Structure elucidation of new compounds from acidic treatment of the progestins gestodene and drospirenone

Gestodene acidic treatment afforded a single rearrangement product, namely 13-beta-ethyl-18,19-dinorpregna-4,14,16-trien-3,20-dione 3, which was originated through HCl-catalyzed Rupe rearrangement. Drospirenone acidic treatment yielded two epimeric lactones by addition of HCl to the 6beta,7beta-cyclopropane ring, namely 7beta-(chloromethyl)-15beta,16beta-methylene-3-oxo-17beta-pregn-4-ene-21,17-carbolactone 4 and 7beta-(chloromethyl)-15beta,16beta-methylene-3-oxo-17alpha-pregn-4-ene-21,17-carbolactone 5. The structure of the compounds was assessed by spectroscopic and crystallographic methods.     

Enhancing survival of photoreceptor cells in vivo using the synthetic progestin Norgestrel

Retinal degenerations such as Retinitis Pigmentosa remain difficult to treat given the diverse array of genes responsible for their aetiology. Rather than concentrate on specific genes, our focus is on identifying therapeutic avenues for the treatment of retinal disease that target general survival mechanisms or pathways. Norgestrel is a synthetic progestin commonly used in hormonal contraception. Here, we report a novel anti-apoptotic role for Norgestrel in diseased mouse retinas in vivo. Dosing with Norgestrel protects photoreceptor cells from undergoing apoptosis in two distinct models of retinal degeneration; the light damage model and the Pde6b(rd10) model. Photoreceptor rescue was assessed by analysis of cell number, structural integrity and function. Improvements in cell survival of up to 70% were achieved in both disease models, indicating that apoptosis had been halted or at least delayed. A speculative mechanism of action for Norgestrel involves activation of survival pathways in the retina. Indeed, Norgestrel increases the expression of basic fibroblast growth factor which is known to both promote cell survival and inhibit apoptosis. In summary, our results demonstrate significant protection of photoreceptor cells which may be attributed to Norgestrel mediated activation of endogenous survival pathways within the retina.     

Base promoted air oxidation of 13beta-ethyl-11-methylenegon-4-en-17-one.

The structure of 13-ethyl-11-methylene-18,19-dinor-17alpha-pregn-4-en-20-yn-16beta,17-diol (3, 16beta-OH desogestrel), a by-product obtained in the last step of the synthesis of desogestrel (1) by reaction of monolithium acetylide-ethylenediamine complex with 13beta-ethyl-11-methylenegon-4-en-17-one (2), is here reported. The structural assignments were supported by NMR 1H-, 13C-, 1H-1H COSY, 1H-13C HSQC, COLOC) and mass spectroscopy, and the configuration at the C-16 and C-17 stereocentres was established by X-ray crystallography. When the same 17-ketoderivative 2 was treated with a non-alkylating base, such as potassium tert-butoxide, instead of the expected 16-hydroxylated ketone, a dimeric product, 13beta-ethyl-16-[2'-(des-D-13"-carboxy-13"beta-ethyl-11"-methylenegon-4"-en-14"-yl)-ethyliden]-11-methylenegon-4-en-17-one (4), was isolated in good yield; it was characterized by NMR, mass, ultraviolet spectroscopy, and chemical transformations. Compounds 3 and 4 originate from the high reactivity of the 16-methylenic position of the 17-keto substrate (2) toward molecular oxygen under basic conditions.     

Clinical and Laboratory Findings in a Trial of Norgestrel,a Low-dose Progestogen-only Contraceptive

Norgestrel, a progestogen-only oral contraceptive, was given continually at a dose of 75 μg/day to 144 women of proved fertility. It was an efficient contraceptive with a failure rate of 2·1% (assessed by the “life-table” method) within the first 12 cycles and 3·6% within the first 30 cycles (or 2·0 conceptions per 100 woman-years when assessed by the Pearl index). The overall conception rate for the entire trial period was 2·1% and 1·3 pregnancies per 100 woman-years respectively. Norgestrel caused a high proportion of irregular and generally short bleeding intervals, about one-fifth of the cycles lasting less than 17 days. This irregularity appeared to be due to individual variance in cycle length between women rather than that between their successive cycles. No confirmed instances of thromboembolism were observed. Norgestrel apparently exerts its contraceptive action by several mechanisms: reduction in the sperm penetrability of the cervical mucus and an impairment of luteal function appear important. The serum concentrations of cholesterol and globulin were significantly reduced in women taking norgestrel. Preliminary observations suggest that on discontinuing the medication fertility is promptly restored. Of the 144 women originally enrolled 57 (40%) withdrew for reasons connected with the method before completing 30 months on trial, over half of them because of the irregular menstrual pattern. Nonetheless, in view of its main clinical and laboratory characteristics and simple mode of administration, norgestrel appears to be a useful alternative to the combined type of pill for women unsuitable for, or unable to tolerate, oestrogen-containing preparations.     

