Synthesis and evaluation of photoreactive probes to elucidate iodide efflux in thyrocytes

Article abstract of DOI:10.1016/j.bmcl.2008.12.008

Four photoreactive analogues of 3-biphenyl-4′-yl-5,6-dihydroimidazo[2,1-b]thiazole were prepared and evaluated as iodide sequestering agents in sodium iodide symporter-expressing cells. One of these new photoactivatable compounds retained biological activ

Full text of DOI:10.1016/j.bmcl.2008.12.008

Bioorganic & Medicinal Chemistry Letters 19 (2009) 825–827
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of photoreactive probes to elucidate
iodide efflux in thyrocytes
*
Séverine Derbré, Nathalie Lecat-Guillet, Florence Pillon, Yves Ambroise
Commissariat à l’Energie Atomique (CEA), Institut de Biologie et Technologies de Saclay (iBiTecS), Service de chimie bioorganique et de marquage, Gif-sur-Yvette F-91191, France
a r t i c l e i n f o
a b s t r a c t
Four photoreactive analogues of 3-biphenyl-40-yl-5,6-dihydroimidazo[2,1-b]thiazole were prepared and
evaluated as iodide sequestering agents in sodium iodide symporter-expressing cells. One of these new
photoactivatable compounds retained biological activity and was further radiolabeled with tritium. This
compound may provide a useful tool for labeling, purification, and identification of target protein respon-
sible for iodide efflux in thyrocytes.
Article history:
Received 31 October 2008
Revised 2 December 2008
Accepted 3 December 2008
Available online 7 December 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Sodium iodide symporter
Radioiodide
Photoactivatable probe
Photoaffinity labeling
The sodium iodide symporter (NIS) is an intrinsic membrane
glycoprotein that catalyzes the active accumulation of iodide into
thyroid follicular cells. NIS-mediated iodide uptake in the thyroid
gland has long provided the basis for selective cell destruction in
benign and malignant thyroid diseases using radioiodine (131I).1
In the light of this experience, it was proposed to extend this strat-
egy to cancer tissues that do not take-up iodide by gene transfer of
the sodium iodide symporter (NIS).2 In recent years, the expression
of functional NIS by gene transfer was successfully achieved in sev-
eral type of cancer using rat or mice models.3 Unfortunately, radi-
oiodine retention within these non-thyroid cells was low due to a
lack of iodide organification. For this reason, this strategy cannot
be applied to humans. Several strategies were proposed to effi-
ciently increase iodide retention in NIS-expressing cells,4 one of
which is to use small organic molecules. Recently, 3-biphenyl-40-
yl-5,6-dihydroimidazo[2,1-b]thiazole (1, Fig. 1) was reported to
enhance iodide retention in NIS-expressing cells.5 Using this com-
pound, the intracellular iodide concentration is increased by a fac-
tor 4–5 in rat thyroid-derived cells (FRTL5). Isotopic flux
experiments showed that compound 1 do not act by activating
NIS function but rather by inhibiting the release of intracellular
iodide. It was suggested that compound 1 may affect the function
of an unidentified anion permeation mediator. We envisioned pre-
paring photoreactive analogues of 1 to identify the protein(s)
responsible for iodide efflux using photoaffinity labeling
techniques.
Photoaffinity labeling is a powerful technique for the identifica-
tion of target proteins and the characterization of ligand-binding
sites.6 Not only the probe must bear a photoactivatable group plus
a chemical tag for the detection of labeled protein(s), but it has to
be biologically active regarding the phenotype of interest. In this
study we report the synthesis of four photoreactive analogues of
compound 1 with a diazonium (2), an azido (3), a benzophenone
(4), or a trifluoromethyldiazirine group (5) (Fig. 1). The compounds
were evaluated for their ability to increase iodide retention in
FRTL5 cells and the benzophenone 4 showed to retain biological
activity. The preparation of tritium-labeled 4 is also described.
For the synthesis of compounds 2 and 3 (Scheme 1), commer-
cially available 40-iodoacetophenone, 2-imidazolidinethione and
iodine were refluxed in EtOH to afford compound 6 in 78% yield.
