Potential bile acid metabolites. 25. Synthesis and chemical properties of stereoisomeric 3alpha,7alpha,16- and 3alpha,7alpha,15-trihydroxy-5beta-cholan-24-oic acids.

Article abstract of DOI:10.1248/cpb.50.1327

Epimeric 3alpha,7alpha,16- and 3alpha,7alpha,15-trihydroxy-5beta-cholan-24-oic acids and some related compounds were synthesized from chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), respectively. The key reaction involved one-step remote oxyfunctionalization of unactivated methine carbons at C-17 of CDCA and at C-14 of UDCA as their methyl ester-peracetate derivatives with dimethyldioxirane (DMDO). After dehydration of the resulting 17alpha- and 14alpha-hydroxy derivatives with POCl(3) or conc. H(2)SO(4), the respective Delta(16)- and Delta(14)-unsaturated products were subjected to hydration via hydroboration followed by oxidation to yield the 3,7,16- and 3,7,15-triketones, respectively. Stereoselective reduction of the respective triketones with tert-butylamine-borane complex afforded the epimeric 3alpha,7alpha,16- or 3alpha,7alpha,15-trihydroxy derivatives exclusively. A facile formation of the corresponding epsilon-lactones between the side chain carboxyl group at C-24 and the 16alpha- (or 16beta-) hydroxyl group in bile acids is also clarified.

Full text of DOI:10.1248/cpb.50.1327

October 2002
Chem. Pharm. Bull. 50(10) 1327—1334 (2002)
1327
Potential Bile Acid Metabolites. 25. Synthesis and Chemical Properties
a a
a a
b
of Stereoisomeric 3 ,7 ,16- and 3 ,7 ,15-Trihydroxy-5 -cholan-24-oic
Acids1)
Takashi IIDA, ,a Masahiro HIKOSAKA,a Genta KAKIYAMA,a Keisuke SHIRAISHI,a
*
Claudio D. SCHTEINGART,b Lee R. HAGEY,b Huong-Thu TON-NU,b Alan F. HOFMANN,b Nariyasu MANO,c
c
Junichi GOTO,c and Toshio NAMBARA
a College of Humanities and Sciences, Nihon University; Sakurajousui, Setagaya-ku, Tokyo 156–8550, Japan: b Department
of Medicine, University of California; San Diego, La Jolla, California 92093, U.S.A.: and c Graduate School of
Pharmaceutical Sciences, Tohoku University; Aobayama, Sendai 980–8578, Japan.
Received April 11, 2002; accepted June 17, 2002
Epimeric 3a,7a,16- and 3a,7a,15-trihydroxy-5b-cholan-24-oic acids and some related compounds were syn-
thesized from chenodeoxycholic acid (CDCA) and ursodeoxycholic acid (UDCA), respectively. The key reaction
involved one-step remote oxyfunctionalization of unactivated methine carbons at C-17 of CDCA and at C-14 of
UDCA as their methyl ester-peracetate derivatives with dimethyldioxirane (DMDO). After dehydration of the re-
sulting 17a- and 14a-hydroxy derivatives with POCl3 or conc. H2SO4, the respective D16- and D14-unsaturated
products were subjected to hydration via hydroboration followed by oxidation to yield the 3,7,16- and 3,7,15-
triketones, respectively. Stereoselective reduction of the respective triketones with tert-butylamine-borane com-
plex afforded the epimeric 3a,7a,16- or 3a,7a,15-trihydroxy derivatives exclusively. A facile formation of the
corresponding e-lactones between the side chain carboxyl group at C-24 and the 16a- (or 16b-) hydroxyl group
in bile acids is also clarified.
Key words remote oxyfunctionalization; dimethyldioxirane; 3a,7a,16-trihydroxy-5b-cholan-24-oic acid; 3a,7a,15-trihydroxy-
5b-cholan-24-oic acid; e-lactone
The 16- and 15-hydroxy derivatives of chenodeoxycholic lithocholic acid (3a-hydroxy-5b-cholan-24-oic acid: LCA)
acid (3a,7a-dihydroxy-5b-cholan-24-oic acid: CDCA; 1), and DCA using a ferrous ion-molecular oxygen system. In
and deoxycholic acid (3a,12a-dihydroxy-5b-cholan-24-oic more recent works, the 15b-hydroxylation of LCA and DCA
acid: DCA), are of sustained interest in synthetic, biological, has been attained by microbiological transformation employ-
physiological, and metabolic studies, because they are major ing Penicillium species, Rhizoctonia solani and Absidia
bile acids in some vertebrates. 3a,12a,16a-Trihydroxy-5b- coerulea14) or Cunninghamella blakesleena St-22.15,16)
cholan-24-oic acid, named as “pythocholic acid”, was iso-
To our knowledge, however, chemical and/or microbiolog-
lated some years ago by Haslewood and Wootton from cer- ical syntheses of 16- and 15-hydroxy derivatives of a primary
tain primitive snakes (boas and pythons), including Cylin- bile acid, CDCA, have hitherto been unreported. For our se-
drophis.2,3) Hagey et al. have recently reported the existence ries of studies on new and uncommon potential bile acids
of “avicholic acid”, 3a,7a,16a-trihydroxy acid, as a primary and their metabolites, we report here an effective, short step
bile acid in many birds such as herons (Ardeidae), pelicans synthesis of epimeric 3a,7a,16- (3, 4) and 3a,7a,15-trihy-
(Pelecanidae), and owls (Tytonidae).4—8) Meanwhile, the droxy-5b-cholan-24-oic acids (5, 6), as well as a related
15a-hydroxy derivative of CDCA (1) and its C-24 sulfonate stereoisomer (18), starting from 1 or ursodeoxycholic acid
analogue have also been shown to be major bile acids in mar- (3a,7b-dihydroxy-5b-cholan-24-oic acid: UDCA; 2), respec-
supials9) and in the liver of hamsters,10) respectively.
tively (Chart 1). In addition, the differences in the chemical
However, a definitive structural determination of the 16- properties between the 16- and 15-hydroxy bile acids were
and 15-hydroxy bile acids, particularly for the stereochemical also clarified.
configuration of a hydroxyl group at the C-16 and -15 posi-
tions, remains uncertain, due to the unavailability of authen- Results and Discussion
tic reference compounds. Unequivocal proof of their identifi-
Introduction of an oxygen-containing function at the C-16
cation requires chemical synthesis and demonstration of and -15 positions of the five-membered D-ring in CDCA (1)
identity of the isolated compounds with synthetic ones. The has not yet been accomplished, probably due to strong shield-
availability of these uncommon bile acids as authentic speci- ings not only by the b-attached C5 alkyl side chain, but also
mens also provides an unique opportunity to study the chem- by the axially-oriented 7a-hydroxyl group.
ical, biological, and physicochemical properties of such or-
ganic molecules.
