Oxaprozin Analogues as Selective RXR Agonists with Superior Properties and Pharmacokinetics

Article abstract of DOI:10.1021/acs.jmedchem.1c00235

The retinoid X receptors (RXR) are ligand-activated transcription factors involved in multiple regulatory networks as universal heterodimer partners for nuclear receptors. Despite their high therapeutic potential in many pathologies, targeting of RXR has only been exploited in cancer treatment as the currently available RXR agonists suffer from exceptional lipophilicity, poor pharmacokinetics (PK), and adverse effects. Aiming to overcome the limitations and to provide improved RXR ligands, we developed a new potent RXR ligand chemotype based on the nonsteroidal anti-inflammatory drug oxaprozin. Systematic structure-activity relationship analysis enabled structural optimization toward low nanomolar potency similar to the well-established rexinoids. Cocrystal structures of the most active derivatives demonstrated orthosteric binding, and in vivo profiling revealed superior PK properties compared to current RXR agonists. The optimized compounds were highly selective for RXR activation and induced RXR-regulated gene expression in native cellular and in vivo settings suggesting them as excellent chemical tools to further explore the therapeutic potential of RXR.

Full text of DOI:10.1021/acs.jmedchem.1c00235

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Article
Oxaprozin Analogues as Selective RXR Agonists with Superior
Properties and Pharmacokinetics
Simone Schierle, Apirat Chaikuad, Felix F. Lillich, Xiaomin Ni, Stefano Woltersdorf, Espen Schallmayer,
Beatrice Renelt, Riccardo Ronchetti, Stefan Knapp, Ewgenij Proschak, and Daniel Merk*
Cite This: J. Med. Chem. 2021, 64, 5123−5136
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ABSTRACT: The retinoid X receptors (RXR) are ligand-
activated transcription factors involved in multiple regulatory
networks as universal heterodimer partners for nuclear receptors.
Despite their high therapeutic potential in many pathologies,
targeting of RXR has only been exploited in cancer treatment as
the currently available RXR agonists suffer from exceptional
lipophilicity, poor pharmacokinetics (PK), and adverse effects.
Aiming to overcome the limitations and to provide improved RXR
ligands, we developed a new potent RXR ligand chemotype based
on the nonsteroidal anti-inflammatory drug oxaprozin. Systematic
structure−activity relationship analysis enabled structural opti-
mization toward low nanomolar potency similar to the well-
established rexinoids. Cocrystal structures of the most active
derivatives demonstrated orthosteric binding, and in vivo profiling revealed superior PK properties compared to current RXR
agonists. The optimized compounds were highly selective for RXR activation and induced RXR-regulated gene expression in native
cellular and in vivo settings suggesting them as excellent chemical tools to further explore the therapeutic potential of RXR.
intermediate micromolar potency and low activation efficacy
with a slight functional preference for the RXRα and RXRβ
subtypes (3: RXRα, EC50 16 μM, 23% act.; RXRβ, EC50 25 μM,
43% act.; RXRγ, EC50 40 μM, 9% act.). As an approved drug,
oxaprozin (3) provided a highly attractive privileged scaffold for
the development of a new RXR ligand chemotype. It has
preferable aqueous solubility (3: 13.3 mg/L; bexarotene (2): 0.3
mg/L20), favorable PK, and clinically confirmed safety. These
features render 3 a superior lead to develop potent RXR
activators with improved characteristics compared to bexar-
otene (2) and other known RXR ligand chemotypes. Following
the concept of selective optimization of side-activities
(SOSA),27 we engaged on oxaprozin (3) as a lead for the
development of a new class of selective RXR agonists and
systematically elucidated its structure−activity relationship
(SAR) as an RXR ligand with analogues 4−49 comprising
minor structural modifications of the NSAID. We succeeded in
designing the RXR agonist 38 and the partial agonist 49 which
exhibit high potency on RXRs and outmatch the reference drug
INTRODUCTION
■
The nuclear retinoid X receptor (RXR) comprises a ligand-
activated transcription factor family existing in three highly
conserved isoforms (RXRα, NR2B1; RXRβ, NR2B2; and RXRγ,
NR2B3).1,2 The RXRs fulfill a central role in the nuclear
receptor superfamily acting not only as universal heterodimer
partners but also as homodimers regulating gene expression.
Several fatty acids and vitamin A metabolites have been
discovered as RXR agonists among which 9-cis retinoic acid
(1, Chart 1) is the most potent ligand.2−5 As the only approved
synthetic rexinoid, bexarotene (2) is used as second-line
treatment in cancer;3,6,7 however, its use has been associated
with severe adverse effects.8,9 Considerable preclinical evidence
suggests a high therapeutic potential of RXR activation to
counteract neurodegenerative diseases such as multiple
sclerosis10−12 and Alzheimer’s disease.13−15 Despite recent
progress in targeting RXRs,2,16−21 new potent RXR agonists are
needed to overcome the limitations of traditional rexinoids, such
as exceptional lipophilicity, poor pharmacokinetics (PK), and
pronounced toxicity,3,22−26 to further probe and exploit the
highly promising pharmacological potential of RXR modulation.
By screening a collection of clinically approved fatty acid
mimetics for nuclear receptor modulation in uniform Gal4
hybrid reporter gene assays, we discovered the nonsteroidal anti-
inflammatory drug (NSAID) oxaprozin (3) as a weak RXR
agonist. The drug activated all three RXR subtypes with
Received: February 6, 2021
Published: April 1, 2021
© 2021 The Authors. Published by
American Chemical Society
https://doi.org/10.1021/acs.jmedchem.1c00235
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Chart 1. RXR Agonists 9-cis Retinoic Acid (1) and Bexarotene (2) and the Lead Compound Oxaprozin (3)
bexarotene (2) in terms of lipophilicity, cytotoxicity, and
pharmakokinetic parameters. The novel RXR ligands induced
RXR-regulated gene expression in native cellular setting and in
mice. Cocrystal structures of the RXRα:28 and RXRα:38
complexes revealed orthosteric binding and favorable occupa-
tion of the canonical RXR ligand-binding site. Oxaprozin-
derived RXR ligands may therefore serve as superior next-
generation RXR agonists offering a chemical tool with highly
improved characteristics over previously reported rexinoids for
pharmacology and mechanistic studies.3
10 and 38 were not accessible via this procedure and were
prepared according to Scheme 2. For this, bromoacetophenone
(132) was converted with succinic acid to ester 133 which
subsequently served as the substrate for oxazole synthesis
according to Pei et al.30 to obtain 10. Suzuki coupling then
enabled the synthesis of 38 from the brominated precursor 134
in good overall yield.
Synthesis of pyrazole derivatives 39−49 was carried out in a
two-step procedure according to Scheme 3. The respective
acetophenones 136−137 were reacted with succinic anhydride
or glutaric anhydride to obtain the diketones 138−140 in a
Claisen condensation with moderate yields. 138−140 were then
treated with phenylhydrazines 141−148 to form pyrazoles 39−
49 in good yields with few exceptions (39, 46, and 49).
Biological Evaluation. The in vitro characterization of 3
and analogues 4−49 for RXR modulation was performed in
uniform Gal4 hybrid reporter gene assays for RXRα, RXRβ, and
RXRγ. In these cellular (HEK293T) in vitro test systems,
chimeric receptors composed of the ligand-binding domain
(LBD) of the respective human nuclear receptor and the DNA-
binding domain (DBD) of the yeast protein Gal4 are employed
to govern expression of a Gal4 responsive firefly luciferase
reporter gene. Additionally, a constitutively expressed renilla
luciferase was used as an internal control for transfection
efficiency, cell growth, and test compound toxicity. All
compounds were characterized relative to the reference RXR
agonist bexarotene (2) whose activity at 1 μM was defined as
100% RXR activation. For comparison, the natural RXR ligand
9-cis RA (1) exhibited 77% (RXRα), 71% (RXRβ), and 75%
(RXRγ) max. relative RXR activation compared to 2 in this
assay.
