Synthesis and cytotoxic effects of 2-thio-3,4-dihydroquinazoline derivatives as novel T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmc.2020.115491

In our previous work, a series of 2-amino-3,4-dihydroquinazoline derivatives using an electron acceptor group was reported to be potent T-type calcium channel blockers and exhibit strong cytotoxic effects against various cancerous cell lines. To investigate the role of the guanidine moiety in the 2-amino-3,4-dihydroquinazoline scaffold as a pharmacophore for dual biological activity, a new series of 2-thio-3,4-dihydroquniazoline derivatives using an electron donor group at the C2-position was synthesized and evaluated for T-type calcium channel blocking activity and cytotoxic effects against two human cancerous cell lines (lung cancer A549 and colon cancer HCT-116). Among them, compound 6g showed potent inhibition of Ca_v3.2 currents (83% inhibition) at 10 μM concentrations. The compound also exhibited IC₅₀ values of 5.0 and 6.4 μM against A549 and HCT-116 cell lines, respectively, which are comparable to the parental lead compound KYS05090. These results indicate that the isothiourea moiety similar to the guanidine moiety of 2-amino-3,4-dihydroquinazoline derivatives may be an essential pharmacophore for the desired biological activities. Therefore, our preliminary work can provide the opportunity to expand a chemical repertoire to improve affinity and selectivity for T-type calcium channels.

Full text of DOI:10.1016/j.bmc.2020.115491

Journal Pre-proofs
Synthesis and Cytotoxic Effects of 2-Thio-3,4-dihydroquinazoline Derivatives
as Novel T-Type Calcium Channel Blockers
Yunchan Nam, Ki Deok Ryu, Changyoung Jang, Yoon Hyoung Moon,
Misong Kim, Dohyeong Ko, Kyung-Sook Chung, Maria A. Gandini, Kyung-
Tae Lee, Gerald W. Zamponi, Jae Yeol Lee
PII:
S0968-0896(20)30312-6
https://doi.org/10.1016/j.bmc.2020.115491
BMC 115491
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
12 October 2019
2 April 2020
3 April 2020
Please cite this article as: Y. Nam, K. Deok Ryu, C. Jang, Y. Hyoung Moon, M. Kim, D. Ko, K-S. Chung, M.A.
Gandini, K-T. Lee, G.W. Zamponi, J. Yeol Lee, Synthesis and Cytotoxic Effects of 2-Thio-3,4-
dihydroquinazoline Derivatives as Novel T-Type Calcium Channel Blockers, Bioorganic & Medicinal Chemistry
(
2020), doi: https://doi.org/10.1016/j.bmc.2020.115491
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover
page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version
will undergo additional copyediting, typesetting and review before it is published in its final form, but we are
providing this version to give early visibility of the article. Please note that, during the production process, errors
may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
©
2020 Published by Elsevier Ltd.

Synthesis and Cytotoxic Effects of 2-Thio-3,4-dihydroquinazoline Derivatives as
Novel T-Type Calcium Channel Blockers
a,1
a,1
a
a
a
Yunchan Nam , Ki Deok Ryu , Changyoung Jang , Yoon Hyoung Moon , Misong Kim ,
a
b
c
b*
Dohyeong Ko , Kyung-Sook Chung , Maria A. Gandini , Kyung-Tae Lee ,
c*
a,d*
Gerald W. Zamponi , Jae Yeol Lee
a
Research Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung
Hee University, Seoul 02447, Republic of Korea
b
Department of Life and Nanopharmaceutical Science, College of Pharmacy, Kyung Hee University,
Seoul 02447, Republic of Korea
c
Department of Physiology and Pharmacology, Hotchkiss Brain Institute and Alberta Children’s
Hospital Research Institute, Cumming School of Medicine, University of Calgary, Calgary T2N 4N1,
Canada
d
KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul
0
2447, Republic of Korea
1
The contribution of the authors is equal.
(Corresponding authors: Tel. +82-2-961-0726; Fax +82-2-961-0443; e-mail: zamponi@ucalgary.ca;
ktlee@khu.ac.kr; ljy@khu.ac.kr)
Abstract: In our previous work, a series of 2-amino-3,4-dihydroquinazoline derivatives using an
electron acceptor group was reported to be potent T-type calcium channel blockers and exhibit strong
cytotoxic effects against various cancerous cell lines. To investigate the role of the guanidine moiety
in the 2-amino-3,4-dihydroquinazoline scaffold as a pharmacophore for dual biological activity, a new
series of 2-thio-3,4-dihydroquniazoline derivatives using an electron donor group at the C2-position
was synthesized and evaluated for T-type calcium channel blocking activity and cytotoxic effects
against two human cancerous cell lines (lung cancer A549 and colon cancer HCT-116). Among them,
compound 6g showed potent inhibition of Ca 3.2 currents (83% inhibition) at 10 µM concentrations.
v
The compound also exhibited IC values of 5.0 and 6.4 µM against A549 and HCT-116 cell lines,
5
0
respectively, which are comparable to the parental lead compound KYS05090. These results indicate
that the isothiourea moiety similar to the guanidine moiety of 2-amino-3,4-dihydroquinazoline
derivatives may be an essential pharmacophore for the desired biological activities. Therefore, our
preliminary work can provide the opportunity to expand a chemical repertoire to improve affinity and
selectivity for T-type calcium channels.
Key Words: T-type calcium channel; 2-thio-3,4-dihydroquinazoline; current inhibition; cytotoxicity
-
1 -

1
. Introduction
2
+
The calcium (Ca ) channel family is divided into three phylogenetic subfamilies: Ca 1 (L-
v
1
type), Ca 2 (P/Q, N, and R-types) and Ca 3 (T-type). Both, Ca 1 and Ca 2 are activated by strong
v
v
v
v
membrane depolarizations whereas Ca 3 is low voltage activated. All calcium channels participate in
v
the activation of intracellular signaling pathways that control many cell processes such as proliferation,
differentiation, mitogenesis, metastasis, and apoptosis.2-7 On the other hand, aberrant calcium channel
activity has been linked to a number of pathological conditions, and consequently calcium channels
8
are important drug targets. This also includes the family of T-type calcium channels (i.e., Ca 3.1,
v
Ca 3.2 and Ca 3.3 which are expressed widely and differentially throughout nerve, muscle and heart,
v
v
with both gain and loss of function mutations in Ca 3 encoding genes being linked to a range of
v
disorders, including epilepsy, movement disorders, and autism.1,9 As a result, there have been
considerable efforts in the development of new T-type calcium channel inhibitors with the goal of
identifying inhibitors that preferentially block T-type over non T-type channels (and even blockers
1
0
with selectivity within the Ca 3 family) to minimize the potential for adverse effects. Indeed, a
v
number of recent studies have identified various pharmacophores acting on T-type calcium channels
selectively, whereas others have yielded compounds that target both Ca 2 and Ca 3 calcium
v
v
channels.11-15
Anomalous regulation of T-type calcium channels has been linked to many cancer processes
2
+
2+
although T-type Ca channel expression levels depend on cancer type, stage, and T-type Ca channel
isoform.16-19 It is possible that survival of cancer cells requires increased basal Ca2+ influx and
remodeled Ca2+ signaling pathways for enhancing proliferation, promoting invasiveness and
conferring chemotherapeutic resistance.20-21 During the past few decades a number of research groups
2
+
22-
have highlighted T-type Ca channels as promising cancer biomarkers and anti-neoplastic targets.
2
5
2
+
T-type Ca channel blockers have been developed with the intent of arresting cancer cell
2
+
proliferation or inducing cancer cell death since the expression of T-type Ca channels in human
-
2 -

2
6
2+
retinoblastoma Y79 cells was first reported in 2002. The pharmacological effects of T-type Ca
channel blockers on cancer cells are supported by the fact that they reduced proliferation due to effects
2
7
on the p53-dependent p21 upregulation pathway halting the G -S progression. Moreover, the T-type
1
blocker mediated decrease in cell survival was shown to involve caspase-dependent apoptosis28,29 or
3
0
autophagy deregulation through p38-MAPK or nutrient stress pathways. The role of T-type channels
3
1
in cancer cell survival is complex due to the intricate nature of the underlying signaling pathways. It
was demonstrated that T-type calcium channel blockers such as mibefradil and pimozide recruit
different cytotoxic pathways to the mitogenic cell lines in which pimozide decreases mRNA levels of
Bcl-2 whereas mibefradil activates caspases.32 Along these lines, our 3.4-dihydroquinazoline
derivative, KYS05090, showed a cytotoxic effect through the induction of autophagy and apoptosis
via ROS generation based on the inhibition of glucose uptake.33
In our previous studies, most structures of 3,4-dihydroquinazoline derivatives consisting of
various groups at the C2-, N3-, C4-positions showed both cytotoxic effects against various cancer cell
2
+
33-38
lines that paralleled T-type Ca channel blocking effects.
The best lead compound in our hands,
KYS05090 with an N’,N’-dimethyl-N-methylpentane-1,5-diamino group at the C2-position, a
biphenyl group at the N3-position, and an N-benzylacetamide group at the C4-position was evaluated
3
9
and synthesized through an optimal design. However, it was limited in commercial chemical utility
and a further modification of the functional group at the C2-position was needed. The limitation had
to be overcome because functional variations into the C2-position should be important for improving
2
+
2+
the selectivity of T-type Ca channels among all Ca channels on the basis of our previous work
reporting selective Ca2+ channel activity according to only C2-position differences between
3
9
KYS05044 and KYS05089 (Figure 1). Therefore, we needed to switch the synthetic pathway to
obtain a surrogate structure with an electron donor group similar to the guanidine moiety. Among a
variety of donor groups, a thiourea group containing a sulfur atom was chosen as a synthetic
intermediate due to the fact that not only can it easily form the final structure through simple SN2
-
3 -

reaction, but also the sulfur version of the final compounds from a thiourea does not affect the original
electronic interaction with targets that has been shown in KYS05090.
O
O
O
O
O
N
H
N
N
N
N
N
NH2
N
N
N
N
N
N
H
KYS05044
KYS05089
KYS05090
Figure 1. Structures of KYS05044, KYS05089 and KYS05090.
2
. Results and discussion
2
.1. Chemistry
The requested core precursor, 3,4-dihydroquinazoline-2-thione, as a surrogate of a
guanidine moiety is an attractive heterocyclic structure, which may be applicable to biological and
4
0
pharmaceutical activities. In this study, we concentrated on an efficient synthetic approach of the 2-
thio-3,4-dihydroquinazoline scaffold and the biological properties to evaluate the scaffold
differentiation by first comparing T-type channel blocking activity of 2-amino-3,4-dihydroquinazoline,
KYS05090 containing a guanidine moiety and chemical variations at C2-position. A series of 2-thio-
3
,4-dihydroquinazoline derivatives as a guanidine-surrogate of KYS05090 was synthesized as
described below (Scheme 1). 2-Nitrocinnamic acid 1 was coupled with benzylamine or 4-
fluorobenzylamine via EDC [1-ethyl-3-(3-dimethylaminopropyl)carbodiimide] amide coupling
4
1
reaction. Compound 2 was reduced with Zn (powder)/NH Cl into compound 3. In the synthesis of
4
compound 4, chemoselective aza-Michael addition in which nitrogen acts as a nucleophile, not sulfur,
was required (Figure 2).
-
4 -

