New antimetastatic hypoxic cell radiosensitizers: Design, synthesis, and biological activities of 2-nitroimidazole-acetamide, TX-1877, and its analogues

Article abstract of DOI:10.1016/S0968-0896(00)00265-0

We designed, based on the molecular orbital (MO) calculation, synthesized, and evaluated the biological activities of the new antimetastatic hypoxic cell radiosensitizer, 2-nitroimidazole-acetamide, TX-1877, and its analogues. Each analogue has an electron-affinic imidazole group, an acetamide group and a certain hydrophilic group to control its biological effect, toxicity, and pharmacokinetics. In in vitro radiosensitization assay, most TX-1877 analogues, which have an electron affinity (EA) of more than 0.9 eV and partition coefficient (P) of more than 0.021, showed satisfactory enhancement ratios (ER > 1.60) at doses of 1 mM. On the other hand, imidazole analogues, such as TX-1908 (EA = 0.67 eV), TX-1910 (EA = -0.34 eV) and TX-1931 (EA = -0.37 eV), which have low electron affinities, had an ER of 1.31 or less. TX-1877 and KIN-806 effectively inhibited tumor regrowth when administered with irradiation in vivo at a dose of 0.4 mg/g. Tumor lung metastasis was inhibited by treatment with either TX-1877 or KIN-806 without irradiation at a dose of 0.4 mg/g. TX-1877 reduced markedly the mean number of metastatic lung nodules in comparison with KIN-806. Moreover, TX-1877 and KIN-806 enhanced macrophage and helper T lymphocyte infiltration for 3 weeks after drug treatment. TX-1877 shows a high EA value and has the C² of HOMO localizing on N-methylamide and the C² of LUMO localizing on 2-nitroimidazole group. The MO data might be useful for designing a bifunctional hypoxic cell radiosensitizer. TX-1877 and its analogues are potential antimetastatic hypoxic cell radiosensitizers, which would improve the efficiency of radiotherapy and quality of life in cancer treatment.

Full text of DOI:10.1016/S0968-0896(00)00265-0

Bioorganic & Medicinal Chemistry 9 (2001) 453±464
New Antimetastatic Hypoxic Cell Radiosensitizers:
Design, Synthesis, and Biological Activities of
2-Nitroimidazole-acetamide, TX-1877, and its Analogues
Soko Kasai,a Hideko Nagasawa,a Mao Yamashita,a Mie Masui,a Hideki Kuwasaka,a
Tomoko Oshodani,a Yoshihiro Uto,a Taisuke Inomata,b Shigenori Oka,c
Seiichi Inayamad,e and Hitoshi Horia,*
aDepartment of Biological Science and Technology, Faculty of Engineering, The University of Tokushima,
Minamijosanjimacho-2, Tokushima 770±8506, Japan
bDepartment of Radiology, Kochi Medical School, Kochi 783±0043, Japan
cResearch & Development Center, Nagase & Co., Ltd., Hyogo 651±2241, Japan
dlnstitute of Oriental Medical Science, Tokyo 155±0032, Japan
eKeio University, School of Medicine, Tokyo 160±8582, Japan
Received 24 July 2000; accepted 2 October 2000
AbstractÐWe designed, based on the molecular orbital (MO) calculation, synthesized, and evaluated the biological activities of the
new antimetastatic hypoxic cell radiosensitizer, 2-nitroimidazole-acetamide, TX-1877, and its analogues. Each analogue has an
electron-anic imidazole group, an acetamide group and a certain hydrophilic group to control its biological e€ect, toxicity, and
pharmacokinetics. In in vitro radiosensitization assay, most TX-1877 analogues, which have an electron anity (EA) of more than
0.9 eV and partition coecient (P) of more than 0.021, showed satisfactory enhancement ratios (ER > 1.60) at doses of 1 mM. On
the other hand, imidazole analogues, such as TX-1908 (EA=0.67 eV), TX-1910 (EA=À0.34 eV) and TX-1931 (EA=À0.37 eV),
which have low electron anities, had an ER of 1.31 or less. TX-1877 and KIN-806 e€ectively inhibited tumor regrowth when
administered with irradiation in vivo at a dose of 0.4 mg/g. Tumor lung metastasis was inhibited by treatment with either TX-1877
or KIN-806 without irradiation at a dose of 0.4 mg/g. TX-1877 reduced markedly the mean number of metastatic lung nodules in
comparison with KIN-806. Moreover, TX-1877 and KIN-806 enhanced macrophage and helper T lymphocyte in®ltration for 3
weeks after drug treatment. TX-1877 shows a high EA value and has the C2 of HOMO localizing on N-methylamide and the C2 of
LUMO localizing on 2-nitroimidazole group. The MO data might be useful for designing a bifunctional hypoxic cell radiosensitizer.
TX-1877 and its analogues are potential antimetastatic hypoxic cell radiosensitizers, which would improve the eciency of radio-
therapy and quality of life in cancer treatment. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
Hypoxic cell radiosensitizers, as electron-anic com-
pounds, have oxygen-mimicking e€ects to tumor
hypoxic cells and ®x the radiation-induced damage in
DNA or other molecules.5 Misonidazole [MISO, 1-
methoxy-3-(2-nitro-1H-imidazolyl)-2-propanol] (Fig. 1)
is a well-known 2-nitroimidazole radiosensitizer. Tested
in clinical trials, its development was discontinued
because of dose-limiting side e€ects such as neurotoxi-
city.6 Etanidazole (SR2508) [N1-(2-hydroxyethyl)-2-(2-
nitro-1H-imidazolyl)acetamide] (Fig. 1) was 3 to 4 times
less toxic than MISO, but had radiosensitizing activity
comparable to MISO. However, a phase III randomized
trial of radiotherapy of head and neck cancer with or
without etanidazole has failed.7 Recently, hypoxic cell
radiosensitizers have been designed not to improve
radiosensitizing activities and hydrophilicities but to
A high rate of tumor cell proliferation increases oxygen
consumption in tumor tissue. Furthermore, abnormal-
ities of structure and function in tumor vessels lead to
decreased oxygen delivery to tumor tissue,1 which
becomes hypoxic and resistant to radiation therapy and
some chemotherapy.2,3 Tumor hypoxia can be exploited
for selective anticancer drug treatment using hypoxic
cell cytotoxins or hypoxic cell radiosensitizers.4
*Corresponding author. Tel.: +81-886-56-7514; fax: +81-886-56-
9164; e-mail: hori@bio.tokushima-u.ac.jp
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(00)00265-0