RESULTS AND DISCUSSION
Chemistry. Oxaprozin analogues 4−7, 10−18, 20−29, and
32−46 were prepared according to Schemes 1−3. 8, 19, and
■
Scheme 1. Synthesis of Oxazole Derivatives 4−7, 11−18, 20−
a
29, and 32−37
Structure−Activity Relationship. We commenced our
systematic SAR evaluation by varying the length of the alkanoic
acid side chain of 3 (Table 1). Analogues 4 and 5 with a
shortened spacer failed to activate RXR while the butyric acid
derivative 6 gained in potency on RXRα and RXRβ by a factor 2
compared to 3 and was equipotent on RXRγ. Further chain
elongation in 7 markedly diminished activation efficacy
highlighting propionic acid (3) and butyric acid (6) as favored
side chain motifs. Additionally, this SAR of the acidic side chain
indicated potential to obtain RXRα/β preference with longer
chains. Next, we probed the importance of the carboxylate group
by replacing it with an amide in 8 which turned out inactive
agreeing with the role of RXR as the fatty acid sensor. The
removal of either phenyl substituent of the central oxazole
moiety in 9 and 10 was also not tolerated. Overall, these results
demonstrated that the scaffold and side chain of 3 were favorable
for optimization toward potent RXR ligands.
a
Reagents and conditions: (a) LiHMDS or NaHMDS, DMF, −4 °C,
3−4 h, 22−93%; (b) R-CN (99−104), I2, oxone, TfOH, reflux, 6 h,
14−87%; (c) LiOH, EtOH, H2O, rt, 12 h (esters 105, 106, 109−113,
115−127, and 129−131) or NaOH, H2O2, DMF, 100 °C, 20 h
(nitriles 107, 108, 114, and 128), 24−89%.
30−31 were previously reported,28 and 9 was commercially
available. Synthesis of oxazole derivatives 4−7, 11−18, 20−29,
and 32−37 followed previously published two- to three-step
procedures29 (Scheme 1). For this, methyl benzoates 50−66
and phenylacetic acids 67−74 were reacted in a Claisen
condensation and decarboxylation to phenylbenzylketones 75−
97 in good to excellent yields except for the 2-methyl (78) and
difluoro (95) derivatives. 98 was commercially available. The
phenylbenzylketones 75−98 were then cyclized with nitriles
99−104 to oxazoles 105−131 in the presence of iodine, TfOH,
and oxidant oxone with varying yields. Alkaline ester (105, 106,
109−113, 115−127, and 129−131) or nitrile (107, 108, 114,
and 128) hydrolysis provided RXR modulators 4−7, 11−18,
20−29, and 32−37 in moderate (nitrile) to good (ester) yields.
We thus retained 3 as a lead compound and focused on minor
structural modifications to enhance potency on RXRs (Table 2).
In order to locate the available space in the RXR ligand-binding
site and identify substituent vectors for further groups, we
systematically introduced methyl and chlorine moieties in all
positions of the 4- and 5-phenyl residues (11−22). On the 5-
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a
Scheme 2. Synthesis of Oxazole Derivatives 10 and 38
a
Reagents and conditions: (a) succinic acid, Et3N, acetone, rt, 12 h, 27%; (b) BF3·Et2O, acetamide, 140 °C, 24 h, 40%; (c) NBS, NH4OAc, ACN,
rt, 2 h, 35%; (d) boronic acid 135, Pd(Ph3)4, Na2CO3, dioxane/H2O (4:1), 100 °C, 12 h, 65%.
a
substituent (12) enhanced potency by more than a factor 30 and
particularly enhanced RXRγ activation efficacy. A 4-chloro
moiety (13) remarkably promoted RXRβ activation efficacy
with less impact on potency. Similarly, a methyl group in 2-
position of the 5-phenyl residue (14) was tolerated while 3- (15)
and 4-methyl (16) modifications enhanced potency with 15 as
the most favored methyl derivative. Following the same strategy
as with 11−16, we then studied the SAR of the 4-phenyl moiety
(17−22) where we observed an equal ranking of potency for 2-,
3-, and 4-substituents but with lower overall activity. This
prompted us to speculate about a potential flipped binding mode
leading to the loss of direct contacts of the oxazole oxygen upon
structural modifications in the 4-phenyl group. The 4-chloro
derivative 19 deviated from this trend suggesting that this
modification might be favored and retained the original binding
mode.
In the systematic sampling of the binding site with chlorine
and methyl substituents, modifications in the 3-position of the 5-
phenyl residue (12 and 15) achieved the highest potency on
RXRs. Thus, we introduced further alternative groups in this
position and observed a distinctive SAR. The 3-dimethylamino
group in 23 was detrimental for potency on RXRs, and while
derivative 24 remained active, its 3-methoxy decoration was less
Scheme 3. Synthesis of Pyrazole Derivatives 39−49
a
Reagents and conditions: (a) succinic anhydride or glutaric
anhydride, LiHMDS, THF, rt, 12 h, 27−28%; (b) NaOAc,
CH3COOH, 70 °C, 3 h or Et3N, MeOH, rt, 16 h (in the case of
49), 17−74%.
phenyl substituent, chlorine was tolerated in 2-position (11) and
favored in 3- (12) and 4-positions (13). Therein, a 3-chloro
a
Table 1. RXR Agonism of 3−10 in Cellular Hybrid Reporter Gene Assays
a
Data are mean S.E.M.; n ≥ 3. Max. rel. activation refers to the activity of reference RXR agonist bexarotene (2) at 1 μM, which was defined as
100% activation. Inactiveno significant activation at the indicated concentration.
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a
Table 2. RXR Agonism of 11−25 in Cellular Hybrid Reporter Gene Assays
a
Compound 3 was included for comparison. Data are the mean S.E.M.; n ≥ 3. Max. rel. activation refers to the activity of reference RXR agonist
bexarotene (2) at 1 μM which was defined as 100% activation. Inactiveno significant activation at the indicated concentration.
favored compared to 12. 3-Trifluoromethyl substitution in 25, in
contrast, further enhanced potency compared to 3-chloro
derivative 12 to double-digit nanomolar EC50 values.
RXRs compared to 12. 3,5-Dichloro substitution was also
strongly favored in terms of activation efficacy. The remaining
2,5-dichloro analogue 29 was significantly less active especially
on the RXRγ subtype indicating potential of this substitution
pattern to obtain subtype preference. Although the series of
monosubstituted derivatives had already suggested preference
for substituents on the 5-phenyl ring, we also transferred the
favored 3,4- (30) and 3,5-dichloro substitution (31) to the 4-
phenyl residue, but as anticipated, both were clearly less active
than 27 and 28. A similar scenario was observed when 3,5-
dichloro substituents were introduced at both phenyl rings (32).
Since 4-chloro substitution on the 4-phenyl residue (19) had
achieved the highest potency among monosubstitutions on this
ring, we combined it with the 3,5-dichlorophenyl substituent in
the 5-position of the oxazole, from which derivative 33 indeed
exhibited higher potency than 32 but did not outmatch 28.
Furthermore, based on our early SAR analysis suggesting
preference for a propanoic acid (3) or a butyric acid (6) side
chain, we studied elongation of the side chain in optimized
derivative 28. The respective butyric acid analogue 34 was
These early SAR results also revealed two trends regarding
structural contributions to activation efficacy. Electron-with-
drawing substituents mostly achieved higher activation efficacy
than the respective alkyl analogues (e.g. 13 vs 16), and
derivatives with substituents on the 4-phenyl residue (17−22)
in addition to being less potent were characterized as partial
RXR agonists with reduced activation efficacy compared to
analogues with modifications on the 5-phenyl motif (11−16).
Substitution in 3- and 4-positions of the phenyl residues had
enhanced activity on RXRs, and chlorine as well as
trifluoromethyl achieved higher potency suggesting that
electron withdrawing substituents were favored. Consequently,
we systematically probed double chloro substitution on the 5-
phenyl ring (26−29, Table 3). 2,3-Dichloro analogue 26 was
inferior to 3-chloro derivative 12 while 3,4-dichloro substitution
(27) achieved equal potency as 12. The 3,5-dichloro isomer 28,
however, increased the potency by an order of magnitude for all
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a
Table 3. RXR Agonism of 26−34 in Cellular Hybrid Reporter Gene Assays
a
Compound 3 was included for comparison. Data are mean S.E.M.; n ≥ 3. Max. rel. activation refers to the activity of reference RXR agonist
bexarotene (2) at 1 μM concentration, which was defined as 100% activation. Inactiveno significant activation at the indicated concentration.
a
Table 4. RXR Agonism of 35−38 in Cellular Hybrid Reporter Gene Assays
a
Compounds 3 and 28 were included for comparison. Data are mean S.E.M.; n ≥ 3. Max. rel. activation refers to the activity of reference RXR
agonist bexarotene (2) at 1 μM concentration, which was defined as 100% activation.
markedly less active than 28, however. These SAR observations
characterized 3,5-disubstitution on the 5-phenyl ring as highly
preferred motif to achieve strong RXR agonism prompting us to
evaluate alternative groups in this substitution pattern (Table 4).