O
O
R4
Ph
R4
Aza-Michael addition
This work
Ph
S
O
HN
N
O
R4
Ph
N
S
N
H
R4
H
HN
NH2
+
O
N
S
O
H
R4
R4
Thia-Michael addition
PhNCS
SH
S
Ph
Ph
N
N
N
H
N
H
Figure 2. The chemoselective aza- and thia-Michael reactions.
So, we screened several solvents and bases to find optimal chemoselective aza-Michael
addition conditions (Table 1). Our first attempt without any base did not provide the desired product
4
a (entry 1 and 2 in Table 1). Moreover, the reaction proceeded very slowly. However, addition of a
base in DMSO solvent improved the yield to around 70% (entry 3 and 4 in Table 1). Additionally, the
reaction time can be reduced also with a high yield (91%) when the reaction temperature was increased
o
up to 80 C (entry 5 and 6 in Table 1). As a result, compound 4, 3.4-dihydroquinazoline-2-thione, was
o
smoothly synthesized via selective aza-Michael addition in our optimal condition (K CO base, 80 C,
2
3
2
h). Subsequently, the 2-thio-3,4-dihydroquinazoline derivatives were synthesized via two successive
4
2
nucleophilic substitution reactions (step d and e in Scheme 1). Isothiocyanates with a biphenyl
moiety, which are not commercially available, were prepared via a two-step reaction as shown below
in Scheme 1: 4-Bromoaniline was coupled with phenyl or 4-fluorophenylboronic acid via Suzuki
4
3
coupling into compound 8. The aniline moiety of 8 was reacted with carbondisulfide (CS ) to afford
2
the desired product 9 through desulfuration of an unstable dithiocarbamate intermediate.44
-
5 -

Table 1. Optimization of reaction conditions for the synthesis of compound 4 a
O
O
N
PhNCS, Base
H
Ph
N
H
o
N
solvent, T ( C)
H N
2
N
S
H
3a
4a
o
base b
yield of 4a (%) c
Entry
solvent
T ( C)
time (h)
1
2
3
4
5
6
Water
DMSO
DMSO
DMSO
DMSO
DMSO
80
r.t.
r.t.
r.t.
40
80
-
12
12
12
12
15
2
0
-
0
DIPEA
K CO
69
73
81
91
2
3
3
3
K CO
2
K CO
2
a
The reaction was conducted with 3a (0.10 g, 0.40 mmol), phenylisothiocyante
b
(
0.052 mL ,0.44 mmol) and base (0.12 mmol) in solvent (5 mL); DIPEA is
c
diisopropylethylamine (Hunig’s base); Isolated yield.
-
6 -

O
O
O
HO
a
N
b
N
H
H
O N
2
X
O N
2
X
H N
2
1
2a: X = H
3a-b
2b: X = F
O
O
N
N
c
H
d
e
R3
R3
H
X
N
X
N
N
S
N
S
Br
H
3
5a-f
4
a-b: R = Ph
3
4
c-d: R = Biphenyl
3
4
e-f: R = 4'-Fluorobiphenyl
O
N
H
R3
N
X
Y =
N
N
,
N
S
Y
6a-l
X
X
Br
f
g
H N
2
H N
2
SCN
7
8
9
8
8
a: X = H
b: X = F
9a: X = H
9b: X = F
Scheme 1. Synthesis of 2-thio-3,4-dihydroquinazoline derivatives (6) and reagent 9. Reagents and
conditions: (a) benzylamine or 4-fluorobenzylamine, EDC HCl, N-hydroxybenzotriazole (HOBt),
DCM/THF (1:1, v/v), rt, 71-78%; (b) Zn, NH Cl, MeOH, reflux, 95- 97%; (c) phenyl isothiocyanate
4
or compound 9, K CO , DMSO, 80 ℃, 80-91%; (d) 1,5-dibromopentane, K CO , DMF, 60 ℃, 60-
2
3
2
3
7
4%; (e) dimethylamine or pyrrolidine, K CO , DMF, 0 ℃ to rt, 22-89%; (f) phenylboronic acid,
2 3
Pd(PPh ) , K CO , EtOH/H O/toluene (3:3:10,v/v/v.), 100 ℃, 76-80%; (g) i) CS , 1,4-
3
4
2
3
2
2
diazabicyclo[2.2.2]octane (DABCO), acetone, rt, ii) di-tert-butyl dicarbonate, DCM, 0 ℃ to rt, 70-
3%.
7
-
7 -

Table 2. Biological activity of 2-thio-3,4-dihydroquinazoline derivatives as sulfur-analogues of
KYS05090
O
N
H
R3
X
N
N
R2
T-type Calcium channel inhibition:
inhibition (at 10 µM)
Cytotoxicity:
IC50 (µM)
%
b
a
Entry
R2
R3
X
(Estimated IC50 in µM)
Ca
v
3.1 Ca 3.2 Ca 3.3
v
v
A549 f HCT-116 g
c
d
e
(α
1G
)
(α1H
)
(α1I)
9
8.0±1.6
0.26)
0.4±19.8
77.01)
89.3±2.1
(3.6)
N
S
3
N
ND h
KYS05090
H
H
6.0
5.6
(
6
6
61.6±10.5
(7.71)
78.3±6.0
(6.19)
3
N
N
6
a
98.1
31.3
(
0.1±13.1
13.50)
40.0±15.3
(57.43)
22.1±13.5
(663.90)
S
3
6
b
F
89.9
27.1
(
4
2
6.5±13.2
18.51)
6.7±15.4
554.90)
3.9±1.7
0.66)
51.8±12.2
(13.18)
76.0±3.2
(3.20)
S
3
N
N
6
c
H
F
138.2
36.8
14.9
38.8
38.8
5.5
(
7.0±7.0
S
3
6
d
inactive
(
(832.30)
9
73.1±19.3
(6.90)
92.7±2.3
(0.81)
S
3
N
6
e
H
(
5
1.5±1.3
69.0±23.7
(276.10)
55.9±21.4
(13.63)
S
3
N
6
f
F
F
19.8
5.0
6.2
6.4
(
14.97)
3
1.0±7.4
83.1±15.4
(3.14)
69.3±7.02
(6.10)
S
3
N
6
g
(
27.38)
6.2±17.3
7.77)
3.9±7.0
2.18)
4.5±2.1
7
83.9±8.6
(2.40)
85.6±4.5
(1.76)
S
3
N
6
h
H
H
F
18.1
17.8
12.6
9.6
(
8
97.5±1.1
(0.26)
68.2±6.6
(5.10)
S
3
N
6
i
F
12.5
7.7
(
4
76.6±9.8
(4.88)
35.1±5.3
(19.78)
S
3
N
6
j
F
(
12.55)
9
7.6±0.8
75.6±12.7
(4.18)
66.7±27.7
(9.06)
S
3
N
F
F
6
k
H
F
17.6
11.3
24.9
9.2
9.3
ND
(
0.25)
6.2±1.0
0.40)
9
84.8±11.5
(2.76)
92.0±4.4
(0.96)
S
3
N
6
l
(
O
H
O
95.9 ± 1.7
(1.34) i
N
O
Mibefradil
ND
ND
N
N
F
a
v
Estimated IC50 values were obtained by measuring the percentage of inhibition of Ca 3 currents by 10 µM compound,
using patch clamp electrophysiology (n≥3) , and then calculating the IC50 by solving the Hill equation Ir=1/(1+[C]/IC50
)
where Ir is the fraction of current remaining after application of the compounds, and [C] is the concentration of the
b
c, d, e
T-type Ca2+ channels (α1G , α1H and α1I expressed in tsA-201 cells; Human non-
f
compound (10 µM) ; MTT assay;
g
h
i
39
small cell lung cancer cells; Human colon cancer cells; Not determined; Reported data by our group.
-
8 -

