Recent syntheses of steroidal oxazoles, oxazolines and oxazolidines

It was found that the introduction of heterocycles to steroids often leads in a change of their physiological activity and the appearance of new interesting biological precursors. Recent developments in the syntheses of steroidal oxazoles, oxazolines


Introduction
2][3] Even after decades of research, the total synthesis of steroid nuclei by improved strategies continues to receive considerable attention.Numerous methods have been exploited for the total synthesis of steroids which are widely distributed in nature and which possess practical medical importance.Research into steroid total synthesis continues to this day. 4,5eterocycles are widespread in drug molecules because they possess hydrogen bond donors and acceptors in a rigid framework, and they can therefore effectively interact with target enzymes and receptors via hydrogen bond formation.They can enhance binding affinity and improve in vitro potency.Heterocycles can modulate the lipophilicity of the drug molecules or improve the aqueous solubility of the compounds, thus providing desired pharmacokinetic and pharmacodynamic properties. 6Heterocyclic compounds are therefore widely applied in pharmaceutical and agrochemical research.
4][15][16][17] Steroids containing heteroatoms have been widely researched and reported. 18][21][22] This article provides an overview of the various synthetic strategies which have been employed to synthesize steroidal derivatives containing oxazoles, oxazolines, and oxazolidines along with interesting biological activities, from the years 1996-2021.To the best of our knowledge and much to our surprise, there are no reports on this subject since 1996. 23

Synthesis of Steroidal Oxazoles
In 2018, Timofeev et al. 24 reported the synthesis of benzoxazolyl derivatives 6-9 (Scheme 1) starting from steroid acids 1, 2, 3, 4, and o-aminophenol.The cyclization step was carried out in the presence of pyridine instead of triethyl amine.Preliminary experiments revealed that cyclization of intermediate amides (shown in brackets) in the presence of strong bases gave target benzoxazoles in low yield, and was accompanied by the formation of a complex mixture of byproducts.If the cyclization step was conducted in the presence of pyridine at 50 °C during 3 h, the yield of benzoxazoles 6-9 was >50%, however, in contrast to the synthesis of oxazolines, all these benzoxazoles were obtained as mixtures of two isomers.Compounds 6-9 in concentrations of 1 μM, 10 μM, and 30 μM did not markedly inhibit CYP17A1 activity.

Scheme 1. Synthesis of steroidal oxazole derivatives 5-9.
In 2019, steroids containing an isoxazole were synthesized by Scherbakov et al. 25 by a dipolar cycloaddition.The influence of solvents on the result of dipolar cycloaddition and solvolysis of bridged nitrosteroids was evaluated.Cytotoxicity testing of estrone analogs on cancer cells of various origins revealed activity against breast, colorectal, prostate, and lung cancer cells.
The starting nitro adduct 11 was synthesized from estrone 10 by the procedure described by Baranovsky et al. 26 with some modifications, in particular, in the synthesis of a mixture of Δ14-and Δ15-ketones, DMF was replaced by dimethylacetamide, which much decreased the reaction time and improved the total yield of product (Scheme 2).
Studying the chemical properties of isoxazolyl steroids 12 and 13 they focused on the reductive transformations of their heterocyclic moiety.The presence of the Δ15 bond in steroids created some difficulties in the selective hydrogenation of this bond.The hydrogenation reaction (Pd/C, H2; Ni/Ra, H2; Pd/C, HCO2NH4) occurred unselectively: along with the saturation of the double bond, they observed ring-opening and intramolecular Michael cyclization of the ring-opening products to form various С-14-С-15-fused derivatives.Thus, to accomplish the intended purpose, they had to exclude the double bond in steroids 12 and 13 from conjugation.As known, sodium borohydride can reduce conjugated systems to saturated alcohols. 27The same result was obtained in the reduction of steroid 12: the reaction resulted in the preferential formation of a 17α epimer (according to the NMR data, the 17α/17β ratio was 1.5:1).Under the reaction conditions, removal of the protective group at С3 was also observed.Therefore, the resulting mixture of products was acetylated to obtain triacetates 18 and 19.They could partially separate these products.Isoxazolines 14 and their 3-hydroxy derivatives 15, unlike isoxazoles, are resistant to Pd-catalyzed hydrogenation, and their reduction afforded steroids 16 and 17, respectively.By repeated chromatography, they could isolate individual isomers of the compound 16.
Treatment of steroid 16 with TsOH leads to the elimination of ethanol from the isoxazoline ring to form isoxazoles 20 and 21 in a high total yield.To synthesize alcohols they made use of Δ15-isoxazolines 14, which were reduced with sodium borohydride.Conjugated reduction of 14 with sodium borohydride, like with steroid 12, resulted in the preferential formation of 17α alcohols (1.5:1) and was accompanied by removal of the protective group at С-3.The mixture of four diastereomers (at С-17 and С-5') was, without separation, acetylated in acidic conditions, where acetylation was accompanied by heteroring aromatization.From the mixture of two epimers of 22 and 23, they isolated 17α epimer.Scheme 2. Synthesis of steroidal oxazole derivatives 18-23.
Steroids 13, 15, and 21 were tested for cytostatic activity against a series of human cancer cell lines, specifically, MCF-7, MDA-MB-231, and T-47D breast, HCT 116 colorectal, 22Rv1 prostate, and A549 lung human cancer cell lines.The concentrations of test compounds in the cytostatic activity tests were varied between 6 and 50 μМ.All tested compounds showed a low cytostatic activity at low concentrations (6-25 μM).Compound 13 showed the highest activity against MCF-7 hormone-dependent breast cancer cells.Compound 15 inhibited the growth of MDA-MB-231 triple-negative breast cancer cells by 18%, while other steroids were less active.Steroids 13, 15, and 21 showed similar activities against HCT 116 cells (~25% growth inhibition).Steroids 13 and 15 did not show any activity against lung cancer cells, while steroid 21 inhibited the growth of this cell line.Steroid 21 was found to be the most active (51% inhibition of T-47D growth).
The reaction of estradiol 24 with acetonitrile led to a spiro-steroid ethanimidic acid analog 25.Then, a nitrospiro-steroid ethanimidic acid 26 was prepared through a reaction of 25 with nitric acid.Finally, 27 was formed via intramolecular displacement of nitro by a hydroxyl group.This reaction involves two reactions mechanism: (a) preparation of an acetamidic acid via reaction of nitrile with the hydroxyl group bound to ring-A; (b) formation of spiro system by the reaction of acetonitrile with the 17-hydroxyl group of steroid derivative.

