3,4-Dihydroquinazoline derivatives inhibit the activities of cholinesterase enzymes

Article abstract of DOI:10.1016/j.bmcl.2017.01.068

A series of 3,4-dihydroquinazoline derivatives consisting of the selected compounds from our chemical library on the diversity basis and the new synthetic compounds were in vitro tested for their inhibitory activities for both acetylcholinesterase (AChE, from electric eel) and butyrylcholinesterase (BChE, from equine serum) enzymes. It was discovered that most of the compounds displayed weak AChE and strong BuChE inhibitory activities. In particular, compound 8b and 8d were the most active compounds in the series against BChE with IC₅₀ values of 45?nM and 62?nM, as well as 146- and 161-fold higher affinity to BChE, respectively. To understand the excellent activity of these compounds, molecular docking simulations were performed to get better insights into the mechanism of binding of 3,4-dihydroquinazoline derivatives. As expected, compound 8b and 8d bind to both catalytic anionic site (CAS) and peripheral site (PS) of BChE with better interaction energy values than AChE, in agreement with our experimental data. Furthermore, the non-competitive/mixed-type inhibitions of both compounds further confirmed their dual binding nature in kinetic studies.

Full text of DOI:10.1016/j.bmcl.2017.01.068

Accepted Manuscript
3,4-Dihydroquinazoline derivatives inhibit the activities of cholinesterase en-
zymes
Byeongyeon Park, Ji Hye Nam, Jin Han Kim, Hyoung Ja Kim, Valentina Onnis,
Gianfranco Balboni, Kyung-Tae Lee, Jeong Ho Park, Marco Catto, Angelo
Carotti, Jae Yeol Lee
PII:
S0960-894X(17)30080-X
http://dx.doi.org/10.1016/j.bmcl.2017.01.068
BMCL 24644
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
18 October 2016
7 January 2017
24 January 2017
Please cite this article as: Park, B., Nam, J.H., Kim, J.H., Kim, H.J., Onnis, V., Balboni, G., Lee, K-T., Park, J.H.,
Catto, M., Carotti, A., Lee, J.Y., 3,4-Dihydroquinazoline derivatives inhibit the activities of cholinesterase enzymes,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.01.068
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3,4-Dihydroquinazoline derivatives inhibit the activities of
cholinesterase enzymes
Byeongyeon Park a, 1, Ji Hye Nam a, 1, Jin Han Kim a, Hyoung Ja Kim b, Valentina Onnis c,
Gianfranco Balboni c, *, Kyung-Tae Lee d, Jeong Ho Park e, Marco Catto f, Angelo Carotti f,*, Jae Yeol
Lee a, g,*
a Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung
Hee University, Seoul 02447, Republic of Korea
b Molecular Recognition Research Center, Future Convergence Research Division, Korea Institute of
Science and Technology, Seoul 02792, Republic of Korea
c Department of Life and Environmental Sciences, Unit of Pharmaceutical, Pharmacological and
Nutraceutical Sciences, University of Cagliari, Via Ospedale 72, I-09124, Cagliari, Italy
d Department of Life and Nanopharmaceutical Science, Kyung Hee University, Seoul 02447,
Republic of Korea
e Department of Chemical & Biological Engineering, Hanbat National University, Daejeon 34158,
Republic of Korea
f Dipartimento di Farmacia—Scienze del Farmaco, Università degli Studi di Bari “Aldo Moro”,via
E. Orabona, 4, I-70125 Bari, Italy
g KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul
02447, Republic of Korea
1 The contributions of the authors are equal.
Corresponding authors:
Tel. +49-70-675-8625; Fax +49-70-675-8612; e-mail: gbalboni@unica.it
Tel. +39-080-544-2782; Fax +39-0805442230; e-mail: angelo.carotti@uniba.it
Tel. +82-2-961-0726; Fax +82-2-961-0443; e-mail; ljy@khu.ac.kr
Keywords: Alzheimer’s disease; cholinesterase inhibitors; 3,4-dihydroquinazolines; molecular
docking study; kinetic study
page 1 / 16

