Open-chain half-bastadins mimic the effects of cyclic bastadins on calcium homeostasis in cultured neurons

Article abstract of DOI:10.1016/j.bmcl.2008.09.080

Constraining the catechol aryl ether moiety of bastadins by incorporation into a macrocyle is not necessary in order to mimic the effects of these marine natural products on neuronal calcium homeostasis. Simple, acyclic analogs that embody the 'western' or 'eastern' parts of bastadins were found to evoke comparable responses with bastadin 5.

Full text of DOI:10.1016/j.bmcl.2008.09.080

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5734–5737
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Open-chain half-bastadins mimic the effects of cyclic bastadins on calcium
homeostasis in cultured neurons
b,c
Elzbieta Zieminska a, Jerzy W. Lazarewicz a, , Elias A. Couladouros
,
*
Vassilios I. Moutsos c, Emmanuel N. Pitsinos c,
*
a Medical Research Centre, Polish Academy of Sciences, Pawinskiego 5, 02-106 Warsaw, Poland
b Chemistry Laboratories, Agricultural University of Athens, Iera Odos 75, GR 118 55 Athens, Greece
c Institute of Physical Chemistry, Natural Products Synthesis & Bioorganic Chemistry Laboratory, NCSR ‘‘DEMOKRITOS”, PO Box 60228, GR 153 10 Aghia Paraskevi, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
Constraining the catechol aryl ether moiety of bastadins by incorporation into a macrocyle is not neces-
sary in order to mimic the effects of these marine natural products on neuronal calcium homeostasis.
Simple, acyclic analogs that embody the ‘western’ or ‘eastern’ parts of bastadins were found to evoke
comparable responses with bastadin 5.
Received 27 August 2008
Revised 22 September 2008
Accepted 23 September 2008
Available online 26 September 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Ryanodine
Calcium channels
Cerebellar granule cells
Bastadins
Diaryl ether
Controlled fluctuations in intracellular calcium cation (Ca2+
)
Although bastadins share the same gross structural features, not
all of them interact with RyR channels. Seemingly subtle differ-
ences in their substitution pattern suffice to alter the response ob-
served.5 Thus, bastarane bastadin 5 is the most active member
concentrations play an important role in many cellular processes
such as muscle contraction, secretion, metabolism, and neuronal
function.1 Ryanodine receptors (RyR) are intracellular ion channels
that mediate the release of calcium ions from internal stores and
have been suggested as pharmacological targets for heart disease
and neurodegenerative diseases.2,3 Three isoforms (RyR1, RyR2,
and RyR3) are expressed by mammalian tissues: RyR1 is present
predominantly in skeletal muscle; RyR2 is found in cardiac muscle
and is the major brain isoform; RyR3 has a wide tissue distribution
and appears to be the major isoform in smooth muscle.2a
Bastadins (Fig. 1) are an ever-growing family of marine natural
products isolated from sponges of the order Verongida.4 Due to
their ability to interact with RyR1 channels, they are considered
useful chemical probes for related biological studies. Most of them
are macrocyclic bis-diaryl ethers and, depending on the relative
orientation of their diaryl ether segments, are further classified
(EC50 = 2.2 lM) and seems to stabilize both open and closed chan-
nel states but has little effect on the apparent sensitivity of the
channel to Ca2+ activation. The isobastarane with the same bro-
mination pattern, bastadin 19, although it competes for the same
binding site, does not mobilize Ca2+ from the channel
(EC50 > 100 lM). The unsaturated bastadin 7 (EC50 = 6.3 lM) fol-
lows closely bastadin 5 in terms of potency while its hydroxylated
relative bastadin 10, in contrast to bastadin 5, stabilizes primarily
the open-channel conformation and over sensitizes it to Ca2+
activation.
Until recently, further structure–activity relationship (SAR)
studies were limited to the use of naturally occurring bastadins
and were hampered by the fact that not all members are readily
available. We have previously reported a general and flexible syn-
thetic strategy towards bastadins, which allows for the synthesis of
both bastaranes and isobastaranes with all possible bromination
patterns.6 Molinski et al. have prepared simplified cyclic analogues
of the ‘western’ hemisphere of bastadin 5 by an alternative strategy
and investigated their effect on RyR1 channels (Fig. 2).7 This study
reiterated the importance of the diarylether unit substitution pat-
tern and illustrated that simpler analogs can retain the activity of
more structurally complex natural bastadins.
either as bastaranes or isobastaranes. They feature unique
a-oxi-
mino amides while the degree of ortho-bromination of the diaryl
ether moieties as well as the oxidation state of C5/C6 varies among
family members.
