Synthesis and Biological Evaluation of Pentacycloundecylamines and Triquinylamines as Voltage-Gated Calcium Channel Blockers

Article abstract of DOI:10.1002/ardp.201500293

Preclinical studies for neurodegenerative diseases have shown a multi-targeted approach to be successful in the treatment of these complex disorders with several pathoetiological pathways. Polycyclic compounds, such as NGP1-01 (7a), have demonstrated the ability to target multiple mechanisms of the complex etiology and are referred to as multifunctional compounds. These compounds have served as scaffolds with the ability to attenuate Ca²⁺ overload and excitotoxicity through several pathways. In this study, our focus was on mitigating Ca²⁺ overload through the L-type calcium channels (LTCC). Here, we report the synthesis and biological evaluation of several novel polycyclic compounds. We determined the IC₅₀ values for both the pentacycloundecylamines and the triquinylamines by means of a high-throughput fluorescence calcium flux assay utilizing Fura-2/AM. The potential of these compounds to offer protection against hydrogen peroxide-induced cell death was also evaluated. Overall, 8-benzylamino-8,11-oxapentacyclo[5.4.0.0^2,6.0^3,10.0^5,9]undecane (NGP1-01, 7a) had the most favorable pharmacological profile with an IC₅₀ value of 86 μM for LTCC inhibition and significant reduction of hydrogen peroxide-induced cell death. In general, the triquinylamines were more active as LTCC blockers than the oxa-pentacycloundecylamines. The aza-pentacycloundecylamines were potent LTCC inhibitors, with 8-hydroxy-N-phenylethyl-8,11-azapentacyclo[5.4.0.0^2,6.0^3,10.0^5,9]undecane (8b) also able to offer significant protection in the cell viability assays.

Full text of DOI:10.1002/ardp.201500293

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Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
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Full Paper
Synthesis and Biological Evaluation of Pentacycloundecylamines
and Triquinylamines as Voltage-Gated Calcium Channel Blockers
1
,2
1
2
2,3
Lois-May Young , Werner J. Geldenhuys , Olwen C. Domingo , Sarel F. Malan
,
1
,4
and Cornelis J. Van der Schyf
1
Department of Pharmaceutical Sciences, Northeast Ohio Medical University, Rootstown, OH, USA
Pharmaceutical Chemistry, School of Pharmacy, North-West University, Potchefstroom, South Africa
School of Pharmacy, University of the Western Cape, Bellville, South Africa
Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, Idaho State University,
Pocatello, ID, USA
2
3
4
Preclinical studies for neurodegenerative diseases have shown a multi-targeted approach to be
successful in the treatment of these complex disorders with several pathoetiological pathways.
Polycyclic compounds, such as NGP1-01 (7a), have demonstrated the ability to target multiple
mechanisms of the complex etiology and are referred to as multifunctional compounds. These
2þ
compounds have served as scaffolds with the ability to attenuate Ca overload and excitotoxicity
2þ
through several pathways. In this study, our focus was on mitigating Ca
overload through the
L-type calcium channels (LTCC). Here, we report the synthesis and biological evaluation of several
novel polycyclic compounds. We determined the IC50 values for both the pentacycloundecylamines
and the triquinylamines by means of a high-throughput fluorescence calcium flux assay utilizing
Fura-2/AM. The potential of these compounds to offer protection against hydrogen peroxide-induced
2,6 3,10 5,9
cell death was also evaluated. Overall, 8-benzylamino-8,11-oxapentacyclo[5.4.0.0 .0 .0 ]undecane
NGP1-01, 7a) had the most favorable pharmacological profile with an IC50 value of 86 mM for LTCC
(
inhibition and significant reduction of hydrogen peroxide-induced cell death. In general, the
triquinylamines were more active as LTCC blockers than the oxa-pentacycloundecylamines. The
aza-pentacycloundecylamines were potent LTCC inhibitors, with 8-hydroxy-N-phenylethyl-8,11-
2
,6 3,10 5,9
azapentacyclo[5.4.0.0 .0 .0 ]undecane (8b) also able to offer significant protection in the cell
viability assays.
Keywords: L-type calcium channel (LTCC) blockers / Multifunctional drugs / Neurodegeneration /
Pentacycloundecylamine / Triquinylamine
Received: August 20, 2015; Revised: January 27, 2016; Accepted: January 27, 2016
DOI 10.1002/ardp.201500293
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
Introduction
such as ischemia, trauma and epilepsy, and in chronic
neurological disorders such as Alzheimer’s disease,
Parkinson’s disease, and Huntington’s disease [1]. For these
conditions, one common key mediator in the neuronal death
2
þ
2þ
Perturbation of Ca
overload have been implicated in acute neurological disorders
homeostasis and subsequent Ca
2
þ
is calcium (Ca ). Although physiological elevation of
2
þ
intracellular Ca
is part of normal cell function and
homeostatic mechanisms exist to maintain the intracellular
2
þ
2þ
2þ
Ca concentration ([Ca
]
i
), excessive influx of Ca together
Correspondence: Dr. Lois-May Young, 7/7 Station Street, Woy
Woy, NSW 2256, Australia.
E-mail: y.loismay@gmail.com
2þ
with Ca
overwhelm Ca -regulatory mechanisms and lead to cell
release from intracellular compartments can
2þ
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Pentacycloundecylamines and Triquinylamines as Calcium Channel Blockers
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death [2]. Apart from the N-methyl-D-aspartate receptor
pentacycloundecylamine 7a served as a lead compound and
R substitution was guided by structure–activity relationship
(SAR) data from previous studies [16]. In short, pentacyclo-
2
þ
(
NMDAR), voltage-gated Ca channels (VGCCs), such as the L,
N, T, and P/Q-type calcium channels have been implicated as a
2þ
2,6 3,10 5,9
major gateway for Ca
entry from the extracellular
[5.4.0.0 .0 .0 ]undecane-8,11-dione (4), also called Cook-
environment [3]. One approach in the treatment of neuro-
son’s “cage” compound, was synthesized by photochemically
initiated intramolecular [2 þ 2] cycloaddition of the Diels–
Alder adduct (3) obtained from the reaction of 1,3-cyclo-
pentadiene (2) with p-benzoquinone (1) [17]. The synthesis
of the oxa-pentacycloundecylamine derivatives (7a–e),
illustrated in Scheme 1, has been described previously by
our group [16c, 18]. These compounds can be obtained by the
reductive amination of 4 with the desired primary amine.
To obtain the oxa-bridge compound, reduction of the
Schiff base/imine intermediate (6) was performed with
sodium borohydride, to facilitate ring closure through a
transannular reaction. In order to obtain the aza-bridged
derivatives (8a–c), reductive amination was done with sodium
cyanoborohydride.
2þ
degenerative diseases would be to block Ca influx through
2þ
the L-type Ca channels (LTCCs). Blockers of these channels
have been shown to be protective in models of acute
neurological disorders such as ischemia [4]. The ability of
known LTCC blockers such as the 1,4-dihydropyridines (DHP)
nimodipine and nitrendipine, the phenylalkylamine (PAA)
verapamil, and the benzothiazepine (BTZ) diltiazem to
2
þ
i
attenuate excitotoxicity caused by an increase in [Ca
well established [5].
] is
Multifunctional or multimechanistic drugs [6] hold an
advantageincasessuchasischemicstrokewherea combination
of therapies has been shown to be beneficial as shown with the
combination of memantine, a polycyclic compound, and
clenbuterol [7]. Such an approach retains the beneficial
therapeutic effect of combining multiple drugs, while simulta-
neously limiting the side-effect profile to that of only one
drug [8], and it is this multimechanistic action that gives these
compounds a unique advantage in the treatment of neurode-
generativedisorders[9].Anexampleofamultimechanisticdrug
isthepolycycliccompound8-benzylamino-8,11-oxapentacyclo-
One of the required pharmacophoric features of polycyclic
LTCC blockers is a bulky, lipophilic scaffold [10a]. The cis,syn,
cis triquinane scaffold (10), which is a thermal-fragmented
derivative of the pentacycloundecane (4), retains the bulki-
̊
̊
3
2
ness (ꢀ10%) of 4 as expressed in molecular volume (4: 498 A
̊
3
and 10: 552 A ) and solvent accessible surface area (4: 325 A
̊
2
and 10: 351 A ) [16a]. The triquinane scaffold, in addition to
contributing a larger surface area to the geometric confor-
mation, was also found to be a less-strained asymmetric
compound with greater flexibility [19]. The cis,syn,cis triqui-
nane system can only be derived through thermal fragmen-
tation and we aimed to optimize and simplify the method for
the synthesis of the triquinylamines by redesigning the
apparatus (Fig. 1) needed to perform the thermal fragmenta-
tion (step a, Scheme 2). The thermal fragmentation was
performed through flash vacuum pyrolysis (FVP), also known
as flash thermolysis. The synthesis for the aza-triquinylamines
(14a–f) is shown in Scheme 2. We utilized a combination of
methods described by several authors for the design and
synthesis of the apparatus [16a, 20]. In short, the thermal
fragmentation of the saturated four-membered ring (4)
2
,6 3,10 5,9
[
5.4.0.0 .0 .0 ]undecane (NGP1-01). Initially characterized
as a LTCC blocker [10], NGP1-01 was later found to also be a
potent uncompetitive NMDAR channel blocker [11] and NGP1-
2þ
0
1 (7a) thus has the ability to modulate Ca influx through
both ion channel types. In addition, NGP1-01 (7a) has also been
shown to be neuroprotective in vivo using the middle cerebral
artery occlusion mouse model of stroke [12], and also offer
protection in a transient model of stroke following reperfu-
sion [13]. Although much is known about pentacycloundecyl-
amines such as 7a (reviewed in Refs. [14] and [15]), further
assessmentasLTCCblockersand theability ofthesecompounds
to offer protection against induced cell death is necessary. The
aim of this paper is to present the synthesis and biological
2,6 3,10 5,9
evaluation for derivatives of the pentacyclo[5.4.0.0 .0 .0 ]-
undecane-8-11-dione (4) scaffold, such as oxa- and aza-bridged
pentacycloundecylamines (7a–e and 8a–c), and the tricyclo-
2,6
afforded the cis,syn,cis triquinane system: tricyclo[6.3.0.0 ]-
undecane-4,9-diene-3,11-dione (9). FVP was carried out in a
quartz vigreux column connected to a substrate tube and a
vacuum line provided with a liquid nitrogen cold trap.
