Design, Synthesis, and Biological Evaluation of Linear Aliphatic Amine-Linked Triaryl Derivatives as Potent Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction with Promising Antitumor Effects in Vivo

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Design, Synthesis, and Biological Evaluation of Linear Aliphatic

Amine-Linked Triaryl Derivatives as Potent Small-Molecule

Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-

Ligand 1 Interaction with Promising Antitumor Eﬀects In Vivo

Jialin Guo,^§Longlong Luo,^§Zhihong Wang, Naijing Hu, Wei Wang, Fei Xie, Erguang Liang, Xinlin Yan,

Junhai Xiao,* and Song Li

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ABSTRACT: A series of novel linear aliphatic amine-linked triaryl

derivatives as inhibitors of PD-1/PD-L1 were designed, synthe-

sized, and evaluated in vitro and in vivo. In this chemical series,

compound 58 showed the most potent inhibitory activity and

binding aﬃnity with hPD-L1, with an IC₅₀value of 12 nM and a

KD value of 16.2 pM, showing a binding potency approximately

2000-fold that of hPD-1. Compound 58 could bind with hPD-L1

on the cellular surface and competitively block the interaction of

hPD-1 with hPD-L1. In a T cell function assay, 58 restored the T cell function, leading to increased IFN-γ secretion. Moreover, in a

humanized mouse model, compound 58 signiﬁcantly inhibited tumor growth without obvious toxicity and showed moderate PK

properties after intravenous injection. These results indicated that 58 is a promising lead for further development of small-molecule

PD-1/PD-L1 inhibitors for cancer therapy.

FDA-approved mAbs targeting PD-1 or PD-L1 (anti-PD-1:

nivolumab, pembrolizumab, and cemiplimab; anti-PD-L1:

atezolizumab, avelumab, and durvalumab) are clinically

available.^7,12In addition, numerous mAbs have entered clinical

trials worldwide.^13,14

INTRODUCTION

■

Cancer immunotherapy activates and enhances pre-existing

anticancer immune responses of the host immune system to

eliminate cancer cells.¹⁻³Undoubtedly, the most remarkable

progress in this ﬁeld is the development of programmed cell

death-1 (PD-1)/programmed cell death-ligand 1 (PD-L1)

checkpoint inhibitors. PD-1 is expressed on the surface of

immune eﬀector cells, including T cells, B cells, natural killer T

cells, monocytes, and dendritic cells. PD-L1 is constitutively

expressed on immune cells and the cells of various tissues,

including the heart, lung, liver, pancreatic islet, vascular

endothelium, atrocities, etc.^4,5Under normal physiological

conditions, the interaction of PD-1 and PD-L1 suppresses the

proliferation, inﬂammatory cytokine secretion, such as that of

IL-2 and interferon-γ (IFN-γ), and cytotoxic activity of T cells

and increases their susceptibility to apoptosis, thereby

maintaining self-tolerance and protecting tissues from un-

necessary damage during immune responses.⁶⁻⁸However,

tumor cells exploit this mechanism to evade immune

surveillance and facilitate their own survival by interaction

between overexpressed PD-L1 on their surface and PD-1 on T

cells.^1,9

Despite the good clinical eﬃcacy, durable responses, and

prolonged survival achieved with mAbs, their intrinsic defects

limit their widespread clinical application. In comparison with

mAbs, small-molecule inhibitors have more appropriate

pharmacokinetic (PK) properties, stability, and oral bioavail-

ability; broader distribution; lower costs of production,

transportation, and storage; enhanced tumor and tissue

penetration; relatively low immunogenicity; and easier reversal

of immune-related adverse events due to their relatively shorter

half-lives, which make them potential alternatives or

complementary drugs to regulate the PD-1/PD-L1 path-

way.¹⁵⁻²³For these reasons, the discovery of small-molecule

inhibitors has become an attractive strategy for inhibiting the

PD-1/PD-L1 interaction, but their development is far behind

that of mAbs.

Received: August 6, 2020

Several clinical trials of humanized monoclonal antibodies

(mAbs) targeting PD-1/PD-L1 have exhibited dramatic

antitumor eﬃcacy and long-term remission in patients.⁹⁻¹¹

Blocking the PD-1/PD-L1 interaction has been one of the

most promising strategies for cancer treatment. To date, six

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A

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Figure 1. Chemical structures of reported small-molecule inhibitors of PD-1/PD-L1.

Figure 2. Design strategy of compounds.

To date, the majority of reported small-molecule inhibitors

targeting the PD-1/PD-L1 pathway remain in the preclinical

stage, and only CA-170 and IMMH-010 have entered phase I

clinical trials. Compounds with triaryl scaﬀolds are the

predominant class of small-molecule inhibitors thus far. The

ﬁrst small-molecule inhibitors of PD-1/PD-L1 with triaryl

scaﬀolds, such as BMS202 and BMS1166, were disclosed by

researchers from Bristol Myers Squibb (BMS) and are shown

in Figure 1. Most of these compounds showed high inhibitory

potency of the PD-1/PD-L1 interaction, with IC₅₀values in

the nanomolar range.²⁴⁻²⁶Further investigation revealed that

the BMS compounds bound to the PD-1 binding site of PD-L1

and induced dimerization of PD-L1, further occluding the PD-

1 binding site of PD-L1.²⁷⁻²⁹Inspired by this remarkable

progress, a series of triaryl scaﬀold derivatives were developed

by other scientists (Figure 1). These derivatives also showed

potential inhibitory activity in biochemical assays.³⁰⁻³⁶Despite

the good biochemical activity of triaryl scaﬀold derivatives, no

further investigation data in vitro and in vivo were disclosed to

exhibit their potential antitumor and druggability properties.

To date, no small-molecule inhibitors have been clinically

approved, nor have their antitumor eﬀects been validated in

clinical trials. The discovery and development of small-

molecule PD-1/PD-L1 inhibitors with novel chemo-types,

relatively high potency, appropriate PK properties, and

optimized therapeutic eﬀects are still needed. Although it is a

challenge to discover a novel inhibitor due to the large, ﬂat,

and hydrophobic interface of PD-1/PD-L1,³⁷further inves-

tigation, including modiﬁcation and structure−activity relation-

ship (SAR) studies, of reported inhibitors will lead to a new

generation of highly potent inhibitors and accelerate the

development of small-molecule inhibitors targeting PD-1/PD-

L1.

In this paper, we designed and synthesized a series of novel

triaryl derivatives with linear aliphatic amine linkers as small-

molecule inhibitors targeting the PD-1/PD-L1 interaction;

investigated their preliminary SARs; and evaluated their

biochemical activities, binding speciﬁcity, aﬃnity, binding

and blocking ability at the cellular level, and eﬀects on T cell

function regulation using reasonable evaluation models in

vitro. Then, we utilized an authentic pharmacodynamic model

to evaluate the antitumor eﬀect of the compounds in vivo. The

results of biological evaluation in vitro and in vivo indicated

that compound 58 was the most promising small-molecule

inhibitor of PD-1/PD-L1 of these compounds, which

remarkably inhibited tumor growth in a humanized mouse

B

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model by inhibiting the PD-1/PD-L1 interaction, leading to

restoration of antitumor immunity.

together via a linker, which makes up the triaryl scaﬀold of the

inhibitors. The biaryl core provides a signiﬁcant contribution

to inhibitor binding though hydrophobic interactions, such as

π−π, π−alkyl, and π−σ interactions. The aryl group generates

a strong π−π stacking with Tyr56 of the PD-L1 protein, which

can tolerate a wide variety of aromatic heterocycles, including

fused heterocycles. The ether-linked group is not essential, but

its presence, especially that of a cyano-substituted aryl group,

increases the binding potency. The extension is usually linked

to the biaryl core via an ether bond, which is not necessary for

inhibitor binding but is helpful for improving PK properties,

especially regarding hydrophilic substituents. The tail is

exposed to the solvent region and forms key hydrogen bond

interactions with PD-L1, which is also required for high

potency.

RESULTS AND DISCUSSION

■

Drug Design and Chemistry. As shown in Figure 2,

reported small-molecule inhibitors with triaryl scaﬀolds shared

structural features with the BMS compounds. According to the

binding modes of BMS compounds with PD-L1,²⁷⁻²⁹with

representative binding modes shown in Figure 3, the SARs of

On the basis of this SAR information, the exploration of

linkers is likely a feasible way to develop novel inhibitors and

further investigate the SARs of small-molecule inhibitors with

triaryl scaﬀolds. Compared to other reported linkers, aliphatic

amines are signiﬁcantly diﬀerent in geometry, and their

introduction will generate inhibitors with diﬀerent molecular

conformations. Aliphatic amines have hydrogen bond donors

and acceptors, which probably form undiscovered interactions

with the Ala121 residue of PD-L1. In addition, the hydro-

philicity of aliphatic amines is probably advantageous in terms

of providing drug-like properties. On the basis of these

considerations, three aliphatic amine linkers were designed to

develop novel small-molecule inhibitors, as shown in Figure 2.

The syntheses of 48−75 are outlined in Scheme 1. The

Suzuki−Miyaura coupling of 10 with phenylboronic acid or

(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)boronic acid yielded

11a or 11b, respectively. Paths A and B were designed to

Figure 3. Representative binding mode of BMS compounds with PD-

L1. BMS202 (green) is located in the hydrophobic cleft formed by

dimeric PD-L1 (the PDB code for dimeric PD-L1 protein is 5J89).

these reported small-molecule inhibitors with triaryl scaﬀolds

were analyzed. The aryl group and biaryl core are linked

a

Scheme 1. Syntheses of Compounds 48−75

a

Reagents and conditions: (a) R₆B(OH)₂, PdCl₂dppf, CsOAc, argon, THF, reﬂux, 23−24 h, and 98%. (b) PBr₃, DCM, 0 °C, 20 min, and 66−86%.

(c) o-Phthalimide, Ph₃P, DEAD, argon, anhydrous THF, 0 °C−rt, 24 h, and 92−97%. (d) H₄N₂·H₂O, EtOH, reﬂux, 3−6 h, and 96−99%. (e)

K₂CO₃, MeCN/DMF, 60 °C−reﬂux, 10−18 h or 35 min (microwave), and 20−57%. (f) Pd(OAc)₂, BINAP or s-phos, Cs₂CO₃, argon, dioxane, 85

°C−reﬂux, 20−24 h, and 16−78%. (g) (1) R₄−NH₂, anhydrous DMF/MeOH/DCE, AcOH, rt, and 12−24 h; (2) NaBH(OAc)₃, 12−24 h, and 8−

79%.

C

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a

Scheme 2. Syntheses of Compounds 87−91

a

Reagents and conditions: (a) PhB(OH)₂, PdCl₂dppf, CsOAc, argon, THF, reﬂux, 36 h, and 52−95%. (b) NBS, BPO, CCl₄, reﬂux, 10−11 h, and

62−65%. (c) Phthalimide potassium salt, DMF, 80 °C, 1.5 h, and 60% (two steps from 77a). (d) H₄N₂·H₂O, EtOH, reﬂux, 3 h, and 98%. (e)

K₂CO₃, MeCN/DMF, 60 °C−reﬂux, 7.5−11 h, and 12−25%. (f) Pd(OAc)₂, BINAP, Cs₂CO₃, dioxane, argon, reﬂux, 16 h, and 67%. (g) (1) D-

serine, anhydrous DMF/MeOH, AcOH, 35 °C, and 24 h; (2) NaBH(OAc)₃, 24 h, and 22−70%.

a

Scheme 3. Syntheses of Compounds 93, 94, and 100

a

Reagents and conditions: (a) PhSO₂OMe, KOH, THF, 35 °C, 40 h, and 63%. (b) (1) R₄−NH₂, anhydrous DMF/DCE, AcOH, rt, and 24 h; (2)

NaBH(OAc)₃, 12−14 h, and 38−60%. (c) 5-(Chloromethyl)nicotinonitrile, K₂CO₃, MeCN, 60 °C, 10 h, and 84%. (d) MsCl, pyridine, anhydrous

DMF/DCM, 0 °C−rt, 24 h, and 63%. (e) 98, K₂CO₃, MeCN/DMF, 30 °C, 24 h, and 31%. (f) (1) D-serine, anhydrous DMF/MeOH, AcOH, 35

°C, and 24 h; (2) NaBH(OAc)₃, 24 h, and 23%.

synthesize the key intermediates 30−47 using aryl halides 19−

29 and aryl amines 15−18, respectively. Intermediates 15−29

were purchased or synthesized as described in the

Experimental Section. As described in path B, 12a-b were

prepared from 11a-b using phosphorus tribromide, and then,

K₂CO₃-promoted N-alkylation of 12a-b and 15−18 was

D

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Table 1. Inhibitory Activities of Compounds 48−52 toward the PD-1/PD-L1 Interaction

a

b

Data are displayed as averages of triplicate assays SD. BMS202 reference value: IC₅₀= 18 nM.

performed to obtain the key intermediates 30−34. As

described in path A, the syntheses of 14a-b employed the

Mitsunobu reaction of 11a-b with o-phthalimide followed by a

hydrazinolysis reaction. The subsequent Buchwald−Hartwig

cross-coupling reaction of 14a-b and 19−29 provided the key

intermediates 35−47. The target molecules 48−75 were

generated by reductive amination using NaBH(OAc)₃and the

appropriate amines.

The syntheses of 87−91 are shown in Scheme 2.

Compounds 78a-b were transformed from 76a-b and phenyl-

boronic acid using the Suzuki−Miyaura coupling reaction

followed by free radical-mediated bromination. By adopting

synthetic routes similar to those of intermediates 30−47,

intermediates 82−86 were generated, which were transformed

to desired compounds 87−91 using a NaBH(OAc)₃-mediated

reductive amination.

activities of the PD-1/PD-L1 interaction were evaluated at the

biochemical level using a homogeneous time-resolved

ﬂuorescence (HTRF) assay. BMS202 was used as a positive

control in the assay. As shown in Table 1, compounds 48−52

exhibited moderate inhibitory activities toward the PD-1/PD-

L1 interaction, with IC₅₀values in the low- to mid-single-digit

micromolar ranges. Comparisons of 52 with 49, 50, and 51

indicated that hydrophilic improvement of the tail resulted in

higher inhibitory potency. In comparison with 49, 48 showed

comparable activity, indicating that aromatic heterocycles were

also tolerated in this region. Notably, BMS202 showed an IC₅₀

value of 0.175 μM in our HTRF assay, which was

approximately 10-fold less than the value reported in the

BMS patent.²⁵Based on the above results, further

optimizations were performed to improve inhibitor potency

and investigate their SARs in a subsequent experiment.

The syntheses of 93, 94, and 100 are shown in Scheme 3.

The N-alkylation of intermediate 30 and PhSO₂OMe

generated intermediate 92. Target molecules 93 and 94 were

provided by NaBH(OAc)₃-mediated reductive amination of 92

and the appropriate amines. Compound 96 was synthesized by

K₂CO₃-promoted O-alkylation of starting material 95 and 5-

(chloromethyl)nicotinonitrile, which was transformed to

benzyl chloride 97 using MsCl. N-alkylation of 97 and 98

was performed to aﬀord intermediate 99. Finally, the

NaBH(OAc)₃-mediated reductive amination of 99 and D-

serine provided desired compound 100.

The geometric diﬀerences of the linkers had a signiﬁcant

impact on the locations of the aryl group and ether-linked

group, which aﬀected their interaction with the PD-L1

homodimer and therefore the activity of the compounds.

Based on this consideration, a series of compounds with

diverse aryl groups and ether-linked groups were developed

and evaluated. As summarized in Table 2, most of these

compounds exhibited attractive inhibitory activity against the

PD-1/PD-L1 interaction, with IC₅₀values in the nanomolar

range. Compound 58 displayed the most potent activity, with

an IC₅₀value of 12 nM. Comparisons of 52, 53, 54, and 55

indicated that substitution at the aryl group was more

beneﬁcial to inhibitory activity than nonsubstitution; more-

over, 2-Me substitution was more beneﬁcial than 3-Me and 3-

OMe substitution. Similar rules could be observed by

Biochemical Assay of PD-1/PD-L1 Blockade and SAR

Study. At the beginning of our research, compounds 48−52,

which have diﬀerent aryl groups, ether-linked groups, and tails,

were synthesized to validate our design strategy. The inhibitory

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Table 2. Inhibitory Activities of Compounds 53−69 toward the PD-1/PD-L1 Interaction

a

Data are displayed as averages of triplicate assays SD.

comparing 58, 59, and 60. Compound 61 with 2-Cl

substitution displayed a slightly lower inhibitory activity than

that of 58, with an IC₅₀value of 26 nM, suggesting that 2-Cl

substitution was a useful modiﬁcation. In general, the (3-

cyanopyridin-5-yl)methyl moiety was the most preferred

moiety for the ether-linked group region. Replacing the (3-

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cyanopyridin-5-yl)methyl moiety with methyl, ethyl, benzyl,

(pyridin-3-yl)methyl, and (3-cyanobenzene-5-yl)methyl moi-

eties yielded 65, 66, 56, 57, and 55, respectively, which showed

reduced eﬃcacy. Comparisons of 58 with 55 and 57 revealed

that the N atom contributed slightly more than −CN to the

high potency of the (3-cyanopyridin-5-yl)methyl moiety. Based

on previous reports, the introduction of the 2,3-dihydro-1,4-

benzodioxinyl moiety in the extension region resulted in the

movement of Tyr56, which made the binding pocket of PD-L1

accessible to solvent on both sides and changed a deep

hydrophobic cleft into a deep, hydrophobic tunnel.²⁷There-

fore, compounds 67−69 were synthesized to examine the

impact of the introduced 2,3-dihydro-1,4-benzodioxinyl moiety

on the eﬃcacy of the compounds. Compounds 68 and 69

exhibited lower inhibitory activities than those of their

precursor compounds (58 and 61), while compound 67 was

more potent than compound 52.

potent, indicating that two hydrogen bond donors were

required for high eﬃcacy.

To increase the hydrophobicity of the biaryl core, replacing

methyl with −Br and −CF₃provided 87−89 and 90−91,

respectively. As shown in Table 4, compounds 87−89

Table 4. Inhibitory Activities of Compounds 87−91 toward

the PD-1/PD-L1 Interaction

The hydrophilic tail provided a signiﬁcant contribution to

the observed high potencies by providing hydrogen bond

interactions with PD-L1. A series of hydrophilic moieties

bearing diverse structures were introduced to yield compounds

70−75, as shown in Table 3. Compounds 70 and 72−75

Table 3. Inhibitory Activities of Compounds 70−75 toward

the PD-1/PD-L1 Interaction

a

Data are displayed as averages of triplicate assays SD.

exhibited potencies comparable to those of their corresponding

precursor compounds 52, 58, and 59. However, 90 and 91

exhibited remarkably reduced eﬃcacies compared with those

of 58 and 59, indicating that a strong electron-withdrawing

group was unfavorable.

a

Data are displayed as averages of triplicate assays SD.

Compounds 93, 94, and 100 were prepared to investigate

whether other linear aliphatic amines were also desirable as

linkers. As shown in Table 5, compound 94 was remarkably

less potent than its precursor compound 50; compound 93 was

clearly inactive at concentrations up to 100 μM. The results

indicated that using a N-substituted linear aliphatic amine as a

linker was inappropriate. The N-substituted molecule probably

formed an unfavorable conformation. In comparison to 59,

exhibited promising eﬃcacy as PD-1/PD-L1 blockades, with

IC₅₀values below 50 nM, indicating that diverse scaﬀolds, such

as linear, branched, and cyclic chains, and diverse hydrogen

bond donors, such as hydroxyl, carboxyl, and amide moieties,

were tolerated in the tail region. This discovery provided room

to improve the potency and PK properties of the compounds.

Compared to 70 and 74, compound 71 was signiﬁcantly less

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compounds that led to disruption of the interaction of PD-1

and PD-L1, a speciﬁcity assay was performed using the biolayer

interferometry (BLI) technique. BLI is a sensitive and label-

free technology for measuring biomolecular interactions.

Human PD-L1/Fc and PD-1/Fc were loaded on an anti-

human IgG Fc capture (AHC) sensor chip. The binding of the

selected compound 52 with human PD-L1 (hPD-L1) and PD-

1 (hPD-1) was measured at a concentration of 500 nM. As

shown in Figure S1, 52 bound to PD-L1 and not to PD-1. To

further verify whether the binding of compound 52 with hPD-

L1 was speciﬁc, the binding of 52 with human PD-L2, CD47,

TIM-3, CD27, and LAG-3 was examined. As shown in Figure

S1, compound 52 showed no binding with these captured

proteins on the sensor chip. These results indicated that this

series of compounds speciﬁcally bound to hPD-L1.

Table 5. Inhibitory Activities of Compounds 93, 94, and 100

toward the PD-1/PD-L1 Interaction

Binding aﬃnity is one of the most important indicators for

evaluating the eﬃcacy of binding. To evaluate the eﬃcacy of

small-molecule inhibitors that will have to compete with the

natural protein partner of PD-L1, the binding aﬃnities of 52,

58, 59, 64, BMS202, and hPD-1 to hPD-L1 were measured.

The kinetic curves of association and dissociation are shown in

Figure 4, in which the compounds show dose-dependent

kinetic characteristics. The kinetic parameters and calculated

binding aﬃnities are summarized in Table 6. Analyses of the

resulting binding kinetics revealed that the small-molecule

inhibitors displayed high binding aﬃnity with hPD-L1, with

KD values in the nanomolar to picomolar ranges. Compound

58 exhibited the most potent aﬃnity, with a KD value of 16.2

pM, which was 43 times greater than that of BMS202. hPD-1

exhibited >18-fold lower potency than those of the selected

a

Data are displayed as averages of triplicate assays SD.

compound 100 displayed decreased activity, with an IC₅₀value

of 191 nM, indicating that the direct linkage of an amine to the

hydrophobic biaryl core was probably inappropriate.

Binding Speciﬁcity and Aﬃnity of Compounds to

Human PD-L1. To identify the binding target of these

Figure 4. Association and dissociation kinetic curves of samples at seven diﬀerent concentrations with hPD-L1. (A) hPD-1, (B) BMS202, (C)

compd 52, (D) compd 58, (E) compd 59, and (F) compd 64.

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cells were incubated with hPD-1, and then, binding was

detected with an APC-labeled anti-human IgG antibody and

measured by ﬂow cytometry. The presence of small-molecule

inhibitors would disrupt the binding of CHO-K1-hPD-L1 and

hPD-1, leading to a change in the percentage of cells bound

with hPD-1 (PCB). This change reﬂected the binding and

blocking ability of the small-molecule inhibitors.

Table 6. The Kinetic Parameters and Binding Aﬃnities of

Samples with hPD-L1

a

samples

K_a(M⁻¹s⁻¹

)

K_dis(s⁻¹

)

KD (M)

hPD-1

1.54 × 10⁴

1.44 × 10⁶

6.51 × 10⁵

2.17 × 10⁶

8.49 × 10⁵

1.29 × 10⁶

5.03 × 10⁻⁴

1.02 × 10⁻³

1.18 × 10⁻³

3.53 × 10⁻⁵

2.51 × 10⁻⁴

6.64 × 10⁻⁴

3.26 × 10⁻⁸

7.06 × 10⁻¹⁰

1.81 × 10⁻⁹

1.62 × 10⁻¹¹

2.96 × 10⁻¹⁰

5.15 × 10⁻¹⁰

BMS202

compd 52

compd 58

compd 59

compd 64

The binding and blocking ability of selected compounds

BMS202, 52, 58, 59, and 64 were measured in the cell-based

PD-1/PD-L1 blocking assay at a concentration of 500 nM. The

binding of CHO-K1-hPD-L1 cells and hPD-1 without an

inhibitor was used as a positive control. The binding of normal

CHO-K1 cells and hPD-1 was used as a negative control. As

shown in Figure 6, the positive control group showed a higher

PCB than that of the negative control group, indicating that

the model was successful, and the increase in percentage was

mediated by the PD-1/PD-L1 interaction. The groups with the

synthesized compounds showed lower PCB values than that of

the positive control group, clearly suggesting that the

compounds had the ability to bind with hPD-L1 on the

surface of CHO-K1 cells and competitively block the

interaction of hPD-1 and hPD-L1. The calculated blocking

eﬃcacies of BMS202, 52, 58, 59, and 64 were 99.2, 96.1, 97.5,

95.9, and 98.6%, respectively, which exhibited their potent

blocking eﬃcacy at the cellular level.

T Cell Function Assay. PD-1 and PD-L1 are the key

coinhibitory signaling molecules that regulate the T cell

function. Therefore, further functional veriﬁcation of the

compounds targeting the PD-1/PD-L1 interaction was

required to evaluate their ability to regulate the T cell

function, such as cytokine IFN-γ secretion. A coculture system

of human peripheral blood mononuclear cells (PBMCs) and

hPD-L1 was used to evaluate the eﬀects of the compounds on

T cell function regulation. PBMCs stimulated by anti-CD3 and

anti-CD28 stimulatory antibodies could secrete IFN-γ. PD-L1

suppressed the T cell function by ligation with PD-1, leading to

decreased production of IFN-γ. The compounds that inhibited

the interaction of PD-1/PD-L1 could restore the T cell

function, leading to enhanced production of IFN-γ. The

immunological functions of selected compounds 52, 58, 59,

and 64 were evaluated using this coculture system. As shown in

Figure 7, compounds 52, 59, and 64 showed no ability to

promote IFN-γ secretion at a concentration of 0.1 μM. At 1

μM, 59 and 64 promoted the production of IFN-γ, which was

increased by 42.5 and 40.9%, respectively, while 52 still

a

Data are generated from seven diﬀerent concentrations. k_a,

association constant; k_dis, dissociation constant.

small-molecule inhibitors, with a KD value of 32.6 nM. The

results demonstrated that these small-molecule inhibitors have

high eﬃcacy in competing with PD-1, the natural protein

partner of PD-L1. These results were also consistent with the

results from the HTRF assay.

