Discovery of Second Generation RORγ Inhibitors Composed of an Azole Scaffold

Article abstract of DOI:10.1021/acs.jmedchem.8b01567

Starting from a previously reported RORγ inhibitor (1), successive efforts to improve in vivo potency were continued. Introduction of metabolically beneficial motifs in conjunction with scaffold hopping was examined, resulting in discovery of the second generation RORγ inhibitor composed of a 4-(isoxazol-3-yl)butanoic acid scaffold (24). Compound 24 achieved a 10-fold improvement in in vivo potency in a mouse CD3 challenge model along with significant anti-inflammatory effects in a mouse dermatitis model.

Full text of DOI:10.1021/acs.jmedchem.8b01567

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Brief Article
Discovery of the Second Generation ROR#
Inhibitors Composed of an Azole Scaffold
Masayuki Kotoku, Takaki Maeba, Shingo Fujioka, Masahiro Yokota, Noriyoshi Seki, Keisuke Ito,
Yoshihiro Suwa, taku ikenogami, Kazuyuki Hirata, Yasunori Hase, Yoshiaki Katsuda, Naoki Miyagawa,
Kojo Arita, Kota Asahina, Masato Noguchi, Akihiro Nomura, Satoki Doi, Tsuyoshi Adachi, Paul
Crowe, Haiyan Tao, Scott Thacher, Hiromasa Hashimoto, Takayoshi Suzuki, and Makoto Shiozaki
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01567 • Publication Date (Web): 18 Feb 2019
Downloaded from http://pubs.acs.org on February 18, 2019
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Suzuki, Takayoshi; Kyoto Prefectural University of Medicine , Graduate
School of Medical Science, Kyoto Prefectural University of Medicine
Shiozaki, Makoto; JAPAN TOBACCO INC., Central Pharmaceutical
Research Institute
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Discovery of the Second Generation ROR Inhibitors Composed of an
Azole Scaffold
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Masayuki Kotoku,*,†,⊥ Takaki Maeba,† Shingo Fujioka,† Masahiro Yokota,† Noriyoshi Seki,†
Keisuke Ito,† Yoshihiro Suwa,† Taku Ikenogami,† Kazuyuki Hirata,†Yasunori Hase,† Yoshiaki
Katsuda,§ Naoki Miyagawa,§ Kojo Arita,§ Kota Asahina,¶ Masato Noguchi,† Akihiro Nomura,†
Satoki Doi,† Tsuyoshi Adachi,† Paul Crowe,‡ Haiyan Tao,‡ Scott Thacher,‡ Hiromasa
Hashimoto,† Takayoshi Suzuki,⊥ and Makoto Shiozaki*,†
†Chemical Research Laboratories, §Biological Pharmacological Research Laboratories, ¶Drug Metabolism &
Pharmacokinetics Research Laboratories, Central Pharmaceutical Research Institute, Japan Tobacco Inc., 1-1,
Murasaki-cho, Takatsuki, Osaka, 569-1125, Japan
⊥
Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 1-5 Shimogamo-hangi-cho, Sakyo-
ku, Kyoto 603-0823, Japan
‡Orphagen Pharmaceuticals, 11558 Sorrento Valley Road, Suite 4, San Diego, California, 92121, United States
KEYWORDS. ROR Autoimmune diseases, Th1, Th2, Th17, ROR, CD3 challenge model, improved PK profiles, LogD
ABSTRACT: Starting from a previously reported ROR
inhibitor (1), successive efforts to improve in vivo potency
were continued. Introduction of metabolically beneficial
motifs in conjunction with scaffold hopping was examined,
resulting in discovery of the second generation ROR
inhibitor composed of a 4-(isoxazol-3-yl)butanoic acid
scaffold (24). Compound 24 achieved a 10-fold improvement
in in vivo potency in a mouse CD3 challenge model along
with significant anti-inflammatory effects in a mouse
dermatitis model.
