Steroid Ring Research Articles

Structure and conformation of 2β,3β-epoxyestr-5(10)-en-1,4,17-trione, spectroscopic and X-ray crystallographic studies

Colourless single crystals were isolated as by-product during the synthesis of steroidal A-ring substituted 1,4-quinones (and epoxyquinols), as synthetic products with antibiotic and antitumor properties. Its structure was proposed by comparing IR, UV, 1H and 13C NMR spectra to the ones of quinone 2 and independently determined by an X-ray analysis, as 2β,3β-epoxyestr-5(10)-en-1,4,17-trione. Crystals belong to the monoclinic system with space group P2 1 : a=6.767(3) A ̊ , b=7.097(5) A ̊ , c=15.748(5) A ̊ , β=97.070(5)°, V=750.6(8) A ̊ 3, Z=2, D x=1.329 Mg m −3, μ( Mo K α)=0.093 mm −1. The structure was solved by direct methods and refined to a final R=0.064 for 1729 reflections with I>2 σ( I). The steroidal skeleton with chiral centre at C13 possesses the S configuration defining β-orientation of O2 atom bridging C2 and C3 atoms and β-oriented methyl group bonded to C13 atom. The best plane through the ring A and epoxy ring plane form an angle of 89.6(2)°. Conformational analysis of the steroid rings are performed by calculating the ring puckering parameters and asymmetry factors. The conformation of the A ring is screw-boat 1S 6, ring B half-chair 4H 3, ring C chair 1C 4, and the five-membered D ring is half-chair 2H 1 conformation. The crystal structure is stabilised by the weak C–H⋯O hydrogen bonds and van der Waals interaction.

Cascade radical cyclisations leading to steroid ring constructions. Regio- and stereo-chemical studies using ester- and fluoro-alkene substituted polyene acyl radical intermediates

A study of the factors affecting the regio- and stereo-selective outcomes of consecutive 6-endo-trig cyclisations of polyene acyl-radical intermediates, leading to decalone, perhydrophenanthrone, and steroid ring constructions, has been carried out. Thus, whereas the E-substituted diene selenyl ester 7 underwent sequential cyclisations in the presence of Bu3SnH-AIBN leading to the trans-decalone 8 exclusively, the corresponding Z- and E-isomers of the methoxycarbonyl-substituted diene 16, under similar conditions, gave rise to a 2∶1 mixture of the trans- and cis-decalones 17a and 17b respectively in 62–73% yield. Cyclisation of the triene selenoate 30 led to a single tricyclic product in 57% yield whose cis,syn,trans relative stereochemistry 32 was established by X-ray diffraction analysis. When solutions of the trienyne selenoates 41a–c in benzene were treated with Bu3SnH–AIBN they each underwent cascades of three 6-endo-trig followed by a 5-exo-dig cyclisation leading to the full steroid ring systems 42, 45, and 47 respectively in 20–40% yields. The stereochemistries of the major steroid diastereoisomers resulting from 41a and 41c were established as trans,anti,trans,anti,cis, e.g.47, following X-ray crystallographic analysis of the corresponding dione 44 produced from 42d and 47 after ozonisation. In each of the cyclisations leading to 42 and 45 varying amounts of other bicyclic products tentatively assigned as 43 and 46 respectively, resulting from a competing radical pathway involving first a 10-endo-trig macrocyclisation of the corresponding acyl radical intermediate onto the C9–C10 olefin in 43/46, followed by a 5-exo-trig cyclisation of the resulting radical intermediate onto the proximal C13–C14 double bond, were produced concurrently. Finally, when the fluoro-alkene selenoate 56 was treated with Bu3SnH–AIBN, a complex mixture of polycyclic products resulted, from which only the indanone 57 could be separated and characterized. The origins of the differing regio- and stereo-selective outcomes in the aforementioned radical cascades are briefly considered.

