European Journal of Medicinal Chemistry: Yan Zhu, Nannan Sun, Mingcheng Yu, Huimin Guo, Qiong Xie, Yonghui Wang

European Journal of Medicinal Chemistry 182 (2019) 111589
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of aryl-substituted indole and indoline derivatives as RORgt

agonists
Yan Zhu 1, Nannan Sun 1, Mingcheng Yu, Huimin Guo, Qiong Xie**, Yonghui Wang*
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China
a r t i c l e i n f o a b s t r a c t
Article history: A series of aryl-substituted indole and indoline derivatives were discovered as novel RORgt agonists by a
Received 5 June 2019 scaffold-based hybridization of the reported RORgt agonists 1 and 2. SAR studies on the core structures,
Received in revised form the RHS hydrophilic side chains and the LHS hydrophobic aryl groups of a hybrid compound 3 led to the
25 July 2019
identification of potent RORgt agonists with improved drug-like properties. Compound 14 represented a
Accepted 5 August 2019
Available online 6 August 2019
high potency lead with an EC50 of 20.8 ± 1.5 nM, the (S)-enantiomer (EC50 ¼ 16.1 ± 4.5 nM) of which was
17 times more potent than the (R) counterpart (EC50 ¼ 286 ± 30.4 nM) in RORg dual FRET assay. The cell-
based GAL4 reporter gene assay also suggested 14 as the most active compound which exhibited an EC50
Keywords:
Indoles
of 247 ± 33.1 nM and a maximum activation percentage of 133%. Moreover, 14 showed high metabolic
Indolines stability (t1/2 ¼ 113 min) in mouse liver microsome and had improved aqueous solubility at pH 7.4
RORgt agonists compared to the parent compounds. Furthermore, 14 was found to be orally bioavailable and demon-
Cancer immunotherapy strated excellent in vivo pharmacokinetics in mice. Present studies indicate that 14 deserves further
Metabolic stability investigation in tumor animal models as a potential candidate of RORgt agonist for cancer
Aqueous solubility immunotherapy.
© 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction that promote the differentiation and activation of Th17 and

Tc17 cells have shown promising therapeutic effects for cancer
The T cell specific isoform of retinoic acid receptor-related immunotherapy [11e13].
orphan receptor gamma (RORg), known as RORgt, is a nuclear re- Starting from the discovery of RORa/g agonist SR1078 [14], more
ceptor (NR) specifically expressed in thymocytes. RORgt serves as a and more small molecule agonists of RORgt have been reported
key transcription factor to drive the differentiation of CD4þ cells [13,15e18] within researches on RORgt inverse agonists. We pre-
into interleukin-17 (IL-17) producing T helper 17 (Th17) cells [1], viously reported the discovery and complex crystal structures of
which are implicated in the pathology of various inflammatory and several N-aryl amide-based RORgt agonists (Fig. 1), such as tertiary
autoimmune diseases [2,3]. In addition to their major role in the amine (PDB: 4NIE) [19], thiazole amide (PDB: 4XT9) [20], thiazole
field of autoimmune diseases [4,5], RORg antagonists were found to ketone amide (PDB: 5YP5) [21] and biaryl amide (PDB: 5YP6) [21].
be effective in treating castration-resistant prostate cancer in a few It was noticed that minor changes of RORgt inverse agonists at the
articles [6,7]. Recent studies have revealed the presence of part interacting with activation function 2 (AF2) of the ligand
Th17 cells in tumor microenvironment [8] and the active involve- binding domain (LBD) of RORg could lead to functional switch from
ment of Th17 cells [9] and CD8þ cytotoxic T (Tc17) cells [10] in inhibition to activation [13,17]. AF2 is a functional site of RORgt-LBD
protective tumor immunity by majorly producing IL17. Therefore, around helix 11 (H11) and helix 12 (H12), which was stabilized by
RORgt has become a promising target with the potential of His479eTyr502ePhe506 p-p cluster interactions [13]. RORgt in-
strengthening immune system to combat cancer. RORgt agonists verse agonists break the hydrogen bond between His479eTyr502
and weaken the stabilization of AF2. Conversely, RORgt agonists
stabilize the AF2 conformation by forming hydrophobic in-
* Corresponding author. teractions with Trp317, His479 and Tyr502, thus RORgt are easier to
** Corresponding author. recruit coactivator peptides [17]. The discovery of RORgt agonists
E-mail addresses: qxie@fudan.edu.cn (Q. Xie), yonghuiwang@fudan.edu.cn from RORgt inverse agonists has been reported for some cases.
(Y. Wang). However, reports about the structure-activity relationship (SAR) of
1
These authors contributed equally in this work.
https://doi.org/10.1016/j.ejmech.2019.111589
0223-5234/© 2019 Elsevier Masson SAS. All rights reserved.
2 Y. Zhu et al. / European Journal of Medicinal Chemistry 182 (2019) 111589
Fig. 1. Reported N-aryl amide-based RORgt agonists. (Parts of the agonist structures interacting with the AF2 domain of RORg LBD were presented in blue.). (For interpretation of
the references to colour in this figure legend, the reader is referred to the Web version of this article.)
RORgt agonists remain limited. Lycera's N-arylsulfonylbenzoxazine compound 2 (LYC-55716, Figs. 2

Scripps reported the SAR study of a series of N-arylsulfony- and 3) [23] was the first drug candidate that entered phase 1/2
lindoline RORgt agonists and found that an ether linker was clinical trials for the treatment of solid tumors alone or in combi-
preferred for RORg agonism [22]. A representative compound 1 nation with programmed cell death protein 1 (PD-1) inhibitors [24].
(Figs. 2 and 3) exhibited good RORg agonism activity Compound 2 was undoubtedly a potent RORgt agonist
(EC50 ¼ 30.2 ± 4.2 nM, percent maximum activation (% (EC50 ¼ 30.1 ± 2.9 nM, %Max ¼ 152%) and showed high metabolic
Max) ¼ 118%) in our RORg dual fluorescence resonance energy stability (t1/2 > 145 min) in MLM. However, compound 2 has a high
transfer (dual FRET) assay. However, compounds of this series molecular weight (MW) value of 603.53 and a high CLogP value of
showed poor metabolic stability (t1/2 ¼ 2.9 min for 1) in mouse liver 6.76 which may need further optimization.
microsome (MLM) and had high lipophilicity (CLogP ¼ 5.98 for 1). Docking modes of 1 and 2 in RORgt LBD indicated that both left-
Fig. 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 RORgt LBD (PDB ID: 4NIE).
(For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Y. Zhu et al. / European Journal of Medicinal Chemistry 182 (2019) 111589 3
Fig. 3. Hybridization strategy for the design of aryl-substituted indole/indolines as RORgt agonists.
hand side (LHS) moieties, namely the benzyl ether group and the the 2- or 3- position of the preferred indole/indoline core structures
phenyl group, occupied the same hydrophobic pocket near AF2 via alkylene linkers of different length, leading to compounds 7e19.
domain (Fig. 2, the structural superimposition of compounds 1 and According to the docking results of selected indole/indoline de-
2 in complex with RORgt LBD is shown in Supplementary Fig. S5). rivatives in RORgt LBD, which indicated that the RHS parts partic-
We hypothesized that replacing the 2-chloro-6-fluorobenzyl ether ipated in hydrogen-bonding interactions with hydrophilic residues,
moiety of 1 with the 3-(difluoromethoxy)-5-fluorophenyl moiety of we designed compounds 20e29 with varied polar RHS groups
2, resulting in a hybrid compound 3 (Fig. 3), could possibly maintain substituted on 2-position of the indoline scaffold. In contrast, the
RORgt activity based on the docking results of 3 (Fig. 2 and Fig. S6). LHS parts were involved in hydrophobic contacts with AF2.
Since there are two likely metabolic soft spots in compound 1, Therefore, compounds 30e39 with different lipophilic substituents
namely the two benzylic positions [22], compound 3 with one of on the LHS aryl group were designed and synthesized (Fig. 4).
the metabolic spots the benzyl ether group removed might have
improved its metabolic stability. Thus compound 3 was prepared
and tested in RORg dual FRET and MLM assays. To our delight, 2.2. Chemistry
compound 3 showed high RORg agonism activity with an EC50 of
50.6 ± 4.6 nM and a maximum activation of 159%. However, the Synthetic procedures for the target compounds 3e39 were
metabolic stability of 3 was a little better than that of 1 but outlined in Scheme 1. Sulfonylation of commercially available 6-
remained low (t1/2 ¼ 11.4 min). Therefore, compound 3 needed bromoindole/indoline/indazole/benzo[d]imidazole (3ae6a) with
further structural modifications on the indoline ring to improve the 3-(trifluoromethyl)benzenesulfonyl chloride afforded the desired
metabolic stability. In this paper, starting from the hybrid com- intermediates 3be6b. A subsequent palladium-catalyzed Suzuki
pound 3, a series of aryl-substituted indole and indoline com- coupling reaction of 3be6b with 3-(difluoromethoxy)-5-
pounds were designed and synthesized by changing the indoline- fluorophenyl boronic acid pinacol ester yielded the corresponding
like core structures, the right-hand side (RHS) 2- or 3-hydrophilic compounds 3e6. Intermediates 7ce19c were obtained by the same
side chains and the LHS 6-aryl groups (Fig. 3). Studies on the SAR, procedures of sulfonylation and Suzuki coupling reaction from
binding modes, in vitro metabolic stability, solubility and in vivo 7ae19a, the synthesis of which was presented in Supporting In-
pharmacokinetic (PK) profiles of these compounds were further formation. Final hydrolysis of 7ce19c in THF by LiOH gave the
investigated to develop novel RORgt agonists as potential thera- carboxylic acid compounds 7e19. Amide condensation of com-
peutic agents for cancer immunotherapy. pound 14 with a set of cyclic secondary amines under the condition
of HATU and DIEA afforded the targeted compounds 20e29. A
2. Results and discussion palladium-catalyzed Suzuki coupling reaction using substituted
bromobenzene and 30a, which was prepared from 14b, gave in-
2.1. Compound design termediates 30be39b. Subsequent hydrolysis of 30be39b by LiOH
yielded the desired compounds 30e39. Enantiomers (S)-14 and (R)-
We first optimized the core structures based on the principle of 14 were obtained by chiral HPLC separation of the racemate com-
bioisosterism, by replacing the indoline of 3 with indole (4), inda- pound 14. Besides, the absolute configurations were determined
zole (5) and benzo[d]imidazole (6) moieties. After the optimal core according to the optical rotation values of (S)-14 and (R)-14
structures identified, carboxylic acid side chains were attached to compared with that of compound 2.
Fig. 4. Target compounds with changes on core structures (3e6), RHS hydrophilic side chains (7e29) and LHS hydrophobic aryl groups (30e39).
2.3. Structure-activity relationship the RORg dual FRET assay (Table 1). With indole replacing the
indoline in 3, compound 4 showed a two-fold reduction in RORgt
The synthesized compounds were evaluated in RORg dual FRET activity (EC50 ¼ 99.4 ± 44.7 nM) and a slight drop in efficacy (%
assay according to the basal level activity changes, which is suitable Max ¼ 125%) relative to 3. On the other hand, the indole compound
for the activity and function assessment of both agonists and in- 4 showed improved metabolic stability in MLM, with a t1/2 of
verse agonists [19]. Compounds in each set having good RORgt 39.1 min, 3-times longer than that of 3. When the core structure
agonism activities were subjected to metabolic stability assay in was replaced by indazole (5) or benzo[d]imidazole (6), both RORgt
MLM and cell-based GAL4 luciferase reporter gene assay. potency and efficacy reduced. In view of the RORgt activity and
Initially, a set of compounds with different 5-, 6-membered metabolic stability, we chose indole in 4 and indoline in 3 as
bicyclic cores (3e6) were designed, synthesized and evaluated in preferred core structures for the following RHS optimization.
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, 3e12 h; e) Pd(dppf)Cl2, KOAc, 1,4-Dioxane, N2, 100 C, 12 h.
After identifying the preferred core structures, we explored SAR maximum activation response, 2-substituted indolines (%
of the RHS moiety of 4 and 3. Introduction of a carboxylic acid Max ~ 120%) outperformed 3-substituted indolines (%Max ~ 100%).
tether to the selected indole/indoline cores was expected to Compounds 14 and 17 with a side chain of acetic acid tethered at
maintain RORgt activity and make improvement in metabolic sta- the 2- and the 3- position, respectively, exhibited the highest RORgt
bility. Compounds 7e19 with carboxylic acid tethered by alkylene potency and efficacy among each subset of the indoline series. In
chains of different length to the 2- or 3- position of indole/indoline the next MLM stability test, 2-substituted indolines (t1/2 > 60 min)
were designed and synthesized. Results of their RORgt activity, were obviously more stable than 3-substituted indolines (t1/
metabolic stability and cell-based gene transcription activity were 2 < 50 min). It seems that metabolic stability increases with an or-
summarized in Table 2. Indole-2-carboxylic acid analogue 7 der of the linker length (n) of 2 < 3 < 1, so does the cell-based ac-
showed essentially the same RORgt potency and efficacy as 4 while tivity. Taking both the cell-based RORgt activity and metabolic
the 2-acetic acid derivative 8 exhibited a lower RORgt activity. stability into account, compound 14 (EC50 ¼ 247 ± 33.1 nM, %
Elongating the linker by adding ethylene resulted in 2-propionic Max ¼ 133%, t1/2 ¼ 113 min) stood out among the indoline series.
acid compound 9, which was 3-fold more active than 4 in acti- We separated two enantiomers of 14 by chiral HPLC, and (S)-
vating RORgt. Further elongation of the linker from ethylene to enantiomer (EC50 ¼ 16.1 ± 4.5 nM) showed 17 times higher potency
propylene (10) slightly lowered the RORgt activity relative to 9. than (R)-enantiomer (EC50 ¼ 286 ± 30.4 nM) in RORgt dual FRET
Switching the carboxylic acid tethers to 3-position (11e13) reduced assay. The activity of (S)-14 on GAL4 luciferase reporter gene assay
the RORgt potency and efficacy comparing with their 2-position (EC50 ¼ 201 ± 48.9 nM, %Max ¼ 144%) was correlated with the dual
counterparts (8e10). Among the indole series, 9 stood out with FRET result, which both suggested (S)-14 as the optimal
an EC50 of 25.5 ± 11.6 nM and a maximum activation of 124%, and enantiomer.
was then subjected to in vitro metabolic stability assay which Compound 16 showed good cell-based RORgt activity
indicated a high stability of 9 in MLM (t1/2 > 145 min) like Lycera's (EC50 ¼ 422 ± 43.2 nM, %Max ¼ 146%) and metabolic stability (t1/
compound 2. However, in cell-based reporter gene assay, 9 only 2 ¼ 93.3 min) as well, but it had a high CLogP of 6.61. We tried to
displayed activity at micromolar level with an EC50 of modify the RHS butyric acid of 16 with different amide-connected
1710 ± 396 nM, 16-fold less potent than 2 (EC50 ¼ 102 ± 4.9 nM). polar groups, including ketones, alcohols, carboxylic acids, ethers
This could be explained by the increased lipophilicity (CLogP and and sulfone. All designed compounds (20e29) had lower CLogP
CLogD) of 9 which may affect cell permeability. values compared to 16 and showed equivalent or higher maximum
All carboxylic acid-tethered indoline compounds (14e19) activation response in RORgt dual FRET assay (Table 3). As different
showed high RORgt potency with EC50s ranging from 14.6 nM to groups substituted on R2, their RORgt activation potencies
31.3 nM, no matter tethered at 2- or 3- position. As for the increased by the order of alcohol (21) < ether (27) < ketone (20, 24,
Table 1
SAR of core structures.
Compd Core CLogPa RORg dual FRET MLM Stabilityd t1/2 (mins)
EC50 (nM)b %Maxc
1 (Scripps' agonist) 5.98 30.2 ± 4.2 118 2.9

