Angiogenesis inhibitor TX-1898: Syntheses of the enantiomers of sterically diverse haloacetylcarbamoyl-2-nitroimidazole hypoxic cell radiosensitizers

Article abstract of DOI:10.1016/j.bmc.2004.06.039

(R)- and (S)-Epichlorohydrins were used to prepare the enantiomers of sterically diverse haloacetylcarbamoyl-2-nitroimidazoles that function as hypoxic cell radiosensitizers. The synthetic design allowed for introduction of a side chain of varying bulk th

Full text of DOI:10.1016/j.bmc.2004.06.039

Bioorganic & Medicinal Chemistry 12 (2004) 4917–4927
Angiogenesis inhibitor TX-1898: syntheses of the enantiomers of
sterically diverse haloacetylcarbamoyl-2-nitroimidazole hypoxic cell
radiosensitizers
Cheng-Zhe Jin,a,f Hideko Nagasawa,a Mariko Shimamura,b Yoshihiro Uto,a
Seiichi Inayama,c Yoshio Takeuchi,d Kenneth L. Kirke and Hitoshi Horia,*
aDepartment of Biological Science and Technology, Faculty of Engineering, University of Tokushima, Tokushima 770-8506, Japan
bMedical Research and Development Center, The Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 113-8613, Japan
cInstitute of Oriental Medical Sciences, 2-6-3 Ebisunishi, Shibuya-ku, Tokyo 155-0021, Japan
dFaculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Sugitani 2630, Toyama 930-0194, Japan
eLaboratory of Bioorganic Chemistry, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Health,
DHHS, Bethesda, MD 20892, USA
fPharmaceutical School of the University of Yanbian, Yanji 133000, China
Received 14 June 2004; revised 28 June 2004; accepted 28 June 2004
Available online 27 July 2004
Abstract—(R)- and (S)-Epichlorohydrins were used to prepare the enantiomers of sterically diverse haloacetylcarbamoyl-2-nitroim-
idazoles that function as hypoxic cell radiosensitizers. The synthetic design allowed for introduction of a side chain of varying bulk
that permitted an examination of the steric effects on enantio-discrimination in biological assay systems. The single stereocenter
also connected the two pharmacophores––a 2-nitroimidazole moiety critical to hypoxic cell radiosensitization, and a halo-
acetylcarbamoyl group to function as an anti-angiogenesis pharmacophore. In the chick embryo chorioallantoic membrane
(CAM) assay, the R-enantiomers possessing the bulky p-tert-butylphenyl group showed higher anti-angiogenic activity than the cor-
responding S-enantiomers, while there were no differences in the activity between the enantiomers containing the less bulky methyl
and tert-butyl groups. Among the compounds we report, R-p-tert-butylphenyl-bromoacetylcarbamoyl-2-nitroimidazole, TX-1898,
was found to be the most promising candidate for further development of as anti-angiogenic hypoxic cell radiosensitizer.
Ó 2004 Elsevier Ltd. All rights reserved.
1.Introduction
thelial growth factor (VEGF), basic fibroblast growth
factor (bFGF), platelet-derived growth factor (PDGF),
insulin-like growth factor (IGF), and interleukin-8 (IL-
8). Many enzymes are involved, including such as the
matrix metalloproteinases (MMPs), thrombin and
methionine aminopeptidase-2 (MetAP-2), urokinase-
type plasminogen activator (uPA), and others. Various
anti-angiogenic drugs have been developed that target
several related growth factor receptors such as VEGF
receptors (VEGFR-1 and VEGFR-2), bFGF receptor,
and PDGF receptor. Indeed, some of these agents, such
as SU5416,4 ZD6474,5 SU6668,6 are now in clinical tri-
als as anti-angiogenic drugs. Our own interest in this
area is focused on small molecule anti-angiogenic agents
related to the naturally-occurring angiogenic inhibitor
fumagillin, and its semi-synthetic analog TNP-470, one
of the first substances to be recognized to as having an
anti-angiogenic effect.7–10
Angiogenesis is required for tumor growth and meta-
stasis and, therefore, represents an exciting target for
cancer treatment. Angiogenesis is a complex process
involving a number of distinct steps, such as endothelial
cell migration, proliferation, formation of capillary
tubes in endothelial cells, their invasion, and metastasis.
These steps are tightly regulated by pro- and anti-angio-
genic factors.1–3 Tumor-related angiogenesis is a multi-
step process that is initiated through the activity of
various pro-angiogenic factors, such as vascular endo-
Keywords: Angiogenesis inhibitor; Hypoxic cell radiosensitizer; Halo-
acetylcarbamoyl-2-nitroimidazole; Enantiomers.
*
Corresponding author. Tel.: +81-88-656-7514; fax: +81-88-656-
9164; e-mail: hori@bio.tokushima-u.ac.jp
0968-0896/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2004.06.039

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C.-Z. Jin et al. / Bioorg. Med. Chem. 12 (2004) 4917–4927
In contrast to anti-angiogenic agents, tumor hypoxia in-
duces the pro-angiogenic genes to stimulate up-regula-
tion of angiogenic and tumor cell survival factors,
giving rise to tumor proliferation, radioresistance, angi-
ogenesis, and metastasis.11 Therefore, hypoxia in solid
tumor is one of the targets for the improvement of
chemo/radio-therapeutic efficacy. We propose that anti-
angiogenic drugs that also function as hypoxic cell radi-
osensitizers will have synergistic interactions between
the hypoxic cytotoxic/hypoxic cell radiosensitizing effect
and anti-angiogenic activity leading to destruction of
hypoxia-initiated neovasculature.
hypoxic cell radiosensitizers, TX-1877,17 an anti-meta-
static hypoxic cell radiosensitizer, anti-angiogenic hyp-
oxic cytotoxin, TX-402,18 a protein tyrosine kinase
inhibitor TX-1123,19 and anti-angiogenic/heparin-bind-
ing arginine dendrimer TX-1943 and TX-194420 (Fig. 1).
We also published previously that the development of
racemic bromoacetylcarbamoyl-2-nitroimidazoles such
as TX-1845 and TX-1846 as strong radiosensitizers
and potent angiogenic inhibitors.21 The molecular chi-
rality of hypoxic cell radiosensitizers generally has little
or no effect on in vitro activity.22,23 In contrast we have
found that there is a significant effect of absolute con-
figuration on anti-angiogenic activity and that a single
enantiomer separated from the racemate, often has a
higher anti-angiogenic activity than the racemate. For
example the R-enantiomer of the thienopyridine SR-
We have reported the molecular design and synthesis of
several 2-nitroimidazole hydroxamate and hydroxamic
acid analogs. These include KIN-841,12 KIN-806,13
KIN-804,14,15 and KIN-844,16 which are bifunctional
Figure 1. Chemical structures of KIN- and TX-series compounds.

