Design of dantrolene-derived probes for radioisotope-free photoaffinity labeling of proteins involved in the physiological Ca2+ release from sarcoplasmic reticulum of skeletal muscle

Article abstract of DOI:10.1016/j.bmcl.2005.01.041

Bifunctional dantrolene derivatives have been synthesized as probes for radioisotope-free photoaffinity labeling with the aim of elucidating the molecular mechanism of skeletal muscle contraction. GIF-0430 and GIF-0665 are aromatic azido-functionalized derivatives that were designed to selectively inhibit physiological Ca²⁺ release (PCR) from sarcoplasmic reticulum (SR) in mouse skeletal muscle without a strong effect on Ca²⁺-induced Ca²⁺ release (CICR). These photoaffinity probes consist of either an azidomethyl or an ethynyl group, respectively, which could function as a tag for introduction of an optional detectable marker unit by an appropriate chemoselective ligation method after the photo-cross-linking operation. Actually, the former probe worked to photolabel its target proteins specifically as confirmed by subsequent fluorescent visualization.

Full text of DOI:10.1016/j.bmcl.2005.01.041

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1289–1294
Design of dantrolene-derived probes for
radioisotope-free photoaffinity labeling of proteins involved in
the physiological Ca2+ release from sarcoplasmic reticulum of
skeletal muscle
Takamitsu Hosoya,a,b Toshiyuki Hiramatsu,a,b Takaaki Ikemoto,a Hiroshi Aoyama,b
Tatsuro Ohmae,b Makoto Endoc and Masaaki Suzukia,b,
*
aDivision of Regeneration and Advanced Medical Science, Gifu University Graduate School of Medicine, Yanagido 1-1,
Gifu 501-1194, 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 21 December 2004; accepted 13 January 2005
Abstract—Bifunctional dantrolene derivatives have been synthesized as probes for radioisotope-free photoaffinity labeling with the
aim of elucidating the molecular mechanism of skeletal muscle contraction. GIF-0430 and GIF-0665 are aromatic azido-function-
alized derivatives that were designed to selectively inhibit physiological Ca2+ release (PCR) from sarcoplasmic reticulum (SR) in
mouse skeletal muscle without a strong effect on Ca2+-induced Ca2+ release (CICR). These photoaffinity probes consist of
either an azidomethyl or an ethynyl group, respectively, which could function as a tag for introduction of an optional detectable
marker unit by an appropriate chemoselective ligation method after the photo-cross-linking operation. Actually, the former probe
worked to photolabel its target proteins specifically as confirmed by subsequent fluorescent visualization.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Dantrolene (1) is a muscle relaxant known as an efficient
drug used clinically for the treatment of malignant
hyperthermia (MH),1,2 a genetic disease triggered by
excessive release of Ca2+ from the sarcoplasmic reticu-
lum (SR), the intracellular Ca2+ store, into the cyto-
plasm of skeletal muscle cells. This unusual Ca2+
release occasionally occurs when skeletal-type ryanodine
receptor (RyR1), a Ca2+-releasing channel on the SR
membrane, has been activated by inhalational anesthet-
ics. Compound 1 is considered to relieve MH by sup-
pressing this abnormal Ca2+ release.1c,3 It has also
been widely used as an analytical tool to regulate intra-
cellular Ca2+ concentration, particularly in the study of
the excitation–contraction (E–C) coupling of skeletal
muscle.4 In skeletal muscle, physiological Ca2+ release
(PCR) from SR is controlled by RyR1, whose function
is regulated by a signal from the dihydropyridine recep-
tor (DHPR), a voltage sensor in the cell membrane.5
Although there is an established consensus that DHPR
and RyR1 locate face-to-face together in the triad junc-
tion, the question of whether the signal from DHPR is
transmitted to RyR1 directly or indirectly via undeter-
mined molecules, is still controversial.4,6 Additionally,
RyR1 mediates Ca2+-induced Ca2+ release (CICR) in
addition to working as a PCR channel in skeletal mus-
cle, although its physiological significance is yet to be
solved.4,5b,c,6
In order to elucidate the molecular mechanism involved
in the Ca2+-related processes of skeletal muscle cells
more precisely, we have developed several efficient
chemical probes based on the structural modification
of 1, a non-selective inhibitor for both PCR and
CICR.7–9 Of these, the radioisotope (RI)-labeled
probes, [125I]GIF-0082 ([125I]-2) and [125I]GIF-0276