As outlined in Charts 2 and 3, a key strategic point in the
present work was to make use of 17a- and 14a-hydroxy in-
Chemical and/or microbiological syntheses of some of 16- termediates (9a, 10a), which were attained in one-step from
and 15-hydroxy bile acids have been reported by several the methyl ester-diacetate derivatives (7a, 8a) of 1 and 2, re-
groups of workers. Beque et al.11) have reported that the ther- spectively. Application of our recent works17,18) involving a
molysis of peroxy 5b-cholanoic acid in n-octane gives a highly efficient, stereoselective remote oxyfunctionalization
mixture of 16a- and 16b-hydroxy-5b-cholanes. Kimura et of unactivated methine carbons in steroids with a powerful
al.12,13) have reported chemical synthesis of 15a-hydroxy oxidant, dimethyldioxirane (DMDO), has led to direct inser-
* To whom correspondence should be addressed. e-mail: takaiida@chs.nihon-u.ac.jp
© 2002 Pharmaceutical Society of Japan

1328
Vol. 50, No. 10
Chart 1
tion of an oxygen atom at the methine protons of C-17 in 7a trum showed the 21-methyl proton at 1.69 ppm as a singlet,
and of C-14 in 8a. Thus, when 7a was treated with a freshly while two olefinic quaternary carbon signals were observed
prepared solution of DMDO (0.32 mol) in CHCl3 for 24 h at at 122.6 and 144.2 ppm in the 13C-NMR. Elimination of
room temperature, the desired 17a-hydroxy ester (9a) was the 14a-hydroxyl group in the ester 10a was successfully
isolated in 13% yield, after careful purification of the oxida- achieved by treating with conc. H2SO4 in dioxane at room
tion product by normal-phase (NP) medium-pressure liquid temperature overnight to give the D14-ester (13a) exclusively
chromatography (MPLC) on silica gel, eluting with CHCl3– (73% isolated yield).
methanol (99 : 1, v/v). Both the experimental and work-up
Hydroboration, followed by oxidation with alkaline hydro-
procedures for the DMDO reaction were very simple and gen peroxide of the D16- and D14-esters (11a, 13a) would be
straightforward as described in detail in the Experimental expected to yield the corresponding 16a- and 15a-hydroxy
section. A similar remote oxyfunctionalization was also at- products, respectively.21) In preliminary experiments, when
tained, when the ester-peracetate (8a) was subjected to the dicyclohexylborane, disiamylborane or 9-borabicyclo[3.3.1]-
DMDO oxidation to give the 14a-hydroxy ester (10a) in nonanone (9-BBN) was employed as a hydroborating reagent
12% isolated yield. The observed difference in the regiose- to avoid simultaneous reduction of the methyl ester at C-
lectivity of oxyfunctionalization between 7a and 8a was ex- 24,12,13,22) each reagent was completely inert at 0 °C and/or
plained in terms of the steric environment as well as the de- room temperature against 11a and 13a, probably because of
gree of electron density of the target methine carbons in the a steric hindrance of the bulky dialkylboranes. By changing
substrates.17,18)
the reagent to less bulky diborane (B2H6),23) however, the hy-
Subsequent treatment of the ester (9a) in pyridine with droboration-oxidation reaction of 11a and 13a proceeded
phosphoryl chloride (POCl3) at 65 °C for 24 h led to elimina- smoothly at an elevated temperature of 50 °C and/or room
tion of the 17a-hydroxyl group. The resulting dehydration temperature for 2 h,24,25) but was accompanied by simultane-
product, which consisted of a mixture of two components ous reduction of the methyl ester at C-24 as well as hydroly-
having essentially identical mobility on TLC, was efficiently sis of the acetoxyl groups at C-3 and C-7, to produce the
separated by NP-MPLC on a column of silica gel, eluting 3a,7a,16a,24- and 3a,7b,15a,24-tetrols (14, 90% and 15,
with a mixture of toluene–Et2O (9 : 1, v/v). The less polar, 72%) in good isolated yields, respectively. The a-configura-
major component (41% isolated yield) was characterized as tion of a hydroxyl group at C-16 in 14 and at C-15 in 15 was
1
the D16-ester (11a),19) and the more polar, minor component based on the comparison of their H-NMR chemical shifts
(30%) as the D17(20)-isomers (12a). The assignment of the and signal multiplicity with those of related compounds (i.e.,
(E)-configuration (i.e., cis relationship between 18- and 21- 3a, 5a) as described below in detail.
1
methyl groups) to 12a followed from a previous H-NMR
Eventually, the desired stereoisomeric 3a,7a,16- and
finding20); the 21-methyl proton in the (E)-isomer of 17(20)- 3a,7a,15-trihydroxy esters (3a—6a) were prepared from the
dehydrocholesterol appears at 1.68 ppm, whereas that in the tetrols 14 and 15, via the intermediates, 3,7,16- and 3,7,15-
(Z)-isomer occurs at 1.55 ppm. For 12a the H-NMR spec- trioxo esters (16a, 17a), respectively. Thus, the preparation of
1

October 2002
1329
Chart 2
Chart 3
16a (or 17a) was carried out by oxidation of 14 (or 15) with conditions employed, either a- or b-alcohols, or the epimeric
Jones reagent at room temperature for 30 min and subsequent mixture may be obtained. Our exploratory experiments in
re-esterification of the resulting 3,7,16- (or 3,7,15-) trioxo which reduction of the 3,7,16-triketone (16a) with various
acid.
reducing agents such as liq. NH3/Li, Zn(BH4)2, NaBH4/
Stereoselective reduction of an oxo group to a hydroxyl in PdCl2, tert-amyl alchohol/K, or K-Selectride for preparing
steroids has been studied extensively by many investiga- methyl 3a,7a,16-trihydroxy-5b-cholan-24-oate stereoselec-
tors.26,27) Depending on both the reducing agents and reaction tively, gave an unsatisfactory result, i.e., a complicated mix-

1330
Vol. 50, No. 10
COOCH3
Table 1. 1H-NMR Chemical Shifts of Stereoisomeric Methyl 3a,7a,16- and 3a,7a,15-Trihydroxy-5b-cholan-24-oates (3a—6a)
18-CH3
19-CH3
21-CH3
3b-H
7b-H
15-H
16-H
3a
4a
Dd
5a
6a
Dd
0.67 (s)
0.87 (s)
0.20
0.67 (s)
0.95 (s)
0.28
0.89 (s)
0.91 (s)
0.02
0.90 (s)
0.95 (s)
0.05
0.95 (d, 5.4)
0.97 (d, 6.3)
0.02
0.91 (d, 5.6)
0.94 (d, 5.2)
0.03
3.43 (br m)
3.46 (br m)
0.03
3.46 (br m)
3.48 (br m)
0.02
3.84 (m)
3.88 (m)
0.04
4.03 (m)
4.18 (m)
0.15
4.01 (d, 2.7)
4.50 (br m)
0.50
3.66 (s)
3.70 (s)
0.04
3.67 (s)
3.67 (s)
0.00
4.03 (m)
4.31 (t, 5.4)
0.28
sϭsinglet, dϭdoublet, tϭtriplet, mϭmultiplet, br mϭbroad multiplet; values in parentheses refer to coupling constant (J in Hz); Dd value means the difference in the chemi-
cal shifts between 4a vs. 3a and 6a vs. 5a.
Table 2. 13C-NMR Chemical Shifts of Stereoisomeric Methyl 3a,7a,16- with tert-C4H9NH2·BH3, respectively. Usual alkaline hydrol-
and 3a,7a,15-Trihydroxy-5b-cholan-24-oates (3a—6a)
ysis of the individual 3a,7a,16- and 3a,7,15-trihydroxy es-
ters (3a—6a, 18a) with methanolic potassium hydroxide, fol-
Carbon
3a
4a
Dda)
5a
6a
Dda)
lowed by acidification with H2SO4 afforded the correspond-
ing free acids (3—6, 18) nearly quantitatively. The physical
and spectral properties of 3a,7a,16a-triols (3a) were virtu-
ally consistent with those reported recently.7,8)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
35.3
30.6
72.1
39.4
41.6
34.1
68.3
38.8
32.7
35.0
20.3
40.0
43.9
47.5
36.2
76.7
66.3
13.1
22.8
33.8
18.6
30.8
31.0
175.0
51.5
35.2
30.6
71.9
39.7
41.4
34.8
68.3
39.0
32.8
35.0
20.2
39.8
42.4
48.3
34.8
72.1
61.9
12.8
22.8
29.3
17.9
30.2
30.2
175.8
51.9
Ϫ0.1
0
Ϫ0.2
0.3
Ϫ0.2
0.7
0
0.2
0.1
0
Ϫ0.1
Ϫ0.2
Ϫ1.5
0.8
Ϫ1.4
Ϫ4.6
Ϫ4.4
Ϫ0.3
0
Ϫ4.5
Ϫ0.7
Ϫ0.6
Ϫ0.8
0.8
35.3
30.5
71.9
40.1
41.4
33.4
67.7
39.0
33.8
34.8
20.6
40.3
43.8
58.0
72.4
39.3
54.1
13.1
22.8
34.8
18.0
30.8
30.8
174.7
51.5
35.2
30.7
72.0
39.9
41.4
35.4
68.0
35.2
33.1
35.2
20.5
41.0
42.3
56.0
70.5
41.8
54.8
14.5
22.7
35.1
18.3
30.9
30.9
174.7
51.5
Ϫ0.1
0.2
0.1
Ϫ0.2
0
2.0
Determination of the position and stereochemistry of a 16-
and 15-hydroxyl group in five-membered D-ring in bile acids
has been unclear for a long time, probably owing to the un-
availability of authentic reference compounds. We have now
in hand a complete set of the four possible stereoisomeric
3a,7a,16- and 3a,7a,15-trihydroxy compounds (3a—6a).
