Steroid Skeleton Research Articles

Triazole-estradiol analogs: A potential cancer therapeutic targeting ovarian and colorectal cancer

1,2,3-triazoles have continuously shown effectiveness as biologically active systems towards various cancers, and when used in combination with steroid skeletons as a carrier, which can act as a drug delivery system, allows for a creation of a novel set of analogs that may be useful as a pharmacophore leading to a potential treatment option for cancer. A common molecular target for cancer inhibition is that of the Epidermal Growth Factor Receptor/Mitogen Activated Protein Kinase pathways, as inhibition of these proteins is associated with a decrease in cell viability. Estradiol-Triazole analogs were thus designed using a molecular modeling approach. Thirteen of the high scoring analogs were then synthesized and tested in-vitro on an ovarian cancer cell line (A2780) and colorectal cancer cell line (HT-29). The most active compound, Fz25, shows low micromolar activity in both the ovarian (15.29 ± 2.19 µM) and colorectal lines (15.98 ± 0.39 µM). Mechanism of action studies proved that Fz25 moderately arrests cells in the G1 phase of the cell cycle, specifically inhibiting STAT3 in both cell lines. Additionally, Fz57 shows activity in the colorectal line (24.19 ± 1.37 µM). Inhibition studies in both cell lines show inhibition against various proteins in the EGFR pathway, namely EGFR, STAT3, ERK, and mTOR. To further study their effects as therapeutics, Fz25 and Fz57 were studied against drug efflux proteins, which are associated with drug resistance, and were found to inhibit the ABC transporter P-glycoprotein. We can conclude that these estradiol-triazole analogs provide a key for future studies targeting protein inhibition and drug resistance in cancer.

Docking-based analysis and modeling of the activity of bile acids and their synthetic analogues on large conductance Ca2+ activated K channels in smooth muscle cells.

The objective of this study was to perform docking-based analysis of bile acid binding on the protein complex of channels and to derive neural network that predicts the influence of bile acids and their synthetic analogues on the activity of BK(Ca) channels in smooth muscle cells based on descriptors for bile acids and their synthetic analogues and on their already published activities using patch-clamp techniques. Ligands for molecular docking were optimized using computer routine for minimization of energy by using the force field MMFF94 via Chem3D 15.0 and ligands and protein channel complex were prepared in AutoDockTools 1.5.6. AutoDock Vina 4.0 software was used for blind docking; processing and verification of the obtained results was performed via Discovery Studio 4.0. Neural network was derived using descriptors for bile acids and their synthetic analogues and their already published activities on calcium-activated K+ channels in smooth muscle cells (ChemDraw Professional 15.0, Dragon 6 software). Molecular docking was performed for: lithocholic acid, deoxycholic acid, 5β-cholanoic acid, 3β-hydroxi-5β-cholanoic acid, henodeoxycholic acid, ursocholic acid and α-muricholic acid. Neural network model Multiple layer perceptron is derived, having 0.9259 training performances and 0.3673 test performances, training error 0.0073 and test error 0,1607. Model was tested for henodeoxycholic, ursocholic and α-muricholic acid, and internal validation of the model is performed. Molecular docking suggested that the pharmacophore for maximizing the activity of BK(Ca) channels in the steroid skeleton of bile acids is the C3 quasi-axial α-OH group and the C24 carboxyl function. Derived neural network model successfully predicted activities of tested bile acids on Ca2+ activated K+ channels in smooth muscle cells.

Investigations on the Degradation of the Bile Salt Cholate via the 9,10-Seco-Pathway Reveals the Formation of a Novel Recalcitrant Steroid Compound by a Side Reaction in Sphingobium sp. Strain Chol11.