Synthesis of 8,14-Secosteroids from (S)-(+)-Carvone

A total synthesis of functionalised 8,14-seco steroids with five- and six-membered D rings is described. The synthesis is based on the transformation of (S)-carvone into a steroidal AB ring moiety with a side chain at C-9 that allowed the creation of a nitrile oxide at this position. The nitrile oxides were coupled with cyclic enones or enol derivatives of 1,3-diketones, and reductive cleavage of the obtained cycloadducts gave the desired products. The formation of a twelve-membered ring compound was observed in cycloaddition of one of the nitrile oxides with cyclopentenone as the result of an intramolecular ene-reaction followed by retroaldol reaction.

Synthesis and anti-HIV activity of cosalane analogues incorporating nitrogen in the linker chain

Introduction of an amido group or an amino moiety into the alkenyl linker chain of cosalane (1) provided a new series of analogues 3–8. The new compounds were evaluated as inhibitors of the cytopathic effect of HIV-1 and HIV-2 in cell culture. The replacement of the 1′ and 2′ carbons in the linker chain of 1 by an amido group was generally tolerated. The length of the linker chain and the stereochemistry of the substituent at C-3 of the steroidal ring had significant effects on the antiviral activity and potency. Incorporation of an amino moiety into the linker completely abolished the anti-HIV activity. There are several steps in the HIV replication cycle that have been proposed as targets for the development of therapeutic agents (De Clercq, E. J. Med. Chem.1995, 38, 2491; De Clercq, E. Pure Appl. Chem.1998, 70, 567). However, currently approved anti-HIV drugs are only directed against the viral enzymes reverse transcriptase or protease (Carpenter, C. C. J.; Fischl, M. A.; Hammer, S. M.; Hirsch, M. S.; Jacobsen, D. M.; Katzenstein, D. A.; Montaner, J. S. G.; Richman, D. D.; Saag, M. S.; Schooley, R. T.; Thompson, M. A.; Vella, S.; Yeni, P. G.; Volberding, P. A. JAMA1998, 280, 78). Drugs capable of interfering with other steps of the virus life cycle will be highly valuable in the antiretroviral therapy of AIDS, as they will have different patterns of resistance mutations than the drugs currently used clinically. In addition, their utilization in combination with other therapeutic agents could provide more potent drug ‘cocktails’ capable of completely suppressing virus replication. Consequently, there is an urgent need for the discovery of clinically useful anti-HIV agents possessing novel mechanisms of action.

Triterpenoide. XVII. 3,28-Dimethoxy-3,11,28-trioxo-2,3-seco-18β-olean-12-en-2,9α-olid

The structure of the title compound, C 32 H 46 O 7 , previously derived by spectroscopic methods, is confirmed. Steroidal ring C has a slightly distorted sofa form and all other six-membered rings have a chair conformation. Rings B and C are trans-fused, and rings D and E are cis-fused. The lactone ring has a conformation intermediate between envelope and half-chair.

Synthesis of deuterium-labeled tetrahydrocortisol and tetrahydrocortisone for study of cortisol metabolism in humans

A method is described for the preparation of multi-labeled tetrahydrocortisol (3α,11β,17α,21-tetrahydroxy-5β-[1,2,3,4,5- 2H 5]pregnan-20-one, THF-d 5), allo-tetrahydrocortisol (3α,11β,17α,21-tetrahydroxy-5α-[1,2,3,4,5- 2H 5]pregnan-20-one, allo-THF-d 5), and tetrahydrocortisone (3α,17α,21-trihydroxy-5β-[1,2,3,4,5- 2H 5]pregnane-11,20-dione, THE-d 5) containing five non-exchangeable deuterium atoms in the steroid ring A. Reductive deuteration at C-1, C-2, C-3, C-4, and C-5 of prednisolone or prednisone was performed in CH 3COOD with rhodium (5%) on alumina under the deuterium atmosphere. The isotopic purities of the labeled compounds as [ 2H 5]-form were estimated to be 86.17 atom%D for THF-d 5, 74.46 atom%D for allo-THF-d 5 and 81.90 atom%D for THE-d 5, based on the ion intensities in the region of the molecular ion of methoxime-trimethylsilyl (MO-TMS) derivatives measured by GC-MS.

