The remote-oxyfunctionalization of unactivated carbons in (5β)-3-oxobile acids by 2,6-dichloropyridine N-oxide catalyzed by ruthenium-porphyrin and HBr: A direct lactonization at C-20

Article abstract of DOI:10.1039/b314965j

Remote-oxyfunctionalization induced by 2,6-dichloropyridine N-oxide (DCP N-oxide) as an oxygen donor and a (5,10,15,20-tetramesitylporphyrinate) ruthenium(n) carbonyl complex (Ru-porphyrin) and HBr as catalysts was examined for a series of methyl ester-peracetylated derivatives of (5β)-3-oxobile acids. Using the DCP-N-oxide/Ru-porphyrin/HBr system, 5β-hydroxylation predominated for the substrates having a 12-acetoxyl substituent due to steric hindrance, but the presence of a 7-acetoxyl substituent decreased the reactivity of the 5β-position allowing for the competitive (20S)-20- oxyfunctionalization, subject to electronic constraints. A variety of novel 5β-hydroxylation and (20S)-24,20-γ-lactonization products, as well as their double-oxyfunctionalization and dehydration products, were obtained in one-step. The alkaline hydrolysis of the γ-lactones gave the corresponding stereoselective (20S)-20-hydroxy-carboxylic acids.

Full text of DOI:10.1039/b314965j

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The remote-oxyfunctionalization of unactivated carbons in
(5ꢀ)-3-oxobile acids by 2,6-dichloropyridine N-oxide catalyzed by
ruthenium–porphyrin and HBr: a direct lactonization at C-20†
Shoujiro Ogawa,a Takashi Iida,*a Takaaki Goto,b Nariyasu Mano,b Junichi Gotob and
Toshio Nambarab
a Department of Chemistry, College of Humanities and Sciences, Nihon University,
Sakurajousui, Setagaya, Tokyo 156-8550, Japan. E-mail: takaiida@chs.nihon-u.ac.jp
b Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama,
Sendai 980-8578, Japan
Received 21st November 2003, Accepted 2nd February 2004
First published as an Advance Article on the web 1st March 2004
Remote-oxyfunctionalization induced by 2,6-dichloropyridine N-oxide (DCP N-oxide) as an oxygen donor and a
(5,10,15,20-tetramesitylporphyrinate) ruthenium() carbonyl complex (Ru-porphyrin) and HBr as catalysts was
examined for a series of methyl ester-peracetylated derivatives of (5β)-3-oxobile acids. Using the DCP-N-oxide/
Ru-porphyrin/HBr system, 5β-hydroxylation predominated for the substrates having a 12-acetoxyl substituent due to
steric hindrance, but the presence of a 7-acetoxyl substituent decreased the reactivity of the 5β-position allowing for
the competitive (20S)-20-oxyfunctionalization, subject to electronic constraints. A variety of novel 5β-hydroxylation
and (20S)-24,20-γ-lactonization products, as well as their double-oxyfunctionalization and dehydration products,
were obtained in one-step. The alkaline hydrolysis of the γ-lactones gave the corresponding stereoselective (20S)-20-
hydroxy-carboxylic acids.
of various drugs and other biologically active compounds by
using metalloporphyrin moieties.
The recent successful use of a powerful new oxidant
system consisting of 2,6-dichloropyridine (DCP) N-oxide as
an oxygen-donating reagent and a (5,10,15,20-tetramesityl-
porphyrinate) ruthenium() carbonyl complex [Ru-porphyrin,
Introduction
Recent efforts in the organic synthesis of bioactive compounds
have been focused on the development of so-called “biomimetic
compounds”, “artificial enzymes”, “enzyme mimics” and/or
the “remote-oxyfunctionalization” of unactivated hydro-
carbons via non-microbial and non-enzymic methods.1 For
Ru(TMP)CO] and HBr as catalysts prompted us to apply this
these purposes, a variety of versatile new oxygen-transfer
method to bile acids.5 We describe here the remote-oxyfunc-
reagents and/or catalysts have been reported by many groups of
tionalization of a series of the methyl ester-peracetylated deriv-
investigators.
atives of 3-oxbile acids with or without an acetoxyl substituent
The cytochrome P-450 oxidase-dependent system efficiently
at the C-7 and/or C-12 positions in the 5β-nucleus using this
catalyzes the key transformation of unactivated carbon atoms
DCP N-oxide/ruthenium porphyrin/HBr system.
of cholesterol to give steroid hormones and related bio-
molecules in vivo.2 Therefore, the regio- and stereoselective
remote-oxyfunctionalization (e.g., hydroxylation and ketoniz-
ation) of unactivated carbons, related to the cytochrome P-450
process, is of particular interest in obtaining bioactive steroids
from abundantly available sterols or bile acid sources avoiding
a multistep synthesis. Recent advances in the remote-oxy-
functionalization reactions of unactivated positions in steroids
have been reviewed by Reese.3 These include the radical relay
reaction (Breslow reaction), the hypohalite reaction, nitrite
photolysis (Barton reaction), ketone irradiations, the nitrene
reaction, the Hofmann–Löffler–Freytag reaction, the lead
tetraacetate reaction, dimethyldioxirane (DMDO), penta-
fluorodialkyloxaziridine, anodic oxidation, the free radical
decomposition of peracids, ceric ammonium nitrate, peroxide
reactions, chromic oxide, hypervalent organoiodine reagents,
and porphyrins.