Synthesis of Steroidal Oxazolines
In 2014, a series of novel D-ring substituted isoxazoline and oxazoline derivatives of dehydroepiandrosterone and pregnenolone, respectively were synthesized by Banday et al. 30 and screened for anticancer activity against a panel of human prostate cancer cell lines.From the data, it was found that all the compounds are having promising anticancer activity especially against LNCaP and DU-145 cell lines and the compound 33e was found to be the most active in this study.
The starting ketone 28 upon condensation with ethyl cyanoacetate in boiling toluene, in the presence of ammonium acetate was transformed to Knoevenagel adduct 29 presumably as a mixture of E and Z isomers (89% yield) (Scheme 4).The condensation product was reduced to the saturated alcohol 30 (97% yield).Hydroxyl group in compound 30 was then protected as its tetrahydropyranyl (THP) ether and the derivative 31 was subjected to reduction with an excess of neat di-isobutyl aluminum hydride (DIBAL) for a prolonged period resulting in the formation of two products.The major product was the required one 32 formed in almost 50% yield.The intermediate 32 served as an activated olefin having a great potential for the construction of a large number of carbocyclic and heterocyclic analogs across ring D preferably through dipolar cycloadditions.The same was done for the preparation of isoxazoline derivatives 33 (a-f) by employing the cycloaddition of aromatic nitrile oxides across the olefin 32.Though the cycloaddition could conceptually lead to the formation of two regioisomers, only one regioisomer was isolated.
The in vitro cytotoxicity studies of various steroidal isooxazoline and oxazoline derivatives revealed that these derivatives are cell-specific as they were found to be active mostly against the LNCaP (androgen dependent) compared to PC-3 and DU-145 (androgen-independent) prostate cancer cell lines.As compounds 33a, 33e, and 39a were found to be more active than other analogs, it can be assumed that electron-donating groups affect the activity.Scheme 5. Synthesis of D-ring substituted pregnenolone oxazolines.
In 2017, bile acid derivatives with an oxazoline ring in the side chain on steroid skeleton were synthesized by Bjedov et al. 33 and their antitumor activity against human cancer cell lines was investigated.Protection of carboxylic group in the form of oxazoline can resolve low solubility of bile salt in ether solvents.The synthesis of bile oxazoline derivatives is described in Scheme 6.The sequence included formylation of 40 and 41, subsequent condensation of the carboxylic group with 2-amino-2-methyl-1-propanol to 44 and 45 using EEDQ as coupling agent followed by cyclization to oxazolines 46 and 47 by treatment with thionyl chloride.After deformylation 12-oxo and 3-oxo oxazolines 48 and 49 were synthesized in good yields.Scheme 6. Synthesis of oxazoline derivatives.
Oxazolines 48 and 49 exhibited moderate antitumor activity toward the HeLa cell line but were 2-3-fold more active than cholic acid.The authors noticed interesting structure-activity relations.Thus, altering the carboxylic group to the oxazoline group has a positive effect on cytotoxicity.This observation could influence future work in the design and synthesis of bile acid derivatives.
Timofeev et al. 24 also described in 2018 the synthesis of oxazolinyl derivatives of [17(20)E]-21-norpregnene 53-55 (Scheme 7).As reported earlier, tosylate 50 34 was transformed to compound 51 by treatment with methanol in the presence of CH3COONa in 85% yield.Formation of 6β-methoxy-3α,5α-cyclo fragment was accompanied by transesterification of the 21-carboxylic group (additional experiments showed that ethyl esters of pregn-17(20)-en-21-oic acid are transformed to related methyl esters).The methyl ester 51 was subjected to alkaline hydrolysis and obtained acid 52 was subjected to reaction with triphenylphosphine, carbon tetrachloride, and ethanolamine, in the presence of triethylamine, to give oxazoline 53 in 62% yield.Oxazolines 54 and 55 were prepared from compound 53 in high yields by acid-catalyzed methanolysis, or treatment with HCl in tetrahydrofuran, respectively.Scheme 7. Synthesis of oxazoline derivatives 54 and 55.
For the preparation of oxazoline 61 comprising seco-A pregnene moiety, ketosteroid 56 34 was chosen as starting compound (Scheme 8).Selective epoxidation of Δ 4 with hydrogen peroxide under alkaline conditions led to compound 57 (a mixture of 4α,5α-and 4β,5β-epoxides in a ratio of 2:1) in 83% yield, which without separation of isomers was subjected to Eshenmoser fragmentation.Treatment of the compound 57 with p-tosyl hydrazine in dichloromethane -acetic acid mixture led to ethyl ester 59, which was subjected to alkaline hydrolysis, and the resulted acid 60 was transformed to the oxazoline 61 in 48% yield according to the procedure mentioned above.
