Discovery of novel small-molecule inhibitors of pd-1/pd-l1 interaction via structural simplification strategy

Article abstract of DOI:10.3390/molecules26113347

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer tr

Full text of DOI:10.3390/molecules26113347

molecules
Article
Discovery of Novel Small-Molecule Inhibitors of PD-1/PD-L1
Interaction via Structural Simplification Strategy
Hongbo Zhang , Yu Xia , Chunqiu Yu , Huijie Du , Jinchang Liu , Hui Li , Shihui Huang , Qihua Zhu 1,2,
1
2
2
2
2
2
2
Yungen Xu 1 * and Yi Zou *
,2,
2,
1
2
State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, China;
zhanghongbo397@sina.com (H.Z.); zhuqihua@vip.126.com (Q.Z.)
Jiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry,
China Pharmaceutical University, Nanjing 210009, China; xiayujacob0213@gmail.com (Y.X.);
ycq20151025@163.com (C.Y.); duhuijie1210@163.com (H.D.); 13022509809@163.com (J.L.);
15536369056@163.com (H.L.); hsh17314973301@126.com (S.H.)
*
Correspondence: xyg@cpu.edu.cn (Y.X.); zouyi@cpu.edu.cn (Y.Z.); Tel.: +86-025-8618-5303 (Y.X.)
Abstract: Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-
L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several
monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer treatment. Despite
this achievement, mAbs-based therapies are struggling with limitations including poor tissue and
tumor penetration, long half-life time, poor oral bioavailability, and expensive production costs,
which prompted a shift towards the development of the small-molecule inhibitors of PD-1/PD-L1
pathways. Even though many small-molecule inhibitors targeting PD-1/PD-L1 interaction have
been reported, their development lags behind the corresponding mAb, partly due to the challenges
of developing drug-like small molecules. Herein, we report the discovery of a series of novel
inhibitors targeting PD-1/PD-L1 interaction via structural simplification strategy by using BMS-1058
as a starting point. Among them, compound A9 stands out as the most promising candidate with
excellent PD-L1 inhibitory activity (IC50 = 0.93 nM, LE = 0.43) and high binding affinity to hPD-L1
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Zhang, H.; Xia, Y.; Yu, C.;
Du, H.; Liu, J.; Li, H.; Huang, S.; Zhu,
Q.; Xu, Y.; Zou, Y. Discovery of Novel
Small-Molecule Inhibitors of
(KD = 3.64 nM, LE = 0.40). Furthermore, A9 can significantly promote the production of IFN-γ in a
PD-1/PD-L1 Interaction via
Structural Simplification Strategy.
Molecules 2021, 26, 3347. https://
doi.org/10.3390/molecules26113347
dose-dependent manner by rescuing PD-L1 mediated T-cell inhibition in Hep3B/OS-8/hPD-L1 and
CD3-positive T cells co-culture assay. Taken together, these results suggest that A9 is a promising
inhibitor of PD-1/PD-L1 interaction and is worthy for further study.
Academic Editor: Silvia Schenone
Keywords: small molecule PD-1/PD-L1 inhibitor; PD-1/PD-L1; structural simplification strategy
Received: 3 May 2021
Accepted: 25 May 2021
Published: 2 June 2021
1
. Introduction
Programmed cell-death receptor (PD-1) and programmed cell death-ligand 1 (PD-L1)
inhibitors have achieved significant success in the oncology community both preclinically
and clinically [ ]. PD-L1, typically expressed on the surface of tumor cells, is bound
to PD-1 on tumor-infiltrating lymphocytes, thereby inhibiting T-cell functions and con-
secutively inducing tumor immune escape [ ]. Consequently, blocking PD-1/PD-L1
interactions has become a prospective approach in cancer treatment, which can restore
immune system and elicit a favorable tumor-specific T cells response [ ]. In the past few
years, several monoclonal antibodies (mAbs) targeting PD-1 or PD-L1 have been approved
in clinical use by the U.S. Food and Drug Administration (FDA) [ ], exhibiting signifi-
cant benefits with durable clinical responses and acceptable treatment-related toxicities
in several types of solid tumors [10 12]. Although these mAbs have transformed cancer
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9
–
immunotherapy forever, they still exhibit several disadvantages such as very long half-life,
immune-related adverse effects (irAEs), low permeability, immunogenicity, complex pro-
duction process, and exorbitant treatment costs from another perspective [13]. Among the
reported immunological adverse events, the overall incidences of all-grade adverse events
4
.0/).
Molecules 2021, 26, 3347. https://doi.org/10.3390/molecules26113347
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 3347
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were 66.0% and of grade 3 or higher adverse events were 14.0%. The most common side
effects are endocrinological disturbances, dermatologic manifestations, and gastrointestinal
disturbances. Apart from this, the most common grade 3 or higher adverse events were
fatigue, anemia, and aspartate aminotransferase increase [14,15]. Compared to mAbs,
small-molecule drugs have attracted tremendous attentions in immuno-oncology in recent
years, due to their better pharmacokinetic properties and diffusion rates and convenient
manufacture [16].
Recently, several small molecules derived from 2-methyl-3-biphenyl methanol scaffold
were disclosed as effective inhibitors of PD-1/PD-L1 interaction (Figure 1) [17–21]. Most
of the inhibitors show moderate to excellent activities of blocking PD-1/PD-L1 protein–
protein interaction (PPI) with IC50 values between picomolar to nanomolar level through
europium (Eu) allophycocyanin (APC) homogeneous time-resolved fluorescence (HTRF)
−
binding assay. The first co-crystal complex structure of small-molecule BMS-202 and hPD-
L1 reveals that this compound induces and stabilize the formation of PD-L1 homodimers,
leading to the blockade of PD-1/PD-L1 interaction [20,22,23].
Figure 1. Structures of the reported compounds with binding PD-1/PD-L1 interaction. Biphenyl moiety is colored by red.
IC50 values are established by the HTRF binding assay.
Since small molecules for immuno-oncology therapeutics have not been approved yet,
future research should continue to focus on the discovery of novel small molecules activat-
ing the immune system with orally bioavailable compounds to combat cancer. Although
some compounds bearing biphenyl moiety and their bivalent derivatives can inhibit the
interaction of PD-1 with PD-L1 with IC50 values of
have inherent disadvantages such as high molecular weight (MW), molecular polarity,
and degree of dissociation, which might be responsible for the poor pharmacokinetic (PK)
≤
10 nM [22–24], most of the inhibitors
profile in preclinical and clinical studies [20,25,26]. Therefore, it still needs further studies
to design small-molecule inhibitors of PD-1/PD-L1 interaction with better physicochemical
properties. In the present study, we designed and synthesized a novel series of 2-(2-methyl-
0
[
1,1 -biphenyl]-3-yl) pyridine derivatives targeting PD-1/PD-L1 interaction via structural
simplification strategy. During our design process, several structural indices, including
MW, ALogP, and polar surface area (PSA), were considered to monitor their potential drug
ability. The ligand efficiency (LE), a measure for the activity normalized by the number of
non-H atoms, was also used as a valuable criterion for evaluating the quality of molecular
design. Note that utilization of this metric during the overall drug design process to control
the balance of molecular size and potency will significantly improve success rates [27].
Fortunately, our research leads to the identification of compound A9 that shows wonderful
inhibitory activity at molecular level with a high LE value (HTRF IC50 = 0.93 nM, LE = 0.43
;

Molecules 2021, 26, 3347
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surface plasmon resonance (SPR) K = 3.64 nM, LE = 0.40). In addition, A9 potently in-
D
duces the release of IFN-
γ in a Hep3B/OS-8/hPD-L1 and primary T cell co-culture system,
deserving further investigation on its role in restoring immune surveillance.
2. Results and Discussion
2.1. Design and Screening
In our pursuit of novel small molecule inhibitors targeting the PD-1/PD-L1 pathway,
we focused our effort on designing molecules with better drug-likeness property. BMS-1058
IC50 = 0.48 nM) and BMS-202 (IC50 = 18 nM), which inhibited the PD-1/PD-L1 interaction
(
in HTRF binding assay, were chosen as the template compounds for structural optimization.
Structural analysis of the X-ray crystal complex of BMS-202 and hPD-L1 showed that it
comprises three essential pharmacophores: (1) The biphenyl core, the minimal fragment
essential for the hPD-L1 binding; (2) the polar moiety, the fragment interacting with
the polar residues in solvent-oriented region; (3) the linker connecting these two regions
(Figure 2A). We noticed that BMS-1058, with excellent IC50 values of 0.48 nM but possessing
high MW and PSA value, has two polar parts, located at opposite ends of this molecule.
Thus, it provided us a clue to design compounds by removing one side of the polar parts
of BMS-1058, leading to the discovery of compound A1 (Figure 2B). It has moderate IC50
value of 113.6 nM but shows higher ligand-binding efficiency (LE = 0.38) than that of
BMS-1058 (LE = 0.24) and BMS-202 (LE = 0.33) (Table 1), which gave us a valuable starting
point for further optimization. In order to rationally design more potent inhibitors without
reducing LE value, the in-silico docking prediction was conducted using Glide module in
Schrödinger [28]. As shown in Figure 2C, the biphenyl core of A1 anchors at the bottom
of the pocket, and two methyl groups form hydrophobic interactions with neighboring
hydrophobic residues. In addition, one of the phenyl rings forms a T-stacking interaction
with the sidechain of Tyr56. The protonated piperidin-4-ol moiety faces solvent-exposed
A
environment and interacts with Asp122 and Tyr56 by ionic and cation-π interactions,
A
B
respectively. On the basis of this analysis, optimization of A1 would be employed by the
following considerations: Substitutes on the distal phenyl ring, the linker, and the polar
group in the tail.
Table 1. Activities of compounds A1–A4 in inhibition of the PD-1/PD-L1 interaction.
Inhibitory Rate (%)
IC50 (nM) a
ALogP b
LE c
Compd.
R1
Linker
MW
PSA b
1
µM
0.1 µM
A1
A2
CH3
F
90
56
113.6
N.D.
339.22
382.24
2.38
2.09
33.90
27.91
0.38
ND
6.7
8.5
A3
A4
F
F
96.2
98.0
74.5
18.4
405.21
406.21
5.05
4.86
32.70
45.59
0.35
0.36
71.0
21.9
0.48 d
18.0
BMS1058
BMS202
a
753.33
419.22
4.04
4.04
132.41
72.48
0.24
0.33
The data were generated from two independent experiments. b Estimated physicochemical parameters by Discovery Studio [29]. c The
results were calculated by the equation: LE =
value is taken from the patent [18].
−
1.35 logIC50/Nheavy atoms, Nheavy atoms is the number of non-H atoms. d This IC50

Molecules 2021, 26, 3347
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Figure 2. (A) Crystal structure of PD-L1 (chain A, yellow; chain B, purple)/BMS-202(white) complex (PDB code: 5J89).
Receptor surfaces, covering atoms that are close to the current ligand, are colored by solvent accessibility surface (SAS). The
default color spectrum used is green–white–blue, and small values (green) correspond to buried residues, whereas large
values (blue) correspond to exposed residues. This picture was drawn by Discovery Studio Visualizer [29]. (
simplification strategy of this work. ( ) Putative binding modes of A1 in the binding pocket of PDL1 dimer structure.
Dashed lines represent the inter-interaction between A1 and protein, specifically, green ones indicate hydrogen bond, dark
green ones indicate cation– interaction, purple ones indicate ionic interaction, and sky-blue ones indicate interaction.
B) Strctural
C
π
π–π
On closer inspection, however, it becomes clear that the binding conformation of
aliphatic chain linker may be unfavorable for ligand binding owing to the adverse con-
formational entropy effects [30]. Thus, as the initial step, compounds with several cyclic
linkers were prepared, where the methyl group of the distal phenyl ring were also re-
placed by the fluoro group in order to reduce its electron density (Table 1). Although the
replacement using piperidine linker leads to a significant drop in potency, compounds
with methoxy substituted benzene and pyridine rings do exhibit a significant improvement
in activity as well as LE values (A3 IC : 18.4 nM, LE: 0.35; A4 IC : 21.9 nM, LE: 0.36).
50
50
Docking studies suggest that A4 establishes an additional–π interaction with Tyr56 com-
B
pared with A1, and the methoxy group fills the hydrophobic cavity defined by Ile54 and
B
BVal68 (Figure 3), which may contribute to the ligand binding. Although A3 and A4 show
a certainly decrease in activity compared with BMS-1058, they have higher LE values and
proper drug-likeness properties such as ALogP and PSA, which encouraged us to make
further modifications. Given the lower electron density of the central pyridine ring and
solubility property, A4 was chosen to explore new molecules blocking PD-1/PD-L1 axis.

