New DNA binding ligands as a model of chromomycin A3

Article abstract of DOI:10.1016/j.bmcl.2004.07.043

Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this study, new DNA-binding ligands were designed to mimic Chromomycin A₃ (CRA₃), which contains a hydroxylated tetrahydroanthracene

Full text of DOI:10.1016/j.bmcl.2004.07.043

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4855–4859
New DNA binding ligands as a model of chromomycin A3
Shuhei Imoto,a,b Yoshinari Haruta,a Kyouichi Watanabea and Shigeki Sasakia,b,
*
aGraduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan
bCREST, Japan Science and Technology Agency, Kawaguchi Center Building, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan
Received 7 June 2004; revised 20 July 2004; accepted 21 July 2004
Available online 7 August 2004
Abstract—Small molecules with DNA-binding affinity within the minor groove have become of great interest. In this study, new
DNA-binding ligands were designed to mimic Chromomycin A3 (CRA3), which contains a hydroxylated tetrahydroanthracene
chromophore substituted with di and trisaccharides. The trisaccharide part of CRA3 that is supposed to contribute to form the
Mg2+-coordinated dimer was expected to be mimicked by a simple alkyl group attached to the chromophore part as new model
compounds. The present study has successfully demonstrated that the new ligands form Mg2+-coordinated dimer complexes to
exhibit DNA-binding affinity.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Chromomycin A3 1 (CRA3) is a potent antitumour anti-
biotic, and is a part of the aureolic acid group including
plicamycin, mithramycin and olivomycins.1 It has been
shown that CRA3 binds in the minor groove of GC-rich
regions of duplex DNA to show inhibition against
DNA-dependent DNA polymerases as well as RNA
polymerases.2 Although the aureolic family of drugs
are not currently used for therapeutic purposes, the
new compounds of this family are reevaluated as poten-
tial candidates in cancer therapy.3 CRA3 contains a
hydroxylated tetrahydroanthracene chromophore sub-
stituted with various sugars. Studies on the structure–
activity relationship have shown that the sugar parts
contribute to DNA-binding properties as well as biolog-
ical activities.4 The aglycone of CRA3, chromomyci-
none, does not bind to DNA.5 It has been established
by structural studies with NMR and X-ray crystallo-
graphy that CRA3 binds in the minor groove of the
DNA as an Mg2+-coordinated dimer and G-specific
hydrogen bonds between CRA3 and DNA provide the
G-rich sequence specificity. It has been also shown from
the crystal structure that Mg2+ adopts an octahedral
coordination to O1 and O9 atoms of two chromophores
and two water molecules, as schematically shown in 2.
The keto-phenol structure of O1 and O9 is responsible
for Mg2+ binding, and mutual stackings of the aromatic
part of the chromophore of one monomer with the C–D
glycosidic linkage of the other monomer stabilize dimer
structure.6 Triethyleneglycol was used to mimic the C/D/
E trisaccharide part for the formation of Mg2+-coordi-
nated dimer, but the role of the triethyleneglycol part
for DNA binding of the model compound remains
unclear.7 We hypothesized that an alkyl chain would
produce higher stacking interaction to stabilize the
dimer structure complexed with Mg2+,8 and designed a
new model compound 3. In this study, the hexyl group
was chosen as an alkyl chain to mimic the C–D part
stacking with the aromatic part (Fig. 1). Herein we
describe that the model compound 3 binds to DNA as
an Mg2+-coordinated dimer.
The synthesis of 3 is shown in Scheme 1. We planned to
construct the peri-hydroxy aromatic skeleton by strong-
base-induced [4 + 2] cycloaddition of homophthalic
anhydrides with a-sulfinyl-substituted derivatives of eno-
lizable enones, which was developed by KitaÕs group.9
Homophthalic anhydride derivative 6 was obtained
from the corresponding homophthalic acid derivative
5.10 The a-sulfinyl-substituted cyclohexenone 8 was syn-
thesized from commercially available (R)-3-methylcyclo-
hexanone. The disulfinylated derivative 7 was obtained
via a two-step reaction by regioselective sulfinylation
with LDA–PhS–SPh,11 followed by reaction with potas-
sium tert-butoxide and PhSSO2Ph. Oxidation of 7 with
an equivalent of m-CPBA produced the 2-phenylthio-
cyclohexenone, which was further exposed to another
equivalent of m-CPBA to furnish the enone 8 in good
Keywords: Chromomycin A3; DNA minor groove binder.
*
Corresponding author. Tel.: +81-92-6426615; fax: +81-92-
6426876; e-mail: sasaki@phar.kyushu-u.ac.jp
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.07.043

