Discovery of 1-{4-[3-Fluoro-4-((3 S,6 R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (GNE-3500): A Potent, Selective, and Orally Bioavailable Retinoic Acid Receptor-Related Orphan Receptor C (RORc or RORγ) Inver

Article abstract of DOI:10.1021/acs.jmedchem.5b00597

Retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) is a nuclear receptor that plays a major role in the production of interleukin (IL)-17. Considerable efforts have been directed toward the discovery of selective RORc inverse agonists

Full text of DOI:10.1021/acs.jmedchem.5b00597

Article
pubs.acs.org/jmc
Discovery of 1‑{4-[3-Fluoro-4-((3S,6R)‑3-methyl-1,1-dioxo-6-phenyl-
[1,2]thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone
(GNE-3500): a Potent, Selective, and Orally Bioavailable Retinoic Acid
Receptor-Related Orphan Receptor C (RORc or RORγ) Inverse Agonist
Benjamin P. Fauber,*,† Olivier Rene,† Yuzhong Deng,‡ Jason DeVoss,§ Celine Eidenschenk,§
́
́
Christine Everett,∥ Arunima Ganguli,⊥ Alberto Gobbi,† Julie Hawkins,⊥ Adam R. Johnson,∥ Hank La,‡
Justin Lesch,§ Peter Lockey,⊥ Maxine Norman,⊥ Wenjun Ouyang,§ Susan Summerhill,⊥
and Harvey Wong‡
‡
§
∥
†Discovery Chemistry, Drug Metabolism and Pharmacokinetics, Immunology, Biochemical Pharmacology, Genentech,
Inc., 1 DNA Way, South San Francisco, California 94080, United States
⊥Discovery Biology, Argenta, Units 7-9 Spire Green Centre, Flex Meadow, Harlow, Essex CM19 5TR, U.K.
S
* Supporting Information
ABSTRACT: Retinoic acid receptor-related orphan receptor C (RORc, RORγ, or NR1F3) is a nuclear receptor that plays a
major role in the production of interleukin (IL)-17. Considerable efforts have been directed toward the discovery of selective
RORc inverse agonists as potential treatments of inflammatory diseases such as psoriasis and rheumatoid arthritis. Using the
previously reported tertiary sulfonamide 1 as a starting point, we engineered structural modifications that significantly improved
human and rat metabolic stabilities while maintaining a potent and highly selective RORc inverse agonist profile. The most
advanced δ-sultam compound, GNE-3500 (27, 1-{4-[3-fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-
ylmethyl)-phenyl]-piperazin-1-yl}-ethanone), possessed favorable RORc cellular potency with 75-fold selectivity for RORc
over other ROR family members and >200-fold selectivity over 25 additional nuclear receptors in a cell assay panel. The
favorable potency, selectivity, in vitro ADME properties, in vivo PK, and dose-dependent inhibition of IL-17 in a PK/PD model
support the evaluation of 27 in preclinical studies.
Our group has previously reported the discovery and op-
timization of tertiary sulfonamide RORc inverse agonists,23−26
as exemplified by 1 (Scheme 1). Compound 1 was a potent
RORc inverse agonist in a biochemical assay (EC50 = 30 nM),
GAL4 human transcription cell assay (EC50 = 130 nM), and
human peripheral blood mononuclear cell (PBMC) assay
(EC50 = 800 nM).25 Compound 1 was also >77-fold selective
for RORc over other NRs as assessed by a panel of GAL4 human
transcription cell assays.25 When 1 was incubated with human or
rat liver microsomes (HLM or RLM), however, it displayed high
hepatic clearance (CLhep) values in both species (>70% of liver
blood flow),25 thereby limiting its utility to in vitro experiments.
INTRODUCTION
■
The nuclear receptor (NR) retinoic acid receptor-related orphan
receptor C (RORc or RORγ, also known as NR1F3)1 is an
important transcription factor involved in the production and
regulation of the pro-inflammatory cytokine interleukin (IL)-17.2
Antibodies inhibiting the production or activity of IL-17 family
cytokines3,4 have demonstrated proof of concept in clinical trials
for the treatment of psoriasis,5−7 rheumatoid arthritis (RA),8
ankylosing spondylitis,9 and uveitis.10 RORc also plays a role in
the regulation of IL-2211 and granulocyte macrophage colony
stimulating factor (GM-CSF)12 as well as the production of
innate lymphoid cells (ILCs)13,14 and γδ T cells.15 On the basis
of the influence of RORc over multiple inflammatory pathways,
it has been proposed that RORc is a potentially valuable molec-
ular target for the treatment of inflammatory diseases.16−22
Received: April 17, 2015
© XXXX American Chemical Society
A
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a
Scheme 1. Identification of the Human and Rodent in Vitro Metabolites of 1
a
Compound 1 was completely metabolized in both human and rat liver microsomes over the course of the 1 h in vitro studies. Metabolites are
shown as a percentage of all detected compounds (LC-MS/MS method) at the end of the studies. The studies were conducted using nonisotopic 1.
Metabolite identification (MetID) studies with either HLM
or RLM revealed that 1 (Scheme 1) was completely consumed
during the 1 h incubation period and two metabolites were
formed. The major metabolite in both the HLM and RLM
studies was the sulfonamide N-dealkylation product 2 (human =
81%, rat = 92%), and oxidation of the piperazine ring (M + 16)
provided the minor metabolite 3 (human = 19%, rat = 8%).
Metabolite 2 was inactive in the RORc SRC1 biochemical
assay (EC50 > 10 μM). On the basis of the MetID data, we
devised a strategy to address the N-dealkylation metabolism
and improve the metabolic stability of compounds related to 1
while also maintaining favorable RORc inverse agonist
potency.
(bromomethyl)benzene, followed by Buchwald−Hartwig ami-
nation27 of the aryl-bromide with N-acetylpiperazine, led to an
in situ cyclization reaction28 to form 6. Compound 6 was the
only product of the reaction.
Syntheses of the sultam analogues originated from the cor-
responding amino-halides (7−8, Scheme 3) or amino-alcohols
(9−11),29 and the sultam rings were assembled according to
the method of Lee et al.30 Compounds 7−8 were reacted with
phenylmethanesulfonyl chloride and triethylamine to yield
the secondary sulfonamides, followed by treatment with two
equivalents of base to facilitate cyclization to the sultam ring
intermediates (12−13). In an analogous manner to 7−8, com-
pounds 9−11 were reacted with two equivalents of phenyl-
methanesulfonyl chloride and triethylamine, followed by
treatment of the crude reaction products with NaCl in hot
DMF to form the alkyl chloride intermediates. The crude alkyl
chloride intermediates were reacted with two equivalents of
base to form the sultam ring intermediates (14−16). Com-
pounds 12−16 were exposed to sodium hydride and 4-bromo-
1-(bromomethyl)-2-fluorobenzene to provide the crude
N-benzyl sultam intermediates. The N-benzyl intermediates
were then carried forward into a Buchwald−Hartwig amina-
tion27 with N-acetylpiperazine to provide the sultam products
as mixtures of enantiomers and diastereomers (17−21). The
stereoisomers of the δ-sultam products were separated by
chiral supercritical fluid chromatography (SFC) to provide the
enantiopure products (22−29). The absolute stereochemistry
of the (6R)-phenyl δ-sultam ring in 23 was assigned by single-
crystal X-ray analysis (see Supporting Information). The
absolute stereochemistry of the 6-phenyl substituent on the
(3R)- and (3S)-methyl δ-sultam products (24−27) were assigned
by NMR analysis.31 We obtained a single-crystal X-ray struc-
ture of 27 to further confirm the absolute stereochemistry of
the (6R)-phenyl δ-sultam ring (see Supporting Information).
The X-ray data for 27 was in agreement with the stereo-
chemistry we had originally assigned by NMR.31 Repeated
attempts to crystallize either 28 or 29 for X-ray analysis were
unsuccessful. Thus, the stereochemistry of the 6-phenyl
substituent in 28 and 29 was assigned based on their relative
optical rotations to 22 and 23, respectively (i.e., optical
RESULTS AND DISCUSSION
■
The synthesis of 1 was described in our lab’s previous article.25
Compound 6 (Scheme 2) was synthesized through the
a
Scheme 2. Synthesis of 6
a
Reagents and conditions: (a) BnSO2Cl, EtN(i-Pr)2, DCM, 23 °C,
46%; (b) 1-bromo-4-(bromomethyl)benzene, NaH, DMA, 75 °C; (c)
N-acetylpiperazine, RuPhos Pd G1 MTBE adduct, RuPhos, NaOt-Bu,
1,4-dioxane, 100 °C, 14% over 2 steps.
sulfonylation of 2-amino-2-methylpropanenitrile (4) with
phenylmethanesulfonyl chloride to provide the secondary
sulfonamide intermediate (5). Benzylation of the secondary
sulfonamide under basic conditions using 1-bromo-4-
B
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a
Scheme 3. Syntheses of Sultam Analogues
a
Reagents and conditions: (a) BnSO2Cl, Et3N, THF, 0 → 23 °C; (b) n-BuLi, (i-Pr)2NH, phenanthroline, THF, −78 °C, 42−59% over 2 steps;
(c) BnSO2Cl, Et3N, THF, 0 → 23 °C; (d) NaCl, DMF, 80 °C; (e) n-BuLi, (i-Pr)2NH, phenanthroline, THF, −78 °C, 21−42% over 3 steps;
(f) 4-bromo-1-(bromomethyl)-2-fluorobenzene, NaH, DMF, 0 °C; (g) Pd(OAc)2, RuPhos, Cs2CO3, N-acetylpiperazine, 1,4-dioxane, 80 °C,
16−73% over 2 steps; (h) chiral column SFC purification.
Table 1. Structure−Activity Relationships and in Vitro Metabolism
a
Inhibition of human RORc-LBD recruitment of the SRC1 coactivator peptide where the EC50 values are reported as means (all standard deviations
were <55% of the mean EC50 values) for ≥3 separate titrations; percent efficacy “%eff” is the maximal efficacy observed in the assay at the highest
test concentration of 10 μM, and the %eff values are reported as means (all standard deviations were <4% of the mean %eff values); negative %eff
b
c
denotes inverse agonism of the basal activity of apo-RORc-LBD in this assay format. Calculated Log P (cLogP) value.55 Ligand-lipophilicity
efficiency (LLE) was calculated using the equation: LLE = (RORc SRC1 pEC50) − cLogP.34,35 Predicted human clearance extrapolated from in
vitro human liver microsome (HLM) experiments. Predicted rat clearance extrapolated from in vitro rat liver microsome (RLM) experiments.
d
e
comparison),32 in addition to their biochemical potencies and
metabolic stabilities in relation to stereoisomers 22−27. The
syntheses of compounds 3025 (Table 1) and 3123 (Table 2)
were described in our lab’s previous manuscripts.
