The synthetic progestin norgestrel acts to increase LIF levels in the rd10 mouse model of retinitis pigmentosa.

PURPOSE: Retinal degenerative conditions affect thousands of people worldwide. Retinitis pigmentosa (RP) is among the most common, but it is currently incurable. It is characterized by the progressive death of photoreceptor cells, eventually leading to blindness. Neurotrophic factors play an important role in such retinopathies, and much research has been performed on their use as treatments. Our group previously demonstrated the ability of the synthetic progestin norgestrel to rescue photoreceptors from cell death, the mechanism of which is believed to include upregulation of the neurotrophic factor basic fibroblast growth factor (bFGF). The objective of the present study was to investigate whether the protection provided by norgestrel is likely to be mediated by other neurotrophins. METHODS: The 661W photoreceptor cells and retinal explants from P30 to P40 wild-type (wt) C57BL/6 mice were treated with norgestrel over time. Homozygous rd10/rd10 mice that mimic the human form of RP were fed either a control or a norgestrel-containing diet. Changes in neurotrophic factor expression in response to norgestrel were detected with real-time PCR, western blotting, or immunofluorescence staining. Using specific siRNA, leukemia inhibitory factor (Lif) expression was knocked down in 661W photoreceptor cells that were stressed by serum starvation. Cells were treated with norgestrel followed by measurement of cell viability with (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) assay. RESULTS: LIF, a potent neuroprotective cytokine, was found to be upregulated in response to norgestrel in vitro and in vivo. Upregulation of LIF in degenerating rd10 retinas coincided with preservation of the photoreceptor layer. We also found LIF was necessary for the norgestrel-mediated rescue of stressed photoreceptor cells from cell death in vitro. CONCLUSIONS: LIF was upregulated in response to norgestrel in all models studied and is necessary for the protective effects of norgestrel in vitro. The increase in LIF expression in rd10 mice undergoing retinal degeneration was concurrent with rescue of the photoreceptor cell layer. These results highlight the ability of norgestrel to induce prosurvival molecules in the compromised retina, underlining norgestrel's potential as a viable drug for treatment of RP.

Steroidal silicon side-chain analogues as potential antifertility agents.

A number of silicon-substituted analogues of ethynylestradiol that exhibit modified and enhanced biological activities have been synthesized. Particularly noteworthy are a group of [(trialkylsilyl)ethynyl]estradiol analogues that exhibit high antifertility potency and markedly reduced estrogenic activity. The best compounds synthesized are 17 alpha-[(triethylsilyl)ethynyl]estradiol (5) and 17 alpha-[(tert-butyldimethylsilyl)ethynyl]estradiol (33), which show a separation of antifertility from estrogenic activity in the rat. The results of structure-activity studies indicate a good correlation between the observed biological activities and the calculated van der Waals volumes of the three variable silicon substituents.

Potential antifertility agents. 6. Synthesis and biological activities of optical isomers of 4 beta-(p-methozyphenyl)-2 beta-methylcyclohexane-alpha-carboxylic acid and related compounds.

PIP: The optical isomers of the title compound were synthesized and the biological potencies of the two enantiomers were compared. There was essentially no difference in their hypocholesterolemic activities, as had been predicted, and little or no difference between their uterotropic potencies. The approximately equal uterotropic activities seen with the enantiomers is explained in terms of stereochemical requirements at the receptor level for an estrogenic response. A working model of an estrogenic receptor is proposed. An accompanying paper provides support for the proposed model.^ieng

Contraceptive agents from cycloaddition reactions of diarylcyclopropenones and diarylthiirene 1, 1-dioxides.

The potential for compounds with antifertility activity from the reactions of diphenylcyclopropernone (1) and 2, 3-diphenylthiirene 1, 1-dioxide (2) with enamines is described. In certain instances, a marked dissociation of antifertility from estrogenic activity was possible. Two series were studied extensively, one was stilbene amides (7) and the other stilbene amino ketones (8). The latter series (8) afforded several materials from which, on further biological work-up, was singled out compound 21 as a potent antifertility agent in rats and hamsters.

PIP: The antifertility activity from the reactions of diphenylcyclopropene (1) and 2,3-diphenylthiirene 1,1 dioxide (2) with enamines was investigated in laboratory rodents. A considerable dissociation of antifertility from estrogenic activity was observed in certain instances. Stilbene amides (7) and stilbene aminoketones (8) we re extensively studied. The stilbene aminoketone group provided several materials of which compound 21 was found to be a highly potent antifertility agent. The preparation of the materials is described.

Antifertility agents. 12. Structure-activity relationship of 3,4-diphenylchromenes and -chromans.

