Discovery of [1,2,4]Triazolo[1,5- a]pyridine Derivatives as Potent and Orally Bioavailable RORγt Inverse Agonists

Article abstract of DOI:10.1021/acsmedchemlett.9b00649

The retinoic acid receptor-related orphan nuclear receptor γt (RORγt), a promising therapeutic target, is a major transcription factor of genes related to psoriasis pathogenesis such as interleukin (IL)-17A, IL-22, and IL-23R. On the basis of the X-ray cocrystal structure of RORγt with 1a, an analogue of the known piperazine RORγt inverse agonist 1, triazolopyridine derivatives of 1 were designed and synthesized, and analogue 3a was found to be a potent RORγt inverse agonist. Structure-activity relationship studies on 3a, focusing on the treatment of its metabolically unstable cyclopentyl ring and the central piperazine core, led to a novel analogue, namely, 6-methyl-N-(7-methyl-8-(((2S,4S)-2-methyl-1-(4,4,4-trifluoro-3-(trifluoromethyl)butanoyl)piperidin-4-yl)oxy)[1,2,4]triazolo[1,5-a]pyridin-6-yl)nicotinamide (5a), which exhibited strong RORγt inhibitory activity and a favorable pharmacokinetic profile. Moreover, the in vitro and in vivo evaluation of 5a in a human whole-blood assay and a mouse IL-18/23-induced cytokine expression model revealed its robust and dose-dependent inhibitory effect on IL-17A production.

Full text of DOI:10.1021/acsmedchemlett.9b00649

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Letter
Discovery of [1,2,4]Triazolo[1,5-a]pyridine Derivatives as
Potent and Orally Bioavailable ROR#t Inverse Agonists
Ryota Nakajima, Hiroyuki Oono, Sakae Sugiyama, Yohei Matsueda, Tomohide Ida, Shinji
Kakuda, Jun Hirata, Atsushi Baba, Akito Makino, Ryo Matsuyama, Ryan D White, Ryan P. Wurz,
Youngsook Shin, Xiaoshan Min, Angel Guzman-Perez, Zhulun Wang, Antony Symons, Sanjay
K. Singh, Srinivasa Reddy Mothe, Sergei Belyakov, Anjan Chakrabarti, and Satoshi Shuto
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00649 • Publication Date (Web): 27 Feb 2020
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Discovery of [1,2,4]Triazolo[1,5-a]pyridine Derivatives as Potent and
Orally Bioavailable RORt Inverse Agonists
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Ryota Nakajima,*,$ Hiroyuki Oono,$ Sakae Sugiyama,$ Yohei Matsueda,$ Tomohide Ida,$ Shinji
Kakuda,$ Jun Hirata,$ Atsushi Baba,$ Akito Makino,$ Ryo Matsuyama,$ Ryan D. White,† Ryan Ρ.
Wurz,‡ Youngsook Shin,‡ Xiaoshan Min,ϲ Angel Guzman-Perez,† Zhulun Wang,♦ Antony Symons,∾
Sanjay K. Singh, ꜟ Srinivasa Reddy Mothe, ꜟ Sergei Belyakov, ꜟ Anjan Chakrabarti,ꜟ and Satoshi
Shuto*,§, ⱱ
$Teijin Institute for Bio-medical Research, Teijin Pharma Limited, 4-3-2 Asahigaoka, Hino, Tokyo 191-8512, Japan
†Department of Medicinal Chemistry, Amgen Discovery Research, Amgen Inc., 360 Binney Street, Cambridge,
Massachusetts 02142, United States
Departments of ‡Medicinal Chemistry, Amgen Discovery Research, Amgen Inc., One Amgen Center Drive, Thousand
Oaks, California 91320-1799, United States
Departments of ♦Biologics, ∾Inflammation & Oncology Research and ϲMolecular Engineering, Amgen Discovery
Research, Amgen Inc., 1120 Veterans Boulevard, South San Francisco, California 94080, United States
ꜟAMRI Singapore Research Centre, Pte. Ltd., 61 Science Park Road, #05-01 The Galen, Science Park III, Singapore
117525
ⱱFaculty of Pharmaceutical Sciences and §Center for Research and Education on Drug Discovery, Hokkaido University,
Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
KEYWORDS nuclear receptor, RORt, triazolopyridine, inverse agonist
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ABSTRACT: The retinoic acid receptor-related orphan nuclear receptor gamma t (RORt), a promising therapeutic target, is
a major transcription factor of genes related to psoriasis pathogenesis such as interleukin (IL)-17A, IL-22, and IL-23R. Based
on the X-ray cocrystal structure of RORt with 1a, an analog of a known piperazine RORt inverse agonist (1),
triazolopyridine derivatives of 1 were designed and synthesized, indicating analog 3a as a potent RORt inverse agonist.
