Discovery, synthesis, biological evaluation and molecular docking study of (R)-5-methylmellein and its analogs as selective monoamine oxidase A inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.03.060

(R)-5-Methylmellein (5-MM), the major ingredient in the fermented mycelia of the medicinal fungus Xylaria nigripes (called Wuling Shen in Chinese)? was found to be a selective inhibitor against monoamine oxidase A (MAO-A) and might play an important role in the clinical usage of this edible fungus as an anti-depressive traditional Chinese medicine (TCM). Based on the discovery and hypothesis, a variety of (R)-5-MM analogs were synthesized and evaluated in vitro against two monoamine oxidase isoforms (MAO-A and MAO-B). Most synthetic analogs showed selective inhibition of MAO-A with IC₅₀ values ranging from 0.06 to 29 μM, and compound 13aR is the most potent analog with high selectivity (IC₅₀, MAO-A: 0.06 μM; MAO-B: >50 μM). Interestingly, the enzyme kinetics study of 13aR indicated that this ligand seemed to bind in the MAO-A active site according to so-called “tight-binding inhibition” mode. The molecular docking study of 13aR was thereafter performed in order to rationalize the obtained biological results.

Full text of DOI:10.1016/j.bmc.2019.03.060

Accepted Manuscript
Discovery, synthesis, biological evaluation and molecular docking study of
(R)-5-methylmellein and its analogs as selective monoamine oxidase A inhibi-
tors
Chao Huang, Juan Xiong, Hui-Da Guan, Chang-Hong Wang, Xinsheng Lei, Jin-
Feng Hu
PII:
S0968-0896(18)31749-8
https://doi.org/10.1016/j.bmc.2019.03.060
BMC 14852
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 October 2018
7 March 2019
31 March 2019
Please cite this article as: Huang, C., Xiong, J., Guan, H-D., Wang, C-H., Lei, X., Hu, J-F., Discovery, synthesis,
biological evaluation and molecular docking study of (R)-5-methylmellein and its analogs as selective monoamine
oxidase A inhibitors, Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.03.060
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Graphical Abstract
Discovery, synthesis, biological evaluation
and molecular docking study of (R)-5-
methylmellein and its analogs as selective
monoamine oxidase A inhibitors
Leave this area blank for abstract info.
Chao Huanga, Juan Xionga, Hui-Da, Guanb, Chang-Hong Wangb, Xinsheng Leia,* and Jin-Feng Hua,*
a School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, China
b Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, No. 1200 Cailun
Road, Shanghai 201203, China
(R)-5-Methylmellein
The optimal compound (13aR)
Up to 42 synthetic analogs
isolated from Xylaria nigripes
MAO-A, IC50: 4.6 µM
MAO-B, IC50: 38.5 µM
MAO-A, IC50: 0.06 µM
MAO-B, IC50: > 50 µM

Bioorganic & Medicinal Chemistry
Discovery, synthesis, biological evaluation and molecular docking study of (R)-5-
methylmellein and its analogs as selective monoamine oxidase A inhibitors
Chao Huanga, Juan Xionga, Hui-Da Guanb, Chang-Hong Wangb, Xinsheng Leia,  and Jin-Feng Hua,
a School of Pharmacy, Fudan University, No. 826 Zhangheng Road, Shanghai 201203, China
b Institute of Chinese Materia Medica, Shanghai University of Traditional Chinese Medicine, No. 1200 Cailun Road, Shanghai 201203, China
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
(R)-5-Methylmellein (5-MM), the major ingredient in the fermented mycelia of the medicinal
fungus Xylaria nigripes (called Wuling Shen in Chinese)¸ was found to be a selective inhibitor
against monoamine oxidase A (MAO-A) and might play an important role in the clinical usage of
this edible fungus as an anti-depressive traditional Chinese medicine (TCM). Based on the
discovery and hypothesis, a variety of (R)-5-MM analogs were synthesized and evaluated in
vitro against two monoamine oxidase isoforms (MAO-A and MAO-B). Most synthetic analogs
showed selective inhibition of MAO-A with IC50 values ranging from 0.06 to 29 µM, and
compound 13aR is the most potent analog with high selectivity (IC50, MAO-A: 0.06 µM; MAO-
B: > 50 µM). Interestingly, the enzyme kinetics study of 13aR indicated that this ligand seemed
to bind in the MAO-A active site according to so-called “tight-binding inhibition” mode. The
molecular docking study of 13aR was thereafter performed in order to rationalize the obtained
biological results.
Available online
Keywords:
Natural product
5-Methylmellein
Synthetic analogs
Monoamine oxidase
Inhibitor
2009 Elsevier Ltd. All rights reserved.
———
 Xinsheng Lei. Tel.: +0-086-21-51980128; fax: +0-086-21-51980128; e-mail: leixs@fudan.edu.cn
 Jin-Feng Hu. Tel.: +0-086-21-51980172; fax: +0-086-21-51980172; e-mail: jfhu@fudan.edu.cn

1. Introduction
we hypothesized that the enantiomer (R)-5-MM (9aR) from
Wuling Capsule might be the active inhibitor to demonstrate anti-
depressive effect.
Monoamine oxidases (МАОs, EC 1.4.3.4, amine−oxygen
oxidoreductase),
a
family of flavin-containing amine
oxidoreductases, are mostly found in the central nervous system
(CNS) and located in the mitochondrial outer membrane1. Their
enzymatic function is to catalyze the oxidative deamination of
monoamine neurotransmitters, such as dopamine, serotonin, and
norepinephrine2. There are two types of MAOs in mammals,
MAO-A and MAO-B, defined by the cysteine amino acid
(Cys406 in MAO-A and Cys397 in MAO-B) bound covalently to
their co-factor flavin adenine dinucleotide (FAD)3. Despite the 70%
sequence identity in their compositions, the two isoenzymes still
show significant difference in their three dimensional structures4,
substrate specificities, sensitivities to inhibitors5 and distributions
in tissues and cells6.
Due to their abilities to degrade neurotransmitters, MAOs
have been demonstrated to be enormously valuable therapeutic
targets in neuropharmacology. In addition, the relationship
between neurotransmitter level and MAOs activity proves to be
linked with neurological and psychiatric disorders7. Currently,
MAO-A inhibitors are used for the treatment of anxiety and
depression, while MAO-B inhibitors could potentially be used as
therapeutic agents in Parkinson’s and Alzheimer’s diseases8.
Although some MAO-A inhibitors, including phenelzine,
isocarboxazid, tranylcypromine, iproniazid, moclobemide and
toloxatone (Fig. 1)9,10, have made great contributions to the
depressive patients, their clinic usage showed some limitation
due to potential side effects, food and drug interactions, and
introduction of other classes of drugs11. As a result, the
development of new generation of MAO-A inhibitors attracted
significant attention recently, aiming at safer and better-tolerated
therapeutic agents12.
Figure 1. The structures of some representative MAO inhibitors
including approved drugs and reported natural products. I: irreversible; R:
reversible; A: MAO-A; B: MAO-B. Compounds 1–4, 9aR and 9bR were
isolated from X. nigripes.
An innegligible source for novel lead molecules in this aspect
is to investigate natural products, especially those herbal
ingredients from folk medicine used to treat various neurological
disorders. Xylaria nigripes (Koltz.) Sacc. (family Xylariaceae),
under the folklore name of Wuling Shen in Chinese, is a precious
and editable medicinal fungus used in traditional Chinese
medicine (TCM) as a nerve tonic agent for a long history13.
Although wild fungus is hard to acquire due to its growth around
abandoned termite (Odontotermes formosanus) nests, its
fermented alternative can be manufactured in large scale. Notably,
Wuling Capsule, made from fermented product, was approved in
1999 by the China Food and Drug Administration (CFDA) as a
First Grade New TCM in the treatment of insomnia, anxiety, and
depression14.
The isocoumarin core structure of six natural products isolated
from X. nigripes is quite popular in nature, and has attracted
significant attention in the field of drug discovery due to their
interesting and diversified biological activities18. Moreover, the
bioisosteres of this heterocyclic scaffold are also privileged
structures for the rational discovery of new MAO inhibitors
(Figure 1), such as coumarin (esuprone), flavones (quercetin) and
chroman (epicatechin gallate)19. Herein, we describe the
synthesis and structure-activity-relationship (SAR) around
compound 9aR.
2. Results and discussion
2.1. Chemistry
In recent years, the fermented mycelia of X. nigripes was
studied regarding their active chemical constituents and their
anti-depression mechanisms. In our previous work, some novel
natural products were obtained from the EtOH extract of
fermented mycelia of X. nigripes. Unfortunately, these minor
components, such as the spiroketal pyrrole-derived alkaloids (i.e.,
xylapyrrosides), did not exhibit any anti-depressive activities in
our well-established assays using neurological disorder model15a-b
.
Interestingly, (R)-5-methylmellein (5-MM)15c-f (9aR, Figure 1),
the major secondary metabolite (yield 0.026%) of X. nigripes,
was found to display promising anti-depression efficacy in the
primary forced swimming test (FST, @ 1 mg/kg) and tail
suspension test (TST, @ 3 mg/kg) using mouse anti-depressive
models16. It was reported that 5-MM, isolated from unidentified
fungal strain 8082, could inhibit mouse brain MAOs in a dose-
dependent manner with an IC50 value of 1.06 µM17. The absolute
configuration at the stereo center was not defined in the paper, so
Reagents and conditions: a) SOCl2, t-BuNH2/THF, 96%; b) n-BuLi, TMEDA,
(R)-(+)-propylene oxide, THF, -78 °C, 47%; c) p-TsOH·H2O/toluene, reflux,
60%; d) BBr3/CH2Cl2, -78 °C to 0 °C, 96%; e) NIS, FeCl3/CH3CN, rt, 60%; f)
1.0 M ZnEt2, Pd(dppf)Cl2, 1,4-dioxane, 110 °C, 63%
Scheme 1. The synthesis of (R)-methyl mullein (9aR) and C5-
substituted analogs.
Despite the known synthesis of racemic 5-MM20, its
asymmetric version has not been reported. Inspired by J.
Clayden’s work on the asymmetric synthesis of (R)-mellein21, our

asymmetric total synthesis of (R)-5-MM was efficiently
accomplished in 4 steps, as shown in Scheme 1. Starting from the
acid 5a, the amide 6a was prepared through the coupling of the
corresponding acyl chloride and t-BuNH2. 6a was next
ortholithiated with n-BuLi and subsequently reacted with the
optical active epoxide [(R)-(+)-propylene oxide] to afford the
chiral alcohol 7aR, which was transformed into (R)-5-MM (9aR)
via cyclization and deprotection. The overall yield was up to 26%
with an excellent enantiomeric excess (ee: 98%).
reactions, like reduction, oxidation, condensation/oxidation25 or
reductive amination, 9pR could be transformed into 9qR, 9rR,
9sR and 9tR, respectively (Scheme 5). In addition, 9aR was
subjected to nitration to introduce nitro group at the C7 position,
and the desired product 9uR was further reduced to the amine
9wR. Upon N-alkylation or N-amidation, 9wR was smoothly
converted into 9xR and 9yR, respectively.
In order to probe the influence of the stereo configuration of
C-3 on the inhibition of MAOs, the enantiomer 9aS was prepared
using the same approach, where the ortholithiated species from
6a reacted with (S)-(–)-propylene oxide. Similarly, (R)-mellein,
(S)-mellein and (R)-5-ethylmellein (9bR, 9bS, 9eR) were also
successfully obtained (Scheme 1).
o
Reagents and conditions: a) NaNO2, HCl, KI, H2O/acetone (1/1), 7–10 C,
85%; b) 2,4-pentanedione, CuI, Cs2CO3, DMSO, 120 °C, 49%; c)
BBr3/CH2Cl2, -78 °C to 0 °C, 51%.
Reagents and conditions: a) Cl2CHOCH3, TiCl4, anhydrous CH2Cl2, -10 °C,
85%; b) H2, 10% Pd/C, EtOH/AcOEt (1/1), rt, 93%; c) NaClO2,30% H2O2,
NaH2PO4, THF/H2O (1/1), rt, 51%; d) NH2OH•HCl, FeCl3, DMF, 160 °C,
43%; e) p-anisidine, p-toluenesulfonic acid; NaBH(OAc)3, ClCH2CH2Cl, rt,
66%; f) [NO2]+BF4–, CH2Cl2, -20 °C, 36%; g) H2, 10% Pd/C, EtOH/CH2Cl2
(1/1), rt, 79%; h) Me2SO4, NaOH, DMF/H2O (1/2), rt, 16%; i) acryloyl
chloride, Et3N, CH2Cl2, rt, 46%.
Scheme 2. The synthesis of the aromatized analog (9c).
The attempt to aromatize 8aR or 8bR with a variety of
oxidants failed to introduce a carbon-carbon double bond, thus
the aromatized products (8c and 9c) were prepared via an iodo
intermediate (11)22 as depicted in Scheme 2.
Scheme 5. The synthesis of the analogs modified at C7-position.
Reagents and conditions: a) n-BuLi, TMEDA, THF, -78 °C, for 7fR with
(2R)-(+)-1,2-epoxybutane, 36%, for 7g with 1,2-epoxy ethane, 45%, for 7h
with 1,1-dimethylethylene oxide, 18%; b) p-TsOH·H2O/toluene, reflux, 8fR:
77%, 8g: 43%, 8h: 42%; c) BBr3/CH2Cl2, -78 °C to 0 °C, 9fR: 85%, 9g, 90%,
9h: 94%.
Scheme 3. The synthesis of C3-substituted analogs.
Reagents and conditions: a) MeI, K2CO3, DMF/H2O (1/2), rt, 85%; b)
EDCI/HOBt, amine (Et2NH or 2-morpholinoethanamine), CH2Cl2, rt, ii.
BBr3/CH2Cl2, -78 °C to 0 °C, for 10bR: 71%, for 10eR: 67%; c) i. SOCl2, ii.
Me2NH (aqueous solution), rt, for 10aR: 72%; d) EDCI/HOBt, amines,
CH2Cl2, rt, for 10cR: 52%, for 10dR: 53% (based on 9rR).
In order to probe the bulky tolerance at the C3-position, a
series of analogs (9fR, 9g, 9h) modified at this position were
prepared as shown in Scheme 3.
With readily available 9aR in hand, a series of C7-halo
Scheme 6. The synthesis of several C7-substituted analogs with
nitrogen-containing side chain
23
analogs (9iR, 9jR and 9kR) were synthesized using SO2Cl2
,
NBS and NIS as halogenating reagents, respectively. As shown
in Scheme 4, the iodide 9kR was coupled with several
organozinc reagents through Negishi coupling reaction to afford
C7-alkylated analogs (9lR–9oR).
Considering the importance of amide or amine fragments in
the known MAO inhibitors, several amide or amine fragments
were introduced into the side chain, through amidation with the
acid 9rR, resulting in the production of 10aR–10eR, as shown in
Scheme 6.
Reagents and conditions: a) FeCl3/CH3CN, halogenating reagents, rt, for 9iR
with SO2Cl2, 87%, for 9jR with NBS, 96%, for 9kR with NIS, 94%; b)
o
Pd(dppf)Cl2, 1,4-dioxane, 110 C, for 9lR with Me2Zn: 81%, for 9mR with
Et2Zn: 63%; for 9nR with n-PrZnI: 31%, for 9oR with n-BuZnI: 17%.
Scheme 4. The synthesis of C7-halo and C7-alkyl analogs
In order to introduce polar substituents to C7-position, 9aR
was initially formylated at the C7 position to provide 9pR
through Friedel-Crafts reaction24, and through some conventional

