Synthesis and in vitro cholesterol dissolution by 23- and 24-phosphonobile acids

doi:10.1016/j.steroids.2005.03.008

Steroids

Volume 70, Issue 10, September 2005, Pages 681-689

https://doi.org/10.1016/j.steroids.2005.03.008 Get rights and content

Abstract

A new class of 23- and 24-phosphonobile acids have been synthesized from bile acid and their in vitro cholesterol-dissolving efficiency have been estimated. 24-Phosphonobile salts (PBSs) are slightly more efficient in solubilizing cholesterol than 23-PBSs and natural bile salts. The cholesterol solubilizing power is influenced by the structure of PBSs, and is considerably reduced with an increase in the bulk pH.

Introduction

Bile acids, which are essential for many physiological functions, are end-products of cholesterol metabolism. The physiological functions of bile salts result from their interesting physico-chemical properties, which have been studied extensively [1]. The glycine/taurine conjugates of bile acids form mixed micelles with phospholipids [2], [3], which solubilizes cholesterol. Impeding this function may cause the over saturation of cholesterol in the bile leading to its precipitation. The accumulation of cholesterol micro-crystals in the presence of glycoproteins leads to gallstone formation or cholelithiasis [4]. Chenodeoxy [5] and ursodeoxycholic acids [6] have been used with success for the dissolution of cholesterol gallstones. Two of the limitations of bile acid therapy are: (a) they undergo 7-dehydroxylation giving rise to hepatotoxic lithocholic acid and (b) low efficacy and prolonged treatment periods. There is growing interest in recent years to find a better gallstone-dissolving agent, which would be resistant to 7-dehydroxylation. The design of a few synthetic analogs of bile acids described in the literature has addressed this issue. N-methylated (sarcosine) glycocholate has been shown to possess therapeutic value, and is resistant to deconjugation and dehydroxylation [7]. The 7-methylated chenodeoxy and ursodeoxy derivatives were shown to be completely resistant to 7-dehydroxylation in hamsters [8]. Sulfonobile salts, side chain-modified analogs of natural bile salts, are resistant to 7-dehydroxylation, and do not interfere with endogenous bile acid synthesis [9], [10]. We have recently reported the synthesis of novel 23-phosphonobile acids [11]. In this paper, we report the synthesis of new 23- and 24-phosphonobile acids and a study of their in vitro cholesterol solubilizing efficiency as a model study for gallstone dissolution.

Section snippets

Materials and methods

Cholic, deoxycholic, chenodeoxycholic and lithocholic acids were purchased from Aldrich and were used as such. Ursodeoxycholic acid was also a commercial sample. Precoated silica gel glass plates with UV indicator on (0.25 mm thickness, Sigma–Aldrich) were used for thin layer chromatography and Liebermann–Burchard reagent was used as the spray reagent to visualize the steroids. Acme 100–200 mesh silica gel was used for gravity column chromatography. All the solvents used were of laboratory grade

Synthesis

The syntheses of 23-phosphonobile acids were accomplished as shown in Scheme 1. In addition, the one-carbon homologs were also synthesized starting from bile acids (Scheme 2). Cholic acid (CA), deoxycholic acid (DCA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA) and lithocholic acid (LCA) were converted to their corresponding performyl derivatives by heating with formic acid.

For the syntheses of 23-phosphonobile acids, formyl protected bile acids were subjected to a modified

Conclusions

We synthesized a new class of bile acid analogs. In vitro cholesterol solubilizing efficiency of 23- and 24-phosphonobile salts has been studied. The 24-PBSs solubilize cholesterol slightly better than 23-PBSs and bile salts. We believe that our preliminary results are encouraging to further explore a better medicinal therapy for cholesterol gallstone dissolution. The aggregation and gelation properties of these new compounds will be published elsewhere.

Acknowledgments

We thank the Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore for financial support of this work. Ponnusamy Babu thanks CSIR for fellowship.

References (18)

M.C. Carey
Physical-chemical properties of bile acids and their salts
B. Borgström et al.
Roles of bile acids in intestinal lipid digestion and adsorption
S. Kuroki et al.
Synthesis of potential cholelitholytic agents: 3α, 7α, 12α-trihydroxy-7β-methyl-5β-cholanic acid, 3α, 7β, 12α-trihydroxy-7α-methyl-5β-cholanic acid, 3α, 12α-dihydroxy-7ɛ-methyl-5β-cholanic acid
J Lipid Res
(1985)
T. Mikami et al.
Metabolism of sulfonate analogs of ursodeoxycholic acid and their effects on biliary bile acid composition in hamsters
J Lipid Res
(1993)
U. Maitra et al.
First Synthesis of phosphonobile acids and preliminary studies on their aggregation properties
Steroids
(2003)
F. Cortese et al.
Synthesis of conjugated bile acids. II. Glycodesoxycholic acid
J Biol Chem
(1936)
M.J. Armstrong et al.
The hydrophobic-hydrophilic balance of bile salts. Inverse correlation between reverse-phase high performance liquid chromatographic mobilities and micellar cholesterol-solubilizing capacities
J Lipid Res
(1982)
A.F. Hofmann
Bile acid science (cholanology) at the dawn of a new millennium: past progress and challenges for the future
M.C. Carey
Formation and growth of cholesterol gallstones: the new synthesis

There are more references available in the full text version of this article.

