Synthesis of small molecule inhibitors of the orphan nuclear receptor steroidogenic factor-1 (NR5A1) based on isoquinolinone scaffolds

Article abstract of DOI:10.1016/j.bmcl.2008.03.027

Three synthetic routes were developed for structure-activity relationship (SAR) studies of HTS-derived isoquinolinone inhibitor probes for the orphan nuclear receptor steroidogenic factor-1 (NR5A1). Among the new analogs reported herein, 31 and 32 have im

Full text of DOI:10.1016/j.bmcl.2008.03.027

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2628–2632
Synthesis of small molecule inhibitors of the orphan
nuclear receptor steroidogenic factor-1 (NR5A1) based
on isoquinolinone scaffolds
Joshua Roth,a Franck Madoux,b Peter Hodderb,c and William R. Rousha,*
aDepartment of Chemistry, Scripps Florida, 5353 Parkside Drive, RE-2, Jupiter, FL 33458, USA
bLead Identification, The Scripps Research Institute Molecular Screening Center, Scripps Florida, 5353 Parkside Drive,
RF-1, Jupiter, FL 33458, USA
cDepartment of Molecular Therapeutics, Scripps Florida, 5353 Parkside Drive, RF-1, Jupiter, FL 33458, USA
Received 10 February 2008; revised 7 March 2008; accepted 7 March 2008
Available online 14 March 2008
Abstract—Three synthetic routes were developed for structure–activity relationship (SAR) studies of HTS-derived isoquinolinone
inhibitor probes for the orphan nuclear receptor steroidogenic factor-1 (NR5A1). Among the new analogs reported herein, 31
and 32 have improved potency, lower cellular toxicity, and improved selectivity compared to the initial HTS-derived leads 1 and 2.
Ó 2008 Elsevier Ltd. All rights reserved.
Nuclear receptors are transcription factors that regulate
gene expression through the binding of endogenous li-
gands.1 Many nuclear receptors have been extensively
studied, and several have proven to be important targets
for treatment of a range of human diseases.2 However,
natural or unnatural ligands are not known for a sub-
set of nuclear receptors, the so-called ‘orphan’ nuclear
receptors, which have limited efforts to determine the
pharmacology and therapeutic potential of these recep-
tors.1 As part of a broader program to develop an
understanding of the pharmacology of relatively unex-
plored orphan receptors, the Scripps Research Insti-
tute’s Molecular Library Screening Center has
performed high-throughput screens of several orphan
receptors, among them the steroidogenic factor-1 (SF-
1, also known as NR5A1).3
ment of adrenals and gonads.6 Due to the potential
role SF-1 plays in the regulation of steroid hormone syn-
thesis including adrenal androgen and gonadal testoster-
one synthesis, selective control of this receptor could
result in therapeutic treatment of metastatic prostate
cancer.7 Additionally, the involvement of SF-1 in energy
metabolism suggests relevancy in controlling obesity.8
Thus, the development of selective small-molecule bio-
logical probes of SF-1 is an important objective. Phos-
pholipids have been found in the ligand-binding
domain of human SF-19 and recently the first small mol-
ecules with the ability to modulate the activity of this
transcription factor were described.10
Approximately 65,000 compounds were screened for
SF-1 inhibition by the Molecular Library Screening
Center Network (MLSCN) at The Scripps Research
Institute.3,11 All initial hits were counterscreened against
the retinoic acid receptor-related orphan receptor a
(RORa), a phylogenetically distant nuclear receptor,2
in order to eliminate promiscuous as well as non-selec-
tive compounds. This led to the identification of two
mid-nanomolar SF-1 selective inhibitors 1 (PubChem
SID 7970631) and 2 (SID 7969543; Fig. 1).3,11 Accord-
ingly, isoquinolinones 1 and 2 were selected as starting
points for the development of SF-1 small molecule
probes. We have developed and report herein three
Steroidogenic factor-1 (SF-1) has been implicated in sex
determination during development and in formation of
steroidogenic tissues.4 SF-1 is involved in endocrine
function throughout life with expression in the pituitary,
testes, ovaries, and adrenal gland.5 Knockout mice exhi-
bit male to female sex reversal and impaired develop-
Keywords: Steroidogenic factor-1 (NR5A1); Orphan nuclear receptor;
Isoquinolinone scaffold; SF-1 inhibitor; MLSCN.
