; PUBERTY GENESIS OF FEMALES-OFFSPRING RATS BORN TO MOTHERS WITH FETOPLACENTAL INSUFFICIENCY.

The purpose of this scientific work was to investigate the development of puberty in females-offspring born to mothers of different age with fetoplacental insufficiency (FPI) and to evaluate efficacy of base and combined drug therapy during pregnancy. Negative influence of FPI on the puberty genesis of females-offspring born to mothers of different reproductive age is considered to be the results of the investigation. In particular, the increased anogenital distance, which is the sign of estrogen deficiency, has been observed in females-offspring born to reproductively young mothers with FPI. Females-offspring born to reproductively matured mothers with FPI have demonstrated more negative changes of reproductive system development. That is, body mass and anogenital distance increasing amid accelerated sexual development have been detected. The increasing of testosterone level has caused inadequate ovaries stimulation which has led to steroid genesis disturbances. During histological investigation of ovarian structure of pubertal rats born to mothers of both groups of age, the decreasing of follicles density, the disturbance in follicles types ratio - early secondary follicles were prevailed, declining folliculogenesis reserve and increased number of atretical follicles have been observed. The introduction of pharmaceutical composition to pregnant rats of both groups of age amid fetoplacental insufficiency leads to stronger normalization of reproductive system development in females-offspring than using of drug of comparison.

Synthesis of potential metabolites of (S)-(-)-bromofosfamide.

(S)-(-)-Bromofosfamide, a newly obtained anticancer agent, recently became a subject of phase I clinical trials in Poland. With the aim to study its metabolism in humans using phosphorus nuclear magnetic resonance a group of potential metabolites of this agent was synthesized.

Synthesis, in vitro metabolic studies, and antitumour activity of methyl analogues of ifosfamide.

Synthesis of 2-chloro-1,1-dimethylethyl and 2-chloro-2,2-dimethylethyl analogues of ifosfamide was performed via aziridine intermediate. In vitro metabolic activation showed that both compounds are metabolised at a rate similar to the parent drug. However, their anticancer activity against L1210 leukaemia in mice was lower as compared with ifosfamide. The reduction of antitumour efficiency of examined analogues is probably caused by a lower ability to cross-link DNA by their final, active metabolites.

Synthesis, chemical and enzymatic reactivity, and toxicity of dithymidylyl-3',5'-phosphorofluoridate and -phosphorothiofluoridate.

Dithymidylyl-3',5'-phosphorofluoridate and phosphorothiofluoridate were obtained by fluorinolysis of the P--Se bond in appropriate bisdimethoxytrityl selenomethyl esters. These compounds, which are hydrolytically unstable, are not inhibitors of snake venom, spleen phosphodiesterases and alkaline phosphatase. Neither compound is highly toxic.

(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.

Nucleophilic N1-->N3 rearrangement of 5'-O-trityl-O2,3'-cycloanhydrothymidine.

5'-O-Trityl-O2,3'-cycloanhydrothymidine (1) heated at 150 degrees C in the presence of O,O-diethyl phosphate or O,O-diethyl phosphorothioate anions undergoes rearrangement into N3-isomer (2); its structure was established by both advanced NMR methods and X-ray crystallographic studies. The most probable mechanism of 1-->2 rearrangement relies upon reversibility of glycosidic bond cleavage process.

Malaria merozoite surface protein antisense oligodeoxynucleotides lack antisense activity but function as polyanions to inhibit red cell invasion.

The effects on malaria parasite growth of antisense and sense oligodeoxynucleoside phosphorothioates based on a merozoite surface protein mRNA was examined. Specific antisense effects of the oligonucleotides could not be demonstrated over three cycles of schizogony or when added as a complex with cationic liposomes. Antisense and sense oligonucleotides however, inhibit merozoite invasion of red blood cells at similar concentrations to dextran sulphate, a polyanion, as determined by microscopy and [3H]hypoxanthine incorporation into DNA. Neutralisation of the negative charge on the oligonucleotides by binding to cationic lipid liposomes, prevented the inhibition of merozoite invasion. We postulate that oligonucleotides because of their polyanionic nature interfere with the binding of merozoites to receptors on red blood cells and that polyanions may be useful in malaria therapy.