The structure-activity relationships (SAR) studies in 3a, focusing on the treatment of its metabolically unstable cyclopentyl
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ring
and
the
central
piperazine
core,
led
to
a
novel
analog,
namely
6-methyl-N-(7-methyl-8-(((2S,4S)-2-methyl-1-(4,4,4-trifluoro-3-(trifluoromethyl)butanoyl)piperidin-4-yl)oxy)-[1,2,4]triaz
olo[1,5-a]pyridin-6-yl)nicotinamide (5a), which exhibited strong RORt inhibitory activity and a favorable pharmacokinetic
profile. Moreover, the in vitro and in vivo evaluation of 5a in a human whole-blood assay and a mouse IL-18/23-induced
cytokine expression model revealed its robust and dose-dependent inhibitory effect on the IL-17A production.
The retinoic acid receptor-related orphan nuclear
receptor gamma t (RORt) is a major transcription factor of
genes related to psoriasis pathogenesis such as interleukin
(IL)-17A, IL-22, and IL-23R.1-2 Therapies blocking IL-17A
or IL-23R have successfully improved skin lesions in
patients with moderate-to-severe psoriasis,3-7 thus
rendering the RORt inhibition a promising therapeutic
target. After T0901317 has been reported for its effective,
albeit unselective binding to ROR,8 many RORt
antagonists (inverse agonists) have been developed,9-23
while several are already being clinically investigated as
promising targets for the treatment of autoimmune
diseases24. Generally, nuclear receptors are proteins with
highly conserved ligand binding domains (LBDs), which
are structurally composed of alpha helices that form a large
lipophilic pocket responsible for binding small lipophilic
ligands such as retinoid derivatives, fatty acids, cholesterol,
and other lipophilic hormones and vitamins.25 Thus, one of
the main challenges for drug delivery in this target class is
the lipophilicity balance, which is required for strong LBD
binding potency, while the metabolism associated with
lipophilic small-molecule ligands should be minimized to
afford favorable drug-like properties.
H
N
N
N
O
N
F
O
1
Molecular Weight = 453
cLogD7.4 = 3.78
Figure 1. A representative RORt ligand, used as reference in
the current study.
Based on a previous report describing a series of
piperazine RORt ligands,26 compound 1 was selected as a
starting point for further investigation, mainly due to its
moderately low molecular weight (453) and lipophilicity
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(cLogD7.4 = 3.78). The optimization of 1 resulted in a new
displayed moderate inhibitory activity in the reporter gene
assay and good liver microsomal stability29. This
triazolopyridine derivative demonstrated
triazolopyridine derivative (3a) (Table 1) that exhibited
inverse RORt agonistic activity. Further optimization of 3a
by modifying its metabolically unstable cyclopentyl ring
and the central piperazine ring led to a new derivative,
6-methyl-N-(7-methyl-8-(((2S,4S)-2-methyl-1-(4,4,4-triflu
oro-3-(trifluoromethyl)butanoyl)piperidin-4-yl)oxy)-[1,2,4
]triazolo[1,5-a]pyridin-6-yl)nicotinamide (5a)(Table 3),
which was indicated as the best candidate for further
studies. Specifically, compound 5a exhibited potent RORt
inhibitory activity, good pharmacokinetic (PK) profile in a
mouse cassette dosing study, and dose-dependent
a
nitrogen-containing bicyclic ring was tolerable for the
RORt inhibitory activity, which lead us into detailed
investigation with other chemo types of triazolopyridine
analogs.
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inhibition of the IL-17A production in
a
mouse
IL-18/23-induced cytokine expression model. Herein, the
results of these studies are described.
An X-ray cocrystal structure of human RORt (PDB ID
6O3Z) with 1a (Figure 2C), an analog of the piperazine
RORt ligand 1, was successfully obtained27-28
. As
illustrated in Figure 2A, the cyano group of 1a formed
hydrogen bonds with Arg367 and Leu287, while the amide
NH group with Phe378. Furthermore, a hydrogen bond was
developed between the fluorobenzene ring and the
hydroxyl group of Ser404, where a small space around the
positions 4 and 5 of the ring was detected (Figure 2B).
Considering this additional space in the pocket, the phenyl
Figure 2. X-ray cocrystal structure of RORt (PDB ID 6O3Z)
with compound 1a. A: RORt-LBD and compound 1a are
depicted in gray and green, respectively. Green dashes
represent the hydrogen bond interactions. B: The red circle
indicates the small space around the positions 4 and 5 of the
fluorobenzene ring of 1a. The surface of the RORt-LBD site
(gray) and the surface of 1a (green) were calculated from the
X-ray structure. C: Molecular structure of 1a.
ring of compound
1
could be replaced by
a
nitrogen-containing bicyclic ring to simultaneously
decrease the overall lipophilicity and facilitate the
hydrogen bond interactions with Ser404, allowing thus a
more detailed investigation of this residue. The previous
report by Hintermann and co-workers described a low
To that end, two series of analogs bearing
N-([1,2,4]triazolo[4,3-a]pyridin-7-yl)amide
and
N-([1,2,4]triazolo[1,5-a]pyridin-6-yl)amide moieties were
synthesized. Since it has been reported that RORt is
constitutively active in the absence of an endogenous
ligand,30 all compounds were evaluated in a luciferase
lipophilic
triazolopyridine
derivative
having
N-([1,2,4]triazolo[4,3-a]pyridin-6-yl)amide moiety, which
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reporter gene assay without a control agonist ligand to
the [1,2,4]triazolo[1,5-a]pyridine ring were well tolerated
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assess their RORt inverse agonistic activity.
for the inhibition of the RORt transcriptional activity.
