Synthesis of Hsp90 inhibitor dimers as potential antitumor agents

Article abstract of DOI:10.1016/j.bmc.2008.04.070

Structure-based drug design was used to systematically synthesize PU3-dimers. The cytotoxicity of PU3 dimers 6 against breast cancer cell lines was evaluated, and their potency increased as the length of the bridging linker increased. Among the compounds

Full text of DOI:10.1016/j.bmc.2008.04.070

Bioorganic & Medicinal Chemistry 16 (2008) 5862–5870
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of Hsp90 inhibitor dimers as potential antitumor agents
a,
Kazuhiro Muranaka a, Akiko Sano b, Satoshi Ichikawa a, , Akira Matsuda
*
*
a Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan
b Discovery & Development Laboratory I, Hanno Research Center, Taiho Pharmaceutical Co., Ltd, Hannou 357-8527, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Structure-based drug design was used to systematically synthesize PU3-dimers. The cytotoxicity of PU3
dimers 6 against breast cancer cell lines was evaluated, and their potency increased as the length of the
bridging linker increased. Among the compounds tested, 6e with a C-20 linker was the most potent and
exhibited a 20- to 30-fold increase in activity compared with that of the parent compound 5. Western
blot analyses of the cell lysates treated with 6c revealed that 6c resulted in the concentration-dependent
degradation of the Hsp90 client protein Her2, which is consistent with other Hsp90 inhibitors.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 2 April 2008
Revised 28 April 2008
Accepted 29 April 2008
Available online 1 May 2008
Keywords:
Heat shock protein
Hsp90
PU3
Anticancer
Structure-based drug design
1. Introduction
minal dimerization domain that contains tetratricopeptide repeat-
binding motifs. Geldanamycin12 (GDM, 1) and radicicol13 (RDC, 2)
The heat shock protein 90 (Hsp90) family is a group of molecu-
lar chaperons that play a key role in the folding of polypeptide
chains in the crowded environment of the cell and that operate un-
der normal conditions as well as under various kinds of stress.1–4
Polypeptides folded by Hsp90 are called as client proteins, and in-
clude a growing list of over 100 members.5 Since Hsp90 and other
molecular chaperons are overexpressed in many human cancers, it
has been suggested that they could be of prognostic value in breast,
renal and endometrial malignancies.6 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.7,8 Cancer cells
acquire sets of functional capacities known as the six hallmarks
of cancer.9 Moreover, many of the Hsp90 client proteins, for exam-
ple, Bcr-abl, Akt, C-Raf, CDK4, Her2, and the estrogen receptor (ER),
are oncogenic and are also considered hallmarks of the disease.10,11
Inhibition of Hsp90 leads to degradation of these various client
proteins by a proteasome. The simultaneous combinatorial deple-
tion of many cancer-causing client proteins and the modulation
of all of the hallmarks of cancer are the major advantages of
Hsp90 inhibitors. 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-ter-
are natural products and are known to be strong inhibitors of the
Hsp90 N-terminal domain (775 nM for GDM, 2.7 nM for RDC).14
These natural products are competitive with an ATP, which is a
natural substrate of the Hsp90 N-terminal domain.15–17 They exhi-
bit growth inhibitory activity against a range of cancer cell lines,
including human breast cancer cells such as MCF-7 (49 nM for
GDM, 23 nM for RDC). Although GDM and RDC have certain draw-
backs as potential drugs, the 17-allylamino derivative (17-AAG, 3)
and the 17-dimethylaminoethyl derivative (17-DMAG, 4) of GDM
are currently in phase II studies as single agents, and combination
studies with cytotoxic agents are also underway.18 The 8,9-disub-
stituted purine class of molecules, for example, PU3 (5),19 is the
first fully synthetic Hsp90 inhibitor with the ability to bind at
the ATP-binding site of the N-terminal domain. This class of syn-
thetic inhibitors is also active in cells causing degradation of Erb
B2 kinase, ER and C-Raf kinase; however, these activities are min-
imal compared to those of GDM and RDC20 (Fig. 1).
When a target molecule is a homodimer, one of the strategies
used in developing a high affinity ligand to this target is to make
a divalent ligand by combining two ligands with an appropriate
linker.21 With this approach, a multiplier effect would be expected
since the conformational restriction by cross-linking monomers re-
duces the free energy associated with the restriction of the trans-
lational and rotational freedom of the ligand and the target. A
recently reported X-ray crystal structure of the full length Hsp90
dimer23 revealed that the distance between each ATP-binding site
of the N-terminal domains is at least 25 Å, which corresponds to a
linear 20 carbon chain in length. Knowing that Hsp90 exists
* 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).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.070

K. Muranaka et al. / Bioorg. Med. Chem. 16 (2008) 5862–5870
5863
2. Results and discussion
O
R
O
2.1. Molecular design
OH
Cl
O
N
O
H
In order to observe more easily the impact of the dimerization
of ligands in developing divalent inhibitors, we selected PU3, a
low affinity N-terminal inhibitor. We planned to systematically
prepare the dimers of PU3 6a–e by varying the length of the linker
enough to cover the distance of the two ATP-binding sites of the N-
terminal domains. The X-ray crystal structures of the PU3 and
Hsp90 complex24 indicated that the terminal alkyl moiety at the
N9-position of PU3 was exposed to the solvent-accessible region
and did not participate in the binding to Hsp90. Therefore, it was
decided that the terminus of the alkyne could be the position
where the linker was attached. After considering water-solubility
and the increase in molecular weight caused by dimerization, we
chose an amine as the pivotal functional group connecting PU3
and the linker. We also prepared the GDM dimers 7a–c, including
several known compounds, in order to compare their biological
activity with that of the PU3 dimers and to reinvestigate the activ-
ity of the GDM dimers.
O
O
HO
MeO
O
OH
OMe
O
O
H2N
radicicol (4)
geldanamycin (1): R = OMe
17-AAG (2): R = HNCH2CH=CH2
17-DMAG (3): R = HNCH2CH2NMe2
MeO
OMe
OMe
NH2
N
N
N
N
2.2. Chemistry
PU3 derivative (5)
The synthesis of the PU3 dimers 6a–e is outlined in Scheme 1.
The hydroxyl group of 4-pentyn-1-ol (8) was temporarily pro-
tected with a TBDPS group to afford 9 in quantitative yield. Depro-
tonation of the terminal alkyne moiety of 9 with n-BuLi followed
by substitution of the resulting lithium acetylide with methyl chlo-
roformate afforded the corresponding methyl ester. Deblocking the
TBDPS group of this compound with TBAF in the presence of acetic
acid gave 10 in good yield. The absence of acetic acid in the depro-
tection step caused the further cyclization of 10 and furnished a
stable 2-methoxycarbonylmethylidenetetrahydrofurane.25 The
Mitsunobu reaction of 10 and 8-(3,4,5-trimethoxybenzyl)ade-
nine26 11 in toluene/CH2Cl2 gave the desired N9-substituted ade-
nine derivative 12 as the major product along with the
corresponding N3- and N7-isomers. The methoxycarbonyl group
of 12 was reduced to the hydroxymethyl group with DIBAL-H pro-
viding the alcohol 13 in 75% yield. Installation of two PU3 moieties
Figure 1. Structures of Hsp90 N-terminal domain inhibitors.
predominantly as dimers, researchers have prepared several di-
mers of GDM and evaluated their biological activity.22 For example,
the four-carbon tethered GDM dimer 7a exhibited the greatest
inhibitory activity for the series of compounds and demonstrated
high selectivity toward the Her2 family of receptors. Given the dis-
tance between the ATP-binding sites of the N-terminal domains, it
is unlikely that the four-carbon tethered GDM dimer 7a would act
as a divalent ligand against the Hsp90 dimer. The linker connecting
the two ligands would have to be lengthened in the divalent li-
gands in order to develop divalent ligands for the Hsp90 N-termi-
nal domains. Herein we describe the design, synthesis and the
biological evaluation of a series of dimeric compounds of PU3
6a–e (Fig. 2).