2 (LYC-55716) 6.76 30.1 ± 2.9 152 >145
3 6.07 50.6 ± 4.6 159 11.4
4 6.95 99.4 ± 44.7 125 39.1
5 6.30 205 ± 35 75 -e
6 6.17 122 ± 101 75 -e
a
Calculated by Discovery Studio 3.0.
b
EC50 value was expressed as Mean ± SD, n ¼ 2.
c
Percent maximum activation.
d
Mouse liver microsome stability test.
e
“-” means not determined.
25) < carboxylic acid (22, 28) < sulfone (29). The more unique were 2.4. Binding mode study
23 with a 2-oxa-6-azaspiro[3.3]heptanyl group and 26 with a 4-
hydroxypiperidinyl group, which represented more active com- The binding mode of (S)-14 in RORgt LBD was revealed by
pounds within the ether and alcohol series, respectively, and were docking study (Fig. 5). In the most possible binding mode, the 3-
subjected to metabolic stability and GAL4 reporter gene assays (difluoromethoxy)-5-fluorophenyl moiety in the LHS of the indo-
along with carboxylic acids (22, 28) and sulfone (29). Results were line core locates in the functional site around H11 and H12, forming
summarized in Table 3. The cell-based activities of two carboxylic p-p stacking interactions with His479 and Trp317 and stabilizing
acids (22, 28) and alcohol (26) dropped dramatically with EC50s of the AF2 domain. This moiety occupies in almost the same place as
4e6 mM, although the metabolic stability of carboxylic acids was that in the initial compound 2 (LYC-55716), which is believed to be
acceptable (t1/2 30e66 min) while the alcohol metabolized very the functional group for our compounds possessing the RORgt
quickly. To our surprise, 23 was fairly unstable in MLM and was agonist activity (shown as Fig. 5b). In the RHS, the acetic acid group
destroyed within one minute (t1/2 < 1 min), although it showed formed hydrogen bonds with the main chain of His323. Besides, the
submicromolar RORgt activity in GAL4 reporter gene assay 3-trifluoromethyl substituted phenylsulfony moiety is in a hydro-
(EC50 ¼ 752 ± 100 nM). The most potent sulfone compound 29 was phobic cavity, forming p-p stacking interactions with Phe377 and
1.3 times more active than 16 in GAL4 reporter gene assay, dis- providing preferred intermolecular interactions with surrounding
playing an EC50 of 322 ± 51.4 nM. Unfortunately, the half-life of 29 hydrophobic residues in the hydrophobic site near Phe377 and
in MLM was as low as 2 min, which prevent it from further Phe378.
development.
Based on the above SAR studies on core structure and RHS 2.5. Drug-like properties and PK study
moiety, 14 was selected as the template to make LHS modifications.
We first made cyclization at the 3- and 4- position of the phenyl The solubility of 1, 2, 3 and 14 in aqueous solution at the con-
group, yielding benzo[d][1,3]dioxole and 2,3-dihydrobenzofuran dition of pH 7.4 was subsequently evaluated. Results were shown in
derivatives (30e32), which were less potent than 14 in RORgt Table 5. Compounds 1 and 3 showed relatively poor solubility
dual FRET assay (Table 4). Changes were then made on the 3- (<2 mM) because they have high lipophilicity and are lack of ionic or
difluoromethoxyl group by halogens (33, 34), trifluoromethyl ionizable group. The aqueous solubility of compounds 2
group (35) and alkoxyl groups (36e39), but no activity enhance- (S ¼ 37.5 mM) and 14 (S ¼ 69.4 mM) dramatically increased due to
ment was observed as shown in Table 4. Introduction of an iso- the introduction of ionic carboxylic acid groups. Compound 14 was
propoxy group (38) caused the greatest activity reduction in this 2 times more soluble than compound 2, which could be explained
series probably due to increased size of the substituent, which may by the decreased MW and CLogP (5.83)/CLogD (4.39) values of 14.
interfere with interactions between the LHS of 38 and the AF2 of The PK of 14 was investigated in mice following intravenous (IV,
RORgt LBD. 1 mg/kg) and oral (PO, 5 mg/kg) administration. Compound 14
Table 2
SAR of RHS moiety.
Compd Core þ RHS n CLogP/CLogDa RORg dual FRET MLM Stabilityd t1/2 (mins) GAL4e
b c
EC50 (nM) %Max EC50 (nM)b Emax(%)
2 6.76/5.32 30.1 ± 2.9 152 >145 102 ± 4.9 141

7 0 6.87/5.41 74.3 ± 21.1 128 -f -f -f
8 1 6.91/5.46 125 ± 4.4 66 e e e
9 2 6.94/5.47 25.5 ± 11.6 124 >145 1710 ± 396 74
10 3 7.40/5.98 45.3 ± 19.9 95 e e e
11 1 6.62/5.17 133 ± 17.8 62 e e e
12 2 7.07/5.61 43.6 ± 29.7 71 e e e
13 3 7.53/6.07 93.8 ± 22.6 74 e e e
14 1 5.83/4.39 20.8 ± 1.5 127 113 247 ± 33.1 133

(S)-14 1 5.83/4.39 16.1 ± 4.5 124 e 201 ± 48.9 144
(R)-14 1 5.83/4.39 286 ± 30.4 93 e e e
15 2 6.15/4.70 31.3 ± 9.1 123 60.9 2320 ± 1560 172
16 3 6.61/5.18 25.2 ± 3.4 117 93.3 422 ± 43.2 146
17 1 5.57/5.02 21.8 ± 1.2 109 45.6 e e
18 2 6.02/4.57 14.6 ± 2.2 97 24.5 e e
19 3 6.48/5.29 26.2 ± 11.6 95 34.2 e e
a
b
c
d
e
GAL4 reporter gene assay.
f
showed low in vivo clearance (CL ¼ 0.573 L/h/kg), a moderate half- mesh) was used for column chromatography. The purity of all test
life of 4 h and a high oral bioavailability of 100% (see Table S4 and compounds was assessed by HPLC and area % purity was measured
Fig. S7). Overall, compound 14 demonstrated excellent in vivo at 254 nm. The HPLC analyses were performed using a Agilent 1260
pharmacokinetics in mice consistent with its in vitro properties instrument. Elution was done with a gradient of 5e90% solvent B
(aqueous solubility and metabolic stability). (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 mm) column at
3. Conclusions 3.0 mL/min. High-resolution MS (HRMS) was analyzed by a TOF
analyzer. The ion source is electrospray ionization (ESI). 1H NMR
In conclusion, we have discovered a series of aryl-substituted spectra were recorded at 400 MHz and 13C NMR spectra were
indole and indoline derivatives as novel RORgt agonists by a recorded at 600 MHz. Chemical shifts in 1H NMR spectra are re-
scaffold-based hybridization strategy. SAR studies on the core ported in parts per million (ppm) on the d scale from an internal
structures, RHS hydrophilic side chains and LHS hydrophobic aryl standard of CDCl3 (7.26 ppm). Data are reported as follows: chem-
groups of the hybrid compound 3 led to the identification of potent ical shift (d ppm), multiplicity (s ¼ singlet, d ¼ doublet, t ¼ triplet,
RORgt agonists with improved drug-like properties. Compound 14 q ¼ quartet, m ¼ multiplet, br ¼ broad), coupling constant in hertz
was found to have good RORgt activities in both dual FRET and GAL4 (Hz), and integration. Chemical shifts of 13C NMR spectra are re-
reporter gene assays. Compound 14 showed high metabolic stability ported in ppm from the central peak of CDCl3 (77.0 ppm) on the
in MLM and had improved aqueous solubility (2-fold relative to 2) at d scale.
pH 7.4. Furthermore, 14 demonstrated excellent oral bioavailability
and in vivo PK profile in mice. Present studies suggest 14 as a po- 4.2. Chemical synthesis
tential RORgt agonist candidate which deserves further investiga-
tion in tumor animal models for cancer immunotherapy. 4.2.1. The synthesis of compounds 1e2
Compounds 1 and 2 were synthesized following the published
4. Experimental procedures [22,25], and were characterized by 1H NMR, 13C NMR
and MS.
4.1. Materials and methods
4.2.1.1. 6-((2-Chloro-6-fluorobenzyl)oxy)-1-((3-(trifluoromethyl)
All commercially available reagents were used without further phenyl)sulfonyl)indoline (1). Yield: 81%, white solid, purity: 98%. 1H
purification unless otherwise stated. The reactions were monitored NMR (400 MHz, DMSO‑d6) d (ppm) 8.15 (d, J ¼ 7.5 Hz, 1H), 8.09 (d,
by thin-layer chromatography (TLC) analysis. Silica gel (200e300 J ¼ 7.1 Hz, 1H), 8.02 (s, 1H), 7.84 (t, J ¼ 6.6 Hz, 1H), 7.51 (dd, J ¼ 14.4,
Table 3
SAR of the R2 of RHS moiety.
Compd RHS (R2) CLogPa RORg dual FRET MLM Stabilityd t1/2 (mins) GAL4e
EC50 (nM)b %Maxc EC50 (nM)b Emax (%)
16 6.61 25.2 ± 3.4 117 93.3 422 ± 43.2 146
20 4.88 78.8 ± 13.7 130 -f -f -f
21 4.92 244 ± 17.4 141 e e e
22 5.07 49.3 ± 21.6 138 65.9 4050 ± 459 100
23 4.93 47.9 ± 3.0 128 0.7 752 ± 100 104
24 5.13 52.3 ± 18.5 136 e e e
25 5.38 80.4 ± 1.0 136 e e e
26 5.05 106 ± 9.7 127 0.7 6040 ± 1010 100
27 5.30 209 ± 90.6 128 e e e
28 5.71 57.1 ± 6.8 116 30.1 5440 ± 3360 178
29 5.03 43.4 ± 19.9 126 2.0 322 ± 51.4 104
a
b
c
d
e
GAL4 reporter gene assay.
f
7.8 Hz, 1H), 7.43 (d, J ¼ 7.8 Hz, 1H), 7.33 (t, J ¼ 8.2 Hz, 1H), 7.11e7.07 J ¼ 9.7 Hz, 1H), 7.24 (s, 1H), 7.13 (d, J ¼ 9.6 Hz, 1H), 6.86 (d, J ¼ 8.5 Hz,
(m, 2H), 6.71 (d, J ¼ 8.1 Hz, 1H), 5.15 (s, 2H), 3.99 (t, J ¼ 6.7 Hz, 2H), 1H), 4.32 (d, J ¼ 12.5 Hz, 1H), 3.39 (d, J ¼ 13.5 Hz, 2H), 1.74 (d,
2.82 (t, J ¼ 7.1 Hz, 2H); 13C NMR (150 MHz, DMSO‑d6) d (ppm) 162.1, J ¼ 4.2 Hz, 2H), 1.02 (s, 3H), 1.00 (s, 3H); 13C NMR (150 MHz,
160.4, 158.0, 141.8, 137.2, 135.3 (d, J ¼ 4.8 Hz), 131.6 (d, J ¼ 9.8 Hz), DMSO‑d6) d (ppm) 178.1, 163.6, 162.0, 152.5, 147.0, 142.8 (d,
130.4 (d, J ¼ 2.5 Hz), 129.9 (q, J ¼ 32.8 Hz), 125.9, 125.6, 124.8, 123.9, J ¼ 9.6 Hz), 138.7, 131.5, 130.9, 130.6, 130.3, 125.2, 123.4, 122.9, 121.8,
123.3 (d, J ¼ 3.4 Hz), 122.1, 121.8 (d, J ¼ 17.7 Hz), 114.6 (d, 118.0, 116.0 (t, J ¼ 258.4 Hz), 112.2, 109.6 (d, J ¼ 22.2 Hz), 104.8 (d,
J ¼ 22.5 Hz), 110.5, 101.7, 61.4 (d, J ¼ 3.0 Hz), 50.8, 26.4; MS (ESI) m/z J ¼ 25.5 Hz), 69.8, 47.9, 41.3, 25.3, 24.5; MS (ESI) m/z 604.1 [M þ H]þ.
486.1 [M þ H]þ.
4.2.2. General procedure for the synthesis of compounds 3e6
4.2.1.2. (S)-3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-4-((3-(tri- Step 1: To a vial were added intermediates 3ae6a (1.0 eq), DMAP
fluoromethyl)phenyl)sulfonyl)-3,4-dihydro-2H-benzo[b] [1,4]oxazin- (20 mol%, 0.2 eq), DIEA (3.0 eq) and DCM (2 mL). After the reaction
2-yl)-2,2-dimethylpropanoic acid (2). Yield: 67%, white solid, pu- mixture was cooled to 0 C by ice-water bath, 3-(trifluoromethyl)
rity: 95%. [a]23 D þ147
(c ¼ 0.2, CH Cl ). 1H NMR (400 MHz,
2 2 benzenesulfonyl chloride (1.2 eq) was subsequently added drop-
DMSO‑d6) d (ppm) 12.28 (s, 1H), 8.14 (d, J ¼ 7.7 Hz, 1H), 7.95 (dd, wise. Then the reaction mixture was stirred at room temperature
J ¼ 8.2, 6.4 Hz, 3H), 7.85 (t, J ¼ 7.9 Hz, 1H), 7.60e7.42 (m, 2H), 7.34 (d, for 12 h. After completion of the reaction, the resulting mixture was
Table 4 4.2.2.1. 6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-

