In vitro and in vivo degradation of programmed cell death ligand 1 (PD-L1) by a proteolysis targeting chimera (PROTAC)

Article abstract of DOI:10.1016/j.bioorg.2021.104833

Immunotherapy via immune checkpoints blockade has aroused the attention of researchers worldwide. Inhibition of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has been one of the most promising immunotherapy strategies. Several neutralizing antibodies targeting this interaction have been developed, which have already achieved considerable clinical success. Additionally, numerous pharmaceutical companies have been committed to develop small molecules which could block the interaction between PD-1 and PD-L1. In this study, a novel PROTAC molecule 21a was developed, and effectively induced the degradation of PD-L1 protein in various malignant cells in a proteasome-dependent manner. Moreover, compound 21a could significantly reduce PD-L1 protein levels of MC-38 cancer cells in vivo, by which promoted the invasion of CD8⁺ T cells and inhibited the growth of MC-38 in vivo. This PROTAC molecule could be used as a novel and alternative strategy for cancer immunotherapy.

Full text of DOI:10.1016/j.bioorg.2021.104833

Bioorganic Chemistry 111 (2021) 104833
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
In vitro and in vivo degradation of programmed cell death ligand 1 (PD-L1)
by a proteolysis targeting chimera (PROTAC)
a
,
1
a
,
1
a
,
1
a
,
1
a
a
Yubo Wang , Yuanyuan Zhou , Sheng Cao , Yue Sun , Zhiqiang Dong , Chen Li ,
a
a
c
c
d
b,*
Haoran Wang , Yuhong Yao , Haiyan Yu , Xiangyi Song , Ming Li , Jiefu Wang
Mingming Wei , Guang Yang , Cheng Yang
,
a,
*
a,
*
a
a
The State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai University, Tianjin 300071, PR China
b
Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin’s Clinical Research Center for Cancer, Key Laboratory
of Cancer Prevention and Therapy, Tianjin 300060, PR China
c
Tianjin Pacific Chemical & Pharmaceutical Co., LTD, Tianjin 300350, PR China
d
Cangzhou Institutes for Food and Drug Control, Cangzhou 061000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Immunotherapy via immune checkpoints blockade has aroused the attention of researchers worldwide. Inhibi-
tion of the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction has been one of
the most promising immunotherapy strategies. Several neutralizing antibodies targeting this interaction have
been developed, which have already achieved considerable clinical success. Additionally, numerous pharma-
ceutical companies have been committed to develop small molecules which could block the interaction between
PD-1 and PD-L1. In this study, a novel PROTAC molecule 21a was developed, and effectively induced the
degradation of PD-L1 protein in various malignant cells in a proteasome-dependent manner. Moreover, com-
pound 21a could significantly reduce PD-L1 protein levels of MC-38 cancer cells in vivo, by which promoted the
PROTACs
PD-L1
Immunotherapy
BMS-37 derivatives
+
invasion of CD8 T cells and inhibited the growth of MC-38 in vivo. This PROTAC molecule could be used as a
novel and alternative strategy for cancer immunotherapy.
1
. Introduction
Immune checkpoint blocking antibodies targeting the programmed
the mentioned small molecule inhibitors are currently undergoing
different phases of clinical trials, (For instance, at the time of writing this
article, CA-170, CA-327 and BMS-986189 were under Phase 1 clinical
evaluation process), which could offer various synergistic immuno-
therapy strategies.
death protein 1 (PD-1)/programmed cell death ligand 1 (PD-L1) inter-
action or cytotoxic-T-lymphocyte-associated protein 4 (CTLA4) have
achieved considerable clinical success, which benefit a subset of patients
with a broad spectrum of cancers [1–6]. Until now, several monoclonal
antibodies blocking the interaction between PD-1 and PD-L1 immune
checkpoint proteins (Anti-PD-1: nivolumab, cemiplimab and pem-
brolizumab. Anti-PD-L1: atezolizumab, avelumab and durvalumab), and
one anti-CTLA4 antibody (ipilimumab) have been approved by the U.S.
Food and Drug Administration (FDA). While several others are currently
undergoing various stages of clinical trials [4–11].
Alternatively, we proposed a method that small molecule induced
the degradation of PD-L1 protein in this article, which may be a novel
strategy for cancer immunotherapy. The proteolysis-targeting chimeras
(PROTACs) technique was initially reported by Crews and his coworkers
[15]. Practically, PROTAC molecules refer to hetero-bifunctional small
ligands conjugated via a proper chemical linker. These molecules
recruited E3 ubiquitin ligases to target proteins, resulting in consequent
degradation of the target proteins by the ubiquitin-proteasome system
(UPS). Thus, PROTAC molecules have been recognized as a valuable tool
for the knockdown of target proteins to treat tumors. Numerous in-
hibitors targeted tumor driver-dependent proteins (e.g., MDM2 [16], AR
Recently, a few series of small molecules, for the instance, macro-
cyclic peptides, peptides, peptidomimetics, and nonpeptides structures,
targeting the PD-1/PD-L1 interaction were reported [12–14]. Some of
*
Corresponding authors.
E-mail addresses: jwang05@tmu.edu.cn (J. Wang), mingmingshengwu@163.com (M. Wei), Guang.yang@nankai.edu.cn (G. Yang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bioorg.2021.104833
Received 22 December 2020; Received in revised form 18 February 2021; Accepted 15 March 2021
Available online 19 March 2021
0
045-2068/© 2021 Published by Elsevier Inc.

Y. Wang et al.
Bioorganic Chemistry 111 (2021) 104833
[
17], CDK6 [18], CDK9 [19], BET [20], BRDs [21], PARP-1 [22] and
ethers analogues was prepared by using the CRBN ligand connected with
diverse types of linkers (For the forward synthesis and characterization,
please see supplementary information). For instance, formation of
arylamine bonds yielded 11a-f analogues. The sonogashira coupling
reaction yielded compounds 18a-c and 21a-c. Arylether bonds were
formed to furnish 30a-g. Overall, 19 PROTAC molecules (That is 11a-f,
18a-c, 21a-c and 30a-g) were prepared with various linkers (Table 1–2).
ALK [23]) have been developed into PROTAC molecules. However, very
limited PROTAC molecules mediating the degradation of GPCRs (e.g.,
FLT3 [24]) have been reported. In fact, most of the reported PROTAC
molecules were designed similar to the structure of ligands binding to
the intracellular domains of target proteins.
Unlike previous studies, the principal challenge encountered in this
study was the designs of a PD-L1 PROTAC stems, because of the fact that
there has only a few numbers of promising ligands binding to the
intracellular domain of PD-L1 [25]. Nonetheless, PD-L1 is circulating
and constantly self-renewed from the cytoplasm to the cell membrane.
