Second-Generation Dual FXR/sEH Modulators with Optimized Pharmacokinetics

Article abstract of DOI:10.1021/acs.jmedchem.1c00831

Non-alcoholic steatohepatitis (NASH) presents as an epidemic chronic liver disease that is closely associated with metabolic disorders and involves hepatic steatosis, inflammation, and fibrosis as key factors. Despite the enormous global prevalence of NASH, effective pharmacological interventions are lacking. Based on the hypothesis that the multifactorial condition NASH may benefit from combined multiple modes of action for enhanced therapeutic efficacy, we have previously developed dual FXR activators/sEH inhibitors (FXRa/sEHi) and observed remarkable antifibrotic effects upon their use in rodent NASH models. However, these first-generation FXRa/sEHi were characterized by moderate metabolic stability and short in vivo half-life. Aiming to overcome these pharmacokinetic drawbacks, we have systematically studied the structure-activity and structure-stability relationships of the chemotype and obtained second-generation FXRa/sEHi with improved pharmacokinetic parameters. With high plasma exposure, a half-life greater than 5 h, and similar dual potency on the intended targets, 13 presents as a substantially optimized FXRa/sEHi for late-stage preclinical development.

Full text of DOI:10.1021/acs.jmedchem.1c00831

pubs.acs.org/jmc
Article
Second-Generation Dual FXR/sEH Modulators with Optimized
Pharmacokinetics
Moritz Helmstädter, Astrid Kaiser, Steffen Brunst, Jurema Schmidt, Riccardo Ronchetti, Lilia Weizel,
Ewgenij Proschak, and Daniel Merk*
Cite This: J. Med. Chem. 2021, 64, 9525−9536
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Non-alcoholic steatohepatitis (NASH) presents as
an epidemic chronic liver disease that is closely associated with
metabolic disorders and involves hepatic steatosis, inflammation,
and fibrosis as key factors. Despite the enormous global prevalence
of NASH, effective pharmacological interventions are lacking.
Based on the hypothesis that the multifactorial condition NASH
may benefit from combined multiple modes of action for enhanced
therapeutic efficacy, we have previously developed dual FXR
activators/sEH inhibitors (FXRa/sEHi) and observed remarkable
antifibrotic effects upon their use in rodent NASH models.
However, these first-generation FXRa/sEHi were characterized by
moderate metabolic stability and short in vivo half-life. Aiming to
overcome these pharmacokinetic drawbacks, we have systemati-
cally studied the structure−activity and structure−stability relationships of the chemotype and obtained second-generation FXRa/
sEHi with improved pharmacokinetic parameters. With high plasma exposure, a half-life greater than 5 h, and similar dual potency
on the intended targets, 13 presents as a substantially optimized FXRa/sEHi for late-stage preclinical development.
Chart 1. FXR agonists OCA (1) and GW4064 (2), Dual
FXR Agonist/sEH Inhibitor 3 and Its Metabolites 3a−c
INTRODUCTION
■
Despite enormous global efforts to develop effective
pharmacological treatments for non-alcoholic steatohepatitis
(NASH), there is still no drug available to counter this severe
chronic liver disorder.1 NASH, which mainly arises from
overnutrition and unhealthy lifestyle, is characterized by a triad
of hepatic pathologies, namely steatosis, inflammation, and
fibrosis in liver.2−5 As hepatic manifestation of the metabolic
syndrome, the complex of non-alcoholic fatty liver disease
(NAFLD)/NASH has an alarming global prevalence and can
progress to liver cirrhosis and hepatocellular carcinoma as
potentially lethal consequences.1,4,5 Effective therapeutic
interventions for this pathology are, hence, urgently needed.
There are a number of drugs in late-stage clinical development
to treat NASH1 with the farnesoid X receptor (FXR) agonist
obeticholic acid6 (OCA, 1) as a frontrunner (Chart 1) despite
recent drawbacks.7 In addition, a number of descendants of the
widely used FXR agonist GW4064 (2)8 follow in earlier stages
of clinical trials.1 Although FXR agonists hadpromising efficacy
in phase 2 trials,9,10 however, no approval has been granted yet.
As outlined above, NASH is a highly multifactorial disease.
Designed polypharmacology11 has therefore been proposed as
a potentially superior approach to treat NASH. By
simultaneously addressing multiple therapeutic modes of
action, designed multitarget agents may achieve additive or
even synergistic efficacy. In an attempt to establish such a
Received: May 7, 2021
Published: June 24, 2021
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536
9525

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
concept for experimental NASH treatment, we have previously
developed a potent and balanced dual FXR agonist and soluble
epoxide hydrolase (sEH) inhibitor (FXRa/sEHi).12
Scheme 1. Synthesis of 4−9
These two modes of action were chosen for the
polypharmacological approach based on preclinical and clinical
observations on their individual therapeutic potential in NASH
and their complementary effects. FXR is a liver-protective
transcription factor, and its activation has been shown to
exhibit pronounced antisteatosic and some anti-inflammatory
effects.9,13−15 Inhibition of the sEH appeared very suitable to
complement these therapeutic effects of FXR activation for its
well-documented anti-inflammatory,16−18 antifibrotic,17−19 and
antiapoptotic20 properties. The sEH is a downstream enzyme
of the cytochrome P450 (CYP) epoxygenase pathway and
degrades epoxyeicosatrienoic acids (EET) to the correspond-
ing diols.21 Inhibition of the sEH leading to increased EET
levels proved beneficial for the treatment of various
inflammatory diseases and has shown efficacy in rodent
models of NASH.16,19,22−26 Indeed, our dual FXRa/sEHi 312
has demonstrated promising anti-NASH effects in three
preclinical models of the disease as preventive27 and curative28
intervention. Therein, 3 especially revealed pronounced
antifibrotic activities,27,28 which is particularly attractive since
fibrosis is considered a major prognostic factor of NASH
progression.4,5
a
Reagents and conditions: (a) 4-tert-butylbenzoic acid (33), EDC·
HCl, 4-DMAP, CHCl3, 50 °C, 16 h, yield: 16−66%; and (b)
tetrahydrofuran (THF), pyridine, 0−66 °C, 2−16 h, yield: 15−60%.
a
Scheme 2. Synthesis of 11−24 and 26−29
The FXRa/sEHi 3 was obtained by rational design of a
minimal dual pharmacophore and subsequent structural
optimization.12 It comprises a designed dual activity profile
with high potency on FXR (pEC50 7.7) and sEH (pIC50 8.4)
but has pharmacokinetic liabilities due to moderate metabolic
stability resulting in a half-life below 1 h. By LC-MS analysis,
we found that metabolic conversion of 3 mainly leads to the
metabolites 3a−c via sulfonamide hydrolysis and hydroxylation
(Chart 1).12 Since the efficacy of 3 in various rodent models of
NASH and other fibrotic diseases has confirmed great
therapeutic potential of dual FXR/sEH modulation, we
aimed to overcome the pharmacokinetic limitations of 3 and
generate a second-generation derivative with improved
metabolic stability while retaining dual potency. To achieve
this objective, we have systematically elucidated the structure−
activity and structure−stability relationships of the FXRa/sEHi
chemotype and focused on incorporating modifications with
the potential to stabilize or replace the metabolic soft spots. In
particular, we probed metabolic stabilization of the sulfona-
mide and the oxidation-sensitive tert-butyl moiety with
sterically demanding electron-withdrawing and bioisosteric
groups. This challenging multi-objective optimization yielded a
second-generation FXRa/sEHi with substantially improved
pharmacokinetics as intended.
a
Reagents and conditions: (a) EDC·HCl, 4-DMAP, CHCl3, 50 °C, 16
h, yield: 10−87%; and (b) THF, mesyl chloride (73), pyridine, reflux,
2−16 h, yield: 5−69%.
a
Scheme 3. Synthesis of Aniline Precursors 30−32
RESULTS AND DISCUSSION
■
Chemistry. Second-generation derivatives 4−29 of the
FXRa/sEHi chemotype 3 were prepared according to Schemes
1−8. Benzylamines 30−32 and benzoic acid derivatives 33−51
served as building blocks for the synthesis of benzamides 52−
72 using EDC·HCl and 4-DMAP as coupling agents for amide
bond formation. Benzamides 52−72 were then reacted with
sulfonyl chlorides 73−77 in the presence of pyridine to yield
second-generation derivatives 4−9 (Scheme 1), 11−24, and
26−29 (Scheme 2).
a
Reagents and conditions: (a) LiAlH4, THF, rt, 18 h, yield: 60−75%.
cially available, and 45−47 were synthesized in a palladium-
catalyzed hydroxycarbonylation reaction adapted from Wu et
al.29 using aryl bromides 81−83, Pd(OAc)2, XantPhos, EDC·
HCl, triethylamine, and formic acid. Benzoic acid derivative 48
was accessible via hydrolysis of benzonitrile 84 using KOH
(Scheme 4).
The required anilines 30−32 were obtained by reduction of
commercially available benzonitriles 78−80 with LiAlH4
(Scheme 3). Benzoic acid precursors 33−44 were commer-
9526
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
a
Scheme 4. Synthesis of Benzoic Acid Precursors 45−48
Scheme 7. Synthesis of 10
a
Reagents and conditions: (a) Pd(OAc)2, XantPhos, EDC·HCl, NEt3,
formic acid, DMF, 50 °C, 20 h, yield: 48−79%; and (b) KOH, H2O,
EtOH, reflux, 48 h, yield: 36%.
