Excellent acceleration of the Diels-Alder reaction by microwave irradiation for the synthesis of new fluorine-substituted ligands of NMDA receptor.

A series of 6,11-ethanobenzo[b]quinolizinium derivatives was synthesized through the Diels-Alder reaction between azoniaanthracne and the corresponding 1,1-disubstituted olefin. After a systematic investigation for achieving rapid synthesis, it was found that the reaction is accelerated in polar media such as H2O and trifluoroethanol. In particular, excellent acceleration was effected by microwave irradiation. The new fluorine-substituted ligands thus obtained exhibited potential affinity toward NMDA receptors.

Simultaneous determination of tegafur and 5-fluorouracil in serum by GLC using nitrogen-sensitive detection.

A sensitive assay of both tegafur (I) and 5-fluorouracil (5-FU) using GLC with a nitrogen-phosphorus-sensitive detector is described. The drugs were extracted from rabbit serum with ethyl acetate and methylated with diazomethane. Linearity was obtained over the concentration ranges of 3.13-200 micrograms/ml for I and 0.0313-2 micrograms/ml and 10-50 ng/ml for 5-FU. The detection limits of I and 5-FU in serum were 50 and 8 ng/ml, respectively. The serum concentrations of the drugs determined by the present method closely agreed with those obtained by spectrophotometry for I and microbial assay for 5-FU.

Studies to increase the antitumor activity of 1-(tetrahydro-2-furanyl)-5-fluorouracil and its metabolic aspects by combined administration with L-cysteine.

The plasma and liver concentrations of both 1-(tetrahydro-2-furanyl)-5-fluorouracil (FT) and 5-fluorouracil (5-FU), an active metabolite of FT, increased very markedly after administration of FT (500 mg/kg, p.o.) combined with L-cysteine (L-CYS, 500 mg/kg, i.p. or p.o.) when compared to FT alone in rats. On the other hand, the oral acute toxicity of FT was also enhanced with the combined administration of FT and L-CYS. There was no difference in the in situ absorption rate of FT from the small intestine between rats treated with L-CYS (500 mg/kg, i.v.) and vehicle-treated controls. The inhibition of the disappearance of FT and the increase of the formation of 5-FU was observed in vitro after incubation of FT with liver microsomes from rats treated with L-CYS (500 mg/kg, p.o.) when compared to vehicle-treated controls. The presence of L-CYS significantly inhibited the in vitro degradation of 5-FU by non-treated rat liver homogenate. In the drug metabolizing enzyme activity of rat liver microsomes, aniline p-hydroxylase activity was inhibited, but aminopyrine N-demethylase activity was conversely activated by the combined administration of FT and L-CYS, but not by FT alone; furthermore, no change of cytochrome P-450 content was observed. In sarcoma 180 bearing mice, the oral antitumor activity of FT in combination with L-CYS (500 mg/kg, i.p. or p.o.) was about 1.1-2.0 times higher than that of FT alone. It was concluded from these findings that the drug metabolizing enzymes in liver involved in the conversion of FT into an active metabolite, 5-FU, are influenced by the combined administration of FT and L-CYS to give an increased organ level of 5-FU; and this resulted in the enhancement of the antitumor activity of FT.

Transport of L-cysteine and reduced glutathione through biological membranes.

Comparative studies were carried out on in vivo and in situ absorption in rats and in vitro uptake by red cells of L-cysteine (CySH) and reduced glutathione (GSH). After oral administration of CySH, the plasma and liver CySH levels and liver GSH level significantly increased, but the plasma GSH level did not. In contrast to the results with CySH, when GSH given orally at a dose equivalent to that of CySH either on a weight or molar base, no increase in the levels of GSH was observed at either dose level. In a rat small intestine recirculation system in situ, CySH added to the recirculation perfusate disappeared progressively with time from the perfusate, indicating that transportation occurred across the intestinal wall, but with GSH such disappearance was not observed. CySH was taken up well by rabbit erythrocytes in vitro, but GSH was not. It was concluded, therefore, that CySH passes through biological membranes and serves as a good source of SH groups, whereas GSH is ineffective when given orally because of its poor absorption from the digestive tract and/or poor ability to permeate through the membrane.

Virilizing effect of testosterone and its metabolites on the urovaginal septum of female fetal rats.

