Synthesis and biological evaluation of oxazole derivatives as T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2010.05.030

T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neuropathic pain. In this study, as a part of our ongoing efforts to develop potent T-type calcium channel blockers, we designed oxazole derivatives subs

Full text of DOI:10.1016/j.bmcl.2010.05.030

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4219–4222
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of oxazole derivatives as T-type calcium
channel blockers
Jie Eun Lee a,b, Hun Yeong Koh b, Seon Hee Seo a, Yi Yeon Baek a, Hyewhon Rhim a, Yong Seo Cho a,
Hyunah Choo a, , , Ae Nim Pae a, ,
*
*
a Life/Health Division, Korea Institute of Science and Technology, PO Box 131, Cheongryang, Seoul 130-650, Republic of Korea
b Department of Chemistry, Inha University, Nam-gu, Incheon 402-751, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
T-type calcium channel is one of therapeutic targets for the treatment of cardiovascular diseases and neu-
ropathic pain. In this study, as a part of our ongoing efforts to develop potent T-type calcium channel
blockers, we designed oxazole derivatives substituted with arylpiperazinylalkylamines. The oxazoles
were synthesized in a convenient convergent synthetic method, and biologically evaluated against a1G
(CaV3.1) T-type calcium channel. Among total 41 oxazole compounds synthesized, the most active one
Received 16 March 2010
Revised 11 May 2010
Accepted 12 May 2010
Available online 15 May 2010
was the compound 10-35 with an IC50 value of 0.65
l
M, which is comparable with that of mibefradil.
Keywords:
T-type calcium channel
HTS assay
Ó 2010 Elsevier Ltd. All rights reserved.
a1G Subtype
Neuropathic pain
Oxazole
Calcium is an essential signaling molecule with fundamental
physiological roles that range from activation of calcium-depen-
dent enzymes to the secretion of neurotransmitters and hor-
mones.1,2 The most abundant route of calcium entry into
electrically excitable cells is by opening of voltage-gated calcium
channels.1,2 The highest organisms express several different types
of voltage-gated calcium channels, which are grossly divided into
two categories: high-voltage activated (HVA) and low-voltage acti-
vated (LVA) calcium channels.3 Members of the HVA channel fam-
ily include the N-, P-, Q-, L-, and R-types and typically require large
membrane depolarizations to open.3 LVA calcium channels, also
known as T-types, are widely expressed in various types of neurons
and can be activated by a weak depolarization near the resting
membrane potential and regulate the excitability and electrore-
sponsiveness of neurons under physiological conditions near rest-
ing states.3
reported that T-type calcium channels play crucial roles in the con-
trol of pain which are caused by hyperexcitable neurons.7 The role
of T-type calcium channels in pain has been addressed using spe-
cific genetic modulation of T-type calcium channel isoforms. Com-
pared with wild-type mice, CaV3.1 knockout (a1GÀ/À) mice were
observed, in which, after L5 spinal nerve ligation, spontaneous pain
responses were reduced and a threshold for paw withdrawal was
increased in response to mechanical stimulation.8 CaV3.2 antisense
treatment resulted in major anti-nociceptive and anti-hyperalgesic
effect, suggesting that CaV3.2 plays a major pronociceptive role in
acute and chronic pain states.9 Together, the results of these two
studies suggest that blocking T-type calcium channels should re-
duce nociceptive pain and neuropathic pain. T-type calcium chan-
nel blockers such as ethosuximide and mibefradil were reported to
reverse neuropathic pain in animal models.10
Since mibefradil, a selective T-type calcium channel blocker,
approved for the treatment of angina pectoris and hypertension
(Fig. 1), was withdrawn from the market in 1998 due to unfavor-
able drug–drug interaction,4,5 there have been efforts to discover
novel T-type calcium channel blockers.6 According to accumula-
tion of new findings on T-type calcium channels, it has been
O
O
O
F
N
N
* Corresponding authors. Tel.: +82 2 958 5157; fax: +82 2 958 5189 (H.C.); tel.:
+82 2 958 5185; fax: +82 2 958 5189 (A.N.P.).
HN
E-mail addresses: hchoo@kist.re.kr (H. Choo), anpae@kist.re.kr (A.N. Pae).
These authors contributed equally to this work.
