Synthesis and DNA damaging ability of enediyne-polyamine conjugates

Article abstract of DOI:10.1016/j.tetlet.2003.12.139

Polyamine-enediyne conjugates were synthesized and exhibited potent DNA damaging ability under physiological conditions. The extent of their activity was shown to depend upon the polyamine length that regulates the DNA binding affinity of the conjugates,

Full text of DOI:10.1016/j.tetlet.2003.12.139

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 1955–1959
Synthesis and DNA damaging ability of enediyne–polyamine
conjugates
Ichiro Suzuki,* Akira Shigenaga, Hisao Nemoto and Masayuki Shibuya
Faculty of Pharmaceutical Sciences, University of Tokushima, Sho-machi 1-78, Tokushima 770-8505, Japan
Received 24 October 2003; accepted 22 December 2003
Abstract—Polyamine–enediyne conjugates were synthesized and exhibited potent DNA damaging ability under physiological
conditions. The extent of their activity was shown to depend upon the polyamine length that regulates the DNA binding affinity of
the conjugates, and enhanced DNA damaging activities were observed under slightly acidic conditions.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Polymethylene polyamines, such as spermidine and
spermine are natural products and have interesting
biological activities. Recently, due to their significant
biological activities, a variety of artificially designed
polyamines and their conjugates have been developed.1
Since polyamines interact with the phosphate moiety of
nucleic acid purely by charge interactions and by
hydrogen bonding in their cationic form, these com-
pounds constitute a class of DNA binding molecules.
The increasing interest in hybrid molecules composed of
polyamines and known anticancer drugs, intercalates
and other biological active agents is due to efforts to
enhance the activity and specificity of these drugs.2 In
addition, a polyamine transporter specifically mediates
the uptake of extracellular polyamines into cells and this
transporter is up-regulated in tumor cells, which require
large amounts of polyamines; therefore polyamines
themselves continue to attract significant attention as
potential anticancer drugs.3 Recently, we developed
artificially designed enediyne drugs possessing charac-
teristic triggering devices.4 In the course of our studies,
we investigated regulation of the reactivity of a,3-dide-
hydrotoluene biradicals which are prone to act as zwit-
ter ionic species having low bioactivities,5 and reported
that a,3-didehydrotoluene biradicals possessing electron
withdrawing groups at the benzylic position exhibited
enhanced radical characteristics.6b;c Previously, we
reported the design, synthesis, and DNA cleaving
activities of enediynes that we designed possessing a
cyanohydrin moiety as a triggering device (Fig. 1).6a
Here, we report the synthesis and DNA damaging
ability of the enediyne drugs 1–4 possessing a mono-
amine, a diamine, a triamine, and a tetraamine module,
respectively, as a DNA binding group (Fig. 2).
Enediynes 1 and 5 were synthesized from a common
precursor 8, as shown in Scheme 1. The THP-protected
cyanohydrin 6 was treated with LDA and pro-
pargylbromide followed by a Pd-mediated coupling
OAc
O
CN
Ph
Ph
Ph
DNA Strand
Cleavage
O
R
R
R
Figure 1.
* Corresponding author. Tel.: +88-633-9534; fax: +88-633-9517; e-mail: isuzuki@ph.tokushima-u.ac.jp
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.12.139

