Small molecule tertiary amines as agonists of the nuclear hormone receptor Rev-erbα

Article abstract of DOI:10.1016/j.bmcl.2012.04.126

The structure-activity relationship study of a small molecule Rev-erbα agonist is reported. The potency and efficacy of the agonists in a cell-based assay were optimized as compared to the initial lead. Modest mouse pharmacokinetics coupled with an improved in vitro profile make 12e a suitable in vivo probe to interrogate the functions of Rev-erbα in animal models of disease.

Full text of DOI:10.1016/j.bmcl.2012.04.126

Bioorganic & Medicinal Chemistry Letters 22 (2012) 4413–4417
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Small molecule tertiary amines as agonists of the nuclear hormone
receptor Rev-erb
a
Youseung Shin, Romain Noel, Subhashis Banerjee, Douglas Kojetin, Xinyi Song, Yuanjun He, Li Lin,
⇑
Michael D. Cameron, Thomas P. Burris, Theodore M. Kamenecka
Department of Molecular Therapeutics and Translational Research Institute, The Scripps Research Institute, Scripps Florida, 130 Scripps Way #A2A, Jupiter, FL 33458, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure–activity relationship study of a small molecule Rev-erba agonist is reported. The potency
and efficacy of the agonists in a cell-based assay were optimized as compared to the initial lead. Modest
mouse pharmacokinetics coupled with an improved in vitro profile make 12e a suitable in vivo probe to
Received 17 February 2012
Revised 25 April 2012
Accepted 27 April 2012
Available online 8 May 2012
interrogate the functions of Rev-erb
a
in animal models of disease
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Nuclear hormone receptor
Agonists
Drug discovery
Medicinal chemistry
SAR
Rev-erb
a
was originally identified as an orphan nuclear hor-
Recently, the first nonporphyrin synthetic ligand for Rev-erba,
mone receptor based on its canonical domain structure.1 Rev-erbb
GSK4112/SR6452/1 (Fig. 1) was identified.15,16 This ligand acts as
an agonist, mimicking the action of heme and resets the circadian
rhythm in a phasic manner. It also represses expression of glucone-
ogenic genes in liver cells and reduces glucose output in primary
hepatocytes. 1 was identified in a fluorescence resonance energy
transfer (FRET) assay that significantly and specifically enhances
was identified based on its homology to other nuclear receptors
(NR) and has an overlapping pattern of expression with Rev-erba.
Rev-erbs have particularly high expression in the liver, adipose tis-
sue, skeletal muscle and brain2–4 and are expressed in a circadian
manner in these tissues.5–8 The Rev-erbs are unique within the
NR superfamily in that they lack the typical C-terminal AF2 domain
(helix 12), which is required for coactivator protein binding.
Although these receptors lack the ability to activate transcription
of target genes due to their inability to recruit transcriptional coac-
tivator proteins, both have been shown to be effective repressors of
transcription due to their ability to recruit transcriptional core-
pressor proteins such as NCoR and HDAC3.9,10 It has been recently
demonstrated that the porphyrin heme functions as a ligand for
the Rev-erb
recapitulated this data showing that SR6452 was able to modulate
the interaction of either Rev-erb or Rev-erbb with an NCoR CoRNR
box peptide using Luminex technology.17,18 SR6452 dose-depen-
dently increased the interaction of both Rev-erb and Rev-erbb with
a-NCoR interaction with an EC50 value of 0.40 lM. We
a
a
the NCoR peptide, indicating that the ligand modulates the activity
of both Rer-erb subtypes. Direct binding of an analog (12e) to
Rev-erb
The compound was reported to show no activity on related nu-
clear hormone receptors (LRH1, SF1, FXR, or ROR ) using the same
FRET assay and no activity on LXR or LXRb in reporter-gene as-
a
was also confirmed by circular dichroism analysis.18
Rev-erba
and Rev-erbb.9–12 Heme binds reversibly and specifically
to the ligand binding domain (LBD) of Rev-erb. Binding induces a
conformational change in the LBD that results in the ability of
the receptor to recruit NCoR and thus repress target gene tran-
a
a
says. Unfortunately, the pharmacokinetic profile of 1 in rodents
was poor hampering its use as an in vivo tool. Additionally, 1
had modest potency and limited efficacy in a cellular assay
(in-house data). With the goal of interrogating the function of
scription. The nuclear hormone receptors, Rev-erba and Rev-erbb,
regulate a number of physiological functions including the circa-
dian rhythm, glucose and lipid metabolism, adipogenesis, and cel-
lular differentiation.13,14 The observation that these NRs are ligand
regulated suggests that development of synthetic ligands may be
possible.
Rev-erba in animal models of disease, we needed a more potent
compound with improved potency and efficacy and an adequate
in vivo profile. Based on trisubstituted amine 2, we initiated the
structure–activity relationships (SAR) study described herein.
Based on the lead structure 1, the three portions of the molecule
were individuallymodified in a step-wisefashion investigating R, R1,
⇑
Corresponding author.
E-mail address: kameneck@scripps.edu (T.M. Kamenecka).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.04.126