0.3
Ϫ3.8
Ϫ0.7
0.4
Ϫ0.1
0.7
Ϫ1.5
Ϫ2.0
Ϫ1.9
2.5
0.7
1.4
Ϫ0.1
0.3
0.3
1
Tables 1 and 2 show the H- and 13C-NMR spectral data for
3a—6a, respectively. Differentiation between the 16a- and
1
16b-epimers (3a vs. 4a) was made by the H-NMR spectra,
in which the 18-methyl signal in 4a resonates at lower-field
by 0.20 ppm than that of 3a,28) due to the pseudo-1,3-diaxial
relationship. Also, the 16a-H (br m) in 4a appeared at much
lower field by 0.50 ppm than the corresponding 16b-H (d) in
1
3a. Similar H-NMR differences in the chemical shifts and
signal multiplicity between the 15a- and 15b-epimers (5a,
6a) were observed for the 18-methyl12—14) [0.67 ppm (s) in 5a
and 0.95 ppm (s) in 6a], 15-H10,14) [4.03 ppm (m) in 5a and
4.31 ppm (t) in 6a] and 7b-H [4.03 ppm (m) in 5a and
4.18 ppm (m) in 6a] resonances.
0.1
0.1
0
0
COOCH3
0.4
a) Difference in the chemical shifts between 4a vs. 3a and 6a vs. 5a.
In the 13C-NMR spectra, the C-3 (71.9—72.1 ppm) and C-
7 (67.7—68.3 ppm) carbon signals in 3a—6a had essentially
ture. However, when tert-butylamine–borane complex (tert- identical chemical shifts and the values were found to be
C4H9NH2·BH3)27) was applied for the reduction of 16a, the very similar to those of the parent methyl 3a,7a-dihydroxy-
reduction reaction proceeded cleanly in refluxing CH2Cl2 for 5b-cholan-24-oate.29) However, the C-16 in 3a (76.7 ppm)
12 h to give a mixture of 3a,7a,16a- and 3a,7a,16b-trihy- and 4a (72.1 ppm) and the C-1510,16) in 5a (72.4 ppm) and 6a
droxy esters (3a, 4a) in an approximate ratio of 1 : 2. The two (70.5 ppm) much differed from one another, depending upon
epimers could be separated cleanly by NP-MPLC on silica the position and stereochemistry of the hydroxyl groups. In
gel, eluting with EtOAc–methanol (95 : 5, v/v). The above this connection, differences in the chemical shifts of the
finding may suggest that the reagent attacks from a less hin- neighboring b-carbons (C-15, -17 in 3a and 4a; C-14, -16 in
dered a-side of the D-ring in 16a to yield 4a as the major 5a and 6a) and g-carbons (C-13, -20 in 3a and 4a; C-8, -13
product. Analogously, reduction of 3,7,15-triketone 17a with in 5a and 6a) between the 16- or 15-epimeric pairs are also
tert-C4H9NH2·BH3 also afforded a similar epimeric mixture noteworthy.
of 3a,7a,15a- and 3a,7a,15b-trihydroxy esters (5a, 6a), ac-
Meanwhile, the mass spectra of 3a—6a measured as their
companied by the simultaneous formation of 3a,7b,15b-tri- trimethylsilyl (TMS) ether derivatives are shown in Table 3.
hydroxy ester (18a) in an approximate ratio of 1 : 1 : 1. The The spectral pattern of each of the epimeric pairs was very
three diastereomers were successfully resolved by reversed- similar to each other, but it much differed between 3a,7a,16-
phase (RP)-MPLC on a C18-bonded silica gel, eluting with (3a, 4a) and 3a,7a,15-triols (5a, 6a). For example, the base
methanol–water (8 : 2—7 : 3, v/v). Thus, of the eight possible peak constituted of an ion at m/z 368 (MϪ3TMSOH) in the
stereoisomeric 3,7,16- or 3,7,15-trihydroxy compounds, es- 3a,7a,16-triols which is absent in the spectra of 3a,7a,15-
sentially only the two or three desired 16- or 15-epimeric ones and of an ion at m/z 458 (MϪ2TMSOH) in the 3a,7a,15-
mixtures were efficiently obtained by reduction of 16a or 17a triols.

October 2002
1331
Table 3. Mass Spectral Fragment Ions of Stereoisomeric Methyl
O-24,16b-lactone (23), respectively, based on the chromato-
3a,7a,16- and 3a,7a,15-Trihydroxy-5b-cholan-24-oates (3a—6a)a)
1
graphic behaviors, H-NMR and mass fragmentation pat-
terns. The above result indicates that, under the usual acid-
catalyzed conditions, intramolecular esterification between
the side chain carboxyl group at C-24 and the hydroxyl
Relative intensity (%)
Major ions
3a
4a
5a
6a
1
group at C-16 in 3 and 4 occurred preferentially. In the H-
638
623
548
533
458
443
368
364
353
329
253
M
MϪCH3
3
14
16
3
11
17
1
NMR spectra of 22 and 23, no methyl ester signal occurrs at
ca. 3.65 ppm. On the other hand, the mass spectra of the
trimethylsilyl (TMS) ether derivatives of 22 and 23 showed
intense fragments at m/z 444 and 3547) originating from the
loss of one and two trimethylsilanol molecules (TMSOH)
from the molecular ion (Mϩ, m/z 534, Ͻ1%), and a diagnos-
tic peak at m/z 253 originating from the further loss of the
lactone ring (C5H9O2) from the ion at m/z 354. In addition,
ions at m/z 429 and 339 arising from the sequential loss of
CH3 from the ions at m/z 444 and 354 always accompanied in
the mass spectra. A similar lactonization of the side chain
carboxyl group was also occurred by treating 17a-hydroxy
acid (21), derived from 9a by alkaline hydrolysis, with p-
toluenesulfonic acid in EtOAc to give the corresponding
d-lactone, 3a,7a-dihydroxy-5b-cholane O-24,17a-lactone
(24).
MϪTMSOH
MϪTMSOHϪCH3
MϪ2TMSOH
MϪ2TMSOHϪCH3
MϪ3TMSOH
21
3
4
100
17
44
44
100
20
100
100
18
16
9
MϪ3TMSOHϪCH3
MϪ3TMSOHϪS.C.
35
34
22
30
35
20
68
a) Measured as the trimethylsilyl ether derivatives; TMSOH, trimethylsilanol; S.C.,
side chain.
DMDO, a powerful oxidant, which was successfully used
for one-step remote-oxyfunctionalization of 7a and 8a to 9a
and 10a, respectively, was also an attractive agent for the
stereoselective epoxidation of a double bond.30) By treatment
of D16- and D15-esters (11a, 13a) with a solution of DMDO
in CHCl3, epoxidation reaction proceeded rapidly under mild
conditions (30 min at room temperature) to give the corre-
sponding 16a,17a- and 14a,15a-epoxides (19a, 20a), re-
spectively, in a high isolated yield of 92—94%. The stereo-
chemistry of the resulting epoxides was determined on the
On the contrary, 15a- and 15b-hydroxy acids (5, 6, 18)
were found to be completely insensitive to lactonization and
afforded the expected methyl esters with methanol-conc. HCl
(5a, 6a, 18a). Since there was no evidence for differentiating
between 16- and 15-hydroxy bile acids, the above observa-
tion may be usefully utilized for their characterization.
