Design, synthesis and biological evaluation of isoxazole-containing biphenyl derivatives as small-molecule inhibitors targeting the programmed cell death-1/ programmed cell death-ligand 1 immune checkpoint

Article abstract of DOI:10.1007/s11030-021-10208-4

Monoclonal antibodies targeting the programmed cell death-1/ programmed cell death-ligand 1 (PD-1/PD-L1) immune checkpoint have achieved enormous success in cancer immunotherapy. But the antibody-based immunotherapies carry a number of unavoidable deficie

Full text of DOI:10.1007/s11030-021-10208-4

Molecular Diversity
https://doi.org/10.1007/s11030-021-10208-4
ORIGINAL ARTICLE
Design, synthesis and biological evaluation of isoxazole‑containing
biphenyl derivatives as small‑molecule inhibitors targeting
the programmed cell death‑1/ programmed cell death‑ligand 1
immune checkpoint
Peiyu Zhu1 · Jian Zhang1 · Yifei Yang2 · Lixun Wang1 · Jinpei Zhou2 · Huibin Zhang1
Received: 17 January 2021 / Accepted: 10 March 2021
© The Author(s), under exclusive licence to Springer Nature Switzerland AG 2021
Abstract
Monoclonal antibodies targeting the programmed cell death-1/ programmed cell death-ligand 1 (PD-1/PD-L1) immune
checkpoint have achieved enormous success in cancer immunotherapy. But the antibody-based immunotherapies carry a
number of unavoidable defciencies such as poor pharmacokinetic properties and immunogenicity. Small-molecule PD-1/
PD-L1 inhibitors ofer the superiority of complementarity with monoclonal antibodies and represent an appealing alterna-
tive. A novel series of isoxazole-containing biphenyl compounds were designed, synthesized and evaluated as PD-1/PD-L1
inhibitors in this paper. The structure–activity relationship of the novel synthesized compounds indicated that the ring-closure
strategy of introducing isoxazole could be employed and the 3-cyanobenzyl group was signifcant for the inhibitory activity
against the PD-1/PD-L1 protein–protein interactions. Molecular docking studies were performed to help understand the bind-
ing mode of the small-molecule inhibitor with the PD-L1 dimer. In particular, compound II-12 was a promising anti-PD-1/
PD-L1 inhibitor with the IC50 value of 23.0 nM, providing valuable information for future drug development.
Graphic abstract
Keywords Cancer immunotherapy · PD-1/PD-L1 immune checkpoint · Small-molecule inhibitor · Molecular docking ·
Inhibitory activity
Introduction
Peiyu Zhu and Jian Zhang are contributed equally
Cancer is a major public health problem worldwide and the
American Cancer Society estimated that in 2020, about 1.8
million new cancer cases and 600 thousand cancer deaths
would be occurred in the USA [1]. Currently, in addition to
surgery, chemotherapy, radiotherapy and targeted therapy,
cancer immunotherapy is promising and has provided a
* Jinpei Zhou
jpzhou668@163.com
* Huibin Zhang
Zhanghb80@cpu.edu.cn
Extended author information available on the last page of the article
Vol.:(0123456789)
1 3

Molecular Diversity
novel powerful weapon against cancer. Immune checkpoint
therapies, especially therapies targeting PD-1/PD-L1 pro-
teins, have achieved great breakthroughs in clinical practice
of numerous malignancies and led the development of tumor
immunity [2, 3]. Notably, The Nobel Prize in Physiology
or Medicine 2018 was awarded for the discovery of cancer
therapy by inhibition of negative immune regulation [4].
The programmed cell death-1 (PD-1) is a member of
the CD28 superfamily of immunoreceptors [5] involved in
regulating T cell activation [6] and is highly expressed on
activated T cells, Treg cells, natural killer T cells, B cells
and myeloid cells to maintain immune homeostasis as a
negative regulator [7]. Programmed cell death-ligand 1 (PD-
L1) is one ligand of PD-1 and regulates the development,
maintenance, and function of induced regulatory T cells [8].
The receptor-ligand interactions between PD-1 and PD-L1
would inhibit T cell proliferation and promote tumor growth
[9, 10]. Accordingly, the PD-1/PD-L1 immune checkpoint
could be an ideal target to restore T cell activation for cancer
immunotherapy.
exemplifed by the introduction of the 3-cyanobenzyl group
(BMS-1166) [19] and the polar hydrophilic side chain
(BMS-3054) [20]. Though such compounds have not entered
clinical research, they ofer an important reference for the
subsequent development of small-molecule PD-1/PD-L1
inhibitors. Researchers in Incyte Corporation developed
a series of compounds based on BMS biphenyl scafolds
and the representative compounds with the IC50 value less
than 10 nM are shown in Fig. 1. For example, compound
4, characterized by an amide linkage [21], and compound
5 containing the benzo[d]oxazole moiety via a ring fusion
strategy [22], would potently inhibit the PD-1/PD-L1 inter-
action. Besides, the symmetry-based ligand design was also
applied to provide the compound 6, which showed excellent
inhibitory activities against PD-1/PD-L1 [23].
Zak and his team revealed the characterization of the
interactions of BMS compounds with the PD-1/PD-L1
axis in 2016 [24]. Structural analysis indicated the small
molecule, BMS-202, could induce and stabilize a dimeric
protein complex of PD-L1 in order to dissociate the recep-
tor-ligand interactions of PD-1/PD-L1 [25]. It was shown
that BMS-202 bound at a hydrophobic cavity formed upon
PD-L1 dimerization and provided hydrophobic and electro-
static interactions to support the binding, consisting of the
hydrophobic π-π, π-alkyl and π-σ interactions with Tyr56,
Ala121 and Met115 provided by the biphenyl moiety, a π-π
stacking interaction made by the methoxy-pyridine moiety
with Tyr56 and electrostatic interactions created via the
extended N-(2-aminoethyl) acetamide moiety. In comparison
with BMS-202, BMS-1166 exhibited more efective activity
due to additional interactions with PD-L1 provided by the
introduced 3-cyanobenzyl group [26].
Recent researches have proven that the blockade of PD-1/
PD-L1 can remove a barrier to the function of T cells and
restore antitumor immunity [11]. Antibodies targeting the
PD-1/PD-L1 immune checkpoint have obtained encourag-
ing results for the clinical treatment of multiple cancers
[12]. Six anti-PD-1/PD-L1 monoclonal antibodies target-
ing either PD-1 (Nivolumab, Pembrolizumab and Cemipli-
mab) or PD-L1 (Durvalumab, Avelumab and Atezolizumab)
have received the US Food and Drug Administration (FDA)
approval. Unavoidably, immune-related adverse events due
to the breaking of immune self-tolerance [13], occurred in
the process of cancer treatment. And other intrinsic draw-
backs of antibodies, including low antitumor responses,
long half-life, poor stability and high cost, also hindered
the clinical therapy [12]. Given that small molecules ben-
eft from their abilities of convenient oral administration,
stability, membrane permeability and non-immunogenicity,
the PD-1/PD-L1 inhibitors based on the small-molecule
approach become a promising alternative for the treatment
of patients with tumors [14]. Thus, small-molecule PD-1/
PD-L1 immune checkpoint inhibitors have been in the rapid
development [15]. Two anti-PD-1/PD-L1 small-molecule
compounds, CA-170 [16] and INCB-086550 [17] (their
structures are undisclosed), have entered into the clinical
trials of solid tumors and lymphomas.
To further explore the potential binding mode formed
by the small-molecule inhibitor with the PD-L1 dimer, we
conducted a molecular docking study on BMS-3054, using
BMS-1166 as a control in the dimeric PD-L1 binding site.
As shown in Fig. 2, BMS-3054 ftted well in the hydro-
phobic pocket and bound to the PD-L1 dimer in a similar
pattern with BMS-1166. Additionally, the π-π stacking inter-
action created by the distal phenyl moiety with Tyr56 was
converted to a π-π interaction with Met115. The propoxy
induced a conformational change of Tyr56, and the con-
nected morpholine group was exposed to the solvent region,
forming an enlarged interaction interface. Moreover, the
nitrogen atom of the morpholine ring could form hydrogen
bonds with Asp122 through water molecules, which was
probably responsible for the improved activity.
Scientists from Bristol-Myers Squibb (BMS) disclosed
the frst class of small-molecule PD-1/PD-L1 inhibitors
with substituted biphenyl scafolds in patent applications.
The substituted biphenyl derivatives are claimed to inhibit
the PD-1/PD-L1 protein–protein interactions well, and the
inhibitory activity of BMS-202 (the active compound in the
patent) is 18 nM [18]. Afterward, more modifcations and
optimizations were made to improve inhibitory activity, as
Based on the above knowledge of PD-1/PD-L1 inhibi-
tors, the core biphenyl scafold was retained by taking
BMS-3054 as the lead compound and the modifcation
was focused on the introduction of isoxazole via the ring-
closing strategy as illustrated in Fig. 3. The isoxazole ring
was expected to induce the fip of sidechain of Tyr56,
1 3

Molecular Diversity
CN
CN
HO
N
O
N
OH
O
O
O
O
O
NH
H
N
O
N
O
N
O
O
O
O
OH
O
Cl
Cl
1
2
3
( BMS-202 )
IC50 = 18 nM [18]
( BMS-1166 )
IC50 = 1.4 nM [19]
( BMS-3054 )
IC50 = 0.21-10 nM [20]
OH
OH
NH
O
Cl
N
H
H
N
N
N
OH
N
H
N
N
O
N
H
H
H
N
N
N
O
HO
O
4
5
6
( INCB-965800 )
IC50 ≤ 10 nM [21]
( INCB-986707 )
IC50 ≤ 10 nM [22]
( INCB-1008418 )
IC50 ≤ 10 nM [23]
Fig. 1 Chemical structures of known small-molecule inhibitors of PD-1/PD-L1 pathway and reported IC50 data
which would make the PD-L1 binding cleft opening, and
form valuable π-π interactions with Tyr56 to enhance
the inhibitor-PD-L1binding. Considering that the amide
has been proven to be a privileged linker fragment in the
design of PD-L1 inhibitors [21] and the substituent on
the isoxazole ring should be solvent exposed and well
tolerated, we introduced (R)-pyrrolidin-3-ol, piperidin-
4-ylmethanol or piperidin-4-ol connected to the isoxa-
zole ring with the linker of an amide bond, to improve the
binding with the PD-L1 dimer. Meanwhile, it had been
proven in BMS patents the introduction of 3-cyanoben-
zyl might strengthen the inhibitory activity [19, 20], so
two classes of compounds were designed to explore the
efects of the 3-cyanobenzyl group at R1 on the inhibitory
activities. Moreover, the polar hydrophilic side chain at
R3 with the ability to form hydrogen bonds with PD-L1
was required for high activity, and we delved the infu-
ence of hydrophilic substituents to the inhibitory potency
via introducing diferent polar tail groups. Overall, we
designed and synthesized 23 compounds and then evalu-
ated their inhibitory activity against the PD-1/PD-L1 pro-
tein–protein interaction by a time-resolved fuorescence
resonance energy transfer (TR-FRET) assay to investigate
the structure–activity relationships (SAR).
Fig. 2 a Superposition of PD-L1 dimer with BMS-3054 (green) and
BMS-1166 (yellow); b interactions between BMS-3054 and PD-L1
dimer (PDB ID: 6R3K)
1 3

