Design, synthesis, and biological evaluation of novel T-Type calcium channel antagonists

Article abstract of DOI:10.1016/j.bmcl.2004.05.011

This paper describes the synthesis of several novel T-type calcium channel antagonists that inhibit calcium influx into the cell, which in turn regulates unknown aspects of the cell cycle pathway that are responsible for cellular proliferation. A library of compounds was synthesized anda brief structure activity relationship will be described. From these studies we have identified a compound (1) that displays anti-proliferative activity in the low micromolar range across a variety of cancer cell lines.

Full text of DOI:10.1016/j.bmcl.2004.05.011

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3691–3695
Design, synthesis, and biological evaluation of novel
T-Type calcium channel antagonists
William F. McCalmont,a,* Tiffany N. Heady,a Jaclyn R. Patterson,a
Michael A. Lindenmuth,a Doris M. Haverstick,b Lloyd S. Grayb and
Timothy L. Macdonalda
aDepartment of Chemistry, University of Virginia, Charlottesville, VA 22904-4319, USA
bDepartment of Pathology, University of Virginia, Charlottesville, VA 22904-4319, USA
Received 28 April 2004; revised 7 May 2004; accepted 7 May 2004
Available online
Abstract—This paper describes the synthesis of several novel T-type calcium channel antagonists that inhibit calcium influx into the
cell, which in turn regulates unknown aspects of the cell cycle pathway that are responsible for cellular proliferation. A library of
compounds was synthesized and a brief structure–activity relationship will be described. From these studies we have identified a
compound (1) that displays anti-proliferative activity in the low micromolar range across a variety of cancer cell lines.
Ó 2004 Elsevier Ltd. All rights reserved.
Calcium is ubiquitous in all living organisms and req-
uisite for a variety of physiological processes.1–5 Calcium
has been linked to cell cycle regulation6–8 and hence to
cellular proliferation via increases in intracellular cal-
cium concentrations.9;10 For instance, calcium is essen-
tial for the activation of early genes, which leads the cell
into G1 phase; a rise in intracellular calcium is also
thought to initiate the replication of DNA at the G1/S
interphase.10 It has been demonstrated that depletion of
intracellular calcium arrests the cell cycle in the G0/G1
and S interphases.11 Regulation of the changes in
intracellular calcium has been proposed to be via a T-
type calcium channel.12 This may be an important tool
to control the cell cycle signaling pathway in order to
manage certain disease states, such as cancer, where the
cell cycle is aberrant.
to the T-type calcium channel, and thereby control cel-
lular proliferation.
We first examined a variety of known L-type calcium
channel blockers for their ability to inhibit the calcium
influx pathway (Fig. 1). These initial studies have illu-
minated common structural motifs that were observed
in some of the more active compounds (Fig. 2). Com-
mon areas of importance include heterocyclic rings that
contain a tertiary nitrogen, aromatic domains, and ether
linkages. By combining these common structural motifs
we were able to envision a series of compounds to syn-
thesize. This new series of molecules would be tested for
their potential to block T-type calcium channels and
ultimately cellular proliferation. The data for these
studies are presented within the following text.
We have recently demonstrated that by blocking cal-
cium uptake, cellular proliferation is inhibited.13 In light
of these findings; we have set out to rationally design a
library of compounds that could regulate certain aspects
of this calcium influx pathway, specifically those related
Synthesis of all the compounds shown in Table 1 are
described in Scheme 1. As outlined in Scheme 1, a so-
dium borohydride reduction of any benzophenone
derivative afforded the corresponding benzhydrol
(Scheme 1).14;15 Subsequently (
L)-proline methyl ester
ðn ¼ 1Þ, or the o-, m-, or p-piperidine ethyl ester iso-
mers16 ðn ¼ 2Þ were coupled to a variety of aryl acetic
acid derivatives using either dicyclohexylcarbodiimide
(DCC) or (benzotriazol-1-yloxy)tripyrrolidinophospho-
nium hexafluorophosphate (PyBOP) to afford the
Keywords: Calcium channel blockers; Cancer; T-type calcium chan-
nels; Cellular proliferation.
* Corresponding author. Tel.: +1-434-9240595; fax: +1-434-9822302;
e-mail: wfm3s@virginia.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.011

