The Discovery of Novel ACA Derivatives as Specific TRPM2 Inhibitors that Reduce Ischemic Injury Both in Vitro and in Vivo

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The Discovery of Novel ACA Derivatives as Speciﬁc TRPM2 Inhibitors

that Reduce Ischemic Injury Both In Vitro and In Vivo

Han Zhang,^#Peilin Yu,^#Hongwei Lin, Zefang Jin, Siqi Zhao, Yi Zhang, Qingxia Xu, Hongwei Jin,

Zhenming Liu, Wei Yang,* and Liangren Zhang *

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ABSTRACT: The transient receptor potential melastatin 2 (TRPM2) channel is associated with ischemia/reperfusion injury,

inﬂammation, cancer, and neurodegenerative diseases. However, the limit of speciﬁc inhibitors impedes the development of TRPM2-

targeted therapeutic agents. To discover more potent and selective TRPM2 inhibitors, 59 N-(p-amylcinnamoyl) anthranilic acid

(ACA) derivatives were synthesized and evaluated using calcium imaging and electrophysiology approaches. Systematic structure−

activity relationship studies resulted in some potent compounds inhibiting the TRPM2 channel with sub-micromolar half-maximal

inhibitory concentration values. Among them, the preferred compound A23 exhibited TRPM2 selectivity over TRPM8 and TRPV1

channels as well as phospholipase A2 and showed neuroprotective activity in vitro. Following pharmacokinetic studies, A23 was

further evaluated in a transient middle cerebral artery occlusion model in vivo, which signiﬁcantly reduced cerebral infarction. These

data indicate that A23 might serve as a useful tool for TRPM2-related research as well as a lead compound for the development of

therapeutic agents for ischemic injury.

participates in many calcium-dependent physiological and

pathophysiological processes, such as pro-inﬂammatory

cytokine production in immune cells, endothelial permeability,

insulin release by pancreatic β-cells, warmth sensation, and cell

death.^7,8Cell death is the most outstanding and common

consequence of TRPM2 channel activation, and its excessive

activation can lead to cytotoxicity due to Ca²⁺overload, which

is involved in ischemia/reperfusion (I/R)-induced damage,

Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral

sclerosis, neurovascular dysfunction, cardiovascular disorders,

diabetes, chronic kidney disease, liver injury, cancer, and

inﬂammatory diseases.⁹⁻¹³

INTRODUCTION

■

The transient receptor potential (TRP) channel family is one

of the largest cation channel families, which exhibits a large

variety of functional properties and plays diverse cellular and

physiological roles.^1,2In mammals, 28 diﬀerent TRP proteins

have been identiﬁed, which are often grouped into six TRP

subfamilies: canonical (TRPC), vanilloid (TRPV), melastatin

(TRPM), ankyrin (TRPA), polycystin (TRPP), and mucolipin

(TRPML), according to amino acid sequence relatedness.³

The TRPM subfamily comprises eight subtypes, which can be

divided into four pairs based on their amino acid sequence

homology: TRPM1/TRPM3, TRPM2/TRPM8, TRPM4/

TRPM5, and TRPM6/TRPM7.⁴

The TRPM2 channel, ﬁrst isolated from the human brain in

1998, is a cation channel permeable for calcium (Ca²⁺).⁵It

exhibits widespread distribution in a number of tissues and

cells, with the highest expression in the brain and a relatively

high expression in bone marrow cells and macrophages.⁶Given

its role in Ca²⁺mobilization as a crucial endogenous sensor of

reactive oxygen species (ROS), the TRPM2 channel

Received: December 10, 2020

Published: March 30, 2021

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Figure 1. Chemical structures of representative inhibitors. (A) Chemical structures and inhibitory activities of representative TRPM2 inhibitors.

(B) Chemical structures and inhibitory activities of SB366791. (C) Design of ACA derivatives as TRPM2 inhibitors. ACA was divided into pentyl

aromatic ring moiety (Class A), linker moiety (Class B), and carboxyl aromatic ring moiety (Class C).

Ischemic stroke is the second leading cause of death after

ischemic heart disease and accounts for 9% of deaths

worldwide.¹⁴Intravenous thrombolysis and endovascular

intervention are clinically applicable treatments for stroke,

but both have a narrow therapeutic window and might cause a

reperfusion injury. Therefore, current measures to protect the

brain against stroke damage are insuﬃcient.^15,16ROS have

been implicated in brain injury after an ischemic stroke and can

damage cellular macromolecules leading to autophagy,

apoptosis, and necrosis. Moreover, the restoration of blood

ﬂow leads to a reperfusion injury that can largely be attributed

to a second burst of ROS generation, which is an important

cellular mechanism for the progressive decline in cognitive

function and I/R-induced brain damage.¹⁷The TRPM2

channel has been extensively scrutinized for its role in

mediating delayed neuronal cell death. A number of studies

using various in vitro and in vivo I/R models have gathered

substantial evidence to support a critical role for the TRPM2

channel in mediating neuronal cell death due to reperfusion,

leading to I/R-induced brain damage.^12,18−20Even though

there is evidence that TRPM2 appears to be a viable target for

therapeutic interventions for ischemic stroke, preclinical

studies have been limited by the lack of TRP-subtype speciﬁc

and potent inhibitors. Therefore, the development of speciﬁc

TRPM2 inhibitors has garnered increasing attention for both

academic functional studies and clinical applications.

methylphenyl) aminobenzoic acid (4, 3-MFA), N-(p-amylcin-

namoyl) anthranilic acid (5, ACA), and 2-aminoethoxydi-

phenyl borate (6, 2-APB).²¹⁻²⁵However, most of these

compounds lack speciﬁcity, especially considering that the

TRPM8 channel has a high homology with TRPM2.²⁶Marine-

derived scalaradial (7) was identiﬁed as a TRPM2 inhibitor,

but it has indirect eﬀects on the channel.²⁷We recently

discovered 2,3-dihydroquinazolin-4(1H)-one derivative D9

(8), which showed a half-maximal inhibitory concentration

(IC₅₀) value of 3.7 μM against TRPM2 and did not aﬀect the

TRPM8 channel.²⁸In addition, two more potent TRPM2

inhibitors were recently identiﬁed. JNJ-28583113 (9) is the

most potent TRPM2 inhibitor in vitro, but it has stability

diﬃculties in vivo.²⁹tatM2NX is a cell-permeable peptide

inhibitor of TRPM2, which exhibits neuroprotective eﬀects in

vivo, but its druggability needs further evaluation.³⁰Several

endogenous ligand analogues are thought to selectively inhibit

TRPM2, but poor membrane permeability and stability limit

their application.^31,32Currently, the number of reported

TRPM2 inhibitors is limited; hence, there is an urgent need

to develop novel small-molecule TRPM2 inhibitors with

improvements in both potency and selectivity, which will not

only provide molecular tools for better understanding of the

channel functions in cell biology but will also lead to potential

clinical therapeutic agents for ischemic injury and other related

diseases.

The chemical structures and inhibitory activities of

representative TRPM2 inhibitors are summarized in Figure

1A. Several initially discovered TRPM2 inhibitors have

inhibitory activities at micromolar levels, including econazole

(1), clotrimazole (2), ﬂufenamic acid (3, FFA), 2-(3-

ACA is a well-described inhibitor that is widely used in

studies on TRPM2. The administration of ACA decreases

hippocampal cell death and infarct size in ischemic stroke,

improves insulin sensitivity in adipose tissue during Angio-

tensin II-induced hypertension, and attenuates the neuro-

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a

Scheme 1. Synthesis of Compounds A1−A33

a

Reagents and conditions: (a) NaOH, THF, ethanol, H₂O, reﬂux, 4 h; (b) 2,2-dimethyl-1,3-dioxane-4,6-dione, toluene, reﬂux, 3 h; (c) Pd(PPh₃)₄,

K₂CO₃, dioxane, H₂O, 105 °C, 24 h, (d) piperidine, toluene, reﬂux, 3 h, then rt, 1 h.

inﬂammation of okadaic acid-induced neurodegenera-

tion.^9,33−36However, the inhibitory eﬀect of ACA on the

TRPM2 channel is nonspeciﬁc. It is often used as a broad-

spectrum inhibitor for the characterization of phospholipase

A2 (PLA2)-mediated pathways in early studies and also blocks

other TRP channels (TRPM8, TRPC3, TRPC6, and

TRPV1).^24,37,38The inhibitory eﬀect of ACA on the TRP

channel is in the following order: TRPM2, TRPM8 > TRPC6

> TRPV1. A side-by-side comparison of ACA with the TRPV1

selective antagonist SB366791 (Figure 1B) showed that N-

phenylcinnamamide forms the core for a class of TRP channel

inhibitors.³⁹Therefore, the speciﬁcity of the N-phenyl-

cinnamamide structures to a particular TRP channel might

be achieved through a speciﬁc set of substituents.

shown in Figure 1C, we divided ACA brieﬂy into the pentyl

aromatic ring moiety (Class A), linker moiety (Class B), and

carboxyl aromatic ring moiety (Class C). The structure of each

moiety was modiﬁed separately to investigate the inﬂuence of

diﬀerent structural fragments on the inhibitory activity against

the TRPM2 channel, to obtain a thorough understanding of

the structure−activity relationship (SAR). The general

synthetic routes for preparation of Classes A, B, and C are

described in Schemes 1−8.

a

Scheme 2. Synthesis of Compound B1

In this study, we report a successful case of modiﬁcation of

the known TRPM2 inhibitor ACA, which led to the discovery

of novel potent and selective inhibitors of the TRPM2 channel.

Among the 59 synthesized compounds, the promising

compound A23 inhibited the TRPM2 channel with a sub-

micromolar IC₅₀value and displayed selectivity against the

TRPM8 and TRPV1 channels as well as PLA2. Further

pharmacological and pharmacokinetic (PK) experiments

revealed that A23 had marked neuroprotective eﬀects against

oxygen glucose deprivation/reperfusion (OGD/R)-induced

cell death in vitro and transient middle cerebral artery

occlusion (tMCAO)-induced ischemic stroke in vivo and

exhibited a high plasma exposure. Beyond facilitating the

discovery of novel speciﬁc TRPM2 inhibitors, the ﬁnding of

this study will provide a new tool for studying the TRPM2

molecular mechanisms in cell physiology and pathophysiology

and might promote therapeutic advances for ischemic injury

and other related diseases.

a

Reagents and conditions: (a) Pd(OAc)₂, Et₃N, CH₃CN, reﬂux, 12 h.

a

Scheme 3. Synthesis of Compound B2

a

Reagents and conditions: (a) NaOH, ethanol, rt, 20 min.

Methyl 2-aminobenzoate (10) was hydrolyzed with sodium

hydroxide to give 2-aminobenzoic acid (11a), which then was

reacted with 2,2-dimethyl-1,3-dioxane-4,6-dione in toluene to

form carboxyacetamidobenzoic acid (12a). Biphenylcarbalde-

hyde derivatives (15a−15t) were synthesized by the Suzuki

coupling reaction from substituted benzboric acids (13a−13s)

and 4-bromobenzaldehyde derivatives (14a−14c). Com-

pounds A1−A33 were prepared from 12a through the

RESULTS AND DISCUSSION

■

Chemistry. Structural optimization was performed on ACA

to discover more active inhibitors of the TRPM2 channel. As

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a

Scheme 4. Synthesis of Compound B3

a

Reagents and conditions: (a) SOCl₂, reﬂux, 4 h; (b) 11a, Et₃N, THF, 0 °C, 15 min, then 60 °C, 3 h.

a

Scheme 5. Synthesis of Compound B4

a

Reagents and conditions: (a) acetone, rt, 2 h.

a

Scheme 6. Synthesis of Compounds C1−C17

a

Reagents and conditions: (a) 2,2-dimethyl-1,3-dioxane-4,6-dione, toluene, reﬂux, 3 h; (b) piperidine, toluene, reﬂux, 3 h, then rt, 1 h.

a

Scheme 7. Synthesis of Compound C18

a

Reagents and conditions: (a) acetic anhydride, reﬂux, 2 h.

Knoevenagel reaction with commercially available benzalde-

hyde derivatives (16a−16m) or synthesized biphenylcarbalde-

hydes 15a−15t by the catalysis of piperidine (Scheme 1).^28,40

Coupling of the 1-iodo-4-pentylbenzene (17) with 2-

vinylbenzoic acid (18) under the Heck conditions produced

compound B1 (Scheme 2).⁴¹

Compound B2 was prepared through an aldol condensation

between 4-pentylbenzaldehyde (16n) and 2-acetylbenzoic acid

(19) in ethanol (Scheme 3).⁴²

Treatment of 4-pentylbenzoic acid (20) with thionyl

chloride yielded 4-pentylbenzoyl chloride (21), which then

was reacted with 11a in the presence of catalytic amounts of

triethylamine to form compound B3 (Scheme 4).

As aforementioned, aminobenzoic acid derivatives (11b−

11n) were reacted with 2,2-dimethyl-1,3-dioxane-4,6-dione to

produce carboxyacetamidobenzoic acid derivatives (12b−

12n). Subsequently, piperidine-catalyzed Knoevenagel reac-

tions of 12b−12n and benzaldehyde derivatives (15q, 16m, or

16n) provided compounds C1−C17 (Scheme 6).⁴⁰

A dehydration reaction was accomplished by combining

compound A13 with acetic anhydride to form compound C18

(Scheme 7).

Knoevenagel reactions of benzaldehyde derivatives (16m or

16n) and malonic acid produced cinnamic acids (24a, 24b),

which then were reacted with thionyl chloride to yield

cinnamoyl chlorides (25a, 25b). Compounds C19−C22

were prepared from 2,3-dihydroquinolin-4(1H)-one or

1,2,3,4-tetrahydro-5H-benzo[b]azepin-5-one with compound

25a or 25b in the presence of triethylamine (Scheme 8).

4-Pentylaniline (22) was reacted with isobenzofuran-1,3-

dione (23) in acetone to aﬀord compound B4 (Scheme 5).⁴³

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a

Scheme 8. Synthesis of Compounds C19−C22

a

Reagents and conditions: (a) malonic acid, piperidine, toluene, reﬂux, 3 h, then rt, 1 h; (b) SOCl₂, reﬂux, 4 h; (c) 2,3-dihydroquinolin-4(1H)-one

or 1,2,3,4-tetrahydro-5H-benzo[b]azepin-5-one, Et₃N, THF, 0 °C, 15 min, then 60 °C, 3 h.

Figure 2. Inhibitory activity of 30 μM compounds A1−A33, B1−B4, and C1−C22 against the TRPM2 channel preliminarily evaluated through a

calcium imaging method. The concentration of agonist (H₂O₂) is 1 mM. Data are presented as the mean SD from at least three independent

experiments (n ≥ 3), and ***P < 0.005, **P < 0.01, *P < 0.05, test groups compared to the control group, one-way ANOVA, Dunnett’s multiple

comparison test.

Inhibitory Activity and SAR. A total of 59 compounds

(Figure S1) were synthesized, and their biological activities

were investigated. Fluorescent calcium imaging was used to

preliminarily evaluate the inhibitory activity of these 59

compounds in human embryonic kidney (HEK) 293T cells

stably overexpressing the human TRPM2 channel. As

measured by intracellular Fluo 3 ﬂuorescence, 38 compounds

signiﬁcantly suppressed hydrogen peroxide (H₂O₂)-induced

Ca²⁺inﬂux after being incubated with cells for 30 min at a

concentration of 30 μM (Figure 2). Among them, 34

compounds reduced the Ca²⁺ﬂuorescence increase by ∼50%

compared with vehicle-treated cells. Their inhibitory activity

against the TRPM2 channel was conﬁrmed in whole-cell

patch-clamp experiments (Figure 3, Table S1). At the

concentration of 1 μM, 22 compounds inhibited more than

45% of TRPM2 currents, which were further tested at 0.3 μM.

The concentration-dependent response curves of compounds

that blocked more than 45% of TRPM2 currents at the

concentration of 0.3 μM were plotted to calculate IC₅₀values.

Six compounds, that is, A22, A23, A30, C7, C12, and C16,

gave IC₅₀values of 398−987 nM (Figure 4, Table S1). The

TRPM2 nonspeciﬁc inhibitor ACA was used as a positive

control with an IC₅₀value of 3.67 μM.

