Selective PPARδ Modulators Improve Mitochondrial Function: Potential Treatment for Duchenne Muscular Dystrophy (DMD)

Article abstract of DOI:10.1021/acsmedchemlett.8b00287

The X-ray structure of the previously reported PPARδ modulator 1 bound to the ligand binding domain (LBD) revealed that the amide moiety in 1 exists in the thermodynamically disfavored cis-amide orientation. Isosteric replacement of the cis-amide with five-membered heterocycles led to the identification of imidazole 17 (MA-0204), a potent, selective PPARδ modulator with good pharmacokinetic properties. MA-0204 was tested in vivo in mice and in vitro in patient-derived muscle myoblasts (from Duchenne Muscular Dystrophy (DMD) patients); 17 altered the expression of PPARδ target genes and improved fatty acid oxidation, which supports the therapeutic hypothesis for the study of MA-0204 in DMD patients.

Full text of DOI:10.1021/acsmedchemlett.8b00287

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Letter
Selective PPAR# Modulators Improve Mitochondrial Function:
Potential Treatment for Duchenne Muscular Dystrophy (DMD)
Bharat Lagu, Arthur F Kluge, Effie Tozzo, Ross Fredenburg, Eric L. Bell, Matthew M Goddeeris,
Peter Dwyer, Andrew Basinski, Ramesh S Senaiar, Mahaboobi Jaleel, Nirbhay Kumar Tiwari,
Sunil K Panigrahi, Narasimha Rao Krishnamurthy, Taisuke Takahashi, and Michael A Patane
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00287 • Publication Date (Web): 31 Jul 2018
Downloaded from http://pubs.acs.org on August 1, 2018
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ACS Medicinal Chemistry Letters
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Selective PPARδ Modulators Improve Mitochondrial Function: Po-
tential Treatment for Duchenne Muscular Dystrophy (DMD)
Bharat Lagu,*§ Arthur F. Kluge,§ Effie Tozzo,§ Ross Fredenburg,§ Eric L. Bell,§ Matthew M. Goddeeris,§ Peter Dwyer,§ Andrew Basinski,§
Ramesh S. Senaiar,≠ Mahaboobi Jaleel,≠ Nirbhay Kumar Tiwari,≠ Sunil K. Panigrahi,≠ Narasimha Rao Krishnamurthy,≠ Taisuke
Takahashi,¶ Michael A. Patane§
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§Mitobridge, Inc. (a wholly owned subsidiary of Astellas Pharma.), 1030 Massachusetts Avenue, Cambridge MA 02138.
≠Aurigene Discovery Technologies, Ltd., Bengaluru and Hyderabad, India.
¶Astellas Pharma., Tsukuba, Japan.
.
KEYWORDS PPARδ modulators, Duchenne Muscular Dystrophy (DMD), mdx mouse, imidazoles, cisꢀamide
ABSTRACT: The xꢀray structure of the previously reported PPARδ modulator 1 bound to the ligand binding domain (LBD) revealed that
the amide moiety in 1 exists in the thermodynamically disfavored cisꢀamide orientation. Isosteric replacement of the cisꢀamide with fiveꢀ
membered heterocycles led to the identification of imidazole 17 (MAꢀ0204), a potent, selective PPARδ modulator with good pharmacokiꢀ
netic properties. MAꢀ0204 was tested in vivo in mice and in vitro in patientꢀderived muscle myoblasts (from Duchenne Muscular Dystroꢀ
phy (DMD) patients); 17 altered the expression of PPARδ target genes and improved fatty acid oxidation, which supports the therapeutic
hypothesis for the study of MAꢀ0204 in DMD patients.
DMD is an Xꢀlinked recessive genetic disease involving severe
muscle wasting that results from cycles of muscle degeneraꢀ
tion/regeneration.1,2 DMD patients carry mutations in the DMD
gene, which encodes for dystrophin, a cellular protein scaffold
that is critical for muscle membrane integrity.3 In addition to
weakened structural integrity, DMD is also characterized by mitoꢀ
chondrial impairment with evidence of decreased fatty acid oxidaꢀ
tive phosphorylation.4ꢀ7
(LBD) of the PPARδ receptor revealed that the carbonyl oxygen
and the Nꢀmethyl group is in a cis relationship.13 A transꢀamide
has been reported to be thermodynamically favored over a cisꢀ
amide.16 Cisꢀamides are uncommon even in the peptide literature,
especially for amino acids other than proline.17 Molecular meꢀ
chanicsꢀbased energy minimization and conformational analysis
of 1 predicted that the trans isomer is favored by 1.3 kcal/mol
over the cis isomer.18 Therefore, we reasoned that a molecule in a
“locked cisꢀamide” conformation would improve binding affinity
for PPARδ. Herein, we disclose a series of compounds representꢀ
ed by general structure 2, wherein 5ꢀmembered heterocyclic rings
(Figure 1) serve as isosteric replacements of the cisꢀamide bond.
