Synthesis and evaluation of pyrrole polyamide-2′-deoxyguanosine 5′-phosphate hybrid

Article abstract of DOI:10.1080/15257770.2013.774014

Pyrrole polyamide-2′-deoxyguanosine 5′-phosphate hybrid (Hybrid 4) was synthesized and evaluated in terms of the inhibition of mouse mammary carcinoma FM3A cell growth. Hybrid 4 was found to exhibit dose-dependent inhibition of cell growth.

Full text of DOI:10.1080/15257770.2013.774014

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Synthesis and Evaluation of Pyrrole
Polyamide- 2′-Deoxyguanosine 5′-
Phosphate Hybrid
Etsuko Kawashima a , Yasuhiro Nakanishi b , Yusuke Terui b , Hideyuki
Tomitori b , Keiko Kashiwagi b , Yusuke Ohba a & Kazuo Kamaike a
a School of Pharmacy, Tokyo University of Pharmacy and Life
Sciences, Hachioji, Tokyo, Japan
b Faculty of Pharmaceutical Sciences, Chiba Institute of Science,
Choshi, Chiba, Japan
Version of record first published: 05 Apr 2013.
To cite this article: Etsuko Kawashima , Yasuhiro Nakanishi , Yusuke Terui , Hideyuki Tomitori , Keiko
Kashiwagi , Yusuke Ohba & Kazuo Kamaike (2013): Synthesis and Evaluation of Pyrrole Polyamide- 2′-
Deoxyguanosine 5′-Phosphate Hybrid, Nucleosides, Nucleotides and Nucleic Acids, 32:4, 196-205
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Nucleosides, Nucleotides and Nucleic Acids, 32:196–205, 2013
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2013.774014
SYNTHESIS AND EVALUATION OF PYRROLE POLYAMIDE-
2ꢀ-DEOXYGUANOSINE 5ꢀ-PHOSPHATE HYBRID
Etsuko Kawashima,1 Yasuhiro Nakanishi,2 Yusuke Terui,2 Hideyuki Tomitori,2
Keiko Kashiwagi,2 Yusuke Ohba,1 and Kazuo Kamaike1
1School of Pharmacy, Tokyo University of Pharmacy and Life Sciences Hachioji,
Tokyo, Japan
2Faculty of Pharmaceutical Sciences, Chiba Institute of Science, Choshi, Chiba, Japan
Pyrrole polyamide-2ꢁ-deoxyguanosine 5ꢁ-phosphate hybrid (Hybrid 4) was synthesized and eval-
2
uated in terms of the inhibition of mouse mammary carcinoma FM3A cell growth. Hybrid 4 was
found to exhibit dose-dependent inhibition of cell growth.
Keywords Pyrrole polyamide-2ꢁ-deoxyguanosine 5ꢁ-phosphate hybrid; minor groove
binder; anticancer agent
INTRODUCTION
The precise control of specific gene expression represents the ulti-
mate aim of gene therapy. We previously designed and synthesized nucle-
osides (Hybrid 1 and Hybrid 2) linked to a pyrrole polyamide[1] minor
groove binder (MGB) comprising modified distamycin A, which possess
high affinity for the AAATT sequence, as lead compounds for potential
application as gene therapy agents (Figure 1).[2] The pyrrole polyamide
moiety of 2ꢁ-deoxyguanosine hybrids was linked at the 2-exocyclic amino
group, which is positioned in the minor groove of a DNA duplex. The ds-
DNA binding ability of hybrids was investigated by analysis[3] of circular
dichroism (CD) spectra and melting temperature (Tm) values using sev-
eral DNA duplexes. It was shown that Hybrid 2 possessed specific DNA
binding ability and stabilized DNA duplexes [e.g., 5ꢁ-d(CGCAAATTGGC)-
3ꢁ/3ꢁ-d(GCGTTTAACCG)-5ꢁ duplex : Hybrid 2 = 1 : 1, ꢀTm = 5.6◦C].[2]
Received 4 February 2013; accepted 4 February 2013.