Sublimation of the substrate 4, which was contained in a
borosilicate glass tube, was achieved at 150°C under vacuum
of 1 Torr. The sublimate traveled through the quartz vigreux
column, which was heated to a temperature of 650°C under
vacuum of 1 Torr. The condensate was deposited in a specially
designed freeze fall, that was cooled with liquid nitrogen, and
2,6
[
6.3.0.0 ]undecane-3,11-dione scaffold, such as the aza-
triquinylamines (14a–f). The latter was obtained by reductive
amination of the flash vacuum pyrolysis product of 4. The IC50
values for these compounds as LTCC blockers were determined
and the ability of these compounds to attenuate peroxide-
induced cell death in PC12 cells by means of the LDH (lactate
dehydrogenase) and Trypan blue staining assays was evaluated.
2
,6
afforded the tricyclo[6.3.0.0 ]undecane-4,9-diene-3,11-
dione (9). We designed and built the tube furnaces to heat
the sublimation tube, and all the glass components and
quartz column were custom made. Our improvements on the
design attributed to a more safe and effective synthesis,
increasing in yield from 64.7% previously reported [16a] to
81.7% and delivered a throughput rate of 1 g every 30 min.
Results and discussion
Chemistry
A series of aza-pentacycloundecylamines (8a–c) and aza-
triquinylamines (14a–f) to supplement the series of oxa-
pentacycloundecylamines (7a–e) was synthesized. The
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O
Scheme 1. Synthesis of compounds 7 and 8.
Reagents and conditions: (a) benzene, 5°C; (b)
a
b
acetone, UV, 6 h; (c) THF, 5°C, NH –R; (d) benzene,
2
+
D, –H
MeOH, NaBH
2
O, 1 h; (e) MeOH, THF, NaBH
4
; (f) HOAc,
3
CN, 12 h. The synthesis of 19 was
O
O
O
achieved by selective decarboxylation of (4) by
means of Huang–Minlon reaction followed by
reductive amination with NaBH . Compound (19)
4
was previously synthesized by Geldenhuys et al.,
and the synthesis is described in the original
paper [19].
O
1
2
3
4
O
c
5
6
d
OH
NH
N
R
R
O
O
e
f
8
7
OH
NH
R
O
N
R
7
a
R =
R =
8a
8b
8c
R =
7
b
R =
R =
O
7
c
R =
-C H
7
15
NO2
7
7
d
e
R =
R =
1
9
N
NH2
The temperature was measured with K-type thermocouples
and regulated with a Shinko Ramp/Soak Auto tune PID
controller (Wika Instruments, Johannesburg, South Africa).
Before pyrolysis commenced, the entire apparatus was
flushed with nitrogen gas and then evacuated to 1 Torr by
a high-capacity rotary vane oil pump. This step is essential for
successful sublimation and to eliminate tar formation.
The product of thermolysis (9) was hydrogenated over 10%
reduction of the diketone (10) was accomplished with sodium
borohydride to facilitate ring closure through a transannular
reaction. The aim was to compare the two scaffolds
(triquinane vs. pentacycloundecane) by adding similar sub-
stituents. For both series, we included compounds where the
aromatic moiety was replaced with an aliphatic side chain (8c,
14d, and 19) to explore the functional role of the aromatic
moiety in isolation, since it is reasoned that activities for these
compounds as LTCC blockers are dominated by the lipophilic
amine substituent with the polycyclic scaffold only contribut-
ing a larger surface area to the geometric conformation [16c].
Several compounds that contain electron withdrawing or
donating moieties (7c, 7d, and 14e) were also included and
the chain length was altered to assess binding within the
channel pore (7b, 8b, 14b, and 14c). Detailed descriptions of
2,6
Pd-C catalyst to yield tricyclo[6.3.0.0 ]undecane-3,11-dione
10). The aza-triquinylamine compounds (14a–f) were
obtained by reductive amination (Scheme 2, steps d–f) of
0 with the desired primary amine side chains. The aza-bridge
(
1
compound was obtained by reduction of the imine interme-
diate (13) with sodium cyanoborohydride. To obtain the oxa-
2,6
bridge 3-hydroxy-3,11-oxatricyclo[6.3.0.0 ]undecane (11),
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Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
Pentacycloundecylamines and Triquinylamines as Calcium Channel Blockers
ARC HH PHARM
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Figure 1. Schematic representation of the apparatus for flash vacuum pyrolysis (FVP).
a
b
c
OH
O
O
O
O
O
O
11
O
4
9
10
d
H
H
f
e
H
H
OH
N
O
N
O
NH
R
R
R
1
4
13
12
1
4a
R =
R =
14d
R =
O
N
1
4b
1
4e
R =
R =
1
4c
R = (CH2)3
1
4f
Scheme 2. Synthesis of compounds 11 and 14. Reagents and conditions: (a) D, 650°C, 1 Torr, 45 min; (b) EtOAc, 2 atm H
0 min; (c) MeOH, THF, NaBH ; (d) THF, 5°C, NH –R, 6 h; (e) benzene, D, –H O, 1 h; (f) MeOH, THF, NaBH CN, 18 h.
2
, 10% Pd/C,
4
4
2
2
3
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all the synthesis performed and structure elucidation are
given in the Experimental section.
Table 1. The IC50 (mM) values for the inhibition of
þ
2þ
K -induced Ca influx as determined experimentally and
predicted logP for the series of 8,11-oxapentacyclounde-
cylamines (7a–d); 8,11-azapentacycloundecylamines (8a–c),
and 3,11-azatricycloundecylamines (14a–f).
Fluorescence measurement of calcium influx
To examine the ability of these compounds to modulate
calcium influx through the VGCC, and to determine their IC50
Compound
IC50 (mM)
logP
2
þ
values, we developed a high-throughput fluorescence Ca
flux assay in undifferentiated rat pheochromocytoma cells
7a
7b
7c
86
51
42
0.580
35
8
7
74
310.9
0.398
68
2.11
2.4
1.95
–
(
PC12 cells) loaded with Fura-2/AM. Depolarizing the cell
þ
membrane by increasing the extracellular [K ]
increase the influx of Ca through the VGCC [21]. PC12 cells
0
is known to
2
þ
7d
8
8
8
1
1
1
a
b
c
4a
4b
4c
1.85
2.14
2.78
3.67
3.96
3
4.06
3.51
2.45
responded to the addition of 50 mM KCl, with a rapid
2
þ
i
increase in [Ca
] (Supporting Information Fig. SA.1(a–t)).
Depolarization with 50 mM KCl in the absence of any test
compound served as the control for normal physiological
function and experiments were done in triplicate. Several
known LTCC blockers were evaluated to serve as references
14d
14e
14f
2
þ
for the amplitude of Ca influx suppression and to confirm
11
5
that LTCC were involved. The reference compounds gave a
2
þ
concentration-dependent suppression of Ca
influx (Sup-
Experimental values are means of at least three experiments
n ¼ 3). IC50 values of reference compounds were calculated to
porting Information Fig. SA.1(a–d)). For this assay, the IC50
values for reference compounds were determined to be
(
be: nimodipine 0.008 mM (n ¼ 6), nitrendipine 0.002 mM
0
.002 mM (nitrendipine), 0.008 mM (nimodipine), 0.328 mM
(
(
n ¼ 3), verapamil 0.328 mM (n ¼ 3), and diltiazem 0.449 mM
n ¼ 3).
