Synthesis and DNA damaging ability of enediyne model compounds possessing photo-triggering devices

Article abstract of DOI:10.1016/j.bmcl.2004.02.096

Enediyne model compounds possessing photo-triggering devices were developed. These enediynes afforded biradicals by UV irradiation and showed DNA cleaving activity. The DNA damage was confirmed to be mainly caused by the biradical, not singlet oxygen.

Full text of DOI:10.1016/j.bmcl.2004.02.096

Bioorganic & Medicinal Chemistry Letters 14 (2004) 2959–2962
Synthesis and DNA damaging ability of enediyne model compounds
possessing photo-triggering devices
Ichiro Suzuki,* Shinsaku Uno, Yuko Tsuchiya, Akira Shigenaga, Hisao Nemoto and
Masayuki Shibuya
Faculty of Pharmaceutical Sciences, University of Tokushima, Sho-machi 1-78, Tokushima, Japan
Received 30 January 2004; accepted 28 February 2004
Abstract—Enediyne model compounds possessing photo-triggering devices were developed. These enediynes afforded biradicals by
UV irradiation and showed DNA cleaving activity. The DNA damage was confirmed to be mainly caused by the biradical, not
singlet oxygen.
Ó 2004 Elsevier Ltd. All rights reserved.
For many decades, DNA damage induced by photo-
irradiation has attracted considerable attention and
several types of DNA photocleavers have been exploi-
ted. Since the photochemical processes of these agents
are generally compatible with other biological processes,
they have significant potential as biological probes of
DNA, and drugs for photochemotherapy in which
ultraviolet, visible, and infrared lights are used together
with administered photoactive agents.1 Now with the
recent rapid progress of laser and fiber technology it is
becoming possible to introduce an intense beam of these
radiations into the depths of the human body and the
importance of photochemotherapy has been corre-
spondingly increasing.2 Since Shiraki and Sugiura
reported the photocleavage of DNA by Dynemicin A in
1990, this new class of photocleaver has attracted a great
deal of attention from researchers, and several enediyne
model compounds bearing photo-triggering devices
have been developed.3 In the course of our studies for
developing enediyne model compounds generating bio-
active carbon biradicals through a characteristic trig-
gering action,4 we have also investigated the design and
synthesis of enediynes activated photochemically. Here,
we report the synthesis and DNA cleaving abilities of
the enediyne model compounds 1, 2a, and 2b that we
designed, activated by photo-irradiation and, by com-
parison with reference compounds 3 and 4, we also
confirm that the DNA damage was mainly caused by the
biradical. The molecular design of targeting arenediynes
is depicted in Figure 1. Photochemical removal of the
protecting group on the cyanohydrin moiety initiates the
reaction cascade including regeneration of the carbonyl
group and isomerization to an arenyne–allene, which
cycloaromatizes to generate the a,3-didehydrotoluene
biradical.5
Initially, the syntheses of the alkynes 9a and 9b were
undertaken as shown in Scheme 1. Hydroxy ester 56 was
reacted with 2-nitrobenzyl chloromethyl ether7 giving
acetal 6, which was treated with aq NH3 in EtOH
affording amide and the following dehydration reaction
using Burgess reagent8 gave nitrile 9a. Carbonate 9b was
also synthesized from ester 5. THP protection of 5
afforded acetal 7, which was converted to nitrile 8 in two
steps. The removal of the THP protection followed by
the introduction of the carbonate moiety using p-nitro-
phenyl 2-(2-nitrophenyl)propyl carbonate9 afforded 9b.
Diiodobenzene was coupled with methyl propargyl ether
in the presence of Pd catalyst affording arenyne 10, and
the sequential coupling reaction of 10 with 9a or 9b
under the Sonogashira conditions gave enediynes 1 and
2a. Enediyne 2b was also synthesized from 11 in a sim-
ilar fashion to the synthesis of 2a.
First, we examined the cycloaromatization reactions of
enediynes 1, 2a, and 2b in the presence of 1.1 equiv of
Et3N and excess amounts (50 equiv) of 1,4-cyclohex-
adiene under UV irradiation (k > 365 nm) in degassed
MeOH. The results are summarized in Table 1. While
carbonate 2a and 2b were photolyzed completely within
30 min, a considerably longer reaction time (0.5 h vs
17 h) was needed for the deprotection of acetal 1.9b In
* Corresponding author. Tel.: +81-088-633-9534; fax: +81-088-633-
9517; e-mail: isuzuki@ph.tokushima-u.ac.jp
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.02.096

