(S)-Stereoisomer of telomestatin as a potent G-quadruplex binder and telomerase inhibitor

Article abstract of DOI:10.1039/c0ob00513d

Total synthesis of the (S)-stereoisomer of telomestatin (1) was accomplished. (S)-Telomestatin exhibited potency four times that of the natural product, (R)-telomestatin, which was the most potent telomerase inhibitor previously reported. In the circular dichroism spectral analysis of the complexes possessing randomly structured single-stranded d[TTAGGG]₄ oligonucleotide, (S)-telomestatin, like (R)-telomestatin, induced an antiparallel G-quadruplex structure. The melting temperature (T_m) value of the (S)-isomer complex was greater than that of the (R)-telomestatin complex. Therefore, it is concluded that the stereochemistry of the thiazoline of telomestatin is important to the binding ability of a G-quadruplex binder, and (S)-telomestatin as a G-quadruplex binder is more potent than the natural product.

Full text of DOI:10.1039/c0ob00513d

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PAPER
(S)-Stereoisomer of telomestatin as a potent G-quadruplex binder and
telomerase inhibitor†
Takayuki Doi,*a Kazuaki Shibata,b Masahito Yoshida,a Motoki Takagi,c Masayuki Tera,d Kazuo Nagasawa,d
Kazuo Shin-yae and Takashi Takahashi*b
Received 29th July 2010, Accepted 6th September 2010
DOI: 10.1039/c0ob00513d
Total synthesis of the (S)-stereoisomer of telomestatin (1) was accomplished. (S)-Telomestatin
exhibited potency four times that of the natural product, (R)-telomestatin, which was the most potent
telomerase inhibitor previously reported. In the circular dichroism spectral analysis of the complexes
possessing randomly structured single-stranded d[TTAGGG]4 oligonucleotide, (S)-telomestatin, like
(R)-telomestatin, induced an antiparallel G-quadruplex structure. The melting temperature (Tm) value
of the (S)-isomer complex was greater than that of the (R)-telomestatin complex. Therefore, it is
concluded that the stereochemistry of the thiazoline of telomestatin is important to the binding ability
of a G-quadruplex binder, and (S)-telomestatin as a G-quadruplex binder is more potent than the
natural product.
quadruplex binding ability than hexaoxazole derivatives.6 There-
fore, it is important that the macrocyclic structure of telomestatin
comprises seven continuous oxazole rings. We also reported the
total synthesis of telomestatin (R)-1 and determined the absolute
Introduction
Telomestatin (R)-1, isolated from Streptomyces anulatus 3533-
SV4, possesses a unique structure comprising eight continuous
configuration of the natural product.7,8 Next we investigated the
effect of the stereocentre of the thiazoline in telomestatin on the
binding ability to the G-quadruplex structure of the telomere. We
synthesized the (S)-stereoisomer of telomestatin, and based on
the melting temperature (Tm) values of ss-telo24 G-quadruplex
structure and a telomeric repeat-amplification protocol (TRAP)
assay, we observed that the enantiomer is more potent than
the natural product (Fig. 1). During total synthesis of the (S)-
stereoisomer, significant epimerization of the modified cysteine
residue was observed in the macrolactamization, though the
stereogenic center and the activated ester moiety are connected
through conjugation by a trisoxazole unit.
heterocyclic rings, such as a thiazoline, two methyloxazoles,
and five oxazoles in a macrocycle, and it exhibits extremely
potent telomerase inhibitory activity.1 Telomestatin binds itself
to the G-quadruplex basket structure of telomere, composed of
repeated (TTAGGG)n sequences, to stabilize the antiparallel G-
quadruplex form,2 inhibiting the telomerase that elongates telom-
ere sequences.3 Since most cancer cells rely on telomerase for their
survival, it is an attractive target in anticancer drug design.4 Studies
on G-quadruplex binding molecules as telomerase inhibitors re-
vealed that they exhibited potential anticancer activity.4,5 Recently,
we observed that a macrocyclic heptaoxazole comprising an amino
group side chain selectively interacted with telomere to induce a
conformational change to an antiparallel G-quadruplex structure,
and a more planar heptaoxazole derivative exhibits more potent G-
aGraduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-
aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan. E-mail: doi_taka@
mail.pharm.tohoku.ac.jp; Fax: +81-22-795-6864; Tel: +81-22-795-6865
bDepartment of Applied chemistry, Tokyo Institute of Technology, 2-12-1
Ookayama, Meguro, Tokyo, 152-8552, Japan
cJapan Biological Informatics Consortium (JBIC), 2-4-7 Aomi, Koto-ku,
Tokyo, 135-0064, Japan
dDepartment of Biotechnology and Life Science, Faculty of Technology,
Tokyo University of Agriculture and Technology (TUAT), 2-24-16 Naka-
machi, Koganei, Tokyo, 184-8588, Japan
Fig. 1 Structures of telomestatin (R)-1 and its enantiomer (S)-1.
