Novel isoxazoline-linked 1,3,4-thiadiazole hybrids as anticancer agents: Design, synthesis, biological evaluation, molecular docking, and molecular dynamics simulation.

In the current study, natural (R)-carvone was utilized as a starting material for the efficient synthesis of two series of isoxazoline derivatives bearing the 1,3,4-thiadiazole moiety. The new compounds were obtained in good yields and were characterized by 1 H and 13 C NMR and HRMS analysis. The newly synthesized monoterpenic isoxazoline 1,3,4-thiadiazole and their thiosemicarbazone intermediate derivatives were evaluated for their anticancer activity in four cancer cell lines (HT-1080, A-549, MCF-7, and MDA-MB-231). Most of the synthesized compounds exhibited moderate to high anticancer effects. Compound 13c showed the highest anticancer activity with IC50 values ranging from 19.33 ± 1.81 to 34.81 ± 3.03 µM. Further investigation revealed that compounds 12e and 13c could inhibit the cell growth of HT-1080 and MCF-7 cells by inducing apoptosis through caspase-3/7 activation. The apoptotic effect was accompanied by an S phase and G2/M cell cycle arrest for 13c and 12e, respectively. Compounds 12e and 13c were assessed in silico using molecular docking and molecular dynamics. We found that compound 13c is moderately active against the caspase-3 protein, which triggers apoptosis via intrinsic and extrinsic routes, making compound 13c a promising candidate to activate the proapoptotic protein (caspase-3).

Synthesis of some novel coumarin isoxazol sulfonamide hybrid compounds, 3D-QSAR studies, and antibacterial evaluation.

With the progressive and ever-increasing antibacterial resistance pathway, the need for novel antibiotic design becomes critical. Sulfonamides are one of the more effective antibiotics against bacteria. In this work, several novel sulfonamide hybrids including coumarin and isoxazole group were synthesized in five steps starting from coumarin-3-carboxylic acid and 3-amino-5-methyl isoxazole and assayed for antibacterial activity. The samples were obtained in good to high yield and characterized by FT-IR, 13C-NMR, 1H-NMR, CHN and melting point techniques. 3D-QSAR is a fast, easy, cost-effective, and high throughput screening method to predict the effect of the compound's efficacy, which notably decreases the needed price for experimental drug assay. The 3D-QSAR model displayed acceptable predictive and descriptive capability to find r2 and q2 the pMIC of the designed compound. Key descriptors, which robustly depend on antibacterial activity, perhaps were explained by this method. According to this model, among the synthesized sulfonamide hybrids, 9b and 9f had the highest effect on the gram-negative and gram-positive bacteria based on the pMIC. The 3D-QSAR results were confirmed in the experimental assays, demonstrating that our model is useful for developing new antibacterial agents. The work proposes a computationally-driven strategy for designing and discovering new sulfonamide scaffold for bacterial inhibition.

Simple heteroaryl modifications in the 4,5-diarylisoxazol-3-carboxylic acid scaffold favorably modulates the activity as dual mPGES-1/5-LO inhibitors with in vivo efficacy.

Microsomal prostaglandin E2 synthase-1 (mPGES-1), 5-lipoxygenase (5-LO) and 5- lipoxygenase-activating protein (FLAP) are key for biosynthesis of proinflammatory lipid mediators and pharmacologically relevant drug targets. In the present study, we made an attempt to explore the role of small heteroaromatic fragments on the 4,5-diarylisoxazol-3-carboxylic acid scaffold, which are selected to interact with focused regions in the active sites of mPGES-1, 5-LO and FLAP. We report that the simple structural variations on the benzyloxyaryl side-arm of the scaffold significantly influence the selectivity against mPGES-1, 5-LO and FLAP, enabling to produce multi-target inhibitors of these protein targets, exemplified by compound 18 (IC50 mPGES-1 = 0.16 µM; IC50 5-LO = 0.39 µM) with in vivo efficacy in animal model of inflammation. The computationally modeled binding structures of these new inhibitors for three targets provide clues for rational design of modified structures as multi-target inhibitors. In conclusion, the simple synthetic procedure, and the possibility of enhancing the potency of this class of inhibitors through structural modifications pave the way for further development of new multi-target inhibitors against mPGES-1, 5-LO and FLAP, with potential application as anti-inflammatory agents.

Synthesis, Characterization, and Biological Properties of Steroidal Ruthenium(II) and Iridium(III) Complexes Based on the Androst-16-en-3-ol Framework.