Molecular Diversity
CN
O
N
OH
R1
O
R3
ringclosure
O
O
R2
O
N
A
Cl
O
Cl
BMS-3054
3-cyanobenzyloxy
H
R1
R3
polar hydrophilic
O
chains
O
R2
piperidin-4-ylmethanol
O
N
Cl
R
(
)-pyrrolidin-3-ol
piperidin-4-ol
Target Compounds
Fig. 3 Design of the target compounds
serine methyl ester was synthesized after reductive amina-
tion and should be hydrolyzed under the condition shown
in step j (ii) to obtain the corresponding target compound.
Scheme 2 illustrates the synthesis of target compounds in
Class II. The synthesis was started from chlorinated reac-
tion of 2,4-dihydroxybenzaldehyde (19) with N-chlorosuc-
cinimide (NCS) to aford 20. Similar to 16, intermediate 21
was generated in the nucleophilic substitution reaction of
20 with 14. Next the hydroxy moiety of 21 was substituted
by 3-(bromomethyl) benzonitrile to prepare 22, which in
turn reacted with bis(pinacolato)diboron to obtain the borate
ester intermediate 23. The subsequent steps were similar to
the corresponding procedures shown in Scheme 1. Target
compounds II-1~II-14 were obtained by adopting a similar
coupling methodology between the borate ester intermediate
23 and the key intermediates 11a-c, followed by reductive
aminations using NaBH3CN and appropriate amines.
Results and discussion
Chemistry
The Class I compounds were synthesized as depicted in
Scheme 1. The starting material, 3-bromoacetophenone
(7), was condensed with diethyl oxalate to obtain interme-
diate 8. Next 9 was prepared in the presence of hydroxy-
lamine hydrochloride via ring-closure condensation reac-
tion with intermediate 8. A subsequent hydrolysis reaction
mediated by lithium hydroxide monohydrate yielded the
corresponding acid (10), which then was treated by ami-
dation with diferent amines to achieve key intermedi-
ates 11a-c. On the other hand, (3-bromo-2-methylphenyl)
methanol (13) was delivered by the reduction of 3-bromo-
2-methylbenzoic acid (12). The conversion of intermedi-
ate 13–14 was achieved in the chlorinated reaction with
POCl3. Alkylation of 3-chloro-4-hydroxybenzaldehyde
(15) with benzyl chloride (14) gave rise to 16. Next 16
was reacted with bis(pinacolato)diboron, getting the borate
ester intermediate 17. After that, intermediates 18a-c
were prepared via palladium-catalyzed Suzuki coupling
reaction of 17 and the key intermediates 11a–c, respec-
tively. Finally, reductive aminations of 18a-c with difer-
ent amines were carried out to aford target compounds
I-1 ~ I-9. Notably, if the substituent at R3 of the target
compound was D-serine or L-serine, the reactant amine
used in step j (i) was D-serine methyl ester hydrochloride
or L-serine methyl ester hydrochloride, respectively. The
PD‑1/PD‑L1 inhibitory activity and SAR study
All the synthesized small-molecule inhibitors were evalu-
ated for their anti-PD-1/PD-L1 activity, and the results are
summarized in Table 1 and 2. Inhibition ratio at 0.5 μM
and IC50 of compounds was measured with TR-FRET
method to verify inhibitory activity. BMS-202 showed
excellent activity against PD-1/PD-L1 interactions with
an IC50 value of 18.7 nM, which was comparable to the
reported value (IC50, 18 nM) [18].
1 3

Molecular Diversity
O
O
O
O
O
Br
O
Br
O
OH
Br
c
Br
a
b
N
N
O
O
O
7
8
9
10
O
R2
R2
11a
11b
=
=
R2
piperidin-4-ylmethanol
Br
d
R
N
(
)-pyrrolidin-3-ol
O
O
11c R2
piperidin-4-ol
=
11a-c
H
HO
Cl
15
O
O
H
Br
Br
Br
e
h
f
g
OH
Cl
Br
OH
O
Cl
14
12
13
16
O
R2
O
Br
O
N
O
H
O
B
H
O
11a-c
O
O
O
R2
Cl
N
O
i
Cl
17
18a-c
R2
R2
18a
18b
18c R2
=
=
piperidin-4-ylmethanol
R
(
)-pyrrolidin-3-ol
=
piperidin-4-ol
R3
O
j
O
R2
N
O
Cl
I-1~I-9
Scheme 1 Synthetic route of Class I compounds I-1~I-9. Rea-
gents and conditions: a diethyl oxalate, C2H5ONa, EtOH, 0℃ to rt;
b hydroxylamine hydrochloride, EtOH, 80℃; c LiOH·H2O, MeOH/
H2O, rt; d amines, HATU, DIPEA, DMF, rt; e BH3-THF, 0℃ to
rt; f POCl3, NMP, 0℃ to rt; g 3-chloro-4-hydroxybenzaldehyde,
K2CO3, DMF, 70℃; h bis(pinacolato)diboron, Pd(dppf)Cl2CH2Cl2,
CH3COOK, 1,4-Dioxane, 80℃; i Pd(PPh3)4, K3PO4, DME, 80℃; j
(i) amines, (CH3COO)3BHNa, CH3COOH, 1,2-dichloroethane, rt; for
compound I-2, I-3, I-5, I-6, I-8, I-9 (ii) LiOH·H2O, MeOH/H2O, rt
the dimeric PD- L1 in a similar pattern with BMS-1166.
More details in Fig. 4b revealed that the hydrophobic
cleft was tolerant to the introduced isoxazole ring and
important interactions of II-9 with amino acid residues
of PD-L1 contributed to the inhibitor binding to PD-L1.
The distal phenyl moiety and the Cl-substituted phe-
nyl formed π-π stacking interaction with Met115 and
Ala121, respectively, providing strong stabilization for
the inhibitor binding. The hydroxyl group and N-atom
Molecular docking studies
To elucidate the binding mode of the target compounds
with PD-L1 dimeric protein complex and validate our
design strategy, molecular docking studies on repre-
sentative molecules, II-9 (IC50, 33.2 nM) and II-12 (IC50,
23.0 nM), were carried out. Figure 4a shows the binding
overlay of the II-9/PD-L1 and BMS-1166/PD-L1 struc-
tures, which demonstrated that compound II-9 bound to
1 3

Molecular Diversity
Br
H
Cl
OH
Cl
O
O
O
OH
O
OH
Cl
O
14
b
H
H
H
a
c
d
Br
Br
O
O
HO
HO
Cl
19
21
20
22
O
N
O
N
O
R2
Br
N
O
O
O
11a-c
H
O
B
H
O
e
O
R2
O
O
N
O
Cl
Cl
R2
=
piperidin-4-ylmethanol
24a
24b
24c
R2
R2
24a-c
R
=
23
(
)-pyrrolidin-3-ol
N
=
piperidin-4-ol
O
R3
f
O
R2
O
N
O
Cl
II-1~II-14
Scheme 2 Synthetic route of Class II compounds II-1~II-14. Rea-
gents and conditions: a NCS, CH3COOH, 80℃; b NaHCO3, KI,
DMF, 40℃; c 3-(bromomethyl)benzonitrile, K2CO3, DMF, 70℃; d
bis(pinacolato)diboron, Pd(dppf)Cl2.CH2Cl2, CH3COOK, 1,4-Diox-
ane, 80℃; e Pd(PPh3)4, 1 mol/L Na2CO3, toluene, EtOH, 80℃; f (i)
amines, CH3BNNa, CH3COOH, MeOH/DMF, rt; for compound II-7,
II-14 (ii) LiOH·H2O, MeOH/H2O, rt
of piperidin-4-ol made hydrogen bonds with Phe19 and
Ala121, respectively. 2-Amino-2-(hydroxymethyl) pro-
pane-1,3-diol was accessible to the solvent region and
formed stable interactions with Gln66. Disappointingly,
the expected π-π stacking interaction created by isoxazole
was not observed. But 3-cyanobenzyl formed additional
π-stacking with the dimeric PD-L1 and the cyano-group
of the 3-cyanobenzyl moiety might interact with Arg125
via a hydrogen bond, accounting for the improvement of
anti-PD-1/PD-L1 activity of Class II compounds when
introducing 3-cyanobenzyl group.
acid residue Asp122 induced the conformational changes of
PD-L1, resulting in the π-π stacking interaction and the π-H
interaction created by isoxazole with Tyr56 and Asp122,
which explicated why compound II-12~II-14 showed more
excellent inhibitory activities among the Class II compounds
and explained the reason for the signifcant increase in activ-
ity of compound I-5 or I-6 substituted by (R)-pyrrolidin-3-ol
compared with other compounds in Class I. Similarly, the
protonated amino-group and hydroxyl-group of the 2-amino-
2-(hydroxymethyl) propane-1,3-diol formed H-bonds with
Lys124 and Asp122, respectively. Moreover, the interactions
between the cyano-group of the introduced 3-cyanobenzyl
moiety and Arg125 would also enhance the inhibitor-PD-L1
binding and improve anti-PD-1/PD-L1 activities (II-14 (IC50,
24.5 nM) v.s. I-6 (IC50, 100.6 nM)), confrming the important
role of the 3-cyanobenzyl moiety in the drug design.
As to the docking study of II-12 in Fig. 5, it was obvious
that II-12 and BMS-1166 could be well superimposed and
II-12 matched well to the PD-L1 hydrophobic cleft in a simi-
lar pattern with compound II-9. Notably, compared to II-9,
the interaction of (R)-pyrrolidin-3-ol of II-12 with the amino
1 3

Molecular Diversity
Table 1 PD-1/PD-L1 inhibitory
activity of Class I target
compounds
PD-1/PD-L1
Compound
R2
R3
Inh%
IC50
(0.5μM)a
(nM)b
I-1
I-2
5.3 1.6
NT
NT
21.7 2.5
I-3
I-4
I-5
20.7 3.1
20.3 1.4
79.1 7.7
NT
NT
112.8 8.7
I-6
I-7
I-8
77.5 9.6
9.8 2.2
100.6 9.2
NT
23.9 3.6
16.5 2.8
NT
I-9
NT
BMS-202c
18.7 0.3
NT Not test.aInhibition ratio values at 0.5 μM for anti-PD-1/PD-L1 activities presented are the
mean SD values of three independent determinations. bIC50 values for anti-PD-1/PD-L1 activi-
ties presented are the mean SD values of three independent determinations.cUsed as the positive
control compound
1 3

