Small molecule amides as potent ROR-γ selective modulators

Article abstract of DOI:10.1016/j.bmcl.2012.11.025

The structure-activity relationship study of a diphenylpropanamide series of ROR-γ selective modulators is reported. Compounds were screened using chimeric receptor Gal4 DNA-binding domain (DBD)-NR ligand binding domain cotransfection assay in a two-step format. Three different regions of the scaffold were modified to assess the effects on repression of ROR-γ transcriptional activity and potency. The lead compound 1 exhibits modest mouse pharmacokinetics and an acceptable in vitro profile which makes it a suitable in vivo probe to interrogate the functions of ROR-γ in animal models of disease.

Full text of DOI:10.1016/j.bmcl.2012.11.025

Bioorganic & Medicinal Chemistry Letters 23 (2013) 532–536
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Small molecule amides as potent ROR-c selective modulators
Pasha M. Khan, Bahaa El-Dien M. El-Gendy, Naresh Kumar, Ruben Garcia-Ordonez, Li Lin, Claudia H. Ruiz,
⇑
Michael D. Cameron, Patrick R. Griffin, 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 diphenylpropanamide series of ROR-c selective modulators
is reported. Compounds were screened using chimeric receptor Gal4 DNA-binding domain (DBD)-NR
ligand binding domain cotransfection assay in a two-step format. Three different regions of the scaffold
Received 22 September 2012
Revised 29 October 2012
Accepted 7 November 2012
Available online 22 November 2012
were modified to assess the effects on repression of ROR-
c transcriptional activity and potency. The lead
compound 1 exhibits modest mouse pharmacokinetics and an acceptable in vitro profile which makes it a
suitable in vivo probe to interrogate the functions of ROR-
c
in animal models of disease.
Keywords:
ROR-c selective
Ó 2012 Elsevier Ltd. All rights reserved.
Diphenylpropanamide
SAR
Nuclear hormone receptor
Nuclear hormone receptors (NR) are a highly conserved group
of transcription factors that regulate a range of metabolic, endo-
crine and immunologic disorders including cancer, inflammation,
diabetes and atherosclerosis. Members of the nuclear receptor
(NR) superfamily are characterized by a highly conserved DNA
binding domain and a ligand binding domain.1 Fourty-eight NRs
have been identified in humans and are comprised of classic ste-
roid receptors, RXR heterodimer receptors and xenobiotic recep-
tors. Approximately half of the human NRs are characterized as
ligand activated transcription factors regulating the expression of
target genes, whereas a large number of these receptors are still
classified as orphan due to lack of a characterized natural ligand.2
Y
Z
N
N
O
OH
O
OH
A
O
O
X
B
R
MeO
OMe
2
1
Figure 1. SR-9805 (1) and analogs as ROR-
c
modulators.
Retinoic acid receptor-like orphan receptors
a and c (RORs) are
O
O
examples of such orphan nuclear receptors that play critical roles
CO2H
OH
in immunity, cellular metabolism and circadian rhythms.3
a
The retinoic receptor-related orphan receptor
c (ROR-c) and its
isoform ROR- t play a critical role in differentiation of Th17 cells
c
MeO
OMe
MeO
OMe
X
and secretion of inflammatory cytokines such as Interleukin 17
(IL-17).4 Th17 cells have been identified as key mediators for im-
mune responses of a wide variety of autoimmune diseases such
as multiple sclerosis (MS), Crohn’s disease, and rheumatoid arthri-
tis (RA). While cholesterol and cholesterol sulfate have been put
X
3
4
5
R1
N
R2
O
OH
b
forward as the natural ligands for ROR-
for ROR-
still remain debatable.5 We have shown that various
oxysterols that bind to ROR- also bind to ROR- and regulate their
activity.6 We have also demonstrated that synthetic LXR agonist
a, the endogenous ligands
MeO
OMe
c
X
a
c
6
Scheme 1. Reagents and conditions: (a) TFA,
60 °C, 2 h.
l
W, 30 min, 120 °C; (b) R1R2NH, THF,
T0901317 binds and modulates ROR-a . Recently we were
and -c 7
able to identify SR-33358a (synthetic ROR-
nist), SR-10018b (dual ROR-
, - synthetic inverse agonist), SR-
a-selective inverse ago-
⇑
Corresponding author.
E-mail address: kameneck@scripps.edu (T.M. Kamenecka).
a
c
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.11.025