Experimental
1
basis of the H chemical shifts and signal patterns of the
Melting points (mp) were determined on a micro hot-stage apparatus and
are uncorrected. IR spectra were obtained on a Bio Rad FTS-7 FT-IR spec-
trometer (Philadelphia, U.S.A.) as KBr tablets. 1H- and 13C-NMR spectra
were obtained on a JEOL JNM-EX 270 FT NMR instrument (Tokyo, Japan)
at 270 and 68.80 MHz, respectively, with CDCl3 or CDCl3 containing 10—
30% DMSO-d6 as the solvent; chemical shifts are expressed as d ppm rela-
tive to tetramethylsilane. Electron impact low-resolution mass spectra (LR-
MS) were recorded on a JEOL JMC-Automass 150 gas chromatograph-mass
spectrometer at 70 eV. High-resolution mass spectra (HR-MS) was obtained
on a JEOL JMS-LCmate double focusing magnetic mass spectrometer
equipped with an electrospray ionization (ESI) or an atmospheric pressure
chemical ionization (APCI) under the positive ion mode (PIM) or the nega-
tive ion mode (NIM). The apparatus used for MPLC consisted of a Shima-
mura YRD-880 RI-detector (Tokyo, Japan) and an uf-3040S chromato-
16b-H (3.32 ppm as s) in 19a and 15b-H (3.20 ppm as s)
in 20a, in comparison with those reported for analogous
16a,17a-epoxy-5a-cholestane-3b,5a-diol31) and methyl 3a-
cathyloxy-14a,15a-epoxy-5b-cholan-24-oate.30) Subsequent
hydrogenolysis of 19a and 20a would be expected to yield
3a,7a-diacetoxy-16a-hydroxy and 3a,7a-diacetoxy-15a-
hydroxy esters, respectively.32) However, attempted reductive
cleavage of 19a and 20a with H2 in the presence of PtO2 in
EtOAc under atmospheric pressure was unsuccessful and
found to be completely inert against the conditions em-
ployed.
As expected, esterification of 16a- and 16b-hydroxy acids graphic pump using silica gel 60 (230—400 mesh, Nakalai Tesque, Kyoto,
Japan) as the NP adsorbent or ODS-AM 120-S50 as the RP adsorbent
(YMC Co. Ltd., Kyoto, Japan). TLC was performed on pre-coated silica gel
(3, 4) dissolved in methanol with a solution of diazomethane
in ether afforded the corresponding methyl esters (3a, 4a)
(0.25 mm layer thickness; E. Merck, Germany) using hexane–EtOAc–acetic
acid mixtures (80 : 20 : 1—20 : 80 : 1, v/v/v) as the developing solvent.
A solution (0.32 mol/l) of DMDO in CHCl3 was prepared according to lit-
erature method using oxone® and acetone.17,18) All compounds were dried by
azeotropic distillation before use in reactions.
Methyl 3a,7a-Diacetoxy-17a-hydroxy-5b-cholan-24-oate (9a) To a
solution of CDCA methyl ester–diacetate (7a) (2.0 g) in CH2Cl2 (10 ml) was
added a freshly prepared solution of DMDO (0.35 mol/l; 24 ml) in CHCl3
with an ice-bath cooling. The mixture was left to stand at room temperature
for 12 h, and excess amounts of the reagent and solvent were evaporated
under reduced pressure. The above procedure was repeated (two runs, total
reaction time, 24 h), and the reaction product, which consisted of four major
components (9a and two monohydroxy and one dihydroxy compounds), was
poured into a column of silica gel (70—230 mesh, 60 g). Elution with ben-
zene–EtOAc (4 : 1, v/v) afforded the starting compound 7a; 790 mg (40%).
Continued elution with the same solvent system gave a mixture of two
monohydroxy components (750 mg).
1
quantitatively, according to chromatographic and H-NMR
analyses. However, conventional treatment of the same acid 3
(or 4) with p-toluenesulfonic acid or conc. HCl in methanol
always produced a mixture of two components, one (a more
polar component) of which was in accord with the methyl
ester 3a (or 4a). According to the finding of Haslewood et
al.,2,33) pythocholic acid, 3a,12a,16a-trihydroxy-5b-cholan-
24-oic acid, forms readily the corresponding pythocholic lac-
tone by dissolving the free acid in NaOH solution, acidified
with H2SO4, and then warmed to 70 °C for 15 h. When each
of the acids 3 and 4 dissolved in EtOAc was treated with a
catalytic amount of p-toluenesulfonic acid or conc. HCl for
2 h at room temperature, a single reaction product, which was
identical with the respective less polar component mentioned
above, was formed, and their structures were characterized as
the corresponding e-lactones, 3a,7a-dihydroxy-5b-cholane
The monohydroxy fraction (see above) was then subjected to NP-MPLC
on silica gel (230—400 mesh, 40 g). Elution with CHCl3–methanol mixture
(99 : 1, v/v) provided two well-separated components. The less polar com-
O-24,16a-lactone (22)7) and 3a,7a-dihydroxy-5b-cholane pound (314 mg, 15%) was identified as the desired 17a-hydroxy ester 9a,

1332
Vol. 50, No. 10
which was recrystallized from EtOAc–hexane as colorless prisms: mp,
A more polar component was recrystallized from aqueous methanol as
157—160 °C (lit.,17) mp, 157—160 °C). IR, nmax cmϪ1: 1728 (ester CϭO), colorless thin plates and identified as the desired D16-ester 11a: yield, 48 mg
1
3545 (OH). H-NMR, d: 0.74 (s, 3H, 18-CH3), 0.91 (d, 3H, Jϭ7.0 Hz, 21- (42%); mp, 109—111 °C (lit.,19) mp, 109—110 °C). IR, nmax cmϪ1: 1735
CH3), 0.94 (s, 3H, 19-CH3), 2.02 and 2.06 (s, each 3H, 3a-, 7a-OCOCH3), (ester CϭO). 1H-NMR, d: 0.73 (s, 3H, 18-CH3), 0.97 (s, 3H, 19-CH3), 1.03
3.67 (s, 3H, COOCH3), 4.59 (br m, 1H, 3b-H), 4.90 (br d, 1H, Jϭ2.7 Hz, 7b- (d, 3H, Jϭ7.0 Hz, 21-CH3), 2.02 and 2.05 (s, each 3H, 3a-, 7b-OCOCH3),
H). LR-MS, m/z: 446 (4%, MϪAcOH), 428 (19%, MϪAcOHϪH2O), 386 3.66 (s, 3H, COOCH3), 4.60 (br m, 1H, 3b-H), 4.97 (br d, 1H, Jϭ2.7 Hz, 7b-
(100%, MϪ2AcOH), 368 (82%, MϪ2AcOHϪH2O), 353 (18%, MϪ H), 5.28 (br s, 1H, 16-H). LR-MS, m/z: 428 (9%, MϪAcOH), 413 (15%,
2AcOHϪH2OϪCH3), 332 (91%), 313 [35%, MϪAcOHϪH2OϪside chain MϪAcOHϪCH3), 368 (19%, MϪ2AcOH), 353 (27%, MϪ2AcOHϪCH3),
(S.C.)], 286 (12%, MϪAcOHϪH2OϪS.C.Ϫring D.), 253 (37%, 313 (34%, MϪAcOHϪS.C.), 281 (34%), 253 (100%, MϪ2AcOHϪS.C.).
