Search

Following the revelation of the presence of vitamin D in fish liver oils and of estrogenic hormones in pregnancy urine in the 1920s, active interest in the steroids began in England. Most of this interest originated from the studies of Ian Heilbron at Liverpool and of Otto Rosenheim at the National Institute for Medical Research in London.

Dioxo-5α-androstanes having one keto-group in a terminal ring (at position 3 or 2, or at position 17) and the second at a middle ring position (7 or 11) are hydroxylated in the other terminal ring (at 17 or 16, or at 3 or 2) by the fungi Wojnowicia graminis and Ophiobolus herpotrichus. Efficient transformations include the 2α-hydroxylation of 5α-androstane-7,17-dione (with W. graminis), the 16-substitution of the 3,11-dione (with O. herpotrichus), and the 17β-hydroxylation of 3,7-dioxygenated substrates (with both fungi).

Mono- and di-ketones and keto-alcohols derived from 5α-androstane have been incubated with the fungus Daedalea rufescens, a Basidiomycete species not previously reported as a steroid hydroxylator. All but one of the dioxygenated substrates, and the monoketones with the keto-group in a central ring, are hydroxylated fairly rapidly. The cleanest hydroxylations occur with 7-oxygenated androstanes and lead to the 3β,16β-dihydroxy-derivatives.Incubation of 3,3-ethylenedioxy-5α-androstan-7-one (in which hydroxylation is accompanied by reduction of the 7-keto-group) followed by hydrolysis of the product gives 7α,16β-dihydroxy-5α-androstan-3-one in 57% yield.

A series of monofluoro- and gem-difluoro-5α-androstanones and the parent ketones were incubated, under standard conditions, with the fungi named in the title. The results may be rationalised by comparing the positions of the fluorine atoms in the substrates with those of the favoured hydroxylation sites in the parent ketones. With few exceptions hydroxylation does not occur at, or adjacent to, the carbon to which a fluorine substituent is attached even though one of these centres is a favoured site (in the parent ketone). In such cases hydroxylation is usually diverted to an alternative position. Where the favoured site is more distant from the fluorine substituent(s) the behaviour of a fluoro-ketone resembles that of its parent. Hydroxylation of several fluoro-ketones by Aspergillus ochraceus gives the 11 α-hydroxy-derivatives cleanly and in yields which are satisfactory for preparative work.

Dioxygenated androstanes are readily hydroxylated by the title fungi. Although complex mixtures are generally formed, 3β-hydroxy-5α-androstan-7-one is converted efficiently into its 12α-hydroxy-derivative (51% yield) by S. racemosum. The poor steroid recoveries of incubations involving 3,17-dioxygenated substrates and A. regnieri are improved by using a medium containing cobalt(II) sulphate: under such conditions 3β-hydroxy-5α-androstan-17-one gives 3β,9α-dihydroxy-5α-androstan-17-one in 49% yield.

The sequence of microbiological reactions involved in the 19-hydroxylation of 5α-androstan-17-one has been established. When a solution of 5α-androstan-17-one in dimethyl sulphoxide is incubated with Calonectria decora, the initial 1β,6α-dihydroxylation is followed by oxidation of the 1β-hydroxy-group and then by hydroxylation at C-19 to give 6α,19-dihydroxy-5α-androstane-1,17-dione in 36% yield. This compound is readily transformed (by chemical methods) into 5α,10β-estrane-1,6,17-trione.C-19 hydroxylation occurs also with certain substituted 17-oxo-5α-androstanes and 17-oxo-3α,5-cyclo-5α-androstanes.

From cyclododecanone a series of bicyclic alcohols and ketones containing 15–16 carbon atoms have been synthesised for use as substrates in microbiological hydroxylations.

Structural investigation of the products obtained by microbiological hydroxylation of substrates in the androstane, pregnane, and œstrane series has involved examining the 1H n.m.r. spectra of 344 steroids, 243 of them being new compounds. Mild oxidation of the products gives polyketones in which the positions of the oxo-groups are characterised by the chemical and solvent shifts of the 18-H and 19-H signals. This information is supplemented by the chemical shifts of the products' 18-H and 19-H signals, and by the positions and form of their CH–OH resonances: the latter are especially useful in establishing the configurations of the hydroxy-groups.

The following new compounds have been prepared: 2α-, 4α-, 4β-, 6β- and12α-hydroxy-5α-androstanes; the acetates of these alcohols and of the 1α-, 1β-, 2β-, 6α-, 12β-, 15α- and 15β-hydroxy-5α-androstanes; 3α- and 3β-hydroxy-5α-androst-1-ene and 1β-hydroxy-5α-androst-2-ene; a series of esters and ethers derived from the 3-hydroxy-5α-androstanes. Some methoxy-5α-androstanes were obtained conveniently from the alcohols with diazomethane.