C.-Z. Jin et al. / Bioorg. Med. Chem. 12 (2004) 4917–4927
4919
25989 possessed more potent in vitro and in vivo anti-
angiogenic properties than the corresponding racemate,
and it inhibited metastatic dissemination and growth.24
Also the R,R-enantiomer of dihydrobenzofuran lignan
showed more potent anti-angiogenic activity than the
S,S-isomer in the chick embryo chorioallantoic mem-
brane (CAM) assay.25 Molecular shape analysis indi-
cates that there is overlap of target compounds with
TNP-470 an anti-angiogenesis drug now in clinical trials,
depending on steric bulk and absolute configuration.
Based on these considerations, we felt it was important
to examine the effects of absolute configuration on a series
of haloacetylcarbamoyl-2-nitroimidazole radiosensitiz-
ers, compounds that, in racemic form, have shown prom-
ising anti-angiogenic and anti-proliferation activity.21
molecular orbitals (LUMOs). The largest region of orbi-
tal coefficient distribution in the LUMO of all TX-com-
pounds is localized on the chloroacetylcarbamoyl
moiety. With TNP-470, the next LUMO (NLUMO) is
localized on this moiety, while the LUMO of this com-
pound is localized on the isoprenyl moiety. Thus, in
the TX-compounds, the orbital coefficient distributions
of the LUMOs are similar to that of NLUMO of
TNP-470. In contrast, the optimized geometries of the
TX-compounds were quite similar to that of TNP-470.
The relative positions of the chloroacetylcarbamoyl
moiety and large alkoxy group of TX-compounds clo-
sely approximate the locations of the chloro-
acetylcarbamoyl and isoprenyl epoxide moieties of
TNP-470. In particular, based on orientation of the
chloroacetylcarbamoyl group, TX-1897, possessing p-
tert-butylphenyl group, closely resembled the molecular
structure of TNP-470. In contrast, after geometry op-
timizations using the same method, the corresponding
(S)-enantiomers was not superimposed on the molecular
structure of TNP-470. TX-1898 had a molecular struc-
ture similar to TX-1897, but possessing lower ELUMO
value ((ELUMO =ꢀ0.05804hartree (ꢀ1.57869eV)) than
In this study we demonstrate the effectiveness of
epichlorohydrin as a chiral scaffold wherein the single
stereocenter connects a haloacetylcarbamoyl, 2-nitroim-
idazolyl, and diverse alkyl or aryl groups of varying
steric dimensions. We examined the individual enantio-
mers of haloacetylcarbamoyl-2-nitroimidazoles as pro-
tease inhibitors, as anti-angiogenic agents in vitro
using a rat lung endothelial (RLE) cell proliferation
assay and a chick embryo CAM assay, and also their
activities as hypoxic cell radiosensitizers.
that
of
TX-1897
(ELUMO =ꢀ0.04915hartree
(ꢀ1.33688eV)) as shown in Figures 3 and 2.
2.2. Synthesis
2.Results
As shown in Scheme 1, (R)- and (S)-tert-butyl glycidyl
ether [(R)- and (S)-1], and (R)- and (S)-p-tert-butylphe-
nyl glycidyl ether [(R)- and (S)-2] were prepared from
the corresponding (S)- and (R)-epichlorohydrins
(99.9% pure, 98.9% ee; 99.6% pure, 98.6% ee, respec-
tively) by reaction with tert-butanol using a catalytic
amount of borontrifluoride ether complex, and p-tert-
butylphenol in aqueous sodium hydroxide, respectively.
The yields ranged from 54% to 90%.
2.1. Molecular modeling
We carried out geometry and molecular orbital optimi-
zations of the (R)-enantiomers of chloroacetyl-
carbamoyl-2-nitroimidazoles possessing three different
alkyl or arylalkyl groups of varying steric bulk–methyl,
tert-butyl, and p-tert-butylphenyl. The results were com-
pared to TNP-470 using the B3LYP hybrid density func-
tional theory based the GAUSSIAN 98 programs.26
Following the procedure we used for the preparation
the methyl derivatives [(R)- and (S)-3], the enantiomers
of 2-hydroxy-3-(2-nitro-1H-imidazolyl)-propyl ethers
[(R)- and (S)-4, 5], were prepared by reaction of the
Figure 2 shows their chemical structures, optimized geo-
metries, and the energies of their lowest unoccupied
Figure 2. LUMO of the geometrically optimized (R)-chloroacetylcarbamoyl-2-nitroimidazoles with the B3LYP hybrid density functional in
conjunction with the 6-31G(d) basis set using the GAUSSIAN 98 suite of program.

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C.-Z. Jin et al. / Bioorg. Med. Chem. 12 (2004) 4917–4927
sodium carbonate. Treatment of the enantiomerically
pure propanols [(R)- and (S)-3, 4, 5] with highly purified
chloro- or bromo-acetyl isocyanates for 30min in
CH2Cl2 gave the chiral haloacetylcarbamoyl-2-nitroimid-
azoles derivatives in good yield (72–98%) as shown in
Scheme 2.
The structures and yields of the single enantiomers of
haloacetylcarbamoyl-2-nitroimidazoles are shown in
Table 1. The methyl derivatives (TX-1863, TX-1878,
TX-1866, and TX-1879) were re-synthesized following
the procedure described in our previous paper.27
2.3. Biological activity
Figure 3. LUMO of the geometrically optimized (R)-bromo-
acetylcarbamoyl-2-nitroimidazoles with the B3LYP hybrid density
functional in conjunction with the 6-31G(d) basis set using the GAUS-
SIAN 98 suite of program.
2.3.1. Serine protease inhibitory activity. 28 Serine pro-
teases, such as thrombin29 and urokinase-type plasmino-
gen activator (uPA),30 and metalloproteases MMPs
participate in tumor growth and angiogenesis, and sev-
eral tyrosine kinases, such as VEGF, PDGF, and Met
enantiomerically pure glycidyl ethers [(R)- and (S)-1, 2],
with 2-nitroimidazole in the presence of ethanolic
p-tert-butylphenol
tert-butanol
O
O
O
Cl
O
O
O
O
NaOH aq
BF3 Et2O
(S)-2
(S)-1
R-(-)-Epichlorohydrin
(99.6% pure, 98.6% ee)
p-tert-butylphenol
tert-butanol
BF3 Et2O
O
Cl
O
NaOH aq
O
(R)-2
(R)-1
S-(+)-Epichlorohydrin
(99.9% pure, 98.9% ee)
Scheme 1. Synthesis of single enantiomeric alkyl (aryl) glycidyl ethers.
