Discovery of carboxyl-containing biaryl ureas as potent RORγt inverse agonists

Article abstract of DOI:10.1016/j.ejmech.2020.112536

GSK805 (1) is a potent RORγt inverse agonist, but a drawback of 1 is its low solubility, leading to a limited absorption in high doses. We have explored detailed structure-activity relationship on the amide linker, biaryl and arylsulfonyl moieties of 1 trying to improve solubility while maintaining RORγt activity. As a result, a novel series of carboxyl-containing biaryl urea derivatives was discovered as potent RORγt inverse agonists with improved drug-like properties. Compound 3i showed potent RORγt inhibitory activity and subtype selectivity with an IC₅₀ of 63.8 nM in RORγ FRET assay and 85 nM in cell-based RORγ-GAL4 promotor reporter assay. Reasonable inhibitory activity of 3i was also achieved in mouse Th17 cell differentiation assay (76percent inhibition at 0.3 μM). Moreover, 3i had greatly improved aqueous solubility at pH 7.4 compared to 1, exhibited decent mouse PK profile and demonstrated some in vivo efficacy in an imiquimod-induced psoriasis mice model.

Full text of DOI:10.1016/j.ejmech.2020.112536

Journal Pre-proof
Discovery of carboxyl-containing biaryl ureas as potent RORγt inverse agonists
Nannan Sun, Yafei Huang, Mingcheng Yu, Yunpeng Zhao, Ji-An Chen, Chenyu Zhu,
Meiqi Song, Huimin Guo, Qiong Xie, Yonghui Wang
PII:
S0223-5234(20)30508-0
DOI:
https://doi.org/10.1016/j.ejmech.2020.112536
EJMECH 112536
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 March 2020
Revised Date: 23 May 2020
Accepted Date: 3 June 2020
Please cite this article as: N. Sun, Y. Huang, M. Yu, Y. Zhao, J.-A. Chen, C. Zhu, M. Song, H. Guo,
Q. Xie, Y. Wang, Discovery of Carboxyl-containing Biaryl Ureas as Potent RORγt Inverse Agonists,
European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112536.
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Graphical abstract:

Discovery of Carboxyl-containing Biaryl Ureas as Potent RORγt Inverse Agonists
1
1
Nannan Sun , Yafei Huang , Mingcheng Yu, Yunpeng Zhao, Ji-An Chen, Chenyu
Zhu, Meiqi Song, Huimin Guo, Qiong Xie*, Yonghui Wang*
a
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826
Zhangheng Road, Shanghai 201203, China
*
Corresponding authors:
Tel & Fax: +86-21-5198 0122, E-mail address: qxie@fudan.edu.cn (Q. Xie);
Tel & Fax: +86-21-5198 0118, E-mail address: yonghuiwang@fudan.edu.cn (Y.
Wang)
1
These authors contributed equally in this work.

Abstract
GSK805 (1) is a potent RORγt inverse agonist, but a drawback of 1 is its low
solubility, leading to a limited absorption in high doses. We have explored detailed
structure-activity relationship on the amide linker, biaryl and arylsulfonyl moieties of
1
trying to improve solubility while maintaining RORγt activity. As a result, a novel
series of carboxyl-containing biaryl urea derivatives was discovered as potent RORγt
inverse agonists with improved drug-like properties. Compound 3i showed potent
RORγt inhibitory activity and subtype selectivity with an IC of 63.8 nM in RORγ
5
0
FRET assay and 85 nM in cell-based RORγ-GAL4 promotor reporter assay.
Reasonable inhibitory activity of 3i was also achieved in mouse Th17 cell
differentiation assay (76% inhibition at 0.3 ꢀM). Moreover, 3i had greatly improved
aqueous solubility at pH 7.4 compared to 1, exhibited decent mouse PK profile and
demonstrated some in vivo efficacy in an imiquimod-induced psoriasis mice model.
Keywords: biaryl ureas; RORγt; inverse agonists; Th17 cells; autoimmune diseases

1
.
Introduction
The retinoic acid receptor related orphan receptor (ROR) is a member of the
nuclear receptor family of transcription factors. There are three subtypes of ROR,
known as RORα, RORβ and RORγ. RORγ exists in two isoforms, RORγ (RORc1)
and RORγt (RORc2). RORγt presents mainly in thymus, and it plays a key role in the
proliferation of T helper (Th) 17 cells and the production of the pro-inflammatory
1
cytokine IL-17. IL-17 is highly associated with the pathogenesis of autoimmune
2
,3,4
diseases,
thus RORγt is implicated in some autoimmune diseases including
multiple sclerosis (MS), rheumatoid arthritis (RA), psoriasis, and inflammatory bowel
5
–10
diseases (IBD).
RORγt inverse agonists, which can down-regulate RORγt gene transcription by
recruiting corepressors or blocking coactivator recruitment, have potential utility in
reducing Th17 cell differentiation and therefore can be developed as therapeutic
11–14
agents for the treatment of IL-17 mediated autoimmune diseases.
The
development of RORγt inverse agonists has been dated back to the year of 2011,
when digoxin, SR1001 and ursolic acid were reported to have transactivation
1
5-17
inhibitory activity of RORγt.
been pushed into clinical studies for the treatment of autoimmune diseases, especially
Up to now, several RORγt inverse agonists have
1
8–20
of psoriasis.
discovery stage, among them, GSK805 is an early reported one.
excellent in vitro activities as well as good oral exposure and CNS penetration,
Additionally, many promising candidates are in preclinical and
2
1–24
Though having
2
1
GSK805 (1, Figure 1A) was used just as a tool compound for RORγt biological
studies. The most likely reason could be its high hydrophobicity (ALogPꢁ=ꢁ7.5 in our
calculation) and poor solubility (1.25 ꢀg/mL in our solubility test), which might affect
its absorption in higher doses and prevent it to be further developed.
We aimed to identify novel RORγt inverse agonists which have a reduced
lipophilicity compared to 1 while maintaining good RORγt inhibitory activity. The
predicted binding mode of 1 indicates that the biaryl moiety acts as a functional group
by forming hydrophobic interaction with Tyr502 and His479 and affecting AF2
domain, and the other parts of molecule, the linker amide and the sulfonyl moiety, can
form hydrogen bonds with surrounding polar residues, such as Arg367 and Leu287
(Figure 1B). We had a hypothesis that introducing some polar groups located on the
linker and/or the sulfone moiety may reduce logP of 1 while maintaining the RORγt

activity. Guided by insights from the binding mode of 1, we carried out a series of
structural modifications, which were made on the linker, the right-hand side (RHS)
and the left-hand side (LHS) moieties of 1. In this paper, we describe design,
synthesis and biological evaluation of carboxyl-containing biaryl urea derivatives as
novel RORγt inverse agonists with improved drug-like properties. The binding mode
of the representative compound is also discussed.
Cl
O
O
S
O
O
F C Cl
N
3
H
1
(GSK805)
A
B
Figure 1. (A) Chemical structure of 1 (GSK805). (B) The binding mode of 1 (orange
stick) in RORγt ligand binding domain (LBD) revealed by docking study. (PDB:
2
5
26
5
NU1 . In 5NU1, structure of the ligand is similar to that of 1, and the overlay of
these two compounds is shown in Figure S1.) In this binding mode, the biaryl moiety
of 1 provides hydrophobic interactions with Tyr502 and His479 and π-π interaction
with Phe378, the sulfonyl moiety forms hydrogen bonds with Arg367 and Leu287,
and the linker amide forms hydrogen bond interaction with the backbone of Phe378.
2
.
Results and discussion
2
.1 Compound design

Based on the structure of 1, we made modifications on linker, RHS and LHS and
used ALogP to estimate logP (Figure 2). Firstly, we designed a few compounds (2-7)
with different linkers, such as reverse amide, urea, amine and ether, trying to identify
a new and suitable linker to connect the LHS and RHS. The urea linker was identified
as the optimal linker as the urea 3 had decent RORγt activity and a slightly lower
ALogP value. Then a number of urea-based compounds were designed to improve
RORγt inhibitory activity and solubility through the RHS and LHS modifications. In
RHS, some polar bioisosteric groups of the sulfone moiety were introduced (3a-3w),
trying to keep the hydrogen bond formed with Arg367 and Leu287 and increase the
hydrophilicity of compounds. Compounds 8-15 with different substituted biaryl
moieties in LHS were designed for fine-tuning of the interaction with the hydrophobic
sub-pocket around His479 in RORγt-LBD.