*
Corresponding author. Tel.: +1 561 799 8880; fax: +1 561 799
8955; e-mail: roush@scripps.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.027

J. Roth et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2628–2632
2629
Me
Me
with agents less reactive than a-halopropionate was
poor, with N-alkylation of 5 competing with phenol
O-alkylation with alkyl halides. This led us to develop
the more convergent second-generation synthesis sum-
marized for 14 in Scheme 2.
EtO
EtO
O
O
O
O
O
O
O
O
N
N
N
H
N
H
O
O
O
1
2
O
Aldehyde 10, available from the Claisen rearrangement
of m-salicaldehyde allyl ether,13 was alkylated in high
Figure 1. SF-1 inhibitors identified via ultra high-throughput screening
of the MLSCN library.
OH
routes for the synthesis of 1 and 2 that enable different
aspects of the SAR of this SF-1 inhibitor series to be
examined. Among the analogs reported here, 31 and
32 have improved SF-1 inhibitor potency, lower cellular
toxicity, and possess improved selectivity compared to
the initial leads 1 and 2.
O
O
O
O
c
a, b
H2N
+
N
H
CHO
CO2H
11
10
12
O
O
O
O
O
O
O
d
H
N
N
Our initial strategy for synthesis of analogs of 1 and 2
focused on sequential alkylations of an isoquinolinone
core (Scheme 1). Treatment of commercially available
5-hydroxyquinoline with peroxyacetic acid provided
N-oxide 4. Heating of the N-oxide 4 in acetic anhydride
gave a diacyl derivative that was not isolated but con-
verted directly to 5-hydroxyisoquinolinone 5.12 Selective
alkylation of 5 at the phenolic position by using ethyl
( )-2-bromopropionate (NaH, DMF, 25 °C, 80%) gave
propionic ester 6. Subsequent N-alkylation of 6 with
tert-butyl bromoacetate (Cs2CO3, DMF, 25 °C, 80%)
provided diester 7. Acidic deprotection of the tert-butyl
ester and subsequent coupling (EDC, DMAP, CH2Cl2)
of the derived carboxylic acid with either benzyl amine
8 or aniline 9 provided authentic samples of 1 and 2,
which were used to confirm the results of the MLSCN
SF-1 inhibitor screen.
N
H
N
H
O
O
O
13
14
Scheme 2. Reagents and conditions: (a) iodobutane, acetone, K2CO3,
60 °C, 4 h (60–85%); (b) 1.5 equiv H2CrO4, acetone, 1 h (98%); (c) 9,
EDC, DMAP, CH2Cl2 (83%); (d) i—O3, CH2Cl2, ꢀ78 °C; ii—PPh3,
iii—cat. I2, CH2Cl2 (45%).
Me
OH
EtO2C
O
b
a
CHO
CO2H
10
15
Me
Me
EtO
EtO
O
O
O
c
O
OH
O
O
H2N
+
or d
OR
O
O
N
While the sequence summarized in Scheme 1 permitted
us to synthesize a number of analogs, this route proved
less than ideal especially for synthesis of isoquinolinone
ether analogs owing to its linear nature. Especially prob-
lematic is that the chemoselectivity of alkylations of 5
OR
O
17a, R = t-Bu
17b, R = H
16
7, R = t-Bu
18, R = H
Scheme 3. Reagents and conditions: (a) ethyl ( )-2-bromopropionate,
K2CO3, acetone, 60 °C, 4 h; (b) i—O3, CH2Cl2, ꢀ78 °C; ii—Me2S, 1 h,
23 °C; (c) tert-butyl glycine hydrochloride (17a), 3 equiv Et3N, AcOH
to achieve pH 3–5, benzene, 100 °C, sealed tube, 12 h (67%); (d) glycine
(17b, 3 equiv), benzene, 100 °C, 12 h, sealed tube (94%).
