Design, Synthesis, and Evaluation of a Novel Series of Indole Sulfonamide Peroxisome Proliferator Activated Receptor (PPAR) α/γ/δ Triple Activators: Discovery of Lanifibranor, a New Antifibrotic Clinical Candidate

Article abstract of DOI:10.1021/acs.jmedchem.7b01285

Here, we describe the identification and synthesis of novel indole sulfonamide derivatives that activate the three peroxisome proliferator activated receptor (PPAR) isoforms. Starting with a PPARα activator, compound 4, identified during a high throughput screening (HTS) of our proprietary screening library, a systematic optimization led to the discovery of lanifibranor (IVA337) 5, a moderately potent and well balanced pan PPAR agonist with an excellent safety profile. In vitro and in vivo, compound 5 demonstrated strong activity in models that are relevant to nonalcoholic steatohepatitis (NASH) pathophysiology suggesting therapeutic potential for NASH patients.

Full text of DOI:10.1021/acs.jmedchem.7b01285

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Article
Design, synthesis and evaluation of a novel series of indole sulfonamide
Peroxisome Proliferator Activated Receptors (PPAR)#/#/# triple
activators: discovery of lanifibranor a new anti-fibrotic clinical candidate
Benaissa Boubia, Olivia Poupardin, Martine Barth, Jean Binet, Philippe Peralba, Laurent Mounier, Elise
Jacquier, Emilie Gauthier, Valerie Lepais, Maryline Chatar, Stephanie Ferry, Anne Thourigny, Fabrice Guillier,
Jonathan Llacer, Jerome Amaudrut, Pierre Dodey, Olivier Lacombe, Philippe Masson, Christian Montalbetti,
Guillaume Wettstein, Jean Michel Luccarini, Christiane Legendre, Jean Louis Junien, and Pierre Broqua
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01285 • Publication Date (Web): 15 Feb 2018
Downloaded from http://pubs.acs.org on February 16, 2018
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Design, synthesis and evaluation of a novel
series of indole sulfonamide Peroxisome
Proliferator Activated Receptors (PPAR) α/γ/δ
triple activators: discovery of lanifibranor a new
antiꢀfibrotic clinical candidate.
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Benaïssa Boubia , Olivia Poupardin , Martine Barth , Jean Binet, Philippe Peralba, Laurent
†
†
†
†
†
‡
Mounier , Elise Jacquier , Emilie Gauthier , Valérie Lepais , Maryline Chatar , Stéphanie Ferry ,
†
†
†
Anne Thourigny, Fabrice Guillier , Jonathan Llacer , Jérome Amaudrut , Pierre Dodey, Olivier
†
†
†
†
Lacombe , Philippe Masson , Christian Montalbetti , Guillaume Wettstein , JeanꢀMichel
†
†
†
Luccarini , Christiane Legendre, JeanꢀLouis Junien , Pierre Broqua
†
Inventiva 50 rue de Dijon 21121 Daix, France.
‡
Novalix Boulevard Sebastien Brant Bioparc 67405 Illkirch, France.
ABSTRACT
Here we describe the identification and synthesis of novel indole sulfonamide derivatives that
activate the three peroxisome proliferator activated receptor (PPAR) isoforms. Starting with a
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PPAR
α
activator, compound 4, identified during a high throughput screening (HTS) of our
proprietary screening library, a systematic optimization led to the discovery of lanifibranor
(IVA337) 5, a moderately potent and well balanced pan PPAR agonist with an excellent safety
profile. In vitro and in vivo, compound 5 demonstrated strong activity in models that are relevant
to NonAlcoholic SteatoHepatitis (NASH) pathophysiology suggesting therapeutical potential for
NASH patients.
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INTRODUCTION
NonAlcoholic SteatoHepatitis (NASH) is a highly prevalent multifactorial and multiꢀstep
disease associated with increasing risk of cardiovascular mortality and development of severe
1
,2
liver conditions such as cirrhosis and hepatocellular carcinoma. NASH is characterized by
histopathological changes in the liver which include steatosis, inflammation, ballooning, necroꢀ
inflammation and perisinusoidal fibrosis. Fibrosis stage is the strongest predictor for allꢀcause
3
and diseaseꢀspecific mortality in NASH patients. Although the pathogenesis of NASH is not
fully understood, there is a consensus that metabolic disorders and hepatic steatosis play a key
role in the initiation of the disease.
Peroxisome proliferatorꢀactivated receptors (PPARs) are members of the nuclear receptor
4
family. They play not only an important role in the regulation of lipid and glucose metabolism,
5,6
but also in the regulation of inflammation and fibrogenesis. Extensive efforts were deployed to
identify potent and selective agonists which could potentially be pharmacologically useful in
7
metabolic diseases. Two chemical families, thiazolidinediones represented by rosiglitazone 1a
and pioglitazone 1b and fibrates represented by fenofibrate 2a and bezafibrate 2b (Figure 1), are
8
,9,10
well described in the literature as respectively glucose and lipid regulators.
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All the three PPAR subtypes PPAR
α (NR1C1), PPARδ (NR1C2) and PPARγ (NR1C3) are
ligandꢀactivated nuclear receptors, which exhibit isotype specific tissue expression and cellular
1
1,12
function.
The PPAR
α
isoform is highly expressed in hepatocytes, where it facilitates fatty
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13,14
acid transport betaꢀoxidation and dampen inflammatory response.
The PPARδ isoform is
found in most liver cells types (hepatocytes, hepatic stellate cells (HSC) and Küpffer cells) and
contributes to the regulation of glucose and lipid metabolism while showing antiꢀinflammatory
15,16
properties especially through the promotion of M2 stage Küpffer cells polarization.
The
PPARγ isoform is highly expressed in the adipose tissue where it promotes adipocyte
differentiation, increases glucose uptake, promotes storage of triglycerides, decreases plasma
free fatty acids (FFA) and induces the secretion of the antiꢀinflammatory cytokine adiponectin.
