Towards to hENT1-nucleoside transporter selective imaging agents. Synthesis and in vitro evaluation of the radiolabeled SAENTA analogues

Article abstract of DOI:10.1016/j.bmcl.2009.07.017

Three new potential hENT₁ inhibitors suitable for labeling with PET/SPECT radioisotopes were prepared from an advanced intermediate 4. They were tested for their capability to inhibit binding of SAENTA-fluorescein to HL60 leukemia cells in flow

Full text of DOI:10.1016/j.bmcl.2009.07.017

Bioorganic & Medicinal Chemistry Letters 19 (2009) 5151–5154
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Bioorganic & Medicinal Chemistry Letters
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Towards to hENT1-nucleoside transporter selective imaging agents.
Synthesis and in vitro evaluation of the radiolabeled SAENTA analogues
Boris D. Zlatopolskiy, Agnieszka Morgenroth, Elizaveta A. Urusova, Cornelia Dinger,
*
Thomas Kull, Manuela Pape, Gerhard Glatting, Sven N. Reske
Klinik für Nuklearmedizin, Ulm University, Albert-Einstein-Allee 23, D-89081 Ulm, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Three new potential hENT1 inhibitors suitable for labeling with PET/SPECT radioisotopes were prepared
from an advanced intermediate 4. They were tested for their capability to inhibit binding of SAENTA-fluo-
rescein to HL60 leukemia cells in flow cytometry assay and SAENTA-I (5) was determined to be the most
active compound. 131I-5 showed high hENT1-specific binding (up to 54% ID) to 6 from 7 tested tumor cell
lines and was chosen for further in vivo study.
Received 27 April 2009
Revised 2 July 2009
Accepted 2 July 2009
Available online 9 July 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Nucleoside transporters
Molecular imaging
hENT1 inhibitor
Radiotracer
Nucleoside analogues are widely used for treatment of different
types of tumors. Nucleosides are hydrophilic and can enter cells
only by active transport via nucleoside transporters.1 hENT1-nucle-
oside transporters2 belong to the subfamily of concentrative nucle-
oside transporters, which are characterized by high sensitivity to
inhibition with p-nitrobenzylmercaptopurine riboside (NBMPR).
They play an important role in the intracellular transport of the dif-
ferent nucleosides such as Ara-C, gemcitabine, cytarabine, fludara-
bine, and others.3 Several studies showed correlation between
sensitivity of different cancers, such as pancreatic, ampullary, lung,
and breast cancer towards treatment with nucleoside-based drugs
and hENT1 expression level.4 Moreover, in several cases immuno-
histochemistry for hENT1 was predictive for patient response on
nucleoside chemotherapy.5 Furthermore, significantly more hENT1
binding sites were found in breast, liver, stomach, and colorectal
tumor tissues as in adjacent normal tissues.6 SAENTA-fluorescein
(50-[(fluorescein-5-ylcarbonyl)-6-aminohexanoyl-(2-aminoethyl)]-
N6-(4-nitrobenzyl)-50-thioadenosine) and similar fluorescent
probes are routinely used to assess the abundance of hENT1 on cell
surface by flow cytometry.7 However, radiotracers suitable for
in vivo molecular imaging of hENT1 were not described up to
now. Here we describe our preliminary results in this regard.
Originally, a conjugate between 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid (DOTA) and SAENTA (DOTA–SAENTA, 1)
was chosen as a promising candidate. Indeed, DOTA conjugates
can be labeled with different radiometal ions such as 68Ga, 64Cu,
and 111In to enable PET or SPECT imaging. SAENTA derivatives usu-
ally have nanomolar affinity to hENT1. They are not internalized
and further metabolized by cells and, therefore, can be used to quan-
tify hENT1 transporters localized at the outer leaflet of the cellular
membrane which are suitable for the intracellular transport of
nucleosides.8
Cystamine was doubly acylated with N-Boc-protected 6-amino-
hexanoic acid (BocAhxOH) to give known 29 in 68% yield (Scheme
1). The latter was reduced with tributylphosphane to give the cor-
responding thiol, which was alkylated directly without purification
with 20-chloroadenosine10 to furnish 3 in 39% yield on two steps,
which in turn was benzylated at 1-position with p-nitrobenzylbro-
mide to give after the dimethylamine mediated 1?6 benzyl shift11
and the acidolytic cleavage of the N-Boc-protecting groups an ad-
vanced intermediate, H2N–SAENTA (4), as a trifluoroacetate (57%
on three steps). Finally, 4 was acylated with DOTA-ONp12 to give
after HPLC purification conjugate 1 in 39% yield. Similarly, acyla-
tion of 4 with 5-carboxyfluorescein using EDC/HOBt gave SAEN-
TA-fluorescein6 (40%), which was used for in vitro study.
DOTA–SAENTA was labeled with 111In (Scheme 1) to give 111In-
1 in 95% radiochemical yield (rcy) and in high radiochemical purity
(rp), which was tested for binding to hENT1-positive cells. Surpris-
ingly, we failed to observe significant cell-bound radioactivity in
this study. Experiments on competitive binding of
1 and
SAENTA-fluorescein to HL60 cells showed, that DOTA–SAENTA as
well as In-1 lacked affinity to hENT1 transporters (data not shown).
We reasoned that the absence of binding may be due to the bulk-
iness and/or the net charge of the macrocyclic chelator.
* Corresponding author. Tel.: +49 731 500 61300; fax: +49 731 500 61302.
E-mail address: sven.reske@uniklinik-ulm.de (S.N. Reske).
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.07.017

