Chemically stable, lipophilic prodrugs of phosphoramide mustard as potential anticancer agents

Benzyl phosphoramide mustard (3), 2,4-difluorobenzyl phosphoramide mustard (4), and methyl phosphoramide mustard (5) were examined as lipophilic, chemically stable prodrugs of phosphoramide mustard (2). These phosphorodiamidic esters are designed to undergo biotransformation by hepatic microsomal enzymes to produce 2. The rate of formation of alkylating species, viz., 2, from these prodrugs and their in vitro cytotoxicity toward mouse embryo Balb/c 3T3 cells were comparable to or better than that of cyclophosphamide (1). Preliminary antitumor screening against L1210 leukemia in mice, however, suggests that these prodrugs are devoid of any significant antitumor activity in vivo.     

Design, synthesis, and biological evaluation of cyclic and acyclic nitrobenzylphosphoramide mustards for E. coli nitroreductase activation

In efforts to obtain anticancer prodrugs for antibody-directed or gene-directed enzyme prodrug therapy using E. coli nitroreductase, a series of nitrobenzylphosphoramide mustards were designed and synthesized incorporating a strategically placed nitro group in a position para to the benzylic carbon for reductive activation. All analogues were good substrates of E. coli nitroreductase with half-lives between 2.9 and 11.9 min at pH 7.0 and 37 degrees C. Isomers of the 4-nitrophenylcyclophosphamide analogues 3 and 5 with a benzylic oxygen para to the nitro group showed potent selective cytotoxicity in nitroreductase (NTR) expressing cells, while analogues 4 and 6 with a benzylic nitrogen para to the nitro group showed little selective cytotoxicity despite their good substrate activity. These results suggest that good substrate activity and the benzylic oxygen are both required for reductive activation of 4-nitrophenylcyclophosphamide analogues by E. coli nitroreductase. Isomers of analogue 3 showed 23,000-29,000x selective cytotoxicity toward NTR-expressing V79 cells with an IC(50) as low as 27 nM. They are about as active as and 3-4x more selective than 5-aziridinyl-2,4-dinitrobenzamide (CB1954). The acyclic 4-nitrobenzylphosphoramide mustard ((+/-)-7) was found to be the most active and most selective compound for activation by NTR with 170,000x selective cytotoxicity toward NTR-expressing V79 cells and an IC(50) of 0.4 nM. Compound (+/-)-7also exhibited good bystander effect compared to 5-aziridinyl-2,4-dinitrobenzamide. The low IC(50), high selectivity, and good bystander effects of nitrobenzylphosphoramide mustards in NTR-expressing cells suggest that they could be used in combination with E. coli nitroreductase in enzyme prodrug therapy.     

Kinetics of phosphoramide mustard hydrolysis in aqueous solution

Hydrolysis of phosphoramide mustard was investigated using HPLC, 31P NMR, and GC-MS with specific deuterium labels. The hydrolysis of phosphoramide mustard in sodium phosphate buffers was found to follow apparent first-order kinetics. The rate of hydrolysis was temperature and pH dependent, being slower under acidic conditions. The hydrolysis was not catalyzed by hydroxyl ion, and its pH dependence appeared to be the result of a change in the mechanism of hydrolysis at different pH values. At a pH value approximately above the pKa of the phosphoramide mustard nitrogen, the major hydrolytic pathway of phosphoramide mustard was via the formation of the aziridinium ion, followed by nucleophilic attack. At pH values below its pKa, cleavage of the P-N bond predominated. At pH 7.4, the formation of an aziridinium ion was followed by a rapid hydrolysis to yield the monohydroxy and, subsequently, the dihydroxy products. The hydrolysis at this pH was adequately described by consecutive first-order kinetics. Seven species in the hydrolytic mixture have been identified as intact phosphoramide mustard, N-(2-chloroethyl)-N-(2-hydroxyethyl)phosphorodiamidic acid, N,N-bis-(2-hydroxyethyl)phosphorodiamidic acid, phosphoramidic acid, phosphoric acid, N,N-bis-(2-chloroethyl)amine, and N-(2-chloroethyl)-N-(2-hydroxyethyl)amine by GC-MS with the aid of deuterium labels. Phosphoramide mustard was found to be stabilized by chloride ion. The stabilization was linearly related to the chloride ion concentration, and the mechanism was found to be via the formation of phosphoramide mustard from the aziridinium and chloride ions. Phosphoramide mustard was significantly more stable in human plasma and in 5% human serum albumin as compared to aqueous buffers, an observation that may be important in vivo.     