The stereo-configuration at the C3-position appeared to have
little effect on the activity and selectivity, because both (R)- and
(S)-5-MM (IC50 of MAO-A/-B for 9aR vs 9aS: 4.6/38.5 µM vs
4.1/40.9 µM) showed almost the same activity and selectivity,
and the similar results were also found in the case of 9bR and
9bS (IC50 of MAO-A/-B for 9bR vs 9bS: 6.2/~50 µM vs 7.5/24.0
µM). However, the aromatized product 9c displayed obviously
reduced activity (IC50 of MAO-A vs -B: 23.4 vs > 50 µM).
Further analysis suggested that one suitably sized group at sp3-
hybrized C3 position might be required to keep both good
activity and selectivity. Removal of C3 methyl group (9g)
maintained similar activity of MAO-A in spite of an evident
sacrifice in selectivity (IC50 of MAO-A vs -B: 3.2 vs 5.5 µM).
Additionally, dimethyl substitution at the C3 position was
unbeneficial to both MAO-A and MAO-B inhibition because 9h
lost both MAO-A and -B inhibition. However, replacement of
methyl with ethyl group (9fR) could keep the activity against
MAO-A, and enhance selectivity (IC50 of MAO-A vs -B: 8.0 vs >
50 µM).
Reagents and conditions: a) MeI, K2CO3, DMF/H2O (1/2), rt, 85%; b) i.
Pd(PPh3)4, K2CO3, ArB(OH)2, 1,4-Dioxane for MeO-11aR to MeO-11fR;
PdCl2(PPh3)2, CuI, 4-(tri-n-butylstannyl)pyridine, CsF, DMF; for MeO-11hR;
for 12aR‒13aR, Pd(OAc)2, dppf, CuCl, ArB(OH)2, CsCO3, DMF, 110 °C;
Pd(PPh3)4, K2CO3, ArB(OH)2, toluene/ethanol/H2O (10/1/0.2), 90 oC for
13bR; ii. BBr3/CH2Cl2, -78 °C to 0 °C, two steps for 11aR: 75%; for 11bR:
58%; for 11cR: 42%; for 11dR: 54%; for 11eR: 58%; for 11fR: 56%; for
11hR: 14%; for 12aR: 7%, for 12bR: 10%, for 12cR: 7%; for 13aR: 13%,
for 13bR: 28%; c) H2 (1 atm), 10% Pd/C, MeOH, rt, 92%; d) FeCl3/CH3CN,
NIS, rt, 94%.
The SAR at C5 position revealed that only small substituent
(Me and H) was permitted, with 9aR and 9bR displaying similar
activity and selectivity. Larger or hydrophilic substituent, such as
Et (9eR), CO2H (1), OH (2) and CH2OH (4), resulted in a loss of
activity against MAO-A or a reduced selectivity over MAO-B.
As a result, methyl group is the most optimal at the C-5 position.
Scheme 7. The synthesis of several C7-substituted analogs containing
aryl or heteroaryl fragments
Various aryl and heteroaryl groups were introduced to C-7
position to probe the potential π-π interaction. As shown in
Scheme 7, these analogs (11aR–11hR, 12aR–12cR, and 13aR–
13bR) were prepared through Pd-catalyzed coupling reactions
with 7-iodo derivatives (9kR and 9zR).
The investigation on the C7-position indicated that this site
was tolerable to a variety of substitutions. For example,
compared with the parent compound 9aR, halogen or C1–4 short
chain alkyl group introduced into this site (9iR–9qR) gave
comparable activity against MAO-A as well as good selectivity
over MAO-B. The polar substituents, such as NO2, NH2 and
NMe2 (9uR–9xR), also seemed to be tolerated. Interestingly,
both CO2H and CN substitutions (9rR–9sR) led to total loss of
both MAO-A and MAO-B activities.
2.2. Biological activity
(R)-5-MM and the synthetic analogs were evaluated for
inhibitory activities using commercially available human MAO-
A and MAO-B, with clorgyline and pargyline as reference
inhibitors of MAO-A or MAO-B, respectively. The enzymatic
inhibition assay was performed using kynuramine as the substrate
for MAOs and 4-hydroxyquinoline as a marker. The inhibitory
activities were analyzed using the sensitive UPLC-ESI-MS/MS
method26 with acetyl aminophenol as internal standard, by which
the quantity of 4-hydroxyquinoline was measured compared with
the blank. The results were shown in Table 1 and Table 2, and
the selectivity index data (SI, MAO-A over MAO-B) were also
listed in the tables if necessary.
Although amido group with small, bulky or hydrophilic chain
side was always unbeneficial to the activities, leading to a
dramatic loss of inhibition (9yR, 10aR–10eR), arylaminomethyl
group (9tR) could keep the activity against MAO-A along with a
better selectivity, implying that there might be a large space or π-
π interaction in this binding pocket.
Further SAR exploration was performed on the aryl groups at
the C7-position (Table 2). The introduction of hydrophobic
benzene ring, such as Me and -CF3 (11aR–11cR), significantly
enhanced the activity against MAO-B (IC50, increased up to 1.0
µM from 38.4 µM of 9aR) rather than that of MAO-A. However,
benzene ring bearing polar substituents, such as p-OH, and p-
NH2, m-NO2, evidently enhanced the activity against MAO-A
with an improved selectivity over MAO-B (11dR–11fR).
Notably, m-NH2 on benzene ring (11gR) could further enhance
the activity against MAO-A with an excellent selectivity over
MAO-B (IC50 of MAO-A vs -B: 0.2 vs > 50 µM). The
substitution with heterocycles was also tolerated at this position.
3-Pyridyl at the C7-position (12aR) provided the optimal potency
and an excellent selectivity (IC50 of MAO-A vs -B: 0.2 vs > 50
µM), compared with 2- and 4-pyridyl (11hR, 12bR).
Interestingly, the pyrimidyl analog (12cR, IC50 of MAO-A vs -B:
0.14 vs > 50 µM) was comparable to the 3-pyridyl analog (12aR).
Further analog of 12cR with ethyl substitution at the stereocenter
led to the most optimal compound 13aR (IC50 of MAO-A vs -B:
0.06 vs > 50 µM).
As shown in tables, the synthetic (R)-5-MM (9aR) exhibited
inhibitory activity against MAO-A with about 10-fold selectivity
over MAO-B, almost the same result compared with the natural
product isolated from Wuling Capsule (IC50, MAO-A: 4.6 µM;
MAO-B: 38.5 µM). Although (R)-5-MM was indeed a selective
MAO-A inhibitor, it was less potent than the known MAO-A
inhibitor clorgyline (IC50, MAO-A: 0.025 µM). Most analogs
exhibited comparable inhibition against MAO-A with IC50 values
in the 0.06–29 µM range and high selectivity over MAO-B
ranging from 1.7-fold to more than 830-fold. Among them,
compound 13aR was the most potent and selective MAO-A
inhibitor (IC50, MAO-A: 0.06 µM; MAO-B: > 50 µM).
2.3 Structure activity relationship (SAR) study
Based on a variety of (R)-5-MM analogs and their MAO
inhibitory activities, we could obtain some insights on the
structure-activity relationship. As shown in Table 1, masking the
phenolic hydroxyl group of 9aR with methyl group resulted in a
dramatic loss in activity of both MAO-A and MAO-B,
exemplified as 8aR, 8aS and 8bR, implying that the essential
hydroxyl group (as a hydrogen-bonding donator) might form
hydrogen-bonding interaction with MAO.

Table 1. Inhibitory activities of synthesized compounds against monoamine oxidases.
Compound
C3,4-bond R1
R2
R3
R4
R5
IC50 (µM)a
(MAO-A)
4.6 ± 0.0
> 50
IC50 (µM)a
(MAO-B)
38.5 ± 1.4
> 50
SIc
8.3
9aRd
8a
H
Me
Me
Me
H
Me
Me
H
H
H
Me
H
Me
Me
H
H
Me
H
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-
H
–
g
Me
H
-
–
–
–
–
=
g
8aS
Me
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
-
H
> 50
> 50
-
g
8bR
9aS
Me
H
H
> 50
> 50
-
Me
Me
Me
Me
Me
H
H
4.1 ± 0.1
23.4 ± 0.1
8.0 ± 0.7
3.2 ± 0.3
> 50
40.9 ± 5.6
> 50
10.0
> 2.0
> 6.2
1.7
9ce
H
-
9fR
Et
H
> 50
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
9g
H
H
5.5 ± 0.6
> 50
g
9h
Me
Me
H
H
-
9bR
9bS
9eR
1f
H
6.2 ± 0.3
7.5 ± 0.4
＞ 50
~ 50
8.1
H
H
24.0 ± 0.9
16.1 ± 1.4
＞ 50
3.2
Et
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
-
H
< 0.3
g
COOH
OH
CH2OH
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
Me
-
H
＞ 50
-
2f
H
17.1 ± 1.2
＞ 50
＞ 50
＞ 2.9
4f
H
＞ 50
-
g
9iR
Cl
2.2 ± 0.1
28.6 ± 1.2
15.9 ± 0.6
3.8 ± 0.1
2.9 ± 0.3
4.5 ± 0.4
18.3 ± 0.3
19.2 ± 0.5
＞ 50
＞ 50
> 22
> 1.7
> 3.1
> 13
> 17
> 11
> 2.7
> 2.6
9jR
Br
＞ 50
9kR
9lR
I
＞ 50
Me
＞ 50
9mR
9nR
9oR
9qR
9rR
9sR
9tR
Et
＞ 50
＞ 50
＞ 50
＞ 50
＞ 50
＞ 50
n-Pr
n-Bu
CH2OH
g
CO2H
-
g
CN
＞ 50
-
NHCH2(4-MeO-C6H4)
4.4 ± 0.2
15.8 ± 1.3
4.1 ± 0.4
13.6 ± 0.8
＞ 50
>11
3.1
＞ 50
＞ 50
＞ 50
＞ 50
＞ 50
＞ 50
＞ 50
＞ 50
＞ 50
＞ 50
9uR
9wR
9xR
9yR
10aR
10bR
10cR
10dR
10eR
Clorgylineb
Pargylineb
NO2
NH2
> 11
> 3.6
NMe2
g
NHCOCH=CH2
-
g
CONMe2
＞ 50
-
g
CONEt2
＞ 50
-
g
CO-[N(CH2CH2)2O]
＞ 50
-
g
CO-[N(CH2CH2)2NMe]
＞ 50
-
g
COCH2CH2-[N(CH2CH2)2O]
＞ 50
-
g
g
-
-
0.025 ± 0.01
-
-
g
g
-
-
-
-
-
5.0 ± 0.6
-
b
c
d
aAll the values are expressed as the mean ± SEM of triplicate determinations; Positive control; SI = [IC50 MAO-B (µM)] / [IC50 MAO-A (µM)]; Both the
sample isolated from Wuling capsule and synthetic sample displayed almost the same values; C3,4-double bond; fThe structures of the compounds (1, 2 and 4)
e
are shown in Figure 1; g means not determined.

