Design, synthesis and biological activities of 2,3-dihydroquinazolin-4(1H)-one derivatives as TRPM2 inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.04.045

Transient receptor potential melastatin 2 (TRPM2), a Ca²⁺-permeable cationic channel, plays critical roles in insulin release, cytokine production, body temperature regulation and cell death as a reactive oxygen species (ROS) and temperature sensor. However, few TRPM2 inhibitors have been reported, especially TRP-subtype selective inhibitors, which hampers the investigation and validation of TRPM2 as a drug target. To discover novel TRPM2 inhibitors, 3D similarity-based virtual screening method was employed, by which 2,3-dihydroquinazolin-4(1H)-one derivative H1 was identified as a TRPM2 inhibitor. A series of novel 2,3-dihydroquinazolin-4(1H)-one derivatives were subsequently synthesized and characterized. Their inhibitory activities against the TRPM2 channel were evaluated by calcium imaging and electrophysiology approaches. Some of the compounds exhibited significant inhibitory activity, especially D9 which showed an IC₅₀ of 3.7 μM against TRPM2 and did not affect the TRPM8 channel. The summarized structure-activity relationship (SAR) provides valuable insights for further development of specific TRPM2 targeted inhibitors.

Full text of DOI:10.1016/j.ejmech.2018.04.045

Accepted Manuscript
Design, synthesis and biological activities of 2,3-dihydroquinazolin-4(1H)-one
derivatives as TRPM2 inhibitors
Han Zhang, Huan Liu, Xiao Luo, Yuxi Wang, Yuan Liu, Hongwei Jin, Zhenming Liu,
Wei Yang, Peilin Yu, Liangren Zhang, Lihe Zhang
PII:
S0223-5234(18)30385-4
10.1016/j.ejmech.2018.04.045
EJMECH 10394
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 31 January 2018
Revised Date: 6 April 2018
Accepted Date: 21 April 2018
Please cite this article as: H. Zhang, H. Liu, X. Luo, Y. Wang, Y. Liu, H. Jin, Z. Liu, W. Yang, P. Yu,
L. Zhang, L. Zhang, Design, synthesis and biological activities of 2,3-dihydroquinazolin-4(1H)-one
derivatives as TRPM2 inhibitors, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.04.045.
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ACCEPTED MANUSCRIPT

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Design, synthesis and biological activities of 2,3-dihydroquinazolin-4(1H)-one derivatives as
TRPM2 inhibitors
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Han Zhang a1, Huan Liu b1, Xiao Luo a, Yuxi Wang a, Yuan Liu a, Hongwei Jin a, Zhenming Liu a, Wei
Yang b, Peilin Yu c*, Liangren Zhang a*, Lihe Zhang a
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a State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences,
Peking University, Beijing 100191, PR China
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b Department of Neurobiology, Key Laboratory of Medical Neurobiology of the Ministry of Health of
China, Zhejiang University School of Medicine, Hangzhou, Zhejiang 310058, P. R. China
c Department of Toxicology, School of Public Health, Zhejiang University, Hangzhou, Zhejiang
310058, P. R. China
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* Corresponding authors:
E-mail: liangren@bjmu.edu.cn or yupeilin@zju.edu.cn
1 These authors contributed to the work equally.
Abstract
Transient receptor potential melastatin 2 (TRPM2), a Ca2+-permeable cationic channel, plays
critical roles in insulin release, cytokine production, body temperature regulation and cell death as a
reactive oxygen species (ROS) and temperature sensor. However, few TRPM2 inhibitors have been
reported, especially TRP-subtype selective inhibitors, which hampers the investigation and validation
of TRPM2 as a drug target. To discover novel TRPM2 inhibitors, 3D similarity-based virtual
screening method was employed, by which 2,3-dihydroquinazolin-4(1H)-one derivative H1 was
identified as a TRPM2 inhibitor. A series of novel 2,3-dihydroquinazolin-4(1H)-one derivatives were
subsequently synthesized and characterized. Their inhibitory activities against the TRPM2 channel
were evaluated by calcium imaging and electrophysiology approaches. Some of the compounds
exhibited significant inhibitory activity, especially D9 which showed an IC50 of 3.7 µM against
TRPM2 and did not affect the TRPM8 channel. The summarized structure-activity relationship (SAR)
provides valuable insights for further development of specific TRPM2 targeted inhibitors.
Key words:
TRPM2; 2,3-dihydroquinazolin-4(1H)-ones; inhibitors; SAR; virtual screening

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1. Introduction
Transient receptor potential (TRP) channels belong to a novel family of cationic ion channels
with strong impacts on cellular functions and signaling pathways [1]. The mammalian TRP channel
superfamily is composed of 28 members that are grouped into six subfamilies based on their
homology of amino acid sequences [2,3]. Transient receptor potential melastatin (TRPM), a
subfamily of TRP channels, contains eight subtypes that can be divided into four pairs based on their
homology: TRPM1/TRPM3; TRPM2/TRPM8; TRPM4/TRPM5 and TRPM6/TRPM7 [4]. The
TRPM2 channel is a multifunctional nonselective cation channel that is expressed in a range of
tissues including brain, bone marrow, spleen, heart, liver and lung [2,4]. As a ROS sensor, TRPM2
plays important roles in a variety of cells, contributing to cellular functions including insulin release,
cytokine production, cell motility, endothelial hyperpermeability and cell death [5,6]. Moreover, it
also functions as an important temperature sensor in a subpopulation of hypothalamic neurons,
monitoring internal body temperature [7,8]. Therefore, the TRPM2 channel has been reported to be
involved in many pathological processes such as ischemia/reperfusion injury [9], diabetes [10],
neurodegeneration [11], cardiovascular diseases [12] and chronic inflammation [5,13]. Mice
knockout studies have shown that inhibition of TRPM2 reduced CA1 pyramidal neuronal death and
memory impairment in a bilateral common carotid artery occlusion (BCCAO) model [14], reduced
inflammatory markers in bleomycin-induced lung inflammation [13], and attenuated inflammatory
and neuropathic pain in various pain models [15]. Therefore, development of TRPM2 inhibitors will
not only provide direct molecular tools for the pharmacological researches related to this channel, but
also lead to therapeutic advances for diseases mediated by this channel.
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Activation of the TRPM2 channel has been widely studied, which provides us information for
the design of its inhibitors. The C terminus of TRPM2 channel has a NUDT9 homology (NUDT9-H)
domain that is homologous to the NUDT9 adenosine diphosphate ribose (ADPR) hydrolase (∼50%
similarity) [6]. And the TRPM2 channel has been proved to be primarily activated by binding of
ADPR to the NUDT9-H domain [16], of which the binding pocket has been revealed by one of our
recent work [17]. Besides, as a redox-sensitive channel, TRPM2 can also be activated by agents that
produce reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), although the mechanism
of action remains debatable [18].
Several TRPM2 inhibitors have been reported with inhibitory activities at micromolar levels,
among which are imidazole or anthranilic acid derivatives, such as econazole, clotrimazole [19],
flufenamic acid (FFA) [20,21], 2-(3-methylphenyl) aminobenzoic acid (3-MFA) [21],
N-(p-amylcinnamoyl) anthranilic acid (ACA) [22] and 2-aminoethoxydiphenyl borate (2-APB) [23]
(Fig. 1). However, most of these compounds lack specificity, especially considering that the TRPM8

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channel has high homology with TRPM2. For instance, although 20 µM ACA completely blocked
both ADPR-induced whole-cell currents and H2O2-induced Ca2+ signals (IC50 = 1.7 µM) in HEK293
cells transfected with human TRPM2, it also blocked the TRPM8 channel with an IC50 value of 3.9
µM (Ca2+ signals) [22]. In addition, several nucleotide analogues (Fig. 1) such as AMP [24,25],
8-bromoadenosine 5′-diphosphoribose (8-Br-ADPR) [26] and a series of synthesized ADPR
analogues (i.e., 8-phenyl-2′-deoxy-ADPR and our recently discovered compound 8a) [27,28] have
also been reported as TRPM2 inhibitors. They usually function as competitive inhibitors to block the
binding of ADPR to NUDT9-H domain [24-26]. For instance, AMP inhibits TRPM2-mediated
currents with an IC50 of about 70 µM as a negative feedback regulator [6,24,25]. Although these
nucleotide analogues are thought to selectively inhibit TRPM2, due to multiple hydroxyl groups and
negatively charged pyrophosphate group, its poor membrane permeability limited their application
[28,29]. Recently, scalaradial was identified as the active component in an extract of Cacospongia
that showed strong TRPM2 inhibitory activity, but it was an indirect effect on the channel [30].
Therefore, discovery of novel TRPM2 inhibitors is required to better understand the channel
functions and for the potential therapeutic development of this class of agents.
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3D similarity-based virtual screening is one of the ligand-based virtual screening (LBVS)
methods, which has resulted in the identification of many active compounds in drug discovery
programs [31]. In this study, AMP was used as the query structure in 3D similarity-based virtual
screening method to achieve scaffold hopping. As a result, 2,3-dihydroquinazolin-4(1H)-one
derivative H1 was discovered to be a novel TRPM2 inhibitor. A variety of H1 derivatives were
further designed by modification of substituted groups or positions on the scaffold to elucidate the
structure-activity relationship (SAR) and discover the most active compounds. The TRPM2
inhibitory activities of all synthesized compounds were assessed by calcium imaging and
electrophysiology approaches. In addition, the inhibitory activities of the most active compounds
against TRPM8 were also evaluated. Herein, we report the synthesis and biological investigation of
these compounds, which will definitely promote the discovery of novel specific TRPM2 inhibitors.

