From ApoA1 upregulation to BET family bromodomain inhibition: Discovery of I-BET151

Article abstract of DOI:10.1016/j.bmcl.2012.01.125

The discovery, synthesis and biological evaluation of a novel series of 7-isoxazoloquinolines is described. Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepatic cell line HepG2.

Full text of DOI:10.1016/j.bmcl.2012.01.125

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2963–2967
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
From ApoA1 upregulation to BET family bromodomain inhibition: Discovery
of I-BET151
⇑
Olivier Mirguet , Yann Lamotte, Frédéric Donche, Jérôme Toum, Françoise Gellibert, Anne Bouillot,
Romain Gosmini, Van-Loc Nguyen, Delphine Delannée, Jonathan Seal, Florence Blandel,
Anne-Bénédicte Boullay, Eric Boursier, Sandrine Martin, Jean-Marie Brusq, Gael Krysa,
Alizon Riou, Rémi Tellier, Agnès Costaz, Pascal Huet, Yann Dudit, Lionel Trottet, Jorge Kirilovsky,
Edwige Nicodeme
Lipid Metabolism Discovery Performance Unit, GlaxoSmithKline, 25 avenue du Québec, 91951 Les Ulis Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 8 February 2012
The discovery, synthesis and biological evaluation of a novel series of 7-isoxazoloquinolines is described.
Several analogs are shown to increase ApoA1 expression within the nanomolar range in the human hepa-
tic cell line HepG2.
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
ApoA1 upregulators
7-Isoxazoloquinolines
BET family
Bromodomains inhibitors
Atherosclerosis is a leading cause of death in Western societies.
Currently, statins are widely used for the treatment of atheroscle-
rosis. However, despite their extensive usage, there still remains a
significant unmet need for treatments directed at preventing or
limiting the effects of atherosclerosis. Apolipoprotein A1 (ApoA1)
is a major protein component of the high density lipoprotein
(HDL). Increased levels of HDL cholesterol have been correlated
with a decreased risk for atherosclerosis and overexpression of
ApoA1 is known to raise HDL cholesterol levels.1 Thus, upregula-
tion of ApoA1 is considered to be one of the most promising
approaches for the development of new therapies for
atherosclerosis.2
concentration of compound resulting in a 70% increase in the lucif-
erase activity (ApoA1 Luc EC170).5
Herein we report the discovery of a series of highly potent isox-
azoloquinolines which may be of potential therapeutic value for
the treatment of atherosclerosis. A summary of the in vitro SAR
studies around the quinoline ring leading to the discovery of cy-
clized analogs with enhanced potency and PK profile is described.
GW694481 (1, Fig. 1) was identified as a robust hit from this HTS
campaign and substructure searches within the GSK compound
collection quickly showed us that the dimethylisoxazole moiety
at position 7 of the quinoline ring was crucial for activity (box A,
We previously reported the discovery of BZD compounds such
as GSK525762A (I-BET or I-BET762) as ApoA1 upregulators3,4
(Fig. 1). In parallel to the lead optimization of the BZD series,4
a
N
N
HTS approach using a transcriptional cellular assay was carried
out to identify novel chemical templates: HepG2 cells were trans-
fected with a plasmid encoding firefly luciferase under the control
of the human ApoA-1 promoter region (ꢀ1162, +232) and the
30-UTR of the human ApoA-1 gene (+1037, +1091), and compounds
were screened for their ability to increase ApoA1 expression in this
stably transfected HepG2 cell line. Potency was defined as the
N
NH
N
O
NH
N
MeO
NH2
O
O
N
Cl
GSK525762A, I-BET
ApoA1 Luc EC170 = 0.2 µM
GW694481, 1
ApoA1 Luc EC170 = 0.5 µM
⇑
Corresponding author.
E-mail address: olivier.x.mirguet@gsk.com (O. Mirguet).
