Synthesis of Hydroxy Acids of Dinorcholane and 5 β-Cholane

Enantiopure α-hydroxy acids (AHAs) are versatile building blocks in organic synthesis. They are used for obtaining pharmaceutically and biologically active substances, such as vitamins. α-Hydroxy acids are easily synthesized from cyanohydrins. Thus, molecules with cyanohydrin substructures are interesting intermediates for the synthesis of AHAs. Usually, cyanohydrins are prepared by the addition of a cyanide group to the carbonyl carbon of aldehydes or ketones resulting in enantiomeric mixtures of optically active cyanohydrins when aldehydes or asymmetric ketones are employed. However, if a chiral centre already exists next to the carbonyl carbon, two possible diastereomeric products are formed, and they are not of equal amounts. As part of our on-going studies on the synthesis and reactions of steroid monomers and dimers, we now report on the development of a convenient and general method for the synthesis of diasteriomerc (55:45) dinorcholane hydroxy acids and 5β-cholane hydroxy acid using readily available dinorcholanal and 5β-cholane3α,24-diol as the starting materials. Results and Discussion


Introduction
Enantiopure α-hydroxy acids (AHAs) are versatile building blocks in organic synthesis.They are used for obtaining pharmaceutically and biologically active substances, such as vitamins.α-Hydroxy acids are easily synthesized from cyanohydrins.Thus, molecules with cyanohydrin substructures are interesting intermediates for the synthesis of AHAs. 1 Usually, cyanohydrins are prepared by the addition of a cyanide group to the carbonyl carbon of aldehydes or ketones resulting in enantiomeric mixtures of optically active cyanohydrins when aldehydes or asymmetric ketones are employed.However, if a chiral centre already exists next to the carbonyl carbon, two possible diastereomeric products are formed, and they are not of equal amounts.As part of our on-going studies on the synthesis and reactions of steroid monomers and dimers, [2][3][4][5][6][7][8] we now report on the development of a convenient and general method for the synthesis of diasteriomerc (55:45) dinorcholane hydroxy acids and 5β-cholane hydroxy acid using readily available dinorcholanal and 5β-cholane-3α,24-diol as the starting materials.

