Synthesis and SAR studies of a novel series of T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmc.2006.01.005

For the novel, potent, and selective T-type Ca²⁺ channel blockers, a series of sulfonamido-containing 3,4-dihydroquinazoline derivatives were prepared and evaluated for their blocking actions on T- and N-type Ca²⁺ channels. Among the

Full text of DOI:10.1016/j.bmc.2006.01.005

Bioorganic & Medicinal Chemistry 14 (2006) 3502–3511
Synthesis and SAR studies of a novel series of T-type
calcium channel blockers
Seong Jun Park,a,b Sung Jun Park,c Min Joo Lee,c Hyewhon Rhim,a Yoonjee Kim,a
Jung-Ha Lee,d Bong Young Chungb and Jae Yeol Leec,*
aLife Sciences Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
bDepartment of Chemistry, Korea University, 1-Anamdong, Seoul 136-701, Republic of Korea
cResearch Institute for Basic Sciences and Department of Chemistry, College of Sciences,
Kyung Hee University, 1 Hoegi-Dong, Seoul 130-701, Republic of Korea
dDepartment of Life Science, Sogang University, Shinsu-1-Dong, Seoul 121-742, Republic of Korea
Received 12 December 2005; revised 30 December 2005; accepted 4 January 2006
Available online 24 January 2006
Abstract—For the novel, potent, and selective T-type Ca2+ channel blockers, a series of sulfonamido-containing 3,4-dihydroquinaz-
oline derivatives were prepared and evaluated for their blocking actions on T- and N-type Ca2+ channels. Among them, 9c
(KYS05064, IC50 = 0.96 0.22 lM) was found to be as potent as Mibefradil and also showed the highest selectivity for T-type
Ca2+ channel with no effect on N-type Ca2+ channel.
ꢀ 2006 Published by Elsevier Ltd.
1. Introduction
channels in physiological processes was limited by two
factors: a lack of potent and selective T-type channel
blockers and a lack of information about T-type chan-
nels at the molecular level. Until recently, three genes
encoding T-type Ca2+ channel pore-forming subunits
were identified and designated Cav3.1 (a1G), Cav3.2
(a1H), and Cav3.3 (a1I).12–15 However, only limited pro-
gress has been made to date in the quest to identify both
potent and selective compounds, except Kurtoxin
(IC50 = 15 nM) and Mibefradil (IC50 = 1 lM) for T-type
channel blockade.16–18
Voltage-gated calcium channels (VGCC) mediate the
entry of calcium ions into excitable cells and are also in-
volved in a variety of calcium-dependent processes,
including muscle contraction, hormone or neurotrans-
mitter release, gene expression, cell motility, cell divi-
sion, and cell death. Voltage-gated calcium channels
fall into two major functional classes: high voltage-acti-
vated (HVA) and low voltage-activated (LVA) chan-
nels.1–4 Of them, T-type calcium channels are transient
or low voltage-activated channels and found in cardiac
myocyte membranes, the sinoatrial node, Purkinje cells
of the heart, and the central nervous system.
Our discovery and initial optimization of the screening
hit compound KYS05001 (7a, IC50 = 1.16 0.04 lM)
leading to potent compound, KYS05041 (9a,
IC50 = 0.17 0.08 lM), KYS05042 (9e, IC50 = 0.11
0.06 lM), and KYS05044 (IC50 = 0.56 0.10 lM) have
recently been disclosed as shown in Figure 1.19,20 While
both KYS05041 and KYS05042 have potent blocking
effects, they showed less selectivity for T-type channel
over N-type channel. Although KYS05044 has both
acceptable potency and selectivity for T-type channel,
in addition, it had a synthetic limitation due to the dif-
ficulty in separating the regioisomers in the course of
synthesis as reported previously.20 We thus set out to
identify new lead compounds with higher potency and
selectivity for T-type channel than KYS05041/2 via the
T-type channels are strongly associated with the genera-
tion of rhythmical firing patterns in the mammalian
CNS.5–7 Furthermore, many reports have suggested that
T-type channels are implicated in pathogenesis of epilep-
sy and neuropathic pain.8–11 In spite of many of these
researches, however, investigation of the role of T-type
Keywords: T-type calcium channel blockers; Selectivity; Potency;
3,4-Dihydroquinazoline; Sulfonamido group.
*
Corresponding author. Tel.: +82 2 9610726; fax: +82 2 9663701;
e-mail: ljy@khu.ac.kr
0968-0896/$ - see front matter ꢀ 2006 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2006.01.005

S. J. Park et al. / Bioorg. Med. Chem. 14 (2006) 3502–3511
3503
O
O
O
N
N
H3CO
O
O
H
H
S
N
N
N
N
H
N
N
R1
N
N
N
N
NH2
H
KYS05001(7a)
KYS05041(9a): R1 = CH3
KYS05042(9e): R1 = F
KYS05044
Figure 1. 3,4-Dihydroquinazoline derivatives as T-type Ca2+ channel blockers.
in 71–97% yields, which were finally coupled with the
respective phenylsulfonyl chloride in the presence of
pyridine to provide the sulfonamido-3,4-dihydroquinaz-
oline derivatives (9a–h) in 12–94% yields.
O
N
O
O
H
S
R2
R1
N
H
N
2.2. Channel blocking effects
R4
N
The in vitro calcium channel blocking activities of all
synthetic derivatives were determined in T-type channels
stably expressed in Xenopus oocytes (a1H) and HEK293
cells (a1G). As preliminary assays, all synthetic com-
pounds (100 lM) were evaluated for their inhibitory ef-
fects on a1H T-type Ca2+ channels expressed in Xenopus
oocytes by a two-electrode voltage clamp method.21 The
compounds were again re-evaluated for the blocking ef-
fects on a1G T-type Ca2+ channels expressed in HEK293
cells at 10 lM concentration by whole-cell patch-clamp
methods.22 Next, these compounds were screened
against a1B N-type Ca2+ channels (high voltage-activat-
ed Ca2+ channels) stably expressed in HEK293 cell for
the evaluation of ion channel selectivity. In vitro block-
ing effects of all compounds are summarized in Table 1,
and the previously reported compounds (7a, 7e, 8a, 9a,
and 9e) and Mibefradil were also tabulated with data
for comparison.
Figure 2. SAR study via the modifications of R1, R2, and R4
substituents.
modification of R1, R2, and R4 substituents of sulfon-
amido-containing 3,4-dihydroquinazoline as shown in
Figure 2.
2. Results and discussion
2.1. Chemistry
As shown in Scheme 1, a series of KYS05041/2 ana-
logues for SAR study were easily prepared by the same
procedure as described previously by our group.19,20
With respect to R2 position, ethyl and phenyl groups
were introduced, respectively, and the R1 position was
introduced by secondary amines such as dimethylamine
and piperidine. Finally, the group of R4 position was
retained as methyl and fluoro substituents, respectively,
as reported previously.19
Against the a1H T-type Ca2+ channel (Xenopus oocyte),
new synthetic compounds showed broad spectrum of
inhibitory activities (% inhibition in >10 to 97.2 range).
Of them, 2-piperidino-3-ethyl derivatives (9c and 9g)
containing sulfonamido groups showed improved inhib-
itory activities (92.0 and 97.2%, respectively) compared
to both Mibefradil (86%) and the parent compounds:
9a (84.7%) and 9e (80.5%). Meanwhile, some com-
pounds such as 7c, 7d, and 8d showed lower blocking ef-
fects (>10%), which result cannot be properly explained
at this stage. Next, all of compounds including the par-
ent compounds (9a and 9e) were re-evaluated in
HEK293 cells (a1G) at lower concentration (10 lM).
Compared with the primary inhibitory activity, their
profiles had a similar inhibitory trend to those against
Xenopus oocyte (a1H) with values of 34.4–92.3% inhibi-
tion range with the exception of some compounds (7b,
8b, and 8c), which had greater blocking effects (77.5–
83.8%) than expected on the primary activity data.
Among new synthetic derivatives, 9c, the second potent
compound against the a1H T-type Ca2+ channel (Xeno-
pus oocyte), was nearly equipotent (91.3 0.6% inhibi-
Carbodiimides (4a,b), an intermediate of this reaction,
were prepared in good yields by the reaction of ureas
(3a,b) with Ph3PÆBr2 and Et3N. The ureas (3a,b) were
prepared in moderate to good yields (66–96%) from
the reaction of the corresponding isocyanate with 2-
aminocinnamate, which was quantitatively prepared
through the reduction of methyl 2-nitrocinnamate (1)
with SnCl2 in ethyl acetate. The regioselective addition
of secondary amines such as piperidine and dimethyl-
amine into the carbodiimides (4a, b) followed by subse-
quent intramolecular conjugate addition to an a,b-
unsaturated ester afforded the corresponding 3,4-dihy-
droquinazoline derivatives (5a–d) in 68–88% yields.