Correlations of alkylating activity and mutagenicity in bacteria of cytostatic drugs

Alkylating activity of cytostatic drugs was studied in relation to their mutagenicity and toxicity in E. coli WP2 uvrA. Four classes of directly acting cytostatic drugs were studied: nitrogen mustards (nitrogen mustard, melphalan, chlorambucil and phosphoramide mustard, a metabolite of cyclophosphamide), ethyleneimine derivatives (Thio-TEPA, TEPA and triethylenemelamine), busulfan, and halogenated nitrosoureas. The reference compounds included methyl methanesulfonate, ethyleneimine and methylnitrosourea. Guanosine alkylation was determined by fluorometry. The rate of guanosine and nitrobenzylpyridine alkylation agreed well. Nitrogen mustard derivatives and triethylenemelamine were the most potent alkylating agents among the cytostatic drugs; nitrogen mustard was 5 to 10 times more active than methyl methanesulfonate. Ethyleneimine derivatives, busulfan and the nitrosoureas were relatively weak alkylating agents. Nitrogen mustard and triethylenemelamine were the most potent mutagens to bacteria; they were also among the most toxic drugs studied.     

Covalent sequestration of phosphoramide mustard by metallothionein--an in vitro study.

Acquired drug resistance is one of the most important problems in cancer chemotherapy. One of the proposed mechanisms for these phenomena is the sequestration of alkylating agents by metallothionein in vivo. This research shows that metallothionein can covalently sequester phosphoramide mustard, the active form of cyclophosphamide in vitro. On-line electrospray mass spectrometry reveals that it is phosphoramide, not nornitrogen mustard that alkylates metallothionein, although the metallothionein/nornitrogen mustard adduct was isolated as the major adduct. Tandem mass spectrometric experiments were performed on an isolated drug-modified tryptic peptide. The alkylation occurred predominantly at Cys48 of metallothionein. These results provide further evidence that overexpression of metallothionein can detoxify the active form of the drugs.     

Synthesis and antiviral evaluation of alkoxyalkyl esters of acyclic purine and pyrimidine nucleoside phosphonates against HIV-1 in vitro

Alkoxyalkyl esters of cidofovir, an acyclic nucleoside phosphonate, have been shown to have antiviral activities several orders of magnitude greater than unmodified cidofovir against cytomegalovirus, herpes simplex virus, vaccinia, cowpox, ectromelia and adenoviruses in vitro. Hexadecyloxypropyl-cidofovir is orally bioavailable and active in lethal animal models of vaccinia, cowpox, ectromelia and cytomegalovirus. To see if this strategy is also applicable to other acyclic nucleoside phosphonates, we have converted several phosophonomethoxyethyl purines and pyrimidines to their hexadecyloxypropyl, octadecyloxyethyl and oleyloxyethyl esters and compared their activity against HIV-1 with the activity of the respective unmodified acyclic nucleoside phosphonates. The hexadecyloxypropyl esters of phosphonomethoxyethyl-adenine, phosphonomethoxyethyl-2,6-diaminopurine and phosphonomethoxyethyl-N(6)-cyclopropyl-diaminopurine were 3-5 orders of magnitude more active against HIV-1 in vitro than the parent nucleotides. The EC(50) values for these compounds were in the 10-20 pM range with selective indexes of 1,250 to >4,000. The acyclic pyrimidine phosphonates were generally inactive against HIV-1 in vitro. Phosphonomethoxyethyl-cytosine and phosphonomethoxyethyl-5-fluorocytosine were inactive against HIV-1. Surprisingly, hexadecyloxypropyl-phosphonomethoxyethyl-5-fluorocytosine was active against HIV-1 with a submicromolar EC(50) and a selective index of 174. Esterification of acyclic nucleoside phosphonates with alkoxyalkyl moieties may represent a general approach for increasing antiviral activity and selectivity of this class of antivirals.     