Molecular Diversity
spectrometer. Mass spectrometry was measured with Hewl-
ett-Packard 1100 LC/MSD spectrometer.
Conclusion
In summary, the development of small-molecule PD-1/
PD-L1 inhibitors has emerged as an attractive approach.
We designed and synthesized two series of isoxazole-con-
taining biphenyl derivatives, and their anti-PD-1/PD-L1
potency was evaluated through a TR-FRET assay. Impor-
tantly, compound II-12, containing (R)-pyrrolidin-3-ol
connected to the isoxazole ring via an amide bond and
the hydrophilic tail, 2-amino-2-(hydroxymethyl) propane-
1,3-diol at R3, exerted the most promising activity with the
IC50 value of 23.0 nM. Furthermore, molecular docking
studies were performed to further verify the rationality
of the ring-closure strategy of introducing isoxazole and
explain the structure–activity relationship.
Ethyl 4‑(3‑bromophenyl)‑2, 4‑dioxobutanoate (8)
To a solution of sodium ethoxide (0.376 g, 5.53 mmol) dis-
solved in 10 mL absolute ethanol was dropped 3-bromoace-
tophenone (1.00 g, 5.02 mmol) at 0℃ and stirred for one
hour. Diethyl oxalate (1.02 mL, 7.54 mmol) was added, and
then, the mixture was reacted at room temperature for 12 h.
The reaction solution was quenched by ice water, adjusted
to pH 2 with 1 mol/L aqueous HCl and extracted with
ethyl acetate. The organic phase was combined, dried over
Na2SO4 and concentrated. The residue was purifed by silica
gel column chromatography (petroleum ether/ethyl acetate:
50/1~30/1) to obtain 8 (1.11 g, 74%). It was obtained as yel-
low liquid. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 8.20 (s,
1H), 8.07 (d, J=7.7 Hz, 1H), 7.89 (d, J=7.7 Hz, 1H), 7.54
(t, J = 7.9 Hz, 1H), 7.13 (s, 1H), 4.32 (q, J= 7.1 Hz, 2H),
1.32 (t, J=7.1 Hz, 3H).
The future research should focus on structural optimiza-
tions of II-12. It is necessary to further explore the struc-
tural activity relationship to identify other promising PD-1/
PD-L1 inhibitors possessing higher activities. Additionally,
the feasibility of the ring-closure strategy should be explored
more deeply, such as the replacement of the isoxazole ring
with other fve-membered aromatic heterocycles, which
might also be well tolerated and make vital interactions
with PD-L1 hydrophobic cleft. Furthermore, biological
evaluations about pharmacokinetic and pharmacodynamic
properties of the promising PD-1/PD-L1 inhibitors should
be carried out to provide valuable information for future
investigation.
Ethyl 5‑(3‑bromophenyl) isoxazole‑3‑carboxylate (9)
To a solution of 8 (1.00 g, 3.35 mmol) dissolved in 10 mL
absolute ethanol was added hydroxylamine hydrochloride
(0.466 g, 6.71 mmol) at room temperature and stirred at
80℃ for an hour. The reaction mixture was cooled to room
temperature and then extracted with ethyl acetate. The
organic phase was combined, dried over Na2SO4 and con-
centrated. The residue was purifed by silica gel column
chromatography (petroleum ether / ethyl acetate: 12/1) to
Consequently, the novel compounds involving the isox-
azole ring we designed pointed out a new perspective to
promote the development of small-molecule PD-1/PD-L1
inhibitors, and the representative compound II-12 could
be optimized as a leading compound in the follow-up drug
design.
1
obtain 9 (0.82 g, 83%). It was obtained as white solid. H
NMR (300 MHz, DMSO-d6) δ (ppm): 8.20 (s, 1H), 7.97
(d, J = 7.9 Hz, 1H), 7.75 (d, J= 8.1 Hz, 1H), 7.64 (s, 1H),
7.52 (t, J = 7.9 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 1.34 (t,
J=7.1 Hz, 3H).
Experimental section
5‑(3‑Bromophenyl) isoxazole‑3‑carboxylic acid (10)
Chemistry
The intermediate 9 (0.500 g, 1.69 mmol) was dissolved
in mixed solvent of 6 mL methanol and 3 ml water. Then
LiOH·H2O (0.213 g, 5.07 mmol) was added to the solution.
The solution was heated to 70℃ and stirred for 2 h. The
reaction mixture was cooled to room temperature, concen-
trated and adjusted to pH 2 with 1 mol/L aqueous HCl. The
precipitate was fltrated, washed with water and then dried
to obtain 10 (0.43 g, 94%). It was obtained as white solid. 1H
NMR (300 MHz, DMSO-d6) δ (ppm): 14.17 (s, 1H), 8.18 (s,
1H), 7.96 (d, J=7.7 Hz, 1H), 7.74 (d, J=8.0 Hz, 1H), 7.53
(dd, J=14.0, 6.0 Hz, 2H).
All materials and reagents used in the experiment are all
commercially available analytical or chemically purity prod-
ucts. Unless otherwise specifed, they were used directly
without treatment. Thin-layer chromatography (TLC) was
performed on 0.20 mm Silica Gel 60 F254 plates (Qingdao
Ocean Chemical Factory, Shandong, China). The melting
point was determined by the Tianjin Analytical Instrument
Factory RY-1 melting point meter, with the thermometer not
calibrated. Nuclear magnetic resonance (NMR) spectra were
recorded on a Bruker spectrometer (300 MHz /400 MHz)
with TMS as an external reference and reported in parts
per million. The infrared spectrum was compressed with
KBr tablets and recorded by Shimadzu FTIR-8400S infrared
1 3

Molecular Diversity
Table 2 PD-1/PD-L1 inhibitory
activity of Class II target
compounds
PD-1/PD-L1
Compound
R2
R3
Inh%
IC50
(0.5μM)a
(nM)b
79.8 2.9
159.7 3.5
310.0 6.6
II-1
63.5 11.6
49.7 7.9
II-2
491.1 13.8
103.3 4.3
279.1 16.9
NT
II-3
II-4
II-5
II-6
90.7 0.9
64.7 6.7
19.3 5.2
82.6 3.7
93.7 2.7
96.3 3.9
92.5 2.4
97.1 7.4
46.5 4.2
33.2 3.6
72.6 2.4
II-7
II-8
II-9
II-10
1 3

Molecular Diversity
Table 2 (continued)
91.6 2.7
70.3 1.8
23.0 2.1
24.8 2.7
II-11
II-12
11-13
99.2 0.1
99.2 1.4
98.8 0.9
24.5 1.6
18.7 0.3
11-14
BMS-202c
NT Not test.aInhibition ratio values at 0.5 μM for anti-PD-1/PD-L1 activities presented are the
mean SD values of three independent determinations. bIC50 values for anti-PD-1/PD-L1 activi-
ties presented are the mean SD values of three independent determinations.cUsed as the positive
control compound
Fig. 5 a Superposition of PD-L1 dimer with II-12 (green) and BMS-
Fig. 4 a Superposition of PD-L1 dimer with II-9 (green) and BMS-
1166 (yellow); b interactions between II-9 and PD-L1 dimer (PDB
ID: 6R3K)
1166 (yellow); b interactions between II-12 and PD-L1 dimer (PDB
ID: 6R3K)
1 3

Molecular Diversity
(5‑(3‑Bromophenyl) isoxazol‑3‑yl)(4‑(hydroxymethyl)
piperidin‑1‑yl)methanone (11a)
4‑((3‑Bromo‑2‑methylbenzyl) oxy)‑3‑chlorobenzaldehyde
(16)
A mixture of 10 (1.5 g, 5.6 mmol), piperidin-4-ylmethanol
(0.709 g, 6.16 mmol) and HATU (2.55 g, 6.71 mmol) was
added in 15 mL N, N-Dimethylformamide. N, N—Diiso-
propylethylamine (DIPEA) was dropped, and the mixture
was reacted at room temperature for 2 h. The reaction mix-
ture was extracted with ethyl acetate. The organic phase was
combined, dried over Na2SO4 and concentrated. The residue
was purifed by silica gel column chromatography (dichlo-
romethane/methanol: 150/1) to obtain 11a (0.90 g, 44%).
It was obtained as slight yellow solid. 1H NMR (300 MHz,
DMSO-d6) δ (ppm): 8.15 (s, 1H), 7.93 (d, J=7.8 Hz, 1H),
7.74 (d, J = 8.7 Hz, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.39 (s,
1H), 4.59 – 4.42 (m, 2H), 3.93 (d, J=13.6 Hz, 1H), 3.28 (s,
2H), 3.12 (t, J=11.6 Hz, 1H), 2.83 (t, J=11.5 Hz, 1H), 1.74
(dd, J=23.4, 12.9 Hz, 3H), 1.19 – 1.07 (m, 2H).
A mixture of 3-chloro-4-hydroxybenzaldehyde (15) (2.85 g,
18.22 mmol) and potassium carbonate (5.04 g, 36.45 mmol)
was added in 20 mL N, N-Dimethylformamide. In total, 14
(4.20 g, 19.13 mmol) was added and the mixture was reacted
at 70℃ for 2 h. The reaction mixture was dropped into
50 mL ice water, and the precipitate was fltered, washed
with water and then dried to obtain 16 (5.92 g, 95%). It was
obtained as white solid. 1H NMR (300 MHz, DMSO-d6) δ
(ppm): 9.88 (s, 1H), 7.98 (s, 1H), 7.92 (d, J=8.4 Hz, 1H),
7.63 (d, J = 8.0 Hz, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.20 (t,
J=7.7 Hz, 1H), 5.39 (s, 2H), 2.41 (s, 3H).
3‑Chloro‑4‑((2‑methyl‑3‑(4,4,5,5‑tetramethyl‑1,3,2‑dioxab‑
orolan‑2‑yl)benzyl)oxy)benzaldehyde (17)
A mixture of 16 (5.10 g, 15.02 mmol), bis(pinacolato)dibo-
ron (5.72 g, 22.53 mmol), 1,1′-Bis (diphenylphosphino)
ferrocene-palladium (II) dichloride dichloromethane com-
plex (0.613 g,0.751 mmol) and potassium acetate (2.95 g,
30.03mml) in 80 mL 1,4-dioxane was purged with N2 and
then stirred at 80 °C for 12 h. The reaction mixture was
cooled to room temperature and fltrated via kieselguhr.
Then, the filtrate was extracted with ethyl acetate. The
combined organic phase was dried over Na2SO4 and con-
centrated. The residue was purifed by silica gel column
chromatography (petroleum ether/ethyl acetate: 20/1) to
obtain 17 (5.447 g, 94%). It was obtained as brownish yel-
low liquid. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 9.87
(d, J=7.4 Hz, 1H), 8.04 – 7.84 (m, 2H), 7.70 – 7.44 (m, 3H),
7.22 (d, J=7.7 Hz, 1H), 5.33 (d, J=7.2 Hz, 2H), 2.57 – 2.49
(m, 3H), 1.31 (d, J=7.3 Hz, 12H).
(3‑Bromo‑2‑methylphenyl)methanol (13)
To a solution of 3-bromo-2-methylbenzoic acid (12) (5.00 g,
23.25 mmol) dissolved in 50 mL anhydrous tetrahydrofuran
was dropped BH3.THF (35 mL, 34.88 mmol) under 0 °C
and then stirred for 5 h at room temperature. The reaction
solution was quenched by methanol and poured into about
100 mL 1 mol/L hydrochloric acid solution. The mixture
was extracted with ethyl acetate. The organic phase was
combined, dried over Na2SO4 and concentrated to obtain
13 (4.58 g, 98%). It was obtained as white solid. 1H NMR
(300 MHz, DMSO-d6) δ (ppm): 7.48 (d, J = 7.9 Hz, 1H),
7.38 (d, J = 7.4 Hz, 1H), 7.11 (t, J = 7.7 Hz, 1H), 5.25 (t,
J=5.4 Hz, 1H), 4.52 (d, J=5.2 Hz, 2H), 2.30 (s, 3H).
3‑Chloro‑4‑((3′‑(3‑(4‑(hydroxymethyl)piperidine‑1‑car‑
bonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)meth‑
oxy)benzaldehyde (18a)
1‑Bromo‑3‑(chloromethyl)‑2‑methylbenzene (14)
To a solution of 13 (4.58 g, 22.78 mmol) dissolved in 30 mL
1-methyl-2-pyrrolidinone (NMP) was dropped phosphorus
oxychloride (10.48 g, 68.34 mmol) under 0 °C and stirred for
3 h at room temperature. The reaction solution was quenched
by water and adjusted to pH 7 with aqueous NaHCO3. The
mixture was extracted with ethyl acetate. The organic phase
was combined, dried over Na2SO4 and concentrated to
obtain 14 (4.68 g, 94%). It was obtained as colorless liquid.
1H NMR (300 MHz, DMSO-d6) δ (ppm): 7.61 (d, J=8.0 Hz,
1H), 7.44 (d, J=7.1 Hz, 1H), 7.15 (dd, J=9.6, 5.5 Hz, 1H),
4.85 (d, J=4.0 Hz, 2H), 2.43 (d, J=3.9 Hz, 3H).
A mixture of 11a (0.555 g, 1.52 mmol), 17 (0.881 g,
2.28 mmol), Tetrakis (triphenylphosphine)palladium
(0.088 g,0.076 mmol) and potassium phosphate tribasic
(0.645 g, 3.04mml) in 15 mL 1,2-ethanediol dimethyl ether
was purged with N2 and then stirred at 80 °C for 12 h. The
reaction mixture was cooled to room temperature and fl-
trated via kieselguhr. Then the fltrate was extracted with
ethyl acetate. The combined organic phase was dried over
Na2SO4 and concentrated. The residue was purifed by silica
gel column chromatography (dichloromethane/methanol:
120/1) to obtain 18a (0.696 g, 84%). It was obtained as
slight yellow solid. 1H NMR (300 MHz, DMSO-d6) δ (ppm):
9.89 (s, 1H), 8.00 (d, J = 1.9 Hz, 1H), 7.95 (d, J = 2.0 Hz,
1H), 7.92 (d, J=2.1 Hz, 1H), 7.88 (s, 1H), 7.69 – 7.56 (m,
1 3

