(S)-(-)-Bromofosfamide (CBM-11): synthesis and antitumor activity and toxicity in mice.

(S)-(-)-Bromofosfamide (CBM-11), an enantiomerically pure bromo analog of ifosfamide, was found to be potent against several model tumors in mice. Therapeutic indices of CBM-11 were more favorable as compared to those received for ifosfamide.

The library of p-nitrophenyl esters of oligopeptides as a model to study the anticancer drug-peptide conjugates activated during tumor angiogenesis.

An indexed library of oligopeptide p-nitrophenyl esters immobilized via methoxy-1,3,5-triazine scaffold on the cellulose support, was synthesized by the step by step procedure and by segment coupling, involving 2-chloro-4,6-dimethoxy-1,3,5-triazine as a condensing reagent. The substrate specificity towards homogenate supernatant of mouse lung cancer LL2 cells has been studied.

The library of p-nitrophenyl esters of oligopeptides as a model to study the anticancer drug-peptide conjugates activated during tumor angiogenesis.

An indexed library of oligopeptide p-nitrophenyl esters immobilized via methoxy-1,3,5-triazine scaffold on the cellulose support, was synthesized by the step by step procedure and by segment coupling, involving 2-chloro-4,6-dimethoxy-1,3,5-triazine as a condensing reagent. The substrate specificity towards homogenate supernatant of mouse lung cancer LL2 cells has been studied.

Pharmacokinetic-stereoselective differentiation of some isomeric analogues of ifosfamide.

Three bromine-ifosfamide analogues: racemic chlorobromofosfamide (+/-)-(R,S)-1, its levorotatory enantiomer (-)-(S)-2 and racemic bromofosfamide (+/-)-(R,S)-3 showed considerable stereoselective differences in their pharmacokinetics and bioavailability depending on the route of administration and regimen of dosage studies in rats. Remarkable differences in the AUCi.p. parameters (212, 54, 89 mumol x min x ml-1, respectively) were demonstrated in comparison with a standard ifosfamide (158 mumol x min x ml-1). However, the AUCp.o. established for (+/-)-(R,S)-1 and (-)-(S)-2 were similar and different from the value measured for (+/-)-(R,S)-3. A wide variability in the determined parameters after i.p. injection and similarities after p.o. administration were finally confirmed by the AUCp.o./AUCi.p. mean ratio which equaled 0.24; 0.79;, 0.54, respectively, as well as by different bioavailability data. The results showed that the pharmacokinetic bioequivalance between i.v. and p.o. treatment is possible to approach by adjustment of fractionated oral dosage.

Effects of alkylating metabolites of ifosfamide and its bromo analogues on DNA of HeLa cells.

The in vitro cytotoxicity and mechanism of action of three alkylating compounds: an active metabolite of ifosfamide (1, isophosphoramide mustard, N,N'-bis(2-chloroethyl)phosphorodiamidic acid) and its bromo substituted analogues, 2 (one chlorine atom replaced by bromine atom) and 3 (two chlorine atoms replaced by bromine atoms), were studied in cultured HeLa cells. Alkaline elution analysis of cellular DNA demonstrated the presence of concentration- and time-dependent interstrand crosslinks, DNA-protein crosslinks and alkali-labile sites (ALSs) in HeLa cells following a 1 hr exposure to the compounds. The bromo analogues were more cytotoxic than 1 and exhibited higher crosslinking potency. The time-course of crosslink formation and removal for the three compounds was similar. ALSs in DNA were produced by all tested drugs but 3 exhibited exceptionally high activity and was able to induce two kinds of alkali-labile lesion (fast- and slow-appearing) whereas 1 and 2 generated only slow-appearing ones. The results suggest that 1 and 2 are more specific in their reaction with DNA in that they produced a lesser variety of lesions than 3. A potential advantage of 2 over 1 seems to be its higher DNA interstrand crosslinking activity.

Synthesis and antitumor activity of analogues of ifosfamide modified in the N-(2-chloroethyl) group.