([125I]-3), whose structures include azido- and trifluo-
romethyl-diazirinyl-benzylated units, respectively, were
designed to capture and identify the target proteins
of 1 using a photoaffinity labeling method.9a,10,11
*
Corresponding author. Tel./fax: +81 058 293 2635; e-mail: suzu-
kims@biomol.gifu-u.ac.jp
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.041

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T. Hosoya et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1289–1294
Regarding the design of RI-free photoaffinity probes
O
O2N
based on the dantrolene structure, we conceived to con-
vert the efficient radiolabeled probes, [125I]-2 and [125I]-
3,9a to the corresponding RI-free structures. Thus, we
designed GIF-0430 (4) and GIF-0666 (5), where the
125I group of [125I]-2 and [125I]-3 is switched to an azi-
domethyl group as a latent detectable group. We envis-
aged that biological activity and target specificity would
be maintained because the azidomethyl group does not
differ considerably from an iodo group in terms of its
size and the polarity.13
N—Na+
O
N
N
O
dantrolene (1)
O
O2N
R1
N
O
N
N
O
R2
125I: [125I]GIF-0082 ([12 5I]-2)
R1 = N3, R2 = I: GIF-0082 (2)
R2
=
We also planned to prepare GIF-0665 (6) and GIF-0667
(7) with an ethynyl group instead of an azidomethyl
group. The ethynyl function was designed to be used
for Huisgen 1,3-dipolar cycloaddition15 with an azido
group, generally described as Ôclick chemistryÕ,17 a con-
venient and efficient bioconjugation technique, which
has been developed recently. Indeed, the azido function
in the ethynyl-substituted phenyl azide 8 undergoes pho-
toreaction cleanly to give didehydroazepine 10 via a
phenylnitrene species 9 (Scheme 1).18 Furthermore, we
confirmed that a trifluoromethyldiazirinyl group, in-
stead of the azido function, also works well in the photo-
reactive function in the presence of an ethynyl group
(Scheme 2). Thus, photoirradiation of methanol solu-
tion of 11 by UV light with a wavelength of 365 nm
R1 = -CCF3, R2 = I: GIF-0276 (3)
R2 = 12 5I: [125 I]GIF-0276 ([125 I]-3)
N
N
1
R
= N3, R2 = CH2N3: GIF-0430 (4)
1
R
= -CCF3, R2 = CH2N3: GIF-0666 (5)
N
N
R1 = N3, R2 = -C≡CH: GIF-0665 (6)
R1 , R2 = -C≡
=
CH:GIF-0667 (7)
-CCF3
N
N
The corresponding non-radiolabeled compounds, GIF-
0082 (2) and GIF-0276 (3), showed a highly selective
inhibitory effect on PCR without affecting CICR.9a
Actually, we have succeeded in the photolabeling of tar-
get proteins using [125I]-2 and the characterization of the
primary structure of one target protein purified from an
independent muscle preparation using molecular weight
stratification by SDS-PAGE analysis of photolabeled
mixtures.12
ring
expansion
N
N3
N
h
ν
8
9
10
In the course of this study, using RI-labeled molecular
probes as described above, we learned several disadvan-
tages in the RI-photoaffinity labeling method, including:
(1) the inaccessibility of photolabeled proteins to direct
analysis, (2) the requirement of special care and facility
to avoid the radiation exposure, and (3) the inherent
instability of RI probes, for example, half-life decay time
of 125I-radionuclide is 60 days. In this context, we re-
cently developed an efficient method of RI-free photo-
Scheme 1. Photoreaction of 1-azido-3-ethynylbenzene (8).18
N
N
THPO
CF
THPO
CF3
CF3
3
3
h
ν
affinity labeling using
a
probe with compact
11
CH3OH
bifunctional unit consisting of a photoreactive function
and an aliphatic azido group.13 We chemically showed
that the latter is relatively photo-stable and, therefore,
can be utilized as a tag to introduce detectable markers
such as fluorescent or biotin units after the photo-cross-
linking operation, using azide-specific bioconjugation
reactions such as Staudinger–Bertozzi ligation14 and
Huisgen 1,3-dipolar cycloaddition.15 This method is
more advantageous than the preceding ones16 because
it only requires the introduction of an additional azi-
domethyl group to the original ligand, thereby allowing
more flexibility in designing probe candidates without
much influence on its intrinsic biological activity and
selectivity. Indeed, the utility of this method was suc-
cessfully demonstrated by the efficient fluorescent detec-
tion of photolabeled HMG-CoA reductase using a
bifunctional cerivastatin derivative.13 Described herein
is the synthesis of bifunctional dantrolene derivatives
suitable for elucidating proteins involved in the PCR
process by the RI-free photoaffinity labeling method.