Synthesis and antiimplantation activity of variously substituted 2,2-dialkyl-3,4-diphenylchromenes and 3,4-cis- and trans-chromans derived from them are described. Pregnancy-inhibiting activity in rats was exhibited by a number of these compounds, which was particularly marked in the case of 3,4-trans-3-phenyl-4-p-(beta-pyrrolidinoethoxy)-phenyl-7-methoxychroman (32), the corresponding 2,2-dimethyl analog 34, and 3-phenyl-4-p-(beta-pyrrolidinoethoxy)phenyl-7-methoxychromene (26). The structure-activity relationship of these compounds is discussed.

Production of antisera against contraceptive steroids.

A four step synthesis of 6-(0-carboxymethyl) oximinoethynylestradiol is reported. This compound, 6-(0-carboxymethyl) oximinomestranol, the 3-(0-carboxymethyl) oximes of norethindrone and norgestrel and the 3-hemisuccinate of ethynylestradiol were synthesized and conjugated with bovine serum albumin. Rabbits were immunized at 3 dose levels of haptene (20, 66 and 200 nmoles) and eight weeks later with a booster containing 66 nmoles of haptene. The antibody titer and association constant of responding rabbits was nearly independent of dose although most antibody production occurred after the booster injection. Antibodies to mestranol crossreacted more than 100 percent with ethynylestradiol and to a small extent with norethindrone and norgestrel.

PIP: The methods of synthesis of the 6-(0-carboxymethyl) oxime derivative of 6-oxoethynylestradiol-17beta, the 3-succinyl derivative of ethiny estradiol-17beta and the 3-carboxymethyl oxime derivative of norethindrone and norgestrel as well as the synthesis of the corresponding bovine serum albumin (BSA) conjugates required for the specific antisera for mestranol, ethinyl estradiol, norethindrone, and norgestrel in rabbits are described. The synthesis of 6-(0-carboxymethyl) oxime derivative involves a 4-step synthesis and a chromatographic purification which is an improvement over past methods. Conjugation to BSA was by the carbodiimide method. Response of immunization was measured in terms of titer, association constant, and cross-reactivity as a function of time after immunization with 3 dose levels of each antigen. Titer response was independent of dose for the original injection. Ethiny estradiol-3-conjugates had very low titers.

Chemical synthesis and bioassay of anordrin and dinordrin I and II.

The chemical synthesis of 2 alpha, 17 alpha-diethynyl-A-nor-5 alpha-androstane-2 beta, 17 beta-diol dipropionate (Anordrin) and the corresponding diacetate is reported. Similarly, the preparation of the 2 alpha, 17 alpha-diethynyl-A-nor-5 alpha-estrane-2 beta, 17 beta-diol, its diacetate and dipropionate (Dinordrin I), along with the corresponding 2 beta-epimer (Dinordrin II) from 17 beta-hydroxy-A-nor-5 alpha=estran-2-one is described. In rat uterotrophic activity bioassay, the slope of ethynylestradiol differed significantly from the slopes of the other three compounds, thus vitiating potency estimates with this reference compound. Dinordrin I was 20 times more potent than Anordrin and considerably more potent then Dinordrin II. The single-dose oral antifertility effect in rats generally paralleled uterotrophic activity. Immediate postovulatory contraceptive effectiveness was assessed in adult cycling female baboons given two doses daily for 4 days. Both Anordrin and Dinordrin I showed antifertility activity worthy of further study. Moreover, a definite luteolytic effect, with depression of both plasma estrogen and progesterone levels, was observed with these two steroids.

Estimation of impurity profiles of drugs and related materials. Part 16: Identification of the side-products of the ethinylation step in the synthesis of contraceptive gestogens.

A new apolar impurity (3,17 alpha-diethinyl-13-ethyl-3,5-gonadiene-17-ol, IIb) was detected and identified in norgestrel with the aid of thin-layer and high-performance chromatography and spectroscopic techniques. IIb is the product of the acid-catalysed dehydration of an overethinylated side product (Ib) of the ethinylation step in the synthesis of norgestrel. IIb can be determined by thin-layer densitometry and high-performance liquid chromatography. Another impurity (17 alpha-ethinyl-13-ethyl-4-gonene-17-ol, IV), originating from a side product of the Birch reduction step in the synthesis of norgestrel was also detected and identified. The spot of IV overlaps with that of IIb in the TLC system of USP XXIII but can be separated and quantification by more selective TLC systems and by gas chromatography.

Choosing contraceptive steroids and doses.