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W. F. McCalmont et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3691–3695
F
F
F
F
Cl
Cl
N
N
N
N
N
N
N
CH3
O
N
N
N
H3C
CH3
N
H
CH3
Chlorcyclizine
Meclizine
Flunarizine
Cinnarizine
Lidoflazine
H
H
N
H3C
N
CH3
O
H3C
CH3
O
CN
CH3
N
OCH3
OCH3
O
CH3
CH3
H3CO
H3CO
O
H3C
H3C
N
CH3
CH3
CH3
O
O
O
O
N
O
N
Verapamil
Nicardipine
Isradipine
OCH3
CH3
H3C
N
S
N
HN
CH3
O
CH3
CH3
CH3
O
N
S
O
N
H
N
H3C
CH3
Prenylamine
Figure 1. Initial L-type calcium channel blockers studied in anti-proliferation assay.
Ethopropazine
Fendiline
Diltiazem
aromatic rings
N
Synthesis of compounds 9a–c and 9d–f in Table 1, fol-
lowed the same synthesis pathway as depicted in Scheme
1, however, p-piperidine ethyl ester and m-piperidine
ethyl ester, respectively, were used in place of o-piper-
idine ethyl ester.
ether linkage
O
N
Cl
tertiary nitrogen
ether linkage
aromatic rings
O
Bepridil
Measurement of cellular proliferation was carried out
using Jurkat (T-cell), LNCaP (prostate), or MDA-361
(breast) tumor cells. All assays were carried out in
triplicate in a standard flat bottom 96 well tissue culture
plate in the presence of drug or vehicle (DMSO). Cells
were grown for 48 h at 37 °C in a CO2 incubator. Rel-
ative cell growth was determined with the CellTiter 96
aqueous cell proliferation assay (Promega, Madison
WI), as described by the manufacturer using an auto-
mated plate reader. Results were calculated in a blinded
fashion and are the means of triplicate determinations.
MeO
heterocyclic rings
N
H
N
tertiary nitrogen
1
Perhexiline
Figure 2. Bepridil and perhexiline, two of the initially screened com-
pounds with areas of importance indicated with red boxes lead to the
development of the structure shown, which is a T-type calcium channel
antagonist, VCP11177 (1).
Measurement of changes in intracellular calcium
([Ca2þ]i) concentration: Cells were incubated in growth
media containing 1 lM of the acetoxy-methyl ester of
the Ca2þ sensitive fluorescent dye indo-1 (indo-1/AM;
Molecular Probes, Eugene OR) for 1 h at 37 °C. Cells
were washed three times in buffer A [10 mM HEPES
(pH 7.4) 1 mM MgCl2, 3 mM KCl, 1 mM CaCl2,
140 mM NaCl, 0.1% glucose, and 1% fetal bovine serum]
and suspended to a final concentration of 106 cells/ mL.
Before stimulation with thapsigargin,12 cells were
warmed to 37 °C. Changes in [Ca2þ]i were monitored in
an SLM 8100C spectrofluorometer (SLM/Aminco;
Urbana IL).
amides 6. A dual reduction of both the amide and ester
functionalities using lithium aluminum hydride afforded
the amino alcohols 7. The final step used p-toluene-
sulfonic acid to achieve the condensation of the benz-
hydrol and the alcohol, thus affording the final products
8 ðn ¼ 1Þ,17 and 9 ðn ¼ 2Þ.18 In the case of compound 10,
in Table 1, the N-methyl amine was afforded by a
reductive amination using proline methyl ester, formal-
dehyde, and sodium cyanoborohydride. The synthesis
after this point was performed according to Scheme 1.

