Synthesis of proline derivatives of bile acids and their evaluation as organocatalysts in the asymmetric direct aldol reaction

Article abstract of DOI:10.1016/j.tetasy.2007.05.023

A new family of bile acid derived organocatalysts was obtained by linking l- or d-proline to amino derivatives of cholic and deoxycholic acids, which were used to promote the asymmetric direct aldol reaction between acetone and 4-nitrobenzaldehyde. Both t

Full text of DOI:10.1016/j.tetasy.2007.05.023

Tetrahedron: Asymmetry 18 (2007) 1364–1375
Synthesis of proline derivatives of bile acids and their evaluation
as organocatalysts in the asymmetric direct aldol reaction
a,b
b
b,
*
Gian Luigi Puleo, Matteo Masi and Anna Iuliano
a
Scuola Normale Superiore di Pisa, Piazza dei Cavalieri 7, 56126 Pisa, Italy
Dipartimento di Chimica e Chimica Industriale, Universit a` di Pisa, Via Risorgimento 35, 56126 Pisa, Italy
b
Received 25 April 2007; accepted 23 May 2007
Available online 20 June 2007
Abstract—A new family of bile acid derived organocatalysts was obtained by linking L- or D-proline to amino derivatives of cholic and
deoxycholic acids, which were used to promote the asymmetric direct aldol reaction between acetone and 4-nitrobenzaldehyde. Both the
activity and enantioselectivity of the organocatalytic systems were dependent not only on the position of the proline moiety on the cho-
lestanic backbone and its absolute configuration, but also on the presence of free hydroxyl group on the steroidal skeleton. The cholic
acid derivative bearing a D-prolinamide moiety at the 12-position and free hydroxyl groups at the 3- and 7-positions emerged as the best
organocatalytic system giving complete conversion of the substrate, even when using only 2% of catalyst loading and ee up to 80%.
ꢀ
2007 Elsevier Ltd. All rights reserved.
1
. Introduction
aldolase enzymes,8 which catalyze enantioselective C–C
bond forming reactions by an enamine pathway. Taking
into account these considerations, and following our long-
standing interest in the use of bile acid derivatives in enan-
The enantioselective formation of C–C bonds promoted by
chiral organic molecules (asymmetric organocatalysts)
represents an attractive approach for the synthesis of enan-
tiomerically enriched products. Organocatalysts are usually
endowed with inertness and robustness, so that demanding
reaction conditions, such as an inert atmosphere or abso-
1
9
tioselective processes, we became interested in developing
proline containing bile acids, where, because of their con-
cave structure, caused by the cis junction of the A and B
cyclohexane rings (Fig. 1), the cholestanic backbone and
the appended proline should form a chiral cleft that can
help the enantioselection. In addition the presence of free
hydroxyl groups can constitute a further advantage by con-
trolling, via hydrogen bonds, the position of the substrate
in the cavity of the organocatalyst.
2
lute solvents, are not required. These features make the
use of organocatalysts advantageous instead of organic
ligands in transition metal complexes to promote asymmet-
ric transformations. For these reasons, since List and
Barbas have reported the use of L-proline as an organo-
3
catalyst for the direct asymmetric aldol reaction, the last
seven years have witnessed an explosive growth with
4
regards to asymmetric organocatalytic methods. Much
1
2
effort has been devoted towards the synthesis of new organo-
catalysts and, in this frame, a great deal of interest has been
addressed to the development of proline containing systems
to be used in the asymmetric formation of C–C bonds via
COOH
A
B
OH
7
3
R
5
OH
an enamine pathway, such as aldol and Mannich reac-
6
7
1
1
a R= OH cholic acid
b R=H deoxycholic acid
tions or conjugate additions. The interest in the develop-
ment of this kind of system lies in the consideration that
some molecular architectures, where proline is linked, form
a chiral cleft that can mimic the active site of the class I
Figure 1. Cholic and deoxycholic acid structure.
Since in the transition state of the reaction, proceeding via
enamine pathway, a hydrogen bond is present between an
acidic proton of the catalyst and an acceptor group on the
*
Corresponding author. Tel.: +39 0502219231; fax: +39 0502219260;
e-mail: iuliano@dcci.unipi.it
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.05.023

G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
1365
substrate,10 we judged that the proline moiety had to be
linked to the cholestanic skeleton by means of an amide
bond. This required that one of the hydroxyl groups of bile
acids was selectively transformed into an amino group. In
addition, since it is known that the enantioselectivity of
the bile acid derivatives depends not only on the nature
of the appended moieties, but also on their position on
the cholestanic backbone, L- and D-proline containing
derivatives at the 3-, 7- and 12-positions were synthesized,
in order to find the best position and the best match be-
tween the stereochemistry of the bile acid and the absolute
configuration of proline for asymmetric organocatalysis.
The synthesis of analogous systems having free hydroxyl
groups was also designed in order to evaluate the effect
of the presence of these groups on asymmetric organocatal-
ysis. The activity and enantioselectivity of this new class of
organocatalysts (Fig. 2) were assayed in the asymmetric
direct aldol reaction.
11 were transformed in the corresponding oximes, under
1
1
standard reaction conditions, which were eventually re-
duced, with complete stereoselectivity, to the amino deriv-
atives by means of catalytic hydrogenation followed by
1
1
reaction with Zn in acetic acid.
The proline containing bile acids 2a–b, 3a–b, 4a, 5a–b were
obtained by starting from the corresponding amino deriva-
tives in two steps (Scheme 2). By reacting 12, 13, 16, 17
with L- or D-Boc-proline in the presence of isobutylchloro-
9
d
1
2
formate at room temperature the corresponding prolina-
mides of bile acids were obtained in 50% yield, after
chromatographic purification. The final products were
obtained in quantitative yield by removing the Boc protect-
ing group by means of trifluoroacetic acid in dichlorometh-
1
3
ane solution.
To obtain analogous systems with free hydroxyl groups,
the acetyl moieties of compounds 2b, 4a and 5b were
removed (Scheme 3). The hydrolysis of the 3-acetyl group
2
2
2
3
3
4
4
5
5
5
a R = OAc R1 = L-Pro R2 = H
b R = OAc R1= D-Pro R2 = H
c R = OH R1 = D-Pro R2 = H
of 2b was performed by reacting 2b with HCl in methanol
O
11a
R1
at room temperature.
These reaction conditions
performed well because the 3-acetyl group is equatorial
and affords 2c in quantitative yield. This experimental
procedure cannot be used for removing the acetyl groups
at the 7- and 12-positions, since they are axial and then
less reactive. Therefore, to obtain 4b and 5c, the hydroly-
sis was carried out with sodium methoxide in methanol at
a R = L-Pro R1 = OH
b R = D-Pro R1=OH
R2 = H
R2 = H
OMe
a R = R2 = OAc R1 = D-Pro
b R = R2 = OH
R1 = D-Pro
a R = R1 = OAc R2 = L-Pro
b R = R1 = OAc R2 = D-Pro
R
R2
c R = R1 = OH
R2 = L-Pro
1
4
room temperature. Under these reaction conditions, the
methylester group is stable and 22a and 23a can be ob-
tained 46% and 93% yield, respectively, after chromato-
graphic purification. Removing the Boc protecting
group by means of trifluoroacetic acid in dichlorometh-
Figure 2. Structure of bile acid derived organocatalysts.
1
.1. Synthesis of organocatalysts
1
3
The amino derivatives of the deoxycholic acid, from which
ane solution, gave products 4b and 5c in quantitative
organocatalysts 2a–c and 3a–b can be prepared, were syn-
yield.
1
1
thesized as previously described. The synthetic route to
2- and 7-amino derivatives of cholic acid is reported in
1
1.2. Direct asymmetric aldol reaction
Scheme 1 and it is similar for the two compounds, requir-
ing the oxidation of 7 or 12 hydroxyl groups to ketones
that can be transformed into oximes, easily reduced to ami-
Organocatalysts 2–5 were assayed in the direct asymmetric
aldol reaction between acetone and 4-nitrobenzaldehyde.
The effects of the different reaction parameters, such as sol-
vent, temperature, catalyst loading and the presence of
additives, were investigated using 2b as an organocatalytic
system with the results obtained, listed in Table 1. All reac-
tions were stopped when conversion of the substrate was
complete or did not proceed further, as judged by TLC
analysis. The first run, performed by mixing p-nitrobenzal-
dehyde (0.25 mmol) and 2b (0.025 mmol) in acetone, used
1
1
no groups. In fact, due to the axial position of the 7 and
2 hydroxyl groups, the Mitsunobu transformation did not
1
work; in addition, due to the asymmetric structure of
the cholestanic backbone, the catalytic hydrogenation of
an oxime at these positions proceeds with complete
1
1a
stereoselectivity.
To obtain ketone 8, the carboxylic function of cholic acid
was transformed in methylester, after which the 3- and 7-
hydroxyl groups were acetylated by reacting 6 with acetic
3
b
as reaction solvent, at room temperature gave complete
conversion of the substrate in 48 h and the condensation
product was obtained in 27% ee (entry 1). However,
together with the aldol product, appreciable amounts of
other condensation products together with the product
coming from the dehydration of the aldol were obtained,
and their formation was observed even when performing
the reaction at 0 ꢁC. These problems could be avoided by
using a different reaction procedure, which involves the
preactivation of the organocatalysts obtained by reacting
1
1g
anhydride and pyridine: under these experimental condi-
tions the 12-OH group does not react and 7 was obtained
in 72% yield after chromatographic purification. The oxi-
dation of the 12-OH group, performed with potassium
1
1a
dicromate,
gave 8 in quantitative yield. Ketone 11 was
obtained in four steps from cholic acid, which was reacted
1
1
with methylacetate to give 9 in 75% yield after chro-
matographic purification. The selective oxidation of the
1
1a
5c
7
-hydroxy group with NBS in acetone solution
to give
2b and acetone for 1 h, then adding the aldehyde.
1
0 in quantitative yield, which was then reacted with acetic
anhydride and triethylamine in the presence of DMAP giv-
ing 11 in 88% yield. Both 12- and 7-keto derivatives 8 and
Under these experimental conditions at 0 ꢁC, complete
conversion of the aldehyde was reached in 48 h, with