O
O
H
RO
H
RO
X
OH
N
H
O
N
NH
NO
N
a
1
b
1
N
N
N
NO
NO
2
2
2
(S)-3: R= methyl
(S)-4: R= tert-butyl
TX-1878: R= methyl X=Cl
TX-1879: R= methyl X=Br
(S)-5: R= p-tert-butylphenyl
TX-1881: R= tert-butyl X=Cl
TX-1883: R= tert-butyl X=Br
TX-1899: R= p-tert-butylphenyl X=Cl
TX-1900: R= p-tert-butylphenyl X=Br
O
O
RO
OH
RO
H
X
H
N
H
O
NH
NO
N
N
N
a
b
2
2
N
N
2
NO
NO
2
2
(R)-3: R= methyl
(R)-4: R= tert-butyl
(R)-5: R= p-tert-butylphenyl
TX-1863: R= methyl X=Cl
TX-1866: R= methyl X=Br
TX-1880: R= tert-butyl X=Cl
TX-1882: R= tert-butyl X=Br
TX-1897: R= p-tert-butylphenyl X=Cl
TX-1898: R= p-tert-butylphenyl X=Br
Scheme 2. Synthesis of single enantiomer of haloacetylcarbamoyl-2-nitroimidazoles; (a1): (S)-alkyl glycidyl ether/anhydrous EtOH, Na2CO3, reflux;
(b1): haloacetylisocyanate/anhydrous CH2Cl2, rt; (a2): (R)-alkyl glycidyl ether/anhydrous EtOH, Na2CO3, reflux; (b2): haloacetylisocyanate/
anhydrous CH2Cl2, rt.

C.-Z. Jin et al. / Bioorg. Med. Chem. 12 (2004) 4917–4927
Table 1. Enantiomeric haloacetylcarbamoyl-2-nitroimidazole radiosensitizers
4921
*
R1O
N
N
R2O
H
NO2
Radiosensitizer
Configuration
R1
R2
Yield (%)
TX-1863
TX-1878
TX-1866
TX-1879
TX-1880
TX-1881
TX-1882
TX-1883
TX-1897
TX-1899
TX-1898
TX-1900
R
S
R
S
R
S
R
S
R
S
R
S
Methyl
Methyl
Methyl
Methyl
t-Butyl
t-Butyl
t-Butyl
t-Butyl
CONHCOCH2Cl
CONHCOCH2Cl
CONHCOCH2Br
CONHCOCH2Br
CONHCOCH2Cl
CONHCOCH2Cl
CONHCOCH2Br
CONHCOCH2Br
CONHCOCH2Cl
CONHCOCH2Cl
CONHCOCH2Br
CONHCOCH2Br
96
90
98
96
73
72
77
74
95
91
98
93
p-t-Butylphenyl
p-t-Butylphenyl
p-t-Butylphenyl
p-t-Butylphenyl
tyrosine kinase31 are also involved in tumor angiogene-
sis. Therefore, we examined the inhibitory activities of
the individual single enantiomers of haloacetyl-
carbamoyl-2-nitroimidazoles as inhibitors of serine pro-
teases, porcine pancreatic (PP) elastase. As shown in
Table 2, compounds containing the more bulky p-tert-
butylphenyl group were more active than those possess-
ing the corresponding tert-butyl and methyl groups.
Regarding the haloacetyl group, bromoacetyl deriva-
tives showed more active than the corresponding chloro-
acetyl derivatives, as described previously.21 It is
noteworthy that the R-isomers were more active than
the corresponding S-isomers. Compound TX-1898,
which has the bulky p-tert-butylphenyl group, the
bromoacetyl group, and the 2-nitroimidazole moiety ap-
pended to the R-stereocenter was the most potent PP
elastase inhibitor among the compounds tested here.
Table 3. Inhibition of single enantiomeric haloacetylcarbamoyl-2-
nitroimidazole radiosensitizers on RLE cell proliferation
Ether moiety
Halogen
TX-No
IC50 (lM)
R-Isomer
S-Isomer
Me
Cl
Br
Cl
Br
Cl
Br
TX-1863
TX-1878
TX-1866
TX-1879
TX-1880
TX-1881
TX-1882
TX-1883
TX-1897
TX-1899
TX-1898
TX-1900
325
20
275
17
Me
t-Bu
71
80
t-Bu
6.3
6.9
6.5
0.9
p-t-BuPh
p-t-BuPh
7.5
1.4
2.3.2. RLE cell proliferation inhibitory activity.32 The
potencies of inhibition of proliferation of RLE cells in
vitro are shown in Table 3. The rank order of activity
(IC50 values) of the compounds tested was: TX-1897
(7.5lM)>TX-1880 (71lM)>TX-1863 (325lM), TX-
1899 (6.5lM)>TX-1881 (80lM)>TX-1878 (275lM),
TX-1898
(1.4lM)>TX-1882
(6.3lM)>TX-1866
(20lM), TX-1900 (0.9lM)>TX-1883 (6.9lM)>TX-
1879 (17lM). The p-tert-butylphenyl bromo-
acetylcarbamoyl derivatives, TX-1900 and TX-1898,
showed the most potent activity.
There was no significant difference between the activities
of the individual enantiomers. The potency of inhibition
increased with the increasing size of the ether moiety
(methyl<tert-butyl<p-tert-butylphenyl). The bromo-
acetylcarbamoyl derivatives were about 5–10 times more
effective than the corresponding chloroacetylcarbamoyl
derivatives.
Table 2. Inhibition of single enantiomeric haloacetylcarbamoyl-2-
nitroimidazole radiosensitizers on porcine pancreatic elastase
Ether moiety
Halogen
TX-No
Ki (lM)a
R-Isomer
S-Isomer
Me
Cl
Br
Cl
Br
Cl
Br
TX-1863
TX-1878
TX-1866
TX-1879
TX-1880
TX-1881
TX-1882
TX-1883
TX-1897
TX-1899
TX-1898
TX-1900
1180
66
1750
220
557
55
Me
2.3.3. Anti-angiogenic activity.33 Anti-angiogenic activi-
ties of the compounds that were measured using the
chick embryo CAM assay are shown in Table 4. All
compounds tested showed high angiogenic inhibitory
activity. In particular, the p-tert-butylphenyl-bromo-
acetylcarbamoyl derivatives, TX-1898 and TX-1900,
inhibited angiogenesis by more than 80% at their lowest
dose of 5lg/pellet. Both enantiomers of all halo-
acetylcarbamoyl-2-nitroimidazoles examined exhibited
potent than TNP-470. In compounds possessing the
t-Bu
260
42
t-Bu
p-t-BuPh
p-t-BuPh
30
56
9
14
a Substrate: succinyl-Ala-Ala-Ala-pNA.

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C.-Z. Jin et al. / Bioorg. Med. Chem. 12 (2004) 4917–4927
Table 4. Antiangiogenic activity of enantiomeric haloacetyl-
carbamoyl-2-nitroimidazoles in CAM assay
Table 5. Radiosensitizing effects and log P of enantiomeric halo-
acetylcarbamoyl-2-nitroimidazole radiosensitizers on EMT6/KU cells
Ether
moiety
Halogen TX-No
lg/pellet
Inhibition (%)
Radiosensitizer
log Pa
ER (10lM)
calcd/obsd
R-Isomer
S-Isomer
R-Isomer S-Isomer
TX-1880
TX-1881
TX-1882
TX-1883
TX-1897
TX-1899
1.45/1.14
1.45/1.13
1.57/1.22
1.57/1.21
3.68/3.16
3.68/3.31
1.76
Me
Cl
Br
Cl
Br
TX-1863 100
TX-1878 100
80
1.75
1.83
83
1.82
Me
TX-1866 100
TX-1879 100
100
100
55
1.73 (1lM)b
t-Bu
t-Bu
TX-1880
TX-1881
TX-1882
TX-1883
TX-1897
TX-1899
TX-1898
TX-1900
TNP-470
10
10
10
10
10
10
5
1.90
1.75 (1lM)
53
61
TX-1898
TX-1900
3.79/2.90
3.79/3.25
1.80
77
1.74
p-t-BuPh Cl
p-t-BuPh Br
64
a log P are calculated by Pallas 3.0 and measured in n-octanol-phos-
phate buffer (pH=7.4).
b Measured at 1lM because of cytotoxicity at 10lM without
irradiation.