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Fig. 1. Chemical structures of some TRPM2 inhibitors.
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2. Results and discussion
2.1. Virtual screening and biological evaluation
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In an effort to discover novel inhibitors of TRPM2, 3D shape- and electrostatic-based
similarity virtual screening approaches were employed (Fig. 2A). The optimized conformation of
AMP (Fig. 2B) was used as query to search SPECS (http://www.specs.net/) and PKU_CNCL
(http://www.pkucncl.cn/) databases by ROCS 3.1.2 (OpenEye Scientific Software, Inc) program. As
a result, the top 3,000 compounds were retained based on ‘Shape Tanimoto’ score in each structure.
EON 2.1.0 (OpenEye Scientific Software, Inc) was then employed to calculate the electrostatic
distribution similarity of the retained compounds according to the original query, and the top 300
compounds were retained based on the ‘ET_pb’ score in each structure. In the end, 600 compounds
were clustered and visually inspected. After the synthetic feasibility was evaluated, 24 compounds
(Fig. S1, Table S1) were selected for biological evaluation.
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Fluorescent calcium imaging was used to preliminarily evaluate the inhibitory activities of

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these 24 compounds in HEK293 cells stably expressing human TRPM2. After incubated with cells
for 30 min at a concentration of 30 µM, compounds H1 and H18 significantly reduced H2O2-induced
Ca2+ fluorescence increase (Fig. S2). Whole-cell patch-clamp recordings were then performed on H1
to further validate its inhibitory activity. When added intracellularly and simultaneously with 500 µM
ADPR, H1 showed no inhibition on ADPR induced TRPM2 currents at either low (30 µM) or high (1
mM) concentrations (Fig. S3). In contrast, extracellular addition of H1 at 30 µM resulted in a
reduction of TRPM2 current by 55% (Fig. S4). These results indicated H1 was a novel extracellular
inhibitor of the TRPM2 channel.
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AMP is one of the hydrolysis products of ADPR, and potentially represents an autoregulatory
negative feedback mechanism of TRPM2 activity. The inhibitory activity of AMP is thought to be the
result of a competition for the NUDT9-H domain [24,25]. Since H1 was identified by 3D similarity
virtual screening based on AMP structure, it should in theory have a similar mode of action as AMP
that acts at the intracellular NUDT9-H domain. However, H1 only inhibits the TRPM2 current
extracellularly, which led us to hypothesize that there are also extracellular binding sites for AMP on
the TRPM2 channel. Thus, whole-cell patch-clamp recordings were used to investigate this
hypothesis. A concentration-dependent extracellular inhibition by AMP on the TRPM2 currents was
indeed observed (Fig. S5).
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Previous studies including ours indicated several metals or compounds inhibited TRPM2
activity through the extracellular pore region of TRPM2 [32-34]. Considering that H995A is a key
residue involved in several metals induced TRPM2 inhibition or inactivation, while D964A has no
effect on TRPM2 function. To determine whether H1 and AMP interacted with the extracellular pore
region of the TRPM2 channel, we detected the effects of H1 and AMP on mutants of these two
residues as previously described [32]. Similar to wild-type (WT) TRPM2 channel, the inhibition by
H1 for the D964A mutant was rapid and about 50%. In contrast, in cells expressing the H995A
mutant channel, substantial inhibition did not occur in response to extracellular H1 over several
minutes (Fig. 2D). On average, H1 resulted in a reduction of ADPR-induced currents by 40 % and 4 %
for the D964A and H995A mutant channel, respectively. Residual currents were abolished by
subsequent application of pH = 5. We also tested the inhibitory effect of AMP in the D964A and
H995A mutant channels, and it was similar to that of H1 (Fig. 2E). These results implied that the
His995 residue was an important molecular determinant conferring on the TRPM2 channel the

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sensitivity to inhibition by extracellular AMP and H1, suggesting that both these two compounds
shared the same mechanism in inhibiting TRPM2 through interacting with the extracellular pore
region.
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Fig. 2. 3D similarity-based virtual screening and inhibition of the TRPM2 current by H1 in HEK293 cells stably
expressing human TRPM2. (A) Flow chart illustrating the virtual screening process. (B) The ROCS query
generated from optimized conformation of AMP. (C) Chemical structure of H1 is divided into four moieties
according to different rings. (D) and (E) Representative 500 µM ADPR-induced currents for WT or indicated
mutants with extracellular treatment of H1 and AMP in whole-cell electrophysiology recordings. Data were
presented as mean ± SEM from six independent experiments (n = 6), and NS were presented as no significant
difference for indicated comparisons, ***P < 0.005.
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2.2. Chemistry

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Further structural optimizations were performed on H1 to discover more active extracellular
inhibitors of the TRPM2 channel. Starting from the molecular structure of H1, we divided H1 into
four moieties A, B, C and D according to the different rings for structural modifications (Fig. 2C).
The general synthetic routes for preparation of H1, Class A, B, C and D are described in Scheme 1-
Scheme 9.
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Treatment of anthranilic acids (1a-1q, 1w, 1x) with EDC•HCl and NH4Cl or methyl
anthranilates (2r-2v) with NH3•H2O afforded anthranilic diamides (3a-3x) [35]. Compounds H1,
A1−A23 were obtained at 45%−67% yields from 3a−3x and commercially available
3-phenyl-1H-pyrazole-4-carbaldehyde (4a) through cyclocondensation reaction under the catalysis of
cyanuric chloride (Scheme 1, Scheme 2) [36].
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Scheme 1. Synthesis of compounds H1, A1−A21. Reagents and conditions: (a). EDC•HCl, HOBt, NH4Cl, DIPEA,
DMSO, r.t., 15 h; (b). NH3•H2O, 100 °C, 12 h; (c). 4a, cyanuric chloride, CH3CN, MeOH or DMSO, r.t., 0.5-2 h.

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Scheme 2. Synthesis of compounds A22, A23. Reagents and conditions: (a). EDC•HCl, HOBt, NH4Cl, DIPEA,
DMSO, r.t., 15 h; (b). 4a, cyanuric chloride, DMSO, r.t., 0.5-2 h.
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Cyclocondensation reactions were accomplished from 4a and 5a or 5b by the catalysis of
2-morpholinoethanesulfonic acid in aqueous 50% ethanol to form compounds A24 or A25,
respectively (Scheme 3) [37].
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Scheme 3. Synthesis of compounds A24, A25. Reagents and conditions: (a). 2-morpholinoethanesulfonic acid, 50 %
EtOH (aq), 60 °C, 3 h.
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2-Aminobenzenesulfonamide (6) was reacted with 4a or 4e in the presence of Amberlyst-15
under ultrasound irradiation (40 KHz) to afford compounds B1 or B2, respectively (Scheme 4) [38].
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Scheme 4. Synthesis of compounds B1, B2. Reagents and conditions: (a). Amberlyst-15, MeOH, r.t., 40 min.
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Reactions of 3a-3d, 3t or 3w with 4a under the catalysis of CuO at 120 °C produced
compounds B3-B8 (Scheme 5).
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Scheme 5. Synthesis of compounds B3-B8. Reagents and conditions: (a). CuO, DMA, 120 °C, 24 h.
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Treatment of substituted bromotoluene (7a-7e) with 1H-imidazole-4-carbaldehyde in the
presence of anhydrous Na2CO3 and tetra-n-butylammonium iodide produced 8a-8e [39], which were
then reacted with 3a using cyanuric chloride as catalyst to afford compounds C1-C6 (Scheme 6).
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Scheme 6. Synthesis of compounds C1-C6. Reagents and conditions: (a). anhydrous Na2CO3,
tetra-n-butylammonium iodide, THF, refluxed, 3 h; (b). 3a, cyanuric chloride, DMSO, r.t., 0.5-2 h.
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Commercially available 1-(4-chlorobenzyl)-1H-imidazole-5-carbaldehyde was reacted with 3a
in DMSO under the catalysis of cyanuric chloride to afford compound C7 (Scheme 7).