Abstract: A series of 3,4-dihydroquinazoline derivatives consisting of the selected compounds
from our chemical library on the diversity basis and the new synthetic compounds were in vitro
tested for their inhibitory activities for both acetylcholinesterase (AChE, from electric eel) and
butyrylcholinesterase (BChE, from equine serum) enzymes. It was discovered that most of the
compounds displayed weak AChE and strong BuChE inhibitory activities. In particular, compound
8b and 8d were the most active compounds in the series against BChE with IC50 values of 45 nM
and 62 nM, as well as 146- and 161-fold higher affinity to BChE, respectively. To understand the
excellent activity of these compounds, molecular docking simulations were performed to get better
insights into the mechanism of binding of 3,4-dihydroquinazoline derivatives. As expected,
compound 8b and 8d bind to both catalytic anionic site (CAS) and peripheral site (PS) of BChE
with better interaction energy values than AChE, in agreement with our experimental data.
Furthermore, the non-competitive/mixed-type inhibitions of both compounds further confirmed
their dual binding nature in kinetic studies.
Alzheimer’s disease (AD) is a fatal progressive neurodegenerative disorder that has no cure
to date. It is the cause of 60 to 70% of senile dementia.1 Although the complete etiology of AD is
still not clear, several pathological events play an important role in the onset and progress of this
disease such as amyloid-β (Aβ) oligomerization and plaques formation,2 oxidative stress,3
decreasing levels of acetylcholine (ACh),4 and τ-protein aggregation.5 Among these conditions,
low levels of ACh appear to be a critical element in the development of cognitive and
neurodegenerative disorders in AD patients.6 In the synapses, acetylcholinesterase (AChE, EC
3.1.1.7) and butyrylcholinesterase (BChE, EC 3.1.1.8) are mainly responsible for the hydrolysis of
ACh into choline and acetic acid, an essential process allowing for the control of the cholinergic
transmission.7-8 In a healthy brain, AChE predominates and BChE is considered to play a minor
role in regulating ACh levels. On the other hand, it was found that BChE activity increases in CNS
during the development of AD, while AChE activity decreases at the same time.9-10 Therefore,
targeting AChE and BChE may be one promising approach in treating AD. To date, several
cholinesterase inhibitors such as tacrine (withdrawn),11 donepezil,12 rivastigmine13 and
galantamine14 have been approved for the treatments of AD.15 Although they offered some
cognitive improvements in AD, several adverse drug reactions such as nausea, diarrhea, dizziness,
page 2 / 16

and vomiting have also been associated with cholinesterase inhibitors.16 Therefore, new safer
cholinesterase inhibitors with minimal adverse effects and/or multi-target activity are urgently
warranted.17-18
N
N
NH
CO2H
N
N
N
OH
2-Carboxyl-3,4-dihydroquinazoline
IC50 (eeAChE): 344 M
IC50 (esBChE): > 1000 M
Vasicine
Deoxyvasicinone
IC50 (eeAChE): 3 M
IC50 (esBChE): 0.1 M
IC50 (eeAChE): 2 M
IC50 (esBChE): 0.04 M
O
N
O
O
N
N
NH
OH
N
N
OH
Pegamin
Vasicinone
Deoxyvasicinone
IC50 (eeAChE): 155 M
IC50 (esBChE): 106 M
IC50 (eeAChE): 77 M
IC50 (esBChE): 10 M
IC50 (eeAChE): 35 M
IC50 (esBChE): 18 M
N
N
N
N
N
i
ii
IC50 (eeAChE): 8 M
IC50 (esBChE): 4 M
IC50 (eeAChE): 28 M
IC50 (esBChE): 2 M
Figure 1. 3,4-Dihydroquinazoline derivatives showing anticholinesterase activities, wherein
eeAChE is eel AChE and esBChE is equine serum BChE.
3,4-Dihydroquinazoline scaffold characterizes well-known alkaloids showing different
types of biological activities such as inhibition of trypanothione reductase (TryR),19
anticonvulsant20 and antidepressant21 activities, T-type calcium channel blocking activity,22 and
especially anticancer activity.23 In addition, the moderate or strong inhibitory activities of 3,4-
dihydroquinazoline derivatives towards AChE and BChE were also identified as shown in Figure
1.24-25 Based on this anticholinesterase activity, a series of 3,4-dihydroquinazoline derivatives,
page 3 / 16