eNational Institute of Advanced Industrial Science and Technology (AIST),
2-4-7 Aomi, Koto-ku, Tokyo, 135-0064, Japan
Results and discussion
† Electronic supplementary information (ESI) available: 1H and 13C
NMR spectra of (2S,14S)–7, (2S,14S)–6, (2S,14R)–5, (S)–8, 1H–1H
COSY spectrum of (S)–8, and 1H NMR spectrum of (S)–1. See DOI:
10.1039/c0ob00513d
In our previous synthesis of (R)-1,7 we coupled (R)-2 and (S)-
3 using PyBroP9 to obtain (2R,14S)-4 (Scheme 1). Removal
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Scheme 2 Macrolactamization of (2S,14S)-7.
that the significant epimerization occurred at the C2 position
during the macrolactamization. In the cyclization of intermediate
7A produced from (2S,14S)-7 (Scheme 3), the reaction proceeded
slowly by conformational restriction and then the epimerization
at the C2 position where the proton is rather acidic by conjugation
with the activated ester formed at the C-terminus through a
trisoxazole unit, would proceed leading to (2R,14S)-5. It should
be noted that there was a significant difference in the reactivity
between the cyclization intermediates 4A and 7A, whose single
Scheme 1 Macrolactamization of (2R,14S)-4 in the synthesis of (R)-1.
of the acetonide and Boc groups with acid, basic hydro-
lysis of the methyl ester, and macrolactamization using
DPPA,10 1-hydroxybenzotriazole (HOBt), N,N-diisopropylethyl-
amine (DIEA), and 4-(dimethylamino)pyridine (DMAP) pro-
ceeded smoothly, leading to the desired (2R,14S)-5 in 48%
yield. In the reaction, we observed the formation of a small
amount of its diastereomer, (2S,14S)-6. The ratio of (2R,14S)-
5 to (2S,14S)-6 was 90 : 10, as determined by high-performance
liquid chromatography (HPLC) peak area (UV 214 nm).
Initially, we attempted to synthesize (S)-1 utilizing (S)-2 instead
of (R)-2 as the coupling unit. Acylation of (S)-2 with (S)-3 using
PyBroP provided (2S,14S)-7 in 86% yield. The acetonide and Boc
groups were removed by treatment with acid, and methyl ester
was hydrolyzed under basic conditions (Scheme 2). However, the
macrolactamization did not proceed smoothly, and the product
obtained in 3 d was a 72 : 28 mixture of diastereomers (2S,14S)-
6 and (2R,14S)-5 in 19% combined yield. As epimerization was
not observed either before or after the cyclization reaction, the
epimerization should proceed during the macrolactamization.11
To identify which stereocentre was epimerized, we measured the
HPLC-circular dichroism (CD) of the cyclized products obtained
from both (2R,14S)-4 and (2S,14S)-7 (Schemes 1 and 2). The for-
mer products (Scheme 1) included 5 at retention time (tR) 16.3 min
and 6 at tR 19.2 min in a ratio of 90 : 10 (UV 214 nm). The major
product obtained was (2R,14S)-5, which exhibited a (-) CD signal
(UV 254 nm).12 In the HPLC-CD analysis of the products obtained
from (2S,14S)-7 (Scheme 2), the same peaks were observed in a
ratio of 28 : 72 (UV 214 nm) for 5 and 6 described above, and
exhibited (-) CD signals, respectively (UV 254 nm). As both peaks
for 5 at tR 16.3 min exhibited (-) CD signals, the stereochemistry
of the minor diastereomer 5 obtained from (2S,14S)-7 (Scheme 2)
was identical with (2R,14S)-5 in Scheme 1. As a result, it was found
Scheme 3 Process of the epimerization at the C2 position in the
macrolactamization.
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Table 1 Optimization of macrolactamization of (2S,14R)-4a
Entry
Reagents
Additive
Concentration
Yield (%)
Ratio of (2S,14R)-5 : (2R,14R)-6
1
2
3
4
5
6
DPPA–HOBt
DPPA–HOBt
HATU
PyBOP–HOBt
DPPA–HOBt
DPPA–HOBt
DMAP
DMAP
DMAP
DMAP
—
3 mM
1 mM
3 mM
3 mM
3 mM
3 mM
48
35
32
16
trace
68
90 : 10
88 : 12
86 : 14
84 : 16
—
DMAPO
93 : 7
a The reaction was performed in CH2Cl2–DMF (1 : 1) at room temperature for 3 d.
stereocentres are of differing absolute configurations. Therefore,
According to our previous report, the seventh oxazole ring was
we altered the synthetic route in the synthesis of (S)-1.
formed from (2S,14R)-5 to provide (S)-8, which underwent thia-
zoline formation leading to (S)-1 (Tf2O/Ph3PO/anisole/CH2Cl2)
(Scheme 5).7,16 (S)-1 was isolated by reversed-phase HPLC and its
1H NMR spectrum was measured by LC-NMR in solution right
after purification because it formed complexes after concentration,
probably due to intermolecular interaction on the basis of p–p
stacking of the oxazole rings. The spectral data of (S)-1 were
identical to those of the natural product (R)-1, except for the
opposite signal of its CD spectrum (Fig. 2).
