A. G. J. Sedee

Isolation, identification and densitometric determination of norethisterone-4/gb,5/gb-epoxide after photochemical decomposition of norethisterone

From the literature it is known that oral contraceptives (OCs) in combination with light can cause side effects. The authors have identified norethisterone-4beta5beta-epoxide as 1 of the photoproducts of norethisterone (NE) a progestogenic component of the OC upon irradiation with ultraviolet light of mainly 300 nm. As part of the research into the cause of those light-induced side effects a method was developed for the quantification of this NE-4beta5beta-epoxide. With the use of impregnated self-charring TLC plates a linear relationship was obtained between the densitometric signal and the spot content of NE NE-4beta5beta-epoxide and mestranol (ME) in the ranges of 0.5-20.0 mcg 0.5-20.0 mcg and 1.2-30.0 mcg respectively. The relative standard deviation is about 2%. The detection limit is low about 0.1 mcg. This densitometric method will be appropriated to determine the OC components NE and ME with some advantages over other assays. After 30 minutes irradiation with 300 nm light at 37 degrees Celsium of a 1.67x10-4M solution of NE in phosphate-buffered saline (pH 7.4) containing 10% ethanol about 23% of the total quantity of photoproducts was NE-4beta5beta-epoxide. Kept in the dark for 4 hours at 37 degrees Celsius the percentage was raised to 34% without further decomposition of NE. This NE-4beta5beta-epoxide may be responsible for some light-induced side effects of the pill. There are some reasons to suspect this photochemically formed epoxide to be the cause of some of the nondermatological side effects of NE-containing OCS. (authors)

A synthesis for p-quinol compounds from phenols fused with other saturated rings

Abstract Starting with a bicyclic or steroidal phenol, a p-quinol compound has been synthesized without side-products with use of a photosensitizer.

Interaction of the photosensitization products of oestrone with DNA: Comparison with the horseradish peroxidase catalyzed reaction

The interaction with DNA of [4-14C]oestrone upon photosensitization with hematoporphyrin (HP) as a photosensitizer has been investigated. By means of Sephadex LH-20 gel filtration and extraction with dichloromethane it was found that, after irradiation (lambda greater than 425 nm) of a solution of HP, DNA and [4-14C]oestrone 21% of the radiolabel was associated with DNA. If DNA was added after irradiation 23% was bound to DNA, whereas 25% of the oestrone remained after photoreaction under the conditions applied. The binding occurs via the reactive 10 beta-hydroperoxy-1,4-estradien-3,17-dione, which is the only product after photosensitization of oestrone. The hydroperoxide has a strong interaction with DNA compared with that of other steroids. By repeated precipitation with 5 M NaCl and ethanol the association can be broken. It is reported, that binding of oestrone to protein induced by both photosensitization and horseradish peroxidase (HRPO)/H2O2 is irreversible, but that the amount of binding to DNA is dependent on the method of determination. However, neither the hydroperoxide nor its reduced product, a p-quinol, is intermediate or product in the HRPO catalyzed reaction of oestrogens. The tight association of the hydroperoxide product of oestrone with DNA, which may proceed via hydrogen bonding between the -OOH group and oxygen atoms of the backbone phosphate groups or of the furanose ring, might be a cause of chemical modification of DNA and of mutagenic effects.

Structure elucidation of two isomeric steroids: photolytical and thermal reaction products from norethisterone studied by two-dimensional nuclear magnetic resonance.

AbstractTwo-dimensional nuclear magnetic resonance was used for the structure elucidation of two isomeric photoproducts of norethisterone, a commonly used progestogen in oral contraceptives. The predominant one of the two isolated products derived from photochemical decomposition of norethisterone upon irradiation with UV-B light (280–320 nm) was 5α,17β-dihydroxy-19-nor-17α-pregn-20-yn-3-one. The minor photoproduct appeared to be the analogous 5β-isomer,i.e. 5β,17β-dihydroxy-19-nor-17α-pregn-20-yn-3-one. The latter compound was also obtained from the thermal reduction of norethisterone-4β,5β-epoxide using aluminium amalgam in isopropanol. Two-dimensional NMR appeared to be superior to mass and IR spectrometry in identifying the isomers.