1366
G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
O
O
O
OMe
OH
OMe
OH
OH
OH
9
1a
6
7
8
a
b
AcO
OH
HO
OH
HO
OH
c
d
O
O
O
OMe
OMe
OH
OMe
OH
10
AcO
AcO
O
AcO
AcO
OAc
OAc
e
f
O
OAc
OMe
O
11
O
OAc
g
O
O
OMe
OMe
HO
OAc
OMe
N
1
5
14
OH
AcO
N
AcO
h
O
O
OMe
OAc
NH2
1
7
16
AcO
NH2
AcO
OAc
Scheme 1. Synthesis of amine derivatives of cholic acid. Reagents and conditions: (a) AcOMe, TsOH, H
c) NBS, acetone/H O, rt, 30 h; (d) Ac O, Py, toluene, rt, 24 h; (e) Ac O, DMAP, Et N, THF, rt, 24 h; (f) K
HCl, NaOAc, MeOH, reflux, 3 h; (h) PtO ÆxH O, AcOH, H (2 bar), rt, 6 d, then Zn powder, rt, 12 h.
2
O, reflux, 24 h; (b) MeI, DBU, CH
2
Cl
2
, rt, 12 h;
OH
(
2
2
2
3
2
Cr , H O, AcOH, rt, 20 h; (g) NH
O
2 7
2
2
2
2
2
neither condensation products nor dehydration product
being observed. In addition the aldol was obtained in
slightly higher ee, unfortunately associated to lower sub-
strate conversion (entry 11). Increasing the temperature
in the presence of this solvent mixture gave complete
conversion of the substrate, but lower enantioselectivity
(entry 12). The use of trifluoroacetic acid as an additive
4
6% ee (entry 2). Further lowering of the reaction temper-
ature, as well as increasing the catalyst loading, caused the
enantioselectivity to decrease (entries 3 and 4). The use of a
lower amount of catalyst did not improve the enantioselec-
tivity, even if the activity of the catalyst remained un-
changed, with complete conversion being reached in 48 h
did not afford positive results, either in DMF-H O solution
2
(entry 14) where no reaction took place, or in acetone
(entry 15), where a very low conversion of the substrate
and 36% ee of the product were obtained.
(
entry 5). Changing the solvent gave, in general, inferior
results both in terms of substrate conversion and enantio-
selectivity (entries 6–13). In fact, when using CHCl or
On the basis of these results, for comparative purposes, the
other organocatalysts were assayed under the experimental
conditions which afforded the best results in terms of con-
version and enantioselectivity, that is, the use of acetone as
solvent at 0 ꢁC with the pre-activation of the catalyst. The
results are listed in Table 2.
3
toluene, complete conversion of the substrate was ob-
served, although 96 h were required in toluene solution
and in both cases the ees were lower (entries 6 and 9). Even
worse results were obtained when using THF or DMSO
(
entries 8 and 10), whereas a DMF–water mixture gave a

G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
1367
N
NH
NH
Boc
NH
O
OMe
O
OMe
O
NH2
O
O
OMe
1
8a L-Pro
8b D-Pro
2a L-Pro
2b D-Pro
AcO
H2N
12
AcO
AcO
1
O
O
O
OH
OH
OH
OMe
OMe
OMe
13
HN
1
9a L-Pro
HN
3a L-Pro
1
9b D-Pro
3b D-Pro
O
O
a
b
N
HN
Boc
N
O
HN
Boc
O
O
O
NH2
NH
O
NH
OMe
OMe
OMe
2
2
0a D-Pro
4a D-Pro
AcO
AcO
OAc
AcO
OAc
OAc
AcO
AcO
OAc
OAc
1
6
O
O
O
OAc
OMe
OMe
OMe
5
a L-Pro
b D-Pro
1a L-Pro
5
NH2
AcO
21b D-Pro
NH
NH
17
O
O
N
HN
Boc
Scheme 2. Synthesis of 2a–b, 3a–b, 4a and 5a–b. Reagents and conditions: (a) (L)- or (D)-Boc-Pro, isobutylchloroformate, NMM, CH
2
Cl
2
, 0 ꢁC, 26 h; (b)
CH Cl , TFA, rt, 15 min.
2
2
The activity of the different organocatalysts was compa-
rable, with complete conversion of the substrate being
reached in 48 h in all cases. Compound 2a, which possesses
an L-proline moiety linked at the 12-position of the deoxy-
cholic acid, gave a remarkably lower enantioselectivity
uration, suggesting a scarce influence of the cholestanic
moiety on the asymmetric induction, which seems depen-
dent only on the aminoacid moiety. The use of 2c, the ana-
logue of 2b possessing a free OH group at the 3-position of
the deoxycholic moiety, did not give better results than 2b
(entry 8), suggesting that a free OH group at this position
does not help the enantioselectivity of the reaction, by con-
trolling the position of the substrate at the inner of the cav-
ity of the catalyst. This is likely due to the high distance
between the moieties linked at positions 3 and 12 of the
cholestanic backbone, which prevents interaction of the
3-OH group with a substrate that lies near the 12-position,
where the active moiety of the catalyst is located. Even the
use of 4a, the analogue of 2b, bearing a D-proline moiety
linked to the 12-position of 3,7-diacetyl cholic acid, did
not work better than 2b, affording the aldol product in
42% ee. On the contrary, the use of 4b, the analogue of
4a bearing two free OH groups at positions 3 and 7, gave
the best enantioselectivity (entry 10). The higher ee can
be explained by taking into account that groups at posi-
tions 7 and 12 are closer than groups at positions 3 and
(
entry 2), suggesting that a mismatched couple can be
obtained when L-proline is linked at the 12-position. How-
ever the sense of asymmetric induction remained
unchanged, with the (S)-enantiomer being obtained with
both the diastereoisomers (entries 1 and 2): this result sug-
gests that the sense of asymmetric induction depends
mainly on the stereochemistry of the cholestanic backbone.
A lower enantioselectivity is obtained when the proline
moiety is linked at the 3-position of deoxycholic acid, as
in 3a and 3b (entries 3 and 4), suggesting that, as observed
with other kind of bile acid derivative,9g derivatization at
the 3-position affords less enantioselective chiral auxilia-
ries. Again, the matched couple is constituted by the diaste-
reoisomer bearing D-proline, although, in this case, the
difference in the extent of asymmetric induction is smaller.
The sense of asymmetric induction changes in passing from
one to another diastereoisomer, suggesting that it depends
on the aminoacid moiety. Very unusual results were
obtained when using, 5a and 5b as organocatalysts, where
the L- or D-proline moiety is linked at the 7-position of the
cholic acid. Both diastereoisomers gave products with the
same ee (entries 5 and 6) but opposite absolute config-
1
5
12, therefore the 7-OH group can interact with a sub-
strate reacting with the group linked at 12-position, by con-
trolling its position inside the chiral cavity and then
affording higher asymmetric induction. This result sug-
gested that the behaviour of 4b could be different from that
one exhibited by the other organocatalysts and prompted