(
verapamil), and 0.449 mM (diltiazem). We then proceeded to
evaluate the ability of the polycyclic amine compounds,
which included the oxa-pentacycloundecylamines (7a–e),
aza-pentacycloundecylamines (8a–c), and aza-triquinyl-
2
þ
amines (14a–f), to suppress Ca influx after depolarization
with 50 mM KCl (Supporting Information Fig. SA.1(e–t)) and
calculated their IC50 values. The IC50 values are summarized
in Table 1 and the log concentration–response graphs are
presented in Fig. 2a–t. According to their inhibitory potency,
the test compounds fell into three categories, with 14c and
therefore these compounds might act in a similar manner as
(S)-Bay K8644 and FPL 64176 (LTCC agonist or activators) [22],
2þ
by prolonging Ca channel current activation and deactiva-
tion and/or increasing the probability of channel opening and
the mean channel open time. This observation was also made
when we evaluated the lipophilic scaffolds 4 and 10 (data not
shown). The pentacycloundecane (4) had no activity, while
the triquinane-dione (10) had a slight potentiating effect but
7
d (IC50 value of 0.398 and 0.580 mM, respectively) being the
most potent and matching the IC50 values of the reference
compounds. Compounds 8b, 8c, 14e, and 14f fell into the
second category with IC50 values ranging from 5 to 11 mM;
and the remainder fell into the third category with IC50
values ranging from 35 to 86 mM. All of the compounds
tested had higher potency as LTCC blockers than the lead
compound 7a (NGP1-01, IC50 ¼ 86 mM).
no antagonistic activity. For compound 7e,
a possible
explanation for lack of activity could be drawn from an
observation made in a study by Malan et al. [16c], in which the
author concluded that compounds such as 7e with low-
calculated energy minima where the oxa-bridged pyridin-
4-ylmethylamine side chain folds back onto the polycyclic
structure displayed poor inhibition of calcium current in a
whole cell patch clamp experiment. Thus, the geometric
orientation could be unfavorable for binding especially since
its counterpart, the triquinane, 14f with the aza-bridged
pyridin-4-ylmethyl substituent showed favorable activity
(IC50 ¼ 5 mM). We also observed that for most compounds,
the graphs illustrate an inhibitory potency which increased in
a dose-dependent manner between 10 and 200 mM. However,
between 0.0001 and 1 mM we observed a slight potentiating
When comparing the dose–response profiles of the
synthesized compounds to the reference compounds
(
Fig. 2a–q), several observations can be made. All of the
þ
2þ
compounds were able to block K -induced Ca influx in a
dose-dependent manner. Compound 14c (IC50 ¼ 0.398 mM),
an aza-triquinylamine with a phenyl propyl substituent had a
similar inhibition profile to nimodipine and nitrendipine.
Compounds 7d and 14f, with the second and third lowest IC50
values (0.580 and 5 mM, respectively) had similar inhibition
profiles to verapamil and diltiazem. For compounds 7e, 11,
and 19, we observed an agonist-like sigmoidal dose–response
profile (Fig. 2r–t). These were compounds that demonstrated
a potentiating effect. A possible explanation could be that
neither compound 11 nor 19 had substituents that could
extend to interact with binding sites within the channel pore;
2
þ
2þ
effect which could be due to Ca -induced Ca
release
(CICR). These compounds may not have the ability at lower
2
þ
concentrations to block KCl-induced increases in [Ca
which has been shown to release Ca
i
] ,
2þ
from intracellular
stores [23]. We will attempt to further elaborate on this
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Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
Pentacycloundecylamines and Triquinylamines as Calcium Channel Blockers
ARC HH PHARM
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þ
2þ
Figure 2. The dose–response relationship of (a–q) indicates antagonist inhibition of K -induced Ca influx. The dose–response
þ
relationship of (r–t) indicates a potentiating effect after K -induced depolarization. Results are presented as the mean ꢀ STDEV
(n ¼ 3–6). Where error bars are not shown, these are covered by the point itself.
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L.-M. Young et al.
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Figure 2. Continued.
observation in future experiments. This biphasic profile was
also described by another group that evaluated the
derivatives were more active than their aromatic counter-
parts. The aliphatic heptylamine (8c) derivative had the
highest potency of the aza-pentacycloundecylamines and for
the aza-triquinylamines, the aliphatic derivative (14d) was
slightly more active than the unsubstituted benzylamine
(14a). In general, it was observed that substitution in the
meta position (7c, 7d, and 14e) increased the activity when
compared to the unsubstituted derivatives (7a and 14a).
Structures with substitution in the meta position with an
electron-withdrawing moiety such as the nitro group (7d)
were more active than the methoxy-substituted compound.
The electron deficient 4-substituted pyridine (14f) was
1
,2,4-oxadiazol-5-one derivatives, and also attributed their
observations to CICR from the sarcoplasmic reticulum [24].
For all three series of compounds, the unsubstituted
benzylamine derivatives (7a, 8a, and 14a) were found to be
the least active. Increases in chain length (7b, 8b, and 14c)
2
þ
increased the inhibition of Ca
influx and the aza-
triquinylamine derivative (14c), with a phenylpropyl substit-
uent, had the highest potency. This correlated with previous
findings that attributed this increase in activity to an increase
in volume and lipophilicity [16c]. Overall the aliphatic
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Pentacycloundecylamines and Triquinylamines as Calcium Channel Blockers
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observed to be more active for the aza-triquinanes when
compared with (14a) and also more active than the meta-
methoxy substituent.
investigate whether our compounds, which have the ability to
regulate calcium influx through the LTCC, would offer
protection against H O -induced cell death.
2 2
Measurement of cell viability
Estimation of LDH release
2þ
It is well known that an increase in [Ca
]
i
plays an important
Plasma membrane damage in PC12 cells was evaluated by
measuring the amount of intracellular LDH released in the
media (Fig. 3). To assess inherent toxicity, compounds were
role in the mechanism of apoptosis and necrosis, which can be
induced by oxidative stress [25]. We performed cell viability
studies utilizing the LDH and Trypan blue staining assays to
2 2
evaluated in the absence of H O . Compound 14c caused
+
+
0
0
0
0
0
0
0
0
0
0
.45
.40
.35
.30
.25
.20
.15
.10
.05
.00
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
*
*
*
+
0.6
.5
.4
.3
.2
.1
.0
+
0
0
0
0
0
0
0
0
.45
.40
.35
.30
.25
.20
.15
.10
.05
.00
0
+
0
*
*
0
0
0
0
0
0
Figure 3. The effect of selected compounds on LDH release after cell injury induced by 200 mM H
2
O
2
. Data are mean ꢀ SEM (n ¼ 3).
þ
Data were subjected to an ANOVA analysis, followed by Dunn’s post-test and significance was defined as p < 0.05 compared to
ꢁ
control-untreated cells and p < 0.05 compared to 200 mM H
2 2
O .
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259

Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
L.-M. Young et al.
ARC HH PHARM
Archiv der Pharmazie
ꢁ
significant LDH release (p < 0.05), but only at the highest
concentration tested (100 mM). The aberrant cytotoxicity
observed for compound 14c, which also had the lowest IC50
value (IC50 ¼ 0.398 mM) as a LTCC blocker, was more than what
(1 and 10 mM, p < 0.05). Compound 7a was the most active,
displaying no significant inherent toxicity, and demonstrating
2 2
the ability to significantly reduce H O -induced LDH release at
all three concentrations evaluated.
2 2
was observed for the control of H O treatment alone.
Compounds 7a and 8b caused some LDH release at 100 mM,
although this was found not to be significant. In general,
H O -induced LDH release was attenuated in the presence of
2 2
Trypan blue exclusion
Trypan blue staining assays were performed to verify results
from the LDH assays. Cell viability was assessed by criterion of
Trypan blue exclusion (Fig. 4). The failure to exclude Trypan
blue dye reflects a loss of plasma membrane integrity
associated with necrosis. The results corresponded well to
selected compounds. Compound 7a showed significant
attenuation of LDH release at concentrations (1, 10, and
ꢁ
1
00 mM, p < 0.05) as did compound 8b at concentrations
100
100
7
5
2
5
0
5
75
*
50
25
0
*
*
*
*
+
+
0
1
00
100
75
50
25
0
7
5
*
5
0
*
*
+
2
5
+
0
Figure 4. Assessment of percentage cell viability by means of Trypan blue uptake and exclusion. The effect of compounds alone was
assessed by counting viable cells and comparing to control of untreated viable cells. The ability of selected compounds to offer
protection against cell injury induced by 200 mM H O was assessed by counting stained non-viable cells and comparing to the control
2 2
of cells treated with 200 mM H
analysis, followed by Dunn’s post-test and significance was defined as p < 0.05 compared to 200 mM H
2
O
2
. Data are mean ꢀ SEM (n ¼ 3, counted two fields per repeat). Data were subjected to an ANOVA
ꢁ
2
O
2
.