MeO
MeO
OMe
MeO
OMe
OMe
NH2
N
NH2
N
6a: n= 1 (C4)
6b: n = 2 (C8)
6c: n = 3 (C12)
6d: n = 4 (C16)
6e: n = 5 (C20)
N
N
N
N
N
N
H
N
n N
H
O
H
N
O
H
O
N
n
O
N
7a: n = 1 (C4)
7b: n = 3 (C12)
7c: n = 4 (C16)
N
H
H
O
O
OMe
MeO
OH
MeO
O
OH
OMe
O
O
O
NH2
H2N
Figure 2. Structures of dimers of Hsp90 N-terminal domain inhibitors.

5864
K. Muranaka et al. / Bioorg. Med. Chem. 16 (2008) 5862–5870
MeO
OMe
OMe
1) BuLi, THF, −78 ˚C
then ClCO2Me, 94%
2) TBAF, AcOH, THF
0 ˚C, 91%
NH2
N
N
N
OR
OH
N
H
11
MeO2C
PPh3, DIAD
toluene−CH2Cl2
82%
TBDPSCl, imidazole
DMF, quant.
8: R = H
9: R = TBDPS
10
MeO
OMe
OMe
MeO
OMe
OMe
NH2
N
NH2
N
14a-e, Bu3P=CHCN
toluene, 100 ˚C
N
N
N
N
N
N
R
R
N
n
N
12: R = CO2Me
13: R = CH2OH
DIBAL−H, CH2Cl2
0 ˚C, 75%
R
N
N
N
*Ns = 4-nitrobenzenesulfonyl
N
MeO
MeO
NH2
OMe
PhSH, K2CO3
DMF
15a: R = Ns, n = 1 (40%)
6a: R = H, n = 1 (34%)
6b: R = H, n = 2 (66%)
6c: R = H, n = 3 (73%)
6d: R = H, n = 4 (33%)
6e: R = H, n = 5 (76%)
15b: R = Ns, n = 2 (39%)
15c: R = Ns, n = 3 (24%)
15d: R = Ns, n = 4 (46%)
15e: R = Ns, n = 5 (19%)
MeO
OMe
OMe
MeO
OMe
OMe
MeO
OMe
OMe
NH2
NH2
NH2
N
N
N
N
N
N
N
N
N
N
N
N
O
R
N
NHNs
18: R = Ns
19: R = H
O
17
3
H
16
N
N
N
N
O
R
Ns
H
O
Scheme 1. Synthesis of dimers of PU3.
on the alkyldiamines was conducted by a Mitsunobu reaction
using the corresponding N,N0-bis-4-nitrobenzenesulfonyl (Ns)27
derivatives 14a–e, which were prepared by simple sulfonylation
of the alkyldiamines (Scheme 2). Optimization of the reaction con-
ditions was first examined with 14a. When Ph3P and DIAD were
used, only a trace amount of the desired 15a was obtained. Careful
analysis of the reaction revealed the formation of the N-alkylhydr-
azine derivative 17, which was formed by the nucleophilic substi-
tution of the triphenylphosphonium intermediate by hydrazine-
N,N0-diisopropylcarbonate, a side-product of the reaction. The for-
mation of 17 consumed the electrophile to give the N,N0-bis-Ns
amide. In order to increase the nucleophilicity of the sulfonamide,
the 2,4-dinitrobenzenesulfonamide derivative was used instead of
14a; however no improvement was observed. The Mitsunobu-type
reaction with cyanomethylene tributylphosphorane (CMBP), which
was developed by Tsunoda et al., allows the use of nucleophiles
having a pKa higher than 13.28 In addition, there are no nucleo-
philes such as hydrazine-N,N0-diisopropylcarbonate present in
NsCl, Et3N
CH2Cl2
14a: n = 1 (70%)
14b: n = 2 (71%)
14c: n = 3 (81%)
14d: n = 4 (86%)
14e: n = 5 (46%)
NsHN
H2N
n NHNs
n NH2
Scheme 2. Preparation of alkyldiamines protected with 4-nitrobenezenesulfonyl group.

K. Muranaka et al. / Bioorg. Med. Chem. 16 (2008) 5862–5870
5865
icity was related to Hsp90 inhibition, 5 and 6c were incubated with
MCF-7 breast cancer cells for 24 h. As can be seen from Western
blot analyses of the protein lysates, 6c resulted in the concentra-
tion-dependent degradation of the Hsp90 client protein Her2
(Fig. 3). The dimer 6c exhibited higher activity than that of 5,
and the amount of the Her2 degradation activity is in good accor-
dance with the cytotoxic data. Although moderate, Hsp70 levels in-
creased in a concentration-dependent manner, which is consistent
with other Hsp90 inhibitors. Since actin is not dependent on the
Hsp90 protein folding machinery, actin levels remained un-
changed. Of importance is the effect of dimerization. The mono-
PU3 derivative 19, which was obtained by deprotection of the Ns
group of 18, exhibited weak cytotoxic activity (IC50 = 55.1 lM for
MCF-7, 13.1 lM for SKBr3). This result indicated that the dimer 6
could act as a divalent ligand for Hsp90, as we expected, although
more detailed mechanistic study is necessary. These molecules
contain two nitrogen atoms within the linker, which are proton-
ated under physiological conditions to form ammonium ions that
increase water-solubility. The fact that the dimers 6 exhibited cli-
ent degradation activity indicates that the newly synthesized di-
mers reach the target Hsp90 inside the cells even though they
are relatively large molecules.
GDM, DMF
7a: n = 1 (C4, 98%)
7b: n = 3 (C12, 95%)
7c: n = 4 (C16, 84%)
H2N
n NH2
Scheme 3. Synthesis of GDM dimers.
the reaction. When the N,N0-bis-Ns amides 14a were treated with
CMBP at 100 °C, the desired 15a was produced in 40% yields to-
gether with the monomeric compound 16. Similarly, compounds
15b–e were also obtained in acceptable yields (19–46%). Finally
the Ns groups were removed by thiophenol and K2CO3 in DMF to
afford the desired dimers 6a–e (33–76%).
As described above, the GDM dimers 7a–c with various lengths
of linker were also synthesized in order to compare their biological
activity with that of the PU3 dimers 6a–e. In a manner similar to
that reported previously,22 simple treatment of GDM with 1,4-
butanediamine, 1,12-dodecanediamine, and 1,16-hexadecanedi-
amine gave the corresponding GDM dimers, respectively, in high
yields (84–98%, Scheme 3).
2.3. Biological activity
The cytotoxicity of the GDM dimers 7a–c was also evaluated.
The dimers 7a–c exhibited cytotoxicity against MCF-7 cells within
the same range (IC50 = 0.31 lM for 7a, 0.27 lM for 7b, 0.15 lM for
7c). However, it is unknown whether they act as divalent ligands,
since the cytotoxicity of 7a–c was less potent than that of the par-
ent GDM.
The cytotoxic activity of the series of synthesized dimers was
evaluated against the MCF-7 and SKBr3 human breast cancer cell
lines, as shown in Table 1. Under conditions where the IC50 for
the parent PU3 5 exhibited the previously reported activity
(51.6 lM for MCF-7, 28.9 lM for SKBr3), the monomer 13 showed
no cytotoxicity at 100 lM. As for the activity of the PU3 dimers,
the results revealed that the longer the length of the linker, the
more potent the cytotoxicity. The IC50s for 6e possessing a C-20 lin-
ker were 1.38 lM (MCF-7) and 1.29 lM (SKBr3), respectively, and a
20- to 30-fold increase in cytotoxicity was observed when com-
pared with that of 5. To determine whether the observed cytotox-
2.4. Conclusion
The PU3-dimers were systematically synthesized and their bio-
logical properties were evaluated according to a structure-based
drug design. It was found that by increasing the length of the
bridging linker, the PU3 dimers became more cytotoxic against hu-
man breast cancer cell lines. The IC50s for 6e with a C-20 linker
exhibited a 20- to 30-fold increase in cytotoxicity compared with
that of the parent compound. In this study, PU3, a relatively weak
inhibitor of the N-terminal domain, was used as a model. If more
potent inhibitors were used, it conceivably would develop more
potent dimeric inhibitors. If the N-terminal domain dimers 6 syn-
thesized in this study actually indeed acts as divalent inhibitors, it
would be theoretically possible to capture the different conforma-
tions of the Hsp90 dimer. Namely, it is expected that these dimers
could modulate the functions of Hsp90 leading to an investigation
of its dynamic-functional relationship.
