Synthesis and pharmacological evaluation of new 16-methyl pregnane derivatives

The pharmacological activity of several new pregnane derivatives 15-19 were determined on gonadectomized male hamster flank organs, seminal vesicles and in vitro conversion of testosterone (T) to dihydrotestosterone (DHT) as 5alpha-reductase inhibitors. Steroids 15-19 decreased the diameter of the pigmented spot in the flank organs as compared to the T treated animals; in this model, steroids 16 and 19 showed a higher activity than the commercially available finasteride 3. Injection of T increased the weight of the seminal vesicles. Compounds 15-19 when injected together with T decreased the weight of the seminal vesicles thus showing an antiandrogenic effect. The trienone 19 exhibited a considerably higher activity than finasteride. Steroids 15-19 inhibited the in vitro metabolism of [3H]T to [3H]DHT in seminal vesicles homogenates of gonadectomized male hamsters. Compounds 18 and 19 showed a much higher antiandrogenic effect than finasteride. This enhancement of the biological activity could probably be attributed to the coplanarity of the steroidal skeleton as previously observed by our group. The high antiandrogenic activity of the epoxy compound 16 is probably the result of the ring opening of the oxiran ring with the nucleophilic part of the enzyme 5alpha-reductase thus leading to a stable adduct with concomitant deactivation of this enzyme.     

Antiandrogenic effect of 16-substituted, non-substituted and D-homopregnane derivatives

The pharmacological activities of 12 pregnane derivatives (4-15) were determined on gonadectomized male hamster flank organs and seminal vesicles as antiandrogens and as 5alpha-reductase inhibitors. The results from this study indicate that subcutaneous injection of testosterone for 3 d increased the diameter of the pigmented spot in the flank organs, whereas finasteride when injected with testosterone decreased the size of the spot significantly when steroids 4-15 were injected together with testosterone, the diameter of the flank organs of gonadectomized male hamsters, decreased significantly (p<0.005) compared to testosterone. Compound 11 was the most active steroid and reduced the diameter of the pigmented spot more than the other synthesized steroids or finasteride. Subcutaneous injections of testosterone to gonadectomized animals restore the seminal vesicle size lost upon castration. Injection of testosterone plus finasteride decreased significantly the weight of these glands (p<0.005). Steroids 5-15 when injected with testosterone decreased the weight of the seminal vesicles compared to testosterone. Finasteride is a good inhibitor of the conversion of testosterone to dihydrotestosterone (DHT) (low formation of DHT) measured as pmole of DHT/g of protein/h. Steroids 6-15 inhibited the conversion of testosterone to DHT as compared to testosterone however finasteride and 10 appeared to be the most effective compounds. Castration increases the protein content of the seminal vesicles (control) expressed as microg/mg of tissues. Testosterone tends to decrease it significantly, as did compounds 4, 5, 7, 9, and 15. We demonstrated that DHT as well as cyproterone acetate and steroids 5, 6, 8, 9, 11, and 14 at increasing non radioactive steroid concentration, inhibited the binding of [3H]DHT to cytosolic androgen receptor (AR), as indicated by its Ki values. However, 4, 7, 10, 12, and 13 did not have any inhibitory effect.     

Novel 17 substituted pregnadiene derivatives as 5 alpha-reductase inhibitors and their binding affinity for the androgen receptor