Table 2. IC50 values of C7-aryl or heteroaryl analogs against monoamine oxidases.
Compound
R3
Ar
H
IC50 (µM)a
(MAO-A)
4.6 ± 0.0
IC50 (µM)a
(MAO-B)
38.5 ± 1.4
1.0 ± 0.1
SIc
9aR
Me
Me
8.3
0.1
11aR
10.5 ± 1.5
11bR
11cR
Me
Me
12.4 ± 2.8
13.7 ± 3.5
4.1 ± 0.3
1.0 ± 0.3
0.3
0.1
11dR
11eR
Me
Me
1.1 ± 0.1
0.8 ± 0.1
38.9 ± 5.9
21.0 ± 2.7
38
26
11fR
11gR
Me
Me
3.0 ± 0.4
＞ 50
＞ 50
> 17
0.20 ± 0.0
> 250
11hR
12aR
12bR
12cR
Me
Me
Me
Me
2.7 ± 0.2
0.20 ± 0.1
2.1 ± 0.1
0.14 ± 0.3
＞ 50
> 50
> 50
> 50
> 18
> 250
> 24
> 350
13aR
13bR
Et
Et
0.06 ± 0.04
0.3 ± 0.1
> 50
> 50
> 830
> 167
d
d
Clorgylineb
Pargylineb
-
-
-
-
0.025 ± 0.005
-
-
d
d
-
5.0 ± 0.6
-
aAll the values are expressed as the mean ± SEM of triplicate determinations; bPositive control; cSI = [IC50 MAO-B (µM)]/[IC50 MAO-A (µM)]; d “-” means not
determined.
A
B
Figure 2. (A) The binding mode of 13aR (green sticks) with MAO-A (PDB code: 2Z5Y); (B) The binding mode of 13aR (yellow stick) with MAO-B (PDB
code: 6FVZ). The pictures were generated by Pymol.

2.4 The kinetic study of enzyme MAO-A and the mode of
MAO-A inhibition
800
600
400
200
0
Currently reversibility is an important characteristic of MAO
inhibitors due to a number of potential side effects from
irreversible MAO inhibitors11. Thus, the kinetic study of enzyme
MAO-A and the mode of MAO-A inhibition were subsequently
carried out with some representative compounds such as 11gR,
12aR, 12cR and the most potent compound (13aR) using
reported protocols8e. From the substrate-dependent kinetic
experiments, both the corresponding progression curves and the
Linewever-Burk plots were generated. The initial rates of the
MAO-A-catalyzed oxidation of kynuramine were measured at
eight different substrate concentrations in the absence and in the
presence of two different concentrations of the tested compounds.
The results are depicted in Fig. 3.
0
200
400
600
800 1000 1200
[S] (μM)
Figure 4. Effect of substrate (S) concentration on the IC50 values for
13aR.
We applied a protocol by plotting IC50 values of 13aR at
different kynuramine concentration from 50 µM to 1000 µM. As
shown in Fig. 4, the linear correlation between IC50 values and
substrate concentration indicated that our compound was in full
agreement with tight-binding competitive inhibition, confirming
our compound should bind in the MAO-A active site.
Based on the kinetic data of the enzyme MAO-A reactions,
the linear Eadie-Hofstee plots exhibited that MAO-A catalyzed
reactions were conformed by Michaelis-Menten equation
(Fig.3A). The enzyme kinetic analysis was also consistent with
the experimental results using the purified recombinant human
MAO-A. The Km and Vmax values for MAO-A were calculated
to be 87.79 ± 6.76 μ M and 492.00±17.52 μM/min/mg,
respectively.
2.5 Molecular docking study
With this insight in mind, in order to explain these biological
results, a preliminary molecular docking study was performed
using the most potent and selective compound 13aR as the ligand
and HRM/MAO-A complex structure (PDB code: 2Z5Y) as the
reference due to the structural similarity of the ligand HRM.19a
Aiding by Schrodinger (Maestro suite) and Pymol, one possible
binding mode of the best compound 13aR and MAO-A was
described as shown in Figure 2A.
The Lineweaver-Burk plots of the inhibitory activities of the
representative compounds (13aR, 12aR, 12cR and 11gR) on
hMAO-A indicated a noncompetitive mechanism. As shown in
Figure 3B-E, all the lines are linear and intersect on the x-axis,
showing a decrease of substrate Vmax with constant Km. These
results appeared to be similar to another natural product luteolin,
which is known as a weak noncompetitive hMAO-A inhibitor27.
Similar to HRM, 13aR would stay into the same pocket of
MAO-A, and the binding site was close to co-factor FAD. Two
critical hydrogen bonds between 13aR and MAO-A were
observed: one from the phenol hydroxyl as hydrogen bond donor
and Phe208 residue as hydrogen bond acceptor; and the other
from the amino of the Gln215 residue as hydrogen bond donor
and the carbonyl oxygen atom of the lactone as hydrogen bond
acceptor. Both hydrogen bonds should give essential contribution
to the activity of (R)-5-MM and its analogs. In addition, the
pyrimidine ring at the C-7 position of 13aR appeared to have a
– interaction with the phenyl ring of Phe208, making extra
contribution to the activity.
To explain the selective inhibition of compound 13aR toward
hMAO-A, molecular docking study of 13aR with hMAO-B
(PDB code: 6FVZ)8a was carried out. As shown in Figure 2B,
13aR displayed an opposite orientation in hMAO-B, leading to
the loss of two important hydrogen bonds and one –
interaction with Phe208 in the binding site of hMAO-B. On the
other hand, the pyrimidine ring in 13aR can not only form one
hydrogen bond with Tyr188, but also establish one –
interaction with Tyr435 in the pocket of hMAO-B. However, the
docking score of 13aR with hMAO-A and hMAO-B were -9.824
and -8.873 respectively, which means stronger binding affinity of
13aR to the binding sites of hMAO-A than hMAO-B.
Figure 3. The kinetic study (A) and the mode of MAO-A inhibition with
the representative compounds including 13aR (B), 12aR (C), 12cR (D) and
11gR (E).
3. Conclusion
Recently, it was reported8a, some chromone analogs could
display non-competitive behavior when ligands bind to the
enzyme active site in a way that is defined as “tight-binding
inhibition”28. In this case, the standard steady-state kinetic model
used to describe the mechanism of inhibition is no longer valid.
Instead, higher concentrations of substrate are needed in order to
compete with the inhibitor in terms of binding to the enzyme
active site. To figure out the exact mechanism of our compounds,
we measured IC50 values of 13aR using different substrate
concentrations.
In continuation of our efforts to investigate the active
chemical constituents of the fermented mycelia of X. nigripes and
their anti-depression mechanisms, we discovered (R)-5-MM was
a selective inhibitor against MAO-A. A variety of analogs around
(R)-5-MM were synthesized and evaluated in vitro for their
inhibitory potency against MAO-A and MAO-B, with most of
them displaying selective MAO-A inhibitory activities in the
0.06–29 µM range. Particularly, compound 13aR was the most
potent MAO-A inhibitor with high selectivity (IC50, MAO-A:

0.06 µM; MAO-B: > 50 µM). The enzyme kinetics study of
13aR indicated that this ligand seemed to bind in the MAO-A
active site according to so-called “tight-binding inhibition” mode.
The molecular docking study of 13aR was thereafter performed
in order to rationalize the obtained biological results, which
supported one possible binding mode of the potent inhibitor with
MAO-A.
(PE/EtOAc 3/1). mp 130–132 °C. [α]20D -165 (c 0.40, CHCl3). IR
(film) νmax: 3285, 2964, 2925, 1637, 1468, 1365, 1261, 1082 cm-1.
1H NMR (CDCl3, 400 MHz) δ 7.11 (1H, d, J = 8.2 Hz), 6.69 (1H,
d, J = 8.2 Hz), 5.85 (1H, s), 4.69 (1H, s), 3.77 (3H, s), 3.72 (1H,
s), 2.86 (1H, d, J = 13.9 Hz), 2.62–2.51 (1H, m), 2.26 (3H, s),
1.44 (9H, s). ESI-MS (m/z): 280.2 [M+H]+.
4.1.3. General procedure for the synthesis of compounds
(8aR–bR, 8fR, 8g and 8h).
4. Experimental
To a solution of the raw materials (7aR–bR, 7fR, 7g or 7h)
(2.32 mmol) in toluene (30.0 mL) was added p-TsOH·H2O (662
mg, 3.48 mmol). The reaction mixture was refluxed and stirred
for 2 h. The resulting mixture was concentrated in vacuo. The
residue was purified by silica gel chromatography.
General. All chemicals were purchased from Adamas, SCRC,
Alfa Aesar, and used without further purification. Deuterated
solvents were purchased from Cambridge Isotope Laboratories.
All non-aqueous reactions were carried out using oven-dried (110
°C) or heat gun dried glassware under a positive pressure of
anhydrous argon unless otherwise noted. THF and
dichloromethane were purified by distillation and dried by
passage over activated 4 Å molecular sieves under an argon
atmosphere. 1H, 13C and 19F NMR data were recorded on a
Varian Model Mercury 400 MHz or Bruker 600 MHz
spectrometers using solvent signals (CDCl3: δH 7.26/δC 77.0;
CFCl3: δF 0) as references. 1H NMR chemical shifts (δ) are given
in ppm (s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet) downfield from Me4Si. LC-MS data were recorded on
an Agilent 1260/6120 quadrupole LC/MS spectrometer, and high
resolution mass spectra obtained on an AB SCIEX Triple TOFTM
5600+ mass spectrometer. IR spectra were recorded on a Nicolet
AVATAR 360 FT-IR spectrometer; Optical rotations were
acquired on a Rudolf Autopol IV automatic polarimeter.
4.1.3.1. (R)-N-(tert-butyl)-2-(2-hydroxypropyl)-6-methoxy-3-
methylbenzamide (8aR). White solid (yield: 60%); Rf = 0.44
(PE/EtOAc 10/1). mp: 89–90 °C. [α]20 -234 (c 0.20, CHCl3). IR
D
(film) νmax: 2954, 2924, 1682, 1673, 1463, 1375, 1236, 1219,
1
1118, 807 cm-1. H NMR (400 MHz, CDCl3) δ 7.30 (d, J = 8.4
Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 4.48 (s, 1H), 3.90 (s, 3H), 2.86
(d, J = 16.3 Hz, 1H), 2.73–2.60 (m, 1H), 2.21 (s, 3H), 1.48 (d, J =
5.8 Hz, 3H). ESI-MS (m/z): 207.2 [M+H]+.
4.1.4. General procedure for the synthesis of compounds
(9aR–bR, 9aS, 9bS, 9fR, 9g and 9h).
To a solution of the raw materials (8aR–bR, 8fR, 8g and 8h)
(7.12 mmol) in anhydrous CH2Cl2 (20.0 mL) was added dropwise
boron tribromide (437 µL, 4.46 mmol) at -78 °C. After 1 h, the
reaction mixture was raised at 0 °C and stirred for 1 h. The
resulting mixture was quenched with saturated aqueous NH4Cl,
and extracted with CH2Cl2. The combined extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo.The
residue was purified by silica gel chromatography.
4.1.1. General procedure for the synthesis of compounds (6a
and 6b)
The appropriate benzoic acid (5a or 5b) (19.9 mmol) was
converted into its corresponding acid chloride by treatment with
thionyl chloride (22.0 mL, 284 mmol), and this intermediate was
reacted with tert-butylamine (8.0 mL, 74.0 mmol) at room
temperature for 0.5 h. The reaction mixture was concentrated
under reduced pressure and dissolved in CH2Cl2, washed with
water, dried over Na2SO4, filtered, and concentrated in vacuo.
The residue was purified by silica gel column chromatography
with petroleum ether (PE)/EtOAc to yield pure product.
4.1.4.1. (R)-8-hydroxy-3,5-dimethylisochroman-1-one (9aR).
White solid (yield: 96%); Rf = 0.23 (PE/EtOAc 20/1). ee% =
98%. [α]20D -110 (c 0.20, CHCl3). mp: 127–128 oC. IR (film) νmax
:
1
2923, 1662, 1377, 1366, 1294, 803 cm-1. H NMR (400 MHz,
CDCl3) δ 11.00 (s, 1H), 7.29 (d, J = 8.7 Hz, 1H), 6.81 (d, J = 8.5
Hz, 1H), 4.69 (s, 1H), 2.95 (d, J = 16.6 Hz, 1H), 2.78–2.65 (m,
1H), 2.20 (s, 4H), 1.55 (d, J = 6.1 Hz, 3H). ESI-MS (m/z): 193.1
[M+H]+.
4.1.1.1. N-(tert-butyl)-2-methoxy-5-methylbenzamide (6a).
Colorless liquid (yield: 96%); Rf = 0.43 (PE/EtOAc 20/1). IR
(film) νmax: 3395, 2925, 1659, 1540, 1495, 1393, 1246, 1025, 810
4.1.4.2. (R)-8-hydroxy-3-methylisochroman-1-one (9bR).
20
1
cm-1. H NMR (CDCl3, 400 MHz) δ 7.11 (1H, d, J = 8.2 Hz),
White solid (yield: 96%); Rf = 0.21 (PE/EtOAc 20/1). [α]D
-
99.1 (c 0.20, CHCl3). IR (film) νmax: 2955–2854, 1681, 1464,
6.74–6.66 (1H, m), 5.85 (1H, s), 4.69 (1H, s), 3.77 (3H, s), 3.72
(1H, s), 2.86 (1H, d, J = 13.9 Hz), 2.62–2.51 (1H, m), 2.26 (3H,
s), 1.44 (9H, s), 1.00 (3H, t, J = 7.2 Hz). ESI-MS (m/z): 202.3
[M+H]+.
1
1377, 1292, 1237, 1220, 1119, 807 cm-1. H NMR (400 MHz,
CDCl3) δ 11.02 (brs, 1H), 7.40 (t, J = 7.9 Hz, 1H), 6.88 (d, J =
8.4 Hz, 1H), 6.68 (d, J = 7.4 Hz, 1H), 4.72 (m 1H), 2.92 (d, J =
7.1 Hz, 2H), 1.52 (d, J = 6.3 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δ 169.9, 162.1, 139.4, 136.1, 117.9, 116.2, 108.3, 76.1,
34.6, 20.7. ESI-MS (m/z): 179.1 [M+H]+.
4.1.2. General procedure for the synthesis of compounds
(7aR–bR, 7fR, 7g and 7h).
To a solution of 6a or 6b (4.83 mmol) in anhydrous THF
(20.0 mL) was added TMEDA (1.6 mL, 10.6 mmol) at -78 °C.
To the resulting mixture was added dropwise a solution n-BuLi
in hexane (2.5 M in hexane, 19.3 mmol) at -78 °C. After 2 h, to
the resulting mixture was added dropwise the epoxide (5.80
mmol) at -78 °C. The reaction mixture was stirred at -78 °C for 7
h. The resulting mixture was quenched with saturated aqueous
NH4Cl, concentrated in vacuo and extracted with CH2Cl2. The
combined extracts were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica gel
chromatography.
4.1.4.3. (S)-8-hydroxy-3,5-dimethylisochroman-1-one (9aS).
White solid (yield: 95%). Rf = 0.23 (PE/EtOAc 20/1). ee% =
86%. [α]20D 95.9 (c 0.20, CHCl3). mp: 126–128 oC. IR (film) νmax
:
3408, 2925, 1597, 1451, 1384, 1295, 1206, 1130, 1089, 1053,
803 cm-1. 1H NMR (400 MHz, CDCl3) δ 11.00 (s, 1H), 7.29 (d, J
= 8.7 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 4.69 (s, 1H), 2.95 (d, J =
16.6 Hz, 1H), 2.78–2.65 (m, 1H), 2.20 (s, 4H), 1.55 (d, J = 6.1
Hz, 4H). ESI-MS (m/z): 193.1 [M+H]+.
4.1.4.4. (S)-8-hydroxy-3-methylisochroman-1-one (9bS).
White solid (yield: 96%); Rf = 0.21 (PE/EtOAc 20/1). [α]D20 85.3
(c 0.20, CHCl3). IR (film) νmax: 2955–2854, 1681, 1464, 1377,
1
1292, 1237, 1200 , 1119, 807 cm-1. H NMR(400MHz, CDCl3) δ
4.1.2.1. (R)-N-(tert-butyl)-2-(2-hydroxypropyl)-6-methoxy-3-
methylbenzamide (7aR). White solid (yield: 47%); Rf = 0.39
11.02 (br s, 1H), 7.40 (t, J = 7.9 Hz, 1H), 6.88 (d, J = 8.4 Hz,