Figure 1. Previously described ApoA1 upregulator GSK525762A (I-BET) and HTS hit
GW694481, 1.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2012.01.125

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O. Mirguet et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2963–2967
profile at both 2C9 and 3A4 (cf. 14 vs 13). (3)—addition of an extra
B
substituent on the quinoline ring (15 to 18) demonstrated that
only a 6-OMe on the quinoline ring maintains potency (cf. 14 vs
16). When applied in the carboxamide subseries (R1 = CONH2),
we observed an increase in ApoA1 upregulation with this addi-
tional 6-OMe (cf. 9 vs 19 and 13 vs 20). Increasing the size of the
ortho substituent on R2 led to potent compounds (cf. 21, 22, 26
and 27) and an improvement of P450 profile for 22 and 27. One
of the preferred compounds we identified within this subseries is
compound 28, which has a quinoline as the R2 substituent, despite
the fact that CYP inhibition is still in the low micromolar range.
In order to eliminate the CONH2 that is the likely cause of the
P450 liability and at the same time freezing a postulated intramo-
lecular hydrogen bond between C3 carboxamide and C4 NH, we
envisaged synthesis of some imidazolone analogues (Fig. 3). Com-
pounds 29 to 38 could be readily obtained from compounds in
Table 1 using either PIFA (phenyliodine bis(trifluoroacetate)) to
perform a Hoffman rearrangement of the carboxamide com-
pounds8 or a DDPA-mediated Curtius rearrangement9 from the
carboxylic acid starting materials as depicted in Scheme 2.
The key SAR findings for the imidazolone subseries are summa-
rized in Table 2 and show that when R2 is a substituted phenyl ring,
cyclization did not improve but only maintained potency (cf. 29
and 30 vs 19 and 20), nor abolished the CYP liability with the re-
moval of the primary carboxamide (cf. 30 vs 20). But when cycliza-
tion was applied to compounds where R2 is benzylic, a 10-fold
improvement of potency was observed (cf. 32 vs 24). The CYP inhi-
bition could be alleviated by introducing a pyridin-2-ylmethyl sub-
stituent at R2 (cf. 33 vs 32) but with some loss in potency on ApoA1
upregulation. Introduction of a chiral racemic methyl group at the
R2 benzylic position further improved potency on ApoA1 without
any effect on the CYP inhibition (cf. 34 vs 32). Chiral HPLC separa-
tion of the racemic compound 34 led to enantiomers 35 and 36,
with 35 being the most active compound on ApoA1 upregulation
identified within this chemical series. A slight improvement on
the P450 profile was obtained with compound 37 and its chiral
ethyl group. Combination of the pyridin-2-ylmethyl substituent
and the benzylic chiral methyl group led to the identification of
compound 38 (I-BET151)10 which displayed high potency on
ApoA1 upregulation and relatively low CYP inhibition.
NH
4
O
C
NH2
3
7
N
O
N
A
Figure 2. SAR overview of HTS hit.
Fig. 2). Both positions 3 and 4 (boxes B and C respectively) were
open for optimization. The only issue identified with this hit com-
pound was an undesirable micromolar inhibition of CYPs 2C9 and
3A4 to eliminate.6
A representative synthesis of these 7-isoxazoloquinolines is de-
picted in Scheme 1: Introduction of the isoxazole ring via a Suzuki
coupling followed by reduction of the nitro group led to an aniline
intermediate. Using Gould-Jacobs conditions,7 condensation of the
aromatic amine with diethyl 2-(ethoxymethylene)malonate and
thermal cyclisation led to the formation of the quinolin-4-one.
Saponification of the ethyl ester followed by a POCl3-mediated
chlorination gave a dichloro intermediate that was readily trans-
formed into a 3-chloro-4-carboxamide by addition of ammonia at
0 °C. Thermal condensation with the desired amine R2NH2 led to
ApoA1 upregulators 1, 6–13, 19–23 and 25–28. To access carbox-
ylic acids 3, 14–18, the ethyl ester was converted to the corre-
sponding chloro intermediate with POCl3. Thermal condensation
with the desired amine R2NH2 followed by saponification afforded
the desired acids. The latter were subsequently transformed into
amide 4, 5 and 24 using standard amide bond formation protocols.
