Discovery of phenyl-linked symmetric small molecules as inhibitors of the programmed cell death-1/programmed cell death-ligand 1 interaction

Article abstract of DOI:10.1016/j.ejmech.2021.113637

Programmed cell death-1/programmed cell death ligand 1 (PD-1/PD-L1) is one of the most promising targets in the field of immune checkpoint blockade therapy. Beginning with our exploration of linkers and structure-activity relationship research, we found that the aromatic ring could replace the linker and aryl group to maintain the satisfactory activity of classic triaryl scaffold inhibitor. Based on previous studies, we designed and synthesized a series of C₂-symmetric phenyl-linked compounds, and further tail optimization afforded the inhibitors, which displayed promising inhibitory activity against the PD-1/PD-L1 interaction with IC₅₀ value at the single nanomolar range (C13–C15). Further cell-based PD-1/PD-L1 blockade bioassays indicated that these C₂-symmetric molecules could significantly inhibit the PD-1/PD-L1 interaction at the cellular level and restore T cells' immune function at the safety concentrations. The discovery of these phenyl-linked symmetric small molecules showed the potential of simplified-linker and C₂-symmetric strategy and provided a basis for developing symmetric small molecule inhibitors of PD-1/PD-L1 interaction. Moreover, C13 and C15 performed stable binding modes to PD-L1 dimeric after computational docking and dynamic simulation, which may serve as a good starting point for further development.

Full text of DOI:10.1016/j.ejmech.2021.113637

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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Discovery of phenyl-linked symmetric small molecules as inhibitors of
the programmed cell death-1/programmed cell death-ligand 1
interaction
,
,
,
Yizhe Wu a 1, Yu Zhang a 1, Yu Guo a 1, Zhichao Pan a, Shichun Zhong a, Xinxin Jin a,
Weihao Zhuang a, Sikang Chen a, Jian Gao a, Wenhai Huang b, Xiaowu Dong a
,
, c, d
Jinxin Che a
, c, *
a Hangzhou Institute of Innovative Medicine, Institute of Drug Discovery and Design, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou,
310058, PR China
b Key Laboratory of Neuropsychiatric Drug Research of Zhejiang Province, Hangzhou Medical College, Hangzhou, 310013, PR China
c Innovation Institute for Artificial Intelligence in Medicine of Zhejiang University, Hangzhou, 310018, PR China
d Cancer Center, Zhejiang University, Hangzhou, 310058, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Programmed cell death-1/programmed cell death ligand 1 (PD-1/PD-L1) is one of the most promising
targets in the field of immune checkpoint blockade therapy. Beginning with our exploration of linkers
and structure-activity relationship research, we found that the aromatic ring could replace the linker and
aryl group to maintain the satisfactory activity of classic triaryl scaffold inhibitor. Based on previous
studies, we designed and synthesized a series of C2-symmetric phenyl-linked compounds, and further
tail optimization afforded the inhibitors, which displayed promising inhibitory activity against the PD-1/
PD-L1 interaction with IC50 value at the single nanomolar range (C13eC15). Further cell-based PD-1/PD-
L1 blockade bioassays indicated that these C2-symmetric molecules could significantly inhibit the PD-1/
PD-L1 interaction at the cellular level and restore T cells' immune function at the safety concentrations.
The discovery of these phenyl-linked symmetric small molecules showed the potential of simplified-
linker and C2-symmetric strategy and provided a basis for developing symmetric small molecule in-
hibitors of PD-1/PD-L1 interaction. Moreover, C13 and C15 performed stable binding modes to PD-L1
dimeric after computational docking and dynamic simulation, which may serve as a good starting
point for further development.
Received 18 April 2021
Received in revised form
1 June 2021
Accepted 5 June 2021
Available online 11 June 2021
Keywords:
Immune checkpoint
PD-1/PD-L1
Phenyl-linker
C2-symmetric small molecules
PD-1/PD-L1 interaction inhibitors
IFN-g secretion
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
corresponding ligand/receptor interaction [4,5]. It plays an impor-
tant role in maintaining self-immune tolerance and regulating the
Immunotherapy has been widely used in the treatment of
various cancers as a potential subversive strategy [1]. Cancer
immunotherapy, such as cancer vaccines, adoptive T-cell therapy,
and immune checkpoint blockade, which aims to fight tumors by
stimulating the immune system, has become one of the fastest-
growing areas of cancer therapy [2,3]. As an inhibitory pathway
in the immune system, immune checkpoints are regulated by the
duration and amplitude of physiological immune response so as to
avoid damage to normal tissues caused by the immune system
[5,6]. Among them, programmed cell death-1/programmed cell
death ligand 1 (PD-1/PD-L1) is one of the most promising targets in
the field of immune-oncology [7]. PD-1 is mainly expressed in
activated T cells and B cells, and its function is to inhibit cell over-
activation, which is a normal homeostasis mechanism of the im-
mune system [8]. The tumor microenvironment will induce the
infiltrating T cells to overexpress PD-1, while the tumor cells
overexpress PD-L1 and PD-L2, leading to the continuous activation
of the PD-1 pathway in the tumor microenvironment [8,9].
Therefore, even if T cells recognize tumor cells and the tumor an-
tigen binds to T cell receptors, the function of T cells will be
* Corresponding author. Hangzhou Institute of Innovative Medicine, Institute of
Drug Discovery and Design, College of Pharmaceutical Sciences, Zhejiang Univer-
sity, Hangzhou, 310058, PR China.
E-mail address: chejx@zju.edu.cn (J. Che).
These authors contributed equally to this work.
1
https://doi.org/10.1016/j.ejmech.2021.113637
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