OH
OH
OH
a
b,c
d
N
NH
N
O
O
4
3
5
O
O
Me
Me
Me
Me
Me
OEt
OEt
EtO
EtO
e
EtO
O
O
O
O
O
O
a
b,c
OH
O
O
O
f, g
O
O
NH
N
N
R
O
O
OtBu
N
H
20
19
O
O
O
O
O
Me
6
7
1, 2
Me
OEt
OEt
O
O
O
O
H2N
O
O
O
O
d, e
H2N
N
N
N
OtBu
H
O
O
O
21
8
1
9
2
22
Scheme 1. Reagents and conditions: (a) peroxyacetic acid, CH2Cl2
(99%); (b) acetic anhydride, 140 °C, 4 h, then concentrate; (c) NaOMe,
MeOH (60%, 2 steps); (d) NaH, DMF, 30 min, then ethyl ( )-2-
bromopropionate (80%); (e) Cs2CO3, DMF, tert-butyl bromoacetate
(80%); (f) 2 M HCl in Et2O and EtOH; (g) EDC, DMAP, CH2Cl2, 8 or
9 (45%, 2 steps).
Scheme 4. Reagents and conditions: (a) i—TBSOTf, Et3N, THF; ii—
OsO4 (2%), NMO, t-BuOH, acetone; (b) NaIO4, acetone, H2O, THF,
t-BuOH (78% from 19); (c) glycine tert-butyl ester hydrochloride,
benzene, Et3N and AcOH until pH 3–5, 100 °C sealed tube 12 h (21%);
(d) 2 M HCl in Et2O and EtOH (99%); (e) 9, EDC, DMAP, CH2Cl2
(55%).

2630
J. Roth et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2628–2632
yield with a variety of electrophiles (use of iodobutane is
illustrated in Scheme 2). The resulting product was then
oxidized using chromic acid to give acid 11.14 Standard
peptide coupling of 11 with glycinamide 12 (EDC,
DMAP, CH2Cl2, 83%) provided diamide 13, which
was then converted to SF-1 inhibitor analog 14 via
Table 1. SF-1 inhibitor activity of analogs of 1 and 2 (all IC50 data are in lM)
Me
EtO2C
O
O
N
R
N
H
O
Compound
R
IC50 dataa (lM)
SF-1
RORa
VP16
Cell toxb
1
2
See Figure 1
See Figure 1
–(CH2)3CH3
0 26 0.06
0.76 0.10
9.71 5.63
1.77 0.16
1.06 0.03
0.68 0.12
4.71 0.86
0.36 0.03
0.53 0.20
0.34 0.02
0.20 0.04
0.20 0.11
0.19 0.01
0.11 0.04
0.36 0.05
>33
>33
>33
>33
>33
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
40.2 3.7
Inactive
Inactive
37.0 0.7
>33
23
24
25
26
27
28
29
30
31
32
33
34
35
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
>33
Cyclohexyl
Benzyl
3.22 0.34
1.68 0.43
>33
p-Fluorobenzyl
–CH2-2-furanyl
phenylethyl
>33
p-Nitrophenyl
p-Methylphenyl
p-Methoxyphenyl
p-(F3C)Ophenyl
p-Ethoxyphenyl
p-Ethylphenyl
p-n-Butylphenyl
Inactive
36.5 6.0
Inactive
Inactive
22.6 2.0
25.0 3.9
30.7 10.7
Inactive
Inactive
24.2 5.6
>33
Inactive
Inactive
OR'
O
O
N
N
O
H
O
Compound
R0
SF-1
RORa
VP16
Cell toxb
14
36
37
38
–CH2(CH2)2CH3
–CH2CH3CH2CH3
–CH2CH3(CH2)3CH3
–C(CH3)2CO2Et
13.7 1.3
7.86 0.22
0.86 0.09
0.59 0.12
21.5 3.5
16.5 2.2
23.1 6.1
Inactive
24.1 1.9
17.5 1.9
20.7 8.0
>33
>33
Inactive
Inactive
Inactive
Me
O
EtO2C
O
O
N
R"
O
Compound
R00
SF-1
RORa
VP16
Cell toxb
40
41
42
43
–CH2CH2CONH–
–CH(Me)CONH–
–CH(CH2Ph)CONH–
–CH2CON(Me)–
8.46 1.67
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
3.57 0.89
EtO2C
O
O
O
O
Me
N
N
H
O
Compound
SF-1
RORa
VP16
Cell toxb
Inactive
22
Inactive
Inactive
Inactive
a IC50 value >33 indicates that curve did not fit, and that IC50 was manually assigned to be >33 lM. ‘Inactive’ means <50% inhibition at 100 lM.
