Novel T-type calcium channel blockers: Dioxoquinazoline carboxamide derivatives

Article abstract of DOI:10.1016/j.bmc.2006.09.051

T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pains. Since the withdrawal of mibefradil, a T-type calcium channel blocker, there have been a lot of efforts to develop T-type calcium chann

Full text of DOI:10.1016/j.bmc.2006.09.051

Bioorganic & Medicinal Chemistry 15 (2007) 365–373
Novel T-type calcium channel blockers: Dioxoquinazoline
carboxamide derivatives
Mi Na Jo,a Hee Jeong Seo,a Yoonji Kim,a Seon Hee Seo,a Hyewhon Rhim,a
Yong Seo Cho,a Joo Hwan Cha,b Hun Yeong Koh,c Hyunah Chooa,*, and Ae Nim Paea,*,
aLife Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
bAdvanced Analysis Center, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
cDepartment of Chemistry, Inha University, Nam-gu, Incheon 402-751, Republic of Korea
Received 31 July 2006; revised 22 September 2006; accepted 23 September 2006
Available online 10 October 2006
Abstract—T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pains.
Since the withdrawal of mibefradil, a T-type calcium channel blocker, there have been a lot of efforts to develop T-type calcium
channel blockers. A small molecule library of dioxoquinazoline carboxamide derivatives containing 155 compounds was designed,
synthesized, and biologically evaluated for T-type calcium channel blocking activity. Among those compounds synthesized, the
compound 1n shows the most potent T-type calcium current blocking activity with an IC50 value of 1.52 lM, which is comparable
to that of mibefradil.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
report that the effect of mibefradil on blood pressure
and small vessel myogenic tone is mediated by the
CaV1.2 L-type calcium channel.5 Therefore, it becomes
very controversial that the cardiovascular effect of mibe-
fradil results from the blockade of T-type calcium
channel.
T-type calcium channel blockers have drawn more
attention since mibefradil was approved for the treat-
ment of angina pectoris and hypertension.1 It reduced
blood pressure and heart rate without suppressing myo-
cardial contraction. Mibefradil also had beneficial
effects in various animal models of heart failure and im-
proved survival rate in a rat chronic myocardial infarc-
tion model. This was possible by accompanied
antiarrhythmic effects through T-type calcium channel
blockade.2 Unfortunately, the drug had to be withdrawn
from the market due to drug interaction at the cyto-
chrome P-450 3A4 enzyme which was unrelated to
T-type calcium channel blockade.3 However, there were
some stark findings reported that a genetic loss-of-func-
tion model, a mouse lacking the CaV3.2 T-type calcium
channel, surprisingly demonstrated that calcium influx
through VSM (vascular smooth muscle) T-type calcium
channels is an essential mediator of normal relaxation,
not contraction, of coronary arteries.4 There is another
On the other hand, T-type calcium channels play cru-
cial roles in the control of neuropathic pains which
are caused by hyperexitable neurons.6 The role of
T-type calcium channels in pain has also been ad-
dressed using specific genetic modulation of T-type cal-
cium channel isoforms. CaV3.1 knockout mice were
shown to have an increased sensitivity to visceral pain,
but no difference in acute pain thresholds or responses
to a cutaneous pain simulus compared to wild-type
mice.7 CaV3.2 antisense knockdown reduced T-type
currents in small and medium-sized DRG (dorsal root
ganglion) cells.8 The antisense treatment resulted in
major anti-nociceptive and anti-hyperalgesic, suggest-
ing that CaV3.2 plays a major pronociceptive role in
acute and chronic pain states. Together, the results of
these two studies suggest that the different T-type calci-
um channel isoforms can be anti-nociceptive or prono-
ciceptive. Perhaps the major problem with elucidating
the exact role of T-type calcium channels in neuropath-
ic pain is the current lack of highly specific pharmaco-
logical agents.
Keywords: T-type calcium channel blockers; Dioxoquinazoline
carboxamide derivatives.
*
Corresponding authors. Tel.: +82 2 958 5185; fax: +82 2 958 5189
(A.N.P); tel.: +82 2 958 5157; fax: +82 2 958 5189 (H.C.); e-mail
addresses: hchoo@kist.re.kr; anpae@kist.re.kr
These co-corresponding authors contributed equally to this work.
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.09.051

366
M. N. Jo et al. / Bioorg. Med. Chem. 15 (2007) 365–373
F
O
O
H
OMe
O
N
N
O
HO
N
N
O
F
O
H
S
N
N
N
H
N
F
N
N
NH
Me
F
U-92032
Mibefradil
KYS05042
Figure 1. T-type calcium channel blockers.