Cited by (18)

Haemolytic activity of formyl- and acetyl-derivatives of bile acids and their gramine salts
2017, Steroids
Citation Excerpt :
Bile acids and their derivatives are also attractive compounds for synthetic chemists because they have a large, rigid skeleton and possess chemically different polar hydroxyl groups. It is often necessary to protect this hydroxyl groups using, for example, acetate or formate as protecting moieties [8–10]. According to Tsemg at al. [11], the formyl groups on these compounds are quite stable to various reaction conditions.
Bile acids (lithocholic: LCA, deoxycholic: DCA and cholic: CA) and their formyl- and acetyl-derivatives can be used as starting material in chemical synthesis of compounds with different biological activity strongly depended on their chemical structures. Our previous studies showed that biological activity of bile acids salts with gramine toward human erythrocytes was significantly different from the activity of bile acids alone. Moreover, gramine effectively modified the membrane perturbing activity of other steroids. As a continuation of our work, the haemolytic activity of formyl- and acetyl-substituet bile acids as well as their gramine salts was studied in vitro. The structures of new compounds were confirmed by spectral (NMR, FT-IR) analysis, mass spectrometry (ESI-MS) as well as PM5 semiempirical methods. The results shown that the haemolytic activity of formyl- and acetyl-LCA and DCA was significantly higher in comparison with their native forms at the whole concentration range. At high concentration, formyl derivative of CA was as effective as LCA and DCA derivatives whereas at lower concentration its haemolytic activity was at the level of original acid. The acetyl-CA was not active as membrane perturbing agents. Furthermore, gramine significantly decreased the membrane-perturbing activity of hydrophobic bile acids derivatives. The results obtained with the cellular system are in line with physicochemical calculation.
Self gelating isoracemosol A, new racemosaceramide A, and racemosol E from Barringtonia racemosa
2017, Natural Product Research
Phytochemical investigation into the CHCl₃ extract of the fruits of Barringtonia racemosa resulted in the isolation of two new metabolites along with isoracemosol A and betulinic acid as known metabolites. The new compounds were characterised as phytosphingosine-type ceramide [(2S,3S,4R)-2-[(2R)-2-hydroxyhexadecanoyl amino]-hexacos-8(E)-ene-1,3,4-triol, 1] and racemosol E [21β-acetoxy-22α-(2-methylbutyroxy)-olean-12-ene-3β,16α,28-triol, 2] on the basis of extensive spectroscopic data analysis and chemical modifications. In addition, the self-gelating property of isoracemosol A (3) was investigated for the first time, which leads to the unexpected agglomerated porous-like morphology.
An efficient synthesis of 7α,12α-dihydroxy-4-cholesten-3-one and its biological precursor 7α-hydroxy-4-cholesten-3-one: Key intermediates in bile acid biosynthesis
2013, Steroids
Citation Excerpt :
The 19-H3 signal (singlet) in 1a was appreciably shifted downfield by 0.19 ppm and resonated at 1.19 ppm, compared to that in 10a (1.00 ppm), whereas a signal appearing at 5.82 ppm as a singlet was assigned to the 4-H. These observations indicated that 1a has the 3-oxo-Δ 4-7α-hydroxy structure in the A/B-ring juncture [15]. Furthermore, the occurrence of the two quaternary 13C signals at 198.7 and 167.4 ppm and the tertiary 13C signal at 126.9 ppm in the 13C-NMR spectrum of 1a also indicated the presence of the conjugated enone moiety.
This paper describes a method for the chemical synthesis of 7α,12α-dihydroxy-4-cholesten-3-one (1a) and its biological precursor, 7α-hydroxy-4-cholesten-3-one (1b), both of which are key intermediates in the major pathway of bile acid biosynthesis from cholesterol. The principal reactions involved were (1) building of the cholesterol (iso-octane) side chain by 3-carbon elongation of the cholane (iso-pentane) one, (2) oxidation sequence to transform the 3α-hydroxy group of the steroidal A/B-ring to the desired 4-en-3-one system, and (3) appropriate protection strategy for hydroxy groups in the positions at C-7 and C-12 in the steroid nucleus. The absolute structure of 1a and 1b were confirmed by NMR and X-ray crystallography. The targeted compounds 1a and 1b, prepared in 11 steps from 2a and 2b respectively, should be useful for biochemical studies of bile acid biosynthesis or clinical studies of bile acid metabolism, as plasma levels of 1b (also termed C4) have been shown to correlate highly with the rate of bile acid biosynthesis in man.
Synthesis, characterization and biological activity of hydroxyl- bisphosphonic analogs of bile acids
2012, European Journal of Medicinal Chemistry
Bisphosphonates (BPs) are now the most widely used drugs for diseases associated with increased bone resorption, such as osteoporosis, and tumor bone diseases. A significant drawback of the BPs is their poor oral absorption that is enhanced by the presence of bile acid substituents in the bisphosphonate framework, with no toxic effects. A straightforward synthesis of bile acid-containing hydroxy-bisphosphonates and a full characterization of these pharmaceutically important molecules, including an evaluation of affinity and the mechanism of binding to hydroxyapatite, is presented. The biological activity of bile acid-containing bisphosphonate salts was determined using the neutral-red assay on the L929 cell line and primary cultures of osteoclasts. The bioactivity of the new compounds was found superior than bisphosphonates of established activity.