Oxidation of 4-isopropylbenzoic acid (85) using KMnO4 in
the presence of pyridine yielded benzoic acid derivative 49,
which subsequently served as a precursor for the synthesis of
benzoic acid derivative 50 using H2SO4/MeOH and LiOH/
H2O/MeOH in a two-step procedure (Scheme 5).
a
Reagents and conditions: (a) di-tert-butyl dicarbonate, THF, 60 °C,
yield: 83%; (b) NBS, AIBN, CHCl3, reflux, 4 h, yield: 77%; (c) NaN3,
DMF, 80 °C, 16 h; (d) PPh3, H2O, THF, rt, 12 h, yield: 24%; (e)
EDC·HCl, 4-DMAP, CHCl3, 50 °C, 16 h; (f) trifluoroacetic acid,
methylene chloride, yield: 49%; and (g) THF, mesyl chloride (73),
pyridine, reflux, 16 h, yield: 14%.
a
Scheme 5. Synthesis of Benzoic Acid Precursors 49 and 50
derivative 95, which was then esterified to 4-(3-acetoxyoxetan-
3-yl)benzoic acid (96). An amide bond formation with 30
using EDC·HCl and 4-DMAP then yielded 97, which was
reacted with mesylchloride (73) in the presence of pyridine to
obtain 98 before basic cleavage of the acetyl group afforded 25
(Scheme 8).
a
Reagents and conditions: (a) KMnO4, H2O, pyridine, reflux, 17 h,
yield: 46%; (b) H2SO4, MeOH, 80 °C, 2 h; and (c) LiOH, H2O,
MeOH, THF, 3 h, yield: 82%.
a
Scheme 8. Synthesis of 25
The benzoic acid precursor 51 was prepared in a
nucleophilic aromatic substitution of methyl 4-fluorobenzoate
(86) using azetidine hydrochloride, followed by a hydrolysis of
the methylester in 87 to 51 (Scheme 6).
a
Scheme 6. Synthesis of Precursor 51
a
Reagents and conditions: (a) azetidine hydrochloride, K2CO3,
a
Reagents and conditions: (a) n-BuLi, 3-oxetanone (94), THF, −78
DMSO, reflux, 72 h; and (b) LiOH, H2O, EtOH, 16 h, yield: 42%
over two steps.
°C, 1.5 h, yield: 48%; (b) acetyl chloride, DCM, NEt3, reflux, 16 h,
yield: 70%; (c) EDC·HCl, 4-DMAP, CHCl3, 50 °C, 16 h, yield: 49%;
(d) mesyl chloride (73), THF, pyridine, reflux, 16 h, yield: 55%; and
(e) LiOH, H2O, EtOH, THF, 5 h, yield: 48%.
The second-generation FXRa/sEHi candidates 10 and 25
were not accessible via the general route outlined in Scheme 1
and had to be synthesized using protecting groups. Di-tert-
butyl dicarbonate enabled the protection of 3,5-dichloro-4-
methylaniline (88), which was then used for the synthesis of
90 by radical bromination. A two-step Staudinger reaction
using sodium azide and triphenylphosphine yielded the Boc-
protected aminomethylaniline 91, which was further reacted to
the respective tert-butylbenzamide 92 using 4-tert-butylbenzoic
acid (33), EDC·HCl, and 4-DMAP and subsequent Boc-
cleavage with trifluoroacetic acid. Mesylation of 92 using
mesylchloride (73) and pyridine then afforded 10 (Scheme 7).
A lithium halogen exchange using n-BuLi and 4-
bromobenzoic acid (93) followed by a reaction with 3-
oxetanone (94) in a two-step procedure yielded benzoic acid
Biological Evaluation. FXR modulation by 4−29 was
determined in a cellular reporter gene assay in transiently
transfected HeLa cells using the heterodimer of the full-length
human FXR and retinoid X receptor α (RXRα) to control the
reporter gene expression. Both receptors were constitutively
overexpressed (CMV promoter). Firefly luciferase under the
control of the human FXR response element from the
promoter region of the bile salt export protein (BSEP) served
as reporter gene,30 and constitutively expressed (SV40
promoter) renilla luciferase was used to normalize for
transfection efficiency and test compound toxicity. Recombi-
nant sEH protein and the fluorogenic substrate (3-
phenyloxiranyl)acetic acid cyano-(6-methoxynapthalen-2-yl)-
9527
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 1. Biological Activity and Microsomal Stability of 3, Its Metabolite 3a, and Derivatives 4−10
a
Data are the mean standard error of the mean (SEM), n ≥ 3. Inactive: no statistically significant activity at the indicated concentration.
Intrinsic clearance (RLM) in mg∗μL-1∗min−1.
b
methyl ester (PHOME) were used to characterize sEH
This initial SAR evaluation demonstrated that even small
structural modifications in the alkylsulfonamide (4−7) or in
the substitution pattern of the neighboring benzene moiety
(8−10) resulted in a pronounced loss of activity on FXR. We
therefore refrained from further SAR studies on the
sulfonamide residue and focused our attention on the
metabolically labile tert-butylbenzamide.
inhibitory potency of 3−29.31,32 To capture metabolic stability,
we incubated compounds 3−29 with rat liver microsomes
(RLMs) and quantified the remaining amount of non-
metabolized compound after 15, 30 and 60 min. The in
vitro half-life was then determined using a logarithmic-linear
transformation of the remaining amount of non-metabolized
compound versus time. Intrinsic clearance was calculated
based on in vitro half-life and normalized for the protein
concentration in the microsomal reaction mixtures.33,34
Structural Optimization. As our first attempt to stabilize
the dual modulator chemotype against metabolic decay, we
focused on the sulfonamide motif of 3 (Table 1) that is
hydrolyzed in the formation of metabolite 3a. During the
development of the FXRa/sEHi 3,12 the sulfonamide motif
turned out highly favorable to achieve the desired poly-
pharmacological profile in terms of balanced dual potency but
also to obtain sufficient solubility. Even its inversion or
replacement by an amide was poorly tolerated. Additionally, 3
has exhibited strong therapeutic effects in several rodent
models27,28 suggesting the chemotype as favorable and
prompting us to conserve its scaffold as far as possible. To
prevent hydrolysis of the methylsulfonamide, we hence
introduced sterically more demanding ethyl- (4), isopropyl-
(5), and cyclopropyl- (6) sulfonamides.
We commenced the structural optimization of tert-
butylbenzamide toward improved metabolic stability by
replacing the tert-butyl motif with known bioisosteres that
have been validated as less labile in previous examples35,36
(Table 2). The trifluoromethylcyclopropyl analogue 11 indeed
revealed markedly higher stability against microsomal degra-
dation and was well tolerated by sEH but the potency on FXR
dropped by a factor of 27. The bicyclo[1.1.1]pentane
derivative 12 exhibited similarly diminished potency on FXR
as 11, demonstrating that the tert-butyl motif is highly favored
by this nuclear receptor. Additionally, 12 was less active on
sEH than 3 and its microsomal stability represented only a
weak improvement over the lead. Trifluoromethyl analogue 13
comprised the most preferable overall profile in this first series.
It retained full inhibitory potency on sEH and revealed high
microsomal stability. Potency of 13 on FXR only dropped
slightly by a factor of 7 compared to 3. We also probed the
replacement of the tert-butyl motif of 3 by a dimethylamine
(14) or a methylether (15), which were both markedly less
active on FXR than 3 while retaining high potency on sEH.
Methoxy derivative 15 revealed preferably high microsomal
stability.
We hypothesized that the pronounced loss in the activity
observed for dimethylamine 14 and methylether 15 might
result from their smaller substituent size prompting us to
follow-up on these modifications with larger amine and ether
residues (Table 2).
The SAR of cyclic amine substituents (16−19) indeed
revealed a correlation between the substituent size and activity
on FXR. Although the azetidine derivative 16 was a very weak
FXR agonist, the pyrrolidine (17) and piperidine (18)
analogues exhibited nanomolar potency on FXR. Regarding
sEH inhibition, the differences in activity were less pronounced
among amine derivatives 14 and 16−18 and covered an
However, while these modifications were well tolerated by
sEH, potency on FXR dropped substantially and microsomal
stability was not enhanced. Replacing the methylsulfonamide
(3) by a trifluoromethylsulfonamide (7) under the assumption
that electron-withdrawing fluorine might boost metabolic
stability was not tolerated by FXR either. Aiming to stabilize
the sulfonamide through reduced electron density in the
neighboring benzene ring, we varied its substitution pattern
with fluorine substituents.
When chlorine (3) was replaced by fluorine (8), the stability
still decreased and the potency on FXR was lost. A second
fluorine substituent (9) caused a strong increase in microsomal
stability but failed to reinstate the activity on FXR. The 3,5-
dichloroderivative 10 exhibited intermediate stability but was
neither tolerated by FXR nor favored by sEH.
9528
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a
Table 2. Biological Activity and Microsomal Stability of 11−29
a
Lead compound 3 for comparison. Data are the mean SEM, n ≥ 3. Inactive: no statistically significant activity at the indicated concentration.
Intrinsic clearance (RLM) in mg∗μL−1∗min−1.
b
intermediate nanomolar range. The more polar morpholino
Following up on methyl ether derivative 15, which possessed
very high microsomal stability, we first combined the ether
moiety with the favored trifluoromethyl substituent of 13 in
trifluoromethoxy analogue 20, which retained high stability
and was a potent sEH inhibitor, but the FXR agonism
decreased markedly compared to 3. We then extended the size
derivative 19 was not favored by FXR and sEH. The
microsomal stability and on-target activity of 14 and 16−19
revealed opposing trends, wherein the least active compounds
16 and 19 comprised the highest stability.
9529
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Figure 1. In vitro profiling of dual FXR/sEH modulators 11, 13, and 23. (a) 11, 13, and 23 were nontoxic up to 100 μM in HepG2 cells in a WST-
1 assay. Data are the mean SEM; n = 3. (b) 11, 13, and 23 were selective over related nuclear receptors except for weak PPARα and PPARγ
activation by 11 at 10 μM. Heatmap shows mean relative nuclear receptor activation vs reference agonists (1 μM); n ≥ 2. (c) 11, 13, and 23 (at 1
μM) modulated FXR-regulated gene expression in HepG2 cells with induction of BSEP and PPARα, and downregulation of SREBP1c. CYP7A1
expression was not affected. Data are mean SEM relative mRNA expression determined by the 2−ΔΔCt method with GAPDH as a reference gene;
#
n = 4. p < 0.1, *p < 0.05, **p < 0.01, ***p < 0.001 (t-test vs DMSO-treated cells).
of the ether substituent from methyl (15) over ethyl (21) to
isopropyl (22) and observed increasing FXR agonism with
larger substituent size while sEH inhibitory potency was less
affected but slightly increased, too. Microsomal stability,
despite dropping with replacement of methyl by larger alkyl
residues, remained superior compared to 3. Oxygen-containing
tert-butyl mimicking motifs, therefore, appeared to be
promising. We hypothesized that increasing polarity in this
region could further enhance microsomal stability and inverted
the ether motif to the tertiary alcohol 23, which indeed was
exceptionally stable against degradation by liver microsomes.