1H), 6.68 (d, J = 7.4 Hz, 1H), 4.72 (m, 1H), 2.92 (d, J = 7.1 Hz,
2H), 1.52 (d, J = 6.3 Hz, 3H). ESI-MS (m/z): 179.1 [M+H]+.
(400 MHz, CDCl3) δ 10.99 (s, 1H), 7.55 (t, J = 7.9 Hz, 1H), 6.91
(d, J = 8.3 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.27 (s, 1H), 2.27
(s, 3H). ESI-MS (m/z): 177.1 [M+H]+.
4.1.4.5.
(R)-3-ethyl-8-hydroxy-5-methylisochroman-1-one
(9fR). White solid (yield: 85%); Rf = 0.32 (PE/EtOAc 20/1).
[α]D20 -87.9 (c 0.20, CHCl3). mp: 83–85 oC. IR (film) νmax: 2963–
2925, 1674, 1609, 1478, 1385, 1219, 1172, 1126, 1043, 826 cm-1.
1H NMR (400 MHz, CDCl3) δ 11.00 (s, 1H), 7.28 (d, J = 8.5 Hz,
1H), 6.81 (d, J = 8.5 Hz, 1H), 4.52–4.41 (m, 1H), 2.92 (dd, J =
16.6, 3.3 Hz, 1H), 2.73 (dd, J = 16.6, 11.7 Hz, 1H), 2.19 (s, 3H),
1.98–1.86 (m, 1H), 1.86–1.75 (m, 1H), 1.10 (t, J = 7.5 Hz, 3H).
13C NMR (150 MHz, CDCl3) δ 160.5, 137.8, 137.1, 124.9, 115.6,
108.3, 80.2, 29.7, 28.0, 18.1, 9.3. ESI-MS (m/z): 207.1 [M+H]+,
HRESI-MS (m/z): [M+H]+ Calcd. for C12H15O3: 207.1016 found:
207.1017.
4.2.3. The procedure for the synthesis of compound (9c).
The same as 4.1.3.
4.2.3.1. 8-hydroxy-3-methyl-1H-isochromen-1-one (9c). White
1
solid (yield: 51%); Rf = 0.25 (PE/EtOAc 5/1). H NMR (400
MHz, CDCl3) δ 10.99 (s, 1H), 7.55 (t, J = 7.9 Hz, 1H), 6.91 (d, J
= 8.3 Hz, 1H), 6.79 (d, J = 7.7 Hz, 1H), 6.27 (s, 1H), 2.27 (s,
3H). ESI-MS (m/z): 177.1 [M+H]+.
4.3.1. General procedure for the synthesis of compounds
(9dR,9iR–kR).
To a solution of 9aR or 9bR (0.52 mmol) in CH2Cl2 (10.0
mL) was added NXS (X = Br, I) (0.57 mmol) and FeCl3 (4.20
mg, 0.03 mmol) at room temperature, then stirred for 1 h. The
resulting mixture was quenched with saturated aqueous NH4Cl,
and extracted with CH2Cl2. The combined extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography (PE/EtOAc
20/1), giving 9dR, 9jR and 9kR, respectively. 9iR was prepared
according to the known method23.
4.1.4.6. 8-hydroxy-5-methylisochroman-1-one (9g). White
solid (yield: 90%); Rf = 0.30 (PE/EtOAc 20/1). IR (film) νmax
:
2955–2924, 1667, 1605, 1588, 1475, 1408, 1384, 1263, 1246,
1206, 1165, 1130, 1043, 829 cm-1. 1H NMR (400 MHz, CDCl3) δ
10.93 (s, 1H), 7.29 (d, J = 8.5 Hz, 1H), 6.82 (d, J = 8.5 Hz, 1H),
4.56 (t, J = 6.1 Hz, 2H), 2.95 (t, J = 6.1 Hz, 2H), 2.20 (s, 3H). 13
C
NMR (150 MHz, CDCl3) δ 160.6, 137.9, 137.3, 124.9, 115.8,
108.5, 67.5, 24.8, 18.0. ESI-MS (m/z):179.1 [M+H]+, HRESI-MS
(m/z): [M+H]+ Calcd. for C10H11O3: 179.0703 found: 179.0700.
4.3.1.1.
(R)-8-hydroxy-5-iodo-3-methylisochroman-1-
4.1.4.7. 8-hydroxy-3,3,5-trimethylisochroman-1-one (9h).
White solid (yield: 94%) Rf = 0.28 (PE/EtOAc 20/1). IR (film)
νmax: 2983–2925, 1669, 1607, 1477, 1387, 1373, 1247, 1220,
1180, 1112, 1045, 834 cm-1. 1H NMR (400 MHz, CDCl3) δ 11.13
(s, 1H), 7.29 (d, J = 8.5 Hz, 1H), 6.81 (d, J = 8.5 Hz, 1H), 2.89
(s, 2H), 2.18 (s, 3H), 1.48 (s, 6H). 13C NMR (150 MHz, CDCl3) δ
160.4, 138.0, 136.0, 125.4, 115.6, 107.7, 81.4, 36.4, 27.5, 18.1.
ESI-MS (m/z): 207.1 [M+H]+, HRESI-MS (m/z): [M+H]+ Calcd.
for C12H15O3: 207.1016 found: 207.1014.
one(9dR). White solid (yield: 20%). Rf = 0.39 (PE/EtOAc 20/1).
[α]20 -65.0 (c 0.30, CHCl3). mp: 144–146 C. IR (film) νmax
:
o
D
3407, 2930, 1676, 1598, 1451, 1384, 1295, 1210, 1130, 1093,
1054, 801 cm-1. H NMR (400 MHz, CDCl3) δ 11.26 (s, 1H),
1
7.83 (d, J = 8.9 Hz, 1H), 6.73 (d, J = 8.8 Hz, 1H), 4.83–4.59 (m,
1H), 3.07 (dd, J = 16.9, 3.1 Hz, 1H), 2.81 (dd, J = 16.7, 11.8 Hz,
1H), 1.57 (d, J = 6.3 Hz, 4H). 13C NMR (150 MHz, CDCl3) δ
162.6, 145.5, 142.0, 118.7, 110.0, 84.9, 75.5, 40.0, 20.7. ESI-MS
(m/z): 304.0 [M+H]+.
4.2.1. The procedure for the synthesis of compound (11).
4.3.1.2. (R)-7-chloro-8-hydroxy-3,5-dimethylisochroman-1-
The 2-amino-6-methoxybenzoic acid 10 (2.0 g, 11.9 mmol)
was added to the solution with ice water (28.0 mL), HCl (10 M,
7.2 mL) and acetone (10.0 mL). The suspension was cooled to 0
oC followed by the addition of sodium nitrite (1.66 g, 24.0 mmol)
one (9iR). White solid (yield: 87%). Rf = 0.17 (PE/EtOAc 25/1).
o
[α]20 11.4 (c 0.30 CHCl3). mp: 93–95 C. IR (film) νmax:2955–
D
2853, 1682, 1480, 1455, 1293, 1216, 1201, 1168, 803, 764 cm-1.
1H NMR (400 MHz, CDCl3) δ 11.54 (brs, 1H), 7.40 (s, 1H),
4.77–4.63 (m, 1H), 2.93 (dd, J = 16.6, 3.4Hz, 1H), 2.71 (dd, J =
16.6, 11.6Hz, 1H), 2.19 (s, 3H), 1.56 (d, J = 6.2 Hz, 4H). 13C
NMR (150 MHz, CDCl3) δ 169.9, 156.0, 137.6, 135.8, 125.7,
120.0, 109.3, 75.7, 31.7, 20.8, 17.9. ESI-MS (m/z): 227.1,
[M+H]+. HRESI-MS (m/z): [M+H]+ Calcd. for C11H12ClO3:
227.0469, found: 227.0468.
o
in 12.0 mL of water. The reaction was stirred for 30 min at 0 C.
Then KI (3.98 g, 24.0 mmol) was added to the solution. The
reaction was heated to 90 oC for 6 h. The reaction was cooled and
quenched with saturated aqueous NH4Cl, and extracted with
CH2Cl2. The combined extracts were washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel chromatography.
4.3.1.3. (R)-7-bromo-8-hydroxy-3,5-dimethylisochroman-1-
one (9jR). White solid (yield: 96%); Rf = 0.20 (PE/EtOAc 20:1).
4.2.1.1.
8-methoxy-3-methyl-1H-isochromen-1-one
(11).
1
o
[α]20 -69.1 (c 0.20, CHCl3). mp: 165–167 C. IR (film) νmax
:
White solid (yield: 85%); Rf = 0.24 (CH2Cl2/EtOAc 1/1). H
NMR (400 MHz, CDCl3) δ 12.17 (s, 1H), 11.35 (s, 1H), 7.41 (t, J
= 8.4 Hz, 1H), 6.72 (d, J = 8.5 Hz, 1H), 6.49 (d, J = 8.3 Hz, 1H),
4.07 (s, 3H). ESI-MS (m/z):278.1 [M+H]+.
D
2953–2851, 1662, 1366, 1377, 1294, 802, 770 cm-1. H NMR
(400 MHz, CDCl3) δ 11.66 (br s, 1H), 7.57 (s, 1H), 4.89–4.56 (m,
1H), 2.92 (dd, J = 3.2, 16.8 Hz, 1H), 2.69 (dd, J = 11.7, 16.8 Hz,
1H), 2.20 (s, 3H), 1.56 (d, J = 6.3 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δ 169.7, 159.2, 146.7, 137.7, 127.1, 108.2, 82.6, 75.6,
31.7, 20.8, 17.8. ESI-MS (m/z): 270.0 [M+H]+. HRESI-MS (m/z):
[M+H]+ Calcd. for C11H12BrO3: 270.9964, found: 270.9962.
1
4.2.2. The procedure for the synthesis of compounds (8c).
Acetylacetone (1.1 mL, 10.8 mmol) was added to
a
suspension of 11 (1.50 g, 5.40 mmol), CuI (103 mg, 0.54 mmol)
and Cs2CO3 (1.76 g, 5.40 mmol) in anhydrous DMSO (10 mL) in
a 50 mL Schlenk tube under an Argon atmosphere and stirred at
120 °C overnight. The resulting mixture was quenched with
saturated aqueous NH4Cl, and extracted with CH2Cl2. The
combined extracts were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica gel
chromatography.
4.3.1.4. (R)-8-hydroxy-7-iodo-3,5-dimethylisochroman-1-one
(9kR). White solid (yield: 94%). Rf = 0.24 (PE/EtOAc 15/1).
o
[α]20 -63.5 (c 0.30, CHCl3). mp: 164–168 C. IR (film) νmax
:
D
2953–2851, 1660, 1389, 1364, 1293, 1216, 804 cm-1. H NMR
(400 MHz, CDCl3) δ 11.84 (br s, 1H), 7.79 (s, 1H), 4.77–4.61 (m,
1H), 2.92 (dd, J = 3.2,16.8 Hz, 1H), 2.69 (dd, J = 16.8, 11.7 Hz,
1H), 2.18 (s, 3H), 1.55 (d, J = 6.3 Hz, 3H).13C NMR (150 MHz,
CDCl3) δ 169.7, 159.2, 146.7, 137.7, 127.1, 108.2, 82.6, 75.5,
1
4.2.2.1.
8-methoxy-3-methyl-1H-isochromen-1-one
(8c).
Yellow solid (yield: 49%); Rf = 0.28 (PE/EtOAc 2.5/1). 1H NMR