In skeletal muscle, PPARδ elicits a transcriptional cascade reꢀ
sulting in increased fatty acid oxidation while sparing glucose
utilization, which enables muscle to function for a longer duration
without decreasing systemic glucose levels or increasing muscle
lactic acid.8 We hypothesized that engaging the PPARδ transcripꢀ
tional cascade with a selective PPARδ modulator would provide
functional improvements to DMD muscle.
The selectivity for PPARδ is considered to be crucial to mitiꢀ
gate the undesirable effects (such as hepatotoxicity, myopathy,
edema, cardiac toxicity etc.) observed with some modulators of
PPARα (fibrates),PPARγ (thiazolidinediones) and the dual
PPARα/γ (e.g. Muraglitazar) in clinical and/or preꢀclinical studꢀ
ies.9,10 While GW501516,11 a known PPARδ modulator, has
shown tumorogenic effects in mice.12 As per the company webꢀ
sites, two modulators of PPARδ, Seladelpar and Elafibranor, have
cleared the twoꢀyear carcinogenicity studies in rats. This suggests
that tumorogenic potential is not a class effect for the PPAR modꢀ
ulators.
Benzamide series
Heterocyclic series
O
cis-amide
O
O
X
W
Y
N
V
Z
Me
Me
HO2C
O
HO2C
O
Me
Me
1
2
PPARδ EC50 = 37 nM
PPARα EC50 = 6,100 nM
We recently disclosed the potent and selective PPARδ modulaꢀ
tor 1 (Figure 1), which displayed significantly improved pharmaꢀ
cokinetic properties relative to previously reported compounds.13ꢀ
(b)
Figure 1. (a) Xꢀray structure of 1 bound to PPARδ LBD; (b) Isoꢀ
steric replacement of cisꢀamide with 5ꢀmembered heterocycles.
15
The xꢀray structure of 1 bound to the ligand binding domain
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The heterocyclic cores required for compounds 2a-g were syntheꢀ
Zn(OTf)2 , toluene, MW (irradiation), 140°C; c) BBr3, DCM, RT;
d) Furanꢀ2ꢀboronic acid, Pd(PPh3)4, Na2CO3, DME, EtOH, H2O,
90°C; e) Methyl (E)ꢀ6ꢀbromoꢀ4ꢀmethylhexꢀ4ꢀenoate, K2CO3,
DMF, 65°C; f) LiOH.H2O, THF, EtOH, H2O, RT
sized using known synthetic procedures (see supporting inforꢀ
mation for the detailed procedures). Synthesis of 2g is shown in
Scheme 1 as a representative example.
1
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PPARδ, PPARα and PPARγ activities of these compounds
were assessed using transactivation assays.19 All the compounds
were screened for exposure in mice following an intravenous dose
of 3 mg/kg. The results are shown in Table 1.
All of the heterocyclic compounds (except compound 2f) in Table
1 show greater affinity (EC50 <10 nM) for PPARδ receptor than
the original amide lead 1, supporting our hypothesis regarding the
use of heterocycles as cisꢀamide isosteres. All the compounds
were found to selectively activate PPARδ (>1,000ꢀfold less potent
for PPARα and PPARγ receptors). Unlike compound 1, all the
compounds (except 2b) shown in Table 1 have plasma clearances
that exceeds hepatic blood flow in mice (>85 mL/min/kg), short
halfꢀlives (<1 h) and low plasma exposures (AUC < 1,000
ng.h/mL) when dosed intravenously in mice. Compound 2b has
the highest plasma exposure among the compounds shown in
Table 1 but is 5ꢀ10ꢀfold less potent for PPARδ compared to the
remaining compounds. Although 2d and 2g showed comparable
PPARδ activity and plasma clearance values, imidazole 2g was
selected for further optimization based on the superior solubility
properties (data not shown). Our previous work in the benzamide
series (like compound 1) demonstrated that modifications of the
hexenoic acid side chain significantly influence the observed
pharmacokinetic (PK) properties.13,14 Therefore, similar chemistry
exploration was undertaken in the imidazole series (e.g., 2g).
