Preparation of steroidal hormones with an emphasis on transformations of phytosterols and cholesterol

Contents

Introduction - Hormonal steroids

Hormonal steroids - first isolation

Hormonal steroids from steroidal sapogenins of Dioscoreae

Hormonal steroids by total synthesis

Chemical cleavage of the C17-side chain in cholesterol - early routes to steroidal hormones fromcholesterol

Enzymatic cleavage of the C17-side chain in cholesterol, sitosterol, stigmasterol,

campesterol and analogs

References

Preparation of steroidal hormones with an emphasis on transformations of phytosterols and cholesterol - a review

Yosef Al Jasem¹, Mubashar Khan², Ahmed Taha³ and Thies Thiemann⁴

¹ Department of Chemical and² Petroleum Engineering, Faculty of Engineering,³ Central University Library and ⁴ Department of Chemistry, Faculty of Science, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates

Abstract: Today, there is a big market for steroidal hormones as well as for their derivatives. This review traces the development in steroidal production from the first milligram-scale isolation of the products to their semi-synthesis from sapogenins, their total synthesis and finally their microbial enzymatic preparation from phytosterols.

Keywords: steroids, hormones, cholesterol, phytosterols, C-17 side chain cleavage, biotransformation, enzymes.

Introduction - Hormonal steroids

Steroids form an integral part of the mammalian endocrinal system. Some of the better known and important steroids are estradiol (1, E2), testosterone (2), progesterone (3, P4), aldosterone (4) and cortisol (5) (Figure 1). As the major estrogen, estradiol (1, E2) serves as a growth hormone for tissues of reproductive organs, being co-responsible for the development of secondary sex characteristics in females, and has an important effect of maintaining bone structure. Progesterone (3, P4) as the major progestogen is involved in the female menstrual cycle. It supports pregnancies and is involved in embryogenesis. Testosterone (2) as the major androgen is co-responsible for the development of secondary sex characteristics in males and helps regulate sperm production. Also, it has anabolic properties. Aldosterone (4) as a mineralocorticoid helps regulate the blood pressure and influences the salt and water balance in the body. It supports Na+ and water retention, but lowers the K+ plasma levels. Lastly, cortisol (5) as a glucocorticoid regulates blood sugar and aids fat, carbohydrate and protein metabolism.

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Figure 1. Important steroidal hormones

As an example of the production of hormonal steroids in humans, let us look at the simplified schematic of the biogenesis of estradiol. In the human body, estradiol (1, E2) is produced from cholesterol as virtual all human hormonal steroids are (Scheme 1). A pivotal intermediate in the synthesis is androstene-3,17-dione (11), some of which is reduced to testosterone (2) by the 17 -hydroxysteroid dehydrogenase (17 -HSD) (Scheme 1). An aromatase (estrogen synthase) converts androstenedione (11) to estrone (19, E1), where the 19-methyl group of androstenedione (11) is eliminated oxidatively (Scheme 2). 17 -HSD can convert estrone (19) to estradiol (1) (Scheme 2).

A second pathway takes us from testosterone (2) directly to estradiol (1), a conversion mediated by aromatase (Scheme 1). Androstenedione (11), which is also a very important intermediate in the synthetic production of steroidal hormones from phytosterols (plant derived steroids) and cholesterol, is derived from pregnenolone (7). Pregnenolone (7) can be converted to either progesterone (3) using both 3 -hydroxysteroid dehydrogenase (3 -HSD) and delta 4-5-isomerase. These conversions lead into the delta-4-pathway, where progesterone (3) transforms to 17 -hydroxyprogesterone (10) via 17 -hydroxylation, and 10 converts to androstenedione (11) by 17,20-lyation. In this regard, the human cytochrome enzyme P45scc (CYP17A1) has both 17 -hydroxylase and 17, 20-lyase activities (Scheme 1).