Subsequent Suzuki–Miyaura cross-coupling reaction with the
R
N
N
N
N
S
S
1
N3
N2
2: R =
4: R =
3: R =
5: R =
O
N
N
F3C
* Corresponding author. Tel.: +33 169 08 24 70; fax: +33 169 08 79 91.
E-mail address: yves.ambroise@cea.fr (Y. Ambroise).
Figure 1. Structure of compound 1 and its photoreactive analogues 2–5.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.12.008

826
S. Derbré et al. / Bioorg. Med. Chem. Lett. 19 (2009) 825–827
For the preparation photoprobe 4 (Scheme 2), 4-acetylbenz-
I
S
I
i
ophenone (8) was first synthesized using a Suzuki–Miyaura type
coupling reaction from benzoyl chloride and 4-acetylphenylbo-
ronic acid using a general procedure described elsewhere.9 Com-
pound 8 was then reacted with 2-imidazolidinethione and iodine
in boiling EtOH to afford the desired product 4 in 75% overall yield
after DCM/Na2S2O3 and DCM/NaHCO3 extraction work-up followed
by flash chromatography (DCM then DCM/MeOH 95:5).10
N
S
HN NH
N
+
O
6
H2N
H2N
ii
O
B
6
+
N
N
Photoprobe 5 was prepared by means of standard procedures
with slight modifications (Scheme 3).11 Starting from the commer-
cially available 40-bromoacetophenone, carbonyl protection fol-
lowed by Br/Li exchange and subsequent acylation by ethyl
trifluoroacetate gave the expected intermediate 10. The ketone
was converted into its oxime 11 with hydroxylamine hydrochlo-
ride in pyridine and ethanol. This oxime was next reacted with to-
syl chloride and triethylamine with catalytic DMAP in
dichloromethane to afford the corresponding O-tosyl oxime 12.
Conversion of 12 to diaziridine 13 was brought about with ammo-
nia under pressure in a sealed tube. Oxidation of the diaziridine to
diazirine 14 was achieved with iodine and triethylamine in meth-
anol. Finally ketone deprotection with 2 N HCl in THF/H2O afforded
15 that was reacted with 2-imidazolidinethione and iodine in boil-
ing EtOH to afford the desired photoprobe 5 after filtration and
subsequent DCM/Na2S2O3 and DCM/NaHCO3 extraction work-up.
Compound 5 was sufficiently pure (>98%) for biological assays.12
The biological properties of photoreactive dihydroimidazo
[2,1-b]thiazole derivatives 2–5 and reference compound 1 were
investigated for their ability to enhance iodide entrapment in rat
thyroid-derived cells (FRTL5). Briefly, the monolayer cell culture
was incubated in the dark in the presence of compounds 1–5
O
S
7
3
CF3CO2 , N2
iii
N3
iv
N
N
N
N
S
2
S
Scheme 1. Reagents and conditions: (i) I2 (2 equiv), EtOH, reflux, 16 h, 78% yield;
(ii) Pd(PPh3)4 (0.1 equiv), K3PO4 (2 equiv), DMF, 80 °C, 16 h, 82% yield; (iii) NaNO2
(1.2 equiv), TFA, +4 °C, 30 min, 90% yield; (iv) NaN3 (10 equiv), TFA, +4 °C to rt, 2 h,
93% yield.
pinacol ester of 4-aminophenylboronic acid afforded the aniline
derivative 7 in 82% yield. The diazonium salt 2 was obtained from
this amine after reaction with sodium nitrite in trifluoroacetic
acid.7 Compound 2 was isolated after simple solvent evaporation
(58% overall yield). Compound 2 was converted into the azido
derivative 3 by the addition of sodium azide to the acidic diazo-
nium salt solution. Compound 3 was isolated after DCM/NaHCO3
extraction work-up (53% overall yield).8 1H NMR and LC-MS
showed that 2 and 3 purities were >90%. For biological assays
and analytical purposes, 2 and 3 were further purified using pre-
(0.1–200 l lM) for 120 min before cell-trapped
M) and Na125I (10
iodide was measured by scintillation counting (a detailed experi-
mental procedure is reported elsewhere).5 The results showed that
photoreactive probes 2, 3, and 5 were inactive, whereas the benzo-
phenone-containing analogue 4 retained biological activity (Fig. 2).
The increase of iodide entrapment was concentration-dependent
parative HPLC (X-bridge C18—5
l
m—19 ꢀ 150 mm).
