Efficient synthesis of Hsp90 inhibitor dimers as potential antitumor agents

Article abstract of DOI:10.1016/j.bmc.2010.05.075

The PU-H58-dimers 13a-15b were efficiently synthesized and their biological properties were evaluated. The copper-catalyzed alkyne azide coupling was effective in simultaneously linking three components via a triazole formation to afford the target dimers

Full text of DOI:10.1016/j.bmc.2010.05.075

Bioorganic & Medicinal Chemistry 18 (2010) 5732–5737
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Efficient synthesis of Hsp90 inhibitor dimers as potential antitumor agents
a,
Hironori Sekiguchi a, Kazuhiro Muranaka a, Akiko Osada b, Satoshi Ichikawa a, , Akira Matsuda
*
*
a Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
b Discovery & Development Laboratory I, Hanno Research Center, Taiho Pharmaceutical Co., Ltd, 1-27 Misugidai, Hanno, Saitama 357, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The PU-H58-dimers 13a–15b were efficiently synthesized and their biological properties were evaluated.
The copper-catalyzed alkyne azide coupling was effective in simultaneously linking three components via
a triazole formation to afford the target dimers. These synthesized dimers exhibited binding affinity to
the N-terminal domain of Hsp90, cytotoxicity, and client degradation activity although these activities
were comparative or weak comparable with that of the parent compound.
Received 7 May 2010
Revised 25 May 2010
Accepted 26 May 2010
Available online 9 June 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
Hsp90
Antitumor agent
Copper-catalyzed alkyne azide coupling
1. Introduction
the first fully synthetic Hsp90 inhibitors with the ability to bind
at the ATP-binding site of the N-terminal domain. This class of syn-
Cancer cells acquire sets of functional capacities known as the
six hallmarks of cancer1 and consequently it is difficult to treat
cancer cells by malfunction of a single cancer-causing target in cur-
rent cancer chemotherapy. The heat shock protein 90 (Hsp90) fam-
ily is a group of molecular chaperones2–5 that plays a key role in
the folding of polypeptide chains called client proteins.6 Many of
the Hsp90 client proteins, for example, Bcr-abl, Akt, C-Raf, CDK4,
Her2, and the estrogen receptor (ER), are oncogenic and are also
considered hallmarks of the disease.7,8 Increased expression of
Hsp90 helps the cancer cell manage not only the stress caused
by mutation and overexpression of oncogenes but also the stress
due to hypoxia, nutrient deprivation and acidosis.9,10 Inhibition
of Hsp90 leads to degradation of these various client proteins by
the proteasome. The simultaneous combinatorial depletion of
many cancer-causing client proteins and the modulation of all of
the hallmarks of cancer are the major advantages of Hsp90 inhib-
itors. Hsp90 exists predominantly as a dimer in the cell, with each
subunit being made up of three functional domains: an N-terminal
ATP-binding domain; a middle domain; and a C-terminal dimeriza-
tion domain. A natural product, geldanamycin,11 is known to be a
strong ATP-competitive inhibitor of the Hsp90 N-terminal do-
main,12–15 and the 17-allylamino derivative (17-AAG) and the
17-dimethylaminoethyl derivative (17-DMAG) are currently in
phase II clinical studies.16 The 8,9-disubstituted purine class of
molecules, for example, PU3 (Fig. 1) (1)17 and PU-H58 (2a),18 are
thetic inhibitors is also active in cells causing degradation of Erb B2
kinase, ER, and C-Raf kinase.
We have recently synthesized PU3-dimers 3 according to a
structure-based drug design using the X-ray crystal structure of
the full length Hsp90 dimer and found that by increasing the
* Corresponding authors. Tel.: +81 11 706 3763; fax: +81 11 706 4980 (S.I.); tel.:
+81 11 706 3228; fax: +81 11 706 4980 (A.M.).
E-mail addresses: ichikawa@pharm.hokudai.ac.jp (S. Ichikawa), matuda@-
pharm.hokudai.ac.jp (A. Matsuda).
Figure 1. Structure of PU-3, PU-H58, and its dimer.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.05.075

H. Sekiguchi et al. / Bioorg. Med. Chem. 18 (2010) 5732–5737
5733
length of the bridging linker, the PU3 dimers became more cyto-
toxic against human breast cancer cell lines.19 Compound 3, with
a C-20 linker, exhibited a 20ꢀ30-fold increase in cytotoxicity com-
pared with that of PU3. However the preparation of these analogs
was not feasible in terms of the yields and product isolation to pur-
sue further optimization. Herein we describe the efficient synthesis
of Hsp90 inhibitor dimers using a copper-catalyzed alkyne azide
coupling (CuAAC). Biological evaluations of the synthesized ana-
logs are also described.
give the corresponding thiol, which was difficult to isolate because
of its susceptibility to air oxidation. Therefore the thiol was
sequentially oxidized by I2 in MeOH to give the disulfide 5 in
72% over two steps. After 5 was reduced with Bu3P and H2O, the
resulting thiol was reacted with 8-bromo-20-deoxyadenosine (6)
in the presence of K2CO3 in DMF to give 7 in 97% yield. Bromination
of the methylenedioxyphenyl ring by NBS gave 8 selectively in 50%
yield. Acid-catalyzed hydrolysis of the glycosidic bond of 8 affor-
ded 9 in 69% yield. Mitsunobu reaction of 9 with 4-pentyn-1-ol
or 5-hexyn-1-ol in CH2Cl2 gave the desired N9-substituted adenine
derivatives 2a or 2b as the major products along with the corre-
sponding N3-isomers. This was confirmed by NOE experiments,
where the correlation of the H-60 proton (1.7%) in 2a was observed
upon irradiation at the methylene protons connected to the N-9
position and no correlation was observed between amino protons
at the N-6 position and the methylene protons. With the alkyne
units in hand, we next examined the CuAAC linking two molecules
of 2 with the bis(azido)ethylene glycol linkers 10–12. The linkers
10–12 were prepared from the corresponding diols 17–19 by con-
ventional methods as shown in Scheme 2. Two types of conditions
for CuAAC were used to prepare the dimmers, namely Cu powder,
CuSO4, and tris(benzyltriazolylmethyl)amine22 (TBTA) ligand in
DMF–MeCN–tBuOH–H2O (conditions A); and CuSO4, sodium ascor-
bate, and TBTA in DMF–H2O (conditions B). Under both sets of con-
ditions, the reaction proceeded effectively at both termini of the
linker, and the desired dimers 13a–15b were obtained in good
yields (47–95%).