Figure 2. Design of novel RORγt inverse agonists. Target compounds with different
linkers (2~7), urea linker with RHS (3a~3w) and LHS (8~15) modifications.
2
.2 Chemistry
Synthetic procedures for the target biaryl urea compounds were outlined in Scheme
and Scheme 2. General synthesis of non-carboxyl-containing biaryl urea derivatives
1
was shown in Scheme 1. The aryl boric acids 16 were coupled with 4-nitro substituted
aryl halides 17 by Suzuki reaction to obtain biaryl intermediates 18. The biaryl amines
1
9 were obtained by reduction of the nitro group of 18. Intermediates 3-1 reacted with
Raney Ni under H to afford different substituted benzylamines intermediate 3-2.The
2

resulting
,6-dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-amine (19-1) in the presence of
triphosgene to produce biaryl urea derivatives (3, 3a~3g, 3k).
benzylamines
3-2
reacted
with
2
Scheme 1. General synthetic procedure for biaryl urea derivatives 3, 3a, 3b, 3c, 3d, 3e,
f, 3g, 3k: Reaction conditions: (a) Pd (dba) , Tri-tert-butylphosphine
3
2
3
tetrafluoroborate, K CO or Na CO , 110 °C; (b) SnCl , hydrochloric acid, ethanol,
2
3
2
3
2
6
0 °C; (c) Raney Ni, H , MeOH; (d) triphosgene, N, N-diisopropylethylamine
2
(DIPEA), DCM, 0°C-rt.
A versatile synthesis of the general structures of carboxyl-containing biaryl urea
derivatives was developed and outlined in Scheme 2. The synthesis started with
-bromo substituted aryl acids 20, which reacted with SOCl in methanol to afford the
4
2
corresponding methyl esters 21. Esters 21 in the presence of Pd(PPh ) , Zn(CN) and
3
4
2
DMF produced 4-cyano substituted aryl esters 22, which reacted with Raney Ni or
Pd/C under H to afford intermediate 4-aminomethyl substituted aryl esters 23. Biaryl
2
amines 23 reacted with esters 19 in the presence of triphosgene to produce urea esters
2
4, which under alkaline condition were converted into biaryl urea- carboxylic acids
(3h~3j, 3l~3w, 8~15).
Synthetic procedures of 2~7 were described in the supporting information.

Scheme 2. General synthetic procedures for biaryl urea-carboxylic acid derivatives
h-3j, 3l~3w, 8~15. Reagents and conditions: (a) SOCl , MeOH, 0 °C-rt; (b)
3
2
Pd(PPh ) , Zn(CN) , DMF, 155 °C, MW; (c) Raney Ni, H , MeOH; (d) triphosgene,
3
4
2
2
DIPEA, DCM, 0 °C-rt; (e) NaOH or LiOH, ethanol/H O, rt.
2
2
.3 Structure-activity relationship (SAR)
All the synthesized compounds were evaluated by FRET assay, an important in
vitro biochemical assay measuring RORγt-coactivator (steroid-receptor coactivator 1,
SRC1) recruitment inhibitory activity of the new compounds. The details of FRET
assay description are shown in Experimental 4.3.1. In order to identify a new and
better linker which has the potential to improve the hydrophilicity of the compounds,
we firstly designed and synthesized compounds (2~7) with different linkers between
biaryl and arylsulfonyl moieties (Table 1). Compound 2 with a reversed amide linker
relative to 1 showed a lowered RORγt inverse agonist activity (IC = 112.4 nM).
5
0
With a nitrogen atom inserted between amide carbonyl and CH of 1 to form a urea
2
linker, compound 3 showed a similar RORγt inverse agonist activity (IC = 46.3 nM).
5
0
Extending the length of the linker by adding an oxygen atom in the linker of 1
declined the activity (4 with an IC of 99.0 nM). Removal of the carbonyl of the
5
0
amide linker in 1 led to compound 5 with an improved activity (IC = 21.6 nM) but
5
0

higher hydrophobicity (ALogP value of 8.4) compared with that of 1. The RORγt
activity of both 6 and 7 dramatically decreased, probably because the hydrogen bond
between the linker and Phe378 was broken when the NH in the linker of 5 was
replaced by O or CH . Considering decent RORγt activity, relatively low
2
hydrophobicity and the importance of urea in improving aqueous solubility and
2
7
permeability, we selected the urea linker in 3 as the preferred linker for further LHS
and RHS optimization.
Table 1. SAR of different linkers.
b
RORγ FRET IC (nM)
a
50
Compd.
Linker
ALogP
7.5
c
(% max. inhibition)
1
(GSK805)
35.2 ± 14.0 (116)
2
3
4
7.5
7.4
7.3
112.4 ± 0.1 (152)
46.3 ± 7.5 (119)
99.0 ± 16.7 (112)
5
8.4
21.6 ± 2.5 (126)
6
7
8.6
8.1
426.1 ± 15.2 (140)
4411 ± 1363 (143)
a
ALogP value is calculated by Discovery Studio 3.5.
IC value is the average of at least two determinations.
b
c
5
0
28, 29
Percent max. inhibition measured against activation by a surrogate agonist.

We then focused on SAR exploration of RHS arylsulfone moiety with urea linker
fixed in the compounds. According to the binding mode, the arylsulfone moiety
locates in a polar pocket around Arg367 and Leu287, which is a more polar-tolerable
site than other locations in the binding side. The results from SAR exploration of RHS
were summarized in Table 2. Replacing the sulfone moiety in 3 with different
bioisosteric groups (3a-3g) led to a decreased RORγt activity, although the
hydrophilicity of these compounds was improved (all ALogPs < 7.4, except for 3d).
Then we introduced a carboxyl group as a replacement of the sulfone moiety trying to
improve the hydrophilicity of compounds as well as keep the hydrogen bond formed
with polar residues around Arg367 and Leu287. As expected, there was a
hydrophobicity reduction for the carboxyl-containing compounds that had lower
ALogD values than 3. Varying length of carboxylic acids (3h, 3i, 3j and 3w) indicates
that length of carboxyl substituent is very important for the activity and acetic acid is
optimal (IC : 63.8 nM for 3i vs. 1096 nM for 3h, 841.4 nM for 3j, 521.7 nM for 3w).
5
0
Besides, the RORγt activity of compound 3k dropped dramatically while replacing
the acetic acid by ethanediol. Introducing substituents on the α-position of the
carboxylic acid such as hydroxyl (3l), methyl (3m), fluoro (3n, 3o), the RORγt
activity decreased. The larger the size of substituent, the weaker the activity, with an
order of –CH < -OH < -F/-F < –H. Introduction of substituent on the phenyl ring of
3
2
the RHS such as fluoro (3p, 3q), chloro (3r) or methyl (3s, 3t) also lowered the
activity. Though introduction of N on the phenyl ring of the RHS (3u, 3v) could
increase the polarity of the compound, the activity showed a sharp decline. Overall,
acetic acid seemed the best replacement of alkyl sulfone with maintained activity and
improved hydrophilicity.
Table 2. RHS SAR of the urea compounds

a
RORγ FRET IC (nM)
5
0
1
5
6
c
Compd.
R
R
R
X
ALogD
b
(
% max. inhibition)
46.3 ± 7.5 (119)
69.0 ± 9.5 (136)
275.6 ± 28.1 (165)
99.8 ± 14.0 (144)
199.6 ± 52.1 (134)
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
CH
7.4
7.0
6.2
6.4
7.7
6.4
6.3
6.2
5.7
5.7
6.2
3
3
a
3
b
3
c
3
d
3
e
936.8 ± 143.1 (156)
2521 ± 234.1 (216)
138 ± 26.0 (214)
3
f
3
g
3
h
1096 ± 153.4 (207)
63.8 ± 1.4 (183)
3
i
3
j
841.4 ± 228.5 (216)
H
H
CH
6.4
3
k
584.3 ± 49.5 (170)
H
H
H
H
H
H
CH
CH
CH
5.0
6.2
6.0
3
l
278.2 ± 72.6 (194)
541.1 ± 4.7 (208)
123.8 ± 29.0 (222)
3
m
3
n