Cytoplasmic PD-L1 has overall two sources: newly produced and
2.2. Biology
2.2.1. Western blotting assays evaluated the degradation of PD-L1 protein
induced by PROTAC molecules
“
recycled” from the cell membrane by endocytosis [26]. Based on these
To assess the effects of PROTAC molecules on the degradation of PD-
L1 protein, western blotting assays were performed in MC-38 cells, a
C57BL/6 murine colon adenocarcinoma cell line, which bears high level
of PD- L1 protein and efficiently responses to PD-L1 antibody in vivo
[31]. In our preliminary studies, PROTAC molecules induced the
degradation of PD-L1 protein in a time-dependent manner, and treat-
ment for 48 h was the most significant at a low concentration (data not
shown). Hence, MC-38 cells were treated with different PROTAC mol-
ecules for 48 h, and followed by western blotting assays for PD-L1
protein level. As shown in Fig. 3A and B, two compounds, i.e., 11d
and 21a exerted the most efficient impact on the degradation of PD-L1
protein in MC-38 cancer cells among all the synthesized analogues.
facts, it was stipulated that PROTAC molecules can be synthesized and
screened depending on the ligand binding extracellular domain. Hope-
fully, the selected PROTAC molecule could induce the degradation of
either newly produced or “circulating” PD-L1 protein in the cytoplasm,
which may also block the PD-L1 protein transport to the cell membrane.
Taken together, this kinds of PROTAC molecules may result in a sig-
nificant reduction of PD-L1 protein level not only in total but also cell
membrane (Fig. 1). We believe that the success of this proposal will
provide a supplementary strategy to design small molecular PROTACs
which decrease the membrane proteins involved in cancer progression.
2
. Results and discussion
2
.2.2. Preliminary toxicity evaluation of compounds 11d and 21a
Based on the results of western blotting assays, two compounds (11d
2
.1. Chemistry
and 21a) were identified with adequate effects on the degradation of
PD-L1 protein in vitro. However, compound 11d may not be suitable for
subsequent biological studies due to its strong cytotoxic activity which
induced cancer cell death at a low concentration, such as IC50 = 1.233 ±
0.11 or 4.233 ± 0.78 for B16F10 or MC-38 cells respectively (the other
data were shown in supplement information). Its safety profile in vivo is
the most concerned factor. First and foremost, the cell proliferation in-
hibition assay was performed on normal human and mouse cells in vitro,
including human embryonic kidney 293, human hepatic L-O2 and mu-
rine fibroblast NIH-3T3 lines (Table 3). Compound 11d attenuated the
growth of all normal cells at a concentration approximately equated
with its average anticancer potency, whereas compound 21a exerted a
considerably less potent anti-proliferation effect on all normal cells.
Furthermore, C57BL/6 mice were administered both compounds. To
be specific, animals were intraperitoneally injected with a single dose of
the aforementioned compounds (50 mg/Kg, n = 5). All the mice
administered compound 11d died within 24 h, confirming the extreme
toxicity profile of this compound. On the other hand, mice treated with
compound 21a were effectively tolerated. Therefore, compound 21a is
safer and more suitable for in-depth investigation.
To test the hypothesis proposed by the authors, a small diaryl ether
molecule named “BMS-37”, which was discovered to bind the extra-
cellular domain of PD-L1, initially acted as a ligand to PD-L1 owing to its
highly binding potency to PD-L1 [27,28]. Compound BMS-37 was
developed as first in class small molecule inhibitor against PD-L1
described in Bristol-Myers-Squibb’s patents [27,28]. The reported IC50
of BMS-37 against PD-L1/PD-1 was 6 nM. Previous reports elucidated
the binding mode between BMS-37 and PD-L1 through the X-ray
structure of co-crystals (PBD code: 5j89) [29,30]. It is suggested that the
diaryl ether core of BMS-37 is located in a deep, hydrophobic pocket
with the flexible ethanediamine side chain pointed out of the protein.
These results indicated useful structure-activity relationship (SAR) of
this class inhibitors and provide general designing ideas for conjugation
with CRBN ligands (Fig. 2). The ethanediamine side chain seems a
promising modification site for connection of linkers as well as CRBN
ligand (Scheme 1).
Based on the above designing principle, a focused library of diaryl
2
.2.3. Compound 21a induced the degradation of PD-L1 protein via
proteasome in various cancer cell lines
Since compound 21a was selected as an active PROTAC molecule, it
was subsequently tested for PD-L1 degradation in different cancer cell
lines, including Human hematological malignant cells (Skno-1, HL-60,
Kasumi-1), human breast cancer cells MCF-7, murine bladder tumor
cells MB-49, human colon cancer cells SW-480 and human prostate
cancer cells PC-3. All of the selected cancer cells were treated with 21a
for 48 h, and followed by western blotting assays. Results indicated that
compound 21a could induce significant decrease of PD-L1 protein in a
pan-spectrum cancer cell lines (Fig. 4A).
To verify whether the decrease of PD-L1 protein caused by treatment
with compound 21a is a PROTAC-mediated degradation, the PD-L1
levels in cycloheximide (CHX)-treatment cells were first detected. As
illustrated in Fig. 4B, compound 21a promoted the degradation of PD-L1
protein in the absence of confounding synthesis, indicating that the
decrease in PD-L1 protein level was not attributed to mRNA reduction.
Subsequently, Skno-1 and PC-3 cells were pre-treated with thalidomide,
Fig. 1. Cellular renewal of PD-L1 and proposed degradation of PD-L1 by
PROTAC molecule.
2

Y. Wang et al.
Bioorganic Chemistry 111 (2021) 104833
Fig. 2. Co-crystal structure of BMS-37/PD-L1 complex.
O
O
O
H
N
N
H
O
-
BMS 37
O
NH
O
O
NH
O
O
N
O
O
O
H
O
O
O
N
N
O
N
H
N
H
O
Linker
O
O
21a
Scheme 1. Design and Screening of PROTAC molecules targeting PD-L1.
MG132 and BMS-37 before PROTAC 21a, and the total PD-L1 degra-
dation was impeded (Fig. 4C). All of the results pointed out that com-
pound 21a mediated the degradation of PD-L1 protein in a proteasome-
dependent manner.
Immumohistochemical analysis of the tumor tissues suggested that
treatment with compound 21a lead to a remarkable degradation of PD-
+
L1 protein, and improved invasion of CD8 T cells into tumor tissues
(Fig. 6E). Moreover, the gene expression of GzmA, GzmB, IFN-γand Prf1
were also upregulated in tumor tissues, which were the critical media-
+
2
.2.4. Compound 21a mediated the degradation of PD-L1 protein in the
cytoplasm
In order to investigate whether the PD-L1 protein was cleaved in the
tors of CD8 T cells cytotoxicity. (Fig. 6F). These results demonstrate
that intravenous administration of compound 21a can considerably
induce the degradation of the PD-L1 protein and suppress MC-38 tumor
proliferation via immune activation in vivo.
cytoplasm, the detection of PD-L1 protein in cytoplasm and membrane
was carried out. As delineated in Fig. 5A, compound 21a efficiently
induced the degradation of PD-L1 protein in the cytoplasm in a time-
dependent manner. Consequently, the reduction of PD-L1 protein on
the cell membrane was later to the reduction in cytoplasm. In accor-
dance with the above results, the PD-L1 protein was co-located with the
fluorescence-labeled compound 21a within the cytoplasm (Fig. 5B).
These results were consistent with our original design.