Molecular Diversity
4H), 7.49 (d, J=7.7 Hz, 1H), 7.36 (s, 1H), 7.34 – 7.31 (m,
1H), 5.42 (s, 2H), 4.51 (dd, J = 14.9, 9.3 Hz, 2H), 3.95 (t,
J=7.1 Hz, 1H), 3.28 (t, J=5.5 Hz, 2H), 3.12 (t, J=11.8 Hz,
1H), 2.82 (t, J=11.4 Hz, 1H), 2.25 (s, 3H), 1.83 – 1.71 (m,
2H), 1.68 (s, 1H), 1.16 – 1.07 (m, 2H).
1H), 7.64 (t, J=7.6 Hz, 2H), 7.55 (d, J=7.5 Hz, 1H), 7.24
– 7.19 (m, 2H), 5.44 (s, 2H), 5.35 (s, 2H), 2.52 (s, 3H), 1.31
(s, 12H).
3‑((4‑Chloro‑2‑formyl‑5‑((3′‑(3‑(4‑(hydroxymethyl)piperi‑
dine‑1‑carbonyl) isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3
‑yl) methoxy)phenoxy)methyl) benzonitrile (24a)
5‑Chloro‑2,4‑dihydroxybenzaldehyde (20)
2,4-Dihydroxybenzaldehyde (19) (0.20 g, 1.45 mmol)
was dissolved in 5 mL acetic acid. N-Chlorosuccinimide
(0.213 g, 1.59 mmol) was added into the solution, and then,
the solution was stirred at 80℃ overnight. The reaction
solution was cooled to room temperature, poured into about
50 mL ice water, adjusted to pH 7 with aqueous NaHCO3
and extracted with ethyl acetate. The organic phase was
combined, dried over Na2SO4 and concentrated. The residue
was purifed by silica gel column chromatography (petro-
leum ether/ethyl acetate: 30/1~20/1) to obtain 20 (0.124 g,
50%). It was obtained as white solid. 1H NMR (400 MHz,
DMSO-d6) δ (ppm): 11.38 (s, 1H), 10.87 (s, 1H), 9.97 (s,
1H), 7.60 (s, 1H), 6.58 (s, 1H).
The intermediate 24a was prepared using similar procedures
as described for 18a. It was obtained as slight yellow solid
(0.828 g, 88%). 1H NMR (400 MHz, DMSO-d6) δ (ppm):
10.24 (d, J=1.7 Hz, 1H), 8.04 (s, 1H), 7.94 (d, J=7.7 Hz,
1H), 7.86 (dd, J= 16.1, 10.0 Hz, 3H), 7.73 (d, J= 1.7 Hz,
1H), 7.65 (dd, J=7.5, 5.2 Hz, 2H), 7.57 (d, J=6.9 Hz, 1H),
7.50 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 1.8 Hz, 3H), 7.28 (s,
1H), 5.44 (d, J = 10.3 Hz, 4H), 4.00 (s, 2H), 3.78 (s, 3H),
2.27 (s, 3H), 1.79 (s, 2H), 1.39 (s, 2H).
(S)‑1‑(3‑chloro‑4‑((3′‑(3‑(4‑(hydroxymethyl)piperi‑
dine‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3
‑yl)methoxy)benzyl)piperidine‑2‑carboxylic acid (I‑1)
4‑((3‑Bromo‑2‑methylbenzyl)oxy)‑5‑chloro‑2‑hydroxyben‑
zaldehyde (21)
A mixture of 18a (0.15 g, 0.275 mmol), L (-)-Pipeco-
linic acid (0.053 g, 0.413 mmol) and acetic acid (0.016 g,
0.275 mmol) in 6 ml 1,2-dichloroethane was stirred at room
temperature for 0.5 h. Then sodium triacetoxyborohydride
(0.175 g, 0.826 mmol) was added, and the mixture was
stirred for 24 h. The reaction mixture was extracted with
dichloromethane/water. The combined organic phase was
dried over Na2SO4 and concentrated. The residue was puri-
fed by silica gel column chromatography (dichlorometh-
ane/methanol: 20/1~10/1) to obtain I-1 (0.066 g, 36%). It
The intermediate 21 was prepared using similar procedures
as described for 16 with sodium bicarbonate replacing
potassium carbonate. 0.1 equiv potassium iodide was added
to the solution to promote the reaction. It was obtained as
white solid (4.24 g, 69%). 1H NMR (400 MHz, DMSO-d6)
δ (ppm): 11.16 (s, 1H), 10.05 (s, 1H), 7.72 (s, 1H), 7.66 (s,
1H), 7.51 (s, 1H), 7.21 (s, 1H), 6.84 (s, 1H), 5.33 (s, 2H),
2.41 (s, 3H).
1
was obtained as white solid. m. p.: 120–124℃; H NMR
(400 MHz, DMSO-d6) δ (ppm): 7.94 (d, J = 7.8 Hz, 1H),
7.87 (s, 1H), 7.65 (t, J = 7.7 Hz, 1H), 7.56 (d, J = 7.4 Hz,
1H), 7.49 (d, J=7.5 Hz, 1H), 7.44 (s, 1H), 7.37 – 7.33 (m,
2H), 7.29 (t, J = 5.7 Hz, 3H), 6.67 (s, 1H), 5.26 (s, 2H),
4.49 (d, J = 12.8 Hz, 1H), 3.95 (d, J = 13.1 Hz, 1H), 3.84
(d, J = 13.4 Hz, 1H), 3.49 (d, J = 13.4 Hz, 1H), 3.28 (d,
J = 5.7 Hz, 2H), 3.14 – 3.07 (m, 2H), 2.84 (dd, J = 27.1,
12.2 Hz, 2H), 2.25 (s, 3H), 1.82 (d, J =13.2 Hz, 2H), 1.70
(d, J=8.9 Hz, 3H), 1.49 (s, 4H), 1.35 (d, J=10.6 Hz, 1H),
1.24 (d, J=5.9 Hz, 1H), 1.11 (d, J=5.7 Hz, 2H); 13C NMR
(75 MHz, DMSO-d6) δ (ppm): 173.47, 172.02, 169.79,
159.76, 159.36, 153.54, 142.78, 141.59, 135.78, 134.41,
132.11, 131.36, 130.19, 130.01, 129.92, 129.80, 128.51,
126.92, 126.80, 126.21, 124.85, 121.72, 114.38, 101.21,
69.64, 65.80, 64.57, 58.12, 49.66, 47.05, 42.15, 38.70,
29.78, 28.79, 24.34, 22.96, 22.27, 16.22; IR(KBr, cm−1)
ν: 3387, 2927, 2858, 1630, 1501, 1459, 1412, 1390, 1290,
1253, 1064, 1009, 980, 794, 701; MS (ESI) m/z: calculated
for C37H40ClN3O6 [M+H]+: 658.26, found: 658.56.
3‑((5‑((3‑Bromo‑2‑methylbenzyl)oxy)‑4‑chloro‑2‑formyl‑
phenoxy)methyl)benzonitrile (22)
The intermediate 22 was prepared using similar procedures
as described for 16. It was obtained as slight yellow solid
1
(4.45 g, 87%). H NMR (400 MHz, DMSO-d6) δ (ppm):
10.23 (s, 1H), 8.04 (s, 1H), 7.86 (dd, J=9.4, 8.3 Hz, 2H),
7.72 (s, 1H), 7.68 – 7.60 (m, 2H), 7.50 (d, J=7.1 Hz, 1H),
7.20 (dd, J= 15.3, 7.4 Hz, 2H), 5.42 (d, J= 10.9 Hz, 4H),
2.42 (s, 3H).
3‑((4‑Chloro‑2‑formyl‑5‑((2‑methyl‑3‑(4,4,5,5‑tetra‑
methyl‑1,3,2‑dioxaborolan‑2‑yl)benzyl)oxy) phenoxy)
methyl)benzonitrile (23)
The intermediate 23 was prepared using similar procedures
as described for 17. It was obtained as slight yellow solid
1
(3.65 g, 91%). H NMR (400 MHz, DMSO-d6) δ (ppm):
10.22 (s, 1H), 8.04 (s, 1H), 7.89 – 7.82 (m, 2H), 7.71 (s,
1 3