Molecules 2021, 26, 3347
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Figure 3. (A) The optimization strategy of the linker part. (B) Putative binding modes of A4 in the binding pocket of PD-L1
dimer structure (PDB code: 5J89). Dashed lines represent the inter-interaction between A4 and protein, specifically, green
ones indicate hydrogen bond, purple ones indicate ionic interaction, and sky-blue ones indicate π–π interaction.
Continuing to search for ways to improve the affinity, we turned our attention to the
modification of the polar moiety in the tail, and a series of compounds were designed and
synthesized. The results of biological activities are summarized in Table 2. Compounds
A5
piperidine-4-carboxylic acid, respectively, are somewhat better tolerated than compound
A6 with piperidine-3-carboxamide moiety. To our delight, the introduction of liner
, A7, and A8, decorated with 4-aminocyclohexan-1-ol, piperidine-4-carboxamide, and
β-
alaninamide to the polar tail (A9, IC50 = 0.93 nM) leads to an increase in the activity of
more than 23-fold as compared with A4. Our CADD studies demonstrate that secondary
aliphatic amine in the
β-alaninamide, which is protonated at pH 7.4, forms a strong
attractive charge interaction with Asp122. This kind of strong interaction was observed
A
in all the top-ranked docking poses. The amine group of an amide simultaneously forms
two hydrogen bonds with Tyr56 and Tyr123, and its carbonyl oxygen also interacts with
B
A
AArg125 by a hydrogen bonding interaction, possibly contributing to the high affinity
Figure 4A). Compared with BMS-202 (Figure 4B), A9 had similar spatial tendencies within
(
the binding interface of dimer-PD-L1. However, more residues have participated in the
formation of strong polar interactions between A9 and PD-L1. Based on the biological data
results of A9, β-alaninamide group has been proved to be the most suitable R substituent.
3
Figure 4. (A) Putative binding modes of A9 in the binding pocket of PD-L1 dimer structure (PDB code: 5J89); (B) binding
modes of BMS-202 in the binding pocket of PD-L1 dimer structure (PDB code: 5J89). Dashed lines represent the inter-
interaction between A9 or BMS-202 and protein, specifically, green ones indicate hydrogen bond, purple ones indicate ionic
interaction, and sky-blue ones indicate π-π interaction.

Molecules 2021, 26, 3347
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Table 2. Activities of compounds A5–A18 in inhibition of the PD-1/PD-L1 interaction.
Compd.
A5
R2
R3
IC50 (nM) a
MW
ALogP b
PSA b
LE c
2-F
14.3
420.22
4.29
58.96
0.35
A6
2-F
121.3
433.22
5.04
68.44
0.30
A7
A8
2-F
2-F
10.7
20.6
0.93
10.54
6.18
51.5
1.94
1.75
0.97
60.0
866.0
790.9
433.22
434.20
393.19
389.21
409.16
443.18
375.19
393.19
393.19
411.18
427.15
461.17
3.35
2.50
2.77
3.05
3.23
3.50
2.56
2.77
2.77
2.97
3.43
3.71
69.65
66.69
81.81
81.81
81.81
81.81
81.81
81.81
81.81
81.81
81.81
81.81
0.34
0.33
0.43
0.38
0.39
0.31
0.43
0.41
0.43
0.33
0.28
0.25
A9
2-F
A10
A11
A12
A13
A14
A15
A16
A17
2-CH3
2-Cl
2-CF3
H
3-F
4-F
2,4-di-F
4-Cl-2-F
A18
4-CF -2-F
3
a
The data were generated from two independent experiments. b Estimated physicochemical parameters by Discovery Studio [29]. c The
results were calculated by the equation: LE = −1.35 logIC50/Nheavy atoms, Nheavy atoms is the number of non-H atoms.
Decorating the distal phenyl ring with various substituents provided further structure-
activity relationships (SARs). The replacement of 2-fluoro by the bulkier 2-methyl (A10
IC50 = 10.54 nM) or 2-trifluoromethyl group (A12, IC50 = 51.5 nM) leads to a decrease in the
,
activity of more than 10-fold as compared with A9. The chloro substituted compound (A11
,
IC50 = 6.18 nM) exhibited slightly higher inhibitory activity compared to A10. As expected,
the unsubstituted phenyl ring A13 (A13, IC50 = 1.94 nM) yields an approximately 3-fold

Molecules 2021, 26, 3347
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increase in potency over compound A11, which, however, does not reach the potency
of 2-fluoro derivative A9. Moving the 2-fluoro to the 3-or 4-position shows comparable
activity with A9, which indicates that the position of mono substituted fluoro has no
significant effect on activity. This may be attributed by the more favorable
π–π interaction
between the electron-deficient fluorobenzene of these compounds and the residue Tyr56.
A
However, the activity is dramatically reduced as for disubstituted derivatives A16
A16, IC50 = 60.0 nM; A17, IC50 = 866.0 nM; A18, IC50 = 790.9 nM). This is probably due
to the unfavorable steric interactions between atom pairs in close contacts. Although the
–A18
(
conformation of Tyr56 can be moved to accommodate to the size of substituent group on
A
the distal phenyl ring, the binding affinity cannot be compensated for the energy loss by
conformational change of Tyr56. In general, we have obtained the target compound A9
,
A
which has potential activity and moderate water solubility (126.37
BMS-202 89.99 g/mL at pH 7.4), and we would conduct subsequent biological activity
evaluations of A9.
µg/mL at pH 7.4 vs.
µ
2.2. Biophysical Characterization of Compounds Binding to Recombinant Human PD-L1 (hPD-L1)
To provide definitive evidence of direct binding to PD-L1 with small-molecule in-
hibitors, an SPR-based binding assay was performed using Biacore T200 instrument, which
facilitates measurement of the kinetic parameters of ligand–protein complex formation.
In this determination, hPD-L1/Fc was loaded on a Series S Sensor Chip Protein A. A9
and A15, with similar bioactivities in HTRF assay, were gradually subjected to this assay,
and their kinetic parameters and the binding affinities were determined with the aid of
Biacore evaluation software. Analyses of the resulting data reveals that both A9 and A15
can efficiently interact with the immobilized hPD-L1 protein, as demonstrated by the
concentration-dependent responses for association and dissociation, respectively (Figure 5,
Table S1). The equilibrium dissociation constant (K ) could be calculated and shows that
D
compound A9 binds hPD-L1 with an excellent K value of 3.64 nM, 5-fold greater than
D
that of A15 (K = 19.9 nM). Noteworthily, their kinetic measurements demonstrate that
D
A9 and A15 have nearly the same association rate but show conspicuous differences in
dissociation rates that may lead to selection of A9 with a slower off-rate as a more effective
blocker of PD-1/PD-L1 interaction.
0
2
.3. Non-Specific Toxicity of (2-Methyl-[1,1 -Biphenyl]-3-yl)Pyridine Derivatives
Considering that the toxicity of the compound might strongly interfere with the assay
if the tested compounds could block PD-1/PD-L1 interaction to activate T cells for killing
tumors, we examined their cell toxicities of MDA-MB-231 and Jurkat T cells in MTT assay.
As shown in Table 3, compounds A9, A11, A13, A14, and A15 do not directly kill tumor
cells and normal T cells. Furthermore, Jurkat T cells were used to simulate primary T
cells for investigating the immune toxicity of the treated compounds with the regular
concentrations within 48 h. This assay also shows that A9, A11, and A15 exhibit lower cell
toxicity, in which their EC50 values are much higher than their respective IC50 values at the
molecular level.
Table 3. Cytotoxic of inhibitors of the PD-1/PD-L1 interaction.
EC50 (µM)/MDA-MB-231
EC50 (µM)/Jurkat
Compound
Toxicity
Toxicity
A9
11.1
7.1
>10
>10
10.2
7.6
5.5
7.73
>10
5.5
A11
A13
A14
A15

Molecules 2021, 26, 3347
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A
A9
5
-1 -1
K : 5.38×10 M s
on
-3
-1
K : 1.96×10 s
off
B
A15
5
-1 -1
K : 5.05×10 M s
on
-2
-1
K : 1.01×10 s
off
Figure 5. Association and dissociation kinetic curves of compounds A9
hPD-L1.
(A) and A15 (B) at different concentrations with
2.4. IFN-γ Production Effect of A9 in T-Cell Function Assay
Considering the excellent potency in biochemical assays and acceptable drug-likeness
properties, we concluded that A9 is optimal, and would be chosen for further study at T
cell function assay. It is well-known that the expression of interferon- (IFN- ) in the tumor
microenvironment is increased by a productive T-cell response against tumor-associated
antigens [ 31 33]. However, it will be prevented once PD-L1 is overexpressed on tumor
surface through PD-1/PD-L1 interaction. In the presence of PD-1/PD-L1 blockers, the
γ
γ
6, –
interaction between PD-1 and PD-L1 will be destroyed, and the expression of IFN-
be restored by activated T cells.
γ would
To evaluate if compound A9 could restore the T cells-mediated immunity responses,
which was previously repressed by the PD-1/PD-L1 pathway, Hep3B cells, engineered
+
to stably express OS-8 and hPD-L1, were co-cultured with primary CD3 T Cells in the
presence of many PD-1/PD-L1 blockers (Figure 6). A PD-1 monoclonal antibody Keytruda
and small molecule BMS-202 were synchronously used as positive controls. Obviously, A9
can promote the dose-dependent release of IFN-
the promoting effect on IFN- production of A9 at 5
at 5 g/mL and is significantly higher than that of BMS-202. This result implies that A9
γ
in this co-culture system. Impressively,
γ
µM was comparable to that of Keytruda
µ
can restore T cells-mediated immune responses by blocking the PD-1/PD-L1 interaction in
a tumor microenvironment.

Molecules 2021, 26, 3347
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+
Figure 6. Effects of A9 on IFN-
assay. A9 and BMS-202, and three independent experiments for blank and Keytruda. Data are shown
as mean SD (A9 and BMS-202: n = 2; Blank and Keytruda: n = 3), * p < 0.05, ** p < 0.01, *** p < 0.001
vs. blank group.
γ expression in a Hep3B/OS-8/hPD-L1 and CD3 T-cell co-culture
±
2
.5. Chemistry
The synthesis of compounds A1 and A2 is shown in Scheme 1. The key intermediate
was prepared through the Sandmeyer reaction of the starting material and reacted with
by Williamson ether synthesis. Then, the intermediate
was obtained by N-alkylation reaction with piperidin-4-ol, which was converted to the
8
7
Br(CH ) Br to obtain intermediate
9
2
3
10
desired compound A1 through Suzuki coupling reaction with 2-methylphenyl boronic acid
.
◦
Scheme 1. Regents and conditions: (a) 1M H SO , 30% NaNO , Toluene, 0–100 C, 1 h, 89.6%; (b) Br(CH ) Br, K CO , ace-
2
4
2
2 3
2
3
◦
◦
tone, 65 C, overnight, 81.7%; (c) piperidin-4-ol, acetonitrile, DIPEA, 80 C, overnight, 80.2%; (d) 2-methylphenylboronic acid,
Pd(PPh ) , K CO , 1,4-dioxane/H O (V:V = 10/1), 80 C, 8 h, 82%; (e) BH -THF, THF, 0 C then r.t, 12 h, 97.0%; (f) SOCl ,
reflux, 2 h, 98.0%; (g) 4-piperidinone, acetonitrile, DIPEA, 80 C, overnight, 72.1%; (h) piperidin-4-ol, HOAc, CH Cl /MeOH,
◦
◦
3
4
2
3
2
3
2
◦
2
2
◦
r.t, 2 h; NaBH(OAc) , r.t., 8 h, 58.3%; (i) 2-fluorophenylboronic acid, Pd(PPh ) , K CO , 1,4-dioxane/H O(V:V = 10:1), 80 C,
3
3 4
2
3
2
8
h, 89.3%.

