Avicholic acid: A lead compound from birds on the route to potent TGR5 modulators

Article abstract of DOI:10.1021/ml200256d

Grounding on our former 3D QSAR studies, a knowledge-based screen of natural bile acids from diverse animal species has led to the identification of avicholic acid as a selective but weak TGR5 agonist. Chemical modifications of this compound resulted in the disclosure of 6α-ethyl-16-epi-avicholic acid that shows enhanced potency at TGR5 and FXR receptors. The synthesis, biological appraisals, and structure-activity relationships of this series of compounds are herein described. Moreover, a thorough physicochemical characterization of 6α-ethyl-16-epi-avicholic acid as compared to naturally occurring bile acids is reported and discussed.

Full text of DOI:10.1021/ml200256d

Letter
pubs.acs.org/acsmedchemlett
Avicholic Acid: A Lead Compound from Birds on the Route to Potent
TGR5 Modulators
Roberto Pellicciari,*,† Antimo Gioiello,† Paola Sabbatini,† Francesco Venturoni,† Roberto Nuti,†
Carolina Colliva,‡ Giovanni Rizzo,§ Luciano Adorini,§ Mark Pruzanski,§ Aldo Roda,‡
and Antonio Macchiarulo†
†Dipartimento di Chimica e Tecnologia del Farmaco, Universita
̀
di Perugia, Via del Liceo 1, 06123 Perugia, Italy
‡Dipartimento di Scienze Farmaceutiche, Alma Mater Studiorum, Universita
§Intercept Pharmaceuticals, New York, New York 10013, United States
̀
di Bologna, Via Belmeloro 6, 40126 Bologna, Italy
S
* Supporting Information
ABSTRACT: Grounding on our former 3D QSAR studies, a knowledge-
based screen of natural bile acids from diverse animal species has led to the
identification of avicholic acid as a selective but weak TGR5 agonist. Chemical
modifications of this compound resulted in the disclosure of 6α-ethyl-16-epi-
avicholic acid that shows enhanced potency at TGR5 and FXR receptors. The
synthesis, biological appraisals, and structure−activity relationships of this
series of compounds are herein described. Moreover, a thorough
physicochemical characterization of 6α-ethyl-16-epi-avicholic acid as compared to naturally occurring bile acids is reported
and discussed.
KEYWORDS: bile acid, TGR5, avicholic acid, 16-epi-avicholic acid, FXR, structure−activity relationship, CMC
ile acid (BA)-activated receptors are attractive targets for
drug discovery efforts.1−5 The chief members of this
agonist in vivo.6,7 Via TGR5 activation, INT-777 (3) is able to
stimulate type 2 iodothyronine deiodinase (D2) activity in
brown adipose tissue (BAT) and muscle, as well as induce the
release of glucagon-like protein 1 (GLP-1) in enteroendocrine
cells. While these results have provided the first proof-of-
concept that modulating TGR5 may represent a novel viable
strategy for the treatment of metabolic disorders such as type 2
diabetes,7 further aspects of TGR5 activation still remain poorly
understood. These include, in particular, the inhibition of
hepatic inflammatory responses,8,9 the protection of liver
against the production of reactive oxygen species in sinusoidal
endothelial cells,10 and, more recently, the release of nitric
oxide in enteric neurons leading to the control of intestinal
motility.11
B
family, namely, farnesoid X receptor (FXR) and TGR5, have
been implicated in a number of liver and metabolic diseases,
including cholestasis, nonalcoholic steatohepatitis (NASH),
obesity, and type II diabetes, to name a few. In the framework
of the development of ligands for BA-activated receptors,
starting from cholic acid (CA, 1) as a lead compound, we have
recently reported the design, synthesis, and pharmacological
effects of INT-777 (3, Figure 1) as a potent and selective TGR5
As a continuation of our efforts aimed at providing potent
and selective chemical tools with tailored pharmacokinetic
properties to probe the functions of BA activated receptors in
different tissues,6,12−17 we have been devising further chemical
modifications of the BA side chain and nucleus. To this end, we
hypothesized that the diverse BA biosynthetic pathways across
animal species could be a potential source of novel lead
compounds. Starting from our previous results of three-
dimensional quantitative structure−activity relationship studies
(3D QSAR) that evidenced how different regions around the
BA scaffold affect TGR5 activity (Figure 2),18 we thus
investigated naturally occurring BAs from 677 vertebrate
Received: October 26, 2011
Accepted: February 6, 2012
Published: February 6, 2012
Figure 1. Natural and semisynthetic BA derivatives.