Furthermore, although 3a improved the human LM
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stability
(human
CLint = 0.032 mL/min/mg),
the
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methyl-substituted triazolopyridine derivatives 2b and 3b
exhibited a lower in vitro activity, probably due to steric
repulsions in the binding pocket. Moreover, the X-ray
analysis results implied that only unsubstituted
triazolopyridine rings are acceptable, because the available
space at the 4 and 5 positions of the fluorobenzene ring of
1a is small. Therefore, compound 3a, which exhibited the
most improved LM stability and high potency, was further
optimized.
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Table 1. Structure-activity relationships (SAR) of the
triazolopyridine series.
H
H
N
N
N
N
N
R
N
O
N
N
O
N
N
N
R
N
O
N
O
N
2a-b
3a-b
Luc IC50
Human LM CLint
a
Compd
R
cLogD7.4
(nM)b
14
(mL/min/mg) c
0.088
1
-
3.78
2.57
2.53
2.37
2.32
2a
2b
3a
3b
H
590
8000
41
0.059
To elucidate the PK profile of 3a, this compound was
incubated in human hepatocytes and its metabolites were
explored by mass spectrometry (MS). During the MS
analysis, no glutathione adducts were detected, suggesting
Me
H
0.032
Me
3200
b
aCalculated by Pipeline Pilot 17.2. Luciferase reporter gene
assay used for the evaluation of the RORt transcriptional
activity inhibition. The employed HEK293T cells were
transfected with GAL4-NR-luciferase plasmids and the activity
was evaluated using the Dual-GLOTM Luciferase Assay
System. cMetabolic stability in liver microsome (LM).
a
low tendency toward the formation of reactive
metabolites, while no metabolic soft spots on the central
triazolopyridine core could be observed, supporting thus
the suitability of the triazolopyridine moiety as a lead
scaffold. In contrast, oxidative adducts were detected on
the cyclopentyl ring, which prompted us to further
optimize analog 3a.
Moreover, all the synthesized triazolopyridine analogs
were designed to achieve a lower lipophilicity than
compound 1 (cLogD7.4 = 3.78). As outlined in Table 1, the
cLogD7.4 values of the novel analogs were successfully
decreased by approximately one unit compared to 1.
However, the [1,2,4]triazolo[4,3-a]pyridine derivative 2a
displayed reduced inhibitory activity in the reporter gene
A series of 3a analogs were designed by exploring
cyclopentyl ring alternatives and acyclic chains bearing
fluorine atoms that could potentially block the metabolism.
The SAR results of the cyclopentyl ring modifications in
analog 3a are summarized in Table 2. Although the
substitution of the cyclopentyl ring by two fluorine atoms
(4a) improved the cLogD7.4 and the LM stability, the
reporter gene inhibitory activity of RORt was slightly
decreased (IC50 = 130 nM). Similarly, the replacement of
assay
(IC50 = 590 nM),
whereas
the
[1,2,4]triazolo[1,5-a]pyridine derivative 3a retained an
excellent inhibitory activity (IC50 = 41 nM), comparable to
that of compound 1, indicating that the nitrogen atoms in
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the cyclopentyl ring by a pyran ring (4b) significantly
nM) and LM stability to 3a. Given that the trifluoromethyl
4
5
6
7
8
decreased the inhibitory activity. Moreover, the phenyl
derivative 4c had similar LM stability (human
CLint = 0.021 mL/min/mg) compared to 3a, but its
inhibitory activity was slightly lower (IC50 = 79 nM). The
cyclopentyl ring of 3a was then replaced by branched or
unbranched alkyl chains, resulting in derivatives 4d-4g.
The inhibitory activity of the isopropyl analog (4d) was
slightly decreased (IC50 = 230 nM), whereas the isobutyl
derivative 4e had comparable inhibitory activity (IC50 = 59
group is often used instead of a methyl group to decrease
the oxidative metabolism or to increase the steric size, the
two methyl groups of 4e were replaced by trifluoromethyl
groups (4f), resulting in similar inhibitory activity
(IC50 = 50 nM) and improved LM stability. Moreover, based
on the results of the mono-trifluoromethyl derivative 4g
with an IC50 value of 240 nM, it was proven that the double
substitution was more favorable for the inhibitory activity.
Hence, compound 4f was selected for further investigation
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Table 2. SAR of the 3a derivatives.
H
N
N
N
O
N
R1
N
N
N
O
Human LM CLint
(mL/min/mg)c
0.088
a
Compound
R1
-
cLogD7.4
Luc IC50 (nM)b
1
3.78
2.37
14
41
3a
0.032
F
F
4a
1.61
130
<0.010
O
4b
4c
1.05
2.27
10000
79
<0.010
0.021
4d
4e
4f
1.82
2.07
2.61
2.07
230
59
0.24
0.020
CF3
CF3
CF3
50
<0.010
<0.010
4g
240
b
aCalculated by Pipeline Pilot 17.2. Luciferase reporter gene assay to assess the RORγt transcriptional activity inhibition. The
employed HEK293T cells were transfected with GAL4-NR-luciferase plasmids and the activity was evaluated using the
Dual-GLOTM Luciferase Assay System. cMetabolic stability in liver microsome.
