[(125)I]-N-[(3-azido-5-iodo)benzyl]dantrolene and [(125)I]-N-[[3-iodo-5-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl]dantrolene: photoaffinity probes specific for the physiological Ca(2+) release from sarcoplasmic reticulum of skeletal muscle.

Article abstract of DOI:10.1016/S0960-894X(02)00701-1

In order to capture and identify key molecules that regulate the release of Ca(2+) from the sarcoplasmic reticulum (SR) of skeletal muscle, we designed specific photoaffinity probes based on the structural modification of dantrolene. Thus, GIF-0082 and GIF-0276 possessing azido- and trifluoromethyldiazirinyl-benzyl groups, respectively, at the hydantoin moiety were found to have a highly selective inhibitory effect on physiological Ca(2+) release (PCR) without affecting Ca(2+)-induced Ca(2+) release (CICR). Successful realization of the sharp discrimination between PCR and CICR has led to the creation of [(125)I]GIF-0082 and [(125)I]GIF-0276, which were synthesized by substituting a stannyl group with (125)I in the corresponding phenylstannane precursors.

Full text of DOI:10.1016/S0960-894X(02)00701-1

Bioorganic & Medicinal Chemistry Letters 12 (2002) 3263–3265
[125I]-N-[(3-Azido-5-iodo)benzyl]dantrolene and [125I]-N-{[3-Iodo-
5-(3-trifluoromethyl-3H-diazirin-3-yl)]benzyl}dantrolene:
Photoaffinity Probes Specific for the Physiological Ca2+ Release
from Sarcoplasmic Reticulum of Skeletal Muscle
Takamitsu Hosoya,a,b Hiroshi Aoyama,b Takaaki Ikemoto,c Toshiyuki Hiramatsu,b
Yasutaka Kihara,c Makoto Endoc and Masaaki Suzukia,b,
*
aDivision of Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Gifu University, Yanagido 1-1,
Gifu 501-1193, Japan
bDepartment of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
cDepartment of Pharmacology, Saitama Medical School, Moroyama-machi, Saitama 350-0495, Japan
Received 25July 2002; accepted 27 August 2002
Abstract—In order to capture and identify key molecules that regulate the release of Ca2+ from the sarcoplasmic reticulum (SR) of
skeletal muscle, we designed specific photoaffinity probes based on the structural modification of dantrolene. Thus, GIF-0082 and
GIF-0276 possessing azido- and trifluoromethyldiazirinyl-benzyl groups, respectively, at the hydantoin moiety were found to have a
highly selective inhibitory effect on physiological Ca2+ release (PCR) without affecting Ca2+-induced Ca2+ release (CICR). Suc-
cessful realization of the sharp discrimination between PCR and CICR has led to the creation of [125I]GIF-0082 and [125I]GIF-0276,
which were synthesized by substituting a stannyl group with 125I in the corresponding phenylstannane precursors.
# 2002 Published by Elsevier Science Ltd.
Muscle contraction is provoked by the release of Ca2+
into the cytoplasm from sarcoplasmic reticulum (SR),
the intracellular Ca2+ store.1 In skeletal muscle, physio-
logical Ca2+ release (PCR) from SR is controlled by the
ryanodine receptor (RyR1), a Ca2+-releasing channel
on the SR membrane, whose function is directed by the
signal from the dihydropyridine receptor (DHPR), a
voltage sensor in the cell membrane.1,2 However, the
precise molecular mechanism by which Ca2+ is released,
in particular the question of whether the signal from
DHPR is transmitted to RyR1 directly or via unde-
termined molecules, remains unclear.3,4 The possibility
that proteins may act as regulatory molecules in such a
bioprocess is still under debate.3,4 RyR1 not only plays
specifically on PCR or CICR would be useful to eluci-
date the process by which Ca2+ is released. Therefore,
we have designed specific photoaffinity probes5 capable
of discriminating PCR from CICR by the structural
modification of dantrolene (1).