13
h
ν
insertion
365 then 302 nm
12 min each
into CH3OH
CH3O
H
benzyl azide
CuSO4
CH3OH—H2O
(9:1)
THPO
sodium ascorbate
TBTA (15)
rt, 15 h
azide—alkyne
cycloaddition
CH3O
H
THPO
CF3
14
N
N
N
N
N
N
N
Ph
12
TBTA (15)
47% yield (2 steps)
Scheme 2. Photoreaction of ethynyl-functionalized phenyltrifluoro-
methyldiazirine 11 in methanol followed by cycloaddition with benzyl
azide (THP = tetrahydropyran-2-yl).

T. Hosoya et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1289–1294
1291
(UVP, UVL-56, 6W) for 12 min and then with a wave-
length of 302 nm (UVP, UVM-57, 6W) also for
12 min,19 followed by successive treatment of the mix-
ture with benzyl azide in the presence of CuSO4, sodium
ascorbate, and the triazole ligand TBTA (15)20 to pro-
mote the azide-alkyne [3+2] cycloaddition, afforded the
desired triazole 12 at a 47% overall yield. This result
clearly shows that the carbene species 13 was generated
by the photoreaction of 11, which underwent an inser-
tion reaction with methanol to give 14.21
N
N
N N
THPO
CF3
CF3
X
c)
e)
GIF-0667
(7)
R
27: R = I
a)
b)
28: R = C≡CSiMe3
30: X = OH
31: X = Br
d)
29: R = C≡CH
Scheme 6. Synthesis of GIF-0667 (7). Reagents and conditions: (a)
Me3SiC„CH, (Ph3P)2PdCl2, CuI, Et2NH, rt, 13 h, 77%; (b) KOH,
MeOH, 0 ꢁC to rt, 0.5 h, 9l%; (c) pyridinium p-toluenesulfonate,
EtOH, 50 ꢁC, 19 h, 98%; (d) CBr4, PPh3, CH2Cl2, rt, 2.5 h, 82%; (e) 1,
DMF, 0 ꢁC to rt, 2 h, 85%.
All of the newly designed compounds, 4–7, were pre-
pared by coupling the corresponding bifunctional benzyl
bromides with 1 (Schemes 3–6).9a Thus, starting from
the diazido-functionalized alcohol 16,13 its hydroxy
group was successively mesylated and brominated to af-
ford benzyl bromide 18, which was coupled with 1 to
give 4 (Scheme 3).22 Similarly, the corresponding triflu-
oromethyldiazirinyl derivative 5 was derived from the
bifunctional unit 19,13 whose THP group was removed,
followed by bromination of the resulting hydroxy group
to afford 22, which was then linked with 1 to furnish 5
(Scheme 4). On the other hand, the terminal acetylenic
compounds, 6 and 7, were synthesized based on the
use of Sonogashira-type coupling reactions.23 Thus,
the former was synthesized starting from 23,9a which
was coupled with trimethylsilylacetylene to provide 24
(Scheme 5). Here, the Pd-catalyzed reaction under
Cu(I) salt-free conditions23b was preferable to avoid
the side reaction of silylacetylene with the azido group.