Moreover, 23 emerged as the first stabilized analogue of 3 with
double-digit nanomolar potency on FXR. The 2-hydroxypropyl
substituent of 23 was less favored by sEH but still exhibited
submicromolar inhibitory potency. When the hydroxyl group
was methylated in 24, the activity on sEH was strongly
enhanced but the microsomal stability dropped dramatically.
Additionally, 24 as well as hydroxyoxetane 25 failed to activate
FXR, which again highlighted the challenge of replacing the
tert-butyl motif without loss of activity on FXR. Despite its
weaker sEH inhibitory potency, 23 therefore emerged as
another substantial improvement over 3. Overall, fluorinated
tert-butyl mimetics such as 11 and 13 had shown favorable
profiles prompting us to study further fluorine-containing alkyl
substituents in this region (Table 2). 1,1-Difluoroethyl (26)
and 2,2,2-trifluoroethyl (27) as well as the merged
pentafluoroethyl (28) analogues exhibited strong sEH
inhibition, while FXR agonism compared to the trifluorome-
thylcyclopropyl derivative 11 and the smaller trifluoromethyl
analogue 13 was decreased. Interestingly, microsomal stability
varied markedly among the fluorinated derivatives. Although
the trifluoromethyl derivative 13 was very stable, pentafluor-
oethyl (28) and especially 2,2,2-trifluoroethyl (27) substitu-
ents resulted in significantly lower stability. The 1,1-
difluoroethyl (26) as well as the trifluoromethylcyclopropyl
(11) derivatives comprised intermediate but sufficient stability.
The pentafluorosulfanyl moiety has been proposed as
another suitable bioisosteric replacement of tert-butyl and
trifluoromethyl groups.37 We incorporated this motif in the
dual modulator chemotype (29) and indeed observed
improved microsomal stability compared to the tert-butyl
lead compound 3; 29 also retained high potency on sEH but
was inactive on FXR.
In our endeavor to design second-generation dual
modulators of FXR and sEH with improved metabolic stability,
we discovered three structural modifications (11, 13, 23) that
retained sufficient potency on the protein targets and
simultaneously generated a substantial improvement in micro-
somal stability. Achieving high potency on FXR emerged as the
greatest challenge in this multi-objective optimization. In an
attempt to combine the most favorable modifications and to
obtain further optimization, we designed the fused compound
30 representing a combination of 11 and 23; 30 was
synthesized but the 1-hydroxycyclopropyl substituent turned
out unstable and underwent a rearrangement to an ethylmethyl
ketone.38 Hence, 11, 13, and 23 evolved as the most favorable
second-generation dual FXR activators/sEH inhibitors for
further in vitro and in vivo profiling.
In Vitro and In Vivo Profiling. All three dual modulators
11, 13, and 23 exhibited no toxic effects in HepG2 cells up to
100 μM concentration as determined in a WST-1 assay (Figure
1a) and were highly selective over related nuclear receptors at
10 μM apart from weak PPARα and PPARγ activation by 11
9530
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(Figure 1b). Aqueous solubility was sufficient for 11 (2.9 mg/
L) and 13 (5.7 mg/L) and high for 23 (48 mg/L).
preventive27 and curative28 intervention. These results from
animal studies have confirmed our polypharmacology hypoth-
esis and characterized dual FXR/sEH modulation as an
attractive strategy to treat NASH. However, while being highly
potent on the intended targets, the original dual modulator 3 is
characterized by a short half-life and moderate plasma levels in
vivo leading to the need of twice-daily dosing. Aiming to
overcome this pharmacokinetic liability, we have further
studied the structure−activity and structure−stability relation-
ships of the dual modulator chemotype in a multi-objective
optimization endeavor. Based on the known metabolic
degradation pathways of 3, we have first probed stabilization
of the sulfonamidophenyl motif, but modifications in this
region were poorly tolerated by FXR and failed to provide
substantial metabolic stabilization. Structural variations of the
labile tert-butyl motif were more suitable to stabilize the
scaffold against microsomal degradation but retaining high
potency on FXR presented as a challenge, which agrees with
the privileged role of the tert-butylphenyl motif in partial FXR
agonists.39−43 In vitro, replacement of tert-butyl motif by
trifluoromethylcyclopropyl (11), trifluoromethyl (13), and
hydroxyisopropyl (23) residues emerged as most favorable
modifications to retain dual potency and achieve metabolic
stabilization. Based on its potency, preferable selectivity, and
robust effects on FXR-regulated gene expression in hepato-
cytes, we chose the second-generation dual modulator 13 for
in vivo pharmacokinetic profiling, the results of which
confirmed the intended improvement over the original
compound 3. With peak plasma levels of 4.9 μg/mL and a
half-life above 5 h, 13 overcomes the pharmacokinetic
liabilities of 3 and retains the attractive polypharmacological
activity profile of the lead. These characteristics render 13
suitable for late-stage preclinical development to move the
therapeutic concept of dual FXR/sEH modulation toward
clinical trials.
To confirm FXR target engagement in a native and
orthogonal cellular setting, we treated FXR-expressing human
hepatocytes (HepG2) with 11, 13, or 23 (each at 1 μM) and
studied the effects on FXR-regulated gene expression by
quantitative real-time polymerase chain reaction (qRT-PCR).
The endogenous FXR agonist chenodeoxycholic acid (CDCA)
served as the positive control. In line with their partial agonism
in the BSEP-based reporter gene assay, 13 and 23 induced
BSEP mRNA expression with low efficacy compared to CDCA,
whereas 11 exhibited very weak BSEP induction (Figure 1c).
11 and 13 efficiently promoted the expression of peroxisome
proliferator-activated receptor α (PPARα) and downregulated
sterol regulatory element-binding protein 1c (SREBP1c), while
23 had no observable effect on both FXR-regulated genes.
Similar to our observations of weak effects of the lead
compound 312,28 and other partial agonists39−41 on cholester-
ol-7α-hydroxylase (CYP7A1) expression, 11, 13, and 23 did
not suppress CYP7A1.
The second-generation FXRa/sEHi 13 consistently ex-
hibited potent partial FXR agonism in the reporter gene
assay and in hepatocytes and potently inhibited sEH.
Additionally, 13 revealed the most favorable selectivity profile
among nuclear receptors and comprised the desired high
stability against microsomal degradation. With these character-
istics, 13 met all criteria of our objective to develop a potent
FXRa/sEHi resembling the biological activity profile of 3 with
improved metabolic stability. Hence, we determined the PK
profile of 13 in mice, which remarkably confirmed the
envisioned improved pharmacokinetics (Figure 2). A single
EXPERIMENTAL SECTION
■
Chemistry. General. All chemicals and solvents were of reagent
grade and were used without further purification unless otherwise
specified. All reactions were conducted in oven-dried glassware under
argon atmosphere and in absolute solvents. NMR spectra were
recorded on a Bruker AV 500, Bruker AV 400, Bruker AV 300, or a
Bruker am250xp spectrometer (Bruker Corporation, Billerica, MA).
Chemical shifts (δ) are reported in ppm relative to tetramethylsilane
(TMS) as reference. Multiplicity is reported: s, singlet; d, doublet; dd,
doublet of doublets; t, triplet; and m, multiplet. Approximate coupling
constants (J) are given in hertz (Hz). Mass spectra were obtained on a
VG Platform II (Thermo Fischer Scientific, Inc., Waltham, MA) using
electrospray ionization (ESI). High-resolution mass spectra were
recorded on a MALDI LTQ ORBITRAP XL instrument (Thermo
Fisher Scientific). Preparative HPLC was performed on a Shimadzu
preparative LC-20A Prominence (Shimadzu, Kyoto, Japan) under the
following conditions: column, Luna (10 μm C18(2) 100 Å; 250 ×
21.2 mm2; Phenomenex, Torrance, CA); mobile phase, isocratic
50:50 acetonitrile/H2O + 0.1 formic acid for 30 min at a flow rate of
21 mL/min, and UV detection at 245 and 280 nm. Compound purity
was analyzed on a Waters 600 Controller HPLC (Waters, Milford,
MA) using a Waters 2487 dual absorbance detector and a Waters 717
plus Autosampler or a VWR Chromaster (VWR, Radnor, PA) with a
5160 pump system, using a DAD 5430 and a 5260 Autosampler both
equipped with a MultoHigh100 RP18-5 μm 250 × 4 mm2 column
(CS-Chromatographie Service GmbH, Langerwehe, Germany) using
a gradient (H2O + 0.1% formic acid/MeOH 80:20 isocratic for 5 min
to MeOH after additional 45 min and MeOH for additional 10 min)
at a flow rate of 1 mL/min or a gradient (H2O + 0.1% formic acid/
MeOH 60:40 isocratic for 5 min to MeOH after additional 25 min
Figure 2. Pharmacokinetic profile and parameters of 13 in mice after
a single oral dose of 10 mg/kg. Lead compound 3 for comparison.
The pharmacokinetic profile of 3 in mice has been determined
previously12 under equal conditions as for 13 (vehicle, dose,
administration route).
oral 10 mg/kg dose of 13 resulted in a maximum plasma level
of 4.9 μg/mL and a half-life of 5.2 h, presenting as a
pronounced improvement in exposure over the lead compound
3 (cmax 1.2 μg/mL, t1/2 0.7 h).