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S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
have chemical and biological activities by introducing
additional functional groups, such as alkylating groups
or enzyme chelating groups, to their side-chain
moieties.8À10 On the other hand, tumor hypoxia induced
the production of vascular endothelial growth factor
(VEGF) mediating angiogenesis, which is essential for
tumor progression and metastasis.11,12 Therefore, the
combination of radiosensitization and antiangiogenesis
targeting of tumor hypoxia can be a powerful anti-
cancer therapeutic. We have previously reported that
the antiangiogenic hypoxic cell radiosensitizer, haloace-
tylcarbamoyl-2-nitroimidazole, showed strong anti-
angiogenic activity in chicken embryo chorioallantoic
membranes (CAM) assay.13 Furthermore, we devel-
oped a hypoxic cell radiosensitizer, KIN-806 [N1,N1-
dimethyl-2-(2-nitro-1H-1-imidazolyl)acetamide, Ro 7-
1052] (Fig. 1), which has cell-di€erentiation inducing,
antimetastatic and immunopotentiating activities.14À16
KIN-806 has electron-anic nitroimidazole with an
N,N-dimethylacetamide group in its side-chain. TX-
1877 [N1-(2-hydroxyethyl)-N-1-methyl-2-(2-nitro-1H-
imidazolyl)acetamide], which was designed based on the
structure of KIN-806 as the lead compound, showed
more hydrophilicity and stronger antimetastatic activity
than KIN-806.17 In this paper, we give a full account of
the design, synthesis, MO calculation, and in vitro
radiosenstizing activity of TX-1877 analogues, and
the in vivo biological activities of TX-1877 in
comparison with those of KIN-806.
Results
Design and synthesis
The antimetastatic hypoxic cell radiosensitizer TX-1877
was designed based on the structure of KIN-806 to be
more hydrophilic. TX-1877 has electron-anic nitro-
imidazole with N-methylacetamide consisting of the
hydroxyethylamine instead of N-methylamine of KIN-
806 in its side-chain. TX-1877 has more potent in vivo
antitumor and antimetastatic activities than KIN-806
(described below). We designed TX-1877 analogues,
including sec-alcohol (TX-1902 and TX-1909), polyol
(TX-1892 and TX-1904), ester (TX-1907 and TX-1911),
morpholino (TX-1921), piperazino (TX-1920), and desoxy
(TX-1914) analogues (Fig. 2).
TX-1877 was synthesized from methyl 2-(2-nitro-1H-1-
imidazolyl)acetate (1), as a key intermediate, treated
with N-methylethanolamine in anhydrous methanol for
26 h at room temperature, in 84% yield (Scheme 1). The
1H NMR spectrum of TX-1877 showed that there were
two resonance forms of N-methyl amide at room tem-
perature. The sec-alcohol and polyol analogues, TX-
1902, TX-1909, and TX-1892, were synthesized from 1
treated with their appropriate amines in methanol at
room temperature in 72, 85 and 88% yield, respectively.
Though TX-1904 was not a€orded in the same condi-
tion, it was obtained in 71% yield by adding the potas-
sium carbonate at 30±35 ꢀC. Ester analogues, such as
TX-1907 and TX-1911, were synthesized from TX-1877
using anhydrous acetic acid or pyridine±sulfur trioxide
complex in dry pyridine in 91 and 78% yield, respec-
tively. Syntheses of the morphorino, piperazino, and
desoxy analogues, TX1921, TX-1920, and TX-1914,
were performed using the corresponding neat amines
(4±16 mol eq.) at room temperature in 83, 79, and 91%
yield, respectively. Imidazole analogues, such as TX-
1908, TX-1910, and TX-1931, were synthesized from the
corresponding methyl esters 2, 3 and 4 with N-methyl-
ethanolamine in 86, 56, and 51% yield, respectively. In
the syntheses of 2 and 3, imidazole and 4-methylimida-
zole were decomposed by using sodium methoxide,
whereas they were obtained when other bases, such as
triethylamine and potassium tert-butoxide, were used,
as shown in Scheme 1. Most TX-1877 analogues were
synthesized in good yields, and their structures and
purities were identi®ed based on physical and spectral
1
data obtained by H NMR, FTIR, and HRMS. Struc-
tures and melting points of these compounds are shown
in Fig. 2 and Table 1. The partition coecients were
measured to estimate their toxicities and pharmacoki-
netics (Table 1).18
Figure 1. Structures of TX-1877, KIN-806, MISO, and etanidazole.

S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
455
Figure 2. Structures of hypoxic cell radiosensitizer TX-1877 analogues.
Scheme 1. Syntheses of 2-nitroimidazole hypoxic cell radiosensitizers TX-1877, TX-1908, TX-1910 and TX-1931.

456
S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
Molecular orbital calculation
the whole molecule, whereas the sums of C2 values of
HOMO on the N-methylamide group were 0.77 and
0.80 in TX-1877 and KIN-806, respectively. The sum of
C2 values of LUMO on the 4-nitroimidazole group was
0.86 in TX-1908. On the other hand, the desni-
troimidazole analogue (TX-1910) had higher C2 values
of LUMO on the N-methylamide group (Me: 0.046, C
(carbonyl): 0.475, N: 0.072, O: 0.012) and the total value
was 0.60, while it was as low as 0.047 was on the imi-
dazole ring. Similarly, the 4-methylimidazole analogue
(TX-1931) had higher C2 values of LUMO on the N-
methylamide group (Me: 0.046, C (amide): 0.475, N:
0.072, O: 0.240) with a total of 0.83. The distribution of
C2 values of HOMO showed a trend opposite to that of
LUMO in all compounds. It was remarkable that TX-
1920, which had the highest ER (1.94), showed C2
values of HOMO as high as 0.71 on the N-methyl
nitrogen of piperazine.
Electron anity is an indicator of radiosensitizing
activity as well as hydrophobicity.19 We previously
reported that the electron anity [EA=ÀLUMO (the
lowest unoccupied MO)] determined by MO calculation
correlated with the radiosensitizing activity.20 To pre-
dict their hypoxic cell radiosensitizing activities, we cal-
culated the LUMO energy values (Eigen values) and
measured partition coecients (P) of TX-1877
analogues (Table 1). The EAs depended on the hetero-
aromatic groups of radiosensitizers. The EAs of 2-
nitroimidazole analogues of TX-1877 were more than
0.9 eV and that of 4-nitroimidazole analogue (TX-1908)
was 0.67 eV. On the other hand, the desnitroimidazole
analogue (TX-1910) and 4-methylimidazole analogue
(TX-1931) had EAs as low as À0.34 and À0.37 eV,
respectively. We also calculated the square of atomic
orbital coecients (C2) of LUMO and HOMO (the
highest occupied MO) of TX-1877 analogues to esti-
mate the e€ects of MO on biological activity (see
Experimental). The C2 values of TX-1877, KIN-806,
TX-1910 and TX-1931 are depicted in their respective
molecular diagrams (Fig. 3).
Electrochemical measurements of TX-1877 analogues
Cyclic voltametry (CV) was performed with TX-1877
analogues in an aqueous solution containing 0.1 M
phosphate bu€er (pH=7.4)±0.5% (v/v) DMSO, and the
obtained cathodic peak potentials versus Ag/AgCl
expressed as Epc were as follows: À661 mV for TX-1877,
À664 mV for TX-1904, À641 mV for etanidazole,
À653 mV for MISO, À793 mV for TX-1908, and no
peak for TX-1910 and TX-1931, respectively (Table 1).
All of the 2-nitroimidazole analogues were found to
have similarly high reduction potentials (À641 to
À664 mV), suggesting that they were reduced with elec-
tron transfer reaction more easily than 4-nitro (TX-
1908), 4-methyl (TX-1931), and desnitroimidazole (TX-
1910) analogues.
The C2 value of LUMO of TX-1877 is higher on the
2-nitroimidazole group (N1: 0.189, C2: 0.169, N3:
0.154, C4: 0.04, C5: 0.195, and NO2: 0.128) than that on
the amide group of the side-chain. KIN-806 also
showed the distribution of LUMO (N1: 0.189, C2:
0.170, N3: 0.153, C4: 0.04, C5: 0.195, and NO2: 0.128)
on the 2-nitroimidazole group. In both compounds, the
sums of the C2 values of LUMO on 2-nitroimidazole
were 0.96 out of a total C2 value of LUMO of 1.0 for
Table 1. Physical data and in vitro radiosensitizing activities of TX-
1877 analogues
Radiosensitizing activity
In vitro radiosensitizing activities of TX-1877 analogues
were measured at doses of 1 mM in EMT6/KU single
cells under hypoxic conditions. The enhancement ratios
(ERs) and the partition coecients of TX-1877 ana-
logues are shown in Table 1. Radiosensitizing activities
of KIN-806 (ER=1.91), TX-1877 (ER=1.75), TX-1902
(ER=1.61), TX-1907 (ER=1.66), TX-1914 (ER=1.76),
TX-1920 (ER=1.94), and TX-1921 (ER=1.87) were
comparable to MISO (ER=1.72). Polyol analogues such
as TX-1892 (ER=1.29) and TX-1904 (ER=1.31), and
sulfonate TX-1911 (ER=1.11) have low radio-
sensitizing activity, because of their higher hydro-
philicities (P<0.02). These results showed that in vitro
ERs of most compounds were related to their partition
coecients except for 4-nitro, desnitro, and 4-methylimi-
dazole analogues, such as TX-1908 (ER=1.31), TX-1910
(ER=1.18), and TX-1931 (ER=0.89).
d
Compound
Mp
(ꢀC)
Pa
ERb
(1 mM)
EAc
(eV)
Epc
(mV)
KIN-806
TX-1877
TX-1892
TX-1902
TX-1904
TX-1907
TX-1909
TX-l911
TX-1914
TX-1920
TX-1921
MISO
130±130.5
83±85
58±60
159±160
108±110
63±65
94±96
81±89
62±63
0.16
1.91
1.75
1.29
1.61
1.31
1.66
1.50
1.11
1.76
1.94
1.87
1.72e
1.72e
0.94
±
0.056
0.021
0.053
0.013
0.74
0.064
0.002
0.32
1.00 À661 (À449)
1.06
1.00
±
±
1.07 À664 (À452)
0.94
1.13
1.09
0.92
0.99
1.04
±
±
±
±
±
±
Amorphous 0.645
114±115
113±114e
165±166e
0.612
0.422e
0.048e
0.93 À653 (À441)
Etanidazole
1.16 À641 (À429)
TX-1908
TX-1910
TX-1931
112±113
123±125
119±120
0.051
0.017
0.088
1.31
1.18
0.89
0.67 À793 (À581)
À0.34
n.d.f
n.d.f
À0.37
aPartition coecient (n-octanol/water) measured at pH 7.4 (0.1 M
phosphate).
Tumor growth inhibition and antimetastatic activity of
TX-1877 and KIN-806
bFor EMT6/KU cells at the dose of 1 mM.
cElectron anit, EA=ÀELUMO (eV).
dThe cathodic peak potential (Epc) was evaluated versus Ag/AgCl.
Parentheses represent the potentials versus NHE (normal hydrogen
electrode), E(NHE)=E(Ag/AgCl) + 212 mV.
eData from ref 14.
Tumor growth inhibition by TX-1877 and KIN-806 is
shown in Figure 4. TX-1877 with R (radiation) and
KIN-806 with R eciently suppressed tumor regrowth
fn.d., not determined due to the absence of electric response.