Docking Analysis. Docking analysis was carried out to

examine the binding mode of 58 with PD-L1 using GOLD

software. The docking results showed that 58 docked well in

the hydrophobic cleft formed by the PD-L1 dimer (Figure 5A)

and superimposed well with BMS202 (Figure 5B). The serine

tail of 58 was solvent-accessible and formed three hydrogen

bonds with the Asp122 and Lys124 residues. The ether-linked

nicotinonitrile group formed hydrogen bonds and π−π

interactions with Arg125 and Tyr123 residues. The distal

phenyl of the biaryl core formed a T-shaped π−π interaction

with Tyr56. In addition, 58 established multiple key

interactions with the Tyr56 residue of another monomer.

The oxygen atom of the ether, linker, and aryl groups formed

hydrogen bonds and π−σ and π−π interactions with Tyr56,

respectively. Notably, the amine of the linker also formed a

hydrogen bond with Ala121, which showed the contribution of

the linker to inhibitor binding. The docking results indicated

that 58 and the BMS compounds shared a similar PD-L1

binding mode, but the novel amine linker of 58 generated an

additional interaction, indicating that the exploration of linkers

was a useful strategy to develop novel inhibitors of PD-1/PD-

L1.

Cell-Based PD-1/PD-L1 Blocking Assay. The binding

and blocking ability of small-molecule inhibitors on the cellular

level are crucial to their inhibitory eﬃciency. To investigate

this, a cell-based PD-1/PD-L1 blocking assay was performed

using hPD-L1-overexpressing CHO-K1 cells (CHO-K1-hPD-

L1) and hPD-1 by ﬂow cytometry. Brieﬂy, CHO-K1-hPD-L1

Figure 5. (A) Proposed binding mode of compound 58 with dimeric PD-L1 (the PDB code for dimeric PD-L1 protein is 5J89). (B) Binding

overlap of compound 58 (green) and BMS202 (pink) in the binding site.

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Figure 6. Representative ﬂow cytometry plots: (A) negative control, (B) positive control, (C) BMS202, (D) compd 52, (E) compd 58, (F) compd

59, and (G) compd 64. The data in the graphs represent the percentage of cells bound with hPD-1.

increased the IFN-γ level by 56.8%. Because of the probable

maximal dose eﬀect, treatment with 1 μM 58 or BMS202 did

not remarkably increase the IFN-γ level compared with

treatment at 0.1 μM.

Evaluation of Cytotoxicity. Cytotoxicity signiﬁcantly

aﬀects cellular functions, leading to inaccurate experimental

results. To obtain accurate results, the cytotoxic eﬀects of the

tested compounds on human PBMCs were evaluated by the

CellTiter-Glo luminescent cell viability assay. As shown in

Figure 8, BMS202 and 48 showed high cytotoxicity to human

PBMCs in a dose-dependent manner, with a killing rate of

approximately 100% at a concentration of 100 μM, while 52,

58, 59, and 64 showed no cytotoxicity up to 100 μM. At a

concentration of 6.3 μM, all of the tested compounds showed

no eﬀect on cell growth, indicating that the compound

Figure 7. Eﬀects of compounds BMS202, 52, 58, 59, and 64 on IFN-γ

secretion. Data are displayed as averages of quadruplicate assays

concentrations used in the T cell function assay were

appropriate. The compounds were further evaluated for

cytotoxicity to Raji and RAW 264.7 cells. As shown in Figure

8, BMS202 and 48 also displayed high cytotoxicity to Raji and

RAW 264.7 cells, with EC₅₀values of 13.51 and 13.84 μM and

10.08 and 6.97 μM, respectively. 52, 58, 59, and 64 were not

toxic to RAW 264.7 cells at a concentration below 100 μM.

For Raji cells, 58 and 64 were not toxic at relatively low

SEM. *p < 0.05, **p < 0.01, and ns = no signiﬁcance.

showed no eﬃcacy because of the low inhibitory activity of

PD-1/PD-L1. BMS202 signiﬁcantly elevated the production of

IFN-γ at concentrations of 0.1 and 1 μM, indicating that the

assay system worked well. Compound 58 signiﬁcantly

promoted IFN-γ secretion at a concentration of 0.1 μM,

which was increased by 72.9%. Treatment with 1 μM 58

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Figure 8. Cytotoxicity of compounds against human PBMCs (A), Raji cells (B), and RAW 264.7 cells (C). The experiment was performed in

triplicate, and the values are expressed as the mean SEM.

Figure 9. In vivo antitumor eﬀect of compound 58 on a PD-1 humanized mouse model. (A) Tumor growth inhibition (the tumor volumes were

measured and calculated once every 3−4 days). (B) Comparison of the ﬁnal tumor weights in each group after 3 weeks of treatment. (C) Body

weight of each group during the treatment period. The body weight was measured once every 3−4 days. (D) Resulting tumors excised from the

animals after treatment. All data are presented as the mean SEM (6 female mice in each group). *p < 0.05.

concentrations and became toxic at relatively high concen-

trations, while 52 and 59 were not toxic.

were administered by intraperitoneal injection once daily for 3

consecutive weeks at a dose of 15 mg/kg. As shown in Figure

9, the tumor volume of the vehicle group increased quickly and

reached 2173.52 mm³after 21 days. Compound 58 and

BMS202 signiﬁcantly suppressed the tumor growth, with

tumor growth inhibition (TGI) values of 61.7 and 71.1%,

respectively. Compound 58 exhibited comparable antitumor

eﬃcacy in vivo when compared with BMS202, but BMS202

showed unspeciﬁc toxicity in the cytotoxic evaluation. In all

experimental groups, no signiﬁcant ﬂuctuations in the body

weight or unusual behaviors of mice were observed during the

whole treatment period. All experimental treatments were well

tolerated without mortality. These results conﬁrmed the

eﬃcacy of compound 58 in vivo as a small-molecule inhibitor

targeting the PD-1/PD-L1 interaction for cancer therapy.

In Vivo Antitumor Eﬃcacy. Murine and human PD-1

share a sequence identity of 64%, and 77% of the PD-L1

sequence identity is shared.³⁸To evaluate antitumor eﬃcacy

accurately, human PD-1 and PD-L1 are required in the murine

model. On the other hand, based on the mechanism of action,

the pharmacodynamic model of PD-1/PD-L1 inhibitors

requires mice with a normal immune system. Based on the

above considerations, PD-1-humanized mice and a PD-L1-

genetically engineered MC38 mouse colon cancer cell line

(mouse PD-L1 knockout, human PD-L1 knock-in) were used

to establish a pharmacodynamic model.

On the basis of the excellent activities in vitro, compound 58

was selected to evaluate antitumor eﬃcacy in vivo. BMS202

was used as a positive control. Compound 58 and BMS202

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Preliminary Evaluation for Pharmacokinetic Proﬁles.

Because of the potent PD-1/PD-L1 inhibition in vitro and

antitumor activity in vivo, an in vivo PK study was performed

to preliminarily evaluate the druggability of 58 in male Sprague

Dawley rats administered with 4 mg/kg iv (n = 6) and 15 mg/

kg po (n = 6). The corresponding PK parameters are

summarized in Table 7.

eﬃcacy for competing with the natural protein partner of PD-

L1. The cell-based PD-1/PD-L1 blocking assay indicated that

compound 58 could bind with hPD-L1 on the cellular surface

and competitively block the interaction of hPD-1 with hPD-L1.

In the T cell function assay, 58 restored the T cell function,

leading to increased IFN-γ secretion. Evaluation of cytotoxicity

showed that 58 was not toxic. Moreover, compound 58

signiﬁcantly inhibited tumor growth in a humanized mouse

model without obvious toxicity at a dose of 15 mg/kg. The

results indicated the promising use of compound 58 as an

alternative or complementary drug to mAbs for cancer

treatment. Compound 58 showed moderate PK properties

after intravenous injection in the PK study but did not exhibit

oral bioavailability.

In conclusion, these studies provide biological evaluation

models that may serve as useful tools for the development of

small-molecule inhibitors of PD-1/PD-L1 and discovered

compound 58, which could be employed as a promising lead

for further development of small-molecule inhibitors of PD-1/

PD-L1 for cancer therapy. Currently, further optimization

eﬀorts to achieve new, potent small-molecule inhibitors of PD-

1/PD-L1 with favorable oral PK proﬁles are underway in our

laboratory.

Table 7. Pharmacokinetic Parameters of Compound 58

c

dosing route

a

PK parameters

iv (4 mg/kg)

po (15 mg/kg)

b

Cl_{_obs}(mL/min/kg)

T_1/2(h)

C₀(ng/mL)

AUC_last(h·ng/mL)

AUC_Inf(h·ng/mL)

AUC_{_%Extrap_obs}(%)

MRT_{Inf_obs}(h)

AUC_last/D (h·mg/mL)

V_{ss_obs}(L/kg)

74.5 10.1

1.38 0.16

4370 1040

905 117

NA

908 117

0.283 0.124

0.486 0.069

226 29

2.17 0.35

a

b

The values are expressed as the mean SD. NA = not available.

c

Formulation: 25% MPD/10% RH40/65% water; 6 male rats in each

group.

EXPERIMENTAL SECTION

■

General Chemistry. Reagents and solvents were purchased from

commercial suppliers and used without further puriﬁcation. All the

reactions were monitored by TLC using silica gel TLC plates

(GF254). Silica gel (200−300 mesh) was used for chromatography.

Melting points were determined on a Buchi melting point apparatus

After iv administration of 58, the values of C₀and AUC_Inf

achieved in plasma were 4370 ng/mL and 908 h·ng/mL,

respectively, which indicated moderate systemic exposure. In

addition, 58 showed a half-life (T_1/2) of 1.38 h, with a

clearance (Cl_{_obs}) of 74.5 mL/min/kg and a volume of

distribution (V_{ss_obs}) of 2.17 L/kg. These results indicated that

58 showed moderate PK properties via intravenous injection,

which provided a foundation for further development.

Unfortunately, po administration of 58 exhibited very low

exposures, with a plasma concentration below the quantiﬁable

limit (0.5 ng/mL), indicating oral unavailability.

1

(M-565). The H and ¹³C NMR spectra were recorded on a JEOL-

ECA400 spectrometer, with TMS as an internal standard at ambient

temperature. All chemical shifts are reported in parts per million

(ppm). All coupling constants were reported in Hertz. The ESI-MS

was recorded on an Agilent TOF G6230A mass spectrometer. The

purity of all ﬁnal compounds was determined by HPLC on an Agilent

1260 system and was 95% or higher. HPLC Method A was performed

on an Agilent ZORBAX SB-C8 column (4.6 mm × 250 mm, 5 μm)

with ammonium acetate aqueous solution (10 mM) containing 5%

MeCN as mobile phase A and MeCN containing 5% ammonium

acetate aqueous solution (10 mM) as mobile phase B at a ﬂow rate of

1 mL/min. The column temperature was maintained at 50 °C, and

the UV wavelength for detection was 220 nm. The gradient program

was as follows: 0−100% B (0−25 min), 100% B (25−30 min), and

0% B (30.01−35 min). HPLC Method B was performed on an

Agilent ZORBAX Eclipse Plus-C18 column (4.6 mm × 100 mm, 3.5

μm) with 100% MeCN as mobile phase A and water (0.1%

triethylamine and H₃PO₄buﬀer solution, pH = 3) as mobile phase B

at a ﬂow rate of 1 mL/min. The column temperature was maintained

at 35 °C, and the UV wavelength for detection was 220 nm. The

gradient program was as follows: 60−5% B (0−10 min), 5% B (10−

18 min), and 60% B (18.01−23 min). The ﬁnal compounds were

puriﬁed by preparative high-performance liquid chromatography on

an Agilent 1260 Inﬁnity system at ambient temperature. The method

was performed on a Phenomenex Gemini-C18 column (30 mm × 100

mm, 5 μm) with 100% MeCN as mobile phase A and water as mobile

phase B at a ﬂow rate of 20 mL/min. The UV wavelength for

detection was 254 nm. The gradient program was as follows: 40−

100% A (0−25 min) and 40% A (25.01−30 min).

CONCLUSION

■

The PD-1/PD-L1 interaction has emerged as a promising

target for cancer therapy. The approval of mAbs to inhibit the

PD-1/PD-L1 interaction has proven the clinical signiﬁcance of

PD-1/PD-L1 inhibitors. Due to their advantages compared

with mAbs, the discovery and development of small-molecule

inhibitors of PD-1/PD-L1 have been of recent interest in

medicinal chemistry. To develop novel small-molecule

inhibitors of PD-1/PD-L1 for cancer therapy, a series of

novel linear aliphatic amine-linked triaryl derivatives were

designed, synthesized, and evaluated for their activities in vitro

and in vivo. In the biochemical assay of PD-1/PD-L1 blockage,

this novel chemical series showed potent inhibitory activities,

with IC₅₀values in the nanomolar to micromolar ranges. SAR

analysis indicated that hydrogen bond interactions with the tail

were required for high potency; strong electron-withdrawing

hydrophobic substituents in the biaryl core and N-substituents

in the amine linker were unfavorable to inhibitory activity. A

binding speciﬁcity assay indicated that this series of

compounds speciﬁcally bound to PD-L1 and not to PD-1 or

other checkpoints. Among the synthesized compounds,

compound 58 was the most potent, with an IC₅₀value of 12

nM. Compound 58 also exhibited the most potent binding

aﬃnity with hPD-L1, with a KD value of 16.2 pM, which was

approximately 2000-fold that of hPD-1, indicating its high

General Procedure A for Suzuki−Miyaura Coupling Re-

action. At an argon atmosphere, halogenated benzene (1 equiv),

phenylboronic acid (1.15−1.5 equiv) or (2,3-dihydrobenzo[b][1,4]-

dioxin-6-yl)boronic acid (1.25 equiv), CsOAc (2.5 equiv), and

PdCl₂dppf (0.1 equiv) were suspended in THF. The mixture was

stirred at reﬂux for 23−36 h. Then, the mixture was cooled,

concentrated in vacuo, diluted with water, and extracted with DCM.

The combined organic phase was washed with water and

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concentrated in vacuo. The crude product was puriﬁed by column

chromatography.

132.2, 129.5, 126.5, 125.6, 123.5, 122.6, 118.3, 116.8, 64.5, 39.9, 16.6.

ESI-MS: m/z = 386.14 [M + H]⁺.

(2-Methyl-[1,1′-biphenyl]-3-yl)methanamine (14a). 13a (11.21 g,

34.27 mmol) and N₂H₄·H₂O (85% w/w in water, 3.63 g, and 61.68

mmol) were suspended in EtOH (210 mL), stirred at reﬂux for 3 h,

and then cooled to rt. To the reaction mixture, 6 N HCl (40 mL) was

added and stirred for 30 min at reﬂux. The mixture was cooled to 0

°C and ﬁltered. The ﬁlter cake was washed with EtOH a few times.

The combined ﬁltrate was alkalized (pH = 9−10), used 1 N NaOH at

0 °C, and extracted with DCM/MeOH (10:1). The combined

organic phase was washed with H₂O once and concentrated to

dryness in vacuo to give 14a (6.5 g, 96% yield) as a colorless oil,

which was used in the next step without further puriﬁcation. ¹H NMR

(400 MHz, CDCl₃): δ 7.43−7.37 (m, 2H), 7.36−7.31 (m, 2H),

7.30−7.27 (m, 2H), 7.24 (t, J = 7.6 Hz, 1H), 7.15 (dd, J = 7.6, 1.2 Hz,

1H), 3.93 (s, 2H), 2.22 (s, 3H), 1.79 (s, 2H). ¹³C NMR (100 MHz,

CDCl₃): δ 142.9, 142.3, 141.3, 133.1, 129.4, 128.8, 128.1, 126.8,

126.4, 125.7, 44.6, 16.1. ESI-MS: m/z = 198.13 [M + H]⁺.

(2-Methyl-[1,1′-biphenyl]-3-yl)methanol (11a). According to the

general procedure A, 10 (2.51 g, 12.5 mmol) and phenylboronic acid

(1.5 equiv) were used. The crude product was puriﬁed on a silica gel

column (PE/EA = 10:1−7:1) to give 11a (2.44 g, 98% yield) as a

1

brown solid. H NMR (400 MHz, CDCl₃): δ 7.44−7.32 (m, 4H),

7.32−7.27 (m, 2H), 7.26−7.23 (m, 1H), 7.20 (dd, J = 7.6, 1.2 Hz,

1H), 4.77 (s, 2H), 2.24 (s, 3H), 1.70 (s, 1H). ¹³C NMR (100 MHz,

CDCl₃): δ 142.9, 142.1, 139.2, 133.6, 129.6, 129.4, 128.1, 126.9,

126.8, 125.6, 64.0, 15.9. ESI-MS: m/z = 181.11 [M − OH]⁺.

(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-

methanol (11b). According to the general procedure A, 10 (3.25 g,

16.16 mmol) and (2,3-dihydrobenzo[b][1,4]dioxin-6-yl)boronic acid

(1.25 equiv) were used. The crude product was puriﬁed on a silica gel

column (PE/EA = 8:1−5:1) to give 11b (4.05 g, 98% yield) as a

1

colorless oil. H NMR (400 MHz, CDCl₃): δ 7.36 (dd, J = 7.6, 1.2

Hz, 1H), 7.22 (t, J = 7.6 Hz, 1H), 7.17 (dd, J = 7.6, 1.6 Hz, 1H), 6.89

(d, J = 8.4 Hz, 1H), 6.81 (d, J = 2.0 Hz, 1H), 6.76 (dd, J = 8.0, 2.0 Hz,

1H), 4.76 (s, 2H), 4.30 (s, 4H), 2.25 (s, 3H), 1.66 (s, 1H). ¹³C NMR

(100 MHz, CDCl₃): δ 143.0, 142.6, 142.3, 139.3, 135.5, 133.7, 129.6,

126.6, 125.6, 122.6, 118.3, 116.9, 64.5, 64.1, 16.0. ESI-MS: m/z =

257.12 [M + H]⁺.

3-(Bromomethyl)-2-methyl-1,1′-biphenyl (12a). PBr₃(0.36 mL,

3.81 mmol) was added dropwise to a solution of 11a (1.51 g, 7.62

mmol) in DCM (30 mL) at 0 °C and stirred for further 20 min. The

mixture was quenched by ice and extracted with DCM. The combined

organic phase was washed with a saturated solution of NaHCO₃and

H₂O, dried over anhydrous Na₂SO₄, ﬁltered, and concentrated to

dryness in vacuo to give 12a (1.32 g, 66% yield) as a colorless oil,

which was used in the next step without further puriﬁcation. ¹H NMR

(400 MHz, CDCl₃): δ 7.44−7.39 (m, 2H), 7.38−7.32 (m, 2H),

7.32−7.27 (m, 2H), 7.22 (d, J = 2.8 Hz, 1H), 7.21 (s, 1H), 4.60 (s,

2H), 2.30 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 143.4, 141.8,

136.4, 134.9, 130.8, 129.4, 129.3, 128.2, 127.0, 125.9, 33.3, 16.2. ESI-

MS: m/z = 181.10 [M − Br]⁺.

6-(3-(Bromomethyl)-2-methylphenyl)-2,3-dihydrobenzo[b][1,4]-

dioxine (12b). According to a similar manner as described for the

synthesis of 12a, 11b (513 mg, 2.0 mmol) was used to obtain 12b

(550 mg, 86% yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃):

δ 7.35−7.29 (m, 1H), 7.22−7.15 (m, 2H), 6.93−6.89 (m, 1H), 6.82−

6.80 (m, 1H), 6.78−6.74 (m, 1H), 4.59 (s, 2H), 4.31 (s, 4H), 2.32 (s,

3H). ¹³C NMR (100 MHz, CDCl₃): δ 143.1, 142.8, 142.7, 136.35,

135.2, 135.1, 130.8, 129.2, 125.9, 122.6, 118.3, 116.9, 64.5, 33.3, 16.2.

ESI-MS: m/z = 239.11 [M − Br]⁺.

(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylphenyl)-

methanamine (14b). According to a similar manner as described for

the synthesis of 14a, 13b (4.88 g, 12.66 mmol) was used to obtain

14b (3.20 g, 99% yield) as a colorless oil. ¹H NMR (400 MHz,

CDCl₃): δ 7.31 (dd, J = 7.6, 1.2 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H),

7.12 (dd, J = 7.6, 1.2 Hz, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.80 (d, J =

2.0 Hz, 1H), 6.75 (dd, J = 8.4, 2.0 Hz, 1H), 4.30 (s, 4H), 3.91 (s,

2H), 2.23 (s, 3H), 1.56 (s, 2H). ¹³C NMR (100 MHz, CDCl₃): δ

143.0, 142.5, 142.2, 141.5, 135.7, 133.2, 128.8, 126.2, 125.7, 122.6,

118.3, 116.8, 64.5, 44.7, 16.1. ESI-MS: m/z = 256.13 [M + H]⁺.

3-((5-Amino-2-formylphenoxy)methyl)benzonitrile (15). 2-Hy-

droxy-4-nitrobenzaldehyde (4.81 g, 28.78 mmol), K₂CO₃(5.97 g,

43.20 mmol), 3-(bromomethyl)benzonitrile (6.21 g, 31.68 mmol),

and (n-Bu)₄N⁺·I⁻(50 mg, 0.14 mmol) were suspended in DMF (40

mL) and stirred for 4 h at rt. The reaction mixture was added to ice-

water (900 mL). The precipitate was ﬁltered, washed with water, and

dried in vacuo. Then, the precipitate was suspended in cyclohexane,

washed using an ultrasonic cleaner, and ﬁltered to obtain 3-((2-

formyl-5-nitrophenoxy)methyl)benzonitrile (15-1) (8.05 g, 99%

yield) as a yellowish solid, which was used in the next step without

1

further puriﬁcation. H NMR (400 MHz, DMSO-d₆): δ 10.49 (s,

1H), 8.09 (t, J = 1.6 Hz, 2H), 7.98−7.91 (m, 3H), 7.86 (d, J = 7.6 Hz,

1H), 7.66 (t, J = 7.8 Hz, 1H), 5.51 (s, 2H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 188.8, 160.1, 151.7, 137.5, 132.3, 132.0, 130.9, 129.8,

129.6, 128.6, 118.7, 115.9, 111.6, 109.1, 69.5. ESI-MS: m/z = 281.06

[M − H]⁻.

15-1 (8.05 g, 28.52 mmol), Fe (5.59 g, 99.82 mmol), and NH₄Cl

(2.29 g, 42.81 mmol) were suspended in EtOH/H₂O (4/1, 100 mL)

and stirred for 1.5 h at reﬂux. The reaction mixture was ﬁltered. The

ﬁltrate was concentrated to dryness in vacuo. The residue was

suspended in water and ﬁltered to give 15 (6.48 g, 90% yield) as an

orange solid, which was used in the next step without further

puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆): δ 10.05 (s, 1H), 8.16−

7.34 (m, 5H), 6.60−6.06 (m, 4H), 5.21 (s, 2H). ¹³C NMR (100

MHz, DMSO-d₆): δ 185.3, 162.5, 156.8, 138.5, 132.1, 131.7, 130.8,

130.2, 129.8, 118.7, 113.9, 111.5, 107.0, 95.8, 67.9. ESI-MS: m/z =

253.10 [M + H]⁺.

3-((5-Amino-2-formyl-3-methoxyphenoxy)methyl)benzonitrile

(16). 2-Methoxy-4-nitrobenzaldehyde (2.05 g, 11.32 mmol), 2-amino-

4-chlorobenzoic acid (0.97 g, 5.66 mmol), 1-ﬂuoro-2,4,6-trimethyl-

pyrdinium triﬂate (4.91 g, 16.98 mmol), Pd(OAc)₂(249 mg, 1.11

mmol), and TsOH (3.89 g, 22.59 mmol) were suspended in AcOH

(80 mL) and stirred for 10 min at rt. Then, the mixture was stirred for

further 24 h at 90 °C and concentrated to dryness in vacuo. The

residue was puriﬁed by column chromatography to obtain 2-hydroxy-

6-methoxy-4-nitrobenzaldehyde (16-1) (1.5 g, 67% yield) as a

yellowish solid. ¹H NMR (400 MHz, CDCl₃): δ 12.06 (s, 1H),

10.41 (s, 1H), 7.39 (dd, J = 2.0, 0.8 Hz, 1H), 7.23 (d, J = 2.0 Hz, 1H),

4.03 (s, 3H). ESI-MS: m/z = 198.05 [M + H]⁺.

2-((2-Methyl-[1,1′-biphenyl]-3-yl)methyl)isoindoline-1,3-dione

(13a). At an argon atmosphere, DEAD (9.06 g, 52.00 mmol) was

added dropwise to a solution of 11a (7.36 g, 37.14 mmol), o-

phthalimide (6.56 g, 44.57 mmol), and PPh₃(13.64 g, 52.00 mmol)

in anhydrous THF (170 mL) at 0 °C. After completion of addition,

the mixture was stirred for 24 h at rt, and then concentrated to

dryness in vacuo. The crude product was puriﬁed on a silica gel

column (PE/DCM = 3:1−2:1) to give 13a (11.21 g, 92% yield) as a

1

white solid. H NMR (400 MHz, CDCl₃): δ 7.92−7.86 (m, 2H),

7.77−7.72 (m, 2H), 7.43−7.38 (m, 2H), 7.36−7.31 (m, 1H), 7.30−

7.26 (m, 2H), 7.24−7.21 (m, 1H), 7.20−7.12 (m, 2H), 4.94 (s, 2H),

2.34 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 168.3, 143.0, 142.1,

134.6, 134.2, 133.4, 132.2, 129.5, 129.5, 128.1, 126.9, 126.7, 125.6,

123.5, 39.8, 16.6. ESI-MS: m/z = 328.13 [M + H]⁺.

