Nonpeptidic quinazolinone derivatives as dual nucleotide-binding oligomerization domain-like receptor 1/2 antagonists for adjuvant cancer chemotherapy

Article abstract of DOI:10.1016/j.ejmech.2020.112723

Nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD1/2) receptors are potential immune checkpoints. In this article, a quinazolinone derivative (36b) as a NOD1/2 dual antagonist was identified that significantly sensitizes B16 tumor-bearing mice to paclitaxel treatment by inhibiting both nuclear factor κB (NF-κB) and mitogen-activated protein kinase inflammatory signaling that mediated by NOD1/2.

Full text of DOI:10.1016/j.ejmech.2020.112723

European Journal of Medicinal Chemistry 207 (2020) 112723
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Nonpeptidic quinazolinone derivatives as dual nucleotide-binding
oligomerization domain-like receptor 1/2 antagonists for adjuvant
cancer chemotherapy
,
,
,
Yao Ma a 1, Jingshu Yang b 1, Xiduan Wei b 1, Yameng Pei b, Jingjia Ye b, Xueyuan Li b,
Guangxu Si b, Jingyuan Tian b, Yi Dong a **, Gang Liu b
,
, *
a Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, 2A Nanwei Rd, Xicheng Dist, Beijing, 100050, PR China
b School of Pharmaceutical Sciences, Tsinghua University, Haidian Dist, Beijing, 100084, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Nucleotide-binding oligomerization domain-containing protein 1 and 2 (NOD1/2) receptors are potential
immune checkpoints. In this article, a quinazolinone derivative (36b) as a NOD1/2 dual antagonist was
identified that significantly sensitizes B16 tumor-bearing mice to paclitaxel treatment by inhibiting both
Received 5 May 2020
Received in revised form
31 July 2020
Accepted 2 August 2020
Available online 14 August 2020
nuclear factor
NOD1/2.
k
B (NF-
k
B) and mitogen-activated protein kinase inflammatory signaling that mediated by
© 2020 Elsevier Masson SAS. All rights reserved.
Keywords:
Quinazolinone
Dual NOD1/2 antagonists
Retroamide
B16 tumor
1. Introduction
as a ‘gut immune checkpoint’ based on the evidence that NOD2
curtails cyclophosphamide (CTX)-induced cancer immuno-
surveillance by limiting the relocation of microbes [10]. Ferri’s
group demonstrated the NOD1 receptor as a putative therapeutic
target in inflammation-mediated colon cancer metastasis. The
experimental results revealed that NOD1 activation increased
colorectal cancer cell adhesion, migration and metastasis via the
p38 mitogen activated protein kinase pathway [11].
In 2011, we first reported a conjugate of paclitaxel (MTC-220, 1)
and a muramyl dipeptide (MDP) derivative that was superior to PTX
alone for treatment of Lewis lung carcinoma (LLC) in mice. MDP is
the natural ligand for NOD2, however, we found that the prototype
for MTC-220 (compound 2) is responsible for remodeling the tumor
microenvironment (TME) by inhibiting NOD2 signaling and
limiting PTX-induced cancer immunosurveillance in mice [12,13].
In addition, conjugates (3 and 4) of docetaxel (DTX) and MDP de-
rivatives were designed as another chemical class of NOD1 antag-
onists for breast cancer treatment [14]. Effective antagonists for
cancer adjuvant treatment should therefore antagonize both NOD1
and NOD2 signaling. Pharmacologically targeting both the NOD1
and NOD2 signaling pathways is expected to have a broad thera-
peutic utility for cancer treatment. Small molecules quinazolinones
(5) and 2-aminobenzimidazoles (6) were discovered and identified
Currently, immunotherapy primarily aims to artificially stimu-
late the immune system to improve its natural ability to stop or
eliminate cancer [1]. Therefore, the identification and evaluation of
novel immune targets has become an important goal of research.
Because the cytosolic nucleotide-binding oligomerization domain-
containing protein 1 and 2 (NOD1 and NOD2) receptors are
important components of the innate immune system, they have
been pharmacologically targeted to enhance the immune response
against cancer cells [2e7]. Many NOD1/2 agonists have been
discovered: Mifamurtide, for example, has already been approved
for treating high-grade, nonmetastatic, resectable osteosarcoma
after surgical excision in children to young adults [8]. However, a
shift has occurred in recent with the emerging knowledge that NOD
antagonists facilitate the chemotherapy of some cancers [9]. For
instance, Zitvogel and coworkers have identified the NOD2 receptor
* Corresponding author.
** Corresponding author.
E-mail addresses: dongyi@imm.ac.cn (Y. Dong), gangliu27@tsinghua.edu.cn
(G. Liu).
1
These authors contributed equally.
https://doi.org/10.1016/j.ejmech.2020.112723
0223-5234/© 2020 Elsevier Masson SAS. All rights reserved.

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Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
as NOD1 antagonists in 2011 and 2014, respectively [15e17]. The
NOD2 selective antagonist (7) is an imidazole derivative that was
disclosed via high-throughput screening [18]. Jakopin’s group re-
ported in 2016 a dual NOD1/2 antagonist (Fig. 1, 8) based on an
indole scaffold [19,20]. Compound 9 was the first dual NOD1/2
antagonist based on a 1,4-benzodiazepine-2,5-dione derivative
which sensitized chemotherapy with PTX to LLC in animals [21]. In
this article, we perform the synthesis and evaluation of quinazoli-
none derivatives aiming to identify novel NOD1/2 dual antagonists.
We have demonstrated compound 36b as a novel NOD1/2 dual
antagonist that can significantly sensitize B16 tumor-bearing mice
to PTX treatment.
2.2. Chemistry
The synthetic route to the quinazolinones in Tables 1e3 is
illustrated in Scheme 1. 3-Chlorobenzoic acid (16) underwent a
nitration by treating with KNO3 and concentrated H2SO4 to give
intermediate 17, which further reacted with phenol derivatives to
afford the intermediates (18a-18e), followed by amidation to pre-
pare the corresponding amides (19a-19g). The nitro group of 19
was then reduced in the presence of Fe powder and formic acid, and
the cyclization intermediates (20a-20g) were gained in one-pot.
Removal of the formyl group by treating with a concentrated HCl
gave the intermediates 21a-21g, which underwent amidation to
give the 14a-14b and 22a-22t. Intermediates of 23a and 23b were
attained after hydrolysis of 14a and 22n, and subsequent amidation
of 23a and 23b gave the desired 24a-24e.
The synthesis of the retroamide quinazolinone analogues is
illustrated in Scheme 2. Commercially available dimethyl 2-
aminoterephthalate (25) underwent an iodination to give the in-
termediate 26 that further reacted with phenylboronic acid de-
rivatives via Suzuki coupling to afford the biphenyl intermediates
27a-27c. Subsequent hydrolysis yielded the corresponding acids
28a-28c. Treatment with formamide resulted in the quinazolinone
intermediates 29a-29c, which were subsequently transformed into
the desired compounds 30a-30j through amidation with a variety
of amine analogues. Compounds 28a-28c reacted with triphosgene
to afford cyclic intermediates 31a-31b that underwent amidation
by treating with methyl glycinate hydrochloride to give the in-
termediates 32a-32b. Cyclization and amidation provided the
compounds 34a-34f, and 34f was hydrolyzed to afford the acid 35.
Subsequent amidation gave the final analogues 36a-36b.
2. Results and discussion
2.1. The discovery of quinazolinone analogues 14 via screening
Initially, we performed a screening of a small quinazolinone li-
brary (approximately 50 compounds) that was previously estab-
lished by our group aiming to identify novel NOD1/2 antagonists
[22]. The library included fused-quinazolinone compounds (10, 11),
spiro-quinazolinone compounds (12) and other structurally diverse
quinazolinones with different substitutions at the C2 position
(13e15) (Fig. 2). All the compounds were tested at a concentration
of 5
mM. The results revealed that compounds 10e13 and 15
exhibited relatively weak antagonistic percentage of NOD1/2 by
either C12-iE-DAP or MDP stimulation with less than 50% inhibi-
tion. However, quinazolinones bearing free substitution at the C2
position (14a,14b) displayed interesting antagonistic activities with
their IC50 (NOD1/2) of 1.15/0.99
m
M and IC50 (NOD1/2) of ND/
2.3. Structure-activity relationship (SAR) and structure-metabolism
relationship (SMR) studies
0.8 M), respectively, to both NOD1/2 by C12-iE-DAP or MDP
m
stimulated HEK-Blue hNOD1/2 cells. Subsequently, the compounds
of 3,6,7-substituents (R3, R5, R4) of 14 are considered for further
structure-activity relationship (SAR) and structure-metabolism
relationship (SMR) investigation (Fig. 2).
In R4, removal of 4-CF3 (22a) of 4-CF3-benzyloxy ether or
replacement of 4-CF3 with 4-CN (22b), 4-F (22c) or 4-Cl (22d) of 14a
led to a decrease in activity, which indicates that 4-CF3 is necessary
Fig. 1. Representative reported NOD1/2 antagonists.

Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
3
Fig. 2. Identification of hit compounds 14a and 14b via screening of quinazolinone derivatives.
for NOD 1/2 signaling antagonistic activity (Table 1). Acceptable
NOD1 inhibitory percentage (70.88%) was observed by 22e in which
the 4-CF3-benzyloxy ether (22a) was replaced by 4-CF3-phenol
ether, however, still reduced the ability that inhibit C12-iE-DAP
simulation of NOD1 signaling with the replacements of 4-CF3 by 4-
CN (22f) or 4-F (22g) too. It is noteworthy that a complete loss of
antagonistic ability was observed for NOD1 signaling when R4 was
directly replaced by a 4-CN-phenyl (22h) or 4-CF3-phenyl (22i)
without an aliphatic linker.
14a are potential metabolic sites, the metabolic stability of
analogue 22o was tested in mouse liver microsomes stability
assessment. Unfortunately, it gained a short T1/2 of 9.6 min.
The OMe ester of R6 was then amidated by cyclohexylamines
(Table 2). All collected analogues including 24a-24e maintained
high NOD1/2 inhibitory activity. Compound 24e with IC50 values of
0.54 mM (NOD1) and 0.50 mM (NOD2) was chosen to test metabolic
stability in mouse liver microsomes. Unfortunately, a short T1/2
(12.9 min) was observed.
In R5, without 3-N,N-dimethylamino group, it resulted in
significantly decreased their antagonistic activity, i.e. 22j, 22k. 2-
N,N-dimethylamino (22l) or 4-N,N-dimethylamino (22m) substi-
tution showed similar or less NOD1 antagonistic activities,
respectively, in comparison with 14a. It was interesting that
replacement of N,N-dimethylamino with circinal morpholinyl
group (22n, 22o) retained their high inhibitory percentage of NOD1
signaling. Since the benzylmethylene and N,N-dimethylamino in
Evaluation of 14b analogues (Table 3) showed that compounds
22p-22t without N3-substitution had relatively weak activity
against NOD1/2, and their metabolic stability were not improved
either (i.e. 22t, T1/2 ¼ 10.1 min in vitro, IC50 ¼ 2.48
mM for NOD1).
A series of 6-biphenyl-7-retroamide analogues of 14a were
designed and synthesized (Table 4) [23,24]. In R5, we placed
halogen atoms including F, Cl, and CF3-substitutions to replace N,N-
dimethylamino group. Compound 30d bearing the 4-CF3-phenol

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Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
Table 1
14a0s derivatives at R4 and R5 a
.
b
c
Compd.
R4
R5
NOD1/2 inhibition %
IC50 (NOD1/2) mM (T1/2) min
14a
3-N,N-dimethylamino
85.44 1.85/76.86 1.62
1.15 0.12/0.99 0.06
22a
22b
22c
3-N,N-dimethylamino
3-N,N-dimethylamino
3-N,N-dimethylamino
47.61 3.94/35.00 3.14
55.69 0.14/58.66 1.29
33.38 7.88/42.47 18.76
ND
ND
ND
22d
22e
3-N,N-dimethylamino
3-N,N-dimethylamino
69.03 1.93/ND
70.88 1.70/ND
4.31 0.36/ND
2.65 0.14/ND
22f
3-N,N-dimethylamino
3-N,N-dimethylamino
48.04 1.85/ND
33.46 1.50/ND
ND
ND
22g
22h
22i
3-N,N-dimethylamino
3-N,N-dimethylamino
13.06 6.44/ND
31.70 3.94/ND
ND
ND
22j
H
H
50.21 0.85/ND
28.20 6.71/ND
ND
ND
22k
22l
2-N,N-dimethylamino
4-N,N-dimethylamino
87.86 1.25/ND
68.37 0.82/ND
1.65 0.11/ND
3.87 1.3/ND
22m
22n
22o
3-morpholinyl
3-morpholinyl
88.92 0.33/ND
91.17 0.28/ND
0.65 0.12/0.55 0.05
1.38 0.11/ND (9.6)
a
All compounds were tested for C12-iE-DAP (50 ng/mL)-stimulated HEK-Blue hNOD1 cells and MDP (100 ng/mL)-stimulated HEK-Blue hNOD2 cells. ND: not determined.
b
c
The compounds were tested at 5.0
In mouse liver microsomes.
mM.

Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
5
Table 2
14a0s derivatives at R5 and R6 a
.
b
c
Compd.
R5
R6
NOD1/2 inhibition %
IC50 (NOD1/2) mM (T1/2) min
14a
OMe
85.44 1.85/76.86 1.62
1.15 0.12/0.99 0.06
0.26 0.14/1.75 0.20
0.21 0.12/1.11 0.07
0.27 0.24/1.18 0.17
0.14 0.17/0.66 0.15
24a
24b
24c
24d
24e
87.48 1.15/92.36 0.45
89.14 1.18/94.35 0.6
82.01 1.6/87 0.69
90.38 1.66/94.15 0.75
88.25 0.55/ND
0.54 0.04/0.50 0.05 (12.9)
a
All compounds were tested for C12-iE-DAP (50 ng/mL)-stimulated HEK-Blue hNOD1 cells and MDP (100 ng/mL)-stimulated HEK-Blue hNOD2 cells. ND: not determined.
b
c
The compounds were tested at 5.0
In mouse liver microsomes.
mM.
ether group exhibited high NOD1 antagonistic activity, while sub-
stitutions with 4-chlorophenyl (30b) or phenyl (30a and 30c) led to
substantially decreased NOD1 inhibition. Interestingly, 3-
chlorophenyl substitution in R5 (30a-30d) showed higher NOD1
antagonistic activity compared with 3-fluorophenyl (30e-30g) or 3-
CF3-phenyl substitution (30h-30j). In R3, replacement of H with
glycine (34a-34f) showed similar NOD1 antagonistic activity. It was
worth noting that incorporation of a free carboxylic acid group at
N3 position (35) resulted in a complete loss of NOD1/2 antagonistic
activity possibly due to its negative charge which prevented the
compound from interaction with NOD1/2 receptors [20]. It was
again that N3 piperidinyl amidation (36a) resulted in lowest IC50
exhibit a comparable contribution, however, 3-halo aryl group is
favor due to its metabolic stability in mouse liver microsomes. In
the C7 position, the 4-CF3-phenyl substituent and the alkoxy linker
significantly contribute to the antagonistic activity. The retroamide
fragment maintains antagonistic activity and slightly improves the
metabolic stability. All results are clearly reflected in 36b that is
continuously investigated in next.
2.4. Biological profiling
The NOD1 and NOD2 inhibition effects of 36b were dose-
dependent, following
a nonlinear semi logarithmic model
values of 0.80 mM (NOD1) and 0.80 mM (NOD2), respectively, in this
(Fig. 4A). The sulforhodamine B (SRB) assay and interference test of
SEAP (secreted embryonic alkaline phosphatase) were simulta-
neously performed to exclude cytotoxicity (Fig. 4B) and off targeted
SEAP binding activity of 36b (Fig. S1). Compound 36b showed the
promising inhibitory activity of both NOD1-and NOD2-induced NF-
study. Compound 36a showed a slightly improvement of metabolic
stability in vitro with T1/2 of 15.6 min, however, difluorination of the
piperidyl group (36b) resulted in an acceptable T1/2 of 67.6 min with
dual NOD1/2 antagonistic activities for IC50 values of 1.13
mM
(NOD1) and 0.77 M (NOD2), respectively. Further investigations
m
k
B activation in HEK293 cells (IC50 ¼ 1.13/0.77
mM, respectively)
involved with pharmacokinetic studies in vivo will be developed as
the reference for compound optimization.
with no observed cytotoxicity at the tested concentrations.
Activation of NOD1 and NOD2 drives innate inflammatory re-
sponses that are primarily dependent on the activation of RIP2. The
A summary of SAR and SMR are shown in Fig. 3. It is firstly
appeared that a free C2 position of the quinazolinone scaffold is
necessary, for instance, any C2 substitution of the quinazolinone
analogues, including alkylation, arylation, spiro- or fused quina-
zolinones, precludes antagonistic NOD1/2 signaling activity. Free
N3-position decreased the activity in which an addition amidation
is useful, in particular a difluorinated cyclohexylamine is optimal
for its abilities of both acceptable IC50 values to NOD1/2 and
metabolic stability in mouse microsomes. For the C6 position,
phenoxy and phenyl substitutions show similar activities, and 3-
N,N-dimethylamino, 3-morpholinyl and 3-halo aryl substituents all
phosphorylation of RIP2 stimulates the NF-
pathways and finally increases the release of cytokines in the
macrophage, such as IL6, TNF- and IL-8 [7]. To determine whether
36b inhibited one or both of these pathways in a NOD1-or NOD2-
dependent manner, the effect on total IkB and phosphorylated
IKK , p65, p38, JNK and ERK levels were assessed in THP-1 cells
kB and MAPK signaling
a
a
a/b
with C12-iE-DAP or MDP stimulation, respectively (Fig. 5A and B).
C12-iE-DAP at 500 ng/mL could induce NOD1 activation and
resulted in increases of phospho-RIP2, phospho-p38, and phospho-
JNK expression. A decrease in the level of IkBa and a barely

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Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
Table 3
14b derivatives of combined R4, R5 substituents a
.
b
c
Compd.
R4
R5
NOD1/2 inhibition %
IC50 (NOD1/2)
mM (T1/2) min
14b
50.40 0.54/53.96 1.74
ND/0.80 0.06
22p
59.15 1.41/58.52 0.52
5.64 2.33/4.77 0.57
22q
22r
22s
30.38 19.43/19.33 4.78
19.41 5.64/13.11 2.57
37.63 3.98/ND
9.33/ND
ND
ND
22t
65.37 1.36/ND
2.48 0.39/ND (10.1)
a
All compounds were tested for C12-iE-DAP (50 ng/mL)-stimulated HEK-Blue hNOD1 cells and MDP (100 ng/mL)-stimulated HEK-Blue hNOD2 cells. ND: not determined.
b
c
The compounds were tested at 5.0
In mouse liver microsomes.
mM.
discernible increase in phospho-ERK, consistent with our previous
reports, were also observed [12,21]. Pretreatment of cells with
quinazolinone focused library were found to show potential NOD1/
2 antagonistic activities. Structural and metabolic site modifica-
tions led to the discovery of a dual NOD1/2 antagonist 36b with its
ability in significant improvement in terms of therapeutic efficacy
in B16-bearing mice compared with PTX treatment alone. These
results enrich the number of lead compounds in antagonizing
NOD1/2 signaling for adjuvant cancer chemotherapy.
36b at 1.0
mM and 10
m
M prevented the increases in p-RIP2, p-IKK
a/
b
, p-p65, p-p38, and p-JNK and the degradation of IkBa
. Because
C12-iE-DAP gave only weak induction of p-ERK, the reduction of
these levels by 36b was difficult to discern. Similarly, 36b treatment
markedly inhibited the MDP-induced phosphorylation of RIP2,
IKKa/b, p65, p38 and JNK as well as the degradation of IkBa in a
dose-dependent manner in THP-1 cells. Compound 36b was also
able to block NOD1-and NOD2-mediated inflammatory cytokine
secretion in THP-1 cells. As shown in Fig. 5C and D, pretreatment of
THP-1 cells with 36b consistently and dose-dependently reduced
4. Experimental
4.1. General
the transcription of IL-6, TNF-a and IL-8 stimulated by C12-iE-DAP
THF, MeOH, DCM and other commercial reagents were pur-
chased from domestic corporations and used without further pu-
rification. Silica gel for column chromatography and analytical thin
layer chromatography (TLC) plates were phased from Qingdao
Haiyang Chemical and Special Silica Gel Co, Ltd. The automatic LC-
MS analysis was also performed on a Waters SQ Advantage mass
spectrometer equipped with an UPLC system and an eluent splitter
(5% eluent was split into the MS system). High-resolution LC-MS
was carried out by Agilent LC/MSD TOF using a column of Agilent
or MDP, respectively. These data suggest that 36b inhibits both the
NOD1 and NOD2 signaling pathways in human immune cells.
To determine whether 36b affected similar sensitization of PTX
in vivo, we tested the efficacy of PTX in combination with 36b in B16
tumor-bearing mice that mimic human melanoma. As shown in
Fig. 6, the combination therapy significantly reduced tumor growth
compared with PTX treatment alone (p < 0.001), and the tumor
weight inhibitory rate increased from 64.07% to 85.46%. Compared
with the vehicle group, the combination of 36b with PTX showed
significant improvement in terms of therapeutic efficacy in B16-
bearing mice.
ZORBAX SB-C18 (rapid resolution, 3.5
m
m, 2.1 ꢀ 30 mm) at a flow of
0.40 mL/min. The solvent was MeOH/water (75:25 (v/v)), contain-
ing 5 mmol/L ammonium formate. The ion source is electrospray
ionization (ESI).
3. Conclusions
Proton nuclear magnetic resonance (1H NMR) and carbon nu-
clear magnetic resonance (13C NMR) spectroscopy were performed
on Bruker Advance 400 NMR spectrometer. Unless otherwise noted
In summary, two hit derivatives discovered by the screening of a

Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
7
Scheme 1. Synthesis of quinazolinone derivatives. Reagents and conditions: (i) KNO3, H2SO4, 80e130 ꢁC, 6 h; (ii) phenol derivatives, NaHCO3, H2O, 110 ꢁC, 2 h; (iii) methyl glycinate
hydrochloride, or NH3$H2O, N,N0-diisopropylcarbodiimide (DIC), THF, rt; (iv) Fe, HCOOH, 100 ꢁC, 5 h; (v) EtOH, concentrated HCl; (vi) acid chloride, Et3N, THF; (vii) LiOH, THF/H2O,
0.5 h; (viii) HOSu, DIC, amine derivatives THF; rt.
below, all compounds were >95% pure by analytical HPLC.
filtration and recrystallized with a mixed solution of EtOH and H2O
(EtOH/H2O ¼ 1:5) to give the intermediate 17 as yellow powder
(44% yield). 1H NMR (400 MHz, DMSO‑d6)
d 14.61 (s, 1H), 8.86 (s,
4.1.1. General procedure for the synthesis of compounds 14a, 14b,
20a-20t, 24a-24e (Scheme 1)
1H), 8.30 (s, 1H).
4.1.1.1. Synthesis of intermediate 17. To the solution of 3-
chlorobenzoic acid 16 (20.0 g, 128.2 mmol) in sulfuric acid
(240 mL), KNO3 (33.0 g, 2.6 equiv.) was added by portions at room
temperature. After KNO3 was completely dissolved, the reaction
mixture was heated to 80 ꢁC and stirred 30 min s. After continu-
ously stirred for at 2 h at 110 ꢁC and at 130 ꢁC respectively, the
starting material was completely consumed. Then the reaction
mixture was cooled to room temperature and poured into ice
(660 g) slowly to afford the yellow solid which was collected by
4.1.1.2. Synthesis of intermediates 18a-18e. To a solution of 17 (4.9 g,
19. 8 mmol) in H2O (36 mL), NaHCO3 (3. 6 g, 42.3 mmol) and phenol
derivatives (1.1 equiv.) were added. The reaction mixture was
heated to 110 ꢁC and reflux for 2 h, then cooled to room tempera-
ture and treated with an aqueous solution of 6 N HCl at 0 ꢁC to
afford the crude intermediates (18a-18e) which were collected by
filtration in 60%e70% yield. These compounds were dried and used
for next step without further purification.

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Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
Scheme 2. Preparation of the retroamide quinazolinone analogues. Reagents and conditions: (i) N-iodosuccinimide, HOAc, rt, 24 h; (ii) phenylboronic acid derivatives, Pd(PPh3)4,
Na2CO3, 80 ꢁC; (iii) LiOH, THF/H2O, 0.5 h; (iv) HCONH2, 160 ꢁC, 5 h; (v) triphosgene, THF, reflux; (vi) methyl glycinate hydrochloride, Et3N, H2O, rt; (vii) HCOOH, 110 ꢁC, 5 h; (viii)
amine derivatives, HATU; (ix) HOSu, DIC, amine derivatives THF; rt.
4.1.1.3. Synthesis of intermediates 19a-19g. To a solution of in-
termediates 18a-18e (13.1 mmol) in THF (120 mL), amino acid hy-
drochloride (13.8 mmol, 1.1 equiv.) or aqueous ammonia (10 mL)
and DIC (3.1 mL, 19.7 mmol) were added. The reaction mixture was
stirred at room temperature for 4 h and filtered. The filtrate was
concentrated and the reaction residue was dissolved in DCM and
washed by water. The aqueous layer was extracted by DCM, the
organic layer was combined and washed with brine and dried over
anhydrous MgSO4. DCM was then removed under reduced pressure
and the residue was purified with flash column chromatography on
silica gel to give the intermediates 19a-19g in the yield of 70%e85%.
4.1.1.5. Synthesis of intermediates 21a-21g. To a solution of 20a-20g
(1 mmol) in EtOH (5 mL), conc. HCl (0.5 mL) was added. The re-
action mixture was stirred at room temperature for 12 h. Then
diethyl ether (50 mL) was added to give the white solid 21a-21g in
the yield of 70%e78%.
4.1.1.6. Synthesis of 14a, 14b, 22a-22t. To a solution of 21a-21g
(0.1 mmol) and triethylamine (69.6 mL, 0.5 mmol) in THF (4 mL), a
mixture of acyl chlorides (0.2 mmol, 2 equiv.) in THF (1 mL) was
added. The reaction mixture was stirred at room temperature for
0.5 h and concentrated under reduced pressure. The residue was
purified with flash column chromatography on silica gel to give the
compounds 14a, 14b, and 22a-22t in the yield of 50%e88%.
4.1.1.6.1. Methyl 2-(6-(3-(dimethylamino)phenoxy)-4-oxo-7-(2-
((4-(trifluoromethyl)benzyl) oxy)acetamido)quinazolin-3(4H)-yl)ac-
etate (14a). Yellow powder (78% yield), mp: 165e166 ꢁC. 1H NMR
4.1.1.4. Synthesis of intermediates 20a-20g. To a solution of 19a-19g
(1.0 mmol) in acetic acid (10 mL), iron powder (20.0 mmol, 20
equiv.) was added. The reaction mixture was stirred at 90 ꢁC for
1.5he2.5 h, and cooled to room temperature. 20 mL of DCM was
added, and the mixture was filtered. The filtrate was concentrated,
and the residue was purified with flash column chromatography on
silica gel to give the intermediates 20a-20g in the yield of 30%e40%.
(400 MHz, DMSO‑d6) d 9.67 (s, 1H), 8.66 (s, 1H), 8.30 (s, 1H), 7.57 (s,
4H), 7.36 (s,1H), 7.27e7.23 (m,1H), 6.64 (dd, J ¼ 8.4,1.9 Hz,1H), 6.53
(s, 1H), 6.38 (dd, J ¼ 7.9, 1.5 Hz, 1H), 4.78e4.76 (m, 4H), 4.33 (s, 2H),
3.68 (s, 3H), 2.89 (s, 6H). 13C NMR (100 MHz, DMSO‑d6)
d 168.5,

Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
9
Table 4
a
6-Biphenyl-7-retroamide analogues
.
b
c
Compd.
R5
Cl
R4
R3
H
NOD1/2 inhibition %
64.36 1.11/ND
IC50 (NOD1/2)
mM (T1/2) min
30a
1.34 0.33/ND (22.9 min)
30b
Cl
H
41.48 4.17/ND
ND
30c
30d
30e
Cl
Cl
F
H
H
H
6.60 6.10/ND
89.96 0.23/ND
56.14 1.45/ND
ND
0.84 0.08/0.84 0.09
4.11 0.62/ND
30f
F
H
43.51 4.18/ND
ND
30g
30h
30i
F
H
H
H
ꢂ1.81 8.27/ND
72.44 1.86/ND
34.95 2.41/ND
ND
CF3
CF3
4.39 0.45/ND
ND
30j
CF3
H
4.06 3.59/ND
ND
34a
34b
F
F
64.04 1.84/ND
65.31 1.53/ND
3.46 0.27/ND
3.24 0.30/ND
34c
34d
34e
F
ꢂ0.98 8.22/ND
82.36 0.6/ND
58.17 1.89/ND
ND
Cl
Cl
1.12 0.20/ND
1.64 0.44/ND
(continued on next page)

