Synthesis and activity evaluation of a series of cholanamides as modulators of the liver X receptors

Article abstract of DOI:10.1016/j.bmc.2018.01.025

The Liver X receptors (LXRs) are members of the nuclear receptor family, that play fundamental roles in cholesterol transport, lipid metabolism and modulation of inflammatory responses. In recent years, the synthetic steroid N,N-dimethyl-3β-hydroxycholenamide (DMHCA) arised as a promising LXR ligand. This compound was able to dissociate certain beneficial LXRs effects from those undesirable ones involved in triglyceride metabolism. Here, we synthetized a series of DMHCA analogues with different modifications in the steroidal nucleus involving the A/B ring fusion, that generate changes in the overall conformation of the steroid. The LXRα and LXRβ activity of these analogues was evaluated by using a luciferase reporter assay in BHK21 cells. Compounds were tested in both the agonist and antagonist modes. Results indicated that the agonist/antagonist profile is dependent on the steroid configuration at the A/B ring junction. Notably, in contrast to DMHCA, the amide derived from lithocholic acid (2) with an A/B cis configuration and its 6,19-epoxy analogue 4 behaved as LXRα selective agonists, while the 2,19-epoxy analogues with an A/B trans configuration were antagonists of both isoforms. The binding mode of the analogues to both LXR isoforms was assessed by using 50 ns molecular dynamics (MD) simulations. Results revealed conformational differences between LXRα- and LXRβ-ligand complexes, mainly in the hydrogen bonding network that involves the C-3 hydroxyl. Overall, these results indicate that the synthetized DMHCA analogues could be interesting candidates for a therapeutic modulation of the LXRs.

Full text of DOI:10.1016/j.bmc.2018.01.025

Accepted Manuscript
Synthesis and activity evaluation of a series of cholanamides as modulators of
the liver X receptors
Mario D. Martínez, Alberto A. Ghini, M. Virginia Dansey, Adriana S. Veleiro,
Adali Pecci, Lautaro D. Alvarez, Gerardo Burton
PII:
S0968-0896(17)32309-X
https://doi.org/10.1016/j.bmc.2018.01.025
BMC 14179
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 November 2017
18 January 2018
26 January 2018
Please cite this article as: Martínez, M.D., Ghini, A.A., Dansey, M.V., Veleiro, A.S., Pecci, A., Alvarez, L.D.,
Burton, G., Synthesis and activity evaluation of a series of cholanamides as modulators of the liver X receptors,
Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.2018.01.025
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Synthesis and activity evaluation of a series of cholanamides
as modulators of the liver X receptors
Mario D. Martínez,a,c Alberto A. Ghini,a,c M. Virginia Dansey,b,c Adriana S. Veleiro,a,c Adali
Pecci,b,d Lautaro D. Alvareza,c and Gerardo Burtona,c
aUniversidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de
Química Orgánica, Buenos Aires, Argentina.
bUniversidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de
Química Biológica, Buenos Aires, Argentina.
cCONICET – Universidad de Buenos Aires, UMYMFOR, Buenos Aires, Argentina.
dCONICET – Universidad de Buenos Aires, IFIBYNE, Buenos Aires, Argentina.
*To whom correspondence should be addressed. Fax: (54-11) 4576-3385; e-mail:
burton@qo.fcen.uba.ar
1

Abstract
The Liver X receptors (LXRs) are members of the nuclear receptor family, that play fundamental
roles in cholesterol transport, lipid metabolism and modulation of inflammatory responses. In
recent years, the synthetic steroid N,N-dimethyl-3-hydroxycholenamide (DMHCA) arised as a
promising LXR ligand. This compound was able to dissociate certain beneficial LXRs effects
from those undesirable ones involved in triglyceride metabolism. Here, we synthetized a series of
DMHCA analogues with different modifications in the steroidal nucleus involving the A/B ring
fusion, that generate changes in the overall conformation of the steroid. The LXRα and LXRβ
activity of these analogues was evaluated by using a luciferase reporter assay in BHK21 cells.
Compounds were tested in both the agonist and antagonist modes. Results indicated that the
agonist/antagonist profile is dependent on the steroid configuration at the A/B ring junction.
Notably, in contrast to DMHCA, the amide derived from lithocholic acid (2) with an A/B cis
configuration and its 6,19-epoxy analogue 4 behaved as LXRα selective agonists, while the 2,19-
epoxy analogues with an A/B trans configuration were antagonists of both isoforms. The binding
mode of the analogues to both LXR isoforms was assessed by using 50 ns molecular dynamics
(MD) simulations. Results revealed conformational differences between LXR- and LXR-
ligand complexes, mainly in the hydrogen bonding network that involves the C-3 hydroxyl.
Overall, these results indicate that the synthetized DMHCA analogues could be interesting
candidates for a therapeutic modulation of the LXRs.
Keywords: Liver X Receptors, Steroid Amides, Molecular Dynamics, DMHCA
2

1. Introduction
The liver X receptors (LXRs) are established mediators of lipid-inducible gene expression and
their activation regulates important steps in cholesterol, fatty acid and bile acid metabolism.1-3
There are two isoforms of the LXRs, LXR and LXR. LXRexpression predominates in
metabolically active tissues such as liver, small intestine, kidney, macrophages and adipose
tissue, whereas LXR is more ubiquitously expressed with particular high levels in the brain.
Many potential applications targeting LXRs have been proposed for the treatment of several
diseases such as atherosclerosis, type 2 diabetes and Alzheimer disease.4-6 However, LXR
agonists elevate liver triglycerides by increasing transcriptional levels of genes that regulate
lipogenesis, such as sterol regulatory element binding transcription factor 1 (SREBP-1c) and fatty
acid synthase (FAS), leading to hepatotoxicity.7 Thus development of therapeutically useful LXR
agonists may require a separation of favorable effects on cholesterol reverse transport from the
unfavorable effects on triglyceride metabolism. One approach to achieve this goal takes
advantage of the predominant expression of the LXR isoform in the liver, by designing
selective agonists for the  isoform that should have a reduced effect on elevating liver
triglycerides. However, activation of LXR in macrophages is required for reduction of
atherosclerotic plaques and for cholesterol efflux.3 An alternate approach seeks selectivity
through differential gene regulation probably associated with selective recruitment of specific
coactivators or corepressors.8 Thus LXR subtype-specific ligands and novel ligands that possess
tissue-specific agonist/antagonist properties might provide drug candidates with improved
therapeutic profiles.
The endogenous LXR ligands are cholesterol metabolites that include additional oxidized
moieties at the side chain. Examples are the 24(S),25-epoxycholesterol, 24(S)-hydroxycholesterol
3

and 22(R)-hydroxycholesterol that are agonist for both LXRs.9,10 Other oxidized derivatives of
cholesterol as 25(R)-cholestenoic acid also activate the LXRs.11 The synthetic ligand N,N-
dimethyl-3-hydroxycholenamide (DMHCA, 1) is an agonist of both LXRs that shows tissue and
gene-selective differential regulation of the ATP-binding cassette transporter 1 (ABCA1) and
SREBP-1c both in vitro and in vivo.12 Cell-based studies indicate that DMHCA enhances
cholesterol efflux in macrophages without stimulating lipogenesis. This steroidal amide neither
increases plasmatic VLDL or LDL-cholesterol nor liver lipid accumulation,12 and on long-term
administration it significantly reduced atheroma formation in apoE-null mice, without increasing
the hepatic triglyceride (TG) levels.13 Moreover, it has been reported that DMHCA exhibits
antiinflammatory effects in animal models without inducing liver lipid accumulation or liver
injury.14
Recently, we reported that the removal of the C-25 methyl group of cholestenoic acid, gives
rise to a drastic change from agonism to inverse agonism in the LXRs response and proposed that
this change may originate in conformational differences of the steroid side chain that affect
coactivator and corepressor recruitment, by interfering with the anchoring of helix 12 in the
agonist conformation.15 Similar effects have been observed by changing the orientation of
substituents on the side chain (e.g. 22R and 22S-hydroxycholesterol).16 However little is known
on the effect of changes of the overall conformation of the steroid nucleus on the LXRs activity
profile except that some 3-hydroxy-5H steroids are agonists of the LXRs (e.g.
hyodesoxycholic acid and a fluorinated analogue).17 Specifically we were interested in evaluating
the effect of changes in the A/B ring fusion and the presence of oxygenated substituents with well
defined orientations in that part of the steroid nucleus.
4