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Scheme 1. Schematic representation of the biogenesis cascades of steroidal hormones

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Scheme 2. In vivo conversion of androst-4-ene-3,17-dione (AD, 11) to estradiol (1)

This leaves to be answered how pregnenolone (7) is produced from cholesterol (6). To understand this C-17 side chain cleavage from 6 to 7 is also of fundamental importance for the synthetic (eg., the industrial) production of steroidal hormones from phytosterols and cholesterol. In the human body, the conversion of cholesterol to pregnenolone (7) is catalysed by the cholesterol side-chain cleavage enzyme P45scc. The reaction proceeds in three steps. First, a hydroxylation step of cholesterol (6) yields 22 R -hydroxycholesterol (20). A second hydroxylation step gives 20 ,22 R -dihydroxycholesterol (21) (Scheme 3).

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Scheme 3. In vivo conversion of cholesterol (6) to pregnenolone (7)

Both of these steps are monooxygenase steps involving electron transfer from NADPH to enzyme P450scc via two electron transfer proteins - adrenodoxin and adrenodoxin reductase. A final step cleaves the 1,2-diol in 21 to furnish pregnenolone (7) with isocaproic acid as an isolable side product in humans¹. In vitro, isocaproaldehyde (22) (see Scheme 3) and other products such as isohexanol have been found². This may in part indicate follow-up reactions of the isocaproaldehyde (4-methylpentanal, 22), ie.,through oxidation or reduction.

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Figure 2. Structure of equilin (23), equilenin (24), androsterone (25), medroxyprogesterone (26)

Hormonal steroids - first isolation

Androsterone (25) was first isolated from the urine of policemen³. Quickly, however, it became evident that natural isolation of hormonal steroids would not satisfy the demand, as, for instance, in 1931, 15.000 L of urine yielded only 15 mg of pure androsterone (25)³. Similarly, 4 tons of sows' ovaries were needed to isolate 12 mg of estradiol (1)⁴,⁵, while extraction from 625 kg of sows’ ovaries produced 20 mg of progesterone (3)⁶. Also, estrone was isolated from pregnant female human urine as well as from mare’s urine. 100 kg of bull’s testicles yielded 10 mg of testosterone (2) and 100 kg of adrenal glands from 20000 cows yielded 75 mg of cortisone and 55 mg of hydrocortisone. Aldosterone (4) was also isolated from adrenal glands⁷. While these isolations were of fundamental importance for the general understanding of the structure and function of steroidal hormones, it did not contribute much to a method of production, at a scale that these compounds would be needed in future years. In this respect, however, it has to be noted that the estrogens of Premarin®, a hormone replacement drug containing mainly the sulfates of estrone, equilin (23), and equilenin (24) have been produced from the urine of pregnant horses from the early 1940s until today. The same is true for Prempro®, which includes additionally the synthetic medroxyprogesterone acetate (26). In the 1930s, efforts were devoted to the wet-chemical transformation of cholesterol into steroidal hormones. Today, natural steroids and their synthetic derivatives find use as contraceptives, hormone replacement agents, muscle enhancers, and anti-inflammatory agents in rheumatic arthritis. By 1975, the total revenue from steroid sales is estimated to have been $ 3 billion⁸ a. By 2011, the revenues from topical corticosteroids alone account for $ 900 million⁸ b.

Hormonal steroids from steroidal sapogenins of Dioscoreae

One such natural source is diosgenin (27), a steroidal sapogenin, first isolated by Tsukamoto from the yam Dioscorea tokoro (Makino)⁹, but which can also be found in the tubers of other species of the Dioscoreae (Dioscorea villosa, D. mexicana and D. composita). Diosgenin (27) is also a constituent of many other plants such as Trillium erectum ¹⁰, Solanum lyratum ¹¹, Tribulus terrestis ¹² and Paris polyphylla ¹³. The hydrogenated form of diosgenin, tigogenin (28), can be found in Chlorogalum pomeridianum bulbs¹⁴, Tribulus terrestis ¹⁵, and Solanum paniculatum ¹⁶. Hecogenin (29) as a third such sapogenin was extracted from the sisal plant Agave sisalana ¹⁷. In the so-called Marker synthesis, diosgenin (27) could be transformed to progesterone (3)¹⁸,¹⁹ (Scheme 4), which was used in the first combined oral contraceptive pills²⁰. While Dioscorea plants grow in various countries such as India, South Africa and China²¹, the large-scale production of steroidal hormones by semi-synthesis from diosgenin (27) and related sapogenins left a mark on the Mexican chemical industry⁸ and led to the founding of Syntex in Mexico²²,²³. Due to protection and limited availability of Dioscorea plants, however, the price for diosgenin (27) increased dramatically over time, so that other sources needed to be found, especially in regions outside of China.

