Synthesis and biological evaluation of fluoro-substituted 3,4-dihydroquinazoline derivatives for cytotoxic and analgesic effects

Article abstract of DOI:10.1016/j.bmc.2017.07.010

As a bioisosteric strategy to overcome the poor metabolic stability of lead compound KYS05090S, a series of new fluoro-substituted 3,4-dihydroquinazoline derivatives was prepared and evaluated for T-type calcium channel (Ca_v3.2) block, cytotoxi

Full text of DOI:10.1016/j.bmc.2017.07.010

Accepted Manuscript
Synthesis and biological evaluation of fluoro-substituted 3,4-dihydroquinazo-
line derivatives for cytotoxic and analgesic effects
Jin Han Kim, Hui Rak Jeong, Da Woon Jung, Hong Bin Yoon, Sun Young Kim,
Hyoung Ja Kim, Kyung-Tae Lee, Vinicius M. Gadotti, Junting Huang, Fang-
Xiong Zhang, Gerald W. Zamponi, Jae Yeol Lee
PII:
S0968-0896(17)30436-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.07.010
BMC 13846
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
2 March 2017
4 July 2017
6 July 2017
Please cite this article as: Kim, J.H., Jeong, H.R., Jung, D.W., Yoon, H.B., Kim, S.Y., Kim, H.J., Lee, K-T., Gadotti,
V.M., Huang, J., Zhang, F-X., Zamponi, G.W., Lee, J.Y., Synthesis and biological evaluation of fluoro-substituted
3
,4-dihydroquinazoline derivatives for cytotoxic and analgesic effects, Bioorganic & Medicinal Chemistry (2017),
doi: http://dx.doi.org/10.1016/j.bmc.2017.07.010
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Synthesis and biological evaluation of fluoro-substituted 3,4-
dihydroquinazoline derivatives for cytotoxic and analgesic effects
a, 1
Jin Han Kim , Hui Rak Jeong , Da Woon Jung , Hong Bin Yoon , Sun Young Kim , Hyoung Ja
a, 1
a
a
a
b
c,*
d
d
d
Kim , Kyung-Tae Lee , Vinicius M. Gadotti , Junting Huang , Fang-Xiong Zhang , Gerald W.
d,*
Zamponi , Jae Yeol Lee
a, e,*
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 Nanopharmaceutical Science, Kyung Hee University, Seoul 02447,
Republic of Korea
d
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
e
KHU-KIST Department of Converging Science and Technology, Kyung Hee University, Seoul
0
2447, Republic of Korea
1
The contributions of the authors are equal.
Corresponding authors:
Tel. +82 2 9610860; Fax +82 2 9620860; e-mail: ktlee@khu.ac.kr
Tel. +1 403 220 8687; Fax +1 403 210 8106; e-mail: zamponi@ucalgary.ca
Tel. +82-2-961-0726; Fax +82-2-961-0443; e-mail; ljy@khu.ac.kr
Keywords: T-type calcium channel; 3,4-dihydroquinazoline; bioisostere; cytotoxic activity;
inflammatory pain; liver microsomal stability
Abstract: As a bioisosteric strategy to overcome the poor metabolic stability of lead compound
KYS05090S, a series of new fluoro-substituted 3,4-dihydroquinazoline derivatives was prepared
and evaluated for T-type calcium channel (Ca
stability. Among them, compound 8h (KCP10068F) containing 4-fluorobenzyl amide potently
blocked Ca 3.2 currents (>90 % inhibition) at 10 µM concentration and exhibited cytotoxic effect
IC50 = 5.9 µM) in A549 non-small cell lung cancer cells that was comparable to KYS05090S.
v
3.2) block, cytotoxic effects and liver microsomal
v
(
Furthermore, 8h showed approximately a 2-fold increase in liver metabolic stability in rat and
human species compared to KYS05090S. Based on these overall results, 8h (KCP10068F) may
therefore represent a good backup compound for KYS05090S for further biological investigations
as novel cytotoxic agent. In addition, compound 8g (KCP10067F) was found to partially protect
v
from inflammatory pain via a blockade of Ca 3.2 channels.
page 1 / 21

1. Introduction
2
Calcium (Ca ) as a second messenger plays a vital role in cellular physiology and
+
1
biochemistry, such as proliferation, differentiation, growth, cell death and apoptosis. Thus,
-4
alterations in calcium signaling can cause defects in cell growth and are associated with certain types
5
of cancer. A number of research groups have suggested a potential role for voltage-activated Ca
-7
2+
8
-14
channels, in particular T-type (Ca
v
3), in the regulation of tumor growth and progression.
This T-
2
+
15
v v v v
type Ca channel family (Ca 3) contains three members: Ca 3.1, Ca 3.2 and Ca 3.3. A growing
2
number of reports show that T-type Ca channels are abnormally overexpressed in many types of
+
1
6-17
2+
Therefore, T-type Ca channel blockers can be
human cancers compared to normal cells.
1
8-19
regarded as new tools in cancer therapies.
Figure 1. The proposed metabolic pathways and identified metabolites of KYS05090S in human,
monkey, dog, rat and mouse liver microsomes.
We have already reported that 3,4-dihydroquinazoline derivatives exhibited both strong T-
page 2 / 21