The methyl ester group of compounds (5a–d) was hydro-
lyzed with LiOH in THF-H2O at 70 ꢁC to provide the
free carboxylic compounds (6a–d) in quantitative yields,
which were directly coupled with benzylamine by using
EDC and HOBT to give the amides 7a–h in 25–88%
yields. Hydrogenation of the nitro group of some com-
pounds (7e–h) using 10% Pd(C) in MeOH afforded N-
(4-aminobenzylamido)-3,4-dihydroquinazolines (8a–d)
tion)
inhibition), 7a (90.1 2.3% inhibition), and 7e
comparable
to
Mibefradil
(95.9 1.7%
(92.3 1.3% inhibition) against the a1G T-type Ca2+

3504
S. J. Park et al. / Bioorg. Med. Chem. 14 (2006) 3502–3511
O
O
O
O
a
b
c
OCH3
OCH3
OCH3
OCH3
NO2
NH2
NH
N C N R2
O
NH
R2
1
2
3a, R2 = Ph
3b, R2 = Et
4a-b
O
O
O
CH3O
HO
N
H
R2
R1
R2
R1
R2
R1
d
e
f
g
N
N
R3
N
for 7e-h
N
N
N
5a, R1 = piperidino
6a-d
7a-d, R3 = H
7e-h, R3 = NO2
5b, R1 = dimethylamino
5c, R1 = piperidino
5d, R1 = dimethylamino
O
N
O
N
O
O
H
H
R2
N
S
R2
h
H2N
N
N
H
R4
N
R1
N
R1
8a-d
9a-d, R4 = CH3
9e-h, R4 = F
Scheme 1. Reagents and conditions: (a) SnCl2Æ2H2O, EtOAc, 70 ꢁC; (b) R2NCO, benzene, rt; (c) Ph3PÆBr2, Et3N, CH2Cl2, 0 ꢁC; (d) R1H, THF, rt; (e)
LiOHÆH2O, THF/H2O (1:1), 60 ꢁC; (f) p-R3-BnNH2, HOBT, EDC, rt; (g) 10% Pd(C), MeOH, rt; (h) p-R4-PhSO2Cl, pyridine, 0 ꢁC to rt.
channel (HEK293 cell). With respect to the IC50 values,
however, all of the compounds not only showed a broad
range of potent efficacy (IC50 value: 0.35 0.07 to
24.16 3.48 lM range) but also were less potent than
the parent compounds (9a and 9e, IC50 = 0.17 0.08
and 0.11 0.06 lM, respectively). A few of the com-
pounds (7b, 7f, 8b, 8c, 9b, and 9f) were more than 2-fold
potent than Mibefradil as shown in Table 1. In particu-
lar, compound 9c (IC50 = 0.96 0.22 lM) was found to
be as potent as Mibefradil (IC50 = 1.34 0.49 lM).
the cultured HEK293 cells were treated with each com-
pound at a concentration of 10 and 100 lM, respectively.
At this time, the cells treated with a solvent, that is, 0.1%
DMSO, were used as a negative control and the cells
treated with H2O2 inducing cytotoxicity were used as a
positive control. As a result, most compounds showing
50% or more inhibitory effect on HEK293 cells did not
show any cytotoxicity at both concentrations but only
compound 9f showed cytotoxicity at a high concentra-
tion (100 lM). Therefore, these findings mean that com-
pound 9c would really be regarded as a new promising
lead compound for selective T-type Ca2+ channel blocker
with respect to the IC50 value and channel selectivity.
With respect to channel selectivity (T/N-type channel),
the replacement of R1 and/or R2 group with dimethyl-
amino and/or ethyl groups, respectively, in general
resulted in an improvement in selectivity as for most
compounds, except for some compounds (8d, 9b, and
9f) as shown in Table 1. However, their selectivities were
not acceptable (T/N: <10-fold) except for compounds
8b, 8c, 9c, and 9h. In addition, the parent and most po-
tent compounds (9a and 9e) also exhibited lower selec-
tivity for T-type channels by only 6.4- and 3.3-fold,
respectively. As for the selective compounds, compound
8b showed more improved selectivity (32.2-fold) for
T-type channel. In particular, compounds 9c and 9h
exerted no effect on N-type channels and thus exhibited
the specific selectivity. This result indicates that com-
pounds 9c and 9h preferentially block T-type Ca2+ chan-
nel. Meanwhile, compound 9c was equipotent with
Mibefradil, while compound 9h (KYS05071) was about
3-fold less potent than Mibefradil as for the blocking
activity against T-type Ca2+ channel. In order to analyze
cytotoxicities of all synthesized compounds in HEK293
cells, finally, MTT assay was conducted as follows:23
In conclusion, the replacement of phenyl group of R2
position in our original series (9a and 9e) of T-type
Ca2+ channel blocker with ethyl group resulted in a
new lead compound of potent and selective T-type
Ca2+ channel blocker, 9c (KYS05064), in comparison
with Mibefradil and our original compounds (9a and
9e). Encouraged by these findings, the pharmacokinetic
profiles for 9c (KYS05064) are in progress and will be
announced in the future.
3. Experimental
3.1. Chemistry
Melting points were determined with a Thomas-Hoover
capillary melting point apparatus and have not been
corrected. The IR spectra were performed on a Perkin-
Elmer 16F-PC FT-IR in KBr pellets. 1H NMR and

S. J. Park et al. / Bioorg. Med. Chem. 14 (2006) 3502–3511
Table 1. In vitro calcium channel blocking effects of 3,4-dihydroquinazolines
3505
O
O
N
H
N
H
O
O
R2
R1
S
R2
R1
R3
N
N
N
H
N
R4
N
Compounds
(library code)
R1
R2
R3
R4
Xenopus oocyte
(T-type: a1H
% Inhibition
HEK293 cells (T-type: a1G
)
HEK293 cell
Selectivity
(T/N-type)
)
(N-type: a1B
% Inhibitiona
(10 lM)
)
b
% Inhibitiona
IC50
(lM)
(100 lM)
(10 lM)
7a (KYS05001)
7b (KYS05073)
7c (KYS05074)
7d (KYS05075)
7e (KYS05034)
7f (KYS05055)
7g (KYS05062)
7h (KYS05068)
8a (KYS50040)
8b (KYS05056)
8c (KYS05063)
8d (KYS05069)
9a (KYS05041)
9b (KYS05057)
9c (KYS05064)
9d (KYS05070)
9e (KYS05042)
9f (KYS05058)
9g (KYS05065)
H
H
H
H
77.0
27.7
>10
>10
91.9
61.1
69.6
30.9
66.8
46.7
15.1
>10
84.7
30.6
92.0
81.9
80.5
84.4
97.2
90.1 2.3
81.7 0.5
59.3 1.9
34.4 1.4
92.3 1.3
87.8 2.2
79.1 0.8
61.9 3.6
43.5 4.5
83.8 1.7
77.5 2.2
38.6 4.9
89.9 1.3
71.2 4.5
91.3 0.6
81.3 0.8
89.0 2.3
73.8 3.7
89.5 2.5
1.16 0.04
0.49 0.13
4.58 2.14
24.16 3.48
2.34 0.36
0.35 0.07
0.87 0.02
2.17 0.62
13.02 1.87
0.38 0.15
0.40 0.08
12.70 2.50
0.17 0.08
0.63 0.04
0.96 0.22
1.12 0.06
0.11 0.06
0.43 0.15
1.04 0.25
28.1 1.7
8.3 0.9
3.2
9.8
N
–N(CH3)2
–CH2CH3
–CH2CH3
9.3 0.5
6.3
N
–N(CH3)2
27.6 0.5
75.3 2.4
9.5 2.6
1.2
NO2
NO2
NO2
NO2
NH2
NH2
NH2
NH2
1.2
N
–N(CH3)2
9.2
–CH2CH3
–CH2CH3
18.4 3.1
11.9 2.6
11.9 1.3
2.6 1.4
4.3
N
–N(CH3)2
5.2
3.7
N
–N(CH3)2
32.2
12.5
3.8
–CH2CH3
–CH2CH3
6.2 0.4
N
–N(CH3)2
10.2 1.0
14.1 2.1
11.3 1.3
No blockingc
8.9 1.8
CH3
CH3
CH3
CH3
F
6.4
N
–N(CH3)2
6.3
–CH2CH3
–CH2CH3
>100
9.1
N
–N(CH3)2
27.2 1.5
29.1 1.4
10.7 1.1
3.3
N
–N(CH3)2
F
2.5
–CH2CH3
–CH2CH3
F
8.4
N
9h (KYS05071)
–N(CH3)2
F
78.1
62.7 2.3
4.10 1.08
No blockingc
>100
Mibefradil
86.0
95.9 1.7
1.34 0.49
67.6 1.2
1.4
a % Inhibition value ( SE) was obtained by repeated procedures (n P 4).
b IC50 value was determined from the dose–response curve.
c No blocking means that the inhibition was less than 1%.
