Nonacidic Farnesoid X Receptor Modulators

Article abstract of DOI:10.1021/acs.jmedchem.7b00903

As a cellular bile acid sensor, farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glucose homeostasis, and liver protection. Clinical results have validated FXR as therapeutic target in hepatic and metabolic diseases. To date, potent FXR agonists share a negatively ionizable function that might compromise their pharmacokinetic distribution and behavior. Here we report the development and characterization of a high-affinity FXR modulator not comprising an acidic residue.

Full text of DOI:10.1021/acs.jmedchem.7b00903

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Brief Article
Non-acidic farnesoid X receptor modulators
Daniel Flesch, Sun-Yee Cheung, Jurema Schmidt, Matthias Gabler, Pascal Heitel, Jan S Kramer, Astrid
Kaiser, Markus Hartmann, Mara Lindner, Kerstin Lüddens-Dämgen, Jan Heering, Christina Lamers,
Hartmut Lűddens, Mario Wurglics, Ewgenij Proschak, Manfred Schubert-Zsilavecz, and Daniel Merk
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00903 • Publication Date (Web): 27 Jul 2017
Downloaded from http://pubs.acs.org on July 27, 2017
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Journal of Medicinal Chemistry
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Non-acidic farnesoid X receptor modulators
Daniel Flesch1‡, Sun-Yee Cheung1‡, Jurema Schmidt1‡, Matthias Gabler1, Pascal Heitel1, Jan Kra-
mer1, Astrid Kaiser1, Markus Hartmann1, Mara Lindner3, Kerstin Lüddens-Dämgen2, Jan Heer-
ing3, Christina Lamers1, Hartmut Lüddens2, Mario Wurglics1, Ewgenij Proschak1, Manfred Schu-
bert-Zsilavecz1, Daniel Merk1*
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1 Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am
Main, Germany
2 Department of Psychiatry and Psychotherapy, University Medical Center Mainz, D-55131 Mainz, Germany
3 Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group Translational Medicine and
Pharmacology TMP, Theodor-Stern-Kai 7, D-60596 Frankfurt am Main, Germany.
KEYWORDS: nuclear receptors, bile acid receptors, non-alcoholic steatohepatitis, NASH, metabolic syndrome
ABSTRACT: As a cellular bile acid sensor farnesoid X receptor (FXR) participates in regulation of bile acid, lipid and glu-
cose homeostasis as well as liver protection. Clinical results have validated FXR as therapeutic target in hepatic and meta-
bolic diseases. To date, potent FXR agonists share a negatively ionizable function that might compromise their pharma-
cokinetic distribution and behavior. Here we report the development and characterization of a high-affinity FXR modula-
tor not comprising an acidic residue.
The nuclear farnesoid X receptor (FXR) acts as ligand-
activated transcription factor sensing bile acids and regu-
lating their synthesis, metabolism, secretion and reup-
take.1–3 Moreover, the receptor is involved in glucose and
lipid metabolism and bile acids are increasingly being
considered as signaling molecules of whole body energy
homeostasis.3 Therefore, pharmacological FXR modula-
tion by agonistic, antagonistic or gene-selective ligands
appears to possess extraordinary therapeutic value. FXR
agonists are intensively evaluated in clinical trials for non-
alcoholic steatohepatitis (NASH) with the steroidal can-
didate obeticholic acid (1a) that was recently approved for
primary biliary cholangitis leading the pipeline.4 Its clini-
cal development has revealed anti-diabetic and weight-
lowering effects4,5 which led to initiation of a variety of
trials with 1a for metabolic diseases. In addition, preclini-
cal data also point to a versatile role of FXR in inflamma-
tion and possible future indications in this field.6–9
non-acidic bile alcohols have been identified as FXR ago-
nists but few demonstrate low micromolar potency.20,21 An
exception is 1c with 0.15 µM potency but its urethane
moiety seems unfavorable for further development.22
3
(EC50 = 0.3 µM)23 constitutes the most potent non-
steroidal, non-acidic FXR agonist thus far.
Chart 1: FXR ligands: Endogenous FXR agonist CDCA
(1) and semi-synthetic derivatives 1a-1c. Non-
steroidal FXR agonists 2a-d and partial agonist 3.
Lead structure 4 and minimal lead pharmacophore 6.