Results and Discussion
The cyanation reaction of carbonyl compounds is one of the most powerful procedures for the synthesis of cyanohydrins or cyanohydrin trimethylsilyl ethers.These products can easily be converted to a variety of other acid derivatives, such as α-hydroxy carboxylic acids, α-hydroxy aldehydes, β-amino alcohols and α-amino acids. 1 Several useful cyanating reagents are available, but among them, trimethylsilyl cyanide (TMSCN) is one of the most effective and safe cyanating sources for nucleophilic addition to carbonyl compounds. 9hen an achiral nucleophile adds to an achiral aldehyde, a chiral centre is formed, which is a racemic mixture.However, when a chiral centre is present next to the carbonyl carbon, two possible diastereomeric products are formed.Thus, the reaction of 3-oxo-23,24-dinorchol-4-en-22-al (1) with TMSCN produced a diastereomeric mixture (55:45) of 22-cyanohydrin silyl ethers (2A and 2B) (Scheme 1).
The FABMS spectrum of the mixture revealed the [M+H] + and [M+Na] + ions, respectively, at m/z 428 and 450.The presence of a C≡N moiety was evident from the IR absorption band at 2229 cm -1 .Among the 1 H NMR signals, the two most important peaks were at d 0.17 (s, 9H, 3 × Me), 4.42 and 4.38 (bd, 1H), which confirmed the presence of a TMS unit at C-22.In the 13 C NMR spectrum of this mixture, apart from the signals confirming the above findings, the most revealing signals were a pair of signals at d 64.8 and 65.0 for the oxymethine (C-22, 2A and 2B) and another pair of signals at d 120.1 and 118.3 for the C≡N carbon of two diastereomeric isomers 2A and 2B.The diastereomeric ratio was determined by 13 C NMR spectroscopic integration.
The diastereomeric 22-cyanohydrin silyl ethers (2A and 2B) were refluxed 18 h using NH 3 (25%) in THF and the reaction mixture was washed with dil.HCl to obtain the 22-cyanohydrin.The products obtained were found to be a diastereomeric mixture of 22-cyanohydrins 3A and 3B (Scheme 1).A portion of the mixture was subjected to preparative reversed-phase HPLC resulting in the isolation of pure 3A and 3B.The isomer 3A was eluted first with the retention time of 17.1 min, followed by the isomer 3B having the retention time of 18.3 min.The diastereomeric ratio (55:45) was obtained from the HPLC signals.The compound 3A and 3B had different melting points, and they were 230 o C and 189-190 o C, respectively.
In the 1 H NMR of 3A, the most important peak was a doublet at d 4.53 (J 4.1 Hz) for the oxymethine at C-22, indicating the presence of a cyanohydrin unit at C-22.The 13 C NMR showed characteristic signals for the carbons, C-22 and C≡N at d 64.9 and 122.4,respectively. 3The 1 H NMR of 3B was similar to that of 3A with the exception that the resonance for the C-22 oxymethine (d 4.48, J 4.1 Hz) was slightly less deshielded than 3A.The 13 C NMR of 3B was also similar to that of 3A with the exception that the characteristic signals for the carbons, C-22 and C≡N were observed at d 65.2 and 118.4,respectively.Unambiguous assignment of all 1 H and 13 C resonances were achieved by 1 H-13 C HSQC and 1 H-13 C HMBC experiments.The [M+H] + and [M+Na] + ions of both isomers were observed, respectively, at m/z 356 and 378 in their FABMS spectra.The presence of OH and C≡N functionalities, in both cases, was supported from the IR absorption bands at 3336 and 2235 cm -1 , respectively.Acid hydrolysis of the diastereomeric mixture 3A and 3B in MeOH yielded the diastereomeric mixture of 22-hydroxy acids 4A and 4B (Scheme 1).The IR spectrum of this mixture showed the absorption bands characteristic for acid and alcohol hydroxyls at 3453 (acidic O-H), 3383 (alcoholic O-H), and acid carbonyl at 1720 cm -1 .In the As outlined in Scheme 2, 3-oxo-5β-cholane-24-hydroxy acid (9) was synthesized from the diol 5 in 4 steps.In the first step, diol 5 was oxidized using PCC in DCM to obtain 5β-cholanal 6. 10 The FABMS spectrum of 6 revealed the [M+H] + and [M+Na] + ions, respectively, at m/z 359 and 381.In its 13 C NMR spectrum, the signals at d 213.4 and 203.1 established, respectively, the presence of 3-oxo and C-24 aldehyde functionalities.
3-Oxo-24-cyanohydrin silyl ether 7 was obtained from 6 in MeCN using TMSCN (Scheme 2).The FABMS spectrum of 7 revealed the [M+H] + and [M+Na] + ions at m/z 458 and 480, respectively.In its IR spectrum the absorption band at 2238 cm -1 was indicative of a C≡N stretching.In the 1 H NMR spectrum the signals for the protons associated with C-1 to C-24 of 7 were almost similar to those of 6, except that a signal at d 4.31 for the oxymethine proton of C-24 (instead of an aldehyde proton at d 9.50 in 6) and a 9H singlet for the TMS group were present at d 0.19.The 13 C NMR spectrum also corroborated this fact by showing signals at d 61.9, 120.2 and -0.4,respectively, for the C-24 oxymethine, nitrile and TMS carbons.
The 24-cyanohydrin silyl ether 7 was treated with NH 3 (25%) and refluxed for 18h then washed with dil.HCl to yield 24-cyanohydrin 8 (Scheme 2).The FABMS spectrum of 8 revealed the 408, respectively.In its IR spectrum the absorption bands at 3452 and 2241 cm -1 were indicative of alcohol OH and C≡N stretchings.The 1 H and 13 C NMR spectra of 8 were similar to those of 7 with the exception that there was no signal for a TMS group, and both C-24 oxymethine and nitrile signals were slightly deshielded.
Acid hydrolysis of the 24-cyanohydrin 8 in MeOH yielded 24-hydroxy acid 9 (Scheme 2).The IR spectrum showed the absorption bands characteristic for acid and alcohol hydroxyls, respectively, at 3458 and 3454 cm -1 , and an acid carbonyl at 1728 cm -1 .In the 1 H NMR spectrum of 9, a broad doublet at d 3.62 for an oxymethine proton indicated the presence of a hydroxyl group and an acid group at C-24.The 13 C NMR spectrum displayed a signal at d 162.0 for the acid carbonyl carbon at C-24, and a signal at d 62.4 for the oxymethine (C-24).The FABMS spectrum revealed the [M+H] + and [M+Na] + ions, respectively, at m/z 405 and 427.

General
The starting material 5β-cholane-3α,24-diol (5) was previously synthesized and identified in our lab. 2 3-Oxo-23,24-dinorchol-4-en-22-al (1), pyridinium chlorochromate (PCC) and trimethylsilyl cyanide (TMSCN) were purchased from Aldrich and used as received.The reactions were monitored and the purity of the products was assessed by thin-layer chromatography (TLC) performed on silica gel (Merck type 60) and visualized under UV illumination and/or by I 2 vapor.Melting points of the products were determined on a Gallen-kamp melting point apparatus.Infrared spectra (wave numbers in cm -1 ) were recorded on an ATI Mattson Genesis FTIR spectrophotometer either as KBr pellets or in CHCl 3 .Nuclear magnetic resonance (NMR) spectra were recorded on a Varian Unity INOVA 400 MHz NMR spectrometer.NMR spectra were obtained in CDCl 3 .Chemical shifts (d) are reported in ppm downfield from TMS, using the residual solvent peak (7.25 ppm for 1 H and 77.23 ppm for 13 C) as an internal standard and coupling constants (J) in Hz.Mass spectroscopic analyses were performed at the EPSRC Mass Spectrometry Service at Swansea.

1 H
NMR spectrum, a broad doublet at d 3.68/3.63for an oxymethine proton indicated the presence of a hydroxyl group at C-22 of diastereomeric isomers 4A and 4B.The 13 C NMR spectrum of this mixture displayed a pair of signals at d 161.2/161.0 for the acid carbonyl carbon at C-22, and a pair of signals at d 64.9 and 65.2 for the oxymethine (C-22) of diastereomeric isomers 4A and 4B.The diastereomeric ratio was determined by 13 C NMR spectroscopic integration.The FABMS spectrum revealed the [M+H] + and [M+Na] + ions, respectively, at m/z 375 and 397.