The in vitro antiandrogenic activity of four new progesterone derivatives: 4, 5, 6 and 7 (8 is a known compound) was determined. These compounds were evaluated as 5alpha-reductase inhibitors as well as by their capacity to bind to the androgen receptor in gonadectomized hamster prostate. The IC(50) value was determined using increasing concentrations of 4, 5, 6, 7 and 8 in the presence of [(3)H]T and the microsomal fraction of the hamster prostate containing the 5alpha-reductase enzyme. In this paper we also demonstrated the effect of increasing concentrations of the novel steroids upon [(3)H]DHT binding to the androgen receptors from hamster prostate which produces competition for the androgen receptor sites. The in vitro studies showed that steroids 4, 5, 6, 7 and 8 had an inhibitory activity for the 5alpha-reductase with IC(50) of: 4 (0.17 microM), 5 (0.19 microM), 6 (1 microM), 7 (4.2 microM), and 8 (2.7 microM). On the other hand, the IC(50) value for compounds 4, 5, 6, 7, 8 and DHT showed the following order of affinity for the androgen receptor: 6>7>5>DHT. Surprisingly compounds 4 and 8 did not bind to the androgen receptor. The overall data indicate that all synthesized compounds are inhibitors for the enzyme 5alpha-reductase present in the hamster prostate. In contrast, compounds 5, 6 and 7, which have a cyclohexyl group in the side chain showed a high affinity for the androgen receptor.     

Synthesis and pharmacological evaluation of 4-halo progesterone derivatives as antiandrogen.

The pharmacological activity of eight pregnane derivatives 17-alpha acetoxyprogesterone 9, 17-alpha acetoxy-4, 5-epoxypregnan-3, 20-dione 10, 17-alpha acetoxy-4-chloro-4-pregnene-3, 20-dione 11, 17-alpha acetoxy-4-bromo-4-pregnene-3, 20-dione 12, 17-alpha hydroxy-4-bromo-4-pregnene-3, 20-dione 13, 4-chloro-17-alpha hydroxy-4-pregnene-3, 20-dione 14, 17-alpha benzoyloxy-4-bromo-4-pregnene-3, 20-dione 15 and 17-alpha benzoyloxy-4-chloro-4-pregnene-3, 20-dione 16 was determined. These compounds were evaluated as antiandrogens on gonadectomized hamster seminal vesicles. The pharmacological data in this study indicate that compounds 15 and 16 having a C-17 benzoyloxy moiety showed the highest antiandrogenic activity as measured by the reduction of the weight of the seminal vesicles, followed by the steroids 11 and 12 (17-alpha acetoxy group). The free alcohols 13 and 14 exhibited a lower antiandrogenic activity. Apparently, the ester moiety at C-17 is a necessary requirement for the presence of high antiandrogenic activity. Shows the inhibitory effect on the conversion of testosterone (T) to DHT, of the above described steroids as measured by the amount of produced DHT 2 expressed as pmoles of DHT/g of protein/h. Steroids 11, 12 and 16 showed a much higher inhibitory activity on the conversion of testosterone (T) to dihydrotestosterone (DHT) than presently used finasteride 3.     

New progesterone esters as 5alpha-reductase inhibitors

The pharmacological activity of four new progesterone derivatives: 4-bromo-17alpha-(p-fluorobenzoyloxy)-4-pregnene-3,20-dione (7), 4-bromo-17alpha-(p-bromobenzoyloxy)-4-pregnene-3,20-dione (8), 4-bromo-17alpha-(p-chlorobenzoyloxy)-pregnene-3,20-dione (9) and 4-bromo-17alpha-(p-toluoyloxy)-4-pregnene-3,20-dione (10) was determined. These compounds were evaluated as 5alpha-reductase inhibitors on gonadectomized hamster seminal vesicles and flank organs. The pharmacological data of this study indicate that compounds 7 and 9 having at C-17 p-fluorobenzoyloxy and p-chlorobenzoyloxy ester functions respectively showed the highest antiandrogenic effect as measured by the reduction of the weight of the seminal vesicles. In the flank organ model, the same compounds 7 and 9 exhibited a smaller diameter, 1.8 and 1.0 mm, respectively, than the commercially available finasteride 3 (2.3 mm), thus indicating a higher inhibitory effect on 5alpha-reductase enzyme. Steroid 7 showed a higher inhibitory activity on the conversion of T to DHT (Fig. 3) than the presently used finasteride, thus indicating a higher antiandrogenic effect. The nonsubstituted benzoyloxy ester (compound 15) showed a lower antiandrogenic activity as measured in the seminal vesicles model than the p-substituted benzoyloxy compounds.     