As shown in Figure 2, Figure 3, and Table S1, replacing the

n-pentyl of ACA with hydrogen (A1), chlorine (A2), ethoxyl

(A8), isopropylcarbamoyl (A9), and various alkyl chains (A3−

A7) resulted in no or decreased inhibitory activity, showing

that the inhibitory activity on the TRPM2 channel decreased

with the shortening of the alkyl chain length. When the n-

pentyl of ACA was replaced with six-member N-containing

heterocyclic substitution groups, that is, pyridin-3-yl (A10),

piperidin-1-yl (A11), and morpholinyl (A12), a lower potency

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Figure 3. Inhibitory activity of selected compounds against TRPM2 currents validated by whole-cell patch-clamping. (A) Mean inhibition ratio of

TRPM2 currents by 1 μM of selected compounds. (B) Representative currents of 500 μM ADPR-induced TRPM2 with treatment of 1 μM of

selected compounds. (C) Mean inhibition ratio of TRPM2 currents by 0.3 μM of selected compounds. Data are presented as the mean SD from

six independent experiments (n = 6), and ***P < 0.005, **P < 0.01, *P < 0.05, test groups compared to the ACA group, one-way ANOVA,

Tukey’s multiple comparison test.

than ACA was observed. However, a phenyl-substituted ACA

derivative (A13) showed a comparable inhibitory activity (IC₅₀

= 2.56 μM) when compared with ACA (IC₅₀= 3.67 μM,

Figure 4). These results demonstrated that the replacement of

the n-pentyl of ACA with phenyl was feasible for improving the

potency.

To further investigate the SAR of phenyl-substituted ACA

derivatives and obtain more active compounds, various

substituted derivatives at the biphenyl of the compound A13

were synthesized. The inhibitory activities of compounds that

introduced methyl, methoxyl, ﬂuorine, or triﬂuoromethyl into

2-, 3-, 2′-, 3′-, or 4′-positions of the biphenyl group of

compound A13 were investigated by ﬂuorescence-based Ca²⁺

inﬂux assays and whole-cell patch-clamp experiments (Figure

2, Figure 3, and Table S1), and they showed the following. (1)

When a methyl was introduced to the 3-position (A14), the

inhibitory activity was maintained, but the substitution of a

methyl into the 2-, 2′-, 3′-, or 4′-position (A15, A16, A20, and

A24) increased the inhibitory activity compared with that of

ACA. (2) The introduction of a methoxyl into the 2′- or 3′-

position (A17, A21) showed a higher inhibitory activity than

that of ACA, whereas compound A25 with a methoxyl

substituent at the 4′-position exhibited retained potency

compared with that of ACA. (3) When ﬂuorine or

triﬂuoromethyl was attached to the 2′- or 3′-position, the

inhibitory activity was maintained (A19) or enhanced (A18,

A22, and A23), particularly compounds A22 and A23 with a

substituent at the 3′-position, with IC₅₀values of 830 and 788

nM, respectively (Figure 4). However, the substitution of

ﬂuorine and triﬂuoromethyl into the 4′-position decreased the

inhibitory activity (A26, A27).

These results indicated that the introduction of an

appropriate substituent at the 2, 2′, 3′, or 4′-position of the

biphenyl group of compound A13 could improve the

inhibitory eﬀect. Among them, the 3′-position could tolerate

a more diverse modiﬁcation and obtain more eﬀective

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Figure 4. Dose−Response curves for A13, A22, A23, A30, C7, C12, C16, and ACA. Data are presented as the mean SD from six independent

experiments (n = 6).

compounds than other positions, especially the substitution of

ﬂuorine and triﬂuoromethyl. Therefore, we synthesized and

investigated the inhibitory activities of more compounds

substituted at the 3′-position of the A13 biphenyl group

with a halogen or other electron-withdrawing group including

chlorine, bromine, and a nitro group to discover more eﬃcient

TRPM2 inhibitors (Figure 2, Figure 3, and Table S1).

Compounds A28−A30 expectedly exhibited strong inhibitory

activities, of which the nitro-substituted compound A30 was

the most potent TRPM2 inhibitor with an IC₅₀value of 398

nM (Figure 4). The inhibitory activity against the TRPM2

channel was enhanced with the increase of electron-with-

drawing capacity of 3′-position substituents. Moreover, with

the exception of compound A24 with methyl substitution at

the 4′-position, other substituents at the 4′-position (A25−

A27) weakened the inhibitory activity of compound A13.

Therefore, more alkyl groups including ethyl, isopropyl, and

tert-butyl were introduced to the 4′-position to discover more

potent compounds. For the resulting compounds (A31−A33),

higher inhibitory activities were observed compared with those

of ACA, especially the ethyl-substituted compound A31, which

showed better inhibitory activity than methyl-substituted A24.

These results indicated that the introduction of an alkyl at the

4′-position of the A13 biphenyl group could exhibit improved

inhibitory activity. In brief, a large number of resulting

compounds modiﬁed on the biphenyl group of compound

A13 displayed improved or at least maintained activity. In

particular, the electron-withdrawing group at the 3′-position

was beneﬁcial to increase the inhibitory activity.

The linker part of ACA consists of a carbon−carbon double

bond and an amide bond. As shown in Figure 2, Figure 3, and

Table S1, removing the amide bond (B1) resulted in improved

inhibitory activity compared with ACA, whereas no inhibitory

activity was observed for compound B2, which only removed

the imine group of the amide bond. Meanwhile, the eﬀect of

removing the carbon−carbon double bond on the inhibitory

activity was investigated (B3), which showed that the potency

was retained. Then the position of the carbonyl group and the

imine group in the amide bond of compound B3 was

exchanged, but decreased inhibitory activity was observed

(B4). These results indicated that removing the carbon−

carbon double bond did not aﬀect the TRPM2 inhibitory

activity, and removing amide bond was favorable to the

inhibitory activity.

As indicated in Figure 2, Figure 3, and Table S1, with the

removal of the carboxyl group from the 1-position to the 5- or

6-position of ACA to obtain compounds C1 and C2, decreased

or disappeared inhibitory activity was given, suggesting the

importance of the carboxyl group at the 1-position of ACA for

inhibitory activity against the TRPM2 channel. Therefore, we

retained the carboxyl at the 1-position of ACA and investigated

the biological activities of derivatives with the methyl or

halogen group introduced at the 3-, 4-, or 5-position. When

methyl or chlorine was introduced into the 3-position of ACA,

the inhibitory activity was decreased or disappeared, even if the

beneﬁcial substituent was introduced into the 4- or 5-position

(C3−C6). The introduction of a methyl or chlorine into the 4-

position of ACA increased the inhibitory activity (C7, C10);

particularly, compound C7 with a methyl substituent showed

an IC₅₀value of 467 nM (Figure 4). However, a replacement

of the n-pentyl of C7 and C10 with 3′-nitrobiphenyl or

biphenyl resulted in reduced inhibitory activities (C8, C11). In

addition, compound C9 with two ﬂuorines introduced at the 4-

and 5-positions of ACA showed improved inhibitory activity.

For the 5-position-substituted ACA derivatives, compounds

substituted with a methyl (C12) and bromine (C13) exhibited

strong inhibitory activities, especially C12 with an IC₅₀value of

987 nM (Figure 4). The replacement of the n-pentyl of C13

with biphenyl or 3′-nitrobiphenyl was accompanied by the

replacement of halogen-formed compounds C14−C17.

Among them, biphenyl- and bromine-substituted compound

C16 was the most potent TRPM2 inhibitor with an IC₅₀value

of 410 nM (Figure 4). Since derivatives of 2,3-dihydroquina-

zolin-4(1H)-one and tetrahydrocyclohepta[c]pyrazol were

eﬀective TRPM2 inhibitors, we also investigated the inhibitory

activities of four-, six-, and seven-membered cyclic ACA

derivatives formed by the carboxyl group and the ortho imine

group (C18−C22). The inhibitory activity was decreased or

disappeared, suggesting the importance of a free carboxyl

group for the inhibitory activity. In brief, a large proportion of

resulting compounds with methyl or halogen introduced at the

4- or 5-position of ACA displayed improved inhibition,

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Figure 5. Selectivity evaluations of A23, A30, C7, C12, and C16 at concentrations of 10 and 3 μM on the TRPM8 and TRPV1 channels. (A)

Representative currents of 1 mM menthol-induced TRPM8 with a treatment of 10 μM compound. (B, C) Mean inhibition ratio of TRPM8

currents by 10 or 3 μM compound. (D) Representative currents of 0.01 mM capsaicin-induced TRPV1 with a treatment of 10 μM compound. (E,

F) Mean inhibition ratio of TRPV1 currents by 10 or 3 μM compound. Data are presented as the mean SD from three to six independent

experiments (n = 3−6), and ***P < 0.005, **P < 0.01, *P < 0.05, test groups compared to the control group, one-way ANOVA, Dunnett’s multiple

comparison test.

especially the compounds substituted with methyl and

bromine.

A30, C7, C12, and C16, which showed excellent inhibitory

activities against TRPM2, were selected for further evaluation.

Inhibitory Eﬀects of ACA Derivatives on the TRPM8

and TRPV1 Channels. Before testing the selectivity on other

TRP channels, we evaluated the eﬀect of these six compounds

on the SH-SY5Y cell viability using the Cell Counting Kit-8

(CCK-8) assay. With the exception of compound A22, which

showed 23.7% toxicity against SH-SY5Y cells at 30 μM, the

other ﬁve compounds did not aﬀect cell viability (<10%

toxicity) when incubated with concentrations of 1−30 μM for

24 h (Figure S2). Therefore, we did not conduct further

studies on A22. To evaluate the compound selectivity, the

inhibitory eﬀects of ﬁve compounds (A23, A30, C7, C12, and

C16) on the closely related TRPM8 channel and more

distantly related TRPV1 channel were investigated by whole-

cell patch-clamp recordings. Menthol and capsaicin activated

the TRPM8 and TRPV1 currents, respectively, in overex-

pressed HEK293T cells.

Taken together, the structural modiﬁcation of ACA resulted

in a series of compounds with an improved TRPM2 inhibitory

activity. Our investigation illustrated that (1) replacing the n-

pentyl with a biphenyl group and introducing an appropriate

substituent at the 2-, 2′-, 3′-, or 4′-position of the biphenyl

group could improve the inhibitory eﬀect, such as ﬂuorine at

the 2′-position, alkyl at the 4′-position, particularly electron-

withdrawing groups at the 3′-position of biphenyl; (2) the

removal of the amide bond of the linker part was favorable to

the inhibitory activity, but the improvement was limited

compared with that of Class A and Class C; (3) the

introduction of a methyl and bromine into the 4- or 5-position

of ACA derivatives contributed to improving inhibitory

activity. Moreover, the orientation and electronegativity of

the substituents on the ACA derivatives played key roles in the

inhibitory activity against TRPM2. Compounds A22, A23,

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Figure 6. Inhibitory activity of A23, A30, C7, C12, and C16 at concentrations of 10, 3, and 1 μM against PLA2. Data are presented as the mean

SD from three determinations (n = 3), and ***P < 0.005, *P < 0.05, test groups compared to the corresponding ACA group, one-way ANOVA,

Dunnett’s multiple comparison test.

As indicated in Figure 5A,B, compared with the control, an

extracellular addition of 10 μM A23 and A30 showed mild

inhibitory eﬀects on TRPM8 currents by 22.2% and 14.6%,

respectively, while C7, C12, and C16 inhibited TRPM8

currents by 88.1%, 85.6%, and 93.3%, respectively, which was

even stronger than the inhibitory eﬀect of ACA (62.2%). The

eﬀects of 3 μM compound on TRPM8 currents were tested

and showed that A23 and A30 exhibited better speciﬁcity for

the TRPM2 channel than the ACA treatment (Figure 5C).

The positive control ACA showed a 39.3% inhibition ratio on

TRPM8 currents at 3 μM, consistent with the reported IC₅₀

value of 3.9 μM.²⁴It is noteworthy that both A23 and A30 had

an electron-withdrawing group (triﬂuoromethyl and nitro) at

the 3′-position of biphenyl, while C7, C12, C16 and ACA had

an n-pentyl or unsubstituted biphenyl at this position,

suggesting that the electron-withdrawing group at the 3′-

position of biphenyl was unfavorable for the inhibition of the

TRPM8 channel. In addition, C7, C12, and C16 all had a

methyl or bromine at the 4- or 5-position, which contributed

to improving the inhibitory activity against the TRPM8

channel.

For the TRPV1 channel, both 10 and 3 μM C7 resulted in a

distinct reduction of TRPV1 currents compared with the

control, and the other four compounds showed no signiﬁcant

eﬀects on the TRPV1 currents. In particular, A23 and A30

only suppressed TRPV1 currents by 12.7% and 5.4%,

respectively, at a concentration of 10 μM (Figure 5D−F).

These results suggested that compounds containing an

electron-withdrawing group at the 3′-position of biphenyl

were not conducive to the inhibition of the TRPV1 channel.

Inhibitory Eﬀects of ACA Derivatives on PLA2. As

aforementioned, ACA inhibits not only TRP channels but also

PLA2, which hydrolyzes cell membrane phospholipids to

produce arachidonic acid (AA) and lyso-phospholipids, playing

a key role in the production of inﬂammatory lipid

mediators.^24,39,44,45To investigate the inhibitory eﬀects of

our synthesized ACA derivatives on PLA2, the colorimetric

PLA2 assay was performed on selected compounds A23, A30,

C7, C12, and C16. As shown in Figure 6, A23 and A30

displayed weaker PLA2 inhibitory activities than ACA at 10

μM with 14.8% and 24.6%, respectively. The positive control

ACA showed a 40.0% inhibition ratio of PLA2 at 10 μM,

which was generally consistent with the reported IC₅₀value of

22 μM,⁴⁴whereas C7, C12, and C16 showed no signiﬁcant

diﬀerence in the inhibitory activity compared with ACA at a

concentration of 10 μM, indicating that the selectivity of these

compounds on PLA2 did not improve. At a concentration of 3

μM, only A23 (7.8%) exhibited a lower PLA2 inhibitory

activity than ACA (23.4%). It is worth noting that, at 1 μM,

only a slight inhibitory activity was observed for all active

compounds.

Summarizing the inhibitory activities of the most active

compounds on TRP channels and PLA2, we found that

diﬀerent substituted groups on the ACA derivatives played

critical roles in the selectivity between the TRPM2 channel

and other TRP channels, as well as PLA2. Compounds with an

electron-withdrawing group (triﬂuoromethyl and nitro) at the

3′-position of biphenyl showed speciﬁcity to the TRPM2

channel and have little or no eﬀect on the TRPM8 and TRPV1

currents and PLA2, whereas compounds with methyl or

bromine introduced at the 4- or 5-position were harmful to the

speciﬁcity of the TRPM2 channel. Speciﬁcally, compounds

with a methyl group at the 4- or 5-position of ACA displayed

improved or retained inhibitory activity against the TRPM8

currents and PLA2. In addition, a compound with a methyl

substitution at the 4-position of ACA also inhibited the

TRPV1 currents. As for the replacement of n-pentyl with

unsubstituted biphenyl and the introduction of a bromine at

the 5-position of ACA, the compound showed an inhibitory

eﬀect on the TRPM8 currents and PLA2.

ACA Derivatives Protect SH-SY5Y Cells against OGD/

R Injury. OGD/R provides a cellular model to mimic neuronal

ischemia and excitotoxicity in neurodegenerative diseases such

as ischemic stroke and Alzheimer’s disease.⁴⁶To conﬁrm

OGD/R-induced cell injury and evaluate the protective eﬀects

of ﬁve selected ACA derivatives, we measured the cell viability

by the CCK-8 assay after OGD/R in SH-SY5Y cells. We used

OGD for 8 h followed by replacement with complete culture

medium for another 12 h as an OGD/R model, and edaravone

(EDA), an ROS scavenger that was widely used clinically as a

neuroprotectant for the treatment of cerebral infarction, was

selected as the positive control. The results showed that the

cell viability was obviously decreased to 49.4% in the OGD/R

group compared with the control group, and 1−30 μM EDA

exhibited eﬃcient protective eﬀects on SH-SY5Y cells; in

particular, 3 μM EDA mediated the high upregulation of cell

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Figure 7. SH-SY5Y cell injury induced by OGD/R and the protective eﬀects of ACA derivatives. Data are presented as the mean SD from three

independent experiments (n = 3), and ^###P < 0.005, OGD/R group compared to the control group; ***P < 0.005, **P < 0.01, *P < 0.05, test

groups compared to the OGD/R group, one-way ANOVA, Tukey’s multiple comparison test.