Molecules 2021, 26, 3347
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Intermediate 12, which was prepared from 11 by BH -THF mediated reductive re-
3
action, was reacted with SOCl to afford 13, followed by the reaction with piperidone
2
to provide 14. Then, 14 was converted into 15 by NaBH(OAc) -mediated reductive am-
3
ination reaction. Finally, the Suzuki-coupling reaction was conducted between 15 and
2
-fluorophenylboronic acid to give the target compound A2.
The target compounds A3 19 were prepared according to the methods summarized in
Scheme 2. Commercially, material 7 was coupled with various substituted phenylboronic
acid to give 16a by Suzuki coupling reaction, which was then converted to 17a by the
Sandmayer reaction. The resulting intermediates 17a were directly used to synthesize the
intermediates 19a with 18 via Suzuki coupling reaction, which were finally reacted with
–
–j
–j
–
j
–
k
appropriate amines to yield target compounds A3–19 through the NaBH(OAc) mediated
3
reductive amination reaction.
Scheme 2. Regents and conditions: (
a
) Substituted phenylboronic acid, Pd(PPh ) , K CO , 1,4-
3
4
2
3
◦
dioxane/H O (V:V = 10/1), 80 C, 8 h, 80.2–99.2%; (b) 3M HCl, 30% NaNO , B pin , MeOH, water,
2
2
2
2
◦
◦
0
C
~rt, 40.1–68.8%; (
c)
18a or 18b, Pd(PPh ) , K CO , 1,4-dioxane/H O (V:V = 10/1), 80–90 C,
3
4
2
3
2
1
2 h, 20.8–95.3%; (d) alkyl amine, HOAc, CH Cl /MeOH (V:V = 3/1), r.t., 4–12 h; NaBH(OAc) , r.t.,
2 2 3
8
h, 19.9–64.4%.
3. Materials and Methods
3.1. Synthesis
Starting materials and solvents were purchased from commercial suppliers and used
1
13
without any additional purification. H NMR and C NMR spectra were obtained on
a Bruker AV-300 nuclear instrument or Bruker AVANCE NEO 400 MHz in CDCl or
3
DMSO-d using TMS as internal standard, operating at 300 MHz or 400 MHz and 75 MHz
,
6
respectively. Chemical shifts (δ) are expressed in ppm and coupling constants J are given
in Hz. Analytical thin layer chromatography (TLC) was purchased from Yantai Chemical
Industry Research Institute (Cat. no. HSGF254, Yantai, China). All the reactions were mon-
itored by thin layer chromatography in UV absorbance (254 nm). Flash chromatography
was performed using silica gel (200–300 or 300–400 mesh). Melting points were measured
with an RY-I melting point apparatus. The HPLC analysis was performed on a Shimadzu

Molecules 2021, 26, 3347
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LC-20AT machine with a BDS Hypersil C18 column, and the column temperature was
◦
at 31 C. Mobile phase B (100% Acetonitrile) and mobile phase A (NaH PO and H PO
2
4
3
4
buffer solution, pH = 7.5) were used in a gradient elution program (0 min: 25% (B), 5 min:
5% (B), 12 min: 75% (B), 20 min: 75% (B), 23 min: 25% (B), 25 min: 25% (B)) with a flow
rate of 1.0 mL/min at 254 nM.
2
3.1.1. 3-Bromo-2-Methylphenol (8)
A mixture of 3-bromo-2-methylaniline and H SO (1M, 65 mL) was stirred for 30 min
2
4
at room temperature, then 30% NaNO (2.25 g, 32.60 mmol) was added to the mixture
2
◦
dropwise at 0–5 C. After 30 min, toluene (50 mL) was then added to the reaction mixture
and allowed to stir at 100 C for approximately 1 h. The mixture was extracted with ethyl
◦
acetate (50 mL
×
3) and washed with brine (50 mL
×
2). The combined organic layer
was dried over anhydrous Na SO , filtered, and concentrated in vacuum. The residue
2
4
was purified by silica gel chromatography (Petroleum ether/ethyl acetate = 100/1–60/1)
◦
1
to afford compound
8
(white solid, 4.50 g, yield: 89.6%). m.p. 95.0–98.0 C. H NMR
(
300 MHz, Chloroform-d) 7.19 (dd, J = 8.0, 1.2 Hz, 1H, ArH), 6.97 (t, J = 8.0 Hz, 1H, ArH),
δ
6.76 (d, J = 7.9 Hz, 1H, ArH), 4.82 (s, 1H, OH), 2.38 (s, 3H, CH3).
3.1.2. 1-Bromo-3-(3-Bromopropoxy)-2-Methylbenzene (9)
A mixture of
10.75 g, 53.80 mmol) in acetone (30 mL) was refluxed for 8 h. The mixture was diluted
with water (100 mL), extracted with ethyl acetate (50 mL 3), and washed with brine
8 (5.00 g, 26.90 mmol), K CO (9.28 g, 67.25 mmol), and 1,3-dibromopropane
2 3
(
×
(
50 mL × 3). The combined organic layer was dried over anhydrous Na SO , filtered, and
2
4
concentrated in vacuum. The residue was purified by silica gel chromatography (Petroleum
1
ether = 100%) to afford compound
Chloroform-d)
9
(colorless oil, 6.72 g, yield: 81.7%). H NMR (300 MHz,
7.16 (dd, J = 8.1, 1.2 Hz, 1H, ArH), 7.00 (td, J = 8.1, 0.6 Hz, 1H, ArH), 6.79
δ
(
dd, J = 8.2, 1.2 Hz, 1H, ArH), 4.10 (t, J = 5.7 Hz, 2H, OCH ), 3.62 (t, J = 6.4 Hz, 2H, BrCH ),
2
2
2
.39–2.32 (m, 2H, CH CH CH ), 2.31 (s, 3H, CH ).
2 2 2 3
3
.1.3. 1-(3-(3-Bromo-2-Methylphenoxy)Propyl)Piperidin-4-ol (10)
A mixture of (1.00 g, 3.27 mmol), DIPEA (1.10 mL, 6.54 mmol), and 4-hydroxypiperidine
0.97 g, 3.27 mmol) in MeCN (10 mL) was refluxed for 12 h. The formed precipitates were
filtrated and washed with petroleum ether and less water to obtain compound 10 (yellow
9
(
◦
1
solid, 0.86 g, yield: 80.8%). m.p.138.0–140.0 C. H NMR (300 MHz, DMSO-d )
δ
7.20
dd, J = 8.0, 1.3 Hz, 1H, ArH), 7.13 (t, J = 8.1 Hz, 1H, ArH), 6.99 (d, J = 7.9 Hz, 1H, ArH),
.02–4.96 (m, 1H, OH), 4.09 (t, J = 5.9 Hz, 2H, OCH ), 4.01–3.92 (m, 1H, C OH), 3.77–3.40
m, 2H, CH N), 3.20–2.86 (m, 4H, NCH ), 2.28 (s, 3H, CH ), 2.23–2.08 (m, 2H, CH CH2CH2),
6
(
5
(
H
2
2
2
3
2
2
.08–1.69 (m, 4H, CH CHOH).
2
3.1.4. (3-Bromo-2-Methylphenyl)Methanol (12)
BH -THF (1 M, 35 mL) was dropwise added to a solution of 3-bromo-2-methylbenzoic
3
◦
acid (5.00 g, 23.20 mmol) in dry THF (25 mL) at 0 C under N . The mixture was stirred at
2
an ambient temperature for 12 h. The reaction quenched with 1 M HCl (30 mL), and then
extracted with ethyl acetate (20 mL
ganic layer was dried over anhydrous Na SO , filtered, and concentrated in vacuum to give
×
3), followed by brine (20 mL
×
3). The combined or-
2
4
◦
1
2 without any further purification (white solid, 4.5 g, yield, 97.0%). m.p. 103.0–104.0 C.
1
H NMR (300 MHz, DMSO-d6)
δ
7.49 (d, J = 7.9 Hz, 1H, ArH), 7.39 (d, J = 7.8 Hz, 1H, ArH),
7
.12 (t, J = 8.1 Hz, 1H, ArH), 5.27 (br, 1H, OH), 4.52 (s, 2H, CH ), 2.30 (s, 3H, ArCH ).
2
3
3
.1.5. 1-Bromo-3-(Chloromethyl)-2-Methylbenzene (13)
A solution of 12 (4.50 g, 22.50 mmol) in SOCl (10 mL) was stirred at 80 C for 2 h. The
◦
2
solvent was removed in vacuum to obtain compound 13 (colorless oil, 4.80 g, yield, 98.0%).
1
H NMR (300 MHz, Chloroform-d) δ 7.47 (d, J = 7.0 Hz, 1H, ArH), 7.25 (d, J = 6.8 Hz, 1H,
ArH), 7.04 (t, J = 8.1 Hz, 1H, ArH), 3.90 (s, 2H, CH ), 2.43 (s, 3H, CH ).
2
3

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3.1.6. 1-(3-Bromo-2-Methylbenzyl)Piperidin-4-One (14)
This compound was prepared following the synthetic procedure similar to that of 10
.
1
Compound 14 (colorless oil 0.93 g, yield, 72.1%). H NMR (300 MHz, Chloroform-d)
δ 7.52
(dd, J = 8.0, 1.6 Hz, 1H, ArH), 7.23 (d, J = 6.4 Hz, 1H, ArH), 7.03 (t, J = 7.8 Hz, 1H, ArH)
3
.61 (s, 2H, CH N), 2.77 (t, J = 6.1 Hz, 4H, 2NCH ), 2.51 (s, 3H, CH ), 2.46 (t, J = 6.2 Hz, 4H,
2 2 3
2
CH CO).
2
0
0
3
.1.7. 1 -(3-Bromo-2-Methylbenzyl)-[1,4 -Bipiperidin]-4-ol (15)
A mixture of 14 (400 mg, 1.42 mmol) and 4-hydroxypiperidine (172 mg, 1.71 mmol)
in DCM (10 mL) was stirred for 2 h at room temperature. NaBH(OAc) (2.30 mmol) was
3
◦
added to the mixture slowly at 0 C and stirred for another 8 h. The reaction was adjusted
to pH 7–8 with saturated NaHCO aqueous solution. The mixture was extracted with DCM
3
(
10 mL
×
3) and washed with brine (10 mL
×
3). The combined organic layer was dried
over anhydrous Na SO , filtered, and concentrated in vacuum. The residue was purified by
2
4
silica gel chromatography (dichloromethane/methanol = 100/1–30/1) to afford compound
◦
1
15
(yellow solid, yield, 58.3%). m.p. 102.0–105.0 C. H NMR (300 MHz, Chloroform-d)
δ
7.51 (d, J =7.9Hz, 1H, ArH), 7.23 (d, J = 7.4 Hz, 1H, ArH), 7.03 (t, J = 7.7 Hz, 1H, ArH),
4
.34–4.27 (m, 1H, CHOH), 3.79–3.66 (m, 1H, CHN), 3.47 (s, 2H, ArCH ), 3.02–2.80 (m, 4H,
2
NCH ), 2.46 (s, 3H, ArCH ), 2.44–2.28 (m, 4H, NCH ), 2.05–1.92 (m, 4H, 2CH ), 1.55–1.70
2
3
2
2
(
m, 4H, 2CH2).
3
.1.8. The Synthesis of Compounds 16a–j
Compounds 16a were synthesized by the following procedure. To a solution of
-bromo-2-methylaniline (1.0 mmol), phenylboronic acid (1.2 mmol), K CO (2.8 mmol) in
–
j
3
2
3
1
,4-dioxane/water (1.0 mL/0.1 mL), Pd(PPh ) (0.05 mmol) was added. The mixture was
3 4
◦
stirred at 80 C for 8 h under N atmosphere. The undissolved solid was filtered off, and
2
the filtrate was concentrated under vacuum. The residue was dissolved in ethyl acetate,
and washed with water, followed by brine. The organic layer was dried over anhydrous
Na SO , filtered, and concentrated. The residue was purified by silica gel chromatography
2
4
(
petroleum ether/ethyl acetate = 100/1–80/1) to afford compounds 16a-j.
0
0
2
-Fluoro-2-methyl-[1,1 -biphenyl]-3-amine (16a)
◦
1
Brown solid. Yield, 95.6%. m.p. 61.0–62.0 C. H NMR (300 MHz, DMSO-d )
δ
7.48–
6
7
.35 (m, 1H, ArH), 7.34 – 7.22 (m, 3H, ArH), 6.98 (t, J = 7.7 Hz, 1H, ArH), 6.70 (dd, J = 8.0,
1
.4 Hz, 1H, ArH), 6.42 (dd, J = 7.5, 1.3 Hz, 1H, ArH), 5.00 (s, 2H, NH ), 1.85 (d, J = 1.8 Hz,
2
3
H, ArCH ).
3
0
0
2,2 -Dimethyl-[1,1 -biphenyl]-3-amine (16b)
◦
1
Yellow oil. Yield, 90%. m.p. 52.0–55.0 C. H NMR (400 MHz, Chloroform-d)
.29 (m, 1H, ArH), 7.28–7.22 (m, 2H, ArH), 7.15 (d, J = 7.0 Hz, 1H, ArH), 7.10 (t, J = 7.7 Hz,
H, ArH), 6.75 (dd, J = 7.9, 1.4 Hz, 1H, ArH), 6.63 (dd, J = 7.5, 1.4 Hz, 1H, ArH), 3.75 (s, 2H,
ArNH ), 2.11 (s, 3H, ArCH ), 1.91 (s, 3H, ArCH ).
δ 7.34 –
7
1
2
3
3
0
0
2
-Chloro-2-methyl-[1,1 -biphenyl]-3-amine (16c)
◦
1
Yellow oil. Yield, 93.1%. m.p. 71.5–74.5 C. H NMR (300 MHz, DMSO-d )
δ
7.58–7.46
m, 1H, ArH), 7.43–7.30 (m, 2H, ArH), 7.30–7.18 (m, 1H, ArH), 6.94 (t, J = 7.6 Hz, 1H, ArH),
.67 (dd, J = 8.0, 1.4 Hz, 1H, ArH), 6.32 (dd, J = 7.5, 1.4 Hz, 1H, ArH), 4.96 (s, 2H, NH2),
6
(
6
1
.76 (s, 3H, ArCH ).
3
0
0
2
-Chloro-2-methyl-[1,1 -biphenyl]-3-amine (16d)
◦
1
Yellow solid. Yield, 82.1%. m.p. 87.5–90.0 C. H NMR (300 MHz, DMSO-d )
δ
7.79
6
(
dd, J = 7.9, 1.6 Hz, 1H, ArH), 7.67 (td, J = 7.6, 1.7 Hz, 1H, ArH), 7.56 (t, J = 7.6, 1H, ArH),
7
6
.25 (d, J = 7.5 Hz, 1H, ArH), 6.90 (t, J = 7.7 Hz, 1H, ArH), 6.67 (dd, J = 8.0, 1.4 Hz, 1H, ArH),
.31 (d, J = 7.3 Hz, 1H, ArH), 4.98 (s, 2H, NH ), 1.68 (s, 3H, ArCH ).
2
3
0
2
-Methyl-[1,1 -biphenyl]-3-amine (16e)
1
White solid. Yield, 89.6%. H NMR (400 MHz, Chloroform-d)
δ 7.46–7.40 (m, 2H,
ArH), 7.40–7.32 (m, 3H, ArH), 7.11 (t, J = 7.8 Hz, 1H, ArH), 6.75 (d, J = 7.8 Hz, 2H, ArH),
.82 (s, 2H, NH ), 2.10 (s, 3H, ArCH ).
3
2
3