Bile salts such as cholate are steroid compounds from the digestive tracts of vertebrates, which enter the environment upon excretion, e.g., in manure. Environmental bacteria degrade bile salts aerobically via two pathway variants involving intermediates with Δ1,4- or Δ4,6-3-keto-structures of the steroid skeleton. Recent studies indicated that degradation of bile salts via Δ4,6-3-keto intermediates in Sphingobium sp. strain Chol11 proceeds via 9,10-seco cleavage of the steroid skeleton. For further elucidation, the presumptive product of this cleavage, 3,12β-dihydroxy-9,10-seco-androsta-1,3,5(10),6-tetraene-9,17-dione (DHSATD), was provided to strain Chol11 in a co-culture approach with Pseudomonas stutzeri Chol1 and as purified substrate. Strain Chol11 converted DHSATD to the so far unknown compound 4-methyl-3-deoxy-1,9,12-trihydroxyestra-1,3,5(10)7-tetraene-6,17-dione (MDTETD), presumably in a side reaction involving an unusual ring closure. MDTETD was neither degraded by strains Chol1 and Chol11 nor in enrichment cultures. Functional transcriptome profiling of zebrafish embryos after exposure to MDTETD identified a significant overrepresentation of genes linked to hormone responses. In both pathway variants, steroid degradation intermediates transiently accumulate in supernatants of laboratory cultures. Soil slurry experiments indicated that bacteria using both pathway variants were active and also released their respective intermediates into the environment. This instance could enable the formation of recalcitrant steroid metabolites by interspecies cross-feeding in agricultural soils.

Neurosteroids: Structure-Uptake Relationships and Computational Modeling of Organic Anion Transporting Polypeptides (OATP)1A2.

In this study, we investigated the delivery of synthetic neurosteroids into MCF-7 human breast adenocarcinoma cells via Organic Anionic Transporting Polypeptides (OATPs) (pH 7.4 and 5.5) to identify the structural components required for OATP-mediated cellular uptake and to get insight into brain drug delivery. Then, we identified structure-uptake relationships using in-house developed OATP1A2 homology model to predict binding sites and modes for the ligands. These binding modes were studied by molecular dynamics simulations to rationalize the experimental results. Our results show that carboxylic acid needs to be at least at 3 carbon-carbon bonds distance from amide bond at the C-3 position of the androstane skeleton and have an amino group to avoid efflux transport. Replacement of hydroxyl group at C-3 with any of the 3, 4, and 5-carbon chained terminal carboxylic groups improved the affinity. We attribute this to polar interactions between carboxylic acid and side-chains of Lys33 and Arg556. The additional amine group showed interactions with Glu172 and Glu200. Based on transporter capacities and efficacies, it could be speculated that the functionalization of acetyl group at the C-17 position of the steroidal skeleton might be explored further to enable OAT1A2-mediated delivery of neurosteroids into the cells and also across the blood-brain barrier.

Structural studies of some androstane based prodrugs

Trestolone, methenolone and dihydroboldenone are synthetic anabolic-androgenic steroids and androstane derivatives. The structural features of trestolone acetate, methenolone acetate and dihydroboldenone cypionate have been described. The crystal structures were determined using X-ray single crystal diffraction technique. Steroid skeleton rings were investigated in terms of configurational analysis. Hirshfeld surfaces were generated to highlight the intermolecular interactions within the crystals and Coulomb-London-Pauli method was employed in the evaluation of total lattice energies and various components such as Coulombic, polarization, dispersion and repulsion.

Degradation of Bile Acids by Soil and Water Bacteria.

Bile acids are surface-active steroid compounds with a C5 carboxylic side chain at the steroid nucleus. They are produced by vertebrates, mainly functioning as emulsifiers for lipophilic nutrients, as signaling compounds, and as an antimicrobial barrier in the duodenum. Upon excretion into soil and water, bile acids serve as carbon- and energy-rich growth substrates for diverse heterotrophic bacteria. Metabolic pathways for the degradation of bile acids are predominantly studied in individual strains of the genera Pseudomonas, Comamonas, Sphingobium, Azoarcus, and Rhodococcus. Bile acid degradation is initiated by oxidative reactions of the steroid skeleton at ring A and degradation of the carboxylic side chain before the steroid nucleus is broken down into central metabolic intermediates for biomass and energy production. This review summarizes the current biochemical and genetic knowledge on aerobic and anaerobic degradation of bile acids by soil and water bacteria. In addition, ecological and applied aspects are addressed, including resistance mechanisms against the toxic effects of bile acids.

Synthesis and mesomorphic characterization of some novel steroidal mesogens: A structure–property correlation

The steroidal derivatives are found to be extremely good mesogens since their inception. Because of their inherent chirality, they have the potential to induce a wide variety of liquid crystalline phases, including frustrated phases depending upon the structure of the steroidal skeleton and the substituents attached. In this report, a series of novel monoalkoxy and dialkoxy benzoate derivatives of ergosterol and a few monoalkoxy derivatives of stigmasterol have been synthesized and their mesomorphic property has been investigated. The derivatives exhibited various mesophases including SmA, SmC*, N*, TGB and blue phases. Also, the gelation ability of some of these derivatives with various organic solvents has been examined. Furthermore, the mesomorphism of these derivatives has been compared with the analogous cholesteryl counterparts.