2
.2. Biological evaluation
The synthetic compounds were broadly divided into two classes to compare a structural
3
4
differentiation of the biological activities even though fluorine was added to R and R as bioisosteres.
2
3
2
One consisted of a combination of variable R and fixed R similar to KYS05090 as R (5-
2
3
dimethylaminopentyl), R -alternative (5-pyrrolidinopentyl) and R (biphenyl). The other was a
2
3
2
2
combination of variable R and R -alternatives as R (5-dimethylaminopentyl), R -alternative (5-
3
pyrrolidinopentyl) and R -alternative (phenyl).
T-type Ca2+ channel blocking abilities of all compounds were determined at 10 M
2
+
concentrations using a patch-clamp assay involving heterologously expressed T-type Ca channels.
Human embryonic kidney tsA-201 cells were cultured and transfected using the calcium phosphate
4
5
method as described previously. Cells were plated on glass coverslips and transfected with either 3
μg of human Ca 3.1, Ca 3.2 or Ca 3.3 cDNA. Plasmid encoding enhanced fluorescent protein (pEGFP)
v
v
v
(0.5μg) was included into the transfection mix as a transfection marker. Electrophysiological
recordings were performed according to the whole cell configuration of the patch-clamp technique 72
h after transfection at room temperature. The external recording solution contained (in mM): 20 BaCl2,
1
MgCl , 40 Tetraethylammonium Chloride (TEACl), 65 CsCl, 10 HEPES, and 10 glucose, pH 7.4.
2
Borosilicate glass pipettes were filled with internal solution containing (in mM): 140 CsCl, 2.5 CaCl2,
MgCl , 5 EGTA, 10 HEPES, 2 Na-Adenosinetriphosphate (ATP), and 0.3 Mg-
1
2
Guanosinetriphosphate (GTP), pH 7.3. Compounds were prepared daily in external solution and were
applied locally to cells. Currents were evoked by depolarization from a holding potential of −110 mV
to a test potential of −20 mV. The interpulse interval was 20 s, and the test pulse was 100 ms long.
The effects of the compounds were assessed by normalizing the current amplitude in the presence of
the compound to that observed before adding the drug. Independent experiments were performed using
the vehicle DMSO (0.1%) and no inhibition was observed.
2+
The overall in vitro T-type Ca channel blocking activities and anti-neoplastic effects of
new synthetic compounds are summarized in Table 2 and Figure 3. Data obtained with KYS05090
-
9 -

and mibefradil are also tabulated in Table 2 as a standard as well as a positive control for the activities.
As shown in Figure 3, some compounds similarly inhibited all three T-type channel isoforms, while
some exhibited a preference for Ca 3.2 (6j for example). We focused our analysis predominantly on
v
Ca 3.1 (α ) and Ca 3.2 (α ) since the correlation between Ca 3.3 overexpression and some tumors
v
1G
v
1H
v
4
6
is relatively lower compared with Ca 3.1 and Ca 3.2 channels. A detailed examination of Ca 3.1 and
v
v
v
Ca 3.2 reveals that similar to our previous SAR (structure-activity relationship) results,32 the
v
modification of the N3-position in 2-thio-3,4-dihydroquinazoline core structure corresponded to the
results of 2-amino-3,4-dihydroquinazoline so that most compounds with the biphenyl group at R3
position gave more effective inhibitory activities on both Ca 3.1 and Ca 3.2 transfected cells rather
v
v
than those of other derivatives that contain the phenyl group. Moreover, 6e (93.9±1.7% inhibition), 6k
97.6±0.8% inhibition), and 6l (96.2±1.0% inhibition) were similar or more potent in comparison to
mibefradil (95.9±1.7% inhibition) and KYS05090 (98.0±1.6% inhibition). For Ca 3.2 (α ) blocking
(
v
1H
activity, 6i (97.5±1.1% inhibition) exhibited a greater degree of inhibition than KYS05090. These
results are consistent with our previous SAR studies36,39 even though there was a substitution to a sulfur
atom at the C2-position of 3,4-dihydroquinazoline. However, it was an unexpected result that 6a and
6
b (60.4±19.8 and 60.1±13.1% against Ca 3.1, respectively) provided more active inhibition of Ca 3.1
v v
(
α ) than those of biphenyl derivatives 6f, 6g, and 6j (51.5±1.3, 31.0±7.4, and 44.5±2.1% against
1G
2
+
Ca 3.1, respectively), and 6d mediated exceptionally low T-type Ca channel inhibition (26.7±15.4
v
and 7.0±7.0% against Ca 3.1 and Ca 3.2, respectively).
v
v
2
+
Some T-type Ca channel blockers including small organic molecules and peptide toxins
1
either block the pore or inhibit gating allosterically. T-type channel activity can also be regulated via
second messengers or post-translational modifications that regulate cell surface density.47-48 Although
a direct action of our compounds on the channel are the most likely scenario, we cannot rule out the
possibility that some of the compounds might affect channel trafficking or regulation.
-
10 -

Figure 3. Inhibition of human Ca 3.1 (black bars), human Ca 3.2 (grey bars) and human Ca 3.3 (white
v
v
v
bars) calcium channels. Compounds were applied at a test concentration of 10 μM, and the percentage
of current inhibition determined (n = 3-7).
All prepared 2-thio-3,4-dihydroquinazoline derivatives were evaluated for cytotoxic activity
against A549 (human non-small cell lung cancer cells) and HCT-116 (human colon cancer cells) cell
4
9
3
lines using an MTT assay. There was also a steep SAR with the R group: The compounds (6a-d)
3
having phenyl group at R position exhibited poorer anti-proliferative activities than compounds (6e-
6
l) having biphenyl group at the same position. The IC values of compounds containing a biphenyl
50
3
group at R position against both A549 and HCT-116 cells were at least 3-fold more active than
3
compounds containing a phenyl group at R position. On the other hand, all of these compounds
exhibited greater cytotoxic effects than mibefradil (IC = 24.9 µM) against A549 cell lines. Among
5
0
the synthetic derivatives, 6g was most active against both A549 and HCT-116 cell lines (IC = 5.0
5
0
and 6.5 µM respectively). In addition, it had a better anti-proliferative effect than KYS05090 against
A549 cell lines. In the case of cytotoxicity against HCT-116 cell lines, compounds containing a
3
biphenyl group at R position showed better or similar activities (IC = 5.5 into 9.6 µM) compared
5
0
2
+
with KYS05090 (IC = 5.6 µM) except 6i (IC = 12.5 µM). For some compounds, T-type Ca
5
0
50
channel blocking activity was low, yet they mediated potent cytotoxicity. It is possible that these
compounds might affect cancer cells by acting on another target, or by altering T-type channel
-
11 -

mediated processes that do not involve a reduction in calcium entry. Therefore, further biochemical or
biological studies will be needed to explain the SAR correlation obtained in 10 μM concentrations.
3
. Conclusions
In summary, a series of 2-thio-3,4-dihydroquinazoline was synthesized and biologically
2
+
evaluated to identify their T-type Ca channel (Ca 3.1, Ca 3.2 and Ca 3.3) blocking effects and
v
v
v
cytotoxic effects against A549 (human non-small cell lung cancer cells) and HCT-116 (human colon
cancer cells). These findings add to a large body of recent literatures that reported a range of different
1
0
pharmacophores and their SAR against heterologously expressed T-type calcium channels, although
SAR comparison between our compound series and these previous works is difficult given the wide
range of chemical structures of T-type channel blockers. Within our compound series, a synthetic
optimization of aza-Michael addition reaction via addition of a base led successfully to the synthesis
of 2-thio-3,4-dihydroquinazoline derivatives in good yield (80-91% yield at the aza-Michael addition
3
step). The biological data showed that most compounds having the biphenyl group at R position of 2-
thio-3,4-dihydroquinazoline derivatives were more effective than compounds containing the phenyl
group at the same position with regard to both T-type calcium channel blockade and cytotoxicity,
which is consistent with our previous research of SAR studies. Therefore, these results indicate that
the guanidine moiety of KYS05090 would be not a necessary functional group for both T-type calcium
channel block and cytotoxicity against cancer cell lines, and thus provide the opportunity to expand
the available repertoire of chemical diversities for T-type calcium channel block. In addition, 6g
exhibited better (IC = 5.0 µM against A549 cell) or similar (IC = 6.2 µM against HCT-116 cell)
5
0
50
cytotoxic effect in comparison with the reference compound KYS05090. Thus, 6g could be a backup
compound of KYS05090 for further biological investigations as an anti-neoplastic agent.
-
12 -

4
4
4
. Experimental section
.1. Chemistry
.1.1. General
All solvents and commercially available reagents were used without additional purification.
Reactions were monitored by analytical thin layer chromatography (TLC, Merck, silica gel 60 F )
2
54
with UV light (254 nm). Visualization was performed by incubation with p-methoxyanisaldehyde
PMA) solution and heating with a heat gun. Flash column chromatography was performed using a
(
1
13
silica gel (Merck, 230-400 mesh). NMR spectra ( H and C) were recorded on Bruker Avance 400
MHz spectrometer. High-resolution mass spectra were recorded with JEOL JMS-700 MStation mass
spectrometer (ionization mode: FAB+). HPLC for chemical purities of final compounds 6a-6l was
carried out on Waters 1525, equipped with Waters PDA 2998 (280 nm).
4
.1.2. General preparation of compound 8 43
To a solution of 4-bromoaniline (7, 2.00 g, 11.60 mmol) and phenylboronic acid (1.56 g,
1
2.78 mmol) in a mixed solution of water, ethanol and toluene (30 mL, H O:EtOH:toluene=3:3:10,
2
v/v) were added Pd(PPh ) (0.13 g, 0.12 mmol) and K CO (16.00 g, 116.00 mmol) neatly. The reaction
3
4
2
3
o
mixture was purged with Ar gas. The reaction mixture was stirred at 100 C for 12 h in Ar atmosphere
and concentrated under reduced pressure to give the crude product. The crude product was extracted
with dichloromethane (50 mL) and water (40 mL), dried (MgSO ) and concentrated in vacuo. The
4
residue was purified by flash column chromatography (CH Cl :hexane = 2:5) to provide desired
2
2
product 8.
1,1'-biphenyl]-4-amine (8a, 1.75 g, 80%): H NMR (400 MHz, DMSO-d ) δ 7.53 (2H, d, J = 7.6 Hz),
1
[
6
7
.37 (4H, t, J = 7.6 Hz), 7.21 (1H, t, J = 7.2 Hz), 6.65 (2H, d, J = 8.4 Hz), 5.23 (2H, s).
1
4
'-fluoro-[1,1'-biphenyl]-4-amine (8b, 1. 65 g, 76%): H NMR (400 MHz, CDCl ) δ 7.56 – 7.53 (2H,
3
m), 7.32 (2H, d, J = 8.4 Hz), 7.18 (2H, t, J = 5.0 Hz), 6.63 (2H, d, J = 8.4 Hz), 5.21 (2H, s).
-
13 -

