Propionate Derivatives Research Articles

Biochemical and biological studies with 4-aza-steroidal 5α-reductase inhibitors

A series of 4-aza-3-oxosteroids were found to be good inhibitors of steroid 5 alpha-reductase. Two of these compounds. 17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one (4-MA) and 4-methyl-4-aza-5 alpha-pregnan-3-one-20(s)-carboxylate, inhibit 5 alpha-reductase competitively with testosterone (T) with Ki values of 5 and 1.7 nM, respectively. These 5 alpha-reductase inhibitors also have an affinity to the androgen receptor which is orders of magnitude lower than that of 5 alpha-dihydrotestosterone (DHT), spironolactone and cyproterone acetate. 4-MA decreases the prostatic concentration of DHT and increases that of T in intact male rats and in castrates given T or its propionate derivative. 4-MA is a better inhibitor of T-induced growth than of DHT-induced growth of the prostate and seminal vesicles in castrated rats. It decreases the weight of the prostate and seminal vesicles in intact rats and that of the prostate in dogs. It has no significant antifertility activity in rats. In pregnant rats, 4-MA reduces the ano-genital distance of male fetuses. 4-MA has no significant androgenic, estrogenic, progestational, antiprogestational or antigonadotrophic activity.

Selected Ion Mass Spectrometric Identification of Chlorophenol Residues in Human Urine

A method is presented for the detection and confirmation of trace chlorophenol residues in environmental and biological samples by mass spectrometry with selected ion monitoring (SIM). Propionate and acetate ester derivatives of phenol, cresols, mono-, di-, tri-, and tetrachlorophenols, pentachlorophenol, as well as the internal standard 4,6-dibromo-o-cresol, were prepared directly in aqueous solution, using the appropriate anhydride reagent. The acetate or propionate esters were quantitatively extracted and then separated by gas-liquid chromatography using either an SP-1240DA or OV-101 packed column. Electron impact SIM was carried out by simultaneously monitoring up to 4 diagnostic fragment ions of each chlorinated phenol. The use of SIM eliminated background interference encountered using flame ionization or electron capture detectors, and allowed identification of chlorophenol residues in extracts of urine samples collected from individuals without known exposure to chlorophenols. Phenol concentrations as low as 1.0 pmol/mL urine gave peaks that were readily discernible by SIM.

Selected ion monitoring determination of acetylcholine during methoxypyridoxine seizures.

The neurotransmitter gamma-aminobutyric acid is known to decrease preictally after administration of the potent convulsant methyoxypyridoxine, a competitive inhibitor of glutamate decarboxylase. An attempt was made to determine the effect of this gamma-aminobutyric acid decrease on the cholinergic system. Rabbits were immobilized and artificially ventilated in order to avoid hypoxidosis. Seizures were induced by intravenous injection of 100 mg kg-1 methoxypyridoxine; 40 minutes later the animals were decapitated and discrete brain areas removed. Tissue contents of acetylcholine and choline were estimated by gas chromatographic mass spectrometric analysis of the beta-dimethylaminoethyl acetate and propionate derivatives using deuterated internal standards. Gas chromatographic column optimization resulted in a considerable sensitivity gain. Computerized selected ion monitoring was carried out on the dimethylmethyleneimmonium ions using voltage switching. The use of a computer controlled solvent dump valve was implemented to increase precision. No significant difference was observed in the concentration of acetylcholine in the frontal cortex, cerebellar cortex, septum, hippocampus, or caudate of seizure versus control animals; septal choline increased, however. This suggests that the acetylcholine turnover could be increased during seizure.

Cyanide reactivity of cytochrome c derivatives

The kinetic rates and equilibrium association constants for cyanide binding have been measured for a series of cytochrome c derivatives as a probe of heme accessibility. The series included horse and yeast cytochromes iodinated at Tyr 67 and 74, horse cytochrome formylated at Trp 59 in both a low and high redox potential form, the Met 80 sulfoxide derivative of horse cytochrome and the N-acylisourea heme propionate derivative of tuna cytochrome. Native cytochromes c are well known to bind cyanide slowly in a reaction simply first order both in cytochrome and cyanide up to at least 100 mM in cyanide. The derivatives demonstrate markedly different kinetics which indicate the following conclusions. (1) In spite of chemical modification at different loci, all the derivatives have highly similar reactivity, suggesting common ligation structures and mechanisms for reaction. (2) Compared to native cytochromes, reaction rates are 10–20 fold greater. This is in accord with a more accessible heme crevice, but not a completely opened crevice. For the completely opened case, rate increases are expected to be between three and five orders of magnitude. (3) Reaction rates are either independent of cyanide concentration (zero order) or show only slight variation. A mechanism which accounts for the data over four orders of magnitude in concentration postulates a protein conformation step, opening of the heme crevice, as the rate determining step. This conformation change has a limiting rate of 6 · 10 −2 s −1.

Determination method of bile acids in biological materials by mass fragmentography (author's transl)

A new method was developed for quantitative analysis of deuterium-labelled and endogenous bile acids in human serum, urine, bile and feces. The conventional extraction method was modified to improve the recovery of the sulfated bile acids. The sulfated and nonsulfated bile acids were adsorbed on an Amberlite XAD-2 column, eluted with methanol containing 20 mM NaOH, and separated on a Sephadex LH-20 column. Sulfated bile acids were dissolved in ethanol (pH 1)-acetone (1 : 9, v/v) and solvolysed for 3 hr at 37°. A new alkaline hydrolysis method was also devised ; bile acids were dissolved in 4 N NaOH-methanol (1 : 1, v/v) and hydrolyzed in a screw-capped Pyrex glass tube at 80° for 16 hr. After extraction with ether, bile acids were converted to methyl ester propionate derivatives. The determination of endogenous and deuterium-labelled bile acids was accomplished by gas chromatography-mass fragmentography. The reproducibility of the present method was good (3-8% of relative standard deviation for endogenous bile acid in serum) and 11, 12-2H2-chenodeoxycholic acid was determined up to 1% of the coexisting chenodeoxycholic acid. Endogenous bile acid levels in biological materials were measured and compared with previously reported results. The present method was sensitive and useful for the quantitation of minute amounts of bile acids. Further, this was a recommendable method for the determination of pool size of bile acids by using deuterium-labelled bile acids.

