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

Article abstract of DOI:10.1016/j.steroids.2005.03.008

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.

Full text of DOI:10.1016/j.steroids.2005.03.008

Steroids 70 (2005) 681–689
Synthesis and in vitro cholesterol dissolution by 23- and
24-phosphonobile acids
Ponnusamy Babu, Uday Maitra∗
Department of Organic Chemistry, Indian Institute of Science, Bangalore 560012, India
Received 2 December 2004; received in revised form 23 February 2005; accepted 7 March 2005
Available online 29 April 2005
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.
© 2005 Elsevier Inc. All rights reserved.
Keywords: Bile acids; Cholesterol dissolution; Gallstone dissolution; Phosphonobile acids
1. Introduction
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.
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.
2. Experimental
Abbreviations: PBS, phosphonobile salt; PC, phosphonocholate; PCDC,
phosphonochenodeoxycholate; PDC, phosphonodeoxycholate; PLC, phos-
phonolithocholate; PUDC, phosphonoursodeoxycholate
Correspondingauthorat:ChemicalBiologyUnit, JawaharlalNehruCen-
tre for Advanced Scientific Research, Bangalore 560012, India. Tel.: +91 80
2360 1968; fax: +91 80 2360 0529.
2.1. Materials and methods
Cholic, deoxycholic, chenodeoxycholic and lithocholic
acidswerepurchasedfromAldrichandwereusedassuch. Ur-
sodeoxycholic acid was also a commercial sample. Precoated
silica gel glass plates with UV indicator on (0.25 mm thick-
∗
E-mail address: maitra@orgchem.iisc.ernet.in (U. Maitra).
0039-128X/$ – see front matter © 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2005.03.008

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P. Babu, U. Maitra / Steroids 70 (2005) 681–689
ness, Sigma–Aldrich) were used for thin layer chromatogra-
phy 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 sol-
vents used were of laboratory grade and distilled prior to use.
Dichloromethane and carbon tetrachloride were dried over
CaH2. Melting points were recorded in Buchi B-540 melting
point instrument. All optical rotation values were recorded
on Jasco-DIP-370 polarimeter at 25 ◦C. Infrared spectra were
recorded ona Jasco-70 FTIRspectrometer. Protonandcarbon
NMR spectra were recorded on a Jeol Lambda 300 MHz and
Bruker AMX 400 MHz spectrometers. Phosphorous NMR
was recorded on a Bruker AMX 400 MHz spectrometer.
(100 MHz, CDCl3) δ: 179.90, 161.00, 160.61, 73.69, 73.44,
53.37, 55.04, 43.68, 42.11, 39.91, 39.50, 35.17, 34.42, 34.00,
32.92, 32.82, 31.03, 30.76, 28.30, 26.42, 25.84, 23.20, 21.26,
18.36, 12.09.
2.1.1.5. 3α-Formyloxy-5β-cholan-24-oic acid. Yield 95%;
mp: 136–138 ◦C; H NMR (300 MHz, CDCl3) δ: 8.03 (s,
1
1H), 4.88–4.81 (m, 1H), 2.44–2.19 (m, 2H), 1.98–0.99 (m),
0.93 (s, 3H), 0.92 (d, J = 6.6Hz, 3H), 0.64 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ: 180.29, 160.84, 74.42, 56.43, 55.92,
42.72, 41.88, 40.42, 40.09, 35.76, 35.28, 34.94, 34.55, 32.18,
30.98, 30.73, 28.14, 26.95, 26.61, 26.28, 24.15, 23.29, 20.82,
18.22, 12.03.
2.1.1. A general procedure for the synthesis of
formylated bile acids
2.1.2. General procedure for the synthesis of 24-hydroxy
bile alcohols
Formic acid (20 mL, 517.4 mmol) was added to bile acid
(12.7 mmol) and stirred at 55 ◦C for 20 h. Volatiles were re-
moved under reduced pressure. The free flowing solid was
crystallized from EtOH/H2O (1:1.3, v/v) and dried to give
formylated bile acids [12].
The formylated bile acid (2.26 mmol) was dissolved
in THF (8 mL). To this solution, triethylamine (0.4 mL,
2.9 mmol) was added, followed by the dropwise addition of
ethyl chloroformate (0.28 mL, 2.92 mmol). After stirring for
2 h, the reaction mixture was cooled with ice bath then added
NaBH4 (0.4 g) and methanol (10 mL) dropwise. After stir-
ring at 0 ◦C for 15 min, the reaction mixture was stirred at rt
for another 15 min, diluted with water (20 mL), acidified with
0.2 M HCl (10 mL), extracted with ethyl acetate (3 × 25 mL),
washed with water and dried over anhyd. Na2SO4. The crude-
product was purified on a silica gel column using EtOAc/pet.
ether (35–50:65–50, v/v).
2.1.1.1. 3α, 7α, 12α-triformyloxy-5β-cholan-24-oic acid.
Yield 82%; mp: 202–204 ◦C; 1H NMR (300 MHz, CDCl3) δ:
8.165 (s, 1H), 8.11 (s, 1H), 8.02 (s, 1H), 5.27 (bs, 1H), 5.07
(bs, 1H), 4.76 (m, 1H), 2.44–1.27 (m), 1.18–1.05 (m, 2H),
0.94 (s, 3H), 0.85 (d, J = 6.6 Hz, 3H), 0.76 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ: 179.78, 160.59, 160.50, 75.24, 73.72,
70.66, 47.18, 44.99, 42.94, 40.76, 37.69, 34.68, 34.49, 34.41,
34.25, 31.30, 30.80, 30.37, 28.52, 27.12, 26.54, 25.51, 22.74,
22.31, 17.42, 12.12.
2.1.2.1. 3α, 7α, 12α-triformyloxy-5β-24-hydroxy-cholane.
Yield 77%; 1H NMR (300 MHz, CDCl3) δ: 8.17 (s, 1H), 8.10
(s, 1H), 8.02 (s, 1H), 5.27 (s, 1H), 5.07 (s, 1H), 4.71 (m, 1H),
3.61 (m, 2H), 2.18–1.0 (m), 0.94 (s, 3H), 0.85 (d, J = 6.3 Hz,
3H), 0.75 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 160.53,
75.44, 73.79, 70.76, 63.37, 47.46, 45.05, 43.03, 40.90, 37.84,
35.04, 34.58, 34.34, 31.65, 31.41, 29.33, 28.63, 27.30, 26.64,
25.61, 22.83, 22.35, 17.87, 12.62.