A single injection of 50 microgram testosterone was given to fetal rats on day 17, 18, 19 or 20 of gestation. On day 21, the fetuses were removed from the mother under maternal ether anesthesia, and the length of the urovaginal septum was measured microscopically in female fetuses in order to assess the virilizing effect of testosterone. In fetuses treated with testosterone on day 17, the length of the urovaginal septum was comparable to that of oil-treated littermate controls. In fetuses treated on day 18, the length was significantly abridged compared with controls. In fetuses treated on day 19, the abridgment of the urovaginal septum was most marked. In fetuses treated on day 20, the length of the septum was again comparable to that of controls. The observations suggest that day 19 is the critical day for the virilizing effect of testosterone. Various amounts of testosterone and its metabolites including dihydrotestosterone, androstane-3 beta, 17beta-diol and androstane-3 alpha, 17beta-diol were injected into 19-day-old female fetuses, in order to test the dose relation to the virilizing effects of these steroids in terms of abridgment of the urovaginal septum. As a consequence, it was found that testosterone was the most effective for virilization.

Virilizing effect of methyltestosterone on female descendants in the rat.

Pregnant rats were given daily a subcutaneous injection of methyltestosterone for 4 days from the 17th to the 20th day of gestation, and were allowed to be delivered to their offsprings (F1) which were used for the examination of later reproductive functioning. When observed for 21 weeks after birth, the growth rate of F1 from methyltestosterone-treated groups was higher than that of F1 from the control group. The anogenital distance in 50-microgram-treated F1 females started to become significantly longer on the 14th day and in 5-microgram-treated F1 females on the 28th day after birth than that in F1 from the control. The day on which vaginal opening took place in 50% of females was 34.4 days of age in both the control and the 5 microgram groups, but it delayed until 40.7 days in the 50 microgram group. Furthermore, persistent estrus was observed after about 90 days of age in the 50 microgram group. This persistent estrus disappeared by placing these females with males, resulting no pregnancy. In the 5 microgram group females could be pregnant, but their female fetuses (F2), when examined on the 21st day of gestation, had significantly shortened the length of the urovaginal septum. The observations show that virilization can be induced in the third generation.

Virilizing activities of various steroids in female rat fetuses.

Virilizing activities of nineteen steroids were examined by a method measuring the abridgment of urovaginal septum length of female rat fetuses directly under microscope, following oral administration of the steroids to the mothers for 4 days during the late period of gestation. The characteristics of the virilizing activities of the steroids seemed to depend on their chemical structures. No virilizing activities were observed in progesterone, retroprogesterone, chlormadinone acetate and nandrolone phenpropionate. The introduction of 6-methyl and 17-acetoxy groups into progesterone exhibited a marked virilizing activity. With testosterone and its derivatives, the introduction of 17alpha-ethynyl group into testosterone slightly decreased the virilizing activity compared with the parent compound. On the contrary, the introduction of [3, 2-C] pyrazole ring in addition to the saturation of A-ring strikingly increased the virilizing activity. The most active virilizing steroid in this group was stanozolol followed by oxymetholone, methyl-testosterone and dimethysterone. Testosterone propionate was assumed to be less active than methyltestosterone by this route. With 19-nortestosterone derivatives, the virilizing activities of compounds with 17alpha-ethynyl group were moderate, but the presence of 17alpha-ethyl group caused a marked increase of virilizing activity. While, nandrolone phenpropionate with long side chain at C-17 exhibited no significant virilizing activity. The present results show the possibility of the evaluation on a potential virilizing activity of compounds in the female fetus, and also suggest that a dossociation between the virilizing activity and the androgenic activity may exist among some compounds.

Quantitative evaluation of virilizing activity of steroids by measuring morphological changes in uro-genital region of rats.

A new technique to measure the uro-genital parameters such as the lengths of urovaginal septum, corpora cavernosa and anogenital distance on the sagittal sections of the pelvic region of female fetus of rat under microscope equipped with a micrometer was developed. In the examination of 180 normal female fetuses on the 21st day of gestation, relationships were observed between the fetal body weight and the length of urovaginal septum as well as anogenital distance, but not on the length of corpora cavernosa. Following maternal subcutaneous administration of various doses of 17alpha-methyltestosterone between the 17th and 20th day of gestation, dose-dependent abridgment in urovaginal septum length and extensions in corpora cavernosa length and anogenital distance were observed in female fetus on the 21st day's examination. When these three parametars were calculated on the relative value to the fetal body weight, however, linear relationships against log-dose were observed in all parameters. Among these three parameters the abridgment in urovaginal septum was shown to be the most sensitive. A quantitative assay of virilizing activity of steroids in female fetuses was examined on rats treated subcutaneously with 17alpha-methyltestosterone and norethandrolone. Linear regressions against the log-doses of both steroids were demonstrated in urovaginal septum length, and parallelism was noted between both regression lines. The relative potency of norethandrolone to 17alpha-methyltestosterone calculated on urovaginal septum length was 0.354 with fiducial limits of 0.293-0.44l, and it was suggested that the virilizing activities of steroids can be evaluated quantitatively.