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The DNA alkylating prodrug cyclophosphamide (CPA), alone or in combination with other agents, is one of the most commonly used anti-cancer agents. As a prodrug, CPA is activated by cytochrome P450 2B6 (CYP2B6), which is transcriptionally regulated by the human constitutive androstane receptor (hCAR). Therefore, hCAR agonists represent novel sensitizers for CPA-based therapies. Among known hCAR agonists, compound 6-(4-chlorophenyl)imidazo-[2,1-b]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime (CITCO) is the most potent and broadly utilized in biological studies. Through structural modification of CITCO, we have developed a novel compound DL5016 (32), which has an EC 50 value of 0.66 μM and E MAX value of 4.9 when activating hCAR. DL5016 robustly induced the expression of hCAR target gene CYP2B6, at both the mRNA and protein levels, and caused translocation of hCAR from the cytoplasm to the nucleus in human primary hepatocytes. The effects of DL5016 were highlighted by dramatically enhancing the efficacy of CPA-based cytotoxicity to non-Hodgkin lymphoma cells., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

The prodrugs cyclophosphamide (CP) and ifosfamide (IF) each metabolize to an active alkylating agent through a cytochrome P450-mediated oxidation at the C-4 position. Competing with this activation pathway are enzymatic oxidations at the exocyclic α and α' carbons, which result in dechloroethylation of CP and IF. The incidence of oxidation at one position relative to another is believed to be at least one factor underlying the high degree of interpatient variability in both CP and IF pharmacokinetics. As standards for the mass spectrometry quantification of dechloroethylation, the following were synthesized: (1) [4,4,5,5-(2) H4 ]-2-dechloroethylcyclophosphamide (equivalent to [4,4,5,5-(2) H4 ]-3-dechloroethylifosfamide); (2) [α,α,4,4,5,5-(2) H6 ]-2-dechloroethylcyclophosphamide (equivalent to [α,α,4,4,5,5-(2) H6 ]-3-dechloroethylifosfamide); and (3) [α,α,4,4,5,5-(2) H6 ]-2-dechloroethylifosfamide. The common precursor to all of the target compounds was [2,2,3,3-(2) H4 ]-3-aminopropanol. A one-pot reaction of this compound with POCl3 and unlabeled or labeled 2-chloroethylamine hydrochloride gave the d4 and d6 labeled 2-dechloroethylcyclophosphamides. The construction of the 2-dechloroethylifosfamide from the aminopropanol required five discreet steps. Optimization of the synthetic pathways and stability studies are discussed., (Copyright © 2013 John Wiley & Sons, Ltd.)

In an effort to develop proteolytically activated prodrugs of phosphoramide mustard by prostate-specific antigen (PSA), a series of tetrapeptide (Cbz-Ser-Ser-Phe-Tyr)-conjugated 4-aminocyclophosphamide (4-NH(2)-CPA) isomers were synthesized and evaluated as substrates of PSA. The cleavage of the conjugates by PSA were found to be stereoselective as only the two isomers with 4R-configuration were efficiently cleaved by PSA. The cis-(2R,4R)-isomer was the best substrate of PSA with a half-life of 12min. LC/MS analysis of the incubation solution of this isomer with PSA suggests that 4-NH(2)-CPA is released upon proteolysis and quickly degrades to cytotoxic phosphoramide mustard. These results clarified the stereochemical requirements of PSA on the peptide conjugates of 4-NH(2)-CPA and demonstrated the potential of these conjugates as potential PSA-activated prodrugs targeting prostate cancer.

A novel class of cyclophosphamide spiropiperaziniums was synthesized and evaluated for their in vivo anti-cancer activities against S180 and H22. Most of them exhibited definite activities. Especially, compounds 8b and 8k showed good anti-cancer activities, meanwhile, 8k also showed much lower toxicity than CP. Several interesting structure-activity relationships were revealed.

Cyclic and acyclic nitroaryl phosphoramide mustard analogues were activated by E. coli nitroreductase, an enzyme explored in GDEPT. The more active acyclic 4-nitrobenzyl phosphoramide mustard (7) showed 167 500x selective cytotoxicity toward nitroreductase-expressing V79 cells with an IC(50) as low as 0.4 nM. This is about 100x more active and 27x more selective than CB1954 (1). The superior activity was attributed to its better substrate activity (k(cat)/K(m) 19x better than 1) and/or excellent cytotoxicity of phosphoramide mustard released.