Compounds 13 - 21 were synthesized as shown in Scheme 2. The
results of pharmacological and pharmacokinetic studies are sumꢀ
marized in Table 2.
Table 1. Structure-activity relationship in the 5-membered
heterocyclic series
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AUC
PPARδ PPARα
t ½
CL
Cpd
(ng*h/mL
EC50
EC50
nMa,b
6,100
5,840
7,250
(h)c (mL/min/kg)c
c
nMa
37±5
1.5
)
2.3
0.2
0.8
0.8
0.4
15
180
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300
130
3300
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900
170
380
1
2a
2b
2c
2d
9.5±0.5
1.3±0.5 412±7.1
1.2
8.4
±1.4
59±3.6
13,100
16,800
ND
Scheme 2. General synthesis of compounds 13-21
0.2
0.7
225
720
220
70
2e
2f
1,230
±165
1.0±0.0
0.5
160
300
2g
aTransactivation assay15 In selected cases, compounds were also
assayed in a protein interaction assay.20 Both assays were in good
agreement. EC50 values are an average of at least two experiꢀ
ments (SEM shown unless single determination); bAll the comꢀ
pounds, except 2d, showed EC50 >10,000 nM for PPARγ; PPARγ
c
EC50 for 2d = 5530 nM; Exposure data for compounds dosed i.v.
at 3 mg/kg to CDꢀ1 mice; ND = Not Determined.
Scheme 1. Synthesis of compound 2g.
Reagents and conditions: a) Propꢀ2ꢀynꢀ1ꢀamine, EDCI.HCl,
HOBt, Et3N, DMF, RT; b) (2ꢀmethoxyphenyl)methanamine,
Zn(OTf)2 , toluene, microwave irradiation, 140°C; c) BBr3, DCM,
RT; d) For compounds 13-15: Furanꢀ2ꢀboronic acid, Pd(PPh3)4,
Na2CO3, DME, EtOH, H2O, 90°C ; e) RꢀBr (11a or 11b or 11c),
K2CO3, DMF, 65°C; f) LiOH.H2O, THF, EtOH, H2O, RT
Table 2 (please see at the end of the manuscript)
In general, PPARδ activity was significantly better for imidazꢀ
ole series of compounds (e.g. PPARδ EC50 = 37 nM for 1 comꢀ
pared to PPARδ EC50 = 1 nM for 2g). On the other hand, higher
plasma exposures were observed for the compounds in the benꢀ
zamide series compared to the imidazole series (e.g. AUC = 3300
ng*h/mL for 1 compared to 300 ng*h/mL for 2g). A significant
improvement in plasma clearance and plasma exposures was obꢀ
served when the hexanoic acid region included substitutions at α,
Reagents and conditions: a) Propꢀ2ꢀynꢀ1ꢀamine, EDCI.HCl,
HOBt, Et3N, DMF, RT; b) (2ꢀmethoxyphenyl)methanamine,
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Table 3. Potency, selectivity, ADME, DMPK, PK and Safety
data for 17 (MA-0204)
β and γ positions relative to the carboxylic acid (e.g. 2g, 14 and 15
compared to 13). A similar trend in the pharmacokinetic properꢀ
ties was observed for the benzamide series of compounds.
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Assay
Results
Among the substituents tried as a replacement of the 2ꢀfuryl
moiety (compounds 17-22), 4ꢀtrifluromethoxy group containing
compound 17, stood out based on subꢀnanomolar activity for
PPARδ, good selectivity over the other PPAR subtypes, reduced
plasma clearance and improved plasma exposure.
