Synthesis and evaluation of nitro 5-deazaflavinpyrrolecarboxamide(s) hybrid molecules as novel DNA targeted bioreductive antitumor agents

Article abstract of DOI:10.1016/S0968-0896(97)10036-0

A series of 6-nitro-5-deazaflavins bearing at N(3) or N(10) position the pyrrolecarboxamide(s) group as DNA minor groove binder has been synthesized. These hybrid molecules show similar redox properties to those of 6-nitro-5- deazaflavins with no pyrrolecarboxamide(s) group, suggesting that they generate stable one- and two-electron reduction product(s). Electrolytic reductions of the hybrid molecules were carried out at a controlled potential under anaerobic conditions in the presence of plasmid pBR322 DNA. Significant conversions of the supercoiled circular pBR322 DNA (form I) to the open circular DNA (form II) have been found by treatment with the reductively activated 6-nitro-5-deazaflavin derivatives. Their DNA damaging effects have been found to be enhanced as the number of pyrrolecarboxamide group as the DNA binder increases. Antitumor activities of the hybrid molecules towards KB and L1210 cells were evaluated in vitro. It has been found that the antitumor effects of the compounds on KB cells slightly change and those on L1210 cells decrease as the number of the pyrrolecarboxamide group increases. These results reveal that the combination of 6-nitro-5-deazaflavin molecule with the pyrrolecarboxamide(s) group increase the DNA binding properties of the compounds, giving rise to promoted DNA damaging effects, and also suggest that the combination would affect the capacity of the compounds to act as the substrate for intracellular reductases and/or the cellular uptake of the compounds.

Full text of DOI:10.1016/S0968-0896(97)10036-0

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 301±314
Synthesis and Evaluation of Nitro 5-Deaza¯avin-
pyrrolecarboxamide(s) Hybrid Molecules as Novel DNA
Targeted Bioreductive Antitumor Agents
Yoshitomo Kanaoka,a Yoshihiro Ikeuchi,a Tetsuji Kawamoto,a*
Kiyoshi Bessho,a Naoshige Akimoto,a Yuji Mikata,b* Mamiko Nishida,b
Shigenobu Yano,b Takuma Sasakic and Fumio Yonedaa*
aFaculty of Pharmaceutical Sciences, Kyoto University, Sakyo-ku Kyoto 60601, Japan
bDepartment of Chemistry, Faculty of Science, Nara Women's University, Nara 630, Japan
cDepartment of Experimental Therapeutics, Cancer Research Institute, Kanazawa University, Takaramachi Kanazawa 920, Japan
Received 16 October 1997; accepted 20 October 1997
AbstractÐA series of 6-nitro-5-deaza¯avins bearing at N(3) or N(10) position the pyrrolecarboxamide(s) group as
DNA minor groove binder has been synthesized. These hybrid molecules show similar redox properties to those of 6-
nitro-5-deaza¯avins with no pyrrolecarboxamide(s) group, suggesting that they generate stable one- and two-electron
reduction product(s). Electrolytic reductions of the hybrid molecules were carried out at a controlled potential under
anaerobic conditions in the presence of plasmid pBR322 DNA. Signi®cant conversions of the supercoiled circular
pBR322 DNA (form I) to the open circular DNA (form II) have been found by treatment with the reductively acti-
vated 6-nitro-5-deaza¯avin derivatives. Their DNA damaging e€ects have been found to be enhanced as the number of
pyrrolecarboxamide group as the DNA binder increases. Antitumor activities of the hybrid molecules towards KB and
L1210 cells were evaluated in vitro. It has been found that the antitumor e€ects of the compounds on KB cells slightly
change and those on L1210 cells decrease as the number of the pyrrolecarboxamide group increases. These results
reveal that the combination of 6-nitro-5-deaza¯avin molecule with the pyrrolecarboxamide(s) group increase the DNA
binding properties of the compounds, giving rise to promoted DNA damaging e€ects, and also suggest that the com-
bination would a€ect the capacity of the compounds to act as the substrate for intracellular reductases and/or the
cellular uptake of the compounds. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
reduction8 and the activated drugs induce DNA dama-
ges due to strand breaks9 and helix destabilization,10
which subsequently lead to cytotoxicities11 as well as
antimicrobial activities.12
Hypoxic cells1 present in human tumors represent clin-
ical problems in the chemotherapy of solid tumors
because they limit the eciency of fractionated radio-
therapy2 and are also resistant to many chemother-
apeutic drugs.3 Bioreductive drugs,4 which are cytotoxic
maximally in the absence of oxygen, have attracted
considerable attention as selective cytotoxins towards
hypoxic cells. Among bioreductive drugs, nitrohetero-
aromatic compounds5 have been studied most exten-
sively and used clinically as selective cytotoxins towards
hypoxic cells6 as well as radiosensitizers.7 It is generally
accepted that these drugs undergo enzymatic one-electron
Recently, we have developed nitro 5-deaza¯avins13,14 as
a novel class of nitrohetero-aromatic compounds con-
taining an electrophilic redox coenzyme ring system. It
has been found that a series of nitro 5-deaza¯avins
shows signi®cant antitumor activities13 and that 6- and
8-nitro derivatives which generate stable one-electron
reduction products show marked selective cytotoxicities
towards hypoxic cells.14 We have also demonstrated
that there is signi®cant interaction between a 5-deaza-
¯avin molecule and DNA by using 5-deaza¯avin
derivatives linked to modi®ed oligonucleotides.15 From
*Corresponding author.
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(97)10036-0

302
Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
these points of view, introduction of DNA binding
group into nitro 5-deaza¯avin molecule would be
anticipated to provide higher concentration of the
reductively activated product around DNA as well as to
promote the interaction of 5-deaza¯avin ring system
with DNA, which could give rise to higher antitumor
activities. It should be intriguing to investigate DNA
interaction and antitumor activities of nitro 5-deaza-
¯avin derivatives bearing a DNA binding group. Oligo-
(pyrrolecarboxamides) netropsin, distamycin, and their
analogues have received considerable attention not
solely because of their antitumor and antibiotic activ-
ities but also their non-intercalative and non-covalent
binding to minor groove of AT rich regions in double
stranded B-DNA.16 And recently, the oligo(pyrrole-
carboxamides) derivatives and their analogues have
been introduced most popularly as DNA binding group
into DNA alkylating reagents,17 DNA topoisomerase
inhibitors,18 photosensitizers,19 and radiosensitizers20
for enhancement of their DNA damaging e€ects and
antitumor activities.21
Results and Discussion
Synthesis of nitro 5-deaza¯avin-pyrrolecarboxamide(s)
hybrid molecules
10-Butyl-6-nitro-5-deaza¯avin 11 was prepared by con-
densation of 6-butylaminouracil with 2-¯uoro-6-nitro-
benzaldehyde22 according to Yoneda's method13,23
(74%). Treatment of 11 with tert-butyl bromoacetate in
DMF in the presence of potassium carbonate a€orded
12 (88%). Hydrolysis of 12 with hydrochloric acid gave
13 (85%) which was treated with N,N0-disuccinimidyl
carbonate in the presence of pyridine in acetonitrile to
a€ord 14 (78%). Condensations of 14 with the appro-
priate amines 18b±20b, which were obtained by hydro-
genation of 18a±20a24 (Scheme 3), in the presence of
DMAP in acetonitrile a€orded 2±4 (51±61%) (Scheme 2).
Also 10-(tert-butoxycarbonyl)methyl-3-methyl-6-nitro-5-
deaza¯avin 16 was prepared by condensation of 6-
(tert-butoxycarbonyl)methylamino-3-methyluracil
with 2-¯uoro-6-nitrobenzaldehyde22 (70%). Hydrolysis
of 16 with hydrochloric acid gave 17 (88%) and con-
densations of 17 with the amines 18b±20b24 (Scheme 3)
in the presence of 1,3-diisopropylcarbodiimide in DMF
a€orded 6±8 (26±28%) (Scheme 4).
15
As an extension of bioreductive drug approach, we have
designed a series of 6-nitro-5-deaza¯avins 2±4 and 6±8
bearing a pyrrolecarboxamide(s) at N(3) position or
N(10) position as novel DNA targeted bioreductive
antitumor agents(Scheme 1). Since amongnitro-positional
isomers of nitro 5-deaza¯avin derivatives, 6-nitro-5-
deaza¯avin derivatives show the most potent antitumor
activities as well as the highest selective cytotoxicities
towards hypoxic cells,13,14 the DNA binding group was
introduced into the 6-nitro derivatives. Considering the
cellular uptakes of the compounds,17c,18b alkyl (butyl)
group was employed as C-terminal group of the pyrrole-
carboxamide(s). In the present paper, we wish to
describe synthesis, redox properties, DNA interactions,
and antitumor activities of the 6-nitro-5-deaza¯avin-
pyrrolecarboxamide(s) hybrid molecules.
Compounds 1 and 5 (Scheme 1) with no 1-methylpyr-
role group were also prepared to compare their anti-
tumor activities with those of 2±4 and 6±8, by alkylation
of 11 with N-butylbromoacetoamide in the presence of
potassium carbonate in DMF and by condensation of 6-
(butylcarbamoyl)methylamino-3-methyluracil 22 and 2-
¯uoro-6-nitrobenzaldehyde22 respectively. As reference
compounds, compounds 9 (Scheme 2) and 10 (Scheme 4)
with no nitro group on the 5-deaza¯avin ring and bis(-
pyrrolecarboxamide) derivative 21 (Scheme 3) with no
(nitro) 5-deaza¯avin nucleus were synthesized. These
compounds are suitable for comparison with the nitro
O
N
R
O
R
H
N
H3C
O
N
N
H
N
O
n
N
C4H9
O
N
N
N
O
C4H9
CH3
H
N
H
N
O
n
1; n = 0, R = NO2
2; n = 1, R = NO2
3; n = 2, R = NO2
4; n = 3, R = NO2
9; n = 2, R = H
5; n = 0, R = NO2
6; n = 1, R = NO2
7; n = 2, R = NO2
8; n = 3, R = NO2
10; n = 2, R = H
C4H9
N
O
CH3
Scheme 1.

Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
303
O
N
R
O
R
O
N
R
t-C4H9O2CH2C
HO2CH2C
O
HN
N
N
i
ii
O
N
O
N
N
N
C4H9
C4H9
C4H9
13; R = NO2
25; R = H
11; R = NO2
23; R = H
12; R = NO2
24; R = H
O
O
O
R
O
N
R
H
N
N
O2CH2C
O
N
N
H
N
O
O
iii
iv
C4H9
N
N
N
N
O
C4H9
CH3
C4H9
n
14; R = NO2
26; R = H
2; n = 1, R = NO2
3; n = 2, R = NO2
4; n = 3, R = NO2
9; n = 2, R = H
Scheme 2. Synthesis of compounds 2±4, and 9: i) tert-butyl bromoacetate, K2CO3, DMF, 60 ꢀC; ii) 12N HCl, 60 ꢀC; iii) N,N0-Di-
succinimidyl carbonate, Pyridine, CH3CN, rt; iv) 18b±20b, DMAP, CH3CN, rt.
R
H
N
18a; n = 0, R = NO2, 18b; n = 0, R = NH2
19a; n = 1, R = NO2, 19b; n = 1, R = NH2
20a; n = 2, R = NO2, 20b; n = 2, R = NH2
21 ; n = 1, R = NHCOCH3
N
H
N
O
CH3
C4H9
N
O
CH3
n
Scheme 3.
O
N
R
O
N
R
O
R
H3C
H3C
H3C
N
N
N
i
ii
O
N
O
N
O
N
N
CH2
CO2t-C4H9
CH2
CO2H
H
N
H
N
O
C4H9
N
O
CH3
16; R = NO2
27; R = H
17; R = NO2
28; R = H
n
6; n = 1, R = NO2
7; n = 2, R = NO2
8; n = 3, R = NO2
10; n = 2, R = H
Scheme 4. Synthesis of compounds 6±8 and 10: 1) 12N HCl, 60 ꢀC; ii) 18b±20b, 1,3-Diisopropylcarbodiimide, CH3CN or DMF, rt.

304
Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
5-deaza¯avin containing hybrid molecules 3 and 7 in
terms of antitumor activities.
Table 1. Reduction potentials (Ep (V)) of 6-nitro-5-deaza-
¯avin-pyrrolecarboxamide(s) hybrid molecules*
Ep (V)
Redox properties of nitro 5-deaza¯avin-pyrrolecarbox-
amide(s) hybrid molecules
1
2
3
4
5
6
7
8
0.548
0.534
0.550
0.545
0.520
0.504
0.530
0.530
0.880
0.870
0.880
0.870
0.834
0.822
0.855
0.840
It is well discussed that electron anity11,12 and stability
of one-electron reduction product25 of a bioreductive
drug a€ect signi®cantly the antitumor activity as well as
the selective cytotoxicity towards hypoxic cells. To
investigate the redox properties of the nitro 5-deaza-
¯avin-pyrrolecarboxamide(s) hybrid molecules, the
reduction potentials of compounds 1±8 were measured by
means of cyclic voltammetry.14 The cyclic voltammo-
grams of compounds 4 and 8 are exempli®ed in Figure 1.
As Table 1 shows, the reduction potentials of the hybrid
molecules 2±4 and 6±8 are similar to those of 6-nitro-
5-deaza¯avin derivatives 1 and 5 with no pyrrole-
carboxamide(s) group. Furthermore, the hybrid
molecules have been found to show similar patterns of
cyclic voltammograms14 to those of other 6-nitro-5-
deaza¯avin derivatives with no pyrrolecarboxamide(s)
group, indicating that these hybrid molecules generate
stable one- and two-electron reduction product(s). From
these results, the hybrid molecules could be anticipated
to provide higher concentrations of the stable one-elec-
tron reduction products around DNA, which would
induce greater DNA damages as well as higher anti-
tumor activities.
*All the potentials were measured at 298 K in DMF,
3
1
[Compound]=1.0Â10 (M), [Bu4NClO4]=1.0Â10 (M) ver-
sus an aqueous Ag/AgCl reference electrode under N2.
¯avin chromophore are observed.26 We also demon-
strated that similar spectral changes are found through
the interaction of 5-deaza¯avin ring system with DNA.27
To investigate the interactions of (nitro) 5-deaza¯avin
derivatives 1±10 with DNA, visible spectra of the com-
pounds 1±10 were measured in the presence of calf thy-
mus DNA, poly (dA-dT), and poly (dG-dC),
accordingly. Essentially no signi®cant spectral change
was found in compounds 1 and 5 with no DNA binding
group, however, as the number of the pyrrolecarbox-
amide group increased, signi®cant spectral changes of
compounds 3, 4, 7, 8, 9, and 10 were observed. The
visible spectra of compounds 4 and 8 in the presence of
calf thymus DNA are exempli®ed in Figure 2. As
Figure 2 shows, hypochromicity and red-shift in visible
absorption band characteristic of the 5-deaza¯avin
chromophore have been observed and these spectra
have isosbestic points26 at 464 nm and 460 nm respec-
tively, suggesting that 5-deaza¯avin ring system of these
Interaction of nitro 5-deaza¯avin-pyrrolecarboxamide(s)
hybrid molecules with DNA
It is understood that when a ¯avin (isoalloxazine) ring
system interacts with DNA, hypochromicity and red-
shift of visible absorption band characteristic of the
Figure 1. Cyclic Voltammograms of compounds 4 (a) and 8 (b) at 298 K in DMF, [Compound]=1.0Â10 3 (M), [Bu4NClO4]=
1.0Â10 1 (M) versus an aqueous Ag/AgCl reference electrode under N2.

Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
305
1
compounds interacts with DNA. Thus, the binding
Table 2. Estimated apparent binding constants (KappÂ105 M
)
of compounds 3, 4, 7, 8, 9, and 10
constants of the compounds 3, 4, 7, 8, 9, and 10 to calf
thymus DNA, poly (dA-dT), and poly (dG-dC) were
estimated from the hypochromicity of visible spectra. As
Table 2 shows, compounds 3, 4, and 9 bearing the pyr-
rolecarboxamide(s) at N(3) position bind more strongly
to poly (dA-dT) than to poly (dG-dC) and higher base
pair selectivity has been observed in tris(pyrrolecarbox-
amides) derivative 4 than in bis(pyrrolecarboxamides)
derivatives 3 and 9. In contrast to this result, com-
pounds 7, 8, and 10 possessing the pyrrolecarbox-
amide(s) at N(10) position bind to poly (dA-dT) as
weakly as to poly (dG-dC), showing essentially no base
pair selectivity. These results imply that spatial
arrangement of (nitro) 5-deaza¯avin nucleus and the
DNA binding group a€ects signi®cantly the interaction
of 5-deaza¯avin ring system with DNA. Compounds 9
and 10 containing a 5-deaza¯avin with no nitro group
gave similar spectral changes and binding constants to
those of compounds 3 and 7, suggesting that 5-deaza-
¯avin ring system of these compounds interacts similarly
with DNA regardless of the nitro group.
1
KappÂ105 M
Calf thymus
poly (dA-dT)
poly (dG-dC)
3
4
0.52
0.71
0.62
0.68
0.79
0.70
2.1
3.7
0.39
0.52
0.68
0.65
0.74
0.57
7
0.66
0.72
2.3
8
9
10
0.50
1,2,4-benzotriazine-1,4-di N-oxide derivative29 with
DNA under anaerobic conditions have been studied. It
is conceivable that nitro 5-deaza¯avin derivatives also
undergo enzymatic one-electron reduction to give rise to
antitumor activities, however, interaction of reductively
activated nitro 5-deaza¯avins with DNA has remained
to be studied.
In order to investigate the interaction of reductively
activated nitro 5-deaza¯avin derivatives with DNA,
compounds 1±8 were reduced electrolytically at a con-
trolled potential9,10,28 in the presence of plasmid
pBR322 DNA under anaerobic conditions. The hybrid
molecules 9 and 10 with no nitro group on 5-deaza¯avin
ring were employed as reference compounds. As Figure 3
shows, marked conversions of supercoiled circular DNA
(form I) into the open circular DNA (form II) have been
found by electrolytical reductions of nitro 5-deaza¯avin
derivatives 3, 4, 7, and 830,31. Essentially no DNA
Interaction of reductively activated nitro 5-deaza¯avin-
pyrrolecarboxamide(s) hybrid molecules with DNA
It has been well studied that the one-electron reduction
products25 of bioreductive drugs induce DNA damages
by causing strand breaks9 and helix destabilization,10
which subsequently give rise to cytotoxicities. To inves-
tigate mechanism of biological actions of the bioreductive
drugs, interactions of reduced nitroimidazoles9,10,28 or
Figure 2. Visible spectra of (a) 4, and (b) 8 in the presence of calf thymus DNA at 298 K, in 10 mM Tris±HCl bu€er (pH 7.0) con-
5
taining 20% of DMF, [Compound]=2.1Â10 (M), [calf thymus DNA]=(a) 0, (b) 1.6Â10 5, (c) 3.2Â10 5, (d) 4.8Â10 5, (e)
6.4Â10 5, (f) 8.0Â10 5 (M).

