Synthesis and Biological Evaluation of Falcarinol-Type Analogues as Potential Calcium Channel Blockers

Article abstract of DOI:10.1021/acs.jnatprod.1c00136

A series of enantiomers of falcarinol analogues (2) were synthesized using a chiral 1,1′-binaphth-2-ol (BINOL)-based catalytic system. The neuroprotective effects of falcarinol (1a) and its analogues (2) on PC12 cells injured by sodium azide (NaN3) were investigated. The structure-function relationships and possible mechanism were studied. Pretreatment of PC12 cells with falcarinol analogues (R)-2d and (R)-2i for 1 h following addition of NaN3 and culture in a CO2 incubator for 24 h resulted in significant elevation of cell viability, as determined by a CCK-8 assay and Hoechst staining, with reduction of LDH release and MDA content, increase of SOD activity, and decrease of ROS stress, when compared with the activity of natural falcarinol (1a). These observations indicated that the falcarinol analogues (R)-2d and (R)-2i can protect PC12 cells against NaN3-induced apoptosis via increasing resistance to oxidative stress. For the first time, falcarinol (1a) and its analogue (R)-2i were found to have potential L-type calcium channel-blocking activity, as recorded using a manual patch clamp technique on HEK-293 cells stably expressing hCav1.2 (α1C/β2a/α2δ1). These findings suggest that the mechanism of the L-type calcium channel-blocking activity of falcarinol (1a) and its analogue (R)-2i might be involved in neuroprotection by falcarinol-type analogues by inhibiting calcium overload in the upstream of the signaling pathway.

Full text of DOI:10.1021/acs.jnatprod.1c00136

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Article
Synthesis and Biological Evaluation of Falcarinol-Type Analogues as
Potential Calcium Channel Blockers
Yang Li,# Wan-Li Tan,# Kai Guo, Xiao-Wei Gao, Jun Wei, Dong Yi, Chun Zhang,* and Qin Wang*
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ABSTRACT: A series of enantiomers of falcarinol analogues (2)
were synthesized using a chiral 1,1′-binaphth-2-ol (BINOL)-based
catalytic system. The neuroprotective effects of falcarinol (1a) and
its analogues (2) on PC12 cells injured by sodium azide (NaN3)
were investigated. The structure−function relationships and
possible mechanism were studied. Pretreatment of PC12 cells
with falcarinol analogues (R)-2d and (R)-2i for 1 h following
addition of NaN3 and culture in a CO2 incubator for 24 h resulted
in significant elevation of cell viability, as determined by a CCK-8
assay and Hoechst staining, with reduction of LDH release and
MDA content, increase of SOD activity, and decrease of ROS
stress, when compared with the activity of natural falcarinol (1a).
These observations indicated that the falcarinol analogues (R)-2d
and (R)-2i can protect PC12 cells against NaN3-induced apoptosis via increasing resistance to oxidative stress. For the first time,
falcarinol (1a) and its analogue (R)-2i were found to have potential L-type calcium channel-blocking activity, as recorded using a
manual patch clamp technique on HEK-293 cells stably expressing hCav1.2 (α1C/β2a/α2δ1). These findings suggest that the
mechanism of the L-type calcium channel-blocking activity of falcarinol (1a) and its analogue (R)-2i might be involved in
neuroprotection by falcarinol-type analogues by inhibiting calcium overload in the upstream of the signaling pathway.
alcarinol (1a) is a C17 polyacetylenic chiral alcohol with
Fthe R-configuration, which is distributed widely in food
plants belonging to the family Apiaceae (e.g., carrots, celery,
parsnips, and coriander) and also in medicinal plants from the
family Araliaceae (e.g., Panax ginseng and Panax notoginseng).1
The polyacetylenic falcarinol-type compounds are character-
ized by a conjugated diyne-carbinol structural motif and a long
hydrocarbon chain and have been reported to possess different
bioactivities.2,3 Due to its lipophilic nature and rapid
absorption in humans, compound 1a is believed to have
potential neuroprotective and neurotrophic effects since it may
cross the blood−brain barrier and enter the brain.4 Lu et al.
