Facile synthesis of diazido-functionalized biaryl compounds as radioisotope-free photoaffinity probes by Suzuki-Miyaura coupling

Article abstract of DOI:10.1016/j.bmc.2009.01.070

Suzuki-Miyaura coupling of 3-azido-5-(azidomethyl)phenylboronic acid pinacol ester with various aryl bromides affords corresponding diazido-functionalized biaryl compounds in good yields. This approach provides an easy access to radioisotope-free photoaff

Full text of DOI:10.1016/j.bmc.2009.01.070

Bioorganic & Medicinal Chemistry 17 (2009) 2490–2496
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Facile synthesis of diazido-functionalized biaryl compounds
as radioisotope-free photoaffinity probes by Suzuki–Miyaura coupling
a
a
b
Takamitsu Hosoya a, , Atsushi Inoue , Toshiyuki Hiramatsu , Hiroshi Aoyama ,
*
Takaaki Ikemoto c, Masaaki Suzuki c
a Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology and SORST, Japan Science and Technology Agency (JST),
4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan
b Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
c Center for Molecular Imaging Science, RIKEN, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Suzuki–Miyaura coupling of 3-azido-5-(azidomethyl)phenylboronic acid pinacol ester with various aryl
bromides affords corresponding diazido-functionalized biaryl compounds in good yields. This approach
provides an easy access to radioisotope-free photoaffinity probes possessing biaryl structure. By using
this method, we prepared a novel diazido-functionalized dantrolene analog, which showed selective
inhibitory effect on physiological Ca2+ release (PCR) from sarcoplasmic reticulum (SR) in mouse skeletal
muscle without affecting Ca2+-induced Ca2+ release (CICR).
Received 12 January 2009
Revised 28 January 2009
Accepted 29 January 2009
Available online 5 February 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Photoaffinity labeling
Radioisotope-free
Suzuki–Miyaura coupling
Dantrolene
1. Introduction
We previously established a different protocol for RI-free photo-
affinity labeling.3 In this scheme, an alkyl azido group, which was
Photoaffinity labeling is a useful technique to determine target
biomolecules of a bioactive compound.1 Usually, photoaffinity
probe consist of two functional groups. One is a photoreactive
group, which enables to form a new covalent bond between the
probe and its target molecule by photoreaction. Another one is a
detectable tag to distinguish the photolabeled molecule from unla-
beled ones. On the occasion of preparation of a photoaffinity probe,
these functional groups are required to be introduced to the origi-
nal compound without diminishing its bioactivity to obtain a reli-
able result. Representative photoreactive groups are aromatic
azido, diazirinyl, and benzophenone derivatives, which generate
highly reactive species such as nitrene, carbene, and excited car-
bonyl, respectively, by irradiation of light. The most widely used
indicator is a radioisotope (RI) such as 3H, 14C, 32P, 35S, and 125I.
The use of biotin-anchored probe, photoaffinity biotinylation,2 is
an excellent alternative, which can avoid the use of hazardous
RIs. In this manner, biotin unit is employed as a detectable tag tak-
ing advantage of its extremely high affinity toward avidin, thereby
allowing direct analysis or purification of photolabeled
biomolecules.
proved to be relatively photostable under the photoreactive condi-
tions of aryl azide and trifluoromethyldiazirine, was used as a post-
modifiable group to introduce an arbitrary detectable tag such as a
fluorescent unit. This method is based upon the bioorthogonal
character of azido group as well as some efficient azido-targeting
reactions such as Staudinger–Bertozzi ligation4 and Huisgen 1,3-
dipolar cycloaddition, click chemistry.5,6 Our method only requires
an introduction of an azidomethyl group to the probe with low
polarity and small in size, which minimizes the adverse effects
on the intrinsic biological activity and selectivity of the original
compound. Indeed, the effectiveness of this approach was demon-
strated by the fluorescent detection of photolabeled HMG-CoA
reductase using a diazido-functionalized cerivastatin derivative,
photovastatin CAA1 (1) (Fig. 1).3a We also prepared a diazidoben-
zlyated dantrolene derivative, GIF-0430 (2), which exhibit a selec-
tive inhibitory effect on the physiological Ca2+ release (PCR)
process involved in the contraction of skeletal muscle, and suc-
ceeded in the fluorescent detection of its target proteins.3b In both
studies, 3-azido-5-(azidomethyl)benzyl derivatives were used as
efficient photoaffinity probes (Fig. 1). These probes were designed
that the all three substituents on the benzene ring to be placed in
meta-positions in relation to each other to avoid unnecessary
interactions.
In order to prepare these type of probes, especially those
with biaryl structure, more straightforwardly, we conceived
* Corresponding author. Tel./fax: +81 45 924 5733.