1368
G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
NH
NH
NH
NH
OMe
O
OMe
O
O
O
a
2
c D-Pro
2
b D-Pro
HO
AcO
N
O
HN
Boc
N
O
Boc
O
O
NH
O
OMe
O
NH
NH
OMe
OMe
AcO
OAc 20a D-Pro
22a D-Pro
HO
OH 4b D-Pro
HO
OH
b
c
O
O
OAc
O
OH
OH
OMe
OMe
OMe
AcO
NH
HO
NH
2
1a L-Pro
23a L-Pro
5c L-Pro
HO
NH
O
O
O
N
HN
N
Boc
Boc
Scheme 3. Synthesis of organocatalysts 2c, 4b, 5c. Reagents and conditions: (a) HCl concd, MeOH, rt, 24 h; (b) MeONa/MeOH 10%, rt, 24 h; (c) TFA,
CH Cl , rt, 15 min.
2
2
us to investigate the effect of various reaction parameters
on activity and enantioselectivity of this system. The results
are reported in Table 3.
metric induction was lower. These results suggest the
importance of the role of water in enhancing the catalyst
activity even if the contemporary presence of an organic
1
6
polar solvent is mandatory for the enantioselectivity.
Changing of solvent from acetone to THF or DMF did
not improve the previous result. In fact when using
THF as solvent, the activity dramatically dropped and
the extent of asymmetric induction was lower (entry 2).
On the contrary, the use of DMF did not change the
enantioselectivity to a significant extent, but still worsened
the activity of the catalyst (entry 3), although to a lesser
extent. The use of THF/H O or DMF/H O mixtures
Improvement of the asymmetric induction was obtained
by lowering the temperature to ꢀ20 ꢁC, without the loss
of catalytic activity (entry 9). The best result in terms of
asymmetric induction was reached by lowering the reac-
tion temperature to further ꢀ40 ꢁC (entry 10), but at this
temperature the reaction was slower, affording complete
substrate conversion in 48 h.
2
2
afforded better results in terms of catalyst activity, to give
complete substrate conversion in 24 h instead of 48 (en-
tries 4 and 5). In addition, the presence of water avoided
the formation of by-products coming from the acetone
auto-condensation to give a cleaner reaction. Using the
2. Conclusion
The synthesis of bile acid proline derivatives has allowed us
to obtain a new class of organocatalysts, able to promote
the direct asymmetric aldol reaction. Both activity and
enantioselectivity depended not only on the absolute con-
figuration of the proline moiety but also on the position,
where it is linked on the cholestanic backbone. As already
observed with other type of chiral auxiliaries derived from
bile acids, the derivative bearing the catalyst moiety at the
12-position of the steroid skeleton was the most active and
enantioselective. In addition, the presence of free OH
groups at the 3- and 7-positions of cholic acid afforded
the most efficient organocatalyst, able to promote the
DMF/H O mixture as a reaction solvent allowed us to
2
lower the catalyst loading to 5% without the loss of activ-
ity and with a slight improvement of asymmetric induc-
tion (entry 6). Further lowering of the catalyst loading
to 2% did not give rise to loss of asymmetric induction
and complete conversion of the substrate was still ob-
tained, even if longer reaction time was required (entry
7
). Performing the reaction in water greatly improved
the catalyst activity, to afford complete substrate conver-
sion in 15 h (entry 8): unfortunately the extent of asym-

G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
Table 1. Reaction of p-nitrobenzaldehyde with acetone in the presence of organocatalyst 2b
1369
O
OH O
O
2b
H
+
O N
O2N
2
a
b
c
Entry
Solvent
T (ꢁC)
Time (h)
ee (%)
Conversion (%)
1
2
3
Neat
Neat
Neat
Neat
Neat
rt
0
48
48
48
48
48
48
48
48
96
48
48
48
48
48
120
27
46
34
37
44
32
38
17
29
23
49
36
37
n.d.
14
>99
>99
>99
>99
>99
>99
50
73
>99
26
72
>99
36
0
26
ꢀ20
d
4
0
e
5
0
0
0
0
f
6
CHCl
CH CN
3
f
7
f
3
8
THF
Toluene
DMSO
f
9
0
10
0
rt
0
f
1
1
1
1
1
1
0
1
2
3
4
5
f
H
H
2
O/DMF 2:1
O/DMF 2:1
f
2
f
DMF
f,g
e,g
H
H
2
O/DMF 1:1
O/acetone 4:1
0
2
rt
a
b
c
d
e
f
Reagents and conditions: p-nitrobenzaldehyde (0.5 mmol), acetone (0.5 mL), catalyst (10 mol %); reactions were monitored by TLC.
Determined by HPLC on chiral stationary phase (Chiralpack AS); the absolute configuration of the prevailing enantiomer was (S).
Determined by H NMR.
With 20% of catalyst.
With 5% of catalyst.
1
Solvent: 1 mL. Solvent/acetone ratio 4:1.
With 10% of TFA as additive.
g
Table 2. Reaction of p-nitrobenzaldehyde with acetone in the presence of organocatalyst 2a–c, 3a–b, 4a–b, 5a–c at 0 ꢁC
OH
O
O
O
cat (10%), 0 °C
+
H
48 h
O N
O2N
2
a
b
c
Entry
Catalyst
ee (%)
Absolute configuration
1
2
3
4
5
6
7
8
9
2b
2a
3a
3b
5a
5b
5c
2c
4a
4b
46
12
32
38
21
21
20
41
42
64
S
S
R
S
R
S
R
S
S
S
1
0
a
Reaction conditions: p-nitrobenzaldehyde (0.5 mmol), acetone (solvent), organocatalyst (10 mol %), 0 ꢁC. All the reactions were stopped after 48 h at
complete conversion.
b
c
Determined by HPLC on chiral stationary phase (Chiralpack AS).
4e
Determined by comparison with the literature data.
asymmetric aldol reaction between acetone and 4-nitro-
benzaldehyde with ee up to 80%. These results point out
the importance of the presence of OH groups on the choles-
tanic backbone, able to control the position of the sub-
