Novel Biphenyl Pyridines as Potent Small-Molecule Inhibitors Targeting the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction

Article abstract of DOI:10.1021/acs.jmedchem.1c00010

With the successful clinical application of anti-programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) monoclonal antibodies (mAb), targeting the PD-1/PD-L1 interaction has become a promising method for the discovery of cancer therapy. Due to the inherent limitations of antibodies, it is necessary to search for small-molecule inhibitors against the PD-1/PD-L1 axis. We report the design, synthesis, and evaluation in vitro and in vivo of a series of novel biphenyl pyridines as the inhibitors of PD-1/PD-L1. 2-(((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)ethan-1-ol (24) was found to inhibit the PD-1/PD-L1 interaction with an IC50 value of 3.8 ± 0.3 nM and enhance the killing activity of tumor cells by immune cells. Compound 24 displays great pharmacokinetics (oral bioavailability of 22%) and significant in vivo antitumor activity in a CT26 mouse model. Flow cytometry and immunohistochemistry data indicated that compound 24 activates the immune activity in tumors. These results suggest that compound 24 is a promising small-molecule inhibitor against the PD-1/PD-L1 axis and merits further development.

Full text of DOI:10.1021/acs.jmedchem.1c00010

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Article
Novel Biphenyl Pyridines as Potent Small-Molecule Inhibitors
Targeting the Programmed Cell Death-1/Programmed Cell Death-
Ligand 1 Interaction
Tianyu Wang, Shi Cai, Mingming Wang, Wanheng Zhang, Kuojun Zhang, Dong Chen, Zheng Li,
and Sheng Jiang*
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ABSTRACT: With the successful clinical application of anti-
programmed cell death-1 (PD-1)/programmed cell death-ligand 1
(PD-L1) monoclonal antibodies (mAb), targeting the PD-1/PD-L1
interaction has become a promising method for the discovery of
cancer therapy. Due to the inherent limitations of antibodies, it is
necessary to search for small-molecule inhibitors against the PD-1/
PD-L1 axis. We report the design, synthesis, and evaluation in vitro
and in vivo of a series of novel biphenyl pyridines as the inhibitors of
PD-1/PD-L1. 2-(((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)-
pyridin-3-yl)methyl)amino)ethan-1-ol (24) was found to inhibit the
PD-1/PD-L1 interaction with an IC50 value of 3.8
0.3 nM and
enhance the killing activity of tumor cells by immune cells.
Compound 24 displays great pharmacokinetics (oral bioavailability
of 22%) and significant in vivo antitumor activity in a CT26 mouse
model. Flow cytometry and immunohistochemistry data indicated that compound 24 activates the immune activity in tumors. These
results suggest that compound 24 is a promising small-molecule inhibitor against the PD-1/PD-L1 axis and merits further
development.
cancer, pancreatic cancer, and breast cancer cells, interacts with
PD-1 on T cells to evade immune surveillance.2,15
INTRODUCTION
■
Tumor cells hijack certain immune checkpoint pathways to
suppress the activation of immune cells and thus evade
immune surveillance.1−3 The blockade of immune checkpoint
pathways by specific inhibitors can restore or increase the
antitumor immunity of the body.4,5 With the recent clinical
success of immune checkpoint inhibitors in cancer patients, the
method has become an important branch of cancer therapy.6−8
Blockade of programmed cell death-1 (PD-1) and its ligand−−
programmed cell death-ligand 1 (PD-L1) has achieved the
most remarkable clinical success, and this work was awarded
the Nobel Prize in Physiology or Medicine in 2018.9,10 PD-1,
also known as CD279, is a homologue of CD28 consisting of
288 amino acids. It is expressed on the surface of many types of
immune cells and is upregulated in activated T cells.11,12 The
interaction of PD-1 with its ligand PD-L1 (also known as B7−
H1 or CD274) impedes T cell receptor-mediated signaling and
induces T cell anergy and functional exhaustion.13,14 Under
normal physiological conditions, the PD-1/PD-L1 pathway
provides inhibitory signals to maintain self-tolerance and
protect tissues from the damage of excessive immune
responses. However, the aberrant expression of PD-L1 on
the surface of several types of tumor cells, such as esophageal
Currently, the US Food and Drug Administration (FDA)
has approved six monoclonal antibodies (mAbs) of the PD-1/
PD-L1 pathway, including three PD-1 inhibitors (nivolumab,
pembrolizumab, and cemiplimab) and three PD-L1 inhibitors
(atezolizumab, durvalumab, and avelumab). Both anti-PD-1
and anti-PD-L1 mAbs exhibit promising clinical antitumor
activity in the treatment of melanoma, non-small-cell lung
cancer, and urothelial carcinoma.16 However, mAbs targeting
the PD-1/PD-L1 pathway have several disadvantages including
their long half-life, unchecked immune response, and immune-
related adverse events.17 Moreover, lack of oral bioavailability
and poor permeability of tumor tissues are the disadvantages of
PD-1/PD-L1 therapeutic antibodies used in clinics. A
promising alternative to overcome these limitations is the
Received: January 4, 2021
Published: May 31, 2021
https://doi.org/10.1021/acs.jmedchem.1c00010
© 2021 American Chemical Society
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Figure 1. Chemical structures of the reported small-molecule inhibitors targeting the PD-1/PD-L1 pathway.
Figure 2. (A) Crystal structure of the PD-L1/1 complex (above, PDB code 5J89) and PD-L1/2 (below, PDB code 5N2F); chain A, green; chain B,
blue; hydrogen bonds are shown as yellow dashed lines; (B) pharmacophoric model and design strategy of compounds.
application of small-molecule inhibitors of the PD-1/PD-L1
pathway.18
In the present study, we designed and synthesized novel
biphenyl pyridines as potent small-molecule inhibitors of the
PD-1/PD-L1 pathway. We evaluated their biochemical
activities and investigated the preliminary structure−activity
relationships (SARs). According to detailed structural
modifications, compound 24 was identified as the most potent
inhibitor with an IC50 of 3.8 0.3 nM, as assessed by a time-
resolved fluorescence resonance energy transfer (TR-FRET)
assay. Compound 24 remarkably activates the antitumor
activities of human peripheral blood mononuclear cells
(PBMC) which kill MDB-MB 231 cells. In vivo, compound
24, administered orally, significantly inhibits tumor growth in a
CT26 mouse model and displays significant activation of the
immune microenvironment in the tumor.
During the past few years, small-molecule inhibitors
targeting the PD-1/PD-L1 pathway have made substantial
progress.19 The crystal structures of the human PD-1/PD-L1
complex and small molecules complexed with PD-L1 have
been disclosed, and the small-molecule interaction “hot spots”
on PD-L1 have been identified.20−22 To date, several potent
small-molecule inhibitors targeting PD-L1 have been reported
(Figure 1).23−28 The analysis of the crystal structures of the
PD-L1/inhibitor complex suggests that an aromatic ring
system is the core pharmacophore for PD-L1 binding, but to
date, no small-molecule inhibitors targeting the PD-1/PD-L1
pathway have been approved for cancer treatment, and the
development of marketable inhibitors for clinics remains a
challenging process. In the future, the search for novel small-
molecule inhibitors targeting the PD-1/PD-L1 pathway with
more structural diversity, high potency, and further inves-
tigative data in vitro and in vivo will continue in drug
development.
RESULTS AND DISCUSSION
■
Drug Design and Chemical Synthesis. As shown in
Figure 2A, compounds 1 and 2 insert into a deep cylindrical
and hydrophobic pocket at the interface of the PD-L1 dimer.
The distal phenyl ring within the biphenyl core creates a T-
stacking interaction with Tyr56, and the phenylmethyl ring
A
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a
Scheme 1. Synthesis of Compounds 15−17
a
Reagents and conditions: (a) Bis(pinacolato)diboron, PdCl2(dppf), KOAc, 1,4-dioxane, 130 °C, overnight; (b) Pd(PPh3)4, K2CO3, 1,4-dioxane/
H2O (10:1), 85 °C, 4 h; (c) XPhos Pd G2, K3PO4, bromobenzene, THF/H2O (3:1), 80 °C, overnight; (d) DMP, NaHCO3, DCM/DMF (4/1), rt,
0.5 h; (e) appropriate amine, HOAc, NaBH3CN, CH3OH/CH2Cl2 (1:1), rt, 6−10 h.
a
Scheme 2. Synthesis of Compounds 24−40 and 43
a
Reagents and conditions: (a) MeOH, NaH, THF, 60 °C, 2 h; SOCl2, MeOH, 70 °C, 3 h; (b) bis(pinacolato)diboron, PdCl2(dppf), KOAc, 1,4-
dioxane, 130 °C, overnight; (c) Pd(PPh3)4, K2CO3, 1,4-dioxane/H2O (10:1), 85 °C, 4 h; (d) LiAlH4, THF, rt, 3 h; (e) XPhos Pd G2, K3PO4, R1Br,
THF/H2O (3:1), 80 °C, overnight; (f) DMP, NaHCO3, DCM/DMF (4/1), room temperature (rt), 0.5 h; (g) appropriate amine, HOAc,
NaBH3CN, CH3OH/CH2Cl2 (1:1), rt, 6−10 h; (h) XPhos Pd G2, K3PO4, bromobenzene, THF/H2O (3:1), 80 °C; (i) LiOH, MeOH/THF/H2O;
(j) 2-aminoethanol, HOBT, EDCI, CH2Cl2; (k) XPhos Pd G2, K3PO4, bromobenzene, THF/H2O (3:1), 80 °C, overnight.
enjoys a hydrophobic interaction with Ala121, Met115, and
instead linked the aryl group and biaryl core directly. With the
direct coupling of the aryl and phenylmethyl groups, the aryl
group will provide additional hydrophobic interaction with
AAla121 and AMet115. Being directly connected with the
phenylmethyl unit, the aryl group can rotate through various
A
A
BMet115. The aryl group is involved in π−π stacking with
BTyr56. In order to support the molecule being inserted into
this deep cylindrical cavity, the linker, which links the
phenylmethyl and aryl groups, must be approximatively
coplanar with the aryl group. The solubilizer R4 at the distal
end stretches out of the cavity into the solvent and may
interact with PD-L1 through hydrogen bonds. Most reported
inhibitors appear to follow the pharmacophoric model shown
in Figure 2B. Among the reported inhibitors in Figure 1, ethers
and amides are commonly chosen as the linkers, which are
replaceable and play a limited role in SARs. Accordingly, we
designed biphenyl pyridines as novel small-molecule inhibitors,
as shown in Figure 2B. We abandoned the original linker and
angles, and this allows it to interact more easily with Tyr56
B
through π−π stacking.