Figure 8. Plasma concentration−time proﬁles for A23 i.v. (0.3, 1, and 3 mg/kg) and i.g. (10, 100 mg/kg) administration in mice. Data are

presented as the mean SD, and each measurement was repeated three times (n = 3).

a

Table 1. PK Parameters of A23 in Mice Plasma after Single-Dose Intravenous and Intragastric Administration

dose

T_1/2

T_max

C_max

AUC_0‑t

AUC_0‑∞

CL

MRT

(h)

F%-0.3

F%-1

route

(mg/kg)

(h)

(μg/mL)

(h·μg/mL)

(mL/h/kg)

mg/kg

i.v.

i.g.

0.3

1

10

0.93

1.70

1.42

0.08

0.50

5.07

12.95

58.35

5.20

18.46

156.63

5.45

18.89

160.13

55.0

52.9

62.5

1.19

1.70

2.25

90.4

84.8

a

Each measurement was repeated three times (n = 3).

viability to 81.2% (Figure 7). Compounds A23, A30, C7, C12,

and C16 showed comparable protective activities with EDA at

a concentration of 3 μM compared with the OGD/R group.

Among them, A23 displayed considerable protective activities

when the concentration was only 0.3 μM (68.7%), and A23

induced an increase of cell viability to 80% at a wider range of

concentrations (1, 3, 10, and 30 μM) compared with EDA (3

μM). These results demonstrated that all ﬁve compounds at a

certain concentration exhibited a protective eﬀect on the cell

viability of SH-SY5Y cells subjected to OGD/R; especially,

compound A23 had a wider eﬀective concentration range,

which was between 0.3 and 30 μM.

Besides the administration of EDA, A30 and C7 at 10 and

30 μM displayed some degree of cytotoxicity to SH-SY5Y cells

under a pathological condition (OGD/R), whereas these

compounds did not aﬀect the cell viability under normoxic and

normal sugar conditions (Figure S1). Similar phenomena have

been reported in some literature, including EDA and other

neuroprotective agents.⁴⁷⁻⁴⁹The reason and mechanism are

still unclear and remain to be further studied.

PK Studies of A23 In Vivo. On the basis of its favorable

speciﬁcity to the TRPM2 channel as well as protective activity

in vitro, A23 was further evaluated in in vivo PK studies. After

a single intravenous (i.v.) administration with 3 mg/kg and a

single intragastric (i.g.) administration with 100 mg/kg, the

half-life (T_1/2), maximum concentration (C_max), and area under

the curve (AUC_0−∞) were determined to be 1.34 h, 24.33 μg/

mL, 40.93 h·μg/mL and 3.62 h, 168.17 μg/mL, 1872.62 h·μg/

mL, respectively (Figure 8 and Table S2). There were no

apparent adverse eﬀects observed in mice at a high gavage dose

of 100 mg/kg. However, the oral bioavailability (F%) of A23

was calculated to be greater than 100%, which suggested that

the doses may be outside the linear pharmacokinetic range.

Subsequently, the single administered dose was reduced to

0.3 (i.v.), 1 (i.v.), and 10 mg/kg (i.g.), and the plasma

concentration−time proﬁles and key PK parameters are

summarized in Figure 8 and Table 1. The results indicated

that the PK behavior of A23 for the i.v. (0.3 and 1 mg/kg) and

i.g. (10 mg/kg) administration conformed to the one-

compartment model. After single 0.3 and 1 mg/kg i.v. doses

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Figure 9. Eﬀects of A23 and EDA i.v. administration (3, 1, and 0.3 mg/kg) 24 h after tMCAO in mice. (A) Representative TTC-stained brain slices

of mice intravenous in the control, vehicle, EDA, and A23 (0.3, 1, and 3 mg/kg) treatment groups; magenta: healthy tissue, white: damaged tissue.

(B−D) Statistical analysis of the percentage of infarct volume after tMCAO in the control, vehicle, and 3 (B), 1 (C), and 0.3 (D) mg/kg EDA and

A23 treatment groups. (E−G) Statistical analysis of the neurological scores after tMCAO in the control, vehicle, and 3 (E), 1 (F), and 0.3 (G) mg/

kg EDA and A23 treatment groups. Data are presented as the mean SD, and each measurement was repeated 7−12 times (n = 7−12), and ***P

< 0.005, *P < 0.05, one-way ANOVA, Tukey’s multiple comparison test.

of A23, the T_1/2, clearance rate (CL), and mean residence time

(MRT) values were 0.93 h, 55.0 mL/h/kg, 1.19 and 1.70 h,

52.9 mL/h/kg, and 1.70 h, respectively. At a dose of 10 mg/kg

i.g., A23 was rapidly absorbed with a time-to-maximum-

concentration (T_max) of 0.5 h, a T_1/2of 1.42 h, and an MRT of

2.25 h. These results showed that compound A23 was rapidly

distributed and eliminated, and the clearance of A23 was in an

acceptable range. A23 had high plasma exposure with high

C_maxand AUC_0−∞. When A23 was administrated by the i.v.

route at only 0.3 mg/kg, the time for the eﬀective

concentration to be higher than the IC₅₀value of TRPM2

(0.32 μg/mL) could reach 2−4 h (the concentration of A23 at

2h was 0.81 μg/mL). Furthermore, the oral bioavailability of

A23 with 10 mg/kg (i.g.) administration was 90.4% (calculated

by i.v. 0.3 mg/kg AUC_{0‑t_D}) and 84.8% (calculated by i.v. 1

mg/kg AUC_{0‑t_D}), respectively. Overall, compound A23

appeared to have favorable drug-like PK properties.

A23 Treatment Provides Neuroprotection following

Focal Cerebral Ischemia in Mice. Encouraged by its

excellent PK proﬁles, we studied compound A23 in an in

vivo tMCAO model. Mice were subjected to 90 min of

tMCAO, and control (saline), vehicle (5% dimethyl sulfoxide

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(DMSO)/5% Solutol in saline), EDA (3, 1, and 0.3 mg/kg,

i.v.), or A23 (3, 1, and 0.3 mg/kg, i.v.) was administered at 3 h

after the onset of reperfusion. As shown in Figure 9A, the

infarct volume was calculated using 2,3,5-triphenyltetrazolium

chloride (TTC) staining on 1 mm brain sections 24 h after

tMCAO. A considerable reduction in the infarct volume was

observed with a treatment of 3 mg/kg EDA (39.0 2.4%, n =

8, p < 0.005) and 3 mg/kg A23 (38.9 4.7%, n = 8, p < 0.005)

vitro OGD/R model and in the in vivo tMCAO model, which

were equal to or even better than the widely used

neuroprotectant EDA. Furthermore, the PK studies showed

that A23 exhibited high plasma exposure, which was consistent

with the pharmacodynamic results. Overall, A23, as a potent

and selective TRPM2 inhibitor with neuroprotective activity in

vitro and anticerebral infarction eﬃcacy in vivo, might serve as

a useful molecular tool for further physiology and pathophysi-

ology function studies related to the TRPM2 channel and also

lead to potential clinical therapeutic agents for ischemic injury.

compared with the vehicle group (57.6

4.9%, n = 9),

whereas there was no diﬀerence in infarct volume in the 3 mg/

kg EDA- and 3 mg/kg A23-treated groups (Figure 9B).

Considering the high plasma exposure of A23 at low doses, the

eﬀects of 1 and 0.3 mg/kg A23 on I/R injury following

tMCAO were further investigated. The results showed that the

1 mg/kg EDA-treated group (43.9 2.8%, n = 12) and 0.3

EXPERIMENTAL SECTION

■

Materials and Methods. All of the 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.) was employed to monitor the

reaction progress, and silica column chromatography was performed

to purify the crude product (silica gel 200−300 mesh, Shanghai

mg/kg EDA-treated group (46.2

signiﬁcantly decrease the infarct volume compared with the

control group (52.0 1.7%, n = 7). However, an obvious

2.1%, n = 12) did not

1

Sanpont Co., Ltd.). H NMR spectra and ¹³C NMR spectra were

alleviation in infarct volume was observed in both the 1 mg/kg

A23-treated group (38.5 2.8%, n = 12, p < 0.05) and 0.3

mg/kg A23-treated groups (39.8 1.4%, n = 12, p < 0.005)

recorded at 400 MHz using a Bruker Avance III spectrometer (Bruker

Co.) in a CDCl₃or DMSO-d₆solution with tetramethylsilane as the

internal standard, and chemical shift values were given in parts per

million. The NMR data were processed by the software MestReNova

(ver. 6.1.0, mestrelab research S.L.). The splitting peak was designated

as s, singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet; br,

broad. The high-resolution mass spectrometry (HRMS) was

measured on an FT-MS-Bruker APEX IV mass spectrometer. The

purity of all ﬁnal compounds for biological tests was determined to be

greater than 95% with a high-performance liquid chromatography

(HPLC) analysis (Dione HPLC system consisting of LPG-3400SD

pump and DAD-3000 UV detector). Water was used with 0.1%

formic acid as mobile phase A (2−20%), acetonitrile was mobile

phase B (80−98%); Diamonsil C18 column, 5 μm, 4.6 × 250 mm, 25

°C, 1 mL/min was used.

Chemical Synthesis. General Procedure for the Synthesis of

Compounds 3a−3n. Methyl 2-aminobenzoate 10 (604 mg, 4 mmol)

and sodium hydroxide (480 mg, 12 mmol) were dissolved in H₂O (6

mL), ethanol (6 mL), and tetrahydrofuran (THF) (18 mL). The

reaction mixture was reﬂuxed for 4 h, and the solvent was removed

under reduced pressure. The residue was extracted with ethyl acetate

(EtOAc) twice. The water layer was neutralized with 1 N HCl, and

the resulting precipitate was ﬁltered and dried to aﬀord 2-

aminobenzoic acid 11a. A mixture of 2-aminobenzoic acid 11a or

commercially available aminobenzoic acid derivatives 11b−11n (4

mmol) and 2,2-dimethyl-1,3-dioxane-4,6-dione (576 mg, 4 mmol) in

toluene (5 mL) was reﬂuxed for 3 h. The suspension was cooled,

ﬁltered, washed with toluene, and dried to aﬀord carboxyacetamido-

benzoic acid derivatives 12a−12n.⁴⁰

General Procedure for the Synthesis of Compounds 6a−6t.

Pd(PPh₃)₄(147 mg, 0.13 mol) was added to a degassed mixture of

substituted benzboric acids 13a−13s (1.81 mmol), 4-bromobenzal-

dehyde derivatives 14a−14c (1.27 mmol), K₂CO₃(501 mg, 3.63

mmol) in dioxane (4 mL), and water (2 mL) under argon. The

mixture was heated at 105 °C for 24 h, cooled to room temperature

(rt), 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 petroleum

ether (PE)/EtOAc (6/1) to aﬀord biphenylcarbaldehyde derivatives

15a−15t.²⁸

General Procedure for the Synthesis of Compounds A1−A33,

C1−C17. Piperidine (94 mg, 1.1 mmol) was added to a suspension of

biphenylcarbaldehyde derivatives 15a−15t or commercially available

benzaldehyde derivatives 16a−16m (1.1 mmol) and carboxyacetami-

dobenzoic acid derivatives 12a−12n (1 mmol) in toluene (3 mL).

The mixture was heated to reﬂux for 4 h, followed by being stirred at

room temperature for an additional 1 h and then acidiﬁed with 1 N

HCl. Subsequently, the mixture was extracted with EtOAc three

times. The combined organic layers were washed with brine, dried

compared to the control group (52.0

1.7%, n = 7),

consistent with the PK results. Notably, at 0.3 mg/kg,

compound A23 was signiﬁcantly more eﬀective than EDA in

reducing the infarct volume (39.8 1.4% vs 46.2 2.1%, n =

12, p < 0.05) (Figure 9C,D). Collectively, the infarct volume of

mice in the EDA (3 mg/kg) group and A23 (3, 1, and 0.3 mg/

kg) group was signiﬁcantly improved compared to that of the

vehicle-treated group, and the eﬀect of A23 (0.3 mg/kg) was

preferable to that of EDA (0.3 mg/kg).

To assess the health of the mouse, we evaluated neurological

scores at 24 h after tMCAO. As shown in Figure 9E, the scores

obtained in the groups treated with 3 mg/kg EDA (1.6 0.2, n

= 8, p < 0.05) and 3 mg/kg A23 (1.8 0.2, n = 8, p < 0.05)

were both markedly lower than those of the vehicle-treated

group (2.4 0.5, n = 9), illustrating that A23 had the same

eﬀect as EDA in reducing neurological scores. Neither EDA

nor A23 at a dose of 1 and 0.3 mg/kg showed signiﬁcantly

diﬀerent neurological improvements compared with the

control group (Figure 9F,G). Taken together, compound

A23 showed protective eﬀects against a cerebral infarction

induced by focal cerebral ischemia in mice, which were equal

to or even better than the positive control EDA, suggesting

that A23 is an attractive candidate for the intervention of

ischemic stroke and merits further study.

CONCLUSION

■

We designed and synthesized 59 novel ACA derivatives to

discover more potent and selective TRPM2 inhibitors. Some of

them exhibited a stronger inhibitory activity against the

TRPM2 currents than ACA, particularly compounds A22,

A23, A30, C7, C12, and C16, with IC₅₀values ranging from

398 to 987 nM. More importantly, A23 showed TRPM2

selectivity over TRPM8 and TRPV1 channels as well as PLA2.

SAR studies revealed that compounds containing electron-

withdrawing groups at the 3′-position of biphenyl improved

the potency and speciﬁcity of the TRPM2 channel, while

compounds with a methyl or bromine introduced at the 4- or

5-position contributed to improving inhibitory activity but

were harmful to the speciﬁcity of the TRPM2 channel. These

summarized SARs provide valuable insights for the further

development of speciﬁc TRPM2 inhibitors. Compound A23

exhibited excellent neuroprotective activities both in the in

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over anhydrous Na₂SO₄, ﬁltered, and concentrated under reduced

pressure. The crude product was puriﬁed by silica gel column

chromatography eluting with dichloromethane (DCM)/methanol

(MeOH) (100/3) to aﬀord A1−A33 and C1−C17.⁴⁰

8.01 (d, J = 7.8 Hz, 1H), 7.70−7.57 (m, 4H), 7.17 (t, J = 7.5 Hz, 1H),

6.98 (d, J = 8.6 Hz, 2H), 6.73 (d, J = 15.6 Hz, 1H), 4.09 (q, J = 7.0

Hz, 2H), 1.35 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

170.0, 164.6, 160.6, 141.7, 141.5, 134.5, 131.6, 130.4, 127.3, 123.2,

120.8, 120.1, 117.1, 115.2, 63.7, 15.0. HRMS (ESI-TOF⁻) calcd for

C₁₈H₁₆NO₄[M-H]⁻m/z 310.1079, found 310.1075.

(E)-2-(3-Phenylacrylamido)benzoic Acid (A1). Oﬀ-white solid.

1

Yield: 62%. mp 198−200 °C. H NMR (400 MHz, CDCl₃) δ 11.30

(s, 1H), 8.94 (d, J = 9.0 Hz, 1H), 8.22 (d, J = 7.6 Hz, 1H), 7.83 (d, J =

15.6 Hz, 1H), 7.67 (t, J = 17.2 Hz, 1H), 7.60 (d, J = 4.0 Hz, 2H),

7.41−7.40 (m, 3H), 7.18 (t, J = 7.6 Hz, 1H), 6.65 (d, J = 15.6 Hz,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 172.2, 165.0, 142.9, 142.2,

135.7, 134.6, 131.9, 130.2, 128.9, 128.1, 122.9, 121.7, 120.9, 114.2.

HRMS (electrospray ionization time-of-ﬂight (ESI-TOF⁻)) calcd for

C₁₆H₁₂NO₃[M-H]⁻m/z 266.0817, found 266.0816.

(E)-2-(3-(4-(Isopropylcarbamoyl)phenyl)acrylamido)benzoic

1

Acid (A9). Oﬀ-white solid. Yield: 58%. mp 236−238 °C. H NMR

(400 MHz, DMSO-d₆) δ 12.37 (s, 1H), 8.62 (d, J = 7.9 Hz, 1H), 8.30

(d, J = 7.6 Hz, 1H), 8.05 (d, J = 7.4 Hz, 1H), 7.89 (d, J = 7.9 Hz, 2H),

7.79 (d, J = 7.6 Hz, 2H), 7.64 (d, J = 15.7 Hz, 1H), 7.54 (t, J = 7.6 Hz,

1H), 7.14 (t, J = 7.4 Hz, 1H), 6.93 (d, J = 15.7 Hz, 1H), 4.15−4.06

(m, 1H), 1.18 (d, J = 6.5 Hz, 6H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 170.5, 165.2, 163.9, 141.2, 140.3, 137.3, 136.1, 133.3, 131.7, 128.3,

128.2, 124.8, 123.1, 120.3, 119.8, 41.5, 22.8. HRMS (ESI-TOF⁻)

calcd for C₂₀H₁₉N₂O₄[M-H]⁻m/z 351.1345, found 351.1343.

