Ursodeoxycholic acid amides as novel glucocorticoid receptor modulators

Article abstract of DOI:10.1021/jm100860s

Ursodeoxycholic acid (UDCA) is used for the treatment of hepatic inflammatory diseases. Recent studies have shown that UDCAs biological effects are partly glucocorticoid receptor (GR) mediated. UDCA derivatives were synthesized and screened for ability to

Full text of DOI:10.1021/jm100860s

122 J. Med. Chem. 2011, 54, 122–130
DOI: 10.1021/jm100860s
Ursodeoxycholic Acid Amides As Novel Glucocorticoid Receptor Modulators
Ruchika Sharma,*,†,‡ David Prichard,‡ Ferenc Majer,† Anne-Marie Byrne,‡ Dermot Kelleher,‡ Aideen Long,‡ and
John F. Gilmer*,†
†School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, Dublin 2, Ireland, and ‡Cell Signalling, Institute of Molecular
Medicine, Trinity Centre for Health Science, St. James’s Hospital, Dublin 8, Ireland
Received July 9, 2010
Ursodeoxycholic acid (UDCA) is used for the treatment of hepatic inflammatory diseases. Recent
studies have shown that UDCA’s biological effects are partly glucocorticoid receptor (GR) mediated.
UDCA derivatives were synthesized and screened for ability to induce GR translocation in a high
content analysis assay using the esophageal cancer SKGT-4 cell line. UDCA derivatives induced GR
translocation in a time dependent manner with equal efficacy to that of dexamethasone (Dex) and with
greatly increased potency relative to UDCA. The cyclopropylamide 1a suppressed TNF-R induced
NF-κB activity and it induced GRE transactivation. 1a was unable to displace Dex from the GR ligand
binding domain (LBD) in a competition experiment but was capable of coactivator recruitment in a
time-resolved fluorescence energy transfer assay (TR-FRET). This represents a novel mechanism of
action for a GR modulator. These derivatives could result in a new class of GR modulators.
Introduction
Homodimers can also bind to negative GREs, inhibiting gene
expression.10,11 Yet another pathway involves direct binding
to pro-inflammatory transcription factors such as activator
protein-1 (AP-1) and nuclear factor κ-light-chain-enhancer of
activated B cells (NF-κB).5,12 This “transrepression” effect is
associated with GR monomers.13 There has been in the past
few years an interest in so-called “dissociated steroids” that
can separate transrepression from transactivation, which is
associated with steroid side effects.14-17 Steroid antagonists
also cause receptor dissociation and nuclear translocation but
with limited transcription. GC antagonists are being investi-
gated in a range of indications including, psychotic depres-
sion, diabetes, obesity, Alzheimer’s disease, neuropathic pain,
drug abuse, and glaucoma.18 Thus it is possible to envisage
ligands capable of inducing GR translocation but associated
witha morelimited repertoire of transcriptional activities than
classical GC agonist. Mechanistically, such compounds
should bind nonclassically, influencing GR self-association
and recruitment of transcriptional machinery.19
UDCA(UDCA, 1, Figure 1), a traditionalmedicine, isused
at high dose in the treatment of primary biliary cirrhosis
(PBC) and primary sclerosing cholangitis.20 The biochemical
basis for UDCA’s actions remains somewhat obscure
although it is the subject of extensive investigation. UDCA
exhibits antiapoptotic action in hepatic21,22 and nonhepatic
cell lines,23,24 and it has neuroprotective actions.25-27 It has
putative chemopreventative effects in colon cance,r28 and it
has been shown to attenuate carcinogenic signaling cascades
induced byotherbileacids. Theseincludeinhibitionof protein
kinase C translocation,29 suppression of p38, MAPK, and
Cox-2 expression30 as well as reduction in activity of the
transcription factors AP-1 and NF-κB.31 UDCA treatment
reduces serum antimitochondrial and immunoglobulin G
antibodies.32 It can also suppress interleukin-233 and inter-
feron γ production34 and decrease the hepatocellular and
The glucocorticoid receptor (GRa) is a member of the nuclear
receptor family of eukaryotic transcription regulators.1 Classi-
cal GR agonists (glucocorticoids, GCs) include the endogenous
GR ligand cortisol and its synthetic derivatives. GCs are the
principal therapies in asthma, rheumatoid arthritis, and inflam-
matory bowel disease.2 They also find clinical application in
neurological conditions and in certain types of cancer.3 The GR
is arguably the most important pharmacological target in
medicine.
The unliganded GR resides in the cytoplasm, part of a
multimeric complex consisting of the GR polypeptide and
chaperone proteins hsp90 and immunophilins, FKBP52,
FKBP51, Cyp40, and PP5.4 Ligand binding causes dissocia-
tion of chaperones leading to exposure of nuclear localiza-
tion signals triggering nuclear translocation. Ligand-bound
GR may dimerize. Homodimeric GR has direct and indirect
effects on a wide spectrum of transcriptional activities.5
Binding to glucocorticoid response elements (GREs) followed
by coactivator recruitment results in transactivation of genes.6-9
*To whom correspondence should be addressed. For R.S.: phone,
þ35318963350/8962844; fax, þ35318962810; E-mail, rsharma@tcd.ie.