E-mail address: thosoya@bio.titech.ac.jp (T. Hosoya).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.070

T. Hosoya et al. / Bioorg. Med. Chem. 17 (2009) 2490–2496
2491
F
OH OH
O
O
O2N
RO
ONa
N R
O
N
N
N
O
cerivastatin: R = CH3
photovastatin CAA1 (1): R =
dantrolene: R = H
GIF-0430 (2): R = CH2
CH
2
N3
N3
N3
N3
Figure 1. Structures of cerivastatin, dantrolene, and their corresponding RI-free photoaffinity probes.
the idea of introducing 3-azido-5-(azidomethyl)benzene unit by
2.2. Suzuki–Miyaura coupling
aryl–aryl coupling reaction. In this article, we describe the facile
approach using Suzuki–Miyaura cross-coupling,7 a well-estab-
lished reaction to prepare diverse biaryl compounds, and its
application to the synthesis of a novel diazido-functionalized
dantrolene analog as an RI-free photoaffinity probe with specific
bioactivity.
With a common intermediate 7 in hand, we examined the
cross-coupling reaction with various aryl bromides. First of all,
the conventional reaction conditions for Suzuki–Miyaura coupling
was applied to the reaction of boron reagent 7 with the most sim-
ple aryl bromide, bromobenzene (8a). Namely, the mixture of 8a
and 7 (1.2 equiv) in the presence of 5 mol% Pd(PPh3)4 and K3PO4
(3 equiv) in DMF was heated at 80 °C under nitrogen atmosphere.
The reaction for 3 h gave the desired coupling product in 70% iso-
lated yield (Table 1, entry 1). Under this condition, no side products
such as compounds arose from reduction of azido groups were ob-
tained. The reaction with iodobenzene slightly increased the yield
to 80%. All other reactions examined using various heteroaryl bro-
mide also gave desired coupling products in good yields (Table 1).
For instance, the reaction with pyridyl (entry 2), pyrimidyl (entry
3), thienyl (entry 4), isoquinolinyl (entry 5), and indolyl (entries
6 and 7) substrates, which are typical heteroaromatic compounds
often included in bioactive drug’s structure, have proceeded
smoothly. The reaction was not retarded in the presence of formyl
or phenolic hydroxy groups (entries 4 and 8). The reaction for sub-
strate 8i, which did not proceed under the standard condition, was
found to undergo efficiently by using a different phosphine ligand
such as PCy3 (entry 9).
2. Results and discussion
2.1. Synthesis of the common intermediate
We prepared 3-azido-5-(azidomethyl)phenylboronic acid pina-
col ester (7) as the common intermediate for Suzuki–Miyaura cou-
pling (Scheme 1). Firstly, 3-azido-5-iodobenzyl alcohol (4) was
prepared in four steps from commercially available 3,5-dinitroben-
zyl alcohol (3) as we reported previously.8a The mesylation of 4 fol-
lowed by treatment with sodium azide afforded diazido-
functionalized iodobenzene derivative 6. The Pd(0)-catalyzed cou-
pling reaction of bis(pinacolato)diboron under standard condition9
with 6 gave desired 7.
2.3. Design and synthesis of a novel diazido-functionalized
dantrolene analog
OH
OH
Dantrolene is a hydantoin derivative, which is known as a mus-
cle relaxant used as an only drug for malignant hyperthermia.10,11
In skeletal muscle, dantrolene inhibits two kinds of Ca2+ release
from sarcoplasmic reticulum (SR): physiological Ca2+ release
(PCR) and Ca2+-induced Ca2+ release (CICR). Although the physio-
logical role of the latter mode of Ca2+ release is not well under-
MsCl, Et3N
CH2Cl2
97%
4 steps
Ref. 8a
O2N
NO2
N3
I
3
4
OMs
N3
stood, PCR from SR in skeletal muscle is controlled by a Ca2+
-
bis(pinacolato)diboron
PdCl2(dppf), KOAc
NaN3
releasing channel on the SR membrane, which is called skeletal-
type ryanodine receptor (RyR1), whose function is regulated by a
signal from the dihydropyridine receptor (DHPR), a voltage sensor
in the cell membrane.12 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 undetermined molecules, is still
controversial.13,14 In order to elucidate the molecular mechanism
involved in the Ca2+-related processes of skeletal muscle, we have
been developing chemical probes based on the structural modifica-
tion of dantrolene.3b,8 Of these, the RI-labeled photoaffinity probe,
DMF
DMSO
59%
N3
I
N3
I
quant.
5
6
N3
O
N3
B
O
7
[
125I]GIF-0082 ([125I]-10), whose structure include azidobenzylated
unit showed highly selective inhibitory effect on PCR without
Scheme 1. Synthesis of common intermediate 7 for Suzuki–Miyaura coupling.