4
.1.3. General preparation of Compound 9 44
To a solution of 8a or 8b (10.38 mmol) and DABCO (3.49 g, 31.15 mmol) in acetone (10
mL) was dropwise added CS (20 mL). The reaction mixture was stirred for 1 h at room temperature
2
and then filtered. The filter cake was dissolved in dichloromethane (30 mL). To this solution, were
added di-tert-butyl dicarbonate (2.24 g, 10.28 mmol) and DMAP (4-dimethylaminopyridine ) (0.02 g,
0
.21 mmol) and stirred for 1 h at room temperature. The reaction mixture was extracted with
dichloromethane (45 mL) and water (30 mL), dried with MgSO and evaporated under reduced
4
pressure. Flash column chromatography (DCM:hexane = 1:5) gave the desired product 9.
1
4
-isothiocyanato-1,1'-biphenyl (9a, 1.78 g, 70%): H NMR (400 MHz, CDCl ) δ 7.51 – 7.48 (4H, m),
3
7
.38 (2H, t, J = 7.4 Hz), 7.32 – 7.30 (1H, m), 7.28 – 7.19 (2H, m).
1
4
-fluoro-4'-isothiocyanato-1,1'-biphenyl (9b, 1.74 g, 73%): H NMR (400 MHz, DMSO-d ) 7.75 –
6
7
.73 (4H, ,m), 7.52 (2H, d, J = 8.4 Hz), 7.31 (2H, t, J = 9.0 Hz).
4
.1.4. General preparation of compound 2 41
A solution of 2-nitrocinnamic acid (1, 2.00 g, 10.35 mmol) in THF (15 mL) and DCM (15
mL) was treated with EDC·HCl (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) salt
4.96 g, 25.88 mmol), HOBt (N-hydroxy-benzotriazole) (4.20 g, 31.05 mmol) and benzylamine or 4-
(
fluorobenzylamine (31.05 mmol) neatly. The reaction mixture was stirred for 30 min under ice bath,
further stirred at room temperature overnight and concentrated under reduced pressure. The reaction
mixture was dissolved with a mixed solvent of CH Cl (100 mL) and water (20 mL), extracted with
2
2
CH Cl , dried (MgSO ) and concentrated under reduced pressure. The concentrated residue was
2
2
4
recrystallized with co-solvent of CH Cl , methanol and ethyl ether to give the desired product 2.
2
2
1
(
E)-N-benzyl-3-(2-nitrophenyl)acrylamide (2a, 2.28 g, 78%): H NMR (400 MHz, CDCl ) δ 8.79 (1H,
3
t, J = 5.4 Hz), 8.06 (1H, d, J = 8.0 Hz), 7.79 – 7.73 (3H, m), 7.65 – 7.63 (1H, m), 7.37 – 7.25 (1H, m),
.70 (1H, d, J = 15.6 Hz) 4.42 (2H, d, J = 6.0 Hz).
6
-
14 -

1
(
E)-N-(4-fluorobenzyl)-3-(2-nitrophenyl)acrylamide (2b, 2.21 g, 71%): H NMR (400 MHz, DMSO-
d6) δ 8.78 (1H, s), 8.06 (1H, d, J = 8.0 Hz), 7.76 (3H, m), 7.65 (1H, m), 7.35 (2H, m), 7.18 (2H, t, J =
9
.2 Hz), 6.68 (1H, d, J = 15.6 Hz), 4.40 (2H, d, J = 5.6 Hz).
4
.1.5. General preparation of compound 3
Zinc powder 2 g was stirred with 2% HCl aqueous solution (4 mL) for 30 min. The mixture
was then filtered and washed with water (10 mL) and diethyl ether (10 mL). The filter cake (activated
zinc powder) was dryed in vacuo. To a solution of 2 (3.54 mmol) in methanol (20 mL) were added
purified zinc (1.57 g, 28.3 mmol) and ammonium chloride (0.42g, 7.79 mmol) at room temperature.
The reaction mixture was stirred at reflux for 16 h. The resulting mixture was cooled to room
temperature and filtered through Celite 545. The filtrate was concentrated in vacuo and dissolved with
CH Cl . The organic solution was washed with sat. NaHCO solution, dried (MgSO ) and evaporated
2
2
3
4
in vacuo to provide the desired product 3.
E)-3-(2-aminophenyl)-N-benzylacrylamide (3a, 0.91 g, 95%): H NMR (400 MHz, CDCl ) δ 7.74 (1H,
1
(
3
d, J = 15.6 Hz), 7.29 – 7.19 (6H, m), 7.09 – 7.05 (1H, m), 6.66 (1H, t, J = 7.4 Hz), 6.61 (1H, d, 8.0
Hz), 6.25 (1H, d, J = 15.2 Hz), 5.22 (1H, br s), 4.49 (2H, d, J = 5.6 Hz), 2.79 (2H, br s).
1
(
E)-3-(2-aminophenyl)-N-(4-fluorobenzyl)acrylamide (3b, 1.0 g, 97%) H NMR (400 MHz, DMSO-d )
6
δ 8.51 (1H, t, J = 5.2 Hz), 7.65 (1H, d, J = 15.6 Hz), 7.31 (3H, m), 7.16 (2H, t, J = 8.8 Hz), 7.04 (1H, t, J = 7.2
Hz), 6.68 (1H, d, J = 7.6 Hz), 6.55 (1H, t, J = 7.6 Hz), 6.45 (1H, d, J = 15.6 Hz), 5.39 (2H, s), 4.38 (2H, d, J =
6
.0 Hz).
4
.1.6. General preparation of compound 4
To a solution of compound 3 (0.65 mmol) and phenylisothiocyanate (0.72 mmol) in DMSO
o
(
10 mL) was added K CO (0.03 g,0.20 mmol). The reaction mixture was stirred for 2 h at 80 C and
2
3
poured into a mixed solution of EtOAc (45 mL) and water (60 mL). The extracted combinded organic
-
15 -

layer was dried with MgSO and evaporated under reduced pressure. The crude product was then
4
purified by flash column chromatography using the gradient eluent system (EA: Hex = 1:5 to 1:1) for
4
a and 4b or purified by recrystalization with THF and hexane for 4c – 4f.
1
N-benzyl-2-(3-phenyl-2-thioxo-1,2,3,4-tetrahydroquinazolin-4-yl)acetamide (4a, 0.18 g, 72%): H
NMR (400 MHz, DMSO) δ 11.03 (1H, s), 8.32 (1H, t, J = 5.6 Hz), 7.46 – 7.28 (5H, m) 7.27 – 7.20
(
4H, m), 7.14 (1H, d, J = 7.6 Hz), 7.05 – 6.97 (4H, m), 5.16 (1H, dd, J = 10 Hz and 4 Hz), 4.22 (1H,
dd, J = 15 Hz and 6 Hz), 4.05 (1H, dd, J = 14.6 Hz and 6.2 Hz), 2.77 (1H, dd, J = 13.6 Hz and 4 Hz),
.63 (1H, dd, J = 14 Hz and 10 Hz ).
2
N-benzyl-2-(3-(4-fluorophenyl)-2-thioxo-1,2,3,4-tetrahydroquinazolin-4-yl)acetamide (4b, 0.16
1
g ,61%): H NMR (400 MHz, CDCl ) δ 9.41 (1H, s), 7.38 – 7.28 (5H, m), 7.22 – 7.14 (1H, m), 7.10 -
3
7
.03 (1H, m), 6.93 – 6.81 (6H, m), 5.76 (1H, br s), 5.22 (1H, dd, J = 9.6 Hz and 4.4 Hz ), 6.25 (1H, d,
J = 15.2 Hz), 5.22 (1H, br s), 5.15 (1H, dd, J = 9.6 Hz and 3.6 Hz), 4.16 (1H, dd, J = 14.8 Hz and 6.0
Hz), 4.06 – 3.97 (1H, m), 2.82 (1H, dd, J = 13.6 Hz and 4.0 Hz), 2.59 – 2.56 (1H, m).
2
-(3-([1,1'-biphenyl]-4-yl)-2-thioxo-1,2,3,4-tetrahydroquinazolin-4-yl)-N-benzylacetamide (4c, 0.21
1
g, 71%): H NMR (400 MHz, DMSO-d ) δ 11.07 (1H, s), 8.35 (1H, s), 7.73 – 7.71 (4H, m), 7.50 – 7.49
6
(
4H, m), 7.42 – 7.40 (1H, m), 7.31 (1H, t, J = 8.0 Hz), 7.25 – 7.20 (4H, m), 7.16 – 7.02 (4H, m), 5.21
1H, dd, J = 9.4 Hz and 3.6 Hz), 4.22 (1H, dd, J = 14.9 Hz and 5.9 Hz), 4.09 – 4.04 (1H, m), 2.82 –
(
2
.79 (1H, m), 2.68 – 2.66 (1H, m).
2
-(3-([1,1'-Biphenyl]-4-yl)-2-thioxo-1,2,3,4-tetrahydroquinazolin-4-yl)-N-(4-fluorobenzyl)acetamide
1
(
4d, 0.22 g, 69%): H NMR (400 MHz, DMSO-d ) δ 11.08 (1H, s), 8.36 (1H, t, J = 5.4 Hz ), 7.73 –
6
7
.71 (4H, m), 7.52 – 7.51 (4H, m), 7.50 – 7.49 (1H, m), 7.42 – 7.38 (1H, m), 7.33 – 7.29 (1H, m) 7.15
-
16 -