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Scheme 7. Synthesis of compound C7. Reagents and conditions: (a). cyanuric chloride, DMSO, r.t., 2 h.
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Pyrrole-2-carbaldehyde was reacted with N-bromosuccinimide in THF to give
4-bromo-1H-pyrrole-2-carbaldehyde, which was converted to 4-phenyl-1H-pyrrole-2-carbaldehyde
(9) through Suzuki coupling reaction with phenylboronic acid under the catalytic system of
Pd(PPh3)4 [40]. Subsequently, compound C8 was prepared from 3a and 9 through cyclocondensation
reaction under the catalysis of cyanuric chloride (Scheme 8).
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Scheme 8. Synthesis of compound C8. Reagents and conditions: (a). N-bromosuccinimide, THF, 0 °C, 15 min; (b).
phenylboronic acid, Pd(PPh3)4, K2CO3, dioxane, water, 105 °C, 24 h; (c). 3a, cyanuric chloride, DMSO, r.t., 2 h.
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Methyl aryl ketones (10b-10h) were reacted with semicarbazide hydrochloride and anhydrous
CH3COONa in ethanol to afford semicarbazones (11b-11h), which were then formylated by reacting
with POCl3-DMF complex to form 3-arylpyrazole-4-carboxaldehydes (4b-4h) [41]. Compounds
D1-D12 were obtained from 3a, 3b, 3k or 3s and 4b-4h through cyclocondensation reaction in the
presence of cyanuric chloride (Scheme 9).

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Scheme 9. Synthesis of compounds D1-D12. Reagents and conditions: (a). semicarbazide hydrochloride,
anhydrous CH3COONa, EtOH, reflux, 45 min, then methyl aryl ketone, reflux, 1 h, then r.t., 10 h; (b). POCl3, DMF,
0 °C, 1 h, then semicarbazone, 80 °C, 1.5 h; (c). 3a, 3b, 3k or 3s, cyanuric chloride, MeOH or DMSO, r.t., 2 h.
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2.3. Inhibitory activity and SAR
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Totally, 53 compounds were synthesized and their biological activities were investigated.
Fluorescent calcium imaging was used to evaluate their inhibitory activities preliminarily, and 33 of
them obviously reduced H2O2-induced Ca2+ fluorescence increase (Fig. 3). Of them, 23 compounds
reduced more than 50% Ca2+ fluorescence increase compared with the vehicle-treated cells. Their
inhibitory activities against TRPM2 were then validated using whole-cell patch-clamp recordings
(Table 1). At the concentration of 30 µM, 9 compounds inhibited about 90% TRPM2 currents. Their
inhibitory activities at 3 µM were further tested. The concentration-dependent response curves of
compounds that blocked more than half of the TRPM2 current at the concentration of 3 µM were
plotted to calculate IC50 values. Four compounds, i.e. A1, A10, D1 and D9, gave IC50 values of
3.7-5.1 µM (Fig. 4, Fig. S6). The TRPM2 nonspecific inhibitor ACA was used as a positive control.
As shown in Fig. 3 and Table 1, when the benzene ring of 2,3-dihydroquinazolin-4(1H)-one
was replaced with pyridine, imidazole and thiazole (A22-A25), no inhibitory activity was observed
for the resulting compounds. The introduction of alkyl, nitryl or halogen substituent (Me, NO2, F, Cl,
or Br) into the 5-, 6-, 7- and 8-positions of the 2,3-dihydroquinazolin-4(1H)-one (A2, A4-A7, A17,
A19 and A21) decreased the inhibitory activity compared with H1. However, a strong inhibitory
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activity
(IC50
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8-position
disubstituted
2,3-dihydroquinazolin-4(1H)-one derivative (A1) (6-chloro -8-methyl) (Fig. 4). To further investigate

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the SAR of 6- and 8-position disubstituted 2,3-dihydroquinazolin-4(1H)-one derivatives, these
positions were substituted with different groups (A3, A8-A16, A18, A20). We found compound A10
(6-bromo-8-methyl) and A18 (6-chloro-8-iodo) maintained the inhibitory activity, particularly A10
showed an IC50 value of 4.6 µM (Fig. S6). But the other derivatives exhibited less potency than H1.
These results indicated that introduction of a chlorine or bromine atom at 6-position accompanied by
the presence of an iodine or methyl at 8-position were favorable for improving potency.
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By replacing the carbonyl group at 4-position of the 2,3-dihydroquinazolin-4(1H)-one with
sulfonyl group, compounds B1 and B2 were formed but no inhibitory activity was observed.
Meanwhile, the introduction of carbon-carbon double bonds at the 1- and 2-position of the
2,3-dihydroquinazolin-4(1H)-one was investigated (B3-B8). It was found that the inhibitory
activities were decreased. These results implied that the scaffold of 2,3-dihydroquinazolin-4(1H)-one
is crucial for the inhibitory activity against TRPM2.
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Replacing pyrazole ring at 2-position of the 2,3-dihydroquinazolin-4(1H)-one with imidazole
ring along with the presence of substituted benzyl group at 1’-position of imidazole ring (C1-C6)
also resulted in no inhibitory activity. To maintain the relative position of ring A and B
(2,3-dihydroquinazolin-4(1H)-one) and ring D (substituted benzyl group), compound C7 with
substituted benzyl group at 5’-position of imidazole ring was synthesized, and a decreased inhibitory
activity was observed. Compound C8 with a pyrrole ring exhibited less potency than the pyrazole
ring compound (H1), suggesting the importance of pyrazole ring for the inhibitory activity.
To further study the SAR of pyrazole ring derivatives, various substituted derivatives at
3’-position of the pyrazole ring were investigated (D1-D8). We found that: i) Removing the phenyl
group at 3’-position of pyrazole ring (D3) exhibited lower inhibitory activity than A1. ii) Replacing
the phenyl group at 3’-position of pyrazole ring with naphthyl group (D1) retained the inhibitory
activity (IC50 = 4.0 µM) (Fig. S6). iii) A p-bromophenyl substituent at 3’-position of pyrazole ring
(D7) displayed higher potency than the corresponding o-bromophenyl substituent (D6), which
indicated that compounds containing p-substituted phenyl group could exhibit improved inhibitory
activity. And compound D2 (p-nitrophenyl) showed improved inhibitory activity compared with D7.
These results demonstrated that the introduction of naphthyl or p-nitrophenyl substituent at
3’-position of pyrazole ring retained the inhibitory activity.
The 6, 8-position disubstituted 2,3-dihydroquinazolin-4(1H)-one (6-bromine-8-methyl and

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6-chlorine-8-iodine) and the 3’-position substituted pyrazole ring (naphthyl and p-nitrophenyl) were
combined (D9-D12) to generate more active inhibitors, and compound D9 expectedly exhibited
strong inhibitory activity with an IC50 = 3.7 µM (Fig. S6). Subsequently, we tested the inhibitory
effect of D9 in the D964A and H995A mutant channels. It exhibited less potency in the D964A
mutant TRPM2 channel compared with the WT, and no inhibitory activity was observed in the
H995A mutant TRPM2 channel (Fig. S7). These results suggested that both D964A and H995A were
critically involved in the inhibition by extracellular D9.
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Fig. 3. Inhibition activity of compounds A1-A25, B1-B8, C1-C8 and D1-D8 against the TRPM2 channel
preliminarily evaluated through calcium imaging method. Data were presented as mean ± SEM from at least three
independent experiments, and ***P < 0.005, **P < 0.01, *P < 0.05.
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Fig. 4. Inhibition of the TRPM2 currents by A1 in whole-cell patch-clamp recordings. (A) Representative currents
of 500 µM ADPR-induced TRPM2 with extracellular treatment of compound A1 at different concentrations. (B)
Concentration-dependent inhibitory curve of compound A1. Data were presented as mean ± SEM from six
independent experiments (n = 6).
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Table 1
Inhibition activity of selected compounds against TRPM2 currents validated by whole-cell patch-clamping
30 µM
Inhibition (%)
68.1±1.9%
90.2±2.4%
27.9±2.2%
60.5±4.1%
42.7±5.5%
92.8±0.3%
50.4±4.5%
37.2±7.0%
39.2±1.4%
89.4±3.9%
34.8±0.9%
28.7±2.1%
21.2±1.2%
90.2±4.8%
3 µM
compds
IC50 (µM)
Inhibition (%)
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5.1
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H1
A1
46.4±2.8%
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A2
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A3
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A8
50.1±2.5%
4.6
--
A10
A11
A13
A17
A18
A21
B3
--
--
--
--
--
39.6±6.2%
--
--
--
--
--
--
--
B6
50.9±1.8%
4.0
D1