© 2012 American Chemical Society
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ACS Medicinal Chemistry Letters
Letter
Breslow's remote functionalization was achieved by photol-
ysis of PhICl2 in the presence of methyl 3α-acetoxy-7α-m-
t
iodobenzoyloxy-5β-cholan-24-ate 10a,b in 0.3 mM BuOH/
CH2Cl2 solution to yield the 17-Cl derivative 11a,b, in nearly
quantitative yield. The corresponding C16−17 olefinic inter-
mediate 12a,b was obtained by refluxing 11a,b in pyridine (81
and 72%). Hydroboration-oxidation of 12a,b by borane−THF
complex and alkaline hydrogen peroxide, followed by basic
hydrolysis (NaOH/MeOH), furnished the tetrol 13a,b in
moderate yield (55 and 47%, respectively). Selective oxidation
of the C24 position was achieved by using (2,2,6,6-
tetramethylpiperidin-1-yl)oxyl (TEMPO) in CH2Cl2/H2O
and Aliquat as a phase transfer catalyst to afford the lactone
14a,b in 37 and 30% yield, respectively. Alkali hydrolysis
(NaOH/MeOH) followed by a reverse phase RP-18
chromatography afforded avicholic acid sodium salt (4) and
6α-ethyl-avicholic acid sodium salt (6) in 10 and 6% overall
yield, respectively.
Figure 2. 3D QSAR scheme of TGR5 agonists.18
species19 to identify those that could be endowed with a
suitable scaffold in terms of potential activity.
Our attention, in particular, was attracted by C24 BAs bearing
a hydroxy group at the C16α-position, as determined in the
biliary composition of birds and snakes. Polar groups at this
position were indeed suggested by the QSAR model as favoring
interaction with TGR5 (Figure 2).18 Accordingly, in this work,
we report the synthesis and preliminary evaluation of
3α,7α,16α-trihydroxy-5β-cholan-24-oic acid (avicholic acid,
4), a natural BA first isolated from avian species (Shoebill
stork and herons) in 2002,20 and its derivatives 5−7 as TGR5
ligands (Figure 1).
The corresponding C16-epimers 5 and 7 were prepared
according to Scheme 2.22 Thus, the C16−17 olefinic BA
Scheme 2. Synthesis of 16-epi-Avicholic Acid Derivatives 5
a
and 7
Avicholic acid derivatives 4 and 6 were prepared according to
the synthetic approach reported previously by Mukhopadhyay
(Scheme 1).21 Thus, CDCA (2) or 6-ECDCA (8)12,17 was
a
Scheme 1. Synthesis of Avicholic Acid Derivatives 4 and 6
a
Reagents and conditions: (i) (1) BH3·THF; (2) NaOH, H2O2; (3)
K2CO3, MeOH. (ii) (1) Jones reagent; (2) MeOH, pTSA. (iii) (1) t-
BuNH2BH3, CH2Cl2, reflux; (2) NaOH, MeOH.
derivative 12a,b was submitted to hydroboration-oxidation
reaction using the previously reported protocol and selectively
deprotected at C3 position by using a solution of K2CO3 in
MeOH at room temperature. The 7α-m-iodobenzoyloxy
derivative 15a,b, obtained in 56 and 50% yield, respectively,
was reacted with Jones reagent and esterified at the terminal
carboxylic group by treatment with pTSA in MeOH at room
temperature to furnish 16a,b, in nearly quantitative yield.
