Antibody binding characteristics of geometrical isomers of testosterone 3-(O-carboxymethyl)oxime.

Geometrical isomers of testosterone 3-(O-carboxymethyl)oxime and their histamine derivatives were purified on reverse-phase high pressure liquid chromatography, and their antibody binding characteristics were studied. Using a competitive testosterone enzyme immunoassay, the unfractionated mixture of the oximes showed 75% cross-reactivity with respect to testosterone, whereas the isolated 3Z- and 3E-isomers showed 124% and 26% cross-reactivity, respectively. The cross-reactivity was increased in the histamine derivatives, but the difference in cross-reactivity of the two isomers was reduced. Suppression of the ionization of the carboxyl group by lowering the pH of the incubation mixture in the antigen-antibody binding step raised the cross-reactivity of the mixture of free oximes to 128%, at pH 4.0. Thus, the geometry and ionization state of the carboxymethyl oxime group has a profound effect on the affinity of the isomers for the antibody.     

Radioimmunoassay of contraceptive steroids. II. Synthesis of 6,7-3H-mestranol and ethinylestradiol of high specific activity

This paper describes a simple and convenient method for the preparation of tritiated mestranol and ethinylestradiol of high specific activity as a prerequisite in the development of the radioimmunoassay of the unlabelled compounds. Radioactive mestranol was prepared from estrone-6,7-³H (40 Ci/mmole) by methylation followed by ethinylation and purification by TLC. Radioactive ethinylestradiol could not be made from this mestranol by demethylation with boron tribromide, but it could be made directly from estrone-6,7-³H by ethinylation, removing the unreacted estrone by Girard's reagent T and purifying the product by TLC. Purities of these compounds were checked by TLC in two solvent systems and finally by the isotopic dilution technique. The specific activity of the final products was calculated by determining the loss of tritium during each step of reaction with a double isotopic method.     

Selective 3-(O-carboxymethyl)oxime formation in steroidal 3,20-diones for hapten immunospecificity

The direct one-step synthesis of 3-(O-carboxymethyl)oximes of representative C₂₁-4-pregnen-3,20-diones is reported. The method requires the preparation of a 3-enamine derivative which, serving as an intermediate product, is readily converted to the 3-(O-carboxymethyl)oxime upon addition of one molar equivalent of O-(carboxymethyl)hydroxylamine hemihydrochloride. The reaction appears to be generally applicable for selective 3-(O-carboxymethyl)oxime formation in steroids possessing multi-carbonyl groups, thus facilitating the coupling of steroidal haptens to protein at the C-3 position of the steroid molecule for enhanced immunospecificity. In this manner, antisera to 16α-hydroxyprogesterone and 17α-hydroxy-progesterone were obtained from immunized rabbits and specificity was established by radioimmunoassay.     

Sulphation of contraceptive steroids

The sulphation of a number of contraceptive steroids by rabbit tissue in vitro was investigated. With liver tissue the three synthetic gestagens (norethisterone, norgesterel and lynestrenol) were sulphated at different rates and none was sulphated as rapidly as dehydroepiandrosterone; sulphation occurred at the tertiary 17 beta-hydroxyl group. The synthetic oestrogen ethynyloestradiol was sulphated more rapidly than dehydroepiandrosterone, both mono and disulphates being formed. Of the other tissues studied, sulphation occurred with stomach and lung but not with heart, spleen, muscle, kidney or adipose tissue. These in vitro studies provide confirmation of in vivo findings regarding sulphate conjugates of the synthetic steroids.     

A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(O-carboxymethyl) oximino-[125I]-iodo-histamine as radioligand.

A reliable, convenient and economical radioimmunoassay (RIA) for serum progesterone has been established and tested. This procedure employs diethyl ether extraction followed by RIA utilizing rabbit anti-11 alpha-hydroxyprogesterone 11-hemisuccinyl-bovine serum albumin (progesterone-11 alpha-BSA) serum, progresterone-3-(O-carboxymethyl) oximino-[125I]-iodohistamine (progesterone-3-[125I]) as radioligand and goat anti-rabbit gamma globulin as second antibody. In conjunction with antiprogesterone-11 alpha-BSA serum, the overall assay specificity of the progesterone-3-[125I] RIA is similar to that of the [3H]-progesterone method using dextran-coated charcoal. The results of serum progesterone measurements during the menstrual cycle obtained by the progesterone-3-[125I] RIA appear comparable to those of [3H]-progesterone assays which employ similar anti-progesterone-11 alpha-BSA sera. The progesterone-3-[125I] double-antibody RIA, however, is more convenient and less expensive than the [3H]-progesterone RIA method.     

