A systematic study of determination and validation of finasteride impurities using liquid chromatography

Pharmaceutical use of finasteride (Dilaprost®) has been well documents in the peer-reviewed literature; however, the presence of trace amounts of related substances (impurities) in finasteride may influence the tharapeutic efficacy and safely. Due to limited information available, the objective of this study was to develop a quantification method for the three impurities of finasteride using high performance liquid chromatography (HPLC) with an ultraviolet (UV) detector. The compounds (impurities) of finasteride that are registered with the European Pharmacopeia, which we sought to validate are: -N-(1,1-dimethylethyl)-3-oxo-4-aza-5α-androstane-17β-carboxamide (impurity A), methyl 3-oxo-4-aza-5α-androst-1-ene-17β-carboxylate (impurity B), and -N-(1,1-dimehylethyl)-3-oxo-4-azaandrosta-1,5-diene-17β-carboxamide (impurity C). Analyses were performed using a Nova Pac C₁₈ column for HPLC with isocratic elution. Detection was carried out at 210 nm, the concentration of the three impurities was in the range was 1.5–4.5 μg mL⁻¹ at ambient temperature with a mobile phase of water + acetonitrile + tetrahydrofuran (80:10:10, v/v/v) and the flow rate was 2.0 mL min⁻¹. The recoveries were: 101.35 ± 0.62% (impurity A), 101.60 ± 2.66% (impurity B) and 101.97 ± 2.05% (impurity C). Validation of the method yielded fairly good results as it relates to the precision and accuracy. It is, therefore, concluded that the method would be suitable for not only the separation and determination of processed impurities to monitor the reactions, but also for the quality assurance of finasteride and its related substances.     

Metabolic studies of mesterolone in horses

Mesterolone (1α-methyl-5α-androstan-17β-ol-3-one) is a synthetic anabolic androgenic steroid (AAS) with reported abuses in human sports. As for other AAS, mesterolone is also a potential doping agent in equine sports. Metabolic studies on mesterolone have been reported for humans, whereas little is known about its metabolic fate in horses. This paper describes the studies of both the in vitro and in vivo metabolism of mesterolone in racehorses with an objective to identify the most appropriate target metabolites for detecting mesterolone administration.In vitro biotransformation studies of mesterolone were performed by incubating the steroid with horse liver microsomes. Metabolites in the incubation mixture were isolated by liquid-liquid extraction and analysed by gas chromatography-mass spectrometry (GC-MS) after acylation or silylation. Five metabolites (M1-M5) were detected. They were 1α-methyl-5α-androstan-3α-ol-17-one (M1), 1α-methyl-5α-androstan-3β-ol-17-one (M2), 1α-methyl-5α-androstane-3α,17β-diol (M3), 1α-methyl-5α-androstane-3β,17β-diol (M4), and 1α-methyl-5α-androstane-3,17-dione (M5). Of these in vitro metabolites, M1, M3, M4 and M5 were confirmed using authentic reference standards. M2 was tentatively identified by mass spectral comparison to M1.For the in vivo metabolic studies, Proviron^® (20 tablets × 25 mg of mesterolone) was administered orally to two thoroughbred geldings. Pre- and post-administration urine samples were collected for analysis. Free and conjugated metabolites were isolated using solid-phase extraction and analysed by GC-MS as described for the in vitro studies. The results revealed that mesterolone was extensively metabolised and the parent drug was not detected in urine. Three metabolites detected in the in vitro studies, namely M1, M2 and M4, were also detected in post-administration urine samples. In addition, two stereoisomers each of 1α-methyl-5α-androstane-3,17α-diol (M6 and M7) and 1α-methyl-5α-androstane-3,16-diol-17-one (M8 and M9), and an 18-hydroxylated metabolite 1α-methyl-5α-androstane-3,18-diol-17-one (M10) were also detected. The metabolic pathway for mesterolone is postulated. These studies have shown that metabolites M8, M9 and M10 could be used as potential screening targets for controlling the misuse of mesterolone in horses.     