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Figure 3. Steroidal sapogenins

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Scheme 4. Marker’s degradation of diosgenin (27) to pregnenolone (7) and progesterone (3)

Thereafter, processes developed use solasodine (30) as a starting material²⁴ (eg, Scheme 5). Solasodine has been found in a number of plants of the genus Solanum, where steroidal manufacturers have tried to cultivate such plants as Solanum auriculatum for the purpose of harvesting solasodine (30).

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Scheme 5. High-yielding process converting diosgenin (27) and solasodine (30) into dehydropregnenolone acetate (33-Ac)

However, solasodine (30) has been found to have anti-tumour activities, where a plant preparation (SBP002, Coramsine®), containing solasonine and solamargine (both solasodine glycoalkaloids), is currently in phase-II clinical trial for treating skin cancers²⁵. Thus, in China, where diosgenin (27) is more readily available, a synthetic process has been developed to transform diosgenin (27) into solasodine (30)²⁶ (Scheme 6). At the same time, numerous Chinese patents underline the continuing importance of diosgenin as a source of steroidal hormones. Nevertheless, the worldwide pharmaceutical industry with a global market for steroidal drugs of US$ 4-8 billion had to find an alternative source for the production of hormonal steroids.

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Scheme 6. Solasodine (30) from diosgenin (27)

Hormonal steroids by total synthesis

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Scheme 7. Bachmann’s total synthesis of O -methylequilenin (48)

Early, a synthetic route for equilenin (24), a steroid found in horses, was devised by Bachmann²⁷. Thus, in part, an alternative access to steroidal hormones presented itself with de novo synthesis of steroids such as estradiol and its derivatives. A number of total syntheses²⁸ of research groups from pharmaceutical companies and academic institutions were disseminated in the late 1950s - early 1960s, namely by Velluz of Roussel-Uclaf, Smith of Wyeth Laboratories (and Manchester University), Rufer and Schroeder of the German Schering AG, and Torgov of the USSR Academy of Sciences. Here, only two of the main synthetic strategies can be shown. The first is the Torgov synthesis (Scheme 8).

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Scheme 8. Torgov total synthesis of rac -estrone

Torgov’s synthesis²⁹ was exploited by Jenapharm, where by the 1980s up to 5 tons of steroids were produced per year, after the process had been improved through various modifications as documented by over 100 patents³⁰. Initially, the 3- O -methylestone was separated into its antipodes by diastereomeric of D-acetyl tartaric acid methyl ester³¹ and also as diastereomeric cinchonine salts derived from racemic hemiphthalates³². Bucourt et al. from Roussel-Uclaf let the secosteroid dione52react with L-tartramic acid and separated the two adducts 55a and 55b by their different solubility in methanol in the presence of trace amounts of acetic acid (Figure 4)³³.

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Figure 4. Separation of the antipodes of secosteroid 52by reaction with tartramic acid

Also, an enantioselective reduction of the secosteroid 52 was developed by Torgov et al., and by the Schering steroidal chemists, which gave enantiopure 3- O -methylestrone (Scheme 9)³⁴.

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Scheme 9. Towards enantiopure estrone/estradiol by enantioselective reduction of secosteroid52

In 2007, Corey et al. published a further enantioselective variation of the Torgov estrone synthesis using the enantiotopo-selective Itsuno-Corey reduction of secosteroid 52 (Scheme 10)³⁵.

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Scheme 10. Corey’s enantioselective variant of the Torgov synthesis

Many syntheses of hormonal steroids employed the approach of preparing a suitable enantiomeric enriched 1-hydroxylated tetrahydroindan-5-one system of type 60. For the most part, this involved the desymmetrization of an 2-alkylcyclopenta-1,3-dione derivative.