The remote-oxyfunctionalization of steroids carried out by
Results and discussion
Almost all of the oxygenation reagents previously reported
result predominantly in 5β-hydroxylation in 5β-steroids.3
The use of DMDO as an oxygenation agent, however, results
not only in 5β-hydroxylation, but also in 14α- and 17α-
hydroxylations in the nucleus as well as their double-oxyfunc-
tionalizations depending upon both the presence and the
stereochemical nature of a 7-substituent in the substrate.6
Nevertheless, direct remote-oxyfunctionalization of the unacti-
vated cabons in the side-chain [-CH(CH3)CH2CH2COOH] of
cytochrome P-450 enzymes in vivo, makes use of a heme pros-
thetic group at the active site of the catalyst. As has been
reviewed by Meunier4a and reported by other groups,4b–4f many
attempts have been made to mimic the action of the cyto-
chrome P-450 enzyme systems in the oxygenation and oxidation
† Electronic supplementary information (ESI) available: 13C-NMR
chemical shifts for oxyfunctionalization products 6–23, 26 and 27. See
http://www.rsc.org/suppdata/ob/b3/b314965j/
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 1 3 – 1 0 1 8
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Table 1 Oxidation products of (5β)-3-oxobile acid methyl ester-peracetates with DCP N-oxide/Ru(TMP)CO/HBr
Substrate
Reaction time (t/h)
Products (yield, %)a
Starting compound recovery
Products
1
1
2
3
4
5
12
60
60
60
60
60
31
20
56
43
40
45
6 (30), 7 (3), 8 (17), 9 (19)
6 (33), 7 (6), 8 (19), 9 (22)
10 (25), 11 (19)
12 (19), 13 (8), 14 (21), 15 (9)
16 (18), 17 (29), 18 (3), 19 (10)
20 (36), 21 (5), 22 (5), 23 (9)
a Determined by capillary GC; for conditions, see Experimental section.
C24 (5β)-bile acids is very rare, though the C8H17-alkyl side-
chain in the 5α-cholestane series of steroids is readily oxidized
at the C-25 methine carbon to give the 25-hydroxylated deriv-
atives exclusively.
24-oate (6, 33%), (20S)-3-oxo-5β-cholan-O-24,20-lactone (8,
19%) and (20S)-5β-hydroxy-3-oxocholan-O-24,20-lactone (9,
22%), along with a small amount of methyl 3-oxo-4-cholen-24-
oate (7, 6%).7 The formation of the conjugated enone 7 may
have been derived from the subsequent elimination of the 5β-
hydroxy group in 6 under the reaction conditions used. These
results for 1 differed from those observed by Shingaki et al.,5b
who reported the formation of 5β-hydroxycholan-24-oic acid
(70%) and (20S)-5β,20-dihydroxycholan-24-oic acid (3%) start-
ing with 5β-cholan-24-oic acid using the same oxidizing system;
(20S)-24,20-γ-lactonization products such as 8 and 9 were not
detected at all. The presence of the electron-withdrawing 3-oxo
group in 1, therefore, seems to prevent the 5β-hydroxylation of
the methine carbon at C-5, but accelerate the remote-oxyfunc-
tionalization of the methine carbon at C-20. Nevertheless, the
DCP N-oxide/Ru-porphyrin/HBr system is faster in adding an
oxygen into the 5β C–H bond of 1 to give 6 as one of the main
products.
An exploratory experiment revealed that remote-oxyfunc-
tionalization with the DCP N-oxide/Ru-porphyrin/HBr system
is more effective for methyl 3-oxo-5β-cholan-24-oate (1) than
for methyl 3α-acetoxy-5β-cholan-24-oate under a prolonged
reaction time (at 50 ЊC for 60 h), suggesting that the presence
of the electron-withdrawing 3-oxo group interferes with the
5β-hydroxylation, compared with the 3α-acetoxyl group (see
ref. 7). Based on this finding, a series of the methyl ester-
peracetylated derivatives of (5β)-3-oxobile acids (1–5) with or
without 7- and/or 12-substituents were used as substrates. The
hydroxy groups at C-7 and C-12 were protected by the acetoxyl
group to prevent simultaneous oxidation of the hydroxyls to
ketones. The results are shown in Table 1. Treatment of 1–5
with DCP N-oxide/ruthenium porphyrin/HBr resulted in the
formation of several monofunctionalized products, together
with their double-functionalized ones. In addition, the regio-
selectivity of the O-insertion reaction was significantly influ-
enced by the presence of a 7- or 12-acetoxyl group (see below).
The oxidation of 1 with DCP N-oxide/Ru-porphyrin/HBr
produced three major compounds, 5β-hydroxy-3-oxocholan-
A much different result was obtained for the DCP N-oxide/
Ru-porphyrin/HBr oxidation of methyl 12α-acetoxy-3-oxo-5β-
cholan-24-oate (2). After chromatographic purification of
the reaction products, two major oxidized components were
isolated and their structures identified as methyl 12α-acetoxy-
5β-hydroxy-3-oxocholan-24-oate (10) and methyl 12α-acetoxy-
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 1 3 – 1 0 1 8
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3-oxo-4-cholen-24-oate (11). No 20-oxygenated product was
detected at all. The axial 12α-acetoxyl group in 2 may therefore
prevent the attack of the bulky DCP N-oxide/Ru-porphyrin/
HBr complex, like most other reagents sensitive to steric
hindrance, on the methine carbon at C-20.
due to C-20 appears in the low field region of ca. 88.5 ppm,
while the C-25 signal of the methyl ester at ca. 51.5 ppm is
absent. The GC-MS of (20S)-24,20-γ-lactones exhibited
intense signals consistent with the sequential loss of side-chains
(S.C.) of the C5H7O2 γ-lactone ring (99 a.m.u) from the respect-
ive M, (M Ϫ AcOH), (M Ϫ 2AcOH) and/or related ions,
instead of C6H11O2 (112 a.m.u.) from the C-24 methyl ester.
According to previous findings, the regioselectivity of
the O-insertion reaction using this reagent system depends
upon the steric and electronic effects of the electron-with-
drawing substituents situated in the vicinity of the methine
carbon under consideration.5 Electron-withdrawing 3-oxo
and 7-acetoxyl substituents destabilize the transition state for
the DCP N-oxide/Ru-porphyrin/HBr complex insertion in the
nearby methine carbon at C-5 through unfavorable electrostatic
interactions, and, as a consequence, insertion occurs at C-20.
The interplay of these and perhaps other factors may be more
subtle: the oxyfunctionalization of C-20 in 1 and 3–5 requires
that the 3-oxo and 7-acetoxyl groups cause a large decrease in
the reaction rate at C-5 compared with C-20, either by decreas-
ing the electron density in the equatorial 5β C–H bond more
than it does on the pseudo-axially oriented 20β C–H bond, or
by destabilizing the transition state leading to insertion at C-5
more than the transition state leading to C-20 insertion.