With this purpose we prepared a series of steroidal amides analogues 2-6 (Figure 1),
compounds 2, 3 and 4 present a bent structure at the A/B junction, while compounds 5 and 6 have
flat structures at this junction. The rigid compounds 4-6 were prepared taking into account that
the incorporation of oxygen bridges involving selected atoms of the steroid nucleus provides
conformationally restricted analogues that mimic or change in a controlled way the molecular
shape of the steroid, and consequently the profile activity. They also act as hydrogen bond
acceptors on the  face of the steroid allowing interaction with specific residues in the ligand
binding pocket (LBP) of the receptor.
Figure 1. Structures of DMHCA (1) showing numbering of key atoms and analogues 2-6.
2- Results
2.1. Chemistry
Amides 1-3 were prepared from the readily available carboxylic acids and dimethylamine, in
the presence of benzotriazol-1-yloxy-tris-(dimethylamino)phosphonium hexafluorophosphate
(BOP).18 Compound 4 was obtained from 3-acetoxy-5-cholenic acid methyl ester (7) as shown
in Scheme 1. Introduction of the 6,19-epoxy bridge was carried out by bromohydrin formation
followed by photochemical functionalization of C-19 with diacetoxyiodobenzene to give
5

bromoether 8. Removal of the acetate at C-3 followed by oxidation with pyridinium
chlorochromate gave the 4-3-ketone (9) that was reduced to the 3-alcohol 10 with sodium
borohydride. Hydrolysis of the methyl ester with lithium hydroxide in methanol followed by
acidification gave the carboxylic acid 11 that was reacted with dimethylamine in the presence of
BOP to give amide 4.
Scheme 1: a) i) 7.5% aqueous perchloric acid, N-bromoacetamide, Et2O-THF, 10 ºC, 55 min, ii)
DIB, I2, Cl2CH2, h, 25 ºC, 30 min; b)i) K2CO3/MeOH, THF, 25 ºC, 1h; ii) PCC, BaCO3, 3Å
molecular sieves, Cl2CH2, 25 ºC, 90 min; c) NaBH4, MeOH-Cl2CH2, 25 ºC, 40 min; d) aqueous
5% LiOH, THF-MeOH (3:1), 25 ºC, 2 h; e) i) (benzotriazol-1-yloxy)-tris(dimethylamino)
phosphonium hexafluorophosphate, Cl2CH2-DMF, Et3N, 0 ºC 30 min, then 25 ºC 2 h, ii) water.
Amides 5 and 6 were obtained from bromoether 8 as depicted in Scheme 2. Cleavage of the 6,19-
epoxy bridge with zinc/acetic acid gave compound 12. Acetylation of the 19-hydroxyl, catalytic
hydrogenation (H2, Pd/C 10%, ethyl acetate/ methanol) of the 5,6-double bond followed by
regioselective hydrolysis of the 3-acetate in 13 with K2CO3 in methanol gave alcohol 14 that was
subsequently converted into the 2-steroid 15. Kumar et al. reported the use of
6

Scheme 2: a) activated Zn, 2-propanol, AcOH, 80ºC, 3 h; b)i) Ac2O, py, DMAP, Cl2CH2, 25 ºC,
3 h; ii) H2, Pd/C 10% w/w, EtOAc-EtOH, 50 psi, 25 ºC, 24h; c) K2CO3/MeOH, THF, 25 ºC, 1h;
d)i) (CF3SO2)2O, Cl2CH2, -20 ºC, 30 minutes, ii) sodium acetate, H2O, 5 min; e) Na2CO3/H2O,
MCPBA, Cl2CH2, 0 °C 1 h, then 25 ºC 2 h; f) NaOMe/MeOH, N2, 40 ºC, 4 h; g) i)aqueous 5%
LiOH, THF-MeOH (3:1), 25 ºC, 2 h, ii) (benzotriazol-1-yloxy)-tris(dimethylamino)phosphonium
hexafluorophosphate, Cl2CH2-DMF, Et3N, 0 ºC 30 min, then 25 ºC, 2 h, iii) water; h) i) PCC,
BaCO3, 3Å molecular sieves, Cl2CH2, 25 ºC, 90 min; ii) 1M lithium tri(t-butoxy)aluminum
hydride/THF, 0 °C, 2 h (yield based on recovered compound 5).
trifluoromethanesulfonic anhydride and dimethylaminopyridine (DMAP) or pyridine as an
efficient system for the direct synthesis of various steroidal olefins from the corresponding
secondary alcohols.19 In the case of compound 15 the best results were obtained upon reaction
with trifluoromethanesulfonic anhydride in dichloromethane at -20 °C followed by treatment
with aqueous sodium acetate, without the addition of an amine. Epoxidation of the 2,3-double
bond in 15 with m-chloroperbenzoic acid (MCPBA) gave the 2,3-epoxy steroid 16. The
stereospecific -epoxidation of the alkene takes place because the  face of the A ring is
7

sterically hindered due to the presence of the 19-acetate.20 Deacetylation of 16 with sodium
methoxide in methanol resulted in the intramolecular attack of the 19-alcoxide on C-2 to give the
2,19-epoxy bridge. Hydrolysis of the methyl ester 17 with lithium hydroxide in methanol gave
the 2,19-epoxy acid, that was transformed to the corresponding cholanamide 5 as above. The 3-
hydroxy-2,19-epoxy cholanamide 6 was obtained from 5 by oxidation of the 3-hydroxy group
with pyridinium chlorochromate followed by reduction of the 3-ketone with LiAl(t-BuO)3H in
THF.
2.2. Biological evaluation
In order to evaluate whether the amides 2-6 were able to modulate the LXRs activities a
luciferase reporter assay was performed in BHK21 cells co-transfected with a pRXR together
with either the full length human LXRβ or the full length human LXR expression vectors and
the reporter construct pGL3/LRELuc. DMHCA (1) was moderately active in this assay increasing
basal levels of luciferase activity at 10 μM (Figure 2a) but was a strong antagonist when co-
administered with the synthetic LXR agonist GW3965 (Figure 2b). Compounds 2 and 4 per se
selectively increased basal levels of luciferase activity at 10 μM (Fig. 2a) mediated by the LXR
isoform, but did not exhibit antagonistic activity (Fig. 2b); for the LXR, 2 was a weak agonist
while 4 was a weak antagonist. On the other hand, compound 3 significantly increased luciferase
activity mediated by LXRβ (Fig. 2a) and behaved as a strong antagonist for both LXR isoforms
(Fig. 2b). Compounds 5 and 6 did not affect per se luciferase basal levels but exhibited strong
antagonism for either receptor isoform (Fig. 2b), revealing pure antagonism for both LXR and
LXRβ.
8

Figure 2. LXR activity of DMHCA (1) and analogues 2-6. Steroids were assayed at 10 µM.
Values are expressed as % induction relative to GW3965 1 µM (100%). Means ± S.E. from 3
independent experiments are shown. Differences were determined by one-way ANOVA followed
by Dunnett’s test. * Significantly different (p < 0.05) from their respective control (no steroid
added).
2.3. Molecular dynamics simulation of LXR/ligand complexes.
To gain further insight in the molecular determinants involved in the LXR activity profiles of
the steroidal amides 1-6, we studied their ligand binding modes to both isoforms by using 50 ns
of molecular dynamics (MD) simulations. In the case of the LXRβ, receptor coordinates of the
ligand binding domain (LBD) were taken from the crystal structure of the human LXRβ/24S,25-
epoxycholesterol complex (pdb:1p8d, chain A). Compounds 1-6 were directly introduced into the
ligand binding pocket (LBP) by superimposing the carbon atoms of C and D rings of optimized
structures with the corresponding atoms of 24S,25-epoxycholesterol. In the case of LXRα, since
9

no crystal structures of LXRα/steroid complex has been resolved yet, the starting coordinates of
the human LXRα were taken from the crystal structure of the complex with a non steroidal ligand
(pdb:5avi, chain A). Then the backbone atoms of the LXRα and the LXRβ/ligand complexes
were superimposed, the ligand coordinates were extracted and combined with the LXRα to obtain
the corresponding LXRα/ligand complexes. Visual inspection of LXRα and LXRβ trajectories
shows that the global folding remains essentially intact, and the time-dependent residue
fluctuation (root-mean square deviations, RMSD) measured over the backbone atoms from the
initial structures, reveals that all simulations are reasonably stable (Supplementary material,
Figure S1).
Figure 3. Binding mode of DMHCA (1) and analogues 2-6. Representative snapshots of LXRβ
complexed with 1 (a), 2 (b), 3 (c), 4 (d), 5 (e) and 6 (f). Dotted lines indicate a direct hydrogen
bond interaction between the ligand and the receptor atoms. Values near the dotted lines are the
% Hydrogen Bond Occupancy (HBO) for each interaction (in green for the LXRα and in purple
for the LXRβ complexes).
As a first step, we evaluated the LXRα and LXRβ models by determining the ligand binding
mode of DMHCA (1). The LBDs of LXRα and LXRβ have a 78% of sequence similarity,
although residues forming the LBP are the same in both receptors. Our MD results showed that
10