A range of novel cyclometalated ruthenium(II) and iridium(III) complexes with a steroidal backbone based on androsterone were synthesized and characterized by NMR spectroscopy and X-ray crystallography. Their cytotoxic properties in RT112 and RT112 cP (cisplatin-resistant) cell lines as well as in MCF7 and somatic fibroblasts were compared with those of the corresponding nonsteroidal complexes and the noncyclometalated pyridyl complexes as well as with cisplatin as reference. All steroidal complexes were more active in RT112 cP cells than cisplatin, whereby the cyclometalated pyridinylphenyl complexes based on 5c showed high cytotoxicity while maintaining low resistant factors of 0.33 and 0.50.

Discovery of 2-isoxazol-3-yl-acetamide analogues as heat shock protein 90 (HSP90) inhibitors with significant anti-HIV activity.

The recent burst of explorations on heat shock protein 90 (HSP90) in virus research supports its emergence as a promising target to overcome the drawbacks of current antiviral therapeutic regimen. In continuation of our efforts towards the discovery of novel anti-retroviral molecules, we designed, synthesized fifteen novels 2-isoxazol-3-yl-acetamide based compounds (2a-o) followed by analysis of their anti-HIV activity and cytotoxicity studies. 2a-b, 2e, 2j, and 2l-m were found to be active with inhibitory potentials >80% at their highest non-cytotoxic concentration (HNC). Further characterization of anti-HIV activity of these molecules suggests that 2l has ∼3.5 fold better therapeutic index than AUY922, the second generation HSP90 inhibitor. The anti-HIV activity of 2l is a cell type, virus isolate and viral load independent phenomena. Interestingly, 2l does not significantly modulate viral enzymes like Reverse Transcriptase (RT), Integrase (IN) and Protease (PR) as compared to their known inhibitors in a cell free in vitro assay system at its HNC. Further, 2l mediated inhibition of HSP90 attenuates HIV-1 LTR driven gene expression. Taken together, structural rationale, modeling studies and characterization of biological activities suggest that this novel scaffold can attenuate HIV-1 replication significantly within the host and thus opens a new horizon to develop novel anti-HIV therapeutic candidates.

The synthesis of 16-androstene sulfoconjugates from primary porcine Leydig cell culture.

Increased public interest in the welfare of pigs reared for pork production has led to an enhanced effort in finding alternatives to castration for controlling the unpleasant odour and flavour from heated pork products known as boar taint. The purpose of this study was to investigate the testicular metabolism of androstenone, one of the major components of boar taint. Leydig cells were isolated from mature boars and incubated with radiolabeled androstenone for 10 min, 1 h, 4 h, 8 h, and 12 h. Steroid profiles were analyzed by high-performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS/MS). Sulfoconjugated, but not glucuronidated steroids were produced by Leydig cells. Approximately 85% of androstenone was converted into sulfoconjugated metabolites in Leydig cell incubations after 8 h. This sulfoconjugate fraction included androstenol-3-sulfate and two major sulfated forms of androstenone. Following removal of the sulfate group, these two sulfated forms of androstenone returned the parent compound androstenone, and not a hydroxylated metabolite. These findings provided direct evidence for the testicular production of sulfoconjugated forms of androstenone and androstenol in the boar. The high proportion of sulfoconjugates produced by the Leydig cells emphasizes the importance of steroid conjugation, which serves to regulate the amount of unconjugated steroid hormones available for accumulation in adipose tissue.

Pregn-5-en-3ß-ol and androst-5-en-3ß-ol dicarboxylic acid esters as potential therapeutics for NMDA hypofunction: In vitro safety assessment and plasma stability.

Neurosteroids are endogenous steroidal compounds that can modulate neuronal receptors. N-Methyl-D-aspartate receptors (NMDARs) are glutamate-gated, calcium-permeable ion channels that are of particular interest, as they participate in synaptic transmission and are implicated in various processes, such as learning, memory, or long-term neuronal potentiation. Positive allosteric modulators that increase the activity of NMDARs may provide a therapeutic aid for patients suffering from neuropsychiatric disorders where NMDAR hypofunction is thought to be involved, such as intellectual disability, autism spectrum disorder, or schizophrenia. We recently described a new class of pregn-5-ene and androst-5-ene 3ß-dicarboxylic acid hemiesters (2-24) as potent positive modulators of NMDARs. Considering the recommended guidelines for the early stage development of new, potent compounds, we conducted an in vitro safety assessment and plasma stability screening to evaluate their druglikeness. First, compounds were screened for their hepatotoxicity and mitochondrial toxicity in a HepG2 cell line. Second, toxicity in primary rat postnatal neurons was estimated. Next, the ability of compounds 2-24 to cross a Caco-2 monolayer was also studied. Finally, rat and human plasma stability screening revealed an unforeseen high stability of the C-3 hemiester moiety. In summary, by using potency/efficacy towards NMDARs data along with toxicity profile, Caco-2 permeability and plasma stability, compounds 14 and 15 were selected for further in vivo animal studies.