Synthesis and Crystal Structure of 2-Fluoro-4-methyl-3-oxo-4-aza-5α-androst-1-ene-17β-carboxylic Acid Methyl Ester

Synthesis and Crystal Structure of 2-Fluoro-4-methyl-3-oxo-4-aza-5α-androst-1-ene-17β-carboxylic Acid Methyl EsterAuthorJANG Yin-Zhi XIANG Zuo LIANG Da-Wei (Department of Applied Chemistry, Zhejiang Sci-Tech. University, Hangzhou 310018, China)AbstractThe title compound VII, 2-fluoro-4-methyl-3-oxo-4-aza-5α-androst-1-ene-17β-carboxylic acid methyl ester (C21H30FNO3, Mr = 363.46), was prepared through a seven-step reaction from pregnenolone, and characterized by elemental and single-crystal X-ray diffraction analyses as well as IR, MS and 1H-NMR spectra. It is of monoclinic system, space group P21/c with a = 6.3882(7), b = 9.9033(11), c = 15.4925(17) , β = 91.923(2)°, V = 979.57(19) 3, Z = 2, Dc = 1.232 mg/m3, μ = 0.088 mm-1, F(000)= 392, R = 0.0465, wR = 0.0989 and λ(MoKα) = 0.71073 . The structure indicates that the four cycles (A: C(1)-C(2)-C(3)-N(1)-C(5)-C(10), B: C(5)-C(6)-C(7)- C(8)-C(9)-C(10), C: C(8)-C(14)-C(13)-C(12)-C(11)-C(9), D: C(14)-C(15)-C(16)-C(17)-C(13)) are in chairand trans-configurations. The results of crystal structure determination show that there exist weak intra-molecular hydrogen bonds, resulting in a two-dimensional supramolecular frame-work of the title compound.Keywordsfluoro-sterol, synthesis, crystal structure, supramolecule     

Synthesis of 2-(β-D-ribofuranosyl)pyrimidines,A new class of C-nucleosides

A new C-glycosyl precursor for C-nucleoside synthesis, 2,5-anhydroallonamidine hydrochloride ( 4 ) was prepared and utilized in a Traube type synthesis to prepare 2-(β-D-ribofuranosyl)pyrimidines, a new class of C-nucleosides. The anomeric configuration of 4 was confirmed by single-crystal X-ray analysis. Reaction of 4 with ethyl acetoacetate gave 6-methyl-2-(β-D-ribofuranosyl)pyrimidin-4-(1H)-one ( 5 ). Reaction of 4 with diethyl sodio oxaloacetate gave 2-(β-D-ribofuranosyl)pyrimidin-6(1H)-oxo-4-carboxylic acid ( 6 ). Esterification of 6 with ethanolic hydrogen-chloride gave the corresponding ester 7 which when treated with ethanolic ammonia gave 2-(β-D-ribofuranosyl)pyrimidin-6(1H)-oxo-4-carboxamide ( 8 ). Condensation of 2,5-anhydroallonamidine hydrochloride ( 4 ) with ethyl 4-(dimethylamino)-2-oxo-3-butenoate ( 9 ), gave ethyl 2-(β-D-ribofuranosyl)pyrimidine-4-carboxylate ( 10 ). Treatment of 10 with ethanolic ammonia gave 2-(β-D-ribofuranosyl)pyrimidine-4-carboxamide ( 11 ). Single-crystal X-ray analysis confirmed the β-anomeric configuration of 11. Acetylation of 11 followed by treatment with phosphorus pentasulfide and subsequent deprotection with sodium methoxide gave 2-(β-D-ribofuranosyl)pyrimidine-4-thiocarboxamide ( 14 ). Dehydration of the acetylated amide 12 with phosphorous oxychloride provided 2-(β-D-ribofuranosyl)pyrimidine-4-carbonitrile ( 15 ). Treatment of 15 with sodium ethoxide gave ethyl 2-(β-D-ribofuranosyl)pyrimidine-4-carboximidate ( 16 ), which was converted to 2-(β-D-ribofuranosyl)pyrimidine-4-carboxamidine hydrochloride ( 17 ) by treatment with ethanolic ammonia and ammonium chloride. Treatment of 16 with hydroxylamine yielded 2-(β-D-ribofuranosyl)pyrimidine-4-N-hydroxycarboxamidine ( 18 ). Treatment of 2-(β-D-ribofuranosyl)pyrimidine-4-carboxamide ( 11 ) with phosphorus oxychloride gave the corresponding 5′-phosphate, 19 , Coupling of 19 with AMP using the carbonyldiimidazole activation procedure gave the corresponding NAD analog, 2-(β-D-ribofuranosyl)pyrimidine-4-carboxamide-(5′ ? 5′)-adenosine pyrophosphate ( 20 ).     