SAR of LHS moiety. fluoromethyl)phenyl)sulfonyl)indoline (3). Yield: 52%, white solid,
purity: 94%. 1H NMR (400 MHz, DMSO‑d6) d (ppm) 8.15 (d,
J ¼ 6.6 Hz, 1H), 8.04 (s, 2H), 7.79 (t, J ¼ 7.2 Hz, 1H), 7.63 (s, 1H),
7.58e7.39 (m, 1H), 7.32 (d, J ¼ 5.1 Hz, 2H), 7.24e7.19 (m, 2H), 7.13 (d,
J ¼ 8.9 Hz, 1H), 4.00 (t, J ¼ 6.6 Hz, 2H), 2.88 (t, J ¼ 7.3 Hz, 2H); 13C
NMR (150 MHz, DMSO‑d6) d (ppm) 163.5, 161.9, 152.2 (d,
J ¼ 12.0 Hz), 143.4 (d, J ¼ 9.5 Hz), 141.6, 137.7, 137.2, 133.1, 131.2,
131.0, 130.4, 129.9 (d, J ¼ 32.9 Hz), 126.1, 123.5, 123.3, 116.1 (t,
Compd LHS CLogP/CLogDa RORg dual FRET J ¼ 258.4 Hz), 112.8, 112.3, 110.2 (d, J ¼ 22.3 Hz), 105.3 (d,
EC50 (nM)b %Maxc J ¼ 25.6 Hz), 50.4, 26.9; MS (ESI) m/z 488.1 [M þ H]þ.
14 5.83/4.39 20.8 ± 1.5 127
4.2.2.2. 6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
fluoromethyl)phenyl)sulfonyl)-1H-indole (4). Yield: 48%, white solid,
purity: 98%. 1H NMR (400 MHz, DMSO‑d6) d (ppm) 8.45 (s, 1H), 8.41
30 6.62/5.18 42.6 ± 6.0 90
(d, J ¼ 7.2 Hz, 1H), 8.18 (s, 1H), 8.10 (d, J ¼ 6.7 Hz, 1H), 8.02 (s, 1H),
7.84 (t, J ¼ 7.1 Hz, 1H), 7.72 (d, J ¼ 7.6 Hz, 1H), 7.63 (d, J ¼ 7.7 Hz, 1H),
31 4.59/3.15 87.4 ± 20.5 77 7.46 (d, J ¼ 8.0 Hz, 2H), 7.30 (d, J ¼ 14.8 Hz, 2H), 7.17 (d, J ¼ 9.4 Hz,
1H), 6.94 (s, 1H); 13C NMR (150 MHz, DMSO‑d6) d (ppm) 163.5, 161.9,
152.2 (d, J ¼ 11.9 Hz), 143.6 (d, J ¼ 9.5 Hz), 137.8, 135.0, 134.5, 131.5,
32 4.88/3.44 56.8 ± 21.0 40
131.3, 130.8, 130.7, 130.2 (d, J ¼ 33.0 Hz), 128.1, 123.4, 123.2, 122.3,
116.1 (t, J ¼ 258.2 Hz), 113.0, 111.0, 110.6 (d, J ¼ 22.3 Hz), 110.0, 105.1
33 5.23/3.79 25.7 ± 4.8 101 (d, J ¼ 25.6 Hz); MS (ESI) m/z 486.1 [M þ H]þ.
4.2.2.3. 6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
34 5.69/4.25 42.0 ± 4.8 98 fluoromethyl)phenyl)sulfonyl)-1H-indazole (5). Yield: 42%, white
solid, purity: 98%. 1H NMR (400 MHz, DMSO‑d6) d (ppm) 8.10 (d,
J ¼ 7.7 Hz, 1H), 8.00 (d, J ¼ 8.7 Hz, 2H), 7.95 (d, J ¼ 8.1 Hz, 1H), 7.85
(d, J ¼ 7.8 Hz, 1H), 7.81 (d, J ¼ 8.9 Hz, 1H), 7.57e7.39 (m, 1H), 7.33 (s,
35 5.97/4.53 41.3 ± 5.2 115
1H), 7.31e7.21 (m, 2H), 7.16 (s, 1H); 13C NMR (150 MHz, DMSO‑d6)
d (ppm) 170.1, 163.5, 161.9, 152.3 (d, J ¼ 12.0 Hz), 142.9, 140.7 (d,
J ¼ 9.6 Hz), 140.4, 138.6, 134.5, 131.0, 130.7, 129.8, 126.0, 125.3, 123.3,
36 5.01/3.57 39.3 ± 1.1 88 115.9, 115.8 (t, J ¼ 258.8 Hz), 113.0, 110.5 (d, J ¼ 22.9 Hz), 109.8, 106.8
(d, J ¼ 25.6 Hz); MS (ESI) m/z 487.1 [M þ H]þ.
37 5.36/3.92 32.9 ± 10.9 96 4.2.2.4. 6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-

fluoromethyl)phenyl)sulfonyl)-1H-benzo[d]imidazole (6). Yield: 43%,
white solid, purity: 99%. 1H NMR (400 MHz, CDCl3) d (ppm) 8.43 (s,
38 5.73/4.30 124 ± 63.8 105 1H), 8.32 (s, 1H), 8.20 (d, J ¼ 8.0 Hz, 1H), 7.99 (d, J ¼ 1.3 Hz, 1H), 7.93
(d, J ¼ 7.8 Hz, 1H), 7.86 (d, J ¼ 8.4 Hz, 1H), 7.73 (t, J ¼ 7.9 Hz, 1H), 7.57
(dd, J ¼ 8.4, 1.7 Hz, 1H), 7.18e7.14 (m, 2H), 6.94e6.90 (m, 1H), 6.60 (t,
39 6.94/5.50 31.1 ± 5.7 95
J ¼ 73.0 Hz, 1H); 13C NMR (150 MHz, DMSO‑d6) d (ppm) 163.5, 161.9,
152.2 (d, J ¼ 12.3 Hz), 143.5 (d, J ¼ 10.7 Hz), 143.1 (d, J ¼ 9.7 Hz),
137.6, 136.0, 132.1, 131.8, 131.5, 130.6 (t, J ¼ 16.7 Hz), 124.6, 124.3,
a
123.7, 121.9, 121.2, 116.1 (t, J ¼ 258.3 Hz), 113.3, 110.9 (d, J ¼ 22.5 Hz),
b
EC50 value was expressed as Mean ± SD, n ¼ 2. 110.3, 105.4 (d, J ¼ 25.8 Hz); MS (ESI) m/z 487.1 [M þ H]þ.
c
4.2.3. General procedure for the synthesis of compounds 7e19
diluted with DCM and washed with water. The separated aqueous Step 1: To a vial were added intermediates 7ae19a (1.0 eq),
phase was washed with DCM. The combined organic layers were DMAP (20 mol%, 0.2 eq), DIEA (3.0 eq) and DCM (2 mL). After the
dried over MgSO4, filtered, and concentrated in vacuo. The crude reaction mixture was cooled to 0 C by ice-water bath, 3-(tri-
mixture was purified by column chromatography on silica gel fluoromethyl)benzenesulfonyl chloride (1.2 eq) was subsequently
(petroleum: ethyl acetate (EA) ¼ 10:1e3:1) to afford the desired added dropwise. Then the reaction mixture was stirred at room
products 3be6b. temperature for 12 h. After completion of the reaction, the resulting
Step 2: To a vial were added intermediates 3be6b (1.0 eq), 2-(3- mixture was diluted with DCM and washed with water. The sepa-
(difluoromethoxy)-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2- rated aqueous phase was washed with DCM. The combined organic
dioxaborolane (1.2 eq), Pd(dppf)Cl2 (5 mol%, 0.05 eq), X-Phos layers were dried over MgSO4, filtered, and concentrated in vacuo.
(10 mol%, 0.1 eq), K2CO3 (3.0 eq) and 1,4-dioxane (3 mL). Then the The crude mixture was purified by column chromatography on
reaction mixture was stirred under N2 atmosphere at 100 C for silica gel (petroleum: EA ¼ 10:1e3:1) to afford the desired products
12 h. After completion of the reaction, the resulting mixture was 7be19b.
diluted with EA and washed with water. The separated aqueous Step 2: To a vial were added intermediates 7be19b (1.0 eq), 2-
phase was washed with EA. The combined organic layers were (3-(difluoromethoxy)-5-fluorophenyl)-4,4,5,5-tetramethyl-1,3,2-
dried over MgSO4, filtered, and concentrated in vacuo. The crude dioxaborolane (1.2 eq), Pd(dppf)Cl2 (5 mol%, 0.05 eq), X-Phos
mixture was purified by column chromatography on silica gel (10 mol%, 0.1 eq), K2CO3 (3.0 eq) and 1,4-dioxane (3 mL). Then the
(petroleum: EA ¼ 10:1e5:1) to afford the desired products 3e6. reaction mixture was stirred under N2 atmosphere at 100 C for
Fig. 5. a) Zoomed-in view of (S)-14 in the binding pocket of RORgt LBD. (PDB ID: 4NIE, (S)-14 is in orange stick); b) Structural superimposition of 2 (green stick) with (S)-14 (orange
stick) in RORgt LBD. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Table 5
Summary of the drug-like properties of selected compounds 1e3, 14.
Compds MW CLogP/CLogDa Solubilityb (mM) MLM Stabilityc t1/2 (min) CLint(mic)d