This work was supported in part by a Grant for private universities provided by the Promotion and
Mutual Aid Corporation for Private Schools of Japan.
Address correspondence to Kazuo Kamaike, School of Pharmacy, Tokyo University of Pharmacy
and Life Sciences, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. E-mail: kamaikek@toyaku.ac.jp
196

Synthesis and Evaluation of Pyrrole polyamide-2ꢁ-deoxyguanosine 5ꢁ-phosphate Hybrid 197
FIGURE 1 Pyrrole polyamide-2ꢁ-deoxyguanosine hybrids.
Furthermore, we synthesized oligonucleotide-conjugated pyrrole polyamide-
2ꢁ-deoxyguanosine hybrids and subsequently examined the DNA recogni-
tion and binding ability of modified dsDNA. The formyl functional group
of Hybrid 2 was unstable under conditions employed for DNA oligonu-
cleotide solid phase synthesis. Oligonucleotides 5 conjugated to Hybrid 3
FIGURE 2 Oligonucleotide-conjugated pyrrole polyamide-2ꢁ-deoxyguanosine hybrid. (Color figure
available online).

198
E. Kawashima et al.
were therefore synthesized and investigated for specific binding to ssDNA
(Figure2).[4] From the Tm values and CD spectral analyses, it was found
that oligonucleotides 5 possessed high specific binding to ssDNA oligonu-
cleotides containing the pyrrole polyamide binding sequence, and that the
generated dsDNAs possessed high stability [e.g., 5ꢁ-d(CGMAATTTGGC)-
3ꢁ/3ꢁ-d(GCCTTAAACCG)-5ꢁ, M = Hybrid 3, ꢀTm = 23.0◦C].[4] These results
suggest that Hybrids 2 and 3 may potentially be used to control the ex-
pression of target genes through interaction with the DNA duplex and/or
incorporation into the DNA during biosynthesis. Based on the studies de-
scribed above, we extended our studies to evaluating the biological activity
of the hybrids synthesized. In terms of incorporation of the hybrids into
DNA during biosynthesis, hybrids possessing a phosphate group at the 5ꢁ
position of the sugar moiety were expected to show high biological activity.
Introduction of a phosphate group also offers solubility advantages for drugs
used in water-based solutions. Herein, we report on the synthesis of Hybrid
4 bearing a 5ꢁ-phosphate group and evaluation of the inhibition of mouse
mammary carcinoma FM3A cell growth by Hybrids 1–4 (Figure 1).
RESULTS AND DISCUSSION
Hybrid 4 was efficiently synthesized by condensation of 2ꢁ-deoxy-2-
fluoroinosine derivative 9 with 3-(Py4-amino)propylamine from 10[4] and
conversion to the 5ꢁ-phosphate derivative via 5ꢁ-phosphoramidite derivative
12.
3ꢁ-Acetyl-2ꢁ-deoxy-2-fluoro-O6-[2-(4-nitropheny)ethylinosine (9) was pre-
pared from 3ꢁ,5ꢁ-di-O-acetyl-2ꢁ-deoxy-O6-[2-(4-nitropheny)ethylguanosine
(6)[4] as shown in Scheme 1. Compound 6 was deacetylated with 2:1
conc. NH4OH/1,4-dioxane and then treated with 35% HF/pyridine and
tert-butyl nitrite to give 2-fluoroinosine derivative 7.[5] After conversion
of 7 to 2ꢁ-deoxy-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2-fluoro-O6-[2-(4-nitrophenyl)-
ethyl]inosine (8),[5] 3ꢁ-O-acetylaion of 8 followed by removal of the DMTr
group afforded 9 in good yield.
SCHEME 1 Synthesis of 2ꢁ-deoxy-2-fluoroinosine derivative 9. Reaction conditions: (i) 2:1 conc.