In conclusion, the remote-oxyfunctionalization of the methyl
ester-peracetylated derivatives of 3-oxobile acids with a 12α-
acetoxyl group via DCP N-oxide/Ru-porphyrin/HBr system
resulted in primarily 5β-hydroxylation, while (20S)-24,20-
lactonization predominanted for 3-oxobile acids having 3-oxo
and 7-acetoxyl substituents. A direct, one-step synthesis of the
(20S)-24,20-γ-lactones and their hydrolysis products, (20S)-20-
hydroxylated 3-oxobile acids, from abundant and common bile
acids may be useful for the easy, efficient synthesis of bioactive
steroids such as steroid hormones and cardiotonic steroids.
On the other hand, the addition of a 7-acetoxyl group
in methyl 7α-acetoxy-3-oxo-5β-cholan-24-oate (3) and its
7β-isomer (4) substantially decreased the O-insertion rate at
C-5 relative to 1 and 2, while significantly increasing the inser-
tion rate at C-20 regardless of the axial 7α- or equatorial 7β-
configuration. Thus, when 3 was treated with the DCP N-oxide/
Ru-porphyrin/HBr system, the successful formation of 20-
oxygenated γ-lactones surpassed that of the 5β-hydroxylated
compounds, yielding (20S)-7α-acetoxy-3-oxo-5β-cholan-O-
24,20-lactone (14) and (20S)-7α-acetoxy-5β-hydroxy-3-oxocho-
lan-O-24,20-lactone (15) as the major products (total yield,
30%) and methyl 7α-acetoxy-5β-hydroxy-3-oxocholan-24-oate
(12) and methyl 7α-acetoxy-3-oxo-4-cholen-24-oate (13) as the
minor components (total yield, 27%). Similarly, 4 produced
three variants of 24,20-γ-lactones (total yield, 42%),
i.e., (20S)-7β-acetoxy-3-oxo-5β-cholan-O-24,20-lactone (17),
(20S)-7β-acetoxy-5β-hydroxy-3-oxocholan-O-24,20-lactone
(18) and (20S)-7β-acetoxy-3-oxo-4-cholen-O-24,20-lactone
(19), together with methyl 7β-acetoxy-5β-hydroxy-3-oxo-
cholan-24-oate (16, 18%). Since both the 7α- and 7β-acetoxyl
groups of the 5β-steroid nucleus do not add significant steric
hindrance to the β face of the bulky complex, the considerable
variation in the ratio of the C-5 and C-20-oxidized products in
3 and 4 may be ascribed to the local electron density (electro-
static) of the two electron-withdrawing 3-oxo and 7-acetoxyl
groups around C-5. The intriguing differences between the
results of 2 and 3 (or 4) can, therefore, be rationalized in terms
of electronic effects rather than steric hindrance.5,6 In all cases,
the stereochemical configuration with respect to C-20 was
retained during the oxyfunctionalization of the methine hydro-
gen (C-H) to the resulting γ-lactones.
Methyl 7α,12α-diacetoxy-3-oxo-5β-cholan-24-oate (5), hav-
ing 7α- and 12α-acetoxyl substituents, afforded a 5β-hydroxy
compound (20, 36%) and its dehydrated derivative (21) when
subjected to DCP N-oxide/Ru-porphyrin/HBr oxidation. The
oxidation of 5 was also accompanied by a reasonable amount
(total yield, 14%) of the double-functionalized 5β-hydroxy-3-
oxo-24,20-lactone (22) and 4-ene-3-oxo-24,20-lactone (23). The
combined effect of the respective 7- and 12-acetoxyl groups on
the oxidation at the C-5 and C-20 positions resulted in the
competitive formation of both the 5β- and 20-oxygenated com-
pounds, in analogy with 1.
The formation of (20S)-24,20-γ-lactones (8, 9, 14, 15, 17,
18, 19, 22 and 23) via the DCP N-oxide/Ru-porphyrin/HBr
oxidation of 1 and 3–5 apparently involves an intramolecular
esterification of the carboxyl group at C-24 with the newly
inserted hydroxy group at C-20. Although the exact reaction
mechanism is unclear, the fact that possible intermediary (20S)-
20-hydroxy-24-methyl ester and/or (20S)-20-hydroxy-24-carb-
oxylic acid derivatives were not isolated at all in any of the
reactions implies that the reaction proceeds directly by a
nucleophilic acyl substitution catalyzed by HBr.
Experimental
Materials and methods
Melting points (mp) were determined on an electric micro hot
stage and are uncorrected. Infrared (IR) spectra were obtained
on a Bio Rad FTS-7 FT-IR spectrometer (Philadelphia, USA)
for samples in KBr tablets. Proton (1H) and carbon (13C)
nuclear magnetic resonance (NMR) spectra were obtained on a
JEOL JNM-EX 270 FT NMR instrument (Tokyo, Japan) at
270 and 67.8 MHz, respectively, with CDCl3 containing 0.1%
Me4Si as the solvent. Chemical shifts are expressed as δ (ppm)
relative to Me4Si. 13C-NMR signals corresponding to the
methyl (CH3), methylene (CH2), methine (CH), and quaternary
(C) carbons were differentiated by means of DEPT experi-
ments. Low-resolution mass (LR-MS) spectra were recorded on
a JEOL JMC-Automass 150 gas chromatography-mass spec-
trometry (GC-MS) at 70 eV with an electron ionization (EI)
probe using the positive ion mode (PIM). High-resolution mass
(HR-MS) spectra were performed using JEOL JMS-LCmate
and JMS-700 double-focusing magnetic mass spectrometers
equipped with an electrospray ionization (ESI) probe using the
PIM and with the EI probe under the PIM, respectively. The
resolution of the ESI mass spectrometer was set at 3000, and
the voltages for the electrospray, orifice and ring lens were 2.5
kV, 30 V and 100 V, respectively. The temperatures of the orifice
and desolvating plate were 150 and 250 ЊC, respectively. HR-
MS spectra were also recorded on a JEOL DX-303 (JMS-
AX500) mass spectrometer equipped with an EI probe under
the PIM. A Shimadzu GC-2010 gas chromatograph equipped
with a flame ionization detector was used isothermally at 270 or
300 ЊC fitted with a chemically bonded fused silica capillary
column (25QC3/BPX5; 25 m × 0.32 mm i.d.; film thickness,
0.25 µm; SGE). The apparatus used for medium-pressure liquid
chromatography (MPLC) consisted of a Shimamura YRD-880
Supporting evidence for the structures of the 24,20-γ-
lactones (8 and 17) was achieved by alkaline hydrolysis with
10% methanolic KOH, followed by acidification with 10%
H2SO4 to give the (20S)-20-hydroxy-carboxylic acids (24 and
25). The free acids 24 and 25 were then converted into the corre-
sponding C-24 methyl esters (26 and 27) with diazomethane,
while esterification with p-toluenesulfonic acid in methanol
reproduced the original γ-lactones (8 and 17), demonstrated in
1
the H- and 13C-NMR and GC-MS analyses.6a,8 Of particular
interest is the appearance of a singlet signal occurring at
ca. 1.47 (or 1.38) ppm due to the 21-methyl protons and the
1
absence of a singlet signal at ca. 3.65 ppm in the H-NMR of
the C-24 methyl ester. In the 13C-NMR, a characteristic signal
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 1 3 – 1 0 1 8
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RI-detector (Tokyo, Japan) and a uf-3040s chromatographic
pump using silica gel 60 (230–400 mesh; Nacalai Tesque, Kyoto,
Japan) as the adsorbent and benzene–EtOAc mixtures as the
eluent. Thin-layer chromatography (TLC) was performed on
precoated silica gel plates (0.25 mm layer thickness; E.Merck,
Darmstadt, Germany) using hexane–EtOAc–acetic acid mix-
tures (80 : 20 : 1–20 : 80 : 1, v/v/v) as the developing solvent.