W. F. McCalmont et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3691–3695
Table 1. Structure and biological data for all compounds
3693
Compound
IC50 (lM)
Jurkat
LNCaP
MDA-361
Prolifera- Calcium
tion
Prolifer- Calcium
ation
Prolifera-
tion
Calcium
Influx
Influx
Influx
Cl
MeO
H
1
6
7
4
4
10
10
O
N
H
Cl
Me
N
10
60
30
17
50
47
100
O
Cl
MeO
8a, n ¼ 1
8b, n ¼ 3
10
4
2
1
7.7
3.5
NE
30
30
11
30
10
n
H
N
O
Cl
R
8c, R ¼ H
11
6
<1
10
1
8
30
10
12
10
27
11
13
29
3
30
10
30
H
8d, R ¼ 4-hydroxy
8e, R ¼ 2-methoxy
8f, R ¼ 3-methoxy
3
3.8
5
2.4
3.2
O
N
3
R
8g, R ¼ H
108
18
100
25
33
14
100
10
83
15
20
100
O
MeO
MeO
8h, R ¼ chloro
N
R
R`
H
8i, R ¼ R0 ¼ H
12
13
10
3
10
2
30
10
15
10
29
14
30
20
8j, R ¼ fluoro, R0 ¼ H
8k, R ¼ bromo, R0 ¼ H 40
30
3
15
4
1200
11
100
10
8l, R ¼ R0 ¼ chloro
6
O
N
R
8m, R ¼ R0 ¼ fluoro
8n, R ¼ R0 ¼ chloro
8o, R ¼ R0 ¼ methyl
8p, R ¼ R0 ¼ methoxy
5
3.6
3
5
4
10
4
14
11
40
15
30
25
20
20
MeO
3
11
13
3
15
6
5.1
5
10
R`
O
N
R`
9a, R ¼ R0 ¼ H
8.3
8.4
20
7
5
15
5
10
30
25
20
MeO
H
N
O
9b, R ¼ H, R0 ¼ chloro
9c, R ¼ R0 ¼ chloro
3.3
3.4
10.4
7.1
H
11.1
10
10
R
(continued on next page)