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T. Hosoya et al. / Bioorg. Med. Chem. 17 (2009) 2490–2496
Table 1 (continued)
Table 1
Synthesis of diazido-functionalized biaryl compounds by Suzuki–Miyaura coupling
Entry
9
Time (h)
Yield (%)
N3
N3
Aryl—Br (8a-i)
Pd(PPh3)4 (5 mol%)
K3PO4 (3 equiv.)
N3
9
20
90b
O
DMF
80 °C
N3
B
N3
Aryl
N3
O
7
9a-i
9i
N
NHAc
a
Reaction with iodobenzene.
Reaction conditions: 2 mol% Pd2(dba)3, PCy3 (0.04 equiv), K3PO4 (3 equiv), 1,4-
b
Entry
9
Time (h)
Yield (%)
dioxane–H2O, 100 °C.
N3
1
3
70
O
(80)a
N3
N3
N3
O2N
R
N
9a
9b
9c
O
N
N
N3
N3
N3
O
N3
R = 125I: [125I]GIF-0082 ([125I]-10)
R = CH2N3: GIF-0430 (2)
2
3
4
5
6
3
85
82
75
75
81
O
N
N
N3
X
NH
O
N
N
O
4
N
X = H: azidodantrolene (11)
X = 3H: [3H]azidodantrolene ([3H]-11)
Figure 2. Photoaffinity probes based on dantrolene.
affecting CICR (Fig. 2).8a,c By using this selective inhibitor, we suc-
ceeded in the photolabeling of target protein candidates and char-
acterized the primary structure of one target protein purified from
an independent muscle preparation using molecular weight strat-
ification by SDS–PAGE analysis. To make the direct analyses of
photolabeled proteins possible, we prepared GIF-0430 (2), where
the 125I group of [125I]-10 is switched to an azidomethyl group as
a latent detectable group.3b Compound 2 also showed selective
PCR inhibitory effect and photolabeling experiment using this
probe enabled fluorescent detection of target proteins, which
showed good agreement with the result where RI-probe [125I]-10
was employed. Unexpectedly, the RyR1, which exhibits higher Mr
value (>350,000), was not photolabeled by neither RI-probe
O
3.5
3.5
4
N3
N3
N3
H
S
9d
N3
9e
9f
N
N3
[
125I]-10 nor RI-free probe 2. This is in marked disagreement with
previous reports using tritium labeled azido congener of dantro-
lene, which is referred to as [3H]azidodantrolene ([3H]-11)
(Fig. 2).15 Although the reason for the discrepancy is presently un-
clear, it may be attributed to the lack of an inhibitory effect of
N
Boc
N3
[
125I]-10 and 2 on the CICR mechanism.3b,8a,c Another possible rea-
son is the difference of the position of photoreactive azido groups
between our probes, [125I]-10 and 2, and [3H]-11.
7
3
81
To make these discrepancies clearer, we were interested in pre-
paring the dantrolene analog such as 13, which is a diazido conge-
ner of dantrolene. Because 13 has non-N-substituted hydantoin
moiety unlike [125I]-10 and 2, it is expected to own similar polarity
with dantrolene and [3H]-11. Compound 13 also possesses photo-
reactive azido group on the phenyl ring same as [3H]-11. Therefore
we anticipated that 13 could be used as a different type of RI-free
photoaffinity probe instead of 2. The compound 13 with small sub-
stituents on two meta-positions of the benzene ring was designed
also because this type of compound was expected to show selec-
tive inhibitory effect on PCR. In fact, we previously have shown
N3
N
9g
9h
SO2Ph
N3
O
8
3
83
Ph
N3
N
N
OH

T. Hosoya et al. / Bioorg. Med. Chem. 17 (2009) 2490–2496
2493
N3
125
7
Pd(PPh3)4 (5 mol%)
K3PO4 (3 equiv.)
O
O
O
O
Br
H
DMF
80 °C, 3 h
100
75
50
25
0
N3
H
8j
68%
9j
O
NH
N3
N
O
ClH•H2N
O
NH
12
O
N
N3
N
aq. HCl
DMF
rt, 2 h
O
13
88%
13
6
4
3
3
3
3
3
Scheme 2. Synthesis of diazido-functionalized dantrolene analog 13.
dantrolene GIF-0430
13
azido-
(2)
dantrolene (11)
50 µM
20 µM
50 µM
50 µM
that both 3-nitro and 3-methoxy congeners of dantrolene are
highly selective inhibitor for PCR.8d
Figure 3. Effects of dantrolene, GIF-0430 (2), 13, and azidodantrolene (11) on
twitch contraction (open column) and CICR rate (filled column) of mouse skeletal
muscle. For the methods of biological evaluation, see Ref. 8. The number of
experiments (n) are indicated in the column. The data of dantrolene and GIF-0430
(2) are cited from Refs. 8a, and 3b, respectively.
