Design, synthesis, biological evaluation and structure-activity relationship study of quinazolin-4(3H)-one derivatives as novel USP7 inhibitors

European Journal of Medicinal Chemistry 216 (2021) 1 13291

Contents lists available at ScienceDirect

European Journal of Medicinal Chemistry

journal homepage: h ttp://www.elsevier.com/locate/ejmech

Design, synthesis, biological evaluation and structure-activity

relationship study of quinazolin-4(3H)-one derivatives as novel USP7

inhibitors

Peng Li, Ying Liu, Hua Yang^*, Hong-Min Liu^**

School of Pharmaceutical Sciences, Zhengzhou University, 100 Kexue Avenue, Zhengzhou, Henan, 450001, China

a r t i c l e i n f o

a b s t r a c t

Article history:

Recent research has indicated that the abnormal expression of the deubiquitinase USP7 induces

tumorigenesis via multiple cell pathways, and in particular, the p53-MDM2-USP7 pathway is well un-

derstood. USP7 is emerging as a promising target for cancer therapy. However, there are limited reports

on USP7 inhibitors. Here we report design, synthesis and biological evaluation of novel quinazolin-4(3H)-

one derivatives as potent USP7 inhibitors. Our results indicated that the compounds C9 and C19

Received 10 November 2020

Received in revised form

10 January 2021

Accepted 20 January 2021

Available online 16 February 2021

exhibited the greatest potency against the USP7 catalytic domain, with IC₅₀values of 4.86

m

M and

M for

1.537 M, respectively. Ub-AMC assays further conﬁrmed IC₅₀values of 5.048 M for C9 and 0.595 m

m

Keywords:

C19. MTT assays indicated that gastric cancer MGC-803 cells were more sensitive to these compounds

than BGC-823 cells. Flow cytometry analysis revealed that C9 restricted cancer cell growth at the G0/G1

and S phases and inhibited the proliferation and clone formation of MGC-803 cells. Further biochemical

experiments indicated that C9 decreased the MDM2 protein level and increased the levels of the tumour

suppressors p53 and p21 in a dose-dependent manner. Docking studies predicted that solvent exposure

of the side chains of C9 and C19 would uniquely form hydrogen bonds with Met407 of USP7. Additionally,

C9 exhibited a remarkable anticancer effect in a zebraﬁsh gastric cancer MGC-803 cell model. Our results

demonstrated that quinazolin-4(3H)-one derivatives were suitable as leads for the development of novel

USP7 inhibitors and especially for anti-gastric cancer drugs.

USP7

Inhibitors

Quinazolin-4(3H)-one

SAR

Gastric cancer

Ubiquitination

1. Introduction

ubiquitin-mediated protein degradation. Subsequently, the

ubiquitin-proteasome pathway has drawn increasing attention,

The process of attaching a ubiquitin chain onto a target protein

is called ubiquitination, and this modiﬁcation plays important roles

in controlling the stabilization, function and localization of target

proteins [1]. In 2004, the Nobel Prize in chemistry was awarded to

two Israeli scientists, Aaron Ciechanover [2] and Avram Hershko

[3], and one American scientist, Irwin Rose [4], for the discovery of

and research has indicated that it is involved in immune diseases

[5], mental diseases [6] and tumour genesis [7].

Deubiquitinating enzymes (DUBs) are proteases in the

ubiquitin-proteasome pathway which can remove the ubiquitin

chain from the ubiquitinated target protein and prevent it from

degradation by the 26S proteasome. The human genome encodes

nearly 100 DUBs, which can be classiﬁed into ﬁve types: ubiquitin

C-terminal hydrolase (UCH), ubiquitin-speciﬁc protease (USP),

ovarian tumour domain protease (OTU), Machado-Joseph disease

protein domain protease (MJD) and JAB1/MPN/Mov34 metal-

loenzyme (JAMM) [8]. The de-ubiquitination process is highly

regulated and mediates many important cellular processes, such as

cell cycle [9], protein degradation [10], gene expression [1 1] and

DNA repair [12]. The ubiquitination and de-ubiquitination pro-

cesses work together to maintain the balance of all cellular

pathways.

Abbreviations: BLI, biolayer interferometry; DMF, N, N-dimethylformamide;

DMSO, dimethyl sulfoxide; DUB, deubiquitinating enzyme; EA, ethyl acetate; HATU,

2-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexaﬂuorophosphate; IR,

inhibitory rate; MOE, molecular operating environment; MDM2, murine double

minute 2; MTD, maximum tolerated dose; TLC, thin-layer chromatography; TMS,

tetramethylsilane; TMSOI, trimethylsulfoxonium iodide; TFA, triﬂuoroacetic acid;

TEA, triethylamine; Ub-AMC, ubiquitin-7-amido-4-methylcoumarin; USP7, ubiq-

uitin-speciﬁc-processing protease 7.

* Corresponding author.

** Corresponding author.

USP7 belongs to the USP family and is a papain-like cysteine

E-mail addresses: yanghua@zzu.edu.cn (H. Yang), liuhm@zzu.edu.cn (H.-M. Liu).

https://doi.org/10.1016/j.ejmech.2021.113291

P. Li, Y. Liu, H. Yang et al.

European Journal of Medicinal Chemistry 216 (2021) 113291

protease [8]. In 2002, the mechanism by which USP7 participates in

the ubiquitin-proteasome pathway was illustrated in detail [13,14].

Thereafter, further research indicated that USP7 was involved in

tumour formation by affecting multiple cancer routes [15e18]. In

particular, the p53-MDM2-USP7 pathway attracted much atten-

tion. Both p53 and MDM2 are substrates of USP7, but USP7 shows

higher afﬁnity for MDM2 [19]. This leads to the upregulation of

MDM2 expression, which promotes the ubiquitination of p53 by

serving as E3 ligase [20]. Finally, ubiquitinated p53 is degraded by

the 26S proteasome, preventing its anticancer function. The onco-

genicity of USP7 makes it a potential target for cancer therapy. The

development of USP7 inhibitors has achieved some success. The

reported USP7 inhibitors can also be classiﬁed into ﬁve types based

on their molecular skeletons: substituted thiophene derivatives

[21e24], acridine derivatives [25], quinazolin-4-one derivatives

[26e29], indeno[1,2-b]pyrazine derivatives [30,31] and 2-amino-4-

ethylpyridine derivatives [32,33]. Additionally, some natural

products also exhibit excellent USP7 inhibitory activities [34e37].

Currently, most research concerning USP7 focuses on eluci-

dating its mechanism of action, and limited progress has been

made in developing novel USP7 inhibitors. Here, we reported the

design, synthesis and biological evaluation of quinazolin-4(3H)-one

derivatives as novel potent USP7 inhibitors. First, we investigated

all published cocrystal structures of the USP7 catalytic domain with

small-molecule inhibitors and evaluated their potency and mech-

anisms. We found abundant co-crystal structure data of quinazolin-

4(3H)-one derivatives with the USP7 catalytic domain and thus

selected this co-crystal structure (PDB ID: 5VSB) and analyzed the

binding mode of this substrate [23]. We found that the potency of

the substrate (compound 1, Fig. 1) against the USP7 catalytic

to solvent exposure, whereas the 3-phenylbutanoic acid moiety

was buried in the hydrophobic pocket. However, few reports have

described the structure-activity relationship (SAR) between these

two sites.