Biosynthesis of coenzyme F430 in methanogenic bacteria. Identification of 15,17(3)-seco-F430-17(3)-acid as an intermediate

Coenzyme F430 is a hydroporphinoid nickel complex present in all methanogenic bacteria. It is part of the enzyme system which catalyzes methane formation from methyl-coenzyme M. We describe here that under certain conditions a second nickel porphinoid accumulates in methanogenic bacteria. The compound was identified at 15,17(3)-seco-F430-17(3)-acid. The structural assignment rests on 14C-labelling experiments, fast-atom-bombardment mass spectra, 1H-NMR spectra of the corresponding hexamethyl ester, and ultraviolet/visible spectral comparison with model compounds. In cell extracts and in intact cells of methanogenic bacteria, 15,17(3)-seco-F430-17(3)-acid was converted to F430. These findings indicate that the new nickel-containing porphinoid is an intermediate in the biosynthesis of coenzyme F430.     

Progesterone Attenuates Microglial-Driven Retinal Degeneration and Stimulates Protective Fractalkine-CX3CR1 Signaling

Retinitis pigmentosa (RP) is a degenerative disease leading to photoreceptor cell loss. Mouse models of RP, such as the rd10 mouse (B6.CXBl-Pde6brd10/J), have enhanced our understanding of the disease, allowing for development of potential therapeutics. In 2011, our group first demonstrated that the synthetic progesterone analogue ‘Norgestrel’ is neuroprotective in two mouse models of retinal degeneration, including the rd10 mouse. We have since elucidated several mechanisms by which Norgestrel protects stressed photoreceptors, such as upregulating growth factors. This study consequently aimed to further characterize Norgestrel’s neuroprotective effects. Specifically, we sought to investigate the role that microglia might play; for microglial-derived inflammation has been shown to potentiate neurodegeneration. Dams of post-natal day (P) 10 rd10 pups were given a Norgestrel-supplemented diet (80mg/kg). Upon weaning, pups remained on Norgestrel. Tissue was harvested from P15-P50 rd10 mice on control or Norgestrel-supplemented diet. Norgestrel-diet administration provided significant retinal protection out to P40 in rd10 mice. Alterations in microglial activity coincided with significant protection, implicating microglial changes in Norgestrel-induced neuroprotection. Utilizing primary cultures of retinal microglia and 661W photoreceptor-like cells, we show that rd10 microglia drive neuronal cell death. We reveal a novel role of Norgestrel, acting directly on microglia to reduce pro-inflammatory activation and prevent neuronal cell death. Norgestrel effectively suppresses cytokine, chemokine and danger-associated molecular pattern molecule (DAMP) expression in the rd10 retina. Remarkably, Norgestrel upregulates fractalkine-CX3CR1 signaling 1 000-fold at the RNA level, in the rd10 mouse. Fractalkine-CX3CR1 signaling has been shown to protect neurons by regulating retinal microglial activation and migration. Ultimately, these results present Norgestrel as a promising treatment for RP, with dual actions as a neuroprotective and anti-inflammatory agent in the retina.     

Exploring novel non-Leloir β-glucosyltransferases from proteobacteria for modifying linear (β1->3)-linked gluco-oligosaccharide chains

Over the years several β-glucan transferases from yeast and fungi have been reported, but enzymes with such an activity from bacteria have not been characterized so far. In this work, we describe the cloning and expression of genes encoding β-glucosyltransferase domains of glycosyl hydrolase family GH17 from three species of proteobacteria: Pseudomonas aeruginosa PAO1, P. putida KT2440 and Azotobacter vinelandii ATCC BAA-1303. The encoded enzymes of these GH17 domains turned out to have a non-Leloir trans-β-glucosylation activity, as they do not use activated nucleotide sugar as donor, but transfer a glycosyl group from a β-glucan donor to a β-glucan acceptor. More particularly, the activity of the three recombinant enzymes on linear (β1?→?3)-linked gluco-oligosaccharides (Lam-Glc(4-9)) and their corresponding alditols (Lam-Glc(4-9)-ol) was studied. Detailed structural analysis, based on thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and 1D/2D (1)H and (13)C nuclear magnetic resonance data, revealed diverse product spectra. Depending on the enzyme used, besides (β1?→?3)-elongation activity, (β1?→?4)- or (β1?→?6)-elongation, or (β1?→?6)-branching activities were also detected.     

