Discovery of a Novel Selective Dual Peroxisome Proliferator-Activated Receptor α/δAgonist for the Treatment of Primary Biliary Cirrhosis

Article abstract of DOI:10.1021/acsmedchemlett.9b00189

A novel peroxisome proliferator-activated receptor (PPAR) α/δdual agonist 5c was developed with an EC₅₀ of 8 nM for PPARα, 5 nM for PPARδ, and >300-fold selectivity against PPARγ(EC₅₀ = 2939 nM), respectively. Further ADME and pharma

Full text of DOI:10.1021/acsmedchemlett.9b00189

Letter
pubs.acs.org/acsmedchemlett
Cite This: ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX
Discovery of a Novel Selective Dual Peroxisome Proliferator-
Activated Receptor α/δ Agonist for the Treatment of Primary Biliary
Cirrhosis
Zhigan Jiang,† Xing Liu,‡ Zhiliang Yuan,† Haiying He,*,† Jing Wang,† Xiao Zhang,† Zhen Gong,†
Lijuan Hou,† Liang Shen,† Fengxun Guo,† Jiliang Zhang,† Jianhua Wang,† Deming Xu,† Zhuowei Liu,‡,§
Haijun Li,‡,§ Xiaoxin Chen,*,‡,§ Chaofeng Long,‡,§ Jian Li,† and Shuhui Chen†
†WuXi AppTec (Shanghai) Co., Ltd, 288 FuTe Zhong Road, Shanghai 200131, P. R. China
‡R&D Center, Guangdong Zhongsheng Pharmaceutical Co., Ltd. The Information Area of Xihu Industrial Base, Shilong Town,
Dongguan, Guangdong Province 523325, P. R. China
§Guangdong Raynovent Biotech Co., Ltd., Room 1701-1705, Main Building of Rongyi Tower, No. 5, Xinxi Road, SongShan Lake
Hi-tech Industrial Development Zone, Dongguan, Guangdong Province 523808, P. R. China
S
* Supporting Information
ABSTRACT: A novel peroxisome proliferator-activated receptor (PPAR) α/δ dual agonist 5c was developed with an EC50 of 8
nM for PPARα, 5 nM for PPARδ, and >300-fold selectivity against PPARγ (EC50 = 2939 nM), respectively. Further ADME and
pharmacokinetic studies indicated 5c possessed distinguished in vitro and in vivo profiles. The excellent in vivo efficacy of
compound 5c was demonstrated by the rat primary biliary cirrhosis (PBC) model.
KEYWORDS: Peroxisome proliferator-activated receptor (PPAR), dual agonist, primary biliary cirrhosis (PBC)
rimary biliary cirrhosis (PBC), known as a cholestatic liver
Pdisease, was caused by an impairment of bile production
Figure 3. Selected synthetic PPARs agonists.
Figure 1. Ursodeoxycholic acid (UDCA) and obeticholic acid
(OCA).
and characterized by portal inflammation and slow progressive
destruction of interlobular bile ducts.1 If left untreated, PBC
leads to further hepatic damage, such as fibrosis, cirrhosis, liver
failure, and hepatocellular carcinoma (HCC), which ultimately
results in the requirement of liver transplantation.2 To date,
the therapeutic options approved by FDA for PBC patients are
ursodeoxycholic acid (UDCA)3−7 and obeticholic acid (OCA)
Received: April 24, 2019
Accepted: June 24, 2019
Published: June 24, 2019
Figure 2. Selected synthetic PPARs agonists.