Table 1
Cytotoxic activity of synthesized analogs
Compound
IC50 (lM)
MCF-7
SKBr3
2
5
0.0892
45.9
0.00364
31.8
6a
6b
6c
6d
6e
7a
7b
7c
13
19
>100
30.6
4.68
1.57
1.46
>100
85.7
5.73
3.18
1.31
0.258
0.296
0.150
>100
13.1
1.11
0.628
0.431
>100
55.1
3. Experimental
3.1. General experimental methods
NMR spectra were obtained on a JEOL EX270, JEOL GX270, JEOL
AL400 or JEOL ECA500, and were reported in parts per million (d)
relative to tetramethylsilane (0.00 ppm) as internal standard
otherwise noted. Coupling constant (J) was reported in hertz
(Hz). Abbreviations of multiplicity were as follows; s: singlet, d:
doublet, t: triplet, q: quartet, m: multiplet, br: broad. Data were
presented as follows; chemical shift (multiplicity, integration, cou-
pling constant). Assignment was based on 1H–1H COSY, HMBC and
HMQC NMR spectra. FAB-MS was obtained on a JEOL JMS-HX101
or JEOL JMS-700TZ. Analytical thin layer chromatography (TLC)
was performed on Merck silica gel 60F254 plates. Normal-phase
column chromatography was performed on Merck silica gel 5715
or Kanto Chemical silica gel 60N (neutral). Flash column chroma-
tography was performed on Merck silica gel 60.
Figure 3. Her2 Degradation assay. MCF-7 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, anti-Hsp70 and anti-b-actin.

5866
K. Muranaka et al. / Bioorg. Med. Chem. 16 (2008) 5862–5870
3.1.1. 1-O-tert-Butyldiphenylsilyl-4-pentyn-1-ol (9)
151.0, 150.4, 150.3, 149.7, 144.7, 130.6, 124.4, 105.2, 105.1,
60.4, 55.8, 52.2, 41.4, 34.2, 26.5, 15.5, 14.5; FABMS-LR m/z
440.2 (MH+); FABMS-HR calcd for C22H26N5O5 440.1934, found
440.1934 (MH+).
A solution of 4-pentyn-1-ol (8, 930 lL, 10 mmol) and imidazole
(1.5 g, 22 mmol) in DMF (50 mL) was treated with TBDPSCl
(2.8 mL, 11 mmol) at room temperature for 1 h. The reaction was
quenched with H2O, and the mixture was partitioned between
AcOEt (200 mL) and H2O (2ꢀ 100 mL). The organic layer was
washed with brine (100 mL), dried (Na2SO4), filtered and concen-
trated. The residue was purified by silica gel column chromatogra-
phy (8 ꢀ 10 cm, 10% hexane/AcOEt) to give 9 (3.2 g, 99%) as a
colorless syrup: 1H NMR (270 MHz, CDCl3) d 7.67 (m, 4H, phenyl),
7.40 (m, 6H, phenyl), 3.75 (t, 2H, OCH2, J = 5.9 Hz), 2.35 (dt, 2H,
CH2, J = 7.3, 2.6 Hz), 1.91 (t, 1H, CH, J = 2.6 Hz), 1.80 (tt, 2H, CH2,
J = 7.3, 5.9 Hz), 1.05 (s, 9H, tert-butyl); 13C NMR (125 MHz, CDCl3)
d 135.5, 133.8, 129.6, 127.6, 84.2, 68.3, 62.2, 31.4, 26.8, 19.2,
14.9; FABMS-LR m/z 323.5 (MH+); FABMS-HR calcd for C21H27OSi
323.1831, found 323.1820 (MH+).
3.1.5. 6-Amino-9-(6-hydroxyhex-4-ynyl)-8-(3,4,5-trimethoxy
benzyl)-9H-purine (13)
Diisobutylaluminiumhydride (1.5 M solution in toluene, 280 lL,
0.42 mmol) was added dropwise to a solution of 12 (60 mg,
0.14 mmol) in CH2Cl2 (2 mL) at 0 °C, and the mixture was stirred
at the same temperature for 1 h. The reaction was quenched with
saturated aqueous Rochelle salt (50 mL). The mixture was stirred
at room temperature overnight, and was extracted with CHCl3
(2ꢀ 20 mL). The separated organic layers were washed with brine
(10 mL), dried (Na2SO4), filtered and concentrated. The residue was
purified by silica gel column chromatography (1.5 ꢀ 10 cm, 4%
MeOH/CHCl3) to give 13 (43 mg, 75%) as a pale yellow foam: 1H
NMR (270 MHz, CDCl3) d 8.32 (s, 1H, H-2), 6.47 (s, 2H, phenyl),
5.69 (br s, 2H, 6-NH2), 4.23 (s, 2H, PhCH2), 4.17 (m, 4H, NCH2,
OCH2), 3.82, 3.80 (each s, 9H, 3ꢀ OMe), 2.19 (t, 2H, CH2,
J = 6.6 Hz), 1.87 (m, 2H, CH2); 13C NMR (67.5 MHz, CDCl3) d
153.7, 153.6, 153.5, 153.2, 152.5, 151.5, 151.1, 117.2, 105.9, 60.9,
56.4, 50.9, 42.0, 34.8, 27.9, 17.8, 16.1; FABMS-LR m/z 412.2
(MH+); FABMS-HR calcd for C21H26N5O4 412.1985, found
412.2000 (MH+).
3.1.2. Methyl 6-hydroxy-6-O-tert-butyldiphenylsiloxy-hex-2-
ynoate
Butyl lithium (1.58 M solution in THF, 2.1 mL, 3.3 mmol) was
added dropwise to a solution of 9 (870 mg, 2.7 mmol) in THF
(10 mL) at ꢁ78 °C, and the mixture was stirred at the same temper-
ature for 20 min. Methyl chloroformate (260 lL, 3.3 mmol) was
added dropwise to the mixture, which was stirred at 0 °C for
30 min. The reaction was quenched with saturated aqueous NH4Cl,
and the mixture was partitioned between AcOEt (200 mL) and H2O
(2ꢀ 100 mL). The organic layer was washed with brine (100 mL),
dried (Na2SO4), filtered and concentrated. The residue was purified
by silica gel column chromatography (2 ꢀ 10 cm, 17% hexane/
AcOEt) to give the title compound (960 mg, 94%) as a colorless syr-
up: 1H NMR (270 MHz, CDCl3) d 7.66 (m, 4H, phenyl), 7.40 (m, 6H,
phenyl), 3.76 (s, 3H, OMe), 3.73 (t, 2H, OCH2, J = 5.9 Hz), 2.50 (t, 2H,
CH2, J = 7.3 Hz), 1.81 (tt, 2H, CH2, J = 5.9, 7.3 Hz), 1.04 (s, 9H, tert-
butyl); 13C NMR (125 MHz, CDCl3) d 154.3, 135.7, 133.7, 129.8,
127.8, 89.5, 73.1, 62.1, 52.6, 30.6, 26.9, 19.3, 15.4: FABMS-LR m/z
403.2 (MNa+); FABMS-HR calcd for C23H28O3SiNa 403.1705, found
403.1715 (MNa+).
3.1.6. N,N0-Di-(4-nitrobenzenesulfonyl)-1,4-diaminobutane
(14a)
4-Nitrobenzenesulfonyl chloride (490 mg, 2.2 mmol) was added
to a mixture of 1,4-diaminobutane (1.0 mmol, 100 lL) and triethyl-
amine (310 lL, 2.2 mmol) in CH2Cl2 (10 mL) at 0 °C, and the mix-
ture was stirred at the same temperature for 1 h. The reaction
mixture was allowed to room temperature, and stirred for further
3 h. The reaction was quenched with MeOH, and the solvent was
concentrated in vacuo. The residue was triturated from AcOEt to
give 14a (320 mg, 70%) as a white solid: 1H NMR (270 MHz,
DMSO-d6) d 8.40 (d, 4H, Ns, J = 8.6 Hz), 7.98 (d, 4H, Ns, J = 8.6 Hz),
7.94 (t, 2H, 2ꢀ NH, J = 5.3 Hz), 2.71 (m, 4H, 2ꢀ NHCH2), 1.33 (m,
4H, 2ꢀ CH2); 13C NMR (125 MHz, DMSO-d6) d 149.5, 146.1,
128.0, 127.9, 124.7, 124.5, 42.0, 26.1; FABMS-LR m/z 459.1
(MH+); FABMS-HR calcd for C16H19N4O8S2 459.0644, found
459.0641 (MH+).
