New highly toxic bile acids derived from deoxycholic acid, chenodeoxycholic acid and lithocholic acid

Article abstract of DOI:10.1016/j.bmc.2013.11.029

We have prepared a new panel of 23 BA derivatives of DCA, chenodeoxycholic acid (CDCA) and lithocholic acid (LCA) in order to study the effect of dual substitution with 3-azido and 24-amidation, features individually associated with cytotoxicity in our previous work. The effect of the compounds on cell viability of HT-1080 and Caco-2 was studied using the 3-[4,5-dimethylthizol-2- yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Compounds with high potency towards reduction of cell viability were further studied using flow cytometry in order to understand the mechanism of cell death. Several compounds were identified with low micromolar IC₅₀ values for reducing cell viability in the Caco-2 and HT1080 cell lines, making them among the most potent BA apoptotic agents reported to date. There was no evidence of relationship between overall hydrophobicity and cytotoxicity supporting the idea that cell death induction by BAs may be structure-specific. Compounds derived from DCA caused cell death through apoptosis. There was some evidence of selectivity between the two cell lines studied which may be due to differing expression of CD95/FAS. The more toxic compounds increased ROS production in Caco-2 cells, and co-incubation with the antioxidant N-acetyl cysteine blunted pro-apoptotic effects. The properties these compounds suggest that there may be specific mechanism(s) mediating BA induced cell death. Compound 8 could be useful for investigating this phenomenon.

Full text of DOI:10.1016/j.bmc.2013.11.029

Bioorganic & Medicinal Chemistry 22 (2014) 256–268
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
New highly toxic bile acids derived from deoxycholic acid,
chenodeoxycholic acid and lithocholic acid
Ferenc Májer a, Ruchika Sharma a, Claire Mullins a, Luke Keogh a, Sinead Phipps b, Shane Duggan b,
Dermot Kelleher b, Stephen Keely c, Aideen Long b, Gábor Radics a, Jun Wang a, John F. Gilmer a,
⇑
a School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland
b Cell and Molecular Biology, Institute of Molecular Medicine, Trinity Centre for Health Science, St James’s Hospital, Dublin 8, Ireland
c Molecular Medicine Lab, RCSI, Smurfit Building, Beaumont Hospital, Beaumont, Dublin 9, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2013
Revised 27 September 2013
Accepted 16 November 2013
Available online 23 November 2013
We have prepared a new panel of 23 BA derivatives of DCA, chenodeoxycholic acid (CDCA) and lithocholic
acid (LCA) in order to study the effect of dual substitution with 3-azido and 24-amidation, features
zindividually associated with cytotoxicity in our previous work. The effect of the compounds on cell via-
bility of HT-1080 and Caco-2 was studied using the 3-[4,5-dimethylthizol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) assay. Compounds with high potency towards reduction of cell viability were further
studied using flow cytometry in order to understand the mechanism of cell death. Several compounds
were identified with low micromolar IC50 values for reducing cell viability in the Caco-2 and HT1080 cell
lines, making them among the most potent BA apoptotic agents reported to date. There was no evidence
of relationship between overall hydrophobicity and cytotoxicity supporting the idea that cell death
induction by BAs may be structure–specific. Compounds derived from DCA caused cell death through
apoptosis. There was some evidence of selectivity between the two cell lines studied which may be
due to differing expression of CD95/FAS. The more toxic compounds increased ROS production in
Caco-2 cells, and co-incubation with the antioxidant N-acetyl cysteine blunted pro-apoptotic effects.
The properties these compounds suggest that there may be specific mechanism(s) mediating BA induced
cell death. Compound 8 could be useful for investigating this phenomenon.
Keywords:
Bile acid
Cytotoxicity
Lipophilicity
Deoxycholic acid
Ursodeoxycholic acid
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introductionz
by membrane activation of PLA2, NAD(P)H oxidase, which causes
intracellular ROS increases.6 DCA causes ligand independent acti-
Bile acids (BAs) are oxidative metabolites of cholesterol whose
principal biological function is the solubilisation of enteral nutri-
ents, facilitating their absorption.1 BAs modulate their own biosyn-
thesis, transport and distribution through interactions with cell
surface and nuclear receptors.2 These interactions have long range
influence on metabolism but their evolution was probably a re-
sponse to BA cytotoxicity. Particularly when in their unconjugated
from, BAs can induce cell death in a wide range of mammalian cell
types.3 This phenomenon is relevant to the pathophysiology of
hepatobiliary diseases and to BA promoter effects in colorectal
and esophageal cancer.1,4
BAs cause cell lysis and necrosis–an effect attributed to a non-
specific capacity to disrupt cell membranes. However BAs can also
induce cell death at sublytic concentrations (a property they share
with detergents such Triton-X).5 Sublytic effects are due in part to
ligand independent activation of the extrinsic or death receptor
pathway (CD95/Fas).6 These effects may be mediated inside-out
vation of EGFR and MAP kinases in some cell types, opposing the
apoptotic effects due to Fas activation.7 DCA effects on cell viability
are therefore complex and sometimes self-attenuating. Increases in
ROS can follow mitochondrial damage which can be direct effect
(intrinsic pathway) or an amplification of death receptor activa-
tion. ER stress is also increasingly being investigated as a mecha-
nism of BA induced cell death.6
A primary chemical trigger for these events continues to evade
elucidation but they are widely believed to be non-specific in ori-
gin and are they are usually attributed to membrane perturbations,
consistent with observations about the importance of lipophilicity
to BA toxicity. However it is likely that there are specific effects at
work too. Katona et al. reported the synthesis of enantiomeric BA
pairs of LCA, DCA and CDCA.8 LCA, DCA and CDCA had the same
experimental critical micellar concentration values as their enan-
tiomeric partners (ent LCA, ent DCA, ent CDCA) but the naturally
occurring stereoisomers were more cytotoxic.9 This indicates that
at least some of the pro-apoptotic features of BAs are due to stereo-
specific interactions, most likely with protein receptors. We
recently reported on the properties of extended library of BAs
⇑
Corresponding author. Tel.: +353 1 896 2795; fax: +353 1 896 2793.
E-mail address: gilmerjf@tcd.ie (J.F. Gilmer).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.11.029

F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
257
deliberately enriched with new members of widely differing phys-
icochemical properties.10 A correlation was observed between
cytotoxicity and lipophilicity (r2 >0.6) in this group, which was par-
ticularly strong in homologous subsets (r2 >0.95). However, the
toxicity of LCA, CDCA and DCA was not well predicted by these cor-
relations. The study concluded that lipophilicity is a necessary but
not sufficient property for BAs to exhibit cytotoxicity.
Much of the focus on the cellular effects of BAs has been con-
cerned with explaining and preventing their contribution to hepa-
tobiliary diseases. What if the pro-apoptotic properties of BAs
could be amplified, producing compounds with useful cytocidal
activity? Some of the azide analogs in our previous work exhibited
unexpectedly high cytotoxic activity towards esophageal cells. We
also identified simple amides of UDCA and DCA that were more
toxic than the parent BAs. In the present study we have prepared
and studied 23 new BA analogs integrating features that were indi-
vidually associated with inhibition of cell viability in our previous
work (Fig. 1). The effects of the compounds on cell viability was
characterised using two cell lines with different intrinsic/extrinsic
pathway susceptibility Caco-2 derived from colorectal adenocarci-
noma and HT1080 from a lung fibrosarcoma.
in pyridine was allowed to stand at rt producing the 7,8-ene. Al-
kene 4 was activated at C24 and amidated to give 5 and the 24-
cyclopropylamido compound 6.
A panel of five amido analogs of DCA was synthesized with 3-b-
azido group on the A-ring (7–11) (Scheme 2). The key intermediate
26 was obtained in three steps form DCA, mesylated on position 3
and azide introduced.10,11 Using standard coupling procedures, this
was reacted with each of five amines to afford 3b-azido, 24-amido
(7), 24-cyclopropylamido (8), 24-benzylamido (9), 24-cyclohexy-
lamido (10) and 24-propylamido (11) analogs.
Preparation of LCA derivatives (12–17) was carried out using
similar approaches to those used to obtain the DCA derivatives
(Scheme 3). The alpha azido DCA derivatives 18, 19, which are epi-
mers of 7 and 8, required introduction of the 3-azide with reten-
tion of configuration (Scheme 4). This was achieved using a
modified Mitsunobu on the selectively protected DCA to produce
the b-bromide 28. Azide substitution and deprotection gave 29
which was amidated using HOBt/EDC and the appropriate amine.
We produced some CDCA analogs with 3a- or 3b-azido orienta-
tion without 7-acetoxy protection which had proven resistant to
removal in 1–3 (Scheme 5). To synthesise the 3-b-azido CDCA,
the 3-hydroxyl group of the methyl ester protected CDCA was
regioselectively mesylated in cold DCM using stoichiometric
amount of methanesulfonyl chloride in the presence of two equiv
of triethylamine. The mixture was worked up immediately after
completing the addition of the reagent then the mesylate was con-
verted to azide in the next step. The methyl ester was hydrolysed,
and compounds 20 and 21 obtained through mixed anhydride
(ehtylchloroformate) which was reacted with the appropriate
amine.
2. Synthetic chemistry
A new panel of 23 BA (Fig. 2) derived from DCA, CDCA and LCA
was prepared using synthetic approaches that we described previ-
ously for incorporating 3-azido and 24-amido functionality.10,11
Some elaboration of the BA 7- and 12- positions was also explored.
In order to obtain the 3b-azido, 7
(1–3), first the CDCA was converted to7
a
-acetyloxy derivatives of CDCA
-hydroxyl acetate in two
a
Synthesis of 23, required introduction of the azide group at
position-3 with retention of configuration (Scheme 6). Mitsunobu
conditions on 30 using methanesulfonic acid were used to afford
the 3-b-mesylate (31). This was reacted with sodium azide yield-
ing. The methyl ester group was hydrolysed to obtain the free car-
boxylic acid 23.
steps (Scheme 1). The 3-OH group on 24 was mesylated using
methanesulfonyl chloride in DCM in the presence of Et3N. The
SN2 substitution on the mesylate using sodium azide afforded
the 3b-azido intermediate, which gave 1 following treatment with
aqueous base. The carboxylic acid was activated using N-OH-Su or
HOBt monohydrate, treated with either ammonia or cyclopropyl
amine to obtain 3b-azido, 7a-acetyloxy-24-amido CDCA deriva-
tives 2-3, respectively. We found the OAc group in 1–3 to be highly
resistant to hydrolysis under basic conditions. In order to produce
4–6, CDCA methyl ester was selectively acetylated on position 3,
followed by mesylation of the 7-OH in the presence of tertiary
base. This spontaneously eliminated when the reaction mixture
3. Effects on viability of Caco-2 and HT1080 cell lines
Caco-2, a cell line derived from a human colorectal epithelial tu-
mour or HT1080 lung fibrosarcoma cells at 70% confluence were
treated with the test compounds in serum free media for 1 h and
O
O
O
OH
OH
OH
OH
OH
NH2
–N
N+
HO
N
H
HO
H
EC50, 39 µM
EC50, 97 µM
H
DCA, EC50, 257 µM
O
OH
NH2
–
N
N+
?
N
H
Figure 1. EC50 values for the inhibition of HET-1A (normal esophageal) cell viability by DCA and its amido and 3-azido analogues. The present study sought to characterise the
effects of incorporating both types of modifications into a single novel BA analogue.

