Synthesis and biological activity of 3,4-dihydroquinazolines for selective T-type Ca2+ channel blockers

Article abstract of DOI:10.1016/j.bmcl.2004.10.078

We have synthesized 3,4-dihydroquinazoline derivatives for the potent and selective T-type Ca²⁺ channel blockers and evaluated for their inhibitory activities against two subtypes T-type Ca²⁺ channels and N-type Ca²⁺ chann

Full text of DOI:10.1016/j.bmcl.2004.10.078

Bioorganic & Medicinal Chemistry Letters 15 (2005) 283–286
Synthesis and biological activity of 3,4-dihydroquinazolines
for selective T-type Ca2+ channel blockers
Hyewhon Rhim,a Yong Sup Lee,b Seong Jun Park,a,c
Bong Young Chungc and Jae Yeol Leed,*
aLife Sciences Division, Korea Institute of Science & Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
bCollege of Pharmacy, Kyung Hee University, 1 Hoegi-Dong, Seoul 130-701, Republic of Korea
cDepartment of Chemistry, Korea University, 1-Anamdong, Seoul 136-701, Republic of Korea
dResearch Institute for Basic Sciences and Department of Chemistry, College of Sciences, Kyung Hee University, 1 Hoegi-Dong,
Seoul 130-701, Republic of Korea
Received 16September 2004; accepted 28 October 2004
Available online 21 November 2004
Abstract—We have synthesized 3,4-dihydroquinazoline derivatives for the potent and selective T-type Ca2+ channel blockers and
evaluated for their inhibitory activities against two subtypes T-type Ca2+ channels and N-type Ca2+ channels. Among them,
5b (KYS05044, IC50 = 0.56 0.10 lM) was identified as potent T-type Ca2+ channel blocker with in vitro selectivity profile at mean-
ingful level (T/N-type, SI = >100).
Ó 2004 Elsevier Ltd. All rights reserved.
Native calcium channels have been classified by their
electrophysiological and pharmacological properties as
T-, L-, N-, P/Q- and R-types. L-, N-, P/Q- and R-type
(or high voltage-activated) channels are activated at
more positive potentials and display diverse kinetics
and voltage-dependent properties.1–3 High voltage-acti-
vated channels are activated by relatively strong mem-
brane depolarization and permit Ca2+ influx in
response to action potentials. Consequential secondary
actions include neurotransmitter release. Thus, these
channels establish a major link between neuronal excit-
ability and synaptic transmission. For these reasons,
high voltage-activated channels have been the focus of
both acute and persistent pain transmission studies.4
T-type (or low voltage-activated) channels describe a
broad class of molecules that transiently activate at neg-
ative potentials and are highly sensitive to changes in
resting potential. T-type channels are strongly associ-
ated with the generation of rhythmical firing patterns
in the mammalian CNS.5–7 Furthermore, many reports
have suggested that T-type channels are implicated in
pathogenesis of epilepsy and neuropathic pain.8–11 In
spite of these many researches, however, investigation
of the role of T-type 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 channels 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 lim-
ited progress has been made to date in the quest to iden-
tify both potent and selective compounds except
kurtoxin (IC50 = 15 nM) and mibefradil (IC50 = 1lM)
for T-type channel blockade.16–18 Therefore, we sought
to discover a novel small molecular compound with high
potency and selectivity for T-type channels. As part of
our recent efforts, the initial lead compounds, 3,4-
dihydroquinazoline derivatives (KYS05001, KYS05041
and KYS05042) with IC50 values of sub-micromolar
concentration were identified by random screening of a
chemical library and SAR study as shown in Figure
1.19,20 Herein, we report the identification and synthesis
of 3,4-dihydroquinazoline derivatives as potent blockers
with high selectivity for T-type Ca2+ channels. Our pri-
mary goal was to optimize the potency and channel
selectivity of these compounds focusing mainly on intro-
duction of biphenyl and x-aminoalkyl groups at the 2-
or 3-position, respectively.
Keywords: T-type Ca2+ channel; Mibefradil; 3,4-Dihydroquinazolines;
Channel selectivity; Blockers.
