Long-acting contraceptive agents: design of the WHO Chemical Synthesis Programme

The great demand for improved long-acting injectable steroid contraceptives, particularly in developing countries, and the relative lack of interest from the pharmaceutical industry to develop such products stimulated WHO to launch a synthetic and screening programme to find improved, safe and acceptable injectable preparations. More than 210 esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) and levonorgestrel (D-(-)-13 beta-ethyl-17 alpha-ethynyl-17 beta-hydroxygon-4-en-3-one) have been prepared in university-based research laboratories situated mainly in developing countries, and then screened by NICHHD in animal models. The following three compounds, levonorgestrel butanoate, cyclopropylcarboxylate and cyclobutylcarboxylate, proved to be particularly long-acting when administered as microcrystalline suspensions. The overall strategy of this research and development programme is described.     

Long-acting contraceptive agents: norethisterone esters of monoalkenyl and monoalkynyl acids

The synthesis of nine new esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) is described, with the esterifying acids bearing an acetylenic or olefinic function in a chain of eight or nine carbon atoms, for evaluation as long-acting contraceptive agents.     

Long-acting contraceptive agents: Structure activity relationships in a series of norethisterone and levonorgestrel esters

A large number of esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) and levonorgestrel (D-(-)-13β-ethyl-17α-ethynyl-17β-hydroxygon-4-en-3-one) were synthesized and tested for biological activity. The test employed in these studies was the duration of estrus suppression in cycling mature rats. In the norethisterone series several esters exhibited duration of activity comparable to that of norethisterone enarthate. In the levonorgestrel series the butanoic, cyclobutylcarboxylic and cyclopropylcarboxylic esters were longer acting than medroxyprogesterone acetate (17α-acetoxy-6α-methylpregn-4-ene-3, 20-dione) when prepared as aqueous microcrystalline suspensions.     

Long-acting contraceptive agents: esters of norethisterone with alpha- and/or beta-chain branching

The synthesis of eighteen esters of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) is described. These all possess some form of alpha- and/or beta-substitution in the ester side-chain. The work was undertaken in order to evaluate any long-acting fertility control effect intrinsic in such compounds. A pentamethyl disiloxy ether was also included in the group of substances prepared for testing because of its similar substitution pattern.     

Long-acting contraceptive agents: Aliphatic and alicyclic carboxylic esters of levonorgestrel

Esters of levonorgestrel (13β-ethyl-17α-ethynyl-17β-hydroxygon-4-en -3-one) with a variety of aliphatic and alicyclic carboxylic acids have been prepared and characterised. In tests for the suppression of estrus in rats, esters with short-chain aliphatic acids and with cyclobutane-carboxylic acid were considerably more active than the standard, norethisterone enanthate (17α-ethynyl-17β-hydroxyestr-4-en-3-one). Such esters show great promise for development as long-acting progestogens.     

Long-acting contraceptive agents: Analysis and purification of steroid esters

More than 200 samples of esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) and levonorgestrel (13β-ethyl-17α-ethynyl-17β-hydroxygon-4-en-3-one) have been analysed by a combination of techniques, including high performance liquid chromatography (HPLC). Compounds having a purity below the required limit (99.5%) were purified, mainly by preparative HPLC, prior to formulation and biological evaluation as long-acting progestogens.     

Long-acting contraceptive agents: carbonates and carbamates of norethisterone

The preparation of three carbonates and two carbamates of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) are described. Due to instability of the carbonates and the very low solubility of the carbamates these compounds could not be submitted to biological testing.     

Long-acting contraceptive agents: norethisterone esters of polyunsaturated acids

Some new derivatives of norethisterone (17 alpha-ethynyl-17 beta-hydroxyestr-4-en-3-one) are described in which the 17 beta-hydroxyl group of the steroid is esterified with polyunsaturated aliphatic acids. The potential of these compounds as long-acting contraceptive agents has been evaluated.     