For J.F.G.: phone, þ35318962795; fax, þ35318962793; E-mail,
gilmerjf@tcd.ie.
a Abbreviations: AP-1, activator protein-1; CDCA, chenodeoxy-
cholic acid; DCA, deoxycholic acid; Dex, dexamethasone; GCs, gluco-
corticoids; GR, glucocorticoid receptor; GREs, glucocorticoid response
elements; HRMS, high resolution mass spectrometry; HPLC, high
performance liquid chromatography; HCl, hydrochloric acid; LBD,
ligand binding domain; LPS, lipopolysaccharide; MgSO4, magnesium
sulfate; MeOH, methanol; NF-κB, nuclear factor κ-light-chain-enhan-
cer of activated B cells; PBS, phosphate buffered saline; PLA2IIA,
phospholipase A2 IIA; PBC, primary biliary cirrhosis; PSC, primary
sclerosing cholangitis; TR-FRET, time-resolved fluorescence energy
transfer; SRC1-4, steroid receptor coactivator 1-4; TLC, thin layer
chromatography; TAT, tyrosine aminotransferase; UDCA, ursodeoxy-
cholic acid.
r
pubs.acs.org/jmc
Published on Web 12/15/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 123
Figure 1. Structural formulas of UDCA (1), DCA (2), CDCA (3), and Dex (4).
Scheme 1. Synthesis and Structural Formulas of UDCA analogues 1a-1k
(i) Formic acid/perchloric acid, 47 °C, 3 h; (ii) SOCl2, reflux, 2 h; (iii) RNH2, NEt3, RT; (iv) NaOMe/MeOH, 70 °C, 2 h.
biliary expression of both major histocompatibility complex
class I and class II molecules in patients with PBC.35,36 UDCA
can correct the defective natural killer cell activity by inhibit-
ing prostaglandin E2 production in PBC patients.37 UDCA
has a suppressive effect at the transcriptional level on phos-
pholipase A2 IIA (PLA2IIA) in hepatocytes.
These latter effects at a physiological and biochemical level
are similar to those of GCs.38 There is also a significant body
of biochemical evidence that UDCA can activate GR path-
ways. UDCA has been reported to induce translocation of
the GR and to activate the GR in various different cell
models.39-42 Furthermore, UDCA can suppress NF-κB ac-
tivity via activation of the GR.41 The GR-ligand binding
domain (LBD) is responsible for UDCA-dependent nuclear
translocation of the GR.41 GR knockdown attenuates UD-
CA’s inhibitory effects on PLA2IIA.38
the esophageal SKGT-4 cell line. UDCA weakly induced
translocation of the GR from the cytoplasm to the nucleus
(54% efficacy of Dex 100 nM (4) at 300 μM (Figure 2A-C).
This is a similar level of effect to that reported in other cell
lines. UDCA has threereadily manipulablefunctionalgroups:
the C3-, C7-OH, and C24-COOH (Figure 1). UDCA’s ability
to induce GR trafficking is connected with its C7-β OH group
because its C7-epimer chenodeoxycholic acid (CDCA, 3) and
deoxycholic acid (DCA, 2) do not induce GR trafficking. Our
initial chemistry therefore focused on manipulating the C24-
COOHgroup. This strategy was supported by the observation
that TauroUDCA (TUDCA) can induce GR and mineralo-
corticoid receptor (MR) translocation, illustrating that the
side chain could be modified and that amide derivatives
of UDCA might be active.44,45 We therefore targeted a
small chemically diverse library of UDCA amides (1a-k,
Scheme 1). These were produced in four steps from UDCA by
formyl protection of the steroidal -OH groups, formation of
the corresponding acyl chloride (SOCl2), amidation with the
appropriate amine, and deformylation (NaOMe/MeOH).
We produced another set of UDCA derivatives (1l-1s,
Figure 3) in which the acid group was abolished or migrated
using classical bile acid chemistry.46,47
Analogues 1a-1s were tested in a high content analysis
assay using immunofluorescence to estimate the nuclear to
cytoplasmic ratio (Figure 4). The experiment was carried out
initially at 300 μM for 4 h and repeated at successively lower
concentration where an effect was observed (Table 1). The
amide series (1a-1k) exhibited the highest biochemical effi-
cacy relative to Dex and highest potency. For example, the
cyclopropyl derivative (1a) and 1b, 1c, and 1d had similar
efficacy to Dex as reflected in steady-state nuclear/cytoplas-
mic ratio. 1a-b were the most potent compounds with EC50
values of 7.3 and 5.6 μM, respectively (Figure 2D). Removal
of the benzyl group in 1c maintained GR translocation
efficacy but reduced potencyby 10-fold (50.8 μM). The anilide
(1e) did not induce translocation but the benzylamide (1f),
R-methylbenzylamide (1g) and phenethylamide (1h) showed
Taking these studies and UDCA’s clinical history of mini-
malsideeffectsintoaccount, wedecidedthat itwould make an
excellent lead for the development of a novel class of GR
modulators.