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260

Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
Pentacycloundecylamines and Triquinylamines as Calcium Channel Blockers
ARC HH PHARM
Archiv der Pharmazie
results obtained with the LDH assay (compare Fig. 3 with 4).
When compounds were evaluated in the absence of H to
Concerning the SAR, it is interesting to note that the
compounds evaluated in this study are structurally unrelated
to established LTCC blockers and based on the IC50 values
obtained we were able to draw meaningful conclusions
regarding the SAR. Calculated IC50 values also allowed for
direct comparison of the triquinylamines with the pentacy-
cloundecylamines. For this series of compounds, SAR as LTCC
blockers were influenced by geometric and steric effects.
Modifying the “cage” moiety of the pentacycloundecane
skeleton (4) by thermal fragmentation to obtain the cis,syn,cis
triquinane system (9) did not diminish activity and, in general,
the triquinane compounds were more active as LTCC blockers.
Previous studies demonstrated that altering the geometric
conformation of the polycyclic scaffold by increasing the size
and volume leads to an increase in activity [16c]. This could
explain why the triquinane compounds were more active, as
the triquinane scaffold has a larger size and solvent accessible
surface area (SASA) [19]. The triquinane scaffold shows
promise in the design of novel LTCC blockers. Increase in
activity was also observed for compounds (7b, 8b, and 14c)
with an increase in chain length. As expected activity was
diminished for compounds such as 11 and 19 that did not have
the substituted amino side chain moiety, for activity as a LTCC
blocker resides primarily in the combination of the secondary
amino side chain and the bulky hydrocarbon polycycle [16a, c].
We found that compounds with aliphatic side chains (8c and
14d) were more active than their aromatic counterparts (7a,
8a, and 14a). As shown by calculated logP values (Table 1), the
aliphatic compounds are more lipophilic, which has been
shown to be a contributing factor to an increase in activity
[16b]. Since these compounds lack the aromatic group
necessary for interaction with the DHP binding site [28], we
could speculate that these compounds are pore-blocking
drugs that occlude the transmembrane pore or act as
allosteric modulators. Known LTCC blockers are neither
structurally nor pharmacologically homogeneous, thus our
findings serve as a valuable guide for future optimization
efforts.
2 2
O
assess inherent toxicity, compounds 7a, 7b, and 8b caused a
slight decrease in cell viability at 10 and 100 mM, compared to
the control of viable untreated cells. Compound 14c showed
considerable reductions (97.91%) in cell viability at 100 mM,
ꢁ
compared to the control of viable untreated cells ( p < 0.05),
which indicates cytotoxicity for this compound at higher
concentrations. This observation also correlates with results
from the LDH assay for this compound at the same
concentration. Treatment with 200 mM H O caused signifi-
2 2
cant (73.6%) reduction in cell viability compared to the
control of viable untreated cells. Pretreatment with selected
test compounds generally attenuated H
2 2
O -induced cell
injury when compared to H treatment alone. Of the
2 2
O
compounds evaluated, 7a demonstrated the most favorable
ꢁ
pharmacological profile, with significant reduction ( p < 0.05)
2 2
of H O -induced toxicity at all concentrations evaluated
(
1, 10, and 100 mM). The same observation was made for this
compound with the LDH assay.
Conclusion
We evaluated the ability of polycyclic compounds such as the
pentacycloundecylamine and triquinylamine derivatives to
2
þ
modulate Ca influx through the LTCC by means of a high-
throughput fluorescence assay utilizing Fura-2/AM. This forms
part of an ongoing study into their potential as novel
multifunctional drug candidates in the treatment of neuro-
degenerative disease. For compounds 14c and 7d, the IC50
values (0.398 and 0.580 mM, respectively) and dose–response
profiles were comparable to that of verapamil (IC50 ¼ 0.328
mM) and diltiazem (IC50 ¼ 0.449 mM). However, 14c which had
the lowest IC50 value (0.398 mM) showed toxicity at 100 mM
2 2
when evaluated in the absence of H O in both the LDH and
Trypan blue staining cell viability assays. The IC50 value for
NGP1-01 (7a) was determined to be 86 mM and this compound
had the most favorable pharmacological profile with the
ability to attenuate cell death in both cell viability assays. This
compound also displayed no inherent toxicity. Compounds 7b
and 8b, oxa- and aza-pentacycloundecylamines which both
have phenylethyl substituents, had favorable activity as LTCC
blockers (IC50 ¼ 51 and 8 mM, respectively). These compounds
were also able to offer protection in the cell viability assays.
In general, the aza-pentacycloundecylamines (8a–c) were the
most potent LTCC blockers. In the cell viability assays, compound
Experimental
Chemistry
Chemicals and instrumentation
Reagents were obtained from Sigma–Aldrich (UK and USA),
Merck (Germany), D.H. Chemicals (UK), Acros Organics (USA),
Fluka (Switzerland and USA), and Saarchem (South Africa).
Reaction and elution solvents were purchased from commer-
cial sources. Column chromatography was performed using
either flash chromatography with nitrogen gas on glass
columns [29], or conventional glass columns packed with
8b offered protection at lower concentrations but showed some
toxicity at 100 mM. For future studies, it would be worthwhile to
synthesize more compounds in the aza-pentacycloundecyl-
amines series and more extensively evaluate these compounds
foractivityaspossibleneuroprotectivedrugs[26].Calciumeffects
obtained through the interactions of these structures on
s-receptors and intracellular calcium channels such as inositol-
̊
silica gel, 60 A pore size, mesh 70–230, purchased from
Separations (South Africa) or by flash chromatography
utilizing the Versa Flash station with VersaPak columns,
silica cartridge 40 ꢂ 75 mm, purchased from Supelco (Pretoria,
South Africa). Preparative thin-layer chromatography was
3
1,4,5-triphosphate receptors (IP Rs) or ryanodine receptors
(RyRs), also need to be investigated [27].
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261

Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
L.-M. Young et al.
ARC HH PHARM
Archiv der Pharmazie
̊
performed on precoated silica gel glass plates, 60 A pore size,
diameter 20 ꢂ 20 cm, and layer thickness 2 mm with fluores-
cent indicator for 245 nm, purchased from Separations, and
system with the vacuum pump turned on. The nitrogen inlet
was then closed and the whole apparatus evacuated to 1 Torr
by a high-capacity, rotary vane oil pump. Sublimation was
achieved by sliding the specially designed ceramic thermal
unit over the sublimation tube and heating the sublimation
tube to 150°C, under 1 Torr vacuum. The substrate slowly
migrated through the quartz column which was preheated to
a temperature of 650°C. The condensate that yielded 9 was
deposited as white waxy crystals mixed with black tar in the
liquid nitrogen freeze fall. The pyrolysis was carried out at a
throughput rate of 1 g every 30 min and repeated several
times. The custom-designed apparatus utilized to perform
pyrolysis is illustrated in Fig. 1. The condensate (9) was purified
by recrystallization in cyclohexane to produce white needle-
̊
also on C-18 preparative chromatography glass plates, 60 A
pore size, diameter 20 ꢂ 20 cm, and layer thickness 0.25 mm,
purchased from Whatman Chemical Separation Inc. (USA).
Commonly used abbreviations are DCM (dichloromethane),
EtOAc (ethyl acetate), EtOH (ethanol), Et O (diethyl ether),
2
HOAc (acetic acid), MeOH (methanol), PE (petroleum ether),
and THF (tetrahydrofuran).
Differential scanning calorimetry (DSC) thermograms were
recorded with a Shimadzu DSC-50 instrument (Shimadzu,
Kyoto, Japan). The measurement conditions were as follows:
sample weight, approximately 2 mg; sample holder, alumi-
num crimp cell; gas flow, nitrogen at 35 mL/min; heating rate,
like crystals (yield: 17.15 g, 0.098 mol for 20.98 g pyrolyzed,
1
1
0°C/min. Melting points (m.p.) were measured using a
81.7%). C11
CDCl ): d 7.51–7.30 (m, 2H, H
3.67–3.35 (m, 2H, H , H ), 3.28–3.03 (m, 2H, H
(m, 1H, H7b), 2.06–1.75 (m, 1H, H7a). C NMR (75 MHz, CDCl
d 207.35 (s, 2C, C , C11), 165.93 (d, 2C, C , C10), 133.35 (d, 2C, C
), 53.05 (d, 2C, C , C ), 50.29 (d, 2C, C , C ), 31.36 (t, 1C, C ). IR
H
O
10 2
; m.p. (DSC) 97.23°C. H NMR (300 MHz,
Gallenkamp and Stuart SMP10 melting point apparatus.