58
93
5
82
10
5
81.8
78.1
combine radiosensitizing activity with anti-angiogenic
activity. In our previous report,21 we found the racemic
haloacetylcarbamoyl-2-nitroimidazole hypoxic cell radi-
osensitizers, such as KIN-1800, TX-1835, TX-1836, TX-
1844, TX-1845, and TX-1846, were also potent angio-
genesis inhibitors activity that we ascribe to the presence
of the reactive haloacetylcarbamoyl group. To examine
the structure–activity relationship of a series of single
enantiomeric anti-angiogenic haloacetylcarbamoyl-2-
nitroimidazole hypoxic cell radiosensitizers we exploited
the functionality and stereocenter of epichlorohydrin to
prepare individual enantiomers of haloacetylcarbamoyl-
2-nitroimidazoles. These were adorned with groups of
increasing bulk (methyl, tert-butyl, p-tert-butylphenyl)
through an ether linkage. This allows the study of effects
of lipophilicity and steric factors in the interactions with
angiogenesis-related enzymes, particularly as a function
of absolute stereochemistry.
bulky p-tert-butylphenyl ether moiety, the R-isomers
were more anti-angiogenic than the corresponding S-
isomers. Thus the potencies can be ranked as follows:
TX-1898 (93% at 5lg/pellet)>TX-1900 (82% at 5lg/
pellet), TX-1897 (64% at 10lg/pellet)>TX-1899 (58%
at 10lg/pellet). This trend was more significant for
bromoacetyl derivatives than chloroacetyl derivatives.
There was no difference in the anti-angiogenic activities
of the single enantiomers of the haloacetylcarbamoyl-2-
nitroimidazole radiosensitizers containing the less steri-
cally bulky methyl and tert-butyl ethers. The rank order
of potency is: TX-1863 (80% at 100lg/pellet) and TX-
1878 (83% at 100lg/pellet) or TX-1866 (100% at
100lg/pellet) and TX-1879 (100% at 100lg/pellet).
From this it can be seen that the bulky p-tert-butylphe-
nyl group increases the anti-angiogenic activity of the
haloacetylcarbamoyl-2-nitroimidazoles in the chick em-
bryo CAM assay, but this positive, albeit modest, steric
effect is seen only with the R-enantiomer. In contrast
there was no difference in potencies of the enantiomers
of the compounds possessing the less bulky methyl
and tert-butyl ethers. As expected, the bromoacetyl
derivatives were more potent than the corresponding
chloroacetyl derivatives: the rank order of potency
was TX-1898 (R)>TX-1897 (R), TX-1900 (S)>TX-
1899 (S).
Recently MetAP-2 has been identified as one of the
molecular targets of the anti-angiogenic agent TNP-470
and its ÔparentÕ fumagillin. Sin et al.35 have shown that
fumagillin selectively inhibits the S. cerevisiae MetAP-2
protein in vivo, and Griffith et al.36,37 revealed that
TNP-470 covalently binds to MetAP-2 through reaction
of His 231 with the ring epoxide of TNP-470. From these
results they suggested that MetAP-2 may play a critical
role in the proliferation of endothelial cells and thus
could represent a promising target for the development
of new anti-angiogenic drugs. However, Griffith et al.
2.3.4. In vitro radiosensitizing activity.34 In vitro radio-
sensitizing activities of the compounds tested are shown
in Table 5. All compounds exhibited strong hypoxic cell
radiosensitizing activities at concentrations of 10lM.
Consistent with the results contained in our previous
report, there were no differences of radiosensitizing
activity between stereoisomers.21 The radiosensitizing
enhancement ratio (ER) values of the compounds in this
study also showed a positive correlation with their par-
tition coefficient logP.
did not discuss
a possible role of the chloro-
acetylcarbamoyl group of TNP-470 in its anti-angiogenic
activity.36,37 From the X-ray data of the fumagillin or
TNP-470 complex with MetAP-2, the four different sub-
stituents on cyclohexane ring are shown to be arranged
asymmetrically to fit neatly into the binding pocket of
MetAP-2.38,39 In this arrangement the cyclohexane ring
functions as a tetravalent chiral scaffold. Our molecular
modeling data for the compounds in our present study
show that if the bulky p-tert-butylphenyl were present,
a single stereocenter could function as chiral scaffold to
produce a similar structural array. Thus, the optimized
geometry of molecular structure of TX-1897 possessing
the R-stereocenter closely resembles the optimized geo-
metry of the molecular structure of TNP-470 (Fig. 4).
3.Discussion
As part of our research to develop effective cancer chem-
otherapeutic agents, we have developed compounds that

C.-Z. Jin et al. / Bioorg. Med. Chem. 12 (2004) 4917–4927
4923
TX-1897
TNP-470
cyclohexane ring
as a chiral scaffold
stereocenter
as a chiral scaffold
similar
functionality
O
O
OMe
O
O
O
O
O
H
N
H
N
O
N
H
O
N
H
Cl
Cl
NO2
Figure 4. The molecular similarity of TX-1897 and TNP-470. The similar stereocenter of TX-1897 acts as a chiral scaffold in a similar manner as the
four-chiral stereocenter-cyclohexane ring chiral scafforld of TNP-470. The symbol (ꢁ) indicates the carbon atoms constituting the scaffold.
In contrast, the compounds with the less bulky methyl
(TX-1863) or tert-butyl (TX-1880) groups showed no
similarity with the optimized molecular structure of
TNP-470 (Fig. 2). Differing only in the nature of the halo-
gen, TX-1898 also may be expected to have a molecu-
lar structure similar to TNP-470, in analogy with TX-
1897. Based on these considerations, we reasoned that
TX-1897 and TX-1898 might be potent inhibitors of
MetAP-2, the molecular target of angiogenesis inhibitor
TNP-470. The corresponding S-enantiomers, TX-1899
and TX-1900, cannot be superimposed on the optimized
molecular structure of TNP-470 and thus would be ex-
pected to have lower anti-angiogenic activity in the
CAM assay. We note that a chiral scaffold usually refers
to a platform with multiple functionality and two or
more chiral centers with fixed geometry, example of
which include such structures as indolizidine alkaloids40
and sugars.41 We consider that in chiral haloacetyl-
carbamoyl-2-nitroimidazoles, even the single stereo-
protein 27 (Hsp 27) and cofilin. We plan to evaluate
the effects of our compounds on these proteins.
