Discovery of the programmed cell death-1/programmed cell death-ligand 1 interaction inhibitors bearing an indoline scaffold

Article abstract of DOI:10.1016/j.ejmech.2019.111856

Inhibiting the programmed cell death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1) pathway is an attractive strategy for tumor immunotherapy. Here, a novel series of indoline-containing compounds were developed, among which, A13 was identified as the most promising PD-1/PD-L1 pathway inhibitor. At the biochemical level, A13 demonstrated strong inhibition of the PD-1/PD-L1 interaction, with an IC₅₀ of 132.8 nM. Notably, it exhibited outstanding immunoregulatory activity, and significantly elevated interferon-γ secretion in a Hep3B/OS-8/hPD-L1 and CD3 T cell co-culture model, without significant toxic effect. Therefore, A13 could be employed as a suitable lead compound for further design of non-peptide inhibitors targeting the PD-1/PD-L1 interaction. In addition, the preliminary structure-activity relationships of these new indoline compounds were investigated in this study, providing valuable information for future drug development.

Full text of DOI:10.1016/j.ejmech.2019.111856

Journal Pre-proof
Discovery of the programmed cell death-1/programmed cell death-ligand 1 interaction
inhibitors bearing an indoline scaffold
Mingze Qin, Qi Cao, Xia Wu, Chunyang Liu, Shuaishuai Zheng, Hongbo Xie, Ye Tian,
Jun Xie, Yanfang Zhao, Yunlei Hou, Xian Zhang, Boxuan Xu, Haotian Zhang, Xiaobo
Wang
PII:
S0223-5234(19)31008-6
DOI:
https://doi.org/10.1016/j.ejmech.2019.111856
EJMECH 111856
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 August 2019
Revised Date: 2 November 2019
Accepted Date: 4 November 2019
Please cite this article as: M. Qin, Q. Cao, X. Wu, C. Liu, S. Zheng, H. Xie, Y. Tian, J. Xie, Y. Zhao,
Y. Hou, X. Zhang, B. Xu, H. Zhang, X. Wang, Discovery of the programmed cell death-1/programmed
cell death-ligand 1 interaction inhibitors bearing an indoline scaffold, European Journal of Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.ejmech.2019.111856.
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Discovery of the Programmed Cell Death-1/Programmed Cell
Death-Ligand 1 Interaction Inhibitors Bearing an Indoline Scaffold
a, d, *
a
a
a
a
Mingze Qin
, Qi Cao , Xia Wu , Chunyang Liu , Shuaishuai Zheng , Hongbo
c
a
b
a
a
a
a
Xie , Ye Tian , Jun Xie , Yanfang Zhao , Yunlei Hou , Xian Zhang , Boxuan Xu ,
b, ***
d, **
Haotian Zhang
, Xiaobo Wang
a
Key Laboratory of Structure-Based Drug Design and Discovery, Ministry of
Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China
b
Department of Pharmacology, Shenyang Pharmaceutical University, Shenyang
1
10016, PR China
c
College of Bioinformatics Science and Technology, Harbin Medical University,
Harbin 150086, PR China
d
Chinese People’s Liberation Army Logistics Support Force No. 967 Hospital,
Dalian 116021, PR China
∗
Corresponding author.
∗
∗
∗
Corresponding author.
∗ ∗
Corresponding author.
E-mail address: qinmingze001@126.com (Mingze Qin); wxbbenson0653@sina.com
Xiaobo Wang); zhanghaotian2087@163.com (Haotian Zhang)
(

ABSTRACT
Inhibiting the programmed cell death-1 (PD-1)/programmed cell death-ligand 1
PD-L1) pathway is an attractive strategy for tumor immunotherapy. Here, a novel
(
series of indoline-containing compounds were developed, among which, A13 was
identified as the most promising PD-1/PD-L1 pathway inhibitor. At the biochemical
level, A13 demonstrated strong inhibition of the PD-1/PD-L1 interaction, with an IC50
of 132.8 nM. Notably, it exhibited outstanding immunoregulatory activity, and
significantly elevated interferon-γ secretion in a Hep3B/OS-8/hPD-L1 and CD3 T cell
co-culture model, without significant toxic effect. Therefore, A13 could be employed
as a suitable lead compound for further design of non-peptide inhibitors targeting the
PD-1/PD-L1 interaction. In addition, the preliminary structure-activity relationships
of these new indoline compounds were investigated in this study, providing valuable
information for future drug development.
Key words: tumor immunotherapy; PD-1/PD-L1 interaction inhibitor; low toxicity;
IFN-γ secretion.

1
. Introduction
Immunotherapy is an emerging therapeutic strategy in tumor treatment [1,2]. The
process of immune surveillance is modulated by several co-stimulatory molecules, as
well as co-inhibitory molecules, whose activation leads to immunosuppressive effects
such as T-cell proliferation and cytokine secretion downregulation [3,4]. Under
physiological conditions, this negative feedback regulation of immune response plays
a critical role in maintaining self-tolerance and protecting against autoimmunity [5].
Programmed cell death-1 (PD-1) is a well-documented immune checkpoint
protein expressed on T cells, B cells, natural killer cells, and dendritic cells; its
cognate ligand PD-L1 is highly expressed in several types of malignancies, such as
non-small cell lung cancer (NSCLC), renal cancer, and melanoma [6–10]. Thus, the
abnormal activation of the PD-1/PD-L1 pathway results in tumor-specific immune
cells exhaustion [11,12].
Humanized monoclonal antibodies (mAbs) which target the PD-1/PD-L1 axis
have exhibited durable antitumor responses in clinical application, and six of them
have been approved for tumor treatment by the U.S. Food and Drug Administration
(FDA). Additionally, combinations of these anti-PD-1/PD-L1 mAbs with other
antitumor agents such as chemotherapeutics, targeted drugs, and antibodies targeting
other immune checkpoints, have been extensively investigated [13–16]. The FDA
recently approved the combination of pembrolizumab with pemetrexed and
carboplatin as first-line therapy for metastatic NSCLC patients, providing a valuable
therapeutic regimen [17]. Despite the successful application of anti-PD-1/PD-L1