Half-life of oxazaphosphorines in biological fluids

Plasma AUC and half-life values for cyclophosphamide were determined in rats manipulated to hydroxylate cyclophosphamide at different rates; plasma AUC and apparent half-life values for two pharmacologically important metabolites of cyclophosphamide, viz. 4-hydroxycyclophosphamide/aldophosphamide and phosphoramide mustard, were also determined in these animals. Apparent plasma half-life values for 4-hydroxycyclophosphamide/aldophosphamide and phosphoramide mustard increased with an increase in plasma half-life values for cyclophosphamide. AUC values for cyclophosphamide increased approximately linearly with an increase in its plasma half-life but AUC values for 4-hydroxycyclophosphamide/aldophosphamide and phosphoramide mustard remained approximately constant with an increase in their respective apparent plasma half-life values. Given that the cytotoxic effects of cyclophosphamide are directly proportional to AUC values for 4-hydroxycyclophosphamide/aldophosphamide and/or phosphoramide mustard, we conclude that changes in the rate of cyclophosphamide hydroxylation will not alter the systemic toxic and therapeutic responses to a given dose of cyclophosphamide. Actual half-life values for 4-hydroxycyclophosphamide/aldophosphamide and phosphoramide mustard after the iv infusion of these agents were also determined. A comparison of the actual plasma half-life values for cyclophosphamide (29 min), 4-hydroxycyclophosphamide/aldophosphamide (14 min), and phosphoramide mustard (14 min) with the apparent plasma half-life values obtained for 4-hydroxycyclophosphamide/aldophosphamide (34 min) and phosphoramide mustard (55 min) following cyclophosphamide administration suggests that the major determinant with regard to the apparent plasma half-life of 4-hydroxycyclophosphamide/aldophosphamide is its rate of formation whereas in the case of phosphoramide mustard, an additional determinant, perhaps efflux from the cell, is operative.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorus-nitrogen compounds. 24. Phosphoramide mustard carrier derivatives

Diethylstilbestrol, psoralen, and propranolol were used as potential carrier molecules for selective concentrations of a nitrogen mustard moiety in breast, skin, and lung tissues, respectively. The propranolol derivative gave two racemic mixtures, which were tested to ascertain any differences in anticancer activity. The insertion of a P = O group between the carrier and oncolytic portions offsets the excess lipophilic contribution of the latter and possibly provides for latentiation of alkylating activity. Murine tumor testing of the phosphoramide mustard derivatives and two intermediates indicated that two compounds possessed marginal activity against mammary carcinoma and lymphocytic leukemia.     

The pharmacokinetics of cyclophosphamide, phosphoramide mustard and nor-nitrogen mustard studied by gas chromatography in patients receiving cyclophosphamide therapy

1 Simple, accurate and specific gas-chromatographic methods for the estimation of derivatized phosphoramide and non-nitrogen mustards utilizing alkali-flame ionization detection are described.

2 The pharmacokinetics in plasma of cyclophosphamide, phosphoramide mustard, nor-nitrogen mustard and nitrobenzyl pyridine alkylating activity were investigated following administration of cyclophosphamide by intravenous and oral routes to patients with malignant disease

3 The mean T_½ for cyclophosphamide was 8.88 h (s.d. 1.25 h) and the apparent volume of distribution (V_dβ) was 0.74 l kg^-1 (s.d. 0.16 l kg^-1).