Since the withdrawal of mibefradil and the increased
interest in the T-type calcium channels as a therapeutic
target, there have been significant efforts to develop
selective T-type calcium channel blockers (Fig. 1).9
Herein we report design, synthesis, and biological eval-
uation of novel T-type calcium channel blockers.
yl 2-aminoterephthalate 2 was treated with isocyanates 3
(R1-NCO) in 1,4-dioxane solution under basic condi-
tions to give cyclized dioxoquinazolines 4 in 41–92%
yields (Scheme 2).13 The compounds 4 were hydrolyzed
to the corresponding carboxylic acids 5 in 88–99%
yields.14 In the case of commercially unavailable alkyl
isocyanates such as methyl, propyl, and cyclohexyl
isocyanates, the corresponding amines such as methyl,
propyl, and cyclohexyl amines were used for the synthe-
sis of dioxoquinazolines (Scheme 2). Therefore, dimeth-
2. Results and discussion
2.1. Design of small molecule library
yl 2-aminoterephthalate 2 was converted to the
corresponding isocyanates 6 by treatment with triphos-
gene in toluene under reflux conditions. The isocyanates
6 were treated with alkyl amines under basic conditions
to give urea analogs 7 in 22–50% yields from compounds
2. The urea analogs 7 underwent cyclization and hydro-
lysis simultaneously in a mixture of 10% NaOH and
methanol to give dioxoquinazoline carboxylic acids 5
in 54–98% yields.
Previously, a pharmacophore model was generated by
using in-house hit compounds and mibefradil.10 The
pharmacophore model was used for the virtual screening
of the small molecule library from ChemDiv and May-
bridge2001 in order to obtain new scaffolds of T-type
calcium channel blockers.11 Among the virtual hit com-
pounds, selected compounds were bought and biological-
ly screened, resulting in novel scaffolds of several T-type
calcium channel blockers. A thioxoquinazolinone car-
boxamide derivative is one of the interesting scaffolds
and its derivatives show potent inhibitory activity against
T-type calcium channels (Fig. 2). From the pharmaco-
phore analysis, the sulfur in the thioxoquinazolinone car-
boxamide derivative was found to be acting as a hydrogen
bonding acceptor. Because oxygen atom is a stronger
hydrogen bonding acceptor than sulfur atom,12 a small
molecule library was designed by using dioxoquinazoline
carboxamide derivatives (Scheme 1).
Dioxoquinazoline carboxylic acids 5 were converted to
the corresponding amides 1 by treatment with cyclic
tertiary aminoalkylamines (R2–NH2, Scheme 1). Some
of cyclic tertiary aminoalkylamines are commercially
available. Commercially unavailable amines (R2–
NH2) were synthesized in two steps (Scheme 3). Piper-
idine derivatives 8 underwent SN2 reaction with N-
bromoethylphthalimide or N-bromopropylphthalimide
to give compounds 9 in 27–77% yields.15 Compounds
9 were treated with hydrazine hydrate in ethanol to
afford the desired piperidinoalkylamines 10 in 67–
87% yields.16 To synthesize the desired dioxoquinazo-
line carboxamide derivatives 1, dioxoquinazoline car-
boxylic acids 5 were converted to the corresponding
acyl halides by treating with SOCl2 under reflux con-
ditions or treating with oxalyl chloride and a catalytic
amount of DMF in CH2Cl2 at room temperature
(Scheme 2). Then, treatment of the intermediate acyl
halides with cyclic tertiary aminoalkylamines (R2–
NH2) provided dioxoquinazoline carboxamide deriva-
tives 1 in 5–82% overall yields starting from the car-
boxylic acids 5.17
Two building blocks, R1 and R2 groups, were added to
the novel key scaffold (the dioxoquinazoline) to fulfill
the common features of the pharmacophore model:
one building block R1 is mainly composed of substituted
phenyl and benzyl groups and alkyl groups, and the
other building block R2 is composed of cyclic tertiary
aminoalkyl groups. By combination of various R1 and
R2 groups, a small molecule library of T-type calcium
channel blockers was constructed (Scheme 1).
2.2. Synthesis
The designed T-type calcium channel blockers were syn-
thesized from dimethyl 2-aminoterephthalate 2. Dimeth-
Total 155 compounds of dioxoquinazoline carboxamide
derivatives 1 were synthesized with combination of two
building blocks, R1 and R2 groups.
O
N
2.3. Biological activity
H
N
N
N
S
Cl
H
O
The synthesized compounds 1 of the small molecule li-
brary were biologically evaluated against HEK293 cells
which stably express both T-type calcium channel
CaV3.1 with a1G subunit and potassium channel
VH04 IC50 0.10 μM
Figure 2. A thioxoquinazolinone derivative.

M. N. Jo et al. / Bioorg. Med. Chem. 15 (2007) 365–373
367
O
R1
N
H
N
R2
N
O
H
O
1
F
OMe
F
OMe
R1
=
F
OMe
a
g
c
i
e
b
h
d
f
l
F
OMe
F
OMe
OMe
F
j
k
Cl
CH3
Cl
m
n
o
p
q
R2
=
O
N
N
N
N
i
ii
iii
iv
O
N
N
N
N
N
v
vi
x
vii
viii
N
N
N
ix
xi
Scheme 1. A small molecule library of dioxoquinazoline carboxamide derivatives 1.
