Cholestane tetrol

The syntheses of (22S)-22-hydroxycampesterol and 7/3-hydroxycampesteroI from the 3,5-cyclo-aldehyde (189) have been reported. The two brassino-steroids (194) and (195) were synthesized from ergosteryl tosylate. The 24-nor-5j8-cholestane tetrols (196) and (197) were synthesized by hydrobor-ation-oxidation of the corresponding and A -compounds, and the tetrol (198) was synthesized from cholic acid. ° Lithocholic acid was converted in... 

Chromatographic analysis of the polar sterol fractions of the bile and feces, in conjunction with gas-liquid chromatography (GLC)-mass spectrometry indicated two major components, 5P-cholestane-3a,7a,12a,25-tetrol and 5P-cholestane-3a,7a,12a,23,25-pentol, and a minor component, 5P-cholestane-3a, 7a,12a,24,25-pentol(18-21). Only minute amounts of 5P-cholestane-3a, 7a,12a,23R-tetrol, 5P-cholestane-3a,7a,12a,24R-tetrol, 5P-cholestane-3a,7a, I2a,24S-tetrol and 5P-cholestane-3a,7a,12a,25S,26-pentol have been detected (20-24). The presence of 5P-cholestane-3a,7a,12a,25-tetrol was positively identified by comparison with the synthesized sample prepared in our laboratory (20-22,24-27) (Fig.l). The predominent bile alcohol of the pentol fraction was 5P-cholestane-3a,7a,12a,23,25-pentol, amounting to approximately 80% by weight, while 5P-cholestane-3a,7a,12a,24,25-pentol accounted for approximately 20% of this fraction. The less abundant pentol was shown to be identical with 5P-cholestane-3a,7a,12a,24a,25-pentol, which had been prepared from 5P-cholestane-3a,7a,12a,25-tetrol (20,21). 

The biosynthetic 5p-cholestanetetrol and pentol had the same melting point, TLC and GLC properties, and infrared and mass spectra as the reference compound (19-21). In addition, the recently described two-dimensional H-NMR studies (28-30) from our and other laboratories have further elucidated their structure and stereochemistry. Fig. 2 illustrates 2D-NMR H- H COSY spectra of 5P-cholestane-3a,7a, 12a 25-tetrol (for comparison 2D-NMR H- H COSY spectra of 5P-cholestane-3a,7P, 12a,25-tetrol is also provided). The 13c-DEPT spectra of (24R) and 5P-cholestane-3a,7a,12a,24a,25-pentol and (23R)-5P-cholestane-3a,7a,12a, 24a,25-pentol has been recently described (21). 

The insertion of hydroxyl groups into the 23- or 24-position of 5P-cholestane-3a,7a,12a,25-tetrol was found to be stereospecific. Although all these compounds were potential precursors of bile acid, studies in vivo and in vitro experiments using [3P- H] and (24- C) 5P-cholestane-3a,7a,12a,25-tetrol (46) (Figs.6, 7), (24- C) 5p-cholestane-3a,7a,12a,24R,25-pentol and (24- C) 5P-cholestane-3a,7a,12a,24S,25-pentol demonstrated the existence of a new 25-hydroxylation pathway for the transformation of cholesterol to cholic acid in these patients (2,10). The reaction sequence involved the stereospecific formation of a 24S-hydroxy pentol, 5P-cholestane-3a,7a,12a,24S,25-pentol, 3a7a,12a,25-tetrahydroxy-5P-cholestan-24-one and did not involve SP-cholestanoic acids as intermediates (Fig. 8). The two bile pentols, SP-cholestane-3a,7a,12a,24R, 25-pentol and 5P-cholestane-3a,7a,12a,23R,25-... 

Hvdroxvlation pathway This pathway has been demonstrated in both rat and human liver (2,10,56). It involves the 25-hydroxylation of 5P-cholestane-3a,7a,12a-triol to give 5P-choiestane-3a,7a,12a,25- tetrol (XIV) followed by stereospecific 24S-hydroxylation to yield 5P-cholestane-3a,7a,12a,24S,25-pentol (XV, Fig. 9). The pentoi is then oxidized to 5P-choiestane-3a,7a,12a, 25-tetrahydroxy-5P-cholestan-24-one (XVI) (59,60), which is degraded by... 