Medicinal chemistry approaches have yielded a variety
of synthetic FXR ligands (Chart 1) to exploit the receptor’s
pharmacological potential.10,11 Derived from the physiolog-
ical agonist chenodeoxycholic acid (1, CDCA, EC50 = 8
µM), highly potent steroidal FXR agonists such as 1a and
1b have been developed.12,13 In addition, there are non-
steroidal FXR agonists such as 2a-d and 3. The isoxazole
GW4064 (2a)14 is widely used as reference FXR agonist.
However, several synthetic FXR agonists are biased by
toxicity, poor solubility or insufficient selectivity.15–19
To date, potent non-steroidal FXR agonists constitute
carboxylic acids or contain an ester moiety that is rapidly
hydrolyzed in vivo. Amongst steroidal FXR ligands, some
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Page 2 of 12
The wide presence of a carboxylic acid moiety amongst were added to dehydrate the intermediate N-formamides
1
2
3
4
5
6
7
8
FXR ligands might in part explain their limited specificity
since acidic compounds tend to promiscuously interact
with various fatty acid binding targets, particularly nucle-
ar receptors and G-protein coupled receptors. In addition,
carboxylic acids are susceptible to rapid metabolic con-
version to glucuronides which in turn may hold toxicity
potential. Glucuronides and reactive thioester acyl-CoA
intermediates formed in the glucuronidation process
constitute highly reactive metabolites prone to unspecific
reactions and considerable liver toxicity.24 A non-acidic
FXR activator would avoid these potential reasons for
unselective toxicity and, therefore, we intended to devel-
op a non-steroidal FXR modulator without acidic moiety.
to isocyanides (II) in 4 hours under reflux. After for-
mation of the isocyanide, 2-aminopyridine and aldehyde
as well as methanol and acetic acid were added and the
mixture was stirred for 48 hours to generate the desired
imidazo[1,2-a]pyridines in sufficient yields (7-29%).
Imidazo[1,2-a]pyridines 6-10 & 12-58 and imidazo[1,2-
a]pyrazine 11 (Table 1) were characterized in a full-length
FXR transactivation assay in HeLa cells with an inducible
firefly luciferase under control of the FXR response ele-
ment from bile salt export protein (BSEP). The receptors
hFXR, hRXR as well as a renilla luciferase for normaliza-
tion and toxicity control were constitutively expressed.
Maximum relative FXR activation of the tested com-
pounds refers to the transactivation of 2a at 3 µM defined
as 100%. Assay validation with various reference agonists
yielded results in good agreement with literature29.
9
10
11
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13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
We engaged on 4 as lead structure which was identified
as FXR modulator with an EC50 value of 480 nM in a com-
putational approach25. Preliminary structure activity rela-
tionship (SAR) data25 suggested that replacement of the
lipophilic benzodioxole moiety in 4 by a simple benzene
ring in 5 was possible. Moreover, the substitution pattern
at the pyridine core structure had remarkable impact on
activity. Based on these considerations, we prepared the
unsubstituted benzyl derivative 6, which revealed an EC50
value of 4.0±0.8 µM for FXR activation and was selected
as lead pharmacophore for a systematic SAR study.
As first step of our SAR studies we investigated the sub-
stitution pattern of the imidazopyridine core. As reported
by Achenbach et al.25, methylation in 7-position (5) had
little influence on the activity. In contrast, an additional
methyl group in 6-position (7) improved potency marked-
ly 8-fold whereas methylation in 5-position (8) caused
inactivity. To exploit potential optimization by substitu-
ents in 7-position, we introduced a lipophilic ethyl resi-
due (9) and a polar carboxylic acid moiety (10) but nei-
ther modification was accompanied by a significant im-
provement in potency. Also an additional nitrogen atom
in 7-position of the ring system (11) was not tolerated and
only the 6-position seemed to offer potential for further
optimization. Hence, we studied various residues in this
position (12-17). Amongst halogens (12-15), only chlorine
(13) improved activity, while a 6-methoxy substituent (16)
slightly diminished potency and a 6-phenyl substituent
(17) produced a moderate improvement.