2-(3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)-

isoindoline-1,3-dione (13b). According to a similar manner as

described for the synthesis of 13a, 11b (3.46 g, 13.50 mmol) was

used. The crude product was puriﬁed on a silica gel column (PE/

DCM = 1:1−1:2) to give 13b (5.02 g, 97% yield) as a white solid. ¹H

NMR (400 MHz, CDCl₃): δ 7.89−7.87 (m, 2H), 7.75−7.73 (m, 2H),

7.21−7.16 (m, 1H), 7.14 (d, J = 4.0 Hz, 1H), 7.13 (s, 1H), 6.88 (d, J

= 8.0 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.74 (dd, J = 8.4, 2.0 Hz,

1H), 4.93 (s, 2H), 4.30 (s, 4H), 2.35 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 168.3, 143.0, 142.6, 142.4, 135.5, 134.6, 134.1, 133.5,

According to a similar manner as described for the synthesis of

compound 15-1, 16-1 (684 mg, 3.47 mmol) was used to obtain 3-((2-

formyl-3-methoxy-5-nitrophenoxy)methyl)benzonitrile (16-2) (1 g,

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93% yield) as a yellowish solid, which was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆): δ 10.42

(s, 1H), 8.00 (s, 1H), 7.91−7.80 (m, 2H), 7.66−7.57 (m, 3H), 5.42

(s, 2H), 3.99 (s, 3H).

According to a similar manner as described for the synthesis of

compound 15, 16-2 (900 mg, 2.88 mmol) was used, and the crude

product was puriﬁed by column chromatography to obtain 16 (790

mg, 97% yield) as an orange solid. ¹H NMR (400 MHz, DMSO-d₆): δ

10.09 (s, 1H), 8.03 (s, 1H), 7.88−7.83 (m, 1H), 7.80 (dt, J = 8.0, 1.4

Hz, 1H), 7.62 (t, J = 7.8 Hz, 1H), 6.39 (s, 2H), 5.87 (dd, J = 9.0, 1.4

Hz, 2H), 5.13 (s, 2H), 3.74 (s, 3H). ¹³C NMR (100 MHz, DMSO-

d₆): δ 183.7, 164.2, 161.9, 156.6, 138.8, 131.6, 131.3, 130.3, 129.6,

118.8, 111.3, 104.2, 89.9, 88.9, 67.9, 55.4. ESI-MS: m/z = 283.11 [M

+ H]⁺.

5-((5-Amino-2-formylphenoxy)methyl)nicotinonitrile (17). 2-Hy-

droxy-4-nitrobenzaldehyde (2.47 g, 14.78 mmol) and K₂CO₃(2.15 g,

15.56 mmol) were suspended in DMF (30 mL) and stirred for 30 min

at rt. Then, 5-(chloromethyl)nicotinonitrile (2.48 g, 16.29 mmol) and

NaI (222 mg, 1.48 mmol) were added to the reaction mixture and

stirred for 19 h at rt. The mixture was diluted with water (1 L),

ﬁltered, and washed with water. The ﬁlter cake was dried to give 5-

((2-formyl-5-nitrophenoxy)methyl)nicotinonitrile (17-1) (4.05 g,

97% yield) as a yellowish solid, which was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆): δ 10.50

(s, 1H), 9.06 (d, J = 2.0 Hz, 1H), 9.05 (d, J = 2.0 Hz, 1H), 8.59 (t, J =

2.0 Hz, 1H), 8.12 (d, J = 1.2 Hz, 1H), 8.00−7.93 (m, 2H), 5.57 (s,

2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 189.0, 159.9, 152.2, 152.1,

151.7, 138.8, 132.2, 129.5, 128.6, 116.8, 116.1, 109.1, 109.1, 67.5. ESI-

MS: m/z = 284.07 [M + H]⁺.

According to a similar manner as described for the synthesis of

compound 15, 17-1 (4.75 g, 16.77 mmol) was used to obtain 17

(4.12 g, 97% yield) as an orange solid, which was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆): δ 10.02

(s, 1H), 9.02 (s, 1H), 9.00 (s, 1H), 8.49 (s, 1H), 7.46 (d, J = 8.4 Hz,

1H), 6.43 (s, 2H), 6.26 (s, 2H), 5.24 (s, 2H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 185.5, 162.2, 156.7, 152.2, 151.8, 138.8, 133.0, 130.3,

116.8, 113.9, 109.0, 107.0, 95.8, 66.0. ESI-MS: m/z = 252.09 [M −

H]⁻.

as a yellowish solid. H NMR (400 MHz, CDCl₃): δ 10.41 (s, 1H),

7.66 (s, 1H), 7.18 (s, 1H), 4.12 (q, J = 7.4 Hz, 2H), 2.35 (s, 3H), 1.47

(t, J = 7.4 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 189.1, 159.3,

132.7, 130.1, 129.3, 123.7, 116.7, 64.6, 21.7, 14.5. ESI-MS: m/z =

243.01 [M + H]⁺.

4-Bromo-5-methyl-2-(pyridin-3-ylmethoxy)benzaldehyde (22).

4-Bromo-2-hydroxy-5-methylbenzaldehyde (430 mg, 2 mmol),

K₂CO₃(608 mg, 4.4 mmol), 3-(bromomethyl)pyridine hydrobromide

(556 mg, 2.20 mmol), and NaI (30 mg, 0.2 mmol) were suspended in

MeCN (10 mL) and stirred for 24 h at rt in a dark place. Then,

another K₂CO₃(332 mg, 2.4 mmol) and 3-(bromomethyl)pyridine

hydrobromide (303 mg, 1.20 mmol) were added and stirred for

further 12 h. The mixture was diluted with water and extracted with

EA. The combined organic phase was washed with water,

concentrated in vacuo, and puriﬁed by column chromatography to

1

obtain 22 (180 mg, 29% yield) as a yellowish solid. H NMR (400

MHz, CDCl₃): δ 10.41 (s, 1H), 8.71 (s, 1H), 8.64 (d, J = 4.0 Hz,

1H), 7.83−7.79 (m, 1H), 7.70 (s, 1H), 7.37 (dd, J = 8.0, 4.8 Hz, 1H),

7.29 (s, 1H), 5.17 (s, 2H), 2.38 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 188.6, 158.5, 150.0, 148.9, 135.3, 132.8, 131.3, 132.2,

129.9, 124.1, 123.8, 117.2, 68.5, 21.9. ESI-MS: m/z = 306.01 [M +

H]⁺.

2-(Benzyloxy)-4-bromo-5-methylbenzaldehyde (23). According

to a similar manner as described for the synthesis of compound 19, 4-

bromo-2-hydroxy-5-methylbenzaldehyde (430 mg, 2 mmol) and

benzyl bromide (376 mg, 2.2 mmol) were used to obtain 23 (503

mg, 82% yield) as a yellowish solid, which was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, CDCl₃): δ 10.45 (s,

1H), 7.69 (s, 1H), 7.45−7.33 (m, 5H), 7.28 (s, 1H), 5.14 (s, 2H),

2.36 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 189.0, 159.1, 135.6,

132.8, 130.9, 129.6, 128.9, 128.5, 127.5, 124.2, 117.4, 71.0, 21.9. ESI-

MS: m/z = 327.00 [M + Na]⁺.

5-((5-Bromo-4-chloro-2-formylphenoxy)methyl)nicotinonitrile

(24). According to a similar manner as described for the synthesis of

compound 16, 4-bromo-3-chlorobenzaldehyde (2.53 g, 11.52 mmol)

was used to obtain 4-bromo-5-chloro-2-hydroxybenzaldehyde (24-1)

(1.50 g, 55% yield) as a yellowish solid. ¹H NMR (400 MHz, DMSO-

d₆): δ 11.33 (s, 1H), 10.20 (s, 1H), 7.73 (s, 1H), 7.38 (s, 1H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 188.7, 159.3, 129.0, 128.7, 123.7,

123.2, 122.4. ESI-MS: m/z = 234.90 [M + H]⁺.

According to a similar manner as described for the synthesis of

compound 19, 24-1 (471 mg, 2 mmol) was used to obtain 24 (663

mg, 94% yield) as a yellowish solid. ¹H NMR (400 MHz, DMSO-d₆):

δ 10.32 (s, 1H), 9.04 (d, J = 2.0 Hz, 1H), 9.01 (d, J = 2.0 Hz, 1H),

8.54 (t, J = 2.0 Hz, 1H), 7.85 (s, 1H), 7.82 (s, 1H), 5.45 (s, 2H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 188.0, 158.2, 152.2, 152.0, 138.8,

132.2, 129.3, 128.7, 126.1, 125.0, 119.7, 116.8, 109.1, 67.5. ESI-MS:

m/z = 350.95 [M + H]⁺.

3-((3-Cyanobenzyl)oxy)-4-formyl-5-methylphenyl Triﬂuorome-

thanesulfonate (25). The solution of Tf₂O (2.96 g, 10.5 mmol) in

DCM (15 mL) was added dropwise to a solution of 2,4-dihydroxy-6-

methylbenzaldehyde (1.52 g, 10 mmol), 2,6-lutidine (1.13 g, 10.5

mmol), and DMAP (245 mg, 2 mmol) in DCM (40 mL) at 0 °C.

After completion of addition, the mixture was stirred for 10 h at rt,

diluted with water, and extracted with DCM. The combined organic

phase was washed with water and concentrated in vacuo. The crude

product was puriﬁed by column chromatography to obtain 4-formyl-

3-hydroxy-5-methylphenyl triﬂuoromethanesulfonate (25-1) (1.7 g,

60% yield) as a colorless oil. ¹H NMR (400 MHz, CDCl₃): δ 12.16 (s,

1H), 10.31 (s, 1H), 6.76 (d, J = 2.4 Hz, 1H), 6.67 (d, J = 2.4 Hz, 1H),

2.67 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 194.5, 164.8, 154.4,

145.3, 118.7 (q, J = 319.2 Hz), 118.1, 114.9, 109.1, 18.5. ESI-MS: m/z

= 285.00 [M + H]⁺.

5-((5-Bromo-2-formyl-4-methylphenoxy)methyl)nicotinonitrile

(19). 4-Bromo-2-hydroxy-5-methylbenzaldehyde (430 mg, 2 mmol),

K₂CO₃(304 mg, 2.2 mmol), 5-(chloromethyl)nicotinonitrile (336

mg, 2.2 mmol), and NaI (30 mg, 0.2 mmol) were suspended in

MeCN (6 mL) and stirred for 24 h at rt. The mixture was diluted with

water (18 mL), ﬁltered, and washed with water. The ﬁlter cake was

dried to give 19 (649 mg, 98% yield) as a yellowish solid, which was

1

used in the next step without further puriﬁcation. H NMR (400

MHz, DMSO-d₆): δ 10.37 (s, 1H), 9.03 (d, J = 1.6 Hz, 1H), 9.01 (d, J

= 2.0 Hz, 1H), 8.53 (s, 1H), 7.68 (s, 1H), 7.63 (s, 1H), 5.40 (s, 2H),

2.34 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 189.4, 158.6, 152.7,

152.5, 139.3, 133.1, 132.5, 130.9, 130.0, 124.3, 118.3, 117.3, 116.0,

109.6, 67.6, 21.8. ESI-MS: m/z = 331.01 [M + H]⁺.

3-((5-Bromo-2-formyl-4-methylphenoxy)methyl)benzonitrile

(20). According to a similar manner as described for the synthesis of

compound 19, 4-bromo-2-hydroxy-5-methylbenzaldehyde (645 mg, 3

mmol) and 3-(bromomethyl)benzonitrile (647 mg, 3.3 mmol) were

used. The crude product was suspended in cyclohexane, washed using

an ultrasonic cleaner, and ﬁltered to obtain 20 (801 mg, 81% yield) as

a yellowish solid, which was used in the next step without further

1

puriﬁcation. H NMR (400 MHz, DMSO-d₆): δ 10.36 (s, 1H), 8.01

(s, 1H), 7.85 (t, J = 8.6 Hz, 2H), 7.69−7.57 (m, 3H), 5.35 (s, 2H),

2.33 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 188.7, 158.2, 137.9,

132.2, 132.0, 131.8, 130.9, 130.2, 129.8, 129.5, 123.7, 118.7, 117.8,

111.5, 69.1, 21.3. ESI-MS: m/z = 330.00 [M + H]⁺.

4-Bromo-2-ethoxy-5-methylbenzaldehyde (21). 4-Bromo-2-hy-

droxy-5-methylbenzaldehyde (660 mg, 3.07 mmol), K₂CO₃(467

mg, 3.38 mmol), and CH₃CH₂I (527 mg, 3.38 mmol) were

suspended in MeCN (15 mL) and stirred for 24 h at 60 °C. The

mixture was diluted with water and extracted with EA. The combined

organic phase was washed with water, concentrated in vacuo, and

puriﬁed by column chromatography to obtain 21 (537 mg, 72% yield)

25-1 (220 mg, 0.77 mmol), K₂CO₃(128 mg, 0.92 mmol), 3-

(bromomethyl)benzonitrile (159 mg, 0.81 mmol), and (n-Bu)₄N⁺·I⁻

(15 mg, 0.04 mmol) were suspended in MeCN (8 mL) and stirred for

6 h at rt. The mixture was diluted with water and extracted with

DCM. The combined organic phase was washed with water and

concentrated in vacuo. The crude product was puriﬁed by column

chromatography to obtain 25 (286 mg, 93% yield) as a yellowish

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solid. H NMR (400 MHz, CDCl₃): δ 10.64 (s, 1H), 7.78−7.61 (m,

152.2, 151.8, 142.1, 141.5, 138.8, 136.9, 133.4, 133.0, 130.1, 129.2,

128.8, 128.2, 127.0, 126.9, 125.5, 116.9, 114.2, 109.0, 105.7, 94.6,

66.1, 45.0, 15.9. ESI-MS: m/z = 434.19 [M + H]⁺.

3H), 7.60−7.52 (m, 1H), 6.80 (s, 2H), 5.20 (s, 2H), 2.63 (s, 3H),

(isomer ratio = 5:1). ¹³C NMR (100 MHz, CDCl₃): δ 190.2, 162.4,

152.3, 145.3, 136.6, 132.4, 131.7, 130.8, 129.9, 123.3, 118.7 (q, J =

319.6 Hz), 118.3, 117.2, 113.2, 104.0, 70.0, 21.8. ESI-MS: m/z =

400.03 [M + H]⁺.

3-((5-Cyanopyridin-3-yl)methoxy)-4-formyl-5-methylphenyl Tri-

ﬂuoromethanesulfonate (26). 25-1 (852 mg, 3 mmol), K₂CO₃(954

mg, 6.90 mmol), 5-(chloromethyl)nicotinonitrile hydrochloride (624

mg, 3.30 mmol), and NaI (45 mg, 0.3 mmol) were suspended in

MeCN (24 mL), stirred for 12 h at rt, and stirred for further 9 h at 45

°C. The mixture was diluted with water and extracted with EA. The

combined organic phase was washed with water and concentrated in

vacuo. The crude product was puriﬁed by column chromatography to

5-Chloro-2-methoxy-4-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

amino)benzaldehyde (33). According to a similar manner as

described for the synthesis of 30, 11a (418 mg, 1.6 mmol), 4-

amino-5-chloro-2-methoxybenzaldehyde (compd 18, 312 mg, 1.68

mmol), and K₂CO₃(265 mg, 1.92 mmol) were suspended in MeCN

(15 mL) and stirred for 18 h at reﬂux. The crude product was puriﬁed

on a silica gel column (PE/DCM = 1:1.5) to give 33 (277 mg, 47%

1

yield) as a yellowish solid. H NMR (400 MHz, CDCl₃): δ 10.14 (s,

1H), 7.79 (s, 1H), 7.47−7.40 (m, 2H), 7.40−7.34 (m, 1H), 7.34−

7.28 (m, 3H), 7.28−7.23 (m, 2H), 6.14 (s, 1H), 5.22 (br, 1H), 4.48

(s, 2H), 3.87 (s, 3H), 2.26 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

186.7, 162.9, 149.7, 143.4, 141.7, 135.3, 133.9, 130.0, 129.4, 129.0,

128.2, 127.6, 127.1, 126.0, 115.5, 111.9, 93.0, 55.8, 46.6, 16.4. ESI-

MS: m/z = 366.13 [M + H]⁺.

1

obtain 26 (780 mg, 65% yield) as a yellowish solid. H NMR (400

MHz, CDCl₃): δ 10.61 (s, 1H), 8.93 (s, 2H), 8.10 (s, 1H), 6.85 (s,

2H), 5.25 (s, 2H), 2.64 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

189.7, 161.9, 152.5, 152.3, 151.9, 145.6, 138.3, 131.6, 123.3, 118.7 (q,

J = 319 Hz), 117.7, 116.1, 110.5, 104.0, 67.7, 21.7. ESI-MS: m/z =

401.03 [M + H]⁺.

3-((5-((3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)-

amino)-2-formylphenoxy)methyl)benzonitrile (34). According to a

similar manner as described for the synthesis of 30, 11b (550 mg, 1.72

mmol), 15 (435 mg, 1.72 mmol), and K₂CO₃(262 mg, 1.90 mmol)

were suspended in MeCN/DMF (10 mL/3 mL) and stirred for 11 h

at reﬂux. The crude product was puriﬁed on a silica gel column (PE/

3-((2-Formyl-5-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)amino)-

phenoxy)methyl)benzonitrile (30). 11a (490 mg, 1.88 mmol), 15

(567 mg, 2.25 mmol), and K₂CO₃(392 mg, 2.84 mmol) were

suspended in MeCN/DMF (15 mL/2 mL) and stirred for 11 h at

reﬂux. Then, the mixture was cooled, concentrated in vacuo, diluted

with water, and extracted with EA. The combined organic phase was

washed with water and concentrated in vacuo. The crude product was

puriﬁed on a silica gel column (DCM/EA = 50:1) to give 30 (460 mg,

57% yield) as a yellowish solid. ¹H NMR (400 MHz, CDCl₃): δ 10.23

(s, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.72−7.66 (m, 2H), 7.66−7.60 (m,

1H), 7.51 (t, J = 7.6 Hz, 1H), 7.48−7.40 (m, 2H), 7.40−7.35 (m,

1H), 7.33−7.27 (m, 3H), 7.25−7.21 (m, 2H), 6.32 (dd, J = 8.8, 2.0

Hz, 1H), 6.09 (d, J = 2.0 Hz, 1H), 5.14 (s, 2H), 4.67 (br, 1H), 4.40

(d, J = 4.8 Hz, 2H), 2.23 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

187.0, 162.7, 154.8, 143.3, 141.8, 138.0, 135.9, 133.8, 131.8, 131.4,

131.2, 130.5, 129.8, 129.7, 129.4, 128.2, 127.5, 127.1, 125.9, 118.6,

116.1, 112.8, 106.1, 94.9, 68.9, 46.5, 16.3. ESI-MS: m/z = 433.19 [M

+ H]⁺.

3-((2-Formyl-3-methoxy-5-(((2-methyl-[1,1′-biphenyl]-3-yl)-

methyl)amino)phenoxy)methyl)benzonitrile (31). 11a (290 mg,

1.11 mmol), 16 (330 mg, 1.17 mmol), K₂CO₃(184 mg, 1.33

mmol), and DMF (5 mL) were added to a sealed vial. The mixture

was heated to 85 °C and held for 35 min in a microwave reactor. After

being cooled to room temperature, the reaction mixture was diluted

with H₂O and extracted with DCM. The combined organic phase was

washed with water and concentrated in vacuo. The crude product was

puriﬁed on a silica gel column (PE/EA = 1:1) to give 31 (150 mg,

29% yield) as a yellowish solid. ¹H NMR (400 MHz, CDCl₃): δ 10.32

(s, 1H), 7.78 (d, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.59 (dt, J = 7.6, 1.4

Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.45−7.40 (m, 2H), 7.40−7.34 (m,

1H), 7.32−7.27 (m, 3H), 7.24 (d, J = 2.0 Hz, 1H), 7.23 (s, 1H), 5.78

(dd, J = 18.2, 1.8 Hz, 2H), 5.11 (s, 2H), 4.69 (br, 1H), 4.39 (s, 2H),

3.85 (s, 3H), 2.24 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 186.4,

164.6, 162.8, 154.8, 143.3, 141.8, 138.2, 135.9, 133.8, 131.5, 131.2,

130.1, 129.9, 129.5, 129.3, 128.2, 127.6, 127.1, 125.9, 118.7, 112.6,

106.3, 89.1, 88.3, 69.1, 55.8, 46.4, 16.3. ESI-MS: m/z = 463.19 [M +

H]⁺.

1

EA = 2:1) to give 34 (170 mg, 20% yield) as a yellowish solid. H

NMR (400 MHz, CDCl₃): δ 10.23 (s, 1H), 7.74 (d, J = 8.4 Hz, 1H),

7.71−7.66 (m, 2H), 7.63 (d, J = 7.8 Hz, 1H), 7.51 (t, J = 7.8 Hz, 1H),

7.25−7.22 (m, 1H), 7.22−7.17 (m, 2H), 6.91 (d, J = 8.4 Hz, 1H),

6.81 (d, J = 2.0 Hz, 1H), 6.76 (dd, J = 8.2, 2.2 Hz, 1H), 6.31 (dd, J =

8.6, 1.8 Hz, 1H), 6.07 (d, J = 1.6 Hz, 1H), 5.14 (s, 2H), 4.64 (s, 1H),

4.38 (s, 2H), 4.31 (s, 4H), 2.24 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 187.2, 162.8, 154.9, 143.2, 142.8, 142.8, 138.1, 135.9,

135.2, 134.0, 132.0, 131.5, 131.3, 130.6, 130.0, 129.8, 127.4, 126.0,

122.6, 118.7, 118.3, 117.1, 116.17, 112.9, 106.2, 95.0, 69.0, 64.6, 46.6,

16.4. ESI-MS: m/z = 491.20 [M + H]⁺.

General Procedure B for Buchwald−Hartwig Coupling

Reaction. At an argon atmosphere, electrophile/amines = 1:1.2

equiv or 1.2:1 equiv, Cs₂CO₃(1.5 equiv), BINAP (0.2 equiv), and

Pd(OAc)₂(0.1 equiv) were suspended in dioxane. The mixture was

stirred at 85 °C or reﬂux for 16−24 h. Then, the mixture was cooled,

concentrated in vacuo, diluted with water, and extracted with DCM or

EA. The combined organic phase was washed with water and

concentrated in vacuo. The crude product was puriﬁed by column

chromatography.

5-((2-Formyl-4-methyl-5-(((2-methyl-[1,1′-biphenyl]-3-yl)-

methyl)amino)phenoxy)methyl)nicotinonitrile (35). According to

the general procedure B, 14a (296 mg, 1.5 mmol) and 19 (596 mg,

1.8 mmol) were used and stirred for 20 h at reﬂux. The crude product

was puriﬁed on a silica gel column (PE/EA = 1.5:1) to give 35 (300

1

mg, 45% yield) as a yellowish solid. H NMR (400 MHz, CDCl₃): δ

10.21 (s, 1H), 8.84 (t, J = 1.8 Hz, 2H), 8.05 (t, J = 2.0 Hz, 1H), 7.61

(s, 1H), 7.46−7.42 (m, 2H), 7.39−7.35 (m, 1H), 7.33−7.30 (m, 2H),

7.27−7.24 (m, 3H), 6.08 (s, 1H), 5.18 (s, 2H), 4.50 (br, 1H), 4.44 (s,

2H), 2.25 (s, 3H), 2.13 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ

185.6, 161.1, 153.5, 151.9, 151.7, 142.0, 141.6, 138.4, 136.9, 133.1,

132.8, 129.2, 129.1, 128.4, 128.1, 126.8, 125.8, 125.3, 116.8, 115.2,

113.6, 108.9, 93.3, 66.3, 44.8, 16.9, 15.9. ESI-MS: m/z = 448.21 [M +

H]⁺.

3-((2-Formyl-3-methyl-5-(((2-methyl-[1,1′-biphenyl]-3-yl)-

methyl)amino)phenoxy)methyl)benzonitrile (36). According to the

general procedure B, 14a (99 mg, 0.5 mmol) and 25 (200 mg, 0.5

mmol) were used and stirred for 24 h at reﬂux. The crude product

was puriﬁed on a silica gel column (DCM/EA = 200:1) to give 36

(131 mg, 59% yield) as a yellowish solid. ¹H NMR (400 MHz,

CDCl₃): δ 10.46 (s, 1H), 7.69−7.64 (m, 2H), 7.63−7.59 (m, 1H),

7.53−7.46 (m, 1H), 7.46−7.40 (m, 2H), 7.39−7.34 (m, 1H), 7.32−

7.27 (m, 3H), 7.24 (d, J = 2.0 Hz, 1H), 7.23 (s, 1H), 6.10 (s, 1H),

5.99 (d, J = 2.0 Hz, 1H), 5.11 (s, 2H), 4.53 (br, 1H), 4.38 (d, J = 3.2

Hz, 2H), 2.57 (s, 3H), 2.23 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

188.9, 164.2, 153.3, 145.0, 143.2, 141.8, 138.1, 136.0, 133.8, 131.8,

131.4, 130.4, 129.8, 129.6, 129.3, 128.2, 127.5, 127.0, 125.9, 118.6,

5-((2-Formyl-5-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)amino)-

phenoxy)methyl)nicotinonitrile (32). According to a similar manner

as described for the synthesis of 30, 11a (2.48 g, 9.50 mmol), 17

(2.52 g, 9.95 mmol), and K₂CO₃(1.58 g, 11.4 mmol) were suspended

in MeCN/DMF (51 mL/17 mL) and stirred for 10 h at 60 °C. The

crude product was puriﬁed on a silica gel column (DCM/EA = 10:1)

to give 32 (1 g, 24% yield) as a yellowish solid. ¹H NMR (400 MHz,

DMSO-d₆): δ 10.06 (s, 1H), 9.00 (d, J = 2.0 Hz, 1H), 8.98 (d, J = 2.0

Hz, 1H), 8.48 (t, J = 2.0 Hz, 1H), 7.51 (d, J = 8.8 Hz, 1H), 7.49−7.43

(m, 2H), 7.40−7.35 (m, 2H), 7.33−7.30 (m, 2H), 7.28 (dd, J = 7.6,

1.2 Hz, 1H), 7.23 (t, J = 7.6 Hz, 1H), 7.14 (dd, J = 7.4, 1.4 Hz, 1H),

6.38 (s, 1H), 6.37 (s, 1H), 5.29 (s, 2H), 4.41 (d, J = 5.2 Hz, 2H), 2.20

(s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 185.7, 162.2, 155.8,

O

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Article

114.4, 112.8, 108.6, 93.3, 69.1, 46.3, 22.5, 16.3. ESI-MS: m/z = 447.17

[M + H]⁺.