S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
457
at 20 days after the treatment (0.4 mg/g). The tumor
volume of TX-1877 without R was slightly reduced but
that of KIN-806 was not reduced. In the group only
irradiated, tumor regrowth was observed 9 days after
the treatment, whereas tumor regrowth for TX-1877
with R was not observed (data not shown). Antimeta-
static activity of TX-1877 and KIN-806 is shown in
Figure 5. TX-1877 and KIN-806 reduced the mean
numbers of metastatic lung nodules even without irra-
diation and they markedly reduced the mean numbers
Figure 3. The molecular diagram, orbital energy of LUMO (ELUMO) and HOMO (EHOMO), and total energy of hypoxic cell radiosensitizers TX-
1877, KIN-806, TX-1908, TX-1910, TX-1931, and TX-1920. The C2 values of LUMO (italic) and HOMO are shown in the molecular diagram.

458
S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
of metastatic lung nodules within 22 days after the irra-
diation. TX-1877 has more potent antimetastatic activ-
ity than KIN-806 in the absence of irradiation.
806 with an N-methylamide group, although the in vitro
radiosensitizing activity of TX-1877 (ER=1.75) was
higher than that of etanidazole (ER=1.72), but lower
than that of KIN-806 (ER=1.91), as shown in Table 1.
We expected that TX-1877 would not pass through the
blood±brain barrier (BBB) because of its high hydro-
philicity (P=0.056) which was close to that of etanida-
zole (P=0.048) and, therefore, show less neurotoxicity.
In in vitro radiosensitization experiments, most 2-
nitroimidazole analogues showed potent radio-
sensitizing activities (ER>1.6) except highly hydro-
Mononuclear cell in®ltration in tumor tissue
Table 2 shows the intensity of the macrophage, B cell,
helper T lymphocyte, and killer T cell in®ltration in
tumor tissue at 1, 2, and 3 weeks after the various
treatments. Macrophage in®ltration was moderately or
markedly enhanced by TX-1877 (0.4 mg/g) and KIN-
806 (0.4 mg/g) during 3 weeks after the treatment. When
combined with radiation, macrophage in®ltration was
not detected at 1 week, because the tumor volume was
too small. We considered that the macrophages con-
tributed to antitumor activity. Helper T cell in®ltration
was slightly enhanced by TX-1877 and KIN-806, but
helper T cell were not detected in control and following
radiation of 30 Gy. Killer T cell in®ltration was very
slightly or moderately enhanced by TX-1877 and KIN-
806, respectively. B cell in®ltration was slightly or mod-
erately enhanced by TX-1877 and KIN-806.
<
philic compounds (P 0:021). TX-1911 showed the
ˆ
lowest radiosensitizing activity (ER=1.11) among
them, because it could be little incorporated into tumor
cells due to hydrophilic sulfonate group. Similarly,
hydrophilic polyol analogues, such as TX-1892 and TX-
1904, also showed low ER values, 1.29 and 1.31,
respectively. TX-1920, having an N-methylpiperazine
group, had the highest radiosensitizing activity
(ER=1.94). Stratford et al. reported that weak basic
hypoxic cell radiosensitizers were localized adjacent to
the DNA due to their ionic interactions.22 Because TX-
1920 is a weak basic and hydrophobic compound (cal-
culated pKa=7.53 and P=0.645) and has a C2 value of
HOMO as high as 0.712 on the nitrogen of N-methyl-
piperazine (Fig. 3), it could be easily incorporated into
tumor cells, thereby interacting with DNA.
Discussion
Hypoxic cells in solid tumors not only are a major
problem for radiation therapy but also lead to resistance
to most anticancer drugs, accelerate malignant progres-
sion, and increase metastasis.4,21 Therefore, hypoxic
cells are important targets for cancer chemotherapy.
The major goal of our research is to design low-toxicity
bifunctional hypoxic cell radiosensitizers which have
biological response modi®er (BRM) activity, such as
antimetastatic, antiangiogenic,13 immunopotentiating as
well as radiosensitizing activity. We previously designed
a bifunctional hypoxic cell radiosensitizer KIN-806
which suppressed tumor metastasis and enhanced
immunoresponse.15,16 A new antimetastatic hypoxic cell
radiosensitizer TX-1877 with an N-hydroxyethylamide
group was designed to be more hydrophilic than KIN-
The electron anity measured by one-electron reduc-
tion potential or EA is thought to be an indicator of
radiosensitizing activity as well as hydrophobicity.19,20
We calculated the MO of TX-1877 analogues. All
2-nitroimidazole analogues showed high EAs between
0.92 and 1.16 eV. The radiosensitizing activities were
mainly dependent on hydrophobicity when they had the
same heteroaromatic group. The most hydrophobic TX-
1920 (P=0.645) gave the highest ER (1.94). We also
designed 4-nitro- (TX-1908), desnitro- (TX-1910), and
4-methyl- (TX-1931) imidazole analogues with the same
side-chain as TX-1877, which have lower electron a-
nities than 2-nitroimidazole analogues, to estimate the
Figure 4. Relative volume of tumor treated with TX-1877 and KIN-
806. TX-1877 (0.4 mg/g) and KIN-806 (0.4 mg/g) were administered to
C3H/He mice (i.p.). The tumor volume was measured 20 days after the
treatment. The relative tumor volume was compared to the tumor
volume (1.00) at the beginning of the treatment. Each bar represents
the mean Æ s.d. for 15 mice. Statistical signi®cance was determined
using Student's t test. *p < 0.001 versus control; +p < 0.001 versus
KIN-806.
Figure 5. Metastatic lung nodules treated with TX-1877 and KIN-806.
Metastatic lung nodules were counted 22 days after the treatment. TX-
1877 (0.4 mg/g) and KIN-806 (0.4 mg/g) were administered to C3H/He
mice (i.p.). Each bar represents the mean Æ s.d. for 15 mice. Statistical
signi®cance was determined using Student's t-test. *p < 0.001 versus
control; **p < 0.002 versus control; +p < 0.001 versus KIN-806.