The haloacetyl functionality appears to be critical to
activity, as we have previously suggested.21 In this re-
gard, Kim et al.43 recently reported that in calf pulmo-
nary artery endothelial cells, TNP-470 showed at least
10,000 times higher anti-proliferation activity than did
fumagillol (deschloroacetylcarbamoyl TNP-470). The
effect of chloroacetylcarbamoyl group was more pro-
nounced in fumagillol derivatives with the methoxy
group at its 5-position. From these results, we consider
that in TNP-470, the four functional groups at 3, 4, 5,
and 6-position of stereo-scaffold cyclohexane ring ori-
ented the molecular geometry to maximize interactions
with angiogenesis-related enzymes such as MetAP-2.
KimÕs results support our previous conclusion that the
haloacetylcarbamoyl group makes an important contri-
bution as an anti-angiogenic pharmacophore.21 In addi-
tion, Satchi-Fainaro et al.44 recently reported the
synthesis of the N-(2-hydroxypropyl)methacrylamide
(HPMA) copolymer-TNP-470 as an anti-angiogenic
agent shown in Figure 5. The synthesis involved conju-
gation of TNP-470 to HPMA copolymer-Gly-Phe-Leu-
Gly-ethylenediamine by displacement of the terminal
chlorine of TNP-470. This indicates that in TNP-470,
the chloroacetylcarbamoyl group is more reactive than
the epoxide on the cyclohexane ring, for general nucleo-
philic substitution reaction. However, the copolymer
showed potent activity, but now not possessing a chloro-
acetyl group. In addition, that interaction of an electro-
phile with an enzyme nucleophile is more influenced by
proper orientation at the active site that the relative
reactivities. We feel these results support our previ-
ous suggestion, that the haloacetylcarbamoyl group
center functioned as a chiral scaffold for their
biological activities including anti-angiogenic activity.
The results of biological evaluation showed that the ef-
fect of stereochemistry varied with the system being
studied. In the PP elastase inhibitory assay the R-iso-
mers were more active than the corresponding S-iso-
mers. In particular, TX-1898 and TX-1897 possessing
the bulky p-tert-butylphenyl group showed impressive
activity. In the CAM assay the R-isomers were slightly
more active than the S-isomers. All p-tert-butylphenyl-
haloacetylcarbamoyl-2-nitroimidazole compounds, TX-
1898, TX-1900, TX-1897 and TX-1899, were more
potent in the chick embryo CAM assay than the
haloacetylcarbamoyl-containing fumagillin synthetic
analog, TNP-470 (Table 4). This high activity, in com-
bination with their potent radiosensitizing activity,
makes them particularly promising as candidates for
development as bifunctional chemotherapeutic agents.
O
Me
OMe
In interactions of our compounds with enzymes or
receptors involved with angiogenesis, the presence of
the bulky p-tert-butylphenyl group apparently is
required before significant stereo-differentiation is
observed. The preference for the R-isomer is seen most
clearly in inhibition of PP elastase. Other targets can
be considered. For example, Keezer et al.42 have recently
reported that TNP-470 targeted the endothelial cell
cytoskeleton through altered regulation of heat shock
H
O
O
O
H
O
N
H
NH
HN
HPMA copolymer
Gly-Phe-Leu-Gly
CO
Figure 5. The structure of HPMA copolymer-TNP-470 (modified Fig.
1 in Ref. 44).

4924
C.-Z. Jin et al. / Bioorg. Med. Chem. 12 (2004) 4917–4927
represents an important pharmacophore and can
play an important part in angiogenesis-related
biotransformation.
ature for 17h, and the organic layer was washed with
water and separated. The aqueous layer was extracted
with ethyl ether, the organic fraction was dried with
anhydrous sodium sulfate and evaporated in vacuo to
give a colorless liquid.
In summary, we have carried out syntheses of the indi-
vidual enantiomers of haloacetylcarbamoyl-2-nitroimid-
azole as anti-angiogenic hypoxic cell radiosensitizers.
The synthetic design allows introduction of diversity in
the side-chain ether function, as illustrated by the intro-
duction of methyl, tert-butyl, p-tert-butylphenyl in this
series. The data from biological evaluation suggest that
4.3.1. (R)-tert-Butyl glycidyl ether ((R)-1). (14.7g, 90%),
28
D
1
½aꢂ +3.81 (c 1.4, CHCl3); H NMR (CDCl3) d 1.21 (s,
9H), 2.63 and 2.81 (m, each 1H), 3.11 (m, 1H), 3.43
and 3.53 (m, each 1H). IR (cmꢀ1): 2976, 1472, 1365,
1252, 1236, 1197, 1090, 1023, 908, 875, 843. Anal. Calcd
for C7H14O2: C, 64.58; H, 10.84. Found: C, 64.48; H,
10.96.
the
(R)-p-tert-butylphenyl-bromoacetylcarbamoyl-2-
nitroimidazole hypoxic cell radiosensitizer, TX-1898,
may be a very promising candidate for further develop-
ment as an anti-angiogenic hypoxic cell radiosensitizer.
4.3.2. (S)-tert-Butyl glycidyl ether ((S)-1). (13.7g, 84%),
28
D
1
½aꢂ ꢀ3.84 (c 1.2, CHCl3); H NMR (CDCl3) d 1.21
(s, 9H), 2.63 and 2.81 (m, each 1H), 3.11 (m, 1H), 3.42
and 3.53 (m, each 1H). IR (cmꢀ1): 2974, 1473, 1365,
1253, 1194, 1087, 1022, 973, 874, 747. Anal. Calcd for
C7H14O2: C, 64.58; H, 10.84. Found: C, 64.42; H, 10.66.
4.Experimental
4.1. General procedures
1H NMR spectra were recorded on a JEOL JNM-
EX400 spectrometer (400MHz) with tetramethylsilane
as the internal standard. Chemical shifts were reported
in ppm. Coupling constants were reported in Hz. IR
spectra were reported in KBr pellet on a Perkin–Elmer
1600 spectrometer. High-resolution mass spectra
(HRMS) were measured on a JEOL JMS-SX102A mass
spectrometer using a fast atom bombardment (FAB)
and EI. Reaction was monitored by analytical thin-layer
chromatograpy (TLC) with use of Merck silica gel
60F254 glass plates and Merck aluminum oxide 60F254
neutral (Type E). Column chromatography was per-
formed on Merck silica gel 60 (230–400 mesh). Optical
rotations were determined on JASCO DIP-370 digital
polarimeter. All melting points were determined with a
micromelting point measurement apparatus (MP-j 3
model) and were uncorrected. Elemental analysis was
performed with a Yanako CHN Corder MT-5. All the
chemicals were purchased from Wako Pure Chemical
Industries, Ltd (Osaka, Japan). (S)-(+)-epichlorohydrin
(99.9% pure, 98.9% ee) and (R)-(ꢀ)-epichlorohydrin
(99.6% pure, 98.6% ee) was provided from DAISO
Co., Ltd (Osaka, Japan).
