Synthesis of pseudellone analogs and characterization as novel T-type calcium channel blockers

Article abstract of DOI:10.3390/md16120475

T-type calcium channel (Ca_v3.x) blockers are receiving increasing attention as potential therapeutics for the treatment of pathophysiological disorders and diseases, including absence epilepsy, Parkinson's disease (PD), hypertension, cardiovascular diseases, cancers, and pain. However, few clinically approved Ca_v3.x blockers are available, and selective pharmacological tools are needed to further unravel the roles of individual Ca_v3.x subtypes. In this work, through an efficient synthetic route to the marine fungal product pseudellone C, we obtained bisindole alkaloid analogs of pseudellone C with a modified tryptophan moiety and identified two Ca_v3.2 (2, IC₅₀ = 18.24 μM; 3, IC₅₀ = 6.59 μM) and Ca_v3.3 (2, IC₅₀ = 7.71 μM; 3, IC₅₀ = 3.81 μM) selective blockers using a FLIPR cell-based assay measuring Ca_v3.x window currents. Further characterization by whole-cell patch-clamp revealed a preferential block of Ca_v3.1 activated current (2, IC₅₀ = 5.60 μM; 3, IC₅₀ = 9.91 μM), suggesting their state-dependent block is subtype specific.

Full text of DOI:10.3390/md16120475

marine drugs
Article
Synthesis of Pseudellone Analogs
and Characterization as Novel T-type Calcium
Channel Blockers
Dan Wang, Pratik Neupane, Lotten Ragnarsson, Robert J. Capon * and Richard J. Lewis *
Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, the University of Queensland,
Brisbane, Qld 4072, Australia; dan.wang@imb.uq.edu.au (D.W.); p.neupane@imb.uq.edu.au (P.N.);
l.ragnarsson@imb.uq.edu.au (L.R.)
*
Correspondence: r.capon@imb.uq.edu.au (R.J.C.); r.lewis@imb.uq.edu.au (R.J.L.); Tel.: +617-3346-2979 (R.J.C.);
617-3346-2984 (R.J.L.)
+
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 8 November 2018; Accepted: 26 November 2018; Published: 28 November 2018
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: T-type calcium channel (Ca 3.x) blockers are receiving increasing attention as potential
V
therapeutics for the treatment of pathophysiological disorders and diseases, including absence
epilepsy, Parkinson’s disease (PD), hypertension, cardiovascular diseases, cancers, and pain.
However, few clinically approved Ca 3.x blockers are available, and selective pharmacological tools are
V
needed to further unravel the roles of individual Ca 3.x subtypes. In this work, through an efficient
V
synthetic route to the marine fungal product pseudellone C, we obtained bisindole alkaloid analogs
of pseudellone C with a modified tryptophan moiety and identified two Ca 3.2 (
2
, IC50 = 18.24
M) selective blockers using a FLIPR
cell-based assay measuring Ca 3.x window currents. Further characterization by whole-cell patch-clamp
µM;
V
3
, IC50 = 6.59 µM) and Ca 3.3 (2, IC50 = 7.71 µM; 3, IC50 = 3.81 µ
V
V
revealed a preferential block of Ca 3.1 activated current (
2, IC50 = 5.60
µM;
3, IC50 = 9.91
µM),
V
suggesting their state-dependent block is subtype specific.
Keywords: Ca 3.x blockers; bisindole alkaloid; marine fungal product; pseudellone C
V
1
. Introduction
Alkaloids produced by marine animals are mostly potent cytotoxins evolved for defense [1].
Marine indole alkaloids have been widely explored for their therapeutic potentials, providing potential
new drug leads for the treatment of a wide range of diseases including cancer, neurological disorders,
and parasitic infections [2,3]. However, the underlying pharmacological targets and mechanisms of
the bioactive indole alkaloids remain largely undefined. Triptans, a family of tryptamine-based drugs
used for the abortive treatment of migraine headaches, have been well established and characterized
as selective agonists of 5-HT1 B and 5-HT1 D serotonin receptors [
4]. As a result, pharmacological
studies on indole alkaloids mainly focus on the discovery of novel serotonin receptor agonists [
5].
Compounds with an indole moiety have been explored for their activities on voltage-gated ion channels
previously, and a series of tremorgenic indole alkaloids have been reported to potently block potassium
channels in smooth muscle [6]. In a more recent study, a novel aryl indole compound was identified
as a potent and selective blocker of N-type Ca 2.2, and showed robust in vivo efficacy in inflammatory
V
and neuropathic rat pain models [7].
In 2015, Liu et al.
) from the marine fungus Pseudallescheria ellipsoidea along with two other epipolythiopiperazine
alkaloids [ ]. Following this discovery, in 2017 Sathieshkumar et al. reported the total synthesis
of pseudellone C (44% overall yield) [
isolated a tryptophan-derived bisindole alkaloid pseudellone C
(1
8
9
], although the biological activity of these natural products
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Mar. Drugs 2018, 16, 475
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remains unstudied. Here, we describe an efficient total synthesis of pseudellone C and several
new bisindole alkaloid analogs. To investigate their pharmacological potential, we explored their
activity in voltage-gated calcium channels (VGCCs) using FLIPR cell-based assays, and further
characterized two potent low voltage-activated (LVA) T-type calcium channel (Ca 3.x) blockers by
V
whole-cell patch-clamp using an automated electrophysiology platform, QPatch 16 X. For the first
time, we identified two “pseudellone” bisindole alkaloids as potent and selective Ca 3.x blockers,
V
demonstrating their potential as leads for the development of new analgesic and antiepileptic agents.
