Discovery of aryl-substituted indole and indoline derivatives as RORγt agonists

Article abstract of DOI:10.1016/j.ejmech.2019.111589

A series of aryl-substituted indole and indoline derivatives were discovered as novel RORγt agonists by a scaffold-based hybridization of the reported RORγt agonists 1 and 2. SAR studies on the core structures, the RHS hydrophilic side chains and the LHS hydrophobic aryl groups of a hybrid compound 3 led to the identification of potent RORγt agonists with improved drug-like properties. Compound 14 represented a high potency lead with an EC₅₀ of 20.8 ± 1.5 nM, the (S)-enantiomer (EC₅₀ = 16.1 ± 4.5 nM) of which was 17 times more potent than the (R) counterpart (EC₅₀ = 286 ± 30.4 nM) in RORγ dual FRET assay. The cell-based GAL4 reporter gene assay also suggested 14 as the most active compound which exhibited an EC₅₀ of 247 ± 33.1 nM and a maximum activation percentage of 133%. Moreover, 14 showed high metabolic stability (t_1/2 = 113 min) in mouse liver microsome and had improved aqueous solubility at pH 7.4 compared to the parent compounds. Furthermore, 14 was found to be orally bioavailable and demonstrated excellent in vivo pharmacokinetics in mice. Present studies indicate that 14 deserves further investigation in tumor animal models as a potential candidate of RORγt agonist for cancer immunotherapy.

Full text of DOI:10.1016/j.ejmech.2019.111589

Journal Pre-proof
Discovery of aryl-substituted indole and indoline derivatives as RORγt agonists
Yan Zhu, Nannan Sun, Mingcheng Yu, Huimin Guo, Qiong Xie, Yonghui Wang
PII:
S0223-5234(19)30723-8
DOI:
https://doi.org/10.1016/j.ejmech.2019.111589
EJMECH 111589
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 5 June 2019
Revised Date: 25 July 2019
Accepted Date: 5 August 2019
Please cite this article as: Y. Zhu, N. Sun, M. Yu, H. Guo, Q. Xie, Y. Wang, Discovery of aryl-substituted
indole and indoline derivatives as RORγt agonists, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.111589.
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Graphic Abstract

Discovery of Aryl-Substituted Indole and Indoline Derivatives as RORγt Agonists
Yan Zhu#, Nannan Sun#, Mingcheng Yu, Huimin Guo, Qiong Xie*, Yonghui Wang*
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng
Road, Shanghai 201203, China
# These authors contributed equally in this work.
*Corresponding authors:
Tel & Fax: +86-21-51980122, E-mail address: qxie@fudan.edu.cn (Q. Xie);
Tel & Fax: +86-21-5198 0118, E-mail address: yonghuiwang@fudan.edu.cn (Y. Wang)

Abstract:
A series of aryl-substituted indole and indoline derivatives were discovered as novel RORγt
agonists by a scaffold-based hybridization of the reported RORγt agonists 1 and 2. SAR studies on
the core structures, the RHS hydrophilic side chains and the LHS hydrophobic aryl groups of a
hybrid compound 3 led to the identification of potent RORγt agonists with improved drug-like
properties. Compound 14 represented a high potency lead with an EC50 of 20.8 ± 1.5 nM, the
(S)-enantiomer (EC50 = 16.1 ± 4.5 nM) of which was 17 times more potent than the (R)
counterpart (EC50 = 286 ± 30.4 nM) in RORγ dual FRET assay. The cell-based GAL4 reporter
gene assay also suggested 14 as the most active compound which exhibited an EC50 of 247 ± 33.1
nM and a maximum activation percentage of 133%. Moreover, 14 showed high metabolic stability
(t1/2 = 113 mins) in mouse liver microsome and had improved aqueous solubility at pH 7.4
compared to the parent compounds. Furthermore, 14 was found to be orally bioavailable and
demonstrated excellent in vivo pharmacokinetics in mice. Present studies indicate that 14 deserves
further investigation in tumor animal models as a potential candidate of RORγt agonist for cancer
immunotherapy.
Keywords: indoles; indolines; RORγt agonists; cancer immunotherapy; metabolic stability;
aqueous solubility

1. Introduction
The T cell specific isoform of retinoic acid receptor-related orphan receptor gamma (RORγ),
known as RORγt, is a nuclear receptor (NR) specifically expressed in thymocytes. RORγt serves
as a key transcription factor to drive the differentiation of CD4+ cells into interleukin-17 (IL-17)
producing T helper 17 (Th17) cells[1], which are implicated in the pathology of various
inflammatory and autoimmune diseases[2, 3]. In addition to their major role in the field of
autoimmune diseases[4, 5], RORγ antagonists were found to be effective in treating
castration-resistant prostate cancer in a few articles[6, 7]. Recent studies have revealed the
presence of Th17 cells in tumor microenvironment[8] and the active involvement of Th17 cells[9]
and CD8+ cytotoxic T (Tc17) cells[10] in protective tumor immunity by majorly producing IL17.
Therefore, RORγt has become a promising target with the potential of strengthening immune
system to combat cancer. RORγt agonists that promote the differentiation and activation of Th17
and Tc17 cells have shown promising therapeutic effects for cancer immunotherapy[11-13].
Starting from the discovery of RORα/γ agonist SR1078[14], more and more small molecule
agonists of RORγt have been reported [13, 15-18] within researches on RORγt inverse agonists.
We previously reported the discovery and complex crystal structures of several N-aryl
amide-based RORγt agonists (Figure 1), such as tertiary amine (PDB: 4NIE)[19], thiazole amide
(PDB: 4XT9)[20], thiazole ketone amide (PDB: 5YP5)[21] and biaryl amide (PDB: 5YP6)[21]. It
was noticed that minor changes of RORγt inverse agonists at the part interacting with activation
function 2 (AF2) of the ligand binding domain (LBD) of RORγ could lead to functional switch
from inhibition to activation[13, 17]. AF2 is a functional site of RORγt-LBD around helix 11 (H11)
and helix 12 (H12), which was stabilized by His479–Tyr502–Phe506 π-π cluster interactions.[13]
RORγt inverse agonists break the hydrogen bond between His479–Tyr502 and weaken the
stabilization of AF2. Conversely, RORγt agonists stabilize the AF2 conformation by forming
hydrophobic interactions with Trp317, His479 and Tyr502, thus RORγt are easier to recruit
coactivator peptides.[17] The discovery of RORγt agonists from RORγt inverse agonists has been
reported for some cases. However, reports about the structure-activity relationship (SAR) of
RORγt agonists remain limited.
Scripps reported the SAR study of a series of N-arylsulfonylindoline RORγt agonists and found
that an ether linker was preferred for RORγ agonism[22]. A representative compound 1 (Figures

2a and 3) exhibited good RORγ agonism activity (EC50 = 30.2 ± 4.2 nM, percent maximum
activation (%Max) = 118%) in our RORγ dual fluorescence resonance energy transfer (dual FRET)
assay. However, compounds of this series showed poor metabolic stability (t1/2 = 2.9 mins for 1) in
mouse liver microsome (MLM) and had high lipophilicity (CLogP = 5.98 for 1). Lycera’s
N-arylsulfonylbenzoxazine compound 2 (LYC-55716, Figures 2b and 3)[23] was the first drug
candidate that entered phase 1/2 clinical trials for the treatment of solid tumors alone or in
combination with programmed cell death protein 1 (PD-1) inhibitors[24]. Compound 2 was
undoubtedly a potent RORγt agonist (EC50 = 30.1 ± 2.9 nM, %Max = 152%) and showed high
metabolic stability (t1/2 > 145 mins) in MLM. However, compound 2 has a high molecular weight
(MW) value of 603.53 and a high CLogP value of 6.76 which may need further optimization.
Figure 1. Reported N-aryl amide-based RORγt agonists. (Parts of the agonist structures interacting
with the AF2 domain of RORγ LBD were presented in blue.)
Docking modes of 1 and 2 in RORγt LBD indicated that both left-hand side (LHS) moieties,
namely the benzyl ether group and the phenyl group, occupied the same hydrophobic pocket near
AF2 domain (Figure 2, the structural superimposition of compounds 1 and 2 in complex with
RORγt LBD is shown in Supplementary Figure S5). We hypothesized that replacing the
2-chloro-6-fluorobenzyl ether moiety of 1 with the 3-(difluoromethoxy)-5-fluorophenyl moiety of
2, resulting in a hybrid compound 3 (Figure 3), could possibly maintain RORγt activity based on
the docking results of 3 (Figure 2 and Figure S6). Since there are two likely metabolic soft spots in
compound 1, namely the two benzylic positions [22], compound 3 with one of the metabolic spots
the benzyl ether group removed might have improved its metabolic stability. Thus compound 3
was prepared and tested in RORγ dual FRET and MLM assays. To our delight, compound 3

showed high RORγ agonism activity with an EC50 of 50.6 ± 4.6 nM and a maximum activation of
159%. However, the metabolic stability of 3 was a little better than that of 1 but remained low (t1/2
= 11.4 mins). Therefore, compound 3 needed further structural modifications on the indoline ring
to improve the metabolic stability. In this paper, starting from the hybrid compound 3, a series of
aryl-substituted indole and indoline compounds were designed and synthesized by changing the
indoline-like core structures, the right-hand side (RHS) 2- or 3-hydrophilic side chains and the
LHS 6-aryl groups (Figure 3). Studies on the SAR, binding modes, in vitro metabolic stability,
solubility and in vivo pharmacokinetic (PK) profiles of these compounds were further investigated
to develop novel RORγt agonists as potential therapeutic agents for cancer immunotherapy.
Figure 2. Docking poses of Scripps’ agonist 1 (yellow stick), LYC-55716 2 (green stick) and the
hybrid compound 3 (magenta stick) in the binding pocket of RORγt LBD (PDB ID: 4NIE).