The analogues were tested in a time-resolved fluorescence
biochemical assay that monitored the ability of the human
RORc ligand binding domain (LBD) to bind to a coactivator
peptide derived from steroid receptor coactivator (SRC)-1.33
Compounds that disrupted the recruitment of the SRC1
coactivator peptide and decreased the basal level of RORc
signaling were inverse agonists. We also monitored the ligand-
lipophilicity efficiency (LLE)34,35 of the analogues with a goal
of maintaining an LLE value comparable to or superior to that
of 1 (LLE = 4.3). Tracking the LLE values ensured that we
C
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Table 2. Structure−Activity Relationships and in Vitro Metabolism of Sultams
a
Inhibition of human RORc-LBD recruitment of the SRC1 coactivator peptide where the EC50 values are reported as means (all standard deviations
were <36% of the mean EC50 values) for ≥3 separate titrations; percent efficacy “%eff” is the maximal efficacy observed in the assay at the highest
test concentration of 10 μM, and the %eff values are reported as means (all standard deviations were <5% of the mean %eff values); negative %eff
b
c
denotes inverse agonism of the basal activity of apo-RORc-LBD in this assay format. Calculated Log P (cLogP) value.55 Ligand-lipophilicity
efficiency (LLE) was calculated using the equation: LLE = (RORc SRC1 pEC50) − cLogP.34,35 Predicted human hepatic clearance extrapolated
from in vitro human liver microsome (HLM) experiments. Predicted rat hepatic clearance extrapolated from in vitro rat liver microsome (RLM)
d
e
experiments.
were maintaining efficient binding interactions with the human
RORc-LBD as we evolved the analogues and avoided the
inclusion of unnecessary lipophilicity.
Other teams have successfully addressed the metabolic
cleavage of N-alkyl groups on tertiary sulfonamides by lowering
the lipophilicity of the N-alkyl substituent.36 Unfortunately, this
D
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approach was not tolerated in our tertiary sulfonamide series
and led to compounds devoid of detectable RORc inverse
agonist biochemical activity with no notable improvements in
microsomal stability. We hypothesized that the major
metabolite of 1 (Scheme 1) arose from oxidation of the
N-alkyl group to generate an unstable aminal intermediate,
followed by cleavage of the C−N bond to form 2. Replacement
of the hydrogen atoms on the N-alkyl carbon atom adjacent
to the nitrogen with methyl groups should block this pro-
cess. Indeed, when we synthesized the N-tert-butyl sulfonamide
analogue (30, Table 1), this structural change abated the
microsome-mediated cleavage of the N-tert-butyl group.
Instead, we observed a new metabolite in which cleavage of
the N-benzylic group of 30 was the major metabolite in MetID
studies, and 30 provided no improvement in HLM and RLM
CLhep values as compared to 1.
In addition to 30, we explored other metabolically stable tert-
butyl isosteres.37 During the attempted synthesis of a N-(2-
methylpropanenitrile)sulfonamide analogue, we observed the
formation of a 4-amino-2,3-dihydroisothizaole 1,1-dioxide
product (6, Table 1). Although 6 had no detectable RORc in-
verse agonist activity in our biochemical assay (EC50 > 10 μM),
it possessed favorable HLM and RLM values (CLhep = 5 and
8 mL/min/kg, respectively). This result led us to evaluate other
cyclic sulfonamide (sultam) analogues as potential metabol-
ically stable and potent RORc inverse agonists.
scaffold in which to test this hypothesis. Compound 17 main-
tained moderate clearance values in the HLM and RLM assays
(CLhep = 13 and 31 mL/min/kg, respectively), but 17 had no
RORc inverse agonist activity up to a 10 μM concentration
in our biochemical assay. Encouraged by the favorable CLhep
values for 17 in comparison to 31, we synthesized the 6-phenyl
δ-sultam analogue (18). Compound 18 displayed moderate
clearance values in the HLM and RLM assays (CLhep = 13 and
29 mL/min/kg, respectively) and was also a RORc inverse
agonist in our biochemical assay (EC50 = 350 nM). Separation
of 18 into its two respective enantiomers provided 22 and 23.
Compound 22 displayed high clearance values in the HLM and
RLM assays (CLhep = 18 and 47 mL/min/kg, respectively),
whereas 23 displayed moderate clearance values in the HLM
and RLM assays (CLhep = 9 and 16 mL/min/kg, respectively).
Compound 23 was also 7-fold more potent than 22 in the
RORc SRC1 biochemical assay (EC50 = 180 nM and 1.2 μM,
respectively). To our delight, the more potent 6-phenyl
δ-sultam enantiomer was also the more metabolically stable
enantiomer. We hypothesized that the improved metabolic
stability of 23 over 22 was potentially due to a steric clash of
the (6R)-phenyl δ-sultam in 23 with the cytochrome P450
(CYP) that was responsible for the increased metabolic turn-
over of 22.
On the basis of the encouraging metabolic stability and
potency of 23, we envisioned a refined strategy to accurately
define the stereochemistry of the (6R)-phenyl group of the
δ-sultam ring in future analogues while also improving the
RORc inverse agonist potency of 23. Introduction of a
3-methyl group with known absolute stereochemistry on the
δ-sultam ring allowed such an opportunity.31 Molecular
modeling of the binding mode of 23 (Figure 1)38 suggested
that the 3-position of the δ-sultam ring provided access to a
region of the ligand binding pocked filled by the N-alkyl
substituent of 31 and other previously disclosed anlogues.23,25
The assay results for the (3R)-methyl-6-phenyl δ-sultam ana-
logues 24 and 25 (Table 2) confirmed the initial observations
made with 22 and 23. Compound 24 had poor HLM and RLM
stabilities (CLhep = 18 and 47 mL/min/kg, respectively) and
We utilized a fluorinated benzylic core in subsequent
analogues that built on the promising results of 6, as we have
previously shown that inclusion of the fluorine atom provided
improvements in RORc biochemical and cellular potencies.23
We were aware of the lipophilic environment in the RORc
ligand binding pocket surrounding the benzylic sulfonamide
moiety of 31 (Table 2 and Figure 1) based on our previous
modest RORc inverse agonist biochemical potency (EC50
=
5.4 μM). Compound 25 had moderate HLM and RLM
stabilities (CLhep = 13 and 27 mL/min/kg, respectively) and
was a potent RORc inverse agonist in the SRC1 biochemical
assay (EC50 = 17 nM). The trend of the (6R)-phenyl group
being the preferred δ-sultam stereoisomer was further
supported with the assay results of the (3S)-methyl-6-phenyl
δ-sultam analogues 26 and 27. Although 26 had an improved
RORc inverse agonist potency (EC50 = 94 nM) as compared
with 24, its 3-methyl stereochemical matched molecular
pair,39 26 had poor HLM and RLM stabilities (CLhep = 18
and 44 mL/min/kg, respectively). The (3S)-methyl-(6R)-
phenyl δ-sultam analogue 27 (GNE-3500, 1-{4-[3-fluoro-4-
((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-
phenyl]-piperazin-1-yl}-ethanone) provided a very favorable
Figure 1. X-ray costructure of the human RORc-LBD (gray) and a
tertiary sulfonamide RORc inverse agonist ligand (magenta lines)
(PDB: 4WQP) modeled with 23 (yellow sticks).38 The surface of the
RORc-LBD ligand binding pocket is shown as an opaque surface (red
and blue = polar residues, gray and yellow = lipophilic residues) and
culled to reveal the bound (magenta lines) and modeled (yellow
sticks) ligands. The arrow (black) illustrates a potential vector from
the 3-position of the δ-sultam ring to fill the same region occupied by
the i-butyl group of the RORc inverse agonist ligand (magenta lines)
in the RORc-LBD X-ray costructure.
overall profile with moderate HLM and RLM stabilities (CLhep
=
10 and 21 mL/min/kg, respectively) and potent RORc inverse
agonist biochemical activity (EC50 = 12 nM). Compound 27
also displayed a favorable LLE value (LLE = 4.2) that was
comparable to 1.
The 3,3-dimethyl-6-phenyl δ-sultam enantiomers were tested
to further probe the role of the 3-substituent in improving
the RORc biochemical potency. The trend of one 6-phenyl
δ-sultam isomer being more potent and metabolically stable
RORc structure-based drug design campaigns.23,25 We
envisioned that removal of the somewhat polar 4-amino
group found in the 2,3-dihydroisothizaole 1,1-dioxide ring of
6 could potentially improve the RORc affinity of closely related
analogues. A 5-phenyl γ-sultam (17) provided a simplified
E
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a
Table 3. In Vitro ADME Properties
b
b
c
compd
human PPB (%bound)
rat PPB (%bound)
MDCK Papp A→B (10−6 cm/s)
MDCK Papp B→A (10−6 cm/s)
solubility (μM)
31
23
25
26
27
29
98
94
95
96
95
98
98
95
96
96
95
96
22
27
16
23
24
24
29
19
16
32
11
21
22
43
34
20
34
17
a
b
See the Supporting information for experimental details associated with each assessment. Madin−Darby canine kidney (MDCK) cell permeability
assay to assess membrane permeability (Papp); A→B, apical-to-basolateral; B→A, basolateral-to-apical.41 Aqueous kinetic solubility at pH 7.4
c
(measured in a high-throughput assay).
a
than the other also held true with these analogues. Compound
28 (Table 2) was less potent (RORc SRC1 EC50 > 10 μM) and less
metabolically stable (HLM and RLM CLhep = 18, 35 mL/min/kg,
respectively) than 29 (RORc SRC1 EC50 = 82 nM; HLM and
RLM CLhep = 13, 33 mL/min/kg, respectively).