3.1.3. Methyl 6-hydroxyhex-2-ynoate (10)
A mixture of methyl 6-hydroxy-6-O-tert-butyldiphenylsiloxy-
hexy-2-ynoate (960 mg, 2.5 mmol), TBAF (1.0 M solution in THF,
3.0 mL, 3.0 mmol) and AcOH (350 lL, 6.1 mmol) in THF (25 mL)
was stirred at 0 °C for 3 h. After AcOH (0.5 mL) was added, the
mixture was concentrated in vacuo. The residue was purified by
silica gel column chromatography (2 ꢀ 10 cm, 20% hexane/
AcOEt) to give 10 (330 mg, 91%) as a pale yellow syrup: 1H
NMR (270 MHz, CDCl3) d 3.75 (s, 3H, OMe), 3.75 (t, 2H, OCH2,
J = 6.3Hz), 2.48 (t, 2H, CH2, J = 6.9 Hz), 1.83 (tt, 2H, CH2,
J = 6.9, 6.3 Hz); 13C NMR (125 MHz, CDCl3) d 154.2, 89.1, 72.9,
60.8, 52.5, 30.1, 15.0; FABMS-LR m/z 143.1 (MH+); FABMS-HR
calcd for C7H10O3 143.0708, found 143.0710 (MH+).
3.1.7. N,N0-Di-(4-nitrobenzenesulfonyl)-1,8-diaminooctane
(14b)
Compound 14b (720 mg, 71%) was obtained from 1,8-diam-
inoctane (290 mg, 2.0 mmol) as described for the synthesis of
14a: 1H NMR (270 MHz, DMSO-d6)
d
8.41 (d, 4H, Ns,
J = 8.8 Hz), 8.02 (d, 4H, Ns, J = 8.6 Hz), 7.94 (t, 2H, 2ꢀ NH,
J = 5.9 Hz), 2.77 (m, 4H, 2ꢀ NHCH2), 1.33 (m, 4H, 2ꢀ CH2),
1.12 (m, 8H, 4ꢀ CH2); 13C NMR (125 MHz, DMSO-d6) d 149.5,
146.2, 128.1, 127.9, 124.7, 124.5, 42.5, 28.9, 28.3, 25.8; FAB-
MS-LR m/z 515.2 (MH+); FABMS-HR calcd for C20H27N4O8S2
515.1270, found 515.1277 (MH+).
3.1.4. 6-Amino-9-(5-methoxycarbonylpent-4-ynyl)-8-(3,4,5-
trimethoxybenzyl)-9H-purine (12)
A
suspension of 11 (200 mg, 0.64 mmol), 10 (120 mg,
3.1.8. N,N0-Di-(4-nitrobenzenesulfonyl)-1,12-diaminododecane
(14c)
0.83 mmol), and PPh3 (370 mg, 1.4 mmol) in toluene/CH2Cl2
(6.5:1) was treated with DIAD (610 lL, 3.2 mmol) at room tem-
perature for 2 h. The solvent was removed in vacuo, and the res-
idue was purified by flash silica gel column chromatography
(1.5 ꢀ 10 cm, 2% MeOH/CHCl3) to give 12 (240 mg, 82%) as a yel-
low foam: 1H NMR (400 MHz, CDCl3) d 8.31 (s, 1H, H-2), 6.48 (s,
2H, phenyl), 5.58 (br s, 2H, 6-NH2), 4.24 (s, 2H, PhCH2), 4.18 (t,
2H, NCH2, J = 7.3 Hz), 3.82, 3.80 (each s, 9H, 3ꢀ OMe), 3.77 (s,
3H, OMe), 2.33 (t, 2H, CH2, J = 6.8 Hz), 2.00 (tt, 2H, CH2, J = 7.3,
Compound 14c (1.4 g, 81%) was obtained from 1,12-diaminod-
odecane (3.0 mmol, 600 mg) as described for the synthesis of
14a: 1H NMR (270 MHz, DMSO-d6) d 8.40 (d, 4H, Ns, J = 9.2 Hz),
8.02 (d, 4H, Ns, J = 9.2 Hz), 7.93 (t, 2H, 2ꢀ NH, J = 5.6 Hz), 2.75
(m, 4H, 2ꢀ NHCH2), 1.30 (m, 4H, 2ꢀ CH2), 1.09 (m, 16H, 8ꢀ
CH2); 13C NMR (125 MHz, DMSO-d6) d 149.5, 146.3, 128.1, 128.0,
124.7, 124.5, 42.5, 29.0, 28.9, 28.8, 28.4, 25.9; FABMS-LR m/z
571.2 (MH+); FABMS-HR calcd for C24H35N4O8S2 571.1896, found
571.1888 (MH+).
6.8 Hz); 13C NMR (67.5 MHz, CDCl3)
d 154.1, 153.2, 152.1,

K. Muranaka et al. / Bioorg. Med. Chem. 16 (2008) 5862–5870
5867
3.1.9. N,N0-Di-(4-nitrobenzenesulfonyl)-1,16-
diaminohexadecane (14d)
temperature and stirred for further 8 h. The reaction was quenched
with MeOH (5 mL), and the solvent was removed in vacuo. The res-
idue was triturated from AcOEt and CHCl3 to give 14 (150 mg, 46%)
as a white solid: 1H NMR (500 MHz, DMSO-d6) d 8.40 (d, 4H, 2ꢀ Ns,
J = 8.1 Hz), 8.02 (d, 4H, 2ꢀ Ns, J = 8.1 Hz), 7.94 (t, 2H, 2ꢀ NH,
J = 5.7 Hz), 2.77 (m, 4H, 2ꢀ NCH2, J = 5.7, 6.3 Hz), 1.31 (m, 4H, 2ꢀ
CH2, J = 6.3 Hz), 1.20–1.12 (br m, 32H, 16ꢀ CH2); 13C NMR
(100 MHz, DMSO-d6) d 149.5, 146.4, 128.1, 124.7, 42.6, 29.1, 29.0,
28.5, 26.0; FABMS-LR m/z 683.4 (MH+); FABMS-HR calcd for
A solution of p-toluenesulfonyl chloride (2.2 g, 11.6 mmol) in
CH2Cl2 (10 mL) was added dropwise to a mixture of 1,16-hexadec-
anediol (1.0 g, 3.8 mmol), Et3N (2.1 mL, 15.2 mmol) and Me3NꢂHCl
(73 mg, 0.76 mmol) in CH2Cl2 (30 mL) at 0 °C, and the mixture was
stirred at the same temperature for 30 min. The reaction was
quenched with MeOH, and whole the mixture was concentrated
in vacuo. The residue was partitioned between CHCl3 and H2O,
the organic layer was washed with brine, dried (Na2SO4), filtered
and concentrated. The residue was triturated from AcOEt/hexane
to give O,O0-ditosyl-1,16-hexadecanediol (2.0 g, 92%) as a white so-
lid: 1H NMR (500 MHz, CDCl3) d 7.78 (d, 4H, 2ꢀ Ts, J = 8.6 Hz), 7.33
(d, 4H, 2ꢀ Ts, J = 8.6 Hz), 4.01 (t, 4H, 2ꢀ OCH2, J = 6.5 Hz), 2.44 (s,
6H, 2ꢀ Me), 1.61 (m, 4H, 2ꢀ CH2), 1.27 (m, 4H, 2ꢀ CH2), 1.21 (m,
20H, 10ꢀ CH2); 13C NMR (125 MHz, CDCl3) d 144.6, 133.2, 129.8,
127.8, 70.7, 29.6, 29.5, 29.4, 29.3, 28.9, 28.8, 25.3, 21.6. A solution
of O,O0-ditosyl-1,16-hexadecanediol (1.1 g, 2.0 mmol) in DMF
(10 mL) was treated with NaN3 (390 mg, 6.0 mmol) at 60 °C for
9 h. The mixture was partitioned between AcOEt (100 mL) and
H2O (3ꢀ 100 mL), and the organic layer was washed with brine,
dried (Na2SO4), filtered and concentrated. A solution of the residue
in THF/H2O (9:1; 20 mL) was treated with PPh3 (1.1 g, 4.4 mmol) at
room temperature for 12 h. The mixture was concentrated in va-
cuo, and the residue was triturated from AcOEt to give 1,16-diami-
nohexadecane (280 mg, 55% in two steps) as a white solid. A
mixture of 7 (200 mg, 0.78 mmol) and triethylamine (260 lL,
1.9 mmol) in CH2Cl2 (10 mL) was treated with 4-nitro-
benzenesulfonyl chloride (380 mg, 1.7 mmol) at 0 °C, and the mix-
ture was stirred at room temperature for 3 h. The reaction was
quenched with MeOH (5 mL), and the mixture was concentrated
in vacuo. The residue was triturated from AcOEt to give 14d
(420 mg, 86%) as a white solid: 1H NMR (500 MHz, DMSO-d6) d
8.40 (d, 4H, 2ꢀ Ns, J = 8.6 Hz), 8.02 (d, 4H, 2ꢀ Ns, J = 8.6 Hz), 7.94
(t, 2H, 2ꢀ NH, J = 5.8 Hz), 2.77 (dt, 4H, 2ꢀ NCH2, J = 5.8, 6.9 Hz),
1.33 (m, 4H, 2ꢀ CH2, J = 6.9 Hz), 1.15 (m, 24H, 12ꢀ CH2); 13C
NMR (125 MHz, DMSO-d6) d 149.5, 146.4, 128.2, 128.0, 124.8,
124.5, 42.5, 29.1, 28.9, 28.5, 25.9; FABMS-LR m/z 627.3 (MH+); FAB-
MS-HR calcd for C28H43N4O8S2 627.2522, found 627.2516 (MH+).