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S. Imoto et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4855–4859
using ChiralPak-AS as a column, and their stereochem-
istry was determined by 1H COSY and NOESY
spectra.13
The Mg2+-binding properties of the compounds were
investigated by measurement of the UV spectra (Fig.
2) in methanol. The UV spectra of 3(S) was changed
by the addition of MgCl2 to produce a hyperchromic ef-
fect around 420nm (Fig. 2A) with the isosbestic points.
Stoichiometry of the complex was evaluated by the Job
plot, and the 2:1 3(S) to Mg2+ complex was proven
(Fig. 2B). The ability of the dimer complex of
3(S) with Mg2+ resembles the TEG-chromophore conju-
gate previously reported.7 It has been also shown that
the 3(R) isomer forms the 2:1 3(R) to Mg2+ complex.
The UV titration data (Fig. 2A) was analyzed based
on the 2:1 complex to produce a stability constant of
Ks = 1.0 · 106 Mꢀ1. In contrast, the Job plots with the
nonalkylated compound 10 (Fig. 2C) were not clear
compared to Figure 2B, probably because 10 forms a
mixture of 1:1 and 2:1 complexes. These results have
shown that the alkyl chain of 3 contributes to the forma-
tion of the dimer complex.
We also analyzed the complex compositions by positive
ESI-MS measurements. The mass spectrum of 3(S) in
the presence of MgCl2 indicated 2:1 ligand to Mg2+
complexes (m/z = 707) in addition to 1:1 complexes
(429, 415, 401) (Fig. 3B). In the MS spectra of 10
with MgCl2, there are the major peaks (m/z = 345,
331, 317) due to the 1:1 ligand to Mg2+ complexes to-
gether with the minor peak corresponding to the 2:1
complex (Fig. 3C). There is a small tendency for the for-
mation of 2:1 complexes with Ca2+ (Fig. 3D). These
results by the positive ESI-MS measurements agree with
those obtained by UV measurements in that 3(S) forms
2:1 complexes with MgCl2. It is also indicated that the
alkyl chain of 3(S) plays an important role for dimer
formation. The 3(R) isomer produced similar ESI-MS
spectrum.
Figure 1. Chromomycin A3 (CRA3, 1) and the schematic structure of
Mg2+-coordinated dimer (2).
yield. The [4 + 2] cycloaddition was performed by using
6 and 8 in the presence of NaH in refluxing dioxane to
produce 9 in 20–30% yield. After a number of attempts,
we found that molecular sieves catalyze the [4 + 2] cyclo-
addition to produce 9 in good yield.12 The hexyl group
was introduced by aldol reaction, followed by deprotec-
tion of the benzyl ethers by hydrogenolysis with H2-pal-
ladium hydroxide to give 11. Dehydroxylation was
accomplished under acidic condition, and the produced
exo-olefin was hydrogenated with H2-palladium hydrox-
ide to furnish a mixture of 3(S) and 3(R) isomers. The
diastereomers were separated by normal phase HPLC
Scheme 1. Reagents and conditions: (a) NaH, BnBr, 93%; (b) KOH, EtOH/H2O, 96%; (c) TMS ethoxyacetylene, 91%; (d) PhS–SPh, LDA, THF,
˚
ꢀ78ꢁC to rt, 82%; (e) PhSSO2Ph, tert-BuOK, THF, 0ꢁC, 77%; (f) m-CPBA, CH2Cl2, ꢀ50ꢁC, quant.; (g) m-CPBA, CH2Cl2, ꢀ60ꢁC, 73%; (h) MS 4A,
rt, 75%; (i) Pd(OH)2, H2 gas, quant.; (j) TBSOTf, TEA, CH2Cl2, quant.; (k) hexanal, BF3Et2O, ꢀ70 ꢁC, quant.; (l) Pd(OH)2, H2 gas, 55%; (m) TsOH,
benzene, 40ꢁC, 56%; (n) Pd(OH)2, H2 gas, 96%.

