Design, synthesis and the structure-activity relationship of agonists targeting on the ALDH2 catalytic tunnel

Article abstract of DOI:10.1016/j.bioorg.2020.104166

ALDH2, a key enzyme in the alcohol metabolism process, detoxifies several kinds of toxic small molecular aldehydes, which induce severe organ damages. The development of novel Alda-1 type ALDH2 activators was mostly relied on HTS but not rational design so far. To clarify the structure–activity relationship (SAR) of the skeleton of Alda-1 analogs by synthesis of the least number of analogs, we prepared 31 Alda-1 analogs and 3 isoflavone derivatives and evaluated for their ALDH2-activating activity. Among these, the ALDH2-activating activity of mono-halogen-substituted (Cl and Br) N-piperonylbenzamides 3b and 3 k, and non-aromatic amides 8a-8c, were 1.5–2.1 folds higher than that of Alda-1 at 20 μM. The relationship between binding affinity in computer aided molecular docking model and the ALDH2-activating activity assays were clarified as follows: for Alda-1 analogs, with the formation of halogen bonds, the enzyme-activating activity was found to follow a specific regression curve within the range between ?5 kcal/mol and ?4 kcal/mol. For isoflavone derivatives, the basic moiety on the B ring enhance the activating activity. These results provide a new direction of utilizing computer-aided modeling to design novel ALDH2 agonists in the future.

Full text of DOI:10.1016/j.bioorg.2020.104166

BioorganicChemistry104(2020)104166
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis and the structure-activity relationship of agonists targeting
on the ALDH2 catalytic tunnel
Ming-Che Cheng, Wei-Chi Lo, Yu-Wen Chang, Shoei-Sheng Lee, Chia-Chuan Chang⁎
School of Pharmacy, College of Medicine, National Taiwan University, Taipei 100, Taiwan, ROC
A R T I C L E I N F O
A B S T R A C T
Keywords:
ALDH2, a key enzyme in the alcohol metabolism process, detoxifies several kinds of toxic small molecular
aldehydes, which induce severe organ damages. The development of novel Alda-1 type ALDH2 activators was
mostly relied on HTS but not rational design so far. To clarify the structure–activity relationship (SAR) of the
skeleton of Alda-1 analogs by synthesis of the least number of analogs, we prepared 31 Alda-1 analogs and 3
isoflavone derivatives and evaluated for their ALDH2-activating activity. Among these, the ALDH2-activating
activity of mono-halogen-substituted (Cl and Br) N-piperonylbenzamides 3b and 3 k, and non-aromatic amides
8a-8c, were 1.5–2.1 folds higher than that of Alda-1 at 20 μM. The relationship between binding affinity in
computer aided molecular docking model and the ALDH2-activating activity assays were clarified as follows: for
Alda-1 analogs, with the formation of halogen bonds, the enzyme-activating activity was found to follow a
specific regression curve within the range between −5 kcal/mol and −4 kcal/mol. For isoflavone derivatives,
the basic moiety on the B ring enhance the activating activity. These results provide a new direction of utilizing
computer-aided modeling to design novel ALDH2 agonists in the future.
ALDH2
Aldehyde metabolism
Alda-1
ALDH2 agonist
1. Introduction
be the primary reason leading to the dysfunctional aldehyde metabo-
lism [12]. The side chain of residue Glu487 at the oligomerization
Aldehyde dehydrogenase 2 (ALDH2) is an important enzyme in-
volved in the metabolic process of alcohol. Ethanol is oxidized into
acetaldehyde by alcohol dehydrogenase (ADH) in human liver, and
ALDH2 further converted it into carboxylic acid to prevent human body
from the damage caused by the elevation of acetaldehyde [1]. ALDH2
not only degrades acetaldehyde but also other endogenous and exo-
genous toxic aldehydes, such as acrolein, 2-furaldehyde, 4-HNE (4-hy-
droxynonenal) and DOPAL (3,4-Dihydroxyphenylacetaldehyde) [2-4].
ALDH2 widely present in the human body which requires high mi-
tochondrial oxidative phosphorylation, especially in the liver, cardio-
vascular system and central nervous system [5].
domain formed two hydrogen bonds with Arg264 and Arg475, these
interactions stabilized the dimer interface of ALDH2 and fixed the
NAD+ binding domain at a suitable position [13,14]. However, the
Glu487Lys mutation made the protein dimer loss of the structural in-
tegrity and finally led to a positional disorder of αG (amino acids
247–260), which plays an important role in NAD+ binding activity
[11]. The X-ray crystallography showed the helix αG of ALDH2*2 were
3 Å shifted into the NAD+-binding cleft compared with the wild type
ALDH2 which was the major reason that cause the functional loss of the
mutated enzyme [12].
Alda-1 (N-piperonyl-2,6-dichlorobenzamide) was first identified as
a potent ALDH2 agonist via a series of high-throughput screening (HTS)
in 2008. Alda-1 alone enhanced the aldehyde oxidation about 2 folds
for the wild-type ALDH2 and 10 folds for ALDH2*2 at 10 μM, respec-
tively [11]. In an animal study, treatment of Alda-1 reduced the in-
farction size by 60% in the ischemia/reperfusion (I/R) injury model [7].
Therefore, Alda-1 showed a strong potential of being a cardiovascular
agent candidate [15-17]. However, the mechanisms for the activation
of ALDH2 by Alda-1 are still unknown. In order to investigate the
manner of Alda-1 in activating ALDH2, a co-crystal structure of Alda-1
in complex with ALDH2 and ALDH2*2 had been reported [11]. As
About 560 million of Asians (nearly 8% of the total population in
the world) are carrying the ALDH2*2 allele, and such metabolic defect
is related to several severe diseases, such as cancers and cardiovascular
diseases [6-9]. Elevated ALDH2 level resulted in the reduction of
myocardial injury by increasing of oxidative stress in an animal model
[10]. In the enzymatic studies, the Michaelis-Menten constant (KM) and
catalytic constant (kcat) which represented the turnover efficacy of
ALDH2*2 were 200-fold higher and 10-fold lower than those of wild
type ALDH2, respectively [11]. According to the crystallography of
mutated ALDH2, the Glu487Lys mutation of ALDH2*2 was founded to
⁎ Corresponding author.
E-mail address: chiachang@ntu.edu.tw (C.-C. Chang).
https://doi.org/10.1016/j.bioorg.2020.104166
Received 1 May 2020; Received in revised form 30 June 2020; Accepted 30 July 2020
Availableonline25August2020
0045-2068/©2020PublishedbyElsevierInc.

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
Fig. 1. (a) Alda-1 binding pose in the substrate entrance of ALDH2 (PDB ID: 3INL). (b) Important amino acid residues in the catalytic domain and the Alda-1 binding
cavity of ALDH2.
shown in Fig. 1a, the catalytic domain of ALDH2 was shaped as a tunnel
and could be divided into three portions, including the NAD+ binding
domain, the substrate-binding domain and the substrate entrance. The
co-crystal structure also indicated that the most stable binding site of
Alda-1 was located at the substrate entrance. The benzamide moiety
was bounded within a hydrophobic pocket comprised of Val120,
Met124, Phe292 and Val458. Inside the binding cavity, a significant
hydrogen bond was formed between the amide nitrogen and the car-
bonyl oxygen on the main chain of Asp457. Besides, the piperonyl
moiety of Alda-1 was sandwiched between Phe296 and Phe459 and
were 7–8 Å apart from the catalytic triad of ALDH2 including Cys302
and Glu268 (Fig. 1b).
back to wild-type like (i.e. ALDH2), thus enhancing the binding affinity
between NAD+ and ALDH2*2 [18].
Based on the aforementioned interaction analysis, several potent
ALDH2 agonists had also been identified via HTS in the previous reports
(Fig. 3) [19]. The results revealed that the N-benzylamide scaffold had
excellent potential for designing novel ALDH2 activator. However, the
development of novel ALDH2 agonists were highly relied on the
screening results of non-systematic-designed compounds so far, and the
structure–activity relationship (SAR) of Alda-1 were not totally estab-
lished. Herein, we designed and synthesized 31 N-benzylamide deri-
vatives based on the theoretical ligand–protein interaction analysis of
Alda-1 in complex with ALDH2 and provided an exhaustive SAR ana-
lysis for a rational approach to novel ALDH2 modulator design.
The X-ray crystallography of Alda-1 in complex with ALDH2 and
ALDH2*2 suggested that binding with Alda-1 also lead to the con-
formational change of the catalytic domain. As shown in Fig. 2a, a
significant shifted of αG in the apo form of ALDH2*2 (PDB code: 1ZUM)
[13] was observed compared to the apo form of ALDH2 (Fig. 2b, PDB
code: 3N80). However, the rotation of Phe459 in the co-crystal struc-
ture of ALDH2*2 binary complex with Alda-1 was also observed
(Fig. 2c, PDB code: 3INL). The relative position of Phe296, Phe459, and
the αG of ALDH2*2 binary complex with Alda-1 were highly similar
with that of the apo form of ALDH2. We assumed that π-π interaction
and hydrophobic interaction were the major interactions that made the
aromatic side chain of Phe459 to be parallel to the piperonyl moiety of
Alda-1. Such effects might be associated to the restoration of the ori-
ginal position of the αG helix, altered the interaction mode between
ALDH2*2 and NAD+, and then decreased the KM value of ALDH2*2. A
plausible mechanism of action had been reported: Alda-1 enhanced the
catalytic efficiency by limiting the diffusion of substrates molecules out
of NAD+ tunnel [11], it also reset the conformation of catalytic domain
2. Results and discussions
2.1. Design of Alda-1 analogs
The aforementioned computer-aided molecular docking model of
protein (i.e. ALDH2, PDB code: 3INJ) in complex with 31 designed li-
gands (3a-3k, 6a, 6c-6g, 8a-8c, 11a-11e, 22, 24, 27a-27b, 29a-29c)
had been applied to evaluate their interactions. As shown in Fig. 4,
starting from the scaffold of Alda-1, the modifications on directions I to
IV were focused on the B part (i.e. the benzamide moiety on the right)
(direction I), the P part (i.e. the benzyl moiety on the left) (direction II),
the interchange of the carbonyl positions on the linkage (direction III),
and replacement of the amide linkage by non-proton donated isosteres
(direction IV), respectively. The modifications on the direction I (i.e.
3a-3k) were used to verify the roles of different substituents (including
halogen, methyl and hydroxy groups) on the 2 and 6 positions of B part,
Fig. 2. Crystal structure of (a) Alda-1 binding domain of ALDH2*2, apo form (PDB code: 1ZUM) (b) Alda-1 binding domain of ALDH2, apo form (PDB code: 3N80) (c)
Alda-1 binding domain of ALDH2*2 in complex with Alda-1 (PDB code: 3INL).
Fig. 3. Chemical structure of four reported potent ALDH2 agonists identified by high-throughput screening (HTS).
2