Synthesis of novel 17-triazolyl-androst-5-en-3-ol epimers via Cu(I)-catalyzed azide-alkyne cycloaddition and their inhibitory effect on 17α-hydroxylase/C17,20-lyase.

The regioselective Cu(I)-catalyzed 1,3-dipolar cycloaddition of 17α- and 17ß-azidoandrost-5-en-3ß-ol epimers (3b and 5b) with different terminal alkynes afforded novel 1,4-substituted triazolyl derivatives (8a-k and 9a-k). For the preparation of 5'-iodo-1',2',3'-triazoles (8m-n and 9m-n), an improved method was developed, directly from steroidal azides and terminal alkynes, in reaction mediated by CuI and ICl as iodinating agents. Acetolysis and subsequent hydrolysis of 8n and 9n yielded 5'-hydroxy-1',2',3'-triazoles 8o and 9o. The inhibitory effect of 8a-o, 9a-o, 3, and 5 on rat testicular C17,20-lyase was investigated by means of an in vitro radioincubation technique. The results revealed that the C-17 epimers of steroidal triazoles influence the C17,20-lyase effect. Inhibitors were found only in the 17α-triazolyl series (8a-o), whereas in the C-17 azide pair the 17ß compound (5b) was more potent.

Metabolic study of methylstenbolone in horses using liquid chromatography-high resolution mass spectrometry and gas chromatography-mass spectrometry.

Methylstenbolone (2,17α-dimethyl-5α-androst-1-en-17ß-ol-3-one) is a synthetic anabolic and androgenic steroid (AAS) sold as an oral 'nutritional supplement' under the brand names 'Ultradrol', 'M-Sten' and 'Methyl-Sten'. Like other AASs, methylstenbolone is a prohibited substance in both human and equine sports. This paper describes the studies of the in vitro and in vivo metabolism of methylstenbolone in horses using LC/HRMS, GC/MS and GC/MS/MS. Phase I in vitro metabolic study of methylstenbolone was performed using homogenised horse liver. Hydroxylation was the only biotransformation observed. Six in vitro metabolites were detected including four mono-hydroxylated metabolites, namely 16α/ß-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), 6-hydroxymethylstenbolone (M1d), and two dihydroxylated methylstenbolone metabolites (M2c-M2d). An in vivo experiment was carried out using two retired thoroughbred geldings. Each horse was administered with 100 mg methylstenbolone supplement by stomach tubing daily for three consecutive days. Methylstenbolone and 14 metabolites were detected in the post-administration urine samples. The proposed in vivo metabolites included 16α/ß-hydroxymethylstenbolone (M1a, M1b), 20-hydroxymethylstenbolone (M1c), two dihydroxylated methylstenbolone (M2a, M2b), 17-epi-methylstenbolone (M3), methasterone (M4), 2,17-dimethylandrostane-16,17-diol-3-one (M5), dihydroxylated and reduced methylstenbolone (M6), 2α,17α-dimethylandrostane-3α,17ß-diol (M7), 2,17-dimethylandrostane-3,16,17-triol (M8a-M8c) and 2,17-dimethylandrostane-2,3,16,17-tetraol (M9), formed from hydroxylation, reduction and epimerisation. Methylstenbolone and ten of its metabolites could be detected in post-administration plasma samples. The highest concentration of methylstenbolone detected in urine was about 10-36 ng/mL at 3-4 h after the last administration, while the maximum concentration in plasma was about 0.4-0.7 ng/mL at 1 h after the last administration. For controlling the misuse of methylstenbolone, M8c and M9 gave the longest detection time in urine, while M4, M5 and M6 were the longest detecting analytes in plasma. They could be detected for up to 5 and 4.5 days respectively in urine and plasma. Apart from 16α/ß-hydroxymethylstenbolone (M1a, M1b), the methylstenbolone metabolites reported herein have never been reported before.