Infrared (IR) spectra were recorded on a Nicolet 470 FT-IR
spectrophotometer. Oily products were applied on the KBr
disks as a thin film. Mass spectra (MS) were recorded on an
analytical VG 7070E mass spectrometer. Ionization was
induced by means of electron impact at 70 eV. HS-MS were
recorded on a Thermo Scientific LTQ Orbitrap XL. Nuclear
magnetic resonance (NMR) spectra were acquired on a Varian
3
5
, H
9
), 6.17–5.50 (m, 2H, H
, H ), 2.45–2.10
):
4
, H10),
6
8
1
2
1
3
3
3
4
5
,
C
9
1
2
6
8
7
ꢃ1
(KBr, cm ): nmax 1720, 1639. MS (EI, 70 eV) m/z: calc. for
C
þ
11
10
H O
2
: 174.1959, found: 174 (M ), 146, 131, 117, 91, 66, 39,
10 2
28. HR-MS m/z: calc. for C11H O : 174.1959, found: 174.0759.
As discussed by Young et al. [19], this compound was also
characterized by means of single crystal X-ray analysis.
1
Gemini 300 with H spectra recorded at a frequency of
00.075 MHz and C spectra at 75.462 MHz. Chemical shifts
13
3
are reported in parts per million (ppm) relative to the internal
standard, tetramethylsilane (TMS). The following abbrevia-
tions were used to indicate multiplicities of the respective
signals: s, singlet; bs, broad singlet; d, doublet; t, triplet; q,
quartet; m, multiplet.
2
,6
Tricyclo[6.3.0.0 ]undecane-3,11-dione (10)
2
,6
Tricyclo[6.3.0.0 ]undecane-4,9-diene-3,11-dione
(3.09 g,
0.0177 mol) was dissolved in 500 mL of dry EtOAc and
reduction was done with 10% Pd-C (300 mg) at a pressure
of 2 atm (H ) for 40 min. The catalyst was removed by filtration
2
1
Synthesis
through Celite and the solvent was removed in vacuo.
Recrystallization from cyclohexane produced 10 as needle-like
white/colorless crystals (yield: 3.01 g, 0.0169 mol, 95.42%).
Physical characterization for this product correlate with that
A general method for the synthesis of aza-pentacycloundecyl-
amines (8a–c) and aza-triquinylamines (14a–f) to supplement
a series of oxa-pentacycloundecylamines (7a–e) is described in
the text and is illustrated in Schemes 1 and 2.
reported in literature [16a, 20a]. C11
H
14
O
2
; m.p. (DSC) 95.82°C.
): d 2.97–2.70 (m, 4H, H , H , H , H ),
2.49–2.12 (m, 5H, H4a, H4b, H7b, H10a, H10b), 2.12–1.94 (m, 2H,
1
H NMR (300 MHz, CDCl
3
1
2
6
8
2
,6 3,10 5,9
Pentacyclo[5.4.0.0 .0 .0 ]undecane-8,11-dione (4)
2,6 3,10 5,9
The pentacyclo[5.4.0.0 .0 .0 ]undecane-8,11-dione (4)
was synthesized according to the well-established method
previously described [17]. The physical characterization, data
not presented, correlated with the physical characteristics
described by Cooksen et al. [17].
H
5b, H9b), 1.72–1.54 (m, 2H, H5a, H9a), 1.30–1.10 (m, 1H, H7a).
1
3
C NMR (75 MHz, CDCl
, C ), 43.44 (d, 2C, C
10), 26.01 (t, 2C, C , C
3
): d 218.59 (s, 2C, C
, C ), 38.54 (t, 1C, C
). IR (KBr, cm ): nmax 1736, 1417. MS (EI,
3
, C11), 55.97 (d, 2C,
4
), 37.02 (t, 2C, C ,
C
C
1
2
6
8
7
ꢃ1
5
9
þ
70 eV) m/z: calc. for C11
14
H O
2
: 178.2277, found: 178 (M ), 122,
9
6, 79, 66, 55, 39, 28. HR-MS m/z: calc. for C11
H
14
O
2
: 178.2277,
2
,6
Tricyclo[6.3.0.0 ]undecane-4,9-diene-3,11-dione (9)
We found, also reported in literature [20b], that optimal yield
was achieved when the starting compound (4) was sublimated
using a small sample spread as a thin film over the entire
inner surface of the sublimation tube. Pentacyclo-
found: 178.1072. As discussed by Young et al. [19], this
compound was also characterized by means of single crystal
X-ray analysis.
General method for the synthesis of compounds 8a–c as
derivatives of azahexacyclo[5.4.1.0 .0 .0 ]dodecan-
2
,6 3,10 5,9
2,6 5,9 8,11
[
5.4.0.0 .0 .0 ]undecane-8,11-dione (4) (1 g, 0.006 mol)
was dissolved in a minimal amount of DCM and spread over
the surface of the sublimation tube using a heat gun to
evaporate the DCM. The sublimation tube was connected to
the vigreux quartz column by interspersing Teflon joint tape.
Nitrogen gas (can also use argon) was flushed through the
8-ol (8) by reductive amination of pentacyclo-
2,6 3,10 5,9
[5.4.0 .0 .0 ]undecane-8,11-dione (4)
2
,6 3,10 5,9
Pentacyclo[5.4.0.0 .0 .0 ]undecane-8,11-dione (4) was
dissolved in anhydrous THF (50 mL) and cooled to 5°C on an
external ice bath. An equimolar quantity of the primary amine
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Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
Pentacycloundecylamines and Triquinylamines as Calcium Channel Blockers
ARC HH PHARM
Archiv der Pharmazie
1
3
was slowly added stoichiometrically, while magnetically
stirring the solution. The reaction was monitored on TLC till
completion. The carbinol amine formed as a white precipi-
tate, which was retrieved by means of filtration. This product
was dehydrated under Dean–Stark conditions for approxi-
mately 1 h using 100 mL anhydrous benzene. The solvent was
removed in vacuo to afford the Schiff base (5) as yellow oil,
which was dissolved in a mixture of HOAc (15 mL) and MeOH
H
9
, H10), 1.93–1.42 (AB-q, 2H, J ¼ 10.5 Hz, H4a, H4b). C NMR
(75 MHz, CDCl ): d 140.34 (s, 1C, C14), 128.55 (d, 2C, C16, C18),
128.38 (d, 2C, C15, C19), 128.29 (d, 1C, C17), 126.03 (s, 1C, C11),
65.12 (d, 1C, C ), 55.07 (d, 1C, C10), 50.76 (d, 1C, C ), 48.98
(t, 1C, C12), 45.56 (t, 1C, C ), 44.73 (d, 1C, C ), 43.10 (d, 1C, C ),
42.36 (d, 1C, C ), 41.66 (d, 1C, C ), 41.59 (d, 1C, C ), 41.52 (t, 1C,
13), 35.13 (d, 1C, C ). MS (EI, 70 eV) m/z: 279 (M ), 246, 188,
3
8
1
4
9
5
2
3
6
þ
C
7
131, 91, 77, 28. HR-MS m/z: calc. for C19H21NO: 279.3761,
(
250 mL). Reduction was carried out by slowly adding
found: 279.1701.
NaBH CN (2 g, 0.032 mol), in one molar excess, over a period
of 5 min. The reaction mixture was allowed to stir overnight at
room temperature. The solvent was removed in vacuo and
3
2,6 3,10 5,9
N-Heptyl-3-hydroxy-4-azahexacyclo[5.4.1.0 .0 .0
.
8,11
0
]dodecane (8c)
H
2
O (100 mL) was added. While stirring, solid NaHCO
added portion-wise until the evolution of carbon dioxide
ceased. Excess NaHCO (2 g) was added and the mixture was
extracted with DCM (4 ꢂ 50 mL). The combined organic phases
were washed with H
O (2 ꢂ 100 mL), dried with anhydrous
MgSO , and filtered. The solvent was removed in vacuo. The
3
was
This compound was prepared according to the general
method for derivatives of 8 described in above text. Equimolar
2
,6 3,10 5,9
3
quantities of the pentacyclo[5.4.0.0 .0 .0 ]undecane-
8,11-dione (3 g, 0.017 mol) and heptylamine (1.98 g,
0.017 mol) were used. The resulting product was purified by
2
4
2
means of flash column chromatography (SiO ; PE/EtOAc/EtOH
compounds were purified by means of flash column
chromatography or crystallization.
5:4:1 as eluent) to yield white crystals (yield: 2.031 g,
1
0.007 mol, 43.13%).
C
18
H27NO; m.p. 100°C;
H
NMR
(
300 MHz, CDCl ): d 6.30–5.70 (bs, 1H, OH), 3.65–3.42 (t, 1H,
3
2
,6 3,10 5,9
N-Benzyl-3-hydroxy-4-azahexacyclo[5.4.1.0 .0 .0
.
