A direct stereoselective preparation of a fish pheromone and application of the zinc porphyrin tweezer chiroptical protocol in its stereochemical assignment.

A two-step stereoselective preparation of a goldfish pheromone, 17α,20ß-dihydroxy-4-pregnen-3-one, is reported from the readily available cortexolone in 64% overall yield. The (20S)-epimer was also synthesized in three steps from cortexolone with an overall yield of 47%. A microscale chiroptical technique based on a host/guest complexation mechanism between the substrate and a dimeric metalloporphyrin host (tweezer) was used to confirm the stereochemical assignment, while Density Functional Theory (DFT) calculations were employed to explain the high stereoselectivity induced by the 17α-hydroxyl and C18-methyl groups.

Derivatization of 11-alpha-hydroxyprogesterone using phosphoramidite chemistry.

The well-known phosphoramidite chemistry routinely used in solid-phase oligonucleotide synthesis is exploited here in the preparation of steroid conjugates in solution. The applicability of the method is tested by conjugating one nucleosidic and one non-nucleosidic phosphoramidite building block to 11-alpha-hydroxyprogesterone. The suitability of one of the conjugates synthesized (5) for a competitive binding assay is also demonstrated.

Biocatalytic synthesis of 4-pregnen-20,21-diol-3-one, a selective inhibitor of human 5alpha-reductase type II.

Biocatalysis, the conversion of substrates into valuable products by the use of enzymes, has some striking advantages in comparison to standard organic chemistry for drug synthesis. By biocatalysis, substrates that contain several identical reactive groups at different positions can be converted with high regio-selectivity and enantio-selectivity. In this study, an E. coli isolate (E132) was identified which was able to convert the steroid desoxycorticosterone into the product 4-pregnen-20,21-diol-3-one in real terms. The product was purified from the cell culture supernatant by HPLC and its structure was demonstrated by mass spectrometry and NMR spectroscopy. It was tested on inhibition of human 5alpha-reductases type I and type II. At a concentration of 10 microM, inhibition was 49.0% for type I and 81.8% for type II, whereas there was no inhibition of human aromatase (CYP19) at 20 microM and human 17alpha-hydroxylase-C17,20-lyase (CYP17) at 2.5 microM detectable. The IC50 value of 4-pregnen-20,21-diol-3-one for human 5alpha-reductase type II was determined to be 1.56 microM.

Guidelines for analytical method development and validation of biotechnological synthesis of drugs. Production of a hydroxyprogesterone as model.

In connection with biotechnological synthesis of pharmaceutical drugs, validated methods for quantification of both product and substrate at different time intervals are essential for proper calculation of rate coefficients. In this field, there still exist no guidelines for analytical validation, unlike the situation in the bioanalytical field. Therefore, in this study the detailed guidelines by FDA for bioanalytical method validation were applied to a typical biotechnological process; the enzymatic synthesis of 9alpha-hydroxyprogesterone in E. coli using progesterone as substrate. The process liquid was extracted and analyzed using an HPLC-DAD system. The quality control (QC) samples of the product demonstrated excellent precision (C.V.<1.5%) and accuracy between 99.3 and 107%. The study showed that the recommendations and the validation terms for bioanalytical methods can be used also for biotechnological production, but with some important exceptions. The tolerances (C.V. values) of the validation terms should be much narrower; the internal standard (I.S.) must be present in the process liquid before the start of the process and must be much different in structure from the substrate (so as not to participate in the biotechnological process). In addition, the selectivity must be checked very frequently during the process due to the changes in the blank process liquid with time.

Facile C21 functionalization through a novel functional group transfer reaction in 16 alpha,17 alpha-epoxy-3 beta-hydroxypregn-5-en-20-one and its applications.

A novel functional group transfer reaction in 16 alpha,17 alpha-epoxy-3 beta-hydroxypregn-5-en-20-one by treatment with dry HCl affords 21-chloro-3 beta-hydroxy-pregn-5,16-dien-20-one, which has been utilized to obtain a number of C21-substituted derivatives.