Simple synthesis of target compounds 76-82 is presented on Scheme 10.Transformation of acetylated acid 75 to related homologous acid 81 was performed by Arndt-Eistert reaction 37 : compound 75 was treated with oxalyl chloride, then resulting acyl chloride 75a was treated with an excess of diazomethane to obtain diazo ketone 80, which rearranged into acid 81 under heating with aqueous dioxane in the presence of Ag + ions.3β-Acetylated acids 74, 75, and 81 were transformed to oxazolines 76, 77, 82 according to the procedure described by Kostin et al. 38 .Acids 74 and 75 were also converted to benzoxazoles 78 and 79 according to the procedure described by Timofeev et al. 24 The synthesized compounds inhibited the growth of prostate carcinoma LNCaP and PC-3 cells at 96 h incubation; the potency of 2′-(3β-hydroxyandrosta-5,16-dien-17-yl)-4′,5′-dihydro-1′,3′-oxazole (76) was superior and could inspire further investigations of this compound as a potential anti-cancer agent.
IK-3 was obtained by chemical reduction of 85 (IK-1) as described in Scheme 12.
The inhibitory activity, evaluated through the formation of thiobarbituric acid reactive substances (TBARS) and the conjugated diene, was compared with that of -tocopherol and 2,2,6,6-tetramethylpiperidine-1-yloxy (TEMPO).In each model system IK-1 and IK-2 exhibited an IC50 of 8 mM and reduced the formation of TBARS and conjugated diene, showing IK-1 a potency comparable to -tocopherol and higher than TEMPO.Moreover, IK-1 and, to a lesser extent IK-2, reduced the lipid peroxidation induced in the microsomes by the water-soluble azo-initiator (2-methylpropionamidine) dihydrochloride (AMPH), indicating the IK-1 and IK-2 ability as chain-breaking antioxidants.The hydroxylamine 4',4'-dimethylspiro (5-cholestane-3,2'-oxazolidin)-3'hydroxide (IK-3) was completely inactive as an inhibitor of lipid peroxidation in heat pre-treated microsomes and liposomes.However, in microsomes, it was active since it was oxidized to the corresponding nitroxyl radical IK-1.The more lipophilic, IK-1, confirms that a good affinity for cell membranes improves the activity of lipid peroxidation inhibitors.
In 2012, a new diversity-oriented synthesis (DOS) methodology was developed by Poirier et al. 45 to generate different types of oxazinones, oxazolidinones from β-amino diols, which can be easily obtained from ketones (Scheme 15).Considering a large number of natural products and synthetic templates bearing a ketone functionality, this DOS methodology opens the door to the exploration of chemical space regions around the ketone and will favor the discovery of bioactive compounds.
The estrone, first protected as a methoxymethyl ether at the 3-position, was submitted to an aldol condensation reaction with benzaldehyde by using KOH in refluxing ethanol (1st level of diversity) to give corresponding enone 104 in high yield.Epoxidation of enone 104 was, however, more problematic and needed investigation.Therefore, the authors decided to stereoselectively reduce the enone to generate the corresponding allylic alcohol 105 and then use it as directing the group to favor epoxidation.Enone 104 was thus reduced by using sodium borohydride in methanol and exclusively gave 17β-OH 105.This simple modification in the synthetic sequence of reaction order, by performing first the reduction of the 17-ketone and then the epoxidation, resolved the epoxidation problem.High yield of epoxides 106a and 106b were obtained by using m-CPBA in DCM with a ratio of α-/β-epoxides (35:65).This ratio is explained by the directing effect of the 17β-OH group, which favors the attack of the peracid from the β-face of the olefin.Aminolysis was done using a microwave heating approach described for the opening of hindered epoxides. 46It was found that 3 equiv of the amine in ethanol at 180 °C gave a complete reaction.The opening of β-epoxide 106b was easier (90 min) than the opening of α-epoxide 106a (8 h).Scheme 14. Synthesis of steroidal oxazolidines 100-103.
The challenge was to generate a five-membered ring considering that the formation of a six-membered ring was much faster.Prior oxidation of the 17β-OH group of 107a to block the formation of the six-membered ring seemed to be the best option.However, selective oxidation of alcohol in the presence of a secondary amine was not obvious.Since the secondary amines are known to easily form imine and N-oxide byproducts with the usual oxidizing reagents 47 , the authors used the polymeric version of 2-iodoxybenzoic acid (PS-IBX) to oxidize the preformed TFA-amine salt in DCM. 48The oxidation was completely selective and compound 108 was obtained in quantitative yield by simple filtration.In that manner, the sequence of reactions from intermediate 106 could be completely compatible with solution-phase parallel chemistry to rapidly generate libraries of azacycle derivatives.They were also able to selectively obtain five-membered ring 109 from amino diol 107b reacting with triphosgene in the presence of DIPEA.In fact, formation of the six-membered ring was not possible considering the opposite position of the 16α-amine and 17β-OH group.Scheme 15.Synthesis of steroidal oxazolidines 108 and 109.