2
. Results
2
.1. Synthesis of Pseudellone C and Its Bisindole Alkaloid Analogs
0
The synthesis of 2,2-bis (3,3 -indolyl) propionic acid (
4
) (85% yield) was achieved by
a Friedel–Crafts condensation of indole and pyruvic acid [10]. As tryptophan is well known
as a precursor for a wide range of pharmacologically important indole alkaloids [11 12], we employed
a one-pot synthesis of deoxy-pseudellone C ( ) by first coupling with tryptophan methyl ester
to yield (38%) [ ], followed by LiOH-mediated hydrolysis to yield the free acid 3a (85% yield),
then DCC-initiated amide coupling of 3a with methylamine to yield deoxy-pseudellone C (3a
37% yield, albeit with racemization about the single chiral center), and finally, DDQ oxidation of to
yield pseudellone C (1) (50% yield) (Scheme 1).
,
2
4
3
9
)
(
2
Scheme 1. Synthesis of pseudellone C (
1
) and analogs
2
,
3
,
3a, and
4. (a) MeCN, FeCl , r.t., 12 h,
3
◦
8
5%; (
b
) DCC, benzotriazole, TEA, DMF, 60 C, 1 h, 38%; (
c) LiOH, MeOH/H O (1:1), r.t., 5 h, 85%;
2
◦
d) DCC, methylamine, benzotriazole, DMF, 60 C, 1 h, 37%; (e) DDQ, MeOH/H O (1:1), 50%.
2
(
2
.2. Evaluation of VGCC Activities of the Synthetic Compounds Using FLIPR Cell-Based Assays
Pseudellone C ( ), along with its bisindole alkaloid analogs 3a, and its bisindole substrate 4,
were evaluated for activity on VGCCs using FLIPR cell-based assays (see Table 1).
1
2, 3,

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Table 1. Effects of pseudellone C-based bisindole compounds on low voltage-activated (LVA) and high
voltage-activated (HVA) calcium channels.
LVA Ca s (IC (µM) N = 3)
HVA Ca s (IC (µM) N = 3)
V 50
V
50
Compounds
Ca 3.1
Ca 3.2
Ca 3.3
Ca 2.2
Ca 1
V
V
V
V
V
1
2
3
53.49 ± 2.72
76.36 ± 5.39
58.57 ± 2.62
122.23 ± 5.96
72.28 ± 0.41
43.96 ± 1.60
18.24 ± 0.49
6.59 ± 0.66
26.59 ± 1.23
7.71 ± 0.23
3.81 ± 1.08
90.05 ± 4.99
119.17 ± 8.66
82.55 ± 7.60
55.29 ± 5.58
31.32 ± 3.40
146.70 ± 1.72
179.30 ± 19.68
322.00 ± 21.63
33.66 ± 2.62
39.76 ± 3.98
132.03 ± 1.63
264.30 ± 11.89
3
a
80.65 ± 6.21
132.17 ± 6.13
4
0
The 2,2-bis (3,3 -indolyl) propionic acid
4
and the bisindole alkaloid 3a showed poor inhibition
showed good potency
of all the Ca2 responses. In contrast, methyl-L-tryptophan-analog
+
3
and selectivity for Ca 3.3 currents measured in T-type window current assays with an IC value
V
50
of 3.81
±
1.08
µ
M
(n = 3), which was >8-fold better than its potency at high voltage-activated
also potently blocked Ca 3.2 responses with an IC value of 6.59 0.66 (n = 3).
), which had tryptophan moiety stabilized with a methyl
amide group, had a 2-fold reduced potency for Ca 3.3 window currents with an IC value
(HVA) Ca s:
3
±
µM
V
V
50
Comparatively, deoxy pseudellone C (
2
V
50
of 7.71
of 18.24
±
0.23
µ
M
M
(n = 3) and a ~3-fold reduced potency for Ca 3.2 window currents with an IC value
V
50
±
0.49
µ
(n = 3), compared to
3
. The natural product pseudellone C (
1), which was generated
by oxidation of the methylene group of the tryptophan moiety of deoxy pseudellone C (
further reduced potency for Ca 3.3 compared to
. The fluorescent Ca2+ responses before and after
, and their representative concentration response curves, are presented
in Figure 1 (2) and Figure 2 (3), respectively.
2), had >3-fold
2
V
addition of compounds
2 and 3
Figure 1.
of compound
) representative fluorescent traces of the Ca 3.3 window current before and after addition of compound 2;
(A) Representative fluorescent traces of the Ca 3.2 window current before and after addition
V
2; (B) the concentration-response curve for compound 2 (IC50 = 18.16 µM, n = 4);
(C
V
and (D) the concentration response curve for compound 2 (IC50 value of 7.83 µM, n = 4).

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Figure 2.
addition of compound
n = 4); (
compound
(
A
) Representative fluorescent traces of the Ca 3.2 window current before and after
V
3; (B) the concentration-response curve for compound 3 (IC50 = 5.77 µM,
C
) representative fluorescent traces of the Ca 3.3 window current before and after addition of
V
3
; and (
D) the concentration response curve for compound
3
(IC50 value of 3.05
µM, n = 4).