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inhibited, thus unable to kill tumor cells.
binding pattern and thus significantly blocking the interaction
between PD-1 and PD-L1 [35], but few C2-symmetric small mole-
cule immune checkpoint inhibitors have been reported.
As an effective way to block protein-protein interaction, several
clinical trials of humanized monoclonal antibody (mAb) against
PD-1/PD-L1 have shown satisfactory anti-tumor effects [10,11]. In
the last decade, six mAbs against PD-1 or PD-L1 (anti-PD-1: nivo-
lumab, pembrolizumab, and cemiplimab; anti-PD-L1: atezolizu-
mab, avelumab, and durvalumab) have been approved by the FDA
[12,13]. In addition, many mAbs are still in clinical trials worldwide
[13,14]. Notably, PD-1/PD-L1 mAbs have shown significant clinical
efficacy in various tumors, including melanoma, lung cancer,
Hodgkin's lymphoma, urothelial carcinoma, bladder cancer, and
etc., [15e20]. The clinical use of these antibodies has proved that
PD-1/PD-L1 are promising targets. However, at present, there is still
a lack of small molecule drugs acting by the blockade on PD-1/PD-
L1 interaction [21]. As a potential complementary strategy of
antibody therapy, the development of small molecule PD-1/PD-L1
blockers has been gradually put on the agenda [22].
Recently, a series of inhibitors, including macrocyclic peptides,
peptide analogs, and non-peptide small molecules, have been
developed to provide pharmacokinetic (PK) properties, stability,
and oral bioavailability different from antibodies [12,21], of which
BMS986189 (BMS), CA-170 (Aurigene), INCB086550 (Incyte),
IMMH-010 (Chasesun), GS-4224 (Gilead) and MAX-10181 (Max-
inovel) have entered clinical trials [7,23]. To date, compounds with
triaryl scaffolds are the dominant class of small molecule inhibitors
of PD-1/PD-L1 interaction [24]. Bristol Myer Squibb (BMS) is the
first company to unveil small molecule inhibitors of PD-1/PD-L1
with triaryl scaffold, such as BMS-202 and BMS-8 (Fig. 1) [25,26].
Incyte corporation retained the biphenyl part and replaced the
methylene ether structure with the amide bond (Fig. 1), showing
significant inhibitory activity against PD-1/PD-L1 binding with IC50
values in the nanomolar range [27,28]. It is not difficult to find that
the small molecule inhibitors with such triaryl scaffold are mostly
composed of biarylcore group, aryl group, linker connecting them,
and tail [29e31]. Interestingly, as the bridge between the biaryl
core and the aryl ring, the linker does not show the key interaction
with PD-L1 in some crystal structures and docking models [32e34].
However, the effect of different linkers on the activity of inhibitors
is significant, which gives rise to our interest in this part. Moreover,
recent studies have shown that a series of C2-symmetric molecules
could induce the dimerization of PD-L1, resulting in an antiparallel
In this study, we found that the linker seemed not necessary for
the blocking efficiency of PD-1/PD-L1 interaction. The
“linker þ aryl” motif can be replaced with a six-membered aro-
matic ring, and the resulted compounds maintained satisfactory
activities. Moreover, we designed and synthesized a series of C2
symmetric compounds based on our newly found inhibitors, and
some of the compounds displayed promising inhibitory activity
against the PD-1/PD-L1 interaction with IC50 value at the single
nanomolar range. The cell-based PD-1/PD-L1 blockade bioassays
indicated that these C2-symmetric molecules could significantly
inhibit the PD-1/PD-L1 interaction at the cellular level and restore
the immune function of T cells at the safety concentrations. The
discovery of these phenyl-linked symmetric small molecules
showed the potential of simplified-linker and C2-symmetric strat-
egy, providing a basis for the development of symmetric small
molecule inhibitors of PD-1/PD-L1 interaction.
2. Results and discussion
2.1. Design strategy
Through the summary of several previous research [29e31,35],
we found that most triaryl scaffold small molecules with good PD-
L1 binding ability shared common pharmacophore characteristics.
The aryl region contributed significantly to the binding of inhibitors
through hydrophobic interactions, such as
p-p, p-alkyl and p-s
interactions (Fig. 2A). As the bridge between the biaryl core and the
aryl ring, the linker did not show the key interaction with PD-L1 in
some crystal structures (Fig. 2A) [32e35]. However, the effect of
different linkers on the activity of inhibitors is significant, which
gave rise to our interest in this part. Therefore, a classic triaryl
scaffold molecule (A1) from Incyte Corporation [28] was employed
as a lead compound, and the exploration of the “linker þ aryl” motif
was carried out in this research (Fig. 2B). Considering the advan-
tages of C2-Symmetric strategy in enhancing the ability of small
molecules to inhibit the PD-1/PD-L1 interaction, it was applied in
the optimization and the activity influenced by tail was
investigated.
Fig. 1. Representative small molecule inhibitors targeting PD-L1.
2

