Some physicochemical properties of the antitumor drug thiotepa and its metabolite tepa as obtained by density functional theory (DFT) calculations.

Density functional theory (DFT) using the B3LYP functional was applied to elucidate the molecular properties of the antitumor drug thiotepa and its main metabolite tepa. Aqueous solvent effects were introduced using the conductor-like polarizable continuum model (CPCM). The protocol for calculating the pK (a) values obtained with different cavity models was tested on a series of aziridine and phosphoramide compounds. An efficient computational scheme has been identified that uses the CPCM model of solvation with a universal force field (UFF) cavity. The method has been used to evaluate the basicities of thiotepa and its metabolite. Our calculations show that the basicities of the aziridine moiety of thiotepa and tepa are dramatically reduced compared to free aziridine, indicating that highly acidic media are needed to produce substantial yields of the N-protonated form of the drug. Finally, the mechanisms of reaction of the drug and its metabolite are discussed based on our theoretical results. The calculations reproduce the experimental trends very satisfactorily.

A search for new metabolites of N,N',N''-triethylenethiophosphoramide.

An attempt was made to unravel the metabolic profile of the alkylating agent N,N',N''-triethylenethiophosphoramide (thioTEPA). thioTEPA and its metabolite N,N',N-triethylenephosphoramide (TEPA) were quantified in urine of treated patients by gas chromatography with selective nitrogen/phosphorous detection. Total alkylating activity was assessed by p-nitrobenzylpyridine reactivity. The total alkylating activity exceeded the amount of thioTEPA and TEPA, indicating the presence of other alkylating metabolites. Solid-phase extraction and liquid-liquid extractions followed by gas chromatography-mass spectrometry analysis revealed the conversion of an aziridinyl function of TEPA into a beta-chloroethyl moiety. This metabolite, N,N'-diethylene-N''-2-chloroethylphosphoramide, was quantified by gas chromatography with selective nitrogen/phosphorous detection and accounted for only 0.69% of the administered dose. Large volumes of urine were concentrated with solid-phase extraction and fractionated with high-performance liquid chromatography. Alkylating activity was determined for each 2-ml fraction and showed the presence of an alkylating compound eluting between 8 and 12 ml. The fractions with alkylating activity were collected, evaporated under a stream of nitrogen at room temperature to dryness, reconstituted in methanol, and subjected to fast atom bombardment-mass spectrometry and fast atom bombardment-tandem mass spectrometry. A new metabolite was found with a molecular mass of 352 Da, the same as that of thioTEPA-mercapturate. thioTEPA-mercapturate is likely the result of glutathione conjugation, after which the glutathione adduct loses two amino acid residues in separate stages. The fragmentation pattern and chromatographic properties of this new metabolite were identical to those of the reference, thioTEPA-mercapturate, which was obtained by incubation of thioTEPA with N-acetylcysteine at pH 11 and 95 degrees C for 30 min. Quantification of thioTEPA-mercapturate was carried out by liquid chromatography-mass spectrometry. The thioTEPA-mercapturate levels in urine accounted for 12.3% of the administered dose and exceeded the amount of TEPA, which was previously assumed to be the main metabolite of thioTEPA. The total excreted amount of thioTEPA and its metabolites accounts for 54-100% of the total alkylating activity, indicating the presence of still other alkylating metabolites.

Biological activity of some thiophosphamide derivatives of alkaloids with respect to influenza virus.

Certain newly synthesized compounds belonging to the class of alkaloid ethylenimines, and those known but not subjected to the test for antiviral selection, were studied. The in vitro, in ovo and in vivo experiments detected the substances (Ukrain, amitozin, colchamine-thiotepa, thiotepa) with developed antiviral effect relative to the influenza A/H3 N2/virus strain Port Chalmers 1/73. In the work we used, 1, 10 and 100 ID50 in 0,1 ml. The virus was injected at 1, 10 and 100 EID50 per embryo. The assessment in vivo was carried out on mice of 16-18 g weight. The LD50 for mice having been determined by the probit-analysis method, therapeutic concentrations were used at 1/10 of the LD50. The preparations were found to possess an inhibitory action for the virus, along with insignificant toxicity for the systems studied. Antigrippe action was revealed by the T alkilic agent, in this case thiophosphamide, in all systems. The comparative study of the main component of the alkaloid sum, and of 2 products of their modification (amitozin and Ukrain), showed promise great for creating remedies using such methods and materials. Especially worthy of attention was the Ukrain preparation, which may be recommended as a potential antiviral remedy with the aim of widening the choice of preparations with an antiviral action.