2.1.1.2. 3α, 12α-diformyloxy-5β-cholan-24-oic acid. Yield
89%; mp: 189–192 ◦C; 1H NMR (300 MHz, CDCl3) δ: 8.11
(s, 1H), 8.01 (s, 1H), 5.23 (s, 1H), 4.81 (m, 1H), 2.36 (m, 1H),
1.92–0.97 (m), 0.90 (s, 3H), 0.82 (d, J = 6.0 Hz, 3H), 0.73 (s,
3H); 13C NMR (75 MHz, CDCl3) δ: 180.18, 160.67, 160.53,
75.91, 74.04, 49.18, 47.28, 44.93, 41.65, 35.52, 34.70, 34.58,
34.12, 33.94, 32.01, 30.89, 30.40, 27.27, 26.70, 26.39, 25.83,
25.66, 25.36, 22.86, 17.36, 12.26.
2.1.2.2. 3α, 12α-diformyloxy-5β-24-hydroxy-cholane. Yi-
eld 92%; 1H NMR (300 MHz, CDCl3) δ: 8.13 (s, 1H), 8.02
(s, 1H), 5.25 (s, 1H), 4.87–4.79 (m, 1H), 3.60 (t, J = 6.3 Hz,
2H), 1.9–1.0 (m), 0.92 (s, 3H), 0.83 (d, J = 5.7 Hz, 3H), 0.74
(s, 3H); 13C NMR (100 MHz, CDCl3) δ: 160.60, 160.48,
75.94, 73.96, 62.79, 49.18, 47.51, 44.90, 41.67, 35.54, 34.96,
34.61, 34.13, 33.91, 32.05, 31.66, 29.12, 27.34, 26.74, 26.37,
25.851, 25.64, 23.38, 22.81, 17.73, 12.22.
2.1.1.3. 3α, 7α-diformyloxy-5β-cholan-24-oic acid. Yield
95%; mp: 184–185 ◦C; 1H NMR (400 MHz, CDCl3) δ: 8.06
(s, 1H), 8.01 (s, 1H), 5.02 (s, 1H), 4.74–4.69 (m, 1H),
2.41–2.33 (m, 1H), 2.28–2.10 (m, 1H), 2.0–1.1 (m), 0.93
(s, 3H), 0.92 (d, J = 6.0 Hz, 3H), 0.64 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ: 179.93, 160.73, 74.07, 71.42, 55.78,
50.14, 42.75, 41.04, 39.46, 37.96, 35.24, 34.81, 34.62, 34.03,
31.51, 30.96, 30.73, 27.93, 26.77, 23.49, 22.64, 20.65, 18.27,
11.74.
2.1.2.3. 3α, 7α-diformyloxy-5β-24-hydroxy-cholane. Yield
1
66%; H NMR (300 MHz, CDCl3) δ: 8.08 (s, 1H), 8.03 (s,
1H), 5.03 (s, 1H), 4.76–4.69 (m, 1H), 3.63–3.59 (m, 2H),
2.1–1.0 (m), 0.96 (s, 3H), 0.96–0.92 (unresolved d, 3H), 0.65
(s, 3H);13CNMR(75 MHz, CDCl3)δ:160.73, 160.70, 73.98,
71.35, 63.39, 55.82, 55.02, 42.56, 40.88, 39.34, 37.79, 35.44,
34.69, 34.48, 33.89, 31.69, 31.39, 29.20, 28.00, 26.64, 23.42,
22.57, 20.53, 1.55, 11.64.
2.1.1.4. 3α, 7β-diformyloxy-5β-cholan-24-oic acid. Yield
96%; mp: 170–171 ◦C; 1H NMR (400 MHz, CDCl3) δ: 8.00
(s, 1H), 7.96 (s, 1H), 4.92–4.85 (m, 1H), 4.83–4.75 (m, 1H),
2.41–2.33 (m, 1H), 2.27–2.19 (m, 1H), 1.86–1.0 (m), 0.97
(s, 3H), 0.91 (d, J = 6.4 Hz, 3H), 0.67 (s, 3H); 13C NMR

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683
2.1.2.4. 3α, 7β-diformyloxy-5β-24-hydroxy-cholane. Yield
82%; H NMR (300 MHz, CDCl3) δ: 8.03 (s 1H), 7.99 (s,
2.1.3.3. 3α, 7α-diformyloxy-5β-23-iodo-24-nor-cholane:.
Yield 99%; H NMR (300 MHz, CDCl3) δ: 8.07 (s, 1H),
1
1
1H), 4.94–4.79 (m, 2H), 3.60 (t, J = 6.6 Hz, 2H), 1.88–1.04
(m), 0.99 (s, 3H), 0.93 (d, J = 6.3 Hz, 3H), 0.69 (s, 3H);
13C NMR (75 MHz, CDCl3) δ: 161.01, 160.61, 73.60, 73.35,
63.33, 55.25, 55.06, 43.48, 41.94, 39.78, 39.73, 39.34, 35.35,
34.27, 33.85, 32.78, 32.65, 31.73, 29.22, 28.36, 26.27, 25.76,
23.11, 21.13, 18.63, 11.96.
8.02 (s, 1H), 5.03 (s, 1H), 4.76–4.68 (m, 1H), 3.32–3.27
(m, 1H), 3.12–3.06 (m, 1H), 2.18–1.01 (m), 0.95 (s, 3H),
0.93 (d, J = 6.0 Hz, 3H), 0.67 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ: 160.66, 74.05, 71.38, 55.68, 50.15, 42.84, 41.03,
40.25, 39.46, 37.95, 37.09, 34.83, 34.63, 34.05, 31.51,
27.98, 26.79, 23.49, 22.67, 20.66, 17.87, 11.76, 4.83.
2.1.3.4. 3α, 7β-diformyloxy-5β-23-iodo-24-nor-cholane.
Yield 91%; 1H NMR (400 MHz, CDCl3) δ: 8.02 (s, 1H), 7.98
(s, 1H), 4.93–4.86 (m, 1H), 4.85–4.77 (m, 1H), 3.33–3.27
(m, 1H), 3.12–3.06 (m, 1H), 2.03–1.98 (m, 2H), 1.87–1.07
2.1.2.5. 3α-Formyloxy-5β-24-hydroxy-cholane. Yield
70%; 1H NMR (300 MHz, CDCl3) δ: 8.04 (s, 1H), 4.89–4.80
(m, 1H), 3.61 (t, J = 5.1 Hz, 2H), 2.0–0.98 (m), 0.94 (s, 3H),
0.93 (d, J = 6.6 Hz, 3H), 0.65 (s, 3H); 13C NMR (75 MHz,
CDCl3) δ: 160.82, 74.42, 63.58, 56.47, 56.16, 42.69, 41.90,
40.44, 40.12, 35.76, 35.55, 34.94, 34.56, 32.19, 31.82,
29.38, 28.28, 26.98, 26.62, 26.31, 24.18, 23.31, 20.83,
18.62, 12.03.
