Discovery of tert-amine-based RORγt agonists

Article abstract of DOI:10.1016/j.ejmech.2021.113704

The nuclear receptor retinoic acid receptor-related orphan receptor gamma-t (RORγt) is a transcription factor regulating Th17 cell differentiation and proliferation from naive CD4⁺ T cells. Since Th17 cells have demonstrated the antitumor efficacy by eliciting remarkable activation of CD8⁺ T cells, RORγt agonists could be applied as potential small molecule therapeutics for cancer immunotherapy. Based on the previously reported RORγt agonist 1 and its resolved co-crystal structure, a series of new tertiary amines were designed, synthesized and biologically evaluated, yielding optimal moieties with improved chemical properties and biological responses. The combination of these optimal moieties resulted in identification of novel RORγt agonists such as 8b with further elevated RORγt agonism responses at a target-based level as well as in cell-based assays, which provided some structural knowledge for further optimization of RORγt agonists as small molecule therapeutics for cancer immunotherapy.

Full text of DOI:10.1016/j.ejmech.2021.113704

European Journal of Medicinal Chemistry 224 (2021) 113704
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Discovery of tert-amine-based RORgt agonists
,
,
Ruomeng Qiu a 1, Mingcheng Yu a 1, Juwen Gong a, Jinlong Tian a, Yafei Huang a,
Yonghui Wang a *, Qiong Xie a
,
, b, **
a Department of Medicinal Chemistry, School of Pharmacy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China
b Fudan Zhangjiang Institute, Shanghai, 201203, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The nuclear receptor retinoic acid receptor-related orphan receptor gamma-t (RORgt) is a transcription
factor regulating Th17 cell differentiation and proliferation from naive CD4þ T cells. Since Th17 cells have
Received 19 April 2021
Received in revised form
4 July 2021
Accepted 12 July 2021
Available online 14 July 2021
demonstrated the antitumor efficacy by eliciting remarkable activation of CD8þ T cells, ROR
could be applied as potential small molecule therapeutics for cancer immunotherapy. Based on the
previously reported ROR t agonist 1 and its resolved co-crystal structure, a series of new tertiary amines
gt agonists
g
were designed, synthesized and biologically evaluated, yielding optimal moieties with improved
chemical properties and biological responses. The combination of these optimal moieties resulted in
Keywords:
RORgt agonists
Tertiary amines
Th17 cell differentiation
Cancer immunotherapy
Small molecule drugs
identification of novel ROR
target-based level as well as in cell-based assays, which provided some structural knowledge for further
optimization of ROR t agonists as small molecule therapeutics for cancer immunotherapy.
© 2021 Elsevier Masson SAS. All rights reserved.
gt agonists such as 8b with further elevated RORgt agonism responses at a
g
1. Introduction
(ROR
is the key transcription factor to drive naive CD4þ T cells differen-
tiated into Th17 cells [13,14]. Similar to other NR modulators, ROR
orthosteric ligands can display divergent mode of actions by
inducing conformational change of the ligand binding domain
(LBD) [14e21]. From the perspective of RORgt LBD promoting or
reducing co-activator recruitment, the ligands can be classified as
agonists, inverse agonists and neutral antagonists [22,23]. Since
gt) is a member of nuclear receptor (NR) superfamily, and it
Cancer immunotherapy aims to exploit the immune system to
attack tumors [1]. The immune suppression response of tumor can
be altered by activating CD8þ T cells through various mechanisms,
including enzymes, receptors and cytokine signaling pathways
[2e4]. T helper 17 (Th17) cells, a subset of CD4þ T helper cells
characterized by interleukin-17A (IL-17A) production, have been
well-described in pathological processes of various human auto-
immune diseases [5,6]. Recent study shows that Th17 cells and IL-
17A expression have been found in a myriad of human tumors
[7e11]. It has been demonstrated that Th17 cells have potential
antitumor efficacy by triggering residential immunity responses to
elicit remarkable activation of CD8þ T cells [12].
gt
ROR
g
t agonists have been shown to increase basal activity of ROR
gt
and promote Th17 cell differentiation, development of ROR
gt ag-
onists can be considered as a promising approach for cancer
immunotherapy [24,25]. In clinical practice, cintirorgon (LYC-
55716), a small molecule ROR
gt agonist developed by Lycera, has
entered Phase 2 trial, supporting the feasibility of ROR
g
t agonists
The retinoic acid receptor-related orphan receptor gamma-t
being applied as orally bioavailable antitumor immunotherapeutic
[26].
During structure-activity relationship (SAR) study of ROR
verse agonists, GSK scientists serendipitously discovered a tertiary
amine ROR t agonist 1 [17]. A co-crystal structure of ROR t LBD
with agonist 1 was then resolved (PDB: 4NIE, resolution of 2.01 Å).
The co-crystal structure revealed that the left-hand side (LHS)
benzyl group of agonist 1 stabilized the hydrophobic site near
Tyr502 of activation function 2 (AF2) domain (also known as H12),
gt in-
* Corresponding author.
g
g
** Corresponding author. Department of Medicinal Chemistry, School of Phar-
macy, Fudan University, 826 Zhangheng Road, Shanghai, 201203, China.
E-mail addresses: yonghuiwang@fudan.edu.cn (Y. Wang), qxie@fudan.edu.cn
(Q. Xie).
1
R.Q. and M.Y. contributed equally to this work. R.Q., M.Y., Q.X., and Y.W. drafted
the manuscript, conceived the experiments, oversaw the experimental design, and
provided study supervision. R.Q., M.Y., J.G., J.T., and Y.H. carried out the synthesis
and characterization of the compounds. M.Y. carried out the Th17 differentiation
assay. All authors reviewed the manuscript.
whose conformation was crucial for RORgt acting in an agonism
manner by recruiting downstream co-activator peptide. Besides,
https://doi.org/10.1016/j.ejmech.2021.113704
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
adding 4-substituted bulky moieties at LHS phenyl ring of agonist 1
resulted in discovery of inverse agonists within the same chemical
scaffold. The agonist 1 has good in vitro biochemical activity
(EC50 ¼ 7.8 nM,% maximum activation (% max act.) ¼ 98.8% in our
corresponding anilines (11). Aldehyde intermediates (13a) were
obtained through amide formation, followed by aldehyde depro-
tection reaction. Intermediates (14) were synthesized through
reductive amination. The following alkylation with alkyl bromides
yielded the target compounds. For compound 6c, further reduction
reaction was required using compound 1. Specially, the preparation
of compound 6b started with a reversed amide (13b) formation
reaction using 4-formylbenzoic acid (12) and (4-(ethylsulfonyl)
phenyl)methanamine as substrates. Then it followed the remaining
synthetic procedures depicted in Scheme 1.
The synthesis of compounds 2f-2h, 4b-4g, 5a-5c, 6a, 7a-7e and
8a-8c is depicted in Scheme 2. The secondary amine intermediates
(16) were obtained through reductive amination. Then a nucleo-
philic reaction with commercially available benzyls furnished the
nitro intermediates (18). The reduction reaction followed by amide
or urea formation afforded the target compounds.
RORgt dual fluorescence resonance energy transfer (dual FRET)
assay). However, agonist 1 was not able to promote mouse Th17
(mTh17) cell differentiation above basal activity, indicating that the
molecular activity of agonist 1 was not sufficient to trigger cellular
responses. Further study was expected to improve agonism of 1 and
discover new ROR
In this paper, based on the co-crystal structure of agonist 1 with
ROR t LBD and mode of action, detailed SAR exploration and
optimization of each region of agonist 1 has been carried out. A
series of tertiary amines as ROR t agonists (Fig. 1) were designed,
synthesized and biologically evaluated. By combining the dominant
moieties of each region, the ROR t agonists such as 8b with
gt agonists with good cell-based activity.
g
g
g
improved cell-based activities were identified as a potential lead
compound for further optimization.
Compounds 4a and 4h-4k were prepared via synthetic path-
ways shown in Scheme 3. Secondary amine intermediates (20)
were obtained through nucleophilic reaction. A nucleophilic reac-
tion and reduction reaction followed to afford amines (22). Then
the target compounds were obtained through amide formation.
2. Results and discussion
The synthesized compounds were then evaluated in RORgt dual
2.1. Design and synthesis
FRET assay, Gal4 cell-based reporter gene assay, and mouse Th17
(mTh17) differentiation assay.
In order to learn how the chemical modifications of each region
on agonist 1 influence the binding mode and potential function-
ality, compounds with moieties of varying properties on LHS re-
gion, N-substituent (R1), amino position (n, m), central phenyl ring
(X, Y), linker and right-hand side (RHS) region were designed
(Fig. 1) and synthesized following different synthetic procedures
(Schemes 1e3).
2.3. Modifications of LHS
The SAR of the LHS of agonist 1 was first explored (Table 1). The
synthesized compounds were assessed at a target-based level by
ROR
% max act. implied efficacy as complementary parameters of ROR
agonism. Substitution on the LHS phenyl ring (2a-2e) almost had no
impact on the ROR t potency of the compounds, according to their
gt dual FRET assay, where the EC50 values signified potency and
gt
2.2. Chemistry
g
The synthesis of tert-amine-based compounds 1, 2a-2e, 3a-3g,
6b, and 6c is depicted in Scheme 1. The commercially available
aldehydes (9) were first protected by ethylene glycol. Then the
nitro-containing intermediates (10) were reduced to afford the
EC50 values. However, for ortho-substituted compounds (2a-2c),
the % max act. values decreased from 96.2% to 52.5% in the order of
-F > -CH3 > -Cl. Similarly, the -Cl substitution position on the LHS
phenyl ring (2c-2e) notably decreased RORgt agonism response in
Fig. 1. Compound design for SAR exploration based on agonist 1.
2

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
Scheme 1. General synthesis route 1a.
a Reagents and conditions: (a) TsOH, glycol, and toluene under reflux (b) PtO2, H2, NaHCO3 and MeOH at r.t. (c) DIPEA, HATU, 2-(4-(ethylsulfonyl)phenyl)acetic acid (23) and DCM at
r.t. followed by 1 N HCl at r.t. (d) DIPEA, HATU, (4-(ethylsulfonyl)phenyl)methanamine and DCM at r.t. (e) n-propylamine, and DCM at r.t. followed by NaBH4, and MeOH at r.t. (f)
Bromine-substituted alkyl or aryl compounds, K2CO3 and DMF at 100 ꢀC (g) BF3$THF at 0 ꢀC then r.t..
the order of ortho > meta > para. These results indicated that both
size and position of the substituents on LHS would interfere with
AF2 domain and thus reduce co-activator recruitment. Replacing
phenyl ring with hetero-aryl ring (2f-2h) decreased both potency
and efficacy, indicating the negative role of these heteroatom-
compounds exhibited improved potency and % max activation (3a-
3d). Compound 3d had a slightly decreased EC50 and higher
maximum activation response (EC50 ¼ 22.4 nM, % max act. ¼ 106%)
compared to agonist 1 (EC50 ¼ 7.80 nM, % max act. ¼ 98.8%), indi-
cating certain size of LHS moiety is needed to stabilize AF2 domain
containing moieties in RORgt agonism.
that maintains RORgt agonism. However, when hetero-atoms
We then replaced LHS phenyl ring with a series of cycloalkyl
moieties. With the increasing size of these moieties, the
substituted at LHS cycloalkyl ring (3e, 3f, 3g), the activation level
notably decreased, likely due to the increased hydrophilicity
Scheme 2. General synthesis route 2a.
a Reagents and conditions: (a) amines (R1NH2), and DCM at r.t. followed by NaBH4, and MeOH at r.t. (b) substituted 4-nitrobenzyl bromides, K2CO3 and DMF at 100 ꢀC (c) PtO2, H2,
NaHCO3 and MeOH at r.t. (d) DIPEA, HATU, 2-(4-(ethylsulfonyl)phenyl)acetic acid (23)/acids and DCM at r.t. (e) triphosgene, DIPEA, and THF at 0 ꢀC, followed by (4-(ethylsulfonyl)
phenyl)methanamine in THF at r.t..
3