mAbs, their intrinsic disadvantages such as severe immune-related adverse effects and
poor drug diffusion prompted the development of small-molecule inhibitors [18].
Figure 1. Chemical structures of representative non-peptide inhibitors of the
PD-1/PD-L1 interaction.
Recently, Bristol-Myers Squibb (BMS) investigators developed a series of
non-peptide inhibitors with a tricyclic scaffold such as compounds 1, 2, and 3 (Fig. 1)
[
19,20]. It has been reported by the Holak group that compound 2 induces PD-L1
dimerization, and thus inhibits the PD-1/PD-L1 interaction [21]. The structural
characterization of 3 revealed that its 2,3-dihydro-1,4-benzodioxinyl group induced a
conformational change involving the Tyr56 of one PD-L1 protein, forming an
enlarged hydrophobic tunnel for inhibitor binding [21]. However, inhibitor 3 failed to
antagonize the repression of PD-1/PD-L1 pathway on T cell activation in the cellular
assay [22]. Compound 4, which contained an additional 3-cyanobenzyl moiety, was a
more promising inhibitor. It displayed a high level of biochemical and cellular activity,
and further investigation on it is awaited. In addition to the BMS inhibitors, Incyte
Corporation researchers developed several compounds characterized by an amide
linkage group such as 5 and 6, which potently inhibited the PD-1/PD-L1 interaction at

the biochemical level [23,24]. However, their effects on immunity in response to
tumor need to be further explored in experimental models. Notably, more
comprehensive investigations addressing the toxic, pharmacokinetic, and
pharmacodynamic properties of these reported inhibitors are necessary. In addition,
the identification of novel inhibitors, especially those with verified activity on
immune activation is urgently needed.
In this paper, the discovery of a novel series of indoline-containing inhibitors
targeting the PD-1/PD-L1 interaction is reported. Particularly, compound A13
displayed outstanding inhibition of the PD-1/PD-L1 interaction and restoration of the
immune response repression, making it a promising lead compound for future drug
development.
2
. Results and discussion
2
.1. Chemistry
The synthesis of compounds A1–4 is shown in Scheme 1. The key intermediates,
a and 9b, which contain an isoindolin-1-one scaffold, were prepared through the free
9
radical-mediated bromination of the starting reagents 7a and 7b, followed by
cyclization with 2-methyl-[1,1'-biphenyl]-3-amine [25,26]. The Suzuki–Miyaura
coupling of 9a and 9b with 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane yielded
1
0a and 10b, which were oxidized by NaIO to form the aldehydes 11a and 11b,
4
respectively [23]. Through the NaBH CN-mediated reductive amination reaction, 11a
3
and 11b were converted to the desired compounds, A1–4 [27].

Scheme 1. Reagents and conditions: (a) NBS, BPO, CCl , reflux, 10 h, 67–71%; (b)
4
2
4
1
-methyl-[1,1'-biphenyl]-3-amine, HOAc, 135 °C, 12 h, 73–75%; (c)
,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane, PdCl (dppf), K CO ,
,4-dioxane/H O (4/1), 60 °C, 9 h, 54–61%; (d) OsO , NaIO , 1,4-dioxane/H O (5/1),
2
2
3
2
4
4
2
rt, 2 h, 90–93%; (e) appropriate amine, HOAc, NaBH CN, CH OH/CH Cl (1/1), rt,
3
3
2
2
7
–22 h, 7–16%.
The preparation of compounds A5–8, A11–14, and A21–31 is shown in Scheme
. The classical Suzuki–Miyaura coupling of 12 with phenylboronic acid or
-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane
2
2
yielded 13a and 13b, which were then reduced by NaBH CN to generate 14a and
3
1
4b, respectively. The amidation of 14a and 14b with appropriate carboxylic acids
readily afforded 15a–c and 15f, and the O-alkylation of the intermediates 15c and 15f,
which contain a hydroxyl group, provided good yields of the intermediates 15d, 15e,
and 15g [24,28]. The desired compounds A5–8, A11–14, and A21–31 were then
produced by reacting 15a–g with appropriate amines in the presence of NaBH CN.
3

Scheme 2. Reagents and conditions: (a) phenylboronic acid, Pd(PPh ) , K CO ,
3
4
2
3
1
,4-dioxane/H O
(4/1),
60
°C,
10
h,
73%,
or
2
2
-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane,
PdCl (dppf), K CO , 1,4-dioxane/H O (2/1), 100 °C, 2 h, 70%; (b) NaBH CN, HOAc,
2
2
3
2
3
rt, 2 h, 73%–80%; (c) appropriate benzoic acids, HATU, DIPEA, DMF, rt, 3 h, 81%–
6%, or 4-formyl-3-hydroxybenzoic acid, EDCI, CH Cl , rt, 3 h, 72%–81%; (d) CH I,
8
2
2
3
K CO , DMF, rt, 2 h, 76%–87%; (e) 3-(bromomethyl)benzonitrile, Cs CO , DMF, 75
2
3
2
3
°
C, 2 h, 74%; (f) appropriate amine, HOAc, NaBH CN, CH OH/CH Cl (1/1) or
3 3 2 2
DMF, rt, 8–20 h, 3–21%.
Scheme 3 shows the synthetic route of compounds A9, A10, and A15–20. The
amides 16a–d were generated via a method similar to that used to synthesize 15a, by
reacting 4-phenylindoline (14a) with appropriate carboxylic acids. Subsequently, the
intermediates 16a–d were converted to the desired compounds A9, A10, and A15–20,
through a three-step reaction including the Suzuki–Miyaura coupling, olefin
oxidization, and reductive amination with appropriate amines.