C
32H51N4O8S2 683.3141, found 683.3134 (MH+).
3.1.11. N,N0-Di-(4-nitrobenzenesulfonyl)-PU3-C4-dimer (15a)
A mixture of 13 (85 mg, 0.20 mmol), 14a (45 mg, 0.098 mmol)
and cyanomethylene tributylphosphorane (CMBP, 71 mg,
0.30 mmol) in toluene (0.5 mL) was stirred at 100 °C for 2 h. After
cooling, the solvent was removed in vacuo. The residue was puri-
fied by preparative TLC (5% 3ꢀ MeOH/CHCl3) to give 15a (48 mg,
40%) as a brownish foam and 16 (26 mg, 31%) as a brownish foam.
Data for 15a: 1H NMR (400 MHz, CDCl3) d 8.29 (s, 2H, 2ꢀ H-2), 8.26
(d, 4H, 2ꢀ Ns, J = 8.8 Hz), 7.97 (d, 4H, 2ꢀ Ns, J = 8.8 Hz), 6.42 (s, 4H,
2ꢀ phenyl), 5.89 (br s, 4H, 2ꢀ 6-NH2), 4.17 (s, 4H, 2ꢀ PhCH2), 4.0 (s,
4H, 2ꢀ H-d), 3.97 (t, 4H, J = 7.3 Hz), 3.81, 3.80 (each s, 18H, 6ꢀ
OMe), 3.23 (m, 4H), 1.88 (t, 4H, J = 6.9 Hz), 1.63 (tt, 4H, J = 7.3,
6.9 Hz), 1.59 (m, 4H); 13C NMR (67.5 MHz, CDCl3) d 180.6, 154.3,
153.1, 151.9, 151.1, 150.1, 149.5, 144.1, 136.9, 130.6, 128.5,
123.5, 118.0, 116.7, 105.2, 84.2, 60.4, 55.8, 44.7, 41.7, 36.0, 34.3,
27.3, 23.4, 22.9, 15.6; FABMS-LR m/z 1245.2 (MH+); FABMS-HR
calcd for C58H64N14O14S2 1245.4246, found 1245.4246 (MH+). Data
for 16: 1H NMR (270 MHz, CDCl3) d 8.34 (d, 2H, Ns, J = 8.6 Hz), 8.31
(s, 1H, H-2), 8.20 (d, 2H, Ns, J = 8.9 Hz), 8.05 (d, 2H, Ns, J = 8.9 Hz),
7.92 (d, 2H, Ns, J = 8.6 Hz), 6.71 (br s, 1H, NsNH), 6.41 (s, 2H, Ph),
5.94 (br s, 2H, 6-NH2), 4.17 (s, 2H, PhCH2), 4.03 (t, 2H, J = 6.3 Hz),
3.84 (s, 2H), 3.82 (s, 9H, 3ꢀ OMe), 3.05 (m, 4H), 1.89 (m, 4H),
1.70 (m, 2H), 1.53 (m, 2H); 13C NMR (67.5 MHz, CDCl3) d 184.5,
155.1, 154.1, 152.9, 152.5, 152.4, 152.2, 151.3, 150.5, 147.3,
146.5, 145.1, 131.4, 129.3, 128.6, 124.9, 124.4, 109.3, 84.9, 73.1,
61.4, 56.8, 56.5, 46.3, 46.1, 43.0, 37.8, 35.3, 27.9, 26.6, 24.9, 16.6;
FABMS-LR m/z 852.2 (MH+); FABMS-HR calcd for C37H42N9O11S2
852.2445, found 852.2447 (MH+).
3.1.12. N,N0-Di-(4-nitrobenzenesulfonyl)-PU3-Ns-C8-dimer
(15b)
3.1.10. N,N0-Di-(4-nitrobenzenesulfonyl)-1,20-diaminoeicosane
(14e)
Compound 15b (44 mg, 39%) was obtained from 13 (75 mg,
0.18 mmol), 14b (45 mg, 0.087 mmol) and CMBP (84 mg,
0.35 mmol) as described for the synthesis of 15a: 1H NMR
(270 MHz, CDCl3) d 8.29 (s, 2H, 2ꢀ H-2), 8.26 (d, 4H, 2ꢀ Ns,
J = 8.8 Hz), 7.97 (d, 4H, 2ꢀ Ns, J = 8.8 Hz), 6.42 (s, 4H, 2ꢀ phenyl),
5.75 (br s, 4H, 2ꢀ 6-NH2), 4.17 (s, 4H, 2ꢀ PhCH2), 4.07 (s, 4H),
3.97 (t, 4H, J = 7.3 Hz), 3.80 (s, 18H, 6ꢀ OMe), 3.17 (t, 4H,
J = 7.1 Hz), 1.85 (t, 4H, J = 6.9), 1.61 (tt, 4H, J = 6.9, 7.3 Hz), 1.52
(m, 4H), 1.26 (m, 8H); 13C NMR (67.5 MHz, CDCl3) d 154.2, 153.2,
152.0, 151.0, 150.0, 149.5, 144.5, 136.9, 130.6, 128.4, 123.4,
118.1, 105.2, 83.9, 72.8, 60.4, 56.9, 55.8, 45.8, 41.6, 36.0, 34.3,
28.4, 27.5, 26.8, 25.7, 15.5; FABMS-LR m/z 1301.1 (MH+); FABMS-
HR calcd for C62H73N14O14S2 1301.4872, found 1301.4892 (MH+).
A
mixture of 1,20-eicosanediol (500 mg, 1.6 mmol), Et3N
(0.90 mL, 6.4 mmol) and Me3NHCl (30 mg, 0.32 mmol) in CH2Cl2
(30 mL) was treated with MsCl (0.37 mL, 4.8 mmol) at 0 °C for
10 min. The reaction was quenched with H2O (30 mL), and the
mixture was extracted with CHCl3 (2ꢀ 50 mL). The organic layers
were washed with brine (30 mL), dried (Na2SO4), filtered and con-
centrated. The residue was triturated from hexane to give 1,20-
O,O0-dimethanesulfonyl eicosanediol (340 mg, 0.72 mmol, 45%) as
a white solid: 1H NMR (400 MHz, CDCl3) d 4.21 (t, 4H, 2ꢀ OCH2,
J = 6.8 Hz), 3.00 (s, 6H, Ms2), 1.73 (tt, 4H, 2ꢀ CH2, J = 6.8, 7.3 Hz),
1.40–1.25 (br m, 32H, 16ꢀ CH2). A solution of 1,20-O,O0-dim-
ethanesulfonyl eicosanediol (240 mg, 0.50 mmol) in DMF
(5.0 mL) was treated with NaN3 (98 mg, 1.5 mmol) at 60 °C for
12 h. The mixture was partitioned between AcOEt (100 mL) and
H2O (2ꢀ 50 mL), The organic layer was washed with brine, dried
(Na2SO4), filtered and concentrated. A solution of the residue
(140 mg) in THF/H2O (9:1, 5 mL) was treated with PPh3 (220 mg,
0.84 mmol) at room temperature for 6 h. The mixture was concen-
trated in vacuo, and the residue was triturated from AcOEt to give
1,20-diaminoeicosane (60 mg, 50% over two steps) as a white solid.