[Alkylation of nucleic acid components by ethyleneimine derivatives. I. Alkylation of bases]. / Alkilirovanie komponentov nukleinovykh kislot proizvodnymi étilenimina. I. Alkilirovanie osnovanii.

In the course of investigating the reaction conditions of the nucleic acid components alcylation, the interaction of thioTEPA (N,N',N''-triethylenethiophosphoamide) with hydrochloric and perchloric acids was studied, perchloric acid increasing the alkylation products yield. HPLC and UV spectroscopy were used to isolate and identify products of nucleic bases alkylation by ethylenimine and its derivatives (thioTEPA and monoaziridinediethylphosphate). It is shown that under neutral conditions phosphoaminoethylation takes place, whereas under slightly acidic conditions products of aminoethylation are formed.

In the search for new anticancer drugs XI. Anticancer activity of nitroxyl labeled phosphoric N,N;N',N';N",N"-tris[1,2-ethanediyl]triamide(TEPA) and phosphorothioic N,N;N',N';N",N"-tris[1,2-ethanediyl]triamide(thio-TEPA) derivatives.

In order to further evaluate the effect of the nitroxyl moiety on the anticancer activity of nitroxyl labeled analogues of phosphoric N,N;N',N';N",N"-tris[1,2-ethanediyl]triamide (TEPA) and phosphorothioic N,N;N',N';N",N"-tris[1,2-ethanediyl]triamide (thio-TEPA), the activity of these compounds was compared in vivo, using murine lymphoid leukemia L1210, with the reduced forms of the drugs, i.e. the hydroxylamines and amine congeners. At optimum dose, all compounds were active. However, the nitroxyl containing compounds were more active than the corresponding reduced forms. An admixture of 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl had no effect on the activity of thio-TEPA. Consequently, the nitroxyl moiety must be an integral part of the anticancer drug's structure in order to influence that drug's performance.

On the search for new anticancer drugs 14: the plasma pharmacokinetics and tissue distribution of spin-labeled thio-TEPA (SL-O-TT).

We defined the plasma and tissue concentrations and pharmacokinetics of SL-O-TT, a spin-labeled analog of thio-TEPA, in 35-44-g male Swiss Webster mice that had received spin-labeled thio-TEPA at a dosage of 10 mg/kg. Concentrations of spin-labeled thio-TEPA in ethyl acetate extracts of tissue and plasma were determined by gas-liquid chromatography and electron spin resonance spectroscopy. Plasma concentrations of spin-labeled thio-TEPA declined in a biexponential fashion that was well described by the equation: Ct = 21.5e-0.276t + 2.30e-0.026t indicating a half-life alpha of 2.5 min and a half-life beta of 26.6 min. After 2 h there was still spin-labeled thio-TE-PA in plasma, but not in tissues. In tissues, no spin-labeled thio-TEPA was detected with gas-liquid chromatography 15 min after injection, but with electron-spin resonance label was found in lung and skeletal muscle. The main metabolite of spin-labeled thio-TEPA is spin-labeled TEPA, where oxidative desulfurization is invoked as the main metabolic mechanism. Reduction of the spin label to the hydroxylamine was also observed with time.

[Effect of thiophosphamide, its paramagnetic analogs and the iminoxyl radical on the division of tumor cells]. / Vliianie tiofosfamida, ego paramagnitnykh analogov i iminoksil'nogo radikala na delenie opukholevykh keltok.

The authors have studied the effect of thiophosphamide its paramagentic analogs and iminoxyl radical on cell reproduction of Ehrlich ascites tumor. It was shown that the effect of paramagnetic analogs is made up of a sum of injuries produced by ethylenimine and iminoxyl portions of molecules. One of the analogs involved proves to be more active than thiophosphamide.

Toxicity, antitumor activity, and pharmacokinetics of spin-labeled thioTEPA analogs.

Two spin-labeled analogs of thioTEPA, containing an iminoxyl radical, have been shown to have significant antitumor activity in several experimental tumor systems, combined with a marked decrease in toxicity compared with the parent compound, thioTEPA. Walker 256 carcinosarcoma, Guérin carcinoma, and Schweitz erythromyelosis showed complete regression using one of these analogs. Pharmacokinetic studies have shown that these compounds possess particular affinity towards tumor tissue.