Finally, reduction of the keto groups with tert-butylamine−
borane complex in CH2Cl2 at reflux, followed by hydrolysis
with NaOH in MeOH and purification by medium pressure
liquid chromatography, afforded the desired 3α,7α,16β-
trihydroxy-5β-cholan-24-oic acid sodium salt (5) and
3α,7α,16β-trihydroxy-6α-ethyl-5β-cholan-24-oic acid sodium
salt (7) in 15 and 11% overall yield, respectively. Compounds
4−7 were unstable in the free acid form, spontaneously reacting
to give the corresponding lactone derivatives. Therefore, the
compounds were prepared and tested as sodium salts.
a
Reagents and conditions: (i) (1) MeOH, pTSA; (2) Ac2O, NaHCO3,
THF. (ii) 3-I-benzoylchloride, CaH2, BnEt3N+Cl−. (iii) PhICl2, t-
BuOH 0.3 M, hυ. (iv) Py, reflux. (v) (1) BH3·THF; (2) NaOH, H2O2;
(3) KOH, MeOH. (vi) NCS, TEMPO, Aliquat, CH2Cl2/H2O. (vii)
NaOH, MeOH.
treated with p-toluensulfonic acid (pTSA) and MeOH at room
temperature to get the corresponding methyl ester analogues,
which were selectively protected at the C3 position by reaction
with acetic anhydride in the presence of NaHCO3 in refluxing
THF (quantitative yield). The 3-acetoxy intermediate 9a,b thus
formed was functionalized at the C7 position with a 3-iodo-
benzoyl moiety by reaction with 3-iodo-benzoylchloride in the
presence of CaH2 and BnEt3N+Cl− in toluene to afford 10a,b in
good yield (80 and 74%, respectively).
Avicholic acid and its derivatives 4−7 were evaluated, along
with CDCA (2) and LCA as reference controls, for their ability
to activate TGR5 and FXR using homogeneous time-resolved
fluorescence (cell-based) assay and ALPHAscreen assay,
respectively (Table 1). The results indicate that avicholic acid
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ACS Medicinal Chemistry Letters
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Table 1. TGR5 and FXR Activities of Natural and
Semisynthetic BAs
Table 2. Physicochemical Properties of BAs
a6,15
a
b
c
Ws
CMC
STCMC
Log
d
albumin
b
c
BA
(μM)
(mM)
(dyn/cm)
PA
binding (%)
TGR5
FXR
CA (1)6
273
32
9.2
3.0
2.0
48
46
50
1.1
2.2
1.4
50
93
62
d
d
BA
EC50
efficacy
EC50
>100
13.0 0.7
>100
>100
efficacy
CDCA (2)
CA (1)
13.6 0.7
6.7 0.3
101
105
166
194
148
198
120
0
62
INT-777
(3)
99
CDCA (2)
INT-777 (3)
0.82 0.04
18
4
7
188
120
10
50
53
1.1
1.6
58
83
4
5
6
7
160
25
8
1
4
0
5.9
55
22
3
132
121
210
a
Ws: water solubility as protonated species in 0.1 M HCl water
b
87
1
solution. CMC: critical micellar concentration determined in 0.15 M
NaCl water solution. STCMC: surface tension at CMC in 0.15 M NaCl
water solution. Log PA (octanol/water partition coefficient of the
c
0.65 0.03
11.5 0.6
d
a
Data represent mean values
SDs of at least three independent
b
ionized species): lipophilicity.