In this work, a series of bile acid derivatives (Scheme 1) were
synthesized in order to test this hypothesis. Chronic inflam-
mation in the esophagus has been associated with the patho-
genesis of esophageal adenocarcinoma.43 Hence we screened
our derivatives for the ability to modulate the GR in an
esophageal cancer cell line. We have shown that modification
at the C24-position of UDCA leads to compounds endowed
with the ability to induce GR translocation in the low micro-
molar range. These may act as leads for development of GR
modulators with new transcriptional profiles. The increased
potency of these analogues makes them useful tools for
further characterizing UDCA’s effects on the GR and eluci-
dating its mechanism of action.
Results and Discussion
We initially established the ability of UDCA to induce
translocation of the GR from the cytoplasm to the nucleus in

124 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Sharma et al.
Figure 2. UDCA derivatives induce GR translocation. SKGT-4 cells were treated with (A) DMSO 1%, (B) Dex (100 nM) as a positive control,
(C) UDCA (300 μM), or (D) 1a (100 μM). Cells were fixed with paraformaldehyde 4% in PBS after 4 h. The GR was identified using a mouse
monoclonal GR antibody (green.) and cells were stained with Hoechst (blue) and phalloidin (red) to identify the nucleus and actin cytoskeleton
respectively. Cells were imaged using the GE IN Cell Analyzer 1000. Original magnification ꢀ10.
Table 1. EC50 Values for GR Translocation of UDCA (1) and Selected
Modified Bile Acids a
EC50
(μM, 95% CI)
compd
(% efficacyb)
1
298.7 (151-432) (54)
7.3 (1-14) (100)
5.6 (2-10) (100)
1a
1b
1c
1d
1f
50.8 (29-88) (100)
25.3 (17-31) (100)
109.5 (60-149) (49)
123.5 (57-191) (49)
109.0 (42-168) (53)
101.0 (44-168) (70)
130.0 (25-181) (59)
105.3 (54-161) (58)
99.7 (51-154) (59)
Figure 3. UDCA analogues 1l-s.
1g
1h
1l
1n
1p
1q
a Compounds less active than UDCA are omitted. EC50 values for
translocation after 4 h of treatment were determined from six point
concentration-effect curves generated using nonlinear regression models
Table 1. EC50 values for GR translocation of UDCA and selected
modified bile acids. b Relative to Dex (100 nM).
Shortening the side chain but maintaining the acid func-
tionality resulted in compounds with equal efficacy to UDCA
but enhanced potency, norUDCA (1p) (EC50 of 105 μM) and
bisnorUDCA (1q) (EC50 of 99.7 μM). The UDCA alcohol
(1o) did not induce translocation, but conversion to the nitrile
(1l) enhanced potency (EC50 of 100 μM, 70%). The bisnorni-
trile (1n) (EC50 of 130 μM, 59%) showed similar efficacy and
potency to 1l although the nornitrile (1m) did not induce
translocation. UDCA methyl and benzyl esters (1r, 1s) did not
induce GR translocation.
Having shown that UDCA amides are moderately potent
inducers of GR translocation, we investigated whether similar
amide derivatives of DCA and CDCA could also induce
translocation of the GR. The cyclopropyl and N-benzylpiper-
azine derivatives of DCA (2) (3R, 12R OH) and UDCA’s
Figure 4. Analysis of GR translocation induced by UDCA deriva-
tives. SKGT-4 cells were treated with DMSO 1%, Dex 100 nM as a
positive control, UDCA 300 μM, or bile acid derivatives as indicated. All
bile acid derivatives were initially screened at 300 μM. Cells were fixed,
stained, and imaged as described, Original magnification, ꢀ10. GR
translocation was measured using the Investigator software package.
Analysis was based on the ratio of the intensity of the GR within the
nucleus and the intensity in the cytoplasm. Values are normalized to
nuclear to cytoplasmic ratio of Dex 100 nM and are expressed as the
mean ( SEM of two experiments performed in triplicate, * p < 0.05.
equal efficacy to UDCA with increased potency, 109.5, 123.5,
and 109.0 μM, respectively. Extending the spacer by one
carbon abolished activity (1i).

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 125
Figure 5. DCA and CDCA amides 2a-b, 3a-b.
Figure 6. DCA and CDCA amides 2a-b, 3a-b do not induce GR
translocation. SKGT-4 cells were (a) untreated or (b) treated with
Dex 100 nM as a positive control, (c) cyclopropyl amide derivatives
of UDCA, DCA, and CDCA (10 μM) (1-3a) or (d) benzylpiper-
idine derivatives of UDCA, DCA, and CDCA (10 μM) (1-3b) for 4
h and fixed, stained, and analyzed as described previously. Values
are normalized to nuclear to cytoplasmic ratio of Dex (100 nM) and
are expressed as the mean ( SEM of two experiments performed in
triplicate, * p < 0.05.