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Table 3. RORt inhibitory activity and lipophilicity of analogs 5a and 5b.
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H
N
N
O
N
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O
N
CF3
CF3
N
N
O
O
Human LM CLint
(mL/min/mg)c
a
Luc IC50 (nM)b
N
Compound
cLogD7.4
1
-
-
3.78
2.61
14
50
0.088
4f
<0.010
O
O
5a
2.67
2.67
51
0.010
N
0.028
5b
1500
N
aCalculated by Pipeline Pilot 17.2. Luciferase reporter gene assay to assess the RORγt transcriptional activity inhibition. The
employed HEK293T cells were transfected with GAL4-NR-luciferase plasmids and the activity was evaluated using the
Dual-GLOTM Luciferase Assay System. cMetabolic stability in liver microsome.
b
To improve the activity of 4f, the 2-methylpyridine
moiety was replaced by substituted pyridines or other
heteroaryl rings (data not shown). However, this structural
modification did not improve either the inhibitory activity
or the lipophilicity of the obtained analogs. Therefore, the
piperazine moiety of 4f was replaced by piperidine to
retain low lipophilicity values, while introducing an ether
linkage between the piperidine ring and the
triazolopyridine ring. Cis- (5a) and trans-piperidine (5b)
analogs were thus obtained with cLogD7.4 values of 2.67
(Table 3). Interestingly, 5a maintained good RORt
inhibitory activity (IC50 = 51 nM) and LM stability (human
CLint = 0.010 mL/min/mg) compared to 4f, whereas the
inhibitory activity of 5b was insignificant. Furthermore, the
in vitro absorption, distribution, metabolism, and excretion
(ADME) profiles of compounds 1, 3a, 4f, and 5a were
assessed (Table 4). Compounds 3a, 4f, and 5a displayed
better microsomal stability than compound 1 (human CLint
= 0.032, <0.010, and 0.010 mL/min/mg, respectively)
along with decreased plasma protein binding (PPB) rates
(77.3%, 86.1%, and 91.8%, respectively). Moreover,
compounds 4f and 5a were sufficiently soluble in an
aqueous medium. Although 3a and 4f exhibited low Caco-2
permeability in the apical-to-basolateral (A to B) direction,
5a displayed efficient permeability in the same evaluation
system.
Table 4. In vitro ADME profiles of compounds 1, 3a, 4f, and 5a.
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Caco-2 Papp
LM CLint (mL/min/mg)a
PPB (%)b
human
Solubility
(×10−6cm/s)d
Compound
(M)c
human
0.088
0.032
<0.010
0.010
mouse
0.11
mouse
95.7e
78.8
A to B
B to A
55
1
99.7
77.3
86.1
91.8
71
40
2.5
9.6
3a
4f
5a
0.011
< 0.010
190
200
170
46
76.7
36
0.030
86.5
31
51
b
c
aMetabolic stability in liver microsome. Plasma protein binding using human or mouse plasma. The second fluid of the
Disintegration Test of the Japanese Pharmacopoeia (pH 6.8) was used. dPermeability in the apical-to-basolateral (A to B) direction
and vice versa (B to A). eCompound 1 might be unstable in the mouse PPB assay.
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Table 5. PK profile of compounds 1, 3a, 4f, and 5a in a mouse cassette dosing study.a
BA (%) AUC p.o. (nM*h) CL/CLu (L/h/kg)
VDss (L/kg)
t1/2 p.o. (h)
t1/2 i.v. (h)
Compound
1
48
47
490
840
2.0/46
1.1/5.3
1.1/4.5
0.15/1.1
1.6
0.77
1.1
0.7
1.2
1.1
3.6
0.7
1.4
1.0
3.9
3a
4f
5a
110
120
2300
15000
0.80
a2 mol/kg p.o. dose and 1 mol/kg i.v. dose.
The in vivo PK profile of compounds 1, 3a, 4f, and 5a was
investigated in mice using cassette dosing (Table 5).
Compared to compound 1, compounds 4f and 5a showed
improved bioavailability (BA > 100%), notably lower
unbound clearance (CLu = 1.1 L/h/kg for 5a), and higher
area under the curve (AUC) (around 31-fold higher than 1
in the case of 5a) and t1/2 (>3 h for 5a). Owing to the
favorable in vitro activity and ADME profile, analog 5a was
further investigated.
IL-17A production in a dose-dependent manner with an
IC50 value of 130 nM (Figure 3A). The in vivo potency of 5a
toward the modulation of the cytokine production through
the suppression of the differentiation and activation of
Th17 and Th1/17 cells was assessed using an in vivo
mouse IL-18/23-induced cytokine expression model.