reported the neurotrophic effects of 1a on PC12 cells and the
mechanism involved in the neurite outgrowth of these cells.5
Furthermore, the protective effect of 1a on sodium nitroprus-
side (SNP)-induced neuronal apoptosis was investigated in
primary cultured cortical neurons.6 The potential mechanism
suggested that 1a can protect neurons against exogenous nitric
oxide (NO)-induced toxicity via regulating apoptotic and
antiapoptotic proteins, where the deleterious effects of NO
production have been associated with neurodegenerative
conditions such as Alzheimer’s disease (AD).6 The protective
effect of 1a on Aβ25−35-induced neuronal apoptosis was also
verified, and its antiapoptotic mechanism involved the
inhibition of calcium influx and free-radical generation.7
These results raise the possibility that 1a might reduce
neurodegeneration in the AD brain. Compound 1a was also
identified as a reversible agonist of the cannabinoid receptor
CB1 and is capable of selectively alkylating the anandamide
binding site.8 Falcarindiol (1b), with two stereogenic hydroxy
groups, was found to show a potent allosteric modulatory
action on GABAA receptors, possibly by modulating agonist
binding and desensitization, but 1a possessed a higher affinity
for GABAA receptors and also a substantially different
pharmacological action than 1b.9,10 Oenanthotoxin (1c) and
dihydrooenanthotoxin (1d), with fully conjugated diyne-diene
moieties and delocalized hydroxy groups, were both found to
show a complex blocking mechanism of GABAA receptors.11
The inhibitory effects of 1c and 1d on GABAergic currents are
strongly dependent on polarity.12 These findings indicate that
falcarinol-type analogues might affect the functioning of the
central nervous system by interfering with receptors for key
neurotransmitters. L-Type calcium channels mainly exist in the
cell body of central neurons and proximal dendrites. The
Received: February 9, 2021
© XXXX American Chemical Society and
American Society of Pharmacognosy
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mechanism of calcium homeostasis suggests that the calcium
disturbances in AD could lead to calcium overloading in the
mitochondria, which results in the opening of mitochondrial
permeability-transition pores and the release of calcium
stored.13,14 Accordingly falcarinol (1a) and its analogues also
may interact with L-type calcium channels as potential targets
for treating calcium dysregulation.
substituted 1,3-diyne to a variety of aromatic and aliphatic
aldehydes, as shown in Scheme 1. The catalysis using BINOL
in combination with Ti(OiPr)4, Zn powder, and EtI avoids the
direct use of the spontaneously combustible ZnEt2, making it
possible to conduct a much safer operation in large scale of
industrial production under very mild reaction conditions.15,18
This method was applied to the preparation of falcarinol
analogues in this investigation for the first time. As shown in
Table 1, the optically active conjugated diynecarbinols with a
phenyl group, including ortho-, meta-, or para-substituents,
were obtained in excellent enantioselectivities and high yields
(entries 1−11). High enantioselectivities and high yields were
observed for the addition of a TIPS-substituted alkyne to α-
branched aliphatic aldehydes and α,β-unsaturated aldehydes
(Table 1, entries 12−16). The R- or S-configuration of each
product was determined by comparing the optical rotation and
HPLC data with literature values (Table 1).15,19,20
The rat PC12 pheochromocytoma cell line is a well-
established model for the investigation of neuronal cell
responses of differentiation, proliferation, and survival. It was
reported that falcarinol (1a) has neuroprotective effects on
PC12 cells.5 An excess of NO is viewed as a deleterious factor
involved in various neurodegenerative disorders.21,22 In the
present study, a model of PC12 cell injury induced by NaN3 as
a NO donor was established using a CCK-8 assay. The survival
rate of PC12 cells exhibited a dose-dependent relationship with
NaN3, as shown in Figure S1, Supporting Information. When
PC12 cells were treated with 0 and 36 mM NaN3 for 24 h,
their survival rates were about 100% in the control group and
50% in the model group. As shown in Table S1, Supporting
Information, pretreatment of the cells with 1−32 μM falcarinol
(1a) for 1 h following addition of NaN3 and culturing in a CO2
incubator for 24 h resulted in an increase of cell viability, and
2−8 μM falcarinol (1a) protected well PC12 cells from injury
by NaN3, with a ca. 59% survival rate, while higher
concentrations (16 and 32 μM) of 1a led to a gradual
decrease in its protective effect.
Our group recently conducted the asymmetric synthesis of
several chiral falcarinol analogues, (R)-2, with a conjugated
diynol moiety and a triisopropylsilyl (TIPS) group, using a
chiral amino alcohol [(1S,2S)-2-N,N-dimethylamino-1-(p-
nitrophenyl)-3-(tert-butyldimethylsilyloxy)propan-1-ol)]-based
catalytic process, which exhibited potential antiproliferative
activity against certain cancer cell lines.15 Several optically
active falcarinol analogues were also prepared using this chiral
1,1′-binaphth-2-ol (BINOL)-based catalytic system.15−17 Since
the TIPS (C9 + Si) group of these compounds is lipophilic,
similar to that of the linear chain (C10) of falcarinol (1a), these
falcarinol analogues also may possess neuroprotective effects.