Subsequent deprotection of the trimethylsilyl group of
24 followed by bromination gave 26, which was coupled
with 1 to afford 6. Similarly, 7 was prepared from the
iodide 2713 in a similar five step sequence (Scheme 6).
N3
X
c)
We evaluated the effects of newly synthesized dantrolene
derivatives 4–7 on two kinds of Ca2+ release, PCR and
CICR. The effects on PCR were determined by compar-
ing the twitch tension of intact mouse skeletal muscle at
room temperature before and 15 min after treatment
with the derivatives.8,9,24 Effects on CICR were evalu-
ated by measuring the rates of Ca2+ release induced by
1 lM Ca2+ in saponin-treated skinned muscle fibers of
mouse skeletal muscle under the same condition in our
previous reports.8,9,24 The final concentration of each
derivative in both experiments was fixed at 50 lM to
compare the efficacy between the derivatives. The results
are shown in Figure 1 with the comparison of 1 (20 lM),
GIF-0430 (4)
N3
16: X = OH
17: X = OMs
18: X = Br
a)
b)
Scheme 3. Synthesis of GIF-0430 (4). Reagents and conditions: (a)
MsCl, Et3N, CH2Cl2, 0 ꢁC, 3.5 h, 94%; (b) KBr, DMF, rt, 13 h, 99%;
(c) 1, DMF, rt, 22 h, 79%.
N
N
CF3
X
d)
GIF-0666 (5)
125
100
75
N3
19: X = OTHP
20: X = OH
21: X = OMs
22: X = Br
a)
b)
c)
Scheme 4. Synthesis of GIF-0666 (5). Reagents and conditions: (a)
pyridinium p-toluenesulfonate, EtOH, 55 ꢁC, 12 h, 94%; (b) MsCl,
Et3N, CH2Cl2, 0 ꢁC, 1.5 h, 99%; (c) KBr, DMF, 0 ꢁC to rt, 13 h, 95%;
(d) 1, DMF, rt, 17 h, 78%.
50
25
N3
N3
13
6
14
7
3
3
4
3
3
3
3
3
3
3
HO
Br
c)
d)
GIF-0665 (6)
0
dantrolene GIF-0082 GIF-0276 GIF-0430 GIF-0666 GIF-0665 GIF-0667
(1) (2) (3) (4) (5) (6) (7)
R
23: R = I
a)
b)
Figure 1. Effects of dantrolene (1) (20 lM) and its derivatives 2–7
(50 lM) on twitch contraction (open column) and CICR (filled
column) of mouse skeletal muscle. The magnitude of twitch contrac-
tion and the rates of Ca2+ release in the presence of 1 or its derivatives
were normalized to the control value (100%) obtained in the absence of
these compounds. The data for compounds 1–3 are cited from Ref. 9a.
The numbers of experiments (n) are indicated in the column.
24: R = C≡CSiMe3
25: R = C≡CH
26
Scheme 5. Synthesis of GIF-0665 (6). Reagents and conditions: (a)
Me3SiC„CH, Pd2(dba)3, (n-Bu)4NOAc, DMF, rt, 0.5 h, 64%; (b)
KOH, MeOH, 0 ꢁC to rt, 0.5 h, 88%; (c) CBr4, PPh3, CH2Cl2, rt, 0.5 h,
88%; (d) 1, DMF, 0 ꢁC to rt, 2 h, 61%.

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T. Hosoya et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1289–1294
2, and 3.9a As shown, azido-functionalized probes 4 and
6 had a selective inhibitory effect on PCR without
strongly affecting CICR. Although their effects were
slightly decreased compared to those of 2 and 3, both
compounds certainly inhibited PCR to a similar extent,
thereby indicating that these probes are potentially use-
ful in selective photolabeling of target molecules in-
volved in PCR. In contrast, the inhibitory effect of
trifluoromethyldiazirinyl compounds 5 and 7 on PCR
decreased considerably compared with 4 and 6. Thus,
we can conclude that the introduction of an azido group
as a photoreactive unit is a better choice than a trifluo-
romethyldiazirinyl group when designing dantrolene-de-
rived RI-free photoaffinity probes.