(m), 0.99 (s, 3H), 0.92 (d, J = 6.4 Hz, 3H), 0.70 (s, 3H); 13
C
NMR (100 MHz, CDCl3) δ: 160.92, 160.53, 73.62, 73.42,
55.38, 54.96, 43.78, 42.12, 40.25, 39.92, 39.52, 36.97,
34.44, 34.01, 32.93, 32.85, 28.31, 26.43, 25.82, 23.22,
21.25, 17.97, 12.01, 5.1.
2.1.3. General procedure for the preparation of
formyl-23-iodo-24-nor-cholanes
2.1.3.5. 3α-Formyloxy-5β-23-iodo-24-nor-cholane. Yield
76%; 1H NMR (300 MHz, CDCl3) δ: 8.02 (s, 1H), 4.87–4.80
(m, 1H), 3.32–3.25 (m, 1H), 3.12–3.04 (m, 1H), 2.06–1.05
(m), 0.92 (s, 3H), 0.90 (d, J = 6.0 Hz, 3H), 0.65 (s, 1H);
13C NMR (75 MHz, CDCl3) δ: 160.73, 74.33, 56.41, 55.82,
42.79, 41.84, 40.39, 40.28, 40.07, 37.15, 35.72, 34.91,
34.54, 32.17, 28.15, 26.93, 26.60, 26.26, 24.11, 23.30,
20.79, 17.81, 12.03, 5.16.
In a 100 mL two-necked rb flask fitted with a reflux
condenser and a pressure equalizing dropping funnel,
formylated bile acid (5.36 mmol) and lead tetraacetate (5.0 g,
11.4 mmol) were suspended in CCl4 (15 mL). The mixture
was refluxed and irradiated with two 100 W tungsten lamps.
After 5 min, I2 (2.7 g, 10.6 mmol) in CCl4 (60 mL) was added
through the dropping funnel until violet color persisted.
Heating and irradiation was continued for an additional 1 h.
The reaction mixture was filtered through celite, washed
with 10% sodium thiosulphate solution (50 mL), water
(20 mL) and brine (25 mL), dried over anhyd. Na2SO4 and
concentrated. The crude-product was purified by column
chromatography on silica gel (2.8 cm × 19.5 cm) using
EtOAc/CHCl3 (2–5:98–95, v/v) to yield 23-iodo deriva-
tives.
2.1.4. General procedure for the synthesis of formyl
24-iodo cholanes
To a solution of PPh3 (0.59 g, 2.3 mmol) and imi-
dazole (0.16 g, 2.3 mmol) in dichloromethane (10 mL) at
0 ◦C, iodine (0.58 g, 2.3 mmol) was added in portions
over 20 min. The formylated cholanol (0.72 g, 1.5 mmol)
in dichloromethane (5 mL) was added and warmed to rt.
After 36 h, the reaction mixture was diluted with CHCl3
(30 mL), washed with 10% aq. sodium thiosulphate solu-
tion (30 mL), and water (20 mL) dried over anhyd. Na2SO4.
The crude-product was purified on silica gel column using
EtOAc/CHCl3 (2–5:98–95, v/v).
2.1.3.1. 3α, 7α, 12α-triformyloxy-5β-23-iodo-24-nor-
cholane. Yield 60%; 1H NMR (300 MHz, CDCl3) δ: 8.16 (s,
1H), 8.10 (s, 1H), 8.02 (s, 1H), 5.28 (bs, 1H), 5.07 (bs, 1H),
4.76–4.68 (m, 1H), 3.33–3.26 (m, 1H), 3.07 (dd, J = 8.4 Hz,
J = 7.8 Hz, 1H), 2.20–1.24 (m, 23H), 1.194–1.090 (m, 2H),
0.952 (s, 3H), 0.844 (d, J = 6.0 Hz, 3H), 0.782 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ: 160.49, 160.47, 160.39, 75.13,
73.63, 70.57, 47.02, 45.05, 42.88, 40.72, 39.74, 37.64,
36.42, 34.45, 34.38, 34.22, 31.27, 28.49, 27.13, 26.52,
25.49, 22.29, 16.94, 12.10, 4.69.
2.1.4.1. 3α, 7α, 12α-triformyloxy-5β-24-iodo-cholane.
1
Yield 95%; H NMR (400 MHz, CDCl3) δ: 8.16 (s, 1H),
8.10 (s, 1H), 8.02 (s, 1H), 5.26 (s, 1H), 5.08 (s, 1H),
4.75–4.68 (m, 1H), 3.21–3.08 (m, 2H), 2.18–1.05 (m), 0.95
(s, 3H), 0.85 (d, J = 6.4 Hz, 3H), 0.76 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ: 160.49, 75.35, 73.76, 70.21, 47.36,
45.08, 43.02, 40.90, 37.84, 36.59, 34.53, 34.34, 31.41, 30.20,
28.63, 27.29, 26.64, 25.60, 22.82, 22.35, 17.97, 12.16, 7.32.
2.1.3.2. 3α, 12α-diformyloxy-5β-23-iodo-24-nor-cholane.
1
Yield 79%; H NMR (300 MHz, CDCl3) δ: 8.12 (s, 1H),
8.03 (s, 1H), 5.25 (s, 1H), 4.83 (m, 1H), 3.30 (m, 1H), 3.05
(m, 2H), 2.05–0.95 (m), 0.92 (s, 3H), 0.83 (d, J = 6.3 Hz, 3H),
0.76 (s, 3H); 13C NMR (75 MHz, CDCl3) δ: 160.61, 160.47,
75.91, 74.04, 49.23, 47.20, 45.06, 41.68, 39.91, 36.55, 35.56,
34.63, 33.99, 32.06, 27.36, 26.73, 26.44, 25.86, 25.70, 23.37,
22.91, 16.97, 12.31, 4.85.
2.1.4.2. 3α, 12α-diformyloxy-5β-24-iodo-cholane. Yield
73%; H NMR (300 MHz, CDCl3) δ: 8.14 (s, 1H), 8.03 (s,
1H), 5.25 (s, 1H), 4.87–4.80 (m, 1H), 3.23–3.08 (m, 2H),
1.90–1.03 (m), 0.93 (s, 3H), 0.84 (d, J = 6.3 Hz, 3H), 0.75 (s,
3H); 13C NMR (100 MHz, CDCl3) δ: 160.57, 160.48, 76.04,
74.10, 49.31, 47.54, 45.08, 42.09, 36.64, 35.69, 34.74,
1

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P. Babu, U. Maitra / Steroids 70 (2005) 681–689
34.59, 34.29, 34.08, 32.19, 30.29, 27.50, 26.85, 26.54,
25.96, 25.80, 23.50, 22.96, 12.38, 7.50.