Molecular interactions of new pregnenedione derivatives

The in vitro inhibitory activity of five new progesterone derivatives: 17alpha-hydroxy-16beta-methylpregna-1,4,6-triene-3,20-dione 1; 16beta-methyl-17alpha-toluoyloxypregna-1,4,6-triene-3,20-dione 2; 17alpha-hydroxy-6-methylenepregn-4-ene-3,20-dione 3; 6-methylene-17alpha-toluoyloxypregn-4ene-3,20-dione 4 and 17alpha-(p-bromobenzoyloxy)-6-methylenepregn-4-ene-3,20-dione 5 was determined. These compounds were evaluated as 5alpha-reductase inhibitors as well as antagonists for the androgen receptor. Compounds 1, 2, 3, 4 and 5 showed the following inhibitory activity for the 5alpha-reductase enzyme with IC(50) values of: 1 (1.65 microM), 2 (10 microM), 3 (19 nM), 4 (100 nM) and 5 (100 nM). The results of this study also showed the effect of increasing concentrations of the novel steroids upon [(3)H]dihydrotestosterone binding to androgen receptors from male hamster prostate. The K(i) values for compounds 1, 2, 3, 4, 5 and dihydrotestosterone showed the following order of affinity for the androgen receptor: 4>5>dihydrotestosterone>2>3>1. The overall data indicated that all synthesized compounds 1, 2, 3, 4 and 5 are inhibitors of the 5alpha-reductase enzyme present in the hamster prostate. In addition compounds 1, 2, 3, 4 and 5 also presented an affinity for the androgen receptor.     

Evaluation of new pregnane derivatives as 5alpha-reductase inhibitor

The objective of this study was to synthesize several new pregnane derivatives and evaluate them as antiandrogens. From the commercially available 16-dehydropregnenolone acetate (7), two new steroidal compounds were synthesized: 17alpha-hydroxy-17beta-methyl-16beta-phenyl-D-homoandrosta-1,4,6-triene-3,20-dione (18) and 17alpha-acetoxy-17beta-methyl-16beta-phenyl-D-homoandrosta-1,4,6-triene-3,20-dione (19). The 5alpha-reductase inhibitory effect of the new compounds 18 and 19 together with the previously synthesized intermediates 7, 8, 13, 16, and 17 was determined in three different models: gonadectomized hamster flank organs diameter size, incorporation of [1,2-(14)C]sodium acetate into lipids in flank organs and conversion of [3H]testosterone (T) to [3H]dihydrotestosterone (DHT) by Penicillium crustosum. The evaluation of these steroids was carried out with three different controls: one group was treated with vehicle, the second with T and the third group with T plus finasteride. The pharmacological results from this work demonstrated that T significantly increases the diameter of the pigmented spot on the flank organs (p<0.05) as well as the incorporation of labeled sodium acetate into lipids in gonadectomized hamster flank organs (from 0.125 to 0.255 nmol per gland). In this study we also observed that broth of Penicillium crustosum converted [3H]T to [3H]DHT in a manner comparable to that of the flank organs. All experiments indicated that finasteride as well as steroids 7, 8, 13, 16-19 reduced significantly the conversion of T to DHT in P. crustosum. These compounds also decrease the size of the pigmented spot in the flank organs as well as reducing the incorporation of radiolabeled sodium acetate into lipids; T and the control sample (treated with vehicle only) were used for comparison. Apparently the presence of the 4,6-diene-3,20-dione moiety and also the C-17 ester group produce a higher inhibitory effect on the parameters used. PPThe data from this study indicated also that the three models used for the pharmacological evaluation exhibited comparable results.     