In this report, we selected compound 1 as the lead and control

compound and preserved the quinazolin-4(3H)-one binding to the

4-hydroxypiperidine moiety as core requirements. The chemical

modiﬁcation of the lead mainly focused on C-7 of the quinazolin-

4(3H)-one ring and the 3-phenylbutanoic moiety.

2. Results and discussion

2.1. Chemistry

The synthetic procedures of quinazolin-4(3H)-one derivatives

are summarized in Scheme 1. N-Boc-piperidine-4-one reacted with

trimethylsulfoxonium iodide to form the key intermediate A2.

Then, 2-amino-4-chlorobenzoic acid (B1) in formamide was heated

at 150 ^ꢀC for 12 h to form 7-chloroquinazolin-4(3H)-one (B2). The

intermediates A2 and B2 were heated at 80 ^ꢀC in N, N-dime-

thylformamide (DMF) for 12 h to form the intermediate B3, whose

protecting group Boc was removed with the addition of triﬂuoro-

acetic acid (TFA) to form the intermediate B4. Finally, B4 was

reacted with different substituted formic acids to form the target

products (B5eB31). Meanwhile, replacement of B1 with C1 by the

same reaction route led to the formation of the key intermediates

C5 and C6, which were further subjected to a Cu-catalyzed Buch-

wald-Hartwig coupling reaction, leading to the synthesis of the

target products (C7eC19).

domain was moderate, with an IC₅₀of 12 mM, which was suitable

2.2. SAR investigations

for further development as a lead molecule. Meanwhile, the co-

crystal structure indicated that the binding of quinazolin-4(3H)-

one to the 4-hydroxypiperidine moiety was extremely important

for its activity. The C-7 of the quinazolin-4(3H)-one ring was open

We applied our optimized screening method based on previous

reports [26] to test the lead compound B5 (Fig. S1). This compound

exhibited moderate inhibitory activity against the USP7 catalytic

Fig. 1. Design strategy of quinazolin-4(3H)-one derivatives as potent USP7 inhibitors.

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European Journal of Medicinal Chemistry 216 (2021) 113291

Scheme 1. Synthesis of compounds B5eB31 and C7eC19. Reagents and conditions: (a) NaH (60% in mineral oil), dry DMSO, 0 ^ꢀC, 4 h; (b) 150 ^ꢀC, 12 h; (c) K₂CO₃, DMF, 80 ^ꢀC, 12 h;

(d) TFA, r.t., 4 h; (e) HATU, TEA, DMF, r.t., 5 h; (f) N₂, 5 mol% CuI, 20 mol% Ligand ¼ (2-hydroxyphenyl)(morpholino)methanone, K₃PO₄, dry DMF, 90 ^ꢀC, 18 h.

domain, with an IC₅₀of 23.05

m

M. Further optimization of the

remarkably increased the USP7 inhibitory activity (B13,

IC₅₀¼ 6.788 M, Fig. S1), whereas introducing a Br atom to C-7 of

the quinazolin-4(3H)-one ring did not remarkably affect the

M, Fig. S1).

The optimization of the quinazolin-4(3H)-one moiety at C-7 led

to the formation of the derivatives C7eC19, (Fig. S2). Hydrophobic

groups were not favoured, and the introduction of hydrophilic

groups remarkably increased the USP7 inhibitory activity (C8,

substituent bound at N-1 of the piperidine ring led to the formation

of other derivatives, which are summarized in Fig. S1. Introduction

of heterocycle to N-1 of the piperidine ring decreased or directly

m

inhibitory activity (B15, IC₅₀¼ 27.41

m

abrogated USP7 inhibitory activity (B7, B8 and B9; IC₅₀> 50

mM; IR

<50%, Fig. S1). Hydrophobic groups were favoured (B6, IR ¼ 18.59%

vs B14, IR ¼ 42.24%, Fig. S1), whereas strong hydrophilic groups

directly abrogated activity (B24, IR ¼ 0.8%, Fig. S1). Interestingly, the

C-2 and C-4 of benzoic acid substitutes were preferred to polar

IC₅₀¼ 15.31

m

M, C7, IC₅₀¼ 9.8

m

M and C9, IC₅₀¼ 4.86

mM, Fig. S2).

groups (B18, IC₅₀¼ 46.3

m

M and B26, IR ¼ 58.34%, Fig. S1), but steric

Introduction of a hydrophobic group decreased the inhibitory ac-

tivity (C10, IR ¼ 45.93%, C11, IR ¼ 49.91% and C12, IR ¼ 49.13%,

Fig. S2). Notably, bulky steric groups were not tolerated (C9,

polar groups were not favoured (B12, IR ¼ 4.29%, Fig. S1). The

introduction of a Br atom to the benzoic acid substitute remarkably

increased USP7 inhibitory activity (B17, IR

IC₅₀¼ 46.3 M, Fig. S1). Meanwhile, shortening the alkyl chain or

introducing a steric group to N-1 of the piperidine ring abrogated

its activity (B29, B30, IC₅₀> 50 M, Fig. S1). Introducing a hetero-

atom or steric group to the 3-phenylpropanoic acid moiety

decreased USP7 inhibitory activity (B11, IR 22.71%; B23,

¼

46.53%; B18,

IC₅₀¼ 4.86

strong hydrophilic groups decreased the inhibitory activity (C14,

M, Fig. S2). Introduction of a racemic methyl group to

the 3-phenylpropanoic acid moiety binding N-1 of the piperidine

ring led to the formation of C19, the most potent compound

m

M vs C13, IC₅₀¼ 36.95

mM, Fig. S2), and introduction of

m

IC₅₀¼ 16.42

m

¼

(IC₅₀¼ 1.537

mM, Fig. S2), whereas introduction of a lipophilic

IR ¼ 9.83%, Fig. S1). Increasing the rigidity of the 3-phenylpropanoic

acid moiety also decreased the inhibitory activity (B27, IR ¼ 32.64%,

group at C-7 of the quinazolin-4(3H)-one moiety enhanced the

lipophilicity of the whole molecule with the help of the racemic

methyl group, which led to decreased inhibitory activity (C15,

Fig. S1). Conversely, introducing

a racemic methyl group

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European Journal of Medicinal Chemistry 216 (2021) 113291

IC₅₀¼ 5.977

m

M vs C19, IC₅₀¼ 1.537

m

M, Fig. S2). The SAR analyses

derivatives exhibited moderate inhibitory activity against gastric

cancer cells. To further illustrate their underlying biochemical

mechanism, we used MGC-803 cells and performed Western blot

analysis. We used C9 as a target compound, C12 as a negative

control compound and B5 as a reference. First, we investigated the

effect of C12 and B5 on USP7 expression level in MGC-803 cells, the

results of which are summarized as Fig. 3. The reference drug B5

inhibited USP7 activity in MGC-803 cells, whereas C12 had no ef-

fect. Interestingly, C12 still exhibited moderate inhibitory activity

against MGC-803 and BGC-823 cells, which indicate its inhibitory

effect against other cellular proteins. Next, we investigated the

inhibitory mechanism of one of the most potent compounds, C9,

the results of which are summarized in Fig. 4. The results indicated

that USP7 protects MDM2 from degradation and maintains high

protein levels of MDM2. In contrast, C9 inhibited USP7 activity and

led to a decreased level of MDM2 in a time-dependent manner.