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90.4±1.5%
16.9±5.1%
17.0±4.9%
17.9±4.0%
51.0±7.2%
10.3±2.9%
--
43.4±3.6%
--
--
D2
D3
--
--
--
D5
--
--
D6
--
--
D7
--
--
D8
48.7±5.1%
42.1±3.7%
19.9±4.8%
34.9±7.1%
75.9±6.8%
3.7
--
D9
--
D10
D11
D12
ACA
--
--
--
--
89.1±3.2%
0.32
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Percent inhibition at a test concentration are mean ± SEM from six determinations (n = 6).
“--” not determined.
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4
2,3-Dihydroquinazolin-4(1H)-one derivatives possess a chiral center on C2 position. To study
the influence of chirality on the TRPM2 inhibitor properties, we performed chiral separation on
racemic A1 using chiral high-performance liquid chromatography (HPLC) and obtained two
enantiomers of A1 (A1-a: [α]2D0 = -165.3 and A1-b: [α]2D0 = +164.0). We then characterized the
absolute configuration of A1-a and A1-b by comparing experimental and calculated electronic
circular dichroism (ECD) spectra [42]. As indicated in Fig. 5A, the calculated ECD spectrum of
R-A1 was similar to the experimental ECD spectrum of A1-a. Therefore, the absolute configuration
of A1-a was confirmed to be R-A1. Similarly, the absolute configuration of A1-b was determined to
be S-A1. Whole-cell patch-clamp recordings were performed on A1, R-A1 and S-A1, respectively, at
concentration of 30 µM. A1, R-A1 and S-A1 showed similar inhibitory activity against TRPM2 (Fig.
5B), suggesting the chirality had no influence on the inhibitory activity.
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Fig. 5. Determination of the absolute configurations of two enantiomers of A1 and inhibition of the TRPM2
currents by A1, R-A1 and S-A1 in whole-cell patch-clamp recordings. (A) Determination of the absolute
configurations of two enantiomers of A1 via a comparison of the calculated ECD spectra for R-A1 and S-A1 with
the experimental ECD spectra for A1-a and A1-b. Calculated ECD spectra for R-A1 at the B3LYP/6-31G* level in
MeOH solution with the IEFPCM model (green line); calculated ECD spectra for S-A1 at the B3LYP/6-31G* level
in MeOH solution with the IEFPCM model (red line); experimental spectra for A1-a in MeOH (dark blue line);
experimental spectra for A1-b in MeOH (yellow line). (B) Representative currents with extracellular treatments of
30 µM A1 (purple), 30 µM R-A1 (green) and 30 µM S-A1 (red) showed similar inhibitory activity. Data were
presented as mean ± SEM from six independent experiments (n = 6), and NS were presented as no significant
difference for indicated comparisons.
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Taken together, we found that naphthyl at 3’-position, methyl at 6-position and chlorine or
bromine at 8-position of the 2,3-dihydroquinazolin-4(1H)-one were favorable for the inhibitory
activity. Moreover, the chirality had no influence on inhibitory activity. Our investigation confirmed

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that 2,3-dihydroquinazolin-4(1H)-one was a crucial scaffold, and the size and orientation of the
substituents on the scaffold played key roles in the inhibitory activity against TRPM2.
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5
2.4. Inhibitory activity of Compounds A1, A10, D1 and D9 on the TRPM8 channel
The TRPM8 channel is the closest related channel of TRPM2, and they share approximately
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42% sequence identity [2]. The known non-nucleoside TRPM2 inhibitors such as 2-APB and ACA
could also block the TRPM8 channel [22,43]. To evaluate the selectivity of A1, A10, D1 and D9, the
inhibitory activities of these compounds (30 µM) on the TRPM8 channel were tested by whole-cell
patch-clamp recordings. TRPM8 currents overexpressed in HEK293 cells were activated by menthol
and blocked by 2-APB.
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As shown in Fig. 6, compared with the control, extracellular addition of D1 and D9 showed no
significant effect on the TRPM8 currents, while A1 and A10 resulted in a mild reduction of TRPM8
current by 27% and 19%, respectively. The intracellular effects of these compounds were also tested,
in which their applications in pipette exhibited similar effects to that of the extracellular treatments
(Fig. S8). It is noteworthy that both D1 and D9 had a naphthyl group at the 3’-position of pyrazole
ring, while A1 and A10 each had a phenyl group at this position, suggesting that the bulky naphthyl
group was unfavorable for the inhibition of TRPM8 channel, and the substitute group at 3’-position
of pyrazole ring played critical roles in the selectivity between the TRPM2 and TRPM8 channels.
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Fig. 6. Selectivity evaluations of extracellular 30 µM A1, A10, D1 and D9 on the TRPM8 channel. (A)
Representative currents with extracellular treatments of 30 µM compounds. (B) The effect of control with its
agonist (1 mM menthol) and inhibitor (0.1 mM 2-APB) on the TRPM8 channel (n = 5). (C) Mean peak currents

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from recordings of (A) and (B), data were presented as mean ± SEM from five independent experiments (n = 5),
and ***P < 0.005.
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4
3. Conclusion
5
In this study, we discovered a potent TRPM2 inhibitor 2,3-dihydroquinazolin-4(1H)-one
derivative H1 by 3D similarity-based virtual screening. Fifty-three H1 derivatives were designed and
synthesized. Some of them exhibited strong inhibitory activity against the TRPM2 currents,
especially compound D9 with an IC50 value of 3.7 µM. More importantly, this compound showed
specificity to the TRPM2 channel without affecting the TRPM8 current. In addition, the summarized
SAR provided insights for the further development of specific TRPM2 inhibitors.
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4. Experimental section
4.1. Materials and methods
All of chemicals and solvents used were obtained from commercial sources. The solvents were
dried by standard procedures. Thin layer chromatography (Silica gel GF254, Qingdao Haiyang
Chemical Co., Ltd, Qingdao, China) was employed to monitor reaction process, and silica column
chromatography was carried out to purify crude product (Silica gel 200−300 mesh, Shanghai
1
13
Sanpont Co., Ltd, Shanghai, China). H NMR spectra and C NMR spectra were recorded at 400
MHz using Bruker Avance III spectrometer (Bruker CO., Switzerland) in CDCl3 or DMSO-d6
solution with tetramethylsilane as internal standard and chemical shift values were given in ppm. The
NMR data was processed by software MestReNova (Ver.6.1.0, mestrelab research S.L.). The splitting
peak was designed as s, singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet; br, broad. The
high resolution mass (HRMS) was measured on FT-MS-Bruker APEX IV mass spectrometer.
4.2. Chemical synthesis
4.2.1. General procedure for the synthesis of compounds 3a−3x
The syntheses of compounds 3a-3x were mainly referred to literature method [35].
A mixture of 1a-1q, 1w, 1x (2 mmol), EDC•HCl (575 mg, 3 mmol), HOBt (446 mg, 3.3 mmol),
NH4Cl (348 mg, 6.5 mmol) and DIPEA (2.3 mL, 13 mmol) in DMSO (7 mL) was stirred at room
temperature for 15 h. The mixture was extracted with EtOAc three times, and the combined organic
layers were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under

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reduced pressure to afford 3a-3q, 3w, 3x.
A mixture of 2r-2v (2 mmol) and NH3•H2O (25-28 wt%, 80 mmol) in sealed tube was heated
at 100 °C for 12 h. The mixture was cooled to room temperature and extracted with EtOAc three
times. The combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered
and concentrated under reduced pressure to afford 3r-3v.
4.2.2. General procedure for the synthesis of compounds 4a−4h
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8
The syntheses of compounds 4a-4h were mainly referred to literature method [41].
A mixture of semicarbazide hydrochloride (2 g, 17.9 mmol) and anhydrous CH3COONa (2 g,
9
10
11
12
13
14
15
16
17
18
24.4 mmol) in EtOH (20 mL) was refluxed for 45 min and filtered while hot. 10a-10h (16.6 mmol)
was added to the filtrate, and the mixture was refluxed for 1 h. The mixture was stirred at room
temperature for an additional 10 h, and the precipitate that formed was filtered and dried to afford
11a-11h.
POCl3 (3.6 mL, 39 mmol) was added dropwise to dry DMF (8.4 mL, 108 mmol) in ice bath,
and the mixture was stirred at 0 °C for 1 h, followed by the addition of 11a-11h (17 mmol) in small
portions. The mixture was heated at 80 °C for 1.5 h and quickly poured onto ice (100 mL), then
treated with 30% aqueous NaOH to adjust pH = 8-9, stirred for 30 min and neutralized with conc.
HCl. After stirred at room temperature for additional 18 h, the resulting precipitate was filtered,
washed with water, recrystallized from water and dried to afford 4a-4h.
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4.2.3. General procedure for the synthesis of compounds H1, A1−A23
Cyanuric chloride (0.15 mmol) was added to a mixture of 3a-3x (1 mmol) and 4a (206 mg, 1.2
mmol) in CH3CN, MeOH or DMSO (3 mL). The mixture was stirred at room temperature for 0.5-2 h.
After completion, the mixture was extracted with EtOAc three times. The combined organic layers
were washed with brine, dried over anhydrous Na2SO4, filtered and concentrated under reduced
pressure. The crude product was purified by silica gel column chromatography eluting with
CH2Cl2/MeOH (30/1) to afford H1, A1-A23.
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31
4.2.3.1.
2-(3-Phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (H1)
CH3CN was employed as the solvent. Off-white solid. Yield: 66%. Mp 149−151 °C. 1H NMR
(400 MHz, DMSO-d6) δ 13.24 (s, 0.5H), 13.09 (s, 0.5H), 8.16 (s, 1H), 8.04 (s, 0.5H), 7.75 (s, 0.5H),
7.71–7.59 (m, 3H), 7.53–7.32 (m, 3H), 7.26 (t, J = 7.6 Hz, 1H), 6.93 (s, 1H), 6.76–6.71 (m, 2H), 5.79
13
(d, J = 17.3 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ 164.6, 149.1, 133.6, 130.4, 129.3, 128.8,