Monoclonal Antibodies That Distinguish Between Two Related Digitalis Glycosides, Ouabain and Digoxin

The exogenous digitalis glycosides, ouabain and digoxin, have been widely used in humans to treat congestive heart failure and cardiac arrhythmias. Several reports have also pointed to the existence of endogenous ouabain- and digoxin-like compounds, but their precise roles in mammalian physiology and various disorders of the circulation are not clear. In an attempt to produce specific Abs for the purification and identification of endogenous ouabain-like compounds, somatic cell fusion was used to produce mAbs specific for ouabain. Our attempts to produce ouabain-specific mAbs were unsuccessful when ouabain was coupled to exogenous proteins such as bovine gamma-globulins, BSA, and human serum albumin. However, when ouabain was coupled to an Ab of A/J mice origin and the same strain of mouse was used for immunization with ouabain-Ab conjugate, three Abs (1-10, 5A12, and 7-1) specific for ouabain were obtained. In assays of fluorescence quenching and saturation equilibrium with tritiated ouabain, Ab 1-10 exhibited 200 nM affinity for ouabain. These three mAbs are distinguished from existing Abs to ouabain and digoxin by their specificity for ouabain and lack of cross-reactivity with digoxin. Specificity studies showed that the loss of cross-reactivity was correlated with the presence of a hydroxyl group at either position 12beta (digoxin) or 16beta (gitoxin) of the steroid ring. These Abs can be used to develop assays for detection and characterization of ouabain-like molecules in vivo.

Isolation and Characterization of Human Monoclonal Antibodies to Digoxin

Fab preparations of sheep polyclonal anti-digoxin Abs have proven useful for reversal of the toxic effects of digoxin overdoses in patients. Unfortunately, the use of foreign species proteins in humans is limited because of the potential for immunological responses that include hypersensitivity reactions and acute anaphylaxis. Immunization of recently developed transgenic mice, whose endogenous micro heavy and kappa light chain Ig genes are inactivated and which carry human Ig gene segments, with a digoxin-protein conjugate has enabled us to generate and isolate eight hybridoma cell lines secreting human sequence anti-digoxin mAbs. Six of the mAbs have been partially characterized and shown to have high specificity and low nanomolar affinities for digoxin. In addition, detailed competition binding studies performed with three of these mAbs have shown them to have distinct differences in their digoxin binding, and that all three structural moieties of the drug, the primary digitoxose sugar, steroid, and five-member unsaturated lactone ring, contribute to Ab recognition.

Synthesis of two analogues of brassinolide, possible plant growth promoting steroids

The synthesis of two isomeric brassinosteroids, analogues of brassinolide, containing the 2β,3β diol system typical of ecdysteroids and a five-membered lactone bonded to the steroid ring B is described. The synthesis leads to both lactones via a metal hydride reduction of the Diels-Alder adduct of ergosterol.

Effects of Steroids and Vitamin D3 on the Permeability of Liposomal Bilayer Membranes

Molecular interactions between L-α-dipalmitoylphosphatidylcholine (DPPC) and steroids differing in the number of double bonds in a monolayer and bilayer were examined by measurement of surface pressure, permeability and microviscosity. The steroids used were 7-dehydrocholesterol (7-DHC), cholesterol (Chol) and β-cholestanol (β-Chol) along with Vitamin D3 (VD3) having an opened steroid B-ring. The limiting molecular area for the steroids in the DPPC-steroid mixed monolayer increased with the number of double bonds in the steroid ring in the order β-Chol<Chol<7-DHC. Liposomes were formed with steroids at steroid mole fractions less than 0.5 and VD3 less than 0.3. The permeability of the liposomes containing steroids decreased with the number of double bonds in the steroid ring and steroid limiting molecular area. That of liposomes containing VD3 with limiting molecular area exceeding that of steroids was higher than for liposomes containing steroids. The effects of steroids or VD3 on the permeability of Liposomal bilayer membranes cannot be adequately explained based on the number of double bonds or limiting molecular area, although Demel et al. have shown a correlation between the effects of sterols on the permeability of liposomes and molecular area of sterols in monolayers. The permeability of liposomal bilayer membranes containing steroids or VD3 was found in this study straightly decrease with microviscosity near the bilayer center. The permeability of liposomal bilayer membranes may thus be considered to related to microviscosity near the bilayer center, which can be evaluated based on fluorescence polarization determined with the fluorescent probe, 12AS.