Compound 5a was orally administered once 3 h prior to
the IL-18/23 injection, and the IL-17A level in blood was
measured 4 h after the injection of IL-18/23. As illustrated
in Figure 3B, 5a achieved a dose-dependent inhibition of
the IL-17A production in 3, 10, 30, and 100 mg/kg doses.
A human whole-blood assay of compound 5a was
conducted to assess its inhibitory activity against the T-cell
receptor-dependent production of IL-17A in human whole
blood. It was proven that compound 5a suppressed the
In summary, a new triazolopyridine derivative (3a) was
identified as a potent RORt inhibitor. Optimization of the
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stimulated using anti-CD3 and anti-CD28 monoclonal
metabolic soft spots on the cyclopentyl ring of 3a, followed
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antibodies, IL-18, and IL-23, followed by treatment with 5a.
The level of IL-17A in the culture medium was measured after
two days. B: In vivo mouse IL-18/23-induced cytokine
expression model. Mice were administered orally once with a
vehicle (0.5% MC400) or 5a. After 3 h, the mice were
administered intraperitoneally with 2 g of mIL-18 (Medical &
Biological Laboratories) and 1 g mIL-23 (R&D Systems). A
blood sample was collected 4 h later and the IL-17A
concentration in the plasma was measured.
by optimization of the piperazine ring, led to a novel
analog,
namely
6-methyl-N-(7-methyl-8-(((2S,4S)-2-methyl-1-(4,4,4-triflu
9
oro-3-(trifluoromethyl)butanoyl)piperidin-4-yl)oxy)-[1,2,4
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]triazolo[1,5-a]pyridin-6-yl)nicotinamide
(5a).
5a
exhibited potent RORt inhibition in a luciferase reporter
gene assay as well as in a human whole-blood assay
measuring IL-17A release. Moreover, a robust PK profile
allowed the in vivo evaluation in a IL-18/23-induced
cytokine expression mouse model, where 5a significantly
inhibited the IL-17A production in a dose-dependent
manner.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Synthetic
experimental,
compound
characterization,
crystallography studies and assay descriptions (PDF).
AUTHOR INFORMATION
Corresponding Author
*For R.N.; phone: +81-3-3506-4216. Fax:
A
+81-3-3506-4217. E-mail: r.nakajima@teijin.co.jp
*For S.S.; phone/fax: +81-11-706-3769. e-mail:
shu@pharm.hokudai.ac.jp.
Present Addresses
$R.N.; Teijin Limited, 2-1, Kasumigaseki 3-chome, Chiyoda-ku,
Tokyo 100-8585, Japan.
ꜟS.K.S.; AWAK Technologies, 3 Tuas Lane, Singapore 638612.
ꜟS.R.M.; Polymer Engineering Characterization, Institute of
Chemical and Engineering Sciences, Agency of Science,
Technology and Research, 1 Pesek Road, Jurong Island,
Singapore 627833.
B
mean ± SEM (n=6), ***P<0.001, vs Vehicle (Dunnett test)
Figure 3. A: Human whole-blood assay (n = 2). Human whole
blood was diluted in half with RPMI 1640TM and was
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ꜟS.B.; Wintershine Health and Skincare Pte Ltd., 23 Goldhill Pl.,
(3) Hueber, W.; Patel, D. D.; Dryja, T.; Wright, A. M.; Koroleva, I.;
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Bruin, G.; Antoni, C.; Draelos, Z.; Gold, M. H.; Psoriasis Study, G.;
Durez, P.; Tak, P. P.; Gomez-Reino, J. J.; Rheumatoid Arthritis Study
Group; Foster, C. S.; Kim, R. Y.; Samson, C. M.; Falk, N. S.; Chu, D. S.;
Callanan, D.; Nguyen, Q. D.; Uveitis Study, G.; Rose, K.; Haider, A.;
Di Padova, F. Effects of AIN457, a fully human antibody to
interleukin-17A, on psoriasis, rheumatoid arthritis, and uveitis.
Sci. Transl. Med. 2010, 2, 52ra72.
Singapore 308931.
ꜟA.C.; Syngene International Limited, Biocon Park SEZ,
Bommasandra IV Phase, Jigani Link Road, Bangalore 560099,
India.
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Author Contributions
These authors contributed equally.
Notes
(4) Leonardi, C.; Matheson, R.; Zachariae, C.; Cameron, G.; Li, L.;
Edson-Heredia, E.; Braun, D.; Banerjee, S. Anti-Interleukin-17
Monoclonal Antibody Ixekizumab in Chronic Plaque Psoriasis. N.
Engl. J. Med. 2012, 366, 1190-1199.
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We thank Keiko Shimada for her help on computational
chemistry. We thank Mariko Hirano, Yukiko Enomoto and Akiko
Takeuchi for the pharmacological discussion. We thank Kevin
Gaida for the whole-blood assay development and support, Shon
Booker for chemistry optimization, and Steve Thibault and Haoda
Xu for the protein supply.