Antisense oligodeoxyribonucleotide against the MLL-LTG19 chimeric transcript inhibits cell growth and induces apoptosis in cells of an infantile leukemia cell line carrying the t(11;19) chromosomal translocation.

To clarify the role of the multiple lineage leukemia gene-leukemia translocation gene of chromosome 19 (MLL-LTG19) protein in leukemogenesis, we synthesized antisense oligodeoxyribonucleotide (ODN) against the fused region of the MLL-LTG19 chimeric transcript and treated KOCL33 cells carrying the t(11;19) translocation with antisense ODN. The antisense ODN inhibited cell growth and induced apoptosis in KOCL33 cells but not in Daudi cells, which have no t(11;19). The levels of MLL-LTG19 mRNA and MLL-LTG19 protein in KOCL33 cells treated with antisense ODN were shown to decrease with time by reverse transcription-PCR and Western blot analysis. These results suggest that the MLL-LTG19 fusion protein contributes to cell proliferation and malignant transformation in infantile acute leukemia cells carrying the t(11;19) translocation.

Synthesis, biochemical and biological studies on oligonucleotides bearing a lipophilic dimethoxytrityl group.

Dimethoxytritylphosphono-oligonucleotide conjugates have been prepared. They are totally resistant to nucleases present in human serum and do not affect cleavage of a complementary oligoribonucleotide by RNase H. Conjugates possessing a phosphate backbone gave better antisense inhibition of expression of plasminogen activator inhibitor type-1 within endothelial cells as compared with unconjugated oligonucleotides.

Synthesis and interaction with thymidylate synthase of 5'-dithiophosphate and 5'-fluorothiophosphate of thymidine.

Thymidine-5'-fluorothiophosphate, dTMP(S)-F, was synthesized by the oxathiaphospholane, and thymidine 5'-dithiophosphate, dTMPS2, by the dithiaphospholane, method. To estimate the role of 5'-phosphate group ionization in binding of pyrimidine nucleotides by thymidylate synthase, dTMP(S)-F was studied as an inhibitor of mouse tumour (L1210) enzyme, and its inhibitory properties were compared with those of dTMPS2, a close dTMP analogue. While dTMPS2 proved to be an inhibitor, competitive vs dUMP, with K(i)app = 94 microM, the 5'-fluorothiophosphate congener displayed no activity, indicating that the enzyme requires for binding the presence of a dianionic 5'-phosphate group in a nucleotide.

Anti-sense oligodeoxynucleoside phosphorothioates nonspecifically inhibit invasion of red blood cells by malaria parasites.

Anti-sense and sense oligodeoxynucleoside phosphorothioates, based on analysis of the secondary structure of Plasmodium falciparumdihydrofolate reductase-thymidylate synthase mRNA, were synthesized. Their effects on P. falciparum growth in vitro were examined by microscopy and [3H] hypoxanthine incorporation. Both anti-sense and sense oligodeoxynucleoside phosphorothioates inhibit invasion of red blood cells by merozoites and this is interpreted as being caused by their polyanionic nature. Specific anti-sense effects of the oligonucleoside phosphorothioates could not however be demonstrated.

Antimalarial activities of oligodeoxynucleotide phosphorothioates in chloroquine-resistant Plasmodium falciparum.

