Discovery of tetrahydro-cyclopenta[b]indole as selective LXRs modulator

Article abstract of DOI:10.1016/j.bmcl.2009.01.109

A series of tetrahydro-cyclopenta[b]indoles modulating the activity of the liver-X-receptor (LXR) were derived from a high throughput screening hit. The potency and selectivity for LXRβ versus LXRα was improved. One compound, administered to wild-type mic

Full text of DOI:10.1016/j.bmcl.2009.01.109

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1654–1657
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of tetrahydro-cyclopenta[b]indole as selective LXRs modulator
*
Hassen Ratni , Denise Blum-Kaelin, Henrietta Dehmlow, Peter Hartman, Philippe Jablonski,
Raffaello Masciadri, Cyrille Maugeais, Angelique Patiny-Adam, Narendra Panday, Matthew Wright
F. Hoffmann-La Roche Ltd, Pharmaceuticals Division, Preclinical Research, CH-4070 Basel, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of tetrahydro-cyclopenta[b]indoles modulating the activity of the liver-X-receptor (LXR) were
Received 15 January 2009
Revised 30 January 2009
Accepted 30 January 2009
Available online 6 February 2009
derived from a high throughput screening hit. The potency and selectivity for LXRb versus LXR
a was
improved. One compound, administered to wild-type mice modestly increased plasma HDL-cholesterol
with no change in plasma triglycerides (TG) and reduced effects on liver TG content compared to
T0901317. This novel series of LXR agonists shows promise to improve therapeutic efficacy with reduced
potential to increase TG.
Keywords:
LXR
Ó 2009 Elsevier Ltd. All rights reserved.
Selective modulators
HDL-cholesterol
TG
The liver-X-receptors, LXR
a
and LXRb, are nuclear hormone
To identify completely novel starting points, we performed a
high throughput screening (HTS) campaign of the Roche compound
receptors that bind oxysterols and inactivate expression of genes
regulating cholesterol and lipid metabolism.1 The LXRs directly in-
duce the expression of the transmembrane lipid/cholesterol trans-
porters ABCA1 and ABCG1 in cholesterol-loaded macrophages and
liver and intestinal cells, promoting cholesterol efflux and forma-
tion of high density lipoprotein particles (HDL).2 Numerous clinical
and epidemiological studies have shown that HDL-cholesterol lev-
els are inversely related to the risk for coronary artery disease
(CAD).3 Drugs that activate LXR have the potential to increase
HDL-C and cellular cholesterol efflux and are thus expected to be
atheroprotective.4 Nonselective LXR agonists, however lead to the
undesired activation of triglyceride (TG) synthesis in the liver by
upregulation of Srebp-1C.5 The identification of LXR modulators
devoid of this undesired side effect remains a major challenge for
drug development. Current efforts focus on the identification of
LXRb-selective agonists,6 partial or gene-specific LXR activators,7
or compounds with more favorable PK/PD properties.8
OH
OH
F3C
CF3
F3C
CF3
T0901317
Anilino-hexafluoro-
isopropanols
Cl
R3
N
N
O
CF3
S
R4 R2
O
Figure 1. T0901013 and aniline–hexafluoroisopropanols series.
R3
N
R1
R3
N
R1
3
R2
N
R2
2
O
One of the most studied LXR agonists is T0901317, a potent but
S
nonselective LXRa
/b co-agonist (Fig. 1).9 T0901317 has been
3
O
O
4
2
Cl
6
shown to increase HDL-c and to reduce atherosclerosis in a mouse
model10 but also to significantly increase liver triglyceride synthe-
sis.5 We previously reported the preparation of analogs of
T0901317. A new series of aniline–hexafluoro-isopropanols was
derived that retained significant HDL-raising activity with reduced
potential to increase liver TG levels compared to T0901317
(Fig. 1).11
9
OEt
R3
N
H
R1
R3
1
N
R2
R3
R1
4
R1
N
R2
N
R2
6
5
* Corresponding author. Tel.: +41 616882748.
E-mail address: hasane.ratni@roche.com (H. Ratni).
Figure 2. HTS hit 1, and initial structural modification.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.109