(5) Langley, R. G.; Elewski, B. E.; Lebwohl, M.; Reich, K.; Griffiths,
C. E.; Papp, K.; Puig, L.; Nakagawa, H.; Spelman, L.; Sigurgeirsson,
B.; Rivas, E.; Tsai, T. F.; Wasel, N.; Tyring, S.; Salko, T.; Hampele, I.;
Notter, M.; Karpov, A.; Helou, S.; Papavassilis, C.; Group, E. S.;
Group, F. S. Secukinumab in Plaque Psoriasis--Results of Two
Phase 3 Trials. N. Engl. J. Med. 2014, 371, 326-338.
ABBREVIATIONS
(6) Papp, K. A.; Reich, K.; Paul, C.; Blauvelt, A.; Baran, W.; Bolduc,
C.; Toth, D.; Langley, R. G.; Cather, J.; Gottlieb, A. B.; Thaci, D.;
Krueger, J. G.; Russell, C. B.; Milmont, C. E.; Li, J.; Klekotka, P. A.;
Kricorian, G.; Nirula, A. A Prospective Phase III, Randomized,
Double-Blind, Placebo-Controlled Study of Brodalumab in Patients
with Moderate-to-severe Plaque Psoriasis. Br. J. Dermatol. 2016,
175, 273-286.
BA, bioavailability; RORt, retinoic acid receptor-related orphan
receptor t.
REFERENCES
(1) Ivanov, I. I.; McKenzie, B. S.; Zhou, L.; Tadokoro, C. E.;
Lepelley, A.; Lafaille, J. J.; Cua, D. J.; Littman, D. R. The Orphan
Nuclear Receptor RORgammat Directs the Differentiation
Program of Proinflammatory IL-17+ T Helper Cells. Cell 2006,
126, 1121-1133.
(7) Farahnik, B.; Beroukhim, K.; Zhu, T. H.; Abrouk, M.;
Nakamura, M.; Singh, R.; Lee, K.; Bhutani, T.; Koo, J. Ixekizumab for
the Treatment of Psoriasis: A Review of Phase III Trials. Dermatol.
Ther. 2016, 6, 25-37.
(2) Annunziato, F.; Cosmi, L.; Santarlasci, V.; Maggi, L., Liotta F.,
Mazzinghi, B.; Parente, E.; Filì, L.; Ferri, S.; Frosali, F.; Giudici, F.;
Romagnani, P.; Parronchi, P.; Tonelli, F.; Maggi, E.; Romagnani, S.
Phenotypic And Functional Features Of Human Th17 Cells. J. Exp.
Med. 2007, 204, 1849-1861.
(8) Kumar, N.; Solt, L. A.; Conkright, J. J.; Wang, Y.; Istrate, M. A.;
Busby, S. A.; Garcia-Ordonez, R. D.; Burris, T. P. Griffin, P. R. The
Benzenesulfoamide T0901317
[N-(2,2,2-trifluoroethyl)-N-[4-[2,2,2-trifluoro-1-hydroxy-1-(triflu
9
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ACS Medicinal Chemistry Letters
Page 10 of 12
1
2
3
4
oromethyl)ethyl]phenyl]-benzenesulfonamide] is a Novel Retinoic
(15) Hirata, K.; Kotoku, M.; Seki, N.; Maeba, T.; Maeda, K.;
5
6
7
8
Acid Receptor-related Orphan receptor-alpha/gamma Inverse
Agonist. Mol. Pharmacol. 2010, 77, 228-236.
Hirashima, S.; Sakai, T.; Obika, S.; Hori, A.; Hase, Y.; Yamaguchi, T.;
Katsuda, Y.; Hata, T.; Miyagawa, N.; Arita, K.; Nomura, Y.; Asahina,
K.; Aratsu, Y.; Kamada, M.; Adachi, T.; Noguchi, M.; Doi, S.; Crowe,
P.; Bradley, E.; Steensma, R.; Tao, H.; Fenn, M.; Babine, R.; Li, X.;
Thacher, S.; Hashimoto, H.; Shiozaki, M. SAR Exploration Guided
by LE and Fsp3: Discovery of a Selective and Orally Efficacious
RORγ Inhibitor. ACS Med. Chem. Lett. 2016, 7, 23-27.
(9) Cyr, P.; Bronner, S. M.; Crawford, J. J., Recent Progress on
Nuclear Receptor RORgamma Modulators. Bioorg. Med. Chem. Lett.
2016, 26, 4387-4393.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(10) Qiu, R.; Wang, Y. Retinoic Acid Receptor-Related Orphan
Receptor t (RORt) Agonists as Potential Small Molecule
Therapeutics for Cancer Immunotherapy. J. Med. Chem. 2018, 61,
5794-5804.
(16) Olsson, R. I.; Xue, Y.; von Berg, S.; Aagaard, A.; McPheat, J.;
Hansson, E. L.; Bernstrom, J.; Hansson, P.; Jirholt, J.; Grindebacke,
H.; Leffler, A.; Chen, R.; Xiong, Y.; Ge, H.; Hansson, T. G.; Narjes, F.
Benzoxazepines Achieve Potent Suppression of IL-17 release in
Human T-Helper 17 (TH 17) Cells Through an Induced-fit Binding
Mode to the Nuclear Receptor RORgamma. ChemMedChem 2016,
11, 207-216.