under human clinical trials or have even been launched quite
INTRODUCTION
recently.4-6
In 2005, more than two decades after the discovery of Th1
Among molecules that participate in the Th17 signaling,
and Th2 cells, the classical understanding of the helper T cell
ROR has been widely recognized as a most attractive entity
theory was revised and an early developmental pathway that
since it functions as a master regulator of Th17 cell
diverged from the Th1 and Th2 lineages was confirmed as the
development.7 Various compounds targeting the orphan
Th17 lineage.1,2 Although pre-Th1 cells are believed to be
nuclear receptor have been reported,8-16 but only a limited
present and to take a part as common intermediates of Th1
number of modulators of this target have progressed into
and Th17 cells, Th17 cells were shown to arise directly from
human clinical trials. This drug discovery program has been
CD4+ helper T cells in the presence of interleukin (IL)-23.
challenged by the need to balance good potency, high
These studies confirmed that three distinct lineages were
specificity and favorable pharmacokinetic (PK) profiles.
initiated from effector CD4+ helper T cells.3 The new findings
Recently, we identified a novel series of ROR inhibitors. The
suggested that direct inhibition of Th17 cell differentiation
lead compound suffered from poor metabolic stability as well
and proliferation could be a rational approach for the
as insufficient selectivity, but these characteristics were
treatment of autoimmune diseases, and several biologic
considerably improved during the course of the
medications blocking the Th17 cell function are currently
investigation.17 Despite our improvements, the in vivo
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potency of the best compound 1 was still unsatisfactory with
Table 1. Effects of R1 Groups on Potency and ADME
Profiles
an ED50 value of 30 mg/kg , which compelled us to pursue
the generation of a more advanced inhibitor.
Me
Me
H
Me
H
Me
5
O
N
Me
N
O
N
N
N
N
Me
6
7
N
Me
N
Me
R1
N
O
Me
Caco2
Pappc
(10-6 cm/sec)
8
9
a
b
EC50
M)
MS
Caco2
Pappc
(10-6 cm/sec)
LogD
compd
R1
H
a
b
EC50
MS
(%)
(%)
(

compd
LogD
4.3
(µM)
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
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41
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43
44
45
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47
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49
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60
2
3
1.7
3.9
3.5
3.1
3.3
3.5
2.9
3.4
3.5
3.8
27
21
88
85
39
91
44
65
45
36
11
0.034
61
20
1
(CH2)2OH
(CH2)2NH2
0.44
>20
19
4
0.2
0.8
1.3
0.2
0.8
1.5
7.0
a
Figure 1. Profiles of the first generation inhibitor 1. Human
ROR luciferase (LUC) assay. Values of EC50 are mean values
determined from at least 3 replicates. Positive control OR-
1050 showed EC50 value of 0.19 ±0.048 (mean ±SD), n=19.18
bMetabolic stability (MS) in human liver microsomes after 60
5
(CH2)2NMe2
6
(CH2)2NHAc
(CH2)2COOH
(CH2)2CONH2
(CH2)2CONHMe
(CH2)2CONMe2
11
7
>20
16
8
9
6.4
2.7
10
minutes of incubation. Value is the remaining
% of
compound 1. cApparent permeability coefficients.
aValues of EC50 are mean values determined from at least 3
replicates. Positive control OR-1050 showed EC50 value of
0.19 ±0.048 (mean ±SD), n=19.18 bMS in human liver
In the preceding paper, the drug-likeness properties
represented by ligand efficiency (LE) and the fraction of sp3
carbons (Fsp3) attained their respective benchmark
scores,19,20 thus indicating another approach was needed to
overcome the moderate in vivo potency. Herein, we report
the successful efforts in our ROR program focused on the
improvement of PK profiles, which led to discovery of the
second generation inhibitors composed of an azole scaffold.
microsomes.
Compounds 3–10 are racemic.
cApparent
permeability
coefficients.