H. Ratni et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1654–1657
1655
library on the LXRb receptor. Among several hits was a promising
drug-like 2,3,4,9-tetrahydro-1H-carbazole derivative 1, with mod-
est in vitro potency; LXR : IC50/EC50: 2.60/3.10 M; LXRb IC50/
EC50: 0.51/3.60 M (Fig. 2). We first evaluated the substitution
pattern of the aromatic moiety of the tetrahydro-carbazole which
revealed that small and lipophilic substituents such as a chloro
atom or a methyl moiety in position 6 were preferred for binding
affinity. The alkylation or acylation of the nitrogen of 1 was not
tolerated.
O
S
a
l
O
( ) 8: R= Me; n=1
( ) 9: R= H; n=2
( ) 10: R= Et; n=2
( ) 11: R= F; n=1
OMe
l
R
Cl
H
O
( )n
N
H
Figure 4. Influence of the quaternary center substitution.
In order to increase the chemical tractability of the hit, as well
as to reduce the number of stereogenic centers, we prepared addi-
tional analogs including carbazoles 2, carbolines 3, indoles 4 and 5
and anilines 6 (Fig. 2). All these modifications led to a dramatic loss
of binding affinity.
Table 1
Binding affinity (IC50) and transactivation potency (EC50) for aza and substituted aryl
derivatives ( )-12–25
We therefore decided to conserve the tricyclic fused
arrangement, and turned toward investigation of the impact
of the size of the saturated ring on receptor binding affinity,
O
O
Ar
O
n
S
S
O
OMe
OMe
preparing corresponding
(Fig. 3).
5 and 7 member ring derivatives
( )
R
F
Cl
Cl
N
H
H
O
O
The 5,6,7,8,9,10-hexahydro-cyclohepta[b]indole 7 was inactive,
but the 1,2,3,4-tetrahydro-cyclopenta[b]indole ( )-8 showed im-
proved affinity to the LXRs. We prepared and tested both diastere-
oisomers and determined that only the (RS, SR) retained activity. In
addition, the compound ( )-8 showed about 3–4-fold selectivity
N
N
H
H
( ) 12-24
( ) 25
Compds
n
R
Ar
LXRa
LXRb
for LXRb versus LXR
scriptional transactivation.
a in both receptor binding affinity and tran-
IC50
EC50
IC50
EC50
( )-12
( )-13
( )-14
( )-15
( )-16
( )-17
( )-18
( )-19
( )-20
( )-21
( )-22
( )-23
( )-24
( )-25
2
2
2
2
2
2
2
1
1
1
1
1
1
1
Me
Me
Me
Me
Me
F
F
F
F
F
p-F-Ph
3.3
5.81
5.8
3.3
0.37
>40
Encouraged by these results, we then examined the influ-
ence of the substitution pattern at the quaternary center
(Fig. 4).
The replacement of the methyl moiety by hydrogen ( )-9 com-
pared to 1, resulted in loss of affinity, presumably due to an epi-
merization of the stereogenic center. Increasing the size of the
substituent from a methyl to an ethyl ( )-10, was not tolerated,
most likely due to steric hindrance. We prepared the fluoro analog
( )-11, in order to avoid epimerization and to minimize steric hin-
drance compared to ( )-8. This was indeed the optimal substituent
3,4-diCl-Ph
m-Cl-Ph
o-Cl-Ph
o-OMe-Ph
m-Cl-Ph
m-F-Ph
m-OMe-Ph
m-Cl-Ph
m-F-Ph
m-Br-Ph
3-Pyridyl
2-Pyridyl
Ph
87.9
0.82
0.54
2.25
0.10
0.073
0.062
1.21
0.049
0.10
20.0
12.2
2.58
31.2
0.15
0.80
3.42
0.031
0.021
0.056
0.70
0.026
0.030
0.50
3.95
0.40
2.18
2.08
2.30
2.17
1.39
1.07
0.84
1.35
0.85
1.07
2.75
4.23
1.70
1.53
3.16
1.81
0.43
0.16
0.19
0.36
0.15
0.34
0.27
0.37
F
F
F
F
as indicated by a further improvement in potency (LXR
a: IC50/
EC50: 0.09/0.48 M; LXRb IC50/EC50: 0.050/0.043 M) with a gain
l
l
IC50 and EC50 are expressed in
lM and are the average of at least two experiments.
in selectivity for LXRb to ten-fold in transcriptional transactivation.
We next turned our focus on the substitution pattern of the aryl
sulfonyl moiety and the preparation of aza-analog of ( )-11 (Table
1).
The introduction of a para substituent on the phenyl sulfonyl
moiety led to a drastic loss of affinity (e.g., ( )-12). Ortho substitu-
tion decreased the affinity (( )-15–16). While meta substitution
(( )-17–22) was tolerated there was no improvement in either
the affinity or physicochemical properties of the molecules. Pyridyl
derivatives (( )-23–24) showed a marked decrease in binding
affinity. For further modifications we therefore kept the unsubsti-
tuted phenyl as the preferred aryl moiety. The aza-derivative
( )25 (analog of ( )-11) was much less potent.
Table 2
Stability of esters ( )-8 and ( )-11 towards esterase in plasma
Compds
Rat plasma
Mice plasma
Hydrolysis after 2 h (%)
Hydrolysis after 2 h (%)
( )-8
( )-11
0
24
0
4
Only the corresponding carboxylic acids were formed as new products.
Unlike the compound ( )-8 (neopentylic ester) which was com-
pletely stable toward hydrolysis in rat plasma (Table 2), the analo-
gous fluoro compound ( )-11 showed a significant hydrolysis after
only 2 h. The fluoro atom in the
a position to the ester moiety
apparently contributed to its instability.
O
To retain stability, a bioisosteric replacement of the ester moi-
ety appeared mandatory. We thus turned our attention to standard
replacements, such as amides, [1,3,4]-oxadiazoles and [1,2,4]-oxa-
diazoles (Table 3) known to be stable.12
In this series, we found that a diverse group of alternative
hydrogen bond acceptor moieties was tolerated. Tertiary amides
were more potent than secondary amides (( )-26b versus ( )-
26a) and the oxadiazoles analogs (types 27 and 28) could be fur-
ther substituted with either lipophilic or polar residues (e.g., ( )-
27c and ( )-28c). The racemic derivatives were resolved using
O
S
O
OMe
S
O
Cl
Cl
H
O
OMe
O
N
N
H
H
7
( ) 8
Inactive
LXRα (IC50/EC50): 1.10 / 1.60 μM
LXRβ (IC50/EC50): 0.35 / 0.25 μM
Figure 3. Saturated ring size modification.