Molecular Diversity
(3‑Chloro‑4‑((3′‑(3‑(4‑(hydroxymethyl)piperidine‑1‑car‑
bonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)meth‑
oxy)benzyl)‑D‑serine ( I‑2)
1.70 (d, J = 10.8 Hz, 2H), 1.09 (dd, J = 12.3, 5.5 Hz, 2H);
13C NMR (101 MHz, DMSO-d6) δ (ppm): 169.78, 159.77,
159.34, 153.86, 131.61, 130.17, 129.76, 126.16, 121.78,
114.45, 101.21, 69.62, 65.82, 62.95, 61.20, 49.10, 47.05,
42.18, 38.71, 29.78, 28.78, 16.19; IR(KBr, cm−1) ν: 3383,
2928, 2863, 1627, 1504, 1447, 1413, 1336, 1291, 1258,
1084, 1062, 1036, 987, 782, 699; MS (ESI) m/z: calculated
for C34H36ClN3O7 [M+H]+: 634.22, found: 634.58.
A mixture of D-serine methyl ester hydrochloride (0.086 g,
0.550 mmol) and sodium acetate (0.046 g, 0.550 mmol)
in 6 ml 1,2-dichloroethane was stirred at room tempera-
ture for 0.5 h. Then 18a (0.15 g, 0.275 mmol) and acetic
acid (0.034 g, 0.550 mmol) were added, and the mixture
was stirred at room temperature for 0.5 h. Then sodium
triacetoxyborohydride (0.175 g, 0.826 mmol) was added,
and the mixture was stirred for 24 h. The reaction mixture
was extracted with dichloromethane/water. The combined
organic phase was dried over Na2SO4 and concentrated. The
residue was purifed by silica gel column chromatography
(dichloromethane/methanol: 60/1~40/1), and 125 mg white
solid was obtained (yield%=72%). After that, to a solution
of the obtained solid (0.125 g, 0.197 mmol) dissolved in
methanol/water (4 mL/1 mL) was added lithium hydroxide
monohydrate (0.025 g, 0.592 mmol), and the reaction mix-
ture was stirred for 5 h at room temperature. The mixture
was concentrated and adjusted to pH 4–5 with acetic acid.
The precipitate formed was fltered, washed with water and
then dried to obtain I-2 (0.103 g, 84%). It was obtained as
white solid. m. p.: 158–162℃; 1H NMR (400 MHz, DMSO-
d6) δ (ppm): 7.93 (s, 1H), 7.87 (s, 1H), 7.64 (s, 1H), 7.56
(s, 2H), 7.49 (s, 1H), 7.32 (d, J=25.4 Hz, 5H), 5.26 (s, 2H),
4.49 (d, J=10.3 Hz, 1H), 3.94 (s, 3H), 3.66 (d, J=11.3 Hz,
2H), 3.28 (s, 2H), 3.13 (s, 2H), 2.82 (s, 1H), 2.24 (s, 3H),
1.77 (d, J = 11.9 Hz, 1H), 1.69 (s, 1H), 1.11 (s, 1H); 13C
NMR (75 MHz, DMSO-d6) δ (ppm): 169.77, 159.76,
159.34, 153.69, 142.75, 141.57, 135.71, 134.37, 132.12,
131.38, 130.19, 129.92, 128.44, 126.91, 126.79, 126.18,
124.85, 121.73, 114.43, 101.20, 69.61, 65.80, 62.94, 61.47,
49.28, 47.05, 42.15, 38.69, 29.77, 28.77, 16.20; IR(KBr,
cm−1) ν: 3373, 2929, 2863, 1627, 1504, 1448, 1413, 1338,
1290, 1258, 1083, 1063, 1036, 987, 802, 699; MS (ESI)
m/z: calculated for C34H36ClN3O7 [M+H]+: 634.22, found:
634.58.
(5‑(3′‑((2‑Chloro‑4‑(((2‑hydroxyethyl)amino)methyl)phe‑
noxy)methyl)‑2′‑methyl‑[1,1′‑biphenyl]‑3‑yl)isoxazol‑3‑yl)
(4‑(hydroxymethyl)piperidin‑1‑yl)methanone (I‑4)
I-4 was prepared using similar procedures as described for
I-1. It was obtained as white solid (0.070 g, 43%). m. p.:
72–76℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.94 (d,
J=7.6 Hz, 1H), 7.87 (s, 1H), 7.65 (t, J=7.7 Hz, 1H), 7.55
(d, J = 7.2 Hz, 1H), 7.48 (d, J = 11.7 Hz, 2H), 7.38 – 7.32
(m, 2H), 7.29 (d, J=9.6 Hz, 3H), 5.25 (s, 2H), 4.61 (s, 1H),
4.49 (d, J=13.6 Hz, 2H), 3.95 (d, J=12.5 Hz, 1H), 3.73 (s,
2H), 3.49 (s, 2H), 3.28 (s, 2H), 3.12 (t, J = 12.0 Hz, 1H),
2.82 (t, J = 13.0 Hz, 1H), 2.61 (t, J= 5.5 Hz, 2H), 2.25 (s,
3H), 1.79 (s, 1H), 1.70 (d, J = 11.1 Hz, 2H), 1.12 (s, 2H);
13C NMR (101 MHz, DMSO-d6) δ (ppm): 169.80, 159.78,
159.36, 153.23, 141.60, 135.84, 134.38, 132.12, 130.61,
129.04, 128.45, 126.92, 126.82, 126.19, 124.87, 114.50,
101.21, 65.82, 59.62, 51.27, 50.55, 49.07, 47.06, 42.16,
38.71, 29.79, 28.79, 16.21; IR(KBr, cm−1) ν: 3438, 2927,
2851, 1631, 1500, 1448, 1412, 1389, 1250, 1060, 1006, 785,
700; MS (ESI) m/z: calculated for C33H36ClN3O5 [M+H]+:
590.23, found: 590.33.
(3‑Chloro‑4‑((3′‑(3‑((R)‑3‑hydroxypyrrolidine‑1‑carbonyl)
isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)methoxy)
benzyl)‑D‑serine (I‑5)
I-5 was prepared using similar procedures as described
for I-2. It was obtained as white solid (0.127 g, 67%). m.
1
p.: 176–180℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
7.96 (d, J = 7.5 Hz, 1H), 7.90 (s, 1H), 7.65 (t, J = 7.6 Hz,
1H), 7.55 (d, J=8.4 Hz, 2H), 7.49 (d, J=7.5 Hz, 1H), 7.44
(s, 1H), 7.40 – 7.24 (m, 4H), 5.27 (s, 2H), 5.08 (s, 1H),
4.34 (s, 1H), 3.92 (dd, J = 25.8, 13.2 Hz, 2H), 3.79 (dd,
J=21.7, 10.8 Hz, 2H), 3.63 (dd, J=14.0, 6.9 Hz, 2H), 3.51
(dd, J=33.6, 12.8 Hz, 2H), 3.13 (s, 1H), 2.25 (s, 3H), 1.93
(dd, J=31.7, 22.5 Hz, 2H); 13C NMR (101 MHz, DMSO)
δ 169.49, 160.01, 158.19, 153.41, 142.31, 141.01, 135.44,
133.88, 131.67, 131.18, 129.77, 129.34, 126.47, 125.73,
124.43, 121.35, 114.07, 101.35, 69.36, 69.19, 67.57, 62.38,
60.66, 56.43, 46.28, 44.63, 29.06, 15.77. IR(KBr, cm−1)
ν: 3372, 3056, 2953, 2882, 1625, 1503, 1443, 1411, 1370,
1296, 1259, 1065, 992, 886, 803, 784, 699, 606; MS (ESI)
(3‑Chloro‑4‑((3′‑(3‑(4‑(hydroxymethyl)piperidine‑1‑car‑
bonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)meth‑
oxy)benzyl)‑L‑serine (I‑3)
I-3 was prepared using similar procedures as described
for I-2. It was obtained as white solid (0.141 g, 92%). m.
1
p.: 152–156℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
7.94 (d, J = 7.7 Hz, 1H), 7.87 (s, 1H), 7.64 (d, J = 7.7 Hz,
1H), 7.55 (d, J = 6.1 Hz, 2H), 7.48 (d, J = 7.5 Hz, 1H),
7.40 – 7.22 (m, 5H), 5.27 (s, 2H), 4.49 (d, J = 12.7 Hz,
1H), 3.91 (dd, J=27.7, 13.2 Hz, 3H), 3.70 – 3.60 (m, 2H),
3.28 (d, J=5.6 Hz, 2H), 3.12 (t, J =11.7 Hz, 2H), 2.82 (t,
J = 11.6 Hz, 1H), 2.24 (s, 3H), 1.78 (d, J= 13.0 Hz, 1H),
1 3

Molecular Diversity
m/z: calculated for C32H32ClN3O7 [M+H]+: 606.19, found:
606.84.
(3‑Chloro‑4‑((3′‑(3‑(4‑hydroxypiperidine‑1‑carbonyl)
isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)methoxy)
benzyl)‑D‑serine (I‑8)
(3‑Chloro‑4‑((3′‑(3‑((R)‑3‑hydroxypyrrolidine‑1‑carbonyl)
isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)methoxy)
benzyl)‑L‑serine (I‑6)
I-8 was prepared using similar procedures as described
for I-2. It was obtained as white solid (0.070 g, 65%). m.
p.: 154–158℃; 1H NMR (400 MHz, DMSO-d6) δ 7.94 (d,
J=7.8 Hz, 1H), 7.87 (t, J=2.0 Hz, 1H), 7.69 – 7.60 (m, 2H),
7.54 – 7.42 (m, 3H), 7.36 (td, J=7.2, 6.4, 4.0 Hz, 3H), 5.28
(s, 2H), 4.94 (s, 1H), 4.01 (s, 3H), 3.98(d, J=14.8 Hz, 1H),
3.73 (d, J=28.3 Hz, 3H), 3.68 (d, J=6.0 Hz, 2H), 3.50 (s,
3H), 2.24 (s, 3H), 2.05 – 1.93 (m, 1H), 1.76 (d, J=12.0 Hz,
1H), 1.40 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ (ppm):
169.79, 159.69, 159.35, 153.74, 142.76, 141.58, 135.72,
134.38, 131.43, 130.18, 129.98, 129.80, 126.92, 126.79,
126.19, 121.73, 114.46, 101.25, 69.61, 65.57, 62.83, 61.39,
49.20, 44.49, 35.13, 34.20, 16.21; IR(KBr, cm−1) ν: 3382,
3060, 2963, 1609, 1504, 1448, 1414, 1340, 1290, 1260,
1157, 1063, 873, 802, 700, 609; MS (ESI) m/z: calculated
for C33H34ClN3O7 [M+H]+: 620.21, found: 620.48.
I-6 was prepared using similar procedures as described for
I-2. It was obtained as white solid (0.139 g, 68%). m. p.:
180–184℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.96
(d, J = 7.6 Hz, 1H), 7.90 (s, 1H), 7.65 (t, J= 7.6 Hz, 1H),
7.55 (d, J = 9.8 Hz, 2H), 7.48 (d, J = 7.3 Hz, 1H), 7.44 (s,
1H), 7.40 – 7.25 (m, 4H), 5.27 (s, 2H), 5.09 (s, 1H), 4.34
(s, 1H), 3.92 (dd, J = 25.4, 13.2 Hz, 2H), 3.84 – 3.70 (m,
2H), 3.68 – 3.61 (m, 2H), 3.51 (dd, J=33.0, 12.7 Hz, 2H),
3.14 (s, 1H), 2.25 (s, 3H), 2.01 – 1.92 (m, 1H), 1.85 (s, 1H);
13C NMR (75 MHz, DMSO-d6) δ 169.44, 160.35, 158.38,
153.63, 142.39, 141.24, 135.32, 134.06, 131.41, 130.01,
129.89, 129.47, 126.53, 126.45, 125.86, 121.43, 114.12,
101.38, 69.49, 69.29, 67.68, 62.50, 60.62, 56.55, 46.38,
44.75, 31.90, 15.86. IR (KBr, cm-1) ν: 3383, 3060, 2951,
2889, 1626, 1504, 1443, 1410, 1370, 1290, 1258, 1063,
991, 886, 804, 781, 698, 609; MS (ESI) m/z: calculated for
C32H32ClN3O7 [M+H]+: 606.19, found: 606.84.
(3‑Chloro‑4‑((3′‑(3‑(4‑hydroxypiperidine‑1‑carbonyl)
isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)methoxy)
benzyl)‑L‑serine (I‑9)
I-9 was prepared using similar procedures as described for
I-2. It was obtained as white solid (0.063 g, 75%). m. p.:
(S)‑1‑(3‑chloro‑4‑((3′‑(3‑(4‑hydroxypiperidine‑1‑carbo
nyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)methoxy)
benzyl)piperidine‑2‑carboxylic acid (I‑7)
1
154–158℃; H NMR (400 MHz, DMSO-d6) δ 7.94 (dd,
J = 7.8, 1.5 Hz, 1H), 7.86 (d, J = 1.9 Hz, 1H), 7.69 – 7.61
(m, 2H), 7.47 (dd, J = 11.8, 7.3 Hz, 3H), 7.39 – 7.32 (m,
3H), 5.28 (s, 2H), 4.94 (s, 1H), 4.03 (s, 3H), 3.82 – 3.73
(m, 3H), 3.70 (dd, J =11.7, 6.3 Hz, 2H), 3.50 (s, 3H), 2.24
(s, 3H), 1.76 (d, J = 15.5 Hz, 2H), 1.47 – 1.32 (m, 2H).
13C NMR (75 MHz, DMSO-d6) δ 170.27, 159.69, 159.36,
153.34, 142.78, 141.11, 135.83, 134.37, 131.29, 130.15,
129.81, 129.29, 126.93, 126.79, 126.20, 121.67, 114.44,
101.25, 69.65, 65.57, 63.21, 61.60, 49.07, 44.92, 35.13,
34.19, 16.21. IR (KBr, cm−1) ν: 3420, 2962, 1609, 1504,
1448, 1406, 1342, 1290, 1260, 16,063, 987, 874, 802,
699, 608; MS (ESI) m/z: calculated for C33H34ClN3O7
[M + H]+: 620.21, found: 620.68.
I-7 was prepared using similar procedures as described for
I-1. It was obtained as white solid (0.159 g, 44%). m. p.:
82–86℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.94 (d,
J=7.6 Hz, 1H), 7.86 (s, 1H), 7.66 (t, J=7.7 Hz, 1H), 7.62
– 7.52 (m, 2H), 7.52 – 7.39 (m, 3H), 7.40 – 7.25 (m, 3H),
5.32 (s, 2H), 4.43 (d, J=13.2 Hz, 1H), 4.07 (d, J=61.5 Hz,
4H), 3.78 (d, J=3.6 Hz, 2H), 3.56 (d, J=48.8 Hz, 1H), 3.31
(d, J=3.6 Hz, 2H), 2.97 (s, 1H), 2.26 (s, 3H), 2.18 (s, 1H),
1.75 (s, 6H), 1.53 (s, 1H), 1.46 – 1.34 (m, 2H); 13C NMR
(101 MHz, DMSO-d6) δ (ppm): 169.80, 159.72, 159.36,
154.99, 142.75, 141.66, 135.52, 134.48, 133.34, 132.16,
132.12, 130.30, 129.81, 128.57, 126.92, 126.82, 126.23,
124.89, 123.26, 122.04, 114.62, 101.26, 69.79, 65.56, 44.48,
35.13, 34.19, 21.97, 16.24; IR(KBr, cm−1) ν: 3428, 2928,
2860, 2550, 1676, 1635, 1504, 1457, 1414, 1291, 1264,
1202, 1139, 1065, 1027, 997, 799, 722, 700, 598; MS (ESI)
m/z: calculated for C36H38ClN3O6 [M+H]+: 644.24, found:
644.46.
3‑((4‑Chloro‑2‑(((2‑hydroxyethyl)amino)methyl)‑5‑((3′‑(3
‑(4‑(hydroxymethyl)piperidine‑1‑carbonyl)isoxazol‑5‑yl)‑
2‑methyl‑[1,1′‑biphenyl]‑3‑yl)methoxy)phenoxy)methyl)
benzonitrile (II‑1)
II-1 was prepared using similar procedures as described
for I-1 with sodium cyanoborohydride replac-
ing sodium triacetoxyborohydride and methanol/N,
N-dimethylformamide(v/v = 4/1) replacing 1,2-dichloro-
ethane. It was obtained as white solid (0.041 g, 19%).m.
1 3