10
Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
Table 4 (continued )
b
c
Compd.
R5
Cl
R4
R3
NOD1/2 inhibition %
89.62 0.53/ND
IC50 (NOD1/2)
mM (T1/2) min
34f
0.89 0.09/1.00 0.07
35
Cl
Cl
Cl
21.97 6.91/ND
93.25 0.40/ND
91.37 0.65/ND
ND
36a
36b
0.80 0.08/0.80 0.10 (15.6)
1.13 0.13/0.77 0.09 (67.7)
a
All compounds were tested for C12-iE-DAP (50 ng/mL)-Stimulated HEK-Blue hNOD1 Cells and MDP (100 ng/mL)-Stimulated HEK-Blue hNOD2 Cells. ND: not determined.
b
c
The compounds were tested at 5.0
In mouse liver microsomes.
mM.
Fig. 3. SAR and SMR’s summary of quinazolinone derivatives.
Fig. 4. (A) Compound 36b inhibits C12-iE-DAP-induced or MDP-induced NF-kB activation. HEK-Blue hNOD1 and HEK-Blue hNOD2 cells were preincubated with different con-
centrations of 36b for 3 h and then stimulated with C12-iE-DAP (50 ng/mL) and MDP (100 ng/mL) for an additional 20 h. SEAP was quantified as described in the Experimental
Section. Data are presented as the mean standard deviation (SD) (n ¼ 3). (B) Compound 36b had no or little effect on cell growth. Following SEAP quantification, cells were fixed
with 80% TCA at 4 ꢁC for 1 h and stained with SRB in 1% acetic acid for 10 min. Then, the plates were washed five times and air-dried at room temperature (r.t.). Bound stain was
subsequently solubilized with 10 mM Tris base (150
mL), and the absorbance was measured with a spectrophotometer at 515 nm. The inhibition rate of cell growth was calculated by
the following formula: [(CeT)/C] ꢀ 100 (C, OD of control group; T, OD of compound-treated group). Data are shown as the mean SD (n ¼ 3).
168.4, 159.2, 155.8, 152.2, 147.2, 146.6, 144.3, 142.3, 134.2, 130.4,
128.3, 128.0, 127.8, 125.1 (q, J ¼ 3.7 Hz), 116.9, 116.7, 110.6, 109.1,
106.6, 103.6, 71.6, 69.7, 52.4, 47.1, 39.9. HRMS (ESI): m/z (M þ Hþ)
calcd for C29H28F3N4O6, 585.1956, found: 585.1950.

Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
11
Fig. 5. Compound 36b suppresses inflammation via NOD1 and NOD2 activation. Serum-starved THP1 cells were pretreated with or without 36b (1.0
mM or 10
mM) for 1 h prior to
(A) C12-iE-DAP (500 ng/mL) or (B) MDP (20
by Western blotting. -Actin was used as a loading control. (C, D) THP-1 cells were treated with or without 36b (1.0
(500 ng/mL) or (D) MDP (20 g/mL) for 90 min. The mRNA levels of IL-6, TNF- and IL-8 were determined by qRT-PCR. Data are presented as the mean standard deviation (SD)
(n ¼ 6): (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 vs C12-iE-DAP- or MDP-treated group; (#) p < 0.05, (##) p < 0.01, (###) p < 0.001 vs the vehicle group.
mg/mL) treatment for 30 min. The levels of I
k
B
a
and total and phosphorylated RIP2, IKK
a/b, p65, JNK, p38, and ERK1/2 were determined
b
m
M or 10 M) for 1 h and then stimulated with (C) C12-iE-DAP
m
m
a

12
Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
Fig. 6. Compound 36b improves the antitumor efficacy of PTX in B16 tumor-bearing model. (A) Schematic timeline and treatment for 36b and PTX in B16 tumor-bearing model. (B)
Compound 36b has no effect on the body weight of mice. (C) Compound 36b improves the efficacy of PTX in inhibiting B16 tumor growth. 11 days after tumor implantation, tumor
volumes were measured and presented as the mean SEM (n ꢃ 6). (D) Mice were sacrificed and tumors were removed and weighed. (E) Tumor weight inhibition rate was
calculated by the following formula: [(C ꢂ T)/C] ꢀ 100 (C, tumor weight of vehicle group; T, tumor weight of treated group). (F) Image of the tumors. Data are shown as the
mean SEM: (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 vs the PTX group; (△) p < 0.05, (△△) p < 0.01, (△△△) p < 0.001 vs 36b group; (#) p < 0.05, (##) p < 0.01, (###) p < 0.001
vs the vehicle group.
4.1.1.6.2. N-(6-(3-(dimethylamino)phenoxy)-4-oxo-3,4-
dihydroquinazolin-7-yl)-2-((4-(trifluoromethyl)benzyl)oxy)acet-
amide (14b). White powder (72% yield), mp: 191e192 ꢁC. 1H NMR
1H), 7.27 (dd, J ¼ 10.1, 6.2 Hz, 4H), 6.65 (dd, J ¼ 8.3, 1.9 Hz, 1H), 6.54
(s, 1H), 6.41 (dd, J ¼ 7.9, 1.6 Hz, 1H), 4.78 (s, 2H), 4.65 (s, 2H), 4.27 (s,
2H), 3.68 (s, 3H), 2.90 (s, 6H). 13C NMR (100 MHz, DMSO‑d6)
d 168.7,
(400 MHz, DMSO‑d6)
d
12.20 (br s,1H), 9.62 (s,1H), 8.61 (s,1H), 8.03
168.4,159.2,155.7,152.2,147.2,146.7,144.3,137.2,134.1,130.5,128.3,
127.8, 127.7, 116.8, 116.6, 110.4, 109.2, 106.8, 103.7, 72.7, 69.4, 52.4,
47.1. HRMS (ESI): m/z (M þ Hþ) calcd for C28H29N4O6, 517.2082,
found: 517.2081.
(s, 1H), 7.56 (s, 4H), 7.35 (s, 1H), 7.2e7.23 (m, 1H), 6.63 (dd, J ¼ 8.1,
1.6 Hz, 1H), 6.51 (s, 1H), 6.37 (d, J ¼ 8.1 Hz, 1H), 4.75e4.72 (m, 2H),
4.32e4.29 (m, 2H), 2.89 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
d
168.4, 159.9, 156.0, 152.2, 146.2, 144.8, 142.3, 133.8, 130.4, 129.7,
4.1.1.6.4. Methyl 2-(7-(2-((4-cyanobenzyl)oxy)acetamido)-6-(3-
(dimethylamino)phenoxy)- 4-oxo quinazolin-3(4H)-yl)acetate (22b).
White powder (80% yield), mp: 199e200 ꢁC. 1H NMR (400 MHz,
127.9, 127.8, 125.1 (d, J ¼ 3.9 Hz), 118.1, 116.9, 110.6, 109.0, 106.6,
103.6, 103.6, 71.6, 69.7. HRMS (ESI): m/z (M þ Hþ) calcd for
C
26H24O4N4F3, 513.1744, found: 513.1742.
4.1.1.6.3. Methyl 2-(7-(2-(benzyloxy)acetamido)-6-(3-(dimethy-
DMSO‑d6)
d
9.64 (s, 1H), 8.64 (s, 1H), 8.30 (s, 1H), 7.68 (d, J ¼ 8.1 Hz,
2H), 7.55 (d, J ¼ 8.1 Hz, 2H), 7.35 (s, 1H), 7.28e7.23 (m, 1H), 6.64 (dd,
lamino)phenoxy)-4-oxo quinazolin-3(4H)-yl)acetate (22a). Red
J ¼ 8.3, 2.1 Hz, 1H), 6.49e6.47 m, 1H), 6.36 (dd, J ¼ 7.9, 1.8 Hz, 1H),
powder (78% yield), mp: 155e156 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
4.78 (s, 2H), 4.75 (s, 2H), 4.33 (s, 2H), 3.68 (s, 3H), 2.90 (s, 6H). 13
C
d
9.63 (s, 1H), 8.66 (s, 1H), 8.30 (s, 1H), 7.37e7.35 (m, 2H), 7.33 (s,
NMR (101 MHz, DMSO‑d6) d 168.5, 168.4, 159.2, 155.8, 152.2, 147.2,

Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
13
146.7, 144.3, 143.2, 134.2, 132.2, 130.4, 127.9, 118.7, 117.0, 116.7, 110.7,
4.1.1.6.10. Methyl
2-(7-(4-cyanobenzamido)-6-(3-(dimethyla-
110.3, 109.1, 106.6, 103.6, 71.6, 69.7, 52.4, 47.1. HRMS (ESI): m/z
(M þ Hþ) calcd for C29H28N5O6, 542.2010, found: 542.2013.
4.1.1.6.5. Methyl 2-(6-(3-(dimethylamino)phenoxy)-7-(2-((4-
mino)phenoxy)-4-oxoquinazolin
Yellow powder (78% yield), mp: 178e180 ꢁC. 1H NMR (400 MHz,
DMSO‑d6)
ꢂ3(4H)-yl)acetate
(22
h).
d
10.36 (s,1H), 8.34 (d, J ¼ 19.6 Hz, 2H), 8.07 (d, J ¼ 8.3 Hz,
fluorobenzyl)oxy)acetamido)-
(22c). White powder (85% yield), mp: 173e174 ꢁC. 1H NMR
(400 MHz, DMSO‑d6) 9.62 (s, 1H), 8.65 (s, 1H), 8.29 (s, 1H), 7.33 (s,
4-oxoquinazolin-3(4H)-yl)acetate
2H), 8.02 (d, J ¼ 8.3 Hz, 2H), 7.40 (s, 1H), 7.26e7.21 (m, 1H), 6.60 (dd,
J ¼ 8.3, 2.0 Hz, 1H), 6.51 (s, 1H), 6.39 (dd, J ¼ 7.9, 1.5 Hz, 1H), 4.80 (s,
d
2H), 3.69 (s, 3H), 2.89 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 168.4,
1H), 7.31e7.24 (m, 2H), 7.23e7.18 (m, 2H), 7.14e7.05 (m, 1H), 6.63
(dd, J ¼ 8.3, 2.1 Hz, 1H), 6.53e6.4 (m, 1H), 6.39 (dd, J ¼ 7.9, 1.9 Hz,
1H), 4.78 (s, 2H), 4.67 (s, 2H), 4.29 (s, 2H), 3.68 (s, 3H), 2.90 (s, 6H).
164.8,159.2,156.1,152.1,149.3,147.1,143.8,138.2,134.6,132.5,130.3,
128.7, 121.6, 118.2, 118.1, 114.2, 111.2, 108.9, 107.0, 103.8, 52.4, 47.2.
HRMS (ESI): m/z (M þ Hþ) calcd for C27H24N5O5, 498.1772, found:
498.1771.
13C NMR (101 MHz, DMSO‑d6)
d
168.6, 168.4, 162.1 (d, J ¼ 243.8 Hz),
159.2, 155.7, 152.2, 147.2, 146.8, 144.2, 140.3 (d, J ¼ 7.3 Hz), 134.1,
130.4, 130.2 (d, J ¼ 8.3 Hz), 123.4 (d, J ¼ 2.7 Hz), 117.0, 116.6, 114.5 (d,
J ¼ 21.0 Hz), 114.1 (d, J ¼ 21.6 Hz), 110.4, 109.2, 106.8, 103.7, 71.8,
69.5, 52.4, 47.1. HRMS (ESI): m/z (M þ Hþ) calcd for C28H28FN4O6,
535.1987, found: 535.1989.
4.1.1.6.11. Methyl 2-(6-(3-(dimethylamino)phenoxy)-4-oxo-7-(4-
(trifluoromethyl)benzamido)
Yellow powder (70% yield), mp: 190e192 ꢁC. 1H NMR (400 MHz,
DMSO‑d6)
quinazolin-3(4H)-yl)acetate
(22i).
d
10.32 (s, 1H), 8.35 (d, J ¼ 28.7 Hz, 2H), 8.11 (d, J ¼ 8.0 Hz,
2H), 7.91 (d, J ¼ 8.2 Hz, 2H), 7.40 (s, 1H), 7.26e7.20 (m, 1H), 6.60 (dd,
4.1.1.6.6. Methyl 2-(7-(2-((4-chlorobenzyl)oxy)acetamido)-6-(3-
(dimethylamino)phenoxy)- 4-oxoquinazolin-3(4H)-yl)acetate (22d).
White powder (83% yield), mp: 148e149 ꢁC. 1H NMR (400 MHz,
J ¼ 8.4, 1.8 Hz, 1H), 6.52 (s, 1H), 6.40 (d, J ¼ 7.9 Hz, 1H), 4.80 (s, 2H),
3.69 (s, 3H), 2.88 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
d 168.4,
165.0, 159.3, 156.1, 152.1, 149.3, 147.1, 143.8, 138.0, 134.6, 131.5, 130.3,
128.8,125.4 (dd, J ¼ 7.4, 3.6 Hz),122.5,121.4,118.0,111.1,108.9,107.0,
103.9, 52.4, 47.2. HRMS (ESI): m/z (M þ Hþ) calcd for C27H24F3N4O5,
541.1693, found: 541.1697.
DMSO‑d6)
d
9.62 (s, 1H), 8.66 (s, 1H), 8.30 (s, 1H), 7.36 (d, J ¼ 8.4 Hz,
3H), 7.27 (d, J ¼ 8.2 Hz, 3H), 6.65 (dd, J ¼ 8.4, 2.1 Hz, 1H), 6.52 (t,
J ¼ 2.2 Hz, 1H), 6.38 (dd, J ¼ 7.9, 1.8 Hz, 1H), 4.78 (s, 2H), 4.64 (s, 2H),
4.28 (s, 2H), 3.68 (s, 3H), 2.91 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
4.1.1.6.12. Methyl2-(4-oxo-6-phenoxy-7-(2-((4-(trifluoromethyl)
d
168.6, 168.4, 159.2, 155.8, 152.2, 147.2, 146.7, 144.3, 136.4, 134.1,
benzyl)oxy)acetamido)
Yellow powder (80% yield), mp: 170e172 ꢁC. 1H NMR (400 MHz,
DMSO‑d6) 9.70 (s, 1H), 8.67 (s, 1H), 8.32 (s, 1H), 7.65e7.54 (m, 4H),
quinazolin-3(4H)-yl)acetate
(22j).
132.3, 130.5, 129.4, 128.3, 116.8, 116.6, 110.6, 109.1, 106.7, 103.6, 71.7,
69.5, 52.4, 47.1. HRMS (ESI): m/z (M þ Hþ) calcd for C28H28ClN4O6,
551.1692, found: 551.1696.
4.1.1.6.7. Methyl 2-(6-(3-(dimethylamino)phenoxy)-4-oxo-7-(4-
(4-(trifluoromethyl)phenoxy) butanamido)quinazolin-3(4H)-yl)ace-
tate (22e). White powder (82% yield), mp: 161e165 ꢁC. 1H NMR
d
7.51e7.45 (m, 2H), 7.35e7.25 (m, 2H), 7.19 (d, J ¼ 7.7 Hz, 2H), 4.76 (d,
J ¼ 17.1 Hz, 4H), 4.33 (s, 2H), 3.68 (s, 3H). 13C NMR (101 MHz,
DMSO‑d6)
d 168.6, 168.4, 159.2, 155.0, 147.5, 146.4, 144.6, 142.3,
134.5, 130.5, 128.4, 128.0, 125.6, 125.1 (q, J ¼ 3.7 Hz), 122.9, 119.9,
117.2, 116.7, 111.0, 71.7, 69.8, 52.4, 47.2. HRMS (ESI): m/z (M þ Hþ)
calcd for C27H23F3N3O6, 542.1534, found: 542.1536.
(400 MHz, DMSO‑d6) d 9.92 (s, 1H), 8.59 (s, 1H), 8.26 (s, 1H), 7.63 (d,
J ¼ 8.7 Hz, 2H), 7.27 (s, 1H), 7.27e7.21 (m, 1H), 7.10 (d, J ¼ 8.6 Hz,
2H), 6.62 (dd, J ¼ 8.3, 2.2 Hz, 1H), 6.51e6.48 (m, 1H), 6.36 (dd,
J ¼ 7.8, 1.9 Hz, 1H), 4.77 (s, 2H), 4.12 (t, J ¼ 6.3 Hz, 2H), 3.67 (s, 3H),
4.1.1.6.13. Methyl 2-(4-oxo-6-phenoxy-7-(4-(4-(trifluoromethyl)
phenoxy)butanamido)
White powder (78% yield), mp: 152e154 ꢁC. 1H NMR (400 MHz,
DMSO‑d6)
quinazolin-3(4H)-yl)acetate
(22k).
2.90 (s, 6H), 2.74e2.70 (m, 2H), 2.07 (dd, J ¼ 13.7, 6.8 Hz, 2H). 13
C
NMR (101 MHz, DMSO‑d6)
d
172.1, 168.4, 161.4, 161.1, 159.2, 156.0,
d
9.99 (s, 1H), 8.62 (s, 1H), 8.29 (s, 1H), 7.63 (d, J ¼ 8.7 Hz,
152.1, 147.7, 146.9, 145.5, 144.0, 135.2, 130.3, 126.9 (q, J ¼ 3.7 Hz),
118.4, 116.4, 114.9, 110.2, 109.0, 107.3, 104.1, 67.4, 52.4, 47.1, 32.8,
24.4. HRMS (ESI): m/z (M þ Hþ) calcd for C30H20F3N4O6, 599.2112,
found: 599.2114.
2H), 7.50e7.44 (m, 2H), 7.29e7.24 (m, 2H), 7.16 (d, J ¼ 7.8 Hz, 2H),
7.10 (d, J ¼ 8.6 Hz, 2H), 4.77 (s, 2H), 4.11 (t, J ¼ 6.3 Hz, 2H), 3.68 (s,
3H), 2.72 (t, J ¼ 7.2 Hz, 2H), 2.11e2.02 (m, 2H). 13C NMR (101 MHz,
DMSO‑d6)
d
172.1, 168.4, 161.4, 159.2, 155.3, 147.2 (d, J ¼ 7.1 Hz),
4.1.1.6.8. Methyl
2-(7-(4-(4-cyanophenoxy)butanamido)-6-(3-
144.4, 135.6, 130.3, 126.9 (q, J ¼ 3.7 Hz), 125.9, 124.9, 123.2, 121.6,
120.2, 118.7, 116.4, 114.9, 111.0, 67.3, 52.4, 47.1, 32.8, 24.4. HRMS
(ESI): m/z (M þ Hþ) calcd for C28H25F3N3O6, 556.1690, found:
556.1677.
(dimethylamino)phenoxy)- 4-oxoquinazolin-3(4H)-yl)acetate (22f).
Yellow powder (71% yield), mp: 124e125 ꢁC. 1H NMR (400 MHz,
DMSO‑d6)
d
9.91 (s, 1H), 8.59 (s, 1H), 8.26 (s, 1H), 7.74 (d, J ¼ 8.8 Hz,
2H), 7.27 (s, 1H), 7.23 (d, J ¼ 8.1 Hz, 1H), 7.09 (d, J ¼ 8.8 Hz, 2H), 6.61
(dd, J ¼ 8.4, 2.1 Hz, 1H), 6.49 (t, J ¼ 2.1 Hz, 1H), 6.36 (dd, J ¼ 7.9,
1.7 Hz, 1H), 4.77 (s, 2H), 4.13 (t, J ¼ 6.3 Hz, 2H), 3.67 (s, 3H), 2.90 (s,
6H), 2.72 (t, J ¼ 7.2 Hz, 2H), 2.08 (dd, J ¼ 13.4, 6.7 Hz, 2H). 13C NMR
4.1.1.6.14. Methyl 2-(6-(2-(dimethylamino)phenoxy)-4-oxo-7-(2-
((4-(trifluoromethyl)benzyl) oxy)acetamido)quinazolin-3(4H)-yl)ac-
etate (22l). Red powder (59% yield), mp: 62e64 ꢁC. 1H NMR
(400 MHz, DMSO‑d6) d 9.77 (s, 1H), 8.67 (s, 1H), 8.29 (s, 1H), 7.57 (d,
(101 MHz, DMSO‑d6)
d
172.1, 168.4, 162.0, 159.2, 156.0, 152.1, 147.7,
J ¼ 6.7 Hz, 4H), 7.26 (dd, J ¼ 11.2, 4.1 Hz, 1H), 7.12 (dd, J ¼ 8.1, 1.4 Hz,
2H), 7.07 (s, 1H), 7.03 (dd, J ¼ 10.8, 4.4 Hz, 1H), 4.77 (d, J ¼ 5.4 Hz,
4H), 4.37 (s, 2H), 3.67 (s, 3H), 2.61 (s, 6H). 13C NMR (101 MHz,
146.9,144.0,135.2,134.2,130.3,119.2,118.4,116.4,115.5,110.2,109.0,
107.3, 104.1, 102.7, 67.5, 52.4, 47.1, 40.0, 32.7, 24.3. HRMS (ESI): m/z
(M þ Hþ) calcd for C30H30N5O6, 556.2191, found: 556.2193.
4.1.1.6.9. Methyl 2-(6-(3-(dimethylamino)phenoxy)-7-(4-(4-
fluorophenoxy)butanamido)-
(22g). White powder (80% yield), mp: 201e202 ꢁC. 1H NMR
(400 MHz, DMSO‑d6) 9.90 (s, 1H), 8.59 (s, 1H), 8.26 (s, 1H),
DMSO‑d6)
d 168.5, 168.4, 159.2, 147.1, 146.4, 145.2, 145.2, 144.0,
142.3, 133.6, 129.6, 128.0, 126.6, 125.5, 125.1 (q, J ¼ 3.7 Hz), 122.6,
121.9,119.1,116.7,116.6,108.6, 71.7, 69.7, 52.4, 47.1, 42.6. HRMS (ESI):
m/z (M þ Hþ) calcd for C29H28F3N4O6, 585.1956, found: 585.1954.
4.1.1.6.15. Methyl 2-(6-(4-(dimethylamino)phenoxy)-4-oxo-7-(4-
(4-(trifluoromethyl)phenoxy) butanamido)quinazolin-3(4H)-yl)ace-
tate (22m). White powder (54% yield), mp: 134e136 ꢁC. 1H NMR
4-oxoquinazolin-3(4H)-yl)acetate
d
7.28e7.22 (m, 2H), 7.08 (dd, J ¼ 10.9, 4.4 Hz, 2H), 6.95e6.91 (m, 2H),
6.62 (dd, J ¼ 8.4, 2.3 Hz, 1H), 6.51e6.49 (m, 1H), 6.37 (dd, J ¼ 7.9,
2.0 Hz, 1H), 4.77 (s, 2H), 3.99 (t, J ¼ 6.3 Hz, 2H), 3.67 (s, 3H), 2.90 (s,
6H), 2.70 (t, J ¼ 7.3 Hz, 2H), 2.06e2.01 (m, 2H). 13C NMR (101 MHz,
(400 MHz, DMSO‑d6) d 9.93 (s, 1H), 8.60 (s, 1H), 8.25 (s, 1H), 7.63 (d,
J ¼ 8.6 Hz, 2H), 7.14 (s, 1H), 7.11 (d, J ¼ 8.5 Hz, 2H), 7.03 (d, J ¼ 9.0 Hz,
DMSO‑d6) d 172.2,168.4,159.2,156.0,152.1,147.7,146.9,144.0,135.2,
2H), 6.80 (d, J ¼ 9.0 Hz, 2H), 4.75 (s, 2H), 4.13 (t, J ¼ 6.3 Hz, 2H), 3.67
130.3, 118.4, 116.3, 115.9, 115.7, 115.7, 115.6, 110.2, 109.0, 107.3, 104.2,
(s, 3H), 2.92 (s, 6H), 2.75 (t, J ¼ 7.2 Hz, 2H), 2.13e2.04 (m, 2H). 13
C
67.5, 52.4, 47.1, 32.9, 24.6. HRMS (ESI): m/z (M þ Hþ) calcd for
NMR (101 MHz, DMSO‑d6) d 172.1, 168.4, 161.4, 159.2, 148.8, 148.2,
C
29H29FN4O6, 549.2144, found: 549.2146.
146.7, 144.8, 143.6, 134.7, 126.9 (q, J ¼ 3.6 Hz), 125.9, 123.2, 121.6,
121.0 (d, J ¼ 32.0 Hz), 118.2, 116.2, 114.9, 113.5, 108.7, 99.6, 67.4, 52.3,