O
O
CO2Me
R1
O
R1
O
a
f, g
N
N
+
R1 NCO
H
N
MeO2C
NH2
RO2C
b
N
H
R2
N
H
3
O
2
4 R = Me
5 R = H
1
c
e
CO2Me
NCO
CO2Me
NH
d
MeO2C
MeO2C
O
NH
R1
6
7
Scheme 2. Reagents and conditions: (a) TEA, 1,4-dioxane, reflux, 41–92%, (b) 10% NaOH/1,4-dioxane (1:4), rt, 88–99%, (c) triphosgene, toluene,
reflux, (d) R1–NH2, TEA, 1,4-dioxane, reflux, 22–50% (two-step yields), (e) 10% NaOH/MeOH (1:3), reflux, 54–98%, (f) SOCl2, reflux or (COCl)2,
cat. DMF, CH2Cl2, rt, (g) R2–NH2, CH2Cl2, rt, 5–82% (two-step yields).
Kir2.1.18 Two assay methods were employed: FDSS6000
assay and patch clamp assay. FDSS6000 assay was devel-
oped for high throughput screening and applied to the
whole small molecule library of dioxoquinazoline carbox-
amide derivatives 1.19 Patch clamp assay using a single
cell is more sensitive and more accurate. However, the
patch clamp assay records the currents of the single cell
one by one so that the work is laborious. The whole

368
M. N. Jo et al. / Bioorg. Med. Chem. 15 (2007) 365–373
R
mibefradil (entry 16). Among the para-substituents such
O
R
R
as chloro, fluoro, and methoxy, the chloro-substituent
shows the most potent activity. The compound 1e with
a para-chlorobenzyl group is more active than the com-
pound 1d with a para-fluorobenzyl group. The com-
pound 1g is more active than the compounds 1f and
1h. Compounds 1i, 1l, and 1n are more active than the
compounds 1j, 1k, and 1m, respectively. For the R2
building block, most R2 groups are piperidine-based
groups except compounds 1a and 1b, which have a pyrr-
olidinoethyl group. The compound 1a shows potent T-
type calcium current blocking activity with an IC50 value
of 2.46 lM. In most cases, the compounds 1i–1o with
piperidinopropyl groups (vi–viii, Scheme 1) show slight-
ly better blocking activity than the compounds 1c–1h
with piperidinoethyl groups (ii–iv). The compounds 1i,
1m, and 1n are more active than the compounds 1e,
1f, and 1g, respectively. Among the substituents on
piperidine-based R2 groups such as hydrogen (ii and
vi), methyl (iii and vii), and ethyl (iv and viii), ethylpi-
peridine-based R2 groups (iv and viii) show better block-
ing activity. The compounds 1f, 1m, and 1n with
ethylpiperidine-based R2 groups (iv and viii) are more
active than the compounds 1d, 1k, and 1l, respectively.
However, the compound 1g with an ethylpiperidinoethyl
group (iv) is slightly less active than the compound 1e
with a piperidinoethyl group (ii). It is consistent with
the SAR results that the compound 1n, which has best
R1 and R2 building blocks, shows the most potent
T-type calcium current blocking activity.
a
b
N
n
N
N
n
HN
H2N
O
8
10
9
R = H, Me and Et
R = H, Me and Et
n = 1 and 2
R = H, Me and Et
n = 1 and 2
Scheme 3. Reagents and conditions: (a) N-bromoethylphathalimide or
N-bromopropylphthalimide, K2CO3, CH3CN, reflux, 27–77%; (b)
hydrazine hydrate, EtOH, reflux, 67–87%.
synthesized 155 compounds were primarily screened by
FDSS6000 assay to obtain % inhibition at a certain molar
concentration (Table 1). From the FDSS6000 results, 15
compounds with the most T-type current blocking activ-
ity were selected and screened by patch clamp assay to ob-
tain IC50 values (Table 2).
From the high throughput screening by FDSS6000 as-
say, several structure–activity relationships were ob-
tained (Table 1): (1) one of the building blocks, R1
group, prefers to have substituted benzyl groups (g–l,
p, and q) instead of substituted phenyl groups (a–f).
Among the substituted benzyl groups, para-substituents
(fluoro i, chloro q, and methoxy l) were preferred to
ortho- or meta-substituents for activity. Among the alkyl
groups, a cyclohexyl group m shows good T-type calci-
um current blocking activity and was selected for the
patch clamp assay rather than a methyl or a propyl
group (n or o) of relatively small size. (2) The other
building block, R2 group, prefers piperidine-based
groups (ii–iv and vi–viii) to morpholine-based groups (i
and v) or a piperazine-based group (xi). The compounds
1 with morpholine-based groups (i and v) or a pipera-
zine-based group (xi) show little T-type calcium current
blocking activity. (3) There was no significant difference
in T-type calcium current blocking activity between pip-
eridinoethyl groups (ii–iv) and piperidinopropyl groups
(vi–viii).