S. Imoto et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4855–4859
4857
Figure 2. The UV spectrum change of 3(S) by the addition of MgCl2 (A), and Job plot with 3(S) (B) and 10 (C).
Figure 3. Analysis of the complexes by ESI-MS measurements: (A) 3(S) only; (B) 3(S) + MgCl2; (C) 10 + MgCl2, (D) 3(S) + CaCl2.
The binding affinity of the ligand with the duplex DNA
was evaluated by a competitive displacement assay using
fluorescent ethidium bromide (ETBr). The emission
intensity of ethidium bromide is increased by binding
with duplex DNA, and is inhibited by another competi-
tive binder. Since competitive DNA binders displace
DNA-bound ETBr to decrease emission regardless of
their binding modes, although the binding mode of ETBr
is intercalation, displacement assay with ETBr is widely
used for primary estimation of the binding affinity of
the compounds.14 In this study, self-complementary 12
base pair DNA duplexes, CT12 and CA12, were used
for measurement of the binding affinity.15 CA12 includes
an AATT region, while CT12 has a GCGC region at the
middle of the duplex. Displacement assay was carried out
at 25ꢁC using a solution of the duplex DNA (CT12 or
CA12, 1lM) in 20mM Tris–HCl buffer. ETBr was effec-
tively displaced by 3(S) in the buffer containing 1mM
MgCl2 (Fig. 4A), clearly indicating the relatively high
affinity of 3(S) to DNA. Conversely, displacement was
not observed with use of 10 in the buffer containing
MgCl2 (Fig. 4B), or with use of 3(S) in the buffer contain-
ing NaCl instead of MgCl2 (Fig. 4C), showing low DNA-
binding affinity of the ligands under these conditions.
Moderate displacements were observed with 3(S) in the
buffer containing CaCl2 (Fig. 4D). These results have
indicated that the DNA-binding affinity of 3(S) corre-
lates with the tendency of dimer complex formation
mediated by Mg2+, and that the alkyl chain of 3(S) is also
an important contributor for DNA binding. The 3(R)

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S. Imoto et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4855–4859
Figure 4. The ethidium bromide displacement assay with 3(S) and 10: (A) 3(S) in the presence of 1mM MgCl2, (B) 10 in the presence of 1mM MgCl2,
(C) 3(S) in the presence of 2mM NaCl, (D) 3(S) in the presence of 1mM CaCl2. One micromolar each of CT12 and ethidium bromide were used. The
ligand concentration was increased from 0 to 10lM. kex = 546nm.
Table 1. Concentrations to displace 50% DNA-bound ethidium
bromide (IC50, lM)a
isomer showed almost the same results in the ethidium
displacement assay. Concentrations of the ligand to inhi-
IC50 (lM)b
bit 50% of fluorescence intensity of ETBr are expressed
as IC50 values, and correspond to the relative binding
affinity of the ligand (Table 1). Selective binding of
Hoechst 3325816 to the AATT region of DNA has been
proven in this ethidium displacement assay (the IC50
values for CT12 and CA12; >20 vs 1lM, respectively).
Displacement profile of CRA3 is somewhat com-
plexed, because only one of two ethidium bromide mol-
ecules bound to CT12 was displaced. This is probably
because the limited region of CT12 is tightly bound with
CRA3.17 The stereochemistry of 3 did not affect the bind-
ing affinity, and the ligand 3 showed slightly weaker
binding affinity to CA12 than Hoechst 33258. The IC50
values of the new ligands are not different towards
CT12 or CA12, and sequence selectivity of the new
ligands was not clear in this ethidium displacement
assay.18
Compounds
CT12c
CA12d
CRA3
3(S)
3e
9
3
3
2.5
3(R)
3(R + S)
3
3.5
3
No inhibition
10
11
No inhibition
6.5
2.5
4.5
2.5
1
12
Hoechst 33258
>20
a 1lM each of the duplex DNA and ethidium bromide were used in
20mM Tris–HCl containing 1mM MgCl2 at pH8.0. kex = 546nm,
k
em = 590nm.
b Concentration to displace 50% DNA-bound ethidium bromide is
expressed as the dimer complex except for Hoechst 33258.
0
c CT12: (5 CGTAGCGCTACG)2.
0
d CA12: (5 CGCGAATTCGCG)2.
e IC50 value to displace one of two ethidium bromide molecules bound
to CT12.17
In conclusion, we have developed the new DNA binding
molecules as a model of Chromomycin A3. The model
ligands 3(S and R) have shown a tendency to form 2:1