13C NMR spectra were recorded on a Varian Unity Plus
300 (300 MHz) spectrometer or a Bruker Avance 300
(300 MHz) spectrometer, using TMS as the internal
standard; the chemical shifts (d) are reported in parts
per million and coupling constant (J) values are given
in hertz (Hz). Signal multiplicities are represented by: s
(singlet), d (doublet), t (triplet), q (quartet), br s (broad
singlet), m (multiplet), and dd (double doublet). Low-
resolution and high-resolution mass spectra (FABMS,
positive ion mode) were obtained using a JEOL 700
mass spectrometer. The progress of all reactions was
monitored using TLC on precoated silica gel plates
(Merck Silica Gel 60 F254). The chromatograms were
viewed under UV light at 254 and 365 nm. For column

3506
S. J. Park et al. / Bioorg. Med. Chem. 14 (2006) 3502–3511
chromatography, Merck Silica Gel (230–400 mesh) was
used. Chemicals were purchased from Sigma-Aldrich,
TCI, Acros, and Fluka.
7.28 (2H, t, J = 7.8 Hz, Ph), 7.12 (1H, t, J = 7.5 Hz,
Ph), 6.97 (1H, t, J = 7.8 Hz, Ph), 6.58 (1H, d,
J = 15.3 Hz, –CH@CH-CO2Me), 3.73 (3H, s, –OCH3);
13C NMR (75 MHz, DMSO) d 167.4, 153.5, 140.5,
140.3, 138.5, 131.4, 129.5, 127.8, 126.8, 124.6, 124.4,
122.7, 119.5, 118.9, 52.2.
3.2. Methyl 2-nitrocinnamate (1)
To
a
solution of 2-nitrocinnamic acid (1.99 g,
10.3 mmol) in CH3OH (100 mL) was dropped concd
H2SO4 (0.17 mL, 3.06 mmol) at room temperature,
and the reaction mixture was stirred at reflux overnight
and then allowed to cool to room temperature. A satu-
rated solution of NaHCO3 was added, and the mixture
was extracted with dichloromethane (3·), dried
(MgSO4), filtered, and solvent evaporated in vacuo to
give the desired product as a colorless solid. Flash col-
umn chromatography (n-hexane/EtOAc = 5:1) gave
2.07 g (97%) of product (1) as a yellow solid: mp
75 ꢁC; IR (KBr) 1718, 1636, 1520, 1432, 1346, 1198,
3.4.2. Compound 3b. 66%; 1H NMR (300 MHz, DMSO)
d 8.25 (1H, s, –NH-CO–), 7.86 (1H, d, J = 15.9 Hz, –
CH@CH-CO2Me), 7.74 (2H, dd, J = 7.8 and 6.9 Hz,
Ph), 7.35 (1H, t, J = 7.7 Hz, Ph), 7.06 (1H, t,
J = 7.5 Hz, Ph), 6.55 (1H, d, J = 15.3 Hz, –CH@CH-
CO2Me), 3.75 (3H, s, –OCH3), 3.18–3.09 (2H, m,
–HN-CH2-CH3), 1.11–1.06 (3H, m, –HN-CH2-CH3);
13C NMR (75 MHz, DMSO) d 167.5, 155.9, 140.8,
139.5, 131.4, 127.7, 127.1, 123.8, 123.7, 118.9, 52.2,
34.8, 16.1.
974, 756 cmꢀ1
;
1H NMR (300 MHz, CDCl3) d 8.10
3.5. General preparation of compounds (4a–b)
(1H, d, J = 15.9 Hz, –CH@CH-CO2Me), 8.03 (1H, d,
J = 7.5 Hz, Ph), 7.70–7.63 (2H, m, Ph), 7.56 (1H, m,
Ph), 6.37 (1H, d, J = 15.9 Hz, –CH@CH-CO2Me), 3.83
(3H, s, –OCH3); 13C NMR (75 MHz, CDCl3) d 166.5,
148.6, 140.4, 133.9, 130.9, 130.7, 129.4, 125.2, 123.1,
52.3.
To a solution of urea (3a or b) (20.4 mmol) and triethyl-
amine (61.2 mmol) in CH2Cl2 (100 mL) was added di-
bromotriphenylphosphorane (30.6 mmol) at 0 ꢁC and
stirred at the same temperature for 1 h. The mixture
was extracted with dichloromethane (3·), dried
(MgSO4), filtered, and the solvent evaporated in vacuo.
Flash column chromatography (n-hexane/EtOAc) gave
product (4a or b).
3.3. Methyl 2-aminocinnamate (2)
To
a solution of 2-nitrocinnamate (1) (202 mg,
0.975 mmol) in EtOAc (20 mL) was added SnCl2Æ2H2O
(1.11 g, 4.87 mmol) at room temperature, and the solu-
tion was stirred at reflux for 1 h. The resulting mixture
was basified with NaHCO3 to pH 8–9 and filtered.
The filtrate was extracted with EtOAc (3·), dried
(MgSO4), filtered, and solvent evaporated in vacuo to
give the desired product as a colorless solid. Flash col-
umn chromatography (n-hexane/EtOAc = 5:1) gave
161 mg (93%) of product (2) as a yellow solid: mp
67 ꢁC; IR (KBr) 3365, 2364, 1704, 1622, 1330, 1198,
3.5.1. Compound 4a. 75%; mp 54 ꢁC; IR (KBr) 2142,
1
1716, 1484, 1172, 756, 59 cmꢀ1; H NMR (300 MHz,
CDCl3) d 8.18 (1H, d, J = 16.2 Hz, –CH@CH-CO2Me),
7.56 (1H, d, J = 7.8 Hz, Ph), 7.36–7.29 (3H, m, Ph), 7.25
(1H, d, J = 7.8 Hz, Ph), 7.20–7.13 (4H, m, Ph), 6.52 (1H,
d, J = 16.2 Hz, –CH@CH-CO2Me), 3.80 (3H, s,
–OCH3); 13C NMR (75 MHz, CDCl3) d 167.5, 140.5,
138.4, 138.0, 134.3, 131.3, 129.8, 129.0, 127.8, 126.1,
126.0, 125.9, 124.6, 119.6, 52.0.
1
1
3.5.2. Compound 4b. 66%; H NMR (300 MHz, CDCl3)
756 cmꢀ1; H NMR (300 MHz, CDCl3) d 7.86 (1H, d,
J = 15.9 Hz, –CH@CH-CO2Me), 7.40 (1H, d,
J = 7.5 Hz, Ph), 7.19 (1H, t, J = 7.2 Hz, Ph), 6.78 (1H,
t, J = 7.8 Hz, Ph), 6.72 (1H, d, J = 7.5 Hz, Ph), 6.38
(1H, d, J = 15.9 Hz, –CH@CH-CO2Me), 4.02 (2H, br
s, –NH2), 3.82 (3H, s, –OCH3); 13C NMR (75 MHz,
CDCl3) d 168.0, 145.9, 140.6, 131.6, 128.3, 120.1,
119.2, 117.9, 117.0, 51.9.
d 8.11 (1H, d, J = 16.5 Hz, –CH@CH-CO2Me), 7.55–
7.52 (m, 1H, Ph), 7.34–7.29 (m, 3H, Ph), 6.48 (1H, d,
J = 15.9 Hz, –CH@CH-CO2Me), 3.81 (3H, s, –OCH3),
3.49 (2H, q, J = 7.2 Hz, –N@C@N-CH2-CH3), 1.37
(3H, t, J = 7.2 Hz, –N@C@N-CH2-CH3); 13C NMR
(75 MHz, CDCl3) d 167.5, 140.7, 140.5, 130.9, 128.3,
127.5, 124.7, 118.7, 51.6, 41.8, 17.0.
