Discovery of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as potent RORγt inverse agonists

Article abstract of DOI:10.1016/j.bmc.2015.07.068

A novel series of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as RORγt inverse agonists was discovered. Binding mode analysis of a RORγt partial agonist (2c) revealed by co-crystal structure in RORγt LBD suggests that the inverse agonists do not directly interfere with the interaction between H12 and the RORγt LBD. Detailed SAR exploration led to identification of potent RORγt inverse agonists such as 3m with a pIC₅₀ of 8.0. Selected compounds in the series showed reasonable activity in Th17 cell differentiation assay as well as low intrinsic clearance in mouse liver microsomes.

Full text of DOI:10.1016/j.bmc.2015.07.068

Accepted Manuscript
Discovery of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as potent RORγt inverse
agonists
Yonghui Wang, Ting Yang, Qian Liu, Yingli Ma, Liuqing Yang, Ling Zhou,
Zhijun Xiang, Ziqiang Cheng, Sijie Lu, Lisa A. Orband-Miller, Wei Zhang,
Qianqian Wu, Kathleen Zhang, Yi Li, Jia-Ning Xiang, John D. Elliott, Stewart
Leung, Feng Ren, Xichen Lin
PII:
S0968-0896(15)00652-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.07.068
BMC 12497
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 May 2015
29 July 2015
30 July 2015
Please cite this article as: Wang, Y., Yang, T., Liu, Q., Ma, Y., Yang, L., Zhou, L., Xiang, Z., Cheng, Z., Lu, S.,
Orband-Miller, L.A., Zhang, W., Wu, Q., Zhang, K., Li, Y., Xiang, J-N., Elliott, J.D., Leung, S., Ren, F., Lin, X.,
Discovery of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as potent RORγt inverse agonists, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.07.068
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Bioorganic & Medicinal Chemistry
Discovery of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as potent RORt inverse
agonists
Yonghui Wanga,, Ting Yangb, Qian Liub, Yingli Mab, Liuqing Yangb, Ling Zhoub, Zhijun Xiangb, Ziqiang
Chengb, Sijie Lub, Lisa A. Orband-Millerb, Wei Zhangb, Qianqian Wub, Kathleen Zhangb, Yi Lib, Jia-Ning
Xiangb, John D Elliottb, Stewart Leungb, Feng Renb, and Xichen Linb,*
aSchool of Pharmacy, Fudan University, 826 Zhangheng Road, Pudong, Shanghai 201203, China
bResearch and Development, GlaxoSmithKline, No. 3 Building, 898 Halei Road, Pudong, Shanghai 201203, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A novel series of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as RORt inverse agonists was
discovered. Binding mode analysis of a RORt partial agonist (2c) revealed by co-crystal
structure in RORt LBD suggests that the inverse agonists do not directly interfere with the
interaction between H12 and the RORt LBD. Detailed SAR exploration led to identification of
potent RORt inverse agonists such as 3m with a pIC50 of 8.0. Selected compounds in the series
showed reasonable activity in Th17 cell differentiation assay as well as low intrinsic clearance in
mouse liver microsomes.
Available online
Keywords:
RORt inverse agonists
Th17 cell differentiation
Crystal structure
2009 Elsevier Ltd. All rights reserved.
Thiazole ether amides
1. Introduction
1). Subsequent SAR exploration on general structure of N-(4-
aryl-thiazol-2-yl) amide (2) led to a number of potent RORt
T helper (Th) 17 cells, a lineage of CD4+ effector T cells
characterized by the production of IL-17A and IL-17F, are
pathogenic in human autoimmune inflammatory diseases,
including multiple sclerosis (MS).1-4 Differentiation of Th17 cells
is controlled by the transcription factor retinoic acid receptor-
related orphan receptor-gamma-t (RORt).5,6 RORt is a member
of the nuclear receptor (NR) superfamily, and its functional
activity is realized through the recruitment of transcriptional co-
activators or co-repressors as the consequence of the ligand
binding to the ligand binding domain (LBD).5 RORt inhibitors
has potential utility in reducing the activity of Th17 cells and
therefore can be developed as therapeutic agents for the treatment
of Th17-mediated autoimmune diseases.7-13
inverse agonists including thiazole ketone amides (2, R2 =
substituted acyl groups).27 In this paper, we report the discovery
of another novel N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides as
potent RORt inverse agonists (2, R2 = aryloxy groups).
Figure 1. RORt HTS hit 1 and general structure of N-(4-aryl-
thiazol-2-yl) amides 2
2. Result and discussion
A few small molecule inhibitors against RORt have been
reported in literature.14 Digoxin,15 SR100116 and Ursolic acid17
were firstly reported to inhibit RORt and ameliorate EAE in
mice via intraperitoneal administration. Afterwards, more small
molecular RORt inhibitors18-26 were disclosed and some of them
were shown to suppress Th17 cell differentiation in vitro.
Recently, we reported the identification of a novel RORt inverse
agonist hit (1) through high throughput screening (HTS) (Figure
SAR of the thiazole amides was first explored with 5-
substitution on the thiazole ring (Table 1).27,28 RORt potency
(pXC50) and maximum response (%max) were measured in both
FRET and dual FRET assays.29,30 While the FRET assay is useful
to identify antagonists, the dual FRET assay in which a surrogate
agonist is not added allows the detection of both agonists and
inverse agonists based on the changes in the basal level of RORt
activity.30 As we observed before,27 addition of small alkyl
———
 Corresponding author. Tel.: +86-21-5198-0118; fax: +86-21-5198-0118; e-mail: yonghuiwang@fudan.edu.cn (Wang, Y.); Tel.: +86-21-
6159-0718; fax: +86-21-6159 0730; e-mail: xichen.2.lin@gsk.com (Lin, X.)

groups such as Me (2a) and Et (2b) at 5-position of the thiazole
ring did not change much of RORt activity in both FRET and
dual FRET assays. However, replacing the small alkyl moiety
with a bulky phenyl group (2c) increased RORt potency and
decreased much of maximum percentage of inhibition in FRET
assay. Furthermore, 2c activated RORt with a pEC50 of 5.5 and
maximum percentage activation of 150% in dual FRET assay.
Data in both assays indicated that 2c is a partial agonist. Other 5-
phenyl substituted thiazole amides such as 2d show similar
partial agonist profile, indicating the potential association of
RORt partial agonism with the bulky phenyl substituent at 5-
thiazole ring.
inverse agonists. The relative binding modes of the two
compounds 2d and 2f proposed using molecular docking were
shown in Figure 3. Phenyl ether 2f showed better RORt
activities than benzyl compound 2e, and served as a new starting
point for further optimization.
To understand the binding mode of the thiazole amides in
RORt LBD and guide activity and property optimization of the
series, we attempted co-crystallization of the thiazole amides
with RORt LBD. To our delight, a crystal structure of RORt
LBD in complex with 2c was resolved at a resolution of 2.0 Å
(PDB accession code: 4XT9), as shown in Figure 2.28 The overall
binding motif of 2c in RORt LBD is similar to a previously
reported RORt agonist.30 Two H-bonding interactions were
formed: one between the ligand sulfone moiety with Arg367 as
well as the backbone NH of Leu287, and the other between the
ligand linker amide NH and a backbone carbonyl of Phe377.
Two ligand phenyl rings are in π-π stacking with Phe377 and
Phe388, respectively. In addition, the thiazole ring shows π-π
stacking with Phe378. The phenyl group at 5-position of the
thiazole ring occupies the hydrophobic pocket near AF2 domain
(H12) and thus contributes to the activation of RORt by
stabilizing the AF2 domain toward steroid receptor co-activator
(SRC) recruitment. Comparing to a full RORt agonist such as
20-hydroxycholesterol (pdb accession code: 3KYT), the 5-phenyl
moiety only partially occupies the inner hydrophobic pocket
which makes 2c a partial agonist (Figure 2).
Figure 2. Co-crystal structure of RORt LBD complexed with
2c (in orange) and with 20-hydroxycholesterol (in blue)
Table 1. SAR of N-(4-aryl-5-substituted-thiazol-2-yl)-amides
Figure 3. Structural overlay of 2d (in orange stick) and 2f (in
green stick) in RORt LBD
A series of phenyl ether analogs were designed and prepared
to explore SAR of the LHS moiety to improve potency of the
amides. The SAR of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides
was summarized in Table 2. Addition of a substituent such as Cl
(3a), CF3 (3b) and OMe (3c) to 2-position of the O-linked phenyl
ring improved RORt potency. Interestingly, with the increase in
size of the 2-substituent, the maximum percentage of inhibition
decreased. While 3a is an inverse agonist, 3c exhibited partial
agonist activity as evidenced in dual FRET assay. To understand
the difference, we analyzed the predicted binding modes of 3a
and 3c shown in Figure 4. The overlay shows that the 2-methoxy
group of 3c occupies more area of the inner hydrophobic pocket
near AF2 domain than the Cl moiety in 3a. This better
occupation stabilizes the AF2 domain further, and makes 3c a
partial agonist. The finding that a minor structural change to a
tertiary sulfonamide RORt ligand led to distinct mechanisms of
action was recently reported.31 We previously showed that
increasing size of a substituent on the phenyl ring of a tertiary
amine RORt agonist could convert the agonist to an inverse
agonist.30 In this paper, it is the first time we observed that
increasing size of a substituent on the phenyl ring of a thiazole
ether RORt inverse agonist could convert the inverse agonist to
an agonist.