*
Corresponding author. Tel.: +82 2 9610726; fax: +82 2 9663701;
e-mail: ljy@khu.ac.kr
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.10.078

284
H. Rhim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 283–286
O
O
OCH3
H3C
CH3
O
N
H
N
H
H
N
O
O
O
S
N
N
N
H
N
N
CH3
F
N
N
R1
N
N
KYS05001 (IC
µM)
mibefradil (IC50 = 1.34 0.49 µM)
KYS05041: R1 = CH3 (IC50 = 0.17 0.08 µM)
KYS05042: R1 = F (IC50 = 0.11 0.06 µM)
50 = 1.16 0.49
Figure 1. Structures of some T-type Ca2+ channel blockers.
The 3,4-dihydroquinazoline derivatives (5–9) containing
both biphenyl and x-aminoalkyl groups were derived
from the intermolecular aza-Wittig reaction as shown
in Scheme 1. The iminophosphorane derivative (3) was
directly prepared in 94% yield by the AppelÕs method
(PPh3–C2CI6–Et3N reagent system) from methyl 2-ami-
nocinnamate (2),21 which was derived from commer-
(5a:5b = 4.6:1) could be separated by silica gel column
chromatography (using a small amount of ammonia
solution) and their structures could be completely eluci-
1
dated by H NMR technique such as NOESY (the data
not shown here), respectively, as illustrated in Scheme 1.
Then, each x-amino group of compounds 5a and 5b was
protected with di-tert-butoxycarbonate to afford the car-
bamate 6a and 6b in 83% and 59% yield, respectively.
The hydrolysis of compounds 6a and 6b with LiOH at
70°C provided the corresponding carboxylic com-
pounds 7a and 7b in quantitative yield. Coupling of 7a
and 7b with benzylamine in the presence of EDC and
HOBT afforded the respective benzyl amides 8a and 8b
in 67% and 80% yield, respectively.22 Finally, deprotec-
tion of tert-butoxy group was carried out by 50% TFA
in CH2Cl2 to provide the corresponding 3,4-dihydro-
quinazoline derivatives (9a and 9b) containing x-amino-
alkyl and biphenyl groups in 95% and 74% yields.
cially available 2-nitrocinnamic acid (1) using
procedure earlier.19
a
In the next step, the intermolecular aza-Wittig reaction
of iminophosphorane (3) with 4-biphenylyl isocyanate
(1.5equiv) in dry benzene at room temperature for 3h,
followed by the reaction with 1,5-diaminopentane
(2.0equiv) at room temperature for 1h afforded the
3,4-dihydroquinazoline derivative (5a and 5b) as a
mixture of regioisomers in 45% yield via tandem intra-
molecular conjugate addition. Two regioisomers
O
O
O
OH
ref. 19
OMe
OMe
PPh3
PPh3, TEA, C2Cl6
NO2
NH2
N
benzene, reflux, 3 h
(94%)
1
2
3
O
OMe
1,5-diaminopentane
benzene, rt, 1 h
4-biphenylyl isocyanate
N
O
C N
benzene, rt, 3 h
(89%)
4
NHR1
O
NOESY
R2
R2
N
N
+
N
N
H
N
N
NHR1
H
5a (37%)
6a (83%)
5b (8%) ; R1 = H,
R2 = OCH3
(tert-BOC)2O
CH3CN, rt, 1 h
6b (59%); R1 = tBOC, R2 = OCH3
LiOH, THF-H2O (1:1)
70 oC, 2 h
7a (97%)
8a (67%)
9a (95%)
7b (99%); R1 = tBOC, R2 = OH
PhCH2NH2, HOBT, EDC
THF-CH2Cl2 (1:1), rt, 48 h
8b (80%); R1 = tBOC, R2 = PhCH2HN
50% TFA in CH2Cl2
0 oC to rt, 1 h
9b (74%); R1 = H,
R2 = PhCH2HN
Scheme 1. Synthesis of 3,4-dihydroquinazoline derivatives.

