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Seven reagents of different types having in common a C–Hal and a CO electrophilic centre have been used in a study of their reactions with 2-aminothiazoles. Three reagents, CHBrAc2 and ROCHBrCO2Et (R = Me, Ph), gave imidazo[2,1-b]thiazoles, thus providing useful routes to the 5-acetyl and 5-ethoxycarbonyl derivatives. Unexpectedly, the solvent (acetone) was involved in the reaction of the fourth reagent, CHBr(CO2Et)2, with 2-aminothiazole which led to 5,5-di(ethoxy;carbonyl)-6,6-dimethyl-5,6-dihydroimidazo[2,1-b]thiazole (yield 81%). With the last three reagents (AcCHBrNO2, BzCHBrCN and ICH2CO2C6H4NO2-p) the outcome was simpler, viz., the formation of 2-amidothiazoles.It is proposed that electrophilic attack by the endo-N of the 2-aminothiazole is the first step in all cases. This occurs at the C–Hal centre of the first four reagents and is followed by cyclisation to the exo-N. In the last three electrophiles the presence of groups well suited to leaving as stabilised anions favours addition to the CO group; the intermediates so formed subsequently isomerise to the more stable exo-N substituted products.

Fifteen imidazo[2,1-b]thiazoles containing a range of substituents at position 6 have been used in studies of electrophilic substitution at position 5. Acid-catalysed deuteriation, was examined quantitively and, by making certain assumptions, values for the rate constants (k1) of the deuteriation process were obtained. A 3-methyl group increases k1 by a factor of ca. 3; for different 6-substituents the k1-values are in the order Me > But∼ C6H4OMe-p > Ph. Bromination occurred preferentially at position 5 even with substrates containing 6-substituents (e.g., 2-thienyl) which themselves have a reactive nucleus. Although 6-alkyl-5-nitrosoimidazothiazoles are unstable and cannot be prepared by the standard method (sodium nitrite and acetic acid), the 6-t-butyl compounds were obtained by a procedure in which the work-up involves very mild treatment. The 5-formyl derivatives show extensive mesomerism, and in solution they adopt the conformation having the carbonyl oxygen syn to N(4). In one compound, the 4,6-dimethyl-5-carbaldehyde, the anti rotamer is present as the minor component. Imidazo[2,1-b]thiazoles are easily converted into 5-trifluoroacetyl compounds and 5-thiocyanates.

Two methods have been used for epimerising the 3-hydroxy-group of 19-norergosta-5,7,9-trien-3β-ol. Although the 3β- and 3α-alcohols show a close resemblance in many of their properties it is possible to distinguish between them by examining the 19F n.m.r. spectra of their α-methoxy-α-trifluoromethylphenylacetates and the 1H n.m.r. spectra of their complexes with shift reagents. Purification of the alcohols is most reliably achieved by crystallising their 3,5-dinitrobenzoates. Measurement of optical rotation is the best method for the quantitative analysis of mixtures of the epimers.

Known methods were developed to give convenient general procedures for preparing N-acyl-N′-mono and -N′,N′-disubstituted thioureas from acid chlorides, and related N-imidoyl thioureas from imidoyl chlorides. In the products from three acid chlorides and ammonium thiocyanate the acyl isothiocyanates did not appear to be accompanied by the isomeric thiocyanates.Treatment of N-(anilino)benzylidene-N′,N′-disubstituted thioureas with chloroacetone in the presence of triethylamine leads to 5-acetyl-4-phenyl -2-(N,N-disubstituted amino)thiazoles. In contrast, the corresponding N-benzoyl thioureas form only small amounts of these compounds; the main products are the 5-benzoyl-4-methyl isomers, and this unexpected outcome requires a revision of the literature. It is thought that formation of the 5-benzoyl-4-methylthiazoles involves N–C(4) fission of a cyclic intermediate to give an open-chain intermediate in which nucleophilic attack can occur at either the acetyl or the benzoyl group. One of the latter intermediates was generated directly from 2-acetyl-2-bromoacetophenone and N-methyl-N-phenylthiourea, and found to give the 5-benzoyl-4-methyl - and 5-acetyl-4-phenyl-thiazoles as the major and minor products, respectively.

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.

In order to establish the structures of compounds described as Δ4-thiazolin-2-ones ten sets of heterocycles (each comprising a 4- or 5-substituted Δ4-thiazolin-2-one, the N-methyl derivative, and the corresponding 2-methoxy-thiazole) have been examined by i.r., u.v., and 1H and 13C n.m.r. spectrometry. The i.r. results established that in solution the parent compounds exist entirely or predominantly as the 2-oxo-forms. This conclusion is supported by the 1H n.m.r. evidence, but the u.v. and 13C n.m.r. data do not give a clear distinction between the possible 2-oxo and 2-hydroxy-structures.