DMHCA (1) reached a stable binding in both LXRs. The original position of the steroid is
conserved, and polar groups contacted the polar residues of the LBP (Fig. 3a). At the steroid side
chain, the amide carbonyl forms a strong and persistent hydrogen bond with His421 (LXRα) or
His435 (LXRβ). Thus, this group appears to play a role similar to the oxygen atom of the epoxy
group of 24S,25-epoxycholesterol, restricting the orientation of this histidine that is crucial to
stabilize the agonist conformation of the receptor. At the other end of the steroid molecule, three
polar residues around the 3β-hydroxyl take part in a hydrogen bond network, in which water
molecules also participate. In the case of LXRβ, the calculation of the hydrogen bond occupancy
(HBO) showed that the interaction with the 3β-hydroxyl is similar for the three residues, Asn239,
Glu281 and Arg319 (Fig. 3a). Instead, the direct interaction between the ligand and Asn225 and
Glu267 was markedly reduced in the LXRα, with the 3β-hydroxyl more exposed to form
hydrogen bonds with water molecules (Table 1). Nevertheless, the superposition of representative
snapshots of the LXRα/1 and LXRβ/1 complexes shows a similar orientation of the four polar
residues contacting the ligand (Fig. 4a).
11

Figure 4. Superposition of representative snapshots of LXRα (orange) and LXRβ (cyan)
complexes with 1 (a), 4 (b) and 6 (c). Arg: Arg305 (LXRα) or Arg319 (LXRβ); Asn: Asn225
(LXRα) or Asn239 (LXRβ); Glu: Glu267 (LXRα) or Glu281 (LXRβ) and His: His421 (LXRα) or
His435 (LXRβ).
The ligand binding mode of analogues with a bent structure (2-4) exhibited a similar
recognition of the amide group in the steroidal side chain (Fig. 3b-d). However, a marked
difference in the interactions with the 3α-hydroxyl group is observed. Thus, because of the
12

overall bent conformation of the steroid nucleus, the 3α-hydroxyl group moves away from
Asn225/Asn239 and Arg305/Arg319, while maintaining hydrogen bonds of variable frequency
with Glu267/Glu281. In addition, a very persistent interaction is formed between the 3α-hydroxyl
and a polar residue (Ser264/Ser278) positioned close to the  face of the steroid. In the case of
compounds 3 and 4, the O6 atom interacts with the backbone atoms of other LBP residues,
Phe315/Phe329 and Leu316/Leu330, respectively. These interactions were more frequent in the
LXRβ receptor. On the other hand, the calculated HBO between water molecules and the 3α-
hydroxyl of these analogues indicates that this ligand group is more accessible for water
molecules in the LXRα than in the LXRβ (Table 1).
Table 1. Percent Hydrogen Bond Occupancy (HBO) between oxygen atoms of ligands and water
molecules in the  and  isoforms of the LXR.a
Compound
1
2
3
4
5
6
Acceptor Donor
α
β
α
β
α
β
α
β
α
β
α
β
O3b
H2O
94 82
69 28
69 41
60 14
91
52
62
52
60
53
53
54
70
15
16
-
20
15
10
-
99 97
81 82
93 68
52 20
O3b
H2O
O6b
H2O
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
O6b
H2O
a The HBO was calculated using 3.0 Å and 135° for the distance and angle cutoffs respectively.
bLigand Atoms
Thus, when representative snapshots of LXRα/4 and LXRβ/4 complexes are superimposed,
differences are evident in the conformation (and relative position) of asparagine and glutamic
acid residues (Fig. 4b) and could explain the observed selectivity towards the  isoform. The
introduction of an equatorial hydroxyl at C-6 in compound 3, attenuates the above differences in
13

the interactions with both isoforms (Fig. 3c, Table 1) and in the activity profile which resembles
that of DMHCA (Fig. 2).
As in the case of the torsioned analogues (2-4), the amide carbonyl of the analogues with a flat
conformation (5 and 6), also forms a strong and very frequent hydrogen bond with the
His421/His435 (Fig. 3e,f). However, while the 3α-hydroxyl group of compound 5 only interacts
sporadically with Asn225/Asn239 and Glu267/Glu281, the 3β-hydroxyl group of compound 6
forms more frequent hydrogen bonds with Asn225/Asn239, Arg305/Arg319 and Phe315/Phe329.
The HBO values for these interactions are larger in the LXRβ complexes (Fig. 3e,f). Moreover,
while the accessibility of water molecules to the 3α-hydroxyl of compound 5 is similar for both
receptor isoforms, the solvation of the 3β-hydroxyl group of compound 6 is larger in the LXRα/6
complex (Table 1). On the other hand, the oxygen atoms of the 2,19-epoxy bridge of
compounds 5 and 6 interact with Arg305/Arg319 in both LXRs. The superposition of
representative snapshots of the LXRα/6 and LXRβ/6 complexes shows small variations in the
conformation of polar residues interacting with the ligand (Fig. 4c), consistent with the similar
activity profile of this compound with both isoforms.
3. Discussion
Taking the partial agonist DMHCA (1) as starting point, structural changes that bend the A
ring towards the  face of the steroid nucleus as in 2 and 4 resulted in pure agonists, selective for
the LXR isoform. On the other hand, bending the A ring towards the  face as in 5 and 6 gave
strong antagonists for both isoforms, with 6 being completely devoid of any agonistic activity.
Despite the similarity of the ligand binding pockets of both isoforms, MD simulations show
differences between LXR- and LXR-ligand complexes, in the hydrogen bonding networks that
14

involve the C-3 hydroxyl that correlate with differential activity on each isoform. In compounds
2 and 4 that exhibit selectivity towards the LXR, the 3-hydroxyl moves away from
Leu316/330 and binds strongly to Ser264/276 increasing the H-bonding interaction with Glu281
in the LXR. The oxygen bridge in 4 adds a further interaction with Phe315/329, stronger for the
LXR. In compounds 5 and 6, the hydrogen bonding networks with LXR and LXR are
similar, which correlates with the similar activity profile observed for both isoforms. The oxygen
bridge adds a specific interaction with Arg305/319 that is reinforced by the 3-hydroxyl in the
case of compound 6
Interestingly, although all compounds bind to the LXRs restricting the His421 (LXRα) or
His435 (LXRβ) to give the so called “agonist conformation” of helix-12, changes in the A/B
region of the steroid nucleus were able to switch from agonistic to antagonistic activity.
4. Conclusion
Although most ligands designed to regulate LXR activity are non-steroidal, the synthetic
steroid DMHCA has emerged in recent years as very promising candidate, due to its ability to
dissociate the beneficial from the undesirable effects. We showed here that certain structural
modifications at the steroidal nucleus of DMHCA considerably alter the LXR response, in some
cases selectively. The precise understanding of the molecular basis that governs this selectivity
would be very valuable for the development of drugs with improved activity profile. Although
further studies are still needed to fully assess whether these analogues conserve the dissociative
properties of DMHCA, we consider that they may constitute a new scaffold to achieve a
propitious LXR modulation. Moreover, since these new compounds also present marked
15

differences among their agonist/antagonist profile, they arise as useful tools for gaining further
insight on LXR-mediated signaling networks.
5. Experimental
5.1 Chemistry
5.1.1. General
Mps were taken on a Fisher-Johns apparatus and are uncorrected. NMR spectra were recorded
on an Avance II 500 NMR spectrometer (1H at 500.13 MHz, 13C at 125.77 MHz). Chemical
shifts are given in ppm downfield from TMS as internal standard, J values are given in Hz.
Multiplicity determinations and 2D spectra (COSY, NOESY, HSQC and HMBC) were obtained
using standard Bruker software. Exact mass spectra were obtained using a Bruker micrOTOF-Q
II mass spectrometer, equipped with an ESI source operating in positive mode. Flash
chromatography was carried out on silica gel 60, 0.0040-0.0063 mm, Merck 9385. Medium
Pressure Liquid Chromatography (MPLC) was carried out on a Buchi Sepacore purification
system equipped with two pumps of 10 bar maximum pressure. Thin layer chromatography (tlc)
analysis was performed on silica gel 60 F254 (0.2 mm thick). The homogeneity of all compounds
was confirmed by tlc and 1H NMR. Solvents were evaporated at reduced pressure and ca. 40–50
°C.
5.1.2. N,N-dimethyl-3-hydroxy-5-cholenamide (1)
General procedure: To a solution of 3-hydroxy-5-cholenic acid methyl ester (50 mg, 0.13
mmol) in dry DMF (0.3 mL), dimethylamine hydrochloride (16 mg, 0.2 mmol), triethylamine (56
L, 0.4 mmol) and a solution of (benzotriazol-1-yloxy) tris(dimethylamino)phosphonium
hexafluorophosphate (89 mg, 0.2 mmol) in dry dichloromethane (0.3 mL) were successively
16