FRETa pIC50
(% max)b
dual FRETa
pXC50 (% max)
Cpd
R1
R2
H
1
2,5-di-Cl
6.0 (116)
6.0 (91)c
2a
2b
2c
2,5-di-Cl
Me
Et
5.4 (79)
5.6 (115)
6.8 (56)
5.9 (46)
6.1 (87)
6.3 (102)
5.7 (58)c
5.9 (76)c
5.5 (150)d
5.4 (150)d
6.7 (42)c
7.2 (50)c
2,5-di-Cl
2,5-di-Cl
Ph
2d
2ee
2fe
H
H
H
Ph
CH2Ph
OPh
aData are the average of at least two determinations; b% max
inhibition measured against activation by the surrogate agonist;
cpIC50 (% max inhibition); pEC50 (% max activation); compounds
d
e
2e and 2f were 7d and 7e in ref. 27, respectively.
Previously, we reported that when 5-substituents on the
thiazole ring are benzyl (2e) and phenoxy (2f), the compounds
become inverse agonists.27 This inverse agonism can be
explained by the binding mode of 2c or 2d. Insertion of a CH2 or
O linker between the thiazole ring and 5-phenyl group would
force the phenyl group away from the inner hydrophobic pocket
and thus may not stabilize the AF2 domain enough, leading to
The compounds with the same substituents at 4-position of the
O-linked phenyl ring (3d-3f) showed slightly lower RORt
activity in FRET assay and similar RORt activity in dual FRET

Table 2. SAR of N-(4-aryl-5-aryloxy-thiazol-2-yl)-amides
a
a
e
Compd
R1
R2
FRET pIC50
Dual FRET pXC50
(%max)
Th17 pIC50
Mouse Cint.
(ml/min/g)
0.46
(%max)b
H
H
2-Cl
7.0 (112)
7.9 (58)c
5.8
6.0
3a
3b
2-CF3
7.1 (101)
7.2 (49)
7.8 (52)c
<0.36
2.37
H
2-OMe
7.8 (33)d
6.6
3c
H
H
4-Cl
4-CF3
6.8 (91)
6.8 (117)
6.5 (97)
8.1 (31)c
7.7 (60)c
7.7 (45)c
7.4 (57)c
7.9 (52)c
7.5 (53)c
7.9 (40)c
6.4 (85)c
7.7 (79)c
7.7 (61)c
5.8
6.2
<5
5.4
5.8
5.7
6.6f
-
<0.36
< 0.36
0.39
<0.36
-
3d
3e
3f
H
4-OMe
H
4-Cl
6.6 (115)
6.7 (106)
7.2 (107)
7.3 (98)
3g
3h
3i
3-Cl
H
3-Cl
2-CF3
<0.36
<0.36
-
3-CN
3-CH2NMe2
3-CN
3-CN
2-CF3
3j
3kg
2-CF3
5.9 (120)
7.5 (111)
8.0 (110)
2-Cl, 4-CF3
2-CF3, 4-Cl
6.4
6.8
<0.36
<0.36
3l
3m
aData are the average of at least two determinations; b% max inhibition measured against activation by the surrogate agonist; cpIC50 (% max inhibition); dpEC50
(% max activation); eunless noted, n=1; fn=3; gobtained as TFA salt
assay. Introduction of a Cl group at 3- or 4-position of 4-phenyl
attached to thiazole ring (3g and 3h) increased RORt activity
over 2f. With a CF3 at the 2-position of 5-phenoxy and an
additional substituent at 3-position of 4-phenyl, 3i (3-Cl) and 3j
(3-CN) maintained good RORt activities over 3b (3-H).
However, addition of a polar group such as CH2NMe2 (3k)
lowered the RORt activity by more than one log unit. Based on
above findings, we designed 3l and 3m by combining the best
substituents on 4-phenyl and 5-phenoxy rings. As expected, both
compounds showed excellent RORt activity. For example,
compound 3m exhibited a pIC50 of 8.0 and 7.7 in FRET assay
and dual FRET assay, respectively.
3. Chemistry
Synthetic route for preparation of phenyl ether analogs 3 is
shown in Scheme 1.32 The 4-arylthiazol-2-amines 5 were
prepared from aryl methyl ketones 4 through a one pot iodination
and cyclization cascade reaction. Bromination of 5 followed by a
nucleophilic addition with substituted phenols, formed 4-aryl-5-
aryloxythiazol-2-amines 7. Amide formation of amines 7 and
acid 8 afforded the targeted compounds 3.
Scheme 1. Reagents and conditions: (a) iodine, thiourea, 100 °C;
(b) CuBr2, acetonitrile; or NBS, THF; (c) substituted phenol,
Cs2CO3, acetone, 55 °C; (d) EDC, HOBt, DCM
Figure 4. Structural overlay of 3c (in cyan stick) and 3a (in
pink stick) in RORγt LBD
4. Conclusion
In summary, we have discovered N-(4-aryl-5-phenyl-thiazol-
2yl)-amides as novel RORt inverse agonists. The binding mode
of the partial agonist 2c was experimentally determined through
co-crystallization with RORt LBD, which served as the basis for
explanation of RORt agonism and inverse agonism determined
using dual FRET assay. Compound 3m was discovered as a
potent RORt inverse agonist with pIC50 of 8.0 in FRET assay
and pIC50 of 6.8 in Th17 assay. In addition, selected compounds
The representative compounds in the series were selected for
evaluation in the mouse Th17 cell differentiation assay.27,29 Most
of the compounds showed reasonable Th17 cellular activity
(Table 2). Compound 3m showed a pIC50 of 6.8 in Th17 assay.
We also examined metabolic stability of representative
compounds in mouse liver microsomes. As shown in Table 2, the
intrinsic clearance is generally low for most of the compounds.

in the series also demonstrated good metabolic stability.
However, compared to the biaryl amide RORt inverse agonists
recently disclosed,33 the current thiazole ether compounds did
have high Th17 cellular potency that is believed to be important
for achieving in vivo efficacy in any Th17-mediated disease
model. Further optimization of the series to improve Th17
cellular potency is undergoing.
of solvent under vacumn, the obtained crude was purified by
flash chromatography eluting with EtOAc:Petro = 0:1 to 1:1. The
collected fractions were concentrated and redissolved in THF.
The sample was submitted to MDAP for further purification to
give the desired product (1.24 g, 22% yield). 1H NMR (600 MHz,
DMSO-d6) δ ppm 12.65 (br. s., 1H), 7.94 (d, J = 2.6 Hz, 1H),
7.86 (d, J = 8.4 Hz, 2H), 7.78 (s, 1H), 7.62 (d, J = 8.4 Hz, 2H),
7.60 (d, J = 8.4 Hz, 1H), 7.45 (dd, J = 8.4, 2.6 Hz, 1H), 3.96 (s,
2H), 3.28 (q, J = 7.3 Hz, 2H), 1.10 (t, J = 7.3 Hz, 3H); 13C NMR
(151 MHz, DMSO-d6) δ ppm 169.3, 157.6, 144.5, 141.4, 137.6,
134.8, 132.7, 132.4, 130.9, 130.0, 129.4, 128.4, 114.6, 49.7, 41.8,
7.6; LCMS: rt = 3.74 mins, MH+ = 455.0; HRMS (ESI): Mass
calculated for C19H17Cl2N2O3S2 [M+H]+, 455.0058. Found
[M+H]+, 455.0053.
5. Experimental
5.1 Chemistry
1H NMR spectral data were recorded on a Bruker 400 NMR
spectrometer operating at 400 MHz. CDCl3 is deuteriochloroform,
DMSO-d6 is hexadeuteriodimethylsulfoxide. Chemical shifts are
given in parts per million () downfield from the NMR solvent.