A suspension of 1,20-diaminoeicosane (150 mg, 0.48 mmol), Et3N
(0.28 mL, 2.0 mmol) and 4-nitrobenzenesulfonyl chloride was stir-
red at 0 °C for 10 min. The reaction mixture was allowed to room
3.1.13. N,N0-Di-(4-nitrobenzenesulfonyl)-PU3-C12-dimer (15c)
Compound 15c (17 mg, 29%) and 18 (18 mg, 41%) were obtained
from 13 (42 mg, 0.10 mmol), 14c (26 mg, 0.046 mmol) and CMBP
(36 mg, 0.15 mmol) as described for the synthesis of 15a. Data
for 15c: 1H NMR (270 MHz, CDCl3) d 8.28 (s, 2H, 2ꢀ H-2), 8.26
(d, 4H, 2ꢀ Ns, J = 8.6 Hz), 7.98 (d, 4H, 2ꢀ Ns, J = 8.6 Hz), 6.41 (s,
4H, 2ꢀ phenyl), 5.92 (br s, 4H, 2ꢀ 6-NH2), 4.16 (s, 4H, 2ꢀ PhCH2),
4.08 (s, 4H), 3.96 (t, 4H, J = 7.3 Hz), 3.81, 3.80 (each s, 18H, 6ꢀ
OMe), 3.16 (t, 4H, J = 7.2 Hz), 1.87 (t, 4H, J = 6.8 Hz), 1.61 (tt, 4H,
J = 7.3, 6.8 Hz), 1.51 (m, 4H), 1.24–1.19 (m, 16H); 13C NMR

5868
K. Muranaka et al. / Bioorg. Med. Chem. 16 (2008) 5862–5870
(125 MHz, CDCl3) d 154.9, 153.8, 152.6, 151.6, 150.7, 150.1, 145.2,
137.5, 131.2, 129.1, 124.0, 118.7, 105.7, 84.5, 73.5, 61.0, 56.4, 46.6,
42.2, 36.6, 34.9, 29.6, 29.3, 28.1, 27.7, 26.6, 16.2; ESI-LR m/z 1379.8
(MNa+); ESI-HR calcd for C66H80N14O14S2Na 1379.5318, found
1379.5334 (MNa+). Data for 18: 1H NMR (270 MHz, CDCl3) d
8.34–8.29 (m, 4H, Ns), 8.26 (s, 1H, H-2), 8.05–7.98 (m, 4H, Ns),
6.63 (t, 1H, NsNH, J = 6.3 Hz), 6.42 (s, 4H, phenyl), 5.89 (br s, 4H,
6-NH2), 4.15 (s, 2H, PhCH2), 4.08 (s, 2H), 3.93 (t, 2H, J = 7.3 Hz),
3.81 (s, 9H, 3ꢀ OMe), 3.19 (t, 2H, J = 6.9 Hz), 3.02 (m, 2H,
J = 6.3 Hz), 1.88 (m, 2H), 1.81 (m, 2H), 1.62 (m, 2H), 1.40 (m, 2H),
1.21 (m, 8H); 13C NMR (125 MHz, CDCl3) d154.9, 153.8, 152.2,
151.5, 150.8, 150.1, 150.0, 146.5, 145.1, 137.5, 131.1, 129.0,
128.4, 124.5, 124.1, 118.7, 105.7, 84.5, 73.6, 61.0, 56.4, 46.7, 43.3,
42.1, 36.9, 34.9, 29.7, 28.9, 28.8, 28.2, 27.7, 26.4, 26.3, 16.1; FAB-
LR m/z 908.3 (MH+); FAB-HR calcd for C41H50N9O11S2 908.3071,
found 908.3068 (MH+).
23.2, 16.0: FABMS-LR m/z 875.3 [(Mꢁ2HCl)H+]: FABMS-HR calcd
for C46H59N12O6 875.4681, found 875.4683 [(Mꢁ2HCl)H+].
3.1.17. PU3-C8-dimer (6b)
Compound 6d (20 mg, 66%) was obtained from 15b (40 mg,
0.031 mmol) as described for the synthesis of 6a: 1H NMR
(500 MHz, D2O) d 8.24 (s, 2H, 2ꢀ H-2), 6.53 (s, 4H, 2ꢀ phenyl),
4.22 (s, 4H, 2ꢀ PhCH2), 4.19 (t, 4H, J = 7.4 Hz), 3.67 (s, 4H), 3.66
(s, 12H, 4ꢀ OMeꢀ), 3.60 (s, 6H, 2ꢀ OMe), 2.89 (t, 4H, J = 7.5 Hz),
2.13 (m, 4H), 1.68 (t, 4H, J = 7.4, 6.9 Hz), 1.40 (tt, 4H, J = 7.5,
8.0 Hz), 1.03 (m, 4H), 0.97 (m, 4H): 13C NMR (125 MHz, D2O) d
156.0, 153.5, 150.4, 149.8, 145.0, 136.8, 132.2, 117.7, 107.3, 88.7,
70.8, 61.5, 56.7, 46.9, 43.3, 37.1, 34.2, 28.7, 28.0, 26.3, 25.9, 16.0:
ESIMS-LR m/z 931.48 [(Mꢁ2HCl)H+]: ESIMS-HR calcd for
C
50H67N12O6 931.5307, found 931.5300 [(Mꢁ2HCl)H+].
3.1.18. PU3-C12-dimer (6c)
3.1.14. N,N0-Di-(4-nitrobenzenesulfonyl)-PU3-C16-dimer (15d)
Compound 15d (72 mg, 46%) was obtained from 13 (100 mg,
0.24 mmol), 4d (69 mg, 0.11 mmol) and CMBP (106 mg,
0.44 mmol) as described for the synthesis of 15a: 1H NMR
(400 MHz, CDCl3) d 8.26 (s, 2H, 2ꢀ H-2), 8.24 (d, 4H, 2ꢀ Ns,
J = 9.1 Hz), 7.96 (d, 4H, 2ꢀ Ns, J = 9.1 Hz), 6.39 (s, 4H, 2ꢀ phenyl),
6.17 (br s, 4H, 2ꢀ 6-NH2), 4.14 (s, 4H, 2ꢀ PhCH2), 4.07 (s, 4H),
3.95 (t, 4H, J = 7.3 Hz), 3.79 (s, 6H, 2ꢀ OMe), 3.77 (s, 12H, 4ꢀ
OMe), 3.15 (t, 4H, J = 7.2 Hz), 1.86 (t, 4H , J = 6.8 Hz), 1.58 (tt, 4H,
J = 7.3, 6.8 Hz), 1.49 (m, 4H, J = 7.2 Hz), 1.23 (m, 4H), 1.20 (m,
20H); 13C NMR (100 MHz, CDCl3) d 155.0, 153.7, 152.5, 151.4,
150.5, 150.0, 145.1, 137.4, 131.2, 129.0, 124.0, 118.6, 105.6, 84.4,
77.4, 73.4, 61.0, 56.4, 46.5, 42.1, 36.5, 34.8, 29.7, 29.6, 29.5, 29.2,
28.1, 27.5, 26.5, 16.1; ESIMS-LR m/z 1413.41 (MH+);
ESIMS-HR calcd for C70H89N14O14S2 1413.6124, found 1413.60925
(MH+).