Hvdroxvlation pathway An alternative explanation for the bile acid synthetic defect in CTX has been proposed by Oftebro and colleagues which starts via 26-hydroxylation of 5P-cholestane-3a,7a,12a-triol (IX, Fig. lOa and 10b). In this pathway the mitochondrial fraction of both human and rat liver contains a 26-hydroxylase enzyme (63) which can convert 5P-cholestane-3a,7a,12a-triol (IX ) to 5P-cholestane-3a,7a,12a,26-tetrol (XI) (Fig. 10a and 10b ). This tetrol is oxidized to 3a,7a,12a-trihydroxy-5P-cholestan-26-oic acid (THCA, XII) by liver cytosol (2,64). Further hydroxylation at C-24 forms varanic acid (XIV) and its side chain is shortened with oxidation at C-24 to yield cholic acid (X,Fig. 10 a). These investigators demonstrated diminished mitochondrial 26-hydroxylation of 5p-cholestane-3a,7a,12a-triol and 5P-cholestane-3a,7a-diol, possible precursors for cholic acid and chenodeoxycholec acid in CTX liver. As a consequence, neither 26-hydroxylated intermediates can be formed so that total primary bile acid synthesis would be diminished. Accordingly, the accumulation of 5P-cholestane-3a,7a,12a,25-tetrol arises from 25-hydroxylation of 5P-cholestane-3a,7a,12a-triol by the alternative microsomal 25-hydroxylation mechanism. 

The two-dimensional H- H chemical shift correlated (2D NMR, COSY-45) data for isomeric 5P-cholestane-3a,7a,12a, 25-tetrol and 5P-cholestane-3a,7P, 12a,25-tetrol were acquired at a sweep width of 3200 Hz using a standard pulse sequence (collection of 256 free induction decays, FlDs) as described previously (28-30). A transform size of 2K x 2K data points was obtained after zerofilling. 

Ichimiya, H., Yanagisawa, J. and Nakayama, F. (1984). Significance of bile alcohols in urine of a patient with cholestasis identification of Sp-cholestane-3a,7a,12a,26,27-pentol (5p-bufoI) and 5P-cholestane-3a,7a, 12a,26-tetrol (27-deoxy-5P-cyprinol). Chem. Pham. Bull. 32 2874-2877. 

Yashuara, M., Kuramoto, T. and Hoshita, T. (1978). Identification of 5P-cholestane-3a,7a,12a,23P-tetrol, 5P-cholestane-3a,7a,12a,24a-tetrol and 5P-cholestane-3a,7a,12a,24P-tetroI in cerebrotendinous xanthomatosis. Steroids. 31 333-345. 

Cholic acid has been converted into 5/S-cholestane-3a,7a,12a,25-tetrol and the 3a,7a,12a,24,25-pentols. Extension of the side-chain involved conversion of the acid via the diazo-ketone (361) into a homocholanic ester derivative (362). Grignard reaction then gave the 25-hydroxy-cholestane (363). Dehydration followed by hydroxylation (OSO4) afforded the 24,25-diols with a little of the 25,26-diol. ... 

Hydroxylation pathway. The mitochondrial fraction of both human and rat liver contains a 26-hydroxylase enzyme (B23), which can convert 5fi-cholestane-3a, 7a, 12a-triol (V) to 5ff-cholestane-3a,7o,12o,26-tetrol (VII) (Fig. 4). This tetrol is oxidized to 3a,7a,12a-trihydroxy-5p-cholestan-26-oic acid (THCA, VIII) by liver cytosol (C5). Further hydroxylation at C-24 forms varanic acid (IX) and its side chain is then shortened with oxidation at C-24 to yield cholic acid (X) (Fig. 4). 

Other clinical signs consist of progressive neurologic dysfunction, cataracts, and premature atherosclerosis (SI). The disease is inherited as an autosomal recessive trait, but is usually only detected in adults when cholesterol and cholestanol have accumulated over many years (S2). Biochemical features of the disease include striking elevations in tissue levels of cholesterol and cholestanol and the presence of unusual bile acids, termed bile alcohols, in bile. These bile alcohols are mainly 5 -cholestane-3a,7a,12a,24S, 25-pentol, Sp-diolestane-3a,7a,12a,23 ,25-pentol and 5P-du)lestane-3a,7a,12a,25-tetrol (S2). As chenodeoxycholic acid is deficient in the bile of patients with CTX, it was postulated that early bile salt precursors are diverted into the cholic acid pathway and 12a-hydroxy bile alcohols with an intact side chain accumulate because of impaired cleavage of the cholesterol side chain and decreased bile acid production (S2). HMG-CoA reductase and cholesterol 7a-hydroxylase activity are elevated in subjects with CTX (N4, N5), so that sufficient 7a-hydroxycholesterol should be available for bile acid synthesis. 