Chemical synthesis of the (25R)- and (25S)-epimers of 3α,7α, 12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding glycine and taurine conjugates
2011, Chemistry and Physics of Lipids
Citation Excerpt :
Our attempt to apply the reduction of 11 with LiAlH4 was discouraging; low yields of the desired 12 were obtained after a laborious chromatographic purification process, due to the concurrent formation of partial hydrolysis products at positions C-3, C-7 and/or C-12. However, a more mild reduction of 11 using the less basic NaBH4, in the presence of triethylamine and ethyl chloroformate (Babu and Maitra, 2005) in methanol at room temperature, proceeded smoothly to give the 3,7,12-triformyl-24-ol 12 in good yield (84%), without the problems of partial hydrolysis of protecting groups. Alkylation of alkyl halides with malonic diesters is a promising way to extend steroid side chains (Starchenkov et al., 2000; Komatsubara, 1954; Hayatsu, 1957).
The (25R)- and (25S)-epimers of C₂₇ 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid as well as their corresponding N-acylamidate conjugates with glycine or taurine were prepared starting from cholic acid in 14 steps. The principal reactions involved were (1) reduction of a key intermediary C₂₄ allo-cholic acid performate with NaBH₄/triethylamine/ethyl chloroformate, (2) iodination of the resulting 3,7,12-triformyloxy-5α-cholan-24-ol with I₂/triphenylphosphine; (3) nucleophilic substitution of the iodo derivative with diethylmethyl malonate/NaH; and (4) hydrolysis of the resulting 3,7,12-triformyloxy-25-methyl-26,27-diethyl ester with KOH, followed by decarboxylation of the geminal dicarboxylic acid with LiCl. N-Acylamidation of the resulting (25R)/(25S)-3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid mixture with glycine or taurine afforded the corresponding epimeric mixtures of the glycine and taurine conjugates. The (25R)- and (25S)-epimers of the three variants of unconjugated and conjugated 3α,7α,12α-trihydroxy-5α-cholestan-27-oic acid were efficiently separated by HPLC on a reversed-phase C₁₈ column and their structural characteristics, particularly the chiral center at C-25, delineated using ¹H and ¹³C NMR. These synthetic compounds should be useful as authentic reference standards for establishing their presence in bile as well as being useful in studies on the biosynthesis of allo-bile acids from cholesterol.
Synthesis and olfactory activity of unnatural, sulfated 5β-bile acid derivatives in the sea lamprey (Petromyzon marinus)
2011, Steroids
Citation Excerpt :
The analogous compounds 9b and 9c, derived from deoxycholic acid (10b) and chenodeoxycholic acid (10c) respectively, were synthesized using a very similar protocol. Lithocholic acid (10d) was converted to the primary alcohol 12d using a one-flask/two-reaction sequence (Scheme 2), presumably via the THP-ester 11d [19]. The primary alcohol 12d was sulfated (SO3·Py, Et3N) to give the THP-ether 13d, which was converted to the secondary alcohol 9d (pPTS, EtOH).
A variety of unnatural bile acid derivatives (9a–9f) was synthesized and used to examine the specificity with which the sea lamprey (Petromyzon marinus) olfactory system detects these compounds. These compounds are analogs of petromyzonol sulfate (PS, 1), a component of the sea lamprey migratory pheromone. Both the stereochemical configuration at C5 (i.e., 5α vs. 5β) and the extent and sites of oxygenation (hydroxylation or ketonization) of the bile acid derived steroid skeleton were evaluated by screening the compounds for olfactory activity using electro-olfactogram recording. 5β-Petromyzonol sulfate (9a) elicited a considerable olfactory response at sub-nanomolar concentration. In addition, less oxygenated systems (i.e., 9b–9e) elicited olfactory responses, albeit with less potency. The sea lamprey sex pheromone mimic 9f (5β-3-ketopetromyzonol sulfate) was also examined and found to produce a much lower olfactory response. Mixture studies conducted with 9a and PS (1) suggest that stimulation is occurring via similar modes of activation, demonstrating a relative lack of specificity for recognition of the allo-configuration (i.e., 5α) in sea lamprey olfaction. This attribute could facilitate design of pheromone analogs to control this invasive species.

View all citing articles on Scopus

View full text

Synthesis and in vitro cholesterol dissolution by 23- and 24-phosphonobile acids

Abstract

Introduction

Section snippets

Materials and methods

Synthesis

Conclusions

Acknowledgments

J Lipid Res

J Lipid Res

Steroids

J Biol Chem

J Lipid Res

Bile acid science (cholanology) at the dawn of a new millennium: past progress and challenges for the future

Formation and growth of cholesterol gallstones: the new synthesis