(mL/min/mg)
1 (Scripps' agonist) 485.87 5.98/5.98 0.08 2.9 482

2 (LYC-55716) 603.53 6.76/5.32 37.5 >145 <9.6
3 487.06 6.07/6.07 1.48 11.4 121
14 545.45 5.83/4.39 69.4 113 12.3
a
b
Kinetic aqueous solubility at pH 7.46.
c
d
Intrinsic clearance per mg microsomal protein per mL.
12 h. After completion of the reaction, the resulting mixture was J ¼ 8.0 Hz, 1H), 7.62 (d, J ¼ 7.7 Hz, 1H), 7.56 (d, J ¼ 8.1 Hz, 1H), 7.43 (d,
diluted with EA and washed with water. The separated aqueous J ¼ 8.2 Hz, 1H), 7.11 (d, J ¼ 14.8 Hz, 2H), 6.87 (d, J ¼ 9.1 Hz, 1H), 6.68
phase was washed with EA. The combined organic layers were (s, 1H), 6.58 (t, J ¼ 73.2 Hz, 1H), 4.19 (s, 2H); 13C NMR (150 MHz,
dried over MgSO4, filtered, and concentrated in vacuo. The crude CDCl3) d (ppm) 174.3, 164.1, 162.5, 152.1 (d, J ¼ 9.0 Hz), 144.5 (d,
mixture was purified by column chromatography on silica gel J ¼ 9.3 Hz), 139.7, 137.0, 136.3, 133.9, 130.7 (d, J ¼ 2.6 Hz), 130.3,
(petroleum: EA ¼ 10:1e5:1) to afford the desired products 7ce19c. 129.9, 129.2, 124.0 (d, J ¼ 3.3 Hz), 123.5, 121.6, 115.6 (t, J ¼ 261.5 Hz),
Step 3: To a vial were added intermediates 7ce19c (1.0 eq), LiOH 114.3, 113.0, 112.8, 111.4 (d, J ¼ 22.2 Hz), 106.2 (d, J ¼ 25.1 Hz), 34.7;
aq. (2N, 2.0 eq) and THF (2 mL). After that the reaction mixture was MS (ESI) m/z 544.1 [M þ H]þ.
stirred at room temperature for 5 h. After completion of the reac-
tion, the pH of solution was adjusted to about 3 by HCl (2N). Then 4.2.3.3. 3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
the resulting mixture was diluted with EA and washed with water. fluoromethyl)phenyl)sulfonyl)-1H-indol-2-yl)propanoic acid (9).
The separated aqueous phase was washed with EA. The combined Yield: 78%, pale yellow solid, purity: 98%. 1H NMR (400 MHz, CDCl3)
organic layers were dried over MgSO4, filtered, and concentrated in d (ppm) 8.29 (s, 1H), 8.10 (s, 1H), 7.90 (d, J ¼ 6.4 Hz, 1H), 7.80 (d,
vacuo. The crude mixture was purified by column chromatography J ¼ 6.3 Hz, 1H), 7.58 (t, J ¼ 6.9 Hz, 1H), 7.50 (d, J ¼ 7.3 Hz, 1H), 7.43 (d,
on silica gel (petroleum: EA ¼ 1:1e1:8) to afford the desired J ¼ 7.4 Hz, 1H), 7.17 (d, J ¼ 13.3 Hz, 2H), 6.88 (d, J ¼ 7.8 Hz, 1H), 6.69
products 7e19. (t, J ¼ 73.7 Hz, 1H), 6.52 (s, 1H), 3.37 (d, J ¼ 16.9 Hz, 2H), 2.88 (d,
J ¼ 17.8 Hz, 2H); 13C NMR (150 MHz, CDCl3) d (ppm) 176.2, 164.1,
4.2.3.1. 6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- 162.5, 152.2 (d, J ¼ 11.5 Hz), 144.7 (d, J ¼ 9.3 Hz), 141.1, 139.7, 137.6,
fluoromethyl)phenyl)sulfonyl)-1H-indole-2-carboxylic acid (7). 136.0, 132.1 (d, J ¼ 33.7 Hz), 130.6, 130.3, 130.0, 129.7 (d, J ¼ 5.8 Hz),
Yield: 76%, white solid, purity: 97%. 1H NMR (400 MHz, CD3OD) 129.4, 123.6, 121.1, 115.7 (t, J ¼ 261.5 Hz), 114.3, 113.3, 111.4 (d,
d (ppm) 8.38 (s, 1H), 8.30 (s, 2H), 7.96 (d, J ¼ 7.6 Hz, 1H), 7.75 (t, J ¼ 22.1 Hz), 109.9, 106.1 (d, J ¼ 25.1 Hz), 32.9, 24.9; MS (ESI) m/z
J ¼ 8.0 Hz, 1H), 7.71 (d, J ¼ 8.3 Hz, 1H), 7.58 (d, J ¼ 8.1 Hz, 1H), 7.30 (d, 558.1 [M þ H]þ.
J ¼ 10.3 Hz, 1H), 7.26 (d, J ¼ 7.1 Hz, 2H), 7.00 (s, 1H), 6.89 (t,
J ¼ 64.7 Hz, 1H); 13C NMR (150 MHz, CD3OD) d (ppm) 163.6, 162.0, 4.2.3.4. 4-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
152.2 (d, J ¼ 11.6 Hz), 143.8 (d, J ¼ 9.4 Hz), 138.9, 137.8, 137.4, 130.4 fluoromethyl)phenyl)sulfonyl)-1H-indol-2-yl)butanoic acid (10).
(d, J ¼ 33.4 Hz), 129.9, 129.8 (d, J ¼ 2.7 Hz), 129.6, 128.2, 123.6, 123.5, Yield: 71%, white solid, purity: 96%. 1H NMR (400 MHz, CDCl3)
123.0, 122.4, 121.7, 115.6 (t, J ¼ 258.9 Hz), 115.4, 112.9, 112.8, 109.9 (d, d (ppm) 8.31 (s, 1H), 8.08 (s, 1H), 7.82 (dd, J ¼ 21.7, 7.8 Hz, 2H), 7.57
J ¼ 22.7 Hz), 104.8 (d, J ¼ 25.6 Hz); MS (ESI) m/z 530.1 [M þ H]þ. (t, J ¼ 7.9 Hz, 1H), 7.50 (d, J ¼ 8.1 Hz, 1H), 7.42 (d, J ¼ 8.1 Hz, 1H), 7.18
(d, J ¼ 13.2 Hz, 2H), 6.86 (d, J ¼ 9.1 Hz, 1H), 6.69 (t, J ¼ 73.3 Hz, 1H),
4.2.3.2. 2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- 6.51 (s, 1H), 3.11 (t, J ¼ 7.4 Hz, 2H), 2.50 (t, J ¼ 7.1 Hz, 2H), 2.18e2.11
fluoromethyl)phenyl)sulfonyl)-1H-indol-2-yl)acetic acid (8). (m, 2H); 13C NMR (150 MHz, CDCl3) d (ppm) 177.6, 164.1, 162.5,
Yield: 73%, pale yellow solid, purity: 93%. 1H NMR (400 MHz, CDCl3) 152.2 (d, J ¼ 11.6 Hz), 144.8 (d, J ¼ 9.3 Hz), 142.1, 139.8, 137.7, 135.8,
d (ppm) 8.20 (s, 1H), 8.08 (s, 1H), 8.00 (d, J ¼ 8.0 Hz, 1H), 7.82 (d, 132.1 (d, J ¼ 34.1 Hz), 130.5, 130.3, 129.9, 129.3, 123.5, 123.4 (d,
J ¼ 3.1 Hz), 121.0, 115.8 (t, J ¼ 261.4 Hz), 114.3, 113.4, 111.4 (d, (dd, J ¼ 16.3, 9.7 Hz, 2H); 13C NMR (150 MHz, CDCl3) d (ppm) 175.6,
J ¼ 22.1 Hz), 110.1, 106.1 (d, J ¼ 25.3 Hz), 32.9, 28.4, 23.9; MS (ESI) m/ 164.3, 162.6, 152.3 (d, J ¼ 10.8 Hz), 143.8 (d, J ¼ 9.4 Hz), 141.3, 139.3,
z 572.1 [M þ H]þ. 138.9, 133.0, 130.1, 129.8, 125.8, 124.9, 124.1, 117.2, 115.6 (t,
J ¼ 259.5 Hz), 114.1, 111.3 (d, J ¼ 22.4 Hz), 106.5 (d, J ¼ 24.7 Hz), 62.3,
4.2.3.5. 2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- 34.5, 30.9, 29.7; MS (ESI) m/z 560.1 [M þ H]þ.
fluoromethyl)phenyl)sulfonyl)-1H-indol-3-yl)acetic acid (11).
Yield: 75%, pale yellow solid, purity: 95%. 1H NMR (400 MHz, CDCl3) 4.2.3.10. 4-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
d (ppm) 8.19 (s, 1H), 8.12 (s, 1H), 8.07 (d, J ¼ 7.4 Hz, 1H), 7.81 (d, fluoromethyl)phenyl)sulfonyl)indolin-2-yl)butanoic acid (16).
J ¼ 6.2 Hz, 1H), 7.65 (s, 1H), 7.62e7.57 (m, 2H), 7.46 (d, J ¼ 7.4 Hz, Yield: 74%, white solid, purity: 97%. 1H NMR (400 MHz, CDCl3)
1H), 7.15 (d, J ¼ 10.1 Hz, 2H), 6.89 (d, J ¼ 8.4 Hz, 1H), 6.60 (t, d (ppm) 7.89 (s, 1H), 7.84e7.74 (m, 3H), 7.55 (t, J ¼ 6.6 Hz, 1H), 7.26 (d,
J ¼ 73.3 Hz, 1H), 3.78 (s, 2H); 13C NMR (150 MHz, CDCl3) d (ppm) J ¼ 5.7 Hz, 1H), 7.12 (q, J ¼ 14.8 Hz, 3H), 6.88 (d, J ¼ 8.0 Hz, 1H), 6.60 (t,
175.7, 164.1, 162.5, 152.2 (d, J ¼ 11.5 Hz), 144.3 (d, J ¼ 9.2 Hz), 139.0, J ¼ 72.5 Hz, 1H), 4.32 (t, J ¼ 10.4 Hz, 1H), 2.72 (dd, J ¼ 15.9, 8.8 Hz, 1H),
136.8, 135.4, 132.1 (q, J ¼ 33.9 Hz), 130.7 (d, J ¼ 2.6 Hz), 130.4, 130.4, 2.55 (d, J ¼ 16.4 Hz, 1H), 2.44e2.35 (m, 2H), 1.83e1.63 (m, 4H); 13C
129.9, 125.7, 123.9 (d, J ¼ 3.2 Hz), 123.3, 120.3, 115.6 (t, J ¼ 261.5 Hz), NMR (150 MHz, CDCl3) d (ppm) 179.0, 164.1, 162.5, 152.2 (d,
115.4, 114.4, 112.1, 111.5 (d, J ¼ 22.2 Hz), 106.3 (d, J ¼ 25.2 Hz), 60.5; J ¼ 11.6 Hz), 143.9 (d, J ¼ 9.2 Hz), 141.6, 139.3, 139.1, 132.9, 131.7 (q,
MS (ESI) m/z 544.1 [M þ H]þ. J ¼ 33.5 Hz), 130.0, 129.9, 129.7 (d, J ¼ 2.4 Hz), 125.8, 124.6, 124.1, 124.1,
116.5, 115.6 (t, J ¼ 259.5 Hz), 114.1, 111.2 (d, J ¼ 22.2 Hz), 106.4 (d,
4.2.3.6. 3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- J ¼ 25.2 Hz), 63.0, 35.7, 34.0, 33.4, 20.2; MS (ESI) m/z 574.1 [M þ H]þ.
fluoromethyl)phenyl)sulfonyl)-1H-indol-3-yl)propanoic acid (12).
Yield: 76%, pale yellow solid, purity: 94%. 1H NMR (400 MHz, CDCl3) 4.2.3.11. 2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
d (ppm) 8.17 (s, 1H), 8.12 (s, 1H), 8.03 (d, J ¼ 7.4 Hz, 1H), 7.80 (d, fluoromethyl)phenyl)sulfonyl)indolin-3-yl)acetic acid (17).
J ¼ 7.8 Hz, 1H), 7.59 (dd, J ¼ 13.6, 8.1 Hz, 2H), 7.46 (d, J ¼ 10.7 Hz, 2H), Yield: 75%, pale yellow solid, purity: 94%. 1H NMR (400 MHz, CDCl3)
7.16 (d, J ¼ 14.3 Hz, 2H), 6.89 (d, J ¼ 9.0 Hz, 1H), 6.60 (t, J ¼ 73.2 Hz, d (ppm) 8.12 (s, 1H), 8.00 (d, J ¼ 7.6 Hz, 1H), 7.86 (d, J ¼ 7.3 Hz, 1H),
1H), 3.05 (t, J ¼ 7.2 Hz, 2H), 2.78 (t, J ¼ 7.3 Hz, 2H); 13C NMR 7.81 (s, 1H), 7.65 (t, J ¼ 7.9 Hz, 1H), 7.20 (d, J ¼ 3.7 Hz, 2H), 7.14 (dd,
(150 MHz, CDCl3) d (ppm) 175.7, 164.3, 162.7, 152.2 (d, J ¼ 10.5 Hz), J ¼ 17.3, 8.0 Hz, 2H), 6.90 (d, J ¼ 9.0 Hz, 1H), 6.59 (t, J ¼ 73.0 Hz, 1H),
144.4 (d, J ¼ 9.0 Hz), 139.2, 136.7, 135.7, 132.3, 132.0, 130.7, 130.6 (d, 4.24e4.19 (m, 1H), 3.81 (dd, J ¼ 11.1, 5.6 Hz, 1H), 3.65e3.58 (m, 1H),
J ¼ 2.6 Hz), 130.3, 129.8, 123.9, 123.1, 122.3, 120.1, 115.6 (t, 2.66 (dd, J ¼ 17.2, 4.6 Hz, 1H), 2.30 (dd, J ¼ 16.9, 9.9 Hz, 1H); 13C NMR
J ¼ 261.7 Hz), 114.4, 112.3, 111.5 (d, J ¼ 22.2 Hz), 106.3 (d, J ¼ 25.1 Hz), (150 MHz, CDCl3) d (ppm) 175.5, 164.1, 162.5, 152.2 (d, J ¼ 11.2 Hz),
32.9, 20.0; MS (ESI) m/z 558.1 [M þ H]þ. 143.9 (d, J ¼ 9.2 Hz), 142.0, 140.3, 138.1, 133.8, 132.0 (d, J ¼ 33.6 Hz),
130.2 (d, J ¼ 9.5 Hz), 125.0, 124.4, 123.9, 123.6, 122.1, 115.6 (t,
4.2.3.7. 4-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- J ¼ 261.7 Hz), 114.2, 113.5, 111.3 (d, J ¼ 22.2 Hz), 106.6 (d, J ¼ 25.1 Hz),
fluoromethyl)phenyl)sulfonyl)-1H-indol-3-yl)butanoic acid (13). 56.1, 38.8, 36.0; MS (ESI) m/z 546.1 [M þ H]þ.
Yield: 72%, white solid, purity: 96%. 1H NMR (400 MHz, CDCl3)
d (ppm) 8.17 (s, 1H), 8.13 (s, 1H), 8.05 (d, J ¼ 6.4 Hz, 1H), 7.81 (d, 4.2.3.12. 3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
J ¼ 6.3 Hz, 1H), 7.60 (dd, J ¼ 17.4, 8.0 Hz, 2H), 7.45e7.41 (m, 2H), 7.16 fluoromethyl)phenyl)sulfonyl)indolin-3-yl)propanoic acid (18).
(d, J ¼ 10.9 Hz, 2H), 6.88 (d, J ¼ 7.8 Hz, 1H), 6.61 (t, J ¼ 73.2 Hz, 1H), Yield: 78%, pale yellow solid, purity: 96%. 1H NMR (400 MHz, CDCl3)
2.77 (t, J ¼ 7.1 Hz, 2H), 2.42 (t, J ¼ 6.8 Hz, 2H), 2.12e1.98 (m, 2H); 13C d (ppm) 8.12 (s, 1H), 8.00 (d, J ¼ 7.7 Hz, 1H), 7.85 (d, J ¼ 7.5 Hz, 1H),
NMR (150 MHz, CDCl3) d (ppm) 178.5, 164.1, 162.5, 152.2 (d, 7.79 (s, 1H), 7.64 (t, J ¼ 7.8 Hz, 1H), 7.20 (s, 2H), 7.13 (d, J ¼ 14.1 Hz,