The smaller 3,5-difluoro analogue 35 was considerably less
active than 28 demonstrating that not only electrostatic but also
steric characteristics affect potency in this region. Among
aliphatic substituents, the smaller 3,5-dimethyl analogue 36 as
well as the bulky 3,5-di-tert-butyl substitution pattern in 37
achieved high agonist potencies on the RXRs but were inferior to
28. Dimethyl derivative 36, interestingly, revealed a slight
preference for RXRβ. The 3,5-bis(trifluoromethyl) analogue 38
evolved as a highly favored RXR agonist with equal potency as
28 on RXRα and RXRβ and enhanced activity on RXRγ. With
EC50 values of 0.05, 0.04 and 0.05 μM, respectively, on RXRα,
RXRβ, and RXRγ, 38 evolved as an exceptionally strong and
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balanced RXR activator exhibiting high activation efficacy on all
three isoforms. With this activity profile, 38 equals the reference
agonist bexarotene (2) in RXR agonist potency.
28 and 38 evolved as very attractive novel RXR modulators
and hence were broadly profiled for their pharmacological
potential. Aqueous solubility of 28 (89.2 0.2 mg/L) and 38
(145.8
0.6 mg/L) was markedly higher compared to the
reference RXR agonist bexarotene (2, 0.3 mg/L20) demonstrat-
ing improved physicochemical properties. While bexarotene (2)
and 28 exhibited pronounced cytotoxicity against HepG2 cells,
38 was nontoxic up to 100 μM concentration (Figure 1a)
suggesting that the toxic effects of 2 and 28 were not RXR-
mediated.
38 was highly selective for RXRs over related nuclear
receptors (Figure 1b) and did neither activate the retinoic acid
receptor (RARα), the peroxisome proliferator-activated recep-
tors (PPAR), the liver X receptors (LXR), the farnesoid X
receptor (FXR), and the constitutive androstane receptor
(CAR) nor counteract their (intrinsic) activity. No cyclo-
oxygenase inhibition was detected for 38 at 10 μM (Figure 1c)
demonstrating that the original activity of the lead compound
oxaprozin (3) was designed out. Moreover, the lead compound
3 and the optimized RXR agonists 28 and 38 exhibited favorable
stability against microsomal degradation suggesting preferable
metabolic stability (Figure 1d). Therein, 38 was the most stable
compound exceeding the lead 3, and in vivo pharmacokinetic
analysis of 38 after oral administration (10 mg/kg) confirmed a
very favorable PK profile for this RXR agonist with a half-life of
more than 3 h and no liver accumulation (Figure 1e,f).
To confirm target engagement in native settings, we then
studied the effects of 38 on RXR-regulated gene expression in
the human hepatocyte cell line HepG2 (Figure 1g). At 1 μM
concentration, 38 caused a robust mRNA induction of the RXR
heterodimer-regulated cholesterol and phospholipid transporter
ATP-binding cassette A1 (ABCA1) as well as the transcription
factor sterol regulatory element-binding protein 1c (SREBP1c)
confirming RXR modulation in this cellular setting. In mice, a
single 10 mg/kg oral dose of 38 promoted hepatic expression of
the RXR heterodimer-regulated genes ABCA1, ApoE, and
SREBP1c (Figure 1h) demonstrating RXR activation by 38 in
vivo. Thus, 38 qualifies as a potent next-generation RXR agonist
tool with highly favorable characteristics and efficient target
engagement in vitro and in vivo.
To gain structural insights into RXR modulation by 28 and 38
as representatives of the new RXR agonist chemotype, we solved
the cocrystal structures of the RXRα-28 (pdb id 7B88) and
RXRα-38 (pdb id 7B9O) complexes (Figure 2). Both RXR
agonists 28 and 38 bind to the canonical orthosteric ligand-
binding site within the RXR LBD and engage in an ionic
interaction with Arg316 via the carboxylic acid motif which is
also present in classical rexinoids.3,32 As observed in the early
SAR elucidation, the chain length between the oxazole and
carboxylic acid group is favorable. Additionally, the carboxylate
residue of 38 forms a water mediated H-bond with the backbone
of Leu309. Apart from these two strong polar interactions, no
further directed contacts between the ligand and the RXR LBD
are obvious. In particular, no interactions are observed for the
central oxazole. The 3,5-bis(trifluoromethyl)phenyl group of 38
is favorably bound in a highly lipophilic cavity formed by Ile268,
Phe313, Val342, Ile345, Phe346, and Phe439. The unsub-
stituted 4-phenyl motif points toward a tunnel between Trp305,
Asn306, and Leu433 where an additional unoccupied space
seems to be available in the binding site.
Figure 1. In vitro and in vivo profiling of oxazole-based RXR agonists 28
and 38. (a) 28, similar to the reference RXR agonist bexarotene (2),
exhibits pronounced cytotoxicity at concentrations above 10 μM. 38, in
contrast, revealed no toxic effect up to 100 μM concentration. Data are
mean S.E.M. relative cell viability (HepG2) determined by the WST-
1 assay; n = 4. (b) 38 at 10 μM concentration exclusively activated
RXRs and was inactive on the retinoic acid receptor (RARα),
peroxisome proliferator-activated receptors (PPARs), liver X receptors
(LXRs), farnesoid X receptor (FXR), and constitutive androstane
receptor (CAR) demonstrating strong selectivity. The heatmap shows
mean relative nuclear receptor activation compared to reference
agonists; n ≥ 3. (c) 38 at 10 μM concentration did not affect COX-1
activity also demonstrating selectivity over the original protein target of
the lead compound 3. Data are mean SD relative COX-1 activity; n =
2. (d) Stability of the lead compound 3 as well as the derivatives 28 and
38 against microsomal degradation was favorably high with RXR
agonist 38 comprising the highest stability. Data are mean S.E.M.
remaining compound [%] after the indicated incubation time; n = 3. (e)
In mice, 38 revealed favorable PK after oral administration (10 mg/kg).
Data are mean S.E.M.; n = 3. (f) 38 showed no hepatic accumulation
in mice. Data are mean S.E.M.; n = 3. (g) In human hepatocytes
(HepG2), 38 promoted the mRNA expression of RXR heterodimer-
regulated genes ABCA1 and SREBP1c. Data are mean S.E.M.; n = 4.
#p < 0.1, *p < 0.05 (t-test vs DMSO-treated cells). (h) A single oral dose
of 10 mg/kg 38 induced the hepatic expression of RXR-regulated genes
ABCA1, ApoE, and SREBP1c in mice. Data are mean S.E.M. relative
mRNA expression 3 h after administration of vehicle (n = 3) or 38 (n =
3).
Comparison of the RXRα-38 cocrystal structure (pdb id
7B9O) with RXRα bound to the natural ligand 9-cis retinoic acid
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Figure 2. Co-crystal structures of the RXRα LBD complexed with 28 and 38. (a) Superimposition of both complexes demonstrates a similar biding
mode of 28 (pdb id 7B88) and 38 (pdb id 7B9O). Binding modes of 28 (b) and 38 (c) reveal similar engagement in a strong polar contact with Arg316.