strate, that allows the reaction to take place into the
chiral cleft formed by the steroidal skeleton and the ap-
pended proline moiety, giving rise to higher levels of asym-
metric induction. Finally, the possibility to obtain complete
substrate conversion with a very low catalyst loading (2%)
makes this system very appealing also for other reaction
proceeding via enamine pathway: work is in progress on
this topic.

1370
G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
Table 3. Reaction of p-nitrobenzaldehyde with acetone in the presence of organocatalyst 4b
O
OH O
O
4b
H +
O N
O2N
2
a
b
c
Entry
Catalyst (mol %)
T (ꢁC)
Time (h)
Solvent
Conversion (%)
ee (%)
1
2
3
4
5
6
7
8
9
10
10
10
10
10
5
2
5
5
5
0
0
0
0
0
0
0
0
48
48
48
48
24
24
48
15
24
48
Acetone
THF
DMF
>99
8
53
>99
>99
>99
>99
>99
>99
>99
64
45
65
67
64
67
65
27
75
80
d
THF/H
DMF/H
DMF/H
DMF/H
2
O
d
2
2
2
O
O
O
d
d
2
H O
DMF/H
DMF/H
d
d
ꢀ20
2
O
O
1
0
ꢀ40
2
a
b
c
Reagents and conditions: p-nitrobenzaldehyde (0.5 mmol), acetone (0.5 mL); reactions were monitored by TLC.
Determined by H NMR.
Determined by HPLC on chiral stationary phase (Chiralpack AS); the absolute configuration of the prevailing enantiomer was (S).
2 2
Solvent/H O 2:1: the same results were obtained using solvent/H O 1:2.
1
d
3
. Experimental
3.3. General procedure for oxime synthesis
3
.1. General procedures and materials
Trihydrated sodium acetate (5.5 equiv) and hydroxylamine
hydrochloride (1.8 equiv) were added to a solution of
ketone 8 or 11 (1.5 g, 1 equiv) in methanol (35 mL). The
reaction mixture was stirred at reflux for 4.5 h. The solvent
was removed under vacuum and the residue dissolved in
CH Cl . The organic solution was washed with water then
TLC analyses were performed on Silica Gel 60 sheets; flash
chromatography separations were carried out on columns
using Silica Gel 60 (230–400 mesh). Toluene was refluxed
over sodium and distilled before the use. THF was refluxed
over Na/K alloy and distilled before use. CH Cl , triethyl-
2
2
dried over Na SO . The solvent was evaporated under
2
2
2
4
amine, pyridine and N-methylmorpholine were refluxed
reduced pressure and the crude product recrystallized by
methanol to afford chemically pure oxime.
over CaH and distilled before use. MeOH was refluxed over
2
magnesium methoxide and distilled before the use. Acetic
anhydride was distilled before the use. NBS was recrystal-
lized by boiling water before the use. Zn powder was washed
with concentrated HCl, water, acetone and diethylether be-
fore the use. MeONa was prepared immediately before the
use with dry MeOHa and metallic Na at 0 ꢁC. Glacial acetic
acid was used. Unless otherwise specified, the reagents were
used without any purification. Methyl-3a-amino-12a-hy-
3
.3.1. Methyl 3a,7a-diacetyloxy-12-oxime-5b-cholan-24-
22
oate 14. Yield 1.60 g, 97%. Mp 94–96 ꢁC. ½aꢁ ¼ þ130:0
D
1
(c 1.00, CH Cl ). H NMR (300 MHz, CDCl , d): 0.91 (s,
2 2 3
3
2
H, CH ), 0.92 (d, 3H, 21-CH ), 1.00 (s, 3H, CH ), 0.90–
3 3 3
.40 (m, 28H, steroidal CH and CH ), 2.00 (s, 3H, 3-
2
CH CO), 2.03 (s, 3H, 7-CH CO), 3.35 (br dd, 1H,
NOH), 3.64 (s, 3H, CH OCO), 4.57 (m, 1H, 3-CH), 4.92
3
3
3
9
b
1
3
droxy-5b-cholan-24-oate 12, methyl 3a-acetyloxy-12a-
(br d, 1H, 7-CH). C NMR (75 MHz, CDCl , d): 12.17,
3
9
b
amino-5b-cholan-24-oate 13, methyl 3a,7a-diacetyloxy-
1
3
4
9.17, 19.93, 21.63, 21.75, 22.31, 23.76, 26.78, 28.16,
0.73, 31.53, 34.67, 35.22, 35.63, 35.83, 36.03, 38.21,
0.92, 46.99, 49.75, 51.68, 53.68, 70.92, 73.99, 165.94
1
1g
1
1
2a-keto-5b-cholan-24-oate 8,
methyl 3a,12a-diacetyl-
1a
oxy-7-keto-5b-cholan-24-oate 11 were obtained as previ-
ously described and matched the reported characteristics.
(
1
C@NOH), 170.51 (acetate C@O), 170.85 (acetate C@O),
ꢀ
1
74.94 (24C@O). IR (KBr, cm ): 2949, 2868, 1734 (br
C@O), 1714 (C@O), 1557, 1537, 1502, 1472, 1457, 1437,
376, 1361, 1250, 1230, 1169, 1059, 1023, 963, 917.
3
.2. Instrumentation
1
1
13
H and C NMR spectra were recorded in CDCl on a
3
Varian Gemini-300 300 MHz NMR spectrometer, using
TMS as external standard. The following abbreviations
are used: s = singlet, d = doublet, dd = double doublet,
t = triplet, m = multiplet, br = broad. HPLC analysis were
performed on a JASCO PU-980 intelligent HPLC pump
equipped with JASCO UV-975 detector. Optical rotations
were measured with a JASCO DIP-360 digital polarimeter.
Melting points were taken using a Kopfler Reichert-Jung
apparatus and are uncorrected. IR spectra were recorded
on a Perkin–Elmer 1710 spectrophotometer.
3.3.2. Methyl 3a,12a-diacetyloxy-7-oxime-5b-cholan-24-
22
oate 15. Yield 3.30 g, 80%. Mp 94–96 ꢁC. ½aꢁ ¼ þ12:0
D
1
(c 1.00, CH Cl ). H NMR (300 MHz, CDCl , d): 0.75 (s,
2
2
3
3H, CH ), 0.82 (d, 3H, 21-CH ), 1.05 (s, 3H, CH ), 0.90–
3
3
3
2.40 (m, 28H, steroidal CH and CH ), 2.00 (s, 3H, 3-
2
CH CO), 2.12 (s, 3H, 12-CH CO), 3.10 (br dd, 1H,
3
3
NOH), 3.66 (s, 3H, CH OCO), 4.68 (m, 1H, 3-CH), 5.10
3
1
3
(t, 1H, 12-CH). C NMR (75 MHz, CDCl , d): 12.75,
3
17.86, 21.54, 21.60, 23.18, 24.79, 25.96, 26.48, 27.49,
27.61, 31.04, 31.21, 32.80, 34.10, 34.78, 35.16, 37.44,