added at 0 ºC under an argon atmosphere. The reaction mixture was stirred at 0 ºC for 30 min and
then at 25 ºC for 2 hours. Water (5 mL) was added and the mixture was extracted with
dichloromethane. The extract was washed with 1 N HCl and water, dried over anhydrous sodium
sulphate and the solvent evaporated. The residue was purified by MPLC (flow rate: 20 mL/min,
hexane-ethyl acetate 60:40hexane-ethyl acetate 30:70) to give (40 mg, 75 %) identical (NMR)
to that reported.21
5.1.3. N,N-dimethyl-3-hydroxy-5H-cholanamide (2)
Amide 2 (51.0 mg, 95%) was obtained from lithocholic acid (50 mg, 0.133 mmol) (see general
procedure); m.p. 168-170 ºC; 1H NMR (500 MHz, CDCl3): δ 3.61 (1H, m, H-3), 3.00 (3H, s,
CON(CH3)2), 2.93 (3H, s, CON(CH3)2), 2.34 (1H, m, H-23a), 2.19 (1H, m, H-23b), 1.95 (1H, m,
H-12β), 1.86 (1H, m, H-16α), 1.84 (1H, m, H-6), 1.78 (2H, m, H-22a and H-1β), 1.75 (1H, m,
H-4α), 1.65 (1H, m, H-2α), 1.56 (1H, m, H-15α), 1.50 (1H, m, H-4β), 1.43 (1H, m, H-20), 1.40
(1H, m, H-7β), 1.38 (3H, m, H-8, H-9 and H-11), 1.37 (1H, m, H-5), 1.33 (1H, m, H-2β), 1.32
(1H, m, H-22b), 1.28 (1H, m, H-16), 1.25 (1H, m, H-6), 1.23 (1H, m, H-11), 1.14 (1H, m, H-
12α), 1.10 (1H, m, H-17), 1.05 (1H, m, H-7α), 1.04 (2H, m, H-15β and H-14), 0.95 (1H, m, H-
1α), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.91 (3H, s, H-19), 0.64 (3H, s, H-18), 13C NMR (125 MHz,
CDCl3): δ 173.7 (C-24), 71.9 (C-3), 56.5 (C-14), 56.1 (C-17), 42.7 (C-13), 42.1 (C-5), 40.4 (C-9),
40.2 (C-12), 36.5 (C-4), 35.8 (C-8), 35.6 (C-20), 35.3 (C-1), 34.6 (C-10), 31.2 (C-22), 30.5 (C-2),
30.3 (C-23), 28.2 (C-16), 27.2 (C-6), 26.4 (C-7), 24.2 (C-15), 23.4 (C-19), 20.8 (C-11), 18.5 (C-
21), 12.5 (C-18); ESI-HRMS m/z: calcd for C26H45NNaO2 [M+Na]+ 426.3343, found 426.3336.
5.1.4. N,N-dimethyl-3,6-dihydroxy-5H-cholanamide (3)
Amide 3 (29.0 mg, 92%) was obtained from hyodeoxycholic acid (30.0 mg, 0.075 mmol) (see
general procedure); m.p. 188-190 ºC; 1H NMR (500 MHz, CDCl3): δ 4.05 (1H, dt, J = 12.3 and
17

4.7 Hz), 3.62 (1H, m, H-3), 3.01 (3H, s, CON(CH3)2), 2.94 (3H, s, CON(CH3)2), 2.37 (1H, m, H-
23a), 2.22 (1H, m, H-23b), 1.98 (1H, m, H-12β), 1.92 (1H, m, H-4α), 1.90 (1H, m, H-16α), 1.80
(1H, m, H-1β), 1.79 (1H, m, H-22a), 1.70 (1H, m, H-2α), 1.64 (1H, m, H-7β), 1.68 (1H, m, H-5),
1.60 (1H, m, H-15α), 1.46 (1H, m, H-20), 1.44 (1H, m, H-8), 1.42 (2H, m, H-4β and H-11),
1.38 (1H, m, H-9), 1.34 (2H, m, H-2β) and H-22b), 1.32 (1H, m, H-16), 1.18 (1H, m, H-11),
1.17 (1H, m, H-12α), 1.15 (2H, m, H-17 and H-7α), 1.13 (1H, m, H-14), 1.09 (1H, m, H-15β),
1.06 (1H, m, H-1α), 0.93 (3H, d, J = 6.6 Hz, H-21), 0.90 (3H, s, H-19), 0.64 (3H, s, H-18), 13C
NMR (125 MHz, CDCl3): δ 173.7 (C-24), 71.5 (C-3), 68.0 (C-6), 56.1 (C-14), 56.0 (C-17), 48.4
(C-5), 42.8 (C-13), 39.9 (C-12), 39.8 (C-9), 37.3 (C-10), 35.9 (C-1), 35.6 (C-20), 35.0 (C-7), 34.8
(C-8), 31.1 (C-22), 30.3 (C-23), 30.2 (C-2), 29.3 (C-4), 28.1 (C-16), 24.2 (C-15), 23.5 (C-19),
20.7 (C-11), 18.4 (C-21), 12.0 (C-18); ESI-HRMS m/z: calcd for C26H46NNaO3 [M+Na]+
442.3292, found 442.3285.
5.1.5. 3-Acetoxy-5-bromo-6,19-epoxycholanic acid methyl ester (8)
3-Hydroxy-5-cholenic acid methyl ester (3.70 g) was acetylated with acetic anhydride (37.0
mL) and pyridine (75.0 mL) to give the corresponding 3-acetate 7. To a stirred solution of this
compound in ether (50.0 mL) and THF (19.0 mL), cooled to 10 ºC was added 7.5% aqueous
perchloric acid (6.0 mL) followed by N-bromoacetamide (2.00 g) in 8 portions during a 25 min
period at 10-15 ºC, protected from light. After 30 min at room temperature, 1% aqueous sodium
thiosulphate was added, and the mixture was poured into dichloromethane/methanol (10:1 v/v).
Extractive workup gave the 5-bromo-6-hydroxy derivative (5.0 g). The crude bromohydrin
was dissolved in recently distilled dichloromethane (435 mL) and diacetoxyiodobenzene (4.1 g)
and iodine (2.94 g) were added. The reaction mixture was vigorously stirred while irradiating
with a 300 W tungsten lamp (5000 lumen) for 30 min at 25 ºC. After filtration, the solution was
18

diluted with dichloromethane, washed with aqueous sodium thiosulfate, and the solvent
evaporated. Purification of the residue by flash chromatography (hexane-ethyl acetate 80:20)
gave the bromoether 8 (amorphous solid, 3.25 g, 64% from 3-hydroxy-5-cholenic acid methyl
ester); 1H NMR (500 MHz, CDCl3): δ 5.19 (1H, m, H-3), 4.05 (1H, d, J = 4.7 Hz, H-6), 3.98 (1H,
d, J = 8.4 Hz, H-19a), 3.73 (1H, d, J = 8.3 Hz, H-19b), 3.66 (3H, s, OCH3), 2.34 (1H, m, H-23a),
2.30 (1H, m, H-4), 2.25 (1H, m, H-4β), 2.21 (1H, m, H-23b), 2.03 (3H, s, CH3COO), 1.99 (2H,
m, H-7β and H-2β), 1.97 (1H, m, H-12β), 1.88 (1H, m, H-15), 1.79 (1H, m, H-22a), 1.69 (1H,
m, H-7), 1.66 (1H, m, H-9), 1.64 (1H, m, H-1), 1.61 (1H, m, H-8), 1.52 (1H, m, H-16 and H-
1), 1.51 (1H, m, H-2); 1.41 (1H, m, H-20), 1.39 (1H, m, H-11), 1.32 (1H, m, H-22b), 1.28
(1H, m, H-15), 1.23 (2H, m, H-12 and 1H, m, H-14), 1.15 (1H, m, H-11), 1.13 (1H, m, H-
17), 0.90 (3H, d, J = 6.5 Hz, H-21), 1.02 (1H, m, H-16β), 0.69 (3H, s, H-18); 13C NMR (125
MHz, CDCl3): δ 174.7 (C-24), 170.3 (3-CH3COO), 74.5 (C-5), 82.3 (C-6), 70.0 (C-3), 67.5 (C-
19), 55.7 (C-17), 54.3 (C-14), 51.5 (OCH3), 48.6 (C-9), 43.2 (C-13), 45.8 (C-10), 39.7 (C-12),
41.3 (C-4), 35.3 (C-20), 23.2 (C-1), 33.3 (C-8), 31.03 (C-23), 32.8 (C-7), 30.95 (C-22), 27.9 (C-
2), 28.1 (C-16), 23.4 (C-15), 21.6 (C-11), 21.3 (3-CH3COO), 18.2 (C-21), 12.4 (C-18); ESI-
HRMS m/z: calcd for C27H41BrNaO5 [M+Na]+ 547.2030, found 547.2033.
5.1.6. 3-Oxo-6,19-epoxy-4-cholenic acid methyl ester (9)
To a solution of compound 8 (475 mg, 0.90 mmol) in THF (12.7 mL), K2CO3 (128 mg) in
methanol (25.5 mL) was added and the mixture was stirred 1 h at 25 ºC. Water was added and the
reaction mixture was neutralized with 1 N HCl (3.2 mL), concentrated to half of its volume and
extracted with ethyl acetate. The organic layer was washed with water, dried with sodium sulfate
and the solvent evaporated under vacuum. A suspension of pyridinium chlorochromate (302 mg,
1.39 mmol), barium carbonate (279 mg, 0.442 mmol) and 3Å molecular sieves (441 mg) in
19

