Synthesis and biological evaluation of 1,4-diazepane derivatives as T-type calcium channel blockers

Article abstract of DOI:10.1016/j.bmcl.2010.03.084

We have synthesized and biologically evaluated 1,4-diazepane derivatives as T-type calcium channel blockers. In this study, we discovered compound 4s, a potential T-type calcium channel blocker with good selectivity over hERG and N-type calcium channels. In addition, it exhibited favorable pharmacokinetic characteristics for further investigation of T-type calcium channel related diseases.

Full text of DOI:10.1016/j.bmcl.2010.03.084

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2705–2708
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and biological evaluation of 1,4-diazepane derivatives as T-type
calcium channel blockers
Su Jin Gu a,b, Jae Kyun Lee a, Ae Nim Pae a, Hye Jin Chung a, Hyewon Rhim a, So Yeob Han b, Sun-Joon Min a,
**
,
*
Yong Seo Cho a,
a Life/Health Division, Korea Institute of Science and Technology, PO Box 131, Cheongyang, Seoul 130-650, Republic of Korea
b Department of Chemistry and Nano Science, Ewha Womans University, Seoul 130-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We have synthesized and biologically evaluated 1,4-diazepane derivatives as T-type calcium channel
blockers. In this study, we discovered compound 4s, a potential T-type calcium channel blocker with good
selectivity over hERG and N-type calcium channels. In addition, it exhibited favorable pharmacokinetic
characteristics for further investigation of T-type calcium channel related diseases.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 24 December 2009
Revised 15 March 2010
Accepted 25 March 2010
Available online 28 March 2010
Keywords:
T-type calcium channel blockers
1,4-Diazepane
Pharmacokinetics
Low-voltage-activated calcium channels, also known as T-type
calcium channels, play a crucial role in the regulation of neuronal
excitability, in both the central and peripheral nervous systems.1
Unlike other types of calcium channels, they comprise only a
To overcome this hurdle, we tried to find a new structural motif
using the 3D ligand based pharmacophore model, which previously
had been established by the hypothesis approach (HipHop) imple-
mented in the CATALYST program.8 As a result, we designed the
1,4-diazepane derivatives 4a–s and 9a–s having two hydrophobic
aromatic components on both sides of the 1,4-diamines, Figure 1.
pore-forming
subtypes.2 Three different genes encode the a1 subunit of T-type
calcium channels, termed a1G 1H, and 1I, respectively, each with
a1 subunit that is different from the calcium channel
,
a
a
its own distinct functional and pharmacological profile.3 Mibefradil
(Posicor, Hoffman-La Roche), the first selective T-type calcium
channel blocker, was approved by the FDA for the treatment of
hypertension and angina pectoris;4 however, it was withdrawn
from the US market in 1998 due to its interaction with the cyto-
chrome P-450 3A4 enzyme which proved to be unrelated to T-type
calcium channel blockage.5 Recent studies have shown that inap-
propriate regulation of T-type calcium channels involves several
pathophysiological diseases, such as epilepsy, pain, hypertension,
congestive heart failure, and cancer.6 Therefore, more potent and
selective inhibitors are required to determine the fundamental
function of T-type calcium channels in these disease states.
During the course of our program to develop a potential T-type
calcium channel blocker, we have found that 1,3-dioxoisoindoline
derivatives showed high potency and excellent selectivity against
the T-type calcium channel over the N-type calcium channel.7
However, further studies revealed that this particular molecular
scaffold did not represent an acceptable pharmacological profile.
* Corresponding author. Tel.: +82 2 958 5173; fax: +82 2 958 5189 (S.-J.M).
** Corresponding author. Tel.: +82 2 958 5156.
E-mail addresses: sjmin@kist.re.kr (S.-J. Min), ys4049@kist.re.kr (Y.S. Cho).
