 protein levels. Immunocytochemistry revealed loss of intact microtubule structure in cells treated with 5f and 5k, and western blot analysis revealed that these conjugates accumulated more tubulin in the soluble fraction. Moreover, the conjugates caused apoptosis of the cells that was confirmed by mitochondrial membrane potential and Annexin V-FITC assay. Molecular docking studies indicated that these conjugates occupy the colchicine binding site of the tubulin protein.

Click chemistry-assisted synthesis of triazolo linked podophyllotoxin conjugates as tubulin polymerization inhibitors.

A series of new triazolo linked 4ß-amidopodophyllotoxin conjugates (9a-l) were synthesized using click chemistry and evaluated for their antitumor activity against four human cancer cell lines. Among them, two compounds (9c and 9j) showed significant anticancer activity with IC50 values of 0.9 and 0.07 µM, respectively. Biological studies are conducted into the cell-cycle distribution of these conjugates inducing G2/M-phase arrest, apart from an increase in the levels of caspase-3 proteins, followed by apoptotic cell death. A tubulin polymerization assay analysis showed that these compounds effectively inhibit microtubule assembly in HeLa cells and, moreover, Hoechst 33258 and Immunohistochemistry staining suggest that these compounds induce cell death by apoptosis. The docking studies showed that compounds 9c and 9j interact and bind efficiently with the tubulin protein at the colchicine site.

2-aryl benzimidazole conjugate induced apoptosis in human breast cancer MCF-7 cells through caspase independent pathway.

Apoptosis is a representative form of programmed cell death, which has been assumed to be critical for cancer prevention. Thus, any agent that can induce apoptosis may be useful for cancer treatment and apoptosis induction is arguably the most potent defense against cancer promotion. In our previous studies, 2-aryl benzimidazole conjugates were synthesized and evaluated for their antiproliferative activity and one of the new molecule (2f) was considered as a potential lead. This lead molecule showed significant antiproliferative activity against human breast cancer cell line, MCF-7. The results of the present study revealed that this compound arrested the cell cycle at G2/M phase. Topoisomerase II inhibition assay and Western blot analysis suggested that this compound effectively inhibits topoisomerase II activity which leads to apoptotic cell death. Apoptosis induction in MCF-7 cells was further confirmed by loss of mitochondrial membrane potential (∆Ψm), release of cytochrome c from mitochondria, an increase in the level of apoptosis inducing factor (AIF), generation of reactive oxygen species (ROS), up regulation of proapoptotic protein Bax and down regulation of anti apoptotic protein Bcl-2. Apoptosis assay using Annexin V-FITC assay also suggested that this compound induced cell death by apoptosis. However, compound 2f induced apoptosis could not be reversed by Z-VAD-FMK (a pan-caspase inhibitor) demonstrated that the 2f induced apoptosis was caspase independent. Further, 2f treatment did not activate caspase-7 and caspase-9 activity, suggesting that this compound induced apoptosis in breast cancer cells via a caspase independent pathway. Most importantly, this compound was less toxic towards non-tumorigenic breast epithelial cells, MCF-10A. Furthermore, docking studies also support the potentiality of this molecule to bind to the DNA topoisomerase II.

Investigation of the apoptotic pathway induced by benzimidazole-oxindole conjugates against human breast cancer cells MCF-7.

In our previous studies, benzimidazole-oxindole conjugates were synthesized and evaluated by National Cancer Institute (NCI) for their cytotoxic activity and the new molecules like 5c and 5p were considered as potential leads. These conjugates arrested the cell cycle at G2/M phase and inhibited tubulin polymerization. These observations prompted us to investigate the apoptotic mechanism induced by these lead molecules against human breast cancer cells (MCF-7). Studies like measurement of mitochondrial membrane potential (ΔΨm), generation of reactive oxygen species (ROS) and Annexin V-FITC assay revealed that these compounds induced mitochondrial mediated (intrinsic apoptotic pathway) apoptosis in human breast cancer cells. It was further confirmed by western blot analysis of pro apoptotic protein Bax, anti apoptotic protein Bcl-2, cytochrome c release, caspase-9 activity and cleavage of PARP.

Benzo[b]furan derivatives induces apoptosis by targeting the PI3K/Akt/mTOR signaling pathway in human breast cancer cells.