RESULTS AND DISCUSSION
The ligand binding pocket of ROR is heavily hydrophobic,21
thus suggesting that an increase in in vitro potency could be
theoretically achievable by accommodation of lipophilic
motifs in the binding pocket. However, such a tactic may
sacrifice metabolic stability of the ligand, therefore pursuing
such van der Waals interactions was not a practical option in
Figure 2. (A) Docking result of 7. The docked 7 (white) was
superimposed with a first generation ROR inhibitor (yellow)
in complex with ROR (PDB code 5AYG).24
this case. Alternatively, implementation of
a
polar
functionality outside the binding pocket is a viable strategy
and a number of successful examples have been reported in
the literature.22,23 The X-ray co-crystal structure of our first
generation inhibitor17 suggested a substituent at the -
position of the propanoyl moiety would project toward the
outside the pocket (Figure 2), so we introduced substituents
bearing a functional group at the terminus and examined
their effects on metabolic stability, as well as other
pharmacokinetic parameters.
We undertook
a
systematic analysis of their
physicochemical characteristics. If the nature of the binding
pocket was taken into consideration, the lipophilicity of the
compounds could affect the binding affinity toward ROR
Actually, significant reduction of LUC potency was observed
once LogD of these compounds fell below a threshold value
(roughly 3.3), suggesting
a possible cut-off value of
lipophilicity to achieve good binding (Figure 3).
To conduct such modifications, compound 2, a surrogate
analogue of 1 served as a model compound in view of the
synthetic tractability. Varieties of substituents allocating a
functional group at the terminus were examined (Table 1),
and the alkyl amines (4, 5) as well as the alkanoic acid (7)
were identified as effective boosters to improve metabolic
stability. Unfortunately, their potencies, as measured by the
LUC assay to determine EC50, were significantly worse even
though favorable binding was presumed based on the crystal
structure of compound 1. We hypothesized that factors other
than the presence of the substituents themselves were
hampering the accommodation of the new compounds in the
binding pocket.
Figure 3. A threshold value of LogD. Light pink dots
represent other close congeners whose structure and profiles
were listed in the section of supporting information.
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Driven by such a speculation, we decided to synthesize
Table 2. Discovery of a Potent and Metabolically Stable
Scaffold
more lipophilic analogues bearing the propanoic acid
substituent, the most metabolically beneficial substituent
listed in Table 1. Initially, compound 11 composed of an
isoxazole scaffold was synthesized, in which the ethylene
motif as well as the aniline part were simultaneously
modified with an aim to minimize plausible metabolic sites.
As expected, 11 showed an increased LogD, exceeding the
hypothetical cut-off value. This compound displayed both
decent LUC potency and good metabolic stability. We
became confident that 4-(isoxazole-3-yl)butanoic acid is a
desirable platform, and further optimization of 11 was
conducted to identify the second generation ROR inhibitors
composed of an azole core scaffold.
Cl
H
N
O
O
N
Me
Ar
O
Me
Me
O-Na+
Caco2
Pappd
(10-6 cm/sec)
b
c
EC50
MS
compd
Ar
LogD
(%)
80
74
54
75
82
82
54
90
78
(µM)
0.044
0.014
0.012
0.66
11a
12
13
14
15
16
17
18
19
4.8
3.9
3.8
3.7
4.0
3.8
3.8
3.6
3.4
16
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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36
28
32
28
34
34
25
10
O
N
N
O
O
N
S
0.12
To improve the potency of 11 as measured by the LUC assay,
we focused on the benzene part on the central isoxazole core.