3.735 (d, J = 9.0 Hz, 6H), 1.9–1.0 (m, 26 H), 0.93 (s, 3H), 0.84
(d, J = 6.6 Hz, 3H), 0.75 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ: 160.45, 160.33, 75.69, 73.81, 61.27 (d, J = 7 Hz), 49.03,
46.76, 44.79, 44.54, 35.41, 35.32 (d, J = 14 Hz), 34.49, 34.00,
33.83, 31.90, 27.78, 27.72, 27.17, 26.60, 26.28, 25.72, 25.54,
23.24, 22.15, 21.70 (d, J = 135 Hz), 17.05, 16.30 (d, J = 6 Hz),
12.19; 31P NMR (162 MHz, CDCl3) δ: 32.81; MALDI-TOF
MS: calc. for M + Na+, M + K+: 564.0, 579.9; obsd. 563.7,
579.8.
2.1.4.3. 3α,
7α-diformyloxy-5β-24-iodo-cholane. Yield
1
87%; H NMR (300 MHz, CDCl3) δ: 8.08 (s, 1H), 8.03 (s,
1H), 5.03 (s, 1H), 4.77–4.69 (m, 1H), 3.23–3.08 (m, 2H),
2.1–1.0 (m), 0.95 (s, 3H), 0.93 (d, J = 6.6 Hz), 0.66 (s, 3H);
13C NMR (75 MHz, CDCl3) δ: 160.70, 73.99, 71.32, 55.70,
50.06, 42.62, 40.91, 39.35, 37.82, 36.75, 34.97, 34.72,
34.51, 33.92, 31.42, 30.22, 28.05, 26.68, 23.46, 22.62,
20.54, 18.67, 11.68, 7.78.
2.1.5.3. Diethyl 3α, 7α-diformyloxy-24-nor-5β-cholane-23-
1
phosphonate. Yield 75%; H NMR (300 MHz, CDCl3) δ:
2.1.4.4. 3α,
7β-diformyloxy-5β-24-iodo-cholane. Yield
8.09 (s, 1H), 8.03 (s, 1H), 5.03 (s, 1H), 4.72 (m, 1H),
4.14–4.02 (m, 4H), 2.18–1.00 (m), 0.95 (bs, 6H), 0.68 (s, 3H);
13C NMR (100 MHz, CDCl3) δ: 160.56, 73.87, 71.18, 61.39
(d, J = 5.0 Hz), 55.18, 50.00, 42.59, 40.91, 39.31, 37.83, 35.83
(d, J = 15.9 Hz), 34.70, 34.51, 33.90, 31.40, 27.96, 27.78,
26.67, 23.37, 22.55, 21.89 (d, J = 133.5 Hz), 20.53, 17.98,
16.38 (d, J = 4.3 Hz), 11.66; 31P NMR (162 MHz, CDCl3) δ:
32.84.
1
86%; H NMR (300 MHz, CDCl3) δ: 8.03 (s, 1H), 7.98 (s,
1H), 4.94–4.79 (m, 2H), 3.23–3.08 (m, 2H), 2.0–1.0 (m),
0.99 (s, 3H), 0.92 (d, J = 6.6 Hz), 0.69 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ: 160.86, 160.48, 73.45, 73.23, 55.17,
54.84, 43.46, 41.88, 39.68, 39.28, 36.65, 34.79, 34.23,
33.80, 32.73, 32.62, 30.18, 28.34, 26.24, 25.71, 23.09,
21.09, 18.67, 11.94, 7.70.
2.1.4.5. 3α-Formyloxy-5β-24-iodo-cholane. Yield 94%; 1H
NMR (300 MHz, CDCl3) δ: 8.04 (s, 1H), 4.9–4.8 (m, 1H),
3.24–3.1 (m, 2H), 2.0–1.0 (m), 0.94 (s, 3H), 0.92 (d,
J = 6.6 Hz, 3H), 0.65 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ: 160.80, 74.40, 56.46, 56.03, 42.71, 41.90, 40.44, 40.09,
36.83, 35.76, 35.07, 34.94, 34.56, 32.19, 30.35, 28.28, 26.96,
26.62, 26.31, 24.18, 23.31, 20.83,18.71, 12.03, 7.91.
2.1.5.4. Diethyl 3α, 7β-diformyloxy-24-nor-5β-cholane-23-
phosphonate. Yield 88%; H NMR (300 MHz, CDCl3) δ:
1
8.02 (s, 1H), 7.97 (s, 1H), 4.94–4.80 (m, 2H), 4.12–4.05
(m, 4H), 1.87–1.02 (m), 0.98 (s, 3H), 0.91 (d, J = 6.0 Hz,
3H), 0.68 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 160.88,
160.51, 73.53, 73.33, 61.49 (d, J = 5.3 Hz), 55.27, 54.50,
43.56, 42.02, 39.82, 39.40, 35.83 (d, J = 16.1 Hz), 34.35,
33.91, 32.85, 32.75, 28.18, 28.02, 26.35, 25.76, 23.13, 22.03
(d, J = 139.7 Hz), 21.18, 18.09, 16.39 (d, J = 4.2 Hz), 12.04;
31P NMR (162 MHz, CDCl3) δ: 32.92; LRMS-ESI: calc. for
M + Na+: 563.3; obsd. 563.7.
2.1.5. General procedure for the synthesis of diethyl 23-
and 24-phosphonocholanates
In a 50 mL rb flask fitted with a reflux condenser, formy-
lated iodocholane (1.9 mmol) in P(OEt)3 (1.6 mL, 9.5 mmol)
was heated at 160 ◦C under nitrogen. After 6 h, excess
P(OEt)3 and other volatiles were removed under vacuum.
The product was purified by column chromatography on sil-
ica gel using EtOAc/CHCl3 (30:70, v/v), yielding phospho-
nocholanate a viscous liquid.
2.1.5.5. Diethyl
3α-formyloxy-24-nor-5β-cholane-23-
phosphonate. Yield 75%; 1H NMR (300 MHz, CDCl3)
δ: 8.00 (s, 1H), 4.81–4.78 (m, 1H), 4.11–3.96 (m, 4H),
1.94–1.04 (m), 0.90 (s, 3H), 0.88 (d, J = 9.0 Hz, 3H), 0.61 (s,
3H); 13C NMR (75 MHz, CDCl3) δ: 160.89, 74.49, 61.58 (d,
J = 7 Hz), 56.53, 55.61, 42.81, 41.00, 40.53, 40.18, 36.15 (d,
J = 17 Hz), 35.88, 35.15, 34.67, 32.30, 28.17, 28.13, 27.07,
26.72, 26.39, 24.25, 23.41, 22.17 (d, J = 139 Hz), 20.92,
18.13, 16.57 (d, J = 6.2 Hz), 12.17; 31P NMR (162 MHz,
CDCl3) δ: 33.35; MALDI-TOF MS: calc. for M + Na+:
519.3; obsd. 519.7.