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Fig. 2. (A) Crystal structures of classic triaryl scaffold inhibitor (BMS-1001, PDB ID: 5NIU) and C2-symmetric inhibitor (PDB ID: 6RPG) binding to PD-L1 dimeric; (B) The design
strategy of the novel phenyl-linked symmetric small molecules as inhibitors of the PD-1/PD-L1 interaction.
2.2. Chemistry
intermediate 3a-3d was obtained by Suzuki coupling of (1,4-
Benzodioxan-6-yl)boronic acid (1) with different substituted bro-
mobenzene (2a-2d). The intermediate 5 was prepared from 4-
bromobenzaldehyde by Suzuki coupling with borate ester.
Almost all series of compounds involve several important in-
termediates, the synthesis of which was shown in Scheme 1. The
3

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Scheme 1. Reagents and conditions: (a) XPhos-Pd G2, K3PO4, THF; (b) Pd (dppf)Cl2, KOAc, Bis(pinacolato)diboron, 1,4-dioxane; (c) NaH, CH3I, DMF; (d) DIPEA, HBTU, corresponding
acid fragments, DCM,; (e) NaBH3CN, HAc, 2-aminoethanol, DCM,; (f) LiOH, THF/H2O; (g) Et3N, 2-aminoethanol, DCM,; (h) i) intermediate 3b, EtOH; ii) TMSCF3, TBAT, THF; iii) HCl,
MeOH; (i) Pd(PPh3)4, K2CO3, intermediate 5, THF/H2O,; (j) Pd(PPh3)4, X-Phos, Cs2CO3, intermediate 3a, 1,4-dioxane; (k) (n-Bu)4NHSO4, NaOH, 4-bromomethylbenzoyl chloride, DCM.
We designed compounds A2-A12 to explore the linker's role in
the interaction with PD-L1. The synthesis of compounds A1-A12 is
shown in Scheme 1. Carboxyl and sulfonic acid substituted aromatic
hydrocarbons 6a-6d, and intermediate 3b was condensed and then
obtained A1-A3 and A5 via a reductive amination reaction. Com-
pound A4 was obtained from the intermediate 3b by amino
methylation, condensation, and reductive amination reaction. The
intermediate 3c was hydrolyzed to obtain 10 and condensed with
4-aminobenzyl alcohol to obtain 11. The intermediate 11 was
formed into benzyl bromide, and then nucleophilic substitution
was carried out to obtain A6. 4-(diethoxymethyl)benzaldehyde (12)
and intermediate 3b were dehydrated to obtain enamine, which
reacted with TMSCF3, and then via de-protection and reductive
amination reaction to obtain A7. Bromine and amino-substituted
pyridine (14a, 14b) were coupled with intermediate 5 to obtain
15a and 15b and then reacted with intermediates 3a to yield 16a
and 16b by the classical Suzuki-Miyaura coupling reaction. Through
the Na(CN)BH3-mediated reductive amination reaction, 16a, and
16b were converted to the desired compounds A8-A9. Intermediate
17a-17c reacted with (1,4-benzodioxan-6-yl)boronic acid (1) to
yield 18a-18c by coupling reaction and reacted with acyl chloride to
obtain the intermediate 19a-19c, and finally, nucleophilic substi-
tution was carried out to obtain A10-A12.
B1eB12 was designed for further optimization to validate linker
and aryl group interact with PD-L1 as a whole motif, while
B13eB16 was designed to explore the influence of conformational
constraints on tails. The synthesis route of compounds B1eB7 is
shown in Scheme 2. The intermediate 21 was obtained by coupling
reaction, which was oxidized to 22. Then the intermediate 23 was
obtained by CeN coupling with the intermediate 3a. In the last step,
the desired compounds B1 were prepared from 23 under Na(CN)
BH3-mediated reductive amination conditions as described above.
4