H
F
H
H
F
CH
CH
CH
CH
CH
6.1
5.9
5.9
6.4
6.2
6.2
4.8
5.2
6.6
3
o
100.3 ± 8.5 (205)
239 ± 9.4 (192)
3
p
q
H
3
232.5 ± 62.5 (126)
433.8 ± 297.8 (198)
995.5 ± 228.5 (223)
397.5 ± 91.7 (196)
390.1 ± 96.3 (214)
1783 ± 103.2 (203)
521.7 ± 52.3 (194)
Cl
CH3
H
H
H
3
r
s
3
CH3 CH
3
t
H
H
H
N
N
3
u
H
3
v
H
CH3 CH
3
w
a
b
a
IC value is the average of at least two determinations.
Percent max. inhibition measured against activation by the surrogate agonist.
ALogD value is calculated by Discovery Studio 3.5.
5
0
Based on the above SAR studies on linker and RHS moiety, 3i was selected as the
template to make LHS modification. As the biaryl moiety is fully explored in the
2
1
biaryl amide series before, we selected a few substituents which were proven to be
privileged on the biaryl rings, trying to increase hydrophilicity of the compounds
(Table 3). The activity of compound 8 dropped dramatically as the substituents on
2
3
4
biphenyl rings were removed, indicating the R , R and R substituents are needed.
3
With only the R substituent of 3i removed, the resulting compound 9 showed over
ten-fold reduction in RORγt activity (IC ꢁ=ꢁ771.5ꢁnM) relative to 3i. Just replacing
5
0
3
the -Cl group on R by methyl (11) and polar -CN group (12), compound 11 could
barely remain activity (IC ꢁ=ꢁ81.6ꢁnM) while 12 had an activity decrease
5
0
2
3
(
IC ꢁ=ꢁ334.3ꢁnM). Replacing both R and R by methyl, compound 13 decreased in
50
3
2
activity compared to 11. Above SAR of R and R substituent demonstrates that both

–
Cl substituent is preferred and polar substituent groups are inappropriate. The
3
2
activity of compound 15 with –F on both R and R fells sharply, which further
4
supports this observation. Besides, R substituent should not be polar group too.
Replacing the -OCF group by a more polar -OCF H group (10 and 14) lowered
3
2
activity.
Table 3. LHS SAR of urea compounds
a
RORγ FRET IC (nM)
5
0
c
Compd.
R4
R2
R3
ALogD
b
(% max. inhibition)
3
i
-OCF
3
-Cl
H
-Cl
H
5.7
3.7
5.0
4.4
5.5
4.9
5.3
4.0
4.8
63.8 ± 1.4 (183)
> 30000 (153)
8
H
9
-OCF
3
-Cl
-Cl
-Cl
-Cl
-CH
-CH
-F
H
771.5 ± 30.6 (189)
80.5 ± 11.1 (178)
81.6 ± 0.6 (178)
334.3 ± 17.8 (191)
226.2 ± 43.8 (233)
345.3 ± 0.4 (170)
1
0
1
2
3
4
5
-OCF
2
H
-Cl
-CH
1
1
1
1
1
-OCF
-OCF
-OCF
3
3
-CN
3
-CH
-CH
-F
3
3
3
3
-OCF
H
2
3
-OCF
995.7 ± 192.8 (156)
3
a
b
c
IC value is the average of at least two determinations.
5
0
Percent max. inhibition measured against activation by the surrogate agonist.
ALogD value is calculated by Discovery Studio 3.5.
2
.4 Binding mode study
The binding mode of the representative carboxyl-containing biaryl urea RORγt
inverse agonist 3i with RORγt-LBD was showed in Figure 3. The binding pose of 3i
with RORγt was similar to that of 1 (GSK805). The urea linker in the skeleton of 3i

formed two strong hydrogen bonds with the main chain of Phe378 (3.0 Å, 2.7 Å) in
the active site of RORγt-LBD, which had one more hydrogen bond than that of the
amide linker in 1. In RHS, the carboxyl group of 3i formed hydrogen bond with the
sidechain of Arg367 (3.6 Å) and the main chain of Leu287 (3.6 Å), which was
important to the RORγt binding affinity as the sulfonyl moiety in 1. In the other side,
the biaryl moiety of 3i resided in the hydrophobic region of the binding site and
formed hydrophobic interaction with His479. Besides, π-π interaction was formed by
biaryl with Phe378. Overlay of 3i with 1 (Figure 3) showed that, though linker in the
core part of 1 and 3i is different, both the biaryl group in the LHS and the
hydrogen-bonding polar groups in the RHS almost occupied the same position. The
similar activity of 1 and 3i in FRET assay was in accord with their binding modes
aforementioned. The binding modes of 1 and 3i in 2D are shown in Figure S2.
A
B
Figure 3. (A) Binding mode of 3i (blue stick) in RORγt-LBD revealed by docking
study (PDB: 5NU1). (B) Overlay of 3i (blue stick) with 1 (orange stick).
2
.5 Solubility, cell-based activity and selectivity
We next evaluated the solubility of the representative carboxyl-containing biaryl
urea RORγt inverse agonist 3i. Reduced lipophilicity (ALogD of 5.7) and much
-
1
improved aqueous kinetic solubility (100 ꢀg·mL at pH 7.4, Table 4) were achieved
for 3i, 80-times higher than that of 1. Then we used RORγ-GAL4 reporter gene assay
and a mouse Th17 cell differentiation assay to test the compounds’ ability to inhibit
RORγt transcription and IL-17 production. The details of the two assays are shown in

Experimental 4.3.2 and 4.3.3. The activities of the compounds tested in the cellular
assay were essentially consistent with those in the FRET assay. In RORγ-GAL4
reporter gene assay, 3i exhibited high transcriptional inhibitory activity with an IC50
of 85 nM. Also, a mouse Th17 cell differentiation assay was performed to evaluate
the functional activity of 3i. It is good to see that compound 3i has a comparable
activity as 1 with an inhibition of 76% at 0.3ꢁꢀM. Compound 3i was evaluated
towards other members of the ROR family to have sub-type selectivity over RORγt.
As shown in Table 4, 3i did not bind to RORα nor RORβ, showing excellent ROR
sub-type selectivity.
Table 4. In vitro activity and selectivity profile for compounds 1 and 3i
mTh17, %inh
Sol.
RORγ-GAL4 RORα-GAL4 RORβ-GAL4
Compd.
at
0.3 ꢀM
-
1 a
c
c
c
(
ꢀg·mL )
IC (nM)
IC (ꢀM)
IC (ꢀM)
5
0
50
50
b
1
1
.25
84
76
-
-
-
(
GSK805)
3
i
100
85
>10
>10
a
b
c
Sol.: solubility in aqueous buffer at pH 7.4.
Results in mouse Th17ꢁcell differentiation assay
Results in HEK293 cells transiently transfected with GAL4-RORγ-, GAL4-RORα-
and GAL4-RORβ-luciferase plasmids.
2
.6 Pharmacokinetic (PK) study of compound 3i
With good RORγt inhibitory activity and selectivity, Th17 differentiation and
transcriptional inhibition, and favorable solubility profile, compound 3i was selected
for further studies to assess its in vivo PK profile. The PK of 3i was investigated in
mice following intravenous (i.v., 1 mg/kg) and oral (p.o., 5 mg/kg) administration.
After oral administration of 3i, the maximum concentration in plasma (Cmax
ng/mL) was reached after 1 h and sufficient plasma exposure was observed (AUC =
412.44 ng·h/mL). Compound 3i also showed a moderate to low clearance (CL = 785
= 609.67
2
mL/h/kg), half-life of 3.6 h and an acceptable oral bioavailability of 38% (see Table 5).
Overall, compound 3i demonstrated good in vivo PK in mice consistent with its in