b Cellular toxicity determined by the CellTiterGlo assay (Ref. 18).

J. Roth et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2628–2632
2631
ozonolysis of the vinyl group and dehydration of the
100
80
60
40
20
0
hemiaminal that is formed in situ. In some cases, the tar-
geted isoquinolinone was obtained directly from stan-
dard workup of the ozonolysis reaction. However, in
other cases the dehydration of the hemiaminal did not
occur spontaneously. Therefore, as a standard proce-
dure, a catalytic amount of I2 iodine was added to a
solution of the crude hemiaminal in CH2Cl2 to promote
dehydration and aromatization.
SF-1
RORα
VP16
Cell tox
In order to probe the SAR of the glycine spacer con-
nected to the isoquinolinone nitrogen, we demonstrated
that condensation of hydroxylactone 16 (accessible from
ozonolysis of acid 15) and glycine tert-butyl ester 17a
provides ester 7 (Scheme 3). Deprotection of 7 provides
carboxylic acid 18, which is an intermediate in the se-
quence summarized in Scheme 1. Alternatively, amino
acids such as glycine (17b), alanine and phenylalanine
can be coupled directly with 16 to give 18, thereby obvi-
ating the need for use of the tert-butyl ester protecting
group. Subsequent coupling of carboxylic acid 18 with
a range of amines and anilines, using the conditions
summarized in Scheme 1, greatly facilitated SAR studies
of this amide substituent.
-20
10-9 10-8 10-7 10-6 10-5 10-4 10-3
[Compound 32] (M)
Figure 2. Titration curves for 32 versus SF-1 (h), RORa (D), VP16
($), and the cytotoxicity assays (Æ). Data represent means of three
independent experiments with the calculated standard deviation.
ylphenyl (30), p-methoxyphenyl (31), p-trifluorometh-
oxyphenyl (32), and p-ethoxyethyl (33) amides. These
analogs displayed SF-1 inhibitor activity in the
100–200 nM range, a 3- to 6-fold improvement over 2.
All substitutions of the R0 unit spanning the isoquinoli-
none and amide units led to substantial or complete loss
of SF-1 activity (analogs 40–43), as did moving the alkoxy
substituent from the meta position in 1–2 to the para posi-
tion in 22. However, clear evidence of a productive SAR
for the isoquinolinone alkoxy substituent has been dem-
onstrated by analogs 14 and 36–39. The stereochemistry
of the ether substituent in 1 and 2 does not appear to be
critical as 38, with a –C(Me2)CO2Et ether at this position,
is more potent than 2. In addition, an analog of 2 with a –
CH2CO2Et aryl ether (39, not shown), was deemed to be
‘inactive’ in the original HTS campaign.3 These data sug-
gest that hydrophobicity at this position is important for
SF-1 inhibitor activity.