Interestingly, as indicated in Figure 2A,B and Figure 3, compound
3 showed potent partial
blocking of the Ca 3.2 window current. Also, pseudellone C is a weak calcium channel blocker
V
that showed partial blocking of Ca 3.1 and Ca 2.2. Partial blocking of Ca 3.x has been indicated to be
V
V
V
useful for the treatment of epileptic behaviors by reducing synchronized oscillations of thalamocortical
neurons [13 14]. Currently, compound analogs are being designed for potential in rodent models of
absence epilepsy and pain.
,
3
Figure 3.
calcium channels; (
Here, produced 63.56%
N-type Ca 2.2, and produced 75.47%
n = 3) inhibition for Ca 3.2.
(
A
) pIC50 values for bisindole alkaloids
1
,
2
,
1
3
, and 3a inhibition of T-, N- and L-type
, and 3a inhibition of calcium channels.
9.18% (n = 3) inhibition for
2.29% (n = 3) inhibition for Ca 3.1 and 56.91% 7.74%
B) Emax values for bisindole alkaloids
,
2
,
3
1
±
2.80% (n = 3) inhibition for Ca 3.1 and 39.26%
±
V
3
±
±
V
V
(
V

Mar. Drugs 2018, 16, 475
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2
.3. Electrophysiological Characterization of the Selective Ca 3.x Blockers in QPatch Assays
V
We also examined the effects of compounds
the automated electrophysiology platform QPatch 16 X (Figures 4–6). Surprisingly, both compounds
showed preferential blocking of Ca 3.1 currents ( IC50 = 5.60 0.26 M (n = 5);
and of Ca 3.2 and Ca 3.3 whole-cell
only showed partial block of the Ca 3.1 current, with an Emax
2 and 3 on the Ca 3.x by whole-cell patch-clamp using
V
2
:
±
µ
V
3
: IC50 = 9.91
currents (Emax value
value of 70.31
±
3.00
µ
M
(n = 6)). Unlike the full inhibition by
95), and
2
3
V
V
≥
2
3
V
±
2.13 (n = 6) and 82.84
±
2.55 (n = 5), respectively. Although 2 and 3 did not show
comparably good potency for the Ca 3.3 peak current compared to their effects on the Ca 3.3 window
V
V
current, low concentrations (200 nM) of 2 and 3 both showed weak inhibition (<10%) of the Ca 3.3
V
peak current.
It is noteworthy that both
2 and 3 had a significant voltage-dependent effect on steady-state
inactivation of Ca 3.2, shifting half-maximal inactivation V to hyperpolarized potentials by ~6 mV
V
50
and 11 mV, respectively (Figure 7). On average, control V50 (−65.40 ± 1.91 mV, n = 9) was shifted
to 76.36 1.56 mV (n = 5, p < 0.01) in the presence of compound , whereas in the presence of
the V50 was shifted to 72.40 1.83 mV (n = 4, p < 0.05). In contrast, the voltage-dependence of
steady-state inactivation for Ca 3.1 and Ca 3.3 was not significantly shifted in the presence of and 3.
−
±
3
2
−
±
2
V
V
Figure 4. Inhibition of Ca 3.1 current by compounds
2
and
3
. (
A
) Concentration-response curves
SEM.
20 mV) from a holding potential
of −90 mV before and after perfusions of 0.2 µM and 5.6 µM of compounds 2 and 3, as indicated.
V
of compounds
2
and
3
on recombinant hCa 3.1 channels (n = 5–6). Data are means
±
V
(
B) Representative ICa during 200 ms depolarizations to Vmax
(
−
Figure 5. Inhibition of Ca 3.2 current by compounds
2
and
3
. (
A
) Concentration-response curves
SEM.
20 mV) from a holding potential
of −90 mV before and after perfusions of 0.2 µM and 5.6 µM of compounds 2 and 3, as indicated.
V
of compounds
2
and
3
on recombinant hCa 3.2 channels (n = 4–5). Data are means
±
V
(
B) Representative ICa during 200 ms depolarizations to Vmax
(
−

Mar. Drugs 2018, 16, 475
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Figure 6. Inhibition of Ca 3.3 current by compounds
2
and
3
.
(
A
) Concentration-response
SEM.
10 mV) from a holding potential of
90 mV before and after perfusions of 0.2 µM and 5.6 µM of compounds 2 and 3, as indicated.
V
curves of compounds
2
and
3
on recombinant hCa 3.3 channels (n = 5). Data are means
±
V
(
B) Representative ICa during 200 ms depolarizations to Vmax
(
−
−
Figure 7. Comparison of the steady-state inactivation kinetics of Ca 3.2 before (black) and after (red)
V
the addition of 0.6
voltages in 5-mV increments, followed by 60-ms test pulses to −20 mV. (A) Normalized I/Imax under
conditioning steps from 110 mV to 40 mV (n = 5), along with representative elicited I under
potentials from 95 mV to 60 mV before (black) and after (red) the addition of 0.6
represent the SEM. ( ) Normalized I/Imax under conditioning steps from 110 mV to
along with representative elicited ICa under potentials from 95 mV to 60 mV before (black) and after
red) the addition of 0.6 µM of 2. Error bars represent the SEM.