17-Epimerization of 17 alpha-methyl anabolic steroids in humans: metabolism and synthesis of 17 alpha-hydroxy-17 beta-methyl steroids.

The 17-epimers of the anabolic steroids bolasterone (I), 4-chlorodehydromethyltestosterone (II), fluoxymesterone (III), furazabol (IV), metandienone (V), mestanolone (VI), methyltestosterone (VII), methandriol (VIII), oxandrolone (IX), oxymesterone (X), oxymetholone (XI), stanozolol (XII), and the human metabolites 7 alpha,17 alpha-dimethyl-5 beta-androstane-3 alpha,17 beta-diol (XIII) (metabolite of I), 6 beta-hydroxymetandienone (XIV) (metabolite of V), 17 alpha-methyl-5 beta-androst-1-ene-3 alpha,17 beta-diol (XV) (metabolite of V), 3'-hydroxystanozolol (XVI) (metabolite of XII), as well as the reference substances 17 beta-hydroxy-17 alpha-methyl-5 beta-androstan-3-one (XVII), 17 beta-hydroxy-17 alpha-methyl-5 beta-androst-1-en-3-one (XVIII) (also a metabolite of V), the four isomers 17 alpha-methyl-5 alpha-androstane-3 alpha,17 beta-diol (XIX) (also a metabolite of VI, VII, and XI), 17 alpha-methyl-5 alpha-androstane-3 beta,17 beta-diol (XX), 17 alpha-methyl-5 beta-androstane-3 alpha,17 beta-diol (XXI) (also a metabolite of V, VII, and VIII), 17 alpha-methyl-5 beta-androstane-3 beta,17 beta-diol (XXII), and 17 beta-hydroxy-7 alpha,17 alpha-dimethyl-5 beta-androstan-3-one (XXIII) were synthesized via a 17 beta-sulfate that spontaneously hydrolyzed in water to several dehydration products, and to the 17 alpha-hydroxy-17 beta-methyl epimer. The 17 beta-sulfate was prepared by reaction of the 17 beta-hydroxy-17 alpha-methyl steroid with sulfur trioxide pyridine complex. The 17 beta-methyl epimers are eluted in gas chromatography as trimethylsilyl derivatives from a capillary SE-54 or OV-1 column 70-170 methylen units before the corresponding 17 alpha-methyl epimer. The electron impact mass spectra of the underivatized and trimethylsilylated epimers are in most cases identical and only for I, II, and V was a differentiation between the 17-epimers possible. 1H nuclear magnetic resonance (NMR) spectra show for the 17 beta-methyl epimer a chemical shift for the C-18 protons (singlet) of about 0.175 ppm (in deuterochloroform) to a lower field. 13C NMR spectra display differences for the 17-epimeric steroids in shielding effects for carbons 12-18 and 20. Excretion studies with I-XII with identification and quantification of 17-epimeric metabolites indicate that the extent of 17-epimerization depends on the A-ring structure and shows a great variation for the different 17 alpha-methyl anabolic steroids.     

Determination of the post-translational modifications of salivary acidic proline-rich proteins

Human salivary acidic proline-rich proteins were analyzed by electrospray-ion trap mass spectrometry and by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. All acidic-PRP isoforms share a common N-terminal region, which contains a pyroglutamic acid residue at the N-terminus, and two phosphorylation sites on Ser 8 and 22. At the same time, HPLC-MS spectra revealed isoforms of PRP-1 and PRP-3 having a different number of phosphoserine residues, namely, a mono-phosphorylated form of PRP-1 and PRP-3 and a tri-phosphorylated form of PRP-1. The analysis of the masses of tryptic digests suggested that the third phosphate residue should be located on Ser 17. Another protein with a mass of 30,923 amu was detected along the HPLC pattern and MS data of its tryptic digest suggested that it corresponds to the dimer of Pa, the isoform of PRP-1 with a substitution Arg-Cys at 103 position. Finally, structural identification is pending for another post-translational modification of acidic-PRP that provides an increase of 111-114 amu.     

