Microbiological synthesis of stereoisomeric 7(α/ß)-hydroxytestololactones and 7(α/ß)-hydroxytestolactones.

Microbiological synthesis of 7α- and 7ß-hydroxy derivatives of testololactone and testolactone was developed based on bioconversion of dehydroepiandrosterone (DHEA) by fungus of Isaria fumosorosea VKM F-881 with subsequent modification of the obtained stereoisomers by actinobacteria. The first stage included obtaining of the stereoisomers of 3ß,7(α/ß)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones in the preparative amounts. Then the conversion of 7-hydroxylated D-lactones obtained by selected actinobacteria of Nocardioides simplex VKM Ac-2033D, Saccharopolyspora hirsuta VKM Ac-666, and Streptomyces parvulus MTOC Ac-21v was studied. Under the transformation of 3ß,7α-dihydroxy-17a-oxa-D-homo-androst-5-en-17-one and its corresponding 7ß-stereoisomer by N. simplex VKM Ac-2033D and S. hirsuta VKM Ac-666 the 7α- and 7ß-hydroxy-17a-oxa-D-homo-androst-4-ene-3,17-dione (7α- and 7ß-hydroxytestololactone), 7α- and 7ß-hydroxy-17a-oxa-D-homo-androsta-1,4-diene-3,17-dione (7α- and 7ß-hydroxytestolactone) were obtained with molar yields in a range of 60.3-90.9 mol%. The crystalline products of 7α-hydroxytestololactone, 7α-hydroxytestolactone, and their corresponding 7ß-hydroxy stereoisomers were isolated, and their structures were confirmed by mass spectrometry and 1H-NMR spectroscopy analyses. The strain of Str. parvulus MTOC Ac-21v transformed 3ß,7(α/ß)-dihydroxy-17a-oxa-D-homo-androst-5-en-17-ones into the corresponding 3-keto-4-ene analogs and did not show 3-ketosteroid 1(2)-dehydrogenase activity. The activity of actinobacteria towards steroid D-lactones was hitherto unreported.The results contribute to the knowledge of metabolic versatility of actinobacteria capable of transforming steroid substrates and may be applied in the synthesis of potential aromatase inhibitors.

MolTarPred: A web tool for comprehensive target prediction with reliability estimation.

Molecular target prediction can provide a starting point to understand the efficacy and side effects of phenotypic screening hits. Unfortunately, the vast majority of in silico target prediction methods are not available as web tools. Furthermore, these are limited in the number of targets that can be predicted, do not estimate which target predictions are more reliable and/or lack comprehensive retrospective validations. We present MolTarPred ( http://moltarpred.marseille.inserm.fr/), a user-friendly web tool for predicting protein targets of small organic compounds. It is powered by a large knowledge base comprising 607,659 compounds and 4,553 macromolecular targets collected from the ChEMBL database. In about 1 min, the predicted targets for the supplied molecule will be listed in a table. The chemical structures of the query molecule and the most similar compounds annotated with the predicted target will also be shown to permit visual inspection and comparison. Practical examples of the use of MolTarPred are showcased. MolTarPred is a new resource for scientists that require a more complete knowledge of the polypharmacology of a molecule. The introduction of a reliability score constitutes an attractive functionality of MolTarPred, as it permits focusing experimental confirmatory tests on the most reliable predictions, which leads to higher prospective hit rates.

Phytosterols as precursors for the synthesis of aromatase inhibitors: Hemisynthesis of testololactone and testolactone.

Using ß-sitosterol and stigmasterol as precursor materials, a concise and efficient hemisynthesis of aromatase inhibitors: testololactone and testolactone was accomplished in a well-established reaction scheme. It involves highly effective Oppaneur oxidation of both ß-sitosterol as well as stigmasterol to generate the required enone moiety in ring 'A' of the desired steroid system. The Oppaneur oxidation products of both ß-sitosterol and stigmasterol were then subjected to oxidative cleavage of the side chain to produce 4-androstene-3,17-dione. Baeyer-Villiger oxidation of 4-androstene-3,17-dione using m-CPBA yielded testololactone. Dehydrogenation of 4-androstene-3,17-dione using phenylselenyl chloride in ethyl acetate followed by selenoxide elimination with H2O2 in dichloromethane furnished androstenedienone. Baeyer-Villiger oxidation of the resulting androstenedienone yielded the desired testolactone (overall yield 33%). This expeditious reaction scheme may be exploited for the bulk production of aromatase inhibitors (especially testolactone marketed under the brand name Teslac) from the most abundant and naturally occurring phytosterols like ß-sitosterol.

Baeyer-Villiger oxidation of DHEA, pregnenolone, and androstenedione by Penicillium lilacinum AM111.

The Baeyer-Villiger monooxygenase (BVMO) produced by Penicillium lilacinum AM111, in contrast to other enzymes of this group known in the literature, is able to process 3beta-hydroxy-5-ene steroid substrates. Transformation of DHEA and pregnenolone yielded, as a sole or main product, 3beta-hydroxy-17a-oxa-d-homo-androst-5-en-17-one, a new metabolite of these substrates; pregnenolone was transformed also to testololactone. Testololactone was the only product of oxidation of androstenedione by P. lilacinum AM111. Investigations of the time evolution of reaction progress have indicated that the substrates stimulate activity of BVMO(s) of P. lilacinum AM111.