Finally, in order to probe the substitution pattern of the
isoquinolinone nucleus of 1 and 2, a third synthetic
strategy was developed starting from commercially
available hydroxyindanones (Scheme 4); the synthesis
of 22 starting from 19 is illustrative. Thus, after alkyl-
ation of 5-hydroxyindanone with ethyl ( )-2-bromopro-
pionate (K2CO3, acetone, 60 °C, 4 h), indanone 19 was
treated with TBSOTf and Et3N15 to give the silyl enol
ether which, without isolation, was treated with N-meth-
ylmorpholine N-oxide (NMO) and catalytic osmium
tetroxide.16 The resulting a-hydroxyindanone 20 was
cleaved using NaIO4 to give the hydroxylactone, which
was condensed with glycine tert-butyl ester under condi-
tions summarized in Scheme 3. Subsequent coupling of
the carboxylic acid derived from deprotection of 21 with
aniline 9 provided the regioisomeric isoquinolinone ana-
log 22.
All analogs were further assessed in another transactiva-
tion assay (Gal4-VP16)17 to confirm that any SF-1 activ-
ity measured was specific to that receptor and not due to
transactivation assay artifacts (e.g., promiscuous inhibi-
tion of the reporter system); cellular cytotoxicity was also
determined (CellTiterGlo assay18; see Table 1).3 The titra-
tion curves for compound 32 are shown in Figure 2. This
compound showed significant activity only in the SF-1 as-
say; no activity in the RORa, Gal4-VP16 and cytotoxicity
assays were detected at concentrations up to 99 lM.
SF-1 inhibition activity of analogs of 1 and 2 is summa-
rized in Table 1, along with comparative inhibition data
versus RORa. All assays were performed as described in
Ref. 3. Briefly, luciferase-format functional assays were
used to measure inhibition of constitutively active SF-
1(LBD)-Gal4 or RORa(LBD)-Gal4 chimeric con-
structs. Compounds 1, 2, 22–35 and 40–43 were studied
as racemates, or as mixtures of diastereomers (41, 42).
In summary, we have developed three routes for the syn-
thesis of substituted isoquinolinones that has enabled us
to explore the SAR of isoquinolinone inhibitors of the
orphan nuclear receptor steroidogenic factor-1 (SF-1).
Of those analogs described herein, 31 and 32 have im-
proved potency and selectivity versus other nuclear
receptor targets compared to the HTS leads 1 and 2.
Initial efforts focused on replacing the oxygenated ben-
zylic amide or anilide units of 1 and 2, but introduction
of alkyl amides as in 23 and 24 led to significant loss of
SF-1 activity. Replacement of the highly oxygenated
benzylic amide unit of 1 with potentially more metabol-
ically stable benzyl (25) or p-fluorobenzyl (26) amides
led to 5- to 50-fold loss of SF-1 activity. However, intro-
duction of a 2-phenylethyl amide (28) yielded a 300 nM
SF-1 inhibitor. Still better results were obtained by
replacing the potentially metabolically sensitive anilide
unit of 2 with other aniline amides, such as the p-meth-
Acknowledgments
This research was supported by the NIH Molecular Li-
brary Screening Center Network (MLSCN) Grant No.

2632
J. Roth et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2628–2632
Zambetti, G. P.; Figueiredo, B. C.; Heckert, L. L.; Lalli,
E. Mol. Endocrinol. 2007, 21, 2968.
U54MH074404 (Dr. Hugh Rosen, Principal Investiga-
tor). Lina DeLuca and Pierre Baillargeon are thanked
for compound management. Dr. Louis Scampavia is
thanked for quality control of SF-1 inhibitor analogs by
LC–MS. The construct for the Gal4-VP16 assays was gen-
erously provided by Prof. Michael Conkright. Prof. Pat
Griffin is thanked for numerous helpful discussions.
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Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.bmcl.
2008.03.027.
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