O
OH
B
O
with an EC50 value of 50
tration was increased by a factor 1.8 and reached a factor 2.5 at
200 M of 4. For comparison EC50 value for compound 1 was calcu-
lated to be 20
M, with an increase of intracellular [I–] by a factor
2.5, whereas the maximum iodide entrapment was reached at
50
l
M.13 At this concentration iodide concen-
i
HO
Cl
O
+
l
8
O
l
O
S
l
M (ꢀ3.9). It has to be noted that even though compound 4 is
ii
less active than reference compound 1, it can be used in photo-
affinity labeling experiments. The bell-like shape of the curve cor-
responding to compound 1 was reported to be due to a cytotoxic
8
HN NH
+
N
N
S
4
effect when high compound concentrations are used (>100 l
M).5
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4Cl2 (0.04 equiv), K3PO4 (2 equiv),
toluene, 100 °C (2.5 h) then 75 °C (14 h), 83% yield; (ii) I2 (2 equiv), EtOH, reflux,
16 h, 91% yield.
The photodecomposition of 4 was tested in MeCN using a 100 W
UV lamp (Black-RayÒ B100AP, k = 365 nm). The light-induced
O
v
O
O
O
O
O
O
R=H, 11
i
ii
iii
iv
CF3
CF3
NOR
R=Tosyl, 12
Br
Br
O
9
10
O
N N
O
O
O
O
vi
vii
viii
F3C
CF3
CF3
N N
CF3
N
N
S
HN NH
N N
13
14
15
5
Scheme 3. Reagents and conditions: (i) ethylene glycol (2.5 equiv), PTSA (0.05 equiv), benzene, reflux, 16 h, 39% yield; (ii) n-BuLi (1.5 equiv), THF, ꢁ78 °C, 3 h then CF3CO2Et
(2 equiv), THF, ꢁ78 °C to rt, 14 h, 80% yield; (iii) NH2OHꢂHCl (1.1 equiv), pyridine/EtOH 2:1, 60 °C, 14 h, quant; (iv) tosyl chloride (1.1 equiv), DMAP (0.05 equiv), TEA (2.5
equiv), DCM, +4 °C to rt, 1 h, 85% yield; (v) NH3 liq., ꢁ78 °C to rt, 20 h, 91% yield; (vi) I2 (1.3 equiv), TEA (3 equiv), MeOH, rt, 1 h, 53% yield; (vii) 2 N HCl, THF/H2O 1:1, rt, 14 h,
95% yield; (viii) 2-imidazolidinethione (1.5 equiv), I2 (1.5 equiv), EtOH, reflux, 8 h, 48% yield.

S. Derbré et al. / Bioorg. Med. Chem. Lett. 19 (2009) 825–827
827
In conclusion, four new photoreactive probes were designed in
order to target proteins responsible for iodide efflux in NIS-
expressing cells. The benzophenone-based photoaffinity probe
was found to be biologically active. This compound was further la-
beled with tritium, thus providing a tool for detection of the photo-
labeled targets.
0.2
0.1
0
Acknowledgment
This work was supported by CEA-iBiTecS.
-7
-6
-5
-4
-3
References and notes
Log C (M)
1. Mazzaferri, E. L. In The Thyroid: A Fundamental and Clinical Text; Braverman, L.
E., Utiger, R. D., Eds., 7th ed.; Lippincott-Raven: Philadelphia, 1996; pp 922–
945.
2. (a) Spitzweg, C.; Morris, J. C. Clin. Endocrinol. 2002, 57, 559; (b) Riesco-
Eizaguirre, G.; Santisteban, P. Eur. J. Endocrinol. 2006, 155, 495.
3. For recent examples, see: (a) Hervé, J.; Cunha, A. S.; Liu, B.; Valogne, Y.; Longuet,
M.; Boisgard, R.; Brégerie, O.; Roux, J.; Guettier, C.; Calès, P.; Tavitian, B.;
Samuel, D.; Clerc, J.; Bréchot, C.; Faivre, J. Hum. Gene Ther. 2008, 19, 915; (b)
Chen, R. F.; Li, Z. H.; Pan, Q. H.; Zhou, J. J.; Tang, Q. B.; Yu, F. Y.; Zhou, Q. B.;
Wang, J.; Chen, J. S. Pancreatology 2007, 7, 505.
