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Title: Chemical and microbiological studies of alicyclic systems
Author: Clark, Ian Maxwell
ISNI:       0000 0001 3554 1841
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1972
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In the microbiological studies initiated in this department nine years ago, an attempt has been made to gain more understanding of the processes relating structural variations in a steroidal substrate with the positions of micro-biological hydroxylation. To this end, oxygenated steroids (mainly androstanes) have been prepared and incubated with selected micro-organisms. The primary purpose of the present work was to synthesise 5α-androstan-15-one [1], 5α,14β-androstan-15-one [2], 5α,13α-androstan-15-one [3], and a series of simple oxygenated androstanes of unnatural configuration, and to incubate these compounds with the fungus Calonectria decora. A related aim was to augment the utility of n.m.r. spectroscopy in elucidating the structures of steroid metabolites by a revision and an extension of the incremental C-methyl shift data recently published from this department. Synthesis of substrates By modifying the procedures of Djerassi et al. and Nambara et al., the isomeric 15-ketones [1] and [3] were prepared in satisfactory yields (Scheme 1). A key factor in the improved yields of 5α-androstan-15-one [1] was the development of conditions for an unusual in situ diimide reduction of 5α-androst-16-en-15β-ol [4]. Three new syntheses of 5α-androstan-15-one [1] were devised (Schemes 2, 3, and 4), all giving yields comparable with the previously reported method. The alternative new chemical synthesis of 5α-androstan-15-one [1] is outlined in Scheme 2. The other two utilised the hydroxylating capacity of the fungi Aspergillus ochraceus and Penicillium urticae (Scheme 3) or Calonectria decora (Scheme 4); this work demonstrated that with a judicious choice of substrate and conditions, microbiological hydroxylation can often be effectively employed in synthetic work. Associated with the work in Scheme 3, optimum conditions for large scale fermentations with the fungus A.ochraceus were developed, and a synthesis of 5α-androstane-3,12-dione [5] much superior to that previously reported was devised. Associated with the work in Scheme 4 were syntheses of the substrates 12β-hydroxy-5α,14β-androstan-15-one [6], 15α-hydroxy-5α-androstan-12-one [7], 5α-androstane-12,15-dione [8], and 5α,14β-androstane-12,15-dione [9]. A high-yield synthesis of 5α,14β-androstan-15-one [2] is shown in Scheme 5. Syntheses of 5β-androstan-3-one [10], A-nor-(5→6β)abeo-androstan-5-one [12] and its 3β-hydroxy derivative [11] followed from the simple modification of established procedures, and are outlined in Scheme 6. Hydroxylations with C.decora The structures of the products of incubations with this fungus were assigned after an examination of the spectra of the compounds and their derivatives, and where possible, conversion of the products to known compounds. The incubation of 5α-androstan-15-one [1] with C.decora (Scheme 7) constituted the final experiment in a series related to the hydroxylation of 5α-androstane mono-ketones, where the position of the carbonyl group had been varied systematically around the androstane skeleton. It was surprising that in the hydroxylation of 5α-androstan-15-one [1], the major product was isolated as the 14β-isomer. The results for the whole series have been discussed in a recent paper, and are not repeated here. The result of incubating 5α,14β-androstan-15-one [2] (Scheme 8) prompted a more systematic study into the hydroxylation of substrates of unnatural configuration. Significant results in this work are summarised in Table 1. Only those groups introduced or modified by the fungus are listed in the Product(s) column. No clear-cut conclusions could be made from a consideration of these results and a few isolated examples from previous work; with the binding site out of the plane of the steroid skeleton, unpredictable variations (usually a spatial restriction) in the pattern of hydroxylation were found to occur. Related topics In connection with the microbiological and mass spectral studies in this laboratory, it was necessary to characterise the complete series of mono-oxygenated 5α-androstanes. A number of the alcohols (including a few of unnatural configuration) were synthesised and a series of acetates were prepared; the principal constants for all 5α-androstane mono-alcohols and their acetate derivatives were tabulated, for later reference in the microbiological work. Some aspects of the mass spectra of the acetates were examined, and an interesting feature was the expulsion of a fragment of 29 mass units, especially dominant in 5α-androstane 2-acetates. It has been difficult to assess the contribution of the C14-methyl group in the initial mass spectral fragmentation of triterpene compounds. With the availability of suitable precursors, it was hoped to examine this effect in the [M-15] peak of 14-methyl-5α,14β-androstane [13] and its 14-methyl-d3 analogue [14], prepared as in Scheme 9. It was found that in these compounds, the initial fragmentation leading to the [M-15] peak was dominated (>90%) by a loss of the 14-methyl group. The development of unusual in situ conditions for the reduction with diimide of 5α-androst-16-en-15β-ol [4] is shown in Scheme 1. In an extension of this work, a number of readily available A- and D-ring olefins and allylic alcohols were reacted under the neutral conditions ("P") described by Corey et al. Within the limited range studied, it was found that facile reduction is unique to the 16-olefin of the 5α,14α-androstane series. N.m.r. spectroscopy of steroids The structural determination of steroid metabolites has been very dependent on n.m.r. spectroscopy; a consideration of the angular methyl signals in CDCl3, of the position and multiplicity of >CH-OR proton signals, and of the difference of angular methyl signals of derived polyketones in CCl4 and benzene has usually provided sufficient data to define the structure of a new metabolite. The most recent refinement of Zürcher's original treatment comes from a study of 411 oxygenated steroids prepared in this department, and other notable contributions have been made by French and Japanese groups. An additional 300 steroids (mostly new, and many of unnatural configuration) have been characterised in this department, and some anomalies, usually arising from structural interactions, have become apparent in the published data. In the present work, use has been made of the now complete series of mono-oxygenated 5α-androstanes to devise a new set of C-methyl incremental values. Account has been taken of interacting functional groups, and many anomalies have been accommodated. A number of previously unrecorded shift values have also been deduced. To facilitate use, the new set of incremental values has been printed in tabular form, and will appear in Part VIII of the Oxford series, "Microbiological Hydroxylotion of Steroids".
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available