2. Results and discussion
2.1. Chemistry
We planned to systematically prepare the dimers of PU-H58
(2a), which was reported to be a better ligand of the Hsp90 N-ter-
minal domain than PU-3, and its homolog (2b) by varying the
length of the linker enough to cover the distance (ca. 25 Å) of the
two ATP-binding sites of the N-terminal domains.20 Considering
water solubility, we chose an ethylene glycol repeat as the linker
connecting the two N-terminal ligands. The CuAAC21 was chosen
for the reaction linking the linker and the two N-terminal ligands,
since a variety of functional groups are well tolerated in the CuAAC,
which proceeds even in aqueous media in good yields, thereby
making it suitable for linking polar molecules and/or macromole-
cules. This strategy allowed us to prepare the target molecules
without using any protecting groups during the synthesis. The syn-
thesis of the dimers 13a–15b is outlined in Scheme 1. 3,4-Methyl-
enedioxyphenyl magnesium bromide (4) was treated with sulfur to
2.2. Biological activity
The biological activity of the synthesized compounds was then
evaluated, and the results are summarized in Table 1. The binding
affinity of the compounds to the N-terminal domain of human
Hsp90a was first evaluated by competitive binding to a biotinyla-
ted geldanamycin, which was quantified by Alpha screen. The IC50
values of 16a,b, which are reference monomeric analogues with a
triazole substituent, were 0.49
lM and 0.51
lM, respectively, and
they exhibited a similar potency to 2a,b (0.3
lM for 2a, 1.4 lM
for 2b). This indicated that introducing the triazole moiety did
not influence the binding of the PU-H58 moiety to the N-terminal
domain. All the dimers also retained affinity to the N-terminal do-
main, which was regarded as a monomeric state of Hsp90a. It was
observed that homologation reduced the binding affinity in all
cases. The cytotoxic activity of these analogues was next evaluated
against the SKBr3 human breast cancer cells. All the synthesized
dimers showed moderate cytotoxicity with IC50s ranging from
1.4ꢀ5.6
cases (15a) compared to the parent 2a, which exhibited the previ-
ously reported activity (0.33 M for SKBr3). To determine whether
lM, and their activity was comparable or reduced in some
l
the observed cytotoxicity was related to Hsp90 inhibition, degra-
dation of the Hsp90 client protein Her2 upon treatment with the
compounds was evaluated. Thus, compounds were incubated with
SKBr-3 breast cancer cells for 24 h, and the protein lysates were
analyzed by Western blot with the anti-Her2 antibody. The
amount of Her2 was normalized by the expression level of GAPDH,
which is not dependent on the Hsp90 protein folding machinery.
The dimers 13a–15b did exhibit the Her2 degradation activity,
which was well-correlated to the cytotoxicity. These results clearly
indicated that the dimers exhibited cytotoxicity against SKBR-3
breast cancer cells through Hsp90 inhibition. Contrary to our pre-
vious study,19 the length of the linker did not correlate to the cyto-
toxicity or to the client degradation activity, in spite of the length
of the linkers (approximate distance between the two triazole rings
for 13, 14, or 15 is 22, 25, or 29 Å in linear length, respectively)
being enough to fit the two ATP-binding sites of the N-terminal do-
mains of the closed form Hsp90 (ca. 25 Å). This may be attributed
Scheme 1.

5734
H. Sekiguchi et al. / Bioorg. Med. Chem. 18 (2010) 5732–5737
Scheme 2.
based on 1H–1H COSY, HMBC, and HMQC NMR spectra. FAB-MS
Table 1
Biological evaluation of the dimers
was obtained on a JEOL JMS-HX110. Analytical thin layer chroma-
tography (TLC) was performed on Merck silica gel 60F254 plates.
Normal-phase column chromatography was performed on Merck
silica gel 5715. Flash column chromatography was performed on
Merck silica gel 60.
Compound
IC50 (lM)
Hsp90
a
Her2
Cytotoxicity
(SKBR-3)c
bindinga
degradationb
2a
2b
0.30
1.4
0.65
5.1
0.79
3.4
1.1
3.1
0.49
0.51
0.46
2.6
6.3
5.2
7.1
4.7
9.2
4.2
3.9
2.9
0.33
1.4
3.8
3.0
5.6
5.0
4.8
4.8
1.8
1.4
13a
13b
14a
14b
15a
15b
16a
16b
4.1.1. Di-(3,4-methylenedioxyphenyl) disulfide (5)
A solution of 3,4-methylenedioxyphenylmagnesium bromide 4
(1.0 M solution in toluene/THF, 1:1, 95 mL, 95 mmol) in THF
(480 mL) was treated with sulfur (3.0 g, 95 mmol) at 0 °C for 1 h.