J ¼ 11.7 Hz), 144.5 (d, J ¼ 9.2 Hz), 139.1, 136.5, 135.8, 132.1 (q, 2H), 6.89 (d, J ¼ 9.4 Hz, 1H), 6.59 (t, J ¼ 73.2 Hz, 1H), 4.06 (t,
J ¼ 33.9 Hz), 131.0, 130.6 (d, J ¼ 2.7 Hz), 130.3, 129.8, 123.8 (d, J ¼ 9.8 Hz, 1H), 3.71 (dd, J ¼ 10.4, 4.8 Hz, 1H), 3.30e3.23 (m, 1H), 2.33
J ¼ 3.3 Hz), 123.7, 123.1, 123.1, 120.3, 115.6 (t, J ¼ 261.5 Hz), 114.4, (t, J ¼ 7.3 Hz, 2H), 2.05e1.87 (m, 2H); MS (ESI) m/z 560.1 [M þ H]þ.
112.2, 111.5 (d, J ¼ 22.2 Hz), 106.2 (d, J ¼ 25.2 Hz), 33.1, 24.1, 23.8; MS
(ESI) m/z 572.1 [M þ H]þ. 4.2.3.13. 4-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
fluoromethyl)phenyl)sulfonyl)indolin-3-yl)butanoic acid (19).
4.2.3.8. 2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- Yield: 79%, white solid, purity: 97%. 1H NMR (400 MHz, CDCl3)
fluoromethyl)phenyl)sulfonyl)indolin-2-yl)acetic acid (14). d (ppm) 8.12 (s, 1H), 8.01 (d, J ¼ 6.7 Hz, 1H), 7.84 (d, J ¼ 6.8 Hz, 1H),
Yield: 76%, white solid, purity: 99%. 1H NMR (400 MHz, CDCl3) 7.79 (s, 1H), 7.64 (t, J ¼ 6.7 Hz, 1H), 7.19e7.12 (m, 4H), 6.88 (d,
d (ppm) 7.96 (s, 1H), 7.90 (d, J ¼ 8.1 Hz, 1H), 7.85e7.81 (m, 2H), 7.59 J ¼ 9.0 Hz, 1H), 6.59 (t, J ¼ 73.2 Hz, 1H), 4.10 (t, J ¼ 9.4 Hz, 1H),
(t, J ¼ 7.6 Hz, 1H), 7.17e7.13 (m, 4H), 6.89 (d, J ¼ 9.0 Hz, 1H), 6.59 (t, 3.73e3.70 (m, 1H), 3.21 (s, 1H), 2.33 (s, 2H), 1.62 (s, 4H); 13C NMR
J ¼ 73.1 Hz, 1H), 4.65 (t, J ¼ 9.5 Hz, 1H), 3.17 (dd, J ¼ 16.2, 3.6 Hz, 1H), (150 MHz, CDCl3) d (ppm) 178.6, 164.0, 162.4, 152.1 (d, J ¼ 11.5 Hz),
3.02 (dd, J ¼ 16.7, 9.3 Hz, 1H), 2.81e2.71 (m, 2H); 13C NMR 144.0 (d, J ¼ 9.1 Hz), 141.9, 139.6, 138.0, 135.2, 131.8 (q, J ¼ 33.6 Hz),
(150 MHz, CDCl3) d (ppm) 174.4, 164.1, 162.5, 152.2 (d, J ¼ 11.2 Hz), 130.2, 130.1, 130.0, 125.1, 124.3, 123.3, 115.6 (t, J ¼ 261.4 Hz), 114.0,
143.9 (d, J ¼ 9.1 Hz), 141.3, 139.6, 138.8, 131.9 (d, J ¼ 33.5 Hz), 131.3, 113.1, 111.2 (d, J ¼ 22.2 Hz), 106.4 (d, J ¼ 25.2 Hz), 55.8, 39.5, 34.0,
130.1 (d, J ¼ 4.2 Hz), 126.0, 124.5, 124.2 (d, J ¼ 3.2 Hz), 123.9, 122.0, 33.5, 24.7; MS (ESI) m/z 574.1 [M þ H]þ.
115.6 (t, J ¼ 261.7 Hz), 115.5, 114.1, 111.3 (d, J ¼ 22.2 Hz), 106.5 (d,
J ¼ 25.3 Hz), 59.3, 41.0, 34.5; HRMS (ESI): m/z [M þ H]þ calcd for 4.2.4. General procedure for the synthesis of compounds 20e29
C24H18F6NO5S: 546.0804, found: 546.0809. 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
4.2.3.9. 3-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- mixture was stirred at 30 C for 3e12 h. After completion of the
fluoromethyl)phenyl)sulfonyl)indolin-2-yl)propanoic acid (15). reaction, the resulting mixture was diluted with DCM and washed
Yield: 77%, white solid, purity: 96%. 1H NMR (400 MHz, CDCl3) with water. The separated aqueous phase was washed with DCM.
d (ppm) 7.84 (d, J ¼ 6.6 Hz, 2H), 7.79 (t, J ¼ 6.1 Hz, 2H), 7.54 (t, The combined organic layers were dried over MgSO4, filtered, and
J ¼ 7.8 Hz, 1H), 7.27 (d, J ¼ 7.3 Hz, 1H), 7.16 (t, J ¼ 12.6 Hz, 3H), 6.89 concentrated in vacuo. The crude mixture was purified by column
(d, J ¼ 8.9 Hz, 1H), 6.60 (t, J ¼ 73.2 Hz, 1H), 4.45 (d, J ¼ 7.0 Hz, 1H), chromatography on silica gel (petroleum: EA ¼ 8:1e1:1) to afford
2.80e2.71 (m, 1H), 2.68e2.59 (m, 2H), 2.49 (d, J ¼ 16.4 Hz, 1H), 1.98 the desired products 20e29.
4.2.4.1. 1-(2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- d (ppm) 7.93 (s, 1H), 7.90 (d, J ¼ 7.3 Hz, 1H), 7.84 (d, J ¼ 3.3 Hz, 1H),
fluoromethyl)phenyl)sulfonyl)indolin-2-yl)acetyl)azetidin-3-one (20). 7.81 (d, J ¼ 7.4 Hz, 1H), 7.59 (t, J ¼ 6.9 Hz, 1H), 7.23 (s, 1H), 7.16e7.13
Yield: 80%, white solid, purity: 96%. 1H NMR (400 MHz, CDCl3) (m, 3H), 6.88 (d, J ¼ 8.9 Hz, 1H), 6.59 (t, J ¼ 73.2 Hz, 1H), 4.75 (t,
d (ppm) 7.95 (s, 1H), 7.91 (d, J ¼ 8.1 Hz, 1H), 7.84 (s, 1H), 7.82 (d, J ¼ 8.8 Hz, 1H), 3.97e3.82 (m, 4H), 3.19e3.02 (m, 2H), 2.84 (dd,
J ¼ 7.6 Hz, 1H), 7.60 (t, J ¼ 7.7 Hz, 1H), 7.15 (dd, J ¼ 13.9, 5.7 Hz, 4H), J ¼ 11.0, 5.6 Hz, 1H), 2.68 (ddd, J ¼ 23.1, 18.5, 8.4 Hz, 3H); 13C NMR
6.89 (d, J ¼ 9.0 Hz, 1H), 6.59 (t, J ¼ 73.3 Hz, 1H), 4.99 (d, J ¼ 14.9 Hz, (150 MHz, CDCl3) d (ppm) 209.0 (d, J ¼ 14.6 Hz), 169.1 (d,
1H), 4.87 (d, J ¼ 19.0 Hz, 2H), 4.73 (d, J ¼ 13.8 Hz, 2H), 3.10e3.02 (m, J ¼ 21.2 Hz), 165.7, 164.1, 162.4, 152.2 (d, J ¼ 11.2 Hz), 143.8 (d,
2H), 2.85 (dd, J ¼ 12.4, 5.9 Hz, 1H), 2.69 (dd, J ¼ 15.5, 8.8 Hz, 1H); 13C J ¼ 9.1 Hz), 141.3, 139.5, 138.5, 131.7 (d, J ¼ 6.4 Hz), 130.1, 131.7 (d,
NMR (150 MHz, CDCl3) d (ppm) 193.8, 169.6, 165.8, 164.1, 162.5, J ¼ 7.5 Hz), 124.4, 124.1, 123.8, 122.0, 115.6 (t, J ¼ 261.5 Hz), 115.2 (d,
152.2 (d, J ¼ 11.5 Hz), 143.8 (d, J ¼ 9.3 Hz), 141.3, 139.6, 138.4, 131.5, J ¼ 17.3 Hz), 114.1, 111.2 (d, J ¼ 22.2 Hz), 106.5 (d, J ¼ 25.2 Hz), 59.9 (d,
130.1, 126.1, 124.5, 124.2, 123.8, 122.0, 115.6 (t, J ¼ 261.6 Hz), 115.1, J ¼ 12.9 Hz), 52.0, 43.4, 42.0 (d, J ¼ 35.5 Hz), 40.6, 34.9 (d,
114.1, 111.2 (d, J ¼ 22.3 Hz), 106.5 (d, J ¼ 25.1 Hz), 59.9, 40.2, 38.6, J ¼ 2.7 Hz); MS (ESI) m/z 613.1 [M þ H]þ.
34.6; MS (ESI) m/z 599.1 [M þ H]þ.
4.2.4.6. 1-(2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
4.2.4.2. 2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(trifluoromethyl)phenyl)sulfonyl)indolin-2-yl)acetyl)piperidin-4-one
fluoromethyl)phenyl)sulfonyl)indolin-2-yl)-1-(3-hydroxyazetidin-1- (25). Yield: 77%, white solid, purity: 94%. 1H NMR (400 MHz, CDCl3)
yl)ethanone (21). Yield: 65%, pale yellow solid, purity: 95%. 1H NMR d (ppm) 7.95 (s, 1H), 7.91 (d, J ¼ 7.9 Hz, 1H), 7.85 (s, 1H), 7.83 (d,
(400 MHz, CDCl3) d (ppm) 7.92 (s, 1H), 7.89 (d, J ¼ 6.6 Hz, 1H), 7.82 J ¼ 7.8 Hz, 1H), 7.61 (t, J ¼ 7.6 Hz, 1H), 7.29e7.25 (m, 1H), 7.18e7.13
(s, 1H), 7.79 (d, J ¼ 7.8 Hz, 1H), 7.58 (t, J ¼ 7.2 Hz, 1H), 7.23 (d, (m, 3H), 6.90 (d, J ¼ 8.6 Hz, 1H), 6.61 (t, J ¼ 73.2 Hz, 1H), 4.77 (dd,
J ¼ 8.0 Hz, 1H), 7.14 (d, J ¼ 9.8 Hz, 3H), 6.87 (d, J ¼ 9.0 Hz, 1H), 6.59 (t, J ¼ 12.8, 6.5 Hz, 1H), 4.05e3.99 (m, 1H), 3.87e3.63 (m, 3H), 3.22 (dd,
J ¼ 73.1 Hz, 1H), 4.69e4.64 (m, 2H), 4.42e4.17 (m, 2H), 3.96 (dddd, J ¼ 15.9, 3.0 Hz, 1H), 3.09 (dd, J ¼ 16.5, 9.4 Hz, 2H), 2.63e2.43 (m,
J ¼ 48.9, 37.9, 10.0, 3.8 Hz, 3H), 3.02e2.94 (m, 1H), 2.85e2.76 (m, 5H); 13C NMR (150 MHz, CDCl3) d (ppm) 206.2, 168.5, 165.7, 164.1,
2H), 2.48 (dd, J ¼ 14.9, 9.6 Hz, 1H); 13C NMR (150 MHz, CDCl3) 162.4, 152.1 (d, J ¼ 11.7 Hz), 143.8 (d, J ¼ 9.1 Hz), 141.3, 139.4, 138.5,
d (ppm) 169.7 (d, J ¼ 28.6 Hz), 165.8, 164.1, 162.4, 152.1 (d, 131.8, 130.1 (d, J ¼ 11.8 Hz), 126.1, 124.4, 124.1, 123.8, 122.0, 115.5 (t,
J ¼ 11.4 Hz), 143.9 (d, J ¼ 6.0 Hz), 141.2, 139.4, 138.6 (d, J ¼ 7.8 Hz), J ¼ 261.5 Hz), 115.2, 114.0, 111.2 (d, J ¼ 22.2 Hz), 106.4 (d, J ¼ 25.1 Hz),
131.9 (d, J ¼ 7.8 Hz), 130.1 (d, J ¼ 2.0 Hz), 126.1, 124.4 (d, J ¼ 4.3 Hz), 60.3, 53.6, 44.0, 38.5, 34.9; MS (ESI) m/z 627.1 [M þ H]þ.
124.1, 123.8, 122.0, 115.6 (t, J ¼ 261.4 Hz), 115.2 (d, J ¼ 11.8 Hz), 114.0,
111.2 (d, J ¼ 22.1 Hz), 106.4 (d, J ¼ 26.5 Hz), 61.1 (d, J ¼ 13.0 Hz), 60.1, 4.2.4.7. 2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
59.9 (d, J ¼ 13.0 Hz), 57.7 (d, J ¼ 22.5 Hz), 38.8 (d, J ¼ 5.0 Hz), 34.5; fluoromethyl)phenyl)sulfonyl)indolin-2-yl)-1-(4-hydroxypiperidin-1-
MS (ESI) m/z 601.1 [M þ H]þ. yl)ethanone (26). Yield: 66%, pale yellow solid, purity: 97%. 1H NMR
(400 MHz, CDCl3) d (ppm) 7.78 (dd, J ¼ 7.9, 5.1 Hz, 2H), 7.70 (d,
4.2.4.3. 1-(2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- J ¼ 7.3 Hz, 2H), 7.55e7.48 (m, 1H), 7.14 (d, J ¼ 7.2 Hz, 1H), 7.06e7.03
fluoromethyl)phenyl)sulfonyl)indolin-2-yl)acetyl)azetidine-3- (m, 3H), 6.77 (d, J ¼ 9.2 Hz, 1H), 6.54 (t, J ¼ 73.2 Hz, 1H), 4.61 (t,
carboxylic acid (22). Yield: 88%, white solid, purity: 97%. 1H NMR J ¼ 9.6 Hz, 1H), 3.96e3.87 (m, 2H), 3.57e3.51 (m, 9H), 2.65e2.46 (m,
(400 MHz, CDCl3) d (ppm) 7.92e7.88 (m, 2H), 7.82 (s, 1H), 7.79 (d, 3H); 13C NMR (150 MHz, CDCl3) d (ppm) 167.6 (d, J ¼ 6.4 Hz), 163.8,
J ¼ 7.4 Hz, 1H), 7.57 (t, J ¼ 7.5 Hz, 1H), 7.23 (d, J ¼ 8.0 Hz, 1H), 162.2, 151.9 (d, J ¼ 11.3 Hz), 143.8 (d, J ¼ 7.9 Hz), 141.1, 139.0, 138.4,
7.18e7.10 (m, 3H), 6.87 (d, J ¼ 9.0 Hz, 1H), 6.59 (t, J ¼ 73.2 Hz, 1H), 132.0, 131.5 (d, J ¼ 66.0 Hz), 130.0 (d, J ¼ 2.9 Hz), 129.8 (d,
4.69 (s, 1H), 4.28 (dd, J ¼ 54.5, 29.6 Hz, 3H), 3.47e3.12 (m, 1H), J ¼ 11.7 Hz), 126.0 (d, J ¼ 5.8 Hz), 124.1, 123.7, 121.8, 115.4 (t,
2.99e2.93 (m, 1H), 2.79 (t, J ¼ 18.8 Hz, 2H), 2.47 (dd, J ¼ 17.1, 8.5 Hz, J ¼ 261.2 Hz), 115.1, 113.8, 110.9 (d, J ¼ 22.1 Hz), 106.0 (d, J ¼ 25.2 Hz),
1H), 2.27 (dd, J ¼ 27.8, 21.0 Hz, 1H); 13C NMR (150 MHz, CDCl3) 66.0 (d, J ¼ 22.2 Hz), 60.3 (d, J ¼ 6.0 Hz), 42.6 (d, J ¼ 14.0 Hz), 40.1 (d,
d (ppm) 170.0, 164.1, 162.4, 152.2 (d, J ¼ 11.3 Hz), 143.9 (d, J ¼ 9.1 Hz), J ¼ 4.3 Hz), 38.7 (d, J ¼ 12.9 Hz), 34.6 (d, J ¼ 8.5 Hz), 34.1 (d,
141.2 (d, J ¼ 10.5 Hz), 139.4, 138.6 (d, J ¼ 6.4 Hz), 131.9, 131.7, 130.1, J ¼ 15.0 Hz), 33.4 (d, J ¼ 5.5 Hz); MS (ESI) m/z 629.1 [M þ H]þ.
130.0, 126.1, 124.5, 124.1, 123.8, 122.0, 115.6 (t, J ¼ 261.4 Hz), 115.3,
114.0, 111.2 (d, J ¼ 22.3 Hz), 106.4 (d, J ¼ 24.8 Hz), 59.9 (d, 4.2.4.8. 2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
J ¼ 18.4 Hz), 45.4, 38.4 (d, J ¼ 20.2 Hz), 34.5 (d, J ¼ 18.2 Hz), 31.9; MS fluoromethyl)phenyl)sulfonyl)indolin-2-yl)-1-morpholinoethanone