Table 4. Single Dose Rat in Vivo PK Properties
e
f
g
CLp
Vd
Cmax
t1/2
h
compd (mL/min/kg) (L/kg)
(μM)
AUC (μM·h) (h)
F%
b
23
25
27
29
13
19
12
15
2.9
2.5
3.3
2.0
0.6
0.6
0.5
0.4
3.9
1.0
2.5
1.3
3.3
1.6
3.5
1.6
74
>99
55
c
Compounds 23, 25, 26, 27, 29, and 31 were evaluated in a
suite of in vitro ADME assays40 to assess their human and rat
plasma-protein binding (PPB), Madin−Darby canine kidney
(MDCK) cellular permeability,41 and aqueous kinetic solubility
at pH 7.4 (Table 3). We were encouraged to see that the
favorable in vitro ADME profiles of the tertiary sulfonamide
compounds exemplified by 31 (Table 3) were maintained in
the δ-sultam subseries. The (6R)-phenyl δ-sultam analogues 23,
25, 27, and 29 all possessed reasonable human and rat PPB
values (%bound = 95−98% across both species) while also
maintaining high apparent permeability (Papp(A→B) = 16−27 ×
10−6 cm/s) with minimal efflux (0.5 < (Papp(A→B)/Papp(B→A)) < 2)
in the MDCK assay. The kinetic aqueous solubility values of
23, 25, 27, and 29 at pH 7.4 (20−43 μM) were also compara-
ble to 31 (21 μM). Compound 26, the stereochemical molec-
ular matched pair of 27, also possessed reasonable human and
rat PPB values (%bound = 96% in both species), favorable ap-
parent permeability (Papp(A→B) = 23 × 10−6 cm/s), and suitable
aqueous solubility (34 μM). Thus, the δ-sultam subseries
possessed favorable in vitro properties regardless of the stereo-
chemistry at the 3-methyl or 6-phenyl substituents.
The (6R)-phenyl δ-sultam analogues 23, 25, 27, and 29, all
of which possessed favorable in vitro CLhep values, were pro-
gressed into single dose rat experiments to assess their in vivo PK
profiles (Table 4). These δ-sultam analogues all possessed low-to-
moderate in vivo clearance values (CLp = 12−19 mL/min/kg),
moderate volumes of distribution (Vd = 2.0−3.3 L/kg), and
reasonable oral bioavailability values (F% = 36−99%). Overall,
there was a very favorable in vitro/in vivo correlation (IVIVc)
with these four analogues, with only 2-fold variability between
the in vitro RLM CLhep (Table 1) and rat in vivo CLp values.
Compound 27 possessed the most favorable in vivo profile in
this set of analogues with low clearance (CLp = 12 mL/min/kg),
a modest volume of distribution (Vd = 3.3 L/kg), and reasonable
oral bioavailability (F% = 55%). We also found that 27 did not
inhibit the major CYP isoforms in vitro up to compound
concentrations of 10 μM.42
d
d
36
a
See the Supporting Information for experimental details associated
with each assessment. Data reported are the means from the dosing
cohorts (male Sprague−Dawley rats, n = 3/dose). Dosed at 1.5 mg/kg
po (75/25 solution of DMSO/H2O) and 0.5 mg/kg iv (50/25/25
solution of DMSO/PEG400/saline). Dosed at 1.5 mg/kg po (40/60
suspension of DMSO/MCT) and 0.5 mg/kg iv (28/50/22 solution of
DMSO/PEG400/saline). Dosed at 1.5 mg/kg po (37/63 suspension
of DMSO/MCT) and 0.5 mg/kg iv (25/50/25 solution of DMSO/
b
c
d
e
f
PEG400/saline). Observed plasma clearance (CLp). Volume of
g
h
distribution (Vd). Maximum plasma concentration (Cmax). Oral
bioavailability (F%) was calculated according to the equation: F% =
(dose normalized AUCpo)/(dose normalized AUCiv). CLp, Vd, and t1/2
were derived from an iv study and Cmax, AUC, and F% were derived
from a po study.
reporter assays of human farnesoid X receptor (FXR), liver
X receptor (LXR)-α, LXRβ, and pregnane X receptor (PXR) in
both agonist mode (no agonist ligand added) and antagonist
mode (using T0901317 [N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-
trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzene-
sulfonamide] as an exogenous ligand).33 Compound 31 was a
selective RORc inverse agonist with >130-fold selectivity for
RORc over the other NRs in our cell assay panel (Table 5).
Compound 23 possessed moderate RORc inverse agonist
potency in the cell assay (EC50 = 670 nM), with no notable
activity against any of the other RORs or NRs in the small cell
panel. We were encouraged by this initial outcome with 23 as
the δ-sultam subseries did not impart any additional off-target
activities over that previously observed with the tertiary
sulfonamide series (e.g., 31).
This initial observation was confirmed with compounds 25
(RORc EC50 = 46 nM) and 27 (RORc EC50 = 47 nM), as both
compounds displayed 75-fold selectivity for RORc over the
other ROR isoforms and >200-fold selectivity over the other
NRs in the cell assay panel. Compound 29, which was less
potent in the RORc biochemical assay than 25 and 27,
displayed modest RORc cellular potency (EC50 = 480 nM).
None of these compounds displayed agonist mode activity
against the NRs in the small cell assay panel at concentrations
up to 10 μM. Compound 27 was also profiled against a larger
cell assay panel of 21 additional NRs,43 where it displayed no
dose-dependent agonist or antagonist activity against the NRs
at concentrations up to 10 μM, further demonstrating the high
selectivity of 27 for RORc (>200-fold).
We profiled 31 and the (6R)-phenyl δ-sultam analogues 23,
25, 26, 27, and 29 in a series of HEK-293 cell GAL4-ROR-LBD
construct transcriptional reporter assays (Table 5). We profiled
the three isoforms of human ROR (RORc, RORb, and RORa)
by monitoring the suppression of their basal transcriptional
activity in the absence of any exogenous agonist.33 To assess
the NR cellular selectivity of the potent RORc inverse agonists,
we also tested these compounds in a small panel of cellular
F
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a
Table 5. RORc Potency and Selectivity Profiles in GAL4 Human Transcription Assays
RORc cell
compd EC50 (μM) [%max]
RORb cell
RORa cell
FXR cell
LXRα cell
LXRβ cell
PXR cell
EC50 (μM) [%max] EC50 (μM) [%max] EC50 (μM) [%max] EC50 (μM) [%max] EC50 (μM) [%max] EC50 (μM) [%max]
b
b
b
31
23
25
26
27
29
0.073 [97]
0.67 [93]
0.046 [96]
0.11 [94]
0.047 [99]
0.48 [97]
16.7 [64]
>20 [43]
>10 [−6]
>10 [37]
>10 [−10]
>10 [4]
>10 [3]
>10 [−10]
>10 [13]
>10 [0]
>10 [−70]
>10 [34]
>10 [51]
9.9 [55]
0.092 [94]
3.5 [83]
3.3 [86]
>10 [44]
>10 [25]
6.1 [75]
5.4 [74]
7.3 [69]
>10 [−30]
>10 [−20]
>10 [−10]
>10 [−20]
>10 [−20]
>10 [8]
>10 [1]
>10 [−40]
>10 [15]
>10 [9]
>10 [−10]
>10 [−5]
>10 [22]
>10 [−150]
a
All assays were conducted in HEK293 cells transiently transfected with GAL4-NR-luciferase plasmids. These NR assays monitored the suppression
of basal transcriptional activities (e.g., no agonist ligand was applied, only test compound was added), an outcome consistent with inverse agonist
activity of the ligands.33 All EC50 values are reported as means (all standard deviations were <54% of the mean EC50 values) for ≥4 separate titrations
for the RORs and ≥2 separate titrations for the LXRs, FXR, and PXR; the maximum percent inhibition “%max” was observed at the highest test
concentration of 10 μM, unless otherwise noted, and the %max values are reported as means (all standard deviations were <17% of the mean %max
values). In this table, positive %max indicates suppression of basal reporter signal, whereas negative %max denotes increased transcription relative to
b
DMSO-treated cells. For this compound, the %max represents inhibition at the highest test concentration of 20 μM.
a
Table 6. Potency in Human IL-17 and IFNγ Production Assays
compd
IL-17AA EC50 (μM) [%max]
IFNγ EC50 (μM) [%max]
CTG EC50 (μM) [%max]
31
25
27
0.36 [79]
0.79 [69]
0.45 [82]
>20 [45]
>20 [6]
>20 [18]
>20 [1]
>20 [45]
>20 [23]
a
All assay EC50 values are reported as means (all standard deviations were <77% of the mean EC50 values) for ≥3 separate titrations; the maximum
percent inhibition “%max” was observed at the highest test concentration of 20 μM and are reported as means (all standard deviations were <10% of
the mean %max value). All assays were conducted using peripheral blood mononuclear cells (PBMCs) isolated from human whole blood.33
Interferon gamma (IFNγ) and CellTiter-Glo (CTG) readouts were obtained to monitor for inhibition of non-TH17 cell cytokine production as well
as adverse off-target effects on cell physiology, respectively.33
Compound 26 was profiled in our NR cell assay panel
(Table 5), and it possessed moderate RORc and RORb inverse
Before assessing whether our molecules possessed favorable
activity in vivo, we established in vitro murine cell assays to
complement the human cell data we collected. Human NR1F3
(RORc) and murine NR1F3 (RORγ) exist in two distinct splice
variants: RORc1/RORc2 for humans and RORγ1/RORγt for
mice.1,19,46,47 The splice variants within each species differ only
in the lengths of their N-terminal sequences. The LBDs of the
slice variants within each species share identical sequence
homology. Our δ-sultam RORc inverse agonists cannot discern
one RORc splice variant over another (e.g., RORc1 vs RORc2)
because they were designed to target the human RORc-LBD.
Murine RORγ and human RORc share an 88% sequence
homology.48 Because of the sequence difference between
human and murine NR1F3, we assessed our potent and
selective human RORc inverse agonist 27 in murine in vitro
IL-17 assays. This approach ensured that 27 could adequately
suppress the production of IL-17 in mouse cells prior to pro-
gressing into murine in vivo models.
Because 27 provided an appealing profile in the human
PBMC assay, we explored its ability to inhibit IL-17 production
in various murine cell-based assays. To do so, we generated
mouse TH17 cells from female C57BL/6J mice using two
different conditions: (1) a mixture of IL-1β, IL-6, and IL-23,
and (2) a combination of transforming growth factor (TGF)-β
and IL-6. In the presence of IL-1β, IL-6, and IL-23, murine
TH17 cells are defined as “pathogenic” cells.49 In addition to
producing IL-17, pathogenic TH17 cells also produce IFNγ and
are involved in autoimmunity and inflammation. In the pres-
ence of the TGF-β and IL-6 conditions, murine TH17 cells
coexpress IL-10 and are defined as “suppressive” cells.49
Pathogenic and suppressive TH17 cells produce three IL-17
subtypes: IL-17AA, -AF, and -FF, which all signal through the
same receptor (IL-17RA·IL-17RC). As shown in Table 7,
murine CD4+ T cells produce IL-17AF at a higher concen-
tration than IL-17AA or -FF, regardless of the stimulus. CD4+
agonist activities (RORc EC50 = 110 nM, RORb EC50
=
92 nM).44 To our knowledge, this is the first reported ligand
with nearly equivalent RORb and RORc inverse agonist cellular
potencies.45 The selectivity profile of 26 was a stark contrast to
the highly selective RORc inverse agonist profile of 27, given
that these two compounds are nearly identical except for their
differing stereochemistry at the 6-phenyl group on the δ-sultam
ring. It is our hypothesis that the conformation of the (6S)-
phenyl δ-sultam ring in 26 may play a role in its enhanced
RORb activity, but we do not have any crystallographic
evidence to support this theory.