4414
Y. Shin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4413–4417
Table 1
Nitrothiophene analogs
Cl
O
Compound
R
aMax Inh
0.82
EC50
2.3
(lM)
R1
R2
1
N
R
N
S
O2N
S
O
4
H
1.2
bNT
NT
O2N
5a
0.81
N
1
2
5b
5c
1.1
NT
NT
Figure 1. GSK4112/SR6452 lead.
N
0.82
S
and R2 as in 2. The analogs 4–11 were synthesized in straightforward
fashion starting from commercially available starting materials
(Scheme 1). In one instance, reductive amination of t-butyl glycine
(3) with p-chlorobenzaldehyde afforded secondary amine 4. Func-
tionalization of the third amine substituent was carried out by a sec-
ond reductive amination or sulfonylation or acylation as described
to give products 5a–e. Alternatively, reductive amination of t-butyl
glycine and 5-nitrothiophenecarboxaldehyde (6) uneventfully
afforded secondary amine 7. This could then be converted to final
products 8 in a similar fashion. Lastly, the 5-nitrothiophenecarbox-
aldehyde and p-chlorobenzylamine (9) could be condensed to yield
amine 10, which was converted to final products 11.
5d
5e
0.93
0.88
NT
NT
S
N
NH
a
Results are average of two or more experiments. Value = fold change relative to
DMSO control at 10 M compound.
l
b
NT = not tested. All standard deviations 625%.
es, however none of them showed any improvement with regards
to efficacy (Table 1). One might argue that the 4-pyridyl analog
(5a) and the benzothiazole analog (5c) were equally efficacious
as 1, however these early compounds were not fully titrated. Com-
pounds 4, 5b and 5d showed no repression. Temporarily unsuc-
cessful in replacing the nitrothiophene ring, we moved on to
investigate the other two portions of the molecule.
Efforts were then focused on replacing the p-chlorobenzyl
group (Table 2). The compounds shown are only a subset of those
actually made however they are representative of the group.
Substitutions on the benzyl group had modest effects on efficacy
(8a–d) as did the naphthyl analogs (8e–f), however this did not
translate into an improved EC50. Converting the amine to an amide
or sulfonamide showed improved efficacy, and 8i was the first
Compounds were screened in a cell-based luciferase assay in a
two-step format.18–20 Cells were co-transfected with an expression
plasmid harboring full-length Rev-erb
driven by the Bmal1 promoter. Compounds were first screened at
two concentrations (1 M and 10 M) to determine the effect on
a and a luciferase reporter
l
l
repression of Bmal1 transcription. Rev-erb is a transcriptional
repressor. Rev-erb agonists lead to recruitment of co-repressors,
which leads to repression of transcription. Maximum inhibition
at 10 l
M is reported.21 The lower the value, the more efficacious
the agonist is at repressing transcription. A value of 1.0 effectively
means no repression. Compounds that appeared efficacious at
compound synthesized with an EC50 <1 lM. This represented a
10 lM were then fully titrated in an eleven-point dose–response
format to generate EC50 values. In our in-house cell-based assay,
GSK4112/SR6452/1 showed only modest potency and minimal
efficacy (Table 1).
Given the potential for issues with the nitrothiophene residue
in vivo, we assessed replacement of this group first. Several small
heterocycles and carbocycles were tried as nitrothiophene isoster-
nice improvement over 1. Unfortunately, we were unable to assess
the in vivo characteristics of 8i as this analog could not be detected
in the mass spectrometer due to poor ionization under a number of
conditions.
Finally, we began to modify the third segment of 1 and looked
to modify the acetic ester side chain (Table 3). We found that the
X
NH
CO2tBu
N
R
b,c or d
b,c or d
a
H2N
O2N
CO2tBu
Cl
Cl
CO2tBu
4
7
5a-e
3
CHO
X
S
S
S
R1
e
N
NH
CO2tBu
O2N
O2N
CO2tBu
6
8a-i
O2N
O2N
R2
Cl
Cl
Cl
S
S
X
N
f
b,c or d
H
N
H2N
9
10
11a-r
X=bond,CO,SO2,CONH,CO2
Scheme 1. Reagents and conditions: (a) NaBH(OAc)3, HOAc, Cl(CH2)2Cl, 4-Cl-PhCHO; (b) RCHO, NaBH(OAc)3, HOAc, Cl(CH2)2Cl; (c) RCOCl, TEA; (d) RSO2Cl, TEA; (e)
NaBH(OAc)3, HOAc, Cl(CH2)2Cl, H2NCH2CO2tBu; (f) 5-nitrothiophenecarboxaldehyde, NaBH(OAc)3, HOAc, Cl(CH2)2Cl.

