Synthesis of bicyclic enediynes that possess a photosensitive triggering device and exhibit strong DNA cleaving activity

Article abstract of DOI:10.1039/c0cc01286f

The synthesis of a bicyclic enediyne capable of photosensitive triggering and conjugated to a pyrrole-imidazole polyamide is described. Using UV irradiation, this hybrid molecule is shown to exhibit 100-fold stronger potency for DNA cleavage, in an in vitro assay, as compared to the enediyne without the DNA-localizing pyrrole-imidazole.

Full text of DOI:10.1039/c0cc01286f

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Synthesis of bicyclic enediynes that possess a photosensitive triggering
device and exhibit strong DNA cleaving activityw
Hiroshi Tanaka,*a Yoshikazu Tanaka,a Masafumi Minoshima,b Sho Yamaguchi,a
a
Shinichiro Fuse,a Takayuki Doi,z Susumu Kawauchi,c Hiroshi Sugiyamab and
Takashi Takahashi*a
Received 7th May 2010, Accepted 21st June 2010
DOI: 10.1039/c0cc01286f
The synthesis of a bicyclic enediyne capable of photosensitive
triggering and conjugated to a pyrrole-imidazole polyamide is
described. Using UV irradiation, this hybrid molecule is shown
to exhibit 100-fold stronger potency for DNA cleavage, in
an in vitro assay, as compared to the enediyne without the
DNA-localizing pyrrole-imidazole.
formation by irradiation of a photosensitive triggering device
of stable enediynes or enediyne precursors is an alternative,
and effective, method for the control of biradical formation.
Nicolaou and co-workers first demonstrated the photo-
induced cycloaromatization of a dynemycin analogue possessing
an o-nitrobenzyl ether as a photosensitive triggering device.8
Popik and co-workers recently reported on a cyclopropenone-
containing enediyne precursor.9 The cyclopropenone acts as a
precursor of acetylene. Herein, we report on the synthesis of
bicyclic enediynes that possess photosensitive triggering
devices and exhibit strong DNA cleavage activity.
DNA-cleaving molecules not only serve as anticancer drug
candidates; they also are useful as chemical probes for the
modulation of cell functions.1 Enediyne antitumor antibiotics
are a family of natural products exhibiting DNA cleavage
activity.2 These products are stable in their natural form and
undergo Masamune–Bergman cyclization of the enediyne
moiety in a 9- or 10-membered ring to provide a benzene
biradical after chemical activation.3 The benzene biradical
abstracts the hydrogen in a DNA sugar backbone and results
in DNA cleavage. There are many reports on the synthesis of
artificial enediyne derivatives, which can generate a biradical
by chemical activation.4 We have reported on the synthesis of
9- and 10-membered enediyne precursors, which undergo
b-elimination under ambient conditions to generate highly
reactive enediynes.5 However, the spatial and temporal control
of enediyne reactivity towards cycloaromatization is difficult
to achieve.
We designed the trans-fused bicyclic 10-membered enediyne
1 possessing an o-nitrobenzyl ether as a photosensitive triggering
device with an azido group as a connecting device (Scheme 1).
The alkyloxy substituents of the o-nitrobenzyl group improved
the sensitivity toward UV irradiation.10 UV irradiation of the
enediyne 1 promoted cleavage of the o-nitrobenzyl ether to
provide the trans-fused hemiketal 3. After epimerization of the
trans-fused hemiketal 3 to the cis-fused hemiketal 4, the
cis-fused hemiketal 4 could undergo cycloaromatization to
generate a p-benzene biradical. Transition state analysis of
Masamune–Bergman cyclization of the trans- and the
cis-fused hemiketals 3 and 4 to 5 and 6 based on DFT
calculation at the B3LYP/6-31G(d) level by Gaussian 0311
indicated that transition states TS-B of Bergman cyclization of
the cis-fused enediyne 4 would be more favorable than TS-A
of the trans-fused enediyne 3 (DDH = 24.9 kJ molꢀ1). These
results suggested that the photopromoted cleavage of the ketal
can initiate the generation of a biradical from 1. In addition,
copper-catalyzed Huisgen [3+2] cycloaddition can use the
azido group for coupling with DNA binding molecules 2 that
possess a terminal alkyne.12,13
Photopromoted cycloaromatization is an effective way to
control the biradical formation of enediynes.6 This involves
the use of stable precursors in the dark, which generate a
biradical by irradiation at an appropriate wavelength. The
direct UV irradiation of the enediyne moiety enables the
acceleration of its cycloaromatization.7 However, the efficiency
of the activation method is low. On the other hand, biradical
a Department of Applied Chemistry, Graduate School of Science and
Engineering, Tokyo Institute of Technology, 2-12-1-H-101
Ookayama, Meguro, Tokyo 152-8552, Japan.