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Scheme 11. Desymmetrization of an 2-alkylcyclopenta-1,3-dione derivative by enzymatic reduction

This could be carried out by an enzymatic reduction at the stage of the 2-alkylcyclopenta- 1,3-dione as shown in Bellet’s synthesis of 61 (Scheme 11)³⁶. In other cases, tetrahydroindan-5-on-1-ols were resolved into antipodes by reacting first an auxiliary group to the molecule. Thus, rac -62, obtained by NaBH4 reduction of 65, was reacted with phthalic anhydride and the resulting ester-acid was resolved using brucine(Scheme 12)³⁷. In further instances, the symmetric cyclopenta-1,3-diones were reacted with molecules which had already a controlled stereocenter introduced into their structure.

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Scheme 12. Separation of optical antipodes by brucinium phthalates

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Scheme 13. Enantioselective synthesis of Hajos-Parrish diketone 65 and other tetrahydroindandiones with chiral amino acids as organocatalyst

A big step forward was the possibility of an early organic catalytic reaction using proline in the intramolecular Aldol reaction to produce the Hajos-Parrish diketone 65 in an enantioselective manner (Scheme 13)³⁸. The chiral organocatalyst used was proline, where R - and S -proline led to the respective enantiomers of the Aldol reaction products. Not only 2-butan-3-onyl-2-methylcyclopenta-1,3-dione but also other 2-butan-3-onyl-2-alkyl / arylcyclopenta-1,3-diones could be used as starting materials in the reaction. Moreover, the butanonyl residue of the starting material could be elaborated further. Also, the Michael addition with the 2-alkylcyclopenta-1,3-dione as Michael acceptor and the subsequent intramolecular Aldol reaction could be run in one-pot, as shown in Scheme 12 in the transformation of 63a and 66 to 67, where S-phenylalanine was used as the organocatalyst. This text-book reaction was used as a key-step in the synthesis of (+)-3- O -methylestrone 54³⁹ and of a number of optically active 19-norsteroids such as 19-nortestosterone 76⁴⁰ (Scheme 14).

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Scheme 14. Use of chiral Hajos-Parrish diketone as a building block in the syntheses to O -protected estra-1,3,5(10),9(11)-tetraen-3,17 -diol (73) and to nortestosterone 76

Sauer et al. also used diketone 65a-Et as their starting material for norgestrel (81)⁴¹, used in hormonal contraceptives.

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Scheme 15. Synthesis of norgestrel (81) from Hajos-Parrish-type diketone 65a-Et

Chemical cleavage of the C17-side chain in cholesterol - early routes to steroidal hormones from cholesterol

At the same time as some of the earliest total syntheses towards steroids, efforts were devoted to finding ways to prepare hormonal steroids from cholesterol (6) and plant phytosterols.Among these sterols, -sitosterol (82), stigmasterol (83) and campesterol (84) (Figure 5) are abundant in soy bean oil, rape seed and paper pulp industrial waste. Lastly, for the most part the human body provides itself also with hormonal steroids through the enzymatic degradation of the C17-side chain of cholesterol (6).

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Figure 5. Structures of -sitosterol (82), stigmasterol (83), and campesterol (84)

At first, efforts were undertaken to chemically cleave the C17-side chain in cholesterol (6) at high reaction temperatures. Initially, these reactions were carried out to elucidate the structure of cholesterol. Thus, Diels subjected cholesterol (6) to a reaction over palladized charcoal at 400oC to yield chrysene (85)⁴²,⁴³, albeit in poor yield, at 500oC a mixture of chrysene (85) and naphthalene⁴⁴,⁴⁵.

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Scheme 16. C-17-side chain cleavage in cholesterol (6) under pyrolysis conditions

Later, also experiments on carbon alone and on a mixture of sulfur and carbon were shown to transform cholesterol (6) to chrysene (85)⁴⁶. Nevertheless, these reactions gave mixtures of products, often in poor yield, where sulfur is known to insert into cyclic carbon structures. An analogous reaction of cholesterol over selenium, albeit at 300oC, was more successful to yield cyclopentanophenanthrene 86, subsequently named Diels hydrocarbon. This derivative could be prepared independently starting from 9,10-dihydrophenanthrene⁴⁷.

The formation of the polycyclic aromatic compounds was believed to be the basis of the cancerogenocity of the pyrolysis oils of cholesterol (6) and caused concern, when reviewing the fate of food sterols, when subjected to heating in cooking processes.