Molecular Diversity
p.: 68–72℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.12
– 7.91 (m, 2H), 7.91 – 7.73 (m, 3H), 7.64 (d, J = 7.9 Hz,
2H), 7.53 (s, 1H), 7.48 (d, J = 10.6 Hz, 2H), 7.40 – 7.23
(m, 3H), 7.13 (s, 1H), 5.30 (d, J = 9.0 Hz, 4H), 4.80 (s,
1H), 4.59 – 4.44 (m, 2H), 3.95 (d, J = 11.7 Hz, 1H), 3.85
(s, 2H), 3.53 (s, 2H), 3.28 (s, 2H), 3.13 (t, J =12.2 Hz, 1H),
2.82 (t, J = 12.2 Hz, 1H), 2.71 (s, 2H), 2.26 (s, 3H), 1.77
(d, J = 14.3 Hz, 1H), 1.69 (s, 2H), 1.12 (s, 2H); 13C NMR
(101 MHz, DMSO-d6) δ (ppm): 169.77, 159.77, 159.33,
156.25, 154.27, 142.76, 141.63, 138.94, 135.64, 134.57,
132.85, 132.25, 132.12, 131.46, 131.36, 130.27, 129.80,
128.78, 126.89, 126.82, 126.21, 124.87, 119.16, 113.33,
111.99, 101.22, 100.90, 70.03, 69.41, 65.80, 59.11, 50.45,
47.05, 45.92, 42.15, 38.70, 29.78, 28.78, 16.32; IR(KBr,
cm−1) ν: 3386, 2922, 2851, 2229, 1632, 1578, 1503, 1448,
1410, 1297, 1164, 1085, 1035, 960, 893, 859, 792, 687;
MS (ESI) m/z: calculated for C41H41ClN4O6 [M + H]+:
721.3, found: 721.3.
1
p.: 165–169℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
8.00 – 7.92 (m, 2H), 7.84 (dd, J= 13.2, 5.6 Hz, 3H), 7.64
(dt, J=11.3, 7.8 Hz, 2H), 7.56 – 7.43 (m, 3H), 7.37 – 7.26
(m, 3H), 7.14 (s, 1H), 5.30 (d, J = 12.9 Hz, 4H), 4.51 (t,
J=14.6 Hz, 2H), 3.95 (d, J=13.1 Hz, 2H), 3.90 – 3.82 (m,
1H), 3.28 (d, J = 5.8 Hz, 2H), 3.16 – 3.09 (m, 1H), 3.04
(s, 1H), 2.89 (s, 1H), 2.86 – 2.79 (m, 1H), 2.73 (s, 1H),
2.26 (s, 3H), 1.91 (s, 1H), 1.78 (d, J= 12.8 Hz, 1H), 1.70
(d, J = 11.5 Hz, 2H), 1.55 (s, 3H), 1.42 (s, 1H), 1.12 (dd,
J = 13.0, 5.6 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ
(ppm): 169.78, 159.78, 159.36, 156.81, 154.87, 142.77,
141.67, 138.80, 135.56, 134.59, 133.01, 132.29, 132.10,
131.67, 130.35, 130.27, 129.79, 128.81, 126.89, 126.84,
126.21, 124.89, 119.11, 113.58, 112.00, 101.20, 100.91,
70.07, 69.53, 65.80, 47.04, 42.16, 38.70, 36.24, 31.41,
29.78, 28.77, 22.51, 16.31; IR(KBr, cm−1) ν: 3415, 2953,
2870, 2232, 1677, 1654, 1618, 1504, 1449, 1413, 1306,
1207, 1140, 1033, 838, 800, 722, 605; MS (ESI) m/z: cal-
culated for C45H45ClN4O7 [M+H]+: 789.3, found: 789.4.
N‑(2‑((5‑chloro‑2‑((3‑cyanobenzyl)oxy)‑4‑((3′‑(3‑(4‑(hydrox
ymethyl)piperidine‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,
1′‑biphenyl]‑3‑yl)methoxy)benzyl)amino)ethyl)acetamide
(II‑2)
3‑((4‑Chloro‑2‑(((1,3‑dihydroxy‑2‑(hydroxymethyl)pro‑
pan‑2‑yl)amino)methyl)‑5‑((3′‑(3‑(4‑(hydroxymethyl)piper‑
idine‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3
‑yl)methoxy)phenoxy)methyl)benzonitrile (II‑4)
II-2 was prepared using similar procedures as described
for II-1. It was obtained as white solid (0.130 g, 77%). m.
II-4 was prepared using similar procedures as described
for II-1. It was obtained as white solid (0.097 g, 42%). m.
1
p.: 58–62℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
1
7.95 (d, J= 9.3 Hz, 2H), 7.89 – 7.80 (m, 4H), 7.68 – 7.60
(m, 2H), 7.51 (dd, J = 16.7, 7.5 Hz, 2H), 7.41 (s, 1H),
7.35 (s, 1H), 7.30 (dd, J=10.5, 7.6 Hz, 2H), 7.10 (s, 1H),
5.28 (d, J = 10.8 Hz, 4H), 4.56 – 4.45 (m, 2H), 3.95 (d,
J = 12.5 Hz, 1H), 3.71 (s, 2H), 3.28 (d, J = 5.2 Hz, 2H),
3.13 (t, J=10.3 Hz, 3H), 2.84 (dd, J=23.9, 13.6 Hz, 1H),
2.58 (s, 2H), 2.26 (s, 3H), 1.74 (d, J=6.4 Hz, 1H), 1.70 (d,
J=11.3 Hz, 2H), 1.12 (s, 2H); 13C NMR (101 MHz, DMSO-
d6) δ (ppm): 169.79, 169.73, 159.80, 159.36, 155.88, 153.63,
142.80, 141.64, 139.13, 135.76, 134.54, 132.73, 132.20,
132.10, 131.37, 130.42, 130.28, 129.78, 128.75, 126.91,
126.85, 126.18, 124.87, 119.15, 113.45, 112.02, 101.23,
101.09, 70.05, 69.36, 65.83, 48.44, 47.05, 46.46, 42.17,
38.81, 38.72, 29.79, 28.78, 23.09, 16.31; IR(KBr, cm−1)
ν: 3410, 2926, 2852, 2230, 1637, 1578, 1504, 1460, 1411,
1291, 1164, 1091, 1036, 960, 887, 858, 793, 687; MS (ESI)
m/z: calculated for C43H44ClN5O6 [M+H]+: 762.3, found:
762.4.
p.: 84–88℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
7.95 (d, J = 11.3 Hz, 2H), 7.90 – 7.78 (m, 3H), 7.68 – 7.58
(m, 2H), 7.50 (dd, J = 12.0, 7.5 Hz, 2H), 7.43 (s, 1H), 7.38
– 7.25 (m, 3H), 7.07 (s, 1H), 5.27 (d, J = 15.0 Hz, 4H),
4.57 – 4.45 (m, 2H), 4.29 (s, 3H), 3.95 (d, J = 13.0 Hz,
1H), 3.74 (s, 2H), 3.40 (s, 6H), 3.28 (s, 2H), 3.13 (t,
J = 12.3 Hz, 1H), 2.84 (dd, J = 25.3, 13.1 Hz, 1H), 2.26 (s,
3H), 1.78 (d, J = 13.1 Hz, 1H), 1.70 (d, J = 10.6 Hz, 2H),
1.12 (s, 2H); 13C NMR (101 MHz, DMSO-d6) δ (ppm):
169.78, 159.77, 159.34, 155.67, 153.39, 142.78, 141.61,
139.10, 135.76, 134.54, 132.69, 132.14, 132.11, 131.27,
130.61, 130.26, 130.20, 129.79, 128.75, 126.90, 126.82,
126.17, 124.86, 119.15, 113.37, 111.94, 101.21, 101.11,
70.01, 69.28, 65.82, 61.50, 47.06, 42.16, 38.71, 29.78,
28.78, 16.30; IR(KBr, cm−1) ν: 3415, 2925, 2871, 2230,
1631, 1578, 1504, 1459, 1406, 1293, 1165, 1036, 960,
857, 793, 688; MS (ESI) m/z: calculated for C43H45ClN4O8
[M + H]+: 781.3, found: 781.4.
(S)‑1‑(5‑chloro‑2‑((3‑cyanobenzyl)oxy)‑4‑((3′‑(3‑(4‑(hydrox
ymethyl)piperidine‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,
1′‑biphenyl]‑3‑yl)methoxy)benzyl)piperidine‑2‑carboxylic
acid (II‑3)
(5‑Chloro‑2‑((3‑cyanobenzyl)oxy)‑4‑((3′‑(3‑(4‑(hydroxymet
hyl)piperidine‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑bip
henyl]‑3‑yl)methoxy)benzyl)proline (II‑5)
II-3 was prepared using similar procedures as described
for II-1. It was obtained as white solid (0.115 g, 49%). m.
II-5 was prepared using similar procedures as described
for II-1. It was obtained as white solid (0.109 g, 53%). m.
1 3