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
Fig. 4. Design of 31 Alda-1 analogs in directions I-IV, which were modified on the B part, P part, the position of carbonyl group and the linkage, respectively.
when interacting with the outer edge of the binding conical. In addi-
tion, to investigate the importance of the aromatic ring on the B
part, the benzene ring that was replaced by three non-aromatic carbon
chains with 4 to 5 atoms (i.e. 8a-8c; see Table 1) were also designed.
For direction II, the piperonyl moiety (P part) was replaced by a series
of aromatic and non-aromatic functional groups (6a, 6c-6g; compound
6d was replaced by non-aromatic substituent) to investigate their ef-
fects on the hydrophobic and aromatic amino acid residues in the
binding site. Finally, the importance of the hydrogen bond between the
ligand and Asp457 was investigated via two approaches: interchange of
the carbonyl group and replacement of the amide linkage by non-hy-
drogen-bond-donating amide bioisosteres.
Table 1
Relative ALDH2-activating activity and calculated binding affinity of com-
pounds modified on B part (3a-3k and 8a-8c).
2.2. Synthesis of Alda-1 analogs
The synthetic route of compounds 3a-3k, 6a-6g and 8a-8c is de-
picted in Scheme 1. The EDC-HOBt reaction was applied to couple
various carboxylic acids with piperonyl amine to give 3a-3k and 8a-8c.
For 6a, 6c-6g, primary amines 5a-5f were prepared from the corre-
sponding aldehydes 4a-4f through reduction of the aldehyde oxime
intermediates by zinc dust. Next, these amines were treated with aryl-
chlorides to achieve the amide linkage of Alda-1 analogs, with total
yields among 52% to 96% after purification.
Entry
R1
R2
Activation (%)a
Calcd. ΔS (Kcal/mol)b
3a
H
H
< 10
67.1
< 10
21.0
26.3
13.7
28.8
18.6
< 10
74.3
34.8
−4.49
−4.68
−4.99
−4.61
−4.95
−4.87
−4.79
−4.58
−4.67
−4.65
−4.81
3b
Cl
I
H
3c
H
3d
Me
F
H
3e
F
3f
Cl
Cl
OH
OH
Br
Cl
F
3g
Me
H
3i
For compounds 11a-11e and 22 (see Scheme 2), protocatechuic
acid (9a), gallic acid (9b) and caffeic acid (20) were used as the starting
materials, respectively, to give the corresponding precursors (10a-10c
and 21). Because the phenolic hydroxy groups were unable to perform a
nucleophilic attack to the dibromomethane when the carboxylic group
was un-protected, the carboxyl group of 9 and 20 were protected by
methyl ester via Fisher esterification before the methylenedioxy moiety
was introduced. Finally, the EDC-HOBt coupling reaction was applied
to condense the benzoic acid precursors with benzylamines to give the
target products 11a-11e and 22.
3j
OH
H
3k
Alda-1 (3h)
Cl
Entry
R
Activation rate (%)a
62.0
Calcd. ΔS (Kcal/mol)b
8a
−4.06
8b
65.3
−4.22
In order to investigate the roles of hydrogen bonding between the
ligands and Asp457, a series of non-hydrogen-bond-donating com-
pounds, including of one ester (24), two tertiary amides (27a-27b) and
three 1,2,4-oxadiazole compounds (29a-29c), were synthesized (see
Scheme 3) (for the synthesis scheme, synthesis procedure and chemical
properties of 24, 29a and 29b: see p. 28–29, p. 32–33 and p. 35–36 of
Supplementary Information). Next, piperonyl aldehyde 25 was
8c
52.0
34.8
−4.09
−4.81
Alda-1 (3h)
a
b
Compared with the control group (DMSO control as 0%, see Section 4.2).
Calculated by Molecular Operating Environment (MOE) 2008.
3

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
Scheme 1. Preparation of 3a-3k, 6a-6g and 8a-8c. a. EDC-HCl, HOBt, TEA, THF, rt, 8–12 h; b. hydroxylamine hydrochloride, NaOAc, EtOH, rt, 12 h; c. zinc dust,
37% HCl(aq), EtOH, 70 ℃, 2 h; d. acyl chlorides, pyridine, ACN, rt, 8 h; e. 10% Pd/C, H2, cat. HCl(aq), MeOH, 24 h.
converted into amines 26a and 26b by reductive amination, and the
two amines were further treated with 2-chlorobenzoyl chloride (pre-
pared by Vilsmeier-Haack reaction) to give tertiary amides 27a-27b.
Finally, the end products 29a-29c were synthesized from aldehyde 25
via two intermediates (piperonyl nitrile and amidoxime 28), which
were prepared by reported oxidative process [20] and hydroxyamina-
tion, respectively.
one acidic phenolic proton, was able to bind into the same binding site
as Alda-1 and inhibit the enzyme activity through blocking the entrance
of the substrates [18,21,22]. The ligand–protein interaction analysis
showed that the phenolic group on the ring C is proximal to Cys302
(3.8 Å) and the water molecule (3.4 Å) when binding to ALDH2
(Fig. 5a). Furthermore, ring A of daidzin were also sandwiched between
Phe296 and Phe459 similar to the P part of Alda-1, and the sugar
moiety (SG) formed hydrogen bond with Asp457 and Phe459 which
were located at the substrate entrance (Fig. 5b).
2.3. Design and synthesis of the isoflavone analogs
The reported catalytic mechanism of ALDH2 included four steps
(steps 1–4 in the pink region of Fig. 6), the formation of thioester bond
Daidzin, which was a potent natural ALDH2 inhibitors consisting of
Scheme 2. Preparation of 11a-11e and 22. a. H2SO4, MeOH, reflux, 3 h; b. CH2Br2, K2CO3, ACN, DMF, N2 atmosphere, 85 ℃, 18 h; c. NaOH, H2O, EtOH, N2
atmosphere, 75 ℃, 1.5 h; d. amines, EDC-HCl, HOBt, TEA, THF, rt, 8–12 h; e. MeI, K2CO3, ACN, DMF, N2, 85 ℃, 6 h.
4

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
Scheme 3. Preparation of Alda-1 analogs
27a-27b and 29b. a. amine hydrochlorides,
TEA, THF, 50-70 ℃, 4 h; b. 10% Pd/C, H2,
THF, rt, 12 h; c. 2-chlorobenzoyl chloride,
pyridine, ACN, rt, 24 h; d. I2, 28% NH3(aq)
,
ACN, rt,
4 h; e. hydroxylamine hydro-
chloride, Na2CO3, isopropyl alcohol, H2O,
reflux, 6 h; f. butyryl chloride, pyridine, 80
℃, 6 h.
Cys302
Glu268
3.8Å
Daidzin
SG
3.4Å
4.9Å
B
C
H2O
A
Phe459
Phe296
(a)
(b)
Fig. 5. (a) The docking model of ALDH2 in complex with daidzin. (b) The 2D-interaction diagram of ALDH2 in complex with daidzin, the structure of daidzin was
divided into A-C ring and sugar moiety (SG).
Reported
Proposed
Fig. 6. The reported catalytic cycle of ALDH2 (pink region) and the proposed inhibition/activation mechanism of daidzin (34, calculated pKa ~ 8.96) and basic
isoflavone analogs 33c (calculated pKb ~ 3.90, light blue region). (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
5

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
Fig. 7. Converting daidzin (34) from an ALDH2 inhibitor to an activator by decreasing binding affinity and modification on the ring B.
in the step 4 is important for the hydride transportation in the catalytic
cycle [23], and the activation of the thiol group on Cys302 in step 1 is a
key step to form the thioester intermediate. According to the simulation
results, the pKa value of the thiol group on Cys302 is 10.17 (calculated
by MarvinView 20.9; 8.14 in the reference data [24]), which is closed to
that of daidzin (pKa ~ 8.96, calculated by MarvinView 20.9). Inspired
by these studies and evidence, we assumed that daidzin could inhibit
the catalytic cycle of ALDH2 not only by blocking the substrate en-
trance but also interrupting the proton transportation process in step 1
through donating an acidity proton to the catalytic domain. Based on
this hypothesis, when the basic moiety (e.g. pyridinyl group; see the
light blue region of Fig. 6) of a ligand was entering the substrate en-
trance and approaching Cys302, the ALDH2 activity might be enhanced
by assisting the proton transportation in step 1.
2.4. Molecular docking analysis and biological evaluation of Alda-1 analogs
2.4.1. The in vitro evaluation results of 3a-3k and 8a-8c
The docking scores and relative ALDH2-activating activity values
were listed in Tables 1-3, and the docking poses of all compounds were
selected based on the binding mode of Alda-1 in the co-crystal structure
(Fig. 8). In the present study, the effects of B part (see Fig. 4) was first
clarified. In Alda-1, the B part is mainly composed of two chloro groups
and a benzene ring. Thus in the present study, both of the two groups
(i.e. the 2- and/or 6- and the benzene) were replaced by other sub-
stituents. The results for the replacement of the 2- and/or 6- sub-
stituents were shown in upper part of Table 1: compounds 3a, 3c and 3j
revealed no ALDH2-activating activity; the ALDH2-activating activities
of 3b and 3k were about 1.9 and 2.1 folds stronger than Alda-1, re-
spectively. As the results for the replacement of the benzene, as shown
in lower part of Table 1, compounds 8a-8c, in which the benzene ring
of B part were replaced by acyl-groups, demonstrated ALDH2-activating
activities that were 1.5–1.9 folds stronger than that of Alda-1.
Therefore, we use daidzin as a template to design and synthesis
three isoflavone analogs (33a-33c) to investigate the effect of acidity in
the catalytic center of ALDH2 (see Fig. 7). First, we replaced SG with
methyl group to decrease the binding affinity of ligand. Second, the
acidic substituent on ring C was replaced by basic moiety (i.e. pyridinyl
group). And analog 33a was also prepared as the neutral ligand for
comparison.
2.4.2. Relationship between the formation of halogen bond and ALDH2-
activating activity
The synthetic route of isoflavone analogs was depicted in Scheme 4,
and the core structure of these isoflavones were synthesized by the re-
ported methods [25]. Briefly, paeonol (30) was heated with di-
methylformamide dimethoxyacetal (DMFDMA) to produce the enamine
31, then cyclized the enamine moiety with hydroxyl group at meta po-
sition by iodination to give 32. Finally, the Suzuki-Miyaura reaction was
applied to couple various of arylboronic acids with 32 to give 33a-33c.
The B part of the Alda-1 analogs, when docked on the active site,
was located at the outer edge of the binding site, and the 2- and/or 6-
halogen moiet(y/ies) may form halogen bonds within a specific limit
distance (normally < 3.6 Å) horizontal (nucleophilic end) and vertical
(electrophilic end) to the C-X bond [26]. In the docking pose of ALDH2
binary complex with Alda-1, as shown in Fig. 9a, the formation of two
possible halogen bonds were observed: one was between the lone-pair
Scheme 4. Preparation of isoflavone analogs 33a-33c. a. DMFDMA, neat, N2 atmosphere, 110 ℃, 2 h; b. I2, MeOH, 60 ℃, 18 h; c. arylboronic acids, Pd(OAc)2, PPh3,
K2CO3, ACN, H2O, 100 ℃, 24 h.
6