In the present study are reported the asymmetric synthesis of
(R)- and (S)-falcarinol analogues 2 with (S)- or (R)-BINOL-
based catalysts, the neuroprotective effects of 1a and its
analogues 2 against NaN3-induced toxicity on PC12 cells,
structure−function relationships, and mechanistic investiga-
tions including lactate dehydrogenase (LDH) release,
malondialdehyde (MDA) content, superoxide dismutase
(SOD) activity, and reactive oxygen species (ROS) stress, as
well as the potential calcium channel-blocking activity.
Next, the protective effects of (R)- and (S)-falcarinol
analogues 2a−p against NaN3-induced toxicity on PC12 cells
were investigated using an established CCK-8 assay. These
results of cell viability from 32 samples at various
concentrations (5, 10, 20, 40, 80, 100 μM) are listed in
Table S2, Supporting Information, as the treatment group
relative to the control group and the model group. As shown in
RESULTS AND DISCUSSION
■
(R)- and (S)-Falcarinol analogues 2a−p were synthesized
using the readily available C2-symmetric (S)- and (R)-BINOL
ligand system, to catalyze the asymmetric addition of TIPS-
Scheme 1. Asymmetric Synthesis of (R)- and (S)-Falcarinol Analogues 2 Using the (S)- and (R)-BINOL-Based Catalytic
System
B
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Table 1. Products (2a−p) from Asymmetric Addition of Diynes to Aldehydes in the Presence of (S)- and (R)-BINOL
C
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Table 1. continued
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Table 1. continued
a
b
The R-configuration of the product was determined by comparing the optical rotation and HPLC data with literature values.15,19,20 Each ee value
1
was determined by H NMR spectroscopy after using a chiral derivatization reagent (see Supporting Information).
Table S2, 32 falcarinol analogues exhibited different cell
viabilities relative to the control group, and lower concen-
trations (5−10 μM) of 2, in general, gave higher cell survival
rates. Compared with the falcarinol (1a) positive control group
(Table S1, Supporting Information), the analogues (R)-2d and
(R)-2i had better neuroprotective effects on the injured cell
model, with 83% (76%) and 60% (67%) cell viabilities at 5 μM
(10 μM) (Table S3, Supporting Information). In addition, the
individual analogues (R)-2k and (R)-2n also exhibited
neuroprotective activities at a higher concentration of 20 μM
(40 μM), with 61% (65%) and 66% (65%) cell viabilities
(Table S3, Supporting Information).
confirmed that the lipophilic group is indispensable for the
neuroprotective effects of analogues (R)-2i (60−67%) and
(R,R)-6 (64−66%), with ether a TIPS group or a linear
aliphatic chain under low concentration levels (5−10 μM).
Moreover, analogue (R)-2i, with a TIPS group (52−58%), led
to a higher potential for cell survival than (R,R)-6, with a linear
aliphatic chain (31−52%), at high concentration levels (40−
100 μM). Additionally, (R)-2i, containing a benzene ring, also
displayed a higher neuroprotective effect than (R)-4 and (R)-5,
containing a C10 linear aliphatic chain, as a synthetic
intermediate of the falcarindiol analogue (R,R)-6 (Figure
S2b, Supporting Information).
The cell viabilities of falcarinol analogues 2 and possible
structure−activity relationships were compared (Figure 1). In
Figure 1A, the benzene ring-substituted analogues (R)-2i
resulted in higher cell viabilities than the vinyl, styryl, and α-
branched aliphatic substituted analogues 2l−p at 5 μM. In
turn, the aromatic-substituted analogues 2a−k, (R)-2d, with a
meta-fluoro substituent, displayed higher neuroprotective
effects for PC12 cells injured by NaN3 than compounds 2a
and 2g, with either an ortho- or para-fluoro substituent, as
shown in Figure 1B, in addition to compounds 2e, 2f, and 2k
containing a meta-chloro, bromo, or methoxy group in the
phenyl ring. Comparison of both enantiomers of certain
falcarinol analogues demonstrated that (R)-2d, (R)-2i, and
(R)-2k gave much greater survival rate values in the injured
PC12 cell model than their opposite enantiomers (S)-2d, (S)-
2i, and (S)-2k, as shown in Figure 1C.