Scheme 1: Preparation of of imidazo[1,2-a]pyridines
6-10 & 12-58 and imidazo[1,2-a]pyrazine 11 in a multi-
component Groebke-Blackburn reaction
R1
R2
N
R1
R2
NH2
X
X
R4
(a)
NC
N
N
R4
O
R5
61a-c
II
NH
R5
R3
60a-ah
R3
59a-l
6-58
R5 NH
(c)
(b)
Using 7 as lead, we then studied the SAR of the benzyl-
amino residue in 3-position. Side chain elongation up to a
butyl spacer (18-20) was tolerated but only phenylpropyl
derivative 19 displayed a 2-fold improvement and even
replacing the benzylamino moiety of 7 by a benzyl residue
(21) had little impact on potency. Methylation in benzylic
position of 7 leading to enantiomers (R)-22 and (S)-22
diminished activity 6-fold and 2-fold, respectively, con-
sistent with a preference for the latter stereoisomer. A 4-
methyl (23) and a 4-methoxy moiety (24) on the substitu-
ent`s aromatic ring slightly promoted FXR agonistic activ-
ity suggesting further optimization may be possible with
other suitable substituents of the aryl moiety.
H
R5 NH2
O
62a-d
I
(a) MeOH, AcOH, rt, 17-48 h (b) ethyl formate, neat, reflux,
3 h (c) CH2Cl2, PPh3, CCl4, NEt3, reflux, 4 h.
Imidazo[1,2-a]pyridines 6-10 & 12-58 and imidazo[1,2-
a]pyrazine 11 were prepared in a Groebke-Blackburn
three-component reaction26,27 (Scheme 1). To study the
structure-activity relationships (SAR) of the compound
class on FXR, we introduced a broad range of structural
variations in all parts of the template by changing either
2-aminopyridine (59a-l), aldehyde (60a-ah) or isocyanide
(61a-c) building block of the multi-component reaction.
The reaction was handled in a one-pot procedure in dry
methanol under acidic catalysis. Due to limited commer-
cial availability of the isocyanide component, we also
employed a method for in situ isocyanide generation from
the respective amines (62a-d) and imidazo[1,2-a]pyridine
formation in one pot.28 In this procedure, primary amines
62a-d were refluxed in ethyl formate for 3 hours to form
N-formamides as intermediates (I). Subsequently, the
mixture was diluted with methylene chloride and tri-
phenylphosphine, tetrachloromethane and triethylamine
Next, we addressed the SAR of the thiophene ring in 2-
position of the imidazopyridine scaffold. Thiophene (7)
replacement by phenyl (25) was accompanied by a slight
increase in potency whereas the saturated cyclohexyl
derivative 26 was inactive underlining the necessity of an
aromatic residue in this position. Methylation of the new-
ly introduced phenyl residue was tolerated in 2-position
(27) and slightly enhanced potency in 3- (28) and 4-
position (29) suggesting the possibility of further optimi-
zation with suitable substituents especially in 4-position.
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Journal of Medicinal Chemistry
Table 1: Structure-activity relationship of imidazo[1,2-a]pyridines 4-10 & 12-58 and imidazo[1,2-a]pyrazine 11 on
FXR (maximum relative activation corresponds to the activity of 2a at 3 µM which was defined as 100%).
1
2
3
4
5
6
7
8
9
hFXR
(mean ± SEM; n ≥ 3)
#
4
X
C
R1
R2
R3
-H
R4
R5
benzo[d][1.3]dioxol-
5-ylmethylamino
-NHCH2Ph
EC50 [µM]
0.4825