Based on the previous exploration, this location was
considered to favor a flattened architecture, so a set of
smaller-sized aromatic compounds were examined as
benzene alternates. As shown in Table 2, various heterocycles
were applied and isoxazole derivatives (12, 13) showed
improved LUC potency while the transformation to the 2, 5-
disubstituted oxazole (14) or thiazole (15) resulted in
significant reductions of the potency. Given the hydrophobic
nature of the pocket, several subsites in the pocket were
presumed to disfavor a polar environment, and we observed
a tendency that the presence of a hetero atom led to
reduction of the potency. Additionally, two regioisomeric
thiazole analogues (16, 17) showed contrasting outcomes in
LUC potency. The terminal isobutyl group of each compound
must project toward a different location due to the presence
of a longer bond length, particularly the C-S bond, and the
terminal substituent of 16 was likely positioned at the
mismatched site while 17 allocated the same substituent at
the right location.
N
N
3.8
S
N
O
S
N
0.017
0.062
0.056
N
S
N
N
aSalt-free form. bEC50 are mean values determined from at
least 3 replicates. Positive control OR-1050 showed EC50 value
of 0.19 ±0.048 (mean ±SD), n=19.18 cMS in human liver
microsomes. dApparent permeability coefficients.
Table 3. Optimization of Terminal Substituents
R3
H
O
N
R2
O
N
O
O
R4
N
O
- Na+
a
b
EC50
(µM)
MS
compd
R2
Me2CHCF2
R3
R4
LogD
(%)
20
21
22
23
24
25
26
27
Cl
Cl
Cl
Cl
Cl
Cl
Cl
F
Me 0.007 4.0
Me 0.008 4.3
Me 0.008 4.4
Me 0.007 4.2
74
79
78
84
96
77
90
107
Me2CHCH2CH2
Me2CHCH2CF2
Me3CCH2
Once the 3, 5-disubstituted isoxazole was identified as a
favorable motif for the Ar part with the best balance between
LUC potency and metabolic stability, the final optimization
was conducted with 12 by modulating both terminal
substituents (Table 3). Firstly, an additional carbon atom was
introduced at the isobutyl moiety, and a slight increase in
LUC potency was observed regardless of the difference in the
location (21, 23). In order to further improve metabolic
Me3CCH2
Cl
0.009 4.2
Me3CCF2
Me 0.006 4.3
Me3CCF2
F
0.006 4.0
0.007 4.2
Me3CCF2
Cl
aValues of EC50 are mean values determined from at least 3
replicates. Positive control OR-1050 showed EC50 value of
0.19 ±0.048 (mean ±SD), n=19.18 bMS in human liver
microsomes.
stability, halogen atoms were incorporated.
A simple
fluorination of the substituents on the isoxazole had virtually
no effect on the stability (20, 22 and 25), while di-halo-
anilide analogues showed better metabolic stability (24, 26
and 27).
Since the LUC potency and the metabolic stability had been
improved by our modifications to the first generation
structure 1, we selected compound 24 to conduct in vivo
studies. Firstly, the experimental allergic encephalomyelitis
(EAE) model was selected to assess the pharmacodynamic
(PD) effects of 24 since Th17-derived IL-17 is recognized as a
major mediator in EAE. Mice were immunized by an addition
of myelin oligodendrocyte glycoprotein (MOG), complete
Freund’s adjuvant and pertussis toxin (PTX), then IL-17
production was further boosted by restimulation with anti-
CD3 antibody.17 At 8 h after the oral administration, the
plasma IL-17 level of each mouse was analyzed and dose-
dependent suppression of IL-17 production was observed
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(Figure 4A). The ED50 value of 24 was calculated as 3 mg/kg,
triazole core, the method was almost identical to the one
previously reported17 except for including extra processes for
pendant group modifications. Meanwhile, the second
generation ROR inhibitors typified by the isoxazole core
structure were synthesized through alkynones or 4-
cyclopropyl-3,5-di-alkylated ioxazoles, which we believe is an
efficient strategy for synthesizing fully-substituted isoxazoles
in a regio-selective manner.27
achieving roughly a 10-fold improvement in comparison to 1
in this pharmacodynamic (PD) model. To further confirm
the value of this compound, disease-modifying effects were
evaluated in the IL-23-induced mouse dermatitis model.25,26
Dermal inflammation was induced by IL-23 injection, and
mice were subjected to multiple oral dosing once daily for
eleven days. The ear thickness of each mouse was measured
on day 1, 5, 8, 10 and 12, and significant reduction of ear
swelling was observed for compound treatment groups
(Figure 4B).