1
7,18
Activation of PPARγ increases insulin sensitivity of several organs including the liver.
PPAR and PPARδ are expressed at different extent in HSC, a primary cell driver of liver
fibrosis. It is recognized that PPAR plays a critical role in keeping HSC in a quiescent nonꢀ
Both
γ
γ
1
9,20
fibrogenic state.
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rosiglitazone (1a)
rioglitazone (1b)
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fenofibrate (2a)
bezafibrate (2b)
muraglitazar (3a)
tesaglitazar (3b)
Figure 1. PPARs agonists. 1a: rosiglitazone and 1b: pioglitazone representing Thiazolidinedione
family. 2a: fenofibrate and 2b: bezafibrate representing Fibrate family. 3a: muraglitazar and 3b:
tesaglitazar representing PPAR α/γ dual agonists.
It can be speculated that combining PPARα, PPARδ and PPARγ activation may bring an
innovative and efficacious therapeutic approach to prevent the development and progression of
NASH by addressing a large spectrum of parameters involved in the disease.
Many of the PPAR agonists that were developed and discontinued are very potent activators of
the PPARs and unbalanced relatively to the different isoforms targeted. This potentially could
lead to a large spectrum of undesirable side effects as this strong potency would regulate the
expression of a wide range of genes and activate many pathways some of which will be
associated with toxicity. Conversely, moderately potent PPAR agonists such as 2a, 2b and 1b
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21,22,23
demonstrated good tolerability and safety in a large population of patients.
In our work, we
chose to focus on moderately potent and wellꢀbalanced modulators of the three PPARs isoforms
to target the NASHꢀrelated cells and pathways of interest while optimizing the therapeutic
margin.
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Our initial indole hit 4 was identified from a high throughput screening campaign of our
proprietary compound collection using a human PPAR transactivation assays. This indole
derivative showed a good agonist activity on PPAR
α (half maximal effective concentration
(EC )=260 nM, maximum efficacy (Emax)=101%) but an unbalanced pan PPAR profile with
5
0
very low potency and efficacy on both PPAR
EC =1558 nM, Emax=43%).
δ (EC =1912 nM, Emax=56%) and PPARγ
50
(
5
0
4
lanifibranor 5
Mouse db/db (10 and 30mg/kg)
Mouse db/db (10 and 30mg/kg)
No significant dose effect
Desired dose effect
Figure 2. From HTS hit to clinical candidate.
The structure of 4 offers similarities with the existing pharmacophoric scaffold, frequently
described in PPAR agonist literature, which consists in an acidic head group that is attached to a
hydrophobic tail group through a linker with various length and flexibility. As mentioned above
there is a clear unmet medical need in the therapeutic area of fibrotic disease like NASH. The
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objective of the optimization effort was the discovery of novel pan PPAR agonist drugs with an
improved safety margin compared to the known PPAR
γ
and dual PPAR
α/
γ
compounds. This
2
4
program led to the discovery of lanifibranor (IVA337) 5 a moderately potent and well balanced
pan PPAR agonist with an excellent safety profile which is presently undergoing phase 2 clinical
trials in NASH.
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RESULTS AND DISCUSSION
Chemistry.
Synthetic routes to the indole compounds shown in Table 1ꢀ6 are outlined in Scheme 1ꢀ4.
Palladiumꢀcatalyzed Sonogashira cross coupling between substituted iodoꢀnitrobenzenes and
various alkynes afforded the nitroalkynes 6, which were subsequently reduced in presence of
tin(II) chloride (SnCl ) to provide the corresponding aminoalkynes 7 (Scheme 1). Sulfonylation
2
of the anilines were conducted in presence of the desired sulfonyl chloride using pyridine as
solvent. Indoles 9 were obtained by cyclisation of the alkyne precursors 8 in presence of copper
2
5
acetate (Cu(OAc) ), at reflux of dichloroethane (CH Cl ) or under microwave irradiations at
2
2
2
1
30ꢀ150°C. Deprotection of the esters with lithium hydroxide (LiOH), in a mixture of
tetrahydrofuran (THF) and water, yielded the desired carboxylic acid compounds 4 and 10.
When the nitro starting material was not available, the synthesis was started from the
corresponding commercially available iodoaniline bearing the desired substitutions (scheme 2).
The sulfonylation reaction was carried out with various aryl sulfonyl chlorides in pyridine. Most
of the time, over reaction was observed yielding a mixture of disulfonylated and
monosulfonylated compounds. Conveniently direct treatment of the crude mixture with a 3 M
potassium hydroxide (KOH) solution allowed to convert the undesired bisꢀcapped product back
to the wanted iodosulfonamide 11. Sonogashira cross coupling in the presence of
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bis(triphenylphosphine)palladium(II) dichloride (Pd(PPh ) Cl ), copper iodide (CuI) and
3
2
2
diethylamine (Et N) in dimethylformamide (DMF) under microwave heating led directly to the
2
indoles 12. Final saponification provided derivatives 13.
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The synthesis sequence depicted in Scheme 3 started with a Sonogashira reaction between
substituted commercially available iodoanilines and tertꢀbutyl hexꢀ5ꢀynoate. Compounds 14
2
2
were treated with Cu(OAc) , under microwave conditions at 130°C for 40 minutes, to provide
2
indoles 15. Deprotonation with sodium hydride (NaH) followed by addition of arylsulfonyl
chlorides gave the corresponding sulfonyle indoles, which were subsequently deprotected with
trifluoroacetic acid (TFA) to readily afford derivatives 16.
Compound 22 was prepared according to the synthetic pathway depicted in Scheme 4.