which were originally identified as potent T-type Ca2+ channel blockers by our group,22-23 were
selected from our in-house compound library (for compound 7a-7b and 8a-8f in Table 1) or newly
synthesized (for compound 8g-8l in Table 1), and evaluated for their inhibitory activity towards the
ChEs. To better understand the enzyme binding mode and inhibition mechanisms, molecular
modeling and kinetic studies were performed, making the design of better ChE inhibitors possible.
O
O
R1
R1
a
OH
OCH3
NO2
NO2
R2
R2
1
2
b
O
R1
O
OCH3
R1
Br
R1
c
d
e
OCH3
NH
R2
R3
NH2
NH2
O
N
R2
R2
H
3
4
5
R5
O
O
O
R1
CH3O
R1
R3
N
H
OCH3
N C N R3
f
g
N
R1
R3
R4
N
R2
N
R4
R2
N
R2
6
7
8
Scheme 1. Reagents and conditions: (a) H2SO4, MeOH, 70 ℃, 12 h, >99%; (b) Zn, NH4Cl, MeOH,
reflux, 70 ℃, 12 h, 98%; (c) Pd(OAc)2, methyl acrylate, (o-tol)3P, Et3N, ACN, reflux, 48 h, 23-
70%; (d) R3-NCO (commercially available or in situ prepared), toluene, rt to 100 ℃, 3 h, 40-70%;
(e) PPh3·Br2, Et3N, CH2Cl2, 0℃, 3 h, 40-55%; (f) amine (R4-NH2), toluene, rt, 1 h, 25-96%; (g) R5-
BnNH2, TBD, 40℃, 12 h, 40-80%.
New F-substituted 3,4-dihydroquinazoline derivatives (8g-8l) were prepared using Heck
reaction and the procedure described previously by our group as shown in Scheme 122-23: 2-
page 4 / 16

Bromoaniline (3) was coupled with methyl acrylate under Heck reaction condition [Pd(OAc)2, (o-
tol)3P, Et3N] to provide the intermediate 4 in 23-70% optimized yields, which was coupled with
isocyanate (R3-NCO: commercially available or in situ prepared from the corresponding carboxylic
acid R3-CO2H via Curtius rearrangement) to provide a urea compound 5. The dehydration of 5 with
Ph3P·Br2 and Et3N provided a carbodiimido compound 6. Subsequently, the coupling reaction of 6
with amine compound (R4-NH2) afforded the 3,4-dihydroquinazoline ester compound 7 as a
racemic mixture. The treatment of 7 with R5-benzylamine and 1,5,7-triazabicyclo[4.4.0]dec-5-ene
(TBD) as a catalyst under solvent-free condition afforded the final amide compound (8).
The spectrophotometric Ellman’s test26 for the in vitro inhibition assay of AChE from
electric eel and BChE from equine serum was followed as previously described.27 The IC50 values
for AChE/BChE inhibitions are summarized in Table 1, together with those of donepezil used as
reference compound. According to AChE inhibitory activity results, some of the compounds
displayed moderate anti-AChE activity (IC50 values of 1.2 to 8.0 M). Among them, compound 8h
showed good activity (IC50 = 1.2 M) against AChE but much less than donepezil (IC50 = 0.020
M), a selective AChE inhibitor.28 Interestingly, ester compound 7a-b and amide compound 8a
containing short phenyl (or c-hexyl) ring and 4-methylpiperazine ring at R3 and R4 positions
exhibited low activity (IC50 = >10 M). The noticeable structure-activity relationship could not be
established for AChE inhibitions due to a variety of IC50 values of compounds as shown in Table 1.
In respect to BChE inhibition, most of compounds except 7a and 8a generally displayed remarkable
selective behavior against BChE and exhibited broad IC50 values ranging from 6.1 to 0.045 M
against BChE (Table 1). In particular, compound 8b and 8d provided the best IC50 values of 0.045
and 0.062 M, respectively, while showing IC50 values of 6.6 and 10 M for AChE inhibition,
revealing 146- and 161-fold selectivity for BChE over AChE, respectively. Through a brief analysis
and structural comparison, we found that the inhibitory activity against BChE depends mainly on
the properties of the substituent at 2-position of 3,4-dihydroquinazoline scaffold, that are: the linker
page 5 / 16