5152
B. D. Zlatopolskiy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5151–5154
NH2
N
N
N
a
N
b, c
S
S
2
O
S
BocNH
COHN
5
2
BocNH
COHN
5
2
H2N
cystamine
2
3
HO OH
NH
N
N
N
NO2
DOTA-SAENTA
g
1
N
d, e, f
S
O
TFA H2N
COHN
5
2
h
(4)
SAENTA-fluorescein
TFA H2N-SAENTA
HO OH
i
1
111In-1
CO2H
O
HO2C
CO
N
N
N
O
DOTA =
5-FAM =
HO
HO2C
CO2H
N
OH
O
Scheme 1. Reagents and conditions: (a) BocAhxOH, EDC, HOBt, DIEA, CH2Cl2, 0 °C to rt, 16 h, 68%; (b) Bu3P, 90% MeOH, 1 h; (c) MeONa, then 50-chloroadenosine, reflux, 4 h,
39% on two steps; (d) p-NO2BnBr, mol. sieves 4 Å, DMF, 4 days; (e) Me2NH in EtOH, 6 h (f) TFA (2.5% TIS, 2.5% H2O), 0 °C, 10 min, 57% on three steps; (g) DOTAONp, 2,4,6-
collidine, MeCN/H2O, 3 days, 39%; (h) 5-FAM, EDC, HOBt, 2,4,6-collidine, DIEA, DMA, 0 °C to rt, 16 h, 40%; (i) 111InCl3, acetate buffer (pH 5.0), 85 °C, 3 min, rcy 95%, rp 95%.
131I
COONSu
Me3Sn
COONSu
b
c
a
d
131I-SAENTA-I
SAENTA-F
SAENTA-I
4
6
131I-5
5
Scheme 2. Reagents and conditions: (a) m-IBzOSu, DIEA, 2,4,6-collidine, DMA, 2 days, 28%; (b) p-fluorobenzoyl chloride, HOBt, Et3N, 2,4,6-collidine, CH2Cl2/DMF, 16 h, 0 °C to
rt, 44%; (c) Na131I, chloroamine T, MeOH/AcOH, 5 min; (d) 4, borate buffer (pH 8), 2 h, rcy 22% two steps, rp > 98%.
Accordingly, we prepared SAENTA derivatives with small neu-
tral and lipophilic side chain. Compound 4 was acylated with
N-succimidyl m-iodobenzoate (m-IBzOSu) or prepared in situ 1-
benzotriazolyl p-fluorobenzoate to give SAENTA-I (5) and SAEN-
TA-F (6) in 28% and 44% yield, respectively (Scheme 2). They were
tested regarding inhibition of the hENT1-specific SAENTA-fluores-
cein binding to HL60 leukemia cells by flow cytometry. Gratify-
ingly, 5 and 6 inhibited SAENTA-fluorescein binding to HL60 cells
(Fig. 1) with 5 as a more potent compound. It led to full inhibition
of SAENTA-fluorescein binding at equimolar concentration. The
IC50 values (concentration of inhibitor that inhibited 50% of SAEN-
TA-fluorescein binding) 1.4 1.0 nM and 5.1 1.0 nM for 5 and 6,
Figure 1. Flow cytometric competition studies of binding of SAENTA-fluorescein with SAENTA-I (5) (a–c), and with SAENTA-F (6) (d–f) using HL60 cells. The binding of
SAENTA-fluorescein (50 nM) was examined in the presence of 5: 5 nM (a), 25 nM (b), 50 nM (c) or in the presence of 6: 5 nM (d), 25 nM (e), 50 nM (f). Dashed lines—negative
control; bold lines—binding of SAENTA-fluorescein preincubated with 5 or 6; filled histogram—binding of SAENTA-fluorescein.

