Chandrakant Bagul

Design and Synthesis of C3-Pyrazole/Chalcone-Linked Beta-Carboline Hybrids: Antitopoisomerase I, DNA-Interactive, and Apoptosis-Inducing Anticancer Agents

A series of β‐carboline hybrids bearing a substituted phenyl and a chalcone/(N‐acetyl)‐pyrazole moiety at the C1 and C3 positions, respectively, was designed, synthesized, and evaluated for anticancer activity. These new hybrid molecules showed significant cytotoxic activity, with IC50 values ranging from <2.0 μM to 80 μM, and the structure–activity relationships (SAR) associated with substitutions at positions 1 and 3 of these hybrids was clearly addressed. Further, induction of apoptosis was confirmed by Annexin V‐FITC, Hoechst staining, and DNA fragmentation analysis. In addition, DNA photocleavage studies proved that two of the hybrids, (E)‐1‐(furan‐2‐yl)‐3‐(1‐(4‐(trifluoromethyl)phenyl)‐9H‐pyrido[3,4‐b]indol‐3‐yl)prop‐2‐en‐1‐one (7 d) and 1‐(3‐(furan‐2‐yl)‐5‐(1‐(4‐(trifluoromethyl)phenyl)‐9H‐pyrido[3,4‐b]indol‐3‐yl)‐4,5‐dihydro‐1H‐pyrazol‐1‐yl)ethanone (8 d) could effectively cleave pBR322 plasmid DNA upon irradiation with UV light. Active hybrid 8 d inhibited DNA topoisomerase I activity efficiently and preserved DNA in the supercoiled form. To further corroborate the biological activities, as well as to understand the nature of the interaction of these hybrids with DNA, spectroscopic studies were also performed. Unlike simple β‐carboline alkaloids, the binding mode of these new hybrid molecules with DNA was not similar, and both biophysical as well as molecular docking studies speculated a combilexin‐type of interaction with DNA. Further, an in silico study of these β‐carboline hybrids revealed their drug‐like properties.

Design and synthesis of dithiocarbamate linked β-carboline derivatives: DNA topoisomerase II inhibition with DNA binding and apoptosis inducing ability.

A series of new β-carboline-dithiocarbamate derivatives bearing phenyl, dithiocarbamate and H/methyl substitutions at position-1, 3 and 9, respectively, were designed and synthesized. These derivatives 8a-l and 13a-l and their starting precursors (7 a-d and 12 a-d) have been evaluated for their in vitro cytotoxic activity on selected human cancer cell lines. Among the derivatives tested, 7 c, 12 c, 8 a, 8 d, 8 i, 8 j, 8 k, 8l and 13 d-l exhibited considerable cytotoxicity against most of the tested cancer cell lines (IC50<10μM). Interestingly, most of the derivatives (8 a-l and 13a-l) exhibited enhanced activity than their precursors (7 a-d and 12 a-d), which indicates that the combination of dithiocarbamate with β-carboline enhances the cytotoxicity of 8 a-l and 13 a-l. Moreover, the derivatives 8 j and 13 g exhibited significant cytotoxic activity with IC50 values of 1.34 μM and 0.79 μM on DU-145 cancer cells, respectively. Further, the induction of apoptosis by these derivatives was confirmed by Annexin V-FITC and Hoechst staining assays. However, both biophysical as well as molecular docking studies suggested a combilexin-type of interaction between these derivatives and DNA, unlike simple β-carbolines. With a view to understand their mechanism of action, DNA topoisomerase II (topo II) inhibition assay was also performed. Overall, the present study emphasizes the importance of linking a dithiocarbamate moiety to the β-carboline scaffold for exhibiting profound activity.

Design, synthesis and biological evaluation of imidazo[1,5-a]pyridine–PBD conjugates as potential DNA-directed alkylating agents

A series of novel imidazo[1,5-a]pyridine–PBD conjugates were synthesized and evaluated for their antitumor activity in breast cancer cell line (MCF-7). Interestingly, all the compounds showed enhanced DNA binding ability. These conjugates showed significant antitumor activity as deduced by MTT cell proliferation assay and amongst them, compounds 13f and 13g exhibit promising antitumor activity. G2/M phase cell cycle arrest was observed on the treatment of breast cancer cells (MCF-7) with 2 μM concentration of these compounds. Moreover, accumulation of cells in the G0 (apoptotic) phase was observed upon increase of their concentration to 4 μM. These compounds also induced the expression of proteins involved in apoptosis and DNA damage such as p53, p21 and γ-H2AX. In silico binding studies of compound 13g with DNA was performed to understand the mode of interactions and we observed that compound 13g binds well with the minor groove of DNA.