Design, synthesis, biological evaluation and docking studies of new 3-(4,5-dihydro-1H-pyrazol/isoxazol-5-yl)-2-phenyl-1H-indole derivatives as potent antioxidants and 15-lipoxygenase inhibitors.

New candidates of 3-(4,5-dihydro-1H-pyrazol/isoxazol-5-yl)-2-phenyl-1H-indole derivatives (4-7) were designed combining the pyrazoline/isoxazoline heterocycles and 2-phenylindole to explore its potential as 15-lipoxygenase (15-LOX) inhibitors. The design of the new derivatives was based on utilizing the antioxidant properties of pyrazoline, 2-phenylindole and the good 15-LOX inhibition properties of indolylpyrazoline. The derivatives were synthesized adopting simple and laboratory friendly reaction conditions to give the target compounds in quantitative yields. The resulting indolylpyrazolines/isoxazolines were evaluated as antioxidants against 2,2-diphenyl-1-picrylhydrazyl (DPPH), nitric oxide (NO) and superoxide dismutase (SOD); indolylpyrazoline (4b) was the most potent antioxidant against SOD assay (IC50 = 1.78 µM) to be superior to ascorbic by 2 folds. Consistently, (4b) was the most potent inhibitor when tested against Soybean 15-LOX (IC50 = 3.84 µM) excelling quercetin as standard inhibitor by 1.8 folds. Some of the new derivatives were docked into the active binding site of human 15-LOX (PDB entry 4NRE) emphasizing the most potent derivative (4b) and the least potent one (4c). Docking solutions of compounds (4b), (4c), (5b) and (6c) revealed that (4b) was the only compound that got stabilized into the catalytic pocket of enzyme by π-cation interaction with the catalytic Fe+ and formation of one hydrogen bond with Ile 676 amino acid. Other derivatives including the least potent one variably got stabilized into the active binding pocket by π-cation interaction with the catalytic Fe+ but failed to form hydrogen bond with Ile 676. For the future optimization of the generated inhibitors, (i) antioxidant activity against SOD, (ii) the inhibitor stabilization by π-cation interaction with the catalytic Fe+3 and (iii) formation of hydrogen bond with Ile 676 should be regarded.

Biotransformation of DHEA into 7α,15α-diOH-DHEA.

3ß,7α,15α-Trihydroxy-5-androsten-17-one (7α,15α-diOH-DHEA) is a key intermediate of the novel oral contraceptive "Yasmin" (widely used in birth control pills and postmenopausal hormone replacement therapy pills; the active ingredient is drospirenone). It can be synthesized from dehydroepiandrosterone (DHEA) by microbial dihydroxylation at the C7 and C15 positions. Here we describe the method of bioconversion from DHEA into 7α,15α-diOH-DHEA by Colletotrichum lini. Using 6 g/L DHEA as a substrate, the DHEA conversion and the 7α,15α-diOH-DHEA molar yield were 72.6% and 51.2%, respectively.

Pd-mediated cross-coupling of C-17 lithiated androst-16-en-3-ol - access to functionalized arylated steroid derivatives.

Herein, we report on Pd-mediated cross-coupling of vinyllithium steroids and aryl bromides to introduce various substituted aryls at C-17 of steroidal frameworks based on the structure of epi-androsterone. Compared to other C-C cross-couplings, this method turned out to be an easy and competitive access to biologically interesting C-17 modified steroids.

Structures of human steroidogenic cytochrome P450 17A1 with substrates.