The cyclization precursor (2S,14R)-4 (Scheme 4) was prepared
from the coupling of (S)-2 and acid (R)-3, the same as its enan-
tiomer (2R,14S)-4.7 After removal of the acetonide and Boc groups
and hydrolysis of the methyl ester, the macrolactamization of
(2S,14R)-4 was investigated (Table 1). Higher dilution conditions
(1 mM) were not effective in improving the yield and suppressing
the epimerization (entry 2 vs. entry 1). The use of HATU13 or
PyBOP14–HOBt instead of DPPA–HOBt resulted in a lower yield
and considerable epimerization (14–16%) was observed (entries
3 and 4). The reaction did not proceed in the absence of DMAP
(entry 5). Interestingly, the addition of 4-(dimethylamino)pyridine
oxide (DMAPO) (1 equiv) significantly increased the yield to
68% and suppressed the epimerization to 7% (entry 6). It has
been reported that the use of 2-methyl-6-nitrobenzoic anhydride
(MNBA)–DMAPO instead of MNBA–DMAP is quite effective
in both macrolactonization and peptide coupling.15 Our results
represent that the addition of DMAPO is also effective for
activation of the HOBt ester in the macrolactamization via
formation of the intermediate 4B. The 4B is enough active for
macrolactamization and the proton at the C2 position in 4B could
be less acidic than that in 4A.
Scheme 5 Total synthesis of (S)-1.
Because synthetic intermediates (S)-8 and (R)-87 as well as
telomestatin (S)-1 and (R)-17 were available to us, their inhibitory
activities against telomerase were evaluated by a modified TRAP
assay with an internal standard.1,17,18 Interestingly, (S)-1 exhibited
potent inhibitory activity with an IC50 value of 5 nM while the IC50
value of (R)-1 was 20 nM (Table 2). The synthetic intermediate (S)-
8 is also equally as potent as the natural product (R)-1, whereas
the potency of (R)-8 decreased to one-fourth of that of (R)-1.
The results are in good agreement with our previous observation
that the compounds having seven continuous oxazole rings in a
Table 2 Inhibitory activity of synthetic compounds against telomerase
Compound
IC50 (nM)
(S)-1
(R)-1
(S)-8
(R)-8
5
20
20
80
Scheme 4 Macrolactamization of (2S,14R)-4.
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Fig. 2 CD spectra of telomestatins, natural product (orange), synthetic (R)-1 (green), and (S)-1 (blue).
macrocycle possess extremely potent inhibitory activity against
telomerase.6 Interestingly, (S)-stereoisomers 1 and 8 are more
potent than the corresponding (R)-isomers; thus, suggesting that
the stereochemistry at the C2 position of telomestatin probably
controls the shape of the molecule binding to the G-quadruplex
structure of telomere sequences.
Next, we carried out CD analyses using randomly structured,
single-stranded d[TTAGGG]4 24-mer (ss-telo24) oligonucleotide
to investigate the G-quadruplex structure induced by (S)-1 and
(R)-1. As previously reported, ss-telo24 was induced into an
antiparallel G-quadruplex structure with (R)-1 on the basis of CD
spectra analysis, including 292 nm positive and 262 nm negative
signals (Fig. 3(b)).2c,6 In the presence of (S)-1, ss-telo24 was again
induced into an antiparallel G-quadruplex structure (Fig. 3(a)).
Therefore, it was revealed that antiparallel G-quadruplex structure
was induced by telomestatin regardless of the stereochemistry of
the thiazoline.
The stability of the antiparallel G-quadruplex structure induced
by telomestatin was investigated using the Tm values obtained
from the CD melting curves at 292 nm molar ellipticity intensity
(Fig. 4).6 The Tm values of the complexes with (S)-1 and (R)-1
were observed to be 62.3 and 56.3 ◦C, respectively. Thus, (S)-
1 stabilizes the telomeric G-quadruplex structure more potently
than (R)-1 does, and this is coincident with the results described
in the TRAP assay.
Fig. 3 CD spectra of ss-telo24 (10 mM) in Tris-HCl buffer (50 mM, pH
7.6) with 1 (10–50 mM). (a) (S)-1; (b) (R)-1.
Conclusion
of a telomestatin derivative to the G-quadruplex telomere by
molecular modeling and by NMR analysis is underway in our
laboratory.
We synthesized the (S)-stereoisomer of telomestatin by over-
coming rare epimerization at the C2 position in the macrolac-
tamization; as compared to the natural product, the (S)-isomer
exhibited four-fold potent inhibitory activity against telomerase.
Comparison of the Tm value of ss-telo24 and changes in the CD
spectra in the presence of both enantiomers also suggested that
the (S)-isomer is a stronger G-quadruplex binder for telomere
than the natural product, telomestatin, which is the most potent
compound reported to date. Further study of the binding structure
Experimental section
General procedures
NMR spectra were recorded on a JEOL Model ECP-400
(400 MHz for 1H, 100 MHz for 13C) or a Varian 500 MHz NMR
390 | Org. Biomol. Chem., 2011, 9, 387–393
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was washed with 3 M HCl, saturated aqueous NaHCO3 and brine,
dried over MgSO4 and filtered. After removal of the solvent, the
residue was purified by column chromatography on silica gel eluted
with 0–30% ethyl acetate in CHCl3 to give bis-trisoxazole amide 4
or 7.