Scheme 3. Reagents and conditions: (a) appropriate benzoic acids, HATU, DIPEA,
DMF, rt, 3 h, 78%–87%; (b) 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane,
PdCl (dppf), K CO , 1,4-dioxane/H O (4/1), 100 °C, 7 h, 36%–45%, or
2
2
3
2
4
,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane,
dicyclohexyl(2',6'-dimethoxy-[1,1'-biphenyl]-2-yl)phosphine,
toluene/EtOH/H O (3/3/1), 90 °C, 5 h, 53%. (c) OsO , NaIO , 1,4-dioxane/H O (5/1),
Pd (dba) ,
2 3
K PO ,
3
4
2
4
4
2
rt, 2 h, 82–91%; (d) appropriate amine, HOAc, NaBH CN, CH OH/CH Cl (1/1), rt,
3
3
2
2
1
2
2
0–19 h, 5–25%.
.2. Bioactivity and discussion
.2.1. Discovery of the indoline-containing compounds A5–8
The Holak group has elucidated the crystal structures of PD-L1 complexed with
BMS inhibitors, facilitating the development of inhibitors with a similar mechanism
of action [21,22,29]. Accordingly, a pharmacophoric model for non-peptide
PD-1/PD-L1 pathway inhibitors was established in our previous study [30].
Regarding this model, hydrophobic interactions such as the π-π interactions formed
between the “Core group” and “Aryl group” with PD-L1 residues significantly
contributed to inhibitor binding. A tail group with the ability to form hydrogen bonds

with PD-L1 was also required for high activity. Thus, the modification of the linkage
group was considered a feasible way to discover novel inhibitors. A ring fusion
strategy was utilized by taking compound 1 as the lead compound (Fig. 2).
Accordingly, different scaffolds, including isoindolin-1-one (mode a) and indoline
(mode b), were developed in the initial study. Based on them, compounds A1–8,
which have an aminoethanol or N-(2-aminoethyl)acetamide tail group incorporated at
different positions of the phenyl ring, were synthesized as proof-of-concept
compounds.
Figure 2. Design strategy of compounds A1–8.
The inhibitory activities of compounds A1–8 against the PD-1/PD-L1 interaction
were evaluated using the well-established homogenous time-resolved fluorescence
(
HTRF) assay. The biochemical data presented as IC values are shown in Table 1. In
50
general, the isoindolin-1-one derivatives A1–4, displayed unfavorable activity in
inhibition of the PD-1/PD-L1 interaction. Among them, compounds A1 (IC , 9378
5
0
nM) and A3 (IC , 7286 nM) exhibited weak activity, and A2 and A4 were almost
5
0
inactive at up to 10 µM concentration. Contrarily, promising activity was exhibited by

all the indoline-based compounds, A5–8, and their preliminary structure-activity
relationships (SARs) were determined. Compound A7, which bears an aminoethanol
tail group at the 4-position of the phenyl ring, exhibited strong activity, with an IC50
of 191.6 nM. Shifting its tail group from the 4- to 3-position yielded A5 (IC , 344
5
0
nM), which showed significantly reduced efficacy. Loss of activity could be observed
by comparing A6 (IC , 744.9 nM) with A8 (IC , 622.5 nM). These results clearly
5
0
50
indicated that the indoline could serve as a suitable scaffold for developing
PD-1/PD-L1 interaction inhibitors, and a hydrophilic tail group incorporated at the
4
-position of the phenyl ring was more desirable.
Table 1. Activities of compounds A1–8 in inhibition of the PD-1/PD-L1
interaction.
PD-1/PD-L1
Compound
R
a
IC (nM)
5
0
A1
A2
A3
A4
9378 ± 465.7
>10000
6
-
-
6
-
7286 ± 397.5
>10000
5
5
-

A5
A6
A7
A8
344 ± 35.8
744.9 ± 56.3
191.6 ± 10.2
622.5 ± 29.5
3
-
-
3
-
4
4
-
a
The data are generated from two independent experiments.
2
.2.2. Structural optimization of the indoline precursors
Based on these new indoline precursors, a detailed structural optimization
campaign was performed to further explore their SARs and improve their potency.
BMS inhibitor 1 was used as the positive control. It exhibited high level of activity in
inhibition of the PD-1/PD-L1 interaction, with an IC of 91.8 nM (Table 2).
5
0
First, compounds A9–20, which have a modified phenyl ring, were synthesized
and evaluated to explore the SARs of this region. It was observed that the introduction
of neither an electron-donating, nor electron-withdrawing group, led to a significant
effect on inhibitory activity against the PD-1/PD-L1 interaction. Compound A13
(
IC , 132.8 nM), which contains a methoxy group, displayed slightly more potent
50
activity as compared with A7 (IC , 191.6 nM), while compounds A9 (IC , 212.3
5
0
50
nM), A11 (IC , 304.4 nM), and A15 (IC , 353.1 nM), with a methyl, hydroxyl, and
5
0
50
chlorine group, respectively, were slightly less active, indicating that the electron
density of the phenyl ring was not a determinant of inhibitory activity at the
biochemical level. Shifting the chlorine group from the R - (A15) to R -position
2
1
(
A17, IC , >10000 nM) dramatically reduced the activity. The new R substituent
5
0
1

was probably sterically repulsive to the hydrogen atom at the 2’-position of indoline
moiety, leading to the formation of an unfavorable molecular conformation. These
results suggested that further modification of this region was inappropriate. Among
these compounds, A13, which has a methoxy group, exhibited the most promising
activity. Surprisingly, with an additional methoxy group at the R -position, A19,
3
showed remarkably decreased activity (IC , 4766 nM).
5
0
Compound A14 (IC , 219.7 nM), which contains a N-(2-aminoethyl)acetamide
5
0
group, was less active than A13, which has an aminoethanol tail group. Decrease in
activity was also observed when A10 (IC , 457.1 nM), A12 (IC , 601.5 nM), and
5
0
50
A16 (IC , 589 nM) were compared with A9 (IC , 212.3 nM), A11 (IC , 304.4 nM),
5
0
50
50
and A15 (IC , 353.1 nM), respectively, indicating that the aminoethanol fragment
5
0
might be one of the preferred moieties for the tail group region.
Table 2. Activities of compounds A9–20 in inhibition of the PD-1/PD-L1
interaction.
PD-1/PD-L1
Compound
R1
R2
R3
R
a
IC (nM)
5
0
A9
H
H
CH
CH
3
3
H
H
212.3 ± 16.2
457.1 ± 18.9
A10