J ¼ 5.3, H11), 3.00–2.80 (m, 1H, H12a), 2.80–2.10 (m, 9H, H
1 2
, H ,
8
,11
0
]dodecane (8a)
H
H
H
3
, H
5
, H
6
, H
7
, H
9
, H10, H12b), 1.87–1.40 (AB-q, J ¼ 10.5 Hz, 2H,
This compound was prepared according to the general
4a, H4b), 1.60–1.40 (m, 2H, H13a, H13b), 1.36–1.10 (m, 8H, H14a
,
method for derivatives of 8 described in above text. Equimolar
14b, H15a, H15b, H16a, H16b, H17a, H17b), 0.96–0.70 (t, 3H,
2
,6 3,10 5,9
13
quantities of the pentacyclo[5.4.0.0 .0 .0 ]undecane-
J ¼ 6 Hz, H18a, H18b, H18c). C NMR (75 MHz, CDCl
(s, 1C, C11), 64.69 (d, 1C, C ), 54.56 (d, 1C, C10), 50.51 (t, 1C, C12),
46.59 (d, 1C, C ), 45.75 (t, 1C, C ), 44.44 (d, 1C, C ), 43.26 (d, 1C,
), 41.91 (d, 2C, C , C ), 41.64 (d, 1C, C ), 41.56 (d, 1C, C ),
3
): d 179.86
8
0
,11-dione (5 g, 0.029 mol) and benzylamine (3.08 g,
.029 mol) were used. The resulting product was purified by
means of crystallization from benzene to yield white crystals
yield: 0.882 g, 0.003 mol, 11.58%). C18 19NO; m.p. 159°C.
8
1
4
9
C
5
2
6
3
7
(
H
31.74 (t, 1C, C16), 29.06 (t, 1C, C15), 27.76 (t, 1C, C13), 27.59
(t, 1C, C14), 22.56 (t, 1C, C17), 14.01 (q, 1C, C18). IR (KBr, cm
1
ꢃ1
H NMR (300 MHz, CDCl
3
): d 7.70–7.00 (m, 5H, H14, H15, H16
,
)
H
H
H
H
17, H18), 5.10–4.30 (bs, 1H, OH), 3.90–3.40 (AB-q, 2H, J ¼ 13.4,
nmax: 3194, 2961, 2868, 1475, 1333, 1296, 1149. MS (EI, 70 eV)
þ
12a, H12b), 3.35–3.20 (t, 1H, J ¼ 5.3, H11), 2.85–2.20 (m, 8H, H
, H , H , H , H , H
4a, H4b). C NMR (75 MHz, CDCl
1
,
m/z: 273 (M ), 271, 256, 242, 228, 213, 200, 156, 129, 91, 77, 29.
HR-MS m/z: calc. for C18 27NO: 273.4131, found: 273.2171.
2
3
5
1
6
7
9
, H10), 1.90–1.37 (AB-q, 2H, J ¼ 10.5 Hz,
H
3
3
): d 138.87 (s, 1C, C13), 128.61
(
1
(
4
1
9
d, 2C, C15, C17), 128.36 (d, 2C, C14, C18), 128.26 (d, 1C, C16),
26.89 (s, 1C, C11), 64.78 (d, 1C, C ), 54.95 (d, 1C, C10), 51.63
t, 1C, C12), 50.72 (d, 1C, C ), 45.58 (t, 1C, C ), 44.60 (d, 1C, C ),
), 41.69 (d, 1C, C ), 41.31 (d,
General method for the synthesis of compounds 14a,c–f as
2,6
8
derivatives of azatricyclo[6.3.0.0 ]undecane (14) by
2,6
1
4
9
amination of tricyclo[6.3.0.0 ]undecane-3,11-dione (10)
2
,6
2.99 (d, 1C, C
5
), 42.20 (d, 2C, C
2
, C
6
3
Tricyclo[6.3.0.0 ]undecane-3,11-dione was dissolved in an-
hydrous THF (30 mL), stirred and cooled to 5°C on an external
ice bath. An equimolar quantity of the desired primary amine
was slowly added stoichiometrically, while the mixture was
stirred for approximately 6 h at low temperature. The reaction
was monitored on TLC till completion. The solvent was
removed in vacuo to yield the Schiff base (12) as a yellow oil.
This product was dehydrated under Dean–Stark conditions for
approximately 1 h using 40 mL anhydrous benzene. Benzene
was removed in vacuo to afford the imine (13). The resulting
oil was dissolved in anhydrous MeOH (20 mL) and anhydrous
THF (75 mL). Reduction was carried out by the addition of
ꢃ1
C, C
7
). IR (KBr, cm ): nmax 3130, 2961, 2834, 1454, 1322, 1108,
þ
21, 729. MS (EI, 70 eV) m/z: 265 (M ), 173, 131, 91, 77, 28. HR-
MS m/z: calc. for C18
H19NO: 265.3496, found: 265.1545.
2
,6 3,10
N-Phenylethyl-3-hydroxy-4-azahexacyclo[5.4.1.0 .0
0
This compound was prepared according to the general
method for derivatives of 8 described in above text. Equimolar
5
,9 8,11
.0 ]dodecane (8b)
2,6 3,10 5,9
quantities of the pentacyclo[5.4.0.0 .0 .0 ]undecane-
,11-dione (4.065 g, 0.023 mol) and phenylethylamine
2.83 g, 0.023 mol) were used. The resulting product was
purified by means of flash column chromatography (SiO , PE/
EtOAc/DCM/EtOH 1:1:1:2 as eluent) to yield white crystals
yield: 2.064 g, 0.007 mol, 32.13%). C19 21NO; m.p. 150°C;
8
(
2
3
NaBH CN, in molar excess and stirred overnight (18 h) at room
temperature. The solvent was removed in vacuo and the
resulting dark-colored oil was suspended in approximately
50 mL of distilled water in a separation funnel. The product
was extracted with DCM (4 ꢂ 20 mL) and the combined DCM
fractions were washed with distilled water (2 ꢂ 50 mL). The
(
H
1
H NMR (300 MHz, CDCl
3
): d 7.40–7.10 (m, 5H, H15, H16, H17
,
H
3
18, H19), 5.90–4.60 (bs, 1H, OH), 3.70–3.40 (t, 1H, J ¼ 5.3, H11),
1 2 3 5 6 7
.20–2.30 (m, 12H, H12a, H12b, H13a, H13b, H , H , H , H , H , H
,
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263

Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
L.-M. Young et al.
ARC HH PHARM
Archiv der Pharmazie
organic phase was dried over anhydrous CaSO
4
and filtered.
C
C
3
, C11), 53.55 (d, 2C, C
), 40.26 (t, 1C, C14), 37.07 (t, 1C, C
, C10), 32.62 (t, 2C, C , C ). IR (KBr, cm ): nmax 2944,
1
, C
2
), 51.05 (t, 1C, C12), 47.11 (d, 2C, C
6
,
The solvent was removed in vacuo. The compound was
purified with sequences of column chromatography, flash
column chromatography, and/or preparative thin-layer
chromatography.
8
7
), 34.35 (t, 1C, C13), 33.83
ꢃ1
(t, 2C, C
1444. HR-MS m/z: calc. for
281.2222.
4
5
9
20
C H27N: 281.4351, found:
2
,6
2,6
N-Benzyl-3,11-azatricyclo[6.3.0.0 ]undecane (14a)
This compound was prepared according to the general
method for derivatives of 14 as described in the above text.
N-Cyclohexylmethyl-3,11-azatricyclo[6.3.0.0 ]undecane
(14d)
This compound was prepared according to the general
method for derivatives of 14 as described in the above text.
Equimolar quantities of the tricyclo[6.3.0.0 ]undecane-3,11-
dione (2.721 g, 0.0153 mol) and cyclohexylmethylamine
(1.7304 g, 0.0153 mol) were used. The resulting light brown
oil was purified with column chromatography sequences. The
2,6
Near equimolar quantities of the tricyclo[6.3.0.0 ]undecane-
,11-dione (2.0838 g, 0.0117 mol) and benzylamine (1.333 g,
.0124 mol) were used. The resulting yellow/brown oil was
purified with column and flash column chromatography
sequences. The first purification step utilized column chro-
2
,6
3
0
matography (SiO
purification step utilized flash column chromatography (SiO
n-hexane/EtOAc/DCM 3:1:1 as eluent). The third purification
step also utilized column chromatography (SiO ; PE/Et O 5:1
2
; PE/EtOAc/EtOH 5:4:2 as eluent). The second
first purification step utilized column chromatography (SiO
PE/CHCl /EtOAc 1:1:1 as eluent). The second purification step
utilized column chromatography (SiO ; PE/THF 5:1 as eluent)
2
;
2
;
3
2
2
2
at lowered temperature (5°C). Purification afforded com-
as eluent) at lowered temperature (5°C). Purification afforded
pound as white waxy substance (yield: 0.106 g, 0.0004 mol,
1
light yellow-colored oil (yield: 0.393 g, 0.002 mol, 13.27%).