–
–
7.13 (1H, m), 7.10 – 6.99 (6H, m), 5.20 (1H, dd, J = 9.4 Hz and 3.6 Hz), 4.22 – 4.17 (1H, m), 4.08
4.01 (1H, m), 2.82 – 2.78 (1H, m), 2.67 – 2.61 (1H, m).
N-benzyl-2-(3-(4'-fluoro-[1,1'-biphenyl]-4-yl)-2-thioxo-1,2,3,4-tetrahydroquinazolin-4-yl)acetamide
1
(
4e, 0.19 g , 62%): H NMR (400 MHz, DMSO-d ) δ 11.04 (1H, s), 8.34 (1H, s), 7.80 – 7.75 (4H, m),
6
7
9
.71 – 7.74 (3H, m), 7.50 – 7.39 (3H, m), 7.25 – 7.19 (1H, m), 7.10 – 7.01 (6H, m), 5.20 (1H, dd, J =
.4 Hz and 3.6 Hz), 4.18 – 4.16 (1H, m), 4.09 – 4.04 (1H, m), 2.80 – 2.74 (1H, m), 2.65 – 2.59 (1H,
m).
2
-(3-(4'-fluoro-[1,1'-biphenyl]-4-yl)-2-thioxo-1,2,3,4-tetrahydroquinazolin-4-yl)-N-(4-
1
fluorobenzyl)acetamide (4f, 0.20 g, 63%): H NMR (400 MHz, DMSO-d ) δ 11.07 (1H, s), 8.35 (1H,
6
s), 7.85 – 7.76 (4H, m), 7.65 – 7.62 (2H, m), 7.50 – 7.39 (3H, m), 7.25 – 7.05 (7H, m), 5.19 (1H, dd,
J = 9.4 Hz and 3.6 Hz), 4.20 – 4.16 (1H, m), 4.10 – 4.06 (1H, m), 2.79 – 2.70 (1H, m), 2.64 – 2.60
(1H, m).
4
.1.7. General preparation of compound 5
To a solution of 1,5-dibromopentane (0.07 mL, 0.52 mmol) and K CO (0.07 g, 0.52 mmol)
2
3
in DMF (6 mL) was added dropwise 4 (0.26 mmol). The reaction mixture was stirred at room
temperature for 7 h. The reaction mixture was then poured into a mixed solution of ethyl ether (30 mL)
and water (15 mL). The exracted organic layer was dried with MgSO , evaporated in vacuo and
4
purified by flash column chromatography (EA: hexane = 1:3) to give the desired product 5.
N-benzyl-2-(2-((5-bromopentyl)thio)-3-phenyl-3,4-dihydroquinazolin-4-yl)acetamide (5a, 0.2 g, 70%):
1
H NMR (400 MHz, DMSO-d ), δ 8.35 (1H, t, J = 5.6 Hz), 7.43 – 7.32 (5H, m), 7.28 – 7.21 (4H, m),
6
7
5
–
.09 – 7.07 (3H, m), 7.01 (1H , t, J = 7.2 Hz), 6.96 (1H, d, J = 7.2 Hz), 5.08 (1H, dd, J = 5.2 Hz and
.6 Hz), 4.26 – 4.10 (2H, m), 3.53 (2H , t, J = 6.6 Hz), 3.15 – 3.08 (1H, m), 2.99 – 2.92 (1H, m), 2.71
2.66 (1H, m), 2.57 – 2.51 (1H, m), 1.86 – 1.79 (2H, m), 1.64 – 1.58 (2H, m), 1.48 – 1.43 (2H, m).
-
17 -

2
0
7
-(2-[(5-bromopentyl)thio]-3-phenyl-3,4-dihydroquinazolin-4-yl)-N-(4-fluorobenzyl)acetamide (5b,
1
.22 g, 64%): H NMR (400 MHz, DMSO-d ), δ 8.21 (1H, t, J = 5.6 Hz), 7.48 – 7.40 (4H, m), 7.32 –
6
.21 (4H, m), 7.11 – 7.13 (3H, m), 7.09 (1H , t, J = 7.2 Hz), 6.87 (1H, d, J = 7.2 Hz), 5.11 (1H, dd, J
=
5.2 Hz and 5.6 Hz), 4.22 – 4.15 (2H, m), 3.46 (2H , t, J = 6.6 Hz), 3.22 – 3.20 (1H, m), 2.94 – 2.90
(1H, m), 2.72 – 2.53 (2H, m), 1.83 – 1.75 (2H, m), 1.59 – 1.54 (2H, m), 1.46 – 1.44 (2H, m).
2
-(3-([1,1'-biphenyl]-4-yl)-2-((5-bromopentyl)thio)-3,4-dihydroquinazolin-4-yl)-N-benzylacetamide
1
(
5c, 0.24 g, 67%): H NMR (400 MHz, CDCl ) δ 7.52 – 7.49 (4H, m), 7.40 – 7.56 (2H, m), 7.31 – 7.26
3
(3H, m), 7.22 – 7.16 (5H, m), 7.02 (2H, d, J = 2.0 Hz), 7.00 – 6.95 (2H, m), 5.71 (1H, s), 5.15 (1H, dd,
J = 5.6 Hz and 6 Hz), 4.27 – 4.18 (2H, m), 3.34 – 3.30 (2H, m), 3.24 – 3.22 (1H, m), 3.04 – 3.02 (1H,
m), 2.69 – 2.65 (1H, m), 2.49 – 2.46 (1H, m), 1.83 – 1.78 (2H, m), 1.63 – 1.58 (2H, m), 1.50 – 1.44
(2H, m).
2
-(3-([1,1'-biphenyl]-4-yl)-2-((5-bromopentyl)thio)-3,4-dihydroquinazolin-4-yl)-N-(4-
1
fluorobenzyl)acetamide (5d, 0.22 g, 69%): H NMR (400 MHz, CDCl ) δ 7.62 – 7.60 (4H, m), 7.50 –
3
7
5
.46 (2H, m), 7.40 – 7. 38 (3H, m), 7.33 – 7. 30 (2H, m), 7.09 – 7.03 (4H, m), 6.98 – 6.94 (2H, m),
.62 (1H, s), 5.23 (1H, t, J = 7.0 Hz), 4.34 – 4.27 (2H, m), 3.43 (2H, t, J = 6.8 Hz), 3.30 – 3.26 (1 H,
m), 3.12 – 3.05 (1H, m), 2.79 – 2.75 (1H, m), 2.63 – 2.60 (1H, m), 1.94 – 1.89 (2H, m), 1.74 – 1.69
3H, m), 1.62 – 1.55 (3H, m).
(
N-benzyl-2-(2-[(5-bromopentyl)thio]-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)-3,4-dihydroquinazolin-4-
1
yl)acetamide (5e, 0.18 g, 60%): H NMR (400 MHz, CDCl ) δ 7.58 – 7.53 (4H, m), 7.39 (2H, d, J =
3
8
8
2
.0 Hz), 7.32 – 7.18 (5H, m), 7.26 – 7.11 (4H, m), 7.07 –7.06 (2H, m), 5.76 (1H, s), 5.23 (1H, t, J =
.0 Hz), 4.34 – 4.31 (1H, m), 3.44 – 3.41(1H, m), 3.27 – 3.24 (1H, m), 3.05 – 3.01 (1H, m), 2.80 –
.75 (1H, m), 2.62 – 2.56 (1H, m), 1.93 – 1.88 (2H, m), 1.73 – 1.68 (2H, m), 1.60 – 1.54 (2H, m).
-
18 -

2
-(2-[(5-bromopentyl)thio]-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)-3,4-dihydroquinazolin-4-yl)-N-(4-
1
fluorobenzyl)acetamide (5f, 0.20 g, 74%), H NMR (400 MHz, CDCl ) δ 7.58 – 7.53 (4H, m), 7.38
3
(
2H, d, J = 8.4 Hz), 7.32 – 7.24 (2H, m), 7.16 (2H, t, J = 8.8 Hz), 7.09 – 7.02 (4H, m), 6.96 (1H, t, J =
.4 Hz), 5.67 (1H, s), 5.21(1H, t, J = 7.6 Hz), 4.34 – 4.27 (2H, m), 3.43 (2H, t, J = 6.8 Hz), 3.27 – 3.25
1H, m), 3.06 – 3.03 (1H, m), 2.75 – 2.73 (1H, m), 2.62 – 2.58 (1H, m), 1.94 – 1.88 (2H, m), 1.74 –
8
(
1
.70 (2H, m), 1.60 – 1.55 (3H, m).
4
.1.8. General preparation of compound 6
To a solution of 5 (0.19 mmol), K CO (0.05 g, 0.38 mmol) in DMF (5 mL) was added
2
3
dropwise dimethylamine (ca. 2 M in THF solution, 0.95 mL) or pyrrolidine ( 0.03 mL,0.38 mmol) at
0 oC. The reaction mixture was then warmed to room temperature and stirred for 3 h. The reaction
mixture was treated with EtOAc (20 mL) and water (30 mL). The extracted organic layer was dried
with MgSO and concentrated under reduced pressure to provide a crude product, which was purified
4
by flash column chromatography (DCM:MeOH:NH OH= 100:3:1 into 100:9:1) to afford the desired
4
product 6.
N-benzyl-2-(3-phenyl-2-[(5-(pyrrolidin-1-yl)pentyl)]thio)-3,4-dihydroquinazolin-4-yl)acetamide (6a,
1
0
5
–
2
.05 g, 50%): H NMR (400 MHz, CDCl ) δ 7.30 – 7.1 (10H, m), 7.00 – 6.93 (4H, m), 5.74 (1H, s),
3
.09 (1H, t, J = 8 Hz), 4.27 (1H, dd, J = 14.6 Hz and 5.6 Hz), 4.18 (1H, dd, J = 14.8 and 5.2 Hz), 3.12
3.05 (1H, m), 2.96 – 2.89 (1H, m), 2.66 (1H, dd, J = 12.8 Hz and 5.6 Hz), 2.50 (1H, s), 2.46 (4H, s),
.38 (2H, t, J = 7.6 Hz), 1.71 (4H, s), 1.58 – 1.57 (2H, m), 1.47 (2H, t. J = 7.2 Hz), 1.35 (2H, t, J = 7.2
1
3
Hz); C NMR (100 MHz, CDCl ) δ 169.1, 157.3, 142.6, 141.7, 137.7, 129.2, 128.6, 128.5, 127.8,
3
1
2
27.4, 127.4, 127.2, 125.6, 125.0, 124.5, 123.2, 60.3, 56.3, 54.1, 43.6, 41.9, 31.3, 29.1, 28.2, 26.8,
+
3.3; Purity: 96%; HRMS (FAB+): m/z estimated for C H N OS [M+H] 527.2839, found: 527.2845.
3
2
39
4
-
19 -