NH4OH/1,4-dioxane, rt, 12 hours. (ii) 35% HF-pyridine, tert-butyl nitrite, −30◦C ∼rt, 2 hours. (iii)
DMTr-Cl (1.1 eq.), pyridine, rt, 4 hours. (iv) 1. Ac2O, pyridine, rt, 3 hours; 2. 2% Cl3CCOOH, 1:3
CH3OH/CH2Cl2, rt, 1 hour.

Synthesis and Evaluation of Pyrrole polyamide-2ꢁ-deoxyguanosine 5ꢁ-phosphate Hybrid 199
Treatment with 9 and 10 in the presence of Et3N afforded 11 in 63% yield
through removal of the Fmoc group of 10 and condensation with the amine
derivative (Py4–NH2). Compound 11 was converted to 5ꢁ-phosphoramidite
derivative 12 using a general method.[6] Phosphoramidite 12 was hydrolyzed
with 0.35 M acetic acid in H2O–CH3CN and oxidized with 0.02 M I2 in
H2O-pyridine-THF. The resulting phosphate derivative was treated with 1 M
NaOH in H2O–CH3OH to effect removal of the 2-cyanoethyl, acetyl (Ac),
and (4-nitrophenyl)ethyl (NPE) protecting groups and finally neutralized
using an ion exchange resin. Hybrid 4 was obtained as a precipitate from
the neutralized solution in 88% yield based with 12 (Scheme 2).
SCHEME 2 Synthesis of Hybrid 4. reaction conditions: (i) 1:5 Et3N/DMF, 60◦C, 10 hours. (ii)
[(iPr)2N]2POCE (1.5 eq.), 1H-tetrazole (1.1 eq.), CH2Cl2, rt, 1 hour. (iii) 1. 0.35 M AcOH/H2O-
CH3CN, rt, 30 min; 2. 0.02 M l2/H2O-pyridine-THF, rt, 20 minutes; 3. 1 M NaOH/H2O-CH3OH, rt,
17 hours; 4. Dowex 50 W × 8 (H+ form).
Inhibition of mouse mammary carcinoma FM3A cell growth by pyrrole
polyamide compounds (Hybrids 1–4 and distamycin A) was then evalu-
ated.[7] FM3A cells (3 × 104 cells/mL) were cultured in ES medium supple-
mented with 2% heat-inactivated fetal calf serum at 37◦C in an atmosphere
of 5% CO2. Cells were grown for 2 days in the absence (control) or presence
of pyrrole polyamide compounds (Hybrids 1–4 and distamycin A) (10 and
30 μM). Cell viability (percent of control) is shown in Figure 3. Hybrid 1,
a 2ꢁ-deoxyganosine derivative bearing pyrrole polyamide through an amide
bond (–N 2HCO–) using an aminopropionyl linker, and distamycin A had
no effect on cell growth. On the other hand, Hybrids 2–4, comprising 2ꢁ-
deoxyganosine bearing pyrrole polyamides through an N 2-alkyl bond using
an aminopropyl linker, induced dose-dependent inhibition of cell growth.
In particular, Hybrid 4 exhibited the highest inhibition compared with the
other Hybrids. We previously reported that oligonucleotides 5 conjugated to
Hybrid 3 possessed high specific binding to ssDNA oligonucleotides contain-
ing the pyrrole polyamide binding sequence, and that the generated dsDNAs
possessed high stability.[4] These results suggest that Hybrids 2–4 bearing

200
E. Kawashima et al.
FIGURE 3 Effect of pyrrole polyamide compounds (Hybrids 1–4 and distamycin A) on the growth of
mouse mammary carcinoma FM3A cells.Cells were grown for 2 days in the absence or presence of pyrrole
polyamide compounds.
pyrrole polyamide using an aminopropyl linker inhibited cell growth by in-
corporation into DNA during DNA replication and biosynthesis through a
process involving binding of the pyrrole polyamide moieties to the gener-
ated DNA duplexes, and that Hybrid 4 bearing a 5ꢁ-phosphate group is a
suitable substrate for biosynthesis. Further studies investigating the effect
of linker length and hybrids involving imidazole polyamide[1] are currently
underway.