PIP: The more than 145 pharmacologically different oral contraceptive (OC) products available throughout the world are based on a wide range of synthetic estrogens and progestogens. The active substance of all the estrogens is ethinyl estradiol, to which the other compounds are metabolized. Attempts to use natural estrogens have been unsuccessful. The more numerous synthetic progestogens are of 3 main types: pregnanes, used in OCs only in Eastern Europe and the People's Republic of China, and gonanes and estranes which are used worldwide. In the US only 2 estrogens are available, ethinyl estradiol (EE) and mestranol, which rapidly metabolizes to EE. The pharamacologic literature on the 2 is confusing, but the compound of 1st choice on theoretical grounds is EE, which is used by all the manufacturers of low-dose OCs. The choice of progestogens is between several estranes and 1 gonane, norgestrel, a totally synthetic steroid, unlike the estranes, which are commonly manufactured by chemical alteration of the plant steroid diosgenin. The original synthesis of norgestrel produces a racemate: an equal mixture of 2 enantiomers (2 molecules with identical composition but mirror images of each other). The naming of the enantiomers has caused much confusion. All the hormonal activity of norgestrel resides in the enantiomer named levonorgestrel by the World Health Organization but also correctly called d-norgestrel or d(-)-norgestrel by chemists. Although levonorgestrel replaced racemic norgestrel in OCs worldwide at least 5 years ago, it is only now being introduced in the US. OCs should be chosen according to the essential pharmacologic princple of exposing a person to the lowest effective dose of the drug. 3 obvious criteria for selecting among OCs are contraceptive efficacy, cycle control, and systemic risk effects and safety. A low dose OC should be chosen for general use whenever possible. The contraceptive efficacy of low-dose levongestrel/ethinyl estradiol is very high, cycle control is good and improves after the initial cycles, and laboratory tests have disclosed minimal systemic effects.^ieng

[Study on asymmetric synthesis of steroids. XVI. Synthesis of dl-3-oxa-A-nor steroid].

A synthesis of racemic 3-oxa-A-nor steroid, 8, is reported. The conjugated addition of anion 5 of 5'-methylfuryl dithiane with 4, an intermediate formed from the racemic dionesulfone 3 afforded 6, which was converted to 7 by cyclization under acidic condition. Desulfurization of 7 with tri-n-bu-tyltin hydride gave the title compound 8, which is an intermediate of the target molecule 1b.

Enantioselective total synthesis of the oral contraceptive desogestrel by a double Heck reaction.

A novel enantioselective total synthesis of the oral contraceptive desogestrel (2) is described, in which the tetracyclic steroid core is formed by a sequence of two consecutive Heck reactions. Conversion of the known enantiopure diketone 7 led to the chiral bicycle 6 which was used for a diastereoselective intermolecular Heck reaction with vinyliodide 5 to give 15. In the following intramolecular Heck reaction, the tetracyclic ring system was formed to give 4, from which the synthesis of desogestrel (2) was furnished.

The mother of the pill.

PIP: The first synthesis of an active ingredient of the pill was performed on October 15, 1951, at Syntex in Mexico City. These preliminary results, obtained in late 1951, encouraged the submission of a synthetic progestin, norethindrone (19-nor-17alpha-ethynyltestosterone), to a number of outside investigators for more extensive biological scrutiny. Norethynodrel, together with many other steroids synthesized in the Searle laboratories, as well as Syntex's norethindrone, were examined by Pincus and collaborators for ovulation inhibition in animals and humans. Contrary to predictions, orally effective steroid ovulation inhibitors became the most widely used method of reversible birth control in most parts of the world some 40 years after their first synthesis. Toward the end of the 1960s, at least 13 international pharmaceutical companies (9 of them US) had active research and development programs dedicated to new advances in the field of contraception. Every drug to which a woman or man is exposed to for long periods of time (e.g., vaccines, systemic contraceptives, cholesterol-lowering agents, antihypertensives) in the end has to pass through large-scale postmarketing experiments. The most damaging was the requirement for 6-year toxicology in beagle dogs, which resulted in enormous development costs. Another setback was a 1986 judgment in Georgia against Ortho Pharmaceutical Company for the amount of $5,151,030 for alleged birth defects caused by the use of its spermicide Ortho-Gynol in spite of overwhelming epidemiological evidence against such a cause-effect relationship. Mifepristone (RU-486) is clearly the most significant new development in birth control as an important alternative to conventional abortion. A priority list of six new contraceptive methods for future development includes a spermicide with antiviral properties, a once-a-month menses inducer, a reliable ovulation predictor, easily reversible male sterilization, a male contraceptive pill, and an antifertility vaccine.^ieng