Synthesis of 2,5 diol of A-nor-5 alpha-androstan-17 beta- ols and A-nor-5 beta-androstan-17 beta-ols

The synthesis of A-nor-5β-androstane-2α,5,17β-triol ( 8 ), A-nor-5β-androstane-2β,5,17β-triol ( 10 ), A-nor-5α-androstane-2β,5,17β-triol ( 20 ), A-nor-5α-androstane-2β,5,17β-triol ( 22 ) and of their 17-O-benzoyl derivatives is described, using A-nor-testosterone ( 1 ) as starting material.     

Steroids and sex hormones. Quassinoid bitter principles. I. 1-oxo-2-methoxy-4 alpha-methyl-17 beta-hydroxy-delta2-5 alpha-androstene as a model for the ring a structure of quassine]

Starting from 3-oxo-17β-hydroxy-Δ¹-5α-androstene (2b) the preparation of 1-oxo-2-methoxy-4α-methyl-17β-hydroxy-Δ²-5α-androstene (9), a compound with the ring A structure of quassine (1) is described. The key problem of the reaction sequence is shown to be the monomethylation at C(4).     

Cytotoxic Terpenes from the Stems of <i>Dipterocarpus obtusifolius</i> Collected in Cambodia

From the stems of Dipterocarpus obtusifolius, five new triterpenes, 3-oxo-20-hydroxy-30α-methyl,17(29)α-epoxy-28-norlupane (1), 3-oxo-20-hydroxy-30β-methyl-17(29)α-epoxy-28-norlupane (2), 3,20-dioxo-28,29-norlupan-17α-ol (3), 27-demethyl-20(S)-dammar-23-ene-20-ol-3,25-dione (4), and 3-epi-cecropic acid (5) together with 13 known compounds including diterpene, sesquiterpenes and triterpenes were isolated and characterized. All isolates were tested for their cytotoxicities against a small panel of human cancer cell lines. Of the tested compounds, compounds 4-11 were found to be cytotoxic against one or more human cancer cell lines.     

Steroids and sex hormones. The synthesis of 1-beta, 4-beta-oxido-3-aza-17-beta-hydroxy-A-homo-5-alpha-androstane

A sterically controlled transformation of 3-oxo-17β-acetoxy-Δ^1?-α-androstene ( 2 ) into 1β,4β-oxido-3-aza-17β-hydroxy-A-homo-5α-androstane ( 16 ) is described. With the exception of two conversions [ 14 → 15 (60%); 15 → 16 (50%)], the yields of the remaining seven steps are higher than 75% each. The reaction sequence will serve as a model for an analogous partial synthesis of samandarine ( 1 ).     

Photochemical Rearrangement of a Steroidal α, β-Epoxylactam. Photochemical reactions,XII [1]

The UV. irradiation of 17β-hydroxy-4α, 5α-epoxy-2-azaandrostan-3-one ( 1 ) yields 17β-hydroxy-2-aza-10 (5 → 4-abeo)-4ζ (H)-androsta-3,5-dione ( 3 ).     