(11) Pandya, V. B.; Kumar, S.; Sachchidanand.; Sharma, R.;
Desai, R. C. Combating Autoimmune Diseases with Retinoic Acid
Receptor-related Orphan Receptor- (ROR or RORc) Inhibitors:
Hits and Misses. J. Med. Chem. 2018, 61, 10976-10995.
(12) van Niel, M. B.; Fauber, B. P.; Cartwright, M.; Gaines, S.;
Killen, J. C.; Rene, O.; Ward, S. I.; de Leon Boenig, G.; Deng, Y.;
Eidenschenk, C.; Everett, C.; Gancia, E.; Ganguli, A.; Gobbi, A.;
Hawkins, J.; Johnson, A. R.; Kiefer, J. R.; La, H.; Lockey, P.; Norman,
M.; Ouyang, W.; Qin, A.; Wakes, N.; Waszkowycz, B.; Wong, H. A
Reversed Sulfonamide Series of Selective RORc Inverse Agonists.
Bioorg. Med. Chem. Lett. 2014, 24, 5769-5776.
(17) Rene, O.; Fauber, B. P.; Barnard, A.; Chapman, K.; Deng, Y.;
Eidenschenk, C.; Everett, C.; Gobbi, A.; Johnson, A. R.; La, H.;
Norman, M.; Salmon, G.; Summerhill, S.; Wong, H. Discovery of
Oxa-sultams as RORc Inverse Agonists Showing Reduced
Lipophilicity, Improved Selectivity and Favorable ADME
Properties. Bioorg. Med. Chem. Lett. 2016, 26, 4455-4461.
(18) Ouvry, G.; Bouix-Peter, C.; Ciesielski, F.; Chantalat, L.;
Christin, O.; Comino, C.; Duvert, D.; Feret, C.; Harris, C. S.; Lamy, L.;
Luzy, A. P.; Musicki, B.; Orfila, D.; Pascau, J.; Parnet, V.; Perrin, A.;
Pierre, R.; Polge, G.; Raffin, C.; Rival, Y.; Taquet, N.; Thoreau, E.;
Hennequin, L. F. Discovery of Phenoxyindazoles and
(13) Chao, J.; Enyedy, I.; Van Vloten, K.; Marcotte, D.; Guertin, K.;
Hutchings, R.; Powell, N.; Jones, H.; Bohnert, T.; Peng, C. C.; Silvian,
L.; Hong, V. S.; Little, K.; Banerjee, D.; Peng, L.; Taveras, A.; Viney, J.
L.; Fontenot, J. Discovery of Biaryl Carboxylamides as Potent RORγ
Inverse Agonists. Bioorg. Med. Chem. Lett. 2015, 25, 2991-2997.
(14) Wang, Y.; Cai, W.; Cheng, Y.; Yang, T.; Liu, Q.; Zhang, G.;
Meng, Q.; Han, F.; Huang, Y.; Zhou, L.; Xiang, Z.; Zhao, Y. G.; Xu, Y.;
Cheng, Z.; Lu, S.; Wu, Q.; Xiang, J. N.; Elliott, J. D.; Leung, S.; Ren, F.;
Lin, X. Discovery of Biaryl Amides as potent, Orally Bioavailable,
and CNS Penetrant RORt Inhibitors. ACS Med. Chem. Lett. 2015, 6,
787-792.
Phenylthioindazoles as RORgamma Inverse Agonists. Bioorg. Med.
Chem. Lett. 2016, 26, 5802-5808.
(19) Kummer, D. A.; Cummings, M. D.; Abad, M.; Barbay, J.;
Castro, G.; Wolin, R.; Kreutter, K. D.; Maharoof, U.; Milligan, C.;
Nishimura, R.; Pierce, J.; Schalk-Hihi, C.; Spurlino, J.; Urbanski, M.;
Venkatesan, H.; Wang, A.; Woods, C.; Xue, X.; Edwards, J. P.; Fourie,
A. M.; Leonard, K. Identification and Structure Activity
10
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(24) Sun, N.; Guo, H.; Wang, Y. Nuclear Retinoic Acid
Relationships of Quinoline Tertiary Alcohol Modulators of RORt.
4
5
6
7
8
Bioorg. Med. Chem. Lett. 2017, 27, 2047-2057.
Receptor-related Orphan Receptor Gamma-t (RORt) Inhibitors in
Clinical Development for the Treatment of Autoimmune Diseases:
a Patent Review (2016-present). Expert Opin Ther Pat. 2019, 29,
663-674.
(20) Barbay, J. K.; Cummings, M. D.; Abad, M.; Castro, G.;
Kreutter, K. D.; Kummer, D. A.; Maharoof, U.; Milligan, C.;
Nishimura, R.; Pierce, J.; Schalk-Hihi, C.; Spurlino, J.; Tanis, V. M.;
Urbanski, M.; Venkatesan, H.; Wang, A.; Woods, C.; Wolin, R.; Xue,
X.; Edwards, J. P.; Fourie, A. M.; Leonard, K. 6-Substituted
Quinolines as RORgammat Inverse Agonists. Bioorg. Med. Chem.
Lett. 2017, 27, 5277-5283.