The reaction mixture was allowed to room temperature and stirred
for additional 4 h. The reaction was quenched with 5 N HCl, and the
mixture was extracted three times with AcOEt. The collected organic
layers were washed with H2O and brine, dried (Na2SO4), filtered and
concentrated in vacuo. The residue was purified by silica gel column
chromatography (/ 7 ꢁ 14 cm, 5% AcOEt/hexane) to give the
mixture of 5 and 3,4-methylenedioxybenzenethiol. The mixture of
5 and 3,4-methylenedioxybenzenethiol in MeOH (200 mL) was trea-
ted with I2 (5.1 g, 20 mmol) at room temperature for 2 h. The reac-
tion was quenched with saturated aqueous Na2S2O3, and the
mixture was partitioned between AcOEt and H2O. The organic layer
was washed with brine, dried (Na2SO4), filtered and concentrated in
vacuo. The residue was purified by silica gel column chromatogra-
phy (/ 7 ꢁ 14 cm, 5% AcOEt/hexane) to give 5 (11 g, 72%) as a yellow
oil: 1H NMR (500 MHz, CDCl3) d 7.00 (d, 2H, H-2, J = 1.8 Hz), 6.94 (dd,
2H, H-6, J = 1.8, 7.7 Hz), 6.72 (d, 2H, H-5, J = 7.7 Hz), 5.97 (s, 4H,
OCH2O); 13C NMR (100 MHz, CDCl3) d 148.3, 148.2, 129.9, 124.8,
111.1, 108.7, 101.6; EIMS-LR m/z = 306 (M)+; EIMS-HR calcd for
C14H10O4S2 306.0021, found 306.0016.
a
Binding of biotinylated geldanamycin to the N-terminal domain of human
Hsp90a was evaluated in the presence of drugs by Alpha screen.
b
SKBR-3 cells were plated 2 ꢁ 105 cells/well and seeded for 24 h. After 24 h
incubation in the presence of drugs or DMSO, lysates were prepared using M-PERÒ
reagent. Clarified protein lysates were analyzed by Western blotting with anti-Her2
and anti-GAPDH.
c
Cells were cultured in the presence of test compounds for 72 h and then fixed
with glutaraldehyde. Fixed cells were stained in 0.05% crystal violet solution. The
dye was extracted and the absorbance was read at 540 nm on a microplate reader.
to the reduced membrane-permeability of the dimers because of
the physicochemical properties of the ethylene glycol linker.
3. Conclusion
The PU-H58-dimers 13a–15b were efficiently synthesized and
their biological properties were evaluated. The CuAAC was effec-
tive in simultaneously linking three components via triazole
formation to afford the target dimers having relatively high molec-
ular weight. The synthesized dimers exhibited binding affinity to
the N-terminal domain of Hsp90, cytotoxicity, and client degrada-
tion activity although these activities were comparable or weak
compared with that of the parent compound. However, the length
of the linker did not correlate to cytotoxicity and client degradation
activity. This may be attributed to the reduced membrane-perme-
ability of the dimers because of the physicochemical properties of
the ethylene glycol linker. Optimization of the linker will be neces-
sary to improve the cytotoxicity to develop potent Hsp90
inhibitors.
4.1.2. 9-(2-Deoxy-b-D-ribo-pentofuranosyl)-8-(3,4-methylenedi-
oxyphenylthio)adenine (7)
A solution of 5 (7.7 g, 25 mmol) in DMF (250 mL) was treated
with tributylphosphine (6.2 mL, 25 mmol) and H2O (0.68 mL,
38 mmol) at room temperature for 1 h. Potassium carbonate (10 g,
75 mmol) and 623 (12 g, 35 mmol) were added to the mixture, which
was stirred for additional 2 h. The mixture was concentrated in va-
cuo, and the residue was triturated from MeOH and CHCl3 to give
7 (14 g, 97%) as a pale yellow solid: 1H NMR (500 MHz, DMSO-d6)
d 8.08 (s, 1H, H-2), 7.47 (br s, 2H, NH2), 7.02 (s, 1H, H-200), 6.94 (s,
2H, H-500 and H-600), 6.48 (dd, 1H, H-10, J = 6.3, 8.3 Hz), 6.04 (s, 2H,
OCH2O), 5.52 (dd, 1H, OH-50, J = 4.0, 8.3 Hz), 5.29 (d, 1H, OH-30,
J = 4.6 Hz), 4.42 (m, 1H, H-30), 3.88 (m, 1H, H-40), 3.66 (dt, 1H, H-50,
J = 4.0, 12.3 Hz), 3.49 (ddd, 1H, H-50, J = 4.0, 8.3, 12.3 Hz), 3.04
(ddd, 1H, H-20b, J = 5.8, 8.3, 13.0 Hz), 1.98 (ddd, 1H, H-20a, J = 2.9,
6.3, 13.0 Hz); 13C NMR (125 MHz, DMSO-d6) d 155.5, 152.4, 150.0,
148.2, 148.0, 145.0, 125.7, 122.7, 119.9, 111.8, 109.3, 101.8, 88.5,
85.7, 71.4, 62.3, 37.8; ESIMS-LR m/z = 426 (M+Na)+; ESIMS-HR calcd
for C17H17N5O5SNa 426.0848, found 426.0864.