Y. Shin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4413–4417
4415
M)
Table 2
p-Cl-Benzyl analogs
Table 3
Ester analogs
Compound
R1
aMax Inh
0.82
EC50
2.3
(
lM)
Compound
R2
aMax Inh
EC50 (l
tBuO2C
MeO2C
HO2C
H2NOC
NC
1
0.82
0.80
0.85
0.75
0.79
2.3
3.9
bNT
3.0
2.5
1
Cl
11a
11b
11c
11d
8a
0.58
2.5
O2N
8b
8c
8d
1.1
bNT
2.9
2.0
11e
11f
0.70
0.45
8.8
5.5
0.68
0.77
MeO
F
F3C
S
11g
11h
0.50
1.0
4.0
NT
N
8e
0.60
2.5
MeO2S
N
O
8f
0.60
0.50
NT
3.0
11i
0.40
7.8
O
O
S
O
11j
0.30
0.33
7.4
1.8
8g
BOC
N
O
11k
O
8h
0.48
0.37
2.6
0.8
11l
0.40
0.50
2.6
2.5
O
S
O
O
O
S
11m
8i
a
Results are average of two or more experiments. Value = fold change relative to
DMSO control at 10 M compound.
NT = not tested. All standard deviations 625%.
a
Results are average of two or more experiments. Value = fold change relative to
M compound.
NT = not tested. All standard deviations 625%.
l
b
DMSO control at 10
l
b
Table 4
Piperidine and pyrrolidine analogs
t-butyl ester residue was not important for activity, as the corre-
sponding methyl ester (11a), primary amide (11c), and nitrile
(11d) were all equipotent. Attempts to replace the ester group with
aryl and heteroaryl residues (11e–g) were slightly misleading as
Compound
11k
R2
aMax Inh
0.33
EC50
1.8
(lM)
BOC
N
improved efficacy at 10 lM did not translate into an improved
11n
0.80
bNT
N
BOC
BOC
EC50. Saturated ring systems were accommodated (11i–k), how-
ever only one showed improved cellular EC50 (11k). Amides and
sulfonamides (11l–m) showed nice improvements in efficacy,
however these analogs also displayed only modest EC50’s. The most
potent and efficacious analog identified was carbamate 11k. In an
effort to further improve the activity of 11k, we investigated ring
size and linker length (Table 4).
The first aspect investigated was to see if the methyl pyrrolidine
side chain was optimal. We examined other five- and six-mem-
bered ring isomers (11n–r) with and without the methylene linker
between the nitrogen atom and the ring. These analogs were syn-
thesized simply via reductive amination with the corresponding
ketone or aldehydes. All products are racemic at this stage, how-
ever, they could be made in enantiomeric fashion if desired. This
might also lead to improvements in potency. The methyl pyrroli-
dine group in 11k was certainly better than either isomer of the
piperidines (11n–o), however the analogs lacking the methylene
spacer appeared to be equipotent (11p–r).
N
11o
11p
11q
0.90
0.31
0.14
NT
NT
NT
BOCN
BOCN
BOCN
11r
0.24
1.6
a
Results are average of two or more experiments. Value = fold change relative to
DMSO control at 10 M compound.
l
b
NT = not tested. All standard deviations 625%.
equally efficacious and considerably more potent than 11k. Urea
12i and sulfonamide 12m were the most potent analogs synthe-
sized. Amide 12c was equipotent to 11k. Removal of the BOC group
and substitution with alkyl groups (12a–b) led to a substantial
drop in efficacy. Clearly the additional hydrogen bond acceptors
are important for activity.
As a secondary screen of in vitro activity, select compounds
were tested in a Gal4-Rev-erb LBD cotransfection assay. 12e
dose-dependently increased the Rev-erb-dependent repressor
activity assessed in HEK293 cells expressing a chimaeric Gal4
We next considered modifications to the carbamate group in
11k (Table 5). Deprotection of the t-butoxycarbonyl group (BOC)
in 11k by exposure to acid was uneventful (Scheme 2). Installation
of the R3-substituent via standard chemistry afforded products 12.
Slightly smaller carbamates (12e,f) showed similar efficacy at
10
lM, but with nearly threefold improvement in EC50’s. The corre-
sponding ureas (12g–i) and sulfonamides (12j, l–m) were also