Figure 1. Designed structures of T-type calcium channel blocker and the structural
mapping of in silico pharmacophore with 4a.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.03.084

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S. J. Gu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2705–2708
Comparing these compounds with Mibefradil, we hypothesized
4-trifluoromethyl group on the terminal phenyl ring, turned out
to be the best with an IC50 value of 1.23 M. Additionally, we found
that compound 4s was highly selective for the T-type over the N-
type channel and the hERG channel. For comparison purposes,
we also evaluated the inhibitory activity of Mibefradil against
those channels and found that it inhibited the T-type, hERG, and
that the benzimidazoyl 1,4-diazepane might be acting as an inhib-
itory element. Thus, we planned to synthesize a series of new com-
pounds by changing the position of the carbonyl group and
replacing the substituents on the terminal phenyl group. In this
study, we report our progress on the synthesis and biological eval-
uation of these 1,4-diazepane-based T-type calcium channel
inhibitors.
The synthesis of 1,4-diazepane derivatives is illustrated in
Scheme 1. The anilines 1a–s were reacted with chloroacetyl chlo-
ride to give the chloro amides 2a–s, which were subjected to SN2
displacement with 1,4-diazepane to afford the amines 3a–s. The
first series of target compounds 4a–s9 were obtained by coupling
the amines 3a–s with 2-chloromethyl benzimidazole 6, prepared
from condensation of the diamine 5 with chloroacetic acid in the
presence of 5 N hydrochloric acid.
The second set of compounds, 9a–s, was prepared following a
reaction sequence similar to the first one. Alkylation of methyl bro-
moacetate with the anilines 1a–s provided the esters 7a–s. The
1,4-diazepanes 8a–s were generated by microwave-assisted amide
formation10 of 7a–s with either the free or the N-Boc-protected
1,4-diazepane. In the latter case, the additional removal of the
Boc protective group was required to produce the corresponding
amines. Finally, the treatment of 8a–s with 2-chloromethyl benz-
imidazole 6 under basic conditions furnished the final diazepane
derivatives 9a–s.
l
L-type channels with IC50 values of 1.34, 1.40 and 1.34 lM, respec-
tively. These results indicated that in terms of selectivity, com-
pound 4s is far superior to Mibefradil and in terms of potency, it
is just as active. Compound 9q, which had the highest % of inhibi-
tion value in the FDSS assay, was tested with regard to the T-type
and hERG channels. However, it gave a disappointingly lower effi-
cacy and, in particular, poor selectivity. As a result, we discontin-
ued investigation of this compound.
In order to determine the potential of compound 4s as a lead
compound for therapeutic targets, we subjected it to a pharmaco-
kinetic profile assay. The pharmacokinetic data for 4s after intrave-
nous and oral administration in rats are presented in Table 3. Based
on our analysis of its pharmacokinetic parameters, we discovered
that compound 4s was absorbed acceptably, eliminated relatively
slowly, and possessed a good oral bioavailability of 43%. The con-
siderably high value of the mean volume of distribution suggests
that it tends to bind to tissue components or plasma proteins.
Although the brain to plasma ratio in oral administration was only
moderate, the significant brain penetration characteristic of 4s (B/P
ratio = 0.44) via intravenous administration indicated that the
compound could be a viable candidate for treatment of CNS
disorders.
In summary, we have synthesized and evaluated two series of
1,4-diazepane derivatives 4a–s/9a–s as potential T-type calcium
channel blockers. Using the FDSS HTS system, we rapidly screened
the title compounds and selected several having high potency. On
comparing the biological activities of Mibefradil, we identified the
potent and highly selective T-type calcium channel blocker 4s,
which displays an excellent pharmacokinetic profile in rats. These
results suggest that the 1,4-diazepane analogue 4s will be a poten-
tial therapeutic candidate for the treatment of various neurological
diseases related to the T-type calcium channel without cardiovas-
cular side effects.