The synthesis of biphenyl pyridines 15−17 is described in
Scheme 1. The preparation of 2,2′-(2-methyl-1,3-phenylene)-
bis(4,4,5,5-tetramethyl-1,3,2-dioxaborolane) (11) was accom-
plished by borylation of the commercially available 1,3-
dibromo-2-methylbenzene (10). Compound 11 reacted with
9a or 9b under Suzuki−Miyaura coupling conditions to afford
the intermediate 12a or 12b, respectively. Suzuki−Miyaura
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coupling of 12a or 12b with bromobenzene yielded 13a or
13b. Then, 13a or 13b was oxidized to aldehydes 14a or 14b
by Dess−Martin periodinane. Finally, the target compounds
15−17 were generated by reductive amination using
NaBH3CN and the appropriate amines.
In view of the excellent activity of compound 24, the
alkoxypyridine fragment was retained, and further modifica-
tions were conducted by replacing the solubilizer tail on the
pyridine. A series of hydrophilic moieties were introduced,
yielding compounds 26−32, as shown in Table 2. All the
The synthesis of biphenyl pyridine compounds 24−40 and
43 is shown in Scheme 2. The methylation and methyl ester
formation of the commercially available 2,6-dichloronicotinic
acid led to the formation of intermediate 19. Compound 20
was generated from 11 and 19 using the Suzuki−Miyaura
coupling reaction. Using LiAlH4, 20 was reduced to alcohol
(21). 21 reacted with phenylboronic acid or a substituted
phenylboronic acid and yielded 22a−22d. By adopting
synthetic routes similar to those used for 15−17, target
compounds 24−39 were generated. Compound 40 was
produced by Suzuki−Miyaura coupling of 20 with bromo-
benzene. Compound 43 was generated from intermediate 20
by hydrolysis and subsequent acylation.
Table 2. SARs of Compounds 26−32 by PD-1/PD-L1 TR-
FRET Assay
Biochemical Evaluation and Structure Optimization.
Initially, compounds 15−17, 24, and 25 were synthesized in an
effort to validate our design strategy. The activities of inhibitors
targeting the PD-1/PD-L1 pathway were evaluated by using
the well-established TR-FRET assay. The resulting IC50 values
are presented in Table 1. As shown in Table 1, biphenyl
Table 1. SARs of Compounds 15−17, 24, and 25 by PD-1/
PD-L1 TR-FRET Assay
a
The data are displayed as averages of duplicate assays SD.
biphenyl alkoxypyridine compounds (24−32) displayed
promising activities against the PD-1/PD-L1 interaction, with
almost all the IC50 values below 20 nM, again demonstrating
the potent efficacy of biphenyl pyridines as PD-1/PD-L1
inhibitors. Among these, compounds 24 and 28−31,
containing an alcohol group, showed somewhat better
inhibitory activities of PD-1/PD-L1, with IC50 values below
10 nM. Optimization of the tail group indicated that the
solubilizer tail containing a hydroxyl group may contribute
more hydrogen bond-like interactions with PD-L1.
In further investigations, compounds 33−35 which have
diverse extensions of the distal phenyl ring within the biphenyl
core were synthesized. In view of the T-stacking interaction
with ATyr56, we chose a suitable substituent that can maintain
this interaction. As shown in Table 3, compounds 33−35
displayed good inhibition of the PD-1/PD-L1 interaction, and
compound 35 with 2,3-dihydro-1,4-benzodioxinyl moiety
a
The data are displayed as averages of duplicate assays SD.
pyridine compounds 15−17 exhibited excellent inhibitory
activities against the PD-1/PD-L1 interaction, with IC50 values
of 130.6 9.6, 34.6 2.4, and 52.8 5.2 nM, respectively.
Comparisons of 15 with 16 and 17 indicated that shifting the
solubilizing tail of biphenyl pyridines from the 4- to the 5-
position led to obviously increased activity. When a methoxy
group was introduced to the pyridine ring, sharply increased
activities of compounds 24 and 25 were observed, indicating
that methoxy attached to the pyridine is beneficial. Compound
24 displayed potent activity, with an IC50 value of 3.8 0.3
nM, approximately 4 times that of the activity of the positive
compound (1). Preliminary biochemical evaluation demon-
strated that biphenyl pyridine compounds lacking a linker
exhibited improved activity over that of the reported inhibitors
of the PD-1/PD-L1 pathway. This is consistent with the
pharmacophoric model in Figure 2B.
showed better activity (IC50, 7.9
1.1 nM). However, all
these compounds exhibited lower activities than the precursor
compound 24. Because the introduction of the 2,3-dihydro-
1,4-benzodioxinyl moiety facilitated the binding pocket of PD-
L1 as a deep, enlarged hydrophobic tunnel, we further
investigated the activity of 2,3-dihydro-1,4-benzodioxinyl
analogues. Compared with the biphenyl compounds 25, 26,
30, and 32, compounds 36−39 exhibited almost equal
inhibitory activity, as shown in Table 3, but 24 was still the
most potent compound. Replacement of the aminoethanol
group of compound 24 with a methyl ester and reaction with
N-(2-hydroxyethyl)acetamide yielded compounds 40 and 43,
which had however sharply decreased activities, with the IC50
values of 326.1 18.5 and 327 12.1 nM, respectively.
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ATyr56, and the phenylmethyl provides hydrophobic inter-
actions with Ala121, Met115, and Met115. As anticipated,
Table 3. SARs of Compounds 33−40 and 43 by PD-1/PD-
L1 TR-FRET Assay
A
A
B
the pyridine group, linking to phenylmethyl directly without a
linker, participated in π−π stacking with BTyr56 and in
hydrophobic interactions with AAla121, providing a strong
stabilization of the binding with the PD-L1 dimer. The
aminoethanol tail of 24 interacts with AAsp122 and BGln66 via
two hydrogen bonds and contributes to the high binding
affinity to dimeric PD-L1.
Antitumor Immunity of PBMCs Activated by Com-
pound 24. To evaluate the potency of compound 24 inducing
antitumor immunity, we investigated the effects of compound
24 on the killing of MDB-MB 231 cells by human PBMC.
Tumor cell viability was detected by the lactate dehydrogenase
(LDH) release. As shown in Figure 4, compound 24 at a
Figure 4. Compound 24 enhances the killing by PBMCs of MDA-
MB-231 cells. Tumor cell viability was detected by LDH release at
MDA-MB-231 cells/PBMCs (1:10). All data are presented as mean
SD, **P < 0.05 vs PBMC group, n = 3.
a
The data are displayed as averages of duplicate assays SD.
Docking Analysis of Compound 24 with the PD-L1
Dimer. To investigate the binding mode of biphenyl pyridine
compounds with the PD-L1 dimer, an analysis of the docking
of compound 24 with dimeric PD-L1 was conducted. The
docking results showed that 24 entered into the deep
hydrophobic cleft formed by dimeric PD-L1 (Figure 3).
Consistent with the binding mode of compounds 1 and 2 with
PD-L1, the distal phenyl creates a T-stacking interaction with
concentration of 1.5 μM is not toxic toward MDB-MB 231
cells, and PBMCs alone failed to show antitumor activity to
MDB-MB 231 cells. When PBMCs were incubated with
different concentrations of compound 24, dead tumor cells
were produced by the treatment with PBMCs and 24.
Compound 24 significantly activated the antitumor immunity
of PBMCs in a dose-dependent manner, with the estimated
EC50 value of ∼100 nM. Dead tumor cells increased upon
Figure 3. Docking analysis of compound 24 with the PD-L1 dimer (chain A, green; chain B, blue). The PDB code for the dimeric PD-L1 protein is
5J89.
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treatment with 375 nM compound 24 (53%), which is higher
than 5 μg/mL durvalumab (49%) and 500 nM compound 1
(44%).
In Vivo Pharmacokinetic Properties of Compound 24.
We further evaluated the pharmacokinetic (PK) properties of
compound 24 in Sprague-Dawley (SD) rats. The primary PK
parameters of compound 24 via intravenous (iv) and oral (per
os, po) routes are shown in Table 4. After the iv administration
indicated that compound 24 exhibited good PK properties and
excellent potency of oral administration, which are rarely seen
in the reported small-molecule inhibitors targeting the PD-1/
PD-L1 pathway in Figure 1.
In Vivo Antitumor Activity of Compound 24. To assess
the antitumor activity of compound 24, we evaluated its in vivo
antitumor efficacy using a CT26 mouse model in BALB/c
mice. After the tumor volume reached approximately 50 mm3,
the mice were treated either with a vehicle control or with
compound 24 (40, 80 mg/kg) by oral administration once a
day for 2 weeks. The results of tumor growth inhibition (TGI)
in different groups at different days after treatment are shown
in Figure 5A. Compound 24 inhibited tumor growth in a dose-
dependent manner and did not cause any body weight loss or
mortality during treatment (Figure 5B), indicating that all
doses of 24 were well tolerated. Based on the final tumor
weights, groups treated with compound 24 were significantly
superior to those treated with the vehicle control (Figure
5C,D). Doses of 40 and 80 mg kg−1 day−1 significantly
decreased the final tumor weight, with TGI values of 60 and
67%, respectively, compared with an untreated vehicle group.
These results confirmed the efficacy of compound 24 in vivo as
an oral small-molecule inhibitor of the PD-1/PD-L1 pathway.
Dissociated tumors were digested into single cells and
analyzed with flow cytometry after being dyed with different
tumor-infiltrating lymphocyte (TIL) markers. As shown in
Figure 6A,B, the level of cytotoxic T cells (CD8+ cells) in
tumor tissue increased distinctly after treatment with
compound 24 in a dose-dependent manner, when compared
to the vehicle control group. When treated with compound 24,
the ratio of CD4+/CD8+ cells was obviously less than that in
the vehicle control (Figure 6C). We also analyzed tumor
Table 4. Primary PK Parameters of Compound 24 in SD
Rats
a
PK parameters
iv (3 mg/kg)
po (25 mg/kg)
AUC(0−t) (ng/mL·h)
Cmax (ng/mL)
t1/2 (h)
430.5 49.8
1233 461.7
4.2 1.6
787.4 103.5
192.0 87.8
6.4 2.8
Tmax
0.03
11.5 1.1
78.6 41.0
0.69 1.1
28.8 6.6
249.3 66.5
22
Cl (L/h/kg)
Vz (L/kg)
F (%)
a
AUC, area under the curve; t1/2, half-life; Tmax, time of maximum
plasma concentration; Cmax, maximum plasma concentration; Cl,
plasma clearance; Vz, apparent distribution volume; F, oral
bioavailability. n = 4, mean SD.
of compound 24 (3 mg/kg), the values of AUC(0−t) and Cmax
of compound 24 are 430.5 49.8 ng/mL·h and 1233 461.7
ng/mL, respectively. The plasma clearance (Cl) and half-life
(t1/2) of compound 24 are 11.5 1.1 L/h/kg and 4.2 1.6 h,
respectively. When compound 24 was administrated orally (25
mg/kg), the half-life (t1/2) of compound 24 reached 6.4 2.8
h. Moreover, compound 24 exhibited markedly excellent oral
exposure, with oral bioavailability (F) of 22%. These results
Figure 5. In vivo antitumor activity of compound 24 against CT26 tumor in mice. (A) Tumor volume changes during treatment. (B) Body weight
changes of mice during treatment. (C) Weight of the excised tumors of each group. (D) Image of the excised tumors in each group after treatment.