Characterization of C-26 aminotransferase, indispensable for steroidal glycoalkaloid biosynthesis.

Steroidal glycoalkaloids (SGAs) are toxic specialized metabolites found in members of the Solanaceae, such as Solanum tuberosum (potato) and Solanum lycopersicum (tomato). The major potato SGAs are α-solanine and α-chaconine, which are biosynthesized from cholesterol. Previously, we have characterized two cytochrome P450 monooxygenases and a 2-oxoglutarate-dependent dioxygenase that function in hydroxylation at the C-22, C-26 and C-16α positions, but the aminotransferase responsible for the introduction of a nitrogen moiety into the steroidal skeleton remains uncharacterized. Here, we show that PGA4 encoding a putative γ-aminobutyrate aminotransferase is involved in SGA biosynthesis in potatoes. The PGA4 transcript was expressed at high levels in tuber sprouts, in which SGAs are abundant. Silencing the PGA4 gene decreased potato SGA levels and instead caused the accumulation of furostanol saponins. Analysis of the tomato PGA4 ortholog, GAME12, essentially provided the same results. Recombinant PGA4 protein exhibited catalysis of transamination at the C-26 position of 22-hydroxy-26-oxocholesterol using γ-aminobutyric acid as an amino donor. Solanum stipuloideum (PI498120), a tuber-bearing wild potato species lacking SGA, was found to have a defective PGA4 gene expressing the truncated transcripts, and transformation of PI498120 with functional PGA4 resulted in the complementation of SGA production. These findings indicate that PGA4 is a key enzyme for transamination in SGA biosynthesis. The disruption of PGA4 function by genome editing will be a viable approach for accumulating valuable steroidal saponins in SGA-free potatoes.

Antiproliferative and antimetastatic characterization of an exo-heterocyclic androstane derivative against human breast cancer cell lines

Cancer in general, and specifically gynaecological neoplasms, represents a major public health issue worldwide. Based on the effect of sex hormones on breast tumorigenesis and prognosis, as well as on the development of breast cancer metastases, modification of the steroid skeleton is a hotspot of research for novel anticancer agents. Numerous recent studies support that minor modifications of the androstane skeleton yield potent antiproliferative and antimetastatic drug candidates. The aim of the present study was to assess the antitumor and antimetastatic properties, as well as the mechanism of action of a D-ring-modified exo-heterocyclic androstadiene derivative named 17APAD. The test compound was found to be highly selective towards human breast cancer-derived cell lines (MCF-7, T47D, MDA-MB-361, MDA-MB-231) compared to non-cancerous fibroblast cells (NIH/3T3), and exerted superior effect compared to the clinically applied reference drug cisplatin. Changes in MCF-7- and MDA-MB-231 cell morphology and membrane integrity induced by the test substance were assessed by fluorescent double staining. Cell cycle disturbances were analyzed by flow cytometry, and concentration-dependent alterations were detected on breast cancer cell lines. Mitochondrial apoptosis induced by the test compound was demonstrated by JC-1 staining. Inhibitory effects on metastasis formation, including the inhibition of migration, invasion and intravasation were investigated in 2D and 3D models. Significant anti-migratory and anti-invasive effects on MCF-7 and MDA-MB-231 cells were detected after 24 h exposure in 2D wound healing and Boyden-chamber assays. The anti-intravasative properties of 17APAD were evident after 4 h of incubation in a co-culture 3D circular chemorepellent-induced defects (CCID) assay, and the level of inhibition at concentrations ≥2 µM was comparable to that exerted by the focal adhesion kinase inhibitor defactinib. Single cell mass cytometry revealed that chemosensitive subpopulations of MDA-MB-231 cells engaged to apoptosis were less positive for EGFR, CD274, and CD326, while the percentage of cells positive for GLUT1, MCT4, Pan-Keratin, CD66(a,c,e), Galectin-3 and TMEM45A increased in response to 17APAD treatment. Finally, the novel androstane analogue 17APAD had an outstanding inhibitory effect on tumour growth in the 4T1 orthotopic murine breast cancer model in vivo after 2 weeks of intraperitoneal administration. These findings support that substitution of the androsta-5,16-diene framework with a N-containing heterocyclic moiety at C17 position yields a molecular entity rational to be considered for design and synthesis of novel, effective antitumor agents, and 17APAD is worth further investigation as a promising anticancer drug candidate.