31.7, 20.8, 17.7. ESI-MS (m/z): 319.0 [M+H]+. HRESI-MS (m/z):
2H), 2.17 (s, 3H), 1.58 (m, 2H), 1.54 (d, J = 6.3 Hz, 3H), 1.38
(dt, J = 14.7, 7.3 Hz, 2H), 0.93 (t, J = 7.3 Hz, 3H). 13C NMR (150
MHz, CDCl3) δ 170.8, 158.7, 138.1, 134.2, 129.4, 124.0, 107.5,
75.5, 31.8, 31.7, 29.1, 22.6, 20.9, 18.0, 13.9. ESI-MS (m/z):
249.2 [M+H]+, HRESI-MS (m/z): [M+H]+ Calcd. for C17H18NO3:
249.1485 found: 249.1487.
[M+H]+ Calcd. for C11H12IO3: 318.9826, found: 318.9825.
4.3.2. General procedure for the synthesis of compounds
(9eR and 9lR–9oR).
To a solution of 9kR or 9dR (0.24 mmol) in 10.0 mL of
anhydrous 1,4-dioxane charged with argon was added organic
zinc reagent (0.72mmol) and Pd(dppf)Cl2 (27.0 mg, 0.04 mmol).
The reaction mixture was refluxed under argon until TLC
analysis showed complete consumption of the starting material.
The resulting mixture was quenched with saturated aqueous
NH4Cl, concentrated in vacuo and extracted with CH2Cl2. The
combined extracts were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica gel
chromatography.
4.4.1. The procedure for the synthesis of compound (9pR).
4.4.1.1. (R)-8-hydroxy-7-formyl-3,5-dimethylisochroman-1-
one (9pR). Yellow solid (yield: 85%). Rf = 0.25 (PE/EtOAc 8/1).
o
[α]20 -137 (c 0.20, CHCl3). mp: 152–154 C. IR (film) νmax
:
D
2955–2858, 1659, 1613, 1453, 1478,1402, 1392, 1260, 1183,
1139, 1038, 806 cm-1. 1H NMR (400 MHz, CDCl3) δ 11.72 (s,
1H), 10.44 (s, 1H), 7.84 (s, 1H), 4.81–4.65 (m, 1H), 3.00 (dd, J =
17.2, 3.4 Hz, 1H), 2.77 (dd, J = 17.3, 11.5 Hz, 1H), 2.24 (s, 3H),
1.57 (d, J = 6.3 Hz, 3H). 13C NMR (150MHz, CDCl3) δ 188.3,
169.7, 162.8, 144.8, 135.6, 125.6, 122.6, 109.5, 75.3, 32.3, 20.8,
18.0. ESI-MS (m/z): 221.0 [M+H]+, 243.0 [M+Na]+, HRESI-MS
(m/z): [M+H]+ Calcd. for C11H13O3: 221.0808, found: 221.0806.
4.3.2.1.
(R)-5-ethyl-8-hydroxy-3-methylisochroman-1-one
(9eR). White solid (yield: 63%). Rf = 0.27 (PE/EtOAc 30/1).
[α]20 -67.0 (c 0.20, CHCl3). mp: 91–93 oC. IR (film) νmax: 2963,
D
1
2927, 1674, 1478, 1385, 1220, 1130, 1071, 836 cm-1. H NMR
(400 MHz, CDCl3) δ 11.04 (s, 1H), 7.31 (d, J = 8.6 Hz, 1H), 6.85
(d, J = 8.6 Hz, 1H), 4.75–4.61 (m, 1H), 3.00 (dd, J = 16.4, 3.1
Hz, 1H), 2.75 (dd, J = 16.4, 11.5 Hz, 1H), 2.54 (q, J = 7.5 Hz,
2H), 1.55 (d, J = 6.3 Hz, 3H), 1.15 (t, J = 7.5 Hz, 3H). 13C NMR
(150 MHz, CDCl3) δ 160.6, 136.6, 136.4, 131.1, 116.0, 108.1,
75.4, 31.4, 25.0, 20.9, 14.9. ESI-MS (m/z): 207.1 [M+H]+.
4.4.2. The procedure for the synthesis of compounds (9qR).
A mixture of 9pR (300.0 mg, 1.36 mmol), 10% Pd/C (30.0
mg) in mixture solution of EtOH and EtOAc (1:1) (10 mL) was
stirred under H2 at room temperature and atmospheric pressure
for 3.5 h. The resulting mixture was quenched with saturated
aqueous NH4Cl, then filtered, concentrated in vacuo and
extracted with CH2Cl2. The combined extracts were washed with
brine, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by silica gel chromatography.
4.3.2.2. (R)-8-hydroxy-3,5,7-trimethylisochroman-1-one 9lR.
Rf = 0.26 (PE/EtOAc 40/1). White solid (yield: 81%). [α]20D -103
o
(c 0.20, CHCl3). mp: 178–180 C. IR (film) νmax: 2921, 1724,
1
1663, 1622, 1468, 1385, 1295, 1187, 1134, 1027, 801 cm-1. H
4.4.2.1.
(R)-8-hydroxy-7-(hydroxymethyl)-3,5-
NMR (400 MHz, CDCl3) δ 11.22 (s, 1H), 7.16 (s, 1H), 4.74–4.57
(m, 1H), 2.91 (dd, J = 16.4, 3.2 Hz, 1H), 2.68 (dd, J = 16.4, 11.7
Hz, 1H), 2.21 (s, 3H), 2.16 (s, 3H), 1.53 (d, J = 6.3 Hz, 3H). 13C
NMR (150 MHz, CDCl3) δ 170.7, 158.8, 138.9, 134.1, 124.6,
124.0, 107.3, 75.6, 31.8, 20.9, 17.9, 15.3. ESI-MS (m/z):207.2
[M+H]+, HRESI-MS (m/z): [M+H]+ Calcd. for C12H15O3:
207.1016 found: 207.1014.
dimethylisochroman-1-one (9qR). White solid (yield: 93%). Rf =
0.22 (PE/EtOAc 30/1). [α]20D -89.6 (c 0.30, CHCl3). mp: 122–124
oC. IR (film) νmax: 3397, 2924, 1664, 1618, 1452, 1478,1430,
1
1386, 1295, 1182, 1138, 1058, 1019, 804 cm-1. H NMR (400
MHz, CDCl3) δ 11.35 (s, 1H), 7.33 (s, 1H), 4.68 (s, 3H), 2.94
(dd, J = 16.6, 2.7 Hz, 1H), 2.71 (dd, J = 16.5, 11.7 Hz, 1H), 2.52
(s, 1H), 2.19 (s, 3H), 1.54 (d, J = 6.3 Hz, 3H). 13C NMR (150
MHz, CDCl3) δ 170.5, 158.3, 136.9, 136.3, 127.2, 124.7, 107.9,
75.7, 61.0, 31.8, 20.6, 18.0. ESI-MS (m/z): 221.1 [M-H]-,
HRESI-MS (m/z): [M+Na]+ Calcd. for C12H15O4: 245.0784,
found: 245.0784.
4.3.2.3. (R)-7-ethyl-8-hydroxy-3,5-dimethylisochroman-1-one
(9mR). White solid (yield: 63%). Rf = 0.24 (PE/EtOAc 30/1).
[α]20D -84.6 (c 0.30, CHCl3). mp: 70–72 oC. IR (film) νmax: 2962–
2926, 1667, 1616, 1456, 1386, 1292, 1244, 1182, 1139, 1048,
1
807 cm-1. H NMR (400 MHz, CDCl3) δ 11.24 (s, 1H), 7.17 (s,
4.4.3. The procedure for the synthesis of compounds 9rR.
1H), 4.73–4.60 (m, 1H), 2.91 (brd, J = 16.3 Hz, 1H), 2.73–2.60
(m, 3H), 2.18 (s, 3H), 1.54 (d, J = 6.1 Hz, 3H), 1.20 (t, J = 7.4
Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.8 , 158.6, 137.4,
134.1, 130.6, 124.1, 107.5, 75.5, 31.8, 22.6, 20.9, 18.0, 13.8. ESI-
MS (m/z): 221.2 [M+H]+, HRESI-MS (m/z): [M+H]+ Calcd. for
C13H17O3: 221.1172, found: 221.1177.
To a solution of (9pR) (100.0 mg, 0.46 mmol) in THF/H2O
(1/1, v/v) (20.0 mL) was added Na2HPO4 (327 mg, 2.73 mmol),
30% H2O2 (0.14 mL, 1.36 mmol), NaClO2 (123 mg, 1.36 mmol),
stirred at room temperature for 2 h. The resulting mixture was
quenched with saturated aqueous Na2S2O3, concentrated in vacuo
and extracted with CH2Cl2. The combined extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography.
4.3.2.4. (R)-8-hydroxyl-3,5-dimethyl-7-propylisochroman-1-
one (9nR). White liquid (yield: 31%). Rf = 0.19 (PE/EtOAc 30/1).
[α]20D -74.6 (c 0.10, CHCl3). 1H NMR (400 MHz, CDCl3) δ 11.24
(s, 1H), 7.15 (s, 1H), 4.73–4.59 (m, 1H), 2.91 (dd, J = 16.6, 3.3
Hz, 1H), 2.69 (dd, J = 16.5, 11.8 Hz, 1H), 2.58 (t, J = 7.8 Hz,
3H), 2.17 (s, 3H), 1.64–1.59 (m, 2H), 1.54 (d, J = 6.2 Hz, 3H),
0.95 (t, J = 7.3 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.8,
158.7, 138.2, 134.2, 129.1, 124.0, 107.5, 75.5, 31.8, 31.5, 22.7,
20.9, 18.0, 14.0. ESI-MS (m/z): 235.2 [M+H]+, HRESI-MS (m/z):
[M+H]+ Calcd. for C14H19O3: 235.1329, found: 235.1336.
4.4.3.1.
(R)-8-hydroxy-3,5-dimethyl-1-oxoisochromane-7-
carboxylic acid (9rR). White solid (yield: 51%). Rf = 0.34
(PE/EtOAc 1/3). IR (film) νmax: 3277, 2963–2851, 1732, 1698,
1667, 1607, 1392, 1380, 1267, 1208, 1190, 1132, 1075, 821 cm-1.
1H NMR (CDCl3, 400 MHz) δ 13.22 (1H, s), 8.21 (1H, s), 4.78
(1H, m), 3.05 (1H, d, J = 17.2 Hz), 2.82 (1H, dd, J = 17.2, 11.8
Hz), 2.28 (3H, s), 1.61 (3H, d, J = 6.3 Hz). 13C NMR (CDCl3, 150
MHz) δ 170.4, 164.5, 158.8, 143.9, 141.0, 127.04, 115.4, 109.0,
76.0, 32.0, 20.8, 18.0. ESI-MS (m/z): 237.1 [M+H]+, HRESI-MS
(m/z): [M+H]+ Calcd. for C16H16NO3: 237.0758 found: 237.0756.
4.3.2.5. (R)-7-butyl-8-hydroxy-3,5-dimethylisochroman-1-one
(9oR). Gray liquid (yield: 17%). Rf= 0.26 (PE/EtOAc 30/1).
[α]20 -81.4 (c 0.10, CHCl3). IR (film) νmax: 2928, 1668, 1456,
D
1386, 1225, 1181, 1139, 807 cm-1. 1H NMR (400 MHz, CDCl3) δ
11.24 (s, 1H), 7.15 (s, 1H), 4.66 (s, 1H), 2.91 (dd, J = 16.5, 2.8
Hz, 1H), 2.69 (dd, J = 16.5, 11.7 Hz, 1H), 2.60 (t, J = 7.4 Hz,
4.4.4. The procedure for the synthesis of compound (9sR).
4.4.4.1.
(R)-8-hydroxy-3,5-dimethyl-1-oxoisochromane-7-
carbonitrile (9sR). White solid (yield: 43%). Rf = 0.21