Figure 1. Structure of mibefradil.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.05.030

4220
J. E. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4219–4222
Previously, we reported isoxazole derivatives substituted with
activities of oxazole derivatives with arylpiperazinylalkylamines
substituents.
arylpiperazinylalkylamines as T-type calcium channel blockers.6e
Their activities depended on the substituents on isoxazoles, aryl
moieties of arylpiperazine, and alkyl chains. In this study, to obtain
better pharmacological activities against T-type calcium channel,
we introduced an oxazole group instead of the isoxazole group
and more diverse aryl moieties at arylpiperazines. By using an oxa-
zole scaffold, structurally unaccessible positions of the isoxazole
ring can be substituted with various functional groups, which
would allow extensive structure–activity relationship study. Here-
in, we report the synthesis and T-type calcium channel inhibitory
The strategy to synthesize the designed oxazole derivatives is a
convergent synthesis, where the separately prepared aryl-
piperazinylalkylamine part (Scheme 1) and the oxazole part
(Scheme 2) were combined through reductive amination to afford
the desired oxazole derivatives (Scheme 3).
Arylpiperazinylalkylamines 4 were synthesized starting from
arylpiperazines 1 in two steps. Various arylpiperazines underwent
alkylation reaction with N-(2-bromoethyl)phthalimide (n = 1) or
N-(3-bromopropyl)phthalimide (n = 2) to afford compounds 3 in
O
R1
R1
Br
K2CO3
N
N
N
O
+
n
NH
CH3CN, reflux
40~80%
N
N
O
n
1
2
3
O
H2NNH2 .H2O
EtOH, reflux
>90%
R1
N
N
NH2
n
4
Scheme 1. Synthesis of arylpiperazinylalkylamines.
O
O
O
O
O
H2N R2
N
O
HDNIB
R2
OEt
OEt
ODNs
EtO
CH3CN, reflux
2days
CH3CN, reflux 1hr
O
or microwave 80W
30s, 45~48%
2
5
6
7a
R = methyl
7b R2 = propyl
DIBAL-H
CH2Cl2, -78oC
91~95%
OH
I
NO2
O
O
S
O
HDNIB =
NO2
DMSO, (COCl)2, TEA
N
N
CH2Cl2, -78oC
44~93%
R2
R2
O
HO
O
O
9a R2 = methyl
8a R2 = methyl
2
2
9b
8b
R = propyl
R = propyl
Scheme 2. Synthesis of oxazole parts.
R1
N
H
N
i) NaBH(OAc)3
CH2Cl2
N
R
N
+
R2
2
O
HCl
N
N
n
O
NH2
O
n
N
ii) HCl etherate
11~81%
4
9
10
R1
Scheme 3. Convergent synthesis of desired oxazole derivatives 10.

J. E. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4219–4222
4221
Theoxazoleparts, oxazolealdehydes 9, weresynthesizedstarting
from ethyl benzoylacetate 5 in four steps. The benzoylacetate 5 was
treated with hydroxyl(2,4-dinitrobenzensulfonyloxy)iodobenzene
(HDNIB), which was easily prepared from diacetoxyiodobenzene
and 2,4-dinitrobenzensulfonic acid, to afford an intermediate 6 in-
stalled with a good leaving group, 2,4-dinitrobenzenesulfonate
(ODNs) (Scheme 2).11 The intermediate 6 was condensed with acet-
amide in acetonitrile under reflux conditions to give one oxazole 7a
with R2 as methyl group in 45% yield, while reaction with butyra-
mide under microwave irradiation afforded the other oxazole 7b
with R2 as propyl group in 48% yield. The oxazoles 7 underwent
reductionbytreatingwithDIBAL-Hto affordthecorresponding alco-
hols 8a and 8b in 91–95% yields. Swern oxidation of the alcohols 8
provided the corresponding aldehydes 9a and 9b in 44–93% yields.
N
H
N
R2
O
HCl
N
N
11-1 R2 = propyl
2
11-2
R = methyl
Figure 2. Benzhydrylpiperazine derivatives 11.
40–80% yields. The compounds 3 were converted to the corre-
sponding amines 4 by treatment with hydrazine in refluxing EtOH
in over 90% yields (Scheme 1).