Testosterone 5 alpha-reductase inhibitors, menaquinone 7 produced by a Bacillus and phenazine methosulfate.

Menaquinone 7 (MW: 649, C46H64O2), a natural electron acceptor for steroid ring A dehydrogenations, produced by Bacillus sp. SNU-299, was isolated as a rat prostate testosterone 5 alpha-reductase inhibitor with an IC50 value of 4.0 x 10(-5) M from the cultured broth. Phylloquinone was as active as the purified microbial metabolite with an IC50 value of 6.6 x 10(-4) M. On the basis of this evidence, the inhibitory activities of electron carriers, menadione, phenazine methosulfate, and 2,6-dichlorophenolindophenol, for rat prostate testosterone 5 alpha-reductase were tested, and the IC50 values were 3.1 x 10(-6) M, 4.9 x 10(-8) M, 8.9 x 10(-5) M, respectively. A product of the 5 alpha-reductase enzyme reaction and an electron and proton carrier, NADP+, inhibited the 5 alpha-reduction by rat prostate testosterone 5 alpha-reductase with an IC50 value of 9.2 x 10(-5) M. However, the inhibition effect of a proton carrier, carbonylcyanide-m-chlorophenylhydrazone, for rat prostate testosterone 5 alpha-reductase was substantially inactive.

Configurational and conformational analyses of α-methylene-γ-butyro steroidal spirolactones

The configurational and conformational analyses of α-methylene-γ-butyro steroidal spirolactones have been accomplished using 2D COSY, NOESY, ROESY and HETEROCOSY in conjunction with 1D 1H NMR. The analyses indicate that the major or the only spirolactones formed at positions 2, 3 and 6 of the steroids have the steroidal ring bearing the spirolactone in a chair conformation with an axial disposition of the oxygen atom attached to the spirocentre. The corresponding minor spirolactones have equatorial configurations. Steroids with spirolactones at positions 16 and 17 have β and α dispositions, respectively, of the oxygen atom attached to the spirocentre.

The binding of 3,6-disubstituted bile salts to human serum albumin induces conformational change on the molecule of this protein

The binding of 3,6-hydroxy and keto disubstituted bile salts to human serum albumin was studied using differential scanning calorimetry, fluorescence spectroscopy and circular dichroism. The bile salts assayed did not produce any modification in the shape of the albumin thermogram, its thermal unfolding process in their presence being reversible; however, an increase in the enthalpy of unfolding and in the T m was observed in the presence of 3,6-diketo and 3-hydroxy-6-keto bile salts. These two derivatives induced a negative circular dichroism spectrum of the protein around 280–290 nm, quenched the native fluorescence of the buried tryptophan of albumin and induced energy transfer between 1 aniline-8-naphthalene sulfonate and the buried tryptophan 214 of albumin. The presence of a keto group at C 6 in the steroid ring of the bile salts plays an important role in producing slight movement of the albumin domains, increasing the distance between domains I and II.

The hydroxylation of steroidal ring D lactones by Cephalosporium aphidicola

The microbiological hydroxylation of 17a-oxa-d-homo-5α-androstane-3,17-dione, the 3β-alcohol and the 13α-methyl analogue by Cephalosporium aphidicola takes place predominantly at C-7α in contrast to other hydroxylations by this organism.