vitro properties.
Table 5. PK profile of 3i in mice
Parameters/species Male CD-1 Mice
Route of administration
Tmax (hr)
IV
/
PO
1.00
609.67
2412.44
/
Cmax (ng/mL)
AUC0-last (ng·h/mL)
Vss (ml/kg)
1550.00
1267.25
1775.46
3.04
T1/2 (hr)
3.63
/
CL_obs (mL/hr/kg)
F (%)
785.55
/
38.07
2
.6 In vivo efficacy study on imiquimod-induced psoriasis mice model
On the basis of the encouraging Th17 inhibition activity and mouse PK profile,
compound 3i was employed for in vivo efficacy evaluation in an imiquimod
IMQ)-induced psoriasis mice model. Methotrexate (MTX) was chosen as the
(
reference and positive control. Compound 3i was orally administered (p.o.) twice
daily at 25 mg/kg. As shown in Figure 4, the treatment with compound 3i brought in a
significant reduction in clinical severity of psoriasis as measured through the ear
erythema, back skin erythema and scaliness scales.

Figure 4. Effects of 3i in imiquimod-induced psoriasis mice model. IMQ-induced
skin inflammation in mice phenotypically resembles psoriasis. BALB/c mice were
treated daily with IMQ cream on the shaved back skin. Compound 3i was
administered orally twice daily at 25 mg/kg and MTX once daily at 0.4 mg/kg.
Erythema, thickness and scaling of the back skin were scored daily on a scale from 0
to 4 according to Psoriasis Area and Severity Index (PASI). The total score is the sum
of erythema, thickness and scaliness scales.
3
Conclusions
In summary, we have discovered a series of biaryl urea derivatives as potent RORγt
inverse agonists by structure modification on 1 (GSK805). SAR explorations on the
amide linker, RHS arylsulfone and LHS biaryl moieties of 1 led to the identification
of potent carboxyl-containing biaryl urea RORγt inverse agonists with improved
drug-like properties. Compound 3i had greatly improved aqueous solubility and was
found to have good activities in RORγ FRET assay, mouse Th17 cell differentiation
assay and cell based RORγ-GAL4 promotor reporter assay. Furthermore, 3i
demonstrated excellent in vivo PK profile in mice and good in vivo efficacy in an
IMQ-induced psoriasis mice model. Present studies suggest 3i as a potential RORγt
inverse agonist candidate which deserves further evaluation in treatment of
autoimmune diseases.

4
. Experimental
4
.1. Materials and methods
All the reagents used were commercially available and were used without further
purification unless otherwise indicated. Microwave reaction was conducted with a
Biotage Initiator™ microwave synthesizer. Melting point was recorded by WRS-1B
digital instrument. Analytical thin-layer chromatography and preparative thin-layer
chromatography were performed with silica gel plates using silica gel 60 GF254
1
(
Qingdao Haiyang Chemical Co., Ltd., Qingdao, Shandong Province, China). H
NMR spectra were recorded in DMSO-d , methanol-d or CDCl solution on a Bruker
6
4
3
4
00 MHz spectrometer, with chemical shifts expressed in parts per million using
tetramethylsilane (TMS) as an internal standard. Mass spectroscopy was carried out
on Electrospray ionization (ESI) instruments or MALDI-TOF (Bruker).
4
4
.2. Synthesis
.2.1 Procedure for the synthesis of compound 1, 2, 4~7, is provided in Support
Information.
.2.1.1
4
N-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)-2-(4-(ethylsulfonyl)phenyl
)
1
acetamide (1). Off white solid. HPLC purity of 95.3%. H NMR (400 MHz, CDCl ) δ
3
7
.90 (d, J = 8.3 Hz, 2H), 7.70 (s, 2H), 7.66 (s, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.53-7.47
(m, 1H), 7.43-7.37 (m, 2H), 7.28-7.24 (m, 1H), 3.85 (s, 2H), 3.17 (q, J = 7.4 Hz, 2H),
1
3
1
1
.33 (t, J = 7.4 Hz, 3H). C NMR (151 MHz, CDCl ) δ 167.85, 146.16, 140.40,
3
38.29, 136.39, 134.84, 131.40, 129.65, 129.50, 128.87, 127.89, 125.99, 119.76,
+
1
18.25, 50.13, 43.31, 6.72. MS (ESI) m/z: 548.0 [M + H] .
4
2
.2.1.2
,6-Dichloro-N-(4-(ethylsulfonyl)benzyl)-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-carb
1
oxamide (2). Off white solid (76.9%). HPLC purity of 97.3%. mp 161.9-163.8 °C. H
NMR (400 MHz, CDCl ) δ 7.82 (s, 2H), 7.77-7.75 (d, J = 8.3 Hz, 2H), 7.47-7.45 (d, J
3
=
8.3 Hz, 2H), 7.45-7.43 (d, J = 1.7 Hz, 1H), 7.36-7.33 (t, J = 7.5 Hz, 2H), 7.21-7.19
(d, J = 1.5 Hz, 1H), 4.70-4.68 (t, 1H), 4.70-4.68 (d, J = 6.0 Hz, 2H), 3.06-3.01 (q, J =