Compound 6c (17 mg, 73%) was obtained from 15c (30 mg,
0.022 mmol) as described for the synthesis of 6a: 1H NMR
(500 MHz, D2O) d 8.25 (s, 2H, 2ꢀ H-2), 6.55 (s, 4H, 2ꢀ phenyl),
4.20 (s, 4H, 2ꢀ PhCH2), 4.18 (t, 4H, J = 7.5), 3.69 (s, 4H), 3.66 (s,
12H, 4ꢀ OMe), 3.60 (s, 6H, 2ꢀ OMe), 2.91 (t, 4H, J = 7.5 Hz), 2.17
(t, 4H, J = 7.4 Hz), 1.66 (tt, 4H, J = 7.4, 7.5 Hz), 1.42 (tt, 4H, J = 7.5,
6.9 Hz), 1.05 (tt, 4H, J = 6.9, 8.0 Hz), 0.89 (tt, 4H, J = 8.0, 7.5 Hz),
0.77 (m, 4H, J = 7.5 Hz), 0.67 (m, 4H): 13C NMR (125 MHz, D2O) d
155.9, 153.7, 150.5, 150.3, 145.6, 137.0, 132.3, 117.9, 107.3, 88.9,
61.6, 56.8, 46.7, 43.4, 37.0, 34.3, 29.6, 29.4, 29.1, 28.2, 26.5, 25.8,
16.1: ESIMS-LR m/z 987.58 [(Mꢁ2HCl)H+]: ESIMS-HR calcd for
C
54H75N12O6 987.5933, found 987.5935 [(Mꢁ2HCl)H+].
3.1.19. PU3-C16-dimer (6d)
Compound 6d (14 mg, 33%) was obtained from 15d (57 mg,
0.040 mmol) as described for the synthesis of 6a: 1H NMR
(500 MHz, D2O) d 8.36 (s, 2H, 2ꢀ H-2), 6.65 (s, 4H, 2ꢀ phenyl),
4.30 (s, 4H, 2ꢀ PhCH2), 4.29 (t, 4H, J = 8.0 Hz), 3.82 (s, 4H), 3.76
(s, 12H, 4ꢀ OMe), 3.69 (s, 6H, 2ꢀ OMe), 3.05 (t, 4H, J = 8.0 Hz),
2.28 (t, 4H, J = 6.9 Hz), 1.79 (tt, 4H, J = 8.0, 6.9 Hz), 1.57 (tt, 4H,
J = 8.0, 7.4 Hz), 1.20 (tt, 4H, J = 7.4, 6.9 Hz), 1.05 (tt, 4H, J = 8.0,
6.9 Hz), 0.95 (m, 4H, J = 8.0 Hz), 0.83–0.80 (m, 12H): 13C NMR
(125 MHz, D2O) d 156.0, 153.6, 150.3, 149.6, 144.7, 136.8, 132.1,
117.7, 107.2, 88.8, 70.9, 61.65 56.7, 46.4, 43.3, 36.8, 34.2, 29.6,
29.5, 29.4, 29.2, 28.7, 28.1, 26.3, 25.5, 16.0: ESIMS-LR m/z 1043.6
[(Mꢁ2HCl)H+]: ESIMS-HR calcd for C58H83N12O6 1043.6559, found
1043.6584 [(Mꢁ2HCl)H+].
3.1.15. N,N0-Di-(4-nitrobenzenesulfonyl)-PU3-C20-dimer (15e)
Compound 15e (30 mg, 19%) was obtained from 13 (100 mg,
0.24 mmol), 14e (75 mg, 0.11 mmol) and CMBP (84 mg,
0.35 mmol) as described for the synthesis of 15e: 1H NMR
(500 MHz, CDCl3) d 8.28 (s, 2H, 2ꢀ H-2), 8.25 (d, 4H, 2ꢀNs,
J = 8.6 Hz), 7.97 (d, 4H, 2ꢀ Ns, J = 8.6 Hz), 6.41 (s, 4H, 2ꢀ phenyl),
5.97 (br s, 4H, 2ꢀ 6-NH2), 4.16 (s, 4H, 2ꢀ PhCH2), 4.08 (s, 4H),
3.96 (t, 4H, J = 7.4 Hz), 3.80 (s, 6H, 2ꢀ OMe), 3.79 (s, 12H, 4ꢀ
OMe), 3.16 (t, 4H, J = 6.9 Hz), 1.87 (t, 4H, J = 6.9 Hz), 1.60 (tt, 4H,
J = 7.4, 6.9 Hz), 1.52 (m, 4H, J = 6.9 Hz), 1.25–1.22 (m, 32H); 13C
NMR (125 MHz, CDCl3) d 154.9, 153.7, 152.6, 151.5, 150.6, 150.0,
145.2, 137.4, 131.2, 129.0, 124.0, 118.6, 105.7, 84.4, 73.5, 61.0,
56.4, 46.6, 42.1, 36.6, 34.8, 29.8, 29.7, 29.6, 29.3, 28.1, 27.6, 26.6,
16.1; ESIMS-LR m/z 1491.65 (MNa+); ESIMS-HR calcd for
3.1.20. PU3-C20-dimer (6e)
Compound 6e (16 mg, 76%) was obtained from 15e (27 mg,
0.018 mmol) as described for the synthesis of 6a: 1H NMR
(500 MHz, D2O) d 8.34 (s, 2H, 2ꢀ H-2), 6.62 (s, 4H, 2ꢀ phenyl),
4.26 (m, 8H), 3.81 (s, 4H), 3.72 (s, 12H, 4ꢀ OMe), 3.61 (s, 6H, 2ꢀ
OMe), 3.04 (t, 4H, J = 7.5 Hz), 2.22 (m, 4H), 1.71 (m, 4H,
J = 6.9 Hz), 1.61 (m, 4H, J = 7.5 Hz), 1.22 (m, 4H), 1.14 (m, 4H),
1.06 (m, 4H), 0.98 (m, 24H); 13C NMR (125 MHz, D2O) d 155.8,
153.6, 150.3, 150.0, 144.7, 136.8, 132.0, 117.6, 107.1, 88.8, 70.8,
61.2, 56.6, 46.3, 43.3, 36.8, 34.3, 30.0, 29.8, 29.6, 29.2, 28.1, 26.6,
25.7, 16.1; ESIMS-LR m/z 1099.7 [(Mꢁ2HCl)H+]; ESIMS-HR calcd
for C62H91N12O6 1099.7185, found 1099.7148 [(Mꢁ2HCl)H+].
C
74H96N14O14S2Na 1491.6570, found 1491.6570 (MNa+).
3.1.16. PU3-C4-dimer (6a)
Thiophenol (17 lL, 0.17 mmol) was added to a mixture of 15a
(35 mg, 0.17 mmol) and K2CO3 (20 mg, 0.15 mmol) in DMF
(0.30 mL), and the mixture was stirred at room temperature for
6 h. The mixture was directly applied to a silica gel column (NH sil-
ica, 1 ꢀ 7 cm, 2% MeOH/CHCl3). Appropriate fractions were evapo-
rated, and the residue was further purified by C18 reverse phase
column chromatography (1 ꢀ 5 cm, 1 N aqueous HCl/MeOH = 1:1).
Appropriate fractions were evaporated, and the residue was lyoph-
ilized to give 6a (9 mg, 34%) as dihydrochloride salt: 1H NMR
(500 MHz, D2O) d 8.22 (s, 2H, 2ꢀ H-2), 6.53 (s, 4H, 2ꢀ phenyl),
4.16 (s, 4H, 2ꢀ PhCH2), 4.13 (t, 4H, J = 7.4 Hz), 3.69 (s, 4H), 3.66
(s, 12H, 4ꢀ OMe), 3.59 (s, 6H, 2ꢀ OMe), 2.95 (m, 4H), 2.08 (t, 4H,
J = 6.9 Hz), 1.60 (tt, 4H, J = 6.9, 7.4 Hz), 1.57 (m, 4H): 13C NMR
(125 MHz, D2O) d 155.2, 153.6, 151.1, 150.5, 146.9, 136.8, 132.5,
117.8, 107.1, 90.6, 76.2, 61.8, 56.7, 46.1, 43.2, 37.2, 34.2, 28.0,
3.1.21. PU3-C12-monomer (19)
Compound 19 was (22 mg, 43%) was obtained from 18 (75 mg,
0.077 mmol) as described for the synthesis of 6a: 1H NMR
(500 MHz, D2O) d 8.39 (s, 1H, H-2), 6.71 (s, 2H, phenyl), 4.35 (m,
2H, PhCH2), 4.32 (t, 2H, J = 7.4 Hz), 3.80 (s, 6H, 2ꢀ OMe), 3.80 (s,
2H), 3.74 (s, 3H, OMe), 3.04 (t, 2H, J = 7.4 Hz), 2.95 (t, 2H,
J = 7.4 Hz), 2.30 (t, 2H, J = 6.8 Hz), 1.71 (tt, 2H, J = 7.4, 6.8 Hz),
1.57 (m, 4H), 1.25 (m, 2H), 1.19 (m, 4H), 1.06–0.94 (m, 10H); 13C

K. Muranaka et al. / Bioorg. Med. Chem. 16 (2008) 5862–5870
5869
NMR (125 MHz, D2O) d 156.0, 153.6, 150.4, 149.7, 136.9, 132.2,
Acknowledgments
117.7, 107.2, 88.7, 61.5, 56.7, 46.6, 43.3, 40.1, 36.9, 34.2, 29.4,
29.3, 29.2, 29.0, 28.9, 28.1, 27.4, 26.4, 25.7, 16.0; ESIMS-LR m/z
594.4 [(Mꢁ2HCl)H+]; ESIMS-HR calcd for C33H52N7O3 594.4132,
found 594.4130 [(Mꢁ2HCl)H+].