The PI3K/Akt/mTOR signaling pathway plays a key regulatory function in cell survival, proliferation, migration, metabolism and apoptosis. Aberrant activation of the PI3K/Akt/mTOR pathway is found in many types of cancer and thus plays a major role in breast cancer cell proliferation. In our previous studies, benzo[b]furan derivatives were evaluated for their anticancer activity and the lead compounds identified were 26 and 36. These observations prompted us to investigate the molecular mechanism and apoptotic pathway of these lead molecules against breast cancer cells. Benzo[b]furan derivatives (26 and 36) were evaluated for their antiproliferative activity against human breast cancer cell lines MCF-7 and MDA MB-231. These compounds (26 and 36) have shown potent efficiency against breast cancer cells (MCF-7) with IC50 values 0.057 and 0.051µM respectively. Cell cycle analysis revealed that these compounds induced cell cycle arrest at G2/M phase in MCF-7 cells. Western blot analysis revealed that these compounds inhibit the PI3K/Akt/mTOR signaling pathway and induced mitochondrial mediated apoptosis in human breast cancer cells (MCF-7).

Combretastatin linked 1,3,4-oxadiazole conjugates as a Potent Tubulin Polymerization inhibitors.

A new class of combretastatin linked 1,3,4-oxadiazoles were designed, synthesized and screened for their cytotoxic activity against five human cancer cell lines such as HeLa, DU-145, A549, MDA-MB-231 and B16. These compounds showed significant cytotoxicity with IC50 values in the range 0.118-54.32µM. Conjugate 5m displayed potent antiproliferative activity against DU-145 cell line. Flow cytometric analysis revealed that these compounds arrested the cell cycle in G2/M phase. Moreover, the tubulin polymerization assay and immunofluorescence analysis indicate that 5m exhibits potent inhibitory effect on the tubulin assembly. Further, DNA fragmentation and Hoecst staining assays confirm that 5m induces apoptosis. Molecular docking studies and competitive binding assay indicated that 5m effectively bind at the colchicine binding site of the tubulin.

Design, synthesis and antiproliferative activity of the new conjugates of E7010 and resveratrol as tubulin polymerization inhibitors.

A new class of (E)-N-phenyl-3-styrylpyridin-2-amine conjugates were designed and synthesized on the basis of E7010 and resveratrol scaffolds. These conjugates were evaluated for their antiproliferative activity in four human cancer cell lines with GI50 values ranging from 2.1 µM to 20 µM. Two of the conjugates RSV-1 and RSV-11 were found to possess 13-fold higher GI50 values than resveratrol and 1 to 2 fold higher GI50 values than E7010 against the human cervical HepG2 cancer line. They displayed high potency and selectivity in a panel of NCI 60 human cancer cell lines. Based on the GI50 values against the panel of 60 NCI cancer cell lines and dock scores from the molecular modelling studies, we selected RSV-1 and RSV-11 for tubulin polymerization and mechanistic studies. Furthermore, RSV-1 and RSV-11 compounds inhibited the assembly of tubulin by strongly binding to the colchicine-binding site. The G2/M-phase is arrested in HepG2 cells as assessed by flow cytometry. Structure based studies, western blotting and immunofluorescence experiments demonstrated that RSV-1 and RSV-11 depolymerize microtubules in the HepG2 cell line, resulting in an accumulation of G2/M cells.

Investigation of the mechanism and apoptotic pathway induced by 4ß cinnamido linked podophyllotoxins against human lung cancer cells A549.