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128.6, 128.0, 118.0, 116.1, 115.1, 60.7. HRMS (ESI−TOF+) calcd for C17H15N4O [M+H]+ m/z
291.1240, found 291.1242.
4.2.3.2.
4
5
6
7
8
9
6-Chloro-8-methyl-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A1)
1
MeOH was employed as the solvent. Off-white solid. Yield: 54%. Mp 175−177 °C. H NMR
(400 MHz, DMSO-d6) δ 13.17 (s, 0.5H), 13.01 (s, 0.5H), 8.31 (s, 1H), 7.94 (s, 0.5H), 7.68−7.58 (m,
2H), 7.50−7.48 (m, 1.5H), 7.37 (dt, J = 39.2, 7.1 Hz, 2H), 7.21 (d, J = 1.8 Hz, 1H), 6.26 (d, J = 29.6
13
Hz, 1H), 5.79 (d, J = 34.6 Hz, 1H), 2.05 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 163.6, 145.7,
133.7, 129.0, 128.6, 126.6, 124.7, 121.6, 117.8, 117.4, 60.3, 17.1. HRMS (ESI−TOF+) calcd for
10
11
C18H16N4OCl [M+H]+ m/z 339.1013, found 339.1012.
4.2.3.3.
12
13
14
15
16
17
6-Chloro-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A2)
CH3CN was employed as the solvent. Off-white solid. Yield: 65%. Mp 149−151 °C. 1H NMR
(400 MHz, DMSO-d6) δ 13.26 (s, 0.5H), 13.12 (s, 0.5H), 8.35 (s, 1H), 8.06 (s, 0.5H), 7.77 (s, 0.5H),
7.70−7.59 (m, 3H), 7.51−7.31 (m, 3H), 7.31 (dd, J = 8.7, 2.5 Hz, 1H), 7.16 (s, 1H), 6.79 (d, J = 8.7
13
Hz, 1H), 5.83 (d, J = 26.6 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ 163.4, 147.8, 133.3, 129.3,
128.8, 128.6, 127.1, 121.7, 117.5, 117.1, 60.5. HRMS (ESI−TOF+) calcd for C17H14N4OCl [M+H]+
18
19
m/z 325.0856, found 325.0864.
4.2.3.4.
20
21
22
23
24
6,8-Dimethyl-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A3)
CH3CN was employed as the solvent. Off-white solid. Yield: 51%. Mp 154−156 °C. 1H NMR
(400 MHz, DMSO-d6) δ 13.20 (s, 0.5H), 13.05 (s, 0.5H), 8.16 (s, 1H), 7.79−7.70 (m, 3H), 7.43 (d,
J = 26.5 Hz, 4H), 7.01 (s, 1H), 5.83 (d, J = 44.3 Hz, 2H), 2.20 (s, 3H), 2.05 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 165.0, 144.7, 135.4, 129.0, 128.5, 126.8, 125.7, 123.9, 118.2, 116.7, 60.5, 20.6,
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26
17.2. HRMS (ESI−TOF+) calcd for C19H19N4O [M+H]+ m/z 319.1559, found 319.1559.
4.2.3.5.
27
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30
5-Chloro-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A4)
CH3CN was employed as the solvent. Off-white solid. Yield: 57%. Mp >320 °C. 1H NMR (400
MHz, DMSO-d6) δ 13.27 (s, 0.5H), 13.12 (s, 0.5H), 8.33 (s, 1H), 8.06 (s, 0.5H), 7.77 (s, 0.5H), 7.67
(d, J = 7.1 Hz, 1H), 7.59−7.50 (m, 2H), 7.45−7.33 (m, 2H), 7.24 (s, 1H), 7.19 (t, J = 7.9 Hz, 1H),

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6.75 (d, J = 7.1 Hz, 2H), 5.66 (d, J = 14.9 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 162.1, 152.0,
134.4, 133.3, 128.9, 128.5, 121.1, 116.6, 114.6, 113.3, 59.8. HRMS (ESI−TOF+) calcd for
3
4
C17H14N4OCl [M+H]+ m/z 325.0856, found 325.0855.
4.2.3.6.
5
6
5-Methyl-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A5)
CH3CN was employed as the solvent. Off-white solid. Yield: 61%. Mp 269−271 °C. 1H NMR
(400 MHz, DMSO-d6) δ 13.21 (s, 0.5H), 13.07 (s, 0.5H), 8.04−7.75 (m, 2H), 7.70−7.58 (m, 2H),
7.52−7.32 (m, 3H), 7.09 (t, J = 8.4 Hz, 1H), 6.80 (s, 1H), 6.65−6.52 (m, 2H), 5.63 (d, J = 24.5 Hz,
1H), 2.51 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 165.2, 150.6, 140.9, 132.3, 128.8, 128.6, 121.8,
117.4, 114.7, 113.6, 60.2, 22.4. HRMS (ESI−TOF+) calcd for C18H17N4O [M+H]+ m/z 305.1402,
7
8
9
10
11
12
found 305.1402.
4.2.3.7.
13
14
15
16
17
8-Bromo-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A6)
CH3CN was employed as the solvent. Off-white solid. Yield: 53%. Mp 238−240 °C. 1H NMR
(400 MHz, DMSO-d6) δ 13.20 (s, 0.5H), 13.05 (s, 0.5H), 8.51 (s, 1H), 7.88−7.60 (m, 5H),
13
7.47−7.41 (m, 3H), 6.74 (t, J = 7.5 Hz, 1H), 6.16−5.95 (m, 2H). C NMR (101 MHz, DMSO-d6) δ
163.2, 145.4, 136.8, 129.1, 128.6, 127.7, 119.5, 118.4, 118.2, 108.9, 60.1. HRMS (ESI− TOF+) calcd
18
19
for C17H14N4OBr [M+H]+ m/z 369.0351, found 369.0352.
4.2.3.8.
20
21
22
23
24
8-Nitro-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A7)
1
DMSO was employed as the solvent. Yellow solid. Yield: 58%. Mp 248−250 °C. H NMR
(400 MHz, DMSO-d6) δ 13.26 (s, 0.5H), 13.10 (s, 0.5H), 8.86 (s, 1H), 8.23−8.09 (m, 3H),
7.95−7.64 (m, 2H), 7.59−7.36 (m, 4H), 6.89 (t, J = 7.9 Hz, 1H), 6.18 (d, J = 43.9 Hz, 1H). 13C NMR
(101 MHz, DMSO-d6) δ 161.9, 143.5, 135.8, 132.9, 130.7, 129.0, 128.5, 119.4, 118.2, 117.0, 59.7.
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26
HRMS (ESI−TOF+) calcd for C17H14N5O3 [M+H]+ m/z 336.1097, found 336.1101.
4.2.3.9.
27
28
29
30
6-Fluoro-8-methyl-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A8)
1
DMSO was employed as the solvent. Off-white solid. Yield: 56%. Mp 263−265 °C. H NMR
(400 MHz, DMSO-d6) δ 13.22 (s, 0.5H), 13.05 (s, 0.5H), 8.38 (s, 1H), 7.98−7.61 (m, 3H),
7.50−7.35 (m, 3H), 7.26 (dd, J = 8.9, 3.0 Hz, 1H), 7.12−7.09 (m, 1H), 6.04 (d, J = 23.1 Hz, 1H), 5.78