As shown in Scheme 2, the compound 13 could be easily pre-
pared by applying above-mentioned synthetic method of diazido-
functionalized biaryl compounds. Thus, Suzuki–Miyaura coupling
of commercially available 5-bromo-2-furaldehyde (8j) with diazi-
do boronic acid pinacol ester 7 afforded desired coupling product
9j in 68% yield.16 Finally, treatment of furaldehyde 9j with 1-amin-
ohydantoin hydrochloride (12) under acidic condition furnished
dantrolene analog 13.
molecules. Indeed, by using this method, we prepared a novel diaz-
ido-functionalized dantrolene analog 13, which showed selective
inhibitory effect on PCR from SR in mouse skeletal muscle without
affecting CICR. Actual photolabeling studies using 13 along with
design and synthesis of different kinds of RI-free photoaffinity
probes based on this strategy are currently in progress.
2.4. Evaluation of effects on Ca2+ release
We examined the effect of diazido-functionalized dantrolene
analog 13 on two kinds of Ca2+ release from SR of mouse skeletal
muscle, PCR and CICR, according to the methods we previously re-
ported.8 The effects on PCR were estimated by comparing the ten-
sion of twitch contraction of intact mouse skeletal muscle at room
temperature before and after treatment with the analogs.8 The ef-
fects on CICR were evaluated at room temperature by measuring
the rates of CICR in saponin-treated skinned muscle fibers of
mouse skeletal muscle by using Fura-2 as a Ca2+ indicator under
4. Experimental
4.1. Chemistry
General: All chemical reagents were used as received. Analytical
thin-layer chromatography (TLC) was performed on precoated
(0.25 mm) silica-gel plates (MERCK, Silica Gel 60 F254, Cat. No.
1.05715). Column chromatography was conducted using silica-
gel (Kanto Chemical Co., Inc., Silica Gel 60 N, spherical neutral,
Mg2+-free conditions at 1 M Ca2+ 8
l . For comparison, we also pre-
pared azidodantrolene (11)15a and evaluated its effect on PCR
and CICR. The effects of these compounds including those of dan-
trolene and GIF-0430 (2) are shown in Figure 3 with normalized
values. As can be seen, compound 13 showed moderate but selec-
tive inhibitory effect on PCR with almost no effect on CICR, which is
mesh 40–50 lm, Cat. No. 37563-85). Melting points (mp) were
measured on a YANACO MP-J3 instrument and are uncorrected.
1H and 13C NMR were obtained with Varian UNITY Plus 400 or Var-
ian MERCURY 300 spectrometers. CDCl3 (CIL) and DMSO-d6 (CIL)
were used as solvents for obtaining NMR spectra. Chemical shifts
(d) are given in parts per million (ppm) downfield from (CH3)4Si
(d 0.00 for 1H NMR in CDCl3) or solvents (d 7.27 and 2.49 for 1H
NMR in CDCl3 and DMSO-d6, respectively, and d 77.0 and 39.5 for
13C NMR in CDCl3 and DMSO-d6, respectively), as internal refer-
ences with coupling constants (J) in hertz (Hz). The abbreviations
s, d, m, and br signify singlet, doublet, multiplet, and broad, respec-
tively. IR spectra were measured by diffuse reflectance method on
a Shimadzu IRPrestige-21 spectrometer attached with DRS-8000A
with the absorption band given in cmÀ1. Elemental analyses were
performed with a YANACO CHN CORDER MT-5 at the Center for
Advanced Materials Analysis (Suzukakedai), Technical Depart-
ment, Tokyo Institute of Technology. Mass spectra (MS) and
high-resolution mass spectra (HRMS) were measured on a JEOL
JMS-700 mass spectrometer under electron impact ionization (EI)
or positive fast atom bombardment (FAB+) conditions at the Center
comparable to GIF-0430 (2). On the other hand, 50
dodantrolene (11) inhibited both PCR and CICR significantly to a
similar extent of that of 20 M dose of dantrolene. This result indi-
lM dose of azi-
l
cates that the novel probe 13 can be used as promising photo-
affinity probe to elucidate selectively the target protein involved
in the PCR mechanism and possibly can make some contribution
to clarify the mechanism of action of dantrolene on Ca2+ releasing
processes in skeletal muscle cells.
3. Conclusion
In summary, we have established a simple method to prepare
diazido-functionalized biaryl compounds by Suzuki–Miyaura
coupling. This method would allow rapid access to RI-free photo-
affinity probes of bioactive compounds especially with phenyl–
heteroaryl structure, which are commonly-seen in drug-like

2494
T. Hosoya et al. / Bioorg. Med. Chem. 17 (2009) 2490–2496
for Advanced Materials Analysis (Suzukakedai), Technical Depart-
ment, Tokyo Institute of Technology.
1165, 1206, 1288, 1308, 1339, 1369, 1429, 1470, 1587, 2108,
2932, 2980, 3431; HRMS (EI) m/z 300.1508 (M+, C13H17BN6O2 re-
quires 300.1506).