Figure 7. UDCA derivatives 1a,b,d show similar translocation
kinetics to Dex. SKGT-4 cells were treated with UDCA derivatives
100 μM or Dex at indicated concentration for 15, 30, 60, 120, or 240
min. Cells were fixed, stained, and analyzed for GR translocation as
described previously. Values are normalized to nuclear to cytoplas-
mic ratio of Dex 100 nM and are expressed as the mean of two
experiments performed in triplicate, p < 0.05 relative to DMSO
1%, for 1a (120, 240 min), 1b (60, 120, 240 min), 1d (120, 240 min),
Dex 100 pM (120, 240 min), Dex 100 nM (30, 60, 120, 240 min).
epimer CDCA (3) (3R, 7R OH) were prepared, Figure 5. The
DCA and CDCA amides (2a-b, 3a-b) did not induce
translocation at 10 μM (Figure 6). This indicates that the
orientation of the OH groups on the steroid nucleus exerts a
strong influence on the capacity of similar amides to induce GR
translocation and it is consistent with a range of observations
about the effects of UDCA compared with DCA and CDCA.
The latter BAs promote apoptosis, whereas UDCA is anti-
apoptotic, due in part to its ability to cause GR translocation.45
The differing biological effects of DCA and CDCA relative to
UDCA are attributed to the uninterrupted hydrophobic β-face
of the former pair, although it is unclear whether this affects a
macroscopic biophysical property or protein binding.
We examined the time course for GR translocation in the
SKGT-4 cell line following treatment with analogues 1a, 1b,
1d, and Dex to help characterize the kinetics of the effect.
Following treatment with the UDCA amides produced a
trend toward increased nuclear GR evident at 15 min; this
achieved significance in 60 min in the case of 1b and 120 min
for 1a and 1d. The time course of a ligand-dependent protein
event such as GR translocation is a function of the affinity of
the ligand for the protein and ultimately ligand concentration.
Therefore, Dex (100 nM) induced significant and maximum
translocation of the GR at 30 min consistent with reported
values, whereas at 100 pM, nuclear trafficking kinetics were
more leisurely, with ratios reaching significance at 120 min,
Figure 7. Overall, the time-course experiments indicated a
direct effect on GR distribution rather than one downstream
of protein synthesis. Furthermore, pretreatment of SKGT-4
cells with cycloheximide, an inhibitor of protein biosynthesis,
did not affect 1a induced GR translocation (Supporting Infor-
mation (SI)). Cycloheximide did not induce translocation of
the GR by itself. Hence 1a induced GR translocation occurs
Figure 8. UDCA derivatives induce co-activator recruitment. A
Lanthascreen TR-FRET GR coactivator assay was used to deter-
mine if the UDCA derivatives were capable of coactivator recruit-
ment. Dex and 1a were incubated at RT for 3 h with a GST-tagged
GR-LBD and a mixture of the fluorescein labeled coactivator
peptide and the terbium labeled anti-GST antibody. The TR-FRET
ratio was calculated by dividing the emission signal at 520 nm by the
emission signal at 495 nm. Values represent the mean ( SEM of
three experiments performed in duplicate, normalized to positive
control, Dex (1 μM).
independent of protein translation and in a similar time frame
to Dex, albeit at a lower rate.
Even though UDCA and its active amides do not satisfy the
classical GR pharmacophore, the most obvious explanation
for their activity and time-course was ligand binding at the
GR active site. However, selected active compounds (1a, 1p,
1m, 1h) did not displace radiolabeled Dex in competition
experiments at concentrations relevant to their ability to
translocate. UDCA (10-50 μM) was also unable to displace
Dex, consistent with previous studies.39,41

126 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Sharma et al.
Figure 9. (A) UDCA derivatives transactivate the GR. SKGT-4 cells were transiently transfected with a mixture of an inducible glucocorticoid
responsive firefly luciferase reporter and constitutively expressing Renilla construct (40:1). Cells were treated with increasing concentrations of
Dex or 1a for 16 h. Cells were then lysed and assayed for luciferase activity. Firefly luciferase activity was normalized to the internal vector
control, renilla luciferase, to control for transfection efficiency. (B) UDCA derivatives decrease NFκB activity. HEK-293 cells were transiently
transfected with an inducible NFκB responsive firefly luciferase reporter gene construct and a constitutively expressing β-galactosidase
construct. After 24 h, the cells were treated with TNF-R 10 ng/μL alone or with TNF-R 10 ng/μL and varying concentrations of Dex or 1a. After
16 h, the cells were lysed and the lysates assayed for firefly luciferase activity. Firefly luciferase activity was normalized to β-galactosidase
activity to control for transfection efficiency. Values are expressed as the mean ( SEM of three experiments performed in duplicate, normalized
to untreated control, * p < 0.05 relative to untreated control.
We next employed a time-resolved fluorescent energy
transfer assay (TR-FRET) to try to detect an effect by UDCA
or 1a on GR coactivator recruitment which might indicate
physical contact with the GR ensemble leading to conforma-
tional changes in the region of the LBD (Figure 8). 1a (300 μM)
increased the TR-FRET ratio to a significant extent, whereas
UDCA (300 μM) did not. This result for UDCA is consistent
with previous reports.41 The DCA and CDCA compound 2a
and 3a were unable to increase the TR-FRET ratio in the
coactivator recruitment assay (data not shown).