MϪ2AcOHϪH2OϪS.C.), 226 (40%, MϪ2AcOHϪH2OϪS.C.Ϫpart of ring HR-MS (ESI-PIM) Calcd for C29H44NaO6 [MϩNa]ϩ: 511.3036. Found: m/z,
D.), 211 (30%, MϪ2AcOHϪH2OϪS.C.Ϫring D.).
The more polar compound was recrystallized from EtOAc–hexane to give
511.3038.
Methyl 3a,7b-Diacetoxy-5b-chol-14-en-24-oate (13a) A solution of
methyl 3a,7a-dihydroxy-5b-hydroxycholan-24-oate as a colorless amor- the 14a-hydroxy ester 10a (250 mg) in 5% conc. H2SO4-dioxane (w/w,
phous solid: yield, 550 mg, 27%; mp, 156—158 °C (lit.,18) mp, 158— 50 ml) was allowed to stand overnight at room temperature. The reaction
159 °C). IR, nmax cmϪ1: 1735 (ester CϭO), 3475 (OH). 1H-NMR, d: 0.65 (s, product was extracted with Et2O, and the combined extract was washed suc-
3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 0.92 (d, 3H, Jϭ7.3 Hz, 21-CH3), 2.03 cessively with water, 5% NaHCO3, and saturated brine, dried over Drierite,
and 2.07 (s, each 3H, 3a-, 7a-OCOCH3), 3.67 (s, 3H, COOCH3), 4.92 (br d,
1H, Jϭ2.4 Hz, 7b-H), 5.02 (br m, 1H, 3b-H). LR-MS, m/z: 428 (19%,
and evaporated. The oily residue was chromatographed on a column of silica
gel (70—230 mesh, 20 g). The fraction eluted by benzene–EtOAc (7 : 3, v/v)
MϪAcOHϪH2O), 386 (100%, MϪ2AcOH), 368 (81%, MϪ2AcOHϪ afforded the title compound which resisted crystallization: yield, 175 mg
H2O), 353 (17%, MϪAcOHϪH2OϪCH3), 332 (91%), 313 (35%, MϪ (73%). IR, nmax cmϪ1: 1739 (ester CϭO). 1H-NMR d: 0.92 (s, 3H, 18-CH3),
AcOHϪH2OϪS.C.), 286 (13%, MϪAcOHϪH2OϪS.C.Ϫpart of ring D.), 0.93 (d, 3H, Jϭ5.9 Hz, 21-CH3), 0.97 (s, 3H, 19-CH3), 2.00 and 2.01 (s,
271 (92%, MϪ2AcOHϪS.C.), 253 (37%, MϪ2AcOHϪH2OϪS.C.), 226 each 3H, 3a-, 7b-OCOCH3), 3.67 (s, 3H, COOCH3), 4.67 (br m, 1H, 3b-H),
(40%, MϪ2AcOHϪH2OϪS.C.Ϫpart of ring D.), 211 (30%, MϪ2AcOHϪ 5.01 (br s, 1H, 15-H), 5.05 (br m, 1H, 7a-H). LR-MS, m/z: 428 (9%,
H2OϪS.C.Ϫring D.).
MϪAcOH), 368 (62%, MϪ2AcOH), 353 (42%, MϪ2AcOHϪCH3), 314
(43%), 253 (100%, MϪ2AcOHϪS.C.). HR-MS (ESI-PIM) Calcd for
Methyl 3a,7b-Diacetoxy-14a-hydroxy-5b-cholan-24-oate (10a) UDCA
methyl ester-diacetate 8a (2.0 g) was subjected to the remote-oxyfunctional- C29H44NaO6 [MϩNa]ϩ: 511.3036. Found: m/z, 511.3035.
ization with DMDO and processed as described for the preparation of 9a to
3a,7a,16a,24-Tetrahydroxy-5b-cholane (14) To a stirred solution of
yield an oily residue, which was consisted of seven components by GC. The the D16-ester 11a (100 mg) in dry THF (3 ml) a solution of BH3·THF
oil was chromatographed on a column of silica gel (70—230 mesh, 60 g). (1.0 mol; 1.3 ml) was added gradually, and the mixture was stirred at 50 °C
Elution with benzene–EtOAc (9 : 1, v/v) afforded the least polar 8a and then
for 2 h under a stream of N2. After cooling the solution, 3 N-NaOH (1 ml)
methyl 3a,7b-diacetoxy-16-oxo-5b-cholan-24-oate.17) Continued elution and then 30% H2O2 (1 ml) were added with an ice-bath cooling, and the
with benzene–EtOAc (7 : 3, v/v) gave a mixture of two monohydroxy com- mixture was stirred overnight at room temperature. The resulting solution
pounds (470 mg), which were then purified by NP-MPLC on silica
gel (230—400 mesh, 20 g). Elution with hexane–benzene–EtOAc (5 : 3 : 2, The combined EtOAc layer was washed with saturated brine and evapo-
v/v/v) provided a less polar component, which was characterized as the de- rated. Recrystallization of the product from EtOAc–hexane gave the
was acidified with 10% HCl and the reaction product extracted with EtOAc.
sired 14a-hydroxy ester 10a. Although this compound was homogeneous 3a,7a,16a,24-tetrahydroxy-5b-cholane 14 as a colorless amorphous solid:
according to TLC and GC, it resisted crystallization: yield, 278 mg (13%). yield, 72 mg (90%); mp, 184—187 °C. IR, nmax cmϪ1: 3332 (OH). 1H-NMR
IR, nmax cmϪ1: 1737 (ester CϭO), 3518 (OH). 1H-NMR, d: 0.79 (s, 3H, 18- (CDCl3ϩCD3OD, 1 : 1, v/v), d: 0.69 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3),
CH3), 0.90 (d, 3H, Jϭ6.2 Hz, 21-CH3), 0.98 (s, 3H, 19-CH3), 2.00 and 2.02 3.46 (1H, br m, 3b-H), 3.54 (2H, br m, 24-H2), 3.79 (m, 1H, 7b-H), 3.94 (m,
(s, each 3H, 3a-, 7b-OCOCH3), 3.67 (s, 3H, COOCH3), 4.66 (br m, 1H, 3b- 1H, 16b-H). LR-MS (as the TMS ether), m/z: 682 (Ͻ1%, M), 592 (19%,
H), 5.15 (br m, 1H, 7a-H). LR-MS, m/z: 428 (8%, MϪAcOHϪH2O), 368 MϪTMSOH), 577 (9%, MϪTMSOHϪCH3), 565 (10%), 502 (33%,
(24%, MϪ2AcOHϪH2O), 353 (9%, MϪ2AcOHϪH2OϪCH3), 314 (9%), MϪ2TMSOH), 455 (15%), 433 (12%, MϪTMSOHϪS.C.), 412 (42%,
281 (5%), 253 (100%, MϪ2AcOHϪH2OϪS.C.), 239 (10%), 212 (25%).