µM of
2
and
3. Steps lasting 1 s were given directly from
−
110 mV to the indicated
−
−
Ca
−
−
µ
M of
−
3. Error bars
40 mV (n = 4),
B
−
−
−
(
3
. Discussion
In this study, we demonstrated that two pseudellone C-derived bisindole alkaloids,
are potent and selective Ca 3.x current blockers that showed preferential inhibition of Ca 3.3
2
and 3,
V
V
window currents measured in FLIPR assays and Ca 3.1 whole-cell currents measured in QPatch
V
assays. The FLIPR window current assay applied to Ca 3.x was permissive of a window current
V
resulting from incomplete inactivation [15]. The specific Ca 3.x window current made Ca 3.x
V
V
2+
a privileged gate for the entry of extracellular Ca during secretion, neurotransmission, and cell
proliferation [16 17]. Therefore, the inhibition of window currents in Ca 3.x should now be targeted for
the development of analgesic, antiepileptic, and anticancer drugs. Novel Ca 3.x blockers that showed
,
V
V
prominent experimental analgesic and antiepileptic efficacy [14,15,18] such as TTA-A2, Z941, and Z944,
all showed better potency for T-type window currents than the activated current generated from
the channel-closed state [14,15].
As mentioned above, tryptophan is the precursor for a wide range of pharmacologically
important indole alkaloids [11,12]. Tryptophan-derived indole alkaloids have been receiving wide
attention in drug discovery for their promising therapeutic potentials, represented by the powerful

Mar. Drugs 2018, 16, 475
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chemotherapy drugs vinblastine and vincristine [19
–21] and the antipsychotic and antihypertensive
drug reserpine [22
via oxidation of the intermediate
The acid 3a was generated from hydrolysis of
of and L-tryptophan methyl ester. The resulting suite of compounds have been evaluated for activity
–
24]. In short, the marine fungal natural product pseudellone C was produced
, which in turn was obtained from amidation of the carboxylic acid 3a
, which was in turn obtained from amide coupling
2
.
3
4
on VGCCs using FLIPR cell-based assays, and
whole-cell currents in QPatch assays.
2 and 3 were further explored for effects on Ca 3.x
V
Both compounds
2 and 3 inhibited Ca 3.3 currents in the FLIPR window current assay, but to
V
our surprise, they did not show comparable potency for Ca 3.3 whole-cell currents in QPatch
V
assays, where affinity is determined by interactions with the resting state of the channel. In contrast,
2
and
3 both showed >7-fold better potency for Ca 3.1-activated currents than Ca 3.1 window currents,
V V
whereas for Ca 3.2, these two compounds did not show any preference for Ca 3.2 window currents or
V
V
Ca 3.2-activated currents. Moreover, by measuring the voltage dependence of steady-state inactivation
V
of Ca 3.x, the addition of both
2
and
3 significantly shifted the half-maximal inactivation V50 of Ca 3.2
V
V
to more hyperpolarized potentials, whereas they did not significantly alter Ca 3.1 and Ca 3.3 channel
V
V
inactivation. In brief, these two new Ca 3.x blockers preferentially blocked either the window or
V
activated current depending on the subtype and exclusively modulated channel inactivation kinetics
for Ca 3.2, which may help in differentiating subtype-specific properties in further pharmacological
V
studies on T-type Ca 3.x.
V
In summary, through a five-step total synthesis of marine fungal product pseudellone C,
we achieved several bisindole alkaloid analogs of pseudellone C, two of which later revealed promising
and selective inhibition of T-type Ca 3.x currents. For the first time, naturally sourced bisindole
V
alkaloids were characterized as Ca 3.x blockers. Moreover, the exquisite differential inhibition of
V
compounds
2 and 3 for the three T-type subtypes makes them useful pharmacological tools to probe
the roles of individual Ca 3.x subtypes, and they could be subjected to further structural modification for
V
the development of analgesic, antiepileptic, and anticancer drug candidates through targeting Ca 3.x.
V
4
. Materials and Methods
4
.1. General Procedure for Chemical Synthesis
All reagents were used as purchased from sigma Aldrich without further purification.
Anhydrous solvents were used. Flash column chromatography was performed using ethylacetate
EtOAc) and petroleum ether as solvent. Preparative HPLC was used for the purification of
(
1
13
the compounds using water and acetonitrile containing 0.01% TFA as solvent. H and C NMR
spectra were recorded on Bruker (600 MHz) spectrometers. Data for H NMR spectra are reported
1
as chemical shift (ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
dd = doublet of doublets, dt = doublet of triplets, br s = broad singlet), and coupling constant (J in Hz).
Electrospray ionization mass spectrometry (ESIMS) experiments were carried out on a LC/MSD
(
quadrupole) instrument in both positive and negative modes. High-resolution ESIMS spectra were
obtained on a micrOTOF mass spectrometer by direct injection in MeCN at 3
formate clusters as an internal calibrant.
µL/min using sodium
4
.1.1. Synthesis of 2,2-di(1H-indol-3-yl)propanoic Acid (4)
To a solution of indole (2.34 g, 20 mmol) in anhydrous CH CN (20 mL), we added pyruvic
3
acid (1.32 g, 30 mmol, 1.5 equiv.) and FeCl (5 mol % 162 mg). The mixture was stirred under
3
nitrogen at room temperature for 12 h until TLC revealed the disappearance of the starting material.