2
0-25
6
type calcium blocking and anti-cancer effects.
An optimization study resulted in the lead
2
compound KYS05090S for non-clinical studies. We explored the in vitro metabolic stability of
2
7-
KYS05090S in mouse, rat, dog, and human liver microsomes using a single point metabolic assay.
2
8
Briefly, KYS05090S was incubated at a concentration of 1.0 µM with 1.0 mg/mL protein at 37 °C
for 30 min. It showed lower metabolic stability with less than 30% remaining in liver microsomes of
three species except dog (Table 1). In vivo pharmacokinetic (PK) studies of KYS05090S led to very
low oral bioavailability in dog (F, 1.27%) and monkey (F, 2.01%) animals together with the
formation of primary metabolites as shown in Fig. 1 (unpublished results).
a
Table 1. In vitro liver microsomal stability of KYS05090
%
remaining after 30 min incubation
Compound
Human
16.6
Dog
Rat
Mouse
27.6
KYS05090S
> 100
24.2
a
The metabolic stability was expressed as percent remaining compound
after 30 minutes incubation in liver microsomal enzyme.
To overcome this issue, herein, we decided to insert a fluorine atom as a bioisostere of
hydrogen atom into various regions of 3,4-dihydroquinazoline scaffold hoping that the small fluorine
atom will not impair the binding of new synthetic 3,4-dihydroquinazoline derivatives to the target T-
2
type Ca channel. The rapid oxidative metabolism by the liver enzymes, in particular the P450
+
cytochromes, is often found to limit bioavailability. A frequently employed strategy to circumvent
2
9
this problem is to block the reactive site by the introduction of a fluorine atom. In fact, this
bioisosteric strategy is one of the most commonly used methods for developing new compounds with
improved pharmacodynamics and/or pharmacokinetic properties and thus has led to many successful
2
9-31
drugs.
2
. Results and discussions
.1. Chemistry
2
A series of fluoro-substituted 3,4-dihydroquinazoline derivatives (8) except 8f in Table 2
page 3 / 21

3
was prepared using a Heck reaction (for 8dand 8j) and the procedure described previously by our
2
2
0, 33
group.
New compound 8f (KCP10066F) was easily synthesized according to the procedure as
Cl into 2-
shown in Scheme 1: Methyl 2-nitrocinnamate 1 was reduced with Zn (powder)/NH
4
aminocinnamate 2, which was coupled with isocyanate (in situ prepared from 4-fluorobiphenyl-4-
carboxylic acid 3 via Curtius rearrangement) to provide a urea 4. The dehydration of 4 with
3 2 3
Ph P·Br and Et N provided a carbodiimido 5, which was subsequently coupled with piperazine
compound 6 afforded 3,4-dihydroquinazoline ester 7. The treatment of 7 with 5-fluorobenzylamine
and 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD) as a catalyst afforded the final compound 8f
(
KCP10066F) under solvent-free condition.
Scheme 1. Reagents and conditions: (a) Zn, NH
toluene, rt to 100 ℃, 9 h, 54%; (c) PPh3·Br , Et
9%; (e) 4-fluorobenzylamine, TBD, 40 ℃, 12 h, 87%.
page 4 / 21
4
Cl, MeOH, reflux, 12 h, 98%; (b) DPPA, Et
3
N,
2
3
N, CH Cl , 0℃, 12 h, 38%; (d) MeOH, rt, 4 h,
2
2
6

2.2. Biological evaluation
All compounds were tested for their abilities to block transiently expressed human Ca
v
3.2
3
3.2) currents by using whole-cell patch clamp recordings in HEK cells. Most of compounds
4
(
hCa
v
except 8a and 8f potently blocked Ca
v
3.2 currents (>90 % inhibition) at 10 µM concentration (Fig.
4
A). Compound 8a (KCP10048F) with a relatively short 4-isopropylphenyl ring at R position was
2
almost inactive, which was consistent with the reported result of SAR studies on the 3.4-
3
dihydroquinazoline scaffold. In contrast, compound 8d (KCP10060F) and 8g (KCP10067F)
5
completely blocked Ca 3.2 currents and their IC50 values determined from the fit to the dose-
v
response curve were 5.6 and 5.1 µM, respectively, when cells were held at a hyperpolarized
potential of -110 mV [Fig. 2C and 2D]. While the dose-response curve for compound 8d could be
fitted with a Hill coefficient of 0.8, that for compound 8g required a Hill coefficient of 0.14. This
suggests that block by 8g required the interaction of more than one drug molecule with the channel
and negative cooperativity. With respect to their channel selectivity, the two compounds showed
poor Ca
v
3.2 (T‐type)/Ca
v
2.2 (N‐type) and (T‐type)/Ca 1.2 (L‐type) calcium channel selectivity (Fig.
v
2
B), suggesting that they are broad spectrum inhibitors of calcium channel activity.
page 5 / 21

a
Table 2. The biological activities of new synthetic 3,4-dihydroquinazoline derivatives (8)
b,c
d
T-type (Ca
v
3.2)
A549
Code
1
R
2
R
3
R
4
R
5
R
No
IC50
IC50
(KCP)
%inhibition
(
µM)
(µM)
H
N
N
8
8
a
10048F
H
H
N
F
<10
5.7
O
N
N
N
N
N
N
N
H
b
10057F
10059F
10060F
10061F
10066F
10067F
10068F
10069F
H
H
F
H
H
F
H
F
H
F
F
F
F
F
F
H
>90
>90
>95
>90
<80
>95
>90
>90
≥90
≥90
8.7
2.2
5.1
7.1
8.7
7.6
5.9
5.9
6.0
6.0
8
c
F
N
8
8
d
e
N
N
5.6
5.1
H
H
H
H
H
H
H
H
H
H
H
H
F
8
f
g
h
F
N
N
N
N
N
(CH
(CH
(CH
2
)
)
)
2
N
8
8
2
2
2
2
N
N
N
N
N
N
N
N
8
i
2 2
(CH )
8
j
10070F
2 2
(CH )
e
N
KYS05090S
H
3.6
a
b
c
Values represent the mean of three independent experiments; Ca 3.2 (α1H); Whole-cell patch clamp recording in
v
d
HEK cells; Non-small cell lung cancer cells; Dihydrochloride salt was used as a parent compound.
e
page 6 / 21