Sulfonylation of 7b with 4ꢀfluoroꢀ3ꢀnitroꢀbenzenesulfonyl chloride in a mixture of pyridine and
2
ꢀ(dimethylamino)pyridine (DMAP) lead to 17, which was cyclized in presence of Cu(OAc) to
2
the indole 18. Nucleophilic substitution of the fluorine atom by aqueous ammonia led to 19. The
benzimidazole moiety 21 was obtained by reduction of the nitro group with iron powder in acetic
acid followed by cyclisation in presence of formic acid. Finally, hydrolysis with LiOH in a THFꢀ
water mixture afforded compound 22.
a
Scheme 1. Synthesis of indole derivatives via nitroalkyne intermediates
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e
c
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8a-m
9a-m
4, 10b-m
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6
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6
a, 7a
R = Cl
R = Cl
R = Cl
R = OMe Y = (CH
Y = (CH
Y = (CH
Y = (CH
2
)
2
8a, 9a, 4
R = Cl
Y = (CH
Y = (CH
2 4
Y = (CH )
2 2
)
Ar = Ph
Ar = Ph
Ar = Ph
Ar = Ph
1
1
b, 7b
c, 7c
d, 7d
2 3
)
8b, 9b, 10b
8c, 9c, 10c
8d, 9d, 10d
R = Cl
2 3
)
1
1
2 4
)
R = Cl
1
1
2
)
3
R = OMe Y = (CH
2 3
)
1
1
6b, 7b
R = Cl
Y = (CH
2
)
3
8e, 9e, 10e
8f, 9f, 10f
R = Cl
Y = (CH )
2 3
Ar =
Ar =
1
1
6b, 7b
R = Cl
Y = (CH
2
)
3
R = Cl
2 3
Y = (CH )
1
1
6
b, 7b
b, 7b
R = Cl
Y = (CH
2
)
3
8g, 9g, 10g
8h, 9h, 10h
8i, 9i, 10i
R = Cl
Y = (CH )
2 3
Ar =
Ar =
Ar =
Ar =
Ar =
Ar =
Ar =
1
1
6
R = Cl
Y = (CH
2
)
3
R = Cl
2 3
Y = (CH )
1
1
6b, 7b
R = Cl
Y = (CH
2
)
3
R = Cl
2 3
Y = (CH )
1
1
6b, 7b
6b, 7b
6b, 7b
R = Cl
Y = (CH
2
)
3
8j, 9j, 10j
R = Cl
2 3
Y = (CH )
1
1
R = Cl
Y = (CH
2
)
3
8k, 9k, 10k
8l, 9l, 10l
R = Cl
2 3
Y = (CH )
1
1
R = Cl
Y = (CH
2
)
3
R = Cl
2 3
Y = (CH )
1
1
6e, 7e
R = CF
3
Y = (CH
2
)
3
8m, 9m,10m R = CF
3
2 3
Y = (CH )
a
(
a) alkyne, Pd(PPh ) , CuI, TEA, DMF; (b) SnCl , AcOEt, EtOH; (c) ArSO Cl, pyridine; (d)
3 4 2 2
Cu(OAc) , 1,2ꢀdichloroethane reflux or microwave (130°Cꢀ150°C); (e) LiOH.H O, THF/H O.
2
2
2
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a
Scheme 2. Synthesis of indole derivatives via halogenosulfonamide intermediates
b
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c
a
11a, 11c-e
12a, 12c-e
13a-d, 5
1
12a R1 = Cl
-
1
11a
R
= Cl
= Cl
= CH
Ar = Ph
Ar = Ph
Ar = Ph
Y = CH
2
OCH
2
Ar = Ph
13a
13b
13c
R
= Cl
= Cl
= CH
Y = CH
Y = (CH
2 3
Y = (CH )
2
OCH
2
Ar = Ph
Ar = Ph
Ar = Ph
1
1
1
1
1a
1c
R
R
2
)
2
C(CH )
3 2
1
12c R1 = CH
12d R1 = Cl
12e R1 = Cl
1
R
3
3
Y = (CH
2 3
)
Ar = Ph
Ar =
R
3
1
1d R1 = Cl
Ar =
Ar =
Y = (CH
2 3
)
13d R1 = Cl
Y = (CH )
2 3
Ar =
Ar =
1
1e R1 = Cl
Y = (CH
2 3
)
Ar =
5
R1 = Cl
2 3
Y = (CH )
a
(
a) (i)ArSO Cl, pyridine, (ii) KOH, dioxane, reflux; (b) alkyne, Pd(PPh ) Cl , CuI,
2
3
2
2
diethylamine, DMF, microwave 130°C; (c) LiOH.H O, THF/H O.
2
2
a
Scheme 3. Synthesis of indole derivatives via indole NH intermediates
b
a
c
1
15a R1 = CF3
1
1
4a
4b
R
= CF
= Cl
3
R1 = CF
16a
16b
3
3
, Ar = Ph
1
1
R
15b
R
= Cl
1
R
R
= CF
, Ar = m-OMe-Ph
, Ar = p-OMe-Ph
1
1
1
1
1
1
1
1
1
1
1
1
6c
= CF
3
6d R1 = CF
, Ar = o-OMe-Ph
, Ar = m-Cl-Ph
, Ar = p- Cl-Ph
, Ar = o- Cl-Ph
, Ar = m-OCF
, Ar = p-OCF
3
1
6e
6f
6g
6h
6i
R
= CF
= CF
= CF
= CF
= CF
3
3
3
3
3
1
R
1
R
1
R
3
-Ph
-Ph
1
R
3
1
6j
6k
R
= Cl, Ar = m-OMe-Ph
1
R = Cl, Ar = p-OMe-Ph
6l R1 = Cl, Ar = m-Me-Ph
1
6m
R
= Cl, Ar = p-Me-Ph
a
(
a) tertꢀbutyl hexꢀ5ꢀynoate, Pd(PPh ) Cl , CuI, diethylamine, DMF, microwave 130°C; (b)
3 2 2
Cu(OAc) , 1,2ꢀdichloroethane, reflux or microwave (150°C); (c) (i) NaH, ArSO Cl, DMF (ii)
2
2
TFA, CH Cl .