Distinct metabolic handling of 3beta-hydroxy-17a-oxa-D-homo-5alpha-androstan-17-one by the filamentous fungus Aspergillus tamarii KITA: Evidence in support of steroid/hydroxylase binding hypothesis.

Aspergillus tamarii KITA transforms progesterone in to testololactone in high yield through a sequential four-step enzymatic pathway which also has the flexibility to transform a range of steroidal substrates. This study has investigated the further metabolism of testololactone and a range of fully saturated steroidal lactone analogues. In contrast to testololactone, which even after 120 h incubation did not undergo further metabolism, the lactone analogues entered the minor hydroxylation pathway. Uniquely, after forming 3beta-hydroxy-17a-oxa-D-homo-5alpha-androstan-17-one (48 h) 4 distinct positions on the steroid skeleton were monohydroxylated (11beta, 6beta, 7beta, 11alpha) which geometrically relate to the four binding positions (normal, reverse, inverted normal and inverted reverse) possible within the steroidal hydroxylase(s). This is the first evidence demonstrating the four possible steroid/hydroxylase(s) binding interactions with a single molecule that has previously been hypothesized with a single organism. In addition a rare 1beta-monohydroxylation was observed, this may be indicative of dehydration generating 1-ene functionality in A. tamarii rather than dehydrogenation as reported in man and microorganisms. The importance of these findings in relation to steroid/hydroxylase binding interactions is discussed.

Fate of novel Quasi reverse steroidal substrates by Aspergillus tamarii KITA: bypass of lactonisation and an exclusive role for the minor hydroxylation pathway.

The fungus Aspergillus tamarii transforms progesterone to testololactone in high yield through a flexible four-step enzymatic pathway. To date no studies have investigated the effect of transposition of steroidal functionality between ring-A and ring-D in order to determine the effect on steroidal metabolism. A series of novel quasi reverse steroids (7-9) were synthesised through Linz and Schafer oxidation where 14-en-16-one functionality is generated on ring-D of the steroid. To retain parity with the normal series ring-D functionality was substituted onto ring-A of the analogues. All of the analogues (7-9) were handled through a minor 11beta-hydroxylation pathway with no lactones being formed. In previous studies testololactone is produced within the first 12 h of metabolism. A time course experiment demonstrated that the transformation of these steroids initiated with the formation of a 3beta-hydroxy group after which (48-96 h) hydroxylation through a minor pathway occurred, indicating that this hydroxylase was only then being induced. This is in contrast to the normal fungal metabolism of xenobiotic steroidal substrates where they are primarily hydroxylated. Furthermore, ring-D hydrogenation is reported for the first time as is reverse metabolism on this pathway. All metabolites were isolated by column chromatography and were identified by 1H and 13C NMR spectroscopy, DEPT analysis and other spectroscopic and crystallographic data.

Effect of aromatase inhibitor, TZA-2209, on the prostate of androstenedione-treated castrated dogs: changes in prostate volume and histopathological findings.

To determine whether the inhibition of estrogen-related effect in the prostate would be of value in the management of benign prostate hyperplasia (BPH), we examined the effect of TZA-2209, a new steroidal aromatase inhibitor, on the prostate in three of six castrated beagles that received 75 mg/week androstenedione. The three other animals served as controls. Sequential measurements of prostate volume by transrectal ultrasonography showed that the volume in TZA-treated dogs was significantly decreased compared with that in the controls. Prostatic aromatase activity was suppressed by TZA administration. Histopathologically, the stromal component was increased and glands were atrophied by androstenedione treatment. TZA administration increased the volume of the glands. Immunohistochemical detection of estramustine-binding protein showed more positive staining of the protein in the glands that were increased in volume by TZA administration. We concluded that the aromatase inhibitor effectively antagonized the estrogen-related stromal changes, however, this action was accompanied by stimulation of the glandular component due to the accumulation of androgens, the substrate of the aromatase. In the light of these findings, we suggest the simultaneous treatment for the androgen-glandular component route in the prostate is necessary for the effective management of BPH.

Omega 1-decoupled 1H homonuclear shift-correlated nuclear magnetic resonance spectroscopy (COSYDEC) applied to steroids.

Problems of cross-peak overlap in two-dimensional 1H homonuclear shift-correlated (COSY) spectra of steroids can often be avoided by use of the omega 1-decoupled COSY (COSYDEC) method. The selection of experimental parameters is discussed, and COSYDEC spectra are illustrated for 17a-oxa-D-homoandrost-4-ene-3,17-dione (testololactone), testosterone, and 17 alpha-hydroxyprogesterone. In a good case, a COSYDEC spectrum obtained at 250 MHz allows cross-peak recognition and assignment with facility comparable to that available only at 500 MHz for normal COSY spectra.