Abbreviations for NMR data are as follows: s = singlet, d =
doublet, t = triplet, q = quartet, m = multiplet, dd = doublet of
doublets, dt = doublet of triplets, app = apparent, br = broad. J
indicates the NMR coupling constant measured in Hertz. LCMS
(Agilent 1200SL-6110) analysis was conducted for all assayed
compounds on either acidic or basic conditions: 1) acidic
condition refers to water containing 0.05 % TFA / acetonitrile as
mobile phase on Agilent SB-C18 column (1.8 µm, 4.6 x 30 mm),
with MS and photodiode array detector (PDA). The following
conditions were used: a gradient from 5 to 95% in 4 min (or 6
min) and held at 95% for 1 min; UV detection at 214 and 254 nm;
a flow rate of 1.5 ml/min; full scan; mass range from 100 to 1000
amu; 2) basic condition refers to water containing 10 mM
NH4HCO3 aqueous/acetonitrile as mobile phase on Waters
XBridge C18 column (3.5 µm, 4.6 x 50 mm) with MS and
photodiode array detector (PDA). The following conditions were
used: a gradient from 5 to 95% in 5 min and held at 95% for 1
min; UV detection at 214 and 254 nm; a flow rate of 2 ml/min;
full scan; mass range from 100 to 1000 amu. All the assayed
compounds possess ≥ 95% purity determined using LC/MS
analysis. Flash column chromatography was performed on ISCO
or Biotage using a pre-packed silica gel column, a detector with
UV wavelength at 254 nm and 280 nm. MDAP equipped with
2489 UV detector, 2767 sample manager, 2545 pump, and 3100
single quadrupole mass spectrometer was performed on Sunfire
Prep C18 column (5 μm, 19 mm × 50 mm) using water
containing 0.05% TFA/acetonitrile as mobile phase. The
following conditions were used: a gradient from 5% to 95% in 15
min and held in 95% for 3 min; a flow rate of 30 mL/min.
5.1.2 Synthesis of N-[4-(2,5-dichlorophenyl)-5-methyl-1,3-
thiazol-2-yl]-2-[4-(ethylsulfonyl)phenyl]acetamide (2a)
5.1.2.1
2,5-dichloro-N-methyl-N-
(methyloxy)benzamide. To a solution of 2,5-dichlorobenzoic acid
(1.54 g), HOBt (1.85 g), N,O-dimethylhydroxylamine
[(methylamino)oxy]methane (0.74 g) and EDC (2.32 g) in DCM
(30 mL) stirred in air at room temperature was added a solution
of triethylamine (2.25 mL) in DCM (30 mL) dropwise during 5
mins. The reaction mixture was stirred at 20 °C overnight. The
organic phase was washed with brine (2*50 mL) and water (50
mL). The resulting mixture was dried over sodium sulphate and
evaporated in vacuo to give the crude product, which was
purified by chromatography (eluting with EtOAc:Petro = 10%) to
give the title compound (1.3 g) as a white solid. MS(ES+) m/z
233.9 (MH+).
5.1.2.2
1-(2,5-dichlorophenyl)-1-propanone. To
a
solution of 2,5-dichloro-N-methyl-N-(methyloxy)benzamide (806
mg) in THF (15 mL) stirred under nitrogen at 0 °C was added a
solution of bromo(ethyl)magnesium (1377 mg) in ether (15 mL)
dropwise during 1 min. The reaction mixture was stirred at 0 °C
for 3 hours. The organic phase was washed with 5% citric acid
solution (10 mL), brine (50 mL) and water (50 mL). The
resulting mixture was dried over sodium sulphate and evaporated
in vacuo to give the crude product, which was purified by
chromatography (eluting with Petro) to give the title compound
(626 mg) as a colourless liquid. MS(ES+) m/z 230.0 (MH+).
5.1.2.3
4-(2,5-dichlorophenyl)-5-methyl-1,3-thiazol-2-
amine. To a solution of 1-(2,5-dichlorophenyl)-1-propanone (900
mg), thiourea (675 mg) and iodine (1125 mg) in 1,4-dioxane (6
mL). The reaction mixture was stirred at 100 °C for 2 days. The
organic phase was washed with saturated sodium bicarbonate
solution (50 mL), brine (50 mL) and water (100 mL). The
resulting solution was dried over sodium sulphate and evaporated
in vacuo to give the crude product, which was purified by flash
chromatography (eluting with EtOAc:Petro = 15%) to afford the
title compound (186 mg) as a brown solid. MS(ES+) m/z 259.0
(MH+).
5.1.1 Synthesis of N-[4-(2,5-dichlorophenyl)-1,3-thiazol-2-
yl]-2-[4-(ethylsulfonyl)phenyl]acetamide (1)
5.1.1.1
4-(2,5-dichlorophenyl)-1,3-thiazol-2-amine.
mixture of 1-(2,5-
Iodine (18.8 g) was added to
a
dichlorophenyl)ethanone (10.7 mL) and thiourea (11.3 g). The
resulting mixture was heated in an oil bath at 100 °C overnight.
After cooling, the reaction mixture was triturated with diethyl
ether (about 50 mL) to remove any unreacted iodine and 1-(2,5-
dichlorophenyl)ethanone. The solid residue was put in cold
distilled water (100 mL) and treated with ammonium solution to
pH 9-10. The precipitated thiazole was collected and used
directly into the next step without further purification. MS(ES+)
m/z 245.0 (MH+).
5.1.2.4
N-[4-(2,5-dichlorophenyl)-5-methyl-1,3-
(2a).
thiazol-2-yl]-2-[4-(ethylsulfonyl)phenyl]acetamide
A
solution of [4-(ethylsulfonyl)phenyl]acetic acid (159 mg), EDC
(178 mg) and HOBt (142 mg) in DCM (10 mL) stirred at room
temperature was added a solution of 4-(2,5-dichlorophenyl)-5-
methyl-1,3-thiazol-2-amine (120 mg) in DCM (10 mL). The
reaction mixture was stirred at 20 °C overnight. The organic
phase was washed with brine (2*50 mL) and water (50 mL), then
dried over sodium sulphate. After filtration, the solution was
evaporated in vacuo to give the crude product, which was
purified by flash chromatography (eluting with DCM) to give the
desired compound (102 mg, 45% yield) as a white solid. 1H NMR
5.1.1.2
N-[4-(2,5-dichlorophenyl)-1,3-thiazol-2-yl]-2-
(1). [4-
[4-(ethylsulfonyl)phenyl]acetamide
(Ethylsulfonyl)phenyl]acetic acid (5.4 g), 4-(2,5-dichlorophenyl)-
1,3-thiazol-2-amine (2.9 g), EDC (4.5 g) and HOBT (3.6 g) were
added subsequently into a 100 mL round bottom flask.
Dichloromethane (DCM) (50 mL) was added. The reaction
mixture was stirred at room temperature overnight. After removal

(600 MHz, DMSO-d6) δ ppm 12.44 (br. s., 1H), 7.86 (d, J = 8.4
Hz, 2H), 7.61 (dd, J = 8.5, 4.7 Hz, 3H), 7.50-7.53 (m, 1H), 7.48-
7.50 (m, 1H), 3.92 (s, 2H), 3.28 (q, J = 7.3 Hz, 2H), 2.20 (s, 3H),
1.10 (t, J = 7.4 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) δ ppm
168.9, 154.8, 142.0, 141.5, 137.6, 136.1, 132.0, 131.9, 131.8,
130.8, 130.2, 128.4, 124.7, 49.7, 41.8, 11.7, 7.6; LCMS: rt = 3.72
mins, MH+ = 469.0; HRMS (ESI): Mass calculated for
C20H19Cl2N2O3S2 [M+H]+, 469.0214. Found [M+H]+, 469.0212.
over anhydrous Na2SO4, and concentrated in vacuo to afford 1,3-
diphenylpropan-1-one (2.2 g) as a yellow solid. MS(ES+) m/z 211
(MH+).
5.1.6.3
2-bromo-1,3-diphenylpropan-1-one.
A
mixture of 1,3-diphenylpropan-1-one (2.2 g) and copper(II)
bromide (3.8 g) in chloroform (20 mL) and ethyl acetate (20 mL)
was stirred at reflux for 2 hours. Solvent was removed, and the
residue was partitioned between water (20 mL) and EtOAc (50
mL). The organic phase was dried and concentrated to give 2-
bromo-1,3-diphenylpropan-1-one (2.6 g) as a yellow solid.
MS(ES+) m/z 289 (MH+).
5.1.3 N-[4-(2,5-dichlorophenyl)-5-ethyl-1,3-thiazol-2-yl]-2-
[4-(ethylsulfonyl)phenyl]acetamide (2b). The compound (53.6
mg, 24% yield) was synthesized by the same method as
1
compound 2a. H NMR (400 MHz, DMSO-d6) δ ppm 12.46 (br.
5.1.6.4
4-phenyl-5-(phenylmethyl)-1,3-thiazol-2-
s., 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.61 (d, J = 8.8 Hz, 3H), 7.45-
7.55 (m, 2H), 3.92 (s, 2H), 3.28 (q, J = 7.3 Hz, 2H), 2.56 (q, J =
7.3 Hz, 2H), 1.08-1.13 (m, 6H); 13C NMR (101 MHz, DMSO-d6)
δ ppm 168.9, 154.9, 141.5, 141.1, 137.5, 136.4, 132.2, 132.0,
131.9, 131.9, 131.7, 130.8, 130.3, 128.4, 49.7, 41.8, 20.0, 16.5;
LCMS: rt = 3.89 mins, MH+ = 483.0; HRMS (ESI): Mass
calculated for C21H21Cl2N2O3S2 [M+H]+, 483.0371. Found
[M+H]+, 483.0376.
amine. A mixture of 2-bromo-1,3-diphenylpropan-1-one (3.5 g)
and thiourea (1.1 g) in ethanol (30 mL) was heated at reflux for 2
hours. Solvent was removed in vacuo. The residue was washed
with EtOAc and water, filtered and dried to afford 4-phenyl-5-
(phenylmethyl)-1,3-thiazol-2-amine (1.7 g) as a yellow soild.