H. Rhim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 283–286
285
Table 1. In vitro calcium channel blocking effects of 3,4-dihydroquinazolines (5, 6, 8 and 9)
NHR1
O
O
R2
R2
N
N
N
N
H
N
N
H
NHR1
a-series
b-series
Compounds
(library code)
R1
R2
Xenopus oocyte
T-type (a1H
HEK293 cell
T-type (a1G
HEK293 cell
N-type (a1B
Selectivity
(T/N-type) at 10lM
)
)
)
% Inhibition
(100lM)
% Inhibition
(10lM)a
IC50
(lM)b
% Inhibition
(10lM)a
5a (KYS05043)
5b (KYS05044)
6a (KYS05045)
6b (KYS05046)
8a (KYS05047)
8b (KYS05048)
9a (KYS05049)
9b (KYS05050)
Mibefradil
H
CH3O
CH3O
CH3O
CH3O
94.6840.130 1.06.09
7.5 0.7
82.5 0.7
88.5 0.4
86.5 0.5
11.2
H
97.0
96.5
92.2
91.2
84.2
97.0
80.1
86.0
0.56 0.10
0.37 0.08
0.68 0.18
No blockingc
94.9 1.7
98.6 1.3
>100
0.9
0.9
tBOC
tBOC
tBOC
tBOC
H
PhCH2NH
PhCH2NH
PhCH2NH
PhCH2NH
88.5 0.60.17 0.03
30.1 1.1
2.9
88.3 1.5
91.8 1.9
83.8 1.4
95.9 1.7
0.16 0.02
16.6 0.7
15.2 2.4
8.3 1.8
67.6 1.2
5.3
6.0
10.1
1.4
0.14 0.01
0.13 0.01
1.34 0.49
H
a % Inhibition value ( SE) was obtained by repeated procedures (n P 4).
b IC50 value was determined from the dose–response curve.
c ÔNo blockingÕ means that the inhibition was less than 1%.
The in vitro calcium channel blocking activities of each
pair of 3,4-dihydroquinazoline derivatives were deter-
mined in T-type channels stably expressed in Xenopus
oocytes (a1H) and HEK293 cells (a1G). As preliminary
assays, all synthetic compounds (100lM) were evalu-
ated for their inhibitory effects on a1H T-type Ca2+ chan-
nels expressed in Xenopus oocytes by a two-electrode
voltage clamp method.23 The compounds were again
re-evaluated for the blocking effects on a1G T-type
Ca2+ channels expressed in HEK293 cells at 10M con-
centration by whole-cells patch clamp methods.24 The
molar concentrations (IC50) of test compounds required
to produce 50% inhibition of a1G T-type currents were
determined from fitting raw data into dose–response
curves. In vitro blocking effects of all pairs of 3,4-
dihydroquinazoline derivatives except compounds 7a
and 7b are summarized in Table 1.
lows. First, their profiles had a similar inhibitory trend
to those against Xenopus oocyte (a1H) with values of
82.5–91.8% inhibition range. Second, the compound 9a
(KYS05049), the most potent compound against the
a1H T-type Ca2+ channel (Xenopus oocyte) was nearly
equipotent (91.8 1.9% inhibition) comparable to mibe-
fradil (95.9 1.7% inhibition) against the a1G T-type
Ca2+ channel (HEK293 cell). With respect to the IC50
values, however, all pairs of compounds showed more
potent efficacy (IC50 value 0.13 0.01 to 0.68 0.18lM
range) than mibefradil (IC50 = 1.34 0.49lM) and also
each pair showed similar IC50 values against the a1G T-
type Ca2+ channel (HEK293 cell). Compared to mibefra-
dil, one pair of compounds 9a and 9b in this series was
most potent (IC50 = 0.14 0.01 and 0.13 0.01lM)
showing a 10-fold increase in potency, as well as the other
pair of compounds 8a and 8b (IC50 = 0.17 0.03 and
0.16 0.02 lM), as shown in Table 1. The above two
pairs of compounds were shown to possess the common
benzyl amide group at 4-position, which consistent with
our previous research result.19,20
Against the a1H T-type Ca2+ channel (Xenopus oocyte),
most pairs of compounds showed high inhibitory activ-
ities (% inhibition in 80.1–97.0 range) except carboxylic
acid compound pair 7a (37.4%) and 7b (77.3%) com-
pared to mibefradil (86%), a reference compound. This
inhibitory trend was very interesting compared with
our initial work, which showed that compounds having
a benzyl amide group at 4-position of parent ring gener-
ally showed more potent efficacies than compounds pos-
sessing a simple ester.19 This biological result implies
that the present series of compounds may have a differ-
ent binding pattern on a1H T-type channels from that of
our initial compound.