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0
0
3
-Fluoro-2-methyl-[1,1 -biphenyl]-3-amine (16f)
1
Yellow oil. Yield, 88.0%. H NMR (400 MHz, DMSO-d )
δ
7.44 (td, J = 8.1, 6.1 Hz, 1H,
6
ArH), 7.25–7.15 (m, 1H, ArH), 7.12–7.04 (m, 2H, ArH), 6.94 (t, J = 7.8 Hz, 1H, ArH), 6.67
dd, J = 8.0, 1.5 Hz, 1H, ArH), 6.42 (dd, J = 7.5, 1.4 Hz, 1H, ArH), 5.05 (s, 2H, NH ), 1.93 (d,
(
2
J = 1.9 Hz, 3H, CH ).
3
0
0
4
-Fluoro-2-methyl-[1,1 -biphenyl]-3-amine (16g)
◦
White solid. Yield, 88.0%. m.p. 76.5–78.0 C. 1H NMR (300 MHz, DMSO-d )
δ
7.32–
6
7
.23 (m, 3H, ArH), 7.20 (d, J = 8.8 Hz, 1H, ArH), 6.93 (t, J = 7.7 Hz, 1H, ArH), 6.66 (d,
J = 7.9 Hz, 1H, ArH), 6.40 (d, J = 7.5 Hz, 1H, ArH), 4.94 (s, 2H, NH2), 1.92 (s, 3H, ArCH3).
0
0 0
,4 -Difluoro-2-methyl-[1,1 -biphenyl]-3-amine (16h)
2
◦
1
Yellow solid. Yield, 88.9%. m.p. 81.5–83.0 C. H NMR (300 MHz, DMSO-d )
δ
6
7
6
.38–7.27 (m, 2H, ArH), 7.16 (td, J = 8.5, 2.2 Hz, 1H, ArH), 6.97 (t, J = 7.8 Hz, 1H, ArH),
.70 (dd, J = 8.1, 1.3 Hz, 1H, ArH), 6.40 (d, J = 7.4 Hz, 1H, ArH), 5.01 (s, 2H, NH ), 1.84 (d,
2
J = 1.8 Hz, 3H, ArCH ).
3
0
0
0
4
-Chloro-2 -fluoro-2-methyl-[1,1 -biphenyl]-3-amine (16i)
1
Yellow oil. Yield, 99.2%. H NMR (300 MHz, DMSO-d )
δ 7.52 (dd, J = 9.8, 1.9 Hz, 1H,
6
ArH), 7.37 (dd, J = 8.3, 2.0 Hz, 1H, ArH), 7.35–7.28 (m, 1H, ArH), 6.99 (t, J = 7.7 Hz, 1H,
ArH), 6.72 (dd, J = 8.0, 1.4 Hz, 1H, ArH), 6.41 (dd, J = 7.5, 1.3 Hz, 1H, ArH), 5.03(s, 2H,
NH ), 1.85 (d, J = 1.8 Hz, 3H, ArCH ).
2
3
0
0
0
2
. -Fluoro-2-methyl-4 -(trifluoromethyl)-[1,1 -biphenyl]-3-amine (16j)
Yellow oil. Yield, 80.6%. H NMR (400 MHz, DMSO-d )
1
δ 7.74 (dd, J = 9.8, 2.1 Hz, 1H,
6
ArH), 7.64 (dd, J = 8.0, 2.1 Hz, 1H, ArH), 7.51 (t, J = 7.6 Hz, 1H, ArH), 6.99 (t, J = 7.8 Hz, 1H,
ArH), 6.73 (d, J = 8.0 Hz, 1H, ArH), 6.42 (dd, J = 7.5, 1.4 Hz, 1H, ArH), 5.04 (bs, 2H, NH2),
1.84 (d, J = 1.9 Hz, 3H, ArCH3).
3.1.9. The Synthesis of Compounds 17a–j
Compounds 17a–j were synthesized by the following procedure. HCl (24.84 mL, 3 M
)
was added to a solution of 16a (4.00 g, 19.9 mmol) in MeOH (40 mL), followed by the
addition of H O (20 mL) at room temperature. The mixture was stirred for 30 min, and
2
◦
NaNO (9.92 mL, 2.2 M) was added dropwise very slowly at 0–5 C. After being stirred for
2
another 30 min, B pin (15.12 g, 59.7 mmol) in MeOH (40 mL) was added to the mixture
2
2
solution and warmed to room temperature for 2 h. Then the mixture was extracted with
DCM (50 mL 3), washed with brine (50 mL 2), and dried over anhydrous Na SO .
×
×
2
4
The organic layer was concentrated in vacuum and purified by column chromatography
on silica gel (petroleum ether/ethyl acetate = 250/1–150/1) to the target compound.
0
0
2
-(2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (17a)
◦
1
Yellow solid. Yield, 60.4%. m.p. 94.0–95.0 C. H NMR (300 MHz, DMSO-d )
δ
7.70
dd, J = 5.4, 3.7 Hz, 1H, ArH), 7.53–7.36 (m, 1H, ArH), 7.34–7.25 (m, 5H, ArH), 2.27 (d,
J = 1.4 Hz, 3H, ArCH ), 1.31 (s, 12H, CH ).
6
(
3
3
0
0
2
-(2,2 -Dimethyl-[1,1 -biphenyl]-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (17b)
Colorless oil. Yield, 40.1%. H NMR (300 MHz, DMSO-d )
1
δ
7.67 (dd, J = 7.4, 1.8 Hz,
6
1
H, ArH), 7.34–7.21 (m, 4H, ArH), 7.15 (dd, J = 7.5, 1.8 Hz, 1H, ArH), 7.10–6.95 (m, 1H,
ArH), 2.17 (s, 3H, ArCH ), 1.99 (s, 3H, ArCH ), 1.33 (s, 12H, CCH ).
3
3
3
0 0
-(2 -Chloro-2-methyl-[1,1 -biphenyl]-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (17c)
2
◦
1
Light yellow solid. Yield, 62.9%. m.p. 101.0–103.0 C. H NMR (300 MHz, DMSO-d )
6
δ
7.71 (dd, J = 7.3, 1.8 Hz, 1H, ArH), 7.60 (dd, J = 5.8, 3.5 Hz, 1H, ArH), 7.48–7.41 (m, 2H,
ArH), 7.33–7.25 (m, 2H, ArH), 7.21 (dd, J = 7.6, 1.8 Hz, 1H, ArH), 2.22 (s, 3H, ArCH ), 1.34
3
(
s, 12H, CH3).
,4,5,5-Tetramethyl-2-(2-methyl-2 -(trifluoromethyl)-[1,1 -biphenyl]-3-yl)-1,3,2-dioxab
orolane (17d)
Colorless oil. Yield, 68.8%. H NMR (300 MHz, DMSO-d )
0
0
4
1
δ
7.83 (dd, J = 7.9, 1.6 Hz,
6
1H, ArH), 7.79–7.70 (m, 2H, ArH), 7.68–7.61 (m, 1H, ArH), 7.29 (d, J = 7.5 Hz, 1H), 7.25–7.13
(
m, 2H, ArH), 2.12 (s, 3H, ArH), 1.31 (s, 12H, CH ).
3
0
4,4,5,5-Tetramethyl-2-(2-methyl-[1,1 -biphenyl]-3-yl)-1,3,2-dioxaborolane (17e)