MS(ES+) m/z 267 (MH+).
5.1.6.5
2-[4-(ethylsulfonyl)phenyl]-N-[4-phenyl-5-
(2e).
(phenylmethyl)-1,3-thiazol-2-yl]acetamide
[4-
5.1.4 N-[4-(2,5-dichlorophenyl)-5-phenyl-1,3-thiazol-2-yl]-2-
[4-(ethylsulfonyl)phenyl]acetamide (2c). The compound (102 mg,
42% yield) was synthesized by the same method as compound 2a.
1H NMR (600 MHz, DMSO-d6) δ ppm 12.69 (br. s., 1H), 7.87 (d,
J = 8.4 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.53-7.57 (m, 1H),
7.47-7.53 (m, 2H), 7.28-7.33 (m, 2H), 7.22-7.28 (m, 1H), 7.15 (d,
J = 7.3 Hz, 2H), 3.98 (s, 2H), 3.28 (q, J = 7.3 Hz, 2H), 1.10 (t, J
= 7.3 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) δ ppm 169.3,
156.0, 141.3, 141.1, 137.6, 136.9, 132.1, 132.1, 131.8, 131.6,
130.9, 130.5, 129.4, 129.0, 128.4, 128.3, 49.7, 41.8, 7.6; LCMS:
rt = 4.00 mins, MH+ = 531.2; HRMS (ESI): Mass calculated for
C25H21Cl2N2O3S2 [M+H]+, 531.0371. Found [M+H]+, 531.0377.
(Ethylsulfonyl)phenyl]acetic acid (89 mg), 4-phenyl-5-
(phenylmethyl)-1,3-thiazol-2-amine (104 mg), EDC (90 mg) and
HOBt (71.7 mg) were added into a 10 mL vial. Dichloromethane
(DCM) (5 mL) was added. The reaction mixture was stirred at
RT over 3 days. DCM was removed. The obtained crude was
redissolved in DMF and submitted to MDAP for purification.
Solvent was evaporated in vacuo to give 2-[4-
(ethylsulfonyl)phenyl]-N-[4-phenyl-5-(phenylmethyl)-1,3-
thiazol-2-yl]acetamide (119 mg, 63% yield) as a white solid. 1H-
NMR (400 MHz, DMSO-d6) δ ppm 1.02 (t, J = 7.4 Hz, 3H), 3.20
(q, J = 7.4 Hz, 2H), 3.83 (s, 2H), 4.16 (s, 2H), 7.09-7.19 (m, 3H),
7.24 (t, J = 7.3 Hz, 2H), 7.25-7.31 (m, 1H), 7.39 (t, J = 7.5 Hz,
2H), 7.48-7.58 (m, 4H), 7.77 (d, J = 8.3 Hz, 2H), 12.44 (s, 1H);
13C NMR (101 MHz, DMSO-d6) δ ppm 168.9, 155.5, 145.3,
141.5, 140.5, 137.5, 135.2, 130.7, 129.1, 129.0, 128.7, 128.5,
128.4, 128.1, 127.0, 126.3, 49.7, 41.8, 32.4, 7.6; LCMS: rt = 3.83
mins, MH+ = 477.3; HRMS (ESI): Mass calculated for
C26H25N2O3S2 [M+H]+, 477.1307. Found [M+H]+, 477.1315.
5.1.5 N-(4,5-diphenyl-1,3-thiazol-2-yl)-2-[4-
(ethylsulfonyl)phenyl]acetamide (2d). The compound (79 mg,
38% yield) was synthesized by the same method as compound 2a.
1H NMR (600 MHz, DMSO-d6) δ ppm 12.68 (br. s., 1H), 7.87 (d,
J = 8.1 Hz, 2H), 7.63 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 7.0 Hz,
2H), 7.26-7.39 (m, 8H), 3.97 (s, 2H), 3.28 (q, J = 7.3 Hz, 2H),
1.10 (t, J = 7.3 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) δ ppm
169.2, 156.1, 144.3, 141.5, 137.6, 135.1, 132.2, 130.8, 129.7,
129.4, 128.9, 128.8, 128.5, 128.4, 128.2, 126.1, 49.7, 41.8, 7.6;
LCMS: rt = 3.80 mins, MH+ = 463.2; HRMS (ESI): Mass
calculated for C25H23N2O3S2 [M+H]+, 463.1150. Found [M+H]+,
463.1153.
5.1.7 Synthesis of 2-[4-(ethylsulfonyl)phenyl]-N-[4-phenyl-5-
(phenyloxy)-1,3-thiazol-2-yl]acetamide (2f)
5.1.7.1
5-bromo-4-phenyl-1,3-thiazol-2-amine. To a
mixture of thiourea (1.147 g) and 2-bromo-1-phenylethanone (3
g) was added ethanol (100 mL). The reaction mixture was heated
up to reflux (oil bath temperature: 80 °C) for 2.5 hours. The
reaction was cooled down to RT. Ethanol was removed under
vacuum to afford the intermediate as a white solid. Copper(II)
bromide (8.42 g) and acetonitrile (40 mL) were added. The
mixture was stirred at RT for 3 days. Acetonitrile was removed
under vacuum. EtOAc was added. The obtained mixture was
washed with diluted ammonia three times until no blue color was
observed in the aqueous phase. The organic phase was then
washed with brine and dried over Na2SO4. After filtration and
concentration, the crude product was purified by flash
chromatography (EtOAc : PE = 0:1 to 1:0) to afford 5-bromo-4-
phenyl-1,3-thiazol-2-amine (2 g). MS(ES+) m/z 255 (MH+).
5.1.6 Synthesis of 2-[4-(ethylsulfonyl)phenyl]-N-[4-phenyl-5-
(phenylmethyl)-1,3-thiazol-2-yl]acetamide (2e)
5.1.6.1
mixture
N-methoxy-N-methyl-3-phenylpropanamide. A
3-phenylpropanoic acid (5 g), N,O-
of
dimethylhydroxylamine (3.9 g), HOBt (6.1 g), EDC (7.7 g) and
Et3N (9.3 mL) in dichloromethane (DCM) (80 mL) was stirred at
25 °C overnight. The mixture was washed with 1 M HCl, sat.
NaHCO3 and brine successively. The organic phase was dried
over anhydrous Na2SO4. After filtration, the filtrate was
concentrated in vacuo, and the residue was purified by flash
chromatography (EtOAc : PE = 3:1) to afford the desired product
(4.8 g) as a yellow solid. MS(ES+) m/z 194 (MH+).
5.1.7.2
4-phenyl-5-(phenyloxy)-1,3-thiazol-2-amine.
5.1.6.2
1,3-diphenylpropan-1-one. To a solution of N-
(4.5 g) in
To a mixture of 5-bromo-4-phenyl-1,3-thiazol-2-amine (213.3
mg), phenol (102 mg) and cesium carbonate (409 mg) was added
acetone (3 mL). The reaction mixture was heated to 55 °C and
stirred for 7 hours. Solvent was removed. The residue was
purified by flash chromatography (EtOAc : PE = 0:1 to 1:4) to
afford the desired product (172 mg). MS(ES+) m/z 269 (MH+).
methoxy-N-methyl-3-phenylpropanamide
tetrahydrofuran (THF) (60 mL) stirred at 0 °C was added a
solution of phenylmagnesium bromide (34 mL) (1 M in THF)
dropwise. After stirring at 0 °C for 3 hours, the reaction mixture
was quenched with sat. NH4Cl solution, and then extracted with
EtOAc (80 mL). The organic phase was washed with brine, dried

5.1.7.3
2-[4-(ethylsulfonyl)phenyl]-N-[4-phenyl-5-
(2f).
(m, 2H), 3.95 (s, 2H), 3.27 (q, J = 7.4 Hz, 2H), 1.10 (t, J = 7.4 Hz,
3H); 13C NMR (151 MHz, DMSO-d6) δ ppm 169.3, 157.2, 149.5,
143.5, 141.3, 137.6, 135.1, 132.8, 130.9, 130.5, 129.2, 128.4,
128.3, 128.2, 126.9, 118.3, 49.7, 41.7, 7.6; LCMS: rt = 4.09 mins,
MH+ = 509.1; HRMS (ESI): Mass calculated for C25H22ClN2O4S2
[M+H]+, 513.0710. Found [M+H]+, 513.0715.
(phenyloxy)-1,3-thiazol-2-yl]acetamide
[4-
(Ethylsulfonyl)phenyl]acetic acid (150 mg), 4-phenyl-5-
(phenyloxy)-1,3-thiazol-2-amine (176 mg), EDC (151 mg) and
HOBt (121 mg) were added into a 10 mL vial. DCM (5 mL) was
added. The reaction mixture was stirred at RT for 3 days. DCM
was removed. The obtained crude was redissolved in DMF and
submitted to MDAP for purification. Solvent was evaporated in
vacuo to give the desired product (41 mg, 13% yield) as a white
5.1.122-[4-(ethylsulfonyl)phenyl]-N-(4-phenyl-5-{[4-
(trifluoromethyl)phenyl]oxy}-1,3-thiazol-2-yl)acetamide
(3e).