3.4. General preparation of compounds (3a–b)
3.6. General preparation of compounds (5a–d)
To a solution of methyl 2-aminocinnamate (2) (6.35 g,
35.8 mmol) in benzene (150 mL) was added phenyl or
ethyl isocyanate (43.0 mmol) at room temperature, and
the solution was stirred for 12 hour. The solid residue
was washed with ether to give phenyl or ethyl substitut-
ed urea (3a, b) as a white solid.
To a solution of carbodiimide (4a, b) (2.17 mmol) in
benzene (20 mL) was added piperidine or dimethylamine
(2.60 mmol) at room temperature, and the solution was
stirred for 2 h. The mixture was extracted with dichloro-
methane (3·), dried (MgSO4), filtered, and solvent evap-
orated in vacuo. Flash column chromatography
(CH2Cl2/MeOH) gave product (5a–d).
3.4.1. Compound 3a. 96%; mp 184 ꢁC; IR (KBr) 3346,
1
3278, 1724, 1650, 1548, 1322, 1172, 758, 672 cmꢀ1; H
3.6.1. Compound 5a. 80%; mp 109 ꢁC; 1H NMR
(300 MHz, CDCl3) d 7.28–7.17 (4H, m, Ph), 7.09–7.01
(3H, m, Ph), 6.97–6.89 (2H, m, Ph), 5.10 (1H, dd,
J = 10.8 and 4.5 Hz, –CH2-CH-N–), 3.79 (3H, s,
–OCH3), 3.42 (4H, s, piperidinyl), 2.85 (1H, dd, J = 15.3
NMR (300 MHz, DMSO) d 8.94 (1H, s, –NH-CO–),
8.49 (1H, s, –NH-CO–), 7.89 (1H, d, J = 15.9 Hz,
–CH@CH-CO2Me), 7.76 (2H, d, J = 7.8 Hz, Ph), 7.46
(2H, d, J = 8.4 Hz, Ph), 7.39 (1H, t, J = 8.1 Hz, Ph),

S. J. Park et al. / Bioorg. Med. Chem. 14 (2006) 3502–3511
3507
and 10.8 Hz, –CO-CH2–), 2.52 (1H, dd, J = 15.5 and
4.7 Hz, –CO-CH2–), 1.55–1.50 (2H, m, piperidinyl),
1.43–1.40 (4H, m, piperidinyl); 13C NMR (75 MHz,
CDCl3) d 172.2, 153.2, 146.3, 144.4, 129.4, 128.6, 126.1,
124.9, 124.1, 123.1, 122.6, 122.4, 61.2, 52.0, 47.0, 39.8,
25.7, 25.0; HRMS (FAB, M+H) calcd for C22H26N3O2:
364.2025, found: 364.2019.
column chromatography (CH2Cl2/MeOH) gave product
(7a–h).
3.7.1. Compound 7a. 74%; mp 168 ꢁC; 1H NMR
(300 MHz, CDCl3) d 7.71 (1H, br s, –CO–NH-CH2-
Ph), 7.35–7.31 (2H, m, Ph), 7.29–7.19 (5H, m, Ph),
7.16–7.03 (5H, m, Ph), 6.96–6.92 (2H, m, Ph), 5.18
(1H, dd, J = 10.1 and 5.0 Hz, –CH2-CH-N–), 4.53
(1H, dd, J = 14.4 and 6.2 Hz, –NH-CH2-Ph), 4.42 (1H,
dd, J = 14.4 and 6.2 Hz, –NH-CH2-Ph), 3.17 (4H, br s,
piperidinyl), 2.68 (1H, dd, J = 14.0 and 10.1 Hz, –CO-
CH2–), 2.23 (1H, dd, J = 14.1 and 5.0 Hz, –CO-CH2–),
1.37–1.33 (2H, m, piperidinyl), 1.18 (4H, br s, piperidi-
nyl); 13C NMR (75 MHz, CDCl3) d 170.4, 153.9,
146.1, 143.2, 138.6, 129.3, 128.8, 128.5, 128.4, 127.7,
127.0, 125.2, 124.9, 124.4, 123.2, 122.8, 122.3, 61.1,
47.4, 43.9, 41.9, 25.3, 24.7; HRMS (FAB, M+H) calcd
for C28H31N4O: 439.2498, found: 439.2534.
1
3.6.2. Compound 5b. 88%: H NMR (300 MHz, CDCl3)
d 7.26–7.18 (4H, m, Ph), 7.04–6.99 (3H, m, Ph), 6.94–
6.88 (2H, m, Ph), 5.10 (1H, dd, J = 10.7 and 4.6 Hz,
–CH2-CH-N–), 3.75 (3H, s, –OCH3), 2.88 (6H, s, –N-
Me2), 2.83 (1H, dd, J = 15.1 and 10.7 Hz, –CO-CH2–),
2.50 (1H, dd, J = 15.0 and 4.5 Hz, –CO-CH2–); 13C
NMR (75 MHz, CDCl3) d 171.9, 153.5, 145.8, 144.0,
129.3, 128.3, 125.3, 124.7, 123.8, 122.8, 122.0, 121.8,
61.2, 51.8, 39.7, 37.6.
3.6.3. Compound 5c. 68%: 1H NMR (300 MHz,
CDCl3) d 7.16 (1H, m, Ph), 7.21–7.09 (2H, m, Ph),
7.02–6.91 (2H, m, Ph), 4.62 (1H, dd, J = 10.2 and
4.5 Hz, –CH2-CH-N–), 3.67 (3H, s, –OCH3), 3.36–
3.27 (5H, m, piperidinyl and –N-CH2-CH3), 3.08
(1H, m, –N-CH2-CH3), 2.61 (1H, dd, J = 14.7 and
9.9 Hz, –CO-CH2–), 2.34 (1H, dd, J = 15.1 and
4.6 Hz, –CO-CH2–), 1.63 (6H, br, piperidinyl), 1.00
(3H, t, J = 7.2 Hz, –N-CH2-CH3); 13C NMR
(75 MHz, CDCl3) d 171.7, 157.2, 144.4, 128.0, 126.3,
124.1, 122.7, 122.4, 55.3, 51.4, 47.2, 39.9, 25.8, 24.8,
14.0.
1
3.7.2. Compound 7b. 30%; H NMR (300 MHz, CDCl3)
d 7.36–7.24 (10H, m, Ph), 7.20–7.08 (4H, m, Ph), 7.00–
6.95 (1H, m, Ph), 5.24 (1H, dd, J = 10.5 and 5.0 Hz,
–CH2-CH-N–), 4.54 (1H, d, J = 5.9 Hz, –CH2-NH–),
4.49 (1H, d, J = 5.9 Hz, –CH2-NH–), 2.78 (6H, s,
–NMe2), 2.73 (1H, dd, J = 14.3 and 10.5 Hz, –CO-
CH2), 2.30 (1H, dd, J = 14.3 and 5.0 Hz, –CO-CH2–);
13C NMR (75 MHz, CDCl3) d 170.1, 154.1, 145.1,
140.9, 138.5, 129.8, 128.8, 128.7, 128.6, 127.7, 126.5,
125.1, 125.0, 123.5, 122.9, 121.8, 61.8, 44.0, 42.2, 38.8,
30.0.
1
3.6.4. Compound 5d. 68%; H NMR (300 MHz, CDCl3)
1
3.7.3. Compound 7c. 65%; H NMR (300 MHz, CDCl3)
d 7.21–7.11 (2H, m, Ph), 7.02–6.91 (2H, m, Ph), 4.61
(1H, dd, J = 10.5 and 4.8 Hz, –CH2-CH-N–), 3.67
(3H, s, –OCH3), 3.21 (1H, m, –N-CH2-CH3), 3.13 (1H,
m, –N-CH2-CH3), 2.96 (6H, s, –NMe2), 2.59 (1H, dd,
J = 14.7 and 9.9 Hz, –CO-CH2–), 2.34 (1H, dd,
J = 15.1 and 4.7 Hz, –CO-CH2–), 1.00 (3H, t,
J = 7.2 Hz, –N-CH2-CH3); 13C NMR (75 MHz, CDCl3)
d 171.5, 157.4, 144.1, 128.0, 125.7, 124.2, 122.5, 122.2,
55.5, 51.3, 46.9, 39.9, 38.4, 13.8.
d 8.07 (1H, br s, –CO-NH-CH2–), 7.36–7.25 (5H, m,
Ph), 7.11–6.82 (4H, m, Ph), 4.81 (1H, dd, J = 9.1 and
5.6 Hz, –CH2-CH-N–), 4.53 (1H, dd, J = 14.5 and
5.9 Hz, –CH2-NH–), 4.41 (1H, dd, J = 14.5 and
6.0 Hz, –CH2-NH–), 3.35–2.97 (6H, m, piperidinyl and
–N-CH2CH3), 2.33 (1H, dd, J = 14.3 and 9.1 Hz, –CO-
CH2), 2.10 (1H, dd, J = 14.3 and 5.7 Hz, –CO-CH2–),
1.43 (6H, br s, piperidinyl), 1.02 (3H, t, J = 7.2 Hz,
–N-CH2CH3); 13C NMR (75 MHz, CDCl3) d 170.1,
157.3, 142.5, 138.5, 128.5, 128.2, 128.0, 127.3, 127.1,
124.4, 123.1, 121.8, 55.3, 47.3, 43.5, 41.9, 25.7, 24.6,
14.3.
