Oxazaphosphorines

The historical significance of the mustard class of cytotoxics cannot be underestimated in that this class of compounds revealed that organic synthesis could provide chemotherapeutically valuable materials. Although the exact mechanism of cytotoxicity of the mustards was not fully appreciated, medicinal chemists appreciated the necessity of the beta-chloroethylamine group for in vivo activity. As a result, the desire to generate compounds that would either mask the reactivity of the mustard group and/or use properties specific to the cancerous cells to increase the selectivity of these compounds versus normal tissue became the desired profile for potential chemotherapeutics. The oxazaphosphorine class of cytotoxics was originally devised with both of the above properties in mind.

Initial profiling of cancerous cells to determine potential differences versus nontransformed cells revealed that cancer cells possessed increased expression of phosphoamidases. Therefore, a compound that conjugated the mustard nitrogen to a phosphate group would not only yield a more chemically robust analog, but also afforded a substrate for cleavage by phosphoamidases. The net result was the discovery of cyclophosphamide (Cytoxan, 12) shown in Figure 5, and the structurally related ifosfamide (Ifex, 13). The synthesis of 12 begins with condensation of phosphoryl chloride (POCl3) with bis(2-chloroethyl)amine hydrochloride (14) providing 15. Subsequent treatment of the intermediate phosphoramide with 3-aminopropanol (16) affords 12.

The development of 12 would have been severely hampered in the current paradigm of drug discovery in that only compounds that are highly active (cytotoxic, antiproliferative, induce apoptosis, etc.) versus transformed cell lines in vitro are further progressed to in vivo xenograft murine models of cancer. Cyclophosphamide is only weakly cytotoxic versus transformed cells in vitro. Nevertheless, administration of 12 in vivo to either animals, or subjects with sensitive tumors, provides dramatic antineoplastic effects. The initial rationalization for the in vivo activity of 12 was based on phosphatase or phosphoamidase cleavage of the P-N bond, which would release the mustard group. This hypothesis has been subsequently proven incorrect (for a summary of the development of oxazaphosphorine-based agents see 23). The accepted mechanism for cyclophosphamide's impressive in vivo activity is shown in Figure 6. Upon administration of 12, metabolic activation occurs via the cytochrome P450 monooxygenase system of the liver to afford 4-hydroxycyclophosphamide (17), a common oxidative precursor in all of the oxazaphosphorine therapeutics. The specific P450 isoforms responsible for the generation of 17 are species dependent. The human P450 isoforms involved in the metabolism of 12 are CYP2B6, CYP3A4, and CYP2C9, with CYP2B6 being primarily responsible for cyclophosphamide's metabolism in vivo.24 The intermediate 17 simultaneously affords the desired cytotoxic metabolites (vide infra) and allows for cell permeability.25 The highly unstable aminal of 17 undergoes spontaneous and reversible ring opening to afford aldophosphamide 18, which affords either inactive or cytotoxic metabolites depending on subsequent chemical or enzymatic modifications. The generation of the inactive metabolites occurs if 18 is oxidized by either alcohol dehydrogenase yielding 19, or by aldehyde dehydrogenase (ALDH1) affording the corresponding acid 20. Alternatively, nonenzymatic elimination of the phosphoryl group from the aldehyde simultaneously generates phosphoramide mustard (21) and acrolein (22) The former is responsible for the generation of interstrand DNA crosslinks, and the latter results in additional DNA lesions,26 and has been implicated in the urotoxicity, principally hemorrhagic cystitis, witnessed with the use of 12.

CI Cl Cl

Cl 14 Cl 15 16 12 Cl 13 Cl Figure 5 Synthesis of cyclophosphamide (12), and the structure of ifosfamide (13).

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