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(d, J = 11.3 Hz, 1H), 2.10 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 164.0, 155.3 (d, JCF = 235.3
Hz), 143.5, 139.8, 128.9, 128.5, 127.0, 121.6 (d, JCF = 23.2 Hz), 117.9, 110.6 (d, JCF = 23.2 Hz), 60.5,
17.3. 19F NMR (376 MHz, DMSO-d6) δ -126.03 (d, J = 94.0 Hz). HRMS (ESI−TOF+) calcd for
4
5
C18H16N4OF [M+H]+ m/z 323.1308, found 323.1304.
4.2.3.10.
6
7
6, 8-Dichloro-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A9)
1
DMSO was employed as the solvent. Off-white solid. Yield: 61%. Mp 149−151 °C. H NMR
8
(400 MHz, DMSO-d6) δ 13.20 (s, 0.5H), 13.04 (s, 0.5H), 8.62 (d, J = 7.7 Hz, 1H), 7.92−7.67 (m,
2H), 7.61 (d, J = 10.9 Hz, 3H), 7.52−7.34 (m, 3H), 6.71 (dd, J = 46.5, 8.1 Hz, 1H), 5.93 (dd, J = 33.1,
7.3 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 162.1, 143.5, 132.8, 130.1, 129.2, 128.8, 128.7,
128.0, 126.4, 118.1, 60.1. HRMS (ESI−TOF+) calcd for C17H13N4OCl2 [M+H]+ m/z 359.0466, found
9
10
11
12
13
359.0464.
4.2.3.11.
14
15
16
17
18
6-Bromo-8-methyl-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A10)
1
DMSO was employed as the solvent. Off-white solid. Yield: 66%. Mp 153−155 °C. H NMR
(400 MHz, DMSO-d6) δ 8.37 (s, 1H), 7.86−7.63 (m, 4H), 7.48−7.34 (m, 4H), 6.34 (s, 1H), 5.82 (s,
1H), 2.08 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 163.5, 146.1, 136.3, 129.0, 128.6, 127.7, 126.9,
117.9, 117.8, 109.1, 60.2, 17.1. HRMS (ESI−TOF+) calcd for C18H16N4OBr [M+H]+ m/z 383.0507,
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20
found 383.0497.
4.2.3.12.
21
22
23
24
25
26
6-Bromo-8-methoxy-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A11)
1
DMSO was employed as the solvent. Off-white solid. Yield: 59%. Mp 243−245 °C. H NMR
(400 MHz, DMSO-d6) δ 13.16 (s, 0.5H), 13.01 (s, 0.5H), 8.34 (s, 1H), 7.81 (dd, J = 57.9, 5.3 Hz,
1H), 7.64 (s, 2H), 7.45−7.36 (m, 4H), 7.12 (d, J = 2.1 Hz, 1H), 6.25 (s, 1H), 5.84 (s, 1H), 3.77 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 163.0, 148.0, 138.2, 129.0, 128.7, 121.5, 118.0, 117.1, 116.7,
108.7, 60.5, 56.6. HRMS (ESI−TOF+) calcd for C18H16N4O2Br [M+H]+ m/z 399.0388, found
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28
399.0388.
4.2.3.13.
29
8-Bromo-6-methyl-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A12)

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DMSO was employed as the solvent. Off-white solid. Yield: 47%. Mp 141−143 °C. H NMR
(400 MHz, DMSO-d6) δ 13.19 (s, 0.5H), 13.01 (s, 0.5H), 8.45 (d, J = 10.9 Hz, 1H), 7.87−7.61 (m,
3H), 7.54−7.34 (m, 5H), 5.94 (d, J = 12.8 Hz, 1H), 5.83 (d, J = 5.1 Hz, 1H), 2.23 (s, 3H). 13C NMR
(101 MHz, DMSO-d6) δ 163.4, 143.2, 137.1, 129.9, 129.2, 128.9, 128.5, 127.9, 118.3, 109.0, 60.3,
5
6
20.1. HRMS (ESI−TOF+) calcd for C18H16N4OBr [M+H]+ m/z 383.0507, found 383.0498.
4.2.3.14.
7
8
6, 8-Difluoro-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A13)
1
DMSO was employed as the solvent. Off-white solid. Yield: 45%. Mp 237−239 °C. H NMR
9
(400 MHz, DMSO-d6) δ 13.21 (s, 0.5H), 13.07 (s, 0.5H), 8.51 (d, J = 8.7 Hz, 1H), 8.01 (s, 0.5H),
7.72−7.69 (m, 1.5H), 7.61 (d, J = 7.3 Hz, 1H), 7.51 (t, J = 7.5 Hz, 1H), 7.45−7.33 (m, 3H), 7.28 (d, J
= 8.6 Hz, 1H), 6.82 (d, J = 17.5 Hz, 1H), 5.83 (d, J = 26.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6)
10
11
12
13
14
15
δ 162.5, 153.9 (dd, JCF = 237.4, 11.1 Hz), 150.3 (dd, JCF = 247.5, 10.1 Hz), 149.4, 134.1 (dd, JCF
=
14.1, 2.0 Hz), 129.2, 128.7, 118.7 (d, JCF = 17.2 Hz), 117.4 (d, JCF = 14.1 Hz), 109.0 (d, JCF = 23.2
Hz), 108.6, 108.4 (d, JCF = 6.1 Hz), 108.1, 60.6 (d, JCF = 29.3 Hz). 19F NMR (376 MHz, DMSO-d6) δ
-123.67 (d, J = 60.2 Hz), -128.46 (d, J = 48.9 Hz). HRMS (ESI−TOF+) calcd for C17H13N4OF2
16
17
[M+H]+ m/z 327.1057, found 327.1060.
4.2.3.15.
18
19
20
21
22
23
24
25
8-Bromo-6-fluoro-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A14)
1
DMSO was employed as the solvent. Off-white solid. Yield: 47%. Mp 256−258 °C. H NMR
(400 MHz, DMSO-d6) δ 13.21 (s, 0.5H), 13.04 (s, 0.5H), 8.66 (d, J = 10.3 Hz, 1H), 7.90−7.61 (m,
4H), 7.53−7.34 (m, 4H), 6.10 (d, J = 53.0 Hz, 1H), 5.90 (d, J = 34.2 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 162.4, 154.8 (dd, JCF = 239.4, 10.1 Hz), 149.0, 142.5 (d, JCF = 12.1, Hz), 139.3, 134.1,
129.3, 128.9, 128.6, 128.0, 124.2 (d, JCF = 26.3 Hz), 128.6 (dd, JCF = 32.3, 6.1 Hz), 118.0 (d, JCF
=
11.1 Hz), 113.5 (d, JCF = 23.2 Hz), 109.4 (dd, JCF = 29.3, 9.1 Hz), 60.2 (d, JCF = 28.3 Hz). 19F NMR
(376 MHz, DMSO-d6) δ -123.83 (d, J = 127.8 Hz). HRMS (ESI−TOF+) calcd for C17H13N4OFBr
26
27
[M+H]+ m/z 387.0257, found 387.0259.
4.2.3.16.
28
29
30
6, 8-Diiodo-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A15)
1
DMSO was employed as the solvent. Off-white solid. Yield: 53%. Mp 151−153 °C. H NMR
(400 MHz, DMSO-d6) δ 13.23 (s, 0.5H), 13.03 (s, 0.5H), 8.61 (s, 1H), 8.04−7.58 (m, 5H), 7.43 (t, J