experiments. Units are μM for EC50 and % of 10 μM LCA (EC50: 5
μM) value for efficacy. Units are μM for EC50 % of 10 μM CDCA (2)
value for efficacy. Efficacy values are here used as a measure of
c
d
The detergent power of BA, that is, the tendency to form
micelles, is the main concern for this detergent-like molecule.24
The critical micellar concentration (CMC) and the surface
tension at CMC (STCMC) values are indeed correlated with
potential (cyto)toxicity of BAs. Molecules with high CMC and
STCMC values are poor detergents and therefore poorly toxic to
biological membranes. Furthermore, the lipophilicity (Log PA)
describes the passive diffusion of BAs across biological
membranes, whereas the albumin binding reflects the amount
of compound that is protein-bound in the blood. The CMC of
7 is higher than CDCA (2) and 3, while being lower than CA
(1) and avicholic acid (4). This relatively high CMC value
combines with a high STCMC value, suggesting a low micellar
aggregation number and poor detergency for 7 that may favor
low toxicity.
fluorescence change.
(4) is a weak TGR5 agonist, with no activity at FXR.
Interestingly, its C16-epimer 5 shows an increased potency by 1
order of magnitude toward TGR5 accompanied by FXR
activity, suggesting the preference of the hydroxyl group to
form hydrogen bond interactions when placed on the
equatorial position of the steroidal nucleus. This finding is
also in agreement with the results of the QSAR model,18
indicating that the equatorial position of C16 as favored for
polar interactions with TGR5 (Figure 3).
Among trihydroxy BAs, compound 7 shows the highest
lipophilicity value (Log PA). This is likely ascribed to the
presence of the ethyl moiety in the C6α position that combines
with the shift of the hydroxyl group from C12α of INT-777 (3)
to C16β of 7. Interestingly, the shift of the hydroxyl from C12α
of CA (1) to C16α of 4 does not play a role in affecting
lipophilicity, as evidenced by the same Log PA values reported
in Table 2. The percent albumin binding of 7 is higher than CA
(1), INT-777 (3), and avicholic acid (4). Again, this may be
due to the presence of the 6α-ethyl moiety and the C16β
hydroxyl group. Interestingly, the value of albumin binding of 7
is compatible with a relatively fast hepatic uptake, similar to
naturally occurring BAs such as CA (1).25 The physicochemical
properties of compound 7, including poor detergency and
adequate hydrophilicity, are in agreement with previous
structure−property relationship studies carried out on a large
number of natural and synthetic BAs.26 The 16β orientation of
the hydroxyl group, in particular, confers to the molecule a low
detergency that, combined with the relatively high lipophilicity
due to the presence of the 6α ethyl group, may further facilitate
the intestinal absorption by passive mechanism.
Compound 7 is fully conjugated by the mouse liver with
taurine (unpublished data), in contrast with INT-777 (3) that
is only partially conjugated with taurine after the C23-methyl
isomerization. Akin to naturally occurring taurine-conjugated
bile salts,27 the taurine-conjugated compound 7 may be
additionally absorbed by the terminal ileum with an active
mechanism. This would likely increase its bioavailability and
modify its pharmacokinetics and biodistribution in comparison
with INT-777 (3).
Figure 3. Alignment of 6α-ethyl-avicholic acid (6, panel a) and 6α-
ethyl-16-epi-avicholic acid (7, panel b) according to the 3D QSAR
scheme. The hydroxyl group of C16 nicely fits the polar region of
interaction when located on the equatorial position (β) of the steroidal
nucleus rather than the axial (α) position.
The 6α-ethyl derivatives 6 and 7 were synthesized to assess
whether the insertion of this alkyl group at the 6α position of
the parent derivatives 4 and 5 would have improved the
potency toward TGR5 and/or FXR, according to previously
reported SARs.6,15 As shown in Table 1, in the case of 6α-ethyl-
16-epi-avicholic acid (7), the introduction of the alkyl group
markedly increased the potency at TGR5 by about 2 orders of
magnitude, while showing a slight improvement of FXR
activity. In the case of 6α-ethyl-avicholic acid (6), however,
this effect is partially reversed, with enhanced agonistic activity
toward FXR rather than TGR5.