DCP N-oxide was prepared according to the procedure
of Rousseau and Robins.9 The method of Lindsey et al. was
used for the preparation of 5,10,15,20-tetrakis-(2,4,6-tri-
methylphenyl)-porphyrin.10 The ruthenium porphyrin complex,
Ru(TMP)CO, was prepared by a slight modification of the
method of Rillema et al.11
Ϫ CH2CO, 8), 271 (M Ϫ H2O Ϫ S.C., 71), 244 (M Ϫ H2O Ϫ
S.C. Ϫ part of ring D, 60), 229 (M Ϫ H2O Ϫ S.C. Ϫ ring D,
100), 201 (31); HR-MS (EI-PIM), Calc. for C24H36O4 [M]ϩ:
388.2614. Found: m/z, 388.2592.
Oxidation products of methyl 12ꢁ-acetoxy-3-oxo-5ꢀ-cholan-24-
oate 2
Methyl 12ꢁ-acetoxy-5ꢀ-hydroxy-3-oxocholan-24-oate 10.
Isolated from the reaction product of 2 as a colorless amorph-
ous solid (Fr. 2); crystallized from toluene–hexane; mp 129–
1
133 ЊC; IR νmax/cmϪ1 1723 (C᎐O), 3505 (OH); H-NMR δ 0.77
᎐
(3H, s, 18-H3), 0.80 (3H, d, J 6.2 Hz, 21-H3), 0.98 (3H, s, 19-H3),
2.07 (3H, s, COCH3), 3.66 (3H, s, COOCH3), 5.14 (1H, br m,
12β-H); LR-MS m/z 462 (Mϩ, <1%), 419 (6), 402 (M Ϫ AcOH,
3), 387 (M Ϫ AcOH Ϫ CH3, 18), 384 (M Ϫ AcOH Ϫ H2O, 11),
332 (100), 287 (M Ϫ AcOH Ϫ S.C., 7), 269 (M Ϫ AcOH Ϫ
H2O Ϫ S.C., 21), 251, (M Ϫ AcOH Ϫ 2H2O Ϫ S.C., 8), 227
(M Ϫ AcOH Ϫ H2O Ϫ S.C. Ϫ ring D, 10), 217 (35), 209
(M Ϫ AcOH Ϫ 2H2O Ϫ S.C. Ϫ ring D, 26); HR-MS (ESI-
PIM), Calc. for C27H42O6Na [M ϩ Na]ϩ: 485.2879. Found: m/z,
485.2848.
General procedure for the oxidation using DCP N-oxide/Ru-
porphyrin/HBr
To a magnetically stirred benzene solution (3 ml) of bile acid
(1.3 mmol) and molecular sieves (850 mg, 4 Å), DCP N-oxide
(630 mg, 3.8 mmol), Ru(TMP)CO (5 mg, 5.5 µmol) and HBr
(50 µl) were successively added, and the mixture was stirred at
50 ЊC for 60 h for 1–5 (the reaction was monitored by TLC).
The reaction product was extracted with benzene, and the com-
bined extracts were washed with water, dried with Drierite, and
evaporated to dryness under reduced pressure. The residue was
chromatographed on a column of silica gel (60 g) eluting with
benzene–EtOAc (8 : 2–5 : 5, v/v) mixtures and then by MPLC
on silica gel (230–400 mesh, 21 g) eluting with benzene–EtOAc
(9 : 1, v/v).
Methyl 12ꢁ-acetoxy-3-oxo-4-cholen-24-oate 11. Isolated from
the reaction product of 2 as a colorless amorphous solid (Fr. 1);
crystallized from ethanol–hexane; mp 120–123 ЊC [lit.13a mp,
124–126 ЊC]; IR νmax/cmϪ1 1711, 1753 (C᎐O), 1654, 2965 (C᎐C);
᎐
᎐
1H-NMR δ 0.79 (3H, s, 18-CH3), 0.80 (3H, s, J 6.5 Hz, 21-CH3),
1.16 (3H, s, 19-CH3), 3.66 (3H, s, COOCH3), 5.12 (1H, m, 12β-
H), 5.74 (1H, s, 4-CH); LR-MS m/z 444 (Mϩ, 1%), 402 (1),
384 (M Ϫ AcOH, 32), 369 (M Ϫ AcOH Ϫ CH3, 11), 269
(M Ϫ AcOH Ϫ S.C., 100), 261 (26), 251, (M Ϫ AcOH Ϫ H2O Ϫ
S.C., 20), 231 (24), 227 (M Ϫ AcOH Ϫ S.C. Ϫ ring D, 25), 211
(29), 209 (M Ϫ AcOH Ϫ H2O Ϫ S.C. Ϫ ring D, 26).