For 38, an additional water-mediated H-bond with the backbone of Leu309 is observed. Both the 3,5-dichlorophenyl residue of 28 and the 3,5-
bis(trifluoromethyl)phenyl motif of 38 are accommodated in a hydrophobic cavity between Ile268, Val342, Ile345, Phe346, and Phe439, while the
unsubstituted 5-phenyl substituent points toward an unoccupied tunnel between Trp305, Asn306, and Leu433. (d) Superimposition of RXRα co-
crystal structures bound to 38 (pdb id 7B9O) and 9-cis retinoic acid (1, pdb id 3OAP31) demonstrates occupation of the same binding site and similar
interaction with Arg316 as well as water-mediated H-bonding to Leu309 for the natural (1) and synthetic (38) RXR agonists. The 3,5-
bis(trifluoromethyl)phenyl motif of 38 binds to the same lipophilic cavity as the terminal trimethylcyclohexene residue of 1, while the unsubstituted 4-
phenyl ring of 38 overlaps with the 7-methyl group in the side chain of 1.
(1, pdb id 3OAP31) revealed no notable differences in the
overall RXR conformation. The Y-shaped, trisubstituted oxazole
derivative 38 and the L-shaped lipid 1 occupy the same binding
site (Figure 2d). The 3,5-bis(trifluoromethyl)-decorated 5-
phenyl motif of 38 is bound to the same region of the RXR
ligand-binding site as the lipophilic trimethylcyclohexene
residue of 1. The unsubstituted 4-phenyl substituent of 38 fills
the binding region of the 7-methyl group in the side chain of 1
with 38 further protruding toward helices H11 and H12. The
carboxylic acid motifs of the natural and the synthetic RXR
agonist align well and both form polar contacts with Arg316 and
the backbone of Leu309 the latter of which is water mediated.
Both the RXRα-28 and RXRα-38 complex structures
indicated no direct contacts of the oxazole motif suggesting a
possibility for its replacement by alternative 5-membered
heterocycles to gain a further solubility improvement. Moreover,
extension of the 4-phenyl residue with substituents in the 3- or 4-
position seemed to hold potential for further optimization.
Following these observations, we designed pyrazole analogues
39−44 (Table 5). Transferring all structural features of oxazole
28 to pyrazole 39 produced a potent RXR agonist with
intermediate activation efficacy despite an approx. 10-fold loss in
potency compared to 28. Introduction of a 3-chloro substituent
on the free phenyl motif in 40 was not well tolerated while a 4-
chlorine atom (41) retained high potency and reinstated full
RXR agonism. 4-Fluoro (42), 4-cyano (43), 4-methoxy (44),
and 4-methyl (45) substituents were less favored. In an attempt
to further increase polarity, we also replaced the free phenyl ring
by an N-methyl pyrazole (46) but this modification resulted in a
marked loss in RXR agonism further highlighting that increasing
polarity of RXR ligands has limitations.
Eventually, we combined the obtained SAR knowledge to
design potentially further optimized pyrazole-based RXR
modulators (Table 5). For the oxazole-based RXR ligands, the
3,5-bis(trifluoromethyl) substitution pattern (38) was slightly
superior to the 3,5-dichloro analogue (28) and hence, we
transferred this motif to the most active pyrazole 41. The
resulting compound 47 exhibited potent RXR agonism but was
inferior to 41 in terms of RXR activation efficacy. Chain
elongation to butyric acid (6) was another compatible
modification with potential for further optimization. However,
the butyric acid analogue 48 of the most active pyrazole-based
RXR agonist 41 was slightly less active, while concomitant
removal of the 4-chloro substituent in 49 was favored on all RXR
subtypes with moderate activation efficacy rendering 49 a potent
partial RXR agonist.
In addition to the pan-RXR agonist 38 comprising high
potency and desirable properties, the pyrazole-based RXR
agonist 41 and the pyrazole-based partial agonist 49 emerged as
further attractive oxaprozin-inspired RXR modulators with a
novel scaffold. Especially, the balanced and high potency
combined with a moderate approx. 25% activation efficacy
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a
Table 5. RXR Agonism of 39−49 in Cellular Hybrid Reporter Gene Assays
a
Data are mean S.E.M.; n ≥ 3. Max. rel. activation refers to the activity of reference RXR agonist bexarotene (2) at 1 μM concentration, which
was defined as 100% activation. Inactiveno significant activation at the indicated concentration.
qualify 49 as the most suitable partial RXR agonist tool of the
presented compound class. As for 38, in vitro profiling of 41 and
49 revealed low toxicity (Figure 3a) and high selectivity over
related transcription factors (Figure 3b). Stability against
microsomal degradation (Figure 3c) was exceptionally high,
and in vivo PK of 41 and 49 (Figure 3d) were preferable, too,
with a half-life greater than 3 h and high plasma concentrations
(∼35 μg/mL) for both compounds. The RXR modulator profile
of the pyrazole-based RXR ligands observed in the reporter gene
assay was confirmed in native cellular setting with strong
induction of RXR-regulated gene expression in HepG2 cells by
RXR agonist 41 and a weaker effect for the partial agonist 49
(Figure 3e). RXR modulation by 41 and 49 was also observed in
mice (Figure 3f). After oral administration of 10 mg/kg 41,
hepatic expression of RXR heterodimer-regulated ABCA1,
ApoE, and SREBP1c was markedly enhanced, while 10 mg/kg
49 caused a weaker effect aligning with the compound’s partial
agonist profile. With favorable PK and target engagement in vitro
and in vivo, pyrazoles 41 and 49 evolve as highly attractive novel
RXR modulators.
overcome these obstacles and provide a new generation of RXR
modulators with improved characteristics, we have sought for
superior RXR agonist lead structures among approved drugs and
discovered oxaprozin (3) as attractive lead candidate for RXR
ligand development. Additionally, the observation of marked
RXR activation by oxaprozin (3) may have clinical relevance.
The NSAID is typically administered in 600−1200 mg doses
which produce plasma levels of up to around 100 mg/L (0.34
mM).33 Thus, 3 reaches plasma concentrations an order of
magnitude above its EC50 values for RXR activation. Oxaprozin
(3) is used, among others, to treat rheumatoid arthritis and was
found to reach high 100 μM concentrations also in the synovial
fluid which is considered as the primary site of action for
NSAIDs in arthritis.33 Involvement of RXR and RXR
heterodimer activation in the clinical anti-inflammatory effects
of oxaprozin (3) can, hence, be speculated. Of note, an anti-
inflammatory activity in arthritis has been shown, for example,
for the activation of PPARγ, LXR, and VDR, which form
permissive RXR heterodimers that can be activated by RXR
agonists.34−36
Using oxaprozin (3) as a lead, we have followed the concept of
SOSA and systematically optimized 3 to potent RXR
modulators while conserving the original scaffold of the
NSAID. The SAR of oxaprozin-derived RXR activators is
summarized in Chart 2. This approach resulted in the discovery
of 38 as a selective RXR agonist with comparable potency as
bexarotene (2) but a remarkably improved physicochemical,
cytotoxicity, and PK profile (Table 6). This optimized
oxaprozin-derived RXR agonist 38 binds to the canonical RXR
CONCLUSIONS
■
RXR activation is a well-established strategy for cancer
treatment and several lines of evidence also point out the
remarkable therapeutic potential of RXR modulation in
neurodegeneration. The available RXR agonists, however,
have major limitations including enormous lipophilicity, poor
PK, high cytotoxicity, and adverse effects, as illustrated by
bexarotene (2), the only approved synthetic rexinoid. To
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Chart 2. SAR Summary of Oxaprozin-Derived RXR Agonists
and Structure of Optimized Derivatives 28, 38, 41, and 49
Table 6. Summarized Characteristics of Novel RXR Agonists
a
38 and 49 Compared to Bexarotene (2)
2
38
49
EC50 [μM]
(RXRα/β/γ)
0.03/0.02/0.03
0.05/0.04/0.05
0.25/0.35/1.6
rel. efficacy
(RXRα/β/γ)
100/100/100%
78/81/100%
26/26/28%
Cytotoxicity
solubility (H2O)
log P
t1/2
cmax
IC50 ∼20 μM
0.3 mg/L
6.9
1.2−1.8 h
0.6−1.2 μg/mL
IC50 >100 μM
146 1 mg/L
4.6
>3 h
33 μg/mL
IC50 >100 μM
21 1 mg/L
5.3
>3 h
35 μg/mL
a
In vitro activity data37 as well as physicochemical20 and PK
Figure 3. In vitro and in vivo profiling of the pyrazole-based RXR
agonists 41 and 49. (a) 41 and 49 were nontoxic up to 100 μM
concentration. Data are mean S.E.M. relative cell viability (HepG2)
determined by the WST-1 assay; n = 4. (b) 41 and 49 at 10 μM
concentration were selective over RARα, PPARs, LXRs, FXR, and
CAR. Heatmap shows mean relative nuclear receptor activation
compared to reference agonists; n ≥ 3. (c) Stability of the pyrazole-
based RXR agonists 41 and 49 against microsomal degradation
remarkably exceeded the lead compound 3. Data are mean S.E.M.