P. M. Khan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 532–536
533
Table 1
Table 2
Effect of substituent R1 and R2 on ROR-
c
modulation
Effect of substituent R3 on ROR-
c
modulation
MeO
OH
OH
O
N
R3
OMe R1 R2
O
Compound R1
R2
Binding
assay IC50
(nM)
% Repression @
Ma (GAL4 assay
a
10
l
O
O
IC50
)
Compound R3
Binding assay
IC50 (nM)
% Repression @ 10 l
Ma
1
57
95 (76 nM)
N
(GAL4 assay IC50
)
NTb
NTb
17
11
O
1
3,5-diOMe
3,5-diOMe
4-Et
4-OEt
4-OPr
3-Me
57
95 (76 nM)
22
6a
6b
N
7a
7b
7c
7d
7e
7f
NTb
NTb
NTb
NTb
NTb
NTb
NTb
HN
95 (>1
95 (>1
96 (>1
96 (>1
95 (>1
95 (>1
lM)
lM)
lM)
lM)
lM)
lM)
N
6c
NTb
4
OH
OH
4-Me
3-OPh
N
7g
6d
NTb
0
1
OH
O
O
N
7h
NTb
93 (>1
lM)
O
N
N
6e
6f
CO2Et NTb
O
NTb
68 (>1
57
lM)
Ph
O
a
Results are average of at least three replicates. Value = fold change relative to
DMSO control at 10 M compound.
NT = not tested. These compounds were tested at 10
assay and only the compounds which showed more than 50% repression of ROR-
transcriptional activity at 1 lM were run in the binding assay. All standard devia-
tions 620%.
6g
NTb
N
l
b
lM and 1 lM in the GAL4
F
c
6h
NTb
23
N
Ph
OH
6i
6j
NTb
NTb
8
N
N
Table 3
Effect of substituent R4, on ROR-
c
modulation
83 (>1 lM)
CF3
MeO
OH
NTc
NTc
N
6k
6l
NA
NA
HN
N
MeO
O
O
R4
N
6m
NA
NTc
Compound R4
Binding assay % Repression @ 10 l
Ma
OMe
IC50 (nM)
(GAL4 assay IC50
)
HN
N
6n
6o
NA
NA
NTc
NTc
1
3,4-
57
95 (76 nM)
Methylenedioxy
4-OMe
2-OMe
6p
8
9
10
11
96
95 (114 nM)
96 (265 nM)
27
0
0
106
NTb
NTb
NTb
6p
96
95 (114 nM)
N
H
See Scheme 2
See Scheme 2
a
Results are average of at least three replicates. Value = fold change relative to
DMSO control at 10 M compound.
NT = not tested. These compounds were tested at 10
assay and only the compounds which showed more than 50% repression of ROR-
transcriptional activity at 1 M were run in the binding assay.
l
OH
N
b
l
M and 1 lM in the GAL4
NTb
96 (>1 lM)
c
19
O
l
c
For these compounds the binding assay was run prior to the GAL4 assay and if
the compounds did not show any binding, no GAL4 cell based assay was performed
for these compounds (NA = No Activity). All standard deviations 620%.
a
Results are average of at least three replicates. Value = fold change relative to
DMSO control at 10 M compound.
NT = not tested. These compounds were tested at 10
assay and only the compounds which showed more than 50% repression of ROR-
l
b
lM and 1 lM in the GAL4
c
10788c (dual ROR- synthetic agonist) and SR-22118d (ROR-
a, -c c
transcriptional activity at 1 lM were run in the binding assay. All standard devia-
selective modulator). Natural products such as digoxin and ursolic
acid have also been shown to inhibit Th17 cell differentiation, but
their potential as candidates for further development is rather lim-
ited due to narrow therapeutic index and selectivity issues over
other receptors such as glucocorticoid receptors.9 While corticoste-
roids are highly efficacious in managing autoimmune disorders,
they also induce some serious side effects such as osteoporosis, ret-
inopathy and diabetes. In light of these facts, development of new
tions 620%.
with improved therapeutic profiles. Herein, we present our SAR ef-
forts to characterize SR-9805/1 (Fig. 1) and its analogs as potent
ROR-c
selective modulators.10
Based on the lead compound 1 four regions of this scaffold were
modified in a step-wise fashion in order to investigate the effects of
substituents X, Y, Z, and R on the activity of these compounds.
Compounds represented by general structure 6 were synthesized
ROR-c selective modulators that specifically control IL-17 expres-
sion and Th17 cell differentiation will provide drug-like molecules