306
Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
Figure 3. Agarose gel electrophoresis patterns of plasmid pBR322 DNA after treatment of reductivity avtivated nitro 5-deaza¯avin
derivatives 1±8. Lane 1±9: Control, 8, 7, 6, 5, 4, 3, 2, 1. (The reductive activations of the compounds 1±8 were carried out at a constant
potential of 0.95 V versus an aqueous Ag/AgCl under N2 in 1.5 mM sodium citrate bu€er (pH 7.0) containing 25% of DMF).
[Compound]=1.0Â10 3 (M), [DNA]=5.0Â10 5 (M/base pair).
damage was observed in the electrolysis in the absence
of the nitro 5-deaza¯avin derivatives or even in the
presence of the hybrid molecules 9 and 10.32 These
experimental results strongly support that activation of
6-nitro-5-deaza¯avin derivatives by electrolytic reduc-
tion leads to DNA damage, suggesting that enzymatic
one-electron reduction of 6-nitro-5-deaza¯avin deriva-
tives could induce DNA damage. The conversion of
form I DNA into form II appears to be enhanced as the
number of pyrrolecarboxamide group increases. The
above results suggest that the higher concentrations of
reductively activated compounds would be provided
around DNA owing to the promoted DNA binding
properties of the compounds.
As compounds 1, 5, and other compounds reported in
our previous paper13 show, nitro 5-deaza¯avin deriva-
tives with no pyrrolecarboxamide(s) group generally
show more potent antitumor activities towards L1210
cells than KB cells (IC50(L1210)<IC50(KB)). Interest-
ingly enough, it has been found that there is a tendency
that the hybrid molecules 1±8 maintain similar anti-
tumor activities towards KB cells and show less anti-
tumor e€ects on L1210 cells as the number of the
pyrrolecarboxamide group increases. Considering the
results of interaction of reductively activated hybrid
molecules with DNA (vide supra), compounds 3, 4, 7,
and 8 bearing the increasing number of pyrrolecarbox-
amide group would be anticipated to show more potent
antitumor activities than compounds 1, 2, 5, and 6
bearing only one or no pyrrolecarboxamide group, if the
same concentrations of reductively activated compounds
are gained around DNA in tumor cells. However, the
In vitro antitumor activities of nitro 5-deaza¯avin-pyrrole-
carboxamide(s) hybrid molecules
To evaluate antitumor activities of the hybrid molecules
and other reference compounds, compounds 1±10 and
21 were tested for in vitro antitumor e€ects on human
oral epidermoid carcinoma KB cells and murine leuke-
mia L1210 cells by the MTT assay developed by Car-
michael.33,34 Mitomycin C and netropsin were also
employed as reference compounds. As Table 3 shows,
the 6-nitro-5-deaza¯avin-pyrrolecarboxamide(s) hybrid
molecules 2±4 and 6±8 have been found to show more
potent antitumor activities than netropsin and the bis-
(pyrrolecarboxamides) 21. The hybrid molecules 9 and
10 composed of 5-deaza¯avin with no nitro group and
bis(pyrrolecarboxamides) showed no signi®cant anti-
tumor activities. These results suggest that nitro 5-deaza-
¯avin nucleus is more relevant to the antitumor e€ects
of the compounds than the pyrrolecarboxamide(s)
group. It has been found that change in the number of
the pyrrolecarboxamide of compounds 1±8 a€ects
slightly the antitumor potencies of the hybrid molecules
towards KB cells. In contrast, the antitumor e€ects of
compounds 1±8 on L1210 cells decreased as the number
of the pyrrolecarboxamide increased.
Table 3. IC50 Values of 6-nitro-5-deaza¯avin-pyrrolecarbox-
amide(s) hybrid molecules and other reference compounds on
KB and L1210 cells growth in vitro
IC50 (mM)*
KB cells
L1210 cells
1
6.1
10.7
8.4
1.8
10.3
2
3
0.5
45.4
4
6.6
6.5
5
0.4
9.3
6
4.3
7
23.5
8.1
73.1
69.2
8
9
10
>100
>100
>100
0.4
>100
>100
>100
0.6
21
Mitomycin C
Netropsin
>100
>100
*IC50 (mM) was given as the concentration at 50% inhibition of
cell growth.

Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
307
antitumor activities of the compounds did not heighten
despite the increase in the number of the pyrrole-
carboxamide group. These results imply that introduc-
tion of pyrrolecarboxamide(s) group into 6-nitro-5-
deaza¯avin molecule would enhance the direct interac-
tion of nitro 5-deaza¯avin nucleus with DNA, however,
would a€ect rather adversely cellular uptakes of the
compounds and/or capacities of the compounds to act
as substrates for intracellular reductases.
powder: mp 253±256 ꢀC. IR (KBr) 1709, 1676 1618,
1
1524 cm 1. H NMR (Me2SO-d6) d 0.99 (3H, t, J ˆ 7:4
Hz), 1.38±1.62 (2H, m), 1.62±1.83 (2H, m), 4.71 (2H,
m), 8.09 (1H, t, J ˆ 7:8 Hz), 8.20 (1H, d, J ˆ 7:8 Hz),
8.33 (1H, d, J ˆ 8:8 Hz), 9.11 (1H, s), 11.33 (1H, s).
Anal. calcd for C15H14N4O4: C, 57.32; H, 4.49; N,
17.83. Found: C, 57.22; H, 4.47; N, 17.73.
3-(tert-Butoxycarbonyl)methyl-10-butyl-6-nitro-5-deaza-
¯avin (12). A mixture of 10-butyl-6-nitro-5-deaza¯avin
(11; 1.57 g 5.0 mmol), tert-butyl bromoacetate (1.95 g,
10.0 mmol), and well pulverized K2CO3 (2.07 g,
15.0 mmol) in 15 mL of DMF was warmed at 60 ꢀC for
1 h under argon atmosphere. The solvent was evapo-
rated under reduced pressure and the residue was
extracted with chloroform. The organic layer was
washed with brine once and was dried over MgSO4. The
solvent was evaporated under reduced pressure and the
resulting yellow solid was recrystallized from ethyl
Conclusion
We ®rst synthesized 6-nitro-5-deaza¯avin-pyrrole-
carboxamide(s) hybrid molecules as novel DNA tar-
geted bioreductive antitumor agents and evaluated their
redox properties, interactions with DNA, and in vitro
antitumor activities. And we ®rst demonstrated the
interaction of reductively activated 6-nitro-5-deaza¯avin
derivatives with DNA. Introduction of DNA binding
pyrrolecarboxamide(s) group into 6-nitro-5-deaza¯avin
molecule enhances DNA binding properties of the
compounds, giving rise to marked DNA damage.
Although antitumor activities of the present hybrid
molecules did not heighten, we consider that further
modi®cation of the compounds would promote cellular
uptakes as well as reductive activations of the com-
pounds to a€ord the more potent antitumor agents
which would be useful for chemotherapy of solid tumors.
acetate to a€ord 1.89 g (88%) of 12 as yellow crystals:
1
mp 208±210 ꢀC. IR (KBr) 1736, 1660, 1624, 1531 cm
.
1H NMR (CDCl3) d 1.03 (3H, t, J ˆ 7:2 Hz), 1.49 (9H,
s), 1.53±1.71 (2H, m), 1.71±1.93 (2H, m), 4.74 (2H, s),
4.80 (2H, br), 7.89±8.03 (2H, m), 8.07 (1H, dd, J ˆ 6:8,
2.4 Hz), 9,41 (1H, s). Anal. calcd for C21H24N4O6: C,
58.87; H, 5.65; N, 13.08. Found: C, 58.84; H, 5.74; N,
12.92.
10-Butyl-6-nitro-5-deaza¯avin-3-acetic acid (13). A solu-
tion of 3-(tert-butoxycarbonyl)methyl-10-butyl-6-nitro-
5-deaza¯avin (12; 0.64 g, 1.5 mmol) in 5 mL of 12 N
hydrochloric acid was warmed at 60 ꢀC with vigorous
stirring for 1 h under argon atmosphere. The solvent
was evaporated to dryness under reduced pressure and
the residue was recrystallized from ethanol±water to
Experimental
Melting points were taken using a Mettler thermosystem
FP80HT and are uncorrected. Infrared (IR) spectra
.
were recorded on
a
Shimadzu IR-400 spectro-
a€ord 0.49 g (85%) of 13 (obtained as 13 0.5 H2O) as
photometer. Proton nuclear magnetic resonance (1H
NMR) spectra were recorded on a JEOL FX200
(200 MHz) spectrometer in CDCl3 or Me2SO-d6 with
tetramethylsilane (TMS) as an internal standard and
chemical shifts are given in ppm. Low-resolution FAB
(fast atom bombardment) mass spectra (MS (FAB)) and
high-resolution FAB mass spectra (HRMS (FAB)) were
recorded on JEOL JMS-HX/HX110A. Column chro-
matography was carried out on silica±gel (Kieselgel 60,
70±230 mesh, Merck).
yellow powder: mp 123±125 ꢀC. IR (KBr) 1743, 1711,
1
1655, 1620, 1531 cm
.
1H NMR (Me2SO-d6) d 0.98
(3H, t, J ˆ 7:4 Hz), 1.38±1.62 (2H, m), 1.62±1.84 (2H,
m), 4.57 (2H, s), 4.62±4.88 (2H, m), 8.14 (1H, t, J ˆ 8:4
Hz), 8.25 (1H, d, J ˆ 7:4 Hz), 8.40 (1H, d, J ˆ 8:8 Hz),
.
9.22 (1H, s). Anal. calcd for C17H16N4O6 0.5H2O: C,
53.54; H, 4.49; N, 14.69. Found: C, 53.70; H, 4.64; N,
14.40.
10-Butyl-6-nitro-5-deaza¯avin-3-acetic acid
N-hydroxysuccinimide ester (14). To a suspension of 10-
butyl-6-nitro-5-deaza¯avin-3-acetic acid (13; 372 mg,
1.00 mmol) in 3 mL of acetonitrile was added pyridine
(0.16 mL, 2.00 mmol) and N,N0-disuccinimidyl carbo-
nate (512 mg, 2.00 mmol) and the reaction mixture was
stirred at room temperature under argon atmosphere
for 16 h. As the reaction proceeded, the suspension
turned into yellow solution. The solvent was evaporated
under reduced pressure and the residue was extracted
with chloroform. The organic layer was washed with
10-Butyl-6-nitro-5-deaza¯avin (11). A mixture of 6-
butylaminouracil23 (1.83 g, 10.0 mmol) and 2-¯uoro-6-
nitrobenzaldehyde22 (1.69 g, 10.0 mmol) in 25 mL of
DMF was heated at 120 ꢀC for 2 h under argon atmo-
sphere. The reaction mixture was poured onto 500 mL
of ice-water and the precipitate was collected by ®ltra-
tion. The precipitate was dried under reduced pressure
at room temperature and recrystallized from chloro-
form±ethanol to a€ord 2.33 g (74%) of 11 as yellow