* Corresponding authors. Fax: +48 22668 54 23 (J.W.L); tel.: +30 2106503789;
fax: +30 2106511766 (E.N.P.).
E-mail addresses: jerzyl@cmdik.pan.pl (J.W. Lazarewicz), pitsinos@chem.demok-
ritos.gr (E.N. Pitsinos).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.080

E. Zieminska et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5734–5737
5735
OH
OH
OH
Y1
OH
Y1
western
part
Bastadin Y1 Y2 Y3
W
Z
N
H
N
O
O
a, b, a
4
7
11
14
H
H
H
Br Br
Br H
H Br
Z
O
Br
Br
Br
O
W
Br Br H
H2N
BocHN
BzHN
BzHN
Br
O
8
H
Br Br
Y1
1, Y1 = Br, H
2a, Y1 = Br
2b, Y1 = H
10
12
17
H
Br H
HO
Br
Y3
Br Br H
OH
H
H
Br
Y2
O
OH
Br
OH
O
Br
5
6
H
Br Br
eastern
part
Br Br Br
Br
OBn
O
Br
OH
N
H
c, d, e
9
H H Br
N
15
16
18
Br Br H
Br H Br
OH
Br
Br
Bastaranes
N
BnO
N
OH
H
Br H
N
BuHNOC
CONHBu
N
CO2H
CO2Me
western
part
Br
H
N
O
OH
O
3
4
Y2
Bastadin Y2 Y3
N
Scheme 1. Reagents and conditions: (a) BzCl, Et3N, benzene; (b) TFA, CH2Cl2; (c)
LiOH, MeOH/THF/H2O; (d) BuNH2, EDC, HOBt, iPr2EtN, DMF/CH2Cl2, 0 °C–rt; (e)
BBr3, thioanisole, 0 °C–rt.
13
19
20
21
H Br
OH
Br
Y3
HO
Br Br
O
O
Br H
eastern
part
H
H
Br
N
H
N
initial level of fluorescence was measured using confocal micros-
copy (Fig. 3).11 Synthetic bastadin 5, which in the control experi-
ments demonstrated identical calcium mobilizing potential with
the natural, commercially available product (results not shown),
induced a substantial increase in the FLUO-3 fluorescence, indicat-
ing its ability to release calcium from the intracellular stores.8
From the three analogs tested, 4 induced the most potent calcium
transients comparable with the effects of bastadin 5 and even more
dynamic. Analogue 2b was less potent, whereas analog 2a had
practically no effect on the neuronal free calcium level.
OH
Isobastaranes
Figure 1. Some naturally occurring bastadins.
O
R1 = NO2, NH2, N3,
O
NH
Br, I, H
HN
NH
R2 = H, Br
R2
O
R2
Increases of intracellular Ca2+ concentration may arise from
intracellular stores calcium release and/or from extracellular Ca2+
influx. Thus, in order to characterize bastadin-induced changes in
calcium homeostasis, apart from measurements of the intracellular
calcium level detected with FLUO-3 fluorescence, we monitored
also the influx of extracellular calcium to neurons employing
45Ca2+ isotope.12 As presented in Fig. 4, application of bastadin 5
significantly increased uptake of 45Ca2+ in the cultured neurons.
Moreover the effect of the acyclic analogs on calcium accumulation
mirrored the picture observed for their effect on the intracellular
O
Br
R1
O
Br
R1
OH
OH
R
1 = Br; R2 = H
R
1 = Br; R2 = H
μM)
(IC50 = 6
(EC50 = 11 μM)
Figure 2. Simplified cyclic analogs of bastadin 5 prepared by Molinski et al. and
their effect on RyR1 channels.7
Subsequently, we have investigated the effect of several syn-
thetic bastadins, including bastadins 5 and 10, on the RyR2 recep-
tors employing primary cultures of rat cerebellar granule neurons
and observed that they elicit responses similar to the ones previ-
ously identified on skeletal RyR1 receptors.8 Intrigued by the above
mentioned structure–activity relationships, we examined and re-
port herein the effect of simple acyclic analogues of ‘eastern’ and
‘western’ parts of bastadins (2a, 2b, and 4; Scheme 1) on calcium
homeostasis in cultured neurons.