EC50 = 0.4 nMa
Human PPARδ
Human PPARα and EC50 = 6,990±1,030 nM and >100,000
nM respectivelya
PPARγ
Mouse
PPARδ and Rat
PPARδ
EC50 = 7.9 nM and 10 nMb
No activity in Eurofins PanLabs Lead
Profiling Screen® of 68 molecular targets
up to 10ꢀM. No activity (up to 10ꢀM)
for androgen, progesterone and glucoꢀ
corticoid receptors.c
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Selectivity
%Plasma
Binding:
Protein
Mouse/Rat/Human = 99.5/99.6/99.9
Cacoꢀ2 permeability A to B = 36; B to A= 41 (Efflux ratio
(cm/sec, Eꢀ06)
CYP450 inhibition
Mutagenicity
1.2)
>10 ꢀM for CYPs except 2C9 (56%
inhibition @ 10 ꢀM)
Nonꢀmutagenic in miniꢀAmes test
1 mg/kg IV: t½ = 2.7 h; CL = 33
mL/min/kg; Vss = 5.8 L/kg ; AUC = 503
ng*h/mL
3 mg/kg PO: t½ = 3.4 h; Cmax =
510 ng/mL; AUC = 630 ng*h/mL; %F =
42
Figure 2. Superposition of the xꢀray structures of compound 1
(magenta) and compound 13 (cyan) bound to the ligand binding
domain (LBD) of PPARδ receptor
Mouse PKd
Rat PKe
The xꢀray structure of compound 13 bound to the LBD of
PPARδ (Figure 2) was obtained at 2Å resolution.21 As anticipatꢀ
ed, the imidazole ring in 13 superimposes nicely with the cisꢀ
amide bond in the protein bound xꢀray structure of 1. Almost all
the interactions observed between 1 and the protein (His287,
His413, Thr253 and Tyr437) are conserved in the xꢀray structure
for 13. One of the nitrogen atoms in the imidazole ring of 13
forms a water mediated interaction with Leu304 as was observed
for the cisꢀamide in the xꢀray structure for 1.13
1 mg/kg IV: t½ = 6.1 h ; Vss = 1.8 L/kg
;CL
=
10 mL/min/kg; AUC
=
1,590 ng*h/mL
3 mg/kg PO: t½ = 4.6 h; Cmax =
1,089 ng/mL; AUC = 4,920 ng*h/mL;
%F = 90
3 mg/kg IV: t½ = 6.1 h; CL =
0.4 mL/min/kg; Vss = 3.2 L/kg ; AUC =
8,400 ng*h/mL
10 mg/kg PO: t½ = 7.3 h: Cmax =
1,660 ng/mL; AUC = 3,840 ng*h/mL;
%F = 13
The binding interactions with Val312 and Ile328 have been
postulated to impart selectivity for a known PPARδ selective
modulator, GW0742.22 Overlay of the crystal structures of 13
with GW0742 (PDB ID: 3TKM) reveals a significant downward
movement of the Ile328 residue in the structure for 13, even
though the Val312 conformation is conserved in both structures.
This movement is possibly necessitated by the presence of the
benzyl moiety on the imidazole ring. Ile328 in PPARδ is replaced
by methionine residues in PPARα and PPARγ. Changes in the
Ile328 region and the waterꢀmediated interaction with Leu304
potentially contribute to the improved PPARδ selectivity for the
imidazole series of compounds.
Monkey PKf
aTransactivation assay; bData from Indigo Bioscience assay; cData
obtained at Aurigene Discovery Technologies Pvt. Ltd.; Male
CDꢀ1 mice (n = 3); Male Wistar rats (n = 3); Male cynomolgus
monkeys (n = 3)
d
e
f
For compound 17 (MAꢀ0204) to be considered as a potential
treatment for Duchene Muscular Dystrophy (DMD), it was necesꢀ
sary to demonstrate that it met at least three criteria: (1) elicit a
response in the target tissue, i.e., skeletal muscle, (2) affect
changes in the PPARδꢀresponsive genes in DMD patient myꢀ
oblasts and (3) affect beta oxidation of fatty acid in patient myꢀ
oblasts. The results from theses in vivo and ex-vivo experiments
are described below.
Compound 17 was profiled in multiple in vitro and in vivo asꢀ
says (Table 3). Compound 17 is >10,000ꢀfold selective for actiꢀ
vation of PPARδ over PPARα and PPARγ receptors. The PPARδ
potency was 10ꢀfold less for mouse and rat PPARδ receptors than
for the human PPARδ receptor; this affect is well documented for
PPAR modulators.11 Compound 17 is selective (EC50 >10 ꢀM)
over other nuclear receptors, for example: androgen, progesterꢀ
one and glucocorticoid receptors. Compound 17 is also inactive
in a panel of 68 receptors and transporters that are known to pose
safetyꢀrelated challenges in the clinic. Compound 17 exhibits
high protein binding to mouse plasma, good permeability and low
potential for efflux. Compound 17 did not show mutagenic potenꢀ
tial or inhibit major cytochrome P450 enzymes (IC50 ≥10 ꢀM).