Figure 3. Hybridization strategy for the design of aryl-substituted indole/indolines as RORγt
agonists.
2. Results and Discussion
2.1 Compound design
We first optimized the core structures based on the principle of bioisosterism, by replacing the
indoline of 3 with indole (4), indazole (5) and benzo[d]imidazole (6) moieties. After the optimal
core structures identified, carboxylic acid side chains were attached to the 2- or 3- position of the
preferred indole/indoline core structures via alkylene linkers of different length, leading to
compounds 7–19. According to the docking results of selected indole/indoline derivatives in
RORγt LBD, which indicated that the RHS parts participated in hydrogen-bonding interactions
with hydrophilic residues, we designed compounds 20–29 with varied polar RHS groups
substituted on 2-position of the indoline scaffold. In contrast, the LHS parts were involved in
hydrophobic contacts with AF2. Therefore, compounds 30–39 with different lipophilic
substituents on the LHS aryl group were designed and synthesized (Figure 4).

Figure 4. Target compounds with changes on core structures (3–6), RHS hydrophilic side chains
(7–29) and LHS hydrophobic aryl groups (30–39).
2.2 Chemistry
Synthetic procedures for the target compounds 3–39 were outlined in Scheme 1. Sulfonylation of
commercially available 6-bromoindole/indoline/indazole/benzo[d]imidazole (3a–6a) with
3-(trifluoromethyl)benzenesulfonyl chloride afforded the desired intermediates 3b–6b. A
subsequent
palladium-catalyzed
Suzuki
coupling
reaction
of
3b–6b
with

3-(difluoromethoxy)-5-fluorophenyl boronic acid pinacol ester yielded the corresponding
compounds 3–6. Intermediates 7c–19c were obtained by the same procedures of sulfonylation and
Suzuki coupling reaction from 7a–19a, the synthesis of which was presented in Supporting
Information. Final hydrolysis of 7c–19c in THF by LiOH gave the carboxylic acid compounds 7–
19. Amide condensation of compound 14 with a set of cyclic secondary amines under the
condition of HATU and DIEA afforded the targeted compounds 20–29. A palladium-catalyzed
Suzuki coupling reaction using substituted bromobenzene and 30a, which was prepared from 14b,
gave intermediates 30b–39b. Subsequent hydrolysis of 30b–39b by LiOH yielded the desired
compounds 30–39. Enantiomers (S)-14 and (R)-14 were obtained by chiral HPLC separation of
the racemate compound 14. Besides, the absolute configurations were determined according to the
optical rotation values of (S)-14 and (R)-14 compared with that of compound 2.
Scheme 1. General synthetic procedures for the aryl-substituted indole/indoline compounds.
Reagents and conditions: a) DMAP, DIEA, DCM, rt, 12 h; b) Pd2(dba)3, X-Phos, K2CO3,
1,4-Dioxane, N2, 100 °C, 12 h; c) LiOH, THF, rt, 5 h; d) HATU, DIEA, DCM, 30 °C, 3-12 h; e)
Pd(dppf)Cl2, KOAc, 1,4-Dioxane, N2, 100 °C, 12 h.
2.3 Structure-activity relationship
The synthesized compounds were evaluated in RORγ dual FRET assay according to the basal

level activity changes, which is suitable for the activity and function assessment of both agonists
and inverse agonists.[19] Compounds in each set having good RORγt agonism activities were
subjected to metabolic stability assay in MLM and cell-based GAL4 luciferase reporter gene
assay.
Initially, a set of compounds with different 5-, 6-membered bicyclic cores (3-6) were designed,
synthesized and evaluated in the RORγ dual FRET assay (Table 1). With indole replacing the
indoline in 3, compound 4 showed a two-fold reduction in RORγt activity (EC50 = 99.4 ± 44.7 nM)
and a slight drop in efficacy (%Max = 125%) relative to 3. On the other hand, the indole
compound 4 showed improved metabolic stability in MLM, with a t1/2 of 39.1 mins, 3-times
longer than that of 3. When the core structure was replaced by indazole (5) or benzo[d]imidazole
(6), both RORγt potency and efficacy reduced. In view of the RORγt activity and metabolic
stability, we chose indole in 4 and indoline in 3 as preferred core structures for the following RHS
optimization.
Table 1. SAR of core structures
MLM Stabilityd
t1/2 (mins)
RORγ dual FRET
Compd
Core
CLogP a
EC50 (nM)b
%Max c
(Scripps’ agonist)
5.98
6.76
30.2 ± 4.2
118
2.9
1
(LYC-55716)
30.1 ± 2.9
50.6 ± 4.6
152
159
>145
2
6.07
6.95
6.30
11.4
39.1
3
99.4 ± 44.7
205 ± 35
125
75
4
5
e
-

e
6.17
122 ± 101
75
-
6
a Calculated by Discovery Studio 3.0. b EC50 value was expressed as Mean ± SD, n=2. c
Percent maximum activation. d Mouse liver microsome stability test. e “-” means not
determined.
After identifying the preferred core structures, we explored SAR of the RHS moiety of 4 and 3.
Introduction of a carboxylic acid tether to the selected indole/indoline cores was expected to
maintain RORγt activity and make improvement in metabolic stability. Compounds 7-19 with
carboxylic acid tethered by alkylene chains of different length to the 2- or 3- position of
indole/indoline were designed and synthesized. Results of their RORγt activity, metabolic stability
and cell-based gene transcription activity were summarized in Table 2. Indole-2-carboxylic acid
analogue 7 showed essentially the same RORγt potency and efficacy as 4 while the 2-acetic acid
derivative 8 exhibited a lower RORγt activity. Elongating the linker by adding ethylene resulted in
2-propionic acid compound 9, which was 3-fold more active than 4 in activating RORγt. Further
elongation of the linker from ethylene to propylene (10) slightly lowered the RORγt activity
relative to 9. Switching the carboxylic acid tethers to 3-position (11-13) reduced the RORγt
potency and efficacy comparing with their 2-position counterparts (8-10). Among the indole series,
9 stood out with an EC50 of 25.5 ± 11.6 nM and a maximum activation of 124%, and was then
subjected to in vitro metabolic stability assay which indicated a high stability of 9 in MLM (t1/2
145 mins) like Lycera’s compound 2. However, in cell-based reporter gene assay, 9 only displayed
activity at micromolar level with an EC50 of 1710 ± 396 nM, 16-fold less potent than 2 (EC50
>
=
102 ± 4.9 nM). This could be explained by the increased lipophilicity (CLogP and CLogD) of 9
which may affect cell permeability.
All carboxylic acid-tethered indoline compounds (14-19) showed high RORγt potency with EC50s
ranging from 14.6 nM to 31.3 nM, no matter tethered at 2- or 3- position. As for the maximum
activation response, 2-substituted indolines (%Max ~ 120%) outperformed 3-substituted indolines
(%Max ~ 100%). Compounds 14 and 17 with a side chain of acetic acid tethered at the 2- and the
3- position, respectively, exhibited the highest RORγt potency and efficacy among each subset of
the indoline series. In the next MLM stability test, 2-substituted indolines (t1/2 > 60 mins) were
obviously more stable than 3-substituted indolines (t1/2 < 50 mins). It seems that metabolic