anhydrous dichloromethane (34.8 mL) was stirred for 5 min under an Ar atmosphere at 25 ºC. A
solution of the residue obtained above in anhydrous dichloromethane (23.2 mL) was added and
the stirring continued at 25 ºC for 90 min. The reaction was diluted with ether, percolated through
celite eluting with ethyl acetate and the solvent was evaporated. The resulting solid was purified
by flash chromatography (hexane-ethyl acetate 90:10  70:30) to give compound 9 (amorphous
solid, 245 mg 0.61 mmol, 68% from 8). 1H NMR (500 MHz, CDCl3): δ 5.81 (1H, s, H-4), 4.69
(1H, d, J = 5.2 Hz, H-6), 4.21 (1H, d, J = 8.1 Hz, H-19a), 3.66 (3H, s, OCH3), 3.50 (1H, d, J =
8.1 Hz, H-19b), 2.36 (2H, m, H-2), 2.35 (1H, m, H-23a), 2.23 (1H, m, H-23b), 2.21 (1H, m, H-
1), 2.07 (1H, m, H-7β), 2.05 (1H, m , H-12β), 1.88 (1H, m, H-16α), 1.84 (1H, m, H-8), 1.81
(1H, m, H-1), 1.80 (1H, m, H-22a), 1.58 (2H, m, H-11 and H-9), 1.52 (1H, m, H-15α), 1.45
(1H, m, H-11), 1.43 (1H, m, H-20), 1.32 (1H, m, H-22b), 1.31 (1H, m, H-16β), 1.25 (1H, m, H-
7α), 1.24 (1H, m, H-12α), 1.15 (2H, m, H-15β and H-14), 1.12 (1H, m, H-17), 0.92 (3H, d, J =
6.5 Hz, H-21), 0.75 (3H, s, H-18); 13C NMR (125 MHz, CDCl3): δ 199.0 (C-3), 174.7 (C-24),
172.2 (C-5), 114.8 (C-4), 77.3 (C-6), 75.6 (C-19), 55.6 (C-17), 54.7 (C-14), 51.5 (OCH3), 50.2
(C-9), 46.0 (C-10), 43.5 (C-13), 41.2 (C-7), 39.4 (C-12), 35.3 (C-20), 33.7 (C-8), 33.3 (C-2), 31.0
(C-23), 30.9 (C-22), 28.1 (C-16), 26.5 (C-1), 24.1 (C-11), 23.7 (C-15), 18.2 (C-21), 12.3 (C-18);
ESI-HRMS m/z: calcd for C25H36NaO4 [M+Na]+ 423.2506, found 423.2510.
5.1.7. 3-Hydroxy-6,19-epoxy-4-cholenic acid methyl ester (10)
Sodium borohydride (17.4 mg, 0.46 mmol) was added to a solution of compound 9 (90.0 mg,
0.23 mmol) in dichloromethane (2.0 mL) and methanol (2.0 mL) at 0 ºC. The reaction mixture
was stirred for 40 min at 25 ºC, acidified (pH 6) with 1N HCl and concentrated to a third of its
volume. Water was added to the residue and then extracted with dichloromethane. The resulting
solid was purified by flash chromatography (hexane-ethyl acetate 90:10  70:30) to give
20

compound 10 (amorphous solid, 76.0 mg, 84%). 1H NMR (500 MHz, CDCl3): δ 5.48 (1H, d, J =
2.4 Hz, H-4), 4.45 (1H, d, J = 4.9 Hz, H-6), 4.07 (1H, d, J = 7.7 Hz, H-19a), 3.66 (3H, s, OCH3),
3.32 (1H, d, J = 7.7 Hz, H-19b), 2.34 (1H, m, H-23a), 2.23 (1H, m, H-23b), 2.00 (1H, m , H-
12β), 1.98 (1H, m, H-1α), 1.93 (1H, m, H-2), 1.89 (1H, m, H-7β), 1.86 (1H, m, H-16α), 1.79
(1H, m, H-22a), 1.78 (1H, m, H-8), 1.54 (1H, m, H-11), 1.52 (1H, m, H-2), 1.50 (1H, m, H-
15α), 1.46 (1H, m, H-9), 1.42 (1H, m, H-20), 1.32 (2H, m, H-1β and H-22b), 1.29 (1H, m, H-
16β), 1.27 (1H, m, H-11), 1.18 (2H, m, H-7α and H-12α), 1.14 (1H, m, H-14), 1.12 (1H, m, H-
15β), 1.10 (1H, m, H-17), 0.93 (3H, d, J = 6.5 Hz, H-21), 0.74 (3H, s, H-18); 13C NMR (125
MHz, CDCl3): δ 174.7 (C-24), 149.6 (C-5), 115.5 (C-4), 77.1 (C-6), 75.3 (C-19), 67.6 (C-3), 55.7
(C-17), 55.0 (C-14), 51.5 (OCH3), 50.2 (C-9), 44.3 (C-10), 43.3 (C-13), 39.7 (C-12), 39.6 (C-7),
35.3 (C-20), 34.3 (C-8), 31.05 (C-23), 30.95 (C-22), 29.0 (C-2), 28.1 (C-16), 25.1 (C-1), 23.6 (C-
15), 22.9 (C-11), 18.2 (C-21), 12.3 (C-18); ESI-HRMS m/z: calcd for C25H38NaO4 [M+Na]+
425.26623, found 425.26616.
5.1.8. 3-Hydroxy-6,19-epoxy-4-cholenic acid (11)
To a solution of compound 10 (20 mg, 0.05 mmol) in a mixture of THF-MeOH 3:1 (2.2 mL),
aqueous 5% lithium hydroxide was added (0.24 mL). After stirring at 25 ºC for 2 h, water was
added (3.5 mL), the mixture was acidified to pH 3 with 1N HCl, concentrated to a third of its
volume and extracted with ethyl acetate. The organic layer was washed with water, dried with
sodium sulfate and the solvent evaporated. The resulting solid was purified by MPLC (flow rate:
20 mL/min, (hexane-ethyl acetate 70:30 50:50) to give acid 11 (amorphous solid, 13.0 mg,
67%). 1H NMR (500 MHz, CDCl3): δ 5.46 (1H, d, J = 2.4 Hz, H-4), 4.44 (1H, d, J = 4.8 Hz, H-
6), 4.38 (1H, m, H-3), 4.06 (1H, d, J = 7.7 Hz, H-19a), 3.30 (1H, d, J = 7.7 Hz, H-19b), 2.39 (1H,
m, H-23a), 2.26 (1H, m, H-23b), 2.00 (1H, m , H-12β), 1.97 (1H, m, H-1α), 1.94 (1H, m, H-2),
21