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
Table 2
Table 3
The relative ALDH2-activating activity and calculated binding affinity of
compounds modified on P part (6a and 6c-6g).
The relative ALDH2-activating activity and calculated binding affinity of
compounds modified on linkage (11a-11e, 22, 27a-27b and 29b).
Entry
R1
R2
Cl
R3
Activation rate (%)a
< 10
Entry
6a
R1
R2 R3 Activation (%)a Calcd. ΔS (Kcal/mol)b
11a
Cl
Cl
H
Cl
Cl
31.1
25.4
−5.42
−6.79
11b
11c
11d
H
F
Cl
F
< 10
< 10
< 10
6c
6d
6e
6f
Cl
H
Cl
Cl
Cl
16.7
42.4
20.7
–
Cl
Cl
−4.76
−6.00
Cl
11e
22
Cl
Cl
Cl
Cl
19.2
6g
H
Cl
Cl
37.1
34.8
−5.06
−4.81
Alda-1 (3h)
Cl
< 10
a
b
Compared with the control group (DMSO control as 0%, see Section 4.2).
Calculated by Molecular Operating Environment (MOE) 2008.
electron of Met124 sulfur (as an electron donor; horizontal to the C-
chloro A bond; calculated distance: 3.3 Å) and chloro group A, and
another was the amide hydrogen on the main chain of Phe459 (as an
electron acceptor; vertical to the C-chloro A bond; calculated distance:
3.5 Å). On the other side, the carbonyl group on the main chain of
Tyr456 was also found to be close to the chloro group B (calculated
distance: 4.4 Å). However, it was vertical to the C-chloro B bond
therefore in the incorrect direction. Formation of halogen bonds in the
docking results of Alda-1 and the rest 10 Alda-1 analogs (3a-3k)
modified on the B part might be utilized to explain their percentage of
activation. In Fig. 3b, the hindrance of the iodo group on the B part of
3c lead the ligands to bind with ALDH2 in an unfavored binding pose
and lose their ALDH2-activating activity. Nevertheless, the in-
tramolecular hydrogen bond formed between the hydroxy group on the
B part and the carbonyl on the linkage of 3i and 3j were lack of any
halogen moieties to interact with Met124, Phe459 or Tyr456. There-
fore, the chloro group A of Alda-1 was thought to be acted as an anchor
to fix the B part at a suitable binding pose. As shown in Fig. 9c, two
possible binding poses may exist, and if halogen bond between the
chloro group B (ClB) and the carbonyl on the main chain of Tyr456 was
formed, the B part would be twisted and the two important halogen
bonds (with Met124 and Phe459) will disappear, and the ALDH2-acti-
vating activity will decrease. Such proposition could explain why the
activation (%) of the mono-halogen-substituted analogs 3b and 3k
(67.1% and 74.3%, respectively; see Table 1) were 1.9 folds and 2.1
folds more than di-chloro Alda-1 (34.8%), while iodo-substituted Alda-
1 analog 3c shown no ALDH2-activating activity.
Entry
Linkage
R
Activation rate (%)a
17.3
27a
27b
< 10
29b
13.8
34.8
Alda-1 (3h)
b24, 29a and 29b were insoluble even in 3.5% (v/v) DMSO aqueous solution, so
their data were not shown.
a
Compared with the control group (DMSO control as 0%; see Section 4.2).
2.4.4. In vitro evaluation and discussion of 11a-11e, 22, 24, 27a-27b and
29a-29c
In the enzyme activation assay, the compounds which been shifted
the carbonyl position (11a-11e and 22) demonstrated no ALDH2-acti-
vating activity, and no meaningful molecular docking results were ob-
served in the computer model. Following, synthesized non-hydrogen-
bond-donating Alda-1 analogs (24, 27a-27b, and 29a-29c) were too
lipophilic to be dissolved in 2% (v/v) DMSO aqueous solution.
2.4.3. In vitro evaluation and discussion of 6a, 6c-6 g
The results of modification on the P part were shown in Table 2. Six
compounds 6a, and 6c-6g were synthesized. Five out of the six de-
monstrated similar activities with that of Alda-1, and 6d, which was
without an aromatic ring on the P part, demonstrated a weaker ALDH2-
activating activity and binding affinity. These results suggested that the
aromatic ring on P part was important for the ALDH2-activating ac-
tivity, there presence π-π interaction between ligand, Phe296 and
Phe459.
Fig. 8. Overlay of ALDH2 (PDB code: 3INL) with Alda-1 (yellow) and 3a-3 k,
6a, 6c-6 g, 8a-8c, 24 (light blue). (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
7

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
Fig. 9. (a) Binding pose of Alda-1 in the co-
crystal structure, showing two halogen bonds
of chloro A with Met124 and Phe459, and the
distance between chloro B and carbonyl of
Tyr456, which was not a halogen bond. (b)
Binding pose of 3c, showing no halogen bond
formation at the outer edge of the binding
site. (c) Two possible binding poses (left: ClB
on the upper of the paper surface; right: ClA
on the upper of the paper surface) the rotation
of carbonyl-benzene bond of Alda-1, showing
two (horizontal: Met124; vertical: Tyr456)
and one (vertical: Phe459) halogen bonds
between the B part and the indicated proximal
amino acid(s), respectively.
Chloro B
b
a
3.3 Å
Met124
Met124
5.1 Å
4.4 Å
Chloro A
3.5 Å
Tyr456
Tyr456
Phe459
Phe459
(c)
Table 4
Isoflavone analogs and the relative ALDH2-activating activity.
Entry
Ar
R
Activation/
inhibition rate
(%)a
Calcd. ΔS Calcd.
(Kcal/
mol)
pKa/
b
pKb
33a
33b
Me
Me
7.9/–
−8.7
−8.0
–/–
Fig. 10. Scatter plot for the calculated ΔS and the relative ALDH2 activation
(%) for the 21 designed ALDH2 ligands. The suggested ΔS for ALDH2 ligands
was in a range between −5 kcal/mol and −4 kcal/mol. The regression curves
were linear and exponential-like in the regions lower than −5 kcal/mol and the
suggested ΔS range, respectively. (Blue: Alda-1 analogs with halogen bonding
formation; purple: Alda-1 analogs without aromatic ring on B part; red: iso-
flavone derivatives; light green: Alda-1 analogs without halogen bonding for-
mation; light blue: Alda-1 analogs with fluoro substituents). (For interpretation
of the references to color in this figure legend, the reader is referred to the web
version of this article.)
–/19.4
8.96/–
33c
Me
22.3/–
−8.0
–/3.90
8.96/–
Daidzin (34)
β-Glucosyl –/63.6
−10.1
a
b
Compared with the control group (DMSO control as 0%, see Section 4.2).
The pKa values were calculated by ChemAxon-MarvinView 20.9.
Therefore, in the present study, these compounds were examined for
the ALDH2-activating assay in 3% (v/v) DMSO aqueous solution.
However, analogs 24, 29a and 29c were dissolve poorly even in 3.5%
(v/v) DMSO aqueous solution, these compounds were not able to run
the in vitro evaluation. Beside, 27a-27b and 29b demonstrated weak
ALDH2-activating activity. Comparing the bio-assay results of 27a-27b
and 29b with the docking model, we surmised that the formation of the
hydrogen bond between the ligand and Asp457 significantly affected
the ALDH2-activating activity.
the enzyme activity just like it mentioned in the previous study [27].
Surprisingly, replacement of the phenolic moiety by a pyridinyl group
converted the isoflavone analogs from an inhibitor (33b: 19.4% in-
hibition) to an activator (33c: 22.3% activation). These results can be a
powerful proof of our hypothesis and provide us a new direction for
design novel ALDH2 activator.
2.4.6. Analysis of the relationship between ligands binding affinity and
ALDH2-activating activity
The relationship between the calculated binding affinity (ΔS) and
the relative ALDH2-activating activity (%) of the ligands was illustrated
in a scatter plot, as shown in Fig. 10, and thirteen compounds were
absent because of lacking calculated ΔS. Interestingly, fifteen out of
twenty-one compounds were in the ΔS range between −5 kcal/mol and
−4 kcal/mol. When these compounds were categorized by their
structural characteristics (as the color of the dot annotated in the le-
gends of Fig. 10), we found that non-halogen bonds formation (light
green) and the compounds with fluoro group (light blue) were in the
lower part of the region. In the suggested region, an exponential-like
regression curve was fitted for the dots of the compounds capable of
2.4.5. In vitro evaluation and discussion of isoflavoids 33a-33c and 34
Following the aforementioned hypothesis (see Section 2.3), the
synthetic isoflavone analogs 33a-33c were examined for their ALDH2-
activating activity to validate the importance of the microenvironment
in the catalytic domain of ALDH2. The molecular docking model also
showed that the phenolic hydroxy group of 33a and daidzin, and the
nitrogen on the pyridinyl group of 33c were all proximal to Cys302
with a distance around 3–4 Å. The following assay results were shown
in table 4. 7-Methoxyisoflavone (33a) demonstrated weak activation
activity toward ALDH2, and isoformononetin (33b) slightly inhibited
8

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
forming halogen bonds with Met124 and Phe459; in the range lower
than −5 kcal/mol, a linear regression curve was fitted. This might
because the ligands with a strong binding affinity toward ALDH2 will
become a blocker of the enzyme. Therefore, we suggested that binding
affinity of ligands should between −5 to −4 kcal/mol, which limited
the substrate at the catalytic domain and allowed the free diffusion of
the end products.
at 25 ℃ to evaluate the initial NADH production rate of each group, and
obtained the activation/inhibition rate data by compared to the control
group. The enzymatic analyzed data were carried out via SpetraMax
Paradigm multi-mode microplate detection platform (Molecular Device,
San Jose, CA, USA).
4.3. Instrumentation and chemicals
2.4.7. Structure-activity relationship of two chemical skeletons (Alda-1 and
isoflavone)
All reagents and solvents were purchased from commercial sources
including: Sigma-Aldrich, Alfa-Aesar, Merck, J.T. Baker, Fluka, Acros,
and TCI chemicals. These chemicals were used without further pur-
ification. Analytical TLC was performed using aluminum plates coated
with a 0.25 mm thickness of silica gel (60 F254). Column chromato-
Four SARs were observed: First, the B part should contain at least
one chloro, or bromo group on ortho positions to form halogen bond
with Met124 and Phe459, and the mono-halogen-substituted analogs
revealed a stronger ALDH2-activating activity than that of Alda-1 (ex-
cept 3c, which demonstrated no activity). Second, the suggested
binding affinity for ligands was in the range between −5 kcal/mol and
−4 kcal/mol. Third, the aromatic moieties on P part that capable to
interacting with the side chain of Phe296 and Phe459 were important
for activating ALDH2 activity [although in the case absent of an aro-
matic ring in the P part, only one compound (6d) was prepared], and
the amide linkage that formed hydrogen bond with Asp457 was es-
sential for the ALDH2 agonists. Finally, the basic moiety that proximal
to Cys302 of the catalytic triad (about 3–4 Å) assisted the hydrogen
transportation process and enhance the ALDH2-activing activity.
1
graphy was performed using silica gel (40–63 mesh). H NMR spectra
were recorded on 200 MHz (DPX-200) (Bruker, Karlsruhe, Germany) or
400 MHz (AV-400) spectrometer in CDCl3 or methanol‑d4 (all signals
are reported in ppm with the internal signal at 7.24 ppm for CDCl3 and
3.30 ppm for methanol‑d4 as standard) at 25 °C. 13C NMR spectra were
recorded on the same spectrometers in CDCl3 or methanol‑d4 (all sig-
nals are reported in ppm with the internal signal at 77.0 ppm for CDCl3
and 49.0 ppm for methanol‑d4) at 25 °C. HRMS analyses were per-
formed on a microTOF orthogonal ESI-TOF mass spectrometer (Bruker
Daltonik, Bremen, Germany). Melting points were determined by
MetTemp melting point measurement apparatus (Laboratory Devices,
Cambridge, MA, USA).
3. Conclusion
4.4. Chemistry
Thirty-one Alda-1 analogs and three isoflavone derivatives were
prepared in the four aforementioned directions and tested for their in
vitro ADLH2-activating activity. Four Alda-1 analogs (3b, 3 k and 8a-
8c) were found to have higher activities than Alda-1. Further in-
vestigations on the SAR of the two types of skeletons (Alda-1 and iso-
flavone) clarified the importance of a specific range of the calculated
binding affinity in the computer-aided molecular docking, the proposed
formation of halogen bond(s), and the basic moiety on the B ring of
isoflavone derivatives. In conclusion, the SAR for Alda-1 type ALDH2
agonists was established, and these results may provide new directions
for the design of novel ALDH2 agonists in the future.
4.4.1. Procedure for synthesis of compounds 3a-3 k
To a solution of corresponding carboxylic acid (1 mmol), EDC-HCl
(268.4 mg, 1.4 mmol), HOBt (189.2 mg, 1.4 mmol) and triethylamine
(195 μL, 1.4 mmol) in dry THF (5 mL) were stirred for 15 min at room
temperature, and the piperonylamine (150 μL, 1.2 mmol) was then
added into the solution. The reaction mixture was stirred for 8–12 h at
room temperature, and monitored by thin-layer chromatography. The
resulting mixture was concentrated by reduced pressure and dissolve
into EtOAc (15 mL) then partitioned by 1 N HCl aqueous solution
(3 × 10 mL) and concentrated NaHCO3 aqueous solution (3 × 10 mL),
sequentially. The organic layers were dried over anhydrous MgSO4,
filtered, and concentrated under reduced pressure. The residue was
recrystallized from the solution of CHCl3-Hexane to give the amides 3a-
3 k.
4. General experiment
4.1. 3D computer docking model of the ALDH2 modulators
Crystal structure of ALDH2 (PDB ID: 3 N80), ALDH2*2 (PDB code:
1ZUM), ALDH2 bounded with Alda-1 (PDB code: 3INJ) and ALDH2*2
bounded with Alda-1 (PDB code: 3INL), were downloaded from protein
data bank and modified by PyMOL 2.0.0. Small molecules were pre-
pared and minimized the structure energy by ChemBio3D ultra. The
final docking data were carried out via Molecular Operating
Environment 2008 and visualized using PyMOL 2.0.0. And the chemical
properties of ligands (i.e. pKa) were calculated via MarvinView 20.9.
4.4.2. Procedure for synthesis of compounds 5a-5f
To a solution of corresponding aldehydes (1 mmol), hydroxylamine
hydrochloride (140.0 mg, 2 mmol), NaOAc (164.1 mg, 2 mmol) in
EtOH (5 mL) were stirred for 12 h at room temperature. The resulting
mixture was concentrated by reduced pressure and suspension in ice
water, the precipitates was then filtrated and washed by ice water.
Following, the precipitates were mixed with activated zinc dust
(500 mg) in EtOH (5 mL), then 0.2 mL 37% HCl(aq) was added into the
mixture slowly under ice bath and stirred for 2 h under 70 ℃. The pH
value of resulting mixture was adjusted to 8–9 by concentrated NaHCO3
solution, then filtrated by a short pad of celite. The solution was dried
over anhydrous MgSO4, filtered, and concentrated under reduced
pressure to give target amines 5a-5f.
4.2. Assay of ALDH2-activating/inhibiting activity
The purified ALDH2 were purchased from Prospec-Tany
TechnoGene Ltd. (Ness-Ziona, Israel). All the synthetic Alda-1 and
isoflavone analogs were assayed for ALDH2-activating/inhibiting ac-
tivity in a standard reaction mixture comprised of 100 mM tris, pH 8.0,
with 0.06–0.10 μM ALDH2, 2 mM acetaldehyde (Merck, Darmstadt,
Germany), 2 mM NAD+ (Acros, Geel, Belgium), 10 mM 2-mercap-
toethanol (Sigma-Alderich, St. Louis, MI, US), and 100 mM KCl (Showa,
Tokyo, Japan). All assays were included a final concentration of 2% (v/
v) DMSO (J.T. Baker, Center Vally, PA, USA) as a co-solvent and 20 μM
of sample compounds for Alda-1 analogs, and only DMSO [final con-
centration: 2% (v/v)] were add into the mixture for the control group.
Then, all mixture was monitored at an excitation wavelength of 340 nm
4.4.3. Procedure for synthesis of compounds 6a-6 g
Corresponding amines (5a-5f) (0.5 mmol) and pyridine (1 mL) were
mixed in dry ACN (5 mL) under ice bath. The prepared acyl chlorides
were then added into the mixture and stirred for 8 h at room tem-
perature. The resulting mixture was concentrated by reduced pressure
and dissolve into EtOAc (15 mL) then partitioned by 1 N HCl aqueous
solution (3 × 10 mL) and concentrated NaHCO3 aqueous solution
(3 × 10 mL), sequentially. The organic layers were dried over anhy-
drous MgSO4, filtered, and concentrated under reduced pressure. The
9