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.9b00189
ACS Med. Chem. Lett. XXXX, XXX, XXX−XXX

ACS Medicinal Chemistry Letters
Letter
Scheme 1. General Procedure for the Synthesis of
Compounds 5a−5d
Scheme 2. Synthesis of Compound 10
Scheme 3. Synthesis of Compound 17a and 17b
Figure 4. Predicted binding modes of 5c in LBD of the three PPAR
isoforms. (a,b) Compound 5c (green) in the three subtypes of PPAR
receptor. (c−e) Different residues in the binding pocket of PPARα
(green), PPARδ (blue, ) and PPARγ (yellow). The protein backbone
is shown in cartoon, residues are in stick, and ligands are in stick-and-
ball. The figures were generated by Pymol package (The PyMOL
̈
Molecular Graphics System, Version 1.8 Schrodinger, LLC)
(Figure 1).8−11 These treatments have been demonstrated to
delay the progression of PBC; however, up to 40% of patients
have an incomplete response to UDCA monotherapy.12
Higher dosage of OCA was found to be related with the
pruritus. A recent safety alert warning from FDA indicates that
the excessive dosing of OCA may be associated with increased
risks of liver injury and death.13 These considerations
emphasize the unmet medical need for this indication.
As effective targets for the therapy of cholestatic diseases,
nuclear receptors are critically involved in the regulation of
hepatic transporters which regulate a series of bile formation
and secretion.14 One of these nuclear receptors is peroxisome
proliferator-activated receptors (PPARs). Three subtypes of
PPARs, namely, PPARα, PPARδ, and PPARγ, have been
known as modulators of many genes implicated in metabolic
Table 1. Evalutation of in Vitro Activities and Selectivities of PPARα/δ Dual Agonists
a
EC50 (nM)
b
b
b
entry
compound
PPARα (% activation)
PPARδ (% activation)
PPARγ (% activation)
γ/α (EC50 ratio)
γ/δ (EC50 ratio)
c
1
2
3
4
5
6
7
8
GW7647
L-165,041
Troglitazone
GFT505
5a
5c
5b
10
5d
5 (100%)
ND
ND
ND
ND
ND
/
/
/
9
/
367
/
/
17
/
/
/
/
18
/
588
/
/
91
/
/
61 (100%)
ND
115 (72%)
ND
5 (79%)
ND
215 (100%)
2116 (93%)
ND
2939 (110%)
ND
ND
457 (78%)
ND
225 (117%)
>10,000 (5%)
8 (92%)
385 (85%)
2821 (78%)
27 (134%)
8974 (50%)
3423 (85%)
ND
9
10
11
5 (73%)
ND
17a
17b
ND
ND
/
a
b
Data generated using nuclear hormone receptor (NHR) assay. For PPARα, δ, and γ % activation, data were generated comparing with GW7647,
c
L-165,041, and troglitazone, respectively. ND: no data.
B
DOI: 10.1021/acsmedchemlett.9b00189
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ACS Medicinal Chemistry Letters
Letter
Table 2. ADME Profile of Compound 5c
study
result
Cl of microsomal (human/rat) mL/min/kg
plasma stability (human/rat) remaining % @ 2 h
plasma protein binding (human/rat) %
<9.6/<9.6
88%/96%
>99.9%/
>99.9%
bidirectional permeability across Caco-2 cell monolayer (A to 0.5/0.2/0.4
B/B to A/efflux ratio) 10−6 cm/s
Table 3. In Vitro Safety Profile of Compound 5c
Figure 5. In vivo efficacy study in ANIT supplemented diet induced
rat PBC model. Eight-day α-naphthylisothiocyanate supplemented
diet provoked rat PBC model. Serum total bilirubin change (A),
serum direct/indirect bilirubin change (B,C), inflammatory score
change (D), fibrosis score change (E). Data were presented as mean
SEM; **p < 0.01 vs vehicle group, *p < 0.05 vs vehicle group,
analyzed by unpaired t test.