129.2, 128.5, 128.4, 128.2, 126.9, 125.4, 125.4, 116.8, 114.5, 111.3,

109.0, 95.2, 66.7, 44.6, 15.9. ESI-MS: m/z = 468.13 [M + H]⁺.

2-Methoxy-5-methyl-4-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

amino)benzaldehyde (41). According to the general procedure B,

14a (473 mg, 2.40 mmol) and 4-bromo-2-methoxy-5-methylbenzal-

dehyde (compound 29, 458 mg, 2.0 mmol) were used and stirred for

24 h at reﬂux. The crude product was puriﬁed on a silica gel column

(PE/EA = 7:1−5:1) to give 41 (405 mg, 59% yield) as a yellowish

solid. ¹H NMR (400 MHz, DMSO-d₆): δ 9.97 (s, 1H), 7.47−7.41 (m,

2H), 7.40−7.32 (m, 2H), 7.33−7.25 (m, 3H), 7.22 (t, J = 7.6 Hz,

1H), 7.11 (dd, J = 7.2, 1.2 Hz, 1H), 6.69 (t, J = 5.8 Hz, 1H), 6.05 (s,

1H), 4.54 (d, J = 5.6 Hz, 2H), 3.71 (s, 3H), 2.25 (s, 3H), 2.14 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 185.2, 162.7, 153.6, 142.1,

141.6, 137.2, 132.9, 129.2, 128.7, 128.4, 128.2, 126.8, 126.3, 125.4,

114.4, 113.1, 91.9, 55.3, 44.8, 16.8, 16.0. ESI-MS: m/z = 346.18 [M +

H]⁺.

2-(Benzyloxy)-5-methyl-4-(((2-methyl-[1,1′-biphenyl]-3-yl)-

methyl)amino)benzaldehyde (42). According to the general

procedure B, 14a (367 mg, 1.86 mmol) and 23 (473 mg, 1.55

mmol) were used and stirred for 24 h at reﬂux. The crude product

was puriﬁed on a silica gel column (PE/EA = 7:1) to give 42 (337 mg,

52% yield) as a yellowish solid. ¹H NMR (400 MHz, CDCl₃): δ 10.29

(s, 1H), 7.60 (d, J = 0.8 Hz, 1H), 7.45−7.41 (m, 2H), 7.40−7.33 (m,

5H), 7.33−7.29 (m, 3H), 7.29−7.26 (m, 1H), 7.25−7.23 (m, 2H),

6.14 (s, 1H), 5.12 (s, 2H), 4.39 (s, 2H), 4.34 (br, 1H), 2.22 (s, 3H),

2.10 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 187.6, 162.5, 152.8,

143.3, 141.8, 136.6, 135.9, 133.8, 129.8, 129.7, 129.4, 128.7, 128.2,

128.1, 127.5, 127.2, 127.0, 125.9, 115.3, 114.5, 93.6, 70.5, 46.7, 16.5,

16.3. ESI-MS: m/z = 422.24 [M + H]⁺.

5-((2-Formyl-3-methyl-5-(((2-methyl-[1,1′-biphenyl]-3-yl)-

methyl)amino)phenoxy)methyl)nicotinonitrile (37). According to

the general procedure B, 14a (311 mg, 1.58 mmol) and 26 (760 mg,

1.89 mmol) were used and stirred for 20 h at 85 °C. The crude

product was puriﬁed on a silica gel column (DCM/MeOH = 100:1)

1

to give 37 (305 mg, 43% yield) as a yellowish solid. H NMR (400

MHz, DMSO-d₆): δ 10.27 (s, 1H), 9.00 (d, J = 2.0 Hz, 1H), 8.96 (d, J

= 2.0 Hz, 1H), 8.45 (t, J = 2.0 Hz, 1H), 7.49−7.42 (m, 2H), 7.42−

7.34 (m, 1H), 7.34−7.29 (m, 2H), 7.29−7.18 (m, 3H), 7.14 (dd, J =

7.4, 1.4 Hz, 1H), 6.27 (s, 1H), 6.16 (s, 1H), 5.26 (s, 2H), 4.39 (d, J =

5.2 Hz, 2H), 2.41 (s, 3H), 2.19 (s, 3H). ESI-MS: m/z = 448.20 [M +

H]⁺.

2-Methoxy-6-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)amino)-

nicotinaldehyde (38). BOC₂O (553 mg, 2.54 mmol) was added to a

solution of 14a (1 g, 5.07 mmol) and DABCO (57 mg, 0.51 mmol) in

DCM (30 mL) at rt and stirred overnight. The mixture was diluted

with n-hexane (30 mL). The precipitate was ﬁltered and washed with

n-hexane/DCM (1:1) to yield 1,3-bis((2-methyl-[1,1′-biphenyl]-3-

1

yl)methyl)urea (660 mg, 62% yield) as a white powder. H NMR

(400 MHz, DMSO-d₆): δ 7.46−7.40 (m, 2H), 7.40−7.34 (m, 1H),

7.31−7.26 (m, 3H), 7.23 (t, J = 7.4 Hz, 1H), 7.09 (dd, J = 7.2, 1.6 Hz,

1H), 6.39 (t, J = 5.8 Hz, 1H), 4.29 (d, J = 6.0 Hz, 2H), 2.14 (s, 3H).

ESI-MS: m/z = 421.21 [M + H]⁺.

At an argon atmosphere, 1,3-bis((2-methyl-[1,1′-biphenyl]-3-yl)-

methyl)urea (907 mg, 2.16 mmol), 6-chloro-2-methoxynicotinalde-

hyde (compd 27, 814 mg, 4.75 mmol), Cs₂CO₃(2.10 g, 6.48 mmol),

s-phos (266 mg, 0.65 mmol), and Pd(OAc)₂(73 mg, 0.33 mmol)

were suspended in dioxane (25 mL) and stirred for 24 h at reﬂux.

Then, the mixture was cooled, concentrated in vacuo, diluted with

water, and extracted with DCM. The combined organic phase was

washed with water, and the precipitate was ﬁltered out. The ﬁltrate

was concentrated in vacuo and puriﬁed by silica gel column

chromatography (PE/EA = 8:1) and preparative thin-layer

chromatography (PE/ DCM/EA = 10:5:1) to give 38 (364 mg,

5-Methyl-4-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)amino)-2-

(pyridin-3-ylmethoxy)benzaldehyde (43). According to the general

procedure B, 14a (571 mg, 2.90 mmol) and 22 (740 mg, 2.42 mmol)

were used and stirred for 24 h at reﬂux. The crude product was

puriﬁed on a silica gel column (PE/acetone = 3:1) to give 43 (386

1

mg, 38% yield) as a yellowish solid. H NMR (400 MHz, CDCl₃): δ

10.23 (s, 1H), 8.65 (s, 1H), 8.59 (d, J = 4.4 Hz, 1H), 7.77 (d, J = 8.0

Hz, 1H), 7.60 (s, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.40−7.35 (m, 1H),

7.34−7.27 (m, 4H), 7.27−7.25 (m, 2H), 6.14 (s, 1H), 5.20−5.08 (m,

2H), 4.43 (s, 2H), 4.40 (br, 1H), 2.25 (s, 3H), 2.11 (s, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 187.2, 162.0, 152.8, 149.6, 148.7, 143.3,

141.7, 135.8, 135.1, 133.8, 132.1, 130.0, 129.8, 129.3, 128.2, 127.5,

127.0, 125.9, 123.7, 115.2, 114.9, 93.2, 68.0, 46.7, 16.5, 16.3. ESI-MS:

m/z = 423.21 [M + H]⁺.

1

25% yield) as a pale-yellow solid. H NMR (400 MHz, CDCl₃): δ

10.07 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.45−7.39 (m, 2H), 7.38−

7.33 (m, 1H), 7.33−7.27 (m, 3H), 7.25−7.19 (m, 2H), 6.05 (d, J =

8.4 Hz, 1H), 5.32 (br, 1H), 4.65 (s, 2H), 3.98 (s, 3H), 2.24 (s, 3H).

¹³C NMR (100 MHz, CDCl₃): δ 187.1, 166.0, 160.8, 143.2, 141.9,

139.1, 136.4, 133.9, 129.7, 129.4, 128.2, 127.7, 127.0, 125.8, 116.0,

103.9, 53.4, 44.7, 16.4. ESI-MS: m/z = 333.14 [M + H]⁺.

3-((2-Formyl-4-methyl-5-(((2-methyl-[1,1′-biphenyl]-3-yl)-

methyl)amino)phenoxy)methyl)benzonitrile (44). According to the

general procedure B, 14a (548 mg, 2.78 mmol) and 20 (766 mg, 2.32

mmol) were used and stirred for 24 h at reﬂux. The crude product

was puriﬁed on a silica gel column (PE/acetone = 5:1) to give 44

(382 mg, 37% yield) as a yellowish solid. ¹H NMR (400 MHz,

CDCl₃): δ 10.25 (s, 1H), 7.68 (s, 1H), 7.67−7.58 (m, 3H), 7.48 (t, J

= 7.6 Hz, 1H), 7.46−7.41 (m, 2H), 7.39−7.35 (m, 1H), 7.34−7.28

(m, 2H), 7.28−7.23 (m, 3H), 6.08 (s, 1H), 5.14 (s, 2H), 4.46 (br,

1H), 4.41 (d, J = 4.8 Hz, 2H), 2.24 (s, 3H), 2.12 (s, 3H). ¹³C NMR

(100 MHz, CDCl₃): δ 187.2, 161.8, 152.8, 143.4, 141.7, 138.2, 135.7,

133.8, 131.8, 131.4, 130.5, 130.2, 130.0, 129.6, 129.4, 128.3, 127.5,

127.1, 126.0, 118.6, 115.3, 115.0, 112.9, 93.3, 69.2, 46.7, 16.5, 16.3.

ESI-MS: m/z = 447.21 [M + H]⁺.

2-Ethoxy-5-methyl-4-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

amino)benzaldehyde (45). According to the general procedure B,

14a (591 mg, 3.0 mmol) and 21 (608 mg, 2.50 mmol) were used and

stirred for 24 h at reﬂux. The crude product was puriﬁed on a silica gel

column (PE/EA = 7:1) to give 45 (350 mg, 39% yield) as a yellowish

solid. ¹H NMR (400 MHz, CDCl₃): δ 10.22 (s, 1H), 7.58 (d, J = 0.8

Hz, 1H), 7.46−7.40 (m, 2H), 7.40−7.34 (m, 1H), 7.34−7.27 (m,

3H), 7.26 (d, J = 3.6 Hz, 1H), 7.24 (s, 1H), 6.09 (s, 1H), 4.44 (d, J =

4.0 Hz, 2H), 4.39 (br, 1H), 4.09 (q, J = 6.8 Hz, 2H), 2.26 (s, 3H),

2.09 (s, 3H), 1.43 (t, J = 6.8 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃):

δ 187.7, 162.9, 152.8, 143.3, 141.8, 136.0, 133.9, 129.9, 129.6, 129.4,

128.2, 127.8, 127.0, 125.9, 115.1, 114.1, 92.7, 64.1, 46.7, 16.5, 16.3,

14.7. ESI-MS: m/z = 360.21 [M + H]⁺.

4-Methoxy-6-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)amino)-

nicotinaldehyde (39). According to a similar manner as described for

the synthesis of 38, 1,3-bis((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

urea (660 mg, 1.57 mmol) and 6-chloro-4-methoxynicotinaldehyde

(compd 28, 448 mg, 2.61 mmol) were used. The crude product was

puriﬁed by silica gel column chromatography (PE/EA = 2.5:1) and

preparative thin-layer chromatography (DCM/MeOH = 40:1) to give

1

39 (143 mg, 16% yield) as a pale-yellow solid. H NMR (400 MHz,

CDCl₃): δ 10.04 (s, 1H), 8.38 (s, 1H), 7.44−7.39 (m, 2H), 7.38−

7.33 (m, 1H), 7.31 (dd, J = 6.8, 2.4 Hz, 1H), 7.29−7.26 (m, 2H),

7.25−7.20 (m, 2H), 5.81 (s, 1H), 5.77 (br, 1H), 4.59 (d, J = 5.2 Hz,

2H), 3.89 (s, 3H), 2.24 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

187.3, 168.0, 163.3, 153.6, 143.3, 141.8, 135.9, 134.0, 129.9, 129.3,

128.2, 127.6, 127.0, 125.9, 114.1, 86.5, 55.5, 45.3, 16.4. ESI-MS: m/z

= 333.16 [M + H]⁺.

5-((4-Chloro-2-formyl-5-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

amino)phenoxy)methyl)nicotinonitrile (40). According to the

general procedure B, 14a (354 mg, 1.80 mmol) and 24 (527 mg,

1.50 mmol) were used and stirred for 24 h at 85 °C. The crude

product was puriﬁed on a silica gel column (PE/EA = 3:1−2:1) to

give 40 (345 mg, 49% yield) as a yellowish solid. ¹H NMR (400 MHz,

DMSO-d₆): δ 10.05 (s, 1H), 8.93 (d, J = 2.0 Hz, 1H), 8.86 (d, J = 2.0

Hz, 1H), 8.37 (t, J = 2.0 Hz, 1H), 7.61 (s, 1H), 7.49−7.43 (m, 2H),

7.41−7.35 (m, 1H), 7.35−7.31 (m, 2H), 7.19−7.14 (m, 1H), 7.11 (s,

1H), 7.11−7.08 (m, 2H), 6.29 (s, 1H), 5.27 (s, 2H), 4.58 (d, J = 6.0

Hz, 2H), 2.22 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 185.4,

160.8, 151.9, 151.8, 150.2, 142.1, 141.5, 138.5, 136.4, 132.7, 132.6,

P

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5-((5-((3-(2,3-Dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)-

amino)-2-formyl-4-methylphenoxy)methyl)nicotinonitrile (46). Ac-

cording to the general procedure B, 14b (420 mg, 1.64 mmol) and 19

(654 mg, 1.97 mmol) were used and stirred for 24 h at 85 °C. The

crude product was puriﬁed on a silica gel column (PE/EA = 1:1) to

give 46 (325 mg, 39% yield) as a yellowish solid. ¹H NMR (400 MHz,

DMSO-d₆): δ 10.06 (s, 1H), 8.92 (d, J = 2.0 Hz, 1H), 8.83 (d, J = 2.4

Hz, 1H), 8.33 (t, J = 2.0 Hz, 1H), 7.38 (d, J = 0.8 Hz, 1H), 7.13−7.08

(m, 2H), 7.07−7.05 (m, 1H), 6.92 (d, J = 8.0 Hz, 1H), 6.81 (d, J =

2.0 Hz, 1H), 6.77 (dd, J = 8.2, 2.2 Hz, 1H), 6.67 (t, J = 5.8 Hz, 1H),

6.09 (s, 1H), 5.21 (s, 2H), 4.47 (d, J = 5.6 Hz, 2H), 4.29 (s, 4H), 2.23

(s, 3H), 2.14 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 185.6,

161.1, 153.5, 151.9, 151.7, 142.9, 142.4, 141.5, 138.4, 136.9, 134.7,

133.1, 132.9, 129.1, 128.4, 125.5, 125.2, 122.2, 117.8, 116.8, 116.7,

115.2, 113.6, 108.9, 93.3, 66.3, 64.1, 44.8, 16.9, 16.0. ESI-MS: m/z =

506.20 [M + H]⁺.

7.10 (dd, J = 7.5, 1.1 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H), 6.35 (d, J =

2.0 Hz, 1H), 6.15 (dd, J = 8.0, 2.0 Hz, 1H), 6.03 (t, J = 5.6 Hz, 1H),

5.10 (s, 2H), 4.23 (d, J = 5.6 Hz, 2H), 3.57 (s, 2H), 3.11 (q, J = 6.4

Hz, 2H), 2.52−2.50 (m, 2H), 2.18 (s, 3H), 1.81 (br, 1H), 1.76 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 169.0, 156.6, 149.2, 141.9,

141.7, 139.4, 138.2, 133.1, 131.9, 131.4, 130.6, 130.2, 129.7, 129.2,

128.3, 128.2, 126.9, 126.8, 125.3, 118.8, 116.2, 111.4, 103.9, 97.3,

67.8, 48.2, 47.5, 45.6, 38.8, 22.6, 15.8. ESI-HRMS: m/z calculated for

+

C₃₃H₃₅N₄O₂[M + H]⁺, 519.2755; found, 519.2754.

3-((2-(((2-Hydroxyethyl)amino)methyl)-5-(((2-methyl-[1,1′-bi-

phenyl]-3-yl)methyl)amino)phenoxy)methyl)benzonitrile (50). Ac-

cording to a similar manner as the general procedure C, 30 (108 mg,

0.25 mmol) was reacted with 2-aminoethanol (31 mg, 0.5 mmol) in

DCE (3 mL) and stirred for 12 h at rt. After completion of

NaBH(OAc)₃addition, the mixture was stirred for further 12 h. The

crude product was puriﬁed by silica gel column chromatography

(DCM/MeOH = 10:1−8:1) and recrystallization (EA/n-hexane) to

give 50 (30 mg, 25% yield) as a pale-yellow solid. mp 173.1−174.2

°C. HPLC purity: 98.56% (method B). ¹H NMR (400 MHz, DMSO-

d₆): δ 7.90 (s, 1H), 7.79 (d, J = 7.6 Hz, 2H), 7.59 (t, J = 8.0 Hz, 1H),

7.45 (t, J = 7.2 Hz, 2H), 7.38−7.35 (m, 1H), 7.31−7.29 (m, 3H),

7.19 (t, J = 7.6 Hz, 1H), 7.10 (d, J = 7.2 Hz, 1H), 6.96 (d, J = 8.0 Hz,

1H), 6.36 (s, 1H), 6.15 (d, J = 7.8 Hz, 1H), 6.03 (t, J = 5.6 Hz, 1H),

5.10 (s, 2H), 4.46 (br, 1H), 4.23 (d, J = 4.8 Hz, 2H), 3.58 (s, 2H),

3.44 (t, J = 5.2 Hz, 2H), 2.53 (t, J = 6.0, 2H), 2.18 (s, 3H), 1.90 (br,

1H). ¹³C NMR (100 MHz, DMSO-d₆): δ 156.7, 149.1, 141.9, 141.7,

139.4, 138.2, 133.1, 132.0, 131.4, 130.6, 130.2, 129.7, 129.2, 128.3,

128.2, 126.9, 126.8, 125.3, 118.7, 116.3, 115.4, 111.3, 103.8, 97.3,

67.8, 60.5, 51.1, 47.9, 45.6, 15.8. ESI-HRMS: m/z calculated for

5-((4-Chloro-5-((3-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)-2-

methylbenzyl)amino)-2-formylphenoxy)methyl)nicotinonitrile

(47). According to the general procedure B, 14b (418 mg, 1.64 mmol)

and 24 (480 mg, 1.37 mmol) were used and stirred for 24 h at reﬂux.

The crude product was puriﬁed on a silica gel column (PE/EA = 3:2)

1

to give 47 (559 mg, 78% yield) as a yellowish solid. H NMR (400

MHz, DMSO-d₆): δ 10.04 (s, 1H), 8.93 (d, J = 2.0 Hz, 1H), 8.85 (d, J

= 2.0 Hz, 1H), 8.36 (t, J = 2.0 Hz, 1H), 7.60 (s, 1H), 7.14−7.03 (m,

4H), 6.92 (d, J = 8.2 Hz, 1H), 6.80 (d, J = 2.0 Hz, 1H), 6.76 (dd, J =

8.2, 2.2 Hz, 1H), 6.26 (s, 1H), 5.25 (s, 2H), 4.56 (d, J = 6.0 Hz, 2H),

4.29 (s, 4H), 2.22 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 185.3,

160.8, 151.9, 151.8, 150.2, 142.9, 142.4, 141.6, 138.5, 136.3, 134.6,

132.8, 132.6, 128.5, 128.4, 125.3, 125.1, 122.1, 117.8, 116.7, 116.7,

114.4, 111.3, 108.9, 95.2, 66.6, 64.1, 44.6, 15.9. ESI-MS: m/z = 526.19

[M + H]⁺.

+

C₃₁H₃₁N₃NaO₂[M + Na]⁺, 500.2308; found, 500.2307.

3-((2-(((1,3-Dihydroxypropan-2-yl)amino)methyl)-5-(((2-methyl-

[1,1′-biphenyl]-3-yl)methyl)amino)phenoxy)methyl)benzonitrile

(51). According to a similar manner as the general procedure C, 30

(108 mg, 0.25 mmol) was reacted with 2-amino-1,3-propanediol (46

mg, 0.5 mmol) in DCE (2 mL) and DMF (0.5 mL). The crude

product was puriﬁed by recrystallization (EA/n-hexane) to give 51

(92 mg, 72% yield) as a pale-yellow solid. mp 137.6−138.5 °C. HPLC

purity: 97.72% (method B). ¹H NMR (400 MHz, DMSO-d₆): δ 7.92

(s, 1H), 7.82−7.78 (m, 2H), 7.58 (t, J = 8.0 Hz, 1H), 7.48−7.42 (m,

2H), 7.40−7.34 (m, 1H), 7.32−7.27 (m, 3H), 7.19 (t, J = 7.6 Hz,

1H), 7.10 (dd, J = 7.6, 1.2 Hz, 1H), 6.97 (d, J = 8.4 Hz, 1H), 6.35 (d,

J = 2.0 Hz, 1H), 6.15 (dd, J = 8.0, 1.6 Hz, 1H), 6.02 (t, J = 5.2 Hz,

1H), 5.10 (s, 2H), 4.38 (br, 1H), 4.23 (d, J = 5.6 Hz, 2H), 3.62 (s,

2H), 3.36 (ddd, J = 26.2, 10.8, 5.6 Hz, 4H), 2.55 (p, J = 5.6 Hz, 1H),

2.18 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 157.2, 149.7, 142.4,

142.2, 139.8, 138.7, 133.6, 132.5, 131.9, 131.2, 130.8, 130.1, 129.7,

128.8, 128.7, 127.4, 127.3, 125.8, 119.3, 117.0, 111.8, 104.3, 97.8,

68.3, 61.7, 60.6, 46.3, 46.1, 16.3. ESI-HRMS: m/z calculated for

General Procedure C for Reductive Amination Reaction.

Aldehydes (1 equiv), amines (2 equiv), and AcOH (2 equiv) were

suspended in an appropriate solvent and stirred for 24 h at 35 °C.

After addition of NaBH(OAc)₃(3 equiv), the mixture was stirred for

further 24 h. Then, the mixture was diluted with water and extracted

with DCM/MeOH (10:1). The combined organic phase was washed

with water and concentrated to dryness in vacuo. The crude product

was puriﬁed by preparative high-performance liquid chromatography.

N-(2-(((2-Methoxy-6-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

amino)pyridin-3-yl)methyl)amino)ethyl)acetamide(48). According

to a similar manner as the general procedure C, 38 (157 mg, 0.47

mmol) was reacted with N-(2-amino-ethyl)acetamide (97 mg, 0.94

mmol) in DCE (4 mL) and DMF (1 mL). After completion of

NaBH(OAc)₃addition, the mixture was stirred for further 12 h. The

reaction yielded 48 (85 mg, 43% yield) as a yellowish solid. mp

1

119.2−120.3 °C. HPLC purity: 96.00% (method B). H NMR (400

MHz, CDCl₃): δ 7.72 (s, 1H), 7.47−7.39 (m, 3H), 7.39−7.32 (m,

1H), 7.32−7.27 (m, 3H), 7.24−7.16 (m, 2H), 6.54 (br, 1H), 5.94 (d,

J = 8.0 Hz, 1H), 4.79 (t, J = 5.2 Hz, 1H), 4.46 (d, J = 5.2 Hz, 2H),

3.93 (s, 3H), 3.88 (s, 2H), 3.54−3.47 (m, 2H), 2.95 (t, J = 4.8 Hz,

2H), 2.22 (s, 3H), 2.02 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ

171.3, 161.6, 158.3, 143.0, 142.2, 142.1, 137.0, 133.8, 129.5, 129.4,

128.1, 127.5, 126.9, 125.7, 100.2, 97.9, 53.4, 47.0, 46.5, 45.0, 36.2,

+

C₃₂H₃₃N₃NaO₃[M + Na]⁺, 530.2414; found, 530.2413.

(2-((3-Cyanobenzyl)oxy)-4-(((2-methyl-[1,1′-biphenyl]-3-yl)-

methyl)amino)benzyl)-^D-serine (52). According to the general

procedure C, 30 (216 mg, 0.50 mmol) was reacted with ^D-serine

(105 mg, 1.0 mmol) in DMF (2 mL), which yielded 52 (180 mg, 69%

yield) as a yellowish solid. mp 173.5−174.4 °C. HPLC purity: 98.15%

+

1

23.2, 16.3. ESI-HRMS: m/z calculated for C₂₅H₃₀N₄NaO₂[M +

(method A). H NMR (400 MHz, DMSO-d₆): δ 8.31 (br, 2H), 7.99

Na]⁺, 441.2261; found, 441.2259.