258
F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
O
O
X
X
1, X= OH
2, X= NH2
3, X= HN
4, X= OH
5, X= NH2
6, X= HN
N3
HO
OAc
H
H
7-8 Alkene series
CDCA 3-beta azide-7-acetate series
O
O
OH
X
X
DCA beta azide series
H
N3
N3
LCA beta azide series
H
12, X= OH
13, X= NH2
7, X= NH2
8, X= HN
14, X= HN
15, X= HNPr
16, X= HN
17, X= HN
9, X= HN
Ph
10, X= HN
11, X= HNPr
Ph
O
O
X
OH
X
18, X= NH2
19, X= HN
N3
DCA alpha azide series
H
N3
OH
H
CDCA alpha azides
O
X
23, X= OH
20, X= OH
21, X= NH2
22, X=NH
N3
OH
H
CDCA beta azides
Figure 2. Structural formulae of the BA analogs studied.
24 h. Initial measurements of effects on cell viability were per-
formed at 50 M using the 3-[4,5-dimethylthizol-2-yl]-2,5-diphe-
DCA.8 Replacement of the 3-OH group with b-azide in the case
of LCA decreased the IC50 for inhibition of Caco-2 cell viability
l
nyltetrazolium bromide (MTT) assay. IC50 (CC50) values were
subsequently estimated by determining the effect on cell viability
over 24 h (n = 6) over eight concentration levels (Fig. 3, Table 1).
IC50 values were also estimated for LCA, DCA and CDCA. BA induced
cell death is attributed to necrotic effects at high concentration and
to apoptotic events at lower concentration. We used flow cytome-
try to determine the relative amounts of necrosis and apoptosis in-
duced by selected compounds from the new panel of BA
derivatives. Apoptosis was detected by the signature presence of
phosphatidylserine (PS) on the cell surface, necrotic cells by the
permeability to the ionic propidium iodide (PI).12 Early apoptotic
cells are therefore positive for annexin V which binds PS but not
permeable to PI; late apoptotic effects are positive for annexin V
and PI permeable. Meanwhile, necrotic cells become permeable
to PI without being positive to annexin V. Live/healthy cells are po-
sitive for neither annexin V nor PI. The protocol provided an indi-
cation of the relative contributions of apoptotic and necrotic effects
to the reduced viability observed in the MTT assay.12 Compounds
7–11, 13, 14, 19 were assessed in this way at seven concentration
levels along with LCA, DCA and CDCA (Fig. 4).
from 56 to 6.3 lM; that of CDCA from 106 to 43.7 lM. In all
three BA cases, the effect of replacing hydroxyl with azide was
significantly amplified by primary amido or cyclpropylamido
(CPA) substitution at C24. The most toxic compounds were the
3b- and
a
-azides of the DCA cyclopropyl amide 8 and 19 (IC50
M in Caco-2). These are among
1.9 M in HT1080 and 19, 2.3
l
l
the most potently cytotoxic BAs to be reported to date. Results
from the two epimeric pairs 8/19 and 20/22 indicate little differ-
ence in toxicity potential between alpha and beta orientations of
the 3-azide in the DCA and CDCA series. In all three series
(CDCA, LCA and DCA), the most cytotoxic compounds feature a
primary amide or CPA. Interestingly, we reported that CPA sub-
stitution in UDCA produced a glucocorticoid receptor agonist
which inhibits NFkB signalling whereas the corresponding CDCA
and DCA analogues were without activity.13 The n-propyl amide
of DCA azide (11) was toxic but its LCA analogue (15) was not.
Surprisingly, the more liphophilic but bulkier cyclohexyl and
benzyl amides in the DCA and LCA series (9, 10, 14, 17) were
not toxic. The LCA analogues (12–17) were less toxic than their
DCA analogues (7–11) which is not consistent with the relative
toxicities of the parents. Furthermore, the alkenyl compounds
5 and 6 were not more toxic than their more hydrophilic CDCA
analogues (20–21).
Replacement of the BA C3
a-OH with azide functionality
significantly increased cytotoxicity in the case of LCA and CDCA-
we have reported similar effects of replacement in the case of

F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
259
O
O
O
OH
OMe
OH
i-iii
iv-vi
N3
OAc
HO
OAc
HO
OH
H
H
H
1
2
viii
CDCA
24
vii
3
i, ix
O
O
O
OMe
OH
NH2
vii
x,vi
4
6
AcO
OH
5
HO
H
HO
H
H
viii
25
Scheme 1. Synthetic approaches to 1–6 from CDCA. Reagents and conditions: (i) MeOH, HCl, reflux, 8 h, 98%; (ii) Ac2O, DMAP, pyridine, rt, 8 h, 67%; (iii), AcCl, MeOH, 0 °C to
rt, 20 h, 91%; (iv) Et3N, MsCl, DCM, 0 °C, 30 min, 96%; (v) NaN3, DMPU, 50 °C, 5 d, 85%; (vi) 2 M NaOH, MeOH, reflux, 2 d, 95%; (vii) N-OH-Su, DCC, THF/MeCN, rt, overnight,
then aq. NH3, DMF, rt, 30 min, 93–98%; (viii) HOBt, EDC, DMF, N2, 0 °C, 10 min, then cyclopropylamine, rt, 24 h, 43–90%; (ix) Ac2O, pyridine, 16 h, 76%; (x) MsCl, pyridine,
DCM, RT, 8 h, 93%.
i-iii
iv-vi
vii
3
viii
Scheme 2. Synthetic approaches to 7–11 from DCA. Reagents and conditions: (i) MeOH, HCl, reflux, 8 h, 98%; (ii) Formic acid, perchloric acid (cat), 60 °C, 20 min, 88%; (iii)
NaHCO3, H2O/MeOH, 0 °C, 3 h, 94%; (iv) Et3N, MsCl, DCM, 0 °C, 30 min, 92%; (v) NaN3, DMPU, 50 °C, 5 d, 91%; (vi) 2 M NaOH, MeOH, reflux, 2 d, 95%; (vii) ClCOOEt, Et3N, DCM,
0 °C, 30 min, rt, 8 h, then NH3 (2 equiv), 67%; (viii) HOBt, EDC, DMF, N2, 0 °C, 10 min, then appropriate amine, rt, 24 h, 37–47%.
O
O
O
OH
OH
NH2
v
i-iv
13
N3
HO
12
H
H
N3
H
LCA
vi
14-17
Scheme 3. Synthetic approaches to 12–17 from LCA. Reagents and conditions: (i) MeOH, HCl, reflux, 8 h, 98%; (ii) Et3N, MsCl, DCM, 0 °C, 1.5 h, 80%; (iii) NaN3, DMPU, 50 °C, 5
d, 96%; (iv) 2 M NaOH, MeOH, reflux, 2 d, 96%; (v) ClCOOEt, Et3N, DCM, 0 °C, 30 min, rt, 8 h, then NH3 (2 equiv), 72%; (vi) HOBt, EDC, DMF, N2, 0 °C, 10 min, then appropriate
amine, rt, 24 h, 56–83%.

260
F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
O
O
O
OF
OH
OH
OMe
OH
NHR
iv
i
ii-iii
26
Br
N3
N3
H
H
H
R=H, 18, (37%)
R=cyclopropyl, 19, (73%)
28
29
Scheme 4. Synthetic approaches to DCA 3
(iv) 2 M NaOH, MeOH, reflux, 8 h, 82%; (iv) HOBt, EDC, DMF, N2, 0 °C, 10 min, then, cyclopropyl amine, rt, 24 h, 37–73%.
a-azido amides 18, 19 from 26. Reagents and conditions: (i) NBS, PPh3, THF, ꢀ18 °C, >rt over 8 h, 95%; (ii) NaN3, DMPU, rt, 4 d, 89%;
O
O
O
OH
NHR
OMe
i-iii
iv
N3
OH
N3
OH
HO
OH
H
H
H
20 (69%)
R=H, 21, (72%)
R=cyclopropyl, 22, (87%)
30
Scheme 5. Synthesis of 3-beta azido derivatives of CDCA 20, 21. Reagents and conditions: (i) Et3N MsCl, DCM, 0 °C, 93%; (ii) NaN3, DMPU, 65 °C, 6 d, 76%; (iii) NaOH, MeOH, rt,
1 d, 69%; (iv) ClCOOEt, Et3N in dioxane for 10 min at 0 °C then appropriate amine 8 h at rt, 72–87%.
O
O
OH
OMe
ii-iii
i
30
N3
OH
MsO
OH
H
H
23
31
Scheme 6. Reagents and conditions: Synthesis of 23: PPh3, Et3N, MsOH, DIAD, anh THF, 0 °C, 2 d, 45 °C, 15%; (ii) NaN3, DMPU, 50 °C, 4 d, 79%, (iii) aq. 2 M NaOH/MeOH (1:1),
rt, 24 h, 88%.
contrast 13, which is related to LCA caused significant amounts
of necrosis. The data, which were collected for a range of the
new derivatives, indicated that the cell viability reductions ob-
served previously with the MTT assay were generally due to apop-
tosis. Figure 4 also shows the analysis of the relative effects of 12
on Caco-2 and HT1080 cell lines consistent with the difference ob-
100
50
0
20
21
22
23
served using the MTT approach (IC50 Caco-2 >250
lM, for HT1080
21.3 M). In general, the fibrosarcoma cell line (HT1080) was more
l
susceptible to apoptosis induction to the BA analogues than the
Caco-2 cell line. The differences were striking for 11–13 (e.g., 13,
IC50 13.1 lM HT1080, >500 lM Caco-2).An important difference
between the two cell lines that could be relevant to this is that
FAS receptor/CD95 is not functionally active in the colorectal cell
line (Caco-2), whereas it is in HT1080 cells.6,14,15 A possible expla-
nation for the greater effects in the colorectal cell line of the LCA-
derived compounds (in particular) is that their apoptotic actions in
HT1080 cells are mediated through the extrinsic pathway. By
implication, the remaining compounds act through other mecha-
nisms such as direct mitochondrial (intrinsic) or ER stress. Further-
more the specific discrepancy in the case of 12 and 13 suggests that
these compounds have a different mechanism of action to those
where potency across the two cell lines was similar. Compounds
capable of inducing cell death specifically through the extrinsic
pathway are promising for drug development because tumour cells
tend not to become desensitised to death receptor signals.16 In-
creased ROS is a prominent BA effect on various cell lines and this
0.5
1.0
1.5
2.0
Log concentration (µM)
Figure 3. Example MTT concentration response curves for HT1080 cells incubated
with 20–23 for 24 h. (n = 6 at 8 concentration levels).
Necrosis was not a prominent feature of the cytometric analysis
of cell populations treated with high concentrations of DCA or
CDCA (up to 500
l
M), though there were some signs of necrosis
M). Compound 8 in-
in populations treated with LCA (50–100
l
duced cell death almost exclusively through apoptosis (Fig. 4). This
is significant because apoptosis is anti-inflammatory and cancer
chemopreventative whereas necrosis is cancer promoting. Necrosis
is less likely to selectively affect malignant populations. By