stability increases with an order of the linker length (n) of 2 < 3 < 1, so does the cell-based activity.
Taking both the cell-based RORγt activity and metabolic stability into account, compound 14
(EC50 = 247 ± 33.1 nM, %Max = 133%, t1/2 = 113 mins) stood out among the indoline series. We
separated two enantiomers of 14 by chiral HPLC, and (S)-enantiomer (EC50 = 16.1 ± 4.5 nM)
showed 17 times higher potency than (R)-enantiomer (EC50 = 286 ± 30.4 nM) in RORγt dual
FRET assay. The activity of (S)-14 on GAL4 luciferase reporter gene assay (EC50 = 201 ± 48.9
nM, %Max = 144%) was correlated with the dual FRET result, which both suggested (S)-14 as the
optimal enantiomer.
Table 2. SAR of RHS moiety
RORγ dual FRET
GAL4 e
MLM
Stabilit
y d t1/2
CLogP/
CLogD a
Compd
Core+RHS
n
%Ma
EC50 (nM)b
x c
EC50
(nM) b
Emax
(%)
(mins)
6.76/5.32
30.1 ± 2.9
152
>145
102 ± 4.9
141
2
f
f
f
0
1
2
3
6.87/5.41
6.91/5.46
6.94/5.47
7.40/5.98
74.3 ± 21.1
125 ± 4.4
128
66
-
-
-
7
8
-
-
-
25.5 ± 11.6
45.3 ± 19.9
124
95
>145 1710 ± 396 74
9
10
-
-
-
-
-
-
-
-
-
11
12
1
2
6.62/5.17
7.07/5.61
133 ± 17.8
43.6 ± 29.7
62
71
3
7.53/6.07
93.8 ± 22.6
74
-
-
-
13

1
1
1
5.83/4.39
5.83/4.39
5.83/4.39
20.8 ± 1.5
16.1 ± 4.5
286 ± 30.4
127
124
93
113
247 ± 33.1 133
201 ± 48.9 144
14
(S)-14
(R)-14
-
-
-
-
2320 ±
1560
2
6.15/4.70
31.3± 9.1
123
60.9
172
15
3
1
2
6.61/5.18
5.57/5.02
6.02/4.57
25.2 ± 3.4
21.8± 1.2
14.6± 2.2
117
109
97
93.3
45.6
24.5
422 ± 43.2 146
16
17
18
-
-
-
-
3
6.48/5.29
26.2± 11.6
95
34.2
-
-
19
b
c
a Calculated by Discovery Studio 3.0. EC50 value was expressed as Mean ± SD, n=2. Percent
e
f
maximum activation. d Mouse liver microsome stability test. GAL4 reporter gene assay. “-”
means not determined.
Compound 16 showed good cell-based RORγt activity (EC50 = 422 ± 43.2 nM, %Max = 146%)
and metabolic stability (t1/2 = 93.3 mins) as well, but it had a high CLogP of 6.61. We tried to
modify the RHS butyric acid of 16 with different amide-connected polar groups, including ketones,
alcohols, carboxylic acids, ethers and sulfone. All designed compounds (20-29) had lower CLogP
values compared to 16 and showed equivalent or higher maximum activation response in RORγt
dual FRET assay (Table 3). As different groups substituted on R2, their RORγt activation potencies
increased by the order of alcohol (21) < ether (27) < ketone (20, 24, 25) < carboxylic acid (22, 28)
< sulfone (29). The more unique were 23 with a 2-oxa-6-azaspiro[3.3]heptanyl group and 26 with
a 4-hydroxypiperidinyl group, which represented more active compounds within the ether and
alcohol series, respectively, and were subjected to metabolic stability and GAL4 reporter gene
assays along with carboxylic acids (22, 28) and sulfone (29). Results were summarized in Table 3.
The cell-based activities of two carboxylic acids (22, 28) and alcohol (26) dropped dramatically
with EC50s of 4 to 6 µM, although the metabolic stability of carboxylic acids was acceptable (t1/2
30~66 mins) while the alcohol metabolized very quickly. To our surprise, 23 was fairly unstable in
MLM and was destroyed within one minute (t1/2 < 1 min), although it showed submicromolar

RORγt activity in GAL4 reporter gene assay (EC50 = 752 ± 100 nM). The most potent sulfone
compound 29 was 1.3 times more active than 16 in GAL4 reporter gene assay, displaying an EC50
of 322 ± 51.4 nM. Unfortunately, the half-life of 29 in MLM was as low as 2 minutes, which
prevent it from further development.
Table 3. SAR of the R2 of RHS moiety
RORγ dual FRET
GAL4 e
MLM
Compd
RHS (R2)
CLogP a
Stability d
EC50
EC50
Emax
(%)
%Max c
(nM)b
(nM) b
t
1/2 (mins)
25.2 ±
117
3.4
422 ±
6.61
4.88
4.92
93.3
146
16
20
21
43.2
78.8 ±
130
13.7
f
f
f
-
-
-
244 ±
141
-
-
-
17.4
49.3 ±
138
21.6
4050 ±
459
5.07
65.9
100
22
47.9 ±
128
3.0
752 ±
100
4.93
5.13
5.38
0.7
104
23
24
25
52.3 ±
136
18.5
-
-
-
-
-
-
80.4 ±
136
1.0
N
O
O
106 ±
127
9.7
6040 ±
1010
5.05
5.30
0.7
-
100
-
26
27
209 ±
128
-

90.6
57.1 ±
6.8
5440 ±
3360
28
29
5.71
116
126
30.1
2.0
178
104
43.4±19
.9
322 ±
51.4
5.03
b
c
a Calculated by Discovery Studio 3.0. EC50 value was expressed as Mean ± SD, n=2. Percent
d
e
f
maximum activation. Mouse liver microsome stability test. GAL4 reporter gene assay. “-”
means not determined.
Based on the above SAR studies on core structure and RHS moiety, 14 was selected as the
template to make LHS modifications. We first made cyclization at the 3- and 4- position of the
phenyl group, yielding benzo[d][1,3]dioxole and 2,3-dihydrobenzofuran derivatives (30-32),
which were less potent than 14 in RORγt dual FRET assay (Table 4). Changes were then made on
the 3-difluoromethoxyl group by halogens (33, 34), trifluoromethyl group (35) and alkoxyl groups
(36-39), but no activity enhancement was observed as shown in Table 4. Introduction of an
isopropoxy group (38) caused the greatest activity reduction in this series probably due to
increased size of the substituent, which may interfere with interactions between the LHS of 38 and
the AF2 of RORγt LBD.
Table 4. SAR of LHS moiety
RORγ dual FRET
CLogP/
CLogD a
Compd
LHS
EC50 (nM)b
%Max c
14
5.83/4.39
20.8 ± 1.5
127
30
31
6.62/5.18
4.59/3.15
42.6 ± 6.0
90
77
87.4 ± 20.5

32
33
4.88/3.44
5.23/3.79
56.8 ± 21.0
25.7 ± 4.8
40
101
34
35
36
37
38
5.69/4.25
5.97/4.53
5.01/3.57
5.36/3.92
5.73/4.30
42.0 ± 4.8
41.3 ± 5.2
39.3 ± 1.1
32.9 ± 10.9
124 ± 63.8
98
115
88
96
105
39
6.94/5.50
31.1 ± 5.7
95
b
c
a Calculated by Discovery Studio 3.0. EC50 value was expressed as Mean ± SD, n=2. Percent
maximum activation.
2.4 Binding mode study
The binding mode of (S)-14 in RORγt LBD was revealed by docking study (Figure 5). In the most
possible binding mode, the 3-(difluoromethoxy)-5-fluorophenyl moiety in the LHS of the indoline
core locates in the functional site around H11 and H12, forming π-π stacking interactions with
His479 and Trp317 and stabilizing the AF2 domain. This moiety occupies in almost the same
place as that in the initial compound 2 (LYC-55716), which is believed to be the functional group
for our compounds possessing the RORγt agonist activity (shown as Figure 5b). In the RHS, the
acetic acid group formed hydrogen bonds with the main chain of His323. Besides, the
3-trifluoromethyl substituted phenylsulfony moiety is in a hydrophobic cavity, forming π-π
stacking interactions with Phe377 and providing preferred intermolecular interactions with