The photochemical decomposition of the progestogenic 19-norsteroid, norethisterone, in aqueous medium

Norethisterone, a contraceptive 19-norsteroid, was decomposed in aqueous medium (pH 7.4) by UV-B radiation (280-320 nm). This 4-en-3-oxo-19-norsteroid was not prone to the skeletal rearrangement reactions usually observed in steroids possessing a C10-methyl group. Under the reaction conditions applied, products were formed by addition of molecules, such as solvent molecules or a second steroid molecule, and by reduction of the double bond. The prevalence of addition type reactions may have consequences for the application of norethisterone-like steroids in subdermal contraceptive devices.

Photosensitized irreversible binding of estrone to protein via a hydroperoxide intermediate: An explanation of (photo-)allergic side-effects of estrogens

After irradiation (lambda greater than 425 nm) for 15 min of a solution of [4-14C]-estrone, albumin and the photosensitizer hematoporphyrin in phosphate buffer, more than 30% of the radioactivity could not be extracted. When the protein was added after irradiation, irreversible binding also occurred. Sephadex gel filtration showed that the radiolabel was bound to albumin as well as to the photosensitizer. A 10 beta-hydroperoxide is the reactive intermediate in this binding. Inasmuch as phenolic steroids coupled to proteins have been used for the induction of estrogenic-specific antibodies, the irreversible binding observed between estrone and albumin by photosensitization might be an explanation for (photo)allergic disorders associated with estrogens.

IRREVERSIBLE BINDING OF CHLORDIAZEPOXIDE TO HUMAN PLASMA PROTEIN INDUCED BY UV‐A RADIATION

Abstract— In view of the phototoxicity of chlordiazepoxide (Librium®) the kinetics of the reaction in the presence of plasma proteins was studied for chlordiazepoxide upon UV‐A irradiation and for its oxaziridine in the dark. Two different methods were used to determine the extent of irreversible binding to protein (up to ∼ 50% was found for both situations). Kinetic data support the conclusion that the formation of oxaziridine from photoexcited chlordiazepoxide is the basic event making chlordiazepoxide phototoxic. The half life of oxaziridine is about 30 min even in the presence of a high concentration of plasma proteins, is in agreement with previous in vivo results, showing covalent binding not only to biomolecules of the UV‐A irradiated skin but also to those of inner organs, e.g. liver and kidneys.

Assignment of the 1H and 13C nuclear magnetic resonance spectra of norethisterone using two-dimensional nuclear magnetic resonance spectroscopy

The information obtained from 2D J-resolved and 2D spin-echo-correlated spectroscopy at 500 MHz allows the complete assignment of the 1H n.m.r. spectrum of norethisterone. In turn, the 1H n.m.r. assignment readily leads to the assignment of the 13C n.m.r. spectrum of this compound by means of a two-dimensional hetero-shift correlation experiment.

Investigation into the effects of the photodecomposition of norethisterone by UV-B light on Salmonella typhimurium TA98 and the rat.

In view of the light-induced side-effects of the oral contraceptive pill and the development of injectable and subdermal contraceptive devices, the effects of norethisterone irradiated with UV-B light (280-320 nm) on the microorganism Salmonella typhimurium TA98 and male Wistar rats have been investigated. The observed cytotoxic effect of the photo-products on Salmonella typhimurium TA98 may in part be caused by the irreversible binding of steroid to protein from this bacterium, of which binding is in line with previous experiments. However, after intraperitoneal or percutaneous administration of 4-14C-norethisterone to rats, followed by irradiation with UV-B light, no significantly higher level of radioactivity was observed in blood, organ material of the skin, kidney or liver, on extraction or dialysis of the samples.