Oxidation products of methyl 3-oxo-5ꢀ-cholan-24-oate 1
Methyl 5ꢀ-hydroxy-3-oxocholan-24-oate 6. Isolated from the
reaction product of 1 as a colorless amorphous solid (Fr. 2);
crystallized from aq. acetone; mp 170–172 ЊC; IR νmax/cmϪ1
1702, 1741 (C᎐O), 3382 (OH); 1H-NMR δ 0.68 (3H, s, 18-CH ),
᎐
3
Oxidation products of methyl 7ꢁ-acetoxy-3-oxo-5ꢀ-cholan-24-
oate 3
0.91 (3H, d, J 6.5 Hz, 21-CH3), 1.00 (3H, s, 19-CH3), 3.67
(3H, s, COOCH3); LR-MS m/z 386 (M Ϫ H2O, 23%), 355
(M Ϫ 2H2O Ϫ CH3, 3), 329 (M Ϫ H2O Ϫ CH3 Ϫ CH2CO, 6),
229 (M Ϫ H2O Ϫ S.C. Ϫ ring D, 100), 211 (M Ϫ 2H2O Ϫ S.C. Ϫ
ring D, 24); HR-MS (ESI-PIM), Calc. for C25H40O4Na
[M ϩ Na]ϩ: 427.2824. Found: m/z, 427.2810.
Methyl 7ꢁ-acetoxy-5ꢀ-hydroxy-3-oxocholan-24-oate 12. Iso-
lated from the reaction product of 3 as a colorless amorphous
solid (Fr. 2); crystallized from EtOAc–hexane; mp 177–180 ЊC;
1
IR νmax/cmϪ1 1732, 1719 (C᎐O), 3348 (OH); H-NMR δ 0.69
᎐
(3H, s, 18-H3), 0.93 (3H, d, J 6.2 Hz, 21-H3), 1.01 (3H, s, 19-H3),
2.05 (3H, s, COCH3), 3.67 (3H, s, COOCH3), 5.01 (1H, m,
7β-H); LR-MS m/z 384 (M Ϫ AcOH Ϫ H2O, 16%), 269
(M Ϫ AcOH Ϫ H2O Ϫ S.C., 38), 249 (M Ϫ AcOH Ϫ 2H2O Ϫ
S.C., 12), 227 (M Ϫ AcOH Ϫ H2O Ϫ S.C. Ϫ ring D, 14); HR-
MS (ESI-PIM), Calc. for C27H42O6Na [M ϩ Na]ϩ: 485.2879.
Found: m/z, 485.2891.
Methyl 3-oxo-4-cholen-24-oate 7. Isolated from the reaction
product of 1 as a colorless amorphous solid (Fr. 1); crystallized
from aq. acetone; mp 124–126 ЊC [lit.12 mp, 125–126 ЊC]; IR
νmax/cmϪ1 1677, 1736 (C᎐O), 1616, 3008 (C᎐C); 1H-NMR δ 0.71
(3H, s, 18-CH3), 0.91 (3H, s, J 6.2 Hz, 21-CH3), 1.18 (3H, s,
19-CH3), 3.67 (3H, s, COOCH3), 5.73 (1H, s, 4-CH); LR-MS
m/z 386 (M, 24%), 371 (M Ϫ CH3, 5), 344 (M Ϫ CH2CO, 8), 329
(M Ϫ CH3 Ϫ CH2CO, 8), 313 (4), 271 (M Ϫ S.C. Ϫ ring D, 14),
229 (M Ϫ S.C. Ϫ ring D, 100), 211 (M Ϫ H2O Ϫ S.C. Ϫ ring D,
21).
᎐
᎐
Methyl 7ꢁ-acetoxy-3-oxo-4-cholen-24-oate 13. Isolated from
the reaction product of 3 as colorless prisms (Fr. 1); crystallized
from aq. methanol; mp 207–208 ЊC [lit.13b mp, 206–208 ЊC]; IR
νmax/cmϪ1 1732 (C᎐O), 1663, 3069 (C᎐C); 1H-NMR δ 0.71 (3H,
᎐
᎐
(20S )-3-Oxo-5ꢀ-cholan-O-24,20-lactone 8. Isolated from the
reaction product of 1 as a noncrystalline substance (Fr. 3); IR
s, 18-H3), 0.92 (3H, d, J 6.2 Hz, 21-H3), 1.20 (3H, s, 19-H3),
2.03 (3H, s, COCH3), 3.66 (3H, s, COOCH3), 5.00 (1H, m, 7β-
H), 5.69 (1H, s, 4-CH); LR-MS m/z 429 (M Ϫ CH3, 2%),
384 (M Ϫ AcOH, 16), 369 (M Ϫ AcOH Ϫ CH3, 5), 269
(M Ϫ AcOH Ϫ S.C., 30), 227 (M Ϫ AcOH Ϫ S.C. Ϫ ring D,
20).
1
νmax/cmϪ1 1735, 1771 (C᎐O); H-NMR δ 0.84 (3H, s, 18-CH ),
᎐
3
1.03 (3H, s, 19-CH3), 1.46 (3H, s, 21-CH3); LR-MS m/z 372
(M, 37%), 354 (M Ϫ H2O, 24), 339 (M Ϫ H2O Ϫ CH3, 63),
321 (M Ϫ 2H2O Ϫ CH3, 8), 302 (87), 273 (M Ϫ S.C., 51), 246
(M Ϫ S.C. Ϫ part of ring D, 95), 231 (M Ϫ S.C. Ϫ ring D, 95),
213 (M Ϫ H2O Ϫ S.C. Ϫ ring D, 100); HR-MS (EI-PIM), Calc.
for C24H36O3 [M]ϩ: 372.2664. Found: m/z, 372.2642.