1956
I. Suzuki et al. / Tetrahedron Letters 45 (2004) 1955–1959
OAc
CN
OAc
CN
OAc
CN
Ph
Ph
Ph
NH2•HCl
N
H•HCl
NH2•HCl
N
H•HCl
N
H•HCl
NH2•HCl
1
2
3
OAc
OAc
CN
CN
Ph
Ph
H•HCl
N
NH2•HCl
N
H•HCl
N
H•HCl
NHCHO
5
4
Figure 2.
OTHP
CN
OAc
CN
OTHP
CN
(a), (b)
(c), (d)
Ph
Ph
Cl
Cl
6
7
8
OAc
CN
OAc
CN
(e)
(f)
Ph
Ph
NHBoc
R
9
1: R = NH2
(g)
5: R = NHCHO
Scheme 1. Reagents and conditions: (a) LDA /THF-HMPA, )78 ꢁC, then propargylbromide, )78 ꢁC, 43%; (b) (Z)-dichloroethylene, Pd(PPh3)4, CuI,
n-BuNH2/C6H6, rt, 96%; (c) p-TsOH/MeOH, rt; (d) EDC, AcOH, DMAP/CH2Cl2, 0 ꢁC, 78% (two steps); (e) N-Boc-1-amino-3-butyne, Pd(PPh3)4,
CuI, Et2NH/C6H6, rt, 95%; (f) HCl/1,4-dioxane, rt, 88%; (g) EDC, HCO2H, DMAP/CH2Cl2, rt, 89%.
reaction with (Z)-dichloroethylene to give enyne 7. After
removal of the THP protection using p-TsOH, the
resulting alcohol was acetylated to give enyne 8. The
common precursor 8 was coupled with N-Boc-1-amino-
3-butyne7 under the Sonogashira condition to afford
enediyne 9, which was treated with HCl to give enediyne
1. The reference enediyne 5, the amide group of which is
incapable of playing the role of a DNA binding group,
was prepared by formylation of enediyne 1.
subsequent reduction using NaBH4 and Boc protection
afforded polyamine–alkynes 11a–11c. The common pre-
cursor 8 was coupled with polyamine–alkynes 11a–11c in
the presence of Pd catalyst to give enediynes 12a–12c,
which were treated with HCl in dioxane affording tar-
geting enediynes 2–4, as depicted in Scheme 3.
DNA cleaving assays of synthetic enediynes 1–5 were
carried out using Col E1 plasmid DNA in phosphate
buffer solutions (pH 6.0 and 9.0) in the presence of the
enediyne drugs (10–1000 lM) and the mixture was incu-
bated for 24 h at 37 ꢁC. The resultant DNA fragments
were separated by electrophoresis on agarose gel and
visualized by ethidium bromide staining. The results are
summarized in Figure 3 (pH 6.0) and Figure 4 (pH 9.0).
Polyamine–enediyne conjugates 2–4 were synthesized
similarly to enediyne 1 using polyamine–alkynes 11a–11c,
which were prepared as described in Scheme 2. The parent
aldehydes 10a,8 10b,9 and 10c10 were treated with 1-
amino-3-butyne to give the corresponding aldimines and
O
(a), (b), (c)
R
R
N
N
H
N
Boc
Boc
Boc
10a: R =H
11a: R =H (50%)
10b: R = (CH2)4NHBoc
11b: R = (CH2)4NHBoc (28%)
10c: R = (CH2)4NBoc(CH2)3NHBoc
11c: R = (CH2)4NBoc(CH2)3NHBoc (70%)
Scheme 2. Reagents and conditions: (a) 1-amino-3-butyne, MgSO4/CHCl3, rt; (b) NaBH4/MeOH, 0 ꢁC; (c) (Boc)2O, aq NaOH (1 M)/1,4-dioxane, rt.

I. Suzuki et al. / Tetrahedron Letters 45 (2004) 1955–1959
1957
OAc
OAc
CN
CN
(b)
Ph
(a)
Ph
8
R
R
N
N
N
N
H•HCl
H•HCl
Boc
Boc
2: R =H (77%)
3: R = [(CH2)4NH2]•HCl (65%)
4: R = [(CH2)4NH(CH2)3NH2]•2HCl (66%)
12a: R =H (58%)
12b: R = (CH2)4NHBoc (73%)
12c: R = (CH2)4NBoc(CH2)3NHBoc (68%)
Scheme 3. Reagents and conditions: (a) 12, Pd(PPh3)4, CuI, Et2NH/C6H6, rt; (b) HCl/1,4-dioxane, rt.
100
80
60
40
20
0
10
100
Concentration (µM)
500
1000
Enediyne 1
Enediyne 2 Enediyne 3
Enediyne 4
Enediyne 5
Figure 3. DNA cleaving assay using the enediyne drugs 1–5 at pH 6.0. Col E1 DNA (12.5 lg/ml) was incubated for 24 h at 37 ꢁC with the enediyne
drugs (10, 100, 500 and 1000 lM) in pH 6.0 phosphate buffers, and analyzed by electrophoresis (1% agarose gel, ethidium bromide staining). Results
are presented as mean values ꢀ SD of three runs. A control reaction mixture without the addition of any drug was incubated and the mean value of
three runs was used as the background to be subtracted from the obtained values.
100
80
60
40
20
0
50
100
500
Concentration (µM)
Enediyne 1
Enediyne 2
Enediyne 3
Enediyne 4
Enediyne 5
Figure 4. DNA cleaving assay using enediyne drugs 1–5 at pH 9.0. Col E1 DNA (12.5 lg/ml) was incubated for 24 h at 37 ꢁC with the enediyne drugs
(50, 100, and 1000 lM) in pH 9.0 phosphate buffer, and analyzed by electrophoresis (1% agarose gel, ethidium bromide staining). Results are
presented as mean values ꢀ SD of three runs. A control reaction mixture without the addition of any drug was incubated and the mean value of three
runs was used as the background to be subtracted from the obtained values.