4 The decline in plasma concentration of phosphoramide mustard was biphasic, the longer T_½ being 8.68 h (s.d. 2.50 h). This was not significantly different from that of cyclophosphamide. This could indicate that the true biological T_½ for phosphoramide mustard is identical with or shorter than that of cyclophosphamide.

5 The plasma concentrations of phosphoramide mustard following cyclophosphamide doses of known therapeutic efficacy are probably insufficient to produce important cytotoxic effects. This suggests that if phosphoramide mustard is the major alkylating metabolite derived from cyclophosphamide, it is transported in the blood in precursor form.

6 The mean plasma T_½ of nor-nitrogen mustard was 3.31 h (s.d. 1.60 h) which was significantly different from that of cyclophosphamide.

7 The mean plasma T_½ of the nitrobenzylpyridine alkylating activity was 9.81 h (s.d. 4.18 h) and did not significantly differ from that of cyclophosphamide. Although the area under the plasma alkylating activity concentration, time curve is related to the T_½ of cyclophosphamide, the alkylating activity does not reflect the concentrations of the two plasma metabolites measured.

     

Alkylation of guanosine by phosphoramide mustard, chloromethine hydrochloride and chlorambucil

Guanosine was reacted in vitro with phosphoramide mustard, chloromethine hydrochloride, and chlorambucil. The products were isolated by HPLC and characterized by UV and fluorescence spectroscopy, and C-8 tritium exchange. The primary products were 7-alkylguanosines according to such evidence. Phosphoramide mustard had 1/10 of the apparent alkylation activity of two other mustards. The primary 7-alkylguanosines were unstable at pH 7.4 and 37 degrees; t1/2 were 3 min. for chloromethine hydrochloride, 2.7 hrs for chlorambucil and 3.0 hrs for phosphoramide mustard. Both dechlorination at the unbound arm of the mustard and imidazole ring opening og guanosine appeared to account for such instability.     

Development of novel quinone phosphorodiamidate prodrugs targeted to DT-diaphorase

A series of naphthoquinone and benzimidazolequinone phosphorodiamidates has been synthesized and studied as potential cytotoxic prodrugs activated by DT-diaphorase. Reduction of the quinone moiety in the target compounds was expected to provide a pathway for expulsion of the phosphoramide mustard alkylating agent. All of the compounds synthesized were excellent substrates for purified human DT-diaphorase (k(cat)/K(m) = 3 x 10(7) - 3 x 10(8) M(-1) s(-1)). The naphthoquinones were toxic to both HT-29 and BE human colon cancer cell lines in a clonogenic assay; however, cytotoxicity did not correlate with DT-diaphorase activity in these cell lines. The benzimidazolequinone analogues were 1-2 orders of magnitude less cytotoxic than the naphthoquinone analogues. Chemical reduction of the naphthoquinone led to rapid expulsion of the phosphorodiamidate anion; in contrast, the benzimidazole reduction product was stable. Michael addition of glutathione and other sulfur nucleophiles provides an alternate mechanism for activation of the naphthoquinone phosphorodiamidates, and this mechanism may contribute to the cytotoxicity of these compounds.     

1,2-benzisoxazole phosphorodiamidates as novel anticancer prodrugs requiring bioreductive activation

Several 1,2-benzisoxazole phosphorodiamidates have been designed as prodrugs of phosphoramide mustard requiring bioreductive activation. Enzymatic reduction of 1,2-benziosoxazole moiety is expected to result in the formation of imine intermediate due to the cleavage of the N-O bond. The imine should then be spontaneously hydrolyzed to a ketone metabolite, thereby facilitating base-catalyzed beta-elimination of cytotoxic phosphoramide mustard. As expected, the proposed prodrugs 4, 9, and 12 were at least 3-5-fold more potent cytotoxins than control compounds 5 and 15, which lack in the phosphoramide mustard group. Upon incubation with phenobarb-induced rat liver S-9 fraction, compounds 4, 9, and 12 underwent extensive NADPH-dependent metabolism with concomitant generation of alkylating activity under both hypoxic and oxic conditions. Corresponding ketone metabolites were detected for 9 and 15. NADPH-dependent bioreduction of 15 to its ketone metabolite 16 was located in the microsomal fraction and inhibited by SKF-525A and pCMBA. Compared with phenobarb-induced rat liver microsomal fraction, incubation of 15 with rat or human p450 reductase microsomes showed moderate generation of 16. Microsomal cytochrome p450 and/or p450 reductase appear to be involved in the reductive metabolism of 1,2-benzisoxazole moiety under hypoxic as well as oxic conditions.     