The solvent was removed in vacuo, and the residue was purified by flash column chromatography
1
(
30% EtOAc/petroleum ether) to give
4
(2.58 g, 85%) as a reddish solid. H NMR (600 MHz, DMSO-d ):
6
δ
10.85 (s, 2H), 7.33 (d, J = 8.09, 1H), 7.31 (d, J = 8.02, 1H), 7.03 (s, 1H), 7.02 (s, 1H), 7.00 (t, J = 8.09, 2H),
.81 (t, J = 8.02, 2H), 1.97 (s, 3H). C NMR (150 MHz, DMSO-d6): δ 176.1, 136.7, 125.9, 123.0, 120.7,
13
6

Mar. Drugs 2018, 16, 475
8 of 12
+
1
20.5, 118.1, 117.9, 111.4, 29.7, 25.9. HRMS (ESI) calcd for C19 H16 N O [M + Na] 327.1104 found
2 2
m/z = 327.1096.
4
.1.2. Synthesis of L-Tryptophan Methyl Ester
To a solution of L-tryptophan (0.204 g, 1 mmol) in anhydrous MeOH (5 mL), we added thionyl
◦
chloride (0.236 g, 2 equiv.) at 0 C in dropwise and stirred for 12 h at room temperature until TLC
revealed the disappearance of the starting material. The solvent was removed in vacuo and the residue
was purified by flash column chromatography (45% ethyl acetate in petroleum ether) to give
5
1
(0.163 g, 75%) as a white solid. H NMR (600 MHz, DMSO-d6):
δ
11.14 (s, 3H), 8.66 (br s, 2H),
7
4
1
.51 (d, J = 8.12, 1H), 7.36 (d, J = 8.12, 1H), 7.25 (s, 1H), 7.08 (t, J = 7.42, 1H), 7.00 (t, J = 7.19, 1H),
.19 (t, J = 5.84, 1H), 3.63 (s, 3H) 3.36–3.27 (m, 2H). 13C NMR (150 MHz, DMSO-d6):
169.7, 136.1,
26.8, 124.9, 121.1, 118.6, 117.9, 111.5, 106.3, 52.6, 52.5, 26.0. HRMS (ESI) calcd for C H15 N O
δ
1
2
2
2
+
[M + H] 219.1128 found m/z = 219.1137.
4
.1.3. Synthesis of Methyl (2,2-di(1H-indol-3-yl)propanoyl)-L-tryptophanate (3)
A solution of (0.304 g, 1 mmol) in DMF (3 mL) with DCC (200 mg, 1.5 mmol) and HOBT
4
(
(
135 mg, 1 mmol) was stirred at room temperature. After 15 min, L-methyl ester tryptophan
0.218 g, 1 mmol) and TEA (404 mg, 4 mmol) were added, and the reaction mixture was stirred
◦
at 60 C for 1 h. After the completion of the reaction, ice-cold water was used to quench the reaction
and the mixture was extracted with ethyl acetate (3
and the residue was purified via flash column chromatography (40% EtOAc in petroleum ether)
×
15). The extract was concentrated in vacuo,
3
0
1
to yield
Acetone-d , ppm):
7
7
6
3
as an off-white solid (38%). Here, [
10.11 (s, 1H), 10.06 (s, 1H), 9.76 (s, 1H), 7.41 (d, J = 8.15, 1H), 7.39 (d, J = 8.15, 1H),
.32 (d, J = 8.15, 1H), 7.26 (d, J = 9.16, 1H), 7.26 (s, 1H), 7.24 (d, J = 8.03, 1H), 7.22 (d, J = 8.03, 1H),
α
]D
=
−
21 (c = 0.01, methanol). H NMR (600 MHz,
δ
6
.05–7.00 (m, 4H), 6.93 (t, J = 7.06, 1H), 6.81 (t, J = 8.03, 1H), 6.79 (t, J = 7.13, 1H), 6.59 (d, J = 7.13, 1H),
.10 (s, 1H), 4.77–4.74 (m, 1H), 3.57 (s, 3H), 3.10–2.96 (m, 2H), 2.85 (s, 3H). 13C NMR (150 MHz,
Acetone-d6):
δ 175.2, 173.0, 138.3, 137.4, 128.1, 127.1, 124.8, 124.6, 124.1, 122.1, 122.0, 121.9, 121.9, 121.5,
1
19.6, 119.5, 119.3, 118.8, 112.4, 112.3, 112.1, 109.8, 53.8, 52.1, 47.85, 28.0, 26.1. HRMS (ESI) calcd for C
3
1
+
H28 N O [M + Na] 527.2054 found: m/z = 527.2054.
4
3
4
.1.4. Synthesis of (2,2-di(1H-indol-3-yl)propanoyl)-L-tryptophan (3a)
A solution of (0.035 g, 0.07 mol) in 1:1 (MeOH/H O) and LiOH (3.22 mg, 2 equiv.) was stirred
for 3 h at pH 3 to yield a white solid that was filtered, dried, and purified by flash column
3
2
30
chromatography to yield 3a (29 mg, 85%) as a white solid. Here, [
α
]D
=
−
14 (c = 0.01, methanol).