4.4. General procedure for the synthesis of (R)-or (S)-p-
tert-butylphenyl glycidyl ether [(R/S)-2]
T o (R)-or (S)-epichlorohydrin (9.25g, 100mmol) heated
to 64°C was added to a solution of p-tert-butylphenol
(7.51g, 50mmol) and sodium hydroxide (2.1g,
52.5mmol, in 16mL H2O) over 8h, with vigorous stir-
ring. The crude product that separated from the aque-
ous layer and washed with H2O, and the water layer
was extracted with ethyl ether. The organic layer was
washed with NaCl (satd) aq, dried with anhydrous so-
dium sulfate, and evaporated in vacuo. The residue
was purified by silica gel column chromatography with
hexane and acetyl acetate, to give the product as a col-
orless liquid.
4.4.1. (S)-p-tert-Butylphenyl glycidyl ether ((S)-2).
28
D
(5.54g, 54%), ½aꢂ +1.53 (c 1.2, CHCl3); 1H NMR
(CDCl3) d 1.30 (s, 9H), 2.73 and 2.88 (each m, each
1H), 3.33 (m, 1H), 3.95 and 4.17 (m, each 1H), 6.84
and 7.29 (dd, J=5.5, 5.9Hz, each 2H). IR (cmꢀ1):
2962, 1609, 1514, 1457, 1364, 1247, 1186, 1037, 917,
829. Anal. Calcd for C13H18O2: C, 75.69; H, 8.80.
Found: C, 75.41; H, 8.67.
4.2. Molecular modeling methods
4.4.2. (R)-p-tert-Butylphenyl glycidyl ether ((R)-2).
28
D
The molecular geometries and molecular calculations
were performed with the B3LYP hybrid density func-
tional in conjunction with the 6-31G** basis set using
the GAUSSIAN 98 suite of programs.26
(1.57g, 51%), ½aꢂ ꢀ0.95 (c 1.2, CHCl3); 1H NMR
(CDCl3) d 1.30 (s, 9H), 2.74 and 2.89 (each m, each
1H), 3.35 (m, 1H), 3.93 and 4.15 (m, each 1H), 6.84
and 7.29 (dd, J=5.5, 5.9Hz, each 2H). IR (cmꢀ1):
2953, 1610, 1512, 1461, 1364, 1292, 1256, 1184, 1117,
1041, 825. Anal. Calcd for C13H18O2: C, 75.69; H,
8.80. Found: C, 75.85; H, 8.99.
4.3. General procedure for synthesis of (R)-or (S)-tert-
butyl glycidyl ether [(R/S)-1]
To a solution of tert-butanol (37.1g, 500mmol) and
boron trifluoride ether complex (46–49%) (88.7mg,
0.625mmol) in anhydrous CH2Cl2 (100mL), (S)-or
(R)-epichlorohydrin (11.6g, 125mmol) was added. After
being stirred until no starting material remained, the
reaction mixture was evaporated in vacuo. Sodium
hydroxide solution (7mL, 20%) was added to the resi-
due. The resulting mixture was stirred at room temper-
4.5. General procedure for synthesis of (R)- or (S)-
alkoxy-1-(2-nitroimidazolyl)-2-propanols
To a suspension of 2-nitroimidazole (1.69g, 15mmol)
and sodium carbonate (853mg, 8mmol) in anhydrous
EtOH (50mL) was added R- or S-alkyl glycidyl ether
(31mmol). After being refluxed for 6h, the reaction mix-
ture was filtered and washed with CH2Cl2. The filtrate

C.-Z. Jin et al. / Bioorg. Med. Chem. 12 (2004) 4917–4927
4925
was evaporated in vacuo to give a semi-solid residue,
4.6. General procedure for synthesis of (2R)- or (2S)-
haloacetylcarbamoyl-2-nitroimidazoles
which was purified by column chromatography on silica
gel with CH2Cl2 and MeOH to afford (R)- or (S)-alk-
oxy-1-(2-nitroimidazolyl)-2-propanols (TX-1888, TX-
1860, TX-1894, TX-1893, TX-1896, and TX-1895).
To a solution of (R)- or (S)-3-substituted-1-(2-nitroimid-
azolyl)-2-propanols (TX-1894, TX-1893, TX-1895 or
TX-1896, 2.1mmol) in anhydrous CH2Cl2 (10mL),
was added haloacetyl isocyanate (either chloroacetyl iso-
cyanate or bromoacetyl isocyanate) (4.2mmol), pre-
pared immediately before use. After being refluxed for
30min, the reaction mixture was filtered and the filtrate
was washed with CH2Cl2. The residue remaining after
evaporation in vacuo was purified by silica gel column
chromatography with CH2Cl2 and AcOEt, to give
(2R)- or (2S)-haloacetylcarbamoyl-2-nitroimidazoles
(TX-1863, TX-1878, TX-1866, TX-1879, TX-1880,
TX-1881, TX-1882, TX-1883, TX-1897, TX-1898, TX-
1899, TX-1900).
4.5.1. TX-1888((S)-3). Yield, 82% as yellow semi-solid,
28
D
1
½aꢂ +5.79 (c 0.3, CH3OH); H NMR (CDCl3) d 3.30
(s, 3H), 3.44 and 3.51 (m, 2H), 4.16 (m, 1H), 4.37 and
4.71 (m, 2H), 7.05 and 7.22 (s, 2H). IR (cmꢀ1): 3425,
2932, 1541, 1490, 1362, 1280, 1124, 838. Anal. Calcd
for C7H11N3O4: C, 41.79; H, 5.51; N, 20.89. Found:
C, 42.05; H, 5.47; N, 21.08.
4.5.2. TX-1860 ((R)-3). Yield, 81% as yellow semi-solid,
28
D
½aꢂ ꢀ8.86 (c 0.3, CH3OH), 1H NMR (CDCl3) d 3.30 (s,
3H), 3.40 and 3.53 (m, 2H), 4.16 (m, 1H), 4.42 and 4.67
(m, 2H), 7.15 and 7.26 (s, 2H). IR (cmꢀ1): 3425, 2935,
1541, 1491, 1362, 1283, 1122, 838. Anal. Calcd for
C7H11N3O4: C, 41.79; H, 5.51; N, 20.89. Found: C,
42.00; H, 5.36; N, 21.84.
4.6.1. TX-1863. Yield, 96% as a yellow solid, mp 100–
28
D
1
101°C; ½aꢂ +27.9 (c 0.3, CH3OH); H NMR (CDCl3)
d 3.40 (s, 3 H), 3.62(m, 2H), 4.36(d, J=15.4Hz, 2H),
4.59 and 4.92 (dd, J=14.4Hz, 2H), 5.32 (m, 1H), 7.14
and 7.28 (s, 2H). IR (cmꢀ1): 3424, 1791, 1718, 1541,
1490, 1363, 1267, 1102, 839, 771, 632. HRMS (FAB)
m/z (MH+): calcd for C10H13ClN4O6, 321.0573; found,
321.0601.