3.7. General preparation of compounds (7a–h)
To a solution of ester compound (5a–d) (0.8 mmol) in
THF (5 mL) and H2O (5 mL) was added LiOH/H2O
(4.00 mmol). The mixture was stirred at 70 ꢁC for 2 h.
The volatiles were removed in vacuo and acidified with
1 N HCl to pH 3–4. The resulting mixture was extracted
with CH2Cl2 (3·), dried (MgSO4), filtered, and solvent
evaporated in vacuo to give the intermediate (6a–d) as
a white solid. Benzylamine or 4-nitrobenzylamine
1
3.7.4. Compound 7d. 28%; H NMR (300 MHz, CDCl3)
d 8.87 (1H, br s, –CO-NH-CH2–), 7.30–7.17 (6H, m,
Ph), 6.99–6.77 (3H, m, Ph), 4.73 (1H, dd, J = 9.4 and
4.6 Hz, –CH2-CH-N–), 4.48 (1H, dd, J = 14.5 and
5.5 Hz, –CH2-NH–), 4.36 (1H, dd, J = 14.5 and
5.5 Hz, –CH2-NH–), 3.16–3.00 (2H, m, –N-CH2CH3),
2.60 (6H, s, –NMe2), 2.29 (1H, dd, J = 13.9 and
9.4 Hz, –CO-CH2–), 1.98 (1H, dd, J = 13.9 and 4.6 Hz,
–CO-CH2–), 0.91 (3H, t, J = 6.6 Hz, –N-CH2CH3); 13C
NMR (75 MHz, CDCl3) d 170.4, 157.8, 142.6, 138.9,
128.7, 128.6, 128.3, 127.6, 127.1, 124.9, 123.3, 121.8,
55.9, 47.5, 43.8, 42.1, 39.3, 14.5.
(2.543 mmol),
HOBt
(N-hydroxy-benzotriazole)
(2.543 mmol), and EDC (N-ethyl-N0-dimethylaminopro-
pyl-carbodiimide hydrochloride) (2.237 mmol) were
added to a solution of 6a–d in CH2Cl2 (15 mL) and
THF (15 mL) at 0 ꢁC. The solution was allowed to
warm to room temperature and stirred overnight. The
solvents were removed in vacuo, and the resulting mix-
ture was basified with 1 N NaOH to pH 10–11. The
organics were extracted with CH2Cl2 (3·), dried
(MgSO4), filtered, and solvent evaporated in vacuo to
give the desired product as a colorless liquid. Flash
3.7.5. Compound 7e. 80%; IR (KBr) 3192, 2932, 2848,
1668, 1552, 1486, 1430, 1344, 1282, 756 cmꢀ1 1H
;
NMR (300 MHz, CDCl3) d 8.58 (1H, br s, –CO-NH-
CH2–), 8.15 (2H, d, J = 8.7 Hz, –CH2-C4H4-NO2),
7.49 (2H, d, J = 8.1 Hz, –CH2-C6H4-NO2), 7.27–7.20

3508
S. J. Park et al. / Bioorg. Med. Chem. 14 (2006) 3502–3511
(2H, m, Ph), 7.15–7.02 (4H, m, Ph), 6.95–6.87 (3H, m,
Ph), 5.23 (1H, dd, J = 8.9 and 6.0 Hz, –CH2-CH-N–),
4.67 (1H, dd, J = 13.7 and 6.2 Hz, –CH2-NH–), 4.58
(1H, dd, 13.7 and 6.2 Hz, –CH2-NH–), 3.10 (4H, br s,
piperidinyl), 2.58 (1H, dd, J = 14.5 and 8.9 Hz, –CO-
CH2–), 2.32 (1H, dd, J = 14.5 and 6.1 Hz, –CO-CH2–),
1.35 (2H, br s, piperidinyl), 1.13 (4H, br s, piperidinyl);
13C NMR (75 MHz, CDCl3) d 170.9, 154.3, 147.4, 146.5,
145.9, 143.1, 129.5, 129.0, 128.5, 127.0, 125.4, 124.7,
124.0, 123.1, 123.0, 122.0, 60.8, 47.5, 43.2, 41.6, 25.2,
24.6; HRMS (FAB, M+H) calcd for C28H30N5O3:
484.2349, found: 484.2341.
3.8.1. Compound 8a. 97%; IR (KBr) 3218, 2930, 2850,
1648, 1550, 1480, 1430, 1350, 1282, 732 cmꢀ1 1H
;
NMR (300 MHz, CDCl3) d 7.26–7.22 (2H, m, Ph),
7.20–7.11 (4H, m, Ph), 7.07–7.02 (3H, m, Ph), 6.96–
6.90 (2H, m, Ph), 6.60–6.56 (2H, m, Ph), 6.37 (1H, br
s, –CO-NH-CH2–), 5.17 (1H, dd, J = 9.9 and 5.3 Hz,
–CH2-CH-N–), 4.32 (2H, d, J = 5.7 Hz, –CH2-NH–),
3.51 (2H, br s, –C4H4-NH2), 3.26 (4H, br s, piperidinyl),
2.57 (1H, dd, J = 14.1 and 9.9 Hz, –CO-CH2), 2.31 (1H,
dd, J = 14.1 and 5.3 Hz, –CO-CH2–), 1.43 (2H, br s,
piperidinyl), 1.26 (4H, br s, piperidinyl); 13C NMR
(75 MHz, CDCl3) d 169.7, 153.3, 145.7, 145.2, 141.0,
129.5, 129.2, 128.2, 128.1, 126.7, 124.8, 124.6, 123.2,
123.1, 121.5, 115.0, 61.2, 47.6, 43.2, 41.7, 24.8, 24.2;
HRMS (FAB, M+H) calcd for C28H32N5O: 454.2607,
found: 454.2654.
1
3.7.6. Compound 7f. 88%; H NMR (300 MHz, CDCl3)
d 8.14–8.10 (2H, m, Ph), 7.39 (2H, d, J = 8.7 Hz, Ph),
7.30–7.13 (4H, m, Ph), 7.08–7.03 (3H, m, Ph), 6.98–
6.89 (2H, m, Ph), 5.20 (1H, dd, J = 9.5 and 6.0 Hz,
–CH2-CH-N–), 4.59 (1H, d, J = 15.5 Hz, –NH-CH2–),
4.36 (1H, d, J = 15.5 Hz, –NH-CH2–), 2.78 (6H, s,
–NMe2), 2.63 (1H, dd, J = 13.8 and 9.5 Hz, –CO-
CH2–), 2.43 (1H, dd, J = 13.8 and 6.0 Hz, –CO-CH2–);
13C NMR (75 MHz, CDCl3) d 170.6, 154.4, 147.0,
145.8, 145.0, 142.7, 129.4, 128.4, 128.3, 126.3, 125.0,
124.4, 123.6, 122.7, 122.2, 121.9, 61.1, 42.7, 41.2, 37.8.
1
3.8.2. Compound 8b. 95%; H NMR (300 MHz, CDCl3)
d 7.35 (1H, t, J = 5.6 Hz, –CO-NH-CH2), 7.26–7.20 (2H,
m, Ph), 7.14–6.99 (6H, m, Ph), 6.92–6.83 (2H, m, Ph),
6.57–6.54 (2H, m, Ph), 5.20 (1H, dd, J = 9.9 and
5.1 Hz, –CH2-CH-N–), 4.41 (1H, dd, J = 14.3
and 5.6 Hz, –CH2-NH–), 4.32 (1H, dd, J = 14.3 and
5.6 Hz, –CH2-NH–), 3.67 (2H, br s, –C6H4-NH2), 2.66
(6H, s, –NMe2), 2.50 (1H, dd, J = 14.3 and 9.9 Hz,
–CO-CH2), 2.25 (1H, dd, J = 14.3 and 5.1 Hz, –CO-
CH2–); 13C NMR (75 MHz, CDCl3) d 170.0, 153.9,
145.7, 145.6, 143.1, 129.4, 129.2, 128.0, 126.2, 124.9,
123.9, 122.3, 122.2, 121.9, 115.0, 61.2, 43.3, 41.6, 37.9.