2.4. Discussion
Calcium channels are divided into high voltage activated
(N-, L-, P/Q, and R-type) and low voltage activated (T-
type) channels based on the amount of cellular depolar-
ization required for activation. Like other calcium chan-
nels, T-type calcium channels are composed of an
a1-subunit and auxiliary b- and a2-d-subunits. The a1-
subunits form the channel pore, sense the membrane
voltage, and define the basic pharmacological properties
of the channel. Calcium channel blockers including a
selective T-type calcium channel blocker, mibefradil,
are assumed to bind with high affinity to the channel
pore, resulting in the blockade of calcium entry.20
According to the FDSS6000 assay results, 15 com-
pounds were selected and biologically evaluated against
T-type calcium channel to obtain IC50 values with
patch-clamp assay method. The results are shown in
Table 2. The selected 15 compounds (1a–1o) show mod-
erate to potent T-type calcium current blocking activity
with IC50 values from 1.52 to 16.87 lM. Compound 1b
was the only example with a cyclohexyl group as R1
group instead of a substituted benzyl group, which
shows T-type current blocking activity with an IC50 val-
ue of 7.96 lM. The position of the substituents in a ben-
zyl group prefers para-position instead of ortho- or
meta-position. The compound 1d with a para-fluoroben-
zyl group as R1 group shows more potent blocking
activity against T-type calcium channel than the com-
pound 1c with a meta-fluorobenzyl group. The com-
pound 1n with a para-chlorobenzyl group is more
active than the compound 1o with an ortho-chlorobenzyl
group. The compound 1n shows the most potent T-type
calcium channel blocking activity among this series of
compounds 1 with an IC50 value of 1.52 lM, which is
comparable to the activity of the positive control,
In our efforts to identify novel T-type calcium channel
blockers with a new scaffold, a small molecule library
of dioxoquinazoline carboxamide derivatives 1 was de-
signed based on the pharmacophore model. Total 155
compounds were synthesized and biologically evaluated
by FDSS6000 assay and patch-clamp assay. The com-
pound 1n shows the most potent inhibitory activity
against T-type calcium channel, which was identified
as a novel T-type calcium channel blocker. Our designed
compounds were derived from the pharmacophore mod-
el which has six common features with mibefradil and
in-house hit compounds. Therefore, the synthesized
compounds with inhibitory activity are also expected
to have binding affinity to the channel pore composed
of a1-subunits to inhibit calcium entry. It was expected
that the compound 1n would be much more active than
our original lead compound VH04, because oxygen

M. N. Jo et al. / Bioorg. Med. Chem. 15 (2007) 365–373
Table 1 (continued)
369
Table 1. %inhibition of T-type calcium current of synthesized com-
pounds by FDSS6000 assaya
Entry
R1
R2
% inhibition
at 100 lM
%inhibition
at 10 lM
O
R1
O
b
57
58
e
f
v
—
N
H
N
v
5.2
b
R2
N
H
59
60
g
h
i
v
—
O
b
v
—
1
b
61
62
v
—
j
v
3.7
Entry
R1
R2
% inhibition
at 100 lM
%inhibition
at 10 lM
63
64
k
l
v
28.9
36.0
36.3
9.2
v
b
—
1
a
b
c
d
e
f
i
65
66
m
n
o
p
q
g
h
i
v
2
i
3.8
b
v
3
i
—
67
68
v
2.2
7.6
9.7
8.7
b
4
i
—
v
b
—
5
i
69
70
v
6
i
5.8
b
—
vi
b
—
7
g
h
i
i
71
72
vi
8
i
7.9
12.8
vi
14.9
b
9
i
73
74
j
vi
—
b
—
b
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
j
i
k
l
vi
—
k
l
i
2.8
75
76
vi
35.2
10.6
b
i
—
m
p
q
a
b
c
d
e
f
vi
b
—
b
—
m
n
o
p
q
a
b
c
d
e
f
i
77
78
vi
b
i
—
vi
34.8
b
—
b
i
—
79
80
vii
vii
vii
vii
vii
vii
vii
vii
vii
vii
vii
vii
vii
vii
vii
vii
vii
viii
viii
viii
viii
viii
viii
viii
viii
viii
ix
ix
ix