2
2
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2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 4. Synthesis of compound 22
b
c
a
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7b
17
18
19
e
f
d
20
21
22
a
(
a) 4ꢀfluoroꢀ3ꢀnitroꢀbenzenesulfonyl chloride, pyridine, DMAP, CH Cl ; (b) Cu(OAc) , 1,2ꢀ
2 2 2
dichloroethane microwave (150°C); (c) NH OH, dioxane; (d) Fe, AcOH 60°C; (e) HCOOH,
4
1
00°C; (f) LiOH.H O, THF/H O.
2 2
In vitro structure activity relationship (SAR).
Compounds were evaluated for human PPARs in vitro potency and efficacy using a cell based
Galꢀ4 transactivation assays in CVꢀ1 simian in origin with SV40 gene (COSꢀ7) cells transfected
with either PPAR
α, PPAR
δ
, or PPAR
γ
ligand binding domains (LBD). Emax was measured
2
6
27
relatively to fenofibric acid for PPAR
α
, 1a for PPAR
γ
and GW501516 for PPAR
δ
.
Investigating the chain linker.
To establish basic SAR information, we started the optimization with analogs bearing different
types of linker between the indole moiety and the acidic head group (Table 1). Modification of
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the linker led to noticeable changes in the PPAR subtype activity. As already mentioned the two
carbon atom unit linker, compound 4 imparted potent PPAR
PPAR and ones. Extending to a three carbons atom chain 10b led to a decreased PPAR
activity, and increased both PPAR and ones. The lengthening to a four carbon atoms unit
chain 10c, or the introduction of a heteroatom in the chain 13a led to a total loss of activity. As
could be expected from previously described PPAR selective compounds, the addition of a
substituent at the α position of the carbonyl group, indeed, resulted in a selective PPAR
α transactivation activity, but week
δ
γ
α
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
δ
γ
α
α
agonist
1
3b. Finally compound 10b with a three carbons atom chain demonstrated the best balanced pan
PPAR profile in terms of potency, with partial efficacy (Emax in the range of 50%).
a
Table 1. Transactivation activity: analogues of compound 4 with modification of the Y linker
hPPAR
α
hPPAR
δ
hPPARγ
compd
Y
EC (nM) Emax (%) EC (nM) Emax (%) EC (nM) Emax (%)
50
50
50
4
(CH
(CH
(CH
2
2
2
)
)
)
2
3
4
260 ± 34
101 ± 14
51 ± 19
< 20
1912 ± 75
56 ± 18
46 ± 10
< 20
1558 ± 1
43 ± 3
46 ± 10
<20
1
0b
10c
3a
3b
956 ± 290
nd
645 ± 21
284 ± 97
nd
nd
nd
nd
1
CH
2
OCH
2
nd
< 20
< 20
nd
< 20
1
(CH
2
)
2
C(CH
3
)
2
160 ± 24
90 ± 39
< 20
910 ± 135
32 ± 13
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Emax = maximum activation in percent of control. compd = compound. nd = not determined.
All EC were mean of at least two independent experiments performed in triplicates.
50
Investigating the substitution of the indole.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
To further explore the activity and pan PPAR profile of this series, the investigation focused
onto the substitution of the indole ring (Table 2). First the 5 position was investigated. Only
hydrophobic substituents, such as 5ꢀChloro 10b, 5ꢀtrifluoromethyl 16a and 5ꢀmethyl 13c led to
active compounds. The trifluoromethyl (CF ) substitution 16a yielded a dual PPARα/γ agonist
3
compound, very close in term of potency and efficacy to the parent compound 4 but still with a
less balanced pan PPAR profile compared to compound 10b which remained the best profile at
this stage. On the contrary the 5ꢀmethoxy 10d was totally inactive.
Additionally a series of more hindered alkoxy chains (data not shown) were prepared but
unfortunately these modifications were completely detrimental for pan PPAR activity.
The other positions of the indole scaffold were also investigated (data not shown). The 6ꢀ
position turned out to be unfavorable for PPAR activity, while the 4ꢀposition led to a completely
misbalanced profile.
1
Table 2. Transactivation activity: analogues of compound 4 with modification of the R
a
substituent
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6
7
hPPAR
α
hPPAR
δ
hPPARγ
1
compd
R
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
EC (nM) Emax (%) EC (nM) Emax (%) EC (nM) Emax (%)
50
50
50
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
0b
6a
13c
Cl
956 ± 290
1198 ± 406
1300 ± 28
nd
51 ± 19
48 ± 12
46 ± 29
<20
645 ± 21
46 ± 10
49 ± 15
45 ± 20
<20
284 ± 97
46 ± 10
50 ± 9
38 ± 22
<20
1
CF
3
>5000
>5000
nd
417 ± 195
284 ± 80
nd
CH
3
1
0d
OCH
3
a
Emax = maximum activation in percent of control. compd = compound. nd = not determined.
All EC were mean of at least two independent experiments performed in triplicates.
50
Investigating the nature of the aryl sulfonamide.
Our efforts were first focused on the phenyl sulfonamide substitutions (Table 3). Substitutions
in the ortho position (16d, 16g), tend mostly to drastically reduce the agonistic activity against
all three subtypes. The meta and para positions of the phenyl were explored using electronꢀ
donating and electronꢀwithdrawing groups and led to active compounds leaning toward PPAR
γ
activity. Furthermore no difference was observed between electronꢀdonating 16b and electronꢀ
withdrawing 16e groups in the meta position. The same tendency is observed for para
substituted compounds 16c, 16f and 16i.
It was therefore concluded that meta and para substituted compounds were quite similar in
term of profile and no electronic effect could be observed on this part of the molecule.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
As described in Table 1, the three carbon atom spacer chain led to moderately potent compounds
presenting however the desired balanced pan PPAR agonist activity. Similar profiles are
obtained when the C5 indole position is substituted by CF or Chloro.
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Finally it clearly appears that meta and para substitutions were the best options towards the
wanted balanced profile.
