Rajesh Kakadiya

Design, synthesis and antitumor evaluation of phenyl N-mustard-quinazoline conjugates

A series of N-mustard-quinazoline conjugates was synthesized and subjected to antitumor studies. The N-mustard pharmacophore was attached at the C-6 of the 4-anilinoquinazolines via a urea linker. To study the structure-activity relationships of these conjugates, various substituents were introduced to the C-4 anilino moiety. The preliminary antitumor studies revealed that these agents exhibited significant antitumor activity in inhibiting various human tumor cell growths in vitro. Compounds 21b, 21g, and 21h were selected for further antitumor activity evaluation against human breast carcinoma MX-1 and prostate PC-3 xenograft in animal model. These agents showed 54-75% tumor suppression with low toxicity (5-7% body-weight changes). We also demonstrate that the newly synthesized compounds are able to induce DNA cross-linking through alkaline agarose gel shift assay and inhibited cell cycle arrest at G2/M phase.

Synthesis and biological activity of stable and potent antitumor agents, aniline nitrogen mustards linked to 9-anilinoacridines via a urea linkage

To improve the chemical stability and therapeutic efficacy of N-mustard, a series of phenyl N-mustard linked to DNA-affinic 9-anilinoacridines and acridine via a urea linker were synthesized and evaluated for antitumor studies. The new N-mustard derivatives were prepared by the reaction of 4-bis(2-chloroethyl)aminophenyl isocyanate with a variety of 9-anilinoacridines or 9-aminoacridine. The antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro without cross-resistance to taxol or vinblastine and showed potent antitumor therapeutic efficacy in nude mice against human tumor xenografts. It also showed that 24d was capable of inducing marked dose-dependent levels of DNA cross-linking by comet assay and has long half-life in rat plasma.

Potent DNA-directed alkylating agents: Synthesis and biological activity of phenyl N-mustard-quinoline conjugates having a urea or hydrazinecarboxamide linker.

A series of N-mustard-quinoline conjugates bearing a urea or hydrazinecarboxamide linker was synthesized for antitumor evaluation. The in vitro cytotoxicity studies revealed that compounds with hydrazinecarboxamide linkers were generally more cytotoxic than the corresponding urea counterparts in inhibiting human lymphoblastic leukemia and various solid tumor cell growths in culture. The therapeutic efficacy against human tumor xenografts in animal model was studied. It was shown that complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft by 17a, i and 18c, d was achieved. In the present study, it was revealed that both linkers are able to lower the chemically reactive N-mustard pharmacophore and thus the newly synthesized conjugates possess a long half-life in rat plasma. Moreover, the new N-mustard derivatives are able to induce DNA cross-linking either by modified comet assay or by alkaline agarose gel shift assay.

Potent antitumor bifunctional DNA alkylating agents, synthesis and biological activities of 3a-aza-cyclopenta[a]indenes.

A series of bifunctional DNA interstrand cross-linking agents, bis(hydroxymethyl)- and bis(carbamates)-8H-3a-azacyclopenta[a]indene-1-yl derivatives were synthesized for antitumor evaluation. The preliminary antitumor studies revealed that these agents exhibited potent cytotoxicity in vitro and antitumor therapeutic efficacy against human tumor xenografts in vivo. Furthermore, these derivatives have little or no cross-resistance to either Taxol or Vinblastine. Remarkably, complete tumor remission in nude mice bearing human breast carcinoma MX-1 xenograft by 13a,b and 14g,h and significant suppression against prostate adenocarcinoma PC3 xenograft by 13b were achieved at the maximum tolerable dose with relatively low toxicity. In addition, these agents induce DNA interstrand cross-linking and substantial G2/M phase arrest in human non-small lung carcinoma H1299 cells. The current studies suggested that these agents are promising candidates for preclinical studies.

Novel antitumor indolizino[6,7-b]indoles with multiple modes of action: DNA cross-linking and topoisomerase I and II inhibition.