Figure 2. (A) Percentage of whole cell current inhibition of Ca
application of the compound series (n = 3 per compound); (B) Percentage of whole cell current
inhibition of Ca 3.2 (T‐type), Ca 2.2 (N‐type), and Ca 1.2 (L‐type) in response to 10 µM
v
3.2 (T‐type) in response to 10 µM
v
v
v
application of compound 8d (KCP10060F – three bars at the left side) and 8g (KCP10067F – three
bars at the right side) (n = 3 per channel); (C) Dose‐response relation for 8d (KCP10060F); (D)
Dose‐response relation for 8g (KCP10067F) block of Ca
v
3.2 (T-type). The IC50 values from the
fitted curve were 5.61±1.81 and 5.11±1.32 µM (n = 3 per dose), respectively. The hill coefficients
for compounds 8d and 8g were 0.8 and 0.14, respectively.
v
Current–voltage relationship experiments for both compounds were carried out for Ca 3.2
channels transiently transfected into HEK-293 cells by stepping to a series of test potentials ranging
from −60 mV to +60 mV in 10 mV steps (lasting for 100 ms). Fig. 3A and 3B display averaged IV-
traces from cells before and after the application of 5 µM 8d (KCP10060F) and 8g (KCP10067F),
respectively. Each compound caused a prominent reduction of peak current density compared to the
control across a range of test potentials. However, neither compound 8d (KCP10060F) nor 8g
(
KCP10067F) caused a shift in the half-activation potential.
page 7 / 21

Figure 3. (A) and (B): Effects of 8d (KCP10060F) and 8g (KCP10067F) on the current-voltage
relation of Ca 3.2 (T-type), respectively. The half activation potential (Va1/2) from the fitted curves
v
was not affected by the blockers (‐29.82±1.12 mV and ‐29.94±1.06 mV before after the application
of 8d (KCP10060F, 5 µM, n = 3), (p = 0.90, paired t‐test); ‐23.49±0.75 mV and ‐24.71±1.61 mV
before and after the application of 8g (KCP10067F, 5 µM, n = 3), (p = 0.27, paired t‐test); (C) and
(
D): Effects of 8d (KCP10060F) and 8g (KCP10067F) on the steady-state inactivation curve of
Ca 3.2 (T-type), respectively. The half inactivation potential from the fitted curves was shifted from
48.12±0.62 mV to -57.43±2.76 mV after the application of 8d (KCP10060F, 5 µM, n = 6), (p =
v
‐
0.03, paired t‐test), whereas there was no effect of 8g (‐46.36±0.85 mV and -49.29±2.18 mV before
and after the application of 8g (KCP10067F, 5 µM, n = 3), (p = 0.38, paired t‐test), respectively.
A two-step voltage-clamp protocol was applied for determining the effects of the
v
compounds on steady-state inactivation of Ca 3.2. This involved an inactivating pre-pulse period
that varied from −110 mV to -20 mV. The test current amplitude at each pulse potential was
normalized to the maximal amplitude of this current (I/Imax). Data were fitted to the Boltzmann
equation. As shown in Fig. 3C and 3D, 5 µM of 8d (KCP10060F) but not 8g (KCP10067F) shifted
the voltage-dependence of the inactivation toward more negative potentials (from ‐48.12±0.62 mV
to -57.43±2.76 mV for 8d). This suggests that 8d interacts with the inactivated state of the channel,
suggesting that this compound will show an enhanced blocking affect at typical neuronal resting
page 8 / 21

potentials and may display use-dependent properties.
All derivatives were evaluated for anti-proliferative activity against A549 (non-small cell
3
6
lung cancer cells) using MTT assay. The overall biological results for the inhibition of calcium
influx and the cytotoxicity on human cancer cells were summarized in Table 2 together with those
of KYS05090S as a positive control. The compounds exhibited different degrees of cytotoxic
activity (IC50 = <10 µM) in A549 cell lines, with some being more potent than KYS05090S.
Interestingly, compound 8a (KYS10048F) was as active as KYS05090S irrespective of its lower T-
type channel blocking effect. Compound 8c (KCP10059F) containing a 4-fluorobiphenylyl ring at
4
R and fluorine atom at R was most active (IC50 = 2.2 µM) and 3-fold more active than
5
KYS05090S (IC50 = 6.0 µM). Compound 8d (KCP10060F) and 8g (KCP10067F) had an IC50 of
5.1 and 7.6 µM, respectively, against A549 cell lines.
We also explored the in vitro metabolic stability of selected compounds in rat and human
3
7
liver microsomes using a single point metabolic assay. Among them, 5 compounds (8c-8e, 8h and
8
j) and 3 compounds (8e, 8h and 8j) exhibited improved liver metabolic stability in rat (48-76%
remaining after 30 min incubation) and human (42-54% remaining after 30 min incubation),
5
respectively, compared to KYS05090S. It can be assumed that a fluorine atom (that is, R
substituent) at the para-position of the benzyl amide and a 4-cyclohexylphenyl ring instead of the
3
biphenyl ring as the R substituent are the main factors contributing to the metabolic stability. A
possible explanation is these two structural changes can block the para-hydroxylation which can
occur to both the phenyl ring of benzyl amide group and the phenyl ring of the biphenyl group.
Among the tested compounds, therefore, 8h (KCP10068F) containing both 4-fluorobenzyl amide
and 4-cyclohexylphenyl ring showed the best metabolic stability, which is approximately a 2-fold
increase in liver metabolic stability in two species compared to KYS05090S as shown in Table 3.
page 9 / 21