CONCLUSIONS
■
Following the hypothesis that the treatment of the multi-
factorial pathology NAFLD/NASH would benefit from
combined multiple modes of action, we have previously
developed a potent dual FXR/sEH modulator chemotype.12 In
vivo characterization of the original dual FXR activator/sEH
inhibitor 3 has demonstrated remarkable therapeutic efficacy in
toxin- and diet-induced rodent models of NASH as a
9531
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
and MeOH for additional 10 min) at a flow rate of 1 mL/min with
UV detection at 245 and 280 nm. All compounds used for biological
characterization had a purity ≥95%. Precursors 30 and 52 and
compound 3 have been reported previously.12
1.00 equiv) were added dropwise. Then, EDC·HCl (0.058 g, 0.301
mmol, 0.200 equiv) and triethylamine (0.305 g, 3.02 mmol, 2.00
equiv) were added and the mixture was stirred at 50 °C for 20 h. After
cooling to room temperature, aqueous hydrochloric acid (10%, 10
mL) was added and the mixture was extracted with EtOAc (3 × 30
mL). The combined organic layers were extracted with a saturated
aqueous solution of Na2CO3 (3 × 30 mL). Then, the combined
aqueous layers were brought to pH 1 with concentrated aqueous
hydrochloric acid and extracted with EtOAc (3 × 30 mL). The
combined organic layers were dried over Na2SO4 and the solvent was
evaporated in vacuum to obtain 45 as a beige solid (0.177 g, 51%). 1H
NMR (300 MHz, DMSO-d6) δ = 12.89 (s, 1H), 7.98−7.90 (m, 2H),
7.62−7.54 (m, 2H), 1.44−1.33 (m, 2H), 1.22−1.14 (m, 2H). 13C
NMR (75 MHz, DMSO-d6) δ = 166.87, 140.02, 131.18, 130.86,
129.41, [131.78, 128.15, 124.52, 121.15], [28.16, 27.75, 27.32, 26.88],
[9.81, 9.77, 9.74, 9.71].
4-(2-Hydroxypropan-2-yl)benzoic Acid (49). 4-Isopropylbenzoic
acid (85, 0.700 g, 4.26 mmol, 1.00 equiv) was dissolved in a mixture
of H2O (15 mL) and pyridine (15 mL). After stirring at 100 °C for 1
h, KMnO4 (2.02 g, 12.8 mmol, 3.00 equiv) was added and the
resulting reaction mixture was stirred at 100 °C for 16 h. After cooling
to room temperature, aqueous sodium hydroxide solution (10%, 50
mL) was added and the mixture was filtered. The filtrate was brought
to pH 1 using concentrated aqueous hydrochloric acid and extracted
with EtOAc (3 × 30 mL). The combined organic layers were dried
over Na2SO4 and the solvent was evaporated in vacuum. Further
purification was performed by column chromatography using hexane/
acetone (9:2) as the mobile phase to obtain 49 as a colorless solid
(0.352 g, 46%). 1H NMR (400 MHz, DMSO-d6) δ = 7.87 (d, J = 8.2
Hz, 2H), 7.57 (d, J = 8.2 Hz, 2H), 5.16 (s, 1H), 1.43 (s, 5H). 13C
NMR (101 MHz, DMSO-d6) δ = 167.31, 155.50, 128.92, 128.57,
124.69, 70.74, 31.66.
N-(4-Amino-2-chlorobenzyl)-4-(1-(trifluoromethyl)cyclopropyl)-
benzamide (55). 4-(1-(Trifluoromethyl)cyclopropyl)benzoic acid
(45, 0.153 g, 0.665 mmol, 1.00 equiv), 1-(4-amino-1-chlorophenyl)-
methanamine (30, 0.312 g, 1.99 mmol, 3.00 equiv), and 4-DMAP
(0.081 g, 0.665 mmol, 1.00 equiv) were dissolved in a mixture of
CHCl3 (25 mL) and DMF (2.5 mL). After cooling the reaction
mixture to 0 °C, EDC·HCl (0.382 g, 1.99 mmol, 3.00 equiv) was
added and the mixture was stirred at 0 °C for 30 min and at 50 °C for
16 h. Then, aqueous hydrochloric acid (10%, 30 mL) was added and
the phases were separated. The aqueous layer was brought to pH 12
using Na2CO3 and extracted with EtOAc (3 × 30 mL). The combined
organic layers were dried over Na2SO4 and the solvent was removed
in vacuum. The crude product was purified by column chromatog-
raphy using n-hexane/EtOAc (4:3) as the mobile phase to obtain 55
as a yellow solid (0.125 g, 51%). 1H NMR (300 MHz, acetone-d6) δ =
7.95−7.90 (m, 2H), 7.57 (d, J = 8.1 Hz, 2H), 7.15 (d, J = 8.3 Hz,
1H), 6.72 (d, J = 2.3 Hz, 1H), 6.58 (dd, J = 8.3, 2.3 Hz, 1H), 4.85 (s,
1H), 4.54 (d, J = 5.6 Hz, 2H), 1.39 (dd, J = 6.9, 5.0 Hz, 2H), 1.18−
1.12 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ = 166.17, 149.60,
[138.79, 138.78], [135.95, 132.33, 128.71, 125.08], 134.96, 132.91,
131.39, 130.23, 127.94, 122.79, 114.07, 113.09, 40.60, [28.58, 28.14,
27.70, 27.26], [10.21, 10.18, 10.15, 10.12].
N-(4-Amino-2-chlorobenzyl)-4-(trifluoromethyl)benzamide (57).
4-(Trifluoromethyl)benzoic acid (35, 0.200 g, 0.912 mmol, 1.00
equiv), 1-(4-amino-1-chlorophenyl)methanamine (30, 0.429 g, 2.74
mmol, 3.00 equiv), and 4-DMAP (0.111 g, 0.912 mmol, 1.00 equiv)
were dissolved in a mixture of CHCl3 (30 mL) and DMF (3 mL).
After cooling the reaction mixture to 0 °C, EDC·HCl (0.525 g, 2.74
mmol, 3.00 equiv) was added and the mixture was stirred at 0 °C for
30 min and at 50 °C for 16 h. Then, aqueous hydrochloric acid (10%,
30 mL) was added and the phases were separated. The aqueous layer
was brought to pH 12 using Na2CO3 and extracted with EtOAc (3 ×
30 mL). The combined organic layers were dried over Na2SO4 and
the solvent was removed in vacuum. The crude product was purified
by column chromatography using n-hexane/EtOAc (4:3) as the
mobile phase to obtain 57 as a yellow solid (0.126 g, 42%). 1H NMR
(300 MHz, DMSO-d6) δ = 9.02 (t, J = 5.2 Hz, 1H), 8.07 (d, J = 8.2
Hz, 2H), 7.84 (d, J = 8.2 Hz, 2H), 7.04 (d, J = 8.3 Hz, 1H), 6.63 (d, J
4-(1-(Trifluoromethyl)cyclopropyl)-N-(2-chloro-4-
(methylsulfonamido)benzyl)benzamide (11). N-(4-Amino-2-chlor-
obenzyl)-(4-(1-(trifluoromethyl)cyclopropyl))benzamide (55, 0.104
g, 0.282 mmol, 1.00 equiv) was dissolved in a mixture of THF (28
mL) and pyridine (2.8 mL). Methanesulfonyl chloride (73, 0.323 g,
2.82 mmol, 10.0 equiv) was added dropwise. The reaction mixture
was stirred at reflux for 16 h. Then, aqueous hydrochloric acid (10%,
30 mL) was added and the phases were separated. The aqueous layer
was extracted with EtOAc (3 × 30 mL). The combined organic layers
were dried over Na2SO4 and the solvent was removed in vacuum. The
crude product was purified by column chromatography using n-
hexane/EtOAc (1:1) as the mobile phase to obtain 11 as a colorless
1
solid (0.052 g, 41%). H NMR (500 MHz, DMSO-d6) δ = 9.93 (s,
1H), 9.04 (s, 1H), 7.91 (d, J = 8.3 Hz, 2H), 7.57 (d, J = 8.1 Hz, 2H),
7.32 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 2.1 Hz, 1H), 7.15 (dd, J = 8.4,
2.1 Hz, 1H), 4.48 (d, J = 5.7 Hz, 2H), 3.02 (s, 3H), 1.38 (s, 2H), 1.17
(s, 2H). 13C NMR (126 MHz, MeOD) δ = 169.74, 141.13, 139.98,
135.45, 134.93, 132.85, 132.52, 131.10, 128.53, [130.97, 128.80,
126.64, 124.47], 121.72, 119.59, 42.16, 39.37, [29.48, 29.21, 28.95,
28.68], [10.45,10.44, 10.43, 10.42]. HRMS (MALDI): m/z calcd
469.05710 for C19H18ClF3N2O3SNa, found 469.05613 [M + Na]+.
4-(Trifluoromethyl)-N-(2-chloro-4-(methylsulfonamido)benzyl)-
benzamide (13). N-(4-Amino-2-chlorobenzyl)-4-(trifluoromethyl)-
benzamide (57, 0.126 g, 0.383 mmol, 1.00 equiv) was dissolved in
a mixture of THF (25 mL) and pyridine (2.5 mL). Methanesulfonyl
chloride (73, 0.439 g, 3.83 mmol, 10.0 equiv) was added dropwise.
The reaction mixture was stirred at reflux for 16 h. Then, aqueous
hydrochloric acid (10%, 25 mL) was added and the phases were
separated. The aqueous layer was extracted with EtOAc (3 × 25 mL).
The combined organic layers were dried over Na2SO4 and the solvent
was removed in vacuum. The crude product was purified by column
chromatography using n-hexane/EtOAc (1:1) as the mobile phase to
1
obtain 13 as a colorless solid (0.042 g, 27%). H NMR (500 MHz,
acetone-d6) δ = 8.71 (s, 1H), 8.40 (t, J = 5.9 Hz, 1H), 8.07−8.00 (m,
2H), 7.75−7.66 (m, 2H), 7.36 (d, J = 8.4 Hz, 1H), 7.31 (d, J = 2.3
Hz, 1H), 7.15 (dd, J = 8.4, 2.3 Hz, 1H), 4.57 (d, J = 5.8 Hz, 2H), 2.93
(s, 3H).13C NMR (126 MHz, acetone-d6) δ = 166.50, 139.65, [139.12
139.11, 139.10, 139.09], 134.21, [133.47, 133.22, 132.96, 132.70],
132.63, 131.13, 128.98, [128.21, 126.05, 123.88, 121.72], [126.27,
126.24, 126.21, 126.18], 121.16, 119.34, 41.68, 39.68. HRMS
(MALDI): m/z calcd 407.04385 for C19H19ClF3N2O3S, found
407.04364 [M + H]+.