A11
A12
A13
A14
A15
A16
A17
A18
A19
H
H
H
H
H
H
Cl
Cl
H
H
OH
OH
OCH
OCH
Cl
H
H
304.4 ± 27
601.5 ± 30.2
132.8 ± 6.9
219.7 ± 7.6
353.1 ± 39.3
589 ± 29.6
>10000
H
3
3
H
H
Cl
H
H
H
H
H
>10000
OCH
OCH
OCH
OCH
4766 ± 212.3
3
3
3
3
A20
1
7258 ± 297.5
91.8 ± 8.6
a
The data are generated from two independent experiments.
With the preliminary SARs determined, compounds A21–25 were then prepared
to explore the effects of the tail group on their PD-1/PD-L1 interaction inhibitory
activity (Table 3). The methylation of the hydroxyl group of A13 decreased the ability
of the compound to inhibit the PD-1/PD-L1 interaction (A21, IC , 349.7 nM).
5
0
Compound A22, which has a hydroxymethyl group attached to the pyrrolidine
moiety, showed a 4.67-fold decrease in activity as compared with A13, suggesting
that the orientation of the tail group played a crucial role on inhibitor activity.

Compound A25 (IC , 277.6 nM), which bears a serine tail group, exhibited favorable
5
0
activity, but was still less potent than A13. Disappointingly, compounds A23 (IC ,
5
0
1
887 nM) and A24 (IC , 1059 nM), which have a proline and pipecolinic acid
50
moiety, respectively, exhibited sharply reduced efficacy.
Based on previous reports on compound 4, the introduction of the 3-cyanobenzyl
moiety resulted in additional π-stacking and hydrogen bond interactions with the
dimeric PD-L1, creating an improved ligand binding [22]. Compounds A26–29 were
prepared to examine whether the 3-cyanobenzyl group was also tolerated by the
newly synthesized indoline-based compounds. The results revealed that all these
compounds displayed significantly decreased activity (IC values: 572.1–3281 nM)
5
0
as compared to their parent compounds (A13, A14, A24, and A25).
It has been proven that the 2,3-dihydro-1,4-benzodioxinyl group is desirable as
terminal moiety in BMS inhibitors and their structural analogues, which induced the
conformational change of Tyr56 to form an enlarged hydrophobic tunnel [21]. In this
new chemical series, compounds A30 (IC , 225.2 nM) and A31 (IC , 415.9 nM),
5
0
50
which contain a 2,3-dihydro-1,4-benzodioxinyl group, also exhibited attractive
activity against the PD-1/PD-L1 interaction, but were less potent than the phenyl
compounds A13 and A14, respectively.
Table 3. Activities of compounds A21–31 in inhibition of the PD-1/PD-L1
interaction.

PD-1/PD-L1
Compound
R4
Ph
R2
R
a
IC (nM)
5
0
A21
OCH
3
3
349.7 ± 17.5
619.9 ± 47
A22
A23
Ph
Ph
OCH
OCH
1887 ± 132.2
3
A24
A25
A26
Ph
Ph
Ph
OCH
OCH
3
3
1059 ± 26.5
277.6 ± 19.7
572.1 ± 29
A27
A28
A29
Ph
Ph
Ph
1064 ± 85.5
1330 ± 36.7
3281 ± 226.5
A30
A31
OCH
OCH
3
3
225.2 ± 25.6
415.9 ± 11.5

1
91.8 ± 8.6
a
The data are generated from two independent experiments.
2
.2.3. Unspecific toxicity of selected inhibitors
The immunoregulatory activity of the PD-1/PD-L1 interaction inhibitors is
strongly affected by their toxicity. Thus, compounds A7, A9, A13, A14, and A30,
which showed promising activity at the biochemical level, were evaluated for toxicity,
using the CCK-8 assay. Jurkat T cells which were proven to be favorable surrogates
of primary T cells in the analysis of immune toxicity, were treated with the indicated
concentrations of the tested compounds for 72 h. The results demonstrated that
compounds A13 and A14 displayed lower toxicity as compared with A7, A9, and A30,
with EC values of 44.7 and 39.8 µM, respectively (Table S1).
50
2
.2.4. Immunoregulatory activity based on IFN-γ secretion
Combining inhibitor activity and toxicity, A13 and A14 were further assessed for
ability to restore the immunity repressed by the PD-1/PD-L1 pathway activation.
Hep3B cells, which were engineered to stably express OS-8 (anti CD-3 single-chain
variable fragment) and human PD-L1, were co-cultured with primary CD3 T cells.
The indicated concentrations of A13 and A14 were added, and interferon-γ (IFN-γ)
levels were determined after 72 h of co-culture. As shown in Figure 3, both
compounds A13 and A14 promoted IFN-γ secretion in a dose-dependent manner.
Particularly, outstanding activity was exhibited by A13, which significantly promoted
IFN-γ secretion even at the lowest concentration. Upon treatment with 5 µM A13, the
IFN-γ level was increased by 316.59%, which was 3.21-fold more remarkable than

that induced by 1 (98.69%). At a 20 µM concentration, the IFN-γ level was increased
by 609.61%.
Figure 3. Effects of selected compounds on IFN-γ production in
a
Hep3B/OS-8/hPD-L1 and CD3 T cell co-culture model. Data are shown as mean ±
SEM, *p<0.05, **p<0.01, *** p<0.001 vs control group.
2
.2.5. Docking analysis of A13 with the dimeric PD-L1
To elucidate the binding mode of these indoline-based inhibitors, the docking
analysis of A13 with the dimeric PD-L1 was conducted. The 2/PD-L1 complex (PDB:
N2D) was employed as the template. As shown in Figure 4A, A13 matched well to
5
the hydrophobic cleft formed by the PD-L1 dimer. For the distal phenyl and
methoxy-phenyl moieties, π−π interactions with the Tyr56 of each monomer, were
formed, which provided strong stabilization for inhibitor binding. Other interactions
such as π−alkyl and π−anion interactions with Met115 and Asp122 also contributed
to this binding mode. The aminoethanol fragment interacted with Asp122 and Lys124