Caution: Azido-containing compounds are presumed to be
potentially explosive. Although we have never experienced such
an explosion with azido-functionalized compounds used in this
study, all manipulations should be carefully carried out behind a
safety shield in a hood.
4.1.4. 3-Azido-5-(azidomethyl)biphenyl (9a)
4.1.4.1. A representative procedure for Suzuki–Miyaura
coupling.
390 mol) in DMF (3.5 mL) was successively added bromoben-
zene (8a) (51.0 mg, 325 mol), Pd(PPh3)4 (17.4 mg, 15.1 mol),
and potassium phosphate n-hydrate (191 mg, 900 mol) at room
Under N2 atmosphere, to a solution of 7 (117 mg,
l
4.1.1. 3-Azido-5-iodobenzyl methanesulfonate (5)
l
l
To a solution of 3-azido-5-iodobenzyl alcohol (4)8a (2.69 g,
9.78 mmol) in CH2Cl2 (80 mL) was successively added triethyl-
amine (3.40 mL, 24.4 mmol) and methanesulfonyl chloride
(1.50 mL, 19.4 mmol) at 0 °C. After stirring for 45 min at the same
temperature, to the mixture was added water and the mixture was
extracted with CH2Cl2 (Â3). The combined organic extracts were
washed with brine (Â1), dried (Na2SO4), filtered, and concentrated
under reduced pressure. The residue was purified by silica-gel col-
umn chromatography (n-hexane/EtOAc = 2/1) to give 5 (3.35 g,
97.0%) as a pale yellow solid; mp 77–79 °C; TLC Rf = 0.32 (n-hex-
ane/EtOAc = 3/1); 1H NMR (400 MHz, CDCl3) d 3.02 (s, 3H), 5.14
(s, 2H), 7.01 (dd, 1H, J = 1.6, 2.0 Hz), 7.39 (dd, 1H, J = 1.6, 2.0 Hz),
7.51 (dd, 1H, J = 1.6, 1.6 Hz); 13C NMR (75.5 MHz, CDCl3) d 38.2,
68.9, 94.8, 118.3, 128.4, 133.5, 137.0, 141.9; IR (KBr, cmÀ1) 513,
530, 698, 737, 754, 804, 837, 864, 907, 930, 955, 970, 991, 1173,
1213, 1263, 1298, 1337, 1381, 1439, 1572, 1593, 2118, 3026,
3406; HRMS (EI) m/z 352.9340 (M+, C8H8IN3O3S requires
352.9331).
l
temperature. The mixture was heated at 80 °C and stirred for
3 h. After cooling to room temperature, to the mixture was added
2 M aqueous HCl solution and extracted with EtOAc (3). The com-
bined organic extracts were successively washed with saturated
aqueous NaHCO3 solution (Â1), water (Â3), and brine (Â1), dried
(Na2SO4), filtered and concentrated under reduced pressure. The
residue was purified by silica-gel column chromatography (n-
hexane/EtOAc = 15/1) to give 9a (57.1 mg, 70.2%) as a pale yellow
oil; TLC Rf = 0.75 (n-hexane/EtOAc = 3/1); 1H NMR (400 MHz,
CDCl3) d 4.41 (s, 2H), 6.96 (dd, 1H, J = 1.4, 1.4 Hz), 7.20 (dd, 1H,
J = 1.4, 1.7 Hz), 7.30 (dd, 1H, J = 1.4, 1.7 Hz), 7.37–7.59 (m, 5H);
IR (KBr, cmÀ1) 696, 725, 762, 851, 1261, 1325, 1346, 1423, 1454,
1497, 1576, 1595, 2100, 2926, 3036, 3061; MS (EI) m/z 250.
4.1.5. 3-[3-Azido-5-(azidomethyl)phenyl]pyridine (9b)
Colorless oil; TLC Rf = 0.25 (n-hexane/EtOAc = 3/1); 1H NMR
(400 MHz, CDCl3) d 4.44 (s, 2H), 7.03 (dd, 1H, J = 1.5, 1.7 Hz), 7.18
(dd, 1H, J = 1.7, 1.7 Hz), 7.26 (dd, 1H, J = 1.5, 1.7 Hz), 7.40 (ddd,
1H, J = 0.9, 4.9, 7.9 Hz), 7.86 (ddd, 1H, J = 1.5, 2.3, 7.9 Hz), 8.64
(dd, 1H, J = 1.5, 4.9 Hz), 8.83 (dd, 1H, J = 0.9, 2.3 Hz); IR (KBr,
cmÀ1) 712, 806, 851, 1022, 1184, 1261, 1327, 1346, 1402, 1445,
1485, 1595, 2104; MS (EI) m/z 251.