N-(4-fluorobenzyl)-2-(3-phenyl-2-[(5-(pyrrolidin-1-yl)pentyl]thio)-3,4-dihydroquinazolin-4-
1
yl)acetamide (6b, 0.02 g, 22 %): H NMR (400 MHz, CDCl ) δ 7.29 – 7.16 (5H, m), 7.07 (1H, d, J =
3
7
.6 Hz), 7.00 – 6.93 (4H, m), 6.82 (2H, t, J = 8.4 Hz), 6.62 (1H, s), 5.11 (1H, dd, J = 8.4 Hz and 6.0
Hz), 4.25 (1H, dd, J = 14.8 Hz and 6.4 Hz), 4.12 (1H, dd, J = 14.8 Hz and 5.6 Hz), 3.03 – 2.92 (6H,
m), 2.85 – 2.80 (2H, m), 2.71 (1H, dd, J = 13.6 Hz and 5.6 Hz), 2.55 (1H, dd, J = 13.6 Hz and 8.4 Hz),
1
3
1
.91 (4H, s), 1.75 (2H, t, J = 3.6 Hz), 1.62 (2H, t, J = 7.2 Hz), 1.48 – 1.30 (2H, m); C NMR (100
MHz, CDCl ) δ 169.1, 157.1, 142.4, 141.7, 134.0, 129.5, 129.4, 129.2, 128.5, 127.3, 125.7, 125.2,
3
1
24.5, 123.1, 115.3, 115.1, 60.2, 55.4, 53.6, 42.6, 41.7, 30.6, 28.7, 25.6, 25.5, 23.3; Purity: 93%;
+
HRMS (FAB+): m/z estimated for C H FN OS [M+H] 545.2745, found: 545.2750.
3
2
38
4
N-benzyl-2-(2-[(5-(dimethylamino)pentyl]thio)-3-phenyl-3,4-dihydroquinazolin-4-yl)acetamide (6c,
1
0
6
.05 g, 49%): H NMR (400 MHz, CDCl ) δ 7.31 – 7.11 (10H, m), 7.00 (2H, d, J = 6.0 Hz), 6.97 –
3
.94 (2H, m), 5.68 (1H, s), 5.10 (1H, dd, J = 8.2 Hz and 6.4 Hz), 4.28 (1H, dd, J = 15.2 Hz and 5.6
Hz), 4.19 (1H, dd, J = 14.8 Hz and 5.2 Hz), 3.10 – 3.05 (1H, m), 2.96 – 2.93 (1H, m), 2.67 (1H, dd, J
13.2 Hz and 6.0 Hz), 2.48 (1H, dd, J = 13.2 Hz and 8.4 Hz), 2.22 (2H, t, J = 7.6 Hz), 2.16 (6H, s),
=
1
3
1
1
5
.61 – 1.57 (2H, m), 1.45 – 1.40 (2H, m), 1.35 – 1.30 (2H, m); C NMR (100 MHz, CDCl ) δ 169.0,
3
57.2, 142.6, 141.7, 137.6, 129.2, 128.6, 127.8, 127.4, 127.3, 127.2, 125.5, 125.0, 124.5, 123.2, 60.3,
9.3, 45.0, 43.7, 42.0, 31.1, 29.7, 29.1, 26.6, 26.4; Purity: 95%; HRMS (FAB+): m/z estimated for
+
C H N OS [M+H] 501.2683, found: 501.2686.
3
0
37
4
N-(4-fluorobenzyl)-2-(2-[(5-(dimethylamino)pentyl]thio)-3-phenyl-3,4-dihydroquinazolin-4-yl)
1
acetamide (6d, 0.03 g, 32 %): H NMR (400 MHz, DMSO-d ) δ 8.36 (1H, t, J = 6.0 Hz), 7.43 – 7.33
6
(
5H, m), 7.26 (1H, t, J = 7.2), 7.14 – 7.06 (5H, m), 7.00 (1H, t, J = 7.2 Hz), 6.94 (1H, d, J = 6.8 Hz),
.07 (1H, dd, J = 8.4 Hz and 5.6 Hz), 4.22 (1H, J = 15.2 Hz and 6.0 Hz), 4.11 (1H, dd, J = 15.2 Hz
and 5.6 Hz), 3.13 – 3.10 (1H, m), 2.96 – 2.93 (1H, m), 2.68 (1H, dd, J = 13.6 Hz and 5.2 Hz), 2.56 –
.52 (1H, m), 2.21 (2H, t, J = 7.2 Hz), 2.13 (6H, s), 1.61 – 1355 (2H, m), 1.43 – 1.37 (2H, m), 1.35 –
5
2
-
20 -

1
3
1
.24 (2H, m); C NMR (100 MHz, CDCl ) δ 169.0, 157.2, 142.6, 141.7, 129.5, 129.4, 129.2, 128.6,
3
1
27.3, 127.2, 125.5, 124.9, 124.5, 123.3, 115.5, 115.2, 60.3, 59.4, 45.2, 43.0, 42.0, 31.2, 29.1, 26.9,
+
2
6.5; Purity: 96%; HRMS (FAB+): m/z estimated for C H FN OS [M+H] 519.2588,
3
0
36
4
found:519.2594.
N-benzyl-2-(3-([1,1'-biphenyl]-4-yl)-2-([5-(pyrrolidin-1-yl)pentyl]thio)-3,4-dihydroquinazolin-4-yl)
1
acetamide (6e, 0.03 g, 25%): H NMR (400 MHz, CDCl ) δ 7.50 (4H, t, J = 7.6 Hz), 7.36 (2H, t, J =
3
7
4
–
=
.6 Hz), 7.30 – 7.02 (8H, m), 7.01 – 6.98 (2H, m), 6.95 (2H, s), 5.77 (1H, s), 5.13 (1H, t, J = 8.0 Hz),
.29 (1H, dd, J = 14.8 Hz and 6.0 Hz), 4.20 (1H, dd, J = 14.8 Hz and 5.2 Hz) 3.13 – 2.97 (1H, m), 2.95
2.4 (1H, m), 2.68 (2H, dd, J = 13.4 Hz and 6.0 Hz), 2.51 – 2.48 (1H, m), 2.44 (4H, s), 2.37 (2H, t, J
1
3
7.6 Hz), 1.69 (4H, s), 1.65 – 1.59 (2H, m), 1.49 – 1.45 (2H, m), 1.38 – 1.32 (2H, m); C NMR (100
MHz, CDCl ) δ 169.1, 157.2, 141.8, 141.7, 140.1, 139.9, 137.7, 128.9, 128.6, 128.5, 127.8, 127.5,
3
1
9
27.4, 127.1, 125.5, 125.1, 124.6, 123.3, 60.4, 56.3, 54.1, 43.7, 41.9, 31.3, 29.1, 28.3, 26.9, 23.4; Purity:
+
3%; HRMS (FAB+): m/z estimated for C H N OS [M+H] 603.3152, found: 603.3158.
3
8
43
4
N-(4-fluorobenzyl)-2-(3-([1,1'-biphenyl]-4-yl)-2-([5-(pyrrolidin-1-yl)pentyl]thio)-3,4-
1
dihydroquinazolin-4-yl)acetamide (6f, 0.04 g, 30%): H NMR (400 MHz, CDCl ) δ 7.50 – 7.48 (4H,
3
m), 7.37 – 7.11 (7H, m), 7.98 – 6.85 (6H, m), 5.96 (1H, s), 5.13 (1H, s), 4.24 – 4315 (2H, m), 3.07
(1H, m), 3.00 (1H, m), 2.69 – 2.67 (1H, m), 2.53 – 2.45 (7H, m), 1.73 (4H, s), 1.62 – 1.53 (5H, m),
1
3
1
1
5
.36 (2H, s); C NMR (100 MHz, CDCl ) δ 169.1, 157.1, 141.8, 141.7, 140.1, 139.9, 133.6, 129.5,
3
29.4, 128.8, 128.6, 127.8, 127.5, 127.3, 127.1, 125.5, 125.1, 124.6, 123.3, 115.4, 115.2, 60.4, 56.2,
4.1, 42.9, 41.9, 31.2, 29.7, 29.1, 27.9, 26.6, 23.3; Purity: 98%; HRMS (FAB+): m/z estimated for
+
C H FN OS [M+H] 621.3058, found:621.3063.
3
8
42
4
N-(4-fluorobenzyl)-2-(3-([1,1'-biphenyl]-4-yl)-2-([5-(dimethylamino)pentyl]thio)-3,4-
1
dihydroquinazolin-4-yl)acetamide (6g, 0.08 g, 75%): H NMR (400 MHz, CDCl ) δ 7.60 (4H, t, J =
3
-
21 -

7
6
4
.6 Hz), 7.47 (2H, t, J = 7.6 Hz), 7.39 (3H, d, J = 8.0 Hz), 7.31 – 7.21 (2H, m), 7.10 – 7.04 (4H, m),
.95 (2H, t, J = 8.4 Hz), 5.96 (1H, s), 5.23 (1H, t, J = 7.6 Hz), 4.36 (1H, dd, J = 14.8 Hz and 5.6 Hz),
.26 (1H, dd, J = 14.8 Hz and 5.2 Hz), 3.21 – 3.16 (1H, m), 3.13 – 3.08 (1H, m), 2.78 (1H, dd, J = 13.6
Hz and 5.6 Hz), 2.60 (1H, dd, J = 13.2 Hz and 8.4 Hz), 2.39 (2H, t, J = 6.0 Hz), 2.30 (6H, s), 1.74 –
1
3
1
1
1
.70 (2H, m), 1.58 (2H, s), 1.48 – 1.43 (2H, m); C NMR (100 MHz, CDCl ) δ 169.2, 163.3, 160.8,
3
57.2, 141.8, 141.7, 140.1, 139.9, 133.6, 129.5, 129.4, 128.9, 128.6, 127.8, 127.6, 127.4, 127.1, 125.5,
25.1, 124.6, 123.3, 115.4, 115.2, 60.4, 59.5, 45.4, 42.9, 41.8, 31.3, 29.2, 27.1, 26.7; Purity: 98%;
+
HRMS (FAB+): m/z estimated for C H FN OS [M+H] 595.2901, found: 595.2903.
3
6
40
4
N-benzyl-2-(3-([1,1'-biphenyl]-4-yl)-2-([5-(dimethylamino)pentyl]thio)-3,4-dihydroquinazolin-4-yl)
1
acetamide (6h, 0.10 g, 89%): H NMR (400 MHz, CDCl ) δ 7.60 (4H, t, J = 7.6 Hz), 7.47 (2H, t, J =
3
7
.6 Hz), 7.41 – 7.38 (3H, m), 7.31 – 722 (5H, m), 7.11 (2H, d, J = 5.6 Hz), 7.06 – 7.05 (2H, m), 5.78
1H, s), 5.24 (1H, dd, J = 8.0 Hz and 6.0 Hz), 4.40 (1H, dd, J = 14.4 Hz and 6.0 Hz), 4.31 (1H, dd, J
14.8 Hz and 5.2 Hz), 3.21 – 3.19 (1H, m), 3.09 – 3.06 (1H, m), 2.79 (1H, dd, J = 13.2 Hz and 6.0
Hz), 2.59 (1H, dd, J = 13.2 Hz and 8.4 Hz), 2.34 (2H, t, J = 7.2 Hz), 2.27 (6H, s), 1.74 – 1.69 (2H, m),
(
=
1
3
1
1
6
.55 – 1.52 (2H, m), 1.48 – 1.42 (2H, m); C NMR (100 MHz, CDCl ) δ 169.1, 157.1, 141.8, 141.7,
3
40.2, 139.9, 137.7, 128.9, 128.6, 128.5, 127.8, 127.5, 127.4, 127.3, 127.1, 125.5, 125.1, 124.6, 123.3,
0.4, 59.5, 45.3, 43.7, 42.0, 31.3, 29.2, 27.0, 26.7; Purity: 97%; HRMS (FAB+): m/z estimated for
+
C H N OS [M+H] 577.2996, found: 577.3001.
3
6
41
4
N-benzyl-2-(3-(4'-fluoro-[1,1'-biphenyl]-4-yl)-2-([5-(pyrrolidin-1-yl)pentyl]thio)-3,4-
1
dihydroquinazolin-4-yl)acetamide (6i, 0.06 g, 50%): H NMR (400 MHz, CDCl ) δ 7.58 – 7.52 (4H,
3
m), 7.39 (2H, d, J = 7.6 Hz), 7.31 – 7.21 (5H, m), 7.17 – 7.11 (4H, m), 7.06 (2H, d, J = 3.6 Hz), 5.98
(1H, s), 5.23 (1H, dd, J = 8.0 Hz and 7.6 Hz), 4.40 (1H, dd, J = 14.4 Hz and 6.0 Hz), 4.30 (1H, dd, J
=
14.8 Hz and 5.2 Hz), 3.19 – 3.06 (2H, m), 2.82 – 2.74 (5H, m), 2.64 – 2.58 (3H, m), 1.87 (4H, s),
1
3
1
.74 – 1.61 (4H, m), 1.48 – 1.44 (2H, m); C NMR (100 MHz, CDCl ) δ 169.2, 163.8, 161.3, 157.1,
3
-
22 -