Figure 4. (A) and (B): Dose-dependent effect of intrathecally delivered 8g (KCP10067F) on the first
and second phases of formalin-induced pain in mice (n = 7-9), respectively; (C) and (D): Effect of 8g
delivered intrathecally to Ca
v
3.2 null mice in the first and second phases of the formalin test (n = 6-
7
), respectively. Each bar represents the mean±SEM and is representative of 2 independent
experiments. Asterisks denote the significance relative to the control group (A, B) or yet to the wild
type group treated with vehicle (C, D) (*p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA
followed by Tukey’s test).
v
Ca 3.2 calcium channels are also a potential target for pain therapeutics. The ability of 8g
(
KCP10067F) to inhibit inflammatory pain responses was thus also examined, using approaches
3
8-40
that have been described by us in detail previously.
The formalin test involves the injection of
formalin into the hindpaw, which triggers two distinct phases of nocifensive behavior (i.e., biting
and licking of the paw), whose durations can be determined in the absence and the presence of the
compounds. Increasing doses of compound 8g mediated analgesic effects in both nocifensive
page 10 / 21

phases in the formalin test (Fig. 4A and 4B), as evident from a shortening of the nocifensive
response duration. This effect was ablated in mice lacking Ca 3.2 channels (Fig. 4C and 4D),
indicating that the physiological effects of compound 8g were mediated by action on Ca 3.2, rather
than on another target such as Ca 2.2 or Ca 1.2. Compound 8g (KCP10067F) was also partially
v
v
v
v
effective in reversing mechanical hypersensitivity in mice subjected to intraplantar injection of
complete Freund’s Adjuvant (CFA) (Fig. 5), where a small 8g-induced increase in mechanical paw
withdrawal was observed. Altogether, these data indicate that compound 8g partially protects from
inflammatory hypersensitivity.
a
Table 3. In vitro liver microsomal stability of selected compounds
%
remaining after 30 min incubation
Code
Entry
(KCP)
Rat
Human
29.2
20.1
42.1
20.3
54.0
44.2
27.3
9.0
8
c
10059F
10060F
10061F
10067F
10068F
10070F
64.5
47.9
65.7
22.3
70.1
75.8
37.8
8
d
8
e
8
8
g
h
8
j
KYS05090S
b
Verapamil
a
The metabolic stability was expressed as percent remaining
compound after 30 minutes incubation in liver microsomal enzyme;
Reference compound.
b
page 11 / 21

Figure 5. Blind analysis of the time course of mechanical hyperalgesia of CFA-injected mice
treated with either vehicle or 8g (KCP10067F). Each circle represents the mean ± SEM (n = 8-11),
and is representative of 2 independent batches of mice. (*p < 0.05, ***p < 0.001, two-way ANOVA
followed by Tukey’s test). Animals injected with CFA significantly differed from the sham treated
group (p < 0.001).
3. Conclusions
In this study, the introduction of fluorine atom into lead compound KYS05090S via a
bioisosteric strategy led to a series of fluoro-substituted 3,4-dihydroquinazoline derivatives, which
2
were evaluated for T-type Ca channel blocking ability and in vitro cytotoxic activity against A-
+
2
+
49 cells. Most of the novel 3,4-dihydroquinazoline derivatives displayed good T-type Ca channel
5
blocking effects (≥90% inhibition at 10 µM), along with potent inhibition of L-type and N-type
channels, and cytotoxic activity (IC50 = <9 µM), especially compound 8h (KCP10068F) which
exhibited particularly good cytotoxic activity (IC50 = 5.9 µM) comparable to the reference
compound KYS05090S with improved human and rat liver microsomal stability. Thus, compound
8
h (KCP10068F) may represent a good backup compound for KYS05090S for further biological
investigations as cytotoxic agents. In addition, compound 8g (KCP10067F) was found to partially
protect from inflammatory pain via the blockade of Ca
v
3.2 channel.
page 12 / 21