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1
2
= 24.7 Hz, 3H), 5.98 (d, J = 38.3 Hz, 2H). C NMR (101 MHz, DMSO-d6) δ 162.1, 149.3, 147.5,
136.4, 134.0, 129.8, 129.3, 128.9, 128.5, 128.4, 128.1, 119.2, 119.0, 118.4, 87.0, 81.0, 60.0. HRMS
3
4
(ESI−TOF+) calcd for C17H13N4OI2 [M+H]+ m/z 542.9179, found 542.9179.
4.2.3.17.
5
6
7
8
9
6-Iodo-8-methyl-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A16)
1
DMSO was employed as the solvent. Off-white solid. Yield: 61%. Mp 226−228 °C. H NMR
(400 MHz, DMSO-d6) δ 13.22 (s, 0.5H), 13.06 (s, 0.5H), 8.33 (s, 1H), 7.98−7.61 (m, 4H),
7.50−7.36 (m, 4H), 6.34 (d, J = 19.2 Hz, 1H), 5.82 (d, J = 24.6 Hz, 1H), 2.05 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ 163.4, 146.5, 141.8, 133.9, 129.2, 128.8, 128.6, 128.0, 127.0, 128.0, 60.3, 16.9.
10
11
HRMS (ESI−TOF+) calcd for C18H16N4OI [M+H]+ m/z 431.0369, found 431.0365.
4.2.3.18.
12
13
14
15
16
17
8-Methyl-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A17)
CH3CN was employed as the solvent. Off-white solid. Yield: 63%. Mp 262−264 °C. 1H NMR
(400 MHz, DMSO-d6) δ 13.20 (s, 0.5H), 13.04 (s, 0.5H), 8.20 (s, 1H), 7.96−7.55 (m, 4H),
7.50−7.37 (m, 3H), 7.16 (d, J = 6.4 Hz, 1H), 6.71−6.67 (m, 1H), 6.06 (d, J = 25.5 Hz, 1H), 5.79 (d, J
13
= 27.7 Hz, 1H), 2.06 (s, 3H). C NMR (101 MHz, DMSO-d6) δ 164.8, 146.8, 134.4, 128.9, 128.6,
128.0, 125.8, 123.7, 118.0, 116.5, 60.4, 17.3. HRMS (ESI−TOF+) calcd for C18H17N4O [M+H]+ m/z
18
19
305.1402, found 305.1404.
4.2.3.19.
20
21
22
23
24
6-Chloro-8-iodo-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A18)
1
DMSO was employed as the solvent. Off-white solid. Yield: 50%. Mp 267−269 °C. H NMR
(400 MHz, DMSO-d6) δ 13.23 (s, 0.5H), 13.03 (s, 0.5H), 8.66 (d, J = 6.8 Hz, 1H), 7.85−7.58 (m,
5H), 7.53−7.34 (m, 3H), 6.03−5.89 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 162.3, 141.7, 129.3,
129.0, 128.5, 127.6, 123.3, 118.3, 118.0, 86.2, 60.2. HRMS (ESI−TOF+) calcd for C17H13N4OClI
25
26
[M+H]+ m/z 450.9823, found 450.9823.
4.2.3.20.
27
28
29
30
7-Bromo-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A19)
CH3CN was employed as the solvent. Off-white solid. Yield: 59%. Mp 238−240 °C. 1H NMR
(400 MHz, DMSO-d6) δ 13.27 (s, 0.5H), 13.12 (s, 0.5H), 8.28 (s, 1H), 8.06 (s, 0.5H), 7.76 (s, 0.5H),
7.58−7.37 (m, 3H), 7.50−7.37 (m, 3H), 7.19 (s, 1H), 6.94 (s, 1H), 6.88 (dd, J = 8.3, 1.5 Hz, 1H), 5.85

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(d, J = 23.3 Hz, 1H). C NMR (101 MHz, DMSO-d6) δ 163.8, 150.1, 130.1, 129.3, 128.8, 128.6,
127.1, 120.8, 117.1, 115.0, 60.6. HRMS (ESI−TOF+) calcd for C17H14N4OBr [M+H]+ m/z 369.0351,
3
4
found 369.0352.
4.2.3.21.
5
6
6, 8-Dibromo-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A20)
1
DMSO was employed as the solvent. Off-white solid. Yield: 67%. Mp 156−158 °C. H NMR
7
8
(400 MHz, DMSO-d6) δ 13.19 (s, 0.5H), 13.02 (s, 0.5H), 8.63−7.88 (m, 2H), 7.81−7.60 (m, 4H),
7.50−7.35 (m, 3H), 6.41 (d, J = 57.3 Hz, 1H), 5.93 (d, J = 36.0 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 162.0, 144.8, 138.2, 129.9, 128.9, 128.6, 119.1, 118.2, 109.9, 109.2, 60.1. HRMS
(ESI−TOF+) calcd for C17H13N4OBr2 [M+H]+ m/z 446.9456, found 446.9450.
4.2.3.22.
9
10
11
12
13
14
15
16
17
18
6-Fluoro-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one (A21)
CH3CN was employed as the solvent. Off-white solid. Yield: 62%. Mp 104−106 °C. 1H NMR
(400 MHz, DMSO-d6) δ 13.25 (s, 0.5H), 13.10 (s, 0.5H), 8.35 (s, 1H), 8.05 (s, 0.5H), 7.77 (s, 0.5H),
7.70−7.59 (m, 2H), 7.53−7.34 (m, 4H), 7.20−7.15 (m, 1H), 6.92 (s, 1H), 6.81−6.77 (m, 1H), 5.78 (d,
J = 26.1 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 163.7, 155.5 (d, JCF = 235.3 Hz), 145.8, 128.8,
19
128.6, 121.0 (d, JCF = 23.2 Hz), 117.4, 116.9, 116.8, 113.1 (d, JCF = 22.2 Hz), 60.8. F NMR (376
MHz, DMSO-d6) δ -126.23. HRMS (ESI−TOF+) calcd for C17H14N4OF [M+H]+ m/z 309.1152, found
19
20
309.1152.
4.2.3.23.
21
22
23
24
25
2-(3-Phenyl-1H-pyrazol-4-yl)-2,3-dihydropyrido[2,3-d]pyrimidin-4(1H)-one (A22)
1
DMSO was employed as the solvent. Off-white solid. Yield: 48%. Mp 178−180 °C. H NMR
(400 MHz, DMSO-d6) δ 13.20 (s, 0.5H), 13.09 (s, 0.5H), 8.36 (s, 1H), 8.16 (d, J = 4.1 Hz, 1H),
7.96−7.66 (m, 5H), 7.45−7.39 (m, 3H), 6.78−6.75 (m, 1H), 5.96 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 163.7, 158.8, 153.1, 136.3, 128.9, 128.7, 118.0, 114.6, 110.4, 59.6. HRMS (ESI− TOF+)
26
27
calcd for C16H14N5O [M+H]+ m/z 292.1198, found 292.1201.
4.2.3.24.
28
29
30
2-(3-Phenyl-1H-pyrazol-4-yl)-2,3-dihydropyrido[3,4-d]pyrimidin-4(1H)-one (A23)
1
DMSO was employed as the solvent. Yellow solid. Yield: 47%. Mp 200−202 °C. H NMR
(400 MHz, DMSO-d6) δ 13.28 (s, 0.5H), 13.13 (s, 0.5H), 8.52 (s, 1H), 8.17 (s, 1H), 8.07 (s, 0.5H),

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3
7.98 (d, J = 4.4 Hz, 1H), 7.77 (s, 0.5H), 7.70−7.59 (m, 2H), 7.53−7.35 (m, 4H), 7.23 (s, 1H), 5.89 (d,
J = 26.8 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 163.1, 144.0, 139.1, 138.5, 129.3, 128.6, 120.9,
120.3, 117.3, 60.5. HRMS (ESI−TOF+) calcd for C16H14N5O [M+H]+ m/z 292.1198, found 292.1201.
4
5
4.2.4. General procedure for the synthesis of compounds A24, A25
A mixture of 4a (172 mg, 1 mmol), 5a or 5b (1 mmol) and 2-morpholinoethanesulfonic acid
6
7
(20 mg, 0.1 mmol) in 50% aq EtOH (10 mL) was heated at 60 °C for 3 h, and the solvent was
removed under reduced pressure. The residue was extracted with EtOAc three times, and the
combined organic layers were washed with brine, dried over anhydrous Na2SO4, filtered and
concentrated. The crude product was purified by silica gel column chromatography eluting with
CH2Cl2/MeOH (30/1) to afford A24 or A25, respectively.
8
9
10
11
4.2.4.1.
12
13
14
15
2-(3-Phenyl-1H-pyrazol-4-yl)-2,3,4,9-tetrahydro-1H-purin-6(5H)-one (A24)
1
Off-white solid. Yield: 43%. Mp 273−275 °C. H NMR (400 MHz, DMSO-d6) δ 13.57 (s,
1H), 12.80 (s, 1H), 9.15 (s, 1H), 8.43 (s, 1H), 7.69−7.63 (m, 4H), 7.55−7.44 (m, 4H). 13C NMR (101
MHz, DMSO-d6) δ 161.4, 151.6, 148.2, 136.2, 129.3, 129.0, 117.6, 117.2. HRMS (ESI−TOF+) calcd
16
17
for C14H13N6O [M+H]+ m/z 281.1151, found 281.1158.
4.2.4.2.
18
19
20
21
22
23
2-(3-Phenyl-1H-pyrazol-4-yl)-2,3-dihydrothieno[2,3-d]pyrimidin-4(1H)-one (A25)
Yellow solid. Yield: 41%. Mp 245−247 °C. 1H NMR (400 MHz, DMSO-d6) δ 13.73 (s, 1H),
8.51−8.45 (m, 2H), 7.97 (s, 1H), 7.68 (d, J = 6.4 Hz, 2H), 7.57−7.50 (m, 3H), 7.32 (d, J = 8.9 Hz,
2H), 7.20 (d, J = 5.6 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ 163.7, 155.6, 153.4, 129.4, 129.2,
129.1, 128.8, 119.4, 116.4. HRMS (ESI−TOF+) calcd for C15H13N4OS [M+H]+ m/z 297.0810, found
297.0811.
24
25
4.2.5. General procedure for the synthesis of compounds B1, B2
A mixture of 4a or 4e (1.2 mmol), 6 (172 mg, 1 mmol) and Amberlyst-15 (10%, w/w) in
26
27
28
29
30
31
MeOH (3 mL) underwent ultrasound irradiation (40 KHz) at room temperature for 40 min, and
EtOAc was added to the reaction. The mixture was filtered, and the filtrate was washed with brine,
dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product
was purified by recrystallization from EtOAc:PE:MeOH = 3:2:1 to afford B1 or B2, respectively.
4.2.5.1.
3-(3-Phenyl-1H-pyrazol-4-yl)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (B1)