On the basis of their favorable RORc cell potency and
selectivity values, compounds 31, 25, and 27 were progressed
into human PBMC cytokine production assays33 to assess their
abilities to inhibit the T cell receptor-dependent production of
IL-17 (Table 6). The IL-17 family contains six members, IL17-
A, -B, -C, -D, -E, and -F, and these family members can exist as
homodimers and heterodimers.19 The forms of IL-17 most
relevant to T helper (TH)-17-mediated inflammatory diseases
are IL-17AA, -AF, and -FF.19 Therefore, we chose to monitor
the production of IL-17AA in our human PBMC assay. Com-
pounds 31, 25, and 27 displayed modest inhibition of IL-17AA
production in the human PBMC assay (EC50 = 350, 650, and
370 nM, respectively). It is also noteworthy that none of the
compounds showed any activity in the interferon (IFN)-γ or
CellTiter-Glo (CTG) assays, demonstrating that the com-
pounds were not indiscriminately suppressing cytokine produc-
tion nor were they grossly cytotoxic. We were encouraged that
25 and 27 displayed comparable human PBMC potencies to
31, further demonstrating that the δ-sultam subseries could
achieve similar potency to the tertiary sulfonamide series of
RORc inverse agonists.23,25,26
G
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Journal of Medicinal Chemistry
Article
a
Table 7. Murine IL-17 Production Assay Results with 27
CD4+ T cells
splenocytes
IL-1β/IL-6/IL-23 stimulus
IL-1β/IL-6/IL-23 stimulus
TGF-β/IL-6 stimulus
IL-17 AA
IL-17 AF
IL-17 FF
4500
IL-17 AA
16000
IL-17 AF
IL-17 FF
IL-17 AA
IL-17 AF
IL-17 FF
ND
b
max IL-17 production (ng/mL)
8500
0.45
87
49000
0.95
85
36000
0.53
96
2600
0.27
90
870
1.1
95
3700
3.6
c
EC50 of 27 (μM)
0.47
99
1.8
99
%max inhibition with 27 at 10 μM
94
a
b
See the Supporting Information for the murine IL-17 production assay protocols. ND = not detected. Experiments conducted in the absence of 27
c
to determine the maximum production of murine IL-17 under various conditions. All EC50 values are reported as means (all standard deviations
were <15% of the mean EC50 values) for ≥2 separate titrations using cells from female C57BL/6J mice.
T cells are not the only source of IL-17. In the presence of
IL-1β, IL-6, and IL-23, murine innate lymphoid cells (ILCs)
produce IL-17 as well.50 To further explore the production of
IL-17 by ILCs, we treated female C57BL/6J mouse splenocytes
with a mixture of IL-1β, IL-6, and IL-23 (Table 7). Under these
conditions, IL-17AF was produced at higher concentration than
IL-17AA, and IL-17FF production was undetectable by ELISA.
We were pleased to see that 27 inhibited the production of all
three IL-17 subtypes in murine CD4+ T cells, regardless of
stimulus (EC50 = 0.27−1.8 μM, Table 7). Compound 27 also
inhibited the production of IL-17AA and -AF by ILCs in the
murine splenocyte assay (EC50 = 1.1−3.6 μM).
After observing that 27 inhibited IL-17 production in vitro in
human PBMC and murine cellular assays, its attractive selec-
tivity profile and metabolic stability prompted us to investigate
its ability to suppress IL-17 production in vivo in a PK/PD
model. Although 27 had favorable HLM, RLM, and in vivo
rat PK clearance values, the mouse liver microsome (MLM)
clearance was moderate-to-high (CLhep = 60 mL/min/kg). A
single dose mouse PK study (Table 8) where 27 was orally
was then dosed orally as an MCT suspension at 3, 10, 30, 100,
and 200 mg/kg. The study animals were stimulated 1 h later
with iv administration of mouse IL-1β and IL-23 recombinant
protein to spur the production of IL-17.53 Three hours later,
blood was drawn and systemic levels of serum IL-17FF were
measured by cytokine ELISA, and the total plasma concen-
trations of 27 were also determined at this time point. Inhibi-
tion of the IL-17 pathway was observed all for doses of 27
(Figure 2a), with dose-proportional inhibition of IL-17 and a
maximal PD response (89% inhibition) at the 200 mg/kg dose.
Overall, the PK/PD relationship suggested that a significant
attenuation of IL-17FF production could be achieved at
doses of 30, 100, and 200 mg/kg. All doses achieved dose-
proportional increases in exposure as assessed by the total
plasma concentration of 27 at the end of the study (Figure 2b).
Anti-p40 antibody, which neutralizes IL-12 and IL-23 cytokines,
was used as a positive control and resulted in near complete
(>99%) inhibition of IL-17FF production. An isotype control
antigen recognizing ragweed was used as the negative control
in this study, and it matched the inhibition achieved with the
vehicle control (PBS).
Table 8. High Dose Mouse in Vivo Oral PK Properties
of 27
All doses of 27 in the mouse IL-17 PK/PD study resulted in
total plasma concentrations of 27 (Figure 2b) that exceeded the
in vitro IL-17FF EC50 value in the murine CD4+ T cell assay
a
b
c
compd
pretreatment with 1-ABT
AUC (μM·h)
Cmax (μM)
conducted under IL-1β, IL-6, and IL-23 conditions (EC50
=
0.47 μM, Table 7).54 However, 27 only demonstrated sig-
nificant inhibition of IL-17FF production in the PK/PD study
at total plasma concentrations ≥8 μM (e.g., 30, 100, and
200 mg/kg doses, Figure 2a). The total plasma concentration of
≥8 μM corresponded to a free-drug concentration of ≥0.32 μM
in mice. This result suggested that a total drug plasma con-
centration in excess of the murine CD4+ T cell EC50 assay value
may be required to demonstrate in vivo inhibition of IL-17FF
production. This hypothesis correlates with a total plasma
concentration coverage of the EC90 value of 27 in the murine
CD4+ T cell assay conducted under IL-1β, IL-6, and IL-23
conditions (EC90 = 7 μM), as was achieved at the 30, 100, and
200 mg/kg doses in the IL-17 PK/PD study (Figure 2b). In
sum, these results suggest that inhibition of murine IL-17FF
production in vivo may require a total plasma drug concen-
tration that covers the in vitro murine IL-17FF production
EC90 assay value. It is unknown if this same relationship will
hold true for the in vivo suppression of human IL-17
production with selective RORc inverse agonists.
27
27
no
58.7
371
12.3
32.8
yes
a
Compound dosed at 100 mg/kg po (MCT suspension). See the
Supporting Information for experimental details associated with each
assessment. Data reported are the means from the dosing cohorts
b
(female CD-1 mice, n = 4/dose). Mice treated with 15 mg/kg ip of
c
1-ABT 2 h prior to dosing with compound. Maximum plasma
concentration (Cmax) from a po study.
administered as a 100 mg/kg 0.2% (v/v) methylcellulose
Tween 80 (MCT) suspension resulted in low exposure (AUC =
58.7 μM·h, Cmax = 12.3 μM), further corroborating the MLM
clearance value. Compound 27 exhibited similar PPB values
(Table 3) across human, rat, and mouse (murine %PPB(bound)
=
96%), thus the low exposure in mouse cannot be attributed to
differences in PPB values.51 In an effort to improve the murine
in vivo oral exposure of 27, mice in the PK study were treated
with 1-aminobenzotriazole (1-ABT) 2 h prior to dosing with 27
to disrupt the CYP metabolism of 27.52 Thus, oral administration
of 27 as a 100 mg/kg MCT suspension to mice pretreated
with 1-ABT provided a substantial increase in exposure (AUC =
371 μM·h, Cmax = 32.8 μM). This approach was then applied to a
mouse PK/PD study to increase the exposure of 27.
CONCLUSION
■
In conclusion, we have evolved a previously disclosed tertiary
sulfonamide series of RORc inverse agonists into a related yet
distinct δ-sultam series. The (6R)-phenyl δ-sultam analogues
demonstrated favorable HLM and RLM profiles, a good IVIVc,
For the PK/PD study, mice were treated with 1-ABT 2 h
prior to dosing with 27 (see Supporting Information for the
details of the mouse IL-17 PK/PD experiment). Compound 27
H
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was carried out with prepacked SiO2 cartridges from either ISCO or
SiliCycle on an ISCO CombiFlash chromatography system using
gradient elution. Optical rotation data were recorded on a Rudolph
Research Analytical Autopol V polarimeter. The reported specific
rotations were recorded at the designated temperatures, light source
wavelengths, concentrations (g/100 mL), and in the designated
solvents. NMR spectra were recorded on a Bruker Avance 400, Bruker
DPX 400M, or Bruker Avance III 400 or 500 NMR spectrometers and
internally referenced to SiMe4. The following abbreviations are used:
br = broad signal, s = singlet, d = doublet, dd = doublet of doublets,
t = triplet, q = quartet, and m = multiplet. Preparative HPLC was per-
formed on a Polaris C18 column (50 mm × 21 mm, 5 μm), a Waters
Sunfire OBD Phenomenex Luna Phenyl Hexyl column (150 mm ×
19 mm), or a Waters Xbridge Phenyl column (150 mm × 19 mm),
eluting with mixtures of H2O/CH3CN or H2O/CH3OH, optionally
containing a modifier (0.1% v/v formic acid or 10 mM ammonium
bicarbonate). Stereoisomers of the final products were separated
on a PIC-100 SFC system using CO2/MeOH + 0.1% v/v NH4OH
mobile phases and a Chiralpak AD column (21 mm × 150 mm, 5 μm).
Low-resolution mass spectra were recorded on a Sciex 15 mass
spectrometer in ES+ mode, a Micromass ZQ single quadrapole LC-MS
in ES+, ES− mode, or a Quattro Micro LC-MS-MS in ES+, ES− mode.