length (8a vs. the other compounds) and the flexibility (8g-8i: 2-aminoethyl-piperazine ring vs. 8j-
8l: 1,5-pentanediamine chain) and the type of terminal amine (8b and 8d vs. 8c).
Table 1. Inhibitory activities of 3,4-dihydroquinazoline derivatives (7 and 8) against electric eel
acetylcholinesterase (eeAChE) and equine serum butyrylcholinesterase (esBChE).a
eeAChE
esBChE
R1 R2
R3
R4
R5
Entry
IC50 (M)
IC50 (M)
N
N
N
H
H
H
H
H
H
H
H
H
F
H
H
H
H
H
H
H
H
H
F
-
-
≥10
>10
7a
7b
8a
8b
8c
8d b
8e
8f
NH2
≥10
0.14±0.01
>10
N
N
N
H
H
H
H
F
>10
N
6.6±0.8
4.1±0.5
≥10
0.045±0.004
5.3±0.1
N
N
N
NH2
N
0.062±0.002
2.6±0.2
N
N
(CH2)2
(CH2)2
N
≥10
N
N
N
N
OCH3
F
>10
6.1±0.4
F
6.5±0.2
1.2±0.4
4.4±0.6
>10
0.24±0.04
0.38±0.02
0.40±0.09
1.47±0.47
2.13±0.69
2.04±0.53
2.3±0.1 d
8g
8h
8i
N
N
N
N
F
H
H
H
H
H
H
H
F
F
N
N
N
N
(CH2)2
(CH2)2
(CH2)2
N
F
8j
N
N
N
F
8.0±1.6
>10
8k
8l
N
F
Donepezil c
0.020±0.002 d
a
b
c
Values represent the mean of two/three independent experiments; Tested as dihydrochloride salt; Positive control
for acetylcholinesterase inhibitory assay; d Data taken from reference [28].
With respect to the inhibitory activity against AChE/BChE inhibitions, it can be concluded
that the different activities of 3,4-dihydroquinazoline compounds against AChE and BuChE may be
resulted from the different hydrophobic gorges of active center in two enzymes. At the base of the
gorge in AChE, the binding of the substrate is represented by two phenylalanine residues (Phe288,
page 6 / 16

and Phe290) whose aromatic residues protrude into the gorge. In BuChE, these residues are
replaced by two smaller amino acid residues Leu286 and Val288 as shown in Figure 2.29 This
change of amino acid sequence creates a larger space within the deepest area of the gorge of
BuChE.30-31 Therefore, higher BuChE inhibitory activities of tested compounds may be related to
the larger active site gorge of BuChE than that of AChE.32
Figure 2. Active site gorges of (a) H. sapiens BChE (hBChE; PDB code 1P0I) and (b) Torpedo
californica AChE (TcAChE; PDB code 1EA5). The dotted red box indicates two important amino
acids at the base of the gorge in two enzymes, respectively. This figure was taken from reference
[29].
To detect and better understand the main interaction underlying the ChEs inhibition,
molecular docking studies were performed on the two most active compound 8b and 8d, as well as
donepezil as a reference, by using the recently crystallized structure of hAChE (PDB code: 4EY7)31
and hBChE (PDB code: 4TPK)33 based on the findings that the overall identity of 88 and 90%
resulted for eeAChE and hAChE, and esBChE and hBChE, respectively. Moreover no significant
difference was detected for the amino acid sequences within the active sites of enzymes from
human and non-human sources.34 The docking studies were performed using Molegro Virtual
Docker (MVD) 2013.6.0.1 for Windows.35 We identified one cavity with volume (171 Å3 for 4EY7
page 7 / 16