1656
H. Ratni et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1654–1657
Table 3
The metabolic stability of (+)-27b was in a medium range
(in vitro microsomal clearance: human/mouse: 10/59 mL/min/
kg). This compound had no affinity for the hERG channel or for
the cytochrome 3A4.
Binding affinity (IC50) and transactivation potency (EC50) for the ester bioisosters of
the types 26–28
The synthetic approach used for the preparation of ( )-11,
(Scheme 1) is representative for the preparation of all deriva-
tives.13 A Michael addition of benzenesulfonyl-acetic acid methyl
ester 29 to cyclopentenone yielded 30 as a racemic mixture of dia-
stereoisomers. Regioselective fluorination produced the two dia-
stereoisomers (RR, SS)-31 and (RS, SR)-31 that could then be
readily separated by column chromatography. A modified Fisher-
Indole reaction between the 4-chlorophenyl hydrazine and the
cyclopentanone (RS, SR)-31 yielded ( )-11 by a regioselective
reaction.
The ester ( )-11 could be converted into the amide ( )-26a
upon treatment with methylamine in ethanol. The amide ( )-26b
was obtained from ( )-26a by alkylation with methyl iodide after
initial BOC protection of the NH moiety from the indole and then
deprotection.
O
R1
S
O
N
N
N
R3
R
N
Cl
F
R =
H
O
O
N
O
R2
N
H
26a,b
27a-d
28a-d
Compds
R1
R2
R3
LXR
a
LXRb
IC50 EC50
IC50
EC50
( )-26a
( )-26b
( )-27a
( )-27b
(+)-27b
(-)-27b
( )-27c
( )-27d
( )-28a
( )-28b
( )-28c
( )-28d
H
—
—
H
Me
Me
Me
—
—
—
—
—
—
—
—
Me
2.70
1.79
2.30
0.92
0.59
2.20
0.29
0.30
0.21
1.63
1.55
1.09
0.77
1.50
1.35
1.85
0.43
0.89
1.09
1.26
0.37
0.16
3.87
0.17
0.49
0.065
0.069
0.31
0.083
0.15
0.027
—
0.40
0.11
0.17
0.24
0.20
0.46
0.092
0.076
0.21
—
Me
—
—
—
—
—
—
—
—
—
—
Cyclo-propyl
N(Me)2
Oxadiazole derivatives of the types 27 were prepared using
the route described Scheme 1. First the ester moiety from ben-
zenesulfonyl-acetic acid methyl ester 29 was converted into
—
—
—
—
CO2Me
N(Me)2
CN
[1,3,4] substituted oxadiazoles via
a hydrazide intermediate.
0.029
0.10
0.013
—
0.12
—
Then a sequential Michael addition, fluorination, separation of
the diastereoisomers and Fisher-Indole reaction afforded the cor-
IC50 and EC50 are expressed in
l
M and are the average of at least two experiments.
responding
derivatives.
substituted
tetrahydro-cyclopenta[b]indole
The pharmacological effects of (+)-27b on HDL-cholesterol
and triglyceride levels were evaluated on C57BL/6J mice (Table
4). The compound was administered once a day for five days by
intraperitoneal injection. The plasma and liver were collected
2 h after the last dose for analysis. Ip administration of
T0901317 produced a non-significant increase in plasma HDL
and TG of about 20% but a 3.7-fold increase in liver TG content.
In contrast, (+)-27b increased HDL-c at all doses reaching a
maximum of +36% at the 100 mg dose, with no change in plas-