Further biochemical exploration of C9 in MGC-803 cells indicated

that it blocked the p53-MDM2-USP7 pathway (Fig. 5). The experi-

mental results also revealed that C9 inhibited USP7 activity and led

to a decreased level of MDM2. This indirectly led to the upregula-

tion of p53 expression and that of its downstream protein, p21.

Meanwhile, biolayer interferometry (BLI) assays also conﬁrmed the

high binding afﬁnity of C9 with the USP7 catalytic domain, with a

K_Dof 7.7 ꢁ 10^ꢂ5M (Fig. S5). Therefore, C9 was considered a USP7

inhibitor and exerted effects on MGC-803 cells via interference in

the p53-MDM2-USP7 pathway.

of quinazolin-4(3H)-one derivatives are summarized in Fig. 2.

Finally, we selected the most potent compounds in Fig. S2 and

screened them using Ub-AMC assays, and the results of which are

summarized in Table S1. These experiments further conﬁrmed the

high efﬁciency of our designed compounds.

2.3. Cellular effects of quinazolin-4(3H)-one derivatives against

gastric cancer cells

Most of the USP7 inhibitors that we reviewed exhibited enzy-

matic activity, but few exhibited cellular activity. The reported USP7

inhibitors were effective in HCT116 cells [18e28], but whether they

were effective against other cancer cell lines was unknown.

Therefore, we tested our derivatives against two gastric cancer cell

lines, MGC-803 and BGC-823. The inhibitory activities of quinazo-

lin-4(3H)-one derivatives against these two cell lines are summa-

rized in Table S2. We also appended the inhibitory activity against

the catalytic domain of USP7 for ease of understanding.

We found that quinazolin-4(3H)-one derivatives exhibited

moderate inhibitory activity against the cell lines, especially MGC-

803, which was more sensitive to compounds B17, B18 and B19

(Table S2). The most potent compounds against the cell lines and

the USP7 catalytic domain were B18 and C8. The most potent

compounds against the cell lines were B23 and B25. Interestingly,

C9 exhibited high potency against the USP7 catalytic domain but

low potency MGC-803 and BGC-823 cells. However, C8, which was

the less potent against the USP7 catalytic domain, exhibited mod-

Additionally, to further prove the efﬁciency of our designed

compounds in the p53-MDM2-USP7 pathway in gastric cancer

cells, we tested the effects of C15, C16, C19 against BGC-823 cells

(Fig. 6). These compounds also affected the expression levels of

USP7-related proteins in a concentration-dependent manner.

erate inhibitory activity against MGC-803 (IC₅₀¼ 32.13

mM,

Table S2) and BGC-823 cells (IR ¼ 54.01%, Table S2). On comparing

the structures of C8 and C9, we found that the inhibitory activity

against gastric cancer cells was correlated with the lipophilicity of

these compounds. Of note, increasing the hydrophilicity of com-

pounds increases the USP7 inhibitory activity, whereas increasing

the lipophilicity of compounds increases the inhibitory activity

against gastric cancer cells. Remarkably, the reference compound

B5 exhibited no inhibitory activity against either gastric cancer cell

line. Flow cytometry and clone formation experiments (Fig. S3 and

Fig. S4) also indicated that C9 inhibited the proliferation of MGC-

803 cells and induced their death by restricting the cell cycle at the

G0/G1 and S phases.

2.5. Molecular docking studies

To determine why C19 exhibited the highest USP7 inhibitory

activity, we selected C13, C16, C17 and C19 as ligands for molecular

modelling research. These compounds had different structures and

USP7 inhibitory activities. We used the co-crystal structure (PDB ID:

5VSB) as a model and the ligand B5 (Fig. S1) as a reference. First, we

compared the binding mode of the highest score conformation of

these compounds, summarized in Fig. S6. The input conformation

of the compound was generated in molecular operating environ-

ment (MOE) 2014 and further optimized using the ‘QuickPrep’

function. This optimization result was used as the input confor-

mation. The co-crystal structure of the USP7 catalytic domain was

2.4. Biochemical characterization of C9

Cell experiments indicated that quinazolin-4(3H)-one

Fig. 2. SAR analysis of quinazolin-4(3H)-one derivatives.

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European Journal of Medicinal Chemistry 216 (2021) 113291

Fig. 3. Analysis of the USP7 inhibitory mechanism against MGC-803 cells. (a) Structures and USP7 inhibitory activity of C12 and B5. (b) Western blot results of cells treated with C12

and B5. (c) Inhibitory activity of C12 and B5 against MGC-803 cells.

also optimized using the ‘QuickPrep’ function, and the solvent and

the same copy of protein were deleted. The optimized result was

used as the ﬁnal model for molecular modelling. We chose the

better-overlapped conformation of C16 with the co-crystal ligand

as the research target. It was essential to keep the quinazolin-

4(3H)-one bound to the 4-hydroxypiperidine moiety in the proper

conformation for USP7 inhibitory activity. The scores and activities

of these compounds are summarized in Table S3. The side chains of

C16 and C19 at C-7 of the quinazolin-4(3H)-one moiety had the

same orientation and solvent exposure contact area; further

investigation of the ligand interaction of C16 and C19 revealed the

important hydrogen bond interaction with residue Met407 (Fig. 7).

Other common important interactions included hydrogen bonds

with residues Phe409, Arg408, Gln297, Val296, Asp295 and Tyr465

and the hydrophobic interaction of the 3-phenylbutanoic acid

moiety was strong, which further affected the arrangement of the

whole molecule in the binding pocket. This was supported by the

binding mode of C13 with the co-crystal ligand (Fig. 8).

2.6. In vivo anti-tumour effect of compound C9

Currently, studies on USP7 inhibitors mainly focus on cell-level

evaluations, and few animal model data are disclosed. Based on the

previous evaluation, we evaluated the anti-tumour effect of C9 in a

zebraﬁsh gastric cancer MGC-803 cell model. First, we tested the

maximum tolerated dose (MTD) to determine the administered

concentration (Table S4). The data indicated that C9 did not induce

the zebraﬁsh death and toxicity phenotype at 25.0 and 50.0 ng per

ﬁsh; however, the death rates reached 30.0% and 36.7% when the

administered doses were 100 ng per ﬁsh and 200 ng per ﬁsh,

respectively. Subsequently, the MTD of C9 in zebraﬁsh was deter-

mined as 50.0 ng per ﬁsh. Then, we divided the zebraﬁsh model

into three groups: a control group (0 ng per ﬁsh), a medium-dose

group (16.7 ng per ﬁsh, 1/3 MTD) and a high-dose group (50.0 ng

per ﬁsh, MTD). The drug was injected directly into tumour tissues.

The results of the zebraﬁsh model are summarized in Table S5 and

Fig. S9. The results indicated that C9 exhibited a remarkable anti-

tumour effect in vivo.

moiety with the binding pocket. Interestingly, no

p-p stacking

interaction was observed, perhaps due to the special conformation

of C19. In particular, the phenyl ring plane of the 3-phenylbutanoic

acid moiety was perpendicular to the phenyl ring plane of Phe409.