1
3.7.7. Compound 7g. 67%; H NMR (300 MHz, CDCl3)
d 9.19 (1H, t, J = 5.4 Hz, –CO-NH-CH2–), 8.11 (2H, d,
J = 8.7 Hz, –CH2-C4H4-NO2), 7.57 (2H, d, J = 9.0 Hz,
–CH2-C6H4-NO2), 7.18–7.07 (4H, m, Ph), 4.89 (1H,
dd, J = 10.7 and 4.1 Hz, –CH2-CH-N–), 4.49 (1H, dd,
J = 13.4 and 4.7 Hz, –CH2-NH–), 4.43 (1H, dd,
J = 13.4 and 4.7 Hz, –CH2-NH–), 3.48–3.20 (6H, m,
piperidinyl and –N-CH2CH3), 2.89 (1H, dd, J = 14.8
and 10.9 Hz, –CO-CH2–), 2.08 (1H, dd, J = 14.6 and
4.1 Hz, –CO-CH2–), 1.52 (6H, br s, piperidinyl), 1.07
(3H, t, J = 7.2 Hz, –N-CH2CH3); 13C NMR (75 MHz,
CDCl3) d 177.3, 170.1, 156.7, 146.3, 138.4, 128.6,
128.5, 127.2, 124.4, 124.3, 123.5, 120.2, 55.1, 47.5,
42.6, 41.2, 25.3, 24.1, 23.3, 14.2.
1
3.8.3. Compound 8c. 83%; H NMR (300 MHz, CDCl3)
d 8.24 (1H, br s, –CH2-NH-CO–), 7.08–7.03 (3H, m,
Ph), 6.97–6.84 (3H, m, Ph), 6.57 (2H, d, J = 8.1 Hz,
–CH2-C6H4-NO2), 4.78 (1H, dd, J = 9.2 and 5.6 Hz,
–CH2-CH-N–), 4.37 (1H, dd, J = 14.3 and 6.1 Hz,
–CH2-NH–), 4.24 (1H, dd, J = 14.3 and 5.6 Hz, –CH2-
NH–), 3.27 (1H, m, –N-CH2CH3), 3.18–3.01 (5H, m,
piperidinyl and –N-CH2CH3), 2.26 (1H, dd, J = 14.3
and 9.2 Hz, –CO-CH2–), 2.01 (1H, dd, J = 14.3 and
5.6 Hz, –CO-CH2–), 1.43 (6H, br s, piperidinyl), 0.98
(3H t, J = 6.9 Hz, –N-CH2CH3); 13C NMR (75 MHz,
CDCl3) d 169.5, 157.0, 145.3, 142.0, 129.0, 128.1,
127.4, 127.0, 123.9, 122.6, 121.1, 114.5, 76.4, 54.6,
46.8, 42.6, 41.3, 25.2, 24.1, 13.9.
1
3.7.8. Compound 7h. 25%; H NMR (300 MHz, CDCl3)
d 9.26 (1H, t, J = 6.0 Hz, –CO-NH-CH2–), 8.12 (2H, d,
J = 8.7 Hz, Ph), 7.62–7.55 (3H, m, Ph), 7.17–7.09 (3H,
m, Ph), 4.85 (1H, dd, J = 10.8 and 4.1 Hz, –CH2-CH-
N–), 4.49–4.46 (2H, m, –CH2-NH–), 3.28–3.22 (2H, m,
–N-CH2CH3), 3.05 (6H, s, –NMe2), 2.83 (1H, dd,
J = 14.3 and 10.9 Hz, –CO-CH2), 2.05 (1H, dd,
J = 14.4 and 4.1 Hz, –CO-CH2–), 1.06 (3H, t,
J = 7.2 Hz, –N-CH2CH3); 13C NMR (75 MHz, CDCl3)
d 169.6, 155.9 147.0 146.3, 134.3, 129.1, 128.6, 126.4,
125.7, 124.4, 123.4, 119.1, 55.9, 48.1, 42.6, 41.3, 40.6,
14.0.
1
3.8.4. Compound 8d. 71%; H NMR (300 MHz, CDCl3)
d 8.13 (1H, br s, –CO-NH-CH2–), 7.14–7.07 (4H, m,
Ph), 6.97 (2H, d, J = 4.1 Hz, Ph), 6.50 (2H, d,
J = 8.3 Hz, Ph), 4.78 (1H, dd, J = 10.7 and 4.4 Hz,
–CH2-CH-N–), 4.36 (1H, dd, J = 14.1 and 6.2 Hz,
–CH2-NH–), 4.19 (1H, dd, J = 14.1 and 5.5 Hz, –CH2-
NH–), 3.19–3.13 (2H, m, –N-CH2CH3), 2.76 (6H, s,
–NMe2), 2.41 (1H, dd, J = 14.2 and 10.9 Hz, –CO-
CH2–), 2.02 (1H, dd, J = 14.2 and 4.4 Hz, –CO-CH2–),
0.98 (3H, t, J = 7.1 Hz, –N-CH2CH3); 13C NMR
(75 MHz, CDCl3) d 170.2, 157.9, 146.0, 142.9, 129.8,
128.5, 128.3, 127.1, 124.9, 123.1, 121.9, 115.3, 55.9,
47.5, 43.4, 42.2, 39.3, 14.5.
3.8. General preparation of compounds (8a–d)
To a solution of 4-nitrobenzylcarbamoyl-substituted
3,4-dihydroquinazoline compounds (7e–h) (2.87 mmol)
in MeOH (40 mL) was added 10% Pd (C) (0.28 g), and
the mixture was stirred for 2 h under H2 atmosphere.
The resulting mixture was filtered with Celite 545 and
the solvent was evaporated in vacuo. Flash column
chromatography (CH2Cl2/MeOH) of the residue gave
product (8a–d).
3.9. General preparation of compounds (9a–h)
To a stirred solution of 4-aminobenzylcarbamoyl-substi-
tuted 3,4-dihydroquinazoline compounds (8a–d)

S. J. Park et al. / Bioorg. Med. Chem. 14 (2006) 3502–3511
3509
(0.446 mmol) in CH2Cl2(10 mL) were added pyridine
(1.34 mmol) and p-toluenesulfonyl chloride or 4-fluoro-
benzensulfonyl chloride (0.535 mmol) sequentially. The
mixture was allowed to stir at room temperature for
24 h and then H2O was added. The resulting mixture
was extracted with CH2Cl2 (3·), dried (MgSO4), filtered,
and solvent evaporated in vacuo. Flash column chroma-
tography (CH2Cl2/MeOH) gave product (9a–h).
CH2–), 2.17 (3H, s, –SO2-C6H4-CH3), 2.00 (1H, m,
–CO-CH2–), 0.89 (3H, t, J = 6.9 Hz, –N-CH2CH3); 13C
NMR (75 MHz, CDCl3) d 169.0, 155.1, 143.0, 136.3,
134.5, 132.7, 129.2, 129.0, 128.5, 127.0, 126.0, 125.7,
124.2, 120.6, 118.5, 55.6, 47.9, 42.4, 41.2, 40.3, 21.1,
13.7.
1
3.9.5. Compound 9e. 73%; H NMR (300 MHz, CDCl3)
d 7.66–7.62 (2H, m, Ph), 7.28–7.23 (2H, m, Ph), 7.20–
7.06 (9H, m, Ph), 7.04–6.90 (4H, m, Ph), 6.72 (1H, br,
–CO-NH-CH2–), 5.19 (1H, dd, J = 9.8 and 6.0 Hz,
–CH2-CH-N–), 4.41 (1H, dd, J = 14.8 and 6.1 Hz,
–Ph-CH2-NH–), 4.24 (1H, dd, J = 14.8 and 5.5 Hz,
–Ph-CH2-NH–), 3.28 (4H, br s, piperidinyl), 2.74 (1H,
dd, J = 14.1 and 9.8 Hz Hz, –CO-CH2), 2.44 (1H, dd,
J = 14.1 and 6.0 Hz, –CO-CH2–), 1.39 (2H, br s, piperid-
inyl), 1.25 (4H, br s, piperidinyl); 13C NMR (75 MHz,
CDCl3) d 170.1, 166.7, 153.9, 145.3, 141.7, 135.9,
135.0, 129.9, 129.7, 129.3, 128.9, 128.4, 126.7, 125.2,
124.8, 123.2, 121.5, 116.3, 116.0, 61.4, 47.9, 43.3, 42.0,
25.2, 24.6; HRMS (FAB, M+H) calcd for
C34H35FN5O3S: 612.2445, found: 612.2436.