MϪ2TMSOH), 343 (23%, MϪ2TMSOHϪS.C.), 329 (26%), 281 (28%),
A more polar component eluting with hexane–benzene–EtOAc (5 : 1 : 4, 253 (100%, MϪ3TMSOHϪS.C.), 239 (49%). HR-MS (ESI-NIM) Calcd for
v/v/v) afforded (20S)-3a,7b-diacetoxy-5b-cholane O-24,20-lactone, which C24H41O4 [MϪH]Ϫ: 393.3005. Found: m/z, 393.2997.
was recrystallized from aqueous methanol as colorless amorphous solid:
3a,7b,15a,24-Tetrahydroxy-5b-cholane (15) The D14-ester 13a (150
yield, 190 mg (10%); mp, 202—204 °C (lit.,17) mp, 202—204 °C). IR, nmax mg) was treated with BH3–THF, followed by alkaline hydrogen peroxide as
1
cmϪ1: 1743, 1771 (CϭO). H-NMR, d: 0.84 (s, 3H, 18-CH3), 0.98 (s, 3H, described for the preparation of 14. Recrystallization of the product from
19-CH3), 1.45 (s, 3H, 21-CH3), 2.03 and 2.05 (s, each 3H, 3a-, 7b- EtOAc gave the 3a,7b,15a,24-tetrahydroxy-5b-cholane 15 as a colorless
OCOCH3), 4.68 (br m, 1H, 3b-H), 4.76 (br m, 1H, 7a-H). LR-MS, m/z: 414
amorphous solid: yield, 87 mg (72%); mp, 172—174 °C. IR, nmax cmϪ1
:
1
(14%, MϪAcOH), 354 (85%, MϪ2AcOH), 339 (41%, MϪ2AcOHϪCH3), 3251 (OH). H-NMR, d: 0.72 (s, 3H, 18-CH3), 0.94 (d, 3H, Jϭ5.6 Hz, 21-
313 (10%), 300 (10%, MϪAcOHϪS.C.ϪCH3), 288 (8%, MϪAcOHϪ CH3), 0.96 (s, 3H, 19-CH3), 3.60 (br m, 4H, 3b-, 7a-, 24-H2), 4.07 (br m,
S.C.Ϫpart of ring D.), 281 (7%), 253 (56%), 241 (19%), 228 (94%, MϪ 1H, 15b-H). LR-MS (as the TMS ether), m/z: 682 (Ͻ1%, M), 592 (4%,
2AcOHϪS.C.Ϫpart of ring D.), 227 (100%), 213 (75%, MϪ2AcOHϪ MϪTMSOH), 577 (15%, MϪTMSOHϪCH3), 502 (25%, MϪ2TMSOH),
S.C.Ϫring D.).
487 (7%, MϪ2TMSOHϪCH3), 433 (100%, MϪTMSOHϪS.C.), 327
Methyl 3a,7a-Diacetoxy-5b-chol-16-en-24-oate (11a) To the 17a-hy- (64%), 253 (19%, MϪ3TMSOHϪS.C.), 207 (32%). HR-MS (ESI-NIM)
droxy ester (9a; 120 mg) dissolved in dry pyridine (3 ml) POCl3 (1 ml) was Calcd for C24H41O4 [MϪH]Ϫ: 393.3005. Found: m/z, 393.2994.
added dropwise. After the mixture was stirred at 65 °C for 24 h, it was
Methyl 3,7,16-Trioxo-5b-cholan-24-oate (16a) Jones reagent (1 ml)
cooled in an ice-bath, ice chips were added gradually, and the reaction prod- was added dropwise to a stirred solution of the 3a,7a,16a,25-tetrol 14
uct was extracted with CH2Cl2. The combined extract was washed with 10% (100 mg) in acetone (13 ml) under 10 °C, and the mixture was stirred for
HCl and water, dried over Drierite, and evaporated. The oily residue, which 30 min at room temperature. Methanol (2 ml) was added, and the oxidation
consisted of two components as evidenced by TLC (four developments by product extracted with EtOAc. The combined EtOAc layer was washed with
benzene–Et2O; 9 : 1, v/v), was subjected to NP-MPLC on a column of silica
gel (230—400 mesh, 12 g) and elution with toluene–Et2O (9 : 1, v/v) gave a
saturated brine, dried over Drierite, and evaporated. After re-esterification of
the residue with methanol and p-toluenesulfonic acid, the product was chro-
less polar component, which was recrystallized from methanol as colorless matographed on a column of silica gel (70—230 mesh, 10 g). Elution with
needles and characterized as the D17(20)-ester 12a: yield, 35 mg (30%); mp, benzene–EtOAc (3 : 2, v/v) and recrystallization of the eluate from EtOAc–
1
119—121 °C. IR, nmax cmϪ1: 1737 (ester CϭO). H-NMR, d: 0.82 (s, 3H, hexane gave the title compound 16a as colorless thin plates: yield, 99 mg
18-CH3), 0.94 (s, 3H, 19-CH3), 1.69 (s, 3H, 21-CH3), 2.03 and 2.04 (s, each (94%); mp, 148—150 °C. IR, nmax cmϪ1: 1709 (ketone CϭO), 1735 (ester
1
3H, 3a-, 7a-OCOCH3), 3.66 (s, 3H, COOCH3), 4.59 (br m, 1H, 3b-H), 4.94 CϭO). H-NMR, d: 0.85 (s, 3H, 18-CH3), 1.00 (d, 3H, Jϭ6.2 Hz, 21-CH3),
(br d, 1H, Jϭ2.4 Hz, 7b-H). LR-MS, m/z: 428 (10%, MϪAcOH), 413 (15%, 1.34 (s, 3H, 19-CH3), 3.67 (s, 3H, COOCH3). LR-MS, m/z: 416 (3%, M),
MϪAcOHϪCH3), 368 (17%, MϪ2AcOH), 353 (26%, MϪ2AcOHϪCH3), 401 (38%, MϪCH3), 369 (35%), 329 (22%), 287 (100%). HR-MS (ESI-
313 (26%, MϪAcOHϪS.C.), 281 (100%), 253 (66%, MϪ2AcOHϪS.C.). PIM) Calcd for C25H36NaO5 [MϩNa]ϩ: 439.2460. Found: m/z, 439.2469.
HR-MS (ESI-PIM) Calcd for C29H44NaO6 [MϩNa]ϩ: 511.3036. Found: m/z,
511.3034.
Methyl 3,7,15-Trioxo-5b-cholan-24-oate (17a) The 3a,7b,15a,24-
tetrol 15 (110 mg) was converted to the 3,7,15-trioxo ester 17a by the

October 2002
1333
method as described for the preparation of 16a. Recrystallization of the
product from EtOAc–hexane gave 17a as colorless amorphous solid: yield,
101 mg (87%); mp, 178—180 °C. IR, nmax cmϪ1: 1712 (ketone CϭO), 1739
(ester CϭO). 1H-NMR, d: 0.75 (s, 3H, 18-CH3), 1.01 (d, 3H, Jϭ6.5 Hz, 21-
CH3), 1.31 (s, 3H, 19-CH3), 3.68 (s, 3H, COOCH3). LR-MS, m/z: 416 (37%,
M), 401 (100%, MϪCH3), 301 (23%). HR-MS (ESI-PIM) Calcd for
C25H36NaO5 [MϩNa]ϩ: 439.2460. Found: m/z, 439.2448.
0.97 (d, 3H, Jϭ5.9 Hz, 21-CH3), 3.43 (br m, 1H, 3b-H), 3.84 (m, 1H, 7b-H),
4.49 (br m, 1H, 16a-H). HR-MS (ESI-NIM) Calcd for C24H39O5 [MϪH]Ϫ:
407.2797. Found: m/z, 407.2827.
3a,7a,15a-Trihydroxy-5b-cholan-24-oic Acid (5) The ester 5a (50 mg),
hydrolyzed by the usual manner, recrystallized from acetone–hexane as a
colorless amorphous solid of 5: yield, 42 mg (86%); mp, 203—205 °C. IR,
1
nmax cmϪ1: 1705 (ester CϭO), 3209 (OH). H-NMR (CDCl3ϩ10% DMSO-
Methyl 3a,7a,16a-Trihydroxy-5b-cholan-24-oate (3a) and Methyl
3a,7a,16b-Trihydroxy-5b-cholan-24-oate (4a) tert-Butylamine–borane
complex (100 mg) was added to a stirred solution of the 3,7,16-triketone
(100 mg) in CH2Cl2 (8 ml), and the mixture was refluxed overnight. After
cooling the mixture, 10% HCl (3 ml) added and the solution stirred at room
temperature for 30 min. The CH2Cl2 layer was washed with 5% NaHCO3
and water, dried over Drierite, and evaporated. The oily residue, which con-
sisted essentially of two components on TLC, was chromatographed on a
column of silica gel (70—230 mesh, 80 g). Elution with EtOAc–methanol
(19 : 1, v/v) provided two well-separated fractions. The less polar fraction
was identified as the 3a,7a,16b-trihydroxy ester (4a), which resisted crys-
tallization: yield, 44 mg (43%); viscous oil. IR, nmax cmϪ1: 1721 (ester
CϭO), 3358 (OH). (1H-, 13C-NMR and LR-MS, see Tables 1—3). HR-
MS (ESI-PIM) Calcd for C25H42NaO5 [MϩNa]ϩ: 445.2930. Found: m/z,
445.2925.