Preparation and antigenic properties of 5alpha-dihydrotestosterone-11-(O-carboxymethyl) oxime-BSA conjugate.

The C-11 (O-carboxymethyl) oxime derivative of 5-alphadihydrotestosterone (5alphaDHT) has been prepared. Due to steric hindrance at C-11, a novel two step procedure was used to introduce the (O-carboxymethyl) oxime at this position. Condensation of this oxime to bovine serum albumin afforded a conjugate which produced anti-5alphaDHT sera inoculated rabbits. Apart from a 30% cross reaction with testosterone, the antisera was reasonably specific for 5alphaDHT.     

The beagle dog and contraceptive steroids.

The mandatory use of beagle dogs for chronic toxicological testing of contraceptive steroids has eliminated some useful products from the market, and stopped development of new compounds. Mammary tumour formation in beagle bitches is induced by some progestogens, including progesterone, but not in other laboratory species, such as rodents or monkeys. Most beagle mammary tumours induced by progestogens are of the benign mixed type, though a small minority are malignant. As all the evidence to date suggests that oral contraceptives have a protective effect on the occurrence of breast neoplasms in women, the beagle findings are irrelevant to the human situation. Possible reasons for the unique response of the beagle to progestogens are discussed.     

Synthesis and stereochemistry of C-3- and C-7-linked (O-carboxymethyl)-oximino- and hemisuccinamido derivatives of 5 alpha-dihydrotestosterone.

The 3-(O-carboxymethyl)oximino derivative of 17β-hydroxy-5α-androstan-3-one (5α-dihydrotestosterone) was prepared. Thin-layer chromatography of the corresponding methyl ester showed the presence of two syn (60%) and anti (40%) geometrical isomers of the oxime chain to the C-4 position, which were characterized by ¹³C nmr. The 3β-hemisuccinami-do-5α-androstan-17β-ol was obtained after selective saponification with potassium carbonate of the 17β-hemisuccinate group of the 3,17-dihemi-succinoylated derivative of the previously described 3β-amino-5α-androstan-17β-ol. This 3β-hemisuccinamide was purified as the corresponding methyl ester-17β-acetate and was regenerated after saponification. The 3,3'-ethylenedioxy-7-oxo-5α-androstan-17β-yl acetate was obtained in quantitative yield by catalytic hydrogenation over 10% palladium-oncharcoal of the Δ⁵-7-oxo precursor in a dioxane-ethanol mixture containing traces of pyridine. The exclusive 5α-configuration of this hydrogenated product was established from nmr data and was confirmed by the synthesis of methyl 3,3'-ethylenedioxy-7-oxo-5β-cholan-24-oate as 5β-H-reference compound. The preceding 5α-H-7-ketone was converted into the 7-(O-carboxymethyl)oximino derivative (syn isomer to the C-6 position, exclusively) which was esterified into the corresponding methyl ester. The selective hydrolysis of the 3-ethyleneketal group was achieved by a short treatment with a formic acid-ether 1:1 (v/v) mixture at 20°C. Saponification of the latter reaction product with ethanolic potassium hydroxide gave the 7-(O-carboxymethyl)oximino-17β-hydroxy-5α-androstan-3-one derivative, which was characterized as the corresponding methyl ester. The reduction of the oxime of the 5α-H-7-ketone with sodium in ethanol or with lithium-aluminium hydride gave respectively the 7β-amine or the 7α-amine as the major product. The 7β- and 7α-configurations were established from nmr spectra of the corresponding 7-acetamido derivatives. The 7β- and 7α-hemisuccinamido derivatives were prepared from the mixture of 7β- and 7α-amines, as described above for 3-derivatives and were isolated after thin-layer chromatography of the methyl esters, followed by saponification of the corresponding 17β-acetates.     