O
O2N
I
N
O
N
N
O
GIF-0162 (32)
OH
O
O
O
O
CH3O
Ph2P
H
N
H
Figure 2. SDS-PAGE analysis of rabbit TC fraction (1.0 mg/mL, 20 lg
total proteins) photolabeled with 5 lM of GIF-0430 (4) in the absence
or presence of dantrolene (1), its derivative, GIF-0162 (32) or GIF-
0082 (2) (20 lM), followed by Staudinger–Bertozzi ligation with GIF-
0373 (33) (500 lM) overnight. The photograph was taken with a
monochrome CCD camera (ATTO, Printgraph AE-6914) and visual-
ized on a UV transilluminator (UVP, TLW-20, 365 nm, 8 W · 4). For
comparison, Coomassie brilliant blue (CBB)-stained proteins con-
tained in the TC fraction are shown in lane 1. The arrowheads and the
attached numbers (Mr · 103) indicate apparent values of the fluores-
cent high molecular weight marker (Sigma).
N
O
O
OH
GIF-0373 (33)
With promising RI-free photoaffinity probes in hand, we
conducted actual photolabeling experiment using the
most promising diazido-functionalized probe 4 in terms
of inhibitory effect and selectivity, according to the pro-
tocol in our previous report.13 In Figure 2 is shown the
SDS-PAGE analysis of terminal cisternae (TC) fraction
of rabbit skeletal muscle25 (1.0 mg/mL, 20 lg total pro-
teins) photolabeled with 4 (5 lM) in the absence or pres-
ence of 1, its derivative, GIF-0162 (32)26 or 2 (20 lM),
followed by Staudinger–Bertozzi ligation with GIF-
0373 (33) (500 lM, incubated overnight), a fluorescein-
anchored triarylphosphine derivative. Photo-irradiation
was performed at room temperature for 180 s using a
portable UV lamp (UVP, UVG-54, 254 nm, 6 W, from
a distance of 1 cm). Consequently, two sharp fluorescent
bands, which exhibited apparent low Mr values
(<29,000), were observed (lane 3).27 Since 33 alone did
not react with any protein (lane 2), these bands can be
considered as candidate target proteins of 4. Further-
more, these clearly visualized protein bands were indis-
tinct or almost invisible when the photolabeling was
performed under the coexistence of an excess amount
of the PCR inhibitor, 1, 32,26 or 2 (lanes 4–6, respec-
tively), where the replacement of 4 with such a com-
pound would have taken place. This fact indicates that
there exists a specific interaction between 4 and the la-
beled proteins. Unexpectedly, the RyR1, which exhibits
higher Mr value (>350,000), was not captured by 4. This
is in marked disagreement with previous reports using
tritium labeled azido congener of 1.11 Although the rea-
son for the discrepancy is presently unclear, it may be
attributed to the lack of an inhibitory effect of 4 on
the CICR mechanism as shown in Figure 1.28
In summary, we designed and synthesized novel bifunc-
tional dantrolene derivatives, which exhibit an selective
inhibitory effect on the PCR process involved in the con-
traction of skeletal muscle, to extend the utility of the
newly devised RI-free photoaffinity labeling method.13
The actual photolabeling experiment using the most
promising probe served to detect candidate target pro-
teins of the dantrolene derivative. The direct determina-
tion of their structures and subsequent functional
studies will be provided in later reports.