(E)-2-(3-(4-(Pyridin-3-yl)phenyl)acrylamido)benzoic Acid (A10).

(E)-2-(3-(4-Chlorophenyl)acrylamido)benzoic Acid (A2). Oﬀ-

white solid. Yield: 64%. mp 223−225 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 13.63 (s, 1H), 11.35 (s, 1H), 8.61 (d, J = 8.4 Hz, 1H),

8.02 (d, J = 7.8 Hz, 1H), 7.79 (d, J = 8.3 Hz, 2H), 7.65−7.61 (m,

2H), 7.51 (d, J = 8.3 Hz, 2H), 7.19 (t, J = 7.6 Hz, 1H), 6.94 (d, J =

15.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.9, 164.1, 141.2,

140.4, 135.0, 134.5, 133.9, 131.6, 130.4, 129.4, 123.7, 123.4, 120.9,

117.4. HRMS (ESI-TOF⁻) calcd for C₁₆H₁₁NO₃Cl [M-H]⁻m/z

300.0427, found 300.0427.

1

Oﬀ-white solid. Yield: 61%. mp 256−258 °C. H NMR (400 MHz,

DMSO-d₆) δ 12.66 (br s, 1H), 8.96 (s, 1H), 8.64−8.60 (m, 2H), 8.14

(d, J = 7.9 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 7.86−7.80 (m, 4H),

7.67 (d, J = 15.7 Hz, 1H), 7.53−7.50 (m, 2H), 7.12 (t, J = 7.6 Hz,

1H), 6.90 (d, J = 15.7 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ

170.7, 164.0, 149.3, 148.1, 141.2, 140.4, 138.7, 135.2, 134.8, 134.5,

133.1, 131.8, 129.3, 127.7, 124.4, 123.9, 122.9, 120.2, 43.9, 22.6, 22.2.

HRMS (ESI-TOF⁻) calcd for C₂₁H₁₅N₂O₃[M-H]⁻m/z 343.1083,

found 343.1081.

(E)-2-(3-(p-Tolyl)acrylamido)benzoic Acid (A3). Oﬀ-white solid.

1

Yield: 65%. mp 199−201 °C. H NMR (400 MHz, DMSO-d₆) δ

13.63 (s, 1H), 11.34 (s, 1H), 8.61 (d, J = 8.4 Hz, 1H), 8.01 (d, J = 7.8

Hz, 1H), 7.65−7.58 (m, 4H), 7.26 (d, J = 7.8 Hz, 2H), 7.18 (t, J = 7.6

Hz, 1H), 6.83 (d, J = 15.6 Hz, 1H), 2.35 (s, 3H). ¹³C NMR (101

MHz, DMSO-d₆) δ 170.0, 164.4, 141.8, 141.4, 140.4, 134.4, 132.1,

131.6, 130.0, 128.6, 123.2, 121.8, 120.8, 117.3, 21.4. HRMS (ESI-

TOF⁻) calcd for C₁₇H₁₄NO₃[M-H]⁻m/z 280.0974, found 280.0974.

(E)-2-(3-(4-Ethylphenyl)acrylamido)benzoic Acid (A4). Oﬀ-white

(E)-2-(3-(4-(Piperidin-1-yl)phenyl)acrylamido)benzoic Acid

1

(A11). Brown solid. Yield: 61%. mp 215−217 °C. H NMR (400

MHz, DMSO-d₆) δ 13.58 (br s, 1H), 11.27 (s, 1H), 8.64 (d, J = 8.4

Hz, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.63−7.50 (m, 4H), 7.16 (t, J = 7.5

Hz, 1H), 6.94 (d, J = 8.6 Hz, 2H), 6.60 (d, J = 15.5 Hz, 1H), 3.28 (s,

4H), 1.58 (s, 6H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.0, 164.9,

152.8, 142.2, 141.8, 134.4, 131.6, 130.1, 123.9, 122.9, 120.6, 117.8,

116.9, 115.0, 48.8, 25.4, 24.4. HRMS (ESI-TOF⁻) calcd for

C₂₁H₂₁N₂O₃[M-H]⁻m/z 349.1552, found 349.1549.

1

solid. Yield: 51%. mp 176−178 °C. H NMR (400 MHz, DMSO-d₆)

δ 13.62 (s, 1H), 11.34 (s, 1H), 8.61 (d, J = 8.4 Hz, 1H), 8.01 (d, J =

8.7 Hz, 1H), 7.67−7.59 (m, 4H), 7.29 (d, J = 7.3 Hz, 2H), 7.18 (t, J =

5.9 Hz, 1H), 6.83 (d, J = 15.4 Hz, 1H), 2.66 (q, J = 15.2 Hz, 2H),

1.21 (t, J = 15.1, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.0,

164.4, 146.6, 141.8, 141.4, 134.4, 132.4, 131.6, 128.8, 128.7, 123.3,

121.9, 120.8, 117.3, 28.5, 15.8. HRMS (ESI-TOF⁻) calcd for

C₁₈H₁₆NO₃[M-H]⁻m/z 294.1130, found 294.1126.

(E)-2-(3-(4-Morpholinophenyl)acrylamido)benzoic Acid (A12).

1

Yellow solid. Yield: 59%. mp 239−241 °C. H NMR (400 MHz,

DMSO-d₆) δ 13.61 (s, 1H), 11.29 (s, 1H), 8.65 (d, J = 8.3 Hz, 1H),

8.02 (d, J = 7.7 Hz, 1H), 7.63−7.53 (m, 4H), 7.16 (t, J = 7.5 Hz, 2H),

6.96 (d, J = 8.4 Hz, 1H), 6.64 (d, J = 15.5 Hz, 1H), 3.73 (s, 4H), 3.21

(s, 4H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.0, 164.8, 152.6,

142.1, 141.7, 134.5, 131.6, 130.0, 125.1, 123.0, 120.7, 118.5, 116.9,

114.8, 66.4, 47.8. HRMS (ESI-TOF⁻) calcd for C₂₀H₁₉N₂O₄[M-H]⁻

m/z 351.1345, found 351.1342.

(E)-2-(3-(4-Propylphenyl)acrylamido)benzoic Acid (A5). Oﬀ-

white solid. Yield: 51%. mp 117−119 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 13.61 (br s, 1H), 11.34 (s, 1H), 8.62 (d, J = 8.4 Hz,

1H), 8.01 (d, J = 7.7 Hz, 1H), 7.65−7.59 (m, 4H), 7.26 (d, J = 7.8

Hz, 2H), 7.18 (t, J = 7.4 Hz, 1H), 6.83 (d, J = 15.6 Hz, 1H), 2.58 (t, J

= 7.4 Hz, 2H), 1.65−1.55 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 169.9, 164.4, 145.0, 141.8, 141.4,

134.5, 132.4, 131.6, 129.4, 128.7, 123.3, 121.8, 120.9, 117.3, 37.6,

24.3, 14.1. HRMS (ESI-TOF⁻) calcd for C₁₉H₁₈NO₃[M-H]⁻m/z

308.1287, found 308.1289.

(E)-2-(3-(4-Isopropylphenyl)acrylamido)benzoic Acid (A6). Oﬀ-

white solid. Yield: 58%. mp 162−164 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 11.36 (s, 1H), 8.62 (d, J = 8.2 Hz, 1H), 8.02 (dd, J =

7.9, 1.3 Hz, 1H), 7.66−7.62 (m, 3H), 7.60−7.59 (m, 1H), 7.31 (d, J =

8.1 Hz, 2H), 7.18 (t, J = 7.2 Hz, 1H), 6.82 (d, J = 15.6 Hz, 1H),

2.97−2.87 (m, 1H), 1.21 (d, J = 6.9 Hz, 6H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 170.1, 164.3, 151.1, 141.8, 141.5, 134.4, 132.5, 131.6,

128.7, 127.3, 123.2, 121.9, 120.8, 117.1, 33.8, 24.0. HRMS (ESI-

TOF⁻) calcd for C₁₉H₁₈NO₃[M-H]⁻m/z 308.1287, found 308.1284.

(E)-2-(3-(4-(tert-Butyl)phenyl)acrylamido)benzoic Acid (A7). Oﬀ-

white solid. Yield: 46%. mp 191−193 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 11.36 (s, 1H), 8.62 (d, J = 8.3 Hz, 1H), 8.02 (dd, J =

7.9, 1.3 Hz, 1H), 7.67−7.59 (m, 4H), 7.45 (d, J = 8.4 Hz, 2H), 7.20−

7.16 (m, 1H), 6.83 (d, J = 15.6 Hz, 1H), 1.29 (s, 9H). ¹³C NMR (101

MHz, DMSO-d₆) δ 170.0, 164.3, 153.3, 141.7, 141.5, 134.5, 132.1,

131.6, 128.5, 126.2, 123.2, 122.0, 120.8, 117.1, 35.0, 31.4. HRMS

(ESI-TOF⁻) calcd for C₂₀H₂₀NO₃[M-H]⁻m/z 322.1443, found

322.1443.

(E)-2-(3-([1,1′-Biphenyl]-4-yl)acrylamido)benzoic Acid (A13). Oﬀ-

white solid. Yield: 66%. mp 248−250 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 11.39 (s, 1H), 8.65 (d, J = 8.2 Hz, 1H), 8.04 (d, J = 7.6

Hz, 2H), 7.83 (d, J = 7.8 Hz, 2H), 7.75−7.72 (m, 4H), 7.67−7.62 (m,

2H), 7.50 (t, J = 15.2 Hz, 2H), 7.40 (t, J = 17.2 Hz, 1H), 7.20 (t, J =

16.4 Hz, 1H), 6.94 (d, J = 15.6 Hz, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 169.9, 164.3, 142.0, 141.3, 139.8, 134.4, 134.1, 131.6,

129.5, 129.3, 128.4, 127.6, 127.2, 123.4, 122.9, 120.9, 117.5, 117.4.

HRMS (ESI-TOF⁻) calcd for C₂₂H₁₆NO₃[M-H]⁻m/z 342.1130,

found 342.1130.

(E)-2-(3-(3-Methyl-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

1

(A14). Oﬀ-white solid. Yield: 39%. mp 223−225 °C. H NMR (400

MHz, DMSO-d₆) δ 13.75 (br s, 1H), 8.64 (d, J = 7.8 Hz, 1H), 8.10

(d, J = 5.5 Hz, 1H), 7.83 (d, J = 15.4 Hz, 1H), 7.75−7.67 (m, 3H),

7.57 (s, 1H), 7.47−7.38 (m, 5H), 7.03 (s, 1H), 6.71 (d, J = 15.5 Hz,

1H), 2.47 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 171.4, 163.8,

141.4, 141.3, 139.8, 138.1, 137.2, 132.9, 131.9, 131.6, 129.4, 129.3,

128.2, 127.2, 127.0, 125.0, 124.8, 122.3, 119.3, 110.0, 20.0. HRMS

(ESI-TOF⁻) calcd for C₂₃H₁₈NO₃[M-H]⁻m/z 356.1287, found

356.1284.

(E)-2-(3-(2-Methyl-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

1

(A15). Oﬀ-white solid. Yield: 42%. mp 187−189 °C. H NMR (400

MHz, DMSO-d₆) δ 13.68 (br s, 1H), 11.39 (s, 1H), 8.64 (d, J = 8.3

Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.66−7.61 (m, 3H),

7.46 (t, J = 7.3 Hz, 2H), 7.40−7.36 (m, 3H), 7.26 (d, J = 7.9 Hz, 1H),

7.19 (t, J = 7.6 Hz, 1H), 6.93 (d, J = 15.6 Hz, 1H), 2.28 (s, 3H). ¹³C

(E)-2-(3-(4-Methoxyphenyl)acrylamido)benzoic Acid (A8). Oﬀ-

white solid. Yield: 57%. mp 164−166 °C. ¹H NMR (400 MHz,

DMSO-d₆) δ 13.64 (s, 1H), 11.31 (s, 1H), 8.63 (d, J = 8.4 Hz, 1H),

3988

https://doi.org/10.1021/acs.jmedchem.0c02129

J. Med. Chem. 2021, 64, 3976−3996

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

NMR (101 MHz, DMSO-d₆) δ 167.0, 164.3, 143.4, 141.6, 141.4,

141.1, 135.9, 134.5, 133.9, 131.6, 130.6, 129.3, 128.8, 127.7, 126.4,

123.3, 122.7, 120.9, 117.3, 20.6. HRMS (ESI-TOF⁻) calcd for

C₂₃H₁₈NO₃[M-H]⁻m/z 356.1287, found 356.1289.

MHz, DMSO-d₆) δ 11.43 (s, 1H), 8.66 (d, J = 8.3 Hz, 1H), 8.03 (d, J

= 7.7 Hz, 1H), 7.82−7.74 (m, 4H), 7.70−7.60 (m, 2H), 7.56−7.46

(m, 3H), 7.21−7.16 (m, 2H), 6.93 (d, J = 15.6 Hz, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 170.0, 164.2, 163.2 (d, J_CF= 244.4 Hz),

142.2 (d, J_CF= 7.1 Hz), 141.4, 141.2, 140.4, 134.6, 134.5, 131.6,

131.4, 131.3, 129.3, 127.7, 123.3, 123.1 (d, J_CF= 3.0 Hz), 120.8,

117.2, 115.0 (d, J_CF= 21.2 Hz), 113.8 (d, J_CF= 22.2 Hz). ¹⁹F NMR

(376 MHz, DMSO-d₆) δ −112.5. HRMS (ESI-TOF⁻) calcd for

C₂₂H₁₅NO₃F [M-H]⁻m/z 360.1036, found 360.1034.

(E)-2-(3-(3′-(Triﬂuoromethyl)-[1,1′-biphenyl]-4-yl)acrylamido)-

benzoic Acid (A23). Oﬀ-white solid. Yield: 54%. mp 226−228 °C. ¹H

NMR (400 MHz, DMSO-d₆) δ 13.56 (br s, 1H), 11.39 (s, 1H), 8.64

(d, J = 8.4 Hz, 1H), 8.02 (d, J = 7.9 Hz, 3H), 7.83 (q, J = 7.8 Hz, 4H),

7.86−7.80 (m, 3H), 7.62 (t, J = 7.8 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H),

6.96 (d, J = 15.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 169.9,

164.2, 141.3, 141.1, 140.8, 140.2, 134.8, 134.4, 131.6, 131.2, 130.5,

(E)-2-(3-(2′-Methyl-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

1

(A16). Oﬀ-white solid. Yield: 45%. mp 218−219 °C. H NMR (400

MHz, DMSO-d₆) δ 11.47 (s, 1H), 8.68 (d, J = 8.3 Hz, 1H), 8.03 (d, J

= 7.8 Hz, 1H), 7.75−7.67 (m, 3H), 7.60 (t, J = 7.7 Hz, 1H), 7.36 (d, J

= 7.5 Hz, 2H), 7.25−7.14 (m, 5H), 6.90 (d, J = 15.6 Hz, 1H), 2.22 (s,

3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.1, 164.3, 143.4, 141.5,

141.4, 141.0, 135.1, 134.5, 133.5, 131.6, 130.9, 123.0, 129.8, 128.5,

128.0, 126.5, 123.3, 122.8, 120.8, 117.2, 20.6. HRMS (ESI-TOF⁻)

calcd for C₂₃H₁₈NO₃[M-H]⁻m/z 356.1287, found 356.1281.

(E)-2-(3-(2′-Methoxy-[1,1′-biphenyl]-4-yl)acrylamido)benzoic

1

Acid (A17). Oﬀ-white solid. Yield: 55%. mp 217−219 °C. H NMR

(400 MHz, DMSO-d₆) δ 13.69 (br s, 1H), 11.39 (s, 1H), 8.65 (d, J =

8.4 Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.76 (d, J = 7.8 Hz, 2H), 7.70−

7.61(m, 2H), 7.55 (d, J = 7.7 Hz, 2H), 7.39−7.33 (m, 2H), 7.19 (t, J

= 7.6 Hz, 1H), 7.13 (d, J = 8.2 Hz, 1H), 7.05 (t, J = 7.4 Hz, 1H), 6.91

(d, J = 15.6 Hz, 1H), 3.78 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 170.0, 169.5, 164.3, 156.7, 141.6, 141.4, 140.3, 134.5, 133.4, 131.6,

130.7, 130.2, 129.5, 128.4, 123.3, 122.6, 121.3, 120.9, 117.3, 112.3,

56.0. HRMS (ESI-TOF⁻) calcd for C₂₃H₁₈NO₄[M-H]⁻m/z

372.1236, found 372.1238.