Glycosyl bis-porphyrin conjugates: synthesis and potential application in PDT

Syntheses of new glycosylated neutral and cationic porphyrin dimers linked at the meso-position via a flexible hydrocarbon chain are described. A detailed 1H and 13C NMR study allows their complete structural elucidation. The UV-visible, fluorescence and MALDI mass spectra are also presented. Photocytotoxicities of these compounds against K562 leukaemia cell line are compared to those of Photofrin II.     

Isolation and characterization of a novel uronic acid-containing acidic glycosphingolipid from the ascidian Halocynthia roretzi

A novel uronic acid-containing glycosphingolipid (UGL-1) was isolated from the ascidian Halocynthia roretzi. UGL-1 was prepared from chloroform-methanol extracts and purified by the use of successive column chromatography on DEAE-Sephadex, Florisil, and Iatrobeads. Chemical structural analysis was performed using methylation analysis, gas chromatography, gas chromatography-mass spectrometry, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry, and 1H-NMR spectra. The chemical structure of UGL-1 was determined to be a glucuronic acid-containing glycosphingolipid, Galbeta1-4(Fucalpha1-3)GlcAbeta1-1Cer. The ceramide component was composed of C16:0 and C18:0 acids and C16-, C17-, and C18-phytosphingosines as major components.     

Isolation and structure of a novel pyridine derivative from the Algerian newt, Pleurodeles waltlii

Since its discovery more than 20 years ago the structure of a strongly fluorescent compound called "pleurodeles blue" has remained unknown. Isolation of this pigment has been carried out by successive column chromatographies including epichlorohydrin-triethanolamine-Sephadex and phospho-Sephadex. The structural elucidation of a novel pyridone N-glycoside, 1-beta-D-glucopyranosylpyrid-2(1H)-one-6-acetic acid, is based on a detailed study of its high-resolution mass spectra, 1H and 13C-NMR spectra and its hydrolysis to yield D-glucose.     

Metabolism of progesterone and testosterone by a Bacillus sp.

Microbial transformations by a Bacillus sp. were employed as a means of preparing potentially important derivatives of progesterone and testosterone. Each microbial metabolite was subjected to structure elucidation employing ¹H and ¹³C nmr, mass spectral and cd analysis. Hplc was used for the determination of the percentages of the metabolites formed. The progesterone metabolites were characterised as 14-hydroxy-4-pregnene-3,20-dione (II), 14-hydroxy-5 α -pregnane-3,6,20-trione (III)., 11 α — hydroxy-5 α — pregnane-3, 6,20-trione (IV) and 11 α, 14-dihydroxy-4-pregnene-3,20-dione (V). The testosterone analogs were identified as 4-androstene-3,17-dione (VII), 17 β-hydroxy-5 α -androstene-3,6-dione (VIII), 14-hydroxy-4-androstene-3,17-dione (IX) and 14, 17 β-dihydroxy-4-androsten -3-one (X)₁. The availability of the metabolites enabled complete elucidation of their ¹³C nmr spectra.     

Detection of designer steroid methylstenbolone in “nutritional supplement” using gas chromatography and tandem mass spectrometry: Elucidation of its urinary metabolites

The use of “nutritional supplements” containing unapproved substances has become a regular practice in amateur and professional athletes. This represents a dangerous habit for their health once no data about toxicological or pharmacological effects of these supplements are available. Most of them are freely commercialized online and any person can buy them without medical surveillance. Usually, the steroids intentionally added to the “nutritional supplements” are testosterone analogues with some structural modifications.In this study, the analyzed product was bought online and a new anabolic steroid known as methylstenbolone (2,17α-dimethyl-17β-hydroxy-5α-androst-1-en-3-one) was detected, as described on label. Generally, anabolic steroids are extensively metabolized, thus in-depth knowledge of their metabolism is mandatory for doping control purposes. For this reason, a human excretion study was carried out with four volunteers after a single oral dose to determine the urinary metabolites of the steroid. Urine samples were submitted to enzymatic hydrolysis of glucuconjugated metabolites followed by liquid–liquid extraction and analysis of the trimethylsilyl derivatives by gas chromatography coupled to tandem mass spectrometry. Mass spectrometric data allowed the proposal of two plausible metabolites: 2,17α-dimethyl-16ξ,17β-dihydroxy-5α-androst-1-en-3-one (S1), 2,17α-dimethyl-3α,16ξ,17β-trihydroxy-5α-androst-1-ene (S2). Their electron impact mass spectra are compatible with 16-hydroxylated steroids O-TMS derivatives presenting diagnostic ions such as m/z 231 and m/z 218. These metabolites were detectable after one week post administration while unchanged methylstenbolone was only detectable in a brief period of 45 h.     