Figure 2. Dose-response curves of 1 (j), 2 (e), 3 (D), 4 (h) and 5 (s) in FRTL5 cells.
FRTL5 cells in 96-Isoplates (Perkin-Elmer) were incubated at 20 °C for 120 min with
1–5 (1 ꢀ 10ꢁ7 to 2 ꢀ 10ꢁ4 M) and Na125I (10
lM, 0.2 lCi per well). Cells were
washed at +4 °C before ethanol (30 lL) was added. Cell-associated radioactivity was
determined after addition of the scintillation cocktail. Results are shown for one
experiment representative of at least three independent experiments with mean
values standard deviation (n = 3). NaClO4-mediated inhibition of iodide uptake
was tested as a control in each plate (IC50 = 1 ꢀ 10ꢁ7 M, 100%).
4. Kogai, T.; Taki, K.; Brent, G. A. Endocr. Relat. Cancer 2006, 13, 797.
5. Lecat-Guillet, N.; Ambroise, Y. ChemMedChem 2008, 3, 1211.
6. (a) Sadakane, Y.; Hatanaka, Y. Anal. Sci. 2006, 22, 209; (b) Dormán, G.;
Prestwich, G. D. Trends Biotechnol. 2000, 18, 64.
O
O
ii
i
4
7. Characterization data for compound 2: 1H NMR (400 MHz, TFA-d at 11.50 ppm)
d 3.91 (s, 1H, NH), 4.41–4.49 (m, 4H), 6.89 (s, 1H), 7.61 (d, J = 8.4 Hz, 2H), 7.78
(d, J = 8.4 Hz, 2H), 8.07 (d, J = 8.8 Hz, 2H), 8.49 (d, J = 8.8 Hz, 2H); MS (ESI+) m/z
N
N
I
N
R
N
S
(%) 278 (100) [MꢁN2]+; IR (KBr)
m
in cmꢁ1 3107, 2267, 1680, 1581, 1205, 1140,
S
802, 724; UV (EtOH) kmax 272 nm; Rf = 0.50 (C18—MeOH/H2O 50:50).
8. Characterization data for compound 3: 1H NMR (400 MHz, CDCl3 at 7.26 ppm) d
3.92 (t, J = 8.8 Hz, 2H), 4.28 (t, J = 8.8 Hz, 2H), 5.78 (s, 1H), 7.12 (d, J = 8.4 Hz,
2H), 7.50 (d, J = 8.4 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.4 Hz, 2H); 13C
NMR (100 MHz, CDCl3 at 77.0 ppm) d 48.4, 60.1, 98.6, 119.5, 126.8, 127.2,
128.6, 129.6, 136.7, 136.9, 139.7, 140.6, 170.4; MS (ESI+) m/z (%) 320 (100)
R = H, 4
R = 3H, [3H]-4
16
Scheme 4. Reagents and conditions: (i) N-iodosuccinimide (1.5 equiv), MeCN, rt,
2 h, 32% yield; (ii) 10% Pd/C, TEA, H2 or 3H2 (1.8 atm), MeOH, rt, 12 h.
[M+H]+; IR (KBr)
m
in cmꢁ1 3120, 2121, 1588, 1491, 1371, 1274, 837, 815; UV
(EtOH) kmax 254 nm; Rf = 0.15 (AcOEt/cHex/MeOH 7:2:0.5).
9. Urawa, Y.; Ogura, K. Tetrahedron Lett. 2003, 44, 271.
breakdown of 4 was irreversible and time-dependent, with t1/2 of
12 min at a distance of 5 cm from the mixture.