All final compounds were purified to >95% chemical and optical
purity, as assayed by either: (a) HPLC (Waters Acquity UPLC column
21 mm × 50 mm, 1.7 μm) with gradient of 0−90% CH3CN (con-
taining 0.04% v/v TFA) in 0.1% v/v aqueous TFA, with UV detection
at λ = 254 and 210 nm as well as CAD detection with an ESA Corona
detector, (b) HPLC (Phenomenex Luna C18 (2) column 4.6 mm ×
100 mm, 5 μm) with gradient of 5−95% CH3CN in H2O (with
0.1% v/v formic acid in each mobile phase), with UV DAD detection
between λ = 210 and 400 nm, (c) HPLC (Waters Xterra MS C18
column 4.6 mm × 100 mm, 5 μm) with gradient of 5−95% CH3CN in
H2O (with 10 mM ammonium bicarbonate in the aqueous mobile
phase), with UV DAD detection between λ = 210 and 400 nm, (d)
HPLC (Supelco, Ascentis Express C18 or Hichrom Halo C18 column
4.6 mm × 150 mm, 2.7 μm) with gradient of 4−100% CH3CN in H2O
(with 0.1% v/v formic acid in each mobile phase), with UV DAD
detection between λ = 210 and 400 nm, or (e) HPLC (Phenomenex,
Gemini NX C18 column 4.6 mm × 150 mm, 3 μm) with gradient of
4.5−100% CH3CN in H2O (with 10 mM ammonium bicarbonate in
the aqueous mobile phase), with UV DAD detection between λ = 210
and 400 nm.
Figure 2. Mouse IL-17 PK/PD results for 27. Six-to-eight week old
female C57BL/6J mice were administered 15 mg/kg of 1-ABT (MCT
suspension) 2 h prior to compound administration. Compound 27
was dosed po (MCT suspension) at five different doses (3, 10, 30, 100,
200 mg/kg) with five mice per dose cohort. One hour after 27 was
dosed, the mice were stimulated with 300 ng of IL-1β and 100 ng of
IL-23. Blood was collected 3 h poststimulation and was analyzed for
the serum IL-17FF concentration by ELISA (means SEM) and the
The synthesis of compound 1 was described previously.25
plasma concentration of 27 (means
SEM). An isotype control
N-(2-Cyanopropan-2-yl)-1-phenylmethanesulfonamide (5). To a
solution of 2-amino-2-methylpropanenitrile (4, 0.12 g, 1.4 mmol) in
DCM (1.5 mL) was added N,N-di-iso-propylethylamine (0.29 mL,
1.7 mmol), followed by phenylmethanesulfonyl chloride (0.32 g,
1.7 mmol). The reaction was stirred at ambient temperature for 16 h.
The mixture was diluted with DCM (10 mL), washed with water
(5 mL) and brine (5 mL), dried over MgSO4, filtered, and purified by
silica gel column chromatography (0−100% EtOAc in heptane) to
provide the title compound (0.15 g, 46% yield). 1H NMR (400 MHz,
CDCl3) δ 7.51−7.43 (m, 2H), 7.43−7.36 (m, 3H), 4.48 (s, 2H), 4.27
(s, 1H), 1.68 (s, 6H). LCMS ES+ m/z = 256 [M + NH4]+.
N-(4-(4-Acetylpiperazin-1-yl)benzyl)-5-phenyl-4-amino-3,3-di-
methyl-2,3-dihydroisothiazole-1,1-dioxide (6). Step 1. To a solution
N-(2-cyanopropan-2-yl)-1-phenylmethanesulfonamide (5, 0.16 g,
0.65 mmol) in DMA (3 mL) was added NaH (60% in mineral oil,
31 mg, 0.78 mmol), and the reaction was stirred at ambient tem-
perature for 30 min. 1-Bromo-4-(bromomethyl)benzene (0.18 g,
0.72 mmol) was added to the reaction and the mixture was stirred at
75 °C for 16 h. The reaction was quenched with water (1 mL), diluted
with EtOAc (10 mL), washed with water (5 mL) and brine (5 mL),
dried over MgSO4, filtered, concentrated, and purified by silica gel
column chromatography (0−100% EtOAc in heptane) to provide the
N-benzylated intermediate (91 mg) that was carried on to the next
step as the crude intermediate. LCMS ES+ m/z = 408 [M + H]+.
Step 2. A vial was charged with the N-benzylated intermediate from
step 1 (91 mg, 0.22 mmol), chloro-(2-dicyclohexylphosphino-2′,6′-di-
iso-propoxy-1,1′-biphenyl)[2-(2-aminoethyl)phenyl]palladium(II)
antigen recognizing ragweed was used as the negative control and anti-
p40 antibody was used as a positive control in the study. A Dunnett’s
test comparison of the IL-1β + IL-23 isotype control animals versus
the animals treated with 27 provided the following statistical p-value:
*p < 0.05.
and potent RORc inverse agonist activity in biochemical and
cellular assays. Conversely, the (6S)-phenyl δ-sultam analogues
demonstrated moderate-to-high CLhep values in HLM and
RLM studies and poor RORc inverse agonist activity. It was
also notable that one of the (6S)-phenyl δ-sultam analogues
(26) exhibited selectivity for RORb and RORc. The most
advanced (6R)-phenyl δ-sultam compound (27) possessed
favorable RORc cellular potencies, with 75-fold selectivity for
RORc over other ROR family members and >200-fold
selectivity over 25 additional nuclear receptors in a cell assay
suite. The favorable potency, selectivity, in vitro ADME
properties, in vivo PK, and dose-dependent inhibition of
IL-17 in a PK/PD model support the evaluation of 27 in
preclinical studies.
EXPERIMENTAL SECTION
Chemistry. General. All chemicals were purchased from
commercial suppliers and used as received. Flash chromatography
■
I
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Journal of Medicinal Chemistry
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methyl-tert-butyl ether adduct (16 mg, 0.022 mmol), 2-dicyclohex-
ylphosphino-2′,6′-di-iso-propoxybiphenyl (11 mg, 0.022 mmol), and
sodium tert-butoxide (33 mg, 0.33 mmol) and purged with nitrogen
gas for 2 min. A solution of N-acetylpiperazine (43 mg, 0.33 mmol)
in 1,4-dioxane (1 mL) was added to the reaction, and the mixture
was stirred at 100 °C for 16 h. The reaction was filtered through
diatomaceous earth, concentrated under reduced pressure, and purified
by reverse-phase HPLC to give provide the title compound (43 mg,
3 steps). 1H NMR (400 MHz, DMSO-d6) δ 7.52−7.25 (m, 5H), 6.78
(s, 1H), 4.03 (dd, J = 12.8, 3.3 Hz, 1H), 2.63−2.52 (m, 1H), 2.15−
2.02 (m, 1H), 1.76−1.67 (m, 2H), 1.36 (s, 3H), 1.21 (s, 3H). LCMS
ES+ m/z = 240 [M + H]+.
1-{4-[4-(1,1-Dioxo-5-phenyl-isothiazolidin-2-ylmethyl)-3-fluoro-
phenyl]-piperazin-1-yl}-ethanone (17). Compound 12 (0.15 g,
0.76 mmol) was subjected to general procedure B to provide the
title compound (53 mg, 16% yield over 2 steps). 1H NMR (400 MHz,
DMSO-d6) δ 7.52−7.35 (m, 5H), 7.26 (t, J = 8.6 Hz, 1H), 6.79 (d, J =
11.0 Hz, 2H), 4.65−4.52 (m, 1H), 4.20 (d, J = 14.5 Hz, 1H), 4.06 (d,
J = 14.4 Hz, 1H), 3.64−3.50 (m, 4H), 3.29−3.08 (m, 6H), 2.63−
2.52 (m, 1H), 2.49−2.42 (m, 1H), 2.04 (s, 3H). LCMS ES+ m/z = 432
[M + H]+.
1
14% yield over 2 steps). H NMR (400 MHz, DMSO-d6) δ 7.49 (d,
J = 7.2 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H), 7.29 (dd, J = 15.9, 8.1 Hz,
3H), 6.92 (d, J = 8.7 Hz, 2H), 6.41 (s, 2H), 4.20 (s, 2H), 3.57 (s, 4H),
3.18−3.11 (m, 2H), 3.11−3.02 (m, 2H), 2.03 (s, 3H), 1.32 (s, 6H).
LCMS ES+ m/z = 455 [M + H]+.
1-{4-[4-(1,1-Dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-3-fluoro-
phenyl]-piperazin-1-yl}-ethanone (18). Compound 13 (0.30 g,
1.4 mmol) was subjected to general procedure B to provide the title
General Procedure A: Formation of Sultam Rings 12−16. The
sultam rings were prepared in a multistep process according to the
literature procedure of Lee et al.30
1
compound (0.39 g, 62% yield over 2 steps). H NMR (400 MHz,
General Procedure B: Formation of Products 17−21. Step 1. The
sultam rings obtained by general procedure A (12−16, 1.0 equiv)
were combined with 4-bromo-1-(bromomethyl)-2-fluorobenzene
(1.1 equiv) and DMF (0.2 M) at 0 °C. Sodium hydride (60% in
mineral oil, 1.2 equiv) was added to the reaction portionwise. The
resultant mixture was stirred at ambient temperature for 2 h. Water
was added, and the reaction was diluted with EtOAc, washed
with brine, dried over MgSO4, filtered, and purified by silica gel
column chromatography (0−60% EtOAc/heptane) to provide the
N-benzylated intermediates to be used directly in the next step.
Step 2. The N-benzylated intermediates obtained in step 1 (1.0 equiv)
were combined with Pd(OAc)2 (0.05 equiv), 2-dicyclohexylphosphine-
2′,6′-di-iso-propoxy-1,1′-biphenyl (0.10 equiv), and Cs2CO3 (1.5 equiv)
in a vial and purged with nitrogen gas for 2 min. 1,4-Dioxane (0.3 M)
and N-acetylpiperazine (1.5 equiv) were added to the vial, and the
reaction was stirred at 80 °C for 2−16 h. The reaction was filtered
through diatomaceous earth, concentrated under reduced pressure, and
purified by reverse-phase HPLC to provide the title compounds.
5-Phenylisothiazolidine 1,1-dioxide (12). 2-Bromoethanamine
hydrobromide (7, 6.4 g, 32 mmol) was subjected to a multistep pro-
cess according to the literature procedure of Lee et al.30 to produce the
DMSO-d6) δ 7.49−7.31 (m, 5H), 7.26 (t, J = 8.8 Hz, 1H), 6.86−6.73
(m, 2H), 4.49 (dd, J = 12.6, 3.2 Hz, 1H), 4.35 (q, J = 14.4 Hz, 2H),
3.63−3.51 (m, 4H), 3.46 (t, J = 12.9 Hz, 1H), 3.28−3.19 (m, 2H),
3.19−3.04 (m, 3H), 2.48−2.35 (m, 1H), 2.22−2.07 (m, 1H), 2.04 (s,
3H), 2.01−1.87 (m, 1H), 1.72−1.49 (m, 1H). LCMS ES+ m/z = 446
[M + H]+.