G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
1371
4
2.10, 42.63, 44.59, 45.32, 47.03, 51.74, 73.33, 75.17, 160.59
over 15 min at 0 ꢁC. The reaction mixture was stirred for
26 h. Finally, the reaction mixture was treated with HCl
aq, NaHCO3 aq, NaCl aq then dried over anhydrous
Na SO . The organic phases were concentrated in vacuo
and the residue purified by column chromatography to give
the pure product.
(
1
C@NOH), 170.70 (acetate C@O), 170.80 (acetate C@O),
ꢀ1
74.81 (24C@O). IR (KBr, cm ): 2952, 2867, 1735 (br
C@O), 1650, 1449, 1382, 1243, 1188, 1018, 957, 860, 732,
6
2
4
05.
3
.4. General procedure for reduction of oxime
3.5.1. Methyl 3a-acetyloxy-12a-N-(L-Boc-prolinoyl)amino-
Hydrated PtO (12.5 mol %) was added to a solution of
5b-cholan-24-oate 18a. Purified by chromatography
2
oxime 14 or 15 (usually 3 g, 1 equiv) in glacial acetic acid
(SiO , CH Cl /acetone 92:8). Yield 350 mg, 50%. Mp 68–
2
2
2
2
2
1
(
usually 3 mL) and the mixture was stirred under H2
2 bar) at room temperature for six days. The solid was fil-
70 ꢁC. ½aꢁ ¼ þ31:0 (c 1.00, CH Cl ).
H
NMR
D
2
2
(
(300 MHz, CDCl , d): 0.78 (s, 3H, CH ), 0.86 (d, 3H, 21-
3
3
tered off and powdered Zn (8 equiv) added to the solution,
and concentrated under vacuum to 2 mL. The mixture was
stirred at room temperature for 12 h then the solid was fil-
tered off and washed with acetic acid. After concentration
under reduced pressure, water was added and the aqueous
solution was made basic with KOH pellets. The organic
product was extracted with ethyl acetate and the organic
extracts were dried (Na SO ). The solvent was evaporated
CH ), 0.90 (s, 3H, CH ), 1.00–2.05 (m, 28H, steroidal
3 3
0
0
CH and CH , and 3 and 4 -CH of Boc-Pro), 1.51 (s,
2
2
0
9H, Boc-3CH ), 1.99 (s, 3H, CH CO), 3.47 (m, 2H, 5 -
3
3
CH of Boc-Pro), 3.64 (s, 3H, CH OCO), 4.16 (br s, 1H,
2
3
2’-CH of Boc-Pro), 4.34 (br d, 1H, 12-CH), 4.69 (m, 1H,
1
3
3-CH), 7.41 (br s, NH amide). C NMR (75 MHz, CDCl3,
d): 13.88, 17.32, 21.58, 23.37, 23.92, 24.67, 25.92, 26.30,
26.59, 26.88, 27.57, 28.76 (3CH Boc), 30.97, 31.32, 32.44,
2
4
3
under vacuum affording pure amine.
34.24, 34.93, 35.14, 36.02, 41.85, 44.75, 47.33, 48.49,
50.94, 51.63, 52.87, 60.52, 74.24, 77.44, 80.63, 156.45
3
.4.1. Methyl 3a,7a-diacetyloxy-12a-amino-5b-cholan-24-
(C@O Boc), 170.63 (acetate C@O), 171.00 (amide C@O),
22
ꢀ1
oate 16. Yield 1 g, 81%. Mp 57–58 ꢁC. ½aꢁ ¼ þ35:0 (c
174.92 (24C@O). IR (KBr, cm ): 2959, 2858, 1767
D
1
1
3
2
.00, CH Cl ). H NMR (300 MHz, CDCl , d): 0.70 (s,
(C@O), 1745 (C@O), 1734 (C@O), 1699 (C@O), 1678,
1649, 1633, 1554, 1537, 1520, 1509, 1453, 1245, 1161, 1020.
2
2
3
H, CH ), 0.90 (s, 3H, CH ), 0.95 (d, 3H, 21-CH ), 0.90–
3
3
3
.40 (m, 28H, steroidal CH and CH ), 2.00 (s, 3H, 3-
2
CH CO), 2.04 (s, 3H, 7-CH CO), 3.16 (br t, 1H, 12-CH),
3.5.2. Methyl 3a-acetyloxy-12a-N-(D-Boc-prolinoyl)amino-
5b-cholan-24-oate 18b. Purified by chromatography
(SiO2, CH Cl /acetone 90:10). Yield 266 mg, 49%.
3
3
3
1
2
3
4
1
.64 (s, 3H, CH OCO), 4.56 (m, 1H, 3-CH), 4.86 (br d,
3
1
3
H, 7-CH). C NMR (75 MHz, CDCl , d): 13.75, 17.39,
3
2
2
2
2
1
1.63, 21.84, 22.75, 23.35, 26.96, 27.72, 28.21, 28.87,
1.09, 31.28, 31.50, 34.59, 34.78, 34.95, 35.34, 38.62,
1.20, 41.98, 46.46, 47.96, 51.67, 53.97, 71.14, 74.28,
70.63 (acetate C@O), 170.78 (acetate C@O), 174.70
Mp 61–62 ꢁC. ½aꢁ ¼ þ176:0 (c 1.00, CH Cl ). H NMR
D
2
2
(300 MHz, CDCl , d): 0.77 (s, 3H, CH ), 0.79 (d, 3H, 21-
3 3
CH ), 0.90 (s, 3H, CH ), 0.95–2.60 (m, 28H, steroidal
3
3
0
0
CH and CH , and 3 and 4 -CH of Boc-Pro), 1.52 (s,
2
2
ꢀ
1
0
9H, Boc-3CH ), 2.01 (s, 3H, CH CO), 3.34 (m, 2H, 5 -
3 3
(
24C@O). IR (KBr, cm ): 2949, 2868, 1734 (br C@O),
1
1
710 (C@O), 1683, 1653, 1542, 1507, 1472, 1457, 1436,
CH of Boc-Pro), 3.65 (s, 3H, CH OCO), 4.20 (br d, 1H,
2 3
0
2 -CH of Boc-Pro), 4.37 (br d, 1H, 12-CH), 4.67 (m, 1H,
376, 1366, 1250, 1235, 1169, 1063, 1023, 928.
1
3
3
-CH), 7.57 (br s, NH amide). C NMR (75 MHz, CDCl3,
3
.4.2. Methyl 3a,12a-diacetyloxy-7a-amino-5b-cholan-24-
d): 13.77, 17.46, 21.75, 23.57, 23.96, 24.89, 26.22, 26.65,
26.92, 27.24, 27.73, 28.73 (3CH3 Boc), 31.21, 31.31,
32.49, 34.55, 35.07, 36.25, 42.20, 44.86, 47.51, 48.80,
50.60, 51.72, 52.23, 60.59, 74.77, 74.24, 77.44, 80.65,
170.86 (acetate C@O), 171.05 (amide C@O), 174.82
(24C@O) 181.09 (C@O Boc). IR (KBr, cm ): 2950,
2862, 1738 (br C@O), 1694 (C@O), 1679, 1541, 1526,
1457, 1438, 1398, 1364, 1241, 1167, 1118, 1088, 1024,
980, 886.
22
oate 17. Yield 1.60 g, 55%. Mp 58–60 ꢁC. ½aꢁ ¼ þ68:0
D
1
(
c 1.00, CH Cl ). H NMR (300 MHz, CDCl , d): 0.72 (s,
2 2 3
3
H, CH ), 0.79 (d, 3H, 21-CH ), 0.89 (s, 3H, CH ), 0.90–
3 3 3
2
.40 (m, 28H, steroidal CH and CH ), 2.00 (s, 3H, 3-
2
ꢀ1
CH CO), 2.08 (s, 3H, 12-CH CO), 3.12 (br s, 1H,
CHNH ), 3.64 (s, 3H, CH OCO), 4.54 (m, 1H, 3-CH),
3
3
2
3
1
3
5
1
2
3
7
.07 (br s, 1H, 12-CH). C NMR (75 MHz, CDCl , d):
3
2.35, 17.72, 21.34, 21.54, 22.08, 22.69, 23.03, 25.33,
6.61, 27.39, 27.53, 30.95, 31.18, 31.43, 34.61, 34.82,
4.88, 37.21, 40.56, 42.82, 45.48, 47.54, 48.83, 51.73,
3.84, 74.99, 170.83 (acetate C@O), 170.92 (acetate
3.5.3. Methyl 12a-hydroxy-3a-N-(L-Boc-prolinoyl)amino-
5b-cholan-24-oate 19a. Purified by chromatography
(SiO2, CH Cl /acetone 87:13). Yield 180 mg, 20%.
ꢀ
1
C@O), 174.70 (24C@O), 177.02. IR (KBr, cm ): 2948,
2
2
2
D
2
1
2
1
870, 1734 (br C@O), 1654, 1559, 1475, 1447, 1374, 1251,
Mp 63–65 ꢁC. ½aꢁ ¼ þ65:0 (c 1.00, CH Cl ). H NMR
2
2
026, 717, 611, 583.
(300 MHz, CDCl , d): 0.69 (s, 3H, CH ), 0.93 (s, 3H,
3 3
CH ), 0.98 (d, 3H, 21-CH ), 1.00–2.00 (m, 28H, steroidal
3
3
0 0
CH and CH , and 3 and 4 -CH of Boc-Pro), 1.51 (s,
2 2
9
3
.5. General procedure for prolinamide synthesis
0
H, Boc-3CH ), 1.99 (s, 3H, CH CO), 2.20 (m, 2H, 5 -
3 3
To a solution of N-Boc protected proline (1.1 equiv) in
anhydrous CH Cl , N-methylmorpholine (1.22 equiv) was
CH of Boc-Pro), 3.67 (s, 3H, CH OCO), 3.76 (m, 1H, 3-
2
3
0
CH), 4.00 (br d, 1H, 12-CH), 4.18 (br s, 1H, 2 -CH of
2
2
1
3
added and the mixture cooled to ꢀ20 ꢁC, after which
isobutylchloroformate (1.1 equiv) was added. The reaction
temperature was maintained at ꢀ20 ꢁC for 5 min, then a
CH Cl solution of amine (1 equiv) was added dropwise
Boc-Pro). C NMR (75 MHz, CDCl , d): 12.90, 17.60,
3
23.50, 23.80, 26.30, 27.10, 27.60, 28.00, 28.60, 28.90,
(3CH Boc), 31.10, 31.30, 33.90, 34.30, 35.30, 36.00, 36.20,
3
42.60, 46.70, 47.50, 48.60, 49.40, 51.70, 73.40, 80.60,
2
2