(s, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H), 7.57 (t, J =

8.0 Hz, 1H), 7.47−7.44 (m, 2H), 7.40−7.35 (m, 1H), 7.31 (d, J = 1.2

Hz, 1H), 7.29 (s, 1H), 7.26 (d, J = 7.6 Hz, 1H), 7.19 (t, J = 7.6 Hz,

1H), 7.10 (d, J = 7.6 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.40−6.35

(m, 2H), 6.19 (dd, J = 8.0, 1.6 Hz, 1H), 5.41 (br, 1H), 5.18−5.11 (m,

2H), 4.25 (d, J = 5.2 Hz, 2H), 4.04 (dd, J = 43.6, 12.8 Hz, 2H), 3.78

(dd, J = 11.6, 4.0 Hz, 1H), 3.66 (dd, J = 11.2, 7.2 Hz, 1H), 3.19−3.16

(m, 1H), 2.17 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.1,

157.3, 151.2, 142.0, 141.7, 138.8, 137.9, 133.2, 132.4, 132.3, 131.6,

130.9, 129.6, 129.2, 128.4, 128.2, 126.8, 125.3, 118.8, 111.4, 107.4,

104.0, 96.7, 68.2, 61.9, 59.9, 45.6, 45.4, 15.8. ESI-HRMS: m/z

N-(2-((2-((3-Cyanobenzyl)oxy)-4-(((2-methyl-[1,1′-biphenyl]-3-

yl)methyl)amino)benzyl)amino)ethyl)acetamide (49). According to

a similar manner as the general procedure C, 30 (100 mg, 0.23 mmol)

was reacted with N-(2-aminoethyl)acetamide (47 mg, 0.46 mmol) in

DCE (2 mL) and DMF (0.5 mL). After completion of NaBH(OAc)₃

addition, the mixture was stirred for further 14 h, diluted with water,

and extracted with DCM. The combined organic phase was washed

with water and concentrated to dryness in vacuo. The crude product

was puriﬁed by silica gel column chromatography (DCM/MeOH =

10:1) and recrystallization (EA/n-hexane) to give 49 (42 mg, 35%

yield) as a pale-yellow solid. mp 147.7−148.7 °C. HPLC purity:

95.99% (method B). ¹H NMR (400 MHz, DMSO-d₆): δ 7.90 (s, 1H),

7.80−7.76 (m, 3H), 7.60 (t, J = 7.6 Hz, 1H), 7.45 (t, J = 7.6 Hz, 2H),

7.39−7.34 (m, 1H), 7.31−7.28 (m, 3H), 7.19 (t, J = 7.6 Hz, 1H),

+

calculated for C₃₂H₃₁N₃NaO₄[M + Na]⁺, 544.2207; found,

544.2208.

(2-((3-Cyanobenzyl)oxy)-6-methyl-4-(((2-methyl-[1,1′-biphenyl]-

3-yl)methyl)amino)benzyl)-^D-serine (53). According to the general

Q

https://dx.doi.org/10.1021/acs.jmedchem.0c01329

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

procedure C, 36 (131 mg, 0.29 mmol) was reacted with D-serine (62

mg, 0.58 mmol) in anhydrous DMF (4 mL) and DCE (1 mL), which

yielded 53 (48 mg, 31% yield) as a white solid. mp 167.5−169.0 °C.

HPLC purity: 97.97% (method B). ¹H NMR (400 MHz, DMSO-d₆):

δ 8.20 (br, 2H), 7.98 (s, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 7.8

Hz, 1H), 7.56 (dt, J = 7.6, 1.2 Hz, 1H), 7.49−7.41 (m, 2H), 7.40−

7.34 (m, 1H), 7.32−7.28 (m, 2H), 7.27−7.23 (m, 1H), 7.18 (t, J =

7.6 Hz, 1H), 7.10 (dd, J = 7.6, 1.2 Hz, 1H), 6.29 (t, J = 5.2 Hz, 1H),

6.24 (d, J = 1.6 Hz, 1H), 6.12 (d, J = 1.6 Hz, 1H), 5.37 (br, 1H), 5.13

(dd, J = 14.8, 13.2 Hz, 2H), 4.24 (d, J = 5.6 Hz, 2H), 4.10 (s, 2H),

3.84 (dd, J = 11.6, 4.4 Hz, 1H), 3.61 (dd, J = 11.6, 8.4 Hz, 1H), 3.19

(dd, J = 8.0, 4.4 Hz, 1H), 2.21 (s, 3H), 2.17 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆): δ 167.6, 157.8, 150.4, 142.0, 141.7, 139.5, 138.7,

137.9, 133.2, 132.4, 131.6, 130.9, 129.7, 129.2, 128.4, 128.3, 126.9,

125.3, 118.8, 115.5, 111.4, 106.2, 94.5, 68.2, 62.5, 59.9, 45.2, 42.7,

59.8, 45.5, 45.1, 16.8, 15.8. ESI-HRMS: m/z calculated for

+

C₃₂H₃₄N₂NaO₄[M + Na]⁺, 533.2411; found, 533.2408.

(5-Methyl-4-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)amino)-2-

(pyridin-3-ylmethoxy)benzyl)-^D-serine (57). According to the general

procedure C, 43 (310 mg, 0.73 mmol) was reacted with ^D-serine (154

mg, 1.47 mmol) in anhydrous DMF (8 mL) and anhydrous MeOH (2

mL), which yielded 57 (51 mg, 14% yield) as a white solid. mp

1

196.4−197.9 °C. HPLC purity: 99.61% (method A). H NMR (400

MHz, DMSO-d₆): δ 8.58 (d, J = 1.6 Hz, 1H), 8.47 (dd, J = 4.8, 1.6

Hz, 1H), 8.26 (br, 2H), 7.81 (dt, J = 8.0, 2.0 Hz, 1H), 7.49−7.42 (m,

2H), 7.40−7.35 (m, 1H), 7.34−7.31 (m, 2H), 7.29 (ddd, J = 7.8, 4.8,

0.4 Hz, 1H), 7.21 (dd, J = 7.6, 1.2 Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H),

7.09 (dd, J = 7.4, 1.4 Hz, 1H), 6.98 (s, 1H), 6.15 (s, 1H), 5.69 (t, J =

5.6 Hz, 1H), 5.38 (br, 1H), 5.04 (dd, J = 15.6, 13.2 Hz, 2H), 4.35 (d,

J = 5.2 Hz, 2H), 3.99 (dd, J = 40.8, 13.2 Hz, 2H), 3.78 (dd, J = 11.4,

4.4 Hz, 1H), 3.65 (dd, J = 11.2, 7.2 Hz, 1H), 3.17 (dd, J = 7.0, 4.6 Hz,

1H), 2.21 (s, 3H), 2.10 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ

167.7, 155.8, 148.9, 148.7, 148.1, 141.9, 141.7, 137.8, 135.3, 132.8,

132.7, 132.6, 129.2, 128.1, 126.8, 126.1, 125.3, 123.4, 113.8, 106.9,

95.1, 67.3, 61.8, 59.9, 45.5, 45.1, 16.8, 15.8. ESI-HRMS: m/z

+

19.6, 15.8. ESI-HRMS: m/z calculated for C₃₃H₃₃N₃NaO₄[M +

Na]⁺, 558.2363; found, 558.2363.

(2-((3-Cyanobenzyl)oxy)-6-methoxy-4-(((2-methyl-[1,1′-biphen-

yl]-3-yl)methyl)amino)benzyl)-^D-serine (54). According to the

general procedure C, 31 (189 mg, 0.41 mmol) was reacted with ^D-

serine (86 mg, 0.82 mmol) in anhydrous DMF (4 mL), which yielded

54 (136 mg, 60% yield) as a white solid. mp 157.2−158.5 °C. HPLC

purity: 98.43% (method B). ¹H NMR (400 MHz, DMSO-d₆): δ 8.16

(br, 2H), 7.95 (s, 1H), 7.84 (d, J = 8.0 Hz, 1H), 7.78 (d, J = 8.0 Hz,

1H), 7.56 (t, J = 7.8 Hz, 1H), 7.48−7.43 (m, 2H), 7.40−7.34 (m,

1H), 7.31−7.28 (m, 3H), 7.20 (t, J = 7.6 Hz, 1H), 7.12 (dd, J = 7.2,

1.2 Hz, 1H), 6.42 (t, J = 5.2 Hz, 1H), 6.02 (d, J = 3.2 Hz, 2H), 5.31

(br, 1H), 5.13 (dd, J = 16.4, 13.2 Hz, 2H), 4.28 (d, J = 5.2 Hz, 2H),

4.09 (dd, J = 26.8, 13.2 Hz, 2H), 3.78 (dd, J = 11.2, 4.4 Hz, 1H), 3.73

(s, 3H), 3.62 (dd, J = 11.6, 7.6 Hz, 1H), 3.12 (dd, J = 7.2, 4.4 Hz,

1H), 2.19 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 167.7, 159.5,

158.1, 151.5, 142.1, 141.6, 138.7, 137.8, 133.3, 132.2, 131.5, 130.8,

129.6, 129.2, 128.5, 128.2, 127.2, 126.8, 125.4, 118.7, 111.4, 95.3,

89.3, 88.7, 68.3, 62.1, 59.6, 55.5, 45.4, 15.9. ESI-HRMS: m/z

+

calculated for C₃₁H₃₃N₃NaO₄[M + Na]⁺, 534.2363; found,

534.2360.

(2-((5-Cyanopyridin-3-yl)methoxy)-5-methyl-4-(((2-methyl-[1,1′-

biphenyl]-3-yl)methyl)amino)benzyl)-^D-serine (58). According to

the general procedure C, 35 (750 mg, 1.68 mmol) was reacted with

D-serine (350 mg, 3.36 mmol) in anhydrous DMF (12 mL) and

anhydrous MeOH (4 mL), which yielded 58 (350 mg, 39% yield) as a

white solid. mp 172.3−173.9 °C. HPLC purity: 97.93% (method A).

¹H NMR (400 MHz, DMSO-d₆): δ 8.88 (dd, J = 15.6, 2.0 Hz, 2H),

8.37 (t, J = 2.0 Hz, 1H), 8.34 (br, 2H), 7.48−7.44 (m, 2H), 7.40−

7.35 (m, 1H), 7.35−7.34 (m, 1H), 7.33−7.32 (m, 1H), 7.16 (dd, J =

7.6, 1.8 Hz, 1H), 7.12 (t, J = 7.2 Hz, 1H), 7.07 (dd, J = 7.2, 1.8 Hz,

1H), 7.01 (s, 1H), 6.09 (s, 1H), 5.72 (t, J = 5.6 Hz, 1H), 5.38 (br,

1H), 5.14 (dd, J = 18.8, 13.2 Hz, 2H), 4.35 (d, J = 5.6 Hz, 2H), 4.04

(dd, J = 44.8, 12.8 Hz, 2H), 3.78 (dd, J = 11.2, 4.4 Hz, 1H), 3.64 (dd,

J = 11.4, 7.2 Hz, 1H), 3.19−3.16 (m, 1H), 2.19 (s, 3H), 2.10 (s, 3H).

¹³C NMR (100 MHz, DMSO-d₆): δ 167.9, 155.6, 151.9, 151.6, 148.2,

141.9, 141.7, 138.4, 137.7, 133.4, 133.0, 132.7, 129.3, 128.2, 126.8,

125.9, 125.2, 116.9, 114.0, 108.8, 106.6, 94.8, 66.3, 61.9, 59.9, 45.4,

+

calculated for C₃₃H₃₃N₃NaO₅[M + Na]⁺, 574.2312; found,

574.2313.

(2-((3-Cyanobenzyl)oxy)-5-methyl-4-(((2-methyl-[1,1′-biphenyl]-

3-yl)methyl)amino)benzyl)-^D-serine (55). According to the general

procedure C, 44 (341 mg, 0.76 mmol) was reacted with ^D-serine (161

mg, 1.53 mmol) in anhydrous DMF (7 mL), which yielded 55 (320

mg, 79% yield) as a white solid. mp 177.4−178.6 °C. HPLC purity:

+

45.1, 16.9, 15.8. ESI-HRMS: m/z calculated for C₃₂H₃₂N₄NaO₄[M

+ Na]⁺, 559.2316; found, 559.2313.

1

97.14% (method A). H NMR (400 MHz, DMSO-d₆): δ 8.29 (br,

(2-((5-Cyanopyridin-3-yl)methoxy)-4-(((2-methyl-[1,1′-biphenyl]-

3-yl)methyl)amino)benzyl)-^D-serine (59). According to the general

procedure C, 32 (133 mg, 0.31 mmol) was reacted with ^D-serine (86

mg, 0.82 mmol) in anhydrous DMF (2.5 mL), which yielded 59 (73

mg, 45% yield) as a white solid. mp 153.8−154.6 °C. HPLC purity:

97.20% (method A). ¹H NMR (400 MHz, DMSO-d₆): δ 9.00 (dd, J =

10.4, 2.4 Hz, 2H), 8.51 (t, J = 2.0 Hz, 1H), 7.48−7.44 (m, 2H), 7.39−

7.35 (m, 1H), 7.32−7.31 (m, 1H), 7.31−7.29 (m, 1H), 7.28−7.25

(m, 1H), 7.19 (t, J = 7.6 Hz, 1H), 7.09 (t, J = 8.0 Hz, 2H), 6.41−7.25

(m, 2H), 6.21 (dd, J = 8.4, 1.6 Hz, 1H), 5.32 (br, 1H), 5.20 (dd, J =

19.6, 12.8 Hz, 2H), 4.26 (d, J = 5.2 Hz, 2H), 4.03 (dd, J = 46.4, 13.2

Hz, 2H), 3.77 (dd, J = 11.2, 4.4 Hz, 1H), 3.64 (dd, J = 11.4, 7.2 Hz,

1H), 3.17−3.14 (m, 1H), 2.18 (s, 3H). ¹³C NMR (100 MHz, DMSO-

d₆): δ 167.9, 157.2, 152.4, 151.7, 151.2, 142.0, 141.7, 138.9, 137.8,

133.2, 133.2, 132.5, 129.2, 128.5, 128.2, 126.9, 126.8, 125.3, 116.9,

108.9, 107.4, 104.1, 96.6, 66.1, 61.9, 59.9, 45.5, 45.3, 15.8. ESI-

1H), 7.87 (s, 1H), 7.74−7.68 (m, 2H), 7.47−7.43 (m, 3H), 7.39−

7.35 (m, 1H), 7.33−7.28 (m, 2H), 7.17 (dd, J = 7.2, 1.2 Hz, 1H), 7.13

(t, J = 7.4 Hz, 1H), 7.08 (dd, J = 7.2, 1.6 Hz, 1H), 6.99 (s, 1H), 6.09

(s, 1H), 5.70 (t, J = 5.6 Hz, 1H), 5.38 (br, 1H), 5.12−5.02 (m, 2H),

4.33 (d, J = 5.2 Hz, 2H), 4.02 (dd, J = 40.4, 13.0 Hz, 2H), 3.79 (dd, J

= 11.6, 4.4 Hz, 1H), 3.65 (dd, J = 11.2, 7.2 Hz, 1H), 3.18 (dd, J = 6.9,

4.6 Hz, 1H), 2.19 (s, 3H), 2.10 (s, 3H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 167.8, 155.8, 148.2, 141.9, 141.7, 138.9, 137.8, 132.80,

132.7, 132.0, 131.4, 130.6, 129.5, 129.2, 128.2, 126.8, 126.0, 125.3,

118.7, 113.8, 111.4, 106.7, 95.0, 68.4, 61.9, 59.9, 45.5, 45.1, 16.9, 15.8.

+

ESI-HRMS: m/z calculated for C₃₃H₃₃N₃NaO₄[M + Na]⁺,

558.2363; found, 558.2364.

(2-(Benzyloxy)-5-methyl-4-(((2-methyl-[1,1′-biphenyl]-3-yl)-

methyl)amino)benzyl)-^D-serine (56). According to the general

procedure C, 42 (297 mg, 0.70 mmol) was reacted with ^D-serine

(148 mg, 1.41 mmol) in anhydrous DMF (8 mL) and anhydrous

MeOH (2 mL), which yielded 56 (36 mg, 10% yield) as a white solid.

+

HRMS: m/z calculated for C₃₁H₃₀N₄NaO₄[M + Na]⁺, 545.2159;

found, 545.2157.

1

mp 186.6−187.8 °C. HPLC purity: 98.26% (method B). H NMR

(2-((5-Cyanopyridin-3-yl)methoxy)-6-methyl-4-(((2-methyl-[1,1′-

biphenyl]-3-yl)methyl)amino)benzyl)-^D-serine (60). According to

the general procedure C, 37 (269 mg, 0.60 mmol) was reacted with

D-serine (126 mg, 1.20 mmol) in anhydrous DMF (8 mL) and DCE

(2 mL), which yielded 60 (119 mg, 37% yield) as a white solid. mp

(400 MHz, DMSO-d₆): δ 8.16 (br, 2H), 7.46 (tt, J = 8.2, 1.6 Hz, 2H),

7.41−7.37 (m, 1H), 7.35−7.32 (m, 3H), 7.31 (t, J = 1.2 Hz, 1H),

7.30−7.24 (m, 3H), 7.21 (dd, J = 7.6, 1.6 Hz, 1H), 7.16 (t, J = 7.6 Hz,

1H), 7.10 (dd, J = 7.4, 1.4 Hz, 1H), 6.97 (s, 1H), 6.13 (s, 1H), 5.70

(t, J = 5.6 Hz, 1H), 5.38 (br, 1H), 4.98 (s, 2H), 4.32 (d, J = 5.2 Hz,

2H), 3.99 (dd, J = 36.2, 13.0 Hz, 2H), 3.78 (dd, J = 11.4, 4.4 Hz, 1H),

3.66 (dd, J = 11.4, 7.0 Hz, 1H), 3.17 (dd, J = 7.2, 4.4 Hz, 1H), 2.20 (s,

3H), 2.09 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.0, 156.0,

148.1, 141.9, 141.7, 137.9, 137.1, 132.7, 132.6, 129.2, 128.3, 128.2,

128.1, 127.6, 127.4, 126.8, 126.0, 125.3, 113.6, 106.7, 95.3, 69.6, 61.8,

1

183.5−184.7 °C. HPLC purity: 96.76% (method B). H NMR (400

MHz, DMSO-d₆): δ 9.02 (d, J = 2.4 Hz, 1H), 8.99 (d, J = 2.0 Hz,

1H), 8.49 (t, J = 2.4 Hz, 1H), 8.17 (br, 2H), 7.48−7.42 (m, 2H),

7.41−7.34 (m, 1H), 7.34−7.28 (m, 2H), 7.26 (d, J = 7.6 Hz, 1H),

7.19 (t, J = 7.6 Hz, 1H), 7.10 (dd, J = 7.6, 1.6 Hz, 1H), 6.31 (t, J = 5.6

Hz, 1H), 6.25 (d, J = 1.6 Hz, 1H), 6.14 (d, J = 1.6 Hz, 1H), 5.38 (br,

R

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1H), 5.18 (dd, J = 18.0, 13.2 Hz, 2H), 4.25 (d, J = 5.2 Hz, 2H), 4.10

(s, 2H), 3.82 (dd, J = 11.6, 4.4 Hz, 1H), 3.61 (dd, J = 11.2, 8.4 Hz,

1H), 3.19 (dd, J = 8.0, 4.4 Hz, 1H), 2.22 (s, 3H), 2.17 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 167.7, 157.6, 152.4, 151.7, 150.4,

142.0, 141.7, 139.6, 138.9, 137.9, 133.3, 133.2, 129.2, 128.4, 128.2,

126.8, 125.3, 116.9, 108.9, 106.3, 94.4, 66.2, 62.6, 59.9, 45.2, 42.6,

(2-Methoxy-5-methyl-4-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

amino)benzyl)-^D-serine (65). According to the general procedure C,

41 (257 mg, 0.74 mmol) was reacted with ^D-serine (156 mg, 1.48

mmol) in anhydrous DMF (5 mL), which yielded 65 (159 mg, 49%

yield) as a white solid. mp 189.9−190.8 °C. HPLC purity: 98.03%

(method B). ¹H NMR (400 MHz, DMSO-d₆): δ 8.18 (br, 2H), 7.47−

7.43 (m, 2H), 7.40−7.33 (m, 1H), 7.32−7.26 (m, 3H), 7.19 (t, J =

7.6 Hz, 1H), 7.08 (d, J = 7.5 Hz, 1H), 6.94 (s, 1H), 6.10 (s, 1H), 5.68

(t, J = 5.6 Hz, 1H), 5.36 (br, 1H), 4.41 (d, J = 5.2 Hz, 2H), 3.95 (dd, J

= 27.2, 13.2 Hz, 2H), 3.77 (dd, J = 11.2, 4.4 Hz, 1H), 3.65 (s, 3H),

3.63−3.58 (m, 1H), 3.13 (dd, J = 7.2, 4.4 Hz, 1H), 2.24 (s, 3H), 2.09

(s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.0, 157.1, 148.3,

142.0, 141.7, 138.1, 132.9, 132.6, 129.2, 128.2, 126.8, 126.4, 125.3,

113.2106.1, 93.5, 62.0, 59.8, 55.3, 45.6, 45.2, 16.9, 15.9. ESI-HRMS:

+

19.5, 15.8. ESI-HRMS: m/z calculated for C₃₂H₃₂N₄NaO₄[M +

Na]⁺, 559.2316; found, 559.2317.

(5-Chloro-2-((5-cyanopyridin-3-yl)methoxy)-4-(((2-methyl-[1,1′-

biphenyl]-3-yl)methyl)amino)benzyl)-^D-serine (61). According to

the general procedure C, 40 (436 mg, 0.93 mmol) was reacted with

D-serine (196 mg, 1.86 mmol) in anhydrous DMF (6 mL), which

yielded 61 (106 mg, 20% yield) as a white solid. mp 180.7−182.0 °C.

HPLC purity: 99.63% (method A). ¹H NMR (400 MHz, DMSO-d₆):

δ 8.91 (d, J = 1.6 Hz, 1H), 8.86 (d, J = 2.0 Hz, 1H), 8.37 (s, 1H), 8.17

(br, 2H), 7.46 (t, J = 7.2 Hz, 2H), 7.40−7.34 (m, 2H), 7.34−7.29 (m,

2H), 7.16−7.03 (m, 3H), 6.24 (s, 1H), 6.09 (t, J = 5.8 Hz, 1H), 5.34

(br, 1H), 5.16 (dd, J = 18.4, 13.2 Hz, 2H), 4.43 (d, J = 5.6 Hz, 2H),

4.01 (dd, J = 33.6, 13.2 Hz, 2H), 3.75 (dd, J = 11.2, 4.4 Hz, 1H), 3.63

(dd, J = 11.2, 6.8 Hz, 1H), 3.17 (dd, J = 6.6, 4.6 Hz, 1H), 2.19 (s,

3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.4, 156.2, 151.9, 151.7,

145.5, 142.0, 141.6, 138.5, 137.2, 133.0, 132.7, 131.6, 129.2, 128.3,

128.2, 126.9, 125.6, 125.3, 116.8, 109.3, 109.2, 108.9, 96.3, 66.6, 62.3,

60.1, 44.7, 44.5, 15.8. ESI-HRMS: m/z calculated for

+

m/z calculated for C₂₆H₃₀N₂NaO₄[M + Na]⁺, 457.2098; found,

457.2096.

(2-Ethoxy-5-methyl-4-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

amino)benzyl)-^D-serine (66). According to the general procedure C,

45 (296 mg, 0.82 mmol) was reacted with ^D-serine (173 mg, 1.65

mmol) in anhydrous DMF (5.5 mL), which yielded 66 (290 mg, 79%

yield) as a white solid. mp 196.1−197.3 °C. HPLC purity: 97.12%

(method A). ¹H NMR (400 MHz, DMSO-d₆): δ 8.21 (br, 2H),

7.47−7.43 (m, 2H), 7.40−7.34 (m, 1H), 7.30−7.26 (m, 3H), 7.18 (t,

J = 7.6 Hz, 1H), 7.08 (dd, J = 7.6, 1.2 Hz, 1H), 6.93 (s, 1H), 6.06 (s,

1H), 5.71 (t, J = 5.6 Hz, 1H), 5.41 (br, 1H), 4.40 (d, J = 5.6 Hz, 2H),

4.02−3.84 (m, 4H), 3.78 (dd, J = 11.6, 4.4 Hz, 1H), 3.63 (dd, J =

11.6, 7.6 Hz, 1H), 3.14 (dd, J = 7.2, 4.4 Hz, 1H), 2.23 (s, 3H), 2.09

(s, 3H), 1.24 (t, J = 7.0 Hz, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ

167.7, 156.2, 148.1, 141.9, 141.7, 138.1, 132.8, 132.5, 129.1, 128.2,

128.1, 126.8, 126.2, 125.2, 113.1, 106.4, 94.4, 63.2, 61.9, 59.8, 45.9,

+

C₃₁H₂₉ClN₄NaO₄[M + Na]⁺, 579.1770; found, 579.1766.

((2-Methoxy-6-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)amino)-

pyridin-3-yl)methyl)-^D-serine (62). According to the general

procedure C, 38 (347 mg, 1.04 mmol) was reacted with ^D-serine

(219 mg, 2.08 mmol) in anhydrous DMF (6 mL), which yielded 62

(154 mg, 36% yield) as a white solid. mp 171.9−173.4 °C. HPLC

purity: 98.22% (method A). ¹H NMR (400 MHz, DMSO-d₆): δ 8.17

(br, 2H), 7.46−7.42 (m, 2H), 7.40−7.35 (m, 2H), 7.35−7.30 (m,

1H), 7.31−7.25 (m, 2H), 7.24−7.16 (m, 2H), 7.08 (dd, J = 7.6, 0.8

Hz, 1H), 6.08 (d, J = 8.0 Hz, 1H), 5.46 (br, 1H), 4.50 (d, J = 5.6 Hz,

2H), 3.92 (dd, J = 20.0, 11.2 Hz, 2H), 3.81 (s, 3H), 3.76 (dd, J = 11.6,

4.4 Hz, 1H), 3.64 (dd, J = 11.4, 7.0 Hz, 1H), 3.16 (dd, J = 6.8, 4.4 Hz,

1H), 2.20 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.4, 161.0,

158.1, 141.9, 141.7, 141.5, 138.5, 133.0, 129.2, 128.3, 128.2, 127.1,

126.8, 125.3, 115.5, 99.5, 62.3, 60.0, 52.8, 44.9, 43.2, 16.0. ESI-

+

45.0, 16.8, 15.9, 14.3. ESI-HRMS: m/z calculated for C₂₇H₃₂N₂NaO₄

[M + Na]⁺, 471.2254; found, 471.2254.

(2-((3-Cyanobenzyl)oxy)-4-((3-(2,3-dihydrobenzo[b][1,4]dioxin-

6-yl)-2-methylbenzyl)amino)benzyl)-^D-serine (67). According to the

general procedure C, 34 (162 mg, 0.33 mmol) was reacted with ^D-

serine (69 mg, 0.66 mmol) in anhydrous DMF (3 mL), which yielded

67 (91 mg, 48% yield) as a yellowish solid. mp 172.3−173.5 °C.