Sensitivity of murine B- and T-lymphocytes to oxazaphosphorine and non-oxazaphosphorine nitrogen mustards

The relative sensitivities of murine B- and T-lymphocytes to the oxazaphosphorine nitrogen mustards, cyclophosphamide and ASTA Z 7557, and to the non-oxazaphosphorine nitrogen mustards, melphalan and chlorambucil, in vivo, were determined. B- and T-lymphocytes were defined by selective mitogen-induced proliferation. Lipopolysaccharide (LPS)-induced B-lymphocytes were approximately twice as sensitive to the cytotoxic effects of cyclophosphamide and ASTA Z 7557 as were phytohemagglutinin (PHA)- and concanavalin A (Con A)-induced T-lymphocytes. LPS-induced B-lymphocytes and PHA-induced T-lymphocytes were approximately equisensitive to the cytotoxic action of melphalan and chlorambucil, but the former were somewhat more sensitive to these agents than were Con A-induced T-lymphocytes. The relative sensitivities of murine B- and T-lymphocytes to ASTA Z 7557 and the non-oxazaphosphorine metabolite of cyclophosphamide, phosphoramide mustard, ex vivo, were also determined. LPS-induced B-lymphocytes were approximately twice as sensitive to the cytotoxic action of ASTA Z 7557 as were PHA- and Con A-induced T-lymphocytes. The three mitogen-induced lymphocyte populations were approximately equisensitive to the cytotoxic action of phosphoramide mustard. These observations suggest that the differential effect of cyclophosphamide on murine B- and T-lymphocytes is uniquely exhibited by oxazaphosphorine nitrogen mustards. Furthermore, the results suggest that 4-hydroxycyclophosphamide is the cyclophosphamide metabolite that mediates the differential immunotoxic effect of the parent compound.     

Aldehyde dehydrogenase activity as the basis for the relative insensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorines

The ex vivo sensitivity of murine pluripotent hematopoietic stem cells (CFU-S) and myeloid progenitor cells (CFU-GM) to 4-hydroperoxycyclophosphamide, ASTA Z 7557, phosphoramide mustard, acrolein, melphalan, and cis-platinum was determined in the absence and presence of known (disulfiram, diethyldithiocarbamate, cyanamide) or suspected [ethylphenyl(2-formylethyl)phosphinate] inhibitors of aldehyde dehydrogenase activity. As compared to CFU-GM, CFU-S were less sensitive to the oxazaphosphorine agents, 4-hydroperoxycyclophosphamide and ASTA Z 7557. The two cell populations were approximately equisensitive to acrolein as well as to the non-oxazaphosphorine cross-linking agents, phosphoramide mustard, melphalan and cis-platinum. All four inhibitors of aldehyde dehydrogenase activity potentiated the cytotoxic action of the oxazaphosphorines toward CFU-S; they did not potentiate the cytotoxic action of acrolein or the non-oxazaphosphorines toward these cells. The inhibitors did not potentiate the cytotoxic action of the oxazaphosphorines, non-oxazaphosphorines, or acrolein toward CFU-GM. Pyridoxal, a substrate for aldehyde oxidase, did not potentiate the cytotoxic action of oxazaphosphorines toward CFU-S. Cellular NAD-linked aldehyde dehydrogenases are known to catalyze the oxidation of the major transport form of cyclophosphamide, 4-hydroxycyclophosphamide/aldophosphamide, to an inactive metabolite, carboxyphosphamide. Our observations suggest that (1) aldehyde dehydrogenase activity is an important determinant of the sensitivity of a cell population to the oxazaphosphorines, (2) CFU-GM lack the relevant aldehyde dehydrogenase activity, and (3) the phenotypic basis for the relative insensitivity of CFU-S to oxazaphosphorines is the aldehyde dehydrogenase activity contained by these cells.     