remaining compound [%] after the indicated incubation time; n = 3. (d)
41 and 49 revealed favorable PK in mice after oral administration (10
mg/kg) with a half-life of above 3 h for both compounds. Data are mean
S.E.M.; n = 3. (e) 41 promoted mRNA expression of RXR
heterodimer-regulated genes ABCA1 and SREBP1c in human
hepatocytes (HepG2). In line with its partial RXR agonism, 49 showed
a weaker effect. Data are mean S.E.M.; n = 4. #p < 0.1, *p < 0.05, **p <
0.01 (t-test vs DMSO-treated cells). (f) Single oral doses of 10 mg/kg
41 or 49 induced hepatic expression of RXR-regulated genes ABCA1,
ApoE, and SREBP1c in mice. Data show mean S.E.M. relative mRNA
expression 3 h after administration of vehicle (n = 3), 41 (n = 3), or 49
(n = 3).
parameters for 2 were derived from the literature.22,25,38 PK
parameters refer to p.o. administration. PK parameters refer to rats
for 2 and to mice for 38 and 49. Log P values were calculated using
the ALOGPS2.1 resource.39
with high potency and selectivity as well as superior character-
istics that might enable progress in targeting RXR for several
indications beyond second-line cancer treatment.
EXPERIMENTAL SECTION
■
Chemistry. General. All chemicals and solvents were of reagent
grade and used without further purification unless otherwise specified.
All reactions were conducted in an oven-dried glassware under an argon
atmosphere and in absolute solvents. NMR spectra were recorded on a
Bruker AV 500, Bruker AV 400, or a Bruker am250xp spectrometer
(Bruker Corporation, Billerica, MA, USA). Chemical shifts (δ) are
reported in ppm relative to tetramethylsilane as the reference.
Multiplicity is reported: s, singlet; d, doublet; dd, doublet of doublets;
ddd, doublet of doublet of doublets; t, triplet; tt, triplet of triplets; p,
quintet; m, multiplet. Approximate coupling constants (J) are shown in
hertz (Hz). Mass spectra were obtained on a VG Platform II (Thermo
Fisher Scientific, Inc., Waltham, MA, USA) using electrospray
ionization (ESI). High resolution mass spectra were recorded on a
MALDI LTQ ORBITRAP XL instrument (Thermo Fisher Scientific).
Some compounds were purified by preparative HPLC using a
Shimadzu preparative LC-20A Prominence (Shimadzu, Kyoto,
Japan) with the following conditions: column, Luna (10μ C18(2)
100 Å; 250 × 21.2 mm; Phenomenex, Torrance, CA, U.S.A.); mobile
phase, linear gradient from 50% ACN to 90% within 10 min, 90% ACN
for 5 min, linear gradient from 90% ACN to 50% within 1 min, 50%
ligand-binding site. Together with its strongly reduced lip-
ophilicity, this finding demonstrates that RXRs can accom-
modate orthosteric agonists which are less lipophilic than
traditional rexinoids. From 38, we have also varied the central
oxazole scaffold of the oxaprozin-derived chemotype to a
pyrazole which resulted in a potent RXR agonist (41) and a
partial agonist (49) both of which outmatched bexarotene (2) in
various features, as well (Table 6). Overall, the oxaprozin-
derived RXR ligands provide a new RXR modulator chemotype
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ACN for 5 min at a flow rate of 21 mL/min and UV-detection at 245 nm
and 280 nm. Compound purity was analyzed on a Waters 600
Controller HPLC (Waters, Milford, MA, U.S.A.) equipped with a
Waters 2487 Dual Absorbance Detector and a Waters 717 plus
Autosampler or on a VWR Chromaster (VWR, Radnor, PA, U.S.A.)
equipped with a 5160 pump system, a DAD 5430, a 5260 Autosampler,
and a MultoHigh100 RP18-5 μ 250 × 4 mm 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 and MeOH
for additional 10 min) at a flow rate of 1 mL/min with UV detection at
245 and 280 nm. Only compounds with a purity ≥95% according to the
AUC at UV 245 and 280 nm detection were used for biological testing.
Compounds 8, 19, and 30−31 were published previously.28
4-(5-(3,5-Dichlorophenyl)-1-phenyl-1H-pyrazol-3-yl)butanoic
Acid (49). 7-(3,5-Dichlorophenyl)-5,7-dioxoheptanoic acid (140, 1.0 g,
3.3 mmol, 1.0 equiv) and phenylhydrazine (141, 0.36 g, 3.3 mmol, 1.0
equiv) were dissolved in MeOH (30 mL), and Et3N (0.45 mL, 3.3
mmol, 1.0 equiv) was added. The reaction mixture was stirred at rt for
16 h. The solvent was evaporated in vacuum, aqueous hydrochloric acid
(10%, 30 mL) was added to the residue, and the mixture was extracted
with Et2O (3 × 30 mL). The combined organic layers were dried over
Na2SO4, and the solvents were evaporated in vacuum. The crude
product was purified by column chromatography using EtOAc/hexane
(1:3) as the mobile phase. 49 was obtained as a yellow solid (0.2 g,
17%). 1H NMR (500 MHz, CDCl3): δ 7.38−7.28 (m, 3H), 7.27−7.21
(m, 3H), 7.05 (d, J = 1.9, 2H), 6.35 (s, 1H), 2.78 (t, J = 7.5, 2H), 2.46 (t,
J = 7.4, 2H), 2.05 (p, J = 7.5, 2H). 13C NMR (126 MHz, CDCl3): δ
178.87, 153.14, 141.09, 139.30, 135.18, 133.42, 129.35, 128.35, 128.14,
127.03, 125.37, 107.62, 33.55, 27.36, 24.52. MS (ESI+) m/z: 375.07
([M + H]+). HRMS (MALDI) m/z: calcd for C19H17Cl2N2O2,
375.06616; found, 375.06683 ([M + H]+).
4-Oxo-4-(2-oxo-2-phenylethoxy)butanoic Acid (133). Succinic
acid (1.8 g, 15 mmol, 2.0 equiv) was dissolved in acetone (50 mL),
and Et3N (2.1 mL, 15 mmol, 2.0 equiv) was added. After stirring for 30
min, a solution of 2-bromo-1-phenylethanone (132, 1.5 g, 7.5 mmol,
1.0 equiv) in acetone (30 mL) was slowly added. The reaction mixture
was stirred at rt for 12 h. The solvent was evaporated in vacuum,
aqueous hydrochloric acid (10%, 30 mL) was added to the residue, and
the mixture was extracted with DCM (3 × 30 mL). The combined
organic layers were dried over Na2SO4, and the solvents were
evaporated in vacuum. 133 was obtained as a light yellow solid and
was used without further purification (1.3 g, 73%). 1H NMR (500 MHz,
DMSO-d6): δ 7.71−7.68 (m, 2H), 7.58−7.54 (m, 3H), 5.49 (s, 2H),
2.67 (t, J = 6.6, 2H), 2.54−2.51 (d, J = 7.1, 2H). 13C NMR (126 MHz,
DMSO-d6): δ 192.73, 173.24, 171.76, 171.41, 128.96, 127.81, 127.79,
66.54, 28.66, 28.48. MS (ESI+) m/z: 259.04 ([M + Na]+).