Table 1
Inhibitory activities of oxazole derivatives 10 and 11 against a1G (CaV3.1) T-type calcium channel
N
H
N
R2
N
O
N
H
N
R2
HCl
N
O
N
n
11
HCl
N
R1
10
Entry
Compd
R1
H
R2
%Inhbition at 10
lM
IC50 in lM
1
2
10-1
10-2
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Propyl
Methyl
Propyl
Methyl
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
—
—
54.26
46.49
62.34
54.08
60.07
51.24
55.84
51.34
43.13
48.06
39.73
40.17
51.49
54.62
56.30
58.70
66.68
64.28
67.87
48.13
43.69
54.41
68.94
62.53
64.72
65.88
63.25
54.45
47.78
53.92
41.03
36.35
42.13
44.99
51.63
51.62
55.24
54.59
38.08
71.85
57.32
80.00
10.25 0.91
b
2-F
—
3
4
5
6
7
8
9
10-3
10-4
10-5
10-6
10-7
10-8
10-9
3-F
4-F
2-Cl
3-Cl
3.35 0.26
7.08 0.29
4.37 0.30
1.45 0.9b
2.82 0.04
4.30 0.41
4-Cl
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2-CF3
3-CF3
4-CF3
2,3-DiCH3
2,4-DiCH3
3,4-DiCH3
2-Pyridyla
H
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
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
10-10
10-11
10-12
10-13
10-14
10-15
10-16
10-17
10-18
10-19
10-20
10-21
10-22
10-23
10-24
10-25
10-26
10-27
10-28
10-29
10-30
10-31
10-32
10-33
10-34
10-35
10-36
10-37
10-38
10-39
11-1
b
b
9.20 0.46
2.10 0.09
1.94 0.12
1.30 0.08
1.71 0.16
2.22 0.11
2.22 0.27
b
—
—
b
16.13 3.48
3.03 0.22
3.21 0.20
2.80 0.39
1.75 0.04
1.31 0.05
8.75 5.4
2-CH3
3-CH3
4-CH3
2-OCH3
3-OCH3
4-OCH3
2-CF3
1.19 0.6
130.01 10.4
b
—
—
—
—
b
b
b
3-CF3
4-CF3
0.65 0.3
7.25 3.7
1.78 0.14
3.02 0.18
2,3-DiCH3
3,4-DiCH3
4-Cl
b
—
b
b
b
—
—
7.47 1.69
b
b
11-2
—
Mibefradil
0.83 0.19
a
Pyridyl instead of R1-phenyl.
Not available or determined.
b

4222
J. E. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4219–4222
Arylpiperazinylalkylamines 4 and the oxazole aldehydes 9 were
inhibitory activity against T-type calcium channel. In this oxazole
series, the most active compound 10-35 also has R1 substituent
as 3-CF3. For this reason, it is fair to assume that the aromatic
meta-trifluoromethyl substituent may play an important role in
inhibitory activity of oxazoles as well as isoxazole derivatives.
In summary, oxazole derivatives with arylpiperazinylalkylam-
ines were designed, synthesized, and biologically evaluated against
combined by reductive amination to afford compounds 10 in 11–
81% yields (Scheme 3). Instead of R1-substituted phenyl, benzhy-
dryl group was also introduced with as same procedures as shown
here to afford compounds 11 in Figure 2.
A total of 41 oxazole derivatives 10 and 11 were biologically
evaluated against a1G (CaV3.1) T-type calcium channel in HEK293
cells which stably express both T-type calcium channel CaV3.1
and potassium channel Kir2.1.12 All the synthesized compounds
were screened by fluorescence-based high-throughput screening
(HTS) FDSS6000 assay,13 and the %-inhibitions of Ca2+ current mea-
a1G (CaV3.1) T-type calcium channel. Among total 41 oxazole com-
pounds synthesized, the most active one was the compound 10-35
with an IC50 value of 0.65 lM, which is comparable to that of mibe-
fradil. The compound 10-35 possesses 3-CF3 as R1 substituent,
which correlates well to the results of previous isoxazole series.