2.67%). C18
J ¼ 7.5 Hz, H
2.64 (m, 2H, H12a, H12b), 2.52–2.25 (m, 2H, H
(m, 19H, H4a, H4b, H5a, H5b, H9a, H9b, H10a, H10b, H13, H14a, H14b
H
29N; H NMR (300 MHz, CDCl
, H11), 3.25–3.00 (t, 2H, J ¼ 7.5 Hz, H
3
): d 3.65–3.37 (t, 2H,
, H ), 2.95–
), 2.22–1.60
1
C
18
H
23N; H NMR (300 MHz, CDCl
3
): d 7.40–7.09 (m, 5H, H14
,
3
1
2
H15, H16, H17, H18), 3.77–3.57 (s, 2H, H12a, H12b), 3.27–3.05 (m,
6
, H
8
2
H, H
3
, H11), 2.88–2.69 (m, 2H, H
1
, H
2
), 2.60–2.35 (m, 2H, H
6
,
,
H
8
), 2.00–1.47 (m, 8H, H4a, H4b, H5a, H5b, H9a, H9b, H10a, H10b),
H
15a, H15b, H16a, H16b, H17a, H17b, H18a, H18b), 1.60–1.47 (m, 2H,
13
13
1
(
1
(
.37–1.16 (m, 2H, H7a, H7b). C NMR (75 MHz, CDCl
s, 1C, C13), 128.73 (d, 2C, C14, C18), 127.84 (d, 2C, C15, C17),
26.41 (d, 1C, C16), 73.56 (d, 2C, C , C11), 58.52 (t, 1C, C12), 54.47
d, 2C, C , C ), 47.33 (d, 2C, C , C
, C10), 32.74 (t, 2C, C , C
3
): d 141.03
H
7a, H7b). C NMR (75 MHz, CDCl
, C ), 47.40 (d, 2C, C
40.39 (t, 1C, C12), 40.38 (t, 2C, C15, C17), 39.51 (t, C, C16), 37.22
(t, 2C, C14, C18), 32.36 (t, 2C, C , C10), 32.13 (t, 2C, C , C ), 26.91
(t, 1C, C ). HR-MS m/z: calc. for C18 29N: 259.4296, found:
259.2378.
3
): d 63.08 (d, 2C, C
3
, C11),
60.44 (d, 2C, C
1
2
6
, C ), 45.88 (d, 1C, C13),
8
3
1
2
6
8
), 37.46 (t, 1C, C
). IR (KBr, cm ): nmax 2944, 1458. MS
7
), 34.50 (t, 2C,
4
5
9
ꢃ1
C
4
5
þ
9
7
H
(
EI, 70 eV) m/z: 253 (M ), 224, 170, 162, 91, 65, 28. HR-MS m/z:
calc. for C18 23N: 253.3819, found: 253.1908.
H
2
,6
N-(3-Methoxybenzyl)-3,11-azatricyclo[6.3.0.0 ]undecane
(14e)
2
,6
N-Phenylpropyl-3,11-azatricyclo[6.3.0.0 ]undecane (14c)
This compound was prepared according to the general
method for derivatives of 14 as described in the above text.
This compound was prepared according to the general
method for derivatives of 14 as described in the above text.
In addition to the spectroscopic analysis, this compound was
characterized by means of single crystal X-ray analysis. We
reported the synthesis and characterization for this com-
pound in Young et al. [19].
2,6
Equimolar quantities of the tricyclo[6.3.0.0 ]undecane-3,11-
dione (3.44 g, 0.0193 mol) and phenylpropylamine (2.613 g,
0
.0193 mol) were used. The resulting dark brown oil
was purified with column, flash column, and preparative
plate chromatography sequences. The first purification
2
,6
step utilized column chromatography (SiO
:1 as eluent). The second purification step utilized flash
column chromatography (SiO ; PE/Et O 3:1 as eluent) at
lowered temperatures (5°C). The third purification step
utilized column chromatography (SiO ; DCM as eluent) at
2
; EtOAc/EtOH
N-(Pyridin-4-ylmethyl)-3,11-azatricyclo[6.3.0.0 ]-
undecane (14f)
9
2
2
This compound was prepared according to the general
method for derivatives of 14 as described above. Equimolar
2
,6
2
quantities of the tricyclo[6.3.0.0 ]undecane-3,11-dione
(3.87 g, 0.0217 mol) and pyridinylamine (2.351 g, 0.0217 mol)
were used. The resulting dark red/brown oil was purified with
column and preparative thin-layer chromatography sequen-
ces. The first purification step utilized column chromatogra-
lowered temperature (5°C). The fourth purification step
utilized preparative thin-layer chromatography (stationary
2
phase was SiO ; DCM/PE 9:1 as eluent). Purification afforded
red/brown-colored oil (yield: 0.1871 g, 0.0007 mol, 3.44%).
1
C
H
20
H
27N; H NMR (300 MHz, CDCl
17, H18, H19, H20), 3.40–3.27 (m, 2H, H
, H ), 3.01–2.43 (m, 6H, H
14b), 2.19–2.06 (m, 2H, H12a, H12b), 1.99–1.62 (m, 8H, H4a, H4b
3
): d 7.37–7.08 (m, 5H, H16
,
phy (SiO
utilized preparative thin-layer chromatography (stationary
phase was SiO and EtOAc as eluent). The third purification
2 2
, Et O as eluent). The second purification step
3
, H11), 3.25–3.14 (t, 2H,
J ¼ 7.5 Hz, H
1
2
6
, H
8
, H13a, H13b, H14a
,
,
2
H
H
step utilized preparative thin-layer chromatography
(reverse phase with C-18 absorbent layer; MeOH/ACN 2:1 as
eluent). Purification afforded red/brown-colored oil (yield:
5a, H5b, H9a, H9b, H10a, H10b), 1.56–1.39 (m, 2H, H7a, H7b).
C NMR (75 MHz, CDCl
13
3
): d 141.90 (s, 1C, C15), 128.34 (d, 2C,
1
C
16, C20), 125.81 (d, 2C, C17, C19), 73.11 (d, 1C, C18), 61.73 (d, 2C,
0.22 g, 0.0009 mol, 4%). C17
22
H N
2
; H NMR (300 MHz, CDCl
3
): d
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264

Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
Pentacycloundecylamines and Triquinylamines as Calcium Channel Blockers
ARC HH PHARM
Archiv der Pharmazie
8
H
2
H
.73–7.07 (dd, 4H, H14, H15, H16, H17), 3.74–3.52 (s, 2H, H12a
12b), 3.23–3.12 (m, 2H, H , H11), 2.86–2.67 (m, 2H, H , H ),
.66–2.40 (m, 2H, H , H ), 2.12–1.61 (m, 8H, H4a, H4b, H5a, H5b
9a, H9b, H10a, H10b), 1.57–1.41 (m, 2H, H7a, H7b). C NMR
): d 151.00 (s, 1C, C13), 149.40 (d, 2C, C15, C16),
22.80 (d, 2C, C14, C17), 74.33 (d, 2C, C , C11), 58.05 (t, 1C, C12),
4.57 (d, 2C, C , C ), 47.28 (d, 2C, C , C ), 37.36 (t, 1C, C ), 34.57
, C10), 32.70 (t, 2C, C , C ). IR (KBr, cm ): nmax 2944,
611. MS (EI, 70 eV) m/z: 254 (M ), 225, 180, 162, 120, 91, 67,
,
(0.425 g, 0.0112 mol) was slowly added in molar excess. The
reaction proceeded at room temperature for 1 h. The solvent
was removed in vacuo and the remaining content was
3
1
2
6
8
,
1
3
2
suspended with distilled H O (20 mL) in a separation funnel.
(
75 MHz, CDCl
3
The product was extracted with DCM (2 ꢂ 20 mL) and the
combined DCM fractions were washed with distilled water
(1 ꢂ 20 mL). The organic phase was dried over anhydrous
1
5
3
1
2
6
8
7
ꢃ1
(
t, 2C, C
4
5
9
þ
4
MgSO and filtered. Subsequent in vacuo removal afforded
1
4
2
0.475 g of the crude material, which was purified by means
of column chromatography (SiO ; EtOAc/benzene 3:17 as
eluent). The hemiacetal was obtained as white crystals (yield:
1, 28. HR-MS m/z: calc. for C17
54.1861.