4.5.3. TX-1894 ((S)-4). Yield, 85% as a yellow solid, mp
28
D
95–96°C; ½aꢂ +3.55 (c 0.5, CHCl3);1H NMR (CDCl3) d
1.19 (s, 9H), 3.35 and 3.53 (dd, J=5.2, 4.0Hz, each 1H),
4.07 (m, 1H), 4.45 and 4.62 (dd, J=7.4, 3.6Hz, each
1H), 7.14 and 7.21 (d, J=1.6, 0.8Hz, each 1H). IR
(cmꢀ1): 3384, 2974, 2863, 1534, 1490, 1362, 1287,
1196, 1157, 1063, 964, 836, 792. Anal. Calcd for
C10H17N3O4: C, 49.37; H, 7.04; N, 17.27. Found: C,
49.58; H, 6.84; N, 17.06.
4.6.2. TX-1878. Yield, 90% as a yellow solid, mp 101–
28
D
1
102°C; ½aꢂ ꢀ30.3 (c 0.3, CH3OH); H NMR (CDCl3)
3.42 (s, 3 H), 3.62(m, 2H), 4.36(d, J=15.4Hz, 2H),
4.58 and 4.92 (dd, J=14.4Hz, 2H), 5.32 (m, 1H), 7.14
and 7.28 (s, 2H). IR (cmꢀ1): 3425, 1789, 1717, 1541,
1490, 1364, 1210, 1103, 839, 768, 653. HRMS (FAB)
m/z (MH+): calcd for C10H13ClN4O6, 321.0573; found,
321.0596.
4.5.4. TX-1893 ((R)-4). Yield, 72% as a yellow solid, mp
28
D
1
95–96°C; ½aꢂ ꢀ2.32 (c 0.5, CHCl3); H NMR (CDCl3)
d 1.19 (s, 9H), 3.35 and 3.53 (dd, J=5.0, 4.0Hz, each
1H), 4.07 (m, 1H), 4.46 and 4.62 (dd, J=3.2, 3.6Hz,
each 1H), 7.14 and 7.21 (d, J=1.2Hz, each 1H). IR
(cmꢀ1): 3405, 2974, 2872, 1579, 1492, 1364, 1272,
1195, 1159, 1097, 1061, 1015, 836, 790, 743, 692. Anal.
Calcd for C10H17N3O4: C, 49.37; H, 7.04; N, 17.27.
Found: C, 49.37; H, 6.78; N, 17.05.
4.6.3. TX-1866. Yield, 98% as a yellow solid, mp 108–
28
D
1
109°C; ½aꢂ +39.9 (c 0.3, CH3OH); H NMR (CDCl3)
d 3.42 (s, 3 H), 3.62(m, 2H), 4.13(d, J=12.8Hz, 2H),
4.59 and 4.92 (dd, J=14.4, 14.8Hz, 2H), 5.33 (m, 1H),
7.14 and 7.28 (s, 2H). IR (cmꢀ1): 3388, 1786, 1717,
1541, 1490, 1364, 1221, 1100, 839, 768, 599. HRMS
(FAB) m/z (MH+): calcd for C10H13BrN4O6, 365.0079;
found, 365.0092.
4.5.5. TX-1896 ((S)-5). Yield, 73% as a yellow solid,
28
D
1
mp 183–184°C; ½aꢂ ꢀ9.28 (c 0.2, CHCl3); H NMR
(CDCl3) d 1.29 (s, 9H), 3.97 and 4.09 (dd, J=5.2,
5.6Hz, each 1H), 4.39 (m, 1H,), 4.55 and 4.81 (dd,
J=7.6, 3.6Hz, each 1H), 6.84 and 7.32 (dd, J=2.4Hz,
each 2H), 7.15 and 7.22 (dd, J=0.8Hz, each 1H). IR
(cmꢀ1): 3374, 3128, 2964, 2871, 1579, 1512, 1492,
1364, 1189, 1107, 1025, 825. Anal. Calcd for
C10H17N3O4: C, 60.17; H, 6.63; N, 13.16. Found: C,
59.91; H, 6.45; N, 12.97.
4.6.4. TX-1879. Yield, 96% as a yellow solid, mp 108–
28
D
1
110°C; ½aꢂ ꢀ38.7 (c 0.3, CH3OH); H NMR (CDCl3)
d 3.42 (s, 3 H), 3.62 (m, 2H), 4.15 (d, J=12.8Hz, 2H),
4.59 and 4.92 (dd, J=14.4, 14.8Hz, 2H), 5.32 (m, 1H),
7.14 and 7.28 (s, 2H). IR (cmꢀ1): 3427, 1785, 1720,
1540, 1490, 1364, 1222, 1102, 839, 768, 599. HRMS
(FAB) m/z (MH+): calcd for C10H13BrN4O6, 365.0079;
found, 365.0088.
4.5.6. TX-1895 ((R)-5). Yield, 71% as a yellow solid,
28
D
1
mp 183–184°C; ½aꢂ +8.30 (c 0.2, CHCl3); H NMR
4.6.5. TX-1880. Yield, 73% as a yellow solid, mp 135–
28
D
1
(CDCl3) d 1.30 (s, 9H), 3.97 and 4.11 (dd, J=5.4,
4.2Hz, each 1H), 4.38 (m, 1H), 4.56 and 4.81 (dd,
J=6.9, 3.4Hz, each 1H), 6.84 and 7.32 (dd, J=1.4,
1.8Hz, each 2H), 7.16 and 7.22 (dd, J=1.2Hz, each
1H). IR (cmꢀ1): 3384, 3128, 2964, 2871, 1579, 1512,
1492, 1364, 1246, 1189, 1117, 1082, 1056, 1025, 825.
Anal. Calcd for C10H17N3O4: C, 60.17; H, 6.63; N,
13.16. Found: C, 59.90; H, 6.50; N, 12.87.
136°C; ½aꢂ +49.1 (c 0.3, CH3OH); H NMR (CDCl3)
d 1.20 (s, 9 H), 3.50 and 3.60 (dd, J=10.4Hz, each
1H), 4.38 (d, J=16.4Hz, 2H) 4.59 and 4.92 (dd,
J=14.4, 14.6Hz, 2H), 5.25 (m, 1H), 7.15 and 7.16 (s,
2H). IR (cmꢀ1): 3140, 1789, 1717, 1534, 1490, 1362,
1290, 1091, 841, 769, 636. Anal. Calcd for
C13H19ClN4O6: C, 43.04; H, 5.28; N, 15.44. Found: C,
42.81; H, 5.06; N, 15.14.