2.2.6. Forward synthesis of compound 11d and 21a
Firstly, the core of BMS-37 was prepared. The commercially avail-
able aryl bromide 2 was cross-coupled with Phenylboronic acid to
delivered biaryl compound 3. Etherification by Mitsunobu reaction with
phenol compound 4 to obtain common intermediate 5 in good chemical
yield. On the one hand, for the synthesis of compound 11d, reduction
amination was employed to connect with deprotected amine, which was
derived from the compound 10d. On the other hand, compound 21a was
obtained by sonogashira coupling between terminal alkyne 15a, derived
from the same intermediate 5, and 2-iodo-thalidomide (Scheme 2).
2
.2.5. Compound 21a inhibited tumor growth in vivo
Given these encouraging results, the anticancer activity of compound
2
1a in vivo was assessed for its suppression on MC-38 tumor growth. As
shown in Fig. 6A-D, tumor sizes were significantly diminished in mice
treated with compound 21a (iv, 15 mg/kg per day) in vivo. There was no
noticeable difference in terms of body weight between the two groups,
indicating that compound 21a was not toxic to the tested animals.
3. Conclusion
In summary, a novel PROTAC molecule known as compound 21a
3

Y. Wang et al.
Bioorganic Chemistry 111 (2021) 104833
Table 1
4. Experimental section
.1. General method
All reactions were carried out under an argon atmosphere with dry,
Structure of Compound 11a-f, 18a-c, 21a-c.
4
freshly distilled solvents under anhydrous conditions, unless otherwise
1
noted. Yields refer to chromatographically and spectroscopically ( H
ID of Compounds
n
NMR) homogeneous materials, unless otherwise stated. The used sol-
vents were purified and dried according to common procedures. Other
1
1
1
1
1
1
1a
1b
1c
1d
1e
1f
4
5
6
7
8
9
1
chemicals and solvents were commercially available. H NMR spectra
were obtained by using a Bruker AV 400 or AV 600. Chemical shifts are
reported in parts per million (ppm) relative to either a tetramethylsilane
internal standard or solvent signals. Data are reported as follows:
chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, br = broad, m = multiplet), coupling constants and integration.
1
3
C NMR spectra were recorded using a Bruker AV 400 spectrometer
(
3
100 MHz) using CDCl as the solvent. Chemical shifts (δ) are reported in
parts per million measured relative to the solvent peak. IR spectra were
recorded with a Bio-Rad FTS 6000 Fourier infrared spectrometer. Low
and high-resolution mass spectra were performed in The State Key
Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai
University. Mass spectral data are reported in the form of m/z (intensity
relative to base = 100). Purities of the tested compounds were deter-
mined by HPLC analysis using Agela Technologies HPLC using an Agela
ID of Compounds
n
1
1
1
8a
8b
8c
2
3
4
Technologies MP C18 column (5
μm, 4.6 mm × 50 mm column and
Methanol/water) and were > 95%.
4
4
.2. Synthesis and characterization of compound 11d and 21a
′
.2.1. (2-Methyl-[1,1 -biphenyl]-3-yl) methanol (3)
ID of Compounds
n
A mixture of (3-bromo-2methylphenyl) methanol (1.00 g, 4.96
2
2
2
1a
1b
1c
2
3
4
mmol), Phenylboronic acid (1.21 g, 9.92 mmol) and Pd(dppf)Cl
2
⋅CH
2 2
Cl
(
20.2 mg, 25.0 µmol) in ethanol (3.3 mL) and toluene (10 mL) was under
argon. To the solution sodium bicarbonate, 2 M (10 mL) was added and
◦
the mixture was heated at 80 C for 3 h. Then the mixture was diluted
with EtOAc (100 mL) and washed with saturated NaCl solution (50 mL),
Table 2
2 4
dried over Na SO , concentrated under reduced pressure. The residue
Structure of Compound 30a-g.
was purified by column chromatography on silica gel (hexane/ethyl
acetate 12:1) to afford compound 3 (1.07 g, 98%) as white solid. M.p.
◦
ꢀ 1
1
7
4–76 C; IR (KBr): 3350, 3049, 1600, 1465, 1050,755 cm . H NMR
13
(
400 MHz, CDCl
3
) δ 7.47–7.20 (m, 8H), 4.78 (s, 2H), 2.25 (s, 3H).
) δ 142.9, 142.1, 139.2, 133.6, 129.5, 128.1,
26.8, 125.6, 77.1, 76.7, 64.1, 15.9. HRMS (ESI) calculated for
C
NMR (101 MHz, CDCl
3
1
ID of Compounds
m
n
+
+
C
14
H
14NaO [M + Na] : 221.0937, found 221.0939.
3
3
3
3
3
3
3
0a
0b
0c
0d
0e
0f
1
1
1
1
2
2
2
2
3
5
6
3
4
6
′
4
.2.2. 2,6-Dimethoxy-4-((2-methyl-[1,1 -biphenyl]-3-yl)methoxy)
benzaldehyde (5)
To a solution of 3 (1.00 g, 5.05 mmol) in anhydrous THF (50 mL) was
added 4-hydroxy-2,6-dimethoxybenzaldehyde (1.19 g, 6.56 mmol) and
Triphenylphosphine (2.51 mg, 9.59 mmol). DIAD (1.94 g, 9.59 mmol)
was added slowly under the ice bar. The mixture was stirred at room
temperature for 22 h. The mixture was then concentrated in vacuum.
The residue was purified by column chromatography on silica gel
0g
was developed based on a BMS-37 derivative. This is an example of
membrane protein degradation based on a ligand binding its extracel-
lular domain. Results from the in vitro experiments revealed that com-
pound 21a could efficiently induce the degradation of PD-L1 within
various cancer cell lines in dose-dependent and time-dependent man-
ners. Furthermore, according to the results obtained from in vivo ap-
plications, treatment with compound 21a could significantly reduce PD-
(
hexane/ethyl acetate 5:1) to afford compound 5 (699 mg, 36%) as
◦
white solid. M.p. 161.5–162 C; IR (KBr): 3020, 2928, 1657, 1613, 1610,
585, 1430, 1160 cm . H NMR (400 MHz, CDCl
ꢀ 1 1
1
3
) δ 10.38 (s, 1H),
7
.44–7.25 (m, 8H), 6.21 (s, 2H), 5.16 (s, 2H), 3.89 (s, 6H), 2.28 (s, 3H).
C NMR (101 MHz, CDCl ) δ 187.8, 165.5, 164.2, 143.3, 141.8, 134.2,
1
3
3
1
30.7, 129.4, 128.5, 128.2, 127.1, 125.8, 91.1, 69.6, 56.1, 16.3. HRMS
+
L1 protein levels, facilitate the chemotaxis of CD8 T cells and inhibit
+
+
(
ESI) calculated for C23
H22NaO
4
[M + Na] : 385.1410, found.
the growth of the MC-38 cells. Hence, compound 21a was identified as a
3
85.1409.
promising immunotherapeutic agent, which is worth verifying further.
4
.2.3. 2-(2, 6-Dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione (8)
To a solution of 9-fluorophthalic anhydride 6 (3.30 g, 20.0 mmol) in
CH
3
COOH (100 mL) was added 3-aminopiperidine-2, 6-dione 7 (3.30 g,
2
0.0 mmol). The mixture was refluxed for 12 h. The mixture was then
4

Y. Wang et al.
Bioorganic Chemistry 111 (2021) 104833
Fig. 3. Degradation of PD-L1 protein induced by synthesized PROTAC molecules. MC-38 cells were treated with DMSO or different dilutions of PROTAC molecules
for 48 h, then the cells were harvested. (A) Western blotting analysis of the PD-L1 protein level, and GAPDH was used as reference. (B) Densitometry analysis of
western blotting in (A). The results were expressed as the mean ± SD of three independent experiments, and the statistical significance was determined by performing
Student’s t-test. *P < 0.05, **P < 0.01, ***P < 0.001.
