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S. Chen et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
mimic a benzene ring of TPP by integrated approach.9,10 And the
long hexanoyl linker was used to reduce the interaction between
the cyclopentadienyltricarbonyl 99mTc/Re chelating group and the
phosphonium cation moiety.
uptakes of [99mTc]3 at every time point were all higher than those
of 99mTc-sestamibi in both cell lines. But the uptake differences of
[
99mTc]3 between the two cell lines were less than those of 99mTc-
sestamibi. The cellular uptake of 99mTc-sestamibi in MCF-7 was
6.7-fold higher than that in MCF-7/ADR, while it was 5.6-fold of
As shown in Scheme 1, intermediates 1 and 4 were synthesized
through a Friedel-Crafts reaction according to the previous proto-
col10 from the ferrocene and (cyclopentadienyl)tricarbonyl rhe-
nium reactants, respectively. Then they could react with
triphenylphosphine to obtain the precursor ferrocenyl compound
2 and rhenium complex Re5 at a modest yield respectively. The
aimed compounds were characterized by 1H NMR, 13C NMR, and
HRMS (as shown in the Supporting Information). According to lit-
erature method,11,12 the precursor ferrocenyl compound 2 directly
reacted with the fac-[99mTc(H2O)3(CO)3]+ at 145 °C for 45 min to
yield [99mTc]3 (radiochemical yield: 40–50%, n = 9). Taking the cor-
responding rhenium complex Re5 as identification, [99mTc]3 was
purified by semi-preparative HPLC (Supporting Information), and
the radiochemical purity of [99mTc]3 was >98%. The radiotracer
was stable in vitro, and the radiochemical purity was >95% both
in saline at room temperature and in mouse serum at 37 °C for
4 h. The partition coefficient (log P) in octanol and phosphate-buf-
fered saline (0.05 molÁLÀ1, pH = 7.4) of [99mTc]3 was 1.20 0.02
(n = 3), suggesting that it was a lipophilic complex.
The rhodamine 123 (R123) is a fluorescent probe that can accu-
mulate in cells and selectively transported by P-gp.13,14 In order to
reveal the different P-gp expression level between the MCF-7
(human breast cancer cells) and MCF-7/ADR (an MDR subline) cell
lines, the rhodamine 123 accumulation assay was conducted in
this study (Supporting Information). As shown in Fig. 1, obviously
brighter fluorescence staining was visualized in MCF-7 cells than in
MCF-7/ADR cells, which indicated the high level of P-gp expression
in MCF-7/ADR cells compared to MCF-7 cells.
[
[
99mTc]3 at 3 h incubation. As shown in Fig. 2B, the efflux rate of
99mTc]3 in MCF-7/ADR cell line was significantly higher than that
in the MCF-7 cell line. These results suggested that the cellular
uptake of the radiotracers was associated with the expression level
of MDR proteins. The over expression of MDR proteins contributed
to the efflux of radiotracer, and reduced the radioactivity accumu-
lation in the MCF-7/ADR cell line.
To further investigate whether [99mTc]3 was a substrate of P-
gp/MRP1, inhibiting study was performed by monitoring the radio-
tracer accumulation in MCF-7/ADR cell line (P-gp and MRP1 over-
expression).19 Verapamil is the modulator of P-gp. Cyclosporine A
is the modulator of P-gp and MRP1. And MK571 is the modulator
of MRP1.19 As shown in Fig. 3, the pre-treatment of modulators
verapamil (Vrp, 20
(20
lM), cyclosporine A (CsA, 20 lM) and MK571
l
M) could block the P-gp/MRP1 mediated transportation of
the [99mTc]3 or 99mTc-sestamibi, and significantly increased the
radio-accumulation in cells. These results demonstrated that the
radiotracer [99mTc]3, similar to 99mTc-sestamibi, could be trans-
ported both by P-gp and MRP1.
To determine whether the radiotracer [99mTc]3 localized in the
tumor cells through mitochondrial membrane potential, the cellu-
lar assay was conducted on MCF-7 cell line using carbonyl cyanide
m-chlorophenylhydrazone (CCCP) to depolarize the mitochondrial
membrane potential.15 As shown in Fig. 4, [99mTc]3 and 99mTc-ses-
tamibi displayed 22% and 23% uptake decrease in the MCF-7 cells
in the presence of CCCP (10 lM, 30 min), respectively. It suggested
that [99mTc]3 permeated into tumor cells through the mitochon-
drial membrane potential as 99mTc-sestamibi.16,17
The cellular uptake and efflux experiments of [99mTc]3 were
performed on the MCF-7 and MCF-7/ADR cell lines, using 99mTc-
sestamibi as a reference (Supporting Information). Similar to
99mTc-sestamibi, the cellular uptake of [99mTc]3 at 37 °C was
time-dependent at both cell lines incubating for 0.5, 1, 1.5, 2, 3
and 4 h (Fig. 2A), and the uptake values of radiotracers were
always higher in MCF-7 cell line than the data obtained in MCF-
7/ADR cell line. The cellular uptake of [99mTc]3 in MCF-7 and
MCF-7/ADR cell lines reached a peak at 3 h (280.98 %uptake/mg
protein) and 1.5 h (78.72 %uptake/mg protein) respectively, while
the uptake of 99mTc-sestamibi rose with time. Furthermore, the
The in vivo biodistribution study of [99mTc]3 was performed in
nude mice bearing MCF-7 or MCF-7/ADR tumor xenografts (Sup-
porting Information). As demonstrated in Table 1, the uptake in
MCF-7 tumor was 1.44 0.09 %ID/g at 1 h p.i., approximately 3-
fold higher than that in MCF-7/ADR tumor (0.42 0.06 %ID/g).
The significant accumulation difference agreed well with the cellu-
lar uptake data of [99mTc]3 in the two cell lines. High level of MDR
proteins expression resulted in a rapid excretion of radiotracer
from tumor cells and lower uptake in MCF-7/ADR tumor. However,
[
99mTc]3 also displayed a high radioactivity accumulation and
Scheme 1. (a) anhydrous CH2Cl2, anhydrous AlCl3, 0 °C to room temperature; (b) triphenylphosphine, acetonitrile, 100 °C, reflux; (c) [99mTc(H2O)3(CO)3]+, DMF/H2O = 1/1,
pH = 1, 145 °C, 45 min.
Chen, Shuting