CONCLUSION
We synthesized Hybrid 4 bearing a 5ꢁ-phosphate group and evaluated
the biological activity of Hybrids 1–4. Hybrid 4 showed marked inhibition
of mouse mammary carcinoma FM3A cell growth. Consequently, Hybrid 4
might potentially be useful as a lead compound in the development of novel
anticancer agents.
EXPERIMENTAL
General
Column chromatography was performed on silica gel (Silica gel N60, pur-
chased from Kanto Chemical Co., Inc. Japan) using methanol/chloroform
and hexane/ethyl acetate as eluent. Melting points were determined us-
ing a Yanaco Micro-melting-point apparatus, and are uncorrected. 1H-NMR
and 31P-NMR spectra were recorded on a Brucker DRX 400 spectrometer.
19F-NMR spectra were recorded on a Varian MERCURY 300 spectrometer.
Mass spectra were recorded on a Micromass Q-Tof Ultima API spectrome-
ter. Elemental analyses were determined using an Elemental Vavio EL ap-
paratus. 3ꢁ,5ꢁ-Di-O-acetyl-2ꢁ-deoxy-O6-[2-(4-nitrophenyl)ethylguanosine (6),

Synthesis and Evaluation of Pyrrole polyamide-2ꢁ-deoxyguanosine 5ꢁ-phosphate Hybrid 201
pyrrole amide tetramer 10, and Hybrids 1–3 were prepared as previously
described.[2,4]
2ꢀ-Deoxy-2-fluoro-O6-[2-(4-nitrophenyl)ethyl]inosine (7)
3ꢁ,5ꢁ-Di-O-acetyl-2ꢁ-deoxy-O6-[2-(4-nitrophenyl)ethyl]guanosineime(6)(0.
892 g, 1.78 mmol) was dissolved in 1,4-dioxane (16 mL) and 28% am-
monia/water (19.5 mL) was added to the solution. After stirring for
20 hours, the solution was evaporated to dryness to give 2ꢁ-deoxy-O6-[2-(4-
nitrophenyl)ethyl]guanosine, which was used in the next reaction without
1
ꢁ
ꢁꢁ
purification; H-NMR (ca. 5% CD3OD-CDCl3) δ 2.25 (dd, 1H, J 1 ,2
=
5.7 Hz, J 2 ,2 = 13.5 Hz, H-2ꢁꢁ), 2.95–3.04 (m, 1H, H-2ꢁ), 3.26 (t, 2H, J =
ꢁ
ꢁꢁ
6.9 Hz, –OCH2CH2PhNO2), 3.65–3.69 (m, 1H, H-5ꢁꢁ), 3.96 (dd, 1H, J 4 ,5ꢁ
ꢁ
= 1.5 Hz, J 5 ,5 = 12.6 Hz, H-5ꢁ), 4.18–4.20 (m, 1H, H-4ꢁ), 4.72 (t, 2H, J =
ꢁ
ꢁꢁ
6.9 Hz, –OCH2CH2PhNO2), 4.72–4.74 (m, 1H, H-3ꢁ), 4.95 (s, 2H, NH2),
6.22 (dd, 1H, J 1 ,2 = 9.3 Hz, J 1 ,2 = 5.7 Hz, H-1ꢁ), 7.48 (d, 2H, J = 9.0 Hz,
Ar-H of the NPE group ×2), 7.62 (s, 1H, H-8), 8.16 (d, 2H, J = 9.0 Hz, Ar-H
of the NPE group × 2).