130.3 (q, J_CF= 31.3 Hz), 129.4, 127.9, 126.0, 124.8, 124.1 (q, J_CF

=

273.7 Hz), 123.5 (q, J_CF= 11.1 Hz), 123.3, 120.9, 117.3. ¹⁹F NMR

(376 MHz, DMSO-d₆) δ −61.05. HRMS (ESI-TOF⁻) calcd for

C₂₃H₁₅NO₃F₃[M-H]⁻m/z 410.1004, found 410.1000.

(E)-2-(3-(4′-Methyl-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

1

(A24). Oﬀ-white solid. Yield: 52%. mp 253−255 °C. H NMR (400

MHz, DMSO-d₆) δ 13.66 ((br s, 1H), 11.37 (s, 1H), 8.63 (d, J = 8.4

Hz, 1H), 8.03 (d, J = 7.9 Hz, 1H), 7.82 (d, J = 8.0 Hz, 2H), 7.74−

7.62 (m, 6H), 7.29 (d, J = 7.8 Hz, 2H), 7.20 (t, J = 7.5 Hz, 1H), 6.93

(d, J = 15.6 Hz, 1H), 2.35 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 169.9, 164.3, 141.9, 141.4, 141.3, 137.8, 136.8, 134.5, 133.7, 131.6,

130.1, 129.3, 127.3, 127.0, 123.4, 122.6, 120.9, 117.3, 21.2. HRMS

(ESI-TOF⁻) calcd for C₂₃H₁₈NO₃[M-H]⁻m/z 356.1287, found

356.1271.

(E)-2-(3-(2′-Fluoro-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

1

(A18). Oﬀ-white solid. Yield: 32%. mp 227−228 °C. H NMR (400

MHz, DMSO-d₆) δ 13.70 (s, 1H), 11.40 (s, 1H), 8.63 (d, J = 8.3 Hz,

1H), 8.03 (d, J = 7.9 Hz, 1H), 7.86 (d, J = 8.2 Hz, 2H), 7.71−7.57

(m, 5H), 7.48−7.43 (m, 1H), 7.37−7.31 (m, 2H), 7.20 (t, J = 7.6 Hz,

1H), 6.98 (d, J = 15.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ

170.0, 164.2, 159.6 (d, J_CF= 247.5 Hz), 141.26 (d, J_CF= 3.7 Hz),

137.0, 134.5, 134.4, 131.6, 131.2 (d, J_CF= 3.1 Hz), 130.5, 130.4, 129.8

(d, J_CF= 2.9 Hz), 128.9, 128.1, 128.0, 125.5 (d, J_CF= 3.3 Hz), 123.4,

123.3, 120.9, 117.4, 116.8, 116.6. ¹⁹F NMR (376 MHz, DMSO-d₆) δ

−118.0. HRMS (ESI-TOF⁻) calcd for C₂₂H₁₅NO₃F [M-H]⁻m/z

360.1036, found 360.1035.

(E)-2-(3-(4′-Methoxy-[1,1′-biphenyl]-4-yl)acrylamido)benzoic

1

ac4id (A25). Oﬀ-white solid. Yield: 59%. mp 259−261 °C. H NMR

(400 MHz, DMSO-d₆) δ 13.67 (br s, 1H), 11.46 (s, 1H), 8.63 (d, J =

8.4 Hz, 1H), 8.02 (d, J = 7.8 Hz, 1H), 7.80 (d, J = 8.1 Hz, 2H), 7.72−

7.61 (m, 5H), 7.19 (t, J = 7.5 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.91

(d, J = 15.7 Hz, 1H), 3.81 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 170.0, 164.3, 159.8, 141.7, 141.4, 141.3, 134.4, 133.3, 132.0, 131.6,

129.3, 128.3, 127.0, 123.3, 122.4, 120.9, 117.5, 115.0, 55.7. HRMS

(ESI-TOF⁻) calcd for C₂₃H₁₈NO₄[M-H]⁻m/z 372.1236, found

372.1238.

(E)-2-(3-(2′-(Triﬂuoromethyl)-[1,1′-biphenyl]-4-yl)acrylamido)-

benzoic Acid (A19). Oﬀ-white solid. Yield: 38%. mp 206−208 °C. ¹H

NMR (400 MHz, DMSO-d₆) δ 13.69 (s, 1H), 11.42 (s, 1H), 8.65 (d,

J = 8.2 Hz, 1H), 8.03 (dd, J = 7.9, 1.5 Hz, 1H), 7.86−7.81 (m, 3H),

7.76−7.69 (m, 2H), 7.66−7.61 (m, 2H), 7.44 (d, J = 7.5 Hz, 1H),

7.38 (d, J = 8.1 Hz, 2H), 7.21−7.17 (m, 1H), 6.97 (d, J = 15.6 Hz,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.0, 164.2, 141.3, 141.,

141.2, 140.5, 140.5, 134.5, 134.4, 132.8, 132.4, 131.6, 129.7, 128.7,

128.3, 127.3 (q, J_CF= 30.3 Hz), 126.6 (q, J_CF= 6.1 Hz), 124.6 (q, J_CF

= 274.7 Hz), 123.4, 123.4, 120.9, 117.3. ¹⁹F NMR (376 MHz,

DMSO-d₆) δ −55.3. HRMS (ESI-TOF⁻) calcd for C₂₃H₁₅NO₃F₃[M-

H]⁻m/z 410.1004, found 410.1002.

(E)-2-(3-(4′-Fluoro-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

1

(A26). Oﬀ-white solid. Yield: 57%. mp 224−226 °C. H NMR (400

MHz, DMSO-d₆) δ 13.65 (s, 1H), 11.37 (s, 1H), 8.63 (d, J = 8.4 Hz,

1H), 8.01 (d, J = 7.9 Hz, 1H), 7.83 (d, J = 7.9 Hz, 2H), 7.77 (t, J = 8.6

Hz, 2H), 7.74−7.62 (m, 4H), 7.31 (t, J = 8.6 Hz, 2H), 7.19 (t, J = 7.5

Hz, 1H), 6.95 (d, J = 15.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 169.9, 164.3, 162.6 (d, J_CF= 264.4 Hz), 141.3 (d, J_CF= 5.1 Hz),

140.9, 136.2 (d, J_CF= 5.1 Hz), 134.5, 134.0, 131.6, 129.3, 129.2 (d,

J_CF= 10.1 Hz), 127.5, 123.4, 122.9, 120.9, 117.3, 116.4, 116.2. ¹⁹F

NMR (376 MHz, DMSO-d₆) δ −114.72. HRMS (ESI-TOF⁻) calcd

for C₂₂H₁₅NO₃F [M-H]⁻m/z 360.1036, found 360.1026.

(E)-2-(3-(3′-Methyl-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

1

(A20). Oﬀ-white solid. Yield: 60%. mp 194−196 °C. H NMR (400

MHz, DMSO-d₆) δ 13.64 (s, 1H), 11.38 (s, 1H), 8.65 (d, J = 8.4 Hz,

1H), 8.03 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 7.9 Hz, 2H), 7.73−7.61

(m, 4H), 7.53−7.49 (m, 2H), 7.36 (t, J = 7.6 Hz, 1H), 7.21−7.17 (m,

2H), 6.93 (d, J = 15.6 Hz, 1H), 2.38 (s, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 170.0, 164.3, 142.1, 141.4, 139.7, 138.6, 134.5, 134.0,

131.6, 129.4, 129.3, 129.0, 127.8, 127.5, 124.3, 123.3, 122.8, 120.9,

117.3, 27.6. HRMS (ESI-TOF⁻) calcd for C₂₃H₁₈NO₃[M-H]⁻m/z

356.1287, found 356.1284.

(E)-2-(3-(4′-(Triﬂuoromethyl)-[1,1′-biphenyl]-4-yl)acrylamido)-

benzoic Acid (A27). Oﬀ-white solid. Yield: 62%. mp 174−176 °C. ¹H

NMR (400 MHz, DMSO-d₆) δ 13.65 (br s, 1H), 11.39 (s, 1H), 8.64

(d, J = 8.4 Hz, 1H), 8.02 (d, J = 7.9 Hz, 1H), 7.94 (d, J = 8.0 Hz, 2H),

7.87 (d, J = 8.0 Hz, 2H), 7.80 (d, J = 8.0 Hz, 4H), 7.71−7.61 (m,

2H), 7.19 (t, J = 7.6 Hz, 1H), 6.97 (d, J = 15.6 Hz, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 170.0, 164.2, 143.7, 141.3, 141.1, 140.2,

135.0, 134.5, 131.6, 129.4, 128.6 (q, J_CF= 31.3 Hz), 128.0, 127.9,

126.3 (q, J_CF= 19.8 Hz), 124.8(q, J_CF= 292.9 Hz), 123.4, 123.3,

120.9, 117.3. ¹⁹F NMR (376 MHz, DMSO-d₆) δ −60.97. HRMS

(ESI-TOF⁻) calcd for C₂₃H₁₅NO₃F₃[M-H]⁻m/z 410.1004, found

410.1003.

(E)-2-(3-(3′-Methoxy-[1,1′-biphenyl]-4-yl)acrylamido)benzoic

1

Acid (A21). Oﬀ-white solid. Yield: 52%. mp 259−261 °C. H NMR

(400 MHz, DMSO-d₆) δ 13.67 (s, 1H), 11.39 (s, 1H), 8.63 (d, J = 8.3

Hz, 1H), 8.02 (dd, J = 7.9, 1.2 Hz, 1H), 7.84 (d, J = 8.3 Hz, 2H), 7.76

(d, J = 8.3 Hz, 2H), 7.70−7.62 (m, 2H), 7.41 (t, J = 7.9 Hz, 1H),

7.31−7.26 (m, 2H), 7.21 (t, J = 7.7 Hz, 1H), 6.99−6.94 (m, 2H),

3.84 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.1, 164.2, 160.3,

141.9, 141.5, 141.3,141.2, 134.5, 134.1, 131.7, 130.5, 129.2, 127.6,

123.2, 122.8, 120.8, 119.4, 117.1, 113.8, 112.6, 55.5. HRMS (ESI-

TOF⁻) calcd for C₂₃H₁₈NO₄[M-H]⁻m/z 372.1236, found 372.1227.

(E)-2-(3-(3′-Fluoro-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

(E)-2-(3-(3′-Chloro-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

1

(A28). Yellow solid. Yield: 55%. mp 211−213 °C. H NMR (400

MHz, DMSO-d₆) δ 13.69 (br s, 1H), 11.44 (s, 1H), 8.64 (d, J = 8.4

Hz, 1H), 8.03 (dd, J = 7.9, 1.4 Hz, 1H), 7.87−7.83 (m, 2H), 7.79−

7.75 (m, 3H), 7.72−7.61 (m, 3H), 7.53−7.49 (m, 1H), 7.47−7.44

(m, 1H), 7.19 (t, J = 7.6 Hz, 1H), 6.97 (d, J = 15.6 Hz, 1H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 170.0, 164.2, 141.9, 141.3, 141.1,

1

(A22). Oﬀ-white solid. Yield: 43%. mp 219−221 °C. H NMR (400

3989

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Article

140.3, 134.7, 134.4, 134.3, 131.6, 131.3, 129.4, 128.2, 127.8, 126.9,

125.9, 123.4, 123.3, 120.9, 117. 6. HRMS (ESI-TOF⁻) calcd for

C₂₂H₁₅NO₃Cl [M-H]⁻m/z 376.0740, found 376.0735.

22.4, 14.4. HRMS (ESI-TOF⁻) calcd for C₂₁H₂₂NO₃[M-H]⁻m/z

336.1600, found 336.1599.

(E)-3-Methyl-2-(3-(4-pentylphenyl)acrylamido)benzoic Acid (C3).

1

(E)-2-(3-(3′-Bromo-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

Oﬀ-white solid. Yield: 49%. mp 126−128 °C. H NMR (400 MHz,

1

(A29). Oﬀ-white solid. Yield: 49%. mp 216−218 °C. H NMR (400

DMSO-d₆) δ 12.73 (s, 1H), 9.76 (s, 1H), 7.63 (d, J = 6.9 Hz, 1H),

7.56−7.45 (m, 4H), 7.28−7.23 (m, 3H), 6.88 (d, J = 15.8 Hz, 1H),

2.61 (t, J = 16.4 Hz, 2H), 2.24 (s, 3H), 1.63−1.56 (m, 2H), 1.33−

1.24 (m, 4H), 0.87 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, DMSO-

d₆) δ 168.5, 164.3, 144.8, 140.4, 135.8, 135.7, 134.0, 132.7, 129.4,

129.3, 128.2, 128.0, 126.1, 121.5, 35.4, 31.3, 30.9, 22.4, 18.7, 14.4.

HRMS (ESI-TOF⁻) calcd for C₂₂H₂₄NO₃[M-H]⁻m/z 350.1756,

found 350.1750.

MHz, DMSO-d₆) δ 13.69 (s, 1H), 11.40 (s, 1H), 8.62 (d, J = 8.2 Hz,

1H), 8.02 (dd, J = 7.9, 1.3 Hz, 1H), 7.93 (s, 1H), 7.85 (d, J = 8.4 Hz,

2H), 7.79−7.74 (m, 3H), 7.70−7.59 (m, 3H), 7.45 (t, J = 7.9 Hz,

1H), 7.19 (t, J = 7.6 Hz, 1H), 6.98 (d, J = 16.0 Hz, 1H).¹³C NMR

(101 MHz, DMSO-d₆) δ 168.8, 164.3, 142.2, 141.4, 141.1, 140.3,

134.7, 134.4, 131.6, 131.5, 131.1, 129.7, 129.3, 127.7, 126.2, 123.3,

123.2, 123.0, 120.9, 117.4. HRMS (ESI-TOF⁻) calcd for

C₂₂H₁₅NO₃Br [M-H]⁻m/z 420.0235, found 420.0238.

(E)-3,4-Dimethyl-2-(3-(4-pentylphenyl)acrylamido)benzoic Acid

1

(C4). Oﬀ-white solid. Yield: 54%. mp 168−170 °C. H NMR (400

(E)-2-(3-(3′-Nitro-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

1

(A30). Oﬀ-white solid. Yield: 50%. mp 228−230 °C. H NMR (400

MHz, DMSO-d₆) δ 12.68 (br s, 1H), 9.77 (s, 1H), 7.58−7.47 (m,

4H), 7.27 (d, J = 7.6 Hz, 2H), 7.17 (d, J = 7.9 Hz, 1H), 6.89 (d, J =

15.7 Hz, 1H), 2.60 (t, J = 7.5 Hz, 2H), 2.32 (s, 3H), 2.10 (s, 3H),

1.62−1.57 (m, 2H), 1.33−1.24 (m, 4H), 0.87 (t, J = 6.6 Hz, 3H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 168.5, 164.5, 144.8, 141.8, 140.2,

136.0, 134.7, 132.8, 129.4, 128.2, 127.7, 127.6, 126.7, 121.7, 35.4,

31.4, 30.9, 22.4, 21.0, 15.0, 14.4. HRMS (ESI-TOF⁻) calcd for

C₂₃H₂₆NO₃[M-H]⁻m/z 364.1913, found 364.1911.

MHz, DMSO-d₆) δ 13.67 (br s, 1H), 11.40 (s, 1H), 8.63 (d, J = 8.3

Hz, 1H), 8.47 (s, 1H), 8.23−8.18 (m, 2H), 8.02 (d, J = 7.8 Hz, 1H),

7.88−7.84 (m, 4H), 7.76 (t, J = 8.0 Hz, 1H), 7.68 (d, J = 15.6 Hz,

1H), 7.62 (t, J = 7.3 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 6.96 (d, J =

15.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.0, 164.1, 148.9,

141.3, 141.3, 140.9, 139.4, 135.1, 134.4, 133.7, 131.6, 131.0, 129.5,

127.9, 123.6, 123.4, 122.9, 121.5, 120.9, 117.4. HRMS (ESI-TOF⁺)

calcd for C₂₂H₁₇N₂O₅[M-H]⁻m/z 389.1137, found 389.1132.