5

Y. Wang et al.
Bioorganic Chemistry 111 (2021) 104833
876, 601 cm 1. 1H NMR (400 MHz, CDCl
8.6, 7.0 Hz, 1H), 7.07 (d, J = 7.1 Hz, 1H), 6.86 (d, J = 8.6 Hz, 1H), 6.22
t, J = 5.7 Hz, 1H), 4.91 (dd, J = 12.2, 5.6 Hz, 1H), 4.58 (s, 1H), 3.24 (q,
ꢀ
) δ 8.42 (s, 1H), 7.47 (dd, J =
Table 3
3
Cell proliferation assay data of compounds in three normal cell lines.
(
Compounds
IC50 value(
93
μ
M)
J = 7.0 Hz, 2H), 3.09 (t, J = 3.7 Hz, 2H), 2.89–2.68 (m, 3H), 2.11 (ddt, J
2
L-O2
NIH-3T3
1
3
=
9.5, 4.5, 2.8 Hz, 1H), 1.63 (d, J = 7.6 Hz, 2H), 1.48–1.30 (m, 17H).
C
1
2
1d
1a
2.618 ± 0.96
21.18 ± 4.22
7.54 ± 2.18
4.77 ± 0.35
NMR (100 MHz, CDCl
3
) δ171.3, 169.6, 168.6, 167.7, 156.1, 147.1,
61.01 ± 6.26
15.65 ± 3.17
1
36.2, 132.6, 116.8, 111.5, 110.0, 79.2, 49.0, 42.7, 40.7, 31.5, 30.1,
IC50 was determined from three independent experiments. MTT assay was
adopted to determine the cytotoxic effects of compounds after undergoing the
29.2, 29.0, 28.6, 28.6, 28.6, 26.9, 26.8, 22.9. HRMS (ESI) calculated for
+
+
C
25
H
34
N
4
NaO
6
[M + Na] : 509.2370, found 509.2369.
4
8 h incubation.
′
4
.2.5. 4-((4-((2,6-Dimethoxy-4-((2-methyl-[1,1 -biphenyl]-3-yl)methoxy)
diluted with EtOAc (200 mL) and washed with HCl solution (1 N, 50
mL), dried over Na SO , concentrated under reduced pressure. The
residue was purified by column chromatography on silica gel
dichloromethane/methanol 50:1) to afford compound 8 (4.78 g, 80%)
benzyl)amino)heptyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-
dione (11d)
2
4
The 10d (400 mg, 900 µmol) was treated with TFA (2.00 mL) in
CH Cl (8 mL) at room temperature for 1 h. The mixture was concen-
(
2
2
◦
ꢀ 1
1
as white solid. M.p. 289 C; IR (KBr): 3180, 1725, 1706, 739.6 cm . H
NMR (400 MHz, DMSO‑d
) δ11.17 (s, 1H), 7.95 (ddd, J = 8.3, 7.3, 4.5
Hz, 1H), 7.89–7.65 (m, 2H), 5.17 (dd, J = 12.9, 5.4 Hz, 1H), 2.97–2.76
trated under reduced pressure to afford intermediate compound as dark
green fluorescent viscous liquid compound, which was added directly
into a solution of 5 (296 mg, 900 µmol) in anhydrous dichloroethane
6
1
3
(
m, 1H), 2.63–2.49 (m, 2H), 2.18–1.95 (m, 1H). C NMR (100 MHz,
DMSO‑d ) δ 172.8, 169.7, 166.2, 164.0, 158.1, 155.5, 138.1, 133.5,
23.0, 120.1, 49.1, 30.9, 21.9. HRMS (ESI) calculated for
(18 mL) and adjusted to pH 5–6 with Et N. The resulting mixture was
3
6
3
then added with NaBH(OAc) (152 mg, 2.43 mmol) at room temperature
1
for 4 h. The reaction mixture was diluted with EtOAc (50 mL) and
washed with saturated NaCl solution (25 mL), dried over Na SO
+
+
C
13
H
9
FN
2
NaO
4
[M + Na] : 299.0438, found. 299.0436.
2
4
,
concentrated under reduced pressure to afford compound 11d (423 mg,
4
.2.4. tert-Butyl (4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
◦
6
6%) as Green fluorescent powder. M.p. 122.1–123.5 C; IR (KBr): 2935,
ꢀ
1 1
yl)amino)heptyl)carbamate (10d)
2855, 2360, 1698, 1615, 1419, 1408, 797, 747 cm
. H NMR (400
To a solution of 8 (500 mg, 1.81 mmol) in DMF (6 mL) was added N-
Boc-heptylenediamine 9d (606 mg, 2.70 mmol). DIPEA (466 mg, 3.62
mmol) was added dropwise. The mixture was refluxed for 12 h. Then the
mixture was diluted with EtOAc (30 mL) and washed with saturated
MHz, CDCl
3
) δ 7.48–7.25 (m, 9H), 7.06 (d, J = 7.1 Hz, 1H), 6.85 (d, J =
8
.5 Hz, 1H), 6.23 (s, 2H), 6.20 (t, J = 6.3 Hz, 1H), 5.08 (s, 2H), 4.90 (s,
H), 4.21 (s, 2H), 3.85 (s, 6H), 3.22 (d, J = 6.4 Hz, 2H), 3.00–2.55 (m,
H), 2.26 (s, 3H), 2.15–2.05 (m, 1H), 1.86 (s, 2H), 1.60 (d, J = 7.4 Hz,
1
5
1
3
2 4
NaCl solution (15 mL), dried over Na SO and concentrated under
2
H), 1.31 (dd, J = 7.9, 4.0 Hz, 6H). C NMR (100 MHz, CDCl
3
) δ 170.7 ,
reduced pressure. The residue was purified by column chromatography
1
61.4, 159.4, 146.5, 142.6, 135.6, 134.1, 134.0, 131.9, 130.0, 128.8,
27.8, 127.6, 126.5, 125.2, 116.2, 110.9, 98.9, 90.5, 68.9, 55.4, 48.4,
2.1, 38.6, 30.5, 28.0, 28.2, 26.2, 26.2, 25.0, 22.2, 15.8. HRMS (ESI)
on silica gel (dichloromethane/methanol 15:1) to afford compound 10d
1
(
(
338 mg, 40%) as dark green fluorescent viscous liquid compound. IR
KBr): 3089, 2926, 2856, 2848, 1630, 1541, 1448, 1382, 1248, 1188,
4
+
+
calculated for C43
H
48
N
4
NaO
7
[M + Na] : 755.3415, found 755.3414.