Molecular Diversity
1
p.: 128–132℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
(5‑Chloro‑2‑((3‑cyanobenzyl)oxy)‑4‑((3′‑(3‑(4‑(hydroxymet
hyl)piperidine‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑bip
henyl]‑3‑yl)methoxy)benzyl)‑L‑serine (II‑7)
8.03 (s, 1H), 7.99 – 7.79 (m, 4H), 7.64 (d, J= 7.5 Hz, 2H),
7.51 (s, 3H), 7.40 – 7.24 (m, 3H), 7.14 (s, 1H), 5.41 – 5.21
(m, 4H), 4.49 (d, J = 12.5 Hz, 1H), 4.06 (d, J = 13.2 Hz,
1H), 3.96 (d, J = 13.5 Hz, 2H), 3.49 (s, 1H), 3.28 (s, 2H),
3.17 (d, J = 9.4 Hz, 1H), 3.11 (d, J = 12.2 Hz, 1H), 2.83 (d,
J=11.7 Hz, 1H), 2.79 – 2.71 (m, 1H), 2.26 (s, 3H), 2.10 (s,
1H), 1.93 (s, 1H), 1.78 (d, J = 14.9 Hz, 2H), 1.69 (s, 3H),
1.12 (s, 2H); 13C NMR (101 MHz, DMSO-d6) δ (ppm):
171.92, 159.79, 159.36, 156.54, 154.85, 142.78, 141.66,
138.80, 135.57, 134.58, 132.97, 132.42, 132.29, 132.11,
131.64, 130.34, 130.27, 129.79, 128.79, 126.92, 126.85,
126.22, 124.88, 119.14, 116.94, 113.50, 101.23, 100.90,
70.04, 69.62, 66.86, 65.82, 53.78, 51.66, 47.06, 42.17,
II-7 was obtained by the hydrolysis of II-6 under the base
condition. To a solution of the II-6 (0.11 g, 0.141 mmol)
dissolved in methanol/water (4 mL/1 mL) was added lith-
ium hydroxide monohydrate (0.018 g, 0.424 mmol), and the
reaction mixture was stirred for 5 h at room temperature.
The mixture was concentrated and adjusted to pH 4–5 with
acetic acid. The precipitate formed was fltered, washed
with water and then dried to obtain II-7. It was obtained
as white solid (0.092 g, 85%). m. p.: 134–138℃; 1H NMR
(400 MHz, DMSO-d6) δ (ppm): 8.02 (s, 1H), 7.99 – 7.78
(m, 4H), 7.71 – 7.58 (m, 2H), 7.51 (t, J=11.3 Hz, 3H), 5.39
– 5.22 (m, 4H), 4.49 (d, J=12.4 Hz, 1H), 3.97 (d, J=9.1 Hz,
3H), 3.84 (s, 1H), 3.71 (d, J=10.4 Hz, 1H), 3.67 – 3.60 (m,
1H), 3.28 (d, J=5.3 Hz, 2H), 3.11 (d, J=11.9 Hz, 1H), 2.82
(t, J=11.8 Hz, 1H), 2.26 (s, 3H), 1.78 (d, J=12.7 Hz, 1H),
1.70 (d, J=10.7 Hz, 2H), 1.12 (s, 2H); 13C NMR (101 MHz,
DMSO-d6) δ (ppm): 169.77, 159.77, 159.34, 156.50, 154.78,
142.75, 141.63, 138.71, 135.55, 134.55, 132.94, 132.27,
132.11, 131.99, 131.51, 130.33, 130.24, 129.79, 128.75,
126.90, 126.82, 126.20, 124.86, 119.16, 116.96, 113.38,
111.99, 101.21, 100.77, 70.00, 69.56, 63.00, 61.11, 52.65,
47.05, 44.88, 42.16, 38.71, 29.78, 28.78, 16.31; IR(KBr,
cm−1) ν: 3379, 3050, 2926, 2860, 2229, 1720, 1632, 1507,
1448, 1410, 1337, 1307, 1201, 1163, 1086, 1038, 960, 790,
699; MS (ESI) m/z: calculated for C42H41ClN4O8 [M+Na]+:
787.3, found: 787.3.
38.72, 29.79, 29.03, 28.78, 23.63, 16.33; IR(KBr, cm−1
)
ν: 3408, 3056, 2925, 2867, 2230, 1635, 1504, 1458, 1411,
1386, 1305, 1167, 1089, 1036, 960, 896, 859, 792, 700;
MS (ESI) m/z: calculated for C44H43ClN4O7 [M + Na]+:
797.3, found: 797.4.
methyl(5‑chloro‑2‑((3‑cyanobenzyl)oxy)‑4‑((3′‑(3‑(4‑(hydro
xymethyl)piperidine‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,
1′‑biphenyl]‑3‑yl)methoxy)benzyl)‑L‑serinate (II‑6)
II-6 was prepared using similar procedures as described for
I-2 without the basic hydrolysis, and with sodium cyano-
borohydride replacing sodium triacetoxyborohydride and
methanol/N, N-dimethylformamide(v/v = 4/1) replacing
1,2-dichloroethane. It was obtained as white solid (0.185 g,
1
80%). m. p.: 60–64℃; H NMR (400 MHz, DMSO-d6)
δ (ppm): 7.99 – 7.91 (m, 2H), 7.88 – 7.80 (m, 3H), 7.64
(dt, J = 9.9, 7.8 Hz, 2H), 7.55 – 7.46 (m, 2H), 7.39 (s,
1H), 7.35 (s, 1H), 7.34 – 7.25 (m, 2H), 7.09 (s, 1H), 5.27
(d, J = 4.5 Hz, 4H), 4.90 (s, 1H), 4.54 (t, J= 5.3 Hz, 1H),
4.49 (d, J = 13.0 Hz, 1H), 3.95 (d, J = 13.4 Hz, 1H), 3.76
(d, J = 13.7 Hz, 1H), 3.64 (d, J = 13.8 Hz, 1H), 3.58 (d,
J=5.1 Hz, 2H), 3.55 (s, 3H), 3.28 (t, J=5.6 Hz, 2H), 3.14
(dd, J=22.4, 8.1 Hz, 1H), 2.90 – 2.78 (m, 1H), 2.26 (s, 3H),
1.78 (d, J = 12.8 Hz, 1H), 1.70 (d, J = 11.6 Hz, 2H), 1.18
– 1.06 (m, 2H); 13C NMR (101 MHz, DMSO-d6) δ (ppm):
169.79, 159.80, 159.36, 155.97, 153.71, 142.80, 141.65,
135.75, 132.69, 132.21, 132.11, 131.33, 130.27, 130.24,
129.79, 128.77, 126.91, 126.85, 126.19, 124.87, 113.43,
101.23, 101.05, 70.04, 69.39, 65.83, 62.83, 51.86, 47.05,
45.31, 42.17, 38.72, 29.79, 28.78, 16.31; IR(KBr, cm−1)
ν: 3416, 2927, 2868, 2230, 1736, 1634, 1578, 1503, 1459,
1402, 1292, 1198, 1165, 1088, 1036, 960, 890, 859, 792,
687; MS (ESI) m/z: calculated for C43H43ClN4O8 [M+Na]+:
801.3, found: 801.4.
3‑((4‑Chloro‑2‑(((1,3‑dihydroxypropan‑2‑yl)amino)methy
l)‑5‑((3′‑(3‑(4‑hydroxypiperidine‑1‑carbonyl)isoxazol‑5‑yl)
‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)methoxy)phenoxy)methyl)
benzonitrile (II‑8)
II-8 was prepared using similar procedures as described for
II-1. It was obtained as white solid (0.106 g, 48%). m. p.:
1
74–78℃; H NMR (400 MHz, DMSO-d6) δ (ppm): 8.01
– 7.91 (m, 2H), 7.90 – 7.78 (m, 3H), 7.64 (dt, J = 12.3,
7.7 Hz, 2H), 7.51 (dd, J=17.0, 7.1 Hz, 2H), 7.44 (s, 1H),
7.36 (s, 1H), 7.30 (dd, J=10.9, 7.5 Hz, 2H), 7.10 (s, 1H),
5.28 (d, J = 9.4 Hz, 4H), 4.85 (d, J = 3.6 Hz, 1H), 4.54 (s,
2H), 4.06 – 3.97 (m, 1H), 3.79 (s, 4H), 3.40 (dd, J =15.2,
4.9 Hz, 4H), 3.32 – 3.26 (m, 2H), 2.61 (s, 1H), 2.26 (s, 3H),
1.77 (d, J = 15.0 Hz, 2H), 1.45 – 1.35 (m, 2H); 13C NMR
(101 MHz, DMSO-d6) δ (ppm): 169.80, 159.70, 159.36,
155.98, 153.68, 142.78, 141.62, 139.05, 135.73, 134.55,
132.80, 132.22, 131.41, 130.71, 130.28, 129.80, 128.77,
126.91, 126.82, 126.20, 124.87, 119.15, 113.29, 111.99,
101.27, 100.96, 70.01, 69.34, 65.58, 61.04, 60.65, 44.81,
44.49, 35.14, 34.20, 16.31; IR(KBr, cm−1) ν: 3387, 3060,
2927, 2873, 2229, 1633, 1578, 1504, 1457, 1410, 1295,
1 3

Molecular Diversity
1265, 1226, 1195, 1164, 1064, 1025, 792, 688; MS (ESI)
m/z: calculated for C41H41ClN4O7 [M+H]+: 737.3, found:
737.3.
(S)‑3‑((4‑chloro‑2‑(((2,3‑dihydroxypropyl)amino)methyl)‑
5‑((3′‑(3‑(4‑hydroxypiperidine‑1‑carbonyl)isoxazol‑5‑yl)‑
2‑methyl‑[1,1′‑biphenyl]‑3‑yl)methoxy)phenoxy)methyl)
benzonitrile (II‑11)
3‑((4‑Chloro‑2‑(((1,3‑dihydroxy‑2‑(hydroxymethyl)
propan‑2‑yl)amino)methyl)‑5‑((3′‑(3‑(4‑hydroxypiperidin
e‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)
methoxy)phenoxy)methyl)benzonitrile (II‑9)
II-11 was prepared using similar procedures as described
for II-1. It was obtained as white solid (0.154 g, 69%). m.
1
p.: 120–124℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
8.00 – 7.92 (m, 2H), 7.85 (dd, J= 11.3, 6.3 Hz, 3H), 7.64
(dt, J = 15.8, 7.8 Hz, 2H), 7.51 (dd, J = 16.8, 5.4 Hz, 3H),
7.38 – 7.28 (m, 3H), 7.14 (s, 1H), 5.30 (d, J=10.9 Hz, 4H),
4.98 (s, 1H), 4.86 (d, J=3.3 Hz, 1H), 4.06 – 3.97 (m, 1H),
3.88 (s, 2H), 3.77 (d, J = 11.9 Hz, 2H), 3.66 (s, 1H), 3.38
(dd, J=11.0, 5.2 Hz, 2H), 3.29 (dd, J=10.9, 6.3 Hz, 2H),
2.81 – 2.72 (m, 1H), 2.58 (dd, J= 11.9, 8.3 Hz, 1H), 2.26
(s, 3H), 1.79 (dd, J=19.6, 10.6 Hz, 2H), 1.40 (dt, J=12.4,
8.8 Hz, 2H); 13C NMR (101 MHz, DMSO-d6) δ (ppm):
169.79, 159.70, 159.35, 156.36, 154.41, 142.76, 141.63,
138.90, 135.63, 134.58, 132.90, 132.26, 132.11, 131.58,
131.48, 130.32, 130.28, 129.79, 128.78, 126.90, 126.81,
126.20, 124.87, 119.17, 118.67, 113.32, 111.99, 101.26,
100.87, 70.04, 69.45, 69.35, 65.57, 64.53, 51.42, 46.12,
44.49, 35.13, 34.19, 16.33; IR(KBr, cm−1) ν: 3352, 3060,
2924, 2873, 2230, 1632, 1579, 1504, 1453, 1411, 1299,
1265, 1226, 1164, 1066, 1022, 792, 688; MS (ESI) m/z:
calculated for C41H41ClN4O7 [M+H]+: 737.3, found: 737.3.
II-9 was prepared using similar procedures as described for
II-1. It was obtained as white solid (0.139 g, 60%). m. p.:
1
68–72℃; H NMR (400 MHz, DMSO-d6) δ (ppm): 7.99
– 7.91 (m, 2H), 7.86 (s, 2H), 7.81 (d, J=7.7 Hz, 1H), 7.63
(dt, J=15.9, 7.8 Hz, 2H), 7.55 – 7.41 (m, 3H), 7.37 – 7.27
(m, 3H), 7.08 (s, 1H), 5.28 (d, J = 14.1 Hz, 4H), 4.85 (d,
J=4.0 Hz, 1H), 4.31 (s, 3H), 4.05 – 3.98 (m, 1H), 3.77 (dd,
J=8.0, 4.1 Hz, 4H), 3.42 (s, 6H), 3.33 – 3.27 (m, 2H), 2.26
(s, 3H), 1.79 (s, 2H), 1.40 (dd, J=8.6, 3.6 Hz, 2H); 13C NMR
(101 MHz, DMSO-d6) δ (ppm): 169.80, 159.71, 159.36,
155.77, 153.53, 142.78, 141.62, 139.08, 135.75, 134.55,
132.71, 132.15, 131.29, 130.20, 129.80, 128.77, 126.90,
126.81, 126.18, 124.87, 119.16, 113.35, 111.95, 101.26,
101.09, 70.02, 69.30, 65.59, 44.49, 35.13, 34.19, 16.30;
IR(KBr, cm−1) ν: 3386, 3063, 2928, 2875, 2230, 1631, 1578,
1504, 1458, 1409, 1296, 1265, 1226, 1166, 1066, 1022, 792,
688; MS (ESI) m/z: calculated for C42H43ClN4O8 [M+H]+:
767.3, found: 767.4.
(R)‑3‑((4‑chloro‑2‑(((1,3‑dihydroxy‑2‑(hydroxymethyl)
propan‑2‑yl)amino)methyl)‑5‑((3′‑(3‑(3‑hydroxypyrrolidin
e‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)
methoxy)phenoxy)methyl)benzonitrile (II‑12)
3‑((4‑Chloro‑2‑(((2‑hydroxyethyl)amino)methyl)‑5‑((3′‑(3‑(
4‑hydroxypiperidine‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1
,1′‑biphenyl]‑3‑yl)methoxy)phenoxy)methyl) benzonitrile
(II‑10)
II-12 was prepared using similar procedures as described
for II-1. It was obtained as white solid (0.105 g, 45%). m.
p.: 76–80℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 7.95
(s, 2H), 7.85 (d, J=26.1 Hz, 3H), 7.70 – 7.56 (m, 2H), 7.49
(s, 2H), 7.42 (d, J=6.3 Hz, 2H), 7.30 (s, 2H), 7.07 (s, 1H),
5.27 (d, J = 14.2 Hz, 4H), 5.04 (s, 1H), 4.34 (s, 1H), 4.27
(s, 3H), 3.92 – 3.75 (m, 2H), 3.73 (s, 2H), 3.67 – 3.54 (m,
2H), 3.40 (s, 6H), 2.27 (s, 3H), 1.91 (d, J = 45.5 Hz, 2H);
13C NMR (101 MHz, DMSO-d6) δ (ppm): 169.75, 160.71,
158.68, 155.65, 153.35, 142.77, 141.61, 139.12, 135.76,
134.54, 132.67, 132.12, 131.26, 130.54, 130.20, 129.76,
128.73, 126.88, 126.16, 124.86, 124.18, 119.15, 113.40,
111.96, 101.77, 101.13, 74.26, 70.02, 69.85, 69.28, 68.03,
61.61, 60.68, 46.71, 45.08, 32.28, 16.31; IR(KBr, cm−1)
ν: 3379, 2926, 2881, 2230, 1626, 1578, 1505, 1445, 1407,
1372, 1297, 1236, 1166, 1012, 793, 688; MS (ESI) m/z:
calculated for C41H41ClN4O8 [M+H]+: 753.3, found: 753.4.
II-10 was prepared using similar procedures as described
for II-1. It was obtained as white solid (0.097 g, 50%). m.
1
p.: 82–86℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
8.00 – 7.92 (m, 2H), 7.84 (dd, J= 13.7, 6.2 Hz, 3H), 7.64
(dt, J = 10.5, 7.8 Hz, 2H), 7.51 (dd, J = 17.9, 6.8 Hz, 2H),
7.42 (s, 1H), 7.39 – 7.26 (m, 3H), 7.11 (s, 1H), 5.28 (d,
J=9.4 Hz, 4H), 4.85 (s, 1H), 4.62 (s, 1H), 4.03 (dd, J=16.4,
9.5 Hz, 1H), 3.76 (s, 4H), 3.49 (s, 2H), 3.32 – 3.26 (m,
2H), 2.63 (t, J=5.5 Hz, 2H), 2.26 (s, 3H), 1.79 (s, 2H), 1.40
(dd, J=8.6, 3.6 Hz, 2H); 13C NMR (101 MHz, DMSO-d6)
δ (ppm): 169.80, 159.70, 159.35, 156.01, 153.80, 142.78,
141.63, 139.06, 135.72, 134.55, 132.76, 132.21, 132.11,
131.38, 130.73, 130.27, 129.79, 128.76, 126.91, 126.83,
126.19, 124.87, 121.44, 119.16, 113.36, 112.00, 101.27,
100.98, 70.02, 69.34, 65.58, 60.05, 51.01, 46.56, 44.48,
35.14, 34.20, 16.32; IR(KBr, cm−1) ν: 3371, 3060, 2926,
2870, 2230, 1634, 1606, 1578, 1504, 1453, 1409, 1296,
1265, 1226, 1164, 1066, 1022, 793, 688; MS (ESI) m/z:
calculated for C40H39ClN4O6 [M+H]+: 707.3, found: 707.3.
1 3