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
residue was purified by silica gel column chromatography
(CHCl3:MeOH from 100:0 to 75:25) to give target amides 6a-6f. And 6b
was further stirred with 10% Pd/C and catalytic amount of 37% HCl(aq)
under H2 atmosphere for 24 h to give the final product 6f.
4.4.8. Procedure for synthesis of compounds 29b
To a solution of piperonyl aldehyde (2 mmol), iodine (558.4 mg,
2.2 mmol) and 28% ammonia hydroxide solution (2 mL) in ACN (4 mL)
were stirred for 4 h at room temperature. The excess iodine was
quenched by concentrated Na2S2O3 solution, and the reaction mixture
was concentrated by reduced pressure and the residue were filtrated
and washed by water. Following, the residue was mixed with hydro-
xylamine hydrochloride (140.1 mg, 2.0 mmol), K2CO3 (138.5 mg,
1 mmol) and water (1.5 mL) in isopropyl alcohol (3.5 mL) and refluxed
for 6 h. After cooling down, the resulting mixture was concentrated by
reduced pressure and the residue were washed by water and re-
crystallized by acetone-hexane to give compound 28.
4.4.4. Procedure for synthesis of compounds 10a-10c and 21
To a solution of corresponding phenolic compounds (9a-9b and 20)
(2 mmol) and catalytic amount of concentrated sulfuric acid in MeOH
(5 mL) were refluxed for 3 h. And the reaction mixture was con-
centrated by reduced pressure and the residue were recrystallized by
MeOH. Following, the result crystals were mixed with CH2Br2 (174 μL,
2.5 mmol), K2CO3 (276.8 mg, 2 mmol) and DMF (0.5 mL) in ACN
(5 mL) under N2 atmosphere. The mixture was then heated to 85 ℃ and
stirred for 18 h. After cooling down, the resulting mixture was con-
centrated by reduced pressure and purified by silica gel column chro-
matography (hexane:EtOAc from 85:15 to 60:40) to give target com-
pounds 10a-10b and 21.
Following, compound 28 (1 mmol) and pyridine (1 mL) were mixed
in dry ACN (5 mL). And the prepared butyryl chloride was then added
into the mixture and heated to 80 ℃ for 6 h. The resulting mixture was
concentrated by reduced pressure and dissolve into EtOAc (15 mL) then
partitioned by 1 N HCl aqueous solution (3 × 10 mL) and concentrated
NaHCO3 aqueous solution (3 × 10 mL), sequentially. The organic
layers were dried over anhydrous MgSO4, filtered, and concentrated
under reduced pressure. The residue was recrystallized from the solu-
tion of CHCl3-Hex to give target compounds 29b.
The purified compound 10b (1 mmol) was then stirred with MeI
(124.5 μL, 2.0 mmol), K2CO3 (276.8 mg, 2 mmol) and DMF (0.5 mL) in
ACN (5 mL) under inert atmosphere. Following, the mixture was then
heated to 85 ℃ and stirred for 6 h. After cooling down, the resulting
mixture was concentrated by reduced pressure and suspension in ice
water, the precipitates were then filtrated and recrystallized by CHCl3-
Hexane to give compound 10c.
4.4.9. Procedure for synthesis of compounds 31
To a solution of paeonol (30; 336.2 mg, 2 mmol) and DMFDMA
(750 μL, 5.6 mmol) were sealed in the seal flask under N2 atmosphere
and stirred at 110 ℃ for 2 h. After cooling down, the resulting mixture
was washed by cold MeOH then recrystallized from the solution of
CHCl3-Hex to give target compounds 31.
4.4.5. General procedure for synthesis of compounds 11a-11e and 22
Corresponding compounds (10a-10c and 21) (0.8 mmol) were dis-
solved in EtOH (0.5 mL) and NaOH aqueous solution (pH 10.0, 4.5 mL)
was added to the mixture and heated to 75 ℃ under N2 atmosphere for
1.5 h. After cooling down, the pH value of reaction mixture was ad-
justed to 5–6 by 1 N HCl aqueous solution. 10 mL of distillated water
was then added to the resulting mixture and partitioned with EtOAc
(3 × 10 mL). Combined the organic layers, dried over anhydrous
MgSO4, filtered, and concentrated under reduced pressure. Following,
the solution of resulting solid, EDC-HCl (268.4 mg, 1.4 mmol), HOBt
(189.2 mg, 1.4 mmol) and triethylamine (195 μL, 1.4 mmol) in dry THF
(5 mL) were stirred for 15 min at room temperature, and the benzyla-
mines (1 mmol; prepared by the procedure shown in Section 4.4.2) was
then slowly added into the solution. The reaction mixture was stirred
for 8–12 h at room temperature, and monitored by thin-layer chro-
matography. The resulting mixture was concentrated by reduced pres-
sure and dissolve into EtOAc (15 mL) then partitioned by 1 N HCl
aqueous solution (3 × 10 mL) and concentrated NaHCO3 aqueous so-
lution (3 × 10 mL), sequentially. The organic layers were dried over
anhydrous MgSO4, filtered, and concentrated under reduced pressure.
The residue was recrystallized from the solution of CHCl3-Hex to give
target amides 11a-11e and 22.
4.4.10. Procedure for synthesis of compounds 32
To a solution of 31 (221.0 mg, 1 mmol), iodine (308.4 mg,
1.2 mmol) and MeOH (4 mL) were stirred at 60 ℃ for 18 h. The excess
iodine was quenched by concentrated Na2S2O3 solution, and the reac-
tion mixture was concentrated by reduced pressure and the residue
were filtrated and washed by water. The resulting residue was purified
by silica gel column chromatography (CHCl3:MeOH from 100:0 to
75:25) to give target compound 32.
4.4.11. Procedure for synthesis of compounds 33a-33c
To a solution of 32 (151.0 mg, 0.5 mmol), corresponding aryl-
boronic acid (0.6 mmol), K2CO3 (138.2 mg, 1 mmol), PPh3 (15.5 mg,
6 mol%), Pd(OAc)2 (12.0 mg, 5 mol%), distillated water (2 mL) and
ACN (3 mL) were sealed in the seal flask under N2 atmosphere. The
mixture was heated to 100 ℃ and stirred for 24 h. The pH value of
resulting mixture was adjusted to 5–6 by 1 N HCl solution, then filtrated
by a short pad of celite. The resulting mixture was concentrated by
reduced pressure and purified by silica gel column chromatography
(CHCl3:MeOH from 100:0 to 75:25) to give target compounds 33a-33c.
4.5. Chemical properties
4.4.6. Procedure for synthesis of compounds 26a-26b
To a solution of piperonyl aldehyde (2 mmol), corresponding amine
hydrochlorides (3 mmol) and triethylamine (3 mmol) in THF (8 mL)
were stir at 50 ℃ for 4 h. Following, 10% Pd/C was added into the
reaction mixture and stir under H2 atmosphere at room temperature for
12 h. The resulting mixture was concentrated by reduced pressure and
purified by silica gel column chromatography (CHCl3:MeOH from 100:0
to 75:25) to give target amines 26a-26b.
4.5.1. N-(3,4-Methylenedioxy)benzylbenzamide (3a)
White solid; m.p.: 114-115 ℃
IR (KBr): 3305, 3067, 3002, 2975, 2940, 2894, 2846, 2787, 1974,
1853, 1759, 1629, 1604, 1578, 1499, 1440, 1309, 1251, 1229, 1090,
1043, 940, 821, 769, 720, 697, 640 cm−1
1H NMR (400 MHz, CDCl3) data:
δ 7.76–7.74 (m, 2H), 7.46 (tt, J = 7.3, 1.1 Hz, 1H), 7.38 (t,
J = 7.2 Hz, 2H), 6.80 (d, J = 1.3 Hz, 1H), 6.76 (dd, J = 8.0, 1.4 Hz,
1H), 6.72 (d, J = 7.9 Hz, 1H), 6.63 (br. s, 1H, D2O exchangeable), 5.90
(s, 2H), 4.48 (d, J = 5.6 Hz, 2H)
4.4.7. Procedure for synthesis of compounds 27a-27b
Corresponding amines (26a-26b) (1 mmol) and pyridine (1 mL)
were mixed in dry ACN (5 mL) under ice bath. The prepared acyl
chlorides were then added into the mixture and stirred for 24 h at room
temperature. The resulting mixture was concentrated by reduced
pressure and purified by silica gel column chromatography
(CHCl3:MeOH from 100:0 to 50:50) to give target amides 27a-27b.
13C NMR (50 MHz, CDCl3) data:
δ 167.3 (C), 147.9 (C), 146.9 (C), 134.2 (C), 132.0 (C), 131.5 (CH),
128.5 (CH), 126.9 (CH), 121.1 (CH), 108.4 (CH), 108.3 (CH), 101.0
(CH2), 43.8 (CH2)
10