4-(2-Hydroxypropan-2-yl)-N-(2-chloro-4-(methylsulfonamido)-
benzyl)benzamide (23). N-(4-Amino-2-chlorobenzyl)-4-(2-hydroxy-
propan-2-yl)benzamide (67, 0.103 g, 0.323 mmol, 1.00 equiv) was
dissolved in a mixture of THF (5 mL) and pyridine (5 mL) and
cooled to 0 °C. Methanesulfonyl chloride (73, 0.111 g, 0.969 mmol,
3.00 equiv) was added dropwise at 0 °C. The reaction mixture was
stirred at 0 °C for 8 h. Then, aqueous hydrochloric acid (10%, 25 mL)
was added and the phases were separated. The aqueous layer was
extracted with ethyl acetate (3 × 30 mL). The combined organic
layers were dried over Na2SO4 and the solvents were removed in
vacuum. Further purification was performed by column chromatog-
raphy using n-hexane/EtOAc (5:3) as the mobile phase to obtain 23
as a colorless solid (6 mg, 5%). 1H NMR (500 MHz, methanol-d4) δ
= 7.85−7.82 (m, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.37−7.33 (m, 2H),
7.16 (dd, J = 8.4, 2.3 Hz, 1H), 4.62 (s, 2H), 2.96 (s, 3H), 1.54 (s,
6H). 13C NMR (126 MHz, methanol-d4) δ = 168.80, 153.67, 138.51,
133.49, 129.62, 127.96, 126.87, 126.60, 124.48, 120.35, 118.23, 71.51,
40.70, 37.98, 30.39. HRMS (MALDI): m/z calcd 419.08028 for
C18H21ClN2O4SNa, found 419.08020 [M + Na]+.
4-((1-Trifluoromethyl)cyclopropan-1-yl)benzoic Acid (45). Pd-
(OAc)2 (0.010 g, 0.045 mmol, 0.03 equiv) and Xantphos (0.010 g,
0.045 mmol, 0.03 equiv) were dissolved in DMF (10 mL). Formic
acid (0.400 mL, 10.7 mmol, 7.00 equiv) and 1-bromo-4-(1-
(trifluoromethyl)cycloprop-1-yl)benzene (81, 1.51 mmol, 0.400 g,
9532
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
= 2.2 Hz, 1H), 6.49 (dd, J = 8.3, 2.1 Hz, 1H), 5.30 (s, 2H), 4.40 (d, J
= 5.5 Hz, 2H). 13C NMR (75 MHz, acetone-d6) δ = 166.13, 150.09,
139.50, 134.43, [133.56, 133.13, 132.70, 132.28], 131.67, [130.44,
126.84, 123.23, 119.63], 128.95, [126.21, 126.16, 126.11, 126.06],
124.07, 115.21, 113.88, 41.81.
up in triplicate by SigmaPlot 12.5 (Systat Software GmbH, Erkrath,
Germany) using a four-parameter logistic regression. The assay was
validated with FXR agonists OCA (EC50 = 0.16 0.02 μM, 87 3%
rel. max. act.) and GW4064 (EC50 = 0.51 0.16 μM, 3 μM defined as
100%).
sEH Activity Assay. sEH inhibitory potency was determined in a
fluorescence-based 96-well sEH activity assay using purified
recombinant human enzyme.31 Nonfluorescent (3-phenyloxiranyl)-
acetic acid cyano-(6-methoxynapthalen-2-yl)-methyl ester32,46
(PHOME) was used as a substrate, which is hydrolyzed by sEH to
fluorescent 6-methoxynaphthaldehyde. Purified recombinant human
sEH (in BisTris buffer, pH 7, with 0.1 mg/mL BSA containing a final
concentration of 3 nM sEH and 0.01% Triton-X 100) was
preincubated with test compounds (in DMSO, final DMSO
concentration: 1.6%) for 30 min at room temperature. Then, the
substrate was added (final concentration 60 μM), and hydrolysis of
the substrate was determined by measuring fluorescent product
N-(4-Amino-2-chlorobenzyl)-4-(2-hydroxypropan-2-yl)-
benzamide (67). 4-(2-Hydroxypropan-2-yl)benzoic acid (49, 0.103 g,
0.572 mmol, 1.00 equiv), 1-(4-amino-1-chlorophenyl)methanamine
(30, 0.134 g, 0.857 mmol, 1.50 equiv), and 4-DMAP (0.007 g, 0.057
mmol, 0.10 equiv) were dissolved in a mixture of CHCl3 (25 mL) and
DMF (2.5 mL). After cooling the reaction mixture to 0 °C, EDC·HCl
(0.131 g, 0.656 mmol, 1.20 equiv) was added and the mixture was
stirred at 0 °C for 30 min and at 50 °C for 16 h. Then, aqueous
hydrochloric acid (10%, 30 mL) was added and the phases were
separated. The aqueous layer was brought to pH 12 using Na2CO3
and extracted with EtOAc (3 × 30 mL). The combined organic layers
were dried over Na2SO4 and the solvent was removed in vacuum. The
crude product was purified by column chromatography using n-
hexane/EtOAc (3:5) as the mobile phase to obtain 67 as a yellow oil
formation on a Tecan Infinite F200 Pro (Tecan, λem = 330 nm, λex
=
465 nm) for 30 min (one point per minute). A blank control (no
protein and no compound) as well as a positive control (no
compound) were executed. All experiments were conducted in
triplicate and repeated in three independent experiments. For IC50
calculation, the dose−response curves of increasing compound
concentrations were recorded. IC50 and standard deviation were
calculated from at least three independent experiments set up in
triplicate in GraphPad Prism 7 using a four-parameter logistic
regression.
1
(0.104 g, 57%). H NMR (400 MHz, methanol-d4) δ = 7.83−7.77
(m, 2H), 7.59−7.53 (m, 2H), 7.11 (d, J = 8.2 Hz, 1H), 6.75−6.74 (m,
1H), 6.60−6.57 (m, 1H), 4.53 (s, 2H), 1.54 (s, 6H). 13C NMR (101
MHz, methanol-d4) δ = 170.04, 154.79, 149.84, 134.87, 133.60,
131.30, 129.32, 128.21, 125.77, 116.45, 114.78, 72.87, 42.19, 31.76.
Biological Characterization. BSEP-Based Full-Length FXR
Reporter Gene Assay. Plasmids: pcDNA3-hFXR44 contains the
sequence of human FXR. pSG5-hRXR45 contains the sequence of
human RXRα. pGL3basic (Promega Corporation, Fitchburg, WI)
with a shortened construct of the promotor of the bile salt export
protein (BSEP) cloned into the SacI/NheI cleavage site in front of the
luciferase gene was used as the reporter plasmid.44 pRL-SV40
(Promega) served as a control for normalization of transfection
efficiency and cell growth. Assay procedure: HeLa cells (German
Collection of Microorganisms and Cell Culture GmbH, DSMZ) were
grown in Dulbecco’s modified Eagle’s medium (DMEM) high
glucose, supplemented with 10% fetal calf serum (FCS), sodium
pyruvate (1 mM), penicillin (100 U/mL), and streptomycin (100 μg/
mL) at 37 °C and 5% CO2; 24 h before transfection, HeLa cells were
seeded in 96-well plates with a density of 8000 cells/well; 3.5 h before
transfection, the medium was changed to DMEM high glucose,
supplemented with sodium pyruvate (1 mM), penicillin (100 U/mL),
streptomycin (100 μg/mL), and 0.5% charcoal-stripped FCS.
Transient transfection of HeLa cells with BSEP-pGL3, pRL-SV40,
and the expression plasmids pcDNA3-hFXR and pSG5-hRXR was
carried out using calcium phosphate transfection method; 16 h after
transfection, the medium was changed to DMEM high glucose,
supplemented with sodium pyruvate (1 mM), penicillin (100 U/mL),
streptomycin (100 μg/mL) and 0.5% charcoal-stripped FCS. 24 h
after transfection, the medium was changed to DMEM without
phenol red, supplemented with sodium pyruvate (1 mM), penicillin
(100 U/mL), streptomycin (100 μg/mL), L-glutamine (2 mM), and
0.5% charcoal-stripped FCS, now additionally containing 0.1%
DMSO and the respective test compound or 0.1% DMSO alone as
untreated control. Each concentration was tested in triplicate, and
each experiment was repeated independently at least three times.
Following 24 h incubation with the test compounds, cells were
assayed for luciferase activity using the Dual-Glo Luciferase Assay
System (Promega) according to the manufacturer’s protocol.
Luminescence was measured with a Tecan Infinite M200 lumin-
ometer (Tecan Deutschland GmbH, Crailsheim, Germany) or a
Tecan Spark 10 M luminometer (Tecan). Normalization of
transfection efficiency and cell growth was done by division of firefly
luciferase data by renilla luciferase data multiplied by 1000 resulting in
relative light units (RLU). Fold activation was obtained by dividing
the mean RLU of a test compound at a respective concentration by
the mean RLU of untreated control. Relative activation was obtained
by dividing the fold activation of the test compound at a respective
concentration by the fold activation of FXR full agonist GW4064 at 3
μM. EC50 and standard deviation were calculated with the mean
relative activation values of at least three independent experiments set
Hybrid Reporter Gene Assays. The plasmids pFA-CMV-NR-
LBD47−51 for hTHRα, hTHRβ, hPPARα, hPPARγ, hPPARδ, hRARα,
hRARβ, hRARγ, hLXRα, hLXRβ, hRXRα, hRXRβ, hRXRγ, and
hVDR coding for the Gal4 DNA binding domain fused to the hinge
region and LBD of the canonical isoform of the respective nuclear
receptor, Gal4-repsonsive pFR-Luc (Stratagene, La Jolla, CA;
reporter), and pRL-SV40 (Promega; internal control) were used for
the hybrid reporter gene assays. HEK293T cells (DSMZ) were
cultured in DMEM high glucose, supplemented with 10% FCS,
sodium pyruvate (1 mM), penicillin (100 U/mL), and streptomycin
(100 μg/mL) at 37 °C and 5% CO2 and seeded in 96-well plates (3 ×
104 cells/well) 24 h prior to transfection. Before transfection, the
medium was changed to Opti-MEM without supplements, and
transient transfection with pFR-Luc, pRL-SV40, and one pFA-CMV-
NR-LBD clone was carried out with the Lipofectamine LTX reagent
(Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol.