S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
459
in¯uence of the heteroaromatic ring on their biological
activities. Their ERs decreased with decreasing EA
values. We also evaluated their reduction potentials as
Epc using cyclic voltametry. The data corroborated the
results of MO calculation, which indicated that EA
values were useful to predict the radiosensitizing activ-
ities instead of the reduction potentials (see Table 1). The
C2 values of LUMO of TX-1910 and TX-1931 shifted
from their imidazole groups to the amide groups on their
side-chain more than in 2-nitroimidazole analogues
(Fig. 3). As a result of its decreased EA value and shift of
C2 values of LUMO to the amide group, TX-1931 (EA=
À0.37 eV) showed radioprotective activity (ER=0.89).
group of TX-1877 and KIN-806 plays a role in their
antimetastatic activity. Previously, it was reported that
a chemically reactive species was formed by oxidative
metabolism of N-methyl group.29,30 The C2 value of
HOMO on the N-methyl carbon of TX-1877 and KIN-
806 was 0.083 and 0.024, respectively. These results
suggested that the N-methyl group of TX-1877 is more
susceptible to oxidative metabolism than that of KIN-
806. TX-1877 and KIN-806 exhibited antimetastatic
activities, but the desnitroimidazole analogue (TX-1910)
did not (data not shown). TX-1910 had higher C2 values
of LUMO on its amide group (C: 0.475, N: 0.072, O:
0.012) and higher values of HOMO on its imidazole
group (N1: 0.091, C2: 0.269, N3: 0.005, C4: 0.314, CS:
0.303), which showed the reverse relationship between
the amide group and the nitroimidazole group of TX-
1877 and KIN-806 (see Fig. 3). This indicated that the
N-methyl group of TX-1910 was hard to oxidize.
Therefore, the MO data might be useful for the rational
design of bifunctional hypoxic cell radiosensitizers hav-
ing antimetastatic and immunopotentiating activities.
We are examining antimetastatic activities of various
analogues to investigate the MO and activity relation-
ships. The minimum requirements for a hypoxic cell
radiosensitizer are a high EA (>0.9 eV) and localized
C2 of LUMO on the electron-de®cient heteroaromatic
group, and for BRM, presumably, localized C2 of
HOMO on a functional group of the side-chain. We will
further evaluate the antiangiogenic activities of other
TX-1877 analogues and also examine the relationship
between the antimetastatic and immunopotentiating
activities of TX-1877 analogues.
It was reported that the activation of macrophages by
biological response modi®er (BRM) played an impor-
tant role in its antitumor activity.23 TX-1877 and KIN-
806 enhanced the mononuclear cell in®ltration, espe-
cially macrophages, into the tumor tissue during 3
weeks after the drug treatment, as shown in Table 2.
When combined with radiation, TX-1877 reduced the
tumor growth for 3 weeks and enhanced macrophage
in®ltration at 3 weeks after irradiation. Although the
mechanisms of antimetastatic activities of TX-1877 and
KIN-806 are not completely elucidated, they may be
related to their immunopotentiating activities. Recently,
Kagiya et al. reported that nitrotriazole-type hypoxic
cell radiosensitizer AK-2123 [N1-(2-methoxyethyl)-2-
(3-nitro-1H-1,2,4-triazol-1-yl)acetamide] had antimeta-
static and immunopotentiating activities acting as a
BRM, which enhanced NK cells, macrophages, and
other lymphocytes, and then suppressed the tumor
metastasis.24,25 On the other hand, it was reported that
MISO and etanidazole promoted the formation of
metastasis of Lewis lung carcinoma and B16 melanoma
cells to the lungs and other organs in C57BL mice.26
Although TX-1877 and KIN-806 are very similar to
etanidazole in chemical structure and EA, they inhibited
the lung metastasis without radiation in vivo. It was
reported that the polar solvent, N-methylformamide
(NMF), had antitumor27 and immunopotentiating28
activities. It was suggested that the N-methyl-amide
Conclusion
In the present study, we designed, based on the MO
calculation, and synthesized new antimetastatic hypoxic
cell radiosensitizer TX-1877 analogues. Almost all the
TX-1877 analogues showed potent in vitro radio-
sensitizing activities. TX-1877 and KIN-806 inhibited
Table 2. Mononuclear cell in®ltration in tumor tissue at 1, 2, and 3 weeks after the treatment. TX-1877 (0.4 mg/g) and KIN-806 (0.4 mg/g) were
administered i.p.a
Week
Control
30 Gy
KIN-806
KIN-806 + 30 Gy
TX-1877
TX-1877 + 30 Gy
Macrophage
1
2
3
Ð
+
Ð
Ã
++
+++
++
Ã
Ã
++
+++
+++
Ã
+
+
+++
+++
++
B cell
1
2
3
Helper T cell
Æ
Æ
Ã
+
++
++
Ã
Ã
+
+
+
Ã
+
+
+
+
Ð
Ð
1
2
3
Ð
Ð
Ð
Ã
+
Æ
Æ
Ã
Ã
+
+
Æ
Ã
Ð
Ð
Ð
Ð
++
Killer T cell
1
2
3
+
Æ
Ã
++
+
+
Ã
Ã
++
+
Æ
Ã
Æ
Æ
Ð
Ð
+
+
a
ÃTumor volume was too small to be estimated. À: No in®ltration; Æ: Very slight; +: Slight; ++: Moderate; +++: Marked.