The electrochemical oxidation of anticancer drugs ifosfamide and cyclophosphamide produced in high yield methoxylated analogues of the key hydroxy-metabolites of these oxazaphosphorine prodrugs. The cytotoxicity of these compounds was evaluated, and found to be as high as the hydroxy-metabolite.

Two prototype N-methyl-4-thio-substituted cyclophosphamide (CP) derivatives (5 and 6), prodrugs of 4-hydroxycyclophosphamide (4-HO-CP), were designed to undergo oxidative N-demethylation to release the active alkylating agent. These prodrugs were chemically stable until oxidatively N-demethylated in the presence of hepatic microsomal P-450 enzymes. While the metabolism of 5 was enhanced in the presence of phenobarbital-induced microsomes, 6 was unaffected. Compound 6 was more active than 5 against L1210 leukemia cells grown in mice and exhibited statistically significant activity against the small cell lung cancer panel in the National Cancer Institute anticancer drug screen. Compound 5, like CP (1), was inactive in this screen. Thus, placement of a dithioester at the 4-position of N-methyl-HO-CP as in 6 markedly changes its spectrum of activity and has resulted in a new type of CP-based prodrug with antitumor activity against small cell lung cancer as well as leukemia cells in vitro as shown by their ability to inhibit tumor cell growth at concentrations as low as 10(-6) M.

The synthesis of 4-ureidooxycyclophosphamide, a potential cytostatic, whose cis/trans-isomers can be stereoselectively obtained from 4-hydroxycyclophosphamide and hydroxyurea in DMF in DMF or water, is described. These compounds are remarkably stable at room temperature. For the first time, the two epimers from a derivative of 4-hydroxycyclophosphamide can be NMR-spectroscopically compared with each other.

Cyclization of racemic 3-amino-3-phenyl-1-propranol with bis(2-chloroethyl)phosphoramidic dichloride gave a diastereomeric mixture of 4-phenylcyclophosphamide (3), which was chromatographically separated into the faster and slower eluting components. A combination of 1H/31PNMR and IR spectral data indicated that the faster and slower racemates correspond to cis-3 (mp 129-130 degrees C) and trans-3 (mp 112-114.5 degrees C), respectively. The molecular structure of the former compound was determined by X-ray crystallography and thereby unambiguously established the cis relationship between equatorially disposed phenyl and P = O substituents in a chair conformation. These results confirm the stereochemical assignments for cis- and trans-3 which have been independently deduced by Y. E. Shih, J. S. Wang, and C. T. Chen [Heterocycles, 9, 1277 (1978)]. Anticancer screening tests against L1210 lymphoid leukemia in mice have revealed that, while both diastereomers of 3 afford toxic metabolites, trans-3 led to therapeutic activity and cis-3 did not. The relevance of these findings to results reported for 4-methylcyclophosphamide and cyclophosphamide is briefly discussed.

3-Fluoro-, 3-chloro-, and 3-bromocyclophosphamide were prepared from the reaction of trifluoromethylhypofluorite, sodium hypochlorite, and bromine with the anticancer drug cyclophosphamide. Treatment of cis- and trans-4-phenylcyclophosphamide and 5,6-benzocyclophosphamide with sodium hypochlorite afforded cis- and trans-3-chloro-4-phenylcyclophosphamide and 3-chloro-5,6-benzocyclophosphamide, respectively. 31P-NMR spectroscopy was used to study the reactivity of these compounds: the fluoro derivative was reduced to cyclophosphamide on incubation with mouse liver slices, and the reactivity order for sulfhydryl-induced reduction of the 3-halocyclophosphamides was Br approximately equal to Cl much greater than F. Compared with the therapeutic efficacy of cyclophosphamide against L-1210 and P-388 cancers in mice, 3-fluoro- and 3-chlorocyclophosphamide were less active, although the fluoro derivative was more efficacious than the 3-chloro compound. The individual R and S enantiomers of 3-chlorocyclophosphamide, prepared from (S)- and (R)-cyclophosphamide, respectively, showed no significant difference in therapeutic activity in the P-388 test system.