ix
ix
ix
ix
ix
ix
ix
ix
ix
ix
ix
ix
ix
b
—
b
—
i
b
i
29.6
81
82
—
b
—
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
ii
iii
iii
iii
iii
iii
iii
iii
iii
iii
iv
iv
iv
iv
iv
iv
iv
iv
iv
v
7.2
3.4
83
84
12.3
1.5
12.5
b
—
85
86
g
h
i
13.9
47.8
56.6
12.7
36.9
42.3
48.3
12.5
8.5
b
—
b
—
87
88
g
h
i
6.3
j
64.6
65.0
89
90
k
l
j
3.8
91
92
m
n
o
p
q
g
h
i
k
l
62.9
23.1
93
94
m
n
o
p
q
g
h
i
52.2
18.0
17.6
1.8
40.8
13.4
21.8
95
96
30.0
44.2
9.8
97
98
68.3
99
j
7.6
15.7
29.3
26.1
34.4
44.0
15.4
14.7
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
k
l
b
—
j
m
p
q
a
b
c
d
e
f
k
l
11.0
22.9
21.7
7.9
m
p
q
g
h
i
6.5
4.3
b
31.8
21.8
19.4
33.6
1.6
—
b
—
13.1
10.5
24.9
j
g
h
i
k
l
31.0
33.7
32.3
13.1
53.9
6.9
47.2
b
—
m
p
q
a
b
c
d
j
k
l
27.8
39.4
57.7
6.7
2.8
15.1
9.2
m
n
o
p
v
b
—
v
b
—
v
15.5
(continued on next page)

370
M. N. Jo et al. / Bioorg. Med. Chem. 15 (2007) 365–373
Table 1 (continued)
3. Conclusion
Entry
R1
R2
% inhibition
at 100 lM
%inhibition
at 10 lM
A small molecule library of dioxoquinazoline carboxam-
ide derivatives 1 was designed, synthesized, and biolog-
ically evaluated to develop novel T-type calcium channel
blockers. Among total 155 compounds, the compound
1n shows the most potent T-type calcium current block-
ing activity. Further evaluations of the compound 1n
such as selectivity for other calcium channels, pharma-
cokinetics, and neuronal analgesic effect are in progress.
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
q
a
b
c
d
e
f
ix
x
31.8
b
—
b
x
—
x
5.8
5.8
x
1.5
b
x
—
b
x
—
g
h
i
x
x
9.8
34.2
x
4. Materials and methods
4.1. Experimental
b
—
j
x
k
l
x
13.2
26.8
x
b
m
n
o
p
q
a
b
c
d
e
f
x
—
b
All the commercially available reagents were obtained
from Aldrich and Fluka, and generally used without fur-
x
—
b
x
—
1
ther purification. H NMR and 13C NMR spectra were
x
6.2
23.1
x
obtained on Bruker Advance 400 (or 300) spectrometer.
Nuclear magnetic resonance spectra were acquired at
xi
xi
xi
xi
xi
xi
xi
xi
xi
xi
xi
xi
xi
xi
xi
xi
xi
2.4
b
1
400 (or 300) MHz for H and 100 MHz for 13C NMR.
—
b
—
Infrared spectra were obtained on a Perkin-Elmer
16FPC FT-IR spectrometer using KBr pellet, CHCl3
or neat. GC/MSD was obtained on a Hewlett Packard
5890. HRMS spectra were obtained on a JMS-700 mass
spectrometer (Jeol). Analytical thin layer chromatogra-
phies (TLC) were carried out on precoated silica gel
plates (Merck Kieselgel 60F254, layer thickness
0.25 mm). Flash column chromatographies were con-
ducted with silica gel grade 230–400 mesh (Merck Kiese-
gel 60 Art 9385).
b
—
b
—
b
—
g
h
i
7.3
8.3
25.5
b
—
j
k
l
20.1
20.6
b
—
m
n
o
p
q
b
—
3.4
4.1.1. Methyl 3-(4-chlorobenzyl)-2,4-dioxoquinazoline-7-
carboxylate (4). A solution of dimethyl aminoterephth-
alate (3.54 g, 16.9 mmol) in 1,4-dioxane (60 mL)
was treated with triethylamine (5.8 mL, 42 mmol)
and 1-chloro-4-isocyanatomethyl-benzene (2.9 mL,
22 mmol) at room temperature. The resulting mixture
was heated up to 90 ꢁC and stirred for 3 days. The
reaction mixture was cooled down to room tempera-
ture and the precipitate was filtered, washed with
diethylether, and dried to afford the desired product
5.7
17.4
a % inhibition was obtained at 10 and 100 lM, alternatively.
b Not active.
atom is a better hydrogen bonding acceptor than sulfur
atom. However, the compound 1n is found to be less ac-
tive than the compound VH04 (Fig. 2), which implies
that there might be more important features than hydro-
gen bonding to be addressed.
4
(2.38 g, 6.90 mmol) in 41% yield: 1H NMR
(300 MHz, DMSO-d6) d 11.73 (s, 1H), 8.06 (d,
J = 8.2 Hz, 1H), 7.79 (s, 1H), 7.71 (d, J = 8.0 Hz,
1H), 7.36 (br s, 4H), 5.06 (s, 2H), 3.89 (s, 3H).