All data are presented as mean SD, **P < 0.05 vs control group, n = 5.
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Figure 6. (A) Representative examples for CD4+ and CD8+ cells in dissociated tumors of vehicle control or mice treated with compound 24. (B)
Percentage of CD4+ and CD8+ cells in CD3+ cells of tumor tissue. (C) Ratio of CD4+/CD8+ cells of tumor tissue. All data are presented as mean
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Figure 6. continued
SD, **P < 0.05 vs control group, n = 3. (D) Representative immunohistochemistry determination of CD8+ cell infiltration into tumor tissues. Scale
bars, 50 μM. (E) H&E staining of tumor tissues. Scale bars, 50 μM.
tissues by immunohistochemistry. In agreement with the
results of flow cytometry, significant increased infiltration of
CD8+ cells was observed in tumor tissues treated with
compound 24, compared with those treated with the vehicle
control (Figure 6D). Hematoxylin and eosin (H&E) staining
of tumor tissues revealed that tumor cells treated with 24 had
decreased nucleus-to-cytoplasm ratios, and compound 24
induced tumor shrinkage and inhibited tumor cell growth
(Figure 6E). These data demonstrated that compound 24
enhances the infiltration of CD8+ cells and activated immune
activity in tumors.
We further directly compared the in vivo antitumor efficacy
of compound 24 with the reported PD-L1 inhibitor compound
1 and antimouse PD-L1 antibody using a CT26 mouse model
in BALB/c mice. As shown in Figure S1, at the same dose and
mode of administration (40 mg/kg, orally), compound 24
(TGI = 57%) showed obviously better TGI compared with
compound 1 (TGI = 32%). Moreover, compound 24 showed
better in vivo activity than PD-L1 antibody (TGI = 45%).
Overall, compound 24 exhibited excellent TGI, administered
orally, comparable to PD-L1 antibody, and demonstrated
significantly higher in vivo activity than the previously
described PD-L1 inhibitor compound 1.
EXPERIMENTAL SECTION
■
General Methods. Reagents and solvents were obtained from
commercial sources and used without further purification. All
reactions were carried out in oven-dried glassware. 1H NMR and
13C NMR spectra were recorded on Bruker AV-400 or Bruker AV-500
spectrometers, with TMS as an internal standard. Low-resolution ESI-
MS spectra were recorded on an Agilent 1100 LC−MS spectrometer.
High-resolution mass (HRMS) measurements were performed with
an Agilent QTOF 6520 mass spectrometer, with electron spray
ionization (ESI) as the ion source. Specific rotation measurement was
performed on Anton Paar MCP500. Flash column chromatography
was carried out using a commercially available 200−300 mesh
adsorbent under pressure. The purity of the target compounds was
determined by high-performance liquid chromatography (HPLC),
which was performed on a Shimadzu Labsolutions system with an
Agilent C18 column (4.6 × 150 mm, 5 μm). The gradient mobile
phase consisted of A (90:10 H2O/MeCN) and B (5:95 H2O/MeCN)
at a flow rate of 1.5 mL/min. The UV wavelength for detection was
254 nm. The gradient program was as follows: 35−100% B (0−10
min), 100% B (10−12 min), and 35% B (12−15 min). The purity of
the target compounds was determined to be higher than 95%.
2,2′-(2-Methyl-1,3-phenylene)bis(4,4,5,5-tetramethyl-1,3,2-diox-
aborolane) (11). 1,3-Dibromo-2-methylbenzene (2 g, 8 mmol) and
bis(pinacolato)diborane (4.8 g, 19.2 mmol) were dissolved in dioxane
(25 mL). Then, Pd(dppf)Cl2 (400 mg, 6% mol) and KOAc (4.8 g, 48
mmol) were added. The reaction mixture was degasified with N2,
heated at 130 °C overnight, and detected by TLC. The mixture was
filtered, and the solvent was removed under reduced pressure. The
resulting crude product was purified on a silica gel column, eluting
with EA in PE (1: 20) to obtain 11 (2.26 g) as a white solid, yield
82%. 1H NMR (400 MHz, chloroform-d): δ 7.79 (d, J = 7.4 Hz, 2H),
7.14 (t, J = 7.4 Hz, 1H), 2.74 (s, 3H), 1.34 (s, 24H). ESI-MS m/z: for
CONCLUSIONS
■
Along with the successful clinical application of anti-PD-1/PD-
L1 mAbs, immune checkpoint inhibitors against PD-1/PD-L1
interaction have made them a promising target for cancer
therapy. Although anti-PD-1/PD-L1 mAbs have gained a large
market share, lack of oral bioavailability, emergence of tumor
resistance, and difficult and expensive production of the mAbs
also limit their current clinical application. In contrast, small
molecules in general are more accessible and have higher oral
bioavailability and acceptable half-lives. Consequently, the
development of small-molecule inhibitors of PD-1/PD-L1
interaction is a promising alternative to mAbs.
+
C19H31B2O4 [M + H]+ calcd, 345.2; found, 345.2.
(2-(2-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl)pyridin-4-yl)methanol (12a). (2-Chloropyridin-4-yl)-
methanol (100 mg, 0.7 mmol) and 11 (358 mg, 1.04 mmol) were
dissolved in the solution of 5 mL dioxane/H2O (10: 1). Then,
tetrakis(triphenylphosphine) palladium (20 mg, 10 % mmol) and
K2CO3 (276 mg, 2 mmol) were added. The reaction mixture was
degasified with N2, heated at 85 °C for 4 h, and detected by TLC. The
mixture was filtered, and the solvent was removed under reduced
pressure. The resulting crude product was purified on a silica gel
column, eluting with MeOH in DCM (1: 50) to obtain 12a (180 mg)
We designed and synthesized a series of biphenyl pyridines
as novel inhibitors of the PD-1/PD-L1 interaction. This novel
chemical series showed great inhibitory activity of the PD-1/
PD-L1 blockage in the TR-FRET assay. Among them,
compound 24 was the most promising small-molecule
1
as a white solid, yield 75%. H NMR (400 MHz, chloroform-d): δ
8.62 (d, J = 4.6 Hz, 1H), 7.80 (d, J = 7.4 Hz, 1H), 7.29−7.27 (m,
2H), 7.22−7.18 (m, 2H), 4.79 (s, 2H), 2.45 (s, 3H). ESI-MS m/z: for
+
C19H25BNO3 [M + H]+ calcd, 326.2; found, 326.2.
inhibitor, with the IC50 value of 3.8
0.3 nM. A PBMC
(6-(2-Methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenyl)pyridin-3-yl)methanol (12b). 12b was prepared according to
the preparation method of 12a, yield 81%. 1H NMR (300 MHz,
chloroform-d): δ 8.65 (d, J = 1.3 Hz, 1H), 7.84−7.71 (m, 2H), 7.38
(dd, J = 12.7, 7.8 Hz, 2H), 7.25 (t, J = 7.5 Hz, 1H), 4.76 (s, 2H), 2.47
killing assay indicated that 24 could activate the antitumor
immunity of T cells in PBMCs. Compound 24 exhibited
favorable PK properties with excellent oral bioavailability.
Orally administered compound 24 significantly inhibits tumor
growth in the CT26 mouse model. In addition, flow cytometry
and immunohistochemistry demonstrated that 24 enhances
immune activity in tumors. Compared with the reported PD-
L1 inhibitors in Figure 1, compound 24 exhibited better in
vitro inhibitory activity against PD-1/PD-L1 interaction and
demonstrated significantly improved potency of oral admin-
istration and in vivo antitumor activity, which also indicated its
potency and advantages as an alternative to mAbs. Overall, our
work has demonstrated that 24 has the potential to serve as a
lead compound for further novel small-molecule inhibitors of
the PD-1/PD-L1 system.
+
(s, 3H). ESI-MS m/z: for C19H25BNO3 [M + H]+ calcd, 326.2;
found, 326.2.
(2-(2-Methyl-[1,1′-biphenyl]-3-yl)pyridin-4-yl)methanol (13a).
12a (100 mg, 0.3 mmol) and bromobenzene (96 mg, 0.6 mmol)
were dissolved in the solution of 3 mL THF/H2O (3:1). Then, XPhos
palladacycle Gen. 2 (XPhos Pd G2, 24 mg, 10 % mmol) and K3PO4
(130 mg, 0.61 mmol) were added. The reaction mixture was
degasified with N2, heated at 80 °C overnight, and detected by TLC.
The solvent was removed under reduced pressure. The resulting
crude product was purified on a silica gel column, eluting with MeOH
in DCM (1:50) to obtain 13a (98 mg) as a white solid, yield 87%. 1H
NMR (400 MHz, chloroform-d): δ 8.61 (d, J = 5.2 Hz, 1H), 7.44−
7397
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7.35 (m, 4H), 7.29 (dd, J = 6.8, 4.4 Hz, 3H), 7.18 (dd, J = 5.1, 0.7 Hz,
3H), 4.79 (s, 2H), 2.12 (s, 3H). ESI-MS m/z: for C19H18NO+ [M +
H]+ calcd, 276.1; found, 276.1.
reduced pressure, and the crude product was purified on a silica gel
column, eluting with MeOH in DCM (1:10) to obtain 17 as a white
solid, yield 79%. H NMR (300 MHz, chloroform-d): δ 8.71−8.56
1
(6-(2-Methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)methanol (13b).
13b was prepared according to the preparation method of 13a,
yield 83%. 1H NMR (300 MHz, chloroform-d): δ 8.71−8.64 (m, 1H),
7.80 (dd, J = 8.0, 2.3 Hz, 1H), 7.54−7.26 (m, 9H), 4.79 (s, 2H), 2.16
(s, 3H). ESI-MS m/z: for C19H18NO+ [M + H]+ calcd, 276.1; found,
276.1.
2-(2-Methyl-[1,1′-biphenyl]-3-yl)isonicotinaldehyde (14a). 13a
(90 mg, 0.33 mmol) and NaHCO3 (55 mg, 0.65 mmol) were
dissolved in 5 mL of DCM. To the mixture, DMP (165 mg, 0.39
mmol) was added and stirred at room temperature for 0.5 h. Then,
saturated NaS2O3 aq was added, and the mixture was diluted with
water and extracted with DCM. The combined organic phase was
washed with water and saturated NaCl aq and concentrated in vacuo.
The crude product was purified on a silica gel column, eluting with
EtOAc in PE (1:8) to obtain 14a as a white solid, yield 84%. 1H NMR
(400 MHz, chloroform-d): δ 10.12 (s, 1H), 8.74 (d, J = 4.8 Hz, 1H),
7.61−7.46 (m, 4H), 7.34−7.22 (m, 3H), 7.15 (d, J = 5.2 Hz, 3H),
2.16 (s, 3H). ESI-MS m/z: for C19H16NO+ [M + H]+ calcd, 274.0;
found, 274.0.