Bis-trisoxazole amide (2S,14S)-7. Pale yellow oil. [a]1D8 -50.0
(c 1.08, CHCl3). 1H NMR (400 MHz, DMSO-d6, 80 ◦C) d 8.91 (s,
1H), 8.86 (s, 1H), 8.84 (s, 1H), 8.74 (d, J = 8.22 Hz, 1H), 8.73 (s,
1H), 5.38 (dt, J = 8.22, 7.25 Hz, 1H), 5.16 (dd, J = 6.77, 2.90 Hz,
1H), 4.31 (dd, J = 9.18, 6.77 Hz, 1H), 4.09 (dd, J = 9.18, 2.90 Hz,
1H), 3.87 (s, 3H), 3.29 (dd, J = 13.5, 7.25 Hz, 1H), 3.23 (dd, J =
13.5, 7.25 Hz, 1H), 2.74 (s, 3H), 2.68 (s, 3H), 1.67 (s, 3H), 1.55 (s,
◦
3H), 1.33 (brs, 18H). 13C NMR (100 MHz, DMSO-d6, 80 C) d
164.1, 160.9, 160.5, 159.0, 155.5, 155.3, 154.0, 153.9, 150.4, 150.3,
144.4, 142.1, 140.4, 140.3, 136.2, 133.0, 129.7, 128.8, 124.5, 123.6,
93.8, 78.7, 66.4, 54.2, 51.3, 47.4, 42.0, 30.4, 30.2, 27.5, 10.9, 10.8.
FT-IR (neat) 3302, 3139, 2976, 1708, 1658, 1367, 1160, 1098, 757,
725 cm-1. HRMS (ESI-TOF) calcd. for C39H44N8O12SNa [M+Na]+
871.2692 found 871.2699.
Fig. 4 CD melting curves of ss-telo24 at 292 nm in the presence of 5
equivalents of telomestatin, (S)-1; ꢀ (R)-1.
᭹
instrument in the indicated solvent. Chemical shifts are reported
in units parts per million (ppm) relative to the residual proton
solvent signal for internal tetramethylsilane (7.25 ppm for 1H) for
solutions in CDCl3. NMR spectral data are reported as follows:
chloroform (7.26 ppm for 1H) or chloroform-d (77.1 ppm for
13C), dichloromethane (5.30 ppm for 1H) or dichloromethane-
Bis-trisoxazole amide (2S,14R)-4. Yellow solid. mp (ethyl
◦
acetate) 175–177 C. [a]2D0 +20.6 (c 1.29, CHCl3). 1H NMR
(400 MHz, DMSO-d6, 80 ◦C) d 8.91 (s, 1H), 8.85 (s, 1H), 8.82
(s, 1H), 8.75 (d, J = 7.24 Hz, 1H), 8.73 (s, 1H), 5.37 (dt, J = 7.73,
7.24 Hz, 1H), 5.15 (dd, J = 6.52, 2.41 Hz, 1H), 4.31 (dd, J = 9.42,
6.52 Hz, 1H), 4.08 (dd, J = 9.42, 2.41 Hz, 1H), 3.86 (s, 3H), 3.29
(dd, J = 13.5, 7.73 Hz, 1H), 3.23 (dd, J = 13.5, 7.73 Hz, 1H), 2.73
(s, 3H), 2.67 (s, 3H), 1.66 (s, 3H), 1.54 (s, 3H), 1.32 (brs, 18H).
13C NMR (100 MHz, DMSO-d6, 80 ◦C) d 164.1, 160.9, 160.5,
159.1, 155.5, 155.3, 154.0, 153.9, 150.4, 150.3, 144.4, 142.1, 140.4,
140.2, 136.2, 133.0, 129.7, 128.8, 124.5, 123.6, 93.8, 78.7, 66.4, 54.2,
51.3, 47.4, 42.1, 30.4, 30.2, 27.5, 10.9, 10.8. FT-IR (solid) 3304,
3137, 2975, 1705, 1657, 1365, 1154, 1097, 755, 724 cm-1. HRMS
(ESI-TOF) calcd. for C39H44N8O12SNa [M+Na]+ 871.2692 found
871.2675. Anal. calcd. for C39H44N8O12S: C, 55.18; H, 5.22; N,
13.20, found: C, 55.32; H, 5.42; N, 13.08.
1
d2 (53.8 ppm for 13C), DMSO (2.50 ppm for H) or DMSO-d6
(39.5 ppm for 13C), CH3CN (1.93 ppm for 1H) when internal
standard is not indicated. Multiplicities are reported by the
following abbreviations: s; singlet, d; doublet, t; triplet, q; quartet,
m; multiplet, br.; broad, J; coupling constants in hertz. IR spectra
were recorded on a Perkin Elmer Spectrum One FT-IR spec-
trophotometer. Only the strongest and/or structurally important
absorption is reported as the IR data given in cm-1. Optical
rotations were measured with a JASCO P-1020 polarimeter in
a 0.3 cm f cell with a pathlength of 5 cm. The [a]D values are
given in 10-1 deg cm2 g-1 and the concentrations are given in 100
cm-3. All reactions were monitored by thin-layer chromatography
carried out on 0.2 mm E. Merck silica gel plates (60F-254)
with UV light, visualized by p-anisaldehyde solution, cerium
sulfate or 10% ethanolic phosphomolybdic acid. Merck silica
gel 60 (0.063–0.200 mm) was used for column chromatography.