4. Experimental
4.1. General experimental methods
NMR spectra were obtained on a JEOL ECX400 or JEOL ECA500,
and were reported in parts per million (d) relative to tetramethyl-
silane (0.00 ppm) as internal standard otherwise noted. Coupling
constant (J) was reported in hertz (Hz). Abbreviations of multiplic-
ity were as follows; s: singlet, d: doublet, t: triplet, q: quartet,
m: multiplet, br: broad. Data were presented as follows; chemical
shift (multiplicity, integration, coupling constant). Assignment was
4.1.3. 8-(6-Bromo-3,4-methylenedioxyphenylthio)-9-(2-deoxy-
b-D
-ribo-pentofuranosyl)adenine (8)
A suspension of 7 (10 g, 25 mmol) in 1 M acetate buffer (pH
4.0)/CH3CN (2:1, 750 mL) was treated with NBS (8.9 g, 50 mmol)

H. Sekiguchi et al. / Bioorg. Med. Chem. 18 (2010) 5732–5737
5735
at room temperature for 3 h. The mixture was extracted three
times with CHCl3. The collected organic layers were washed with
saturated aqueous NaHCO3, H2O and brine, dried (Na2SO4), filtered
and concentrated in vacuo. The residue was triturated from hexane
and AcOEt to give 8 (6.1 g, 50%) as a brown solid: 1H NMR (400 MHz,
CDCl3) d 8.11 (s, 1H, H-2), 7.50 (br s, 2H, NH2), 7.37 (s, 1H, H-500), 6.83
(s, 1H, H-200), 6.42 (dd, 1H, H-10, J = 6.3, 8.2 Hz), 6.09 (s, 2H, OCH2O),
5.47 (m, 1H, OH-50), 5.29 (d, 1H, OH-30, J = 3.6 Hz), 4.43 (m, 1H, H-30),
3.88 (m, 1H, H-40), 3.66 (m, 1H, H-50), 3.50 (m, 1H, H-50), 3.11 (ddd,
1H, H-20b, J = 5.9, 8.2, 12.7 Hz), 2.05 (ddd, 1H, H-20a, J = 1.9, 6.3,
12.7 Hz); 13C NMR (100 MHz, DMSO-d6) d 155.5, 152.5, 150.0,
148.6, 147.9, 143.6, 124.3, 120.1, 115.3, 113.1, 111.6, 102.7, 88.4,
85.6, 71.3, 62.2, 37.7; ESIMS-LR m/z = 504 (M+Na)+; ESIMS-HR calcd
for C17H16BrN5O5SNa 503.9953, found 503.9950.
J = 5.0 Hz), 3.64–3.56 (m, 16H, CH2), 2.43 (s, 6H, Me); 13C NMR
(100 MHz, CDCl3) d 144.4, 133.1, 129.9, 128.1, 70.8, 70.7, 70.6,
69.4, 68.8, 21.7; FABMS-LR m/z = 591 (M+H)+; FABMS-HR calcd
for C26H39O11S2 591.1934, found 591.1957.
4.1.8. 3,6,9,12,15,18-Hexaoxaeicosane-1,20-diol ditosylate (21)
Compound 21 (320 mg, quant.) was obtained from heptaethyl-
eneglycol 18 (160 mg, 0.50 mmol) as described for the synthesis of
20: 1H NMR (400 MHz, CDCl3) d 7.77 (d, 4H, Ts, J = 8.2 Hz), 7.32 (d,
4H, Ts, J = 8.2 Hz), 4.13 (t, 4H, TsOCH2, J = 4.9 Hz), 3.67–3.56 (m,
24H, CH2), 2.42 (s, 6H, Me); 13C NMR (100 MHz, CDCl3) d 144.9,
133.1, 129.9, 128.1, 77.5, 77.2, 76.8, 70.9, 70.8, 70.7, 70.6, 69.4,
68.7, 21.7; FABMS-LR m/z = 635 (M+H)+; FABMS-HR calcd for
C28H43O12S2 635.2196, found 635.2186.
4.1.4. 8-(6-Bromo-3,4-methylenedioxyphenylthio)adenine (9)
A solution of 8 (24 mg, 0.050 mmol) in 80% aqueous TFA (2 mL)
was stirred at room temperature for 1 h. The reaction was
quenched with saturated aqueous NaHCO3, and the mixture was
extracted five times with CHCl3. The collected organic layers were
washed with brine, dried (Na2SO4), filtered and concentrated in va-
cuo to give 9 (13 mg, 69%) as a yellow solid, properties of which
were identical in all respects to those for previously reported:18b
1H NMR (400 MHz, DMSO-d6) d 8.06 (s, 1H, H-2), 7.36 (s, 1H, H-
50), 7.22 (br s, 2H, NH2), 6.99 (s, 1H, H-20), 6.10 (s, 2H, OCH2O).
4.1.9. 3,6,9,12,15,18,21-Heptaoxatrieicosane-1,23-diol ditosylate
(22)
Compound 22 (320 mg, 71%) was obtained from octaethylene-
glycol 19 (190 mg, 0.50 mmol) as described for the synthesis of
20: 1H NMR (400 MHz, CDCl3) d 7.78 (d, 4H, Ts, J = 8.5 Hz), 7.33
(d, 4H, Ts, J = 8.0 Hz), 4.14 (t, 4H, TsOCH2CH2, J = 4.6 Hz), 3.67 (t,
4H, TsOCH2CH2, J = 4.6 Hz), 3.62–3.57 (m, 24H, CH2), 2.43 (s, 6H,
Me); 13C NMR (100 MHz, CDCl3) d 144.9, 133.1, 129.9, 128.1,
70.9, 70.7, 70.6, 69.4, 68.8, 21.8; FABMS-LR m/z = 679 (M+H)+; FAB-
MS-HR calcd for C30H47O13S2 679.2474, found 679.2458.