Having demonstrated that the cyclopropylamide derivative
(1a) was able to induce coactivator recruitment, our next step
was to investigate whether treatment with 1a could result in
GR activation. We found that 1a was able to induce GRE
transactivation in a concentration dependent manner, reach-
ing significance at 100 μM (Figure 9A). UDCA could only
induce transactivation at 500 μM, but this did not follow a
pharmacological trend (SI). The ability to attenuate NF-kB
signaling is regarded as pivotal to the therapeutic actions of
classical GCs. We therefore carried out a reporter assay to
determine if 1a could inhibit TNF-R induced NF-κB tran-
scriptional activity in a HEK-293 cell line. We first showed
that 1a caused translocation of the GR in this cell line with
similar potency and biochemical efficacy to the SKGT-4 cell
line. Compound 1a was capable of transrepression of NF-κB
activity in a concentration dependent manner, p < 0.05 at 50
μM, 100 μM (Figure 9B).
The GR and MR are known to have widely overlapping
ligand binding preferences and interrelated transcriptional
activities, indeed, TUDCA causes translocation of both.
However when MR expression was knocked down using
siRNA, 1a was still able to inhibit NF-κB transcriptional
activity, whereas in the GR knock down condition, this effect
was attenuated (SI). Furthermore, treatment with 1a (50 μM)
was not associated with nuclear translocation of the MR.
Therefore, taking together the evidence of translocation ex-
erted by 1a, its ability to induce GRE transactivation and its
GR dependent inhibition of NF-κB transcriptional activity,
an agonist mode of action involving the GR is most likely.
However, thepotentialinvolvement ofother nuclear receptors
merits further study.
UDCA has been shown to induce GR translocation in
multiple cell models41,42,48 at high concentrations (300 μM
range). The mechanism by which UDCA induces GR trans-
location remains unknown despite extensive efforts over the
past decade. Incubation of radiolabeled UDCA with the GR
binding site expressed in a GR fusion protein yielded no
specific binding of UDCA.39 UDCA did not displace tritiated
Dex from the GR binding site; furthermore, specific bind-
ing of tritiated UDCA to GR could not be detected in
CHOpMTGR cells.41 UDCA (10 and 50 μM) was unable to
displace tritiated Dex in our competition binding experiment.
Despite this, UDCA requires the GR-LBD to induce GR
nuclear translocation and for its subsequent biological
effects.41,45,49 Using a series of GR mutants, Miura et al
showed that UDCA influences a broader region of the LBD
than Dex. GR deletion mutants (1-765, 1-750, 1-740) could
not be translocated by Dex but were by UDCA.41 On the
other hand, GR mutant 1-730 was unresponsive to UDCA.
The specific carboxy terminal region of the LBD was identi-
fied as essential for translocation and UDCA’s cytoprotective
effect on TGFβ1 induced apoptosis.49 This mutant lacks
Tyr735 of the steroid binding pocket, which is necessary for
hydrophobic contact between ligands and the GR.50
The ability of UDCA to induce GR activation downstream
of translocation remains a matter of debate as studies are
widely conflicted over its ability to activate GREs.39,41,42,49
Previous investigators failed to show an interaction be-
tween UDCA and the coactivator transcription intermediary
factor-2.41 Other studies have shown that UDCA can have an
additive effect with GCs in inducing gene expression. For
example, UDCA increased Dex induced mRNA levels of
tyrosine aminotransferase (TAT), a hepatocyte-specific mar-
ker of GC action.51 Others have shown that combining
UDCA and Dex can increase the expression of the bicarbo-
nate carrier, AE2, altered expression of which is associated
with PBC. It has been suggested that certain genes contain
only half of a GRE site referred to as GRE cores. Binding of GR
to GRE core sites may require further assistance in addition to
the interaction with GCs which UDCA may provide.52
Similar to UDCA, there is not a defined mechanism for GR
translocation induced by its more active amides. The most

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 127
potent GR translocator 1a was unable to displace tritiated
Dex in a competition binding experiment at similar concen-
tration to its EC50 for translocation. Previous authors have
suggested that UDCA may interact with the cell membrane
and set up a series of cytoplasmic events through secondary
signals, one of which may be GR activation.41,45 Such
an explanation cannot be ruled out for the derivatives
although GR translocation by the amides displayed a similar
time profile to Dex. Furthermore 1a was able to induce
coactivator recruitment in a cell free system. Recruitment of
steroid receptor coactivator 1-4 (SRC1-4) by 1a indicates
that it can induce the necessary conformational change in the
GR-LBD. This recruitment was specific to the UDCA core
nucleus as the corresponding derivatives of DCA and CDCA
did not cause coactivator recruitment.
was performed on Merck Kieselgel 60 particle size 0.040-
0.063 mm. Thin layer chromatography (TLC) was performed on
silica gel Merck F-254 plates. Compounds were visually detected
by absorbance at 254 nm and/or vanillin staining. All test com-
pounds were g95% purity by HPLC.