Mass spectra were obtained on Waters LCT PremierTM XE with
leucine enkephalin (SIGMA) as an external standard. Preparative
reversed-phase HPLC (UV 254 nm) was performed on a Gilson
215 system with Senshu Pak PEGASIL-ODS, 5 mm, 20 ¥ 250 mm
eluted with CH3CN–H2O–CF3CO2H (70 : 30 : 0.1). CD spectra
were recorded on a JASCO-810 spectropolarimeter using a quartz
cell of 1 mm optical path length and an instrument scanning speed
of 100 nm min-1 with a response time of 1 s, and over a wavelength
range of 200–320 nm. The ss-telo24 nucleotide was purchased
from Sigma Genosys and was dissolved in doubly-distilled water
to be used without further purification.
General procedure for the macrocyclization
To a solution of bis-trisoxazole amide 4 or 7 in 1,4-dioxane (10 mL
mmol-1) was added 4 M HCl/1,4-dioxane (60 mL mmol-1) at
0 ◦C. After being stirred at room temperature for 2 h, the reaction
mixture was concentrated in vacuo. The residue was used for the
next reaction without further purification.
To a solution of the residue in MeOH (50 mL mmol-1), THF
(50 mL mmol-1) and H2O (16 mL mmol-1) was added LiOH·H2O
(0.1 M) at 0 ◦C, and then warmed up to room temperature.
After being stirred at the same temperature for 2 h, the reaction
mixture was quenched with 4 M HCl/1,4-dioxane at 0 ◦C, and
concentrated in vacuo. The residue was dried under reduced
pressure for 12 h and used for the next reaction without further
purification.
General procedure for the synthesis of bis-trisoxazole amide
To a solution of the HCl salt of the amine 27 in CH2Cl2
(10 mL mmol-1) was added a solution of the trisoxazole carboxylic
acid 37 in DMF (10 mL mmol-1) and DIEA (9 equiv.) at room
temperature. To the reaction mixture was added PyBroP (1.5
equiv.) at 0 ◦C. After being stirred at room temperature for 2 h, the
reaction mixture was poured into 1 M HCl at 0 ◦C and the aqueous
layer was extracted with ethyl acetate. The combined organic layer
To a solution of a condensing agent (10 equiv., DPPA/HOBT,
HATU or PyBOP/HOBt), DIEA (15 equiv.) and an additive (1
equiv., DMAP or DMAPO) in CH2Cl2 (160 mL mmol-1) and
DMF (80 mL mmol-1) was added dropwise a solution of the
above residue in DMF (80 mL mmol-1) at room temperature under
argon. After being stirred at room temperature for 3 d, the reaction
mixture was poured into 3 M HCl at 0 ◦C and the aqueous layer
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was extracted with ethyl acetate. The combined organic layer was
washed with 3 M HCl, saturated aqueous NaHCO3 and brine,
dried over MgSO4 and filtered. After removal of the solvent, the
residue was purified by column chromatography on silica gel eluted
with 0–5% MeOH in CHCl3 to give macrocyclic lactams 5 and 6.
extract from namalva cells. The reaction buffer contained 20 mM
Tris·HCl (pH 8.3), 63 mM KCl, 1.5 mM MgCl2, 1 mM EGTA,
0.005% Tween 20, 0.01 mg mL-1 T4 gene 32 protein and 0.1 mg
mL-1 BSA supplemented with 50 mM dNTP and 0.3 mM Ts primer.
Reactions were stopped by heating for 2 min at 94 ◦C, and the
samples were placed on ice. The amplification step was done on
ice by adding to the samples 5 mL of PCR solution containing
0.3 mM CX primer and 1.4 units Taq polymerase. The PCRs were
then performed for amplification as follows: 94 ◦C for 2 min and
28 cycles consisting 94 ◦C for 30 s, 50 ◦C for 30 s and 72 ◦C for 60 s.
Telomerase extension products were analyzed by polyacrylamide
gel electrophoresis in 1X Tris-borate-EDTA buffer and the 10%
acrylamide gels were scanned with a Typhoon (GE Healthcare
Bio-Sciences). The extension products were quantified by using
ImageQuant 5.2 software and normalized to the signal of the
extension products in control solvent.
Macrocyclic lactam (2S,14S)-6. White solid. mp (CH2Cl2)
◦
>300 C (decomposed). [a]2D4 -36.1 (c 0.205, CHCl3). H NMR
(400 MHz, CD2Cl2) d 8.49 (d, J = 4.83 Hz, 1H), 8.41 (d, J =
6.28 Hz, 1H), 8.32 (s, 1H), 8.28 (s, 1H), 8.25 (s, 1H), 8.23 (s, 1H),
5.41 (m, 1H), 5.33 (m, 1H), 4.27 (m, 1H), 4.00 (m, 1H), 3.33 (dd,
J = 13.0, 5.32 Hz, 1H), 3.17 (dd, J = 13.0, 3.38 Hz, 1H), 2.73 (s, 3H),
2.68 (s, 3H), 1.21 (s, 9H). 13C NMR (100 MHz, CD2Cl2) d 162.7,
161.6, 161.2, 160.0, 156.7, 156.6, 155.7, 155.1, 151.7, 151.3, 141.3,
141.1, 140.7, 139.2, 137.4, 136.9, 131.5, 130.1, 126.1, 125.1, 65.2,
52.8, 49.1, 42.8, 32.5, 31.0, 12.0. FT-IR (neat) 3370, 3104, 2925,
1666, 1588, 1515, 1166, 1105, 1071, 916, 726, 620 cm-1. HRMS
(ESI-TOF) calcd. for C30H28N8O9SNa [M+Na]+ 699.1592 found
699.1608.