(m, 1H), 7.73 (dd, J = 8.0, 2.3 Hz, 1H), 7.51−7.26 (m, 9H), 3.85 (s,
2H), 3.38 (q, J = 5.7 Hz, 2H), 2.82 (t, J = 5.8 Hz, 2H), 2.15 (s, 3H),
1.97 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 170.6, 159.6, 149.0,
143.2, 142.3, 141.2, 136.2, 133.4, 133.3, 130.1, 129.4, 128.9, 128.2,
126.9, 125.6, 124.2, 50.8, 48.4, 39.2, 23.4, 18.4. HRMS (ESI): for
C23H26N3O+ [M + H]+ calcd, 360.2070; found, 360.2072. HPLC, tR =
5.431 min, purity 97.4%.
Methyl 6-Chloro-2-methoxynicotinate (19). MeOH (830 mg, 26
mmol) was dissolved in 15 mL of THF, and the reaction mixture was
stirred at 0 °C. NaH was added, and the reaction mixture was stirred
for 0.5 h. Then, 18 (1 g, 5.2 mmol) was added to the reaction mixture
and stirred at 60 °C for 2 h. The mixture was cooled to 0 °C and
filtered. The filter cake was dissolved in 20 mL of MeOH, and the
mixture was stirred at 0 °C. SOCl2 (1.9 g, 16 mmol) was added and
stirred for 3 h at reflux. Then, the mixture was cooled, concentrated in
vacuo, diluted with water, and extracted with DCM. The combined
organic phase was washed with water and saturated NaCl aq and
concentrated in vacuo. The crude product was purified on a silica gel
column, eluting with EtOAc in PE (1:50) to obtain 19 as a white
6-(2-Methyl-[1,1′-biphenyl]-3-yl)nicotinaldehyde (14b). 14b was
1
solid, yield 91%. H NMR (400 MHz, chloroform-d): δ 8.13 (d, J =
1
prepared according to the preparation method of 14a, yield 78%. H
7.9 Hz, 1H), 6.96 (d, J = 7.9 Hz, 1H), 4.05 (s, 3H), 3.89 (s, 3H). ESI-
NMR (400 MHz, chloroform-d): δ 10.18 (s, 1H), 9.17 (dd, J = 2.1,
0.8 Hz, 1H), 8.26 (dd, J = 8.1, 2.2 Hz, 1H), 7.64 (dt, J = 8.0, 0.7 Hz,
1H), 7.46−7.32 (m, 8H), 2.20 (s, 3H). 13C NMR (101 MHz,
CDCl3): δ 190.7, 166.0, 151.9, 143.6, 141.9, 140.4, 136.1, 133.5,
131.0, 129.6, 129.4, 128.9, 128.3, 127.1, 125.9, 125.0, 18.6. ESI-MS
m/z: for C19H16NO+ [M + H]+ calcd, 274.0; found, 274.0.
+
MS m/z: for C8H8ClNaNO3 [M + Na]+ calcd, 224.0; found, 224.0.
Methyl 2-Methoxy-6-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-di-
oxaborolan-2-yl)-phenyl)nicotinate (20). 19 (10 g, 50 mmol) and
11 (21.3 g, 62 mmol) were dissolved in 150 mL of 1,4-dioxane/H2O
(10:1). Tetrakis(triphenylphosphine) palladium (5.8 g, 10% mmol)
and K2CO3 (17.5 g, 124 mmol) were added to the mixture, which was
was degasified with N2 and heated at 85 °C for 4 h and detected by
TLC. The reaction mixture was filtered, and the solvent was removed
under reduced pressure. The resulting crude product was purified on a
silica gel column, eluting with EtOAc in PE (1:30) to obtain 20 as a
white solid, yield 65%. 1H NMR (300 MHz, chloroform-d): δ 8.22 (d,
J = 7.7 Hz, 1H), 7.82 (dd, J = 7.4, 1.6 Hz, 1H), 7.45 (dd, J = 7.6, 1.6
Hz, 1H), 7.26 (t, J = 7.5 Hz, 1H), 7.03 (d, J = 7.7 Hz, 1H), 4.05 (s,
3H), 3.92 (s, 3H), 2.56 (s, 3H), 1.36 (s, 12H). ESI-MS m/z: for
2-(((2-(2-Methyl-[1,1′-biphenyl]-3-yl)pyridin-4-yl)methyl)amino)-
ethan-1-ol (15). Compound 14a (60 mg, 0.2 mmol) was dissolved in
DCM (1.5 mL) and MeOH (1.5 mL). 2-aminoethan-1-ol (15 mg,
0.24 mmol) and acetic acid (0.02 mL) were added, and the reaction
mixture was stirred at rt for 1 h. NaCNBH3 (62 mg, 1 mmol) was
added, and the mixture was stirred at rt overnight and detected by
TLC. The solvent was removed under reduced pressure, and the
crude product was purified on a silica gel column, eluting with MeOH
1
in DCM (1: 10) to obtain 15 as a white solid, yield 78%. H NMR
+
C21H26BNaNO5 [M + Na]+ calcd, 406.2; found, 406.2.
(500 MHz, chloroform-d): δ 8.64 (d, J = 5.1 Hz, 1H), 7.41 (d, J = 7.2
Hz, 3H), 7.37−7.33 (m, 4H), 7.31 (t, J = 7.4 Hz, 1H), 7.28−7.26 (m,
1H), 7.26−7.23 (m, 1H), 3.91 (s, 2H), 3.70 (t, J = 5.1 Hz, 2H), 2.85
(t, J = 5.1 Hz, 2H), 2.15 (s, 3H). 13C NMR (126 MHz, CDCl3): δ
161.0, 149.4, 149.0, 143.3, 142.3, 141.5, 133.4, 130.1, 129.5, 128.9,
128.2, 127.0, 125.6, 123.7, 121.2, 61.0, 52.4, 50.7, 18.5. HRMS (ESI):
for C21H23N2O+ [M + H]+ calcd, 319.1805; found, 319.1809. HPLC,
tR = 4.918 min, purity 98.2%.
(2-Methoxy-6-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)phenyl)-pyridin-3-yl)MeOH (21). LiAlH4 (2.1 g, 55 mmol)
was slowly added to 50 mL of THF, and the mixture was stirred at 0
°C. 20 (7 g, 18 mmol) was added to the mixture, and the reaction
mixture was stirred at rt for 3 h. Then, the mixture was concentrated
in vacuo, diluted with water, and extracted with DCM. The combined
organic phase was washed with water and saturated NaCl aq. and
1
concentrated in vacuo to obtain 21 as a white solid, yield 71%. H
2-(((6-(2-Methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)methyl)amino)-
ethan-1-ol (16). Compound 14b (60 mg, 0.2 mmol) was dissolved in
DCM (1.5 mL) and MeOH (1.5 mL). 2-aminoethan-1-ol (15 mg,
0.24 mmol) and acetic acid (0.02 mL) were added, and the reaction
mixture was stirred at rt for 1 h. NaCNBH3 (62 mg, 1 mmol) was
added, and the reaction mixture was stirred at rt overnight and
detected by TLC. The solvent was removed under reduced pressure,
and the crude product was purified on a silica gel column, eluting with
NMR (400 MHz, chloroform-d): δ 7.81 (dd, J = 7.4, 1.6 Hz, 1H),
7.62 (dd, J = 7.4, 1.0 Hz, 1H), 7.45 (dd, J = 7.6, 1.6 Hz, 1H), 7.31−
7.23 (m, 2H), 6.96 (dd, J = 7.3, 1.1 Hz, 1H), 4.70 (d, J = 6.0 Hz, 2H),
4.01 (d, J = 1.1 Hz, 3H), 2.57 (s, 3H), 1.38 (d, J = 1.1 Hz, 12H). ESI-
MS m/z: for C20H26BNaNO4+ [M + Na]+ calcd, 378.2; found, 378.2.
(2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)MeOH
(22a). 22a was prepared from 21 and bromobenzene according to the
preparation method of 13a, yield 79%. 1H NMR (400 MHz, MeOH-
d4): δ 7.80−7.75 (m, 1H), 7.40 (dd, J = 8.0, 6.6 Hz, 2H), 7.34−7.24
(m, 5H), 7.19 (dd, J = 7.6, 1.6 Hz, 1H), 7.04 (d, J = 7.4 Hz, 1H), 4.64
(s, 2H), 3.94 (s, 3H), 2.16 (s, 3H). 13C NMR (101 MHz, CDCl3): δ
160.7, 157.2, 143.3, 142.4, 141.0, 137.0, 133.8, 130.0, 129.5, 129.0,
128.2, 126.9, 125.5, 121.2, 117.4, 61.1, 53.6, 18.7. ESI-MS m/z: for
1
MeOH in DCM (1:10) to obtain 16 as a white solid, yield 73%. H
NMR (400 MHz, chloroform-d): δ 8.63 (d, J = 2.2 Hz, 1H), 7.76 (dd,
J = 8.0, 2.3 Hz, 1H), 7.46−7.26 (m, 9H), 3.87 (s, 2H), 3.69 (t, J = 5.1
Hz, 2H), 2.84 (t, J = 5.1 Hz, 2H), 2.15 (s, 3H). 13C NMR (75 MHz,
CDCl3): δ 159.5, 149.0, 143.2, 142.3, 141.2, 136.3, 133.4, 133.3,
130.1, 129.5, 128.9, 128.2, 126.9, 125.6, 124.3, 61.0, 50.9, 50.8, 18.5.
HRMS (ESI): for C21H23N2O+ [M + H]+ calcd, 319.1805; found,
319.1810. HPLC, tR = 4.079 min, purity 98.9%.
N-(2-(((6-(2-Methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)methyl)-
amino)ethyl)acetamide (17). Compound 14b (60 mg, 0.2 mmol)
was dissolved in DCM (1.5 mL) and MeOH (1.5 mL). N-(2-
aminoethyl)acetamide (24 mg, 0.24 mmol) and acetic acid (0.02 mL)
were added, and the reaction mixture was stirred at rt for 1 h.
NaCNBH3 (62 mg, 1 mmol) was added, and the mixture was stirred
at rt overnight and detected by TLC. The solvent was removed under
+
C20H20NO2 [M + H]+ calcd, 306.1; found, 306.1.
(2-Methoxy-6-(3′-methoxy-2-methyl-[1,1′-biphenyl]-3-yl)-
pyridin-3-yl)MeOH (22b). 22b was prepared from 21 and 1-bromo-3-
methoxybenzene according to the preparation method of 13a, yield
88%. 1H NMR (300 MHz, chloroform-d): δ 7.64 (d, J = 7.3 Hz, 1H),
7.45−7.27 (m, 4H), 7.03 (d, J = 7.3 Hz, 1H), 7.00−6.84 (m, 3H),
4.70 (s, 2H), 4.02 (s, 3H), 3.84 (s, 3H), 2.25 (s, 3H). 13C NMR (75
MHz, CDCl3): δ 160.8, 159.7, 157.4, 144.0, 143.3, 141.3, 137.0,
133.8, 129.9, 129.2, 129.1, 125.5, 122.1, 121.4, 117.4, 115.4, 112.7,
7398
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+
61.0, 55.4, 53.6, 18.5. ESI-MS m/z: for C21H22NO3+ [M + H]+ calcd,
336.2; found, 336.2.