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
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36
37
38
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40
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44
45
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Scheme 1. Synthesis of Compounds 3-27
Me
Me
Me
O
O
Me
N
Me
N
O
(A)
a
O
N
OEt
·TfOH
+
N
OEt
NH2
Me
HN
28
29
30
Me
Me
H
N
O
N
N
c
b
N
Me
3
10
-
31
O
OtBu
O
O
OtBu
O
O
d
e
HO
(B)
Me
Me
Me
Me
Ar1
N
OBn
O
OBn
OBn
32
33
34a
34b
34c
(4-phenyl)
(2-thiazol-4-yl)
(2-thiazol-5-yl)
Cl
H
O
OtBu
O
N
O
O
N
N
Me
Me
f
g
Ar1
Ar1
O
Me
Me
Me
OBn
O-Na+
35a
35b
35c
11*
(4-phenyl)
(4-phenyl)
(2-thiazol-4-yl)
(2-thiazol-5-yl)
16
17
(2-thiazol-4-yl)
(2-thiazol-5-yl)
*Salt-free form
Cl
H
O
OtBu
O
N
O
O
N
HO
N
Me
Ar2
h
i
33
Me
Figure 4. Effect of compound 24 in (A) CD3-induced mouse
PD model. aNon-immunized (neither MOG nor complete
Freund’s adjuvant nor PTX treatment) group. (B) IL-23-
induced mouse dermatitis model. bNon-IL-23-treatment
group.
Me
OBn
OBn
38a
(3-isoxazol-5-yl)
(5-oxazol-2-yl)
(5-thiazoe-2-yl)
(5-(1,2,4-oxadiazol)-2-yl)
(5-thiadiazol-2-yl)
37
38b
38c
38d
38e
j
OH
O
OtBu
O
OtBu
High ROR specificity of 24 was also confirmed by the
absence of inhibitory activity against other nuclear receptors
(EC50>20 M; hROR, mLXR, hRXR, hPPAR, hPPAR) and
the lack of time-dependent CYP inhibition properties
(IC50>50 M; hCYP3A4m, hCYP3A4t, hCYP2C9, hCYP2D6,
hCYP1A2, hCYP2C19). Compound 24 showed greatly
improved PK profiles compared to our first generation
inhibitors (Table 4), suggesting it was indeed a promising
preclinical candidate as a selective ROR inhibitor.
O
N
O
N
HO
N
R5
k
O
Cl
N
OBn
OBn
5
39
40a
40b
40c
40d
40e
(R =Me2CH)
5
(R =Me2CH)
l
m
5
(R =Me3C)
5
(R =Me3C)
R3
5
(R =Me3C)
H
O
N
R2
O
N
n
R4
12 - 15, 18 - 27
O
38a-e, 41a-i
N
OBn
2
3
4
2
3
4
41a
41b
41c
41d
41e
41f
(R =Me2CHCH2, R =Cl, R =Me)
(R =Me2CHCF2, R =Cl, R =Me)
2
3
4
2
3
4
Table 4. PK parameters of 24 in rats
(R =Me2CHCH2CH2, R =Cl, R =Me)
(R =Me2CHCF2, R =Cl, R =Me)
2
3
4
2
3
4
41g
41h
41i
(R =Me3CCH2, R =Cl, R =Me)
(R =Me3CCF2, R =Cl, R =Me)
2
3
4
2
3
4
(R =Me3CCH2, R =Cl, R =Cl)
(R =Me3CCF2, R =Cl, R =F)
iv parameters (1.0 mg/kg)
po parameters (3.0 mg/kg)
2
3
4
(R =Me3CCF2, R =F, R =Cl)
AUCc
MRTd
Fe
a
b
t1/2
(h)
Cltot
(L/h/kg) (L/kg)
0.25 0.76
Vdss
( M•h)

(h)
(%)
Reagents and conditions: (a) Et3N, 1,4-dioxane; (b) (i) tert-
butyl bromoacetate, NaH, THF; (ii) allyl iodide, NaHMDS,
THF; (iii) 4 M NaOH, EtOH; (iv) TFA, CHCl3, MeCN; (v) 2,4-
dimethylaniline, HATU, Et3N, DMF; (c) see supporting
information for detailed protocol to introduce respective
functional groups. (d) (i) (R)-4-benzyloxazolidin-2-one,
WSC•HCl, DMAP, MeCN; (ii) LDA, tert-butyl bromoacetate,
4.2
15
4.9
72.0
aTotal body clearance. bVolume of distribution at steady state.