The effects of the administration of testosterone propionate alone or with phenobarbitone and of testosterone metabolites to neonatal female rats

The effects of graded doses of testosterone propionate administered to female rats on Day 4 of postnatal life have been determined. The incidence of failure of ovulation as adults was related to the dose. With increasing dosage over the range 1–100 μg no significant evidence of a progressive decrease in immunoassayable luteinizing hormone concentration in the plasma or anterior pituitary was obtained. The reported protective action of sodium phenobarbitone when administered with testosterone propionate could not be confirmed. Single injections of 100 μg of testosterone, dihydrotestosterone, 5 α-androstanediol, or a combination of the two latter compounds had no masculinizing effect. When the 17-β propionate derivatives of these compounds were administered at the same dose level only testosterone propionate had a masculinizing effect.

Purification and properties of esterases characteristic of adult rat brain.

1. A method for the partial purification of an esterase fraction, present in the brain of the adult but not the newborn rat, is described. A 54-fold purification was achieved in three steps. 2. When subjected to starch-gel electrophoresis, the purified fraction resolved into three bands of esterase activity. Two of these bands migrate close together and faster than other esterases in the brain. These two esterases are inhibited by p-hydroxymercuribenzoate but not by di-isopropyl phosphorofluoridate. The third band is di-isopropyl phosphorofluoridate-sensitive and migrates just behind the two leading esterases. 3. After treatment with di-isopropyl phosphorofluoridate, to obviate the effects of the di-isopropyl phosphorofluoridate-sensitive esterase, the enzyme preparation hydrolyses alpha-naphthyl acetate, alpha-naphthyl propionate and alpha-naphthyl butyrate, but not cholesteryl acetate. The V(max.) for the naphthyl esters decreased with increase in chain length of the acyl group. The acetate ester is hydrolysed 34 times as fast as the butyrate and about seven times as fast as the propionate derivative. The K(m) values for these three esters, measured at pH7.2 and 37 degrees , are 2.8x10(-4)m, 3.1x10(-4)m and 7.3x10(-5)m for the acetate, propionate and butyrate derivatives respectively. 4. The Hofstee (1952) plots for the kinetic data show a single line, indicating that the two most-rapidly migrating esterases, although electrophoretically separable, are not kinetically distinguishable in the substrate ranges examined.

Structural requirements for the action of steroids as inhibitors of electron transfer

A series of C 21, C 19, and C 18-steroids (including hormonal steroids and derivatives) have been tested as inhibitors of NADH-oxidase and succinate oxidase activities of heart-muscle sarcosomal fragments (Keilin-Hartree preparation). NADH oxidation is much more sensitive to steroids than succinate oxidation, in accordance with electron transfer inhibition in the vicinity of the NADH-flavoprotein site. Steroid activity on NADH-oxidase has been titrated and the 50% inhibitory concentration ( I 50) of each steroid has been determined. Among the steroids tested, the following were the more effective inhibitors, having I 50 values ≦ 1.0 μM 19-norethynyltestosterone acetate, 5-pregnen-3β,20α-diol, 5α-pregnan-3β,20α-diol, 5-pregnen-3β,20β-diol, 5-pregnen-3β,17α,20α-triol, testosterone acetate, testosterone enol diacetate, testosterone benzoate, androstanolone acetate, and 17β-ethynylestradiol benzoate. Comparison I 50 values for steroids differing in a single group (or structure) shows that the following structural features promote the inhibitory potency: (a) a trans A B junction; (b) an alcohol or ketone group at C-3 and/or C-17; (c) an acetyl chain at C-17; (d) 17α-methyl or ethynyl groups at C-17 of 17β-hydroxylated steroids; (e) acetylated or benzoylated hydroxyl groups at C-3 or C-17; (f) α configuration of 3-OH groups in 5α-androstane derivatives and β-OH configuration in 5β-pregnane derivatives; (g) α configuration of hydroxyl groups at C-17; (h) an angular β-methyl group at C-10; (i) a hydroxyl group at C-20 of 5α-pregnane and 5-pregnene derivatives. Decreased inhibitory potency is determined by the following structural features: (j) a cis A B junction; (k) Δ 1 and Δ 4 double bonds; (l) ionizable (hydrophylic) groups at C-3 or C-17; (m) hydroxyl groups at C-17 or C-21 of C 21-steroids; (n) relatively large substituents at C-3 or C-17 (hexahydrobenzoate and propionate derivatives); (o) substituents at the skeleton of the steroid molecule (C-2, C-4, C-6, C-11, C-16) irrespective of group-polarity and configuration. Accordingly, the most effective steroid structure to inhibit electron transfer is a planar and narrow hydrocarbon framework with relatively small, adequately orientated polar end-groups and a large intermediate hydrophobic area. These structural characteristics are consistent with the interaction of steroids with the phospholipid components of the electron transfer chain at the NADH-flavoprotein site. Many of the structural requirements for inhibition of electron transfer resemble those for androgenicity.