The key SAR findings are summarized in Table 1 and show—(1)
the need for a R1 substituent on the quinoline ring, a primary car-
boxamide being optimal for potency in ApoA1 upregulation (cf. 1
vs 2, 3, 4 and 5); when R1 is a carboxylic acid, 3-fold potency is lost
at ApoA1, but the compound is devoid of activity on both CYPs 2C9
and 3A4.—(2) the R2 position tolerates a lot of diversity, aromatic,
aliphatic (23) or benzylic (24 and 25) substituents maintaining
ApoA1 potency. When R2 is a phenyl ring, the optimal position
for substitution is on the ortho carbon (cf. 6 vs 7 and 8). The ortho
position of R2 tolerates a lot of diversity in terms of electronics and
size (cf. 6, 9, 10, 11, 12, and 13). Again, replacement of the primary
carboxamide with a carboxylic acid at R1 improves the P450
Compounds 28 and I-BET151 were selected for further profiling
and were evaluated in rat to determine their pharmacokinetic pro-
file (Table 3). Both compounds were well absorbed and exhibited
R2
O
Cl
N
R3
NH
N
O
R3
R3
R3
CO2Et
CO2Et
R3
a, c
d, e
g
i, f
OH
NH2
O
N
N
H
I
NO2
NO2
O
N
O
N
O
N
b, c
R3
3, 14 to 18
HO
B
f, g, h
Cl
j
OH
R2
R2
O
NH
N
O
NH
O
R3
R3
R3
R5
NH2
NH2
N
i
R4
N
N
O
O
O
N
N
N
1, 6 to 13, 19 to 23, 25 to 28
4, 5 and 24
Scheme 1. Reagents and conditions: (a) (3,5-dimethyl-4-isoxazoyl)boronic acid, cat. Pd(PPh3)4, base, DME, water, 90 °C; (b) 4-iodo-3,5-dimethylisoxazole, cat. Pd(PPh3)4,
sodium carbonate, toluene, EtOH, water, reflux (c) H2, Pd/C, EtOH, rt or SnCl2 hydrate, EtOH/water, reflux; (d) diethyl 2-(ethoxymethylene)malonate, 130 °C; (e)
diphenylether, 260 °C; (f) NaOH, EtOH, reflux; (g) POCl3, 120 °C; (g) NH4OH, THF, 0 °C or NH3 (g), dioxane, 0 °C; (i) R2NH2, neat, 300 °C or R2NH2, solvent, reflux, (j) R4R5NH,
EDCI, HOBt, TEA, DMF or R4R5NH, HATU, TEA, DCM/DMF or (1) oxalyl chloride, cat. DMF, DCM/THF, rt (2) R4R5NH, DIPEA, DCM/THF, rt.

O. Mirguet et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2963–2967
2965
Table 1
SAR data for the isoxazoloquinoline series
R2
NH
R3
R1
N
O
N
Compd
R1
R2
R3
ApoA1 Luc EC170
(l
M)a
CYP inhibition IC50 (lM)
2C9
2.1
3A4
17.0
1
2
3
4
5
6
7
8
CONH2
H
CO2H
Phenyl
Phenyl
Phenyl
Phenyl
Phenyl
2-Tolyl
3-Tolyl
4-Tolyl
2-Fluorophenyl
2-Methoxyphenyl
2-Cyanophenyl
2-Chlorophenyl
2-tert-Butylphenyl
2-tert-butylphenyl
2-tert-Butylphenyl
2-tert-butylphenyl
2-tert-Butylphenyl
2-tert-butylphenyl
2-Fluorophenyl
2-tert-Butylphenyl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
0.52
4.27
1.74
1.44
7.08
0.44
0.60
1.05
0.50
0.39
1.02
1.00
0.63
0.32
1.41
0.28
1.99
1.05
0.30
0.13
>33
>33
CONHCH2Ph
CONMe2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CONH2
CO2H
CO2H
CO2H
CO2H
CO2H
CONH2
CONH2
9
10
11
12
13
14
15
16
17
18
19
20
0.7
10
2.0
13.0
2-Me
6-OMe
6-Me
8-Me
6-OMe
6-OMe
0.6
1.2
4.7
1.9
O
N
21
CONH2
6-OMe
0.09
2.2
9.9
1.9
3.1
O
N
22
CONH2
6-OMe
0.20
23
24
CONH2
CONH2
Cyclohexyl
Benzyl
6-OMe
6-OMe
0.45
1.07
N
25
26
CONH2
CONH2
6-OMe
6-OMe
2.57
0.09
2-Morpholinophenyl
5.1
8.5
1.5
1.8
O
27
28
CONH2
CONH2
N
6-OMe
6-OMe
0.13
0.22
N
2.8
3.9
a
Given EC170 are mean of at least two independent determinations.