It has been reported that falcarinol (1a) not only exhibits
neuroprotective effects on injured cortical neurons but also
promotes neurite outgrowth and the expression of the
differentiation marker of PC12 cells.5−7 Thus, the neuro-
trophic effects of falcarinol (R)-1a and its analogues, (R)-2d,
(R)-2i, and (R)-2k, on PC12 cells were investigated by their
cell viability index in the CCK-8 assay. As shown in Table S4
(Supporting Information), the falcarinol analogues, (R)-2d,
(R)-2i, and (R)-2k, exhibited dose-dependent neurotrophic
effects on PC12 cells in a manner similar to that of falcarinol
(1a). At low concentrations (1−10 μM), analogues (R)-2d,
(R)-2i, and (R)-2k gave cell survival rates of 81−96% at 24 h,
among which (R)-2i exhibited the best neurotrophic effects on
PC12 cells, with a cell viability rate up to 96% at a
concentration of 1 μM.
Hoechst staining was performed to visualize the extent of
cell apoptosis. The neuroprotective effects of (R)-2d and (R)-
2i on apoptotic PC12 cells induced by NaN3 was determined
by Hoechst 33258 staining (Figure S3, Supporting Informa-
tion). As shown in Figure 2, the falcarinol analogue (R)-2d at
concentrations of 5 and 10 μM attenuated NaN3-induced
apoptosis of PC12 cells, and the percentage of positively
stained cells showed good consistency and hallmarks of
apoptotic death such as chromatin condensation and
fragmentation.
Lactate dehydrogenase release into the medium is a good
indicator of the degree of cellular damage. The neuroprotective
effects of falcarinol (1a) and its analogues, (R)-2d and (R)-2i,
on NaN3-induced PC12 cell death was investigated using an
LDH release assay (Table S5, Supporting Information). As
shown in Figure 3, the PC12 cell injury induced by NaN3
resulted in a significant LDH release (412 or 417 U/L).
Pretreatment with falcarinol (1a) (4 μM) reduced the LDH
release to 211 U/L. Concentration of analogues (R)-2d and
(R)-2i from 5 to 10 μM lowered NaN3-induced toxicity in
PC12 cells with LDH release from 132 to 270 U/L, and
analogue (R)-2i at 5 μM protected PC12 cells from NaN3-
induced toxicity with an LDH content down to 132 U/L.
It has been reported that falcarinol (1a) can also alleviate
early-stage neuronal degeneration induced by the toxic
fragment of fibril Aβ25−35. Its mechanism involved the
inhibition of intracellular free-radical generation and extrac-
ellular calcium overload.7 Several studies have shown that NO-
Given the relationship between its lipophilic structure and
neurotrophic effects of (R)-2i, the neuroprotective effects were
tested of the falcarinol analogues (R)-2i and (R)-3 and the
falcarindiol analogue (R,R)-6 in the injured PC12 cell models
by removal of the lipophilic TIPS (C9 + Si) group of (R)-2i
and replacement with a C10 linear aliphatic chain. As shown in
Table 2, the low cellular viability for analogue (R)-3 (48−52%)
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Figure 1. Cell viability of the falcarinol analogues 2 and their structure−activity relationships. (A) Aromatic-substituted (R)-2i and aliphatic-
substituted 2l−p; (B) aromatic substituents containing ortho-, meta-, and para-fluoro phenyl groups, 2a, 2d, and 2g, and containing an electron-
withdrawing group in the meta-position, 2e, 2f, and 2k; (C) aromatic-substituted (S)-2d, (R)-2d, (S)-2i, (R)-2i, (S)-2k, and (R)-2k. x s, n = 3.
*p < 0.05, **p < 0.01 vs model group.
induced toxicity engendered by its reaction with the super-
oxide anion radical yields the highly cytotoxic ROS
peroxynitrite.21 Peroxynitrite then decomposes to form
nitrogen dioxide and hydroxyl radicals, leading to lipid
peroxidation, protein oxidation, and DNA damage.22 MDA
has been studied extensively as an indicator of lipid
peroxidation with manifestations of oxygen toxicity. As
shown in Figure 4, a significant increase of MDA levels (253
nmol/mL) compared to the control group was observed in a
model group of PC12 cell injury induced by NaN3.