Photochemical reactivity of α,β-unsaturated δ-lactams: Synthesis of seco-steroids. Photochemical reactions. XIII [1]

The UV. irradiation of 17 β-hydroxy-2-aza-4-androsten-3-one (1) , N-methyl-17 β-hydroxy-2-aza-4-androsten-3-one (3) , 17 β-hydroxy-4-aza-5 β-androst-1-en-3-one (2) and N-methyl-17 β-hydroxy-4-aza-5 β-androst-1-en-3-one (4) , gives rise to 1,10-seco (from 1 and 3 ) and 5, 10-seco (from 2 and 4 ) steroids.     

Alkyl nitrate nitration of active methylene compounds. Nitration of 1-methyl4-piperidone, 1-methyl-2-pyrrolidinone and 1,3-dimethyl-2-pyrrolidinone

The reaction of 1-methyl-4-piperidone ( 1 ) with amyl nitrate in the potassium tert-butoxidetetrahydrofuran system gave dipotassium 5-methyl-2-oxo-1,3-piperidinedinitronate ( 4 ) in 78% yield. Similar treatment of 1-methyl-2-pyrrolidinone ( 2 ) afforded potassium 3-methyl-2-oxopyrrolidinenitronate ( 7 ) in 85% yield. In contrast, the nitration of 1,3-dimethyl-2-pyrrolidinone ( 3 ) led to opening of the lactam ring with the formation of amyl 2-aza-2-methyl-5-nitrohexanoate ( 10 ) in 40% yield. Acidification of disalt 4 did not cause ring opening but gave the dipolar ion of 1-methyl-3-nitro 4-hydroxy-5-aci-nitro-Δ³-tetrahydropyridinium (6).     

14-methyl steroids. Part 3. Synthesis of (±)-14α-methyl-9β-estradiol and related 14α-methyl-19-norsteroids

Catalytic hydrogenation of a totally synthetic mixture of (±)-3-methoxy-14-methyl-14α-estra-1,3,5( 10), 9(11)-tetraen-17-one(1) and the corresponding 1,3,5(10),8-tetraen-17-one (2) gives a mixture of 14α-methyl-8β,9β-, -8α,9α-, and -8β,9α-estrones, which is converted into the 17β-hydroxy-mixtures. t-Butylation gives a separable mixture of the three isomers, of which (±)-17β-t-butoxy-3 methoxy-14-methyl-9β,14α-estra-1,3,5(10)-triene(6) is the major component. The corresponding 14α-methylestradiols are prepared. A practical synthesis of (±)-14-methyl-14α-estra-1,3,5(10), 6,8-pentaene-3,17β-diol(25) is described, and it is shown that DDQ dehydrogenation of 1,3,5(10),9(11)-tetraenes in this series leads exclusively to the corresponding 1,3,5(10),6,8,11-hexaenes, whereas that of 1,3,5(10),8-tetraenes gives only 1,3,5(10),6,8-pentaenes.     

Synthesis and conversions of 3-(4-amino-5-methyl-4H-1,2,4-triazol-3-ylmethylene)-2-oxo-1,2,3,4-tetrahydroquinoxaline

The reaction of the hydrazone 3a with hydrazine hydrate in DBU/ethanol conveniently gave 3-(4-amino-5-methyl-4H-1,2,4-triazol-3-ylmethylene)-2-oxo-1,2,3,4-tetrahydroquinoxaline 6 . The reactions of 6 with an equimolar and 2-fold molar amount of nitrous acid afforded 3-(α-hydroxyimino-4-amino-5-methyl-4H-1,2,4-triazol-3-ylmethyl)-2-oxo-1,2-dihydroquinoxaline 9 and 3-(α-hydroxyimino-5-methyl-2H-1,2,4-triazol-3-ylmethyl)-2-oxo-1,2-dihydroquinoxaline 10 , respectively, which were converted into the 3-heteroarylisoxazolo[4,5-b]quin-oxalines 13a,b and 11 , respectively. Compound 9 was also cyclized into the 8-quinoxalinyl-1,2,4-triazolo-[3,4-f][1,2,4]triazines 14a,b .     