Synthesis of N-picolylcarboxamides in aminocarbonylation

Palladium-catalysed aminocarbonylation of iodocamphene and steroidal iodoalkenes was carried out in the presence of 2-, 3- and 4-picolylamine, as well as secondary amines possessing 1-picolyl substituent. In general, primary picolylamines require less than 2 h to achieve practically complete conversion. The secondary amines proved to be less reactive, requiring 6–24 h depending on the substrate structure. The corresponding carboxamides were isolated in moderate to excellent yields. The synthesis of α,β-unsaturated carboxamides is based on the synthesis of iodoalkene substrates from enolizable ketones. • Facile synthesis of unsaturated carboxamides in palladium-catalysed aminocarbonylation. • High-yielding functionalization of camphene and steroidal skeletons. • The use of easily available, reactive iodoalkene substrates.

Synthesis of steroid bearing heterocyclic derivatives and biological activity. Review 2014-2020

Steroidal building blocks have drawn research groups attention in many branches of science and technology, such as pharmacological and medical fields, supramolecular chemistry and compounds in nanotechnology, So the recent years have witnessed a wide focus of research directed towards preparing heterocyclic derivatives fused with steroid molecules as a result of the great biological activity these compounds, This review described an outline of the literature reports (2014-2020) of synthesis heterocyclic compounds for steroid molecules fused at rings-A or B or D of steroid skeleton or annealed, Also the review included the biological activity of steroid hetero cyclic as anti breast cancer, anti prostate cancer, antioxidant and antimicrobial.

Synthesis and mesomorphic characterization of novel liquid crystals derived from bioactive natural sterols

Steroids are one of the prominent categories of secondary metabolites that are renowned for their biological activities. The steroidal derivatives are well known for their inherent potential to exhibit diverse mesophases. In this report, we have shown the mesomorphic behavior of some novel 4-mercaptoalkyl benzoates of cholesterol, stigmasterol and ergosterol. This study mainly focuses on the effect of the steroidal skeleton, as well as the effect of replacement of oxygen with the sulfur atom in the side chains on their mesogenic behavior. The cholesteryl analogs were found to show smectic, cholesteric and blue phases; whereas the stigmasterol and ergosterol derivatives were found to be smectogenic in nature. The stigmasterol derivatives were found to exhibit significantly lower clearing temperatures compared to corresponding cholesterol and ergosterol analogs. Also, the replacement of the oxygen atom with sulfur in the cholesteryl derivatives resulted in a considerable reduction in their clearing temperatures.

Hydrophobicity and self‐association (micellization) of bile salts with a lactone or lactam group in a steroid skeleton

AbstractBile acid salts are amphiphiles that have planar polarity: the convex surface (the β side) of their steroid skeleton is hydrophobic, whereas the concave surface (the α side) is hydrophilic. Lactone and lactam derivatives of bile acid salts have reduced hydrophobicity on the convex surface (β side) of the steroid skeleton, but they are still able to build micelles. In the vicinity of their critical micellar concentration (CMC), they probably form Small's primary micelles. In this work, it is shown that retention capacities (reverse‐phase high‐performance liquid chromatography) are linearly dependent on temperature, for each tested bile acid. The parameters of this linear dependence (intercept and slope) can be used to describe the hydrophobicity of the analyzed bile acid salts. The first and the second principal components, derived from the covariance matrix of temperature dependence of retention capacity, can also be used as hydrophobicity parameters of the analyzed bile acid salts. There is a strong correlation between the CMC values and the values of the hydrophobicity parameters of the convex surface (the β side) of the steroid skeleton: the CMC values decrease as the hydrophobicity of the β side of the steroid skeleton decreases.