F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
261
Table 1
is thought to contribute substantially to the pro-apoptotic effects
of the BAs.17 Therefore we measured ROS at 30 min and at 24 h
after exposure to selected BA analogues. There was evidence of
increased ROS at 30 min and this was significant at 24 h in the
cases of 9, 11, 18, 21 and 22 (Fig. 5A). The effect of the compounds
on cell viability was furthermore attenuated when the compounds
were incubated in the presence of the anti-oxidant N-acetyl cys-
teine (NAC) (Fig. 5B). The general trend was significant in the case
of LCA and 18. These data indicate that ROS plays a significant role
in the apoptotic processes initiated by the new BAs, although it is
not clear at which stage.
IC50 values for analogs on cell viability of Caco-2 and HT1080 cells at 24 h or %
remaining at the given concentration (lM)
IC50 (95% confidence interval,
Caco-2
lM)
Compound
HT1080
1
2
3
4
5
6
7
8
15.6 (13.4–17.6)
5.5 (5.2–5.8)
6.2 (2.1–13.2)
>100
nda
nd
nd
nd
nd
nd
3.6 (1.6–8.3)
1.9 (0.8–4.5)
>500
>100
9.5 (4.8–18.9)
10.7 (3.5–27)
6.3 (2.5–15)
>1 mM
>1 mM
60% @ 50
>250
>500
>1 mM
>1 mM
There have been several previous attempts to design BA-based
cytotoxic/anti-microbial agents featuring extensive biochemical
characterization of a relatively small number of compounds.18
The effect of some amino acid and amino acid ester conjugates of
UDCA and CDCA was studied using an extensive range of cancer
cell types including hepatic, breast cancer, leukaemia, prostate
cancer, stomach cancer, cervical and colon cancer cell lines.19–21
CDCA amino acid conjugates have exhibited an anti-cancer effect
on malignant glioblastoma cell lines U-118MG, U-87MG, T98G,
and U-373M. Some of these conjugates exhibited promising effects
in subsequent in vivo models of malignancy and in murine models
of obesity. More recently Kihel et al. studied the proapoptotic ef-
fects of piperazinyl analogues of LCA and CDCA. These were re-
ported to cause cell death in a multiple myeloma cell line with
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
DCA
CDCA
LCA
>100
6.3 (2.5–16)
21.3 (10.5–45)
13.1 (12.4–13.8)
>1 mM
>200
>100
>100
>100
>100
12.6 (11.2–14.2)
2.3 (0.75–7.3)
40.3 (28.3–57)
9.8 (8.1–11.9)
7.6 (5.6–10.8)
43.7 (34–56)
80.3 (49.2–131)
106.0 (98.3–122.6)
56.0 (37.3–84.3)
10.0 (8.0–12.1)
5.7 (4.4–7.4)
23.6 (20.5–27)
6.9 (6.2–7.6)
3.2 (2.3–4.3)
26.2 (24.5–27.8)
110.0 (70.2–171)
130.1 (80.7–210)
23.0 (15–35.5)
the most compound exhibiting LD50 in the l
M range.22
These drug design efforts described above, which target pro-
apoptotic pathways, can be distinguished from BA analogues that
have been deliberately designed to exhibit increased amphilicity
and tendency to self-organization in solution. Such compounds
cause cell death mainly through necrosis. Although these have
a
nd, not determined.
8 HT1080
13 HT1080
100
80
60
40
20
0
100
Q1 Necrotic
Q2 Late Apoptosis
Q3 Alive
Q4 Early Apoptosis
Q1 Necrotic
Q2 Late Apoptosos
Q3 Alive
80
60
40
20
0
Q4 Early Apoptosis
12 HT1080
12 Caco-2
100
80
60
40
20
0
100
80
60
40
20
0
Figure 4. Flow cytometry analysis showing percentage cells (y axis) in each of the indicated stages following treatment of HT1080 cells with 8 and 13 for 24 h. The lower
graphs show a comparison between the effects of 12 on Caco-2 and HT1080 cells.

262
F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
NAC
No NAC
400
300
200
100
0
A
B
*
100
50
0
*
NC 9 10 11 13 14 17 18 20 21 22 23
Figure 5. Plots showing A: Reactive oxygen species production at 24 h in Caco-2 cells treated with BA analogs. Data are shown relative to vehicle control and corrected for cell
viability (MTT data). B: The effect of selected compounds on cell viability (MTT) is shown in the presence and absence of N-acetyl cysteine (NAC), an anti-oxidant Compounds
were tested at 10
l
M in both experiments (n = 3). Natural BAs were evaluated at 100
l
M. (⁄p <0.05 Anova)
limited scope for therapeutically relevant selectivity or effective
distribution in vivo, they can make effective anti-microbial agents.
Examples in this more extensive line of research include analogues
featuring hydrophobic side chains that act through membrane dis-
ruption causing fungal and bacterial cell death. A separate line of
research associated with Bellini’s lab identified simpler
non-amphiphatic BA analogues with interesting antimicrobial
effects.23,24 3-Amino and 24-amido DCA analogues caused pro-
nounced effects on gram negative and positive bacteria as well as
fungi. The molecular mechanism of action of these compounds is
unclear; however they appear to be designed to act through recep-
tor mediated rather than membrane dependent effects.
4.1.1. (4R)-Methyl 4-((3R,5S,7R,10S,13R,17R)-7-acetoxy-3-
hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]
phenanthren-17-yl)pentanoate (24-methyl, 3a-hydroxy, 7a-
acetoxy-5b-cholanoate) (24)
To a solution of CDCA (5 g, 12.7 mmol) dissolved in MeOH
(94 ml), was added dropwise concentrated HCl (37%, 0.53 ml,
6.35 mmol). The mixture was stirred under reflux for 8 h. The sol-
vent was removed under reduced pressure giving the methyl ester
(30) as a white solid (5.07 g, 98%). 1H NMR (400 MHz, CDCl3): d
3.88 (m, 1H, 7b-H), 3.67 (s, 3H, –OCH3), 3.5 (m, 1H, 3b-H,), 0.96
(d, 3H, 21-CH3), 0.92 (s, 3H, 19-CH3), 0.67 (s, 3H 18-CH3). 13C
NMR ppm (100 MHz, CDCl3): d 174.36 (C@O, 24-C), 71.76 (CH,
In summary, by integrating azido and amido functionality into
DCA, we have produced interesting new BA analogues capable of
inducing apoptosis in cancer cells exposed in the low micromolar
range. It will be interesting to explore the SAR of the amido group
further, since our initial work suggests quite tight structural
requirements. The relatively high potency of 8 in this context sug-
gests that its interactions are specific, and although its binding
partner is unidentified, it would appear to be an interesting ligand
for studies into the mechanism of BA cytotoxicity.
3-C), 68.26 (CH, 7-C), 51.09 (CH3, OCH3). IR
(KBr): 3424.28,
m
max
2932.36, 2866.53, 1741.25, 1447.62, 1375.42 cmꢀ1. HRMS: Found:
(M-Na)+ = 429.2987, calculated for C25H42O4Na = 429.2981. CDCA
methyl ester (2 g, 4.92 mmol) and 4-DMAP (120 mg, 0.98 mmol)
were dissolved in anhydrous pyridine (30 ml). Ac2O (14 ml,
148 mmol) was added and the reaction mixture allowed to stir
for 8 h at rt. The mixture was poured into water (200 ml) and the
compound extracted with EtOAc:hexane (1:1) (3 ꢁ 100 ml). The
organic phases were combined and washed with 1 M HCl
(100 ml), water (100 ml) and brine (100 ml). The organic layer
was dried over MgSO4 and filtered. The solvent was removed and
the semi-solid residue was purified by flash column chromatogra-
phy using hexane: EtOAc (3:1) as mobile phase. White foam (67%,
1.617 g). 1H NMR (400 MHz, CDCl3): d 5.12 (m, 1H, 7b-H), 4.83 (sex,
1H, J1 = 4.02 Hz, J2 = 7.02 Hz, J3 = 4.52 Hz, 3b-H), 3.91 (s, 3H, –O-
CH3), 2.48 (s, 3H, 3 –C@OCH3), 2.43 (s, 3H, 7 –C@OCH3), 1.31 (s,
3H, 19-CH3), 1.28 (d, 3H, J = 6.53 Hz, 21-CH3) 0.89 (s, 3H, 18-
CH3). 13C NMR ppm (100 MHz, CDCl3): 173.54 (C@O, 24-C),
169.49 and 169.28 (2C@O, 3-C@OCH3 and 7-C@OCH3), 73.08 and
70.15 (2CH, 3-C and 7-C), 51.08 (CH3, OCH3), 21.17 (CH3, 7-
4. Experimental
4.1. General synthetic methods
All chemicals were purchased from Sigma–Aldrich (Dublin, Ire-
land), except where stated. All the reactions were monitored using
TLC. Uncorrected melting points were measured on a Stuart
Apparatus. Infra-red (IR) spectra were performed on a Perkin Elmer
FT–IR Paragon 1000 spectrometer. 1H and 13C nuclear magnetic
resonance (NMR) spectra were recorded at 27 °C on a Bruker DPX
400 spectrometer (400.13 MHz, 1H; 100.61 MHz, 13C). Coupling
constants are reported in Hertz. For 1H NMR assignments, chemical
shifts are reported: shift value (number of protons, description of
absorption, coupling constant(s) where applicable). Electrospray
ionization mass spectrometry (ESI–MS) was performed in the posi-
tive ion mode on a liquid chromatography time-of-flight mass
spectrometer (Micromass LCT, Waters Ltd, Manchester, UK). The
samples were introduced into the ion source by an LC system
(Waters Alliance 2795, Waters Corporation, USA) in MeCN:water
OC@OCH3), 21.06 (CH3, 3-OC@OCH3). IR
(KBr): 2942.84,
m
max
2872.20, 2848.09, 1734.83, 1466.86, 1438.18, 1378.44, 1364.49,
1248.94 cmꢀ1. HRMS: Found: (M-Na)+ = 513.3208, calculated for
C29H46O6Na = 513.3192. The CDCA diacetate methyl ester (1.55 g,
3.17 mmol) was dissolved in anhydrous MeOH (22 ml) then AcCl
(0.29 ml, 4.078 mmol), dissolved in MeOH (3 ml) was added drop-
wise to the reaction mixture at 0 °C. The mixture was allowed to
reach rt and left stirring for 20 h when it was quenched by the
addition of saturated NaHCO3 (70 ml) and water. The compound
was extracted using EtOAc (3 ꢁ 50 ml) the organic phase was dried
over MgSO4. The organic solvent was removed under reduced pres-
sure and the product formed as a white solid (1.294 g, 91%). 1H
NMR (400 MHz, CDCl3): d 4.86 (m, 1H, 7b-H), 4.12 (m, 1H, 3b-H),
3.65 (s, 3H, –O-CH3), 1.97 (s, 3H, 7 –C@OCH3), 0.91 (s, 3H, 19-
CH3), 0.9 (d, 3H, 21-CH3) 0.63 (s, 3H, 18-CH3). 13C NMR ppm
(100 MHz, CDCl3): d 174.30 (C@O, 24-C), 170.23 (C@O, 7-C@OCH3),
(60:40% v/v) at 200 ll/min. The capillary voltage of the mass spec-
trometer was at 3 kV. The sample cone (de-clustering) voltage was
set at 40 V. For exact mass determination, the instrument was
externally calibrated for the mass range 100 –1000 m/z. A lock
(reference) mass (m/z 556.2771) was used. Mass measurement
accuracies of < 5 ppm were obtained. Compound purity/homoge-
neity was confirmed using a combination of NMR, TLC and HPLC.