A series of 3-(2-chloroethyl)-N-(2-X-ethyl)tetrahydro-2H-1,3,2-oxazaphosphorin -2-amine 2-oxides with various X substituents have been prepared by cyclization of racemic ifosfamide or its enantiomers with sodium hydride and subsequent treatment of intermediary products with hydrobromic acid, diethyl hydrogen phosphate, dibenzyl hydrogen phosphate, p-toluenesulfonic acid, and acetic acid. All of these compounds were tested in vivo against L 1210 lymphoid leukemia in mice. Only bromo analogue 13 and its enantiomers were effective, exceeding the activity of racemic ifosfamide and cyclophosphamide. The therapeutic index of the racemic 13 and its levorotatory enantiomer was about 1.7 times higher than that for ifosfamide and about 2.7 times higher than that for cyclophosphamide.

Antitumor activity of optical isomers of cyclophosphamide, ifosfamide and trofosfamide as compared to clinically used racemates.

The relationship between enantiomeric homogeneity of three oxazaphosphorine drugs: cyclophosphamide, ifosfamide and trofosfamide and their antitumor activity was evaluated by standard screening tests against four in vivo transplantable tumor models: L 1210 and P 388 lymphoid leukemias, Lewis lung carcinoma and 16/C line of mouse mammary adenocarcinoma. It was shown that the stereodifferentiation of anti-tumor effect of enantiomers was not outstanding although quite consistently in favour of levorotatory forms. The only exception was seen for cyclophosphamide enantiomers tested against leukemias where R/+/form was more effective than S/-/or racemate.

5-Fluoro- and 5-chlorocyclophosphamide: synthesis, metabolism, and antitumor activity of the cis and trans isomers.

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.

Stereochemistry of hydrolysis of adenosine 3':5'-cyclic phosphorothioate by the cyclic phosphodiesterase from beef heart.

Adenosine 3':5'-cyclic phosphorothioate, Sp-diastereomer was hydrolyzed by cyclic phosphodiesterase from beef heart in the presence of [18O]water to [18O]adenosine 5'-phosphorothioate. This was phosphorylated by myokinase and pyruvate kinase to [18O]adenosine 5'-(1-thiotriphosphate),Sp-diastereomer. The position of 18O was determined to be in a nonbridging position. This result indicates that the hydrolysis proceeded with inversion of configuration at phosphorus.

Comparative metabolism of 2-[bis(2-chloroethyl)amino]tetrahydro-2-H-1,3,2-oxazaphosphorine-2-oxide (cyclophosphamide) and its enantiomers in humans.

The comparative metabolism of the enantiomers of cyclo phosphamide and of the racemate has been studied in humans. Four patients were each given, sequentially, the racemate, the (+)-enantiomer, and its (-)-antipode. The plasma levels of parent drug and the urinary output (24 hr) of unchanged drug and of two enzymatically produced metabolites, 4-ketocyclophosphamide and carboxyphosphamide, were determined using mass spectrometry-stable isotope dilution. There was no significant difference between the three forms of cyclophosphamide with respect to plasma half-life (beta phase) or in the urinary outputs of the drug or of carboxyphosphamide. The output of 4-ketocyclophosphamide after administration of (+)-cyclophosphamide was significantly greater than that produced from the racemate. Cyclophosphamide recovered from the urine of patients given the racemate was either racemic or only slightly enriched in the (-)-enantiomer. The two enantiomers were almost equally bound to plasma protein. Based on these metabolic studies alone, there is little reason to predict that the enantiomers will differ from each other or from the racemate in their therapeutic effects in humans, but there are other factors, e.g., stereoselective uptake of the intermediary 4-hydroxylated metabolites by neoplastic cells, which could elicit such differences.

X-ray-diffraction study and determination of absolute configuration of the anticancer drug S(--)cyclophosphamide (Endoxan, Cytoxan, NSC-26271).

(--)Cyclophosphamide(1) crystallized from tetrachloromethane in the triclinic space group P1 with cell dimensions a = 10.500 (4) A, b = 10.490 (4) A, c = 10.761 (4) A, alpha = 110.0 (2) degrees, beta = 110.0 (2) degrees, gamma = 108.9 (2) degrees. Three molecules are contained in the unit cell. The X-ray analysis was based on diffractometer measurement of 2635 independent reflections and the structure was solved by Patterson and direct methods. The final R and Rw factors after full-matrix least-squares refinement are 0.0717 and 0.0677, respectively. The absolute configuration is S based on Hamilton's R-factor ratio test. The oxazaphosphorinane ring exists in a chair form with the bis-beta chloroethyl-amino group in equatorial position and about perpendicular to and bisecting the O--P--N plane. The structure is similar to that found in racemic 1 except a different conformation about one ethyl-amino N--C bond.