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.
3.1.22. GMD-C4-dimer (7a)
A mixture of geldanamycin (6.0 mg, 0.011 mmol) and 1,4-diam-
inobutane (0.1 M solution in DMF; 50 lL, 5.0 lmol) in DMF
(0.3 mL) was stirred at room temperature for 14 h. The solvent
was removed in vacuo. The residue was purified by silica gel col-
umn chromatography (0.5 ꢀ 6.0 cm, 33% acetone/CH2Cl2) to give
7a (5.7 mg, 98%) as a purple solid: 1H NMR (500 MHz, CDCl3) d
9.15 (br s, 2H), 7.29 (s, 2H), 6.95 (d, 2H, J = 12.5 Hz), 6.58 (dd, 2H,
J = 12.5, 11.5 Hz), 6.24 (t, 2H, J = 5.7 Hz), 5.90 (m, 2H, J = 8.6 Hz),
5.86 (m, 2H, J = 11.5, 10.3 Hz), 5.19 (s, 2H), 5.00–4.50 (br s, 4H),
4.31 (d, 2H, J = 10.3 Hz), 4.20 (br s, 2H), 3.79–3.54 (m, 6H,
J = 7.4 Hz), 3.45 (d, 2H, J = 8.6 Hz), 3.36 (s, 6H), 3.27 (s, 6H), 2.79–
2.69 (m, 4H, J = 13.7 Hz), 2.37 (dd, 2H, J = 13.7, 10.9 Hz), 2.02 (s,
6H), 1.79 (br s, 14H), 1.72 (m, 2H), 0.99 (d, 6H, J = 7.4 Hz), 0.96
(d, 6H, J = 6.9 Hz); 13C NMR (125 MHz, CDCl3) d 184.0, 181.1,
168.5, 156.2, 144.7, 141.4, 136.1, 135.1, 133.8, 132.9, 127.1,
126.7, 109.0, 109.0, 81.8, 81.6, 81.3, 72.8, 69.7, 57.3, 56.9, 53.9,
45.4, 35.2, 34.6, 32.5, 31.9, 29.4, 28.8, 27.3, 23.1, 13.0, 12.8, 12.6,
0.1; ESIMS-LR m/z 1167.6 (MNa+); ESIMS-HR calcd for
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2008.04.070.
References and notes
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C
60H84N6O16Na 1167.5842, found 1167.5817 (MNa+).
3.1.23. GMD-C12-dimer (7b)
13. Delmotte, P.; Delmotte-Plaque, J. Nature 1953, 171, 344.
Compound 7b (6.0 mg, 95%) was obtained after purification by
silica gel column chromatography (0.5 ꢀ 6.0 cm, 25% acetone/
CH2Cl2) as a purple solid as described for the synthesis of 7a:
1H NMR (500 MHz, CDCl3) d 9.19 (br s, 2H), 7.27 (s, 2H), 6.95
(d, 2H, J = 11.5 Hz), 6.58 (dd, 2H, J = 11.5, 10.9 Hz), 6.30 (t, 2H,
J = 5.2 Hz), 5.92 (d, 2H, J = 9.8 Hz), 5.86 (dd, 2H, J = 10.9,
10.3 Hz), 5.19 (s, 2H), 5.00–4.60 (br s, 4H), 4.39 (m, 2H), 4.29
(d, 2H, J = 10.3 Hz), 3.50 (m, 4H), 3.44 (m, 4H), 3.36 (s, 6H), 3.27
(s, 6H), 2.73 (m, 2H, J = 9.8, 7.4 Hz), 2.65 (d, 2H, J = 13.2 Hz),
2.43 (dd, 2H, J = 13.2, 10.9 Hz), 2.02 (s, 6H), 1.80 (m, 10H),
1.78–1.65 (m, 6H), 1.40 (m, 6H), 1.30–1.25 (m, 10H), 0.99 (d,
6H, J = 7.4 Hz), 0.96 (d, 6H, J = 6.9 Hz); 13C NMR (100 MHz, CDCl3)
d 184.1, 180.7, 168.6, 156.2, 145.0, 141.6, 135.9, 135.1, 133.9,
132.8, 127.1, 126.7, 108.8, 108.4, 81.8, 81.6, 81.3, 72.8, 69.7,
57.3, 56.9, 53.9, 46.0, 35.2, 34.6, 32.5, 31.9, 29.9, 29.8, 29.6,
29.5, 29.4, 29.3, 28.6, 26.9, 23.1, 12.9, 12.8, 12.5, 0.1; ESIMS-LR
m/z 1279.7 (MNa+); ESIMS-HR calcd for C68H100N6O16Na
1279.7094, found 1279.7069 (MNa+).
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3.1.24. GMD-C16-dimer (7c)
Compound 7c (5.5 mg, 84%) was obtained after purification by
silica gel column chromatography (0.5 ꢀ 10 cm, 25% acetone/
CH2Cl2) as a purple solid as described for the synthesis of 7a: 1H
NMR (400 MHz, CDCl3) d 9.19 (br s, 2H), 7.27 (s, 2H), 6.95 (d, 2H,
J = 11.3 Hz), 6.58 (dd, 2H, J = 11.3, 10.9 Hz), 6.30 (t, 2H, J = 5.5 Hz),
5.90 (d, 2H, J = 9.5 Hz), 5.86 (dd, 2H, J = 10.9, 9.5 Hz), 5.19 (s, 2H),
5.00–4.60 (br s, 4H), 4.40 (br s, 2H), 4.29 (d, 2H, J = 9.5 Hz), 3.55
(m, 4H), 3.44 (m, 4H), 3.36 (s, 6H), 3.26 (s, 6H), 2.73 (m, 2H,
J = 9.5, 7.3 Hz), 2.66 (d, 2H, J = 13.2 Hz), 2.43 (dd, 2H, J = 13.2,
10.9 Hz), 2.02 (s, 6H), 1.79 (m, 10H), 1.72 (m, 6H), 1.40–1.26 (m,
24H), 0.99 (d, 6H, J = 7.3 Hz), 0.96 (d, 6H, J = 6.4 Hz); 13C NMR
(100 MHz, CDCl3) d 184.1, 180.7, 168.6, 156.2, 145.0, 141.6,
135.9, 135.1, 134.0, 132.9, 127.0, 126.7, 108.8, 108.4, 81.8, 81.6,
81.4, 72.7, 69.7, 57.3, 56.9, 53.9, 46.0, 41.1, 35.2, 34.6, 32.4, 31.9,
29.9, 29.8, 29.8, 29.7, 29.6, 29.4, 29.3, 28.6, 26.9, 23.1, 12.9, 12.8,
12.5, 0.1; ESIMS-LR m/z 1335.8 (MNa+); ESIMS-HR calcd for
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H.; Fink, A.; Hayes, A.; Howes, R.; Hubbard, R. E.; James, K.; Jordan, A. M.;
Lockie, A.; Martins, V.; Massey, A.; Matthews, T. P.; McDonald, E.; Northfield, C.
J.; Pearl, L. H.; Prodromou, C.; Ray, S.; Raynaud, F. I.; Roughley, S. D.; Sharp, S. Y.;
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2cg9.
C
72H108N6O16Na 1335.7720, found 1335.7705 (MNa+).

5870
K. Muranaka et al. / Bioorg. Med. Chem. 16 (2008) 5862–5870
24. Wright, L.; Barril, X.; Dymock, B.; Sheridan, L.; Surgenor, A.; Beswick, M.;
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785. PDB ID; 1uy6.
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