B. D. Zlatopolskiy et al. / Bioorg. Med. Chem. Lett. 19 (2009) 5151–5154
5153
Table 1
80
70
60
50
40
30
20
10
0
131I-SAENTA
a
KD and Bmax for binding of SAENTA-fluorescein to the used cell lines
131I-SAENTA + NBMPR
b
b,c
Cell line
KD (nM)
Bmax (MFU)
HL60
MCF7
0.26 0.05
n.d.d
0.32 0.08
3.8 0.4
0.91 0.07
0.36 0.05
0.40 0.28
21.1 0.7
n.d.d
Capan1
Panc1
MIA-PaCa2
BX-PC3
SK-PC1
9.5 0.4
49.8 1.7
30.7 0.6
6.7 0.2
2.6 0.4
a
KD—apparent equilibrium dissociation constant for SAENTA-fluorescein at es
nucleoside transporter sites; Bmax—maximum specifically bound SAENTA-fluores-
cein per 104 cells.
b
KD and Bmax values were determined according to Ref. 16.
MFU—mean fluorescence units.
n.d.—not determined.
c
d
t/min
Figure 2. Cell binding of 131I-5 (20 KBq/5 ꢀ 105 cells) to HL60 leukemia, MCF7
ference between the Ki values of 5 and 6 indicates that in case of
SAENTA derived compounds even minor structural changes in
the substituent at the 50 position of adenosine residue can affect
the binding to ENT1 transporters.
breast, and Capan1, Panc1, MIA-PaCa2, BX-PC3, SK-PC1 pancreas tumor cells
depending on pre-incubation with NBMPR (7.2 lM).
131I-5 was prepared by acylation of 4 with 131I-m-IBzOSu, which
in turn was obtained by radioiodination of the stannyl precursor15
(Scheme 2) in 22% radiochemical yield on two steps (Scheme 2). It
was tested for binding to leukemia, breast, and pancreas tumor cell
lines and showed good or moderate time-dependent binding to 6
from 7 tested cell lines (Fig. 2). The cell binding was inhibited up
to >90% by NBMPR demonstrating its hENT1 specificity. Addition-
ally, cell binding of 131I-5 correlated significantly (P < 0.05) with
the binding potential (Bmax/KD) of the ENT1-specific SAENTA-
fluorescein binding (Fig. 3). The respective binding parameters,
the binding affinity (KD), and the capacity (Bmax), were determined
respectively, were obtained by the nonlinear regression analysis of
the inhibitor concentration–fluorescence curves using GraphPad
Prism software. Inhibition constants (Ki) were calculated from
IC50 using the Chaeng–Pristoff equation:13 Ki = IC50/(1 + [S]/KD),
where [S]—concentration of SAENTA-fluorescein, KD—equilibrium
dissociation constant for SAENTA-fluorescein at es nucleoside
transporter sites. The Ki values 0.018 0.016 nM and
0.064 0.025 nM were obtained for 5 and 6, respectively, demon-
strating somewhat stronger binding for 5. The Ki values were even
lower than that of NBMPR [Ki(NBMPR) = 0.1–1.0 nM].13a,14 The dif-
a
b
60
60
50
40
30
20
10
0
50
40
30
20
10
0
0
20
40
60
80
100
0
20
40
60
80
100
Bmax/KD
Bmax /KD
Figure 3. Correlation between the binding potency of SAENTA-fluorescein and the cell binding of 131I-SAENTA-I (131I-5). The following parameters were obtained: (a) after
30 min incubation: slope of linear regression = 0.45 0.09, P = 0.008, R2 = 0.86; (b) after 240 min incubation: slope of linear regression = 0.58 0.18, P = 0.03, R2 = 0.72. Error
bars smaller than symbols are not shown.
b
a
60
50
40
30
20
10
0
10
8
HL60
MIA-PaCa-2
PANC1
6
BX-PC3
Capan1
SK-PC3
4
2
0
0
5
10
15
20
0
5
10
15
20
SAENTA-fluorescein [nM]
SAENTA-fluorescein [nM]
Figure 4. Flow cytometric analysis of the dependence on concentration of ENT1-selective binding of SAENTA-fluorescein. The specific binding data represent the difference
between the total and nonspecific fluorescence. The nonspecific fluorescence was measured in presence of 7.2 M NBMPR.
l

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present we cannot explain variations of the KD values which sug-
gest differences in affinity of the SAENTA-fluorescein binding sites
by different cell lines.
In conclusion, SAENTA-I showed consistently high and hENT1-
specific binding to most tumor cell lines tested. SAENTA-I is there-
fore a hENT1-selective marker which should be further studied for
its suitability for imaging the expression of hENT1 nucleoside
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Professor Dr. S. Hahn (Bochum, Germany) is gratefully acknowl-
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PC1 cells. This work was supported in part by the European Com-
munity’s FP6 funding (MolDiag-PaCa). This publication reflects
only the author’s views. The European Community is not liable
for any use that may be made of the information herein.
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