The human cytochrome P450 17A1 (CYP17A1) enzyme operates at a key juncture of human steroidogenesis, controlling the levels of mineralocorticoids influencing blood pressure, glucocorticoids involved in immune and stress responses, and androgens and estrogens involved in development and homeostasis of reproductive tissues. Understanding CYP17A1 multifunctional biochemistry is thus integral to treating prostate and breast cancer, subfertility, blood pressure, and other diseases. CYP17A1 structures with all four physiologically relevant steroid substrates suggest answers to four fundamental aspects of CYP17A1 function. First, all substrates bind in a similar overall orientation, rising ∼60° with respect to the heme. Second, both hydroxylase substrates pregnenolone and progesterone hydrogen bond to Asn(202) in orientations consistent with production of 17α-hydroxy major metabolites, but functional and structural evidence for an A105L mutation suggests that a minor conformation may yield the minor 16α-hydroxyprogesterone metabolite. Third, substrate specificity of the subsequent 17,20-lyase reaction may be explained by variation in substrate height above the heme. Although 17α-hydroxyprogesterone is only observed farther from the catalytic iron, 17α-hydroxypregnenolone is also observed closer to the heme. In conjunction with spectroscopic evidence, this suggests that only 17α-hydroxypregnenolone approaches and interacts with the proximal oxygen of the catalytic iron-peroxy intermediate, yielding efficient production of dehydroepiandrosterone as the key intermediate in human testosterone and estrogen synthesis. Fourth, differential positioning of 17α-hydroxypregnenolone offers a mechanism whereby allosteric binding of cytochrome b5 might selectively enhance the lyase reaction. In aggregate, these structures provide a structural basis for understanding multiple key reactions at the heart of human steroidogenesis.

Membrane properties of cholesterol analogs with an unbranched aliphatic side chain.

The interactions between cholesterol and other membrane molecules determine important membrane properties. It was shown that even small changes in the molecular structure of cholesterol have a crucial influence on these interactions. We recently reported that in addition to alterations in the tetracyclic ring structure, the iso-branched side chain of cholesterol also has a significant impact on membrane properties (Scheidt et al., 2013). Here we used synthetic cholesterol analogs to investigate the influence of an unbranched aliphatic side chain of different length. The (2)H NMR order parameter of the phospholipid chains and therefore the molecular packing of the phospholipid molecules shows a significant dependence on the sterol's alkyl side chain length, while, membrane permeation studied by a dithionite ion permeation assay and lateral diffusion measured by (1)H MAS pulsed field gradient NMR are less influenced. To achieve the same molecular packing effect similar to that of an iso-branched aliphatic side chain, a longer unbranched side chain (n-dodecyl instead of n-octyl) at C17 of cholesterol is required. Obviously, sterols having a branched iso-alkyl chain with two terminal methyl groups exhibit altered cholesterol-phospholipid interactions compared to analogous molecules with a straight unbranched chain.

Small-molecule RORγt antagonists inhibit T helper 17 cell transcriptional network by divergent mechanisms.

We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.

Synthesis and in-vivo hypolipidemic activity of some novel substituted phenyl isoxazol phenoxy acetic acid derivatives.

The present study was undertaken to evaluate in-vivo hypolipidemic activity of a novel series of 2-methyl-2-(substituted phenyl isoxazol)phenoxyacetic acid derivatives by triton induced hyperlipidemia in rats. The newly synthesized compounds 5a, 5d and 5g showed significant decrease in the serum TCH, TG, LDL and VLDL along with an increase in serum HDL levels as compared to standard drug Fenofibrate. The treated groups also showed significant decrease in the atherogenic index and increase in % protective activity compared to control group.

Metabolism of methylstenbolone studied with human liver microsomes and the uPA⁺/⁺-SCID chimeric mouse model.

Anti-doping laboratories need to be aware of evolutions on the steroid market and elucidate steroid metabolism to identify markers of misuse. Owing to ethical considerations, in vivo and in vitro models are preferred to human excretion for nonpharmaceutical grade substances. In this study the chimeric mouse model and human liver microsomes (HLM) were used to elucidate the phase I metabolism of a new steroid product containing, according to the label, methylstenbolone. Analysis revealed the presence of both methylstenbolone and methasterone, a structurally closely related steroid. Via HPLC fraction collection, methylstenbolone was isolated and studied with both models. Using HLM, 10 mono-hydroxylated derivatives (U1-U10) and a still unidentified derivative of methylstenbolone (U13) were detected. In chimeric mouse urine only di-hydroxylated metabolites (U11-U12) were identified. Although closely related, neither methasterone nor its metabolites were detected after administration of isolated methylstenbolone. Administration of the steroid product resulted mainly in the detection of methasterone metabolites, which were similar to those already described in the literature. Methylstenbolone metabolites previously described were not detected. A GC-MS/MS multiple reaction monitoring method was developed to detect methylstenbolone misuse. In one out of three samples, previously tested positive for methasterone, methylstenbolone and U13 were additionally detected, indicating the applicability of the method.

Insect γ-aminobutyric acid receptors and isoxazoline insecticides: toxicological profiles relative to the binding sites of [³H]fluralaner, [³H]-4'-ethynyl-4-n-propylbicycloorthobenzoate, and [³H]avermectin.