The more polar fraction was characterized as the 16a-epimer (3a) of 4a:
yield, 16 mg (16%); viscous oil. IR, nmax cmϪ1: 1739 (ester CϭO), 3271
(OH). (1H-, 13C-NMR and LR-MS, see Tables 1—3). HR-MS (ESI-PIM)
Calcd for C25H42NaO5 [MϩNa]ϩ: 445.2930. Found: m/z, 445.2928.
Methyl 3a,7a,15a-Trihydroxy-5b-cholan-24-oate (5a) and Methyl
3a,7a,15b-Trihydroxy-5b-cholan-24-oate (6a) The 3,7,15-trioxo ester
17a (140 mg) was treated with tert-C4H9NH2·BH3 as described for the
preparation of 3a and 4a. After being processed analogously, the oily prod-
uct was purified by RP-MPLC on a column of C18-bonded silica gel (16 g).
Elution with methanol–H2O (8 : 2—7 : 3, v/v) afforded the following three
well-separated fractions.
d6), d: 0.72 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3), 0.96 (d, 3H, Jϭ5.4 Hz,
21-CH3), 3.49 (br m, 1H, 3b-H), 4.06 (m, 2H, 7b-, 15b-H). HR-MS (ESI-
NIM) Calcd for C24H39O5 [MϪH]Ϫ: 407.2797. Found: m/z, 407.2819.
3a,7a,15b-Trihydroxy-5b-cholan-24-oic Acid (6) The ester 6a (50 mg),
hydrolyzed by the usual manner, recrystallized from EtOAc as colorless nee-
dles of 6: yield, 40 mg (82%); mp, 130—132 °C. IR, nmax cmϪ1: 1705 (ester
1
CϭO), 3337 (OH). H-NMR (CDCl3ϩ30% DMSO-d6), d: 0.94 (br s, 3H,
18-CH3), 0.94 (br s, 3H, 19-CH3), 0.94 (br s, 3H, 21-CH3), 3.43 (br m, 1H,
3b-H), 4.11 (m, 1H, 7b-H), 4.27 (br m, 1H, 15a-H). HR-MS (ESI-NIM)
Calcd for C24H39O5 [MϪH]Ϫ: 407.2797. Found: m/z, 407.2801.
3a,7b,15b-Trihydroxy-5b-cholan-24-oic Acid (18) The ester 18a
(50 mg), hydrolyzed by the usual manner, recrystallized from aqueous
methanol as a colorless amorphous solid: yield, 39 mg (81%); mp, 126—
1
128 °C. IR, nmax cmϪ1: 1703 (ester CϭO), 3279 (OH). H-NMR (CDCl3ϩ
30% DMSO-d6), d: 0.97 (br s, 3H, 18-CH3), 0.97 (br s, 3H, 19-CH3), 0.97
(br s, 3H, 21-CH3), 3.60 (br m, 1H, 3b-H), 3.73 (br m, 1H, 7a-H), 4.29 (m,
1H, 15a-H). HR-MS (ESI-NIM) Calcd for C24H39O5 [MϪH]Ϫ: 407.2797.
Found: m/z, 407.2781.
Methyl 3a,7a-Diacetoxy-16a,17a-epoxy-5b-cholan-24-oate (19a) To
a solution of the D16-ester (11a; 100 mg) in CH2Cl2 (1.0 ml) was added a
DMDO solution in CHCl3 (0.32 mol/l, 1.5 ml), and the mixture was allowed
to stand at room temperature for 30 min. Excess amounts of the DMDO and
solvent were evaporated under reduced pressure, and the residue was recrys-
tallized directly from hexane to give an analytically pure sample of the title
compound (19a): yield, 95 mg (92%); mp, 111—114 °C. IR, nmax cmϪ1
:
1724 (ester CϭO). 1H-NMR, d: 0.79 (s, 3H, 18-CH3), 0.94 (s, 3H, 19-CH3),
1.01 (d, 3H, Jϭ6.8 Hz, 21-CH3), 2.03 and 2.05 (each s, 6H, 3a-, 7a-
OCOCH3), 3.32 (s, 1H, 16b-H), 3.67 (s, 3H, COOCH3), 4.57 (br m, 1H, 3b-
H), 4.87 (m, 1H, 7b-H). LR-MS, m/z: 504 (3%, M), 444 (5%, MϪAcOH),
429 (11%, MϪ2AcOHϪCH3), 329 (39%, MϪAcOHϪS.C.), 269 (39%,
MϪ2AcOHϪS.C.), 224 (100%). HR-MS (ESI-PIM) Calcd for C29H44NaO7
[MϩNa]ϩ: 527.2985. Found: m/z, 527.2973.
Fr. 1. 3a,7a,15b-Trihydroxy ester (6a): yield, 30 mg (21%); viscous oil.
IR, nmax cmϪ1: 1728 (ester CϭO), 3350 (OH). (1H-, 13C-NMR and LR-MS,
see Tables 1—3). HR-MS (ESI-PIM) Calcd for C25H42NaO5 [MϩNa]ϩ:
445.2930. Found: m/z, 445.2900.
Fr. 2. Recrystallization of the eluate from acetone–hexane gave 3a,7b,15b-
trihydroxy ester (18a) as colorless needles: yield, 26 mg (18%); mp, 203—
205 °C. IR, nmax cmϪ1: 1738 (ester CϭO), 3271 (OH). 1H-NMR, d: 0.94 (d,
3H, Jϭ6.8 Hz, 21-CH3), 0.96 (s, 3H, 18-CH3), 0.98 (s, 3H, 19-CH3), 3.60
(br m, 1H, 3b-H), 3.67 (s, 3H, COOCH3), 3.76 (br m, 1H, 7a-H), 4.29 (t,
1H, Jϭ5.4 Hz, 15a-H). 13C-NMR, d: 14.5 (C-18), 18.4 (C-21), 21.1 (C-11),
23.3 (C-19), 30.2 (C-2), 30.9 (C-22), 31.1 (C-23), 34.1 (C-10), 34.9 (C-1),
34.9 (C-20), 37.2 (C-6), 37.5 (C-4), 38.7 (C-12), 39.1 (C-9), 39.4 (C-8), 41.2
(C-16), 42.3 (C-5), 43.0 (C-13), 51.5 (COOCH3), 55.8 (C-17), 61.1 (C-14),
71.1 (C-15), 71.2 (C-7), 71.3 (C-3), 174.7 (C-24). LR-MS (as the Me-TMS
ether), m/z: 533 (11%, MϪTMSOHϪCH3), 458 (7%, MϪ2TMSOH), 443
(11%, MϪ2TMSOHϪCH3), 433 (81%, MϪTMSOHϪS.C.), 368 (4%, MϪ
3TMSOH), 353 (6%, MϪ3TMSOHϪCH3), 343 (13%, MϪ2TMSOHϪ
S.C.), 314 (13%), 283 (100%), 253 (55%, MϪ3TMSOHϪS.C.). HR-MS
(ESI-PIM) Calcd for C25H42NaO5 [MϩNa]ϩ: 445.2930. Found: m/z,
445.2900.