Early endeavors of chemically converting cholesterol (6) to hormonal steroids such as progesterone have produced the target compounds in poor yield. Thus, the oxidation of cholestenone dibromide 88 with potassium permanganate in a mixture of sulfuric acid and benzene has been reported to give progesterone in 1% yield⁴⁸. An oxidation of the cholesteryl dibromide is also possible, where cholesterol is brominated. Thereafter, without purification.the cholesteryl dibromide 89 is oxidized with potassium permanganate in sulfuric acid. After crude separation of MnO2 formed, the mixture of oxidation products is debrominated with zinc in glacial acetic acid to give progesterone in 0.2% yield⁴⁹. The oxidation of cholestenone (87) with chromic acid in acetic acid to progesterone (3) in unspecified yield has also been communicated⁵⁰ (Scheme 17).

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Scheme 17. Early chemical conversions of cholesterol (6) to progesterone (3)

Cholesterol (6) could be converted to 3 -hydroxyallocholanic acid (91-OH) by oxidation of the intermediate dihydrocholesterol acetate90⁵¹. Subsequently, 90 was transformed to 3 -hydroxynorallocholanic acid by Barbier-Wieland degradation, ie., by reacting 3 -hydroxyallocholanate 91with phenylmagnesium bromide, dehydrating the resulting diphenylcarbinol with iodine in benzene and oxidizing the olefin, thus prepared, with chromic acid⁵². Three further cleavages to 3 -hydroxynorallocholanic acid (93), subsequently to 3 -hydroxybisnorallocholanic acid (94) and then to 3 -allopregnanol-20-one (97), respectively, were affected along the same lines, with the oxidative cleavage of the final olefin 95 carried out with ozone in ethyl acetate at -40oC with a reductive work-up by hydrogenation of the ozonide on Pd/CaCO352. Diphenyl-3- -acetoxyallocholene 92 could be reacted with N -bromosuccinimide (NBS) to diene 96, which could be cleaved with chromic acid to yield 3 -allopregnanol-20-one (97), directly. It must be noted that the procedure was not meant as a synthetic process from cholesterol (6) to hormonal steroids, but rather as a clarification of the biogenesis of cholesterol through degradation of a¹⁴ C labelled cholesterol, prepared enzymatically by incubating rat liver slices with 1-¹⁴ C-acetate and 2-¹⁴ C-acetate⁵².

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Scheme 18. Degradation of cholesterol (6) to allopregnanolone (97) and androstanediol (18) through repeated Barbier-Wieland degradation

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Scheme 19. Marker’s andRuzicka’s syntheses of androsterone (25) and 3-epiandrosterone (25- ) from cholesterol (6)

Tetrahydroergosterol was acetylated and subsequently the C17-side chain was cleaved with chromic acid⁵³. No yield is given, but it is expected to be low for this type of reaction. Similarly -cholestanyl chloride (99a) was treated with CrO3 in acetic acid to give chloroandrosterone (100a) in 2.5% yield⁵⁴ (Scheme 19). A similar sequence was carried out by Ruzicka, with a change in the sequence chlorination - hydrogenation of Marker to hydrogenation - chlorination of the starting material cholesterol (6)⁵¹ (Scheme 19).

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Figure 6. Identified reaction products from the autooxidation of cholesterol in air

It is interesting to note, however, that autooxidation of cholesterol (6) in air can also lead to steroids of the androstane and the pregnane series, namely androst-5-ene-3 ,17 -diol (102), androst-5-en-3- -ol (103), 3 -hydroxyandrost-5-en-17-one (104), pregn-5-ene-3 ,20 -diol (105), pregn-5-en-3 -ol (106), and to 3 -hydroxypregn-5-en-20-one (7)⁵⁵ (Figure 6). It has been suggested that the oxidation proceeds via the 20 - and 25-hydroperoxyderivatives, which can be detected when ultrapure cholesterol is reacted with air at elevated temperatures⁵⁵.

For a time, Jenapharm also used hyodeoxycholic acid (107), derived from hog bile, as anindustrial starting material for pregnenolone (7)³⁰,⁵⁶ (Scheme 20).