1-(4-(3-Fluoro-4-(((3R)-3-methyl-1,1-dioxido-6-phenyl-1,2-thia-
zinan-2-yl)methyl)phenyl)piperazin-1-yl)ethanone (19). Compound
14 (0.15 g, 0.67 mmol) was subjected to general procedure B to pro-
duce the title compound (0.23 g, 73% yield over 2 steps). This com-
pound was carried on directly to the next step and not characterized.
1-(4-(3-Fluoro-4-(((3S)-3-methyl-1,1-dioxido-6-phenyl-1,2-thia-
zinan-2-yl)methyl)phenyl)piperazin-1-yl)ethanone (20). Compound
15 (0.68 g, 3.0 mmol) was subjected to general procedure B to
produce the title compound (0.92 g, 67% yield over 2 steps). This
compound was carried on directly to the next step and not
characterized.
1-(4-(4-((3,3-Dimethyl-1,1-dioxido-6-phenyl-1,2-thiazinan-2-yl)-
methyl)-3-fluorophenyl)piperazin-1-yl)ethanone (21). Compound
16 (0.15 g, 0.63 mmol) was subjected to general procedure B to
produce the title compound (0.30 g, 50% yield over 2 steps). This
compound was carried on directly to the next step and not
characterized.
1
title compound (2.6 g, 42% yield over 2 steps). H NMR (300 MHz,
CDCl3) δ 7.45−7.26 (m, 5H), 4.42 (m, 1H), 4.28 (m, 1H), 3.54−3.49
(m, 2H), 2.85−2.72 (m, 2H). LCMS ES+ m/z = 220 [M + Na]+.
6-Phenyl-1,2-thiazinane-1,1-dioxide (13). 3-Bromoproan-1-amine
hydrobromide (8, 2.2 g, 10 mmol) was subjected to a multistep
process according to the literature procedure of Lee et al.30 to produce
1-{4-[4-((S)-1,1-Dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-3-flu-
oro-phenyl]-piperazin-1-yl}-ethanone (22). Compound 18 (0.15 g,
0.34 mmol) was subjected to SFC purification to yield the title
compound (50 mg, 33% yield). The absolute stereochemistry of the
1
23
the title compound (1.2 g, 59% yield over 2 steps). H NMR (300
title compound was assigned by analogy to 23. [α]D −19° (c 0.18,
1
MHz, DMSO-d6) δ 7.40−7.35 (m, 5H), 6.98 (m, 1H), 4.12 (m, 1H),
3.26−3.20 (m, 2H), 2.40−2.30 (m, 1H), 2.16−2.12 (m, 1H), 1.77−
1.65 (m, 2H). LCMS ES+ m/z = 234 [M + Na]+.
CH3OH). H NMR (400 MHz, DMSO-d6) δ 7.53−7.32 (m, 5H),
7.29−7.22 (m, 1H), 6.85−6.80 (m, 1H), 6.80−6.75 (m, 1H), 4.54−
4.44 (m, 1H), 4.38 (d, J = 14.4 Hz, 1H), 4.32 (d, J = 14.3 Hz, 1H),
3.63−3.52 (m, 4H), 3.52−3.40 (m, 1H), 3.28−3.20 (m, 2H), 3.20−
3.04 (m, 3H), 2.47−2.36 (m, 1H), 2.18−2.07 (m, 1H), 2.04 (s, 3H),
2.02−1.91 (m, 1H), 1.71−1.56 (m, 1H). LCMS ES+ m/z = 446
[M + H]+.
(3R)-3-Methyl-6-phenyl-1,2-thiazinane 1,1-dioxide (14). (R)-3-
Aminobutan-1-ol29 (9, 1.0 g, 11 mmol) was subjected to general pro-
cedure A to produce the title compound (0.83 g, 33% yield over
1
3 steps). H NMR (400 MHz, DMSO-d6, reported as a 3:1 mixture
of diastereomers) δ 7.52−7.30 (m, 5H), 7.10 (d, J = 5.9 Hz, 0.25H),
6.79 (d, J = 9.8 Hz, 0.75H), 4.18 (dd, J = 9.8, 3.9 Hz, 0.25H), 4.02 (dd,
J = 12.8, 3.5 Hz, 0.75H), 3.65−3.52 (m, 0.25H), 3.52−3.34 (m,
0.75H), 2.47−2.26 (m, 1H), 2.24−2.04 (m, 1H), 1.90−1.76 (m, 1H),
1.69−1.54 (m, 0.25H), 1.55−1.34 (m, 0.75H), 1.31 (d, J = 7.1 Hz,
0.75H), 1.15 (d, J = 6.6 Hz, 2.25H). LCMS ES+ m/z = 226 [M + H]+.
(3S)-3-Methyl-6-phenyl-1,2-thiazinane 1,1-dioxide (15). (S)-3-
Aminobutan-1-ol29 (10, 7.5 g, 84 mmol) was subjected to general
procedure A to produce the title compound (4.0 g, 21% yield over 3
1-{4-[4-((R)-1,1-Dioxo-6-phenyl-[1,2]thiazinan-2-ylmethyl)-3-fluoro-
phenyl]-piperazin-1-yl}-ethanone (23). Compound 18 (0.15 g,
0.34 mmol) was subjected to SFC purification to yield the title
compound (75 mg, 50% yield). The absolute stereochemistry of the
title compound was assigned by single-crystal X-ray analysis (see
Supporting Information). [α]D +18° (c 0.17, CH3OH). H NMR
(400 MHz, DMSO-d6) δ 7.49−7.32 (m, 5H), 7.30−7.21 (m, 1H),
6.84−6.80 (m, 1H), 6.80−6.75 (m, 1H), 4.55−4.44 (m, 1H), 4.38 (d,
J = 14.2 Hz, 1H), 4.32 (d, J = 14.4 Hz, 1H), 3.60−3.52 (m, 4H), 3.52−
3.40 (m, 1H), 3.26−3.19 (m, 2H), 3.19−3.04 (m, 3H), 2.48−2.36 (m,
1H), 2.18−2.06 (m, 1H), 2.04 (s, 3H), 2.01−1.92 (m, 1H), 1.69−1.57
(m, 1H). LCMS ES+ m/z = 446 [M + H]+.
23
1
1
steps). H NMR (400 MHz, DMSO-d6, reported as a 4:1 mixture of
diastereomers) δ 7.50−7.31 (m, 5H), 7.09 (d, J = 5.5 Hz, 0.2H), 6.78
(d, J = 9.0 Hz, 0.8H), 4.18 (dd, J = 9.8, 4.0 Hz, 0.2H), 4.02 (dd, J =
12.8, 3.5 Hz, 0.8H), 3.64−3.52 (m, 0.2H), 3.49−3.37 (m, 0.8H),
2.46−2.29 (m, 1H), 2.24−2.08 (m, 1H), 1.91−1.76 (m, 1H), 1.66−
1.55 (m, 0.2H), 1.53−1.38 (m, 0.8H), 1.31 (d, J = 7.1 Hz, 0.6H), 1.15
(d, J = 6.6 Hz, 2.4H). LCMS ES+ m/z = 226 [M + H]+.
3,3-Dimethyl-6-phenyl-1,2-thiazinane 1,1-dioxide (16). 3-Amino-
3-methylbutan-1-ol (11) (2.0 g, 19 mmol) was subjected to general
procedure A to produce the title compound (1.5 g, 33% yield over
1-{4-[3-Fluoro-4-((3R,6S)-3-methyl-1,1-dioxo-6-phenyl-[1,2]-
thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (24). Com-
pound 19 (0.22 g, 0.48 mmol) was subjected to SFC purification to
yield the title compound (53 mg, 24% yield). The absolute stereo-
chemistry was assigned by NMR analysis.31 [α]D +28° (c 0.18,
23
CH3OH). 1H NMR (400 MHz, DMSO-d6) δ 7.49−7.43 (m, 2H), 7.43−
7.29 (m, 4H), 6.81 (dd, J = 8.6, 2.1 Hz, 1H), 6.74 (d, J = 13.9 Hz, 1H),
J
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4.47 (dd, J = 12.0, 3.0 Hz, 1H), 4.41 (d, J = 16.9 Hz, 1H), 4.28 (d, J =
17.1 Hz, 1H), 4.15−4.03 (m, 1H), 3.59−3.52 (m, 4H), 3.22−3.15 (m,
2H), 3.15−3.08 (m, 2H), 2.46−2.35 (m, 1H), 2.13−2.05 (m, 1H),
2.03 (s, 3H), 1.89−1.73 (m, 1H), 1.69−1.60 (m, 1H), 1.08 (d, J =
6.9 Hz, 3H). LCMS ES+ m/z = 460 [M + H]+.
ligand were mixed in coregulator buffer D (Invitrogen PV4420)
containing 5 mM DTT and added to the plate at twice their final
concentrations in a volume of 8 μL. Test ligands at 2× the final
concentration were then added to the wells in 8 μL of coregulator
buffer D containing 5 mM DTT and 4% DMSO. Final incubations
contained 1× coregulator buffer D, 5 mM DTT, test ligand, 2%
DMSO, 50 nM biotinyl-CPSSHSSLTERKHKILHRLLQEGSPS
(American Peptide Company; Vista, CA), 2 nM europium anti-GST
(Cisbio 61GSTKLB), 12.5 nM streptavidin-D2 (Cisbio 610SADAB),
50 mM KF, and 10 nM of bacterially expressed human RORc ligand
binding domain protein containing an N-terminal 6xHis-GST-tag and
residues 241−486 of accession NP_001001523. Ten test ligand
concentrations were tested in duplicate. After the reaction plates were
incubated for 3 h in the dark at room temperature (22−23 °C), the
plate was read on an EnVision plate reader (PerkinElmer) following
the europium/D2 HTRF protocol (ex 320, em 615 and 665, 100 μs
lag time, 100 flashes, 500 μs window). The time-resolved FRET signal
at 665 nm was divided by that at 615 nm to generate the signal ratio of
each well. The signal ratio of wells containing RORc and peptide, but
no test ligand were averaged and set to 0% effect while the signal ratios
of the blank wells containing coactivator peptide but no RORc are
averaged and set to −100% effect.