E-mail: thiroshi@apc.titech.ac.jp; Fax: (+) 81-3-5734-2884;
Tel: (+) 81-3-5734-2471
The synthesis of a bicyclic enediyne bearing an azido group
is shown in Scheme 2. Treatment of the terminal alkene 7 with
butyllithium at ꢀ78 1C for 30 min, followed by addition of
1.5 equivalents of butenoride 8 afforded hemiketal 9 in 78%
yield. The ethoxyethyl ether at the C2 position of 8 is
important for yielding the mono adduct 9. Treatment of the
hemiketal 9 with 1,1,1-trimethoxyethane and a catalytic
amount of PPTS in MeOH at room temperature resulted in
removal of the ethoxyethyl ethers and ketalization with
methanol to provide the methyl ketal 10 in 85% yield. The
methyl ether 10 was converted to the o-nitrobenzyl ether 12 by
treatment with benzyl alcohol 11 under acidic conditions in the
presence of MS5A (47%). The primary alcohol 12 was converted
to bromide 13 in 82% yield. Cyclization of bromoalcohol 13
was examined. A solution of bromoalcohol 12 was slowly
b Department of Chemistry, Graduate School of Science,
Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-Ku,
Kyoto 606-8502, Japan. E-mail: hs@kuchem.kyoto-u.ac.jp;
Fax: (+) 81-75-753-4002; Tel: (+) 81-75-753-3670
c Department of Polymer Chemistry, Graduate School of Science and
Engineering, Tokyo Institute of Technology, 2-12-1-E6-4 Ookayama,
Meguro, Tokyo 152-8552, Japan.
E-mail: skawauchi@polymer.titech.ac.jp; Fax: (+) 81-3-5734-2888;
Tel: (+) 81-3-5734-3757
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/c0cc01286f
z Current Address: Department of Pharmaceutical, Graduate School
of Pharmaceutical Sciences, Tohoku University, 6-3 Aza-aoba,
Aramaki, Aoba-ku, Sendai 980-8578, Japan.
ꢁc
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Scheme 1 Design of a bicyclic enediyne 1 with a photosensitive
triggering device.
added to a suspension of NaH in THF in the presence of a
small amount of H2O at room temperature to provide the
trans- and cis-fused 10-membered cyclic ethers 14a and 15a in
30% and 12% yields, respectively. The relative stereochemistry
of the bicyclic ethers 14a and 15a was determined by comparison
with the 1H NMR spectra of the corresponding methyl
derivatives 14b and 15b, the relative stereochemistries of which
were determined by the NOE experiments.14 Removal of the
TBS ether from 14a and mesylation of the secondary alcohol,
followed by b-elimination of the resulting mesyl ester treated
with DBU at room temperature, provided the trans-fused
10-membered enediyne 1 in 32% yield. The enediyne 1 was
stable enough to stock as an acetonitrile solution at ꢀ20 1C for
more than a month. In addition, it should be noted that the
cis-fused cyclic ether 15a was not converted to the corresponding
cis-fused enediyne by the same procedure.
Scheme 2 Reagents and conditions: (a) butyllithium, THF, ꢀ78 1C,
30 min then 8, THF, 78%; (b) 1,1,1-trimethoxyethane, cat. PPTS,
MeOH, 85%; (c) 11, PPTS, MS5A, CH2Cl2, 47%; (d) CBr4, PPh3,
2,6-lutidine, rt, 82%; (e) NaH, THF, rt, 30% for 14, 12% for 15;
(f) TBAF, THF; (g) MsCl, NEt3, CH2Cl2, 0 1C; and, (h) DBU, THF,
rt. 33% for 1. (i) UV irradiation at 360 nm, THF : H2O (3 : 1), 5 1C,
30 min, then rt, 30 min; (j) UV irradiation at 360 nm, THF-d8 : H2O
(3 : 1), 5 1C, 30 min, then rt, 30 min
of the reaction mixture by a reverse phase HPLC provided the
conjugated enediyne 20 in 60% yield.
The photochemical activation of enediyne 1 was examined.
UV light irradiation (360 nm) to a solution of enediyne 1 in
THF : H2O (3 : 1) for 30 min at 0 1C, followed by stirring at
room temperature for 30 min without UV irradiation provided
the aromatic product 16 in 25% yield instead of the tricyclic
product 17. Use of THF-d8 : H2O (3 : 1) as a solvent for the
reaction resulted in the aromatic product 18 in 25% yield with
62% and 70% deuteration at the 6 and 9 positions, respectively.
However, comparison with the reactivity of the enediynes 3
and 4 towards the cycloaromatization failed because the
difficulty of isolation of 3 and 4.