(ESI) m/z 629.1 [M þ H]þ. (27). Yield: 78%, white solid, purity: 98%. 1H NMR (400 MHz,
DMSO‑d6) d (ppm) 8.08 (t, J ¼ 7.2 Hz, 2H), 7.90 (s, 1H), 7.82 (t,
4.2.4.4. 2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- J ¼ 7.3 Hz, 1H), 7.70 (s, 1H), 7.42 (d, J ¼ 6.3 Hz, 2H), 7.37 (d, J ¼ 9.2 Hz,
fluoromethyl)phenyl)sulfonyl)indolin-2-yl)-1-(2-oxa-6-azaspiro[3.3] 1H), 7.26 (d, J ¼ 9.5 Hz, 2H), 7.18 (d, J ¼ 9.4 Hz, 1H), 3.62e3.50 (m,
heptan-6-yl)ethanone (23). Yield: 71%, white solid, purity: 96%. 1H 5H), 3.49e3.42 (m, 4H), 2.91 (dd, J ¼ 18.9, 10.1 Hz, 3H), 2.71e2.65
NMR (400 MHz, CDCl3) d (ppm) 7.88 (s, 1H), 7.84 (d, J ¼ 7.6 Hz, 1H), (m, 1H); 13C NMR (150 MHz, DMSO‑d6) d (ppm) 167.7, 163.5, 161.9,
7.76 (d, J ¼ 6.9 Hz, 2H), 7.55 (t, J ¼ 7.5 Hz, 1H), 7.20 (d, J ¼ 7.4 Hz, 1H), 152.2 (d, J ¼ 12.2 Hz), 143.3 (d, J ¼ 9.2 Hz), 140.8, 137.8, 137.7, 132.7,
7.11e7.08 (m, 3H), 6.84 (d, J ¼ 8.8 Hz, 1H), 6.57 (t, J ¼ 73.2 Hz, 1H), 131.4, 130.7, 130.5, 129.6 (d, J ¼ 21.9 Hz), 126.4, 124.3, 123.2, 116.0 (t,
4.60 (d, J ¼ 8.0 Hz, 1H), 4.21 (ddd, J ¼ 50.4, 38.3, 9.7 Hz, 3H), 3.62 (d, J ¼ 258.5 Hz), 114.2, 112.7, 110.2 (d, J ¼ 22.3 Hz), 105.3 (d,
J ¼ 4.5 Hz, 4H), 2.95 (dd, J ¼ 16.9, 9.4 Hz, 1H), 2.76 (dd, J ¼ 22.3, J ¼ 25.5 Hz), 65.8, 60.3, 45.2, 41.2, 34.1; MS (ESI) m/z 615.1 [M þ H]þ.
10.4 Hz, 2H), 2.41 (dd, J ¼ 15.1, 9.0 Hz, 1H), 2.21 (s, 1H); 13C NMR
(150 MHz, CDCl3) d (ppm) 169.5, 164.0, 162.3, 152.1 (d, J ¼ 11.2 Hz), 4.2.4.9. 1-(2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri-
143.8 (d, J ¼ 9.1 Hz), 141.1, 139.3, 138.5, 131.8, 130.1, 130.0, 126.0, fluoromethyl)phenyl)sulfonyl)indolin-2-yl)acetyl)piperidine-4-
124.3, 123.9 (d, J ¼ 2.5 Hz), 123.7, 121.9, 115.5 (t, J ¼ 261.5 Hz), 115.0, carboxylic acid (28). Yield: 86%, white solid, purity: 96%. 1H NMR
113.9, 111.1 (d, J ¼ 22.2 Hz), 106.3 (d, J ¼ 25.3 Hz), 80.6, 80.5, 59.9, (400 MHz, CDCl3) d (ppm) 7.93 (s, 1H), 7.90 (d, J ¼ 7.8 Hz, 1H), 7.84 (s,
59.6, 57.3, 38.6, 37.6, 34.5; MS (ESI) m/z 627.1 [M þ H]þ. 1H), 7.80 (d, J ¼ 7.3 Hz, 1H), 7.58 (t, J ¼ 7.2 Hz, 1H), 7.23 (s, 1H),
7.16e7.13 (m, 3H), 6.88 (d, J ¼ 8.2 Hz, 1H), 6.59 (t, J ¼ 73.1 Hz, 1H),
4.2.4.5. 1-(2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- 4.77e4.69 (m, 1H), 4.39 (dd, J ¼ 25.6, 13.1 Hz, 1H), 3.87e3.77 (m,
fluoromethyl)phenyl)sulfonyl)indolin-2-yl)acetyl)pyrrolidin-3-one 1H), 3.22e2.90 (m, 3H), 2.85e2.59 (m, 4H), 2.01 (t, J ¼ 14.7 Hz, 2H),
(24). Yield: 85%, white solid, purity: 95%. 1H NMR (400 MHz, CDCl3) 1.80e1.59 (m, 2H); 13C NMR (150 MHz, CDCl3) d (ppm) 179.1 (d,
J ¼ 15.9 Hz), 168.2 (d, J ¼ 14.9 Hz), 164.1, 162.5, 152.2 (d, J ¼ 11.3 Hz), 4.2.5.3. 2-(6-(2,3-Dihydrobenzofuran-5-yl)-1-((3-(trifluoromethyl)
143.9 (d, J ¼ 9.0 Hz), 141.4, 139.4, 138.7, 132.0, 131.8 (d, J ¼ 33.7 Hz), phenyl)sulfonyl)indolin-2-yl)acetic acid (32). Yield: 79%, white
130.0 (d, J ¼ 15.2 Hz), 126.1 (d, J ¼ 7.2 Hz), 124.3, 124.1, 123.8, 122.0, solid, purity: 96%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.97 (d,
115. 6 (d, J ¼ 261.5 Hz), 115.3, 114.1, 111.2 (d, J ¼ 22.2 Hz), 106.4 (d, J ¼ 8.6 Hz, 1H), 7.86 (dd, J ¼ 22.4, 8.4 Hz, 2H), 7.79 (t, J ¼ 8.2 Hz, 1H),
J ¼ 25.2 Hz), 60.4 (d, J ¼ 1.9 Hz), 44.7 (d, J ¼ 10.8 Hz), 40.8 (d, 7.56 (q, J ¼ 8.2 Hz, 1H), 7.43 (d, J ¼ 8.3 Hz, 1H), 7.34 (t, J ¼ 8.4 Hz, 1H),
J ¼ 11.8 Hz), 34.9 (d, J ¼ 5.6 Hz), 28.1 (d, J ¼ 15.2 Hz), 27.5 (d, 7.23 (d, J ¼ 9.2 Hz, 1H), 7.08 (t, J ¼ 8.3 Hz, 1H), 6.86 (t, J ¼ 8.7 Hz, 1H),
J ¼ 4.4 Hz); MS (ESI) m/z 657.1 [M þ H]þ. 4.63 (q, J ¼ 8.8 Hz, 3H), 3.29 (q, J ¼ 8.6 Hz, 2H), 3.20e3.12 (m, 1H),
3.00e2.92 (m, 1H), 2.81e2.64 (m, 2H); 13C NMR (150 MHz, CDCl3)
4.2.4.10. 2-(6-(3-(Difluoromethoxy)-5-fluorophenyl)-1-((3-(tri- d (ppm) 162.0, 142.2, 141.3, 138.5, 133.2, 129.8 (d, J ¼ 39.0 Hz), 130.1,
fl u o r o m e t h y l ) p h e n y l ) s u l f o n y l ) i n d o l i n - 2 - y l ) - 1 - ( 1,1 - 130.0, 129.9, 129.8 (d, J ¼ 2.5 Hz), 129.7, 129.1, 127.8, 127.1, 125.6,
dioxidothiomorpholino)ethanone (29). Yield: 82%, pale yellow solid, 124.2 (d, J ¼ 10.1 Hz), 123.8, 115.3, 109.5, 71.6, 59.3, 40.9, 34.4, 31.9;
purity: 95%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.95 (s, 1H), 7.90 (d, MS (ESI) m/z 504.1 [M þ H]þ.
J ¼ 7.3 Hz, 1H), 7.84 (s, 2H), 7.61 (t, J ¼ 6.7 Hz, 1H), 7.27 (d,
J ¼ 10.2 Hz, 1H), 7.15 (dd, J ¼ 9.1, 4.6 Hz, 3H), 6.90 (d, J ¼ 8.2 Hz, 1H), 4.2.5.4. 2-(6-(3,5-Difluorophenyl)-1-((3-(trifluoromethyl)phenyl)sul-
6.62 (t, J ¼ 73.2 Hz, 1H), 4.74 (s, 1H), 4.31e4.21 (m, 1H), 4.09e3.92 fonyl)indolin-2-yl)acetic acid (33). Yield: 77%, pale yellow solid,
(m, 3H), 3.24e2.96 (m, 8H); 13C NMR (150 MHz, CDCl3) d (ppm) purity: 94%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.96 (s, 1H), 7.89 (d,
168.5, 165.7, 164.1, 162.4, 152.2 (d, J ¼ 11.3 Hz), 143.6 (d, J ¼ 9.1 Hz), J ¼ 7.8 Hz, 1H), 7.85 (s, 1H), 7.82 (d, J ¼ 7.6 Hz, 1H), 7.59 (t, J ¼ 7.9 Hz,
141.1, 139.6, 138.3, 131.8 (d, J ¼ 33.6 Hz), 131.6, 130.2, 130.0, 126.1, 1H), 7.32e7.28 (m, 1H), 7.15 (d, J ¼ 7.9 Hz, 1H), 7.11 (d, J ¼ 7.1 Hz, 2H),
124.5, 124.1, 115.6 (d, J ¼ 261.5 Hz), 115.1, 114.0, 111.2 (d, J ¼ 22.2 Hz), 6.83 (t, J ¼ 8.7 Hz, 1H), 4.65 (t, J ¼ 8.9 Hz, 1H), 3.17 (dd, J ¼ 16.5,
106.5 (d, J ¼ 25.2 Hz), 60.2, 51.8 (d, J ¼ 17.3 Hz), 44.0, 40.1 (d, 3.9 Hz, 1H), 3.01 (dd, J ¼ 16.8, 9.3 Hz, 1H), 2.81e2.70 (m, 2H); 13C
J ¼ 12.4 Hz), 34.8; MS (ESI) m/z 663.1 [M þ H]þ. NMR (150 MHz, CDCl3) d (ppm) 174.5, 164.2 (d, J ¼ 13.5 Hz), 162.5 (d,
J ¼ 13.3 Hz), 143.7 (d, J ¼ 13.3 Hz), 141.3, 139.6, 138.6, 131.8 (d,
J ¼ 13.3 Hz), 131.2, 130.1, 129.9, 127.6 (d, J ¼ 48.0 Hz), 126.0, 124.4,
4.2.5. The synthesis of compounds 30e39
124.2 (d, J ¼ 3.4 Hz), 123.8, 122.0, 115.5, 110.1 (dd, J ¼ 20.6, 5.1 Hz),
102.9 (t, J ¼ 25.3 Hz), 59.2, 40.9, 34.5; MS (ESI) m/z 498.1 [M þ H]þ.
Step 1. To a vial were added intermediate 14b (1.0 eq), bis(pina-
colato)diboron (1.2 eq), Pd(dppf)Cl2 (5 mol%, 0.05 eq), KOAc (3.0 eq) 4.2.5.5. 2-(6-(3-Chloro-5-fluorophenyl)-1-((3-(trifluoromethyl)
and 1,4-dioxane (3 mL). Then the reaction mixture was stirred phenyl)sulfonyl)indolin-2-yl)acetic acid (34). Yield: 73%, pale yellow
under N2 atmosphere at 100 C for 12 h. After completion of the solid, purity: 97%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.97 (s, 1H),
reaction, the resulting mixture was diluted with EA and washed 7.89 (d, J ¼ 7.9 Hz, 1H), 7.85 (s, 1H), 7.82 (d, J ¼ 8.0 Hz, 1H), 7.59 (t,
with water. The separated aqueous phase was washed with EA. The J ¼ 7.8 Hz, 1H), 7.37 (s, 1H), 7.24 (s, 1H), 7.19 (d, J ¼ 9.7 Hz, 1H), 7.15 (d,
combined organic layers were dried over MgSO4, filtered, and J ¼ 8.0 Hz, 1H), 7.11 (d, J ¼ 8.1 Hz, 1H), 4.65 (t, J ¼ 9.2 Hz, 1H), 3.17 (dd,
concentrated in vacuo. The crude mixture was purified by column J ¼ 16.7, 3.8 Hz, 1H), 3.01 (dd, J ¼ 16.6, 9.3 Hz, 1H), 2.81e2.71 (m, 2H);
13
chromatography on silica gel (petroleum: EA ¼ 10:1e5:1) to afford C NMR (150 MHz, CDCl3) d (ppm) 175.1, 163.0 (d, J ¼ 249.6 Hz),
the desired intermediate 30a. 143.7 (d, J ¼ 8.6 Hz), 141.3, 139.4, 138.6, 135.5 (d, J ¼ 10.9 Hz), 131.9 (d,
Following the Step 2 and Step 3 of the general procedure (4.2.3) J ¼ 33.5 Hz), 131.3, 130.1 (d, J ¼ 4.1 Hz), 128.9 127.2, 126.0, 124.5, 124.2
used for the preparation of 7e19, the desired products 30e39 were (d, J ¼ 2.9 Hz), 123.3, 122.0, 115.5, 115.3 (d, J ¼ 24.9 Hz), 112.7 (d,
obtained using substituted bromobenzene (1.0 eq) and intermedi- J ¼ 22.2 Hz), 59.2, 41.0, 34.5; MS (ESI) m/z 514.1 [M þ H]þ.
ate 30a (1.2 eq) as the starting materials.
4.2.5.6. 2-(6-(3-Fluoro-5-(trifluoromethyl)phenyl)-1-((3-(tri-
fluoromethyl)phenyl)sulfonyl)indolin-2-yl)acetic acid (35).
4.2.5.1. 2-(6-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-1-((3-(tri- Yield: 78%, pale yellow solid, purity: 96%. 1H NMR (400 MHz, CDCl3)
fluoromethyl)phenyl)sulfonyl)indolin-2-yl)acetic acid (30). d (ppm) 7.97 (s, 1H), 7.90 (d, J ¼ 7.6 Hz, 1H), 7.87 (s, 1H), 7.82 (d,
Yield: 85%, white solid, purity: 97%. 1H NMR (400 MHz, CDCl3) J ¼ 7.5 Hz, 1H), 7.60 (d, J ¼ 12.2 Hz, 2H), 7.47 (d, J ¼ 9.2 Hz, 1H), 7.34
d (ppm) 7.96 (s, 1H), 7.90 (d, J ¼ 7.6 Hz, 1H), 7.82 (d, J ¼ 7.2 Hz, 2H), (d, J ¼ 8.0 Hz, 1H), 7.29 (d, J ¼ 6.9 Hz, 1H), 7.18 (d, J ¼ 7.7 Hz, 1H), 4.66
7.59 (t, J ¼ 7.4 Hz, 1H), 7.29 (d, J ¼ 9.0 Hz, 2H), 7.22 (d, J ¼ 7.5 Hz, 1H), (t, J ¼ 9.6 Hz, 1H), 3.18 (dd, J ¼ 16.7, 3.6 Hz, 1H), 3.03 (dd, J ¼ 16.9,
7.15 (d, J ¼ 4.2 Hz, 2H), 4.68e4.61 (m, 1H), 3.18 (d, J ¼ 16.7 Hz, 1H), 9.5 Hz, 1H), 2.82e2.69 (m, 2H); 13C NMR (150 MHz, CDCl3) d (ppm)
3.00 (dd, J ¼ 16.6, 9.4 Hz, 1H), 2.81e2.70 (m, 2H); 13C NMR 175.0, 162.8 (d, J ¼ 247.8 Hz), 143.8 (d, J ¼ 7.3 Hz), 141.4, 139.2, 138.6,
(150 MHz, CDCl3) d (ppm) 175.3, 144.3, 143.4, 141.2, 140.6, 138.7, 133.1 (d, J ¼ 26.4 Hz), 131.9 (d, J ¼ 33.6 Hz), 131.5, 130.1, 130.1, 129.9,
137.0, 133.4, 131.9, 131.7 (d, J ¼ 2.7 Hz), 130.4, 130.1, 130.0, 125.9, 126.1, 124.6, 124.2, 123.8, 122.2 (d, J ¼ 52.6 Hz), 119.8, 117.6 (d,
124.5, 124.2 (d, J ¼ 2.7 Hz), 123.8, 122.6, 122.0, 115.6, 109.7, 108.5, J ¼ 21.8 Hz), 115.57, 111.9 (d, J ¼ 24.3H), 59.2, 41.0, 34.5; MS (ESI) m/z
59.2, 41.1, 34.5; MS (ESI) m/z 542.1 [M þ H]þ. 548.1 [M þ H]þ.
4.2.5.2. 2-(6-(Benzo[d][1,3]dioxol-5-yl)-1-((3-(trifluoromethyl) 4.2.5.7. 2-(6-(3-Fluoro-5-methoxyphenyl)-1-((3-(trifluoromethyl)