Synthesis and biological evaluation of cis-restricted triazole/tetrazole mimics of combretastatin-benzothiazole hybrids as tubulin polymerization inhibitors and apoptosis inducers

A series of colchicine site binding tubulin inhibitors were synthesized by the modification of the combretastatin pharmacophore. The ring B was replaced by the pharmacologically relevant benzothiazole scaffolds, and the cis configuration of the olefinic bond was restricted by the incorporation of a triazole and tetrazole rings which is envisaged by the structural resemblance to a tubulin inhibitor like combretastatin (CA-4). These compounds were evaluated for their antiproliferative activity on selected cancer cell lines and an insight in the structure activity relationship was developed. The most potent compounds (9a and 9b) demonstrated an antiproliferative effect comparable to that of CA-4. Mitotic cell cycle arrest in G2/M phase revealed the disruption of microtubule dynamics that was confirmed by tubulin polymerization assays and immunocytochemistry studies at the cellular level. Western blot analysis revealed that these compounds accumulate more tubulin in the soluble fraction. The colchicine competitive binding assay and the molecular docking studies suggested that the binding of these mimics at the colchicine site of the tubulin is similar to that of CA-4. Moreover, the triggering of apoptotic cell death after mitotic arrest was investigated by studying their effect by Hoechst staining, Annexin-V-FITC assay, mitochondrial membrane potential, ROS generation and caspase-3 activation.

Investigation of the mechanism and apoptotic pathway induced by 4β 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 4β-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.Graphical AbstractPodophyllotoxin 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.

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.Graphical Abstract

Synthesis and biological evaluation of imidazopyridinyl-1,3,4-oxadiazole conjugates as apoptosis inducers and topoisomerase IIα inhibitors

A series of imidazopyridinyl-1,3,4-oxadiazole conjugates were synthesized and investigated for their cytotoxic activity and some compounds showed promising cytotoxic activity. Compound 8q (NSC: 763639) exhibited notable growth inhibition that satisfies threshold criteria at single dose (10μM) on all human cancer cell lines. This compound was further evaluated at five dose levels (0.01, 0.1, 1, 10 and 100μM) to obtain GI50 values ranging from 1.30 to 5.64μM. Flow cytometric analysis revealed that compound 8q arrests the A549 cells in sub G1 phase followed by induction of apoptosis which was further confirmed by Annexin-V-FITC, Hoechst nuclear staining, caspase 3 activation, measurement of mitochondrial membrane potential and ROS generation. Topo II mediated DNA relaxation assay results showed that conjugate 8q could significantly inhibit the activity of topo II. Moreover, molecular docking studies also indicated binding to the topoisomerase enzyme (PDBID 1ZXN).

Aryl-imidazothiadiazole analogues as microtubule disrupting agents

Two series of 2-ethyl-6-(3′,4′,5′-trimethoxyphenyl)-5-aryl and 2-cyclopropyl-6-(3′,4′,5′-trimethoxyphenyl)-5-aryl-imidazothiadiazoles were designed, synthesized and evaluated for anti-proliferative activity in various human cancer cell lines. A common starting material 2-bromo-1-(3-,4-,5-trimethoxyphenyl)-ethanone was employed to generate the two scaffolds. Among them, compounds functionalized with N,N-dimethylaniline (5i and 6i) and napthyl (5k) groups showed significant cytotoxic activity with average IC50 values of 1.7–2.9 μM, respectively. Moreover, the structure–activity relationship was elucidated by incorporating the different substitutions on the aryl moiety at the 5th position of the imidazothiadiazole ring. Treatments with 5i, 6i and 5k arrested cells at the G2/M phase, with cell death proceeding through an apoptotic pathway that was dependent on the activation of caspase-3. Immunocytochemical analysis revealed the loss of intact microtubule structures in treated cells. Treatments with 5i, 6i and 5k manifested increased mRNA and protein levels of the G2/M marker, cyclin B1. Molecular docking studies of the most potent compounds 5i, 5k and 6i showed that these compounds interact and bind efficiently to the active site of tubulin. This was further confirmed by a colchicine competitive binding assay.

Synthesis and Biological Evaluation of Benzo[d][1,3]Dioxol-5-yl Chalcones as Antiproliferating Agents

A series of chalcone derivatives were designed, synthesized, and evaluated for their cytotoxic potential. These molecules have showed promising cytotoxic activity with IC50 values ranging from 5.24 to 63.12 μm. Among them, conjugates 16k, 16m and 16t showed significant antiproliferative activity with IC50 values ranging from 5.24 to 10.39 μm in MDA‐MB‐231 cell line. These compounds were further investigated for their effect on cell membrane blebbing, chromatin condensation, DNA fragmentation, Hoechst staining, annexin V, and cell cycle arrest (G2/M). The Western blot experiments revealed up regulation of pro‐apoptotic Bax and downregulation of antiapoptotic Bcl‐2. The studies also indicated reduction of mitochondrial membrane potential and increase in the levels of caspase‐3 and caspase‐7.

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.