Microbial transformation of 17alpha-ethynyl- and 17alpha-ethylsteroids, and tyrosinase inhibitory activity of transformed products

The microbial transformation of the 17alpha-ethynyl-17beta-hydroxyandrost-4-en-3-one (1) (ethisterone) and 17alpha-ethyl-17beta-hydroxyandrost-4-en-3-one (2) by the fungi Cephalosporium aphidicola and Cunninghamella elegans were investigated. Incubation of compound 1 with C. aphidicola afforded oxidized derivative, 17alpha-ethynyl-17beta-hydroxyandrosta-1,4-dien-3-one (3), while with C. elegans afforded a new hydroxy derivative, 17alpha-ethynyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (4). On the other hand, the incubation of compound 2 with the fungus C. aphidicola afforded 17alpha-ethyl-17beta-hydroxyandrosta-1,4-dien-3-one (5). Two new hydroxylated derivatives, 17alpha-ethyl-11alpha,17beta-dihydroxyandrost-4-en-3-one (6) and 17alpha-ethyl-6alpha,17beta-dihydroxy-5alpha-androstan-3-one (7) were obtained from the incubation of compound 2 with C. elegans. Compounds 1-6 exhibited tyrosinase inhibitory activity, with compound 6 being the most potent member (IC(50)=1.72 microM).     

The chemistry of 9 alpha-hydroxysteroids. 1. Preparation of 9 alpha,17 beta-dihydroxy-17 alpha-ethynylandrost-4-en-3-one

9 alpha-Hydroxyandrost-4-ene-3,17-dione 1, when allowed to react with dipotassium acetylide in tetrahydrofuran, resulted, after chromatographic separation, in 4-methyl-19-norandrosta-4,9-diene-1,17-dione 2, 4 xi-methyl-19-norandrosta-5(10),9(11)-diene-1,17-dione 3, 4-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-4,9-dien-1-one 4, 4 xi-methyl-17 alpha-ethynyl-17 beta-hydroxy-19-norandrosta-5(10),9(11)-dien- 1-one 5, and 17 alpha-ethynyl-17 beta-hydroxy-9,10-secoandrost-4-ene-3,9-dione 6. Selective protection of delta 4-3-ketone of 9 alpha-hydroxyandrost-4-ene-3,17-dione 1 as its dienol methyl ether 7, and subsequent reaction with lithium acetylide-ethylenediamine followed by acidic hydrolysis, afforded 9 alpha,17 beta-dihydroxy-17 alpha- ethynylandrost-4-en-3-one 8.     

Long-acting contraceptive agents: Bile acid esters of norethisterone

The synthesis of the esters of norethisterone (17α-ethynyl-l7β-hydroxyestr-4-en-3-one) with three bile acids and of the cholesteryl carbonate of norethisterone are described.     

Long-acting contraceptive agents: Cyclopropyl and cyclobutyl esters of norethisterone

Several esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) with carboxylic acids containing a cyclopropyl or cyclobutyl ring have been synthesized and the stereochemistries of the side-chains determined.     

Long-acting contraceptive agents: Norethisterone esters of arylcarboxylic acids

The synthesis of esters of norethisterone (17α-ethynyl-17β-hydroxy-estr-4-en-3-one) with acids containing a benzene ring is described, two methods of esterification being compared in terms of yield and convenience. The activities of these esters as long-acting contraceptive agents have been evaluated.     

Long-acting contraceptive agents: Esters of norethisterone with alkoxy- and halogeno-substituted carboxylic acids

The chemical synthesis and physical data of several new esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) are reported, which contain either a chloro- or an alkoxy-group as a substituent in the acid side-chain.     

Long-acting contraceptive agents: The influence of pharmaceutical formulation upon biological activity of esters of norethisterone

17β-esters of norethisterone (17α-ethynyl-17β-hydroxyestr-4-en-3-one) have been formulated as aqueous microcrystalline suspensions and oily solutionsfor administration to rats to assess the length of progestogenic activity. Results show that, for some of the esters, the rate-controlling step in prolonging activity is the rate of drug release from the injected formulation. For these esters, when formulated as suspensions, it is proposed that crystal size and form will have a critical effect upon duration of estrus suppression. The influence of crystal form has been demonstrated with the 4-(butoxy)phenylacetate ester for which two different crystal forms have been identified. The lower melting point, more soluble crystal form shows marked prolongation of action, whereas the other form is ineffective.     