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I. Suzuki et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2959–2962
OH
CN
OR
O
PRPG
hν
CHO
OR
CN
OR
PRPG = Photo-removable Protecting Group
CHO
DNA damage
OR
Me
O
O
O
O
CN
OMe
NO2
CN
O
O2N
OR
1
2a: R = Me; 2b: R = H
O
O
O
OEt
CN
O
O
NO2
OMe
3
4
Figure 1.
NO2
NO2
b, c
OH
O
O
O
O
a
CO2Et
CO2Et
CN
9a (65%)
6 (53%)
5
d
O
OTHP
CO2Et
7 (61%)
OTHP
CN
b, c
O
O
e, f
Me NO2
9b (68%)
CN
8 (80%)
I
j
1 (98%) or 2a (61%)
g
h
OMe
10 (43%)
I
I
I
k
i
I
j, k
2b (87%)
OTBS
OH
11 (65%)
12 (94%)
Scheme 1. Reagents and conditions: (a) 2-Nitrobenzyl chloromethyl ether, NaH/THF, rt. (b) aq NH3/EtOH, rt. (c) Et3NþSO2NꢀCO2Me/THF, rt.
(d) DHP, p-TsOH/C6H6, rt. (e) Amberlyst 15/MeOH, rt. (f) p-Nitrophenyl 2-(2-nitrophenyl)propyl carbonate, Py/CH2Cl2, rt. (g) Methyl propargyl
ether, Pd(PPh3)4, CuI, Et3N/toulene, rt. (h) 2-Propyn-1-ol, Pd(PPh3)4, CuI, Et3N/toulene, rt. (i) TBSCl, imidazole/CH2Cl2, rt. (j) 9a (or 9b),
Pd(PPh3)4, CuI, Et3N/toulene, rt. (k) aq HF/MeCN, 0 °C.
both enediynes 1 and 2a, the reactions proceeded, giving
the products 13a, 14, and 15a accompanied by consid-
erable amounts of unknown polar materials (entries 1
and 2). Adduct 13a was obtained as an isomeric mixture
and then was converted to 16 by catalytic hydrogenation
to confirm the structure. In the cycloaromatization
reactions of 2b, 13b was obtained along with the trace
amounts of 15b (entry 3). 13b and 15b were converted to
17 and 15a, respectively, to confirm the structure. To
exclude any possibility that the cascade reaction was
initiated by the base promoted deprotection of 2, we
prepared reference enediyne 3, bearing a photostable
protecting group. When cycloaromatization of the
reference enediyne 3 was carried out under the same

