Elsevier

Bioorganic & Medicinal Chemistry

Volume 12, Issue 18, 15 September 2004, Pages 4917-4927
Bioorganic & Medicinal Chemistry

Angiogenesis inhibitor TX-1898: syntheses of the enantiomers of sterically diverse haloacetylcarbamoyl-2-nitroimidazole hypoxic cell radiosensitizers

https://doi.org/10.1016/j.bmc.2004.06.039Get rights and content

Abstract

(R)- and (S)-Epichlorohydrins were used to prepare the enantiomers of sterically diverse haloacetylcarbamoyl-2-nitroimidazoles that function as hypoxic cell radiosensitizers. The synthetic design allowed for introduction of a side chain of varying bulk that permitted an examination of the steric effects on enantio-discrimination in biological assay systems. The single stereocenter also connected the two pharmacophores––a 2-nitroimidazole moiety critical to hypoxic cell radiosensitization, and a haloacetylcarbamoyl group to function as an anti-angiogenesis pharmacophore. In the chick embryo chorioallantoic membrane (CAM) assay, the R-enantiomers possessing the bulky p-tert-butylphenyl group showed higher anti-angiogenic activity than the corresponding S-enantiomers, while there were no differences in the activity between the enantiomers containing the less bulky methyl and tert-butyl groups. Among the compounds we report, R-p-tert-butylphenyl-bromoacetylcarbamoyl-2-nitroimidazole, TX-1898, was found to be the most promising candidate for further development of as anti-angiogenic hypoxic cell radiosensitizer.

Introduction

Angiogenesis is required for tumor growth and metastasis and, therefore, represents an exciting target for cancer treatment. Angiogenesis is a complex process involving a number of distinct steps, such as endothelial cell migration, proliferation, formation of capillary tubes in endothelial cells, their invasion, and metastasis. These steps are tightly regulated by pro- and anti-angiogenic factors.[1], [2], [3] Tumor-related angiogenesis is a multistep process that is initiated through the activity of various pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF), and interleukin-8 (IL-8). Many enzymes are involved, including such as the matrix metalloproteinases (MMPs), thrombin and methionine aminopeptidase-2 (MetAP-2), urokinase-type plasminogen activator (uPA), and others. Various anti-angiogenic drugs have been developed that target several related growth factor receptors such as VEGF receptors (VEGFR-1 and VEGFR-2), bFGF receptor, and PDGF receptor. Indeed, some of these agents, such as SU5416,4 ZD6474,5 SU6668,6 are now in clinical trials as anti-angiogenic drugs. Our own interest in this area is focused on small molecule anti-angiogenic agents related to the naturally-occurring angiogenic inhibitor fumagillin, and its semi-synthetic analog TNP-470, one of the first substances to be recognized to as having an anti-angiogenic effect.[7], [8], [9], [10]

In contrast to anti-angiogenic agents, tumor hypoxia induces the pro-angiogenic genes to stimulate up-regulation of angiogenic and tumor cell survival factors, giving rise to tumor proliferation, radioresistance, angiogenesis, and metastasis.11 Therefore, hypoxia in solid tumor is one of the targets for the improvement of chemo/radio-therapeutic efficacy. We propose that anti-angiogenic drugs that also function as hypoxic cell radiosensitizers will have synergistic interactions between the hypoxic cytotoxic/hypoxic cell radiosensitizing effect and anti-angiogenic activity leading to destruction of hypoxia-initiated neovasculature.

We have reported the molecular design and synthesis of several 2-nitroimidazole hydroxamate and hydroxamic acid analogs. These include KIN-841,12 KIN-806,13 KIN-804,[14], [15] and KIN-844,16 which are bifunctional hypoxic cell radiosensitizers, TX-1877,17 an anti-metastatic hypoxic cell radiosensitizer, anti-angiogenic hypoxic cytotoxin, TX-402,18 a protein tyrosine kinase inhibitor TX-1123,19 and anti-angiogenic/heparin-binding arginine dendrimer TX-1943 and TX-194420 (Fig. 1).