HPLC purity: 97.12% (method B). ¹H NMR (400 MHz, DMSO-d₆):

δ 8.33 (br, 2H), 7.99 (s, 1H), 7.88 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 7.6

Hz, 1H), 7.57 (t, J = 8.0 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.15 (t, J =

7.6 Hz, 1H), 7.07 (d, J = 8.0 Hz, 2H), 6.92 (d, J = 8.0 Hz, 1H), 6.80−

6.67 (m, 2H), 6.36−6.34 (m, 2H), 6.19 (d, J = 8.0 Hz, 1H), 5.36 (br,

1H), 5.18−5.10 (m, 2H), 4.28 (br, 4H), 4.23 (d, J = 4.4 Hz, 2H),

4.03 (dd, J = 43.6, 13.2 Hz, 2H), 3.77 (dd, J = 10.8, 4.0 Hz, 1H), 3.65

(dd, J = 11.2, 8.0 Hz, 1H), 3.17−3.14 (m, 1H), 2.18 (s, 3H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 168.4, 157.8, 151.6, 143.4, 142.9,

141.9, 139.2, 138.2, 135.2, 133.8, 132.8, 132.1, 131.4, 130.1, 128.9,

127.1, 125.7, 122.6, 119.3, 118.2, 117.2, 116.0, 111.9, 107.8, 104.4,

97.2, 68.6, 64.6, 62.4, 60.3, 46.1, 45.9, 16.3. ESI-HRMS: m/z

+

HRMS: m/z calculated for C₂₄H₂₇N₃NaO₄[M + Na]⁺, 444.1894;

found, 444.1892.

((4-Methoxy-6-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)amino)-

pyridin-3-yl)methyl)-^D-serine (63). According to the general

procedure C, 39 (203 mg, 0.61 mmol) was reacted with ^D-serine

(160 mg, 1.52 mmol) in anhydrous DMF (4 mL). The reaction

mixture was extracted with 1-butanol. The combined organic phase

was concentrated to dryness in vacuo directly and puriﬁed to give 63

(104 mg, 40% yield) as a white solid. mp 177.6−179.2 °C. HPLC

purity: 98.86% (method A). ¹H NMR (400 MHz, DMSO-d₆): δ 7.81

(s, 1H), 7.45 (t, J = 7.4 Hz, 2H), 7.36 (t, J = 7.2 Hz, 1H), 7.30−7.26

(m, 3H), 7.19 (t, J = 7.4 Hz, 1H), 7.12−7.07 (m, 2H), 6.19 (s, 1H),

5.39 (br, 1H), 4.50 (d, J = 5.2 Hz, 2H), 3.93 (dd, J = 21.2, 13.6 Hz,

2H), 3.81−3.75 (m, 4H), 3.64 (dd, J = 11.4, 7.0 Hz, 1H), 3.18 (dd, J

= 6.8, 4.4 Hz, 1H), 2.17 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ

168.5, 164.4, 161.1, 150.0, 141.9, 141.7, 138.6, 132.9, 129.2, 128.2,

126.8, 126.7, 125.3, 115.5, 106.5, 89.4, 62.4, 60.0, 55.2, 43.6, 43.0,

+

calculated for C₃₄H₃₃N₃NaO₆[M + Na]⁺, 602.2262; found,

602.2266.

(2-((5-Cyanopyridin-3-yl)methoxy)-4-((3-(2,3-dihydrobenzo[b]-

[1,4]dioxin-6-yl)-2-methylbenzyl)amino)-5-methylbenzyl)-^D-serine

(68). According to the general procedure C, 46 (288 mg, 0.57 mmol)

was reacted with ^D-serine (120 mg, 1.14 mmol) in anhydrous DMF (6

mL) and anhydrous MeOH (2 mL), which yielded 68 (98 mg, 29%

yield) as a white solid. mp 198.1−199.6 °C. HPLC purity: 97.28%

(method A). ¹H NMR (400 MHz, DMSO-d₆): δ 8.89 (d, J = 2.0 Hz,

1H), 8.85 (d, J = 2.0 Hz, 1H), 8.36 (t, J = 2.0 Hz, 1H), 8.22 (br, 2H),

7.11 (dd, J = 7.2, 1.6 Hz, 1H), 7.08 (t, J = 7.2 Hz, 1H), 7.03 (dd, J =

7.2, 1.6 Hz, 1H), 7.00 (s, 1H), 6.91 (d, J = 8.0 Hz, 1H), 6.81 (d, J =

2.0 Hz, 1H), 6.77 (dd, J = 8.4, 2.0 Hz, 1H), 6.07 (s, 1H), 5.69 (t, J =

5.6 Hz, 1H), 5.31 (br, 1H), 5.13 (dd, J = 18.8, 13.2 Hz, 2H), 4.32 (d,

J = 5.2 Hz, 2H), 4.28 (s, 4H), 4.03 (dd, J = 44.0, 13.2 Hz, 2H), 3.77

(dd, J = 11.2, 4.4 Hz, 1H), 3.64 (dd, J = 11.6, 7.2 Hz, 1H), 3.16 (dd, J

= 7.2, 4.4 Hz, 1H), 2.20 (s, 3H), 2.10 (s, 3H). ESI-HRMS: m/z

+

15.8. ESI-HRMS: m/z calculated for C₂₄H₂₈N₃O₄[M + H]⁺,

422.2074; found, 422.2074.

(5-Chloro-2-methoxy-4-(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

amino)benzyl)-^D-serine (64). According to the general procedure C,

33 (150 mg, 0.41 mmol) was reacted with ^D-serine (86 mg, 0.82

mmol) in anhydrous DMF (3 mL), which yielded 64 (110 mg, 59%

yield) as a yellowish solid. mp 197.5−199.1 °C. HPLC purity: 99.72%

(method A). ¹H NMR (400 MHz, DMSO-d₆): δ 7.47−7.43 (m, 2H),

7.40−7.34 (m, 1H), 7.30 (s, 2H), 7.29−7.26 (m, 2H), 7.20 (t, J = 7.6

Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 6.23 (s, 1H), 6.05 (t, J = 5.6 Hz,

1H), 4.48 (d, J = 5.6 Hz, 2H), 3.93 (dd, J = 21.6, 13.2 Hz, 2H), 3.75

(dd, J = 11.6, 4.8 Hz, 1H), 3.67 (s, 3H), 3.65−3.60 (m, 1H), 3.15

(dd, J = 6.8, 4.4 Hz, 1H), 2.23 (s, 3H). ESI-HRMS: m/z calculated for

+

calculated for C₃₄H₃₄N₄NaO₆[M + Na]⁺, 617.2371; found,

617.2377.

(5-Chloro-2-((5-cyanopyridin-3-yl)methoxy)-4-((3-(2,3-

dihydrobenzo[b][1,4]dioxin-6-yl)-2-methylbenzyl)amino)benzyl)-^D-

+

C₂₅H₂₇ClN₂NaO₄[M + Na]⁺, 477.1552; found, 477.1553.

S

https://dx.doi.org/10.1021/acs.jmedchem.0c01329

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

serine (69). According to the general procedure C, 47 (340 mg, 0.65

mmol) was reacted with ^D-serine (135 mg, 1.28 mmol) in anhydrous

DMF (6 mL) and anhydrous MeOH (2 mL), which yielded 69 (135

mg, 34% yield) as a white solid. mp 203.2−204.8 °C. HPLC purity:

99.00% (method A). ¹H NMR (400 MHz, DMSO-d₆): δ 8.89 (d, J =

2.0 Hz, 1H), 8.85 (d, J = 2.0 Hz, 1H), 8.36 (t, J = 2.0 Hz, 1H), 8.17

(br, 2H), 7.37 (s, 1H), 7.12−7.01 (m, 3H), 6.92 (d, J = 8.0 Hz, 1H),

6.80 (d, J = 2.0 Hz, 1H), 6.76 (dd, J = 8.2, 2.2 Hz, 1H), 6.22 (s, 1H),

6.07 (t, J = 5.8 Hz, 1H), 5.38 (br, 1H), 5.15 (dd, J = 18.0, 13.2 Hz,

2H), 4.40 (d, J = 5.6 Hz, 2H), 4.28 (s, 4H), 4.01 (dd, J = 33.6, 13.2

Hz, 2H), 3.75 (dd, J = 11.2, 4.4 Hz, 1H), 3.64 (dd, J = 11.6, 6.8 Hz,

1H), 3.18 (dd, J = 6.8, 4.4 Hz, 1H), 2.20 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆): δ 168.4, 156.1, 151.9, 151.6, 145.4, 142.9, 142.4,

141.5, 138.5, 137.1, 134.7, 133.0, 132.8, 131.6, 128.3, 125.4, 125.3,

122.2, 117.8, 116.8, 116.7, 109.4, 109.3, 108.9, 96.4, 66.6, 64.1, 62.3,

60.1, 44.8, 44.5, 15.8. ESI-HRMS: m/z calculated for

59.2, 45.0, 44.9, 32.1, 16.9, 15.8. ESI-HRMS: m/z calculated for

+

C₃₃H₃₄N₄NaO₄[M + Na]⁺, 573.2472; found, 573.2472.

(R)-3-((2-((5-Cyanopyridin-3-yl)methoxy)-5-methyl-4-(((2-meth-

yl-[1,1′-biphenyl]-3-yl)methyl)amino)benzyl)amino)-4-hydroxybu-

tanoic Acid (73). According to the general procedure C, 35 (313 mg,

0.7 mmol) was reacted with (R)-3-amino-4-hydroxybutanoic acid

(167 mg, 1.40 mmol) in anhydrous DMF (6 mL) and anhydrous

MeOH (2 mL), which yielded 73 (30 mg, 8% yield) as a white solid.

1

mp 157.9−159.5 °C. HPLC purity: 98.08% (method B). H NMR

(400 MHz, DMSO-d₆): δ 10.00 (br, 2H), 8.91−8.80 (m, 2H), 8.32

(s, 1H), 7.45 (t, J = 7.4 Hz, 2H), 7.39−7.33 (m, 3H), 7.16−7.04 (m,

3H), 6.97 (s, 1H), 6.08 (s, 1H), 5.66 (t, J = 5.4 Hz, 1H), 5.17−5.07

(m, 2H), 4.34 (d, J = 4.8 Hz, 2H), 3.91 (dd, J = 68.0, 12.8 Hz, 2H),

3.58 (dd, J = 11.6, 4.4 Hz, 1H), 3.47−3.39 (m, 1H), 3.07−3.01 (m,

1H), 2.19 (s, 3H), 2.16−2.13 (m, 2H), 2.11 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆): δ 173.3, 155.5, 152.0, 151.5, 147.8, 141.9, 141.7,

138.4, 137.8, 133.6, 132.7, 132.5, 129.3, 128.2, 126.8, 125.9, 125.2,

116.9, 113.8, 108.8, 108.8, 95.0, 66.2, 60.3, 55.3, 45.1, 42.9, 33.6, 16.9,

+

C₃₃H₃₁ClN₄NaO₆[M + Na]⁺, 637.1824; found, 637.1825.

(S)-4-((2-((5-Cyanopyridin-3-yl)methoxy)-5-methyl-4-(((2-meth-

yl-[1,1′-biphenyl]-3-yl)methyl)amino)benzyl)amino)-3-hydroxybu-

tanoic Acid (70). According to the general procedure C, 35 (313 mg,

0.7 mmol) was reacted with (S)-(+)-4-amino-3-hydroxybutyric acid

(167 mg, 1.40 mmol) in anhydrous DMF (6.5 mL) and anhydrous

MeOH (2 mL), which yielded 70 (97 mg, 25% yield) as a white solid.

+

15.8. ESI-HRMS: m/z calculated for C₃₃H₃₄N₄NaO₄[M + Na]⁺,

573.2472; found, 573.2472.

(2S,4R)-1-(2-((5-Cyanopyridin-3-yl)methoxy)-5-methyl-4-(((2-

methyl-[1,1′-biphenyl]-3-yl)methyl)amino)benzyl)-4-hydroxypyrro-

lidine-2-carboxylic Acid (74). According to the general procedure C,

35 (339 mg, 0.76 mmol) was reacted with ^L-hydroxyproline (199 mg,

1.52 mmol) in anhydrous DMF (6.5 mL), which yielded 74 (150 mg,

35% yield) as a white solid. mp 171.2−172.9 °C. HPLC purity:

95.54% (method A). ¹H NMR (400 MHz, DMSO-d₆): δ 8.91 (d, J =

2.0 Hz, 1H), 8.85 (d, J = 2.0 Hz, 1H), 8.36 (t, J = 2.2 Hz, 1H), 7.47−

7.43 (m, 2H), 7.40−7.35 (m, 1H), 7.34−7.32 (m, 1H), 7.32−7.31

(m, 1H), 7.20 (dd, J = 7.6, 1.6 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.08

(dd, J = 7.6, 1.6 Hz, 1H), 7.01 (s, 1H), 6.11 (s, 1H), 5.71 (t, J = 5.8

Hz, 1H), 5.36 (br, 1H), 5.15 (dd, J = 23.2, 13.6 Hz, 2H), 4.35 (d, J =

5.6 Hz, 2H), 4.28−4.24 (m, 1H), 4.13 (dd, J = 63.0, 13.0 Hz, 2H),

3.71 (t, J = 8.2 Hz, 1H), 3.33 (dd, J = 11.6, 4.8 Hz, 1H), 2.86 (dd, J =

11.6, 3.2 Hz, 1H), 2.20 (s, 3H), 2.11 (s, 3H), 2.08−1.98 (m, 2H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 169.4, 155.7, 151.9, 151.6, 148.3,

141.9, 141.7, 138.5, 137.7, 133.5, 133.4, 132.8, 129.3, 128.2, 128.2,

126.8, 126.1, 125.2, 116.9, 114.2, 108.9, 106.8, 94.9, 68.9, 66.4, 66.4,

59.1, 53.8, 45.2, 37.9, 17.0, 15.9. ESI-HRMS: m/z calculated for

1

mp 134.0−135.4 °C. HPLC purity: 97.39% (method A). H NMR

(400 MHz, DMSO-d₆): δ 8.90 (d, J = 2.0 Hz, 1H), 8.83 (d, J = 2.0

Hz, 1H), 8.29 (t, J = 2.2 Hz, 1H), 7.48−7.42 (m, 2H), 7.40−7.34 (m,

1H), 7.34−7.31 (m, 2H), 7.17 (dd, J = 7.6, 1.6 Hz, 1H), 7.13 (t, J =

7.4 Hz, 1H), 7.07 (dd, J = 7.4, 1.8 Hz, 1H), 6.96 (s, 1H), 6.08 (s,

1H), 5.57 (t, J = 5.6 Hz, 1H), 5.11 (s, 2H), 4.33 (d, J = 5.2 Hz, 2H),

3.88 (qui, J = 5.6 Hz, 1H), 3.76 (dd, J = 17.0, 13.0 Hz, 2H), 2.68 (d, J

= 5.6 Hz, 2H), 2.29 (ddd, J = 32.2, 15.2, 5.6 Hz, 2H), 2.20 (s, 3H),

2.10 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 173.7, 155.3, 151.9,

151.5, 147.4, 141.9, 141.7, 138.3, 137.9, 133.8, 132.7, 132.4, 129.3,

128.2, 126.8, 126.0, 125.2, 121.8, 116.9, 113.9, 110.1, 108.8, 95.2,

66.3, 64.7, 52.9, 45.9, 45.2, 43.5, 17.0, 15.8. ESI-HRMS: m/z

calculated for C₂₉H₂₆N₃O⁺[M − C₄H₈NO₃]⁺, 432.2070; found,

432.2069.

(S)-5-((2-((4-Hydroxy-2-oxopyrrolidin-1-yl)methyl)-4-methyl-5-

(((2-methyl-[1,1′-biphenyl]-3-yl)methyl)amino)phenoxy)methyl)-

nicotinonitrile (71). 71 (38 mg, 10% yield) was obtained from the

synthesis of 70 as a white solid. mp 121.6−122.3 °C. HPLC purity:

99.72% (method A). ¹H NMR (400 MHz, CDCl₃): δ 8.80 (d, J = 1.6

Hz, 2H), 8.08 (t, J = 2.0 Hz, 1H), 7.46−7.41 (m, 2H), 7.39−7.34 (m,

1H), 7.34−7.32 (m, 1H), 7.32−7.31 (m, 1H), 7.30−7.27 (m, 1H),

7.24 (d, J = 1.6 Hz, 1H), 7.23 (s, 1H), 6.96 (s, 1H), 6.15 (s, 1H), 5.09

(s, 2H), 4.55 (d, J = 14.4 Hz, 1H), 4.45 (br, 1H), 4.34 (d, J = 14.4 Hz,

1H), 4.32 (s, 2H), 3.86 (br, 1H), 3.50 (dd, J = 10.8, 5.6 Hz, 1H), 3.20

(dd, J = 10.8, 2.0 Hz, 1H), 2.69 (dd, J = 17.2, 6.8 Hz, 1H), 2.44 (br,

1H), 2.39 (dd, J = 17.4, 2.2 Hz, 1H), 2.25 (s, 3H), 2.08 (s, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 172.4, 155.5, 151.7, 151.7, 147.0, 143.2,

142.0, 138.2, 136.9, 133.8, 133.7, 132.5, 129.6, 129.4, 128.2, 127.5,

127.0, 125.8, 116.5, 115.2, 112.4, 110.1, 95.4, 67.0, 64.5, 55.8, 47.2,

+

C₃₄H₃₄N₄NaO₄[M + Na]⁺, 585.2472; found, 585.2471.

(2-((5-Cyanopyridin-3-yl)methoxy)-5-methyl-4-(((2-methyl-[1,1′-

biphenyl]-3-yl)methyl)amino)benzyl)-^D-asparagine (75). According

to the general procedure C, 35 (313 mg, 0.7 mmol) was reacted with

D-asparagine anhydrous (185 mg, 1.40 mmol) in anhydrous DMF (8

mL), which yielded 75 (180 mg, 46% yield) as a white solid. mp

1

122.3−124.1 °C. HPLC purity: 97.34% (method B). H NMR (400

MHz, DMSO-d₆): δ 8.89 (d, J = 2.0 Hz, 1H), 8.85 (d, J = 2.0 Hz,

1H), 8.34 (t, J = 2.0 Hz, 1H), 7.78 (s, 1H), 7.49−7.43 (m, 2H),

7.39−7.35 (m, 1H), 7.35−7.34 (m, 1H), 7.33−7.32 (m, 1H), 7.16−

7.09 (m, 3H), 7.07 (dd, J = 7.0, 2.2 Hz, 1H), 6.96 (s, 1H), 6.07 (s,

1H), 5.73 (t, J = 5.8 Hz, 1H), 5.16 (s, 2H), 4.34 (d, J = 5.2 Hz, 2H),

4.04 (dd, J = 21.4, 13.0 Hz, 2H), 3.36 (dd, J = 9.2, 4.0 Hz, 1H), 2.73

(dd, J = 16.6, 3.8 Hz, 1H), 2.43 (dd, J = 16.6, 9.0 Hz, 1H), 2.19 (s,

3H), 2.11 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 173.4, 168.2,

155.5, 151.9, 151.5, 148.3, 141.9, 141.7, 138.3, 137.7, 133.4, 132.8,

132.7, 129.3, 128.2, 126.8, 125.8, 125.2, 116.9, 113.9, 108.8, 106.9,

94.9, 66.2, 57.4, 45.5, 45.1, 34.0, 16.9, 15.8. ESI-HRMS: m/z

+

41.5, 40.9, 16.8, 16.3. ESI-HRMS: m/z calculated for C₃₃H₃₃N₄O₃

[M + H]⁺, 533.2547; found, 533.2545.

(2-((5-Cyanopyridin-3-yl)methoxy)-5-methyl-4-(((2-methyl-[1,1′-

biphenyl]-3-yl)methyl)amino)benzyl)-^D-homoserine (72). According

to the general procedure C, 35 (313 mg, 0.7 mmol) was reacted with

D-homocitrulline (167 mg, 1.40 mmol) in anhydrous DMF (6 mL)

and anhydrous MeOH (2 mL), which yielded 72 (230 mg, 60% yield)

as a white solid. mp 193.7−194.8 °C. HPLC purity: 99.84% (method

A). ¹H NMR (400 MHz, DMSO-d₆): δ 8.90 (d, J = 2.0 Hz, 1H), 8.85

(d, J = 2.0 Hz, 1H), 8.35 (t, J = 2.0 Hz, 1H), 8.15 (br, 2H), 7.48−7.42

(m, 2H), 7.40−7.36 (m, 1H), 7.35−7.31 (m, 2H), 7.17−7.10 (m,

2H), 7.07 (dd, J = 6.8, 2.0 Hz, 1H), 7.01 (s, 1H), 6.08 (s, 1H), 5.73

(t, J = 5.6 Hz, 1H), 5.54 (br, 1H), 5.13 (dd, J = 18.0, 13.2 Hz, 2H),

4.35 (d, J = 5.6 Hz, 2H), 4.03−3.89 (m, 2H), 3.56−3.44 (m, 2H),

3.21 (t, J = 6.4 Hz, 1H), 2.19 (s, 3H), 2.11 (s, 3H), 1.93−1.67 (m,

2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 169.6, 155.5, 151.9, 151.5,

148.2, 141.8, 141.7, 138.4, 137.7, 133.4, 132.8, 132.7, 129.2, 128.1,

126.8, 125.9, 125.1, 116.8, 113.9, 109.4, 108.8, 107.0, 94.8, 66.2, 59.9,

+

calculated for C₃₃H₃₃N₅NaO₄[M + Na]⁺, 586.2425; found,

586.2422.

3-Methyl-2-(triﬂuoromethyl)-1,1′-biphenyl (77a). According to

the general procedure A, 76a (10.25 g, 42.88 mmol) and

phenylboronic acid (1.4 equiv) were used. The crude product was

puriﬁed on a silica gel column (PE) to give compound 77a (5.3 g,

1

52% yield) as a colorless oil. H NMR (400 MHz, CDCl₃): δ 7.41−

7.31 (m, 4H), 7.28−7.25 (m, 3H), 7.12−7.08 (m, 1H), 2.57 (q, J =

2.8 Hz, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 142.2 (q, J = 2.9 Hz),

141.9, 137.9 (q, J = 2.2 Hz), 133.5, 132.1, 131.7, 130.4, 130.1, 128.5

(q, J = 2.1 Hz), 127.7, 127.2, 126.8, 125.1 (q, J = 274.8 Hz), 21.3 (q, J

= 4.0 Hz).

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2-Bromo-3-methyl-1,1′-biphenyl (77b). According to the general

procedure A, 76b (5 g, 16.8 mmol) and phenylboronic acid (1.15

equiv) were used. The crude product was puriﬁed on a silica gel

column (PE) to give compound 77b (3.93 g, 95% yield) as a colorless

DMF (17 mL/3.5 mL) to obtain 5-((2-formyl-4-methyl-5-

nitrophenoxy)methyl)nicotinonitrile (81-2) (939 mg, 99% yield) as

1

a yellowish solid. H NMR (400 MHz, DMSO-d₆): δ 10.44 (s, 1H),

9.04 (t, J = 2.0 Hz, 1H), 8.56 (t, J = 2.0 Hz, 1H), 7.91 (s, 1H), 7.82 (s,

1H), 5.47 (s, 2H), 2.45 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ

188.8, 157.9, 152.8, 152.3, 152.0, 138.9, 132.2, 132.1, 127.0, 124.5,

116.8, 109.8, 109.1, 67.4, 17.9. ESI-MS: m/z = 296.07 [M − H]⁻.

According to a similar manner as described for the synthesis of

compound 17, 81-2 (1.46 g, 4.91 mmol) was used. The ﬁltrate was

concentrated to dryness in vacuo, diluted with water, and extracted

with DCM/MeOH (20:1). The combined organic phase was

concentrated to give 81 (1.30 g, 99% yield) as an orange solid,

which was used in the next step without further puriﬁcation. ¹H NMR

(400 MHz, DMSO-d₆): δ 10.03 (s, 1H), 9.02 (d, J = 2.0 Hz, 1H),

8.99 (d, J = 2.0 Hz, 1H), 8.48 (t, J = 2.0 Hz, 1H), 7.33 (s, 1H), 6.32

(s, 1H), 6.19 (s, 2H), 5.22 (s, 2H), 2.01 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆): δ 185.5, 160.6, 154.8, 152.3, 151.8, 138.8, 133.1,

130.1, 116.9, 114.1, 113.8, 109.0, 96.0, 66.1, 16.4. ESI-MS: m/z =

268.11 [M + H]⁺.

5-((2-Formyl-5-(((2-(triﬂuoromethyl)-[1,1′-biphenyl]-3-yl)-

methyl)amino)phenoxy)methyl)nicotinonitrile (82). According to a

similar manner as described for the synthesis of 30, 78a (630 mg, 2

mmol), 17 (532 mg, 2.10 mmol), and K₂CO₃(332 mg, 2.40 mmol)

were suspended in MeCN/DMF (10.5 mL/3.5 mL) and stirred for 11

h at 60 °C. The crude product was puriﬁed on a silica gel column

(DCM/EA = 7:1) to give 82 (120 mg, 12% yield) as a yellowish solid.

¹H NMR (400 MHz, CDCl₃): δ 10.19 (s, 1H), 8.85 (d, J = 2.0 Hz,

2H), 8.05 (t, J = 2.2 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 7.2

Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.44−7.36 (m, 3H), 7.30−7.23 (m,

3H), 6.35 (dd, J = 8.4, 1.6 Hz, 1H), 6.06 (d, J = 2.0 Hz, 1H), 5.16 (s,

2H), 5.04 (t, J = 5.8 Hz, 1H), 4.69 (d, J = 4.8 Hz, 2H). ¹³C NMR

(100 MHz, CDCl₃): δ 186.8, 162.1, 154.4, 152.0, 151.6, 142.9 (q, J =

3.1 Hz), 141.0, 137.8, 137.4, 132.7, 132.0, 131.7, 131.2, 128.3 (q, J =

22.5 Hz), 127.9, 127.9, 127.5, 125.9 (q, J = 29.1 Hz), 124.9 (q, J =

275.2 Hz), 116.3, 116.3, 110.2, 106.6, 94.9, 66.6, 45.34 (q, J = 5.3

Hz). ESI-MS: m/z = 488.14 [M + H]⁺.