1958
I. Suzuki et al. / Tetrahedron Letters 45 (2004) 1955–1959
The results in Figure 3 show that the enediynes having a
polyamine moiety exhibited potent DNA damaging
ability while the reference amide–enediyne 5 showed
only low bioactivity. Even enediyne 1, which has only
one amino group, showed considerably enhanced
activity compared to the reference 5, and the extent of
bioactivity was similar to that of diamine 2. Further,
triamine 3 and tetraamine 4 showed a similar level
of bioactivity, but were more potent than monoamine
1 and diamine 2.
with DNA in a similar fashion, a clear correlation
between the release of EthBr and the DNA cleaving
activity was observed.
In conclusion, we developed enediyne model compounds
having a polyamine module as DNA binding groups.
Our synthesized polyamine–enediyne conjugates showed
potent DNA damaging ability at acidic pH and their
bioactivity depends upon their binding affinity to DNA.
In basic buffer solution, as shown in Figure 4, the bio-
activities of the drugs 1–4 were somewhat reduced
compared to those in acidic buffers, while higher activity
than the reference 5 was still observed. In addition, the
order of bioactivity of enediynes 1–4 was similar to that
observed in acidic buffer solution. Considering the
reaction mechanisms in which the hydrolytic removal of
the acetyl group of the cyanohydrin moiety initiates the
cascade reaction leading to the biradical generation, it is
only reasonable that the reference enediyne 5 showed
relatively higher DNA damaging ability at pH 9.0 than
at pH 6.0. Indeed, when the reactions were monitored by
TLC, the enediyne drugs were still detected after 24 h at
pH 6.0, while they were consumed completely after 24 h
at pH 9.0.11 It is noteworthy that enediynes 1–4 exhib-
ited more potent DNA cleaving activity in acidic buffer
solutions than in basic buffers and their activities were
higher than the reference 5 at both pH. These results
indicated that the polyamine moiety directed the drugs
to DNA efficiently by electrostatic interactions and
hydrogen bonding in their cationic form at pH 6.0. In
contrast, since protonation of the polyamine moiety
should occur only partially at pH 9.0,12 the directivity of
the drugs toward DNA should be reduced and, as a
result, bioactivity diminished. Further, to confirm the
interaction of polyamine–enediyne conjugates 1–4 with
DNA, we performed a fluorescence quenching assay13
based upon the displacement of ethidium bromide
(EthBr) from DNA caused by the conformational
change of DNA induced by binding of the polyamine–
enediyne conjugates 1–4. The results are summarized in
Table 1.
Acknowledgements
This work was supported by Grant-in-Aid for Scientific
Research (No. 13771334) from the Ministry of Educa-
tion, Culture, Sports, Science and Technology, Japan.
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Table 1. Fluorescence quenching assay of polyamine–enediyne con-
jugates 1–4
Enediyne
% Release of EthBra
% Cleavage of DNAb
1
2
3
4
5.39 0.35
5.86 0.37
12.82 0.20
14.40 0.23
5.3 2.6
4.7 2.5
33.7 2.8
38.9 4.3
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b Selected data from Figure 3.

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