ꢁ
ꢁ
ꢁ
ꢁꢁ
A stirred solution of 2ꢁ-deoxy-O6-[2-(4-nitrophenyl)ethyl]guanosine
(0.629 g, 1.51 mmol) in 35% HF/pyridine (7.5 mL) was prepared in an
ice-bath and tert-butyl nitrite (0.54 mL, 4.53 mmol) was added. After stir-
ring for 2 hours, the solution was diluted with ethyl acetate (150 mL)
and then poured slowly over 5% aqueous sodium hydrogen carbonate so-
lution (100 mL). The organic layer was washed with water (100 mL), dried
over anhydrous magnesium sulfate, and evaporated to dryness. The residue
was subjected to chromatographic separation on a column of silica gel us-
ing ethyl acetate/hexane as eluent to give 7 (0.428 g, 67.5% yield) as a
1
white powder; m.p. 143–145◦C; H-NMR (CDCl3) δ2.36 (dd, 1H, J 1 ,2
=
ꢁ
ꢁꢁ
5.7 Hz, J 2 ,2 = 13.1 Hz, H-2ꢁꢁ), 2.97 (m, 1H, H-2ꢁ), 3.32 (t, 2H, J = 6.9 Hz,
–OCH2CH2PhNO2), 3.81 (m, 1H, H-5ꢁꢁ), 3.97 (m, 1H, H-5ꢁ), 4.20 (m, 1H,
H-4ꢁ), 4.80 (m, 1H, H-3ꢁ), 4.85 (t, 2H, J = 6.9 Hz, –OCH2CH2PhNO2), 6.32
ꢁ
ꢁꢁꢁ
(dd, 1H, J 1 ,2 = 9.3 Hz, J 1 ,2 = 5.7 Hz, H-1ꢁ), 7.50 (d, 2H, J = 8.4 Hz,
Ar-H of the NPE group × 2), 7.98 (s, 1H, H-8), 8.18 (d, 2H, J = 8.4 Hz,
Ar-H of the NPE group × 2); 19F-NMR (CDCl3) δ –48.95 (s); ESI-TOF MS
calcd for C18H18N5O6 (M+H)+ 420.1319, found 420.1313; Anal. calcd for
C18H18FN5O6; C, 51.55; H, 4.33; N, 16.70, found. C, 51.35; H, 4.39: N, 16.85.
ꢁ
ꢁ
ꢁ
ꢁꢁ
2ꢀ-Deoxy-5ꢀ-O- (4,4ꢀ-dimethoxytrityl) -2-fluoro-O6-
[2-(4-nitrophenyl)ethyl]inosine (8)
Compound 7 (1.49 g, 3.54 mmol), after azeotropic evaporation from
pyridine (5 mL × 3), was dissolved in dried pyridine (18 mL), and 4,4ꢁ-
dimethoxytrityl chloride (1.32 g, 3.9 mmol) was added to the solution. After
stirring for 12 hours, the mixture was quenched with water (2 mL), diluted

202
E. Kawashima et al.
with chloroform (70 mL), and washed with 5% aqueous sodium hydrogen
carbonate solution (50 mL × 2) and water (50 mL). After drying over an-
hydrous magnesium sulfate, the organic layer was evaporated to dryness,
and the residue was subjected to chromatographic separation on a column
of silica gel using methanol/chloroform as eluent to give 8 (2.39 g, 93.5%
yield) as a white glass; 1H-NMR (CDCl3) δ 2.54 (m, 1H, H-2ꢁꢁ), 2.77 (m, 1H,
H-2ꢁ), 3.32 (t, 2H, J = 6.9 Hz, –OCH2CH2PhNO2), 3.35–3.47 (m, 2H, H-5ꢁ
and –5ꢁꢁ), 3.78 (s, 6H, –OCH3 × 2 of the DMTr group), 4.12 (m, 1H, H-4ꢁ),
4.64–4.68 (m, 1H, H-3ꢁ), 4.83 (t, 2H, J = 6.9 Hz, –OCH2CH2PhNO2), 6.37
(t, 1H, J 1 ,2 = J 1 ,2 = 6.9 Hz, H-1ꢁ), 6.79 (d,4H, J = 10.5 Hz, Ar-H of the
DMTr group), 7.17–7.29 (m, 9H, Ar-H of the DMTr group × 9), 7.38 (d, 2H,
J = 6.9 Hz, Ar-H of the NPE group × 2), 8.03 (s, 1H, H-8), 8.17 (d, 2H, J =
6.9 Hz, Ar-H of the NPE group × 2); 19F-NMR (CDCl3) δ –49.29 (s); ESI-TOF
MS calcd for C39H36FN5O8 (M+H)+ 722.2626, found 722.2597; Anal. calcd
for C39H36FN5O8 + 0.5 H2O; C, 64.10; H, 5.10; N, 9.58, found. C, 63.94; H,
5.21: N, 9.97.