5-((5-(((2-Bromo-[1,1′-biphenyl]-3-yl)methyl)amino)-2-

formylphenoxy)methyl)nicotinonitrile (83). According to a similar

manner as described for the synthesis of 30, 78b (860 mg, 2.64

mmol), 17 (668 mg, 2.64 mmol), and K₂CO₃(437 mg, 3.16 mmol)

were suspended in MeCN/DMF (15 mL/5 mL) and stirred for 11 h

at reﬂux. The crude product was puriﬁed on a silica gel column

(DCM/EA = 7:1) to give 83 (200 mg, 15% yield) as a yellowish solid.

¹H NMR (400 MHz, DMSO-d₆): δ 10.06 (s, 1H), 9.00 (d, J = 1.6 Hz,

1

oil. H NMR (400 MHz, CDCl₃): δ 7.61−7.58 (m, 1H), 7.48−7.31

(m, 6H), 7.26−7.23 (m, 1H), 7.16−7.10 (m, 1H), 2.49 (s, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 143.4, 142.2, 138.9, 129.8, 129.5, 128.7,

127.9, 127.5, 126.8, 125.4, 24.4.

3-(Bromomethyl)-2-(triﬂuoromethyl)-1,1′-biphenyl (78a). BPO

(75% w/w, 32 mg, 0.10 mmol) was added to a solution of 77a

(473 mg, 2.0 mmol) and NBS (356 mg, 2 mmol) in CCl₄(8 mL) and

stirred for 10 h at reﬂux. Then, the mixture was concentrated to

dryness in vacuo, diluted with water, and extracted with DCM. The

combined organic phase was washed with water, dried over anhydrous

Na₂SO₄, and ﬁltered. The ﬁltrate was puriﬁed on a silica gel column

(PE) to give 78a (390 mg, 62% yield) as a white solid. ¹H NMR (400

MHz, CDCl₃): δ 7.61−7.56 (m, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.40−

7.36 (m, 3H), 7.27−7.23 (m, 3H), 4.73 (q, J = 1.6 Hz, 2H). ¹³C

NMR (100 MHz, CDCl₃): δ 142.7 (q, J = 2.9 Hz), 141.1, 137.9 (q, J

= 2.1 Hz), 132.7, 132.2, 131.2, 128.5 (q, J = 2.0 Hz), 127.9, 127.5,

126.4 (q, J = 28.9 Hz), 124.5 (q, J = 275.5 Hz), 30.4 (q, J = 4.5 Hz).

2-Bromo-3-(bromomethyl)-1,1′-biphenyl (78b). According to a

similar manner as described for the synthesis of 78a, 77b (3.93 g,

15.90 mmol) was used to obtain 78b (3.36 g, 98% yield) as a white

solid. ¹H NMR (400 MHz, CDCl₃): δ 7.47−7.42 (m, 2H), 7.41−7.39

(m, 2H), 7.38−7.35 (m, 2H), 7.33 (t, J = 7.6 Hz, 1H), 7.25 (dd, J =

7.6, 2.0 Hz, 1H), 4.71 (s, 2H). ¹³C NMR (100 MHz, CDCl₃): δ

144.4, 141.4, 137.9, 131.5, 130.3, 129.4, 128.1, 127.8, 127.5, 125.0,

34.8.

2-((2-(Triﬂuoromethyl)-[1,1′-biphenyl]-3-yl)methyl)isoindoline-

1,3-dione (79). BPO (75% w/w, 304 mg, 1.26 mmol) was added to a

solution of 77a (4.48 g, 18.96 mmol) and NBS (3.37 g, 18.96 mmol)

in CCl₄(70 mL) and stirred for 11 h at reﬂux. Then, the mixture was

concentrated to dryness in vacuo, diluted with water, and extracted

with DCM. The combined organic phase was washed with water,

dried over anhydrous Na₂SO₄, and ﬁltered. The ﬁltrate was

concentrated to dryness in vacuo and dissolved in DMF (50 mL).

A phthalimide potassium salt (4.05 g, 21.87 mmol) was added to the

above solution and stirred for 1.5 h at 80 °C. The reaction mixture

was concentrated to dryness in vacuo, diluted with water, and

extracted with DCM. The combined organic phase was washed with

water, concentrated in vacuo, and puriﬁed on a silica gel column (PE/

DCM = 1:1) to give 79 (4.55 g, 60% yield) as a white solid. ¹H NMR

(400 MHz, CDCl₃): δ 7.98−7.91 (m, 2H), 7.82−7.77 (m, 2H),

7.43−7.36 (m, 4H), 7.31−7.27 (m, 2H), 7.19 (d, J = 7.6 Hz, 1H),

7.14 (d, J = 7.6 Hz, 1H), 5.19 (s, 2H). ¹³C NMR (100 MHz, CDCl₃):

δ 142.7 (q, J = 2.9 Hz), 141.2, 135.6, 134.4, 132.1, 131.6, 130.9, 128.5

(q, J = 2.2 Hz), 127.8, 127.4, 126.1 (q, J = 27.5 Hz), 125.9, 124.5 (q, J

= 219.0 Hz), 123.7, 39.2 (q, J = 6.5 Hz). ESI-MS: m/z = 382.09 [M +

H]⁺.

1H), 8.97 (d, J = 1.6 Hz, 1H), 8.47 (s, 1H), 7.55−7.50 (m, 2H),

7.50−7.44 (m, 2H), 7.43−7.37 (m, 4H), 7.33 (dd, J = 7.4, 1.4 Hz,

1H), 7.28 (dd, J = 7.2, 1.6 Hz, 1H), 6.34 (d, J = 10.4 Hz, 2H), 5.29 (s,

2H), 4.50 (d, J = 5.6 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ

186.0, 162.3, 155.6, 152.4, 152.0, 143.2, 141.2, 139.0, 138.4, 135.5,

133.2, 130.4, 129.4, 128.3, 128.1, 127.8, 127.7, 123.2, 117.1, 115.7,

114.7, 109.2, 95.0, 66.4, 47.5. ESI-MS: m/z = 498.08 [M + H]⁺.

5-((5-(((2-Bromo-[1,1′-biphenyl]-3-yl)methyl)amino)-2-formyl-4-

methylphenoxy)methyl)nicotinonitrile (84). According to a similar

manner as described for the synthesis of 30, 78b (815 mg, 2.50

mmol), 81 (702 mg, 2.63 mmol), and K₂CO₃(415 mg, 3.0 mmol)

were suspended in MeCN/DMF (12 mL/4 mL) and stirred for 7.5 h

at reﬂux. The crude product was puriﬁed on a silica gel column

(DCM/EA = 10:1−7:1) to give 84 (180 mg, 14% yield) as a

(2-(Triﬂuoromethyl)-[1,1′-biphenyl]-3-yl)methanamine (80). Ac-

cording to a similar manner as described for the synthesis of 14a, 79

(450 mg, 1.18 mmol) was used to obtain 80 (290 mg, 98% yield) as a

1

colorless oil. H NMR (400 MHz, CDCl₃): δ 7.57 (d, J = 7.6 Hz,

1H), 7.51 (t, J = 7.6 Hz, 1H), 7.42−7.33 (m, 3H), 7.29−7.25 (m,

2H), 7.19 (d, J = 7.6 Hz, 1H), 4.08 (q, J = 1.6 Hz, 2H). ¹³C NMR

(100 MHz, CDCl₃): δ 143.1, 142.3 (q, J = 3.0 Hz), 141.6, 131.1,

131.1, 129.4, 128.4 (q, J = 2.0 Hz), 127.8, 127.3, 125.8 (q, J = 28.2

Hz), 125.1 (q, J = 275.5 Hz), 44.7 (q, J = 4.1 Hz). ESI-MS: m/z =

252.09 [M + H]⁺.

5-((5-Amino-2-formyl-4-methylphenoxy)methyl)nicotinonitrile

(81). According to a similar manner as described for the synthesis of

compound 16, 3-methyl-4-nitrobenzaldehyde (1.90 g, 11.52 mmol)

was used to obtain 2-hydroxy-5-methyl-4-nitrobenzaldehyde (81-1)

(942 mg, 45% yield) as a yellowish solid. ¹H NMR (400 MHz,

DMSO-d₆): δ 11.31 (s, 1H), 10.33 (s, 1H), 7.69 (s, 1H), 7.52 (s, 1H),

2.40 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 189.3, 158.7, 152.6,

132.0, 125.4, 122.3, 112.8, 18.2. ESI-MS: m/z = 180.03 [M − H]⁻.

According to a similar manner as described for the synthesis of

compound 17-1, 81-1 (578 mg, 3.19 mmol) was used in MeCN/

1

yellowish solid. H NMR (400 MHz, CDCl₃): δ 10.20 (s, 1H), 8.82

(d, J = 2.0 Hz, 1H), 8.80 (d, J = 2.0 Hz, 1H), 7.99 (t, J = 2.2 Hz, 1H),

7.62 (d, J = 0.4 Hz, 1H), 7.49−7.42 (m, 3H), 7.42−7.37 (m, 2H),

7.36−7.29 (m, 2H), 7.28−7.24 (m, 1H), 5.98 (s, 1H), 5.13 (s, 2H),

4.90 (t, J = 5.8 Hz, 1H), 4.61 (d, J = 6.0 Hz, 2H), 2.19 (s, 3H). ¹³C

NMR (100 MHz, CDCl₃): δ 186.9, 161.1, 152.4, 151.9, 151.5, 144.1,

141.1, 137.7, 137.2, 132.9, 130.8, 129.4, 128.2, 127.9, 127.7, 127.5,

123.6, 116.3, 115.9, 115.5, 115.4, 110.1, 93.5, 66.9, 48.7, 16.5. ESI-

MS: m/z = 512.08 [M + H]⁺.

3-((5-(((2-Bromo-[1,1′-biphenyl]-3-yl)methyl)amino)-2-

formylphenoxy)methyl)benzonitrile (85). According to a similar

manner as described for the synthesis of 30, 78b (400 mg, 1.23

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mmol), 15 (325 mg, 1.29 mmol), and K₂CO₃(203 mg, 1.47 mmol)

were suspended in MeCN/DMF (4.5 mL/2.5 mL) and stirred for 10

h at reﬂux. The crude product was puriﬁed on a silica gel column

(DCM/EA = 70:1) to give 85 (155 mg, 25% yield) as a yellowish

solid. ¹H NMR (400 MHz, CDCl₃): δ 10.24 (s, 1H), 7.74 (d, J = 8.4

Hz, 1H), 7.69−7.60 (m, 3H), 7.50 (t, J = 7.6, 1H), 7.48−7.40 (m,

3H), 7.41−7.34 (m, 2H), 7.32−7.26 (m, 3H), 6.35−6.30 (m, 1H),

6.06 (d, J = 2.0 Hz, 1H), 5.12 (s, 2H), 4.99 (br, 1H), 4.55 (s, 2H).

¹³C NMR (100 MHz, CDCl₃): δ 187.1, 162.6, 154.5, 144.0, 141.2,

(br, 2H), 7.48−7.39 (m, 5H), 7.30 (t, J = 7.6 Hz, 1H), 7.20 (t, J = 7.6

Hz, 2H), 7.03 (s, 1H), 6.03−5.96 (m, 2H), 5.29 (br, 1H), 5.12 (dd, J

= 18.6, 13.4 Hz, 2H), 4.44 (d, J = 5.6 Hz, 2H), 4.03 (dd, J = 40.0, 13.0

Hz, 2H), 3.77 (dd, J = 11.4, 4.2 Hz, 1H), 3.64 (dd, J = 11.0, 7.4 Hz,

1H), 3.17 (dd, J = 6.8, 4.4 Hz, 1H), 2.13 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆): δ 167.8, 155.6, 151.8, 151.6, 147.7, 142.7, 141.1,

139.0, 138.4, 133.3, 133.1, 129.8, 129.3, 128.1, 127.6, 127.3, 122.6,

116.8, 114.0, 108.8, 107.2, 94.7, 66.3, 62.0, 59.9, 47.4, 45.4, 16.9. ESI-

HRMS: m/z calculated for C₃₁H₂₉BrN₄NaO₄⁺[M + Na]⁺, 623.1264;

found, 623.1265.

138.0, 137.4, 131.8, 131.3, 131.2, 130.6, 130.4, 129.7, 129.4, 128.1,

127.8, 127.8, 127.4, 123.6, 118.6, 116.3, 112.8, 106.4, 95.2, 68.9, 48.6.

ESI-MS: m/z = 497.07 [M + H]⁺.

(2-((5-Cyanopyridin-3-yl)methoxy)-5-methyl-4-(((2-(triﬂuoro-

methyl)-[1,1′-biphenyl]-3-yl)methyl)amino)benzyl)-^D-serine (90).

According to the general procedure C, 86 (358 mg, 0.71 mmol)

was reacted with ^D-serine (150 mg, 1.43 mmol) in anhydrous DMF (6

mL) and anhydrous MeOH (2 mL), which yielded 90 (181 mg, 43%

yield) as a white solid. mp 131.5−132.6 °C. HPLC purity: 97.97%

(method A). ¹H NMR (400 MHz, DMSO-d₆): δ 8.89 (d, J = 2.0 Hz,

1H), 8.82 (d, J = 2.0 Hz, 1H), 8.34 (t, J = 2.0 Hz, 1H), 7.51 (t, J = 7.6

Hz, 1H), 7.46−7.38 (m, 4H), 7.31 (d, J = 2.0 Hz, 1H), 7.29 (d, J =

1.6 Hz, 1H), 7.19 (d, J = 7.6 Hz, 1H), 7.05 (s, 1H), 6.09 (t, J = 5.6

Hz, 1H), 5.93 (s, 1H), 5.32 (br, 1H), 5.09 (dd, J = 17.8, 13.2 Hz,

2H), 4.57 (d, J = 4.0 Hz, 2H), 4.03 (dd, J = 40.4, 13.2 Hz, 2H), 3.78

(dd, J = 11.4, 4.4 Hz, 1H), 3.64 (dd, J = 11.6, 7.2 Hz, 1H), 3.19−3.16

(m, 1H), 2.14 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.0,

155.6, 151.8, 151.6, 147.7, 141.6, 141.1, 139.6, 138.4, 133.3, 133.2,

131.4, 130.9, 128.2, 127.9, 127.4, 127.1, 125.1 (q, J = 276 Hz), 124.3

(q, J = 28.4 Hz), 116.8, 114.1, 108.8, 107.1, 94.3, 66.2, 62.0, 59.9,

5-((2-Formyl-4-methyl-5-(((2-(triﬂuoromethyl)-[1,1′-biphenyl]-3-

yl)methyl)amino)phenoxy)methyl)nicotinonitrile (86). According to

the general procedure B, 80 (290 mg, 1.15 mmol) and 19 (459 mg,

1.39 mmol) were used and stirred for 16 h at reﬂux. The crude

product was puriﬁed on a silica gel column (PE/EA = 3:2) to give 86

(388 mg, 67% yield) as a yellowish solid. ¹H NMR (400 MHz,

CDCl₃): δ 10.21 (s, 1H), 8.82 (d, J = 1.6 Hz, 1H), 8.79 (d, J = 2.0 Hz,

1H), 7.99 (t, J = 2.0 Hz, 1H), 7.63 (s, 1H), 7.51−7.46 (m, 2H),

7.46−7.38 (m, 3H), 7.30−7.27 (m, 3H), 5.96 (s, 1H), 5.10 (s, 2H),

4.88 (br, 1H), 4.73 (s, 2H), 2.21 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 186.9, 161.0, 152.3, 151.9, 151.5, 142.9 (d, J = 3.0 Hz),

141.0, 137.7, 137.3, 132.8, 132.0, 131.3, 130.8, 128.3 (q, J = 2.4 Hz),

128.0, 127.6, 127.6, 125.9 (q, J = 29.0 Hz), 124.9 (q, J = 275.4 Hz),

116.3, 115.9, 115.4, 110.1, 93.2, 66.71, 45.47 (q, J = 5.3 Hz), 16.5.

ESI-MS: m/z = 502.15 [M + H]⁺.

+

45.3, 44.0, 16.9. ESI-HRMS: m/z calculated for C₃₂H₂₉F₃N₄NaO₄

(4-(((2-Bromo-[1,1′-biphenyl]-3-yl)methyl)amino)-2-((3-

cyanobenzyl)oxy)benzyl)-^D-serine (87). According to the general

procedure C, 85 (152 mg, 0.31 mmol) was reacted with ^D-serine (64

mg, 0.61 mmol) in anhydrous DMF (3 mL), which yielded 87 (128

mg, 70% yield) as a white solid. mp 169.9−171.0 °C. HPLC purity:

[M + Na]⁺, 613.2033; found, 613.2033.

(2-((5-Cyanopyridin-3-yl)methoxy)-4-(((2-(triﬂuoromethyl)-[1,1′-

biphenyl]-3-yl)methyl)amino)benzyl)-^D-serine (91). According to

the general procedure C, 82 (120 mg, 0.25 mmol) was reacted with

D-serine (52 mg, 0.49 mmol) in anhydrous DMF (3 mL), which

yielded 91 (56 mg, 39% yield) as a white solid. mp 164.9−166.1 °C.

HPLC purity: 97.16% (method B). ¹H NMR (400 MHz, DMSO-d₆):

δ 9.00 (d, J = 2.0 Hz, 1H), 8.99 (d, J = 2.0 Hz, 1H), 8.50 (t, J = 2.0

Hz, 1H), 8.21 (br, 2H), 7.60−7.55 (m, 2H), 7.47−7.37 (m, 3H),

7.31−7.26 (m, 2H), 7.22 (t, J = 4.0 Hz, 1H), 7.10 (d, J = 8.0 Hz, 1H),

6.72 (t, J = 5.6 Hz, 1H), 6.35 (d, J = 1.6 Hz, 1H), 6.13 (dd, J = 8.0,

1.6 Hz, 1H), 5.34 (br, 1H), 5.19 (dd, J = 19.6, 13.2 Hz, 2H), 4.49 (d,

J = 4.0 Hz, 2H), 4.03 (dd, J = 44.0, 13.2 Hz, 2H), 3.76 (dd, J = 11.6,

4.4 Hz, 1H), 3.63 (dd, J = 11.6, 7.2 Hz, 1H), 3.16 (dd, J = 7.2, 4.4 Hz,

1H). ¹³C NMR (100 MHz, DMSO-d₆): δ 167.9, 157.3, 152.3, 151.7,

150.6, 141.7, 141.1, 139.5, 138.9, 133.2, 132.7, 131.4, 131.0, 128.2,

127.9, 127.4, 124.9 (q, J = 275.9 Hz), 124.5 (q, J = 28.4 Hz), 116.9,

108.9, 108.0, 103.9, 96.7, 66.1, 62.0, 59.9, 45.4, 44.1. ESI-HRMS: m/z

1

96.70% (method A). H NMR (400 MHz, DMSO-d₆): δ 8.26 (br,

2H), 7.98 (s, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.79 (d, J = 7.6 Hz, 1H),

7.57 (t, J = 8.0 Hz, 1H), 7.51−7.29 (m, 6H), 7.24 (dd, J = 7.2, 1.6 Hz,

1H), 7.08 (d, J = 8.4 Hz, 1H), 6.64 (t, J = 6.0 Hz, 1H), 6.34 (d, J = 1.6

Hz, 1H), 6.12 (dd, J = 8.0, 1.6 Hz, 1H), 5.33 (br, 1H), 5.18−5.10 (m,

2H), 4.35 (d, J = 5.6 Hz, 2H), 4.03 (dd, J = 40.4, 12.8 Hz, 2H), 3.78

(dd, J = 11.6, 4.0 Hz, 1H), 3.65 (dd, J = 11.2, 7.2 Hz, 1H), 3.18−3.15

(m, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.0, 157.4, 150.6,

142.8, 141.1, 139.1, 138.7, 132.5, 132.3, 131.6, 130.9, 129.8, 129.7,

129.3, 128.1, 127.7, 127.6, 127.4, 122.8, 118.8, 111.4, 107.9, 103.9,

96.8, 68.2, 62.0, 59.9, 47.6, 45.5. ESI-HRMS: m/z calculated for

C₂₈H₂₂BrN₂O⁺[M − C₃H₆NO₃]⁺, 481.0910; found, 481.0911.

(4-(((2-Bromo-[1,1′-biphenyl]-3-yl)methyl)amino)-2-((5-cyano-

pyridin-3-yl)methoxy)benzyl)-^D-serine (88). According to the general

procedure C, 83 (316 mg, 0.63 mmol) was reacted with ^D-serine (133

mg, 1.27 mmol) in anhydrous DMF (6 mL), which yielded 88 (153

mg, 41% yield) as a white solid. mp 169.9−171.0 °C. HPLC purity:

98.04% (method A). ¹H NMR (400 MHz, DMSO-d₆): δ 9.00 (d, J =

2.0 Hz, 1H), 8.98 (d, J = 2.0 Hz, 1H), 8.50 (t, J = 2.0 Hz, 1H), 8.22

(br, 1H), 7.50−7.44 (m, 2H), 7.44−7.40 (m, 1H), 7.40−7.34 (m,

3H), 7.32 (dd, J = 7.8, 1.8 Hz, 1H), 7.24 (dd, J = 7.2, 2.0 Hz, 1H),

7.09 (d, J = 8.4 Hz, 1H), 6.66 (t, J = 5.8 Hz, 1H), 6.37 (d, J = 1.6 Hz,

1H), 6.14 (dd, J = 8.2, 1.8 Hz, 1H), 5.31 (br, 1H), 5.20 (dd, J = 19.6,

13.2 Hz, 2H), 4.37 (d, J = 6.0 Hz, 2H), 4.04 (dd, J = 44.4, 13.2 Hz,

2H), 3.77 (dd, J = 11.2, 4.4 Hz, 1H), 3.63 (dd, J = 11.6, 7.2 Hz, 1H),

3.16 (dd, J = 7.2, 4.4 Hz, 1H). ¹³C NMR (100 MHz, DMSO-d₆): δ

167.8, 157.3, 152.4, 151.7, 150.6, 142.8, 141.1, 139.1, 138.9, 133.2,

132.6, 129.8, 129.2, 128.1, 127.7, 127.6, 127.4, 122.8, 116.9, 108.9,

107.9, 104.0, 96.8, 66.1, 62.0, 59.9, 47.6, 45.5. ESI-HRMS: m/z

+

calculated for C₃₁H₂₇F₃N₄NaO₄[M + Na]⁺, 599.1877; found,

599.1876.

3-((2-Formyl-5-(methyl((2-methyl-[1,1′-biphenyl]-3-yl)methyl)-

amino)phenoxy)methyl)benzonitrile (92). Methyl benzenesulfonate

(65 mg, 0.38 mmol) was added to a suspension of 30 (108 mg, 0.25

mmol) and KOH (85% w/w, 20 mg, and 0.30 mmol) in THF (3 mL)

and stirred for 25 h at 35 °C. Then, a further amount of methyl

benzenesulfonate (13 mg, 0.08 mmol) was added to the reaction

mixture and stirred for 15 h. The mixture was concentrated to dryness

in vacuo, diluted with water, and extracted with DCM. The combined

organic phase was washed with water, dried over anhydrous Na₂SO₄,

and ﬁltered. The ﬁltrate was concentrated to dryness and puriﬁed by

recrystallization (PE/DCM) to give compound 92 (70 mg, 63%

1

yield) as a yellowish solid. H NMR (400 MHz, CDCl₃): δ 10.24 (s,

1H), 7.75 (d, J = 8.8 Hz, 1H), 7.69 (s, 1H), 7.67−7.59 (m, 2H),

7.51−7.41 (m, 3H), 7.41−7.35 (m, 1H), 7.34−7.29 (m, 2H), 7.23−

7.15 (m, 2H), 6.94 (dd, J = 6.4, 2.4 Hz, 1H), 6.38 (dd, J = 8.8, 2.0 Hz,

1H), 6.09 (d, J = 2.4 Hz, 1H), 5.14 (s, 2H), 4.59 (s, 2H), 3.19 (s,

3H), 2.18 (s, 3H). ¹³C NMR (100 MHz, CDCl₃): δ 187.0, 162.4,

155.6, 143.2, 141.7, 138.1, 134.7, 132.7, 131.8, 131.3, 130.9, 130.4,

129.6, 129.4, 129.2, 128.2, 127.0, 125.9, 124.4, 118.6, 115.3, 112.8,

105.5, 94.7, 68.9, 55.0, 39.0, 16.0. ESI-MS: m/z = 447.22 [M + H]⁺.

(2-((3-Cyanobenzyl)oxy)-4-(methyl((2-methyl-[1,1′-biphenyl]-3-

yl)methyl)amino)benzyl)-^D-serine (93). According to the general

+

calculated for C₃₀H₂₇BrN₄NaO₄[M + Na]⁺, 609.1108; found,

609.1111.

(4-(((2-Bromo-[1,1′-biphenyl]-3-yl)methyl)amino)-2-((5-cyano-

pyridin-3-yl)methoxy)-5-methylbenzyl)-^D-serine (89). According to

the general procedure C, 84 (180 mg, 0.35 mmol) was reacted with ^D-

serine (74 mg, 0.70 mmol) in anhydrous DMF (3.5 mL), which

yielded 89 (46 mg, 22% yield) as a white solid. mp 169.1−170.7 °C.

HPLC purity: 97.52% (method A). ¹H NMR (400 MHz, DMSO-d₆):

δ 8.89 (d, J = 1.6 Hz, 1H), 8.83 (d, J = 1.6 Hz, 1H), 8.34 (s, 1H), 8.14

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procedure C, 92 (89 mg, 0.2 mmol) was reacted with D-serine (42

mg, 0.40 mmol) in anhydrous DMF (2 mL). After completion of

NaBH(OAc)₃addition, the mixture was stirred for further 14 h. The

crude product was puriﬁed by preparative high-performance liquid

chromatography to obtain 94 (64 mg, 60% yield) as a yellowish solid.

