J. Le Gal et al. / Journal of Inorganic Biochemistry 105 (2011) 880–886
885
coupled to a gamma detector (radioflow monitor) HERM LB500
(Berthold). LC–MS analysis was carried out on an Agilent 1100 Series
chromatography system, with a Varian Pursuit C18 analytical column,
with a linear gradient system (0% to 15% B) from 0 to 5 min, a linear
gradient system (15% to 30% B) from 5 to 35 min and then an isocratic
system (100% B). The chromatography system was coupled to a Bruker
Esquire HCT ES/MS mass spectrometer. 1H and 13C NMR spectra were
recorded on Bruker AVANCE 250 NMR spectrometers; δ and J are
reported in ppm relative to TMS and Hz, respectively. High-resolution
mass spectrometry (HRMS) was performed on a Q-tof (Micromass).
Microplates were counted using a Perkin-Elemer microplate scintillation
and luminescence counter Topcount NXT.
CHα Cys), 4.42 (s, 2H, CH2–CO NS2), 3.59 (bs, CO–CH2–N–C N), 3.41–
3.24 (m+H2O signal, CH2 Cys+2 CH2–N NS2), 3.03 (m, 4 H, CH2–S
NS2); HRMS: calcd, 749.1008; found, 749.1052.
12f (9.5 mg, 61%): 1HNMR (400 MHz, d6-DMSO): δ 7.82 (d, J=7.2,
2H, Ar CHo), 7.64 (d, J=7.2, 2H, Ar CHm), 4.50–4.55 (m, 1H, CHα Cys),
4.37 (s, 2H, CH2–CO NS2), 3.66 (bs, CO–CH2–N–C N), 3.54–3.20 (m+
H2O signal, CH2 Cys+2 CH2–N NS2), 2.99 (m, 4H, CH2–S NS2); HRMS:
calcd, 783.0618; found, 783.0597.
4.3. [99mTc]oxotechnetium complexes 24a, 25a, 25f
After reduction of modules (10: 2 μmol, 19a, 20a, and 20f: 1 μmol)
as described above, the solvent was removed under a flux of argon and
the product was dissolved in a 35 mM HEPES buffer pH 7.8 to give a
500 μM solution. [99mTc]oxotechnetium gluconate was produced by
mixing solutions of 2 mg ml−1 stannous chloride (2 μL) in HCl 0.1 N,
50 mM gluconate (10 μL), and a [99mTc]pertechnetate solution (90 μL,
about 130 MBq) eluted from a 99Mo/99mTc generator. The resulting
4.2. Oxorhenium complexes
4.2.1. General procedure for complexation
Compound 10 (101 mg, 200 μmol, 1 equiv.) was treated with a
mixture of TFA:TIPS:water 95:2.5:2.5 (10 mL) for 30 min at room
temperature. After evaporation of the solvent in vacuo, toluene was
chased 3 times on the product. The crude product was dissolved in
degassed methanol (3.0 mL) and the solution was neutralized by
addition of TEA. A solution of tributylphosphine 10% in methanol
(750 μL, 1.5 equiv.) was added and the mixture was stirred for 1 h at
room temperature under argon. The solution was divided into 20
portions (10 μmol each) which were used immediately. Eighteen
portions were treated successively with a different thiol (compounds
19–21) reduced in the conditions described above (0.5 equiv. before
reduction), and a solution of tetrabutylammonium tetrachlorooxor-
henate in degassed methanol (7.5 μL of a solution at 117.5 mg/150 μL,
1 equiv.) and TEA (6 μL, 4 equiv.). The pH was adjusted to pH 10 with
TEA. The mixture was stirred for 2 h at room temperature. The
precipitate was isolated by 3 successive centrifugation/washing
(500 μL of methanol) cycles and was purified by elution through a
Sep-Pak Vac 500 mg C18 cartridge (elution by a manual gradient of
water:ACN 100:0 to 0:100% by steps of 5%). The yellow-green
fractions were pooled, diluted with water and freeze-dried to give
92–97% pure oxorhenium complexes as assessed by LC–MS
analysis: 11a (23.1 min, m/z 734.0 (MH+)); 11b (28.3 min, m/z
790.1 (MH+)); 11c (26.8 min, m/z 810.1 (MH+)); 11d (25.2 min, m/z
784.0 (MH+)); 11e (26.5 min, m/z 818.1 (MH+)); 11f (27.9 min, m/z
768.1 (MH+)); 12a (23.5+24.1 min (27+73%), m/z 749.0 (MH+));
12b (28.9 min, m/z 805.1 (MH+)); 12c (28.0 min, m/z 825.0 (MH+));
12d (26.4 min, m/z 799.0 (MH+)); 12e (27.2 min, m/z 833.0 (MH+));
12f (29.0 min, m/z 783.1 (MH+)); 13a (26.0 min, m/z 770.0
(MH+)); 13b (28.5 min, m/z 826.0 (MH+)); 13c (27.4 min, m/z
846.0 (MH+)); 13d (25.9 min, m/z 819.9 (MH+)); 13e (26.5 min,
m/z 854.0 (MH+)); 13f (28.7 min, m/z 804.0 (MH+)).