Conclusions
The present review offers an up-to-date literature on the latest syntheses of steroidal oxazoles, oxazolines and oxazolidinones reported during the last years.Several of these syntheses may be useful, and in particular Poirier et al. 45 offers an attractive, short and efficient preparation of steroidal oxazinones and oxazolidinones.Overall, the interest in steroids and related compounds continue to expand given the diversity of structure and emerging bioactivity inherent in this compound class.

Malika
Ibrahim-Ouali carried out her Ph.D. under the supervision of Professor Gramain in Clermont-Ferrand (France) in 1996.The work was focused on the synthesis of alkaloids.She was a postdoctoral fellow with Prof. Knochel (Humboldt fellowship) in 1996 in Germany where she got her first training in organometallic chemistry.In 1997, she joined Professor H. P Husson's group at the ICSN (Paris) as a postdoctoral researcher.Since 1998, she is currently an assistant professor at Aix-Marseille University and her field of interest remains the total synthesis of natural compounds.Frédéric Dumur received his Ph.D. in chemistry in 2002 from the University of Angers (France) under the supervision of Professor Pietrick Hudhomme.After Post-Doctoral studies at the University of Groningen (The Netherlands), Reims Champagne-Ardennes (France) and Versailles Saint-Quentin-en-Yvelines (France), he joined the Faculty of Sciences at Aix-Marseille University in 2008, where he is currently working as an Associate Professor.His research interests include the synthesis of phosphorescent dopants for OLEDs and photoinitiators of polymerization.He co-authored about 300 publications and 5 book chapters.This paper is an open access article distributed under the terms of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/)