F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
263
71.32 and 70.91 (2CH, 3-C and 7-C), 51.08 (CH3, OCH3), 21.21 (CH3,
7-OC@OCH3). HRMS: Found: (M-Na)+ = 471.3084, calculated for
(98%). 1H NMR (400 MHz, CDCl3): d 4.90 (m, 1H, 7b-H), 3.92 (m,
1H, 3b-H), 2.05 (s, 3H, 7-C@OCH3), 0.97 (s, 3H, 19-CH3), 0.93 (d,
3H, 21-CH3), 0.66 (s, 3H, 18-CH3). 13C NMR ppm (CDCl3): 11.67,
18.21, 20.80, 21.51, 23.04, 23.48, 27.95, 30.45, 30.64, 30.89, 32.44,
33.68, 35.68, 35.12, 36.38, 37.82, 39.46, 42.69, 50.38, 55.65, 58.50,
C27H44O5Na = 471.3086.
4.1.2. (4R)-4-((3S,5S,7R,10S,13R,17R)-7-Acetoxy-3-azido-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-
65.81, 71.47, 170.39, 180.28. IR
(KBr): 3329.05, 2927.43,
m
max
yl)pentanoic acid (3b-azido, 7a-acetoxy-5b-cholanoate) (1)
2101.15, 1732.77 and 1247.18 cmꢀ1 HRMS: Found: [M-H]ꢀ
.
To a stirred solution of the 24 (1.273 g, 2.84 mmol) in anhy-
drous DCM (38 ml) was added Et3N (0.43 ml, 3.105 mmol). Meth-
anesulfonyl chloride (0.331 ml, 4.276 mmol) dissolved in
anhydrous DCM (12 ml) was added dropwise at 0 °C. The mixture
was stirred for 30 min when it was quenched using cooled water
(50 ml). The compound was extracted using DCM (2 ꢁ 40 ml).
The organic phase was washed with brine (100 ml), dried over
MgSO4 and filtered. The solvent was removed using the rotary
evaporator, ensuring the bath temperature was no higher than
40 °C. The flask was placed under high pressure, giving semi-solid
oil as the product (1.43 g, 96%). 1H NMR (400 MHz, CDCl3): d 4.90
(m, 1H, 7b-H), 4.52 (m, 1H, 3b-H), 3.68(s, 3H, –O-CH3), 3.02(s,
3H, –OSO2CH3), 2.08 (s, 3H, 7-C@OCH3), 0.96 (s, 3H, 19-CH3),
0.94 (d, 3H, 21-CH3), 0.67 (s, 3H, 18-CH3). 13C NMR ppm
= 458.3015, calculated for C26H40N3O4 = 458.3019.
4.1.4. (3S,5S,7R,10S,13R,17R)-3-Azido-17-((R)-5-
(cyclopropylamino)-5-oxopentan-2-yl)-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-7-yl
acetate (N-cyclopropyl 3b-azido, 7a-acetoxy-5b-cholan-24-
amide) (3)
To a solution of 1 (0.1 g, 0.217 mmol) in anhydrous DMF (10 ml)
was added HOBt monohydrate (0.044 g, 0.325 mmol) and EDC
(0.047 ml, 0.26 mmol) at 0 °C under N2 atmosphere. After 10 min,
a solution of cyclopropylamine (0.023 ml, 0.322 mmol) dissolved
in anhydrous DMF (0.5 ml) was added slowly to the mixture and left
stirring for 24 h. Once complete, the reaction mixture was poured
into brine (70 ml) and extracted using EtOAc (3 ꢁ 50 ml). The or-
ganic phase was dried over Na2SO4, filtered and the solvent was re-
moved using the rotary evaporator. The crude product was purified
by flash column chromatography using gradient elution (25-100%
EtOAc in hexane). The solvent was removed under reduced pressure
giving white foam (0.047 g, 43%) as product. 1H NMR d (CDCl3): 5.64
(100 MHz, CDCl3):
d 174.69 (C@O, 24-C), 170.43 (C@O, 7-
OC@OCH3), 81.39 (CH, 3-C), 71.01 (CH, 7-C), 52.57 (CH3, –OSO2-
CH3), 51.52 (CH3, OCH3), 21.59 (CH3, 7-C@OCH3). HRMS: Found:
(M-Na)+ = 549.2874, calculated for C28H46O7SNa = 549.2862. To
the mesylate (1.791 g, 3.4 mmol) in DMPU, was added NaN3
(2.21 g, 34 mmol) at 50 °C. The reaction mixture was left stirring
for 5 d and then poured into distilled water (100 ml). EtOAc
(3 ꢁ 100 ml) was used to extract the compound. The organic phase
was washed with brine (100 ml), dried over Na2SO4 and filtered.
The solvent was removed under reduced pressure, the residue
purified by flash column chromatography, using 25% EtOAc in hex-
ane as mobile phase. The product azide was isolated as a white
foam (1.369 g, 85%). The 3b-azido CDCA methyl ester (0.78 g,
1.647 mmol) was dissolved in MeOH (10 ml) and 1 M NaOH
(2 ml) was added dropwise to the solution, until pH ꢂ 14 was at-
tained. The mixture was stirred at 65 °C. The reaction was moni-
tored using TLC analysis and once complete, the mixture was
poured into 2 M HCl (50 ml) and extracted using EtOAc
(3 ꢁ 50 ml). The organic phase was washed with water
(2 ꢁ 100 ml) and brine (100 ml), dried over Na2SO4, filtered and
the solvent removed under the high vacuum to afford the product
as white foam (0.719 g, 95%). White foam. 1H NMR (400 MHz,
CDCl3): d 4.98 (m, 1H, 7b-H), 4.55 (m, 1H, 3b-H), 3.67 (s, 3H, –O-
CH3), 2.04 (s, 3H, 7-C@OCH3), 0.93 (s, 3H, 19-CH3), 0.92 (d, 3H,
(s, 1H, N-H), 4.89 (m, 1H, 7b-H), 3.92 (m, 1H, 3a-H), 2.04 (s, 3H, 7-
OC@OCH3), 0.95 (s, 3H, 19-CH3), 0.92 (d, 3H, 21-CH3), 0.64 (s, 3H,
18-CH3), 0.68 (m, 2H, cyclopropyl-CH2). 13C NMR ppm (CDCl3):
6.86, 11.91, 18.58, 21.03, 21.80, 22.82, 23.29, 23.73, 24.88, 28.30,
30.67, 31.15, 31.89, 32.66, 33.59, 33.90, 35.35, 35.65, 35.89, 36.61,
38.03, 39.69, 42.91, 50.61, 55.97, 58.74, 71.65, 170.55, 175.17.
IR
(KBr): 3429.95, 2938.13, 2100.42, 1734.02, 1651.02 and
m
max
1248.42 cmꢀ1. HRMS: Found: [M+Na]+ = 521.3463, calculated for
29H46N4O3Na = 521.3468.
C
4.1.5. (4R)-Methyl 4-((3R,5R,7R,10S,13R,17R)-3-acetoxy-7-
hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]
phenanthren-17-yl)pentanoate (24-methyl, 3a-acetoxy, 7a-
hydroxy-5b-cholanoate) (25)
To a solution of compound 30 (2 g, 4.9 mmol) in DCM (30 ml),
was added Ac2O (3.25 ml, 34.3 mmol) and pyridine (5.97 ml,
73.8 mmol). After 16 h at rt, TLC analysis showed the formation
of several acetate products with consumption of starting material.
EtOAc (100 ml) was added to the mixture and washed with 1 M
HCl (100 ml) and water (2 ꢁ 100 ml). The organic layer was col-
lected, dried over MgSO4 and filtered. The crude product was puri-
fied by flash column chromatography (hexane: EtOAc, 3:1) and the
solvent was removed under high vacuum to produce a white solid
(1.668 g, 76%). 1H NMR (400 MHz, CDCl3): d 4.82 (m, 1H, 3b-H),
4.10 (1H, 7b-H), 3.91 (s, 3H, –O-CH3), 2.26 (3H, 3-C@O-CH3), 1.18
(d, 3H, 21-CH3), 1.16 (s, 3H, 19-CH3), 0.91 (s, 3H, 18-CH3). 13C
NMR ppm (100 MHz, CDCl3): d 174.33 (C@O, 24-C), 170.38 (C@O,
3-C@OCH3) 74.28 (CH, 7-C), 68.39 (CH, 3-C), 51.44 (CH3, OCH3).
21-CH3), 0.65 (s, 3H, 18-CH3). IR
(KBr): 3329.05, 2927.43,
m
max
2101.15, 1732.77 and 1247.18 cmꢀ1 HRMS: Found: [MH]ꢀ
. =
458.3015, calculated for C26H40N3O4 = 458.3019.
4.1.3. (3S,5S,7R,10S,13R,17R)-17-((R)-5-Amino-5-oxopentan-2-
yl)-3-azido-10,13-dimethylhexadecahydro-1H-cyclopenta[a]
phenanthren-7-yl acetate (3b-azido, 7a-acetoxy-5b-cholan-24-
amide) (2)
Compound 1 (0.15 g, 0.326 mmol) was dissolved in anhydrous
THF (15 ml) and acetonitrile (1.5 ml), then N-OH-Su (0.124 g,
1.07 mmol) and DCC (0.22 g, 1.06 mmol) were added to the mixture
and the reaction was left stirring overnight. The white solid formed
was filtered off and the filtrate was concentrated using the rotary
evaporator and placed under high vacuum. The compound was then
dissolved in DMF and ammonia solution (37%, 0.054 ml) was added.
The white solid precipitated from the mixture when brine (70 ml)
was added. The mixture was filtered using suction filtration. The fil-
trate was washed with water (2 ꢁ 30 ml) and allowed to dry in the
air. Column chromatography was carried out using 50–100% EtOAc
in hexane as the mobile phase. The organic solvent was removed
under reduced pressure leaving behind 0.147 g of white foam
IR
m
(KBr): 3531.43, 2942.98, 2867.01, 1737.24, 1440.12,
max
1379.84, 1364.52, 1250.45 cmꢀ1 HRMS: Found: (M-Na)+
.
= 471.3086, calculated for C27H44O5Na = 471.3086.
4.1.6. (4R)-4-((3R,5R,10S,13R,17R)-3-Hydroxy-10,13-dimethyl-
2,3,4,5,6,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl)pentanoic acid (3
a-hydroxy,
7-ene-5b-cholanoate) (4)
Compound 25 (0.5 g, 1.114 mmol) was dissolved in anhydrous
DCM (15 ml) at 0–4 °C then anhydrous pyridine (0.448 ml,
5.56 mmol) and methanesulfonyl chloride (0.434 ml, 4.4 mmol)
were added to the mixture and stirred for 15 min. The mixture