2.1.5.1. Diethyl 3α, 7α, 12α-triformyloxy-24-nor-5β-
1
cholane-23-phosphonate. Yield 71%; H NMR (300 MHz,
CDCl3) δ: 8.12 (s, 1H), 8.06 (s, 1H), 7.98 (s, 1H), 5.22
(s, 1H), 5.03 (s, 1H), 4.71–4.63 (m, 1H), 4.08–4.00 (m,
4H), 2.15–1.39 (m), 1.33–1.24 (m, 6H), 1.21–1.0 (m), 0.90
(s, 3H), 0.80 (d, J = 6.3 Hz, 3H), 0.71 (s, 3H); 13C NMR
(75 MHz, CDCl3) δ: 160.46, 160.38, 75.06, 73.58, 61.45
(d, J = 6 Hz), 46.63, 44.84, 42.81, 40.65, 37.57, 35.27 (d,
J = 17 Hz), 34.38, 34.30, 34.14, 31.19, 28.40, 27.73, 26.92,
26.43, 25.40, 22.61, 22.19, 21.80 (d, J = 140 Hz), 17.11,
16.33 (d, J = 5 Hz), 12.02; 31P NMR (162 MHz, CDCl3) δ:
32.74; MALDI-TOF MS: calc. for M + Na+, M + K+: 607.6,
623.8; obsd. 607.7, 623.8.
2.1.5.6. Diethyl 3α, 7α, 12α-triformyloxy-5β-cholane-24-
1
phosphonate. Yield 97%; H NMR (300 MHz, CDCl3) δ:
8.01 (s, 1H), 7.96 (s, 1H), 7.87 (s, 1H), 5.11 (s, 1H), 4.92
(s, 1H), 4.56 (m, 1H), 3.95–3.91 (m, 4H), 2.04–0.91 (m),
0.80 (s, 3H), 0.70 (d, J = 6.4 Hz, 3H), 0.61 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ: 160.44, 75.24, 73.64, 70.58,
61.27 (d, J = 5.8 Hz), 47.22, 44.94, 42.88, 40.76, 37.69, 36.59
(d, J = 15.9 Hz), 34.82, 34.47, 34.41, 34.21, 31.29, 28.48,
27.17, 26.52, 25.91 (d, J = 139.7 Hz), 25.47, 22.69, 22.24,
18.99, 18.94, 17.63, 16.37 (d, J = 4.9 Hz), 12.03; 31P NMR
2.1.5.2. Diethyl 3α, 12α-diformyloxy-24-nor-5β-cholane-
23-phosphonate. Yield 80%; 1H NMR (300 MHz, CDCl3) δ:
8.13 (s, 1H), 8.04 (s, 1H), 5.24 (bs, 1H), 4.87–4.80 (m, 1H),

P. Babu, U. Maitra / Steroids 70 (2005) 681–689
685
(162 MHz, CDCl3) δ: 31.69; LRMS-ESI: calc. for M + Na+,
M + K+: 621.3, 637.3; obsd. 621, 637.
2.1.6. General procedure for the synthesis of 23- and
24-phsophonobile acids
Diethyl phosphonate ester (0.26 g, 0.44 mmol) was dis-
solved in dry dichloromethane (3 mL) under nitrogen and
cooled in an ice-water bath. Iodotrimethylsilane (0.14 mL,
0.98 mmol) was added dropwise, while maintaining the tem-
perature 0–5 ◦C, and then warmed to room temperature. After
24 h, the reaction mixture was evaporated in vacuo and the
brownish residue was stirred with aq. NaOH solution (4 mL
of 0.8 M, 3.2 mmol). After 4 h, the basic solution was acidi-
fied with conc. HCl and the precipitated pale yellow product
was crystallized from MeOH/acetone (1:2) to yield phospho-
nobile acid as a white solid.
2.1.5.7. Diethyl 3α, 12α-diformyloxy-5β-cholane-24-phos-
phonate. Yield 83%; 1H NMR (400 MHz, CDCl3) δ:
8.13 (s, 1H), 8.03 (s, 1H), 5.24 (s, 1H), 4.87–4.81 (m,
1H), 4.14–4.04 (m, 4H), 1.91–1.4 (m), 1.34–1.30 (m,
6H), 1.0–0.90 (m), 0.92 (s, 3H), 0.84 (d, J = 6.4 Hz),
0.74 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 160.55,
160.47, 76.01, 74.04, 61.34 (d, J = 5.9 Hz), 49.21, 47.45,
44.97, 41.73, 36.73 (d, J = 16.9 Hz), 35.60, 34.65, 34.18,
33.98, 32.09, 27.39, 26.75, 26.44, 26.00 (d, J = 140.9 Hz),
25.86, 25.70, 23.38, 22.86, 19.08 (d, J = 3.3 Hz), 17.67,
16.40 (d, J = 5.0 Hz), 12.26; 31P NMR (162 MHz, CDCl3)
δ: 30.57; LRMS-ESI: calc. for M + Na+: 577.3; obsd.
578.
2.1.6.1. 3α, 7α, 12α-trihydroxy-24-nor-5β-cholane-23-
phosphonic acid (23-PCA, 1). Yield 65%; mp:
257.4–259 ◦C; [␣]D25 (c 1.85 in MeOH) = +27; FTIR
(KBr): 3426 cm−1 (hydroxyl); 1H NMR (300 MHz, DMSO-
d6) δ: 3.77 (s, 1H), 3.60, (s, 1H), 3.16 (bs, 1H), 1.8–1.13
(m), 0.91 (d, J = 6 Hz, 3H), 0.81 (s, 3H), 0.59 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) δ: 71.00, 70.43, 66.26, 45.66,
41.50, 41.36, 35.79 (d, J = 15 Hz), 35.29, 34.84, 34.36,
30.38, 28.68, 28.52, 27.26, 26.21, 23.99 (d, J = 136 Hz),
22.78, 22.60, 16.81, 12.35; 31P NMR (162 MHz, DMSO-d6)
δ: 29.55; HRMS Q-TOF: calc. for M + Na+: 467.2538; obsd.
467.2505.
