Structural studies on bioactive compounds. Part 29: palladium catalysed arylations and alkynylations of sterically hindered immunomodulatory 2-amino-5-halo-4,6-(disubstituted)pyrimidines.

The immunological agent bropirimine 5 is a tetra-substituted pyrimidine with anticancer and interferon-inducing properties. Synthetic routes to novel 5-aryl analogues of bropirimine have been developed and their potential molecular recognition properties analysed by molecular modelling methods. Sterically challenged 2-amino-5-halo-6-phenylpyrimidin-4-ones (halo = Br or I) are poor substrates for palladium catalysed Suzuki cross-coupling reactions with benzeneboronic acid because the basic conditions of the reaction converts the amphoteric pyrimidinones to their unreactive enolic forms. Palladium-mediated reductive dehalogenation of the pyrimidinone substrates effectively competes with cross-coupling. 2-Amino-5-halo-4-methoxy-6-phenylpyrimidines can be converted to a range of 5-aryl derivatives with the 5-iodopyrimidines being the most efficient substrates. Hydrolysis of the 2-amino-5-aryl-4-methoxy-6-phenylpyrimidines affords the required pyrimidin-4-ones in high yields. Semi-empirical quantum mechanical calculations show how the nature of the 5-substituent influences the equilibrium between the 1H- and 3H-tautomeric forms, and the rotational freedom about the bond connecting the 6-phenyl group and the pyrimidine ring. Both of these factors may influence the biological properties of these compounds.

Effects of passive immunization against testosterone on serum and pituitary gonadotrophins: implications for the relative importance of androgens and inhibin in the control of gonadotrophin secretion in the male rat.

The acute effects of a specific reduction in androgen feedback to the hypothalamus and pituitary gland have been investigated in male rats by passive immunization against testosterone. An ovine antiserum raised against testosterone which had been conjugated through position 3 to bovine serum albumin was employed. Negative feedback control by androgens was effectively reduced by administration of the antiserum, as shown by an increase in levels of LH in the circulation. Immunized animals had a high concentration of testosterone in the circulation of which virtually all was tightly bound to antibody. In normal animals specific increases of serum LH concentration were obtained at all ages using a low dose of antiserum. At higher doses, serum FSH concentration was also increased. The LH response was reduced by anaesthesia and sham-operation. In sham-operated rats given a high dose of antiserum for 3 days the serum concentrations of LH and FSH could not be distinguished from those which followed castration while differences were found in the pituitary contents. It was concluded that testicular androgen provides an important inhibitory feedback control of secretion of FSH as well as that of LH in the adult male rat. Some of the data can best be explained by the action of inhibin as a minor or alternative inhibitor of FSH secretion.

Serum gonadotrophin levels in prepubertally castrated male sheep treated for long periods with propionated testosterone, dihydrotestosterone, 19-hydroxytestosterone or oestradiol.

At different times of the year, groups of wethers were treated with 20 mg testosterone, dihydrotestosterone or 19-hydroxytestosterone propionates/day or 2 mg oestradiol dipropionate/day, or the oil vehicle, for 6 weeks after a 2-week control period. LH and FSH values were determined by radioimmunoassay of serum samples collected at regular intervals. Oestradiol and dihydrotestosterone reduced LH and FSH concentrations whereas 19-hydroxytestosterone and testosterone had no effect.

The effects of long-term administration of either a crude inhibin preparation or an antiserum to FSH on serum hormone levels, testicular function and fertility of adult male rats.

Adult male rats were given either daily injections of ram rete testis fluid for periods of up to 70 days or injections of an antiserum against FSH every 3 days for 90 days. Compared with the control groups, the rats injected with ram rete testis fluid had lowered serum FSH levels, but only at treatment periods of 30 days and less. The levels of LH and testosterone in serum, testicular fluid secretion, sperm counts, testis weights and fertility were not affected by rete testis fluid treatment. The rats injected with anti-FSH serum exhibited an impairment of fertility which was never complete and evident only after 49 days of treatment. After 90 days of anti-FSH treatment, testis weight and free serum FSH were reduced, but sperm counts, testicular fluid secretion and serum levels of LH and testosterone were not affected.