264
F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
was allowed to reach rt and held at that for 8 h. 0.1 M aq. HCl
(80 ml) was added to the mixture and the alkene product was ex-
tracted using DCM (2 ꢁ 60 ml). The DCM layer was washed with
brine (160 ml), dried using MgSO4 and filtered. The solvent was re-
moved with the rotary evaporator and the product 7-alkene-3-ace-
tate methyl ester isolated by flash chromatography (0.450 g, 93%).
1H NMR (400 MHz, CDCl3): d 5.12 (m, 1H, 7-H), 4.75 (m, 1H, 3b-H),
3.68 (s, 3H, –O-CH3), 2.04 (s, 3H, 3-C@OCH3), 0.96 (s, 3H, 19-CH3),
0.94 (d, 3H, 21-CH3), 0.71 (s, 3H, 18-CH3). 13C NMR ppm (100 MHz,
CDCl3): d 174.31 (C@O, 24-C), 170.22 (C@O, 3-OC@OCH3), 136.74
(quaternary C, 8-C), 114.92 (CH, 7-C), 73.23 (CH, 3-C), 51.085
37.80, 40.07, 41.06, 43.92, 45.41, 55.23, 55.97, 71.44, 115.62,
137.43, 175.18. 13C NMR ppm (CDCl3): 175.41 (C@O, C@ONH2),
137.67 (quaternary C, 8-C), 115.86 (CH, 7-C), 71.68 (CH, 3-C),
7.06 (CH2, cyclopropyl-CH2). IR
(KBr): 3444.44, 2931.55 and
m
max
1663.29 cmꢀ1. HRMS: Found: [M+Na]+ = 436.3178, calculated for
27H43NO2Na = 436.3191.
C
4.1.9. (4R)-4-((3S,5R,10S,12S,13R,17R)-3-Azido-12-hydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
17-yl)pentanamide (3b-azido, 12a-hydroxy-5b-cholan-24-
amide) (7)
(CH3, –OCH3), 21.02 (CH3, 3-C@OCH3). IR
(KBr): 2952.47,
3b-Azido DCA (27) (0.150 g, 0.36 mmol) was dissolved in anhy-
drous DCM (13 ml) and Et3N (3 equiv, 1.1 mmol) was added at 0 °C,
followed by the slow addition of ethyl chloroformate (1.2 equiv,
0.43 mmol). After 30 min at 0 °C, aqueous ammonia (35%,
1.2 equiv, 0.43 mmol) was added. The reaction was let warm up
to rt for 8 h. The mixture was poured onto 1 M HCl and extracted
with EtOAc (3 ꢁ 100 ml). The organic phase was collected and
washed with water (100 ml) and brine (100 ml). The organic phase
was dried over Na2SO4, filtered and the solvent removed under re-
duced pressure to form a white foam (0.101 g, 67%). 1H NMR d
m
max
2871.89, 1730.01, 1451.58, 1437.29, 1379.29, 1362.42, 1326.88,
1311.19 cmꢀ1. HRMS: Found: (M-Na)+ = 453.2959, calculated for
C27H42O4Na = 453.2981. To a solution of the alkene methyl ester
(0.7 g, 1.63 mmol) in MeOH (40 ml) was added 2 M NaOH (2 ml)
dropwise to pH ꢂ 14. The mixture was stirred at 60 °C for 1 d then
it was poured into 2 M HCl (50 ml) and extracted with EtOAc
(3 ꢁ 50 ml). The organic phase was washed with water
(2 ꢁ 100 ml) and brine (100 ml), dried over Na2SO4, filtered and
solvent removed in vacuo, leaving a white solid (0.43 g, 71%). 1H
NMR d (CD3OD): 5.14 (m, 1H, 7-H), 3.54 (m, 1H, 3b-H), 3.31 (s,
3H, –O-CH3), 0.98 (d, 3H, 21-CH3), 0.88 (s, 3H, 19-CH3), 0.59 (s,
3H, 18-CH3). 13C NMR ppm (CD3OD): 12.49, 19.08, 22.90, 24.10,
25.24, 29.10, 29.92, 32.18, 32.40, 34.77, 35.92, 37.25, 38.17,
38.43, 41.34, 42.46, 45.02, 56.45, 57.36, 71.98, 116.78, 138.82,
(CD3OD): 3.97 (s, 2H, 12b-H and 3a-H), 1.03 (d, 3H, J = 6.04 Hz,
21-CH3), 0.96 (s, 3H, 19-CH3), 0.71 (s, 3H, 18-CH3). 13C NMR ppm
(CD3OD): 13.33, 17.78, 24.22, 24.99, 25.59, 27.38, 27.82, 28.81,
30.13, 31.38, 31.92, 33.37, 33.70, 34.43, 35.83, 37.07, 37.37,
39.04, 47.74, 48.27, 49.50, 60.37 (CH, 3-C), 74.19 (CH, 12-C),
178.31. IR
(KBr): 3342.10, 2934.10, 2859.74, 1708.32,
1541.26, 1468.23, 1447.14, 1416.48, 1367.02, 1333.67,
1286.98 cmꢀ1 HRMS: Found: [M]ꢀ = 373.2757, calculated for
24H37O3 = 373.2743.
180.05 (C@O, 24-C). IR
(KBr): 3400.40, 2937.50, 2102.52 and
m
max
m
max
1666.29 cmꢀ1. HRMS: Found: (M+Na)+ = 439.3027. HRMS: Found:
[M+Na]+ = 439.3027, calculated for C24H40N4O2Na = 439.3049.
.
C
4.1.10. (4R)-4-((3S,5R,10S,12S,13R,17R)-3-Azido-12-hydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
17-yl)-N-cyclopropylpentanamide (N-cyclopropyl 3b-azido,
4.1.7. (4R)-4-((3R,5R,10S,13R,17R)-3-Hydroxy-10,13-dimethyl-
2,3,4,5,6,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-
cyclopenta[a]phenanthren-17-yl)pentanamide (3
a
-hydroxy, 7-
12a-hydroxy-5b-cholan-24-amide) (8)
ene-5b-cholan-24-amide) (5)
To a stirred solution of 27 (0.15 g, 0.359 mmol) in DMF (10 ml)
To 4 (0.15 g, 0.4 mmol) in anhydrous THF (15 ml) and acetoni-
trile (1.5 ml) was added DCC (0.248 g, 1.2 mmol) and N-OH-Su
(0.138 g, 1.19 mmol). The mixture was left stirring overnight. The
solid was filtered off and the filtrate was concentrated. The product
was dissolved in DMF (5 ml) and ammonia solution (37%, 0.044 ml)
was added. The reaction mixture was stirred at 50 °C and left over-
night, then the mixture was poured into brine (70 ml) and the pre-
cipitate collected by suction filtration. The solid collected on the
filter paper was washed with distilled water (2 ꢁ 30 ml) and the
solid (0.14 g, 93%) was allowed to dry under reduced pressure as
a foam. 1H NMR d (CD3OD): 5.14 (m, 1H, 7-H), 3.45 (m, 1H, 3b-
H), 1.00 (d, 3H, 21-CH3), 0.88 (s, 3H, 19-CH3), 0.59 (s, 3H, 18-
CH3). 13C NMR ppm (CD3OD): 12.48, 19.15, 22.90, 24.10, 25.23,
29.13, 29.92, 32.19, 33.29, 33.66, 34.77, 35.92, 37.42, 38.19,
38.44, 41.36, 42.47, 45.03, 56.47, 57.35, 71.98, 116.78, 138.83,
was added HOBt monohydrate (0.243 g, 1.796 mmol) and EDC
(0.32 ml, 1.796 mmol) at 0 °C and monitored by TLC. When the
intermediate was formed the amine (1.2 equiv) was added at 0 °C
and stirred at rt. When the reaction was complete the mixture
was poured into brine (50 ml) and extracted with EtOAc
(3 ꢁ 50 ml). The organic phase was collected and dried over Na2-
SO4, filtered and the solvent was removed under reduced pressure.
The crude product was purified by column chromatography using
50–100 EtOAc in hexane as gradient mobile phase to give the prod-
uct. A similar general method was used to produce 9–11. Com-
pound 8 was isolated as a light yellow foam (40 mg, 37%). 1H
NMR d (CD3OD): 4.02 and 3.96 (d, 2H, 12b- and 3a-H), 2.64 (m,
1H, CPA-CH), 1.02 (d, 3H, J = 6.52 Hz, 21-CH3), 0.98 (s, 3H, 19-
CH3), 0.72 (m, 2H, CPA-CH2), 0.71 (s, 3H, 18-CH3), 0.49 (m, 2H,
CPA-CH2). 13C NMR ppm (CD3OD): 6.58 (2 ꢁ CH2, CPA), 13.32,
15.13, 17.80, 23.44, 24.22, 24.98, 25.60, 27.39, 27.82, 28.83,
30.13, 31.38, 31.92, 33.43, 34.06, 34.43, 35.84, 37.04, 37.37,
39.04, 47.74, 48.25, 49.50, 60.38 (CH, 3-C), 74.18 (CH, 12-C),
179.95. IR
(KBr): 3327.85, 2929.36, 1626.36 and
1576.41 cmꢀ1. HRMS: Found: [M+Na]+ = 396.2874, calculated for
24H39NO2Na = 396.2878.
m
max
C
178.60 (C@O, 24-C). IR
(KBr): 3302.59, 2937.69, 2102.59 and
m
max
4.1.8. (4R)-N-Cyclopropyl-4-((3R,5R,10S,13R,17R)-3-hydroxy-
10,13-dimethyl-2,3,4,5,6,9,10,11,12,13,14,15,16,17-
tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)
1648.38 cmꢀ1. HRMS: Found: [M+Na]+ = 479.3353, calculated for
27H44N4O2Na = 479.3362.
C
pentanamide (N-cyclopropyl 3
amide) (6)
a-hydroxy, 7-ene-5b-cholan-24-
4.1.11. (4R)-4-((3S,5R,10S,12S,13R,17R)-3-Azido-12-hydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
Compound 6 was produced from 4 using the HOBt/EDC proce-
dure described in 4.1.4 to give a white foam (0.053 g, 90%). 1H
NMR d (CDCl3): 5.66 (d, 1H, N-H), 5.10 (m, 1H, 7-H), 3.62 (1H,
3b-H), 0.92 (d, 3H, 21-CH3), 0.85 (s, 3H, 19-CH3), 0.76 (m, 2H, cyclo-
propyl-CH2), 0.53 (s, 3H, 18-CH3), 0.48 (2H, cyclopropyl-CH2). 13C
NMR ppm (CDCl3): 6.83, 12.09, 18.75, 21.83, 22.77, 23.03, 24.69,
28.18, 28.84, 31.58, 31.86, 33.64, 33.69, 34.86, 36.07, 36.86,
17-yl)-N-benzylpentanamide (N-benzyl 3b-azido, 12
5b-cholane-24-amide) (9)
Compound 9 was produced from 27 as described in 4.1.10.
White foam (70 mg, 39%). 1H NMR d (CDCl3): 7.30 (m, 5H, aro-
matic-H), 5.79 (br s, 1H, NH), 4.45 (m, 2H, benzyl-CH2), 4.01 and
a-hydroxy-
3.97 (d, 2H, 12b- and 3
a-H), 1.00 (d, 3H, J = 6.00 Hz, 21-CH3),
0.96 (s, 3H, 19-CH3), 0.70 (s, 3H, 18-CH3). 13C NMR ppm (CDCl3):