31P nuclear magnetic resonance spectroscopic observation of the intracellular transformations of oncostatic cyclophosphamide metabolites

31P NMR spectroscopy was used to directly monitor, for the first time, the intracellular chemistry of the ultimate active metabolite of cyclophosphamide, namely, phosphoramide mustard. These NMR studies utilized a human histiocytic lymphoma cell line (U937), embedded in agarose gel threads, and perfused with medium containing synthetically derived metabolites (4-hydroxycyclophosphamide, aldophosphamide, and phosphoramide mustard). Metabolites 2 or 3 or both readily crossed the cell membrane; in contrast, the membrane was relatively impermeable to 4. Intracellular concentrations of 4 could, therefore, be attributed primarily to the intracellular fragmentation of 3. Signals suggestive of either carboxyphosphamide or 4-ketophosphamide were not detected. Spectral data were used to calculate a rate constant of (5.4 +/- 0.3) X 10(-3) min-1 for the intracellular disappearance of 4 at 23 degrees C. The intracellular pH was determined to be 7.1 from the chemical shift of the internal inorganic phosphate signal.     

Metabolism-based anticancer drug design

Many conventional anticancer drugs display relatively poor selectivity for neoplastic cells, in particular for solid tumors. Furthermore, expression or development of drug resistance, increased glutathione transferases as well as enhanced DNA repair decrease the efficacy of these drugs. Research efforts continue to overcome these problems by understanding these mechanisms and by developing more effective anticancer drugs. Cyclophosphamide is one of the most widely used alkylating anticancer agents. Because of its unique activation mechanism, numerous bioreversible prodrugs of phosphoramide mustard, the active species of cyclophosphamide, have been investigated in an attempt to improve the therapeutic index. Solid tumors are particularly resistant to radiation and chemotherapy. There has been considerable interest in designing drugs selective for hypoxic environments prevalent in solid tumors. Much of the work had been centered on nitroheterocyclics that utilize nitroreductase enzyme systems for their activation. In this article, recent developments of anticancer prodrug design are described with a particular emphasis on exploitation of selective metabolic processes for their activation.     

31P NMR and chloride ion kinetics of alkylating monoester phosphoramidates

31P NMR spectroscopy was used to study the solvolysis kinetics of a novel series of alkylating monoester phosphoramidates (4a-d) under model physiologic conditions. Halide ion kinetics were used to determine the rate of aziridinium ion formation. The solvolysis rates showed the expected dependence upon substitution at the reactive nitrogen; comparison of 4a with phosphoramide mustard (1a) indicated that replacement of the amino group by alkoxy decreased the solvolysis rate by approximately 10-fold. The rate of conversion of starting compound (4a-d) to solvolysis product was essentially equal to the rate of halide ion release, suggesting that the aziridinium ion is a short-lived intermediate. 1H NMR and 31P NMR kinetics experiments performed in the absence and presence of trapping agent (dimethyldithiocarbamate) confirmed that the aziridinium ion was too short-lived to be observed via NMR. These compounds were also tested for cytotoxicity against L1210 leukemia and B16 melanoma cells in vitro; the monoalkylators 4c and 4d showed no activity, 4a was weakly cytotoxic, and 4b was comparable in activity to phosphoramide mustard.     