Pretreatment with the analogues (R)-2d and (R)-2i at a
concentration of 5 μM decreased greatly the content of MDA
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Table 2. Effects of Various Concentrations of Analogues (R)-2i, (R)-3, (R)-4, (R)-5, and (R,R)-6 on PC12 Cell Injury Induced
a
by NaN3
analogue
control
model
5 μM
10 μM
20 μM
40 μM
80 μM
100 μM
b
b
b
b
b
b
(R)-2i
(R)-3
(R)-4
(R)-5
100 2.2
100 0.7
100 1.8
100 0.9
100 1.9
49.9 1.0
51.5 2.1
50.0 1.0
50.6 0.5
52.0 2.3
60.1 1.2
47.8 2.1
49.4 0.8
50.4 1.0
65.6 1.8
66.6 2.5
52.4 2.5
47.8 1.2
47.9 1.3
63.7 2.4
58.0 1.8
55.0 1.2
46.2 2.4
47.5 1.2
61.3 1.9
58.2 2.9
49.9 1.5
43.5 1.8
46.4 0.6
51.7 2.8
53.0 1.5
47.5 1.2
39.5 1.4
46.4 0.4
36.2 2.4
51.9 1.3
43.5 1.5
37.6 1.6
43.9 1.9
31.3 1.8
b
b
b
b
a
(R,R)-6
a
b
Synthesis of falcarinol and falcarindiol analogues (R)-3, (R)-4, (R)-5, and (R,R)-6 (see the Supporting Information). x s, n = 3. **p < 0.01 vs
control group.
Figure 4. Effects of (R)-2d and (R)-2i on MDA in NaN3-induced
PC12 cells (x s, n = 3; **p < 0.01 vs model group). The MDA
activity for falcarinol (1a) at 4 μM was 68 nmol/mL.
Figure 2. Apoptosis morphology of PC12 cells determined by
Hoechst 33258 staining in the presence of 5 and 10 μM (R)-2d.
Figure 5. Effects of (R)-2d and (R)-2i on SOD in NaN3-induced
PC12 cells (x s, n = 3; **p < 0.01 vs a model group). The activity
for falcarinol (1a) at 4 μM was 213 U/mg pro.
Figure 3. Effects of (R)-2d and (R)-2i on LDH release in NaN3-
induced PC12 cells (x s, n = 3; **p < 0.01 vs a model group). LDH
activity for falcarinol (1a) at 4 μM was 211 U/L.
dose-dependently when PC12 cells were preincubated with
analogues (R)-2d and (R)-2i at concentrations of 5−20 μM
with 124−265 U/mg pro (Table S7, Supporting Information).
These findings indicate that analogues (R)-2d and (R)-2i may
protect PC12 cells against NaN3-induced cell injury, mainly via
increasing resistance to oxidative stress.
It was examined as to whether the protective effect of (R)-2d
and (R)-2i on NaN3-induced oxidative stress is due to their
inhibition on the excessive production of intracellular free
radicals generated by a ROS-dependent up-regulation of an
(53 and 47 nmol/mL, Table S6, Supporting Information),
which shows their higher potential for oxidation resistance
than falcarinol (1a) (68 nmol/mL). Furthermore, superoxide
dismutase is the primary defense system to protect biological
systems from oxidative stress. As shown in Figure 5, exposure
of PC12 cells to NaN3 in the model reduces significantly the
activity of SOD (54 U/mg pro) as compared to the control
group. However, the decrease of SOD activity was attenuated
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endogenous antioxidant system. As shown in Figure 6, the
exposure of PC12 cells to NaN3 in the model resulted in a
those of its analogue, (R)-2i, were 7.7 2.4%, 38.6 6.9%,
and 79.7 8.4%, respectively (Figure S6F, Supporting
Information). These results showed that falcarinol (R)-1a
and its analogue (R)-2i can inhibit rapidly the opening of L-
type calcium channels in a concentration-dependent manner,
with IC50 values of 67.3 and 41.3 μM. Thus, the mechanism of
the neuroprotective effect of the falcarinol-type analogues may
be correlated with L-type calcium channel-blocking activity by
inhibiting calcium overload, which subsequently reduces cell
apoptosis.
EXPERIMENTAL SECTION
■
General Experimental Procedures. All reactions were per-
formed under inert gas conditions unless otherwise specified.
Falcarinol [(R)-1a] was purchased from Shanghai YuanMu. Other
chemicals were from commercial sources and were used directly
without purification. Methylene chloride, tetrahydrofuran, and diethyl
ether were purified by MBraun SPS-800-Systems, and the other
solvents used were dried by standard methods prior to use. Optical
rotations were obtained by using a Hanon P810/P850 automatic
polarimeter with a sodium λ = 589.3 nm filter. The NMR data were
recorded with a Bruker TM400 NMR spectrometer. HRMS (QTOF)
were measured on a SCIES-X500r instrument. HPLC analyses were
performed with a Waters 1525 apparatus using Diacel Chiralcel OD-
H, Chiracel AD-H, and Chiralcel AS-H columns, with detection at λ =
254 or 220 nm on a Waters 2487 instrument.