(20S )-7ꢁ-Acetoxy-3-oxo-5ꢀ-cholan-O-24,20-lactone 14. Iso-
lated from the reaction product of 3 as a colorless amorphous
solid (Fr. 3); crystallized from aq.methanol; mp 187–190 ЊC;
(20S )-5ꢀ-Hydroxy-3-oxocholan-O-24,20-lactone 9. Isolated
from the reaction product of 1 as a colorless amorphous solid
IR νmax/cmϪ1 1H-NMR δ 0.84 (3H, s, 18-H3), 1.05 (3H, s,
;
19-H3), 1.47 (3H, s, 21-H3), 2.04 (3H, s, COCH3), 4.98 (1H, m,
7β-H); LR-MS m/z 430 (Mϩ, 2%), 370 (M Ϫ AcOH, 4), 337
(M Ϫ AcOH Ϫ H2O Ϫ CH3, 38), 299 (32), 271 (M Ϫ AcOH Ϫ
S.C., 20), 229 (M Ϫ AcOH Ϫ S.C. Ϫ ring D, 15), 211
(M Ϫ AcOH Ϫ H2O Ϫ S.C. Ϫ ring D, 36); HR-MS (EI-PIM),
Calc. for C26H38O5 [M]ϩ: 430.2719. Found: m/z, 430.2716.
(Fr. 4); crystallized from acetone–hexane; mp 164–166 ЊC;
1
IR νmax/cmϪ1 1736, 1773 (C᎐O), 3414 (OH); H-NMR δ 0.84
᎐
(3H, s, 18-CH3), 1.01 (3H, s, 19-CH3), 1.47 (3H, s, 21-CH3);
LR-MS m/z 370 (M Ϫ H2O, 47%), 355 (M Ϫ H2O Ϫ CH3, 13),
328 (M Ϫ H2O Ϫ CH2CO, 43), 313 (M Ϫ H2O Ϫ CH3
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 1 3 – 1 0 1 8
1016

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(20S )-7ꢁ-Acetoxy-5ꢀ-hydroxy-3-oxocholan-O-24,20-lactone
15. Isolated from the reaction product of 3 as a colorless
amorphous solid (Fr. 4); crystallized from EtOAc–hexane; mp
m/z 382 (M Ϫ 2AcOH Ϫ H2O, 7%), 330 (53), 267 (M Ϫ 2AcOH
Ϫ H2O Ϫ S.C., 10), 249 (M Ϫ 2AcOH Ϫ H2O Ϫ S.C., 5),
240 (M Ϫ 2AcOH – H2O Ϫ S.C. Ϫ part of ring D, 8), 225
(M Ϫ 2AcOH Ϫ H2O Ϫ S.C. Ϫ ring D, 10), 207 (100); HR-MS
(ESI-PIM), Calc. for C29H44O8Na [M ϩ Na]ϩ: 543.2934.
Found: m/z, 543.2963.
232–234 ЊC; IR νmax/cmϪ1 1762, 1715 (C᎐O), 3449 (OH);
᎐
1H-NMR δ 0.85 (3H, s, 18-H3), 1.03 (3H, s, 19-H3), 1.47 (3H, s,
21-H3), 2.05 (3H, s, COCH3), 5.02 (1H, m, 7β-H); LR-MS m/z
368 (M Ϫ AcOH Ϫ H2O, 7%), 353 (M Ϫ AcOH Ϫ H2O Ϫ CH3,
10), 335 (M Ϫ AcOH Ϫ 2H2O Ϫ CH3, 6), 281 (74), 269
(M Ϫ AcOH Ϫ H2O Ϫ S.C., 32), 255 (32), 227 (M Ϫ AcOH–
H2O Ϫ S.C. Ϫ ring D, 7); HR-MS (ESI-PIM), Calc. for
C26H38O6Na [M ϩ Na]ϩ: 469.2566. Found: m/z, 469.2567.
Methyl 7ꢁ,12ꢁ-diacetoxy-3-oxo-4-cholen-24-oate 21. Isolated
from the reaction product of 5 as a colorless amorphous solid
(Fr. 1); crystallized from aq. methanol; mp 186–189 ЊC [lit.13c
mp, 183–186 ЊC]; IR νmax/cmϪ1 1744 (C᎐O), 1690, 2977 (C᎐C);
᎐
᎐
1H-NMR δ 0.79 (3H, s, 18-H3), 0.81 (3H, d, J 6.2 Hz, 21-H3),
1.19 (3H, s, 19-H3), 2.06, 2.09 (each 3H, s, COCH3), 3.66 (3H, s,
COOCH3), 5.02 (1H, m, 7β-H), 5.11 (1H, m, 12β-H), 5.70
(1H, s, 4-CH); LR-MS m/z 382 (M Ϫ 2AcOH, 7%), 267
(M Ϫ 2AcOH Ϫ S.C., 100), 249 (M Ϫ 2AcOH Ϫ H2O Ϫ S.C.,
11), 240 (M Ϫ 2AcOH Ϫ S.C. Ϫ part of ring D, 8), 225
(M Ϫ 2AcOH Ϫ S.C. Ϫ ring D, 18), 207 (35).
Oxidation products of methyl 7ꢀ-acetoxy-3-oxo-5ꢀ-cholan-24-
oate 4
Methyl 7ꢀ-acetoxy-5ꢀ-hydroxy-3-oxocholan-24-oate 16. Iso-
lated from the reaction product of 4 as colorless thin plates
(Fr. 1); crystallized from EtOAc–hexane; mp 169–171 ЊC; IR
1
νmax/cmϪ1 1731, 1756 (C᎐O), 3423 (OH); H-NMR δ 0.72 (3H,
᎐
s, 18-H3), 0.92 (3H, d, J 6.5 Hz,21-H3), 1.04 (3H, s, 19-H3),
2.00 (3H, s, COCH3), 3.66 (3H, s, COOCH3), 4.62 (1H, br m,
7α-H); LR-MS m/z 384 (M Ϫ AcOH Ϫ H2O, 5%), 281 (15), 267
(M Ϫ AcOH Ϫ H2O Ϫ S.C., 13), 227 (M Ϫ AcOH Ϫ H2O Ϫ
S.C. Ϫ ring D, 5), 207 (100); HR-MS (ESI-PIM), Calc. for
C27H42O6Na [M ϩ Na]ϩ: 485.2879. Found: m/z, 485.2865.