Five hours after transfection, the medium was changed to Opti-MEM
supplemented with penicillin (100 U/mL) and streptomycin (100
μg/mL) additionally containing 0.1% DMSO and the respective test
compound or 0.1% DMSO alone as the untreated control. Each
concentration was tested in duplicate, and each experiment was
repeated independently at least two times. Following overnight (14−
16 h) incubation, cells were assayed for luciferase activity using the
Dual-Glo Luciferase Assay System (Promega) according to the
manufacturer’s protocol. Luminescence was measured with a Tecan
Spark M luminometer (Tecan). Normalization of transfection
efficiency and cell growth was done by division of firefly luciferase
data by renilla luciferase data and multiplying the value by 1000
resulting in relative light units (RLU). Fold activation was obtained by
dividing the mean RLU of the test compound by the mean RLU of
the untreated control. Relative activation refers to fold reporter
activation of a test compound divided by the fold activation of the
respective reference agonist (PPARα: GW7647; PPARγ: pioglitazone;
PPARδ: L165,041; LXRα/β: T0901317; RXRα/β/γ: bexarotene;
RARα/β/γ: tretinoin; VDR: calcitriol; 1 μM each and THRα/β:
3,3′,5-triiodo-L-thyronine; 0.1 μM). All hybrid assays were validated
with the respective reference agonists, which yielded EC50 values in
agreement with the literature.
Microsomal Stability Assay. The solubilized test compound (5 μL,
final concentration 10 μM) was preincubated at 37 °C in 432 μL of
phosphate buffer (0.1 M, pH 7.4) together with 50 μL of NADPH
regenerating system (30 mM glucose-6-phosphate, 4 U/mL glucose-
6-phosphate dehydrogenase, 10 mM NADP, 30 mM MgCl2). After 5
9533
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
min, the reaction was started by the addition of 13 μL of microsome
mix from the liver of Sprague-Dawley rats (Invitrogen; 20 mg
protein/mL in 0.1 M phosphate buffer) in a shaking water bath at 37
°C. The reaction was stopped by adding 500 μL of ice-cold methanol
at 0, 15, 30, and 60 min. The samples were centrifuged at 5000g for 5
min at 4 °C, and the test compound was quantified from the
supernatants by HPLC: the composition of the mobile phase was
adapted to the test compound in a range of MeOH 40−90% and
water (0.1% formic acid) 10−60%; flow rate: 1 mL/min; stationary
phase: Purospher STAR, RP18, 5 μm, 125 × 4, precolumn: Purospher
STAR, RP18, 5 μm, 4 × 4; detection wavelength: 254 and 280 nm;
injection volume: 50 μL. Control samples were performed to check
the test compound’s stability in the reaction mixture: first control was
without NADPH, which is needed for the enzymatic activity of the
microsomes, second control was with inactivated microsomes
(incubated for 20 min at 90 °C), and third control was without the
test compound (to determine the baseline). The amounts of the test
compound were quantified by an external calibration curve. Data are
TTC GAA AGT GCA ATC C-3′ (rev); PPARα: 5′-GCT GTC ACC
ACA GTA GCT TGG A-3′ and 5′-GTG ATG ACC GAG CCA TCT
GA-3′ (rev); BSEP: 5′-CAT GGT GCA AGA AGT GCT GAG T-3′
and 5′-AAG CGA TGA GCA ACT GAA ATG AT-3′ (rev).
In Vivo PK Study. The PK study was performed by the contract
̈
research organization Pharmacelsus (Saarbrucken, Germany). All
experimental procedures were approved by and conducted in
accordance with the regulations of the local animal welfare authorities
(Landesamt fur Gesundheit and Verbraucherschutz, Abteilung
̈
̈
̈
Lebensmittel- and Veterinarwesen, Saarbrucken). Three male CD1
mice (32−34 g body weight, purchased from Janvier Labs, France)
were used in the study. The animals were housed in a temperature-
controlled room (20−24 °C) and maintained in a 12 h light/12 h
dark cycle. Food and water were available ad libitum. The animals
received a single oral dose of 10 mg/kg body weight of the FXRa/
sEHi 13 in water containing 1% HPMC/Tween 80 (99:1). The
animals behaved normal throughout the study and showed no adverse
effects. At four time points (30, 60, 120, and 480 min after application
of 13), plasma samples (80 μl Li-heparin plasma) were obtained from
the mice by puncture of the V. facialis. Compound 13 was quantified
from the plasma samples by LC-MS. Pharmacokinetic analysis was
performed with Kinetica 5.0 software (Thermo Scientific, Waltham).
expressed as the mean
SEM remaining compound from three
independent experiments. In vitro half-life was calculated by a
logarithmic-linear transformation of the remaining amounts of non-
metabolized test compound versus time in GraphPad Prism 7.
Intrinsic clearance was calculated based on the in vitro half-life and
normalized for the protein concentration used in the microsomal
reaction mixtures as described previously.33,34
ASSOCIATED CONTENT
■
sı
* Supporting Information
Cell Viability Assay (WST-1). HepG2 cells were grown in DMEM
high glucose, supplemented with sodium pyruvate (1 mM), penicillin
(100 U/mL), streptomycin (100 μg/mL), and 10% FCS and seeded
in 96-well plates (3 × 104 cells/well). After 24 h, the medium was
changed to DMEM high glucose, supplemented with penicillin (100
U/mL), streptomycin (100 μg/mL), and 1% charcoal-stripped FCS
and cells were incubated with the test compounds at varying
concentrations in DMEM containing 0.1% DMSO, Revlotron as
positive control, or DMEM containing 0.1% DMSO as negative
control. Each sample was set up in duplicate, and each experiment was
performed in three independent repeats. After 48 h, WST-1 reagent
(Roche Diagnostics International AG, Rotkreuz, Schweiz) was added
to each well according to manufacturer’s instructions. After 45 min
incubation, absorption (450 nm/ reference: 620 nm) was determined
with a Tecan Infinite M200 (Tecan Deutschland GmbH) and data
were analyzed according to the manufacturer’s protocol.
Quantification of FXR Regulated Gene Expression by Quantita-
tive Real-Time PCR. FXR target gene quantification was performed as
described previously.30,39 In brief, HepG2 cells were grown in DMEM
high glucose, supplemented with sodium pyruvate (1 mM), penicillin
(100 U/mL), streptomycin (100 μg/mL), and 10% FCS and seeded
in 6-well plates (1 × 106 cells/well). After 24 h, the medium was
changed to MEM supplemented with L-glutamine (2 mM), penicillin
(100 U/mL), streptomycin (100 μg/mL), and 1% charcoal-stripped
FCS. After further 24 h, HepG2 cells were incubated with test
compounds 11, 13, and 23 (1 μM) or CDCA (30 μM) in the same
medium additionally containing 0.1% DMSO, or the medium with
0.1% DMSO alone as untreated control for 8 h, harvested, washed
with cold phosphate-buffered saline (PBS) and then directly used for
RNA extraction with the total RNA Mini Kit (R6834-02, Omega Bio-
Tek, Inc., Norcross, GA). RNA (2 μg) was reverse-transcribed using
the high-capacity cDNA reverse transcription kit (4368814, Thermo
Fischer Scientific, Inc.) according to the manufacturer’s protocol.
FXR-regulated gene expression was then evaluated by quantitative
real-time PCR analysis with a StepOnePlus System (Life Tech-
nologies, Carlsbad, CA) using PowerSYBRGreen (Life Technologies;
12.5 μL/well). Each sample was set up in duplicate and repeated in
four independent experiments. Data were analyzed by the
comparative ΔΔCt method with glycerinaldehyde 3-phosphate
dehydrogenase (GAPDH) as the reference gene. The following
PCR primers were used: GAPDH: 5′-ATA TGA TTC CAC CCA
TGG CA-3′ (fwd) and 5′-GAT GAT GAC CCT TTT GGC TC-3′
(rev); CYP7A1: 5′-CAC CTT GAG GAC GGT TCC TA-3′ (fwd)
and 5′-CGA TCC AAA GGG CAT GTA GT-3′ (rev); SREBP1c: 5′-
GGA GGG GTA GGG CCA ACG GCC T-3′ and 5′-CAT GTC
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00831.
Synthetic procedures and analytical characterization data
for compounds 4−29 and their precursors, as well as
chromatographic purity analysis of 4−29 (PDF)
Molecular Formula strings contains structures of 4−29
and associated activity data (CSV)
AUTHOR INFORMATION
■
Corresponding Author
Daniel Merk − Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, 60438 Frankfurt, Germany;
orcid.org/0000-0002-5359-8128; Phone: +49 69
79829327; Email: merk@pharmchem.uni-frankfurt.de
Authors
Moritz Helmstädter − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, 60438 Frankfurt, Germany
Astrid Kaiser − Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, 60438 Frankfurt, Germany
Steffen Brunst − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, 60438 Frankfurt, Germany
Jurema Schmidt − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, 60438 Frankfurt, Germany
Riccardo Ronchetti − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, 60438 Frankfurt, Germany
Lilia Weizel − Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, 60438 Frankfurt, Germany
Ewgenij Proschak − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, 60438 Frankfurt, Germany;
orcid.org/0000-0003-1961-1859
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00831
Author Contributions
All authors have given approval to the final version of the
manuscript.
9534
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
A.; Megnien, S.; Staels, B.; Sanyal, A.; et al. Elafibranor, an Agonist of
the Peroxisome Proliferator-Activated Receptor-α and -δ, Induces
Resolution of Nonalcoholic Steatohepatitis Without Fibrosis Worsen-
ing. Gastroenterology 2016, 150, 1147−1159e5.