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
HR-ESI-MS: Positive mode: calcd. For C15H13NO3Na (m/z):
101.1 (CH2), 43.8 (CH2)
278.0788; found: 278.0792 ([M + Na]+)
HR-ESI-MS: Positive mode: calcd. For C15H11F2NO3Na (m/z):
314.0599; found: 314.0621 ([M + Na]+)
4.5.2. N-(3,4-Methylenedioxy)benzyl-2-chlorobenzamide (3b)
White solid; m.p.: 102-103 ℃
4.5.6. N-(3,4-Methylenedioxy)benzyl-2-chloro-6-fluorobenzamide (3f)
White solid; m.p.: 114-115 ℃
IR (KBr): 3304, 3074, 3002, 2930, 2893, 2778, 1849, 1644, 1592,
1531, 1504, 1443, 1375, 1309, 1255, 1039, 926, 746 cm−1
1H NMR (400 MHz, CDCl3) data:
IR (KBr): 3260, 3069, 2911, 1848, 1648, 1544, 1489, 1445, 1254,
1040, 928, 899 cm−1
δ 7.62 (dd, J = 7.2, 1.8 Hz, 1H), 7.36 (dd, J = 7.9, 1.5 Hz, 1H, H),
7.32 (td, J = 7.1, 1.8 Hz, 1H), 7.28 (td, J = 7.1, 1.7 Hz, 1H), 6.83 (d,
J = 1.4 Hz, 1H), 6.79 (dd, J = 8.0, 1.4 Hz, 1H), 6.74 (d, J = 7.8 Hz,
1H) 6.51 (br s, 1H, D2O exchangeable), 5.92 (s, 2H), 4.51 (d,
J = 5.6 Hz, 2H)
1H NMR (400 MHz, CDCl3) data:
δ 7.25 (td, J = 8.2, 6.0 Hz, 1H), 7.16 (br. d, J = 8.0 Hz, 2H), 6.99
(td, J = 8.5, 0.9 Hz, 1H), 6.82 (d, J = 1.4 Hz, 1H), 6.77 (dd, J = 7.9,
1.4 Hz, 1H), 6.73 (d, J = 7.9 Hz, 1H), 6.21 (br s, 1H, D2O exchange-
able), 5.91 (s, 2H, O-CH2-O), 4.51 (d, J = 5.7 Hz, 2H)
13C NMR (50 MHz, CDCl3) data:
13C NMR (50 MHz, CDCl3) data:
δ 168.3 (C), 147.9 (C), 147.0 (C), 134.8 (C), 131.4 (C), 131.3 (CH),
130.6 (C), 130.2 (CH), 130.1 (CH), 127.0 (CH), 121.2 (CH), 108.4 (CH),
108.3 (CH), 101.1 (CH2), 44.0 (CH2)
δ 162.2 (C), 159.4 (d, JC-F = 251.5 Hz, C), 147.9 (C), 147.0 (C),
132.3 (d, JC-F = 5.6 Hz, C), 131.1 (d, JC-F = 9.4 Hz, CH), 131.0 (C),
125.5 (d, JC-F = 4.0 Hz, CH), 125.1 (d, JC-F = 21.6 Hz, C), 121.2 (CH),
114.4 (d, JC-F = 22.3 Hz, CH), 108.4 (CH), 108.3 (CH), 101.1 (CH2),
43.8 (CH2)
HR-ESI-MS: Positive mode: calcd. For C15H12ClNO3Na (m/z):
312.0421; found: 312.0398 ([M + Na]+)
HR-ESI-MS: Positive mode: calcd. For C15H11ClFNO3Na (m/z):
330.0304; found: 330.0319 ([M + Na]+)
4.5.3. N-(3,4-Methylenedioxy)benzyl-2-iodobenzamide (3c)
White solid; m.p.: 141–142 ℃
IR (KBr): 3253, 3067, 2992, 2899, 2841, 2779, 1969, 1929, 1848,
1637, 1539, 1503, 1443, 1251, 1036, 932, 806, 728 cm−1
1H NMR (400 MHz, CDCl3) data:
4.5.7. N-(3,4-Methylenedioxy)benzyl-2-chloro-6-methylbenzamide (3 g)
White solid; m.p.: 134–136 ℃
IR (KBr): 3247, 3080, 2877, 2777, 1870, 1639, 1488, 1440, 1248,
1041, 944, 868 cm−1
δ 7.83 (d, J = 7.9 Hz, 1H), 7.39–7.33 (m, 2H), 7.07 (td, J = 7.5,
2.0 Hz, 1H), 6.89 (d, J = 1.2 Hz, 1H), 6.83 (dd, J = 7.9, 1.3 Hz, 1H),
6.76 (d, J = 7.9 Hz, 1H), 5.97 (br s, 1H, D2O exchangeable), 5.94 (s,
2H), 4.52 (d, J = 5.6 Hz, 2H).
1H NMR (400 MHz, CDCl3) data:
δ 7.17–7.15 (m, 2H), 7.07–7.05 (m, 1H), 6.85 (d, J = 1.6 Hz, 1H),
6.80 (dd, J = 7.9, 1.6 Hz, 1H), 6.74 (d, J = 7.9 Hz, 1H), 5.98 (br s, 1H,
D2O exchangeable), 5.93 (s, 2H), 4.53 (d, J = 5.7 Hz, 2H), 2.32 (s, 3H)
13C NMR (50 MHz, CDCl3) data:
13C NMR (50 MHz, CDCl3) data:
δ 169.1 (C), 147.9 (C), 147.1 (C), 141.9 (C), 139.9 (C), 131.3 (C),
131.2 (CH), 128.23 (CH), 128.16 (CH), 121.5 (CH), 108.7 (CH), 108.3
(CH), 101.1 (CH2), 92.4 (C), 44.0 (CH2)
δ 167.1 (C), 147.9 (C), 147.1 (C), 137.1 (C), 136.3 (C), 131.4 (C),
130.5 (CH), 129.8 (CH), 128.5 (CH), 126.7 (CH), 121.4 (CH), 108.6
(CH), 108.3 (CH), 101.1 (CH2), 43.7 (CH2), 19.3 (CH3)
HR-ESI-MS: Positive mode: calcd. For C16H14ClNO3Na (m/z):
326.0544; found: 326.0578 ([M + Na]+)
HR-ESI-MS: Positive mode: calcd. For
403.9730; found: 403.9766 ([M + Na]+)
C15H12INO3Na (m/z):
4.5.4. N-(3,4-Methylenedioxy)benzyl-2-methylbenzamide (3d)
White solid; m.p.: 114-115 ℃
4.5.8. N-(3,4-Methylenedioxy)benzyl-2,6-dichlorobenzamide (Alda-1, 3 h)
White solid; m.p.: 175–176 ℃
IR (KBr): 3281, 3066, 3021, 2921, 2878, 2789, 1964, 1843, 1732,
1627, 1521, 1503, 1487, 1252, 1039, 933, 809, 741 cm−1
1H NMR (400 MHz, CDCl3) data:
IR (KBr): 3258, 3072, 2930, 2899, 2871, 1850, 1641, 1549, 1486,
1430, 1248, 1217, 1039, 932, 789, 694 cm−1
δ 7.31 (d, J = 7.6 Hz, 1H), 7.27 (td, J = 7.6, 1.1 Hz, 1H), 7.19–7.13
(m, 2H), 6.81 (d, J = 1.2 Hz, 1H), 6.77 (dd, J = 8.0, 1.2 Hz, 1H), 6.74
(d, J = 7.9 Hz, 1H), 6.11 (br s, 1H, D2O exchangeable), 5.92 (s, 2H),
4.47 (d, J = 5.8 Hz, 2H), 2.41 (s, 3H)
1H NMR (400 MHz, CDCl3) data:
δ 7.31–7.29 (m, 2H), 7.25–7.21 (m, 1H), 6.89 (d, J = 1.3 Hz, 1H),
6.83 (dd, J = 7.9, 1.3 Hz, 1H), 6.75 (d, J = 7.9 Hz, 1H), 5.94 (s, 2H),
5.93 (br s, 1H, D2O exchangeable), 4.57 (d, J = 5.6 Hz, 2H)
13C NMR (100 MHz, CDCl3) data:
13C NMR (50 MHz, CDCl3) data:
δ 169.8 (C), 147.9 (C), 147.0 (C), 136.1 (C × 2), 132.0 (C), 131.0
(CH), 129.9 (CH), 126.6 (CH), 125.7 (CH), 121.1 (CH), 108.35 (CH),
108.30 (CH), 101.0 (CH2), 43.6 (CH2), 19.8 (CH3)
δ 164.2 (C), 147.9 (C), 147.1 (C), 135.8 (C), 132.3 (C), 131.0 (C),
130.7 (CH), 128.1 (CH × 2), 121.4 (CH), 113.8 (C), 108.6 (CH), 108.3
(CH), 101.1 (CH2), 43.9 (CH2)
HR-ESI-MS: Positive mode: calcd. For
292.0944; found: 292.0966 ([M + Na]+)
C
16H15NO3Na (m/z):
HR-ESI-MS: Positive mode: calcd. For C15H11Cl2NO3Na (m/z):
346.0008; found: 346.0024 ([M + Na]+)
4.5.5. N-(3,4-Methylenedioxy)benzyl-2,6-difluorobenzamide (3e)
White solid; m.p.: 127–128 ℃
4.5.9. N-(3,4-Methylenedioxy)benzyl-2-hydroxybenzamide (3i)
White solid; m.p.: 125–126 ℃
IR (KBr): 3364, 3073, 3041, 3002, 2972, 2940, 2906, 2790, 1954,
1858, 1652, 1524, 1491, 1459, 1250, 1038, 999, 975, 926, 799, 776,
646 cm−1
IR (KBr): 3365, 2923, 2853, 2786, 1851, 1726, 1637, 1590, 1547,
1499, 1484, 1442, 1378, 1353, 1303, 1253, 1231, 1192, 1148, 1122,
1078, 1042, 957, 939, 923, 867 cm−1
1H NMR (400 MHz, CDCl3) data:
1H NMR (200 MHz, CDCl3) data:
δ 7.34 (tt, J = 8.4, 6.4 Hz, 1H), 6.92 (t-like, J = 8.2 Hz, 1H), 6.84
(d, J = 1.3 Hz, 1H), 6.79 (dd, J = 7.9, 1.3 Hz, 1H), 6.75 (d, J = 7.9 Hz,
1H), 6.19 (br s, 1H, D2O exchangeable), 5.93 (s, 2H), 4.54 (d,
J = 5.7 Hz, 2H)
δ 12.33 (br s, 1H, D2O exchangeable), 7.39 (dd, J = 8.6, 1.6 Hz,
1H), 7.33 (ddd, J = 9.7, 8.4, 1.4 Hz, 1H), 6.98 (dd, J = 8.4, 1.2 Hz,
1H), 6.84–6.80 (m, 1H), 6.82–6.78 (m, 1H), 6.78 (dd, J = 8.6, 1.3 Hz,
1H), 6.77 (d, J = 1.2 Hz, 1H) 6.50 (br s, 1H, D2O exchangeable), 5.94
(s, 2H), 4.51 (d, J = 5.6 Hz, 2H)
13C NMR (50 MHz, CDCl3) data:
δ 160.2 (C), 159.9 (dd, JC-F = 250.7, 6.5 Hz, C), 147.9 (C), 147.1
(C), 131.7 (t, JC-F = 10.4 Hz, C), 131.2 (C), 121.1 (CH), 114.1 (t, JC-
F = 20.0 Hz, CH), 112.0 (dd, JC-F = 22.7, 3.2 Hz, CH), 108.3 (CH × 2),
13C NMR (50 MHz, CDCl3) data:
δ 169.7 (C), 161.6 (C), 148.0 (C), 147.3 (C), 134.3 (CH), 133.3 (C),
131.7 (C), 131.1 (CH), 125.3 (CH), 121.3 (CH), 118.6 (CH), 114.0 (C),
11