We also published previously that the development of racemic bromoacetylcarbamoyl-2-nitroimidazoles such as TX-1845 and TX-1846 as strong radiosensitizers and potent angiogenic inhibitors.21 The molecular chirality of hypoxic cell radiosensitizers generally has little or no effect on in vitro activity.[22], [23] In contrast we have found that there is a significant effect of absolute configuration on anti-angiogenic activity and that a single enantiomer separated from the racemate, often has a higher anti-angiogenic activity than the racemate. For example the R-enantiomer of the thienopyridine SR-25989 possessed more potent in vitro and in vivo anti-angiogenic properties than the corresponding racemate, and it inhibited metastatic dissemination and growth.24 Also the R,R-enantiomer of dihydrobenzofuran lignan showed more potent anti-angiogenic activity than the S,S-isomer in the chick embryo chorioallantoic membrane (CAM) assay.25 Molecular shape analysis indicates that there is overlap of target compounds with TNP-470 an anti-angiogenesis drug now in clinical trials, depending on steric bulk and absolute configuration. Based on these considerations, we felt it was important to examine the effects of absolute configuration on a series of haloacetylcarbamoyl-2-nitroimidazole radiosensitizers, compounds that, in racemic form, have shown promising anti-angiogenic and anti-proliferation activity.21

In this study we demonstrate the effectiveness of epichlorohydrin as a chiral scaffold wherein the single stereocenter connects a haloacetylcarbamoyl, 2-nitroimidazolyl, and diverse alkyl or aryl groups of varying steric dimensions. We examined the individual enantiomers of haloacetylcarbamoyl-2-nitroimidazoles as protease inhibitors, as anti-angiogenic agents in vitro using a rat lung endothelial (RLE) cell proliferation assay and a chick embryo CAM assay, and also their activities as hypoxic cell radiosensitizers.

Section snippets

Molecular modeling

We carried out geometry and molecular orbital optimizations of the (R)-enantiomers of chloroacetylcarbamoyl-2-nitroimidazoles possessing three different alkyl or arylalkyl groups of varying steric bulk-methyl, tert-butyl, and p-tert-butylphenyl. The results were compared to TNP-470 using the B3LYP hybrid density functional theory based the gaussian 98 programs.26

Figure 2 shows their chemical structures, optimized geometries, and the energies of their lowest unoccupied molecular orbitals

Discussion

As part of our research to develop effective cancer chemotherapeutic agents, we have developed compounds that combine radiosensitizing activity with anti-angiogenic activity. In our previous report,21 we found the racemic haloacetylcarbamoyl-2-nitroimidazole hypoxic cell radiosensitizers, such as KIN-1800, TX-1835, TX-1836, TX-1844, TX-1845, and TX-1846, were also potent angiogenesis inhibitors activity that we ascribe to the presence of the reactive haloacetylcarbamoyl group. To examine the

General procedures

1H NMR spectra were recorded on a JEOL JNM-EX400 spectrometer (400 MHz) with tetramethylsilane as the internal standard. Chemical shifts were reported in ppm. Coupling constants were reported in Hz. IR spectra were reported in KBr pellet on a Perkin–Elmer 1600 spectrometer. High-resolution mass spectra (HRMS) were measured on a JEOL JMS-SX102A mass spectrometer using a fast atom bombardment (FAB) and EI. Reaction was monitored by analytical thin-layer chromatograpy (TLC) with use of Merck silica

Acknowledgements

This study was supported in part by the Japan–China Sasakawa Medical Fellowship. The author C.-Z.J. thanks ‘The Japan–China Sasakawa Medical Fellowship’, and also thanks Professor Shibata Shoji, Emeritus Professor of Tokyo University, for his helpful advice. The authors thank Dr. Goto Satoru of the Faculty of Pharmaceutical Sciences, The University of Tokushima, for his advice of Molecular modeling, and also Dr. Y. Takekawa, H. Nishitani, and Y. Nishimoto of the School of Medical, The

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