Compound 17 has acceptable PK profiles in mice and rats upon
oral dosing.23 In the aggregate, these properties made 17 (MAꢀ
0204) an excellent candidate for study in pharmacological assays.
To determine if 17 was capable of eliciting a biological effect
in skeletal muscle, mice were dosed orally with MAꢀ0204 and
GW501516 and target gene expression was assessed by qPCR
(quantitative polymerase chain reaction). As shown in Figure 4,
17 increased PPARδ target gene transcription in the muscle, repꢀ
resented here by Pdk4 (pyruvate dehydrogenase kinase 4).
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ASSOCIATED CONTENT
Page 4 of 7
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Supporting Information
1
Supporting Information [experimental procedures, HPLC and H
NMRs, xꢀray crystallographic data as a PDF] is available free of
charge on the ACS Publications website at DOI: XXX.
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AUTHOR INFORMATION
Corresponding Author
9
*
Tel. 1(617)401ꢀ9122. Eꢀmail: Bharat.lagu@astellas.com
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Author Contributions
Figure 4. Compound 17 (MAꢀ0204) engages target gene expresꢀ
sion after oral administration in mice (please see supporting inꢀ
formation for details).
The manuscript was written through contributions of all authors. /
All authors have given approval to the final version of the manuꢀ
script.
In order to test for changes in fatty acid oxidation, primary myꢀ
oblasts isolated from a seventeen month old DMD patient were
utilized. 17 increased PPARδ target genes ANGPTL4 (angiopoiꢀ
etin like protein 4), CPT1a (carnitine palmytoyltransferase 1a),
and PDK4 in a doseꢀdependent manner (Figure 5). Furthermore,
oxidation of the longꢀchain fatty acid palmitic acid was signifiꢀ
cantly elevated at the concentrations of 1.2 nM, 4 nM and 12 nM
(3ꢀ, 10ꢀ, and 30ꢀfold EC50, respectively), which represents a funcꢀ
tional improvement of up to 52% in palmitate utilization (Figure
6).
Notes
The authors are current or former employees of Mitobridge, Inc.,
Astellas Pharma. or Aurigene Discovery Technologies and may
have or currently own shares of these companies.
ACKNOWLEDGMENT
Authors thank B. Mallesh (Aurigene Discovery Technologies) for
technical assistance.
ABBREVIATIONS
ANGPTL4 gene expression
PDK4 gene expression
DMSO, Dimethyl Sulfoxide; BBr3, boron tribromide; DCM, diꢀ
chloromethane; RT, room temperature; h, hour; Pd(PPh3)4,
Tetrakis(triphenylphosphine)palladium(0); Na2CO3, sodium carꢀ
bonate; DME, 1,2ꢀdimethoxyethane; EtOH, ethyl alcohol; Liꢀ
OH.H2O, lithium hydroxide monohydrate; THF, tetrahydrofuran;
EDCI.HCl, ethylcarbodiimide hydrochloride; HOBt, 1ꢀhydroxy
benzotriazole; Et3N, triethylamine; DMF, N,Nꢀdimethyl formaꢀ
mide; Zn(OTf)2, zinc trifluoromethanesulfonate.
20000
15000
10000
5000
0
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15000
10000
5000
0
****
****
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****
+146%
+144%
+143%
+143%
****
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+98%
+98%
****
****
+32%
+36%
O
O
S
M
M
M
M
M
M
n
M
n
M
n
M
n
S
n
n
n
n
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0
4
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4
0
M
.
.
0
0
4
0
.
0
4
.
0
D
D
0
MA-0204
MA-0204
Figure 5. Compound 17 (MAꢀ0204) engages target gene expresꢀ
sion in DMD patient muscle myoblasts. mice (please see supportꢀ
ing information for details).
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Figure 6. Compound 17 (MAꢀ0204) improves fatty acid oxidaꢀ
tion in DMD patient muscle myoblasts mice (please see supportꢀ
ing information for details).