Molecular Diversity
(R)‑3‑((4‑chloro‑2‑(((1,3‑dihydroxypropan‑2‑yl)amino)
methyl)‑5‑((3′‑(3‑(3‑hydro xypyrrolidine‑1‑carbonyl)isoxa
zol‑5‑yl)‑2‑methyl‑[1,1′‑biphenyl]‑3‑yl)methoxy)phenoxy)
methyl)benzonitrile (II‑13)
In vitro assay of PD‑1/PD‑L1 interaction inhibitory activity
Time-Resolved Fluorescence Resonance Energy Transfer
(TR-FRET) assays to evaluate the synthesized small-mole-
cule inhibitors for inhibiting PD-1/PD-L1 interactions were
conducted in a 384-well microplate (PerkinElmer) with a
fnal volume of 20 μL. Inhibitors were frst serially diluted
in DMSO and then added to the 384-well microplate before
the addition of other reaction components. BMS-202 (Sell-
eckchem) was used as the positive compound. PD-1-Eu, PD-
L1-Biotin and Dye labeled acceptor were purchased from
BPS Bioscience. PD-L1-Biotin was diluted in the 1×modi-
fed TR-FRET assay bufer, and 5 μL was added to the com-
pound well and the positive control well, respectively. In
total, 5μL of 1×modifed TR-FRET assay bufer was added
to the negative control well. The components in the micro-
plate were centrifuged at 1000 rpm for 30 s and incubated
with inhibitors for 15 min at room temperature. PD-L1-Eu
and Dye-labeled acceptor were diluted in the 1×modifed
TR-FRET assay bufer, respectively. In total, 5 μL diluted
PD-L1-Eu bufer and 10 μL diluted Dye-labeled acceptor
bufer were added to the microplate. The mixtures were cen-
trifuged at 1000 rpm for 30 s and incubated with inhibitors
for 90 min at room temperature. The 384-well microplate
was centrifuged at 1000 rpm for 30 s. Then the fuorescence
intensity at 665 nm and 620 nm was read on a microplate
reader, and the TR-FRET ratio (665 nm emission/620 nm
emission) was calculated. IC50 value was determined by
ftting the curve of the log of the inhibitor concentration
versus response variable slope using the GraphPad Prism
5 software.
II-13 was prepared using similar procedures as described
for II-1. It was obtained as white solid (0.100 g, 45%). m.
1
p.: 105–109℃; H NMR (400 MHz, DMSO-d6) δ (ppm):
7.96 (d, J=6.4 Hz, 2H), 7.89 (s, 1H), 7.87 – 7.77 (m, 2H),
7.64 (dt, J= 11.5, 7.8 Hz, 2H), 7.51 (dd, J= 18.4, 7.3 Hz,
2H), 7.41 (s, 2H), 7.31 (q, J = 7.6 Hz, 2H), 7.08 (s, 1H),
5.27 (d, J=9.7 Hz, 4H), 5.04 (s, 1H), 4.39 (s, 2H), 4.34 (s,
1H), 3.87 – 3.74 (m, 2H), 3.73 (s, 2H), 3.62 (dd, J =23.2,
11.0 Hz, 2H), 3.38 (dt, J = 15.9, 8.0 Hz, 4H), 2.58 – 2.52
(m, 1H), 2.27 (s, 3H), 2.01 – 1.82 (m, 2H); 13C NMR
(101 MHz, DMSO-d6) δ (ppm): 169.75, 169.69, 160.72,
160.66, 158.69, 158.65, 155.83, 153.38, 142.77, 141.61,
139.12, 135.77, 134.53, 132.74, 132.19, 132.09, 131.35,
130.33, 130.26, 129.77, 128.72, 126.89, 126.84, 126.83,
126.18, 124.86, 123.52, 119.15, 113.29, 111.98, 101.79,
101.75, 101.00, 70.00, 69.84, 69.29, 68.02, 61.59, 60.66,
46.70, 45.13, 45.08, 32.28, 16.32; IR(KBr, cm−1) ν: 3317,
2924, 2851, 2227, 1624, 1579, 1506, 1443, 1410, 1372,
1297, 1197, 1165, 1053, 800, 696; MS (ESI) m/z: calculated
for C40H39ClN4O7 [M+H]+: 723.3, found: 723.4.
(5‑Chloro‑2‑((3‑cyanobenzyl)oxy)‑4‑((3′‑(3‑((R)‑3‑hydroxyp
yrrolidine‑1‑carbonyl)isoxazol‑5‑yl)‑2‑methyl‑[1,1′‑bipheny
l]‑3‑yl)methoxy)benzyl)‑L‑serine (II‑14)
II-14 was prepared using similar procedures as described
for II-6, followed by the basic hydrolysis as described for
II-7. It was obtained as white solid (0.096 g, 86%). m. p.:
134–138℃; 1H NMR (400 MHz, DMSO-d6) δ (ppm): 8.02
(s, 1H), 8.00 – 7.87 (m, 3H), 7.83 (d, J=7.8 Hz, 1H), 7.68
– 7.59 (m, 2H), 7.57 – 7.46 (m, 3H), 7.43 (s, 1H), 7.32 (d,
J=7.7 Hz, 2H), 7.15 (d, J=10.9 Hz, 1H), 5.32 (dd, J=15.0,
12.2 Hz, 4H), 5.11 (s, 1H), 4.34 (s, 1H), 3.98 (d, J=10.5 Hz,
2H), 3.82 – 3.70 (m, 2H), 3.64 (dd, J = 11.4, 6.4 Hz, 2H),
3.58 – 3.46 (m, 2H), 3.19 (s, 1H), 2.26 (s, 3H), 2.00 – 1.80
(m, 2H); 13C NMR (101 MHz, DMSO-d6) δ (ppm): 169.96,
169.75, 160.72, 158.69, 156.55, 154.87, 142.74, 141.63,
138.69, 135.53, 134.55, 132.95, 132.28, 132.10, 131.52,
130.36, 130.24, 129.77, 128.74, 126.88, 126.21, 124.88,
119.16, 116.60, 113.39, 112.00, 101.78, 100.76, 70.01,
69.84, 69.58, 68.01, 62.99, 60.97, 46.71, 45.09, 44.80,
32.28, 16.31; IR(KBr, cm−1) ν: 3306, 3046, 2946, 2889,
2230, 1720, 1632, 1577, 1508, 1444, 1410, 1372, 1338,
1307, 1200, 1163, 1064, 1039, 805, 699; MS (ESI) m/z:
calculated for C40H37ClN4O8 [M+H]+: 759.2, found: 759.3.
Docking studies
All ligand molecules were drawn in ChemDraw 2017 and
saved as sdf style. Then ligands were processed at a sim-
ulated pH of 7.4 2.0 to generate all possible tautomers,
stereoisomers and protonation states and were fnally mini-
mized at the OPLS3e force feld with ligand preparation pro-
tocol of Maestro 11.6. The crystal complex (PDB ID: 6R3K)
was selected as the PD-L1 docking protein. The protein was
opened with Glide generation protocol of Maestro 11.6,
molecules of water around the binding site were removed,
and the bond orders were assigned. Hydrogen atoms were
added. Then a 36-Å box centered on the geometrical center
of the ligand binding site was generated for grid calculation.
The docking studies were processed with the Glide docking
protocol. After docking fnished, only one docking confor-
mation was saved for every compound.
1 3

Molecular Diversity
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inhibitors : skin toxicities and immunotherapy. Am J Clin Derma-
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7816
Supplementary Information The online version contains supplemen-
tary material available at https://doi.org/10.1007/s11030-021-10208-4.
Acknowledgements This study was supported by the Natural Science
Foundation of Jiangsu Province (No. BK 20141349) and the China
National Key HiTech Innovation Project for the R&D of Novel Drugs
(No. 2013ZX09301303-002).
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Compliance with ethical standards
Conflict of Interest The authors have no relevant afliations or fnan-
cial involvement with any organization or entity with a fnancial in-
terest in or fnancial confict with the subject matter or materials dis-
cussed in the manuscript.
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Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
1 3

Molecular Diversity
Authors and Afliations
Peiyu Zhu1 · Jian Zhang1 · Yifei Yang2 · Lixun Wang1 · Jinpei Zhou2 · Huibin Zhang1
1
2
Center of Drug Discovery, Jiangsu Key Laboratory of Drug
Discovery for Metabolic Disease, China Pharmaceutical
University, Nanjing 210009, People’s Republic of China
Department of Medicinal Chemistry, China Pharmaceutical
University, Nanjing 210009, People’s Republic of China
1 3