Cholesterol and Sitosterol, Two Members of the Steroid Family That Differ in Structure by a Single Ethyl Group Yet Maintain the Right Balance between Plant and Animal Kingdoms

Cholesterol and sitosterol are two members of the steroid family, which molecular structures differ only by the presence of an ethyl substituent at the position 24 of the steroid skeleton. Cholesterol is mainly synthesized in large quantities in the liver of animals and only in small quantities by plants. In contrast sitosterol is only synthesized by plants. This tiny difference of an ethyl group between the two steroids structures have induced profound effects on the climatic changes during evolution, but plant sterols still have important effects on cholesterol metabolism on humans.

Analysis of bile acids in human biological samples by microcolumn separation techniques: A review.

Bile acids are a group of compounds essential for lipid digestion and absorption with a steroid skeleton and a carboxylate side chain usually conjugated to glycine or taurine. Bile acids are regulatory molecules for a number of metabolic processes and can be used as biomarkers of various disorders. Since the middle ofthe twentiethcentury, the detection of bile acids has evolved from simple qualitative analysis to accurate quantification in complicated mixtures. Advanced methods are required to characterize and quantify individual bile acids in these mixtures.This article overviews the literature from the last two decades (2000-2020) and focuses on bile acid analysis in various human biological samples. The methods for sample preparation, including the sample treatment of conventional (blood plasma, blood serum, and urine) and unconventional samples (bile, saliva, duodenal/gastric juice, feces, etc.) are shortly discussed. Eventually, the focus is on novel analytical approaches and methods for each particular biological sample, providing an overview of the microcolumn separation techniques, such as high-performance liquid chromatography, gas chromatography, and capillaryelectrophoresis, used in their analysis. This is followed by a discussion on selected clinical applications.

Bile acid bearing poly (vinyl chloride) nanofibers by combination of CuAAC click chemistry and electrospinning process

This study aims to deposit most common bile acids such as lithocholic acid (LCA) and chenodeoxycholic acid (CDCA) bearing poly (vinyl chloride) nanofiber (PVC-LCA and PVC-CDCA) coatings on glass slides surface using a combination method by employing copper (I)-catalyzed azide-alkyne cycloaddition ‘click’ reaction (CuAAC), followed by electrospinning process. The resulted nanofibers and their intermediates are characterized by spectroscopic (FT-IR- 1H-NMR), thermal (TGA and DSC), wettability (WCA) and morphological (SEM) analyses. Many characterizations reveal that the bile acid functionalized PVC-LCA and PVC-CDCA are successfully achieved by CuAAC between azido-functional PVC (PVC-N3) and alkyne end-functional LCA and CDCA (LCA-Alkyne and CDCA-Alkyne) and their nanofibers obtained by electrospinning show efficiently improved thermal behavior, hydrophilicity and morphological features compared to pristine PVC or their granule analogues. Furthermore, the SEM images indicate that the formation of nanoscale, uniform, cylindrical and smooth fibers without free beads. The changeable properties achieved are attributed to the presence of rigid steroid skeleton, triazole rings and free −OH groups on the final polymers. Hence, it is clear that this facile combination procedure is encouraging for scientists working on potential biomedical PVC applications.

Подходы к эстрогенам на основе аддуктов Дильса-Альдера левоглюкозенона с диеном Дэйн

Levoglucosenone has established itself as a good Michael acceptor and a powerful dienophile in Diels-Alder reactions, dipolar cycloaddition and in a number of other transformations. In the Diels-Alder reactions of levoglucosenone with 1,3-dienes, chiral derivatives of cyclohexene are obtained, which are valuable products for the synthesis of natural compounds. We previously studied the reaction of the interaction of levoglucosenone with Dane diene under catalytic, thermal conditions, at ultrahigh pressures and microwave irradiation. It was found that as a result of the reaction, 2 adducts are formed – (1S,2S,15S,17R)-9-methoxy-18,20-dioxapentacyclo[15.2.1.02,15.0.5,14.06,11]icosa-4,6,8, 10-tetraen-16-one and its isomer, the product of the double bond migration is (1S,2S,14S,15S,17R)-9-methoxy-18,20-dioxapentacyclo[15.2.1.02,15.0.5, 14.06,11]icosa-5(14), 6,8,10-tetraen-16-one. In this work, we have developed methods for the transformation of these Diels-Alder adducts in approaches to compounds with a steroid skeleton. Thus, based on the obtained Diels-Alder adducts, optically active hydrazone was synthesized. An optimal method for deoxygenation of a keto group proceeding by aromatization of cycle B in (1S,2S,14S,15S,17R)-9-methoxy-18,20-dioxapenta-cyclo[15.2.1.02,15.0.5,14.06,11]icosa-5(14),6,8,10-tetraen-16-one, converting it to sulfide, followed by boiling in the presence of Raney nickel. The resulting compound, 9-methoxy-18,20-dioxapentacyclo-[15.2.1.02,15.0.5, 14.06,11]icosa-5(14),6,8,10,12-pentaenone, is a promising synthetic block for use in the synthesis of estrogen – equilenin. The biological activity of the synthesized compounds was predicted using the PASS computer program, which resulted in the identification of derivatives that are promising for the study of antacid, anti-seborrheic, embryotoxic, and anti-cancer properties.