max. rel. act. [%]
16 ± 125
-H
-CH3
thiophen-2-yl
5
6
C
C
C
C
C
C
N
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
-CH3
-H
-H
-H
-Et
-COOH
-
-H
-H
-H
-H
-H
-CH3
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
thiophen-2-yl
-Ph
16.2 ± 2.4% @ 30 µM25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
-NHCH2Ph
4.0 ± 0.8
14 ± 1
15 ± 1
7
-CH3
-H
-NHCH2Ph
0.54 ± 0.08
8
-NHCH2Ph
inactive
9
-H
-NHCH2Ph
1.2 ± 0.3
16 ± 1
10
11
-H
-NHCH2Ph
inactive
inactive
inactive
-H
-NHCH2Ph
12
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-F
-NHCH2Ph
13
-Cl
-NHCH2Ph
0.16 ± 0.01
0.50 ± 0.04
0.46 ± 0.01
0.83 ± 0.15
0.20 ± 0.06
1.0 ± 0.2
26 ± 1
14 ± 1
14 ± 1
18 ± 1
17 ± 1
12 ± 1
18 ± 1
17 ± 1
12 ± 1
14 ± 1
12 ± 1
14 ± 1
14 ± 1
16 ± 1
14
-Br
-NHCH2Ph
15
-CF3
-OMe
-Ph
-NHCH2Ph
16
-NHCH2Ph
17
-NHCH2Ph
18
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-NH(CH2)2Ph
-NH(CH2)3Ph
-NH(CH2)4Ph
-CH2Ph
19
0.23 ± 0.09
0.75 ± 0.17
0.81 ± 0.13
3.2 ± 0.5
20
21
(R)-22
(S)-22
23
24
25
26
27
28
29
30
31
(R)-NHCH(CH3)Ph
(S)-NHCH(CH3)Ph
-NHCH2(4-Me-Ph)
-NHCH2(4-MeO-Ph)
-NHCH2Ph
1.3 ± 0.3
0.22 ± 0.03
0.17 ± 0.05
0.28 ± 0.01
cyclohexyl
-NHCH2Ph
inactive
inactive
-(2-Me-Ph)
-NHCH2Ph
0.26 ± 0.02
0.18 ± 0.05
0.13 ± 0.03
0.081 ± 0.011
0.21 ± 0.01
35 ± 1
14 ± 1
14 ± 1
17 ± 1
18 ± 1
-(3-Me-Ph)
-NHCH2Ph
-(4-Me-Ph)
-NHCH2Ph
-(4-Ph-Ph)
-NHCH2Ph
2-naphthyl
-NHCH2Ph
32
33
34
35
36
37
-CH2Ph
-NHCH2Ph
-(CH2)2Ph
-NHCH2Ph
0.31 ± 0.06
1.16 ± 0.44
0.25 ± 0.05
1.27 ± 0.18
0.91 ± 0.17
12 ± 1
13 ± 1
16 ± 1
16 ± 1
16 ± 1
-CH=CH-Ph
-(3-MeO-Ph)
-(4-MeO-Ph)
-(3,4-(MeO)2-Ph)
2,3-dihydro-1,4-
benzodioxan-6-yl
-(4-Cl-Ph)
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
38
C
-H
-CH3
-H
-NHCH2Ph
0.17 ± 0.04
15 ± 1
39
40
41
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-CH3
-Cl
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-H
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2Ph
-NHCH2(4-MeO-Ph)
0.24 ± 0.04
0.17 ± 0.02
14 ± 1
21 ± 1
14 ± 1
20 ± 1
-(4-F-Ph)
-(4-Br-Ph)
0.43 ± 0.05
0.016 ± 0.009
42
43
44
45
46
47
48
49
50
51
-(3-Cl-Ph)
-(2-Cl-Ph)
inactive
-(3,4-Cl2-Ph)
-(3-Cl-4-F-Ph)
furan-2-yl
0.024 ± 0.003
0.026 ± 0.002
0.56 ± 0.05
0.83 ± 0.14
1.9 ± 0.5
19 ± 1
54 ± 1
17 ± 1
17 ± 1
16 ± 1
17 ± 1
19 ± 1
15 ± 1
17 ± 1
19 ± 1
18 ± 1
31 ± 1
27 ± 1
46 ± 1
23 ± 1
thiophen-3-yl
furan-3-yl
N-Me-2-pyrrol-2-yl
4-Me-1,3-thiazol-2-yl
4-pyridyl
1.1 ± 0.3
0.22 ± 0.03
2.0 ± 0.2
52
53
54
55
56
57
58
3-pyridyl
0.52 ± 0.10
0.020 ± 0.007
1.2 ± 0.3
2-pyridyl
2-pyrazinyl
6-Cl-2-pyridyl
4-Cl-2-pyridyl
5-Cl-2-pyridyl
4-Cl-2-pyridyl
0.080 ± 0.001
0.0015 ± 0.0007
0.0074 ± 0.0008
0.00030 ± 0.00001
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Accordingly, 4-biphenyl derivative 30 was significantly substituent had discovered the 4-chloro-2-pyridyl residue
1
2
3
4
5
6
7
8
more potent. In contrast, a 2-naphthyl moiety (31) mim-
icking 3,4-disubstitution failed to increase the activity.
Replacement of the phenyl residue (25) by a benzyl sub-
stituent (32) was not tolerated while further elongation to
a phenethyl moiety (33) retained the potency. The less
flexible cinnamyl substituent in 34 was not favored.