4.1.5. 8-(6-Bromo-3,4-methylenedioxyphenylthio)-9-(pent-4-
ynyl)adenine (2a: PU-H58)
4.1.10. 1,17-Diazide-3,6,9,12,15-pentaoxaheptadecane (10)
A mixture of 20 (120 mg, 0.20 mmol) and Bu4NI (5.5 mg,
0.015 mmol) in DMF (5 mL) was treated with NaN3 (78 mg,
1.2 mmol) at 80 °C for 3 h. After cooling to room temperature, the
solvent was removed in vacuo. The residue was diluted with Et2O
(2 mL), and the insoluble was filtered off. The filtrate was concen-
trated in vacuo, and the residue was purified by silica gel column
chromatography (/ 1 ꢁ 6 cm, 80% AcOEt/hexane) to give 16b
(40 mg, 61%) as a yellow oil: 1H NMR (500 MHz, CDCl3) d 3.67–
3.64 (m, 20H, CH2), 3.37 (t, 4H, N3CH2, J = 5.1 Hz); 13C NMR
(125 MHz, CDCl3) d 70.8, 70.7, 70.1, 50.8; FABMS-LR m/z = 355
(M+Na)+; FABMS-HR calcd for C12H24N6O5Na 355.1706, found
355.1712.
A suspension of 9 (500 mg, 1.4 mmol), 4-pentyn-1-ol (190 lL,
2.1 mmol) and PPh3 (790 mg, 3.0 mmol) in CH2Cl2 (14 mL) was
treated with DIAD (1.4 mL, 6.9 mmol) at room temperature for
1 h. The solvent was removed in vacuo, and the residue was puri-
fied by flash silica gel column chromatography (/ 1 ꢁ 22 cm, 1%
MeOH/CHCl3) to give 2a (400 mg, 68%) as a pale yellow foam: 1H
NMR (500 MHz, CDCl3) d 8.14 (s, 1H, H-2), 7.36 (br s, 2H, NH2),
7.35 (s, 1H, H-50), 6.83 (s, 1H, H-20), 6.08 (s, 2H, OCH2O), 4.21 (t,
2H, CH2CH2CH2C„CH, J = 7.4 Hz), 2.78 (t, 1H, CH2CH2CH2C„CH,
J = 2.6 Hz), 2.20 (dt, 2H, CH2CH2CH2C„CH, J = 2.6, 6.9 Hz), 1.87
(tt, 2H, CH2CH2CH2C„CH, J = 6.9, 7.4 Hz).
4.1.6. 8-(6-Bromo-3,4-methylenedioxyphenylthio)-9-(hex-5-
ynyl)adenine (2b)
Compound 2b (410 mg, 67%) was obtained from 9 (500 mg,
1.4 mmol) and 5-hexyn-1-ol (230 lL, 2.1 mmol) as described for
4.1.11. 1,20-Diazide-3,6,9,12,15,18-hexaoxaeicosane (11)
Compound 11 (64 mg, 57%) was obtained from 21 (190 mg,
0.30 mmol) as described for the synthesis of 10: 1H NMR
(500 MHz, CDCl3) d 3.67–3.63 (m, 24H, CH2), 3.37 (t, 4H, N3CH2,
J = 5.2 Hz); 13C NMR (125 MHz, CDCl3) d 70.8, 70.7, 70.1, 50.8; FAB-
MS-LR m/z = 399 (M+Na)+; FABMS-HR calcd for C14H28N6O6Na
399.1968, found 399.1962.
the synthesis of 2a: 1H NMR (500 MHz, CDCl3) d 8.32 (s, 1H, H-2),
7.07 (s, 1H, H-50), 6.83 (s, 1H, H-20), 5.97 (s, 2H, OCH2O), 5.88 (br s,
2H, NH2), 4.23 (t, 2H, CH2CH2CH2CH2C„CH, J = 7.4 Hz), 2.22 (dt,
2H, CH2CH2CH2CH2C„CH, J = 2.6, 7.4 Hz), 1.93 (t, 1H, CH2CH2-
CH2CH2C„CH, J = 2.6 Hz), 1.90 (tt, 2H, CH2CH2CH2CH2C„CH,
J = 6.9, 7.4 Hz), 1.55 (tt, 2H, CH2CH2CH2CH2C„CH, J = 6.9, 7.4 Hz);
13C NMR (125 MHz, CDCl3) d 154.7, 153.2, 151.7, 149.2, 148.2,
145.7, 123.9, 120.3, 117.2, 113.5, 112.7, 102.5, 83.7, 69.1, 43.5,
29.0, 25.6, 18.2; ESIMS-LR m/z = 446 (M+H)+; ESIMS-HR calcd for
4.1.12. 1,23-Diazide-3,6,9,12,15,18,21-heptaoxatrieicosane (12)
Compound 12 (100 mg, 83%) was obtained from 22 (180 mg,
0.27 mmol) as described for the synthesis of 10: 1H NMR
(400 MHz, CDCl3) d 3.65–3.61 (m, 28H, CH2), 3.35 (t, 4H, N3CH2,
J = 5.4 Hz); 13C NMR (100 MHz, CDCl3) d 70.7, 70.6, 70.1, 50.7; FAB-
MS-LR m/z = 443 (M+Na)+; FABMS-HR calcd for C16H32N6O7Na
443.2230, found 443.2216.
C18H17BrN5O2S 446.0286, found 446.0280.