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W. F. McCalmont et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3691–3695
Table 1 (continued)
Compound
IC50 (lM)
LNCaP
Prolifera- Calcium
Jurkat
MDA-361
Prolifer- Calcium
ation
Prolifera-
tion
Calcium
Influx
Influx
tion
Influx
R
H
MeO
9d, R ¼ R0 ¼ H
13.8
8.8
8
10
10
9.2
6.8
5
15
12
5
17.7
30
30
30
9e, R ¼ H, R0 ¼ chloro
9f, R ¼ R0 ¼ chloro
13
13
O
9.6
H
N
R`
R
MeO
H
R`
9g, R ¼ H, R0 ¼ chloro
9h, R ¼ R0 ¼ chloro
11
9
3
15
5
4
10
7
25
22
30
30
O
N
H
IC50 values are in lM concentrations; NE, no effect.
O
OH
4-chlorobenzhydrol compound (1) showed the best
activity across all of the cell lines in the series. The 4,40-
dichlorobenzhydrol moiety (8n) was made to deduce if
stereochemistry at the tertiary benzhydrol carbon played
a role in whether the compound exhibited biological
activity. Surprisingly 8n showed no appreciable increase
in biological activity to that of the mono-chloro deriv-
ative (1) indicating that the stereochemistry at this cen-
ter may have an overall effect on the biological activity
of the compounds tested. The six membered piperidine
ring systems (9a–h) exhibited no overall increase in
biological activity in all of the cell lines to that of the five
membered pyrrolidine ring systems.
a
Cl
Cl
2
3
OMe
MeO
H
N
O
b
c
+
O
O
OMe
n
O
N
OH
OMe
n
4, n = 1, 2
5
6, n = 1, 2
OMe
In conclusion, we have presented strong data indicating
that cellular proliferation can indeed be regulated by
inhibiting calcium influx via chemical means. By using a
variety of L-type calcium channel blockers we were able
to formulate an active chemical structure that inhibits
T-type calcium channels with greater potency than the
original L-type calcium channel blockers tested.
Refinement of this chemical structure has led to a
greater understanding of the structure–activity rela-
tionship required to inhibit calcium influx and, conse-
quently, cellular proliferation.
OMe
Cl
d
N
N
O
n
OH
n
n = 1, (8); 2, (9)
7 n = 1, 2
Scheme 1. Synthesis of 8a–q and 9a–h. Reagents and conditions:
(a) NaBH4, THF; (b) DCC, CH2Cl2, NEt3; or PyBOP, NEt3, CH2Cl2;
(c) LiAlH4, THF; (d) 3, p-toluenesulfonic acid, toluene.
Table 1 provides the results of assays used to measure
the inhibition of calcium influx and cellular proliferation
by the compounds synthesized as described in Scheme 1.
From this data, a brief structure–activity profile
emerged as follows: The compound that has a methyl
pyrrolidine is less active than compounds with more
bulkier groups attached to the nitrogen (10 compared to
8a–f). The benzhydrol moiety is crucial for activity as
seen from the loss in activity for compounds 8g and h.
Substitutions on one of the phenyl rings of the benz-
hydrol seem to be important for imparting biological
activity, this can be seen from examples 8i–l. The
Acknowledgements
We thank Dr. Mahendra Chordia and Brian Heasley for
all their help and for reviewing this manuscript.
References and notes
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Watanabe, S.; Sato, N.; Kivilaakso, E. Gastroenterology
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phosphate yielded proline amides, or piperidine amides
subsequent double reduction of amide and ester function-
alities furnished amino-alcohols. Final synthesis of ethers
from benzhydrols and N-substituted prolinols or piperid-
inols was achieved with p-toluene sulfonic acid condensa-
tion, using a Dean–Stark.
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14. The convergent synthesis of library of compounds started
from commercially available starting materials such as
substituted benzophenones, (L)-proline methyl ester and
15. The standard reductions employed on the benzophenone
would in no way induce a stereochemical preference on the
forming benzhydrol. Therefore, these compounds were
synthesized as the racemate and were subsequently
submitted for biological assaying as the two possible
diasteriomers.
16. All piperidine isomers were purchased as the racemic
mixture and no further chiral resolutions or chiral
separations were employed to increase the enantiomeric
mixture.
17. Chloro-phenyl)-phenyl-methoxymethyl]-1-[2-(4-methoxy-
1
phenyl)-ethyl]-pyrrolidine (1): H NMR (CDCl3): d 7.21–
7.33 (m, 9H), 6.95–7.01 (d, 2H), 6.74–6.80 (d, 2H), 5.37 (s,
1H), 3.79 (s, 3H), 3.45–3.56 (m, 2H), 3.11–3.23 (m, 2H),
2.65–2.88 (m, 3H), 2.48–2.62 (m, 1H), 2.26–2.40 (q, 1H),
1.88–2.04 (m, 1H), 1.71–1.86 (m, 2H), 1.56–1.71 (m, 1H);
13C NMR (CDCl3): d 158.3, 141.6, 133.1, 130.0, 128.9,
128.8, 128.1, 128.0, 127.5, 127.4, 114.2, 83.8, 73.7, 63.9,
58.4, 55.4, 55.1, 35.0, 29.1, 23.6; MS (m/z): 436.2 (M)þ;
Anal. Calcd for C27H30ClNO2: C, 74.38; H, 6.94; N, 3.21.
Found: C, 74.64; H, 6.88; N, 3.24.
the appropriate phenylacetic acids. The routine synthetic
steps such as reduction of benzophenone with sodium
borohydride afforded benzhydrols. The coupling of a
18. The piperidine analogs, in each case, were submitted as a
mixture of the four possible diasteriomers.