This was common to C16 and C19 (Fig. S7), which was the same as

the co-crystal ligand. Additionally, we overlapped C16, C17 and C19

as co-crystal ligands, as shown in Fig. S8. This indicated that the

lipophilic properties remarkably affected the location of the side

chain at C-7 of the quinazolin-4(3H)-one moiety and that both the

hydrogen bond interaction and the lipophilicity of the side chain

affected the USP7 inhibitory activity.

3. Conclusions

The racemic methyl group maintained the stability of the special

conformation of the ligand, which further enhanced the ligand-

receptor interaction. Without the racemic methyl group, the

interaction of the side chain at C-7 of the quinazolin-4(3H)-one

Here we reported several novel quinazolin-4(3H)-one de-

rivatives as potent USP7 inhibitors. We analyzed the co-crystal

structure (PDB ID: 5VSB) and selected the ligand as a lead com-

pound and reference drug. In combination with structure-based

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European Journal of Medicinal Chemistry 216 (2021) 113291

Fig. 4. Effect of C9 on MDM2 protein levels. (a) C9 inhibited USP7 activity and led to the degradation of MDM2. (b) Anti-tumour effects of C9. (c) Structure and USP7 inhibitory

activity of C9.

drug design, we designed a series of derivatives. We then utilized

the Corey-Chaykovsky epoxidation reaction to synthesize the key

intermediate A2 and Cu-catalyzed Buchwald-Hartwig coupling

reaction to synthesize the ﬁnal products. Further anti-tumour

evaluation indicated that C9 and C19 exhibited the best inhibitory

activity against the catalytic domain of USP7, with IC₅₀values of

4.86

of reference drug B5, which was 23.05

conﬁrmed using the Ub-AMC assay, which revealed the IC₅₀for C9

and C19 as 5.048 M and 0.595 M, respectively. These derivatives

mM and 1.537

mM, respectively, compared with the IC₅₀value

mM. This was further

m

also exhibited moderate inhibitory activity against gastric cancer

cells, although MGC-803 cells were more sensitive to these com-

pounds than BGC-823 cells. In particular, B18 (IC₅₀¼ 30.03

MGC-803; IR ¼ 52.03% for BGC-823; IC₅₀¼ 46.3 M for USP7) and

C8 (IC₅₀¼ 32.13 M for MGC-803; IR ¼ 54.01% for BGC-823;

IC₅₀¼ 15.31 M for USP7) exhibited better inhibitory activity

mM for

m

against both USP7 and gastric cancer cells, whereas the reference

drug B5 exhibited no obvious inhibitory activity against gastric

cancer cells, with IR < 50% at 50 mM. Furthermore, C9 restricted the

cell cycle at the G0/G1 and S phases to inhibit gastric cancer cell

proliferation. Western blot using MGC-803 cells indicated that C9

induced the p53-MDM2-USP7 pathway to interfere with cancer

progression. C9 inhibited USP7 activity, leading to a decreased level

of MDM2; this further increased the levels of the MDM2 down-

stream proteins p53 and p21. BLI assay also conﬁrmed the high

Fig. 5. Effect of C9 on the p53-MDM2-USP7 pathway.

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European Journal of Medicinal Chemistry 216 (2021) 113291

Fig. 6. Effect of C15, C16 and C19 on the p53-MDM2-USP7 pathway in BGC-823 cells.

Fig. 7. Ligand interactions of (a) C16 and (b) C19 with the catalytic domain of USP7 (PDB ID: 5VSB).

afﬁnity of C9 for the USP7 catalytic domain. Additionally, docking

studies for C9 and C19 predicted that the C-7 side chain of the

quinazolin-4(3H)-one mother core located at the solvent exposure

part of the USP7 catalytic domain forms an important hydrogen

bond with Met407, which enhances the ligand-receptor interac-

tion. Finally, C9 exhibited remarkable anti-tumour effects in the

zebraﬁsh gastric cancer MGC-803 cell model. These results indi-

cated that quinazolin-4(3H)-one derivatives could serve as prom-

ising leads for the development of potent USP7 inhibitors and

particularly for anti-gastric cancer drugs.

MHz and 100-MHz spectrometers, respectively. TMS was used as

an internal standard in CDCl₃or dimethyl sulfoxide (DMSO)-d₆.

Chemical shifts were reported as

d ppm values relative to TMS.

Proton coupling patterns were described as singlet (s), doublet (d),

doublet of doublets (dd), triplet (t), triplet of doublets (td), quartet

(q), multiplet (m), and broad (br). High-resolution mass spectra of

all derivatives were recorded on a Waters Q-T Micromass spec-

trometer using electrospray ionization. The melting point (m.p.)

was measured on a microscopic melting point apparatus. The pu-

rity of compounds was tested by Shimadzu LC-20A HPLC with VWD

detector. The HPLC conditions were as follows: Shiseido C18 MG

column, 4.6 mm ꢁ 250 mm, 5

mm; detection wavelength: 254 nm;

ﬂow rate: 1.0 ml/min, eluent: MeOH/H₂O ¼ 40/60 (v/v), and the

4. Experimental section

temperature: 23.5 ^ꢀC.

4.1. General information

Reagents and solvents were purchased from commercial sour-

ces and used directly without further puriﬁcation. Thin-layer

chromatography (TLC) plates were prepared by coating glass

plates with silica gel (Qingdao Haiyang Chemical Co., GF254), and

ultraviolet light (254 nm), and phosphomolybdic acid were used to

visualize the TLC plates. Silica gel (Qingdao Haiyang Chemical Co.,

200e300 mesh) was used in the column chromatography proced-

ure. ¹H NMR and ¹³C NMR spectra were recorded on Bruker 400-

4.2. General procedure for the synthesis of A2, B2eB4, B5eB31

4.2.1. tert-Butyl 1-oxa-6-azaspiro[2.5]octane-6-carboxylate (A2)

Trimethylsulfoxonium iodide (3.094 g,14.06 mmol,1.4 eq) was

dissolved in 15 ml of dry DMSO in an ice bath, and then NaH

(0.562 g, 14.06 mmol,1.4 eq, 60% dispersion in mineral oil) was

added in portions. The solution was stirred at 0 ^ꢀC for 30 min, and

then tert-butyl 4-oxopiperidine-1-carboxylate (2.0 g, 10.04 mmol,

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European Journal of Medicinal Chemistry 216 (2021) 113291

Fig. 8. Binding mode of C13 against the catalytic domain of USP7 (PDB ID: 5VSB). (a) Residues around C13 binding. The red structure represents C13, and the green structure

represents the co-crystal ligand. Other structures represent residues of the receptor. (b) The ligand interactions of C13.

1.0 eq) was added. The reaction mixture was stirred at 0 ^ꢀC for

another 4 h. Then 60 ml of water was added to the reaction mixture,

and it was extracted with EA (3 ꢁ 90 ml). The organic phase was

washed with brine, and dried over anhydrous Na₂SO₄. The solvent

was removed under reduced pressure to yield the crude product,

which was further puriﬁed by ﬂash chromatography, eluting with

PE:EA at a ratio of ¼ 10:1 and then 5:1 to yield the pure product A2

(1.285 g, white solid,60% yield). m.p.56.0e56.9 ^ꢀC. ¹H NMR

29.69, 53.73, 57.15, 79.73, 154.77. HRMS (ESI) m/z calcd for

C

₁₁H₁₉NO₃[MþH]^þ, 214.1365, found: 214.1793.