1
H NMR (600 MHz, Acetone-d6):
δ 10.13 (s, 1H), 10.07 (s, 1H), 9.77 (s, 1H), 7.41–7.38 (m, 2H),
7
6
6
.33–7.30 (m, 2H), 7.29 (d, J = 2.48, 1H), 7.27 (d, J = 8.19, 1H), 7.20 (d, J = 8.04, 1H), 7.03–7.00 (m, 3H),
.98 (d, J = 2.58, 1H), 6.92–6.90 (m, 1H), 6.82–6.79 (m, 1H), 6.77–6.74 (m, 1H), 6.62 (d, J = 7.05, 1H),
13
.13 (d, J = 2.3, 1H), 4.70–4.74 (m, 1H), 3.12–3.03 (m, 2H), 2.06 (s, 3H).
175.5, 173.4, 138.3, 138.2, 137.4, 128.2, 127.1, 127.0, 124.8, 124.6, 124.1, 122.0, 121.98,
21.93, 121.8, 121.4, 119.7, 119.5, 119.4, 119.2, 119.0, 112.3, 112.4, 112.3, 112.0, 110.0, 53.6, 47.8, 28.0, 26.1.
C NMR (150 MHz,
Acetone-d6):
1
δ
+
HRMS (ESI) calcd for C30 H26 N O [M + Na] 513.1897 found m/z = 513.1886.
4
3
4
.1.5. Synthesis of (S)-N-(3-(1H-indol-3-yl)-1-(methylamino)-1-oxopropan-2-yl)-2,2
-
di(1H-indol-3-yl)propanamide (2)
A solution of 3a (0.15 g, 0.29 mmol) in DMF (3 mL) with DCC (200 mg, 1.5 mmol) and HOBT
(
135 mg, 1 mmol) was stirred at room temperature. After 15 min methyl amine (3 equiv., ethanolic
◦
solution) and TEA (404 mg, 4 mmol) was added, and the reaction mixture was stirred at 60 C for 1 h.
After the completion of the reaction, cold water was used to quench the reaction and the mixture
was extracted with EtoAc (3 × 15 mL), and the combined EtOAc concentrated in vacuo with the residue
purified by HPLC (phenomenex Luna C 250 × 21 mm, 10 µm, 20 mL/min elution, 10% to 100%
18

Mar. Drugs 2018, 16, 475
9 of 12
3
0
MeCN/H O with 0.01% TFA as modifier) yielded 2 (54 mg, 37%) as a white solid. Here, [α]
= 0.0
2
D
30
1
(
c = 0.10, methanol) and [
α
]D = 0.0 (c = 0.10, methanol). H NMR (600 MHz, Acetone-d6):
δ
7
6
3
10.17 (s, 1H), 10.07 (s, 1H), 9.89 (s, 1H), 7.45 (d, J = 8.19, 1H), 7.39 (d, J = 8.21, 1H), 7.36–7.33 (m, 2H),
.19 (d, J = 2.45, 1H), 7.10–7.05 (m, 3H), 7.01 (t, J = 7.60, 1H), 6.93 (t, J = 7.60, 1H), 6.89–6.88 (m, 2H),
.72 (d, J = 7.44, 1H), 6.68 (t, J = 7.45, 1H), 6.34 (s, 1H), 6.26–6.19 (m, 1H), 4.68 (q, J = 6.52, 1H),
.33 (s, 1H), 3.13–2.96 (m, 2H) 2.31 (d, J = 4.50, 3H), 2.04 (s, 3H). 3C NMR (150, Acetone-d6):
1
δ
175.4,
1
1
72.5, 138.3, 137.5, 128.3, 126.9, 126.6, 124.9, 124.3, 121.1, 122.0, 121.9, 121.5, 119.53, 119.52, 119.4, 119.2,
+
12.6, 112.5, 112.0, 54.7, 47.8, 28.5, 26.1, 25.9. HRMS (ESI) calcd for C31 H29 N O [M + Na] 526.2213
5
2
found m/z = 526.2228.
4
.1.6. Synthesis of Pseudellone C (1)
A solution of (35 mg, 0.07 mol) in 9:1 MeCN/H O (1 mL) was treated with DDQ
32 mg, 0.14 mmol) in 6 portions over intervals of 10 min, and the resulting mixture
2
2
(
was stirred at room temperature for 3 h, after which the residue was dissolved in EtOAc,
washed with saturated NaCl (5 mL) and water (10 mL), dried over anhydrous Na2 SO4,
and concentrated in vacuo to give residue, which was purified by HPLC (phenomenex
Luna C18 250 × 21 mm, 10 µm, 20 mL/min elution, 10% to 100% MeCN/H2 O with 0.01%
TFA as modifier) to afford pseudellone C (14.4 mg, 97%) as a white solid. Here, [α
]D30 = 0.0 (c = 0.10,
methanol) and 1H NMR (600 MHz, Acetone-d6):
δ
11.17 (s, 1H), 10.25 (s, 1H), 10.21 (s, 1H),
8
7
7
6
.49 (d, J = 3.24, 1H),
8.14 (d, J = 7.44, 1H),
7.59, (d, J = 6.07, 1H), 7.50 (dt, J = 1.03, 8.07, 1H),
.47 (m, 2H), 7.41 (d, J = 8.15, 1H), 7.38 (d, J = 8.14, 1H), 7.29 (d, J = 2.48, 1H), 7.26 (d, J = 2.47, 1H),
.23–7.20 (m, 1H), 7.19–7.15 (m, 1H), 7.10–7.07 (m, 1H), 7.05–7.01, (m, 1H), 6.96–6.91 (m, 1H),
.83–6.81 (m, 1H), 6.45 (d, J = 5.09, 1H), 5.69 (d, J = 5.97, 1H), 2.45 (d, J = 4.87, 3H), 2.14 (s, 3H).