(E)-2-(3-(4′-Ethyl-[1,1′-biphenyl]-4-yl)acrylamido)benzoic Acid

(E)-5-Chloro-3-methyl-2-(3-(4-pentylphenyl)acrylamido)benzoic

1

Acid (C5). Oﬀ-white solid. Yield: 44%. mp 208−210 °C. H NMR

1

(A31). Oﬀ-white solid. Yield: 40%. mp 231−233 °C. H NMR (400

(400 MHz, DMSO-d₆) δ 13.06 (s, 1H), 9.79 (s, 1H), 7.59−7.50 (m,

5H), 7.27 (d, J = 7.5 Hz, 2H), 6.87 (d, J = 15.8 Hz, 1H), 2.60 (t, J =

7.4 Hz, 2H), 2.25 (s, 3H), 1.62−1.55 (m, 2H), 1.34−1.29 (m, 4H),

0.87 (t, J = 6.3 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.2,

164.5, 145.0, 140.7, 138.4, 134.7, 133.3, 132.6, 131.2, 130.2, 129.4,

128.2, 127.3, 121.2, 35.4, 31.4, 30.9, 22.4, 18.4, 14.4. HRMS (ESI-

TOF⁻) calcd for C₂₂H₂₃NO₃Cl [M-H]⁻m/z 384.1366, found

384.1361.

MHz, DMSO-d₆) δ 13.63 (br s, 1H), 11.36 (s, 1H), 8.63 (d, J = 8.4

Hz, 1H), 8.02 (dd, J = 7.9, 1.5 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.74

(d, J = 8.4 Hz, 2H), 7.70−7.62 (m, 4H), 7.33 (d, J = 8.2 Hz, 2H),

7.22−7.18 (m, 1H), 6.94 (d, J = 15.6 Hz, 1H), 2.66 (q, J = 7.5 Hz,

2H), 1.22 (t, J = 7.6 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

170.0, 164.3, 144.1, 142.0, 141.4, 141.3, 137.1, 134.5, 133.7, 131.6,

129.3, 128.9, 127.3, 127.1, 123.3, 122.7, 120.9, 117.3, 28.3, 16.0.

HRMS (ESI-TOF⁻) calcd for C₂₄H₂₀NO₃[M-H]⁻m/z 370.1443,

found 370.1449.

(E)-3-Chloro-2-(3-(4-pentylphenyl)acrylamido)benzoic Acid (C6).

1

Oﬀ-white solid. Yield: 62%. mp 174−176 °C. H NMR (400 MHz,

(E)-2-(3-(4′-Isopropyl-[1,1′-biphenyl]-4-yl)acrylamido)benzoic

DMSO-d₆) δ 13.00 (br s, 1H), 10.01 (s, 1H), 7.73 (d, J = 7.9 Hz,

2H), 7.56−7.51 (m, 3H), 7.37 (t, J = 7.9 Hz, 1H), 7.28 (d, J = 7.9 Hz,

2H), 6.91 (d, J = 15.8 Hz, 1H), 2.61 (t, J = 16.8 Hz, 2H), 1.63−1.55

(m, 2H), 1.33−1.24 (m, 4H), 0.87 (t, J = 6.8 Hz, 3H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 167.4, 164.6, 145.0, 141.0, 134.2, 132.9,

132.6, 132.1, 131.5, 129.4, 129.1, 128.2, 127.5, 121.0, 35.4, 31.4, 30.9,

22.4, 14.4. HRMS (ESI-TOF⁻) calcd for C₂₁H₂₁NO₃Cl [M-H]⁻m/z

370.1210, found 370.1205.

1

Acid (A32). Oﬀ-white solid. Yield: 49%. mp 213-215 °C. H NMR

(400 MHz, DMSO-d₆) δ 13.66 (s, 1H), 11.39 (s, 1H), 8.66 (d, J = 8.3

Hz, 1H), 8.04 (d, J = 7.5 Hz, 1H), 7.81 (d, J = 8.2 Hz, 2H), 7.72−

7.62 (m, 6H), 7.33 (d, J = 8.0 Hz, 2H), 7.19 (t, J = 7.5 Hz, 1H),

7.97−7.90 (m, 1H), 2.95−2.89 (m, 1H), 1.22 (d, J = 6.9 Hz, 6H). ¹³C

NMR (101 MHz, DMSO-d₆) δ 170.0, 164.3, 148.7, 142.0, 141.4,

141.4, 137.3, 134.5, 133.7, 131.6, 129.5, 129.3, 127.4, 127.3, 127.1,

123.3, 122.6, 120.9, 117.3, 33.6, 24.3. HRMS (ESI-TOF⁻) calcd for

C₂₅H₂₂NO₃[M-H]⁻m/z 384.1600, found 384.1595.

(E)-4-Methyl-2-(3-(4-pentylphenyl)acrylamido)benzoic Acid (C7).

1

Oﬀ-white solid. Yield: 63%. mp 190−192 °C. H NMR (400 MHz,

(E)-2-(3-(4′-(tert-Butyl)-[1,1′-biphenyl]-4-yl)acrylamido)benzoic

DMSO-d₆) δ 13.47 (s, 1H), 11.38 (s, 1H), 8.50 (s, 1H), 7.91 (d, J =

8.1 Hz, 1H), 7.64−7.57 (m, 3H), 7.25 (d, J = 8.1 Hz, 2H), 7.00 (dd, J

= 8.1, 1.0 Hz, 1H), 6.80 (d, J = 15.6 Hz, 1H), 2.60 (t, J = 17.0 Hz,

2H), 2.37 (s, 3H), 1.61−1.54 (m, 2H), 1.33−1.24 (m, 4H), 0.86 (t, J

= 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.0, 164.3,

145.2, 145.0, 141.7, 141.6, 132.4, 131.6, 129.3, 128.7, 124.0, 121.9,

120.9, 114.2, 35.4, 31.3, 30.9, 22.4, 22.1, 14.4. HRMS (ESI-TOF⁻)

calcd for C₂₂H₂₄NO₃[M-H]⁻m/z 350.1756, found 350.1754.

(E)-4-Methyl-2-(3-(3′-nitro-[1,1′-biphenyl]-4-yl)acrylamido)-

1

Acid (A33). Oﬀ-white solid. Yield: 41%. mp 237−238 °C. H NMR

(400 MHz, DMSO-d₆) δ 13.67 (br s, 1H), 11.40 (s, 1H), 8.65 (d, J =

8.1 Hz, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.82−7.64 (m, 8H), 7.48 (d, J

= 7.6 Hz, 2H), 7.19 (t, J = 15.5 Hz, 1H), 6.93 (d, J = 15.5 Hz, 1H),

1.31 (s, 9H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.0, 164.3, 150.9,

141.9, 141.4, 141.4, 136.9, 134.5, 133.8, 131.6, 129.3, 127.3, 126.8,

126.3, 123.3, 122.6, 120.9, 117.3, 34.7, 31.5. HRMS (ESI-TOF⁻)

calcd for C₂₆H₂₄NO₃[M-H]⁻m/z 398.1756, found 398.1749.

(E)-3-(3-(4-Pentylphenyl)acrylamido)benzoic Acid (C1). Oﬀ-white

1

benzoic Acid (C8). Yellow solid. Yield: 42%. mp 230−232 °C. H

1

solid. Yield: 52%. mp 259−261 °C. H NMR (400 MHz, DMSO-d₆)

NMR (400 MHz, DMSO-d₆) δ 11.99 ((br s, 1H), 8.47 (d, J = 23.9

Hz, 2H), 8.22−8.15 (m, 2H), 7.93 (d, J = 8.0 Hz, 1H), 7.82 (s, 4H),

7.75 (t, J = 8.0 Hz, 1H), 7.64 (d, J = 15.6 Hz, 1H), 6.97−6.88 (m,

2H), 2.35 (s, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 170.5, 163.9,

148.9, 144.0, 141.4, 141.3, 140.5, 139.3, 135.1, 133.6, 131.7, 131.0,

129.4, 127.9, 123.9, 122.9, 121.5, 120.6, 22.1. HRMS (ESI-TOF⁻)

calcd for C₂₃H₁₇N₂O₅[M-H]⁻m/z 401.1137, found 401.1139.

(E)-4,5-Diﬂuoro-2-(3-(4-pentylphenyl)acrylamido)benzoic Acid

δ 12.99 (s, 1H), 10.37 (s, 1H), 8.32 (s, 1H), 7.95 (d, J = 9.0 Hz, 1H),

7.63−7.54 (m, 4H), 7.46 (t, J = 12.4 Hz, 1H), 7.28 (d, J = 7.6 Hz,

2H), 6.78 (d, J = 15.6 Hz, 1H), 2.61 (t, J = 7.4 Hz, 2H), 1.63−1.55

(m, 2H), 1.34−1.24 (m, 4H), 0.87 (t, J = 6.5 Hz, 3H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 167.6, 164.4, 145.1, 141.0, 140.0, 132.6,

131.8, 129.5, 129,4, 128.3, 124.5, 123.7, 121.5, 120.4, 35.4, 31.3, 30.9,

22.4, 14.4. HRMS (ESI-TOF⁻) calcd for C₂₁H₂₂NO₃[M-H]⁻m/z

336.1600, found 336.1596.

1

(C9). Oﬀ-white solid. Yield: 48%. mp 234−236 °C. H NMR (400

(E)-4-(3-(4-Pentylphenyl)acrylamido)benzoic Acid (C2). Oﬀ-white

MHz, DMSO-d₆) δ 14.11 (br s, 1H), 11.38 (s, 1H), 8.70−8.64 (m,

1H), 7.98−7.93 (m, 1H), 7.63−7.59 (m, 3H), 7.24 (d, J = 8.0 Hz,

2H), 6.81 (d, J = 15.6 Hz, 1H), 2.59 (t, J = 7.6 Hz, 2H), 1.60−1.53

(m, 2H), 1.34−1.22 (m, 4H), 0.85 (t, J = 6.9 Hz, 3H). ¹³C NMR

(101 MHz, DMSO-d₆) δ 168.3, 164.6, 152.5 (dd, J_CF= 251.5, 13.0

1

solid. Yield: 46%. mp 284−286 °C. H NMR (400 MHz, DMSO-d₆)

δ 12.73 (s, 1H), 10.49 (s, 1H), 7.93 (d, J = 8.5 Hz, 2H), 7.83 (d, J =

8.5 Hz, 2H), 7.63−7.53 (m, 3H), 7.26 (d, J = 7.2 Hz, 2H), 6.82 (d, J

= 15.7 Hz, 1H), 2.58 (m, 2H), 1.56 (m, 2H), 1.27 (m, 4H), 0.85 (m,

3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 167.4, 164.5, 145.2, 143.8,

141.4, 132.5, 130.9, 129.4, 128.3, 125.7, 121.3, 118.9, 35.4, 31.3, 30.9,

Hz), 145.7, 144.6 (dd, J_CF= 244.4, 14.9 Hz), 142.5, 139.1 (dd, J_CF

=

10.7, 2.0 Hz), 132.2, 129.3, 128.8, 121.2, 120.1 (d, J = 19.9 Hz),

3990

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114.1, 109.5 (d, J = 23.2 Hz), 35.5, 31.3, 30.9, 22.4, 14.3. ¹⁹F NMR

(376 MHz, DMSO-d₆) δ −128.30 (d, J = 22.6 Hz), −143.63 (d, J =

22.6 Hz). HRMS (ESI-TOF⁻) calcd for C₂₁H₂₀NO₃F₂[M-H]⁻m/z

372.1411, found 372.1410.

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.6, 164.3, 142.1, 141.7,

140.4, 139.7, 136.9, 134.0, 133.6, 129.5, 129.4, 128.4, 127.6, 127.2,

123.1, 122.5, 119.7, 114.8. HRMS (ESI-TOF⁻) calcd for

C₂₂H₁₅NO₃Br [M-H]⁻m/z 420.0235, found 420.0239.

(E)-5-Bromo-2-(3-(3′-nitro-[1,1′-biphenyl]-4-yl)acrylamido)-

benzoic Acid (C17). Oﬀ-white solid. Yield: 45%. mp 278−280 °C. ¹H

NMR (400 MHz, DMSO-d₆) δ 13.56 (s, 1H), 9.19 (s, 1H), 8.58 (d, J

= 9.0 Hz, 1H), 8.44 (s, 1H), 8.22−8.16 (m, 3H), 7.80−7.73 (m, 5H),

7.64−7.55 (m, 2H), 6.82 (d, J = 15.7, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 169.1, 163.8, 148.9, 141.3, 140.4, 140.1, 139.3, 135.2,

134.1, 134.0, 133.7, 131.0, 129.3, 127.9, 125.4, 124.1, 122.9, 121.6,

121.5, 114.2. HRMS (ESI-TOF⁻) calcd for C₂₂H₁₄N₂O₅Br [M-H]⁻

m/z 465.0086, found 465.0083.

(E)-4-Chloro-2-(3-(4-pentylphenyl)acrylamido)benzoic Acid

1

(C10). Oﬀ-white solid. Yield: 62%. mp 174−176 °C. H NMR (400

MHz, DMSO-d₆) δ 11.44 (s, 1H), 8.75 d, J = 8.6 Hz, 1H), 8.01 (d, J =

8.6 Hz, 1H), 7.64−7.59 (m, 3H), 7.25−7.21 (m, 3H), 6.81 (d, J =

15.6 Hz, 1H), 2.58 (t, J = 7.6 Hz, 2H), 1.60−1.52 (m, 2H), 1.32−1.23

(m, 4H), 0.85 (t, J = 7.0 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

169.3, 164.7, 145.5, 142.5, 142.4, 139.0, 133.3, 132.2, 129.3, 128.8,

123.1, 121.4, 120.0, 115.6, 35.5, 31.3, 30.9, 22.4, 14.4. HRMS (ESI-

TOF⁻) calcd for C₂₁H₂₁NO₃Cl [M-H]⁻m/z 370.1210, found

370.1204.

The Synthesis of Compound B1. The synthesis of compound B1

was mainly done according to a literature method.⁴¹

(E)-2-(3-([1,1′-Biphenyl]-4-yl)acrylamido)-4-chlorobenzoic Acid

1

A mixture of 1-iodo-4-pentylbenzene 17 (493 mg, 1.8 mmol), 2-

vinylbenzoic acid 18 (296 mg, 2 mmol), palladium acetate (0.9 mg,

0.004 mmol), and triethylamine (0.3 mL) in 5 mL of acetonitrile was

heated to reﬂux for 12 h. After it cooled, it was diluted with H₂O, and

K₂CO₃powder was added to maintain the pH at 11. The mixture was

extracted with Et₂O three times. The water layer was acidiﬁed to pH 2

with 1 N HCl and extracted with EtOAc three times, and the

combined organic layers were washed with brne, dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated. The crude product

was puriﬁed by silica gel column chromatography eluting with DCM/

MeOH (100/3) to aﬀord compound B1.

(C11). Oﬀ-white solid. Yield: 65%. mp 258−260 °C. H NMR (400

MHz, DMSO-d₆) δ 13.89 (br s, 1H), 11.54 (s, 1H), 8.77 (s, 1H), 8.03

(d, J = 8.8 Hz, 1H), 7.86 (d, J = 12.2 Hz, 2H), 7.77−7.68 (m, 5H),

7.50 (t, J = 13.6 Hz, 2H), 7.40 (t, J = 7.2 Hz, 1H), 7.26 (d, J = 8.6 Hz,

1H), 6.97 (d, J = 10.1 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ

169.2, 164.6, 142.4, 142.2, 141.9, 139.7, 138.7, 133.9, 133.4, 129.5,

129.4, 128.4, 127.6, 127.2, 123.2, 122.5, 120.0, 116.2. HRMS (ESI-

TOF⁻) calcd for C₂₂H₁₅NO₃Cl [M-H]⁻m/z 376.0740, found

376.0742.

(E)-5-Methyl-2-(3-(4-pentylphenyl)acrylamido)benzoic Acid

1

(C12). Oﬀ-white solid. Yield: 59%. mp 174−176 °C. H NMR (400

(E)-2-(4-Pentylstyryl)benzoic Acid (B1). Brown solid. Yield: 74%.

MHz, DMSO-d₆) δ 13.56 (s, 1H), 11.21 (s, 1H), 8.50 (d, J = 8.5 Hz,

1H), 7.82 (d, J = 1.5 Hz, 1H), 7.64−7.56 (m, 3H), 7.44 (dd, J = 8.6,

1.7 Hz, 1H), 7.25 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 15.6 Hz, 1H), 2.59

(t, J = 16.8 Hz, 2H), 2.32 (s, 3H), 1.61−1.54 (m, 2H), 1.33−1.25 (m,

4H), 0.86 (t, J = 6.9 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ

170.0, 164.2, 145.2, 141.5, 139.0, 135.0, 132.5, 132.4, 131.6, 129.3,

128.6, 121.9, 120.9, 117.2, 35.4, 31.3, 30.9, 22.4, 20.7, 14.4. HRMS

(ESI-TOF⁻) calcd for C₂₂H₂₄NO₃[M-H]⁻m/z 350.1756, found

350.1747.