cArea under the curve. dMean residence time. eBioavailability.
ROR inhibitors reported in this manuscript were prepared
according to Scheme 1. Regarding compounds bearing a 1,2,4-
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THF; (iii) H2O2, 4M LiOH, THF; (iv) (R)-1-phenylethan-1-
General. The purity of all of the tested compounds was
determined by HPLC (SHIMADZU Prominence) and was >
95%. The chemistry, experimental information and
spectroscopic data for the target compounds were supplied
in the Supporting Information. The representative procedure
of the final transformation to afford the target compounds
was presented as exemplified by the synthesis of 24.
amine, MTBE; (v) KHSO4, EtOAc, H2O; (vi) MeONHMe•HCl,
WSC•HCl, HOBt•H2O, i-Pr2NEt, MeCN; (e) aryl alkyne, n-
BuLi, THF; (f) (i) MeONH2•HCl, Na2CO3, EtOH; (ii) ICl,
CH2Cl2; (iii) cyclopropylboronic acid pinacol ester, K3PO4,
PdCl2(PPh3)2, DMF; (g) (i)H2, Pd(OH)2/C, MeOH, THF; (ii)
TEMPO, NaClO2, NaClO, MeCN, phosphate buffer (pH 6.8);
(iii) MeI, K2CO3, DMF; (iv) TFA, CHCl3; (v) SOCl2, 2-chloro-
4-methyl aniline, DMA; (vi) 2M NaOH, MeOH, THF; (h) (i) i-
Bu2AlH, toluene, THF; (ii) H2NOH•HCl, 4M NaOH, EtOH,
THF, H2O; (iii) NCS, DMF, toluene; (iv) 3-bromoprop-2-yn-1-
ol, K2CO3, toluene, H2O; (v) cyclopropylboronic acid pinacol
ester, K3PO4, PdCl2(PPh3)2, DMF; (i) see supporting
information for detailed protocol to construct the Ar2 moiety,
heteroaromatic rings; (j) (i) DMP, CHCl3; (ii) NH2OH•HCl,
4M NaOH, EtOH, THF, H2O; (iii) NCS, DMF; (k) alkyl
alkyne-3-ol, K2CO3, toluene, H2O; (l) (i) DMP, CHCl3; (ii)
Deoxo-Fluor®, CH2Cl2; (iii) TFA, toluene; (iv) SOCl2, 2,4-
disubstituted aniline, DMA; (m) (i) alkyl alkyne, K2CO3,
toluene, H2O; (ii) TFA, toluene; (iii) 2,4-disubstituted aniline,
HATU, i-Pr2NEt, DMF; (n) (i) BBr3, CH2Cl2; (ii) TEMPO,
NaClO2, NaClO, MeCN, phosphate buffer (pH 6.8); (iii) 2M
NaOH, EtOH.