4. Experimental section
4
.1. Chemistry
.1.1. General
4
All solvents and commercially available reagents were used without additional purification.
Reactions were monitored by analytical thin layer chromatography (TLC, Merck, silica gel 60 F254
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
®
silica gel (Merck, 230–400 mesh). Melting points were recorded on electrothermal MEL-TEMP 3.0
1
13
capillary melting point apparatus and are uncorrected. NMR spectra ( H and C) were recorded on
Bruker Avance 400 MHz spectrometers. High-resolution mass spectra (ESI, positive/negative ion
mode) were obtained using SYNAPT G2-Si mass spectrometry, Waters.
4.1.2. Synthesis of compounds except 8f (KCP10066f) listed in Table 2 were previously reported by
2
0, 33
our group.
4.1.3. Preparation of compound 2: To a solution of methyl trans-2-nitrocinnamate (1, 3.76 g, 18.14
mmol) in MeOH (100 mL) was added activated Zn powder (8.04 g, 145.16 mmol) and NH Cl (2.14
4
g, 39.32 mmol) at 0℃ and the solution was heated at reflux for 12 h. The resulting mixture was
filtered through celite 545 and evaporated in vacuo to give the crude product. The concentrated
product was poured into EtOAc (100 mL) and stayed under sonicator for 10 min. EtOAc-insoluble
matter was filtered off and the filtrate was washed with sat. NaHCO
3
, dried (MgSO
4
), evaporated in
1
vacuo to give the desired product 2 (3.16 g, 98%) as an orange color solid: mp 61.1-62.2 ℃; H
NMR (400 MHz, CDCl
3
) δ 7.83 (1H, d, J = 15.6 Hz), 7.38 (1H, dd, J = 8 and 1.6 Hz), 7.19 (1H, t, J
=
8 Hz), 6.77 (1H, t, J = 7.6 Hz), 6.72 (1H, dd, J = 8 and 0.8 Hz), 6.35 (1H, d, J = 16.0 Hz), 3.97 (2H,
1
3
br s), 3.80 (3H, s); C NMR (100 MHz, CDCl ) δ 167.7, 145.5, 140.3, 131.3, 128.1, 119.9, 119.0,
3
117.7, 116.8, 51.7; HRMS (ESI+): calcd for C10H12NO (M+H) 178.0868, found: 178.0866.
2
4
.1.4. Preparation of compound 4: To a solution of 4-(4-fluorophenyl)benzoic acid (3, 0.65 g, 3.01
mmol) in distilled toluene (20 mL) was added 97% diphenylphosphonic azide (DPPA) (0.78 mL,
.61 mmol) and Et N (0.89 mL, 6.01 mmol) at rt for Curtius rearrangement. The reaction mixture
3
3
was stirred at rt for 3 h, further stirred at 100 ℃ for 6 h, followed by an addition of a solution of
methyl 2-aminocinnamate (2, 0.53 g, 3.01 mmol) in distilled toluene (20 mL) at rt. The reaction
mixture was stirred at rt overnight and concentrated under reduced pressure to give a solid, which
was washed with MeOH (50 mL) to give the desired product 4 (0.63 g, 54%) as a white solid: mp
1
2
18.3-222.7 ℃; H NMR (400 MHz, DMSO) δ 9.09 (1H, s), 8.57 (1H, s), 7.90 (1H, d, J = 15.6 Hz),
page 13 / 21

7.78 (2H, dd, J = 8.0 and 3.6 Hz), 7.68 (2H, dd, J = 8.8 and 6.0 Hz), 7.58 (4H, dd, J = 15.6 and 8.8
Hz), 7.43-7.39 (1H, m), 7.27 (2H, t, J = 8.8 Hz), 7.15 (1H, t, J = 7.6 Hz), 6.61 (1H, d, J = 15.6 Hz),
1
3
3
.75 (3H, s); C NMR (100 MHz, DMSO) δ 166.7, 162.7, 160.3, 152.7, 139.8, 139.1, 137.8, 136.3,
132.6, 130.7, 128.0, 127.9, 127.1, 127.0, 126.1, 123.9, 123.7, 118.8, 118.5, 115.7, 115.5, 51.5;
+
[M+Na] 413.1277, found 413.1272.
HRMS (ESI+): m/z calcd. for C23
H19FN
2
NaO
3
4
.1.5. Preparation of compound 5: A solution of urea (4, 0.63 g,1.61 mmol) in distilled CH
2
Cl
N (0.67 mL, 4.84 mmol) was added into a solution of dibromotriphenylphosphorane
Cl (10 mL) at 0℃ under Ar. The reaction mixture was stirred at
the same temperature for 12 h and treated with water (30 mL). The mixture was extracted with
CH Cl (3×), dried (MgSO ), and concentrated under reduced pressure. The residue was briefly
2
(20
mL) and Et
3
(
1.02 g, 2.42 mmol) in distilled CH
2
2
2
2
4
purified by flash column chromatography (EtOAc:Hexane = 1:20) to provide the desired product 5
1
0.23 g, 38%) as a yellow oil, which was used for next reaction without further purification: H NMR
(
(
400 MHz, DMSO) δ 8.03 (1H, d, J = 16.0 Hz), 7.89 (1H, d, J = 16.0 Hz), 7.74-7.68 (4H, m), 7.49-
7.43 (2H, m), 7.37 (2H, d, J = 16.0 Hz), 7.32-7.28 (3H, m), 6.72 (1H, d, J = 16.0 Hz), 3.74 (3H, s);
4.1.6. General of compound 7: To a solution of carbodiimide compound (5, 0.51 g, 1.38 mmol) in
MeOH (30 mL) was added a solution of 1-[2-(1-pyrrolidinyl)ethyl]piperazine (6, 0.22 g, 1.18 mmol)
in MeOH (5 mL) and the mixture solution was stirred for 4 h. The mixture was concentrated in vacuo
2 2 4
and subjected to flash column chromatography (CH Cl :MeOH:NH OH = 100:9:1) to provide the
1
desired product (7, 0.46 g, 69%) as a white solid: mp 61.0-64.2 ℃; H NMR (400 MHz, CDCl
.50-7.46 (2H, m), 7.43-7.41 (2H, m), 7.26-7.19 (2H, m), 7.16-7.08 (4H, m), 7.00-6.93 (2H, m), 5.15
1H, dd, J = 10.8 and 4.8 Hz), 3.77 (3H, s), 3.49 (4H, br s), 2.86 (1H, dd, J = 15.2 and 10.8 Hz),
3
) δ
7
(
1
3
2
1
1
3
.64-2.60 (2H, m), 2.57-2.51 (7H, m), 2.40 (4H, br s), 1.78 (6H, s); C NMR (100 MHz, CDCl ) δ
71.9, 163.6, 161.1, 152.4, 144.9, 143.7, 136.4, 135.8, 128.5, 128.4, 128.3, 127.7, 125.8, 124.7,
23.0, 122.6, 122.3, 115.8, 115.5, 60.9, 57.6, 54.6, 53.7, 53.2, 51.9, 45.6, 39.5, 23.4; HRMS (ESI+):
+
[M+H] 556.3088, found 556.3084.
m/z calcd. for C33
H39FN
5
O
2
4.1.7. Preparation of compound 8f (KCP10066F): The reaction mixture containing ester 7 (0.46 g,
0.82 mmol), 1,5,7-triazabicyclo[4,4,0]dec-5-ene (TBD: 0.034 g, 0.25 mmol) as a catalyst, and 4-
fluorobenzylamine (0.25 g, 1.97 mmol) was stirred at 40 ℃ for 12 h under solvent-free condition.
Flash column chromatography (CH
2
Cl
2
:MeOH:NH
4
OH = 100:14:1) of the resultant mixture gave the
1
desired product (8f, 0.46 g, 87%) as a pale yellow solid: mp 85.2-95.4 ℃; H NMR (400 MHz,
CDCl
3
) δ 7.79 (1H, s), 7.45 (2H, dd, J = 8.0 and 5.2 Hz), 7.38 (2H, d, J = 8.4 Hz), 7.28 (2H, dd, J =
8.0 and 5.6 Hz), 7.17 (2H, d, J = 8.4 Hz), 7.12 (1H, d, J = 7.2 Hz), 7.07 (2H, t, J = 8.8 Hz), 7.00 (1H,
d, J = 7.6 Hz), 6.91 (4H, m), 5.24 (1H, dd, J = 8.8 and 4.8 Hz), 4.46 (2H, m), 3.20 (4H, s), 2.63 (4H,
page 14 / 21