(56) as highly optimized building block. These structural
variations were combined in one molecule 58 as exten-
sively optimized and highly potent non-acidic FXR modu-
lator. 58 activated FXR with a subnanomolar EC50 value
(0.30 ± 0.01 nM) but with lower efficacy than reference
FXR agonists such as 1, 1a and 2a (Supporting Figure 1 for
comparison). Isothermal titration calorimetry (ITC, Sup-
porting Figure 2) revealed a Kd value of 37 nM for FXR
binding which is predominantly driven by entropic con-
tributions (ΔS = 98 J/mol·K) with a moderate enthalpic
share (ΔH = -13 J/mol) equivalent to one hydrogen bond.
The discrepancy between cellular EC50 and Kd is best
explained by the absence of coactivators in the ITC meas-
urement that can affect the binding equilibrium.
This preliminary SAR evaluation of the 2-substituent
revealed a phenyl residue (25) as very favorable and indi-
cated optimization potential in 3- and 4-substituents of
this ring. Thus, we studied the substitution pattern in
more detail. In contrast to 3- and 4-methyl residues (28,
29), methoxy groups (35, 36, 37) failed to enhance potency
and 4-methoxy derivative 36 as well as 3,4-dimethoxy
derivative 37 displayed markedly diminished activity. This
loss of potency was not observed for dihydrobenzodioxine
38 which was slightly more active than 25. Introduction of
chlorine in 4-position (39) was tolerated and a smaller 4-
fluorine substituent (40) was slightly more favored while
the larger 4-bromine residue (41) reduced activity. A 3-
chlorine substituent (42) remarkably enhanced potency to
a low two-digit nanomolar EC50 value while 2-chloro-
derivative 43 was inactive and 3,4-dichlorophenyl (44) as
well as 3-chloro-4-fluorophenyl derivative (45) revealed
about equal potency as 42 but no further improvement.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Finally, we studied heterocycles as imidazopyridine-2-
substituents. The 2-thienyl residue of 7 could be replaced
by a 2-furyl moiety (46) without loss of potency. In con-
trast, 3-thienyl (47) 3-furyl (48) as well as N-methyl-2-
pyrryl derivative 49 exhibited lower activity. Introduction
of a nitrogen in thiazole 50 enhanced the potency com-
pared to 7 roughly 2-fold. Pyridine residues generated
much more pronounced effects. 4-Pyridine derivative 51
significantly lost potency while 3-pyridine 52 was slightly
less potent than 25. Introduction of a 2-pyridyl residue
(53) robustly increased the activity more than 10-fold but
this effect was lost in 2-pyrazine derivative 54.
Figure 1. Molecular docking of 58 into the FXR-LBD (PDB
code: 3OKI) indicates a single H-bond between imidaz-
opyridin-N1 and Tyr373 of the FXR-LBD and binding seems
particularly mediated by lipophilic contacts. An intramolecu-
lar H-bond between pyridine and amine moiety stabilizes an
optimal conformation of 58 for binding to FXR.
In summary, we identified several distinct structural
modifications that each remarkably increased potency of
the imidazopyridine derivatives to intermediate nanomo-
lar EC50 values and, potentially, could be combined in one
optimized compound. In particular, a 3-chlorophenyl
residue (42) and a 2-pyridyl substituent (53) caused strong
improvements in potency. Both modifications were com-
patible for combination but resulted in two possible iso-
mers due to the rotatable bond between the imidaz-
opyridine scaffold and its 2-substituent. Consequently, we
combined the structural elements of 42 and 53 in 6-
chloropyridyl derivative 55 and 4-chloropyridyl isomer 56.
5-Chloropyridyl derivative 57 was characterized as well.
55-57 significantly differed in potency on FXR with 4-
chloro-2-pyridyl derivative 56 as most potent isomer.