Simultaneous determination of 15 steroids in rat blood via gas chromatography-mass spectrometry to evaluate the impact of emasculation on adrenal

Adrenal was believed to affect the prostate tumor tissue growth by its secretion of adrenal androgens. However, the mechanisms regulating these effects were not fully understood. In this work, a sensitive and specific method for the determination of 15 steroids in blood via gas chromatography-mass spectrometry in selective ion storage detection mode was established to evaluate the impact of emasculation on adrenal steroids metabolism. Steroids were isolated by solid-phase extraction using Oasis HLB cartridge, and then derivated with heptafluorobutyric anhydride before analysis. The limits of detection were between 0.15 and 1.0 ng/mL and limits of quantification were between 0.62 and 2.6 ng/mL. The recoveries of steroids were above 83%, and both the intra-day and inter-day precisions (RSD%) were lower than 8%. Pregnenolone, progesterone, 17α-hydroxyprogesterone (17αP), 17α-hydroxypregnenolone (17αH), dehydroepiandrosterone (DHEA), estrone, 17β-estradiol, dihydrotestosterone (DHT), testosterone (T), 4-androstenedione (4-A), 1,4-androstadiene-3,17-dione, 11-deoxycortisol, 11-deoxycorticosterone, cortisol and aldosterone were quantified in 156 major male SD rats at 0, 1, 2, 4, 7 days, 2, 4, 6, 8, 10, 12, 14, and 16 weeks following emasculation. T and DHT decreased by 86.2% and 73.4%, respectively in the first 7 days following emasculation, but adrenal androgens (DHEA, 4-A) stabled at the normal level accordingly. Adrenal androgens and their precursors (17αH, 17αP) increased from the 2nd week along with the increase of androgens and the decrease of mineralocorticoids. These facts revealed that adrenal possibly enhanced its function of producing adrenal androgens from the 2nd week responding to the low androgens level induced by emasculation.     

Novel 4-Azapregnene Derivatives as Potential Anticancer Agents: Synthesis,Antiproliferative Activity and Molecular Docking Studies

A series of novel 21E-arylidene-4-azapregn-5-ene steroids has been successfully designed, synthesized and structurally characterized, and their antiproliferative activity was evaluated in four different cell lines. Within this group, the 21E-(pyridin-3-yl)methylidene derivative exhibited significant cytotoxic activity in hormone-dependent cells LNCaP (IC₅₀ = 10.20 µM) and T47-D cells (IC₅₀ = 1.33 µM). In PC-3 androgen-independent cells, the steroid 21E-p-nitrophenylidene-4-azapregn-5-ene was the most potent of this series (IC₅₀ = 3.29 µM). Considering these results, the 21E-(pyridin-3-yl)methylidene derivative was chosen for further biological studies on T47-D and LNCaP cells, and it was shown that this azasteroid seems to lead T47-D cells to apoptotic death. Finally, molecular docking studies were performed to explore the affinity of these 4-azapregnene derivatives to several steroid targets, namely 5α-reductase type 2, estrogen receptor α, androgen receptor and CYP17A1. In general, compounds presented higher affinity to 5α-reductase type 2 and estrogen receptor α.     

Synthesis and pharmacological evaluation of new progesterone esters as 5alpha-reductase inhibitors