Dioxygenated 5α-androstanes are more readily hydroxylated with Calonectria decora than the mono-oxygenated substrates studied previously. Oxygen functions in rings A and D act as primary directing groups and, when a blocking effect by middle ring substituents is allowed for, the pattern of hydroxylation is predictable in most cases.1β-Hydroxy-compounds are obtained in moderate yields from the readily accessible 5α-androstane-6,17- and 7,17-diones.

Hydroxylation of 3β-hydroxy-5α-pregnan-20-one with Aspergillus ochraceus, in shake-flasks or using 10g batch fermentation, gives 1β,3β,11α-trihydroxy-5α-pregnan-20-one as the main product (ca. 50% yield); this trihydroxy-ketone is readily converted into 5α-pregn-1-ene-3,11,20-trione. With some other 20-oxo-5α-pregnanes containing a second oxygen group, the initial 11α-hydroxylation is followed by a variety of processes which lead to mixtures of products.

Diketones and keto-alcohols derived from 5α-androstane are readily converted into monohydroxy-derivatives by Rhizopus nigricans. Varying the positions and oxidation levels of the oxygen functions leads to hydroxylation at different positions. Substrates with one oxygen substituent in each of the terminal rings are attacked at position 11 or 7; those with one group in rings B or C are hydroxylated at position 16 if the second group is in ring A, and at position 3 if the second group is in ring D.The results are rationalised by assuming the presence of three dual-purpose sites on the enzyme surface capable of binding to the steroidal oxygen groups and of hydroxylating those positions of the steroid nucleus which come into their vicinity.

A number of 5,6-diols and -epoxides in the 4,4-dimethyl-chlolestane and -androstane series have been prepared and their reactions under acidic conditions studied. Different products are obtained from the diols and the epoxides and, as expected, the type of oxygen function at C-3 influences the nature of the products formed.Dehydration of the 5α,6α-diol system with an acetate or hydroxyl group at C-3 gives 6-ketones of both the 5α and 5β series and also, with the 4,4-dimethyl-cholestane compounds, 4a-ketones of the A-homo-B-nor series (1).In the case of the 5α,6α-epoxides the 3β-hydroxy-group participates in the rearrangement leading to methyl migration from C-4 to C-5 and eventually to the 6α-hydroxy-4,5-dimethyl-3-oxo derivatives (2). Such participation is obviated when a keto-group is present at C-3, and these 5α,6α-epoxides undergo a rearrangement involving the migration of the C(1)–C(10) bond with the formation of the spirans (3).With the corresponding 5β,6β-epoxides cis C-10 methyl group migration predominates. [graphic omitted]

Of the eleven 5α-androstane monoketones, only two are hydroxylated by Aspergillus ochraceus. 5α-Androstan-3-one (and 5α-estran-3-one and the related Δ4-3-ketones) give 11α-hydroxy- and then 6β,11α-dihydroxy-compounds; 5α-androstan-17-one gives a 7β,11α-dihydroxy-derivative.The predilection of A. ochraceus for 11α-hydroxylation is emphasized by the results with dioxygenated andro-stanes which, although representing a range of structural types, are all hydroxylated efficiently at the 11α-position.

16-Oxo-5α-androstanes are readily obtained from the 17-ketones in three stages, viz., condensation with benzaldehyde to 16-benzylidene-17-ketones, reduction with lithium aluminium hydride–aluminium chloride to 16-benzylidene-androstanes, and ozonolysis. This sequence is not suitable for the 3 → 2 transposition of oxo-groups in ring-A. Here the 2-arylidene-6-ketones are reduced with sodium borohydride and acetylated before ozonolysis: the acetoxy-groups of the 3β-acetoxy-2-ketones are removed by reduction with zinc and acetic acid.With 5α-androstane-3,17-dione the longer route afforded 5α-androstane-2,16-dione in 24% yield overall.

The steroids named in the title have been prepared by routes in which the yields at all stages are higher than 70%. For the ring-A lactams the key stages involve ring opening of 3-oxa- and 3-aza-2,4-diones; attack by nucleophiles occurs selectively at position 4. 17-Aza-5α-androstan-16-one is readily obtained by Beckmann rearrangement of the 16-hydroxyimino-17-ketone to 17-aza-D-homo-5α-androstane-16,17α-dione, followed by Hofmann degradation. The 16-aza-17-oxo-isomer was prepared from the 17-ketone in six stages by a sequence similar to that used previously for other 16-azasteroids.