Apoptosis is essential for normal development and the maintenance of homeostasis. It plays a necessary role to protect against carcinogenesis by eliminating damaged cells. Many studies have demonstrated that the dysregulation of apoptosis results in cancer and this provides an approach to develop therapeutic agents via inducing apoptosis. In our previous studies 4ß-cinnamido linked podophyllotoxin conjugates were synthesized and evaluated for their cytotoxic activity in a panel of five human cancer cell lines and the new molecules like 17a and 17f were considered as potential leads. The cytotoxic activity was comparable to etoposide. These observations prompted us to investigate the mechanism underplaying the cytotoxic activity and apoptotic pathway induced by these compounds in human lung cancer cells A459. The results of the present study revealed that these compounds exhibited DNA topoisomerase IIα inhibition and induced mitochondrial mediated apoptosis. It was further confirmed by Mitochondrial membrane potential, Cytochrome c release, cleavage of poly (ADP-ribose) polymerase (PARP), Reactive oxygen species (ROS) generation, regulation of antiapoptotic protein Bcl-2 and pro apoptotic protein Bax studied by Western blot analysis. Annexin V-FITC assay also suggested that these compounds induced cell death by apoptosis. Pretreatment with N-acetyl-L-cysteine (NAC) prevented the generation of ROS. Further, pretreatment with NAC significantly inhibited 17a and 17f induced apoptosis, suggesting that ROS are the key mediators for 17a and 17f induced apoptosis. These data indicate that these compounds might induce apoptosis in A549 cells through a ROS mediated mitochondrial dysfunction pathway. Moreover, these compounds did not significantly inhibit the noncancerous human embryonic kidney cells, HEK-293. Docking studies also elucidate the potential of these molecules to bind to the DNA topoisomerase II. Podophyllotoxin analogs were investigated for their mechanism and apoptotic pathway against lung cancer cell line, A549. These podophyllotoxin analogs inhibited DNA topoisomerase IIα and induced mitochondrial mediated apoptosis in lung cancer cell line, A549. Western blot analysis suggested that these compounds inhibited the DNA topoisomerase IIα. Studies like, Measurement of mitochondrial membrane potential (∆Ψm), Generation of intracellular reactive oxygen species (ROS) and Annexin V-FITC assay suggested that these compounds induced mitochondrial mediated apoptosis. Pretreatment with N-acetyl-L-cysteine (NAC) suggested that ROS plays a role in 17a and 17f induced apoptosis. Further the apoptotic effect of these compounds was confirmed by western blot analysis of pro apoptotic protein Bax and antiapoptotic protein Bcl-2, Cytochrome c release and cleavage of poly (ADP-ribose) polymerase (PARP). Moreover, these compounds did not significantly inhibit the noncancerous human embryonic kidney cells, HEK-293.

Synthesis of 2-aryl-1,2,4-oxadiazolo-benzimidazoles: Tubulin polymerization inhibitors and apoptosis inducing agents.

A new series of 2-aryl 1,2,4-oxadiazolo-benzimidazole conjugates have been synthesized and evaluated for their antiproliferative activity in the sixty cancer cell line panel of the National Cancer Institute (NCI). Compounds 5l (NSC: 761109/1) and 5x (NSC: 761814/1) exhibited remarkable cytotoxic activity against most of the cancer cell lines in the one dose assay and were further screened at five dose concentrations (0.01, 0.1, 1, 10 and 100 µM) which showed GI50 values in the range of 0.79-28.2 µM. Flow cytometric data of these compounds showed increased cells in G2/M phase, which is suggestive of G2/M cell cycle arrest. Further, compounds 5l and 5x showed inhibition of tubulin polymerization and disruption of the formation of microtubules. These compounds induce apoptosis by DNA fragmentation and chromatin condensation as well as by mitochondrial membrane depolarization. In addition, structure activity relationship studies within the series are also discussed. Molecular docking studies of compounds 5l and 5x into the colchicine-binding site of the tubulin, revealed the possible mode of interaction by these compounds.

DNA-binding affinity and anticancer activity of ß-carboline-chalcone conjugates as potential DNA intercalators: Molecular modelling and synthesis.

A new series of DNA-interactive ß-carboline-chalcone conjugates have been synthesized and evaluated for their in vitro cytotoxicity and DNA-binding affinity. It has been observed that most of these new hybrids have shown potent cytotoxic activities on A-549 (lung adenocarcinoma) cell lines with IC50 values lower than 10 µM. The hybrid 7b is more effective against some of the selected cancer cell lines with IC50 values less than 50 µM. In addition, compounds 7e, 7k, 7p-u has displayed significant elevation in ΔTm of DNA in comparison to Adriamycin, suggesting significant interaction and remarkable DNA stabilization. The DNA intercalation of these new hybrids has been investigated by fluorescence titration, DNA viscosity measurements, molecular docking as well as molecular dynamics and the results are in agreement with the thermal denaturation studies.

Synthesis of 2-anilinopyridyl-triazole conjugates as antimitotic agents.