3-(5-Bromo-4-phenyloxazol-2-yl)propanoic Acid (134). To a
solution of 3-(4-phenyloxazol-2-yl)propanoic acid (10, 0.27 g, 1.2
mmol, 1.0 equiv) in ACN (5 mL), NH4OAc (10 mg, 0.12 mmol, 0.10
equiv) and NBS (0.23 g, 1.3 mmol, 1.05 equiv) were added and the
mixture was stirred at rt for 2 h. The solvent was then removed in
vacuum, the residue was suspended in H2O (50 mL), and the mixture
was extracted with EtOAc (3 × 50 mL). The combined organic phases
were dried over MgSO4, and the solvent was removed in vacuum. The
crude product was purified by column chromatography using EtOAc/
hexane (4:1) + 1% AcOH as the mobile phase. 134 was obtained as a
light orange solid (0.13 g, 35%). 1H NMR (400 MHz, CDCl3): δ 7.94−
7.92 (m, 2H), 7.43 (t, J = 7.5 Hz, 2H), 7.44−7.43 (m, 1H), 3.13 (t, J =
7.3, 2H), 2.94 (t, J = 7.3 Hz, 2H). 13C NMR (101 MHz, CDCl3): δ
176.8, 163.7, 137.0, 130.0, 128.7, 128.5, 126.6, 116.4, 30.5, 23.5. MS
(ESI+) m/z: 297.45 ([M + H]+).
3-(4-Phenyloxazol-2-yl)propanoic Acid (10). 4-Oxo-4-(2-oxo-2-
phenylethoxy)butanoic acid (133, 1.24 g, 5.23 mmol, 1.00 equiv),
acetamide (1.56 g, 26.1 mmol, 5.00 equiv), and BF3·Et2O (0.740 g, 5.23
mmol, 1.00 equiv) were mixed and stirred at 140 °C for 16 h. After
cooling to rt, aqueous hydrochloric acid (10%, 30 mL) was added and
the mixture was extracted with EtOAc (3 × 30 mL). The combined
organic layers were dried over Na2SO4, and the solvent was evaporated
in vacuum. The crude product was purified by column chromatography
using EtOAc/hexane (1:1) + 1% AcOH as the mobile phase. 10 was
obtained as a brown solid (0.46 g, 40%). 1H NMR (400 MHz, CDCl3):
δ 7.82 (s, 1H), 7.70−7.68 (m, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.32−7.28
(tt, J = 7.4, 2.1 Hz, 1H), 3.16 (t, J = 7.3 Hz, 2H), 2.93 (t, J = 7.4 Hz, 2H).
13C NMR (101 MHz, CDCl3): δ 177.2, 163.6, 140.8, 133.6, 130.9,
128.9, 128.2, 125.7, 23.4, 20.9. MS (ESI+) m/z: 218.02 ([M + H]+).
HRMS (MALDI) m/z: calcd for C12H12NO3, 218.08117; found,
218.08112 ([M + H]+).
3-(5-(3,5-Bis(trifluoromethyl)phenyl)-4-phenyloxazol-2-yl)-
propanoic Acid (38). 3-(5-Bromo-4-phenyloxazol-2-yl)propanoic acid
(134, 0.12 g, 0.40 mmol, 1.0 equiv) and Na2CO3 (0.13 g, 1.2 mmol, 3.0
equiv) were dissolved in a degassed 1,4-dioxane/H2O mixture (4:1, 20
mL). Then, (3,5-bis(trifluoromethyl)phenyl)boronic acid (135, 0.10 g,
0.40 mmol, 1.0 equiv) and Pd(PPh3)4 (23 mg, 0.020 mmol, 0.050
equiv) were added. The reaction mixture stirred for 20 h at 100 °C.
Aqueous hydrochloric acid (10%) was added, and the mixture was
extracted with EtOAc (3 × 30 mL). The combined organic layers were
dried over Na2SO4, and the solvent was evaporated in vacuum. The
crude product was purified by column chromatography using EtOAc/
hexane (1:3) + 1% AcOH as the mobile phase followed by HPLC
1
purification. 38 was obtained as a colorless solid (0.11 g, 65%). H
NMR (500 MHz, DMSO-d6): δ 12.38 (s, 1H), 8.12 (s, 1H), 8.08 (s,
2H), 7.61−7.55 (m, 2H), 7.49−7.42 (m, 3H), 3.11 (t, J = 7.1, 2H), 2.82
(t, J = 7.1, 2H). 13C NMR (126 MHz, DMSO-d6): δ 173.12, 163.83,
141.67, 137.29, 131.16, 130.86, 130.71, 129.10, 128.91, 127.75, 125.95,
122.98, 121.86, 30.18, 23.00. MS (ESI+) m/z: 430.24 ([M + H]+).
HRMS (MALDI) m/z: calcd for C20H14F6NO3Na, 430.08724; found,
430.08735 ([M + H]+).
6-(3,5-Dichlorophenyl)-4,6-dioxohexanoic Acid (138). A cooled
(−5 °C) solution of 3′,5′-dichloroacetophenone (136, 3.0 g, 16 mmol,
1.2 equiv) in THF (30 mL) was added to a cooled (−5 °C) solution of
LiHMDS (3.4 g, 20 mmol, 1.5 equiv) in THF (20 mL). The reaction
mixture was stirred at −5 °C for 30 min. Then, a solution of succinic
anhydride (1.3 g, 0.013 mol, 1.0 equiv) in THF (20 mL) was slowly
added. The reaction mixture was stirred at rt for 12 h. The solvent was
evaporated in vacuum, and aqueous hydrochloric acid (10%, 30 mL)
was then added to the residue. The product was extracted with CHCl3
(3 × 30 mL). The combined organic layers were dried over Na2SO4,
and the solvents were evaporated in vacuum. The crude product was
purified by column chromatography using EtOAc/hexane (1:1) as the
3-(1-(4-Chlorophenyl)-5-(3,5-dichlorophenyl)-1H-pyrazol-3-yl)-
propanoic Acid (41). 6-(3,5-Dichlorophenyl)-4,6-dioxohexanoic acid
(138, 0.32 g, 1.1 mmol, 1.0 equiv) and (4-chlorophenyl)hydrazine
(143, 0.16 g, 1.1 mmol, 1.0 equiv) were dissolved in glacial acetic acid;
NaOAc (0.45 g, 5.5 mmol, 5.0 equiv) was added, and the mixture was
stirred at 70 °C for 3 h. After cooling to rt, aqueous hydrochloric acid
(10%, 30 mL) was added, and the mixture was extracted with EtOAc (3
× 30 mL). The combined organic layers were dried over Na2SO4, and
the solvent was evaporated in vacuum. The crude product was purified
by column chromatography using EtOAc/hexane (1:3) + 1% AcOH as
the mobile phase followed by HPLC purification. 41 was obtained as a
yellow solid (0.29 g, 67%). 1H NMR (500 MHz, CDCl3): δ 7.37−7.32
(m, 3H), 7.20 (d, J = 8.8, 2H), 7.08 (d, J = 1.9, 2H), 6.38 (s, 1H), 3.06
(t, J = 7.4, 2H), 2.84 (t, J = 7.4, 2H). 13C NMR (126 MHz, CDCl3): δ
177.41, 152.54, 141.21, 137.82, 135.44, 133.81, 133.12, 129.53, 128.73,
127.09, 126.24, 108.16, 33.31, 23.24. (ESI+) m/z: 395.26 ([M + H]+).
HRMS (MALDI) m/z: calcd for C18H14Cl3N2O2, 395.01154; found,
395.01180 ([M + H]+).
1
mobile phase. 138 was obtained as a colorless solid (1.0 g, 27%). H
NMR (500 MHz, DMSO-d6): δ 7.96−7.88 (m, 2H), 7.83 (s, 1H), 4.36
(s, 2H), 2.76 (t, J = 6.7, 2H), 2.61−2.57 (m, 2H). 13C NMR (126 MHz,
DMSO-d6): δ 200.03, 193.29, 174.95, 137.16, 132.69, 131.62, 127.16,
52.93, 37.73, 29.77. MS (ESI+) m/z: 311.00 ([M + Na]+).