308
Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
brine once and was dried over MgSO4. The solvent was
evaporated under reduced pressure and the residue was
subjected to column chromatography on silica gel (ethyl
acetate as eluent). The yellow solid obtained was
recrystallized from ethyl acetate±ether to a€ord 373 mg
500 mL of ice-water and precipitate appeared was col-
lected by ®ltration. The precipitate was dried under
reduced pressure at room temperature and was recrys-
tallized from chloroform±ethanol to a€ord 2.70 g (70%)
of 16 as yellow crystals: mp 209±212 ꢀC. IR (KBr) 1732,
1659, 1622, 1537 cm 1. 1H NMR (CDCl3) d 1.49 (9H, s),
3.48 (3H, s), 5.57 (2H, br), 7.62 (1H, d, J ˆ 8:6 Hz), 7.93
(1H, t, J ˆ 8:6 Hz), 8.07 (1H, d, J ˆ 7:8 Hz), 9.44 (1H,
s). Anal. calcd for C18H18N4O6: C, 55.96; H, 4.70; N,
14.50. Found: C, 56.02; H, 4.66; N, 14.38.
.
(78%) of 14 (obtained as 14 0.5 H2O) as yellow powder:
1
mp 138±141 ꢀC. IR (KBr) 1743, 1664, 1624, 1531 cm
.
1H NMR (CDCl3) d 1.04 (3H, t, J ˆ 7:2 Hz), 1.41±1.73
(2H, m), 1.73±1.97 (2H, m), 2.84 (4H, s), 4.81 (2H, br),
5.16 (2H, s), 7.91±8.06 (2H, m), 8.09 (1H, dd, J ˆ 6:8,
2.4 Hz),
9.46
(1H,
s).
Anal.
calcd
for
.
C21H19N5O8 0.5H2O: C, 52.72; H, 4.21; N, 14.64.
Found: C, 52.98; H, 4.16; N, 14.43.
3-Methyl-6-nitro-5-deaza¯avin-10-acetic acid (17). A
suspension of 10-(tert-butoxycarbonyl)methyl-3-methyl-
6-nitro-5-deaza¯avin (16; 1.35 g, 3.5 mmol) in 5.6 mL of
12N HCl (70.0 mmol) was warmed at 60 ꢀC with vigor-
ous stirring for 1 h under argon atmosphere. The pre-
cipitate was collected by ®ltration and was washed with
ethanol. The precipitate was dried under reduced pres-
sure at room temperature togive 1.02 g (88%) of 17 as
yellow powder: mp 226±228 ꢀC. IR (KBr) 1718, 1622,
6-(tert-Butoxycarbonyl)methylamino-3-methyluracil (15).
A
mixture of 6-chloro-3-methyluracil (3.21 g,
20.0 mmol), glycine tert-butyl ester hydrochloride
(3.35 g, 20.0 mmol), DBU (3.0 mL, 20.0 mmol), and
N,N-diisopropylethylamine (6.97 mL, 40.0 mmol) in
50 mL of 1-butanol was heated to re¯ux under argon
atmosphere for 3 h. The reaction mixture was cooled at
room temperature and deposited crystals were collected
by ®ltration. The crystals were washed with ethanol and
were dried under reduced pressure at room temperature
to a€ord 3.62 g (71%) of 15 as white crystals. mp 242±
1
1597, 1529 cm 1. H NMR (Me2SO-d6) d 3.27 (3H, s),
5.58 (2H, s), 8.09 (1H, t, J ˆ 8:2 Hz), 8.19±8.33 (2H, m),
9.24 (1H, s). Anal. calcd for C14H10N4O6: C, 50.92; H,
3.05; N, 16.95. Found: C, 50.94; H, 3.01; N, 16.80.
246 ꢀC dec. IR (KBr) 1741, 1709, 1603 cm 1. H NMR
10-Butyl-5-deaza¯avin (23). A mixture of 6-butylamino-
uracil23 (1.83 g, 10.0mmol) and 2-¯uorobenzaldehyde
(1.24 g, 10.0mmol) in 25 mL of DMF was heated at 120 ꢀC
for 3 h under argon atmosphere. The reaction mixture was
cooled and deposited crystals were collected by ®ltration.
RecrystallizationfromDMFa€orded2.36 g(88%)of23as
pale yellow crystals: mp 274±276 ꢀC. IR (KBr) 1695, 1666,
1
(Me2SO-d6) d 1.44 (9H, s), 3.04 (3H, s), 3.82 (2H, d,
J ˆ 5:8 Hz), 4.52 (1H, s), 6.28 (1H, t, J ˆ 5:8 Hz), 10.51
(1H, br). Anal. calcd for C11H17N3O4: C, 51.76; H, 6.71;
N, 16.46. Found: C, 51.54; H, 6.74; N, 16.41.
6-(Butylcarbamoyl)methylamino-3-methyluracil (22). A
mixture of 6-chloro-3-methyluracil (3.21 g, 20.0 mmol),
N-butyl-2-aminoacetamide
1
1612 cm 1. H NMR (Me2SO-d6) d 0.98 (3H, t, J ˆ 7:2
hydrochloride
(3.33 g,
Hz), 1.37±1.60 (2H, m), 1.60±1.84 (2H, m), 4.53±4.88 (2H,
br), 7.48±7.60 (1H, m), 7.88±8.03 (2H, m), 8.19 (1H, d,
J ˆ 7:6 Hz), 9.00 (1H, s), 11.10 (1H, br). Anal. calcd for
C15H15N3O2: C, 66.90; H, 5.61; N, 15.60. Found: C, 66.83;
H, 5.66; N, 15.55.
20.0 mmol), DBU (3.0 mL, 20.0 mmol), and N,N-diiso-
propylethylamine (6.97 mL, 40.0 mmol) in 50 mL of 1-
butanol was heated to re¯ux under argon atmosphere
for 3 h. The reaction mixture was cooled at room tem-
perature and deposited crystals were collected by ®ltra-
tion. The crystals were washed with ethanol and were
dried under reduced pressure at room temperature to
a€ord 3.76 g (74%) of 22 as white crystals: mp 275±
3-(tert-Butoxycarbonyl)methyl-10-butyl-5-deaza¯avin
(24). A mixture of 10-butyl-5-deaza¯avin (23; 2.69 g,
10.0 mmol), tert-butyl bromoacetate (3.90 g, 20.0 mmol),
and well pulverized K2CO3 (4.15 g, 30.0 mmol) in 30 mL
of DMF was warmed at 60 ꢀC for 1 h under argon
atmosphere. The K2CO3 was removed by ®ltration and
the solvent was evaporated under reduced pressure. The
residue was extracted with chloroform and the organic
layer was washed with brine once. The organic layer was
dried over MgSO4 and the solvent was evaporated
under reduced pressure to give yellow solid. The solid
was recrystallized from chloroform±ethanol to a€ord
2.99 g (78%) of 24 as yellow crystals: mp 204±206 ꢀC. IR
279 ꢀC dec. IR (KBr) 1726, 1618, 1568 cm 1. H NMR
1
(Me2SO-d6) d 0.88 (3H, t, J ˆ 6:6 Hz), 1.16±1.52 (4H,
m), 3.03 (3H, s), 3.10 (2H, d, J ˆ 6:2 Hz), 3.65 (2H, d,
J ˆ 3:6 Hz), 4.42 (1H, s), 6.37 (1H, br), 8.05 (1H, br),
10.41 (1H, br). Anal. calcd for C11H18N4O3: C,
51.96; H, 7.13; N, 22.03. Found: C, 52.04; H, 6.91; N,
22.27.
10-(tert-Butoxycarbonyl)methyl-3-methyl-6-nitro-5-deaza-
¯avin (16). A mixture of 6-(tert-butoxycarbonyl)methyl-
amino-3-methyluracil (15; 2.55 g, 10.0 mmol) and
2-¯uoro-6-nitrobenzaldehyde22 (1.69 g, 10.0 mmol) in
25 mL of DMF was heated at 120 ꢀC for 2 h under argon
atmosphere. The reaction mixture was poured onto
1
(KBr) 1741, 1703, 1651, 1620 cm 1. H NMR (Me2SO-
d6) d 1.02 (3H, t, J ˆ 7:2 Hz), 1.37±1.67 (2H, m), 1.49
(9H, s), 1.73±1.94 (2H, m), 4.74 (4H, m), 7.49 (1H, t,
J ˆ 7:8 Hz), 7.67 (1H, d, J ˆ 8:8 Hz), 7.84±7.96 (2H,

Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
309
m), 8.89 (1H, s). Anal. calcd for C21H25N3O4: C, 65.78;
H, 6.57; N, 10.96. Found: C, 65.75; H, 6.39; N, 11.10.
(Me2SO-d6) d 1.45 (9H, s), 3.27 (3H, s), 5.52 (2H, s),
7.57 (1H, t, J ˆ 7:6 Hz), 7.81 (1H, d, J ˆ 8:8 Hz), 7.96
(1H, t, J ˆ 7:6 Hz), 8.26 (1H, d, J ˆ 7:6 Hz), 9.14 (1H,
s). Anal. calcd for C18H19N3O4: C, 63.33; H, 5.61; N,
12.31. Found: C, 63.18; H, 5.47; N, 12.47.
10-Butyl-5-deaza¯avin-3-acetic acid (25).
A suspen-
sion of 3-(tert-butoxycarbonyl)methyl-10-butyl-5-deaza-
¯avin (24; 1.92 g, 5.0 mmol) in 8.2 mL of 12N HCl
(100.0 mmol) was warmed with vigorous stirring at
60 ꢀC for 1 h under argon atmosphere. The reaction
solution was cooled at room temperature and the
deposited crystals were collected by ®ltration. The crys-
tals were washed with puri®ed water and were dried
under reduced pressure at room temperature to a€ord
3-Methyl-5-deaza¯avin-10-acetic acid (28). To a suspen-
sion of 10-(tert-butoxycarbonyl)methyl-3-methyl-5-
deaza¯avin (27; 2.73 g, 8.0 mmol) in 20 mL of ethanol
was added 6.58 mL of 12N HCl (80.0 mmol) dropwise
and the mixture was warmed at 50 ꢀC with vigorous
stirring for 1 h under argon atmosphere. The reaction
mixture was cooled at room temperature and the
precipitate appeared was collected by ®ltration. The
precipitate was washed with puri®ed water and ethanol
and was dried under reduced pressure at room tem-
perature to give 2.15 g (95%) of 28 as pale yellow pow-
.
1.48 g (86%) of 25 (obtained as 25 1.0 H2O) as yellow
crystals: mp 133±136 ꢀC. IR (KBr) 1705, 1641,
1
1618 cm 1. H NMR (Me2SO-d6) d 0.98 (3H, t, J ˆ 7:2
Hz), 1.39±1.63 (2H, m), 1.63±1.84 (2H, m), 4.56 (2H, s),
4.62±4.86 (2H, m), 7.52±7.62 (1H, m), 7.93±8.06 (2H,
m), 8.24 (1H, d, J ˆ 7:8 Hz), 9.09 (1H, s). Anal. calcd
der: mp 225±228 ꢀC. IR (KBr) 1745, 1687, 1621,
1
for C17H17N3O4 1.0H2O: C, 59.12; H, 5.55; N, 12.17.
1535 cm
.
1H NMR (Me2SO-d6) d 3.26 (3H, s), 5.52
.
Found: C, 59.31; H, 5.34; N, 12.25.
(2H, s), 7.56 (1H, t, J ˆ 7:8 Hz), 7.80±8.01 (2H, m), 8.25
(1H, d, J ˆ 7:8 Hz), 9.13 (1H, s). Anal. calcd for
C14H11N3O4: C, 58.95; H, 3.89; N, 14.73. Found: C,
58.75; H, 3.96; N, 14.66.
10-Butyl-5-deaza¯avin-3-acetic acid N-hydroxysuccinimide
ester (26). To a suspension of 10-butyl-5-deaza¯avin-
3-acetic acid (25; 655 mg, 2.00 mmol) in 3 mL of acet-
onitrile was added pyridine (0.49 mL, 6.00 mmol) and
N,N0-disuccinimidyl carbonate (1025 mg, 4.00 mmol)
and the mixture was stirred at room temperature under
argon atmosphere for 3 h. As the reaction proceeded,
the suspension turned into a yellow solution. The sol-
vent was evaporated under reduced pressure and the
residue was extracted with chloroform. The organic
layer was washed with brine once and was dried over
MgSO4. The solvent was evaporated under reduced
pressure and the residue was recrystallized from ethyl
acetate to a€ord 679 mg (80%) of 26 as yellow crystals:
N-Butyl-1-methyl-4-nitropyrrole-2-carboxamide
(18a).
To solution of 1-methyl-4-nitro-2-trichloroacetyl-
a
pyrrole24 (10.0 g, 36.8 mmol) in 50 mL of THF, was
added n-butylamine (2.96 g, 40.5 mmol) dropwise at
0 ꢀC. The solution was stirred at room temperature for
1 h under argon atmosphere. The solvent was evapor-
ated under reduced pressure and the residue was puri-
®ed by column chromatography on silica±gel (ethyl
acetate as eluent) to a€ord 8.1 g (98%) of 18a as pale
yellow needles: mp 100±103 ꢀC. IR (KBr) 1639, 1543,
1
1309 cm
.
1H NMR (CDCl3) d 0.92 (3H, t, J ˆ 7:2
mp 218±221 ꢀC. IR (KBr) 1743, 1709, 1654, 1614,
Hz), 1.29±1.48 (2H, m), 1.48±1.68 (2H, m), 3.39 (2H, dd,
J ˆ 12:9, 6.9 Hz), 3.99 (3H, s), 6.22 (1H, br), 7.10 (1H,
d, J ˆ 1:8 Hz), 7.57 (1H, d, J ˆ 1:8 Hz). Anal. calcd for
C10H15N3O3: C, 53.32; H, 6.71; N, 18.66. Found: C,
53.38; H, 6.63; N, 18.71.
1
1529 cm
.
1H NMR (CDCl3) d 1.03 (3H, t, J ˆ 7:4
Hz), 1.45±1.73 (2H, m), 1.73±1.95 (2H, m), 2.84 (4H, s),
4.79 (2H, br), 5.19 (2H, s), 7.52 (1H, t, J ˆ 7:8 Hz), 7.70
(1H, d, J ˆ 9:2 Hz), 7.86±7.99 (2H, m), 8.95 (1H, s).
Anal. calcd for C21H20N4O6: C, 59.43; H, 4.75; N,
13.20. Found: C, 59.24; H, 4.95; N, 12.97.
N-Butyl-1-methyl-4-(1-methyl-4-nitropyrrole-2-carbox-
amido)pyrrole-2-carboxamide (19a).
A
solution of
N-butyl-1-methyl-4-nitropyrrole-2-carboxamide (18a;
1.0 g, 4.4 mmol) in 10 mL of methanol was hydro-
genated at atmospheric pressure at room temperature in
the presence of 0.1 g of 10% Pd-C for 1 h. The catalyst
was removed by ®ltration and the solvent was evapo-
rated under reduced pressure to give 0.86 g of N-butyl-4-
amino-1-methylpyrrole-2-carboxamide 18b. To a solu-
tion of 18b (0.86 g, 4.4 mmol) and triethylamine
(1.23 mL, 8.8 mmol) in 10 mL of THF, was added
10-(tert-Butoxycarbonyl)methyl-3-methyl-5-deaza¯avin
(27)
A mixture of 6-(tert-butoxycarbonyl)methylamino-3-
methyluracil (15; 2.55 g, 10.0 mmol) and 2-¯uoro-
benzaldehyde (1.37 g, 11.0 mmol) in 25 mL of DMF was
heated at 120 ꢀC for 3 h under argon atmosphere. The
reaction mixture was cooled at room temperature and
deposited crystals were collected by ®ltration. The crys-
tals were washed with ethanol and were dried under
reduced pressure at room temperature to a€ord 2.97 g
1-methyl-4-nitro-2-trichloroacetylpyrrole24
(1.15 g,
4.22 mmol) at 0 ꢀC. The solution was stirred at room
temperature for 16 h under argon atmosphere. The sol-
vent was evaporated under reduced pressure and the
(87%) of 27 as yellow crystals: mp 235±237 ꢀC. IR
1
(KBr) 1741, 1701, 1653, 1620, 1535 cm
.
1H NMR