Synthesis of oxido-bridged analogs of 18-hydroxyprogesterone.

18-Hydroxy-6,19-oxidoprogesterone and 18-hydroxy-11, 19-oxidoprogesterone were synthesized from readily available materials. The functionalization of C-18 was accomplished with phenyliodosodiacetate/iodine, whereas that of C-19 was carried out with the mercuric oxide/iodine system, both under irradiation with visible light. For 18-hydroxy-11,19-oxidoprogesterone, C-19 was functionalized before C-18, whereas the reverse order had to be used for the 6,19-oxido derivative.

A rapid approach to 11 alpha-hemisuccinylprogesterone synthesis.

The synthetic hapten 11 alpha-hemisuccinylprogesterone (11 alpha-hemisuccinyl-pregn-4-ene-3,20-dione), when linked to the appropriate macromolecular carrier, has been used successfully as a solid-phase antigen for progesterone detection in immunoassay. In this study the synthesis of 11 alpha-hemisuccinylprogesterone from 11 alpha-hydroxyprogesterone has been improved by using 4-dimethylaminopyridine (DMAP in refluxing dioxane, a highly nucleophilic polar solvent.

Influence of functional groups on homologous antibody affinity of 11 alpha-hydroxyprogesterone derivatives. I. Synthesis and affinity evaluation.

Syntheses and cross-reactivities with progesterone toward the same specific antibody are reported for a series of amides of 11 alpha-hydroxyprogesterone 11-hemisuccinate. Some hypotheses are made regarding the effects of the chemical structure of the substituents on the immunological properties of derivatives.

Synthesis of deuterium-labeled 17-hydroxyprogesterone suitable as an internal standard for isotope dilution mass spectrometry.

A synthesis is reported of 17-hydroxyprogesterone, labeled with four atoms of deuterium at ring C and suitable for use as an internal standard for isotope dilution mass spectrometry. Base-catalyzed equilibration of methyl 3 alpha-acetoxy-12-oxo-cholanate (III) with 2H2O, followed by reduction of the 12-oxo group by the modified Wolff-Kisher method using [2H]diethylene glycol and [2H]hydrazine hydrate afforded [11,11,12,12,23,23(-2)H]lithocholic acid (V). The Meystre-Miescher degradation of the side chain of V yielded 3 alpha-hydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (X). Oxidation of the 3,20-enol-diacetate of X with perbenzoic acid followed by saponification afforded 3 alpha,17-dihydroxy-5 beta-[11,11,12,12(-2)H]pregnan-20-one (XI). Oxidation of XI with N-bromoacetamide yielded 17-hydroxy-5 beta-[11,11,12,12(-2)H]pregnane-3,20-dione (XII). Bromination of XII followed by dehydrobromination yielded 17-hydroxy-[11,11,12,12(-2)H] progesterone (XIV), consisting of 0.3% 2H0-, 1.1% 2H1-, 8.6% 2H2-, 37.1% 2H3-, 52.1% 2H4-, and 0.8% 2H5-species.

Total synthesis of (+/-)-11 alpha-hydroxyprogesterone by cyclization of a polyunsaturated epoxide.

The total synthesis of a typical 11-hydroxylated steroid, (+/-)-11 alpha-hydroxyprogesterone, was achieved by picric acid-catalyzed tricyclization of a polyunsaturated epoxide appropriately substituted with ketal, hydroxyl, and acetylenic units. This epoxide was prepared by a multistage sequence featuring two successive alkylations of intermediary, monocyclic sulfones. The first sulfone intermediate was obtained by means of a short sequence starting from levulinic acid and diethyl succinate and involving a selective cyclization reaction.

Preparation of 11 alpha-hemisuccinyl-progesterone for immunological purposes and structural determination of the hapten.

Progress in reproductive endocrinology to some extent is due to the application of immunogens produced from synthetic haptens. In this preliminary study the synthesis of the hapten 11 alpha-hemisuccinyl-progesterone (11 alpha-hemisuccinyl-pregn-4-ene-3,20-dione) from 11 alpha-hydroxy-progesterone and its complete structure elucidation by u.v./VIS, i.r., [1H]NMR-spectroscopy and E.I.-mass-spectrometry is described. Estimates of sample purity are given.