460
S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
tumor lung metastasis and enhanced in®ltration by macro-
phages without irradiation. TX-1877 has remarkable anti-
metastatic activity. TX-1877 has a high EA, a moderate
P value, large C2 values of HOMO on N-methylamide,
and large C2 values of LUMO on 2-nitroimidazole
group. These observations suggested that the MO cal-
culation data might be useful for the design of bifunc-
tional hypoxic cell radiosensitizers. Thus, TX-1877 is
expected to have clinical applications as a bifunctional
hypoxic cell radiosensitizer with antimetastatic and
immunopotentiating activities. Furthermore, we will
elucidate the antimetastatic activities of TX-1877
analogues and design more potent TX-1877 analogues.
residue was puri®ed by silica gel column chromato-
graphy (CH2Cl2:MeOH, 9:1) to a€ord 1 (4.2 g, 80%) as
light yellow plates (from CHCl3): mp 95±96 ꢀC (lit.33 mp
1
94±95); H NMR (400 MHz, CDCl3): d 3.82 (s, 3H),
5.14 (s, 2H), 7.09 (s, 1H), 7.21 (s, 1H); IR (KBr) 3142, 3025,
2978, 1736, 1537, 1490, 1425, 1360, 1290, 1249, 1161, 1149,
984, 843, 784, 761, 696cmÀ1; EIMS m/e: 185 (M+).
Synthesis of N1,N1-dimethyl-2-(2-nitro-1H-1-imidazolyl)-
acetamide (KIN-806, Ro 7-1052)
To a solution of 1 (10 g, 54 mmol) in anhydrous MeOH
(50 mL), N,N-dimethylamine in MeOH (112 mL,
270 mmol) was added. The reaction mixture was stirred
for 18.5 h at room temperature. The solvent was evapo-
rated under reduced pressure, and the residue was puri-
Experimental
®ed
by
silica
gel
column
chromatography
General procedures
(CH2Cl2:MeOH, 99:1) to a€ord KIN-806 (9.3 g, 87%)
as pale yellow needles (from acetone): mp 130±130.5 ꢀC
(lit.33 mp 129±130); 1H NMR (400 MHz, CDCl3): d 3.01
(s, 3H), 3.14 (s, 3H), 5.22 (s, 2H), 7.06 (s, 1H), 7.18 (s,
1H); IR (KBr) 3113, 2953, 2359, 1662, 1539, 1510, 1487,
1H and 13C NMR spectra were recorded on a JEOL
JNM-EX400 spectrometer with tetramethylsilane as the
internal standard. Chemical shifts are reported in ppm.
Coupling constants are reported in Hz. IR spectra are
recorded in KBr pellets on a Perkin±Elmer 1600 spec-
trometer. Mass spectra were measured on a Shimazu
GC±MS QP-1000 mass spectrometer using an electron
ionization (EI) method. High-resolution mass spectra
(HRMS) were measured on a JEOL JMS-SX 102A
mass spectrometer using a fast atom bombardment
(FAB) and EI method. Reactions were monitored by
analytical thin-layer chromatography (TLC) with use of
Merck silica gel 60F254 glass plates and Merck alumi-
num oxide 60F254 neutral (Type E). Column chromato-
graphy was performed on Merck silica gel 60 (230±400
mesh) and aluminum oxide 90 active neutral (70±230
mesh). The n-octanol±water partition coecient (P) was
measured by Hitachi U-2000 spectrophotometer
according to the method of Fujita et al.31 All melting
points were determined by Yanagimoto micro melting
point apparatus (MP-S3 model) and are uncorrected.
Elemental analysis was performed with a Yanako CHN
recorder MT-5. All chemicals were purchased from
Wako Pure Chemical Industries, Ltd. (Osaka, Japan),
Kanto Chemical Co. Inc. (Tokyo, Japan), Tokyo Che-
mical Industry Co., Ltd. (Tokyo, Japan), and Sigma-
Aldrich Japan (Tokyo, Japan). The semi-empirical
molecular orbital (MO) calculation was applied by the
PM3 methods of Stewart32 and program MOPAC 97
(Fujitsu WinMOPAC V. 2.0, Fujitsu, Tokyo). Orbital
energy (eV) of LUMO (ELUMO) and HOMO (EHOMO),
C2 value of LUMO and HOMO, and total energy (eV)
were calculated. C2 values of less than 0.05 were
omitted. Electrodes for cyclic voltammetry (CV) were
purchased from BAS (Osaka, Japan).
1365, 1301, 1161, 1138, 917, 843, 820, 794, 629 cmÀ1
;
FABMS m/e: 199 (MH+); HRMS (FAB+) m/e: 199.0840
(MH+ C7H11O3N4 requires 199.0831). MO data: ELU-
2
(LUMO): 0.195 (C-5, imidazole), 0.189
MO=À0.946 eV; C
(N-1, imidazole), 0.170 (C-2, imidazole), 0.153 (N-3,
imidazole), 0.128 (N, NO2), 0.054 and 0.067(O, NO2);
EHOMO=À10.043 eV; C2 (HOMO): 0.625 (N, amide),
0.136 (O, amide); total energy: À2510.27680 eV.
Synthesis of N1-(2-hydroxyethyl)-N-1-methyl-2-(2-nitro-
1H-imidazolyl)acetamide (TX-1877)
A solution of 1 (10.1 g, 50 mmol) and N-methylethanol-
amine (11.4 g, 152 mmol) in anhydrous MeOH (80 mL)
was stirred for 26 h at room temperature. The solvent
was evaporated under reduced pressure, and the residue
was puri®ed by silica gel column chromatography
(CH2Cl2:MeOH, 8:2) to a€ord TX-1877 (9.7 g, 84%) as
a
light yellow solid: mp 100±102 ꢀC; 1H NMR
(400 MHz, CD3OD): d 2.98 and 3.21 (each s, 3H) 3.50
and 3.66 (each t, J=5.6 Hz, 2H), 3.55 and 3.79 (each t,
J=5.2 Hz, 2H), 5.43 and 5.54 (each s, 2H), 7.15 and
7.16 (each s, 1H), 7.37 and 7.40 (each s, 1H); 13C NMR
(100 MHz, CD3OD): d 34.26, 36.25, 51.98, 52.23, 52.42,
59.73, 60.43, 128.16, 129.04, 167.81, 168.14, 188.37; IR
(KBr) 3549, 3124, 2929, 1661, 1532, 1487, 1470, 1367,
1292, 1206, 1160, 1137, 1068, 916, 845, 802, 632 cmÀ1
;
HRMS (FAB+) m/e: 229.0935 (MH+ C8H13O4N4
requires 229.0892). MO data: ELUMO=À1.004 eV;
C2 (LUMO): 0.195 (C-5, imidazole), 0.189 (N-1,
imidazole), 0.169 (C-2, imidazole), 0.154 (N-3, imi-
dazole), 0.128 (N, NO2), 0.054 and 0.067 (O, NO2);
EHOMO=À10.154 eV, C2 (HOMO): 0.639 (N, amide),
0.137 (O, amide); total energy: À2953.44597 eV.
Synthesis of methyl 2-(2-nitro-1H-1-imidazolyl)acetate (1)
To a solution of 2-nitroimidazole (3 g, 26.5 mmol) in
anhydrous DMF (20 mL), sodium methoxide (2 g,
37.0 mmol) was added at 90 ꢀC. Chloroacetic acid
methyl ester (3 g, 27.8 mmol) was added to the reaction
mixture and stirred for 1 h at 100±110 ꢀC. DMF was
evaporated under reduced pressure, and the brown oil
Synthesis of N1,N1-di(2-hydroxyethyl)-2-(2-nitro-1H-imi-
dazolyl)acetamide (TX-1892, SR2555)
A solution of 1 (200 mg, 1.1 mmol) and 2,20-imino-
diethanol (350 mg, 3.3 mmol) in anhydrous MeOH