Fig. 4. Compound 21a induced PD-L1 protein degradation in various cancer cell lines. (A) Various cancer cells were administrated with DMSO or compound 21a for
8 h, and then harvested. PD-L1 protein levels were determined by the western blotting assay. (B) PC-3 and Skno-1 cells treated with cycloheximide were incubated
4
with or without compound 21a for the indicated times, and western blotting assays were conducted to detect PD-L1 protein levels. (C) PC-3 and Skno-1 cells were
pre-incubated with DMSO, BMS-37(10 µM), thalidomide (10 µM) and the proteasome inhibitor MG132 (10 µM) for 2 h, followed by treatment with compound 21a
(
15 µM) for 6 h. Finally, the PD-L1 protein level was determined by western blotting assay. All the experiments were repeated three times independently.
6

Y. Wang et al.
Bioorganic Chemistry 111 (2021) 104833
Fig. 5. Compound 21a induced PD-L1 protein degradation within the cytoplasm. (A) Skno-1 and MC-38 cells were administered 5 M of compound 21a for the
μ
indicated duration. Cytoplasmic and cell membrane PD-L1 protein were extracted using the proteinExt Mammalian Membrane Protein Extraction Kit, and the total
PD-L1 protein was extracted with a RIPA buffer. The Western blotting assay was performed to detect PD-L1 protein levels. (B) MC-38 cells were administrated with 5
μ
M of compound 21a for 6 h. Subsequently, the cells were fixed, immunostained for PD-L1 (red) and analyzed by confocal microscopy. Additionally, compound 21a
emitted green fluorescence after being irradiated by excited light. All the experiments were repeated three times independently. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 6. Effects of compound 21a on MC-38 tumor growth in vivo. C57BL/6 mice were administered subcutaneous injections of MC-38 cells. They were further treated
intravenously with compound 21a for 11 days. (A) Tumor volume curves for MC-38 xenografts, (B) Tumor weight of the different treatment groups at the end of the
treatment. (C) Body weight changes of the mice during the treatment (n = 5). Statistical significance was determined by two-sided unpaired Student’s t-test. **P <
0
.01. (D) Images of the tumors at the end of the treatment. (E) Immunohistochemical staining images of PD-L1 and CD8 for tumor tissue sections and photographs
(
100X). (F) Assessment of the selected gene expressions. Total RNAs were extracted from tumor tissues (n = 5), and gene expressions were assessed by qRT-PCR.
Mean ± SD, n = 5, Student’ t-test, *P < 0.05, **P < 0.01.
7

Y. Wang et al.
Bioorganic Chemistry 111 (2021) 104833
PHENYLBORO ANC I CI D
,
,
PD(DPPF)C
2
LNAHCO
3
PPH
3
DIAD, 4
C
O
O
TOL, ETHANOH
2
L O, , 80
C
THF, 0
O
O
H
OH
OH
BR
90%
56%
O
O
2
3
5
H
2
N
NHBOC
ACONA
HOAC
REFLUX
BOCHN
NH
O
F
F
O
O
80 O
C
NH
O
2
DIPEA, DMF,
40%
+
N
O
O
N
O
O
N
H
NH
80%
NH
O
O
O
O
O
6
7
8
10D
O
NH
O
O
1
2
) TFA, DCM, RT
, ,
) NABH(OAC)
3
5
CLCH
2
CH
2
CL,RT
O
O
O
N
HO
CHO
H
H
6
6% OVE RT W OS TEPS
N
N
O
O
4
O
1
1D
,
,
1
) TFA, CH
2) NABH(OAC)
2
CL
2
RT
5
O
EDCI, DIPEA
H
3
N
HO
MECN, HOBT ,R T
H
CLCHCH CL,RT
N
N
2
2
+
NHBOC
H
H
2
N
BOCHN
O
O
83 %
80%
OVE RT WO
STEPS
O
O
O
12A
13
14
15A
,
ACONA
HOAC
REFLUX
PD(PPH
3
)
2
CL
2
DIPEA
O
I
I
3,
H
N
O
O
O
O
O
N
K
2CO CUI
NH
O
2
15A,
O
N
N
O
DMF, 80
C
+
O
H
O
O
O
N
H
82%
NH
O
O
NH
O
65%
O
O
19
7
20
21A
Scheme 2. Synthesis PROTAC molecules 11d and 21a.
To a solution of N-Boc-ethylenediamine 13 (4.00 g, 24.9 mmol) in
CN (150 mL) was added 12a (3.00 g, 30.5 mmol), EDCI (7.30 g,
8.0 mmol), HoBt (5.16 g, 38.0 mmol) and DIPEA (12.6 mL). The
mixture was stirred at room temperature for 18 h. Then the mixture was
diluted with EtOAc (500 mL) and washed with saturated NaHCO
aqueous solution (200 mL) and 1 N HCl (150 mL), dried over Na SO
CDCl
3
) δ 8.86 (s, 2H), 8.00 (t, J = 5.9 Hz, 1H), 7.42 (m, 3H), 7.37 (m,
CH
3
1H), 7.31 (m, 2H), 7.28 (d, J = 3.3 Hz, 1H), 6.22 (s, 2H), 5.06 (s, 2H),
4.14 (s, 2H), 3.82 (s, 6H), 3.50 (d, J = 4.9 Hz, 2H), 3.04 (t, J = 5.0 Hz,
2H), 2.45 (m, 2H), 2.40 (d, J = 6.4 Hz, 2H), 2.26 (s, 3H), 1.91 (t, J = 2.5
3
Hz, 1H). 13C NMR (100 MHz, CDCl
) δ 172.4, 162.3, 159.9, 143.2,
3
3
2
4
,
134.6, 130.6, 129.4, 128.4, 128.2, 127.0, 125.8, 99.0, 91.1, 69.5, 69.0,
and concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel (dichloromethane/methanol 60:1)
55.9, 46.9, 40.3, 36.0, 34.7, 16.3, 14.7. HRMS (ESI) calculated for
+
+
C
30
H
34
N
2
NaO
4
[M + Na] : 509.2411, found 509.2412.
◦
to afford compound 14a (5.41 g, 83%) as white solid. M.p. 83.5–85.0 C;
ꢀ 1 1
IR (KBr): 3301, 3296, 2980, 1654, 1550, 1284, 860, 635 cm . H NMR
4.2.7. 5-Bromo-2-(2, 6-dioxopiperidin-3-yl)isoindoline-1,3-dione (17)
(
400 MHz, CDCl
3
) δ 6.36 (s, 1H), 4.97 (s, 1H), 3.38 (td, J = 6.5, 5.7, 4.4
To a solution of 16 (4.54 g, 20.0 mmol) in CH
3
COOH (100 mL) was
◦
Hz, 2H), 3.28 (q, J = 5.8 Hz, 2H), 2.53 (td, J = 6.9, 2.5 Hz, 2H), 2.40 (t, J
added 7 (2.20 g, 20.0 mmol). The mixture was refluxed at 75 C for 12 h.
7.0 Hz, 2H), 2.01 (s, 1H), 1.44 (s, 9H). 1 C NMR (100 MHz, CDCl
3
Then the mixture was diluted with EtOAc (200 mL) and washed with
=
3
) δ
1
71.70, 157.05, 83.10, 69.42, 40.89, 40.26, 35.46, 28.49, 15.05. HRMS
2 4
saturated HCl solution (50 mL), dried over Na SO and concentrated
+
+
(
ESI) calculated for C12
H
20
2
N NaO
3
[M + Na] : 263.1366, found
under reduced pressure. The residue was purified by column chroma-
tography on silica gel (dichloromethane/ methanol 50:1) to afford
2
63.1368.