Table 2 reports the physicochemical data of 6α-ethyl-16-epi-
avicholic acid (7) as compared to INT-777 (3) and natural
BAs, including CA (1), CDCA (2), and avicholic acid (4). All
of the studied BA presents the same acidity constant with a pKa
= 5 like all natural occurring BA,23 and the hydroxyl group on
the C16 position does not affect the C24 carboxylic ionization. In
addition, it was found that the water solubility (Ws) of 6α-
ethyl-16-epi-avicholic acid (7) as a protonated acid is slightly
higher than the trihydroxy BA INT-777 (3) and lower than the
parent compound 4.
In conclusion, this work suggests that natural BAs can further
provide additional lead compounds on the route to unravel
physiological and therapeutic implications of TGR5 modu-
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ACS Medicinal Chemistry Letters
Letter
(9) Wang, Y. D.; Chen, W. D.; Yu, D.; Forman, B. M.; Huang, W.
The G-protein coupled bile acid receptor Gpbar1 (TGR5) negatively
regulates hepatic inflammatory response through antagonizing Nuclear
Factor kappaB. Hepatology 2011, DOI: 10.1002/hep.24525.
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Selbach, O.; Haussinger, D.; Kubitz, R. The G-protein coupled bile salt
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Kirkland, J. G.; Pelayo, J. C.; Bunnett, N. W.; Corvera, C. U.
Expression and function of the bile acid receptor GpBAR1 (TGR5) in
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lation. Among these, herein, we have reported avicholic acid
(4) as a selective TGR5 modulator, although showing weak
potency. Chemical manipulations of 4 have led to the discovery
of 6α-ethyl-16-epi-avicholic acid (7) as a potent TGR5 agonist
and moderately active FXR ligand, endowed with an interesting
physicochemical profile. Further modifications of both the
steroidal nucleus and side chain of 7 are currently being
investigated with the aim of bestowing TGR5 selectivity over
FXR and additional potency to this compound.
ASSOCIATED CONTENT
* Supporting Information
■
S
(12) Pellicciari, R.; Fiorucci, S.; Camaioni, E.; Clerici, C.; Costantino,
G.; Maloney, P. R.; Morelli, A.; Parks, D. J.; Willson, T. M. 6alpha-
ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR
agonist endowed with anticholestatic activity. J. Med. Chem. 2002, 45,
3569−3572.
Description of the synthetic procedures, biological methods,
and analytical procedures of all target compounds. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(13) Pellicciari, R.; Costantino, G.; Camaioni, E.; Sadeghpour, B. M.;
Entrena, A.; Willson, T. M.; Fiorucci, S.; Clerici, C.; Gioiello, A. Bile
acid derivatives as ligands of the farnesoid X receptor. Synthesis,
evaluation, and structure-activity relationship of a series of body and
side chain modified analogues of chenodeoxycholic acid. J. Med. Chem.
2004, 47, 4559−4569.
(14) Pellicciari, R.; Gioiello, A.; Costantino, G.; Sadeghpour, B. M.;
Rizzo, G.; Meyer, U.; Parks, D. J.; Entrena-Gaudix, A.; Fiorucci, S.
Back door modulation of the Farnesoid X Receptor: design, synthesis,
and biological evaluation of a series of side chain modified
chenodeoxycholic acid derivatives. J. Med. Chem. 2006, 49, 4208−
4215.
AUTHOR INFORMATION
Corresponding Author
*Tel: +39 075 5855120. Fax: +39 075 5855124. E-mail: rp@
unipg.it.
■
Funding
This work was supported by Intercept Pharmaceuticals (New
York).
Notes
The authors declare no competing financial interest.
(15) Pellicciari, R.; Sato, H.; Gioiello, A.; Costantino, G.;
Macchiarulo, A.; Sadeghpour, B. M.; Giorgi, G.; Schoonjans, K.;
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ACKNOWLEDGMENTS
We thank Erregierre (Bergamo, Italy) for the gift of BAs as
starting material.
■
ABBREVIATIONS
BA, bile acid; FXR, farnesoid X receptor; SAR, structure−
activity relationship
■
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