Acknowledgements
We are grateful to Mr. Kin-ichi Oyama of the 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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Biochem. 1993, 62, 483; (c) Kotzyba-Hibert, F.; Kapfer,
I.; Goeldner, M. Angew. Chem., Int. Ed. Engl. 1995, 34,
1296; (d) Fleming, S. A. Tetrahedron 1995, 51, 12479; (e)
Hatanaka, Y.; Nakayama, H.; Kanaoka, Y. Rev. Hetero-
1
hexane/EtOAc = 1:2); H NMR (400 MHz, DMSO-d6) d
4.48 (s, 2H), 4.49 (s, 2H), 4.68 (s, 2H), 7.07 (br s, 2H), 7.08
(d, 1H, J = 3.6), 7.14 (br s, lH), 7.47 (d, 1H, J = 3.6 Hz),
7.86 (s, 1H), 7.99–8.03 (AA0BB0, 2H), 8.29–8.34 (AA0BB0,
2H); 13C NMR (100 MHz, DMSO-d6 + CDCl3) d 41.3,
48.4, 53.0, 112.1, 115.7, 117.8, 118.0, 123.9, 124.3 (2C),
124.4 (2C), 133.2, 135.0, 138.0, 138.4, 139.9, 146.1, 150.6,
152.2, 152.5, 167.0; IR (KBr, cmꢀ1) 853, 1150, 1250, 1329,
1447, 1512, 1597, 1717, 2109; UV (EtOH, nm) kmax (log e)
294 (4.16), 309 (4.16), 382 (4.50); HRMS (FAB+) m/z
Calcd for C22H17N10O5: 501.1383. Found: 501.1388
[M+H]+. GIF-0666 (5): Yellow solid; TLC Rf = 0.59 (n-
´
atom Chem. 1996, 14, 213; (f) Dorman, G. In Bioorganic
Chemistry of Biological Signal Transduction Topics in
Current Chemistry 211; Waldmann, H., Ed.; Springer:
´
Berlin, 2001; pp 169–225; (g) Dorman, G.; Prestwich, G.
D. Trends Biotechnol. 2000, 18, 64; (h) Hatanaka, Y.;
Sadakane, Y. Curr. Top. Med. Chem. 2002, 2, 271.
11. (a) Parness et al. reported the synthesis of tritium labeled
azido congener of 1 and proposed that it binds to RyR1
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; (c) Paul-Pletzer, K.; Yamamoto, T.; Bhat,
M. B.; Ma, J.; Ikemoto, N.; Jimenez, L. S.; Morimoto, H.;
Williams, P. G.; Parness, J. J. Biol. Chem. 2002, 277,
34918.
1
hexane/EtOAc = 1:2); H NMR (400 MHz, DMSO-d6) d
4.48 (s, 2H), 4.54 (s, 2H), 4.71 (s, 2H), 7.08 (d, J = 3.6 Hz,
1H), 7.23–7.27 (m, 2H), 7.47–7.49 (m, 2H), 7.87 (s, 1H),
8.00–8.04 (AA0BB0, 2H), 8.32–8.34 (AA0BB0, 2H);
13C NMR (100 MHz, DMSO-d6 + CDCl3)
d
27.8
(2J(19F–13C) = 40.1 Hz), 41.1, 48.1, 52.7, 111.9, 115.5,
121.6 (1J(19F–13C) = 275.3 Hz), 124.1 (2C), 124.3 (2C),
125.2, 125.5, 128.5, 129.2, 133.3, 135.0, 137.4, 137.7, 146.2,
150.6, 152.3, 152.5, 167.0; 19F NMR (372 MHz, DMSO-
d6) d 10.6 (s, 3F); IR (KBr, cm1) 603, 656, 694, 704, 749,
752, 764, 795, 855, 922, 970, 994, 1028, 1111, 1150, 1179,
1202, 1246, 1269, 1337, 1374, 1412, 1441, 1480, 1512, 1599,
1721, 1779, 2107; UV (EtOH, nm) kmax (loge) 309 (4.15),
320 (4.13), 381 (4.49); HRMS (FAB+) m/z Calcd for
C24H17F3N9O5: 568.1305. Found: 568.1301 [M+H]+.
12. Unpublished results.
13. Hosoya, T.; Hiramatsu, T.; Ikemoto, T.; Nakanishi, M.;
Aoyama, H.; Hosoya, A.; Iwata, T.; Maruyama, K.;
Endo, M.; Suzuki, M. Org. Biomol. Chem. 2004, 2, 637.
14. (a) Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007; (b)
Kiick, K. L.; Saxon, E.; Tirrell, D. A.; Bertozzi, C. R.

1294
T. Hosoya et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1289–1294
GIF-0665 (6). Yellow solid; TLC Rf = 0.56 (n-hexane/
EtOAc = 1:2); H NMR (400 MHz, DMSO-d6) d 4.33 (s,
(EtOH, nm) kmax (loge) 309 (4.14), 320 (4.11), 382 (4.47);
HRMS (FAB+) m/z Calcd for C25H16F3N6O5: 537.1134.