Computerized Interpretation of the Electron Ionization Mass Spectra of Nucleoside TMS Derivatives

Abstract

A computer program has been developed for the automated interpretation of mass spectra of TMS derivatives of nucleosides found in human urine. The m/z values in the unknown spectrum are compared to m/z values of 3 different ion series commonly observed in the mass spectra of nucleoside TMS derivatives.¹ If a correlation exists, the unknown spectra are marked with color according to the scheme: 1) blue—molecular ion series, 2) red—base ion series and 3) yellow—sugar ion series. The program suggests a structural assignment for each of the marked ions and calculates a series related ion current. The calculated ion current is used to assign the of sugar contained in the unknown nucleoside.     

Isolation of 20 alpha and 20 beta 5 beta-pregnane-3 alpha,17 alpha,20,21-tetrol (hexahydro-Reichstein's compound-S) in a case of congenital adrenal hyperplasia

5β-Pregnane-3α, 17α, 20α, 21-tetrol (l) and 5β-pregnane-3α, 17α 20β, 21-tetrol (II) have been isolated and identified from the urine of a girl with congenital adrenal hyperplasia. The total 5β-pregnane-3α, 17α, 20(α+β),21-tetrol consisted of 60% of I and 40% of II. The final identity of the compounds was established by gas chromatography — mass spectrometry. The mass spectra of the two trimethylsilyl isomers were closely related to each other in contrast to the spectra of five other pairs of C₂₁-C-20(α and β)-hydroxy steroid-trimethylsilyl-ethers. The mass spectra of free I and II also exhibited many common features, but were less similar to each other than their trimethylsilyl derivatives.     

Application of mass spectrometry to structural identification of flavonoid monoglycosides isolated from shoot of lupin (Lupinus luteus L.).

Flavonoid glycosides constitute important group of plant secondary metabolites. This class of natural products play significant role in different physiological processes. A new methodological approach where mass spectrometric techniques are applied to structural studies of this class of compounds is presented. Four flavonoid O-monoglycosides and one C-monoglycoside were isolated from green parts of lupin (Lupinus luteus L.). Several different mass spectrometric techniques were applied to structural elucidation of isolated compounds. Desorption ionization mass spectrometry was used for registration of mass spectra of intact and derivatized (permethylated) flavonoid glycosides. In some cases electron impact mass spectra of permethylated compounds were also recorded. Methylated samples after methanolysis and further derivatization of free hydroxyl groups (methylation or acetylation) were analyzed with gas chromatography-mass spectrometry. Combined information drawn from the registered mass spectra enabled us to define molecular mass, structure of aglycones and sugars, and positions of glycosidic bonds on the aglycon. Structures of four flavonoid monoglycosides were elucidated as follows: genistein 7-O-glucoside (1), genistein 4'-O-glucoside (2), 2'-hydroxygenistein 7-O-glucoside (3), and apigenin or genistein 8-C-glycoside (5). For the fourth O-glycoside (4) only molecular mass and masses of the aglycone and sugar were estimated.     

X-ray diffraction studies on the absolute configuration of alpha- and beta-anordrins

The molecular structures and absolute configurations of alpha- and beta-anordrins are reported. Pure alpha- and beta-epimers were obtained with recrystallization and column chromatography combined with high-pressure liquid chromatography methods; they were identified by high-resolution infrared and mass spectra and 1H and 13C nuclear magnetic resonance. By single crystal x-ray diffraction analysis, the crystals of alpha- and beta-epimers were found to belong to the orthorhombic space groups P2(1)2(1)2(1) and P2(1)2(1)2, respectively. The molecular structures of these two epimers were determined. The absolute configurations were deduced by conformation analysis, 1H nuclear magnetic resonance, and comparison with the absolute configuration of the starting material. The absolute configurations of asymmetric centers of alpha- and beta-epimers were observed to be 2R, 5S, 8R, 9S, 10S, 13S, 14S, 17R, and 2S, 5S, 8R, 9S, 10S, 13S, 14S, 17R, respectively. These results were confirmed by the x-ray diffraction determination of the absolute configuration of 2 alpha,17 alpha-diethynyl-A-nor-5 alpha- androstane-2 beta, 17 beta-diol dichloroacetate.     