4926
C.-Z. Jin et al. / Bioorg. Med. Chem. 12 (2004) 4917–4927
4.6.6. TX-1881. Yield, 72% as a yellow solid, mp 135–
d 1.30 (s, 9H), 4.17 and 4.19 (d, J=5.2Hz, 1H), 4.18
and 4.23 (s, 2H), 4.73 and 5.04 (d, J=14.8, 14.4Hz,
2H), 5.53 (m, 1H), 6.84 and 7.33 (dd, J=6.8, 6.0Hz,
2H), 7.15 and 7.17 (s, 1H). IR (cmꢀ1): 3139, 2957,
1784, 1720, 1538, 1490, 1362, 1287, 1244, 1084, 838,
774, 651, 555. Anal. Calcd for C19H23BrN4O6: C,
47.22; H, 4.80; N, 11.59. Found: C, 47.14; H, 4.68; N,
11.35.
28
D
1
136°C; ½aꢂ ꢀ48.7 (c 0.4, CH3OH); H NMR (CDCl3)
d 1.20(s, 9 H), 3.50 and 3.60 (dd, J=10.4Hz, each
1H), 4.38 (d, J=16.0Hz, 2H) 4.59 and 4.92 (dd,
J=14.4, 14.6Hz, 2H), 5.24 (m, 1H), 7.15 and 7.16 (s,
2H). IR (cmꢀ1): 3144, 1790, 1720, 1543, 1490, 1361,
1285, 1091, 1020, 836, 738, 632. Anal. Calcd for
C13H19ClN4O6: C, 43.04; H, 5.28; N, 15.44. Found: C,
43.16; H, 5.20; N, 15.22.
Acknowledgements
4.6.7. TX-1882. Yield, 77% as a yellow solid, mp 141–
28
D
1
142°C; ½aꢂ +46.8 (c 0.2, CH3OH); H NMR (CDCl3)
This study was supported in part by the Japan–China
Sasakawa Medical Fellowship. The author C.-Z.J.
thanks ÔThe Japan–China Sasakawa Medical Fellow-
shipÕ, and also thanks Professor Shibata Shoji, Emeritus
Professor of Tokyo University, for his helpful advice.
The authors thank Dr. Goto Satoru of the Faculty of
Pharmaceutical Sciences, The University of Tokushima,
for his advice of Molecular modeling, and also Dr. Y.
Takekawa, H. Nishitani, and Y. Nishimoto of the
School of Medical, The University of Tokushima for
their generous help with the radiobiological experi-
ments. Thanks are also due to N. Mikamo, J. Azuma,
A. Yokobori, K. Kiyoi of the Department of Biological
Science and Technology for excellent technical assist-
ance. We also thank M. Nakamura, E. Okayama, K.
Yamashita of our faculty for performing the spectral
measurements. We also thank DAISO Co., Ltd (Osaka,
Japan) for providing (R)- and (S)-epichlorohydrin.
d 1.21 (s, 9 H), 3.52 and 3.60 (dd, J=10.6Hz, each
1H), 4.21 (d, J=13.2Hz, 2H) 4.59 and 4.87 (dd,
J=14.6Hz, 2H), 5.26 (m, 1H), 7.16 and 7.26 (s, 2H).
IR (cmꢀ1): 3124, 1787, 1718, 1542, 1488, 1360, 1291,
1093, 842, 799, 698, 596. Anal. Calcd for
C13H19BrN4O6: C, 38.34; H, 4.70; N, 13.76. Found: C,
38.12; H, 4.58; N, 13.60.
4.6.8. TX-1883. Yield, 74% as a yellow solid, mp 141–
28
D
1
142°C; ½aꢂ ꢀ49.4 (c 0.4, CH3OH); H NMR (CDCl3)
d 1.20 (s, 9 H), 3.51 and 3.60 (dd, J=10.4, 10.6Hz, each
1H), 4.21 (d, J=13.2Hz, 2H) 4.59 and 4.87 (dd,
J=14.6Hz, 2H), 5.26 (m, 1H), 7.16 and 7.26 (s, 2H).
IR (cmꢀ1): 3141, 1786, 1718, 1527, 1488, 1359, 1290,
1091, 840, 596. Anal. Calcd for C13H19BrN4O6: C,
38.34; H, 4.70; N, 13.76. Found: C, 38.35; H, 4.44; N,
13.73.
4.6.9. TX-1897. Yield, 95% as a yellow solid, mp 65–
28
D
1
66°C; ½aꢂ +51.6 (c 0.4, CH3OH); H NMR (CDCl3)
References and notes
d 1.30 (s, 9H), 4.17 and 4.18 (dd, J=4.4, 4.0Hz, each
1H), 4.33 and 4.38 (d, J=15.6, 15.2Hz, 2H), 4.74 and
5.04 (dd, J=14.4Hz, 2H), 5.52 (m, 1H), 6.84 and 7.33
(dd, J=6.4, 6.8Hz, 2H), 7.15 and 7.16 (d, J=0.8Hz,
2H). IR (cmꢀ1): 3278, 2957, 1784, 1725, 1538, 1490,
1362, 1287, 1244, 1084, 838, 769, 651. Anal. Calcd for
C19H23ClN4O6: C, 52.00; H, 5.28; N, 12.77. Found: C,
51.85; H, 5.37; N, 12.54.
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4.6.10. TX-1899. Yield, 91% as a yellow solid, mp 66–
28
D
1
67°C; ½aꢂ ꢀ53.2 (c 0.2, CH3OH); H NMR (CDCl3) d
1.30 (s, 9H), 4.17 and 4.18 (dd, J=4.4, 4.0Hz, each
1H), 4.35 and 4.36 (s, 2H), 4.74 and 5.04 (dd, J=14.8,
14.2Hz, 2H), 5.52 (m, 1H), 6.84 and 7.34 (dd, J=2.2,
2.4Hz, 2H), 7.15 and 7.16 (s, 2H). IR (cmꢀ1): 3278,
2957, 1789, 1725, 1544, 1490, 1362, 1287, 1244, 1084,
838, 769, 651. Anal. Calcd for C19H23ClN4O6: C, 52.00;
H, 5.28; N, 12.77. Found: C, 51.81; H, 5.26; N, 12.49.
4.6.11. TX-1898. Yield, 98% as a yellow solid, mp 73–
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28
D
1
74°C; ½aꢂ +53.7 (c 0.3, CH3OH); H NMR (CDCl3)
d 1.30 (s, 9H), 4.15 and 4.18 (s, 1H), 4.20 and 4.23 (s,
2H), 4.74 and 5.04 (dd, J=14.8, 14.2Hz, 2H), 5.52 (m,
1H), 6.84 and 7.33 (dd, J=6.8, 5.8Hz, 2H), 7.15 and
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1490, 1362, 1287, 1244, 1084, 838, 774, 651, 555. Anal.
Calcd for C19H23BrN4O6: C, 47.22; H, 4.80; N, 11.59.
Found: C, 47.04; H, 4.73; N, 11.39.
4.6.12. TX-1900. Yield, 93% as a yellow solid, mp 74–
28
D
1
75°C; ½aꢂ ꢀ56.8 (c 0.3, CH3OH); H NMR (CDCl3)

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