1
3.9.1. Compound 9a. 94%; H NMR (300 MHz, CDCl3)
d 7.66 (1H, d, J = 8.4 Hz, Ph), 7.58–7.73 (3H, m, Ph),
7.28–7.21 (3H, m, Ph), 7.18–6.95 (12H, m, Ph), 5.19
(1H, dd, J = 10.3 and 5.2 Hz, –CH2-CH-N–), 4.35
(1H, dd, J = 14.5 and 5.8 Hz, –CH2-NH–), 4.24 (1H,
dd, J = 14.5 and 5.8 Hz, –CH2-NH–), 3.28 (4H, br s,
piperidinyl), 2.82 (1H, dd, J = 14.2 and 10.3 Hz, –CO-
CH2–), 2.36 (1H, dd, J = 14.2 and 5.2 Hz, –CO-CH2–),
2.29 (3H, s, –SO2-C6H4-CH3), 1.33 (2H, br s, piperidi-
nyl), 1.20 (4H, br s, piperidinyl); 13C NMR (75 MHz,
CDCl3) d 170.4, 154.0, 144.9, 143.6, 138.9, 136.9,
136.7, 134.7, 129.8, 129.2, 129.0, 128.9, 127.3, 126.2,
125.9, 125,4, 124.4, 124.0, 121.1, 121.0, 61.7, 48.6,
43.2, 41.9, 24.8, 24.2, 21.7; HRMS (FAB, M+H) calcd
for C35H38N5O3S: 608.2695, found: 608.2680.
1
3.9.6. Compound 9f. 38%; H NMR (300 MHz, DMSO)
d 10.3 (1H, br s, Ts-NH–), 8.56 (1H, t, J = 5.3 Hz, –CO-
NH-CH2), 7.82–7.77 (2H, m, Ph), 7.41–7.35 (2H, m,
Ph), 7.30–7.25 (2H, m, Ph), 7.19–6.98 (10H, m, Ph),
6.89 (1H, m, Ph), 5.10 (1H, dd, J = 10.5 and 3.9 Hz,
–CH2-CH-N–), 4.26 (1H, dd, J = 14.5 and 5.0 Hz,
–Ph-CH2-NH–), 4.17 (1H, dd, J = 14.4 and 5.0 Hz,
–Ph-CH2-NH–), 2.66 (6H, s, –NMe2), 2.55 (1H, m,
–CO-CH2–), 2.27 (1H, dd, J = 14.4 and 3.9 Hz, –CO-
CH2–); 13C NMR (75 MHz, DMSO) d 169.3, 165.9,
162.6, 152.9, 145.4, 136.1, 135.9, 135.2, 129.8, 129.6,
129.3, 128.6, 127.9, 126.2, 125.0, 123.7, 121.4, 120.2,
116.6, 116.3, 60.9, 41.8, 40.9, 37.5.
3.9.2. Compound 9b. 60%; 1H NMR (300 MHz, DMSO)
d 10.2 (1H, br s, Ts-NH–), 8.52 (1H, t, J = 5.6 Hz, –CO-
NH-CH2), 7.62 (2H, d, J = 8.1 Hz, Ph), 7.33–7.23 (m,
4H, Ph), 7.15–7.05 (3H, m, Ph), 7.01–6.96 (7H, m,
Ph), 6.82 (1H, m, Ph), 5.08 (1H, dd, J = 10.4 and
4.3 Hz, –CH2-CH-N–), 4.26 (1H, dd, J = 14.7
and 5.8 Hz, –NH-CH2–), 4.17 (1H, dd, J = 14.8 and
5.8 Hz, –NH-CH2–), 2.63 (6H, s, –NMe2), 2.52 (1H,
m, –CO-CH2), 2.33 (3H, s, –SO2-C6H4-CH3), 2.24
(1H, dd, J = 14.0 and 4.3 Hz, –CO-CH2–); 13C NMR
(75 MHz, DMSO) d 169.4, 152.9, 145.8, 143.9, 143.1,
136.7, 136.4, 134.8, 129.6, 129.2, 128.5, 127.7, 126.6,
126.2, 124.8, 123.2, 122.0, 121.5, 121.1, 119.7, 60.8,
41.7, 40.8, 37.2, 20.9.
3.9.7. Compound 9g. 12% ; 1H NMR (300 MHz, CDCl3)
d 8.38 (1H, br, –CO-NH-CH2–), 7.89–7.84 (2H, m, Ph),
7.56 (1H, d, J = 8.4 Hz, Ph), 7.14–7.05 (7H, m, Ph),
7.01–6.95 (2H, m, Ph), 4.94 1H, (dd, J = 10.0 and
5.2 Hz, –CH2-CH-N–), 4.30 (1H, dd, J = 14.7
and 6.1 Hz, –Ph-CH2-NH–), 4.23 (1H, dd, J = 14.6
and 6.1 Hz, –Ph-CH2-NH–), 3.36–3.22 (6H, m, piperid-
inyl and –N-CH2CH3), 2.67 (1H, dd, J = 14.3 and
9.9 Hz, –CO-CH2–), 2.24 (1H, dd, J = 14.3 and 5.1 Hz,
–CO-CH2–), 1.45 (6H, br, piperidinyl), 1.10 (3H, t,
J = 6.9 Hz, –N-CH2CH3); 13C NMR (75 MHz, CDCl3)
d 169.3, 166.5, 163.1, 155.1, 136.2, 135.4, 134.8, 133.6,
129.9, 128.8, 126.5, 125.9, 124.4, 121.1, 118.9, 116.1,
55.3, 48.2, 42.6, 41.4, 25.1, 23.5, 14.0.
1
3.9.3. Compound 9c. 38%; H NMR (300 MHz, CDCl3)
d 8.28 (1H, t, J = 6.0 Hz, –CO-NH-CH2–), 7.72 (2H, d,
J = 8.4 Hz, Ph), 7.63 (1H, d, J = 7.8 Hz, Ph), 7.27–7.02
(9H, m, Ph), 4.99 (1H, dd, J = 10.5 and 4.8 Hz, –CH2-
CH-N–), 4.22 (2H, d, J = 6.0 Hz, –Ph-CH2-NH–),
3.39–3.22 (6H, m, piperidinyl-2H2, 2H6 and –N-
CH2CH3), 2.83 (1H, dd, J = 14.2 and 10.5 Hz, –CO-
CH2–), 2.29 (3H, s, –SO2-C6H4-CH3), 2.22 (1H, dd,
J = 14.2 and 4.8 Hz, –CO-CH2–), 1.60–1.35 (6H, m,
piperidinyl-2H3, 2H4, 2H5), 1.09 (3H, t, J = 7.0 Hz,
–N-CH2CH3); 13C NMR (75 MHz, CDCl3) d 169.3,
155.2, 143.3, 136.5, 136.4, 134.5, 133.8, 129.4, 128.8,
127.2, 126.4, 125.9, 124.4, 120.8, 119.1, 77.2, 55.4,
48.2, 42.6, 41.3, 25.1, 23.5, 21.4, 14.1; HRMS (FAB,
M+H) calcd for C31H38N5O3S: 560.2695, found:
560.2720.