534
P. M. Khan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 532–536
Figure 2. Demonstration of transcriptional repression and binding of compound 1 to ROR-c.
OH
HO2C
OH
O
O
N
O
a
b
CHO
OMe
OMe
OMe
12
10
14
13
O
O
CHO
O
OH
NH
c
H2N
NH2
18
17
16
15
OH
H
N
N
d
O
11
lW, 30 min, 120 °C; (b) (1) H2, Pd-C, 48 h, (2) 3,5-dimethylpiperidine, THF, 40 °C; (c) NaI, TMSCl, DMF, 140 °C; (d) 3,5-
Scheme 2. Reagents and conditions: (a) DCC, DMF,
dimethylpiperidine, THF, 40 °C.
via a two-step protocol starting from commercially available start-
ing materials (Scheme 1). 3,5-Dimethoxyphenol 3 was treated with
various electron rich cinnamic acids 4 in the presence of trifluoro-
acetic acid (TFA) as a solvent to furnish dihydrocoumarins 5. Com-
pound 5 was subjected to ring opening using various amines to
yield the final compounds 6.11
pounds were further evaluated using a competition assay to deter-
mine if they can directly bind to ROR-c 12
As shown in Figure 2B
increasing concentrations of SR-9805 were incubated with 5 nM
.
of [3H]-T0903017 and 1
lg of GST-ROR-c along with Glutathi-
one-YSi beads to determine IC50 as detailed in the methods.12
We started the SAR studies by modifying the amide functional-
ity in the lead compound 1 (substituent R2, Table 1). Compound 1
Compounds were screened using chimeric receptor Gal4 DNA-
binding domain (DBD)-NR ligand binding domain cotransfection
shows 95% repression of ROR-c transcriptional activity at 10 lM
assay in a two-step format. To determine the effect on the ROR-
transcriptional activity, HEK293T cells were cotransfected with
Gal4-ROR- along with a UAS-luciferase plasmid. The cells were
treated for 20 h with the compound and relative change was deter-
mined by normalizing to cells treated with vehicle. Compounds
c
with an IC50 of 76 nM in a GAL4 assay and an IC50 of 57 nM in a
binding assay. Several five and six membered ring amides were
synthesized, however none of them exhibited any improvement
with regards to efficacy (Table 1). For example, analogs with a hy-
droxy substituent on the six member ring (6c, 6d, 6i) showed poor
c
were first screened at two concentrations (1
determine the effect on repression of ROR- transcriptional activity
and the maximum repression at 10 M is reported (Table 1–3). A
l
M and 10
l
M) to
repression of ROR-
Compounds 6f and 6j exhibited modest repression but with
micromolar potency. The high repression of ROR- transcriptional
c transcriptional activity at 10 lM.
c
l
c
high % repression indicates that the compound is more efficacious
at repressing transcription. Compounds that showed more than
activity and nanomolar potency (114 nM) of compound 6p
(Table 1) further validates the importance of the 3,5-dimethyl
piperidine functionality in this scaffold. As far as the substituent
R1 is concerned, modifying 3,4-(methylenedioxy)phenyl to 4-
methoxy phenyl (1 vs 6p) did not have a dramatic effect on the effi-
cacy or potency of these compounds. Modifying substituent R2
from 3,5-dimethylpiperidine (6p) to benzylamine (6k), morpholine
50% repression at 1
response format to generate IC50 values. As shown in Figure 2A, SR-
9805 shows an IC50 of 76 nM on ROR- transcriptional activity. SR-
9805 also did not show any activity on related nuclear hormone
receptors ROR- , LXR- , and FXR (data not shown). These com-
lM were then fully titrated in a ten-point dose
c
a
a