In summary, we have reported the design and synthesis of a seꢀ
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crystallographic information derived from previously disclosed
compounds. The key compound to emerge from these studies, 17
(MAꢀ0204), combined potency and PK properties which enabled
the demonstration that selective the PPARδ modulators may offer
benefits as a therapeutic strategy for DMD.
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e4.
9. Peraza, M. A.; Burdick, A. D.; Marin, H. E.; Gonzalez,
F. J.; Peters, J. M. The Toxicology of Ligands for Peꢀ
roxisome ProliferatorꢀActivated Receptors (PPAR).
Tox. Sci. 2006, 90, 269.
10. Niessen, S. E.; Wolski, K.; Topol, E. J. Effect of Muꢀ
raglitazar on death and major adversecardiovascular
events in patients with typeꢀ2 diabetes mellitus JAMA
2005, 294(20), 2581.
11. Sznaidman, M. L.; Haffner, C. D.; Malloney, P. R.;
Fivush, A.; Chao, E.; Goreham, D.; Sierra, M. L.;
LeGrummelec, C.; Xu, E. H.; Montana, V. G.; Lambert,
M. H.; Willson, T. M.; Oliver, W. R.; Sternbach, D. D.
Novel selective small molecule agonists for peroxisome
proliferatorꢀactivated receptor delta (PPARdelta)ꢀ
synthesis and biological activity. Bioorg. Med. Chem.
Lett. 2003, 13, 1517.
12. Geiger, L. E,; Dunsford, W. S,; Lewis, D. J,; Brennan,
C,; Liu, K. C,; Newsholme, S. J. Rat carcinogenicity
study with GW501516, a PPAR delta agonist. 48th An-
nual Meeting of the Society of Toxicology, 2009, PSꢀ
895.
13. Lagu, B.; Kluge, A. F.; Fredenburg, R. A.; Tozzo, E.;
Senaiar, R. S.; Jaleel, M.; Panigrahi, S. K.; Tiwari, N.
K.; Krishnamurthi, N. R.; Takahashi, T.; Patane, M. A.
Novel highly selective peroxisome proliferatorꢀ
activated receptor delta (PPARδ) modulators with
pharmacokinetic properties suitable for onceꢀdaily oral
dosing. Bioorg Med Chem Lett., 2017, 27(23): 5230ꢀ
5234.
15. Wu, C. C.; Baiga, T. J.; Downes, M.; La Clair, J. J.; Atꢀ
kins, A. R.; Richard, S. B.; Fan, W.; StockleyꢀNoel, T.
A.; Bowman, M. E.; Noel, J. P.; Evans, R. M. Structural
basis for specific ligation of the peroxisome proliferaꢀ
torꢀactivated receptor δ Proc. Natl. Acad. Sci. 2017,
114, E2563.
16. Jorgensen, W. L.; Gao, J. Cisꢀtrans energy difference
for the peptide bond in the gas phase and in aqueous soꢀ
lution. J. Am. Chem. Soc. 1988. 110 (13), 4212ꢀ4216.
17. Weiss, M. A.; Jabs, A.; Hilgenfeld, R. H. “Peptide bond
revisited” Nat. Str. Biol. 1998, 5(8), 676.
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18. Molecular mechanics based energy minimization and
conformational analysis study using Molsoft/MMFF
force field.
19. In the transactivation assay CVꢀ1 cells are transfected
with a PPAR ligand binding domain fused to a GAL4
promoter to generate a hormoneꢀinducible activator. A
test ligand is added and activity is measured in a lucifꢀ
erase assay. See WO2016057660 for further details.
20. In the protein interaction assay the PPAR ligand comꢀ
plexes with a PPAR ligand binding domain fused to an
inactive fragment of galactosidase. This complex comꢀ
plements another inactive galactosidase fragment to
form an active galactosidase enzyme that is read out in a
fluorescence assay. See www.discoverx.com for deꢀ
tails.
21. Coordinates of the Xꢀray structure have been deposited
in the Protein Data Bank (accession code PDB 5ZXI).
22. Batista, F. A. H.; Trivella, D. B. B.; Bernardes, A.;
Gratieri, J.; Oliveira, P. S. L.; Figueira, A. C. M.; Webb,
P.; Polikarpov, I. Structural insights into human peroxiꢀ
some proliferator activated receptor delta (PPARꢀdelta)
selective ligand binding. PLos One 2012, 7, e33643.