Compound 1, which is used clinically in the treatment of
malignant hyperthermia,6,7 inhibits abnormal Ca2+
release from SR8 and inhibits both PCR and CICR in
normal skeletal muscle.3,9 Our goal was to design spe-
cific photoaffinity probes by introducing a photoreactive
functional group either to the phenyl ring or the
hydantoin moiety of 1. Derivatives 2, 3, and 4, with
azido, benzoyl, and 3-trifluoromethyl-3H-diazirin-3-yl
groups on the phenyl ring, respectively, were either
ineffective or non-selective.10,11 On the other hand, the
hydantoin derivatives, GIF-0082 (5) and GIF-0276 (6),
exhibited a specific inhibitory effect on PCR without
affecting CICR, as shown in Figure 1.9,12,13 Here, three
substituents, a photoreactive group, an iodo group
(introduced as a latent 125I group), and a methylene
group, were placed to satisfy meta configurations with
a role as a PCR channel but also can facilitate Ca2+
-
induced Ca2+ release (CICR), although the physio-
logical significance of the CICR process is not clear in
skeletal muscle.2,3 Accordingly, an agent which acts
*Corresponding author. Tel.: +81-58-293-2635; fax: +81-58-293-
2635; e-mail: suzukims@biomol.gifu-u.ac.jp
0960-894X/02/$ - see front matter # 2002 Published by Elsevier Science Ltd.
PII: S0960-894X(02)00701-1

3264
T. Hosoya et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3263–3265
Scheme 2. Synthesis of GIF-0276 (6): (a) DMF, rt, 14.5h, 67%.
probe GIF-0276 (6) was prepared from 1 and known
benzyl bromide 1315 (Scheme 2).12
A conspicuous biological property of 5 and 6 led to the
syntheses of the corresponding 125I-labeled compounds,
[
125I]GIF-0082 ([125I]-5) and [125I]GIF-0276 ([125I]-6).
Since it is better to introduce a radioisotope as late as
possible, we planned to apply conventional radio-
iododestannylation16 at the final stage of the synthesis.
Thus, the iodo derivatives, 5 and 6, were once converted
to the corresponding stannanes, 14 and 15, respectively,
by palladium(0)-catalyzed stannylation with bis(tri-n-
butyltin) (Scheme 3).12 The resulting stannyl precursor
underwent radioiododestannylation16,17 by mixing
about a 600-fold excess of stannanes with commercial
10 mCi Na125I solution (Perkin–ElmerTM, NEZ-033A)
in the presence of Chloramine-T (ꢀ150-fold excess)
followed by standing at room temperature overnight.
An extractive work up with EtOAc followed by pur-
ification by silica-gel column chromatography produced
the desired [125I]-5 and [125I]-6 in 61–97 and 31% radio-
chemical yields, respectively, based on Na125I.18
Figure 1. Effects of dantrolene (1), GIF-0082 (5), and GIF-0276 (6) on
twitch contraction (open column) and CICR rate (filled column) of
mouse skeletal muscle. For the methods of biological evaluation, see
ref 9. The number of experiments (n), indicated in the column, of
compounds 1 and 5 for twitch contraction were increased from that
reported in ref 9b.
one another to avoid unnecessary interactions with
neighboring functional groups.