48.2, 18.8. HRMS (ESI): for C22H25N2O2 [M + H]+, 349.1911;
found, 349.1912. HPLC, tR = 5.012 min, purity 98.3%.
(6-(3-(Benzo[d][1,3]dioxol-5-yl)-2-methylphenyl)-2-methoxypyri-
din-3-yl)MeOH (22c). 22c was prepared from 21 and 5-bromobenzo-
[d][1,3]dioxole according to the preparation method of 13a, yield
72%. 1H NMR (300 MHz, chloroform-d): δ 7.66 (d, J = 7.4 Hz, 1H),
7.41 (dd, J = 7.2, 2.0 Hz, 1H), 7.35−7.27 (m, 2H), 7.05 (d, J = 7.3
Hz, 1H), 6.91−6.80 (m, 3H), 6.03 (s, 2H), 4.73 (s, 2H), 4.04 (s, 3H),
2.27 (s, 3H). 13C NMR (101 MHz, CDCl3): δ 160.8, 157.2, 147.4,
146.6, 142.9, 141.1, 137.1, 136.3, 134.0, 130.1, 128.9, 125.5, 122.8,
121.2, 117.4, 110.2, 108.2, 101.1, 61.2, 53.6, 18.8. ESI-MS m/z: for
N-(2-(((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)-
methyl)amino)-ethyl)acetamide (25). 25 was prepared from 23a and
N-(2-aminoethyl)acetamide according to the preparation method of
1
15, yield 83%. [α]2D0.0 −34.213 (c = 0.5, H2O). H NMR (300 MHz,
chloroform-d): δ 7.57 (d, J = 7.4 Hz, 1H), 7.45−7.28 (m, 8H), 7.02
(d, J = 7.2 Hz, 1H), 4.00 (s, 3H), 3.80 (s, 2H), 3.40 (q, J = 5.6 Hz,
2H), 2.81 (dd, J = 7.0, 4.5 Hz, 2H), 2.25 (s, 3H), 2.00 (s, 3H). 13C
NMR (126 MHz, MeOD): δ 173.6, 162.5, 158.5, 144.6, 143.7, 142.4,
139.9, 134.5, 130.7, 130.4, 129.8, 129.2, 127.9, 126.4, 120.7, 118.3,
+
+
C21H22NO3 [M + H]+ calcd, 350.1; found, 350.1.
54.1, 49.1, 48.6, 39.9, 22.6, 18.8. HRMS (ESI): for C24H28N3O2 [M
+ H]+, 390.2176; found, 390.2178. HPLC, tR = 5.506 min, purity
98.7%.
(6-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-2-
methoxypyridin-3-yl)MeOH (22d). 22d was prepared from 21 and 6-
bromo-2,3-dihydrobenzo[b][1,4]dioxine according to the preparation
((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)-
methyl)-L-serine (26). 26 was prepared from 23a and L-serine
according to the preparation method of 15, yield 39%. 1H NMR (400
MHz, DMSO-d6): δ 7.87 (d, J = 7.5 Hz, 1H), 7.46 (t, J = 7.3 Hz, 2H),
7.41−7.30 (m, 5H), 7.25 (dd, J = 7.5, 1.7 Hz, 1H), 7.16 (d, J = 7.4
Hz, 1H), 3.91 (s, 5H), 3.75−3.67 (m, 3H), 2.17 (s, 3H). 13C NMR
(126 MHz, DMSO): δ 170.9, 160.4, 156.7, 142.6, 141.6, 140.6, 139.5,
132.9, 129.7, 129.2, 128.8, 128.3, 127.1, 125.6, 117.2, 116.2, 63.3,
1
method of 13a, yield 83%. H NMR (500 MHz, chloroform-d): δ
7.64 (d, J = 7.3 Hz, 1H), 7.37 (dd, J = 7.3, 1.8 Hz, 1H), 7.32−7.23
(m, 2H), 7.03 (d, J = 7.4 Hz, 1H), 6.94−6.87 (m, 2H), 6.84 (dd, J =
8.2, 2.0 Hz, 1H), 4.71 (s, 2H), 4.31 (s, 4H), 4.02 (s, 3H), 2.26 (s,
3H). 13C NMR (126 MHz, CDCl3): δ 160.8, 157.4, 143.2, 142.8,
142.7, 141.1, 137.1, 135.9, 134.0, 130.1, 128.9, 125.5, 122.8, 121.2,
118.4, 117.4, 117.0, 64.6, 64.6, 61.3, 53.6, 18.8. ESI-MS m/z: for
+
60.9, 53.5, 44.9, 18.4. HRMS (ESI): for C23H25N2O4 [M + H]+,
+
C22H22NO4 [M + H]+ calcd, 364.1; found, 364.1.
393.1809; found, 393.1815. HPLC, tR = 3.880 min, purity 95.7%.
(S)-5-((((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-
yl)methyl)amino)-methyl)pyrrolidin-2-one (27). 27 was prepared
from 23a and (S)-5-(aminomethyl)pyrrolidin-2-one according to the
preparation method of 15, yield 64%. [α]2D0.0 20.946 (c = 0.5, EtOH).
1H NMR (500 MHz, MeOH-d4): δ 7.70 (d, J = 7.5 Hz, 1H), 7.40 (t, J
2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)nicotinaldehyde
(23a). 23a was prepared from 22a according to the preparation
1
method of 14a, yield 84%. H NMR (400 MHz, chloroform-d): δ
10.45 (d, J = 0.8 Hz, 1H), 8.23 (d, J = 7.7 Hz, 1H), 7.49−7.35 (m,
8H), 7.22 (dd, J = 7.7, 0.8 Hz, 1H), 4.14 (s, 3H), 2.31 (s, 3H). 13C
NMR (101 MHz, CDCl3): δ 189.2, 164.3, 163.8, 143.6, 142.0, 140.3,
138.0, 133.8, 130.9, 129.5, 128.9, 128.3, 127.1, 125.7, 118.1, 116.9,
= 7.5 Hz, 2H), 7.36−7.27 (m, 5H), 7.21 (dd, J = 7.4, 1.5 Hz, 1H),
7.04 (d, J = 7.4 Hz, 1H), 3.97 (s, 3H), 3.83−3.78 (m, 3H), 2.68 (qd, J
= 12.1, 6.2 Hz, 2H), 2.32 (dd, J = 12.4, 7.0 Hz, 2H), 2.28−2.22 (m,
1H), 2.17 (s, 3H), 1.84−1.75 (m, 1H). 13C NMR (126 MHz,
MeOD): δ 180.9, 162.4, 158.3, 144.5, 143.7, 142.4, 139.7, 134.5,
130.7, 130.4, 129.8, 129.2, 128.0, 126.4, 120.9, 118.3, 55.7, 55.3, 54.1,
+
54.1, 18.8. ESI-MS m/z: for C20H18NO2 [M + H]+ calcd, 304.1;
found, 304.1.
2-Methoxy-6-(3′-methoxy-2-methyl-[1,1′-biphenyl]-3-yl)-
nicotinaldehyde (23b). 23b was prepared from 22b according to the
preparation method of 14a, yield 79%. 1H NMR (300 MHz,
chloroform-d): δ 10.41 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.49−7.27
(m, 5H), 7.18 (d, J = 7.7 Hz, 1H), 6.99−6.86 (m, 3H), 4.11 (s, 3H),
3.85 (s, 3H), 2.28 (s, 3H). 13C NMR (75 MHz, CDCl3): δ 188.9,
164.4, 163.9, 159.8, 143.7, 142.4, 140.5, 138.1, 133.9, 130.7, 129.3,
129.0, 129.0, 125.7, 122.1, 118.1, 115.5, 112.8, 55.5, 54.1, 18.6. ESI-
+
54.0, 31.0, 25.9, 18.8. HRMS (ESI): for C25H28N3O2 [M + H]+,
402.2176; found, 402.2180. HPLC, tR = 5.067 min, purity 97.0%.
2-(((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)-
methyl) (methyl)amino)-ethan-1-ol (28). 28 was prepared from 23a
and 2-(methylamino)ethan-1-ol according to the preparation method
1
+
MS m/z: for C21H20NO3 [M + H]+ calcd, 334.1; found, 334.1.
of 15, yield 60%. H NMR (600 MHz, chloroform-d): δ 7.61 (d, J =
7.3 Hz, 1H), 7.45−7.41 (m, 3H), 7.40−7.34 (m, 3H), 7.32 (t, J = 7.5
Hz, 1H), 7.28 (dd, J = 7.6, 1.5 Hz, 1H), 7.03 (d, J = 7.4 Hz, 1H), 4.00
(s, 3H), 3.70 (t, J = 5.4 Hz, 2H), 3.60 (s, 2H), 2.69 (t, J = 5.3 Hz,
2H), 2.30 (s, 3H), 2.27 (s, 3H). 13C NMR (151 MHz, CDCl3): δ
161.3, 156.8, 143.3, 142.5, 141.1, 139.0, 133.8, 130.0, 129.5, 129.0,
128.2, 126.9, 125.5, 118.9, 117.1, 59.0, 58.7, 55.6, 53.6, 42.2, 18.8.
6-(3-(Benzo[d][1,3]dioxol-5-yl)-2-methylphenyl)-2-methoxynico-
tinaldehyde (23c). 23c was prepared from 22c according to the
preparation method of 14a, yield 68%. 1H NMR (500 MHz,
chloroform-d): δ 10.41 (s, 1H), 7.72 (t, J = 7.4 Hz, 1H), 7.42 (d, J =
7.1 Hz, 1H), 7.31 (q, J = 8.3, 7.8 Hz, 2H), 7.17 (d, J = 7.7 Hz, 1H),
6.92−6.76 (m, 3H), 6.01 (s, 2H), 4.10 (s, 3H), 2.27 (s, 3H). 13C
NMR (126 MHz, CDCl3): δ 189.2, 164.3, 163.8, 147.5, 146.8, 143.3,
140.4, 138.1, 134.0, 131.0, 128.8, 125.7, 122.8, 118.1, 117.0, 110.1,
+
HRMS (ESI): for C23H28N2O2 [M + H]+, 363.2067; found,
363.2071. HPLC, tR = 6.184 min, purity 96.3%.
+
108.3, 101.2, 54.1, 18.7. ESI-MS m/z: for C21H18NO4 [M + H]+
(R)-2-(((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)-
methyl)amino)-propan-1-ol (29). 29 was prepared from 23a and
(R)-2-aminopropan-1-ol according to the preparation method of 15,
calcd, 348.1; found, 348.1.