4416
Y. Shin et al. / Bioorg. Med. Chem. Lett. 22 (2012) 4413–4417
Table 5
Carbamates, amides, ureas, and sulfonamides
Compound
R3
aMax Inh
EC50 (lM)
CO2tBu
CH3
11k
12a
12b
12c
12d
12e
12f
0.33
0.95
1.00
0.35
0.55
0.35
0.35
1.8
bNT
NT
CH2Ph
COCH3
CO2Ph
CO2Et
CO2Allyl
1.6
NT
0.70
0.67
12g
12h
0.33
0.35
0.62
0.67
CONHPh
CONHnBu
CONHp-NO2Ph
Figure 2. Cell-based comparison between lead 1 and optimized analog 12e.
12i
0.37
0.30
>1
0.40
0.57
NT
SO2CH3
12j
good. Urea 12h had somewhat better plasma exposure, with re-
duced brain penetration. The reduced brain penetration is not
surprising given the increased polar surface area of the urea. Car-
bamate 12e had slightly better plasma and brain exposure as 1.
Most surprising was sulfonamide 12j which displayed the best
CNS exposure. These trisubstituted amines have several metabolic
soft spots which may contribute to their poor exposure and it’s
not clear yet the liability of the nitrothiophene ring. Interestingly,
upon increasing the dose to 50 mg/kg ip for 12e, plasma exposure
and brain exposure increase significantly, although drug formula-
tion was also modified. With an EC50 = 0.7 lM, given a 50 mg/kg
dose, there is over 10-fold concentration of drug in brain at
t = 8 h.
As can be seen from the curves in Figure 2, we have greatly im-
proved the overall efficacy and potency of compounds in this series
when compared to the lead GSK4112/SR6452/1. Compounds like
12e, 12h, and 12j also have good plasma and brain exposure such
they might represent useful tools to study the function of Rev-erb
in vivo in models of disease. Progress in this area is on-going and
SO2p-Tol
12k
12l
SO2CH2Ph
SO21-Naphthyl
0.40
0.45
0.63
0.45
12m
a
Results are average of two or more experiments. Value = fold change relative to
DMSO control at 10 M compound.
l
b
NT = not tested. All standard deviations 625%.
Cl
Cl
O2N
O2N
a; then b, c,
d, e or f
S
S
N
N
11k
12a-m
N
N
O
R3
X
a
O
X=bond,CO,SO2,CONH,CO2
will be reported in due course.
Scheme 2. Reagents and conditions: (a) TFA, CH2Cl2; (b) NaBH(OAc)3, HOAc,
Cl(CH2)2Cl, R3CHO; (c) R3COCl, TEA; (d) R3SO2Cl, TEA; (e) R3NCO; (f) R3OCOCl, TEA,
CH2Cl2.
Acknowledgement
This work was supported, in whole or in part, by National Insti-
tutes of Health Grants DK080201 and MH093429 (to T. P. B.).
DNA binding domain (DBD): REV-ERB ligand binding domain (LBD)
a
or b and a Gal4-responsive luciferase reporter. Its half-maximum
inhibitory concentration (IC50) against Rev-erb
a was 670 nM, in
References and notes
good correlation with its BMAL data.18
The in vivo properties of several analogs were examined in
mouse (Table 6).22 As Rev-erb
is highly expressed in the central
nervous system (CNS), brain penetration was also evaluated. Mice
were given a 10 mg/kg ip dose of drug, and plasma and brain lev-
els of drug were determined 2 h later. The hydrophobic lead 1
had limited exposure in plasma, although CNS penetration was
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21. All assays are performed as contransfections with the dual glow luciferase and
data are normalized to constitutively active renilla luciferase reporter activity.
This provides normalization for any non-specific effects of the compounds and
we also monitor renilla luciferase activity directly to determine potential
toxicity.
22. CNS exposure was evaluated in C57Bl6 mice (n = 3). Compounds were dosed at
10 mg/kg intraperitoneally and after 2 h blood and brain were collected.
Plasma was generated and the samples were frozen at À80 °C. The plasma and
brain were mixed with acetonitrile (1:5 v:v or 1:5 w:v, respectively). The brain
sample was sonicated with a probe tip sonicator to break up the tissue, and
samples were analyzed for drug levels by LC–MS/MS. Plasma drug levels were
determined against standards made in plasma and brain levels against
standards made in blank brain matrix. All procedures were approved by the
Scripps Florida IACUC.