1372
G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
1
28.70 (C@O Boc), 174.80 (2C methylester C@O, amide
2353, 1733 (br C@O), 1677, 1654, 1560, 1520, 1509, 1452,
1408, 1357, 1250, 1167, 1121, 1021, 802, 611.
ꢀ1
C@O). IR (KBr, cm ): 2956, 2862, 1774 (br C@O),
1
1
730, 1656, 1570, 1566, 1510, 1456, 1450, 1420, 1270,
190, 1170, 1087, 1069, 1038, 890.
3.5.7. Methyl 3a,12a-diacetyloxy-7a-(D-Boc-prolinoyl)-
amino-5b-cholan-24-oate 21b. Purified by flash chromato-
graphy (SiO , CH Cl /acetone 90:10). Yield 170 mg,
3
5
.5.4. Methyl 12a-hydroxy-3a-N-(D-Boc-prolinoyl)amino-
b-cholan-24-oate 19b. Purified by chromatography
2
2
2
2
2
32%. Mp 67–68 ꢁC. ½aꢁ ¼ þ103:0 (c 1.00, CH Cl ).
D
2
2
1
(
SiO , CH Cl /acetone 87:13). Yield 420 mg, 42%. Mp
H NMR (200 MHz, CDCl , d): 0.76 (s, 3H, CH ), 0.81
3 3
2
2
2
2
D
2
1
6
9–71 ꢁC. ½aꢁ ¼ þ102:0 (c 1.00, CH Cl ).
H
(d, 3H, 21-CH ), 0.96 (s, 3H, CH ), 0.90–2.40 (m, 28H, ste-
2
2
3 3
0 0
roidal CH and CH and 3 and 4 -CH of Boc-Pro), 1.54 (s,
2 2
NMR (300 MHz, CDCl , d): 0.67 (s, 3H, CH ), 0.90 (s,
3
3
3
H, CH ), 0.96 (d, 3H, 21-CH ), 1.00–2.00 (m, 28H, steroi-
9H, Boc-3CH ), 2.03 (s, 3H, 3-CH CO), 2.18 (s, 3H, 12-
CH CO), 3.32 (br s, 1H, 7-CH), 3.67 (s, 3H, CH OCO),
3 3
3
3
3 3
0
0
dal CH and CH , and 3 and 4 -CH of Boc-Pro), 1.52 (s,
2
2
0
9
H, Boc-3CH ), 2.01 (s, 3H, CH CO), 2.20 (m, 2H, 5 -
3.94 (br s, 1H, 2’-CH of Boc-Pro), 4.36 (br s, 1H, 12-CH)
3
3
1
3
CH of Boc-Pro), 3.65 (s, 3H, CH OCO), 3.75 (m, 1H, 3-
CH), 3.97 (br d, 1H, 12-CH), 4.18 (br d, 1H, 2 -CH of
Boc-Pro). C NMR (75 MHz, CDCl , d): 12.90, 17.60,
2
4.54 (m, 1H, 3-CH), 6.93 ppm (br s, NH amide).
C
2
3
0
NMR (75 MHz, CDCl , d): 12.39, 17.61, 21.58, 22.74,
3
1
3
22.99, 25.79, 26.98, 27.24, 28.58, 29.30, 30.81, 31.02,
34.63, 34.79, 36.63, 41.35, 43.90, 45.32, 46.60, 47.35,
47.61, 51.61, 74.20, 75.40, 80.42, 170.37, 170.70, 171.18,
3
3.50, 23.80, 26.30, 27.20, 27.60, 28.10, 28.60, 28.90
(
3CH Boc), 31.10, 31.30, 34.00, 34.30, 35.30, 35.90, 36.20,
3
ꢀ
1
4
8
2.60, 46.80, 47.50, 48.60, 49.30, 51.70, 61.30, 73.40,
174.60, 198.11 ppm. IR (KBr, cm ): 2960, 2871, 1738
(br C@O), 1703 (C@O), 1679, 1654, 1526, 1506, 1457,
1437, 1398, 1368, 1245, 1162, 1118, 1083, 1022, 960, 887.
0.60, 171.70 (Boc C@O), 174.80 (methylester C@O, amide
ꢀ
1
C@O). IR (KBr, cm ): 2950, 2870, 1753 (br C@O), 1718,
1
1
682, 1655, 1556, 1598, 1507, 1470, 1437, 1371, 1277, 1190,
169, 1092, 1050, 1025, 898.
3.6. General procedure for deacetylation
3
.5.5. Methyl 3a,7a-diacetyloxy-12a-N-(D-Boc-prolinoyl)-
The Boc protected derivative of the bis-acetylated cholic
acid (1 equiv) was treated with MeONa solution 10% in
MeOH (2.2 equiv) for 6 h at rt. The reaction was quenched
in HCl aq, extracted with CH Cl and dried over Na SO .
The organic phases were concentrated in vacuo and the
crude product was purified by column chromatography.
amino-5b-cholan-24-oate 20a. Purified by flash chromato-
graphy (SiO , CH Cl /acetone 95:5). Yield 236 mg, 33%.
2
2
2
2
D
2
1
Mp 63–64 ꢁC ½aꢁ ¼ þ113:0 (c 1.00, CH Cl ). H NMR
2
2
2
2
2
4
(
300 MHz, CDCl , d): 0.78 (s, 3H, CH ), 0.79 (d, 3H, 21-
3
3
CH ), 0.95 (s, 3H, CH ), 0.95–2.60 (m, 28H, steroidal
CH and CH , and 3 and 4 -CH of Boc-Pro), 1.52 (s,
3
3
0
0
2
2
9
H, Boc-3CH ), 2.06 (s, 3H, 3-CH CO), 2.19 (s, 3H, 7-
3.6.1. Methyl 3a,7a-dihydroxy-12a-N-(D-Boc-prolinoyl)-
amino-5b-cholan-24-oate 22a. Purified by chromatogra-
phy (SiO , CH Cl /acetone 1:1). Yield 210 mg, 46%. Mp
3
3
0
CH CO), 3.36 (m, 2H, 5 -CH of Boc-Pro), 3.68 (s, 3H,
CH OCO), 4.28 (br d, 1H, 2 -CH of Boc-Pro), 4.47 (br d,
3
2
0
3
2
2
2
2
2
1
1
8
1
2
3
4
1
H, 12-CH), 4.57 (m, 1H, 3-CH), 4.94 (br d, 1H, 7-CH)
.04 (br s, NH amide). C NMR (75 MHz, CDCl , d):
97–98 ꢁC. ½aꢁ ¼ þ97:4 (c 1.00, CH Cl ). H NMR
D
2
2
1
3
(300 MHz, CDCl , d): 0.77 (s, 3H, CH ), 0.83 (d, 3H, 21-
3
3 3
3.33, 17.23, 21.56, 21.62, 22.88, 23.24, 24.86, 26.71,
7.10, 27.42, 28.47 (3CH3 Boc), 28.85, 30.77, 30.91,
1.51, 34.49, 34.75, 35.01, 38.32, 41.13, 43.78, 44.65,
7.42, 48.25, 51.57, 51.67, 59.83, 70.75, 74.32, 80.59,
56.38 (C@O Boc), 170.53 (acetate C@O), 170.63 (acetate
CH ), 0.84 (s, 3H, CH ), 0.90–2.40 (m, 28H, steroidal
3 3
0
0
CH and CH , and 3 and 4 -CH of Boc-Pro), 1.45 (s,
2
2
0
9H, Boc-3CH ), 3.37 (m, 2H, 5 -CH of Pro), 3.44 (m,
3
2
1H, 3-CH) 3.63 (s, 3H, CH OCO), 3.83 (br s, 1H, 12-
3
1
3
CH), 4.27 (br d, 1H, 7-CH), 7.10 (br d, NH amide).
C
C@O), 170.78 (amide C@O), 174.57 (24C@O). IR (KBr,
cm ): 2961, 1738, 1703, 1698, 1682, 1537, 1457, 1400,
NMR (75 MHz, CDCl , d): 13.69, 17.58, 19.53, 22.66,
3
ꢀ
1
23.43, 24.72, 26.07, 27.68, 27.87, 28.76, 30.55, 31.09,
1
365, 1260, 1167, 1118, 1024, 935, 802.
31.15, 34.67, 35.03, 35.56, 39.66, 39.87, 41.58, 44.52,
44.64, 47.76, 48.73, 51.67, 52.19, 61.14, 68.20, 71.93,
3
.5.6. Methyl 3a,12a-diacetyloxy-7a-(L-Boc-prolinoyl)-
80.97, 171.03 (Boc C@O), 174.74 (24C@O). IR (KBr,
ꢀ1
amino-5b-cholan-24-oate 21a. Purified by flash chromato-
graphy (SiO , CH Cl /acetone 90:10). Yield 260 mg, 50%.
cm ): 2954, 2870, 1772 (br C@O), 1762, 1650, 1569,
1457, 1366, 1258, 1166, 1118, 1078, 1020, 982.
2
2
2
2
D
2
1
Mp 160–161 ꢁC. ½aꢁ ¼ þ23:0 (c 1.00, CH Cl ). H NMR
2
2
(
300 MHz, CDCl , d): 0.72 (s, 3H, CH ), 0.79 (d, 3H,
3.6.2. Methyl 3a,12a-dihydroxy-7a-N-(L-Boc-prolinoyl)-
amino-5b-cholan-24-oate 23a. Yield 180 mg, 93%. Mp
3
3
2
1-CH ), 0.93 (s, 3H, CH ), 0.90-2.40 (m, 28H, steroidal
3
3
0
0
22
D
1
CH and CH and 3 and 4 -CH of Boc-Pro), 1.49 (s, 9H,
Boc-3CH ), 2.02 (s, 3H, 3-CH CO), 2.15 (s, 3H, 12-
104–108 ꢁC. ½aꢁ ¼ ꢀ3:6 (c 1.00, CH Cl ). H NMR
2
2
2
2
(300 MHz, CDCl , d): 0.66 (s, 3H, CH ), 0.92 (s, 3H,
3
3
3 3
CH CO), 3.39 (br s, 1H, 7-CH), 3.64 (s, 3H, CH OCO),
CH ), 0.96 (d, 3H, 21-CH ), 0.90–2.40 (m, 28H, steroidal
3
3
3 3
0
0 0
CH and CH , and 3 and 4 -CH of Boc-Pro), 1.46 (s,
2 2
4
5
.27 (br s, 1H, 2 -CH of Boc-Pro), 4.52 (m, 1H, 3-CH),
.11 (br s, 1H, 12-CH) 6.93 (br s, NH amide). C NMR
1
3
0
9H, Boc-3CH ), 3.37 (m, 2H, 5 -CH of Pro), 3.49 (m,
3 2
(
75 MHz, CDCl , d): 12.51, 17.77, 21.62, 21.70, 23.05,
1H, 3-CH) 3.65 (s, 3H, CH OCO), 3.92 (br s, 1H, 12-
3
3
0
2
3
4
1
5.76, 27.29, 27.41, 28.73 (3CH Boc), 29.18, 29.49, 31.00,
CH), 3.92 (br s, 1H, 2 -CH of Pro), 4.18 (br d, 1H, 7-
3
1
3
1.39, 34.85, 34.91, 35.01, 36.79, 41.49, 44.17, 45.30,
7.51, 47.93, 51.73, 74.22, 75.51, 170.51 (acetate C@O),
70.80 (acetate C@O), 171.05 (amide C@O), 174.67
CH), 7.18 (br d, NH amide). C NMR (75 MHz, CDCl3,
d): 12.55, 17.27, 22.51, 23.21, 27.42, 27.68, 28.17, 28.55,
30.01, 30.86, 31.28, 32.30, 34.76, 35.40, 36.80, 39.86,
41.64, 42.24, 46.08, 46.65, 46.99, 47.17, 51.48, 59.99,
ꢀ1
(
24C@O), 181.09 (C@O Boc). IR (KBr, cm ): 2948,