S. Imoto et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4855–4859
4859
ligand-to-Mg2+ complexes in methanol. In comparison
with 10 without containing the alkyl chain, it has
been revealed that the alkyl chain is responsible for
the formation of the dimeric complex of 3 with
Mg2+. The DNA binding experiments have indicated
that the alkyl chain of 3 plays a key role for Mg2+-medi-
ated DNA binding. There is no significant difference in
binding properties examined in this study between
the 3(S) and the 3(R) isomer. The present study has
shown that the simple alkyl chain can mimic some roles
of the trisaccharide part for Mg2+-dimer complex
formation and Mg2+-mediated DNA binding. Further
efforts are currently underway to develop new ligands
that bind to DNA more strongly based on the structure
of 3.
9. Iio, K.; Ramesh, N. G.; Okajima, A.; Higuchi, K.;
Fujioka, H.; Akai, S.; Kita, Y. J. Org. Chem. 2000, 65,
89–95.
10. Kim, S.; Fan, G.; Lee, J.; Lee, J. J.; Kim, D. J. Org. Chem.
2002, 67, 3127–3130.
11. Oppolzer, W.; Petrzilka, M. Helv. Chim. Acta 1978, 61,
2755–2762.
12. Details will be published soon.
13. Selected data for compound 3(S) and 3(R). 3(S): 1H
NMR: d (ppm) 16.64 (1H, b s), 10.02 (1H, s), 6.72 (1H, s),
6.46 (1H, d, J = 2.3Hz), 6.36 (1H, d, J = 2.3Hz), 3.04 (1H,
dd, J = 16.0, 3.5Hz), 2.59 (1H, dd, J = 16.0, 7.5Hz), 2.36–
2.33 (1H, m), 2.24–2.19 (1H, m), 1.89–1.83 (1H, m), 1.71–
1.67 (1H, m), 1.37–1.27 (8H, m), 1.08 (3H, d, J = 6.7Hz),
0.87 (3H, t, J = 6.9Hz), IR (cmꢀ1, KBr) 3384, 1635, 1596,
HR-ESIMS (m/z) calcd for C21H27O4 (M+H)+ 343.1904,
1
found 343.1879. 3(R): H NMR: d (ppm) 16.44 (1H, b s),
9.95 (1H, s), 6.74 (1H, s), 6.47 (1H, d, J = 2.3Hz), 6.37
(1H, d, J = 2.3Hz), 2.97 (1H, dd, J = 16.2, 3.6Hz), 2.79
(1H, dd, J = 16.2, 6.7Hz), 2.60 (1H, dt, J = 6.7, 4.0Hz),
2.47–2.43 (1H, m), 1.88–1.84 (1H, m), 1.49–1.30 (9H, m),
Acknowledgements
0.97 (3H, d, J = 6.7Hz), 0.88 (3H, t, J = 6.9Hz), IR (cmꢀ1
,
This work was supported by a Grant-in-Aid for Scien-
tific Research (B) from Japan Society for the Promotion
of Science (JSPS).
KBr) 3384, 1635, 1596, HR-ESIMS (m/z) calcd for
C21H27O4 (M+H)+ 343.1904, found 343.1890. The stere-
ochemistry of 3(S) and 3(R) was determined by 1H COSY
and NOESY as illustrated in Scheme 1.
14. Fox, K. R., Ed.; Drug-DNA Interaction Protocols: Opti-
mal Absorbance and Fluorescence Techniques for Measur-
ing DNA-Drug Interactions; Humana Press: Totowa, NJ,
1997; Vol. 90, pp 195–218.
15. Alam, M. R.; Maeda, M.; Sasaki, S. Bioorg. Med. Chem.
2000, 8, 465–473.
16. A representative DNA minor groove binder with selectiv-
ity to an A3T3 region. Crystal structure of Hoechst 33258
bound to d(CGCAAATTTGCG)2 duplex has been
reported. Spink, N.; Brown, D. G.; Skelly, J. V.; Neidle,
S. Nucl. Acids Res. 1994, 22, 1607–1612.
References and notes
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8. We have reported that the alkyl chains enhance DNA-
binding affinity of small molecules: Sasaki, S.; Shibata, T.;
Torigoe, H.; Shibata, Y.; Maeda, M. Nucleos. Nucleot.
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17. Two ethidium bromide molecules bind to one CT12 or
CA12 duplex. We have observed that some minor groove
binders such as distamycin displace only one of two
ethidium bromide molecules.15
18. DNA-binding affinity of the dimer complex of 3-Mg2+ was
estimated from the IC50 value to be ca. Ks = 2 · 106 Mꢀ1 to
CT12, and 7 · 106 Mꢀ1 to CA12. Several attempts to
estimate DNA binding by footprinting experiments were
not successful so far, probably because of insufficient
DNA-binding affinity of 3. Further modification of 3 for
higher affinity to DNA is now in progress.