1
CD experiments. 6 The CD titration experiment was performed
with a modification of the reported procedure.2c The ss-telo24
oligonucleotide was dissolved in Tris-buffer (50 mM, pH 7.6) and
the solution was heated to 94 ◦C for 5 min, then slowly cooled to
25 ◦C. (S)-1 and (R)-1 were diluted with water from a 10 mM stock
solution to a concentration of 1 mM, respectively. The solution
was titrated into the oligonucleotide samples at 1 mol equiv up
to 5 mol equiv (the 10 mM stock solutions of 1 were prepared
in DMSO). The final DNA concentration was 10 mM, and the
CD spectra are representative of three averaged scans taken at
25 ◦C. The CD melting curves of the ss-telo24 G-quadruplex were
determined from measurements of the CD intensity at 292 nm.
The heating rate was 2.0 ◦C min-1.
Macrocyclic lactam (2S,14R)-5. White solid. mp (CH2Cl2)
◦
>300 C (decomposed). [a]2D7 +7.03 (c 0.730, CHCl3). H NMR
(400 MHz, CD2Cl2) d 8.49 (d, J = 6.28 Hz, 1H), 8.44 (d, J = 7.25 Hz,
1H), 8.29 (s, 1H), 8.27 (s, 1H), 8.25 (s, 1H), 8.22 (s, 1H), 5.39–5.47
(m, 2H), 4.17 (m, 1H), 4.00 (m, 1H), 3.24 (dd, J = 13.0, 5.80 Hz,
1H), 3.16 (dd, J = 13.0, 4.35 Hz, 1H), 2.73 (s, 3H), 2.68 (s, 3H),
1.24 (s, 9H). 13C NMR (100 MHz, CD2Cl2) d 163.0, 161.5, 161.2,
159.8, 156.7, 156.6, 155.7, 155.1, 151.6, 151.2, 141.2, 140.5, 139.2,
137.2, 136.8, 131.4, 130.0, 126.0, 125.1, 65.3, 51.8, 48.6, 42.9, 33.0,
31.0, 12.0. FT-IR (solid) 3362, 3120, 2938, 1665, 1581, 1499, 1180,
1100, 1065, 914, 768, 726, 582 cm-1. HRMS (ESI-TOF) calcd. for
C30H28N8O9SNa [M+Na]+ 699.1592 found 699.1577.
1
Acknowledgements
Total synthesis of the enantiomer of telomestatin (S)-1. The
synthesis was performed according to the procedure previously
reported.7
We thank Prof. Katsuhiko Tomooka (Kyushu Univ.) for kind
help in HPLC-CD analysis. We also thank New Energy and
Industrial Technology Development Organization. This study
was partially supported by a Grant-in-Aid from the Ministry of
Education, Culture, Sports, Science and Technology, Japan (T.D.,
No. 18032028), a Grant-in-Aid for Exploratory Research (K.N.,
No. 21655060), and The Novartis Foundation (Japan) for the
Promotion of Science (K.N.). Dr M. Tera is grateful for JSPS
Research Fellowships for Young Scientists. We also deeply thank
Mr Katsuhiko Kushida (Varian Technologies Japan Ltd.) for the
measurement of the NMR spectrum of (S)-telomestatin.
Cyclic heptaoxazole (S)-8. White solid. mp (CH2Cl2) >300 ◦C
(decomposed). [a]2D2 -13.2 (c 0.370, CHCl3). 1H NMR (400 MHz,
CDCl3) d 8.21 ¥ 2 (each s, 2H), 8.16 (s, 2H), 8.13 (s, 1H), 7.80
(brs, 1H), 5.25 (brs, 1H), 3.23 (dd, J = 13.5, 4.35 Hz, 1H), 3.07
(brd, J = 11.1 Hz, 1H), 2.63 (s, 3H), 2.61 (s, 3H), 1.14 (s, 9H).
13C NMR (100 MHz, CDCl3) d 160.4, 159.9, 156.6, 156.4, 156.2,
155.8, 155.2, 154.9, 150.7, 149.0, 141.8, 139.2, 138.5, 137.6, 137.0,
130.5, 129.7, 129.5, 125.3, 124.5, 70.7, 42.8, 32.3, 30.8, 11.9, 11.6.
FT-IR (neat) 3408, 3123, 2926, 1673, 1588, 1494, 1365, 1176, 1087,
918, 753, 716 cm-1. HRMS (ESI-TOF) calcd. for C30H24N8O8SNa
[M+Na]+ 679.1330 found 679.1316.