22
H N
2
: 254.3700, found:
2
1
0
.081 g, 0.0004 mol, 16%). C11
(300 MHz, CDCl ): d 4.85–4.70 (m, 1H, H11), 3.60–3.10 (bs, 1H,
OH), 3.10–2.95 (m, 1H, H ), 2.82–
), 2.95–2.82 (t, 1H, J ¼ 7.5, H
2.65 (m, 1H, H ), 2.65–2.40 (m, 1H, H ), 2.20–1.40 (m, 10H, H4a
4b, H5a, H5b, H7a, H7b, H9a, H9b, H10a, H10b). C NMR (75 MHz,
CDCl ): d 118.22 (s, 1C, C ), 87.750 (d, 1C, C11), 60.70 (d, 1C, C ),
56.38 (d, 1C, C ), 47.14 (d, 1C, C ), 46.02 (d, 1C, C ), 39.63 (t, 1C,
), 37.85 (t, 1C, C
16 2
H O ; m.p. 95°C. H NMR
Method for the synthesis of N-phenylethyl-3,11-
azatricyclo[6.3.0.0 ]undecane (14b)
Tricyclo[6.3.0.0 ]undecane-3,11-dione was dissolved in an-
hydrous THF (50 mL), stirred and cooled down to 5°C on an
external ice bath. Equimolar quantities of the tricyclo-
3
2
,6
1
2
2,6
6
8
,
1
3
H
3
3
1
2,6
[
6.3.0.0 ]undecane-3,11-dione (0.179 g, 0.001 mol) and
2
6
8
phenylethylamine (0.12 g, 0.001 mol) were slowly added
stoichiometrically, while the mixture was stirred for approxi-
mately 1 h at reduced temperature. The reaction was allowed
to stir for an additional hour at room temperature. The
solvent was removed in vacuo to yield the carbinol amine (12)
as yellow oil. For this reaction, the Schiff base formed
spontaneously from the carbinol amine and we did not
perform Dean–Stark dehydration. The resulting oil was
dissolved in ice cold MeOH (50 mL). Reduction was carried
C
7
4
), 34.52 (t, 1C, C10), 32.57 (t, 1C, C
5
), 32.26
ꢃ1
(t, 1C, C
9
). IR (KBr, cm ) nmax: 3382, 2918, 1463, 1013. MS (EI,
70 eV) m/z: 180 (M ), 163, 120, 96, 91, 79, 67, 53, 41, 27. HR-MS
þ
16 2
m/z: calc. for C11H O : 180.2435, found: 180.1228.
Biological evaluation
Cell culture
Undifferentiated rat pheochromocytoma (PC12) cells
obtained from American Type Culture Collection (ATCC,
Manassas, VA, USA) were used in this study. The PC12 cell
3
out by the addition of NaBH CN in molar excess. The reaction
2þ
mixture was allowed to stir at room temperature for 1 h and
the solvent was removed in vacuo. The resulting oil was
suspended in approximately 20 mL distilled water in a
separation funnel. The product was extracted with DCM
line can be utilized to study Ca influx through the LTCC and
2
þ
can serve as a model for Ca
regulation in the nervous
2
system [30]. The cells were cultured in 75 cm tissue culture-
treated flasks containing RPMI-1640 media (Hyclone, Fisher
Scientific, USA) supplemented with 5% fetal bovine serum,
10% horse serum, 0.5% penicillin/streptomycin/amphotericin
B, and 1% (2.05 mM) L-glutamine. The medium was formu-
(
5 ꢂ 20 mL) and the combined DCM fractions were washed
with distilled water (2 ꢂ 50 mL). The organic phase was dried
over anhydrous MgSO and filtered. The subsequent in vacuo
removal of the solvent afforded a yellow oil. The compound
was purified by means of flash column chromatography (SiO
PE/EtOAc/EtOH 5:4:1 as eluent), which afforded the product as
a light yellow oil (yield: 0.072 g, 0.0003 mol, 26.9%). C19
4
2
lated for use with 5% CO at 37°C. Culture media were
changed every 3 days and cells were sub-cultivated twice a
week.
2
;
H25N;
1
H NMR (300 MHz, CDCl
3
): d 7.30–7.10 (m, 5H, H15, H16, H17
18, H19), 3.30–3.10 (m, 2H, H , H11), 2.90–2.70 (m, 4H, H , H
13a, H13b), 2.60–2.40 (m, 2H, H , H ), 2.00–1.60 (m, 8H, H4a
4b, H5a, H5b, H9a, H9b, H10a, H10b), 1.50–1.30 (m, 2H, H12a
,
Evaluation of calcium influx
5
H
H
H
H
3
1
2
,
PC12 cells were plated (1 ꢂ 10 cells/well) in a black clear-
6
8
,
,
bottom 96-well plates (Costar, Corning, NY, USA) 24 h before
experiments. Experimental techniques were similar to those in
published studies [31]. Cultures were washed and then loaded
13
12b), 1.30–1.20 (m, 2H, H7a, H7b). C NMR (75 MHz, CDCl
3
): d
TM
TM
1
40.60 (s, 1C, C14), 128.69 (d, 2C, C16, C18), 128.20 (d, 2C, C15
,
with 5 mM Fura-2/AM (Invitrogen , Molecular Probes
,
C
5
(
19), 125.66 (d, 1C, C17), 72.96 (d, 2C, C
4.74 (d, 2C, C , C ), 47.36 (d, 2C, C , C ), 34.94 (t, 1C, C13), 34.68
t, 1C, C ), 32.68 (t, 2C, C , C ), 32.66 (t, 2C, C
3
, C11), 55.00 (t, 1C, C12),
Eugene, OR, USA) in Hanks balanced salt solution (HBSS)
2
þ
2þ
1
2
6
8
containing Ca , Mg , and 10% bovine serum albumin (BSA).
Preincubation with compounds to be evaluated was con-
ducted in HBSS at increasing concentrations (0.0001–200 mM)
over a 10 min period before the application of 50 mM KCl.
Fluorescence was read on a BioTech Synergy 4 microplate
reader (Winooski, VT, USA) with the excitation wavelengths
set at 340 and 380 nm and emission wavelength set at 510 nm.
After 10 min of recording, cells were depolarized with a high
concentration of KCl (50 mM). Fluorescence intensity (ratio of
340/380 nm) was normalized for each graph to start at 1 and
7
5
9
4
, C10). IR (KBr,
ꢃ1
þ
cm ) nmax: 2949, 1453, 699. MS (EI, 70 eV) m/z: 267 (M ), 190,
1
76, 162, 151, 91, 67, 27. HR-MS m/z: calc. for C19H25N:
2
67.4085, found: 267.2065.
2
,6
3
-Hydroxy-3,11-oxatricyclo[6.3.0.0 ]undecane (11)
2,6
Tricyclo[6.3.0.0 ]undecane-3,11-dione (0.5 g, 0.0028 mol)
was dissolved in MeOH (50 mL), stirred and cooled down to
5
°C on an external ice bath. The reducing agent NaBH
4
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265

Arch. Pharm. Chem. Life Sci. 2016, 349, 252–267
L.-M. Young et al.
ARC HH PHARM
Archiv der Pharmazie
2þ
plotted as a function of time (sec). The graphs illustrate Ca
done with control of untreated cells. Where compounds were
influx through the LTCC (n ¼ 3) and the inhibitory potency of
the compounds in a dose-dependent manner. Amplitude of
suppression for each concentration was plotted as a dose–
response curve from which the IC50 values could be calculated.
The IC50 values are summarized in Table 1 and the log
concentration–response graphs are presented in Fig. 2a–t.
evaluated in the presence of H , the comparisons were
2
O
2
done with control of 200 mM H O treatment alone. The level
2
2
of statistical significance was taken at p < 0.05. Prism 3.0
(GraphPad, San Diego, CA) statistical software was used to
perform analyses.
Cell viability assays: Measurement of LDH release
Supplemental information available
Apoptosis can be assayed by evaluating the plasma mem-
brane damage of PC12 cells and measuring the amount of
intracellular lactate dehydrogenase (LDH) released in the
media. LDH leakage was measured after a 24 h exposure to
Additional graphs illustrating fluorescence inhibition profiles
are given in Appendix A. Spectroscopic graphs for compound
characterization are given in Appendix B. The InChI codes of
the new compounds are provided in a third Supporting
Information file. This material is available free of charge at
http://onlinelibrary.wiley.com/doi/10.1002/ardp.201500293/
suppinfo.
2
00 mM H
evaluated (1, 10, and 100 mM) was done 30 min prior to
treatment with 200 mM We also evaluated the
compounds in the absence of H at 10 and 100 mM to
2 2
O . A 10 min preincubation with compounds to be
2 2
H O .
2 2
O
assess if these compounds had any inherent toxicity. The
culture supernatant (100 mL) was collected from each well and
We are grateful to the Northeast Ohio Medical University,
the LDH activity was determined using a colorimetric LDH
USA for financial support.
1
cytotoxicity assay kit obtained from Cayman (Ann Arbor, MI,
USA), according to the manufacturer’s protocols. The absor-
The authors have declared no conflicts of interest.
bance was measured at 490 nm using a microplate reader
1
384
(
Molecular Devices SpectraMax 340PC
microplate reader,
Sunnyvale, CA, USA).
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Cell viability assays: Trypan blue exclusion
Conditions and treatments under which this assay was
performed were the same as for the LDH assay and cell
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2 2
H O .
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For the calcium influx assay, all experimental results are
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1
graphs drawn in Excel (Microsoft Office 2003, USA). For the
calculation of IC50 values, the analyses were performed and
graphs were drawn in Prism 3.0 (GraphPad, San Diego, CA),
and results presented as the mean ꢀ SDEV. For the LDH and
Trypan blue staining assays, all experimental results are
presented as the mean ꢀ SEM and statistical significance of
differences between the means was determined by one-way
ANOVA analysis followed by Dunnett’s post hoc test to
compare relevant groups with controls. Where compounds
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