The hydroxylations of seven diketones and keto-alcohols derived from 5α-androstane with Rhizopus arrhizus and Rhizopus circinnans are similar to, but not identical with, those observed previously using Rhizopus nigricans. R. circinnans is useful for introducing a 4α-hydroxy-group into 5α-androstane-11,17-dione.N.m.r. shift values are given for the influence on steroidal 18- and 19-H signals of various single substituents, and of systems containing two or more substituents whose effects are not additive.

Diaporthe celastrina, a fungus not previously reported as being active with steroids, hydroxylates a range of dioxygenated 5α-androstanes generally by attack on the α-face. The sites of hydroxylation and the efficiencies of the processes depend on the positions and oxidation levels of the androstanes' substituents. 5α-Androstane-3,7-dione is converted into 3β,16α-dihydroxy-5α-androstan-7-one (45% yield); 3β-hydroxy-5α-androstan-17-one is hydroxylated in the 9α-position (55%), and 5α-androstane-7,17-dione gives the 3α-hydroxy-derivative (52%).

By utilising the specific hydroxylating capabilities of the fungi Aspergillus ochraceus Wilhelm and Penicillium urticae Bain in combination with standard chemical reactions, 5α-androstan-15α-ol has been obtained from 3α-hydroxy-5α-androstan-17-one in an overall yield of 16%.

Eight 3,20-, 7,20-, and 11,20-dioxygenated 5α-pregnanes have been incubated with the fungi Calonectria decora and Daedalea rufescens, and with three Rhizopus species. In most cases complex mixtures are formed, and the hydroxylations are less satisfactory than those of dioxygenated androstane analogues. Although Rhizopus nigricans leads mainly to the 11α-hydroxylation of 5α-pregnane-3,20-dione, hydroxylation of 3β-hydroxy-5α-pregnan-20-one occurs predominantly at the 7β-position (not at the 11α-position as reported in the literature). The cleanest incubation studied here is that of 5α-pregnane-7,20-dione with Calonectria decora, which gives the 1α,12β-dihydroxy-7,20-dione (30%) and the 12β-hydroxy-1,7,20-trione (19%).Convenient preparations of the 7,20- and 11,20-dioxygenated substrates have been developed.

The hydroxylations set out in the title have been studied and the results compared with those (obtained in earlier work) of hydroxylating the parent 3-, 7-, and 17-ketones. The microbiological effect of a halogeno-substitutent depends on its nature, position, and configuration in the steroid nucleus. The 3α-fluoro-, 3α-chloro-, and 3α-bromo-5α-androstan-17-ones undergo 1β,6α-dihydroxylation with C.decora; while the 3β-chloro- and 3β-bromo-analogues are more reactive (and are also hydroxylated initially in this way), the 3β-fluoro-17-ketone is recovered essentially unchanged. Loss of halogen generally occurs during incubations of 3-halogeno-17-ketones with R.nigricans, the products being 3-oxo- and 3β-hydroxy-compounds. A.ochraceus converts the fluoro-ketones (but not the chloro-ketones) into their 11α-hydroxy- or 7β,11α-dihydroxy-derivatives; the former process has been utilised in the preparation of 3α- and 3β-fluoro-5α-androstane-11,17-dione.

Acetals and enol ethers derived from oxoandrostanes are less reactive than the parent ketones towards the title fungi. None of the derivatives is hydroxylated by Rhizopus nigricans, and only one by Aspergillus ochraceus. With Calonectria decora the acetals generally give patterns of hydroxylation similar to, but less specific than, those of the corresponding ketones. 16,16-Ethylenedioxy-5α-androstane is exceptional in that its hydroxylation with Calonectria decora to a 6α,12β-dihydroxy-product is more efficient than the 6α,11α-dihydroxylation of 5α-androstan-16-one.

Although steroidal monoketones with the carbonyl group in ring B or C are relatively unreactive towards Rhizopus nigricans, 2-, 3-, 16-, and 17-ketones give modest yields of dihydroxy-derivatives. The position of the carbonyl group influences the direction of the hydroxylation process: comparison of the 11,16-dihydroxylation of 3-ketones with the 3,7-dihydroxylation of 17-ketones suggests that a reversal effect is operating.16-Hydroxylation, not previously recorded with this fungus, occurs commonly with the present androstane and estrane derivatives, i.e. steroids lacking side-chains at position 17. Estr-4-en-3-one gives three 16-oxygenated products (total yield 68%), the main one being the 10β,16β-dihydroxy-Δ4-3-ketone.