The preliminary T-type calcium channel blocking activity of the
synthesized compounds against the T-type calcium channel sub-
type a1G exogenously expressed in HEK293 cells was evaluated
using the FDSS6000 HTS system.11 Table
1 summarizes the
in vitro potency of 4a–s and 9a–s. Most of the compounds in the
series 4a–s exhibited greater than 40% inhibitory activity against
the a1G calcium channel at 10 lM concentration whereas the 9a–
s series showed relatively low activity except for 9a and 9q, indi-
cating that the position of the carbonyl group is very important
for the inhibition. The substituent on the phenyl ring had little ef-
fect. Next, we investigated the IC50 values of selected compounds
using the whole-cell patch-clamp method12 and the results are
shown in Table 2. Among those tested, compound 4s, bearing the
O
a
e
R
R
R
Cl
MeO
N
H
H2N
N
H
O
N
1a-s
2a-s
7a-s
f or g
b
HN
HN
O
R
R
N
N
N
H
H
O
3a-s
8a-s
c
h
N
N
N
H
N
N
H
N
O
R
R
N
N
N
N
H
H
O
4a-s
9a-s
NH2
NH2
Cl
N
d
N
H
5
6
Scheme 1. Reagents and conditions: (a) chloroacetyl chloride, CH2Cl2, 0–23 °C, 79–99%; (b) 1,4-diazepane, CH2Cl2, 0 °C, 75–85%; (c) iPr2NEt, 6, DMF, 60 °C; (d) chloroacetic
acid, 5 N HCl, 110–130 °C; (e) methyl bromoacetate, iPr2NEt, DMF, 60 °C, 36–99%; (f) 1,4-diazepane, TBD (1,5,7-triazabicyclo[4.4.0]dec-5-ene), THF, W, 75 °C, 27–64%; (g) (i)
N-Boc-1,4-diazepane, TBD, THF, W, 75 °C, 34–99%; (ii) TFA, CH2Cl2, or 1 N HCl, MeOH, 29–77%; (h) iPr2NEt, DMF, 70 °C, 32–86%.
l
l

S. J. Gu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2705–2708
2707
Table 1
In vitro T-type calcium channel blocking activity of 1,4-diazepane derivatives using FDSS6000 HTS systemsa
Entry
Compounds
R
HEK293 cell % inhibition (10 lM)
4
9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
4a/9a
4b/9b
4c/9c
4d/9d
4e/9e
4f/9f
4g/9g
4h/9h
4i/9i
4j/9j
4k/9k
4l/9l
4m/9m
4/n/9n
4o/9o
4p/9p
4q/9q
4r/9r
4s/9s
2,6-Diethyl-
2,4-Dimethyl-
3,4-Dimethyl-
2,6-Dimethyl-
2-F–
3-F–
4-F–
2-Cl–
3-Cl–
4-Cl–
2-CH3–
3-CH3–
4-CH3–
2-CH3O–
3-CH3O–
4-CH3O–
2-CF3–
3-CF3–
4-CF3–
57.40
47.64
56.22
39.78
32.35
46.35
46.64
56.76
41.66
51.29
51.83
54.79
44.75
59.15
46.07
32.36
41.68
60.07
60.73
78.92
62.37
21.14
16.48
29.48
22.31
7.52
12.77
32.22
40.87
36.85
7.04
8.48
16.36
8.29
À1.54
0.92
17
18
19
66.00
27.29
28.19
Mibefradil
a
% inhibition value was obtained at 10 lM.
Table 2
3. (a) Shin, H.-S.; Cheong, E.-J.; Choi, S.; Lee, J.; Na, H. S. Curr. Opin. Pharmacol.
2008, 8, 33; (b) Ertel, E. A.; Campbell, K. P.; Harpold, M. M.; Hofmann, F.; Mori,
Y.; Perez-Reyes, E.; Schwartz, A.; Snutch, T. P.; Tanabe, T.; Birnbaumer, L.; Tsien,
R. W.; Catterall, W. A. Neuron 2000, 25, 533.
4. (a) Van der Vring, J.; Cleophas, T.; Van der Wall, E.; Niemeyer, M. Am. J. Ther.