Notes and references
Enantiomer of telomestatin (S)-(1). 1H NMR (500 MHz,
CD3CN:D2O = 1 : 1) d 8.57 (s, 1H), 8.49 (s, 1H), 8.41 (s, 3H),
5.85 (t, J = 10.0 Hz, 1H), 4.14 (dd, J = 10.0, 10.0 Hz, 1H), 3.68
(dd, J = 10.0, 10.0 Hz, 1H), 2.63 (s, 3H), 2.61 (s, 3H). HRMS
(ESI-TOF) calcd. for C26H14N8O7SNa [M+Na]+ 605.0598 found
605.0583.
1 K. Shin-ya, K. Wierzba, K. Matsuo, T. Ohtani, Y. Yamada, K.
Fujirhata, Y. Hayakawa and H. Seto, J. Am. Chem. Soc., 2001, 123,
1262–1263.
2 (a) M.-Y. Kim, H. Vankayalapati, K. Shin-ya, K. Wierzba and L.
H. Hurley, J. Am. Chem. Soc., 2001, 124, 2098–2099; (b) F. Rosu, V.
Gabelica, K. Shin-ya and E. De Pauw, Chem. Commun., 2003, 2702–
2703; (c) E. M. Rezler, J. Seenisamy, S. Bashyam, M.-Y. Kim, E. White,
W. D. Wilson and L. H. Hurley, J. Am. Chem. Soc., 2005, 127, 9439–
9447; (d) N. Arnoult, K. Shin-ya and J. A. London˜o-Vallejo, Cytogenet.
Genome Res., 2008, 122, 229–236.
3 A. M. Zahler, J. R. Williamson, T. R. Cech and D. M. Prescott, Nature,
1991, 350, 718–720.
4 (a) S. Neidle, FEBS J., 2010, 277, 1118–1125; (b) A. De Cian, L. Lacroix,
C. Douarre, N. Temime-Smaali, C. Trentesaux, J.-F. Riou and J.-L.
Mergny, Biochimie, 2008, 90, 131–155; (c) J. W. Shay and W. N. Keith,
Br. J. Cancer, 2008, 98, 677–683; (d) L. Oganesian and T. M. Bryan,
TRAP assay. The oligonucleotides, Ts (5¢-AATCCGTC-
GAGCAGAGTT-3¢) and CX primer (5¢-CCCTTACCCTTA-
CCCTAACCCTAA-3¢), were synthesized by Sigma Genosys. The
TRAP reaction was carried out in two steps (elongation and
amplification). Elongation reactions were carried out at 30 ◦C
for 30 min in final volume of 40 mL containing 1 ¥ 104 cells of
392 | Org. Biomol. Chem., 2011, 9, 387–393
This journal is
The Royal Society of Chemistry 2011
©

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BioEssays, 2007, 29, 155–165; (e) Y. Mo, Y. Gan, S. Song, J. Johnston,
X. Xiao, M. G. Wientjes and J. L.-S. Au, Cancer Res., 2003, 63, 579–585;
(f) J. W. Shay and W. E. Wright, Cancer Cell, 2002, 2, 257–265.
5 Recent studies for G-quadruplex ligands: (a) H. Ma, M. Zhang, D.
Zhang, R. Huang, Y. Zhao, H. Yang, Y. Liu, X. Weng, Y. Zhou,
M. Deng, L. Xu and X. Zhou, Chem.–Asian J., 2010, 5, 114–122;
(b) T. P. Garner, H. E. L. Williams, K. I. Gluszyk, S. Roe, N. J.
Oldham, M. F. G. Stevens, J. E. Moses and M. S. Searle, Org.
Biomol. Chem., 2009, 7, 4194–4200; (c) B. Rubis, M. Kaczmarek, N.
Szymanowska, E. Galezowska, A. Czyrski, B. Juskowiak, T. Hermann
and M. Rybezynska, Invest. New Drugs, 2009, 27, 289–296; (d) H.
El-Daly, M. Kull, S. Zimmermann, M. Pantic, C. F. Waller and U.
M. Martens, Blood, 2005, 105, 1742–1749; (e) M. Gunaratnam, C.
Green, J. B. Moreira, A. D. Moorhouse, L. R. Kelland, J. E. Moses
and S. Neidle, Biochem. Pharmacol., 2009, 78, 115–122; (f) J.-H. Tan,
T.-M. Ou, J.-Q. Hou, Y.-J. Lu, S.-L. Huang, H.-B. Luo, J.-Y. Wu, Z.-S.
Huang, K.-Y. Wong and L.-Q. Gu, J. Med. Chem., 2009, 52, 2825–
2835; (g) D.-L. Ma, C.-M. Che and S.-C. Yan, J. Am. Chem. Soc.,
2009, 131, 1835–1846; (h) B. Chu, G. Yuan, J. Zhou, Y. Ou and P. Zhu,
Drug Dev. Res., 2008, 69, 235–241; (i) S. G. Rzuczek, D. S. Pilch, E. J.
LaVoie and J. E. Rice, Bioorg. Med. Chem. Lett., 2008, 18, 913–917;
(j) P. S. Shirude, E. R. Gillies, S. Ladame, F. Godde, K. Shin-ya, I.