The compound (9.6 mg , 5% yield) was synthesized by the same
1
solid. H NMR (600 MHz, DMSO-d6) δ ppm 12.62 (br. s., 1H),
1
method as compound 2f. H NMR (400 MHz, DMSO-d6) δ ppm
7.84-7.88 (m, 4H), 7.62 (d, J = 8.4 Hz, 2H), 7.35-7.43 (m, 4H),
7.27-7.31 (m, 1H), 7.11-7.16 (m, 3H), 3.94 (s, 2H), 3.27 (q, J =
7.3 Hz, 2H), 1.09 (t, J = 7.4 Hz, 3H); 13C NMR (151 MHz,
DMSO-d6) δ ppm 169.2, 158.4, 149.2, 144.2, 141.3, 137.6, 134.7,
133.0, 130.9, 130.7, 129.2, 128.4, 128.2, 126.9, 124.4, 116.6,
49.7, 41.7, 7.6; LCMS: rt = 3.88 mins, MH+ = 479.2; HRMS
(ESI): Mass calculated for C25H23N2O4S2 [M+H]+, 479.1099.
Found [M+H]+, 479.1100.
12.73 (br. s., 1H), 7.86 (d, J = 8.3 Hz, 2H), 7.81 (d, J = 7.5 Hz,
2H), 7.74 (d, J = 8.8 Hz, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.37-7.44
(m, 2H), 7.27-7.35 (m, 3H), 3.96 (s, 2H), 3.28 (q, J = 7.3 Hz, 2H),
1.10 (t, J = 7.4 Hz, 3H); LCMS: rt = 3.79 mins, MH+ = 547.0;
HRMS (ESI): Mass calculated for C26H22F3N2O4S2 [M+H]+,
547.0973. Found [M+H]+, 547.0973.
5.1.132-[4-(ethylsulfonyl)phenyl]-N-(5-{[4-
(methyloxy)phenyl]oxy}-4-phenyl-1,3-thiazol-2-yl)acetamide (3f).
The compound (38.1 mg, 11% yield) was synthesized by the
same method as compound 2f. 1H NMR (400 MHz, DMSO-d6) δ
ppm 12.58 (br. s., 1H), 7.88 (d, J = 7.8 Hz, 2H), 7.85 (d, J = 8.3
Hz, 2H), 7.60 (d, J = 8.1 Hz, 2H), 7.41 (t, J = 7.7 Hz, 2H), 7.26-
7.33 (m, 1H), 7.10 (d, J = 9.0 Hz, 2H), 6.93 (d, J = 9.0 Hz, 2H),
3.92 (s, 2H), 3.71 (s, 3H), 3.27 (q, J = 7.3 Hz, 2H), 1.09 (t, J =
7.3 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ ppm 169.1, 156.3,
152.3, 148.5, 146.0, 141.4, 137.6, 133.4, 133.2, 130.8, 129.1,
128.4, 128.0, 126.8, 118.2, 115.5, 55.9, 49.6, 41.7, 7.6; LCMS: rt
= 3.86 mins, MH+ = 509.1; HRMS (ESI): Mass calculated for
C26H25N2O5S2 [M+H]+, 509.1205. Found [M+H]+, 509.1208.
5.1.8 N-{5-[(2-chlorophenyl)oxy]-4-phenyl-1,3-thiazol-2-yl}-
2-[4-(ethylsulfonyl)phenyl]acetamide (3a). The compound (11.3
mg, 3% yield) was synthesized by the same method as compound
2f. 1H NMR (600 MHz, DMSO-d6) δ ppm 12.66 (br. s., 1H), 7.85
(dd, J = 8.2, 1.8 Hz, 4H), 7.58-7.63 (m, 3H), 7.42 (t, J = 7.8 Hz,
2H), 7.28-7.33 (m, 2H), 7.17-7.20 (m, 1H), 7.14 (d, J = 8.3 Hz,
1H), 3.94 (s, 2H), 3.27 (q, J = 7.3 Hz, 2H), 1.10 (t, J = 7.3 Hz,
3H); 13C NMR (151 MHz, DMSO-d6) δ ppm 169.4, 153.7, 149.2,
143.8, 141.3, 137.6, 134.3, 132.9, 131.3, 130.9, 129.5, 129.2,
128.4, 128.3, 126.8, 125.9, 122.7, 117.6, 49.7, 41.7, 7.6; LCMS:
rt = 4.02 mins, MH+ = 513.2; HRMS (ESI): Mass calculated for
C25H22ClN2O4S2 [M+H]+, 513.0710. Found [M+H]+, 513.0713.
5.1.14N-[4-(4-chlorophenyl)-5-(phenyloxy)-1,3-thiazol-2-yl]-
2-[4-(ethylsulfonyl)phenyl]acetamide (3g). The compound (40.0
mg, 23% yield) was synthesized by the same method as
5.1.9 N-{5-[(2-chlorophenyl)oxy]-4-phenyl-1,3-thiazol-2-yl}-
2-[4-(ethylsulfonyl)phenyl]acetamide (3b). The compound (38.7
mg, 13% yield) was synthesized by the same method as
1
compound 2f. H NMR (600 MHz, DMSO-d6) δ ppm 12.64 (br.
1
compound 2f. H NMR (600 MHz, DMSO-d6) δ ppm 12.73 (br.
s., 1H), 7.86 (dd, J = 8.1, 5.5 Hz, 4H), 7.61 (d, J = 8.4 Hz, 2H),
7.48 (d, J = 8.4 Hz, 2H), 7.36-7.42 (m, 2H), 7.12-7.19 (m, 3H),
3.94 (s, 2H), 3.27 (q, J = 7.3 Hz, 2H), 1.09 (t, J = 7.3 Hz, 3H);
13C NMR (151 MHz, DMSO-d6) δ ppm 169.3, 158.3, 149.2,
145.0, 141.3, 137.6, 133.3, 132.6, 131.9, 130.8, 130.7, 129.3,
128.5, 128.4, 124.7, 116.8, 49.7, 41.7, 7.6; LCMS: rt = 4.14 mins,
MH+ = 513.1; HRMS (ESI): Mass calculated for C25H22ClN2O4S2
[M+H]+, 513.0710. Found [M+H]+, 513.0713.
s., 1H), 7.86 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.1 Hz, 2H), 7.80 (d,
J = 7.7 Hz, 1H), 7.62 (d, J = 8.1 Hz, 3H), 7.41 (t, J = 7.7 Hz, 2H),
7.31 (q, J = 7.1 Hz, 2H), 7.19 (d, J = 8.4 Hz, 1H), 3.95 (s, 2H),
3.27 (q, J = 7.3 Hz, 2H), 1.09 (t, J = 7.3 Hz, 3H); 13C NMR (151
MHz, DMSO-d6) δ ppm 169.4, 155.6, 149.8, 142.4, 141.2, 137.6,
135.6, 135.4, 132.6, 130.8, 129.2, 128.5, 128.4, 127.8 (q, J = 5.5
Hz), 126.8, 124.5, 123.8 (q, J = 271.5 Hz), 118.4 (q, J = 31.5 Hz),
116.7, 49.7, 41.7, 7.6; LCMS: rt = 4.05 mins, MH+ = 547.2;
HRMS (ESI): Mass calculated for C26H22F3N2O4S2 [M+H]+,
547.0973. Found [M+H]+, 547.0966.
5.1.15Synthesis of N-(4-(3-chlorophenyl)-5-phenoxythiazol-2-
yl)-2-(4-(ethylsulfonyl)phenyl)acetamide (3h).
5.1.102-[4-(ethylsulfonyl)phenyl]-N-(5-{[2-
5.1.15.1
2-bromo-1-(3-chlorophenyl)ethanone. To a
(methyloxy)phenyl]oxy}-4-phenyl-1,3-thiazol-2-yl)acetamide (3c).
The compound was synthesized (47.1 mg, 14% yield) by the
same method as compound 2f. 1H NMR (400 MHz, DMSO-d6) δ
ppm 12.54 (br. s., 1H), 7.91 (d, J = 7.8 Hz, 2H), 7.84 (d, J = 8.1
Hz, 2H), 7.59 (d, J = 8.3 Hz, 2H), 7.42 (t, J = 7.6 Hz, 2H), 7.25-
7.33 (m, 1H), 7.13-7.21 (m, 2H), 7.08 (d, J = 8.1 Hz, 1H), 6.85-
6.95 (m, 1H), 3.91 (s, 2H), 3.83 (s, 3H), 3.27 (q, J = 7.3 Hz, 2H),
1.08 (t, J = 7.3 Hz, 3H); 13C NMR (101 MHz, DMSO-d6) δ ppm
169.0, 150.2, 147.7, 147.2, 147.0, 141.4, 137.5, 133.4, 131.9,
130.8, 129.1, 128.4, 127.8, 126.8, 126.1, 121.4, 118.5, 113.9,
56.3, 49.6, 41.7, 7.6; LCMS: rt = 3.77 mins, MH+ = 509.1;
HRMS (ESI): Mass calculated for C26H25N2O5S2 [M+H]+,
509.1205. Found [M+H]+, 509.1210.
solution of 1-(3-chlorophenyl)ethanone (15 g) in chloroform (100
mL) and ethyl acetate (100 mL) was added copper(II) bromide
(21.67 g), and the reaction mixture was refluxed overnight. The
reaction mixture was concentrated to give the desired product (18
g) as a brown oil. MS(ES+) m/z 219 (MH+).
