Elsevier

Bioorganic & Medicinal Chemistry

Volume 16, Issue 17, 1 September 2008, Pages 8109-8116
Bioorganic & Medicinal Chemistry

Synthesis and biological evaluation of glucuronide prodrugs of the histone deacetylase inhibitor CI-994 for application in selective cancer chemotherapy

https://doi.org/10.1016/j.bmc.2008.07.048Get rights and content

Abstract

Two glucuronide prodrugs of the histone deacetylase inhibitor CI-994 were synthesized. These compounds were found to be soluble in aqueous media and stable under physiological conditions. The carbamoyl derivatisation of CI-994 significantly decreased its toxicity towards NCI-H661 lung cancer cells. Prodrug incubation with β-glucuronidase in the culture media led efficiently to the release of the parent drug and thereby restoring its ability to decrease cell proliferation, to inhibit HDAC and to induce E-Cadherin expression.

Graphical abstract

Two glucuronide prodrugs of the histone deacetylase inhibitor CI-994 were synthesized and evaluated as potential selective antitumour agents.

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Introduction

Recently, the inhibition of histone deacetylases (HDACs), a family of enzymes which play a fundamental role in the regulation of gene expression,1 has emerged as a new strategy in cancer chemotherapy. During the last decade, an increasing number of structurally diverse small molecule HDAC inhibitors including hydroxamates, carboxylates, benzamides, thiol derivatives, cyclic peptides and hydrophilic ketones, have been studied as potential therapeutic agents.2, 3, 4, 5 These investigations demonstrated that several HDAC inhibitors exhibited potent antitumour activities in the course of the treatment of solid and haematological malignancies. As a consequence, there are well over 100 clinical trials ongoing with at least 13 different HDAC inhibitors as monotherapy or in combination with other anticancer drugs.6 In October 2006, SAHA 1 (Zolinza™) (Fig. 1) became the first HDAC inhibitor approved by the Food and Drug Administration for the treatment of patients with cutaneaous T-cell lymphoma.

CI-994 2 (Fig. 1) is a potent member of the benzamide class of HDAC inhibitors that has demonstrated significant antitumour activity against a broad spectrum of murine, rat and human tumour models.7, 8, 9 This compound is currently progressing through clinical trials in combination with other standard anticancer agents such as carboplatin, paclitaxel,10 capecitabine11 or gemcitabine.12 Although CI-994 is a promising chemotherapeutic agent, it induces adverse events including thrombocytopenia, anemia and neutropenia. Another major drawback linked to its use in cancer chemotherapy is its lack of aqueous solubility. Within this framework the design of non-toxic hydrophilic prodrugs that could deliver CI-994 predominantly in the vicinity of the tumour seems to be an interesting alternative in order to enhance the therapeutic index of this HDAC inhibitor. Such an approach has recently been proposed for the selective targeting of SAHA.13

Toward this end, we have undertaken the study of the two glucuronide prodrugs 3 and 4 (Fig. 2). Indeed, several glucuronide prodrugs have already been selectively activated by β-glucuronidase, either present in high concentration in necrotic tumour areas (PMT)14 or previously targeted to the tumour sites (ADEPT,15 GDEPT16), and consequently demonstrated superior efficacy in vivo compared to standard chemotherapy.17 These results were attributed to the increased drug deposition and retention in the tumour connected with reduced anticancer agent concentration in normal tissues, considerably lowering the destruction of normal cells.

Prodrug 3 includes a nitrobenzylphenoxy carbamate linker18 which has been employed successfully to release either anticancer drug such as doxorubicine19 or magnetic resonance imaging contrast agent.20 According to these results, this linker should allow easy recognition of 3 by β-glucuronidase and, after enzymatic cleavage of the glycosidic bond should rapidly liberate CI-994 via an 1,6 elimination as depicted in Figure 2.

On the other hand, for prodrug 4 CI-994 is attached directly to the glucuronide moiety through a carbamate functional group. With this design, we anticipated that 4 would be an excellent substrate for the enzyme since N-phenyl β-glucuronyl carbamate was proved to be hydrolyzed by β-glucuronidase at a rate comparable to that of p-nitrophenyl β-glucuronide.21 Thus, enzymatic hydrolysis should conduct to the expulsion of the carbamic acid of the drug that should lead rapidly to the free aniline after decarboxylation (Fig. 2).

In both cases, we hypothesised that (1) the carbamoyl derivatisation of the amino group of CI-994 may prevent its ability to inhibit HDACs and therefore may decrease its toxicity toward normal cells and, (2) the hydrophilic character of the glucuronide moiety should enhance the water solubility of the drug allowing i.v. administration.

Section snippets

Chemistry

Prodrug 3 was prepared in three steps from the readily accessible activated carbonate 519 (Scheme 1). First, CI-994 was condensed with 5 in the presence of pyridine to give the carbamate 6 in 60% yield. The O-acetyl groups of 6 were then deprotected with MeONa in MeOH to afford 7 (40%). Finally, saponification of the methyl ester using NaOH produced the target prodrug 3 in quantitative yield.

In order to access to the requested glucuronide prodrug 4, our initial efforts focused upon the

Conclusion

In summary, we have reported the synthesis of the first CI-994 glucuronide prodrugs to date aimed at β-glucuronidase activation in ADEPT or PMT strategies. Such prodrugs were at least 12.5 times more soluble in aqueous media compared to the parent drug and exhibited good stability under physiological conditions. In contrast with CI-994, prodrugs 3 and 4 have no activity on cell proliferation, histone acetylation and E-Cadherin expression in our cellular model. On the other hand, when incubated

General chemistry methods

All reactions were performed under N2 atmosphere. Solvents used were of HPLC quality and chemicals were of analytical grade. 1H and 13C NMR were performed on an Avance 300 DPX Bruker. The chemical shifts are expressed in part per million (ppm) relative to TMS (δ = 0 ppm) and the coupling constant J in hertz (Hz). NMR multiplicities are reported using the following abbreviations: b, broad; s, singulet; d, doublet; t, triplet; q, quadruplet; m, multiplet.

Optical rotations were measured on a Schmidt + 

Acknowledgements

The authors thank CNRS, La Ligue Nationale contre le Cancer (Comité de Charentes-Maritime) and the Région Poitou-Charentes for financial support of this study.

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