A series of bis(hydroxymethyl)indolizino[6,7-b]indoles and their bis(alkylcarbamates) were synthesized for antitumor studies. These agents were designed as hybrid molecules of β-carboline (topoisomerase inhibition moiety) and bis(hydroxymethyl)pyrrole (DNA cross-linking moiety). The preliminary antitumor studies indicated that these agents exhibited significant cytotoxicity against a variety of human tumor cells in vitro. Treatment of human breast carcinoma MX-1 xenograft-bearing nude mice with compounds 18b and 28c achieved more than 99% tumor remission. We also observed that 18a displayed potent therapeutic efficacy against human lung adenocarcinoma A549 and colon cancer HT-29 xenografts. These results revealed that compound 18a was more potent than irinotecan against HT-29 cells and was as potent as irinotecan against A549 cells in xenograft models. Furthermore, we demonstrated that these derivatives possess multiple modes of action, such as induction of DNA cross-linking, inhibition of topoisomerase I and II, and cell-cycle arrest at the S-phase.

The development of bis(hydroxymethyl)pyrrole analogs as bifunctional DNA cross-linking agents and their chemotherapeutic potential

Bifunctional DNA cross-linking agents are widely used as chemotherapeutic agents in clinics. The advance in the development of these agents as potential antitumor agents has generated various types of bis(hydroxymethyl)pyrrole analogs. In order to develop highly effective anticancer agents, it is necessary to understand the chemophysical properties, structure-activity relationships, therapeutic potency, toxicity/safety, and pharmacokinetics of these DNA cross-linking agents. This review presents an overview of the recent advances in developing various types of bis(hydroxymethyl)pyrrole analogs with potential antitumor activity to provide more information for future drug design and strategies for combination chemotherapy. The rational drug design, chemical syntheses, antitumor activity, mechanism of action, and development of combined chemotherapy regimens, including a DNA repair inhibitor, are discussed.

The novel DNA alkylating agent BO-1090 suppresses the growth of human oral cavity cancer in xenografted and orthotopic mouse models.

Oral cancer is the fourth‐most common cause of death in males and overall the sixth‐most common cause of cancer death in Taiwan. Surgery, radiotherapy and chemotherapy combined with other therapies are the most common treatments for oral cavity cancer. Although cisplatin, 5‐fluorouracil and docetaxel are commonly used clinically, there is no drug specific for oral cavity cancer. Here, we demonstrated that derivatives of 3a‐aza‐cyclopenta[a]indene, a class of newly synthesized alkylating agents, may be drugs more specific for oral cancer based on its potent in vitro cytotoxicity to oral cancer cells and on in vivo xenografts. Among them, BO‐1090, bis(hydroxymethyl)‐3a‐aza‐cyclopenta[a]indene derivative, targeted DNA for its cytotoxic effects as shown by inhibition of DNA synthesis (bromodeoxyuridine‐based DNA synthesis assay), induction of DNA crosslinking (alkaline gel shift assay), and induction of DNA single‐stranded breaks (Comet assay) and double‐stranded breaks (γ‐H2AX focus formation). Following DNA damage, BO‐1090 induced G1/S‐phase arrest and apoptosis in oral cancer cell lines. The therapeutic potential of BO‐1090 was tested in mice that received a xenograft of oral cavity cancer cell lines (SAS or Cal 27 cells). Intravenous injection of BO‐1090 significantly suppressed tumor growth in comparison to control mice. BO‐1090 also significantly reduced the tumor burden in orthotopic mouse models using SAS cells. There was no significant adverse effect of BO‐1090 treatment with this dosage based on whole blood count, biochemical enzyme profiles in plasma and histopathology of various organs in mouse. Taken together, our current results demonstrate that B0‐1090 may have potential as a treatment for oral cavity cancer.