surrounding hydrophobic residues in the hydrophobic site near Phe377 and Phe378.
a)
b)
Figure 5. a) Zoomed-in view of (S)-14 in the binding pocket of RORγt LBD. (PDB ID: 4NIE,
(S)-14 is in orange stick); b) Structural superimposition of 2 (green stick) with (S)-14 (orange stick)
in RORγt LBD.
2.5 Drug-like properties and PK study
The solubility of 1, 2, 3 and 14 in aqueous solution at the condition of pH 7.4 was subsequently
evaluated. Results were shown in Table 5. Compounds 1 and 3 showed relatively poor solubility
(< 2 µM) because they have high lipophilicity and are lack of ionic or ionizable group. The
aqueous solubility of compounds 2 (S = 37.5 µM) and 14 (S = 69.4 µM) dramatically increased
due to the introduction of ionic carboxylic acid groups. Compound 14 was 2 times more soluble
than compound 2, which could be explained by the decreased MW and CLogP (5.83)/CLogD
(4.39) values of 14. The PK of 14 was investigated in mice following intravenous (IV, 1 mg/kg)
and oral (PO, 5 mg/kg) administration. Compound 14 showed low in vivo clearance (CL = 0.573
L/h/kg), a moderate half-life of 4 h and a high oral bioavailability of 100% (see Table S4 and
Figure S7). Overall, compound 14 demonstrated excellent in vivo pharmacokinetics in mice
consistent with its in vitro properties (aqueous solubility and metabolic stability).
Table 5. Summary of the drug-like properties of selected compounds 1-3, 14.
Solubility b MLM Stability c
CLint(mic)
d
Compds
MW
CLogP/CLogD a
(µM)
t1/2 (mins)
(µL/min/mg)
1
485.87
5.98/5.98
0.08
2.9
482
(Scripps’

agonist)
2
603.53
6.76/5.32
37.5
>145
< 9.6
(LYC-55716)
487.06
545.45
6.07/6.07
5.83/4.39
1.48
69.4
11.4
113
121
3
12.3
14
a
b
Calculated by Discovery Studio 3.0. Kinetic aqueous solubility at pH 7.46. c Mouse liver
microsome stability test. d Intrinsic clearance per mg microsome protein per mL.
3. Conclusions
In conclusion, we have discovered a series of aryl-substituted indole and indoline derivatives as
novel RORγt agonists by a scaffold-based hybridization strategy. SAR studies on the core
structures, RHS hydrophilic side chains and LHS hydrophobic aryl groups of the hybrid
compound 3 led to the identification of potent RORγt agonists with improved drug-like properties.
Compound 14 was found to have good RORγt activities in both dual FRET and GAL4 reporter
gene assays. Compound 14 showed high metabolic stability in MLM and had improved aqueous
solubility (2-fold relative to 2) at pH 7.4. Furthermore, 14 demonstrated excellent oral
bioavailability and in vivo PK profile in mice. Present studies suggest 14 as a potential RORγt
agonist candidate which deserves further investigation in tumor animal models for cancer
immunotherapy.
4. Experimental
4.1 Materials and Methods
All commercially available reagents were used without further purification unless otherwise stated.
The reactions were monitored by thin-layer chromatography (TLC) analysis. Silica gel (200-300
mesh) was used for column chromatography. The purity of all test compounds was assessed by
HPLC and area % purity was measured at 254 nm. The HPLC analyses were performed using a
Agilent 1260 instrument. Elution was done with a gradient of 5−90% solvent B (acetonitrile with
0.1% TFA) in solvent A (water with 0.1% TFA) through an Agilent HC-C18(2) (4.6 mm × 150
mm, 5 µm) column at 3.0 mL/min. High-resolution MS (HRMS) was analyzed by a TOF analyzer.
1
The ion source is electrospray ionization (ESI). H NMR spectra were recorded at 400 MHz and

1
13C NMR spectra were recorded at 600 MHz. Chemical shifts in H NMR spectra are reported in
parts per million (ppm) on the δ scale from an internal standard of CDCl3 (7.26 ppm). Data are
reported as follows: chemical shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad), coupling constant in hertz (Hz), and integration. Chemical
shifts of 13C NMR spectra are reported in ppm from the central peak of CDCl3 (77.0 ppm) on the δ
scale.
4.2 Chemical Synthesis
4.2.1 The synthesis of compounds 1–2
Compounds 1 and 2 were synthesized following the published procedures[19, 22], and were
characterized by 1H NMR, 13C NMR and MS.
4.2.1.1 6-((2-Chloro-6-fluorobenzyl)oxy)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indoline (1). Yield:
81%, white solid, purity: 98%. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 8.15 (d, J = 7.5 Hz, 1H),
8.09 (d, J = 7.1 Hz, 1H), 8.02 (s, 1H), 7.84 (t, J = 6.6 Hz, 1H), 7.51 (dd, J = 14.4, 7.8 Hz, 1H),
7.43 (d, J = 7.8 Hz, 1H), 7.33 (t, J = 8.2 Hz, 1H), 7.11 – 7.07 (m, 2H), 6.71 (d, J = 8.1 Hz, 1H),
5.15 (s, 2H), 3.99 (t, J = 6.7 Hz, 2H), 2.82 (t, J = 7.1 Hz, 2H); 13C NMR (150 MHz, DMSO-d6) δ
(ppm) 162.1, 160.4, 158.0, 141.8, 137.2, 135.3 (d, J = 4.8 Hz), 131.6 (d, J = 9.8 Hz), 130.4 (d, J =
2.5 Hz), 129.9 (q, J = 32.8 Hz), 125.9, 125.6, 124.8, 123.9, 123.3 (d, J = 3.4 Hz), 122.1, 121.8 (d,
J = 17.7 Hz), 114.6 (d, J = 22.5 Hz), 110.5, 101.7, 61.4 (d, J = 3.0 Hz), 50.8, 26.4; MS (ESI) m/z
486.1 [M + H]+.
4.2.1.2
(S)-3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-4-((3-(trifluoromethyl)phenyl)sulfonyl)-3,4-dihydro
-2H-benzo[b][1,4]oxazin-2-yl)-2,2-dimethylpropanoic acid (2). Yield: 67%, white solid, purity:
95%. [α]D23 +147° (c = 0.2, CH2Cl2). 1H NMR (400 MHz, DMSO-d6) δ (ppm) 12.28 (s, 1H), 8.14
(d, J = 7.7 Hz, 1H), 7.95 (dd, J = 8.2, 6.4 Hz, 3H), 7.85 (t, J = 7.9 Hz, 1H), 7.60 – 7.42 (m, 2H),
7.34 (d, J = 9.7 Hz, 1H), 7.24 (s, 1H), 7.13 (d, J = 9.6 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 4.32 (d, J
= 12.5 Hz, 1H), 3.39 (d, J = 13.5 Hz, 2H), 1.74 (d, J = 4.2 Hz, 2H), 1.02 (s, 3H), 1.00 (s, 3H); 13C
NMR (150 MHz, DMSO-d6) δ (ppm) 178.1, 163.6, 162.0, 152.5, 147.0, 142.8 (d, J = 9.6 Hz),

138.7, 131.5, 130.9, 130.6, 130.3, 125.2, 123.4, 122.9, 121.8, 118.0, 116.0 (t, J = 258.4 Hz), 112.2,
109.6 (d, J = 22.2 Hz), 104.8 (d, J = 25.5 Hz), 69.8, 47.9, 41.3, 25.3, 24.5; MS (ESI) m/z 604.1 [M
+ H]+.
4.2.2 General procedure for the synthesis of compounds 3–6
Step 1: To a vial were added intermediates 3a–6a (1.0 eq), DMAP (20 mol%, 0.2 eq), DIEA
(3.0 eq) and DCM (2 mL). After the reaction mixture was cooled to 0 °C by ice-water bath,
3-(trifluoromethyl)benzenesulfonyl chloride (1.2 eq) was subsequently added dropwise. Then the
reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the
resulting mixture was diluted with DCM and washed with water. The separated aqueous phase
was washed with DCM. The combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo. The crude mixture was purified by column chromatography on silica gel
(petroleum: ethyl acetate (EA) = 10:1~3:1) to afford the desired products 3b–6b.
Step
2:
To
a
vial
were
added
intermediates
3b–6b
(1.0
(1.2
eq),
eq),
2-(3-(difluoromethoxy)-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
Pd(dppf)Cl2 (5 mol%, 0.05 eq), X-Phos (10 mol%, 0.1 eq), K2CO3 (3.0 eq) and 1,4-dioxane (3
mL). Then the reaction mixture was stirred under N2 atmosphere at 100 °C for 12 h. After
completion of the reaction, the resulting mixture was diluted with EA and washed with water. The
separated aqueous phase was washed with EA. The combined organic layers were dried over
MgSO4, filtered, and concentrated in vacuo. The crude mixture was purified by column
chromatography on silica gel (petroleum: EA = 10:1~5:1) to afford the desired products 3–6.
4.2.2.1 6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indoline (3).
Yield: 52%, white solid, purity: 94%. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 8.15 (d, J = 6.6 Hz,
1H), 8.04 (s, 2H), 7.79 (t, J = 7.2 Hz, 1H), 7.63 (s, 1H), 7.58 – 7.39 (m, 1H), 7.32 (d, J = 5.1 Hz,
2H), 7.24 – 7.19 (m, 2H), 7.13 (d, J = 8.9 Hz, 1H), 4.00 (t, J = 6.6 Hz, 2H), 2.88 (t, J = 7.3 Hz,
2H); 13C NMR (150 MHz, DMSO-d6) δ (ppm) 163.5, 161.9, 152.2 (d, J = 12.0 Hz), 143.4 (d, J =
9.5 Hz), 141.6, 137.7, 137.2, 133.1, 131.2, 131.0, 130.4, 129.9 (d, J = 32.9 Hz), 126.1, 123.5,
123.3, 116.1 (t, J = 258.4 Hz), 112.8, 112.3, 110.2 (d, J = 22.3 Hz), 105.3 (d, J = 25.6 Hz), 50.4,
26.9; MS (ESI) m/z 488.1 [M + H]+.