S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
461
(8 mL) was re¯uxed for 24 h. The solvent was evapo-
rated under reduced pressure, and the residue was puri-
Synthesis of 2-{methyl[2-(2-nitro-1H-1-imidazolyl)acetyl]-
amino}ethyl acetate (TX-1907)
®ed
by
silica
gel
column
chromatography
(CH2C12:MeOH, 9:1) to a€ord TX-1892 (250 mg, 88%)
as a pale yellow solid: mp 78±79 ꢀC (lit.19 not described);
1H NMR (400 MHz, CD3OD): d 3.52 (t, J=5.6 Hz,
2H), 3.62 (t, J=5.6 Hz, 2H), 3.67 (t, J=5.6 Hz, 2H),
3.80 (t, J=5.6 Hz, 2H), 5.55 (s, 2H), 7.15 (s, 1H), 7.32
(s, 1H) ; IR (KBr) 3397, 2943, 1649, 1542, 1500,
1425, 1374, 1295, 1216, 1160, 1142, 1073, 923, 843,
TX-1877 (500 mg, 2.2 mmol) was added to a solution of
anhydrous acetic acid (300 mg, 7.9 mmol) and anhy-
drous pyridine (10 mL). The reaction mixture was stir-
red for 6.5 h, and then solvents were evaporated under
reduced pressure. The residue was puri®ed by silica gel
column chromatography (CH2Cl2:MeOH, 9:1) to a€ord
TX-1907 (542 mg, 91%) as a light yellow solid: mp 80±
82 ꢀC; 1H NMR (400 MHz, CDCl3): d 2.05 and 2.15
(each s, 3H), 3.01 and 3.18 (each s, 3H), 3.62±3.67 (m,
2H), 4.22 and 4.30 (each t, J=6.0 Hz, 2H), 5.21 and
5.32 (each s, 2H), 7.05 and 7.07 (s, 1H), 7.17 (s, 1H);
IR (KBr) 3436, 2942, 1725, 1654, 1484, 1366.7, 1255,
1114, 1043, 837, 790, 649 cmÀ1; HRMS (FAB+) m/e:
271.0998 (MH+ C10H15O5N4 requires 271.1026). MO
data: ELUMO=À0.942 eV; C2 (LUMO): 0.195 (C-5,
imidazole), 0.189 (N-1, imidazole), 0.172 (C-2, imida-
zole), 0.152 (N-3, imidazole), 0.127 (N, NO2), 0.052 and
0.062 (O, NO2); EHOMO=À10.100 eV; C2 (HOMO):
0.639 (N, amide), 0.138 (O, amide); total energy:
À3515.09111 eV.
787, 650 cmÀ1
;
HRMS (FAB+) m/e: 259.0998
(MH+ C9H15O5N4 requires 259.1023). MO data:
ELUMO=À1.062 eV; C2 (LUMO): 0.194 (C-5, imida-
zole), 0.187 (N-1, imidazole), 0.161 (C-2, imidazole),
0.156 (N-3, imidazole), 0.132 (N, NO2), 0.055 and 0.069
(O, NO2); EHOMO=À10.162 eV, C2 (HOMO): 0.646 (N,
amide), 0.147 (O, amide); total energy: À3396.68314 eV.
Synthesis of 1-(3-hydroxytetrahydro-1H-1-pyrrolyl)-2-(2-
nitro-1H-1-imidazolyl)-1-ethanone (TX-1902)
To a solution of 1 (200 mg, 1.1 mmol) in anhydrous
MeOH (7 mL), dl-3-pyrrolidinol (192 mg, 2.2 mmol) was
added. The reaction mixture was re¯uxed for 23 h and
then solvent was evaporated under reduced pressure,
and the residue was puri®ed by silica gel column chro-
matography (CH2Cl2:MeOH, 9:1) to a€ord TX-1902
(190 mg, 72%) as a white solid: mp 169±170 ꢀC; 1H
NMR (400 MHz, CD3OD): d 1.89±2.12 (m, 2H), 3.23±
3.45 (m, 2H), 3.49±3.68 (m, 2H), 4.36±4.48 (m, 1H),
5.25±5.37 (m, 2H), 7.10 (s, 1H), 7.35 (s, 1H); IR
(KBr) 3339, 3128, 2955, 1659, 1542, 1497, 1457, 1422,
1371, 1142, 1103, 799 cmÀ1; HRMS (FAB+) m/e:
241.0938 (MH+ C9H13O4N4 requires 241.0892). MO
data: ELUMOÀ1.005 eV; C2 (LUMO): 0.182 (C-5, imi-
dazole), 0.183 (N-1, imidazole), 0.139 (C-2, imidazole),
0.162 (N-3, imidazole), 0.162 (N, NO2), 0.082 and 0.061
(O, NO2); EHOMO=À9.905 eV; C2 (HOMO): 0.627 (N,
amide), 0.134 (O, amide); total energy: À3072.15287
eV.
Synthesis of methyl 2-(4-nitro-1H-1-imidazolyl)acetate (2)
To a solution of 4-nitroimidazole (510 mg, 4.5 mmol) in
anhydrous DMF (10 mL), sodium methoxide (500 mg,
9.3 mmol) was added at 90 ꢀC, and then chloroacetic
acid methyl ester (573 mg, 5.3 mmol) was added to the
reaction mixture and stirred for 2 h at 110 ꢀC. DMF was
evaporated under reduced pressure and washed with
n-hexane to give a pale yellow residue, which was pur-
i®ed by Celite column chromatography (AcOEt) to
a€ord 2 (670 mg, 80%) as a pale yellow solid : mp 129±
130 ꢀC; H NMR (400 MHz, CD3OD): d 3.21 (s, 3H),
1
4.95 (s, 2H), 7.65 (s, 1H), 8.06 (s, 1H); IR (KBr) 3124,
3022, 297, 1739, 1536, 1489, 1423, 1356, 1291, 1248,
1163, 1142, 983, 914, 841, 786, 765, 656, 645, 592 cmÀ1
;
EIMS m/e: 185 (M+); HRMS (EI+) m/e: 185.0437 (M+
C6H7O4N3 requires 185.0445).
Synthesis of N1-[2-hydroxy-1,1-di(hydroxymethyl)-ethyl]-
2-(2-nitro-1H-1-imidazolyl)acetamide (TX-1904)
Synthesis of N1-(2-hydroxyethyl)-N1-methyl-2-(4-nitro-1H-
1-imidazolyl)acetamide (TX-1908)
To a solution of 1 (200 mg, 1.1 mmol) in anhydrous
DMF (8 mL), 2-amino-2-hydroxymethyl-1,3-propandiol
(240 mg, 2.2 mmol) and potassium carbonate (152 mg,
1.1 mmol) were added. The reaction mixture was stirred
for 1.6 h at 30±35 ꢀC, and then the solvent was evapo-
rated under reduced pressure, and the residue was pur-
i®ed by silica gel column chromatography
(CH2Cl2:MeOH, 8.8:1.2) to a€ord TX-1904 (215 mg,
A solution of 2 (370 mg, 1.9 mmol) and N-methyletha-
nolamine (200 mg, 2.7 mmol) in anhydrous MeOH
(30 mL) was stirred for 23 h at room temperature. The
solvent was evaporated under reduced pressure, and the
residue was puri®ed by silica gel column chromato-
graphy (CH2Cl2:MeOH, 9:1) to a€ord TX-1908
(362 mg, 86%) as a white solid: mp 119±120 ꢀC; 1H
NMR (400 MHz, CD3OD): d 2.994 and 3.16 (each s,
3H), 3.51±3.55 (m, 2H), 3.69 and 3.77 (each t, J=5.6
and 5.2 Hz, 2H), 5.16 and 5.24 (each s, 2H), 7.67 (s,
1H), 8.07 and 8.08 (each s, 1H); IR (KBr) 3295,
3119, 2919, 1648, 1537, 1484, 1407, 1337, 1290, 1114,
71%) as a light yellow solid: mp 119±121 ꢀC; H NMR
1
(400 MHz, CD3OD): d 3.75 (s, 6H), 5.21 (s, 2H), 7.16 (s,
1H), 7.41 (s, 1H); IR (KBr) 3428, 1683, 1542, 1494,
1368, 1295, 1161, 1140, 1051, 923, 843, 778, 650 cmÀ1
;
HRMS (FAB+) m/e: 275.0997 (MH+ C9H15O6N4
requires 275.0947). MO data: ELUMO=À1.074 eV; C2
(LUMO): 0.194 (C-5, imidazole), 0.188 (N-1, imidazole),
0.163 (C-2, imidazole), 0.159 (N-3, imidazole), 0.131 (N,
NO2), 0.064 and 0.069(O, NO2); EHOMO=À10.039 eV;
C2 (HOMO): 0.588 (N, amide), 0.161 (O, amide); total
energy: À3690.46747 eV.
996, 873, 826, 679 cmÀ1
;
HRMS (FAB+) m/e:
228.0865 (M+ C8H13O4N4 requires 228.0859). MO
data: ELUMO=À0.697 eV; C2 (LUMO): 0.389 (C-5,
imidazole), 0.149 (N-1, imidazole), 0.063 (C-2, imi-
dazole), 0.147 (C-4, imidazole), 0.074 (N, NO2);