s), 2.56 (3H, m), 2.44 (2H, d, J = 5.6 Hz), 2.31 (1H, dd, J = 14.4 and 5.2 Hz), 2.11 (4H, s), 1.80 (4H,
1
3
s); C-NMR (100 MHz, CDCl
35.5, 135.1, 134.1, 134.0, 129.3, 129.2, 127.6, 127.5, 126.8, 125.9, 124.3, 122.1, 122.0, 121.3,
14.9, 114.7, 114.6, 114.4, 59.9, 55.7, 53.7, 52.5, 51.8, 45.1, 41.9, 40.6, 22.5; HRMS (ESI+): m/z
3
) δ 169.4, 162.7, 162.4, 160.2, 159.9, 152.4, 144.0, 142.2, 135.6,
1
1
+
43 2 6
calcd. for C39H F N O [M+H] 649.3466, found 649.3464.
4.2. Biological evaluation
4.2.1. Cell culture for MTT assay
Human lung adenocarcinoma A549 cells were obtained from the Korean cell line bank
(
Seoul, Korea). Cells were cultured in RPMI 1640 supplemented with 10% heat-inactivated FBS,
penicillin (100 U/mL) and streptomycin sulfate (100 µg/mL). Cells were cultured at 37 °C in an
atmosphere of 5% CO
2
.
4.2.2. MTT assay
4
The cells (5 × 10 /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 in the cells, was dissolved with 100 µL DMSO. The optical density was measured at 540 nm
by an automatic microplate reader (Molecular Devices Corp., Sunnyvale, CA, USA).
4.2.3. Cell culture and transient transfection for electrophysiology
2
HEK tsA-201 cells were grown to 80∼90% confluence at 37 °C (5 % CO ) in DMEM
medium (Life Technologies, Grand Island, NY, USA), supplemented with 10 % (vol/vol) FBS
HyClone, Thermo Scientific, Pittsburgh, PA, USA), 200 U/ml penicillin, and 0.2 mg/ml
(
streptomycin (Life Technologies). Cells were suspended with 0.25% trypsin/EDTA and plated onto
glass coverslips in 10-cm culture dishes (Corning, Corning, NY, USA) at 10% confluence 24 h
before transfection. Calcium channel (6 µg) and GFP marker (0.6 µg) DNAs were transfected into
cells with calcium phosphate. For Ca
v
2.2 and Ca
v
1.2 channels, cDNA encoding Ca
v
α2δ1 and Ca β1b
v
subunits were co-transfected. Cells were transferred to 30 °C 16–18 h later following transfection
and stored for 2 days before recording.
4
.2.4. Electrophysiology
Cells on a glass coverslip were transferred into an external bath solution of 20 mM BaCl
mM MgCl , 40 mM TEACl, 65 mM CsCl, 10 mM HEPES, 10 mM glucose, pH 7.4. Borosilicate
page 15 / 21
2
, 1
2