o
(PE/EtOAc 5/2). [α]20 -87.8 (c 0.20, CHCl3). mp: 206–208 C.
1H), 4.72–4.56 (m, 1H), 3.79 (s, 2H), 2.85 (dd, J = 16.4, 3.2 Hz,
1H), 2.63 (dd, J = 16.3, 11.5 Hz, 1H), 2.12 (s, 3H), 1.52 (d, J =
6.3 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.9, 148.3, 134.0,
125.3, 124.6, 122.3, 107.5, 76.2, 31.3, 20.9, 18.0. ESI-MS (m/z):
208.1 [M+H]+, HRESI-MS (m/z): [M+H]+ Calcd. for C11H14NO3:
208.0968 found: 288.0966.
D
IR (film) νmax: 2955–2923, 2225, 1670, 1618, 1395, 1369, 1308,
1271, 1199, 1039, 802 cm-1. 1H NMR (400 MHz, CDCl3) δ 11.72
(s, 1H), 7.55 (s, 1H), 4.82–4.65 (m, 1H), 3.01 (dd, J = 17.2, 3.3
Hz, 1H), 2.78 (dd, J = 17.2, 11.5 Hz, 1H), 2.24 (s, 3H), 1.58 (d, J
= 6.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 169.1, 162.0,
143.2, 140.2, 126.1, 115.1, 109.3, 100.1, 75.4, 32.0, 20.8, 18.0.
ESI-MS (m/z): 218.1 [M+H]+, HRESI-MS (m/z): [M+H]+ Calcd.
for C12H12NO3: 218.0812, found: 218.0814.
4.4.8. The procedure for the synthesis of compound (9xR).
To a solution of 9wR (156.0 mg, 0.43 mmol) in DMF/H2O
(15.0 mL, 1/2) was added NaOH (102 mg, 2.54 mmol), the
mixture stirred at room temperature for 0.5 h. Then added
dimethyl sulfate (143 μL, 1.51 mmol) to the mixture and stirred
at room temperature for 0.5 h. The resulting mixture was
quenched with saturated aqueous NH4Cl, concentrated in vacuo
and extracted with CH2Cl2. The combined extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography.
4.4.5. The procedure for the synthesis of compound (9tR).
To a solution of 9pR (70.0 mg, 0.30 mmol) in ClCH2CH2Cl
(20.0 mL) was added p-toluenesulfonic acid (52.0 mg, 0.30
mmol), p-anisidine (106.0 µL, 0.91 mmol), NaBH(OAc)3 (193.0
mg, 0.91mmol). The reaction mixture was stirred at room
temperature for 3.5 h under argon atmosphere. The resulting
mixture was quenched with saturated aqueous NaHCO3,
concentrated in vacuo and extracted with CH2Cl2. The combined
extracts were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
chromatography.
4.4.8.1. (R)-7-(dimethylamino)-8-hydroxy-3,5-dimethyl-3,4-
dihydronaphthalen-1(2H)-one (9xR). Yellow solid (yield: 16%).
Rf = 0.27 (PE/EtOAc 6/1). [α]20D -91.2 (c 0.10, CHCl3). Mp: 153–
o
155 C. IR(film)νmax: 2918–2778, 1660, 1487, 1448, 1385, 1341,
1
4.4.5.1. (R)-8-hydroxy-7-(((4-methoxyphenyl)amino)methyl)-
3,5-dimethylisochroman-1-one (9tR). Yellow solid (yield: 66%).
Rf = 0.29 (PE/EtOAc 6/1). [α]20D -77.4 (c 0.30, CHCl3). mp: 120–
1289, 1235, 1179, 1148, 1051, 804 cm-1. H NMR (400MHz,
CDCl3) δ 11.60 (s, 1H), 6.91 (s, 1H), 4.74–4.50 (m, 1H), 2.90
(dd, J = 16.4, 3.5 Hz, 2H), 2.81 (s, 6H), 2.67 (dd, J = 16.4, 11.6
Hz, 2H), 2.19 (s, 4H), 1.53 (d, J = 6.2 Hz, 3H). 13C NMR (150
MHz, CDCl3) δ 171.1, 154.1, 140.3, 128.8, 126.0, 124.2, 107.9,
75.8, 42.9, 31.6, 20.9, 18.3. ESI-MS (m/z): 236.3 [M+H]+,
HRESI-MS (m/z): [M+H]+ Calcd. for C13H18NO3: 236.1281
found: 236.1283.
o
121 C. IR (film) νmax: 2925, 1664, 1617, 1512, 1455, 1386,
1293, 1364, 1234, 1180, 1084, 1137, 1034, 820 cm-1. H NMR
1
(400 MHz, CDCl3) δ 11.36 (s, 1H), 7.33 (s, 1H), 6.76 (d, J = 8.8
Hz, 2H), 6.62 (d, J = 8.8 Hz, 2H), 4.74–4.60 (m, 1H), 4.30 (s,
2H), 3.73 (s, 3H), 2.91 (dd, J = 16.6, 3.0 Hz, 1H), 2.69 (dd, J =
16.5, 11.7 Hz, 1H), 2.16 (s, 3H), 1.54 (d, J = 6.3 Hz, 3H). 13
C
4.4.9. The procedure for the synthesis of compound (9yR).
NMR (150 MHz, CDCl3) δ 170.6, 158.5, 152.3, 142.2, 137.2,
135.7, 125.8, 124.5, 114.8, 114.6, 107.82, 75.6, 55.7, 43.9, 31.8,
20.9, 18.1. ESI-MS (m/z): 326.1 [M-H]-, HRESI-MS (m/z):
[M+H]+ Calcd. for C19H22NO4: 328.1543 found: 328.1542.
To a solution of 9wR (60.0 mg, 0.29 mmol) in anhydrous
CH2Cl2 (20.0 mL) was added acryl chloride (7.2 µL, 0.87 mmol).
Then the mixture stirred at room temperature for 6 h. The
resulting mixture was quenched with saturated aqueous NH4Cl,
concentrated in vacuo and extracted with CH2Cl2. The combined
extracts were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
chromatography.
4.4.6. The procedure for the synthesis of compound (9uR).
To a solution of nitronium tetrafluoroborate (1.04 g, 7.81
mmol) in anhydrous CH2Cl2 (20.0 mL) was added dropwose the
mixture of 9aR (1.0 g, 5.21 mmol) in anhydrous CH2Cl2 (15.0
o
mL) at -20 C. Then the reaction mixture was stirred at room
4.4.9.1. (R)-N-(8-hydroxy-3,5-dimethyl-1-oxoisochroman-7-
temperature overnight. The resulting mixture was quenched with
saturated aqueous NH4Cl, concentrated in vacuo and extracted
with CH2Cl2. The combined extracts were washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel chromatography.
yl)acrylamide (9yR). White solid (yield: 46%). Rf = 0.26
o
(PE/EtOAc 3/1). [α]20 -96.2 (c 0.30, CHCl3). mp: 126–128 C.
D
IR(film)νmax: 3339, 2925, 1667, 1624, 1530, 1425, 1387, 1372,
1
1263, 1210, 1184, 1139, 1045, 801 cm-1. H NMR (400 MHz,
CDCl3) δ 11.43 (s, 1H), 8.51 (s, 1H), 7.85 (s, 1H), 6.42 (d, J =
16.7 Hz, 1H), 6.37–6.24 (m, 1H), 5.77 (d, J = 9.8 Hz, 1H), 4.68
(s, 1H), 2.91 (dd, J = 16.6, 2.8 Hz, 1H), 2.68 (dd, J = 16.5, 11.8
Hz, 1H), 2.19 (s, 3H), 1.53 (d, J = 5.8 Hz, 3H). 13C NMR (150
MHz, CDCl3) δ 170.5, 163.4, 149.1, 131.2, 131.0, 127.7, 127.6,
125.5, 125.1, 107.3, 76.1, 31.4, 20.8, 18.3. ESI-MS (m/z): 262.2
[M+H]+, HRESI-MS (m/z): [M+H]+ Calcd. for C14H16NO4:
262.1074 found: 262.1076.
4.4.6.1. (R)-8-hydroxy-3,5-dimethyl-7-nitroisochroman-1-one
(9uR). Yellow solid (yield: 36%). Rf = 0.21 (PE/EtOAc 5/1).
[α]20 -197 (c 0.20, CHCl3). mp: 192–194 C. IR (film) νmax
:
o
D
2926, 1674, 1582 1518, 1385, 1340, 1311, 1263, 1179, 1143,
1084, 1079, 764 cm-1. H NMR (400 MHz, CDCl3) δ 12.39 (s,
1
1H), 8.03 (s, 1H), 4.84–4.66 (m, 1H), 3.04 (dd, J = 17.2, 2.7 Hz,
1H), 2.81 (dd, J = 17.1, 11.7 Hz, 1H), 2.28 (s, 3H), 1.59 (d, J =
6.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 169.2, 154.8, 144.4,
136.1, 132.9, 125.3, 110.8, 75.4, 32.1, 20.7, 18.0. ESI-MS
(m/z):236.0 [M-H]-, HRESI-MS (m/z): [M+H]+ Calcd. for
C11H12NO5: 238.0710 found: 238.0711.
4.5.1. The procedure for the synthesis of compound (8rR).
A mixture of 9rR (1.56 mmol), K2CO3 (744.0 mg, 4.68
mmol), and iodomethane (1.80 mmol) in DMF/H2O (1/2, v/v) (10
mL) was stirred for 5 h at room temperature. The resulting
mixture was quenched with saturated aqueous NH4Cl,
concentrated in vacuo and extracted with CH2Cl2. The combined
extracts were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
chromatography.
4.4.7. The procedure for the synthesis of compound (9wR).
The procedure is same as the preparation of 9qR in 4.4.2.
4.4.7.1. (R)-7-amino-8-hydroxy-3,5-dimethylisochroman-1-
one(9wR). Yellow solid (yield: 79%). Rf = 0.26 (PE/EtOAc 4/1).
[α]20 -118 (c 0.20, CHCl3). mp: 172–174 C. IR (film) νmax
:
o
D
3370, 2926, 1664, 1605, 1486, 1385, 1324, 1181, 1138, 1049,
4.5.2. General procedure for the synthesis of compounds
(10aR‒eR).
1
801 cm-1. H NMR (400 MHz, CDCl3) δ 11.04 (s, 1H), 6.74 (s,