7-(3,5-Dichlorophenyl)-5,7-dioxoheptanoic Acid (140). A cooled
(−5 °C) solution of 3′,5′-dichloroacetophenone (136, 2.0 g, 10 mmol,
1.2 equiv) in THF (30 mL) was added to a cooled (−5 °C) solution of
LiHMDS (2.1 g, 13 mmol, 1.5 equiv) in THF (20 mL). The reaction
mixture was stirred at −5 °C for 30 min. Then, a solution of glutaric
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anhydride (0.95 g, 8.3 mmol, 1.0 equiv) in THF (20 mL) was slowly
added. The reaction mixture was stirred at rt for 12 h. The solvent was
evaporated in vacuum, aqueous hydrochloric acid (10%, 30 mL) was
added to the residue, and the mixture was extracted with CHCl3 (3 × 30
mL). The combined organic layers were dried over Na2SO4, and the
solvents were evaporated in vacuum. The crude product was purified by
column chromatography using EtOAc/hexane (1:1) as the mobile
phase. 140 was obtained as a colorless solid (0.7 g, 28%). 1H NMR (500
MHz, DMSO-d6): δ 7.90 (d, J = 1.7, 2H), 7.83 (s, 1H), 3.93 (s, 2H),
2.36 (t, J = 7.3, 2H), 2.10 (t, J = 7.5, 2H), 1.84 (p, J = 7.3, 2H). 13C NMR
(126 MHz, DMSO-d6): δ 98.06, 188.45, 178.41, 134.44, 133.62,
133.41, 127.51, 59.83, 46.76, 33.15, 18.41. MS (ESI+) m/z: 325.06 ([M
+ Na]+).
studied by quantitative real-time PCR analysis with a StepOnePlus
System (Life Technologies, Carlsbad, CA, USA) using PowerSYBR-
Green (Life Technologies; 12.5 μL per well). Each sample was set up in
duplicate and repeated in four independent experiments. Data were
analyzed by the comparative ΔΔCT method with glyceraldehyde-3-
phosphate dehydrogenase (GAPDH) as the reference gene. The
following primers (for the human genes) were used: hGAPDH: 5′-ATA
TGA TTC CAC CCA TGG CA (fw), 5′-GAT GAT GAC CCT TTT
GGC TC (rev); hABCA1: 5′-TTC GCT CTG AGA TGA GCA CCA
(fw), 5′-TTT CAA GCG GGC ATA GAA CAA (rev) and hSREBP-1c:
5′-GGA GGG GTA GGG CCA ACG GCC T (fw), 5′-CAT GTC TTC
GAA AGT GCA ATC C (rev).
Microsomal Stability Assay. The solubilized test compounds [5 μL,
at 10 μM final concentration in phosphate buffer (0.1 M, pH 7.4)] were
preincubated at 37 °C in 432 μL of phosphate buffer (0.1 M, pH 7.4)
together with 50 μL of NADPH regeneration system (30 mM glucose-
6-phosphate, 4 U/mL glucose-6-phosphate dehydrogenase, 10 mM
NADP, and 30 mM MgCl2). After 5 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 addition of
250 μL of ice-cold methanol at 0, 15, 30, and 60 min. The samples were
diluted with 250 μL of DMSO and centrifuged at 10,000g for 5 min at 4
°C. The supernatants were analyzed, and the test compound was
quantified by HPLC: mobile phase: MeOH 83%/H2O 17%/formic
acid 0.1%; flow-rate: 1 mL/min; stationary phase: MultoHigh Phenyl
phase, 5 μm, 250 × 4, precolumn, phenyl, 5 μm, 20 × 4; detection
wavelength: 330 and 254 nm; injection volume: 50 μL. Control samples
were performed to check the test compound’s stability in the reaction
mixture: the first control was without NADPH, which is needed for the
enzymatic activity of the microsomes, the second control was with
inactivated microsomes (incubated for 20 min at 90 °C), the third
control was without the test compound (to determine the baseline).
The amounts of the test compound were quantified by an external
calibration curve, where data are expressed as mean SEM of single
determinations obtained in three independent experiments.
WST-1 Cytotoxicity Assay. The WST-1 assay from Roche (Roche
Diagnostics, Rotkreuz, Switzerland) was performed according to
manufacturer’s protocol. In brief, HepG2 cells were seeded in
DMEM supplemented with 1 mM sodium pyruvate, penicillin (100
U/mL), streptomycin (100 μg/mL), and 10% FCS in 96-well plates (3
× 104 cells/well). After 24 h, medium was changed to DMEM
supplemented with penicillin (100 U/mL), streptomycin (100 μg/
mL), and 1% FCS, additionally containing the test compounds and
0.1% DMSO or 0.1% DMSO alone as negative control. After 48 h, the
WST reagent (Roche) was added to each well according to
manufacturer’s instructions. After 45 min of incubation, the absorbance
(450 nm/reference: 620 nm) was determined with a Tecan Spark M
luminometer (Tecan). Each experiment was repeated four times in
duplicates.
Determination of Aqueous Solubility. Aqueous solubility of
compounds 28, 38, and 49 was determined using Whatman Uniprep
filters (Whatman plc, Maidstone, U.K.). 3 mg of each compound and 2
mL of phosphate buffer (0.1 M, pH 8) were inserted into the Uniprep
vessel, and the mixture was shaken at 37 °C for 1 h. The mixture was
then pressed through the Uniprep filter, and the concentration of the
dissolved test compound in the filtrate was quantified by HPLC
(Waters 600 controller and Waters 2487 dual absorbance detector
equipped with a MultoHigh 100 Phenyl 5 μ, 240 mm × 4 mm column)
using external calibration. The assay was repeated in three independent
experiments.
Biological Characterization. Hybrid Reporter Gene Assays. The
plasmids pFA-CMV-NR-LBD for hPPARα,40 hPPARγ,40 hPPARδ,40
hRARα,20 RARβ,41 hRARγ41 hLXRα,42 hLXRβ,42 hRXRα,43 hRXRβ,41
hRXRγ,41 hFXR,44 hVDR,41 and hCAR41 coding for the hinge region
and LBD of the canonical isoform of the respective nuclear receptor and
pFR-Luc (Stratagene, La Jolla, CA, USA; reporter) and pRL-SV40
(Promega, Madison, WI, USA; internal control) were used for the
hybrid reporter gene assays. HEK293T cells (German Collection of
Microorganisms and Cell Culture GmbH, DSMZ) were cultured 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 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 above-
mentioned plasmids (pFR-Luc, pRL-SV40 and one pFA-CMV-NR-
LBD clone) was carried out with the Lipofectamine LTX reagent
(Invitrogen) 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) addition-
ally 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. For antagonistic characterization, the test respective test
compound was coincubated with the respective reference agonist.
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 Deutschland
GmbH, Germany). 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 test
compound by the mean RLU of the untreated control. Relative
activation refers to fold reporter activity 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; CAR: CITCO; 1
μM each)-treated cells. All hybrid assays were validated with the
respective reference agonists which yielded EC50 values in agreement
with the literature.
Quantification of RXR-Regulated Gene Expression in HepG2 Cells.
HepG2 cells were grown in DMEM high glucose, supplemented with
10% FCS, 1 mM SP, penicillin (100 U/mL), and streptomycin (100
μg/mL) at 37 °C and 5% CO2. Cells were seeded in 6-well plates (2 ×
106 per well). 24 h after seeding, medium was changed to MEM
supplemented with 1% charcoal stripped FCS, penicillin (100 U/mL),
streptomycin (100 μg/mL), and 2 mM L-glutamine. After an additional
24 h, cells were incubated with test compounds dissolved in the same
medium with 0.1% DMSO or medium with 0.1% DMSO alone as the
untreated control for 12 h, harvested, washed with cold phosphate-
buffered saline (PBS), and then directly used for RNA extraction. Total
RNA was extracted using the total RNA Mini Kit (R6834-02, Omega
Bio-Tek, Inc., Norcross, GA, USA). 2 μg of RNA was then reverse-
transcribed into cDNA using the high-capacity cDNA reverse
transcription kit (4368814, Thermo Fisher Scientific, Inc.) according
to the manufacturer’s protocol. FXR-regulated gene expression was
Crystal Structure Determination. Recombinant RXRα LBD was
purified as previously described.2 The protein was incubated with the
inhibitors and GRIP-1 peptide prior to crystallization using sitting drop
vapor diffusion at 20 °C and the conditions described in Table S1.
Diffraction data were collected at SLS X06SA and were processed and
scaled with XDS45 and aimless,46 respectively. Molecular replacement
was performed using Phaser47 and the published coordinates of RXRα.2
Manual model rebuilding alternated with refinement was performed in
COOT48 and REFMAC5.49 The geometric correctness of the final
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pubs.acs.org/jmc
Article
models was verified by MOLPROBITY.50 Data collection and
refinement statistics are summarized in Table S1.