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
Scheme 3. General synthesis route 3a.
Reagents and conditions: (a) Bromine-substituted benzenes or alkylbenzenes, K2CO3, KI, and MeCN at 110 ꢀC, M.W. (b) 1-bromopropane, K2CO3, KI, and MeCN at 110 ꢀC, M.W. (c) Pd/
C, H2, and MeOH at r.t. (d) DIPEA, HATU, 2-(4-(ethylsulfonyl)phenyl)acetic acid (23) and DCM at r.t..
Table 1
LHS SAR of the tertiary amine compounds.
Cmpd.
LHS
RORgt dual FRET
% max act.a
98.8
a
EC50 (nM)
7.80
1
2a
9.40
96.2
2b
2c
11.6
16.9
70.5
52.5
2d
2e
18.7
16.8
42.1
14.7
2f
29.1
40.2
63.4
47.7
2g
2h
>30000
N.D.b
3a
3b
3c
3d
6530
291
16.9
45.1
101
106
109
22.4
3e
79.6
14.0
3f
3g
a
86.2
285
23.0
80.5
All results are an average of at least two runs of individual experiments.
N.D.: no data.
b
4

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
Fig. 2. Predicted binding mode of 2g (in orange) overlay with co-crystal structure of 1 (in magenta) in RORgt LBD (PDB: 4NIE, resolution of 2.01 Å). Key residues of RORgt LBD,
His479 and Tyr502, are displayed in green. Hydrogen bond was highlighted in yellow dash dot.
introduced by these moieties.
destabilization of AF2 domain and decrease of RORgt agonism.
In order to explain the impact of LHS heteroatom alterations on
biological function, we performed the molecular docking study
using compound 2g, which has the identical chemical structure
with 1 except for pyridine moiety substitution on LHS. Despite of
having a similar ligand conformation to 1 as shown by the overlay
2.4. Modifications of N-alkyl moieties
To explore the potential conformational effect of N-alkyl moi-
eties on ROR t agonism, we then focused on the SAR study of N-
g
structure in RORgt LBD (Fig. 2), the nitrogen atom in pyridine
alkyl of the tertiary amines (Table 2). Without N-alkyl substitution
(4a), the potency dropped dramatically. Increasing size of N-alkyl
moieties (4b, 4c, 1) improved both potency and % max activation in
the order of Me < Et < n-Pr. However, further enlarged moieties
(4d-4g) did not influence potency so much but lowered efficacy
compared to agonist 1.
moiety on LHS of 2g formed hydrogen bonding with Tyr502,
competing against the polar interactions between Tyr502 and
His479. Previous studies have shown that Tyr502-His479 associa-
tion in AF2 domain is critical to maintaining RORgt agonism [27].
We hypothesized that the presence of heteroatoms in LHS could
disrupt the polar contacts of Tyr502 with His479, leading to
Based on the structure of agonist 1, the nitrogen atom was left-
shifted or right-shifted (Table 3). For non-N-alkylated agonists (4a,
4h, 4i), right-shifted NH (4h) exhibited better RORgt agonism than
Table 2
the left-shifted NH (4i); besides, both 4h and 4i showed better
activity than 4a, which could be attributed to the adjusted
SAR of N-alkyl of the tertiary amine
Table 3
SAR study of the N-shift of the tertiary amine.
Cmpd.
R1
RORgt dual FRET
EC50a (nM)
% max act.a
4a
4b
4c
1
-H
-CH3
-CH2CH3
>30000
101
36.3
N.D.b
79.0
85.1
98.8
Cmpd
R1
n
m
ROR
g
t dual FRET
7.80
EC50 (nM)
% max act.a
a
N.D.b
56.3
30.0
85.7
4d
4e
11.1
10.8
76.9
66.5
4a
4h
4i
-H
-H
-H
1
2
0
2
1
0
2
0
>30000
55.2
49.3
4j
11.6
4f
14.0
7.10
84.5
82.0
4k
1
0
1
2
1
11.2
7.80
70.1
98.8
4g
a
a
All results are an average of at least two runs of individual experiments.
N.D.: no data.
All results are an average of at least two runs of individual experiments.
N.D.: no data.
b
b
5

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
molecular conformation. For N-alkylated agonists (1, 4j, 4k), similar
to non-N-alkylated agonists, right-shifted N-alkyl agonist (4j) dis-
the Cl group of 5a formed a halogen bonding with Cys320 (high-
lighted in yellow) of H3 in adjacent to AF2 domain (H12) in the
played better RORgt agonism than the left-shifted (4k), but less
RORgt LBD ternary structure. We speculate that the additional
than the compound 1.
halogen bonding of 5a with Cys320 stabilized AF2 domain as a
preferred conformation for downstream co-activator recruitment,
leading to enhanced efficacy. This explanation is also in accord with
the further increase in potency of 5c, as the 2,6-diCl substitution
may endow a better chance to the compound forming halogen
bonding with Cys320.
2.5. Modifications of middle phenyl ring
For the identified tertiary amine ROR
g
t agonists (1, 4e), a Cl
group was introduced to the ortho-position of the middle phenyl
ring (5a, 5b, 5c) (Table 4). Interestingly, the Cl group addition
improved ROR
potency; for the starting compound 1, 2,6-diCl addition further
improved the ROR t agonism, affording a potentially better ROR
g
t efficacy (5a vs. 1；5b vs. 4f) while maintaining the
2.6. Modifications of linker
g
g
t
We next replaced the amide linker with urea, reversed amide
agonist (5c). To obtain the structural insights of the enhanced
and ethylamine (Table 5). The compound with urea linker (6a)
agonism by introducing Cl group, we performed docking study of
achieved better RORgt agonism than 1 although the potency was
5a with co-crystal structure of RORgt LBD and 1 (PDB: 4NIE). As
slightly lowered. The reverse amide (6b) showed decreased RORgt
shown in the overlay structure (Fig. 3), the predicted molecular
conformation of 5a was mostly overlapped with 1, which ensured
hydrogen bonding formation with the key residues in RORgt LBD.
Aside from the intramolecular interactions shown with agonist 1,
maximum activation although the potency was mostly maintained
compared to 1, indicating that the subtle position shift of NH as the
hydrogen bonding donor may affect the stabilization of hydro-
phobic pocket recruiting downstream co-activators that would
determine the potential ROR
was replaced by ethylamine (6c), the compound became an ROR
inverse agonist instead of ROR t agonist, indicating that a flexible
linker would disrupt the preferred molecular conformation for an
ROR t agonist.
gt agonism. When the amide linker
gt
Table 4
g
SAR study of the central phenyl ring.
g
2.7. Modifications of RHS
Cmpd
R1
X
Y
ROR
g
t dual FRET
The SAR of RHS substituents of the tertiary amine compounds
was investigated (Table 6). Replacing ethyl sulfone with methyl
sulfone (7a) decreased the potency, but the RORgt agonism
response increased slightly. Also, considering 7a has slightly low-
ered molecular weight and CLogP value compared to 1, methyl
sulfonyl group is more preferable for further optimization. Intro-
ducing the carboxylic group (7b) in place of ethyl sulfonyl group
lowered the activity dramatically. And smaller substituents like -CN
(7c) and -H (7d) decreased the activity, likely owing to these sub-
EC50 (nM)
7.80
% max act.a
98.8
a
1
-H
-Cl
-Cl
-H
-H
-H
5a
5b
8.70
8.00
120
101
5c
-Cl
-Cl
8.20
117
stituents not able to establish intermolecular forces with the ROR
ligand binding pocket. We also switched the benzene ring of RHS
gt
a
All results are an average of at least two runs of individual experiments.
Fig. 3. (A) Predicted binding mode of 5a (in cyan) overlay with co-crystal structure of 1 (in magenta) in RORgt LBD (PDB: 4NIE, resolution of 2.01 Å). (B) The key intramolecular
interactions are shown in dash line. A halogen bond with Cys320 is highlighted in yellow.
6

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
Table 5
high potency at nanomolar level. The in vitro activities of compound
SAR of the amide linker replacement.
1 and 8a-8c were then evaluated in cell-based ROR
gene assay. Signified by activation maximum percentage on ROR
Gal4 assay, the ROR t agonism efficacy of 8a-8c enhanced from
gt Gal4 reporter
gt
g
59.2% to 79.7% compared to agonist 1 (50.1%). We chose compound
8b as a promising agonist for its overall potency and efficacy. A
mouse Th17 (mTh17) cell differentiation assay that could better
reflect the functionality was then performed to evaluate its bioac-
tivity in promoting IL-17 production. We measured the proportion
of IL-17 produced by mTh17 cells in flow cytometry and determined
the percentage of activation relative to the vehicle with DMSO. In
contrast with compound 1 that had no activity in promoting Th17
differentiation, compound 8b promoted the production of IL-17
with a favorable EC50 value of 37.2 nM and a promising activation
of around 30% above basal level (Fig. 4). By comparing the chemical
structures of 8b with 1, we suspect that the cyclohexyl substitution
on LHS has a more flexible molecular conformation that may in-
crease the efficacy of being internalized by mTh17 cells, in contrast
Cmpd
Linker
ROR
g
t dual FRET
EC50 (nM)
7.80
% max act.a
98.8
a
1
6a
6b
58.0
118
9.20
33.6
84.3
6c
ꢁ51.7
to rigid conformation caused by p-p stacking between the phenyl
group on the LHS of agonist 1.
To describe the drug-like properties of the lead compound, we
assessed the kinetic solubility, predicted cell permeability and
a
All results are an average of at least two runs of individual experiments.
in vitro metabolic stability of 8b in comparison with
1
(supplementary Table S1). Although the solubility of 8b was not
improved, compound 8b showed enhanced predicted cell perme-
ability and improved metabolic stability in mouse liver microsome.
Enhanced cell permeability may partially contribute to the activity
improvement in mTh17 assay. Yet, more efforts are needed for
further optimization of the tertiary amine lead series.
Table 6
SAR of RHS substituents of the tertiary amine.
Cmpd
R2
Z
MW
CLogP
ROR
gt dual FRET
3. Conclusions
EC50 (nM)
7.80
% max act.a
a
Based on previously discovered tertiary amine agonist 1 and its
1
-CH
-CH
464.62
450.60
4.25
3.72
98.8
resolved ROR
designed, synthesized and biologically identified as novel potent
ROR t agonists. Consistently with our previous findings, LHS of
tertiary amines was sensitive and crucial for ROR t agonism. Aside
gt co-crystal structure, a series of tertiary amines were
7a
43.1
102
g
g
7b
7c
7d
7e
-CH
-CH
-CH
-N
416.52
397.52
372.22
465.62
5.10
4.79
5.36
3.37
>30000
346
N.D.b
83.2
46.2
N.D.b
from LHS, SARs of other regions were also explored separately and
led to the discovery of compounds (3d, 5a, 5c, 7a) with better
ROR
optimal moieties resulted in novel ROR
further elevated ROR t agonism at target-based level as well as in
gt agonism or drug-like properties. The combination of these
g
t agonists (8a-8c) with
2170
7100
g
cell-based assay, among which 8b is superior in the mTh17 cell
differentiation assay. This work provided some structural knowl-
edge for further optimization of RORgt agonists as small molecule
a
All results are an average of at least two runs of individual experiments.
N.D.: no data.
b
therapeutics for cancer immunotherapy.
4. Experimental
into pyridine (7e) as an alternative approach to improving the hy-
drophilicity. Although 7e had a lower CLogP value comparable to
4.1. Materials and methods
7a, it did not show significant potency and agonism on RORgt.
All commercially available reagents and solvents were pur-
chased and used without further purification unless otherwise
stated. All reactions were monitored by TLC, using silica gel plates
with UV light visualization. Column chromatography was per-
formed using silica gel (200e300 mesh). 1H NMR data was recorded
on a Bruker 400 MHz NMR system. Chemical shifts in 1H NMR
2.8. Combination of dominant moieties
According to the SAR investigation of LHS region, N-substituents
as well as modifications on the central phenyl ring, linker and RHS
region, we found that substituting the LHS with cyclohexyl moiety,
adding Cl atoms to the central phenyl ring or replacing by methyl
sulfone on RHS could all lead to the compounds with improved
spectra are reported in parts per million (ppm) on the
relative to an internal control (TMS). Data are reported as follows:
chemical shift (
d scale
d
ppm), multiplicity (s ¼ singlet, d ¼ doublet,
RORgt agonism. We thus hypothesized that the combination of
t ¼ triplet, q ¼ quartet, m ¼ multiplet, dd ¼ doublet of doublets,
dt ¼ doublet of triplets, br ¼ broad), coupling constant in hertz (Hz),
and integration. LC-MS data was recorded on Agilent Technologies
6120 quadrupole mass spectrometer. High-resolution mass spectra
(HRMS) were obtained on an AB SCIEX TripleTOF 5600þ mass
these optimized moieties may result in the identification of com-
pounds with greater potency and efficacy in the further bioactivity
evaluation (Table 7). On the RORgt dual FRET assay, 8a-8c achieved
higher % maximum activation compared to 1, which also exhibited
7