These compounds were readily prepared (Scheme 1) from key
synthetic intermediates of our total synthesis effort.6d Thus, benzo-
ylation of amine 1 (1:1 mixture of mono- and dibromo-derivative),
followed by cleavage of the Boc protective group and a second ben-
zoylation furnished a separable mixture of dibenzoylamides 2a and
2b in 76% overall yield.9 On the other hand, hydrolysis of methyl
ester 3 followed by coupling of the resulting diacid with n-butyl-
amine and final benzyl ether cleavage, upon treatment with BBr3
in the presence of thioanisole, yielded derivative 4 in 33% overall
yield.9
Figure 3. Effects of bastadin 5 and analogues on the intracellular calcium level in
The ability of bastadin 5 (positive control) and the acyclic ana-
logues 2a, 2b, and 4 to increase the intracellular calcium concen-
tration in cultured rat cerebellar granule cells10 was evaluated
using FLUO-3, a calcium-sensitive fluorescent probe. After loading
the cells with the fluorescent probe, the compounds were applied
primary cultures of rat cerebellar granule cells. Synthetic bastadin 5 as well as
analogues 2a, 2b, and
incubation. Analog 4 was also applied in the presence of 200
or 50 M FK506 (4 + FK). Results, means SD (n = 15) are presented as percent
4
(all compounds 20
l
M) were applied after 60 s of
lM ryanodine (4 + R)
l
changes in the intensity of fluorescence compared with the basal level (F/F0). The
presented data are from one of three independent experiments using different
cultures that gave qualitatively identical results.
to the incubation medium in equal concentration (20 lM) and the

5736
E. Zieminska et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5734–5737
ortho- to the ether linkage (2a) leads to diminished activity while
introduction of an additional bromine ortho- to the catechol (4)
has the opposite result. Clearly further studies are needed to deci-
pher the effect of aryl substitution pattern.
In conclusion, our results confirm that the calcium-mobilizing
potential of bastadins critically depends on the presence and
location of aryl substituents and indicate that constraining of the
diarylether moiety in a macrocycle is not a prerequisite for activity.
Thus, a major simplification of the structure of bastadins, resulting
in open-chain analogs that conserve the bromocatechol ether
moiety, is possible without obligatory lose of their ability to release
calcium via ryanodine receptors. Some of these compounds, in
particular the one embodying the dibromocatechol ether moiety
found in bastadin 5, retain this activity. This major structural
simplification facilitates the development of novel chemical probes
for the study of RyR channels and at the same time compels further
investigations to illuminate the modulating effect that ‘satellite’
substituents (i.e., local and nature of further aromatic ring
substitution, aliphatic chain length, and substitution pattern)
may exert.
Figure 4. Effects of bastadin 5 and analogues 2a, 2b, and 4 (all compounds 20 lM)
on 45Ca2+ uptake in cultured cerebellar granule cells. Accumulation of 45Ca2+ was
measured after 10 min incubation with the compounds and results (means SD,
n = 6) are presented as percent of control. All results were significantly different
from the control (P < 0.05) as verified with ANOVA test.
Acknowledgments
calcium concentration, that is, the effect of 4 > 2b > 2a, the latter
being very slight.
This study was supported by the Poland-Greece Joint Research
and Technology Program 2005–2007 16189-158e. Prof. A. Giannis
is gratefully acknowledged for his assistance in obtaining HRMS
spectra for the compounds tested.
The fact that the influx of 45Ca2+ to neurons evoked by applica-
tion of bastadin 5 and some of the synthetic analogues correlates
with increases in the intracellular calcium levels could be an
indication that they directly activate the receptor-operated or volt-
age-sensitive calcium channels in neuronal plasma membranes.