Isoxazoline insecticides, such as fluralaner (formerly A1443), are noncompetitive γ-aminobutyric acid (GABA) receptor (GABA-R) antagonists with selective toxicity for insects versus mammals. The isoxazoline target in house fly ( Musca domestica ) brain has subnanomolar affinity for [³H]fluralaner and a unique pattern of sensitivity to isoxazolines and avermectin B(1a) (AVE) but not to fipronil and α-endosulfan. Inhibitor specificity profiles for 15 isoxazolines examined with Musca GABA-R and [³H]fluralaner, [³H]-4'-ethynyl-4-n-propylbicycloorthobenzoate ([³H]EBOB), and [³H]AVE binding follow the same structure-activity trends although without high correlation. The 3 most potent of the 15 isoxazolines tested in Musca [³H]fluralaner, [³H]EBOB, and [³H]AVE binding assays and in honeybee (Apis mellifera) brain [³H]fluralaner assays are generally those most toxic to Musca and four agricultural pests. Fluralaner does not inhibit [³H]EBOB binding to the human GABA-R recombinant ß3 homopentamer, which is highly sensitive to all of the commercial GABAergic insecticides. The unique isoxazoline binding site may resurrect the GABA-R as a major insecticide target.

Protein-binding properties of a designed steroidal lactam compound.

Introducing amide bonds into a steroid nucleus or its side chain may reduce the acute toxicity and enhance the pharmaceutical activity. In this work, a designed steroidal amide compound, named 3ß-hydroxy-17-aza-d-homo-5-androsten-17-one (HAAO), was synthesized and identified. The interactions between HAAO and human serum albumin (HSA) were studied by multiple spectroscopic methods and molecular modeling procedures. It was found that HAAO locates in Sudlow's site I in subdomain IIA of HSA molecules, relying on hydrogen bonds and van der Waals power to form HAAO-HSA complexes at ground state. The number of binding sites, binding constants, enthalpy change (ΔH(θ)), Gibbs free energy change (ΔG(θ)) and entropy change (ΔS(θ)) were calculated at different temperatures based on fluorescence quenching theory and classical thermodynamic equation. The percentages content of the HSA's secondary structures in presence of HAAO were detected by circular dichroism (CD) spectra and compared with those in no presence of HAAO. In addition, the experimental results of both binding site and conformational change were further confirmed by molecular modeling investigation, in which more details of the binding were visually unfolded. The information provided by the study may be useful for designing novel chemotherapeutic drugs and be helpful both in the early stages of drug discovery and in clinical practice.

5α-Androst-16-en-3α-ol ß-D-glucuronide, precursor of 5α-androst-16-en-3α-ol in human sweat.

5α-Androst-16-en-3α-ol (α-androstenol) is an important contributor to human axilla sweat odor. It is assumed that α-andostenol is excreted from the apocrine glands via a H2 O-soluble conjugate, and this precursor was formally characterized in this study for the first time in human sweat. The possible H2 O-soluble precursors, sulfate and glucuronide derivatives, were synthesized as analytical standards, i.e., α-androstenol, ß-androstenol sulfates, 5α-androsta-5,16-dien-3ß-ol (ß-androstadienol) sulfate, α-androstenol ß-glucuronide, α-androstenol α-glucuronide, ß-androstadienol ß-glucuronide, and α-androstenol ß-glucuronide furanose. The occurrence of α-androstenol ß-glucuronide was established by ultra performance liquid chromatography (UPLC)/MS (heated electrospray ionization (HESI)) in negative-ion mode in pooled human sweat, containing eccrine and apocrine secretions and collected from 25 female and 24 male underarms. Its concentration was of 79 ng/ml in female secretions and 241 ng/ml in male secretions. The release of α-androstenol was observed after incubation of the sterile human sweat or α-androstenol ß-glucuronide with a commercial glucuronidase enzyme, the urine-isolated bacteria Streptococcus agalactiae, and the skin bacteria Staphylococcus warneri DSM 20316, Staphylococcus haemolyticus DSM 20263, and Propionibacterium acnes ATCC 6919, reported to have ß-glucuronidase activities. We demonstrated that if α- and ß-androstenols and androstadienol sulfates were present in human sweat, their concentrations would be too low to be considered as potential precursors of malodors; therefore, the H2 O-soluble precursor of α-androstenol in apocrine secretion should be a ß-glucuronide.