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
Table 7
Activities of the compounds with combined chemical moieties.
Cmpd
Structure
ROR
g
t dual FRET
RORg
t Gal4 EC50 (nM)(% max act.)a
EC50 (nM)(% max act.)a
1
7.80 (98.8)
110 (50.1)
277 (59.2)
8a
8b
8c
13.9 (108)
10.5 (111)
33.5 (115)
283 (72.1)
382 (79.7)
a
All results are an average of at least two runs of individual experiments.
Fig. 4. ROR
gt agonist 8b enhanced mTh17 cell differentiation and IL-17 production. (A) Dose-response curve obtained from stimulation of Th17 cells by 8b at different concen-
trations. (B) 8b increased the percentage of CD4þIL-17þ (Th17) cells at a concentration of 250 nM.
spectrometer (AB SCIEX, LLC., Redwood City, CA, USA). The contents
of compounds for biological evaluation were examined with an
Agilent 1260 Infinity LC system (Agilent Technologies, Inc., Santa
Clara, CA, USA) with methanol/water (40:60 to 95:5) as the eluent.
Unless specified, the purity of target compounds was >95%, which
was considered to be pure enough for biological assays. The syn-
thetic procedures, 1H NMR, 13C NMR and HRMS are detailed in the
Supporting Information section.
filtered through celite and evaporated under reduced pressure to
afford desired intermediate 11.
Step 3. To a solution of 4-(1,3-dioxolan-2-yl)aniline (1.0 eq), 2-(4-
(ethylsulfonyl)phenyl)acetic acid (1.2 eq) and HATU (1.2 eq) in DCM
was gradually added DIPEA (3.0 eq). The mixture was stirred at
room temperature for 4 h and was then added 1 N aqueous HCl,
continuing to react for 1 h. The solution was basified by sat. Na2CO3,
washed with brine, dried over Na2SO4 and evaporated under
reduced pressure. Purification by column chromatography through
silica gel afforded desired intermediate 13a. The synthesis of 13b
followed the procedure of 13a.
4.2. Chemical synthesis
4.2.1. General procedure A for the synthesis of compounds (1, 2a-2e,
3a-3g, 6b)
Step 4. To a solution of intermediate 13a or 13b (1.0 eq) and n-
propylamine (2.4 eq) in dry DCM was added anhydrous Na2SO4 and
stirred at room temperature overnight under nitrogen atmosphere.
The mixture was filtered and evaporated under reduced pressure.
The residue re-dissolved in methanol and was added NaBH4 (0.5
eq), stirring at room temperature for 3 h. The reaction was
quenched by saturated Na2CO3 and extracted with EA. The com-
bined organic phase was evaporated to afford intermediate 14.
Step 1. To a solution of 4-nitrobenzaldehyde (1.0 eq) and p-tol-
uenesulfonic acid monohydrate (0.02 eq) in dry toluene was added
ethylene glycol (3.0 eq). The mixture was heated up to reflux for 4 h.
After cooling down, the solvent was removed under reduced
pressure. The residue was purified by column chromatography on
silica gel to afford the intermediate 10.
Step 5. The mixture of 14 (1.0 eq), substituted benzyl bromide (3.0
eq) and anhydrous potassium carbonate (3.0 eq) in DMF was sealed
and heated up to 60 ꢀC for 3 h. The solution was washed with brine
three times and the combined organic phase was evaporated.
Step 2. To a solution of 2-(4-nitrophenyl)-1,3-dioxolane (1.0 eq)
and anhydrous sodium bicarbonate (1.0 eq) in methanol was added
Adams' catalyst. The mixture was degassed and purged with
hydrogen and stirred at room temperature for 4 h. The solution was
8