Positive and negative electrospray ionisation tandem mass spectrometry as a tool for structural characterisation of acid released oligosaccharides from olive pulp glucuronoxylans

Xylo-oligosaccharides with degrees of polymerisation 5-13, formed by partial acid hydrolysis from an extract representative of olive pulp glucuronoxylans (GX), were analysed by electrospray ionisation mass spectrometry (ESI-MS), both in positive and negative modes. The positive spectrum showed the presence of xylo-oligosaccharides in the mass range between m/z 500 and 1500 corresponding to singly [M+Na](+) charged ions of neutral (Xyl(7-9)) and acidic xylo-oligosaccharides (Xyl(5-9)MeGlcA), and doubly [M+2Na](2+) charged ions of Xyl(9-13) and Xyl(7-11)MeGlcA. Ammonium adducts [M+NH(4)](+) were also observed for Xyl(5-9)MeGlcA. The negative spectra showed the contribution of ions in the mass range between m/z 600 and 1400, ascribed to the deprotonated molecules [M-H](-) of Xyl(3-9)MeGlcA. Tandem mass spectrometry (MS/MS) of the major ions observed in the MS spectra was performed. The MS/MS spectra of the [M+Na](+) adducts showed the loss of MeGlcA residues as the major fragmentation pathway and glycosidic fragment ions of Xyl(n) and Xyl(n)MeGlcA structures. The MS/MS spectra of the [M+NH(4)](+) adducts suggests the occurrence of isomers of Xyl(5-9)MeGlcA oligosaccharides with the MeGlcA residue at the reducing end and at the non-reducing end of the molecules, although other structural isomers can also occur. Both glycosidic bond and cross-ring cleavages in the MS/MS spectra of the [M-H](-) ion suggest the occurrence of Xyl(3-9)MeGlcA with the substituting group at the reducing end position of the xylose backbone, as the main fragmentation ions. The results obtained by ESI-MS/MS, both in positive and negative modes, of Xyl(7-13)- and Xyl(5-11)MeGlcA, allow to identify fragmentation patterns of the structural isomers with MeGlcA linked to the terminal xylosyl residues of the oligosaccharides. The occurrence of these higher molecular weight oligosaccharides with a low substitution pattern allows to infer a scatter and random distribution of MeGlcA along the xylan backbone of olive pulp.     

Synthesis and characterization by 1H and 13C nuclear magnetic resonance spectroscopy of 17 alpha-hexanoic derivatives of 5 alpha-dihydrotestosterone and testosterone.

The synthesis and characterization of 17 alpha-(6'-hexanoic acid) derivatives of 5 alpha-dihydrotestosterone and testosterone, useful as ligands for affinity chromatography purification or as precursors for affinity-labeling of androgen-binding proteins, is described. Alkynylation of 3-ethylenedioxy-, 3 beta-hydroxy-, and 3 beta,5-dihydroxy-5 alpha-androstan-17-one precursors with the potassium derivative of 5-hexyn-1-ol led to the corresponding 17 alpha-(6'-hydroxyhex-1'-ynyl) derivatives, which were hydrogenated over 10% Pt-C catalyst to give 17 alpha-(6'-hydroxyhexyl) derivatives. Chromic acid oxidation of the primary hydroxy group of the 3-ethylenedioxy-17-hexyl intermediate into carboxylic acid followed by acid cleavage of the 3-ketal group gave 17 alpha-(5'-carboxypentyl)-5 alpha-dihydrotestosterone, which was also obtained directly by chromic acid oxidation of the 3 beta-hydroxy intermediate. Chromic acid oxidation of the primary hydroxy group of the 3 beta,5 alpha-dihydroxy precursor resulted in a 5 alpha-hydroxy-3-oxo intermediate, which was dehydrated to give 17 alpha-(5'-carboxypentyl)testosterone. The 17 alpha configuration of these derivatives and of synthetic precursors was established by comparing their molecular rotations and their 1H and 13C nuclear magnetic resonance (NMR) spectra including solvent effects, with data reported for 17 alpha- or 17 beta-substituted steroid analogs as well as with 1H and 13C NMR reference data recorded in this work for 17 alpha-ethynyltestosterone, 17 alpha-ethynyl-19-nortestosterone, 17 alpha-ethyl-19-nortestosterone, 17 alpha-methyltestosterone, and 17 alpha-methyl-5 alpha-dihydrotestosterone.