4.1.7. 3,6,9,12,15-Pentaoxaheptadecane-1,17-diol ditosylate
(20)
4.1.13. Dimer (13a)
A mixture of hexaethyleneglycol 17 (140 mg, 0.50 mmol) and
KOH (220 mg, 4.0 mmol) in CH2Cl2 (1.0 mL) was treated with p-
toluenesulfonyl chloride (190 mg, 1.0 mmol) at 0 °C for 1 h. The
reaction was quenched with H2O, and the mixture was extracted
three times with CH2Cl2. The collected organic layers were washed
with brine, dried (Na2SO4), filtered and concentrated in vacuo to
give 20 (300 mg, quant.) as a colorless oil: 1H NMR (400 MHz,
CDCl3) d 7.78 (d, 4H, Ts, J = 8.2 Hz), 7.34 (d, 4H, Ts, J = 8.1 Hz),
4.15 (t, 4H, TsOCH2CH2, J = 5.0 Hz), 3.67 (t, 4H, TsOCH2CH2,
A solution of 2a (34 mg, 0.079 mmol), 10 (12 mg, 0.036 mmol),
CuSO4 (8.6 mg, 0.054 mmol) and tris(benzyltriazolylmethyl)amine
(TBTA, 19 mg, 0.036 mmol) in DMF (1 mL) was treated with copper
powder at room temperature, and the mixture was stirred for over-
night. The solvent was removed in vacuo. The residue in CHCl3
(1 mL) was treated with diamine silica gel for removal of copper
at room temperature for 5 min. Copper powder and diamine silica
gel were filtered off, and the filtrate was concentrated in vacuo. The
residue was purified by silica gel column chromatography

5736
H. Sekiguchi et al. / Bioorg. Med. Chem. 18 (2010) 5732–5737
(/ 1.1 ꢁ (6 + 1) cm, 10% MeOH/CHCl3) to give 13a (20 mg, 47%) as a
colorless oil: 1H NMR (500 MHz, CDCl3) d 8.29 (s, 2H, H-2), 7.49 (s,
2H, triazole), 7.04 (s, 2H, H-50), 6.82 (s, 2H, H-20), 6.08 (br s, 4H,
NH2), 5.96 (s, 4H, OCH2O), 4.47 (t, 4H, NCH2CH2O, J = 5.1 Hz), 4.29
(t, 4H, NCH2CH2CH2, J = 7.4 Hz), 3.81 (t, 4H, NCH2CH2O,
J = 5.1 Hz), 3.57–3.56 (m 16H, OCH2CH2O), 2.74 (t, 4H,
NCH2CH2CH2, J = 8.0 Hz), 2.19 (tt, 4H, NCH2CH2CH2, J = 7.4,
8.0 Hz); 13C NMR (125 MHz, CDCl3) d 154.7, 153.1, 151.7, 149.3,
148.3, 146.4, 146.0, 123.6, 122.2, 120.2, 117.4, 113.5, 112.8,
102.6, 70.6, 70.5, 69.7, 50.2, 43.4, 29.3, 23.0; ESIMS-LR m/z = 1217
(M+Na)+; ESIMS-HR calcd for C46H52Br2N16O9S2Na 1217.1809,
found 1217.1795.
(125 MHz, CDCl3) d 154.8, 153.1, 151.6, 149.3, 148.2, 146.4, 145.7,
123.4, 122.3, 120.1, 117.5, 113.5, 112.9, 102.6, 70.6, 70.5, 69.6,
50.2, 43.4, 29.3, 23.0; ESIMS-LR m/z = 1305 (M+Na)+; ESIMS-HR
calcd for C50H60Br2N16O11S2Na 1305.2333, found 1305.2307.
4.1.18. Dimer (15b)
Compound 15b (31 mg, 79%) was obtained from 2b (29 mg,
0.066 mmol) and 12 (13 mg, 0.030 mmol) as described for the syn-
thesis of 13a: 1H NMR (500 MHz, CDCl3) d 8.29 (s, 2H, H-2), 7.43 (s,
2H, triazole), 7.05 (s, 2H, H-50), 6.82 (s, 2H, H-20), 6.07 (br s, 4H,
NH2), 5.97 (s, 4H, OCH2O), 4.47 (t, 4H, NCH2CH2O, J = 5.2 Hz) 4.23
(t, 4H, NCH2CH2CH2CH2, J = 6.9 Hz), 3.82 (t, 4H, NCH2CH2O,
J = 5.2 Hz), 3.60–3.57 (m, 24H, OCH2CH2O), 2.72 (t, 4H, NCH2CH2-
CH2CH2, J = 8.0 Hz), 1.85 (tt, 4H, NCH2CH2CH2CH2, J = 6.9, 7.4 Hz),
1.68 (tt, 4H, NCH2CH2CH2CH2, J = 7.4, 8.0 Hz); 13C NMR (125 MHz,
CDCl3) d 154.7, 153.1, 151.6, 149.2, 148.2, 147.3, 145.8, 123.7,
122.1, 120.2, 117.4, 113.5, 112.8, 102.6, 70.6, 70.5, 69.7, 50.2, 43.6,
29.4, 26.6, 25.3; ESIMS-LR m/z = 1333 (M+Na)+; ESIMS-HR calcd for
4.1.14. Dimer (13b)
Compound 13b (35 mg, 95%) was obtained from 2b (29 mg,
0.066 mmol) and 10 (10 mg, 0.030 mmol) as described for the syn-
thesis of 13a: 1H NMR (500 MHz, CDCl3) d 8.28 (s, 2H, H-2), 7.41 (s,
2H, triazole), 7.03 (s, 2H, H-50), 6.81 (s, 2H, H-20), 6.15 (br s, 4H,
NH2), 5.95 (s, 4H, OCH2O), 4.46 (t, 4H, NCH2CH2O, J = 5.0 Hz) 4.22
(t, 4H, NCH2CH2CH2CH2, J = 7.2 Hz), 3.81 (t, 4H, NCH2CH2O, J =
5.0 Hz), 3.60–3.55 (m, 16H, OCH2CH2O), 2.71 (t, 4H, NCH2CH2-
CH2CH2, J = 7.2 Hz), 1.84 (tt, 4H, NCH2CH2CH2CH2, J = 7.2, 8.1 Hz),
1.67 (tt, 4H, NCH2CH2CH2CH2, J = 7.2, 8.1 Hz); 13C NMR (125 MHz,
CDCl3) d 154.8, 153.1, 151.6, 149.2, 148.2, 147.3, 145.6, 123.7,
122.1, 120.2, 117.2, 113.4, 112.7, 102.5, 70.6, 70.5, 69.7, 50.2, 43.6,
29.4, 26.6, 25.3; ESIMS-LR m/z = 1245 (M+Na)+; ESIMS-HR calcd for
C52H64Br2N16O11S2Na 1333.2646, found 1333.2611.