Figure 6. Effects of (R)-2d and (R)-2i on ROS level in NaN3-induced
PC12 cells as determined using flow cytometric analysis (x s, n = 3;
**p < 0.01 vs a model group). The ROS level for falcarinol (1a) at 4
μM was 5098.
significant increase of the ROS level (8250) in the PC12 cells,
as determined using flow cytometric analysis and comparison
to the control group (6197) (Table S8, Supporting
Information). The increase of ROS levels could be attenuated
significantly by pretreatment with analogues (R)-2d (3842)
and (R)-2i (3794) at a concentration of 5 μM (Table S8,
Supporting Information), which both exhibited a better
resistance to oxidative stress than falcarinol (1a) (5098).
Calcium overload can activate calcium-dependent proteases
and nitric oxide synthase, destroy mitochondria, and result in
neuronal excitotoxicity.23,24 These effects might derive from
the formation of ion channels within the cell membrane by
toxic fragments of fibril Aβ, fostering a direct leakage of
calcium into cells.25 Some studies have shown that several L-
type calcium channel blockers may alleviate neuronal
degeneration in early-stage AD therapy.26,27 Therefore, in the
above-mentioned mechanism investigation, the falcarinol-type
analogues might act as targeted molecules for L-type calcium
ion channels and play a blocking role in the upstream of the
cell pathway. As shown in Figure 7, the L-type calcium
channel-blocking activity of falcarinol (R)-1a and its analogue
(R)-2i was found for the first time using the manual patch
clamp technique on HEK-293 cells stably expressing hCav1.2
(α1C/β2a/α2δ1). In the presence of 100 μM falcarinol (R)-
1a, the membrane current (ICa‑L) decreased to 38.4% of its
original value, but was restored to 50.8% of this value after
being washed out with the external solution (Figure 7B). In the
presence of 100 μM (R)-2i, the ICa‑L value decreased to 20.3%
of its original value but was not restored after being washed out
(Figure 7D). These results showed that the inhibitory effect of
falcarinol (R)-1a on the ICa‑L value was partially reversible,
Synthesis, Isolation, and Purification of Falcarinol Ana-
logues (2). A typical procedure for the asymmetric addition of diynes
to aldehydes by using (R)- or (S)-BINOL is as follows: Under argon,
Zn powder (5.1 mmol, 10.2 equiv) and (R)- or (S)-BINOL (0.34
mmol, 0.68 equiv) were added to a 25 mL flask equipped with a
mechanical stirrer. Then, EtI (10.2 mmol, 20.4 equiv), Ti(OiPr)4
(0.85 mmol, 1.70 equiv), and an alkyne (1 mmol, 2 equiv) were
added dropwise using a syringe. After 5 min, tetrahydrofuran (1 mL)
was added, and the mixture was stirred at room temperature for 24 h.
Then, diethyl ether (15 mL) was added, followed by the addition of
an aldehyde (0.5 mmol, 1 equiv) after 1 h. The mixture was stirred for
12 h, and then it was quenched with aqueous saturated NH4Cl (5
mL). The solution was extracted with CH2Cl2 (3 × 30 mL), and the
combined organic phase was dried (Na2SO4), filtered, and
concentrated under reduced pressure. The crude product was purified
by column chromatography on silica gel to afford the desired
compound 2.
Culture of PC12 Cells. PC12 cells were provided by Shanghai
Cell Bank of the Chinese Academy of Sciences. Cells were cultured in
RIMP 1640 culture medium (Gibco) containing 10% inactivated fetal
bovine serum (FBS, Sijiqing Biological Engineering Materials Co.,
Ltd., Hangzhou, People’s Republic of China), penicillin (100 IU/
mL), streptomycin (100 mg/mL), and L-glutamine (2 mmol/L) in a
thermostatic incubator (Thermo) (setting: 37 °C, 5% CO2). When
the cell confluence reached 80−90%, cells were digested with 0.25%
trypsin and passaged by a ratio of 1:3.
Cell Viability Assay by the CCK-8 Method. The viability of
PC12 cells was determined using a cell counting kit-8 (CCK-8) assay
(Dojindo Molecular Technologies, Inc., Kumamoto, Japan), accord-
ing to the manufacturer’s instructions. PC12 cells were cultured in 96-
well plates at 37 °C under an atmosphere of 5% CO2 and 95% air. At
the end of the treatment, CCK-8 reagent (10 μL) was added to each
well, and the plates were then incubated at 37 °C for 0.5 to 2 h in the
incubator. Absorbance at a wavelength of 450 nm was measured with
a Spectra Max M3 microplate reader (Molecular Devices, USA). The
mean optical densities (ODs) from four wells in the indicated groups
were used to calculate the cell viability, which was expressed as a
percentage of the cell survival rate when compared with the control
group. All experiments were performed in triplicate and repeated
three independent times.
whereas its analogue (R)-2i irreversibly affected the ICa‑L
.