Several structure–activity relationships were identified
from this study. The R1 group prefers a substituted ben-
zyl group to a substituted phenyl group (Table 1).
Among the benzyl groups, para-substituted benzyl
groups show more inhibitory activity than ortho- or a
meta-substituted benzyl groups and the best substituent
on a benzyl group in this study is a chloro-substituent
(Table 2). The R2 group prefers cyclic tertiary amines
according to the pharamacophore model. Among the
cyclic tertiary amines, an ethylpiperidinopropyl group
shows the best inhibitory activity against T-type calcium
current (Table 2). The tether between the core dioxoqui-
nazoline carboxamide and the tertiary amine prefers a
propyl group. A bulkier group in the tertiary amine is
preferred such as an ethylpiperidino-group instead of a
simple piperidino-group. Further modification of the
compound 1n is under study, based on the information
of the SAR study.
4.1.2. 3-(4-Chlorobenzyl)-2,4-dioxoquinazoline-7-carbox-
ylic acid (5, R1 = 4-chlorobenzyl). Methyl 3-(4-chlorob-
enzyl)-2,4-dioxoquinazoline-7-carboxylate
4
(2.54 g,
7.37 mmol) was dissolved in a 1:4 mixture of 10% NaOH
and 1,4-dioxane, and the resulting mixture was stirred at
room temperature for 1 h. The reaction mixture was
neutralized with concd HCl and the resulting precipitate
was filtered, washed with water, and dried to afford the
1
desired product 5 (2.22 g, 6.71 mmol) in 91% yield: H
NMR (300 MHz, DMSO-d6) d 13.52 (s, 1H), 11.73 (s,
1H), 8.02 (d, J = 8.2 Hz, 1H), 7.78 (s, 1H), 7.69 (d,
J = 8.2 Hz, 1H), 7.36 (br s, 4H), 5.07 (s, 2H); 13C
NMR (100 MHz, DMSO-d6) d 166.80, 162.05, 150.56,
139.92, 137.75, 136.65, 132.21, 131.46, 129.25, 128.77,
128.31, 123.14, 116.82, 43.22.

M. N. Jo et al. / Bioorg. Med. Chem. 15 (2007) 365–373
371
Table 2. IC50 values of T-type calcium channel blocking activity of selected compounds by patch clamp assay
O
R1
N
H
N
R2
N
O
H
O
1
Entry
1
Compound
R1
R2
IC50 (lM)
1a
q
ix
2.46 0.22
7.96 0.61
16.87 2.38
9.23 0.68
2.08 0.36
7.98 0.55
3.38 0.36
5.97 1.23
1.95 0.25
3.11 1.28
4.63 0.53
3.72 0.20
3.31 0.19
1.52 0.23
2.79 0.36
1.43 0.49
N
N
Cl
2
1b
m
h
i
ix
ii
F
3
1c
N
N
N
4
1d
ii
F
5
1e
q
i
ii
Cl
6
1f
iv
F
N
N
N
7
1g
q
l
iv
Cl
8
1h
iv
OMe
Cl
9
1i
q
l
vi
N
N
N
N
10
11
12
13
14
15
16
1j
vi
OMe
1k
i
vii
vii
viii
viii
viii
F
1l
q
i
Cl
1m
F
N
N
N
1n
q
p
Cl
Cl
1o
mibefradil

372
M. N. Jo et al. / Bioorg. Med. Chem. 15 (2007) 365–373
4.1.3.
3-Cyclohexyl-2,4-dioxoquinazoline-7-carboxylic
the desired product 10 (1.6 g, 9.4 mmol) in 73% yield: 1H
NMR (300 MHz, CDCl3) d 2.66–2.61 (m, 2H), 2.35–
2.25 (m, 1H), 2.10–2.06 (m, 2H), 1.57–1.36 (m, 10H),
1.24–1.21 (m, 2H), 0.79 (t, J = 7.4 Hz, 3H); 13C NMR
(100 MHz, CDCl3) d 61.66, 51.50, 51.43, 40.91, 29.35,
28.12, 25.30, 23.32, 23.21, 10.08.
acid (5, R1 = cyclohexyl). A solution of dimethyl 2-amin-
oterephthalate 2 (3.0 g, 14 mmol) and triphosgene (5.1 g,
17 mmol) in toluene (60 mL) was refluxed for 6 h. The
resulting mixture was concentrated to dryness to give
compound 6. The compound 6 was dissolved in 1,4-di-
oxane (60 mL) and treated with triethylamine
(0.2 mL). The reaction mixture was warmed up to
90 ꢁC and stirred for 60 h. The resulting mixture was
cooled to room temperature and diethyl ether
(100 mL) was added to the mixture. The precipitate
was filtered and washed with diethyl ether to give the de-
sired product, dimethyl 2-(3-cyclohexylureido)-tere-
4.1.6. 3-(4-Chlorobenzyl)-N-(3-(2-ethylpiperidin-1-yl)pro-
pyl)-2,4-dioxoquinazoline-7-carboxamide (1n). 3-(4-Chlo-
robenzyl)-2,4-dioxoquinazoline-7-carboxylic acid
5
(90 mg, 0.27 mmol) was dissolved in thionyl chloride
(4 mL) and the resulting solution was refluxed for 2 h.
