S. Iwaki et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2798–2802
2801
reduction, based on a previous report.7 Further, we also examined
the r1 relaxivity change in enzymatic reaction mixtures with differ-
ent microsome concentrations, and a 1.6-fold increment of the r1
relaxivity was observed even when we used more diluted micro-
somes (0.37 mg protein/mL) (Fig. S4). These results indicate that
4NO22MeOSAGd can be biologically reduced under hypoxic condi-
tions, and this reaction provides the basis for MR detection of a
hypoxic state by means of specific contrast enhancement.
4
3.5
3
a
Hypoxia
Normoxia
In conclusion, we have developed a series of Gd3+ complexes of
DO3A-derived ligands bearing a nitrobenzenesulfonamide moiety
(SAGds). These compounds showed a high correlation between
2.5
2
r
the pKa value of the r1 relaxivity and the sum of Hammett
r con-
stants of substituents on the aromatic ring ( ). Among the syn-
R
r
thesized compounds, 4NO22MeOSAGd showed the best
properties as a ‘smart’ MRI contrast agent to detect hypoxic condi-
tions, that is, 4NO22MeOSAGd was reduced selectively under hyp-
1.5
0
60
120
180
240
300
Time (min)
oxic conditions and this was accompanied with
a 1.8-fold
increment of the r1 relaxivity. This enhancement of the r1 relaxivity
is sufficiently large to be detected in T1-weighted MR images. To
our knowledge, 4NO22MeOSAGd is the first 1H MRI contrast agent
that can selectively detect hypoxia. Further studies are under way.
4
3.5
3
b
Hypoxia
Normoxia
Acknowledgments
This work was supported in part by a Grant-in-Aid for Scientific
Research (Specially Promoted Research No. 22000006 to T.N. and
21750135 to T.T.) from the Ministry of Education, Culture, Sports,
Science and Technology of Japan. K.H. was supported by SENTAN
JST, Inoue Foundation for Science, Takeda Science Foundation, the
Research Foundation for Pharmaceutical Science, the Tokyo Bio-
chemical Research Foundation, Konica Minolta Science and Tech-
nology Foundation, The Asahi Glass Foundation and Astellas
Foundation for Research on Metabolic Disorders.
2.5
2
r
1.5
0
60
120
180
240
300
Time (min)
Figure 4. Time-dependent change of r1 relaxivity of 250
(b) 3NO2triMeSAGd in the presence of rat liver microsomes (25
500 L buffer, 1.2 mg protein/mL) under hypoxic (red) or normoxic (blue) condi-
tions. Measurements were performed in 0.1 M Tris–HCl buffer (pH 7.4) containing
0.25% DMSO as a cosolvent and 5 mM NADPH. Error bars show standard deviation.
n = 3.
l
M (a) 4NO22MeOSAGd or
Supplementary data
lL microsomes/
l
Supplementary data (synthetic procedures and characterization
of SAGds, experimental procedures, Figs. S1–S4) associated with
this article can be found, in the online version, at doi:10.1016/
(Fig. 4a). The r1 relaxivities of 4NO22MeOSAGd and 4NH22MeO-
SAGd were 2.1 and 3.6 mMÀ1 sÀ1 at pH 7.4, respectively, and thus
a large enhancement (1.8-fold) of r1 relaxivity after enzymatic
reduction under hypoxic conditions was observed. The generation
of the reduced product, 4NH22OMeSAGd, was confirmed by HPLC
analyses (Fig. S3).
In T1-weighted MR images (1.5 T) of the enzymatic reaction
mixture, significant enhancement of the MR signal was observed
under hypoxic conditions compared with that under normoxic
conditions (Fig. 5). It is difficult to establish precisely the activity
of reductive enzymes in vivo, but the conditions of the above enzy-
matic reaction can be considered similar to those of actual in vivo
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