1.89 (1H, m, H-7β), 1.86 (1H, m, H-16α), 1.81 (1H, m, H-22a), 1.78 (1H, m, H-8), 1.55 (1H, m,
H-11), 1.52 (1H, m, H-2), 1.50 (1H, m, H-15α), 1.46 (1H, m, H-9), 1.45 (1H, m, H-20), 1.33
(1H, m, H-1β), 1.32 (1H, m, H-22b), 1.29 (1H, m, H-16β), 1.28 (1H, m, H-11), 1.18 (2H, m, H-
7α and H-12α), 1.14 (1H, m, H-14), 1.13 (1H, m, H-15β), 1.10 (1H, m, H-17), 0.93 (3H, d, J =
6.6 Hz, H-21), 0.73 (3H, s, H-18); 13C NMR (125 MHz, CDCl3): δ 178.8 (C-24), 149.6 (C-5),
115.4 (C-4), 77.1 (C-6), 75.3 (C-19), 67.6 (C-3), 55.7 (C-17), 55.0 (C-14), 50.2 (C-9), 44.3 (C-
10), 43.4 (C-13), 39.7 (C-12), 39.6 (C-7), 35.3 (C-20), 34.3 (C-8), 30.8 (C-23), 30.7 (C-22), 28.9
(C-2), 28.2 (C-16), 25.1 (C-1), 23.7 (C-15), 22.9 (C-11), 18.2 (C-21), 12.3 (C-18); ESI-HRMS
m/z: calcd for C24H36NaO4 [M+Na]+ 411.25058, found 411.25028.
5.1.9. N,N-Dimethyl-3-hydroxy-6,19-epoxy-4-cholenamide (4)
The steroid acid 11 (25 mg, 0.064 mmol) was treated with a solution of (benzotriazol-1-
yloxy)-tris(dimethylamino)phosphonium hexafluorophosphate in dry dichloromethane as
previously described (General procedure) to give amide 4 after purification by MPLC (flow rate:
20 mL/min, hexane-ethyl acetate 50:500:100) (24.2 mg, 91%); m.p. 192-193 ºC; 1H NMR (500
MHz, CDCl3): δ 5.47 (1H, d, J = 2.4 Hz, H-4), 4.45 (1H, d, J = 4.8 Hz, H-6), 4.05 (1H, d, J = 7.7
Hz, H-19a), 3.31 (1H, d, J = 7.7 Hz, H-19b), 3.02 (3H, s, CON(CH3)2), 2.95 (3H, s,
CON(CH3)2), 2.35 (1H, m, H-23a), 2.21 (1H, m, H-23b), 2.01 (1H, m , H-12β), 1.98 (1H, m, H-
1α), 1.93 (1H, m, H-2), 1.89 (2H, m, H-7β and H-16α), 1.79 (1H, m, H-22a), 1.78 (1H, m, H-8),
1.55 (1H, m, H-11), 1.52 (1H, m, H-2), 1.49 (1H, m, H-15α), 1.46 (2H, m, H-9 and H-20),
1.33 (2H, m, H-1β and H-22b), 1.31 (1H, m, H-16β), 1.28 (1H, m, H-11), 1.18 (2H, m, H-7α
and H-12α), 1.14 (1H, m, H-15β), 1.13 (1H, m, H-14), 1.12 (1H, m, H-17), 0.95 (3H, d, J = 6.6
Hz, H-21), 0.73 (3H, s, H-18); 13C NMR (125 MHz, CDCl3): δ 173.6 (C-24), 149.7 (C-5), 115.4
(C-4), 77.1 (C-6), 75.3 (C-19), 67.6 (C-3), 55.9 (C-17), 55.0 (C-14), 50.2 (C-9), 44.3 (C-10),
22

43.4 (C-13), 39.7 (C-12), 39.6 (C-7), 37.4 and 35.4 (CON(CH3)2, 35.6 (C-20), 34.3 (C-8), 30.4
(C-23), 31.2 (C-22), 29.0 (C-2), 28.2 (C-16), 25.1 (C-1), 23.7 (C-15), 22.9 (C-11), 18.4 (C-21),
12.3(C-18); ESI-HRMS m/z: calcd for C26H41NNaO3 [M+Na]+ 438.2979, found 438.2981.
5.1.10. 3-Acetoxy-19-hydroxy-5-cholenic acid methyl ester (12)
To a solution of 8 (2.70 g, 5.15 mmol) in 2-propanol (275 mL) at 80 ºC, acetic acid (3.8 mL)
and activated Zn (3.40 g) were added. The suspension was stirred for 3 h at 80 ºC. The mixture
was cooled and filtered, the 2-propanol was evaporated under vacuum and the resulting solution
was extracted with dichloromethane. The organic layer was washed with water, dried with
sodium sulfate and the solvent evaporated. The resulting solid was recrystallized from methanol
to give compound 12 (1.77 g, 80 % from 8); m.p. 138-139 ºC; 1H NMR (500 MHz, CDCl3): δ
5.77 (1H, m, H-6), 4.64 (1H, m, H-3), 3.83 (1H, d, J = 10.9 Hz, H-19a), 3.66 (3H, s, OCH3), 3.62
( 1H, bs, H-19b), 2.42 (1H, m, H-4α), 2.35 (1H, m, H-23a), 2.27 (1H, td, J = 13.4 and 1.8 Hz, H-
4β), 2.23 (1H, m, H-23b), 2.02 (2H, m, H-12β and H-7β), 2.03 (3H, s, CH3COO), 1.96 (1H, m,
H-1β), 1.87 (2H, m, H-2α and H-16α), 1.81 (1H, m, H-8), 1.80 (1H, m, H-22a), 1.58 (2H, m, H-
11), 1.57 (1H, m, H-15α), 1.54 (1H, m, H-7α), 1.52 (1H, m, H-2β), 1.43 (1H, m, H-20), 1.33 (1H,
m, H-22b), 1.30 (1H, m, H-16β), 1.16 (1H, m, H-12α), 1.14 (1H, m, H-1α), 1.10 (1H, m, H-17),
1.09 (1H, m, H-15β), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.92 (1H, m, H-9), 0.91 (1H, m, H-14), 0.73
(3H, s, H-18), 13C NMR (125 MHz, CDCl3): δ 174.7 (C-24), 170.5 (3-CH3COO), 134.5 (C-5),
128.3 (C-6), 73.4 (C-3), 62.7 (C-19), 57.5 (C-14), 55.7 (C-17), 51.5 (OCH3), 50.2 (C-9), 42.6
(C-13), 41.6 (C-10), 39.9 (C-12), 38.2 (C-4), 35.4 (C-20), 33.4 (C-8), 33.1 (C-1), 31.2 (C-7),
31.06 (C-23), 31.00 (C-22), 28.1 (C-2 and C-16), 24.0 (C-15), 21.7 (C-11), 21.4 (3-CH3COO),
18.3 (C-21), 12.2 (C-18); ESI-HRMS m/z: calcd for C27H42NaO5 [M+Na]+ 469.2924, found
469.2919.
23

5.1.11. 3,19-Diacetoxy-5H-cholanic acid methyl ester (13)
Acetylation of 12 (2.41 g, 4.91 mmol) in dichloromethane (40 mL) with acetic anhydride (27
mL), pyridine (53.0 mL) and DMAP (1.0 mg) at 25 ºC for 3 h, followed by purification by flash
chromatography (hexane - ethyl acetate 95:5  90:10) gave the 19-acetoxy steroid. To a solution
of the latter product (2.00 g, 4.47 mmol) in ethyl acetate (45.0 mL) and ethanol (45 mL), Pd/C
10% w/w (300 mg) was added and the resulting mixture was hydrogenated at 50 psi and 25 ºC
for 24 h. The reaction mixture was then filtered through a silica gel pad and the filter was washed
with ethyl acetate. The filtrate was evaporated under reduced pressure to give compound 13
(amorphous solid, 1.96 mg, 98 % from 12).1H NMR (500 MHz, CDCl3): δ 4.72 (1H, m, H-3),
4.32 (1H, d, J = 12.1 Hz, H-19a), 4.23 (1H, d, J = 12.1 Hz, H-19b), 3.66 (3H, s, OCH3), 2.33
(1H, ddd, J = 15.5, 10.4 and 5.2 Hz, H-23a), 2.22 (1H, m, H-23b), 2.21 (1H, m, H-1β), 2.07 (3H,
s, 19-CH3COO), 2.02 (3H, s, 3-CH3COO), 1.94 (1H, m, H-12β), 1.85 (1H, m, H-16α), 1.84 (1H,
m, H-2α), 1.78 (1H, m, H-22a), 1.71 (1H, m, H-4α), 1.70 (1H, m, H-7β), 1.57 (1H, m, H-15α),
1.56 (1H, m, H-11), 1.46 (1H, m, H-4β), 1.45 (1H, m, H-8), 1.42 (1H, m, H-20), 1.40 (1H, m,
H-2β), 1.36 (1H, m, H-11), 1.33 (2H, m, H-5 and H-22b), 1.26 (2H, m, H-6 and H-16), 1.08
(2H, m, H-17 and H-15β), 1.07 (1H, m, H-12α), 1.01 (1H, m, H-14), 0.93 (1H, m, H-1α), 0.92
(1H, m, H-7α), 0.90 (3H, d, J = 6.5 Hz, H-21), 0.74 (1H, m, H-9), 0.64 (3H, s, H-18), 13C NMR
(125 MHz, CDCl3): δ 174.7 (C-24), 171.2 (19-CH3COO), 170.7 (3-CH3COO), 73.0 (C-3), 62.7
(C-19), 56.6 (C-14), 55.8 (C-17), 54.1 (C-9), 51.5 (OCH3), 44.7 (C-5), 42.6 (C-13), 40.2 (C-12),
37.9 (C-10), 35.8 (C-8), 35.4 (C-20), 34.2 (C-4), 31.8 (C-7), 31.6 (C-1), 31.04 (C-23), 30.96 (C-
22), 28.1 (C-6), 28.0 (C-16), 27.5 (C-2), 24.1 (C-15), 22.2 (C-11), 21.4 (19-CH3COO), 21.2 (3-
CH3COO), 18.2 (C-21), 12.1 (C-18); ESI-HRMS m/z: calcd for C29H46Na6 [M+Na]+ 513.3187,
found 513.3190.
24