P. M. Khan et al. / Bioorg. Med. Chem. Lett. 23 (2013) 532–536
535
Table 4
In vitro and in vivo properties of selected compounds
Entry
Solublilitya
M) pH 7.4 (
[Plasma]b
(lM)
[Brain]b
(l
M)
b.p.c (%)
p450 % inhibitiond
pH 5.0 (
l
lM)
1A2
2C9
2D6
3A4
1c
8
6p
15.2
6.0
19.5
15.6
12.7
18.0
0.30
NT
NT
0.78
NT
NT
260
NT
NT
3
92
53
65
15
97
70
75
À22
À11
4
À7
NT= Not tested.
a
100 lM solutions of compounds were shaken at pH 7.4 (PBS) and pH 5.0 (Sodium Citrate Buffer) at room temperature for 20 h.
Mice sacrificed at t = 6 h. Brain and plasma levels of drug determined. Mice dosed 10 mg/kg IP in 10:10:80 DMSO:Tween:water.
b.p. = brain penetration.
% Inhibition at 10 lM drug.
b
c
d
(6l), piperidine (6m), cyclopentylamine (6n) or pyrrolidine (6o),
resulted in loss of activity. In fact as long as the substituent R2
was 3,5-dimethyl piperidine the compounds maintained nanomo-
lar potency (1 vs 6p, Table 1 & 8, Table 3).
Efforts were then focused on modifications to the phenol ring
bearing the substituent R3 (Table 2). The compounds shown are
only a subset of those actually synthesized, however they are rep-
resentative of the group. Disubstituted analogs such as 4-Et (7b), 4-
OEt (7c), 3-Me (7e), 4-Me (7f) had modest effect on the % repres-
sion as well as the potency. Bulky substituents such as 2-naphthyl
(7h), 4-OPr (7d) or 3-OPh (7g) were also well tolerated. While
these substitutions provided compounds which showed maximum
at pH 5 and 7.4. Brain penetration of these compounds was also
measured since ROR- is highly expressed in the central nervous
c
system (CNS). Mice were given a 10 mg/kg IP dose of drug, and
plasma and brain levels of drug were determined 6 h later. The lead
compound 1 exhibited limited exposure in plasma, although CNS
penetration was good. Compound 1 also exhibited high % inhibi-
tion of cytochrome 2C9 and 3A4.
The SAR on the ROR-c selective scaffold (Fig. 1) presented here-
in is very tight. Small modifications to its structure result in total
loss of potency. Having the 3,5-dimethyl piperidine as the amine
fragment in the amide moiety is pertinent to the efficacy and po-
tency of these compounds. A wide variety of electron-donating
substituents are tolerated on ring A (Fig. 1) without affecting the
transcriptional repression at 10 lM, IC50’s were significantly right-
shifted compared to 1. The nature of this effect is unclear, but is
also fairly robust. Hence, we conclude that modifications to this
ring are not well tolerated.
% repression of ROR-c transcriptional activity. Ring B is likely most
tolerant to substitution. We also observed that modifying the
amide in compound 1 to an urea functionality (11, Table 3) or
changing the location of the 3,5-dimethoxy phenyl ring system
Modification of substituent R4 also presented a very flat SAR
(Table 3). Modifying the substituent from 3,4-(methylenedi-
oxy)phenyl to 4-OMe (6p) or 2-OMe (8) reduced the potency by
1.5 and 3.5 fold respectively in a GAL4 assay while still maintaining
from the b position to the
Table 3) resulted in complete loss of activity. In summary, this
ROR- selective scaffold provides a starting point for development
of new probes to interrogate the functions of ROR- in animal
a position of the amide carbonyl (10,
c
a high percentage repression of ROR-
c
transcriptional activity.
c
ROR-c
binding was also very good. The R4-substituent also has to
models of disease. Further work focused on the improvement of
efficacy, potency and in vivo profile of these compounds is under-
way and will be reported in due course.
be something other than hydrogen (9) which leads to loss of
activity.
In order to study the effect of amide functionality and the loca-
tion of 3,5-dimethoxy phenyl ring system with respect to the
amide carbonyl, we synthesized analogs 10, 11, and 19 (Scheme 2).
Compound 10 was synthesized via a two-step protocol which
involved coupling of 2-hydroxy-6-methoxybenzaldehyde 12 and
p-tolylacetic acid 13 in the presence of DCC to afford coumarin
14.13 Compound 14 was hydrogenated followed by treatment with
3,5-dimethyl piperidine to afford compound 10. Synthesis of com-
pound 11 also involved a two-step sequence starting with a three
component reaction.14 Treatment of a mixture of 2-naphthol, p-tol-
ualdehyde and urea along with TMSCl/NaI as a promoter resulted
in the naphthoxazinone 18. Compound 18 was treated with 3,5-di-
methyl piperidine to afford compound 11. Compound 19 (synthe-
sis not shown) was made as described in Scheme 1.
Acknowledgment
This work was supported, in whole or in part, Grant MH084512
(PI:H Rosen).
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12. Cell culture and cotransfections: HEK293 cells were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum
at 37 °C under 5% CO2. Reverse transfections were performed in bulk using
15. CNS exposure was evaluated in C57Bl6 mice (n = 3). Compounds were dosed at
10 mg/kg intraperitoneally and after 6 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 LCMS/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.
1 Â 106 cells in 6 cm plates, 3
lg of total DNA in a 1:1 ratio of receptor and
reporter and FuGene6 (Roche) in a 1:3 DNA:lipid ratio. Following day, cells re
plated in 384 well plates at a density of 10,000 cells/well. After 4 h, the cells
were treated with the compound or DMSO as control. The luciferase levels
were assayed following additional 20 h incubation by one-step addition of
BriteLite Plus(Perkin Elmer) and read using an Envision (Perkin Elmer). Data
was normalized as fold change over DMSO treated cells.