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Sodium (S)-4-(4'-cyclopropyl-5-neopentyl-[3,5'-biisoxazol]-3'-
yl)-6-((2,4-dichlorophenyl)amino)-6-oxohexanoate (24)
To a solution of 41d (264 mg, 0.432 mmol) in CH2Cl2 (4.0
mL) was added 1.0 M BBr3 in CH2Cl2 (1.1 mL, 1.08 mmol)
dropwise at −78 °C. The reaction mixture was warmed to
room temperature over 20 min and stirred for another 10 min.
Saturated aqueous NaHCO3 was added to the reaction at 0 °C
and extracted with EtOAc. The organic layer was washed
with brine, and then dried over Na2SO4. After filtration and
removal of the solvent under reduced pressure, the residue
was purified by flash chromatography (EtOAc:n-hexane = 1:4
to 1:1 (v/v)). To a solution of the debenzylated compound
(180 mg, 0.346 mmol) in MeCN (1.3 mL) and 1.0 M phosphate
buffer (pH=6.8, 1.3 mL) were added TEMPO (5.4 mg, 0.0346
mol) and NaClO2 (78 mg, 0.692 mmol). Sodium hypochlorite
solution (90 µL) was added dropwise to the reaction mixture
at 0 °C and stirred at room temperature for 1 h. The reaction
mixture was quenched with 20% aqueous Na2SO3 at 0 °C and
extracted with EtOAc. The organic layer was washed with 1M
HCl, water and brine, and then dried over Na2SO4. After
filtration and removal of the solvent under reduced pressure,
the residue was purified by preparative TLC (CHCl3:MeOH =
12:1 (v/v)) to give the corresponding carboxylic acid (161 mg,
70 % yield). To a solution of this compound (161 mg, 0.302
mmol) in EtOH (1.0 mL) was added 1 M NaOH (302 µL, 0.302
mmol). After stirring at room temperature for 30 min, the
reaction mixture was concentrated under reduced pressure
to give the title compound 24 (168 mg, quantitative).
The binding conformation of 24 in ROR was subsequently
confirmed by the X-ray co-crystal analysis, in which the
metabolically beneficial carboxylate was proven to project
outside the pocket as originally expected (Figure 5).
1H NMR (400 MHz,DMSO-d6) δ: 0.43–0.55 (m, 1H), 0.59–0.72
(m, 1H), 0.85–1.02 (m, 2H), 0.95 (s, 9H), 1.67–1.78 (m, 1H),
1.78–1.98 (m, 4H), 2.76 (s, 2H), 2.82 (dd, J = 15.20, 6.80Hz, 1H),
2.90 (dd, J = 15.20, 8.40Hz, 1H), 3.47–3.59 (m, 1H), 6.73 (s, 1H),
7.34 (dd, J = 8.80, 2.40Hz, 1H), 7.59 (d, J = 2.40Hz, 1H), 7.61 (d,
J = 8.80Hz, 1H), 10.03 (brs, 1H). HRMS m/z: [M−Na+2H]+
calcd for C26H30N3O5Cl2, 534.1557; found, 534.1555. HPLC
purity: 97.2 %. [α]20D -2.5 (c 0.40, MeOH).
Figure 5. Cocrystal structure of 24 in ROR PDB code 6IVX).
CONCLUSION
We have identified potent and metabolically stable ROR
inhibitors composed of an isoxazole scaffold. In order to
overcome a general shortcoming of ROR inhibitors, their
poor metabolic stability, we strategized to introduce
metabolically beneficial functionalities at the outside of the
CD3-induced mouse PD model17
binding
pocket.