14
Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
47.1, 40.4, 32.8, 24.4. HRMS (ESI): m/z (M þ Hþ) calcd for
(dd, J ¼ 8.3, 2.0 Hz, 1H), 6.81e6.78 (m, 1H), 6.55 (dd, J ¼ 7.9, 1.9 Hz,
1H), 4.75 (s, 2H), 4.31 (s, 2H), 3.72e3.69 (m, 4H), 3.13e3.08 (m, 4H).
C
30H30F3N4O6, 599.2112, found: 599.2116.
4.1.1.6.16. Methyl 2-(6-(3-morpholinophenoxy)-4-oxo-7-(2-((4-
13C NMR (101 MHz, DMSO‑d6)
d 168.4, 159.8, 156.0, 153.0, 146.2,
(trifluoromethyl)benzyl)oxy) acetamido)quinazolin-3(4H)-yl)acetate
145.2, 144.8, 142.3 (d, J ¼ 1.0 Hz), 133.9, 130.5, 128.2 (d, J ¼ 31.6 Hz),
127.8, 125.5, 125.1 (q, J ¼ 3.8 Hz), 118.1, 117.0, 111.3, 110.7, 109.8,
106.5, 71.6, 69.7, 65.9, 47.9. HRMS (ESI): m/z (M þ Hþ) calcd for
(22n). White powder (68% yield), mp: 142e144 ꢁC. 1H NMR
(400 MHz, DMSO‑d6) d 9.67 (s, 1H), 8.66 (s, 1H), 8.30 (s, 1H), 7.61 (d,
J ¼ 8.6 Hz, 2H), 7.58 (s, 2H), 7.34 (s, 1H), 7.33e7.28 (m, 1H), 6.87 (dd,
J ¼ 8.3, 2.1 Hz, 1H), 6.82e6.79 (m, 1H), 6.57 (dd, J ¼ 8.0, 2.0 Hz, 1H),
4.77 (d, J ¼ 11.5 Hz, 4H), 4.33 (d, J ¼ 3.4 Hz, 2H), 3.77e3.68 (m, 8H),
C28H26F3N4O5, 555.1850, found: 555.1853.
4.1.1.6.22. N-(6-(3-morpholinophenoxy)-4-oxo-3,4-
dihydroquinazolin-7-yl)-4-(4-(trifluoromethyl)phenoxy)butanamide
3.68 (s, 3H). 13C NMR (101 MHz, DMSO‑d6)
d
168.4 (d, J ¼ 13.2 Hz),
(22t). White powder (88% yield), mp > 210 ꢁC. 1H NMR (400 MHz,
159.2, 155.8, 153.0, 147.3, 146.6, 144.4, 142.3, 134.2, 130.6, 128.4,
128.0, 127.9, 127.7, 125.4, 125.0 (q, J ¼ 3.7 Hz), 116.8 (d, J ¼ 27.5 Hz),
111.4,110.6,109.9,106.5, 71.6, 71.4, 69.7, 65.9, 52.4, 47.9. HRMS (ESI):
m/z (M þ Hþ) calcd for C31H30F3N4O7, 627.2061, found: 627.2069.
DMSO‑d6) d 12.12 (s, 1H), 9.86 (s, 1H), 8.54 (s, 1H), 8.00 (s, 1H), 7.62
(d, J ¼ 8.7 Hz, 2H), 7.30 (d, J ¼ 8.2 Hz, 1H), 7.27 (s, 1H), 7.10 (d,
J ¼ 8.6 Hz, 2H), 6.84 (dd, J ¼ 8.3, 2.0 Hz, 1H), 6.76e6.73 (m, 1H), 6.53
(dd, J ¼ 7.9, 1.9 Hz, 1H), 4.11 (t, J ¼ 6.3 Hz, 2H), 3.73e3.69 (m, 4H),
3.14e3.10 (m, 4H), 2.70 (t, J ¼ 7.3 Hz, 2H), 2.07 (dd, J ¼ 8.6, 5.0 Hz,
4.1.1.6.17. Methyl
2-(6-(3-morpholinophenoxy)-4-oxo-7-(4-(4-
(trifluoromethyl)phenoxy) butanamido)quinazolin-3(4H)-yl)acetate
2H). 13C NMR (101 MHz, DMSO‑d6)
d 172.0, 161.4, 159.8, 156.2, 152.9,
(22O). White powder (60% yield), mp: 178e180 ꢁC. 1H NMR
147.1, 144.9, 144.4, 135.0, 130.4, 128.1, 127.9, 126.9 (q, J ¼ 3.6 Hz),
118.7, 117.8, 114.9, 111.2, 110.5, 110.3, 106.9, 67.4, 66.0, 48.0, 32.7,
24.4. HRMS (ESI): m/z (M þ Hþ) calcd for C29H28F3N4O5, 569.2006,
found: 569.1993.
(400 MHz, DMSO‑d6) d 9.92 (s, 1H), 8.59 (s, 1H), 8.27 (s, 1H), 7.63 (d,
J ¼ 8.7 Hz, 2H), 7.31 (d, J ¼ 8.2 Hz,1H), 7.27 (s, 1H), 7.10 (d, J ¼ 8.7 Hz,
2H), 6.85 (dd, J ¼ 8.4, 2.0 Hz, 1H), 6.76 (s, 1H), 6.54 (dd, J ¼ 8.0,
1.8 Hz, 1H), 4.77 (s, 2H), 4.12 (t, J ¼ 6.3 Hz, 2H), 3.74e3.69 (m, 4H),
3.68 (s, 3H), 3.15e3.10 (m, 4H), 2.72 (t, J ¼ 7.2 Hz, 2H), 2.07 (p,
4.1.1.7. Synthesis of intermediates 23a and 23b. To a solution of 14a
or 22n (1.0 mmol) in THF (6 mL) and H2O (3 mL), LiOH (72 mg,
3 mmol) was added. The reaction mixture was stirred at room
temperature for 0.5 h and adjusted to neutral (pH ¼ 7) by adding
aqueous HCl solution (1 N) to give the white solid 23a-23b in the
yield of 85e90%.
J ¼ 6.8 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 172.1, 168.4, 161.4,
159.2, 156.1, 152.9, 147.5, 147.0, 144.1, 135.3, 130.4, 129.6, 126.9 (q,
J ¼ 3.6 Hz), 121.0 (d, J ¼ 32.0 Hz), 118.5, 116.4, 114.9, 111.3, 110.5,
110.3, 106.9, 67.4, 65.9, 52.34, 48.0, 47.1, 32.7, 24.4. HRMS (ESI): m/z
(M þ Hþ) calcd for C32H32F3N4O7, 641.2218, found: 641.2213.
4.1.1.6.18. N-(6-(3-(dimethylamino)phenoxy)-4-oxo-3,4-
dihydroquinazolin-7-yl)-4-(4-(trifluoromethyl)phenoxy)butanamide
(22p). Red powder (70% yield), mp > 210 ꢁC. 1H NMR (400 MHz,
4.1.1.8. Synthesis of compounds 24a-24e. To a solution of 23a or 23b
DMSO‑d6)
d
12.11 (s, 1H), 9.86 (s, 1H), 8.53 (s, 1H), 7.99 (s, 1H), 7.62
(1.0 mmol) and DIC (464 mL, 3 equiv.) in THF (30 mL), HOSu (345 mg,
(d, J ¼ 8.7 Hz, 2H), 7.27 (s, 1H), 7.27e7.21 (m, 1H), 7.10 (d, J ¼ 8.6 Hz,
2H), 6.61 (dd, J ¼ 8.4, 2.1 Hz, 1H), 6.49 (t, J ¼ 2.2 Hz, 1H), 6.36 (dd,
J ¼ 7.9, 1.8 Hz, 1H), 4.11 (t, J ¼ 6.3 Hz, 2H), 2.90 (s, 6H), 2.71 (t,
J ¼ 7.3 Hz, 2H), 2.08 (dd, J ¼ 13.4, 6.7 Hz, 2H). 13C NMR (101 MHz,
3 equiv.) was added. The reaction mixture was stirred at room
temperature overnight. Then amines (5 equiv.) were added. The
reaction mixture was continually stirred at room temperature for
3 h and filtered. The filtration was concentrated under reduced
pressure, and the residue was purified with flash column chro-
matography on silica gel to give the compounds 24a-24e in the
yield of 45e90%.
DMSO‑d6) d 172.0, 161.4, 159.9, 156.1, 152.1, 147.3, 144.7, 144.4, 134.8,
130.3, 126.9 (q, J ¼ 3.8 Hz), 123.2, 121.2, 120.8, 117.8, 114.9, 110.3,
108.9, 107.3, 104.1, 67.4, 40.0, 32.7, 24.4. HRMS (ESI): m/z (M þ Hþ)
calcd for C27H26F3N4O4, 527.1901, found: 527.1899.
4.1.1.8.1. N-(6-(3-(dimethylamino)phenoxy)-4-oxo-3-(2-oxo-2-
(piperidin-1-yl)ethyl)-3,4-dihydroquinazolin-7-yl)-2-((4-(tri-
fluoromethyl)benzyl)oxy)acetamide (24a). Yellow powder (45%
4.1.1.6.19. 2-((4-cyanobenzyl)oxy)-N-(6-(3-(dimethylamino)phe-
noxy)-4-oxo-3,4-dihydroquinazolin-7-yl)acetamide
(22q). Red
powder (55% yield), mp > 210 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
yield), mp: 85e86 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 9.64 (s, 1H),
d
9.50 (s, 1H), 8.51 (s, 1H), 8.02 (s, 1H), 7.68 (d, J ¼ 8.2 Hz, 2H), 7.52
8.64 (s, 1H), 8.18 (s, 1H), 7.56 (s, 4H), 7.36 (s, 1H), 7.24 (t, J ¼ 8.1 Hz,
1H), 6.63 (d, J ¼ 7.4 Hz, 1H), 6.52 (s, 1H), 6.36 (d, J ¼ 7.6 Hz, 1H), 4.85
(s, 2H), 4.75 (s, 2H), 4.33 (s, 2H), 3.44 (d, J ¼ 24.5 Hz, 4H), 2.89 (s,
(d, J ¼ 8.2 Hz, 2H), 7.34 (s, 1H), 7.25e7.20 (m, 1H), 6.60 (dd, J ¼ 8.4,
2.2 Hz, 1H), 6.44 (t, J ¼ 2.2 Hz, 1H), 6.31 (dd, J ¼ 8.0, 1.9 Hz, 1H), 4.72
(s, 2H), 4.29 (s, 2H), 2.89 (s, 6H). 13C NMR (101 MHz, DMSO‑d6)
6H), 1.59 (s, 4H), 1.44 (s, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 168.4,
d
168.1, 156.5, 152.1, 146.1, 145.1, 143.2, 133.1, 132.2, 130.3, 129.7,
164.4, 159.2, 156.0, 152.2, 148.0, 146.3, 144.5, 142.3, 134.0, 130.4,
129.6, 128.0, 127.8, 125.1 (q, J ¼ 3.7 Hz), 116.9, 116.8, 110.9, 109.0,
106.5, 103.5, 71.6, 69.7, 46.6, 45.2, 42.6, 39.9, 25.8, 25.1, 23.9. HRMS
(ESI): m/z (M þ Hþ) calcd for C33H35F3N5O5, 638.2585, found:
638.2580.
4.1.1.8.2. N-(6-(3-(dimethylamino)phenoxy)-3-(2-(4-
fluoropiperidin-1-yl)-2-oxoethyl)-4-oxo-3,4-dihydroquinazolin-7-
yl)-2-((4-(trifluoromethyl)benzyl)oxy)acetamide (24b). Yellow pow-
der (50% yield), mp: 99e100 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
127.9, 118.7, 118.5, 117.2, 116.6, 111.4, 110.3, 108.7, 106.3, 103.2, 71.6,
69.7. HRMS (ESI): m/z (M þ Hþ) calcd for C26H24N5O4, 470.1823,
found: 470.1821.
4.1.1.6.20. 3-Cyano-N-(6-(3-(dimethylamino)phenoxy)-4-oxo-
3,4-dihydroquinazolin-7-yl)benzamide (22r). Gray powder (50%
yield), mp > 210 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 12.21 (s, 1H),
10.30 (s, 1H), 8.28 (d, J ¼ 5.8 Hz, 2H), 8.20 (d, J ¼ 7.9 Hz, 1H),
8.10e8.03 (m, 2H), 7.76e7.71 (m,1H), 7.42 (s, 1H), 7.25e7.17 (m, 1H),
6.58 (d, J ¼ 8.4 Hz, 1H), 6.50 (s, 1H), 6.37 (d, J ¼ 7.8 Hz, 1H), 2.89 (s,
d
9.65 (s, 1H), 8.65 (s, 1H), 8.18 (s, 1H), 7.56 (s, 4H), 7.36 (s, 1H), 7.24
6H). 13C NMR (101 MHz, DMSO‑d6)
d
164.3, 159.9,156.3, 152.1, 148.8,
(t, J ¼ 8.1 Hz, 1H), 6.64e6.62 (m, 1H), 6.52 (s, 1H), 6.36 (dd, J ¼ 7.8,
1.1 Hz, 1H), 4.90 (d, J ¼ 3.8 Hz, 2H), 4.75 (s, 2H), 4.33 (s, 2H),
3.67e3.61 (m, 1H), 3.56e3.43 (m, 4H), 2.89 (s, 6H), 2.03e1.96 (m,
1H), 1.80 (dd, J ¼ 17.9, 9.4 Hz, 2H), 1.68e1.56 (m, 1H). 13C NMR
144.6, 144.6, 135.3, 135.3, 134.3, 132.7, 131.6, 130.3, 129.8, 121.8,
119.6, 118.2, 111.5, 111.5, 108.7, 106.8, 103.6. HRMS (ESI): m/z
(M þ Hþ) calcd for C24H20N5O3, 426.1561, found: 426.1566.
4.1.1.6.21. N-(6-(3-morpholinophenoxy)-4-oxo-3,4-
dihydroquinazolin-7-yl)-2-((4-(trifluoromethyl)benzyl)oxy)acet-
amide (22s). White powder (60% yield), mp > 210 ꢁC. 1H NMR
(101 MHz, DMSO‑d6)
d 168.4, 164.9, 159.2, 156.0, 152.2, 147.9, 146.4,
144.5, 142.3, 134.0, 130.4, 128.0, 127.8, 125.5, 125.1 (q, J ¼ 3.7 Hz),
122.8, 116.9 (d, J ¼ 14.0 Hz)., 110.9, 109.0, 106.5, 103.5, 88.9, 71.6,
69.7, 46.5, 40.6, 39.9, 31.2. HRMS (ESI): m/z (M þ Hþ) calcd for
(400 MHz, DMSO‑d6)
1H), 7.57 (s, 4H), 7.33 (d, J ¼ 2.1 Hz, 1H), 7.30 (d, J ¼ 8.2 Hz, 1H), 6.86
d 12.19 (s, 1H), 9.62 (s, 1H), 8.61 (s, 1H), 8.03 (s,
C33H34F4N5O5, 656.2491, found: 656.2496.

Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
15
4.1.1.8.3. N-(3-(2-(4,4-difluoropiperidin-1-yl)-2-oxoethyl)-6-(3-
(dimethylamino)phenoxy)-4-oxo-3,4-dihydroquinazolin-7-yl)-2-((4-
(trifluoromethyl)benzyl)oxy)acetamide (24c). Yellow powder (46%
washed with brine and dried over anhydrous MgSO4. Solvent was
removed, and the residue was purified with flash column chro-
matography on silica gel to give intermediates 27a-27c in the yield
of 80e90%.
yield), mp: 102e103 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 9.65 (s, 1H),
8.65 (s, 1H), 8.18 (s, 1H), 7.56 (s, 4H), 7.37 (s, 1H), 7.24 (s, 1H), 6.64 (s,
1H), 6.52 (s, 1H), 6.37 (s, 1H), 4.94 (s, 2H), 4.75 (s, 2H), 4.32 (s, 2H),
3.60 (d, J ¼ 31.1 Hz, 4H), 2.88 (s, 6H), 2.14e1.92 (m, 4H). 13C NMR
4.1.2.3. Synthesis of intermediates 28a-28c. To a solution of 27a-27c
(1.0 mmol) in THF (6 mL) and H2O (3 mL), LiOH (72.0 mg, 3 mmol)
was added. The reaction mixture was stirred at room temperature
for 0.5 h and adjusted to acidity (pH ¼ 2) by adding aqueous HCl
solution (6 N) to give the yellow solid 28a-28c in the yield of
80e90%.
(101 MHz, DMSO‑d6)
d 168.4, 165.2, 159.2, 156.0, 152.2, 147.9, 146.4,
144.5, 142.3, 134.1, 130.4, 129.6, 128.3, 128.0, 127.8, 125.1 (q,
J ¼ 3.7 Hz), 122.5, 116.9 (d, J ¼ 12.1 Hz), 110.9, 109.0, 106.5, 103.5,
71.6, 69.7, 46.5, 41.2, 39.9, 26.6. HRMS (ESI): m/z (M þ Hþ) calcd for
C
33H33F5N5O5, 674.2396, found: 674.2390.
4.1.1.8.4. N-(3-(2-(3,3-difluoropiperidin-1-yl)-2-oxoethyl)-6-(3-
(dimethylamino)phenoxy)-4-oxo-3,4-dihydroquinazolin-7-yl)-2-((4-
4.1.2.4. Synthesis of intermediates 29a-29c. The mixture of 28a-28c
(1.0 mmol) in formamide (10 mL) was heated to 160 ꢁC, and stirred
for 5 h. Then the reaction mixture was concentrated under reduced
pressure, and the residue was purified with flash column chro-
matography on silica gel to give the intermediates 29a-29c in the
yield of 50e60%.
(trifluoromethyl)benzyl)oxy)acetamide (24d). Yellow powder (62%
yield), mp: 106e107 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 9.65 (s, 1H),
8.65 (s, 1H), 8.19 (s, 1H), 7.56 (s, 4H), 7.36 (s, 1H), 7.24 (t, J ¼ 8.0 Hz,
1H), 6.63 (d, J ¼ 7.8 Hz, 1H), 6.52 (s, 1H), 6.37 (d, J ¼ 7.5 Hz, 1H), 4.91
(d, J ¼ 20.1 Hz, 2H), 4.75 (s, 2H), 4.33 (s, 2H), 3.91 (t, J ¼ 11.2 Hz, 1H),
3.78 (t, J ¼ 11.9 Hz, 1H), 3.53 (d, J ¼ 32.2 Hz, 2H), 2.89 (s, 6H),
2.12e2.08 (m, 2H), 1.77 (s, 1H), 1.61 (s, 1H). 13C NMR (101 MHz,
4.1.2.5. Synthesis of compounds 30a-30j. To a mixture of 29a-29c
(0.1 mmol) in THF (10 mL), amines (0.2 mmol) and HATU (114.0 mg,
3.0 mmol) were added. The reaction mixture was stirred at room
temperature overnight and concentrated under reduced pressure.
The residue was purified with flash column chromatography on
silica gel to give the compounds 30a-30j in the yield of 70%e90%.
4.1.2.5.1. 6-(3-chlorophenyl)-4-oxo-N-(2-((4-(trifluoromethyl)
DMSO‑d6)
d
168.4, 165.6 (br s), 159.2 (d, J ¼ 3.1 Hz), 156.0, 152.2,
147.8 (dd, J ¼ 7.5, 1.1 Hz), 146.4, 144.4, 142.3 (d, J ¼ 1.0 Hz), 134.0,
130.4, 128.3, 128.0, 127.8, 125.5, 125.1 (q, J ¼ 3.7 Hz), 122.8, 116.8 (d,
J ¼ 9.5 Hz), 110.9, 109.0, 106.5, 103.5, 71.6, 69.7, 46.6, 46.6, 43.2, 40.8,
39.9, 13.9. HRMS (ESI): m/z (M þ Hþ) calcd for C33H33F5N5O5,
674.2396, found: 674.2399.
benzyl)oxy)ethyl)-3,4-dihydroquinazoline-7-carboxamide
White powder (75% yield), mp: 177e179 ꢁC. 1H NMR (400 MHz,
DMSO‑d6) 12.47 (s, 1H), 8.78e8.74 (m, 1H), 8.19 (s, 1H), 8.05 (s,
(30a).
4.1.1.8.5. N-(3-(2-(3,3-difluoropiperidin-1-yl)-2-oxoethyl)-6-(3-
morpholinophenoxy)-4-oxo-3,4-dihydroquinazolin-7-yl)-2-((4-(tri-
fluoromethyl)benzyl)oxy)acetamide (24e). White powder (88%
d
1H), 7.71 (d, J ¼ 8.0 Hz, 2H), 7.67 (s, 1H), 7.55 (d, J ¼ 8.0 Hz, 2H), 7.47
(d, J ¼ 1.5 Hz, 1H), 7.40 (q, J ¼ 4.4 Hz, 3H), 4.56 (s, 2H), 3.45 (t,
J ¼ 5.7 Hz, 2H), 3.40e3.37 (m, 2H). 13C NMR (101 MHz, DMSO‑d6)
yield), mp > 210 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 9.66 (s, 1H),
8.65 (s, 1H), 8.20 (d, J ¼ 2.6 Hz, 1H), 7.57 (s, 4H), 7.34 (d, J ¼ 2.8 Hz,
1H), 7.31 (d, J ¼ 8.2 Hz, 1H), 6.87 (dd, J ¼ 8.4, 2.1 Hz, 1H), 6.81 (d,
J ¼ 2.0 Hz, 1H), 6.56 (dd, J ¼ 8.0, 2.0 Hz, 1H), 4.94 (s, 1H), 4.89 (s, 1H),
4.75 (s, 2H), 4.33 (s, 2H), 3.91 (t, J ¼ 11.5 Hz, 1H), 3.76 (d, J ¼ 12.2 Hz,
1H), 3.71e3.68 (m, 4H), 3.59e3.55 (m, 1H), 3.52e3.47 (m, 1H),
3.13e3.10 (m, 4H), 2.09 (dd, J ¼ 13.0, 6.7 Hz, 2H), 1.77 (s, 1H), 1.61 (s,
d
167.9, 160.4, 148.0, 146.6, 143.4, 142.5, 141.2, 135.9, 133.0, 130.2,
128.1, 128.0, 127.8, 127.5, 127.1, 127.0, 126.4, 125.7, 125.1 (q,
J ¼ 3.6 Hz), 123.0, 71.0, 68.3. HRMS (ESI): m/z (M þ Hþ) calcd for
C25H20ClF3N3O3, 502.1140, found: 502.1149.
4.1.2.5.2. 6-(3-chlorophenyl)-N-(3-(4-chlorophenyl)propyl)-4-
1H). 13C NMR (101 MHz, DMSO‑d6)
d
168.5, 165.7, 159.2,156.0,153.0,
oxo-3,4-dihydroquinazoline-7-carboxamide (30b). White powder
147.9, 147.9, 146.4, 144.5, 142.3, 134.1, 130.6, 128.3, 128.0, 127.9,
125.5, 125.1 (q, J ¼ 3.7 Hz), 116.9 (d, J ¼ 1.4 Hz), 111.4, 110.9, 109.8,
106.4, 71.6, 69.7, 65.9, 47.9, 46.6, 43.2, 40.9, 29.1, 22.4. HRMS (ESI):
m/z (M þ Hþ) calcd for C35H35F5N5O6, 716.2502, found: 716.2499.
(80% yield), mp: 140e142 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d
12.45
(s, 1H), 8.61e8.56 (m, 1H), 8.19 (s, 1H), 8.06 (s, 1H), 7.67 (s, 1H), 7.49
(s, 1H), 7.45e7.41 (m, 3H), 7.33 (d, J ¼ 8.3 Hz, 2H), 7.18 (d, J ¼ 8.3 Hz,
2H), 3.12 (q, J ¼ 6.7 Hz, 2H), 2.47e2.43 (m, 2H), 1.63 (dd, J ¼ 14.6,
7.3 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 167.6, 160.3, 148.0,
4.1.2. General procedure for the synthesis of compounds 30a-30j,
34a-34f, 35, 36a, 36b
146.5, 142.9, 141.2, 140.6, 135.9, 133.0, 130.4, 130.3, 130.2, 128.2,
128.1, 127.6, 127.2, 126.9, 126.3, 122.9, 38.4, 31.6, 30.3. HRMS (ESI):
m/z (M þ Hþ) calcd for C24H20Cl2N3O2, 452.0927, found: 452.0928.
4.1.2.5.3. 6-(3-chlorophenyl)-4-oxo-N-(3-phenylpropyl)-3,4-
dihydroquinazoline-7-carboxamide (30c). White powder (78%
4.1.2.1. Synthesis of intermediate 26. The mixture of dimethyl 2-
aminoterephthalate 25 (2.1 g, 10 mmol) and N-iodosuccinimide
(2.5 g, 11 mmol) in acetic acid (200 mL) was stirred at room tem-
perature for 24 h, and water (30 mL) was then added. The reaction
mixture was adjusted to neutral (pH ¼ 7) by adding saturated so-
dium bicarbonate solution, and extracted with ethyl acetate. The
organic phase was washed with brine and dried over MgSO4. Sol-
vent was removed under reduced pressure, and the residue was
purified with flash column chromatography on silica gel to give
dimethyl 2-amino-5-iodoterephthalate (26) in the yield of 57%.
yield), mp: 99e100 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 12.45 (s, 1H),
8.62e8.58 (m, 1H), 8.19 (s, 1H), 8.06 (s, 1H), 7.67 (s, 1H), 7.49 (s, 1H),
7.43 (q, J ¼ 4.3 Hz, 3H), 7.30e7.25 (m, 2H), 7.19e7.14 (m, 3H), 3.14 (d,
J ¼ 6.1 Hz, 2H), 2.46 (d, J ¼ 7.8 Hz, 2H), 1.69e1.63 (m, 2H). 13C NMR
(101 MHz, DMSO‑d6)
d 167.6, 160.2, 148.0, 146.5, 142.9, 141.6, 141.2,
135.9, 133.0, 130.2, 128.3, 128.2, 128.1, 127.6, 127.2, 126.9, 126.3,
125.7, 122.8, 38.5, 32.4, 30.4. HRMS (ESI): m/z (M þ Hþ) calcd for
C24H21ClN3O2, 418.1317, found: 418.1307.
4.1.2.2. Synthesis of intermediates 27a-27c. To a solution of 26
(3.4 g, 10 mmol) and phenylboronic acid derivatives (20 mmol, 2
equiv.) in 1, 4-dioxane and H2O (1, 4-dioxane/H2O ¼ 2:1), Pd(PPh3)4
(1.3 g, 1.1 mmol) and Na2CO3 (5.3 g, 50 mmol) were added. The
reaction mixture was heated to 100 ꢁC and stirred overnight under
N2 atmosphere. Then the mixture was filtered and the filtrate was
concentrated under reduced pressure. The reaction residue was
dissolved in DCM, and washed with water. The aqueous phase was
extracted with DCM, and the organic phase was combined and
4.1.2.5.4. 6-(3-chlorophenyl)-4-oxo-N-(3-(4-(trifluoromethyl)
phenoxy)propyl)-3,4-dihydroquinazoline-7-carboxamide
White powder (86% yield), mp: 90e91 ꢁC. 1H NMR (400 MHz,
DMSO‑d6) 8.66e8.63 (m, 1H), 8.19 (s, 1H), 8.06 (s, 1H), 7.69 (s, 1H),
(30d).
d
7.64 (d, J ¼ 8.4 Hz, 2H), 7.48 (s, 1H), 7.42 (dd, J ¼ 6.0, 3.3 Hz, 2H), 7.39
(dd, J ¼ 5.6, 2.7 Hz, 1H), 7.36e7.23 (m, 1H), 7.04 (d, J ¼ 8.8 Hz, 2H),
3.89 (t, J ¼ 6.1 Hz, 2H), 3.27 (dd, J ¼ 12.6, 6.2 Hz, 2H), 1.87e1.79 (m,
2H). 13C NMR (101 MHz, DMSO‑d6)
d 167.7, 161.4, 160.3, 148.0, 146.6,
142.7, 141.2, 135.8, 133.0, 130.2, 128.7, 128.1, 127.5, 127.1, 127.0, 126.9