After concentration to dryness, the resulting acyl chlo-
ride was dissolved in CH2Cl2 (4 mL) and treated with
3-(2-ethylpiperidin-1-yl)propan-1-amine 10 (101 mg,
0.598 mmol) at 0 ꢁC. The reaction mixture was stirred
at room temperature for 2 h. After concentration, the
residue was purified with a 10:1 mixture of CH2Cl2
and methanol by column chromatography on silica gel
to give the desired product 1n (58 mg, 0.12 mmol) in
1
phthalate 7 (1.75 g, 3.22 mmol) in 22% yield: H NMR
(300 MHz, DMSO-d6) d 9.66 (s, 1H), 9.00 (s, 1H), 7.97
(d, J = 8.2 Hz, 1H), 7.51–7.48 (m, 2H), 4.75 (br s, 1H),
3.88 (s, 3H), 3.86 (s, 3H), 2.38 (br s, 2H), 1.82–1.54
(m, 4H), 1.29–1.11 (m, 4H).
Dimethyl 2-(3-cyclohexylureido)-terephthalate 7 (1.51 g,
5.23 mmol) was dissolved in a 1:3 mixture of 10% NaOH
and MeOH, and the reaction mixture was stirred at
70 ꢁC for 3 h. The resulting mixture was cooled to room
temperature and acidified with concd HCl to give a pre-
cipitate. The precipitate was filtered, washed with water,
and dried to give the desired product 5 (0.80 g,
1
44% yield: H NMR (400 MHz, DMSO-d6) d 11.65 (s,
1H), 8.71 (bs, 1H), 7.99 (d, J = 8.3 Hz, 1H), 7.59–7.55
(m, 2H), 7.34 (br s, 4H), 5.06 (s, 2H), 3.26–3.25 (m,
2H), 2.75–2.66 (m, 2H), 2.22 (bs, 1H), 2.12 (bs, 2H),
1.65–1.60 (m, 2H), 1.49–1.37 (m, 6H), 1.24–1.22 (m,
2H), 0.78 (t, J = 7.2 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6) d 165.46, 162.08, 150.66, 141.33, 139.94,
136.72, 132.21, 130.02, 128.78, 128.15, 120.99, 115.77,
115.12, 61.04, 51.53, 50.90, 43.19, 38.65, 29.55, 25.99,
25.62, 23.63, 23.54, 10.03; IR (KBr) 3278, 1720, 1658,
1
2.8 mmol) in 54% yield: H NMR (300 MHz, DMSO-
d6) d 13.47 (s, 1H), 11.50 (s, 1H), 7.99 (d, J = 8.2 Hz,
1H), 7.71 (s, 1H), 7.65 (dd, J = 8.2 Hz, J = 1.3 Hz,
1H), 4.76–4.68 (m, 1H), 2.42–2.34 (m, 2H), 1.81–1.77
(m, 2H), 1.65–1.58 (m, 2H), 1.32–1.16 (m, 4H); 13C
NMR (100 MHz, DMSO-d6) d 166.68, 162.27, 150.61,
139.86, 136.70, 128.38, 122.80, 117.52, 116.42, 53.47,
28.74, 26.43, 25.55.
1649 cmÀ1
;
C28H38ClN4O3 483.2163, found 483.2166.
HR-MS (FAB, M+H) calcd for
4.2. Biological assay
4.1.4. 2-(3-(2-Ethylpiperidin-1-yl)propyl)isoindoline-1,3-
dione (9). A solution of 2-ethylpiperidine (2.63 mL,
19.7 mmol) in acetonitrile (50 mL) was treated with
2-(3-bromopropyl)-isoindol-1,3-dione (4.4 g, 16 mmol)
and K2CO3 (3.4 g, 25 mmol). The resulting mixture
was heated up to 100 ꢁC and stirred for 6 h. After con-
centration, the residue was diluted with CH2Cl2
(100 mL) and washed with water and the organic layer
was dried over MgSO4, filtered, and concentrated. The
residue was purified with a 1:1 mixture of hexane and
EtOAc by column chromatography on silica gel to give
4.2.1. FDSS6000 assay. HEK293 cells which stably ex-
press both a1G and Kir2.1 subunits were grown in Dul-
becco’s modified Eagle’s medium supplemented with
10% (v/v) fetal bovine serum, penicillin (100 U/mL),
streptomycin (100 lg/mL), geneticin (500 lg/mL), and
puromycin (1 lg/mL) at 37 ꢁC in a humid atmosphere
of 5% CO2 and 95% air. Cells were seeded into 96-well
black wall clear bottom plates at a density of 4 · 104
cells/well and were used the next day for high-through-
put screening (HTS) FDSS6000 assay. For FDSS6000
assay, cells were incubated for 60 min at room tempera-
ture with 5 lM fluo3/AM and 0.001% Pluronic F-127 in
a Hepes-buffered solution composed of (in mM): 115
NaCl, 5.4 KCl, 0.8 MgCl2, 1.8 CaCl2, 20 Hepes, and
13.8 glucose (pH 7.4). During the fluorescence-based
FDSS6000 assay, a1G T-type Ca2+ channels were acti-
vated using high concentration of KCl (70 mM) in
10 mM CaCl2 contained Hepes-buffered solution and
the increase in [Ca2+]i by KCl-induced depolarization
was detected. During the whole procedure, cells were
washed using the BIO-TEK 96-well washer. All data
were collected and analyzed using FDSS6000 and relat-
ed software (Hamamatsu, Japan).