Molecules 2021, 26, 3347
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1
Colorless oil. Yield, 55.3%. H NMR (400 MHz, DMSO-d )
δ
7.69 (d, J = 7.8 Hz, 1H,
6
ArH), 7.57–7.40 (m, 1H, ArH), 7.39–7.20 (m, 4H, ArH), 7.03 (s, 1H, ArH), 6.99 (d, J = 7.9 Hz,
1
H, ArH), 2.26 (d, J = 1.3 Hz, 3H, ArCH ), 1.32 (s, 12H, CH ).
3 3
0
0
2
-(2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (17f)
1
White solid. Yield, 56.8%. H NMR (300 MHz, DMSO-d )
δ
7.69 (dd, J = 6.4, 2.5 Hz,
6
1
H, ArH), 7.50 (td, J = 8.1, 6.2 Hz, 1H, ArH), 7.34–7.26 (m, 2H, ArH), 7.26–7.21 (m, 1H,
ArH), 7.19–7.16 (m, 2H, ArH), 2.37 (s, 3H, ArCH ), 1.34 (s, 12H, CH ).
3
3
0
0
2
-(4 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (17g
Yellow solid. Yield, 60.0%. H NMR (300 MHz, DMSO-d )
)
1
δ
7.71–7.63 (m, 1H, ArH),
6
7.38–7.34 (m, 1H, ArH), 7.34–7.29 (m, 2H, ArH), 7.29–7.23 (m, 3H, ArH), 2.35 (s, 3H, ArCH3),
1
.33 (s, 12H, CH ).
3
0 0 0
-(2 ,4 -Difluoro-2-methyl-[1,1 -biphenyl]-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
2
(
17h)
◦
1
Light yellow solid. Yield, 67.5%. m.p. 59.5–61.5 C. H NMR (300 MHz, DMSO-d )
6
δ
7.79–7.68 (m, 1H, ArH), 7.43–7.32 (m, 2H, ArH), 7.28 (d, J = 4.4 Hz, 2H, ArH), 7.19 (td,
J = 8.5, 2.7 Hz, 1H, ArH), 2.28 (d, J = 1.3 Hz, 3H, ArCH ), 1.33 (s, 12H, CH ).
3
3
0 0 0
-(2 ,4 -Difluoro-2-methyl-[1,1 -biphenyl]-3-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
2
(17i)
◦
1
Light yellow solid. Yield, 63.5%. m.p. 91.5–94.5 C. H NMR (300 MHz, DMSO-d )
δ
6
7.72 (q, J = 4.0 Hz, 1H, ArH), 7.57 (dd, J = 9.8, 2.0 Hz, 1H, ArH), 7.41 (dd, J = 8.2, 2.0 Hz, 1H,
ArH), 7.37 (d, J = 7.7 Hz, 1H, ArH), 7.30 (d, J = 4.5 Hz, 2H, ArH), 2.28 (s 3H, ArCH ), 1.34
3
(
s, 12H, CH ).
3
0
0
0
2-(2 -Fluoro-2-methyl-4 -(trifluoromethyl)-[1,1 -biphenyl]-3-yl)-4,4,5,5-tetramethyl-1,3,2-
dioxaborolane (17j)
Brown solid. Yield, 63.5%. m.p. 76.5–78.5 C. H NMR (400 MHz, DMSO-d )
◦
1
δ
7.80 (d,
6
J = 9.8Hz, 1H, ArH), 7.74 (d, J = 6.3Hz, 1H, ArH), 7.70–7.66 (m, 1H, ArH), 7.59–7.52 (m, 1H,
ArH), 7.33–7.28 (m, 2H, ArH), 2.27 (s, 3H, CH ), 1.31 (s, 12H, CH ).
3
3
0
0
0
3
.1.10. 2”-Fluoro-3-Methoxy-2 -Methyl-[1,1 :3 ,1”-Terphenyl]-4-Carbaldehyde (19a)
To a solution of 17 (2.50 g, 8.01 mmol), 18a (1.51 g, 8.81 mmol), and K CO (3.10 g,
2
3
2
2.4 mmol) in 1,4-dioxane/water (25 mL/2.5 mL), Pd(PPh ) (0.78 g, 0.67 mmol) was added,
3
4
◦
and the mixture was allowed to stir at 90 C for 8 h under N atmosphere. The undissolved
2
solid was filtered off, and the filtrate was concentrated under vacuum. The residue was
dissolved in ethyl acetate (25 mL
by brine (25 mL 2). The organic layer was dried over anhydrous Na SO , filtered,
and concentrated in vacuum. The residue was purified by silica gel chromatography
Petroleum ether/ ethyl acetate = 150/1–80/1) to afford compound 19a (white solid 2.45 g,
×
3) and washed with water (25 mL
×
2), followed
×
2
4
(
◦
1
yield: 95.3%). m.p. 82.0–83.5 C. H NMR (400 MHz, DMSO-d )
δ
10.39 (s, 1H, CHO), 7.77
d, J = 7.9 Hz, 1H, ArH), 7.50–7.43 (m, 1H, ArH), 7.41–7.36 (m, 2H, ArH), 7.36–7.30 (m, 3H,
ArH), 7.30–7.24 (m, 1H, ArH), 7.20 (s, 1H, ArH), 7.08 (dt, J = 7.9, 1.0 Hz, 1H, ArH), 3.96 (s,
6
(
3
H, OCH ), 2.21 (s, 3H, ArCH ).
3 3
3.1.11. The Synthesis of Compounds 19b–k
Compounds 19b–k were prepared analogously to compound 16a.
0 0
-(2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxynicotinaldehyde (19b)
White solid powder. Yield, 72.1%. m.p. 78.5–81.5 C. H NMR (300 MHz, DMSO-d )
6
◦
1
δ
6
1
0.32 (s, 1H, CHO), 8.22 (d, J = 7.7 Hz, 1H, ArH), 7.59–7.54 (m, 1H, ArH), 7.51 (d, J = 7.8 Hz
,
1
H, ArH), 7.46 (d, J = 7.3 Hz, 1H, ArH), 7.42 (d, J = 5.4 Hz, 1H, ArH), 7.40–7.31 (m, 4H,
ArH), 4.07 (s, 3H, OCH ), 2.18 (s, 3H, ArCH ).
3
3
0
0
6
-(2,2 -Dimethyl-[1,1 -biphenyl]-3-yl)-2-methoxynicotinaldehyde (19c)
Colorless oil. Yield, 74.5%. H NMR (400 MHz, DMSO-d )
1
δ 10.30 (s, 1H, CHO),
6
8
7
.19 (d, J = 7.7 Hz, 1H, ArH), 7.50 (dd, J = 7.8, 1.5 Hz, 1H, ArH), 7.42–7.36 (m, 2H, ArH),
.35–7.24 (m, 3H, ArH), 7.20 (dd, J = 7.5, 1.5 Hz, 1H, ArH), 7.14 (dd, J = 6.9, 2.0 Hz, 1H,
ArH), 4.04 (s, 3H, OCH ), 2.05 (s, 3H, ArCH ), 2.04 (s, 3H, ArCH3).
3
3

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0
0
6
-(2 -Chloro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxynicotinaldehyde (19d)
1
Colorless oil, Yield, 61.7%. H NMR (300 MHz, DMSO-d )
δ
10.32 (s, 1H, CHO), 8.22
6
(
(
d, J = 7.7 Hz, 1H, ArH), 7.65–7.61 (m, 1H, ArH), 7.57 (dd, J = 7.8, 1.7 Hz, 1H, ArH), 7.54–7.46
m, 2H, ArH), 7.46–7.37 (m, 3H, ArH), 7.28 (dd, J = 7.6, 1.5 Hz, 1H, ArH), 4.07 (s, 3H, OCH3),
2
.12 (s, 3H, ArCH3).
0
0
2-Methoxy-6-(2-methyl-2 -(trifluoromethyl)-[1,1 -biphenyl]-3-yl)nicotinaldehyde (19e
)
1
White solid. Yield, 48.4%. H NMR (300 MHz, DMSO-d )
δ
10.31 (s, 1H, CHO), 8.20
6
(
d, J = 7.7 Hz, 1H, ArH), 7.89 (d, J = 6.6 Hz, 1H, ArH), 7.77 (t, J = 6.7 Hz, 1H, ArH), 7.66 (t,
J = 7.7 Hz, 1H, ArH), 7.56 (dd, J = 7.8, 1.5 Hz, 1H, ArH), 7.52–7.43 (m, 3H, ArH), 7.25 (d,
J = 7.8 Hz, 1H, ArH), 4.05 (s, 3H, OCH ), 2.03 (s, 3H, ArCH ).
3
3
0
2
-Methoxy-6-(2-methyl-[1,1 -biphenyl]-3-yl)nicotinaldehyde (19f)
White solid. Yield, 56.8%. H NMR (300 MHz, DMSO-d )
1
δ
10.42 (s, 1H, CHO), 7.79
6
(
7
d, J = 7.8 Hz, 1H, ArH), 7.54–7.45 (m, 1H, ArH), 7.44–7.25 (m, 6H, ArH), 7.23 (s, 1H, ArH),
.10 (d, J = 7.9 Hz, 1H, ArH), 3.99 (s, 3H, OCH ), 2.04 (s, 3H, ArCH ).
3
3
0 0
-(3 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxynicotinaldehyde (19g)
6
◦
1
White solid. Yield, 68.8%. m.p. 131.0–132.0 C. H NMR (300 MHz, DMSO-d )
δ
6
1
0.31 (s, 1H, CHO), 8.22 (dd, J = 8.2, 5.4 Hz, 1H, ArH), 7.52 (dq, J = 8.9, 3.7, 3.1 Hz, 2H,
ArH), 7.46–7.31 (m, 3H, ArH), 7.26 (t, J = 7.5 Hz, 3H, ArH), 4.06 (s, 3H, OCH ), 2.24 (s,
3
3
H, ArCH ).
3
0
0
6-(3 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxynicotinaldehyde (19h)
◦
1
White solid. Yield, 72.3%. m.p. 46.0–48.0 C. H NMR (300 MHz, DMSO-d )
δ
10.18 (s,
H, ArH), 8.12 (d, J = 7.9 Hz, 1H, ArH), 7.64 (dd, J = 5.7, 3.3 Hz, 1H, ArH), 7.42–7.10 (m, 7H,
ArH), 4.01 (s, 3H, OCH ), 2.33 (s, 3H, ArCH ).
6
1
3
3
0 0 0
6
-(2 ,4 -Difluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxynicotinaldehyde (19i)
◦
1
White solid. Yield, 20.8%. m.p. 119.5–222.0 C. H NMR (400 MHz, DMSO-d )
δ
10.30
6
(s, 1H, CHO), 8.20 (d, J = 7.6 Hz, 1H, ArH), 7.54 (dd, J = 7.8, 1.6 Hz, 1H, ArH), 7.48–7.41(m,
2
H, ArH), 7.40–7.35 (m, 2H, ArH), 7.33 (dd, J = 7.6, 1.6 Hz, 1H, ArH), 7.22 (td, J = 8.5, 2.3 Hz
,
1
H, ArH), 4.04 (s, 3H, OCH ), 2.14 (s, 3H, ArCH ).
3
3
0
0
0
6
-(4 -Chloro-2 -fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxynicotinaldehyde (19j
)
◦
1
White solid. Yield, 29.8%. m.p. 132.0–134.0 C. H NMR (400 MHz, DMSO-d )
δ
10.30
6
(
s, 1H, CHO), 8.20 (d, J = 7.8 Hz, 1H, ArH), 7.61–7.53 (m, 2H, ArH), 7.47–7.40 (m, 3H, ArH),
7
.37 (d, J = 7.6 Hz, 1H, ArH), 7.34 (dd, J = 7.6, 1.6 Hz, 1H, ArH), 4.04 (s, 3H, OCH ), 2.14 (s,
3
3
H, ArCH ).
3
0
0
0
6-(2 -Fluoro-2-methyl-4 -(trifluoromethyl)-[1,1 -biphenyl]-3-yl)-2-methoxy nicotinalde-
hyde (19k)
White solid. Yield, 47.5%. m.p. 62.0–64.0 C. H NMR (400 MHz, DMSO-d )
◦
1
δ
10.30 (s,
6
1
1
1
H, CHO), 8.20 (d, J = 7.6 Hz, 1H, ArH), 7.84 (dd, J = 9.9, 1.9 Hz, 1H, ArH), 7.74–7.69 (m,
H, ArH), 7.65 (t, J = 7.6 Hz, 1H, ArH), 7.58 (dd, J = 7.6, 1.6 Hz, 1H, ArH), 7.46 (t, J = 7.5 Hz,
H, ArH), 7.41-7.32 (m, 2H, ArH), 4.04 (s, 3H, OCH ), 2.15 (s, 3H, ArCH ).
3
3
3.1.12. The Synthesis of Compounds A1 and A2
Compounds A1 and A2 were prepared analogous to compounds 16a–j.
0 0
-(3-((2,2 -Dimethyl-[1,1 -biphenyl]-3-yl)oxy)propyl)piperidin-4-ol hydrochloride (A1).
1
◦
1
White solid. Yield, 82.0%. m.p. 118.0–201.0 C. H NMR (400 MHz, DMSO-d ) δ 7.27
6
(
(
dd, J = 6.6, 1.5 Hz, 2H, ArH), 7.26–7.22 (m, 1H, ArH), 7.19 (t, J = 7.8 Hz, 1H, ArH), 7.09–7.00
m, 1H, ArH), 6.94 (d, J = 8.1 Hz, 1H, ArH), 6.66 (d, J = 7.4 Hz, 1H, ArH), 4.60 (s, 1H, OH),
4
.09–3.99 (m, 3H, OCH ), 3.54–3.42 (m, 2H, NCH ), 2.87–2.72 (m, 2H, NCH -piperidinol),
3 2 2
2
.14 (m, 2H, CH CH2CH ), 1.99 (s, 3H, ArCH ), 1.94 (s, 2H, NCH -piperidinol), 1.83
2
2
3
2
1
3
(
(
s, 3H, ArCH ), 1.73 (s, 2H, CH -piperidinol), 1.44 (s, 2H, CH -piperidinol). C NMR
75 MHz, DMSO-d6) δ 156.82, 142.68, 141.40, 135.61, 130.22, 129.45, 127.72, 126.74, 126.13,
3
2
2
1
24.03, 121.91, 110.52, 65.62, 60.03, 50.72, 47.37, 31.89, 29.75, 19.94(2C), 13.21(2C). ESI-HRMS:
+
m/z [M+H] calculated for C H NO , 340.2277; Found: 340.2268. LCMS: t 9.97 min,
22
31
2
R
purity, 95.1%.
0
0 0 0
-((2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)methyl)-[1,4 -bipiperidin]-4-ol (A2).
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◦
1
Light yellow powder. Yield, 89.3%. m.p. 110.0–113.0 C. H NMR (300 MHz, DMSO-
d )
6
δ
7.52–7.41 (m, 1H, ArH), 7.36–7.25 (m, 4H, ArH), 7.23 (d, J = 7.5 Hz, 1H, ArH), 7.11
(dd, J = 7.5, 1.6 Hz, 1H, ArH), 4.54 (s, 1H, CHO
H), 3.46 (s, 3H, ArCH2N/ CHOH), 2.89
(
d, J = 11.0 Hz, 2H, NCH -piperidine), 2.83–2.69 (m, 2H, NCH -piperidine), 2.36–2.15 (m,
2
2
3
H, CHN, NCH2-piperidine), 2.12 (s, 3H, CH ), 1.99 (t, J = 10.7 Hz, 2H, NCH -piperidine),
3
2
13
1.82–1.61 (m, 4H, CH -piperidine), 1.57–1.18 (m, 4H, CH -piperidine). C NMR (75 MHz,
2
2
DMSO-d6)
δ 161.07, 157.84, 137.64, 136.18, 135.79, 132.11, 129.81, 129.14, 125.51, 124.96,
1
16.01, 115.72, 67.13, 61.95, 61.08, 53.49(2C), 47.13(2C), 35.25(2C), 28.32(2C), 16.09. ESI-
+
HRMS: m/z [M+H] calculated for C H FN O, 383.2499 ; Found: 383.2495. LCMS: t
24
32
2
R
10.69 min, purity, 96.5%.
3
.1.13. The Synthesis of Compounds A3–A8
Compounds A3 A8 were synthesized by the following procedure. A mixture of 19a
or 19b (0.92 mmol), amide (1.11 mmol), and HAc (0.11 mL, 1.84 mmol) in DCM/MeOH
–
(
5 mL/1 mL) was stirred for 4 h at room temperature. NaBH(OAc) (2.30 mmol) was added
3
◦
to the mixture slowly at 0 C and stirred for another 8 h. The reaction was adjusted to
pH 7–8 with saturated NaHCO aqueous solution. The mixture was extracted with DCM
3
(
10 mL × 3) and washed with brine (10 mL
×
3). The combined organic layer was dried
over anhydrous Na SO , filtered, and concentrated in vacuum. The residue was purified
2
4
by silica gel chromatography to afford the product.
0
0 0
1
-((2”-Fluoro-3-methoxy-2 -methyl-[1,1 :3 ,1”-terphenyl]-4-yl)methyl)piperidin-4-ol (A3).
◦
1
White solid. Yield, 66.4%. m.p. 61.5–63.5 C. H NMR (400 MHz, DMSO-d )
δ
7.52–
.43 (m, 1H, ArH), 7.41–7.33 (m, 3H, ArH), 7.32–7.26 (m, 3H, ArH), 7.21 (dd, J = 7.3, 1.9 Hz
H, ArH), 6.92 (d, J = 8.4 Hz, 2H, ArH), 4.57 (s, 1H, OH), 3.81 (s, 3H, OCH ), 3.47 (br,
6
7
1
,
3
3
H, ArCH /piperidine-CH), 2.75 (b r, 2H, piperidine-CH ), 2.11 (b r, 2H, piperidine-CH ),
2
2
2
2
.01 (s, 3H, ArCH ), 1.79–1.63 (m, 2H, piperidine-CH ), 1.50–1.36 (m, 2H, piperidine-CH ).
3
2
2
1
3
C NMR (75 MHz, DMSO-d6)
δ 161.08, 157.85(F-C), 157.45(F-C), 142.66, 141.71, 136.56,
1
1
32.13(d), 132.08, 130.12(d), 129.93, 129.51, 129.29, 126.04, 125.25, 125.02(d), 121.37, 116.12,
15.83, 112.12, 66.71, 55.85 (2C), 55.73, 51.59, 34.82, 18.35 (2C). ESI-HRMS: m/z [M+H]+
calculated for C H FNO , 406.2104; Found: 406.2177. LCMS: t 10.48 min, purity, 98.4%.
2
6
28
2
R
0
0
1
-((6-(2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)piperidin-
-ol (A4).
White solid powder. Yield, 56.4%. m.p. 84.0–86.0 C. H NMR (300 MHz, DMSO-d )
4
◦
1
δ
6
7
7
4
2
.76 (d, J = 7.5 Hz, 1H, ArH), 7.52 (dd, J = 7.6, 1.7 Hz, 1H, ArH), 7.42–7.28 (m, 5H, ArH),
.24–7.16 (m, 1H, ArH), 7.09 (d, J = 7.3 Hz, 1H, ArH), 4.02 (s, 3H, OCH ), 3.77 (dt, J = 8.8,
3
.5 Hz, 1H, C
HOH), 3.61 (s, 2H, ArCH N), 2.90 (dt, J = 10.8, 4.7 Hz, 2H, CH -piperidine),
2
2
.37–2.28 (m, 2H, CH -piperidine), 2.27 (s, 3H, ArCH ), 2.05–1.96 (m, 2H, CH -piperidine),
2
3
2
1
3
1.84 (br, 1H, OH) 1.78–1.68 (m, 2H, CH -piperidine), 1.47–1.36 (m, 2H, CH -piperidine).
C
2
2
NMR (75 MHz, Chloroform-d)
δ 161.16, 155.99, 140.94, 138.50, 136.90, 134.92, 131.75, 130.08,
1
29.69, 129.58, 129.17, 129.06, 125.43, 124.00, 118.64, 117.24, 115.70, 115.40, 67.97, 55.53, 53.49
+
(2C), 51.19, 34.51 (2C),18.05. ESI-HRMS: m/z [M+H] calculated for C H FNO , 407.2182;
26 29 2
Found: 407.2137. LCMS: t 11.76 min, purity, 96.1%.
R
0
0
4
-(((6-(2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)amino)
cyclohexan-1-ol (A5).
White solid. Yield, 51.3%. m.p. 80.0–82.0 C. H NMR (300 MHz, DMSO-d )
◦
1
δ
7.82
d, J = 7.5 Hz, 1H, ArH), 7.54–7.44 (m, 2H, ArH), 7.43–7.33 (m, 4H, ArH), 7.32–7.25 (m,
H, ArH), 7.14 (d, J = 7.3 Hz, 1H, ArH), 4.52 (d, J = 4.5 Hz, 1H, OH), 3.90 (s, 3H, OCH3),
.72 (s, 2H, CH2NH), 3.45–3.40 (m,1H, C OH), 2.43–2.32 (m, 1H, NHC ), 2.13 (s, 3H,
6
(
1
3
H
H
1
3
ArCH ), 1.94–1.78 (m, 4H, CH -cyclohexane), 1.21–1.01 (m, 4H, CH -cyclohexane).
C
3
2
2
NMR (75 MHz, DMSO-d6)
δ 160.55, 157.88(F-C), 155.05(F-C), 141.03, 138.01, 136.75, 134.40,
1
5
32.12,130.12, 130.23, 129.95, 129.3(d), 126.05, 125.04, 122.11, 117.48, 116.11, 115.82, 69.29,
5.91, 53.59, 44.53, 34.32(2C), 31.26(2C), 18.17. ESI-HRMS: m/z [M+H] calculated for
+
C H FN O , 421.2291; Found: 421.2284. LCMS: t 9.46 min, purity, 95.1%.
26
30
2
2
R