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
108.4 (CH), 101.2 (CH2), 43.5 (CH2)
HR-ESI-MS: Positive mode: calcd. For
294.0737; found: 294.0781 ([M + Na]+)
IR (KBr): 3274, 3091, 2955, 2924, 2867, 1667, 1644, 1586, 1557,
1454, 1430, 1385, 1341, 1298, 1192, 1172, 803, 786, 737, 695 cm−1
1H NMR (200 MHz, CDCl3) data:
C
15H13NO4Na (m/z):
δ 7.29–7.16 (m, 3H), 5.80 (br s, 1H, D2O exchangeable), 3.47 (dt,
J = 7.3, 6.0 Hz, 2H), 1.68 (m, H-3′), 1.48 (q, J = 7.3 Hz, 2H), 0.93 (d,
J = 6.5 Hz, 6H)
4.5.10. N-(3,4-Methylenedioxy)benzyl-2,6-dihydroxybenzamide (3j)
White solid; m.p.: 104–105 ℃
IR (KBr): 3603, 3365, 3121, 2903, 1851, 1641, 1589, 1555, 1502,
1447, 1346, 1254, 1152, 1123, 1100, 1076, 1039, 1010, 979, 924, 863,
845, 806, 778, 762, 715, 637 cm−1
13C NMR (50 MHz, CDCl3) data:
δ 164.4 (C), 136.2 (C), 132.2 (C), 130.5 (CH) 128.0 (CH), 38.2
(CH2), 38.0 (CH2), 25.7 (CH), 22.4 (CH3),
1H NMR (200 MHz, Methanol‑d4) data:
HR-ESI-MS: Positive mode: calcd. For C12H15NOCl2Na (m/z):
282.0423; found: 282.0440 ([M + Na]+)
δ 7.13 (t, J = 8.3 Hz, 1H), 6.84 (d, J = 1.4 Hz, 1H), 6.82 (dd,
J = 8.5, 1.4 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.35 (d, J = 8.3 Hz, 2H),
5.91 (s, 2H), 4.48 (s, 2H)
4.5.15. N-(Thiophen-2-ylmethyl)-2-chlorobenzamide (6e)
Brown solid; m.p.: 84–85 ℃
13C NMR (50 MHz, Methanol‑d4) data:
δ 171.7 (C), 161.6 (C), 149.3 (C), 148.2 (C), 134.4 (CH), 133.5 (C),
121.8 (CH), 109.1 (CH), 109.0 (CH), 108.4 (CH), 103.9 (C), 102.3 (C),
102.2 (CH2), 43.6 (CH2)
IR (KBr): 3275, 3049, 1640, 1592, 1529, 1468, 1450, 1430, 1367,
1294, 1258, 1158, 1126, 1061, 1036, 846, 833, 747, 703, 687 cm−1
1H NMR (400 MHz, CDCl3) data:
HR-ESI-MS: Positive mode: calcd. For C15H13NO5Na (m/z):
310.0686; found: 310.0672 ([M + Na]+)
δ 7.60 (dd, J = 7.2, 1.6 Hz, 1H), 7.39–7.22 (m, 4H), 7.04 (d,
J = 3.2 Hz, 1H), 6.95 (dd, J = 5.0, 3.6 Hz, 1H), 6.55 (br s, 1H, D2O
exchangeable), 4.81 (d, J = 5.5 Hz, 2H)
4.5.11. N-(3,4-Methylenedioxy)benzyl-2-bromobenzamide (3 k)
White solid; m.p.: 98–99 ℃
13C NMR (100 MHz, CDCl3) data:
δ 166.1 (C), 140.1 (C), 134.6 (C), 131.5 (CH), 130.7 (C), 130.27
(CH), 130.25 (CH), 127.1 (CH), 126.9 (CH), 126.3 (CH), 125.4 (CH),
38.9 (CH2)
IR (KBr): 3273, 3068, 2884, 2786, 1645, 1534, 1489, 1444, 1377,
1303, 1251, 1223, 1097, 1041, 928, 819 cm−1
1H NMR (400 MHz, CDCl3) data:
HR-ESI-MS: Positive mode: calcd. For C12H10NOSClNa (m/z):
274.0064; found: 274.0058 ([M + Na]+)
δ 7.58–7.26 (m, 4H), 6.88–6.75 (m, 3H), 6.32 (br s, 1H, D2O ex-
changeable), 5.95 (s, 2H), 4.53 (d, J = 5.6 Hz, 2H)
13C NMR (100 MHz, CDCl3) data:
4.5.16. N-(2-chloro)benzyl-2,6-dichlorobenzamide (6f)
Orange solid; m.p.: 155–156 ℃
δ 167.4 (C), 147.9 (C), 147.0 (C), 137.5 (C), 133.3 (CH), 131.4 (C),
131.2 (CH), 129.5 (CH), 127.5 (CH), 121.3 (CH), 119.2 (C), 108.5 (CH),
108.3 (CH), 101.0 (CH2), 41.0 (CH2)
IR (KBr): 3398, 1656, 1623, 1511, 1430, 1409, 1359, 1290, 1247,
1193, 1035, 986, 801, 758, 565 cm−1
HR-ESI-MS: Positive mode: calcd. For C15H12BrNO3Na (m/z):
355.9893; found: 355.9900 ([M + Na]+)
1H NMR (400 MHz, CDCl3) data:
δ 7.60–7.58 (m, 1H), 7.41–7.24 (m, 6H), 6.21 (br. s, 1H, D2O ex-
changeable), 4.78 (d, J = 6.0, 2H)
4.5.12. N-Benzyl-2,6-dichlorobenzamide (6a)
13C NMR (100 MHz, CDCl3) data:
White crystal; 157–158 ℃
δ 164.5 (C), 135.7 (C), 134.8 (C), 133.6 (C), 132.3 (CH), 130.7
(CH), 130.5 (C), 129.5 (CH), 129.2 (C), 128.1 (CH), 127.2 (CH), 41.9
(CH2)
IR (KBr): 3269, 3065, 3030, 2980, 2922, 2875, 1966, 1937, 1878,
1861, 1805, 1786, 1672, 1652, 1604, 1588, 1578, 1548, 1497, 1454,
1431, 1357, 1333, 1292, 1220, 1194, 1168, 1154, 1088, 1079, 1043,
1030, 957, 916, 800, 776, 761, 734, 698 cm−1
1H NMR (400 MHz, CDCl3) data:
HR-ESI-MS: Positive mode: calcd. For C14H10NOCl3Na (m/z):
335.9720; found: 335.9706 ([M + Na]+)
δ 7.38–7.19 (m, 8H), 6.5 (br s, 1H, D2O exchangeable), 4.64 (d,
J = 5.7 Hz, 2H)
4.5.17. N-(3-Methoxy-4-hydroxy)benzyl-2-chlorobenzamide (6 g)
White solid; m.p.: 120–122 ℃
13C NMR (100 MHz, CDCl3) data:
IR (KBr): 3357, 3230, 2996, 2834, 1628, 1603, 1573, 1549, 1524,
1492, 1451, 1363, 1291, 1257, 1213, 1158, 1125, 1042, 987, 848, 813,
719 cm−1
δ 164.3 (C), 137.3 (C), 135.8 (C), 132.3 (C), 130.6 (CH), 128.7
(CH), 128.0 (CH), 128.0 (CH), 127.7 (CH), 44.0 (CH2)
HR-ESI-MS: Positive mode: calcd. For C14H11NOCl2Na (m/z):
302.0110; found: 302.0123 ([M + Na]+)
1H NMR (400 MHz, Methanol‑d4) data:
δ 7.84–7.81 (m, 1H), 7.52–7.42 (m, 3H), 6.94 (d, J = 1.8, 1H),
6.81–6.73 (m, 2H), 4.47 (s, 2H), 3.82 (s, 3H)
4.5.13. N-((4-Dimethylamino)benzyl)-2-chlorobenzamide (6c)
White solid; 122-123 ℃
13C NMR (100 MHz, Methanol‑d4) data:
δ 170.1 (C), 149.0 (C), 146.8 (C), 135.7 (C), 132.7 (CH), 131.3 (C),
129.6 (C), 129.6 (CH), 128.3 (CH), 121.4 (CH), 116.1 (CH), 112.5 (CH),
56.3 (CH3), 44.4 (CH2)
IR (KBr): 3262, 3066, 2914, 2875, 2793, 1960, 1925, 1889, 1859,
1812, 1620, 1593, 1524, 1435, 1349, 1312, 1188, 1128, 1058, 946,
792, 750, 725, 689, 580 cm−1
HR-ESI-MS: Positive mode: calcd. For C15H14NO3ClNa (m/z):
314.0554; found: 314.0529 ([M + Na]+)
1H NMR (400 MHz, CDCl3) data:
δ 7.65 (dd, J = 7.2, 2.0 Hz, 1H), 7.38–7.26 (m, 5H), 6.84 (br s, 2H),
6.36 (br s, 1H, D2O exchangeable), 4.55 (d, J = 5.4 Hz, 2H), 2.95 (s,
6H)
4.5.18. N-(3,4-Methylenedioxy)benzyl-3,3-dimethylacrylamide (8a)
White solid; m.p.: 57–58 ℃
13C NMR (100 MHz, CDCl3) data:
IR (KBr): 3278, 3168, 3063, 2973, 2939, 2907, 2795, 1848, 1671,
1624, 1484, 1493, 1361, 1281, 1250, 1229, 1179, 1102, 1040, 1016,
940, 924, 855, 818, 807, 672 cm−1
δ 166.3 (C), 135.2 (C), 131.3 (C), 130.23 (C), 130.15 (CH), 129.2
(CH), 127.1 (CH), 43.9 (CH2), 41.0 (CH3)
HR-ESI-MS: Positive mode: calcd. For C16H17N2OClNa (m/z):
311.0922; found: 311.0918 ([M + Na]+)
1H NMR (200 MHz, CDCl3) data:
δ 6.90–6.86 (m, 2H), 6.86 (s, 1H, H-2′), 6.05 (s, 2H), 5.92 (br s, 1H,
D2O exchangeable), 5.69 (m, H-2), 4.47 (d, J = 5.2 Hz, 2H), 2.29 (d,
J = 1.2 Hz, 3H), 1.95 (d, J = 1.2 Hz, 3H, 3-Me)
13C NMR (100 MHz, CDCl3) data:
4.5.14. N-Isoamyl-2,6-dichlorobenzamide (6d)
White needle; m.p.: 112–113 ℃
12