However, similarly to our previous studies on bastadins (including
bastadin 5),8 increases in the intracellular calcium levels induced
by the most potent analog 4 were found to be sensitive to high con-
centrations of ryanodine and FK506 (Fig. 3).13 This observation
clearly indicates that ryanodine receptors are the primary targets
for their pharmacological activity. Thus, the influx of extracellular
calcium to bastadin-treated neurons appears to be a secondary
effect resulting from the release of calcium from the ryanodine-
sensitive pool. This release leads, in turn, to stimulation of gluta-
mate release from the glutamatergic cerebellar granule neurons
or otherwise leads to secondary activation of the NMDA receptors,
as it has been recently described for PCB 95.14 Therefore both bast-
adin-induced phenomena, an increase in FLUO-3 fluorescence and
activation of 45Ca2+ uptake, represent different aspects of the same
primary biological phenomenon: the release of calcium via ryano-
dine receptors in neurons.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.09.080.
References and notes
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3. Mattson, M. P. Aging Cell 2007, 6, 337.
4. Peng, J.; Li, J.; Hamann, M. The Alkaloids: Chemistry and Biology 2005, 61, 59.
5. (a) Mack, M. M.; Molinski, T. F.; Buck, E. D.; Pessah, I. N. J. Biol. Chem. 1994, 269,
23236; (b) Franklin, M. A.; Penn, S. G.; Lebrilla, C. B.; Lam, T. H.; Pessah, I. N.;
Molinski, T. F. J. Nat. Prod. 1996, 59, 1121; (c) DiJulio, D. H.; Watson, E. L.;
Pessah, I. N.; Jacobson, K. L.; Ott, S. M.; Buck, E. D.; Singh, J. C. J. Biol. Chem. 1997,
272, 15687; (d) Pessah, I. N.; Molinski, T. F.; Meloy, T. D.; Wong, P.; Buck, E. D.;
Allen, P. D.; Mohr, F. C.; Mack, M. M. Am. J. Physiol. 1997, 272, C601; (e) Chen, L.;
Molinski, T. F.; Pessah, I. N. J. Biol. Chem. 1999, 274, 32603; (f) González, A.;
Kirsch, W. G.; Shirokova, N.; Pizarro, G.; Brum, G.; Pessah, I. N.; Stern, M. D.;
Cheng, H.; Rios, E. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 4380.
6. (a) Couladouros, E. A.; Moutsos, V. I. Tetrahedron Lett. 1999, 40, 7023; (b)
Couladouros, E. A.; Moutsos, V. I. Tetrahedron Lett. 1999, 40, 7027; (c)
Couladouros, E. A.; Moutsos, V. I.; Pitsinos, E. N. ARKIVOC 2003, 15, 92; (d)
Couladouros, E. A.; Pitsinos, E. N.; Moutsos, V. I.; Sarakinos, G. Chem. Eur. J.
2005, 11, 406.
7. Masuno, M. N.; Pessah, I. N.; Olmstead, M. M.; Molinski, T. F. J. Med. Chem. 2006,
49, 4497.
8. Zieminska, E.; Stafiej, A.; Pitsinos, E. N.; Couladouros, E. A.; Moutsos, V.;
Kozlowska, H.; Toczylowska, B.; Lazarewicz, J. W. Neurosignals 2006–2007, 15,
283.
Our newly prepared acyclic analogues embody segments of dif-
ferent naturally occurring bastadins: compound 2a embodies the
‘western’ part of bastadin 16 while compound 2b embodies the
corresponding segment of bastadins 9, 13 and 21; compound 4
embodies the ‘eastern’ part of bastadins 4, 5, 6, 8, 9, 11, 13,16, 17
and 19. It should be noted, however, that recent structure–activity
relationship (SAR) studies by Molinski et al. indicated that the
ketoximino groups present in the natural products are not required
for activity and thus the difference between ‘western’ and ‘eastern’
parts becomes less profound.7 Furthermore, the same studies
strongly support the importance of the shape and size of the mac-
rocyclic half-bastadins. Interestingly, our findings clearly indicate
that constraining the C–O–C–C torsional angle by incorporating
the diarylether in a macrocycle is not a prerequisite for activity.