5.1.12. 3-Hydroxy-19-acetoxy-5H-cholanic acid methyl ester (14)
Compound 13 (1.89 g, 4.23 mmol) in THF (60.0 mL), was treated with K2CO3 (600 mg) in
methanol (120 mL) as previously described for 9. The resulting solid was purified by flash
chromatography (hexane-ethyl acetate 90:10  80:20) to give compound 14 (amorphous solid,
1.26 g, 74%); 1H NMR (500 MHz, CDCl3): δ 4.33 (1H, d, J = 12.1 Hz, H-19a), 4.20 (1H, d, J =
12.1 Hz, H-19b), 3.66 (3H, s, OCH3), 3.65 (1H, m, H-3), 2.34 (1H, ddd, J = 15.5, 10.4 and 5.2
Hz, H-23a), 2.22 (1H, m, H-23b), 2.20 (1H, m, H-1β), 2.06 (3H, s, 19-CH3COO), 1.94 (1H, m,
H-12β), 1.85 (1H, m, H-16α), 1.84 (1H, m, H-2α), 1.78 (1H, m, H-22a), 1.71 (1H, m, H-7β), 1.68
(1H, m, H-4α), 1.59 (1H, m, H-11), 1.57 (1H, m, H-15α), 1.47 (1H, m, H-8), 1.40 (1H, m, H-
20), 1.38 (1H, m, H-4β), 1.34 (1H, m, H-11), 1.32 (1H, m, H-22b), 1.31 (1H, m, H-2β), 1.28
(1H, m, H-16), 1.27 (3H, m, H-5 and H-6), 1.08 (2H, m, H-17 and H-15β), 1.07 (1H, m, H-
12α), 1.00 (1H, m, H-14), 0.90 (1H, m, H-7α), 0.90 (3H, d, J = 6.5 Hz, H-21), 0.89 (1H, m, H-
1α), 0.72 (1H, m, H-9), 0.64 (3H, s, H-18); 13C NMR (125 MHz, CDCl3): δ 174.7 (C-24), 171.2
(19-CH3COO), 70.9 (C-3), 62.8 (C-19), 56.7 (C-14), 55.8 (C-17), 54.3 (C-9), 51.5 (OCH3), 45.0
(C-5), 42.6 (C-13), 40.3 (C-12), 38.4 (C-4), 37.8 (C-10), 35.8 (C-8), 35.4 (C-20), 31.9 (C-7), 31.8
(C-1), 31.6 (C-2), 31.04 (C-23), 30.97 (C-22), 28.2 (C-6), 28.0 (C-16), 24.1 (C-15), 22.3 (C-11),
21.2 (3-CH3COO), 18.2 (C-21), 12.1 (C-18); ESI-HRMS m/z: calcd for C27H44NaO5 [M+Na]+
471.3081, found 471.3089.
5.1.13. 19-Acetoxy-5H-2-cholenic acid methyl ester (15)
Trifluoromethanesulfonic anhydride (75.0 L) was slowly added to a solution of compound 14
(100 mg, 0.22 mmol) in dichloromethane (1.0 mL) at -20 ºC under a N2 atmosphere. After
completion of the addition the reaction was stirred for 30 minutes, then an aqueous solution of
sodium acetate (72.0 mg in 0.1 mL) was added and the reaction was stirred at -20 ºC for 5 min.
25

The reaction mixture was allowed to warm to room temperature and extracted with
dichloromethane and water. The organic layer was washed with water, dried with sodium sulfate
and the solvent evaporated under vacuum. The resulting solid was purified by MPLC (flow rate:
20 mL/min, hexanehexane-ethyl acetate 95:5) to give compound 15 (amorphous solid, 70 mg,
73%). 1H NMR (500 MHz, CDCl3): δ 5.62 (2H, m, H-2 and H-3), 4.31 (1H, d, J = 11.8 Hz, H-
19a), 4.07 (1H, d, J = 11.8 Hz, H-19b), 3.66 (3H, s, OCH3), 2.34 (1H, m, H-23a), 2.30 (1H, m, H-
4β), 2.23 (1H, m, H-23b), 2.03 (3H, s, CH3COO), 1.98 (1H, m, H-12β), 1.96 (1H, m, H-1β), 1.85
(1H, m, H-16α), 1.82 (1H, m, H-1), 1.79 (1H, m, H-22a), 1.74 (1H, m, H-7β), 1.65 (1H, m, H-
4α), 1.60 (1H, m, H-15α), 1.56 (1H, m, H-5), 1.55 (1H, m, H-11), 1.51 (1H, m, H-8), 1.47 (1H,
m, H-11), 1.46 (1H, m, H-6), 1.42 (1H, m, H-20), 1.35 (1H, m, H-6), 1.32 (1H, m, H-22b),
1.28 (1H, m, H-16β), 1.10 (1H, m, H-17), 1.09 (1H, m, H-12α), 1.07 (1H, m, H-15β), 0.97 (1H,
m, H-14), 0.93 (1H, m, H-7α), 0.91 (3H, d, J = 6.5 Hz, H-21), 0.83 (1H, m, H-9), 0.65 (3H, s, H-
18); 13C NMR (125 MHz, CDCl3): δ 174.7 (C-24), 171.2 (19-CH3COO), 126.1-125.5 (C-2 and
C-3), 63.5 (C-19), 56.9 (C-14), 55.8 (C-17), 54.0 (C-9), 51.5 (OCH3), 42.5 (C-13), 41.5 (C-5),
40.2 (C-12), 37.4 (C-10), 36.1 (C-8), 35.4 (C-20), 34.7 (C-4), 31.7 (C-7), 31.04 (C-23), 30.98 (C-
22), 30.4 (C-1), 28.6 (C-6), 28.0 (C-16), 24.1 (C-15), 21.7 (C-11), 21.3 (3-CH3COO), 18.2 (C-
21), 12.0 (C-18); ESI-HRMS m/z: calcd for C27H42NaO4 [M+Na]+ 453.2975, found 453.2965.
5.1.14. 19-Acetoxy-2,3-epoxy-5H-cholanic acid methyl ester (16)
To a solution of 15 (113 mg, 0.262 mmol) in dichloromethane (15 mL) were added Na2CO3
(245 mg) in water (4.9 mL) and 3-chloroperoxybenzoic acid (245mg, 1.42 mmol) in
dichloromethane (9.0 mL), and the mixture was vigorously stirred at 0 °C for 1 h and then at 25
ºC for 2 h. The mixture was diluted with dichloromethane (27 mL) and the aqueous layer was
extracted with dichloromethane. The organic layer was washed with 5% aqueous Na2SO3,
26

saturated aqueous NaHCO3 and water, dried with sodium sulfate and the solvent evaporated. The
resulting solid was purified by MPLC (flow rate: 20 mL/min, hexanehexane-ethyl acetate
75:25) to give compound 16 (amorphous solid, 91.0 mg, 79%); 1H NMR (500 MHz, CDCl3): δ
4.18 (1H, d, J = 11.9 Hz, H-19a), 4.14 (1H, d, J = 11.8 Hz, H-19b), 3.66 (3H, s, OCH3), 3.15
(1H, m, H-2), 3.12 (1H, m, H-3), 2.34 (1H, m, H-23a), 2.22 (1H, m, H-23b), 2.15 (1H, m, H-4β),
2.05 (3H, s, CH3COO), 1.96 (1H, m, H-12β), 1.95 (1H, m, H-1β), 1.84 (2H, m, H-6 and H-
16α), 1.78 (1H, m, H-22a), 1.69 (1H, m, H-7β), 1.61 (1H, m, H-1), 1.58 (1H, m, H-15α), 1.50
(1H, m, H-11), 1.47 (1H, m, H-8), 1.46 (1H, m, H-6), 1.45 (1H, m, H-4α), 1.41 (1H, m, H-20),
1.40 (1H, m, H-11), 1.37 (1H, m, H-16β), 1.31 (1H, m, H-22b), 1.28 (1H, m, H-6), 1.26 (1H,
m, H-5), 1.08 (1H, m, H-12α), 1.07 (1H, m, H-17), 1.03 (1H, m, H-15β), 0.90 (1H, m, H-14),
0.90 (3H, d, J = 6.5 Hz, H-21), 0.86 (1H, m, H-7α), 0.74 (1H, m, H-9), 0.63 (3H, s, H-18); 13C
NMR (125 MHz, CDCl3): δ 174.7 (C-24), 170.9 (19-CH3COO), 63.8 (C-19), 56.7 (C-14), 55.7
(C-17), 53.6 (C-9), 52.7 (C-2), 51.4 (OCH3), 51.3 (C-3), 42.4 (C-13), 39.9 (C-12), 36.4 (C-10),
36.2 (C-8), 36.0 (C-5), 35.3 (C-20), 34.0 (C-4), 31.5 (C-7), 30.99 (C-23), 30.92 (C-22), 29.5 (C-
1), 28.2 (C-16), 28.0 (C-6), 24.1 (C-15), 21.22 (C-11), 21.18 (19-CH3COO), 18.2 (C-21), 12.0
(C-18); ESI-HRMS m/z: calcd for C27H42NaO5 [M+Na]+ 469.2925, found 469.2929.
5.1.15. 3-Hydroxy-2,19-epoxy-5H-cholanic acid methyl ester (17)
To a solution of compound 16 (100 mg, 0.22 mmol) in methanol (2 mL), a solution of sodium
methoxide (60 mg, 1.1 mmol) in methanol (5 mL) was added at 0 ºC under a N2 atmosphere and
the reaction mixture was heated at 40 ºC for 4 h. The mixture was diluted with water (5 mL),
concentrated to a third of its volume by evaporation under reduced pressure and extracted with
dichloromethane. The organic layer was washed with water, dried with sodium sulfate and the
solvent evaporated. The resulting solid was purified by MPLC (flow rate: 20 mL/min,
27