DNA cleavage experiments using enediyne 1 were examined
(Fig. 1a). A Form I DNA was incubated with enediyne 1
(1.0 mM) under UV irradiation (365 and 302 nm) for 5, 10 and
To demonstrate the utility of the enediyne 1, the synthesis of
the 10-membered enediyne 20 conjugated with a hairpin
pyrrole-imidazole polyamide through an o-nitrobenzyl ether
was examined (Scheme 3). The pyrrole-imidazole polyamide
unit acted as an effective miner groove binder.15,16 The enediyne
1 was treated with 1.5 equivalents of the pyrrole-imidazole
polyamide 19 bearing a terminal alkyne in the presence of
CuSO4 (1.0 equiv.) and sodium ascorbate (2.0 equiv.) in tert-
BuOH : H2O (1 : 1) for 4 h at room temperature. Purification
Scheme 3 Synthesis of the hybrid enediyne 20 by the coupling of
bicyclic enediyne 1 with a pyrrole-imidazole polyamide 19.
ꢁc
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molecules, such as pyrrole-imidazole polyamide, by copper-
catalyzed Huisgen [3+2] cycloaddition. The pyrrole-imidazole
polyamide hybrid 20 exhibited more than 100-fold stronger
DNA cleaving activity than 1.
This work was financially supported by NEDO (New
Energy and Industrial Technology Development Organization)
and was partially supported by G-COE program. We thank
Prof. Dr Takenori Kusumi and Dr Haruko Takahashi for
their suggestions.
Notes and references
Fig. 1 Cleavage of pGEX-4T-1 plasmid DNA by enediynes 1 and 20.
(a) Lanes 1 and 5: DNA alone incubated in the dark for 15 min at
23 1C; lanes 2–4: DNA incubated with 1 mM of 1 and UV irradiation
at 302 nm for 5, 10, and 15 min; and, lanes 6–8: DNA incubated with
1 mM of 1 and UV irradiation at 365 nm for 5, 10 and 15 min. (b) Lane
1: DNA alone incubated for 15 min at 23 1C; lane 2: DNA incubated
in the dark with 1 mM of 1; lanes 3–6: DNA incubated with 0, 100,
1 U. Galm, M. H. Hager, S. G. V. Lanen, J. Ju, J. S. Thorson and
B. Shen, Chem. Rev., 2005, 105, 739–758.
2 (a) A. Smith and K. C. Nicolaou, J. Med. Chem., 1996, 39,
2103–2117; (b) Comprehensive Natural Products Chemistry, ed.
D. H. R. Barton and K. Nakanishi, Elsevier, Amsterdam, 1999,
vol. 7.
3 I. H. Goldberg, Acc. Chem. Res., 1991, 24, 191–198.
4 I. A. Maretina and B. A. Trofimov, Russ. Chem. Rev., 2006, 75,
825–845.
i
200, and 500 mM of PrOH and 1 mM of 1 and UV irradiation at
5 (a) T. Doi and T. Takahashi, J. Org. Chem., 1991, 56, 3465–3467;
(b) T. Takahashi, H. Tanaka, H. Yamada, T. Matsumoto and
Y. Sugiura, Angew. Chem., Int. Ed. Engl., 1996, 35, 1835–1837;
(c) Y. Tanaka, S. Fuse, H. Tanaka, T. Doi and T. Takahashi, Org.
Process Res. Dev., 2009, 13, 1111–1121 and references therein.
6 M. Kar and A. Basak, Chem. Rev., 2007, 107, 2861–2890.
7 N. J. Turro, A. Evenzaha and K. C. Nicolaou, Tetrahedron Lett.,
1994, 35, 8089–8092.
302 nm. (c) Lane 1: DNA alone incubated in the dark for 15 min at
23 1C; lanes 2–5: DNA incubated with 0, 0.1, 1.0, and 10 mM of 20 and
UV irradiation at 302 nm; lane 6: DNA incubated with 1 mM of
compound 1 and UV irradiation at 302 nm. (d) Lane 1: DNA alone
incubated in the dark for 15 min at 23 1C; lanes 2 and 3: DNA
incubated with 10 mM of 20 and UV irradiation at 365 nm and 302 for
15 min, respectively.
8 K. C. Nicolaou, W.-M. Dai, S. V. Wendeborn, A. L. Smith,
Y. Torisawa, P. Maligres and C.-K. Hwang, Angew. Chem., Int.
Ed. Engl., 1991, 30, 1032–1036.
9 D. R. Pandithavidana, A. Poloukhtine and P. P. Popik, J. Am.
Chem. Soc., 2009, 131, 351–356.
10 A. Hasan, K.-H. Stengele, H. Giegrich, P. Cornwell, K. R. Isham,
R. A. Sachleben, W. Pfleiderer and R. S. Foote, Tetrahedron, 1997,
53, 4247–4264.