16
Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
(d, J ¼ 4.4 Hz), 126.2, 122.9, 121.0 (d, J ¼ 31.8 Hz), 114.9, 65.4, 35.7,
28.3. HRMS (ESI): m/z (M þ Hþ) calcd for C25H20ClF3N3O3, 502.1140,
found: 502.1143.
4.1.2.5.10. 4-Oxo-N-(3-phenylpropyl)-6-(3-(trifluoromethyl)
phenyl)-3,4-dihydroquinazoline-7-carboxamide (30j). White pow-
der (85% yield), mp: 188e190 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
4.1.2.5.5. 6-(3-fluorophenyl)-4-oxo-N-(2-((4-(trifluoromethyl)
d 12.50 (s, 1H), 8.67e8.62 (m, 1H), 8.21 (s, 1H), 8.10 (s, 1H), 7.77 (d,
benzyl)oxy)ethyl)-3,4-dihydroquinazoline-7-carboxamide
White powder (80% yield), mp: 176e178 ꢁC. 1H NMR (400 MHz,
DMSO‑d6) 12.46 (s, 1H), 8.78e8.74 (m, 1H), 8.19 (s, 1H), 8.06 (s,
(30e).
J ¼ 11.3 Hz, 2H), 7.71 (d, J ¼ 8.2 Hz, 2H), 7.66 (t, J ¼ 7.5 Hz,1H), 7.26 (t,
J ¼ 7.3 Hz, 2H), 7.17 (d, J ¼ 7.1 Hz, 1H), 7.13 (d, J ¼ 7.6 Hz, 2H), 3.11
(dd, J ¼ 12.4, 6.3 Hz, 2H), 2.44 (t, J ¼ 7.6 Hz, 2H), 1.67e1.57 (m, 2H).
d
1H), 7.71 (d, J ¼ 7.9 Hz, 2H), 7.67 (s, 1H), 7.55 (d, J ¼ 7.9 Hz, 2H), 7.40
(dd, J ¼ 14.5, 7.4 Hz, 1H), 7.30e7.25 (m, 2H), 7.21e7.15 (m, 1H), 4.55
(s, 2H), 3.45 (d, J ¼ 5.1 Hz, 2H), 3.38 (d, J ¼ 5.2 Hz, 2H). 13C NMR
13C NMR (101 MHz, DMSO‑d6)
d 167.6, 160.3, 148.1, 146.6, 142.9,
141.6, 140.2, 135.9, 132.6, 129.6, 129.1 (d, J ¼ 31.6 Hz), 128.3 (d,
J ¼ 2.9 Hz), 127.1, 126.4, 125.8, 125.5, 124.8 (q, J ¼ 3.8 Hz), 124.4 (d,
J ¼ 2.9 Hz), 122.9, 122.8, 38.6, 32.4, 30.3. HRMS (ESI): m/z (M þ Hþ)
calcd for C25H21F3N3O2, 452.1580, found: 452.1585.
(101 MHz, DMSO‑d6)
d 167.9, 163.2, 160.8, 160.4, 147.9, 146.6, 143.4,
142.6, 141.5 (d, J ¼ 8.0 Hz), 136.1 (d, J ¼ 1.9 Hz), 130.3 (d, J ¼ 8.4 Hz),
127.8, 127.0, 126.4, 125.7, 125.1 (q, J ¼ 3.5 Hz), 124.6 (d, J ¼ 2.4 Hz),
123.0 (d, J ¼ 7.7 Hz), 115.1 (d, J ¼ 22.2 Hz), 114.4 (d, J ¼ 20.9 Hz), 71.0,
68.3, 38.9. HRMS (ESI): m/z (M þ Hþ) calcd for C25H20F4N3O3,
486.1435, found: 486.1431.
4.1.2.6. Synthesis of intermediates 31-33. To a mixture of 28a-28b
(0.3 mmol) in THF (10 mL), triphosgene (29.7 mg, 0.1 mmol) was
added under N2 atmosphere. The reaction mixture was heated to
50 ꢁC and stirred for 4 h, then concentrated under reduced pressure
to give intermediate 31. To a mixture of 31 in water, glycine methyl
ester hydrochloride (113.0 mg, 0.9 mmol) and triethylamine
4.1.2.5.6. N-(3-(4-chlorophenyl)propyl)-6-(3-fluorophenyl)-4-
oxo-3,4-dihydroquinazoline-7-carboxamide (30f). White powder
(70% yield), mp: 165e167 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 12.45
(s, 1H), 8.59e8.54 (m, 1H), 8.19 (s, 1H), 8.06 (s, 1H), 7.67 (s, 1H), 7.45
(dd, J ¼ 11.0, 4.8 Hz, 1H), 7.33 (s, 1H), 7.31 (d, J ¼ 3.4 Hz, 2H), 7.29 (d,
J ¼ 1.6 Hz, 1H), 7.21e7.18 (m, 2H), 7.17 (s, 1H), 3.15e3.10 (m, 2H),
2.48e2.43 (m, 2H), 1.65e1.59 (m, 2H). 13C NMR (101 MHz,
(125.0 mL, 0.9 mmol) was added. The reaction mixture was stirred at
room temperature for 6 h and concentrated under reduced pres-
sure to give intermediate 32. The mixture of 32 in HCOOH was then
heated to 110 ꢁC and stirred for 5 h. The reaction mixture was
concentrated under reduced pressure and the residue was purified
with flash column chromatography on silica gel to give the in-
termediates 33a-33b in the yield of 50e65%.
DMSO‑d6)
d 167.6, 163.1, 160.7, 160.2, 147.9, 146.4, 142.9, 141.5 (d,
J ¼ 8.0 Hz), 140.6, 136.1 (d, J ¼ 1.9 Hz), 130.3 (d, J ¼ 7.9 Hz), 130.2,
128.1, 126.9, 126.3, 124.6 (d, J ¼ 2.6 Hz), 122.8, 115.2 (d, J ¼ 22.2 Hz),
114.4 (d, J ¼ 21.0 Hz), 38.3, 31.6, 30.2. HRMS (ESI): m/z (M þ Hþ)
calcd for C24H20ClFN3O2, 436.1223, found: 436.1220.
4.1.2.7. Synthesis of compounds 34a-34f. To a mixture of 33a-33b
(0.1 mmol) in THF (10 mL), amine derivatives (0.2 mmol) and HATU
(114 mg, 3.0 mmol) were added. The reaction mixture was stirred at
room temperature overnight and concentrated under reduced
pressure, the residue was purified with flash column chromatog-
raphy on silica gel to give the compounds 34a-34f in the yield of
80e95%.
4.1.2.5.7. 6-(3-fluorophenyl)-4-oxo-N-(3-phenylpropyl)-3,4-
dihydroquinazoline-7-carboxamide (30g). White powder (80%
yield), mp: 116e118 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 12.45 (s,
1H), 8.60e8.56 (m, 1H), 8.19 (s, 1H), 8.07 (s, 1H), 7.67 (s, 1H), 7.45
(dd, J ¼ 14.3, 8.0 Hz, 1H), 7.32e7.29 (m, 2H), 7.28e7.25 (m, 2H),
7.23e7.19 (m, 1H), 7.16 (dd, J ¼ 5.9, 4.7 Hz, 3H), 3.16e3.10 (m, 2H),
2.46 (d, J ¼ 7.8 Hz, 2H), 1.64 (dd, J ¼ 14.6, 7.3 Hz, 2H). 13C NMR
4.1.2.7.1. Methyl
2-(6-(3-fluorophenyl)-4-oxo-7-((2-((4-(tri-
(101 MHz, DMSO‑d6)
d
167.6, 163.1, 160.7, 160.3, 147.9, 146.5, 142.9,
fluoromethyl)benzyl) oxy)ethyl) carbamoyl)quinazolin-3(4H)-yl)ace-
141.6, 141.5 (d, J ¼ 8.1 Hz), 136.1 (d, J ¼ 2.0 Hz), 130.3 (d, J ¼ 8.5 Hz),
128.3 (d, J ¼ 2.9 Hz), 126.9, 126.3, 125.7, 124.6 (d, J ¼ 2.6 Hz), 122.8,
115.2 (d, J ¼ 22.2 Hz), 114.4 (d, J ¼ 20.9 Hz), 38.5, 32.4, 30.4. HRMS
(ESI): m/z (M þ Hþ) calcd for C24H21FN3O2, 402.1612, found:
402.1602.
tate (34a). White powder (89% yield), mp > 210 ꢁC. 1H NMR
(400 MHz, DMSO‑d6)
d 8.80e8.75 (m, 1H), 8.47 (s, 1H), 8.08 (s, 1H),
7.73 (s, 1H), 7.71 (s, 2H), 7.56 (d, J ¼ 7.7 Hz, 2H), 7.41 (d, J ¼ 6.7 Hz,
1H), 7.29 (d, J ¼ 7.9 Hz, 2H), 7.22e7.17 (m, 1H), 4.89 (s, 2H), 4.56 (s,
2H), 3.72 (s, 3H), 3.49e3.40 (m, 4H). 13C NMR (101 MHz, DMSO‑d6)
4.1.2.5.8. 4-Oxo-N-(2-((4-(trifluoromethyl)benzyl)oxy)ethyl)-6-
(3-(trifluoromethyl)phenyl)-3,4-dihydroquinazoline-7-carboxamide
(30 h). White powder (90% yield), mp: 201e203 ꢁC. 1H NMR
d
168.4, 167.7, 159.7, 158.6, 149.0, 147.1, 145.1, 143.4 (d, J ¼ 2.4 Hz),
143.0, 141.3, 136.7, 136.7, 128.1, 127.9, 127.1, 126.5, 125.1 (q,
J ¼ 3.9 Hz), 124.6 (d, J ¼ 2.4 Hz), 121.5, 115.1, 114.5, 71.0, 68.3, 52.6,
47.3, 32.4. HRMS (ESI): m/z (M þ Hþ) calcd for C28H24F4N3O5,
558.1647, found: 558.1646.
(400 MHz, DMSO‑d6)
d 12.50 (br s, 1H), 8.82e8.77 (m, 1H), 8.21 (s,
1H), 8.09 (s, 1H), 7.73 (dd, J ¼ 17.1, 9.1 Hz, 6H), 7.63e7.58 (m, 1H),
7.54 (d, J ¼ 8.0 Hz, 2H), 4.53 (s, 2H), 3.43 (t, J ¼ 5.6 Hz, 2H),
4.1.2.7.2. Methyl 2-(7-((3-(4-chlorophenyl)propyl)carbamoyl)-6-
3.38e3.32 (m, 2H). 13C NMR (101 MHz, DMSO‑d6)
d
167.8, 160.4,
(3-fluorophenyl)-4-oxo
White powder (94% yield), mp: 160e162 ꢁC. 1H NMR (400 MHz,
DMSO‑d6)
quinazolin-3(4H)-yl)acetate
(34b).
148.1, 146.8, 143.3, 142.4, 140.1, 135.7, 132.4, 129.5, 129.5, 129.1 (d,
J ¼ 31.6 Hz), 128.1, 127.8, 127.1, 126.4, 125.5, 125.1 (q, J ¼ 3.9 Hz),
124.7 (d, J ¼ 4.0 Hz), 123.0, 122.8, 70.9, 68.2, 40.1. HRMS (ESI): m/z
(M þ Hþ) calcd for C26H20F6N3O3, 536.1403, found: 536.1407.
4.1.2.5.9. N-(3-(4-chlorophenyl)propyl)-4-oxo-6-(3-(tri-
fluoromethyl)phenyl)-3,4-dihydroquinazoline-7-carboxamide (30i).
White powder (86% yield), mp: 186e188 ꢁC. 1H NMR (400 MHz,
d
8.60 (d, J ¼ 4.8 Hz, 1H), 8.47 (s, 1H), 8.09 (s, 1H), 7.73 (s,
1H), 7.46 (dd, J ¼ 14.5, 7.7 Hz, 1H), 7.31 (dd, J ¼ 16.3, 7.9 Hz, 4H), 7.22
(d, J ¼ 8.7 Hz, 1H), 7.18 (d, J ¼ 8.1 Hz, 2H), 4.89 (s, 2H), 3.72 (s, 3H),
3.13 (dd, J ¼ 12.0, 6.0 Hz, 2H), 2.45 (t, J ¼ 7.5 Hz, 2H), 1.63 (dd,
J ¼ 13.9, 6.9 Hz, 2H). 13C NMR (101 MHz, DMSO‑d6)
d 168.4, 167.5,
163.1, 160.7, 159.7, 149.0, 147.1, 143.3, 141.3 (d, J ¼ 8.1 Hz), 140.6,
136.7 (d, J ¼ 2.2 Hz), 130.4 (d, J ¼ 5.3 Hz), 130.2, 128.2, 127.1, 126.4,
124.7 (d, J ¼ 2.8 Hz), 121.4, 115.3 (d, J ¼ 22.3 Hz), 114.6 (d,
J ¼ 21.0 Hz), 52.5, 47.3, 38.4, 31.6, 30.2. HRMS (ESI): m/z (M þ Hþ)
calcd for C27H24ClFN3O4, 508.1434, found: 508.1433.
DMSO‑d6)
d
12.50 (s, 1H), 8.67e8.62 (m, 1H), 8.21 (d, J ¼ 2.2 Hz, 1H),
8.10 (s, 1H), 7.81e7.74 (m, 3H), 7.72 (d, J ¼ 7.4 Hz, 2H), 7.69e7.64 (m,
1H), 7.32 (d, J ¼ 8.2 Hz, 2H), 7.15 (d, J ¼ 8.0 Hz, 2H), 3.10 (dd, J ¼ 12.3,
6.2 Hz, 2H), 2.42 (t, J ¼ 7.6 Hz, 2H), 1.59 (dd, J ¼ 14.2, 7.0 Hz, 2H). 13
C
NMR (101 MHz, DMSO‑d6)
d
167.6, 160.3, 148.1, 146.6, 142.8, 140.6,
4.1.2.7.3. Methyl 2-(6-(3-fluorophenyl)-4-oxo-7-((3-
140.2, 135.9,132.6,130.3 (d, J ¼ 19.0 Hz), 129.6, 129.1 (d, J ¼ 31.5 Hz),
128.2, 127.1, 126.4, 125.5, 124.8 (q, J ¼ 3.7 Hz), 124.4 (d, J ¼ 2.9 Hz),
123.0, 122.8, 38.4, 31.6, 30.2. HRMS (ESI): m/z (M þ Hþ) calcd for
phenylpropyl)carbamoyl)quinazolin-
White powder (92% yield), mp: 120e121 ꢁC. 1H NMR (400 MHz,
DMSO‑d6) 8.61 (s, 1H), 8.47 (s, 1H), 8.09 (s, 1H), 7.72 (s, 1H), 7.46
3(4H)-yl)acetate
(34c).
d
C
25H20ClF3N3O2, 486.1191, found: 486.1199.
(dd, J ¼ 14.5, 7.6 Hz, 1H), 7.29 (dd, J ¼ 14.9, 7.3 Hz, 4H), 7.18 (dd,
J ¼ 18.7, 8.0 Hz, 4H), 4.89 (s, 2H), 3.72 (s, 3H), 3.14 (dd, J ¼ 12.4,

Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
17
6.3 Hz, 2H), 2.46 (d, J ¼ 7.8 Hz, 2H), 1.70e1.60 (m, 2H). 13C NMR
Solvent was removed under reduced pressure, and the residue was
purified with flash column chromatography on silica gel to give 36a
and 36b in the yield of 80e86%.
(101 MHz, DMSO‑d6)
d 168.4, 167.5, 163.1, 160.7, 159.7, 149.0, 147.1,
143.3, 141.6, 141.3 (d, J ¼ 8.0 Hz), 136.8 (d, J ¼ 1.7 Hz), 130.4 (d,
J ¼ 8.8 Hz), 128.3 (d, J ¼ 2.7 Hz), 127.1, 126.4, 125.8, 124.7 (d,
J ¼ 2.5 Hz), 121.4, 115.3 (d, J ¼ 22.3 Hz), 114.6 (d, J ¼ 20.4 Hz), 52.5,
47.3, 38.5, 32.4, 30.4. HRMS (ESI): m/z (M þ Hþ) calcd for
4.1.2.9.1. 6-(3-chlorophenyl)-4-oxo-3-(2-oxo-2-(piperidin-1-yl)
ethyl)-N-(3-(4-(trifluoromethyl)phenoxy)propyl)-3, 4-
dihydroquinazoline-7-carboxamide (36a). White powder (86%
C
27H25FN3O4, 474.1824, found: 474.1823.
4.1.2.7.4. Methyl
yield), mp: 153e155 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 8.68e8.64
2-(6-(3-chlorophenyl)-4-oxo-7-((2-((4-(tri-
(m, 1H), 8.40 (d, J ¼ 49.8 Hz, 1H), 8.07 (d, J ¼ 8.1 Hz, 1H), 7.73 (d,
J ¼ 8.7 Hz, 1H), 7.65 (d, J ¼ 8.7 Hz, 2H), 7.51e7.48 (m, 1H), 7.44e7.42
(m, 2H), 7.41e7.36 (m, 1H), 7.05 (d, J ¼ 8.7 Hz, 2H), 4.92 (d,
J ¼ 27.1 Hz, 2H), 3.89 (t, J ¼ 6.1 Hz, 2H), 3.72 (s, 2H), 3.46 (dd,
J ¼ 18.5, 13.5 Hz, 4H), 3.31 (q, J ¼ 6.3 Hz, 2H), 1.87e1.81 (m, 2H), 1.54
fluoromethyl)benzyl) oxy)ethyl) carbamoyl)quinazolin-3(4H)-yl)ace-
tate (34d). White powder (80% yield), mp: 169e171 ꢁC. 1H NMR
(400 MHz, DMSO‑d6)
d 8.80e8.76 (m, 1H), 8.47 (s, 1H), 8.08 (s, 1H),
7.72 (s, 2H), 7.71 (s, 1H), 7.56 (d, J ¼ 8.0 Hz, 2H), 7.49 (s, 1H),
7.44e7.39 (m, 3H), 4.89 (s, 2H), 4.56 (s, 2H), 3.72 (s, 3H), 3.45 (t,
J ¼ 5.4 Hz, 2H), 3.42e3.37 (m, 2H). 13C NMR (101 MHz, DMSO‑d6)
(d, J ¼ 62.5 Hz, 4H). 13C NMR (101 MHz, DMSO‑d6)
d 168.3, 167.6 (d,
J ¼ 9.6 Hz), 164.3, 161.4, 159.7 (d, J ¼ 4.6 Hz), 149.0, 147.2 (d,
J ¼ 12.2 Hz), 142.9 (d, J ¼ 28.0 Hz), 141.1 (d, J ¼ 14.3 Hz), 136.3 (d,
J ¼ 31.5 Hz), 133.0, 130.2, 128.1, 127.6 (d, J ¼ 7.0 Hz), 127.1 (d,
J ¼ 11.4 Hz), 126.9 (q, J ¼ 3.7 Hz), 126.3 (d, J ¼ 12.9 Hz), 123.3, 121.6
(d, J ¼ 21.0 Hz), 121.0 (d, J ¼ 32.1 Hz), 114.9, 65.4, 52.5, 47.3, 35.8,
d
168.4, 167.7, 159.7, 149.0, 147.1, 143.3, 142.9, 141.0, 136.5, 133.0,
130.2, 128.1, 128.1, 127.8, 127.7, 127.2 (d, J ¼ 3.7 Hz), 126.5, 125.7,
125.1 (q, J ¼ 3.6 Hz), 123.0, 121.5, 71.0, 68.3, 52.5, 47.3. HRMS (ESI):
m/z (M þ Hþ) calcd for C28H24ClF3N3O5, 574.1351, found: 574.1346.
4.1.2.7.5. Methyl
2-(6-(3-chlorophenyl)-7-((3-(4-chlorophenyl)
28.3, 25.2, 23.9. HRMS (ESI): m/z (M
32H31ClF3N4O4, 627.1980, found: 627.1982.
4.1.2.9.2. 6-(3-chlorophenyl)-3-(2-(3,3-difluoropiperidin-1-yl)-2-
þ
Hþ) calcd for
propyl)carbamoyl)-4-
oxoquinazolin-3(4H)-yl)acetate
(34e).
C
White powder (83% yield), mp: 175e177 ꢁC. 1H NMR (400 MHz,
DMSO‑d6)
d
8.62e8.58 (m, 1H), 8.47 (s, 1H), 8.08 (s, 1H), 7.73 (s, 1H),
oxoethyl)-4-oxo-N-(3-(4-(trifluoromethyl)phenoxy)propyl)-3,4-
dihydroquinazoline-7-carboxamide (36b). White powder (80%
7.50 (s, 1H), 7.46e7.41 (m, 3H), 7.33 (d, J ¼ 8.3 Hz, 2H), 7.18 (d,
J ¼ 8.3 Hz, 2H), 4.89 (s, 2H), 3.72 (s, 3H), 3.13 (dd, J ¼ 12.6, 6.5 Hz,
2H), 2.45 (t, J ¼ 7.6 Hz, 2H), 1.68e1.60 (m, 2H). 13C NMR (101 MHz,
yield), mp: 90e91 ꢁC. 1H NMR (400 MHz, DMSO‑d6)
d 8.68e8.64 (m,
1H), 8.35 (d, J ¼ 5.4 Hz, 1H), 8.06 (s, 1H), 7.73 (s, 1H), 7.65 (d,
J ¼ 8.7 Hz, 2H), 7.49 (s, 1H), 7.45 (dd, J ¼ 9.6, 5.1 Hz, 1H), 7.42e7.38
(m, 2H), 7.05 (d, J ¼ 8.6 Hz, 2H), 5.01 (d, J ¼ 21.1 Hz, 2H), 3.99e3.77
(m, 4H), 3.63e3.58 (m, 1H), 3.53 (dd, J ¼ 6.9, 2.7 Hz, 1H), 3.32e3.24
(m, 2H), 2.20e2.03 (m, 2H), 1.83 (dd, J ¼ 11.8, 5.5 Hz, 3H), 1.67e1.56
DMSO‑d6) d 168.3, 167.4, 159.6, 149.0, 147.1, 143.2, 141.0, 140.6, 136.5,
133.0, 130.3, 130.2, 130.2, 128.1, 128.1, 127.7, 127.2, 127.1, 126.4, 121.4,
52.5, 47.3, 38.4, 31.6, 30.2. HRMS (ESI): m/z (M þ Hþ) calcd for
C
27H24Cl2N3O4, 524.1138, found: 524.1136.
4.1.2.7.6. Methyl
2-(6-(3-chlorophenyl)-4-oxo-7-((3-(4-(tri-
(m, 1H). 13C NMR (101 MHz, DMSO‑d6)
d 167.6, 165.5, 161.40 (d,
fluoromethyl)phenoxy) propyl) carbamoyl)quinazolin-3(4H)-yl)ace-
J ¼ 1.1 Hz), 159.7, 149.5, 147.2, 142.9, 141.1, 136.3, 133.0, 130.2, 128.1,
127.6, 127.2, 127.1, 126.9 (q, J ¼ 3.7 Hz), 126.3, 126.0, 123.3, 121.6,
121.2, 114.9, 65.4, 46.8, 43.3, 40.9, 35.7, 31.7, 28.3, 22.4. HRMS (ESI):
m/z (M þ Hþ) calcd for C32H29ClF5N4O4, 663.1792, found: 663.1791.
tate (34f). White powder (88% yield), mp: 190e192 ꢁC. 1H NMR
(400 MHz, DMSO‑d6)
d 8.70e8.65 (m, 1H), 8.46 (s, 1H), 8.08 (s, 1H),
7.74 (s, 1H), 7.65 (d, J ¼ 8.7 Hz, 2H), 7.50 (s, 1H), 7.47e7.38 (m, 3H),
7.05 (d, J ¼ 8.6 Hz, 2H), 4.89 (s, 2H), 3.89 (t, J ¼ 6.1 Hz, 2H), 3.72 (s,
3H), 3.33e3.28 (m, 2H), 1.87e1.79 (m, 2H). 13C NMR (101 MHz,
4.2. Biology
DMSO‑d6) d 168.3, 167.5, 161.4, 159.6, 149.0, 147.1, 143.1, 141.0, 136.5,
133.0, 130.2, 128.7, 128.1, 127.7, 127.2, 127.1, 126.9 (q, J ¼ 3.7 Hz),
126.4, 121.5, 121.0 (d, J ¼ 32.1 Hz), 114.9, 65.4, 52.5, 47.3, 35.7, 28.3.
HRMS (ESI): m/z (M þ Hþ) calcd for C28H24ClF3N3O5, 574.1351,
found: 574.1341.
4.2.1. Animals
Male C57BL/6 mice (6e8 weeks old) were purchased from Vital
River Experimental Animal Co., Ltd. (Beijing, China). Mice were
housed in the specific pathogen-free conditions with a 12 h light/
dark cycle and free access to food and water in the animal research
center laboratory of Tsinghua University. All animal protocols were
conducted in compliance with the Institute of Animal Care and Use
Committee (IACUC) of Tsinghua University approved by institu-
tional and national guidelines.
4.1.2.8. Synthesis of compound 35. To a mixture of 34f (573 mg,
1.0 mmol) in THF (6 mL) and H2O (3 mL), LiOH (72 mg, 3.0 mmol)
was added. The reaction mixture was stirred at room temperature
for 0.5 h and adjusted to neutral (pH ¼ 7) by adding aqueous HCl
solution (1 N) to give the white solid 35 in the yield of 85%.
4.1.2.8.1. 2-(6-(3-chlorophenyl)-4-oxo-7-((3-(4-(trifluoromethyl)
phenoxy)propyl)carbamoyl) quinazolin-3(4H)-yl)acetic acid (35).
White powder (85% yield), mp: 198e200 ꢁC. 1H NMR (400 MHz,
4.2.2. Cell culture
HEK-Blue hNOD1 cells, HEK-Blue hNOD2 cells were purchased
from InvivoGen (San Diego, CA) and cultured in Dulbecco’s modi-
fied Eagle medium (DMEM) (Life Technologies, CA, USA) with 4.5 g/
l glucose (Life Technologies), 10% (v/v) fetal bovine serum (FBS)
(Gibco, Australia), 1% (v/v) penicillin/streptomycin (Life Technolo-
DMSO‑d6) d 8.67e8.64 (m, 1H), 8.45 (s, 1H), 8.08 (s, 1H), 7.73 (s, 1H),
7.65 (d, J ¼ 8.7 Hz, 2H), 7.50 (s, 1H), 7.43 (dd, J ¼ 5.9, 3.0 Hz, 2H), 7.40
(dd, J ¼ 4.6, 1.9 Hz, 1H), 7.05 (d, J ¼ 8.7 Hz, 2H), 4.76 (s, 2H), 3.89 (t,
J ¼ 6.1 Hz, 2H), 3.29e3.20 (m, 2H), 1.83 (dd, J ¼ 12.6, 6.3 Hz, 2H). 13
C
gies), 100 mg/mL Normocin (InvivoGen, San Diego, CA), and addi-
NMR (101 MHz, DMSO‑d6)
d
169.2, 167.6, 161.4, 159.7, 149.2, 147.2,
tional selective antibiotics according to the manufacturer’s
protocol. B16F10 cell line was purchased from ATCC (Mannassas,
VA, USA) and cultured in DMEM, human monocytic leukemia THP1
cell line was kindly provided by Dr. Wanli Liu (Tsinghua University,
Beijing, China) maintained in RPMI-1640 medium (Life Technolo-
gies), supplemented with 10% (v/v) FBS, 1% (v/v) penicillin/strep-
tomycin. All the cells were grown at 37 ꢁC in a 5% CO2 humidified
incubator and guaranteed to be mycoplasma-free.
142.9, 141.1, 136.3, 133.0, 130.2, 128.1, 127.6, 127.2, 127.1, 126.9 (q,
J ¼ 3.7 Hz), 126.3, 125.9, 121.6, 121.0 (d, J ¼ 32.0 Hz), 114.9, 65.4, 47.4,
35.8, 28.3. HRMS (ESI): m/z (M þ Hþ) calcd for C27H22ClF3N3O5,
560.1195, found: 560.1192.
4.1.2.9. Synthesis of compounds 36a and 36b. To a solution of 35
(1.0 mmol) and DIC (464 mL, 3 equiv.) in THF (30 mL), HOSu (345 mg,
3 equiv.) was added, and the reaction mixture was stirred at room
temperature overnight. Then amine (5 equiv.) was added, and the
mixture was continually stirred at room temperature for 3 h.
4.2.3. Screening assay to identify NOD1/2 dual inhibitors
HEK-Blue hNOD1 cells were seeded in 96-well plates at

18
Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
50,000 cells/well, preincubated with compounds (10
and stimulated with 50 ng/mL (~EC80) C12-iE-DAP (InvivoGen, San,
Diego, CA) for 20 h. As we previously described [21], HEK-Blue
m
M) for 3 h,
36b were formulated in DMSO/Cremophor EL/saline at 5:5:90
(v:v:v). 11 days after implantation, primary tumor growths were
measured using vernier calipers to determine the two orthogonal
axes. The tumor volumes were calculated by formula (1/2) a2b,
where a is the shorter axis and b is the longer axis. 16 days after
implantation, all mice were sacrificed. The primary tumors were
removed, weighed, and photographed. Tumor weight inhibition
was calculated by the following formula: [(CꢂT)/C] ꢀ 100 (C, tumor
weight of vehicle group; T, tumor weight of treated group).
hNOD2 cells were pretreated with different compounds (10 mM)
for 3 h and then stimulated with 100 ng/mL (~EC80) MDP (Inviv-
oGen) for an additional 20 h. SEAP was detected using HEK-Blue
detection (InvivoGen) and measured by a spectrophotometer at
655 nm according to the manufacturer’s instructions. The inhibi-
tion rate (%) was determined using the following formula: inhibi-
tion (%) ¼ {[C12-iE-DAP (OD655) or MDP (OD655) e compounds
(OD655)]/[C12-iE-DAP (OD655) or MDP (OD655)]} ꢀ 100. Once the
inhibition rate (%) was >50%, compounds were retested, and the
SRB assay was conducted to exclude cytotoxicity. The IC50 values
were determined using the GraphPad Prism 7 software [21].
4.2.8. Statistical analysis
Statistical analyses were performed by two tailed Student’s t-
test, P values are indicated in the figure with statistically significant
(P < 0.05). All results were confirmed in at least three independent
experiments. All quantitative data were statistical analyzed with
GraphPad software7.0.
4.2.4. In vitro metabolism and stability study
In stability studies using mouse liver microsomes, the incuba-
tion mixture contained phosphate buffer (100 mM, pH 7.4), liver
Declaration of competing interest
microsomal protein (1.25 mg/mL) and compound (1.25
mM) in a
total volume of 20
m
L. After pre-incubation at 37 ꢁC for 5 min, the
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
reaction was initiated by adding an NADPH generating system
[NADP (1 mM), MgCl2 (10 mM), glucose 6-phosphate (10 mM),
glucose-6-phosphate dehydrogenase (0.5 U/mL)]. The samples
were then collected at 0, 5, 15, 30, 60 and 120 min after incubation,
Acknowledgments
and the reaction was terminated by adding 300 mL Methanol/ACN
(1:1, v/v). The mixtures were then centrifuged, and the superna-
tants were tested by LCeMS/MS assay. These assays were per-
formed by BioDuro Technology Co., Ltd. (Shanghai, China).
We gratefully acknowledge the funding support of grants from
the National Natural Science Foundation of China (Grants
81803358, 81703329, 81273364 and 81773114) and Non-profit
Central Research Institute Fund of Chinese Academy of Medical
Sciences (2019-RC-HL-008).
4.2.5. Quantitative real-time PCR
The expression of mRNA was determined by quantitative
reverse transcriptase-PCR analysis as previously described [21] and
normalized to GAPDH. The following probes from Applied Bio-
systems were used for PCR analysis: human IL-6
(Hs00985639_m1), human IL-8 (Hs00174103_m1), human TNF-
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2020.112723.
a
(Hs01113624_g1), human GAPDH (Hs02758991_g1).
Abbreviations
4.2.6. Western blotting
Cells were lysed in cold whole cell lysis buffer and the protein
expression levels were determined as previously described [21].
Ac
AcOEt
acetyl group
acetic ether
Antibodies phosphor-IKK
a
/b
(Ser176/180) (16A6) (no. 2697S), IKK
a
BMDMs bone marrow derived macrophages
(no. 2682S), IKK (L570) (no. 2678S), phosphor-p65 (Ser536) (no.
b
Bz
benzoyl
3033), phosphor-p38 (no. 4631), p38 (no. 9212S), phosphor-JNK
(no. CST9251S), JNK (no. 9252S), phosphor-RIP2 (no. 14397S),
BZD
1, 4-benzodiazepine-2, 5(H)-dione
C12-ie-DAP lauroyl-g-D-glutamyl-meso-diaminopimelic acid
RIP2 (no. 4142) and I
Signaling Technology (Danvers, USA). Phosphor-ERK1/2 (sc-7383)
and ERK1/2 (sc-94) antibodies were purchased from Santa Cruz
k
B
a
(no. 9242S) were obtained from Cell
CARD
Calcd
CTX
caspase activation and recruitment domain
calculated
cyclophosphamide
Biotechnology (Santa Cruz, CA). AntieNFe
k
B p65 (ab16502), anti-
d
doublet
beta Actin antibody (HRP) (ab49900), goat polyclonal secondary
antibody to rabbit IgG-H&L-HRP (ab6721) and rabbit polyclonal
secondary antibody to mouse IgG-H&L-HRP (ab6728) were from
Abcam (Cambridge, UK). The protein bands were developed by
chemiluminescence (Thermo Scientific, Rockford, USA) with
ChemiDoc XRSþ (Bio-Rad).
DAMPs
DCM
DIC
DMAP
DMF
DMSO
DTX
damage/danger-associated molecular patterns
dichloromethane
N,N0-Diisopropylcarbodiimide
4-dimethylaminopyridine
N, N0-dimethylformamide
dimethyl sulfoxide
docetaxel
4.2.7. Animal experiment
eq
equivalent
C57BL/6 mice were kept in the animal research center labora-
tory of Tsinghua University for 1 week to adapt the environment.
Each mouse was subcutaneously injected with B16F10 melanoma
cells (2 ꢀ 105/0.1 mL in PBS) on the shoulder and left hind limb
towards skeletal muscle. One day after implantation of cells, mice
were divided into 4 groups randomly (n ¼ 7 for each group):
control vehicle, PTX, 36b, PTX combined with 36b. Mice were
received intravenous injections with control vehicle or 36b (50 mg/
kg) every other day while PTX (12 mg/kg) every 4 days. PTX and
ESI-MS
EtOAc
EtOH
Glu
electrospray ionization-mass spectrometry
ethyl acetate
ethanol
glutamic acid
glycine
Gly
HOAc
HOSu
HPLC
HR-MS
acetic acid
N-hydroxysuccinimide
high performance liquid chromatography
high resolution-mass spectrometry

Y. Ma et al. / European Journal of Medicinal Chemistry 207 (2020) 112723
19
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