(11) Proschak, E.; Stark, H.; Merk, D. Polypharmacology by Design:
A Medicinal Chemist’s Perspective on Multitargeting Compounds. J.
Med. Chem. 2019, 62, 420−444.
Notes
The authors declare the following competing financial
interest(s): The authors M.H., J.S., E.P. and D.M are inventors
of the patents WO2018215610A1 and US62/902,771 which
claim compounds reported in this manuscript and their uses.
No other competing interests are declared.
(12) Schmidt, J.; Rotter, M.; Weiser, T.; Wittmann, S.; Weizel, L.;
Kaiser, A.; Heering, J.; Goebel, T.; Angioni, C.; Wurglics, M.; Paulke,
A.; Geisslinger, G.; Kahnt, A.; Steinhilber, D.; Proschak, E.; Merk, D.
A Dual Modulator of Farnesoid X Receptor and Soluble Epoxide
Hydrolase to Counter Nonalcoholic Steatohepatitis. J. Med. Chem.
2017, 60, 7703−7724.
ACKNOWLEDGMENTS
■
This research was financially supported by the Else-Kroener-
Fresenius Foundation. D.M. is grateful for support by the
Aventis Foundation. E.P. thanks Deutsche Forschungsgemein-
schaft (DFG; Heisenberg-Professur PR1405/7-1) for financial
support. R.R. received an “Erasmus Traineeship” from the
University of Perugia.
̀
(13) Gadaleta, R. M.; Cariello, M.; Sabba, C.; Moschetta, A. Tissue-
Specific Actions of FXR in Metabolism and Cancer. Biochim. Biophys.
Acta, Mol. Cell Biol. Lipids 2015, 1851, 30−39.
(14) Pellicciari, R.; Costantino, G.; Fiorucci, S. Farnesoid X
Receptor: From Structure to Potential Clinical Applications. J. Med.
Chem. 2005, 48, 5383−5403.
ABBREVIATIONS USED
■
BSEP, bile salt export protein; CDCA, chenodeoxycholic acid;
CYP7A1, cholesterol-7α-hydroxylase; FXR, farnesoid X
receptor; LXR, liver X receptor; NAFLD, non-alcoholic fatty
liver disease; NASH, non-alcoholic steatohepatitis; OCA,
obeticholic acid; RAR, retinoic acid receptor; PHOME, (3-
phenyloxiranyl)acetic acid cyano-(6-methoxynapthalen-2-yl)-
methyl ester; PK, pharmacokinetic; PPAR, peroxisome
proliferator-activated receptor; qRT-PCR, quantitative real-
time polymerase chain reaction; RAR, retinoic acid receptor;
RLM, rat liver microsomes; RXR, retinoid X receptor; sEH,
soluble epoxide hydrolase; SREBP1c, sterol regulatory
element-binding protein 1c; THR, thyroid hormone receptor;
VDR, vitamin D receptor
(15) Jiang, C.; Xie, C.; Lv, Y.; Li, J.; Krausz, K. W.; Shi, J.; Brocker,
C. N.; Desai, D.; Amin, S. G.; Bisson, W. H.; Liu, Y.; Gavrilova, O.;
Patterson, A. D.; Gonzalez, F. J. Intestine-Selective Farnesoid X
Receptor Inhibition Improves Obesity-Related Metabolic Dysfunc-
tion. Nat. Commun. 2015, 6, No. 10166.
(16) Mangels, N.; Awwad, K.; Wettenmann, A.; Dos Santos, L. R. B.;
Frömel, T.; Fleming, I. The Soluble Epoxide Hydrolase Determines
Cholesterol Homeostasis by Regulating AMPK and SREBP Activity.
Prostaglandins Other Lipid Mediators 2016, 125, 30−39.
(17) Sun, C. C.; Zhang, C. Y.; Duan, J. X.; Guan, X. X.; Yang, H. H.;
Jiang, H. L.; Hammock, B. D.; Hwang, S. H.; Zhou, Y.; Guan, C. X.;
Liu, S. K.; Zhang, J. PTUPB Ameliorates High-Fat Diet-Induced Non-
Alcoholic Fatty Liver Disease via Inhibiting NLRP3 Inflammasome
Activation in Mice. Biochem. Biophys. Res. Commun. 2020, 523, 1020−
1026.
(18) Zhang, C. H.; Zheng, L.; Gui, L.; Lin, J. Y.; Zhu, Y. M.; Deng,
W. S.; Luo, M. Soluble Epoxide Hydrolase Inhibition with T-TUCB
Alleviates Liver Fibrosis and Portal Pressure in Carbon Tetrachloride-
Induced Cirrhosis in Rats. Clin. Res. Hepatol. Gastroenterol. 2018, 42,
118−125.
(19) Harris, T. R.; Bettaieb, A.; Kodani, S.; Dong, H.; Myers, R.;
Chiamvimonvat, N.; Haj, F. G.; Hammock, B. D. Inhibition of Soluble
Epoxide Hydrolase Attenuates Hepatic Fibrosis and Endoplasmic
Reticulum Stress Induced by Carbon Tetrachloride in Mice. Toxicol.
Appl. Pharmacol. 2015, 286, 102−111.
(20) Bettaieb, A.; Nagata, N.; AbouBechara, D.; Chahed, S.;
Morisseau, C.; Hammock, B. D.; Haj, F. G. Soluble Epoxide
Hydrolase Deficiency or Inhibition Attenuates Diet-Induced
Endoplasmic Reticulum Stress in Liver and Adipose Tissue. J. Biol.
Chem. 2013, 288, 14189−14199.
(21) Spector, A. A.; Fang, X.; Snyder, G. D.; Weintraub, N. L.
Epoxyeicosatrienoic Acids (EETs): Metabolism and Biochemical
Function. Prog. Lipid Res. 2004, 43, 55−90.
(22) Arvind, A.; Osganian, S. A.; Sjoquist, J. A.; Corey, K. E.; Simon,
T. G. Epoxygenase-Derived Epoxyeicosatrienoic Acid Mediators Are
Associated With Nonalcoholic Fatty Liver Disease, Nonalcoholic
Steatohepatitis, and Fibrosis. Gastroenterology 2020, 159, 2232−
2234e4.
(23) Raffaele, M.; Bellner, L.; Singh, S. P.; Favero, G.; Rezzani, R.;
Rodella, L. F.; Falck, J. R.; Abraham, N. G.; Vanella, L.
Epoxyeicosatrienoic Intervention Improves NAFLD in Leptin
Receptor Deficient Mice by an Increase in PGC1α-HO-1-PGC1α-
Mitochondrial Signaling. Exp. Cell Res. 2019, 380, 180−187.
(24) Wang, X.; Li, L.; Wang, H.; Xiao, F.; Ning, Q.
Epoxyeicosatrienoic Acids Alleviate Methionine-Choline-Deficient
Diet−Induced Non-Alcoholic Steatohepatitis in Mice. Scand. J.
Immunol. 2019, 90, No. e12791.
REFERENCES
■
(1) Vuppalanchi, R.; Noureddin, M.; Alkhouri, N.; Sanyal, A. J.
Therapeutic Pipeline in Nonalcoholic Steatohepatitis. Nat. Rev.
Gastroenterol. Hepatol. 2021, 373−392.
(2) Buzzetti, E.; Pinzani, M.; Tsochatzis, E. A. The Multiple-Hit
Pathogenesis of Non-Alcoholic Fatty Liver Disease (NAFLD).
Metabolism 2016, 65, 1038−1048.
(3) MacHado, M. V.; Diehl, A. M. Pathogenesis of Nonalcoholic
Steatohepatitis. Gastroenterology 2016, 150, 1769−1777.
(4) Diehl, A. M.; Day, C. Cause, Pathogenesis, and Treatment of
Nonalcoholic Steatohepatitis. N. Engl. J. Med. 2017, 377, 2063−2072.
(5) Tilg, H.; Adolph, T. E.; Moschen, A. R. Multiple Parallel Hits
Hypothesis in Nonalcoholic Fatty Liver Disease: Revisited After a
Decade. Hepatology 2021, 73, 833−842.
(6) Pellicciari, R.; Fiorucci, S.; Camaioni, E.; Clerici, C.; Costantino,
G.; Maloney, P. R.; Morelli, A.; Parks, D. J.; Willson, T. M. 6α-Ethyl-
Chenodeoxycholic Acid (6-ECDCA), a Potent and Selective FXR
Agonist Endowed with Anticholestatic Activity. J. Med. Chem. 2002,
45, 3569−3572.
(7) Mullard, A. FDA Rejects NASH Drug. Nat. Rev. Drug Discovery
2020, 19, 501.
(8) Maloney, P. R.; Parks, D. J.; Haffner, C. D.; Fivush, A. M.;
Chandra, G.; Plunket, K. D.; Creech, K. L.; Moore, L. B.; Wilson, J.
G.; Lewis, M. C.; Jones, Sa.; Willson, T. M. Identification of a
Chemical Tool for the Orphan Nuclear Receptor FXR. J. Med. Chem.
2000, 43, 2971−2974.
(9) Neuschwander-Tetri, B. A.; Loomba, R.; Sanyal, A. J.; Lavine, J.
E.; Van Natta, M. L.; Abdelmalek, M. F.; Chalasani, N.; Dasarathy, S.;
Diehl, A. M.; Hameed, B.; Kowdley, K. V.; McCullough, A.; Terrault,
N.; Clark, J. M.; Tonascia, J.; Brunt, E. M.; Kleiner, D. E.; Doo, E.
Farnesoid X Nuclear Receptor Ligand Obeticholic Acid for Non-
Cirrhotic, Non-Alcoholic Steatohepatitis (FLINT): A Multicentre,
Randomised, Placebo-Controlled Trial. Lancet 2015, 385, 956−965.
(10) Ratziu, V.; Harrison, S. A.; Francque, S.; Bedossa, P.; Lehert, P.;
Serfaty, L.; Romero-Gomez, M.; Boursier, J.; Abdelmalek, M.;
Caldwell, S.; Drenth, J.; Anstee, Q. M.; Hum, D.; Hanf, R.; Roudot,
(25) Liu, Y.; Dang, H.; Li, D.; Pang, W.; Hammock, B. D.; Zhu, Y.