hexanehexane-ethyl acetate 50:50) to give compound 17 (amorphous solid, 70 mg, 80%); 1H
NMR (500 MHz, CDCl3): δ 4.14 (1H, dd, J = 6.1 and 4.9 Hz, H-2), 3.89 (1H, m, H-3), 3.75 (1H,
d, J = 8.1 Hz, H-19a), 3.66 (1H, d, J = 8.1 Hz, H-19b), 3.66 (3H, s, OCH3), 2.34 (1H, m H-23a),
2.21 (1H, m, H-23b), 2.00 (1H, m, H-12β), 1.96 (1H, m, H-1α), 1.85 (1H, m, H-16α), 1.78 (1H,
m, H-22a), 1.70 (1H, m, H-1β), 1.67 (1H, m, H-5), 1.66 (1H, m, H-7), 1.65 (1H, m, H-11b),
1.59 (1H, m, H-15α), 1.52 (2H, m, H-4), 1.49 (1H, m, H-6), 1.41 (1H, m, H-20), 1.34 (1H, m,
H-11), 1.31 (1H, m, H-22b), 1.27 (1H, m, H-16β), 1.16 (1H, m, H-12α), 1.15 (2H, m, H-6 and
H-9), 1.10 (1H, m, H-17) 1.02 (1H, m, H-15β), 0.98 (1H, m, H-14), 0.91 (3H, d, J = 6.6 Hz, H-
21), 0.87 (1H, m, H-8 and H-7), 0.62 (3H, s, H-18); 13C NMR (125 MHz, CDCl3): δ 174.7 (C-
24), 77.6 (C-2), 68.7 (C-3), 66.4 (C-19), 56.5 (C-14), 55.8 (C-17), 51.5 (OCH3), 46.9 (C-10),
46.2 (C-9), 42.3 (C-13), 39.8 (C-5), 39.6 (C-12), 38.5 (C-8), 35.5 (C-4), 35.5 (C-20), 34.8 (C-1),
31.3 (C-7), 31.04 (C-23), 30.96 (C-22), 30.0 (C-6), 28.1 (C-16), 24.1 (C-15), 20.9 (C-11), 18.3
(C-21), 11.8 (C-18); ESI-HRMS m/z: calcd for C25H40NaO4 [M+Na]+ 427.2819, found 427.2806.
5.1.16. N,N-Dimethyl-3-hydroxy-2,19-epoxy-5H-cholanamide (5)
Compound 17 (20 mg, 0.05 mmol) in a mixture of THF-MeOH 3:1 (2.1 mL) was treated with
aqueous 5% lithium hydroxide as previously described for 11. The resulting acid was converted
to the amide 5 following the general procedure described above. The crude material was
crystallized from chloroform/n-hexane to give 5 (20 mg, 89% from 17); m.p. 190-192 ºC; 1H
NMR (500 MHz, CDCl3): δ 4.14 (1H, dd, J = 6.1 and 4.9 Hz, H-2), 3.89 (1H, m, H-3), 3.75 (1H,
d, J = 8.1 Hz, H-19a), 3.66 (1H, d, J = 8.1 Hz, H-19b), 3.00 (3H, s, 24-CON(CH3)2), 2.94 (3H, m,
24-CON(CH3)2), 2.35 (1H, m, H-23a), 2.20 (1H, m, H-23b), 2.01 (1H, m, H-12β), 1.96 (1H, m,
H-1α), 1.88 (1H, m, H-16), 1.78 (1H, m, H-22a), 1.72 (1H, m, H-1β), 1.68 (1H, m, H-5), 1.66
(1H, m, H-7), 1.65 (1H, m, H-11), 1.59 (1H, m, H-15α), 1.53 (2H, m, H-4), 1.50 (1H, m, H-
28

6), 1.44 (1H, m, H-20), 1.35 (1H, m, H-11), 1.33 (1H, m, H-22b), 1.30 (1H, m, H-16β), 1.17
(1H, m, H-12α), 1.16 (2H, m, H-6 and H-9), 1.12 (1H, m, H-17), 1.01 (1H, m, H-15β), 0.98
(1H, m, H-14), 0.94 (3H, d, J = 6.6 Hz, H-21), 0.88 (2H, m, H-8 and H-7), 0.62 (3H, s, H-18);
13C NMR (125 MHz, CDCl3): δ 173.7 (C-24), 77.6 (C-2), 68.7 (C-3), 66.5 (C-19), 56.6 (C-17),
56.0 (C-14), 46.9 (C-10), 46.2 (C-9), 42.3 (C-13), 39.8 (C-5), 39.6 (C-12), 38.5 (C-8), 37.3 (24-
CON(CH3)2), 35.6 (C-20), 35.5 (C-4), 35.4 ((24-CON(CH3)2), 34.8 (C-1), 31.3 (C-7), 31.2 (C-
22), 30.4 (C-23), 30.0 (C-6), 28.1 (C-16), 24.1 (C-15), 20.9 (C-11), 18.5 (C-21), 11.9 (C-18);
ESI-HRMS m/z: calcd for C26H44NO3 [M+H]+ 418.3316, found 418.3311.
5.1.17. N,N-Dimethyl-3-hydroxy-2,19-epoxy-5H-cholanamide (6)
Compound 5 (13 mg, 0.031 mmol) in anhydrous dichloromethane (1.0 mL) was treated with a
suspension of pyridinium chlorochromate (13 mg, 0.060 mmol), barium carbonate (12 mg, 0.019
mmol) and 3Å molecular sieves (19 mg) in anhydrous dichloromethane (1.5 mL) as previously
described for 9. The resulting ketone was dissolved in anhydrous THF (0.5 mL) and a 1 M
solution of lithium tri(t-butoxy)aluminum hydride in anhydrous THF (0.140 mL, 0.140 mmol)
was added at 0 °C under an Ar atmosphere. The resulting solution was stirred for 2 h at 0 °C and
then acidified to pH 2 with a aqueous KHSO4. Extraction with dichloromethane followed by
preparative tlc (toluene-ethyl acetate 40:60) gave compound 6 (5.8 mg, 45%) and compound 5
(2.9 mg, 22%). Compound 6: m.p. 173-175 ºC; 1H NMR (500 MHz, CDCl3): δ 4.12 (1H, d, J =
7.1 Hz, H-2), 3.74 (1H, d, J = 8.0 Hz, H-19b), 3.70 (1H, d, J = 8.3 Hz, H-19a), 3.51 (1H, m, H-
3), 3.00 (3H, s, 24-CON(CH3)2, 2.94 (3H, m, 24-CON(CH3)2), 2.37 (1H, m, H-23a), 2.22 (1H, m,
H-23b), 2.03 (1H, m, H-12β), 2.24 (1H, m, H-1α), 2.02 ( 1H, m, H-4), 1.88 (1H, m, H-16),
1.80 (1H, m, H-22a), 1.14 (1H, m, H-1β), 1.41 (1H, m, H-5), 1.58 (1H, m, H-7), 1.65 (1H, m,
H-11), 1.60 (1H, m, H-15α), 1.13 (1H, m, H-4), 1.70 (1H, m, H-6), 1.46 (1H, m, H-20), 1.40
29