23. All the animal experiments were carried out as per the
guidelines of the Committee for the Purpose of Control
and Supervision of Experiments on Animals
(CPCSEA), Government of India and approved by the
Institutional Animal Ethics Committee (IAEC), Auriꢀ
gene Discovery Technologies Ltd, Bengaluru, India.
14. Lagu, B., Kluge, A. F.; Goddeeris, M. M.; Tozzo, E.;
Fredenburg, R. A.; Chellur, S.; Senaiar, R. S.; Jaleel,
M.; Krishna Babu, R. D.; Tiwari, N. K.; Krishnamurthi,
N. R.; Takahashi, T.; Patane, M. A. Highly selective peꢀ
roxisome proliferatorꢀactivated receptor delta (PPAR )
Table 2. Hexanoic acid region modifications, PPAR activity and selected pharmacokinetic data
Hexanoic acid region
Modifications
CL
AUC
PPARδ EC50
PPARα EC50
t½ (h)b
Cpd
R1
(mL/min/kg)b
(ng*h/mL)b
(nM)a
(nM)a
ꢀꢀꢀ
ꢀꢀꢀ
A
37±5
6,100
2.3
0.5
15
3300
300
1
4ꢀ(2ꢀfuryl)
1.0±0.0
1,230±165
160
2g
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4ꢀ(2ꢀfuryl)
4ꢀ(2ꢀfuryl)
4ꢀ(2ꢀfuryl)
OCF3
B
C
D
A
D
D
D
0.7±0.2
4.5
6,970
ND
0.3
2.4
1.4
3.1
3.3
4.3c
1.4c
270
120
73
180
400
13
14
15
16
17
18
19
1
2
3
4
5
6
7
8
3.7±0.1
0.3±0.1
0.4±0.1
0.8±0.1
5.5±2.8
>100,000
2,290
650
70
240c
1250
440c
752c
9
6,900
±1030
4ꢀOCF3
4ꢀClꢀ3ꢀF
4ꢀCl
25
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
69,000
38c
22c
>100,000
4ꢀMe
4ꢀOCHF2
4ꢀF
D
D
D
20±4.8
13.5
78,500
15,060
ND
ND
ND
ND
ND
ND
ND
ND
ND
20
21
22
39.0
>100,000
aTransactivation assay15 In selected cases, compounds were also assayed in a protein interaction assay.16 Both assays were in good
agreement. EC50 values are an average of at least two experiments (SEM shown unless single determination); bExposure data for comꢀ
pounds dosed i.v. at 3 mg/kg to CDꢀ1 mice; cExposure data for compounds dosed i.v. at 1 mg/kg to CDꢀ1 mice; ND = Not Determined.
All the compounds showed EC50 >10,000 nM for PPARγ.
SYNOPSIS TOC (Word Style “SN_Synopsis_TOC”).
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Selective PPARδ Modulators Improve Mitochondrial Function:
Potential Treatment for Duchenne Muscular Dystrophy (DMD)
Bharat Lagu,*§ Arthur F. Kluge,§ Effie Tozzo,§ Ross Fredenburg,§ Eric L. Bell,§ Matthew M.
Goddeeris,§ Peter Dwyer,§ Andrew Basinski,§ Ramesh S. Senaiar,≠ Mahaboobi Jaleel, ≠ Nirbhay
Kumar Tiwari, ≠ Sunil K. Panigrahi,≠ Narasimha Rao Krishnamurthy,≠ Taisuke Takahashi,¶
Michael A. Patane§
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§
Mitobridge, Inc. (a wholly owned subsidiary of Astellas Pharma.), 1030 Massachusetts Avenue, Cambridge MA
02138.
≠
Aurigene Discovery Technologies, Ltd., Bengaluru and Hyderabad, India.
¶
Astellas Pharma., Tsukuba, Japan.
Graphical Abstract
F
3CO
O
Pre-organization in the
cis-amide conformation
N
O
N
1
N
Me
Me
Me
HO2C
O
HO2C
O
Me
MA-0204
EC = 0.4 nM
δ
cis-amide conformation
PPAR
50
EC = 6,990 nM
α
in protein-bound x-ray
PPAR
50
EC = 37 nM
δ
PPAR
PPAR
Good oral bioavailability
50
EC = 6,100 nM
α
50
1
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