Loss of phosphoserine polar group asymmetry and inhibition of cholesterol transport in Jurkat cells treated with cholesterylphosphoserine

Cholesterylphosphoserine (CPHS) is a synthetic ester of cholesterol showing immunosuppressive activity. In the present study, we have used the T cell line Jurkat to investigate its mechanism of action. CPHS incorporates into cells reaching a molar ratio of 0.23 and 3.9 with the total phospholipid and cholesterol content, without inducing necrosis or apoptosis. CPHS incorporation elicits a dose-dependent binding of fluorescein isothiocyanate-labeled annexin V, suggesting that the steroid distributes in the external leaflet of plasma membrane exposing the phosphoserine group to the external cell environment and inserting the steroid ring into the phospholipid bilayer. In agreement with a preferential steroid association with sphingolipids, CPHS is included in a Triton X-100-insoluble complex when mixed with sphingomyelin and cholesterol. CPHS incorporation inhibits the esterification of low density lipoprotein (LDL)-derived cholesterol, producing a minor influence on the endogenous synthesis of cholesterol and on the acyl-CoA:cholesterol acyltransferase activity. In this effect, CPHS is as potent as progesterone (IC50 of 3.5 μm).▪ It is concluded that the insertion of cholesterylphosphoserine (CPHS) in the Jurkat plasma membrane neutralizes the asymmetric distribution of the phosphoserine group and inhibits the movement of cholesterol to the endoplasmic reticulum. As CPHS is a negatively charged steroid, this last effect may be linked to the perturbation of sphingolipid/cholesterol-based microdomains, proposed to play a role in cholesterol trafficking.—Cusinato, F., W. Habeler, F. Calderazzo, F. Nardi, and A. Bruni. Loss of phosphoserine polar group asymmetry and inhibition of cholesterol transport in Jurkat cells treated with cholesterylphosphoserine.

ChemInform Abstract: A New Approach to Steroid Ring Construction Based on a Novel Radical Cascade Sequence.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Mass Spectrometric Methods for Distinguishing Structural Isomers of Glutathione Conjugates of Estrone and Estradiol

Collisionally activated decompositions (CAD) of [M+H] + ions from two sets (estrone and estradiol) of three isomeric glutathione (GSH) conjugates were studied by using five tandem mass spectrometric methods: (1) low energy (LE) CAD in an ion trap, (2) LE CAD in a triple quadrupole, (3) electrospray ionization (ESI)-source CAD in a tandem four sector, (4) high energy (HE) CAD of both ESI-produced and fast-atom bombardment (FAB)-produced ions in a tandem four-sector mass spectrometer, and (5) metastable-ion decompositions of FAB-produced ions. Four types of fragment ions are produced. The first type, formed from cleavage of the peptide backbone, gives rise to modified b 2, modified y 2, y 2, and b 1 ions. These fragments are observed with all the methods and show that the catechol estrogen attachment is at the cysteine moiety of the GSH. Internal fragment ions are the second type, and they also support that the modification is at cysteine. The third type involves fragmentation of the C–S bond to give an ion containing the steroid bonded to the sulfur. The fourth type of fragment ion is similar to the third but involves oxidation of the steroid ring and reduction of the GSH moiety; it is the most isomer specific of the four. The isomer-specific ions are of relatively low abundance in the product-ion spectra taken on the triple quadrupole and ion trap, but their abundances can be improved by increasing the collision energy. ESI source-CAD and the HE-CAD spectra of the isomers are the most distinctive because abundant product ions of all four types are seen in a single spectrum.

Full length article

Several derivatives of lotoprednol etabonate (1), a soft corticosteroid antiinflammatory drug, are formed during the synthesis and sterilization process. Some of these contaminants of 1 result from side reactions taking place on the steroid ring C including oxidation, dehydration, chlorination and chlorohydroxylation. The products have been identified, synthesized, and fully characterized by 1H and 13C NMR spectroscopy.

Labelling of biogenetic brassinosteroid precursors

A base catalyzed exchange procedure was employed to introduce tritium and deuterium, respectively, into enolizable positions of the steroidal ring system of several putative intermediates of 24R-type brassinosteroids. The method is suitable both for derivatives with protected hydroxyl groups as well as for non-protected compounds. © 1998 John Wiley & Sons, Ltd.

Simple Routes to Keto-norsteroids

Ring A and B keto-norsteroids have been obtained by applying the McMurray low valency titanium cyclization to steroidal seco ring A and ring B nor-keto-esters which were readily available by cleaving the corresponding unsaturated ketones.