Synthesis of 2-[bis(2-chloroethyl)amino]-3,4-dihydro-2H-1,3,2-benzoxazaphosphorin 2-oxide (2), which is a benzo annulated analog of cyclophosphamide [2-bis(2-chloroethyl)aminotetrahydro-2H-1,3,2-oxazaphosphorin 2-oxide (1)], was carried out in order to test for possible increased antitumor activity relative to 1 due to the presence of an oxidatively reactive C-4 benzylic site in 2. A structural isomer of 2, 2-[bis(2-chloroethyl)amino]-1,4-dihydro-2H-3,1,2-benzoxazaphosphorin 2-oxide (3), and cognate systems 2-[bis(2-chloroethyl)amino]-1,2,3,4-tetrahydro-1,3,2-benzodiazaphosphorin 2-oxide (4) and 2-[bis(2-chloroethyl)amino]-4H-1,3,2-benzodioxaphosphorin 2-oxide (5) were also prepared for comparative purposes. In vivo antitumor evaluation in mice against L1210 lymphoid leukemia indicated no significant activity for compound 2. Compounds 3 and 4 were likewise found to be inactive and only marginal activity was exhibited by 5.

Cyclophosphamide (CP), one of the most commonly used cytostatic drugs, is known to be a human carcinogen. In this study, CP represents a model compound for the identification of potential exposure situations in the various phases of its manufacture and hospital use. Ambient air samples were taken in the various phases and analyzed for CP. A low detection limit (0.05 micrograms/m3 for a 1 m3 sample volume) was obtained for CP using the mass spectrometry method (MS) developed for this study. The detection limit was 1 microgram/m3 with the high performance liquid chromatography (HPLC) method. The correlation between the two methods was good. The highest potential exposure situations were found to occur during specific operations at the end of the process. During these operations the airborne CP concentrations may rise as high as several hundred micrograms/m3, and the workers must use supplied-air respirators, gloves and protective clothing. For the measurements in hospital use, air samples were taken from flow hoods. During normal working practices, no measurable amounts of CP could be observed. A filter from a flow hood, however, contained CP in measurable quantities, showing that occasional spilling may occur.

Crystalline 4-(SR)-sulfidocyclophosphamides, sulfido derivatives of activated cyclophosphamide (4-hydroxycyclophosphamide), were synthesized by ozonation of cyclophosphamide and reaction of the intermediate 4-hydroxycyclophosphamide with various thiols (HSR). The products were characterized by elemental analysis, 1H NMR and IR spectroscopy, and mass spectrometry. 1H NMR and polarimetric analysis demonstrated that they consist of racemic cis-isomers that are stable in the crystalline state at room temperature. In aqueous solution these derivatives are hydrolyzed to 4-hydroxycyclophosphamide and the corresponding thiol, with half-lives ranging between 4 and 17 min at 37 degrees C and pH 7. The cytotoxicity of 4-(S-ethyl)- and 4-(S-ethanol)-sulfidocyclophosphamide against Yoshida sarcoma ascites cells and the toxicity in rats were found to be practically identical with those of activated cyclophosphamide. A preliminary evaluation of the curative effect after a single IV injection of 4-(S-ethane)- and 4-(S-ethanol)-sulfidocyclophosphamide in rats bearing Yoshida ascites sarcoma or of 4-(S-ethanol)-sulfidocyclophosphamide in nu/nu mice bearing human breast carcinoma xenografts suggested that these sulfido derivatives possess the same oncostatic efficacy as activated cyclophosphamide itself.

A synthetic study was made on the active metabolite of cyclophosphamide. Ozonolysis of O-(3 butenyl)-N,N-bis(2-chloroethyl)phosphorodiamidate, prepared by reaction of POC13 with 3-buten-1-ol followed by treatment with N,N-bis(2-chloroethyl)amine (nor mustard) and NH3, afforded 2-[bis(2-chloroethyl)amino]-4-hydroperoxytetrahydro-2H-1, 3,2-oxazaphosphorine 2-oxide (4-hydroperoxycyclophosphamide). Deoxygenation of 4-hydroperoxycyclophosphamide by triphenylphosphine yielded 4-hydroxycyclophosphamide in a pure crystalline state. These products exhibited high cytostatic activity in both in vitro and in vivo experiments. The results give confirmatory evidence for the hypothesis that C4-hydroxylation on the 1,3,2-oxazaphosphorinane ring of cyclophosphamide is necessary for its activation.