Further evaluations of the compound 10-35 such as selectivity
for other calcium channels, pharmacokinetics and neuronal anal-
gesic effect are in progress.
sured at 10 lM concentration of the oxazole derivatives are sum-
marized in Table 1. Among those, compounds with over 50%
inhibition of Ca2+ current in FDSS assay were selected for patch-
clamp assays using single cells to evaluate IC50 values.14,15
In general, the %-inhibition results from the fluorescence-based
HTS FDSS6000 assay do not directly correlate with the IC50 values
obtained from patch-clamp assays. In this study, we also observed
that the two compounds 10-23 and 10-19 with the highest %-inhi-
bitions show inhibitory activities with moderate IC50 values of
Acknowledgement
This work was supported by Korea Institute of Science and
Technology.
3.03 lM and 2.22 lM, respectively. On the other hand, two com-
pounds 10-35 and 10-16 with the most potent IC50 values have
low inhibition of T-type calcium channel activity (51.63% and
58.70% inhibitions, respectively). Nevertheless, the filtering pro-
cess using FDSS6000 assay is necessary because compounds active
in both fluorescence-based and electrophysiological methods
could have more possibility to be eventually effective in in vivo
system. The %-inhibition results were used as primary screening
tool to filter compounds for further laborious patch-clamp assays.
A total of 28 compounds were selected to obtain IC50 values
against T-type calcium channel, especially a1G subtype. The IC50
References and notes
1. (a) Clapham, D. E. Cell 2007, 131, 1047; (b) Perez-Reyes, E. Physiol. Rev. 2003, 83,
117.
2. Iftinca, M. C.; Zamponi, G. W. Trends Pharmacol. Sci. 2009, 30, 32.
3. Catterall, W. A.; Goldin, A. L.; Waxman, S. G. Pharmacol. Rev. 2005, 57, 411.
4. (a) McDonough, S. I.; Bean, B. P. Mol. Pharmacol. 1998, 54, 1080; (b) Mishra, S.
K.; Hermsmeyer, K. Circ. Res. 1994, 75, 144.
5. SoRelle, R. Circulation 1998, 98, 831.
6. (a) Park, S. J.; Park, S. J.; Lee, M. J.; Rhim, H.; Kim, Y.; Lee, J.-H.; Chung, B. Y.; Lee,
J. Y. Bioorg. Med. Chem. 2006, 14, 3502; b Furukawa, T.; Yamada, O.; Matsumoto,
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; (g) Oh, Y.; Kim, Y.; Seo, S. H.; Lee, J. K.; Rhim, H.; Pae, A. N.; Jeong,
K. S.; Choo, H.; Cho, Y. S. Bull. Korean Chem. Soc. 2008, 29, 1; see a review: (h)
Lory, P.; Chemin, J. Expert Opin. Ther. Targets 2007, 11, 717.
values are between 0.65 lM and 130.01 lM as shown in Table 1.
There is no huge difference in inhibitory activities according to
the chain length (n = 1 or 2). However, the electronic nature of
the R1 substituents exerted subtle effects on the activities of the
compounds. The compounds with electron-withdrawing groups
such as F, Cl, and CF3 are more potent than those with electron-
donating groups such as OCH3. The position of the substituent also
affects the biological activity. Generally, oxazole analogues with
electron-withdrawing R1 substituents at meta position show good
inhibitory activities against T-type calcium channel. The com-
pound 10-3 with meta-fluoro (3-F) substituent shows better inhib-
itory activity than 10-2 and 10-4 with ortho- and para-fluoro
groups, respectively. By the same token, compounds 10-6, 10-23,
and 10-35 are more potent than the corresponding regioisomers.
Among those, the compound 10-35 with meta-CF3 (3-CF3) shows
7. Flatters, S. J. L. Drugs Future 2005, 30, 573.
8. Na, H. S.; Choi, S.; Kim, J.; Park, J.; Shin, H.-S. Mol. Cells 2008, 25, 242.
9. Bourinet, E.; Alloui, A.; Monteil, A.; Barrère, C.; Couette, B.; Poirot, O.; Pages, A.;
McRory, J.; Snutch, T. P.; Eschalier, A.; Nargeot, J. EMBO J. 2005, 24, 315.
10. (a) Dogrul, A.; Gardel, L. R.; Ossipov, M. H.; Tulunay, F. C.; Lai, J.; Porreca, F. Pain
2003, 105, 159; (b) Flatters, S. J. L.; Bennett, G. J. Pain 2004, 109, 150.