Found: 537.1152 [M+H]+.
1
1H), 4.49 (s, 2H), 4.66 (s, 2H), 7.08 (d, J = 3.6 Hz, 1H),
7.12–7.15 (m, 2H), 7.25 (br s, 1H), 7.48 (d, J = 3.6 Hz,
1H), 7.86 (s, 1H), 8.00–8.04 (AA0BB0, 2H), 8.31–8.34
(AA0BB0, 2H); 13C NMR (100 MHz, DMSO-d6 + CDCl3)
d 40.8, 48.2, 81.2, 81.9, 111.9, 115.5, 119.0, 120.9, 123.4,
124.1 (2C), 124.3 (2C), 127.4, 133.3, 135.0, 138.3, 140.0,
146.2, 150.6, 152.3, 152.5, 167.0; IR (KBr, cmꢀ1) 690, 735,
752, 803, 852, 924, 972, 1140, 1210, 1240, 1300, 1337, 1416,
1439, 1522, 1601, 1723, 1790, 2107; UV (EtOH, nm) kmax
(loge) 295 (4.23), 309 (4.26), 383 (4.55); HRMS (FAB+)
m/z Calcd for C23H16N7O5: 470.1213. Found: 470.1198
[M+H]+. GIF-0667 (7): Yellow solid; TLC Rf = 0.60 (n-
23. (a) Sonogashira, K.; Tohda, Y.; Hagihara, N. Tetrahedron
Lett. 1975, 4467; (b) Urgaonkar, S.; Verkade, J. G. J. Org.
Chem. 2004, 69, 5752.
24. Ikemoto, T.; Endo, M. Br. J. Pharmacol. 2001, 134,
719.
25. TC fraction was isolated from back and leg muscles from
Japanese white rabbit (2–2.5 kg, male) by sucrose density
gradient centrifugation according to the reported protocol
Saito, A.; Seiler, S.; Chu, A.; Fleischer, S. J. Cell Biol.
1984, 99, 875.
26. GIF-0162 (32) is a selective inhibitor for PCR without an
azido function. Normalized values on twitch contraction
and CICR rate of 32 (50 lM) were 36.3 10.2% (n = 3)
and 90.5 4.2% (n = 4), respectively.
27. These photolabeled proteins by 4 were in the same
molecular mass size with those labeled by [125I]-2. The
detailed analyses to compare them are in progress.
28. We prepared the phenyl ring-modified azido congener of 1
and found that it strongly inhibits not only the PCR but
also the CICR, as is the case with 1 (unpublished data).
This implicates that such an azido congener is functionally
different from the derivatives described in the present
study.
1
hexane/EtOAc = 1:2); H NMR (400 MHz, DMSO-d6), d
4.41 (s, 1H), 4.47 (s, 2H), 4.70 (s, 2H), 7.08 (d, J = 3.6 Hz,
1H), 7.31 (br s, 2H), 7.48 (d, J = 3.6 Hz, 1H), 7.59 (br s,
1H), 7.86 (s, 1H), 8.00–8.04 (AA0BB0, 2H), 8.30–8.34
(AA0BB0, 2H); 13C NMR (100 MHz, DMSO-d6 + CDCl3)
d 27.5 (2J(19F–13C) = 40.1 Hz), 40.9, 48.1, 81.4, 81.7,
111.7, 115.4, 121.4 (1J(19F–13C) = 274.5 Hz), 123.2, 124.0
(2C), 124.3 (2C), 125.5, 126.2, 128.6, 132.5, 133.3, 135.0,
137.6, 146.1, 150.5, 152.3, 152.4, 166.9; 19F NMR
(372 MHz, DMSO-d6), d 10.7 (s, 3F); IR (KBr, cmꢀ1
)
690, 735, 752, 803, 852, 924, 972, 1140, 1210, 1240, 1300,
1337, 1416, 1439, 1522, 1601, 1723, 1790, 2107; UV