5.1.15.2
4-(3-chlorophenyl)thiazol-2-amine.
To
a
solution of 2-bromo-1-(3-chlorophenyl)ethanone (28 g) in
ethanol (300 mL) was added thiourea (10.04 g). The reaction
mixture was refluxed overnight. Solvent was removed, and the
residue was triturated with diethyl ether to give the title
compound (23 g) as a white solid. MS(ES+) m/z 211 (MH+).
5.1.15.3
5-bromo-4-(3-chlorophenyl)thiazol-2-amine.
To a solution of 4-(3-chlorophenyl)thiazol-2-amine (1 g) in
tetrahydrofuran (20 mL) was added NBS (0.88 g). After stirring
at room temperature overnight, the mixture was partitioned
between ethyl acetate (30 mL) and water (20 mL). The organic
phase was washed with water (20 mL x 2), dried and
5.1.11N-{5-[(4-chlorophenyl)oxy]-4-phenyl-1,3-thiazol-2-yl}-
2-[4-(ethylsulfonyl)phenyl]acetamide (3d). The compound (47.4
mg, 14% yield) was synthesized by the same method as
compound 2f. H NMR (600 MHz, DMSO-d6) δ ppm 12.66 (br.
s., 1H), 7.84-7.88 (m, 2H), 7.82 (dd, J = 8.4, 1.1 Hz, 2H), 7.62 (d,
1
J = 8.3 Hz, 2H), 7.37-7.43 (m, 4H), 7.28-7.32 (m, 1H), 7.14-7.18

concentrated to give the desired product (1.05 g) as a red liquid.
5.1.18N-(4-(3-((dimethylamino)methyl)phenyl)-5-(2-
(trifluoromethyl)phenoxy)thiazol-2-yl)-2-(4-
MS(ES+) m/z 289 (MH+).
(ethylsulfonyl)phenyl)acetamide trifluoroacetate (3k). The
5.1.15.4
4-(3-chlorophenyl)-5-phenoxythiazol-2-amine.
compound (12mg, 7% yield) was synthesized by the same
To a solution of 5-bromo-4-(3-chlorophenyl)thiazol-2-amine (1.0
g) in acetone (10 mL) was added phenol (0.36 g) and cesium
carbonate (1.35 g). The reaction mixture was stirred at 45 °C
overnight. DCM (100 mL) was added. The obtained solution was
washed with water (100 mL x 3), dried and concentrated. The
residue was purified by flash chromatography (EtOAc : PE = 1:5
to 1:1) to give the desired product (230 mg) as a red liquid.
MS(ES+) m/z 303 (MH+).
1
method as compound 3h. H NMR (600 MHz, DMSO-d6) δ ppm
12.73 (br. s., 1H), 9.82 (br. s., 1H), 7.85-7.90 (m, 4H), 7.82 (d, J
= 7.7 Hz, 1H), 7.62-7.66 (m, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.53
(t, J = 7.7 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.35 (t, J = 7.7 Hz,
1H), 7.24 (d, J = 8.4 Hz, 1H), 4.23 (br. s., 2H), 3.96 (s, 2H), 3.28
(q, J = 7.3 Hz, 2H), 2.68 (br. s., 6H), 1.09 (t, J = 7.3 Hz, 3H);
LCMS: rt = 3.22 mins, MH+ = 604.3; HRMS (ESI): Mass
calculated for C29H29F3N3O4S2 [M+H]+, 604.1552. Found
[M+H]+, 604.1554.
5.1.15.5
2-(4-(ethylsulfonyl)phenyl)acetamide (3h). To a solution of 4-(3-
chlorophenyl)-5-phenoxythiazol-2-amine (230 mg) in
N-(4-(3-chlorophenyl)-5-phenoxythiazol-2-yl)-
5.1.19N-(5-(2-chloro-4-(trifluoromethyl)phenoxy)-4-(3-
cyanophenyl)thiazol-2-yl)-2-(4-(ethylsulfonyl)phenyl)acetamide
(3l). The compound (78mg, 17% yield) was synthesized by the
same method as compound 3h. 1H NMR (400 MHz, DMSO-d6) δ
ppm 12.83 (br. s., 1H), 8.04-8.18 (m, 3H), 7.86 (d, J = 8.3 Hz,
2H), 7.80 (d, J = 7.6 Hz, 1H), 7.58-7.70 (m, 4H), 7.36 (d, J = 8.8
Hz, 1H), 3.97 (s, 2H), 3.28 (q, J = 7.3 Hz, 2H), 1.09 (t, J = 7.3 Hz,
3H); 13C NMR (101 MHz, DMSO-d6) δ ppm 169.6, 156.2, 150.4,
143.8, 141.1, 137.6, 133.6, 133.2, 132.0, 131.2, 130.9, 130.8,
130.1, 128.6 (q, J = 3.7 Hz), 128.4, 126.9 (q, J = 3.7 Hz), 126.1
(q, J = 33.0 Hz), 123.6 (q, J = 271.0 Hz), 123.5, 118.9, 117.5,
112.5, 49.6, 41.7, 7.6; LCMS: rt = 4.21 mins, MH+ = 606.2;
HRMS (ESI): Mass calculated for C27H20ClF3N3O4S2 [M+H]+,
606.0536. Found [M+H]+, 606.0537.
dichloromethane (DCM) (20 mL) was added 2-(4-
(ethylsulfonyl)phenyl)acetic acid (831 mg), EDC (728 mg),
HOBt (140 mg) and DIPEA (589 mg). After stirring at 35 °C for
2 days, the reaction mixture was washed with water (30 mL x 3),
dried and concentrated. The residue was purified by flash
chromatography (EtOAc : PE = 0:1 to 1:3) to give the crude
product. Further purification by preparative HPLC afforded the
desired product (18 mg, 5% yield) as a white solid. 1H NMR (600
MHz, DMSO-d6) δ ppm 12.64 (br. s., 1H), 7.87 (t, J=1.8 Hz, 1H),
7.83-7.87 (m, 2H), 7.81 (dt, J = 7.9, 1.3 Hz, 1H), 7.61 (d, J = 8.3
Hz, 2H), 7.42-7.47 (m, 1H), 7.38-7.42 (m, 2H), 7.36 (ddd, J = 8.0,
2.2, 1.0 Hz, 1H), 7.12-7.20 (m, 3H), 3.94 (s, 2H), 3.27 (q, J = 7.3
Hz, 2H), 1.09 (t, J = 7.3 Hz, 3H); 13C NMR (151 MHz, DMSO-d6)
δ ppm 169.3, 158.3, 149.3, 145.7, 141.3, 137.6, 135.0, 133.9,
132.7, 131.2, 130.9, 130.8, 128.4, 127.9, 126.5, 125.2, 124.8,
116.8, 49.7, 41.7, 7.6; LCMS: rt = 4.14 mins, MH+ = 513.0;
HRMS (ESI): Mass calculated for C25H22ClN2O4S2 [M+H]+,
513.0710. Found [M+H]+, 513.0706.
5.1.20Synthesis
of
N-(5-(2-chloro-4-
(trifluoromethyl)phenoxy)-4-(3-cyanophenyl)thiazol-2-yl)-2-(4-
(ethylsulfonyl)phenyl)acetamide (3m)
5.1.20.1 4-nitro-2-(trifluoromethyl)phenol. Lithium chloride
(2.30 g) was added to a solution of 1-methoxy-4-nitro-2-
(trifluoromethyl)benzene (4 g) in N,N-dimethylformamide (40
mL) at room temperature. The reaction mixture was stirred at
160 °C for 5 hours. After cooling to RT, the solution was diluted
with EtOAc (300 mL). The mixture was washed with water (100
mL x 2) and brine (100 mL), dried and concentrated to give 4-
nitro-2-(trifluoromethyl)phenol (3.5 g) as a yellow solid.