Synthetic oligonucleotides and their chemical modifications have been shown to inhibit viral and cellular gene expression by sequence-specific antisense hybridization to target mRNAs. We now report that oligodeoxynucleotide phosphorothioates and their nuclease-resistant modifications are effective in micromolar and submicromolar concentrations against the growth of both chloroquine-resistant and chloroquine-sensitive strains of Plasmodium falciparum in vitro. Parasitized human erythrocytes were found to be accessible to radioactively labeled oligodeoxynucleotides, whereas the uninfected erythrocytes did not permit any cellular entry of the same compounds. The dihydrofolate reductase-thymidylate synthase gene of P. falciparum was demonstrated to be a good target for sequence-dependent inhibition of plasmodial growth by exogenously administered modified oligonucleotides. The antimalarial activities observed in vitro were identical for chloroquine-sensitive and chloroquine-resistant strains of P. falciparum. The antimalarial activity of oligodeoxynucleotide phosphorothioates is related to sequence complementarity to certain regions of the plasmodial genome as well as to non-sequence-defined activities.

Biotinyl and phosphotyrosinyl phosphoramidite derivatives useful in the incorporation of multiple reporter groups on synthetic oligonucleotides.

Non-nucleosidic phosphoramidite linker units suitable for use on commercial DNA synthesis machines have been designed for the direct incorporation of biotin and a new reporter group, phosphotyrosine, at multiple sites on synthetic oligonucleotides. The units are based on a 3-carbon glyceryl backbone where the reporter group is attached to the 2-O-position through a 3-aminopropyl spacer. 17-mer oligonucleotides were synthesized carrying at the 5'-end 1, 2, 4 or 8 biotinyl units or 1, 2, 4 or 8 phosphotyrosinyl units respectively and used for the detection of DNA on nitrocellulose filters by hybridization. Subsequent incubation of the filters with a monoclonal antibody to the reporter group followed by secondary detection using enhanced chemiluminescence (ECL) resulted in amplification of signal strengths as the number of reporter groups was increased. The results were quantitated by use of a charge couple device (CCD) camera. Spacing of biotin moieties by thymidyl residues resulted in further improvements in signal strengths, whereas similar spacing of phosphotyrosinyl units did not.

The X-ray structure and absolute configuration of the anticancer drug (+)-4-ketocyclophosphamide.

4-Ketocyclophosphamide (4-keto CP), C7H12Cl2N2O3P, monoclinic, P2(1), a = 11.909 (2), b = 10.254 (1), c = 9.873 (1) A, beta = 91.08 (1) degrees, V = 1205.45 (3) A3 Z = 4, Dc = 1.51 Mg-m-3, Cu K alpha, lambda = 1.54178 A, alpha 25 = +53.8 degrees (c = 3.0, MeOH), m.p. 107 degrees C, mu = 61.8 cm-1, F (000) = 564, R = 0.064 for 2961 observed reflexions with I greater than 1.96 sigma(I). Dextrarotatory enantiomer of 4-keto CP has S configuration at the stereogenic center. One of the two crystallographically independent molecules is disordered both in a six-membered ring and in --N(CH2CH2Cl)2 moiety. With the exception of a less populated conformer of a disordered molecule, 4-keto CP molecules adopt a conformation in which 1,3,2-oxazophosphorinane ring is in the sofa form with C(6) deviating from the plane through the remaining five ring atoms while an exocyclic N atom with its three substituents is nearly coplanar with the phosphoryl oxygen atom O(8). In a less populated conformer, the six membered ring takes the form of sofa with C(5) as a flap while an exocyclic N atom and its substituents are oriented toward the P--N(3) bond.

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.

Stereospecific synthesis of chiral metabolites of ifosfamide and their determination in the urine.

The stereospecific synthesis of two chiral metabolites of ifosfamide (2), 4-ketoifosfamide (5) and 2-amino-3-(2-chloroethyl)tetrahydro-2H-1,3, 2-oxazaphosphorine 2-oxide (9), is reported. The absolute configuration of both compounds was assigned on the basis of chemical correlation. In addition, two other achiral metabolites of 2, carboxyifosfamide (6) and IPAM (7), were synthesized. These and other organophosphorus metabolites of ifosfamide were found, by 31P NMR, in the urine of patients to whom racemic 2 was administered. The measurements performed in the presence of optically active lanthanide shift reagent [Eu(tfc)3] showed considerable stereoselectivity of in vivo formation of some chiral metabolites of ifosfamide.