1
Table 3. Transactivation activity: analogues of compound 4 with modification of the R and Ar
a
substituents
hPPAR
α
hPPAR
δ
hPPAR
γ
1
compd
R
Ar
Ph
EC (nM) Emax (%)
EC (nM) Emax (%)
EC (nM) Emax (%)
50
50
50
1
1
0b
6b
Cl
956 ± 290
51 ± 19
37 ± 23
55 ± 2
645 ± 21
1607 ± 453
1259 ± 393
nd
46 ± 10
28 ± 12
24 ± 4
<20
284 ± 97
65 ± 34
46 ± 10
46 ± 14
38 ± 8
CF
CF
CF
CF
CF
CF
CF
mꢀOMeꢀPh 1164 ± 352
3
16c
6d
6e
6f
6g
16h
3
3
3
3
3
3
pꢀOMeꢀPh
oꢀOMeꢀPh
mꢀClꢀPh
pꢀClꢀPh
764 ± 218
>5000
82 ± 19
1
35 ± 23
70 ± 5
1248 ± 447
256 ± 171
629 ± 359
>5000
35 ± 14
49 ±12
36 ± 13
45 ± 1
1
2421 ± 229
2824 ± 326
>5000
2026 ± 149
1650 ± 636
nd
21 ± 3
38 ± 13
<20
1
45 ± 27
104 ± 8
50 ± 19
1
oꢀClꢀPh
mꢀOCF3ꢀPh 2756 ± 287
1140±26
23 ± 11
201±141
39 ± 6
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1
6i
6j
6k
6l
6m
CF
3
pꢀOCF3ꢀPh 1963 ±1425
40 ± 18
40 ± 19
26 ± 10
59 ± 22
36 ± 10
2100 ±1273
1230 ± 449
nd
28 ± 11
43 ± 7
<20
573 ± 292
55 ± 18
32 ± 14
48 ± 10
25 ± 4
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
Cl
Cl
Cl
mꢀOMeꢀPh
pꢀOMeꢀPh
mꢀMeꢀPh
pꢀMeꢀPh
305 ± 35
419 ± 122
1250 ± 281
>5000
1
72 ± 59
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1347 ± 251
1085 ± 78
42 ± 6
29 ± 3
75 ± 25
46 ± 10
30 ± 9
1
Cl
277 ± 117
a
Emax = maximum activation in percent of control. compd = compound. nd = not determined.
All EC were mean of at least two independent experiments performed in triplicates.
5
0
The role of the phenyl group (south part of the molecule), was further investigated by
exploring fused rings systems (Table 4). As expected a fused ring mimicking the ortho
substitution led to a drastic decrease in PPAR activity. For example, 1ꢀnaphtyl sulfonamide
derivative 10g showed a significantly reduced efficacy on PPAR
γ and no activity at all on
PPAR . Efforts were then focused on other fused rings mimicking meta or para substitutions
δ
that were shown to be more in line with our objectives to reach a pan PPAR balanced profile.
Surprisingly 2ꢀnaphtyl 10f showed very low potency on all three PPARs. It was observed, at this
stage, that non conjugated fused ring like benzo[1,3]dioxole 10e and Nꢀmethyl benzomorpholine
1
3d led to very active compounds but with misbalanced potency in favor of the PPARγ. The
conjugated fused ring benzothiazole analogue 5 displayed high efficacy on all PPARs subtypes
and the best balanced profile in term of potency.
Table 4. Transactivation activity: analogues of compound 4 with modifications of the Ar
a
substituent Part1.
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hPPAR
α
hPPAR
δ
hPPAR
γ
compd
Ar
Ph
EC (nM) Emax (%)
EC (nM) Emax (%)
EC (nM) Emax (%)
50
50
50
10b
956 ± 290
410 ± 107
51 ± 19
645 ± 21
46 ± 10
54 ± 6
284 ± 97
16 ± 3
46 ± 10
48 ± 10
1
3d
0e
113 ± 44
697 ± 271
1
790 ± 339
1537 ± 278
nd
77 ± 21
106 ± 30
<20
977 ± 134
866 ± 180
nd
43 ± 9
105 ± 16
<20
71 ± 12
206 ± 71
35 ± 3
45 ± 11
79 ± 15
34 ± 7
36 ± 8
S
N
5
10f
10g
901 ± 435
67 ± 16
nd
<20
265 ± 131
a
Emax = maximum activation in percent of control. compd = compound. nd = not determined.
All experiments were performed in triplicates in at least two independent experiments.
At this stage it was decided to investigate more in depth the potential of the benzothiazole
moiety, by introducing a series of different heteroaromatic rings (Table 5). Unfortunately the 5ꢀ
sulfonylbenzothiazole, isomer 4ꢀ[1ꢀ(1,3ꢀbenzothiazolꢀ5ꢀylsulfonyl)ꢀ5ꢀchloroꢀindolꢀ2ꢀyl]butanoic
acid, of compound 5, was not prepared due to chemical stability issues of the corresponding
sulfonyl chloride intermediate. In order to investigate the influence of substitution at position 2
of the benzothiazole we decided to prepare the methylated derivatives of both benzothiazole
isomers (1,3ꢀbenzothiazolꢀ5ꢀylsulfonyl) compound 10i and 1,3ꢀbenzothiazolꢀ6ꢀylsulfonyl
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compound 10h was prepared. These derivatives ended to be much less efficient than 5 on both
PPAR
(10h)=27%, Emax (10i)=20%, Emax (5)=105%) while keeping some efficacy against
PPAR (Emax (10h)=36%, Emax (10i)=39%, Emax (5)=79%). At last it became clear that there
α (Emax (10h)<20 %, Emax (10i)=22%, Emax (5)=106%) and PPARδ (Emax
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
γ
was no advantage to use the other isomer of the benzothiazole, as the final picture of PPAR
activity was completely unbalanced. Replacing the methyl group on the position 2 of the
benzothiazole with an amino group compound 10j allowed to regain activity on PPAR
PPAR but did also increase the unbalance toward PPAR with EC of 41 nM. Moving to the
corresponding benzoxazole 10k allowed to obtain with a compound well balanced for PPAR
and PPARγ but less potent on PPAR . To conclude on this part all the tested substitutions on the
ꢀposition (10h, 10i, 10j and 10k) led to unbalanced profile and the naked compound 5 remained
α and
δ
γ
50
δ
α
2
the best regarding the wanted profile.