4.2.2.2 6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-indole
1
(4). Yield: 48%, white solid, purity: 98%. H NMR (400 MHz, DMSO-d6) δ (ppm) 8.45 (s, 1H),
8.41 (d, J = 7.2 Hz, 1H), 8.18 (s, 1H), 8.10 (d, J = 6.7 Hz, 1H), 8.02 (s, 1H), 7.84 (t, J = 7.1 Hz,
1H), 7.72 (d, J = 7.6 Hz, 1H), 7.63 (d, J = 7.7 Hz, 1H), 7.46 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 14.8
13
Hz, 2H), 7.17 (d, J = 9.4 Hz, 1H), 6.94 (s, 1H); C NMR (150 MHz, DMSO-d6) δ (ppm) 163.5,
161.9, 152.2 (d, J = 11.9 Hz), 143.6 (d, J = 9.5 Hz), 137.8, 135.0, 134.5, 131.5, 131.3, 130.8,
130.7, 130.2 (d, J = 33.0 Hz), 128.1, 123.4, 123.2, 122.3, 116.1 (t, J = 258.2 Hz), 113.0, 111.0,
110.6 (d, J = 22.3 Hz), 110.0, 105.1 (d, J = 25.6 Hz); MS (ESI) m/z 486.1 [M + H]+.
4.2.2.3
6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-indazole (5).
Yield: 42%, white solid, purity: 98%. 1H NMR (400 MHz, DMSO-d6) δ (ppm) 8.10 (d, J = 7.7 Hz,
1H), 8.00 (d, J = 8.7 Hz, 2H), 7.95 (d, J = 8.1 Hz, 1H), 7.85 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 8.9
Hz, 1H), 7.57 – 7.39 (m, 1H), 7.33 (s, 1H), 7.31 – 7.21 (m, 2H), 7.16 (s, 1H); 13C NMR (150 MHz,
DMSO-d6) δ (ppm) 170.1, 163.5, 161.9, 152.3 (d, J = 12.0 Hz), 142.9, 140.7 (d, J = 9.6 Hz), 140.4,
138.6, 134.5, 131.0, 130.7, 129.8, 126.0, 125.3, 123.3, 115.9, 115.8 (t, J = 258.8 Hz), 113.0, 110.5
(d, J = 22.9 Hz), 109.8, 106.8 (d, J = 25.6 Hz); MS (ESI) m/z 487.1 [M + H]+.
4.2.2.4
6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-benzo[d]imid
azole (6). Yield: 43%, white solid, purity: 99%. 1H NMR (400 MHz, CDCl3) δ (ppm) 8.43 (s, 1H),
8.32 (s, 1H), 8.20 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 1.3 Hz, 1H), 7.93 (d, J = 7.8 Hz, 1H), 7.86 (d, J
= 8.4 Hz, 1H), 7.73 (t, J = 7.9 Hz, 1H), 7.57 (dd, J = 8.4, 1.7 Hz, 1H), 7.18 – 7.14 (m, 2H), 6.94 –
6.90 (m, 1H), 6.60 (t, J = 73.0 Hz, 1H); 13C NMR (150 MHz, DMSO-d6) δ (ppm) 163.5, 161.9,
152.2 (d, J = 12.3 Hz), 143.5 (d, J = 10.7 Hz), 143.1 (d, J = 9.7 Hz), 137.6, 136.0, 132.1, 131.8,
131.5, 130.6 (t, J = 16.7 Hz), 124.6, 124.3, 123.7, 121.9, 121.2, 116.1 (t, J = 258.3 Hz), 113.3,
110.9 (d, J = 22.5 Hz), 110.3, 105.4 (d, J = 25.8 Hz); MS (ESI) m/z 487.1 [M + H]+.
4.2.3 General procedure for the synthesis of compounds 7–19

Step 1: To a vial were added intermediates 7a–19a (1.0 eq), DMAP (20 mol%, 0.2 eq), DIEA
(3.0 eq) and DCM (2 mL). After the reaction mixture was cooled to 0 °C by ice-water bath,
3-(trifluoromethyl)benzenesulfonyl chloride (1.2 eq) was subsequently added dropwise. Then the
reaction mixture was stirred at room temperature for 12 h. After completion of the reaction, the
resulting mixture was diluted with DCM and washed with water. The separated aqueous phase
was washed with DCM. The combined organic layers were dried over MgSO4, filtered, and
concentrated in vacuo. The crude mixture was purified by column chromatography on silica gel
(petroleum: EA = 10:1~3:1) to afford the desired products 7b–19b.
Step
2:
To
a
vial
were
added
intermediates
7b–19b
(1.0
(1.2
eq),
eq),
2-(3-(difluoromethoxy)-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
Pd(dppf)Cl2 (5 mol%, 0.05 eq), X-Phos (10 mol%, 0.1 eq), K2CO3 (3.0 eq) and 1,4-dioxane (3
mL). Then the reaction mixture was stirred under N2 atmosphere at 100 °C for 12 h. After
completion of the reaction, the resulting mixture was diluted with EA and washed with water. The
separated aqueous phase was washed with EA. The combined organic layers were dried over
MgSO4, filtered, and concentrated in vacuo. The crude mixture was purified by column
chromatography on silica gel (petroleum: EA = 10:1~5:1) to afford the desired products 7c–19c.
Step 3: To a vial were added intermediates 7c–19c (1.0 eq), LiOH aq. (2N, 2.0 eq) and THF
(2 mL). After that the reaction mixture was stirred at room temperature for 5 h. After completion
of the reaction, the pH of solution was adjusted to about 3 by HCl (2N). Then the resulting mixture
was diluted with EA and washed with water. The separated aqueous phase was washed with EA.
The combined organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The
crude mixture was purified by column chromatography on silica gel (petroleum: EA = 1:1~1:8) to
afford the desired products 7–19.
4.2.3.1
6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-indole-2-carb
oxylic acid (7). Yield: 76%, white solid, purity: 97%. 1H NMR (400 MHz, CD3OD) δ (ppm) 8.38
(s, 1H), 8.30 (s, 2H), 7.96 (d, J = 7.6 Hz, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H),
7.58 (d, J = 8.1 Hz, 1H), 7.30 (d, J = 10.3 Hz, 1H), 7.26 (d, J = 7.1 Hz, 2H), 7.00 (s, 1H), 6.89 (t, J
= 64.7 Hz, 1H); 13C NMR (150 MHz, CD3OD) δ (ppm) 163.6, 162.0, 152.2 (d, J = 11.6 Hz), 143.8