F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
265
12.96, 17.66, 23.73, 23.77, 24.78, 26.15, 26.63, 27.69, 28.96, 30.30,
30.71, 31.85, 33.45, 33.77, 34.68, 35.35, 36.00, 37.47, 43.83, 46.71,
47.56, 48.54, 58.91 (CH, 3-C), 73.39 (CH, 12-C), 127.73, 128.06,
(C@O, 24-C), 58.85 (CH, 3-C). IR
(KBr): 2937.19, 2089.38 and
max
m
1734.37 cmꢀ1. HRMS: Found: (M-Na)+ = 438.3078, calculated for
C25H41N3O2Na = 438.3096. Thehe 3-b azido LCA methyl ester
128.92, 138.54, 173.52 (C@O, 24-C). IR
(KBr): 3414.05,
. HRMS: Found:
(2.35 g, 5.654 mmol) was subjected to hydrolysis using NaOH in
aqueous methanol as already described and the product isolated
as white foam (2.18 g, 96%).%). 1H NMR d (CDCl3): 3.93 (s, 1H,
m
max
2927.53, 2107.71, 1646.85 and 698.15 cmꢀ1
[M+Na]+ = 529.3544, calculated for C31H46N4O2Na = 529.3518.
3a-H), 3.66 (s, 1H, 7b-H), 0.96 (d, 6H, 19-CH3 and 21-CH3), 0.72
4.1.12. (4R)-4-((3S,5R,10S,12S,13R,17R)-3-Azido-12-hydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
(s, 3H, 18-CH3). 13C NMR ppm (CDCl3): 12.49, 18.76, 22.13, 24.39,
25.24, 25.61, 27.43, 27.69, 29.23, 31.25, 31.83, 32.00, (32.20),
32.30, 36.08, 36.70, 37.06, 38.83, 41.33, 41.45, 43.90, (57.37),
17-yl)-N-cyclohexylpentanamide (N-cyclohexyl 3b-azido, 12
a-
hydroxy-5b-cholane-24-amide) (10)
Compound 10 was produced from 27 as described in 4.1.10.
Light yellow foam (85 mg, 47%). 1H NMR d (CDCl3): 5.32 (br s,
57.44, 57.81, 60.16 (CH, 7-C), 178.21 (C@O, 24-C IR
(KBr):
m
max
3450.29, 2927.53, 2101.43 and 1709.14 cmꢀ1
. Found: [M-
H]ꢀ = 400.2966, calculated for C24H38N3O2 = 400.2964.
1H, NH), 3.99 and 3.95 (d, 2H, 12b- and 3a-H), 3.77 (m, 1H, cyclo-
hexyl-CH), 0.99 (d, 3H, J = 6.52 Hz, 21-CH3), 0.95 (s, 3H, 19-CH3),
0.69 (s, 3H, 18-CH3). 13C NMR ppm (CDCl3): 12.97, 17.69, 23.72,
23.77, 24.77, 25.07, 25.73, 26.16, 26.63, 27.69, 28.93, 30.29,
30.71, 31.93, 33.45, 34.03, 34.68, 35.34, 36.00, 37.47, 46.73,
47.59, 48.23, 48.53, 58.92 (CH, 3-C), 73.38 (CH, 12-C), 172.66
4.1.15. (4R)-4-((3S,5R,10S,13R,17R)-3-Azido-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)
pentanamide (3b-azido, 7a-hydroxy-5b-cholane-24-amide) (13)
Compound 12 was amidated producing 13 as already described
for the conversion of 27 to 7. Compound 13 was isolated as a white
(C@O, 24-C). IR
(KBr): 3434.18, 2932.36, 2102.19 and
1643.62 cmꢀ1. HRMS: Found: [M+Na]+ = 521.3864, calculated for
30H50N4O2Na = 521.3831.
foam (0.071 g, 72%). 1H NMR d (CDCl3): 3.98 (s, 1H, 3
a-H), 3.66 (s,
m
max
1H, 7b-H), 0.96 (d, 6H, 21-CH3 and 19-CH3), 0.68 (s, 3H, 18-CH3).
13C NMR ppm (CDCl3): 12.27, 18.45, 21.20, 24.00, 24.37, 24.91,
26.49, 26.73, 28.38, 30.40, 30.88, 30.95, 31.15, 35.18, 35.51,
35.85, 37.52, 40.33, 40.38, 42.97, 56.16, 56.78, 59.00, 180.32
C
4.1.13. (4R)-4-((3S,5R,10S,12S,13R,17R)-3-Azido-12-hydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
(C@O, 24-C). IR
(KBr): 3436.57, 2927.53, 2101.06 and
1648.37 cmꢀ1. HRMS: Found: [M+Na]+ = 423.3086, calculated for
24H40N4ONa = 423.3100.
m
max
17-yl)-N-propylpentanamide (N-propyl 3b-azido, 12
a-hydroxy-
5b-cholane-24-amide) (11)
C
Compound 11 was produced from 27 as described in 4.1.10.
White foam (0.065 g, 40%). 1H NMR d (CDCl3): 5.47 (br s, 1H,
NH), 3.99 and 3.95 (d, 2H, 12b- and 3a-H), 3.22 (m, 2H, propyl-
CH2), 1.01–0.92 (m, 9H, 21-CH3+propyl-CH3+21-CH3), 0.69 (s, 3H,
18-CH3). 13C NMR ppm (CDCl3): 11.57 (CH3, propyl), 12.98,
17.69, 23.11, 23.73, 23.78, 24.80, 26.17, 26.64, 27.68, 28.98,
29.47, 30.32, 30.73, 31.94, 33.48, 33.86, 34.69, 35.37, 36.02,
37.49, 41.42 (CH2, propyl), 46.73, 47.61, 48.57, 58.93 (CH, 3-C),
4.1.16. (4R)-4-((3S,5R,10S,13R,17R)-3-Azido-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-
N-cyclohexylpentanamide (N-cyclohexyl 3b-azido-5b-cholane-
24-amide) (14)
Compound 14 was produced from 12 using HOBt/EDC in DMF as
already described for compounds 8–11. It was obtained as a light
yellow foam (0.149 g, 83%). 1H NMR d (CDCl3): 5.48 (br s, 1H,
73.41 (CH, 12-C), 173.65 (C@O, 24-C). IR
(KBr): 3425.12,
NH), 3.95 (s, 1H, 3a-H), 3.78 (m, 1H, cyclohexyl-CH), 0.95 (s, 3H,
m
max
2935.22,
2102.60
and
1638.55 cmꢀ1
.
HRMS:
Found:
19-CH3), 0.92 (d, 3H, J = 6.04 Hz, 21-CH3), 0.64 (s, 3H, 18-CH3).
13C NMR ppm (CDCl3): 12.25, 18.59, 21.19, 23.98, 24.36, 24.89,
25.08, 25.74, 26.47, 26.72, 28.46, 30.37, 30.86, 32.08, 33.44,
33.47, 34.14, 35.16, 35.70, 35.83, 37.50, 40.31, 40.38, 42.95,
[M+Na]+ = 481.3507, calculated for C27H46N4O2Na = 481.3518.
4.1.14. (4R)-4-((3S,5R,10S,13R,17R)-3-Azido-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)
pentanoic acid (3b-azido -LCA) (12)
48.21, 56.28, 56.77, 58.98, 172.80 (C@O, 24-C). IR
(KBr)
m
max
3318.76, 2930.15, 2100.76 and 1639.16 cmꢀ1
. HRMS: Found:
LCA (3 g, 7.966 mmol) was dissolved in MeOH and cc. HCl
(0.34 ml, 3.983 mmol) added and the mixture was stirred at reflux.
After the completion of the reaction the solvent was removed and
the residue was dissolved in EtOAc (50 ml) then washed with
water (50 ml) and brine (50 ml). The organic phase was dried over
MgSO4, filtered and the solvent was removed under reduced pres-
sure giving LCA methyl ester as a white solid as product (3.049 g,
98%). To a solution of the LCA methyl ester (3.049 g, 7.807 mmol)
and Et3N (1.24 ml, 8.871 mmol) in anhydrous DCM (50 ml) was
added methanesulfonyl chloride (0.94 ml, 12. 097 mmol) in anh.
DCM (10 ml) dropwise at 0 °C and stirred at rt for 1.5 h. Then the
reaction mixture was washed with 1 M HCl (2 ꢁ 100 ml), water
(100 ml) and brine (100 ml), dried over MgSO4, filtered and the sol-
vent was removed under reduced pressure giving white solid as
product (3.01 g, 80%). 1H NMR (400 MHz, CDCl3): 4.66 (m, 1H,
3b-H), 3.68 (s, 3H, –O-CH3), 3.02 (s, 3H, –SO2-CH3), 0.94 (s, 3H,
19-CH3), 0.93 (d, 3H, J = 6.52 Hz, 21-CH3), 0.65 (s, 3H, 18-CH3).
13C NMR ppm (100 MHz, CDCl3): 174.59 (C@O, 24-C), 82.81 (CH,
[M+Na]+ = 505.3865, calculated for C30H50N4ONa = 505.3882.
4.1.17. (4R)-4-((3S,5R,10S,13R,17R)-3-Azido-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-
N-propylpentanamide (N-propyl 3b-azido-5b-cholane-24-
amide) (15)
Compound 15 was produced from 12 using HOBt/EDC in DMF as
already described for compounds 8–11. It was obtained as a white
foam (0.123 g, 74%). 1H NMR d (CDCl3): 5.48 (br s, 1H, NH), 3.95 (s,
1H, 3a-H), 3.22 (m, 2H, propyl-CH2), 0.95–0.91 (m, 9H, 19-CH3+-
propyl-CH3+21-CH3), 0.64 (s, 3H, 18-CH3). 13C NMR ppm (CDCl3):
11.58, 12.26, 18.58, 21.19, 23.11, 23.98, 24.37, 24.89, 26.47,
26.72, 28.45, 30.38, 30.86, 32.08, 33.98, 35.16, 35.73, 35.83,
37.50, 40.31, 40.38, 41.39, 42.94, 56.28, 56.77, 58.98, 173.74
(C@O, 24-C). IR
(KBr): 3278.17, 2935.82, 2101.57 and
m
max
1645.32 cmꢀ1. HRMS: Found: [M-2H+Na]+ = 463.3401, calculated
for C27H44N4ONa = 463.3413.
3-C), 51.32 (CH3, –O-CH3). The 3-
was converted to the 3-b azide by treating with NaN3 in DMPU
a
mesylate (2.88 g, 6.145 mmol)
4.1.18. (4R)-4-((3S,5R,10S,13R,17R)-3-Azido-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-
N-cyclopropylpentanamide (16 N-cyclopropyl 3b-azido-5b-
cholane-24-amide) (16)
as already described. The yield product was white solid (2.44 g,
96%). 1H NMR (400 MHz, CDCl3): d 3.97 (s, 1H, 3
a-H), 3.68 (s, 3H,
–O-CH3), 0.96 (s, 3H, 19-CH3), 0.93 (d, 3H, J = 6.52 Hz, 21-CH3),
Compound 16 was produced from 12 using HOBt/EDC in DMF as
already described for compounds 8–11. It was obtained as a white
0.66 (s, 3H, 18-CH3). 13C NMR ppm (100 MHz, CDCl3): d 175.24