A favorski reaction using iodosobenzene

Iodosobenzene or iodobenzene diacetate and excess base when reacted with 17β-hydroxy-5α-androstane-3-one (1a) unexpectedly gave a good yield of Favorski acid (3a) and some (3b). 17β-hydroxy-5α-19-norandrostan-3-one (1b) gave mainly the expected dimethylketal of the 2α-hydroxy-3-keto steroid (5).     

一锅法合成新型7-苯基-1,4,5,6,7,8-六氢喹啉酮衍生物

以5-苯基-1,3-环己二酮,醛,乙酰乙酸乙酯(或乙酰丙酮)和乙酸铵为原料,在无水乙醇中经一锅反应合成了14个新型的7-苯基-1,4,5,6,7,8-六氢喹啉酮衍生物,总收率85%~95%,其结构经¹H NMR, ¹³C NMR, IR和HR-MS表征。采用X-ray单晶衍射研究了2-甲基-4,7-二苯基-5-氧代-1,4,5,6,7,8-六氢喹啉顺反异构体(5a和5a′)的晶体结构。结果表明：5a空间群为C2/c, a=9.458 35(19) ,b=19.789 0(4) ,c=11.040 9(2) , α=90°, β=105.614(2)°, γ=90°,V=1 990.28(7) ³, Z=4, μ=0.673 mm^-1, F(000)=824; 5a′空间群为C2/c, a=9.770 2(5) , b=19.981 0(10) , c=10.430 1(4) , α=90°, β=98.361(5)°, γ=90°, V=2 014.51(17) ³, Z=4, μ=0.665 mm^-1, F(000)=824。     

Synthesis of 8-methyl-2-azainosine and related nucleosides

The synthesis of 6-methyl-7-(β-D-ribofuranosyl)imidazo[4,5-d]-v-triazin-4-one (8-methyl-2-azainosine ( 2) ) and 6-methyl-7-(β-D-glucopyranosyl)imidazo[4,5-d]-v-triazin-4-one ( 5 ) by diazotization of 5-amino-1-(β-D-ribofuranosyl)-2-methylimidazole-4-carboxamide ( 1 ) and diazotization of 5-amino-1-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-2-methylimidazole-4-carboxamide ( 3 ), followed by deacetylation of the resulting compound 4 , is described. The preparation of 6-methyl-5-(β-D-ribofuranosyl)imidazo[4,5-d]-v-triazin-4-one ( 10 ) and 6-methyl-5-(β-D-glucopyranosyl)imidazo[4,5-d]-v-triazin-4-one ( 11 ) by glycosylation of 6-methylimidazo[4,5-d]-v-triazin-4-one (8-methyl-2-azahypoxanthine, ( 7) ) is also described. Structural assignments were made on basis of analytical and ¹H-nmr and uv spectral data.     

Bromination and diazo-coupling of pyridinethiones; microwave assisted synthesis of isothiazolopyridine, pyridothiazine and pyridothiazepines

Isothiazolopyridines, pyridothiazines and pyridothiazepines are important compounds that possess valuable biological activities. This paper reports on the synthesis of these compounds using both conventional chemical methods and modern microwave techniques. 3-Bromo-6-hydroxy-4-methyl-2-thioxo-2,3-dihydropyridine-3-carboxamide, 5-arylazo-6-hydroxy-4-methyl-2-thioxo-1,2-dihydropyridine-3-carboxamides, 3,5-bis-arylazo-6-hydroxy-4-methyl-2-thioxo-2,3-dihydropyridine-3-caboxamide, 4-methyl-2,3,6,7-tetra-hydroisothiazolo[5,4-b]-pyridine-3,6-dione, 2,2'-(methylene-bis-(sulfanediyl))bis(4-methyl-6-oxo-1,6-dihydropyridine-3-carboxamide), 2-hydroxy-5-methyl-4H-pyrido[3,2-e][1,3]-thiazine-4,7(8H)-dione and 2-arylmethylene-8-hydroxy-6-methyl-2,3,4,5-tetrahydropyrido-[3,2-f][1,4]thiazepine-3,5-diones have been prepared from 6-hydroxy-4-methyl-2-thioxo-2,3-dihydropyridine-3-carboxamide. Some of these compounds were prepared using microwave-assisted reaction conditions, that provided higher yields in shorter times than the conventional methods.     