phenyl)sulfonyl)indolin-2-yl)acetic acid (31). Yield: 82%, white solid, phenyl)sulfonyl)indolin-2-yl)acetic acid (36). Yield: 77%, white solid,
purity: 95%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.97 (s, 1H), 7.89 (d, purity: 95%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.97 (s, 1H), 7.89 (d,
J ¼ 7.7 Hz, 1H), 7.83 (s, 1H), 7.80 (d, J ¼ 7.6 Hz, 1H), 7.57 (t, J ¼ 7.8 Hz, J ¼ 8.0 Hz, 1H), 7.87 (s, 1H), 7.81 (d, J ¼ 7.8 Hz, 1H), 7.58 (t, J ¼ 7.8 Hz,
1H), 7.22 (d, J ¼ 7.8 Hz, 1H), 7.08 (dd, J ¼ 8.9, 7.8 Hz, 3H), 6.90 (d, 1H), 7.28 (s, 1H), 7.13 (d, J ¼ 7.8 Hz, 1H), 6.89 (d, J ¼ 12.9 Hz, 2H), 6.64
J ¼ 8.4 Hz, 1H), 6.02 (s, 2H), 4.64 (t, J ¼ 9.4 Hz, 1H), 3.16 (dd, J ¼ 16.6, (d, J ¼ 10.5 Hz, 1H), 4.65 (t, J ¼ 9.5 Hz, 1H), 3.87 (s, 3H), 3.17 (dd,
3.6 Hz, 1H), 2.97 (dd, J ¼ 16.5, 9.3 Hz, 1H), 2.77 (dd, J ¼ 16.5, 10.0 Hz, J ¼ 16.7, 3.8 Hz, 1H), 3.00 (dd, J ¼ 16.8, 9.4 Hz, 1H), 2.78 (dd, J ¼ 16.7,
1H), 2.69 (d, J ¼ 17.0 Hz, 1H); 13C NMR (150 MHz, CDCl3) d (ppm) 10.1 Hz, 1H), 2.71 (d, J ¼ 17.0 Hz, 1H); 13C NMR (150 MHz, CDCl3)
174.7, 148.2, 147.4, 141.6, 140.9, 138.7, 134.8, 131.8 (d, J ¼ 33.4 Hz), d (ppm) 175.5, 163.9 (d, J ¼ 245.0 Hz), 161.2 (d, J ¼ 11.6 Hz), 143.1 (d,
130.1, 130.0, 129.9, 129.8 (d, J ¼ 1.7 Hz), 129.6, 125.7, 124.2 (d, J ¼ 9.7 Hz), 141.0, 140.8, 138.7, 131.9 (q, J ¼ 66.0 Hz), 130.7, 130.1 (d,
J ¼ 3.3 Hz), 124.2, 120.8, 115.5, 108.6, 107.7, 101.3, 59.2, 41.0, 34.4; MS J ¼ 9.0 Hz), 130.0, 125.8, 124.5, 124.2, 123.8, 122.0, 115.6, 109.0, 106.5
(ESI) m/z 506.1 [M þ H]þ. (d, J ¼ 22.7 Hz), 100.6 (d, J ¼ 25.1 Hz), 59.2, 55.7, 41.1, 34.5; MS (ESI)
m/z 510.1 [M þ H]þ. for 1 h at room temperature and then read on Envision in LANCE
mode configured for europeum-APC labels.
4.2.5.8. 2-(6-(3-Ethoxy-5-fluorophenyl)-1-((3-(trifluoromethyl)
phenyl)sulfonyl)indolin-2-yl)acetic acid (37). Yield: 72%, white solid, 4.3.2. RORgt GAL4 reporter gene assay
purity: 97%. 1H NMR (400 MHz, DMSO‑d6) d (ppm) 8.09 (dd, J ¼ 18.3, hRORgt LBD coding sequence was inserted into a pBIND
7.1 Hz, 2H), 7.92 (s, 1H), 7.81 (t, J ¼ 7.4 Hz, 1H), 7.65 (s, 1H), 7.37 (d, expression vector (Promega, E1581) to express ROR-GAL4 binding
J ¼ 7.5 Hz, 1H), 7.24 (d, J ¼ 7.1 Hz, 1H), 6.98 (d, J ¼ 9.9 Hz, 1H), 6.94 (s, domain chimeric receptors. This expression vector and a reporter
1H), 6.85 (d, J ¼ 10.0 Hz, 1H), 4.71 (t, J ¼ 6.9 Hz, 1H), 4.12 (q, vector (pGL4.35 which carries a stably integrated GAL4 promoter
J ¼ 6.0 Hz, 2H), 3.00e2.93 (m, 1H), 2.80 (t, J ¼ 14.7 Hz, 2H), driven luciferase reporter gene [luc2P/9XGAL4 UAS/Hygro]) were
2.71e2.65 (m, 1H), 1.36 (t, J ¼ 6.0 Hz, 3H); 13C NMR (150 MHz, co-transfected into HEK293T host cells. Upon agonist binding to the
DMSO‑d6) d (ppm) 171.3, 163.2 (d, J ¼ 242.6 Hz), 160.2 (d, corresponding ROR-GAL4 chimeric receptor, the chimeric receptor
J ¼ 12.1 Hz), 142.6 (d, J ¼ 10.3 Hz), 140.6, 138.8, 137.6, 131.9, 131.3, binds to the GAL4 binding sites and stimulates the reporter gene. In
130.8, 130.4, 129.6 (d, J ¼ 21.9 Hz), 126.1, 124.0, 123.3 (d, J ¼ 2.5 Hz), the present of inverse agonist, agonist will bind competitively to
122.0, 114.0, 109.1, 105.3 (d, J ¼ 22.7 Hz), 100.8 (d, J ¼ 25.0 Hz), 63.6, the nuclear receptor and activate the reporter gene transcription.
59.5, 41.1, 33.7, 14.3; MS (ESI) m/z 524.1 [M þ H]þ. HEK293T cells were cultured in a culture medium composed of
DMEM containing 5% charcoal-treated FBS at 37 C under 5% CO2
4.2.5.9. 2-(6-(3-Fluoro-5-isopropoxyphenyl)-1-((3-(trifluoromethyl) atmosphere, as ATCC recommended. Before assay, the cells were
phenyl)sulfonyl)indolin-2-yl)acetic acid (38). Yield: 70%, white solid, washed with PBS to remove phenol red and suspended in phenol
purity: 98%. 1H NMR (400 MHz, DMSO‑d6) d (ppm) 7.94 (s, 1H), 7.87 red-free medium (phenol red-free DMEM containing 5% charcoal-
(d, J ¼ 7.9 Hz, 1H), 7.83 (s, 1H), 7.78 (d, J ¼ 7.7 Hz, 1H), 7.55 (t, treated FBS and Penicillin-Streptomycin (10000 U/mL) to a proper
J ¼ 7.8 Hz, 1H), 7.23 (d, J ¼ 7.7 Hz, 1H), 7.09 (d, J ¼ 7.8 Hz, 1H), 6.88 (s, concentration. 6 106 HEK293T cells were seeded into a 100 mm
1H), 6.83 (d, J ¼ 9.3 Hz, 1H), 6.59 (d, J ¼ 10.7 Hz, 1H), 4.66e4.55 (m, dish and incubated for 16 h. To a reagent mixture of Trans-IT re-
2H), 3.08 (dd, J ¼ 16.5, 3.6 Hz, 1H), 2.95 (dd, J ¼ 16.8, 9.2 Hz, 1H), agent and Opti-MEM (Invitrogen) was added plasmid DNA (used as
2.69 (dd, J ¼ 13.4, 6.7 Hz, 2H), 1.37 (s, 3H), 1.36 (s, 3H); 13C NMR 0.5 mg/mL stocks), containing 5 mg RORg plasmid and 5 mg pGL4.35
(150 MHz, DMSO‑d6) d (ppm) 172.9, 163.8 (d, J ¼ 244.5 Hz), 159.4 (d, luciferase plasmid. The mixture was added to the cells in the
J ¼ 11.5 Hz), 143.1 (d, J ¼ 9.9 Hz), 141.1, 140.6, 138.8, 131.9 (q, 100 mm dish and incubated for 5e6 h. Test compounds were seri-
J ¼ 67.5 Hz), 131.0, 130.0 (d, J ¼ 20.2 Hz), 129.8 (d, J ¼ 2.7 Hz), 125.7, ally diluted in DMSO to 5e6 doses. LYC-55716 was used as the
124.3, 124.1 (d, J ¼ 3.2 Hz), 123.8, 122.0, 115.6, 110.8, 106.2 (d, positive control and 100% DMSO was used as vehicle control.
J ¼ 22.6 Hz), 102.0 (d, J ¼ 24.8 Hz), 70.5, 59.5, 41.2, 34.4, 21.9 (d, Compounds (25 nL) were transferred into a 384-well plate (white
J ¼ 4.8 Hz); MS (ESI) m/z 538.1 [M þ H]þ. opaque) using Echo550. Then seeded the cells at 15,000 cells/well
into the 384-well plate using phenol red-free DMEM containing 5%
4.2.5.10. 2-(6-(3-(Trifluoromethoxy)phenyl)-1-((3-(trifluoromethyl) charcoal-treated FBS and 0.25 mM ursolic acid. Cells were incubated
phenyl)sulfonyl)indolin-2-yl)acetic acid (39). Yield: 76%, white solid, for 16e20 h at 37 C under 5% CO2 atmosphere. 25 mL of Steady-
purity: 96%. 1H NMR (400 MHz, CDCl3) d (ppm) 7.96 (s, 1H), 7.91 (d, Glo™ Luciferase Assay Reagent was added into each well of the
J ¼ 7.8 Hz, 1H), 7.87 (s, 1H), 7.80 (d, J ¼ 7.4 Hz, 1H), 7.58 (t, J ¼ 7.8 Hz, 384-well plate. Shake the plate (avoiding light) for 5 min on a plate
1H), 7.50 (dd, J ¼ 20.0, 7.5 Hz, 2H), 7.42 (s, 1H), 7.31e7.23 (m, 2H), shaker. Record the luminescence value on Envision 2104 plate
7.15 (d, J ¼ 7.7 Hz, 1H), 4.69e4.67 (m, 1H), 3.16 (d, J ¼ 16.0 Hz, 1H), reader. EC50 values were determined by the nonlinear regression
3.00 (dd, J ¼ 16.6, 9.2 Hz, 1H), 2.76 (dd, J ¼ 27.6, 13.3 Hz, 2H); 13C analysis of dose-response curves.
NMR (150 MHz, CDCl3) d (ppm) 175.4, 149.7, 142.6, 141.2, 140.3,
138.7, 131.8 (d, J ¼ 33.5 Hz), 130.9, 130.1 (d, J ¼ 6.3 Hz), 130.0, 129.9, 4.4. Aqueous solubility determination
128.9, 127.2, 125.9, 125.6, 124.6, 124.2 (d, J ¼ 2.4 Hz), 121.7 (d,
J ¼ 98.1 Hz), 119.9, 119.8, 115.7, 59.4, 41.3, 34.5; MS (ESI) m/z 546.1 Compounds 1e3, 14 were dissolved in DMSO to a concentration
[M þ H]þ. of 10 mM as the stock solutions. These solutions were diluted into
PBS buffer (pH 7.46, 100 mM, with 3.3 mM MgCl2) to a final com-
4.3. Biological assays pound concentration of 100 mM. The samples were incubated at
37 C in water bath for 120 min, followed by filtration. The filtrates
4.3.1. RORg dual FRET assay were then diluted with 70% ACN as needed. To the dilutions was
The assay was performed in an assay buffer consisting of 50 mM added an internal standard solution meanwhile as stop solution.
NaF, 50 mM 3-(N-morpholino)propanesulfonic acid (pH 7.4), LC-MS/MS was used to determine compound concentrations in the
0.05 mM 3-[(3-cholamidopropyl) dimethylammonio]propane- prepared samples. Ketoconazole and nicardipine were tested as the
sulfonate, 0.1 mg/mL bovine serum albumin, and 10 mM dithio- control with solubility of 31.1 mM and 5.01 mM, respectively.
threitol in 384-well plates. The total volume was 25 mL/well. The
europium-labeled SRC1 solution was prepared by adding an 4.5. Microsomal stability assay
appropriate amount of biotinylated SRC and europium labeled
streptavidin into assay buffer, with final concentrations of 20 and Mouse liver microsomes (0.5 mg/mL), PBS and NADPH cofactors
10 nM, respectively. The allophycocyanin (APC)-labeled-LBD solu- were added to the incubation system. The system was pre-
tion was prepared by adding an appropriate amount of biotinylated incubated for 10 min at 37 C, and then test compounds were
RORc-LBD and APC-labeled streptavidin at final concentrations of added to start the reaction at a final concentration of 1 mM. The
20 and 10 nM, respectively. After 15 min of incubation at room reaction was then evaluated at 0, 5, 10, 20, 30 and 60 min and was
temperature, a 20-fold excess of biotin was added and incubated for terminated by the addition of acetonitrile. Samples were centri-
10 min at room temperature to block the remaining free strepta- fuged for 20 min at 4000 rpm at 4 C, and the supernatant was
vidin. Equal volumes of europium-labeled SRC and APC-labeled analysed using HPLC-MS/MS. Percentage of the parent remaining
RORc-LBD were dispensed into 384-well assay plates at 25 mL vol- was calculated considering the percent parent area at 0 min as
ume/well. The 384-well assay plates had 100 nL of test compound 100%, and the peak areas at other time points are converted into
in DMSO predispensed into each well. The plates were incubated corresponding residual amounts according to the control. t1/2 and
CLint (mic) were calculated by equations as follow:
Ct ¼ C0 eke t
1
when Ct ¼ C0 ;
2
Ln2 0:693
T1=2 ¼ ¼
ke ke
0:693 1
CLintðmicÞ ¼
In vitro T1=2 mg=mL microsomal protein in reaction system
mg microsomes g liver
CLintðliverÞ ¼ CLintðmicÞ
g liver kg body weight
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European Journal of Medicinal Chemistry: Yan Zhu, Nannan Sun, Mingcheng Yu, Huimin Guo, Qiong Xie, Yonghui Wang