462
S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
EHOMO=À10.410 eV; C2 (HOMO): 0.614 (N, amide),
0.135 (O, amide); total energy: À2953.57440 eV.
Synthesis of 2-{methyl[2-(2-nitro-1H-1-imidazolyl)acetyl]-
amino}ethyl hydrogen sulfate (TX-1911)
To a solution of TX-1877 (280 mg, 1.2 mmol) in anhy-
drous pyridine (7 mL), pyridine±sulfur trioxide (206 mg,
1.3 mmol) was added and stirred for 2 h at room tem-
perature. The solvent was evaporated under reduced
pressure, and the residue was puri®ed by silica gel col-
umn chromatography (CH2Cl2:MeOH, 4:6) to give TX-
1911 (287 mg, 78%) as a yellow solid: mp 130±132 ꢀC;
1H NMR (400 MHz, CD3OD): d 3.00 and 3.25 (each s,
3H), 3.68 (t, J=5.2 Hz, 1H), 3.77 (t, J=5.2 Hz, 1H),
4.12 (t, J=5.2 Hz, 1H), 4.23 (t, J=5.2 Hz, 1H), 5.45,
5w>5.48 and 5.57 (each s, 2H), 7.14±7.49 (each s,
total 2H); IR (KBr) 3471, 2954, 1654, 1490, 1366,
Synthesis of N1-(2-hydroxypropyl)-2-(2-nitro-1H-1-imid-
azolyl)acetamide (TX-1909)
To a solution of 1 (200 mg, 1.1 mmol) in anhydrous
MeOH (6 mL), dl-isopropanolamine (250 mg, 3.3 mmol)
was added. The reaction mixture was re¯uxed for 15 h.
The solvent was evaporated under reduced pressure,
and the residue was puri®ed by silica gel column chro-
matography (CH2Cl2:MeOH, 9.5:0.5) to a€ord TX-
1909 (213 mg, 85%) as a white solid: mp 125±126 ꢀC;
1H NMR (400 MHz, CD3OD): d 1.15 (d, J=6.4 Hz,
3H), 3.15±3.28 (m, 2H), 3.81±3.85 (m, 1H), 5.18 (s, 2H),
7.16 (s, 1H), 7.44 (s, 1H); IR (KBr) 3307, 3154, 2966,
1666, 1572, 1542, 1495, 1360, 1296, 1260, 1155, 1084,
843, 773, 720 cmÀ1; HRMS (FAB+) m/e: 229.0916
(MH+ C9H12O4N4 requires 229.0892). MO data:
ELUMO=À0.946 eV; C2 (LUMO): 0.192 (C-5, imida-
zole), 0.188 (N-1, imidazole), 0.160 (C-2, imidazole),
0.159 (N-3, imidazole), 0.136 (N, NO2), 0.055 and
0.070 (O, NO2); EHOMO=À10.043; C2 (HOMO): 0.654
(N, amide), 0.162 (O, amide); total energy:
À2953.77667 eV.
1237, 1143, 1067, 1014, 902, 773, 643, 579 cmÀ1
.
Anal. calcd for C8H1207N4Sꢁ 2.5H2O: C, 27.19; H,
3.42; N, 15.85. Found: C, 27.23; H, 3.52; N, 15.43.
MO data: ELUMO=À0.946 eV; C2 (LUMO): 0.194
(C-5, imidazole), 0.188 (N-1, imidazole), 0.163 (C-2,
imidazole), 0.157 (N-3, imidazole), 0.131 (N, NO2),
0.055 and 0.069 (O, NO2); EHOMO=À10.043 eV; C2
(HOMO): 0.661 (N, amide), 0.127 (O, amide); total
energy: À4021.04278 eV.
Synthesis of N1-ethyl-N1-methyl-2-(2-nitro-1H-1-imida-
zolyl)acetamide (TX-1914)
Synthesis of methyl 2-(1H-1-imidazolyl)acetate (3)
To a solution of imidazole (1 g, 14.7 mmol) and chloro-
acetic acid methyl ester (1.6 g, 15.0 mmol) in anhydrous
DMF (40 mL), triethylamine (2.3 g, 22.7 mmol) was
added and stirred for 22 h at 60 ꢀC. The solvent was
evaporated under reduced pressure, and the residue
was puri®ed by silica gel column chromatography
(CH2Cl2:MeOH, 9:1) to a€ord 3 (924 mg, 45%) as col-
orless needles (from CHCl3): mp 55±56 C; H NMR
(400 MHz, CDCl): d 3.79 (s, 3H), 4.71 (s, 2H), 6.95 (s,
1H), 7.09 (s, 1H), 7.50 (s, 1H); IR (KBr) 3471, 2956,
1751, 1512, 1418, 1374, 1299, 1216, 1073, 990, 907, 829,
761, 692, 663, 573 cmÀ1; EIMS m/e: 140 (M+). HRMS
(EI+) m/e: 140.0587 (M+ C6H8O2N2 requires 140.0586).
A solution of 1 (70 mg, 0.35 mmol) in N-ethylmethyl-
amine (0.5 mL, 5.8 mmol) was stirred for 24 h at room
temperature. The reaction mixture was evaporated in
vacuo to give an oil residue, which was puri®ed by silica
gel column chromatography (CH2Cl2:MeOH, 9.5:0.5),
to a€ord TX-1914 (74 mg, 91%) as a white solid: mp
74±75 ꢀC; H NMR (400 MHz, CDCl3): d 1.14 and 1.33
1
ꢀ
1
(each t, J=7.2 Hz, 3H), 2.97 and 3.10 (each s, 3H),
3.40±3.48 (m, 2H), 5.18 and 5.23 (each s, 2H), 7.06 (s,
1H), 7.18 (s, 1H); IR (KBr) 3421, 2968, 1654, 1540,
1508, 1490, 1457, 1414, 1363, 1298, 1163, 1143, 1090,
845, 815, 797, 668 cmÀ1
;
HRMS (FAB+) m/e:
213.0993 (M+ C8H12O3N4 requires 213.0988). MO
data: ELUMO=À0.929 eV; C2 (LUMO): 0.195 (C-5,
imidazole), 0.190 (N-1, imidazole), 0.170 (C-2, imida-
zole), 0.153 (N-3, imidazole), 0.128 (N, NO2), 0.054 and
0.067 (O, NO2); EHOMO=À10.013 eV; C2 (HOMO):
0.655 (N, amide), 0.142 (O, amide); total energy:
À2659.81336 eV.
Synthesis of N1-(2-hydroxyethyl)-N1-methyl-2-(1H-1- imi-
dazolyl)acetamide (TX-1910)
A solution of 3 (900 mg, 8 mmol) and N-methylethanol-
amine (1.27 g, 16.8 mmol) in anhydrous MeOH (45 mL)
was stirred for 15 h at room temperature. The solvent
was evaporated under reduced pressure, and the residue
was puri®ed by silica gel column chromatography
(CH2Cl2:MeOH, 9:1) to a€ord TX-1910 (828 mg, 56%)
as a colorless crystal: mp 145±146 ꢀC; 1H NMR
(400 MHz, CD3OD): d 2.97 and 3.15 (each s, 3H), 3.49±
3.55 (m, 2H), 3.68 and 3.75 (each t, J=5.6 and 5.2 Hz,
2H), 5.03 and 5.12 (each s, 2H), 6.95 (s, 1H), 7.04
and 7.06 (each s, 1H), 7.59 and 7.60 (each s, 1H); IR
(KBr) 3186, 2869, 1647, 1488, 1406, 1335, 1300, 1236,
Synthesis of 1-(4-methylpiperazino)-2-(2-nitro-1H-1-imid-
azolyl)-1-ethanone (TX-1920)
A solution of 1 (200 mg, 1.1 mmol) in N-methylpiper-
azine (0.5 mL, 4.5 mmol) was stirred for 72 h at room
temperature. N-Methylpiperazine was evaporated in
vacuo, and the brown residue was puri®ed by aluminum
oxide (Al2O3 neutral, 70±230 mesh, Merck) column
chromatography (CH2Cl2:MeOH, 95:5) to a€ord TX-
1053, 918, 848, 754 cmÀ1
;
HRMS (FAB+) m/e:
1920 (220 mg, 79%) as an amorphous brown solid: H
1
183.0993 (M+ C8H13O2N3 requires 183.1008). MO
data: ELUMO=À0.346 eV; C2 (LUMO) 0.475 (N,
amide); EHOMO=À9.415 eV; C2 (HOMO): 0.314 (C-4,
imidazole), 0.303 (C-5, imidazole), 0.269 (C-2, imid-
azole), 0.135 (O, amide); total energy: À2222.06461 eV.
NMR(400 MHz, CD3OD): d 2.25 and 2.26 (each s, 3H),
2.37(m, 2H), 2.47 (m, 2H), 3.51 (m, 4H), 5.35 (s, 2H),
7.06 (s, 1H), 7.30 (s, 1H); IR (KBr) 3455, 2944, 2800,
1654, 1540, 1492.0, 1465, 1428, 1366, 1292, 1257, 1237,
1144, 1074, 1036, 1001, 917, 842, 813 783, 650 cmÀ1
;