1

mp 128−130 °C. H NMR (400 MHz, DMSO-d₆) δ 13.04 (s, 1H),

7.92−7.81 (m, 3H), 7.56 (t, J = 7.0 Hz, 1H), 7.46 (d, J = 7.2 Hz, 2H),

7.37 (t, J = 7.0 Hz, 1H), 7.21−7.11 (m, 3H), 2.56 (t, J = 6.8 Hz, 2H),

1.56 (m, 2H), 1.28 (m, 4H), 0.85 (m, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 169.1, 142.8, 138.4, 135.1, 132.3, 131.1, 130.8, 130.1,

129.2, 127.7, 127.1, 127.0, 126.6, 35.4, 31.3, 31.0, 22.4, 14.4. HRMS

(ESI-TOF⁻) calcd for C₂₀H₂₁O₂[M-H]⁻m/z 293.1542, found

293.1533.

The Synthesis of Compound B2. An aqueous solution of NaOH

(1.5 M, 2 mL) was added to a solution of 4-pentylbenzaldehyde 16n

(352 mg, 2 mmol) and 2-acetylbenzoic acid 19 (328 mg, 2 mmol) in

ethanol (EtOH) (2 mL) at 0 °C. After it was stirred at rt for another

20 h, the resultant mixture was poured into icy water and neutralized

by 1 N HCl. The crude product as a solid was collected by ﬁltration

and recrystallized from EtOAc to give compound B2.⁴²

(E)-5-Bromo-2-(3-(4-pentylphenyl)acrylamido)benzoic Acid

1

(C13). Oﬀ-white solid. Yield: 53%. mp 214−216 °C. H NMR (400

MHz, DMSO-d₆) δ 14.00 (br s, 1H), 11.25 (s, 1H), 8.57 (d, J = 9.0

Hz, 1H), 8.07 (s, 1H), 7.79 (d, J = 9.0 Hz, 1H), 7.63−7.58 (m, 3H),

7.24 (d, J = 7.6 Hz, 2H), 6.81 (d, J = 15.6 Hz, 1H), 2.58 (t, J = 7.4 Hz,

2H), 1.58−1.54 (m, 2H), 1.32−1.27 (m, 4H), 0.85 (t, J = 6.5 Hz,

3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.6, 164.4, 145.4, 142.2,

140.5, 136.9, 133.6, 132.3, 129.3, 128.7, 122.9, 121.5, 119.4, 114.7,

35.5, 31.3, 30.9, 22.4, 14.4. HRMS (ESI-TOF⁻) calcd for

C₂₁H₂₁NO₃Br [M-H]⁻m/z 414.0705, found 414.0701.

(E)-2-(3-(4-Pentylphenyl)acryloyl)benzoic Acid (B2). Oﬀ-white

1

solid. Yield: 69%. mp 102−104 °C. H NMR (400 MHz, DMSO-

d₆) δ 8.11 (d, J = 7.8 Hz, 1H), 7.70−7.66 (m, 1H), 7.60−7.56 (m,

1H), 7.46−7.42 (m, 3H), 7.22−7.19 (m, 3H), 7.05−7.01 (m, 1H),

2.63 (t, J = 16.2 Hz, 2H), 1.66−1.58 (m, 2H), 1.36−1.30 (m, 4H),

0.90 (t, J = 6.8 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 197.1,

171.2, 146.3, 146.2, 142.7, 133.07, 131.9, 131.1, 129.6, 129.0, 128.6,

128.0, 127.7, 126.6, 35.9, 31.4, 30.9, 22.59, 14.0. HRMS (ESI-TOF⁻)

calcd for C₂₁H₂₁O₃[M-H]⁻m/z 321.1491, found 321.1490.

The Synthesis of Compound B3. 4-Pentylbenzoic acid 20 (500

mg, 2.6 mmol) and thionyl chloride (3 mL) were heated together

under reﬂux for 4 h. Any unreacted thionyl chloride was removed

under reduced pressure, and the crude product 4-pentylbenzoyl

chloride 21 was used without further puriﬁcation.

(E)-2-(3-([1,1′-Biphenyl]-4-yl)acrylamido)-5-ﬂuorobenzoic Acid

1

(C14). Oﬀ-white solid. Yield: 61%. mp 251−253 °C. H NMR (400

MHz, DMSO-d₆) δ 11.50 (s, 1H), 8.64−8.61 (m, 1H), 7.79 (d, J =

8.2 Hz, 2H), 7.78−7.69 (m, 7H), 7.51−7.45 (m, 3H), 7.38 (t, J = 7.2

Hz, 1H), 6.92 (d, J = 15.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆)

δ 168.8, 164.1, 157.4 (d, J_CF= 242.4 Hz), 141.9, 141.2, 139.7, 137.6

(d, J_CF= 2.0 Hz), 134.0, 129.5, 129.3, 128.4, 127.5, 127.1, 123.0 (d,

J

_CF= 7.1 Hz), 122.7, 120.8 (d, J_CF= 22.2 Hz), 120.4, 117.4 (d, J_CF=

23.2 Hz). ¹⁹F NMR (376 MHz, DMSO-d₆) δ −118.9. HRMS (ESI-

TOF⁻) calcd for C₂₂H₁₅NO₃F [M-H]⁻m/z 360.1036, found

360.1031.

A THF solution of 4-pentylbenzoyl chloride 21 (237 mg, 1.3

mmol) was added dropwise to a mixture of 2-aminobenzoic acid 11a

(274 mg, 2 mmol), catalytic amounts of triethylamine, and dry THF

(4 mL) in an ice bath, and the mixture was stirred at 0 °C for 15 min,

followed by being heated at 60 °C for an additional 3 h. After 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 DCM/MeOH (100/3) to aﬀord

compound B3.

(E)-2-(3-([1,1′-Biphenyl]-4-yl)acrylamido)-5-chlorobenzoic Acid

1

(C15). Oﬀ-white solid. Yield: 57%. mp 260−262 °C. H NMR (400

MHz, DMSO-d₆) δ 14.09 (br s, 1H), 11.37 (s, 1H), 8.63 (d, J = 9.0

Hz, 1H), 7.95 (d, J = 2.6 Hz, 1H), 7.83 (d, J = 8.3 Hz, 2H), 7.75−

7.66 (m, 6H), 7.49 (t, J = 7.5 Hz, 2H), 7.40 (t, J = 7.3 Hz, 1H), 6.95

(d, J = 15.6 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ 168.8,

164.3, 142.0, 141.6, 140.1, 139.7, 133.9, 130.8, 129.5, 129.3, 128.3,

127.5, 127.1, 126.9, 122.6, 122.5, 119.3. HRMS (ESI-TOF⁻) calcd for

C₂₂H₁₅NO₃Cl [M-H]⁻m/z 376.0740, found 376.0741.

(E)-2-(3-([1,1′-Biphenyl]-4-yl)acrylamido)-5-brmobenzoic Acid

1

(C16). Oﬀ-white solid. Yield: 57%. mp 270−272 °C. H NMR (400

MHz, DMSO-d₆) δ 13.97 (br s, 1H), 11.30 (s, 1H), 8.58 (d, J = 9.1

Hz, 1H), 8.08 (s, 1H), 7.84−7.81 (m, 3H), 7.76−7.67 (m, 5H), 7.50

(t, J = 13.7 Hz, 2H), 7.40 (t, J = 7.2 Hz, 1H), 6.96 (d, J = 10.5 Hz,

2-(4-Pentylbenzamido)benzoic Acid (B3). Oﬀ-white solid. Yield:

1

58%. mp 126−128 °C. H NMR (400 MHz, DMSO-d₆) δ 13.77 (s,

1H), 12.16 (s, 1H), 8.73 (d, J = 6.2 Hz, 1H), 8.07 (d, J = 6.1 Hz, 1H),

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7.88 (d, J = 6.4 Hz, 2H), 7.67 (t, J = 16.4 Hz, 1H), 7.42 (d, J = 6.4 Hz,

2H), 7.20 (t, J = 12.6 Hz, 1H), 2.67 (t, J = 9.8, 2H), 1.63−1.60 (m,

2H), 1.35−1.23 (m, 4H), 0.88 (t, J = 11.7, 3H). ¹³C NMR (101 MHz,

DMSO-d₆) δ 170.5, 165.1, 147.6, 141.8, 134.8, 132.4, 131.8, 129.3,

127.5, 123.3, 120.3, 116.8, 35.4, 31.3, 30.8, 22.4, 14.4. HRMS (ESI-

TOF⁻) calcd for C₁₉H₂₀NO₃[M-H]⁻m/z 310.1443, found 310.1448.

The Synthesis of Compound B4. 4-Pentylaniline 22 (148 mg, 1

mmol) was added dropwise to a solution of isobenzofuran-1,3-dione

23 (163 mg, 1 mmol) in acetone (3 mL) at 0 °C. After it was stirred

at rt for another 2 h, the resulting precipitate was ﬁltered,

recrystallized from ethanol, and dried to aﬀord compound B4.⁴³

2-((4-Pentylphenyl)carbamoyl)benzoic Acid (B4). Oﬀ-white solid.

1H), 7.58−7.53 (m, 1H), 7.42 (d, J = 8.1 Hz, 2H), 7.33−7.28 (m,

2H), 7.20 (d, J = 8.1 Hz, 2H), 6.88 (d, J = 15.5 Hz, 1H), 4.41 (t, J =

6.3 Hz, 2H), 2.87 (t, J = 6.3 Hz, 2H), 2.63 (t, J = 12.2 Hz, 2H), 1.67−

1.59 (m, 2H), 1.38−1.30 (m, 4H), 0.91 (t, J = 6.8 Hz, 3H). ¹³C NMR

(101 MHz, CDCl₃) δ 194.0, 165.8, 145.7, 143.9, 143.8, 134.0, 132.3,

129.0, 128.1, 128.0, 125.7, 125.4, 124.0, 117.8, 43.6, 39.7, 35.8, 31.4,

30.9, 22.5, 14.0. HRMS (ESI-TOF⁺) calcd for C₂₃H₂₆NO₂[M + H]⁺

m/z 348.1964, found 348.1959.

(E)-1-(3-([1,1′-Biphenyl]-4-yl)acryloyl)-3,4-dihydro-1H-benzo[b]-

azepin-5(2H)-one (C21). Oﬀ-white solid. Yield: 57%. mp 213−215

°C. ¹H NMR (400 MHz, CDCl₃) δ 8.00 (d, J = 7.7 Hz, 1H), 7.81 (d,

J = 15.5 Hz, 1H), 7.64−7.53 (m, 5H), 7.51−7.35 (m, 6H), 7.28−7.27

(m, 1H), 6.42 (d, J = 15.4 Hz, 1H), 4.98 (m, 1H), 3.23 (m, 1H), 2.78

(m, 2H), 2.34 (m, 1H), 1.94 (m, 1H). ¹³C NMR (101 MHz, CDCl₃)

δ 201.6, 166.1, 142.6, 141.8, 140.1, 135.0, 133.8, 133.4, 130.0, 128.9,

128.5, 128.1, 127.8, 127.4, 127.0, 118.1, 46.5, 40.1, 22.2. HRMS (ESI-

TOF⁺) calcd for C₂₅H₂₂NO₂[M + H]⁺m/z 368.1651, found

368.1642.

(E)-1-(3-(4-Pentylphenyl)acryloyl)-3,4-dihydro-1H-benzo[b]-

azepin-5(2H)-one (C22). Yellow oil. Yield: 44%. ¹H NMR (400 MHz,

DMSO-d₆) δ 7.82 (d, J = 7.6 Hz, 1H), 7.66 (t, J = 10.9 Hz, 1H), 7.59

(d, J = 15.5 Hz, 1H), 7.53−7.51 (m, 1H), 7.34−7.29 (m, 3H), 7.13

(d, J = 7.1 Hz, 2H), 6.33 (d, J = 13.7 Hz, 1H), 2.62 (m, 2H), 2.14−

1.80 (m, 2H), 1.53−1.47 (m, 2H), 1.27−1.19 (m, 4H), 0.81 (t, J =

6.9 Hz, 3H). ¹³C NMR (101 MHz, DMSO-d₆) δ 201.6, 165.6, 145.1,

142.1, 135.0, 133.9, 132.6, 129.5, 129.4, 129.3, 128.3, 118.3, 46.4,

35.4, 31.3, 30.9, 27.7, 22.4, 14.3. HRMS (ESI-TOF⁺) calcd for

C₂₄H₂₈NO₂[M + H]⁺m/z 362.2120, found 362.2116.

Cell Culture and Transfection. The tetracycline-inducible

HEK293T cells were cultured in DMEM/F-12 (Gibco) plus 10%

fetal bovine serum (FBS) with 1 μg/mL tetracycline (Sigma) as

previously described.^50,51SH-SY5Y cells were cultured in DMEM

(Gibco) supplemented with 10% FBS. All cells were cultured at 37 °C

under a humidiﬁed atmosphere containing 5% CO₂. HEK293T cells

were used to transiently express wild-type TRPM8 or TRPV1. The 1

μg of human TRPM8 or mouse TRPV1 and 50 ng of green

ﬂuorescent protein were together transfected into HEK293T cells

using Lipofectamine 2000 (Invitrogen) for 24−48 h before record-

ings. Cells were seeded on coverslips before use.

1

Yield: 84%. mp 172−174 °C. H NMR (400 MHz, DMSO-d₆) δ

12.89 (s, 1H), 10.24 (s, 1H), 7.88 (d, J = 7.6 Hz, 1H), 7.67−7.53 (m,

5H), 7.14 (d, J = 7.8 Hz, 2H), 2.54 (t, J = 7.6 Hz, 2H), 1.60−1.53 (m,

2H), 1.35−1.27 (m, 4H), 0.88 (t, J = 6.6 Hz, 3H). ¹³C NMR (101

MHz, DMSO-d₆) δ 168.0, 167.6, 139.4, 137.4, 132.1, 130.5, 129.9,

129.7, 128.8, 128.2, 120.0, 35.1, 31.3, 31.2, 22.4, 14.4. HRMS (ESI-

TOF⁻) calcd for C₁₉H₂₀NO₃[M-H]⁻m/z 310.1443, found 310.1442.

The Synthesis of Compound C18. Compound A13 (172 mg, 0.5

mmol) and acetic anhydride (3 mL) were heated together under

reﬂux for 2 h. The excess of acetic anhydride was removed under

reduced pressure. The crude product was recrystallized from ethanol

and dried to aﬀord compound C18.

(E)-7-(3-([1,1′-Biphenyl]-4-yl)acryloyl)-7-azabicyclo[4.2.0]octa-

1,3,5-trien-8-one (C18). Yellow solid. Yield: 57%. mp 172−174 °C.

¹H NMR (400 MHz, DMSO-d₆) δ 8.14 (dd, J = 7.8, 1.1 Hz, 1H),

7.96−7.91 (m, 3H), 7.84 (d, J = 16.2 Hz, 1H), 7.76 (t, J = 17.6 Hz,

4H), 7.66−7.59 (m, 2H), 7.50 (t, J = 7.6 Hz, 2H), 7.42 (t, J = 7.3 Hz,

1H), 7.07 (d, J = 16.2 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ

159.3, 157.4, 147.1, 142.3, 141.1, 139.7, 137.3, 134.1, 129.5, 129.4,

128.9, 128.6, 128.4, 127.6, 127.2, 119.7, 117.3, 100.0. HRMS (ESI-

TOF⁺) calcd for C₂₂H₁₆NO₂[M + H]⁺m/z 326.1181, found

326.1187.

General Procedure for the Synthesis of Compounds C19−C22.

Piperidine (376 mg, 4.4 mmol) was added to a suspension of

benzaldehyde derivatives 16m or 16n (4.4 mmol) and malonic acid

(416 mg, 4 mmol) in toluene (6 mL). The mixture was heated to

reﬂux for 4 h, followed by being stirred at room temperature for an

additional 1 h and then acidiﬁed with 1 N HCl. The resulting

precipitate was ﬁltered and dried to aﬀord cinnamic acids 24a and

24b.

Calcium Fluorescent Measurements. Free [Ca²⁺]_iwas

determined using Fluo-3/AM (Invitrogen) as previously described.²⁸

Cells in 96-well plates (3603; Costar) were incubated with 3.5 μM

Fluo-3/AM at 37 °C for 0.5 h and washed with Hanks’ balanced salt

solution, and the ﬂuorescence intensity was measured using a

SynergyMx M5 microplate reader (Molecular Devices) with excitation

at 485 nm and emission at 525 nm. The intensity of the control well,

in which 1 mM H₂O₂was added, was set as 100%, and the data were

normalized as percentages of the control. Each group had three

replicate wells, and all procedures were performed in a dark condition.