1
1
41.9, 141.6, 138.8, 137.9, 136.2, 128.7, 128.6, 128.5, 127.8, 127.6, 127.5, 127.3, 125.6, 125.2, 124.6,
23.2, 115.8, 115.6, 60.4, 56.2, 54.1, 43.5, 41.9, 31.2, 29.7, 29.1, 27.9, 26.7, 23.3; Purity: 93%; HRMS
+
(
FAB+):m/z estimated for C H FN OS [M+H] 621.3058, found: 621.3063.
3
8
42
4
N-(4-fluorobenzyl)-2-(3-(4'-fluoro-[1,1'-biphenyl]-4-yl)-2-([5-(pyrrolidin-1-yl)pentyl]thio)-3,4-
1
dihydroquinazolin-4-yl)- acetamide (6j, 0.03 g, 28%): H NMR (400 MHz, CDCl ) δ 7.46 – 7.41 (4H,
3
m), 7.26 (2H, d, J = 8.0 Hz), 7.19 – 7.17 (1H, m), 7.10 (1H, d, J = 8.0 Hz), 7.06 – 7.02 (2H, m), 6.99
–
6.90 (4H, m), 6.83 (2H, t, J = 7.2 Hz), 6.00 (1H, s), 5.10 (1H, t, J = 7.2 Hz), 4.13 (1H, dd, J = 14.4
Hz and 6.0 Hz), 4.15 (1H, dd, J = 14.8 Hz and 4.8 Hz), 3.14 – 3.07 (1H, m), 2.97 – 2.90 (1H, m), 2.65
(1H, dd, J = 13.6 Hz and 6.0 Hz), 2.47 (1H, dd, J = 13.6 Hz and 8.4 Hz), 2.38 (4H, s), 2.33 (2H, t, J =
1
3
7
.6 Hz), 1.66 (4H, s), 1.61 – 1.56 (2H, m), 1.45 – 1.41 (2H, m), 1.36 – 1.31 (2H, m); C NMR (100
MHz, CDCl ) δ 169.1, 163.8, 163.3, 161.4, 160.9, 157.1, 141.9, 141.7, 138.9, 136.3, 136.2, 133.5,
3
1
1
33.5, 129.5, 129.4, 128.7, 128.6, 128.5, 127.6, 127.3, 125.5, 125.0, 124.6, 123.3, 115.8, 115.6, 115.4,
15.2, 60.4, 56.4, 54.2, 42.9, 41.9, 31.3, 29.7, 29.2, 28.4, 26.9, 23.4; Purity: 96%; HRMS (FAB+):m/z
+
estimated for C H F N OS [M+H] 639.2964, found: 639.2969.
3
8
41
2
4
N-benzyl-2-(2-([5-(dimethylamino)pentyl]thio)-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)-3,4-
1
dihydroquinazolin-4-yl)acetamide (6k, 0.03 g, 25%) H NMR (400 MHz, CDCl ) δ 7.58 – 7.52 (4H,
3
m), 7.38 (2H, d, J = 8.4 Hz), 7.33 – 7.22 (5H, m), 7.17 – 7.11 (4H, m), 7.06 – 7.05 (2H, m), 5.81 (1H,
s), 5.22 (1H, t, J = 8.0 Hz), 4.39 (1H, dd, J = 14.8 Hz and 5.6 Hz), 4.31 (1H, dd, J = 14.8 Hz and 5.2
Hz), 3.25 – 3.18 (1H, m), 3.11 – 3.04 (1H, m), 2.78 (1H, dd, J = 13.2 Hz and 6.0 Hz), 2.58 (1H, dd,
J = 13.2 Hz and 8.4 Hz), 2.30 (2H, t, J = 7.2 Hz), 2.24 (6H, s), 1.73 – 1.69 (2H, m), 1.55 – 1.50 (2H,
1
3
m), 1. 47 – 1.41 (2H, m); C NMR (100 MHz, CDCl ) δ 169.0, 157.0, 141.8, 141.6, 138.9, 137.6,
3
1
5
36.3, 128.7, 128.6, 128.5, 127.8, 127.7, 127.5, 127.4, 125.5, 125.1, 124.6, 123.3, 115.8, 115.6, 60.4,
9.5, 45.2, 43.8, 42.0, 31.3, 29.7, 29.2, 27.0, 26.6; Purity: 98%; HRMS (FAB+):m/z estimated for
+
C H FN OS [M+H] 595.2901, found: 595.2907.
3
6
40
4
-
23 -

N-(4-fluorobenzyl)-2-(2-([5-(dimethylamino)pentyl]thio)-3-(4'-fluoro-[1,1'-biphenyl]-4-yl)-3,4-
1
dihydroquinazolin-4-yl)acetamide (6l, 0.04 g, 38%) H NMR (400 MHz, CDCl ) δ 7.48 – 7.42 (4H,
3
m), 7.28 (2H, d, J = 8.4 Hz), 7.2 – 7.12 (2H, m), 7.05 (2H, t, J = 8.8 Hz), 7.00 – 6.92 (4H, m), 6.85
(
2H, t, J = 8.8 Hz), 5.82 (1H, s), 5.11 (1H, t, J = 7.6 Hz), 4.26 (1H, dd, J = 15.2 Hz and 5.6 Hz), 4.17
1H, dd, J = 14.8 Hz and 5.2 Hz), 3.15 – 3.08 (1H, m), 3.00 – 2.94 (1H, m), 2.66 (1H, dd, J = 13.2 Hz
(
and 6.0 Hz ), 2.48 (1H, dd, J = 13.6 Hz and 8.0 Hz), 2.19 (2H, t, J = 7.2 Hz), 2.13 (6H, s), 1.63 – 1.58
1
3
(
2H, m), 1.42 – 1.38 (2H, m), 1.36 – 1.31 (2H, m); C NMR (100 MHz, CDCl ) δ 169.1, 163.8, 163.3,
3
1
1
2
6
61.4, 160.9, 157.1, 141.8, 141.7, 138.9, 136.3, 136.2, 133.5, 133.4, 129.5, 129.5, 128.7, 128.7, 128.6,
27.7, 127.3, 125.4, 125.0, 124.6, 123.4, 115.8, 115.6, 115.5, 115.3, 60.4, 59.5, 45.3, 43.0, 41.9, 31.3,
+
9.7, 29.2, 27.1, 26.7; Purity: 98%; HRMS (FAB+):m/z estimated for C H F N OS [M+H]
3
6
39
2
4
13.2807, found: 613.2813.
4
4
.2. Biological evalutation
.2.1. Cell Culture for MTT assay
Human non-small cell lung cancer A549 cells and Human colon cancer HCT-116 cells were
obtained from the Korean cell line bank (Seoul, Korea). Cells were cultured in RPMI 1640
supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS), penicillin (100U/Ml) and
o
streptomycin sulfate (100 µg/mL). Cells were cultured at 37 C in an atmosphere of 5% CO2.
4
.2.2. MTT assay
The cells (5x105/mL) were seeded in each well containing 100 µL of the RPMI medium
supplemented with 10% FBS in a 96-well plate. Various concentrations of synthetic compound were
added and incubated for 24 h. MTT (5 mg/mL stock solution) was added and the plates were incubated
for an additional 4 h. The medium was discarded and the formazan blue, which was formed was
measured at 540 nm by an automatic microplate reader (Molecular Devices Corp., Sunnyvale, CA,
USA).
-
24 -

4
.2.3. tsA-201 cell culture and cDNA transfection
Human embryonic kidney tsA-201 cells were cultured and transfected using the calcium
4
3
phosphate method as describe previously. Cells were plated on glass coverslips and transfected with
either 3μg of human Ca 3.1, Ca 3.2 or Ca 3.3 cDNA. pEGFP (0.5μg) was included into the transfection
v
v
v
mix as a transfection marker.
4
.2.4. Electrophysiology
Electrophysiological recordings were performed according to the whole cell configuration of
the patch-clamp technique 72 h after transfection at room temperature. The external
recording solution contained (in mM): 20 BaCl , 1 MgCl , 40 TEACl, 65 CsCl, 10 HEPES,
2
2
and 10 glucose, pH 7.4. Borosilicate glass pipettes were filled with internal solution
containing (in mM): 140 CsCl, 2.5 CaCl , 1 MgCl , 5 EGTA, 10 HEPES, 2 Na-Adenosine-
2
2
Tri-Phosphate (ATP), and 0.3 Mg-GTP, pH 7.3. Drugs were prepared daily in external
solution and were applied locally to cells. The effects of the compounds (10 μM) were
assessed by normalizing the current amplitude in the presence of the compound to that
observed before adding the drug. In dependent experiment was performed using the vehicle
DMSO (0.1%) and no inhibition was observed. Currents were evoked by depolarization from
a holding potential of −110 mV to a test potential of −20 mV. The interpulse interval was 20
s, and the test pulse was 100 ms long.
Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
-
25 -