1
3
7
1
1
.4 Hz, 2H), 1.22-1.18 (d, J = 7.4 Hz, 3H). C NMR (151 MHz, CDCl ) δ 164.36 ,
3
45.78 , 144.07 , 137.14 , 136.20 , 135.24 , 135.08 , 130.76 , 129.88 , 128.57 , 127.66 ,
26.17, 122.28，120.57 , 119.82 ,117.14 , 50.10 , 42.68, 6.69. MS (ESI) m/z: 529.7
-
+
+
[
M-H] . HRMS (ESI ) m/z calcd for C H C F NO S [M + H] : 532.0358; found:
23 18 l2 3 4
5
4
32.0370.
.2.1.3
N-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)-2-(4-(ethylsulfonyl)pheno
xy)acetamide (4). Off white solid (35.3%). HPLC purity of 95.9%. mp 204.8-205.7°C.
1
H NMR (400 MHz, CDCl ) δ 8.26 (s, 1H), 7.92 (d, J = 8.8 Hz, 2H), 7.76 (s, 2H),
3
7
.51-7.47 (t, J = 8.0 Hz ,1H), 7.41-7.37 (t, J = 7.5 Hz, 2H), 7.29-7.26 (d, J = 6.0, 1H),
.16-7.14 (d, J = 8.8 Hz, 2H), 4.72 (s, 2H), 3.15-3.09 (q, J = 7.4 Hz, 2H), 1.31-1.27 (t,
7
1
3
J = 7.4 Hz, 4H). C NMR (151 MHz, CDCl ) δ 164.60, 159.94, 146.18, 136.96 ,
3
1
35.15, 131.78, 131.33, 130.45, 130.21, 129.57, 128.81, 125.99, 119.83, 118.52,
+
+
1
14.68, 66.88, 50.22, 6.90. MS (ESI) m/z: 548.0 [M + H] . HRMS (ESI ) m/z calcd
+
for C H C F NO S [M + H] : 548.0308; found: 548.0312.
2
3
18 l2
3
5
4
2
.2.1.4
,6-Dichloro-N-(4-(ethylsulfonyl)phenethyl)-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-a
1
mine (5). Off white solid (50.1%). HPLC purity of 98.2%. mp 166.6-168.5°C. H
NMR (400 MHz, DMSO-d ) δ 7.83 (d, J = 8.2 Hz, 2H), 7.60 (d, J = 8.2 Hz, 2H),
6
7
2
2
1
1
.57-7.51 (m, 1H), 7.46 (t, J = 7.5 Hz, 2H), 7.34 (dd, J = 7.8, 1.6 Hz, 1H), 6.74 (s,
H), 6.48 (t, J = 5.5 Hz, 1H), 3.39 (dd, J = 12.8, 6.8 Hz, 2H), 3.28 (q, J = 7.3 Hz, 2H),
1
3
.98 (t, J = 7.0 Hz, 2H), 1.10 (t, J = 7.3 Hz, 3H). C NMR (151 MHz, DMSO-d ) δ
6
49.66, 146.35, 145.81, 136.23, 134.30, 132.39, 129.93, 129.62, 127.65, 127.05,
+
20.39, 119.51, 110.59, 49.06, 43.15, 34.28, 7.01. MS (ESI) m/z: 517.8 [M + H] .
+
+
HRMS (ESI ) m/z calcd for C H C F NO S [M + H] : 518.0566; found: 518.0581.
2
3
20 l2
3
3
4
2
.2.1.5
,6-Dichloro-4-(4-(ethylsulfonyl)phenethoxy)-2'-(trifluoromethoxy)-1,1'-biphenyl (6).
1
Off white solid (56.3% ). mp 150.1-152.3°C. H NMR (400 MHz, CDCl ) δ 7.88-7.86
3
(d, J = 8.3 Hz, 2H), 7.51-7.49 (d, J = 8.3 Hz, 2H), 7.48-7.44 (t, J = 7.8Hz, 1H),

7
.38-7.34 (t, J = 7.0 Hz, 2H), 7.26-7.23 (d, J = 1.5 Hz, 1H), 6.95 (s, 2H), 4.24 (t, J =
.4 Hz, 2H), 3.21 (t, J = 6.4 Hz, 2H), 3.12 (q, J = 7.4 Hz, 2H), 1.29 (t, J = 7.4 Hz, 3H).
6
1
3
C NMR (151 MHz, CDCl ) δ 157.94, 145.37, 143.81, 135.36, 135.09, 131.66,
3
1
6
+
29.32, 129.27, 129.10, 127.93, 126.58, 125.98, 119.77, 113.72, 67.63, 50.04, 34.85,
+
+
.87. MS (ESI) m/z: 518.7 [M + H] . HRMS (ESI ) m/z calcd for C H C F O S [M
23
19 l2
3
4
+
H] : 519.0406; found: 519.0412.
.2.1.6
4
2
,6-Dichloro-4-(((4-(ethylsulfonyl)benzyl)oxy)methyl)-2'-(trifluoromethoxy)-1,1'-biph
1
enyl (7). Off white solid (33.3%). HPLC purity of 92.0%. H NMR (400 MHz, CDCl )
3
δ 7.92 (d, J = 8.2 Hz, 2H), 7.60 (d, J = 8.0 Hz, 2H), 7.49 (t, J = 7.0 Hz, 1H), 7.42 (s,
2
H), 7.39 (t, J = 7.0 Hz, 2H), 7.28 (s, 1H), 4.71 (s, 2H), 4.61 (s, 2H), 3.12 (q, J = 7.4
13
Hz, 2H), 1.28 (t, J = 7.4 Hz, 4H). C NMR (151 MHz, CDCl ) δ146.22, 143.47,
3
1
1
39.72, 137.71, 134.84, 133.34, 131.23, 129.60, 129.07, 127.93, 127.37, 126.01,
+
25.97, 119.77, 71.09, 68.16, 50.04, 6.86. MS (ESI) m/z: 519.1[M + H] . HRMS
+
+
(
ESI ) m/z calcd for C H C F O S [M + H] : 519.0406; found: 519.0414.
23 19 l2 3 4
4
.2.2 General procedure for the synthesis of compound (3, 3a~3g, 3k)
The synthesis of different substituted [1,1'-biphenyl]-4-amine intermediate (19).
Different substituted phenylboronic acid 16 (3 mmol, 1.5 eq),
1
-bromo-4-nitrobenzene 17 (2 mmol, 1 eq), Pd (dba) (0.1 mmol, 0.05 eq),
2 3
tri-tert-butylphosphine tetrafluoroborate (0.1 mmol, 0.05 eq), K CO (6 mmol, 3 eq)
2
3
or Na CO (6 mmol, 3 eq), and 1,4-dioxane (10 mL) were added to a microwave vial.
2
3
The mixture was reacted at 100 °C for several hours under microwave. After the
reaction completed, the mixture was cooled to room temperature, then water was
added and the mixture was extracted with ethyl acetate. Solvent was removed under
reduced pressure to afford crude product of 4-nitro-1,1'-biphenyl 18, The crude
product of 18 was dissolved in ethanol (20 mL) and reacted with SnCl (6 mmol, 3 eq)
2
and hydrochloric acid (0.5 mL) at 60°C for several hours. After the reaction
completed, the mixture was purified by column chromatography on silica gel to afford
the [1,1'-biphenyl]-4-amine intermediate (19).

The synthesis of compound (3, 3a~3g, 3k)
Step 1: Intermediate 3-1 (0.51 mmol, 1eq. The synthesis of different substituted
intermediate 3-1 is provided in Support Information.), methanol (10 mL), aqueous
ammonia (0.5 mL, 28% in water) and Raney Ni (100 mg) were added to a 25 mL
single-mouth bottle to react for 30 minutes at room temperature while stirring, then
the obtained mixture was filtered through celite and concentrated in vacuo to give
different substituted benzylamines intermediate 3-2.
Step 2: 2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-amine (19-1, 0.31 mmol,
1
eq), DCM (4 mL), and DIEA (120 mg, 0.93 mmol, 3eq) were added to a 25 mL
single-mouth bottle. The mixture was stirred for 10 min under ice bath, and then
triphosgene (35mg, 0.11 mmol, 0.35 eq) was added, and the reaction was stirred under
ice bath for 30 min, and then 3-2 (0.37 mmol, 1.2 eq) was added, and the reaction was
stirred under ice bath for 30 min, then at room temperature overnight. After the
reaction completed, the mixture was purified by column chromatography on silica gel
to afford the product (3, 3a~3g, 3k).
4
1
.2.2.1
-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)-3-(4-(ethylsulfonyl)benzyl
1
)
urea (3). Off white solid (32%); HPLC purity of 95%. mp 164.0-165.5 °C. H NMR
(
400 MHz, CDCl ) δ 7.67-7.65(d, J = 8.4 Hz, 2H), 7.56 (s, 2H), 7.54 (s, 1H),
3
7
.49-7.43 (m, 1H), 7.39-7.34 (m, 4H), 7.26-7.22 (m, 1H), 6.08-6.05 (t, J = 6.0 Hz,
H), 4.51-4.50 (d, J = 6.0 Hz, 2H), 3.18-3.13 (q, J = 7.4 Hz, 2H), 1.31-1.27 (t, J = 7.4
1
1
3
Hz, 3H). C NMR (151 MHz, CDCl ) δ 154.53 , 145.65 , 139.81 , 135.71 , 134.71 ,
3
1
31.63 , 129.29, 129.16, 127.63, 127.54, 127.26, 125.90 ,120.51, 119.71, 118.90,
+
+
1
16.97 , 50.13 , 42.43 , 6.65. MS (ESI) m/z: 546.7 [M + H] . HRMS (ESI ) m/z calcd
+
for C H C F N O S [M + H] : 547.0467; found: 547.0486.
2
3
19 l2
3
2
4
4
1
.2.2.2
-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)-3-(4-(methylsulfonyl)benz
1
yl)urea (3a). Off white solid (3 %). HPLC purity of 95%. mp 150.4-152.0 °C. H
NMR (400 MHz, CDCl ) δ 7.73-7.71 (d, J = 8.2 Hz, 2H), 7.54 (s, 2H), 7.51-7.42 (m,
3