Molecules 2021, 26, 3347
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0
0
1
-((6-(2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)piperidine-
-carboxamide (A6).
White solid powder. Yield, 41.0%. m.p. 88.0–91.0 C. H NMR (300 MHz, DMSO-d )
3
◦
1
δ
6
7
7
3
.78 (d, J = 7.5 Hz, 1H, ArH), 7.55–7.44 (m, 2H, ArH), 7.42–7.33 (m, 4H, ArH, 1/2CONH2),
.32–7.26 (m, 2H, ArH), 7.16 (d, J = 7.5 Hz, 1H), 6.82 (br, 1H, 1/2CONH ), 3.91 (s, 3H, OCH ),
2
3
.50 (s, 2H, ArCH ), 2.89–2.71 (m, 2H, piperidine-CH ), 2.45–2.33 (m, 1H, piperidine-
2
2
CHCO), 2.23–2.00 (m, 5H, ArCH , piperidine-CH ), 1.84–1.62 (m, 2H, piperidine-CH ),
3
2
2
13
1
.56–1.36 (m, 2H, piperidine-CH2). C NMR (75 MHz, DMSO-d6)
δ
176.04, 161.00, 157.8(C-
F), 155.51(C-F), 140.95, 139.15, 136.76, 134.41, 132.14, 130.28, 129.97, 129.39, 129.16, 126.07,
1
25.09, 119.21, 117.53, 115.9(d), 56.57, 56.05, 53.86, 53.65, 42.77, 27.54, 24.76, 18.19. ESI-
+
HRMS: m/z [M+H] calculated for C H FN O , 434.2244; Found: 434.2238. LCMS: t
26
29
3
2
R
1
2.04 min, purity, 95.6%.
0
0
1-((6-(2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)piperidine-
4
-carboxamide (A7).
White solid powder. Yield, 44.7%. m.p. 194.0–196.0 C. H NMR (300 MHz, DMSO-
7.80 (d, J = 7.5 Hz, 1H, ArH), 7.53–7.45 (m, 2H, ArH), 7.43–7.34 (m, 3H, ArH),
◦
1
d )
δ
6
7
.33–7.26 (m, 2H, ArH), 7.25 (s, 1H, 1/2CONH ), 7.17 (d, J = 7.5 Hz, 1H, ArH), 6.75 (s,
2
1
H, 1/2CONH ), 3.91 (s, 3H, OCH ), 3.54 (s, 2H, ArCH ), 2.91 (m, 2H, piperidine-CH ),
2 3 2 2
2
.18–2.12 (s, 3H, ArCH ), 2.11–1.99 (m, 2H, piperidine-CH ), 1.78–1.54 (m, 4H, piperidine-
3
2
13
CH2). C NMR (75 MHz, Chloroform-d)
δ
177.63, 161.37, 161.16,158.16(C-F) 156.27(C-F),
1
1
40.83, 138.79, 136.90, 134.90, 131.76, 130.12, 129.74, 129.66, 129.52, 129.17(d), 125.43, 124.01,
17.29, 115.53(d), 55.64, 53.51(2C), 53.03, 42.38, 28.70(2C), 18.04. ESI-HRMS: m/z [M+H]+
calculated for C H FN O , 434.2244; Found: 434.2243. LCMS: t 10.05 min, purity, 96.3%.
2
6
29
3
2
R
0
0
1
-((6-(2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)piperidine-
4
-carboxylic acid (A8).
◦
1
White solid powder. Yield, 19.9%. m.p. 218.0–220.0 C. H NMR (300 MHz, DMSO-d )
6
δ
4
2
2
12.27 (Br, 1H, COOH), 7.78 (d, J = 7.3 Hz, 1H, ArH), 7.53–7.44 (m, 2H, ArH), 7.43–7.32 (m,
H, ArH), 7.32–7.24 (m, 1H, ArH), 7.15 (d, J = 7.3 Hz, 1H, ArH), 3.90 (s, 3H, OCH ), 3.49 (s,
3
H, ArCH N), 2.83 (d, J = 12.0 Hz, 2H, piperidine-CH ), 2.32–2.16 (m, 1H, CHCOOH), 2.17–
2
2
.03 (m, 5H, ArCH / piperidine-CH ), 1.83 (d, J = 13.0 Hz, 2H, piperidine-CH ), 1.68–1.54
3
2
2
13
(m, 2H, piperidine-CH2). C NMR (75 MHz, DMSO-d6)
δ
176.78, 160.90, 157.85(C-F),
1
1
55.41(C-F), 140.94, 138.98, 136.75, 134.40, 132.16, 130.28, 130.16, 129.96, 129.35, 129.14,
26.08, 125.07(d), 119.20, 117.59, 115.97(d), 55.66, 53.65(2C), 53.16, 40.76, 28.62(2C),18.19.
+
ESI-HRMS: m/z [M+H] calculated for C H FN O , 435.2084; Found: 435.2806. LCMS:
26
28
2
3
tR 6.52 min, purity, 99.1%.
3
.1.14. The Synthesis of Compounds A9–A18
Compounds A9 A18 were synthesized by the following procedure. A mixture of
aldehyde (0.92 mmol), -alanine amide hydrochloride (1.11 mmol), and Et N (1.84 mmol)
–
β
3
in DCM/MeOH (5 mL/1 mL) was stirred for 12 h at room temperature. NaBH(OAc)
3
◦
(2.30 mmol) was added to the mixture slowly at 0 C and stirred for another 8 h. The
reaction was adjusted to pH 7–8 with saturated NaHCO aqueous solution. The mixture
3
was extracted with DCM (10 mL
organic layer was dried over anhydrous Na SO , filtered, and concentrated in vacuum.
×
3) and washed with brine (10 mL
×
3). The combined
2
4
The residue was purified by silica gel chromatography to afford the product.
0
0
3
-(((6-(2 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)amino)
propenamide (A9)
◦
1
White solid powder. Yield, 54.1%. m.p. 218.0–222.0 C. H NMR (300 MHz, DMSO-d )
6
δ
7.81 (d, J = 7.5 Hz, 1H, ArH), 7.52–7.45 (m, 2H, ArH), 7.45–7.33 (m, 5H, ArH, 1/2CONH2),
7
.33–7.26 (m, 1H, ArH), 7.15 (d, J = 7.3 Hz, 1H, ArH), 6.82 (s, 1H, 1/2CONH ), 3.92 (s, 3H,
2
OCH ), 3.72 (s, 2H, ArCH ), 2.77 (t, J = 6.8 Hz, 2H, NHCH2), 2.29 (t, J = 6.8 Hz, 2H, CH CO),
3
2
2
13
2
.14 (s, 3H, ArCH3). C NMR (75 MHz, DMSO-d6)
δ
172.08, 161.02, 158.13(F-C), 157.85(F-
C), 141.56, 140.48, 136.87, 134.42, 132.12, 130.69,130.34, 130.24, 129.97, 129.01(d), 126.21,