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.

Convenient general procedures have been developed for preparing series of thiazole-4- and -5-carboxylates containing alkyl and halogeno substituents. While both series of esters show i.r. carbonyl doublets caused by rotational isomerism, the more intense absorptions of the 4-carboxylates are the lower wavenumber components, whereas those of the 5-carboxylates are the higher wavenumber components. In both series the stronger bands arise from the thermochemically more stable forms; identification of these forms as the carbonyl O,S-syn-s-trans rotamers is more certain with the 4-carboxylates than with the 5-carboxylates.

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.

4,4-Dimethyl-5α-androstan-2-one and lupan-2-one, preapared from the 3-ketones by general methods for transposing oxo-groups, undergo substitutions at position 3. Acetoxylation and bromination proceed easily and introduce 3α-substituents; formylation to the 3-hydroxymethylene-2-ketones is difficult and gives poor yields even under forcing conditions.

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%).

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.

Suitable, readily available 3,17-dioxygenated 5α-androstanes are hydroxylated efficiently at position 11 by Aspergillus ochraceus and at position 7 by Rhizopus nigricans. These reactions, carried out on a reasonable scale, are the basis of convenient routes to derivatives of 5α-androstane (3,7-, 3,11-, 3,12-, 7,11-, 7,17-, and 11,17-dioxygenated compounds, and 5α-androstan-11-one) which are not readily accessible by chemical means.

Convenient, efficient sequences involving 3α,5-cyclo-intermediates have been developed for obtaining 3,6- and 6,17-dioxygenated 5α-androstanes from the cheap steroid 3β-hydroxyandrost-5-en-17-one. Useful selective reactions of 5α-androstane derivatives are achieved by acetalising diketones in the presence of an ion-exchange resin and by oxidising dihydric alcohols with the Fetizon (silver carbonate) reagent.

The title compounds, required as models in spectrometric work, have been synthesised by a six-stage route from 3-bromofuran-2-carbaldehyde diethyl acetal and the corresponding thiophen acetal.

3β-Hydroxy-5α-androstan-16-one is conveniently prepared from the readily available 3-hydroxy-17-ketone by a simple sequence which gives an overall yield of 53%

The mechanisms of two reactions which give isomeric thiazol-5-yl ketones have been elucidated by work with substrates containing pairs of acyl groups and their deuterium-substituted analogues.

Incubation of certain dioxygenated 5α-androstanes, notably 3,6-hydroxy-ketones, with Leptoporus fissilis gives 16β, 18-dihydroxy- and 16β-monohydroxy-products in combined yields of over 50%.

A convenient procedure for preparing N-mono- and N,N-di-substituted cyanamides from cyanogen bromide has been developed. N,N-Disubstituted thioureas, obtained from the cyanamides, were condensed with α-bromo-α-cyano-ketones to give 5-cyano-2-(N,N-disubstituted amino)thiazoles and with α-bromo-ketones to give 2-(N,N-disubstituted amino)thiazoles. Substitution in the latter products afforded 5-trifluoroacetyl- and 5-nitro-2-(N,N-disubstitutedamino)thiazoles; nitration is greatly facilitated by the presence of a 4-aryl group.The average values of the barriers to rotation of the 2-NR2 groups (ΔG‡298) in the 5-substituted thiazoles were established by variable temperature 1H n.m.r. spectrometry to be 57.4 kJ mol-1(5-NO2), 56.2 (5-COCF3), and 51.5 (5-CN). I.r. examinations showed that the 5-trifluoroacetyl compounds adopt one rotational form preferentially; this is probably the carbonyl O,S-syn-conformation.

The conformational preferences of 2-(N,N-disubstituted amino)thiazoles having a 4-substituent (R1) and a 5-acyl group (R2CO) have been established by i.r. spectrometry and a crystallographic examination. In solution the compounds with R2= aryl and R1= Me or aryl exist predominantly or entirely in the carbonyl O,S-anti arrangement; for those with R1= aryl and R2= Me the syn rotamer is the main form but a small amount (ca. 15%) of the anti rotamer is present.Nitrosation of 4-aryl-2-dimethylaminothiazoles gives 5-nitroso derivatives, and these are probably the first authentic nitrosothiazoles. The barrier to rotation of the dimethylamino group is unexpectedly high (ΔG298‡= 69 kJ mol–1), exceeding even that of the 5-trifluoroacetyl analogues.

Hydroxy-ketones in the 5α-androstane series are converted into fluoro-ketones by diethylaminosulphur trifluoride under mild conditions; acetoxy-ketones give acetoxydifluorides under more vigorous conditions.