2
H), 7.41-7.33 (m, 4H), 7.27-7.23 (m, 2H), 6.02-5.99 (t, J = 6.1 Hz, 1H), 4.49-4.48 (d,
1
3
J = 5.7 Hz, 2H), 3.09 (s, 3H). C NMR (151 MHz, CDCl ) δ 154.65, 146.27, 145.49,
3
1
1
39.72, 137.87, 134.71, 131.61, 129.34, 129.08, 127.62, 127.32, 127.25, 126.86,
-
25.93, 119.70, 117.00, 43.90, 42.49. MS (ESI) m/z: 531.0 [M - H] .
4
4
.2.2.3
-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)benzene
sulfonamide (3b). Off white solid (48 %). HPLC purity of 97.2%. mp 215.6-217.6 °C.
1
H NMR (400 MHz, CD OD) δ 7.86 (d, J = 8.3 Hz, 2H), 7.58 (s, 2H), 7.53-7.47 (m,
3
13
3
H), 7.43-7.36 (m, 2H), 7.28 (d, J = 7.5 Hz, 1H), 4.47 (s, 2H). C NMR (151 MHz,
CD OD) δ 155.37, 146.28, 143.59, 141.66, 140.73, 134.27, 131.60, 129.27, 129.21,
3
-
1
4
4
26.82, 126.55, 125.94, 125.40, 119.46, 116.48, 42.04. MS (ESI) m/z: 531.7 [M – H] .
.2.2.4
-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)-N-meth
ylbenzenesulfonamide (3c). Off white solid (79 %). HPLC purity of 96%. mp
1
1
71.9-173.2 °C. H NMR (400 MHz, DMSO-d ) δ 9.20 (s, 1H), 7.74-7.72 (d, J = 8.2
6
Hz, 2H), 7.66 (s, 2H), 7.59-7.55 (t, J = 8.2Hz, 1H), 7.53-7.43 (m, 4H), 7.44-7.32 (m,
H), 7.07-7.04 (t, J = 5.8 Hz, 1H), 4.39-4.38 (d, J = 5.7 Hz, 2H), 2.38-2.37 (d, J = 4.8
2
1
3
Hz, 3H). C NMR (151 MHz, DMSO-d ) δ 154.71, 145.93, 144.85, 142.01, 137.47,
6
1
33.93, 132.37, 130.44, 129.26, 127.42, 127.31, 126.64, 125.60, 120.65, 120.56,
+
1
18.95, 116.37, 42.28, 28.49. MS (ESI) m/z: 547.8 [M + H] .
4
1
.2.2.5
-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)-3-(4-(ethylsulfonimidoyl)b
1
enzyl)urea (3d). Off white solid (50 %). HPLC purity of 98%. mp 98.7-99.9 °C. H
NMR (400 MHz, CDCl ) δ 8.36 (s, 1H), 7.75 (d, J = 6.9 Hz, 2H), 7.55 (s, 2H), 7.45 (t,
3
J = 7.1 Hz, 1H), 7.39-7.28 (m, 4H), 7.22 (d, J = 6.7 Hz, 1H), 6.68 (s, 1H), 4.44 (d, J =
1
3
4
.4 Hz, 2H), 3.20 (s, 2H), 2.77 (s, 1H), 1.25 (d, J = 7.5 Hz, 3H). C NMR (151 MHz,
CDCl ) δ 145.11, 143.47, 139.72, 137.30, 134.84, 134.34, 131.23, 129.60, 129.07,
3
1
3
1
27.93, 127.37, 126.01, 125.97, 119.77, 71.09, 70.57, 50.04, 6.86. C NMR (151
MHz, CDCl ) δ 154.79, 146.31, 145.54, 140.09, 136.51, 134.65, 131.67, 129.27,
3

1
29.20, 128.13, 127.27, 125.90, 120.62, 119.69, 118.91, 116.71, 52.16, 42.30, 7.30.
+
MS (ESI) m/z: 545.7 [M + H] .
4
.2.2.6
N-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)phen
yl)methanesulfonamide (3e). Off white solid (39 %). HPLC purity of 96%. mp
1
2
20.8-222.2 °C. H NMR (400 MHz, CD OD) δ 7.56 (s, 2H), 7.49 (t, J =28Hz, 1H),
3
7
.44-7.35 (m, 2H), 7.35-7.25 (m, 3H), 7.25-7.16 (m, 2H), 4.34 (s, 2H), 2.90 (s, 3H).
1
3
C NMR (151 MHz, CD OD) δ 155.34, 146.29, 140.82, 136.50, 135.16, 134.25,
3
1
31.60, 129.29, 129.19, 127.47, 126.71, 125.93, 119.90, 119.45, 116.39, 41.99, 37.04.
-
MS (ESI) m/z: 545.7 [M - H] .
4
4
.2.2.7
-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)-N-(met
hylsulfonyl)benzamide (3f). Off white solid (70 %). HPLC purity of 99%. mp
1
1
7
1
1
1
25.5-137.4 °C. H NMR (400 MHz, CD OD) δ 7.88 (d, J = 8.2 Hz, 2H), 7.59 (s, 2H),
3
.55-7.49 (m, 1H), 7.49-7.45 (m, 2H), 7.45-7.36 (m, 2H), 7.29 (dd, J = 7.6, 1.5 Hz,
1
3
H), 4.48 (s, 2H), 3.34 (d, J = 1.7 Hz, 3H). C NMR (151 MHz, CD OD) δ 166.77,
3
55.39, 146.28, 144.82, 140.76, 134.26, 131.60, 130.24, 129.28, 129.21, 127.70,
+
26.80, 126.40, 125.94, 119.46, 116.46, 42.14, 39.56. MS (ESI) m/z: 575.6 [M + H] .
4
.2.2.8
N-((4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)phe
nyl)sulfonyl)acetamide (3g). Off white solid (63%). HPLC purity of 94%. mp
1
2
7
1
31.0-232.2 °C. H NMR (400 MHz, CD OD) δ 7.97 (d, J = 8.3 Hz, 2H), 7.59 (s, 2H),
3
.54 (d, J = 8.2 Hz, 2H), 7.51 (d, J = 8.1 Hz, 1H), 7.45-7.41 (m, 1H), 7.40-7.36 (m,
13
H), 7.29 (d, J = 6.3 Hz, 1H), 4.49 (s, 2H), 1.94 (s, 3H). C NMR (151 MHz,
CD OD) δ 169.13, 155.35, 146.26, 145.43, 140.75, 137.51, 134.25, 131.59, 129.28,
3
1
5
4
1
29.20, 127.41, 126.81, 126.52, 125.93, 119.45, 116.47, 42.02, 21.33. MS (ESI) m/z:
-
73.5 [M - H] .
.2.2.9
-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)-3-(4-(1,2-dihydroxyethyl)