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
Purification by column chromatography through silica gel afforded
the target compounds (1, 2a-2e, 3a-3g, 6b)
4.2.1.6. N-(4-(((4-chlorobenzyl)(propyl)amino)methyl)phenyl)-2-(4-
(ethylsulfonyl)phenyl)acetamide (2e). Yield: 80%, white solid. 1H
NMR (400 MHz, CDCl3)
d
8.20 (s, 1H), 7.78 (d, J ¼ 8.1 Hz, 2H),
7.51e7.41 (m, 4H), 7.26e7.20 (m, 6H), 3.73 (s, 2H), 3.45 (s, 4H), 3.07
(q, J ¼ 7.4 Hz, 2H), 2.31 (t, J ¼ 7.2 Hz, 2H), 1.55e1.40 (m, 2H),
1.25e1.18 (m, 3H), 0.81 (t, J ¼ 7.3 Hz, 3H).MS (ESI) m/z: 499.1 [MHþ].
4.2.1.1. N-(4-((benzyl(propyl)amino)methyl)phenyl)-2-(4-(ethyl-
sulfonyl)phenyl)acetamide (1). Yield: 52%, white solid. 1H NMR
13C NMR (151 MHz, CDCl3)
d 167.83, 141.34, 137.16, 136.52, 132.40,
(400 MHz, DMSO)
d
10.25 (s, 1H), 7.86 (d, J ¼ 8.0 Hz, 2H), 7.62 (d,
J ¼ 8.0 Hz, 2H), 7.56 (d, J ¼ 8.2 Hz, 2H), 7.36e7.20 (m, 7H), 3.80 (s,
2H), 3.48 (d, J ¼ 14.8 Hz, 4H), 3.28 (q, J ¼ 7.3 Hz, 2H), 2.29 (t,
J ¼ 7.1 Hz, 2H), 1.52e1.40 (m, 2H), 1.10 (t, J ¼ 7.3 Hz, 3H), 0.78 (t,
J ¼ 7.3 Hz, 3H). MS (ESI) m/z: 465.2 [MHþ]. 13C NMR (101 MHz,
130.23, 130.04, 129.33, 128.53, 128.28, 119.83, 57.65, 57.40, 55.25,
50.64, 44.04, 29.69, 11.76, 7.38. HRMS (m/z): [MHþ] calcd for
C
27H32ClN2O3S 499.1817; found, 499.1825.
DMSO)
d 168.48, 142.69, 140.15, 138.18, 137.26, 134.98, 130.64,
4.2.1.7. N-(4-(((cyclopropylmethyl)(propyl)amino)methyl)phenyl)-2-
(4-(ethylsulfonyl)phenyl)acetamide (3a). Yield: 78%, white solid. 1H
129.33, 128.90, 128.60, 128.30, 127.18, 119.47, 57.96, 57.57, 55.13,
49.71, 43.42, 20.00, 12.20, 7.62. HRMS (m/z): [MHþ] calcd for
NMR (400 MHz, CDCl3)
d
7.85 (d, J ¼ 8.2 Hz, 2H), 7.59 (d, J ¼ 8.2 Hz,
C
27H33N2O3S 465.2206; found, 465.2210.
2H), 7.55 (d, J ¼ 9.1 Hz, 2H), 7.37 (d, J ¼ 8.2 Hz, 2H), 3.86 (s, 2H), 3.77
(s, 2H), 3.11 (q, J ¼ 7.4 Hz, 2H), 2.68e2.57 (m, 2H), 2.50 (d, J ¼ 6.1 Hz,
2H), 1.57 (dt, J ¼ 14.6, 7.3 Hz, 2H), 1.30e1.26 (m, 3H), 0.87 (t, J ¼
7.3 Hz, 4H), 0.55 (d, J ¼ 7.7 Hz, 2H), 0.14 (d, J ¼ 4.7 Hz, 2H), 0.07 (s,
1H). MS (ESI) m/z: 429.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
4.2.1.2. 2-(4-(ethylsulfonyl)phenyl)-N-(4-(((2-fluorobenzyl)(propyl)
amino)methyl)phenyl)acetamide (2a). Yield: 59%, white solid. 1H
NMR (400 MHz, CDCl3)
d
7.83 (d, J ¼ 8.1 Hz, 2H), 7.66 (s, 1H), 7.51 (d,
d
168.01, 165.79, 141.75, 137.15, 136.69, 130.24, 129.25, 128.50,
J ¼ 8.1 Hz, 2H), 7.48e7.43 (m, 1H), 7.43 (d, J ¼ 8.3 Hz, 2H), 7.29 (d, J ¼
8.2 Hz, 2H), 7.19 (dd, J ¼ 12.9, 6.2 Hz,1H), 7.08 (t, J ¼ 7.2 Hz,1H), 6.98
(t, J ¼ 9.1 Hz, 1H), 3.77 (s, 2H), 3.60 (s, 2H), 3.52 (s, 2H), 3.09 (q, J ¼
7.4 Hz, 2H), 2.35 (t, J ¼ 7.2 Hz, 2H), 1.59e1.43 (m, 2H), 1.24 (s, 3H),
0.81 (t, J ¼ 7.3 Hz, 3H). MS (ESI) m/z: 483.2 [MHþ]. 13C NMR
119.66, 58.81, 56.25, 50.67, 38.63, 31.82, 26.19, 11.86, 7.41. HRMS (m/
z): [MHþ] calcd for C27H39N2O3S 471.2676; found, 471.2681.
4.2.1.8. N-(4-(((cyclobutylmethyl)(propyl)amino)methyl)phenyl)-2-
(4-(ethylsulfonyl)phenyl)acetamide (3b). Yield: 36%, white solid. 1H
(151 MHz, CDCl3)
d 167.79, 141.31, 137.18, 136.45, 131.12, 130.23,
NMR (400 MHz, CDCl3)
d
7.80 (d, J ¼ 7.8 Hz, 2H), 7.51 (s, 1H), 7.47 (d,
129.35, 128.56, 123.87, 119.83, 115.18, 115.03, 57.77, 55.30, 50.64,
50.51, 44.07, 20.01, 11.75, 7.38. HRMS (m/z): [MHþ] calcd for
J ¼ 7.9 Hz, 2H), 7.35 (d, J ¼ 7.9 Hz, 2H), 7.18 (d, J ¼ 16.4 Hz, 2H), 3.72
(s, 2H), 3.41 (s, 2H), 3.05 (q, J ¼ 7.3 Hz, 2H), 2.51e2.39 (m, 1H), 2.25
(t, J ¼ 6.9 Hz, 2H), 1.98e1.88 (m, 2H), 1.78 (dd, J ¼ 17.7, 8.6 Hz, 3H),
1.60e1.45 (m, 2H), 1.42e1.32 (m, 2H), 1.24e1.20 (m, 3H), 0.75 (t, J ¼
7.1 Hz, 3H). MS (ESI) m/z: 443.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
C
27H32FN2O3S 483.2112; found, 483.2115.
4.2.1.3. 2-(4-(ethylsulfonyl)phenyl)-N-(4-(((2-methylbenzyl)(propyl)
amino)methyl)phenyl)acetamide (2b). Yield: 77%, white solid. 1H
d
167.70, 141.13, 137.43, 130.30, 129.62, 128.70, 119.75, 59.85, 58.17,
NMR (400 MHz, CDCl3)
d
7.87 (s, 1H), 7.80 (d, J ¼ 8.2 Hz, 2H), 7.48 (d,
55.65, 50.65, 44.21, 33.50, 27.50, 18.73, 11.76, 7.42, 1.02. HRMS (m/z):
J ¼ 8.1 Hz, 2H), 7.42 (d, J ¼ 8.3 Hz, 2H), 7.39e7.34 (m, 1H), 7.24 (s,
1H), 7.18e7.08 (m, 3H), 3.73 (s, 2H), 3.48 (d, J ¼ 11.5 Hz, 4H), 3.09 (q,
J ¼ 7.4 Hz, 2H), 2.38e2.27 (m, 5H), 1.50 (dd, J ¼ 14.5, 7.2 Hz, 2H),
1.26e1.22 (m, 3H), 0.80 (t, J ¼ 7.3 Hz, 3H). MS (ESI) m/z: 479.2
[MHþ] calcd for C25H35N2O3S 443.2363; found, 443.2375.
4.2.1.9. N-(4-(((cyclopentylmethyl)(propyl)amino)methyl)phenyl)-2-
(4-(ethylsulfonyl)phenyl)acetamide (3c). Yield: 83%, white solid. 1H
[MHþ]. 13C NMR (151 MHz, CDCl3)
d 167.73, 141.29, 137.14, 130.22,
NMR (400 MHz, CDCl3)
d
9.59 (s, 1H), 7.81 (d, J ¼ 7.9 Hz, 2H), 7.64 (d,
130.15, 129.58, 129.39, 128.54, 126.75, 125.53, 119.72, 57.97, 56.65,
J ¼ 7.6 Hz, 2H), 7.57 (d, J ¼ 7.9 Hz, 2H), 7.30 (d, J ¼ 7.9 Hz, 2H), 4.13 (s,
2H), 3.83 (s, 2H), 3.48 (s, 2H), 3.10 (q, J ¼ 7.3 Hz, 2H), 3.00e2.90 (m,
2H), 1.83 (d, J ¼ 17.9 Hz, 2H), 1.81e1.69 (m, 2H), 1.67e1.45 (m, 4H),
1.26 (t, J ¼ 7.4 Hz, 3H), 1.13 (d, J ¼ 7.8 Hz, 2H), 0.90 (t, J ¼ 7.1 Hz, 3H).
55.72, 50.64, 44.05, 29.69, 19.34, 11.86. HRMS (m/z): [MHþ] calcd for
C
28H35N2O3S 479.2363; found, 479.2376.
MS (ESI) m/z: 457.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 168.89,
4.2.1.4. N-(4-(((2-chlorobenzyl)(propyl)amino)methyl)phenyl)-2-(4-
(ethylsulfonyl)phenyl)acetamide (2c). Yield: 46%, white solid. 1H
141.63, 140.19, 137.06, 131.56, 130.32, 128.44, 123.80, 120.33, 57.27,
57.17, 53.69, 50.63, 43.81, 35.27, 31.79, 31.65, 24.90, 24.85, 16.52,
11.01, 7.40. HRMS (m/z): [MHþ] calcd for C26H37N2O3S 457.2519;
found, 457.2537.
NMR (400 MHz, CDCl3)
d
7.85 (d, J ¼ 8.1 Hz, 2H), 7.54 (d, J ¼ 8.0 Hz,
2H), 7.44 (d, J ¼ 7.1 Hz, 2H), 7.37e7.27 (m, 3H), 7.22 (d, J ¼ 7.2 Hz,
1H), 7.17 (d, J ¼ 7.5 Hz, 2H), 3.80 (s, 2H), 3.76e3.44 (m, 4H), 3.10 (q,
J ¼ 7.3 Hz, 2H), 2.51e2.34 (m, 2H), 1.66e1.45 (m, 2H), 1.29e1.26 (m,
3H), 0.82 (t, J ¼ 7.3 Hz, 3H). MS (ESI) m/z: 499.1 [MHþ]. 13C NMR
4.2.1.10. N-(4-(((cyclohexylmethyl)(propyl)amino)methyl)phenyl)-2-
(4-(ethylsulfonyl)phenyl)acetamide (3d). Yield: 89%, white solid. 1H
(151 MHz, CDCl3)
d 167.85, 141.20, 137.34, 134.19, 130.29, 129.73,
129.45, 128.65, 126.88, 119.88, 57.73, 55.32, 54.64, 50.64, 44.15,
29.70, 11.69, 7.41. HRMS (m/z): [MHþ] calcd for C27H32ClN2O3S
499.1817; found, 499.1819.
NMR (400 MHz, CDCl3)
d
7.92 (d, J ¼ 8.7 Hz, 2H), 7.85 (d, J ¼ 8.0 Hz,
2H), 7.70 (d, J ¼ 7.8 Hz, 2H), 7.69e7.61 (m, 2H), 4.02 (s, 2H), 3.10 (dd,
J ¼ 14.9, 7.5 Hz, 3H), 2.00 (M, 1H), 1.92e1.83 (m, 1H), 1.77 (d, J ¼
10.5 Hz, 2H), 1.34e1.26 (m, 8H), 0.99 (M, 4H), 0.93e0.85 (m, 3H),
0.10e0.05 (m, 5H). MS (ESI) m/z: 471.2 [MHþ]. 13C NMR (151 MHz,
4.2.1.5. N-(4-(((3-chlorobenzyl)(propyl)amino)methyl)phenyl)-2-(4-
(ethylsulfonyl)phenyl)acetamide (2d). Yield: 51%, white solid. 1H
CDCl3) d 168.20, 141.51, 137.66, 137.18, 130.33, 130.07, 128.54, 119.91,
NMR (400 MHz, CDCl3)
d
7.81 (d, J ¼ 8.2 Hz, 2H), 7.79 (s, 1H), 7.50 (d,
58.00, 57.64, 54.77, 50.65, 44.06, 18.89, 11.67, 7.41, 7.27, 4.25, 1.02.
HRMS (m/z): [MHþ] calcd for C24H33N2O3S 429.2206; found,
429.2211.
J ¼ 8.1 Hz, 2H), 7.44 (d, J ¼ 8.3 Hz, 2H), 7.33 (s,1H), 7.27 (d, J ¼ 9.8 Hz,
2H), 7.20 (s, 2H), 3.76 (s, 2H), 3.47 (d, J ¼ 2.5 Hz, 4H), 3.09 (q, J ¼
7.4 Hz, 2H), 2.33 (t, J ¼ 7.2 Hz, 2H), 1.54e1.44 (m, 2H), 1.25e1.22 (m,
3H), 0.82 (t, J ¼ 7.3 Hz, 3H). MS (ESI) m/z: 499.1 [MHþ]. 13C NMR
4.2.1.11. N-(4-((((4,4-difluorocyclohexyl)methyl)(propyl)amino)
(151 MHz, CDCl3)
d
167.75, 141.23, 137.24, 136.46, 134.06, 130.23,
methyl)phenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide
(3e).
7.88 (d, J ¼
129.44, 129.38, 128.69, 128.59, 127.01, 127.01, 126.84, 126.84, 119.85,
57.71, 57.60, 55.33, 50.65, 44.10, 29.69, 11.76, 7.39. HRMS (m/z):
[MHþ] calcd for C25H32ClN2O3S 499.1817; found, 499.1820.
Yield: 87%, white solid. 1H NMR (400 MHz, CDCl3)
d
8.2 Hz, 2H), 7.55 (d, J ¼ 8.1 Hz, 2H), 7.41 (d, J ¼ 8.2 Hz, 2H), 7.25 (d,
J ¼ 3.4 Hz, 2H), 3.80 (s, 2H), 3.73e3.69 (m, 1H), 3.47 (s, 2H), 3.11 (q,
9