A series of 2-anilinopyridyl­triazole conjugates (6a­t) were prepared and evaluated for their cytotoxic activity against a panel of three human cancer cell lines. Among them compounds 6q, 6r and 6s showed significant cytotoxic activity with IC50 values ranging from 0.1 to 4.1 µM. Structure­activity relationships were elucidated with various substitutions on these conjugates. Flow cytometric analysis revealed that these compounds arrest the cell cycle at the G2/M phase and induce cell death by apoptosis. The tubulin polymerization assay and immunofluorescence analysis showed that these compounds (6q, 6r and 6s) effectively inhibited the microtubule assembly in human prostate cancer cells (DU-145). The docking studies showed that 6s interacts and binds efficiently with the tubulin protein at the colchicine binding site. This was further confirmed by the colchicine competitive binding assay. Moreover, compounds 6q, 6r and 6s possess anti-tubulin activity both in vitro and within cells as demonstrated by the ratio of soluble versus polymerized tubulin. Further the apoptotic effects of compounds were confirmed by Hoechst staining, caspase 3 activation, annexin-V FITC, mitochondrial membrane potential and DNA fragmentation analysis. Interestingly, these compounds did not affect the normal human embryonic kidney cells, HEK-293.

Synthesis of phenstatin/isocombretastatin-chalcone conjugates as potent tubulin polymerization inhibitors and mitochondrial apoptotic inducers.

A series of phenstatin/isocombretastatin­chalcones were synthesized and screened for their cytotoxic activity against various human cancer cell lines. Some representative compounds exhibited significant antiproliferative activity against a panel of sixty human cancer cell lines of the NCI, with GI50 values in the range of 0.11 to 19.0 µM. Three compounds (3b, 3c and 3e) showed a broad spectrum of antiproliferative efficacy on most of the cell lines in the sub-micromolar range. In addition, all the synthesized compounds (3a­l and 4a­l) displayed moderate to excellent cytotoxicity against breast cancer cells such as MCF-7 and MDA-MB-231 with IC50 values in the range of 0.5 to 19.9 µM. Moreover, the tubulin polymerization assay and immunofluorescence analysis results suggest that some of these compounds like 3c and 3e exhibited significant inhibitory effect on the tubulin assembly with an IC50 value of 0.8 µM and 0.6 µM respectively. A competitive binding assay suggested that these compounds bind at the colchicine-binding site of tubulin. A cell cycle assay revealed that these compounds arrest at the G2/M phase and lead to apoptotic cell death. Furthermore, this was confirmed by Hoechst 33258 staining, activation of caspase 9, DNA fragmentation, Annexin V-FITC and mitochondrial membrane depolarization. Molecular docking studies indicated that compounds like 3e occupy the colchicine binding site of tubulin.

Synthesis of (Z)-(arylamino)-pyrazolyl/isoxazolyl-2-propenones as tubulin targeting anticancer agents and apoptotic inducers.

A new class of pyrazole and isoxazole conjugates were synthesized and evaluated for their cytotoxic activity against various human cancer cell lines. These compounds have shown significant cytotoxicity with lower IC50 values. FACS results revealed that A549 cells treated with these compounds arrested cells at the G2/M phase of the cell cycle apart from activating cyclin B1 protein levels. Particularly, compounds 9a and 9b demonstrated a remarkable inhibitory effect on tubulin polymerization and showed a pronounced inhibitory effect on tubulin polymerization with IC50 values of 1.28 µM and 0.28 µM respectively, whereas nocodazole, a positive control, has shown lower antitubulin activity with an IC50 value of 2.64 µM. Furthermore, these compounds induced apoptosis by loss of mitochondrial membrane potential, propidium iodide (PI) staining and the activation of caspase-3. Results of a fluorescence based competitive colchicine binding assay suggest that these conjugates bind successfully at the colchicine binding site of tubulin. These investigations reveal that such conjugates containing pyrazole with a trimethoxy phenyl ring and indole moieties have potential for the development of newer chemotherapeutic agents.

Synthesis and biological evaluation of cinnamido linked benzophenone hybrids as tubulin polymerization inhibitors and apoptosis inducing agents.

A new class of hybrid molecules containing cinnamide subunit linked to benzophenone as inhibitors of tubulin polymerization were synthesized and evaluated for their anticancer potential. These hybrids exhibit anticancer activity with IC50 values ranging from 0.06 to 16.3µM. Compounds 4f and 4g possessing fluoro and trifluoromethyl on the cinnamido subunit showed significant cytotoxic activity with IC50 values 0.06 and 0.09µM against HeLa cell line, respectively. These compounds showed cell cycle arrest at G2/M phase of the cell cycle and inhibited tubulin polymerization followed by activation of caspase-3 activity and apoptotic cell death. Further in vitro tubulin polymerization assay showed that the level of tubulin inhibition was comparable to that of 2a for the compounds 4f and 4g. Moreover, Hoechst 33258 staining and DNA fragmentation assay suggested that these compounds induce cell death by apoptosis. Overall, the current study demonstrates that the synthesis of benzophenone linked cinnamide subunit conjugates as promising anticancer agents with G2/M arrest and apoptotic-inducing ability via targeting tubulin.