G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
1373
7
1.76, 73.06, 80.77, 171.17 (Boc C@O), 174.73 (24C@O).
1734 (br C@O), 1713, 1678, 1648, 1633, 1628, 1537, 1522,
1507, 1456, 1431, 1376, 1260, 1189, 1169, 1099, 1069,
1038, 802.
ꢀ1
IR (KBr, cm ): 2963, 2880, 1780 (br C@O), 1702, 1684,
1
539, 1401, 1370, 1260, 1162, 1118, 1087, 1048, 920.
3
.7. General procedure for Boc-deprotection
3.7.4. Methyl 12a-hydroxy-3a-N-(D-prolinoyl)amino-5b-
cholan- 24-oate 3b. Yield 360 mg, quantitative. Mp 80–
2
2
1
A solution of amide in CH Cl (5 mL) was treated with a
large excess of TFA (2 mL) and stirred at rt for 15 h. The
85 ꢁC. ½aꢁ ¼ þ54:0 (c 1.00, CH Cl ).
H
NMR
2
2
D
2
2
(300 MHz, CDCl , d): 0.64 (s, 3H, CH ), 0.88 (s, 3H,
3
3
resulting mixture was washed with NaHCO 5% and
CH ), 0.94 (d, 3H, 21-CH ), 1.00–2.00 (m, 28H, steroidal
3 3
3
0 0
CH and CH , and 3 and 4 -CH of Pro), 2.02 (s, 3H,
2 2
extracted with CH Cl (3 · 30 mL). The organic phase
2
2
0
CH CO), 2.95 (m, 2H, 5 -CH of Pro), 3.67 (s, 3H, CH O-
3 2 3
was then washed with brine (3 · 50 mL) and dried over
1
3
anhydrous Na SO . The solvent was evaporated under
CO), 3.73 (m, 1H, 3-CH), 3.96 (br d, 1H, 12-CH).
C
2
4
vacuum affording chemical pure product.
NMR (75 MHz, CDCl , d): 12.90, 17.50, 23.50, 23.80,
3
26.20, 27.20, 27.60, 28.00, 29.00, 30.80, 31.00, 33.50,
3
.7.1. Methyl 3a-acetyloxy-12a-N-(L-prolinoyl)amino-5b-
34.00, 34.20, 35.20, 35.90, 36.10, 42.60, 46.70, 47.40,
cholan-24-oate 2a. Yield 250 mg, 94%. Mp 55–57 ꢁC.
48.70, 48.90, 51.60 (OCH ), 60.70 (C3), 73.40 (C12),
173.60 (amide C@O), 174.80 (24C@O). IR (KBr, cm ):
2939, 2858, 1734 (br C@O), 1718, 1678, 1648, 1638, 1632,
1542, 1522, 1507, 1457, 1437, 1371, 1260, 1189, 1169,
1099, 1069, 1038, 802.
3
2
2
1
ꢀ1
½
aꢁ ¼ þ73:0 (c 1.00, CH Cl ). H NMR (300 MHz,
D
2
2
CDCl , d): 0.79 (s, 3H, CH ), 0.82 (d, 3H, 21-CH ), 0.89
3
3
3
(
s, 3H, CH ), 0.90–2.40 (m, 28H, steroidal CH and CH ,
3
2
0
0
and 3 and 4 -CH of Pro), 1.98 (s, 3H, CH CO), 3.05
2
3
0
m, 2H, 5 -CH of Pro), 3.64 (s, 3H, CH OCO), 3.82 (br
2 3
(
0
m, 1H, 2 -CH of Pro), 4.16 (br d, 1H, 12-CH), 4.68 (m,
1
3.7.5. Methyl 3a,7a-diacetyloxy-12a-(D-prolinoyl)amino-5b-
cholan-24-oate 4a. Yield 30 mg, quantitative. Mp 70–
1
3
H, 3-CH), 8.18 (br d, NH amide). C NMR (75 MHz,
2
D
2
1
CDCl , d): 13.85, 17.52, 21.58, 23.31, 23.99, 26.32, 26.42,
71 ꢁC. ½aꢁ ¼ þ69:5 (c 1.00, CH Cl ).
H
NMR
3
2
2
2
3
5
6.60, 26.92, 27.67, 31.10, 31.26, 32.35, 34.27, 34.93,
5.11, 36.05, 41.86, 44.82, 47.60, 48.97, 51.07, 51.69,
1.83, 60.85, 74.23, 170.66 (acetate C@O), 173.09 (amide
(300 MHz, CDCl , d): 0.77 (s, 3H, CH ), 0.82 (d, 3H, 21-
3
3
CH ), 0.90 (s, 3H, CH ), 0.90–2.40 (m, 28H, steroidal
3
3
0
0
CH and CH , and 3 and 4 -CH of Pro), 2.01 (s, 3H, 3-
2
2
ꢀ
1
0
CH CO), 2.05 (s, 3H, 7-CH CO), 2.99 (m, 2H, 5 -CH of
3 3 2
C@O), 174.84 (24C@O). IR (KBr, cm ): 2940, 2867,
0
1
1
733 (br C@O), 1655, 1517, 1450, 1383, 1361, 1244, 1194,
Pro), 3.62 (s, 3H, CH OCO), 3.78 (dd, 1H, 2 -CH of
3
166, 1100, 1022, 978, 805.
Pro), 4.16 (br d, 1H, 12-CH), 4.52 (m, 1H, 3-CH), 4.88
1
3
(d, 1H, 7-CH), 8.19 (br d, NH amide).
C NMR
3
.7.2. Methyl 3a-acetyloxy-12a-N-(D-prolinoyl)amino-5b-
(75 MHz, CDCl , d): 13.44, 17.45, 21.65, 22.85, 23.36,
3
cholan-24-oate 2b. Yield 174 mg, quantitative. Mp 75-
26.37, 26.63, 27.08, 27.53, 28.86, 30.76, 31.06, 31.38,
34.57, 34.77, 35.02, 35.26, 38.17, 41.04, 44.33, 44.66,
47.71, 48.71, 51.06, 51.64, 60.98, 70.95, 74.30, 170.22 (ace-
tate C@O), 170.61 (acetate C@O), 173.79 (amide C@O),
22
1
7
7 ꢁC. ½aꢁ ¼ þ171:0 (c 1.00, CH Cl ).
H
NMR
D
2
2
(
300 MHz, CDCl , d): 0.79 (s, 3H, CH ), 0.81 (d, 3H, 21-
3
3
CH ), 0.90 (s, 3H, CH ), 0.90–2.40 (m, 28H, steroidal
3
3
0
0
ꢀ1
CH and CH , and 3 and 4 -CH of Pro), 2.02 (s,
H, CH CO), 3.09 (m, 2H, 5 -CH of Pro), 3.65 (s, 3H,
174.64 (24C@O). IR (KBr, cm ): 2954, 2875, 1740
2
2
0
3
(br C@O), 1662, 1652, 1516, 1438, 1365, 1249, 1172,
1099, 1066, 1024, 968, 892.
3
2
0
CH OCO), 3.95 (br s, 1H, 2 -CH of Pro), 4.17 (br d, 1H,
3
1
3
1
2-CH), 4.67 (m, 1H, 3-CH), 8.14 (br d, NH amide).
C
NMR (75 MHz, CDCl , d): 13.87, 17.44, 21.74, 23.53,
3.7.6. Methyl 3a,7a-dihydroxy-12a-(D-prolinoyl)amino-5b-
cholan-24-oate 4b. Yield 100 mg, quant. Mp 98–100 ꢁC.
3
2
3
4
3.99, 26.17, 26.30, 26.74, 26.91, 27.06, 27.77, 31.08,
1.16, 32.51, 34.34, 34.91, 35.12, 36.17, 42.04, 44.77,
7.68, 48.72, 50.98, 51.80, 52.11, 60.86, 74.52, 170.78 (ace-
2
2
1
½aꢁ ¼ þ79:9 (c 1.00, CH Cl ). H NMR (300 MHz,
D
2
2
CDCl , d): 0.81 (s, 3H, CH ), 0.84 (d, 3H, 21-CH ), 0.89
3
3
3
tate C@O), 172.80 (amide C@O), 174.98 (24C@O). IR
(s, 3H, CH ), 0.90–2.40 (m, 28H, steroidal CH and CH ,
3
2
ꢀ1
0 0 0
and 3 and 4 -CH of Pro), 2.98 (m, 2H, 5 -CH of Pro),
2 2
(
KBr, cm ): 2941, 2872, 1737 (br C@O), 1664, 1649,
1
1
556, 1511, 1447, 1383, 1359, 1256, 1228, 1167, 1093,
025, 975, 758.
3.45 (m, 1H, 3-CH) 3.66 (s, 3H, CH OCO), 3.78 (dd, 1H,
3
0
2 -CH of Pro), 3.87 (br s, 1H, 12-CH), 4.19 (br d, 1H, 7-
1
3
CH), 8.17 (br d, NH amide). C NMR (75 MHz, CDCl3,
d): 13.68, 17.55, 22.98, 23.55, 26.56, 27.76, 28.04, 30.89,
31.26, 34.85, 35.24, 39.84, 41.64, 44.73, 47.71, 48.87,
51.69, 61.04, 68.22, 72.11, 173.91 (Boc C@O), 174.77
(24C@O). IR (KBr, cm ): 2957, 2925, 2869, 1739 (br
C@O), 1648, 1523, 1448, 1382, 1261, 1198, 1170, 1078,
992, 801.
3
.7.3. Methyl 12a-acetyloxy-3a-N-(L-prolinoyl)amino-5b-
cholan-24-oate 3a. Yield 150 mg, quant. Mp 68–72 ꢁC.
2
D
2
1
½
aꢁ ¼ þ36:0 (c 1.00, CH Cl ). H NMR (300 MHz,
2
2
ꢀ
1
CDCl , d): 0,67 (s, 3H, CH ), 0,91 (s, 3H, CH ), 0,96 (d,
3 3 3
3
H, 21-CH ), 1.00–2.00 (m, 28H, steroidal CH and CH ,
3
2
0
0
and 3 and 4 -CH of Pro), 1.98 (s, 3H, CH CO), 2.90
2
3
0
m, 2H, 5 -CH of Pro), 3.66 (s, 3H, CH OCO), 3.75 (m,
2 3
(
1
3
1
H, 3-CH), 3.98 (br d, 1H, 12-CH). C NMR (75 MHz,
3.7.7. Methyl 3a,12a-diacetyloxy-7a-(L-prolinoyl)amino-5b-
cholan-24-oate 5a. Yield 120 mg, 74%. Mp 78–79 ꢁC.
CDCl , d): 12.90, 17.60, 23.50, 23.80, 26.30, 27.20, 27.60,
3
2
D
2
1
2
3
5
8.00, 29.00, 30.80, 31.00, 33.50, 34.00, 34.30 35.30,
6.00, 36.20, 42.60, 46.70, 47.40, 47.60, 48.60, 48.90,
½aꢁ ¼ þ84:0 (c 1.00, CH Cl ). H NMR (300 MHz,
2
2
CDCl , d): 0.73 (s, 3H, CH ), 0.80 (d, 3H, 21-CH ), 0.94
3 3 3
1.60 (OCH ), 60.70 (C3), 73.40 (C12), 173.70 (amide
(s, 3H, CH ), 0.90–2.40 (m, 28H, steroidal CH and CH
3
3
2
ꢀ
1
0 0
and 3 and 4 -CH of Pro), 2.03 (s, 3H, 3-CH CO), 2.13
2 3
C@O), 174.90 (24C@O). IR (KBr, cm ): 2939, 2858,