Thus, the catechol aryl ether substitution pattern potentially plays
the decisive role regarding calcium mobilization activity. The avail-
able data would seem to indicate that a para-substituted catechol
ortho-brominated-para-substituted aryl ether, like the one found
in 2b, is required. This pattern is maintained in compounds 2a
and 4. Intriguingly, introduction of an extra bromine substituent
9. Compound 2a: 38% yield from 1; 1H NMR (500 MHz, CDCl3) d 7.76 (d, J 7.2 Hz,
2H), 7.59 (d, J 7.2 Hz, 2 H), 7.46–7.51 (m, 2H), 7.44–7.37 (m, 6H), 7.01 (d, J
8.2 Hz, 1H), 6.84 (d, J 7.9 Hz, 1H), 6.65–6.71 (br m, 1H), 6.24 (s, 1H), 5.97–6.04
(br m, 1H), 5.5–5.8 (br s, 1H), 3.65–3.69 (m, 2H), 3.57–3.61 (m, 2H), 2.89–2.92
(m, 2H), 2.76–2.79 (m, 2H); HRMS (ESI): m/z: 661.01161 [M+Na+],
C30H26Br2N2O4Na requires 661.01366.Compound 2b: 38% yield from 1; 1H
NMR (500 MHz, CDCl3) d 7.73 (d, J 7.4 Hz, 2H), 7.63 (d, J 7.4 Hz, 2H), 7.39–7.55
(m, 7H), 7.05 (d, J 8.1 Hz, 1H), 7.00 (d, J 8.2 Hz, 1H), 6.89–6.91 (m, 2H), 6.57 (s,
1H), 6.34–6.40 (br m, 1H), 6.03–6.10 (br m, 1H), 5.4–5.8 (br s, 1H), 3.66–3.70
(m, 2H), 3.59–3.63 (m, 2H), 2.88–2.91 (m, 2H), 2.78–2.81 (m, 2H); HRMS (ESI):
m/z: 581.10407 [M+Na+], C30H27BrN2O4Na requires 581.10519.Compound 4: 1H
NMR (500 MHz, acetone-d6): d 11.29 (br s, 1H), 10.89 (br s, 1H), 8,77 (br s, 1H),
7.67 (br s, 2H), 7.55 (br m, 1H), 7.31 (br m, 1H), 7.17 (br s, 1H), 6.40 (br s, 1H),
3.98 (br s, 2H), 3.69 (br s, 2H), 3.29 (br m, 2H), 3.21 (br m, 2H), 1.20–1.56 (m,
4H), 0.90 (br m, 6H); HRMS (ESI): m/z: 756.96752 [M+Na+], C26H31Br3N4O6Na
requires 756.96710.

E. Zieminska et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5734–5737
5737
10. Rat cerebellar granule neurons were prepared as described in Schousboe, A.;
Drejer, J.; Hansen, G. H.; Meier, E. Dev. Neurosci. 1985, 7, 252 and cultivated in
modified basal Eagle’s medium containing 25 mM KCl, exactly as described
previously in details.8 The cells were used for experiments on the 7th day
in vitro.
fluorescence was measured for 15 min using LSM 510 computer program
(version 3.2), as described previously.8
12. Accumulation of 45Ca in cultured neurons was determined in the magnesium-
free low potassium Locke 5 buffer. After preincubation for 10 min at 37°C,
45CaCl2 (1
lCi/well) was added together with tested compounds for 10 min.
11. For loading with 16
medium for 30 min. After washing, the cells were incubated in magnesium-
free low potassium Locke 5 buffer. In relevant trials, ryanodine (200 M) or
FK506 (50 M) was added to the medium. The basal fluorescence reflecting the
l
M FLUO-3 AM, the dye was added to the original growth
Then the medium was removed and the cells were washed, dissolved in 0.5 M
NaOH, and radioactivity in neurons was measured with a Wallac 1409 liquid
scintillation counter as described in Makarewicz, D.; Zieminska, E.; Lazarewicz,
J. W. Neurochem. Int. 2003, 43, 273.
l
l
steady-state level of intracellular calcium concentration was monitored, using
the confocal microscope Zeiss LSM 510 with excitation at 488 nm and emission
measured at 530 nm. Then the tested compounds were added and the
13. Ryanodine and FK506 had similar effects on the activity of compound 2b
(Fig. S1 in Supplementary data).
14. Gafni, J.; Wong, P. W.; Pessah, I. N. Toxicol. Sci. 2004, 77, 72.