(d, J = 9.4 Hz), 138.9, 137.8, 137.4, 130.4 (d, J = 33.4 Hz), 129.9, 129.8 (d, J = 2.7 Hz), 129.6,
128.2, 123.6, 123.5, 123.0, 122.4, 121.7, 115.6 (t, J = 258.9 Hz), 115.4, 112.9, 112.8, 109.9 (d, J =
22.7 Hz), 104.8 (d, J = 25.6 Hz); MS (ESI) m/z 530.1 [M + H]+.
4.2.3.2
2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-indol-2-yl)
1
acetic acid (8). Yield: 73%, pale yellow solid, purity: 93%. H NMR (400 MHz, CDCl3) δ (ppm)
8.20 (s, 1H), 8.08 (s, 1H), 8.00 (d, J = 8.0 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.62 (d, J = 7.7 Hz,
1H), 7.56 (d, J = 8.1 Hz, 1H), 7.43 (d, J = 8.2 Hz, 1H), 7.11 (d, J = 14.8 Hz, 2H), 6.87 (d, J = 9.1
Hz, 1H), 6.68 (s, 1H), 6.58 (t, J = 73.2 Hz, 1H), 4.19 (s, 2H); 13C NMR (150 MHz, CDCl3) δ (ppm)
174.3, 164.1, 162.5, 152.1 (d, J = 9.0 Hz), 144.5 (d, J = 9.3 Hz), 139.7, 137.0, 136.3, 133.9, 130.7
(d, J = 2.6 Hz), 130.3, 129.9, 129.2, 124.0 (d, J = 3.3 Hz), 123.5, 121.6, 115.6 (t, J = 261.5 Hz),
114.3, 113.0, 112.8, 111.4 (d, J = 22.2 Hz), 106.2 (d, J = 25.1 Hz), 34.7; MS (ESI) m/z 544.1 [M +
H]+.
4.2.3.3
3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-indol-2-yl)
1
propanoic acid (9). Yield: 78%, pale yellow solid, purity: 98%. H NMR (400 MHz, CDCl3) δ
(ppm) 8.29 (s, 1H), 8.10 (s, 1H), 7.90 (d, J = 6.4 Hz, 1H), 7.80 (d, J = 6.3 Hz, 1H), 7.58 (t, J = 6.9
Hz, 1H), 7.50 (d, J = 7.3 Hz, 1H), 7.43 (d, J = 7.4 Hz, 1H), 7.17 (d, J = 13.3 Hz, 2H), 6.88 (d, J =
7.8 Hz, 1H), 6.69 (t, J = 73.7 Hz, 1H), 6.52 (s, 1H), 3.37 (d, J = 16.9 Hz, 2H), 2.88 (d, J = 17.8 Hz,
2H); 13C NMR (150 MHz, CDCl3) δ (ppm) 176.2, 164.1, 162.5, 152.2 (d, J = 11.5 Hz), 144.7 (d, J
= 9.3 Hz), 141.1, 139.7, 137.6, 136.0, 132.1 (d, J = 33.7 Hz), 130.6, 130.3, 130.0, 129.7 (d, J = 5.8
Hz), 129.4, 123.6, 121.1, 115.7 (t, J = 261.5 Hz), 114.3, 113.3, 111.4 (d, J = 22.1 Hz), 109.9, 106.1
(d, J = 25.1 Hz), 32.9, 24.9; MS (ESI) m/z 558.1 [M + H]+.
4.2.3.4
4-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-indol-2-yl)
1
butanoic acid (10). Yield: 71%, white solid, purity: 96%. H NMR (400 MHz, CDCl3) δ (ppm)
8.31 (s, 1H), 8.08 (s, 1H), 7.82 (dd, J = 21.7, 7.8 Hz, 2H), 7.57 (t, J = 7.9 Hz, 1H), 7.50 (d, J = 8.1

Hz, 1H), 7.42 (d, J = 8.1 Hz, 1H), 7.18 (d, J = 13.2 Hz, 2H), 6.86 (d, J = 9.1 Hz, 1H), 6.69 (t, J =
73.3 Hz, 1H), 6.51 (s, 1H), 3.11 (t, J = 7.4 Hz, 2H), 2.50 (t, J = 7.1 Hz, 2H), 2.18 – 2.11 (m, 2H);
13C NMR (150 MHz, CDCl3) δ (ppm) 177.6, 164.1, 162.5, 152.2 (d, J = 11.6 Hz), 144.8 (d, J =
9.3 Hz), 142.1, 139.8, 137.7, 135.8, 132.1 (d, J = 34.1 Hz), 130.5, 130.3, 129.9, 129.3, 123.5,
123.4 (d, J = 3.1 Hz), 121.0, 115.8 (t, J = 261.4 Hz), 114.3, 113.4, 111.4 (d, J = 22.1 Hz), 110.1,
106.1 (d, J = 25.3 Hz), 32.9, 28.4, 23.9; MS (ESI) m/z 572.1 [M + H]+.
4.2.3.5
2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-indol-3-yl)
acetic acid (11). Yield: 75%, pale yellow solid, purity: 95%. 1H NMR (400 MHz, CDCl3) δ (ppm)
8.19 (s, 1H), 8.12 (s, 1H), 8.07 (d, J = 7.4 Hz, 1H), 7.81 (d, J = 6.2 Hz, 1H), 7.65 (s, 1H), 7.62 –
7.57 (m, 2H), 7.46 (d, J = 7.4 Hz, 1H), 7.15 (d, J = 10.1 Hz, 2H), 6.89 (d, J = 8.4 Hz, 1H), 6.60 (t,
J = 73.3 Hz, 1H), 3.78 (s, 2H); 13C NMR (150 MHz, CDCl3) δ (ppm) 175.7, 164.1, 162.5, 152.2 (d,
J = 11.5 Hz), 144.3 (d, J = 9.2 Hz), 139.0, 136.8, 135.4, 132.1 (q, J = 33.9 Hz), 130.7 (d, J = 2.6
Hz), 130.4, 130.4, 129.9, 125.7, 123.9 (d, J = 3.2 Hz), 123.3, 120.3, 115.6 (t, J = 261.5 Hz), 115.4,
114.4, 112.1, 111.5 (d, J = 22.2 Hz), 106.3 (d, J = 25.2 Hz), 60.5; MS (ESI) m/z 544.1 [M + H]+.
4.2.3.6
3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-indol-3-yl)
propanoic acid (12). Yield: 76%, pale yellow solid, purity: 94%. 1H NMR (400 MHz, CDCl3) δ
(ppm) 8.17 (s, 1H), 8.12 (s, 1H), 8.03 (d, J = 7.4 Hz, 1H), 7.80 (d, J = 7.8 Hz, 1H), 7.59 (dd, J =
13.6, 8.1 Hz, 2H), 7.46 (d, J = 10.7 Hz, 2H), 7.16 (d, J = 14.3 Hz, 2H), 6.89 (d, J = 9.0 Hz, 1H),
6.60 (t, J = 73.2 Hz, 1H), 3.05 (t, J = 7.2 Hz, 2H), 2.78 (t, J = 7.3 Hz, 2H); 13C NMR (150 MHz,
CDCl3) δ (ppm) 175.7, 164.3, 162.7, 152.2 (d, J = 10.5 Hz), 144.4 (d, J = 9.0 Hz), 139.2, 136.7,
135.7, 132.3, 132.0, 130.7, 130.6 (d, J = 2.6 Hz), 130.3, 129.8, 123.9, 123.1, 122.3, 120.1, 115.6 (t,
J = 261.7 Hz), 114.4, 112.3, 111.5 (d, J = 22.2 Hz), 106.3 (d, J = 25.1 Hz), 32.9, 20.0; MS (ESI)
m/z 558.1 [M + H]+.
4.2.3.7
4-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)-1H-indol-3-yl)

butanoic acid (13). Yield: 72%, white solid, purity: 96%.1H NMR (400 MHz, CDCl3) δ (ppm)
8.17 (s, 1H), 8.13 (s, 1H), 8.05 (d, J = 6.4 Hz, 1H), 7.81 (d, J = 6.3 Hz, 1H), 7.60 (dd, J = 17.4, 8.0
Hz, 2H), 7.45 – 7.41 (m, 2H), 7.16 (d, J = 10.9 Hz, 2H), 6.88 (d, J = 7.8 Hz, 1H), 6.61 (t, J = 73.2
Hz, 1H), 2.77 (t, J = 7.1 Hz, 2H), 2.42 (t, J = 6.8 Hz, 2H), 2.12 – 1.98 (m, 2H); 13C NMR (150
MHz, CDCl3) δ (ppm) 178.5, 164.1, 162.5, 152.2 (d, J = 11.7 Hz), 144.5 (d, J = 9.2 Hz), 139.1,
136.5, 135.8, 132.1 (q, J = 33.9 Hz), 131.0, 130.6 (d, J = 2.7 Hz), 130.3, 129.8, 123.8 (d, J = 3.3
Hz), 123.7, 123.1, 123.1, 120.3, 115.6 (t, J = 261.5 Hz), 114.4, 112.2, 111.5 (d, J = 22.2 Hz), 106.2
(d, J = 25.2 Hz), 33.1, 24.1, 23.8; MS (ESI) m/z 572.1 [M + H]+.
4.2.3.8
2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)ace
tic acid (14). Yield: 76%, white solid, purity: 99%. 1H NMR (400 MHz, CDCl3) δ (ppm) 7.96 (s,
1H), 7.90 (d, J = 8.1 Hz, 1H), 7.85 – 7.81 (m, 2H), 7.59 (t, J = 7.6 Hz, 1H), 7.17 – 7.13 (m, 4H),
6.89 (d, J = 9.0 Hz, 1H), 6.59 (t, J = 73.1 Hz, 1H), 4.65 (t, J = 9.5 Hz, 1H), 3.17 (dd, J = 16.2, 3.6
Hz, 1H), 3.02 (dd, J = 16.7, 9.3 Hz, 1H), 2.81 – 2.71 (m, 2H); 13C NMR (150 MHz, CDCl3) δ
(ppm) 174.4, 164.1, 162.5, 152.2 (d, J = 11.2 Hz), 143.9 (d, J = 9.1 Hz), 141.3, 139.6, 138.8,
131.9 (d, J = 33.5 Hz), 131.3, 130.1 (d, J = 4.2 Hz), 126.0, 124.5, 124.2 (d, J = 3.2 Hz), 123.9,
122.0, 115.6 (t, J = 261.7 Hz), 115.5, 114.1, 111.3 (d, J = 22.2 Hz), 106.5 (d, J = 25.3 Hz), 59.3,
41.0, 34.5; HRMS (ESI): m/z [M + H]+ calcd for C24H18F6NO5S: 546.0804, found: 546.0809.
4.2.3.9
3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)pro
panoic acid (15). Yield: 77%, white solid, purity: 96%. 1H NMR (400 MHz, CDCl3) δ (ppm) 7.84
(d, J = 6.6 Hz, 2H), 7.79 (t, J = 6.1 Hz, 2H), 7.54 (t, J = 7.8 Hz, 1H), 7.27 (d, J = 7.3 Hz, 1H),
7.16 (t, J = 12.6 Hz, 3H), 6.89 (d, J = 8.9 Hz, 1H), 6.60 (t, J = 73.2 Hz, 1H), 4.45 (d, J = 7.0 Hz,
1H), 2.80 – 2.71 (m, 1H), 2.68 – 2.59 (m, 2H), 2.49 (d, J = 16.4 Hz, 1H), 1.98 (dd, J = 16.3, 9.7
Hz, 2H); 13C NMR (150 MHz, CDCl3) δ (ppm) 175.6, 164.3, 162.6, 152.3 (d, J = 10.8 Hz), 143.8
(d, J = 9.4 Hz), 141.3, 139.3, 138.9, 133.0, 130.1, 129.8, 125.8, 124.9, 124.1, 117.2, 115.6 (t, J =
259.5 Hz), 114.1, 111.3 (d, J = 22.4 Hz), 106.5 (d, J = 24.7 Hz), 62.3, 34.5, 30.9, 29.7; MS (ESI)
m/z 560.1 [M + H]+.