via hydrogen bonds. Notably, the hydrogen bond formed between the hydroxyl group
and Asp122 indicated that the hydrogen bond donor at the tail group is necessary for
high activity. This observation accounted for the decrease in activity as comparing
A21 with A13. The indoline moiety of A13 created hydrophobic interactions with
Met115 and Ala121, similar with those in the methyl-phenyl group of 2. Additionally,
the indoline moiety interacted with the Tyr123 of PD-L1, which might help to
stabilize the conformation of the inhibitor. Figure 4B demonstrates that compounds
A13 and 2 were well superimposed.
Figure 4. (A) Docking analysis of A13 in the hydrophobic cleft formed by the
dimeric PD-L1. (B) Binding overlap of A13 and 2 in the binding site. The crystal
structure of the dimeric PD-L1 protein was taken from the RCSB Protein Data Bank
(PDB: 5N2D).
3
. Conclusion
Inhibiting the PD-1/PD-L1 interaction using non-peptide small-molecules is of
great potential in tumor treatment, but none of them has entered into clinical trials yet.
The inhibitors discovered by BMS investigators provide a favorable starting point for
the identification of inhibitors with a similar mode of action. Inhibitors with potent

activity, but containing a novel skeleton are eagerly needed, considering that they may
possess distinct pharmacokinetic and pharmacodynamic profiles from the reported
compounds. In this study, a series of novel indoline derivatives were developed from
1
, using a ring fusion strategy. Most of them potently inhibited the PD-1/PD-L1
interaction, with IC values at the nanomolar level, clearly indicating that indoline is
5
0
a suitable scaffold for designing an inhibitor. Among these compounds, A13
displayed the strongest activity, with an IC50 of 132.8 nM. Furthermore, in a
Hep3B/OS-8/hPD-L1 and CD3 T cells co-culture model, it significantly increased
IFN-γ secretion in a dose-dependent manner. Additionally, preliminary SAR studies
were performed on these indoline-based compounds, paving the way for further
development of their structural analogues.
In this study, A13 was slightly less active than the BMS inhibitor 1 in inhibition
of the PD-1/PD-L1 interaction in the HTRF assay, but showed significantly more
potent immunoregulatory activity in a T cell-tumor co-culture model, highlighting
their potential for further investigations. These observations might be partially
ascribed to its low toxicity, which was experimentally addressed using Jurkat T cells.
The future studies should focus on structural optimization of A13. Most
especially, it is necessary to identify its analogues possessing higher affinity with the
dimeric PD-L1, and thus, an improved immunoregulatory activity can be expected.
For the tail group, detailed structural modifications should be made when reserving a
hydrogen bond donor. Physicochemical properties such as solubility can also be tuned
by modifying this fragment. In addition, further investigation on the distal phenyl and

methoxy-phenyl moieties is desirable, diverse heterocyclic fragments may also be
well tolerated in each part of the inhibitor.
In conclusion, this study identified the indoline-based compound A13, as a
promising lead compound in the designing of the PD-1/PD-L1 interaction inhibitors,
facilitating future drug design.
4
. Experimental procedures
4
.1. Chemistry
Reagents and solvents were obtained from commercial sources and used without
further purification. All the reactions were monitored by TLC using silica gel GF/UV
54. Flash chromatography was performed using silica gel (300–400 mesh). Melting
points were determined on a Büchi Melting Point B-540 apparatus (Büchi
2
1
13
Labortechnik, Flawil, Switzerland). The H and C NMR spectra were recorded on a
Bruker AV-400 spectrometer, with TMS as an internal standard. The low resolution
of ESI-MS was recorded on an Agilent 1100 LC-MS spectrometer, and
high-resolution mass spectrometry was performed on an Agilent Accurate-Mass
Q-TOF 6530 in ESI mode. The purities of A1–31 were determined by HPLC, which
were determined to be higher than 95%. The HPLC analysis was performed on an
Agilent 1260 machine with a C18 reverse-phase column (4.6 mm × 250 mm, 5 µm),
the column temperature was 25 °C. Mobile phase A (100% methanol) and mobile
phase B (NaH PO and H PO buffer solution, pH = 2.5) were used in a gradient
2
4
3
4
elution program (0 min, phase A: 5%, phase B: 95%; 25 min, phase A: 95%, phase B:
%) with a flow rate of 1.0 mL/min. UV Detection was conducted at 230 nm.
5

4
4
5
.1.1. General procedure for preparation of intermediates 8a and 8b
.1.1.1. Ethyl 5-bromo-2-(bromomethyl)benzoate (8a). To a solution of ethyl
-bromo-2-methylbenzoate (7a, 10.53 g, 43.51 mmol) and BPO (0.21 g, 0.87 mmol)
in CCl (80 mL) was added NBS (8.08 g, 45.67 mmol) in portions. The mixture was
4
stirred under reflux for 10 h. When TLC showed the completion of the reaction, the
precipitate was filtered off and the filtrate was evaporated to give a residue, which
was purified by column chromatography (petroleum ether) to afford compound 8a
1
(
9.87 g, 70.9%). It was obtained as a white solid. H NMR (600 MHz, DMSO-d ) δ
6
7
.98 (d, J = 2.2 Hz, 1H), 7.82 (dd, J = 8.2, 2.2 Hz, 1H), 7.57 (d, J = 8.3 Hz, 1H), 4.98
+
(
s, 2H), 4.34 (t, J = 7.1 Hz, 2H), 1.35 (m, 3H). ESI-MS m/z: 320.9 [M+H] .
4
.1.1.2. Ethyl 4-bromo-2-(bromomethyl)benzoate (8b). Compound 8b was prepared
from intermediate ethyl 4-bromo-2-methylbenzoate (7b) in a similar manner as
1
described for compound 8a. It was obtained as a white solid. Yield, 67%. H NMR
(
600 MHz, DMSO-d ) δ 7.88 (s, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.69 (d, J = 8.3 Hz,
6
1
3
4
4
H), 4.99 (s, 2H), 4.34 (q, J = 6.7 Hz, 2H), 1.34 (t, J = 6.7 Hz, 3H). ESI-MS m/z:
+
20.9 [M+H] .
.1.2. General procedure for preparation of intermediates 9a and 9b
.1.2.1. 6-Bromo-2-(2-methyl-[1,1'-biphenyl]-3-yl)isoindolin-1-one (9a). A mixture of
intermediate 8a (6.39 g, 19.97 mmol) and 2-methyl-[1,1'-biphenyl]-3-amine (2.81 g,
5.35 mmol) in acetic acid (30 mL) was stirred at 135 °C for 12 h. When TLC
1
showed the completion of the reaction, the reaction mixture was cooled to room
temperature and poured into water. The precipitate was filtered off to give a residue,