2.1.5.8. Diethyl 3α, 7α-diformyloxy-5β-cholane-24-phos-
1
phonate. Yield 98%; H NMR (400 MHz, CDCl3) δ: 8.08
(s, 1H), 8.03 (s, 1H), 5.03 (s, 1H), 4.77–4.69 (m, 1H),
4.12–4.06 (m, 4H), 2.1–1.38 (m), 1.33 (t, J = 7.2 Hz, 6H),
1.25–1.0 (m), 0.95 (s, 3H), 0.93 (d, J = 6.0 Hz, 3H),
0.65 (s, 3H); 13C NMR (100 MHz, CDCl3) δ: 160.59,
74.02, 71.37, 61.34 (d, J = 5.9 Hz), 55.84, 50.11, 42.68,
41.02, 39.43, 37.95, 36.94 (d, J = 16.1Hz), 35.39, 34.79,
34.62, 34.01, 31.50, 28.02, 26.75, 26.12 (d, J = 138.4 Hz),
26.75, 23.46, 22.61, 20.61, 19.07 (d, J = 4.0 Hz), 18.50,
16.43 (d, J = 5.0 Hz), 11.66; 31P NMR (162 MHz, CDCl3)
δ: 31.93; LRMS-ESI: calc. for M + Na+: 577.3; obsd.
578.
2.1.6.2. 3α,
12α-dihydroxy-24-nor-5β-cholane-23-phos-
phonic acid (23-PDCA, 2). Yield 70%; mp: 262–263 ◦C;
[␣]2D5 (c 1.05 in MeOH) = +44; FTIR (KBr): 3400 cm−1
(hydroxyl); 1H NMR (300 MHz, DMSO-d6) δ: 3.77 (s, 1H),
3.36 (m, 1H), 1.90–1.0 (m), 0.90 (d, J = 6.0 Hz, 3H), 0.83
(s, 3H), 0.59 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
71.02, 69.95, 47.44, 45.95, 45.76, 41.61, 36.27, 35.72 (d,
J = 15 Hz), 35.14, 33.80, 32.93, 30.20, 28.59, 27.16, 26.97,
2.1.5.9. Diethyl 3α, 7β-diformyloxy-5β-cholane-24-phos-
1
phonate. Yield 93%; H NMR (300 MHz, CDCl3) δ: 8.03
(s, 1H), 7.99 (s, 1H), 4.94–4.79 (m, 2H), 4.12–4.06 (m, 4H),
2.1–1.37 (m), 1.33 (t, J = 7.2 Hz, 6H), 1.25–1.0 (m), 0.99
(s, 3H), 0.93 (d, J = 6.6 Hz, 3H), 0.68 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ: 160.67, 160.27, 73.34, 73.10, 61.12 (d,
J = 5.9 Hz), 55.10, 54.81, 43.34, 41.82, 39.62, 39.21, 36.69
(d, J = 17.0 Hz), 35.06, 34.14, 33.70, 32.66, 32.53, 28.17,
26.14, 25.80 (d, J = 138.7 Hz), 25.57, 22.93, 20.97, 18.86
(d, J = 3.5 Hz), 18.34, 16.21 (d, J = 5.2 Hz), 11.77; 31P NMR
(162 MHz, CDCl3) δ: 31.87; LRMS-ESI: calc. for M + Na+:
577.3; obsd. 577.7.
26.09, 23.99 (d, J = 135 Hz), 23.48, 23.07, 16.79, 12.46; 31
P
NMR (162 MHz, DMSO-d6) δ: 29.63; HRMS Q-TOF: calc.
for M + Na+: 451.2589; obsd. 451.2577.
2.1.6.3. 3α,
7α-dihydroxy-24-nor-5β-cholane-23-phos-
phonic acid (23-PCDCA, 3). Yield 66%; mp: 226–227 ◦C;
[␣]2D5 (c 2.1 in MeOH) = +9; 1H NMR (400 MHz, DMSO-d6)
δ: 3.64 (s, 1H), 3.22–3.17 (m, 1H), 1.93–0.98 (m), 0.89
(d, J = 6.4 Hz, 3H), 0.85 (s, 3H), 0.62 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ: 70.33, 66.17, 55.24, 49.97, 41.89,
41.44, 35.64 (d, J = 15.3 Hz), 35.31, 34.80, 34.72, 32.30,
30.54, 28.59, 27.72, 23.91 (d, J = 136.5 Hz), 23.12, 22.69,
20.25, 18.04, 11.69; 31P NMR (162 MHz, DMSO-d6) δ:
30.10; HRMS Q-TOF: calc. for M + Na+: 451.2589; obsd.
451.2655.
2.1.5.10. Diethyl 3α-formyloxy-5β-cholane-24-phospho-
nate. Yield 95%; 1H NMR (400 MHz, CDCl3) δ: 8.04
(s, 1H), 4.85 (m, 1H), 4.12–4.07 (m, 4H), 2.0–1.4 (m),
1.35–1.30 (m, 6H), 1.29–1.04 (m), 0.93–0.9 (m, 6H), 0.64
(s, 3H); 13C NMR (100 MHz, CDCl3) δ: 160.67, 74.39,
61.49 (d, J = 4.7 Hz), 56.48, 56.14, 41.95, 40.50, 40.14,
36.97 (d, J = 16.6 Hz), 35.81, 35.41, 34.97, 32.25, 28.21,
26.98, 26.64, 26.28, 26.04 (d, J = 139.2 Hz), 24.14, 23.25,
20.83, 19.06, 18.46, 16.37 (d, J = 3.7 Hz), 11.98; 31P NMR
(162 MHz, CDCl3) δ: 32.24; LRMS-ESI: calc. for M + Na+:
533.3; obsd. 534.
2.1.6.4. 3α,
7β-dihydroxy-24-nor-5β-cholane-23-phos-
phonic acid (23-PUDCA, 4). Yield 76%; mp: 234–235 ◦C;
[␣]2D5 (c 1.44 in MeOH) = +51; 1H NMR (400 MHz, DMSO-
d6) δ: 3.34–3.26 (m, 2H), 1.96–1.09 (m), 0.89 (d, J = 4.8 Hz,
3H), 0.88 (s, 3H), 0.63 (s, 3H); 13C NMR (100 MHz,

686
P. Babu, U. Maitra / Steroids 70 (2005) 681–689
DMSO-d6) δ: 69.74, 69.47, 55.83, 54.38, 49.05, 43.02,
42.18, 39.82, 38.75, 37.67, 37.23, 35.56 (d, J = 15.2 Hz),
34.82, 33.74, 30.20, 28.67, 28.63, 28.10, 26.63, 23.94
(d, J = 132.6 Hz), 23.28, 20.85, 18.18, 12.07; 31P NMR
(162 MHz, DMSO-d6) δ: 29.82; HRMS Q-TOF: calc. for
M + Na+: 451.2589; obsd. 451.2603.