Synthesis of a terphenyl substituted 4-aza-2,3-didehydropodophyllotoxin analogues as inhibitors of tubulin polymerization and apoptosis inducers.

A series of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates (8a-r) were synthesized by a straightforward one-step multicomponent synthesis that demonstrated anticancer activity against five human cancer cell lines (lung, colon, renal, prostate and cervical). All the tested compounds showed potent anticancer activity with IC50 values ranging from 0.87 to 16.59 µM. Among them compounds 8n and 8p showed significant anticancer activity in lung cancer cells with IC50 values 0.91 and 0.87 µM, respectively. Flow cytometric analysis revealed that these compounds induced cell cycle arrest in G2/M phase in A549 cell line leading to caspase-3 dependent apoptotic cell death. The tubulin polymerization assay and immunofluorescence analysis showed that these compounds effectively inhibit microtubule assembly at both molecular and cellular levels in A549 cells. Further, Hoechst staining, DNA fragmentation analysis also suggested that these compounds induced cell death by apoptosis. Overall, the current study demonstrated that the synthesis of terphenyl based 4-aza-2,3-didehydropodophyllotoxin conjugates as promising anticancer agents with G2/M cell cycle arrest and apoptotic-inducing activities via targeting tubulin.

Synthesis and biological evaluation of benzo[b]furans as inhibitors of tubulin polymerization and inducers of apoptosis.

A series of benzo[b]furans was synthesized with modification at the 5-position of the benzene ring by introducing C-linked substituents (aryl, alkenyl, alkynyl, etc.). These compounds were evaluated for their antiproliferative activities, inhibition of tubulin polymerization, and cell-cycle effects. Some compounds in this series displayed excellent activity in the nanomolar range against lung cancer (A549) and renal cell carcinoma (ACHN) cancer cell lines. (6-Methoxy-5-((4-methoxyphenyl)ethynyl)-3-methylbenzofuran-2-yl)(3,4,5-trimethoxyphenyl)methanone (26) and (E)-3-(6-methoxy-3-methyl-2-(1-(3,4,5-trimethoxyphenyl)vinyl)benzofuran-5-yl)prop-2-en-1-ol (36) showed significant activity in the A549 cell line, with IC50 values of 0.08 and 0.06 µM, respectively. G2/M cell-cycle arrest and subsequent apoptosis was observed in the A549 cell line after treatment with these compounds. The most active compound in this series, 36, also inhibited tubulin polymerization with a value similar to that of combretastatin A-4 (1.95 and 1.86 µM, respectively). Furthermore, detailed biological studies such as Hoechst 33258 staining, DNA fragmentation and caspase-3 assays, and western blot analyses with the pro-apoptotic protein Bax and the anti-apoptotic protein Bcl-2 also suggested that these compounds induce cell death by apoptosis. Molecular docking studies indicated that compound 36 interacts and binds efficiently with the tubulin protein.

Synthesis of pyrazolo[1,5-a]pyrimidine linked aminobenzothiazole conjugates as potential anticancer agents.

A series of pyrazolo[1,5-a]pyrimidine linked 2-aminobenzothizole conjugates (6a-t) were synthesized and evaluated for their anticancer activity against five human cancer cell lines. Among them two compounds 6p and 6m showed significant anticancer activity with IC50 values ranging from 2.01 to 7.07 and 1.94-3.46 µM, respectively. Moreover, cell cycle arrest in G2/M and reduction in Cdk1 expression level were observed upon treatment of these compounds and they also induced caspase-3 dependent apoptosis. This was further confirmed by staining as well as DNA fragmentation analysis.

Synthesis and anticancer activity of heteroaromatic linked 4ß-amido podophyllotoxins as apoptotic inducing agents.

A series of different heteroaromatic linked 4ß-amidopodophyllotoxin conjugates (16a-i, 17a-i and 18a-d) were synthesized and evaluated for anticancer activity against five human cancer cell lines. Among the series, one of the compound 17g showed significant antiproliferative activity in A549 (lung cancer) cell line. Flow cytometric analysis showed that 17g arrested the cell cycle in the G2/M phase leading to caspase-3 dependent apoptotic cell death. Further, Hoechst 33258 staining and DNA fragmentation assay also suggests that 17g induces cell death by apoptosis.