GIF-0082 (5) was obtained by alkylation of 1 with ben-
zyl bromide 7, derived from commercial 3,5-dini-
trobenzyl alcohol (8) (Scheme 1).12 Thus, the partial
reduction of one of the nitro groups of 8 by SnCl2 under
acidic conditions resulted in 9. The amino group of 9
was converted to an iodo group by in situ diazo-iodo-
dediazotization to give 10.14 The remaining nitro group
of 10 was changed to an azido group by reduction with
SnCl2 in ethanol followed by diazo-azidodediazotiza-
tion to give 12. Bromination of the benzyl alcohol 12 by
the conventional method using CBr4/PPh3 produced the
benzyl bromide 7 and, finally, the coupling of 1 with 7 in
DMF gave the desired 5.12 Similarly, the diazirinyl-type
Thus, based on the molecular design and biological
evaluation, we succeeded in developing specific photo-
reactive inhibitors for the PCR process, which is
involved in the excitation–contraction (E–C) coupling1,2
in skeletal muscle. The use of these probes for photo-
affinity labeling, and for capturing novel molecules and
determining their structures and functions, will be
reported in due course.19
Scheme 1. Synthesis of GIF-0082 (5): (a) SnCl2, concd. HCl, EtOH,
0ꢁC to rt, 22 h, 57%; (b) NaNO2, AcOH–H2O (9:1), 0 ꢁC; then NaI,
15min, 67%; (c) SnCl 2, EtOH, 70 ꢁC, 2.5h, 91%; (d) NaNO 2, AcOH–
H2O (9:1), 0 ꢁC; then NaN3, 15min, 95%; (e) CBr 4, PPh3, CH2Cl2, rt,
2 h, 95%; (f) 1, DMF, rt, 14.5h, 85%.

T. Hosoya et al. / Bioorg. Med. Chem. Lett. 12 (2002) 3263–3265
3265
K. O.; Bryant, S. H. Naunyn-Schmiedeberg’s Arch. Pharmacol.
1972, 274, 107. (d) Ellis, K. O.; Carpenter, J. F. Naunyn-
Schmiedeberg’s Arch. Pharmacol. 1972, 275, 83.
9. (a) Ikemoto, T.; Endo, M. Br. J. Pharmacol. 2001, 134, 719.
(b) Ikemoto, T.; Hosoya, T.; Aoyama, H.; Kihara, Y.; Suzuki,
M.; Endo, M. Br. J. Pharmacol. 2001, 134, 729.
10. Palnitker et al. recently reported the synthesis of tritium
labeled 2 and identified 160/172 kDa proteins in procine/rab-
bit SR, which are the endogenously cleaved NH2-terminus
parts of RyR1. We independently synthesized 2 and found it
to be a non-selective inhibitor for both Ca2+ release modes as
is 1. (a) Palnitkar, S. S.; Bin, B.; Jimenez, L. S.; Morimoto, H.;
Williams, P. G.; Paul-Pletzer, K.; Parness, J. J. Med. Chem.
1999, 42, 1872. (b) Paul-Pletzer, K.; Palnitkar, S. S.; Jimenez,
L. S.; Morimoto, H.; Parness, J. Biochemistry 2001, 40, 531.
11. We intended to radiolabel these compounds by tritium
according to ref 10a.
12. All new compounds were characterized by spectroscopic
means. 5: Yellow solid; TLC Rf=0.63 (n-hexane/EtOAc=1:2);
1H NMR (400 MHz, CDCl3) d 4.31 (s, 2H), 4.68 (s, 2H), 6.97 (d,
1H, J=3.8), 6.99 (d, 1H, J=3.8), 7.07 (dd, 1H, J=2.0, 1.5Hz),
7.32 (dd, 1H, J=1.5, 2.0 Hz), 7.56 (dd, 1H, J=1.5, 1.5 Hz), 7.86–
7.91 (AA0BB0, 2H), 8.01 (s, 1H), 8.25–8.29 (AA0BB0, 2H); 13C
NMR (100 MHz, CDCl3) d 41.6, 49.0, 94.8, 111.0, 115.7,
119.2, 124.4 (2C), 124.7 (2C), 127.8, 134.1, 135.18, 135.22,
Scheme 3. Synthesis of [125I]GIF-0082 ([125I]-5) and [125I]GIF-0276
([125I]-6): (a) (n-Bu3Sn)2, (Ph3P)4Pd (cat.), DME, 90 ꢁC, (14; 22 h, 55%,
15; 5.5 h, 41%); (b) Na125I, Chloramine-T, DMF–H2O (3:1), room tem-
perature, ([125I]-5; 17 h, 61–97%, [125I]-6; 20 h, 31% radiochemical yields).