1
benzyl)urea (3k). Off white solid (34%). HPLC purity of 98%. mp 71.7-73.8°C. H
NMR (400 MHz, CD OD) δ 7.58 (s, 2H), 7.50 (t, J = 6.2 Hz, 1H), 7.45-7.25 (m, 7H),
3
1
3
4
.67 (s, 1H), 4.63 (s, 1H), 4.38 (s, 2H), 3.59 (s, 2H), 3.30 (s, 1H).
C NMR (151
MHz, CD OD) δ 155.37, 146.29, 140.82, 140.24, 137.92, 134.26, 131.62, 129.30,
3
1
29.19, 126.69, 126.30, 125.93, 125.67, 119.46, 116.42, 73.71, 66.69, 42.33. MS (ESI)
+
m/z: 514.7 [M + H] .
4
.2.3 General procedure for the synthesis of biaryl urea-carboxylic acid derivatives
(3h-3j, 3l-3w, 8-15)
Step 1: 4-Bromo aryl substituted carboxylic acid 20 (8.66 mmol, 1 eq) and
methanol (10 mL) were added to a 25 mL single-mouth bottle, and then thionyl
chloride (0.5 mL) was added to enable the mixture to react at 65 °C for several hours.
After completion of the reaction, the mixture was concentrated in vacuo to remove the
solvent to give the product methyl 4-bromo aryl substituted carboxylic ester (21)
Step 2: Methyl 4-bromo aryl substituted carboxylic ester 21 (4.1 mmol, 1 eq), zinc
cyanide (6.1 mmol, 1.5 eq) and N, N-dimethylformamide (10 mL) were added to a
microwave tube. The mixture was nitrogen sparged for 5 min, then
tetrakis(triphenylphosphine)palladium (0.2 mmol, 0.05 eq) was added, and the
mixture was stirred and heated to 155 °C for 1.5 hours under microwave. After
completion of reaction, the mixture was cooled to room temperature, and extracted
with ethyl acetate for three times. The organic layers were combined, washed 5 times
with water, finally washed with saturated sodium chloride, and concentrated in vacuo
to get a crude product. The crude product was separated by a silica gel column to give
the product methyl 4-cyano aryl substituted carboxylic ester (22).
Step 3: Methyl 4-cyano aryl substituted carboxylic ester 22 (1.05 mmol, 1 eq) was
dissolved in methanol (2 mL), aqueous ammonia (0.5 mL, 28% in water) and Raney
Ni (100 mg) was added, and the mixture was stirred at room temperature under
hydrogen atmosphere. After the reaction completed, the mixture was filtered by celite,
and the solvent was removed under vacuum to obtain intermediate methyl
4
-aminomethyl aryl substituted carboxylic ester 23, which was directly used in the

next reaction.
Step 4：To a solution of different substituted [1,1'-biphenyl]-4-amine intermediate
(
19) (0.15 mmol, 1 eq) and N, N-diisopropylethylamine (0.3 mmol, 3 eq) in DCM (2
mL) was added triphosgene (0.05 mmol, 0.35 eq) at 0 °C. The mixture was stirred at
°C for 10 minutes, then methyl 4-aminomethyl aryl substituted carboxylic ester (23)
0.18 mmol, 1.2 eq) was added. The mixture was stirred at room temperature for 30
minutes and was then purified by column chromatography on silica gel to afford
-((3-([1,1'-biphenyl]-4-yl)ureido)methyl)aryl substituted carboxylic ester
intermediate (24).
0
(
4
Step 5：To a solution of the ester (24) (0.1 mmol, 1 eq) in ethanol/H O (2 mL/1 mL)
2
was added NaOH (0.3 mmol, 3 eq) or LiOH (0.3 mmol, 3 eq). The mixture was
stirred at room temperature until the reaction completed. Ethanol was removed in
vacuum, and pH value of the mixture was adjusted to 3 with 1 N hydrochloric acid to
afford the target products (3h-3j, 3l-3w, 8-15).
4
4
.2.3.1
-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)benzoic
1
acid (3h). Off white solid (24.3%). HPLC purity of 98%. mp 152.4-154.2 °C.
H
NMR (400 MHz, CD OD) δ 8.01 (d, J = 8.2 Hz, 2H), 7.60 (s, 2H), 7.52 (t, J = 7.0 Hz,
3
13
1
H), 7.47- 7.39 (m, 4H), 7.30 (d, J = 7.5 Hz, 1H), 4.48 (s, 2H). C NMR (151 MHz,
CD OD) δ 168.10, 155.39, 145.30, 144.01, 140.81, 134.25, 131.61, 129.30 , 129.18,
3
-
1
4
2
29.02, 126.75, 126.12, 125.92, 119.45, 116.43, 42.21. MS (ESI) m/z: 496.7 [M - H] .
.2.3.2
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)pheny
l)acetic acid (3i). Off white solid (75%). HPLC purity of 99.0%. mp 150.0-152.4 °C.
1
H NMR (400 MHz, CD OD) δ 7.59 (s, 2H), 7.54-7.51 (t, J = 7.1 Hz, 1H), 7.44-7.39
3
1
3
(
m, 2H), 7.33-7.25 (m, 5H), 4.39 (s, 2H), 3.59 (s, 2H). C NMR (151 MHz, CD OD)
3
δ 174.37, 163.83, 155.38, 146.30, 140.87, 137.07, 134.23, 133.59, 131.63, 129.32,
29.16, 128.58, 126.63, 126.46, 125.92, 119.44, 116.40, 42.27. MS (ESI) m/z: 513.1
1
+
+
+
[
M + H] . HRMS (ESI ) m/z calcd for C H C F N O [M + H] : 513.0590; found:
23 17 l2 3 2 4
5
13.0602.

4
3
.2.3.3
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)pheny
l)propanoic acid (3j). Off white solid (84%). HPLC purity of 96%. mp 94.2-96.7°C.
H NMR (400 MHz, CD OD) δ 7.58 (s, 2H), 7.51 (t, J = 11.1, 4.6 Hz, 1H), 7.45-7.37
3
(m, 2H), 7.29 (d, J = 7.7 Hz, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.0 Hz, 2H),
1
3
4
.36 (s, 2H), 2.90 (t, J = 7.6 Hz, 2H), 2.58 (t, J = 7.6 Hz, 2H). C NMR (151 MHz,
CD OD) δ 155.34, 145.30, 140.86, 139.22, 136.42, 134.24, 131.61, 129.31, 129.18,
3
1
27.56, 126.56, 126.53, 125.92, 119.44, 116.35, 42.29, 29.70. MS (ESI) m/z: 526.9
+
[
M + H] .
4
2
.2.3.4
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)pheny
1
l)-2-hydroxyacetic acid (3l). Off white solid (18%). HPLC purity of 85%. H NMR
(
400 MHz, CD OD) δ 7.58 (s, 2H), 7.55-7.42 (m, 3H), 7.42-7.36 (m, 2H), 7.33 (s,
3
13
2
H), 7.28 (d, J = 7.4 Hz, 1H), 4.40 (s, 2H), 2.03 (s, 1H). C NMR (151 MHz,
CD OD) δ 155.10, 146.73, 140.63, 139.30, 134.59, 131.60, 129.32, 129.18, 129.06,
3
-
1
26.72, 126.28, 125.91, 124.17, 119.34, 116.74, 42.25. MS (ESI) m/z: 526.8 [M - H] .
4
2
.2.3.5
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)pheny
l)propanoic acid (3m). Off white solid (56%). HPLC purity of 96%. mp 86.9-99.2°C.
1
H NMR (400 MHz, CD OD) δ 7.58 (s, 2H), 7.50 (td, J = 8.1, 1.4 Hz, 1H), 7.43-7.35
3
(m, 2H), 7.31-7.23 (m, 5H), 4.37 (s, 2H), 3.69 (q, J = 7.1 Hz, 1H), 1.43 (d, J = 7.1 Hz,
1
3
3
1
H). C NMR (151 MHz, DMSO-d ) δ 175.28, 154.62, 145.94, 142.09, 139.75,
6
38.30, 133.91, 132.38, 130.42, 129.29, 127.30, 127.21, 127.10, 125.46, 120.55,
+
1
18.94, 44.24, 42.35, 18.43. MS (ES) m/z: 526.8 [M + H] .
4
2
.2.3.6
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)pheny
1
l)-2-fluoroacetic acid (3n). Off white solid (97%). HPLC purity of 97%. H NMR
(
400 MHz, CD OD) δ 7.59 (s, 2H), 7.51 (t, J = 7.9 Hz, 1H), 7.49-7.43 (m, 2H),
3
7
.43-7.36 (m, 4H), 7.29 (d, J = 7.6 Hz, 1H), 5.82 (d, J = 48.2 Hz, 1H), 4.43 (s, 2H).