I. Suzuki et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2959–2962
Table 1. The cycloaromatization reactions of enediynes 1 and 2
2961
hν (> 365 nm),
3
1,4-CHD (50 eq.)
MeOH
Et N (1.1 eq.)
CHO
OR
CHO
OMe
CHO
OR
OMe
+
1, 2a and 2b
+
13a: R = Me
13b: R = H
14
15a: R = Me
15b: R = H
1) Me SO ,
2
4
aq. NaOH
OMe
H , Pd/C
2) H , Pd/C
2
CHO
OMe
2
O
For 13a
For 13b
16
17
Entry
Enediyne
Time (h)
Products
1
2
3
4
1
17
13aa (8%), 14 (9%), 15a (16%)
13aa (2%), 14 (3%), 15a (9%)
13bb (6%), 15bc (trace)
No reactions
2a
2b
3
0.5
0.5
0.5
Enediynes, Et3N (1.1 equiv), and 1,4-cyclohexadiene (50 equiv) were dissolved in MeOH and photo-irradiated using a MUV-202U (MORITEX,
2000 mW cmꢀ2, k > 365 nm) for the indicated times.
a Compound 13a was obtained as an isomeric mixture and then was converted to 16 by catalytic hydrogenation to confirm the structure.
b Compound 13b was converted to 17 in two steps and the structure was confirmed.
c The structure of 15b was confirmed by converting to 15a using Me2SO4 and aq NaOH.
conditions, enediyne 3 was recovered almost completely
(entry 4). These results indicate that the cascade reaction
was initiated photochemically to form the a,3-didehy-
drotoluene biradical. In addition, the enediyne skeleton
itself might undergo a photo-Bergman cyclization reac-
tion,3d;g however, in our enediyne systems, no product
arising from Bergman cycloaromatization was obtained.
Next, we carried out DNA cleaving assays of synthetic
enediynes. Enediynes 2a and 2b were incubated with
UX174R-1 plasmid DNA in a phosphate buffer solution
(pH 8.0, containing 20% of acetonitrile) and the mixture
was irradiated (k > 365 nm). The resultant DNA frag-
ments were separated by electrophoresis on agarose gel
and visualized by ethidium bromide staining: the results
are summarized in Table 2. Prolonged irradiation time
(30 min vs 60 min) and elevated temperature (25 °C vs
37 °C) were effective in improving the DNA damaging
abilities of 2b (entries 4–6), however, in the case of 2a,
these modifications were not so effective (entries 1–3).
Although 2a and 2b showed a similar degree of facility
in the deprotection step (Table 1, entries 2 and 3) and
disappeared almost completely after the irradiation, 2a
showed only low activity compared with 2b. This dif-
ference would be attributed to the low solubility of 2a in
the buffer solution. Indeed, enediyne 2b could dissolve
A plausible mechanism of the formation of 13–15 is
depicted in Figure 2. Photo-irradiation initiates the
reaction cascade, consisting of the deprotection, iso-
merization, and cycloaromatization processes, giving
the biradical. The resulting biradical abstracts hydrogen
from 1,4-CHD, and finally should afford 13. The
biradical also reacts with adventitious oxygen to give a
peroxide, which would decompose to aldehyde 14.10 In
addition, the intermediary arenyne–allene degrades with
the conjugate addition of solvent MeOH affording 15.
CHO
CHO
CHO
hν
1 and 2
OR
OR
OR
R = H or Me
1,4-CHD
MeOH
15
CHO
OR
H
OOH
[1,4-CHD]•
O2
CHO
OR
14
13
H
Figure 2.