Multidrug-resistant cells overexpressing P-glycoprotein are susceptible to DNA crosslinking agents due to attenuated Src/nuclear EGFR cascade-activated DNA repair activity

We synthesized several novel bifunctional alkylating derivatives of 3a-aza-cyclopenta[a]indene (BO-1012, BO-1005, BO-1099 and BO-1101) that are potent DNA interstrand crosslinking agents. In in vitro cytotoxicity assay, these compounds were more cytotoxic to multidrug-resistant (MDR) cells, such as KBvin10, KBtax50 and CEM/VBL, than their parental cells. Using a xenograft model, BO-1012, at a dose of 5 mg/kg, partially suppressed the growth of parental KB cells but completely suppressed the growth of KBvin10 cells in nude mice. In exploring the possible mechanism, we found that DNA double-strand break (DSB) repair activity in MDR cells, KBvin10 and CEM/VBL, was significantly reduced compared with their parental cells, KB and CEM. Reduced DSB repair activity in KBvin10 cells was likely due to a defect in nuclear translocation of DNA-dependent protein kinase (DNA-PK), a component of the non-homologous end-joining repair machinery. Furthermore, BO-1012-induced DNA-PK translocation from the cytosol into the nucleus in KB cells is associated with the activation of the Src/nuclear epidermal growth factor receptor (EGFR) cascade, which is defective in MDR cells. As knockdown of P-glycoprotein (P-gp) by siRNA reactivated the Src/nuclear EGFR cascade, DNA-PK translocation and DNA repair activity in MDR cells, overexpression of P-gp attenuates the activity of DNA DSB repair through suppression of Src/nuclear EGFR cascade. Therefore, DNA interstrand crosslinking agents may have potential therapeutic use against P-gp-overexpressing MDR cells.

Inhibition of autophagy enhances DNA damage-induced apoptosis by disrupting CHK1-dependent S phase arrest.

DNA damage has been shown to induce autophagy, but the role of autophagy in the DNA damage response and cell fate is not fully understood. BO-1012, a bifunctional alkylating derivative of 3a-aza-cyclopenta[a]indene, is a potent DNA interstrand cross-linking agent with anticancer activity. In this study, BO-1012 was found to reduce DNA synthesis, inhibit S phase progression, and induce phosphorylation of histone H2AX on serine 139 (γH2AX) exclusively in S phase cells. Both CHK1 and CHK2 were phosphorylated in response to BO-1012 treatment, but only depletion of CHK1, but not CHK2, impaired BO-1012-induced S phase arrest and facilitated the entry of γH2AX-positive cells into G2 phase. CHK1 depletion also significantly enhanced BO-1012-induced cell death and apoptosis. These results indicate that BO-1012-induced S phase arrest is a CHK1-dependent pro-survival response. BO-1012 also resulted in marked induction of acidic vesicular organelle (AVO) formation and microtubule-associated protein 1 light chain 3 (LC3) processing and redistribution, features characteristic of autophagy. Depletion of ATG7 or co-treatment of cells with BO-1012 and either 3-methyladenine or bafilomycin A1, two inhibitors of autophagy, not only reduced CHK1 phosphorylation and disrupted S phase arrest, but also increased cleavage of caspase-9 and PARP, and cell death. These results suggest that cells initiate S phase arrest and autophagy as pro-survival responses to BO-1012-induced DNA damage, and that suppression of autophagy enhances BO-1012-induced apoptosis via disruption of CHK1-dependent S phase arrest.

Novel bifunctional alkylating agents, 5,10-dihydropyrrolo[1,2-b]isoquinoline derivatives, synthesis and biological activity.

A series of linear pyrrolo[1,2-b]isoquinoline derivatives was synthesized for antitumor evaluation. The preliminary antitumor studies reveal that both bis(hydroxymethyl) and their bis(alkylcarbamate) derivatives show significant antitumor activity in inhibiting various human tumor cell growth in vitro. 1,2-Bis(hydroxymethyl)-3-methyl-5,10-dihydropyrrolo[1,2-b]isoquinoline (20a) was selected for antitumor studies in animal models. The results show that this agent can induce complete tumor remission or significant suppression in nude mice bearing human breast (MX-1) xenograft and ovarian (SK-OV-3) xenografts, respectively. Alkaline agarose gel shifting assay showed that 20a is able to cross-link with DNA. Studies on the cell cycle inhibition revealed that this agent induces cell arrest at G2/M phase. The results warrant further antitumor investigation against other human tumor growth in animal models.