study
IC50 (μM)
CYP1A2/2C9/2C19/2D6/3A4
hERG
10/20/>50/>50/>50
>30
syndrome.15 PPARα plays a key role not only in lipid
metabolism but also in bile acid synthesis and anti-
inflammatory. The activation of PPARα regulates genes
responsible for bile formation and transportation, such as
CYP7A1 (cytochrome P450 isoform), CYP27A1, UGT1A1,
SULT2A1, MDR3, and ASBT. As a result, NF-κβ (nuclear
factor kappaβ) could also be inhibited via PPARα activation,
which leads to decreased expression of IL-1 (interleukins) and
IL-6.7 A few clinical trials explored the effect of PPARα
agonists, fenofibrate, and bezafibrate (Figure 2), in patients
with PBC and demonstrated positive effects.16−22 The second
subtype is ubiquitously expressed PPARδ.23 Besides fatty acid
catabolism and energy metabolism capacities, the activation of
PPARδ decreases cholesterol absorption and synthesis and
down-regulates cholesterol 7α-hydroxylase, which results in
overt anticholestatic activity.24 For years, synthetic PPARγ
(glitazones) and dual PPARα/γ (glitazars) agonists have been
developed to treat dyslipidaemia and T2DM (type 2 diabetes
mellitus).25,26 Nevertheless, several of them were discontinued
because of PPARγ related weight gain, fluid retention,
hemodilution, and edema effect on an increasing risk of
heart failure in clinical trials.27,28 Given the potential
pharmacological benefit of activating PPARα/δ in cholestasis
and PPARγ related cardiovascular safety concerns, developing
a dual PPARα/δ agonist with high selectivity against PPARγ
may lead to a better solution in the treatment of PBC.
Results and Discussion. GFT505 (Elafibranor) is a
PPARs agonist (Figure 2) for the treatment of NASH
(nonalcoholic steatohepatitis, NCT02704403, phase III) and
PBC (NCT03124108, phase II). Preferential activities on
PPARα and δ were exhibited for GFT505 with no PPARγ-
associated adverse cardiac effects; however, it must be used at
moderate to high dosage (80−120 mg/day) to achieve
sufficient efficacy.29−32 Hence, we describe herein the design
and synthesis of novel PPARα/δ dual agonists to improve
pharmacological activity and selectivity. The rational of drug
design for PPARα/δ dual agonist is shown in Figure 3. A
typical PPARs agonist comprises a carboxylic acid head, a
lipophilic tail, and a linker.33−38 In a variety of different
substructures for head, linker, and tail regions, GFT505 has a
remarkable chalcone group, which was assumed to be
important. Using GFT505 as a lead compound, α,β-
unsaturated ketone group was replaced with isoxazole linkage.
The easily oxidized methylthioyl group was replaced by a more
stable bromine atom, which exhibited similar lipophilicity and
size. By retaining hydrogen bond interaction and molecular
rigidity, and the utilization of a chemical stable substitution, we
expect these modifications would improve not only the activity
and selectivity of PPARα/δ but also pharmacokinetics
properties.
First, compounds with isoxazole linkage were designed and
prepared. Reactions of benzaldehydes with hydroxylamine
afforded benzaldehyde oximes 2a−2c. After the formation of
isoxazole analogues by oxidative cyclizations, compounds 5a−
5d were obtained through simple hydrolysis from ester to acid
(Scheme 1).39 To switch the fibric acid substitution position
on the isoxazole ring, a different pathway was designed for the
preparation of compound 10 (Scheme 2).
The synthesis of polycyclic isoxazole analogues 17a and 17b
was depicted in Scheme 3. SeO2-mediated oxidation trans-
formed 4-hydroxyphenylethanone 12 to 2-oxo-2-phenylacetic
acid 13.40,41 After the chlorination, the α-alkylation of
benzoxepin-5-one proceeded, followed by hydroxylamine
participated annulations. Deoxydation and subsequent hydrol-
ysis reactions provided compounds 17a and 17b, ultimately.
The novel isoxazole analogues were evaluated in a nuclear
hormone receptor (NHR) assay.42 The % activation of all test
compounds was compared to reference compounds that were
normalized to 100%. For PPARα, δ, and γ activities, the
reference compounds were GW7647, L-165,041, and troglita-
zone, respectively. Initially, the structure−activity relationship
Table 4. Pharmacokinetic Parameters of 5c after a Single Intravenous or Oral Dose Administration in Mice and Rats
species
route
dose (mg/kg)
Vdss (L/kg)
Cl (mL/min/kg)
T1/2 (h)
AUC0‑last (nM·h)
Cmax (nM)
Tmax (h)
F (%)
a
C57BL/6 mouse
i.v.