24-Cyclopropyl-3r,7β-dihydroxy-5β-cholanamide (1a). 3R,7β-
Diformyloxy-5β-cholan-24-oic acid (1.0 g, 2 mmol) was dissolved
in thionyl chloride (5 mL) and refluxed at 90 °C for 2 h. After 1 h,
the reaction was cooled to room temperature and the thionyl
chloride was removed in vacuo to leave a sticky solid which was
the acid chloride. This was dissolved in dry DCM (10 mL) and
cyclopropylamine (131 μL, 1.9 mmol) and triethylamine (244 μL,
1.9 mmol) wereaddedon ice. The reactionmixturewas allowed to
warm to room temperature and stirred monitoring for disap-
pearance of the starting material by TLC. After 24 h, the solvent
was removed in vacuo. The residue was dissolved in ethyl acetate
(20 mL) and washed with hydrochloric acid (HCl) (3 ꢀ 20 mL)
for removal of unreacted amines, water (2 ꢀ 20 mL), and brine
solution (2 ꢀ 20 mL). The organic layer was dried and evapora-
tion of solvent gave the cyclopropylamide after chromatographic
elution (hexane:ethyl acetate 1:1). Sodium (0.1 g) was added to
HPLC grade methanol (MeOH) (10 mL) to form an excess
of sodium methoxide. The formyl cyclopropylamide (0.5 g,
1 mmol) was added to this solution and refluxed for 2 h. After
2 h, the reaction was cooled to room temperature and added to
water (100 mL). The amide was extracted with ethyl acetate. The
organic layer was then washed with water (3 ꢀ 20 mL) and
dried (MgSO4). The solvent was removed in vacuo and after
chromatographic elution with ethyl acetate afforded a white solid
(0.4 g, 92%). 1H NMR δ (CD3OD): 0.49 (2-H, m), 0.65 (3-H, s),
0.92 (3-H, d, J = 6.52 Hz), 0.97 (3-H, s), 2.75 (1-H, m), 3.61
(2-H, m), 5.32 (1-H, br). 13C NMRppm (CD3OD): 8.4, 12.4, 18.8,
22.4, 24.0, 25.2, 25.5, 28.0, 29.8, 31.1, 35.3, 36.5, 36.9, 38.1, 38.8,
40.9, 41.7, 44.1, 44.6, 44.9, 56.5, 57.8, 71.1, 175.2. HRMS: found
(M - Na)þ = 454.3297. Melting point 124 °C
Conclusion
We have discovered amido derivatives of UDCA that
causes GR translocation in the low micromolar range in
SKGT-4 cells. These compounds do no bind at the classical
GC binding locus, but they affect coactivator recruitment and
cause transactivation at high concentration. The most potent
compound decreased NF-κB activity in a HEK-293 line with
moderate potency.
Taken together, the data suggests that UDCA amides bind
to the LBD but at a different site to the classical pocket,
binding to which accounts for the actions of established GR
modulators, including dissociated steroids which separate
transactivation from transrepression.53-56 UDCA has well
established but poorly understood effects on cellular function
that appear to be mediated at least in part by the GR. The
amido analogues reported herein amplify this activity by
about 100-fold. They hold potential in therapeutic applica-
tions and in exploring ongoing puzzles associated with the
actions of UDCA.
24-N-Benzylpiperazine-3r,7β-dihydroxy-5β-cholanamide (1b).
1H NMR δ (CD3OD): 0.70 (3-H, s, 18-CH3), 0.95 (6-H, m),
3.41-3.60 (8-H, m), 3.52 (2-H, m). 13C NMR ppm (CD3OD):
12.9, 19.0, 22.5, 24.0, 28.0, 29.8, 31.1, 32.2, 32.5, 35.1, 36.1, 36.8,
38.1, 38.9, 39.2, 40.9, 41.7, 44.2, 44.7, 44.9, 45.3, 56.6, 57.8, 71.1,
80.3, 130.2, 175.5. HRMS: found (M - Na)þ = 573.4097. Melting
point 116 °C.
Experimental Section
Chemistry. Uncorrected melting points were obtained using a
Stuart melting point SMP11 melting point apparatus. IR spec-
tra were obtained using a Perkin-Elmer 205 FT Infrared Para-
gon 1000 spectrometer. Band positions are given in cm-1. Solid
samples were obtained by KBr disk; oils were analyzed as neat
films on NaCl plates. 1H and 13C spectra were recorded at 27 °C
on a Bruker Advance II 600 MHz spectrometer (600.13 MHz
1H, 150.91 MHz 13C) and Bruker DPX 400 MHz FT NMR
spectrometer (400.13 MHz 1H, 100.16 MHz 13C) in either
CDCl3 or CD3OD (tetramethylsilane as internal standard).
For CDCl3, 1H NMR spectra were assigned relative to the
TMS peak at 0.00 δ and 13C NMR spectra were assigned relative
to the middle CDCl3 triplet at 77.00 ppm. For CD3OD, 1H and
13C NMR spectra were assigned relative to the center peaks of
the CD3OD multiplets at 3.30 δ and 49.00 ppm, respectively.