1
3
C NMR (150 MHz, Acetone-d6):
δ 187.0, 175.4, 168.0, 138.4, 138.3, 137.6, 136.7, 127.0, 126.9,
1
26.8, 125.1, 124.9, 124.5, 124.3, 124.2, 123.1, 122.6, 122.2, 122.1, 121.6, 121.4, 119.7, 119.6, 119.5,
+
112.9, 112.7, 112.5, 62.4, 47.9, 26.2, 26.1. HRMS (ESI) calcd for C31 H27 N O [M + Na] 540.2006
5
3
found m/z = 540.2026.
4
.2. Cell Culture and Transient Expression
The human embryonic kidney 293 (HEK 293) cell lines (from Emmanuel Bourinet,
Montpellier, France) stably expressing Ca 3.2 or Ca 3.3 were cultured under 5% carbon dioxide
V
V
◦
at 37 C in Dulbecco’s Modified Eagle Medium (DMEM) Glutamax (Gibco, Life Technologies,
Carlsbad, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS), 100 U/mL
penicillin, 100
µg/mL streptomycin (Gibco, Life Technologies), and 750 µg/mL geneticin (G418)
(
Gibco, Life Technologies). The Chinese Hamster Ovary (CHO) cell lines (Emmanuel Bourinet,
◦
Montpellier, France) expressing Ca 3.1 were cultured under 5% carbon dioxide at 37 C in
V
Alpha Minimum Essential Media (MEM) Glutamax (Gibco, Life Technologies), supplemented with
10% (v/v) fetal bovine serum (FBS) and 300 µg/mL geneticin (G418) (Gibco, Life Technologies).
The human neuroblastoma SH-SY5 Y cells (Victor Diaz, Goettingen, Germany) were cultured under
◦
5
% carbon dioxide at 37 C in RPMI 1640 antibiotic-free medium (Invitrogen, Carlsbad, CA, USA),
supplemented with 15% FBS and 2 mM GlutaMAX
™ (Invitrogen). D-PBS (Gibco, Life Technologies)
was used to wash the cells, and 0.25% Trypsin-EDTA (Gibco, Life Technologies) was used to
2
detach cells from the flask surface. They were split in a ratio of 1:5 (ideally 1000 cells/cm )
when they reached 70%–80% confluence (every 2–3 days). Transiently transfected Ca 3.1HEK293
V
T cells were used in FLIPR Assays. HEK 293 T cells were cultured under 5% carbon dioxide
◦
at 37 C in DMEM supplemented with 10% (v/v) FBS. D-PBS was used to wash the cells, and 0.25%
Trypsin-EDTA was used to detach cells from the flask surface. The cells were split and seeded
at 6 million cells per T175 flask, to reach 70%–80% confluence after 24 h. The next day, 12
DNA of human Ca 3.1 was incubated in 900 L serum-free DMEM with 36 L FuGENE HD
transfection reagent (Promega Corporation, Madison, WI, US) (1:3 DNA/Fugene ratio) for 20 min,
µg
µ
µ
V

Mar. Drugs 2018, 16, 475
10 of 12
and then the mixture was added into the cell flask slowly, drop by drop. After the transfection, the cells
◦
◦
were cultured under 5% carbon dioxide at 37 C for 24 h and then moved to a 28 C incubator.
4
.3. T-Type Calcium Channel Window Current FLIPR Assays
HEK 293 cells stably expressing Ca 3.2 or Ca 3.3 were seeded into 384-well black wall clear
V
V
bottom plates (Corning, Lowell, MA, USA) at a density of 30,000 cells per well. Transiently transfected
Ca 3.1HEK293 T cells were seeded into 384-well black wall clear bottom plates at a density
V
of 60,000 cells per well. Once the cells reached 90%–95% confluence after 24 h, the media were
removed from the wells and replaced with 20
µL of 10% calcium 4 dye (Molecular Devices,
Sunnyvale, CA, USA) in HBSS-HEPES (containing 5 mM KCl, 10 mM HEPES, 140 mM NaCl,
1
0 mM glucose, and 0.5 mM CaCl , pH 7.4) with 0.1% bovine serum albumin (BSA). The cells were
2
◦
incubated for 30 min at 37 C in the presence of 5% carbon dioxide. Each well on the reagent
plates for the first addition contained 15
µL different concentrations of compounds dissolved
in HBSS-HEPES containing 0.1% BSA and <0.1% DMSO and was incubated for 20 min after
loading. Positive and negative controls contained 15
µL of HBSS-HEPES (0.1% BSA) alone.
TETRA
The plates were placed in the FLIPR
(Molecular Devices, Sunnyvale, CA, USA) programmed
to measure maximum fluorescence intensity following a second addition of the agonist 5 mM CaCl2.
The data acquisition parameters were adjusted as follows: Baseline fluorescence of 1500–2000 arbitrary
fluorescence units (AFU), emission wavelength of 515–575 nm, and excitation wavelength of
470–495 nm. HBSS-HEPES (0.1% BSA) was used in the second addition as a negative control.