In this study we report the synthesis and pharmacological evaluation of four new progesterone derivatives; 17alpha-hydroxy-16beta-methylpregna-4,6-diene-3,20-dione 12, 17alpha-cyclopropylcarbonyloxy-16beta-methylpregna-4,6-diene-3,20-dione 13, 17alpha-cyclobutylcarbonyloxy-16beta-methylpregna-4,6-diene-3,20-dione 14, 17alpha-acetoxy-16beta-methylpregna-4,6-diene-3,20-dione 15 and the pregnatriene compound 17alpha-cyclobutylcarbonyloxy-16beta-methylpregna-1,4,6-triene-3,20-dione 16. The pharmacological effect of these compounds was determined in vivo as well as in vitro. The evaluation in vivo was carried out on gonadectomized male hamsters that were injected subcutaneously daily with testosterone (T) and/or finasteride, or with the novel compounds. At the end of the treatments the animals were sacrificed and the prostates were weighed. It was observed that when testosterone (T) and finasteride or compounds 12-16 were injected together, the weight of the prostate decreased significantly as compared to that of the testosterone-treated animals. The 5alpha-reductase inhibitory activity was evaluated in vitro using human prostate homogenates. These experiments showed the following IC50 values: compound 12 (alcohol at C-17) 1.2 x 10(-6) M, 13 (cyclopropyl substituent at C-17) 7.9 x 10(-10) M, 14 (cyclobutyl substituent) 3.2 x 10(-8) M, 15 (acetoxy substituent) 6.3 x 10(-11) M and 16 (cyclobutyl substituent) 3.9 x 10(-6) M. It is evident from these data that when the size of the substituent at C-17 is decreased, the 5alpha-reductase inhibitory activity increases. Apparently, in this biological model, the 5alpha-reductase inhibitory activity depends upon the steric effect of the substituent at C-17. However, the free alcohol 12 showed much lower 5alpha-reductase inhibitory activity.     

Application of an androgen receptor assay for the characterisation of the androgenic or antiandrogenic activity of various phenylurea herbicides and their derivatives.

The potency of different substances for [3H]dihydrotestosterone ([3H]DHT) displacement from the bovine androgen receptor was tested. The phenylurea herbicide linuron and its derivative 3,4-dichloroaniline (3,4-DCA), which are found in sediments and surface waters, are known to displace bound testosterone from the rat androgen receptor. Because 3,4-DCA is rapidly taken up by fish and metabolised into 3,4-dichloroacetanilide (3,4-DCAc), it was investigated whether the displacement effects are attributable to 3,4-DCA or to 3,4-DCAc. The potency of 3,4-DCAc androgen receptor binding was compared with that of several phenylurea compounds. In a radioreceptor assay with calf uterus cytosol as androgen receptor preparation, the specific binding of [3H]DHT, the endogenous ligand, was completely displaceable by increasing concentrations of 3,4-DCAc. The relative binding affinities (RBA) of the various compounds were about 1/10(4) to 1/10(5) of that of DHT. 3,4-DCAc had the relative highest affinity (1.31 x 10(-4)), followed by linuron, 3,4-dichlorophenylurea, flutamide, 3,4-DCA and diuron with the lowest RBA (2.4 x 10(-5)). Hence the metabolism of xenobiotic compounds has to be considered to estimate potential ecotoxiocological effects. This test not only can be used to screen for androgen- and antiandrogen-like substances in environmentally relevant samples such as surface waters, but might also be applied for drug testing and for residue monitoring.     

Novel Cholic-Acid-Type Sterones of Deltocyathus magnificus,a deep-water scleractinian coral from the Loyalty Islands,SW Pacific

Cholic-acid-type 3-keto steroids 1–8 , all isolated from Deltocyathus magnificus ( 2–8 after CH₂N₂ treatment), are the first secondary metabolites obtained from a deep-water scleractinian. Steroids 1–3, 5 , and 7 are new, their main structural oddities being loss of C(24) (see 5 ) or hydroxylation in the side chain (see 3 and 7 ). Steroids 4, 6 , and 8 , from the same coral, were previously known from other marine organisms.     

Synthesis of tricyclic compounds as steroid 5alpha-reductase inhibitors

A series of 4-phenoxybutyric acid derivatives attached to a tricyclic skeleton were prepared and evaluated as 5alpha-reductase inhibitors. Structure activity relationships for these compounds in terms of rat epididymis (type 2) 5alpha-reductase inhibitory activities reveal that 1) the substitution pattern at the 11-position of dibenz[b,e]oxepin influenced potency, 2) higher lipophilicity of the tricyclic skeleton improved potency, whereas the existence of a basic nitrogen atom in this skeleton was detrimental to potency, and 3) isobutyl substitution at the 8 positon of the azepine skeleton was tolerated. Among the tricyclic compounds studied, 4-[3-[5-benzyl-8-(2-methyl)propyl-10,11-dihydrodibenz[b,f]azepine- 2-carboxamido]phenoxy]butyric acid (26) was the most potent inhibitor of rat type 2 5alpha-reductase at 0.1 microM.     