Alternative 5,6 heteroaromatic rings containing compounds, like the benzimidazole 22 and
benzothiadiazole 10l, display low potency or unbalanced profile.
Finally all the produced derivatives demonstrated lower efficacy, potency or unbalanced
profile in comparison to compound 5. These findings prompted us to retain the benzothiazole
substituent on the south part of the molecule as the most preferred option for the desired pan
PPAR profile.
Table 5. Transactivation activity: analogues of compound 4 with modification of the Ar
a
substituent Part 2.
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hPPAR
α
hPPAR
δ
hPPAR
γ
compd
Ar
EC (nM) Emax (%)
EC (nM) Emax (%)
EC (nM) Emax (%)
50
50
50
5
1537 ± 278 106 ± 30
866 ± 180
441 ± 292
1098 ± 114
919 ± 79
1387 ± 317
>5000
105 ± 16
27 ± 7
20 ± 7
82 ± 11
66 ± 16
20
206 ± 71
11±1
79 ± 15
36 ± 6
39 ± 8
74 ± 3
52 ± 19
42
10h
nd
<20
22 ± 3
57 ± 6
44 ± 20
43
10i
2360 ± 127
1263 ± 25
>5000
175 ± 88
41 ± 8
10j
10k
22
378 ± 111
>5000
>5000
10l
>5000
81
>5000
35
153
67
a
Emax = maximum activation in percent of control. compd = compound. nd = not determined.
All EC were mean of at least two independent experiments performed in triplicates. Values
50
based on single experiment run in triplicates are indicated in italics.
Reverting to the core ring investigation, the 5ꢀCF substitution on the indole which was
3
favorable on the earlier phenyl series (16a, Table 2), surprisingly turned out to be similar in term
of potency but less efficient in this more evolved benzothiazole series (10m, Table 6) and only
the 5ꢀChloro analog 5 retained the desired balanced pan PPAR profile.
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1
a
Table 6. Transactivation activity: Exploring the R substituent of compound 5
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
hPPAR
α
hPPAR
δ
hPPAR
γ
1
compd
R
EC (nM) Emax (%)
EC (nM) Emax (%)
EC (nM) Emax (%)
50
50
50
5
Cl
CF
1537 ± 278
1620 ± 430
106 ± 30
82 ± 14
866 ± 180
105 ± 16
79 ± 13
206 ± 71
147 ± 12
79 ± 15
70 ± 7
1
0m
3
1120 ± 161
a
Emax = maximum activation in percent of control. compd = compound. All EC were mean of
5
0
at least two independent experiments performed in triplicates.
Physicochemical properties and pharmacokinetic profiling.
The promising in vitro human results obtained with this novel indolsulfonamides series have
motivated further investigation to demonstrate their potential to become preclinical development
compounds.
The drugꢀlike properties of the most promising compound 5 were examined to determine its
suitability for further investigation.
Aqueous solubility and permeability are two major players that determine oral bioavailability.
Despite its low solubility compound 5, as well as the original hit 4, showed a very strong
permeability profile compared to Warfarin. (Table 7).
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2
2
2
2
2
2
2
2
2
3
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3
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3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 7. In vitro permeability of selected analogs
ꢀ6
Compound
Cacoꢀ2 Mean Papp (x10 cm/s) neat/FASSIF
Solubility (µM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A→B
24
B→A
0.2
Efflux ratio
0.1
5
4
8.7
9
61
14.4
0.7
0.2
Warfarin
39.8
0.02
56
Functional activity: beta-oxidation in human hepatocytes and mouse myoblast cell line.
Functional PPAR activity for all three subtypes was determined in a panel of in vitro assays.
Fatty acid betaꢀoxidation was used as a marker of PPAR
respectively evaluated in human hepatoma cell line (HuH7) and in mouse myoblast cell line
C2C12). The PPAR functional activity was measured in human adipocytes through the
expression of adiponectin and adipocyte protein 2 (aP2) genes, using 1a at 1 µM as reference.
As can be observed in Table 8, compound 5 demonstrated significant activation of PPAR
PPAR as indicated by increasing levels of betaꢀoxidation on HuH7 and C2C12 consistent with
α and PPARδ activation. They were
(
γ
α
and
δ
the observed transactivation data. Compound 5 induced 45% of betaꢀoxidation at 1 µM in HuH7
and a significant effect in C2C12 at 3 µM, while compound 4 showed betaꢀoxidation activation
on HuH7 only at 10 µM and no effect on C2C12. The best dose response profile on betaꢀ
oxidation in both human hepatocytes and mouse myoblasts was observed with compound 5 in
comparison to compound 4. Similar conclusion can be drawn from the induction of PPARγ target
genes adiponectin and aP2 expression (Table 9). A clear dose response effect is observed, despite
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a slight decrease at the highest concentration i.e. 30 µM which could be attributed to partial
compound precipitation.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 8. Cellular activities: beta oxidation in HuH7 and C2C12
compd
HuH7 (% betaꢀoxidation)
C2C12 (% betaꢀoxidation)
(
Conc.)