ma TG, and variable, non-dose dependant effects on liver TG
content that were reduced compared to T0901317. Plasma and
liver exposure of (+)-27b also increase in a dose-dependant
manner.
O
O
O
S
S
O
O
OMe
a
b
O
O
S O
OMe
F
H
O
O
O
O
(RR/SS)-31
29
30
O
S
O
S
OMe
O
c
O
The ability of (+)-27b to increase HDL with a much reduced ef-
fect on liver TG content may be explained by either its slight selec-
+
OMe
Cl
F
F
H
O
H
O
tivity toward LXR-b versus LXR
a an alternative recruitment of
N
transcriptional cofactors to the LXRs with consequential differen-
tial regulation of LXR target genes, or perhaps by differences in
PK profile and tissue exposure. Further studies will be required
to dissect the impact of these potentially contributing
mechanisms.
In summary, we have developed a novel class of LXR agonists
starting from a singleton hit originating from a high throughput
screening campaign. We derived molecules that could be evaluated
in animal models and the in vivo data suggest an alternative profile
compared to non-selective LXR agonist such as T0901317. Further
optimization of the potency and physicochemical properties are re-
quired in this series.
O
H
(RS/SR)-31
( )-11
Scheme 1. Reagents and conditions: (a) cyclopentenone 1.1 equiv, NaOMe
0.2 equiv, MeOH, rt, 85%; (b) NaH 1.1 equiv, (PhSO2)2N–F 1.5 equiv, DMF, 0 °C,
72%; (c) (4-chloro-phenyl)-hydrazineÁHCl, AcOH, 60 °C, 52%.
standard chiral HPLC methods (Chiralpak AD, Heptane/EtOH
80:20) and the (+) enantiomers was always found to be the more
active one (e.g., (+)-27b). The absolute configuration was estab-
lished by X-ray analysis and is depicted in Table 3.
Table 4
Effects on plasma HDL-C and triglyceride levels and liver triglyceride content in male C57B16 J mice after 5 days treatment
Compounds
Dose (mg/kg/day)
HDL-C (%)
Plasma TG (%)
Liver TG (%)
Plasma exposure (ng/ml)
Liver exposure (ng/g)
T0901317
(+)-27b
(+)-27b
(+)-27b
10
10
40
+23
+27
+27
+36*
+20
+9
+9
À2
+270*
+100*
À19
255
180
682
7900
1133
5488
100
+45*
2146
13,173
*
p < 0.05 versus vehicle treated group, n = 6 animals/group.

H. Ratni et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1654–1657
1657
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12. For examples of replacement of an ester by hydrolytically stable bioisoters:
Saunders, J.; Cassidy, M.; Freedman, S. B.; Harley, E. A.; Iversen, L. L.; Kneen, C.;
MacLeod, A. M.; Merchant, K. J.; Snow, R. J.; Baker, R. J. Med. Chem. 1990, 33, 1129.
13. Detailed synthetic procedures for the preparation of all derivatives can be
found in: Dehmlow, H.; Kuhn, B.; Masciadri, R.; Panday, N.; Ratni, H.; Wright,
M. B. U.S. Patent 0215577, 2005.
The authors thanks B. Wolf for performing binding and transac-
tivation assays, H. Isel for the formulation, S. Masur for LC–MS
measurements and A. Guenzi for plasma stability measurement.
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