◦
compound 17 (5.89 g, 82%) as white solid. M.p. > 250 C (decom.); IR
′
ꢀ 1
1
4
.2.6. N-(2-((2,6-Dimethoxy-4-((3-methyl-[1,1 -biphenyl]-4-yl)methoxy)
(KBr): 3195, 1724, 1706.7, 739.4 cm . H NMR (400 MHz, DMSO‑d ) δ
6
benzyl)amino)ethyl)pent-4-ynamide (15a).
11.14 (s, 1H), 8.20–8.04 (m, 2H), 7.86 (d, J = 7.9 Hz, 1H), 5.17 (dd, J =
12.8, 5.4 Hz, 1H), 2.89 (ddd, J = 17.0, 13.8, 5.4 Hz, 1H), 2.66–2.51 (m,
2 2
The solution of 14a (1.18 g, 8.45 mmol) in CH Cl (30 mL) was
1
3
adjusted to pH 5–6 with Et
3
N. Compound 5 (1.50 g, 4.14 mmol) was
2H), 2.07 (ddq, J = 10.5, 5.4, 2.9, 2.5 Hz, 1H). C NMR (100 MHz,
added to the solution at room temperature for 4 h. Then the mixture was
diluted with EtOAc (100 mL) and washed with saturated NaCl solution
6
DMSO‑d ) δ 172.7, 169.6, 166.4, 165.8, 137.6, 133.1, 130.1, 128.5,
126.4, 125.2, 49.2, 30.9, 21.9. HRMS (ESI) calculated for
+
+
(
50 mL), dried over Na
SO
2 4
and concentrated under reduced pressure.
C
13
H
9
BrN
2
NaO
4
[M + Na] : 358.9638, found 358.9639.
The residue was purified by column chromatography on silica gel
(
dichloromethane/methanol 60:1) to afford compound 15a (3.44 g,
◦
8
1
0%) as white solid. M.p. 87.1–88.2 C; IR (KBr): 3058, 2929, 2840,
ꢀ
1 1
716, 1610, 1499, 1197, 1142, 745, 703 cm
. H NMR (400 MHz,
8

Y. Wang et al.
Bioorganic Chemistry 111 (2021) 104833
′
4
.2.8. N-(2-((2,6-Dimethoxy-4-((3-methyl-[1,1 -biphenyl]-4-yl)methoxy)
sulfoxide (DMSO). Optical density (OD) of formazan concentration was
read at 490 nm and 570 nm with THERMO FISHER Multiskan FC. The
concentrations required to inhibit growth by 50% (IC50) were calculated
with GraphPad Prism 7.
benzyl)amino)ethyl)-5-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-
yl)pent-4-ynamide (18a)
To a solution of 17 (210 mg, 620 µmol) in DMF (3 mL) was added
1
5a (300 mg, 620 µmol), Pd(PPh
3
)
2
Cl
2
(43.0 mg, 60.0 µmol), CuI (11.0
◦
mg, 60.0 µmol) and DIPEA (1.00 mL). The mixture was stirred at 50 C
under an argon atmosphere for 12 h. Then the mixture was diluted with
EtOAc (15 mL) and washed with saturated NaCl solution (5 mL), dried
4
.5. CCK-8 assay
Skno-1 cells were seeded in a 96-well plate at a density of 8000 cells/
2 4
over Na SO , concentrated under reduced pressure. The residue was
well in 100
pounds, and incubated for 48 h. After for the incubation, each well of the
plate was added in 10 L of CCK-8 solution and incubated for another
μ
L culture medium supplemented with or without com-
purified by column chromatography on silica gel (dichloromethane/
methanol 15:1) to afford compound 18a (310 mg, 66%) as white solid.
◦
μ
M.p. 145.7–147.1 C; IR (KBr): 2925, 2849, 1717, 1636, 1595, 1420,
ꢀ
1
1
4–6 h in the incubator. The absorbance at 450 nm was measured with
THERMO FISHER Multiskan FC. The concentrations required to inhibit
growth by 50% (IC50) were calculated with GraphPad Prism 7.
1
1
377, 1141, 1113, 732, 707 cm . H NMR (400 MHz, CDCl ) δ 7.95 (s,
3
H), 7.76–7.72 (m, 1H), 7.66–7.60 (m, 2H), 7.44–7.26 (m, 9H), 6.19 (d,
J = 1.3 Hz, 2H), 5.06 (s, 3H), 4.16 (d, J = 3.5 Hz, 2H), 3.82 (s, 6H), 3.63
(
d, J = 5.5 Hz, 2H), 2.99 (t, J = 5.4 Hz, 2H), 2.80 (q, J = 7.4 Hz, 5H),
1
3
2
.59 (t, J = 7.0 Hz, 2H), 2.25 (d, J = 1.3 Hz, 3H), 2.16–2.11 (m, 1H).
) δ 171.8, 171.0, 168.0, 166.9, 166.8, 162.4,
60.0, 143.3, 141.9, 137.5, 134.7, 132.0, 130.8, 130.7, 130.0, 129.5,
C
4
.6. Cell membrane protein and cytoplasmic protein extraction
NMR (100 MHz, CDCl
3
1
1
4
1
The protein was extracted according to the operating steps of the
28.5, 128.3, 127.1, 126.7, 125.9, 123.7, 98.8, 94.8, 91.3, 69.6, 56.1,
9.6, 46.9, 40.3, 35.9, 35.0, 31.5, 29.8, 29.8, 29.5, 22.8, 22.7, 16.4,
ProteinExt Mannalian Membrane Protein Extraction Kit. This step is
improved according to the experimental plan. The treated cells are
processed according to the method of cell passage, and the cell pellet is
obtained by centrifugation. Cell pellets were washed twice with 1 mL
pre-cooled PBS, centrifuged at 1000g for 3 min, and discard the super-
+
+
5.9. HRMS (ESI) calculated for C43
H
42
N
4
NaO
8
[M + Na] : 765.2895,
found 765.2897.
′
4
.2.9. N-(2-((2,6-Dimethoxy-4-((3-methyl-[1,1 -biphenyl]-4-yl)methoxy)
natant carefully. Then added 150 L MPEB I solution to the pellet, shake
μ
benzyl)amino)ethyl)-5-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)pent-4-ynamide (21a)
vigorously for 15 s, and incubated on ice for 2 min, shaking for 2 min
each time, 5 times in total. Next, centrifuged at 16000g for 15 min at
To a solution of compound 20 (140 mg, 370 µmol) in DMF (2 mL)
◦
2
–8 C. The supernatant is cytoplasmic protein and placed it on ice.
was added 15a (180 mg, 370 µmol), Pd(PPh
3
)
2
Cl
2
(25.0 mg, 370 µmol),
Added 30 ul MPEB II to the pellet, resuspended the pellet, shaked
vigorously for 15 s, incubated at low temperature for 30 min, and shakes
CuI (7.10 mg, 370 µmol) and DIPEA (1 mL). The flask was stirred under
◦
the argon at 50 C for 12 h. Then the mixture was diluted with EtOAc
once every 5 min. Then added 300
μ
l MPEB III, shake vigorously for 5 s,
(
15 mL) and washed with saturated NaCl solution (5 mL), dried over
◦
centrifuged at 16000g for 15 min at 2–8 C. Carefully collect the su-
Na SO and concentrated under reduced pressure. The residue was pu-
2
4
pernatant (membrane protein) and store it together with the cyto-
rified by column chromatography on silica gel (dichloromethane/
◦
plasmic protein – 80 C for subsequent experiments.
methanol 10:1) to afford compound 21a (183 mg, 65%) as white solid.