266
F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
foam (0.093 g, 56%). 1H NMR d (CDCl3): 5.66 (br s, 1H, NH), 3.97 (s,
1H, 3 -H), 2.71 (m, 1H, CPA-CH), 0.97 (s, 3H, 19-CH3), 0.93 (d, 3H,
2091.28, 1738.46 and 1245.13 cmꢀ1
.
HRMS: Found: (M-
a
Na)+ = 496.3168, calculated for C27H43N3O4Na = 496.3151. The 3-
azido formate ester of DCA methyl ester (0.101 g) was subjected
to hydrolysis using NaOH in aqueous methanol as described
(0.074 g, 82%). mp: 88 °C. 1H NMR (400 MHz, CDCl3): d 4.01 (s, 1H,
12b-H), 3.35 (m, 1H, 3b-H), 1.01 (d, 3H, J = 6.03 Hz, 21-CH3), 0.94
(s, 3H, 19-CH3), 0.70 (s, 3H, 18-CH3). 13C NMR ppm (100 MHz,
CDCl3): d 179.80 (C@O, 24-C), 73.29 (CH, 12-C), 61.40 (CH, 3-C).
J = 6.52 Hz, 21-CH3), 0.79 (m, 2H, CPA-CH2), 0.66 (s, 3H, 18-CH3),
0.50 (m, 2H, CPA-CH2). 13C NMR ppm (CDCl3): 6.84 (2 ꢁ CH2, CPA),
12.25, 18.57, 21.19, 22.81, 23.99, 24.36, 24.89, 26.47, 26.72, 28.45,
30.38, 30.86, 31.94, 33.65, 35.16, 35.72, 35.83, 37.51, 40.31, 40.38,
42.95, 56.25, 56.77, 58.98 (CH, 3-C), 175.24 (C@O, 24-C). IR
m
max
(KBr): 2932.36, 2102.08 and 1645.39 cmꢀ1
. HRMS: Found:
[M+Na]+ = 463.3399, calculated for C27H44N4ONa = 463.3413.
IR
(KBr): 3440.00, 2941.49, 2866.14, 2090.46, 1709.85,
m
max
1679.00 and 1246.00 cmꢀ1. HRMS: Found: (M-Na)+ = 440.2878, cal-
culated for C24H39N3O3Na = 440.2889.
4.1.19. (4R)-4-((3S,5R,10S,13R,17R)-3-Azido-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-
N-benzylpentanamide (N-benzyl 3b-azido-5b-cholane-24-
amide) (17)
4.1.22. (4R)-4-((3R,5R,10S,12S,13R,17R)-3-Azido-12-hydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
Compound 17 was produced from 12 using HOBt/EDC in DMF as
already described for compounds 8–11. It was obtained as a light
yellow foam (0.123 g, 67%). 1H NMR d (CDCl3): 7.38–7.29 (m, 5H,
aromatic-H), 5.77 (br s, 1H, NH), 4.47 (d, 2H, J = 5.52 Hz, benzyl-
17-yl)pentanamide (3
amide) (18)
a
ꢀazido, 12
a-hydroxy-5b-cholane-24-
The 3-a azido analogue of DCA 29 (0.15 g, 0.359 mmol) was con-
verted to the corresponding primary amide using the HOBt ap-
proach with ammonia solution. The product was isolated as a
white foam (0.055 g, 37%). 1H NMR d (acetone-d6): 4.02 (s, 1H,
12b-H), 3.35 (m, 1H, 3b-H), 1.01 (d, 3H, J = 6.52 Hz, 21-CH3), 0.94
(s, 3H, 19-CH3), 0.70 (s, 3H, 18-CH3). 13C NMR ppm (acetone-d6):
13.12, 17.68, 23.65, 24.55, 27.06, 27.28, 27.87, 28.32, 32.63, 33.08,
33.22, 34.29, 34.98, 36.17, 36.39, 36.96, 43.20, 47.32, 47.66, 48.78,
CH2), 3.97 (s, 1H, 3a-H), 0.97 (s, 3H, 19-CH3), 0.94 (d, 3H,
J = 6.04 Hz, 21-CH3), 0.66 (s, 3H, 18-CH3). 13C NMR ppm (CDCl3):
12.26, 18.57, 21.19, 23.99, 24.37, 24.89, 26.47, 26.72, 28.47,
30.37, 30.86, 32.00, 33.86, 35.16, 35.71, 35.83, 37.50, 40.30,
40.37, 42.95, 43,81, 56.26, 56.77, 58.98 (CH, 3-C), 127.71, 128.05,
128.91, 138.59, 173.58 (C@O, 24-C). IR
(KBr): 3293.28,
m
max
3065.29, 3030.69, 2932.36, 2101.90, 1644.72, 731.74 and
61.97 (CH, 3-C), 72.85 (CH, 12-C), 175.71 (C@O, 24-C). IR
mmax
697.67 cmꢀ1. HRMS: Found: [M+H]+ = 491.3751, calculated for
(KBr): 3382.75, 2939.74, 2091.78 and 1655.26 cmꢀ1. HRMS: Found:
[M+Na]+ = 439.3039, calculated for C24H40N4O2Na = 439.3049.
C31H47N4O = 491.3750.
4.1.20. (4R)-Methyl 4-((3S,5R,10S,12S,13R,17R)-3-bromo-12-
(formyloxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]
4.1.23. (4R)-4-((3R,5R,10S,12S,13R,17R)-3-azido-12-hydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
phenanthren-17-yl)pentanoate (24-methyl 3b-bromo, 12
formyloxy-5b-cholanoate) (28)
a
-
17-yl)-N-cyclopropylpentanamide (24-cyclopropyl-3
12 -hydroxy-5b-cholanamide) (19)
Compound 19 was produced from 29 using the HOBt coupling
a
ꢀazido,
a
Triphenylphosphine (2.135 g) was added to a solution of 26
(1.769 g) in anhydrous THF (80 ml) and NBS (1.449 g) was added
in 3 parts over 1 h at ꢀ18 °C then allowed to warm to rt and stirred
for 1.5 h. The reaction mixture was poured into 1 M HCl solution
(100 ml) and extracted with EtOAc (3 ꢁ 75 ml). The organic layer
was washed with brine (2 ꢁ 100 ml) and after drying over MgSO4
the solvent was removed under reduced pressure. The crude prod-
uct was flash columned (hexane/EtOAc 5:1) to afford the product
as pale white solid (1.919 g, 95%). 1H NMR (400 MHz, CDCl3): d
approach already described (0.119 g, 73%).1H NMR d (CDCl3):
5.66 (s, 1H, NH), 3.99 (m, 1H, 12b-H), 3.35 (m, 1H, 3b-H), 2.71
(m, 1H, CPA-CH), 0.99 (d, 3H, J = 6.00 Hz, 21-CH3), 0.94 (s, 3H, 19-
CH3), 0.78 (m, 2H, CPA), 0.69 (s, 3H, 18-CH3), 0.49 (m, 2H, CPA).
13C NMR ppm (CDCl3): 6.83 (2 ꢁ CH2, CPA), 12.95, 17.63, 22.78,
23.43, 23.78, 26.21, 26.87, 27.20, 27.63, 28.86, 31.74, 32.62,
33.45, 33.85, 34.37, 35.30, 35.56, 36.15, 42.51, 46.65, 47.43,
48.36, 61.44 (CH, 3-C), 73.24 (CH, 12-C), 175.11 (C@O, 24-C). IR
mmax
8.13 (s, 1H, 12-OC@OH), 5.26 (s, 1H, 12b-H), 4.79 (s, 1H, 3
a
-H),
(KBr): 3434.92, 2938.91, 2086.95 and 1650.61 cmꢀ1
. HRMS:
3.68 (s, 3H, –O-CH3), 1.01 (s, 3H, 19-CH3), 0.85 (d, 3H, J = 6.52 Hz,
21-CH3), 0.76 (s, 3H, 18-CH3). 13C NMR ppm (100 MHz, CDCl3):
174.70 (C@O, 24-C), 160.68 (C@O, 12-OC@OH), 76.29 (CH, 12-C),
Found: [M+H]+ = 457.3542, calculated for C27H45N4O2 = 457.3543.
4.1.24. (4R)-4-((3S,5S,7R,10S,13R,17R)-3-Azido-7-hydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)
pentanoic acid (3b-azido, 7a-hydroxy-5b-cholane) (20)
Compound 30 was selectively mesylated at position 3 using a
similar procedure to that already described in 4.1.2. The mesylate
was obtained as a white foam (1.299 g, 93%). 1H NMR (400 MHz,
CDCl3): d 4.54 (m, 1H, 3b-H), 3.87 (s, 1H, 7b-H), 3.68 (s, 3H, –O-
CH3), 3.01 (s, 3H, –SO2-CH3), 0.95–0.93 (d, 6H, 21-CH3 and 19-
CH3), 0.67 (s, 3H, 18-CH3). 13C NMR ppm (100 MHz, CDCl3): d
51.67 (CH3, OCH3). IR
(KBr): 3426.56, 2939.12, 2873.47,
m
max
1739.75, 1720.29 and 1180.89 cmꢀ1
.
HRMS: Found: (M-
Na)+ = 519.2073, calculated for C26H41BrO3Na = 519.2086.
4.1.21. (4R)-4-((3R,5R,10S,12S,13R,17R)-3-Azido-12-hydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
17-yl)pentanoic acid (3
(29)
a-azido, 12aꢀhydroxy-5b-cholanoate)
To a stirred solution of 28 (0.712 g) in DMPU (20 ml) was added
10 equiv of NaN3 (0.904 g) at rt. The mixture was stirred for 4 d then
poured into water (100 ml) and extracted with EtOAc (3 ꢁ 50 ml).
The organic phase was washed with brine (100 ml), dried over
MgSO4, filtered and the solvent was evaporated in vacuum. The
product was separated on a flash column using 0–12% EtOAc in hex-
ane as mobile phase to yield light yellow oil as product (0.589 g,
89%). 1H NMR (400 MHz, CDCl3): d 5.09 (s, 1H, 12b-H), 3.68 (s, 3H,
–O-CH3), 3.29 (m, 1H, 3b-H), 2.11 (s, 3H, 12-C@OCH3), 0.92 (s, 3H,
174.55 (C@O, 24-C), 82.57 (CH, 3-C), 68.73 (CH, 7-C). IR
(KBr): 3552.50, 2942.04, 1737.27, 1348.89, 1170.76 and
926.45 cmꢀ1. HRMS: Found: (M-Na)+ = 507.2758, calculated for
mmax
C26H44O6SNa = 507.2756. The mesylate was converted to azide
using the NaN3/DMPU approach at 65 °C as already described.
The product was isolated following chromatography using hexane:
EtOAc 3:1 as mobile phase to afford colourless semi-solid as prod-
uct (0.86 g, 76%). 1H NMR (400 MHz, CDCl3): d 3.92 (s, 1H, 3
a-H),
3.88 (s, 1H, 7b-H), 3.68 (s, 3H, –O-CH3), 0.95–0.93 (d, 6H, 19-CH3
19-CH3), 0.83 (d, 3H, J = 6.53 Hz, 21-CH3), 0.74 (s, 3H, 18-CH3). 13
C
and 21-CH3), 0.68 (s, 3H, 18-CH3). 13C NMR ppm (100 MHz, CDCl3):
NMR ppm (100 MHz, CDCl3): d 175.07 (C@O, 24-C), 171.04 (C@O,
12-OC@OCH3), 76.27 (CH, 12-C), 61.41 (CH, 3-C), 51.98 (CH3,
d 175.20 (C@O, 24-C), 69.04 (CH, 7-C). IR
(KBr): 3494.84,
m
max
2930.37, 2091.78 and 1739.85 cmꢀ1
Na)+ = 454.3029, calculated for C25H41N3O3Na = 454.3046. The
.
HRMS: Found: (M-
OCH3), 21.79 (CH3, 12-C@OCH3). IR
(DCM): 2941.42, 2867.23,
m
max