1
10
1
0.53
NA
0.16
NA
3.53
NA
0.35
NA
3.6
NA
6.0
NA
10566
63211
105180
561136
NA
52000
NA
NA
0.33
NA
59.8
b
p.o.
i.v.
a
SD rat
53.3
b
p.o.
10
65267
1.33
a
b
Formulation of intravenous dosing was prepared in DMSO/10% HP-β-CD = 5:95 (v/v). Oral formulation was prepared in 0.1% Tween 80/1%
CMC-Na in distilled water.
C
DOI: 10.1021/acsmedchemlett.9b00189
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ACS Medicinal Chemistry Letters
Letter
(SAR) study was set up in PPARα assay. Replacing chalcone
structure with isoxazole ring led to compound 5a with loss of
in vitro activity, while GFT505 showed an EC50 of 225 nM on
PPARα. However, encouraging results were observed when
extended carbon chains were employed. Compounds 5c and
5d are both full agonists, whose potency is 8−28-fold higher
than GFT505 (PPARα EC50 = 8 nM for 5c and 27 nM for 5d).
With the introduction of bromine atom, the activity of 5c was
47-fold higher than 5b (PPARα EC50 = 385 nM for 5b).
Switching the position of fibric acid led to a remarkable
decrease in PPARα potency (PPARα EC50 = 2821 nM for 10).
As a result, the cyclization of phenyl and isoxazole group,
which generated polycyclic isoxazole analogues 17a and 17b,
exhibited no effects on improving activities.
Next, we moved on to the evaluation of the selectivity of
PPARα and δ against PPAR γ (Table 1). PPARα/δ dual
agonist GFT505 showed good potency for both PPARα and δ
(PPARα EC50 = 225 nM, PPARδ EC50 = 115 nM), and
moderate to high selectivity for PPARγ/α and γ/δ (PPARγ
EC50 = 2116 nM, γ/α = 9, γ/δ = 18). Interestingly, selected
isoxazole analogues 5c and 5d, which showed higher PPARα
activity than GFT505, indicated excellent in vitro PPARδ
potency (PPARδ EC50 = 5 nM for 5c, PPARδ EC50 = 5 nM for
5d). Both of them acted as partial PPARδ agonists as GFT505
did. Compound 5c has a EC50 value of PPARγ as 2939 nM,
which means the higher selectivity for PPARα and δ (γ/α =
367, γ/δ = 588) over PPAR γ than that of GFT505. However,
an EC50 = 457 nM for PPARγ was noted for 5d when a
dimethylene group was employed. Since PPARγ activity results
in a series of cardiovascular safety concerns, compound 5d was
not selected for further investigation. The SAR study of the
isoxazole linker group presented that compound 5c was
optimal, providing potent PPARα/δ activities and the highest
level of PPARα/δ selectivity against PPARγ.
The structure of PPAR includes a DNA binding domain
(DBD) and a ligand binding domain (LBD). The small-
molecule agonists can bind to LBD and stabilize an active
conformation form, activating transcription process of the
downstream proteins.43−46 LBD of PPARs consisted of 12 α
helixes. The last helix, called AF-2 segment, can help to bind
with variant cofactors and therefore is very important for
receptor activation. The binding pocket of PPARα and PPAR δ
is mainly in “Y” shape that can be divided into three “arms”
regions. To better understand the SAR and selectivity profile,
these compounds have been modeled in the three PPAR
subtypes by molecular docking. PDB codes of the crystal
structures used in docking are 4CI4 for PPARα, 1GWA for
PPARδ, and 3VJH for PPARγ.17 The detailed interactions of
key compounds with proteins are shown in Figure 4.
It can be seen that 5c can take a “U”-shaped conformation
and occupy the arm I and arm II regions of PPARα. The
carboxylic acid of 5c binds to arm I region, forming H-bond
network to Tyr314, His440, and Tyr464. The dimethyl phenyl
has π−π stacking with Phe318 and His440, meanwhile
interacting with hydrophobic residues in arm I subpocket.