ꢁ
ꢁ
ꢁ
ꢁꢁ
3ꢀ-O-Acetyl-2ꢀ-deoxy-2-fluoro-O6-[2-(4-nitrophenyl)ethyl]inosine
(9)
Compound 8[4,5] (0.375 g, 0.52 mmol), after azeotropic evaporation
from pyridine (5 mL × 3), was dissolved in dried pyridine (18 mL),
and acetic anhydride (0.30 mL, 3.1 mmol) was added to the solution.
After stirring for 3 hours, the solution was evaporated to dryness to give
3ꢁ-O-acetyl-2ꢁ-deoxy-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2-fluoro-O6-[2-(4-nitrophen-
yl)ethyl]inosine (0.397 g), which was used in the next reaction without pu-
1
rification; H-NMR (CDCl3) δ 2.16 (s, 3H, CH3 of the Ac group), 2.63 (m,
1H, H-2ꢁꢁ), 2.87 (m, 1H, H-2ꢁ), 3.32 (t, 2H, J = 6.9 Hz, –OCH2CH2PhNO2),
3.36–3.48 (m, 2H, H-5ꢁ and –5ꢁꢁ), 3.78 (s, 6H, –OCH3 × 2 of the DMTr
group), 4.26 (m, 1H, H-4ꢁ), 4.83 (t, 2H, J = 6.9 Hz, –OCH2CH2PhNO2),
5.48 (m, 1H, H-3ꢁ), 6.40 (dd, 1H, J 1 ,2 = 8.4 Hz, J 1 ,2 = 5.7 Hz, H-1ꢁ), 6.93
(d,4H, J = 11.1 Hz, Ar-H of the DMTr group), 7.20–7.44 (m, 9H, Ar-H of
the DMTr group × 9), 7.50 (d, 2H, J = 8.7 Hz, Ar-H of the NPE group ×
2), 8.11 (s, 1H, H-8), 8.18 (d, 2H, J = 8.7 Hz, Ar-H of the NPE group × 2).
3ꢁ-O-Acetyl-2ꢁ-deoxy-5ꢁ-O-(4,4ꢁ-dimethoxytrityl)-2-fluoro-O6-[2-(4-nitroph-
enyl)ethyl]inosine (0.397 g, 0.52 mmol) was treated with a 2%
Cl3CCOOH/CH2Cl2 (6 mL)-methanol (2 mL) solution by stirring for
1 hour at room temperature. The mixture was neutralized with 5% aqueous
sodium hydrogen carbonate solution (50 mL), diluted with chloroform
(50 mL), and washed with water (50 mL). After drying over anhydrous
magnesium sulfate, the organic layer was evaporated to dryness, and the
residue was subjected to chromatographic separation on a column of silica
gel using methanol/chloroform as eluent to give 9 (0.223 g, 93%) as a white
glass; 1H-NMR (CDCl3) δ 2.13 (s, 3H, CH3 of the Ac group), 2.46 (dd, 1H,
ꢁ
ꢁ
ꢁ
ꢁꢁ

Synthesis and Evaluation of Pyrrole polyamide-2ꢁ-deoxyguanosine 5ꢁ-phosphate Hybrid 203
J 1 , 2 = 5.1 Hz, J 2 , 2 = 13.8 Hz, H-2ꢁꢁ), 3.05 (m, 1H, H-2ꢁ), 3.33 (t, 2H, J
=ꢁ 6.9 Hz, –OCH2CH2PhNO2), 3.88–4.00 (m, 2H, H-5ꢁ and –5ꢁꢁ), 4.25 (m,
ꢁꢁ
ꢁ
ꢁꢁ
1H, H-4ꢁ), 4.85 (t, 2H, J = 6.9 Hz, –OCH2CH2PhNO2), 5.53 (d, 1H, J 2 , 3
=
ꢁ
ꢁ
5.7 Hz, H-3ꢁ), 6.28 (dd, 1H, J 1 , 2 = 9.6 Hz, J 1 , 2 = 5.1 Hz, H-1ꢁ), 7.50 (d,
2H, J = 8.4 Hz, Ar-H of the NPE group × 2), 8.00 (s, 1H, H-8), 8.18 (d, 2H,
J = 8.4 Hz, Ar-H of the NPE group × 2); 19F-NMR (CDCl3) δ –49.18 (s);
ESI-TOF MS calcd for C20H21FN5O7 (M+H)+ 462.1425, found 462.1442;
Anal. calcd for C20H20FN5O7 + 0.5 H2O; C, 51.07; H, 4.50; N, 14.89, found.