glass pipettes (Sutter Instrument Co., Novato, CA, USA) (3–5 MΩ) were filled with internal solution
containing 140 mM CsCl, 2.5 mM CaCl , 1 mM MgCl , 5 mM EGTA, 10 mM HEPES, 2 mM Na-
2
2
ATP, and 0.3 mM Na-GTP, pH 7.3. Whole-cell patch clamp recordings were performed by using an
EPC 10 amplifier (HEKA Elektronik, Bellmore, NY, USA) linked to a personal computer equipped
with Pulse (V8.65) software (HEKA Elektronik). After seal formation, the membrane beneath the
pipette was ruptured and the pipette solution was allowed to dialyze into the cell for 2–5 min before
recording. T-type calcium currents were elicited by depolarization from a holding potential of −110
mV to a test potential of −20 mV for 100 ms with an interpulse interval of 20 s. Voltage-dependent
currents were leak corrected with an online P/4 subtraction paradigm. Data were recorded at 10 kHz
and filtered at 2.9 kHz. Data analysis was performed by using online analysis built in Pulse software,
and graphs were prepared by using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA).
Error bars plotted represent the mean values±standard error.
4.2.5. Animals
Experiments were carried out using either adult male C57BL/6J (wild-type) or CACNA1H
knockout (Ca 3.2 null) mice (20–25g) purchased from Jackson Laboratories. Animals were used at a
v
3
maximum of five mice per cage (30×20×15 cm ) with free access to food and water and temperature
kept at 23±1 °C on a 12 h light/dark cycle (lights on at 7:00 a.m.). Best efforts were made to
minimize animal suffering and distress according to the policies and recommendations of the
International Association for the Study of Pain and the guidelines of the Canadian Council on Animal
Care, all protocols were approved by the Institutional Animal Care and Use Committee. Intrathecal
(
i.t.) injections were given using volumes of 10 µL and performed according to the method described
4
1
39
by Hylden and Wilcox and carried out routinely in our laboratory. All drugs were dissolved in 2%
or less DMSO, whereas control animals received PBS + 2% DMSO. For each test, a different group
of mice were used and only one experiment per mouse was performed. The experimenter was
blinded to the experimental groups when testing the action of 8g (KCP10067F) on CFA-injected
animals.
4.2.6. Formalin Test
Animals were left to acclimatize for at least 60 min before the experiments. They were
injected intraplantarily (i.pl.) in the ventral surface of the right hind paw with 20 µL of a formalin
solution (1.25%) made up in PBS. Immediately after formalin injection, mice were individually
placed into observation chambers and tested from 0 to 5 min (acute nociceptive phase) and 15–30
min (inflammatory phase). Time spent licking or biting the injected paw was considered as a
nocifensive response and recorded with a chronometer. Different doses of 8g (KCP10067F) (0.3 – 3
µg/i.t.) were delivered intrathecally 20 min prior to formalin and the effects of treatments were
page 16 / 21

analyzed.
4.2.7. Persistent inflammatory pain induced by CFA
In order to induce mechanical hyperalgesia in mice triggered by peripheral inflammation, 20
µL of Complete Freund's Adjuvant (CFA) was injected subcutaneously in the plantar surface of the
right hindpaw (i.pl.) and animals in the sham group received 20 µl of PBS in the ipsilateral paw
4
2
accordingly to the method previously reported by Ferreira et al. Animals were treated with either
vehicle (10 µL/i.t.) or 8g (KCP10067F) (3 µg/i.t.) 2 days following CFA injections and their
mechanical withdrawal thresholds were analyzed time-dependently.
4.2.8. Evaluation of Mechanical Hyperalgesia
Mechanical hyperalgesia measurements were taken before injection of CFA and 2 daysafter
CFA and prior to 8g (KCP10067F) delivery (0), and also at different time points (20, 45, 90 min)
after treatment. Measurements were taken using a Dynamic Plantar Aesthesiometer (DPA, Ugo
Basile, Varese, Italy). Mice were individually placed in small enclosed transparent testing chambers
3
20 × 18.5 × 13 cm , length × width × height) on top of a grid floor and allowed to acclimate for a
(
period of at least 90 min. The DPA device was then positioned underneath so thatthe filament was
directly below the plantar surface of the ipsilateral hind paw of the animal and tested three times per
time point.
4.2.9. Statistical analysis
For behavioral experiments, data are presented as mean±SEM and analyzed by either one-
way or two-way analysis of variance (ANOVA) followed by Tukey's test. A value of p < 0.05 was
considered to be significant (*p <0.05; **p <0.01; ***p <0.001; NS = not different).
4.2.10. Microsomal stability
The microsomal clearance of the test compounds was evaluated using a single point
metabolic assay. Incubation mixtures contained 0.5 mg/mL human liver microsomes or 0.5 mg/mL
rat liver microsomes, 1 µM compound in 0.1 M phosphate buffer (pH 7.4) at 37 °C for 5 min. The
reaction was started by adding NADPH regeneration system solution and was incubated at 37 °C for
30min. Acetonitrile solution containing chlorpropamide as an internal standard was added to stop the
reaction. The sample was centrifuged (14,000 rpm, 4 °C) for 5 min. The supernatant was run on a
Shimadzu Nexera XR system and TSQ vantage (Thermo). A Kinetex C18 column (50 × 2.1 mm, 2.6
µm particle size, Phenomenex) was used for the analysis. The mobile phase used was 0.1% formic
acid in water (A) and 0.1% formic acid in acetonitrile (B). Data were processed on Analyst software
(
version 1.6.1) and Xcalibur (version 1.6.1).
page 17 / 21

