Elsevier

Tetrahedron

Volume 63, Issue 43, 22 October 2007, Pages 10637-10645
Tetrahedron

Synthesis and epimerization of phenylalanyl 4-aminocyclophosphamides

https://doi.org/10.1016/j.tet.2007.08.009Get rights and content

Abstract

Peptide and amino acid conjugates of (4R)- and (4S)-4-aminocyclophosphamides (4-NH2-CPA, 3) were designed as prodrug forms of phosphoramide mustard. Four diastereomers of Boc-Phe-4-NH-CPA (6) were synthesized stereospecifically from homoserine (R or S) and the protection strategy was optimized for the homoserine hydroxyl group during the construction of the 1,3,2-oxazaphosphorinane ring. The Phe-4-NH-CPA isomers of the trans-configuration ((2S,4R)- and (2R,4S)-) were found to be less stable than the corresponding isomers of the cis-configuration ((2R,4R)- and (2S,4S)-) and to undergo epimerization of the C-4 chiral center in the presence of 25% TFA used during Boc deprotection. The synthetic route developed should be applicable to the synthesis of a variety of peptide and amino acid conjugates of 4-aminocyclophosphamide.

Graphical abstract

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Novel phenylalanyl 4-aminocyclophosphamide isomers were synthesized and the trans-isomers were found to undergo epimerization to form cis-isomers in the presence of 25% TFA during Boc deprotection.

Introduction

The oxazaphosphorinane cyclophosphamide (1) is an alkylating antitumor agent with activity against a broad spectrum of human cancers including slow-growing solid tumors.1 Over the past four decades, cyclophosphamide has become one of the most frequently used anticancer agents in clinic. The clinical significance as well as the unique conformational and stereochemical aspects of oxazaphosphorinane derivatives have attracted much interest in the chemistry community.2, 3, 4 To elucidate the mechanism of action of cyclophosphamide and to enhance its efficacy as an antitumor agent, numerous oxazaphosphorinane derivatives were synthesized. The mechanism of action of cyclophosphamide has been well understood after decades of investigation.5, 6 Briefly, cyclophosphamide is oxidized by cytochrome P450 enzymes in the liver to 4-hydroxycyclophosphamide (2), which then decomposes into acrolein and the alkylating species phosphoramide mustard.5, 6 Acrolein is responsible for the hemorrhagic cystitis, a major dose-limiting side effect of cyclophosphamide. Tumor-targeted prodrug therapy is one of the strategies explored to improve the therapeutic index of cyclophosphamide.7, 8, 9, 10, 11, 12 In this strategy, phosphoramide mustard is incorporated with a biochemical activation mechanism specifically associated with tumor cells, such as hypoxic reduction, enzymatic action, and receptor recognition. Recently, we have reported for the first time that 4-aminocyclophosphamide (4-NH2-CPA, 3) can be used as a prodrug form of phosphoramide mustard because of its structural similarity to 2 and its spontaneous degradation as a mono-phosphorylated gem-diamine.12 In that communication we had briefly described the synthesis of phenylalanyl 4-aminocyclophosphamide (H-Phe-4-NH-CPA, 4) and its Cbz-protected derivative (5). Herein, we wish to report an alternative synthetic route to 4 through its Boc-protected derivative (6) and an unexpected but interesting epimerization reaction was observed for the trans-isomers of 4 under acidic de-Boc conditions. Protecting strategy for the hydroxyl group of homoserine was also optimized for the construction of the 1,3,2-oxazaphosphorinane ring. The synthetic routes developed can be easily adapted for the synthesis of various 4-aminocyclophosphamide conjugates, especially those of amino acids or peptides.

Section snippets

Results and discussion

The four configurational diastereomers of compounds 46 are referred to as (2R,4R)-, (2R,4S)-, (2S,4R)-, and (2S,4S)- (Fig. 1) according to the C-2 and C-4 absolute configuration. Among them, (2R,4R)- and (2S,4S)- have been referred to as cis-isomers and (2R,4S)- and (2S,4R)- as trans-isomers according to the relative orientation of the C-4 substituent to the oxygen atom of Pdouble bondO bond in the oxazaphosphorinane ring (cis=RR/SS, trans=SR/RS). Only the chair conformations are shown in Figure 1 to

Conclusion

We successfully synthesized Boc-protected phenylalanine-conjugated 4-aminocyclophosphamide (6) isomers in a stereospecific manner from homoserine (R or S) and explored various protection strategies for the hydroxyl group of homoserine for the steps leading to the construction of the 1,3,2-oxazaphosphorinane ring. Among the protecting groups explored, Cbz was the best for the synthesis of the key intermediate 10 prior to the formation of the 1,3,2-oxazaphosphorinane ring and TBDPS can be used as

General

Moisture-sensitive reactions were performed in flame-dried glassware under a positive pressure of nitrogen or argon. Air- and moisture-sensitive materials were transferred by a syringe or cannula under an argon atmosphere. Solvents were either ACS reagent grade or HPLC grade. Tetrahydrofuran was dried over sodium/benzophenone. Triethylamine, dichloromethane, and ethyl acetate were dried over calcium hydride. Pyridine was dried over potassium hydroxide and distilled over calcium hydride. N,N

Acknowledgements

We gratefully acknowledge the financial support of grant SNJ-CCR 700-009 from the State of New Jersey Commission on Cancer Research, a pilot grant from the Gallo Prostate Cancer Center of the Cancer Institute of New Jersey, and grant RSG-03-004-01-CDD from the American Cancer Society.

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    Present address: Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, CA 94143, USA.

    Present address: Department of Drug Metabolism, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA.

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