2.1.6.9. 3α, 7β-dihydroxy-5β-cholane-24-phosphonic acid
(24-PUDCA, 9). Yield 64%; mp: 182–183 ◦C; [␣]D25 (c 1.7 in
MeOH) = +53. 1H NMR (400 MHz, DMSO-d6) δ: 3.36–3.26
(m, 2H), 1.9–1.0 (m), 0.90 (d, J = 6.4 Hz, 3H), 0.88 (s,
3H), 0.64 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
69.55, 69.29, 55.73, 54.69, 42.67, 42.03, 38.59, 37.54, 37.12,
36.52 (d, J = 16.1 Hz), 34.79, 34.69, 33.60, 30.07, 27.83 (d,
J = 135.9 Hz), 28.09, 27.15, 26.50, 23.13, 20.69, 19.19, 18.43,
11.85; 31P NMR (162 MHz, DMSO-d6) δ: 28.67; HRMS Q-
TOF: calc. for M + Na+; M + K+: 465.2746, 481.2485; obsd.
465.2743, 481.2408.
2.1.6.5. 3α-hydroxy-24-nor-5β-cholane-23-phosphonic
acid (23-PLCA, 5). Yield 75%; mp: 226.6–229 ◦C; [␣]D25 (c
0.65 in EtOH) = +30; FTIR (KBr): 3418 cm−1 (hydroxyl);
1H NMR (300 MHz, DMSO-d6) δ: 3.2–3.17 (m, 1H),
1.9–1.0 (m), .87 (bs, 6H), 0.63 (s, 3H); 13C NMR (75 MHz,
DMSO-d6) δ: 69.90, 56.06, 55.26, 42.26, 41.57, 36.29,
35.64 (d, J = 17 Hz), 35.44, 35.19, 34.24, 30.39, 28.69,
27.72, 26.93, 26.21, 24.03 (d, J = 136 Hz), 23.98, 23.31,
20.45, 18.08, 11.96; 31P NMR (162 MHz, DMSO-d6) δ:
29.33; HRMS Q-TOF: calc. for M + Na+; M + K+: 435.2640,
451.2331; obsd. 435.2653, 451.2379.
2.1.6.10. 3α-Hydroxy-5β-cholane-24-phosphonic acid (24-
PLCA, 10). Yield 90%; m. p.: 227–228 ◦C; [␣]D25 (c 1.0 in
EtOH) = +32; 1H NMR (400 MHz, DMSO-d6) δ: 3.40–3.35
(m, 1H), 1.96–1.0 (m), 0.89 (d, J = 7.2 Hz, 3H), 0.88 (s, 3H),
0.63 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 69.80,
56.01, 55.65, 42.19, 41.47, 36.52 (d, J = 16.1 Hz), 36.18,
35.33, 35.07, 34.85, 34.12, 30.25, 27.88 (d, J = 135.5 Hz),
27.71, 26.81, 26.06, 23.73, 23.15, 20.33, 19.24, 18.31, 11.58;
31P NMR (162 MHz, DMSO-d6) δ: 28.72; HRMS Q-TOF:
calc. for M + Na+: 449.2797; obsd. 449.2791.
2.1.6.6. 3α, 7α, 12α-trihydroxy-5β-cholane-24-phosphonic
acid (24-PCA, 6). Yield 60%; mp: 189–190 ◦C; [␣]D24 (c
1
1.68 in MeOH) = +30; H NMR (400 MHz, DMSO-d6) δ:
3.79 (s, 1H), 3.61 (s, 1H), 3.21–3.16 (m, 1H), 2.3–2.11 (m,
2H), 2.0–1.1 (m), 0.93 (d, J = 6.4 Hz, 3H), 0.81 (s, 3H),
0.59 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 70.91,
70.31, 66.20, 66.15, 45.97, 45.59, 41.40, 41.21, 36.65 (d,
J = 5.6 Hz), 35.17, 34.95, 34.70, 34.25, 30.25, 28.42, 27.94
(d, J = 134.5 Hz), 27.20, 26.09, 22.62, 22.44, 19.15, 17.12,
12.16; 31P NMR (162 MHz, DMSO-d6) δ: 28.66; HRMS Q-
TOF: calc. for M + Na+; M + K+: 481.2695, 497.2434; obsd.
481.2664, 497.2411.
2.1.7. Estimation of solubilized cholesterol by enzymatic
assay
The amount of solubilized cholesterol was estimated by
an enzymatic assay [13]. Samples were incubated with a
commercially available cholesterol reagent [14] at 37 ◦C for
10 min, and then absorbance was recorded at 510 nm. The
cholesterol converted was estimated by comparing this ab-
sorbance with that from a standard sample.
2.1.6.7. 3α, 12α-dihydroxy-5β-cholane-24-phosphonic acid
(24-PDCA, 7). Yield 55%; mp: 242–243.5 ◦C; [␣]D24 (c 1.3
in MeOH) = +49; 1H NMR (400 MHz, DMSO-d6) δ: 3.80 (s,
1H), 3.41–3.34 (m, 1H), 1.85–1.0 (m), 0.94 (d, J = 6.8 Hz,
3H), 0.86 (s, 3H), 0.61 (s, 3H); 13C NMR (75 MHz, DMSO-
d6) δ: 71.02, 69.93, 47.48, 46.13, 45.95, 41.62, 36.75 (d,
J = 17.3 Hz), 36.29, 35.68, 35.11, 33.84, 32.95, 30.24, 28.11
(d, J = 135.8 Hz), 28.66, 27.34, 27.01, 26.13, 23.53, 23.13,
19.33, 19.28, 17.24, 12.46; 31P NMR (162 MHz, DMSO-
d6) δ: 28.71; HRMS Q-TOF: calc. for M + Na+; M + K+:
465.2746, 481.2485; obsd. 465.2740, 481.2447.
3. Results and discussion
3.1. Synthesis
The syntheses of 23-phosphonobile acids were accom-
plished 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.
2.1.6.8. 3α, 7α-dihydroxy-5β-cholane-24-phosphonic acid
(24-PCDCA, 8). Yield 75%; mp: 234–235 ◦C; [␣]D25 (c 1.38
in MeOH) = +15; H NMR (400 MHz, DMSO-d6) δ: 3.61
For the syntheses of 23-phosphonobile acids, formyl
protected bile acids were subjected to a modified Huns-
diecker reaction (iododecarboxylation) in the presence of
Pb(OAc)4/I2/hν to yield the corresponding formylated 24-
nor-23-iodo derivatives. The iodo derivatives were refluxed
with triethyl phosphite at 160 ◦C under nitrogen (Arbuzov re-
action) to furnish the corresponding formylated diethyl phos-
phonocholanate esters. The cleavage of the ethyl groups was
achieved by stirring with Me3SiI in CH2Cl2 at rt. The removal
of the TMS ester and the formyl groups were effected in the
same pot by alkaline hydrolysis in MeOH. The basic solution
1
(s, 1H), 3.23–3.15 (m, 1H), 1.9–1.0 (m), 0.89 (d, J = 6.4 Hz,
3H), 0.83 (s, 3H), 0.60 (s, 3H); 13C NMR (100 MHz, DMSO-
d6) δ: 70.20, 66.03, 55.57, 49.86, 41.79, 41.32, 36.53 (d,
J = 15.8 Hz), 35.19, 34.89, 34.65, 34.59, 32.15, 30.39, 27.86
(d, J = 134.9 Hz), 27.72, 22.99, 22.55, 20.12, 19.16, 18.31,
11.49; 31P NMR (162 MHz, DMSO-d6) δ: 28.58; HRMS Q-
TOF: calc. for M + Na+; M + K+: 465.2746, 481.2485; obsd.