S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
463
HRMS (FAB+) m/e: 253.1175 (M+ C10H16O3N5
requires 253.1175). MO data: ELUMO=À0.994 eV; C2
(LUMO): 0.194 (C-5, imidazole), 0.189 (N-1, imidazole),
0.164 (C-2, imidazole), 0.157 (N-3, imidazole), 0.123 (N,
NO2), 0.055 and 0.068 (O, NO2); EHOMO=À9.459 eV;
C2 (HOMO): 0.712 (N, piperazine); total energy:
À3105.28588 eV.
1050, 1009, 970, 810 cmÀ1; FABMS m/e: 198 (MH+).
HRMS (FAB+) m/e: 198.1241 (M+ C9H16O2N3 requires
198.1243). MO data: ELUMO=À0.372 eV; C2 (LUMO):
0.475 (C, amide), 0.240 (O, amide), 0.072 (N, amide);
EHOMO=À9.132 eV; C2 (HOMO): 0.319 (C-4, imida-
zole), 0.186 (C-2, imidazole), 0.256 (C-5, imidazole),
0.140 (N-1, imidazole); total energy: À2371.782600 eV.
Synthesis of 1-morpholino-2-(2-nitro-1H-1-imidazolyl)-1-
ethanone (TX-1921, Ro 07-1113)
Electrochemical measurements
Cyclic voltammetry (CV) was carried out with an ALS/
chi Electrochemical Analyzer Model 660A, BAS, with a
three-electrode system. A glassy-carbon as a working
electrode, Pt-wire as an auxiliary electrode, and Ag/
AgCl (saturated sodium chloride) as a reference elec-
trode were used. The reference electrode was separated
from the bulk of the solution by a glass frit and was
immersed in an aqueous solution containing 0.1 M-
phosphate bu€er (0.1 M, pH=7.4)À0.5% (v/v) DMSO.
All experiments were measured at 298 K in each sample
solution (1 mM, 500 mL) purged with N2 gas. The
cathodic peak potential (Epc) was evaluated versus Ag/
AgCl, in which the sweep rate was 100 mV/s for CV.
The potentials of both reference electrodes Ag/AgCl
and NHE (normal hydrogen electrode) were calibrated
A solution of 1 (200 mg, 1.1 mmol) in morpholine
(0.5 mL, 5.7 mmol) was stirred for 87 h at room tem-
perature. The residue was puri®ed by silica gel column
chromatography (CH2Cl2:acetone, 8:2) to a€ord TX-
1921 (220 mg, 83%) as a light yellow solid: mp 114±
115 ꢀC (lit.33 mp 113.5±115ꢀC); 1H NMR (400 MHz,
CDCl3): d 3.55±3.79 (8H), 5.22 (s, 2H), 7.08 (s, 1H), 7.18
(s, 1H); IR (KBr) 3441, 3147, 2972, 2855, 2362, 1655,
1536, 1491, 1426, 1369, 1292, 1269, 1231, 1215, 1138,
1104, 1065, 1034, 964, 914, 883, 834, 779, 625 cmÀ1
;
HRMS (FAB+) m/e: 240.0859 (M+ C9H12O4N4
requires 240.0845). MO data: ELUMO=À1.074 eV; C2
(LUMO): 0.193 (C-5, imidazole), 0.183 (N-1, imidazole),
0.168 (C-2, imidazole), 0.159 (N-3, imidazole), 0.1280 (N,
NO2), 0.054 and 0.067 (O, NO2); EHOMO=À9.459 eV; C2
(HOMO): 0.609 (N, amide), 0.132 (O, amide); total
energy: À3071.66914 eV.
by
the
AgCl)+212 mV.
following
equation:
E(NHE)=E(Ag/
In vitro radiosensitizing assay
Synthesis of methyl 2-(4-methyl-1H-1-imidazolyl) acetate
(4)
In vitro radiosensitization was measured in EMT6/KU
single cells under hypoxic conditions. Hypoxic cell
radiosensitizer was added to an EMT6/KU cell (5 Â
105 cells/mL) suspension at a dose of 1 mM in test tubes
and treated with 95% N2±5%CO2 gas for 15 min. Local
irradiation of 8±32 Gy was given by 60Co g-rays for
30 min after the administration and then colony forming
assays were performed. Enhancement ratios (ERs) were
determined from the ratio of radiation doses required to
reduce the surviving fraction of EMT6/KU cells to 1%.
Usually, each ER was obtained from survival curves
consisting of four or ®ve points per curve.
To a solution of 4-methylimidazole (410 mg, 5 mmol) in
anhydrous DMF (8 mL), potassium tert-butoxide
(617 mg, 5.5 mmol) was added at 70 ꢀC, then chlor-
oacetic acid methyl ester (596 mg, 5.5 mmol) was added
and stirred for 1 h at 90 ꢀC. The solvent was evaporated
under reduced pressure, and the residue was puri®ed by
silica gel column chromatography (CH2Cl2:MeOH,
9.5:0.5), to a€ord 4 (421 mg, 55%) as a light yellow oil:
1H NMR (400 MHz, CDCl3): d 2.16 and 2.23 (each s,
3H), 3.78 (s, 3H), 4.61 and 4.63 (each s, 2H), 6.65 and
6.81 (each s, 1H), 7.38 and 7.43 (each s, 1H); IR (KBr)
3354, 1748, 1566, 1501, 1437, 1354, 1290, 1225, 1002,
826.1, 779, 702, 661, 620, 573 cmÀ1; EIMS m/e: 154
(M+); HRMS (EI+) m/e: 154.0735 (M+ C7H10O2N2
requires 154.0742).
In vivo radiosensitization, tumor growth inhibition, supp-
ression of lung metastasis, and mononuclear cells in®ltration
In vivo radiosensitizing activity, tumor growth inhibi-
tion, suppression of lung metastasis, and immunopo-
tentiation were evaluated as reported previously.17 In
brief, female C3H/He mice bearing the SCCVII tumor
were used. Fifteen or 10 days after the inoculation of
105 SCCVII tumor cells into the right hind thigh of
the animals at 8 weeks of age, a drug (0.4 mg/g) was
administered i.p. and then local irradiation of 30 Gy
was given by 60Co g-rays for 30 min after the adminis-
tration. The tumor volume was measured at three
perpendicular diameters with 2- or 3-day intervals. The
relative tumor volume was compared to the tumor
volume (1.00) at the beginning of the treatment. Mice
were sacri®ced after the drug treatment to count the
number of metastatic nodules on the surface of the lungs.
All tissues were stained by the ABC method for immuno-
logical evaluation.
Synthesis of N1-(2-hydroxyethyl)-N1-methyl-2-(4-methyl-
1H-1-imidazolyl)acetamide (TX-1931)
A solution of 4 (200 mg, 1.3 mmol) and N-methyletha-
nolamine (196 mg, 2.6 mmol) in anhydrous DMF (3 mL)
was stirred for 23 h at 70±80 ꢀC. The solvent was eva-
porated under reduced pressure, and the residue was
puri®ed by aluminum oxide (Al2O3 neutral, 70±230
mesh, Merck) column chromatography (eluted from
CH2Cl2 to CH2Cl2:MeOH, 9.5:0.5) to a€ord TX-1931
(101 mg, 51%) as a white solid: mp 119±120 ꢀC; 1H
NMR (400 MHz, CD3OD): d 2.18 and 2.20 (each s, 3H),
2.94 and 3.10 (each s, 3H), 3.43±3.54 (m, 4H), 4.66 and
4.86 (each s, 2H), 6.65 (s, 1H), 7.26 (s, 1H); IR (KBr)
3423, 2935, 1653, 1508, 1411, 1341, 129, 1235, 1173, 1129,

464
S. Kasai et al. / Bioorg. Med. Chem. 9 (2001) 453±464
13. Hori, H.; Jin, C.-Z.; Kiyono, M.; Kasai, S.; Shimamura,
Acknowledgements
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The authors thank Dr. Yoshihiro Takekawa and Dr.
Hiroshi Maezawa of the School of Medical Sciences,
The University of Tokushima for generous help with the
radiobiological experiments. The authors also thank
Mrs. Maki Nakamura, Mrs. Emiko Okayama, Mrs.
Kayoko Yamashita and the sta€ of the Center for
Cooperative Research of our Faculty for measurement
of NMR, MS and elemental analysis. We also appreci-
ate Dr. Hiroshi Terada, Dr. Yasuo Shinohara, and Mrs.
Yasuko Yoshioka of the Faculty of Pharmaceutical Sci-
ences of The University of Tokushima, for measurement
of HRMS.
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