6-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-2-
yield 82%. [α]2D0.0 −27.151 (c = 0.5, EtOH). H NMR (600 MHz,
1
methoxynicotin-aldehyde (23d). 23d was prepared from 22d
1
chloroform-d): δ 7.63 (d, J = 7.4 Hz, 1H), 7.45−7.41 (m, 3H), 7.40−
7.35 (m, 3H), 7.32 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 7.5 Hz, 1H), 7.02
(d, J = 7.3 Hz, 1H), 4.01 (s, 3H), 3.90 (d, J = 13.7 Hz, 1H), 3.78 (d, J
= 13.6 Hz, 1H), 3.66 (dd, J = 10.7, 3.9 Hz, 1H), 3.36 (dd, J = 10.7, 6.8
Hz, 1H), 2.91−2.86 (m, 1H), 2.26 (s, 3H), 1.13 (d, J = 6.5 Hz, 3H).
13C NMR (151 MHz, CDCl3): δ 161.1, 156.8, 143.3, 142.4, 141.1,
138.1, 133.7, 130.0, 129.5, 129.0, 128.2, 126.9, 125.5, 120.1, 117.3,
according to the preparation method of 14a, yield 88%. H NMR
(400 MHz, chloroform-d): δ 10.41 (d, J = 0.7 Hz, 1H), 8.19 (d, J =
7.7 Hz, 1H), 7.41 (dd, J = 6.2, 3.0 Hz, 1H), 7.34−7.29 (m, 2H), 7.17
(dd, J = 7.6, 0.7 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.89 (d, J = 2.0 Hz,
1H), 6.83 (dd, J = 8.2, 2.1 Hz, 1H), 4.31 (s, 4H), 4.11 (s, 3H), 2.29
(s, 3H). 13C NMR (101 MHz, CDCl3): δ 189.2, 164.3, 163.7, 143.2,
143.0, 142.8, 140.3, 138.0, 135.4, 133.9, 130.9, 128.7, 125.7, 122.7,
118.3, 118.0, 117.0, 116.9, 64.55, 64.5, 54.1, 18.9. ESI-MS m/z: for
+
65.4, 54.0, 53.6, 45.8, 18.7, 17.2, 17.1. HRMS (ESI): for C23H28N2O2
+
C22H19NaNO4 [M + Na]+ calcd, 384.1; found, 384.1.
[M + H]+, 363.2067; found, 363.2072. HPLC, tR = 7.882 min, purity
97.4%.
2-(((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)-
methyl)amino)ethan-1-ol (24). 24 was prepared from 23a and 2-
aminoethan-1-ol according to the preparation method of 15, yield
71%. 1H NMR (300 MHz, chloroform-d): δ 7.59 (d, J = 7.4 Hz, 1H),
7.46−7.28 (m, 8H), 7.01 (d, J = 7.3 Hz, 1H), 4.00 (s, 3H), 3.82 (s,
2H), 3.73−3.66 (m, 2H), 2.87−2.80 (m, 2H), 2.25 (s, 3H). 13C NMR
(101 MHz, CDCl3): δ 161.2, 156.8, 143.3, 142.4, 141.1, 138.1, 133.8,
130.0, 129.5, 129.0, 128.2, 126.9, 125.5, 120.1, 117.2, 60.9, 53.6, 50.5,
3-(((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)-
methyl)amino)propan-1-ol (30). 30 was prepared from 23a and 3-
aminopropan-1-ol according to the preparation method of 15, yield
79%. 1H NMR (600 MHz, chloroform-d): δ 7.58 (d, J = 7.4 Hz, 1H),
7.42 (q, J = 7.5 Hz, 3H), 7.39−7.33 (m, 3H), 7.31 (t, J = 7.5 Hz, 1H),
7.27 (dd, J = 7.6, 1.6 Hz, 1H), 7.01 (d, J = 7.3 Hz, 1H), 4.00 (s, 3H),
3.86−3.81 (m, 4H), 2.93 (t, J = 5.7 Hz, 2H), 2.25 (s, 3H), 1.77 (dp, J
7399
https://doi.org/10.1021/acs.jmedchem.1c00010
J. Med. Chem. 2021, 64, 7390−7403

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
= 10.4, 5.5, 5.1 Hz, 2H). 13C NMR (151 MHz, CDCl3): δ 161.2,
157.0, 143.3, 142.4, 141.0, 138.4, 133.8, 130.0, 129.5, 129.0, 128.2,
126.9, 125.5, 119.3, 117.2, 64.2, 53.6, 49.3, 48.6, 30.6, 18.8. HRMS
according to the preparation method of 15, yield 87%. 1H NMR (500
MHz, MeOH-d4): δ 7.69 (d, J = 7.4 Hz, 1H), 7.31 (d, J = 7.6 Hz,
1H), 7.25 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 6.8 Hz, 1H), 7.04 (d, J = 7.4
Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.82−6.73 (m, 2H), 4.27 (s, 4H),
3.99 (s, 3H), 3.81 (s, 2H), 3.35 (t, J = 6.3 Hz, 2H), 2.76 (t, J = 6.3 Hz,
2H), 2.18 (s, 3H), 1.95 (s, 3H). 13C NMR (126 MHz, MeOD): δ
173.6, 162.5, 158.6, 144.6, 144.2, 144.1, 142.3, 140.0, 136.6, 134.6,
130.8, 129.6, 126.3, 123.4, 120.5, 119.1, 118.3, 117.9, 65.7, 65.7, 54.1,
49.1, 48.6, 39.8, 22.6, 18.8. HRMS (ESI): for C26H30N3O4+ [M + H]+,
448.2231; found, 448.2238. HPLC, tR = 5.921 min, purity: 99.2%.
((6-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-2-
methoxypyridin-3-yl)methyl)-L-serine (37). 37 was prepared from
23d and L-serine according to the preparation method of 15, yield
35%. [α]2D0.0 −27.248 (c = 0.25, H2O). 1H NMR (400 MHz, DMSO-
d6): δ 7.90 (d, J = 7.5 Hz, 1H), 7.37−7.25 (m, 2H), 7.22−7.17 (m,
1H), 7.13 (d, J = 7.5 Hz, 1H), 6.91 (d, J = 8.2 Hz, 1H), 6.84−6.75
(m, 2H), 4.26 (s, 4H), 4.05 (t, J = 15.8 Hz, 2H), 3.89 (s, 3H), 3.78
(d, J = 9.0 Hz, 3H), 2.16 (s, 3H). 13C NMR (101 MHz, DMSO): δ
171.4, 160.8, 157.1, 143.4, 143.0, 142.5, 140.9, 140.0, 135.1, 133.4,
130.1, 129.0, 126.0, 122.7, 122.6, 118.2, 117.6, 117.3, 64.6, 63.7, 61.2,
53.9, 45.3, 18.9. HRMS (ESI): for C25H29N2O6+ [M + H]+, 451.1864;
found, 451.1863. HPLC, tR = 3.274 min, purity 95.6%.
+
(ESI): for C23H28N2O2 [M + H]+, 363.2067; found, 363.2067.
HPLC, tR = 5.284 min, purity 98.9%.
(R)-1-((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)-
methyl)pyrrolidin-3-ol (31). 31 was prepared from 23a and (R)-
pyrrolidin-3-ol according to the preparation method of 15, yield 56%.
[α]2D0.0 −120.752 (c = 0.25, EtOH). 1H NMR (300 MHz, chloroform-
d): δ 7.70 (d, J = 7.4 Hz, 1H), 7.39 (tdd, J = 10.9, 10.2, 4.8, 2.2 Hz,
6H), 7.32−7.27 (m, 2H), 7.03 (d, J = 7.4 Hz, 1H), 4.37 (tt, J = 4.7,
2.4 Hz, 1H), 3.98 (s, 3H), 3.70 (s, 2H), 3.00 (td, J = 8.6, 4.9 Hz, 1H),
2.81 (dd, J = 10.1, 2.1 Hz, 1H), 2.64 (dd, J = 10.2, 5.2 Hz, 1H), 2.51−
2.37 (m, 2H), 2.30−2.27 (m, 1H), 2.26 (s, 3H), 1.87−1.71 (m, 1H).
13C NMR (151 MHz, CDCl3): δ 161.0, 156.6, 143.3, 142.5, 141.2,
138.7, 133.8, 129.9, 129.5, 129.1, 128.2, 126.9, 125.5, 118.8, 117.2,
+
71.4, 63.2, 53.6, 53.3, 52.8, 35.1, 18.8. HRMS (ESI): for C24H27N2O2
[M + H]+, 375.2067; found, 375.2072. HPLC, tR = 6.330 min, purity
97.8%.
N1-((2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)pyridin-3-yl)-
methyl)ethane-1,2-diamine (32). 32 was prepared from 23a and tert-
butyl (2-aminoethyl)carbamate according to the preparation method
of 15, yield 86%. 1H NMR (600 MHz, MeOH-d4): δ 7.88 (d, J = 7.5
Hz, 1H), 7.41 (t, J = 7.6 Hz, 2H), 7.37−7.28 (m, 5H), 7.23 (dd, J =
7.5, 1.5 Hz, 1H), 7.14 (d, J = 7.5 Hz, 1H), 4.31 (s, 2H), 4.04 (s, 3H),
3.41 (dt, J = 33.3, 6.6 Hz, 4H), 2.18 (s, 3H). 13C NMR (151 MHz,
MeOD): δ 162.5, 161.3, 144.6, 143.5, 142.2, 141.8, 134.5, 131.1,
130.3, 129.8, 129.2, 128.0, 126.5, 118.6, 113.3, 54.5, 47.7, 45.6, 37.0,
18.8. HRMS (ESI): for C22H26N3O+ [M + H]+, 348.2070; found,
348.2073. HPLC, tR = 4.639 min, purity: 98.4%.
2-(((2-Methoxy-6-(3′-methoxy-2-methyl-[1,1′-biphenyl]-3-yl)-
pyridin-3-yl)methyl)-amino)ethan-1-ol (33). 33 was prepared from
23b and 2-aminoethan-1-ol according to the preparation method of
15, yield 67%. 1H NMR (500 MHz, chloroform-d): δ 7.64 (d, J = 7.4
Hz, 1H), 7.40 (d, J = 6.0 Hz, 1H), 7.36−7.26 (m, 3H), 7.04 (d, J =
7.4 Hz, 1H), 6.98−6.88 (m, 3H), 4.01 (s, 3H), 3.91 (s, 2H), 3.84 (s,
3H), 3.74 (t, J = 5.1 Hz, 2H), 2.88 (t, J = 5.0 Hz, 2H), 2.25 (s, 3H).