4.1.19. 8-(6-Bromo-3,4-methylenedioxyphenylthio)-9-[{1-(3-
hydroxypropyl)-1H-1,2,3-triazol-4-yl}propyl]adenine (16a)
A solution of 2a (43 mg, 0.10 mmol), 3-azidopropanol (10 mg,
0.10 mmol), CuSO4 (24 mg, 0.15 mmol) and TBTA (53 mg,
0.10 mmol) in DMF (1 mL) was treated with copper powder at room
temperature, and the mixture was stirred for overnight. The solvent
was removed in vacuo. The residue in CHCl3 (1 mL) was treated with
diamine silica gel for removal of copper at room temperature for
5 min. Copper powder and diamine silica gel were filtered off, and
the filtrate was concentrated in vacuo. The residue was purified by
silica gel column chromatography (/ 1 ꢁ 7 cm, 10% MeOH/CHCl3)
to give 16a (35 mg, 65%) as a white solid: 1H NMR (500 MHz, CDCl3)
d 8.30 (s, 1H, H-2), 7.36, (s, 1H, triazole), 7.06 (s, 1H, H-50), 6.84 (s, 1H,
H-20), 5.98 (s, 2H, OCH2O), 5.86 (br s, 2H, NH2), 4.46 (t, 2H,
NCH2CH2CH2O, J = 6.8 Hz), 4.27 (t, 2H, NCH2CH2CH2C, J = 6.8 Hz),
3.59 (t, 2H, NCH2CH2CH2O, J = 6.3 Hz), 2.78 (t, 2H, NCH2CH2CH2C,
J = 7.4 Hz), 2.20 (tt, 2H, NCH2CH2CH2C, J = 6.8, 7.4 Hz), 2.08 (tt, 2H,
NCH2CH2CH2O, J = 6.3, 6.8 Hz); 13C NMR (125 MHz, CDCl3) d 154.5,
153.1, 151.7, 149.3, 148.3, 146.5, 146.1, 123.4, 121.6, 120.2, 117.6,
113.6, 113.0, 102.6, 58.7, 46.9, 43.3, 32.6, 29.2, 23.0; ESIMS-LR m/
z = 555 (M+Na)+; ESIMS-HR calcd for C20H21BrN8O3SNa 555.0538,
found 555.052.
C48H56Br2N16O9S2Na 1245.2122, found 1245.2143.
4.1.15. Dimer (14a)
Compound 14a (33 mg, 90%) was obtained from 2a (29 mg,
0.066 mmol) and 11 (11 mg, 0.030 mmol) as described for the syn-
thesis of 13a: 1H NMR (500 MHz, CDCl3) d 8.28 (s, 2H, H-2), 7.51 (s,
2H, triazole), 7.03 (s, 2H, H-50), 6.81 (s, 2H, H-20), 6.25 (br s, 4H,
NH2), 5.94 (s, 4H, OCH2O), 4.47 (t, 4H, NCH2CH2O, J = 5.1 Hz), 4.28
(t, 4H, NCH2CH2CH2, J = 6.8 Hz), 3.81 (t, 4H, NCH2CH2O, J = 5.1 Hz),
3.56–3.55 (m 20H, OCH2CH2O), 2.74 (t, 4H, NCH2CH2CH2,
J = 7.5 Hz), 2.18 (tt, 4H, NCH2CH2CH2, J = 6.8, 7.5 Hz); 13C NMR
(125 MHz, CDCl3) d 154.8, 153.1, 151.7, 149.2, 148.2, 146.4, 145.7,
123.5, 122.2, 120.1, 117.4, 113.5, 112.8, 102.6, 70.6, 70.5, 69.6,
50.2, 43.4, 29.3, 23.0; ESIMS-LR m/z = 1261 (M+Na)+; ESIMS-HR
calcd for C48H56Br2N16O10S2Na 1261.2071, found 1261.2044.