11. Lee, J. C.; Choi, H. J.; Lee, Y. C. Tetrahedron Lett. 2003, 44, 123.
12. Kim, T.; Choi, J.; Kim, S.; Kwon, O.; Nah, S. Y.; Han, Y. S.; Rhim, H. Biochem.
Biophys. Res. Commun. 2004, 324, 401.
13. Kim, Y.; Seo, S.; Kim, D.; Rhim, H. The 11th Annual Conference & Exhibition,
Geneva, Switzerland, Sep 11À15 2005; Society for Biomolecular Screening:
Danbury, CT, 2005; P07016.
best inhibitory activity against
a
1G (CaV3.1) T-type calcium channel
14. Rhim, H.; Lee, Y. S.; Park, S. J.; Chung, B. Y.; Lee, J. Y. Bioorg. Med. Chem. Lett.
2005, 15, 283.
with an IC50 value of 0.65
lM which is comparable to that of mibe-
15. Two methods are available: One is a manual patch-clamp assay (EPC 10Ò, HEKA,
Germany) and the other is an automated patch-clamp assay (NPCÒ-16
patchliner, Nanion Technologies, Germany). Either of the patch-clamp assays
was used to obtain IC50 values of the selected oxazole derivatives, irrespective
of kinds of the compounds, because both IC50s of mibefradil from two methods
fradil.16 In the case of compounds 10-15 and 10-26 with meta-sub-
stituents, their activities were only slightly lower than those of
compounds 10-16 and 10-27 with para-substituents. On the other
hand, it is noteworthy that the dimethyl substituted compounds
10-17, 10-18, 10-19, 10-37, and 10-38 show potent activities with
was practically same with IC50 values of 0.86
l
M and 0.83
l
M, respectively.
16. Spectral data: Free form of 10-35: 1H NMR (400 MHz, CDCl3)
d
0.99 (t,
IC50 values of 1.78–3.02 lM, regardless of the substitution pat-
terns. The compound 11-1 with a benzhydrylpiperazine group
J = 7.45 Hz, 3H), 1.70–1.89 (m, 4H), 2.48 (t, J = 7.07 Hz, 2H), 2.59 (br t, J = 5.1 Hz,
4H), 2.72–2.79 (m, 4H), 3.19 (br t, J = 5.1 Hz, 4H), 4.02 (s, 2H), 6.98–7.13 (m,
3H), 7.23–7.36 (m, 2H), 7.38–7.45 (m, 2H), 7.65 (dd, J = 8.3, 1.3 Hz, 2H); 13C
NMR (100 M Hz, CDCl3) d 13.7, 20.6, 26.0, 30.1, 43.7, 78.2, 78.5, 53.0, 57.1,
112.1, 115.9, 118.7, 124.3 (q, J = 272.4 Hz), 127.1, 127.7, 128.7, 129.5, 131.4 (q,
J = 31.7 Hz), 131.8, 136.5, 144.4, 151.3, 163.7; HCl salt form of 10-35: 1H NMR
(400 MHz, MeOD) d 1.06 (t, J = 7.45 Hz, 3H), 1.89 (sxt, J = 7.43 Hz, 2H), 2.21–
2.36 (m, 2H), 2.88 (t, J = 7.45 Hz, 2H), 3.24–3.34 (m, 8H), 3.62–3.75 (m, 2H),
3.84–4.00 (m, 2H), 4.61 (s, 2H), 7.20 (d, J = 7.58 Hz, 1H), 7.25–7.31 (m, 2H),
7.42–7.47 (m, 2H), 7.52 (t, J = 7.33 Hz, 2H), 7.71 (d, J = 7.33 Hz, 2H); HRMS (FAB,
M+1) calcd for C27H34F3N4O 487.2679, found 487.2683.
shows the highest %-inhibition with 71.85%, but shows only mod-
erate activity with an IC50 value of 7.47 l
M. When R2 substituents
are changed from a propyl group to a methyl group, activities are
reduced (entries 39 and 41 in Table 1).
Previously, we reported isoxazole derivatives as T-type calcium
channel blockers,6e in which compounds with 3-CF3–phen-
ylarylpiperazinylalkylamine substituent showed the most potent