(20S )-7ꢁ,12ꢁ-Diacetoxy-5ꢀ-hydroxy-3-oxocholan-O-24,20-
lactone 22. Isolated from the reaction product of 5 as a noncrys-
talline substrate (Fr. 4); IR νmax/cmϪ1 1688, 1719 (C᎐O), 3402
᎐
(OH); 1H-NMR δ 0.88 (3H, s, 18-H3), 1.01 (3H, s, 19-H3), 1.38
(3H, s, 21-H3), 2.08, 2.14 (each 3H, s, COCH3), 5.05 (1H, m, 7β-
H), 5.27 (1H, m, 12β-H); LR-MS m/z 426 (M Ϫ AcOH Ϫ H2O,
3), 366 (M Ϫ 2AcOH Ϫ H2O, 44), 267 (M Ϫ 2AcOH Ϫ H2O Ϫ
S.C., 100), 243 (19), 240 (M Ϫ 2AcOH Ϫ H2O Ϫ S.C. Ϫ part of
ring D, 79), 225 (M Ϫ 2AcOH Ϫ H2O Ϫ S.C. Ϫ ring D, 66);
HR-MS (ESI-PIM), Calc. for C28H40O8Na [M ϩ Na]ϩ:
527.2621. Found: m/z, 527.2652.
(20S )-7ꢀ-Acetoxy-3-oxo-5ꢀ-cholan-O-24,20-lactone 17. Iso-
lated from the reaction product of 4 as a noncrystalline
1
substance (Fr. 2); IR νmax/cmϪ1 1680, 1762 (C᎐O); H-NMR
᎐
δ 0.88 (3H, s, 18-H3), 1.08 (3H, s, 19-H3), 1.46 (3H, s, 21-H3),
2.00 (3H, s, COCH3), 4.97 (1H, br m, 7α-H); LR-MS m/z 370
(M Ϫ AcOH, 21%), 355 (M Ϫ AcOH Ϫ CH3, 17), 300 (58), 281
(66), 271 (M Ϫ AcOH Ϫ S.C., 60), 221 (M Ϫ AcOH Ϫ H2O Ϫ
S.C. Ϫ ring D, 30); HR-MS (ESI-PIM), Calc. for C26H38O5Na
[M ϩ Na]ϩ: 453.2617. Found: m/z, 453.2604.
(20S )-7ꢁ,12ꢁ-Diacetoxy-3-oxo-4-cholen-O-24,20-lactone 23.
Isolated from the reaction product of 5 as colorless prisms
(Fr. 3); crystallized from aq. methanol; mp 251–254 ЊC; IR
νmax/cmϪ1 1711 (C᎐O), 1630, 3000 (C᎐C); 1H-NMR δ 0.94 (3H,
᎐
᎐
s, 18-H3), 1.04 (3H, s, 19-H3), 1.38 (3H, s, 21-H3), 2.07, 2.14
(each 3H, s, COCH3), 5.00 (1H, m, 7β-H), 5.22 (1H, m, 12β-H),
5.71 (1H, s, 4-CH); LR-MS m/z 366 (M Ϫ 2AcOH, 22%),
335 (13), 298 (72), 267 (M Ϫ 2AcOH Ϫ S.C., 100), 240
(M Ϫ 2AcOH Ϫ S.C. Ϫ part of ring D, 81), 225 (M Ϫ 2AcOH
Ϫ S.C. Ϫ ring D, 30), 221 (14); HR-MS (EI-PIM), Calc. for
C28H38O7 [M]ϩ: 486.2618. Found: m/z, 486.2591.
(20S )-7ꢀ-Acetoxy-5ꢀ-hydroxy-3-oxocholan-O-24,20-lactone
18. Isolated from the reaction product of 4 as a colorless
amorphous solid (Fr. 4); crystallized from EtOAc–hexane; mp
233–236 ЊC; IR νmax/cmϪ1 1745, 1779 (C᎐O), 3442 (OH);
᎐
1H-NMR δ 0.90 (3H, s, 18-H3), 1.24 (3H, s, 19-H3), 1.46 (3H, s,
21-H3), 2.04 (3H, s, COCH3), 4.62 (1H, br m, 7α-H), 5.78
(1H, s, 4-CH); LR-MS m/z 368 (M Ϫ AcOH Ϫ H2O, 17%), 353
(M Ϫ AcOH Ϫ H2O Ϫ CH3, 15), 335 (M Ϫ AcOH Ϫ 2H2O Ϫ
CH3, 10), 281 (27), 269 (M Ϫ AcOH Ϫ H2O Ϫ S.C., 32), 255
(32), 227 (M Ϫ AcOH Ϫ H2O Ϫ S.C. Ϫ ring D, 17); HR-MS
(ESI-PIM), Calc. for C26H38O6Na [M ϩ Na]ϩ: 469.2566.
Found: m/z, 469.2563.
(20S )-20-Hydroxy-3-oxo-5ꢀ-cholan-24-oic acid 24 and its C-24
methyl ester 26
A 5% methanolic KOH solution of the lactone 8 (30 mg) was
refluxed for 2 h. Most of the solvent was evaporated under
reduced pressure. The residual oil dissolved in water was care-
fully neutralized with 5% H2SO4 while cooling in an ice bath.
The precipitate was filtered, washed with water, and recrystal-
lized from EtOAc–hexane to give the title compound 24 as a
(20S )-7ꢀ-Acetoxy-3-oxo-4-cholen-O-24,20-lactone 19. Iso-
lated from the reaction product of 4 as a colorless amorphous
solid (Fr. 3); crystallized from EtOAc–hexane; mp 222–226 ЊC;
1
IR νmax/cmϪ1 1701, 1742 (C᎐O), 1680, 2965 (C᎐C); H-NMR
colorless amorphous solid; mp 158–162 ЊC; yield, 25 mg, 70%;
᎐
᎐
1
δ 0.88 (3H, s, 18-H3), 1.05 (3H, s, 19-H3), 1.47 (3H, s, 21-H3),
2.01 (3H, s, COCH3), 4.64 (1H, br m, 7α-H); LR-MS m/z
368 (M Ϫ AcOH, 16%), 353 (M Ϫ AcOH Ϫ CH3, 10), 335
(M Ϫ AcOH Ϫ H2O Ϫ CH3, 5), 281 (17), 269 (M Ϫ AcOH Ϫ
S.C., 17), 255 (15), 227 (M Ϫ AcOH Ϫ S.C. Ϫ ring D, 17);
HR-MS (ESI-PIM), Calc. for C26H36O5Na [MϩNa]ϩ:
451.2460. Found: m/z, 451.2438.
IR νmax/cmϪ1 1735, 1771 (C᎐O), 3410 (OH); H-NMR (as the
᎐
C-24 methyl ester 26 prepared by treating 24 with diazo-
methane) δ 0.82 (3H, s, 18-CH3), 1.25 (3H, s, 19-CH3), 1.25 (3H,
s, 21-CH3), 3.67 (3H, s, COOCH3); LR-MS m/z (as the methyl
ester 26) 384 (M Ϫ H2O, 35%), 371 (M Ϫ H2O Ϫ CH3, 35),
368 (M Ϫ 2H2O, 10), 281 (70), 273 (M Ϫ H2O Ϫ S.C., 3), 255
(M Ϫ 2H2O Ϫ S.C., 7), 221, (100); HR-MS (EI-PIM) (as the
methyl ester 26), Calc. for C25H40O4 [M]ϩ: 404.2927. Found:
m/z, 404.2941.
Oxidation products of methyl 7ꢁ,12ꢁ-diacetoxy-3-oxo-5ꢀ-
cholan-24-oate 5
(20S )-7ꢀ,20-Dihydroxy-3-oxo-5ꢀ-cholan-24-oic acid 25 and its
C-24 methyl ester 27
Methyl
7ꢁ,12ꢁ-diacetoxy-5ꢀ-hydroxy-3-oxocholan-24-oate
20. Isolated from the reaction product of 5 as a colorless
amorphous solid (Fr. 2); crystallized from EtOAc–hexane; mp
Alkaline hydrolysis of the lactone 17 (30 mg) with 5%
methanolic KOH followed by acidification with 5% H2SO4, as
described for the preparation of 24, gave the free acid 25, which
was recrystallized from EtOAc–hexane as a colorless amorph-
ous solid; mp 174–176 ЊC; yield, 18 mg, 66%; IR νmax/cmϪ1
175–177 ЊC; IR νmax/cmϪ1 1772, 1680 (C᎐O), 3399 (OH);
᎐
1H-NMR δ 0.77 (3H, s, 18-H3), 0.82 (3H, d, J 5.4 Hz, 21-H3),
1.00 (3H, s, 19-H3), 2.09, 2.11 (each 3H, s, COCH3), 3.66 (3H, s,
COOCH3), 5.02 (1H, m, 7β-H), 5.16 (1H, m, 12β-H); LR-MS
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 1 3 – 1 0 1 8
1017

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1
(d ) R. Breslow, B. Gabriele and J. Yang, Tetrahedron Lett., 1998, 39,
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Chem. Soc., 1993, 115, 11648; ( f ) A. M. S. Chauhan, A. S.
Kandadai, N. Jain and A. Kumar, Chem. Pharm, Bull., 2003, 51,
1345.
5 (a) H. Ohtake, T. Higuchi and M. Hirobe, Heterocycles, 1995, 40,
867; (b) T. Shingaki, K. Miura, T. Higuchi, M. Hirobe and
T. Nagano, Chem. Commun., 1997, 861; (c) H. Ohtake, T. Higuchi
and M. Hirobe, J. Am. Chem. Soc., 1992, 114, 10660.
6 (a) C. Cerrè, A. F. Hofmann, C. D. Schteingart, W. Jia and
D. Maltby, Tetrahedron, 1997, 53, 435; (b) T. Iida, T. Yamaguchi,
R. Nakamori, M. Hikosaka, N. Mano, J. Goto and T. Nambara,
J. Chem. Soc., Perkin Trans. 1, 2001, 2229.
7 T. Iida, S. Ogawa, K. Shiraishi, G. Kakiyama, T. Goto, N. Mano and
J. Goto, ARKIVOC, 2003(viii), 170.
8 T. Iida, M. Hikosaka, G. Kakiyama, K. Shiraishi, C. D. Schteingart,
L. R. Hagey, H-T. Ton-Nu, A. F. Hofmann, N. Mano, J. Goto and
T. Nambara, Chem. Pharm. Bull., 2002, 50, 1327.
9 R. J. Rousseau and R. K. Robins, J. Heterocycl. Chem., 1965, 2, 196.
10 J. S. Lindsey, I. C. Schreiman, H. C. Hsu, P. C. Kearney and A. M.
Marguerettaz, J. Org. Chem., 1987, 52, 827.
1735, 1771 (C᎐O), 3433, 3445 (OH); H-NMR (as the C-24
᎐
methyl ester 27 prepared by treating 25 with diazomethane)
δ 0.85 (3H, s, 18-CH3), 1.25 (3H, s, 19-CH3), 1.25 (3H, s, 21-
CH3), 3.47 (1H, dt, J1 12.2 Hz, J2 5.4 Hz, 7α-H), 3.67 (3H, s,
COOCH3); LR-MS m/z (as the methyl ester 27) 420 (Mϩ, 4%),
402 (M Ϫ H2O, 10), 384 (M Ϫ 2H2O, 10), 287 (M Ϫ H2O Ϫ
S.C., 18), 272 (M Ϫ 2H2O Ϫ S.C., 100), 211 (47); HR-MS (EI-
PIM) (as the methyl ester 27), Calc. for C25H40O5 [M]ϩ:
420.2876. Found: m/z, 420.2861.
Acknowledgements
We would like to express our thanks to Mr. Daniel C. Aspleaf
for his helpful comments. This work was supported in part by a
Grant-in-Aid for Scientific Research from the Ministry of
Education, Science, Sports and Culture of Japan.
11 (a) D. P. Rillema, J. K. Nagle, L. F. Barringer, Jr. and T. J. Meyer,
J. Am. Chem. Soc., 1981, 103, 56; (b) J. T. Groves and R. Quinn,
Inorg. Chem., 1984, 23, 3844.
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O r g . B i o m o l . C h e m . , 2 0 0 4 , 2, 1 0 1 3 – 1 0 1 8
1018