Solid phase fluoroimmunoassay for 11-deoxycortisol in serum using 21-amino-17-hydroxyprogesterone.

Solid phase fluoroimmunoassay of serum 11-deoxycortisol (17,21-dihydroxy-4-pregnene-3,20-dione) was established using fluorescein isothiocyanate-labelled 11-deoxycortisol and anti-11-deoxycortisol antibody-conjugated polyacrylamide beads. 21-Amino-17-hydroxyprogesterone (21-amino-17-hydroxy-4-pregnene-3,20-dione) was synthesized as a useful derivative for preparing the fluorescent dye conjugate. Serum 11-deoxycortisol was measured with this assay system after extraction and purification by Sephadex LH-20 column chromatography. The minimal amount of 11-deoxycortisol detected was 40 pg/tube and the measurable range was from 0.04 to 5.0 microgram/dl. Intra- and inter-assay coefficients of variation were 8.3% (n=6) and 9.8% (n=5), respectively. 11-deoxycortisol values determined by the present assay correlated well with those determined by radioimmunoassay. The present assay is particularly suitable for estimating the conditions of the pituitary and adrenocortical functions.

Synthesis of potential antiprogestins II.

Alkylated derivatives of 17-acetoxyprogesterone were prepared in order to test the hypothesis that bulky groups in certain positions of the steroid molecule have the effect of transforming progestogens into antiprogestogens. These groups might exert binding influence outside the area occupied by progesterone itself. The compounds were tested for competitive affinity against tritiated progesterone and receptor from rabbit uterus cytosol. The low affinity of all derivatives makes it unlikely that they would be active as antiprogestational agents.

7 alpha- and 7 beta-carboxymethyl-derivatives of 17-hydroxyprogesterone and 11-deoxycortisol. Synthesis and immunogenic properties.

7 alpha- and 7 beta-carboxymethyl-derivatives of 17-hydroxyprogesterone and 11-deoxycortisol have been synthesized. After coupling to bovine serum albumin, they were used to elicit antibodies in rabbits. No major difference in the steroid specificity of the antisera was observed when either 7 alpha- or 7 beta-epimers were used for immunization. In both cases, highly specific antisera were obtained which may possibly be used to assay human plasma 17-hydroxyprogesterone and 11-deoxycortisol without chromatographic purification.

Steroids and related products. XLVI. The regiospecific alpha-hydroxymethylation of saturated carbonyl compounds via preformed enolate ions. An improved synthesis of 17-hdyroxymethyl 20-keto steroids.

As demonstrated for pregnenolone, saturated ketones are conveniently alpha-hydroxymethylated by their transformation into a lithium enolate and by the reaction of the latter with formaldehydr. The 17-hydroxymethylpregnenolone prepared by this method in very good yield was readily converted to 17-hydroxymethylprogesterone; either by selective acetylation in position 17(1) and subsequent Jones oxidation, followed by hydrolysis, or by conversion to the 4,5-dibromo 3-ketone - by bis(tri-n-butyltin)oxide-bromine oxidation or by dibromination and oxidation with N-bromoacetamde - and debromination with zinc and acetic acid.

Synthesis of 18-substituted steroids Part II (1). Improvements in the preparation of 18-hydroxyprogesterone.

18-Hydroxyprogesterone is conveniently prepared from 3beta-acetoxypregn-5-en-20beta-ol by a modified route. 3beta-Acetoxy-18-iodopregn-5-en-20-one, obtained by the hypoiodite-photolysis procedure and oxidation, is treated with methanolic silver acetate to give the 18, 20-epoxy-20-methoxy derivative, which crystallises directly without need for chromatography. Hydrolysis of the 3-acetate, and a modified Oppenauer oxidation, gave 18-hydroxy-progesterone in 24% over-all yield.