1 µM
44 ± 10
5 ± 12
3 µM
10 µM
113 ± 23 120 ± 9 12 ± 9
45 ± 2 96 ± 19 ꢀ6 ± 4
30 µM
1 µM
3 µM
10 µM 30 µM
67 ± 9 55 ± 8
12 ± 12 27 ± 7
5
4
79
37
ꢀ
ꢀ
a
All values are mean of at least 2 independent experiments run in triplicate. Values based on
single experiment run in triplicate are indicated in italics. compd = compound.
a
Table 9. Cellular activities: gene expression of aP2 and Adiponectine in adipocytes
compd 5
0.1 µM
0.3 µM
1 µM
3 µM
10 µM
30 µM
(conc.)
aP2
6 ± 1
7 ± 1
18 ± 4
16 ± 1
47 ± 1
42 ± 8
78 ± 18
62 ± 6
97 ± 3
80 ± 6
70 ± 12
56 ± 3
Adiponectine
a
%
expression vs 1a @ 1 µM accounting for 100%. All values are mean of 3 independent
experiments run in triplicate. compd = compound.
In vivo pharmacokinetic studies.
Encouraged by the promising in vitro results, the pharmacokinetic parameters of the best
candidate, compound 5 were determined in common in strain of laboratory mouse (C57Bl6). 8
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2
2
2
2
2
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2
2
2
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3
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3
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4
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5
5
5
5
5
5
5
5
5
5
6
animals were dosed orally with 5 (10 mg/kg), and plasma samples were collected over a 24 hours
period. The preliminary data suggested a once daily dose.
Interestingly, compound 5 was detected in plasma in the microgram range (maximum serum
concentration that a drug achieves in a specific compartment (Cmax) = 10.7 ꢁg/mL), well above
the EC50 value, which aligns well with the measured solubility, human Caucasian colon
adenocarcinoma (Cacoꢀ2) permeability and absorption data. This overall satisfactory profile
provided solid evidence that this new generation of pan PPAR derivatives were well suited for
evaluation in animal disease models.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 10. Compound 5 Plasma Pharmacokinetic Parameters Following Single Oral Dose (10
mg/kg in methyl cellulose as vehicle) in C57Bl6 mice.
a
a
a
Compound
Cmax (ng/mL)
Tmax (h)
AUCinf
(h·(ng/mL))
5
4
10710
14590
11850
1
1
1
29367
37286
51082
1
0b
a
Single measurement.
In vivo pharmacological studies.
db/db mouse model.
Before moving to the in vivo models it was established that compound 5 exhibited similar
potency and efficacy for human and mouse PPARs as measured in chimeric galactoseꢀresponsive
transcription factor (Galꢀ4) human or mouse PPARꢀmediated reporter gene assays (Table 11).
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28
The antiꢀdiabetic effect of compound 5 was evaluated in db/db mice, an obese rodent model
of type 2 diabetes characterized by severe insulin resistance, hypertriglyceridemia, marked
hyperglycemia.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Table 11. Agonistic Effect of compound 5 on human and mouse PPARs isoforms
PPAR
α
PPAR
δ
PPARγ
EC (nM) Emax (%) EC (nM) Emax (%) EC (nM) Emax (%)
50
50
50
Human 1537 ± 278
Mouse 366 ± 71
106 ± 30
72 ± 14
866 ± 180
1560 ± 279
105 ± 16
131 ± 30
206 ± 71
202 ± 48
79 ± 15
73 ± 17
a
Emax = maximum activation in percent of control. All experiments were performed in
triplicates in at least three independent experiments.
Compounds were administrated orally once daily for 5 days for 5 and 10 days for 4, to
homozygous C57BL/Ksꢀdb male mice (db/db mice). A blood sample was taken from the retroꢀ
orbital sinus before treatment and 4 hours after the last gavage. Triglycerides and glucose levels
were measured after serum collection. The results are expressed in % variation on the final day
relative to the control group (Figure 3).
As would be expected from a pan PPAR agonist, treatment of db/db mouse with compound 5
induced a dose dependent and significant decrease of circulating glucose levels: 40% at 10
mg/kg, and 58% at 30 mg/kg. In the same study abnormal plasma triglycerides levels observed in
this disease model were markedly corrected following treatment with compound 5: 33% at 10
mg/kg, and 45% at 30 mg/kg (Figure 3). On the contrary compound 4 , the initial hit, had nonꢀ
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5
5
5
5
5
5
6
significant effect on triglycerides levels, and moderate decrease of circulating glucose at 10
mg/kg which was improved at 30 mg/kg.
0
1
2
3
4
5
6
7
8
9
0
1
2
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 3. Compounds 4 and 5: Percentage reduction of plasma Glucose and triglycerides in
db/db mice. Mice were dosed qd with 10 or 30 mg/kg for 5 days with 5 and 10 days with 4. *P<
0.05, ***P< 0.001 versus control (oneꢀway analysis of variance followed by Dunnett’s test)
2
9
Carbon tetrachloride (CCl )ꢀinduced liver fibrosis in mice .
4
Liver fibrosis is a critical hallmark of NASH, being associated with longꢀterm overall mortality
in patients. Fibrosis is characterized by an excessive synthesis of ExtraꢀCellular Matrix
composed of collagen and fibronectin. CCl induced liver fibrosis is a wellꢀdocumented model to
4
investigate potential antiꢀfibrotic properties of pharmacological agents, the evaluation of antiꢀ
fibrotic activity being determined by the measurement of collagen deposition in the liver.
Collagen deposition measured by picrosirius red (PSR) staining on liver histological sections
was significantly higher in CCl ꢀvehicle treated mice than in saline injected mice. Treatment
4
with compound 5 at 10 and 30 mg/kg, orally once a day, produced a dose dependent and
significant reduction of collagen deposition. In parallel, and consistently with panPPAR
activation, compound 5 produced a significant reduction in plasma triglycerides levels, a maker
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of PPARα/δ engagement, and a significant elevation of plasma adiponectin levels, a marker of
PPARγ engagement (Figure 4).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
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5
5
5
5
5
5
5
5
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0
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0
1
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1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Effects of compound 5 at 3, 10, 30 mg/kg on collagen deposition, plasma triglycerides
and adiponectin in CCl ꢀinduced liver fibrosis in mice after 3 weeks of treatment. **P< 0.01,
4
*
**P< 0.001 versus vehicle CCl ; ###P<0.001 versus vehicle oil (oneꢀway analysis of variance
4
followed by Dunnett’s test).