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
Step 1. To a solution of aldehyde (1.0 eq) and alkylamine (1.1 eq) in
dry DCM was added Na2SO4 and stirred overnight. The mixture was
filtered and evaporated under reduced pressure. Then, the residue
was re-dissolved in methanol, carefully added NaBH4 (0.5 eq) under
ice bath and was stirred for 1 h at room temperature. The reaction
was quenched by saturated Na2CO3, extracted with EA and dried
over Na2SO4. The combined organic phase was evaporated to afford
the desired intermediate 16.
J ¼ 7.4 Hz, 3H), 2.20 (d, J ¼ 6.7 Hz, 2H), 2.00 (s, 3H), 1.86 (t, J ¼
12.0 Hz, 3H), 1.25 (d, J ¼ 8.3 Hz, 5H), 0.84 (dd, J ¼ 13.6, 6.4 Hz, 4H).
MS (ESI) m/z: 507.2 [MHþ]. 13C NMR (101 MHz, CDCl3)
d 130.34 (s),
128.69 (s), 119.87 (s), 58.80 (s), 56.35 (s), 50.65 (s), 44.19 (s), 33.09
(s), 29.70 (s), 29.33 (s), 27.44 (d, J ¼ 8.6 Hz), 22.70 (s), 11.63 (s), 7.42
(s). HRMS (m/z): [MHþ] calcd for C27H36F2N2O3S 507.2487; found,
507.2487.
4.2.1.12. 2-(4-(ethylsulfonyl)phenyl)-N-(4-((propyl((tetrahydro-2H-
pyran-4-yl)methyl)amino)methyl)phenyl)acetamide (3f). Yield: 55%,
Step 2. A mixture of intermediate 16 (1.0 eq), nitrobenzene in-
termediates (0.5 eq), anhydrous potassium carbonate (1.5 eq) and
acetonitrile was heated up to reflux for 4 h. The solvent was
removed. The residue was re-dissolved, washed with brine and
dried over Na2SO4. Purification by column chromatography
through silica gel afforded intermediate 17.
white solid. 1H NMR (400 MHz, CDCl3)
d
7.88 (d, J ¼ 8.0 Hz, 2H), 7.55
(d, J ¼ 7.7 Hz, 2H), 7.40 (d, J ¼ 8.6 Hz, 2H), 7.23 (d, J ¼ 7.7 Hz, 2H),
3.90 (d, J ¼ 10.9 Hz, 2H), 3.80 (s, 2H), 3.47 (s, 2H), 3.34 (t, J ¼ 11.2 Hz,
2H), 3.19e3.04 (m, 2H), 2.38e2.26 (m, 2H), 2.23e2.16 (m, 2H),
1.48e1.40 (m, 2H), 1.26 (d, J ¼ 6.9 Hz, 3H), 1.12 (m, 3H), 0.83 (t, J ¼
7.3 Hz, 3H).MS (ESI) m/z: 473.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
Step 3. To a solution of intermediate 17 (1.0 eq) in methanol was
added Adams' catalyst. The mixture was degassed and purged with
hydrogen and stirred at room temperature for 1 h. The solution was
filtered through celite and evaporated to afford desired amine 18.
d
167.53, 141.04, 137.47, 130.32, 129.79, 128.72, 119.81, 67.77, 58.71,
56.23, 50.64, 44.19, 31.53, 29.71, 11.70, 7.42, 1.02. HRMS (m/z):
[MHþ] calcd for C26H37N2O4S 473.2469; found, 473.2474.
Step 4. To a solution of 18 (1.0 eq) in DCM was added p-alkyl-
sulfone aryl acetic acid (1.2 eq), HATU (1.2 eq) and DIPEA (1.2 eq).
The mixture was stirred at room temperature for 5 h. Purification
by column chromatography through silica gel afforded desired
compound (2f-2h, 4b-4g, 5a-5c, 6a, 7a-7e, 8a-8c)
4.2.1.13. 2-(4-(ethylsulfonyl)phenyl)-N-(4-((propyl((tetrahydrofuran-
2-yl)methyl)amino)methyl)phenyl)acetamide (3g). Yield: 67%, white
solid. 1H NMR (400 MHz, CDCl3)
d
7.86 (d, J ¼ 8.1 Hz, 2H), 7.53 (d, J ¼
8.1 Hz, 2H), 7.41 (d, J ¼ 8.3 Hz, 2H), 7.26 (d, J ¼ 4.4 Hz, 2H), 3.99 (dd,
J ¼ 12.8, 6.4 Hz,1H), 3.80 (s,1H), 3.77 (s, 2H), 3.74e3.70 (m, 2H), 3.11
(q, J ¼ 7.4 Hz, 3H), 2.46e2.36 (m, 2H), 2.00e1.86 (m, 2H), 1.80 (dt,
J ¼ 13.4, 6.8 Hz, 3H), 1.52e1.41 (m, 3H), 1.26e1.24 (m, 3H), 0.82 (t,
J ¼ 7.3 Hz, 3H). MS (ESI) m/z: 459.2 [MHþ]. 13C NMR (151 MHz,
4.2.2.1. 2-(4-(ethylsulfonyl)phenyl)-N-(4-((propyl(thiophen-2-
ylmethyl)amino)methyl)phenyl)acetamide (2f). Yield: 78%, white
solid. 1H NMR (400 MHz, CDCl3)
d
7.84 (d, J ¼ 10.5 Hz, 2H), 7.80 (s,
CDCl3)
d 167.81, 141.15, 137.39, 130.29, 129.98, 128.67, 119.76, 68.15,
1H), 7.56 (d, J ¼ 7.5 Hz, 2H), 7.47 (d, J ¼ 8.1 Hz, 2H), 7.33 (d, J ¼ 7.8 Hz,
2H), 7.22 (s,1H), 6.98e6.84 (m, 2H), 3.81 (s, 4H), 3.59 (s, 2H), 3.10 (d,
J ¼ 6.3 Hz, 3H), 2.43 (s, 2H), 1.30e1.26 (m, 3H), 0.88e0.80 (m, 3H).
58.38, 57.40, 55.87, 50.65, 44.15, 30.29, 25.32, 19.41, 11.65, 7.42.
HRMS (m/z): [MHþ] calcd for C25H35N2O4S 459.2312; found,
459.2318.
MS (ESI) m/z: 471.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 168.37,
141.46, 137.16, 130.37, 130.27, 128.53, 127.06, 120.10, 57.08, 54.49,
4.2.1.14. 4-((benzyl(propyl)amino)methyl)-N-(4-(ethylsulfonyl)
50.63, 44.00, 29.71, 11.42, 7.40. HRMS (m/z): [MHþ] calcd for
benzyl)benzamide (6b). Yield: 80%, white solid. 1H NMR (400 MHz,
C
25H31N2O3S2 471.1771; found, 471.1775.
CDCl3)
d
7.81 (d, J ¼ 8.0 Hz, 2H), 7.77 (d, J ¼ 8.1 Hz, 2H), 7.48 (dd, J ¼
14.5, 8.0 Hz, 4H), 7.38e7.33 (m, 2H), 7.30 (t, J ¼ 7.4 Hz, 2H),
7.25e7.20 (m, 1H), 6.79 (s, 1H), 4.72 (s, 2H), 3.58 (d, J ¼ 11.4 Hz, 4H),
3.08 (q, J ¼ 7.4 Hz, 2H), 2.38 (t, J ¼ 7.0 Hz, 2H), 1.53 (dd, J ¼ 14.1,
7.2 Hz, 2H),1.25 (t, J ¼ 7.4 Hz, 3H), 0.84 (t, J ¼ 7.3 Hz, 3H). MS (ESI) m/
4.2.2.2. 2-(4-(ethylsulfonyl)phenyl)-N-(4-((propyl(pyridin-2-
ylmethyl)amino)methyl)phenyl)acetamide (2g). Yield: 65%, white
solid. The title compound was synthesized following the procedure
z: 465.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d
167.61, 145.19, 137.08,
for compound 2f. 1H NMR (400 MHz, CDCl3)
d 8.46 (s, 1H), 8.00 (s,
1H), 7.82 (d, J ¼ 6.9 Hz, 2H), 7.65 (s, 1H), 7.55e7.46 (m, 3H), 7.43 (d,
J ¼ 7.1 Hz, 2H), 7.27 (s, 1H), 7.14 (s, 1H), 3.74 (d, J ¼ 16.5 Hz, 4H), 3.59
(s, 2H), 3.09 (d, J ¼ 6.5 Hz, 2H), 2.43 (s, 3H), 1.58e1.45 (m, 2H), 1.27
(s, 1H), 0.92e0.78 (m, 4H). MS (ESI) m/z: 466.2 [MHþ]. 13C NMR
132.30, 128.87, 128.71, 128.43, 128.20, 128.12, 127.03, 126.70, 58.33,
57.95, 55.54, 50.63, 43.17, 20.15, 11.79, 7.38. HRMS (m/z): [MHþ]
calcd for C27H33N2O3S 465.2206; found, 465.2211.
(151 MHz, CDCl3)
d 168.09, 148.73, 141.44, 137.10, 136.79, 130.29,
4.2.1.15. 4-((benzyl(propyl)amino)methyl)-N-(4-(ethylsulfonyl)phe-
nethyl)aniline (6c). A mixture of N-(4-((benzyl(propyl)amino)
methyl)phenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide (1) (80 mg,
0.17 mmol) and BH3 in THF (0.5 mL, 0.5 mmol) was stirred under ice
bath for 30 min, and was then stirred overnight at room temper-
ature. Purification through pTLC (PE:EA 1:1) afforded white solid
129.68, 128.49, 123.03, 122.32, 119.95, 59.24, 58.04, 55.83, 50.63,
43.94, 19.79, 11.64, 7.38. HRMS (m/z): [MHþ] calcd for C26H32N3O3S
466.2159; found, 466.2167.
4.2.2.3. 2-(4-(ethylsulfonyl)phenyl)-N-(4-((propyl(thiazol-2-
(50 mg, yield: 65%). 1H NMR (400 MHz, CDCl3)
d
7.83 (d, J ¼ 8.1 Hz,
ylmethyl)amino)methyl)phenyl)acetamide (2h). Yield: 65%, white
2H), 7.40 (d, J ¼ 8.0 Hz, 2H), 7.36 (d, J ¼ 7.3 Hz, 2H), 7.29 (t, J ¼ 7.3 Hz,
2H), 7.21 (d, J ¼ 8.5 Hz,1H), 7.17 (d, J ¼ 8.1 Hz, 2H), 6.56 (d, J ¼ 8.2 Hz,
2H), 3.56 (s, 2H), 3.48 (d, J ¼ 5.9 Hz, 2H), 3.42 (t, J ¼ 6.8 Hz, 2H), 3.10
(q, J ¼ 7.4 Hz, 2H), 2.99 (t, J ¼ 6.7 Hz, 2H), 2.37 (t, J ¼ 6.8 Hz, 2H), 1.53
(dd, J ¼ 14.3, 7.2 Hz, 2H), 1.27 (t, J ¼ 7.5 Hz, 3H), 0.83 (t, J ¼ 7.3 Hz,
solid. 1H NMR (400 MHz, CDCl3)
d
7.89 (d, J ¼ 7.8 Hz, 2H), 7.68 (s,
1H), 7.55 (d, J ¼ 7.8 Hz, 2H), 7.48e7.41 (m, 2H), 7.40e7.31 (m, 3H),
3.91 (s, 2H), 3.80 (s, 2H), 3.65 (s, 2H), 3.11 (d, J ¼ 7.3 Hz, 2H), 2.48 (s,
2H), 1.62 (s, 1H), 1.31e1.27 (m, 3H), 0.86 (t, J ¼ 7.1 Hz, 4H). MS (ESI)
m/z: 472.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 172.57, 167.86,
3H). MS (ESI) m/z: 451.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d
146.43,
142.13, 141.36, 137.12, 136.76, 135.21, 130.19, 129.31, 128.50, 119.89,
119.38, 57.96, 55.79, 55.23, 50.64, 43.99, 20.30, 11.68, 7.38. HRMS
(m/z): [MHþ] calcd for C24H30N3O3S2 472.1723; found, 472.1729.
146.09, 136.60, 130.05, 129.67, 128.81, 128.51, 128.09, 126.68, 112.76,
57.95, 57.59, 55.08, 50.62, 44.78, 35.54, 20.01, 11.82, 7.46. HRMS (m/
z): [MHþ] calcd for C27H35N2O2S 451.2414; found, 451.2411.
4.2.2.4. N-(4-((benzyl(methyl)amino)methyl)phenyl)-2-(4-(ethyl-
4.2.2. General procedure B for the synthesis of compounds (2f-2h,
4b-4g, 5a-5c, 6a, 7a-7e, 8a-8c)
sulfonyl)phenyl)acetamide (4b). Yield: 77%, white solid. 1H NMR
(400 MHz, CDCl3)
d
8.08 (s, 1H), 7.84 (d, J ¼ 7.8 Hz, 2H), 7.59 (d, J ¼
7.8 Hz, 2H), 7.52 (s, 1H), 7.50 (d, J ¼ 8.0 Hz, 3H), 7.29 (d, J ¼ 10.4 Hz,
10