Although
our
first
generation
Peptides derived from myelin oligodendrocyte glycoprotein
(MOG35–55) were dissolved in D-PBS (−) at concentration of 3
mg/mL, and then emulsified with an equal volume of
complete Freund’s adjuvant. Female C57BL/6 mice were
immunized with the MOG35–55 emulsion by subcutaneous
injection at two sites on the back (75 μg of MOG35–55 per site)
and 0.2 μg/200 μL of pertussis toxin (PTX) in D-PBS (−) was
injected intraperitoneally on day 1. On day 3, the same
amount of PTX was injected. On day 5, 1 μg / 200 μL of the
anti-CD3 antibody dissolved in saline was injected
intravenously to induce IL-17 production. The blood samples
ROR inhibitors were not compatibility to such pendant
groups, those isoxazole analogues realized good potency as
well as high metabolic stability. Furthermore, the
representative compound 24 was proven to achieve both
high ROR specificity and improved in vivo potency.
Compound 24 is expected to be an ideal probe to accelerate
the future research of ROR pharmacology.
EXPERIMENTAL SECTION
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lineage of CD4
T cells regulates tissue inflammation by
were collected 2 hours after the CD3 injection, then the IL-
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relationships of quinoline tertiary alcohol modulators of RORγt.
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Yukawa, T.; Tokuhara, H.; Skene, R.; Sang, B. C.; Hoffman, I. D.;
Snell, G. P.; Uga, K.; Shibata,A.; Igaki, K.; Nakamura, Y.;
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dimethyl-2,3-dihydro‑1H‑inden-5-yl)carbamoyl]-2-methoxy-7,8-
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acid (TAK-828F) as a potent, selective, and orally available novel
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Hawkins, J.; Johnson, A. R.; Liimatta, M.; Lockey, P.; Norman,
M.; Wong, H. Identification of tertiary sulfonamides as RORc
inverse agonists. Bioorg. Med. Chem. Lett. 2014, 24, 2182–2187. (b)
Fauber, B. P.; René, O.; de Leon Boenig, G.; Burton, B.; Deng, Y.;
Eidenschenk, C.; Everett, C.; Gobbi, A.; Hymowitz, S. G.; Johnson,
A. R.; La, H.; Liimatta, M.; Lockey, P.; Norman, M.; Ouyang, W.;
Wang, W.; Wong, H. Reduction in lipophilicity improved the
solubility, plasma–protein binding, and permeability of tertiary
IL-23-induced dermatitis model25,26
Intradermal injections of 250 ng recombinant mouse IL-23
(R&D Systems, Minneapolis, MN) in 20 μL PBS containing
0.1% BSA were performed into the left ear of anesthetized
mice on days 1, 3, 5, 8 and 10. Compound 24 (3 and 30 mg/kg)
or 0.5% MC were administered orally at a volume of 10
mL/kg every day from day 1 to 11. On Days 1, 5, 8, 10 and 12,
the left ear thickness was measured using a digital thickness
gauge (Digimatic Indicator IDA-112M, Mitutoyo Corporation,
Kawasaki, Japan). The increase in the ear thickness from
baseline value (the ear thickness on Day 1) was defined as the
change in the ear thickness.
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ASSOCIATED CONTENT
Supporting Information
The Supporting information is available free of charge on the
ACS Publications website at DOI:
Synthetic procedures, experimental data, docking protocol,
assay procedures and PK profiles of 24 (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*For M.K.: E-mail, masayuki.kotoku@jt.com
*For M.S.: E-mail, makoto.shiozaki@jt.com
ORCID
Masayuki Kotoku: 0000-0003-2245-1673
Makoto Shiozaki: 0000-0001-5519-6547
Funding Sources
The authors declare no competing financial interest.
ABBREVIATIONS
RORγ, retinoic acid receptor-related orphan receptor
gamma; ADME, absorption, distribution, metabolism,
excretion; CYP, cytochrome P450; LXR, liver X receptor;
RXR, retinoid X receptor; PPAR, peroxisome proliferator
activated receptor; DMP, Dess-Martin periodinane; TEMPO,
2,2,6,6-tetramethylpiperidine
[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-
b]pyridinium 3-oxide hexafluorophosphate;
1-oxyl;
HATU,
1-
TfOH,
trifluoromethanesulfonic acid.
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Table of Contents Graphic
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