5.1.16N-(4-(3-chlorophenyl)-5-(2-
(trifluoromethyl)phenoxy)thiazol-2-yl)-2-(4-
(ethylsulfonyl)phenyl)acetamide (3i). The compound (49 mg, 3%
yield) was synthesized by the same method as compound 3h. H
1
NMR (400 MHz, DMSO-d6) δ ppm 12.77 (br. s., 1H), 7.86 (d, J
= 7.8 Hz, 3H), 7.80 (dd, J = 16.4, 7.8 Hz, 2H), 7.57-7.68 (m, 3H),
7.42-7.49 (m, 1H), 7.30-7.41 (m, 2H), 7.24 (d, J = 8.6 Hz, 1H),
3.96 (s, 2H), 3.28 (q, J = 7.3 Hz, 2H), 1.09 (t, J = 7.3 Hz, 3H);
13C NMR (101 MHz, DMSO-d6) δ ppm 169.5, 155.3, 149.9,
143.6, 141.2, 137.6, 135.5, 134.6, 134.0, 133.7, 131.2, 130.8,
128.4, 128.2, 127.9 (q, J = 5.1 Hz), 126.5, 125.2, 124.8, 123.7 (q,
J = 271.0 Hz), 118.5 (q, J = 32.0 Hz), 116.8, 49.6, 41.7, 7.6;
LCMS: rt = 4.26 mins, MH+ = 581.0; HRMS (ESI): Mass
calculated for C26H21ClF3N2O4S2 [M+H]+, 581.0583. Found
[M+H]+, 581.0590.
5.1.20.2
4-amino-2-(trifluoromethyl)phenol.
Zinc
(4.42 g) was added to
a
solution of 4-nitro-2-
(trifluoromethyl)phenol (3.5 g) and ammonium formate (8.52 g)
in methanol (5 mL) and water (5 mL) at room temperature. The
reaction mixture was stirred at 80 °C for 3 hours. After cooling to
room temperature, the mixture was filtered and EtOAc (80 mL)
was added to the filtrate. The organic layer was washed with
water (30 mL) and brine (40 mL), dried, and concentrated to
afford 4-amino-2-(trifluoromethyl)phenol (2.8 g) as a yellow
solid.
5.1.17N-(4-(3-cyanophenyl)-5-(2-
(trifluoromethyl)phenoxy)thiazol-2-yl)-2-(4-
5.1.20.3
4-chloro-2-(trifluoromethyl)phenol. Sodium
(ethylsulfonyl)phenyl)acetamide (3j). The compound (29 mg, 8%
yield) was synthesized by the same method as compound 3h. It
nitrite (0.47 g) in H2O (3 mL) was added dropwise to a solution
of 4-amino-2-(trifluoromethyl)phenol (1 g) in acetonitrile (20 mL)
and hydrochloric acid (10 mL) at 0 °C . After stirring for 0.5 hour,
copper(I) chloride (1.12 g) in 3 M HCl (5 mL) was added to the
mixture. Then the reaction mixture was stirred at 100 °C for 6
hours. After cooling to room temperature, the solution was
diluted with EtOAc (200 mL). The organic phase was washed
with water (100 mL) and brine (50 mL), dried, and concentrated.
The residue was purified by flash chromatography (DCM : PE =
1:1) to afford 4-chloro-2-(trifluoromethyl)phenol (700 mg) as a
brown oil. MS(ES+) m/z 195 (MH+).
1
was isolated as TFA salt. H NMR (600 MHz, DMSO-d6) δ ppm
12.78 (br. s., 1H), 8.14 (s, 1H), 8.11 (d, J = 8.1 Hz, 1H), 7.86 (d,
J = 8.4 Hz, 2H), 7.83 (d, J = 7.7 Hz, 1H), 7.79 (d, J = 7.7 Hz,
1H), 7.62-7.68 (m, 2H), 7.61 (d, J = 8.4 Hz, 2H), 7.36 (t, J = 7.5
Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 3.96 (s, 2H), 3.27 (q, J = 7.3
Hz, 2H), 1.09 (t, J = 7.3 Hz, 3H); 13C NMR (151 MHz, DMSO-d6)
δ ppm 169.6, 155.2, 150.0, 144.4, 141.2, 137.7, 135.5, 133.8,
133.0, 131.8, 131.1, 130.9, 130.7, 130.1, 128.4, 127.9 (q, J = 5.5
Hz), 125.0, 123.7 (q, J = 271.5 Hz), 118.9, 118.6 (q, J = 30.0 Hz),
117.1, 112.4, 49.7, 41.7, 7.6; LCMS: rt = 3.96 mins, MH+ =
572.2; HRMS (ESI): Mass calculated for C27H21F3N3O4S2
[M+H]+, 572.0926. Found [M+H]+, 572.0925.
5.1.20.4
3-(2-amino-5-(4-chloro-2-
(trifluoromethyl)phenoxy)thiazol-4-yl)benzonitrile. 5-Bromo-4-
(3-cyanophenyl)thiazol-2-amine (500 mg) was added to a

solution of 4-chloro-2-(trifluoromethyl)phenol (351 mg) and
Cs2CO3 (872 mg) in acetone (30 mL) at room temperature. The
reaction mixture was stirred at 45 °C for 4 hours. The reaction
mixutre was cooled to room temperature, filtered through celite
and concentrated. The crude product was purified by flash
chromatography (EtOAc : PE = 1:4) to afford 3-(2-amino-5-(4-
chloro-2-(trifluoromethyl)phenoxy)thiazol-4-yl)benzonitrile (170
mg) as a yellow solid. MS(ES+) m/z 396 (MH+).
using standard methods. Typically proteins have between 1-5
biotins. Biotinylated-Peptide was purchased from CPC scientific.
Streptavidin-labeled APC (CR130-150) and Eu-W1024 labeled
Streptavidin (AD0063) were purchased from Perkin Elmer.
DMSO purchased from EMD (MX14561). MOPS (M1254),
Sodium Fluoride (S6521) and CHAPS (C3023) were purchased
from Sigma. DTT (F780-01) was purchased from JT Baker.
Immunopure D-biotin (29129) was purchased from Pierce. BSA
(100350), Frac V, fatty acid free was purchased from Boehringer
Mannheim.
5.1.20.5
N-(5-(4-chloro-2-(trifluoromethyl)phenoxy)-
4-(3-cyanophenyl)thiazol-2-yl)-2-(4-
(ethylsulfonyl)phenyl)acetamide (3m). 3-(2-Amino-5-(4-chloro-
2-(trifluoromethyl)phenoxy)thiazol-4-yl)benzonitrile (170 mg)
was added to a solution of 2-(4-(ethylsulfonyl)phenyl)acetic acid
(147 mg), HOBt (79 mg,), EDC (247 mg) and DIPEA (0.45 mL)
in dichloromethane (15 mL) at room temperature. The reaction
mixture was stirred at 40 °C for 12 hours. The mixture was
cooled to room temperature and concentrated. The residue was
purified by flash chromatography (EtOAc : PE = 1:4 to 1:1) to
afford the crude product (100 mg) as a yellow oil. Further
purification by preparative HPLC to afford the desired product
(28 mg, 11% yield) as a white solid. 1H NMR (600 MHz,
DMSO-d6) δ ppm 12.80 (br. s., 1H), 8.11 (t, J = 1.7 Hz, 1H),
8.06-8.09 (m, 1H), 7.91 (d, J = 2.7 Hz, 1H), 7.84-7.88 (m, 2H),
7.80 (dt, J = 7.9, 1.3 Hz, 1H), 7.64-7.71 (m, 2H), 7.58-7.64 (m,
2H), 7.32 (d, J = 9.0 Hz, 1H), 3.96 (s, 2H), 3.28 (q, J = 7.3 Hz,
2H), 1.10 (t, J = 7.3 Hz, 3H); 13C NMR (151 MHz, DMSO-d6) δ
ppm 169.6, 154.1, 150.4, 143.8, 141.1, 137.7, 135.2, 133.6, 133.5,
132.0, 131.2, 130.9, 130.8, 130.1, 128.8, 128.4, 127.8 (d, J = 5.5
Hz), 122.8 (d, J = 273.3 Hz), 120.1 (d, J = 33.2 Hz), 118.9, 118.9,
112.5, 49.6, 41.7, 7.6; LCMS: rt = 4.20 mins, MH+ = 606.1;
HRMS (ESI): Mass calculated for C27H20ClF3N3O4S2 [M+H]+,
606.0536. Found [M+H]+, 606.0532.
Compound preparation: Compounds were diluted in 100%
neat DMSO at 10 mM. The compounds were then dispensed into
an intermediate plate (polypropylene Greiner PP V-bottom:
781280) to make serial dilutions in 100% neat DMSO.
Approximately 100 nL of the serial dilution was added to the
assay plate (Costar 3573) using a Hummingbird (Genomic
Solutions).
Stock Buffer: A 0.5 M solution of MOPS was made by adding
104 grams of MOPS to 800 mL H2O in a graduated cylinder,
using a calibrated pH meter, add increasing amounts NaOH to
give a final pH of 7.5. This solution was filtered using a Costar
0.2 um filtering apparatus and stored in the refrigerator until
ready to use.
Assay buffer: Add 100 mL of 10x MOPS stock solution to
graduated cylinder bring up to 800 mL. Add 2.09 grams of NaF,
0.03 grams of CHAPS to the flask, 0.1 grams of BSA to the flask.
Make sure all components are dissolved. Add dH2O to give final
volume of 1L. The assay buffer was filtered with a Costar 0.2 um
filtering apparatus. On assay day, DTT was added to the assay
buffer to a final concentration of 10 mM. Fresh DTT solid should
be used.