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
δ 166.7 (C), 151.1 (C), 147.6 (C), 146.6 (C), 132.5 (C), 120.8 (CH),
118.2 (CH), 108.2 (CH), 108.0 (CH), 100.8 (CH2), 42.8 (CH2), 27.0
(CH3), 19.7 (CH3)
IR (KBr): 3338, 3279, 3075, 2996, 2944, 2910, 2786, 2478, 2416,
1926, 1865, 1761, 1635, 1624, 1603, 1550, 1502, 1457, 1419, 1358,
1260, 1061, 1034, 981, 883, 821, 787, 760, 490 cm−1
1H NMR (400 MHz, CDCl3) data:
HR-ESI-MS: Positive mode: calcd. For
256.0944; found: 256.0973 ([M + Na]+)
C13H15NO3Na (m/z):
δ 7.27–7.20 (m, 3H), 6.88 (t, J = 7.9 Hz, 2H), 6.77 (d, J = 8.0 Hz,
1H), 6.44 (br. s, 1H, D2O exchangeable), 5.98 (s, 2H), 4.69 (d,
J = 5.6 Hz, 2H)
4.5.19. N-(3,4-Methylenedioxy)benzyl-2-cyclopentylacetamide (8b)
White solid; m.p.: 110–112 ℃
13C NMR (100 MHz, CDCl3) data:
IR (KBr): 3248, 3071, 2959, 1649, 1629, 1556, 1505, 1444, 1252,
1212, 1039, 926, 811, 645 cm−1
δ 166.3 (C), 161.5 (dd, JC-F = 247.4, 8.0, C), 150.3 (C), 147.9 (C),
129.5 (t, JC-F = 10.3, CH), 128.3 (CH), 121.6 (CH), 113.8 (t, JC-
F = 19.1 Hz, C), 111.4 (dd, JC-F = 18.8, 6.6 Hz, CH), 107.9 (CH), 107.7
(CH), 101.6 (CH2), 32.0 (t, JC-F = 3.7 Hz, CH2)
1H NMR (400 MHz, CDCl3) data:
δ 6.73–6.70 (m, 3H), 5.91 (s, 2H), 5.79 (br s, 1H, D2O exchange-
able), 4.31 (d, J = 5.6 Hz, 2H), 2.22–2.17 (m, 3H), 1.86–1.76 (m, 2H),
1.62–1.49 (m, 4H), 1.17–1.07 (m, 2H)
HR-ESI-MS: Positive mode: calcd. For C15H11NO3F2Na (m/z):
314.0599; found: 314.0624 ([M + Na]+)
13C NMR (100 MHz, CDCl3) data:
δ 172.8 (C), 148.0 (C), 147.0 (C), 132.3 (C), 121.1 (CH), 108.5
(CH), 108.4 (CH), 101.1 (CH2), 43.5 (CH2), 43.1 (CH2), 37.2 (CH), 32.6
(CH2), 25.0 (CH2)
4.5.24. N-(2,6-Dichloro)benzyl-3,4-methylenedioxy-5-hydroxybenzamide
(11d)
White solid; m.p.: 163 ℃ decomposed
HR-ESI-MS: Positive mode: calcd. For
284.1257; found: 284.1251 ([M + Na]+)
C
15H19NO3Na (m/z):
IR (KBr): 3444, 3287, 1937, 1781, 1552, 1459, 1437, 1405, 1390,
1309, 1260, 1220, 1177, 1111, 1074, 1043, 1025, 929, 778, 715,
649 cm−1
4.5.20. N-(3,4-Methylenedioxy)benzylbutyramide (8c)
Pale yellow solid; m.p.: 58–60 ℃
1H NMR (400 MHz, Methaol-d4) data:
δ 7.41–7.26 (m, 3H), 6.87 (d, J = 1.6 Hz, 1H), 6.73 (d, J = 1.7 Hz,
1H), 5.91 (s, 2H), 4.79 (s, 2H)
IR (KBr): 3248, 3071, 2959, 1649, 1629, 1556, 1505, 1444, 1252,
1212, 1039, 926, 811, 645 cm−1
13C NMR (100 MHz, Methaol-d4) data:
1H NMR (400 MHz, Methanol‑d4) data:
δ 170.2 (C), 150.0 (C), 145.4 (C), 139.4 (C), 137.6 (C), 134.4 (C),
131.0 (CH), 129.7 (CH), 129.3 (C), 114.1 (CH), 102.5 (CH2), 99.3 (CH),
41.0 (CH2)
δ 6.76–6.73 (m, 3H), 5.90 (s, 3H), 4.24 (s, 2H), 2.18 (t, J = 7.2,
2H), 1.63 (sextet, J = 7.5H-2), 0.93 (t, J = 7.4 Hz, 3H)
13C NMR (100 MHz, Methanol‑d4) data:
HR-ESI-MS: Positive mode: calcd. For C15H11NO4Cl2Na (m/z):
361.9957; found: 361.9970 ([M + Na]+)
δ 174.5 (C), 147.9 (C), 146.9 (C), 132.7 (C), 120.6 (CH), 107.8
(CH), 107.7 (CH), 101.0(CH2), 42.5 (CH2), 37.7 (CH2), 19.1 (CH2), 12.6
(CH3)
4.5.25. N-(2,6-Dichloro)benzyl-3,4-methylenedioxy-5-methoxybenzamide
(11e)
HR-ESI-MS: Positive mode: calcd. For C12H15NO3Na (m/z):
244.0944; found: 244.0936 ([M + Na]+)
White solid; m.p.: 178–179 ℃
IR (KBr): 3259, 3082, 2940, 2896, 1719, 1620, 1547, 1505, 1462,
1437, 1426, 1366, 1320, 1236, 1192, 1120, 1091, 1038, 925, 862, 779,
767, 664 cm−1
4.5.21. N-(2,6-Dichloro)benzyl-(3,4-methylenedioxy)benzamide (11a)
White solid; m.p.: 172–173 ℃
IR (KBr): 3317, 3092, 3068, 3002, 2963, 2913, 2464, 2414, 1942,
1870, 1789, 1744, 1635, 1614, 1504, 1437, 1421, 1350, 1250, 1039,
930, 874, 764 cm−1
1H NMR (400 MHz, CDCl3) data:
δ 7.37–7.20 (m, 3H), 7.15 (d, J = 1.5 Hz, 1H), 6.86 (d, J = 1.5 Hz,
1H), 6.38 (br s, 1H, D2O exchangeable), 6.04 (s, 2H), 4.94 (d,
J = 5.5 Hz, 2H), 3.93 (s, 3H)
1H NMR (200 MHz, CDCl3) data:
δ 7.38–7.14 (m, 5H), 6.78 (d, J = 8.7 Hz, 1H), 6.30 (br. s, 1H, D2O
exchangeable), 5.99 (s, 2H), 4.91 (d, J = 5.4 Hz, 2H)
13C NMR (50 MHz, CDCl3) data:
13C NMR (100 MHz, CDCl3) data:
δ 166.2 (C), 149.3 (C), 148.7 (C), 143.5 (C), 138.1 (C), 136.2 (C),
133.4 (C), 129.6 (CH), 128.5 (CH), 108.1 (CH), 102.1 (CH2), 100.7
(CH), 56.6 (CH3), 39.8 (CH2)
δ 166.3 (C), 150.3 (C), 147.9 (C), 136.2 (C × 2), 133.5 (C), 129.6
(CH), 128.5 (CH × 2), 128.3 (C), 121.6 (CH), 107.9 (CH), 107.7 (CH),
101.6 (CH2), 39.8 (CH2)
HR-ESI-MS: Positive mode: calcd. For C16H13NO4Cl2Na (m/z):
376.0114; found: 376.0118 ([M + Na]+)
HR-ESI-MS: Positive mode: calcd. For C15H11NO3Cl2Na (m/z):
346.0008; found: 346.0022 ([M + Na]+)
4.5.26. N-(2,6-Dichloro)benzyl- 3,4-methylenedioxycinnamic amide (22)
White solid; m.p.: 157–158 ℃
4.5.22. N-2-Chlorobenzyl-(3,4-methylenedioxy)benzamide (11b)
White solid; m.p.: 109-110 ℃
IR (KBr): 3274, 2923, 2851, 1647, 1614, 1533, 1497, 1444, 1376,
1245, 1190, 1102, 1040, 1020, 969, 935, 848, 813, 783, 766 cm−1
1H NMR (200 MHz, CDCl3) data:
IR (KBr): 3304, 3065, 2895, 2789, 2456, 2364, 1845, 1634, 1617,
1603, 1546, 1458, 1354, 1308, 1259, 1040, 937, 750, 600, 522 cm−1
1H NMR (400 MHz, CDCl3) data:
δ 7.57 (d, J = 15.5 Hz, 1H), 7.36–7.14 (m, 3H), 6.98 (d, J = 1.9 Hz,
1H), 6.96 (dd, J = 8.5, 1.5 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.20 (d,
J = 15.5 Hz, 1H), 5.98 (s, 2H), 4.88 (d, J = 5.5, 2H)
13C NMR (50 MHz, CDCl3) data:
δ 7.42–7.15 (m, 6H), 6.77 (d, J = 7.9 Hz, 1H), 6.65 (br. s, 1H, D2O
exchangeable), 5.98 (s, 2H), 4.64 (d, J = 5.6 Hz, 2H)
13C NMR (50 MHz, CDCl3) data:
δ 165.5 (C), 149.1 (C), 148.2 (C), 135.1 (C), 134.9 (C), 133.5 (C),
131.2 (C), 129.6 (CH), 128.5 (CH), 124.4 (CH), 124. (CH), 124.2 (CH),
118.0 (CH), 108.5 (CH), 106.3 (CH), 101.4 (CH2), 40.1 (CH2)
HR-ESI-MS: Positive mode: calcd. For C17H13NO3Cl2Na (m/z):
372.0165; found: 372.0178 ([M + Na]+)
δ 166.7 (C), 150.4 (C), 147.9 (C), 135.5 (C), 133.6 (C), 130.2 (CH),
129.5 (CH), 128.9 (CH), 128.3 (C), 127.1 (CH), 121.6 (CH), 107.9 (CH),
107.6 (CH), 101.7 (CH2), 42.0 (CH2)
HR-ESI-MS: Positive mode: calcd. For C15H12NO3ClNa (m/z):
312.0398; found: 312.0414 ([M + Na]+)
4.5.27. N-Piperonyl-N-methyl-2-chlorobenzamide (27a)
Yellow oil
4.5.23. N-(2,6-Difluoro)benzyl-(3,4-methylenedioxy)benzamide (11c)
White solid; m.p.: 129–130 ℃
IR (KBr): 1735, 1638, 1502, 1490, 1444, 1402, 1292, 1244, 1188,
13