1
3
C NMR (151 MHz, CD OD) δ 155.45, 146.23, 140.83, 140.15, 134.31, 131.71,
3
1
29.30, 129.18, 126.71, 126.55, 126.23, 126.20, 125.83, 119.44, 116.40, 42.55.MS
-
+
+
(
ESI) m/z: 528.7 [M - H] . HRMS (ESI ) m/z calcd for C H C F N O [M + H] :
23 16 l2 4 2 4
5
4
2
31.0496; found: 531.0509.
.2.3.7
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)pheny
l)-2,2-difluoroacetic acid (3o). Off white solid (71%). HPLC purity of 95%. H NMR
(400 MHz, CD OD) δ 7.62-7.55 (m, 4H), 7.55-7.49 (m, 1H), 7.49-7.43 (m, 3H), 7.40
3
1
3
(
t, J = 7.6 Hz, 2H), 7.30 (t, J = 10.1 Hz, 1H), 4.46 (s, 2H). C NMR (151 MHz,
CD OD) δ 155.49, 146.35, 142.06, 140.90, 134.25, 131.60, 129.30, 129.19, 126.76,
3
13
1
26.45, 125.92, 124.68, 119.32, 116.77, 116.43, 41.94. C NMR (151 MHz, MeOD)
δ 155.49, 146.35, 142.06, 140.90, 134.25, 131.50, 129.30, 129.19, 126.75, 126.45,
+
1
4
2
25.92, 124.48, 119.37, 116.77, 116.43, 41.94. MS (ESI) m/z: 548.8 [M + H] .
.2.3.8
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)-2-flu
orophenyl)acetic acid (3p). Off white solid (90%). HPLC purity of 94%. mp
1
1
33.0-135.5°C. H NMR (400 MHz, CD OD) δ 7.58 (s, 2H), 7.54-7.49 (m, 1H), 7.43
3
(d, J = 7.6 Hz, 1H), 7.40 (s, 1H), 7.37 (d, J = 3.7 Hz, 1H), 7.34 (d, J = 7.9 Hz, 1H),
1
3
7
.31-7.26 (m, 1H), 7.08 (t, J = 9.7 Hz, 2H), 4.44 (s, 2H), 3.61 (s, 2H). C NMR (151
MHz, CD OD) δ 173.49, 160.81, 159.19, 155.24, 146.29, 140.77, 136.03, 134.24,
3
1
31.61, 129.30, 129.18, 128.59, 128.56, 126.71, 125.91, 124.43, 123.80(d, J=15.1 Hz),
+
1
19.44, 116.37, 115.20 (d, J=22.7), 39.58, 36.30. MS (ESI) m/z: 530.8 [M + H] .
+
+
HRMS (ESI ) m/z calcd for C H C F N O [M + H] : 531.0496; found: 531.0508.
2
3
16 l2
4
2
4
4
2
.2.3.9
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)-3-flu
orophenyl)acetic acid (3q). Off white solid (54%). HPLC purity of 98%. mp
1
1
2
7
2
67.8-170.8°C. H NMR (400 MHz, DMSO-d ) δ 12.40 (s, 1H), 9.13 (s, 1H), 7.64 (s,
6
H), 7.57 (t, J = 7.8 Hz, 1H), 7.48 (t, J = 7.0 Hz, 2H), 7.40-7.35 (m, 1H), 7.29 (t, J =
.9 Hz, 1H), 7.07 (t, J = 9.5 Hz, 2H), 6.95 (t, J = 5.9 Hz, 1H), 4.32 (d, J = 5.7 Hz,
1
3
H), 3.57 (s, 2H). C NMR (151 MHz, DMSO-d ) δ 172.17, 160.39, 158.77, 154.58,
6

1
1
5
4
2
45.94, 141.99, 136.13, 133.93, 132.37, 130.41, 129.28, 128.95, 127.28, 125.54,
25.25, 124.60 (d, J=15.1) 120.54, 116.28, 115.92 (d, J=22.7), 36.42. MS (ESI) m/z:
+
30.8 [M + H] .
.2.3.10
-(2-Chloro-4-((3-(2,6-dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)met
hyl)phenyl)acetic acid (3r). Off white solid (76%). HPLC purity of 92%. mp
1
1
7
7
2
1
1
4
2
97.7-201.2°C. H NMR (400 MHz, CD OD) δ 12.51-12.29 (m, 1H), 9.18 (s, 1H),
3
.64 (s, 2H), 7.57 (t, J = 7.0 Hz, 1H), 7.47 (t, J = 7.0 Hz, 2H), 7.39-7.35 (m, 1H),
.33 (d, J = 7.9 Hz, 2H), 7.20 (d, J = 9.0 Hz, 1H), 7.04 (s, 1H), 4.28 (d, J = 5.8 Hz,
1
3
H), 3.66 (s, 2H). C NMR (151 MHz, CD OD) δ 172.37, 155.32, 146.30, 140.79,
3
39.90, 134.25, 133.74, 131.61, 131.25, 131.04, 129.30, 129.18, 127.07, 126.74,
+
26.49, 125.92, 124.99, 119.44, 116.43, 41.70. MS (ESI) m/z: 546.7[M + H] .
.2.3.11
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)-2-me
thylphenyl)acetic acid (3s). Off white solid (96%). HPLC purity of 94%. mp
1
1
12.3-114.8°C. H NMR (400 MHz, CD OD) δ 7.59 (s, 2H), 7.52 (td, J = 7.8, 1.3 Hz,
3
1
7
H), 7.43 (d, J = 7.4 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H),
.20-7.14 (m, 2H), 7.12 (d, J = 8.1 Hz, 1H), 4.35 (s, 2H), 3.62 (s, 2H), 2.31 (s, 3H).
1
3
C NMR (151 MHz, CD OD) δ 173.44, 155.33, 146.30, 140.85, 137.42, 136.40,
3
1
34.24, 131.77, 131.62, 129.60, 129.31, 129.18, 128.23, 126.66, 125.92, 124.02,
+
+
1
19.44, 116.37, 42.28,17.65. MS (ESI) m/z: 526.8 [M+H] . HRMS (ESI ) m/z calcd
+
for C H C F N O [M + H] : 527.0747; found: 527.0760.
2
4
19 l2
3
2
4
4
2
.2.3.12
-(4-((3-(2,6-Dichloro-2'-(trifluoromethoxy)-[1,1'-biphenyl]-4-yl)ureido)methyl)-3-me
thylphenyl)acetic acid (3t). . Off white solid (42%). HPLC purity of 96%. mp
1
1
7
81.1-183.4°C. H NMR (400 MHz, DMSO-d ) δ 14.32-13.80 (m, 1H), 9.06 (s, 1H),
6
.64 (s, 2H), 7.60-7.54 (m, 1H), 7.48 (t, J = 7.0 Hz, 2H), 7.41-7.35 (m, 1H), 7.18 (d, J
=
8.3 Hz, 1H), 7.08-7.02 (m, 2H), 6.83 (t, J = 5.6 Hz, 1H), 4.26 (d, J = 5.6 Hz, 2H),
1
3
3
.49 (s, 2H), 2.27 (s, 3H). C NMR (151 MHz, DMSO-d ) δ 172.59, 154.51, 145.95,
6