The time course of ICa‑L was changed progressively by the
gradient concentrations of 10, 30, and 100 μM of falcarinol
(R)-1a and its analogue (R)-2i, which acted rapidly on the L-
type calcium channel (Figures S4−S6, Supporting Informa-
tion). The inhibition rates by falcarinol (R)-1a at 10, 30, and
Establishment of a Model of PC12 Cell Injury Induced by
NaN3. The injury model was constructed in the following manner. In
brief, the RPMI 1640 medium was removed, and PC12 cells were
100 μM were 7.9
3.2%, 28.1
8.5%, and 61.6
6.7%,
respectively (Figure S6C, Supporting Information), while
H
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Figure 7. L-Type calcium channel-blocking activity of falcarinol (R)-1a and its analogue (R)-2i at a concentration of 100 μM. (A) Time course for
(R)-1a. (B) Current traces for (R)-1a. (C) Time course for (R)-2i. (D) Current traces for (R)-2i.
washed twice with glucose-free Earle’s balanced salt solution (PBS) at
pH 7.5 and then maintained in glucose-free RPMI 1640 without FBS.
Subsequently, neurotoxic damage was induced by adding the
indicated concentration of NaN3 to the cultured cells for 24 h.
Evaluation of Protective Effects of Falcarinol (1a) and Its
Analogues (2). Cells were preincubated with the indicated
concentrations of falcarinol (1a) and its analogues (2) for 1 h prior
to NaN3 treatment and maintained throughout the entire experiment.
Falcarinol (1a) was dissolved in DMSO and was freshly prepared
immediately prior to use. Analogues 2 were dissolved in absolute ethyl
alcohol and were freshly prepared immediately prior to use. The stock
solutions were added directly into the bath solution to achieve the
final concentration. Control cultures were maintained in RPMI 1640
for the same duration under normoxic conditions. The concentrations
of all the reagents were maintained throughout the injury period.
Hoechst 33258 Fluorescence Staining for Morphological
Observations of PC12 Cell Apoptosis. PC12 cells in the
logarithmic growth phase were taken and inoculated in a six-well
culture plate at a density of about 1 × 105 cells per well. After
culturing overnight at 37 °C and in a 5% CO2 incubator, the medium
was discarded, following treatment with NaN3 for 24 h in the
presence or absence of the 5, 10, and 20 μM falcarinol analogues, (R)-
2d and (R)-2i, also including a control group and a model group. The
cells were washed twice with PBS and fixed in 0.5 mL of methanol per
well for 30 min. The fixed cells were incubated with 1 mL of Hoechest
33258 staining solution for 10 min at 37 °C in the dark. After removal
of the staining solution, the cells were washed twice with PBS and
then observed using a fluorescence microscope (FL CD12-002, AMG,
USA), with an excitation wavelength of 350 nm and an emission
wavelength of 461 nm. For cell counts, five random fields (about 100
cells each field) were observed per coverslip. The results were
expressed as percentages of Hoechst-positive nuclei (condensed or
fragmented) relative to the total number of nuclei counted per
coverslip for each experimental condition.
LDH Release, MDA Content, and SOD Activity Assays. The
content of LDH and the activity of SOD and MDA released into the
medium were evaluated with commercial detection kits (NJJC Bio).
In brief, the cells were ruptured using a ultrasonic cell disruptor and
centrifuged at 10 000 rpm for 10 min at 4 °C. The supernatant of each
well was transferred to a fresh flat-bottomed 96-well culture plate and
processed further for enzymatic analysis, as per the manufacturer’s
instructions. Each experiment was repeated three times independ-
ently.
ROS Levels Detected by Flow Cytometry. Production of
intracellular ROS was determined using the fluorescent probe
dichlorofluorescein diacetate (DCFH-DA), which can cross cell
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membranes and is hydrolyzed subsequently by intracellular esterase to
nonfluorescent DCFH (Beyotime Institute of Biotechnology). PC12
cells were seeded in a six-well plate, with a cell density of 1 × l05 cells/
mL. Following treatment with NaN3 for 24 h in the presence or
absence of 5, 10, and 20 μM of the falcarinol analogues (R)-2d and
(R)-2i, the culture medium was changed to fresh RPMI 1640
containing 10 μM DCFH-DA for 30 min in an incubator at 37 °C in
the dark. After being washed three times with PBS, the cells were
observed by flow cytometry (FCM), with an excitation wavelength of
488 nm and an emission wavelength of 535 nm. Each experiment was
performed independently three times.