Methyl 3a,7b-Diacetoxy-14a,15a-epoxy-5b-cholan-24-oate (20a)
This compound was prepared from the D14 ester 13a (100 mg) by the epoxi-
dation method as described in the preparation of 19a: yield, 97 mg (94%);
viscous oil. IR, nmax cmϪ1: 1732 (ester CϭO). 1H-NMR, d: 0.87 (d, 3H,
Jϭ8.1 Hz, 21-CH3), 0.88 (s, 3H, 18-CH3), 0.98 (s, 3H, 19-CH3), 1.99 and
2.01 (each s, 6H, 3a-, 7b-OCOCH3), 3.20 (s, 1H, 15b-H), 3.67 (s, 3H,
COOCH3), 4.65 (br m, 1H, 3b-H), 4.71 (br m, 1H, 7a-H). LR-MS, m/z: 444
(36%, MϪAcOH), 429 (11%, MϪ2AcOHϪCH3), 389 (43%, MϪS.C.), 384
(10%, MϪ2AcOH), 329 (76%, MϪAcOHϪS.C.), 290 (42%), 269 (100%,
MϪ2AcOHϪS.C.). HR-MS (ESI-PIM) Calcd for C29H44NaO7 [MϩNa]ϩ:
527.2985. Found: m/z, 527.2979.
3a,7a-Dihydroxy-5b-cholane O-24,16a-Lactone (22) A solution of
the 3a,7a,16a-trihydroxy acid (3: 20 mg) in EtOAc (2 ml) containing p-
toluenesulfonic acid (2 mg) or conc. HCl (one drop) was left to stand at
room temperatrure for 2 h. The organic layer was washed with saturated
brine, dried over Drierite, and evaporated to give the title compound 22:
yield, 16 mg (81%); viscous oil. IR, nmax cmϪ1: 1727 (ester CϭO), 3456
(OH). 1H-NMR, d: 0.75 (s, 3H, 18-CH3), 0.90 (s, 3H, 19-CH3), 1.03 (d, 3H,
Jϭ6.5 Hz, 21-CH3), 3.46 (br m, 1H, 3b-H), 3.84 (d, 1H, Jϭ2.7 Hz, 7b-H),
4.65 (t, 1H, Jϭ8.1 Hz, 16b-H). LR-MS (as the TMS ether), m/z: 444 (4%,
MϪTMSOH), 429 (3%, MϪTMSOHϪCH3), 354 (68%, MϪ2TMSOH),
339 (13%, MϪ2TMSOHϪCH3), 253 (100%). HR-MS (APCI-PIM) Calcd
for C24H35O2 [MϩHϪ2H2O]ϩ: 355.2637. Found: m/z, 355.2653.
Fr. 3. 3a,7a,15a-Trihydroxy ester (5a): yield, 38 mg (27%); viscous oil.
IR, nmax cmϪ1: 1739 (ester CϭO), 3229 (OH). (1H-, 13C-NMR and LR-MS,
see Tables 1—3). HR-MS (ESI-PIM) Calcd for C25H42NaO5 [MϩNa]ϩ:
445.2930. Found: m/z, 445.2948.
3a,7a,16a-Trihydroxy-5b-cholan-24-oic Acid (3) The ester 3a (40 mg)
was refluxed in 5% methanolic KOH (5.0 ml) for 1 h. Most of the solvent
was evaporated off, and the residue was dissolved in water and acidified with
10% H2SO4 with stirring and ice-bath cooling. The precipitate was collected
by filtration, washed with water, and recrystallized from EtOAc–hexane as a
colorless amorphous solid of 3: yield, 38 mg (98%); mp, 131—134 °C. IR,
3a,7a-Dihydroxy-5b-cholane O-24,16b-Lactone (23) This compound
was prepared from the 3a,7a,16b-trihydroxy acid (4) by the procedure as
described in the preparation of 22: yield, 15 mg (80%); viscous oil. IR, nmax
1
nmax cmϪ1: 1709 (ester CϭO), 3278 (OH). H-NMR (CDCl3ϩ10% DMSO-
d6), d: 0.67 (s, 3H, 18-CH3), 0.89 (s, 3H, 19-CH3), 0.95 (d, 3H, Jϭ5.9 Hz,
21-CH3), 3.44 (br m, 1H, 3b-H), 3.81 (m, 1H, 7b-H), 4.00 (t, 1H, Jϭ5.4 Hz,
16b-H). HR-MS (ESI-NIM) Calcd for C24H39O5 [MϪH]Ϫ: 407.2797.
Found: m/z, 407.2807.
1
cmϪ1: 1720 (ester CϭO), 3429 (OH). H-NMR, d: 0.85 (s, 3H, 18-CH3),
0.92 (s, 3H, 19-CH3), 1.02 (d, 3H, Jϭ6.8 Hz, 21-CH3), 3.48 (br m, 1H, 3b-
H), 3.87 (br d, 1H, Jϭ2.7 Hz, 7b-H), 4.88 (br m, 1H, 16a-H). LR-MS (as the
TMS ether), m/z: 444 (11%, MϪTMSOH), 429 (15%, MϪTMSOHϪCH3),
411 (5%), 354 (100%, MϪ2TMSOH), 339 (16%, MϪ2TMSOHϪCH3),
253 (60%). HR-MS (ACPI-PIM) Calcd for C24H35O2 [MϩHϪ2H2O]ϩ:
355.2637. Found: m/z, 355.2655.
3a,7a,16b-Trihydroxy-5b-cholan-24-oic Acid (4) The ester 4a (50 mg),
hydrolyzed with 5% methanolic KOH and processed as described for the
preparation of 3, yielded the crude acid. Recrystallization from EtOAc–
hexane gave 4 as a colorless amorphous solid: yield, 42 mg (86%); mp,
148—150 °C. IR, nmax cmϪ1: 1712 (ester CϭO), 3356 (OH). 1H-NMR
(CDCl3ϩ10% DMSO-d6), d: 0.86 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3),
3a,7a-Dihydroxy-5b-cholane O-24,17a-Lactone (24) This compound
was prepared from the 3a,7a,17a-trihydroxy acid (21) by the procedure as

1334
Vol. 50, No. 10
described in the preparation 22: yield, 16 mg (82%); mp, 128—131 °C (col- 12) Kimura M., Kawata M., Tohma M., Fijino A., Yamazaki K., Sawaya
orless amorphous solid from acetone–hexane). IR, nmax cmϪ1: 1711 (ester
T., Chem. Pharm. Bull., 20, 1883—1889 (1972).
CϭO), 3456 (OH). 1H-NMR, d: 0.84 (s, 3H, 18-CH3), 0.91 (s, 3H, 19-CH3), 13) Kimura M., Sawaya T., Chem. Pharm. Bull., 25, 1073—1077 (1977).
1.09 (d, 3H, Jϭ6.8 Hz, 21-CH3), 3.46 (br m, 1H, 3b-H), 3.85 (d, 1H, 14) Carlström K., Kirk D. N., Sjövall J., J. Lipid Res., 22, 1225—1234
Jϭ2.7 Hz, 7b-H). LR-MS (as the TMS ether), m/z: 444 (4%, MϪTMSOH),
(1981).
429 (32%, MϪTMSOHϪCH3), 354 (47%, MϪ2TMSOH), 343 (31%), 15) Kulprecha S., Nihira T., Yamada K., Yoshida T., Nilubol N., Taguchi
253 (100%). HR-MS (APCI-PIM) Calcd for C24H35O2 [MϩHϪ2H2O]ϩ:
355.2637. Found: m/z, 355.2651.
H., Appl. Microbiol. Biotechnol., 22, 211—216 (1985).
16) Jodoi Y., Nihira T., Naoki H., Yamada Y., Taguchi H., Tetrahedron, 43,
487—492 (1987).
Acknowledgements This work was supported in part by a Grant-in-Aid 17) Iida T., Yamaguchi T., Nakamori R., Hikosaka M., Mano N., Goto J.,
for Scientific Research from the Ministry of Education, Science, Sports and
Culture of Japan.
Nambara T., J. Chem. Soc. Perkin Trans. 1, 2001, 2229—2236 (2001).
18) Cerrè C., Hofmann A. F., Schteingart C. D., Jia W., Maltby D., Tetra-
hedron, 53, 435—446 (1997).
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