Reactivity of 5,10:8,14-Disecosteroids: An unusual rearrangement of cyclodecene-1,4-dione systems to five-membered-ring spiro-γ-lactones

Upon heating in AcOH, the stereoisomeric (Z)- and (R)-6,9-dioxocyclodex-3-enyl derivatives, 5 and 6 , respectively, obtained by HgO/I₂ oxidation of 5-hydroxy-8-oxo-8,14-seco-5α-androstane-3β,17β-diyl diacetate ( 3 ), undergo an unusual intramolecular rearrangement to give the corresponding unsaturated (5R,9R)- and (5R,9S)-spiro-lactones 7 and 8 , respectively. Hydroxylation of the C?C bond in 7 and 8 , and subsequent glycol cleavage of the resulting diols 9 and 10 afforded the epimeric spiro-lactones (5R,9S)- 11 and (5R,9R)- 14 , respectively, and in both cases, the ring-D-containing fragments 12 and 13 .     

Combination of carbon isotope ratio with hydrogen isotope ratio determinations in sports drug testing

Carbon isotope ratio (CIR) analysis has been routinely and successfully applied to doping control analysis for many years to uncover the misuse of endogenous steroids such as testosterone. Over the years, several challenges and limitations of this approach became apparent, e.g., the influence of inadequate chromatographic separation on CIR values or the emergence of steroid preparations comprising identical CIRs as endogenous steroids. While the latter has been addressed recently by the implementation of hydrogen isotope ratios (HIR), an improved sample preparation for CIR avoiding co-eluting compounds is presented herein together with newly established reference values of those endogenous steroids being relevant for doping controls. From the fraction of glucuronidated steroids 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-Hydroxy-5β-androstane-11,17-dione, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 3β-hydroxy-androst-5-en-17-one (DHEA), 5α- and 5β-androstane-3α,17β-diol (5aDIOL and 5bDIOL), 17β-hydroxy-androst-4-en-3-one and 17α-hydroxy-androst-4-en-3-one were included. In addition, sulfate conjugates of ANDRO, ETIO, DHEA, 3β-hydroxy-5α-androstan-17-one plus 17α- and androst-5-ene-3β,17β-diol were considered and analyzed after acidic solvolysis. The results obtained for the reference population encompassing n?=?67 males and females confirmed earlier findings regarding factors influencing endogenous CIR. Variations in sample preparation influenced CIR measurements especially for 5aDIOL and 5bDIOL, the most valuable steroidal analytes for the detection of testosterone misuse. Earlier investigations on the HIR of the same reference population enabled the evaluation of combined measurements of CIR and HIR and its usefulness regarding both steroid metabolism studies and doping control analysis. The combination of both stable isotopes would allow for lower reference limits providing the same statistical power and certainty to distinguish between the endo- or exogenous origin of a urinary steroid.     

Synthesis and antimicrobial activity of 4-aza-5α-sitostane and the 4-methyl derivative

Synthesis of 4-aza-5α-sitostane (IX), and 4-methyl-4-aza-5α-sitostane (XII) were accomplished through a set of reactions involving oxidative opening of ring A of α,β-unsaturated ketone, ring closure, followed by catalytic hydrogenation and lithium aluminum hydride reduction, respectively. The antimicrobial activity for X and XII is reported.