310
Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
residue was recrystallized from methanol to a€ord
1.15 g (78%) of 19a as yellow powder: mp 222±224 ꢀC.
IR (KBr) 1657, 1628, 1533, 1304 cm
(Me2SO-d6) d 0.90 (3H, t, J ˆ 7:2 Hz), 1.21±1.39 (2H,
m), 1.39±1.57 (2H, m), 3.39 (2H, dd, J ˆ 12:8, 6.8 Hz),
3.81 (3H, s), 3.97 (3H, s), 6.85 (1H, d, J ˆ 2:0 Hz), 7.19
(1H, d, J ˆ 2:0 Hz), 7.58 (1H, d, J ˆ 2:0 Hz), 8.00 (1H,
t, J ˆ 5:6 Hz), 8.16 (1H, d, J ˆ 2:0 Hz), 10.21 (1H, s).
Anal. calcd for C16H21N5O4: C, 55.32; H, 6.09; N,
20.16. Found: C, 55.22; H, 6.18; N, 20.04.
s), 4.79 (2H, br), 5.87 (1H, br), 7.88±8.03 (2H, m), 8.07
(1H, dd, J ˆ 6:8, 2.4 Hz), 9.41 (1H, s). Anal. calcd for
C21H25N5O5: C, 59.01; H, 5.89; N, 16.38. Found: C,
59.04; H, 5.85; N, 16.23.
1
.
1H NMR
N-Butyl-1-methyl-4-[(10-butyl-6-nitro-5-deaza¯avin-3-
yl)-methylcarboxamido]pyrrole-2-carboxamide (2).
A
solution of N-butyl-1-methyl-4-nitropyrrole-2-carbox-
amide (18a; 676 mg, 3.0 mmol) in 10 mL of methanol was
hydrogenated at atmospheric pressure at room tem-
perature in the presence of 30 mg of PtO2 for 1 h. The
catalyst was removed by ®ltration and the solvent was
evaporated under reduced pressure to give 952 mg of
N-butyl-4-amino-1-methylpyrrole-2-carboxamide 18b.
To a solution of 18b (952 mg, 3.0 mmol) in 10 mL of
acetonitrile, was added 10-butyl-6-nitro-5-deaza¯avin-3-
acetic acid N-hydroxysuccinimide ester (14; 1408 mg,
3.0 mmol) and DMAP (366 mg, 3.0 mmol) and the mix-
ture was stirred at room temperature under argon
atmosphere for 16 h. The solvent was evaporated under
reduced pressure and the residue was puri®ed by column
chromatography on silica gel (chloroform: methanol
=10:1 as eluent) to a€ord 1038 mg (61%) of 2 (obtained
N-Butyl-1-methyl-4-[1-methyl-4-(1-methyl-4-nitropyr-
role -2 - carboxamido)pyrrole - 2 - carboxamido]pyrrole - 2-
carboxamide (20a). A solution of N-butyl-1-methyl-4-
(1-methyl-4-nitropyrrole-2-carboxamido)pyrrole-2-carbox-
amide (19a; 0.70 g, 2.0 mmol) in 10 mL of methanol was
hydrogenated at atmospheric pressure at room tem-
perature in the presence of 0.1 g of PtO2 for 1 h. The
catalyst was removed by ®ltration and the solvent was
evaporated under reduced pressure to give 0.63 g of
N-butyl-1-methyl-4-(4-amino-1-methylpyrrole-2-carbox-
amido)pyrrole-2-carbox- amide 19b. To a solution of 19b
(0.63 g, 2.0 mmol) and triethylamine (0.28 mL,
4.0 mmol) in 10 mL of acetonitrile, was added 1-methyl-
4-nitro-2-trichloroacetylpyrrole24 (0.50 g, 1.8 mmol) at
0 ꢀC. The solution was stirred at room temperature for
16 h under argon atmosphere. The solvent was evapo-
rated under reduced pressure and the residue was
recrystallized from methanol to a€ord 0.53 g (62%) of
ꢀ
.
as 2 1.0 H2O) as yellow powder: mp 223±226 C. IR
1
(KBr) 1705, 1650, 1624, 1531 cm 1. H NMR (Me2SO-
d6) d 0.88 (3H, t, J ˆ 7:1 Hz), 0.98 (3H, t, J ˆ 7:4 Hz),
1.20±1.62 (6H, m), 1.63±1.85 (2H, m), 3.15 (2H, dd,
J ˆ 12:2, 6.6 Hz), 3.77 (3H, s), 4.64 (2H, s), 4.78 (2H,
br), 6.68 (1H, d, J ˆ 1:0 Hz), 7.06 (1H, d, J ˆ 1:0 Hz),
7.95 (1H, br), 8.16 (1H, dd, J ˆ 9:0, 7.8 Hz), 8.26 (1H, d,
J ˆ 7:8 Hz), 8.41 (1H, d, J ˆ 9:0 Hz), 9.21 (1H, s), 10.02
20a as yellow powder: mp 215±218 ꢀC. IR (KBr) 1639,
1
1589, 1535, 1311 cm
.
1H NMR (Me2SO-d6) d 0.91
.
(3H, t, J ˆ 7:4 Hz), 1.18±1.39 (2H, m), 1.39±1.58 (2H,
m), 3.18 (2H, dd, J ˆ 12:8, 6.6 Hz), 3.81 (3H, s), 3.88
(3H, s), 3.98 (3H, s), 6.87 (1H, d, J ˆ 1:8 Hz), 7.05 (1H,
d, J ˆ 1:8 Hz), 7.19 (1H, d, J ˆ 1:8 Hz), 7.28 (1H, d,
J ˆ 1:8 Hz), 7.61 (1H, d, J ˆ 1:8 Hz), 7.97 (1H, t,
J ˆ 5:4 Hz), 8.17 (1H, d, J ˆ 1:8 Hz), 9.93 (1H, s), 10.28
(1H, s). Anal. calcd for C22H27N7O5: C, 56.28; H, 5.80;
N, 20.88. Found: C, 56.11; H, 5.88; N, 20.68.
(1H, s). Anal. calcd for C27H31N7O6 1.0H2O: C, 57.13;
H, 5.86; N, 17.27. Found: C, 57.20; H, 5.76; N, 17.08.
N-Butyl-1-methyl-4-[1-methyl-4-[(10-butyl-6-nitro-5-
deaza¯avin - 3 - yl)methylcarboxamido]pyrrole - 2 - carbox-
amido]pyrrole-2-carboxamide (3). A solution of N-butyl-
1-methyl-4-(1-methyl-4-nitropyrrole-2-carboxamido)-
pyrrole-2-carboxamide (19a; 987 mg, 2.84 mmol) in
10 mL of methanol was hydrogenated at atmospheric
pressure at room temperature in the presence of 90 mg
of PtO2 for 1 h. The catalyst was removed by ®ltration
and the solvent was evaporated under reduced pressure
to give 900 mg of N-butyl-1-methyl-4-(4-amino-1-methyl-
pyrrole-2-carboxamido)pyrrole-2-carboxamide 19b. To a
solution of 19b (900 mg, 2.84 mmol) in 10 mL of aceto-
nitrile, was added 10-butyl-6-nitro-5-deaza¯avin-3-
acetic acid N-hydroxysuccinimide ester (14; 1330 mg,
2.84 mmol) and DMAP (346 mg, 2.84 mmol) and the
mixture was stirred at room temperature under argon
atmosphere for 16 h. The solvent was evaporated under
reduced pressure and the residue was puri®ed by column
chromatography on silica gel (chloroform:metha-
nol=10:1 as eluent) to a€ord 1030 mg (54%) of 3 as
3-(Butylcarbamoyl)methyl-10-butyl-6-nitro-5-deaza¯avin
(1). A mixture of 10-butyl-6-nitro-5-deaza¯avin (11;
0.63 g 2.0 mmol), N-butylbromoacetamide (0.78 g,
4.0 mmol), and well pulverized K2CO3 (0.83 g,
6.0 mmol) in 10 mL of DMF was warmed at 60 ꢀC for
1 h under argon atmosphere. The solvent was evapo-
rated under reduced pressure and the residue was
extracted with chloroform. The organic layer was
washed with brine once and was dried over MgSO4. The
solvent was evaporated under reduced pressure and the
resulting yellow solid was recrystallized from chloro-
form-ethanol to a€ord 0.64 g (75%) of 1 as yellow crys-
tals: mp 264±267 ꢀC. IR (KBr) 1709, 1657, 1624,
1
1529 cm 1. H NMR (Me2SO-d6) d 0.92 (3H, t, J ˆ 7:0
Hz), 1.07 (3H, t, J ˆ 7:2 Hz), 1.20±1.73 (6H, m), 1.73±
1.96 (2H, m), 3.29 (2H, dd, J ˆ 13:0, 6.6 Hz), 4.72 (2H,
yellow powder: mp 164±168 ꢀC. IR (KBr) 1707, 1640,
1
1626, 1531 cm
.
1H NMR (Me2SO-d6) d 0.89 (3H, t,

Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
311
J ˆ 7:3 Hz), 0.99 (3H, t, J ˆ 7:3 Hz), 1.28 (2H, m), 1.34
(2H, m), 1.44 (2H, m), 1.49 (2H, m), 3.17 (2H, t, J ˆ 6:6
Hz), 3.79 (3H, s), 3.82 (3H, s), 4.66 (2H, br), 4.78 (2H,
s), 6.83 (1H, s), 6.89 (1H, s), 7.12 (1H, s), 7.16 (1H, s),
7.97 (1H, br), 8.14 (1H, dd, J ˆ 8:6, 7.9 Hz), 8.25 (1H, d,
J ˆ 8:6 Hz), 8.40 (1H, d, J ˆ 7:9 Hz), 9.21 (1H, s), 9.84
(1H, s), 10.09 (1H, s). MS (FAB): m/z: 672 [(M+H)+].
HRMS (FAB) for C33H38N9O7 [(M+H)+] calcd
672.2897, found 672.2912.
Hz), 1.19±1.52 (4H, m), 3.11 (2H, dd, J ˆ 12:2, 6.4 Hz),
3.28 (3H, s), 5.48 (2H, br), 8.02±8.14 (2H, m), 8.22 (1H,
d, J ˆ 8:8 Hz), 8.34 (1H, t, J ˆ 5:6 Hz), 9.23 (1H, s).
.
Anal. calcd for C18H19N5O5 0.5H2O: C, 54.82; H, 5.11;
N, 17.76. Found: C, 54.73; H, 4.88; N, 17.65.
N-Butyl-1-methyl-4-[(3-methyl-6-nitro-5-deaza¯avin-10-
A
yl)methylcarboxamido]pyrrole-2-carboxamide (6).
solution of N-butyl-1-methyl-4-nitropyrrole-2-carbox-
amide (18a; 225 mg, 1.0 mmol) in 3 mL of methanol was
hydrogenated at atmospheric pressure at room tem-
perature in the presence of 20 mg of PtO2 for 1 h. The
catalyst was removed by ®ltration and the solvent was
evaporated under reduced pressure to give 195 mg of
N-butyl-4-amino-1-methylpyrrole-2-carboxamide 18b.
To a solution of 18b (225 mg, 1.0mmol) in 3 mL of aceto-
nitrile, was added 3-methyl-6-nitro-5-deaza¯avin-10-
acetic acid (17; 330 mg, 1.0 mmol) and 1,3-diisopropyl-
carbodiimide (126 mg, 1.00 mmol) and the mixture was
stirred at room temperature under argon atmosphere
for 16 h. The solvent was evaporated under reduced
pressure and the residue was puri®ed by column chro-
matography on silica gel (chloroform : methanol =10:1
as eluent) to give yellow solid. Recrystallization from
N-Butyl-1-methyl-4-[1-methyl-4-[1-methyl-4-[(10-butyl-6-
nitro-5-deaza¯avin-3-yl)methylcarboxamido]pyrrole-2-
carboxamido]pyrrole - 2 - carboxamido]pyrrole- 2 - carbox-
amide (4). A solution of N-butyl-1-methyl-4-[1-methyl-4
-(1-methyl-4-nitropyrrole-2-carboxamido)pyrrole-2-
carboxamido]pyrrole-2-carboxamide
(20a;
1.4 g,
2.98 mmol) in 15 mL of methanol was hydrogenated at
atmospheric pressure at room temperature in the pres-
ence of 140 mg of PtO2 for 1 h. The catalyst was
removed by ®ltration and the solvent was evaporated
under reduced pressure togive 1.3 g of N-butyl-1-methyl-
4-[1-methyl-4-(4-amino-1-methylpyrrole-2-carboxamido)
pyrrole-2-carboxamido]pyrrole-2-carboxamide 20b. To
a solution of 20b (1.3 g, 2.98 mmol) in 15 mL of aceto-
nitrile, was added 10-butyl-6-nitro-5-deaza¯avin-3-acetic
acid N-hydroxysuccinimide ester (14; 1.4 g, 2.98 mmol)
and DMAP (363 mg, 2.98 mmol) and the mixture was
stirred at room temperature under argon atmosphere
for 16 h. The solvent was evaporated under reduced
pressure and the residue was puri®ed by column chro-
matography on silica±gel (chloroform : methanol=10:1
as eluent) to a€ord 1.2 g (51%) of 4 as yellow powder:
mp 211±213 ꢀC. IR (KBr) 1707, 1651, 1618, 1560,
chloroform±ethanol a€orded 134 mg (26%) of
6
.
(obtained as 6 0.5H2O) as yellow powder: mp 169±
172 ꢀC. IR (KBr) 1701, 1653, 1618, 1564, 1533 cm 1. 1H
NMR (Me2SO-d6) d 0.92 (3H, t, J ˆ 7:2 Hz), 1.18±1.36
(2H, m), 1.36±1.53 (2H, m), 3.03±3.22 (2H, m), 3.28
(3H, s), 3.76 (3H, s), 5.67(2H, br), 6.73 (1H, d, J ˆ 1:8
Hz), 7.06 (1H, d, J ˆ 1:8 Hz), 7.97 (1H, t, J ˆ 5:6 Hz),
8.09 (1H, d, J ˆ 8:4 Hz). Anal. calcd for
.
C24H25N7O6 0.5H2O: C, 55.81; H, 5.07; N, 18.98.
1
1527 cm 1. H NMR (Me2SO-d6) d 0.89 (3H, t, J ˆ 7:3
Found: C, 55.88; H, 4.99; N, 19.02.
Hz), 0.99 (3H, t, J ˆ 7:3 Hz), 1.28 (2H, m), 1.34 (2H,
m), 1.44 (2H, m), 1.49 (2H, m), 3.17 (2H, t, J ˆ 6:6 Hz),
3.79 (3H, s), 3.83 (3H, s), 3.84 (3H, s), 4.66 (2H, br),
4.77 (2H, s), 6.85 (1H, s), 6.92 (1H, s), 7.03 (1H, s), 7.13
(1H, s), 7.17 (1H, s), 7.23 (1H, s), 7.97 (1H, br), 8.13
(1H, dd, J ˆ 8:6, 7.9 Hz), 8.25 (1H, d, J ˆ 8:6 Hz), 8.40
(1H, d, J ˆ 7:9 Hz), 9.21 (1H, s), 9.88 (1H, s), 9.91 (1H,
s), 10.10 (1H, s). MS (FAB) m/z: 794 [(M+H)+].
HRMS (FAB) for C39H44N11O8 [(M+H)+] calcd
794.3378, found 794.3369.
N-Butyl-1-methyl-4-[1-methyl-4-[(3-methyl-6-nitro-5-
deaza¯avin-10-yl)methylcarboxamido]pyrrole-2-carbox-
amido]pyrrole-2-carboxamide (7). A solution of N-butyl-
1-methyl-4-(1-methyl-4-nitropyrrole-2-carboxamido)pyr-
role-2-carboxamide (19a; 1000 mg, 2.88 mmol) in 10 mL
of methanol was hydrogenated at atmospheric pressure
at room temperature in the presence of 100 mg of PtO2
for 1 h. The catalyst was removed by ®ltration and the
solvent was evaporated under reduced pressure to give
910 mg of N-butyl-1-methyl-4-(4-amino-1-methylpyr-
role-2-carboxamido) pyrrole-2-carboxamide 19b. To a
solution of 19b (910 mg, 2.88 mmol) in 10 mL of aceto-
nitrile, was added 3-methyl-6-nitro-5-deaza¯avin-10-
acetic acid (17; 950 mg, 2.88 mmol) and 1,3-diisopropyl-
carbodiimide (435 mg, 3.45 mmol) and the mixture was
stirred at room temperature under argon atmosphere
for 16 h. The solvent was evaporated under reduced
pressure and the residue was puri®ed by column chro-
matography on silica gel (chloroform:methanol=10:1 as
10-(Butylcarbamoyl)methyl-3-methyl-6-nitro-5-deaza-
¯avin (5). A mixture of 6-(butylcarbamoyl)methyl-
amino-3-methyluracil (22; 0.51 g, 2.0mmol) and 2-¯uoro-
6-nitrobenzaldehyde22 (0.34 g, 2.0 mmol) in 10 mL of
DMF was heated at 120 ꢀC for 2 h under argon atmos-
phere. The precipitate appeared was collected by ®ltra-
tion and was washed with ethanol. The precipitate was
dried under reduced pressure at room temperature to
.
a€ord 0.55 g (75%) of 5 (obtained as 5 0.5 H2O) as yel-
low powder: mp 267±271 ꢀC. IR (KBr) 1709, 1655, 1620,
eluent) to a€ord 507 mg (28%) of 7 as yellow powder:
mp 213±216 ꢀC. IR (KBr) 1702, 1641, 1626 cm
1
1
1539 cm 1. H NMR (Me2SO-d6) d 0.91 (3H, t, J ˆ 7:2
.

312
Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
1H NMR (Me2SO-d6) d 0.89 (3H, t, J ˆ 7:3 Hz), 1.23
(2H, m), 1.46 (2H, m), 3.10 (3H, s), 3.16 (2H, t, J ˆ 6:6
Hz), 3.79 (3H, s), 3.81 (3H, s), 5.96 (2H, br), 6.81 (1H,
s), 6.93 (1H, s), 7.13 (1H, s), 7.16 (1H, s), 7.97 (1H, br),
8.07 (1H, dd, J ˆ 8:6, 7.9 Hz), 8.22 (1H, d, J ˆ 8:6 Hz),
8.27 (1H, d, J ˆ 7:9 Hz), 9.25 (1H, s), 9.84 (1H, s), 10.53
(1H, s). MS (FAB) m/z: 630 [(M+H)+]. HRMS (FAB)
for C30H32N9O7 [(M+H)+] calcd 630.2428, found
630.2417.
added 10-butyl-5-deaza¯avin-3-acetic acid N-hydroxy-
succinimide ester (26, 424 mg, 1.00 mmol) and DMAP
(122 mg, 1.00 mmol) and the mixture was stirred at
room temperature under argon atmosphere for 16 h.
The solvent was evaporated under reduced pressure and
the residue was puri®ed by column chromatography on
silica gel (chloroform : methanol=10:1 as eluent) to
a€ord 426 mg (68%) of 9 as yellow powder: mp 179±
182 ꢀC. IR (KBr) 1690, 1641, 1618, 1568, 1529 cm 1. 1H
NMR (Me2SO-d6) d 0.89 (3H, t, J ˆ 7:4 Hz), 0.99 (3H,
t, J ˆ 7:2 Hz), 1.20±1.36 (2H, m), 1.36±1.62 (4H, m),
1.62±1.86 (2H, m), 3.16 (2H, dd, J ˆ 12:8, 6.6 Hz), 3.79
(3H, s), 3.83 (3H, s), 4.66 (2H, s), 4.74 (2H, br), 6.83
(1H, d, J ˆ 1:8 Hz), 6.90 (1H, d, J ˆ 1:6 Hz), 7.13 (d,
1H, J ˆ 1:8 Hz), 7.16 (1H, d, J ˆ 1:6 Hz), 7.50±7.63
(1H, m), 7.90±8.08 (3H, m), 8.25 (1H, d, J ˆ 7:6 Hz),
9.10 (1H, s), 9.84 (1H, s), 10.06 (1H, s). MS (FAB) m/z
627 [(M+H)+]. HRMS (FAB) for C33H39N8O5
[(M+H)+] calcd 627.3047, found 627.3051.
N-Butyl-1-methyl-4-[1-methyl-4-[1-methyl-4-[(3-methyl-6
-nitro-5-deaza¯avin-10-yl)methylcarboxamido]pyrrole-2-
carboxamido]pyrrole-2-carboxamido]pyrrole-2-carbox-
amide (8). A solution of N-butyl-1-methyl-4-[1-methyl-
4-(1-methyl-4-nitropyrrole-2-carboxamido)pyrrole-2-
carboxamido]pyrrole-2-carboxamide (20a; 1000 mg,
2.13 mmol) in 10 mL of methanol was hydrogenated at
atmospheric pressure at room temperature in the pres-
ence of 75 mg of PtO2 for 1 h. The catalyst was
removed by ®ltration and the solvent was evaporated
under reduced pressure to give 935 mg of N-butyl-1-
methyl -4 -[1- methyl- 4- (4-amino- 1- methylpyrrole -2-
carboxamido)pyrrole-2-carboxamido]pyrrole-2-carbox-
amide 20b. To a solution of 20b (935 mg, 2.13 mmol) in
10 mL of acetonitrile, was added 3-methyl-6-nitro-5-
deaza¯avin-10-acetic acid (17; 700 mg, 2.12 mmol) and
1,3-diisopropylcarbodiimide (403 mg, 3.20 mmol) and
the mixture was stirred at room temperature under
argon atmosphere for 16 h. The solvent was evaporated
under reduced pressure and the residue was puri®ed by
column chromatography on silica gel (chloroform:metha-
nol=10:1 as eluent) to a€ord 415 mg (26%) of 8 as
N-Butyl-1-methyl-4-[1-methyl-4-[(3-methyl-5-deaza¯avin-
10-yl)methylcarboxamido]pyrrole-2-carboxamido]pyrrole-
2-carboxamide (10). A solution of N-butyl-1-methyl-4-
(1- methyl - 4 - nitropyrrole - 2 - carboxamido)pyrrole - 2-
carboxamide (19a; 347 mg, 1.00 mmol) in 3 mL of
methanol was hydrogenated at atmospheric pressure at
room temperature in the presence of 30 mg of PtO2 for
1 h. The catalyst was removed by ®ltration and the sol-
vent was evaporated under reduced pressure to give
317 mg
of
N-butyl-1-methyl-4-(4-amino-1-methyl-
pyrrole-2-carboxamido) pyrrole-2-carboxamide 19b.
To a solution of 19b (317 mg, 1.00 mmol) in 3 mL of
DMF, was added 3-methyl-5-deaza¯avin-10-acetic acid
(28; 282 mg, 1.00 mmol) and 1,3-diisopropylcarbodiimide
(126 mg, 1.00 mmol) and the mixture was stirred at
room temperature under argon atmosphere for 16 h.
The solvent was evaporated under reduced pressure and
the residue was puri®ed by column chromatography on
silica±gel (chloroform : methanol =10:1 as eluent) to
a€ord 117 mg (20%) of 10 as yellow powder: mp 192±
195 ꢀC. IR (KBr) 1689, 1640, 1620, 1570, 1533 cm 1. 1H
NMR (Me2SO-d6) d 0.89 (3H, t, J ˆ 7:0 Hz), 1.18±1.38
(2H, m), 1.38±1.59 (2H, m), 2.88±3.20 (2H, m), 3.27
(3H, s), 3.79 (3H, s), 3.82 (3H, s), 5.61 (2H, br), 6.82
(1H, s), 6.93 (1H, s), 7.12 (1H, s), 7.16 (1H, s), 7.55 (1H,
t, J ˆ 7:4 Hz), 7.86 (1H, t, J ˆ 8:6 Hz), 7.91±8.05 (2H,
m), 8.26 (1H, d, J ˆ 7:8 Hz), 9.15 (1H, s), 9.84 (1H, s),
10.44 (1H, s). MS (FAB) m/z 585 [(M+H)+]. HRMS
(FAB) for C30H33N8O5 [(M+H)+] calcd 585.2577,
found 585.2567.
yellow powder: mp 261±265 ꢀC. IR (KBr) 1702, 1637,
1
1626, 1560 cm
.
1H NMR (Me2SO-d6) d 0.90 (3H, t,
J ˆ 7:3 Hz), 1.32 (2H, m), 1.46 (2H, m), 3.08 (3H, s),
3.16 (2H, t, J ˆ 6:6 Hz), 3.79 (3H, s), 3.83 (3H, s), 3.84
(3H, s), 5.95 (2H, br), 6.84 (1H, s), 6.85 (1H, s), 6.96
(1H, s), 7.02 (1H, s), 7.16 (1H, s), 7.23 (1H, s), 7.96 (1H,
br), 8.07 (1H, dd, J ˆ 8:6, 7.9 Hz), 8.24 (1H, d, J ˆ 8:6
Hz), 8.27 (1H, d, J ˆ 7:9 Hz), 9.26 (1H, s), 9.87 (1H, s),
9.89 (1H, s), 10.56 (1H, s). MS (FAB) m/z: 752
[(M+H)+].
HRMS
(FAB)
for
C36H38N11O8
[(M+H)+] calcd 752.2908, found 752.2875.
N-Butyl-1-methyl-4-[1-methyl-4-[(10-butyl-5-deaza¯avin-
3-yl)methylcarboxamido]pyrrole-2-carboxamido]pyrrole-
2-carboxamide (9). A solution of N-butyl-1-methyl-4-
(1-methyl-4-nitropyrrole-2-carboxamido)pyrrole-2-car-
boxamide (19a; 347 mg, 1.00 mmol) in 3 mL of methanol
was hydrogenated at atmospheric pressure at room
temperature in the presence of 30 mg of PtO2 for 1 h.
The catalyst was removed by ®ltration and the solvent
was evaporated under reduced pressure to give 317 mg
of N-butyl-1-methyl-4-(4-amino-1-methylpyrrole-2-carb-
oxamido)pyrrole-2-carboxamide 19b. To a solution of
19b (317 mg, 1.00 mmol) in 5 mL of acetonitrile, was
N-Butyl-1-methyl-4-(1-methyl-4-acetamido-pyrrole-2-
carboxamido)pyrrole-2-carboxamide (21). A solution of
N-butyl-1-methyl-4-(1-methyl-4-nitropyrrole-2-carbox-
amido)pyrrole-2-carboxamide (19a; 0.70 g, 2.0 mmol) in
10 mL of methanol was hydrogenated at atmospheric