Acknowledgments
This work was supported by the National Research Foundation (NRF) grant funded by the
Korea government MISIT (No. 2016R1D1A1B03936197) and MIE (No. 2019R1F1A1059332)/ The
Canadian Institutes for Health Research (CIHR) and Alberta Innovates. GWZ is a Canada Research
Chair. MAG holds a CIHR Fellowship.
References and notes
1
2
3
4
5
6
7
8
9
1
1
.
.
.
.
.
.
.
.
.
Zamponi, G. W.; Striessnig, J.; Koschak, A.; Dolphin, A. C. Pharmacol. Rev. 2015, 67, 821.
Berridge, M. J.; Lipp; P.; Bootman, M. D. Nat. Rev. Mol. Cell Biol. 2000, 1, 11.
Berridge, M. J.; Bootman, M. D.; Roderick, H. L. Nat. Rev. Mol. Cell Bio. 2003, 4, 517.
Clapham, D. E. Cell 2007, 131, 1047.
Parkash, J.; Asotra, K. Life Sci. 2010, 87, 587.
Schreiber, R. J. Membr. Biol. 2005, 205, 129.
Weiss, N.; Zamponi, G. W. Int. J. Biochem. Cell Biol. 2019, 108, 34.
Zamponi, G. W. Nat. Rev. Drug Discov. 2016, 15, 19.
Weiss, N; Zamponi, G. W. J. Med. Genet. 2019, 106163.
0. Snutch, T. P.; Zamponi, G. W.; Br. J. Pharmacol. 2017, 175, 2375.
1. Teleb, M.; Rizk, O. H.; Zhang, F.; Fronczek, Fang-Xiong. R.; Zamponi, G. W.; Fahmy, H.
Bioorg. Chem. 2019, 88, 102915.
1
1
1
2. Teleb, M.; Rizk, O. H.; Zhang, F.; Fronczek, Fang-Xiong. R.; Zamponi, G. W.; Fahmy, H.
Bioorg. Chem. 2019, 83, 354.
3. Sairaman, A.; Cardoso, F. C.; Bispat, A.; Lewis, R. J.; Duggan, P. J.; Tuck, K. L. Bioorg. Med.
Chem. 2018, 26, 3046.
4. Wang, D.; Neupane, P.; Ragnarsson, L.; Capon, R. J.; Lewis, R. J. Mar. drugs 2018, 16, 475.
-
26 -

1
1
1
5. Gunaratna, M. J.; Hua, D. H.; Zou, B.; Pascual, C.; Cao, W.; Zhang, M.; Weerasekara, S.;
Nguyen, T. D. T.; Xiao, K.; Xie, X. S. Alkivoc 2019, part iii, 22.
6. Latour, I.; Louw, D. F.; Beedle, A. M.; Hamid, J.; Sutherland, G. R.; Zamponi, G. W. Glia 2004,
4
8, 112.
7. Monteith, G. R.; McAndrew, D.; Faddy, H. M.; Roerts-Thomson. S. J. Nat. Rev. Cancer. 2007,
, 519.
7
1
1
8. Monteith, G. R.; Davis, F. M.; Roberts-Thomson, S. J. J. Biol. Chem. 2012, 287, 31666.
9. Suo, A.; Childers, A.; D'Silva; A.; Petersen, L. F.; Otsuka, S.; Dean, M.; Li, H.; Enwere, E. K.;
Pohorelic, B.; Klimowicz, A.; Souza, I. A.; Hamid, J.; Zamponi, G. W.; Bebb, D. Oncotarget.
2
018, 9, 8573.
2
2
2
2
2
2
2
0. Monteith, G. R.; Prevarskaya, N.; Roberts-Thomson, S. J. Nat. Rev. Cancer 2017, 17, 367.
1. Büsselberg, D.; Florea, A.-M. Cancers 2017, 9, pii: E48.
2. Lastraioli, E.; Iorio, J.; Arcangeli, A. Biochim. Biophys. Acta. 2015, 1848, 2685.
3. Akl, H.; Bultynck, G. Biochim. Biophys. Acta. 2013, 1835, 180.
4. Stewart, T. A.; Yapa, K. T.; Monteith, G. R. Biochim. Biophys. Acta. 2015, 1848, 2502.
5. Chen, Y. F.; Chen, Y. T.; Chiu, W. T.; Shen, M. R. J. Biomed. Sci. 2013, 20, 23.
6. Hirooka, K.; Bertolesi, G. E.; Kelly, M. E.; Denovan-Wright, E. M.; Sun, X.; Hamid, J.; Zamponi,
G. W.; Juhasz, A. E.; Haynes, L. W.; Barnes, S. J. Neurophysiol. 2002, 88, 196.
2
2
2
7. Lu, F.; Chen, H.; Zhou, C.; Liu, S.; Guo, M.; Chen, P.; Zhuang, H.; Xie, D.; Wu, S. Cell Calcium
2
008, 43, 49.
8. Dziegielewska, B.; Brautigan, D. L.; Larner, J. M.; Dziegielewski, J. Mol Cancer Res. 2014, 12,
48.
3
9. Das, A.; Pushparaj, C.; Herreros, J.; Nager, M.; Vilella, R.; Portero, M.; Pamplona, R.; Matias-
Guiu, X.; Martí, R. M.; Cantí, C. Pigment Cell Melanoma Res. 2013, 26, 874.
3
3
3
0. White, E.; Mehnert, J. M.; Chan, C. S. Clin. Cancer Res. 2015, 21, 5037.
1. Dziegielewska, B.; Gray, L. S.; Dziegielewski, J. Pflugers Arch.-Eur. J. Physiol. 2014, 466, 801.
2. Bertolesi, G. E.; Shi, C.; Elbaum, L.; Jollimore, C.; Rozenberg, G.; Barnes, S.; Kelly, M. E. Mol.
-
27 -

Pharmacol. 2002,62, 210.
3
3
3
3
3
3
3
4
3. Rim, H. K.; Cho, S.; Shin, D. H.; Chung, K. S.; Cho, Y. W.; Choi, J. H.; Lee, J. Y.; Lee, K. T.;
Molecules 2014, 19, 9864.
4. Byun, J. S.; Sohn, J. M.; Leem, D. G.; Park, B.; Nam, J. H.; Shin, D. H.; Shin, J. S.; Kim, H. J.;
Lee, K. T.; Lee, J. Y. Bioorg. Med. Chem. Lett. 2016, 26, 1073.
5. Choi, D. L.; Jang, S. J.; Cho, S.; Choi, H. E.; Rim, H. K.; Lee, K. T.; Lee, J. Y. Bioorg. Med.
Chem. Lett. 2014, 24, 1565.
6. Jang, S. J.; Choi, H. W.; Choi, D. L.; Cho, S.; Rim, H. K.; Choi, H. E.; Kim, K. S.; Huang, M.;
Rhim, H.; Lee, K. T.; Lee, J. Y. Bioorg. Med. Chem. Lett. 2013, 23, 6656.
7. Rim, H. K.; Lee, H. W.; Choi, I. S.; Park, J. Y.; Choi, H. W.; Choi, J. H.; Cho, Y. W.; Lee, J. Y.;
Lee, K. T. Bioorg. Med. Chem. Lett. 2012, 22, 7123.
8. Kang, H. B.; Rim, H. K.; Park, J. Y.; Choi, H. W.; Choi, D. L.; Seo, J. H.; Chung, K. S.; Huh,
G.; Kim, J.; Choo, D. J.; Lee, K. T.; Lee, J. Y. Bioorg. Med. Chem. Lett. 2012, 22, 1198.
9. Seo, H. N.; Choi, J. Y.; Choe, Y. J.; Kim, Y.; Rhim, H.; Lee, S. H.; Kim, J.; Joo, D. J.; Lee, J.
Y. Bioorg. Med. Chem. Lett. 2007, 17, 5740.
0. Pillaiyar, T.; Ki-Cheul, L.; Vinay Kumar, S.; Niti, S.; Youngsoo, K.; Sang-Hung, J. Chem.
Pharm. Bull. 2011, 59, 1285.
4
4
1. Regina, G. L.; Piscitelli, F.; Silvestri, R. J. Heterocycl. Chem. 2009, 46, 10.
2. Park, S. J.; Park, S. J.; Lee, M. J.; Rhim, H.; Kim, Y.; Lee, J. H.; Chung, B. Y.; Lee, J. Y.
Bioorg. Med. Chem. 2006, 10, 3502.
4
4
3. Paetzold, E.; Oehme, G. J. Mol. Catal. A-Chem. 2000, 152, 69.
4. Henrik, M.; Jon, S. H.; Michael, P.; Jørn, B. C.; Ulrik, B. Tetrahedron Lett. 2008, 49, 3117.
4
5. Garcia-Caballero, A.; Gandini, M. A.; Huang, S.; Chen, L.; Souza, I. A.; Dang, Y. L.; Stutts, M.
J.; Zamponi, G. W. Mol. Brain 2019, 12, 98.
4
4
6. Sallán, M. C.; Visa, A.; Shaikh, S.; Nàger, M.; Herreros, J.; Cantí, C. Cancer Res. 2018, 78, 603.
7. Iftinca, M. C.; Zamponi, G. W. Trends Pharmacol. Sci. 2009, 1, 32.
-
28 -

4
4
8. Garcia-Caballero, A.; Gadotti, V. M.; Stemkowski, P.; Weiss, N.; Souza, I.A.; Hodgkinson, V.;
Bladen, C.; Chen, L.; Hamid, J.; Pizzocaaro, A.; Deage, M.; Francois, A.; Bourinet, E. Zamponi,
G. W. Neuron 2014, 5, 1144.
9. Rubinstein, L. V.; Shoemaker, R. H.; Paull, K. D.; Simon, R. M.; Tosini, S.; Skehan, P.; Scudiero,
D. A.; Monks, A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
-
29 -