9
10
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21
22
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26
27
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(25) Burris, T. P.; Solt, L. A.; Wang, Y,.; Crumbley, C.; Banerjee, S.;
Griffett, K.; Lundasen, T.; Hughes, T.; Kojetin, D. J. Nuclear
Receptors and their Selective Pharmacologic Modulators.
Pharmacol Rev. 2013, 65, 710-778.
(21) Kono, M.; Ochida, A.; Oda, T.; Imada, T.; Banno, Y.; Taya, N.;
Masada, S.; Kawamoto, T.; Yonemori, K.; Nara, Y.; Fukase, Y.;
Yukawa, T.; Tokuhara, H.; Skene, R.; Sang, B. C.; Hoffman, I. D.;
Snell, G.P.; Uga, K.; Shibata, A.; Igaki, K.; Nakamura, Y.; Nakagawa,
H.; Tsuchimori, N.; Yamasaki, M.; Shirai, J.; Yamamoto, S. Discovery
of [ cis-3-({(5 R)-5-[(7-Fluoro-1,1-dimethyl-2,3-dihydro-1
H-inden-5-yl)carbamoyl]-2-methoxy-7,8-dihydro-1,6-naphthyridi
n-6(5 H)-yl}carbonyl)cyclobutyl]acetic acid (TAK-828F) as a
Potent, Selective, and Orally Available Novel Retinoic Acid
Receptor-related Orphan Receptor t Inverse Agonist. J. Med.
Chem. 2018, 61, 2973-2988.
(26) Han, F.; Lei, H.; Lin, X.; Meng, Q.; Wang, Y. Modulators of the
Retinoid-related Orphan Receptor Gamma (ROR-gamma) for Use
in the Treatment of Autoimmune and Inflammatory Diseases. WO
Patent WO2014/086894A1, 2014.
(27) X-ray cocrystal structure of RORt (PDB ID 5NTP) with
close analog to 1a was reported to have a similar binding mode to
1a.
(28) Kallen, J.; Izaac, A.; Be, C.; Arista, L.; Orain, D.; Kaupmann,
K.; Guntermann, C.; Hoegenauer, K.; Hintermann, S. Structural
States of RORt: X-ray Elucidation of Molecular Mechanisms and
Binding Interactions for Natural and Synthetic Compounds.
ChemMedChem. 2017, 12, 1014-1021.
(22) Gege, C.; Cummings, M. D.; Albers, M.; Kinzel, O.; Kleymann,
G.; Schlüter, T.; Steeneck, C.; Nelen, M. I.; Milligan, C.; Spurlino, J.;
Xue, X.; Leonard, K.; Edwards, J. P.; Fourie, A.; Goldberg, S. D.;
Hoffmann, T. Identification and Biological Evaluation of
Thiazole-based Inverse Agonists of RORt. Bioorg. Med. Chem. Lett.
2018, 28, 1446-1455.
(29) Hintermann, S.; Guntermann, C.; Mattes, H.; Carcache, D. A.;
Wagner, J.; Vulpetti, A.; Billich, A.; Dawson, J.; Kaupmann, K.;
Kallen, J.; Stringer, R.; Orain, D. Synthesis and Biological
Evaluation of New Triazolo- and Imidazolopyridine RORt Inverse
Agonists. ChemMedChem 2016, 11, 2640-2648.
(23) Tian, J.; Sun, N.; Yu, M.; Gu, X.; Xie, Q.; Shao, L.; Liu, J.; Liu, L.;
Wang, Y. Discovery of N-indanyl Benzamides as Potent RORt
Inverse Agonists. Eur. J. Med. Chem. 2019, 167, 37-48.
(30) Solt, L. A.; Burris, T. P. Action of RORs and their Ligands in
(Patho)Physiology. Trends Endocrinol. Metab. 2012, 23, 619-627.
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Discovery of [1,2,4]Triazolo[1,5-a]pyridine Derivatives as Potent and
Orally Bioavailable RORt Inverse Agonists
Ryota Nakajima,*,$ Hiroyuki Oono,$ Sakae Sugiyama,$ Yohei Matsueda,$ Tomohide Ida,$ Shinji
Kakuda,$ Jun Hirata,$ Atsushi Baba,$ Akito Makino,$ Ryo Matsuyama,$ Ryan D. White,† Ryan Ρ.
Wurz,‡ Youngsook Shin,‡ Xiaoshan Min,ϲ Angel Guzman-Perez,† Zhulun Wang,♦ Antony Symons,∾
Sanjay K. Singh, ꜟ Srinivasa Reddy Mothe, ꜟ Sergei Belyakov, ꜟ Anjan Chakrabarti,ꜟ and Satoshi
Shuto*,§, ⱱ
H
H
N
N
O
N
N
N
N
O
N
CF3
CF3
O
N
N
N
O
F
O
Compound 1
5a
Reporter Gene IC50 = 14 nM
cLogD7.4 = 3.78
Reporter Gene IC50 = 51 nM
cLogD7.4 = 2.67
mouse CDPK CLu (L/hr/kg) = 46
mouse CDPK CLu (L/hr/kg) = 1.1
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