Hydrogenolysis of 3-(benzyloxy)cyclophosphamide (10) using Pd/C catalyst and ethyl acetate as solvent leads to the formation of 3-hydroxycyclophosphamide (3, approximately 20%) and cyclophosphamide (1, approximately 10%), accompanied by regioselective hydrogen-exchange reactions at the C-4 and C-5 positions in 3 and 1. A variety of oxidizing reagents and liver microsomal incubation failed to provide evidence (31P NMR) for conversion of 1 into 3, whereas identical incubation of 3 led to its reduction to 1. Compound 3 is stable at pH 6.5-8.2, 37 degrees C, and exhibits anticancer activity comparable to 1 when tested against L1210 leukemia in mice. Data are discussed with regard to a previously reported suggestion that metabolism of 1 may involved oxidation to give 3 followed by rearrangement of 3 to 2.

The primary metabolite of cyclophosphamide (CP, 1), i.e. 4-hydroxy-CP 2, has high pharmacological activity, but it is a very unstable compound. Chemical approaches to the stabilization involved in the substitution of the hydroxy group at the C 4-position, especially by a sulfoalkylthio-moiety. Within this new class of compounds ASTA Z 7557 (2-(bis-(2-chloroethyl]-amino-cis-4-[2-sulfoethyl)-thio)-tetrah ydr o-2H-1,3, 2-oxaza-phosphorine-r-2-oxide cyclohexylamine salt, i.e. cis-4-sulfoethylthio-CP, cis-13) was chosen for further evaluation. Cis-13 was synthesized by condensation of compound 2 and 2-mercapto-ethanesulfonic acid cyclohexylamine salt 14 in aqueous acetone yielding the cis-isomer with high stereoselectivity. It is a white crystalline powder, m.p. 126-134 degrees C, stable at room temperature, with a solubility of 16% in water. The stereochemistry was confirmed by NMR-data and X-ray diffraction. In 0.07 M phosphate buffer at pH 7 and 37 degrees C cis-13 isomerizes to the epimer trans-13, equilibrating at a cis-trans-ratio of 59 to 41 within less than 5 minutes. Simultaneously a rapid initial hydrolysis occurs to 2 and 14 followed by a time period with lower degradation due to the decomposition of 2. The rate of release of 2 increases with decreasing concentration and especially by addition of an oxidant. It could be retarded by addition of the corresponding thiol mesna, sodium 2-mercapto-ethanesulfonate 15, or of another thiol.(ABSTRACT TRUNCATED AT 250 WORDS)

Phenyl ketone phosphorodiamidates [C6H5C(O)CH2CH2OP(O)NHR1NR2R3] were synthesized in conjunction with an ongoing investigation into the effects of substituents on the dynamical solution chemistry of the metabolites of cyclophosphamide (1a). In contrast to aldophosphamide (3a), which readily interconverts with its cyclic isomer 4-hydroxycyclophosphamide (2a), phenylketophosphamide (14a: R1 = H, R2 = R3 = CH2CH2Cl) exhibited an apparent "resistance" toward an intramolecular addition reaction such that 4-hydroxy-4-phenylcyclophosphamide (13a) could not be detected either spectroscopically (31P or 13C NMR) or chemically (NaCN trapping experiment). Control studies that compared the relative reactivities of 14a and methylketophosphamide [20: CH3C(O)CH2CH2OP(O)NH2N-(CH2CH2Cl)2] revealed that the factors that modulate the ring closure/opening reactions were not peculiar to the phenyl group; however, differences between phenyl and methyl profoundly influenced the rates of fragmentation of 14a and 20. 31P NMR spectroscopy was used to determine the rates at which each compound generated a cytotoxic alkylating agent. Under a standard set of reaction conditions [1 M lutidine buffer with added Me2SO (8:2), pH 7.4, 37 degrees C], the half-lives of 2a/3a, 14a, phenylketoifosfamide (14b: R1 = R2 = CH2CH2Cl, R3 = H), phenylketotrofosfamide (14c: R1 = R2 = R3 = CH2CH2Cl), and 20 were 72, 66, 63, 56, and 173 min, respectively. Analogues 14a and 14b exhibited good anticancer activity against a variety of test systems.

3-(1-Oxy-2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (7) was isolated in 36% yield following H2O2-Na2WO4 oxidation of 3-(2,2,6,6-tetramethyl-4-piperidinyl)cyclophosphamide (6), which was synthesized in three steps (25% yield) starting with 4-amino-2,2,6,6-tetramethylpiperidine. Binding of 7 to mouse liver microsomes was investigated by optical and electron spin resonance spectroscopy. Compared with the mouse liver microsomal metabolism of 1, separate incubations of 6 and an ca. 1:1 mixture of 1 and 6 gave approximately 90 and 60% less acrolein, respectively. A spin-labeled metabolite of 7, viz., N-(1-oxy-2,2,6,6-tetramethyl-4-piperidinyl)phosphoramide mustard (9), was synthesized and its intramolecular O-alkylation at pH 7.4, 37 degrees C, was studied by 31P NMR spectroscopy. Compounds 7 and 9 were inactive in screening tests against L1210 lymphoid leukemia in mice.