10. Characterization data for compound 4: 1H NMR (400 MHz, DMSO-d6 at
2.50 ppm) d 3.93 (t, J = 8.0 Hz, 2H), 4.10 (t, J = 8.0 Hz, 2H), 6.39 (s, 1H), 7.57
(dd, J = 8.4 Hz, J0 = 8.0 Hz, 2H), 7.69 (dd, J = 8.0 Hz, J0 = 1.8 Hz, 1H), 7.74 (dd,
J = 8.4 Hz, J0 = 1.8 Hz, 2H), 7.75 (dd, J = 8.4 Hz, J0 = 1.8 Hz, 2H), 7.79 (dd,
J = 8.4 Hz, J0 = 1.8 Hz, 2H); 13C NMR (100 MHz DMSO-d6 at 39.5 ppm) d 48.2,
60.3, 100.7, 126.0, 128.6, 129.5, 130.3, 132.8, 134.0, 136.0, 136.7, 136.9, 168.2,
We envisioned preparing tritium-labeled photoprobe 4 by
introducing the radiolabeled portion by selective tritium reduction
of the corresponding iodoarene. Thus compound 4 was iodinated
by treatment with N-iodosuccinimide in acetonitrile to provide
the monoiodinated compound 16 in 32% yield (Scheme 4) after
DCM/Na2S2O3 and DCM/NaHCO3 extraction work-up followed by
flash chromatography (DCM/TEA 99:1 then DCM/MeOH/TEA
95:5:1). Catalytic dehalogenation of iodoarene 16 with H2, 10%
Pd/C and TEA in MeOH gave 4 in >98% yield (HPLC). The identical
reaction with tritium gas provided the desired radiolabeled photo-
affinity probe [3H]-4 after purification by semi-preparative HPLC
using a Zorbax-C18 column.14 Comparison of HPLC UV traces for
[3H]-4 and 4 showed them to be identical. The radiochemical pur-
ity of [3H]-4 was >98% (analytical radio-HPLC and radio-TLC). The
specific activity of [3H]-4 was determined by ESI-MS to be
350 mCi mmolꢁ1 representing an isotopic enrichment of 1.2%. This
is a surprisingly low value for this type of reaction. However, this
may not be a major drawback in the context of photolabeling
experiments since the effective concentration of 4 is in the range
195.0; MS (ESI+) m/z (%) 307 (100) [M+H]+; IR (KBr)
m
in cmꢁ1 3395, 2868, 1649,
1591, 1276, 931, 859, 700; UV (EtOH) kmax 366 nm (4900 Mꢁ1 cmꢁ1); Rf = 0.54
(DCM/MeOH 9:1). ESI-HRMS calcd for C18H15N2OS ([M+H]+) m/z 307.0900
found 307.0905.
11. Weber, T.; Brunner, J. J. Am. Chem. Soc. 1995, 117, 3084.
12. Characterization data for compound 5: 1H NMR (400 MHz, DMSO-d6 at
2.50 ppm) d 3.87 (t, J = 8.8 Hz, 2H), 4.07 (t, J = 8.8 Hz, 2H), 6.30 (s, 1H), 7.33
(d, J = 8.0 Hz, 2H), 7.69 (d, J = 8.0 Hz, 2H); 13C NMR (100 MHz DMSO-d6 at
39.5 ppm) d 48.0, 60.3, 100.0, 107.8 (q, Jc-f = 275 Hz), 126.9, 127.0, 127.7, 132.1,
135.6, 168.1; MS (ESI+) m/z (%) 311 (100) [M+H]+; IR (KBr)
m
in cmꢁ1 3433,
2939, 2085, 1609, 1514, 1178, 1138, 938, 828, 690; UV (EtOH) kmax 349 nm
(380 Mꢁ1 cmꢁ1); Rf = 0.20 (DCM/MeOH 9:1).
13. The term half-maximal effective concentration (EC50
) refers to the
concentration of compound which induces a response halfway between the
lower signal (i.e., signal at low compound concentrations) and maximum
signal (50 lM for 1 and 200 lM for 4). Experimental data were fitted by
nonlinear regression to the four-parameter sigmoidal Hill equation.
14. For a general procedure of tritiation reactions see: Ambroise, Y.; Pillon, F.;
Mioskowski, C.; Valleix, A.; Rousseau, B. Eur. J. Org. Chem. 2001, 20, 3961.
15. When other non protic solvents were tested (acetone, THF, AcOEt) no reaction
occurred. The same reaction run with deuterium gas in MeOH afforded [2H]-4
with a moderate isotopic enrichment of 16%. For any unknown reasons, this
reaction is subjected to strong isotopic effects.
of 20–200
[3H]-4 failed.15 Finally, [3H]-4 was stable at least 6 months when
stored at +4 °C in MeCN/H2O 1:1 at a concentration of 4 mCi mLꢁ1
lM. All attempts to increase the specific activity of
.