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
5H), 3.81 (s, 2H), 3.68e3.64 (m, 4H), 3.14 (d, J ¼ 7.1 Hz, 4H), 2.24 (d,
J ¼ 11.1 Hz, 4H).MS (ESI) m/z: 437.2 [MHþ]. 13C NMR (151 MHz,
4.2.2.10. N-(4-((benzyl(propyl)amino)methyl)-3-chlorophenyl)-2-(4-
(ethylsulfonyl)phenyl)acetamide (5a). Yield: 47%, white solid. 1H
CDCl3)
d
168.92, 141.66, 137.71, 136.43, 134.32, 130.50, 130.32,
NMR (400 MHz, CDCl3)
d
8.01 (s, 1H), 7.78 (d, J ¼ 8.2 Hz, 2H), 7.61 (s,
130.15, 129.56, 128.49, 128.18, 127.97, 120.38, 55.29, 43.20, 17.33,
12.41, 6.99. HRMS (m/z): [MHþ] calcd for C25H29N2O3S 437.1893;
found, 437.1891.
1H), 7.53 (d, J ¼ 8.4 Hz, 1H), 7.46 (d, J ¼ 8.1 Hz, 2H), 7.33 (d, J ¼
3.1 Hz, 1H), 7.30e7.23 (m, 3H), 7.20 (t, J ¼ 7.1 Hz, 1H), 3.73 (s, 2H),
3.57 (d, J ¼ 13.5 Hz, 4H), 3.09 (q, J ¼ 7.4 Hz, 2H), 2.36 (t, J ¼ 7.2 Hz,
2H),1.57e1.44 (m, 2H),1.25 (t, J ¼ 7.4 Hz, 3H), 0.81 (t, J ¼ 7.3 Hz, 3H).
MS (ESI) m/z: 499.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 168.22,
4.2.2.5. N-(4-((benzyl(ethyl)amino)methyl)phenyl)-2-(4-(ethyl-
141.36, 139.70, 137.24, 136.95, 133.98, 133.56, 130.63, 130.20, 128.65,
128.37, 128.13, 126.78, 120.53, 118.22, 58.42, 55.72, 54.66, 50.64,
43.77, 20.10, 11.82, 7.32. HRMS (m/z): [MHþ] calcd for
sulfonyl)phenyl)acetamide (4c). Yield: 40%, white solid. 1H NMR
(400 MHz, CDCl3)
d
7.85 (d, J ¼ 8.2 Hz, 2H), 7.69 (s, 1H), 7.56 (d, J ¼
8.1 Hz, 2H), 7.45 (d, J ¼ 8.4 Hz, 2H), 7.34 (t, J ¼ 6.5 Hz, 3H), 7.31e7.26
(m, 4H), 3.80 (s, 2H), 3.61 (s, 2H), 3.13 (dd, J ¼ 13.8, 6.3 Hz, 4H), 3.08
(d, J ¼ 7.4 Hz, 2H), 1.08 (t, J ¼ 7.1 Hz, 3H). MS (ESI) m/z: 451.2 [MHþ].
C
27H32ClN2O3S 499.1817; found, 499.1820.
4.2.2.11. N-(4-((benzyl(cyclopropylmethyl)amino)methyl)-3-
chlorophenyl)-2-(4-(ethylsulfonyl)phenyl)acetamide (5b). Yield:
38%, white solid. 1H NMR (400 MHz, CDCl3)
8.06 (s,1H), 7.79 (d, J ¼
13C NMR (151 MHz, CDCl3)
d 169.01, 141.88, 137.40, 136.67, 136.23,
130.53, 129.90, 129.32, 128.57, 128.24, 127.87, 120.58, 57.23, 56.89,
d
55.58, 43.49, 17.58, 12.66, 7.26. HRMS (m/z): [MHþ] calcd for
8.0 Hz, 2H), 7.63 (s, 1H), 7.58 (d, J ¼ 8.4 Hz, 1H), 7.47 (d, J ¼ 8.0 Hz,
2H), 7.37 (s, 1H), 7.32 (d, J ¼ 13.4 Hz,1H), 7.27 (d, J ¼ 8.9 Hz, 2H), 7.21
(t, J ¼ 7.2 Hz, 1H), 3.74 (s, 2H), 3.71 (s, 2H), 3.67 (s, 2H), 3.10 (q, J ¼
7.4 Hz, 2H), 2.33 (d, J ¼ 6.4 Hz, 2H), 1.26 (t, J ¼ 7.4 Hz, 3H), 0.97e0.85
(m, 1H), 0.45 (q, J ¼ 4.8 Hz, 2H), 0.05e0.00 (m, 2H). MS (ESI) m/z:
C
26H31N2O3S 451.2050; found, 451.2053.
4.2.2.6. N-(4-((benzyl(isobutyl)amino)methyl)phenyl)-2-(4-(ethyl-
sulfonyl)phenyl)acetamide (4d). Yield: 60%, white solid. 1H NMR
511.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d
168.28 (s), 141.39 (s),
(400 MHz, CDCl3)
d
7.85 (t, J ¼ 7.2 Hz, 2H), 7.55 (t, J ¼ 7.4 Hz, 2H),
7.45 (t, J ¼ 6.5 Hz, 2H), 7.37e7.27 (m, 6H), 7.23 (s, 1H), 3.79 (d, J ¼
139.76 (s), 137.24 (s), 136.89 (s), 133.95 (s), 133.55 (s), 130.64 (s),
130.21 (s), 128.58 (s), 128.33 (s), 128.13 (s), 126.77 (s), 120.54 (s),
118.24 (s), 58.55 (s), 58.26 (s), 54.42 (s), 50.63 (s), 43.73 (s), 8.35 (s),
7.30 (s), 3.96 (s).
7.0 Hz, 2H), 3.53e3.44 (m, 3H), 3.19e3.01 (m, 2H), 2.83e2.81 (m,
7H), 2.12 (s, 2H), 1.32e1.23 (m, 4H). MS (ESI) m/z: 479.2 [MHþ]. 13
C
NMR (151 MHz, CDCl3)
d 167.89, 141.51, 139.89, 137.17, 136.54,
130.22, 129.35, 128.81, 128.51, 128.10, 126.74, 58.69, 58.22, 50.65,
44.02, 38.64, 26.07, 20.78, 14.19, 7.38. HRMS (m/z): [MHþ] calcd for
4.2.2.12. N-(4-((benzyl(propyl)amino)methyl)-3,5-dichlorophenyl)-
2-(4-(ethylsulfonyl)phenyl)acetamide (5c). Yield: 71%, white solid.
C
28H35N2O3S 479.2363; found, 479.2360.
1H NMR (400 MHz, CDCl3)
d
8.19 (s, 1H), 7.77 (d, J ¼ 8.3 Hz, 2H), 7.55
(s, 2H), 7.45 (d, J ¼ 8.3 Hz, 2H), 7.27 (t, J ¼ 5.8 Hz, 2H), 7.23 (t, J ¼
7.3 Hz, 2H), 7.20e7.15 (m, 1H), 3.80 (s, 2H), 3.74 (s, 2H), 3.54 (s, 2H),
3.11 (q, J ¼ 7.4 Hz, 2H), 2.37 (dd, J ¼ 8.2, 6.5 Hz, 2H), 1.58e1.44 (m,
2H), 1.26 (dd, J ¼ 7.7, 4.7 Hz, 3H), 0.76 (t, J ¼ 7.3 Hz, 3H). MS (ESI) m/
4.2.2.7. N-(4-((benzyl(cyclopropylmethyl)amino)methyl)phenyl)-2-
(4-(ethylsulfonyl)phenyl)acetamide (4e). Yield: 83%, white solid. 1H
NMR (400 MHz, CDCl3)
d
7.86 (d, J ¼ 8.0 Hz, 2H), 7.53 (d, J ¼ 7.7 Hz,
2H), 7.41 (d, J ¼ 8.0 Hz, 2H), 7.37e7.27 (m, 6H), 7.21 (d, J ¼ 7.5 Hz,
1H), 3.78 (s, 2H), 3.61 (d, J ¼ 11.2 Hz, 4H), 3.10 (q, J ¼ 7.4 Hz, 2H),
2.82e2.76 (m, 3H), 2.29 (d, J ¼ 5.6 Hz, 2H), 1.29e1.26 (m, 3H), 0.44
(d, J ¼ 7.3 Hz, 2H). MS (ESI) m/z: 477.2 [MHþ]. 13C NMR (151 MHz,
z: 533.1 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 168.27, 141.04, 140.02,
137.83, 137.11, 137.01, 130.99, 130.21, 128.79, 128.44, 127.90, 126.62,
119.33, 57.92, 55.48, 53.12, 50.70, 43.83, 19.41, 11.88, 7.33. HRMS (m/
z): [MHþ] calcd for C27H31Cl2N2O3S 533.1427; found, 533.1435.
CDCl3)
d 168.12, 141.32, 139.04, 136.25, 136.03, 135.23, 129.59,
128.61, 128.11, 127.59, 127.48, 126.12, 119.29, 57.52, 57.32, 56.90,
49.34, 42.93, 37.86, 7.57, 6.57, 3.31. HRMS (m/z): [MHþ] calcd for
4.2.2.13. 1-(4-((benzyl(propyl)amino)methyl)phenyl)-3-(4-(ethyl-
sulfonyl)benzyl)urea (6a). A mixture of 4-((benzyl(propyl)amino)
methyl)aniline (90 mg, 0.35 mmol), triphosgene (35 mg,
C
28H33N2O3S 477.2206; found, 477.2205.
0.12 mmol) and DIPEA (181 mL, 1.05 mmol) in dry DCM (1 mL) was
4.2.2.8. N-(4-((benzyl(butyl)amino)methyl)phenyl)-2-(4-(ethyl-
stirred under ice bath for 30 min, then (4-(ethylsulfonyl)phenyl)
methanamine (70 mg, 0.35 mmol) was added. The mixture was
stirred at room temperature overnight. Purification by column
chromatography (DCM:MeOH 50:1) through silica gel afforded
sulfonyl)phenyl)acetamide (4f). Yield: 45%, white solid. 1H NMR
(400 MHz, CDCl3)
d
7.85 (d, J ¼ 7.8 Hz, 2H), 7.58e7.46 (m, 3H), 7.41
(d, J ¼ 8.2 Hz, 2H), 7.34e7.26 (m, 5H), 7.22e7.15 (m, 1H), 3.77 (s, 2H),
3.50 (d, J ¼ 11.8 Hz, 4H), 3.15e3.01 (m, 2H), 2.81e2.75 (m, 5H), 2.37
(s, 2H), 1.25e1.23 (m, 2H), 0.82e0.74 (m, 3H). MS (ESI) m/z: 479.2
white solid (90 mg, yield: 53.7%). 1H NMR (400 MHz, CDCl3)
d 7.69
(s, 1H), 7.65 (d, J ¼ 8.2 Hz, 2H), 7.35e7.27 (m, 6H), 7.25e7.16 (m, 5H),
4.31 (d, J ¼ 5.7 Hz, 2H), 3.49 (s, 2H), 3.43 (s, 2H), 3.03 (q, J ¼ 7.4 Hz,
2H), 2.32 (t, J ¼ 7.2 Hz, 2H), 1.54e1.44 (m, 2H), 1.19 (t, J ¼ 7.4 Hz, 3H),
0.81 (t, J ¼ 7.3 Hz, 3H). MS (ESI) m/z: 480.2 [MHþ]. 13C NMR
[MHþ]. 13C NMR (151 MHz, CDCl3)
d 167.65, 141.21, 139.67, 137.34,
136.29, 130.24, 129.38, 58.13, 57.67, 50.66, 44.15, 38.65, 29.11, 20.45,
14.02, 7.40. HRMS (m/z): [MHþ] calcd for C28H35N2O3S 479.2363;
found, 479.2369.
(151 MHz, CDCl3)
d 171.28, 156.21, 146.36, 139.69, 137.55, 136.59,
134.45, 129.42, 128.74, 128.21, 128.14, 127.70, 126.79, 119.59, 58.06,
57.63, 55.23, 50.61, 43.13, 20.01, 11.79, 7.29. HRMS (m/z): [MHþ]
calcd for C27H34N3O3S 480.2315; found, 480.2317.
4.2.2.9. N-(4-((benzyl(cyclobutylmethyl)amino)methyl)phenyl)-2-
(4-(ethylsulfonyl)phenyl)acetamide (4g). Yield: 76%, white solid. 1H
NMR (400 MHz, CDCl3)
d
8.10 (s, 1H), 7.79 (d, J ¼ 8.0 Hz, 2H), 7.48 (t,
J ¼ 8.4 Hz, 2H), 7.43 (d, J ¼ 8.1 Hz, 2H), 7.29 (dd, J ¼ 12.0, 5.3 Hz, 3H),
7.26e7.22 (m, 3H), 7.20 (d, J ¼ 6.9 Hz, 1H), 3.73 (s, 2H), 3.44 (d, J ¼
13.1 Hz, 4H), 3.07 (q, J ¼ 7.4 Hz, 2H), 2.79 (s, 4H), 2.38 (d, J ¼ 7.0 Hz,
2H), 1.81 (dd, J ¼ 18.3, 8.9 Hz, 1H), 1.23 (d, J ¼ 7.2 Hz, 5H). MS (ESI)
4.2.2.14. N-(4-((benzyl(propyl)amino)methyl)phenyl)-2-(4-(methyl-
sulfonyl)phenyl)acetamide (7a). Yield: 55%, white solid. 1H NMR
(400 MHz, CDCl3)
d
9.90 (s, 1H), 7.92 (d, J ¼ 8.1 Hz, 2H), 7.84 (dd, J ¼
13.1, 8.3 Hz, 2H), 7.71 (d, J ¼ 8.5 Hz, 2H), 7.56 (d, J ¼ 8.0 Hz, 4H), 7.37
(d, J ¼ 6.9 Hz, 3H), 4.02 (s, 2H), 3.86 (s, 2H), 3.05 (s, 3H), 2.80e2.62
(m, 2H), 1.80 (dd, J ¼ 16.7, 8.9 Hz, 4H), 0.90 (t, J ¼ 7.4 Hz, 3H). MS
m/z: 491.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 167.61, 141.16, 137.37,
130.24, 129.35, 128.74, 128.65, 128.13, 126.79, 59.88, 58.41, 57.92,
50.66, 44.17, 38.64, 33.76, 27.31, 18.69, 7.40. HRMS (m/z): [MHþ]
calcd for C29H35N2O3S 491.2363; found, 491.2359.
(ESI) m/z: 451.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 165.78, 141.73,
139.16, 136.81, 130.34, 129.99, 129.25, 128.75, 128.12, 127.66, 127.61,
11