New anti-neuroinflammatory steroids against LPS induced NO production in BV2 microglia cells by microbial transformation of isorhodeasapogenin.

Microbial transformation of isorhodeasapogenin (1), the major steroidal sapogenin of Tupistra chinensis, was performed with the fungus Syncephalastrum racemosum (AS 3.264). As a result, nine new biotransformation metabolites (2-10) were isolated and their structures were elucidated by spectroscopic analysis. Hydroxylation, oxidation and glycosylation reactions were observed on the B, C, D and F rings of steroidal skeleton. Substrate (1) and its biotransformed metabolites 2-6, 8-10 were evaluated for their anti-neuroinflammatory effect on the NO accumulation induced by LPS in BV-2 cells. All the tested metabolites were found to have more potential anti-neuroinflammatory activity than the substrate. Especially, metabolites 2, 5 and 6 exhibited significant inhibition on NO production after hydroxylation at C-12 or C-15. Moreover, metabolite 2 dose-dependently reduced the LPS-induced protein expression of iNOS and COX-2.

Structural studies of Trenbolone, Trenbolone Acetate, Hexahydrobenzylcarbonate and Enanthate esters

Estra-4,9,11-trien-17β-ol-3-one, known as trenbolone is a synthetic anabolic-androgenic steroid which belongs to nandrolone group. The structural features of trenbolone base and three esterified forms of trenbolone have been described. The crystal structures of trenbolone acetate and trenbolone hexahydrobenzylcarbonate were determined by X-ray single crystal diffraction and they were shown to belong to the monoclinic P21 space group. The crystal structure of trenbolone enanthate was determined with Parallel tempering-Monte Carlo method and refined with Rietveld procedure and was shown to belong to the monoclinic P21 space group as well. The A, B, C steroid skeleton rings are adopting distorted envelope and intermediate sofa half chair configuration, while the D ring exists in a 13β distorted envelope configuration. The intermolecular interactions were evaluated in terms of lattice energy computation with CLP (Coulomb-London-Pauli) method and intermolecular interactions have been analyzed and described by Hirshfeld surface.

Design, Synthesis, and Biological Evaluation of Two Series of Novel A-Ring Fused Steroidal Pyrazines as Potential Anticancer Agents.

Background: Increasingly, different heterocyclic systems have been introduced into the steroid nucleus to significantly enhance the antitumor activities of steroid molecules. However, in this study, few literature precedents describing the pyrazine heterocyclic-condensed modification to an A-ring of steroid monomers were found, although the pyrazine group is thought to be essential for the potent anticancer activity of clinically relevant drugs and natural steroid dimers. Methods and Results: Two series of novel A-ring fused steroidal pyrazines were designed and efficiently synthesized from commercially available progesterone via key α-ketoenol intermediates. Through a cell counting kit-8 cytotoxic assay of 36 derivatives for three tumor cells, 14 compounds displayed significant antiproliferative activity compared to 5-fluorouracil, especially for human prostatic tumor cells (PC-3) in vitro. Further mechanistic studies indicated that the most active compound, 12n (IC50, 0.93 μM; SI, 28.71), could induce the cell apoptosis of PC-3 cells in a dose-dependent manner and cause cell cycle arrest in the G2/M phase. The molecular docking study suggested that compound 12n fitted the active sites of cytochrome P450 17A1 (6CIZ) well. Conclusions: 12n might serve as a promising lead compound for the development of novel anticancer drugs. This facile ring-closing strategy may provide a novel and promising avenue for the cycloaddition reaction of the steroidal skeleton through α-ketoenol intermediates.