4.1.2. 1-Azido-3-azidomethyl-5-iodobenzene (6)
To a solution of 5 (2.85 g, 8.07 mmol) in DMF (20 mL) was
added sodium azide (2.10 g, 32.3 mmol) at room temperature
and the mixture was stirred for 1.5 h at the same temperature.
To this was added water and the mixture was extracted with EtOAc
(Â3). The combined organic extracts were successively washed
with water (Â3) and brine (Â1), dried (Na2SO4), filtered and con-
centrated under reduced pressure. The residue was purified by sil-
ica-gel column chromatography (n-hexane/EtOAc = 15/1) to give 6
(2.41 g, 99.5%) as a pale yellow oil; TLC Rf = 0.78 (n-hexane/
EtOAc = 3/1); 1H NMR (400 MHz, CDCl3) d 4.31 (s, 2H), 6.93 (dd,
1H, J = 1.5, 2.0 Hz), 7.34 (dd, 1H, J = 1.5, 2.0 Hz), 7.43 (dd, 1H,
J = 1.5, 1.5 Hz); 13C NMR (75.5 MHz, CDCl3) d 53.4, 94.8, 117.9,
127.6, 133.2, 139.1, 141.8; IR (KBr, cmÀ1) 675, 698, 814, 845,
1202, 1246, 1292, 1339, 1433, 1442, 1560, 1595, 2100, 2328,
2359, 3065, 3370; HRMS (EI) m/z 299.9618 (M+, C7H5IN6 requires
299.9620).
4.1.6. 5-[3-Azido-5-(azidomethyl)phenyl]pyrimidine (9c)
Colorless solid; TLC Rf = 0.13 (n-hexane/EtOAc = 3/1); 1H NMR
(400 MHz, CDCl3) d 4.47 (s, 2H), 7.10 (dd, 1H, J = 1.6, 1.6 Hz), 7.17
(dd, 1H, J = 1.6, 1.6 Hz), 7.29 (dd, 1H, J = 1.6, 1.6 Hz), 8.95 (s, 2H),
9.26 (s,1H); IR (KBr, cmÀ1) 681, 692, 725, 854, 1188, 1261, 1356,
1408, 1557, 1607, 2110; MS (EI) m/z 252.
4.1.7. 4-[3-Azido-5-(azidomethyl)phenyl]thiophene-2-
carboxaldehyde (9d)
Pale yellow solid; TLC Rf = 0.60 (n-hexane/EtOAc = 3/1); 1H NMR
(400 MHz, CDCl3) d 4.42 (s, 2H), 6.98 (dd, 1H, J = 1.5, 2.0 Hz), 7.18
(dd, 1H, J = 2.0, 2.0 Hz), 7.31 (dd, 1H, J = 1.5, 2.0 Hz), 7.90 (dd, 1H,
J = 1.4, 1.4 Hz), 8.02 (d, 1H, J = 1.4 Hz), 9.90 (d, 1H, J = 1.4 Hz); IR
(KBr, cmÀ1) 665, 849, 1188, 1221, 1248, 1302, 1339, 1354, 1425,
1541, 1595, 1670, 2108; MS (EI) m/z 284.
4.1.3. 2-[3-Azido-5-(azidomethyl)phenyl]-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (7)
Under N2 atmosphere, to a solution of 6 (2.68 g, 8.93 mmol) in
DMSO (15 mL) were successively added bis(pinacolato)diboron
(3.17 g, 12.5 mmol), potassium acetate (2.45 g, 25.0 mmol), and
4.1.8. 4-[3-Azido-5-(azidomethyl)phenyl]isoquinoline (9e)
Pale yellow solid; TLC Rf = 0.53 (n-hexane/EtOAc = 2/1); 1H NMR
(400 MHz, CDCl3) d 4.46 (s, 2H), 7.11 (dd, 1H, J = 1.5, 1.7 Hz), 7.15
(dd, 1H, J = 1.7, 1.7 Hz), 7.24 (dd, 1H, J = 1.5, 1.7 Hz), 7.67 (ddd,
1H, J = 1.2, 6.8, 8.2 Hz), 7.72 (ddd, 1H, J = 1.6, 6.8, 8.2 Hz), 7.85
(dd, 1H, J = 1.2, 8.2 Hz), 8.07 (dd, 1H, J = 1.6, 8.2 Hz), 8.47 (s, 1H),
9.29 (s, 1H); IR (KBr, cmÀ1) 700, 754, 785, 856, 893, 1209, 1223,
1254, 1275, 1308, 1331, 1344, 1391, 1439, 1591, 2106; MS (EI)
m/z 301.