◦
M.p. 142.1–143.5 C; IR (KBr): 2920, 2840, 1720, 1646, 1597, 1422,
ꢀ
1 1
1
1
2
5
2
1
1
4
C
397, 1151, 1113, 740, 737 cm . H NMR (400 MHz, CDCl ) δ 7.89 (m,
3
4
.7. Western blot analysis
H), 7.75 (dd, J = 6.5, 1.9 Hz, 1H), 7.65 (m, 2H), 7.36 (m, 9H), 6.19 (s,
H), 5.06 (s, 3H), 4.17 (m, 2H), 3.81 (s, 6H), 3.64 (m, 2H), 3.02 (t, J =
.3 Hz, 2H), 2.79 (ddt, J = 18.1, 16.6, 6.3 Hz, 5H), 2.58 (t, J = 6.9 Hz,
Cells were seeded in 6-well culture plates, treated with compound at
indicated concentrations for 48 h, and lysed in RIPA lysis buffer sup-
plemented with protease inhibitors cocktail/PMSF and dephosphorylase
inhibitor NaF. The protein concentration was measured with the BCA
Protein Assay kit (Beyotime Biotechnology, P0012). Equal amounts of
proteins were electrophoresed by SDS-PAGE (10%) under denaturing
conditions and transferred onto the PDVF membranes (Millipore Cor-
poration, IPVH00010). Membranes were blocked in 5% nonfat milk (BD
Difco, 232100) and then incubated with primary antibodies. After being
washed for three times, the membranes were incubated with secondary
antibodies and detected by Luminescent Image Analyzer LSA 4000 (GE,
Fairfield, CO, USA). Antibodies used in this study were: anti-PD-L1 (no.
H), 2.25 (s, 3H), 2.16 (m, 1H). 13C NMR (100 MHz, CDCl
) δ 172.3,
3
71.4, 168.7, 166.7, 166.5, 159.9, 143.2, 138.4, 134.7, 134.1, 132.2,
30.6, 129.4, 128.5, 128.2, 127.0, 125.8, 122.9, 97.7, 91.2, 69.5, 56.6,
9.6, 40.2, 36.4, 35.2, 31.5, 22.8, 16.4, 16.4. HRMS (ESI) calculated for
+
+
43
H
42
4
N NaO
8
[M + Na] : 765.2895, found 762.2899.
4
.3. Cell lines and mice
Cells were cultured in RPMI 1640 (MC-38, B16F10, 4T1, MCF-7,
skno-1, kasumi-1, HL-60, PC3, NIH-3T3, L-O2), DMEM (MB-49, 293)
or IMDM (SW480) medium supplemented with 10% FBS (Procell), 100
6
6248-1-Ig), anti-GAPDH(no. 60004-1-Ig).
◦
U/mL penicillin, and 100
% CO
μ
g/mL streptomycin at 37 C in the presence of
5
2
. Female C57BL/6 mice (6–8 weeks old) were purchased from
Beijing Vital River Laboratory (Beijing, People’s Republic of China). The
4.8. Animal study
mice were housed in a pathogen-free environment that was maintained
◦
at a temperature of 25 C and a relative humidity of 45%–55%.
The animal studies were approved by the Institutional Review Board
of Nankai University. All animal studies were conducted according to
protocols approved by the Animal Ethics Committee of Nankai Univer-
4
.4. MTT assay
sity. MC-38 (5.0 × 10^6 cells/100
μ
L) cells was injected subcutaneously
Adherent cells were seeded in 96-well plates at a density of
into six-week-old C57BL/6 mice, respectively. When the tumor volume
3
3
000–5000 cells/well and cultured for overnight. And then the cells
were treated with different concentrations of synthetic compounds for
reached about 50–100 mm , the mice were randomly divided to vehicle
control, and compounds 21a (15 mg/kg/day, iv, n = 5 for each group)
and were treated as above dose. Tumor volume and mice weight were
measured each day after the initiation of the treatment. Mice were
executed and tumors were harvested after 11 days. Tumor volume =
length*width*width/2.
4
8 h. After 48 h of drug incubation, 15 L of MTT (5 mg/mL, Med-
μ
ChemExpress (MCE), HY-15924) solution was added to each well and
incubation was continued for another 3–4 h. The supernatant was dis-
carded, and formazan crystals were dissolved in 100 L of dimethyl
μ
9

Y. Wang et al.
Bioorganic Chemistry 111 (2021) 104833
4
.9. Statistical analysis
[9] M.J. Smyth, S.F. Ngiow, A. Ribas, M.W.L. Teng, Combination cancer
immunotherapies tailored to the tumour microenvironment, Nat. Rev. Clin. Oncol.
1
3 (2015) 143–158.
All statistical analyses were conducted using Graph prism 7.0 soft-
[
[
[
[
10] S. Farkona, E.P. Diamandis, I.M. Blasutig, Cancer immunotherapy: the beginning of
the end of cancer, BMC Med. 14 (2016) 1–18.
ware. Statistically significant differences were determined by Student’s t
test, and P values less than 0.05 were considered statistically significant
in all cases.
11] S.H. Baumeister, G.J. Freeman, G. Dranoff, A.H. Sharpe, Coinhibitory pathways in
immunotherapy for cancer, Annu. Rev. Immunol. 34 (2016) 539–573.
12] H. Weinmann, Cancer immunotherapy: selected targets and small-molecule
modulators, ChemMedChem. 11 (2016) 450–466.
13] T. Zarganes-Tzitzikas, M. Konstantinidou, Y. Gao, T.A. Holak, D. Krzemien, K. Zak,
G. Dubin, A. D o¨ mling, Inhibitors of programmed cell death 1 (PD-1): a patent
review (2010–2015), Expert Opin. Ther. Pat. 26 (2016) 973–977.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[14] M.-M. Zhan, X.-Q. Hu, X.-X. Liu, B.-F. Ruan, J. Xu, C. Liao, From monoclonal
antibodies to small molecules: the development of inhibitors targeting the PD-1/
PD-L1 pathway, Drug Discovery Today 21 (6) (2016) 1027–1036.
[
15] (a) G.M. Burslem, C.M. Crews, Chem. Rev. 117 (2017) 11269–11301;
(
b) J. Salami, C.M. Crews, Science 355 (2017) 1163–1167.
16] (a) R.P. Wurz, V.J. Cee, J. Med. Chem. 62 (2019) 445–447;
b) Y. Li, J. Yang, A. Aguilar, D. McEachern, S. Przybranowski, L. Liu, C.Y. Yang,
[
Acknowledgments
(
M. Wang, X. Han, S. Wang, J. Med. Chem. 62 (2019) 448–466.
This work was supported by the Fundamental Research Funds for the
Central Universities, Nankai University (735-63201240), the National
Natural Science Foundation of China (NSFC, No. 81703343, 82003186,
[17] X. Han, C. Wang, C. Qin, W. Xiang, E. Fernandez-Salas, C.Y. Yang, M. Wang,
L. Zhao, T. Xu, K. Chinnaswamy, J. Delproposto, J. Stuckey, S. Wang, J. Med.
Chem. 62 (2019) 941–964.