4.2.2. 7-Chloroquinazolin-4(3H)-one (B2)

2-amino-4-chlorobenzoic acid (1.0 g, 5.83 mmol, 1.0 eq) was

suspended in 10 ml of formamide. The mixture was heated at

150 ^ꢀC for 12 h, cooled to room temperature, and ﬁltered under

reduced pressure to yield a crude product, which was washed with

ethanol and dried at room temperature overnight to yield the pure

product B2 (0.744 g, brown solid, 70.7% yield). m.p.202.7e203.0 ^ꢀC.

(400 MHz, Chloroform-d)

J ¼ 13.2, 9.4, 3.7 Hz, 2H), 2.69 (s, 2H), 1.80 (m, J ¼ 13.8, 9.4, 4.5 Hz,

2H), 1.47 (s, 11H). ¹³C NMR (101 MHz, Chloroform-d)

14.11, 28.44,

d

3.72 (m, J ¼ 13.7, 4.9 Hz, 2H), 3.43 (m,

¹H NMR (400 MHz, DMSO‑d₆)

d 12.39 (s, 1H), 8.18e8.09 (m, 2H),

d

8

P. Li, Y. Liu, H. Yang et al.

European Journal of Medicinal Chemistry 216 (2021) 113291

7.73 (d, J ¼ 2.0 Hz, 1H), 7.56 (dd, J ¼ 8.5, 2.1 Hz, 1H). ¹³C NMR

160.12, 169.56. HRMS (ESI) m/z calcd for C₂₃H₂₄ClN₃O₃[MþH]^þ,

(101 MHz, DMSO‑d₆)

d

121.42, 126.33, 126.98, 127.92, 138.87, 146.89,

426.1506, found: 426.1568. LC t_R: 1.971 min, purity 54.74%.

149.83, 160.13. HRMS (ESI) m/z calcd for C₈H₅ClN₂O [MþH]^þ

181.0090, found: 181.0157.

4.2.6. 3-((1-benzoyl-4-hydroxypiperidin-4-yl)methyl)-7-

chloroquinazolin-4(3H)-one (B6)

4.2.3. tert-Butyl 4-((7-chloro-4-oxoquinazolin-3(4H)-yl)methyl)-4-

hydroxypiperidine-1-carboxylate (B3)

Benzoic acid (0.226 g, 1.85 mmol, 1.5 eq) was dissolved in 2 ml of

DMF, and then TEA (0.249 g, 2.46 mmol, 2.0 eq) and HATU (0.935 g,

2.46 mmol, 2.0 eq) were added. The reaction mixture was stirred at

room temperature for 10 min. B4 (0.5 g, 1.23 mmol, 1.0 eq) was

dissolved in 2 ml of DMF and added to the above mixture, followed

by stirring at room temperature for 5 h. Then, 50 ml of water was

added to the mixture, and it was extracted with EA (3 ꢁ 60 ml). The

organic phase was washed with brine and dried over anhydrous

MgSO₄. The solvent was removed under reduced pressure to yield

crude product, which was dissolved in 5 ml of EA, left at room

temperature overnight, ﬁltered to remove the by-product and

concentrated under reduced pressure to yield crude product. The

crude product was further puriﬁed by ﬂash column chromatog-

raphy, eluting with PE:EA at a ratio of ¼ 2:1 and then 1:2 to yield

the pure product B6 (0.415 g, colorless oil, 85% yield). ¹H NMR

B2 (2.0 g, 11.07 mmol, 1.0 eq), A2 (2.597 g, 12.18 mmol, 1.1 eq)

and anhydrous potassium carbonate (10.821 g, 33.21 mmol, 3.0 eq)

in 20 ml of DMF were heated at 80 ^ꢀC for 12 h. Then, 90 ml of water

was added, and it was extracted with EA (3 ꢁ 90 ml). The organic

phase was washed with brine, and dried over anhydrous MgSO₄.

The solvent was removed under reduced pressure to yield crude

product. Next, 10 ml of PE and EA (1:1, v/v) was added to the crude

product, followed by stirring at room temperature for 30 min. This

was ﬁltered under reduced pressure and dried at room temperature

overnight to yield the pure product B3 (3.651 g, white solid, 83.7%

yield). m.p.181.1e182.0 ^ꢀC. ¹H NMR (400 MHz, DMSO‑d₆)

d 8.29 (s,

1H), 8.16 (d, J ¼ 8.6 Hz, 1H), 7.75 (d, J ¼ 2.1 Hz, 1H), 7.58 (dd, J ¼ 8.6,

2.0 Hz,1H), 4.93 (s,1H), 4.00 (s, 2H), 3.66 (d, J ¼ 13.1 Hz, 2H), 3.05 (s,

2H), 1.48 (td, J ¼ 12.4, 10.9, 4.2 Hz, 2H), 1.39 (s, 11H). ¹³C NMR

(400 MHz, DMSO‑d₆)

d

8.31 (s, 1H), 8.16 (d, J ¼ 8.6 Hz, 1H), 7.75 (d,

(101 MHz, DMSO‑d₆)

d

28.05, 34.28, 53.77, 69.13, 78.47, 120.32,

J ¼ 2.1 Hz, 1H), 7.57 (dd, J ¼ 8.5, 2.1 Hz, 1H), 7.47e7.42 (m, 3H), 7.39

(dd, J ¼ 6.7, 3.0 Hz, 2H), 5.05 (s, 1H), 4.21 (s, 1H), 4.04 (s, 2H), 3.27 (s,

126.19, 127.14, 128.42, 138.87, 148.98, 150.39, 153.79, 160.16. HRMS

(ESI) m/z calcd for

394.1509.

C

₁₉H₂₄ClN₃O₄[MþH]^þ394.1455,found:

1H), 3.15 (s, 1H), 1.63 (s, 2H), 1.54 (s, 1H), 1.36 (d, J ¼ 13.6 Hz, 1H). ¹³

C

NMR (101 MHz, DMSO‑d₆) d 168.90, 160.18, 150.38, 148.98, 138.87,

136.30, 129.27, 128.41, 128.34, 127.12, 126.62, 126.19, 120.34, 69.40,

53.85. HRMS (ESI) m/z calcd for C₂₁H₂₀ClN₃O₃[MþH]^þ, m/z:

398.1193, found: 398.1262.

4.2.4. 7-Chloro-3-((4-hydroxypiperidin-4-yl)methyl)quinazolin-

4(3H)-one (B4)

B3 (2.0 g, 5.08 mmol, 1.0 eq) in 6 ml of TFA was stirred at room

temperature for 4 h. Next, 20 ml of water was added to the above

mixture in an ice bath, and this was basiﬁed by the addition of

ammonium hydroxide until a pH of 8.0 was attained. This solution

was stirred at 0 ^ꢀC for 30 min and ﬁltered under reduced pressure.

The precipitate was dried at room temperature overnight to yield

the pure product B4 (1.492 g, white solid, about 100% yield). m.p.