4.2.3.10
4-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)but
anoic acid (16). Yield: 74%, white solid, purity: 97%. 1H NMR (400 MHz, CDCl3) δ (ppm) 7.89 (s,
1H), 7.84 – 7.74 (m, 3H), 7.55 (t, J = 6.6 Hz, 1H), 7.26 (d, J = 5.7 Hz, 1H), 7.12 (q, J = 14.8 Hz,
3H), 6.88 (d, J = 8.0 Hz, 1H), 6.60 (t, J = 72.5 Hz, 1H), 4.32 (t, J = 10.4 Hz, 1H), 2.72 (dd, J =
15.9, 8.8 Hz, 1H), 2.55 (d, J = 16.4 Hz, 1H), 2.44 – 2.35 (m, 2H), 1.83 – 1.63 (m, 4H); 13C NMR
(150 MHz, CDCl3) δ (ppm) 179.0, 164.1, 162.5, 152.2 (d, J = 11.6 Hz), 143.9 (d, J = 9.2 Hz),
141.6, 139.3, 139.1, 132.9, 131.7 (q, J = 33.5 Hz), 130.0, 129.9, 129.7 (d, J = 2.4 Hz), 125.8,
124.6, 124.1, 124.1, 116.5, 115.6 (t, J = 259.5 Hz), 114.1, 111.2 (d, J = 22.2 Hz), 106.4 (d, J =
25.2 Hz), 63.0, 35.7, 34.0, 33.4, 20.2; MS (ESI) m/z 574.1 [M + H]+.
4.2.3.11
2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-3-yl)ace
tic acid (17). Yield: 75%, pale yellow solid, purity: 94%. 1H NMR (400 MHz, CDCl3) δ (ppm)
8.12 (s, 1H), 8.00 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 7.3 Hz, 1H), 7.81 (s, 1H), 7.65 (t, J = 7.9 Hz,
1H), 7.20 (d, J = 3.7 Hz, 2H), 7.14 (dd, J = 17.3, 8.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 1H), 6.59 (t, J =
73.0 Hz, 1H), 4.24 – 4.19 (m, 1H), 3.81 (dd, J = 11.1, 5.6 Hz, 1H), 3.65 – 3.58 (m, 1H), 2.66 (dd,
J = 17.2, 4.6 Hz, 1H), 2.30 (dd, J = 16.9, 9.9 Hz, 1H); 13C NMR (150 MHz, CDCl3) δ (ppm) 175.5,
164.1, 162.5, 152.2 (d, J = 11.2 Hz), 143.9 (d, J = 9.2 Hz), 142.0, 140.3, 138.1, 133.8, 132.0 (d, J
= 33.6 Hz), 130.2 (d, J = 9.5 Hz), 125.0, 124.4, 123.9, 123.6, 122.1, 115.6 (t, J = 261.7 Hz), 114.2,
113.5, 111.3 (d, J = 22.2 Hz), 106.6 (d, J = 25.1 Hz), 56.1, 38.8, 36.0; MS (ESI) m/z 546.1 [M +
H]+.
4.2.3.12
3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-3-yl)pro
panoic acid (18). Yield: 78%, pale yellow solid, purity: 96%. 1H NMR (400 MHz, CDCl3) δ (ppm)
8.12 (s, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.85 (d, J = 7.5 Hz, 1H), 7.79 (s, 1H), 7.64 (t, J = 7.8 Hz,
1H), 7.20 (s, 2H), 7.13 (d, J = 14.1 Hz, 2H), 6.89 (d, J = 9.4 Hz, 1H), 6.59 (t, J = 73.2 Hz, 1H),
4.06 (t, J = 9.8 Hz, 1H), 3.71 (dd, J = 10.4, 4.8 Hz, 1H), 3.30 – 3.23 (m, 1H), 2.33 (t, J = 7.3 Hz,

2H), 2.05 – 1.87 (m, 2H); MS (ESI) m/z 560.1 [M + H]+.
4.2.3.13
4-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-3-yl)but
anoic acid (19). Yield: 79%, white solid, purity: 97%. 1H NMR (400 MHz, CDCl3) δ (ppm) 8.12 (s,
1H), 8.01 (d, J = 6.7 Hz, 1H), 7.84 (d, J = 6.8 Hz, 1H), 7.79 (s, 1H), 7.64 (t, J = 6.7 Hz, 1H), 7.19
– 7.12 (m, 4H), 6.88 (d, J = 9.0 Hz, 1H), 6.59 (t, J = 73.2 Hz, 1H), 4.10 (t, J = 9.4 Hz, 1H), 3.73 –
3.70 (m, 1H), 3.21 (s, 1H), 2.33 (s, 2H), 1.62 (s, 4H); 13C NMR (150 MHz, CDCl3) δ (ppm) 178.6,
164.0, 162.4, 152.1 (d, J = 11.5 Hz), 144.0 (d, J = 9.1 Hz), 141.9, 139.6, 138.0, 135.2, 131.8 (q, J
= 33.6 Hz), 130.2, 130.1, 130.0, 125.1, 124.3, 123.3, 115.6 (t, J = 261.4 Hz), 114.0, 113.1, 111.2
(d, J = 22.2 Hz), 106.4 (d, J = 25.2 Hz), 55.8, 39.5, 34.0, 33.5, 24.7; MS (ESI) m/z 574.1 [M +
H]+.
4.2.4 General procedure for the synthesis of compounds 20–29
To a vial were added 14 (1.0 eq), related secondary amine (1.2 eq), HATU (1.2 eq), DIEA
(3.0 eq) and DCM (2 mL). Then the reaction mixture was stirred at 30 °C for 3-12 h. After
completion of the reaction, the resulting mixture was diluted with DCM and washed with water.
The separated aqueous phase was washed with DCM. The combined organic layers were dried
over MgSO4, filtered, and concentrated in vacuo. The crude mixture was purified by column
chromatography on silica gel (petroleum: EA = 8:1~1:1) to afford the desired products 20–29.
4.2.4.1
1-(2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)
acetyl)azetidin-3-one (20). Yield: 80%, white solid, purity: 96%. 1H NMR (400 MHz, CDCl3) δ
(ppm) 7.95 (s, 1H), 7.91 (d, J = 8.1 Hz, 1H), 7.84 (s, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.60 (t, J = 7.7
Hz, 1H), 7.15 (dd, J = 13.9, 5.7 Hz, 4H), 6.89 (d, J = 9.0 Hz, 1H), 6.59 (t, J = 73.3 Hz, 1H), 4.99
(d, J = 14.9 Hz, 1H), 4.87 (d, J = 19.0 Hz, 2H), 4.73 (d, J = 13.8 Hz, 2H), 3.10 – 3.02 (m, 2H),
13
2.85 (dd, J = 12.4, 5.9 Hz, 1H), 2.69 (dd, J = 15.5, 8.8 Hz, 1H); C NMR (150 MHz, CDCl3) δ
(ppm) 193.8, 169.6, 165.8, 164.1, 162.5, 152.2 (d, J = 11.5 Hz), 143.8 (d, J = 9.3 Hz), 141.3,
139.6, 138.4, 131.5, 130.1, 126.1, 124.5, 124.2, 123.8, 122.0, 115.6 (t, J = 261.6 Hz), 115.1, 114.1,