Molecular docking (Figure 1) of 58 into the FXR ligand
binding domain (LBD) revealed only one H-bond be-
tween imidazopyridin-N1 and the hydroxyl group of
Tyr373 which agrees with the ITC data. Compared to the
binding mode of 1 forming an H-bond towards Tyr373, 58
lacks polar contacts towards Tyr365 and His451, that were
shown to be crucial for helix 12 stabilization30, thus ex-
plaining the lower activation efficacy of 58 (Supporting
Figure 3). Additionally, the side chain amino residue of 58
participates in an intramolecular H-bond with the lone
pair of the 2-pyridyl moiety in 2-position. This intramo-
lecular H-bond was confirmed by QM-based energy min-
imization (Supporting Figure 4). In contrast, QM-based
energy minimization of 24 only differing from 58 by the 2-
thiophen-2-yl residue revealed an interaction between
sulfur and the lone pair of the imidazopyridine core as
described by Beno et al.31, that might weaken the H-bond
towards Tyr373 and explain the lower potency of 24.
As a final optimization step, we combined our SAR
knowledge on the three building blocks of the imidaz-
opyridine scaffold. As 5-substituent, chlorine (13) had
turned out superior to a methyl residue while in 3-
position, the 4-methoxybenzylamino moiety (24) was
favored and finally, extensive SAR studies on the 2-
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Preliminary ADME characterization of 58 revealed a fa-
(BSEP), small heterodimer partner (SHP), organic solute
transporter α (OSTα) and PPARα while indirect FXR tar-
get genes cholesterol 7α hydroxylase (CYP7A1) and sterol-
regulatory element binding protein 1c (SREBP1c) were
repressed confirming FXR activation. Compared to refer-
ence agonists 1, 1a and 2a, 58 tends to have a lower effica-
cy in target gene modulation. In agreement with the re-
sults of the reporter gene assay relying on the FXR re-
sponse element from the BSEP promoter, especially BSEP
and OSTα were less induced by 58 than by 1, 1a and 2a.
Still, the gene expression profile of 58 resembles that of
reference FXR agonists and revealed no gene selectivity.
1
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4
5
6
7
8
vorable logD7.4 of 1.69 (1: 0.53; 1a: 0.81; 2a: 1.01) and an
aqueous solubility of 63.7 ng/ml. A WST-1 (water soluble
tetrazolium) assay in HepG2 cells showed no cytotoxic
effect up to 100 µM. In contrast, acidic FXR agonists 1a
and 2a exhibited toxicity at 50 µM (Supporting Figure 5).
Metabolism studies in HepG2 cells indicated acceptable
stability of 58 with a half-life of approx. 1.5-2.0 h and 12%
remaining compound after 24 h (Supporting Figure 6).
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Figure 3. 58 at a concentration of 10 nM induced the
expression of FXR target genes BSEP, SHP, OSTα and
PPARα and repressed indirect target genes CYP7A1 and
SREBP1c (n = 3; * p < 0.05; ** p < 0.01; *** p < 0.001).
Figure 2. Selectivity screening of 58 on 14 nuclear recep-
tors and membrane bile acid receptor TGR5 revealed no
agonistic or antagonistic activity at 1 µM (n = 3).
As cellular bile acid sensor, FXR accommodates bipolar
ligands with an acidic functionality and a lipophilic back-
bone similar to the common structure of its natural bile
acid ligands.33 Accordingly, synthetic FXR ligands share
this characteristic containing a negative charge at physio-
logical pH and typically forming a neutralizing contact
between their acidic function and Arg335 of FXR. Howev-
er, acidic compounds often encounter ADME issues such
as high plasma protein binding and metabolic formation
of toxic intermediates.34 Here we show that high affinity
binding to and activation of FXR is also feasible with non-
acidic compounds which might be superior for some the-
rapeutic demands and help to reach body-compartments
not accessible to acidic compounds. This might enable
development of CNS-bioavailable FXR modulators to
study the receptors role for example in multiple sclerosis.9
To evaluate selectivity amongst related receptors, 58
was assayed for agonistic and antagonistic activity on
membrane bile acid receptor TGR5 as well as peroxisome
proliferator-activated receptors (PPAR), liver X receptors
(LXR), retinoid X receptors (RXR), retinoic acid receptors
(RAR), vitamin D receptor (VDR), pregnane X receptor
(PXR) and constitutive androstane receptor (CAR). 58
proved highly selective amongst nuclear receptors and
TGR5 with no agonistic or antagonistic activity at 1 µM
corresponding to at least 3000-fold selectivity (Figure 2).