4.2.7. 7-Chloro-3-((4-hydroxy-1-(1H-pyrrole-2-carbonyl)

piperidin-4-yl)methyl)quinazolin-4(3H)-one (B7)

1H-pyrrole-2-carboxylic acid (0.206 g, 1.85 mmol, 1.5 eq) was

dissolved in 2 ml of DMF, and then TEA (0.249 g, 2.46 mmol, 2.0 eq)

and HATU (0.935 g, 2.46 mmol, 2.0 eq) were added. The mixture

was stirred at room temperature for 10 min. B4 (0.5 g, 1.23 mmol,

1.0 eq) was dissolved in 2 ml of DMF and added to the above

mixture, followed by stirring at room temperature for 5 h. Then,

50 ml of water was added to the mixture, and it was extracted with

EA (3 ꢁ 60 ml). The organic phase was washed with brine, and

dried over anhydrous MgSO₄. The solvent was removed under

reduced pressure to yield crude product. This was dissolved in 5 ml

of EA, left at room temperature overnight, ﬁltered to remove the

by-product and concentrated under reduced pressure to yield

crude product, which was further puriﬁed by ﬂash column chro-

matography, eluting with PE:EA at a ratio of ¼ 2:1 and then 1:2 to

yield the pure product B7 (0.214 g, brown solid, 45% yield).

228.5e229.1 ^ꢀC. ¹H NMR (400 MHz, DMSO‑d₆)

d 8.32 (s, 1H), 8.17 (d,

J ¼ 8.6 Hz, 1H), 8.11 (s, 1H), 7.76 (d, J ¼ 2.1 Hz, 1H), 7.59 (dd, J ¼ 8.7,

2.1 Hz, 1H), 5.38 (s, 1H), 4.06 (s, 2H), 3.18 (d, J ¼ 12.5 Hz, 2H),

3.09e2.93 (m, 2H), 1.87e1.71 (m, 2H), 1.60 (d, J ¼ 14.1 Hz, 2H). ¹³

C

NMR (101 MHz, DMSO‑d₆)

d 31.11, 53.51, 67.49, 120.27, 126.25,

127.24, 128.39, 138.99, 148.96, 150.28, 160.21. HRMS (ESI) m/z calcd

for C₁₄H₁₆ClN₃O₂[MþH]^þ294.0931, found: 294.0992.

4.2.5. 7-Chloro-3-((4-hydroxy-1-(3-phenylpropanoyl)piperidin-4-

yl)methyl)quinazolin-4(3H)-one (B5)

3-phenylpropanoic acid (0.278 g, 1.85 mmol, 1.5 eq) was dis-

solved in 2 ml of DMF, and triethylamine (TEA) (0.249 g, 2.46 mmol,

2.0 eq) and HATU (0.935 g, 2.46 mmol, 2.0 eq) were added. The

mixture was then stirred at room temperature for 10 min. B4 (0.5,

1.23 mmol, 1.0 eq) was dissolved in 2 ml of DMF and added to the

above mixture. The reaction mixture was stirred at room temper-

ature for 5 h. Next, 50 ml of water was added, and it was extracted

with EA (3 ꢁ 60 ml). The organic phase was washed with brine, and

dried over anhydrous MgSO₄. The solvent was removed under

reduced pressure to yield crude product. This was puriﬁed by ﬂash

column chromatography, eluting with PE:EA at a ratio of ¼ 2:1 and

then 1:2, to yield the pure product B5 (0.419 g, colorless oil, 80%

m.p.116.8e116.9 ^ꢀC. ¹H NMR (400 MHz, DMSO‑d₆)

d 11.41 (s, 1H),

8.32 (s, 1H), 8.17 (d, J ¼ 8.6 Hz, 1H), 7.76 (d, J ¼ 2.2 Hz, 1H), 7.58 (dd,

J ¼ 8.6, 2.1 Hz, 1H), 6.87 (s, 1H), 6.46 (s, 1H), 6.10 (q, J ¼ 2.8 Hz, 1H),

5.04 (s, 1H), 4.12 (d, J ¼ 13.3 Hz, 2H), 4.04 (s, 2H), 3.30 (s, 2H),

1.66e1.54 (m, 2H), 1.48 (d, J ¼ 13.4 Hz, 2H). ¹³C NMR (101 MHz,

DMSO‑d₆)

d 161.39, 160.18, 150.39, 148.99, 138.89, 128.44, 127.15,

126.21, 124.37, 120.82, 120.34, 111.34, 108.22, 69.50, 53.81, 34.75.

HRMS (ESI) m/z calcd for C₁₉H₁₉ClN₄O₃[MþH]^þ, m/z: 387.1146,

found: 387.1208.

4.2.8. 7-Chloro-3-((4-hydroxy-1-picolinoylpiperidin-4-yl)methyl)

quinazolin-4(3H)-one (B8)

yield). ¹H NMR (400 MHz, DMSO‑d₆)

d 8.30 (s, 1H), 8.16 (d,

J ¼ 8.7 Hz, 1H), 7.74 (s,1H), 7.57 (d, J ¼ 8.7 Hz,1H), 7.25 (d, J ¼ 9.3 Hz,

4H), 7.16 (t, J ¼ 7.4 Hz, 1H), 4.98 (s, 1H), 4.09 (d, J ¼ 13.0 Hz, 1H), 3.99

(s, 2H), 3.63 (d, J ¼ 13.6 Hz, 1H), 3.23 (t, J ¼ 12.2 Hz, 1H), 2.92 (t,

J ¼ 11.6 Hz, 1H), 2.81 (t, J ¼ 7.8 Hz, 2H), 2.71e2.57 (m, 2H), 2.51 (s,

Picolinic acid (0.228 g, 1.85 mmol, 1.5 eq) was dissolved in 2 ml

of DMF, and then TEA (0.249 g, 2.46 mmol, 2.0 eq) and HATU

(0.935 g,2.46 mmol,2.0 eq) were added. The mixture was stirred at

room temperature for 10 min. B4 (0.5 g, 1.23 mmol, 1.0 eq) was

dissolved in 2 ml of DMF and added, followed by stirring at room

temperature for 5 h. Next, 50 ml of water was added to the mixture,

and it was extracted with EA (3 ꢁ 60 ml). The organic phase was

1H), 1.43 (d, J ¼ 15.6 Hz, 4H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 30.87,

33.87, 34.20, 34.89, 36.94, 38.20, 40.80, 53.79, 69.26, 120.32, 125.76,

126.19, 127.10, 128.14, 128.34, 128.39, 138.87, 141.42, 148.97, 150.32,

9

P. Li, Y. Liu, H. Yang et al.

European Journal of Medicinal Chemistry 216 (2021) 113291

washed with brine, and dried over anhydrous MgSO₄. The solvent

was removed under reduced pressure to yield crude product. This

was dissolved in 5 ml of EA, and left at room temperature overnight,

ﬁltered to remove the by-product and concentrated under reduced

pressure to yield crude product, which was further puriﬁed by ﬂash

column chromatography, eluting with PE:EA at a ratio of ¼ 2:1 and

then 1:2 to yield the pure product B8 (0.152 g, brown solid, 31%

1.68e1.56 (m, 2H), 1.44 (s, 2H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

168.89, 160.18, 150.38, 149.63, 148.98, 148.37, 138.87, 128.41,

128.36, 127.13, 126.19, 120.34, 119.57, 111.07, 110.83, 69.45, 55.53,

55.50, 53.86. HRMS (ESI) m/z calcd for C₂₃H₂₄ClN₃O₅[MþH]^þ,

458.1404, found: 458.1477.