and 399 Å3 for 4TPK) containing ligand from the x-ray crystallized structure of each ChE by the
same software, which can automatically detect cavities from protein surfaces. The putative active
site of the enzyme was defined to include residues within a 15 Å radius to this cavity. The docking
wizard of 2013.6.0.1 was used to dock compound 8b and 8d, which were used as two protonated
forms and arbitrary (R)–enantiomer because both (R)– and (S)–enantiomer were found to exert
similar biological activities against T-type Ca2+ channel and cancer cells,36 for both docking
validation and data comparison in the presence of H2O. The most stable docking pose was selected
according to the best MolDock score conformation predicted by the MVD scoring function and
inserted into Table 2. The MolDock score of 8b and 8d with hAChE was found to be -152.4 and -
160.4 kcal/mol, respectively, and was lower than that (-202.7 kcal/mol) of donepezil as a selective
hAChE inhibitor, suggesting a lower binding-affinity of these compounds at hAChE enzyme than
donepezil (Table 2). In the case of hBChE, however, the MolDock score of 8b and 8d with hBChE
was found to be -195.4 and -199.5 kcal/mol, respectively, two values higher than that (-133.8
kcal/mol) of donepezil. The calculated MolDock scores of the 8b and 8d suggested their potent
inhibition and selectivity towards BChE over AChE, which was almost consistent with the
experimental data (Table 2 and Table S1 in Supplementary content).
Table 2. The inhibitory concentration (IC50), calculated MolDock score and selectivity index (S.I.)
of the two most active compound 8b and 8d investigated against AChE and BChE
AChE
BChE
Selectivity
index b
Compound
MolDock Score a
(PDB 4EY7)
MolDock Score a
(PDB 4TPK)
IC50 (M)
IC50 (M)
6.6
~10
0.02
-152.4
-160.4
-202.7
0.045
0.062
2.3
-195.4
-199.5
-133.8
146
161
8b
8d
Donepezil c
0.009
a Unit = kcal/mol; b Selectivity index (S.I.) is the BChE selectivity index defined as IC50 eeAChE/IC50 esBChE ratio; c
Reference compound as a selective AChE inhibitor.
The proposed 2D binding mode of best pose for each compound in the active site of each
page 8 / 16

ChE was calculated using both MVD’s Pose Viewer utility. The overall interactions between the
amino acids in active site gorge of each ChE with the proposed pose for each compound were
illustrated in Figure 3 and 4, respectively. The active site of AChE involves two sites: the peripheral
anionic site (PAS), which corresponds to the peripheral site (PS) for BChE, and the catalytic site.
This peripheral site (P-site) contains a number of aromatic amino acid residues as well as an
aspartate residue that is able to interact with cationic substrates and guide them down the gorge to
the catalytic triad. The catalytic site of AChE consists of two subsites: the esteratic site and the
anionic site. In the esteratic site, a catalytic triad consisting of Glu334, His447 and Ser203 forms a
planar array. In the anionic site, the Trp86 binds trimethylammonium group of ACh. In the overall
catalytic site, the substrate is well positioned to be hydrolyzed into acetic acid and cholin.37-38
In the case of AChE, compound 8b exhibited steric interactions with Tyr72 (PAS), Asp74
(PAS), Tyr124 (PAS: non-favorable), Trp286 (PAS), Phe295 (acyl binding pocket), Phe297 (acyl
binding pocket), Phe338, and Tyr341 (PAS: non-favorable) in the active site of AChE. It also
formed five hydrogen bonds between the nitrogen atom of 3,4-dihydroquinazoline ring with Tyr124
(PAS, 2.17 Å), between the nitrogen atom of 3,4-dihydroquinazoline ring with water (3.42 Å), and
between the nitrogen and oxygen atom of amide and water953 (3.20 and 3.25 Å), between the
oxygen atom of amide and water728 (3.27 Å) [Figure 3(a)]. Compound 8d showed steric
interactions with Tyr72 (PAS), Asp74 (PAS), Leu76, Tyr124 (PAS), Trp286 (PAS), Ser293 (non-
favorable), Phe295 (acyl binding pocket: favorable & non-favorable), Phe297 (acyl binding pocket:
non-favorable), Phe338, Tyr341 (PAS: non-favorable), and His447 (catalytic triad) in the active site
of AChE. It also formed five hydrogen bonds between the oxygen atom of amide with Tyr341 (PAS,
3.29 Å) and water954 (2.60 Å), and between the nitrogen atom of 3,4-dihydroquinazoline ring with
Phe295 (3.00 Å), between two nitrogen atoms of 3,4-dihydroquinazoline ring with water728 (2.84
and 2.96 Å) [Figure 3(b)].
With respect to the docking with BChE enzyme, compound 8b exhibited steric interactions
page 9 / 16