Method 1. The appropriate benzoic acid (9rR) (0.32mmol)
was converted into its corresponding acid chloride by treatment
with thionyl chloride (4 mL, 56.0 mmol), then added this
intermediate to the aqueous solution of amine (0.64 mmol) in
CH2Cl2 (10.0 mL) at room temperature for 1 h. The resulting
mixture was quenched with saturated aqueous NH4Cl,
concentrated in vacuo and extracted with CH2Cl2. The combined
extracts were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was purified by silica gel
chromatography.
J = 5.9 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.0, 166.1,
156.1, 138.7, 136.7, 125.5, 123.8, 108.3, 75.5, 55.2, 54.6, 46.9,
46.0, 41.7, 31.8, 20.8, 18.0. ESI-MS (m/z): 319.2 [M+H]+,
HRESI-MS (m/z): [M+H]+ Calcd. for C17H23N2O4: 319.1652
found: 319.1655.
4.5.2.5. (R)-8-hydroxy-3,5-dimethyl-N-(2-morpholinoethyl)-1-
oxoisochromane-7-carboxamide (10eR). Gray solid (yield: 67%).
Rf = 0.28 (MeOH/CH2Cl2 1/40). [α]20 -67.3 (c 0.20, CHCl3).
D
o
Mp: 160–162 C. IR (film) νmax: 2952–2851, 1672, 1641, 1525,
1454, 1396, 1354, 1255, 1116, 1019, 808 cm-1. H NMR (400
1
Method 2. To a solution of 8rR or 9rR (0.32 mmol) in
MHz, CDCl3) δ 12.63 (s, 1H), 8.50 (s, 1H), 8.26 (s, 1H), 4.84–
4.61 (m, 1H), 3.73 (m, 4H), 3.59 (q, J = 6.0 Hz, 2H), 3.00 (dd, J
= 16.9, 3.3 Hz, 1H), 2.77 (dd, J = 16.9, 11.7 Hz, 1H), 2.59 (t, J =
6.2 Hz, 2H), 2.51 (s, 4H), 2.24 (s, 3H), 1.57 (d, J = 6.3 Hz, 3H).
13C NMR (150 MHz, CDCl3) δ 170.8, 164.1, 158.7, 141.2, 140.2,
125.6, 119.3, 75.6, 67.0, 56.9, 53.3, 36.5, 32.0, 20.8, 18.0. ESI-
MS (m/z): 371.2 [M+H]+, HRESI-MS (m/z): [M+Na]+ Calcd. for
C18H24N2O5Na: 371.1577 found: 371.1587.
anhydrous
CH2Cl2
(20.0
mL)
was
added
1-[3-
(dimethylamino)propyl]-3-ethylcarbodiimide
hydrochloride
(EDCI, 123.0 mg, 0.64 mmol), HOBt (0.96 mmol), amine (0.48
mmol). Then the mixture stirred at room temperature overnight.
The resulting mixture was quenched with saturated aqueous
NH4Cl, concentrated in vacuo and extracted with CH2Cl2. The
combined extracts were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by silica gel
chromatography.
4.6.1. General procedure for the synthesis of compounds
(11aR‒hR, 12aR‒cR, 13aR‒bR).
4.5.2.1.
(R)-8-hydroxy-N,N,3,5-tetramethyl-1-
oxoisochromane-7-carboxamide (10aR). White solid (yield:
72%). Rf = 0.38 (CH2Cl2/EtOAc 10/1). [α]20 -82.7 (c 0.10,
Method 1. General reaction for the synthesis of 11aR‒gR and
13bR or 9zR (0.30 mmol), boronic acid or its pinacol ester (0.39
mmol), K2CO3 (125 mg, 0.90 mmol) and Pd(Ph3P)4 (10 mol%)
were suspended in 1,4-dioxane (10 mL) and water (2 drops) and
vial was sealed tightly. The mixture was irradiated for 1 h at a
pre-selected temperature of 100 °C, using a maximum irradiation
power of 150 W. After the reaction, the vial was cooled to 60 °C
by air jet cooling. The resulting mixture was quenched with
saturated aqueous NH4Cl, concentrated in vacuo and extracted
with CH2Cl2. The combined extracts were washed with brine,
dried over Na2SO4, and concentrated in vacuo. The residue was
purified by silica gel chromatography. Then the intermediate
released free phenyl hydroxyl according to the reaction of 4.1.3,
producing the target compounds.
D
CHCl3). mp: 156–158 oC. IR (film) νmax: 2925, 1669, 1634, 1503,
1454, 1424, 1389, 1259, 1190, 1148, 1068, 1019, 861 cm-1. H
1
NMR (400 MHz, CDCl3) δ 11.34 (s, 1H), 7.35 (s, 1H), 4.75–4.62
(m, 1H), 3.12 (s, 3H), 2.99–2.92 (m, 4H), 2.73 (dd, J = 16.8, 11.5
Hz, 1H), 2.20 (s, 3H), 1.56 (d, J = 6.3 Hz, 3H). 13C NMR (150
MHz, CDCl3) δ 170.1, 167.8, 156.2, 138.6, 136.6, 125.4, 124.5,
108.3, 75.5, 38.2, 34.9, 31.8, 20.8, 18.0. ESI-MS (m/z): 264.1
[M+H]+, HRESI-MS (m/z): 264.1 [M+Na]+ Calcd. for
C14H17NO4Na: 264.1230 found: 264.1233.
4.5.2.2.
(R)-N,N-diethyl-8-hydroxy-3,5-dimethyl-1-
oxoisochromane-7-carboxamide (10bR). White solid (yield:
71%). Rf = 0.28 (CH2Cl2/EtOAc 30/1). [α]20 -68.6 (c 0.20,
D
CHCl3). Mp: 180–182 oC. IR (film) νmax: 2925, 1669, 1620, 1432,
1379, 1362, 1372, 1253, 1210, 1184, 1146, 1072, 810 cm-1. H
1
Method 2. General procedure–The Suzuki-Miyaura reaction
for the synthesis of 13bR in toluene/EtOH/H2O. The mixture was
refluxed at 110 °C overnight. The remaining steps were the
similar to Method 1.
NMR (400 MHz, CDCl3) δ 11.23 (s, 1H), 7.27 (s, 1H), 4.69 (m,
1H), 3.57 (q, J = 6.8 Hz, 2H), 3.22 (q, J = 7.2 Hz, 2H), 2.95 (dd,
J = 16.8, 3.2 Hz, 1H), 2.72 (dd, J = 16.8, 11.6 Hz, 1H), 2.19 (s,
3H), 1.55 (d, J = 6.3 Hz, 3H), 1.24 (t, J = 7.2 Hz, 3H), 1.08 (t, J =
6.8 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.0, 167.2, 156.1,
138.0, 135.9, 125.2, 125.1, 108.3, 75.5, 42.9, 39.1, 31.8, 20.8,
18.0, 14.1, 12.9. ESI-MS (m/z): 292.2 [M+H]+, HRESI-MS (m/z):
[M+Na]+ Calcd. for C16H21NO4Na: 292.1543 found: 292.1542.
Method 3. General procedure–Conventional Stille coupling
reaction for the synthesis of 11gR. 8kR (100 mg, 0.30 mmol),
CuI (11.0 mg, 0.06 mmol), CsF (60.3 mg, 0.40 mmol),
PdCl2(PPh3)2
(17.0
mg,
0.03
mmol),
4-(Tri-n-
butylstannyl)pyridine (100.0 mg, 0.27 mmol), were suspended in
anhydrous DMF (7.0 mL) under argon atmosphere. The mixture
was refluxed at 120 °C overnight. The resulting mixture was
quenched with saturated aqueous NH4Cl, concentrated in vacuo
and extracted with CH2Cl2. The combined extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography.
4.5.2.3.
(R)-8-hydroxy-3,5-dimethyl-7-(morpholine-4-
carbonyl)isochroman-1-one (10cR). White solid (yield: 52%). Rf
= 0.32 (PE/EtOAc 2/3). [α]20 -88.3 (c 0.20, CHCl3). Mp: 312–
D
314 oC. IR (film) νmax: 2957–2925, 1673, 1633, 1457, 1391,
1362, 1268, 1211, 1177, 1139, 1114, 1008, 810 cm-1. H NMR
1
(400 MHz, CDCl3) δ 11.37 (s, 1H), 7.37 (s, 1H), 4.69 (m, 1H),
3.77 (br s, 4H), 3.67 (brs, 2H), 3.33 (s, 2H), 2.96 (dd, J = 16.5,
2.8 Hz, 1H), 2.73 (dd, J = 16.5, 11.8 Hz, 1H), 2.20 (s, 3H), 1.56
(d, J = 6.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.0, 166.3,
156.1, 139.0, 136.9, 125.67, 123.4, 108.3, 75.5, 67.0, 66.8, 47.5,
42.4, 31.8, 20.8, 18.0. ESI-MS (m/z): 306.1 [M+H]+, HRESI-MS
(m/z): [M+H]+ Calcd. for C16H20NO5: 306.1336 found: 306.1341.
Method 4. General procedure–Conventional Suzuki-Miyaura
reaction for the synthesis of 12aR‒13aR. 9kR or 9zR (0.30
mmol), boronic acid or its pinacol ester (0.39 mmol), CuCl (31.3
mg, 0.31 mmol), dppf (18.0 mg, 0.03 mmol), Pd(OAc)2 (7.3 mg,
0.01 mmol), CsCO3 (410.6 mg, 1.27 mmol), were suspended in
anhydrous DMF (7.0 mL) under argon atmosphere. The mixture
was refluxed at 120 °C overnight. The resulting mixture was
quenched with saturated aqueous NH4Cl, concentrated in vacuo
and extracted with CH2Cl2. The combined extracts were washed
with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified by silica gel chromatography.
4.5.2.4. (R)-8-hydroxy-3,5-dimethyl-7-(4-methylpiperazine-1-
carbonyl)isochroman-1-one (10dR). Yellow solid (yield: 53%).
Rf = 0.24 (CH2Cl2/MeOH 40/1). H NMR (400 MHz, CDCl3) δ
11.32 (s, 1H), 7.35 (s, 1H), 4.69 (m, 1H), 3.82 (s, 2H), 3.34 (s,
2H), 2.96 (dd, J = 16.6, 3.5 Hz, 1H), 2.73 (dd, J = 16.6, 12.1 Hz,
1H), 2.48 (s, 2H), 2.39 (s, 2H), 2.31 (s, 3H), 2.20 (s, 3H), 1.56 (d,
1

4.6.1.1. (R)-8-hydroxy-3,5-dimethyl-7-phenylisochroman-1-
4.6.1.6.
(R)-8-hydroxyl-3,5-dimethyl-7-(3-
one (11aR). Yellow solid (yield: 75%). Rf = 0.31 (PE/CH2Cl2
nitrophenyl)isochroman-1-one (11fR). White solid (yield: 56%).
Rf = 0.33 (PE/EtOAc 3/1). [α]20D -85.0 (c 0.30, CHCl3). mp: 245–
247 oC. IR (film) νmax: 2926, 1664, 1619, 1526, 1457, 1385, 1349,
1324, 1197, 1136, 1057, 804 cm-1. 1H NMR (400 MHz, CDCl3) δ
11.68 (s, 1H), 8.44 (s, 1H), 8.19 (d, J = 8.2 Hz, 1H), 7.96 (d, J =
7.7 Hz, 1H), 7.59 (dd, J = 8.2,7.7 Hz, 1H), 7.42 (s, 1H), 4.81–
4.67 (m, 1H), 3.02 (dd, J = 16.7, 3.1 Hz, 1H), 2.80 (dd, J = 16.7,
11.6 Hz, 1H), 2.28 (s, 3H), 1.59 (d, J = 6.2 Hz, 3H). 13C NMR
(150 MHz, CDCl3) δ 170.5, 157.7, 148.3, 138.5, 138.3, 137.8,
135.4, 129.0, 125.8, 125.3, 124.1, 122.1, 108.7, 75.6, 31.9, 20.9,
18.1. ESI-MS (m/z): 312.0 [M-H]-, HRESI-MS (m/z): [M+H]+
Calcd. for C17H14NO5: 314.1023 found: 314.1023.
2/1). [α]20D -111 (c 0.20, CHCl3). mp: 168–170 oC. IR (film) νmax
:
2928, 1663, 1617, 1458, 1421, 1385, 1322, 1263, 1194, 1131,
1
1057, 806 cm-1. H NMR (400 MHz, CDCl3) δ 11.56 (s, 1H),
7.58 (d, J = 7.6 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.40–7.31 (m,
2H), 4.83–4.65 (m, 1H), 2.99 (dd, J = 16.6, 3.1 Hz, 1H), 2.77
(dd, J = 16.6, 11.7 Hz, 1H), 2.25 (s, 3H), 1.58 (d, J = 5.8 Hz,
3H). 13C NMR (150 MHz, CDCl3) δ 170.7, 157.8, 138.7, 136.9,
136.3, 129.2, 128.5, 128.2, 127.4, 124.8, 108.3, 75.5, 31.9, 20.9,
18.1. ESI-MS (m/z):269.2 [M+H]+, HRESI-MS (m/z): [M+H]+
Calcd. for C17H17O3: 269.1172 found: 269.1178.
4.6.1.2. (R)-8-hydroxyl-3,5-dimethyl-7-(p-tolyl)isochroman-1-
one (11bR). White solid (yield: 58%). Rf = 0.35 (PE/CH2Cl2 2/1).
4.6.1.7.
(R)-7-(3-aminophenyl)-8-hydroxyl-3,5-
o
[α]20 -81.1 (c 0.20, CHCl3). mp: 157–159 C. IR (film) νmax
:
dimethylisochroman-1-one (11gR). Gray solid (yield: 92%). Rf =
D
2925, 1723, 1471, 1385, 1317, 1270, 1195, 1151, 1063, 1041,
821 cm-1. 1H NMR (400 MHz, CDCl3) δ 11.53 (s, 1H), 7.47 (d, J
= 7.5 Hz, 2H), 7.37 (s, 1H), 7.25 (d, J = 7.9 Hz, 2H), 4.80–4.59
(m, 1H), 2.99 (dd, J = 16.5, 3.1 Hz, 1H), 2.77 (dd, J = 16.5,11.7
Hz, 1H), 2.40 (s, 3H), 2.25 (s, 3H), 1.58 (d, J = 5.8 Hz, 3H). 13C
NMR (150 MHz, CDCl3) δ 170.7, 157.9, 138.6, 137.1, 136.0,
134.0, 129.1, 128.9, 128.6, 124.7, 75.5, 32.0, 21.2, 20.9, 18.1.
ESI-MS (m/z): 297.1 [M+H]+, HRESI-MS (m/z): [M+H]+ Calcd.
for C18H19O3: 297.1485 found: 297.1496.
0.18 (PE/EtOAc 3/1). [α]20 -75.5 (c 0.10, CHCl3). mp: 160–162
D
oC. IR (film) νmax: 2923–2851, 1661, 1615, 1455, 1417, 1386,
1327, 1257, 1190, 1131, 1056, 806 cm-1. 1H NMR (400 MHz,
CDCl3) δ 11.54 (s, 1H), 7.37 (s, 1H), 7.22 (t, J = 7.8 Hz, 1H),
6.97–6.89 (m, 2H), 6.69 (d, J = 7.7 Hz, 1H), 4.72 (m, 1H), 3.73
(s, 2H), 2.98 (dd, J = 16.6, 3.3 Hz, 1H), 2.76 (dd, J = 16.6, 11.6
Hz, 1H), 2.23 (s, 3H), 1.57 (d, J = 6.3 Hz, 3H). 13C NMR (150
MHz, CDCl3) δ 170.7, 157.8, 146.2, 138.7, 138.0, 136.2, 129.1,
128.7, 124.6, 119.6, 116.1, 114.3, 108.2, 75.5, 31.9, 20.9, 18.1.
ESI-MS (m/z): 284.2 [M+H]+, HRESI-MS (m/z): [M+H]+ Calcd.
for C17H18NO3: 284.1281 found: 284.1287.
4.6.1.3.
(R)-8-hydroxyl-3,5-dimethyl-7-(4-
(trifluoromethyl)phenyl)isochroman-1-one (11cR). Yellow solid
(yield: 42%). Rf = 0.37 (PE/CH2Cl2 2/1). [α]20 -72.7 (c 0.40,
4.6.1.8.
(R)-8-hydroxyl-3,5-dimethyl-7-(pyridine-4-
D
CHCl3). mp: 141–143 oC. IR (film) νmax: 2926, 1667, 1618, 1402,
yl)isochroman-1-one (11hR). White solid (yield: 14%). Rf = 0.25
1
o
1388, 1324, 1195, 1165, 1123, 1069, 1057, 845 cm-1. H NMR
(PE/EtOAc 5/1). [α]20 -48.2 (c 0.10, CHCl3). mp: 142–144 C.
D
(400 MHz, CDCl3) δ 11.63 (s, 1H), 7.73–7.64 (m, 4H), 7.38 (s,
1H), 4.82–4.61 (m, 1H), 3.00 (d, J = 16.7 Hz, 1H), 2.78 (dd, J =
IR (film) νmax: 2955‒2853, 1668, 1615, 1593, 1544, 1455, 1386,
1
1332, 1267, 1195, 1134, 1058, 825 cm-1. H NMR (400 MHz,
16.7, 11.6 Hz, 1H), 2.26 (s, 3H), 1.58 (d, J = 6.3 Hz, 3H). 13
C
CDCl3) δ 11.69 (s, 1H), 8.64 (d, J = 5.2 Hz, 2H), 7.53 (d, J = 5.2
Hz, 2H), 7.41 (s, 1H), 4.81–4.64 (m, 1H), 3.00 (dd, J = 16.7, 3.1
Hz, 1H), 2.78 (dd, J = 16.7, 11.6 Hz, 1H), 2.26 (s, 3H), 1.58 (d, J
= 6.3 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.5, 157.9,
149.7, 144.6, 138.2, 138.1, 125.4, 125.2, 123.8, 108.7, 75.5, 32.0,
20.9, 18.1. ESI-MS (m/z): 270.1 [M+H]+, HRESI-MS (m/z):
[M+H]+ Calcd. for C17H18NO3: 270.1125 found: 270.1133.
NMR(150 MHz, CDCl3) δ 170.6, 157.8, 140.6, 138.5, 137.4,
129.5, 129.4, 127.0, 125.1, 108.6, 75.5, 31.9, 20.9, 18.1. 19F
NMR (376 MHz, CDCl3) δ -62.97. ESI-MS (m/z): 335.0 [M-H]-,
HRESI-MS (m/z): [M-H]- Calcd. for C18H14F3O3: 335.0900
found: 335.0901.
4.6.1.4.
(R)-8-hydroxyl-7-(4-hydroxyphenyl)-3,5-
dimethylisochroman-1-one (11dR). Yellow solid (yield: 54%). Rf
4.6.1.9.
(R)-8-hydroxyl-3,5-dimethyl-7-(pyridine-3-
= 0.28 (PE/EtOAc 2/1). [α]20 -32.1 (c 0.20, CHCl3). mp: 226–
yl)isochroman-1-one (12aR). Yellow solid (yield: 7%). Rf = 0.22
(PE/EtOAc 2/1). [α]20D -146 (c 0.10, CHCl3). mp: 150–152 oC. IR
(film) νmax: 2923, 1662, 1615, 1448, 1385, 1321, 1290, 1185,
1132, 1059, 809 cm-1. 1H NMR (400 MHz, CDCl3) δ 11.59 (br s,
1H), 8.76 (s, 1H), 8.56 (s, 1H), 7.96 (d, J = 7.9 Hz, 1H), 7.38 (s,
1H), 7.34 (t, J = 6.3 Hz, 1H), 4.81–4.62 (m, 1H), 3.00 (d, J =
16.7 Hz, 1H), 2.78 (dd, J = 16.7, 11.7 Hz, 1H), 2.26 (s, 3H), 1.58
(d, J = 6.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.7, 158.1,
149.9 148.6, 138.5, 137.6, 136.9, 132.9, 125.4, 125.1, 123.2,
108.8, 75.8, 32.1, 21.1, 18.3. ESI-MS (m/z): 270.1 [M+H]+,
HRESI-MS (m/z): [M+H]+ Calcd. for C16H16NO3: 270.1125
found: 270.1127
D
o
228 C. IR (film) νmax: 3332, 2923, 1649, 1611, 1578, 1520,
1422, 1392, 1264, 1199, 1152, 1106, 1056, 804 cm-1. H NMR
1
(400 MHz, CDCl3) δ 11.54 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H), 7.35
(s, 1H), 6.90 (d, J = 8.6 Hz, 2H), 5.12 (s, 1H), 4.80–4.63 (m, 1H),
2.98 (dd, J = 16.6, 3.1 Hz, 1H), 2.76 (dd, J = 16.5, 11.6 Hz, 1H),
2.24 (s, 3H), 1.57 (d, J = 6.3 Hz, 3H). 13C NMR (150 MHz MHz,
CDCl3) δ 170.8, 157.8, 155.0, 138.5, 135.8, 130.5, 129.4, 128.1,
124.7, 115.1, 108.3, 75.5, 31.9, 20.9, 18.1. ESI-MS (m/z): 285.0
[M+H]+, HRESI-MS (m/z): [M-H]- Calcd. for C18H16F3O3:
283.0976 found: 283.0976.
4.6.1.5
(R)-7-(4-aminophenyl)-8-hydroxy-3,5-
dimethylisochroman-1-one (11eR). Yellow solid (yield: 58%). Rf
4.6.1.10.
(R)-8-hydroxyl-3,5-dimethyl-7-(pyridine-2-
= 0.29 (PE/EtOAc 2/1). [α]20 -62.3 (c 0.10, CHCl3). Mp: 91–93
yl)isochroman-1-one (12bR). Yellow solid (yield: 10%). Rf =
D
oC. IR (film) νmax: 3372, 2924, 1660, 1612, 1519, 1455, 1385,
0.18 (PE/EtOAc 2/1). [α]20 -120 (c 0.10, CHCl3). mp: 148–150
D
1
1324, 1289, 1261, 1131, 1056, 831 cm-1. H NMR (400 MHz,
oC. IR (film) νmax: 2920, 1661, 1614, 1583, 1443, 1418, 1385,
1
CDCl3) δ 11.52 (s, 1H), 7.40 (d, J = 8.1 Hz, 2H), 7.34 (s, 1H),
6.75 (d, J = 8.1 Hz, 2H), 4.71 (d, J = 5.3 Hz, 1H), 3.68 (br s, 2H),
2.96 (dd, J = 16.5, 3.1 Hz, 1H), 2.75 (dd, J = 16.5, 11.7 Hz, 1H),
2.23 (s, 3H), 1.57 (d, J = 6.2 Hz, 3H). 13C NMR (151 MHz,
CDCl3) δ 170.8, 157.9, 145.8, 138.3, 135.3, 130.2, 128.6, 127.1,
124.6, 114.8, 108.2, 75.5, 31.9, 20.9, 18.1. ESI-MS (m/z): 284.2
[M+H]+, HRESI-MS (m/z): [M+H]+ Calcd. for C17H18NO3:
284.1281 found: 284.1280.
1318, 1258, 1195, 1134, 1008, 807 cm-1. H NMR (400 MHz,
CDCl3) δ 12.35 (s, 1H), 8.67 (s, 1H), 8.04 (d, J = 8.0 Hz, 1H),
7.96 (s, 1H), 7.75 (t, J = 8.0 Hz, 1H), 7.24 (dd, J = 12.2, 5.9 Hz,
1H), 4.71 (dt, J = 10.9, 5.0 Hz, 1H), 2.99 (d, J = 16.7 Hz, 1H),
2.77 (dd, J = 16.7, 11.6 Hz, 1H), 2.26 (s, 3H), 1.56 (d, J = 6.2 Hz,
3H). 13C NMR (150 MHz, CDCl3) δ 169.9, 158.7, 154.5, 148.9,
138.5, 138.2, 136.2, 125.5, 125.0 124.3 ，122.0, 109.1, 75.2,
32.1, 20.9, 18.2. ESI-MS (m/z): 270.1 [M+H]+, HRESI-MS (m/z):
[M+H]+ Calcd. for C16H16NO3: 270.1125 found: 270.1131.