Next, these compounds were screened against a1B N-
type Ca2+ channels (high voltage-activated Ca2+ chan-
nels) stably expressed in HEK293 cells for the evalua-
tion of ion channel selectivity. The inhibitory activity
data of these compounds were also summarized in Table
1. Under our assay condition, mibefradil showed lower
selectivity for a1G T-type channel (T/N-type chan-
nel = 1.4), which is consistent with the recent reported
studies.25 As shown in Table 1, most pairs of com-
pounds, except compounds 5a and 5b, showed similar
and less inhibitory activities against a1B N-type Ca2+
channels, although there is about a twofold difference
with respect to the blocking effect of each other. Com-
pounds 6a (94.9 1.7% inhibition) and 6b
Next, all pairs of compounds, except compounds 7a and
7b, were re-evaluated in HEK293 cells (a1G) at lower con-
centration (10lM). Together with the primary inhibitory
activity, our experimental results are summarized as fol-

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H. Rhim et al. / Bioorg. Med. Chem. Lett. 15 (2005) 283–286
5. Llinas, R.; Yarom, Y. J. Physiol. 1981, 315, 549.
6. Gutnick, M. J.; Yarom, Y. J. Neurosci. Methods 1989, 28,
93.
7. Bal, T.; McCormick, D. A. J. Physiol. 1993, 468, 669.
8. Huguenard, J. R.; Prince, D. A. J. Neurosci. 1994, 14,
5485.
9. Huguenard, J. R. Annu. Rev. Physiol. 1996, 58, 329.
10. Tsakiridou, E.; Bertollini, L.; De Curtis, M.; Avanzini, G.;
Pape, H. Neuroscience 1995, 15, 3110.
11. Hogan, Q. H.; McCallum, J. B.; Sarantopoulos, C.;
Aason, M.; Mynlieff, M.; Kwok, W. M.; Bosnjak, Z. J.
Pain 2000, 86, 43.
12. Cribbs, L. L.; Lee, J.-H.; Yang, J.; Satin, J.; Zhang, Y.;
Daud, A.; Barclay, J.; Williamson, M. P.; Fox, M.; Rees,
M.; Perez-Reyes, E. Circ. Res. 1998, 83, 103.
13. Lee, J.-H.; Daud, A. N.; Cribbs, L. L.; Lacerda, A. E.;
Pereverzev, A.; Klo¨ckner, U.; Schneider, T.; Perez-Reyes,
E. J. Neurosci. 1999, 19, 1912.
14. Perez-Reyes, E.; Cribbs, L. L.; Daud, A.; Lacerda, A. E.;
Barclay, J.; Williamson, M. P.; Fox, M.; Ress, M.; Lee,
J.-H. Nature 1998, 391, 896.
15. Randall, A.; Benham, C. D. Mol. Cell. Neurosci. 1999, 14,
255.
16. Chuang, R. S.; Jaffe, H.; Cribbs, L.; Perez-Reyes, E.;
Swartz, K. J. Nat. Neurosci. 1998, 1, 668.
(98.6 1.3% inhibition) in these pairs showed more
potent efficacy against N-type than T-type channels
and thus less selectivity for T-type channels
(T/N = 0.9-fold, respectively). Meanwhile, compound
9a (KYS05049), the most potent compound against
two isoforms of T-type Ca2+ channel sub-family,
showed low selectivity over N-type channels by only
6.0-fold. Also, compounds 9b (KYS05050), 8a and 8b,
the most potent compounds against T-type channels,
did not show the favorable selectivity for T-type chan-
nels by 10.1-, 2.9- and 5.3-fold, respectively. However,
compound 5b (KYS05044) possessing x-aminoalkyl
group at 3-position and methyl ester group at 4-position
blocked little the current of N-type channels (less than
1%) and thus exhibited the highest selectivity for T-type
over N-type channel (T/N = >100), even though com-
pound 5b (IC50 = 0.56 0.10 lM) was more potent than
mibefradil (IC50 = 1.34 0.49lM) by only 2.4-fold.
More importantly, all compounds, including compound
5b, had no cytotoxicity on HEK293 cells at 10lM con-
centration as confirmed using MTT assay method (data
not shown). Therefore, it is likely that compound 5b
would really be regarded as a new promising lead com-
pound for selective T-type Ca2+ channel blocker with re-
spect to the IC50 value and channel selectivity.26
17. Clozel, J. P.; Ertel, E. A.; Ertel, S. I. J. Hypertens.
1997(Suppl. 15), S17.
18. Mishra, S. K.; Hermsmeyer, K. Circ. Res. 1994, 75,
144.
In conclusion, new series of 3,4-dihydroquinazoline
derivatives containing both biphenyl and x-aminoalkyl
groups as a continuous research for a novel selective
T-type Ca2+ channel blocker were prepared and evalu-
ated for the blocking effects against two isoforms of T-
type Ca2+ channel subfamily and N-type Ca2+ channel.