138.6, 141.8, 147.1, 150.2, 152.6, 153.6, 166.0; IR (KBr, cmꢂ1
)
851, 992, 1130, 1148, 1202, 1238, 1331, 1445, 1510, 1566, 1597,
1723, 1781, 2122; UV (EtOH, nm) lmax (log e) 298 (3.66), 309
(3.65), 381 (3.94). Anal. calcd for C21H14N7O5I: C, 44.15; H,
2.47; N, 17.16. Found: C, 43.93; H, 2.48; N, 17.14. 6: Yellow
solid; TLC Rf=0.35( n-hexane/EtOAc=1:1); 1H NMR
(400 MHz, CDCl3) d 4.30 (s, 2H), 4.69 (s, 2H), 6.97 (d, 1H,
J=3.6 Hz), 6.99 (d, 1H, J=3.6 Hz), 7.24 (dd, 1H, J=1.2,
1.5Hz), 7.50 (dd, 1H, J=1.2, 1.5Hz), 7.86 (dd, 1H, J=1.5,
1.5Hz), 7.86–7.90 (AA 0BB0, 2H), 8.01 (s, 1H), 8.25–8.29
(AA0BB0, 2H); 13C NMR (100 MHz, CDCl3+DMSO-d6) d
26.7 (2J(19F–13C)=41.1 Hz), 40.5, 47.5, 93.8, 110.4, 114.6,
120.6 (1J(19F–13C)=273.1 Hz), 123.2 (2C), 123.5(2C), 125.3,
129.9, 133.0, 133.8, 134.2, 137.4, 137.9, 145.6, 149.3, 151.5,
151.9, 165.3; 19F NMR (372 MHz, CDCl3) d 12.65(s, 3F); IR
(KBr, cmꢂ1) 741, 754, 853, 1150, 1179, 1202, 1246, 1333, 1412,
1441, 1510, 1599, 1728, 1782; UV (EtOH, nm) lmax (log e) 309
(4.24), 323 (4.22), 379 (4.55). Anal. calcd for C23H14N6O5F3I:
C, 43.28; H, 2.21; N, 13.17. Found: C, 43.06; H, 2.21; N, 13.31.
13. The effect for PCR was evaluated by the twitch tension of
intact mouse skeletal muscle. The CICR rates were quantified
by the amount of Ca2+ released using microfluorometry of
Fura-2, a Ca2+ indicator, in skinned fibers prepared from
mouse skeletal muscle. For the methods of biological evalua-
tion, see ref 9.
Acknowledgements
We thank Prof. Dr. Michio Ui, Prof. Dr. Shunji Natori,
Dr. Toshiyuki Fujioka, and Dr. Nobuko Akiyama at
RIKEN Institute for their kind support. We are grateful
to Mr. Kin-ichi Oyama of Nagoya University Chemical
Instrument Center for performing the mass spectra
analysis. This work was supported in part by a Grant-
in-Aid for Creative Scientific Research (No. 13NP0401)
of the Ministry of Education, Culture, Sports, Science
and Technology (MEXT) of Japan.
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17. Before handling the radioisotope, the reproducibility of
the reaction was confirmed by examining iododestannylation
under ‘cold’ conditions similar to the actual conditions used.
18. We confirmed from the TLC analysis that [125I]-5 was
completely decomposed by photoirradiation by UV-light
(254nm, 16W UV-lamp, from the distance of 1 cm) within 60 s
under the actual photolabeling conditions. On the other hand,
[
125I]-6 required rather longer irradiation time (10 min) for
complete decomposition even using the 100 W UV-lamp
(365nm).
8. (a) Endo, M. In Simmons R. M., Ed. Muscular Contrac-
tion; Cambridge University Press, New York, 1992.; p 67. (b)
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19. Actually, we have succeeded in capturing novel molecules
by using these specific photoaffinity probes. Their structures
and functions will be reported separately in due course.