For structural similarity of 6-58 with γ-aminobutyric ac-
id (GABA) receptor agonist zolpidem (Supporting Figure
7) possessing the same imidazo[1,2-a]pyridine scaffold,
cross-reactivity had to be considered. However, zolpidem
neither activated nor competitively antagonized FXR in
our full length FXR reporter gene assay up to 10 µM and
53 up to 10 µM did not bind to GABAA in a radio ligand
binding assay. Thus, replacement of the dimethyl amide
in zolpidem by the benzylamine residue in 6-58 seems to
prevent cross-reactivity on GABAA receptors.
Starting from the moderately potent lead compound 6,
we systematically studied the structure-activity relation-
ship of all parts of the molecular scaffold and identified
specific positions for optimization. Remarkable improve-
ment in potency was achieved by exploiting a potential
intramolecular H-bond through introduction of 2-pyridyl
substituents and a concluding re-combination of all favo-
rable modifications. The resulting non-acidic FXR modu-
lator 58 possesses sub-nanomolar potency and extraordi-
nary selectivity for FXR over nuclear receptors and the
membrane bile acid receptor TGR5. Moreover, the lack of
an acidic moiety can also help to preserve specificity over
To study FXR modulation by 58 in a less artificial set-
ting, we incubated hepatocarcinoma cells (HepG2) with
58 and quantified FXR target gene expression on mRNA
level by qRT-PCR (Figure 3).29,32 58 in 10 nM concentration
induced direct FXR target genes bile salt export protein
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the wide variety of other targets of fatty acid mimetics.34
Corresponding Author
Page 6 of 12
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6
58 robustly modulates FXR target gene expression at a
concentration as low as 10 nM in HepG2 cells with lower
efficacy compared to FXR agonists 1, 1a and 2a. Thus, with
its low toxicity and favorable ADME properties, 58 seems
well suitable for further development.
* P: +49 69 79829327 E: merk@pharmchem.uni-frankfurt.de
Author Contributions
‡These authors contributed equally.
Funding Sources
DF and PH gratefully acknowledge financial support by the
Else-Kroener-Fresenius-Foundation in the graduate school
‘Translational Research Innovation - Pharma (TRIP)’. This
research was supported by Deutsche Forschungsge-
meinschaft (DFG; Sachbeihilfe PR 1405/2-2; Heisenberg-
Professur PR 1405/4-1; SFB 1039 Teilprojekt A07).
Notes
7
8
9
Experimental
Chemistry. General. All target compounds had a purity
of ≥ 95% determined by elemental analysis.
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6-Chloro-2-(4-chloropyridin-2-yl)-N-(4-
methoxybenzyl)imidazo[1,2-a]pyridin-3-amine (58): 4-
Methoxybenzylamine (0.55 g, 4.0 mmol, 1.0 eq) was re-
fluxed in ethyl formate (355 mg, 4.4 mmol, 1.1 eq) for 3 h.
The volatile reaction products were evaporated, CH2Cl2
(10 mL), PPh3 (1.26 g, 4.8 mmol, 1.2 eq), NEt3 (405 mg, 4.0
mmol, 1.0 eq) and CCl4 (615 mg, 4.0 mmol, 1.0 eq) were
added and the mixture was refluxed for 4 h. After cooling
to r.t., methanol (abs., 10 mL), 2-amino-4-chloropyridine
(0.26 g, 2.0 mmol, 0.5 eq), 4-chloropyridine-2-carbalde-
hyde (0.28 g, 2.0 mmol, 0.5 eq) and glacial acetic acid (360
mg, 6.0 mmol, 1.5 eq) were added and the mixture was
stirred at r.t. for 48 h. Solvents were evaporated and the
crude product was purified by column chromatography
(hexane/ethyl acetate = 10:1 to 1:1).28 Yield: 234 mg; 29%.
The authors declare no competing interests.
ACKNOWLEDGMENT
The FXR-LBD protein was a generous gift from Dr. Krister
Bamberg, AstraZeneca R&D, Mölndal, Sweden.
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BSEP, bile salt export protein; CAR, constitutive an-
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RAR, retinoic acid receptor; RXR, retinoid X receptor;
SHP, small heterodimer partner; SREBP, sterol-regulatory
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ASSOCIATED CONTENT
Supporting Information. General procedures, preparation
and analytical data of compounds 6-57, methods for in vitro
characterization and supporting figures. This material is
available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
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figure 2
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Journal of Medicinal Chemistry
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figure 3
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Journal of Medicinal Chemistry
Page 12 of 12
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Table of Contents graphic
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