Salen, G., Shefer, S., Setoguchi, T., and Mosbach, E. H., Bile acid metabolism. Conversion of 5p-cholestane-3o,7a,12o,25-tetrol to cholic add. J. Clin. Invest. 56,226—231... 

H) Steroids (compounds reported —1) A tetrahydroxylated steroid isolated by the Schmitz group from specimens of P. disabethae collected in Puerto Rico was shown by spectral analyses and degradative studies to be 5a-cholestane-3/3,5,6/3,9-tetrol (106)/ ... 

IX) Conversion of 5 -cholestane-3a,7a,12a,26-tetrol into 3a,7a,12a-trihydroxy-5 -cholestanoic acid... 

Cholestane-3a,7a,12a-triol is efficiently 25-hydroxylated in the microsomal fraction of hver from both rat and man [40,41]. Shefer et al. [184] and Salen et al. [185] have shown that 5/8-cholestane-3a,7a,12a,25-tetrol is converted to S S-choles-tane-3a,7a,12a,24a,25-pentol, 5i8-cholestane-3a,7a,12a,24j8,25-pentol, 5j8-choles-tane-3 ,7a,12a,23,25-pentol and 5j8-cholestane-3 ,7a,12a,25,26-pentol in the presence of microsomes fortified with NADPH. In the presence of NAD", 5j8-choles-tane-3a,7a,12a,24, 25-pentol, but not the other 5j8-cholestanepentols formed, is efficiently converted to chohc add by soluble enzymes (Fig. 13). The latter conversion must be assumed to involve formation of acetone. These experiments demonstrate the existence of a new pathway for side-chain degradation in chohc acid synthesis which does not involve hydroxylation at C-26 or the participation of mitochondria. The relative importance of this pathway is a matter of controversy. Salen et al. suggested that this may be the major pathway for biosynthesis of chohc acid in man [185]. The finding that 5i8-cholestane-3a,7a,12a,25-tetrol is converted into chohc add in vivo in rat and man considerably less efficiently than 5j8-choles-tane-3a,7a,12a,26-tetrol does not support this contention [40,186]. 

Fig. 13. Possible mechanism for accumulation of 5 -cholestane-3o ,7a,12a,25-tetrol in patients with CTX.

This rare inherited hpid storage disease is characterized by xanthomas, progressive neurological dysfunction, cataracts and the development of xanthomatous lesions in the brain and lung. In contrast to other diseases with tendon xanthomatosis, plasma cholesterol levels are remarkably low. Large deposits of cholesterol and cholestanol are present in most tissues, and the concentration of cholestanol is 10-100 times higher than normal. Salen and collaborators have made extensive and elegant studies on the various metabolic aspects of this disease [184,185,187-192]. They have conclusively shown that there is a subnormal synthesis of bile acids and that the metabolic defect is an impaired oxidation of the cholesterol side chain. The synthesis of chenodeoxycholic acid is reduced more than that of cholic acid. These patients excrete considerable amounts of bile alcohol in bile and faeces. The bile alcohols have been identified as 5)S-cholestane-3a,7a,12a,25-tetrol, 5 8-cholestane-3a,7a,12a,24,25-pentol and 5/8-cholestane-3 ,7a,12a,23,25-pentol. Two different explanations for the accumulation of these bile alcohols have been presented. 

Salen et al. reported that liver microsomes from 2 patients with CTX had a decreased capacity to 24/8-hydroxylate 5)8-cholestane-3a,7a,12a,25-tetrol [185]. It was suggested that the basic metabolic defect is a relative deficiency of the 24j8-hy-droxylase. To explain the severe metabolic consequences of such a defect, it must be assumed that the 25-hydroxylase pathway is the major pathway in the biosynthesis of cholic acid. This hypothesis does not explain the marked reduction in the biosynthesis of chenodeoxycholic acid. In view of the very low activity of the microsomal 25-hydroxylase towards 5)3-cholestane-3a,7a-diol in human hver [41] it is evident that a 25-hydroxylase pathway cannot be of importance in the normal... 

Deoxy-5a-chimaerol 5a-Cholestane-3a,7a,12a,24-tetrol Some fishes... 

Deoxy-5a-cyprinol 5a-Cholestane-3o,7a,12a,26-tetrol Some fishes some frogs and toads... 

Deoxy-5/8-cyprinol 5/8-Cholestane-3o,7a, 12 a,26-tetrol Some frogs and toads... 

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