PdCl2(dppf)ÁCH2Cl2 (221 mg, 271
lmol) at room temperature. The
mixture was heated at 80 °C and stirred for 4.5 h. After cooling to
room temperature, to the mixture was added 1 M aqueous HCl
solution and extracted with EtOAc (Â3). The combined organic ex-
tracts were successively washed with saturated aqueous NaHCO3
solution (Â1), water (Â3) and brine (Â2), dried (Na2SO4), filtered
and concentrated under reduced pressure. The residue was puri-
fied by silica-gel column chromatography (n-hexane/EtOAc =
20/1) to give 7 (1.59 g, 59.3%) as a pale yellow solid; mp 46–
48 °C; TLC Rf = 0.77 (n-hexane/EtOAc = 3/1); 1H NMR (400 MHz,
CDCl3) d 1.36 (s, 12H), 4.35 (s, 2H), 7.05 (dd, 1H, J = 1.8, 2.2 Hz),
7.45 (dd, 1H, J = 0.4, 2.2 Hz), 7.51 (dd, 1H, J = 0.4, 1.8 Hz); 13C
NMR (75.5 MHz, CDCl3) d 24.8 (4C), 54.1, 84.2 (2C), 121.4, 124.6,
130.7, 136.7, 140.2 (the carbon adjacent to boron was not ob-
served); IR (KBr, cmÀ1) 706, 735, 851, 905, 968, 1113, 1144,
4.1.9. 5-[3-Azido-5-(azidomethyl)phenyl]-1-tert-
butoxycarbonyl-1H-indole (9f)
Pale yellow oil; TLC Rf = 0.80 (n-hexane/CH2Cl2 = 3/2); 1H NMR
(400 MHz, CDCl3) d 1.69 (s, 9H), 4.42 (s, 2H), 6.63 (d, 1H,
J = 3.5 Hz), 6.95 (dd, 1H, J = 1.6, 1.6 Hz), 7.25 (dd, 1H, J = 1.6,
1.6 Hz), 7.35 (dd, 1H, J = 1.6, 1.6 Hz), 7.52 (dd, 1H, J = 1.7, 8.8 Hz),
7.64 (d, 1H, J = 3.5 Hz), 7.75 (d, 1H, J = 1.7 Hz), 8.21 (d, 1H,

T. Hosoya et al. / Bioorg. Med. Chem. 17 (2009) 2490–2496
2495
J = 8.8 Hz); IR (KBr, cmÀ1) 729, 766, 818, 854, 1024, 1086, 1138,
1157, 1240, 1282, 1321, 1340, 1368, 1449, 1479, 1593, 1603,
1732, 2106, 2978; MS (EI) m/z 389.
949, 972, 1022, 1121, 1215, 1229, 1246, 1283, 1319, 1344, 1377,
1406, 1445, 1518, 1558, 1603, 1618, 1726, 1782, 1819, 2108,
3064, 3169; HRMS (FAB+) m/z 366.1060 ([M+H]+, C15H12N9O3 re-
quires 366.1063).
4.1.10. 3-[3-Azido-5-(azidomethyl)phenyl]-1-(phenylsulfonyl)-
1H-indole (9g)
5. Biological evaluation
Pale yellow solid; TLC Rf = 0.50 (n-hexane/EtOAc = 3/1); 1H NMR
(400 MHz, CDCl3) d 4.43 (s, 2H), 7.00 (dd, 1H, J = 1.7, 2.0 Hz), 7.22
(dd, 1H, J = 1.7, 1.7 Hz), 7.31–7.36 (m, 2H), 7.41 (ddd, 1H, J = 0.9,
7.2, 8.2 Hz), 7.46–7.51 (m, 2H), 7.55–7.60 (m, 1H), 7.75 (ddd, 1H,
J = 0.9, 0.9, 8.2 Hz), 7.75 (s, 1H), 7.94–7.98 (m, 2H), 8.08 (ddd, 1H,
J = 0.9, 0.9, 8.2 Hz); IR (KBr, cmÀ1) 571, 592, 685, 729, 746, 972,
1090, 1111, 1134, 1177, 1242, 1277, 1292, 1310, 1371, 1447,
1593, 1607, 2106; MS (EI) m/z 429.
See Ref. 8 and references cited therein.
Acknowledgments
This work was partially supported by Tokyo Tech Award for
Challenging Research and a Grant-in-Aid for Scientific Research
(C) (No. 19510213) of Japan Society for the Promotion of Science
(JSPS). We are grateful to Ms. M. Ishikawa of Center for Advanced
Materials Analysis (Suzukakedai), Technical Department, Tokyo
Institute of Technology, for performing the mass spectra analysis.