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 the Canadian Institutes of Health Research and Canada Research
Chair Program (GWZ).
References and Notes
1
2
3
4
5
6
7
8
. 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 Biol. 2003, 4, 517.
. Clapham, D. E. Cell, 2007, 131, 1047.
. Parkash, J.; Asotra, K. Life Sci. 2010, 87, 587.
. Akl, H.; Bultynck, G. Biochim. Biophys. Acta. 2013, 1835, 180.
. Stewart, T. A.; Yapa, K.T.; Monteith, G. R. Biochim. Biophys. Acta. 2015, 1848, 2502.
. Chen, Y. F.; Chen, Y. T.; Chiu, W. T.; Shen, M. R. J. Biomed. Sci. 2013, 20, 23.
. Taylor, J. T.; Zeng, X. B.; Pottle, J. E.; Lee, K.; Wang, A. R.; Yi, S. G.; Scruggs, J. A.; Sikka, S.
S.; Li M. World J. Gastroenterol. 2008, 14, 4984.
9. Lory, P.; Bidaud, I.; Chemin, J. Cell Calcium 2006, 40, 135.
10. Gray, L. S.; Perez-Reyes, E.; Gamorra, J. C.; Haverstick, D. M.; Shattock, M.; McLatchie, L.;
Harper, J.; Brooks, G.; Heady, T.; Macdonald, T. L. Cell Calcium 2004, 36, 489.
11. Yunker, A. M.; McEnery, M. W. J. Bioenerg. Biomembr. 2003, 35, 533.
12. Monteil, A.; Chemin, J.; Leuranguer, V.; Altier, C.; Mennessier, G.; Bourinet, E.; Lory, P.;
Nargeot, J. J. Biol. Chem. 2000, 275, 16530.
1
1
1
1
1
1
3. Chemin, J.; Monteil, A.; Lory, P. J. Soc. Biol. 2003, 197, 235.
4. Berridge, M. J. Neuron 1988, 21, 13.
5. Perez-Reyes, E.; Lory, P. CNS Neurol. Disord.: Drug Targets 2006, 5, 605.
6. Lastraioli, E.; Iorio, J.; Arcangeli, A. Biochim. Biophys. Acta. 2015, 1848, 2685.
7. Nilius, B.; Carbone, E. Pflügers Arch. 2014, 466, 689.
8. Dziegielewska, B.; Gray, L. S.; Dziegielewski, J. Pflügers Arch. 2014, 466, 801.
page 18 / 21

19. Santoni, G.; Santoni, M.; Nabissi, M. Br. J. Pharmacol. 2012, 166, 1244.
20. Byun, J. S.; Sohn, J. M.; Leem, D. G.; Park, B.; Nam, J. H.; Shin, D. H.; Shin, J. S.; Kim, J. H.;
Lee, K.-T.; Lee, J. Y. Bioorg. Med. Chem. Lett. 2016, 26, 1073.
2
2
2
2
2
2
2
2
2
1. 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.
2. 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.
3. 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.
4. 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.
5. 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.
6. 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.
7. Choi, T. A.; Furimsky, A. M.; Swezey, R.; Bunin, D. I.; Byrge, P.; Iyer, L. V.; Chang, P. Y.;
Abergel, R. J. J. Pharm. Sci. 2015, 104, 1832
8. Di, L.; Kerns, E. H.; Gao, N.; Li, S. Q.; Huang, Y.; Bourassa, J. L.; Huryn, D. M. J. Pharm. Sci.
2
004, 93, 1537.
9. Böhm, H. J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Müller, K.; Obst-Sander, U.; Stahl,
M. ChemBioChem. 2004, 5, 637.
3
3
3
3
0. Shah, P.; Westwell, A. D. J. Enzyme Inhib. Med. Chem. 2007, 22, 527.
1. Müller, K.; Faeh, C.; Diederich, F. Science 2007, 317, 1881.
2. Littke, A. F.; Fu, G. C. Org. Synth. 2005, 81, 63.
3. 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. Bioorg. Med. Chem. Lett. 2017, 27, 1179.
3
3
3
3
4. M'Dahoma, S.; Gadotti, V. M.; Zhang, F. X.; Park, B.; Nam, J. H.; Onnis, V.; Balboni, G.; Lee, J.
Y.; Zamponi, G. W. Pflügers Arch. 2016, 468, 193.
5. 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, 14, 3502.
6. Rubinstein, L. V.; Shoemaker RH.; Paull KD.; Simon RM.; Tosini S;; Skehan P.; Scudiero DA.;
Monks A.; Boyd, M. R. J. Natl. Cancer Inst. 1990, 82, 1113.
7. Di, L.; Kerns, E. H.; Gao, N.; Li, S. Q.; Huang; Y.; Bourassa, J. L.; Huryn, D. M. J. Pharm. Sci.
2
004, 93,1537.
page 19 / 21

3
3
4
8. Gadotti, V. M.; You, H.; Petrov, R. R.; Berger, N. D.; Diaz, P.; Zamponi, G. W. Mol. Pain 2013,
9
, 32.
9. Bladen, C.; McDaniel, S. W.; Gadotti, V. M.; Petrov, R. R.; Berger, N. D.; Diaz, P.; Zamponi, G.
W. ACS Chem. Neurosci. 2015, 6, 277.
0. Gadotti, V. M.; Caballero, A. G.; Berger, N. D.; Gladding, C. M.; Chen, L.; Pfeifer, T.
A.; Zamponi, G. W. Mol. Pain 2015, 11, 12.
41. Hylden, J. L.; Wilcox, G. L. Eur. J. Pharmacol. 1980,67, 313.
42. Ferreira, J.; Campos, M. M.; Pesquero, J. B.; Araujo, R. C.; Bader, M.; Calixto, J. B. Neuro-
pharmacology. 2001, 41, 1006.
page 20 / 21

Graphical Abstract
Synthesis and biological evaluation of fluoro-substituted 3,4-dihydroquinazoline
derivatives for cytotoxic and analgesic effects
Jin Han Kim, Hui Rak Jeong, Da Woon Jung, Hong Bin Yoon, Sun Young Kim,
*
Hyoung Ja Kim, Kyung-Tae Lee , Vinicius M. Gadotti, Junting Huang,
*
Fang-Xiong Zhang, Gerald W. Zamponi , Jae Yeol Lee
*
page 21 / 21