1-{4-[3-Fluoro-4-((3R,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]-
thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (25). Com-
pound 19 (0.22 g, 0.48 mmol) was subjected to SFC purification to
yield the title compound (23 mg, 10% yield). The absolute stereo-
chemistry was assigned by NMR analysis.31 [α]D +50° (c 0.18,
23
1
CH3OH). H NMR (400 MHz, DMSO-d6) δ 7.48−7.43 (m, 2H),
7.43−7.33 (m, 3H), 7.29 (t, J = 8.9 Hz, 1H), 6.85−6.77 (m, 2H), 4.40
(dd, J = 12.4, 3.2 Hz, 1H), 4.37 (s, 2H), 3.60−3.51 (m, 5H), 3.26−
3.19 (m, 2H), 3.19−3.13 (m, 2H), 2.79−2.64 (m, 1H), 2.15−1.99 (m,
5H), 1.66−1.57 (m, 1H), 1.34 (d, J = 7.2 Hz, 3H). LCMS ES+ m/z =
460 [M + H]+.
1-{4-[3-Fluoro-4-((3S,6S)-3-methyl-1,1-dioxo-6-phenyl-[1,2]-
thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (26). Com-
pound 20 (0.21 g, 0.46 mmol) was subjected to SFC purification to
yield the title compound (22 mg, 10% yield). The absolute stereo-
chemistry was assigned by NMR analysis.31 [α]D −45° (c 0.17,
23
1
CH3OH). H NMR (400 MHz, DMSO-d6) δ 7.49−7.43 (m, 2H),
RORc exhibits a basal (constitutive) signal in this assay and test
ligands can increase or decrease the signal ratio from this basal level.
RORc agonists increase the signal ratio in this assay, resulting in a
positive % effect value. Inverse agonists decrease the signal ratio,
resulting in a negative % effect value. The EC50 value was the
concentration of test compound that provided half-maximal effect
(increased or decreased assay signal) and was calculated by Genedata
Screener software (Genedata; Basel, Switzerland) using the following
equation
7.43−7.34 (m, 3H), 7.29 (t, J = 8.9 Hz, 1H), 6.87−6.74 (m, 2H), 4.40
(dd, J = 12.4, 3.2 Hz, 1H), 4.37 (s, 2H), 3.60−3.48 (m, 5H), 3.25−
3.18 (m, 2H), 3.18−3.11 (m, 2H), 2.81−2.61 (m, 1H), 2.17−1.95 (m,
5H), 1.67−1.53 (m, 1H), 1.34 (d, J = 7.1 Hz, 3H). LCMS ES+ m/z =
460 [M + H]+.
1-{4-[3-Fluoro-4-((3S,6R)-3-methyl-1,1-dioxo-6-phenyl-[1,2]-
thiazinan-2-ylmethyl)-phenyl]-piperazin-1-yl}-ethanone (27). Com-
pound 20 (0.21 g, 0.46 mmol) was subjected to SFC purification to
yield the title compound (50 mg, 24% yield). The absolute stereo-
chemistry was assigned by NMR analysis31 and further confirmed by
23
c
n
% effect = S0 + {(Sinf − S0)/[1 + (10log EC /10 ) ]}
50
single-crystal X-ray analysis (see Supporting Information). [α]D
(1)
1
−22° (c 0.17, CH3OH). H NMR (400 MHz, DMSO-d6) δ 7.49−
7.43 (m, 2H), 7.43−7.28 (m, 4H), 6.81 (dd, J = 8.6, 2.1 Hz, 1H), 6.74
(d, J = 13.9 Hz, 1H), 4.48 (dd, J = 12.0, 3.0 Hz, 1H), 4.41 (d, J = 16.9
Hz, 1H), 4.28 (d, J = 17.1 Hz, 1H), 4.18−4.00 (m, 1H), 3.63−3.51
(m, 4H), 3.26−3.15 (m, 2H), 3.15−3.02 (m, 2H), 2.48−2.39 (m, 1H),
2.15−2.05 (m, 1H), 2.03 (s, 3H), 1.92−1.71 (m, 1H), 1.71−1.60 (m,
1H), 1.08 (d, J = 6.8 Hz, 3H). LCMS ES+ m/z = 460 [M + H]+.
(S)-1-(4-(4-((3,3-Dimethyl-1,1-dioxido-6-phenyl-1,2-thiazinan-2-
yl)methyl)-3-fluorophenyl)piperazin-1-yl)ethanone (28). Compound
21 (0.15 g, 0.31 mmol) was subjected to SFC purification to yield the
title compound (57 mg, 38% yield). The stereochemistry of the
6-phenyl substituent was assigned based on the relative specific optical
rotation, biochemical potency, and metabolic stability of the title
where S0 equals the activity level at zero concentration of test
compound, Sinf is the activity level at infinite concentration of test
compound, EC50 is the concentration at which the activity reaches
50% of the maximal effect, c is the concentration in logarithmic units
corresponding to the values on the x-axis of the dose−response curve
plot, and n is the Hill coefficient (the slope of the curve at the EC50).
Cellular Nuclear Receptor (NR) Transcriptional Reporter Assays.
Assays were carried out in 30 μL reaction volumes in black 384-well
tissue culture-treated viewplates (Perkin-Elmer 6007460). Ten test
ligand concentrations were tested in duplicate. The reporter assays for
RORa, RORb, and RORc were carried out in the absence of any
control agonist ligand (inverse agonist mode). The assays for the other
NRs were run in the absence (agonist mode) and presence (antagonist
mode) of T0901317 as a control agonist ligand. HEK293T cells were
cultured in complete medium [DMEM (Gibco 31966) supplemented
with 10% heat-inactivated fetal bovine serum (PAA Laboratories
A15-252)]. On the day of the assay, the cells were transiently
cotransfected in bulk, using 2.5 μL of Lipofectamine 2000 (Invitrogen
11668-019) per μg of DNA, with an expression vector for a GAL4
fusion protein of a human NR (FXR, LXRα, LXRβ, PXR, RORa,
RORb, or RORc; 40 ng/well) subcloned into either pLenti
(Invitrogen) or pFastBacMam,56 a firefly luciferase expression vector
under control of the yeast GAL4 promoter (pFrLuc-GAL4UAS,
Stratagene; 40 ng/well), and a pcDNA3.1 (Invitrogen) expression
vector into which the Renilla luciferase sequence had been subcloned
to generate a vector, pcDNA3.1Ren.Luc (4 ng/well). Then 20 μL of
transfected cells (20000 cells total) were plated per well into 384-well
viewplates, and the plates were placed in a cell culture incubator. Five
hours later, test ligand at 3× the final concentration was added in a
10 μL volume of complete medium containing 0.9% DMSO. The
plates were kept for another 20 h in the cell culture incubator before
the luciferase activity was measured using the Dual-Glo luciferase kit
(Promega E2940). At that time, the plates were allowed to equilibrate
to room temperature before 30 μL of firefly luciferase reagent was
added to each well. Plates were shaken for 15 min before measuring
the firefly luciferase signal on an EnVision plate reader using the
23
compound in relation to stereoisomers 22−27. [α]D −89° (c 0.18,
1
CH3OH). H NMR (400 MHz, DMSO-d6) δ 7.54−7.35 (m, 6H),
6.86−6.78 (m, 1H), 6.78−6.66 (m, 1H), 4.58−4.47 (m, 1H), 4.43 (d,
J = 17.4 Hz, 1H), 4.19 (d, J = 17.5 Hz, 1H), 3.61−3.51 (m, 4H), 3.24−
3.16 (m, 2H), 3.16−3.07 (m, 2H), 2.77−2.58 (m, 1H), 2.17−2.07 (m,
1H), 2.04 (s, 3H), 2.01−1.92 (m, 1H), 1.86−1.75 (m, 1H), 1.42 (s,
3H), 1.14 (s, 3H). LCMS ES+ m/z = 474 [M + H]+.
(R)-1-(4-(4-((3,3-Dimethyl-1,1-dioxido-6-phenyl-1,2-thiazinan-2-
yl)methyl)-3-fluorophenyl)piperazin-1-yl)ethanone (29). Compound
21 (0.15 g, 0.31 mmol) was subjected to SFC purification to yield the
title compound (56 mg, 37% yield). The stereochemistry of the
6-phenyl substituent was assigned based on the relative specific optical
rotation, biochemical potency, and metabolic stability of the title
23
compound in relation to stereoisomers 22−27. [α]D +85° (c 0.18,
1
CH3OH). H NMR (400 MHz, DMSO-d6) δ 7.53−7.34 (m, 6H),
6.86−6.77 (m, 1H), 6.77−6.66 (m, 1H), 4.57−4.48 (m, 1H), 4.43 (d,
J = 17.5 Hz, 1H), 4.19 (d, J = 17.5 Hz, 1H), 3.61−3.51 (m, 4H), 3.23−
3.16 (m, 2H), 3.16−3.07 (m, 2H), 2.77−2.56 (m, 1H), 2.20−2.06 (m,
1H), 2.04 (s, 3H), 2.01−1.92 (m, 1H), 1.88−1.73 (m, 1H), 1.42 (s,
3H), 1.14 (s, 3H). LCMS ES+ m/z = 474 [M + H]+.
The syntheses of compounds 3025 and 3123 were described previously.
Biological Assay Protocols.33 RORc SRC1 Biochemical Assay.
Assays were carried out in 16 μL reaction volumes in black 384 Plus F
Proxiplates (Perkin-Elmer 6008269). All assay components except test
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ultrasensitive luminescence protocol. Finally, 30 μL of Stop & Glo
reagent was added per well to quench the firefly luciferase signal and
allow detection of the Renilla luciferase signal.
temperature. Blood was then diluted in an equal volume of phosphate-
buffered saline (PBS) and 35 mL of the resultant mixture was added to
each Leucosep tube. Tubes were centrifuged at 930g for 20 min at
room temperature, brake off. After centrifugation, the PBMCs were
harvested and washed twice in a total volume of 50 mL of Roswell
Park Memorial Institute (RPMI) medium and then centrifuged at 524g
for 7 min at room temperature, brake on. PBMCs were suspended in
assay medium [Yssel’s T cell medium57,58 (Gemini Bioproducts
400-102) plus 10% human serum (Gemini Bioproducts 100-512)] to
1.25 million cells per mL. On the day before the assay, 50 μL of a
10 μg/mL solution of anti-CD3 (BD Biosciences 555329) in PBS was
added to test wells and stimulated control wells of a 96-well white/
clear bottom polypropylene plate (Corning 3610). Wells to be used
for nonstimulated controls received 50 μL of PBS only. Plates were
incubated overnight at 4 °C. On the day of the assay, the anti-CD3-
coated plates were washed 3 times with 200 μL of PBS using a Biotek
ELx405 automated plate washer. Immediately after plate washing,
160 μL of isolated PBMCs were added to the central wells of the plate;
the perimeter wells received 200 μL of assay medium to reduce
evaporation. Next, test ligands at 10× their final concentration were
added to the plate in 20 μL of RPMI containing 2% DMSO, whereas
nonstimulated and stimulated control wells received only RPMI plus
2% DMSO. Finally, 20 μL of 10 μg/mL anti-CD28 (BD Biosciences
555725) in assay medium were added to test and stimulated control
wells. Nonstimulated control wells received 20 μL of assay medium.