In vitro results demonstrated that compound 5b in this
series exhibited both favorable potency and highest
selectivity for T-type Ca2+ channel in comparison with
mibefradil. Therefore, the discovery of compound 5b
(KYS05044), a novel and selective T-type Ca2+ channel
blocker, is expected to provide impetus for the develop-
ment of new T-type Ca2+ channel drugs as well as the re-
search in the field of electrophysiology.26 Encouraged by
these findings, further evaluation of pharmacokinetics
profiles and neuroprotective properties are in progress.
19. Lee, Y. S.; Lee, B. H.; Park, S. J.; Kang, S. B.; Rhim, H.;
Park, J.-Y.; Lee, J.-H.; Jeong, S.-W.; Lee, J. Y. Bioorg.
Med. Chem. Lett. 2004, 13, 3379–3384.
20. Doddareddy, M. R.; Jung, H. K.; Lee, J. Y.; Lee, Y. S.;
Cho, Y. S.; Koh, H. Y.; Pae, A. N. Bioorg. Med. Chem.
2004, 12, 1605–1611.
21. (a) Appel, R.; Halstenberg, M. In Organophosphorus
Reagents its Organic Synthesis; Cadogan, J. I. G., Ed.;
Academic Press: London, 1979; p 378ff; (b) Okawa, T.;
Osakada, N.; Eguchi, S.; Kakehi, A. Tetrahedron 1997, 53,
16061–16082.
22. (a) Gaucher, A.; Zuliani, Y.; Cabaret, D.; Wakselman, M.;
Mazaleyrat, J.-P. Tetrahedron: Asymmetry 2001, 12, 2571;
(b) Dhaon, M. K.; Olesen, R. K.; Ramasamy, K. J. Org.
Chem. 1982, 47, 1962.
23. Lee, J.-H.; Gomora, J. C.; Cribbs, L. L.; Perez-Reyes, E.
J. Biophys. 1999, 77, 3034.
24. Monteil, A.; Chemin, J.; Bourinet, E.; Mennessier, G.;
Lory, P.; Nargeot, J. J. Biol. Chem. 2000, 275, 6090.
25. (a) Randall, A. D.; Tsien, R. W. Neuropharmacology 1997,
36, 879–893; (b) Randall, A. D. J. Physiol. 1995, 485, 49P;
(c) Gomora, J. C.; Enyeart, J. A.; Enyeart, J. J. Mol.
Pharmacol. 1999, 56, 1192; (d) Nilius, B.; Prenen, J.;
Kamouchi, M.; Viana, F.; Voets, T.; Droogmans, G. Br.
J. Pharmacol. 1997, 121, 547.
Acknowledgements
This study was supported by Vision 21 Program from
Korea Institute of Science and Technology (2E18000-
04-024).
26. Spectral data of the selected compound: 5b (KYS05044):
1H NMR (300MHz, CDCl3) d 7.58–6.91 (13H, m, Ph),
5.10 (1H, t, J = 7.1Hz, –CO–CH2–CH–N–), 3.66 (3H, s,
CH3O–), 3.46–3.30 (2H, m, –N–CH2–CH2–), 2.87 (1H, dd,
J = 7.1, 14.9Hz, –CO–CH–CH–N–), 2.74–2.65 (3H, m,
–CO–CH–CH–N– and –CH2–CH2–NH2), 1.78–1.27 (6H,
m, –CH2–C3H6–CH2–); 13C NMR (75MHz, CDCl3) d
171.5, 147.1, 141.1, 137.0, 135.4, 129.1, 128.9, 128.5, 127.0,
126.9, 125.7, 123.3, 122.3, 115.1, 56.2, 52.1, 47.8, 41.7,
39.1, 32.3, 28.3, 25.0, 24.1; ES-MS (m/z, M + 1) 457;
HRMS (FAB, M + 1) calcd for C28H33N4O2 457.2604,
found 457.2608.
References and notes
1. Cox, B.; Denyer, J. C. Exp. Opin. Ther. Patents 1998, 8,
1237–1250.
2. Williams, M.; Kowaluk, E. A.; Arneric, S. P. J. Med.
Chem. 1999, 42, 1481–1500.
3. Tsien, R. W.; Lipscombe, D.; Madison, D.; Bley, K.; Fox,
M. Trends Neurosci. 1995, 18, 52–54.
4. Matthews, E. A.; Dickenson, A. H. Eur. J. Pharmacol.
2001, 415, 141–149.