13C NMR (126 MHz, CDCl3): δ 161.1, 159.4, 157.7, 143.7, 143.2,
N1-((6-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphen-
yl)-2-methoxypyridin-3-yl)methyl)ethane-1,2-diamine (38). 38 was
prepared from 23d and tert-butyl (2-aminoethyl)carbamate according
1
to the preparation method of 15, yield 77%. H NMR (400 MHz,
chloroform-d): δ 7.62 (d, J = 7.2 Hz, 1H), 7.28 (dd, J = 6.2, 3.3 Hz,
1H), 7.20 (d, J = 3.0 Hz, 2H), 6.97 (d, J = 7.3 Hz, 1H), 6.88−6.80
(m, 2H), 6.76 (d, J = 8.2 Hz, 1H), 4.25 (s, 4H), 4.12 (s, 2H), 3.92 (s,
3H), 3.42 (d, J = 21.5 Hz, 4H), 2.19 (s, 3H). 13C NMR (151 MHz,
CDCl3): δ 161.2, 160.3, 143.1, 142.8, 142.7, 140.8, 140.4, 135.6,
133.9, 130.4, 128.8, 125.5, 122.7, 118.3, 117.6, 116.9, 110.5, 64.5,
+
64.5, 53.8, 46.8, 44.8, 36.4, 18.6. HRMS (ESI): for C24H28N3O3 [M
+ H]+, 406.2125; found, 406.2128. HPLC, tR = 4.183 min, purity
99.1%.
3-(((6-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-
2-methoxypyridin-3-yl)methyl)amino)propan-1-ol (39). 39 was
prepared from 23d and 3-aminopropan-1-ol according to the
preparation method of 15, yield 75%. 1H NMR (300 MHz,
chloroform-d): δ 7.71 (d, J = 7.4 Hz, 1H), 7.37 (dd, J = 6.2, 3.2
Hz, 1H), 7.30−7.27 (m, 2H), 7.05 (d, J = 7.4 Hz, 1H), 6.96−6.81 (m,
3H), 4.32 (s, 4H), 4.06 (s, 2H), 4.03 (s, 3H), 3.77 (d, J = 5.3 Hz,
2H), 3.13 (t, J = 5.6 Hz, 2H), 2.26 (s, 3H), 1.89 (t, J = 5.4 Hz, 2H).
13C NMR (151 MHz, CDCl3): δ 161.1, 159.0, 143.1, 142.8, 142.7,
140.8, 139.0, 133.7, 129.9, 129.2, 129.0, 125.5, 122.0, 117.6, 117.4,
115.2, 112.4, 59.9, 55.3, 53.8, 50.2, 47.8, 18.7. HRMS (ESI): for
+
C23H27N2O3 [M + H]+, 379.2016; found, 379.2018. HPLC, tR
=
4.786 min, purity 96.6%.
2-(((6-(3-(Benzo[d][1,3]dioxol-5-yl)-2-methylphenyl)-2-methoxy-
pyridin-3-yl)methyl)amino)ethan-1-ol (34). 34 was prepared from
23c and 2-aminoethan-1-ol according to the preparation method of
15, yield 70%. 1H NMR (500 MHz, chloroform-d): δ 7.60 (d, J = 7.4
Hz, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.30−7.24 (m, 2H), 7.00 (d, J =
7.3 Hz, 1H), 6.90−6.77 (m, 3H), 6.00 (s, 2H), 4.00 (s, 3H), 3.85 (s,
2H), 3.71 (t, J = 5.2 Hz, 2H), 2.85 (t, J = 5.3 Hz, 2H), 2.25 (s, 3H).
13C NMR (126 MHz, CDCl3): δ 161.2, 157.2, 147.4, 146.6, 142.9,
140.5, 140.0, 135.6, 133.8, 130.4, 128.8, 125.6, 122.7, 118.3, 117.5,
117.0, 113.7, 64.5, 64.5, 62.0, 53.9, 47.9, 47.4, 28.6, 18.8. HRMS
+
(ESI): for C25H29N2O4 [M + H]+, 421.2122; found, 421.2121.
HPLC, tR = 5.330 min, purity 97.2%.
Methyl 2-Methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)nicotinate
(40). 40 was prepared from 21 and bromobenzene according to the
preparation method of 13a, yield 80%. 1H NMR (300 MHz,
chloroform-d): δ 8.26 (d, J = 7.7 Hz, 1H), 7.43 (ddd, J = 7.4, 4.6, 1.6
Hz, 3H), 7.39−7.30 (m, 5H), 7.12 (d, J = 7.7 Hz, 1H), 4.09 (s, 3H),
3.94 (s, 3H), 2.26 (s, 3H). 13C NMR (126 MHz, CDCl3): δ 165.6,
162.1, 161.6, 143.4, 142.0, 141.6, 140.2, 133.7, 130.6, 129.4, 128.8,
141.1, 138.3, 134.0, 133.8, 130.1, 129.0, 125.5, 122.9, 119.6, 117.3,
110.2, 108.2, 101.2, 60.8, 53.7, 50.4, 48.1, 18.8. HRMS (ESI): for
+
C23H25N2O4 [M + H]+, 393.1809; found, 393.1813. HPLC, tR
=
5.226 min, purity 97.1%.
2-(((6-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-
2-methoxypyridin-3-yl)methyl)amino)ethan-1-ol (35). 35 was pre-
pared from 23d and 2-aminoethan-1-ol according to the preparation
method of 15, yield 82%. 1H NMR (500 MHz, MeOH-d4): δ 7.68 (d,
J = 7.3 Hz, 1H), 7.30 (d, J = 6.7 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H),
7.17 (d, J = 5.9 Hz, 1H), 7.02 (d, J = 7.4 Hz, 1H), 6.85 (d, J = 8.2 Hz,
1H), 6.81−6.70 (m, 2H), 4.25 (s, 4H), 3.97 (s, 3H), 3.82 (s, 2H),
3.69 (t, J = 5.5 Hz, 2H), 2.75 (t, J = 5.5 Hz, 2H), 2.17 (s, 3H). 13C
NMR (126 MHz, MeOD): δ 162.5, 158.6, 144.6, 144.1, 144.0, 142.3,
139.9, 136.8, 134.6, 130.8, 129.6, 126.3, 123.4, 120.5, 119.1, 118.2,
128.1, 127.0, 125.6, 116.9, 111.6, 54.3, 52.3, 18.7. HRMS (ESI): for
+
C21H20NO3 [M + H]+, 334.1438; found, 334.1442. HPLC, tR
=
6.392 min, purity 96.4%.
2-Methoxy-6-(2-methyl-3-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)phenyl)-nicotinic Acid (41). 40 (200 mg, 0.52 mmol) and
LiOH (60 mg, 2.6 mmol) were dissolved in 4 mL of THF/MeOH/
H2O (3:2:2), and the mixture was stirred at rt for 1 h. Then, the
mixture was diluted with water and extracted with EtOAc. To the
mixture, 1 N HCl aq was added, and the combined organic phase was
washed with water and saturated NaCl aq and concentrated in vacuo
to obtain 41 as a white solid, yield 93%. 1H NMR (400 MHz, MeOH-
d4): δ 8.26 (d, J = 7.7 Hz, 1H), 7.74 (d, J = 7.5 Hz, 1H), 7.41 (d, J =
7.7 Hz, 1H), 7.22 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 7.7 Hz, 1H), 3.99
(s, 3H), 2.51 (s, 3H), 1.34 (s, 12H). 13C NMR (101 MHz, MeOD): δ
168.2, 163.7, 162.8, 143.4, 143.1, 142.6, 141.0, 137.3, 133.2, 125.9,
117.8, 65.7, 65.7, 61.4, 54.1, 51.7, 48.9, 18.8. HRMS (ESI): for
+
C24H27N2O4 [M + H]+, 407.1965; found, 407.1964. HPLC, tR
=
5.237 min, purity: 96.5%.
N-(2-(((6-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methyl-
phenyl)-2-methoxy-pyridin-3-yl)methyl)amino)ethyl)acetamide
(36). 36 was prepared from 23d and N-(2-aminoethyl)acetamide
7400
https://doi.org/10.1021/acs.jmedchem.1c00010
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
+
118.2, 112.8, 84.9, 54.4, 25.2, 20.2. ESI-MS m/z: for C20H20BNO5
quantification of cell damage, which was evaluated by the release of
LDH. The level of LDH released to the supernatant was detected by
an LDH cytotoxicity assay detection kit (Beyotime, China), following
the manufacturer’s instructions. MDA-MB-231 cell viability was
measured as
[M + H]+ calcd, 370.2; found, 370.2.
N-(2-Hydroxyethyl)-2-methoxy-6-(2-methyl-3-(4,4,5,5-tetra-
methyl-1,3,2-dioxa-borolan-2-yl)phenyl)nicotinamide (42). 41
(100 mg, 0.27 mmol), 2-aminoethan-1-ol (20 mg, 0.33 mmol),
HOBT (54 mg, 0.4 mmol), and EDCI (76 mg, 0.4 mmol) were
dissolved in 10 mL of DCM, and the reaction mixture was stirred at rt
for 2 h. Then, the solvent was removed under reduced pressure. The
resulting crude product was purified on a silica gel column, eluting
with MeOH in DCM (1: 40) to obtain 42 as a white solid, yield 55%.
1H NMR (400 MHz, MeOH-d4): δ 8.42 (d, J = 7.7 Hz, 1H), 7.77
viability (%) =
i
y
LD231+PBMC+24 − LDPBMC+24 − LD231+24
j
z
j
j
z
z
1 −
× 100
j
j
z
z
LD231
k
{
(dd, J = 7.5, 1.5 Hz, 1H), 7.46 (dd, J = 7.6, 1.6 Hz, 1H), 7.27 (d, J =
7.6 Hz, 1H), 7.18 (d, J = 7.7 Hz, 1H), 4.12 (s, 3H), 3.76 (t, J = 5.6
Hz, 2H), 3.59 (t, J = 5.6 Hz, 2H), 2.54 (s, 3H), 1.38 (s, 12H). 13C
NMR (101 MHz, CDCl3): δ 165.6, 161.3, 159.7, 142.5, 141.9, 141.8,
139.7, 136.5, 132.3, 125.0, 118.4, 113.3, 83.8, 62.8, 54.4, 43.1, 25.0,
where LD231+PBMC+24, LDPBMC+24, and LD231+24 represented the
detected LDH absorbency values of MDA-MB-231 cells treated
with PBMCs and different concentrations of compound 24, PBMCs
treated with 1.5 μM compound 24, and MDA-MB-231 cells treated
with 1.5 μM compound 24. LD231 represented the detected LDH
absorbency value of MDA-MB-231 cells without PBMCs or
compound 24 treated but with full lysis at the end of culture by
the lysis buffer.
20.0. ESI-MS m/z: for C20H20BNaNO5 [M + Na]+ calcd, 435.2;
+
found, 435.2.