[
99mTc]oxotechnetium gluconate solution was treated 15 min at 90 °C
with the reduced peptide (100 μL). The complexes were analyzed by
analytical RP-HPLC with a linear gradient system (0% to 15% B) from
0 to 5 min, a linear gradient system (15% to 30% B) from 5 to 35 min
and then an isocratic system (100% B): 24a. tR =19.0 min; 25a.
tR =20.6+21.7 min; 25f. tR =22.0 min.
4.4. Stability of complexes 24a, 25a and 25f in murine plasma
Complexes in murine plasma were analyzed by analytical RP-HPLC
(same gradient as above) as previously reported [12].
4.5. Integrin binding assays
Integrin binding assays were carried out as previously described
[12] using either 1% (αVβ3 and αVβ5) or 5% (αIIbβ3) BSA with 0.06 nM
(αVβ3 and αIIbβ3) or 0.1 nM (αVβ5) [125I]I-echistatin.
Abbreviations
ACN
Boc
Acetonitrile
tert-butyloxycarbonyl
cHex/Hex cyclohexane/hexane
ClHOBT 6-Chloro-1-hydroxy-1H-benzotriazole
DCC
Dicyclohexylcarbodiimide
DCM
DIPEA
EDC
ES/MS
GSH
Dichloromethane
Diisopropylamine
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide
Electrospray mass spectrometry
Glutathione
HATU
2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
4.2.2. Synthesis of complexes 11a, 12a and 12f at the preparative scale
The general procedure was applied to the preparative synthesis of
hit compounds using Compound 10 (10.1 mg, 20 μmol, 1 equiv.) in
degassed methanol (3 mL), compounds 19a (4.5 mg, 0.5 equiv.), 20a
(4.8 mg, 0.5 equiv.) or 20f (5.5 mg, 1 equiv), tributylphosphine (75 μL,
1 equiv.) and tetrabutylammonium tetrachlorooxorhenate (11.7 mg,
1.0 equiv.) and triethylamine (11 μL, 4 equiv.). Three successive
centrifugation/washing (1.0 mL of methanol) cycles and purification
through a Sep-Pak Vac 500 mg C18 cartridge as reported above
afforded the pure oxorhenium complexes.
HEPES
4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
LC–MS Liquid chromatography coupled to a mass spectrometer
PBS
PMA
RGD
RP-HPLC Reverse phase high performance liquid chromatography
SD
TEA
TFA
TIPS
Phosphate buffered saline
Phosphomolybdic acid
Arg-Gly-Asp
Standard deviation
Triethylamine
Trifluoacetic acid
Triisopropyl silane
11a (11.4 mg, 78%): 1HNMR (400 MHz, d6-DMSO, d = doublet,
m = multiplet, s = singlet, bs = broad singlet): δ 7.92 (d, J=7.3, 2H,
Ar CHo), 7.55–7.59 (m, 3H, Ar CHm+CHp), 4.54 (m, 1H, CHα Cys),
4.35 (s, 2H, CH2–CO NS2), 3.57 (bs, CO–CH2–N–C N), 3.46–3.27 (m+
H2O signal, CH2 Cys+2 CH2–N NS2), 2.95 (m, 4H, CH2–S NS2); HRMS:
calcd, 734.0899; found, 734.0964.
Acknowledgements
We thank the Institut National du Cancer (INCa) for the financial
support of this research program (Grant No. PL06-060). We gratefully
acknowledge Dr Carole Gaillard for iodination of echistatin and
Bertrand Czarny for preparation of fresh mice plasma. We are indebted
12a (12.0 mg, 80%): 1HNMR (400 MHz, d6-DMSO): δ 7.67 (d,
J=7.2, 2H, Ar CHo), 7.40–7.24 (m, 3H, Ar CHm+CHp), 4.58 (m, 1H,