good bioavailability. In vivo clearance was found to be high for 28
and moderate for I-BET151 respectively, resulting in limited oral
plasma exposure. Nevertheless, the pharmacokinetic profile
showed that both compounds were suitable for chronic oral
administration.
The presence of an unsubstituted quinoline ring in 28 prompted
us to assess potential routes of metabolism and we determined
that compound 28 would be likely to undergo bioactivation, with
epoxidation and subsequent glutathione trapping seen in both
rat and human hepatocytes (data not shown). Furthermore, 28
was found to be positive in an Ames test,11 thus precluding further
progression. None of these liabilities were observed for I-BET151.
As part of the in vitro profiling of our compounds, we observed
that all compounds active in ApoA1 upregulation displayed anti-
inflammatory properties in non hepatic cells (ex: h-PBMC or
cyclise
R2
O
R2
NH
O
N
R3
R3
4
NH
NH2
3
N
N
O
N
O
N
Figure 3. Freezing likely active confirmation and eliminating Iary carboxamide at
the same time.

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O. Mirguet et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2963–2967
R2
Table 3
NH
N
O
In vivo pharmacokinetic properties for 28 and I-BET151 in CD rats
R3
Parametersa
28
I-BET151
OH
DNAUC0–inf (min kg/L)b
CLbc (mL/min/kg)
Vss (L/kg)
t1/2 (iv, h)
F, po %
12
50
4.0
1.1
59
38
18
2.1
1.7
66
O
N
b
R2
R2
O
a
NH
N
O
Values are means (n = 3).
DNAUC: dose-normalized AUC. Dose vehicle: 1% (w/v) methylcellulose
N
R3
b
R3
NH
NH2
a
(400 cps) (aq).
c
iv dose was 1 h of infusion in DMSO:10% (w/v) Kleptose HPB (2:98).
N
O
N
O
N
29 to 38
evaluated the anti-inflammatory effects of I-BET151 in a model
of LPS-challenged Balb/C mouse looking at a range of plasmatic
Scheme 2. Reagents and conditions: (a) PIFA, CH3CN, rt; (b) DPPA, DMF, 110 °C.
inflammatory markers including IL-6, MCP-1, TNF
a and PAI-1
THP-1 cells). The correlation between the potency of compounds
(Table 4). Following a single oral administration at 10 mg/kg, I-
BET151 displayed broad anti-inflammatory properties compared
to two different classes of anti-inflammatory agents namely pred-
on ApoA1 upregulation and inhibition of TNFa production was
similar suggesting that a common pharmacology was driving the
two effects (See Supplementary data). Figure 4 shows the dose-
nisolone,13 a well-known corticosteroid drug or SB-731445,14
p38 MAPK inhibitor.