4.2.11. 7-Chloro-3-((4-hydroxy-1-(2-phenoxyacetyl)piperidin-4-yl)

methyl)quinazolin-4(3H)-one (B11)

yield). m.p.124.3e124.6 ^ꢀC. ¹H NMR (400 MHz, DMSO‑d₆)

d 8.59 (d,

J ¼ 4.8 Hz, 1H), 8.31 (s, 1H), 8.16 (d, J ¼ 8.6 Hz, 1H), 7.98e7.88 (m,

1H), 7.75 (d, J ¼ 2.2 Hz, 1H), 7.61e7.52 (m, 2H), 7.47 (dd, J ¼ 7.5,

5.1 Hz, 1H), 5.06 (s, 1H), 4.23 (d, J ¼ 13.0 Hz, 1H), 4.09 (d, J ¼ 13.7 Hz,

1H), 4.01 (d, J ¼ 13.7 Hz, 1H), 3.48 (d, J ¼ 13.6 Hz, 1H), 3.32e3.05 (m,

2H), 1.72e1.51 (m, 3H), 1.37 (d, J ¼ 13.6 Hz, 1H). ¹³C NMR (101 MHz,

Phenol (1.0 g, 10.63 mmol, 1.0 eq), ethyl 2-bromoacetate

(2.664 g, 15.95 mmol, 1.5 eq) and potassium carbonate (2.938 g,

21.26 mmol, 2.0 eq) were dissolved in 10 ml of DMF and stirred at

room temperature for 4 h. Then, 60 ml of water was added, and it

was extracted with EA (3 ꢁ 60 ml). The organic phase was washed

with brine, and dried over anhydrous MgSO₄. The solvent was

removed under reduced pressure to yield crude product. This was

dissolved in 10 ml of methanol, and potassium hydroxide (2.385 g,

42.52 mmol, 4.0 eq) and water (5 ml) were added. This was heated

at 80 ^ꢀC for 4 h. The solvent was removed under reduced pressure,

and 20 ml of water was added. The solution was acidiﬁed using 1 M

HCl until the pH reached approximately 3.0 and then extracted

with EA (3 ꢁ 60 ml). The organic phase was washed with brine and

dried over anhydrous MgSO₄.The solvent was removed under

reduced pressure to yield 2-phenoxyacetic acid (1.051 g, colorless

oil, 65% yield), which was used directly in the next step.

DMSO‑d₆)

d 166.59, 160.17, 154.35, 150.37, 148.97, 148.37, 138.89,

137.28, 128.42, 127.15, 126.20, 124.37, 122.77, 120.32, 69.39, 53.83,

42.40, 37.40, 34.90, 34.24. HRMS (ESI) m/z calcd for C₂₀H₁₉ClN₄O₃

[MþH]^þ, m/z: 399.1146, found: 399.1223.

4.2.9. 7-Chloro-3-((4-hydroxy-1-nicotinoylpiperidin-4-yl)methyl)

quinazolin-4(3H)-one (B9)

Nicotinic acid (0.228 g, 1.85 mmol, 1.5 eq) was dissolved in 2 ml

of DMF, and then TEA (0.249 g, 2.46 mmol, 2.0 eq) and HATU

(0.935 g, 2.46 mmol, 2.0 eq) were added, followed by stirring at

room temperature for 10 min. B4 (0.5 g, 1.23 mmol, 1.0 eq) was

dissolved in 2 ml of DMF and added to the above mixture, followed

by stirring at room temperature for 5 h. Then, 50 ml of water was

added to the mixture, and it was extracted with EA (3 ꢁ 60 ml). The

organic phase was washed with brine and dried over anhydrous

MgSO₄. The solvent was removed under reduced pressure to yield

crude product. This was dissolved in 5 ml of EA, and left at room

temperature overnight, ﬁltered to remove the by-product and

concentrated under reduced pressure to yield crude product, which

was further puriﬁed by ﬂash column chromatography, eluting with

PE:EA at a ratio of ¼ 2:1 and then 1:2 to yield the pure product B9

(0.196 g, brown solid, 40% yield). m.p.145.5e145.7 ^ꢀC. ¹H NMR

2-phenoxyacetic acid (0.281 g, 1.85 mmol, 1.5 eq) was dissolved

in 2 ml of DMF, and then TEA (0.249 g, 2.46 mmol, 2.0 eq) and HATU

(0.935 g, 2.46 mmol, 2.0 eq) were added, followed by stirring at

room temperature for 10 min. B4 (0.5 g, 1.23 mmol, 1.0 eq) was

dissolved in 2 ml of DMF and added, followed by stirring at room

temperature for 5 h. Then, 50 ml of water was added to the mixture,

and it was extracted with EA (3 ꢁ 60 ml). The organic phase was

washed with brine, and dried over anhydrous MgSO₄. The solvent

was removed under reduced pressure to yield crude product. This

was dissolved in 5 ml of EA, left at room temperature overnight,

ﬁltered to remove the by-product and concentrated under reduced

pressure to yield crude product, which was further puriﬁed by ﬂash

column chromatography, eluting with PE:EA at a ratio of 2:1 and

then 1:2 s to yield the pure product B11 (0.447 g, colorless oil, 85%

(400 MHz, DMSO‑d₆)

d 8.67e8.59 (m, 2H), 8.30 (s, 1H), 8.16 (d,

J ¼ 8.6 Hz, 1H), 7.84 (d, J ¼ 7.9 Hz, 1H), 7.75 (d, J ¼ 2.1 Hz, 1H), 7.58

(dd, J ¼ 8.6, 2.1 Hz,1H), 7.48 (dd, J ¼ 7.7, 5.0 Hz,1H), 5.06 (s,1H), 4.23

(d, J ¼ 12.8 Hz, 1H), 4.04 (s, 2H), 3.32 (s, 1H), 3.14 (t, J ¼ 12.1 Hz, 1H),

1.66 (d, J ¼ 12.4 Hz, 2H), 1.53 (d, J ¼ 13.6 Hz, 1H), 1.38 (d, J ¼ 13.5 Hz,

yield). ¹H NMR (400 MHz, DMSO‑d₆)

d 8.31 (s, 1H), 8.17 (d,

J ¼ 8.6 Hz, 1H), 7.76 (d, J ¼ 2.1 Hz, 1H), 7.58 (dd, J ¼ 8.7, 2.1 Hz, 1H),

7.27 (t, J ¼ 7.8 Hz, 2H), 6.91 (d, J ¼ 8.4 Hz, 3H), 5.05 (s,1H), 4.86e4.73

(m, 2H), 4.02 (q, J ¼ 13.9 Hz, 3H), 3.64 (d, J ¼ 13.6 Hz, 1H), 3.30 (t,

J ¼ 12.4 Hz, 1H), 2.99 (t, J ¼ 11.7 Hz, 1H), 1.66 (t, J ¼ 11.1 Hz, 1H), 1.46

1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 166.61, 160.18, 150.38, 150.23,

148.98, 147.38, 138.87, 134.52, 132.07, 128.42, 127.13, 126.19, 123.48,

120.35, 69.35, 53.87. HRMS (ESI) m/z calcd for C₂₀H₁₉ClN₄O₃[MþH],

399.1146, found: 399.1218.