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Scheme 20. Hyodeoxycholic acid (107) from hog bile in the synthesis of steroidal hormones

In the mid-1950s, Jenapharm was already producing kilogram quantities of progesterone (3) and deoxycorticosterone acetate (12-Ac), utilizing this process. This was the starting point of a whole production cascade leading also to testosterone (2), cortisone (17), cortisol (5) and prednisone (109), a synthetic corticosteroid drug. One of the key reactions was to differentiate the 3 - and 5 -hydroxy groups in hyodeoxycholic acid (107). This could be done by reacting 107 with phosphorus tribromide in the absence of pyridine followed in a second step by a nucleophilic substitution of an acetate for the bromo function at C-3 in the intermediate to yield methyl 3 -acetoxy cholanate (108).

In 1970, Breslow and Baldwin developed site-specific, template-directed functionalisation reactions in ring D of steroids⁵⁷ -⁵⁹, where 3 -cholestanol and analogous derivatives were esterified with 4-benzoylphenyl containing alkanoic acids of differing chain lengths, and the resulting esters were photolysed. Photolyis would lead to the substituted benzophenone triplet, which would abstract a hydrogen atom from the steroidal framework nearby. It was found that depending on the chain lengths of the attached ester, either macrolide formation would dominate in the case of shorter chain lengths or dehydrogenation would be favored. The site of the initial hydrogen abstraction would also differ, with abstraction occurring at C12 and C14 with shorter chain length of the ester and at C14 and C17 with longer chain esters. When the photolysis was carried out in the presence of chlorinated solvents, the radical pair thus prepared was found to abstract a chloro atom from the solvent to form chlorinated steroids. This fact was utilized by Breslow's group with further, published examples of site-specific, template-directed halogenations of steroidal frameworks. Within this context, they also achieved halogenation at C17⁶⁰,⁶¹.

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Scheme 21. Photochemical chlorination of a 3 -cholestanol derived ester 110 with subsequent dehydrohalogenation to 3 -cholest-16-enol (111)⁶¹

This allowed for the introduction of a double at C17-C20 via dehydrohalogenation. In a number of cases the creation of an unsaturation at C16-C17 competed effectively (Scheme 20), so that in these instances an isomerisation step had to be added to obtain the C17-C20 olefins in good yield. These could be subjected to oxidative cleavage to produce the C17-keto steroids, eg., by ozonolysis. Nevertheless, the sequence of reactions was used effectively to prepare androst-4-en-3,17-dione (11) from cholesterol, -sitosterol and from campesterol (Scheme 22). Best suited base for the dehalogenation reaction with the highest ratio of 17(20)-ene over 16(17)ene formed was found to be 1,5-diazabicyclo[5.4.0]undec- 5-ene (DBU)⁶² (Scheme 22). The reaction was run solventless, where the addition of solvent was found to increase the proportion of 16(17)ene⁶³ (Scheme 23). Also, it was found that an oxo function or acetoxy function in C11 of the steroidal intermediate would direct the dehydrohalogenation involving the C17 -chloro substituent to provide predominantly the 17(20)-enes64. This strategy would be useful for the preparation of corticosteroid derivatives⁶⁴.

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Scheme 22. Use of 1,5-diazabicyclo[5.4.0]undecene (DBU) to furnish steroidal 17(20)-enes after radical template directed chlorination of C17⁶²

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Scheme 23. 4:1 selectivity of steroidal 17(20)-enes vs. 16(17)-enes when using DBU⁶³

Enzymatic cleavage of the C17-side chain in cholesterol, sitosterol, stigmasterol, campesterol and analogs

All the methods of preparation mentioned above were not adequate to address the size of the market for steroidal hormones, which in 2001 was for androsta-1,4-dien-3,17-dione (ADD) and androst-4-ene-3,17-dione (AD) derived compounds over 1000 tons per year⁶⁵. Thus, new resources for steroids needed to be found. Phytosterols were deemed to be such a resource. Already in 1970, Wiechert noted that such phytosterols as -sitosterol are regaining importance as raw materials for steroidal hormones due to the fact that enzymatic degradations of the C-17 side chain of phytosterols became better understood⁶⁶.