F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
267
methyl ester (0.84 g, 1.946 mmol) was subjected to hydrolysis in
methanolic solution of aq. NaOH affording product as a white foam
4.1.28. (4R)-4-((3R,5S,7R,10S,13R,17R)-3-Azido-7-hydroxy-
10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-
17-yl)pentanoic acid (3 -azido, 7 -hydroxy-5b-cholane) (23)
Compound 23 was produced from 31 using the NaN3/DMPU
procedure already described above. The product was separated
by column chromatography using hexane: EtOAc 3:1 as mobile
phase to afford yellow semi-solid (0.098 g, 79%). 1H NMR d
(400 MHz, CDCl3): 3.87 (s, 1H, 7b-H), 3.68 (s, 3H, –O-CH3), 3.16
(s, 1H, 3b-H), 0.95 (d, 6H, 21-CH3 and 19-CH3), 0.67 (s, 3H, 18-
CH3). 13C NMR ppm (100 MHz, CDCl3): d 174.88 (C@O, 24-C),
(0.277 g, 69%). 1H NMR d (CDCl3): 3.93 (s, 1H, 3
a
-H), 3.89 (s, 1H,
a
a
7b-H), 0.96 (d, 6H, 19-CH3 and 21-CH3), 0.69 (s, 3H, 18-CH3). 13C
NMR ppm (CDCl3): 11.98, 18.43, 20.98, 23.35, 23.90, 24.85, 28.34,
30.79, 30.95, 31.18, 32.63, 33.31, 34.23, 35.54, 35.65, 36.97,
39.50, 39.80, 42.94, 50.64, 55.98, 58.95, 68.89 (CH, 7-C),
180.27(C@O, 24-C). IR
(KBr): 3450.29, 2927.53, 2101.43 and
m
max
1709.14 cmꢀ1
.
HRMS: Found: [M]ꢀ = 416.2901, calculated for
C24H38N3O3 = 416.2913.
68.44 (CH, 7-C). IR
(KBr): 3516.06, 2934.95, 2086.95 and
m
max
4.1.25. (4R)-4-((3S,5S,7R,10S,13R,17R)-3-Azido-7-hydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)
1721.20 cmꢀ1. HRMS: Found: (M-Na)+ = 454.3040, calculated for
25H41N3O3Na = 454.3046. The methyl ester was hydrolysed in a
C
pentanamide (3b-azido, 7
a
-hydroxy-5b-cholane-24-amide) (21)
methanolic NaOH solution as already described to give product
as white foam (0.084 g, 88%). 1H NMR d (CDCl3): 3.89 (s, 1H, 7b-
H), 3.18 (s, 1H, 3b-H), 0.96 (d, 3H, J = 6.56 Hz, 21-CH3), 0.93 (s,
3H, 19-CH3), 0.68 (s, 3H, 18-CH3). 13C NMR ppm (CDCl3): 11.98,
18.43, 20.76, 23.06, 23.88, 27.01, 28.34, 30.94, 31.15, 32.95,
34.59, 35.32, 35.54, 35.64, 35.75, 39.56, 39.69, 41.98, 42.90,
50.52, 55.93, 61.55, 68.60 (CH, 7-C), 180.01 (C@O, 24-C). HRMS:
Found: [M-H]ꢀ = 416.2914. HRMS: Found: (M)ꢀ = 416.2914, calcu-
lated for C24H38N3O3 = 416.2913.
Compound 21 was prepared from 20 using the ethyl chlorofor-
mate procedure. White foam (0.071 g, 72%). 1H NMR d (CDCl3):
5.89 (br s, 2H, NH2), 3.92 (s, 1H, 3a-H), 3.88 (s, 1H, 7b-H), 0.96
(d, 6H, 21-CH3 and 19-CH3), 0.68 (s, 3H, 18-CH3). 13C NMR ppm
(CDCl3): 11.97, 18.53, 20.97, 23.34, 23.90, 24.84, 28.42, 30.79,
31.77, 32.61, 32.79, 33.30, 34.27, 35.65, 36.98, 39.49, 39.80,
42.91, 50.64 (CH, 3-C), 55.95, 58.94, 68.78 (CH, 7-C), 176.86
(C@O, 24-C). IR
(KBr): 3436.57, 2927.53, 2101.06 and
1648.37 cmꢀ1. HRMS: Found: [M+Na]+ = 439.3061, calculated for
24H40N4O2Na = 439.3049
m
max
C
4.2. Biochemical methods
4.1.26. (4R)-4-((3S,5S,7R,10S,13R,17R)-3-Azido-7-hydroxy-10,13-
dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-
4.2.1. MTT assay
Cell viability was determined by MTT assay. Cells were seeded
(Caco-2 at 8 ꢁ 103 cells/well and HT1080 at 12 ꢁ 104 cells/well)
into 96-well plates and grown until 70% confluent. Cells were ser-
um starved for 1 h and then treated with test compounds at 0.1–
N-cyclopropylpentanamide (24-cyclopropyl-3b-azido, 7
a-
hydroxy-5b-cholanamide) (22)
Compound 22 was produced from 20 using the ethyl chlorofor-
mate coupling procedure with cyclopropyl amine. White foam
(0.095 g, 87%). 1H NMR d (CDCl3): 6.12 (s, 1H, NH), 3.92 (s, 1H,
1000 lM, under serum free conditions for 24 h. Test articles were
introduced in DMSO at maximum 0.5% at which concentration
no effects on cell viability could be observed. At the end of this
incubation, MTT (0.5 mg/mL) was added and incubated for 3 h at
3a-H), 3.88 (s, 1H, 7b-H), 2.74 (m, 1H, CPA-CH), 0.96 (d, 6H, 21-
CH3 and 19-CH3), 0.80 (m, 2H, CPA), 0.67 (s, 3H, 18-CH3), 0.56
(m, 2H, CPA). 13C NMR ppm (CDCl3): 6.80 (2 ꢁ CH2, CPA), 11.98,
18.56, 20.98, 23.11, 23.35, 23.92, 24.85, 28.41, 30.80, 32.03,
32.61, 33.30, 34.23, 35.65, 35.69, 36.99, 39.50, 39.79, 42.92, 50.62
(CH, 3-C), 55.93, 58.96, 67.28, 68.82 (CH, 7-C), 176.09 (C@O, 24-
37 °C. After 3 h, supernatants were removed, 100 lL of DMSO
was added, and the plates were incubated with shaking for
10 min. The formazan crystals formed inside the viable cells were
solubilized in DMSO and the optical density was read on a plate
reader at 540 nm. The % cell viability was determined as follows:
(optical density of treated cells/optical density of non-treated
cells)⁄100. The experiments were performed a minimum of three
times with three replicates over eight concentration levels.
Resultant concentration effect curves were transformed and
analyzed using GraphPad Prism 5 (San Diego, CA, USA). In order
to evaluate the role of ROS in these observations, MTT assays were
repeated but with co-incubation with NAC (1 mM).
C). IR
(KBr): 3429.43, 2929.05, 2096.61 and 1665.91 cmꢀ1
.
m
max
HRMS: Found: [M+Na]+ = 479.3349, calculated for C27H44N4O2Na
= 479.3362.
4.1.27. (4R)-Methyl 4-((3S,5R,7R,10S,13R,17R)-7-hydroxy-10,13-
dimethyl-3-(methylsulfonyloxy)hexadecahydro-1H-
cyclopenta[a]phenanthren-17-yl)pentanoate (24-Methyl 3b-
(methanesulfonyl)oxy, 7a-hydroxy-5b-cholanoate) (31)
Compound 30 (0.8 g, 1.967 mmol) and PPh3 (1.703 g,
6.492 mmol) were dissolved in anhydrous THF (40 ml) and cooled
to 0 °C. Et3N (0.55 ml, 3.935 mmol) and methanesulfonic acid
(0.27 ml, 4.132 mmol) were added to the mixture at this tempera-
ture. The solution was warmed to 45 °C and DIAD (1.2 ml,
6.099 mmol) was added dropwise and the mixture was stirred un-
til TLC analysis showed the reaction was complete. Then the sol-
vent was removed and the residue was dissolved in EtOAc
(100 ml) which was washed with 1 M HCl (2 ꢁ 100 ml), water
(100 ml) and brine (100 ml). After drying the organic phase over
MgSO4 the solvent was removed and the product was separated
by column chromatography using hexane: EtOAc 3:1, 1:1 and 1:3
as mobile phase. The yield product was odourless semi-solid
4.2.2. Flow cytometry
Cells were grown to 80% confluence in T25 flasks and treated
with test compound for 24 h in serum free medium, control cells
were treated only with vehicle in serum free media for 24 h. Fol-
lowing 24 h treatment cells were removed from T25 flasks and
washed with 1X Binding Buffer, before being re-suspended in
100 ml of 1X binding buffer. A 20 ml aliquot of these cells was
stained using 5 ml annexin V-FITC and 5 ml propidium iodide
and incubated at rt for 15 min in the dark. Following incubation,
samples were diluted in 1X binding buffer and analyzed within
5 min using a BD FACSArray (BD Biosciences, Oxford, UK). Flow
cytometry was performed on double stained cell samples (Caco-2
and HT1080 cells). The instrument was set up to measure the size
(forward scatter), granularity (side scatter) and cell fluorescence.
Antibody binding was measured by analyzing individual cells for
fluorescence. Data were analyzed using BD FACS Array software
and expressed as a percentage of control fluorescence in arbitrary
units. Cells in late stages of apoptosis or those already dead have
lost PM integrity and are permeable to PI whereas annexin V binds
(0.139 g, 15%). 1H NMR (400 MHz, CDCl3): d 5.00 (m, 1H, 3
a-H),
3.88 (s, 1H, 7b-H), 3.68 (s, 3H, –O-CH3), 3.01 (s, 3H, –SO2-CH3),
0.98 (s, 3H, 19-CH3), 0.95 (d, 3H, J = 6.52 Hz, 21-CH3), 0.68 (s, 3H,
18-CH3). 13C NMR ppm (100 MHz, CDCl3): d 175.19 (C@O, 24-C),
81.22 (CH, 3-C), 68.98 (CH, 7-C). IR
(KBr): 3448.15, 2939.07,
m
max
1737.73, 1351.28, 1173.17 and 901.12 cmꢀ1
(M-Na)+ = 507.2690, calculated for C26H44O6SNa = 507.2756.
. HRMS: Found:

268
F. Májer et al. / Bioorg. Med. Chem. 22 (2014) 256–268
5. Powell, A. A.; LaRue, J. M.; Batta, A. K.; Martinez, J. D. Biochem. J. 2001, 356, 481.
6. Barrasa, J. I.; Olmo, N.; Lizarbe, M. A.; Turnay, J. Toxicology in vitro: an
international journal published in association with BIBRA 2013, 27, 964.
7. Qiao, L.; Studer, E.; Leach, K.; McKinstry, R.; Gupta, S.; Decker, R.; Kukreja, R.;
Valerie, K.; Nagarkatti, P.; El Deiry, W.; Molkentin, J.; Schmidt-Ullrich, R.;
Fisher, P. B.; Grant, S.; Hylemon, P. B.; Dent, P. Mol. Biol. Cell 2001, 12, 2629.
8. Katona, B. W.; Cummins, C. L.; Ferguson, A. D.; Li, T.; Schmidt, D. R.;
Mangelsdorf, D. J.; Covey, D. F. J. Med. Chem. 2007, 50, 6048.
9. Katona, B. W.; Anant, S.; Covey, D. F.; Stenson, W. F. J. Biol. Chem. 2009, 284,
3354.
10. Sharma, R.; Majer, F.; Peta, V. K.; Wang, J.; Keaveney, R.; Kelleher, D.; Long, A.;
Gilmer, J. F. Bioorg. Med. Chem. 2010, 18, 6886.
11. Majer, F.; Salomon, J. J.; Sharma, R.; Etzbach, S. V.; Najib, M. N.; Keaveny, R.;
Long, A.; Wang, J.; Ehrhardt, C.; Gilmer, J. F. Bioorg. Med. Chem. 2012, 20, 1767.
12. Walsh, G. M.; Dewson, G.; Wardlaw, A. J.; Levi-Schaffer, F.; Moqbel, R. J.
Immunol. Methods 1998, 217, 153.
13. Sharma, R.; Prichard, D.; Majer, F.; Byrne, A. M.; Kelleher, D.; Long, A.; Gilmer, J.
F. J. Med. Chem. 2011, 54, 122.
14. Favre-Felix, N.; Fromentin, A.; Hammann, A.; Solary, E.; Martin, F.; Bonnotte, B.
J. Immunol. (Baltimore, Md.: 1950) 2000, 164, 5023.
15. Oikonomou, E.; Kothonidis, K.; Taoufik, E.; Probert, E.; Zografos, G.; Nasioulas,
G.; Andera, L.; Pintzas, A. Br. J. Cancer 2007, 97, 73.
16. Villa-Morales, M.; Fernandez-Piqueras, J. Expert Opin. Ther. Targets 2012, 16, 85.
17. Ignacio Barrasa, J.; Olmo, N.; Perez-Ramos, P.; Santiago-Gomez, A.; Lecona, E.;
Turnay, J.; Antonia Lizarbe, M. Apoptosis 2011, 16, 1054.
to cells in early apoptosis and continues to be bound through cell
death. Thus cells in early stage of apoptosis will be annexin V po-
sitive and PI negative (in Q4), cells in late stage of apoptosis (in Q2)
or annexin V Positive and PI positive and finally already dead (in
Q1). Healthy cells are annexin V and PI negative (in Q3).
4.2.3. Measurement of ROS
Human colonic epithelial cancer cells Caco-2 cells at 70–80%
confluence were treated in serum free media with test articles
for 30 min and 24 h. The medium was removed and replaced with
100 l
M 20,70-dichlorfluorescein-diacetate for 30 min at 37 °C. The
solutions were removed and the cells were washed with PBS. The
DCF fluorescence was monitored with an excitation wavelength
of 490 nm and an emission wavelength of 530 nm.
4.2.4. Statistical analysis
Data are presented as group of means with standard error of the
mean of n P3. Statistical analysis of the mean difference between
multiple groups was determined by one-way ANOVA followed by
Tukey–Kramer multiple comparison post-test. A P value <0.05
was considered to be statistically significant. All statistical analyses
were performed using Prism version 4 for Windows, GraphPad
Software, La Jolla California USA.
18. Sharma, R.; Long, A.; Gilmer, J. F. Curr. Med. Chem. 2011, 18, 4029.
19. Kim, N. D.; Im, E.; Yoo, Y. H.; Choi, Y. H. Curr. Cancer Drug Targets 2006, 6, 681.
20. Liu, H.; Qin, C. K.; Han, G. Q.; Xu, H. W.; Ren, W. H.; Qin, C. Y. Cancer Lett. 2008,
270, 242.
21. Park, S. E.; Lee, S. W.; Hossain, M. A.; Kim, M. Y.; Kim, M. N.; Ahn, E. Y.; Park, Y.
C.; Suh, H.; Kim, G. Y.; Choi, Y. H.; Kim, N. D. Cancer Lett. 2008, 270, 77.
22. Brossard, D.; El Kihel, L.; Clement, M.; Sebbahi, W.; Khalid, M.; Roussakis, C.;
Rault, S. Eur. J. Med. Chem. 2010, 45, 2912.
References and notes
23. Bellini, A. M.; Mencini, E.; Quaglio, M. P.; Guarneri, M.; Fini, A. Steroids 1991, 56,
395.
24. Bellini, A. M.; Quaglio, M. P.; Cavazzini, G.; Cecchenini, R. Farmaco Sci. 1984, 39,
305.
1. Hofmann, A. F.; Hagey, L. R. Cell. Mol. Life Sci. 2008, 65, 2461.
2. Porez, G.; Prawitt, J.; Gross, B.; Staels, B. J. Lipid Res. 2012, 53, 1723.
3. Amaral, J. D.; Viana, R. J.; Ramalho, R. M.; Steer, C. J.; Rodrigues, C. M. J. Lipid Res.
2009, 50, 1721.
4. McQuaid, K. R.; Laine, L.; Fennerty, M. B.; Souza, R.; Spechler, S. J. Aliment.
Pharmacol. Ther. 2011, 34, 146.