465.2729, 481.2477.

P. Babu, U. Maitra / Steroids 70 (2005) 681–689
687
Scheme 1.
containing phosphonobile salt was neutralized (pH ∼ 1) and
filtered to yield the 23-phosphonobile acids.
For the synthesis of 24-phosphonobile acids, the formy-
lated bile acids were reduced to the corresponding formyl
protected alcohols in two steps. First a mixed anhydride was
made with ethyl chloroformate in the presence of triethy-
lamine, which was then selectively reduced by NaBH4 in
MeOH. The 24-hydroxy compounds were converted to the
corresponding 24-iodo derivatives using PPh3/I2/imidazole
in dichloromethane at rt [15]. The formation of diethyl esters
of 24-phosphonobile acids and subsequent deprotection
of ethyl and formyl groups were accomplished following
Scheme 2.

688
P. Babu, U. Maitra / Steroids 70 (2005) 681–689
Table 1
Comparison of cholesterol sloubilization efficiency of 23- and 24-PBSs with
those of bile salts
Entry PBS (50 mM) in pH 6.4 (0.1 M Ratio of cholesterol:bile
phosphate buffer), 0.15 M
NaCl at 37 ◦C
salt (Sp)a
23-PBS
24-PBS
BSb
1
2
3
4
PC
1:46 (1)
1:19 (2)
1:20 (3)
1:23 (6)
1:13.5 (7) 1:15.6
(8)c
1:29
PDC
PCDC
PUDC
1:14
1:364
1:238 (4) 1:151 (9)
a
Molar concentration of dissolved cholesterol = 0.05 × Sp; the litho-
cholate analogs (5 and 10) were insoluble under identical conditions.
b
The values are taken from ref. [17] at pH 10.
Under the experimental conditions, a turbid gel was obtained, so the
c
amount of solubilized cholesterol could not be measured.
structure and number of hydroxyl groups present in the PBSs.
Forboth23-and24-PBSs, thesolubilizingpowerdecreasesin
∼
the following order: PDC PCDC > PC ꢀ PUDC. The same
=
trend of cholesterol solubilization is observed for bile salts,
which correlated inversely with hydrophilicity [18]. Interest-
ingly, the 23- and 24-PUDC showed 1.5 and 2.4 times higher
solubilization, respectively, compared to that of UDC. Ki-
netic experiments show a fast initial rate of solubilization,
which slows down with time. For 23-PCDC, cholesterol sol-
ubilization saturates in about 8 h.
3.3. Effect of pH on equilibrium cholesterol solubility
The effect of pH on the equilibrium cholesterol solubility
was examined using 23-PCDC and 23-PUDC as the solubi-
lizing agents. The solubilization experiments were done in
three different buffer solutions (0.1 M): pH 6.4 (phosphate),
7.4 (TRIS) and 10.0 (carbonate) and 0.15 M NaCl. The equi-
librium cholesterol solubility decreases considerably upon
increasing the bulk pH. The same trend was observed for
phosphonocheno and phosphonoursodeoxycholates (Fig. 2).
An increase in the bulk pH increases the CMC of phospho-
Fig. 1. 31P NMR spectra of 23-PBA (a) and 24-PBA (b) in DMSO-d6
(162 MHz). Top to bottom: phosphonocholic, phosphonodeoxycholic, phos-
phonochenodeoxycholic, phosphonoursodeoxycholic and phosphonolitho-
cholic acid.
the same procedures as described for 23-phosphonobile
acids. All the compounds synthesized were character-
ized spectroscopically. The 31P NMR spectra of 23- and
24-phosphonobile acids are shown in Fig. 1.
3.2. In vitro equilibrium cholesterol solubilization by
23- and 24-PBSs
All the equilibrium cholesterol solubilization experiments
were done at 37 ◦C in solutions containing varying concen-
trations of disodium salt of phosphonobile acids and 0.15 M
NaCl in pH 6.4 (0.1 M phosphate) buffer solutions. The sol-
ubilized cholesterol was analyzed using an enzymatic assay
(see Section 2). The equilibrium cholesterol solubility data
for 23- and 24-phosphonobile salts at pH 6.4 (0.1 M potas-
sium phosphate), where the phosphonobile acids are predom-
inantly monoionic, are presented in Table 1. In general, the
equilibrium solubilizing power (Sp), defined as the ratio of
cholesterol:PBS, is slightly higher for 24-PBSs than that of
23-PBSs with the solubilizing power of the corresponding
bile salts (at pH 10.0) [16,17] being in between. The equi-
librium solubility of cholesterol is greatly influenced by the
Fig. 2. Amount of solubilized cholesterol (by 23-PCDC and 23-PUDC,
50 mM) as a function of pH 6.4 (phosphate), 7.4 (TRIS) and 10.0 (carbonate).

P. Babu, U. Maitra / Steroids 70 (2005) 681–689
689
nobile salts. For example, the CMC of 23-PCDC increased
from 2 mM to 7–8 mM when the pH is changed from 6.4 to
10. Hence, the observed decrease in equilibrium solubility is
possibly due to an increase in the CMC, which decreases the
number of micelles formed at a given total concentration of
the bile salt.
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4. Conclusions
We synthesized a new class of bile acid analogs. In
vitro cholesterol solubilizing efficiency of 23- and 24-
phosphonobilesaltshasbeenstudied. The24-PBSssolubilize
cholesterol slightly better than 23-PBSs and bile salts. We be-
lieve 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.
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codesoxycholic acid. J Biol Chem 1936;113:779–85.
Acknowledgments
We thank the Jawaharlal Nehru Centre for Advanced Sci-
entific Research, Bangalore for financial support of this work.
Ponnusamy Babu thanks CSIR for fellowship.
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[14] Accurex, Biomedical Pvt. Ltd., Mumbai. The cholesterol reagent
contains a mixture of cholesterol esterase, cholesterol oxidase, per-
oxidase, 4-aminoantipyrine and phenol in pH 6.8 buffer solution.
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