One fluorescence reading was taken before the second addition. Then the fluorescence readings were
recorded every two seconds for 300 s. Raw fluorescence readings in the form of relative light units were
®
converted to response over baseline using ScreenWorks (Molecular Devices, version 3.2.0.14) software.
Using this approach, pimozide (Ca 3.2, IC = 4.2
±
1.06
µ
M (n = 3); Ca 3.3, IC = 7.71
±
0.70
µM
V
50
V
50
(
n = 3)) and mibefradil (Ca 3.2, IC = 1.02
±
0.14
µ
M (n = 3); Ca 3.3, 2.0
±
0.2 µM (n = 3)) inhibited
V
50
V
Ca 3.x calcium influx with IC s consistent with literature values [25].
V
50
4
.4. HVA Calcium Channel FLIPR Assays
SH-SY5Y cells were seeded into 384-well black wall clear bottom plates at a density of 15,000 cells
per well, resulting in 90%–95% confluence after 24 h. The media were then removed from the wells
and replaced with 20 L of 10% calcium 4 dye (Molecular Devices) in physiological salt solution (PSS)
containing 5.9 mM KCl, 1.4 mM MgCl , 10 mM HEPES, 1.2 mM NaH PO , 5 mM NaHCO , 140 mM
µ
(
2
2
4
3
NaCl, 11.5 mM glucose, and 1.8 mM CaCl , pH 7.4) with 0.1% BSA. As reported [26], for N-type calcium
2
channel FLIPR assays the cells were pre-incubated with 10 µM nifedipine added in the dye to ensure
full inhibition of L-type calcium responses. For L-type calcium channel FLIPR assays, the cells were
pre-incubated with 1
Positive control on the first reagent plate contained 15
containing 1 M CVID and 10 M nifedipine (final concentration) was used as a negative control.
The fluorescence readings were recorded and converted as described previously [26], and agonist
containing 90 mM KCl + 5 mM CaCl was used in the second addition.
µM CVID added in the dye to ensure full inhibition of N-type calcium responses.
µL of PSS (0.1% BSA), whereas PSS (0.1% BSA)
µ
µ
2
4
.5. Whole-Cell Patch-Clamp Electrophysiology
Whole-cell patch-clamp experiments were performed on an automated electrophysiology
platform QPatch 16 X (Sophion Bioscience A/S, Ballerup, Denmark) in single-hole configuration
using 16-channel planar patch chip QPlates (Sophion Bioscience A/S). The extracellular recording
solution contained, in mM: TEACl 157, MgCl 0.5, CaCl 5, and HEPES 10; pH 7.4 adjusted
2
2
with TEAOH; and osmolarity 320 mOsm. The intracellular pipette solution contained, in mM:
CsF 140, EGTA 1, HEPES 10, and NaCl 10; pH 7.2 adjusted with CsOH; and osmolarity 325 mOsm.
Compounds were diluted in extracellular recording solution with 0.1% BSA at the concentrations stated
(DMSO ≤ 0.1%), and the effects of compounds were compared to the control (extracellular solution

Mar. Drugs 2018, 16, 475
11 of 12
with 0.1% BSA) parameters within the same cell. Compounds’ incubation time varied from two
for the highest concentration) to five (for the lowest concentration) minutes by applying the voltage
(
protocol 10–30 times at 10 s intervals to ensure steady-state inhibition was achieved. The effects of
compounds were obtained using 200 ms voltage steps to peak potential from a holding potential
of
lasting 1 s from
to 20 mV. Data were fitted with a single Boltzmann distribution: I/Imax = {1 + exp[V
where V50 is the half-availability voltage and k is the slope factor. Off-line data analysis was performed
using QPatch Assay Software v5.6 (Sophion Bioscience A/S) and Excel 2013 (Microsoft Corporation,
Redmond, WA, USA).
−
90 mV. The steady-state inactivation kinetics of Ca 3.x currents were examined by applying steps
V
−
110 mV to the indicated voltages in 5-mV increments, followed by 60-ms test pulses
−
1
−
−
V ]/k}
,
5
0
4
.6. Data Analysis
Data were plotted and analyzed using GraphPad Prism v7.0 (GraphPad Software Inc.,
San Diego, CA, USA). A four-parameter logistic Hill equation with variable Hill coefficients was fitted
to the data for concentration-response curves. Data are means SEM of n independent experiments.
±
Statistical analysis was performed with a paired Student’s t-test with statistical significance at p < 0.05.
Author Contributions: D.W. and P.N. are joint first authors. Conceptualization, R.J.L. and R.J.C.; methodology,
D.W., P.N., and L.R.; formal Analysis, D.W. and P.N.; chemical Investigation, P.N.; biological investigation, D.W.;
data curation, D.W. and P.N.; writing–original draft preparation, D.W. and P.N.; writing–review and editing, R.J.L.
and R.J.C.; supervision, R.J.L., L.R., and R.J.C; funding acquisition, R.J.L.
Funding: This research was funded by the NHMRC Program, grant number APP1072113, and the APC was funded
by the NHMRC Principal Research Fellowship.
Acknowledgments: The authors thank Emmanuel Bourinet for donating stable cell lines for T-type calcium
channels and Gerald W. Zamponi for donating plasmids for T-type calcium channels.
Conflicts of Interest: The authors declare no conflicts of interest.
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©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
CC BY) license (http://creativecommons.org/licenses/by/4.0/).
(