C, 50.92; H, 4.35: N, 14.82.
ꢁ
ꢁ
ꢁ
ꢁꢁ
Pyrrole Amide Tetramer-2’-O-acetyl-2’-deoxy-O6-[2-(4-
nitrophenyl)ethyl]guanosine Hybrid Derivative
11
Compound 9 (0.242 g, 0.525 mmol) and 10 (0.385 g, 0.5 mmol) were
dissolved in DMF (2 mL), and triethylamine (0.4 mL) was added to the
solution. After stirring for 10 hours at 60◦C, the solution was evaporated.
The residue was subjected to chromatographic separation on a column of
silica gel using methanol/ethyl acetate as eluent to give 11 (0.311 g, 63%)
as a yellow glass; 1H-NMR (CDCl3) δ 1.86 (m, 2H, –CH2CH2CH2–), 2.06 (s,
ꢁ
ꢁ
ꢁꢁ
H-2ꢁꢁ), 3.18–3.24 (m, 1H, H-2ꢁ), 3.22 (t, 2H, J =ꢁ6ꢁ .6 Hz, –OCH2CH2PhNO2),
3H, CH3 of the Ac group), 2.33 (dd, 1H, J 1 ,2 = 5.7 Hz, J 2 ,2 = 13.8 Hz,
3.46–3.58 (m, 4H, –CH2CH2CH2–), 3.80–3.90 (m, 2H, H-5ꢁ and –5ꢁꢁ), 3.86
(s, 3H, CH3), 3.90 (s, 3H, CH3), 3.93 (s, 3H, CH3), 3.97 (s, 3H, CH3),
4.20–4.22 (m, 1H, H-4ꢁ), 4.70 (t, 2H, J = 6.6 Hz, –OCH2CH2PhNO2), 5.53
ꢁ
ꢁ
ꢁ
(d, 1H, J 2 ,3 = 5.4 Hz, H-3 ), 6.12 (dd, 1H, J = 2.7 Hz, J = 3.9 Hz, Py-H), 6.24
(dd, 1H, J 1 ,2 = 9.6 Hz, J 1 ,2 = 5.7 Hz, H-1ꢁ), 6.38 (s, 1H, N2-H), 6.71–6.77
(m, 5H, Py-H × 5), 7.12 (m, 2H, Py-H × 2), 7.14 (s, 1H, Py-H), 7.26 (m, 1H,
CH2NHCO-Py), 7.43 (d, 2H, J = 8.7 Hz, Ar-H of the NPE group × 2), 7.67
(s, 1H, H-8), 7.73 (s, 1H, –NHCO–), 7.82 (s, 1H, –NHCO–), 8.06 (s, 1H,
–NHCO–), 8.11 (d, 2H, J = 8.7 Hz, Ar-H of the NPE group × 2); ESI-TOF
MS calcd for C47H53N14O11 (M+H)+ 989.4018, found 989.3993; Anal. Calcd
for C47H52N14O11+ 1.5 H2O: C, 55.56; H, 5.45; N, 19.30, found. C, 55.49; H,
5.43; N, 19.50.