The evidence that inhibin must exist.

The historical development of the 'inhibin' hypothesis is restated and the recent evidence reviewed. The circumstantial evidence for inhibin now seems very strong, and it probably acts in both sexes. Active materials have been isolated from several sources but it is not clear which is the circulating form of inhibin and a reciprocal relationship between circulating inhibin and FSH levels has not yet been demonstrated. Nevertheless, it appears that we may be optimistic about the existence of inhibin though the importance of its role as a physiological feedback inhibitor is still controversial.

Feedback control by the testis of gonadotrophin secretion: an examination of the inhibin hypothesis.

Spermatogenesis in rats was interrupted by local X-irradiation, heat or ligation of the testicular efferent ducts. A significant and specific rise in the serum level of FSH occurred 5--8 days after ligation of the efferent ducts, reaching twice the value observed in sham-operated controls by 21 days after the operation. After the testes were heated to 43 degrees C for 30 min, the serum levels of both LH and FSH were raised within 3 days and remained so up to 50 days after treatment. After X-irradiation, no changes in the concentration of FSH were observed in the first 21 days after treatment, but the serum levels of both gonadotrophins were increased at 49 days. By comparing the relative increases in the concentrations of FSH and LH after germ cell damage with those occurring after castration, it was evident that testicular androgens could account for only part of the normal feedback control of FSH secretion; at least one third of the inhibition of FSH secretion appeared to be due to non-androgenic sources, presumably 'inhibin'.

Preparation of internally labelled rat pituitary somatotropin (growth hormone).

Rat somatotropin (growth hormone) was labelled biosynthetically by incubating anterior pituitary lobes with radioactive amino acids for 24 h in a simple buffered salts medium containing glucose. The labelled hormone was isolated by preparative polyacrylamide-gel electrophoresis or by chromatography on Sephadex G-100 and then DEAE-cellulose. The labelled material was pure by several criteria and cross-reacted immunologically with unlabelled rat somatotropin. When a mixture of 14C-labelled amino acids was used for labelling the protein, label could be introduced into these same amino acids of somatotropin, though relative specific radioactivities varied considerably. Somatotropin labelled by the procedures described in the present paper was suitable for structural studies and could be used for a variety of other biochemical experiments.

The restricted penetration of iodinated rat FSH and LH into the seminiferous tubules of the rat testis.

The penetration of 125I-iodinated rat follicle-stimulating hormone (FSH; labelled by three different techniques) and luteinizing hormone (LH) through the walls of the seminiferous tubules of the rat testis has been studied by injecting the labelled hormone into rats with the efferent ducts of one testis ligated 16 h before the collection of samples of blood and tissues. The concentration of trichloracetic acid-precipitable and immunoprecipitable radioactivity was measured in blood plasma and rete testis fluid and calculated for the total secreted fluid retained in the testis by the ligature, and for the additional tubular fluid from the ligated testis, separated by centrifugation after decapsulating the testis and dispersing the cells. Very little intact hormone penetrated into the testicular fluids, even 16 h after injection of the labelled hormone, and the volume of distribution in the unligated testis of the trichloracetic acid-precipitable radioactivity was only slightly greater than that for markers known to be confined to the extracellular interstitial fluid. This suggests that the labelled hormones do not penetrate readily through the walls of the semiferous tubules into their lumina. Injected inorganic iodiide and trichloracetic acid-soluble 125I-circulating after the injection of iodinated hormones penetrated more rapidly into the tubules, but had not reached equilibrium between the testicular fluids and blood plasma 16 h after injection. Labelled FSH was reasonably stable in the circulation after injection, but 80% of the 125I was not protein-bound 16 h after injection of labelled LH.