1H), 7.44 (d, J = 1.6 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 5.41 (s, 2H),

4.83 (s, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 189.1, 159.9, 152.4,

152.0, 145.9, 139.0, 132.6, 128.6, 124.2, 121.7, 116.8, 114.0, 109.1,

66.7, 45.3. ESI-MS: m/z = 287.05 [M + H]⁺.

2-Methyl-[1,1′-biphenyl]-3-amine (98). At an argon atmosphere,

3-bromo-2-methylaniline (2.79 g, 15 mmol), phenylboronic acid

(2.74 g, 22.5 mmol), CsOAc (7.20 g, 37.51 mmol), and PdCl₂dppf

(1.10 g, 1.50 mmol) were suspended in THF (48 mL) and stirred for

24 h at reﬂux. Then, the mixture was cooled, concentrated in vacuo,

diluted with water, and extracted with DCM. The combined organic

phase was washed with water and concentrated in vacuo. The crude

product was puriﬁed by column chromatography to give 98 (2.61 g,

1

mp 178.6−179.9 °C. HPLC purity: 97.40% (method B). H NMR

(400 MHz, DMSO-d₆): δ 8.33 (br, 2H), 7.98 (s, 1H), 7.85 (d, J = 8.0

Hz, 1H), 7.77 (d, J = 7.6 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.46 (t, J =

7.6 Hz, 2H), 7.41−7.35 (m, 1H), 7.33−7.29 (m, 2H), 7.17−7.11 (m,

2H), 7.08 (d, J = 7.6 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.37 (d, J =

2.0 Hz, 1H), 6.25 (dd, J = 8.4, 2.0 Hz, 1H), 5.39 (br, 1H), 5.21 (dd, J

= 17.2, 13.2 Hz, 2H), 4.56 (s, 2H), 4.06 (dd, J = 42.4, 12.8 Hz, 2H),

3.78 (dd, J = 11.6, 4.4 Hz, 1H), 3.66 (dd, J = 11.2, 7.2 Hz, 1H), 3.18

(dd, J = 6.8, 4.4 Hz, 1H), 3.04 (s, 3H), 2.14 (s, 3H). ¹³C NMR (100

MHz, DMSO-d₆): δ 168.1, 157.4, 151.2, 142.1, 141.5, 138.8, 136.5,

132.6, 132.3, 132.3, 131.5, 130.9, 129.6, 129.2, 128.2, 128.1, 126.9,

125.3, 124.5, 118.7, 111.4, 107.6, 104.1, 96.3, 68.3, 62.0, 59.9, 53.8,

1

95% yield) as a brown solid. H NMR (400 MHz, CDCl₃): δ 7.41−

7.37 (m, 2H), 7.36−7.27 (m, 3H), 7.07 (t, J = 7.8 Hz, 1H), 6.70 (d, J

= 7.6 Hz, 2H), 3.69 (br, 2H), 2.06 (s, 3H). ¹³C NMR (100 MHz,

CDCl₃): δ 145.0, 143.1, 142.4, 129.4, 128.0, 126.7, 126.2, 120.6,

119.9, 114.1, 14.4. ESI-MS: m/z = 184.12 [M + H]⁺.

+

5-((2-Formyl-5-(((2-methyl-[1,1′-biphenyl]-3-yl)amino)methyl)-

phenoxy)methyl)nicotinonitrile (99). 97 (397 mg, 1.38 mmol), 98

(379 mg, 2.07 mmol), K₂CO₃(229 mg, 1.66 mmol), and NaI (42 mg,

0.28 mmol) were suspended in MeCN/DMF (8 mL/2.5 mL) and

stirred for 24 h at 30 °C. Then, the reaction mixture was diluted with

water and extracted with EA. The combined organic phase was

washed with water, concentrated in vacuo, and puriﬁed on a silica gel

column (PE/EA = 3:2) to give 99 (188 mg, 31% yield) as a yellowish

solid. ¹H NMR (400 MHz, CDCl₃): δ 10.46 (s, 1H), 8.87 (d, J = 2.4

Hz, 1H), 8.85 (d, J = 2.0 Hz, 1H), 8.06 (t, J = 2.2 Hz, 1H), 7.88 (d, J

= 7.6 Hz, 1H), 7.44−7.39 (m, 2H), 7.37−7.29 (m, 3H), 7.17 (d, J =

8.0 Hz, 1H), 7.09 (t, J = 7.8 Hz, 2H), 6.72 (d, J = 7.6 Hz, 1H), 6.46

(d, J = 8.0 Hz, 1H), 5.24 (s, 2H), 4.50 (s, 2H), 4.19 (br, 1H), 2.11 (s,

3H). ¹³C NMR (100 MHz, CDCl₃): δ 188.6, 160.2, 152.1, 151.7,

149.3, 145.8, 142.8, 142.3, 137.9, 132.5, 129.9, 129.4, 128.1, 126.8,

126.3, 124.3, 120.5, 120.0, 119.6, 116.3, 111.0, 110.2, 109.2, 66.9,

48.6, 14.4. ESI-MS: m/z = 434.19 [M + H]⁺.

45.4, 38.4, 15.5. ESI-HRMS: m/z calculated for C₃₃H₃₃N₃NaO₄[M

+ Na]⁺, 558.2363; found, 558.2361.

3-((2-(((2-Hydroxyethyl)amino)methyl)-5-(methyl((2-methyl-

[1,1′-biphenyl]-3-yl)methyl)amino)phenoxy)methyl)benzonitrile

(94). According to the general procedure C, 92 (70 mg, 0.16 mmol)

was reacted with 2-aminoethanol (19 mg, 0.31 mmol) in DCE (2 mL)

and anhydrous DMF (0.5 mL). After completion of NaBH(OAc)₃

addition, the mixture was stirred for further 12 h. The crude product

was puriﬁed on a silica gel column (DCM/MeOH = 25:1−8:1). The

residue was dissolved in anhydrous DCM (3 mL). HCl (1 N solution

in ether, 3 mL) was added dropwise to the above solution and stirred

for 30 min at rt. The precipitate was collected and puriﬁed by

preparative high-performance liquid chromatography to give dihydro-

chloride dihydrate of 93 (34 mg, 38% yield) as a white solid. mp

1

100.6−101.7 °C. HPLC purity: 95.78% (method B). H NMR (400

MHz, DMSO-d₆): δ 9.00 (s, 2H), 7.98 (s, 1H), 7.83 (d, J = 7.6 Hz,

1H), 7.80−7.74 (m, 1H), 7.55 (t, J = 7.8 Hz, 1H), 7.48−7.42 (m,

2H), 7.38−7.34 (m, 1H), 7.32−7.27 (m, 3H), 7.13 (t, J = 7.6 Hz,

1H), 7.07 (dd, J = 7.4, 1.0 Hz, 1H), 6.87 (d, J = 7.2 Hz, 1H), 6.47 (s,

1H), 6.35 (d, J = 5.6 Hz, 1H), 5.59 (br, 6H), 5.22 (s, 2H), 4.60 (s,

2H), 4.04 (t, J = 4.8 Hz, 2H), 3.66 (t, J = 5.4 Hz, 2H), 3.07 (s, 3H),

2.96−2.85 (m, 2H), 2.13 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆):

δ 157.5, 150.8, 142.2, 141.5, 138.8, 136.1, 132.8, 132.7, 132.3, 131.6,

130.9, 129.6, 129.2, 128.2, 126.9, 125.3, 124.9, 118.7, 111.4, 107.6,

104.7, 96.8, 68.3, 56.3, 54.1, 48.3, 44.6, 38.5, 15.7. ESI-HRMS: m/z

(2-((5-Cyanopyridin-3-yl)methoxy)-4-(((2-methyl-[1,1′-biphenyl]-

3-yl)amino)methyl)benzyl)-^D-serine (100). According to the general

procedure C, 99 (278 mg, 0.64 mmol) was reacted with ^D-serine (135

mg, 1.28 mmol) in anhydrous DMF (6 mL) and anhydrous MeOH (2

mL), which yielded 100 (78 mg, 23% yield) as a white solid. mp

1

167.1−168.8 °C. HPLC purity: 98.20% (method A). H NMR (400

MHz, DMSO-d₆): δ 9.02 (d, J = 2.4 Hz, 1H), 8.98 (d, J = 2.0 Hz,

1H), 8.50 (t, J = 2.0 Hz, 1H), 7.43−7.38 (m, 2H), 7.38−7.31 (m,

2H), 7.29−7.24 (m, 2H), 7.20 (s, 1H), 7.03 (d, J = 7.6 Hz, 1H), 6.91

(t, J = 7.8 Hz, 1H), 6.42 (dd, J = 7.6, 0.8 Hz, 1H), 6.37 (d, J = 8.0 Hz,

1H), 5.76 (t, J = 6.0 Hz, 1H), 5.27 (dd, J = 18.8, 12.8 Hz, 2H), 4.37

(d, J = 5.6 Hz, 2H), 4.10 (dd, J = 20.6, 13.4 Hz, 2H), 3.75 (dd, J =

11.2, 4.4 Hz, 1H), 3.63 (dd, J = 11.2, 6.8 Hz, 1H), 3.19 (dd, J = 6.8,

4.4 Hz, 1H), 2.03 (s, 3H). ¹³C NMR (100 MHz, DMSO-d₆): δ 168.9,

156.2, 152.4, 151.8, 146.4, 143.3, 142.4, 141.7, 138.9, 133.1, 131.2,

129.2, 128.0, 126.6, 125.8, 120.2, 119.5, 119.0, 117.7, 116.9, 110.9,

108.9, 108.8, 66.3, 62.6, 60.3, 46.6, 45.3, 14.6. ESI-HRMS: m/z

+

calculated for C₃₂H₃₃N₃NaO₂[M + Na]⁺, 514.2465; found,

514.2460.

5-((2-Formyl-5-(hydroxymethyl)phenoxy)methyl)nicotinonitrile

(96). 2-Hydroxy-4-(hydroxymethyl)benzaldehyde (compound 95,

683 mg, 4.49 mmol), K₂CO₃(683 mg, 4.94 mmol), and 5-

(chloromethyl)nicotinonitrile (755 mg, 4.95 mmol) were suspended

in MeCN (20 mL) and stirred for 10 h at 60 °C. Then, the reaction

mixture was cooled, diluted with H₂O (230 mL), and ﬁltered. The

precipitate was washed with water and dried in vacuo to give 96 (1.01

g, 84% yield) as a yellowish solid, which was used in the next step

without further puriﬁcation. ¹H NMR (400 MHz, DMSO-d₆): δ 10.41

(d, J = 0.8 Hz, 1H), 9.04 (dd, J = 3.2, 2.0 Hz, 2H), 8.55 (t, J = 2.0,

1H), 7.71 (d, J = 7.6 Hz, 1H), 7.29 (d, J = 0.8 Hz, 1H), 7.08 (ddd, J =

8.0, 1.2, 0.8 Hz, 1H), 5.49 (t, J = 5.6 Hz, 1H), 5.39 (s, 2H), 4.59 (d, J

= 5.6 Hz, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 189.1, 160.1,

152.3, 152.2, 151.9, 138.8, 132.9, 128.1, 123.1, 118.8, 116.9, 110.8,

109.1, 66.5, 62.5. ESI-MS: m/z = 269.10 [M + H]⁺.

5-((5-(Chloromethyl)-2-formylphenoxy)methyl)nicotinonitrile

(97). A solution of MsCl (537 mg, 4.69 mmol) in anhydrous DCM (4

mL) was added dropwise to a solution of 96 (838 mg, 3.12 mmol)

and pyridine (494 mg, 6.24 mmol) in anhydrous DCM (20 mL) and

anhydrous DMF (2 mL) at 0 °C. After completion of addition, the

reaction mixture was stirred for 24 h at rt, and then diluted with water

and extracted with DCM. The combined organic phase was washed

with water, concentrated in vacuo, and puriﬁed on a silica gel column

(PE/EA = 2:1) to give 97 (565 mg, 63% yield) as a yellowish solid.

¹H NMR (400 MHz, DMSO-d₆): δ 10.42 (d, J = 0.4 Hz, 1H), 9.04

+

calculated for C₃₁H₃₁N₄O₄[M + H]⁺, 523.2340; found, 523.2338.

Biochemical Assay of PD-1/PD-L1 Blockade. The assay was

performed using the PD-1/PD-L1 binding assay kit (Cisbio,

64CUS000C-1). BMS202 was purchased from MedChemExpress

and used as a positive control. The protocol was performed according

to the instructions of the assay kit. Brieﬂy, 2 μL of compounds (or

diluent buﬀer), 4 μL of PD-L1-Tag1, and 4 μL of PD1-Tag2 were

incubated in 384-well low volume plates at rt for 15 min. Then, 10 μL

of the premixture of two conjugates (5 μL of anti-Tag1-Eu³⁺and 5 μL

of anti-Tag2-XL665) was added to wells. The mixture was incubated

at rt for 2.5 h, and the ﬂuorescence emission was detected at two

diﬀerent wavelengths (665 and 620 nm) on an HTRF reader

(F200Pro, TECAN). The results were calculated from the 665 and

620 nm ﬂuorescence signals and expressed in an HTRF ratio = (665

nm/620 nm) × 10⁴. The inhibitory percentage was calculated from

the HTRF ratio. The IC₅₀value was calculated from inhibitory

percentage.

Binding Speciﬁcity and Aﬃnity Assay. The speciﬁcity assay

was performed by biolayer interferometry (BLI) using the Octet RED

(dd, J = 3.6, 2.0 Hz, 2H), 8.56 (t, J = 2.0 Hz, 1H), 7.76 (d, J = 7.6 Hz,

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system (ForteBio). The proteins were purchased from the

ACROBiosystems Company. Human PD-L1/Fc, PD-1/Fc, PD-L2/

Fc, CD47/Fc, TIM-3/Fc, LAG-3/Fc, and CD27/Fc were diluted to

30 μg/mL using running buﬀer (PBS, 0.02% Tween-20, and 0.1%

BSA) and loaded onto anti-human IgG Fc capture (AHC) biosensors

for 600 s. Compound 52 was diluted to a concentration of 500 nM

(2% DMSO) using running buﬀer. After loading, the biosensors were

baselined, associated in compound 52 for 300 s, and then dissociated

in buﬀer for 300 s. The baseline-corrected binding curves were

analyzed.

To determine the binding aﬃnity, the 10 μg/mL biotinylated

human PD-L1/AVI (ACRO) was loaded onto SA (streptavidin)

biosensors for 300 s (60 s for determination of human PD-1). After

loading, the biosensors were baselined, associated in deﬁned

concentrations of compounds for 180 s (240 s for determination of

human PD-1), and then dissociated in buﬀer for 300 s (600 s for

determination of human PD-1). The assay concentrations of

compounds were 20, 10, 5, 2.5, 1.25, 0.625, 0.3125, and 0 nM. The

assay concentrations of human PD-1/His₆were 500, 250, 125, 62.5,

31.25, 15.63, 7.81, and 0 nM. Each data set was ﬁtted globally to a 1:1

interaction model to determine the kinetic parameters k_aand k_dis.

Apparent aﬃnity was then calculated as a ratio (k_a/k_dis) of these

kinetic constants.

Molecular Docking. The crystal structures of PD-L1 (PDB code

5J89) were obtained from the Protein Data Bank. Molecular docking

was performed by the GOLD 5.3 software. For receptors, hydrogen

atoms were added, and water molecules and bound ligands were

removed. The scoring function was set to ChemPLP. All of the other

parameters used in the docking process were set to default values. The

best binding modes were chosen to analysis according to docking

scores. Figures were created using Pymol version Open-Source 1.6.x.

Flow Cytometry. According to the instructions, the CHO-K1 and

CHO-K1-hPD-L1 cells (ATCC) were resuscitated and subcultured,

when the cell conﬂuence rate exceeded 80%. The cells in the

logarithmic growth phase were digested with trypsin and resuspended

in the complete medium. The cells were placed in a tube, centrifuged

at 1000 rpm for 5 min, and resuspended in FACS staining buﬀer (1 ×

PBS with 2% FBS). 5 × 10⁵cells/tube were incubated with hPD-1/Fc

(ﬁnal concentration, 10 μg/mL) at 4 °C for 30 min. The dilutions of

blocking compounds were added into tubes at a ﬁnal concentration of

500 nM in 100 μL of FACS staining buﬀer. The equal volume of

buﬀer was added into tubes in the negative and positive control

groups. All tubes were incubated at 4 °C for another 30 min. Then,

the cells were washed, resuspended in buﬀer (95 μL), and incubated

with the APC anti-human IgG Fc antibody (5 μL). After incubation,

the cells were washed and resuspended in buﬀer. The ﬂuorescence

intensity and the percentage of cells bound with hPD-1 (PCB) were

analyzed by ﬂow cytometry. The blocking eﬃcacy (%) = [1 − (the

PCB value of compounds groups − the PCB value of negative

groups)/(the PCB value of the positive control − the PCB value of

negative groups)] × 100%.

Determination of IFN-γ Secretion. Peripheral blood mono-

nuclear cells (PBMCs) were isolated from 10 to 15 mL whole blood

of a healthy donor using a human lymphocyte separation ﬂuid (TBD

sciences, Cat. LTS1077). The cells were washed twice with normal

saline and resuspended in buﬀer (RPMI 1640 complete medium

containing 10% FBS), and the density was measured. The PBMCs (1

× 10⁵cells in 50 μL buﬀer) were inoculated in 96-well plates. The

speciﬁc concentrations of the anti-CD3 antibody and anti-CD28

antibody were mixed in a ratio of 1:1. Antibody mixtures (50 μL)

were added to the corresponding wells at the ﬁnal concentrations of

0.8 μg/mL. At the same time, the gradient PD-L1/FC fusion protein

(50 μL/well) was added into the coculture system at a ﬁnal

concentration of 10 μg/mL. Compounds were diluted with buﬀer and

added into the coculture system at ﬁnal concentrations of 0.1 and 1

μM (50 μL/well). The mixtures were cocultured at 37 °C with a 5%

CO₂cell incubator for 72 h. The supernatant was harvested to

determine the expression levels of IFN-γ using the ELISA MAXTM

Standard Set Human IFN-γ kit (BioLegend) according to the

instructions of the manufacturer.

Cytotoxicity Assay. The cell lines Raji (ATCC), Raw 264.7

(ATCC), and PBMCs (isolated from whole blood of a healthy donor)

were resuspended in a complete medium with 10% FBS, and cell

density was measured. The cells were plated into 96-well plates at the

ﬁnal density of 1 × 10⁴cells per well (50 μL) and treated with

compounds at diﬀerent ﬁnal concentrations (100, 50, 25, 12.5, 6.25,

and 3.125 μM) or control buﬀer (containing 0.2% DMSO) for 24 h at

a 37 °C, 5% CO₂cell incubator. After incubation, 100 μL of CellTiter-

Glo Luminescent Solution (Promega) was added to each well and

shaken at room temperature for 10 min. Luminescence was measured

on a GloMax Navigator System with an integration time of 1 s per

well according to the instructions of the manufacturer. The killing rate

(%) = (1 − RLU_compound/RLU_control) × 100%.

In Vivo Pharmacological Study. All animal procedures

described in this study were performed in adherence with the

guidelines as deﬁned by the Animal Care and Use Committee at the

Beijing Institute of Pharmacology & Toxicology. Six-week-old PD-1

humanized female mice (full strain name: C57BL/6J-

Pdcd1^{em1(hPDCD1)/Smoc}) were purchased from the Shanghai Southern

Model Animal Center and placed in the SPF animal feeding room for

1 week. At the same time, the PD-L1 genetically engineered colon

cancer cell line MC38 (mouse PD-L1 knockout and human PD-L1

knock-in) was subcutaneously inoculated into the mouse near the

groin of the hind leg. The amount of inoculated cells was 1 × 10⁶cells

per mouse. The animals are randomly divided into 3 groups (6 mice

in each group) according to the size of the tumor using the random

number table, when the tumor volume reached 50−150 mm³.

BMS202 (15 mg/kg) and compound 58 (15 mg/kg) were

administered at the frequency of once a day for 3 weeks by

intraperitoneal administration. The treatment with equal volume of

buﬀer (0.8% DMSO and 7.5% RH40 in normal saline) was used as

the vehicle control. Tumor volume and body weight were measured

twice every week. The tumor volume was calculated using the

equation (0.5 × L × W²), where L and W refer to the length and

width dimensions of the tumor. At the end of the experiment, the

mice were sacriﬁced with the cervical dislocation method, and the

tumors were stripped and weighted. Tumor growth inhibition (TGI)

was calculated using the equation: TGI = [1 − (T_final− T₀)/(C_final

−

C₀)] × 100%, where T_finaland T₀were the mean tumor volumes of

test groups at the end of the treatment and on the ﬁrst day of

treatment, respectively; C_finaland C₀were the mean tumor volumes of

the vehicle group at the end of the treatment and on the ﬁrst day of

treatment, respectively.

In Vivo Pharmacokinetic Study in Rats. The pharmacokinetic

experiments of 58 in rats were performed by Beijing Pharmaron, Inc.

according to the guidelines of the Animal Care and Use Committee of

the company. PK was evaluated after a single dose of 4 mg/kg

intravenous (iv) and 15 mg/kg oral (po) administration in male

Sprague Dawley rats (n = 6, 6−8 weeks age, and 200−300 g weight).

Dosing solutions were freshly prepared on the day of administration

in 25% MPD/10% RH40/65% water. The time points for blood

sample collection were 0.08 (only for iv), 0.25, 0.5, 1, 2, 4, 8, and 24 h

post dose. Approximately 0.2 mL of blood was collected at each time

point. Each blood sample was transferred into plastic microcentrifuge

tubes containing an anticoagulant of EDTA-K2 and mixed well then

placed on wet ice prior to centrifugation. Blood samples were

centrifuged at 4000g for 5 min at 4 °C to obtain plasma. The samples

were stored in a freezer at −75 15 °C prior to analysis. Plasma

concentrations of the tested compounds were analyzed by the LC−

MS/MS method. The Phoenix WinNonlin 8.0 software was used to

calculate all pharmacokinetic parameters.

Statistical Analysis. The IC₅₀of PD-1/PD-L1 blockade and PK

parameters were expressed as mean

SD. The other ones were

shown as mean SEM. An unpaired t-test was used to determine the

statistical signiﬁcance. *, p < 0.05; **, p < 0.01.

X

https://dx.doi.org/10.1021/acs.jmedchem.0c01329

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

National Science and Technology Major Project (no.

2018ZX09711003-003-001).

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01329.

■

sı

ABBREVIATIONS

■

Results of binding speciﬁcity assay, NMR spectrum of

ﬁnal compounds, and HPLC spectra of key target

compounds (PDF)

PD-1, programmed cell death-1; PD-L1, programmed cell

death-ligand 1; IL-2, interleukin 2; IFN-γ, interferon-γ; mAbs,

monoclonal antibodies; HTRF, homogeneous time-resolved

ﬂuorescence; BMS, Bristol Myers Squibb; BINAP, 2,2′-

bis(diphenylphosphino)-1,1′-binaphthalene; BPO, benzoyl

peroxide; BLI, biolayer interferometry; AHC, anti-human

IgG Fc Capture; hPD-L1, human PD-L1; hPD-1, human

PD-1; k_a, association constant; k_dis, dissociation constant;

CHO-K1-hPD-L1, hPD-L1 overexpressing CHO-K1 cells;

PCB, percentage of cells bound with hPD-1; PBMCs,

peripheral blood mononuclear cells; MPD, 1,2-propanediol;

RH40, cremophor RH40; PE, petroleum ether; EA, ethyl

acetate; SA, streptavidin; FBS, fetal bovine serum; FACS,

ﬂuorescence activated cell sorting; APC, allophycocyanin;

TGI, tumor growth inhibition

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Author

Junhai Xiao − National Engineering Research Center for the

Emergency Drug, State Key Laboratory of Toxicology and

Medical Countermeasures, Beijing Institute of Pharmacology

and Toxicology, Beijing 100850, China; Email: xiaojunhai@

139.com

■

Authors

Jialin Guo − National Engineering Research Center for the

Emergency Drug, State Key Laboratory of Toxicology and

Medical Countermeasures, Beijing Institute of Pharmacology

and Toxicology, Beijing 100850, China; orcid.org/0000-

0001-8056-994X

Longlong Luo − State Key Laboratory of Toxicology and

Medical Countermeasures, Beijing Institute of Pharmacology

and Toxicology, Beijing 100850, China

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Naijing Hu − State Key Laboratory of Toxicology and Medical

Countermeasures, Beijing Institute of Pharmacology and

Toxicology, Beijing 100850, China

Wei Wang − The Aﬃliated Hospital, Changchun University of

Chinese Medicine, Changchun 130021, China

Fei Xie − National Engineering Research Center for the

Emergency Drug, State Key Laboratory of Toxicology and

Medical Countermeasures, Beijing Institute of Pharmacology

and Toxicology, Beijing 100850, China

Erguang Liang − National Engineering Research Center for the

Emergency Drug, State Key Laboratory of Toxicology and

Medical Countermeasures, Beijing Institute of Pharmacology

and Toxicology, Beijing 100850, China

Xinlin Yan − National Engineering Research Center for the

Emergency Drug, State Key Laboratory of Toxicology and

Medical Countermeasures, Beijing Institute of Pharmacology

and Toxicology, Beijing 100850, China

Song Li − National Engineering Research Center for the

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and Toxicology, Beijing 100850, China

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01329

Author Contributions

^§J.G. and L.L. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by grants from the National Natural

Science Foundation of China (no. 81903455), the China

Postdoctoral Science Foundation (no. 2017M623412), and the

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Article Doi

Design, Synthesis, and Biological Evaluation of Linear Aliphatic Amine-Linked Triaryl Derivatives as Potent Small-Molecule Inhibitors of the Programmed Cell Death-1/Programmed Cell Death-Ligand 1 Interaction with Promising Antitumor Effects in Vivo

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