1374
G. L. Puleo et al. / Tetrahedron: Asymmetry 18 (2007) 1364–1375
0
0 0
CH and CH , and 3 and 4 -CH of Pro), 3.04 (m, 2H,
2 2
(
1
s, 3H, 12-CH CO), 3.08 (m, 2H, 5 -CH of Pro) 3.60 (br s,
3
2
0
0
5 -CH of Pro), 3.62 (m, 1H, 3-CH), 3.67 (s, 3H, CH O-
2 3
H, 7-CH), 3.65 (s, 3H, CH OCO), 3.96 (br s, 1H, 2 -CH of
Pro), 4.53 (m, 1H, 3-CH), 5.10 (br s, 1H, 12-CH) 7.95 (br s,
NH amide). C NMR (75 MHz, CDCl , d): 12.48, 17.79,
3
0
CO), 3.84 (br s, 1H, 2 -CH of Pro), 4.18 (br d, 1H, 12-
1
3
13
CH), 8.21 (br d, NH amide). C NMR (75 MHz, CDCl3,
3
2
2
3
5
1
1.62, 21.70, 23.00, 23.17, 25.70, 26.00, 26.08, 27.20,
7.35, 29.18, 31.05, 31.28, 32.03, 34.85, 34.88, 35.01,
5.74, 36.86, 41.46, 44.41, 45.17, 45.97, 47.51, 47.86,
1.74, 60.61, 74.28, 75.51, 76.47, 170.40 (acetate C@O),
70.50 (acetate C@O), 171.08 (amide C@O), 174.67
d): 13.74, 17.39, 23.36, 23.88, 26.16, 26.31, 26.64, 27.08,
27.66, 29.78, 30.57, 30.77, 31.08, 31.17, 34.16, 34.93,
35.12, 36.07, 36.29, 42.07, 44.69, 47.58, 48.79, 50.97,
51.61, 51.76, 60.88, 71.73, 173.28 (24C@O), 174.78 (amide
ꢀ
1
C@O). IR (KBr, cm ) 2923, 2862, 1741, 1654, 1647, 1560,
ꢀ
1
(
24C@O). IR (KBr, cm ): 2925, 2861, 1739, 1734, 1730,
1522, 1449, 1583, 1310, 1261, 1167, 1097, 1034, 802.
1
1
1
718, 1700, 1696, 1684, 1675, 1669, 1663, 1653, 1647,
636, 1559, 1534, 1517, 1499, 1457, 1437, 1395, 1375,
026, 804.
3.8. General procedure for aldol reaction
The organocatalyst (10 mol %) was stirred in 0.5 mL of
acetone for 1 h. p-Nitrobenzaldehyde (75.56 mg, 0.5 mmol)
was added in 0.5 mL of acetone and the mixture was stirred
at the desired temperature for 4 h. This solution was
3
5
8
.7.8. Methyl 3a,12a-diacetyloxy-7a-(D-prolinoyl)amino-
b-cholan-24-oate 5b. Yield 121 mg, quantitative. Mp
2
D
2
1
0–82 ꢁC. ½aꢁ ¼ þ131:0 (c 1.00, CH Cl ). H NMR
2
2
(
300 MHz, CDCl , d): 0.76 (s, 3H, CH ), 0.83 (d, 3H, 21-
3 3
quenched in aqueous NH Cl (1 mL) and extracted with
4
CH ), 0.96 (s, 3H, CH ), 0.90–2.40 (m, 28H, steroidal
CH and CH and 3 and 4 -CH of Pro), 2.02 (s, 3H, 3-
CH CO), 2.15 (s, 3H, 12-CH CO), 3.08 (m, 2H, 5 -CH
of Pro), 3.67 (s, 3H, CH OCO), 3.95 (br s, 1H, 7-CH)
3
3
AcOEt (1 mL), then dried over anhydrous Na SO and
2
4
0
0
2
2
concentrated under reduced pressure. The residue was puri-
fied over a small column of silica gel (AcOEt/hexane 1:1)
and after concentration, analyzed with HPLC (chiralcel
0
3
3
2
3
0
1
3
.96 (br s, 1H, 2 -CH of -Pro), 4.57 (m, 1H, 3-CH), 5.13
AS, 1.0 mL/min flux, hexane/IPA 85:15) and H NMR.
1
3
(
br s, 1H, 12-CH) 8.12 (br s, NH amide) C NMR
(
75 MHz, CDCl , d): 12.34, 17.65, 21.44, 22.81, 25.52,
3
2
3
4
1
5.95, 26.94, 27.25, 29.01, 30.89, 31.07, 31.92, 34.65,
4.79, 35.42, 36.74, 41.26, 44.20, 45.08, 45.92, 47.24,
7.62, 51.62, 60.49, 74.19, 75.35, 170.43 (acetate C@O),
70.48, 188.63 (amide C@O), 174.62 (24C@O). IR (KBr,
References
1
2
. Berkessel, A.; Gr o¨ ger, H. Asymmetric Organocatalysis;
Wiley-VCH: Weinheim, 2005.
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1
cm ): 2964, 2923, 2856, 1743, 1722, 1650, 1637, 1623,
1
1
565, 1544, 1525, 1511, 1479, 1463, 1442, 1380, 1263,
091, 883, 802.
3
.7.9. Methyl 3a,12a-dihydroxy-7a-(D-prolinoyl)amino-5b-
4
cholan-24-oate 5c. Yield 100 mg, quantitative. Mp 128–
2
D
2
1
1
30 ꢁC. ½aꢁ ¼ þ3:7 (c 1.00, CH Cl ).
H
NMR
2
2
2
003, 103, 3401; (c) Organic Synthesis Highlights V; Schmalz,
(
300 MHz, CDCl , d): 0.67 (s, 3H, CH ), 0.94 (s, 3H,
3
3
H.-G., Wirth, T., Eds.; Wiley-VCH: Weinheim, 2003; (d)
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CH and CH and 3 and 4 -CH of Pro), 3.28 (m, 2H, 5 -
CH of Pro), 3.64 (s, 3H, CH OCO), 3.92 (br s, 1H, 2 -
CH of -Pro), 3.95 (br s, 1H, 7-CH) 4.89 (m, 1H, 3-CH),
3
3
0
0
0
0
2
2
2
3
1
3
5
.45 (br d, 1H, 12-CH) 8.89 (br s, NH amide) C NMR
(
75 MHz, CDCl , d): 12.28, 17.40, 22.70, 23.56, 26.18,
3
(
i) List, B. Asym. Synth. 2007, 161–165; (j) Ley, S. V. Asym.
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3
4
7.91, 28.12, 28.23, 29.92, 31.02, 31.26, 31.44, 31.72, 32.56,
5.47, 35.96, 36.33, 37.11, 41.42, 42.25, 42.45, 46.89, 47.71,
8.06, 48.67, 51.68, 60.01, 72.54, 73.51, 166.90 (amide
Synth. 2007, 201–206.
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Shoji, M.; Sakai, K. Angew. Chem., Int. Ed. 2003, 42, 3677–
3680; (b) Cobb, A. J. A.; Shaw, D. M.; Ley, S. V. Synlett
2004, 558–560; (c) Ibrahem, I.; Zou, W.; Engqvist, M.; Xu,
ꢀ
1
C@O), 174.81 (24C@O). IR (KBr, cm ): 2960, 2923,
2
1
864, 1740, 1727, 1698, 1661, 1631, 1562, 1556, 1497, 1463,
454, 1381, 1260, 1196, 1161, 1098, 1051, 1023, 804.
3
.7.10. Methyl 3a-hydroxy-12a-N-(D-prolinoyl)amino-5b-
cholan- 24-oate 2c. A solution of 2b (174 mg, 0.32 mmol)
in MeOH (8 mL) was treated with a large excess of concen-
trated HCl (0.2 mL) and stirred at rt for 24 h. The resulting
4
mixture was washed with NaHCO 5% and extracted with
3
2
CH Cl (3 · 30 mL). The organic phase was then washed
2
2
with brine (3 · 50 mL) and dried over anhydrous Na SO .
2
4
The solvent was evaporated under vacuum to afford chem-
ically pure product (yield 160 mg, quantitative). Mp 79–
6
2
D
2
1
8
0 ꢁC. ½aꢁ ¼ þ69:6 (c 1.00, CH Cl ).
H
NMR
2
2
(
300 MHz, CDCl , d): 0.81 (s, 3H, CH ), 0.85 (d, 3H, 21-
3
3
CH ), 0.91 (s, 3H, CH ), 0.90–2.40 (m, 28H, steroidal
3
3

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takes place in a ‘concentrated organic phase’ constituted by
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