4.6.1.11.
(R)-8-hydroxyl-3,5-dimethyl-7-(pyrimidin-5-
6. Molecular docking
yl)isochroman-1-one (12cR). Gray solid (yield: 7%). Rf = 0.18
The crystal structures of MAO-A (PDB code: 2Z5Y) and
MAO-B (PDB code: 6FVZ) were obtained from the Protein Data
Bank. Molecular modeling studies were performed using
Schrodinger (Maestro suite). Protein structures were prepared by
Protein Preparation Wizard (PrepWizard) of the chr dinger
suite to add missing hydrogen atoms, adjust bond orders, assign
hydrogen bonds, and relax the protein−ligand complex. The other
parameters were accepted as default.
o
(PE/EtOAc 4/1). [α]20 -31.7 (c 0.10, CHCl3). mp: 181–183 C.
D
IR (film) νmax: 2922, 1661, 1618, 1411, 1387, 1323, 1204, 1137,
1
1057, 999, 804 cm-1. H NMR (400 MHz, CDCl3) δ 11.65 (s,
1H), 9.16 (s, 1H), 8.97 (s, 2H), 7.38 (s, 1H), 4.81–4.65 (m, 1H),
3.01 (dd, J = 16.8, 3.3 Hz, 1H), 2.79 (dd, J = 16.8, 11.6 Hz, 1H),
2.27 (s, 3H), 1.58 (d, J = 6.3 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δ 170.3, 157.8, 157.2, 156.6, 138.4, 137.7, 130.8, 125.6,
121.3, 108.8, 75.6, 31.9, 20.9, 18.1. ESI-MS (m/z): 271.1
[M+H]+, HRESI-MS (m/z): [M+H]+ Calcd. for C16H16NO3:
271.1077 found: 271.1079.
Ligand sampling and preparation were performed sing
ig rep of chr dinger by sing the O 2005 force field and
charges. All possible ligand states at pH 7.4 were generated by
Epik. The other parameters were accepted as default.
4.6.1.12. (R)-3-ethyl-8-hydroxy-5-methyl-7-(pyrimidin-5-yl)-
3,4-dihydronaphthalen-1(2H)-One (13aR). White solid (yield:
13%). Rf = 0.39 (PE/EtOAc 4/1). [α]20 -77.3 (c 0.20, CHCl3).
The compounds were then docked using Glide standard
precision (SP) mode, and the top 10 poses were kept for analysis.
The center of the docking receptor grids was defined as the
coordinate of cognate ligands. The other parameters were
accepted as default. The pictures were performed using Pymol
software, and polar interactions were analyzed.
D
o
mp: 153–155 C. IR (film) νmax: 2921–2851, 1662, 1616, 1550,
1412, 1393, 1323, 1201, 1183, 1138, 1040, 806 cm-1. H NMR
1
(400 MHz, CDCl3) δ 11.67 (s, 1H), 9.18 (s, 1H), 8.98 (s, 2H),
7.38 (s, 1H), 4.61–4.46 (m, 1H), 2.99 (dd, J = 16.8, 3.4 Hz, 1H),
2.81 (dd, J = 16.8, 11.6 Hz, 1H), 2.29 (s, 3H), 2.04–1.91 (m, 1H),
1.91–1.80 (m, 1H), 1.13 (t, J = 7.5 Hz, 3H). 13C NMR (150 MHz,
CDCl3) δ 170.4, 157.8, 157.2, 156.6, 138.5, 137.7, 130.8, 125.7,
121.2, 109.0, 80.4, 29.8, 27.9, 18.2, 9.3. ESI-MS (m/z): 283.1
[M-H]-, HRESI-MS (m/z): [M+H]+ Calcd. for C16H22NO4:
285.1234 found: 285.1232.
7. The kenetics study of enzyme MAO-A and the mode of
MAO-A inhibition
The kinetics study of MAO-A, whose substrate was
kynuramine, was represented by Michaelis-Menten kinetic
experiment. The catalytic rates of human MAO-A enzyme were
measured at eight different concentrations of substrate (1.5625,
3.125, 6.25, 12.5, 25, 50, 100 and 200 μM). The corresponding
progression curve and the linear Eadie-Hofstee plot were
generated using GraphPad Prism 5.0 (GraphPad software). In
addition, the maximal velocity (Vmax) and the Michaelis
constant (Km) were calculated using the same software version
5.0.
4.6.1.13.
(R)-7-(4-aminophenyl)-3-ethyl-8-hydroxy-5-
methylisochroman-1-one (13bR). Gray solid (yield: 28%). Rf =
0.23 (PE/EtOAc 4/1). [α]20 -59.3 (c 0.10, CHCl3). mp: 141–143
D
oC. IR (film) νmax: 3367, 2958–2851, 1660, 1613, 1518, 1455,
1
1387, 1287, 1270, 1191, 1126, 1038, 834 cm-1. H NMR (400
MHz, CDCl3) δ 11.54 (s, 1H), 7.40 (d, J = 8.4 Hz, 2H), 7.34 (s,
1H), 6.75 (d, J = 8.4 Hz, 2H), 4.57–4.41 (m, 1H),3.13 (brs, 2H),
2.94 (dd, J = 16.6, 3.3 Hz, 1H), 2.76 (dd, J = 16.6, 11.7 Hz, 1H),
2.23 (s, 3H), 2.00–1.87 (m, 1H), 1.88–1.77 (m, 1H), 1.12 (t, J =
7.4 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 170.9, 157.8, 145.7,
138.2, 135.4, 130.1, 128.5, 127.0, 124.6, 114.8, 108.4, 80.3, 29.7,
28.0, 18.1, 9.4. ESI-MS (m/z): 298.2 [M+H]+, HRESI-MS (m/z):
[M+H]+ Calcd. for C18H20NO3: 298.1438 found: 298.1440.
The mode of MAO-A inhibition by 13aR, 12cR, 11gR and
12aR were investigated by constructing
a set of three
Lineweaver-Burk plots. The first plot was constructed in the
absence of inhibitor, while the other two plots were constructed
in the presence of two different concentrations of the inhibitor
(IC50 value and 2×IC50 value). The substrate kynuramine was
sed at concentrations ranging from 12.5 to 100 μM, while the
final concentration of MAO-A was 0.0025 mg/mL. All enzyme
reactions and catalytic activity measurements were carried out as
described above for the IC50 determinations. Linear regression
analysis was performed using the Prism version 5.0 software
package.
5. Monoamine oxidase (MAO-A and MAO-B) inhibition
assay
Human recombinant MAO-A and MAO-B were purchased
from BD Biosciences (Woburn, MA, USA). Clorgyline and
Deprenyl were used as standard inhibitors of MAO-A and MAO-
B, respectively. Kynuramine was used as substrate for MAO-A
and MAO-B in incubations, and 4-hydroxyquinoline metabolized
from Kynuramine by MAOs was used as marker to evaluate
inhibitive activities of test compounds.
Conflict of interest
The authors confirm that this article content has no conflict of
interest.
The test compounds or reference standard were dissolved in
DMSO, and diluted to different concentration samples (from 100
µM to 0.0125 µM in pH 7.4 buffer solution, 50 µL). The enzyme
reactions were carried out with the samples (50 µL), enzymes
(0.0025 mg/mL, 5 µL) and substrates (25 µM in pH 7.4 buffer
solution, 25 µL) in buffer solution (the final volume: 100 µL) at
Acknowledgements
This research was supported by NSFC grants (No. 81773599,
21472024, 21472021) and a MOST grant (2018ZX09735008-
005). The authors are grateful to Dr. Jie-Fei Cheng for his helpful
discussion.
o
37 C for 10 min, and then quenched with a cooled solution of
acetyl aminophenol in CH3CN (200 µL). After eddy-mixing (1
min) and centrifugation (13000 g, 10 min), 10 µL supernatant
was injected to the UPLC-ESI-MS/MS system to measure the
quantity of 4-hydroxyquinoline related to the blank. The results
for both reference drugs and test compounds were expressed as
IC50 in Table 1 and Table 2, and the selectivity index (SI, MAO-
A over MAO-B) was also given in the tables if necessary.
Supplementary Material
Supplementary data associated with this article can be found
in Supporting Information.

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