which was triturated with ether to afford compound 9a (4.24 g, 73.3%). It was
1
obtained as a white solid. H NMR (600 MHz, DMSO-d ) δ 7.93 (d, J = 1.7 Hz, 1H),
6
7
.87 (dd, J = 8.1, 1.8 Hz, 1H), 7.66 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.5 Hz, 2H), 7.45
(d, J = 8.7 Hz, 1H), 7.42–7.38 (m, 2H), 7.37–7.35 (m, 2H), 7.28 (d, J = 7.5 Hz, 1H),
+
4
4
9
9
7
2
2
4
4
.88 (s, 2H), 2.02 (s, 3H). ESI-MS m/z: 378.0 [M+H] .
.1.2.2. 5-Bromo-2-(2-methyl-[1,1'-biphenyl]-3-yl)isoindolin-1-one (9b). Compound
b was prepared from intermediate 8b in a similar manner as described for compound
1
a. It was obtained as a white solid. Yield, 75%. H NMR (600 MHz, DMSO-d ) δ
6
.95 (s, 1H), 7.76 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.1 Hz, 1H), 7.48 (t, J = 7.5 Hz,
H), 7.44 (d, J = 7.8 Hz, 1H), 7.42–7.35 (m, 4H), 7.28 (d, J = 7.5 Hz, 1H), 4.90 (s,
+
H), 2.02 (s, 3H). ESI-MS m/z: 378.1 [M+H] .
.1.3. General procedure for preparation of intermediates 10a and 10b
.1.3.1. 2-(2-Methyl-[1,1'-biphenyl]-3-yl)-6-vinylisoindolin-1-one (10a). A mixture of
intermediate 9a (3.26 g, 8.65 mmol), 4,4,5,5-tetramethyl-2-vinyl-1,3,2-dioxaborolane
2.67 g, 17.32 mmol), PdCl (dppf) (0.63 g, 0.86 mmol) and K CO (2.38 g, 17.26
(
2
2
3
mmol) in 1,4-dioxane (24 mL) and water (6 mL) was stirred at 60 °C for 9 h under the
N atmosphere. The reaction mixture was cooled to room temperature, the catalyst
2
was filtered off. The filtrate was diluted with water and extracted with CH Cl . The
2
2
organic layer was washed with brine, and concentrated to give a residue, which was
purified by column chromatography (ethyl acetate/petroleum ether, 1/35 to 1/20) to
1
yield 10a (1.53 g, 54.5%). It was obtained as a white solid. H NMR (600 MHz,
DMSO-d ) δ 7.88 (s, 1H), 7.80 (dd, J = 7.9, 1.4 Hz, 1H), 7.65 (d, J = 7.9 Hz, 1H),
6

7
1
2
4
1
.51–7.43 (m, 3H), 7.42–7.34 (m, 4H), 7.27 (dd, J = 7.6, 1.0 Hz, 1H), 6.90 (dd, J =
7.7, 11.0 Hz, 1H), 6.00 (d, J = 17.7 Hz, 1H), 5.36 (d, J = 11.1 Hz, 1H), 4.89 (s, 2H),
+
.03 (s, 3H). ESI-MS m/z: 326.2 [M+H] .
.1.3.2. 2-(2-Methyl-[1,1'-biphenyl]-3-yl)-5-vinylisoindolin-1-one (10b). Compound
0b was prepared from intermediate 9b in a similar manner as described for
1
compound 10a. It was obtained as a white solid. Yield, 61%. H NMR (600 MHz,
DMSO-d ) δ 7.80–7.73 (m, 2H), 7.67 (d, J = 7.9 Hz, 1H), 7.51–7.43 (m, 3H), 7.42–
6
7
1
3
4
4
.34 (m, 4H), 7.27 (d, J = 7.5 Hz, 1H), 6.90 (dd, J = 17.6, 11.0 Hz, 1H), 6.03 (d, J =
7.7 Hz, 1H), 5.43 (d, J = 11.0 Hz, 1H), 4.89 (s, 2H), 2.03 (s, 3H). ESI-MS m/z:
+
26.2 [M+H] .
.1.4. General procedure for preparation of intermediates 11a and 11b
.1.4.1. 2-(2-Methyl-[1,1'-biphenyl]-3-yl)-3-oxoisoindoline-5-carbaldehyde (11a). To
a solution of intermediate 10a (0.6 g, 1.85mmol) in 1,4-dioxane (25 mL) and water (5
mL) was added aqueous solution of osmium tetraoxide (2% w/w in water, 3.5 mL,
0
.28 mmol). Stirring was continued at room temperature for 10 min, then sodium
periodate (1.58 g, 7.38 mmol) was added. The mixture was stirred at room
temperature for another 2 h. Upon the completion of the reaction, the reaction mixture
was poured into water. The precipitate was filtered off to give compound 11a (0.55 g,
1
9
1
0.9%). It was obtained as a gray solid. H NMR (600 MHz, DMSO-d ) δ 10.16 (s,
6
H), 8.32 (s, 1H), 8.20 (d, J = 9.0 Hz, 1H), 7.90 (d, J = 7.9 Hz, 1H), 7.48 (t, J = 7.5
Hz, 3H), 7.42–7.35 (m, 4H), 7.29 (d, J = 7.6 Hz, 1H), 5.03 (s, 2H), 2.04 (s, 3H).
+
ESI-MS m/z: 328.1 [M+H] .