[
18] S. Su, Z. Yang, H. Gao, H. Yang, S. Zhu, Z. An, J. Wang, Q. Li, S. Chandarlapaty,
8
2073691 and 81503019), National Science & Technology Major Proj-
ect “Key New Drug Creation and Manufacturing Program”, China (No.
019ZX09201001). Key Research and Development Program of Tianjin
H. Deng, W. Wu, Y. Rao, J. Med. Chem. 60 (2017) 9860–9873.
[19] C.M. Robb, J.I. Contreras, S. Kour, M.A. Taylor, M. Abid, Y.A. Sonawane, M. Zahid,
D.J. Murry, A. Natarajan, S. Rana, Chem. Commun. 53 (2017) 7577–7580.
2
[
20] C. Qin, Y. Hu, B. Zhou, E. Fernandez-Salas, C.Y. Yang, L. Liu, D. McEachern,
S. Przybranowski, M. Wang, J. Stuckey, J. Meagher, L. Bai, Z. Chen, M. Lin,
J. Yang, D.N. Ziazadeh, F. Xu, J. Hu, W. Xiang, L. Huang, S. Li, B. Wen, D. Sun,
S. Wang, J. Med. Chem. 61 (2018) 6685–6704.
City (No. 19YFZCSY00160) and Key research program directing project
of Cangzhou City (No. 192106010).
[
21] V. Zoppi, S.J. Hughes, C. Maniaci, A. Testa, T. Gmaschitz, C. Wieshofer, M. Koegl,
K.M. Riching, D.L. Daniels, A. Spallarossa, A. Ciulli, J. Med. Chem. 62 (2019)
Appendix A. Supplementary material
6
99–726.
The copies of 1H/13C NMR Spectrum of all compounds and in-
[22]
[
Q. Zhao, T. Lan, S. Su, Y. Rao, Chem. Commun. 55 (2019) 369–372.
23] X. Kong, P. Pan, H. Sun, H. Xia, X. Wang, Y. Li, T. Hou, J. Med. Chem. 62 (2019)
0927–10954.
[24] G.M. Burslem, J. Song, X. Chen, J. Hines, C.M. Crews, J. Am. Chem. Soc. 140
termediates. Biological evaluation of all selected compounds (PDF)
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104833.
1
(
2018) 16428–16432.
[
[
25] B. Cheng, Y. Ren, H. Cao, J. Chen, Discovery of novel resorcinol diphenyl ether-
based PROTAC-like molecules as dual inhibitors and degraders of PD-L1, Eur. J.
Med. Chem. 199 (2020) 112377–112393.
References
26] M.L. Burr, C.E. Sparbier, Y.C. Chan, J.C. Williamson, K. Woods, P.A. Beavis, E.Y.
N. Lam, M.A. Henderson, C.C. Bell, S. Stolzenburg, O. Gilan, S. Bloor, T. Noori, D.
W. Morgens, M.C. Bassik, P.J. Neeson, A. Behren, P.K. Darcy, S.J. Dawson,
I. Voskoboinik, J.A. Trapani, J. Cebon, P.J. Lehner, M.A. Dawson, CMTM6
maintains the expression of PD-L1 and regulates anti-tumour immunity, Nature
549 (2017) 101–105.
[
[
[
[
[
1] S. Topalian, C. Drake, D. Pardoll, Immune checkpoint blockade: a common
denominator approach to cancer therapy, Cancer Cell 27 (4) (2015) 450–461.
2] D.S. Shin, A. Ribas, The evolution of checkpoint blockade as a cancer therapy:
what’s here, what’s next? Curr. Opin. Immunol. 33 (2015) 23–35.
3] P. Sharma, J. Allison, Immune checkpoint targeting in cancer therapy: toward
combination strategies with curative potential, Cell 161 (2) (2015) 205–214.
4] A. Hoos, Development of immuno-oncology drugs from CTLA4 to PD1 to the next
generations, Nat. Rev. Drug Discovery 15 (4) (2016) 235–247.
[27] Chupak, L. S.; Zheng, X. Compounds Useful as Immunomodulators.
WO2015034820, 2015.
[28] L.S. Chupak, M. Ding, S.W. Martin, X. Zheng, P. Hewawasam, T.P. Connolly, N. Xu,
K.-S. Yeung, J. Zhu, D.R. Langley, D.J. Tenney, P.M. Scola, P.A. Mingo, Compounds
useful as immunomodulators. WO2015160641, 2015.
5] K.M. Mahoney, P.D. Rennert, G.J. Freeman, Combination cancer immunotherapy
and new immunomodulatory targets, Nat. Rev. Drug Discovery 14 (8) (2015)
5
61–584.
[29] K.M. Zak, P. Grudnik, K. Guzik, B.J. Zieba, B. Musielak, A. D o¨ mling, G. Dubin, T.
A. Holak, Structural basis for small molecule targeting of the programmed death
ligand 1 (PD-L1), Oncotarget 7 (2016) 30323–30335.
[
[
6] D.N. Khalil, E.L. Smith, R.J. Brentjens, J.D. Wolchok, The future of cancer
treatment: immunomodulation, CARs and combination immunotherapy, Nat. Rev.
Clin. Oncol. 13 (2016) 273–290.
[30] K. Guzik, K.M. Zak, P. Grudnik, K. Magiera, B. Musielak, R. T o¨ rner, L. Skalniak,
A. D o¨ mling, G. Dubin, T.A. Holak, J. Med. Chem. 60 (2017) 5857–5867.
[31] B.J. Hos, M.G.M. Camps, J.V.D. Bulk, E. Tondini, T.C. Ende, D. Ruano, Franken, K.
Janssen, G. M.C., A. Ru, D.V. Filippov, R. Arens. P.A. Van Veelen, N. Miranda, F.
Ossendorp, Identification of a neo-epitope dominating endogenous CD8 T cell
responses to MC-38 colorectal cancer, Oncoimmuninology 2020, 9, e1673125.
7] J.D. Wolchok, H. Kluger, M.K. Callahan, M.A. Postow, N.A. Rizvi, A.M. Lesokhin,
N.H. Segal, C.E. Ariyan, R.A. Gordon, K. Reed, M.M. Burke, A. Caldwell, S.
A. Kronenberg, B.U. Agunwamba, X. Zhang, I. Lowy, H.D. Inzunza, W. Feely, C.
E. Horak, Q. Hong, A.J. Korman, J.M. Wigginton, A. Gupta, M. Sznol, Nivolumab
plus Ipilimumab in Advanced Melanoma, N. Engl. J. Med. 369 (2013) 122–133.
8] I. Melero, D.M. Berman, M.A. Aznar, A.J. Korman, J.L.P. Gracia, J. Haanen,
Evolving synergistic combinations of targeted immunotherapies to combat cancer,
Nat. Rev. Cancer 15 (8) (2015) 457–472.
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