China; orcid.org/0000-0001-9852-5347;
Email: wq_ring@hotmail.com
Chun Zhang − Department of Medicinal Chemistry, School of
Pharmacy, Southwest Medical University, Luzhou 646000,
People’s Republic of China; Email: zc83good@126.com
Authors
Yang Li − Department of Medicinal Chemistry, School of
Pharmacy, Southwest Medical University, Luzhou 646000,
People’s Republic of China
Electrophysiological Manual Patch Clamp Recordings.
Extracellular solution (mM): CsCl 139, BaCl2 5, MgCl2 1, glucose
10, HEPES 10, pH = 7.4, and osmolarity 290−320 mOsm.
Intracellular solution (mM): cesium methanesulfonate 108, EGTA
10, MgCl2 0.4, CaCl2 2, HEPES 24, Na-ATP 4, pH = 7.2, and
osmolarity 280−310 mOsm. The extracellular solution was used to
bathe the recorded cell, while the intracellular solution with
amphotericin B was used to fill the recording pipet. The final
concentration for amphotericin B was 250 μg/mL. Test compounds
and nifedipine were dissolved in 100% ethanol or 100% DMSO to
obtain stock solutions for different test concentrations. Then, the
stock solutions were diluted further with the extracellular solution to
achieve final concentrations for testing. The final ethanol or DMSO
concentration was not more than 1.00% for all compound
concentrations. HEK-293 cells stably expressing hCav1.2 (α1C/
β2a/α2δ1) calcium channels were used for this test. The cells were
cultured in a humidified and air-controlled (5% CO2) incubator at 37
°C and were induced by tetracycline (Sangon Biotech, T0422) at 1
μg/mL for 24 h before testing. All experiments were performed at 24
°C. The recorded cells were incubated in a chamber attached to an
inverted microscope (Nikon Ti-S, Japan) with the external solution at
a flow rate of 1 mL/min. The glass microelectrode resistance ranged
from 2 to 4 MΩ after being filled with the pipet solution. After
establishing the whole cell configuration (perforation for 10 min),
membrane currents (ICa‑L) were recorded by a manual patch clamp
system (Axon Multiclamp 700B, Digidata 1440, Molecular Devices,
USA) and filtered at 10 kHz, and Clampfit 10.7 software was used for
the analysis. State-dependent voltage protocol: from a holding
potential of −90 mV, the voltage was stepped to 0 mV for 50 ms
after 1000 ms prepulses that evoked 20% of channels into an
inactivated state. After this, the voltage was stepped back down to a
−90 mV holding potential. This voltage command protocol was
repeated continuously every 15 s during the test.
Wan-Li Tan − Department of Medicinal Chemistry, School of
Pharmacy, Southwest Medical University, Luzhou 646000,
People’s Republic of China
Kai Guo − Key Laboratory of Medical Electrophysiology,
Ministry of Education, School of Pharmacy, Southwest
Medical University, Luzhou 646000, People’s Republic of
China
Xiao-Wei Gao − Key Laboratory of Medical Electrophysiology,
Ministry of Education, School of Pharmacy, Southwest
Medical University, Luzhou 646000, People’s Republic of
China
Jun Wei − Department of Medicinal Chemistry, School of
Pharmacy, Southwest Medical University, Luzhou 646000,
People’s Republic of China
Dong Yi − Department of Medicinal Chemistry, School of
Pharmacy, Southwest Medical University, Luzhou 646000,
People’s Republic of China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jnatprod.1c00136
Author Contributions
#Y.L. and W.-L.T. contributed equally and are joint first
authors.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by the Sichuan Science and
Technology Program (2019JDTD0016), the Program of the
Education Department of Sichuan Province (14TD0017), and
the Applied Basic Research Program of Science and
Technology Department of Sichuan Province (2021YJ0114).
Statistical Analysis. All statistical analyses were conducted with
SPSS statistical software 17.0. All the values are expressed as means
standard error of the mean. The statistical significance of differences
between groups was determined by one-way analysis of variance
followed by Tukey’s post hoc multiple comparison tests or a Student’s
t test (two means comparison), with p < 0.05 considered to indicate
statistically significant differences. Each experiment consisted of at
least three replicates per condition.
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AUTHOR INFORMATION
Corresponding Authors
Qin Wang − Department of Medicinal Chemistry, School of
Pharmacy and Key Laboratory of Medical Electrophysiology,
Ministry of Education, School of Pharmacy, Southwest
Medical University, Luzhou 646000, People’s Republic of
■
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