15 min. The mixtures were directly analyzed by agarose gel
electrophoresis. The enediyne 1 did not cause DNA cleavage
without UV irradiation (lanes 1 and 5). UV irradiation
promoted Form I DNA cleavage to Form II (lanes 2–4 and
6–8). In addition, no significant differences were observed in
the cleavage of DNA by enediyne 1 regardless of whether UV
irradiation was at 302 or 365 nm. The effect of isopropyl
alcohol as a radical scavenger on the DNA cleavage of 1 is
shown in Fig. 1b. Isopropyl alcohol reduced the generation of
Form II DNA from Form I DNA. These results suggest that
radical species cause DNA cleavage.
11 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
M. A. Robb, J. R. Cheeseman, J. A. Montgomery, Jr.,
T. Vreven, K. N. Kudin, J. C. Burant, J. M. Millam,
S. S. Iyengar, J. Tomasi, V. Barone, B. Mennucci, M. Cossi,
G. Scalmani, N. Rega, G. A. Petersson, H. Nakatsuji, M. Hada,
M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida,
T. Nakajima, Y. Honda, O. Kitao, H. Nakai, M. Klene, X. Li,
J. E. Knox, H. P. Hratchian, J. B. Cross, V. Bakken, C. Adamo,
J. Jaramillo, R. Gomperts, R. E. Stratmann, O. Yazyev,
A. J. Austin, R. Cammi, C. Pomelli, J. W. Ochterski,
P. Y. Ayala, K. Morokuma, G. A. Voth, P. Salvador,
J. J. Dannenberg, V. G. Zakrzewski, S. Dapprich, A. D. Daniels,
M. C. Strain, O. Farkas, D. K. Malick, A. D. Rabuck,
K. Raghavachari, J. B. Foresman, J. V. Ortiz, Q. Cui,
A. G. Baboul, S. Clifford, J. Cioslowski, B. B. Stefanov, G. Liu,
A. Liashenko, P. Piskorz, I. Komaromi, R. L. Martin, D. J. Fox,
T. Keith, M. A. Al-Laham, C. Y. Peng, A. Nanayakkara,
M. Challacombe, P. M. W. Gill, B. Johnson, W. Chen,
M. W. Wong, C. Gonzalez and J. A. Pople, GAUSSIAN 03
(Revision D.02), Gaussian, Inc., Wallingford, CT, 2004.
12 R. Huisgen, Angew. Chem., Int. Ed. Engl., 1963, 2, 565–598.
13 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., Int.
Ed., 2001, 40, 2004–2021.
14 8% NOE between Ha and Hb in 15b was observed.
15 (a) J. W. Trauger, E. E. Barid and P. B. Dervan, Nature, 1996, 382,
559–561; (b) J. M. Gottesfeld, L. Neely, J. W. Trauger, E. E. Baird
and P. B. Dervan, Nature, 1997, 387, 202–205.
16 Synthesis of the pyrrole-imidazole polyamide was performed by
solid phase peptide synthesis following the reported
method:(a) N. R. Wurtz, J. M. Turner, E. E. Baird and
P. B. Dervan, Org. Lett., 2001, 3, 1201–1203; (b) S. Sasaki,
T. Bando, M. Minoshima, K. Shinohara and H. Sugiyama,
Chem.–Eur. J., 2008, 14, 864–870.
DNA cleavage experiments using enediyne 20 were examined.
(Fig. 1c). Incubation of Form I DNA with enediyne 20
at varying concentrations (0, 0.1, 1.0, and 10 mM) under
UV irradiation (302 nm) resulted in cleavage to Form II
(lanes 3–5). Lane 6 showed DNA cleavage of non-hybrid
molecule 1 (1000 mM). These results indicate that the conjugated
enediyne 20 exhibited more than 100-fold stronger DNA
cleavage activity compared with non-conjugated enediyne 1.
We additionally found an unexpected effect of UV wavelength
on the DNA cleavage of 20. UV irradiation at 302 nm was
more effective for the cleavage of DNA by the hybrid enediyne
20 (Fig. 1d). However, the mechanism responsible for the
difference is not yet clear.
In conclusion, we described the synthesis of a hybrid bicyclic
enediyne 20 possessing a photosensitive triggering device and
its pyrrole-imidazole polyamide. The trans-fused bicyclic
enediyne 1 is stable in the dark and undergoes Masamune–
Bergman cyclization after photochemical activation initiated
by cleaving the o-nitrobenzyl ether. The azido moiety of cyclic
enediyne 1 can be used for conjugation with DNA binding
ꢁc
This journal is The Royal Society of Chemistry 2010
5944 | Chem. Commun., 2010, 46, 5942–5944