ꢁ
ꢁ
ꢁ
ꢁꢁ
Pyrrole Amide Tetramer-2ꢀ-deoxyguanosine 5ꢀ-phoasphate Hybrid
(Hybrid 4)
Compound 11 (0.198 g, 0.2 mmol) was dissolved in dichloromethane
(1.6 mL), and cyanoethyl N,N,Nꢁ,Nꢁ-tetraisopropylphosphorodiamidite
(96 μL, 0.3 mmol) and 0.5 M 1H-tetrazole/acetonitrile solution (0.44 mL,
0.22 mmol) were added to the solution. After stirring for 30 min, triethy-
lamine (31 μL, 0.22 mmol) was added. The mixture was evaporated to
dryness and the residue was subjected to chromatographic separation on a

204
E. Kawashima et al.
column of silica gel using 0.1% triethylamine/ethyl acetate/hexane as elu-
ent to give phosphoramidite derivative 12 (a diastereomer mixture) (0.156 g,
66%) as a slightly yellow glass; 31P-NMR (CDCl3) δ 149.83 and 149.94.
Compound 12 (20 mg, 0.168 mmol) was treated with 0.5 M acetic
acid/acetonitrile solution (0.5 mL) and H2O (0.2 mL). After stirring for 30
minutes, the mixture was evaporated. The residue was treated with 0.02 M
I2/H2O-pyridine-THF solution (3 mL). After stirring for 20 minutes, 1 M
NaS2O3/H2O solution was added, and the mixture was evaporated. The
residue was treated with 2 M NaOH/H2O solution (2 mL) and methanol
(3 mL) at room temperature for 17 hours. The solution was neutralized with
Dowex 50 W × 8 (H+ form) and the resin was instantly removed by filtration.
The filtrate was allowed to stand for 1 hour in an ice-bath and Hybrid 4 was
separated by centrifugation as a white powder in 88% yield (13 mg); 1H-NMR
(CD3OD) δ 1.90 (m, 2H, –CH2CH2CH2–), 2.27 (m, 1H, H-2ꢁꢁ), 2.81 (m, 1H,
H-2ꢁ), 3.39 (m, 2H, –CH2CH2CH2–), 3.50 (m, 2H, –CH2CH2CH2–), 3.87
(m, 1H, H-5ꢁꢁ), 3.86 (s, 3H, CH3), 3.91 (s, 6H, CH3 × 2), 3.93 (s, 3H, CH3),
4.04–4.09 (m, 2H, H-4ꢁ and –5ꢁ), 4.62 (m, 1H, H-3ꢁ), 6.09 (m, 1H, Py-H),
6.28 (t, 1H, J 1 ,2 = J 1 ,2 = 6.9 Hz, H-1ꢁ), 6.83 (s, 1H, Py-H), 6.85 (s, 1H,
Py-H), 6.87 (m, 1H, Py-H), 6.93 (m, 2H, Py-H × 2), 7.22 (m, 2H, Py-H ×
2),7.26 (s, 1H, Py-H), 8.04 (s, 1H, H-8), 7.73; 31P-NMR (CD3OD) δ 0.47;
ESI-TOF MS calcd for C37H44N13O11P (M + H)+ 878.3099, found 878.3096.
ꢁ
ꢁ
ꢁ
ꢁꢁ
Biological Activity of Pyrrolepolyamide Compounds
The effect of pyrrole polyamide compounds [Hybrids 1–4 and distamycin
A hydrochloride (Sigma)] on the growth of mouse mammary carcinoma
FM3A[7] cells (Japan Health Science Foundation) was examined. Mouse
mammary carcinoma FM3A cells (3 × 104 cells/mL) were cultured in ES
medium (Wako Pure Chemical Industries, Ltd.) supplemented with 2% calf
serum at 37◦C in an atmosphere of 5% CO2, and containing 0 μM (control),
10 or 30 μM pyrrole polyamide compound (Hybrids 1–4 or distamycin A).
After 2 days, cell viability was determined according to the method of Jones
et al.[8] (Figure 3).
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