Haemodilution, plasma volume expansion and heart weight increase in rats.
Contrasting with first generation PPAR
γ
agonists like 3a, 3b or 1a, compound 5 had no effect
agonists that were
on hematocrit, plasma volume or heart weight. All the 3 other PPAR
γ
evaluated in this study induced a significant reduction in hematocrit levels and a significant
increase in plasma volume after 4 weeks of treatment at the highest dose. These compounds, but
not compound 5 showed marked increase in heart weight after 6 weeks of treatment (Figure 5).
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Effects of compound 5 at 100 and 1000 mg/kg on hematocrit, plasma volume and heart
weight after 4 and 6 weeks of treatment in SpragueꢀDawley rats, comparison with the PPAR
γ
and dual PPARα/γ agonists 3a, 3b and 1a. *P< 0.05, ***P< 0.001 versus control (oneꢀway
analysis of variance followed by Dunnett’s test).
Xray PPAR
γ
structure with compound 5 in comparison to 1a.
structures have been described in the protein data base (PDB). PPAR’s
Numerous PPAR
γ
ligand binging domain classically consists of 12 αꢀhelices and 4 betaꢀstranded sheets. PPARγ’s
3
ligand binding pockets (LBP) is large (1300Å ) with Yꢀor Tꢀshapes. Different binding modes of
ligands are observed in PPARγ’s LBP. Ligands may bind in different locations in the LBP
3
0
31
32
(
3K8S , 3GBK and 4PWL ) and, on some occasions, two ligands can be accommodated at
33
the same time (4BCR ). Compound 1a (dark green) (Figure 6B) is a potent PPAR
γ
agonist. It
3
4
binds in a Uꢀshape conformation, wrapping itself around Helix3 (1FM6 ). The thiazolidinedione
head group, which mimics the usually encountered carboxylic acid moiety present in PPAR’s
35
natural ligands such as fatty acids (e.g. 2VV2 ), interacts directly with Helix12 via multiple
hydrogen bonds. The thiazolidinedione’s nitrogen atom binds to Y473 and its two carbonyl
groups to H323 and H449. Differently from 1a (dark green), compound 5 (purple) (Figure 6A)
does not interact directly with Helix12 even if it stabilizes a conformationally active form of
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PPAR
γ
. It adopts a Tꢀshape conformation partially overlapping with the Uꢀshape of 1a (Figure
6
C) and filling an additional subꢀpocket with specific hydrogen bond interactions between its
carboxylic acid and both E343 and R288.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
C
Figure 6.
A. Coꢀcrystal structure of compound 5 (purple) with PPAR
γ
LBD (6ENQ)
LBD (1FM6)
B. Coꢀcrystal structure of compound 1a (dark green) with PPAR
γ
C. Superposition of compound 5 (purple) and 1a (dark green) in PPARγ LBD
Cofactors recruitment by compound 5 vs. 1a.
It is now known from genetic and pharmacological studies that moderately activating
3
6
compound can lead to better therapeutic outcomes compared to full activation . These
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1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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4
4
4
4
4
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5
5
5
5
5
5
5
5
5
6
observations have been translated into the concept of PPAR partial agonists and selective PPAR
3
7,38
modulators (SPPARMs),
leading to differential cofactor recruitment profiles, and finally
3
9
different gene expression patterns. The cofactor recruitment profile induced by compound 5
and 1a, were evaluated in a timeꢀresolved fluorescence energy transfer (TRꢀFRET) assay using a
panel of coꢀactivators and coꢀrepressors, for the determination and comparison of their respective
potency and efficacy.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Comparatively to 1a, compound 5 displayed significant differences in cofactor recruitment both
in terms of potency (Figure 7B) and efficacy (Figure 7A). For example, the coꢀrepressors nuclear
receptor corepressor 1 (NCoRꢀID1) and silencing mediator of retinoic acid and thyroid hormone
receptor (SMRT) were both “derecruited” by compound 5 and 1a, but with a 10ꢀfold lower
potency for compound 5. EC respectively were 4 097 nM vs. 326 nM for NCoRꢀID1 and 1 140
50
nM vs. 95 nM for SMRT (Table 13).
Major differences in potency were also noticed for nuclear receptor coactivator 3 (NCoA3),
steroid receptor coactivator 1 (SRCꢀ1), PPARγ coactivatorꢀ1α (PGC1α), receptor interacting
protein 140 (RIP140) and prolineꢀrich nuclear receptor coactivator 1 (PNRC1) recruitment.
When considering efficacy, compound 5 showed a partial agonist activity for PGC1α and ligandꢀ
dependent corepressor (LCOR), and a greater efficacy for PNRC1 in comparison to 1a.
Table 13. Coꢀfactors recruitment comparison compound 5 vs 1a
1a
5
Peptide
EC50
Emin/max
Emax
EC50
Emin/max
Emax
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(
nM)
(FI)
0.06
0.1
(%)
100
100
100
100
100
100
(nM)
1140
4097
727
(FI)
0.06
0.1
(%)
100
100
68
SMRTꢀID1 (2339ꢀ2363)
NCoRꢀID1 (2253ꢀ2277)
LCOR (39ꢀ63)
95
326
52
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
15.5
12.2
14.2
2.3
10.6
12.4
13.3
1.5
NCoA3 (607ꢀ631)
NCoA3 (671ꢀ695)
PGC1α (196ꢀ221)
PNRC1 (302ꢀ327)
RIP140 (922ꢀ946)
SRCꢀ1 (619ꢀ643)
114
117
1484
1917
3208
102
93
64
332
NC
NC
316
1.9
1.95
2.3
100
100
100
4111
3274
3408
2.7
1.7
2.6
142
88
111
ACS Paragon Plus Environment
29