1
the desired product 9 (3.8 g, 13 mmol) in 81% yield: H
NMR (300 MHz, CDCl3) d 7.78–7.74 (m, 2H), 7.67–
7.63 (m, 2H), 3.67–3.60 (m, 2H), 2.79–7.64 (m, 2H),
2.43–2.34 (m, 1H), 2.11 (bs, 2H), 1.82–1.77 (m, 2H),
1.55–1.44 (m, 6H), 1.21–1.19 (m, 2H), 0.78 (t,
J = 7.4 Hz, 3H).
4.1.5. 3-(2-Ethylpiperidin-1-yl)propan-1-amine (10). A
solution of 2-(3-(2-ethylpiperidin-1-yl)propyl)isoindo-
line-1,3-dione 9 (3.8 g, 13 mmol) in ethanol (50 mL)
was treated with hydrazine monohydrate (2.5 mL,
50 mmol) at room temperature and the resulting mixture
was refluxed for 1 h. The reaction mixture was cooled
down to room temperature. The precipitate was filtered
off and the filtrate was concentrated. The residue was
diluted with 30 mL EtOAc and the precipitate was fil-
tered off. The filtrate was concentrated to dryness to give
4.2.2. Patch clamp assay. For the recordings of a1G
T-type Ca2+ currents, the standard whole-cell patch-
clamp method was utilized as previously described.21
Briefly, borosilicate glass electrodes with a resistance

M. N. Jo et al. / Bioorg. Med. Chem. 15 (2007) 365–373
373
H.; Yamashita, T. WO2005051402, 2005; (c) Rhim, H.; Lee,
Y. S.; Park, S. J.; Chung, B. Y.; Lee, J. Y. Bioorg. Med.
Chem. Lett. 2005, 13, 283; (d) McCalmont, W. F.; Heady, T.
N.; Patterson, J. R.; Lindenmuth, M. A.; Haverstick, D. M.;
Gray, L. S.; MacDonald, T. L. Bioorg. Med. Chem. Lett.
2004, 14, 3691; (e) Jung, H. K.; Doddareddy, M. R.; Cha, J.
H.; Rhim, H.; Cho, Y. S.; Koh, H. Y.; Jung, B. Y.; Pae, A.
N. Bioorg. Med. Chem. 2004, 12, 3965; (f) Kumar, P. P.;
Stotz, S. C.; Paramashivappa, R.; Beedle, A. M.; Zamponi,
G. W.; Rao, A. S. Mol. Pharmacol. 2002, 61, 649.
10. (a) Doddareddy, M. R.; Jung, H. K.; Cha, J. H.;
Cho, Y. S.; Koh, H. Y.; Chang, M. H.; Pae, A. N.
Bioorg. Med. Chem. 2004, 12, 1613; (b) Doddareddy,
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of 3–4 MX were pulled and filled with the internal solu-
tion contained (in mM): 130 KCl, 11 EGTA, 5 Mg-
ATP, and 10 Hepes (pH 7.4). The external solution con-
tained (in mM): 140 NaCl, 2 CaCl2, 10 Hepes, and 10
glucose (pH 7.4). a1G T-type Ca2+ currents were evoked
every 15 s by a 50 ms depolarizing voltage step from
À100 mV to À30 mV. The molar concentrations of test
compounds required to produce 50% inhibition of peak
currents (IC50) were determined from fitting raw data
into dose–response curves. The current recordings were
obtained using an EPC-9 amplifier and Pulse/Pulsefit
software program (HEKA, Germany).
Acknowledgment
11. Doddareddy, M. R.; Choo, H.; Cho, Y. S.; Rhim,
H. Y.; Koh, H. Y.; Lee, J.-H.; Jeong, S. W.; Pae,
A. N. Bioorg. Med. Chem. 2006, accepted for
publication.
This work was supported by Korea Institute of Science
and Technology (2E18970).
12. Volgel, G. C.; Drago, R. S. J. Am. Chem. Soc. 1970, 92,
5347.
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