The isoxazole linker can provide appropriate geometry to place
the moieties on both sides in arm I and II, respectively. When
the substituent group is moved from position 5 to 4, the
linking angle was changed and significant activity drop (>300
folders, 5c versus 10) was observed. Moreover, the isoxazole
oxygen can catch an H-bond with Thr279, which is one of the
important factors of selectivity against PPARγ. The bromine
phenyl can bind to a highly hydrophobic pocket in arm II
region. The role of bromine here is to better occupy the
binding pocket and form more hydrophobic interactions with
protein. Removing bromine will drop activity over 50-fold (5c
versus 5b). The binding insight of 5c in PPARδ is very close to
that in PPARα (Figure 4a,b).
The binding pocket of PPARγ has less similarity to PPARα
and PPARδ. Different residues of the three subtypes around
active site are shown in Figure 4c,d. As mentioned above,
isoxazole can catch an H-bond interaction to Thr279 in
PPARα and match well to the binding pocket. The residue on
the same position of PPARδ is Thr252. However, there is a big
and polar residue, Arg288, on this position of PPARγ. It can be
seen that 5c has good activity on both PPARα and PPARδ but
has poor activity on PPARγ. When the linker is prolonged by
one more methylene, the activity of PPARα is slightly dropped,
but the activity on PPARγ increased over 6-fold (5c versus
5d). This is because the enhanced flexibility can help take a
better “U”-shaped conformation, reducing steric repulsion to
Arg288.
ADME, PK Results, and Discussion. The most potent and
selective compound 5c had distinguished in vitro ADME and in
vivo PK profiles. Compound 5c was stable in human and rat
microsome and plasma. However, 5c was a highly plasma
protein binding compound, with protein binding above 99.9%
in both human and rat plasma. Compound 5c showed
moderate permeability based on the Caco2 assay (Table 2).
Compound 5c indicated no significant inhibition on five
human CYP isozymes. An in vitro cardiovascular safety
evaluation also exhibited no hERG potassium channel activity
at concentrations up to 30 μM (Table 3).
The pharmacokinetic profile of 5c is presented in Table 4.
Across all tested species, mouse and rat, 5c showed low plasma
clearance. The half-life of 5c was 3.6 h in mouse and 6.0 h in
rat. A remarkable high oral plasma exposure of 5c indicated a
significant absolute oral bioavailability, which was 59.8% in
mouse and 53.3% in rat, respectively.
Pharmacology. Having been identified as a potent and
selective dual PPARα/δ agonist with favorable PK profile, 5c
was further studied in an in vivo efficacy model. A rat model
induced by 8-day ANIT (α-naphthylisothiocyanate) supple-
mented diet provoked increased TB (total bilirubin) and
inflammation parameters. Severe hepatic fibrosis could also be
determined in this model. To evaluate the effect on PBC, a
comparative study of 5c (3 mg/kg/day, once daily) versus
OCA (20 mg/kg/day, once daily) and GFT505 (30 mg/kg/
day, once daily) was performed in this experiment (six animals
per group). By comparison with OCA-20 mg/kg/day and
GFT505-30 mg/kg/day, compound 5c indicated obvious TB
(including DB-direct bilirubin and IB-indirect bilirubin)
lowering effect at only 3 mg/kg/day (Figure 5A−C).
Furthermore, 5c lowered inflammatory score by 30% (from
2.3 to 1.6) and reduced fibrosis area percentage by 28% (3.9%
to 2.8%). No obvious change was observed for inflammatory
score and fibrosis when GFT505-30 mg/kg/day and OCA-20
mg/kg/day were utilized. This was probably due to the lower
dosage of OCA in the study and poorer in vitro potency of
GFT505, respectively. In the meantime, the FXR potency of 5c
was checked in a FXR binding assay to exclude the
combinational efficacy of FXR agonism in the PBC model.
Its IC50 for FXR of 5c was more than 50 μM, and the
selectivity against PPARα and δ is over 6000.