N-(2-Hydroxyethyl)-2-methoxy-6-(2-methyl-[1,1′-biphenyl]-3-yl)-
nicotinamide (43). 43 was prepared from 42 and bromobenzene
PK Study in Male SD Rats. The experimental procedures and
animal use and care protocols were approved by the Institutional
Animal Care and Use Committee (IACUC) of China Pharmaceutical
University, following the guidelines of the Association for Assessment
and Accreditation of Laboratory Animal Care. Male SD rats (n = 4,
240−260 g) were purchased from Veterinary College of Yangzhou
University (Yangzhou, Jiangsu, China). Diet was prohibited for 12 h
before the experiment, but water was freely available. Compound 24
was dissolved in 10% ethanol, 40% PEG-300, and 50% DI water for iv
administration and intragastric gavage. Blood samples (0.35 mL) were
collected from the orbit into heparinized polythene tubes at 0.033,
0.167, 0.5, 1, 2, 3, 5, 8, 12, 16, and 24 h after oral (25 mg/kg) or iv (3
mg/kg) administration of compound 24. Blood samples were
immediately centrifuged at 8000g for 10 min at 4 °C to obtain
plasma. The obtained plasma was stored at −20 °C prior to analysis.
The plasma concentrations of compound 24 were analyzed by LC−
MS/MS. Phoenix WinNonlin was used to calculate all PK parameters.
In Vivo Antitumor Activity Study. The experimental procedures
and animal use and care protocols were approved by the Institutional
Animal Care and Use Committee (IACUC) of China Pharmaceutical
University, following the guidelines of the Association for Assessment
and Accreditation of Laboratory Animal Care. Male BALB/c mice
between 5 and 6 weeks in age were purchased from Veterinary
College of Yangzhou University (Yangzhou, Jiangsu, China). A total
of 5 × 105 CT26 cells were inoculated into the right flank of each
mouse. The vehicle formulation for compound 24 was 10% ethanol,
40% PEG-300, and 50% DI water and was formulated in a single
batch for the entire study. Mice in control groups were treated with
200 μL of vehicle solution only. Mice were treated with vehicle
control and compound 24 (40, 80 mg/kg) by intragastric gavage once
a day for 2 weeks. The tumor volume sizes were measured every 2
days with a caliper and calculated with the formula 0.5 × length ×
width. The mice were sacrificed after the treatment, and tumors were
excised and weighed.
1
according to the preparation method of 13a, yield 71%. H NMR
(400 MHz, chloroform-d): δ 8.55 (d, J = 7.7 Hz, 1H), 8.43 (t, J = 5.6
Hz, 1H), 7.41 (ddd, J = 6.5, 3.5, 1.6 Hz, 3H), 7.38−7.29 (m, 4H),
7.20 (d, J = 7.8 Hz, 1H), 4.12 (s, 3H), 3.83 (t, J = 5.1 Hz, 2H), 3.66
(q, J = 5.4 Hz, 2H), 2.24 (s, 3H). 13C NMR (126 MHz, CDCl3): δ
165.1, 161.0, 159.7, 143.4, 142.0, 141.8, 140.1, 133.6, 130.5, 129.3,
128.9, 128.1, 126.9, 125.6, 118.3, 113.5, 62.0, 54.3, 42.7, 18.6. HRMS
+
(ESI): for C22H23N2O3 [M + H]+, 363.1703; found, 363.1705.
HPLC, tR = 4.526 min, purity 98.4%.
Molecular Modeling. The X-ray structure of PD-L1 was
downloaded from the Protein Data Bank (PDB code: 5J89) into
Schrodinger Maestro 11.5. The protein structure was prepared by
assigning bond orders, adding hydrogens, and generating het states
using Epik. H-bond assignment was optimized using PROPKA, and
water with less than three H bonds and co-crystallized ligand was
removed. Energy minimization with the root-mean-square deviation
value of 0.3 was applied using OPLS3 force field. Compound 24 was
prepared using the OPLS3 force field, and chiralities were determined
from the 3D structure. The docking procedure was performed by
employing the DOCK program using Glide 5.8, and the structural
image was obtained using PyMOL software.
PD-1/PD-L1 TR-FRET Assay. The PD-1/PD-L1 TR-FRET assay
was used to measure the inhibition of PD-1 binding to PD-L1 of the
synthesized compounds. The PD-1/PD-L1 binding assay kits (catalog
#71105 &79311) were purchased from BPS Bioscience. The
experiments were performed according to the instructions of the
manufacturer. In brief, 5 μL of PD-L1-biotin (11 μg/mL) and 5 μL of
prediluted solution with compounds at the indicated concentration
were added and incubated for 15 min at rt. Subsequently, 5 μL of PD-
1-Eu (0.2 μg/mL) and 5 μL of dye-labeled acceptor were added to
reach a total 20 μL volume for the experimental system, which was
then incubated for 90 min at rt. Time-resolved fluorescence intensities
were measured using a Molecular Devices instrument (Perkin Elmer
EnVision). Tb donor emission was measured at 620 nm, followed by
the dye acceptor emission at 665 nm. Data analysis was performed
using the TR-FRET ratio (665 nm emission/620 nm emission), and
inhibition rate % = (Ratio_max-Ratio_sample)/(Ratio_max-Ratio_-
min) × 100, where Ratio_max, Ratio_min, and Ratio_sample
represented the ratios of the positive control, negative control, and
test compounds, respectively. The IC50 values were determined using
GraphPad Prism 6 software.
Flow Cytometry Analysis. Mice were sacrificed and tumors were
harvested. 0.3 mg tumor tissue was isolated and added into a solution
of collagenase (400 units/mL), DNase 1 (100 μg/mL), and
hyaluronidase (0.04 units/mL). The mixture was shaken at 37 °C
for 1 h. Then, the mixture was passed through a 70 μm nylon cell
strainer. The sample was washed three times with PBS (with 2% FBS)
and then resuspended in 1 × 106 cells/mL for flow cytometry analysis.
Antibody (AF488 antimouse CD45, PE/Cy7 antimouse CD3, APC
antimouse CD4, BV510 antimouse CD8, and PE antimouse FOXP3,
all purchased from BioLegend) was added, following the antibody
manufacturer’s recommendations, and incubated for 30 min on ice.
Cells were analyzed on a BD FACS Celesta flow cytometer, and data
were analyzed using FlowJo 10. Cellular events were first gated by
forward and then by side scattering characteristics. The cell counts for
PBMC Killing Assay. Fresh PBMCs were isolated from three
healthy donors. MDA-MB 231 cells (2 × 103 cells/well) were seeded
in 96-well plates 12 h ahead. Different concentrations of compound
24, compound 1 (500 nM), and durvalumab (5 μg/mL; Selleck,
#A2013) were added 2 h ahead. PBMCs (2 × 104 cells/well) were
stimulated with the CD3/CD28/CD2 T cell activator (25 μL/mL,
ImmunoCult, catalog #10970) for 24 h. The stimulated PBMCs were
added to the MDA-MB-231 cells and the test compound and then co-
cultured at 37 °C for 12 h. Cell death was evaluated by the
C D 4 5 +
,
C D 4 5 + C D 3 +
,
C D 4 5 + C D 3 + C D 4 +
,
a n d
CD45+CD3+CD4+FOXP3+ CD45+CD3+-CD8+ were assessed. For
7401
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J. Med. Chem. 2021, 64, 7390−7403

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
quantification, the proportion of each immune subpopulation was
determined by flow cytometry.
Dong Chen − State Key Laboratory of Natural Medicines and
Department of Medicinal Chemistry, School of Pharmacy,
China Pharmaceutical University, Nanjing 210009, China
Zheng Li − Center for Bioenergetics, Houston Methodist
Research Institute, Houston, Texas 77030, United States;
orcid.org/0000-0001-6118-9175
Immunohistochemistry Staining. Isolated tumors were fixed in
4% paraformaldehyde for 24 h at rt, dehydrated, and embedded in
paraffin wax. Before staining with antibody, the tissues were
transferred to slides and deparaffinized. The antimouse CD8 antibody
was added on the slides and incubated at 4 °C overnight for
immunofluorescence staining. After washing with PBS three times,
tissues were incubated with DAPI. Immunofluorescence images were
acquired on a fluorescent inverted microscope (Nikon Eclipse Ts2R).
H&E Staining. The tumor sample was immersed in 4%
paraformaldehyde for 4 h and transferred to 70% ethanol. Individual
lobes of the tumor sample were placed in processing cassettes,
dehydrated through a serial alcohol gradient, and embedded in
paraffin wax blocks. 5 μm thick tissue sections were dewaxed in
xylene, rehydrated through decreasing concentrations of ethanol,
washed in PBS, and stained with H&E. After staining, the sections
were dehydrated through increasing concentrations of ethanol and
xylene. Images were acquired on a fluorescent inverted microscope
(Nikon Eclipse Ts2R).
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00010
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the National Natural Science
Foundation of China (81773559, 21807114, and
82161138005), the Double First-Class University Project
(CPU2018GY03).
ASSOCIATED CONTENT
■
ABBREVIATIONS USED
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00010.
Ala, alanine; Asp, aspartic acid; AUC, area under the curve; Cl,
plasma clearance; Cmax, maximum plasma concentration; ESI,
electron spray ionization; FDA, the US Food and Drug
Administration; Gln, glutamine; H&E, hematoxylin and eosin;
HPLC, high-performance liquid chromatography; HRMS,
high-resolution mass; LDH, lactate dehydrogenase; mAbs,
monoclonal antibodies; Met, methionine; PBMCs, peripheral
blood mononuclear cells; PD-1, programmed cell death-1; PK,
pharmacokinetic; PD-L1, programmed cell death-ligand 1;
Data of PD-1/PD-L1 TR-FRET assay, in vivo antitumor
activity study of compound 24, compound 1 and PD-L1
antibody, binding affinity of compound 1 and 24 to PD-
1
L1 by microscale thermophoresis assay, and H NMR,
13C NMR, and HPLC spectra of the final compounds
(PDF)
SAR, structure−activity relationship; SD, Sprague-Dawley; t1/2
,
Docking of compound 24 with PD-L1 from 5J89 (CSV)
half-life; Tmax, time of maximum plasma concentration; TCR,
T cell receptor; TGI, tumor growth inhibition; TILs, tumor-
infiltrating lymphocytes; TR-FRET, time-resolved fluorescence
resonance energy transfer; Tyr, tyrosine; Vz, apparent
distribution volume; XPhos Pd G2, XPhos palladacycle Gen. 2
Molecular formula strings (PDB)
AUTHOR INFORMATION
■
Corresponding Author
REFERENCES
Sheng Jiang − State Key Laboratory of Natural Medicines and
Department of Medicinal Chemistry, School of Pharmacy,
China Pharmaceutical University, Nanjing 210009, China;
orcid.org/0000-0002-4550-5024; Email: jiangsh9@
gmail.com
■
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Authors
Tianyu Wang − State Key Laboratory of Natural Medicines
and Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, Nanjing
210009, China
Shi Cai − State Key Laboratory of Natural Medicines and
Department of Medicinal Chemistry, School of Pharmacy,
China Pharmaceutical University, Nanjing 210009, China
Mingming Wang − State Key Laboratory of Natural
Medicines and Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, Nanjing
210009, China
Wanheng Zhang − State Key Laboratory of Natural
Medicines and Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, Nanjing
210009, China
Kuojun Zhang − State Key Laboratory of Natural Medicines
and Department of Medicinal Chemistry, School of
Pharmacy, China Pharmaceutical University, Nanjing
210009, China
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