High-resolution mass spectrometry (HRMS) was performed on
a Micromass mass spectrophotometer (EI mode) at the Depart-
ment of Chemistry, Trinity College. High-performance liquid
chromatography (HPLC) was performed on a reversed phase
250 mm ꢀ 4.6 mm Waters Spherisorb ODS-2, 5 μm column
using a Waters Alliance 2695 chromatograph equipped with an
autosampler, column oven, and dual wavelength detector. The
flow rate was 1 mL/min, with a mobile phase consisting of 40%
phosphate buffer pH 2.5 and 60% acetonitrile at time 0 and
grading to 85% acetonitrile at 4 min. Injection volume was
20 μL and areas determined at 254 nm. The isocratic HPLC
method was aqueous phosphate buffer solution pH 2.5 40% and
acetonitrile 60%. Flow rate was 1 mL/min. Flash chromatography
Biochemistry. Chemicals. Dex, UDCA, and all reagents for
chemical synthesis were obtained from Sigma-Aldrich Chemical
Co. (St. Louis, MO, USA). Lipofectamine was purchased from
Invitrogen (Carlsbad, CA, USA). TNF-R was purchased from
R&D Systems and reconstituted with sterile PBS at 100 μg/mL.
Cycloheximide (Calbiochem, Merck Chemicals Ltd. Notting-
ham, UK) was reconstituted with ethanol at a concentration
of 20 mg/mL. Escherichia coli lipopolysaccharide was obtained
from Alexis Biochemicals (San Diego, CA, USA).
Dex was dissolved in DMSO to give a 10 mM stock solution,
and all other bile acids and bile acid derivatives were maintained
as 200 mM stock solutions in DMSO. The compounds were
diluted to the required concentrations with medium with 1%
DMSO in order to maintain solubility.
A Smartpool of predesigned siRNA oligos (ON-TARGET
plus siRNA reagents) targeting the glucocorticoid receptor
(siGR), mineralocorticoid receptor (siMR), scrambled control
siRNA (siScr), and siRNA transfection reagent were purchased
from Dharmacon (Lafayette, CO, USA).
Cell Culture. The SKGT4 cell line, derived from a well-
differentiated adenocarcinoma arising in Barrett’s epithelium
of the distal esophagus, was generously provided by Dr. David
Schrump (Bethesda, MA).57 Cells were maintained in RPMI
1640 medium supplemented with 10% fetal bovine serum and
4 mM L-glutamine (GIBCO-BRL, Grand Island, NY) HEK-
293 cells, an adherent human embryonic kidney cell line, were
grown in Dulbecco’s Modified Eagle’s Medium (DMEM, GIB-
CO, Invitrogen Ltd., Paisley, UK) supplemented with 10%

128 Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1
Sharma et al.
heat-inactivated FBS. Cultures were maintained at 37 °C in a
humidified atmosphere containing 5% CO2.
S-transferase (GST), a terbium-labeled anti-GST antibod,y and
a fluorescein labeled SRC1-4 peptide. Binding of agonist to the
nuclear receptor causes a conformational change in the LBD of
the GR, which results in recruitment of the coactivator peptide.
When the terbium label on the anti-GST antibody is excited at
340 nm, energy is transferred to the fluorescein label on the
coactivator peptide and detected as emission at 520 nm. The
GR-LBD was added to the indicated concentration of Dex, 1a,
2a, and 3a in a 384-well black assay plates (Corning BV Life
Sciences, Fogostraat, Amsterdam, The Netherlands) followed
by addition of a mixture of the fluorescein labeled coactivator
peptide and the terbium labeled anti-GST antibody. The plates
were incubated at room temperature for 3 h and then measured
for TR-FRET activity using the LanthaScreen optic filter
module on the BMG pherastar (Imgen Technologies, VA,
USA). The TR-FRET ratio was calculated by dividing the
emission signal at 520 nm by the emission signal at 495 nm.
Values represent the mean ( SEM of three experiments
performed in duplicate, normalized to positive control,
Dex 1 μM.
GR Translocation Assay. Cells were plated into 96-well plates
at a concentration of 6 ꢀ 104 cells/mL (100 μL volume). Cells
were serum starved for 12 h prior to bile acid treatment. After 48
h, cells were treated with bile acid derivatives in supplement free
medium at the required concentration for the indicated time
points. Control wells were left untreated or treated with 1%
DMSO as a vehicle control or Dex 100 nM as positive control.
After the appropriate treatment time, cells were fixed with 4%
paraformaldehyde/phosphate buffered saline (PBS) then per-
meabilized with 0.1% (v/v) Triton X-100/PBS followed by
blocking with 3% bovine serum albumin in PBS. Cells were
incubated with purified mouse antiglucocorticoid receptor
antibody (BD Transduction Laboratories) then incubated
with AlexaFluor-488-conjugated secondary antibody (Invi-
trogen, Carlsbad, CA, USA). Images were acquired using the
GE IN cell analyzer 1000, original magnification ꢀ10. Four
fields of view per well were acquired using a 10ꢀ objective,
with up to 2000 cells imaged per treatment group, for n=3
experiments.