111.2 (d, J = 22.3 Hz), 106.5 (d, J = 25.1 Hz), 59.9, 40.2, 38.6, 34.6; MS (ESI) m/z 599.1 [M +
H]+.
4.2.4.2
2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)-1-(
3-hydroxyazetidin-1-yl)ethanone (21). Yield: 65%, pale yellow solid, purity: 95%. 1H NMR (400
MHz, CDCl3) δ (ppm) 7.92 (s, 1H), 7.89 (d, J = 6.6 Hz, 1H), 7.82 (s, 1H), 7.79 (d, J = 7.8 Hz, 1H),
7.58 (t, J = 7.2 Hz, 1H), 7.23 (d, J = 8.0 Hz, 1H), 7.14 (d, J = 9.8 Hz, 3H), 6.87 (d, J = 9.0 Hz, 1H),
6.59 (t, J = 73.1 Hz, 1H), 4.69 – 4.64 (m, 2H), 4.42 – 4.17 (m, 2H), 3.96 (dddd, J = 48.9, 37.9,
10.0, 3.8 Hz, 3H), 3.02 – 2.94 (m, 1H), 2.85 – 2.76 (m, 2H), 2.48 (dd, J = 14.9, 9.6 Hz, 1H); 13C
NMR (150 MHz, CDCl3) δ (ppm) 169.7 (d, J = 28.6 Hz), 165.8, 164.1, 162.4, 152.1 (d, J = 11.4
Hz), 143.9 (d, J = 6.0 Hz), 141.2, 139.4, 138.6 (d, J = 7.8 Hz), 131.9 (d, J = 7.8 Hz), 130.1 (d, J =
2.0 Hz), 126.1, 124.4 (d, J = 4.3 Hz), 124.1, 123.8, 122.0, 115.6 (t, J = 261.4 Hz), 115.2 (d, J =
11.8 Hz), 114.0, 111.2 (d, J = 22.1 Hz), 106.4 (d, J = 26.5 Hz), 61.1 (d, J = 13.0 Hz), 60.1, 59.9 (d,
J = 13.0 Hz), 57.7 (d, J = 22.5 Hz), 38.8 (d, J = 5.0 Hz), 34.5; MS (ESI) m/z 601.1 [M + H]+.
4.2.4.3
1-(2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)
acetyl)azetidine-3-carboxylic acid (22). Yield: 88%, white solid, purity: 97%. 1H NMR (400 MHz,
CDCl3) δ (ppm) 7.92 – 7.88 (m, 2H), 7.82 (s, 1H), 7.79 (d, J = 7.4 Hz, 1H), 7.57 (t, J = 7.5 Hz,
1H), 7.23 (d, J = 8.0 Hz, 1H), 7.18 – 7.10 (m, 3H), 6.87 (d, J = 9.0 Hz, 1H), 6.59 (t, J = 73.2 Hz,
1H), 4.69 (s, 1H), 4.28 (dd, J = 54.5, 29.6 Hz, 3H), 3.47 – 3.12 (m, 1H), 2.99 – 2.93 (m, 1H), 2.79
13
(t, J = 18.8 Hz, 2H), 2.47 (dd, J = 17.1, 8.5 Hz, 1H), 2.27 (dd, J = 27.8, 21.0 Hz, 1H); C NMR
(150 MHz, CDCl3) δ (ppm) 170.0, 164.1, 162.4, 152.2 (d, J = 11.3 Hz), 143.9 (d, J = 9.1 Hz),
141.2 (d, J = 10.5 Hz), 139.4, 138.6 (d, J = 6.4 Hz), 131.9, 131.7, 130.1, 130.0, 126.1, 124.5,
124.1, 123.8, 122.0, 115.6 (t, J = 261.4 Hz), 115.3, 114.0, 111.2 (d, J = 22.3 Hz), 106.4 (d, J =
24.8 Hz), 59.9 (d, J = 18.4 Hz), 45.4, 38.4 (d, J = 20.2 Hz), 34.5 (d, J = 18.2 Hz), 31.9; MS (ESI)
m/z 629.1 [M + H]+.

4.2.4.4
2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)-1-(
2-oxa-6-azaspiro[3.3]heptan-6-yl)ethanone (23). Yield: 71%, white solid, purity: 96%.1H NMR
(400 MHz, CDCl3) δ (ppm) 7.88 (s, 1H), 7.84 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 6.9 Hz, 2H), 7.55 (t,
J = 7.5 Hz, 1H), 7.20 (d, J = 7.4 Hz, 1H), 7.11 – 7.08 (m, 3H), 6.84 (d, J = 8.8 Hz, 1H), 6.57 (t, J
= 73.2 Hz, 1H), 4.60 (d, J = 8.0 Hz, 1H), 4.21 (ddd, J = 50.4, 38.3, 9.7 Hz, 3H), 3.62 (d, J = 4.5
Hz, 4H), 2.95 (dd, J = 16.9, 9.4 Hz, 1H), 2.76 (dd, J = 22.3, 10.4 Hz, 2H), 2.41 (dd, J = 15.1, 9.0
Hz, 1H), 2.21 (s, 1H); 13C NMR (150 MHz, CDCl3) δ (ppm) 169.5, 164.0, 162.3, 152.1 (d, J =
11.2 Hz), 143.8 (d, J = 9.1 Hz), 141.1, 139.3, 138.5, 131.8, 130.1, 130.0, 126.0, 124.3, 123.9 (d, J
= 2.5 Hz), 123.7, 121.9, 115.5 (t, J = 261.5 Hz), 115.0, 113.9, 111.1 (d, J = 22.2 Hz), 106.3 (d, J =
25.3 Hz), 80.6, 80.5, 59.9, 59.6, 57.3, 38.6, 37.6, 34.5; MS (ESI) m/z 627.1 [M + H]+.
4.2.4.5
1-(2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)
acetyl)pyrrolidin-3-one (24). Yield: 85%, white solid, purity: 95%.1H NMR (400 MHz, CDCl3) δ
(ppm) 7.93 (s, 1H), 7.90 (d, J = 7.3 Hz, 1H), 7.84 (d, J = 3.3 Hz, 1H), 7.81 (d, J = 7.4 Hz, 1H),
7.59 (t, J = 6.9 Hz, 1H), 7.23 (s, 1H), 7.16 – 7.13 (m, 3H), 6.88 (d, J = 8.9 Hz, 1H), 6.59 (t, J =
73.2 Hz, 1H), 4.75 (t, J = 8.8 Hz, 1H), 3.97 – 3.82 (m, 4H), 3.19 – 3.02 (m, 2H), 2.84 (dd, J = 11.0,
13
5.6 Hz, 1H), 2.68 (ddd, J = 23.1, 18.5, 8.4 Hz, 3H); C NMR (150 MHz, CDCl3) δ (ppm) 209.0
(d, J = 14.6 Hz), 169.1 (d, J = 21.2 Hz), 165.7, 164.1, 162.4, 152.2 (d, J = 11.2 Hz), 143.8 (d, J =
9.1 Hz), 141.3, 139.5, 138.5, 131.7 (d, J = 6.4 Hz), 130.1, 131.7 (d, J = 7.5 Hz), 124.4, 124.1,
123.8, 122.0, 115.6 (t, J = 261.5 Hz), 115.2 (d, J = 17.3 Hz), 114.1, 111.2 (d, J = 22.2 Hz), 106.5
(d, J = 25.2 Hz), 59.9 (d, J = 12.9 Hz), 52.0, 43.4, 42.0 (d, J = 35.5 Hz), 40.6, 34.9 (d, J = 2.7 Hz);
MS (ESI) m/z 613.1 [M + H]+.
4.2.4.6
1-(2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)
acetyl)piperidin-4-one (25). Yield: 77%, white solid, purity: 94%.1H NMR (400 MHz, CDCl3) δ
(ppm) 7.95 (s, 1H), 7.91 (d, J = 7.9 Hz, 1H), 7.85 (s, 1H), 7.83 (d, J = 7.8 Hz, 1H), 7.61 (t, J = 7.6
Hz, 1H), 7.29 – 7.25 (m, 1H), 7.18 – 7.13 (m, 3H), 6.90 (d, J = 8.6 Hz, 1H), 6.61 (t, J = 73.2 Hz,