with Asp70 (PS), Trp82 (CAS), Thr120 (CAS), Gly116 (oxyanion hole), Pro285, Met437, and
Tyr332 (PS) in the active site of BChE. It also formed two hydrogen bonds (2.15 and 3.52 Å,
respectively) between the nitrogen atom of 3,4-dihydroquinazoline ring with Thr120, and between
the hydrogen atom of pyrrolidine ring with water736 [Figure 4(a)]. Compound 8d exhibited the
steric interactions with Asn68 (PS), Asn83, Trp82 (CAS), Thr120 (CAS), Pro285, Leu286 (acyl
binding site), and His438 (catalytic triad) in the active site of enzyme. Furthermore, it formed two
hydrogen bonds between the terminal hydrogen atom of side chain at 2-position of 3,4-
dihydroquinazoline ring with His438 (catalytic triad, 2.61 Å), and between nitrogen atom of 3,4-
dihydroquinazoline ring with water736 (3.20 Å) [Figure 4(b)]. The overall binding models showed
that two compounds were able to interact with both catalytic anionic site (CAS) and peripheral site
(PS) residues in active site of BChE.
Figure 3. (a) and (b) are the proposed 2D binding mode of 8b and 8d, respectively, with hAChE
(PDB ID: 4EY7). Hydrogen bonds and steric interactions are shown in blue and red dotted lines,
respectively. The green and red sphere centered at each atom visualizes the strength of the
favorable and non-favorable (clash) interactions, respectively, for this specific atom with amino
acid residues in the active site.
page 10 / 16

Figure 4. (a) and (b) are the proposed 2D binding mode for 8b and 8d, respectively, with hBChE
(PDB ID: 4TPK). Hydrogen bonds and steric interactions are shown in blue and red dotted lines,
respectively. The green sphere centered at each atom visualizes the strength of the favorable
interactions for this specific atom with amino acid residues in the active site.
To gain further insight into the mechanism of action on BChE of the potent compound 8b
and 8d, Lineweaver–Burk double reciprocal plots were generated (Figure 5).39 The interception of
the lines in the Lineweaver–Burk plot at the x-axis with both increased slope (decreased Vmax) and
intercepts (higher Km) at increasing concentration of the inhibitor, which indicated a non-
competitive/mixed-type inhibition with a Ki value equal to 29±1 and 51±3 nM, respectively. Based
on their kinetic studies, we concluded that both compounds might be able to interact with both the
catalytic active site (CAS) and peripheral site (PS) of BChE, which was also consistent with the
docking results.
page 11 / 16

Figure 5. Lineweaver–Burk plot of inhibition kinetics of (a) 8b and (b) 8d: reciprocals of enzyme
activity (esBChE) versus reciprocals of substrate concentration in the presence of different
concentrations of inhibitor.
In conclusion, in this study a series of 3,4-dihydroquinazoline derivatives was selected
from our compound library and evaluated for its ChE inhibitory activity. These compounds showed
good levels of inhibition against BChE, while inhibiting AChE with less potency. In particular,
compound 8b and 8d exhibited the highest BChE inhibition. Our molecular docking also revealed
that both of compounds would bind both CAS and PS residues in the active site of BChE, which
implicates that these compounds could act as dual binding site (DBS) inhibitors. In addition, kinetic
analysis also indicated that both of compounds might be non-competitive/mixed-type inhibitors
against BChE, in accordance to the docking data. Considering these overall results, our findings
could be extended to design and develop a new selective BChE inhibitor containing 3,4-
dihydroquinazoline scaffold as potential agents for the treatment of AD.
Acknowledgments
This work was supported by Basic Science Research Program through the Ministry of Education of
the Republic of Korea and the National Research Foundation (NRF-2016R1D1A1B03936197) and
by the University of Bari (Fondi di Ateneo 2014-2015). We acknowledge also the Regione
page 12 / 16

Autonoma della Sardegna (L.R. n. 7/2007 CRP-59473 /2012) for funding.
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Graphical Abstract
3,4-Dihydroquinazoline derivatives inhibit the activities of cholinesterase enzymes
Byeongyeon Park, Ji Hye Nam, Jin Han Kim, Hyoung Ja Kim, Valentina Onnis, Gianfranco
Balboni*, Kyung-Tae Lee, Jeong Ho Park, Marco Catto, Angelo Carotti*, Jae Yeol Lee*
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