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European Journal of Medicinal Chemistry 182 (2019) 111589

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

Discovery of aryl-substituted indole and indoline derivatives as RORgt

1. Introduction that promote the differentiation and activation of Th17 and

RORgt agonists remain limited. Lycera's N-arylsulfonylbenzoxazine compound 2 (LYC-55716, Figs. 2

EC50 (nM)b %Maxc

1 (Scripps' agonist) 5.98 30.2 ± 4.2 118 2.9

4 6.95 99.4 ± 44.7 125 39.1

6 6.17 122 ± 101 75 -e

2 6.76/5.32 30.1 ± 2.9 152 >145 102 ± 4.9 141

14 1 5.83/4.39 20.8 ± 1.5 127 113 247 ± 33.1 133

EC50 (nM)b %Maxc EC50 (nM)b Emax (%)

16 6.61 25.2 ± 3.4 117 93.3 422 ± 43.2 146

20 4.88 78.8 ± 13.7 130 -f -f -f

21 4.92 244 ± 17.4 141 e e e

22 5.07 49.3 ± 21.6 138 65.9 4050 ± 459 100

23 4.93 47.9 ± 3.0 128 0.7 752 ± 100 104

24 5.13 52.3 ± 18.5 136 e e e

25 5.38 80.4 ± 1.0 136 e e e

26 5.05 106 ± 9.7 127 0.7 6040 ± 1010 100

27 5.30 209 ± 90.6 128 e e e

28 5.71 57.1 ± 6.8 116 30.1 5440 ± 3360 178

29 5.03 43.4 ± 19.9 126 2.0 322 ± 51.4 104

Table 4 4.2.2.1. 6-(3-(Diﬂuoromethoxy)-5-ﬂuorophenyl)-1-((3-(tri-

37 5.36/3.92 32.9 ± 10.9 96 4.2.2.4. 6-(3-(Diﬂuoromethoxy)-5-ﬂuorophenyl)-1-((3-(tri-

Compds MW CLogP/CLogDa Solubilityb (mM) MLM Stabilityc t1/2 (min) CLint(mic)d

1 (Scripps' agonist) 485.87 5.98/5.98 0.08 2.9 482

4.2.5.2. 2-(6-(Benzo[d][1,3]dioxol-5-yl)-1-((3-(triﬂuoromethyl) 4.2.5.7. 2-(6-(3-Fluoro-5-methoxyphenyl)-1-((3-(triﬂuoromethyl)

CLint (mic) were calculated by equations as follow:

4.6. Mouse PK study References

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