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Scheme 24. Transformation of androsta-1,4-dien-3,17-dione (128) to estrone (19) by flash vacuum pyrolysis

The enzymatic cleavage of phytosterols such as sitosterol builds on the accumulation of androsta-1,4-dien-3,17-dione (128), which can be transformed to other hormonal steroids such as to estrone (19), eg., by flash vacuum pyrolysis. Soybean Oil Deodorizer Destillate (SODD), which is a waste product in the soybean oil production, has been forwarded as a source especially rich inphytosterols, campesterol (83), -sitosterol (81), stigmasterol (82) and brassicasterol (129)⁶⁷ -⁷⁰.

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Figure 7. Structures of brassicasterol (129) and 20 -hydroxymethylpregna-1,4-dien-3-one (130)

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Scheme 25. Pathway of the conversion of -sitosterol with M. spp. (adopted from ref. 73)

One of the earliest viable enzymatic transformations of C17-side chain containing steroids to androsta-1,4-dien-3,17-dione (128) was the conversion of cholesterol with Mycobacteriumphlei in the presence of nickel sulfate⁷¹. The steroid degradation with Mycobacterium strains is a complicated, multienzyme process, incorporating the often unwanted action of 1,2-dihydrogenases and 9 -hydroxylase⁷². In the case of the action of

Mycobacterium spp. on -sitosterol (82), 9 -hydroxylation of accumulated AD (11) and subsequent 1,2-dehydrogenation leads to the instable 9 -hydroxyandrosta-1,4-diene- 3,17-dione (135), which undergoes A-ring aromatization with concurrent cleavage of ring B, leading to the 9(10)-secosteroid 136, which is then metabolized further⁷³ (Scheme 25). Early on, it was noted that the presence of chelating agents such as o -phenanthroline or , '-bipyridyl inhibits the enzymatic ring cleavage, providing the opportunity to transform for instance cholesterol (6) into androst-1,4-diene-3,17-dione (128) in high yield. Other inhibitors of enzymatic ring cleavage were found to be hydroxyquinoline, -naphthol, copper sulfate and nickel sulfate. Thus, cholesterol (6) was incubated with Arthrobacter simplex (IAM 1660) in the presence of 0.8 mM bipyridyl to yield androst-1,4-diene-3,17-dione (128) in 14.9% yield after 44h at 30oC⁷⁴. At some point, however, it was tried to develop strains of bacteria, in which the synthesis of 9 -hydroxylase and 1,2-dihydrogenases is inhibited. This was successful with a number of Mycobacterium strains, and patents followed. Kraychy et al. described the fermentation of steroids of the cholestane and stigmastane series with Mycobacterium sp.NRRL-B-3683, providing a mixture of AD (11), ADD (128), and 20 -hydroxymethylpregna-1,4-dien-3-one (130)⁷⁵. Weber and Kennecke of Schering A.G. patented a microbial degradation of ergosterol to AD (11) and ADD (128) with a mycobacterium, with an example given, where ergosterol was transformed into ADD in 30% yield with 67% of ergosterol unreacted⁷⁶. In the patent, it was expressly stated that the process was to be 9 -hydroxylation inhibitor free. M. fortuitum, NRRL B-8153, obtained through mutation of M. fortuitum, ATC 6842, a non-selective degrader of steroids, by addition of nitrosoguanidin, was found to be a selective degrader.Herein, stigmasterol (83), cholesterol (6), campesterol (84), and sitosterol (82) were transformed into a mixture of AD (11) and ADD (128)⁷⁷. Other patents included the use of M. vaccae for preparing AD from steroids of the cholestane and stigmastane series⁷⁸, the use of a M. fortuitum mutant (B-11359) for preparing AD (11) from steroids sitosterol (82), cholesterol (6), stigmasterol (83), campesterol (84) or a combination thereof (all examples given experimentally in the patent) with the advantage of obtaining very little ADD (128) as by-product. A comprehensive overview of microbial reactions leading to C17-cleavage in cholesterol and phytosterols can be found in Figure 8 and Table 1. Today, more than 1000 tons of steroids are produced annually from phytosterols by microbial transformation, alone. Some of the production streams have been schematized below.

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Scheme 26. Major pathways of microbial production of hormonal steroids from phytosterols and cholesterol (6); adopted from ref. 79

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Figure 8 (Continued)

Acknowledgement: Thies Thiemann thanks the Emirates Foundation for grant 21S023.

Table 1: Microbial transformation of cholesterol and phytosterols with C17-side chain cleavage

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References

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