1999, 6, 229; (b) Clozel, J.; Ertel, E.; Ertel, S. J. Hypertens. Suppl. 1997, 15, S17; (c)
Hermsmeyer, K.; Mishra, S.; Miyagawa, K.; Minshall, R. Clin. Ther. 1997, 19, 18.
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1998, 13, 1787.
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H.; Sui, G.; Wu, C. Cell Calcium 2006, 40, 231; (c) Khosravani, H.; Zamponi, G. W.
Physiol. Rev. 2006, 86, 941; (d) Belardetti, F.; Zamponi, G. W. Curr. Opin. Invest.
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Pharm. Des. 2006, 12, 2189; (f) McCalmont, W. F.; Heady, T. N.; Patterson, J. R.;
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Chem. Lett. 2004, 14, 3691; (g) McCalmont, W. F.; Patterson, J. R.; Lindenmuth,
M. A.; Heady, T. N.; Haverstick, D. M.; Gray, L. S.; Macdonald, T. L. Bioorg. Med.
Chem. 2005, 13, 3821.
Inhibitory activity of selected compounds against T-type calcium, hERG, and N-type
calcium channels
Compounds
T-type (a1G
IC50
Ma
)
hERG IC50
l
,
N-type (a1B
)
,
l
Ma
IC50, l
Ma
4a
4c
4h
4n
4s
9a
9q
2.08 0.22
2.39 0.21
1.47 0.16
12.60 0.87
1.23 0.04
4.00 1.31
3.17 0.84
1.34 0.49
4.97 1.90
28.71 8.71
1.34 0.02
1.32 0.17
1.40 0.29
Mibefradil
a
IC50 value( SD) was obtained from a dose–response curve.
7. (a) Kim, H. S.; Kim, Y.; Doddareddy, M. R.; Seo, S. H.; Rhim, H.; Tae, J.; Pae, A. N.;
Choo, H.; Cho, Y. S. Bioorg. Med. Chem. Lett. 2007, 17, 476; For recently reported
T-type calcium channel blockers, see: (b) Lindsley, C. W.; Rittle, K. E.; Bock, M.
G.; Hartman, G. D.; Uebele, V. N.; Nuss, C. E.; Fox, S. V.; Kraus, R. L.; Doran, S. M.;
Connolly, T. M.; Tang, C.; Ballard, J. E.; Kuo, Y.; Adarayan, E. D.; Prueksaritanont,
T.; Zrada, M. M.; Marino, M. J.; Graufelds, V. K.; DiLella, A. G.; Reynolds, I. J.;
Vargas, H. M.; Bunting, P. B.; Woltmann, R. F.; Magee, M. M.; Koblan, K. S.;
Renger, J. J. J. Med. Chem. 2008, 51, 6471; (c) Shipe, W. D.; Barrow, J. C.; Yang, Z.-
Q.; Lindsley, C. W.; Yang, F. V.; Schlegel, K. S.; Shu, Y.; Rittle, K. E.; Bock, M. G.;
Hartman, G. D.; Tang, C.; Ballard, J. E.; Kuo, Y.; Adarayan, E. D.; Prueksaritanont,
T.; Zrada, M. M.; Uebele, V. N.; Nuss, C. E.; Connolly, T. M.; Doran, S. M.; Fox, S.
V.; Kraus, R. L.; Marino, M. J.; Graufelds, V. K.; Vargas, H. M.; Bunting, P. B.;
Hasbun-Manning, M.; Evans, R. M.; Koblan, K. S.; Renger, J. J. J. Med. Chem. 2008,
51, 3692; (d) Hangeland, J. J.; Cheney, D. L.; Friends, T. J.; Swartz, S.; Levesque, P.
C.; Rich, A. J.; Sun, L.; Bridal, T. R.; Adam, L. P.; Normandin, D. E.; Murugesan, N.;
Ewing, W. R. Bioorg. Med. Chem. Lett. 2008, 18, 474.