Inhibition of Soluble Epoxide Hydrolase Attenuates High-Fat-Diet-
9535
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Induced Hepatic Steatosis by Reduced Systemic Inflammatory Status
in Mice. PLoS One 2012, 7, No. e39165.
(42) Heitel, P.; Faudone, G.; Helmstädter, M.; Schmidt, J.; Kaiser,
ller, S.; Schierle, S.; Pollinger, J.;
A.; Tjaden, A.; Schröder, M.; Mu
̈
(26) Schuck, R. N.; Zha, W.; Edin, M. L.; Gruzdev, A.; Vendrov, K.
C.; Miller, T. M.; Xu, Z.; Lih, F. B.; DeGraff, L. M.; Tomer, K. B.;
Jones, H. M.; Makowski, L.; Huang, L.; Poloyac, S. M.; Zeldin, D. C.;
Lee, C. R. The Cytochrome P450 Epoxygenase Pathway Regulates
the Hepatic Inflammatory Response in Fatty Liver Disease. PLoS One
2014, 9, No. e110162.
(27) Hye Khan, M. A.; Schmidt, J.; Stavniichuk, A.; Imig, J. D.;
Merk, D. A Dual Farnesoid X Receptor/Soluble Epoxide Hydrolase
Modulator Treats Non-Alcoholic Steatohepatitis in Mice. Biochem.
Pharmacol. 2019, 166, 212−221.
(28) Helmstädter, M.; Schmidt, J.; Kaiser, A.; Weizel, L.; Proschak,
E.; Merk, D. Differential Therapeutic Effects of FXR Activation, SEH
Inhibition, and Dual FXR/SEH Modulation in NASH in Diet-
Induced Obese Mice. ACS Pharmacol. Transl. Sci. 2021, 966−979.
(29) Wu, F. P.; Peng, J. B.; Qi, X.; Wu, X. F. Palladium-Catalyzed
Carbonylative Transformation of Organic Halides with Formic Acid
as the Coupling Partner and CO Source: Synthesis of Carboxylic
Acids. J. Org. Chem. 2017, 82, 9710−9714.
(30) Schmidt, J.; Klingler, F.-M.; Proschak, E.; Steinhilber, D.;
Schubert-Zsilavecz, M.; Merk, D. NSAIDs Ibuprofen, Indometacin,
and Diclofenac Do Not Interact with Farnesoid X Receptor. Sci. Rep.
2015, 5, No. 14782.
Merk, D. A Triple Farnesoid X Receptor and Peroxisome Proliferator-
Activated Receptor α/δ Activator Reverses Hepatic Fibrosis in Diet-
Induced NASH in Mice. Commun. Chem. 2020, 3, No. 174.
(43) Helmstädter, M.; Vietor, J.; Sommer, J.; Schierle, S.; Willems,
S.; Kaiser, A.; Schmidt, J.; Merk, D. A New FXR Ligand Chemotype
with Agonist/Antagonist Switch. ACS Med. Chem. Lett. 2021, 12,
267−274.
(44) Steri, R.; Achenbach, J.; Steinhilber, D.; Schubert-Zsilavecz, M.;
Proschak, E. Investigation of Imatinib and Other Approved Drugs as
Starting Points for Antidiabetic Drug Discovery with FXR Modulating
Activity. Biochem. Pharmacol. 2012, 83, 1674−1681.
(45) Seuter, S.; Väisänen, S.; Rådmark, O.; Carlberg, C.; Steinhilber,
D. Functional Characterization of Vitamin D Responding Regions in
the Human 5-Lipoxygenase Gene. Biochim. Biophys. Acta, Mol. Cell
Biol. Lipids 2007, 1771, 864−872.
(46) Wolf, N. M.; Morisseau, C.; Jones, P. D.; Hock, B.; Hammock,
B. D. Development of a High-Throughput Screen for Soluble Epoxide
Hydrolase Inhibition. Anal. Biochem. 2006, 355, 71−80.
(47) Heitel, P.; Achenbach, J.; Moser, D.; Proschak, E.; Merk, D.
DrugBank Screening Revealed Alitretinoin and Bexarotene as Liver X
Receptor Modulators. Bioorg. Med. Chem. Lett. 2017, 27, 1193−1198.
(48) Flesch, D.; Cheung, S.-Y.; Schmidt, J.; Gabler, M.; Heitel, P.;
Kramer, J. S.; Kaiser, A.; Hartmann, M.; Lindner, M.; Lu
̈
ddens-
(31) Blöcher, R.; Lamers, C.; Wittmann, S. K.; Merk, D.; Hartmann,
Dämgen, K.; Heering, J.; Lamers, C.; Luddens, H.; Wurglics, M.;
̈
M.; Weizel, L.; Diehl, O.; Bru
Schader, T.; Göbel, T.; Grundmann, M.; Angioni, C.; Heering, J.;
Geisslinger, G.; Wurglics, M.; Kostenis, E.; Brune, B.; Steinhilber, D.;
̈
ggerhoff, A.; Boß, M.; Kaiser, A.;
Proschak, E.; Schubert-Zsilavecz, M.; Merk, D. Non-Acidic Farnesoid
X Receptor Modulators. J. Med. Chem. 2017, 60, 7199−7205.
(49) Gellrich, L.; Heitel, P.; Heering, J.; Kilu, W.; Pollinger, J.;
Goebel, T.; Kahnt, A.; Arifi, S.; Pogoda, W.; Paulke, A.; Steinhilber,
D.; Proschak, E.; Wurglics, M.; Schubert-Zsilavecz, M.; Chaikuad, A.;
̈
Schubert-Zsilavecz, M.; Kahnt, A.; Proschak, E. N-Benzylbenzamides:
A Novel Merged Scaffold for Orally Available Dual Soluble Epoxide
Hydrolase/Peroxisome Proliferator-Activated Receptor γ Modulators.
J. Med. Chem. 2016, 59, 61−81.
Knapp, S.; Bischoff, I.; Furst, R.; Merk, D. L-Thyroxin and the
̈
Nonclassical Thyroid Hormone TETRAC Are Potent Activators of
PPARγ. J. Med. Chem. 2020, 63, 6727−6740.
(50) Rau, O.; Wurglics, M.; Paulke, A.; Zitzkowski, J.; Meindl, N.;
Bock, A.; Dingermann, T.; Abdel-Tawab, M.; Schubert-Zsilavecz, M.
Carnosic Acid and Carnosol, Phenolic Diterpene Compounds of the
Labiate Herbs Rosemary and Sage, Are Activators of the Human
Peroxisome Proliferator-Activated Receptor Gamma. Planta Med.
2006, 72, 881−887.
(51) Heitel, P.; Gellrich, L.; Kalinowsky, L.; Heering, J.; Kaiser, A.;
Ohrndorf, J.; Proschak, E.; Merk, D. Computer-Assisted Discovery
and Structural Optimization of a Novel Retinoid X Receptor Agonist
Chemotype. ACS Med. Chem. Lett. 2019, 10, 203−208.
(32) Jones, P. D.; Wolf, N. M.; Morisseau, C.; Whetstone, P.; Hock,
B.; Hammock, B. D. Fluorescent Substrates for Soluble Epoxide
Hydrolase and Application to Inhibition Studies. Anal. Biochem. 2005,
343, 66−75.
(33) Obach, R. S. Prediction of Human Clearance of Twenty-Nine
Drugs from Hepatic Microsomal Intrinsic Clearance Data: An
Examination of In Vitro Half-Life Approach and Nonspecific Binding
to Microsomes. Drug Metab. Dispos. 1999, 27, 1350−1359.
(34) Brian Houston, J. Utility of in Vitro Drug Metabolism Data in
Predicting in Vivo Metabolic Clearance. Biochem. Pharmacol. 1994,
47, 1469−1479.
(35) Westphal, M. V.; Wolfstädter, B. T.; Plancher, J.-M.; Gatfield,
J.; Carreira, E. M. Evaluation of Tert-Butyl Isosteres: Case Studies of
Physicochemical and Pharmacokinetic Properties, Efficacies, and
Activities. ChemMedChem 2015, 10, 461−469.
(36) Barnes-Seeman, D.; Jain, M.; Bell, L.; Ferreira, S.; Cohen, S.;
Chen, X. H.; Amin, J.; Snodgrass, B.; Hatsis, P. Metabolically Stable
Tert-Butyl Replacement. ACS Med. Chem. Lett. 2013, 4, 514−516.
(37) Sowaileh, M. F.; Hazlitt, R. A.; Colby, D. A. Application of the
Pentafluorosulfanyl Group as a Bioisosteric Replacement. Chem-
MedChem 2017, 12, 1481−1490.
(38) Kulinkovich, O. G. The Chemistry of Cyclopropanols. Chem.
Rev. 2003, 103 (103), 2597−2632.
(39) Merk, D.; Lamers, C.; Ahmad, K.; Carrasco Gomez, R.;
Schneider, G.; Steinhilber, D.; Schubert-Zsilavecz, M. Extending the
Structure-Activity Relationship of Anthranilic Acid Derivatives as
Farnesoid x Receptor Modulators: Development of a Highly Potent
Partial Farnesoid x Receptor Agonist. J. Med. Chem. 2014, 57, 8035−
8055.
(40) Merk, D.; Sreeramulu, S.; Kudlinzki, D.; Saxena, K.; Linhard,
V.; Gande, S. L.; Hiller, F.; Lamers, C.; Nilsson, E.; Aagaard, A.;
Wissler, L.; Dekker, N.; Bamberg, K.; Schubert-Zsilavecz, M.;
Schwalbe, H. Molecular Tuning of Farnesoid X Receptor Partial
Agonism. Nat. Commun. 2019, 10, No. 2915.
(41) Schierle, S.; Schmidt, J.; Kaiser, A.; Merk, D. Selective
Optimization of Pranlukast to Farnesoid X Receptor Modulators.
ChemMedChem 2018, 13, 2530−2545.
9536
https://doi.org/10.1021/acs.jmedchem.1c00831
J. Med. Chem. 2021, 64, 9525−9536