Molecules 2021, 26, 3347
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1
25.11, 117.70, 115.85, 113.26, 54.12, 44.95, 43.43, 30.87, 18.16. ESI-HRMS: m/z [M+H]+
calculated for C H FN O , 394.1931; Found: 394.1929. LCMS: t 8.19 min, purity, 96.7%
.
23
25
3
2
R
0
0
3-(((6-(2,2 -Dimethyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)amino) prope-
namide (A10).
◦
1
White solid powder. Yield, 55.5%. m.p. 70.0–73.0 C. H NMR (400 MHz, DMSO-d )
6
δ
7.77 (d, J = 7.5 Hz, 1H, ArH), 7.42 (dd, J = 7.6, 1.6 Hz, 2H, ArH), 7.36–7.25 (m, 4H, ArH,
1
/2CONH ), 7.17–7.10 (m, 3H, ArH), 6.82 (s, 1H, 1/2CONH ), 3.89 (s, 3H, OCH ), 3.88 (s,
2
2
3
1
H, NH), 3.70 (s, 2H, ArCH ), 2.74 (t, J = 6.8 Hz, 2H, NHCH2CH ), 2.26 (t, J = 6.8 Hz, 2H,
2
2
13
NHCH CH2), 2.05 (s, 3H, ArCH ), 2.00 (s, 3H, ArCH ). C NMR (75 MHz, DMSO-d6)
δ
2
3
3
1
74.03, 160.68, 155.56, 142.51, 141.86, 140.88, 138.20, 135.65, 133.61, 130.29, 129.58, 129.37,
1
29.18, 127.78, 126.25, 125.92, 120.84, 117.49, 53.58, 47.04, 45.50, 35.68, 20.10, 18.03. ESI-
+
HRMS: m/z [M+H] calculated for C H N O , 390.2182; Found: 390.2183. LCMS: t
24
28
3
2
R
9
.04 min, purity, 95.1%.
0
0
3-(((6-(2 -Chloro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)amino)
propenamide (A11).
White solid powder. Yield, 52.9%. m.p. 49.0–51.0 C. H NMR (300 MHz, DMSO-d )
◦
1
δ
6
7
3
3
.81 (d, J = 7.5 Hz, 1H, ArH), 7.58–7.54 (m, 1H, ArH), 7.50 (s, 1H, 1/2CONH ), 7.46–7.40 (m,
2
H, ArH), 7.40–7.31 (m, 2H, ArH), 7.24–7.02 (m, 2H, ArH), 6.89 (s, 1H, 1/2CONH ), 3.91 (s,
2
H, OCH ), 3.74 (s, 2H, ArCH ), 2.80 (t, J = 6.8 Hz, 2H, NHCH2CH ), 2.33 (t, J = 6.7 Hz, 2H,
3
2
2
13
NHCH CH2), 2.07 (s, 3H, ArCH3). C NMR (75 MHz, DMSO-d6)
δ
174.15, 160.70, 155.26,
2
1
40.83, 140.69, 140.38, 138.15, 134.20, 133.99, 132.82, 131.76, 129.87, 129.71, 129.59, 127.74,
+
1
25.94, 121.13, 117.52, 53.57, 47.11, 45.57, 35.80, 17.99. ESI-HRMS: m/z [M+H] calculated
for C H ClN O , 410.1635; Found: 410.1633. LCMS: t 9.02 min, purity, 98.7%.
2
3
3
25
3
2
R
0
0
-(((2-Methoxy-6-(2-methyl-2 -(trifluoromethyl)-[1,1 -biphenyl]-3-yl)pyridin-3-yl)meth
yl)amino)propenamide (A12).
◦
1
White solid powder. Yield, 57.5%. m.p. 50.5–53.5 C. H NMR (300 MHz, DMSO-d )
6
δ
7.85 (t, J = 7.5 Hz, 2H, ArH), 7.74 (t, J = 7.2 Hz, 1H, ArH), 7.63 (t, J = 7.7 Hz, 1H, ArH),
7
.50 (s, 1H, 1/2CONH ), 7.46 (d, J = 7.7Hz, 1H, ArH), 7.40 (d, J = 7.3 Hz, 1H, ArH), 7.33
2
(t, J = 7.7 Hz, 1H, ArH), 7.15 (dd, J = 7.6, 5.2 Hz, 2H, ArH), 6.90 (s, 1H, 1/2CONH2),
3
.90 (s, 3H, OCH ), 3.82 (s, 2H, ArCH ), 2.85 (t, J = 6.9 Hz, 2H, NHCH2CH ), 2.36 (t,
3
2
2
13
J = 6.8 Hz, 2H, NHCH CH2), 1.98 (s, 3H, ArCH3). C NMR (75 MHz, DMSO-d6)
δ
173.58,
60.77, 155.86, 140.71, 140.43, 140.03, 138.98, 133.93, 132.84, 132.31, 129.91, 129.65, 128.58,
28.16( -CF ), 127.77( -CF ), 127.39( -CF ), 127.00 ( -CF ), 126.47(CF ), 126.39, 125.26,
22.75(CF ), 120.61(CF ), 119.24, 117.59, 53.68, 46.60, 45.06, 34.75, 18.53. ESI-HRMS: m/z
2
1
1
1
β
β
β
β
3
3
3
3
3
3
3
+
[M+H] calculated for C H F N O , 444.1899; Found: 444.1894. LCMS: t 6.68 min,
24 25 3 3 2 R
purity, 95.9%.
0
-(((2-Methoxy-6-(2-methyl-[1,1 -biphenyl]-3-yl)pyridin-3-yl)methyl)amino)propenamide
3
(
A13).
◦
1
White solid powder. Yield, 50.3%. m.p. 127.0–128.0 C. H NMR (400 MHz, DMSO-d )
6
δ
7
3
7.78 (d, J = 7.4 Hz, 1H, ArH), 7.50–7.43 (m, 2H, ArH), 7.43–7.31 (m, 6H, ArH, 1/2CONH2),
.24 (dd, J = 7.4, 1.7 Hz, 1H, ArH), 7.12 (d, J = 7.4 Hz, 1H, ArH), 6.81 (s, 1H, 1/2CONH2),
.90 (s, 3H, OCH ), 3.71 (s, 2H, ArCH ), 2.74 (t, J = 6.8 Hz, 2H, NHCH2CH ), 2.26 (t, J = 6.8
3
2
2
13
Hz, 2H, NHCH CH2), 2.18 (s, 3H, ArCH3). C NMR (100 MHz, Chloroform-d)
δ
175.15,
61.09, 156.99, 143.21, 142.21, 140.93, 138.21, 133.65, 129.94, 129.42(2C), 128.91, 128.08(2C),
26.84, 125.44, 119.35, 117.15, 53.52, 48.30, 44.85, 35.16, 18.64. ESI-HRMS: m/z [M+H]+
2
1
1
calculated for C H N O , 376.2025; Found: 376.2022. LCMS: t 8.51 min, purity, 95.6%.
2
3
26
3
2
R
0
0
3
-(((6-(3 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)amino)
propenamide (A14)
White solid powder. Yield, 48.6%. m.p. 48.0–51.0 C. H NMR (300 MHz, DMSO-d )
◦
1
δ
6
7
7
3
.80 (d, J = 7.5 Hz, 1H, ArH), 7.58–7.48 (m, 1H, ArH), 7.48–7.41 (m, 3H, ArH, 1/2CONH2),
.33–7.18 (m, 4H, ArH), 7.14 (d, J = 7.3 Hz, 1H, ArH), 6.81 (s, 1H, 1/2CONH ), 3.92 (s,
2
H, OCH ), 3.71 (s, 2H, ArCH ), 2.76 (t, J = 6.9 Hz, 2H, NHCH2), 2.28 (t, J = 6.8 Hz, 2H,
3
2
13
CH CONH ), 2.21 (s, 3H, ArCH ). C NMR (75 MHz, DMSO-d ) δ 172.31, 164.00, 161.02,
2
2
3
6
1
60.77, 158.03, 144.37, 141.78, 141.33, 140.85, 133.33, 130.78, 130.19, 129.67, 126.24, 117.71,