Y. Kanaoka et al./Bioorg. Med. Chem. 6 (1998) 301±314
313
pressure at room temperature in the presence of 0.1 g of
PtO2 for 1 h. The catalyst was removed by ®ltration and
the solvent was evaporated under reduced pressure
to give 0.63 g of N-butyl-1-methyl-4-(4-amino-1-methyl-
pyrrole-2-carboxamido) pyrrole-2-carboxamide 19b. To
a solution of 19b (0.63 g, 2.0 mmol), pyridine (0.16 mL,
2.0 mmol), and triethylamine (0.28 mL, 4.0 mmol) in
10 mL of acetonitrile, was added acetic anhydride
(204 mg, 2.0 mmol) at 0 ꢀC. The solution was stirred at
room temperature under argon atmosphere for 1 h. The
solvent was evaporated under reduced pressure and the
residue was recrystallized from methanol to a€ord
Electrolytic reductions of the compounds in the pres-
ence of plasmid DNA (pBR322 (Sigma), 5Â10 5 M/bp)
were carried out at 298 K under argon atmosphere in
the dark at a constant potential of 950 mV for com-
pounds 1±8 and 1100 mV for compounds 9 and 10 in
1.5 mM sodium citrate bu€er containing 20% DMF.
The electrons involved in the reduction process of the
compounds were monitored on Yanaco V10-CM cou-
lometer. After the complete reduction of the compounds,
the reaction solution was incubated at room tempera-
ture overnight. The degradation of form I of pBR322
DNA was analyzed by means of electrophoresis (1%
agarose gel) with standard procedure.
.
0.56 g (74%) of 21 (obtained as 21 1.0 H2O) as pale yel-
low powder: mp 110±112 ꢀC. IR (KBr) 1641, 1583,
1
1535 cm 1. H NMR (Me2SO-d6) d 0.90 (3H, t, J ˆ 7:4
In vitro antitumor activities of nitro 5-deaza¯avin-
pyrrolecarboxamide(s) hybrid molecules
Hz), 1.19±1.39 (2H, m), 1.39±1.59 (2H, m), 1.97 (3H, m),
3.17 (q, 2H, J ˆ 6:2 Hz), 3.80 (3H, s), 3.83 (3H, s), 6.84
(1H, s), 6.85 (1H, s), 7.15 (1H, d, J ˆ 1:6 Hz), 7.17 (1H,
d, J ˆ 1:6 Hz), 7.97 (1H, t, J ˆ 5:6 Hz), 9.82 (1H, s),
The tetrazolium-based semiautomated colorimetric
assay (MTT assay) developed by Carmichael33 was
modi®ed and used for the in vitro assay. 2000 Cells
(human oral epidermoid carcinoma KB cells or murine
leukemia L1210 cells) in 180 mL of RPML-1640 medium
were seeded in a 96-well ¯at bottom microtest plate and
20 mL of drug solutions with graded concentrations were
simultaneously added in triplicate to each well. The
plate was incubated for 3 days at 37 ꢀC in a humidi®ed
atmosphere of 5% CO2. To each well was added 25 ml of
MTT reagent (2 mg/mL in Dulbecco's phosphate bu€-
ered saline without calcium and magnesium). After
another 4 h incubation at 37 ꢀC, the microplate was
centrifuged at 3000 rev./min for 10 min and the medium
was removed by aspiration. To each well was added
0.2 mL of dimethyl sulfoxide and each well was mixed
thoroughly with a mechanical plate mixer to solubilize
the resulting MTT-formazan. Absorbance at 540 nm
(OD540) was measured with a ImmunoReader NJ-2000
(InterMed Japan, Tokyo Japan). By using the OD540 of
each well the 50% inhibitory drug concentration (IC50
value) was determined as was described previously.34
.
9.84 (1H, s). Anal. calcd for C18H25N5O3 1.0H2O: C,
57.28; H, 7.21; N, 18.55. Found: C, 57.22; H, 7.18; N,
18.47.
Redox potentials of the compounds
The reduction peak potentials (Ep) of the compounds
were determined in DMF solutions (1.0Â10 3 M) rela-
tive to the Ag/AgCl electrode using cyclic voltammetry
(Hokuto Denko HB-104 voltage scanner and Hokuto
Denko HA-301 potentiostat using glassy carbon and
platinum wire as the working and auxiliary electrodes
and tetrabutylammonium perchlorate (1.0Â10 1 M) as a
supporting electrolyte, scan rate ꢀ ˆ 100 mVs 1) at
298 K in the dark under argon atmosphere.
DNA-titration by UV-VIS spectra
5
The UV-VIS spectra of the compounds (2.1Â10 (M))
were measured on Shimadzu UV-3100PC spectro-
photometer in the presence of [poly (dA-dT)]2 (Sigma),
[poly (dG-dC)]2 (Sigma), or calf thymus DNA (Sigma)
in 10 mM Tris-HCl bu€er (pH 7.0) containing 20% of
DMF. The apparent binding constants of the com-
pounds (Kapp) were determined according to the method
as was described in ref. 26.
Acknowledgements
We are grateful to Assistant Professor K. Iwai of Nara
Women's University and Professor T. Matsumura-
Inoue of Nara University of Education for helpful dis-
cussion for CV measurement and for helpful discussion
for electrolytic reduction of the compounds. This work
was partly supported by the Sasakawa Scienti®c
Research grant from The Japan Science Society.
Reductive activation of the compounds and plasmid
DNA-cleaving assay
Controlled potential electrolysis was performed using a
three electrode con®guration involving
a platinum
References and Notes
(5 mmꢁ) working electrode, platinum wire counter elec-
trode, and Ag/AgCl reference electrode.9 The working
and counter electrode were separated each other with
sintered glass ®lter to prevent the reoxidation of reduc-
tively activated compounds.
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