NF-KB Assay. HEK-293 cells were transiently cotransfected
with an inducible NF-κB responsive construct, which contains
3 κB elements upstream of a minimal conalbumin promoter
linked to the firefly luciferase gene58 and a constitutively ex-
pressing β-galactosidase construct (Clontech, Saint-Germain-
en-Laye, France) as an internal control to monitor for transfec-
tion efficiency (ratio of 4:1). A reverse transfection procedure
was carried out using lipofectamine as a transfection reagent at a
ratio of 3:2 (lipofectamine:DNA). DNA and lipofectamine were
diluted in OptiMem, and this mixture was added to 48-well
plates to give 500 ng of DNA per well. HEK-293 cells at a density
of 7 ꢀ 105 cells/mL (200 μL) were added to this mixture. After
24 h, the cells were treated with TNF-R 10 ng/μL alone or with
TNF-R 10 ng/μL and varying concentrations of Dex or CPA
(1a) for 16 h. After 16 h, the cells were lysed with Promega lysis
buffer (50 μL) and the lysates assayed for firefly luciferase
activity (Promega Corporation, Madison, WI, USA). The ly-
sates were then assayed for β-galactosidase activity using o-
nitrophenyl-β-D-galactopyranoside as a substrate following
methods described by Sambrook et al.59 Values are expressed
as the mean ( SEM of three experiments performed in dupli-
cate, normalized to untreated control, * p < 0.05 relative to
untreated control.
Radioligand Binding Assay. Human HeLa S3 cells enriched
with GRs were used in HEPES/RPMI-1640 buffer pH 7.2. Cells
(3 ꢀ 106) were incubated with 3 nM [3H] Dex for 120 min at
25 °C. Nonspecific binding was estimated in the presence of Dex
(10 μM). Cells were filtered and washed, and the filters were then
counted to determine [3H] Dex specifically bound. UDCA
derivatives were screened at 10 μM and UDCA at 10 and 50 μM.
Statistical Analysis. Statistical comparison between groups
was carried out using one way ANOVA with Dunnett’s posthoc
correction. Data are graphically represented as the mean (
standard error of the mean (SEM). All data were analyzed using
GraphPad Prism5.
Quantification of GR Translocation Using High Content
Analysis. The GE IN cell analyzer 1000 is a microscope based
screening platform capable of large scale objective analysis of
fluorescently labeled cells using automated image acquisition,
data management, and multiparametric analysis. SKGT-4s
were stained for GR as outlined above. Nuclear to cytoplasmic
ratio was measured using the Investigator software package (GE
Healthcare, Piscataway, NJ, USA). The analysis was based
on the ratio of the intensities of GR within the nucleus and
cytoplasm. The multitarget analysis algorithm was optimized to
detect GR within the nucleus and the cytoplasm using vehicle
only treated cells as negative control and Dex treated cells as
positive control. Hoechst staining enabled identification of the
nucleus. The nuclear to cytoplasmic ratios were determined by
calculating (nuclear Intensity of GR - background intensity)/
(cytoplasmic intensity of GR - background intensity). Values
represent the mean ( SEM of three experiments performed in
triplicate. EC50 values were estimated from concentration-
effect curves generated using nonlinear regression models in
GraphPad Prism5.
GRE Assay. SKGT-4 cells were transiently transfected with a
mixture of an inducible glucocorticoid responsive firefly lucifer-
ase reporter and constitutively expressing renilla construct
(40:1) (SABiosciences Corporation, Executive Way, Frederick,
MD, USA). A reverse transfection procedure was optimized
using fugene HD as a transfection reagent at a ratio of 4:1
(Fugene HD:DNA). DNA and Fugene HD were diluted in
OptiMem, and this mixture was added to 96-well plates to give
100 ng DNA per well. SKGT-4 cells at a density of 7 ꢀ 104 cells/mL
(100 μL) were added to this mixture. After 24 h, the cells were
treated with varying concentrations of Dex or 1a for 16 h. After
16 h, the cells were lysed using passive lysis buffer 50 μL
(Promega Corporation, Madison, WI, USA) and incubated at
room temperature for 15 min. The lysates were then assayed for
firefly and renilla luciferase activity (Promega Dual-Luciferase
Reporter Assay System) using a Victor Perkin-Elmer lumin-
ometer. Values represent the mean ( SEM of three experiments
performed in duplicate, normalized to Dex 100 nM as positive
control.
Acknowledgment. We thank Dr. John O’Brien and Dr.
Manuel Reuther for NMR spectroscopy, Dr. Martin Feeney
and Dr. Dilip Rai for HRMS data, Dr. Sinead Smith
for assistance with plasmids, and Ray Keaveney for HPLC
work. This work was supported by the Health Research
Board [TRA/2007/11, Esophageal Cancer-Toward New
Therapies] and the Embark Initiative (IRCSET Award to
Sharma, 2006).
Time-Resolved Fluorescence Resonance Energy Transfer Assay.
TR-FRET is based on the principle that when a suitable pair
of fluorophores is brought within close proximity of one
another, excitation of the first fluorophore results in energy
transfer to the second fluorophore. This can be detected by an
increase in the fluorescence emission of the acceptor molecule
and a decrease in the fluorescence emission of the donor
molecule. We used a Lanthascreen TR-FRET glucocorticoid
receptor coactivator assay kit (Invitrogen, Carlsbad, CA, USA),
which provides a human GR-LBD tagged with a glutathione-
Supporting Information Available: Characterization and pur-
ity data, biochemistry figures. This material is available free of
charge via the Internet at http://pubs.acs.org.

Article
Journal of Medicinal Chemistry, 2011, Vol. 54, No. 1 129
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