4
.1.4.2. 2-(2-Methyl-[1,1'-biphenyl]-3-yl)-1-oxoisoindoline-5-carbaldehyde (11b).
Compound 11b was prepared from intermediate 10b in a similar manner as described
1
for compound 11a. It was obtained as an off-white solid. Yield, 93%. H NMR (600
MHz, DMSO-d ) δ 10.20 (s, 1H), 8.22 (s, 1H), 8.11 (d, J = 7.7 Hz, 1H), 8.01 (d, J =
6
7
2
4
4
.8 Hz, 1H), 7.49 (t, J = 7.3 Hz, 3H), 7.39 (m, 4H), 7.30 (d, J = 7.5 Hz, 1H), 5.02 (s,
+
H), 2.05 (s, 3H). ESI-MS m/z: 328.1 [M+H] .
.1.5. Procedure for preparation of intermediates 13a and 13b
.1.5.1. 4-Phenyl-1H-indole (13a). A mixture of intermediate 12 (10 g, 51.29
mmol), phenylboronic acid (6.26 g, 51.29 mmol), Pd(PPh ) (1.78g, 1.54 mmol) and
3
4
K CO (21.23 g, 153.94 mmol) in 1,4-dioxane (80 mL) and water (20 mL) was stirred
2
3
at 60 °C for 10 h under the N atmosphere. After that time, the reaction mixture was
2
cooled to room temperature, the catalyst was filtered off. The filtrate was
concentrated, and extracted with CH Cl . The organic layer was washed with brine,
2
2
and evaporated to give a residue, which was purified by column chromatography
(ethyl acetate/petroleum ether, 1/50 to 1/20) to afford intermediate 13a (7.25 g,
1
7
1
3.2%). It was obtained as a white solid. H NMR (600 MHz, DMSO-d ) δ 11.27 (s,
6
H), 7.67 (d, J = 7.3 Hz, 2H), 7.49 (t, J = 7.7 Hz, 2H), 7.42 (t, J = 5.9 Hz, 2H), 7.37
(t, J = 7.4 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 7.08 (d, J = 7.1 Hz, 1H), 6.55 (s, 1H).
+
ESI-MS m/z: 194.1 [M+H] .
4
.1.5.2. 4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-1H-indole (13b). A mixture of
intermediate 12 (1 g, 5.13
mmol), 2-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborol

ane (2.00 g, 7.63 mmol), PdCl (dppf) (0.37 g, 0.51 mmol) and K CO (2.1 g, 15.23
2
2
3
mmol) in 1,4-dioxane (10 mL) and water (5 mL) was stirred at 100 °C for 2 h under
the N atmosphere. The catalyst was filtered off, the filtrate was evaporated to give a
2
residue. The residue was dissolved in CH Cl and washed with water. The organic
2
2
layer was dried and evaporated to provide the crude product, which was purified by
column chromatography (ethyl acetate/petroleum ether, 1/100 to 1/15) to give
1
intermediate 13b (0.91 g, 70.7%). It was obtained as a white solid. H NMR (600
MHz, DMSO-d ) δ 11.22 (s, 1H), 7.40–7.33 (m, 2H), 7.12 (dt, J = 6.1, 4.6 Hz, 3H),
6
7
.00 (d, J = 7.1 Hz, 1H), 6.96 (d, J = 8.2 Hz, 1H), 6.51 (s, 1H), 4.29 (s, 4H). ESI-MS
+
m/z: 252.1 [M+H] .
4
.1.6. General procedure for preparation of intermediates 14a and 14b
4
.1.6.1. 4-Phenylindoline (14a). To a solution of intermediate 13a (1.5 g, 7.77 mmol)
in acetic acid (9 mL) was added NaBH CN (1.47 g, 23.32 mmol) slowly in an ice
3
bath. The mixture was stirred at room temperature for 2 h. After that time, the reaction
mixture was alkalified with aqueous NaOH and extracted with ethyl acetate. The
organic layer was washed with brine and concentrated to give a residue, which was
purified by column chromatography (ethyl acetate/petroleum ether, 1/60 to 1/15) to
1
give compound 14a (1.21 g, 79.8%). It was obtained as a white solid. H NMR (600
MHz, DMSO-d ) δ 7.47–7.40 (m, 4H), 7.36–7.29 (m, 1H), 7.00 (t, J = 7.7 Hz, 1H),
6
6
.59 (d, J = 7.1 Hz, 1H), 6.51 (d, J = 7.7 Hz, 1H), 5.59 (s, 1H), 3.39 (t, J = 8.4 Hz,
+
2
H), 2.97 (t, J = 8.4 Hz, 2H). ESI-MS m/z: 196.1 [M+H] .

4
.1.6.2. 4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)indoline (14b). Compound 14b was
prepared from intermediate 13b in a similar manner as described for compound 14a.
1
It was obtained as a white solid. Yield, 73%. H NMR (600 MHz, DMSO-d ) δ 6.96
6
(
t, J = 7.6 Hz, 1H), 6.91 (s, 1H), 6.89 (t, J = 6.4 Hz, 2H), 6.54 (d, J = 7.6 Hz, 1H),
.48 (d, J = 7.7 Hz, 1H), 5.68 (s, 1H), 4.26 (s, 4H), 3.37 (t, J = 8.3 Hz, 2H), 2.96 (t, J
8.3 Hz, 2H). ESI-MS m/z: 254.1 [M+H]+.
6
=
4
4
.1.7. General procedure for preparation of intermediates 15a–g
.1.7.1. 3-(4-Phenylindoline-1-carbonyl)benzaldehyde (15a). To a solution of
intermediate 14a (1 g, 5.13 mmol), 3-formylbenzoic acid (0.85 g, 5.64 mmol), and
HATU (2.92 g, 7.69 mmol) in DMF (15 mL) was added DIPEA (3.31 g, 25.63
mmol). The mixture was stirred at room temperature for 3 h. After that time, the
solution was poured into water. The precipitate was filtered off to give a residue,
which was triturated with ether to give compound 15a (1.37 g, 81.7%). It was
1
obtained as a white solid. H NMR (600 MHz, DMSO-d ) δ 10.09 (s, 1H), 8.13 (s,
6
1
H), 8.06 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 7.8 Hz, 1H), 7.75 (t, J = 7.6 Hz, 1H), 7.47
(dd, J = 7.9, 5.9 Hz, 5H), 7.41–7.36 (m, 1H), 7.32 (s, 1H), 7.13 (s, 1H), 4.05–3.98 (m,
+
2
H), 3.15 (t, J = 8.1 Hz, 2H). ESI-MS m/z: 328.1 [M+H] .
4
.1.7.2. 4-(4-Phenylindoline-1-carbonyl)benzaldehyde (15b). Compound 15b was
prepared from intermediate 14a in a similar manner as described for compound 15a,
with 4-formylbenzoic acid replace 3-formylbenzoic acid. It was obtained as a white
1
solid. Yield, 86%. H NMR (600 MHz, DMSO-d ) δ 10.10 (s, 1H), 8.04 (d, J = 8.1
6