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
126.75, 119.76, 58.14, 57.72, 55.29, 44.56, 43.94, 38.63, 11.78. HRMS
white solid. 1H NMR (400 MHz, CDCl3)
d
7.92 (d, J ¼ 8.3 Hz, 2H), 7.56
(m/z): [MHþ] calcd for C26H31N2O3S 451.2050; found, 451.2046.
(d, J ¼ 8.2 Hz, 2H), 7.29 (dd, J ¼ 8.4, 2.0 Hz, 1H), 7.26 (s, 2H), 3.82 (s,
2H), 3.62 (s, 2H), 3.06 (s, 3H), 2.39 (s, 2H), 2.22 (d, J ¼ 15.2 Hz, 2H),
1.79 (d, J ¼ 12.4 Hz, 2H), 1.67e1.60 (m, 3H), 1.54e1.42 (m, 3H),
1.21e1.07 (m, 3H), 0.85 (t, J ¼ 7.3 Hz, 3H), 0.82e0.70 (m, 2H). MS
4.2.2.15. 4-(2-((4-((benzyl(propyl)amino)methyl)phenyl)amino)-2-
oxoethyl)benzoic acid (7b). Yield: 62%, white solid. 1H NMR
(400 MHz, CD3OD)
d
7.95 (d, J ¼ 8.0 Hz, 2H), 7.68 (d, J ¼ 8.3 Hz, 2H),
(ESI) m/z: 490.8[MHþ].13C NMR (101 MHz, CDCl3)
d 140.85 (s),
7.49e7.37 (m, 9H), 4.22 (s, 2H), 4.20 (s, 2H), 3.76 (s, 2H), 2.87 (dd, J ¼
9.7, 6.6 Hz, 2H), 1.81e1.68 (m, 2H), 0.86 (t, J ¼ 7.3 Hz, 3H). MS (ESI)
139.58 (s), 130.41 (s), 127.93 (s), 120.47 (s), 118.03 (s), 56.65 (s),
55.71 (s), 44.56 (s), 44.14 (s), 31.82 (s), 26.74 (s), 26.10 (s), 11.85 (s).
HRMS (m/z): [MHþ] calcd for C26H35ClN2O3S 491.2130; found,
491.2132.
m/z: 415.2 [MH-]. 13C NMR (151 MHz, CD3OD)
d 169.54, 168.24,
139.42, 139.03, 130.49, 129.95, 129.77, 129.50, 128.76, 128.56,
128.09, 125.21, 119.33, 56.09, 55.79, 52.76, 42.36, 16.24, 9.14. HRMS
(m/z): [MHþ] calcd for C26H29N2O3 417.2173; found, 417.2176.
4.2.2.21. N-(3,5-dichloro-4-(((cyclohexylmethyl)(propyl)amino)
methyl)phenyl)-2-(4-(methylsulfonyl)phenyl)acetamide
(8c).
7.89 (d, J ¼
Yield: 71%, white solid. 1H NMR (400 MHz, CDCl3)
d
4.2.2.16. N-(4-((benzyl(propyl)amino)methyl)phenyl)-2-(4-
cyanophenyl)acetamide (7c). Yield: 59%, white solid. 1H NMR
8.3 Hz, 2H), 7.54 (s, 2H), 7.52 (d, J ¼ 8.3 Hz, 2H), 3.80 (s, 2H), 3.72 (s,
2H), 3.06 (s, 3H), 2.38 (t, J ¼ 7.2 Hz, 2H), 2.20 (d, J ¼ 6.9 Hz, 2H), 1.74
(d, J ¼ 12.0 Hz, 2H), 1.65e1.57 (m, 3H), 1.50e1.40 (m, 3H), 1.18e1.05
(m, 3H), 0.80 (t, J ¼ 7.3 Hz, 3H), 0.69e0.59 (m, 2H). MS (ESI) m/z:
(400 MHz, CDCl3)
d
7.63 (t, J ¼ 7.8 Hz, 2H), 7.45 (d, J ¼ 8.0 Hz, 2H),
7.40 (d, J ¼ 8.2 Hz, 2H), 7.34 (d, J ¼ 7.4 Hz, 2H), 7.32e7.27 (m, 4H),
7.22 (t, J ¼ 7.2 Hz, 1H), 3.74 (s, 2H), 3.52 (s, 2H), 3.48 (s, 2H), 2.35 (t,
J ¼ 7.3 Hz, 2H), 1.52 (dt, J ¼ 14.5, 0.3 Hz, 3H), 0.83 (t, J ¼ 7.3 Hz, 3H).
524.8[MHþ].13C NMR (101 MHz, CDCl3)
d 167.89 (s), 140.58 (s),
MS (ESI) m/z: 398.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 167.25,
139.57 (s),137.52 (s),137.22 (s),130.41 (s),127.92 (s),119.21 (s), 61.10
(s), 56.24 (s), 53.83 (s), 44.56 (s), 44.07 (s), 35.80 (s), 31.84 (d, J ¼
10.5 Hz), 26.85 (s), 26.13 (s), 22.70 (s), 19.52 (s), 14.12 (s), 11.92 (s).
HRMS (m/z): [MHþ] calcd for C26H34Cl2N2O3S 525.1740; found,
525.1740.
139.75, 139.25, 135.90, 135.66, 131.83, 129.56, 128.69, 128.12, 127.56,
126.18, 119.33, 118.14, 110.40, 57.55, 57.07, 54.72, 43.54, 19.51, 11.22.
HRMS (m/z): [MHþ] calcd for C26H28N3O 398.2227; found,
398.2236.
4.2.2.17. N-(4-((benzyl(propyl)amino)methyl)phenyl)-2-
4.2.3. General procedure C for the synthesis of compounds (4a, 4h-
4k)
phenylacetamide (7d). Yield: 77%, white solid. 1H NMR (400 MHz,
CDCl3)
d
7.37 (d, J ¼ 6.5 Hz, 2H), 7.35e7.30 (m, 6H), 7.30 (s, 1H), 7.28
(d, J ¼ 2.5 Hz, 2H), 7.25 (s, 2H), 7.21 (d, J ¼ 7.2 Hz, 1H), 3.71 (s, 2H),
3.50 (s, 2H), 3.47 (s, 2H), 2.37e2.29 (m, 2H), 1.49 (t, J ¼ 14.5, 7.3 Hz,
3H), 0.81 (t, J ¼ 7.3 Hz, 2H). MS (ESI) m/z: 373.2 [MHþ]. 13C NMR
Step 1. To a solution of nitrobenzene intermediates (1.0 eq), amine
(3.0 eq) and anhydrous potassium carbonate (1.0 eq) in acetonitrile
was added potassium iodide (0.1 eq). The mixture was heated up to
110 ꢀC in microwave reactor for 30 min. Purification by column
chromatography through silica gel afforded the desired interme-
diate 20.
(151 MHz, CDCl3)
d 169.16, 136.37, 134.60, 129.47, 129.24, 129.11,
128.72, 128.10, 127.52, 126.71, 119.70, 58.12, 57.67, 55.27, 44.70,
20.09, 11.77. HRMS (m/z): [MHþ] calcd for C25H29N2O 373.2274;
found, 373.2277.
Step 2. To a solution of compound 20 (1.0 eq), 1-bromopropane
(3.0 eq) and anhydrous potassium carbonate (1.0 eq) in acetoni-
trile was added potassium iodide (0.1 eq). The mixture was heated
up to 110 ꢀC in microwave reactor for 30 min. Purification by col-
umn chromatography through silica gel afforded the desired in-
termediate 21.
4.2.2.18. N-(4-((benzyl(propyl)amino)methyl)phenyl)-2-(5-(ethyl-
sulfonyl)pyridin-2-yl)acetamide (7e). Yield: 89%, white solid. 1H
NMR (400 MHz, CDCl3)
d
10.19 (s, 1H), 8.72 (s, 1H), 7.95 (d, J ¼
8.0 Hz, 1H), 7.66 (d, J ¼ 8.1 Hz, 2H), 7.57 (d, J ¼ 8.0 Hz, 1H), 7.49 (d,
J ¼ 6.9 Hz, 2H), 7.45e7.34 (m, 5H), 4.41e4.30 (m, 2H), 4.30e4.21 (m,
2H), 4.04 (s, 2H), 2.87e2.74 (m, 4H), 1.97e1.86 (m, 2H), 1.22 (t, J ¼
7.4 Hz, 3H), 0.83 (t, J ¼ 7.2 Hz, 3H). MS (ESI) m/z: 466.2 [MHþ]. 13
C
Step 3. To a solution of 21 (1.0 eq) in methanol was added 10% Pd/
C. The mixture was degassed and purged with hydrogen and stirred
at room temperature for 1 h. The solution was filtered through
celite and evaporated under reduced pressure to afford desired
product 22 as white solid.
NMR (101 MHz, CDCl3) d 167.80 (s), 158.59 (s), 145.30 (s), 138.23 (s),
133.22 (d, J ¼ 19.3 Hz), 132.46 (s), 130.21 (s), 128.90 (s), 124.93 (s),
123.17 (s), 119.92 (s), 69.05 (s), 68.84 (s), 63.71 (s), 50.37 (s), 46.18
(s), 29.71 (s), 29.33 (s), 17.20 (s), 14.13 (s), 10.67 (s), 5.96 (s). MS (ESI)
m/z: 466.2 [MHþ]. HRMS (ESIþ) m/z calcd for C26H31N3O3S
[MþH]þ: 466.2159; found: 466.2163.
Step 4. To a solution of 22 (1.0 eq) in DCM was added p-alkyl-
sulfone aryl acetic acid (1.2 eq), HATU (1.2 eq) and DIPEA (1.2 eq).
The mixture was stirred at room temperature for 5 h. Purification
by column chromatography through silica gel afforded desired
compound (4a, 4h-4k)
4.2.2.19. N-(4-(((cyclohexylmethyl)(propyl)amino)methyl)phenyl)-2-
(4-(methylsulfonyl)phenyl)acetamide (8a). Yield: 48%, white solid.
1H NMR (400 MHz, CDCl3)
d
7.90 (dd, J ¼ 7.3, 4.5 Hz, 2H), 7.55 (dd,
J ¼ 7.3, 4.5 Hz, 2H), 7.43 (d, J ¼ 8.4 Hz, 2H), 7.27 (d, J ¼ 8.4 Hz, 2H),
3.79 (s, 2H), 3.50 (s, 2H), 3.04 (s, 3H), 2.33 (t, J ¼ 7.2 Hz, 2H), 2.18 (d,
J ¼ 7.0 Hz, 2H),1.78 (d, J ¼ 12.7 Hz, 2H), 1.70e1.58 (m, 3H), 1.50e1.41
(m, 3H), 1.21e1.05 (m, 3H), 0.83 (t, J ¼ 7.4 Hz, 3H), 0.80e0.70 (m,
4.2.3.1. 2-(4-(ethylsulfonyl)phenyl)-N-(4-(2-(phenyl(propyl)amino)
ethyl)phenyl)acetamide (4k). Yield: 12%, white solid. 1H NMR
(400 MHz, CDCl3)
d
7.78 (d, J ¼ 8.0 Hz, 2H), 7.71 (d, J ¼ 8.2 Hz, 2H),
2H). MS (ESI) m/z: 457.3[MHþ]. 13C NMR (101 MHz, CDCl3)
d
167.63
7.47e7.39 (m, 4H), 7.34 (d, J ¼ 7.3 Hz, 2H), 7.29 (t, J ¼ 7.4 Hz, 2H),
7.21 (t, J ¼ 7.1 Hz, 1H), 7.10e7.04 (m, 1H), 4.68 (d, J ¼ 6.0 Hz, 2H),
3.54 (d, J ¼ 12.7 Hz, 4H), 3.04 (q, J ¼ 7.4 Hz, 2H), 2.38 (t, J ¼ 7.1 Hz,
2H), 1.56e1.42 (m, 2H), 1.28 (dd, J ¼ 15.3, 7.7 Hz, 2H), 1.21 (t, J ¼
7.5 Hz, 3H), 0.87e0.77 (m, 3H). MS (ESI) m/z: 465.2 [MHþ]. 13C NMR
(s), 141.16 (s), 139.43 (s), 136.28 (s), 130.41 (s), 129.51 (s), 127.86 (s),
119.73 (s), 61.03 (s), 58.76 (s), 56.21 (s), 44.55 (s), 44.17 (s), 35.81 (s),
31.77 (s), 26.78 (s), 26.13 (s), 19.91 (s), 11.81 (s). HRMS (m/z): [MHþ]
calcd for C26H36N2O3S 457.2519; found, 457.2524.
(151 MHz, MeOD)
d 170.79, 143.60, 138.47, 137.93, 131.38, 130.29,
4.2.2.20. N-(3-chloro-4-(((cyclohexylmethyl)(propyl)amino)methyl)
phenyl)-2-(4-(methylsulfonyl)phenyl)acetamide (8b). Yield: 76%,
129.50, 121.47, 54.41, 51.29, 49.65, 44.26, 33.87, 21.43, 11.65, 7.62.
HRMS (m/z): [MHþ] calcd for C27H33N3O3S 465.2206; found,
12