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I. Suzuki et al. / Bioorg. Med. Chem. Lett. 14 (2004) 2959–2962
Table 2. DNA cleaving abilities of synthetic enediynes 1–3 and the reference 4
Entry
Enediyne
Time (min)
Temperature (°C)
% Cleavageb;c
1
2
3
4
5
6
7
8
2aa
2aa
2aa
2b
2b
2b
3
3
0
25 ꢁ 3
11
27
60
3
25
12 ꢁ 4
ꢁ 4
0
0
3
7
7
3
25 ꢁ 4
40 ꢁ 3
60
3
25
0
0
0
3
25
ꢁ 7
3
<5
11
4
3
25 ꢁ 2
UX174R-1 DNA (0.46 lg, WAKO Pure Chemical Industries, Ltd) in 20 lL phosphate buffer solution (pH 8.0, containing 20% acetonitrile) with
drugs (500 lM) was incubated and photo-irradiated using a VL-30L (VILBER LOURMAT, 1820 lW cmꢀ2, k > 365 nm) for indicated times at 25 or
37 °C. Immediately, 15 lL samples were loaded into 1% agarose gel. The running buffer was 20 mM TAE (pH 7.8). Electrophoresis was at 50 V for
8 h. After electrophoresis, gel was stained for 1 h in ethidium bromide (1 lg/mL) and de-stained for 5 min in water. Relative amounts of DNA in
Form I, Form II, and Form III were determined by densitometry.
a Enediyne 2a was suspended in phosphate buffer solution.
b Values presented are mean value ꢁ SD of three runs. A control reaction mixture without the addition of any drug was incubated and photo-
irradiated. The mean value of three runs was used as the background to be subtracted from the obtained values.
c No isolable product could be detected on TLC from the reaction mixtures.
740; (g) Evenzahav, A.; Turro, N. J. J. Am. Chem. Soc.
into a buffer solution up to a concentration of 500 lM,
1998, 120, 1835–1841.
while 2a did not dissolve completely even below 100 lM,
4. (a) Suzuki, I.; Wakayama, M.; Shigenaga, A.; Nemoto,
becoming a suspension. The observed DNA cleavage
H.; Shibuya, M. Tetrahedron Lett. 2000, 41, 10019–10023;
(b) Suzuki, I.; Shigenaga, A.; Nemoto, H.; Shibuya, M.
could be caused by the biradical through the photo-
triggered cycloaromatization reaction, however, the
possibility of cleavage being due to the singlet oxygen
that was yielded by the photosensitization of a nitro-
Heterocycles 2001, 54, 571–576; (c) Suzuki, I.; Tsuchiya,
Y.; Shigenaga, A.; Nemoto, H.; Shibuya, M. Tetrahedron
Lett. 2002, 43, 6779–6781.
phenyl moiety could not be excluded. To clarify this
point, we performed a DNA cleaving assay of the ref-
erence compounds 4 under the same conditions.
Although the reference carbonate 4 damaged DNA to
some extent, the degree of cleavage was around 10%
(entry 8) and therefore the potent DNA cleavage
exhibited by enediyne 2b should be mainly attributed to
the oxidative damage caused by the photoreleased
biradicals.11
5. (a) Nagata, R.; Yamanaka, H.; Okazaki, E.; Saito, I.
Tetrahedron Lett. 1989, 30, 4995–4998; (b) Myers, A. G.;
Kuo, E. Y.; Finney, N. S. J. Am. Chem. Soc. 1989, 111,
8057–8059; (c) Saito, I.; Nagata, R.; Yamanaka, H.;
Murahashi, E. Tetrahedron Lett. 1990, 31, 2907–2910; (d)
Hirama, M.; Gomibuchi, T.; Fujiwara, K. J. Am. Chem.
Soc. 1991, 113, 9851–9853; (e) Nicolaou, K. C.; Wende-
born, S.; Maligres, P.; Isshiki, K.; Zein, N.; Ellestad, G.
Angew. Chem., Int. Ed. Engl. 1991, 30, 418–420; (f) Myers,
A. G.; Dragovich, P. S.; Kuo, E. Y. J. Am. Chem. Soc.
1992, 114, 9369–9386; (g) Wenthold, P. G.; Wierschke, S.
G.; Nash, J. J.; Squires, R. R. J. Am. Chem. Soc. 1993,
115, 12611–12612; (h) Wenthold, P. G.; Wierschke, S. G.;
Nash, J. J.; Squires, R. R. J. Am. Chem. Soc. 1994, 116,
7378–7392; (i) Sugiyama, H.; Yamashita, K.; Fujiwara, T.;
Saito, I. Tetrahedron 1994, 50, 1311–1325.
In conclusion, we developed enediyne model compounds
bearing photo-triggering devices and showed that these
synthetic enediynes exhibited potent DNA damaging
activity under UV irradiation conditions. We also clar-
ified that the biradical formation played the important
role in the cleavage of DNA in our systems.12 Further
improvement of the model compounds is now under-
way.
6. Brudermueller, M.; Musso, H. Angew. Chem. 1988, 100,
267–268.
7. Schwartz, M. E.; Breaker, R. R.; Asteriadis, G. T.; deBear,
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References and notes
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11. Mechanistically, the r-radical part of the biradical instead
of the resonance-stabilized p-radical part would be
responsible for this DNA scission, although the alkylation
pathway cannot be ruled out.
3. (a) Shiraki, T.; Sugiura, Y. Biochemistry 1990, 29, 9795–
9798; (b) Wender, P. A.; Zercher, C. K.; Beckham, S.;
Haubold, E. M. J. Org. Chem. 1993, 58, 5867–5869; (c)
Gomibuchi, T.; Fujiwara, K.; Nehira, T.; Hirama, M.
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Evenzahav, A.; Nicolaou, K. C. Tetrahedron Lett. 1994,
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Gomibuchi, T.; Hirama, M. J. Antibiot. 1995, 48, 738–
12. Since the DNA damage caused by 2a was of the similar
degree to the case of 4, we cannot attribute the DNA
damage to only the biradical.