Possible role of acrolein in 4-hydroperoxycyclophosphamide-induced cell damage in vitro

Unlike "conventional" oxazaphosphorines such as cyclophosphamide (CP) and ifosfamide, a relatively new drug termed 4-hydroperoxy-CP (4-HC) degrades spontaneously in water yielding phosphoramide mustard considered to be the activated cytotoxic metabolite. During this degradation a toxic, volatile factor termed acrolein is also formed. In order to examine the possible role of this compound in 4-HC-induced inhibition of tumor cell growth in vitro, 8 different established human tumor cell lines were cultured in the presence of 4-HC or equimolar concentrations of acrolein. It was observed that the cell lines differed widely with respect to sensitivity to these compounds. However, each individual cell line exhibited virtually identical sensitivities to both 4-HC and acrolein. The observation that 2-mercaptoethansulfonate (mesna), which is highly reactive with acrolein but not with phosphoramide mustard, could markedly reduce the cytotoxic activity of 4-HC indicates that acrolein may play an important role in 4-HC induced cell damage in vitro.     

Effects of aldehyde dehydrogenase inhibitors on the ex vivo sensitivity of murine late spleen colony-forming cells (day-12 CFU-S) and hematopoietic repopulating cells to mafosfamide (ASTA Z 7557)

The effects of inhibitors of aldehyde dehydrogenase activity on the sensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorine anticancer agents, e.g. mafosfamide, were examined using two different assay procedures. In the first part of the investigation, the ex vivo sensitivity of murine day-12 spleen colony-forming cells (CFU-S) to mafosfamide was determined in the absence and presence of known inhibitors of aldehyde dehydrogenase activity, viz. diethyldithiocarbamate and cyanamide. These results were compared to those generated for day-8 CFU-S. Day-12 CFU-S were less sensitive to mafosfamide, and to phosphoramide mustard, although the difference in sensitivity to the latter was less marked. Diethyldithiocarbamate and cyanamide each potentiated the cytotoxic action of mafosfamide toward both day-12 and day-8 CFU-S; they did not potentiate the cytotoxic action of phosphoramide mustard toward these cells. Since cellular aldehyde dehydrogenases are known to catalyze the oxidation of 4-hydroxycyclophosphamide/aldophosphamide, the major transport form of mafosfamide, to the relatively nontoxic acid, carboxyphosphamide, the results suggest that intracellular aldehyde dehydrogenase activity is a determinant of the sensitivity of day-12 CFU-S, as well as of day-8 CFU-S, to mafosfamide and other oxazaphosphorines, e.g. cyclophosphamide. In the second part of this investigation, a murine syngeneic bone marrow transplantation model was used to determine the ex vivo sensitivity of murine hematopoietic repopulating cells to mafosfamide in the absence and presence of diethyldithiocarbamate. Specifically, the ability of treated marrow grafts to repopulate the hematopoietic system, and thereby save recipients from the otherwise lethal effect of total body irradiation, was determined. Diethyldithiocarbamate potentiated the cytotoxic action of mafosfamide, but not that of phosphoramide mustard, toward hematopoietic repopulating cells. These observations support our previous contention that aldehyde dehydrogenase activity is an operative determinant with regard to the sensitivity of murine pluripotent hematopoietic stem cells to oxazaphosphorines.     

Design,synthesis, and biological evaluation of indolequinone phosphoramidate prodrugs targeted to DT-diaphorase

A series of 2- and 3-substituted indolequinone phosphoramidate prodrugs targeted to DT-diaphorase (DTD) have been synthesized and evaluated. These compounds are designed to undergo activation via quinone reduction by DTD followed by expulsion of the phosphoramide mustard substituent from the hydroquinone. Chemical reduction of the phosphoramidate prodrugs led to rapid expulsion of the corresponding phosphoramidate anions in both series of compounds. Compounds substituted at the 2-position are excellent substrates for human DTD (k(cat)/K(M) = (2-5) x 10(6) M(-1) s(-1)); however, compounds substituted at the 3-position are potent inhibitors of the target enzyme. Both series of compounds are toxic in HT-29 and BE human colon cancer cell lines in a clonogenic assay. There was a correlation found between cytotoxicity and DTD activity for the 2-series of phosphoramidates; however, there was no correlation between cytotoxicity and DTD activity in the 3-series of compounds. This finding suggests the presence of an alternative mechanism for the activation of these compounds.