Huc and S. Balasubramanian, J. Am. Chem. Soc., 2007, 129, 11890–
11891.
6 (a) M. Tera, K. Iida, H. Ishizuka, M. Takagi, M. Suganuma, T. Doi, K.
Shin-ya and K. Nagasawa, ChemBioChem, 2009, 10, 431–435; (b) M.
Tera, H. Ishizuka, M. Takagi, M. Suganuma, K. Shin-ya and K.
Nagasawa, Angew. Chem., Int. Ed., 2008, 47, 5557–5560.
7 T. Doi, M. Yoshida, K. Shin-ya and T. Takahashi, Org. Lett., 2006, 8,
4165–4167.
8 Synthetic studies for telomestatin: (a) N. Endoh, K. Tsuboi, R. Kim,
Y. Yonezawa and C. Shin, Heterocycles, 2003, 60, 1567–1572; (b) G.
S. Minhas, D. S. Pilch, J. E. Kerrigan, E. J. LaVoie and J. E. Rice,
Bioorg. Med. Chem. Lett., 2006, 16, 3891–3895; (c) M. Tera, Y.
Sohtome, H. Ishizuka, T. Doi, M. Takagi, K. Shin-ya and K. Nagasawa,
Heterocycles, 2006, 69, 505–514; (d) S. K. Chattopadhyay and S. Biswas,
Tetrahedron Lett., 2006, 47, 7897–7900; (e) C. M. Marson and M. Saadi,
Org. Biomol. Chem., 2006, 4, 3892–3893; (f) S. K. Chattopadhyay, S.
Biswas and S. K. Ghosh, Synthesis, 2008, 1029–1032; (g) J. M. Atkins
and E. Vedejs, Org. Lett., 2005, 7, 3351–3354; (h) H. Araki, T. Katoh
and M. Inoue, Tetrahedron Lett., 2007, 48, 3713–3717; (i) E. F. Flegeau,
M. E. Popkin and M. F. Greaney, Org. Lett., 2008, 10, 2717–2720; (j) K.
Shibata, M. Yoshida, T. Doi and T. Takahashi, Tetrahedron Lett., 2010,
51, 1674–1677.
9 PyBroP = bromo-tris-pyrrolidino-phosphonium hexafluorophosphate:
(a) J. Coste, M.-N. Dufour, A. Pantaloni and B. Castro, Tetrahedron
Lett., 1990, 31, 669–672; (b) J. Coste, E. Fre´rot, P. Jouin and B. Castro,
Tetrahedron Lett., 1991, 32, 1967–1970.
10 DPPA = diphenyl phosphorazidate: T. Shioiri, K. Ninomiya and S.
Yamada, J. Am. Chem. Soc., 1972, 94, 6203–6205.
11 Before the macrolactamization, no diastereomeric isomer was observed
in the 1H-NMR spectrum. The diastereomers were easily distinguish-
able by the chemical shift at the C14 position (d 4.22 from 4, d 4.33
from 7). After the macrolactamization, prolonged reaction time did not
affect the diastereomeric ratio of the products.
12 HPLC analysis was performed using Inertsil ODS-3 (4.6 ¥ 75 mm)
equipped with a UV detector (254 nm) and a CD spectropolarimeter
(254 nm) by elution with 40% CH3CN in H2O (1.0 mL min-1).
13 HATU = 2-(1-oxy-7-azabenzotriazol-3-yl)-1,1,3,3-tetramethylguani-
dium hexafluorophosphate: L. A. Carpino, J. Am. Chem. Soc., 1993,
115, 4397–4398.
14 PyBOP = (benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluo-
rophosphate: J. Coste, D. Le-Nguyen and B. Castro, Tetrahedron Lett.,
1990, 31, 205–208.
15 (a) I. Shiina, H. Ushiyama, Y. Yamada, Y. Kawakita and K. Nakata,
Chem.–Asian J., 2008, 3, 454–461; (b) I. Shiina, T. Kikuchi and A.
Sasaki, Org. Lett., 2006, 8, 4955–4958; (c) I. Shiina and M. Hashizume,
Tetrahedron, 2006, 62, 7934–7939; (d) I. Shiina, M. Hashizume, Y.
Yamai, H. Oshiumi, T. Shimazaki, Y. Takasuna and R. Ibuka, Chem.–
Eur. J., 2005, 11, 6601–6608.
16 S. You, H. Razavi and J. W. Kelly, Angew. Chem., Int. Ed., 2003, 42,
83–85.
17 W. E. Wright, J. W. Shay and M. A. Piatyszek, Nucleic Acids Res., 1995,
23, 3794–3795.
18 We always compare the activity of the compounds with that of a
control compound because the mode of action of these derivatives,
which are structurally similar, is the same as that of naturally isolated
telomestatin, and even a TRAP assay is reported as being not reliable for
comparison of the activities of G-quadruplex ligands. A. De Cian, G.
Cristofari, P. Reichenbach, E. De Lemos, D. Monchaud, M.-P. Teulade-
Fichou, K. Shin-ya, L. Lacroix, J. Lingner and J.-L. Mergny, Proc. Natl.
Acad. Sci. U. S. A., 2007, 104, 17347–17352.
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The Royal Society of Chemistry 2011
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