1
3.9.8. Compound 9h. 22%; H NMR (300 MHz, CDCl3)
d 10.0 (1H, br s, Ts-NH–), 8.18 (1H, m, –CO-NH-CH2–
), 7.91–7.79 (3H, m, Ph), 7.51 (1H, d, J = 7.8 Hz, Ph),
7.30–6.95 (8H, m, Ph), 5.02 (1H, dd, J = 10.2 and
4.5 Hz, –CH2-CH-N–), 4.30–4.26 (2H, m, –Ph-CH2-
NH–), 3.29 (2H, q, J = 7.2 Hz, –N-CH2CH3), 2.95
(6H, s, N(CH3)2), 2.86 (1H, dd, J = 14.0 and 10.2 Hz,
–CO-CH2–), 2.35 (1H, dd, J = 14.0 and 4.5 Hz, –CO-
CH2–), 1.13 (3H, t, J = 7.2 Hz, –N-CH2CH3); 13C
NMR (75 MHz, CDCl3) 169.3, 166.5, 163.1, 155.1,
136.2, 135.5, 134.8, 133.6, 130.0, 129.9, 128.8, 126.5,
1
3.9.4. Compound 9d. 55%; H NMR (300 MHz, CDCl3)
d 8.51 (1H, br s, –CO-NH-CH2–), 7.68 (2H, d,
J = 8.1 Hz, Ph), 7.56 (1H, d, J = 7.2 Hz, Ph), 7.11–6.94
(9H, m, Ph), 4.85 (1H, m, –CH2-CH-N–), 4.31–4.00
(2H, m, –CO-NH-CH2–), 3.02–3.00 (2H, m, –N-
CH2CH3), 2.73 (6H, s, –NMe2), 2.76 (1H, m, –CO-

3510
S. J. Park et al. / Bioorg. Med. Chem. 14 (2006) 3502–3511
125.9, 124.4, 121.1, 118.9, 116.1, 115.8, 56.1, 48.5, 43.0,
41.8, 40.7, 14.4; HRMS (FAB, M+H) calcd for
C27H31FN5O3S: 524.2132, found: 524.2141.
on the unfertilized oocytes whose potential was fixed
at ꢀ90 mV, and a blocking percentage was calculated
by comparing the potentials before and after the drug
treatment.
4. Biological data
4.3. Methods for culturing HEK293 cells and measuring
T- and N-type calcium channel activity using an electro-
physiological method22
4.1. Preparation of unfertilized Xenopus oocytes and
cRNA synthesis of a1H T-type calcium channel21
HEK293 cells were cultured in Dulbecco’s modified Ea-
gle’s medium (DMEM) supplemented with 10% fetal bo-
vine serum (FBS) and 1% penicillin/streptomycin (v/v) in
36.5 ꢁC humidified incubator (95% air–5% CO2). The
culture solution was replaced with a fresh medium every
3 to 4 days, and the cultured cells were subjected to sub-
culture every week. At this time, the culture solution was
treated with G-418 (0.5 mg/mL) solution so that only
HEK293 cells expressing a1G T-type calcium channel
can grow. The cells used for T-type calcium channel
activity assay were cultured on a coverslip coated with
poly-L-lysine (0.5 mg/mL) whenever sub-cultured, and
their calcium channel activity was recorded 2 to 7 days
after the cultivation. Current of the T-type calcium chan-
nel at a single cell level was measured according to an
electrophysiological whole-cell patch-clamp method
using EPC-9 amplifier (HEKA, Germany). At this time,
a cell exterior solution [140 mM NaCl, 2 mM CaCl2, and
10 mM HEPES (pH 7.4)] and a cell interior solution
[KCl 130 mM, HEPES 10 mM, EGTA 11 mM, and
MgATP 5 mM (pH 7.4)] were employed. Inward current
caused by the T-type calcium channel activation which
occurred when the cells were converted into a whole-cell
recording mode by stabbing a microglass electrode hav-
ing 3–4 MX resistance which was filled with the cell inte-
rior solution into a single cell and depolarized at ꢀ30 mV
(50 ms duration period) every 10 s with fixing membrane
potential to ꢀ100 mV was measured according to a T-
type calcium channel protocol activated at low current.
In the case of N-type, Ca2+ currents were measured in
HEK293 cells expressing a1B Ca2+ channel using a cell
exterior solution [151 mM TEACl, 5 mM BaCl2,
10 mM HEPES, 1 mM MgCl2, and 10 mM glucose (pH
7.4)] and a cell interior solution [100 mM CsCl, 1 mM
MgCl2, 10 mM HEPES, 10 mM BAPTA, 3.6 mM
MgATP, 0.1 mM GTP, 14 mM phosphocreatine and
50 U/ml creatine phosphokinase (pH 7.4)]. The currents
were evoked every 15 s by a 200 ms depolarizing voltage
step from ꢀ80 to 0 mV.
In order to express a gene encoding T-type calcium
channel a1H (Cav3.2) in unfertilized Xenopus oocytes,
vector (pGEM-HEA) was treated with restriction en-
zyme AflII to obtain a DNA fragment containing 50-ter-
minal region having the T-type calcium channel cDNA
(AF051946), and cRNA having a corresponding se-
quence to that of the fragment was synthesized using
T7 RNA polymerase according to the manufacturer’s
instruction (mMESSAGE mMACHINE kit, Ambion,
Austin, U.S.A.). The synthesized cRNA was quantified
by measuring the OD value with a spectrophotometer.
At this time, unfertilized oocytes were prepared from fe-
male Xenopus laevis (Xenopus I, U.S.A.) according to
the following method. After the frog’s abdomen was in-
cised by about 1 cm, three to four lobes were detached
therefrom with scissors and separated into small pieces
to which several oocytes attached. The small pieces were
hydrolyzed in OR solution (82.5 mM NaCl, 2.5 mM
KCl, 1 mM MgCl2, and 5 mM HEPES, pH 7.6) supple-
mented with collagenase type IA (Sigma, U.S.A.) to re-
move defolliculation. After selecting healthy oocytes
with a dissecting microscope, they were soaked in SOS
solution (100 mM NaCl, 2 mM KCl, 1.8 mM CaCl2,
1 mM MgCl2, 5 mM HEPES, 2.5 mM pyruvate, and
50 lM/mL gentamicin, pH 7.6) for 3 to 4 h to revitalize
them. 2 to 5 ng of cRNA was injected into each oocyte
using a nano-injector, and the oocytes were subjected to
test for examining the electrical properties of the channel
expressed therefrom 4 to 5 days after the injection with
maintaining at 18 ꢁC.
4.2. Examination of electrophysiological property of a1H
T-type calcium channel using a two-electrode voltage
clamping method21
Current of the calcium channel expressed from the
Xenopus unfertilized oocytes was measured according
to a two-electrode voltage clamp method. Current was
measured in 10 mM Ba2+ solution [10 mM Ba(OH)2,
90 mM NaOH, 1 mM KCl, 0.1 mM EDTA, and 5 mM
HEPES, pH was adjusted to 7.4 with methanesulfonic
acid]. At this time, voltage clamp and current measure-
ments were regulated with an amplifier for unfertilized
oocytes (Model OC-725C, Warner Instrument Corp.,
U.S.A.), analog signals were converted into digital sig-
nals using Digidata 2000A (Analog-Digital converter,
Axon Instrument), and acquisition, storage, and analy-
sis of all data were recorded in Pentium IV computer
via pCLAMP8. The data were mainly collected at
5 KHz and filtered at l KHz (Model 902 filter; Fre-
quency devices located at Harverhill, MA, U.S.A.).
The generation of T-type current was occurred by
imposing test electric potential of ꢀ20 mV every 15 s
4.4. Method for screening T-type calcium channel
blockers using an electrophysiological method
In order to confirm whether the cells and methods used
in 4.3 are a suitable screening system for selecting T-type
calcium channel blockers, the results obtained in 4.3
were compared with those of a1G T-type calcium chan-
nel study reported in a public document.22 As a result,
it has been confirmed that since the screening system
of the present invention showed: (1) the maximum acti-
vation at low voltage of ꢀ30 mV, (2) the fast activation-
inactivation of the activated current, and (3) the same
IC50 as those of Ni2+ and Mibefradil known as T-type
calcium channel blockers, it is suitable for screening

S. J. Park et al. / Bioorg. Med. Chem. 14 (2006) 3502–3511
3511
T-type calcium channel blockers. Thus, the candidate
References and notes
compounds were subjected to test for their inhibitory ef-
fects on the T-type calcium channel according to the
screening system of the present invention as follows:
each compound was dissolved in 100% dimethylsulfox-
ide (DMSO) to prepare 10 mM stock solution, and the
inhibitory effect on the T-type calcium channel current
was examined in 10 lM sample solution (containing
0.1% DMSO) prepared by diluting the stock solution
by 1000-fold. The cells were treated with each com-
pound at a concentration of 10 lM for 30–60 s with
the cell exterior solution. Then, the inhibition level of
peak current caused by the compound was calculated
as a percentage.
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This study was supported by Vision 21 Program from
Korea Institute of Science and Technology (2E18800-
05-041) and Brain Research Center of the 21st Century
Frontier Research Program (M103KV010004-03K2201-
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