4.1.16. Dimer (14b)
Compound 14b (35 mg, 92%) was obtained from 2b (29 mg,
0.066 mmol) and 11 (11 mg, 0.030 mmol) as described for the syn-
thesis of 13a: 1H NMR (500 MHz, CDCl3) d 8.28 (s, 2H, H-2), 7.42 (s,
2H, triazole), 7.03 (s, 2H, H-50), 6.82 (s, 2H, H-20), 6.17 (br s, 4H,
NH2), 5.95 (s, 4H, OCH2O), 4.47 (t, 4H, NCH2CH2O, J = 5.2 Hz) 4.22
(t, 4H, NCH2CH2CH2CH2, J = 7.5 Hz), 3.81 (t, 4H, NCH2CH2O,
J = 5.2 Hz), 3.60–3.56 (m, 20H, OCH2CH2O), 2.71 (t, 4H, NCH2CH2-
CH2CH2, J = 7.5 Hz), 1.84 (tt, 4H, NCH2CH2CH2CH2, J = 6.9, 7.5 Hz),
1.67 (tt, 4H, NCH2CH2CH2CH2, J = 6.9, 7.5 Hz); 13C NMR (125 MHz,
CDCl3) d 154.8, 153.1, 151.6, 149.2, 148.2, 147.3, 145.6, 123.7,
122.1, 120.2, 117.3, 113.4, 112.8, 102.6, 70.6, 70.5, 69.7, 50.2, 43.6,
29.4, 26.6, 25.3; ESIMS-LR m/z = 1289 (M+Na)+; ESIMS-HR calcd for
4.1.20. 8-(6-Bromo-3,4-methylenedioxyphenylthio)-9-[{1-(3-
hydroxypropyl)-1H-1,2,3-triazol-4-yl}butyl]adenine (16b)
A solution of 2b (45 mg, 0.10 mmol), 3-azidopropanol (10 mg,
0.10 mmol), CuSO4 (1.9 mg, 0.012 mol) and TBTA (10 mg,
0.019 mmol) in DMF/tBuOH/H2O/CH3CN (4:2:1:1, 2 mL) was trea-
ted with sodium L-ascorbate (8.0 mg, 0.040 mmol) at room tem-
perature, and the mixture was stirred for overnight. The solvent
was removed in vacuo. The residue in CHCl3 (1 mL) was treated
with diamine silica gel for removal of copper at room temperature
for 5 min. Diamine silica gel were filtered off, and the filtrate was
concentrated in vacuo. The residue was purified by silica gel col-
umn chromatography (/ 1.1 ꢁ (6 + 2) cm, 12% MeOH/CHCl3) to
give 16b (36 mg, 66%) as a white solid: 1H NMR (500 MHz, CDCl3)
d 8.31 (s, 1H, H-2), 7.28 (s, 1H, triazole), 7.07 (s, 1H, H-50), 6.86 (s,
1H, H-20), 6.00 (s, 2H, OCH2O), 5.80 (br s, 2H, NH2), 4.46 (t, 2H,
NCH2CH2CH2O, J = 6.9 Hz) 4.24 (t, 2H, NCH2CH2CH2CH2C,
J = 7.5 Hz), 3.60 (t, 2H, NCH2CH2CH2O, J = 5.7 Hz), 2.74 (t, 2H,
NCH2CH2CH2CH2C, J = 7.5 Hz), 2.09 (tt, 2H, NCH2CH2CH2O, J = 5.7,
6.9 Hz), 1.85 (tt, 2H, NCH2CH2CH2CH2C, J = 7.5, 8.0 Hz), 1.70 (tt,
2H, NCH2CH2CH2CH2C, J = 7.5, 8.0 Hz); 13C NMR (100 MHz, CDCl3)
d 154.5, 153.1, 151.7, 149.2, 148.3, 147.5, 146.0, 123.7, 121.4,
120.3, 117.4, 113.5, 112.8, 102.6, 59.0, 46.8, 43.7, 32.6, 29.3, 26.5,
C50H60Br2N16O10S2Na 1289.2384, found 1289.2360.
4.1.17. Dimer (15a)
Compound 15a (31 mg, 81%) was obtained from 2a (29 mg,
0.066 mmol) and 12 (13 mg, 0.030 mmol) as described for the syn-
thesis of 13a: 1H NMR (500 MHz, CDCl3) d 8.27 (s, 2H, H-2), 7.52 (s,
2H, triazole), 7.03 (s, 2H, H-50), 6.82 (s, 2H, H-20), 6.26 (br s, 4H,
NH2), 5.94 (s, 4H, OCH2O), 4.48 (t, 4H, NCH2CH2O, J = 5.2 Hz), 4.27
(t, 4H, NCH2CH2CH2, J = 6.9 Hz), 3.81 (t, 4H, NCH2CH2O, J = 5.2 Hz),
3.57–3.56 (m, 24H, OCH2CH2O), 2.74 (t, 4H, NCH2CH2CH2,
J = 7.4 Hz), 2.17 (tt, 4H, NCH2CH2CH2, J = 6.9, 7.4 Hz); 13C NMR

H. Sekiguchi et al. / Bioorg. Med. Chem. 18 (2010) 5732–5737
5737
25.2; ESIMS-LR m/z = 569 (M+Na)+; ESIMS-HR calcd for
21H23BrN8O3SNa 569.0695, found 569.0669.
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His-tagged N-terminal fragment of human HSP90a (amino acids
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purified HSP90
the presence of different concentrations of test compounds. The
binding of bition-labeled geldanamycin to HSP90 was detected
a and biotin-labeled geldanamycin (InvivoGen) in
a
using the AlphaScreen Histidine Detection Kit (PerkinElmer).
4.3. HER2 degradation assay
HER2 degradation was determined by cell-based ELISA assay.
SK-BR-3 cells were exposed to the compounds for 6 h and then
fixed in 4% paraformaldehyde. Fixed cells were permeabilized in
0.1% Triton-X100. HER2 and GAPDH was probed with anti-HER2
antibody (#2165, Cell signaling Technology) and anti-GAPDH anti-
body (#H86504 M, BioDesign), respectively, and then probed with
IRDye-labeled secondary antibodies (LI-COR Biosciences). Both
proteins were imaged with the Odyssey IR Imaging System (LI-
COR Biosciences).
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4.4. Cytotoxicity assay
Antiproliferative activities of the compounds against SK-BR-3
cells were measured using the crystal violet assay.24 Cells were cul-
tured in the presence of test compounds for 72 h and then fixed
with glutaraldehyde. Fixed cells were stained in 0.05% crystal vio-
let solution. The dye was extracted and the absorbance was read at
540 nm on a microplate reader.
Acknowledgments
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This work was supported financially by a Grant-in-Aid from the
Ministry of Education, Science, Sports, and Culture. We thank Ms. S.
Oka (Center for Instrumental Analysis, Hokkaido University) for
measurement of mass spectra.
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References and notes
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