Pilot In Vivo Profiling. Animals and compound application
Twelve male RjOrl:Swiss (CD-1) mice (38−41 g body weight,
purchased from Janvier Labs, France) were used for the in vivo 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 in-life phase was performed by the
Molecular formula strings with structures and activity data
(CSV)
HPLC traces for all biologically tested compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Daniel Merk − Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, D-60438 Frankfurt, Germany;
orcid.org/0000-0002-5359-8128; Email: merk@
pharmchem.uni-frankfurt.de
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 und Verbraucherschutz, Abteilung
̈
̈
̈
Lebensmittel-und Veterinarwesen, Saarbrucken). Three animals per
group received a single oral dose of 10 mg/kg body weight of either 38,
41, or 49, and three animals received the vehicle. Water containing 1%
hydroxypropyl methylcellulose/Tween 80 (99:1) served as a vehicle.
All animals behaved normal throughout the study and showed no
adverse effects.
Authors
Simone Schierle − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Apirat Chaikuad − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany;
Structural Genomics Consortium, BMLS, Goethe University
Frankfurt, 60438 Frankfurt, Germany; orcid.org/0000-
0003-1120-2209
Felix F. Lillich − Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, D-60438 Frankfurt, Germany
Xiaomin Ni − Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, D-60438 Frankfurt, Germany;
Structural Genomics Consortium, BMLS, Goethe University
Frankfurt, 60438 Frankfurt, Germany
Stefano Woltersdorf − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Espen Schallmayer − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Beatrice Renelt − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Riccardo Ronchetti − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany
Stefan Knapp − Institute of Pharmaceutical Chemistry, Goethe
University Frankfurt, D-60438 Frankfurt, Germany;
Structural Genomics Consortium, BMLS, Goethe University
Frankfurt, 60438 Frankfurt, Germany; orcid.org/0000-
0001-5995-6494
Plasma and tissue sampling
At two time points (30 and 60 min after test compound application),
blood (20 μL) was obtained from the lateral tail vein. Three hours after
test compound application, mice were anesthetized under isoflurane
and then sacrificed by cervical dislocation for tissue collection.
Complete livers were obtained, immediately snap-frozen, and stored
at −80 °C until further evaluation.
Pharmacokinetic Analysis
38, 41, and 49 were quantified from the above-described animal
samples using LC−MS. Pharmacokinetic analysis was performed based
on the obtained plasma concentrations, applying a noncompartment
model using Kinetica 5.0 software (Thermo Scientific, Waltham).
Quantification of mRNA levels from mouse tissue
Liver tissue samples from mice were used to study mRNA expression
of FXR- and PPAR-regulated genes. To homogenize the tissue samples
for qRT-PCR analysis, one-third of each liver was placed on a Falcon
Cell Strainer with 40 μm pore size (BD Bioscience, Erembodegem,
Belgium) in a 50 mL Falcon tube. Every tissue was rinsed with PBS
buffer containing 10% FCS, penicillin (100 U/mL), and streptomycin
(100 μg/mL) and pressed through the cell strainer until 5 mL of cell
suspension had been collected. The samples were centrifuged at 1200
rpm for 10 min at 4 °C. The supernatant was discarded, and the pellets
were washed twice with 2 mL of fresh cold PBS buffer and centrifuged.
After discarding the supernatant, total RNA was extracted using EZA
Total RNA Kit I (Omega BioTek Inc., Norcross, Georgia, USA)
following the Animal Tissue Protocol. 2 μg per sample of the extracted
RNA were reverse-transcribed into cDNA using the High-Capacity
cDNA Reverse Transcription Kit (4368814, Thermo Fisher Scientific,
Inc.) according to the manufacturer’s protocol. mRNA expression was
studied by quantitative real-time PCR analysis with a StepOnePlus
Ewgenij Proschak − Institute of Pharmaceutical Chemistry,
Goethe University Frankfurt, D-60438 Frankfurt, Germany;
orcid.org/0000-0003-1961-1859
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00235
System (Applied Biosystems) based on the 2−ΔC method (Supporting
T
Information Figure 4) or the 2−ΔΔC method (Figure 2) using the
T
Author Contributions
All authors have given approval to the final version of the
manuscript.
Power SYBR Green PCR Master Mix (Life Technologies). Each
biological sample was set up in two technical replicates. GAPDH served
as a reference gene. PCR primers for the murine genes were mGAPDH:
5′-CGA CTT CAA CAG CAA CTC CCA CTC TTC C (fw), 5′-TGG
GTG GTC CAG GGT TTC TTA CTC CTT (rev); mABCA1: 5′-
CGA CCA TGA AAG TGA CAC GC (fw), 5′-AGC ACA TAG GTC
AGC TCG TG (rev); mApoE: 5′-AGA ACT GAC GGC ACT GAT
GG (fw), 5′-CGT AGA TCC TCC ATG TCG GC (rev) and mSREBP-
1c: 5′-ACT GGA CAC AGC GGT TTT GA (fw), 5′-CGG GAA GTC
ACT GTC TTG G (rev).
Notes
The authors declare no competing financial interest.
Cocrystal structure data of the RXRα-28 (pdb 7B88) and
RXRα-38 (pdb 7B9O) complexes are available in the wwPDB.
ACKNOWLEDGMENTS
■
This research was financially supported by the German Research
Foundation (DFG, grants ME5481/1-1 and PR1405/7-1).
D.M. is grateful for support by the Aventis Foundation. R.R.
received an “Erasmus Traineeship” from the University of
Perugia. S.K., A.C., and X.N. are grateful for support by the SGC,
a registered charity (no. 1097737) that receives funds from
AbbVie, Bayer AG, Boehringer Ingelheim, the Canada
Foundation for Innovation, Eshelman Institute for Innovation,
Genentech, Genome Canada through Ontario Genomics
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00235.
Synthetic procedures and analytical characterization data
for compounds 4−49 and intermediates and further
methods for in vitro characterization (PDF)
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Article
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Institute [OGI-196], EU/EFPIA/OICR/McGill/KTH/Dia-
mond, Innovative Medicines Initiative 2 Joint Undertaking
[EUbOPEN grant 875510], Janssen, Merck KGaA (aka EMD in
Canada and US), Merck & Co (aka MSD outside Canada and
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̃
Takeda and Wellcome as well as support from the German
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ABBREVIATIONS
■
ABCA1, ATP-binding cassette transporter A1; ApoE, apolipo-
protein E; CAR, constitutive androstane receptor; COX,
cyclooxygenase; DBD, DNA-binding domain; FXR, farnesoid
X receptor; LBD, ligand-binding domain; LXR, liver X receptor;
NSAID, nonsteroidal anti-inflammatory drug; PK, pharmacoki-
netics; PPAR, peroxisome proliferator-activated receptor; RAR,
retinoic acid receptor; RXR, retinoid X receptor; SAR,
structure−activity relationship; SOSA, selective optimization
of side activities; SREBP1c, sterol regulatory element-binding
protein 1c
(17) Watanabe, M.; Fujihara, M.; Motoyama, T.; Kawasaki, M.;
Yamada, S.; Takamura, Y.; Ito, S.; Makishima, M.; Nakano, S.; Kakuta,
H. Discovery of a “Gatekeeper” Antagonist That Blocks Entry Pathway
to Retinoid X Receptors (RXRs) without Allosteric Ligand Inhibition
in Permissive RXR Heterodimers. J. Med. Chem. 2021, 64, 430−439.
(18) Yukawa-Takamatsu, K.; Wang, Y.; Watanabe, M.; Takamura, Y.;
Fujihara, M.; Nakamura-Nakayama, M.; Yamada, S.; Kikuzawa, S.;
Makishima, M.; Kawasaki, M.; Ito, S.; Nakano, S.; Kakuta, H.
Convenient Retinoid X Receptor Binding Assay Based on Fluorescence
Change of the Antagonist NEt-C343. J. Med. Chem. 2021, 64, 861−870.
(19) Pollinger, J.; Schierle, S.; Gellrich, L.; Ohrndorf, J.; Kaiser, A.;
Heitel, P.; Chaikuad, A.; Knapp, S.; Merk, D. A Novel Biphenyl-Based
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