5.2 Assay description
Assay: RORγ were assayed using the generic protocol
described. To polypropylene costar conical centrifuge tubes, add
assay buffer, an appropriate amount of biotinylated-SRC1(2)
from the 1E-4 M (100 uM) stock solution to give a final
concentration of 4E-8 M (40 nM). To the above biotinylated
SRC1(2) solution, add an appropriate amount of Europium-
labeled streptavidin to give a final concentration of 1E-8 M (10
nM). Invert gently to mix. Incubate 15 minutes at room
temperature. At the same time, but in another polypropylene tube
add an appropriate amount of biotinylated-ROR protein from the
stock solution to give a final concentration of 4E-8 M (40 nM).
To the biotinylated-ROR solution, add an appropriate amount of
APC-labeled streptavidin to give a final concentration of 2E-8 M
(20 nM). Invert gently to mix. Incubate 15 minutes at room
temperature. Following the 15 minute incubations, add 20-fold
excess biotin from the 1E-2 M (10 mM) stock solution. Invert
gently to mix. Incubate 10 minutes at room temperature. Gently
mix the above solutions together to give a final solution
containing 20 nM ROR_10 nM APC and 20 nM SRC1(2)_5nM
SA_EU. Incubate 5 min and use a Thermo Combi Multidrop to
add 25 uL peptide/ROR solution to assay plates containing 100
nL of test compound. Incubate plates for 1 hr at room
temperature, then read on ViewLux in Lance mode for EU/APC.
5.2.1 RORγ FRET Assay
The assays were performed in an assay buffer consisting of 50
mM NaF, 50 mM 3-(N-morpholino)propanesulfonic acid, pH 7.4,
50
µM
3-[(3-
cholamidopropyl)dimethylammonio]propanesulfonate, 0.1 mg/ml
bovine serum albumin, and 10 mM dithiothreitol in 384-well
plates (Greiner 784076, Longwood, FL). The total volume was
10 µl/well. The europium-labeled SRC1 solution was prepared
by adding an appropriate amount of biotinylated SRC and
europiumlabeled streptavidin (PerkinElmer Life and Analytical
Sciences, Waltham, MA) into assay buffer, with final
concentrations of 27 and 3.3 nM, respectively. The APC-labeled-
LBD solution was prepared by adding an appropriate amount of
biotinylated RORγ-LBD and APC-labeled streptavidin (CR130-
100; PerkinElmer Life and Analytical Sciences) at a final
concentration of 33 nM each. After 15 min of incubation at room
temperature, a 20-fold excess of biotin was added to block the
remaining free streptavidin. Equal volumes of europium-labeled
SRC- and APC-labeled RORγ-LBD were then mixed with 0.2
µM surrogate agonist N-(2-chloro-6-fluorobenzyl)-N-((2'-
methoxy-[1,1'-biphenyl]-4-yl)methyl)benzenesulfonamide
and
dispensed into 384-well assay plates at 10 µl volume/well. The
384-well assay plates had 25 nl of test compound in DMSO
predispensed into each well. The plates were incubated for 1 h at
room temperature and then read on ViewLux (PerkinElmer Life
and Analytical Sciences) in LANCE mode configured for
europeum-APC labels.
Data analysis: Raw data was analyzed using ABASE (IBDS)
software. The data was normalized initially using the following
equation: Normalization = 100*((Basal HTRF – value)/(Basal
HTRF – Minimal HTRF). The normalized data was then was fit
to a 4 – parameter logistic equation.
5.2.3 Mouse Th17 cell differentiation assay
5.2.2 RORγ dual FRET assay
CD4+T cells were purified from spleens using anti-CD4
magnetic microbeads (Miltenyi Biotec) and MACS columns
Materials: RAR-related orphan receptor gamma (RORγ)
protein was made at GSK. Proteins were chemically biotinylated

(purity was >95%). CD4+ cells were resuspended in RPMI 1640
complete medium and added to 96-well plates pre-coated with
anti-mCD3 (5 μg/ml) at 105 cell/well in a total volume of 80 μl.
One hundred microliters of a 2×cytokine cocktail and 20 μl of
compounds (100×) were added to the well. The final
concentrations of antibodies and cytokines (all from R&D
Systems, Minneapolis, MN) were as follows: anti-mCD28 (5
μg/ml); anti-mIFN-γ (10 μg/ml); anti-mIL4 (10 μg/ml); mIL-6
(20 ng/ml); hTGF-β1 (5 ng/ml). The culture was incubated in
37°C for 3 days, and supernatants were collected for ELISA. The
mouse IL-17 ELISA was performed according to the
manufacturer's instruction (R&D Systems). The results were
analyzed using Prism software with nonlinear regression to
determine the IC50 value.
with “triangle matcher” placement and “London dG” scoring
function in MOE2013.08 was used in docking. The binding mode
which showed the best structural overlay with that of 2c in
RORγt LBD among the top 30 predicted poses was selected as
the predicted binding mode of the compound docked.
5.5 Metabolic stability study in mouse liver microsomes
In vitro metabolic incubations were carried out in 96-well
polypropylene plates on a waterbath shaker at 37°C. Reaction
mixtures consisted of 0.5µM compound, 0.5mg/mL of
microsomes and 50mM potassium phosphate buffer (pH 7.4). To
initiate the reaction, beta-NADP (0.44mM in incubation),
G6P(5.2mM in incubation) and G6PDH(1.2U/mL in incubation)
were added to give a final volume of 0.8mL. An initial time-
point (t0) was collected and subsequent aliquots were collected at
10, 20, 30, 40, 50, and 60 mins. Reactions were terminated by
transferring 100 µL of the incubation mixture into 200 µL of ice-
cold acetonitrile:methanol: acetic acid containing internal
standard (80:20:1, v/v/v). Precipitated protein was pelleted by
centrifugation and the resultant supernatant was transferred to a
new 96-well popypropylene plate for LC/MS/MS analysis.
Compound remaining as a ratio to internal standard is measured
using specific HPLC-MS/MS methods. The first order
elimination rate constant (k) is determined from non-linear
regression analysis of peak area ratio vs time.
5.3 X-ray crystallography study: protein preparation,
crystallization and structure determination
Human RORγt 263-509 was expressed from a modified
pET21d vector in E. Coli BL21(DE3) cells with a TEV-
cleavable N-terminal hexa-histidine tag.
The cells were
harvested after 12-16 hours of protein expression at 15ºC and
lysed in Nickel Buffer A (30 mM Imidazole, pH 8.0, 150 mM
NaCl).
Cleared lysate was applied to a 5ml HisTrap column (GE
healthcare) at 2ml/min. The column was washed with 5% Nickel
Buffer B (500 mM Imidazole, pH 8.0, 150 mM NaCl) and eluted
with 5% to 100% gradient Nickel Buffer B. The protein was
further purified with a Superdex 200 16/60 column (GE
Healthcare) in the storage buffer (20mM Hepes, pH 7.4, 50 mM
NaCl). 5-fold excess of RORgt activator compound and 2-fold
excess of the SRC2-2 peptide (KEKHKILHRLLQDSS, China
Peptide) were preincubated with purified RORγt LBD on ice for
30 minutes before filter concentrated to 10 mg/ml in storage
buffer.
Notes: All studies were conducted in accordance with the
GSK Policy on the Care, Welfare and Treatment of Laboratory
Animals and were reviewed the Institutional Animal Care and
Use Committee either at GSK or by the ethical review process at
the institution where the work was performed.”
Acknowledgments
Authors thank Wei Cai, Yaobang Cheng, Gang An, Jinqiang
Zhang, Zhong Zhong and Hongtao Lu for their helpful
discussions.
Crystals of RORγt LBD complexed with activator compound
were grown at 20ºC in hanging drops by mixing 1 l of protein
(at 10 mg/ml) and 1 l of a reservoir solution (0.2M Ammonium
Sulfate, 0.1M Bis-Tris, pH 6.5, 14% (w/v) PEG 8000). Crystals
were quickly transferred to reservoir solution supplemented with
20% (v/v) glycerol before flash-frozen in liquid nitrogen. X-ray
diffraction data were collected at the 17U beamline of the
Shanghai Synchrotron Radiation Facility (SSRF). All data were
processed with HKL2000 denzo/scalepack package (Otwinowski
and Minor, 1997).
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Discovery of N-(4-aryl-5-aryloxy-thiazol-2-
yl)-amides as potent RORt inverse agonists
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Yonghui Wanga,, Ting Yangb, Qian Liub, Yingli Mab, Liuqing Yangb, Ling Zhoub, Zhijun Xiangb, Ziqiang
Chengb, Sijie Lub, Lisa A. Orband-Millerb, Wei Zhangb, Qianqian Wub, Kathleen Zhangb, Yi Lib, Jia-Ning
Xiangb, John D Elliottb, Stewart Leungb, Feng Renb, and Xichen Linb,*
aSchool of Pharmacy, Fudan University, 826 Zhangheng Road, Pudong, Shanghai 201203, China
bResearch and Development, GlaxoSmithKline, No. 3 Building, 898 Halei Road, Pudong, Shanghai 201203, China