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1
2
3
4
Off-white solid. Yield: 61%. Mp 226−228 °C. H NMR (400 MHz, DMSO-d6) δ 13.36 (s,
0.5H), 13.16 (s, 0.5H), 8.10–7.94 (m, 2H), 7.69–7.38 (m, 7H), 7.32 (t, J = 7.8 Hz, 1H), 6.90 (d, J =
6.3 Hz, 1H), 6.77 (t, J = 7.6 Hz, 1H), 5.79 (dd, J = 26.9, 10.2 Hz, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 144.2, 133.3, 129.2, 128.1, 124.2, 121.9, 117.2, 116.8, 115.4, 61.6. HRMS (ESI−TOF+)
5
6
calcd for C16H15N4O2S [M+H]+ m/z 327.0910, found 327.0916.
4.2.5.2.
7
3-(3-(4-Chlorophenyl)-1H-pyrazol-4-yl)-3,4-dihydro-2H-benzo[e][1,2,4]thiadiazine 1,1-dioxide (B2)
1
8
9
Off-white solid. Yield: 45%. Mp 254−256 °C. H NMR (400 MHz, DMSO-d6) δ 13.44,
13.24 (s, 1H), 8.10–7.95 (m, 2H), 7.71–7.48 (m, 6H), 7.33 (t, J = 7.8 Hz, 1H), 6.90 (d, J = 6.8 Hz,
1H), 6.77 (t, J = 6.4 Hz, 1H), 5.83–5.72 (m, 1H). 13C NMR (101 MHz, DMSO-d6) δ 147.6, 144.1,
133.3, 133.1, 132.5, 130.4, 129.8, 129.7, 129.2, 124.2, 121.9, 117.3, 116.9, 115.9, 115.5, 61.7.
HRMS (ESI−TOF+) calcd for C16H14N4O2ClS [M+H]+ m/z 361.0521, found 361.0529.
10
11
12
13
14
4.2.6. General procedure for the synthesis of compounds B3-B8
A mixture of 3a-3d, 3t or 3w (1 mmol), 4a (172 mg, 1 mmol) and CuO (4 mg, 0.05 mmol) in
15
16
17
18
19
DMA (3 mL) was heated at 120 °C for 24 h. The mixture was cooled to room temperature and
extracted with EtOAc three times. The combined organic layers were washed with brine, dried over
anhydrous Na2SO4, filtered and concentrated under reduced pressure. The crude product was purified
by silica gel column chromatography eluting with CH2Cl2/MeOH (30/1) to afford B3-B8.
4.2.6.1.
20
21
22
23
24
2-(3-Phenyl-1H-pyrazol-4-yl)quinazolin-4(3H)-one (B3)
1
Off-white solid. Yield: 50%. Mp 267−269 °C. H NMR (400 MHz, DMSO-d6) δ 13.59 (s,
0.5H), 13.41 (s, 0.5H), 12.22 (s, 1H), 8.46 (s, 0.5H), 8.19 (s, 0.5H), 8.12 (d, J = 7.6 Hz, 1H),
7.77−7.70 (m, 3H), 7.48−7.37 (m, 5H). 13C NMR (101 MHz, DMSO-d6) δ 162.5, 149.5, 149.2, 134.8,
129.3, 129.1, 128.6, 128.2, 127.4, 126.5, 126.2, 121.3, 113.4, 113.0. HRMS (ESI−TOF+) calcd for
25
26
C17H13N4O [M+H]+ m/z 289.1089, found 289.1090.
4.2.6.2.
27
28
6-Chloro-8-methyl-2-(3-phenyl-1H-pyrazol-4-yl)quinazolin-4(3H)-one (B4)
1
Off-white solid. Yield: 44%. Mp 272−274 °C. H NMR (400 MHz, DMSO-d6) δ 13.59 (s,
29
30
0.5H), 13.41 (s, 0.5H), 12.37 (s, 1H), 8.55 (s, 0.5H), 8.30 (s, 0.5H), 7.86 (d, J = 3.6 Hz, 1H),
7.68−7.62 (m, 3H), 7.41 (d, J = 36.9 Hz, 3H), 2.10 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 161.9,

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2
3
148.2, 146.8, 138.7, 134.7, 129.9, 129.6, 128.2, 122.6, 122.1, 113.1, 17.1. HRMS (ESI−TOF+) calcd
for C18H14N4OCl [M+H]+ m/z 337.0856, found 337.0854.
4.2.6.3.
4
5
6
7
8
6-Chloro-2-(3-phenyl-1H-pyrazol-4-yl)quinazolin-4(3H)-one (B5)
1
Off-white solid. Yield: 42%. Mp 286−288 °C. H NMR (400 MHz, DMSO-d6) δ 13.49 (s,
1H), 12.38 (s, 1H), 8.35−8.25 (m, 1H), 8.04 (d, J = 3.0 Hz, 1H), 7.77−7.75 (m, 1H), 7.70−7.68 (m,
2H), 7.43−7.41 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 161.6, 149.6, 148.2, 134.9, 130.6, 129.6,
129.3, 128.4, 125.2, 122.4, 113.0. HRMS (ESI− TOF+) calcd for C17H12N4OCl [M+H]+ m/z 323.0700,
9
found 323.0699.
10
4.2.6.4.
11
12
13
14
15
8-Methyl-2-(3-phenyl-1H-pyrazol-4-yl)quinazolin-4(3H)-one (B6)
1
Off-white solid. Yield: 49%. Mp 252−254 °C. H NMR (400 MHz, DMSO-d6) δ 13.57 (s,
0.5H), 13.39 (s, 0.5H), 12.22 (s, 1H), 8.54 (s, 0.5H), 8.29 (s, 0.5H), 7.93 (d, J = 7.7 Hz, 1H), 7.70 (dd,
J = 7.5, 1.8 Hz, 2H), 7.56 (d, J = 7.1 Hz, 1H), 7.45−7.38 (m, 3H), 7.30 (t, J = 17.6 Hz, 1H), 2.14 (s,
3H). 13C NMR (101 MHz, DMSO-d6) δ 162.9, 148.0, 147.7, 135.6, 135.1, 129.5, 128.3, 125.8, 123.8,
16
17
121.1, 113.4, 17.4. HRMS (ESI−TOF+) calcd for C18H15N4O [M+H]+ m/z 303.1246, found 303.1257.
4.2.6.5.
18
19
20
21
22
6, 8-Dimethyl-2-(3-phenyl-1H-pyrazol-4-yl)quinazolin-4(3H)-one (B7)
1
Off-white solid. Yield: 41%. Mp 274−276 °C. H NMR (400 MHz, DMSO-d6) δ 13.53 (s,
0.5H), 13.37 (s, 0.5H), 12.12 (s, 1H), 8.51 (s, 0.5H), 8.26 (s, 0.5H), 7.73−7.68 (m, 3H), 7.45−7.34 (m,
4H), 2.37 (s, 3H), 2.13 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 162.8, 146.9, 146.0, 136.4, 135.5,
135.3, 130.0, 128.3, 123.2, 120.9, 113.5, 21.2, 17.3. HRMS (ESI−TOF+) calcd for C19H17N4O
23
24
[M+H]+ m/z 317.1402, found 317.1396.
4.2.6.6.
25
26
27
28
29
30
2-(3-Phenyl-1H-pyrazol-4-yl)pyrido[2,3-d]pyrimidin-4(3H)-one (B8)
1
Off-white solid. Yield: 40%. Mp 269−271 °C. H NMR (400 MHz, DMSO-d6) δ 13.62 (s,
0.5H), 13.51 (s, 0.5H), 12.49 (s, 1H), 8.88 (d, J = 6.6 Hz, 1H), 8.49−8.24 (m, 2H), 7.74−7.72 (m, 2H),
7.49−7.41 (m, 4H). 13C NMR (101 MHz, DMSO-d6) δ 163.2, 159.5, 156.3, 152.4, 135.8, 129.2,
128.5, 122.2, 116.3, 113.0. HRMS (ESI−TOF+) calcd for C16H12N5O [M+H]+ m/z 290.1042, found
290.1042.