Plates were placed in a humidified cell culture incubator. After 48 h,
the plates were centrifuged at 524g for 5 min at room temperature.
The supernatant fluids (100 μL per well) were transferred to a
96-well V-bottom polypropylene plate (Greiner 655201) and stored at
−80 °C. To assess cell viability, 100 μL of CellTiter-Glo reagent
(Promega G7572) was then added to the original cell plates, which
were incubated for 10 min on a plate shaker. The assay signal in
relative luminescence (RLU) was measured on an EnVision plate
reader with a 0.1 s read time. The mean RLU values of the perimeter
blank wells defined 0% of control, while the mean values from
stimulated control wells defined 100% of control. The % of control
values for wells containing test ligand were calculated using the RLU
data and
Inverse Agonist Format. Transient transfection of RORa, RORb,
and RORc generates a GAL4 reporter signal in the absence of any
added control agonist ligand. This constitutive reporter signal from
transfected cells incubated with complete medium containing 0.3%
DMSO constituted the uninhibited 0% effect and was compared with
cells that were not transfected which defined the −100% effect. The
percent inhibition was calculated with respect to these limits and the
data were fit to eq 1 to generate EC50 values. Compounds that
decreased firefly luciferase expression below the constitutive signal
were potential inverse agonists.
Agonist Format. Activation of the LXRα, LXRβ, FXR, and PXR
reporter signal in transfected cells was measured by an increase in
firefly luciferase expression induced by test compounds. Complete
medium containing 3 μM of T0901317 [N-(2,2,2-trifluoroethyl)-N-[4-
[2,2,2-trifluoro-1-hydroxy-1-(trifluoromethyl)ethyl]phenyl]-benzene-
sulfonamide] in 0.3% DMSO defined the 100% effect activation level,
while the basal activity in the absence of T0901317 set the 0% effect
level. The % effect values were calculated with respect to these limits,
and EC50 values were calculated by fitting the % effect titration data to
% of control = [(test ligand cpm − NSB cpm)
/(TB cpm − NSB cpm)]× 100
(2)
The concentration of test ligand that inhibited binding by 50% (e.g.,
the IC50) was calculated using XLfit by plotting the % of control versus
test ligand concentration and fitting the data to a sigmoidal inhibition
curve equation
% of control = 100/[1 + ([I]/IC50)slope
]
(3)
where [I] was the test ligand concentration. Compounds that in-
creased firefly luciferase expression above the basal level were potential
agonists of that NR. The fold activation by test compounds was
calculated from the raw assay signal by dividing the relative
luminescence units (RLU) value in the presence of test compound
by the RLU in the absence of test compound.
Antagonist Format. Inhibition of agonist-induced activation of
LXRα, LXRβ, FXR, and PXR was measured by a reduction in firefly
luciferase expression in transfected cells by test compounds. Test
compounds were added in the presence of a stimulus (EC80
concentration of T0901317) in complete medium containing 0.3%
DMSO final concentration. The EC80 stimulus concentrations were
1.5, 0.75, 3, and 0.5 μM T0901317 for LXRα, LXRβ, FXR, and PXR,
respectively, and defined the 100% effect level in this assay format. The
basal signal in the absence of T0901317 defined the 0% effect level.
The percent inhibition was calculated with respect to these limits and
IC50 values were determined by fitting the data to eq 1. Compounds
that decreased firefly luciferase expression below the T0901317-
stimulated level were potential antagonists of that NR.
Nonspecific Activity. In addition to measuring the firefly luciferase
signal due to the transfected NR, the potential impact of off-target
activity was also measured in the same wells by monitoring the effect
of test compounds on Renilla luciferase expression. Complete medium
containing 0.3% DMSO defined the constitutive expression 100%
effect signal level. The percent inhibition was calculated with respect
to this control signal. Cells that have not been transfected showed
negligible signal in this assay. Compounds that decreased Renilla
luciferase expression below the 100% effect signal level may have
potential off-target activities that could be responsible for nonspecific
inhibition or activation of the firefly reporter signal. EC50 values were
calculated using eq 1.
% of control = [(test ligand − blank)/(stimulated − blank)]
(4)
× 100
EC50 values (concentration of test ligand that provides half-maximal
inhibition) were calculated using eq 1.
Cytokine ELISAs. Sandwich ELISAs for IL-17 (R&D Systems
DY317) and IFNγ (R&D Systems DY285) were performed on the
thawed supernatants in 96-well half area clear polystyrene plates
(Costar 3690) following the vendor’s protocols. On the day before the
ELISAs, 25 μL of capture antibody diluted 180× in PBS was added to
the ELISA plates. The plates were incubated overnight at room
temperature. On the day of the assay, the plates were washed 3 times
on a Biotek ELx405 automated plate washer with 200 μL of wash
buffer (PBS + 0.05% Tween-20). Immediately after washing, the plates
were blocked with ELISA buffer (PBS + 1% BSA) and incubated for
1 h at room temperature on a plate shaker. During this blocking step,
frozen supernatant samples were thawed for 1 h at room temperature.
After incubation, ELISA plates were washed as before. Next, 25 μL of
supernatant samples were diluted in an equal volume of ELISA buffer,
while 25 μL of buffer blanks and 25 μL of serial dilutions of the
respective cytokine standard prepared in ELISA buffer were added
to their designated wells on the plate. Plates were incubated for 2 h at
room temperature or overnight at 4 °C on a plate shaker. After
incubation, plates were washed as before. Next, 25 μL of detection
antibody diluted 180× in ELISA buffer was added to all wells of the
plate and incubated for 1 h at room temperature on a plate shaker.
Plates were washed and then 25 μL of streptavidin-HRP diluted 200×
in ELISA buffer was added to all wells of the plate. Plates were
incubated for 20 min at room temperature on a plate shaker. Following
incubation, plates were again washed. Then 25 μL of 3,3′,5,5′-
tetramethylbenzidine liquid substrate (Sigma T4444) was added to the
Human Peripheral Blood Mononuclear Cell (PBMC) IL-17, IFNγ,
and Viability Assays. On the day of the assay, PBMCs were isolated
from heparin-treated human whole blood obtained from the Genentech
Research Employee Donation Program. In each assay run, blood from
two or more donors was processed in parallel. First, 15 mL of Ficoll-
Paque (GE Healthcare 17-1440-03) was added to Leucosep tubes
(Greiner 227290) and centrifuged at 524g for 5 min at room
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plates. The plates were incubated for 15 min on a plate shaker. Finally,
25 μL of 1 M phosphoric acid stop solution was added to all wells of
the plate. The absorption at 450 nm was read using a Molecular
Devices SPECTRAmax PLUS384 plate reader. The mean absorption
values for nonstimulated controls defined 0% of control, while the
mean values from stimulated controls wells defined 100% of control.
The % of control in each test compound well was calculated using
eq 4, and EC50 values were calculated using eq 1.
binding; PXR, pregnane X receptor; RA, rheumatoid arthritis;
RAR, retinoic acid receptor; RLM, rat liver microsomes; RLU,
relative luminescence units; ROR, retinoic acid receptor-related
orphan receptor; RPMI, Roswell Park Memorial Institute;
RuPhos, 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl;
RXR, retinoid X receptor; SEM, standard error of the mean;
SFC, supercritical fluid chromatography; SRC, steroid receptor
coactivator; t1/2, half-life; TH, T helper cell; TGF, transforming
growth factor; TR, thyroid receptor; UDPGA, uridine 5′-
diphospho-glucuronosyltransferase; Vd, volume of distribution;
VDR, vitamin D receptor
ASSOCIATED CONTENT
* Supporting Information
■
S
Additional NMR data, assay protocols, and X-ray data (PDF,
CSV) can be found in the Supporting Information. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.5b00597.
REFERENCES
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PDB 4WQP was discussed in this manuscript.
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AUTHOR INFORMATION
Corresponding Author
*Phone: +1-650-467-5773. Fax: +1-650-225-2061. E-mail:
Fauber.Benjamin@gene.com.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Drs. Krista Bowman and Jiansheng Wu, and their
respective Genentech research groups, for performing all
required protein expression and purification activities. We
gratefully acknowledge Yanzhou Liu for NMR support and
Antonio G. DiPasquale for solving the crystal structures of 23
and 27, along with the support of the US NIH Shared
Instrumentation grant S10-RR027172 for the purchase of the
diffractometer used in this research.
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ABBREVIATIONS USED
■
1-ABT, 1-aminobenzotriazole; ADME, adsorption, distribution,
metabolism, and excretion; AR, androgen receptor; AUC, area
under the curve; BSA, bovine serum albumin; CAR,
constitutive androstane receptor; CD, cluster of differentiation;
CLhep, predicted hepatic clearance; CLp, observed plasma
clearance; cLogP, calculated Log P; Cmax, maximum plasma
concentration; CTG, CellTiter-Glo; CYP, cytochrome P450;
DTT, dithiothreitol; ELISA, enzyme-linked immunosorbent
assay; ER, estrogen receptor; F%, oral bioavailability; FRET,
fluorescence resonance energy transfer; FXR, farnesoid X
receptor; GM-CSF, granulocyte-macrophage colony stimulating
factor; GR, glucocorticoid receptor; GSH, glutathione; GST,
glutathione-S-transferase; HLM, human liver microsomes;
HRP, horseradish peroxidase; HTRF, homogeneous time-
resolved fluorescence; IL, interleukin; ILCs, innate lymphoid
cells; IFN, interferon; ip, intraperitoneal; iv, intravenous; IVIVc,
in vitro to in vivo correlation; LBD, ligand binding domain;
LLE, ligand-lipophilicity efficiency; LXR, liver X receptor;
MCT, methylcellulose and Tween; MDCK, Madin−Darby
canine kidney; MetID, metabolite identification; MLM, mouse
liver microsomes; MR, mineralocorticoid receptor; NADPH,
nicotinamide adenine dinucleotide phosphate; NOE, nuclear
Overhauser effect; NR, nuclear receptor; NSB, nonspecific
binding; Papp, apparent permeability; PBMC, peripheral blood
mononuclear cell; PBS, phosphate-buffered saline; PD,
pharmacodynamics; PGR, progesterone receptor; PK, pharma-
cokinetics; po, oral administration; PPB, plasma-protein
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