Synthesis of 6-substituted uridines. synthesis of (R or S)-6-(3-amino-2-carboxypropyl)uridine

Addition of 5-bromo-2′,3′-O-isopropylidene-5′-O-trityluridine (2) in pyridine to an excess of 2-lithio-1,3-dithiane (3) in oxolane at 78° gave (6R)-5,6-dihydro-(1,3-dithian-2-yl)-2′,3′-O-isopropylidene -5′-O-trityluridine (4), (5S,6S)-5-bromo-5,6-dihydro-(1,3-dithian-2-yl)-2′,3′-O-isopropylidene-5′-O-trityluridine (5), and its (5R) isomer 6 in yields of 37, 35, and 10%, respectively. The structure of 4 was proved by Raney nickel desulphurization to (6S)-5,6-dihydro-2′,3′-O-isopropylidene-6-methyl-5′-O-trityluridine (7) and by acid hydrolysis to give D-ribose and (6R)-5,6-dihydro-6-(1,3-dithian-2-yl)uracil (9). Treatment of 4 with methyl iodide in aqueous acetone gave a 30&%; yield of (R,S)-5,6-dihydro-6-formyl-2′,3′-O-isopropylidene-5′-O-trityl-uridine (10), characterized as its semicarbazone 11. Both 5 and 6 gave 4 upon brief treatment with Raney nickel. Both 5 and 6 also gave 6-formyl-2′,3′-O-isopropylidene-5′- O-trityluridine (12) in ～41%; yield when treated with methyl iodide in aqueous acetone containin- 10%; dimethyl sulfoxide. A by-product, identified as the N-methyl derivative (13) of 12 was also formed in yields which varied with the amount of dimethyl sulfoxide used. Reduction of 12 with sodium borohydride, followed by deprotection, afforded 6-(hydroxymethyl)uridine (17), characterized by hydrolysis to the known 6-(hydroxymethyl)uracil (18). Knoevenagel condensation of a mixture of the aldehydes 12 and 13 with ethyl cyanoacetate yielded 38%; of E- (or Z-)6-[(2-cyano-2-ethoxycarbonyl)ethylidene]-2′,3′-O-isopropylidene-5′-O-trityluridine (19) and 10%; of its N-methyl derivative 20. Hydrogenation of 19 over platinum oxide in acetic anhydride followed by deprotection gave R (or S)-6-(3-amino-2-carboxypropyl)uridine (23).     

An Improved Synthesis of N6-(6-Aminohexyl)FAD

Abstract

We report an improved synthesis of N ⁶-(6-aminohexyl)FAD (1) using an efficient one-pot conversion of inosine to the N-trifluoroacetyl protected N ⁶-(6-aminohexyl)adenosine 3. The 5′-O-phosphorylated AMP derivative 4, activated as the imidazolide, was coupled with commercial sodium riboflavin phosphate by using 18-crown-6 in DMF.     

Radioimmunoassay of nafarelin ([ 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum

A procedure which is suitable for the radioimmunoassay (RIA) of nafarelin [( 6-(3-(2-naphthyl)-D-alanine)]-luteinizing hormone-releasing hormone) in plasma or serum at concentrations as low as 50 pg/ml is described. Antiserum was prepared by replacing the pyroglutamyl portion of nafarelin with glutaric acid, coupling the product to keyhole limpet hemocyanin, and immunizing rabbits with the resulting conjugate. At a dilution of 1:30,000 the binding was approximately 50%. The antibodies did not cross react with luteinizing hormone-releasing hormone. For RIA, 125I-labeled analyte was used as the tracer and charcoal was used to separate the free and the bound fractions. No purification of samples was required prior to RIA. Accuracy of the method was assessed by adding known quantities of nafarelin to nafarelin-free plasma and determining the ratio of measured to added analyte. Linear regression analysis for the concentration range 0.050-5.00 ng/ml yielded a regression equation of y = 1.01x - 0.066 and a correlation coefficient of 0.997. At 0.050 ng/ml the CV was 11.3% (interassay). Additional validation was obtained from an in vivo study in which [3H]nafarelin was administered to monkeys and plasma profiles were determined by RIA, by high-performance liquid chromatography (HPLC), and by an HPLC-radiochemical method. The results obtained by RIA agreed well with those obtained by the HPLC methods.     

Synthesis, cytostatic and anti-HCV activity of 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides

An efficient and facile synthesis of a large series of diverse 6-(N-substituted aminomethyl)-, 6-(O-substituted hydroxymethyl)- and 6-(S-substituted sulfanylmethyl)purine nucleosides (55 examples of both ribo- and 2'-deoxyribonucleosides), aimed at identifying novel homologues of natural nucleosides, was developed. The key transformation involved nucleophilic substitutions of Tol-protected 6-(mesyloxymethyl)purine nucleosides by primary or secondary amines, alcoholates or thiolates. While the 2'-deoxyribonucleosides were inactive, the ribonucleosides exerted considerable cytostatic effects and some anti-HCV activity with low selectivity.