4.1.11. 5-[3-Azido-5-(azidomethyl)phenyl]-2-hydroxyphenyl 1-
phenyl-1H-pyrazol-4-yl ketone (9h)
Yellow solid; TLC Rf = 0.53 (n-hexane/EtOAc = 3/1); 1H NMR
(400 MHz, CDCl3) d 4.42 (s, 2H), 6.97 (dd, 1H, J = 1.5, 2.0 Hz),
7.12 (dd, 1H, J = 1.5, 2.0 Hz), 7.18 (dd, 1H, J = 0.4, 8.6 Hz), 7.24
(dd, 1H, J = 1.5, 1.5 Hz), 7.38–7.43 (m, 1H), 7.49–7.55 (m, 2H),
7.72–7.78 (m, 3H), 8.08 (dd, 1H, J = 0.4, 2.3 Hz), 8.21 (d, 1H,
References and notes
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J = 0.7 Hz), 8.53 (d, 1H, J = 0.7 Hz), 12.03 (s, 1H); IR (KBr, cmÀ1
)
758, 835, 953, 1221, 1238, 1288, 1308, 1329, 1348, 1404,
1447, 1460, 1491, 1504, 1541, 1595, 1626, 2104; MS (EI) m/z
436.
4.1.12. 2-Acetamino-5-[3-azido-5-(azidomethyl)phenyl]
pyridine (9i)
Yellow solid; TLC Rf = 0.27 (n-hexane/EtOAc = 3/2); 1H NMR
(400 MHz, CDCl3) d 2.25 (s, 3H), 4.43 (s, 2H), 7.00 (dd, 1H, J = 1.5,
1.8 Hz), 7.14 (dd, 1H, J = 1.5, 1.8 Hz), 7.26 (dd, 1H, J = 1.8, 1.8 Hz),
7.89 (dd, 1H, J = 2.4, 8.7 Hz), 8.17 (s, 1H), 8.29 (dd, 1H, J = 0.6,
8.7 Hz), 8.47 (dd, 1H, J = 0.6, 2.4 Hz); IR (KBr, cmÀ1) 839, 1260,
1308, 1321, 1375, 1452, 1543, 1593, 1607, 1701, 2110, 3021,
3067, 3244; MS (EI) m/z 308.
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4.1.13. 5-[3-Azido-5-(azidomethyl)phenyl]-2-furaldehyde (9j)
Orange solid; TLC Rf = 0.31 (n-hexane/EtOAc = 3/1); 1H NMR
(400 MHz, CDCl3) d 4.43 (s, 2H), 6.90 (d, 1H, J = 3.7 Hz), 7.02
(dd, 1H, J = 1.6, 1.6 Hz), 7.34 (d, 1H, J = 3.7 Hz), 7.41 (dd, 1H,
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NMR (75.5 MHz, CDCl3)
123.2, 131.0, 138.4, 141.6, 152.2, 157.4, 177.3; IR (KBr,
cmÀ1
764, 800, 856, 966, 1030, 1252, 1281, 1315, 1342,
d 53.9, 108.8, 115.2, 119.2, 121.0,
)
1389, 1441, 1516, 1568, 1599, 1674, 2108; MS (EI) m/z 268;
HRMS (FAB+) m/z 269.0789 ([M+H]+, C12H9N6O2 requires
269.0787).
4.1.14. ({5-[3-Azido-5-(azidomethyl)phenyl]furfurylidene}
amino)imidazolidine-2,4-dione (11)
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A solution of 1-aminohydantoin hydrochloride (12) (42.7 mg,
282
l
mol) in 2.0 M aqueous HCl (0.5 mL, 1.0 mmol) was added to
mol) in DMF (1.5 mL) at 0 °C and
a solution of 9j (50.4 mg, 188
l
the mixture was stirred for 2 h at room temperature. To this was
added water (ca. 10 mL) and the precipitate formed was filtered
and washed well with water on a funnel. The collected solid was
dried under reduced pressure to give 11 (60.5 mg, 88.1%) as a
slightly yellow solid; TLC Rf = 0.42 (n-hexane/EtOAc = 1/2); 1H
NMR (400 MHz, DMSO-d6) d 4.43 (s, 2H), 4.56 (s, 2H), 6.97 (d,
1H, J = 3.6 Hz), 7.12 (br s, 1H), 7.28 (d, 1H, J = 3.6 Hz), 7.43 (br s,
1H), 7.57 (br s, 1H), 7.74 (s, 1H), 11.29 (s, 1H); 13C NMR
(75.5 MHz, DMSO-d6 + CDCl3) d 48.7, 52.9, 109.4, 113.7, 114.9,
117.9, 120.0, 131.4, 132.9, 138.4, 140.6, 149.5, 153.0, 153.2,
168.4; IR (KBr, cmÀ1) 440, 465, 606, 687, 741, 779, 810, 856, 912,

2496
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16. Compound 9j was also obtainable by Stille coupling of 5-(tri-n-butylstannyl)-
2-furaldehyde and 1-azido-3-azidomethyl-5-iodobenzene (6) in 84% yield
(5 mol% (Ph3P)2PdCl2/DME, 70 °C, 5 h), which is a general synthetic method of
dantrolene analogs we previously reported. See Ref. 8d.