Analysis of testosterone and dihydrotestosterone from biological fluids as the oxime derivatives using high-performance liquid chromatography/tandem mass spectrometry

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the simultaneous quantitative analysis of dihydrotestosterone (DHT) and testosterone (T) from biological fluids has been developed. Commercially available deuterated analogues were used as internal standards. Steroids were extracted from serum or testicular fluid with hexane/ethyl acetate, evaporated to dryness, and treated with hydroxylamine to form their oxime derivatives. Upon chromatographic separation, the compounds were quantified using selected reaction monitoring (SRM). For T, the [M+H](+) ion at m/z 304 and the fragment ion at m/z 124 were used as the precursor and product ions. For DHT the ion cluster [M+H+ACN](+) at m/z 347 and the dissociated ion [M+H](+) at m/z 306 were used as the precursor and product ions, respectively. The limits of detectability on-column were in the sub-femtomole range for both compounds and the intra-day coefficient of variation (CV) for analysis from serum was less than 7% for both compounds. Given its high reproducibility, sensitivity, and relative simplicity, this assay should be of use in determining androgen levels in biospecimens, particularly in settings where sample quantity or steroid concentration are low.     

Gas chromatography/mass spectrometry based hair steroid profiling may reveal pathogenesis in hair follicles of the scalp

A method of steroid profiling, including androgens, progestins, corticoids and sterols, was developed to evaluate the concentrations of steroids as well as the activities of the enzymes responsible for steroidogenesis in hair by gas chromatography/mass spectrometry. The extraction efficiencies of steroids from the hair matrix were improved by ultrasonication for 1 h at 50 °C. The overall recoveries ranged from 71 to 132%, with a limit of quantification for all analytes ranging from 1 to 50 ng/g. The devised method was used to identify the metabolic changes for both male‐pattern baldness (MPB) and the drug efficiency of dutasteride, which inhibits 5α‐reductase. Increased dihydrotestosterone levels and the dihydrotestosterone/testosterone (DHT/T) ratio, which is responsible for the 5α‐reductase activity, were observed in the MPB patients. A dutasteride treatment resulted in decreases in the DHT and 5α‐androstanedione concentrations and DHT/T ratio in the hair samples. Hair steroid profiling reflects the sebaceous status in the scalp and may be useful for monitoring the metabolic responses to both the disease and drug actions. Copyright © 2011 John Wiley & Sons, Ltd.     

Computer-aided drug design: A free energy perturbation study on the binding of methyl-substituted pterins and N5-deazapterins to dihydrofolate reductase

Summary Molecular dynamics simulation and free energy perturbation techniques have been used to study the relative binding free energies of 8-methylpterins and 8-methyl-N5-deazapterins to dihydrofolate reductase (DHFR). Methyl-substitution at the 5, 6 and 7 positions in the N-heterocyclic ring gives rise to a variety of ring substituent patterns and biological activity: several of these methyl derivatives of the 8-methyl parent compounds (8-methylpterin and 8-methyl-N5-deazapterin) have been identified as substrates or inhibitors of vertebrate DHFR in previous work. The calculated free energy differences reveal that the methyl-substituted compounds are thermodynamically more stable than the primary compounds (8-methylpterin and 8-methyl-N5-deazapterin) when bound to the enzyme, due largely to hydrophobic hydration phenomena. Methyl substitution at the 5 and/or 7 positions in the 6-methyl-substituted compounds has only a small effect on the stability of ligand binding. Furthermore, repulsive interactions between the 6-methyl substituent and DHFR are minimal, suggesting that the 6-methyl position is optimal for binding. The results also show that similarly substituted 8-methylpterins and 8-methyl-N5-deazapterins have very similar affinities for binding to DHFR. The computer simulation predictions are in broad agreement with experimental data obtained from kinetic studies, i.e. 6,8-dimethylpterin is a more efficient substrate than 8-methylpterin and 6,8-dimethyl-N5-deazapterin is a better inhibitor than 8-methyl-N5-deazapterin.     