Hz, 2H), 7.81 (d, J = 7.5 Hz, 2H), 7.52–7.32 (m, 7H), 7.13 (s, 1H), 3.35 (s, 2H), 3.14
+
(
t, J = 8.1 Hz, 2H). ESI-MS m/z: 328.1 [M+H] .
4
.1.7.3. 2-Hydroxy-4-(4-phenylindoline-1-carbonyl)benzaldehyde (15c). To a solution
of intermediate 14a (1.5 g, 7.69 mmol) and 4-formyl-3-hydroxybenzoic acid (1.34 g,
.07 mmol) in CH Cl (20 mL) was added EDCI (1.92 g, 10.00 mmol). The mixture
8
2
2
was stirred at room temperature for 3 h when TLC showed the completion of the
reaction. The reaction mixture was washed with water and brine. The organic layer
was dried and concentrated to give compound 15c (2.13 g, 80.7%). It was obtained as
1
a yellow solid. H NMR (600 MHz, DMSO-d ) δ 11.04 (s, 1H), 10.34 (s, 1H), 8.14 (s,
6
1
3
4
H), 7.77 (d, J = 7.8 Hz, 1H), 7.52–7.30 (m, 6H), 7.19–7.03 (m, 3H), 3.97 (s, 2H),
+
.15 (t, J = 8.0 Hz, 2H). ESI-MS m/z: 344.1 [M+H] .
.1.7.4. 2-Methoxy-4-(4-phenylindoline-1-carbonyl)benzaldehyde (15d). To a solution
of intermediate 15c (0.5 g, 1.46 mmol) and K CO (0.6 g, 4.35 mmol) in DMF (10
2
3
mL) was added CH I (0.62 g, 4.37 mmol) dropwise. The mixture was stirred at room
3
temperature for 2 h. After that time, the mixture was poured into water. The
precipitate was filtered off and dried to give compound 15d (0.45 g, 86.6%). It was
1
obtained as a white solid. H NMR (600 MHz, DMSO-d ) δ 10.41 (s, 1H), 8.17 (s,
6
1
4
2
4
H), 7.81 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 4.2 Hz, 4H), 7.45 (s, 1H), 7.40 (dt, J = 8.6,
.3 Hz, 2H), 7.27 (d, J = 7.3 Hz, 1H), 7.15 (s, 1H), 3.98 (s, 5H), 3.16 (t, J = 8.1 Hz,
+
H). ESI-MS m/z: 358.1 [M+H] .
.1.7.5.
15e).
3-((2-Formyl-5-(4-phenylindoline-1-carbonyl)phenoxy)methyl)benzonitrile
A mixture of intermediate 15c (0.6 g, 1.75 mmol),
(

3
-(bromomethyl)benzonitrile (0.41 g, 2.1 mmol) and Cs CO (1.14 g, 3.5 mmol) in
2 3
DMF (15 mL) was stirred at 75 °C for 2 h. After that time, the solution was cooled to
room temperature and was poured into water. The precipitate was filtered off and
1
dried to give compound 15e (0.59 g, 73.6%). It was obtained as a yellow solid. H
NMR (600 MHz, DMSO-d ) δ 10.48 (s, 1H), 8.15 (s, 1H), 8.04 (s, 1H), 7.88 (d, J =
6
7
.8 Hz, 1H), 7.83 (t, J = 7.2 Hz, 2H), 7.63 (t, J = 7.8 Hz, 1H), 7.52 (s, 1H), 7.47 (d, J
=
4.3 Hz, 5H), 7.39 (dt, J = 8.7, 4.3 Hz, 1H), 7.29 (d, J = 6.6 Hz, 1H), 7.14 (s, 1H),
+
5
4
4
.42 (s, 2H), 3.87 (s, 2H), 3.13 (t, J = 7.9 Hz, 2H). ESI-MS m/z: 459.2 [M+H] .
.1.7.6.
-(4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)indoline-1-carbonyl)-2-hydroxybenzaldeh
yde (15f). Compound 15f was prepared from intermediate 14b in a similar manner as
1
described for compound 15c. It was obtained as a yellow solid. Yield, 72%. H NMR
(
600 MHz, DMSO-d ) δ 11.03 (s, 1H), 10.33 (s, 1H), 8.07 (s, 1H), 7.75 (d, J = 7.9 Hz,
6
1
2
4
4
H), 7.29 (s, 1H), 7.16–7.02 (m, 3H), 6.94 (t, J = 2.1 Hz, 1H), 6.92 (d, J = 2.0 Hz,
+
H), 4.27 (s, 4H), 3.93 (s, 2H), 3.13 (t, J = 8.1 Hz, 2H). ESI-MS m/z: 402.1 [M+H] .
.1.7.7.
-(4-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)indoline-1-carbonyl)-2-methoxybenzaldeh
yde (15g). Compound 15g was prepared from intermediate 15f in a similar manner as
1
described for compound 15d. It was obtained as a white solid. Yield, 76%. H NMR
(
600 MHz, DMSO-d ) δ 10.39 (s, 1H), 7.79 (d, J = 7.8 Hz, 1H), 7.43 (s, 1H), 7.32 (s,
6
1
H), 7.25 (d, J = 6.3 Hz, 1H), 7.19 (s, 1H), 7.07 (s, 1H), 6.94 (d, J = 9.3 Hz, 3H), 4.27
+
(
s, 4H), 3.97 (s, 5H), 3.13 (t, J = 8.2 Hz, 2H). ESI-MS m/z: 416.1 [M+H] .