Molecules 2021, 26, 3347
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+
1
16.33, 114.26, 113.69, 54.11, 45.81, 43.61, 31.17, 18.77. ESI-HRMS: m/z [M+H] calculated
for C H FN O , 394.1931; Found: 394.1926. LCMS: t 4.95 min, purity, 96.1%.
2
3
3
25
3
2
R
0
0
-(((6-(4 -Fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)amino)
propenamide (A15)
◦
1
White solid powder. Yield, 49.3%. m.p. 120.0–122.0 C. H NMR (300 MHz, DMSO-d )
6
δ
7.82 (d, J = 7.5 Hz, 1H, ArH), 7.47–7.42 (m, 4H, ArH, 1/2CONH2), 7.40–7.25 (m, 4H,
ArH), 7.15 (d, J = 7.5 Hz, 1H, ArH), 6.85 (s, 1H, 1/2CONH2), 3.93 (s, 3H, OCH ), 3.76 (s,
3
2
H, ArCH ), 2.80 (t, J = 6.8 Hz, 2H, NHCH2CH ), 2.31 (t, J = 6.8 Hz, 2H, NHCH CH2),
2
2
2
13
2.19 (s, 3H, ArCH3). C NMR (100 MHz, Chloroform-d)
δ
175.34, 163.17(F-C), 161.08(F-C),
1
1
60.17, 156.78, 142.14, 141.07, 138.18(2C), 133.72, 130.96(2C), 129.93, 129.07, 125.50, 119.67,
17.11, 115.09, 114.85, 53.50, 48.27, 44.93, 35.32, 18.61. ESI-HRMS: m/z [M+H] calculated
+
for C H FN O , 394.1931; Found: 394.1931. LCMS: t 8.47 min, purity, 95.6%.
2
3
3
25
3
2
R
0
0
0
-(((6-(2 ,4 -Difluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)methyl)
amino)propenamide (A16)
◦
1
White solid powder. Yield, 43.9%. m.p. 113.0–115.0 C. H NMR (300 MHz, DMSO-d )
6
δ
7.78 (d, J = 7.4 Hz, 1H, ArH), 7.49–7.43 (m, 1H, ArH), 7.43–7.39 (m, 2H, ArH), 7.39–7.33
(
(
m, 2H, ArH), 7.27–7.16 (m, 2H, ArH, 1/2CONH ), 7.12 (d, J = 7.3 Hz, 1H, ArH), 6.80
2
s, 1H, 1/2CONH ), 3.89 (s, 3H, OCH ), 3.64 (s, 2H, ArCH ), 2.74 (t, J = 6.8 Hz, 2H,
2
3
2
1
3
NHCH2CH CO), 2.26 (t, J = 6.8 Hz, 2H, NHCH CH2CO), 2.10 (s, 3H, ArCH3). C NMR
2
2
(100 MHz, DMSO-d6)
δ
174.01, 160.71, 155.38, 141.04, 138.31, 135.85, 134.58, 133.03(d, 2C),
1
3
4
30.38, 130.10, 126.07, 125.59(d), 120.88, 117.46, 112.21(d), 104.38(d, 2C), 53.58, 47.00, 45.49,
+
5.60, 18.09. ESI-HRMS: m/z [M+H] calculated for C H F N O , 412.1837; Found:
23
24
2
3
2
12.1831. LCMS: t 8.92 min, purity, 98.7%.
R
0
0
0
3
-(((6-(4 -Chloro-2 -fluoro-2-methyl-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-3-yl)
methyl)amino)propenamide (A17)
White solid powder. Yield, 46.9%. m.p. 122.0–124.0 C. H NMR (300 MHz, DMSO-
7.80 (d, J = 7.5 Hz, 1H, ArH), 7.56 (d, J = 9.4 Hz, 1H, ArH), 7.50–7.37 (m, 5H,
ArH,1/2CONH ), 7.25 (d, J = 7.5 Hz, 1H, ArH), 7.13 (d, J = 7.3 Hz, 1H, ArH), 6.83 (s, 1H,
◦
1
d )
δ
6
2
1
2
/2CONH ), 3.89 (s, 3H, OCH ), 3.73 (s, 2H, ArCH ), 2.77 (t, J = 5.9 Hz, 2H, NHCH2CH2),
2 3 2
13
.28 (t, J = 6.2 Hz, 2H, NHCH CH2), 2.11 (s, 3H, ArCH3). C NMR (75 MHz, DMSO-d6)
δ
2
1
73.95, 161.05, 160.70, 157.77(F-C), 155.20(F-C), 141.08, 138.23, 135.61, 134.43, 133.69, 133.55,
1
3
4
33.37(d), 130.27, 128.33(d), 126.19, 125.35(d), 121.18, 117.51, 116.66(d), 53.63, 47.04, 45.55,
+
5.76, 18.15. ESI-HRMS: m/z [M+H] calculated for C H ClFN O , 428.1541; Found:
23
24
3
2
28.1537. LCMS: t 9.62 min, purity, 95.2%.
R
0
0
0
3-(((6-(2 -Fluoro-2-methyl-4 -(trifluoromethyl)-[1,1 -biphenyl]-3-yl)-2-methoxypyridin-
3
-yl)methyl)amino)propenamide (A18)
◦
1
White solid powder. Yield, 50.9%. m.p. 137.0–139.0 C. H NMR (300 MHz, DMSO-
d )
6
δ
7.87–7.76 (m, 2H, ArH), 7.71 (dd, J = 8.5, 1.8 Hz, 1H, ArH), 7.64 (t, J = 7.6 Hz, 1H,
ArH), 7.50 (dd, J = 7.7, 1.6 Hz, 1H, ArH), 7.45–7.37 (m, 2H, ArH, 1/2CONH ), 7.30 (dd,
2
J = 7.6, 1.6 Hz, 1H, ArH), 7.13 (d, J = 7.3 Hz, 1H, ArH), 6.80 (s, 1H, 1/2CONH ), 3.89 (s,
2
3
2
H, OCH ), 3.69(s, 2H, ArCH ), 2.74 (t, J = 6.8 Hz, 2H, NHCH2CH ), 2.26 (t, J = 6.8 Hz
,
3
2
2
13
H, NHCH2CH ), 2.12 (s, 3H, ArCH ). C NMR (100 MHz, Chloroform-d)
δ
175.29,
2
3
1
1
1
61.15, 160.63(F-C),158.16(F-C), 156.22, 140.95, 138.15, 135.48, 134.68, 133.49(d), 132.47(d),
31.51(dd), 130.23, 127.44(CF ), 125.69, 124.72(CF ), 122.04(CF ), 121.03, 119.95,119.33(CF ),
3
3
3
3
+
17.17, 113.13(d), 53.52, 48.33, 44.90, 35.35, 18.00. ESI-HRMS: m/z [M+H] calculated for
C H F N O , 462.1805; Found: 462.1799. LCMS: t 9.72 min, purity, 96.8%.
24
24
4
3
2
R
3.2. Pharmacology
3.2.1. HTRF-Based PD-1/PD-L1 Binding Assay
The PD-1/ PD-L1 binding assay kits (Cisbio, Codolet, France, Cat. no. 64ICP01PEG)
were purchased from Cisbio. The experiments were performed according to the manufac-
turer’s guidelines. The HTRF assay used Perkin Elmer EnVision to read the fluorescence
intensity at 665 nm and 615 nm and calculate the HTRF ratio (665 nm emission/615 nm emis-
sion). The IC50 values were calculated by four-parameter logistic curve in Graphpad prism
.

Molecules 2021, 26, 3347
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3.2.2. In Vitro Non-Specific Cytotoxicity Assay
The MDA-MB-231 and Jurkat cell line was provided by the Chinese Academy of
Sciences Cell Bank. The cytotoxicity of the test compounds was determined using the MTT
◦
assay. Briefly, MDA-MB-231 and Jurkat cells were incubated at 37 C in a humidified 5%
CO incubator for 24 h in 96-microwell plates. Then, 100 µL of culture medium containing
2
the test compounds at different concentrations was added to each well and incubated at
◦
3
7 C for another 48 h. The MTT was added and incubated for another 4 h, and the optical
density was detected with a microplate reader at 570 nm. The IC50 values were calculated
according to the dose-dependent curves.
3.2.3. Binding Affinity Assay
SPR analysis was carried out according to the method described by a previous ar-
ticle [23]. The kinetic analysis for the interaction of small molecules with hPD-L1 was
performed on a Biacore T200 (GE Healthcare Bio-sciences, Uppsala, Sweden) using Series S
Sensor Chip Protein A (GE Healthcare Bio-sciences, Uppsala, Sweden, catalog # 29139131-
AA). Briefly, Fc-hPD-L1 (Acro Biosystems, Cambridge, MA, USA, catalog #PD1-H5258) was
+
diluted using 1
#
×
PBS-P buffer (phosphate buffer 1
×
, GE Healthcare Bio-sciences, catalog
28-9950-84, enriched with 0.05% surfactant P20) containing 5% DMSO (Sigma-Aldrich,
St. Louis, MO, America, catalog # D4540) and captured as the ligand (i.e., the target of
small molecules) on protein A chip surface following Biacore T200 build-in protocol for ki-
netic/affinity analysis at a concentration of 10
µg/mL. The contact time and flow rates were
set as 120 s and 10 L/min for the ligand capture. Each experiment was performed using a
µ
pair of flow cells, one with ligand captured and the other kept as reference. A concentration
+
gradient of analytes was freshly prepared in 1
×
PBS-P buffer with 5% DMSO with at
least five concentrations, and the concentration range was optimized according to different
analytes. Analytes could flow through both ligand-captured flow cells and reference flow
cells at the same rate (30 µL/min) and contact time (90 s). Solvent correction was included
to avoid the impact of DMSO on surface plasmon effect during binding analysis. Extra
+
washing of the flow system using 50% DMSO in 1
×
PBS-P buffer was allowed to control
carry over effects. For the regeneration of surface after each cycle (one-time ligand capture
and binding of analyte at a single concentration) was carried out using 10 mM glycine pH
1
3
.5 (GE Healthcare Bio-sciences, Uppsala, Sweden, catalog #BR-1003-54) at a flow rate of
0 µL/min for 30 s. Kinetic analysis was performed at 25 C. Maximum binding response
◦
(R
max) and equilibrium dissociation rate constant (K ) values were calculated using Biacore
D
T200 Evaluation software following the 1:1 binding model.
3.2.4. IFN-γ Production Effect in T Cells Function Assay
The tumor/T cells coculture assay was conducted by ChemPartner [19]. Briefly, Hep3B
cells were engineered to stably express OS-8 (anti-CD3 single chain variable fragment)
and human PD-L1 (hPD-L1). Fresh PBMCs were isolated from a healthy donor by density
+
gradient centrifugation. CD3 T cells were isolated from fresh PBMCs by EasySep Human
T Cells Isolation Kit (negative selection, STEMCELL Technologies, Vancouver, Canada).
Hep3B-OS8- hPD-L1 cells were harvested and treated with 10
µ
g/mL mitomycin C at
◦
4
3
5
7 C for 1.5 h and washed 4 times with PBS. Hep3B-OS8-hPD-L1 and T cells (2.5
×
10 in
4
0
µL and 5
×
10 in 100
µ
L complete media, respectively) were added to the 96-well plates,
final concentration of test compound in 50 L complete
L) were harvested after 72 h of coculture to determine
followed by the addition of 4
media. The supernatants (150
×
µ
µ
IFN-γ levels by ELISA.
3.3. Molecular Modelling
Molecular docking simulation was performed in the Schrödinger suite [28]. The
molecules were washed and processed, the 3D conformation of small molecules were
prepared by LigPrep module. The X-ray crystallographic structure of PD-L1 was obtained
from the RCSB Protein Data Bank (PDB ID: 5J89). The protein was prepared to remove

Molecules 2021, 26, 3347
21 of 23
the water molecules and add hydrogens and minimized using Protein Preparation Wizard
module. Then, the binding site was defined using the original ligand in the complex by
Receptor Grid Generation module. The Glide Docking module in standard precision (SP) was
used to investigate the interactions between compounds and PD-L1. Other parameters
were set as the default. All the figures illustrating the molecular docking results were
visualized and generated using the Maestro or Pymol software.
3.4. Water Solubility Studies
The aqueous solubility was estimated by HPLC. The HPLC analysis was performed
on a Shimadzu LC-20AT machine with a BDS Hypersil C18 column, and the column
◦
temperature was at 28 C. Mobile phase B (100% MeOH) and mobile phase A (100% water)
were used in a gradient elution program (0 min: 30% (B), 2 min: 50% (B), 8 min: 95% (B),
1
2
4 min: 95% (B), 18 min: 70% (B), 20 min: 30% (B)) with a flow rate of 1.0 mL/min at
54 nM. Methanol was used to configure an accurate compounds concentration to calculate
the standard curve. The excess test compound was dissolved in PBS (KH PO 0.24 g + KCl
2
4
0
.2 g + NaCl 8 g + Na HPO ·12H O 3.58 g + 1 L water) and then analyzed by HPLC.
2
4
2
4. Conclusions
The blockade of the PD-1/PD-L1 interaction by small molecules has been highly
anticipated as a promising alternative or complementary therapeutic to mAbs in the field
of cancer immunotherapy. Currently, the field of developing small-molecule inhibitors
of PD-1/PD-L1 interaction is intensively explored and makes great progress. Moreover,
several small molecules, derived from the skeleton of BMS-202, have entered clinical trials
to treat advanced solid tumors. Although several co-crystal complexes of inhibitors binding
to PD-L1 dimer have been reported, which provided a definite guide in the rational design
of PD-L1 small molecular inhibitors, the highly hydrophobic PD-1/PD-L1 binding interface
remain a challenge for designing anti-PD-1/PD-L1 small-molecule inhibitors.
0
In this research, a novel series of (2-methyl-[1,1 -biphenyl]-3-yl) pyridine derivatives
were designed and synthesized as inhibitors targeting PD-1/PD-L1 pathways based on
structural simplification strategy. The representative compound A9 presents significantly
improved potency in biochemical assay compared with BMS-202. Notably, the bioactivity
of A9 is equivalent to that of BMS-1058, but possesses lower MW, PSA, and higher LE value.
Subsequently, we utilized SPR assay to measure the binding affinity and kinetics of A9 to
hPD-L1 (K : 3.64 nM), and the results show that A9 possesses the feature of fast association
D
and slow dissociation rate. Furthermore, small-molecule inhibitor A9 (5
µM) and mAbs
Keytruda (5 g/mL) show comparable promoting effect on production of INF-
µ
γ
in a dose-
dependent manner in Hep3B/OS-8/hPD-L1 and CD3-positive T cells co-culture assay,
implying that A9 may reverse hPD-1/hPD-L1 pathway-mediated immunosuppression.
Taken together, these results suggest that A9 is a promising inhibitor of PD-1/PD-L1
interaction and is worthy for further study.
Supplementary Materials: The following are available online, Table S1: The Kinetic Parameters and
Binding Affinities of compounds A7, A9, A11 and A15 with hPD-L1.
Author Contributions: Designed and synthesized the target compounds, H.Z., Y.X. (Yu Xia) and
H.L.; in vitro HTRF assay, H.Z., S.H. and H.D.; in vitro non-specific cytotoxicity assay, C.Y.; software,
Y.Z.; writing—original draft preparation, H.Z. and J.L.; writing—review and editing, H.Z. and Y.Z.;
funding acquisition, Q.Z. and Y.X. (Yungen Xu). All authors have read and agreed to the published
version of the manuscript.
Funding: This work was supported by the Outstanding Scientific and Technological Innovation
Team Projects of Jiangsu Province, China (2015).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.

Molecules 2021, 26, 3347
22 of 23
Data Availability Statement: The article contains complete data used to support the findings of
this study.
Acknowledgments: Thank you for support of the public platform of the State Key Laboratory of
Natural Medicines, China Pharmaceutical University.
Conflicts of 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.
Sample Availability: Samples of the compounds described in this paper are available from the authors
.
References
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