Derivatization of neutral steroids to enhance their detection characteristics in liquid chromatography–mass spectrometry

This review article underlines the detection-oriented derivatization of neutral steroids in liquid chromatography-mass spectrometry (LC-MS). Steroids have strong biological activity at very low concentrations in target tissues and, therefore, the analysis of steroids in body fluids or tissues is necessary to elucidate the nature of the many endocrine disease processes and thus be useful for diagnosis and treatment. LC-MS has recently been used for steroid analysis because of its specificity and versatility, but the ionization efficiencies of most steroids are relatively low for the different ionization methods. Derivatization enhances the ionization efficiencies of steroids, leading to high sensitivity and specific detection. For electrospray ionization MS the introduction of permanently charged moieties or easily ionizable moieties effectively increases the sensitivity of detection of steroids. The introduction of moieties with proton affinity or electron affinity enhances the analyte signals in positive and negative atmospheric pressure chemical ionization MS, respectively.     

Synthesis of Hydantoin Androgen Receptor Antagonists and Study on Their Antagonistic Activity

Hydroxymethylthiohydantoin, hydroxymethylthiohydantoin, and hydantoin, containing a pyridine group, were synthesized to study their androgen receptor antagonistic activities. Among them, compounds 6a/6c/7g/19a/19b exhibited excellent androgen receptor antagonistic activity, which was consistent with or even superior to enzalutamide. In addition, compounds 19a and 19b exhibited better antiproliferative activity than enzalutamide in prostate cancer cells. The results show that compound 19a has great potential as a new AR antagonist.     

An improved procedure for the preparation of 2-amino-8-alkylpyrido-[2,3-d]pyrimidin-4(3H)-ones (8-alkyl-N5-deazapterins)

We report an improved procedure for the preparation of 8-alkyl-N5-deazapterins which allows clean preparation of all ring-methyl substituted compounds, including 5- and 7-methyl substituted compounds. The procedure was also successfully applied to the preparation of N5-deazapterins with improved yield over previous reports. The uv/visible and pKa data confirm the predicted increased basicity of 8-alkyl-N5-deazapterins compared with the N5-deazapterin parents, and indicate that N5-deazapterins protonate on N8 and 8-alkyl-N5-deazapterins protonate on N3.     

Direct determination of anabolic steroids in pig urine by a new SPME-GC-MS method

A new solid phase microextraction (SPME) method coupled with gas chromatography-mass spectrometry (GC-MS) was developed for rapid determination of four anabolic steroids such as 3α-hydroxy-5α-androstane-17-one (HA), dihydrotestosterone (DHT), androstenedione (AD) and methyltestosterone (MT) in pig urine. SPME was used to extract the four anabolic compounds directly without derivatization. The optimum SPME sampling conditions were based on the home-made carbowax-divinylbenzene (CW-DVB) fiber coating during extraction at 40 °C for 50 min with 0.18 g/mL NaCl solution and 750 rpm stirring speed. The linear ranges of the proposed method were in the range of 8-640 pg/mL for HA and DHT and 16-510 pg/mL for AD and MT, respectively. The detection limits (S/N = 3) were from 2 to 8 pg/mL for the four anabolic steroids. This SPME method provided very high enrichment factors for the four anabolic steroids, which were 1063-fold and 965-fold for HA and DHT at the concentration of 8 pg/mL and 207-fold and 451-fold for AD and MT at the concentration of 16 pg/mL, respectively. The recoveries ranged from 71.3 to 121%, and the RSDs were lower than 12.9%. The method was sensitive and reliable for determination of trace anabolic steroids in biological samples.