(q, J ¼ 10.6, 8.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO‑d₆)

d 165.55,

160.16, 158.03, 150.37, 148.98, 138.90, 129.30, 128.42, 127.17, 126.22,

120.73, 120.33, 114.51, 69.26, 65.87, 53.78, 37.18, 34.80, 34.16. HRMS

(ESI) m/z calcd for C₂₂H₂₂ClN₃O₄[MþNa]^þ, 450.1299, found:

450.1184.

4.2.10. 7-Chloro-3-((1-(3,4-dimethoxybenzoyl)-4-

hydroxypiperidin-4-yl)methyl)quinazolin-4(3H)-one (B10)

3,4-dimethoxybenzoic acid (0.337 g, 1.85 mmol, 1.5 eq) was

dissolved in 2 ml of DMF, and then TEA (0.249 g, 2.46 mmol, 2.0 eq)

and HATU (0.935 g,2.46 mmol,2.0 eq) were added, followed by

stirring at room temperature for 10 min. B4 (0.5 g, 1.23 mmol, 1.0

eq) was dissolved in 2 ml of DMF, and added, followed by stirring at

room temperature for 5 h. Then, 50 ml of water was added to the

mixture, and it was extracted with EA (3 ꢁ 60 ml). The organic

phase was washed with brine and dried over anhydrous MgSO₄.

The solvent was removed under reduced pressure to yield crude

product. It was dissolved in 5 ml of EA, left at room temperature

overnight, ﬁltered to remove the by-product and concentrated

under reduced pressure to yield crude product, which was further

puriﬁed by ﬂash column chromatography, eluting with PE:EA at a

ratio of ¼ 2:1 and then 1:2, to yield the pure product B10 (0.366 g,

white solid, 65% yield). m.p.250.4e250.7 ^ꢀC. ¹H NMR (400 MHz,

4.2.12. 7-Chloro-3-((4-hydroxy-1-(2-nitrobenzoyl)piperidin-4-yl)

methyl)quinazolin-4(3H)-one (B12)

2-nitrobenzoic acid (0.309 g, 1.85 mmol, 1.5 eq) was dissolved in

2 ml of DMF, and then TEA (0.249 g, 2.46 mmol, 2.0 eq) and HATU

(0.935 g, 2.46 mmol, 2.0 eq) were added, followed by stirring at

room temperature for 10 min. B4 (0.5 g, 1.23 mmol, 1.0 eq) was

dissolved in 2 ml of DMF, and added, followed by stirring at room

temperature for 5 h. Next, 50 ml of water was added to the mixture,

and it was extracted with EA (3 ꢁ 60 ml). The organic phase was

washed with brine, and dried over anhydrous MgSO₄. The solvent

was removed under reduced pressure to yield crude product. This

was dissolved in 5 ml of EA, left at room temperature overnight,

ﬁltered to remove the by-product, and concentrated under reduced

pressure to yield crude product, which was further puriﬁed by ﬂash

column chromatography, eluting with PE:EA at a ratio of 2:1 and

1:2 to yield the pure product B12 (0.121 g, white solid, 22.2% yield).

DMSO‑d₆)

d

8.30 (s, 1H), 8.16 (d, J ¼ 8.6 Hz, 1H), 7.75 (d, J ¼ 2.1 Hz,

1H), 7.58 (dd, J ¼ 8.6, 2.1 Hz,1H), 6.97 (d, J ¼ 8.8 Hz, 3H), 5.02 (s, 1H),

4.04 (s, 2H), 3.78 (d, J ¼ 4.6 Hz, 6H), 3.55 (s, 1H), 3.21 (s, 3H),

10

P. Li, Y. Liu, H. Yang et al.

European Journal of Medicinal Chemistry 216 (2021) 113291

m.p.162.1e162.2 ^ꢀC. ¹H NMR (400 MHz, DMSO‑d₆)

d

8.30 (s,1H), 8.17

4.2.15. 7-Bromo-3-((4-hydroxy-1-(3-phenylpropanoyl)piperidin-4-

yl)methyl)quinazolin-4(3H)-one (B15)

(dd, J ¼ 14.2, 8.4 Hz, 2H), 7.84 (t, J ¼ 7.7 Hz, 1H), 7.75 (d, J ¼ 2.0 Hz,

1H), 7.69 (t, J ¼ 7.9 Hz, 1H), 7.58 (dd, J ¼ 8.6, 2.1 Hz, 2H), 5.08 (s, 1H),

4.21 (s, 1H), 4.07 (d, J ¼ 13.6 Hz, 1H), 4.01 (d, J ¼ 13.8 Hz, 1H), 3.21 (s,

2H), 3.20e3.13 (m, 1H), 1.67 (s, 1H), 1.56 (d, J ¼ 13.6 Hz, 2H), 1.34 (d,

The synthetic procedures for B15 were the same as those for C5.

Compound B15, colorless oil, 90% yield. ¹H NMR (400 MHz,

DMSO‑d₆)

d

8.28 (s, 1H), 8.08 (d, J ¼ 8.7 Hz, 1H), 7.90 (s, 1H), 7.70 (d,

J ¼ 13.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO‑d₆)

d

165.06, 160.17,

J ¼ 8.6 Hz, 1H), 7.31e7.13 (m, 5H), 4.96 (s, 1H), 4.09 (d, J ¼ 12.9 Hz,

1H), 3.98 (s, 1H), 3.63 (d, J ¼ 13.6 Hz, 1H), 3.22 (t, J ¼ 12.4 Hz, 1H),

2.97e2.87 (m, 1H), 2.81 (t, J ¼ 7.8 Hz, 2H), 2.69 (d, J ¼ 3.4 Hz, 1H),

2.66e2.57 (m, 2H), 1.41 (q, J ¼ 15.3, 13.6 Hz, 4H). ¹³C NMR (101 MHz,

150.37, 148.98, 138.90, 134.80, 132.68, 130.08, 128.41, 127.90, 127.16,

126.21, 124.67, 120.32, 69.36, 53.80, 36.91, 34.18, 33.77. HRMS (ESI)

m/z calcd for C₂₁H₁₉ClN₄O₅[MþNa]^þ, 465.1044, found: 465.0940.

DMSO‑d₆)

d 169.57, 160.25, 150.27, 149.03, 141.43, 129.89, 129.29,

128.39, 128.35, 128.19, 128.15, 127.82, 125.76, 120.62, 69.26, 53.83,

40.80, 38.20, 36.94, 34.89, 34.21, 33.86, 30.86. HRMS (ESI) m/z calcd

for C₂₃H₂₄BrN₃O₃[MþH]^þ, 470.1001, found: 470.1061.

4.2.13. 7-Chloro-3-((4-hydroxy-1-(3-phenylbutanoyl)piperidin-4-

yl)methyl)quinazolin-4(3H)-one (B13)

3-phenylbutanoic acid (0.304 g,1.85 mmol,1.5 eq) was dissolved

in 2 ml of DMF, and then TEA (0.249 g, 2.46 mmol, 2.0 eq) and HATU

(0.935 g,2.46 mmol,2.0 eq) were added, followed by stirring at