a
dependent inhibition of both IL-6 and TNF
I-BET151 in LPS-stimulated human PBMC.12
a production by
Those intriguing results prompted us to investigate the mecha-
nism of action of our compounds. We recently disclosed the use of
a combination of phenotypic screening, chemoproteomics, and
On the basis of these results, we selected I-BET151 for further
in vivo evaluation in an acute inflammation mouse model. We
Table 2
SAR data for the imidazolone subseries
R2
O
N
NH
O
N
O
N
Compd
R2
ApoA1 Luc EC170
(l
M)a
CYP inhibition IC50
(l
M)
2C9
3A4
29
30
31
32
2-Fluorophenyl
2-tert-Butylphenyl
2-Trifluoromethoxyphenyl
Benzyl
0.76
0.05
0.09
0.06
0.9
2.3
1.2
1.7
2.0
3.7
N
33
0.38
0.03
0.01
0.47
0.03
0.09
9.8
1.3
1.0
12.9
2.0
34
35
1.5
36
37
2.4
9.9
2.4
9.7
N
38 (I-BET151)
a
Given EC170 are mean of at least two independent determinations.

O. Mirguet et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2963–2967
2967
Acknowledgment
We thank Mélanie Barnathan for chiral separation support and
Ann Walker for proof-reading.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2012.01.125.
References and notes
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Chandwani, R.; Marazzi, I.; Wilson, P.; Coste, H.; White, J.; Kirilovsky, J.; Rice, C.
M.; Lora, J. M.; Prinjha, R. K.; Lee, K.; Tarakhovsky, A. Nature 2010, 468, 1119.
4. Mirguet, O.; Ajakane, M.; Boullay, A. B.; Boursier, E.; Brusq, J.-M.; Clement, C.;
Costaz, A.; Daugan, A.; Dudit, Y.; Gosmini, R.; Huet, P.; Martin, S.; Pineau, O.;
Riou, A.; Toum, J.; Trottet, L.; Nicodeme, E. Abstracts of Papers, 241st ACS
National Meeting & Exposition, Anaheim, CA, United States, March 27–31,
2011, MEDI-76.
Figure 4. I-BET151 dose-dependently inhibits IL-6 and TNF
stimulated human PBMC.
a production in LPS-
5. The luciferase cellular assay was used as a primary screen. We also measured
ApoA1 secretion as a secondary assay in the same cell type. A very nice
correlation between the two readouts was observed, indicating that
compounds were truly upregulators of ApoA1 production by hepatic cells.
See Supplementary data for details.
Table 4
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In vivo anti-inflammatory pharmacology in Balb/c mice
Plasma cytokines
6. A combination of our ApoA1 upregulators with statins has been envisaged.
Since Cytochrome P450 (CYP) isoenzymes are involved in the oxidative
metabolism of the statins, with CYP3A4 and CYP2C9 isoenzymes being the
most dominant, we wanted to discharge any risk of drug-drug interactions
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PAI-1 (%)
IL-6 (%)
MCP-1 (%)
TNFa (%)
Prednisolone
SB-731445
I-BET151
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ꢀ35
ꢀ59
ꢀ92
ꢀ75
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biophysical studies that ultimately allowed us to demonstrate that
ApoA1 upregulators from a benzodiazepine series were selectively
antagonizing protein-protein interaction between bromodomains
of BET proteins family and acetylated histones by binding at the
acetylated lysine recognition pocket.15 In the accompanying
paper,16 we demonstrate that I-BET151 and other isoxazolquino-
lines are also capable of inhibiting the interaction between BET
proteins and acetylated histones by the same mechanism as
benzodiazepines.
In summary,
a series of 7-isoxazoloquinoline derivatives
was optimized on an assay of ApoA1 upregulation in HepG2 cells.
This led to the identification of potent compounds displaying low
CYP inhibition and PK properties compatible with oral exposure.
These compounds also displayed anti-inflammatory activities in
various cell assays. In particular, I-BET151 showed a broad anti-
inflammatory profile in a LPS-challenged Balb/C mouse model.
Moreover, this compound was found to be a BET inhibitor whose
complete profile will be disclosed in the following Letter.16