The chemistry of 9 alpha-hydroxy steroids. 2. Epimerization and functionalization of 17 alpha-ethynylated 9 alpha-hydroxy steroids

Practical routes to 9 alpha-hydroxypregnenes were developed by epimerization and hydration of 17 alpha-ethynyl-9 alpha,17 beta-dihydroxyandrost-4-en-3-one. In the three different methods of epimerization which were used, the C-9 alpha hydroxy group was not susceptible to rearrangement or other side reactions. C-21 functionalized 9 alpha-hydroxypregnenes were obtained by introducing a 17 alpha-halogenated ethynyl group into 9 alpha-hydroxyandrost-4-ene-3,17-dione. Epimerization and hydration by the 17 beta-nitrooxy method produced 21-halogenated 9 alpha-hydroxypregnenes, which were further converted into 21-acetoxy-9 alpha-hydroxypregn-4-ene-3,20-dione.     

Synthesis and stereochemistry of 7β- and 7α-amino-, acetamido-, hemisuccinamido- and terephthalamido derivatives of testosterone

The reduction of 3-ethylenedioxy-7-oximino-5-androsten-17β-yl acetate and of its 17β-tetrahydropyranyl ether analog with sodium in ethanol, followed by thin-layer chromatography, allowed the isolation of the corresponding 17β-hydroxy- and 17β-tetrahydropyranyioxy-5-en-7β- and 7α-amines which were also characte-rized as 7-acetamides. The acylation of the two epimeric 17β-hydroxy-5-en-7-amines with succinic anhydride followed by selective saponification of the 17β-hemisuccinate group and diazomethane esterification, gave the corresponding 17β-hydroxy-5-en-7β- and 7α-hemisuccinamido methyl esters characterized also as 17β-acetates. On the other hand, the acylation of the two 17β-tetrahydropyranyl-oxy-5-en-7-amines with the acid chloride of terephthalic acid monomethyi ester led to the more rigid 7β- and 7α-terephthalamido methyl ester side-chains. The acidolysis of the 3-ethyleneketal protecting group of the preceding 5-en-7-N-acyl derivatives regenerated the 4-en-3-oxo function while the 17β-tetrahydropyranyl ether group was cleaved simultaneously into the 17β-alcohol. The four desired 7β- and 7α-hemisuccinamido- and terephthalamido carboxylic side-chain derivatives of 17β-hydroxy-4-androsten-3-one (testosterone) were finally obtained by saponification of the corresponding methyl esters.     

Metabolism of norethynodre, a 19-nor progestin: subcellular localization of enzyme activity

The subcellular distribution of the enzymes involved in the metabolism of norethynodrel (17 alpha-ethynyl-17 beta-hydroxy-estr-5(10)-en-3-one) to the 3alpha and 3beta diols (17 alpha-ethynyl-3alpha (or 3beta-17 beta-dihydroxy-estr-5(10)-ene) and 17 alpha-ethinyl estradiol was studied. The purity of the male rat liver subcellular fractions was evaluated by the use of marker enzymes. Sample sections were viewed by electron microscopy. The data showed that the cytosol fraction contained the highest relative specific activity for the hydroxysteroid dehydrogenases required for the formation of the diols. The cytosol fraction also contained the highest total activity. The enzymes required for the formation of ethinyl estradiol were distributed equally among mitochondrial and microsomal fractions, however, the highest relative specific activity was associated with the heavy microsomal fraction (18,000 g).     

Long-acting contraceptive agents: Levonorgestrel esters of unsaturated acids

Esters of levonorgestrel (13β-ethyl-17β-ethynyl-17β-hydroxygon-4-en-3-one) with a variety of unsaturated carboxylic acids have been synthesized for evaluation as potential long-acting, injectable contraceptive agents.     

Potential long-acting contraceptive agents: esters of testosterone with alkoxy- and halogeno-substituted carboxylic acids

The chemical synthesis and physical data of several new esters of testosterone (17 beta-hydroxyandrost-4-en-3-one), which contain either a halogeno or an alkoxy substituent in the acid chain, are reported.