R. Qiu, M. Yu, J. Gong et al.
European Journal of Medicinal Chemistry 224 (2021) 113704
465.2201.
LANCE mode configured for europeum-APC labels.
4.2.3.2. 2-(4-(ethylsulfonyl)phenyl)-N-(4-(2-(phenylamino)ethyl)
4.4. RORgt GAL4 cell-based reporter gene assay
phenyl)acetamide (4i). Yield: 45%, white solid. 1H NMR (400 MHz,
CDCl3)
d
7.84 (d, J ¼ 8.2 Hz, 2H), 7.57 (s, 1H), 7.52 (d, J ¼ 8.2 Hz, 2H),
hROR
g
t LBD coding sequence was inserted into a pBIND
7.41 (d, J ¼ 8.3 Hz, 2H), 7.20e7.09 (m, 4H), 6.69 (t, J ¼ 7.3 Hz, 1H),
6.59 (d, J ¼ 7.9 Hz, 2H), 3.77 (s, 2H), 3.34 (t, J ¼ 6.9 Hz, 2H), 3.10 (q,
J ¼ 7.4 Hz, 3H), 2.85 (t, J ¼ 6.9 Hz, 2H), 1.24 (d, J ¼ 4.2 Hz, 2H). MS
expression vector (Promega, E1581) to express ROR
gt-GAL4 bind-
ing domain chimeric receptors. This expression vector and a re-
porter vector (pGL4.35 which carries a stably integrated GAL4
promoter driven luciferase reporter gene [luc2P/9XGAL4 UAS/
Hygro]) were co-transfected into HEK293T host cells. Upon agonist
(ESI) m/z: 423.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 167.84, 147.77,
141.19, 137.27, 135.99, 135.73, 130.24, 129.30, 128.60, 120.33, 117.65,
113.12, 50.64, 45.06, 44.05, 34.80, 7.39. HRMS (m/z): [MHþ] calcd for
binding to the corresponding RORgt-GAL4 chimeric receptor, the
C
24H27N2O3S 423.1737; found, 423.1741.
chimeric receptor binds to the GAL4 binding sites and stimulates
the reporter gene. In the present of inverse agonist, agonist will
bind competitively to the nuclear receptor and activate the reporter
gene transcription. HEK293T cells were cultured in a culture me-
dium composed of DMEM containing 5% charcoal-treated FBS at
37 ꢀC under 5% CO2 atmosphere, as ATCC recommended. Before
assay, the cells were washed with PBS to remove phenol red and
suspended in phenol red-free medium (phenol red-free DMEM
containing 5% charcoaltreated FBS and Penicillin-Streptomycin
(10,000 U/mL) to a proper concentration. 6 ꢂ 106 HEK293T cells
were seeded into a 100 mm dish and incubated for 16 h. To a re-
agent mixture of Trans-IT reagent and Opti-MEM (Invitrogen) was
4.2.3.3. 2-(4-(ethylsulfonyl)phenyl)-N-(4-(phenethyl(propyl)amino)
phenyl)acetamide (4j). Yield: 37%, white solid. 1H NMR (400 MHz,
CDCl3)
d
7.86 (d, J ¼ 8.2 Hz, 2H), 7.54 (d, J ¼ 8.1 Hz, 2H), 7.31e7.26
(m, 4H), 7.23e7.19 (m, 1H), 7.17 (d, J ¼ 7.5 Hz, 2H), 6.63 (d, J ¼ 8.4 Hz,
2H), 3.76 (s, 2H), 3.48 (s, 2H), 3.20e3.06 (m, 4H), 2.81 (s, 2H),
1.59e1.46 (m, 2H), 1.30e1.26 (m, 3H), 0.87 (t, J ¼ 7.4 Hz, 3H). MS
(ESI) m/z: 465.2 [MHþ]. 13C NMR (151 MHz, CDCl3)
d 167.44, 141.45,
137.36, 130.25, 128.73, 128.67, 128.56, 126.31, 122.32, 111.90, 50.66,
44.06, 11.39, 7.42. HRMS (m/z): [MHþ] calcd for C27H33N2O3S
465.2206; found, 465.2199.
added plasmid DNA (used as 0.5 mg/mL stocks), containing 5
mg
4.2.3.4. N-(4-((benzylamino)methyl)phenyl)-2-(4-(ethylsulfonyl)
ROR t plasmid and 5 g pGL4.35 luciferase plasmid. The mixture
g
m
phenyl)acetamide (4a). Yield: 66%, white solid. 1H NMR (400 MHz,
was added to the cells in the 100 mm dish and incubated for 5e6 h.
Test compounds were serially diluted in DMSO to 300 nM. Com-
pounds (25 nL) were transferred into a 384-well plate (white
opaque) using Echo550. Then seeded the cells at 15,000 cells/well
into the 384-well plate using phenol red-free DMEM containing 5%
CD3OD)
d
7.87 (d, J ¼ 8.3 Hz, 2H), 7.71e7.65 (m, 1H), 7.62 (d, J ¼
8.1 Hz, 2H), 7.50e7.38 (m, 5H), 7.36e7.20 (m, 3H), 4.19 (d, J ¼ 7.9 Hz,
2H), 3.82 (d, J ¼ 8.6 Hz, 2H), 3.30e3.29 (m, 5H), 3.19 (q, J ¼ 7.3 Hz,
2H). MS (ESI) m/z: 423.2 [MHþ]. 13C NMR (151 MHz, CD3OD)
d
171.15, 143.33, 141.23, 138.58, 131.87, 131.43, 131.07, 130.78, 130.38,
charcoal-treated FBS and 0.25
m
M ursolic acid. Cells were incubated
129.51, 121.55, 52.02, 51.70, 51.28, 44.28, 7.62. HRMS (m/z): [MHþ]
for 16e20 h at 37 ꢀC (white opaque) using Echo550
m
L of Steady-
calcd for C24H27N2O3S 423.1737; found, 423.1740.
Glo™ Luciferase Assay Reagent was added into each well of the
384-well plate. Shake the plate (avoiding light) for 5 min on a plate
shaker. Record the luminescence value on Envision 2104 plate
reader. Activation/Inhibition values of the compounds at 300 nM
were determined by the average value of duplicated tests.
4.2.3.5. 2-(4-(ethylsulfonyl)phenyl)-N-(4-(phenethylamino)phenyl)
acetamide (4h). Yield: 61%, white solid. 1H NMR (400 MHz, CDCl3)
d
7.85 (d, J ¼ 8.2 Hz, 2H), 7.46e7.38 (m, 4H), 7.36 (s, 3H), 7.21 (d, J ¼
8.4 Hz, 2H), 6.58 (d, J ¼ 8.3 Hz, 2H), 3.46 (t, J ¼ 6.9 Hz, 2H), 3.12 (q,
J ¼ 7.5 Hz, 2H), 3.02 (t, J ¼ 6.9 Hz, 2H), 1.92 (dd, J ¼ 15.8, 7.6 Hz, 2H),
1.29 (t, J ¼ 7.4 Hz, 3H). MS (ESI) m/z: 423.2 [MHþ]. 13C NMR
4.5. Mouse Th17 differentiation assay
(151 MHz, CDCl3)
d
167.41, 145.44, 141.39, 139.15, 137.31, 130.24,
CD4þ T cells were purified from mouse splenocytes using a
commercial CD4þ T cell negative selection kit (Invitrogen). The 48-
well plates were wrapped in the presence anti-CD3 (0.25 mg/mL,
Bioxcel), and anti-CD28 (1 mg/mL, Bioxcel) at 0 ꢀC overnight. CD4þ
T cells were skewed to Th17 cells by culturing cells in the presence
128.76, 128.64, 128.62, 126.46, 122.26, 115.57, 113.12, 50.64, 45.24,
44.00, 35.38, 7.41, 0.00.
4.3. RORgt dual FRET assay
of anti-IFNg (10 mg/mL, Bioxcel), anti-IL-4 (10 mg/mL, Bioxcel), TGF-b
The assay was performed in an assay buffer consisting of 50 mM
NaF, 50 mM 3-(N-morpholino)propanesulfonic acid (pH ¼ 7.4),
0.05 mM 3-[(3-cholamidopropyl) dimethylammonio]propane-
sulfonate, 0.1 mg/mL bovine serum albumin, and 10 mM dithio-
(2 ng/mL, Peprotech) and IL-6 (20 ng/mL, Peprotech) for 4 days
before analysis. Compounds or DMSO control were added to the
culture on day 0 of Th17 differentiation at indicated concentrations.
Percentage of IL-17 production from CD4þ T cells were analyzed by
intracellular staining followed by flow cytometry. Dose-response
curves were plotted to determine half-maximal inhibitory con-
centrations (EC50) for the compounds using the GraphPad Prism 5
(GraphPad Software, San Diego CA, USA).
threitol in 384-well plates. The total volume was 25 mL/well. The
europium-labeled SRC1 solution was prepared by adding an
appropriate amount of biotinylated SRC and europium labeled
streptavidin into assay buffer, with final concentrations of 20 and
10 nM, respectively. The allophycocyanin (APC)-labeled-LBD solu-
tion was prepared by adding an appropriate amount of biotinylated
4.6. Molecular docking studies
RORgt-LBD and APC-labeled streptavidin at final concentrations of
20 and 10 nM, respectively. After 15 min of incubation at room
temperature, a 20-fold excess of biotin was added and incubated for
10 min at room temperature to block the remain ning free strep-
tavidin. Equal volumes of europium-labeled SRC and APC-labeled
Molecular docking was carried out using Schrodinger 3.5 soft-
ware package. The co-crystal structure of RORgt LBD (PDB: 4NIE,
resolution of 2.01 Å) was selected and processed using the Protein
Preparation Wizard including water deletion, addition of missing
hydrogen atoms as well as adjustment of the tautomerization and
protonation states of histidine. The compound 3D structures were
subjected to energy minimization with force field (OPLS_2005)
before submitting to the docking procedure. The docking grid was
RORgt-LBD were dispensed into 384-well assay plates at 25 mL
volume/well. The 384-well assay plates had 100 nL of test com-
pound in DMSO predispensed into each well. The plates were
incubated for 1 h at room temperature and then read on Envision in
13

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Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
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Acknowledgements
This work was supported by National Natural Science Founda-
tion of China(Grant Numbers: 81874287; 81973163; 82003577),
Shanghai Bio-pharmaceutical Science and Technology Supporting
Plan of China (Grant Number: 19431900100), National Science and
Technology Major Project (Grant Number: 2018ZX09711002-003-
014), Natural Science Foundation of Shanghai (Grant Number:
19ZR1436700) and China Postdoctoral Science Foundation (Grant
Number: 2019M651383).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113704.
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