Table 3
Mean pharmacokinetic parameters in rat plasma following intravenous (n = 4) and
oral (n = 3) administration of 4s
Intravenous
Oral
Cmax
(
lg/ml)
—
—
2.284 ( 0.6358)
120 (60–120)a
260.3 ( 81.65)
—
0.1048 ( 0.03623)
43.42%
Tmax (min)
T1/2 (min)
Vdss (ml/kg)
B/P ratio
F (%)
340.5 ( 106.1)
2325 ( 731.1)
0.4382 ( 0.2306)
—
Values are presented as mean (standard deviation in parentheses). Cmax, peak
plasma concentration; Tmax, time to reach Cmax; Vdss, apparent volume of distribu-
8. (a) Doddareddy, M. R.; Choo, H.; Cho, Y. S.; Rhim, H.; Koh, H. Y.; Lee, J. H.; Jeong,
S. W.; Pae, A. N. Bioorg. Med. Chem. 2007, 15, 1091; (b) 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.
tion at steady state; F, bioavailability.
a
Median (range) for Tmax
.
9. Spectral data of compound 4s: 1H NMR (CDCl3, 400 MHz) d 9.44 (br s, 1H), 7.67
(d, 2H, J = 8.50 Hz), 7.54 (q, 4H, J = 2.69 Hz), 7.24–7.21 (m, 2H), 3.97 (s, 2H), 3.25
(s, 2H), 2.83–2.79 (m, 8H), 1.86–1.82 (m, 2H); 13C NMR (CDCl3, 100 MHz) d
169.5, 152.2, 140.6, 126.3, 125.8, 125.4, 122.6, 119.1, 63.5, 62.3, 60.4, 56.5, 55.8,
54.7, 29.7, 27.9. LRMS (m/z): [M+H+] calcd for C22H25F3N5O 432.5, Found 432.2.
10. Sabot, C.; Kumar, K. A.; Meunier, S.; Mioskowski, C. Tetrahedron Lett. 2007, 48,
3863.
11. Experimental procedure for the FDSS6000 assay: HEK293 cells which express
both stable a1G and Kir2.1 subunits were grown in Dulbecco’s modified Eagle’s
medium supplemented with 10% (v/v) fetal bovine serum, penicillin (100 U/
Acknowledgment
This work was financially supported by a Grant (2E21010) from
KIST.
References and notes
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 Â 104 cells/well
and were used the next day for the high-throughput screening (HTS) FDSS6000
assay. For the FDSS6000 assay, cells were incubated for 60 min at room
1. Clapham, D. E. Cell 2007, 131, 1047; Perez-Reyes, E. Physiol. Rev. 2003, 83, 117.
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2005, 57, 411; (b) McRory, J. E.; Santi, C. M.; Hamming, K. S. C.; Mezeyova, J.;
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temperature with 5 lM fluo3/AM and 0.001% Pluronic F-127 in a Hepes-

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S. J. Gu et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2705–2708
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, in which a1G T-type Ca2+ channels were activated using
high
concentration of KCl (70 mM) in 10 mM CaCl2 containing a Hepes-buffered
solution, an increase in [Ca2+
by KCl-induced depolarization was detected.
Throughout the entire procedure, cells were washed in a BIO-TEK 96-well
washer. All data were collected and analyzed using FDSS6000 and related
software (Hamamatsu, Japan).
resistance of 3–4 MX were pulled and filled with the internal solution
containing (in mM): 130 KCl, 11 EGTA, 5 Mg–ATP, and 10 Hepes (pH 7.4). The
external solution contained (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
a
]
i
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). For more details, see: Rhim, H.; Lee, Y. S.; Park, S. J.;
Chung, B. Y.; Lee, J. Y. Bioorg. Med. Chem. Lett. 2005, 15, 283.
12. Experimental procedure for the patch-clamp test (electro-physiological recording):
For the recording of a1G T-type Ca2+ currents, the standard whole-cell patch-
clamp method was utilized. Briefly, borosilicate glass electrodes with
a