Electrophysiology. For the inhibitory activity test on the TRPM2

channel, patch-clamp recordings were performed in a whole-cell

conﬁguration at room temperature using Axonpatch 700B (Axon

Instruments). Similar to our previously reported protocol,^28,32cells

were kept in an extracellular solution (ECS) containing (in mM): 145

NaCl, 5.6 KCl, 2MgCl₂, 1.2 CaCl₂, 10 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES), pH 7.2. Electrodes had a

ﬁnal resistance of 3−5 MΩ when ﬁlled with an intracellular solution

(ICS) containing (in mM) 147 NaCl, 1 ethylene glycol tetraacetic

acid (EGTA), 1 MgCl₂, 24 HEPES, pH 7.3. Patch pipettes were

prepared from the Narishige PC-10 puller (Narishige) with

borosilicate glass (Sutter Instrument). The adenosine diphosphate

ribose (ADPR) concentration was ﬁxed at 500 μM in the ICS. Test

compounds were added to the ECS with the indicated concentrations

and perfused for at least 60 s after the ADPR-activated TRPM2

current was stabilized. Acidic ECS (pH 5.0) was applied to block

TRPM2 currents. A change in ECS was achieved using the RSC-160

system (Biologic Science Instruments) in which the solution changing

time was ∼300 ms. The cell was held at 0 mV. Voltage ramps with a

500 ms duration from −100 to 100 mV were applied every 5 s. Data

were acquired at 10 kHz and ﬁltered oﬄine at 50 Hz. The mean of the

Cinnamic acid 24a or 24b (2.6 mmol) and thionyl chloride (3 mL)

were heated together under reﬂux for 4 h. Any unreacted thionyl

chloride was removed under reduced pressure, and the crude product,

cinnamoyl chloride 25a or 25b, was used without further puriﬁcation.

A THF solution of cinnamoyl chloride 25a or 25b (1.3 mmol) was

added dropwise to a mixture of 2,3-dihydroquinolin-4(1H)-one or

1,2,3,4-tetrahydro-5H-benzo[b]azepin-5-one (2 mmol), catalytic

amounts of triethylamine, and dry THF (4 mL) in an ice bath, and

the mixture was stirred at 0 °C for 15 min, followed by being heated

at 60 °C for an additional 3 h. After 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 DCM/

MeOH (100/3) to aﬀord compounds C19−C22.

(E)-1-(3-([1,1′-Biphenyl]-4-yl)acryloyl)-2,3-dihydroquinolin-

1

4(1H)-one (C19). Oﬀ-white solid. Yield: 57%. mp 232−234 °C. H

NMR (400 MHz, DMSO-d₆) δ 7.93 (dd, J = 7.8, 1.4 Hz, 1H), 7.77−

7.70 (m, 7H), 7.68−7.64 (m, 1H), 7.52−7.47 (m, 3H), 7.42−7.33

(m, 2H), 7.16 (d, J = 15.5 Hz, 1H), 4.32 (t, J = 6.2 Hz, 2H), 2.83 (t, J

= 6.2 Hz, 2H). ¹³C NMR (101 MHz, DMSO-d₆) δ 194.3, 165.5,

144.2, 142.4, 142.0, 139.7, 134.6, 134.4, 129.5, 129.2, 128.4, 127.6,

127.4, 127.1, 125.8, 125.5, 124.8, 120.2, 55.4, 44.1. HRMS (ESI-

TOF⁺) calcd for C₂₄H₂₀NO₂[M + H]⁺m/z 354.1494, found

354.1500.

(E)-1-(3-(4-Pentylphenyl)acryloyl)-2,3-dihydroquinolin-4(1H)-

one (C20). Oﬀ-white solid. Yield: 46%. mp 78−80 °C. ¹H NMR (400

MHz, CDCl₃) δ 8.08 (dd, J = 8.0, 1.6 Hz, 1H), 7.82 (d, J = 15.5 Hz,

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ﬁrst three ramps before the channel activation was used for a leak

subtraction of all the current recording. For a speciﬁcity evaluation,

recordings were conducted on the TRPM8 and TRPV1 currents. The

ECS and ICS of TRPM8 and TRPV1 were the same. The ECS

contains (in mM) 130 NaCl, 5 KCl, 10 ^D-glucose, 10 HEPES, 1.2

MgCl₂, and 1.5 CaCl₂, pH 7.4. The ICS contains (in mM) 115 CsCl,

10 EGTA, 2 MgCl₂, 10 HEPES, and 5.7 CaCl₂, pH 7.2. The test

compounds were added to the ECS with indicated concentrations and

perfused for at least 60 s after the TRPM8 or TRPV1 currents were

stabilized. Menthol (1 mM) was added in the ECS to activate the

TRPM8 channel, and capsaicin (0.01 mM) was added in the ECS to

activate the TRPV1 channel. The inhibition ratio (%) was calculated

using this formula: [1 − (residual current after test compound

treatment − residual current after pH 5 or ECS treatment)/(stable

current after agonist treatment − residual current after pH 5 or ECS

treatment)] × 100. The IC₅₀values were derived from ﬁtting the

concentration−response relationships to the Hill equation.

OGD/R Procedure. An I/R injury was induced in vitro using the

OGD/R model as previously described.⁵²OGD was conducted when

the SH-SY5Y cells were seeded in a 96-well plate at a cell density of

∼2 × 10⁴cells per well for 12 h. The complete culture medium was

changed to glucose-free and serum-free DMEM medium and

incubated in a modular incubator chamber (Billups-Rothenberg)

with 95% N₂and 5% CO₂. The chamber was kept in an incubator for

8 h at 37 °C to produce OGD and then cultured under normoxic

conditions (5% CO₂, 95% air) in normal DMEM for an additional 12

h. The test compounds were added to normal DMEM before the

reperfusion phase. The control culture was always maintained in

normal DMEM and placed in the incubator under normoxic

conditions. Each group had three replicate wells.

Cell Viability Assessment. The cell viability was detected with

the CCK-8 cell proliferation assay kit (Beyotime) in a 96-well plate.

Then 2 × 10⁴SH-SY5Y cells per well were seeded and incubated at 37

°C for 12 h. After the indicated drug treatment, the cells were cultured

for a further 24 h. Then the culture medium was changed to one

containing 10% CCK8, and the cells were incubated at 37 °C for 2 h.

For the OGD/R model, the culture medium was changed to one

containing 10% CCK8 after the reperfusion phase. The optical density

(OD) at 450 nm wavelength was measured with a SynergyMx M5

microplate reader (Molecular Devices). The cellular viability (%) was

calculated using this formula: (experimental group OD − blank group

OD mean)/(control group OD mean − blank group OD mean) ×

100. Each group had three replicate wells.

Colorimetric PLA2 Assay. This method has been published and

was used with minor modiﬁcations.⁵³Brieﬂy, PLA2 (Sigma) was

diluted with phosphate-buﬀered saline to a concentration of 4 μg/L.

The enzyme solution (50 μL) was incubated at 37 °C with 5 μL of an

appropriate concentration of inhibitor buﬀer (125 mmol/L Tris).

After 30 min, 50 μL of substrate buﬀer (12.5 mg of soybean

phosphatidylcholine purchased from Sigma, 250 mmol Tris, 8 mmol

CaC1₂, 4 mmol of sodium desoxycholate, and 0.5% Triton X100 in 1

mL of distilled water) was added and incubated for another 60 min.

The liberated free fatty acids were quantitated using the free fatty acid

content assay kit in a 96-well plate according to the manufacturer’s

instructions (Solarbio). The OD at 550 nm wavelength was measured

with the SynergyMx M5 microplate reader (Molecular Devices).

Results are expressed as the percent inhibition compared with the

enzyme activity without inhibitor.

5% DMSO/5% Solutol in saline). After the doses were given, the time

points for the blood sample collection were 0.083, 0.25, 0.5, 1, 2, 4, 6,

8, and 24 h. And after centrifugation, the plasma samples were

extracted with acetonitrile and analyzed by high-pressure liquid

chromatography/tandem mass spectrometry (LC/MS/MS) with an

ethylene bridged hybrid (BEH) C18 column (50 mm × 2.1 mm, 1.7

μm). The compound detection was performed with the mass

spectrometer (XEVO TQ-S micro; Waters Corp.). The PK

parameters were calculated with WinNonlin software 8.0 (Pharsight

Corporation).

tMCAO Model. As we described in our previous studies,^54,55we

used laser Doppler to monitor the cerebral blood ﬂow (CBF) of each

model mouse. Animals with less than an 80% reduction in CBF were

excluded from the study. The tMCAO model is stably reproducible in

our lab. Brieﬂy, the mice were anesthetized with isoﬂurane (5%

induction, 1.5−2% maintenance) in a 30% oxygen/70% nitrous oxide

mix. The right common carotid artery (CCA), internal carotid artery

(ICA), and external carotid artery (ECA) were exposed through a

midline incision of the neck. A 6−0 nylon monoﬁlament suture,

blunted at the tip and coated with 1% poly ^L-lysine, was inserted from

the ECA into the ICA to occlude the origin of the middle cerebral

artery (MCA). After 90 min of the occlusion, the nylon suture was

removed for reperfusion. After the operation, mice were transferred

into an intensive-care chamber with the maintained temperature at 37

°C, until animals woke up completely. The body temperature was

maintained at 37.0 0.5 °C throughout the operation and recovery.

The number of animals excluded from the hemorrhage is less than

10%.

Experimental Groups and Drug Administration. Mice were

randomly divided into four groups as follows: control group (saline),

vehicle group (5% DMSO/5% Solutol in saline), A23 group (0.3, 1,

and 3 mg/kg), and EDA group (0.3, 1, and 3 mg/kg). The drugs were

administered by i.v. infusion (tail vein) at 3 h after the onset of

reperfusion. The control group, vehicle group, A23 group, and EDA

group were injected with the same volume of saline.

Evaluation of Neurological Scores. Neurological scores were

evaluated after 24 h of reperfusion by the examiner who was blinded

to the treatment conditions to assess the health of mouse.⁵⁶The

scores were assigned using the following scale: 0, no neurological

deﬁcits; 1, failure to fully extend the right forepaw; 2, circling to the

right; 3, falling to the right; and 4, absence of spontaneous walking

and depressed levels of consciousness.

Infarct Volume Analysis. After an evaluation of neurological

scores, the mice were sacriﬁced with 10% chloral hydrate injected.

Brain tissues were dissected and stored at −20 °C for 20 min. The

mice were excluded from the study if a subarachnoid hemorrhage was

observed. Then, the brain tissue was removed into the brain slicer

matrix and sliced into ﬁve 1.0 mm thick coronal sections. The brain

slices were incubated for 30 min at 37 °C in 2% TTC solution and

were then immersed in 4% paraformaldehyde overnight. The infarct

volume was measured by a researcher blinded to the experimental

groups using ImageJ software (National Institutes of Health).

Data Analysis. Data are expressed as the mean

standard

deviation (SD) from at least three independent experiments. A

statistical analysis was performed using one-way analysis of variance

(ANOVA), followed by Dunnett’s test or Tukey’s test for multiple

comparisons, with P < 0.05 considered statistically signiﬁcant.

GraphPad Prism 7 software (GraphPad) was used for all statistical

analyses.

Animal and Treatments. C57BL/6 mice were housed under

standard conditions with a 12/12 h light/dark cycle and free access to

food and water. Eight- to 10-week-old male mice that weighed 22−26

g were used in the study and were randomly assigned to each group.

Compound A23 was dissolved in 5% DMSO with 5% Solutol

dissolved in saline for i.v. and i.g. treatments. All animal use

procedures were according to the University Policies on the Use and

Care of Animals. All of the experiments were performed at room

temperature.

ASSOCIATED CONTENT

* Supporting Information

■

sı

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c02129.

Molecular formula strings of tested compounds (CSV)

Structural formulas of all synthesized ﬁnal compounds;

inhibition activity of selected compounds; cell viability

for A22, A23, A30, C7, C12, and C16 on SH-SY5Y

PK Studies. C57BL/6 mice (n = 3/group) were dosed with A23

via the tail vein for i.v. administration (3, 1, and 0.3 mg/kg, 5%

DMSO/5% Solutol in saline) or i.g. administration (10, 100 mg/kg,

3993

https://doi.org/10.1021/acs.jmedchem.0c02129

J. Med. Chem. 2021, 64, 3976−3996

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

cells; pharmacokinetic parameters of A23; ¹H NMR, ¹³

NMR, HRMS, and HPLC data for all synthesized ﬁnal

compounds (PDF)

C

Foundation (7212152 to L.Z.), Key R & D Projects of

Zhejiang Province (2021C03039 to P.Y.), and the Key

Research and Development Program of Ningxia Hui

Autonomous Region (2019BFH02003 to W.Y.).

AUTHOR INFORMATION

Corresponding Authors

■

ABBREVIATIONS USED

AA, arachidonic acid; ACA, N-(p-amylcinnamoyl) anthranilic

acid; 2-APB, 2-aminoethoxydiphenyl borate; AUC, area under

the curve; CCK-8, cell counting kit-8; CL, clearance rate; C_max

maximum concentrations; EDA, edaravone; F%, oral bioavail-

ability; FFA, ﬂufenamic acid; HEK293T, human embryonic

kidney 293 T cells; I/R, ischemia/reperfusion; i.g., intragastric;

i.v., intravenous; 3-MFA, 2-(3-methylphenyl) aminobenzoic

acid; MRT, mean residence time; OGD/R, oxygen glucose

deprivation/reperfusion; PK, pharmacokinetic; PLA2, phos-

pholipase A2; ROS, reactive oxygen species; SAR, structure−

activity relationship; T_max, time-to-maximum-concentration;

■

Wei Yang − Department of Biophysics, and Department of

Neurosurgery of the First Aﬃliated Hospital, Zhejiang

University School of Medicine, Hangzhou 310058, P. R.

China; Email: yangwei@zju.edu.cn

Liangren Zhang − State Key Laboratory of Natural and

Biomimetic Drugs, School of Pharmaceutical Sciences, Peking

University, Beijing 100191, P. R. China; orcid.org/0000-

0002-7362-9497; Email: liangren@bjmu.edu.cn

,

Authors

Han Zhang − State Key Laboratory of Natural and

Biomimetic Drugs, School of Pharmaceutical Sciences, Peking

University, Beijing 100191, P. R. China

Peilin Yu − Department of Toxicology, and Department of

Medical Oncology of Second Aﬃliated Hospital, Zhejiang

University School of Medicine, Hangzhou 310058, Zhejiang,

P. R. China

Hongwei Lin − Department of Biophysics, and Department of

Neurosurgery of the First Aﬃliated Hospital, Zhejiang

University School of Medicine, Hangzhou 310058, P. R.

China

Zefang Jin − State Key Laboratory of Natural and Biomimetic

Drugs, School of Pharmaceutical Sciences, Peking University,

Beijing 100191, P. R. China

Siqi Zhao − State Key Laboratory of Natural and Biomimetic

Drugs, School of Pharmaceutical Sciences, Peking University,

Beijing 100191, P. R. China

Yi Zhang − Department of Biophysics, and Department of

Neurosurgery of the First Aﬃliated Hospital, Zhejiang

University School of Medicine, Hangzhou 310058, P. R.

China

Qingxia Xu − State Key Laboratory of Natural and

Biomimetic Drugs, School of Pharmaceutical Sciences, Peking

University, Beijing 100191, P. R. China

Hongwei Jin − State Key Laboratory of Natural and

Biomimetic Drugs, School of Pharmaceutical Sciences, Peking

University, Beijing 100191, P. R. China

T

_1/2, half-life; tMCAO, transient middle cerebral artery

occlusion; TRPM2, transient receptor potential melastatin 2;

TRPV1, transient receptor potential vanilloid 1.

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https://pubs.acs.org/10.1021/acs.jmedchem.0c02129

Author Contributions

^#H.Z. and P.Y. contributed equally to this work.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This research work was ﬁnancially supported by Key Programs

of National Natural Science Foundation of China (82030108

to W.Y. and L.Z.), National Natural Science Foundation of

China (81673279 to L.Z. and 32071102 to P.Y.), National

Major Special Project on New Drug Innovation of China

(2018ZX09711001-004-005 to W.Y.), Beijing Natural Science

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The Discovery of Novel ACA Derivatives as Specific TRPM2 Inhibitors that Reduce Ischemic Injury Both in Vitro and in Vivo

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