Conclusion. In summary, we described the development of
novel PPARα/δ dual agonists. Compound 5c was found to be
D
DOI: 10.1021/acsmedchemlett.9b00189
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ACS Medicinal Chemistry Letters
Letter
controlled trials of ursodeoxycholic acid in primary biliary cirrhosis.
Gastroenterology 1997, 113, 884−890.
the most potent and highly selective PPARα/δ agonist (PPAR
α/δ/γ, EC50 = 8/5/2939 nM, EC50 γ/α ratio = 367, EC50 γ/δ
ratio = 588, FXR/PPAR > 6000). With good ADME profile, 5c
exhibited excellent in vivo efficacy in a rat PBC model. On the
basis of pharmacological effects described above, compound 5c
was identified as a promising lead compound for further
optimization.
(10) Usual adult dose of obeticholic acid for biliary cirrhosis: (1)
initial dose, 5 mg orally once a day. (2) Maintenance dose: 5 mg
orally once a day. (3) If adequate reduction in ALP (alkaline
phosphatas) and/or total bilirubin is not achieved after 3 months,
increase the dosage to 10 mg orally once a day. (4) Maximum dose:
10 mg/day.
(11) Pellicciari; et al. The Discovery of Obeticholic acid (Ocaliva):
First-in-Class FXR AgonistSuccessful Drug Discovery; Wiley, 2018;
Vol. 3.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.9b00189.
■
S
(12) Pares, A.; Caballeria, L.; Rodes, J. Excellent long-term survival
in patients with primary biliary cirrhosis and biochemical response to
ursodeoxycholic acid. Gastroenterology 2006, 130, 715−720.
(13) The Food and Drug Administration (FDA) warned that OCA
was being incorrectly dosed in some patients with moderate to severe
decreases in liver function in September 21st, 2017. These patients
were receiving excessive dosing, particularly a higher frequency of
dosing than was recommended in the drug label, which would result
in an increased risk of serious liver injury and death. OCA may also be
associated with liver injury in some patients with mild disease who are
receiving the correct dose.
Synthetic procedures, analytical data, and assay protocol
(PDF)
AUTHOR INFORMATION
Corresponding Authors
*E-mail: he_haiying@wuxiapptec.com.
*E-mail: chenzhenyu2000@zspcl.com.
■
(14) Ghonem, N. S.; Assis, D. N.; Boyer, J. L. On fibrates and
cholestasis: a review. Hepatology 2015, 62, 635−643.
ORCID
(15) Polvani, S.; Tarocchi, M.; Tempesti, S.; Bencini, L.; Galli, A.
Peroxisome proliferator activated receptors at the crossroad of
obesity, diabetes, and pancreatic cancer. World J. Gastroenterol.
2016, 22, 2441−2459.
Zhigan Jiang: 0000-0001-7277-330X
Notes
The authors declare no competing financial interest.
(16) Hazzan, R.; Tur-Kaspa, R. Bezafibrate treatment of primary
biliary cirrhosis following incomplete response to ursodeoxycholic
acid. J. Clin. Gastroenterol. 2015, 44, 371−373.
ACKNOWLEDGMENTS
■
The work financially supported by National Major Scientific
and Technological Special Project for “Significant New Drugs
Development” (No. 2018ZX09201001-002-001).
(17) Ohira, H.; Sato, Y.; Ueno, T.; Sata, M. Fenofibrate treatment in
patients with primary biliary cirrhosis. Am. J. Gastroenterol. 2002, 97,
2147−2149.
(18) Iwasaki, S.; Ohira, H.; Nishiguchi, S.; Zeniya, M.; Kaneko, S.;
Onji, M.; Ishibashi, H.; Sakaida, I.; Kuriyama, S.; Ichida, T.; Onishi,
S.; Toda, G. The efficacy of ursodeoxycholic acid and bezafibrate
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ABBREVIATIONS
PK, pharmacokinetics; PO, per os; Cmax, peak concentration;
max, time to peak; CL, clearance; T1/2, half-life; Vd, volume of
distribution; AUC, area under curve; mpk, mg/kg; %F, oral
■
T
bioavailability
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treatment: a new medical approach for PBC patients? J. Gastroenterol.
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