4-Ethoxycarbonyl analogs of cyclophosphamide and its five-membered ring homolog were synthesized utilizing the cyclization method previously described. N,N-Bis(2-chloroethyl)-4-ethoxycarbonyl-1,3,2-oxazaphospholidin-2-amine 2-oxide demonstrated activity against L-1210 lymphoid leukemia whereas N,N-bis(2-chloroethyl)-4-(ethoxycarbonyl)tetrahydro-2H-1,3,2-oxazaphosphorin-2-amine 2-oxide did not. The oxazaphosphorin-2-amine was evaluated against human epidermoid carcinoma of the nasopharynx (cell culture). The results again were negative: ED50 = 2.8 X 10.

In seeking analogues of cyclophosphamide (1) having improved antitumor activity by virtue of accelerated formation of the cytotoxic metabolite phosphoramide mustard, cis and trans isomers of 5-fluoro- and 5-chlorocyclophosphamide (9, 10, 11 and 12, respectively) were synthesized by condensation of the appropriate 3-amino-2-halopropan-1-ol (13 or 26) with N,N-bis(2-chloroethyl)phosphoramidic dichloride (14). The metabolism of the halocyclophosphamides by rat liver microsomes was stereoselective; the cis isomers (9 and 11) were poorly metabolized, whereas the trans isomers (10 and 12) were metabolized with efficiency comparable to that of cyclophosphamide. However, there was no evidence that the yield of phosphoramide mustard produced by the trans analogues were significantly greater than that from cyclophosphamide following microsomal 4-hydroxylation. Hence, the halogen substituents did not accelerate beta-elimination of acrolein from the acyclic aldehydo tautomers. As expected, the poorly metabolized cis-5-fluoride (9) had little activity against the ADJ/PC6 tumor in mice. However, the cis-5-chloride (11) was as active as the trans isomer (12) and each had approximately half the therapeutic index of 1. The trans-5-fluoride (10) was much less active, having an ED90 value some 16-fold that of 1.

4-Methylcyclophosphamide, an analogue of the antitumour agent cyclophosphamide, exists in cis and trans forms, each of which comprises a pair of optical isomers. The extents of metabolism by rat liver microsomes during 20 min were compared for the four steroisomers incubated separately, and for the racemic cis and trans-derivatives in admixture, using mass spectrometry and gas chromatography respectively. In comparative antitumour tests against the ADJ/PC6 plasma cell tumour in mice, the racemic cis and trans forms of 4- and 6-methylcyclophosphamide had similar therapeutic indices. The four stereoisomers of 4-methylcyclophosphamide exhibited an approx. two-fold range in therapeutic index so that there was no marked effect on either metabolism or antitumour activity occasioned by change of configuration either at C-4 or at phosphorus.

Convenient syntheses are described of d4 analogs of cyclophosphamide and some of its metabolites, potential standards for the quantitative analysis of the drug and its metabolites in human body fluids by stable isotope dilution-mass spectrometry. Base-catalyzed H-D exchange on N-nitrosobis(2-hydroxyethyl)amine gave N-nitrosobis(1,1-dideuterio-2-hydroxyethyl)amine from which bis(2-chloro-1,1-dideuterioethyl)amine (nor-HN2-d4) was readily obtained. Established synthetic routes were then used to convert nor-HN2-d4 into d4 analogs of cyclophosphamide [2-[bis(2-chlorethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorine 2-oxide], 4-ketocyclophosphamide [2[BIS(2-chloroethyl)amino]tetrahydro-2H-1,3,2-oxazaphosphorin-4-one 2-oxide], and carboxyphosphamide [2-carboxyethyl N-N-bis(2-chloroethyl)phosphorodiamidate], and these analogs were used in a preliminary investigation into the quantitation of the appropriate components in human plasma and urine. Also prepared were d4 analogs of phosphoramide mustard [N,N-bis(2-chloroethyl)phosphorodiamidic acid (cyclohexylammonium salt)] and 3-(2-chloroethyl)oxazolidone and the methyl and trideuteriomethyl esters of phosphoramide mustard.