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
1085, 1039, 928, 770, 748 cm−1
1H NMR (400 MHz, CDCl3) data:
1H NMR (400 MHz, CDCl3) data:
δ 9.37 (br s, 1H, D2O exchangeable), 8.11 (d, J = 8.9 Hz, 1H), 8.01
(s, 1H), 7.41 (d, J = 8.4 Hz, 2H), 7.02 (dd, J = 8.9 Hz, 1.2, 1H), 7.01
(d, J = 1.2 Hz, 1H), 6.87 (d, J = 8.4 Hz, 2H), 3.95 (s, 3H)
13C NMR (100 MHz, CDCl3) data:
Form A: δ 7.34–7.20 (m, 4H), 6.85–6.52 (m, 3H), 5.88 (s, 2H),
4.77–4.45 (m, 2H), 2.65 (s, 3H)
Form B: δ 7.34–7.20 (m, 4H), 6.85–6.52 (m, 3H), 5.87 (s, 2H),
4.30–4.10 (m, 2H), 2.94 (s, 3H)
δ 174.3 (C), 163.0 (C), 156.7 (C), 156.6 (CH), 151.0 (C), 128.7
(CH), 125.8 (CH), 123.2 (CH), 121.3 (CH), 116.5 (C), 113.8 (CH), 98.9
(CH), 54.4 (CH3)
13C NMR (100 MHz, CDCl3) data:
δ 168.60 (C), 168.56 (C), 148.1 (C), 148.0 (C), 147.1 (C), 136.2 (C),
136.0 (C), 130.5 (CH), 130.4 (CH), 130.2 (CH), 130.1 (CH), 129.8 (CH),
129.7 (CH), 129.65 (C), 128.0 (CH), 127.7 (CH), 127.23 (CH), 127.20
(CH), 121.8 (C), 120.8 (C), 108.8 (C), 108.4 (C), 108.2 (CH), 107.6
(CH), 101.2 (CH2), 101.1 (CH2), 54.2 (CH2), 50.1 (CH2), 35.2 (CH3),
32.1 (CH3)
HR-ESI-MS: Positive mode: calcd. For C16H12O4Na (m/z): 291.0628;
found: 291.0630 ([M + Na]+)
4.5.32. 3-(4-Pyridinyl)-7-methoxychromone (33c)
Yellow solid; m.p.: 152–153 ℃
HR-ESI-MS: Positive mode: calcd. For C16H14NO3ClNa (m/z):
326.0544; found: 326.0540 ([M + Na]+)
IR (KBr): 3219, 3054, 2920, 2851, 1638, 1600, 1506, 1445, 1382,
1322, 1271, 1098, 1053, 942, 892, 824, 796, 779, 696, 540 cm−1
1H NMR (400 MHz, CDCl3) data:
4.5.28. N-Piperonyl-N-isopropyl-2-chlorobenzamide (27b)
Yellow solid; m.p.: 132–134 ℃
δ 8.66 (dd, J = 4.5, 1.6 Hz, 2H), 8.20 (d, J = 8.9 Hz, 1H), 8.07 (s,
1H), 7.63 (dd, J = 4.6, 1.6 Hz, 2H), 7.02 (dd, J = 8.9, 2.3 Hz, 1H), 6.88
(d, J = 2.3 Hz, 1H), 3.92 (s, 3H)
IR (KBr): 1631, 1571, 1502, 1490, 1390, 1258, 1244, 1148, 1048,
941, 808, 760, 647 cm−1
13C NMR (50 MHz, CDCl3) data:
1H NMR (400 MHz, CDCl3) data:
δ 174.7 (C), 164.4 (C), 157.8 (C), 153.5 (CH), 150.6 (C), 149.6
(CH), 127.8 (CH), 123.3 (CH), 121.4 (CH), 118.2 (C), 115.1 (CH), 100.2
(CH), 55.9 (CH3)
δ 7.53–7.52 (m, 1H), 7.33–7.30 (m, 1H), 7.21–7.18 (m, 2H), 7.03 (s,
1H), 6.89 (dd, J = 7.9, 0.9 Hz, 1H), 6.64 (d, J = 7.9 Hz, 1H), 5.83 (s,
2H), 3.84 (s, 2H), 3.13, (sept, J = 6.4 Hz, 1H), 1.22 (d, J = 6.2 Hz, 6H)
13C NMR (100 MHz, CDCl3) data:
HR-ESI-MS: Positive mode: calcd. For C15H12NO3 (m/z): 254.0812;
found: 254.0828 ([M + H]+) calcd. For C15H11NO3Na (m/z):
276.0631; found: 276.0663 ([M + Na]+).
δ 172.2 (C), 147.9 (C), 147.8 (C), 139.3(C), 130.8 (C), 129.9 (CH),
129.2 (CH), 126.4 (CH), 125.2 (CH), 124.2 (CH), 110.7 (CH), 108.3
(CH), 101.2 (CH2), 48.4 (CH2), 47.8 (CH), 18.9 (CH3)
HR-ESI-MS: Positive mode: calcd. For C18H18NO3ClNa (m/z):
354.0867; found: 354.0870 ([M + Na]+)
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
4.5.29. 3-(3,4-Methylenedioxy)phenyl-5-propyl-1,2,4-oxadiazole (29b)
White solid; m.p.: 39–40 ℃
IR (KBr): 2966, 1868, 1800, 1729, 1631, 1579, 1531, 1453, 1389,
1334, 1258, 1238, 1164, 1112, 1038, 937, 880, 821, 745 cm−1
1H NMR (400 MHz, CDCl3) data:
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104166.
δ 7.60 (dd, J = 8.1, 1.6 Hz, 1H), 7.49 (d, J = 1.6 Hz, 1H), 6.87 (d,
J = 8.1 Hz, 1H), 6.00 (s, 2H), 2.87 (t, J = 7.4 Hz, 2H), 1.87 (sextet,
J = 7.4 Hz, 2H), 1.03 (t, J = 7.4 Hz, 3H)
References
13C NMR (100 MHz, CDCl3) data:
δ 179.6 (C), 167.9 (C), 150.0 (C), 148.0 (C), 122.2 (CH), 120.8 (C),
108.6 (CH), 107.4 (CH), 101.5 (CH2), 28.4 (CH2), 20.2 (CH2), 13.6
(CH3)
[1] M.Y. Eng, S.E. Luczak, T.L. Wall, Alcohol Res. Health 30 (2007) 22–27.
[2] V. Bagnardi, M. Rota, E. Botteri, I. Tramacere, F. Islami, V. Fedirko, L. Scotti,
M. Jenab, F. Turati, E. Pasquali, Br. J. Cancer 112 (2015) 580–593.
[3] M.-F. Wang, C.-L. Han, S.-J. Yin, Chem. Biol. Interact. 178 (2009) 36–39.
[4] A.I. Cederbaum, Clin. Liver Dis. 16 (2012) 667–685.
HR-ESI-MS: Positive mode: calcd. For
255.0740; found: 255.0721 ([M + Na]+)
C12H12N2O3Na (m/z):
[5] C.H. Chen, J.C. Ferreira, E.R. Gross, D. Mochly-Rosen, Physiol. Rev. 94 (2014)
1–34.
[6] Li, H.; Borinskaya, S.; Yoshimura, K.; Kal’ina, N.; Marusin, A.; Stepanov, V. A.; Qin,
Z.; Khaliq, S.; Lee, M. Y.; Yang, Y. Ann. Hum. Genet. 73 (2009) 335–345.
[7] C.H. Chen, G.R. Budas, E.N. Churchill, M.H. Disatnik, T.D. Hurley, D. Mochly-
Rosen, Science 321 (2008) 1493–1495.
4.5.30. 7-Methoxyisoflavone (33a)
White solid; m.p.: 137-138 ℃
IR (KBr): 3074, 3021, 2924, 1633, 1600, 1567, 1500, 1483, 1367,
1329, 1290, 1204, 1098, 1052, 1027, 968, 941, 884, 823, 787, 722,
662, 529 cm−1
[8] R.-L. Yu, C.-H. Tan, Y.-C. Lu, R.-M. Wu, Sci. Rep. 6 (2016) 30424.
[9] J.S. Chang, J.-R. Hsiao, C.-H. Chen, J. Biomed. Sci. 24 (2017) 19.
[10] E.R. Gross, V.O. Zambelli, B.A. Small, J.C. Ferreira, C.H. Chen, D. Mochly-Rosen,
Annu. Rev. Pharmacol. Toxicol. 55 (2015) 107–127.
1H NMR (400 MHz, CDCl3) data:
[11] S. Perez-Miller, H. Younus, R. Vanam, C.-H. Chen, D. Mochly-Rosen, T.D. Hurley,
Nat. Struct. Mol. Biol. 17 (2010) 159–164.
δ 8.19 (d, J = 8.9 Hz, 1H), 7.92 (s, 1H), 7.53 (dd, J = 8.3, 1.3 Hz,
2H), 7.43–7.34 (m, 3H), 6.97 (dd, J = 8.9, 2.2 Hz, 1H), 6.83 (d,
J = 2.2 Hz, 1H), 3.88 (s, 3H)
[12] H.N. Larson, J. Zhou, Z. Chen, J.S. Stamler, H. Weiner, T.D. Hurley, J. Biol. Chem.
282 (2007) 12940–12950.
13C NMR (100 MHz, CDCl3) data:
[13] H.N. Larson, H. Weiner, T.D. Hurley, J. Biol. Chem. 280 (2005) 30550–30556.
[14] C.G. Steinmetz, P. Xie, H. Weiner, T.D. Hurley, Structure 5 (1997) 701–711.
[15] J.-M. Guo, A.-J. Liu, P. Zang, W.-Z. Dong, L. Ying, W. Wang, P. Xu, X.-R. Song,
J. Cai, S.-Q. Zhang, J.-L. Duan, J.L. Mehta, D.-F. Su, Cell Res. 23 (2013) 915–930.
[16] K. Matsuo, I. Oze, S. Hosono, H. Ito, M. Watanabe, K. Ishioka, S. Ito, M. Tajika,
Y. Yatabe, Y. Niwa, K. Yamao, S. Nakamura, K. Tajima, H. Tanaka, Carcinogenesis
34 (2013) 1510–1515.
δ 175.7 (C), 164.1 (C), 159.0 (C), 152.7 (CH), 132.0 (C), 129.1
(CH), 128.5 (CH), 128.2 (CH), 127.9 (CH), 125.3 (C), 118.5 (C), 114.7
(CH), 100.2 (CH), 55.9 (CH3)
HR-ESI-MS: Positive mode: calcd. For C16H12O3Na (m/z): 275.0679;
found: 275.0664 ([M + Na]+)
[17] W. Wang, L.-L. Lin, J.-M. Guo, Y.-Q. Cheng, J. Qian, J.L. Mehta, D.-F. Su, P. Luan,
A.-J. Liu, Int. J. Stroke 10 (2015) 1261–1269.
[18] T. Zhang, Q. Zhao, F. Ye, C. Huang, W.-M. Chen, W.-Q. Huang, Free Radic. Res. 52
(2018) 629–638.
4.5.31. Isoformononetin (33b)
White solid; m.p.: 208–210 ℃
[19] D. Mochly-Rosen, C.H. Chen, W.J. Yang, WO patent NO. 2010062308 (2010).
[20] S. Talukdar, J.-L. Hsu, T.-C. Chou, J.-M. Fang, Tetrahedron Lett. 42 (2001)
1103–1105.
IR (KBr): 3215, 3054, 2852, 2362, 1723, 1622, 1586, 1515, 1441,
1379, 1255, 1172, 1098, 1018, 942, 831, 796, 779, 698, 542 cm−1
14

M.-C. Cheng, et al.
BioorganicChemistry104(2020)104166
[21] A.H. Rezvani, D.H. Overstreet, M. Perfumi, M. Massi, Pharmacol. Biochem. Behav.
75 (2003) 593–606.
Theory Comput. 7 (2011) 1951–1961.
[25] K.F. Biegasiewicz, J.S. Gordon, D.A. Rodriguez, R. Priefer, Tetrahedron Lett. 55
(2014) 5210–5212.
[22] K. WingMing, B.L. Vallee, Phytochemistry 47 (1998) 499–506.
[23] V. Koppaka, D.C. Thompson, Y. Chen, M. Ellermann, K.C. Nicolaou, R.O. Juvonen,
D. Petersen, R.A. Deitrich, T.D. Hurley, V. Vasiliou, Pharmacol. Rev. 64 (2012)
520–539.
[26] G. Cavallo, P. Metrangolo, R. Milani, T. Pilati, A. Priimagi, G. Resnati, G. Terraneo,
Chem. Rev. 116 (2016) 2478–2601.
[27] E.D. Lowe, G.-Y. Gao, L.N. Johnson, W.M. Keung, J. Med. Chem. 51 (2008)
4482–4487.
[24] M. Mangold, L. Rolland, F. Costanzo, M. Sprik, M. Sulpizi, J. Blumberger, J. Chem.
15