122
ALONSO MARTINEZ AND DASILVA
unless stated. Flash chromatography is carried out with
silica gel (40–60 μm). 1H- and13C-NMR spectra are
acquired with a Bruker 300 MHz spectrometer at ambient
temperature. Spectral data are reported in parts per mil-
lion (ppm) using residual solvent as a reference. High res-
olution and accurate mass measurements are acquired in
positive mode by flow injection analysis into a Thermo
Scientific Q-Exactive Plus Orbitrap Mass Spectrometer
quenched with cold saturated NaCl and extracted with
ethyl acetate (3 × 20 ml), dried over MgSO4, and the
filtrated solution was partially evaporated under reduced
pressure. The residue was purified by column chromatog-
raphy using a solvent gradient EtOAc/Hexanes (15:85 to
30:70). The tetrazole-protected alkyne Candesartan (5)
was obtained in 55% (yellowish solid, 84 mg). Purity of
98.2% (HPLC, 2 ml/min, AcN/0.1-M ammonium formate
80:20). Melting point: 94ꢁC–98ꢁC. ESI-MS: Calculated for
(San Jose, CA, USA) interfaced with
a
heated
1
electrospray ion source. Sep-Pak C18 plus (360 mg,
Waters) solid-phase extraction cartridges were
preconditioned with 10-ml ethanol followed by 20-ml
C47H40N6O2: 720.3213 Found (MH+): 721.3288. H NMR
(CDCl3): δ: 7.95–6.84 (m, 26H); 5.45 (s, 2H); 4.60 (q,
J = 6.96 Hz, 2H); 4.32 (s, 2H); 3.55–3.45 (m, 2H); 2.42 (td,
J = 6.58, 2.63 Hz, 2H); 1.99 (m, 1H) 1.45 (t, J = 7.05 Hz,
3H). 13C NMR (75 MHz, CDCl3): δ 164.0, 157.6, 141.7,
141.3, 141.4, 140.3, 137.3, 132.5, 130.3, 130.2, 129.9, 129.8,
128.2, 127.5, 126.3, 124.9, 124.2, 121.2, 119.6, 118.4, 82.9,
81.6, 70.7, 69.4, 67.3, 66.4, 45.8, 19.8, 14.7.
water. Analytical HPLC was performed on
a
Phenomenex Luna C18 (2) column (250 × 4.6 mm,
10 μm) with a Waters HPLC composed of 1,515 isocratic
pump, 2,487 dual λ absorbance detector, and a Raytest
Gabi Star radioactivity detector. Two analytical methods
were utilized: Method 1 (2 ml/min, A: water and B:
CH3CN, linear gradient 50% B to 80% during 25 min) and
Method 2 (2 ml/min, 55:45 acetonitrile/water 0.1% TFA).
Radio-TLC chromatograms (solvent system, EtOAc/Hex-
anes/MeOH 80:20:5) were acquired with an AR-2000
radio-TLC imaging scanner (Eckert & Ziegler, Germany).
Automated radiosyntheses were performed with the
Synthra® RNPlus Research (Germany) synthesis module.
Semi-preparative HPLC separations were carried out
within the radiosynthesizer with a Phenomenex Luna
C18 (2) HPLC column (250 × 10 mm, 10 μm) at a flow
rate of 8 ml/min (first purification [18F]6: 55:45 acetoni-
trile/water 0.1% TFA and second purification [18F]7:
45:55 acetonitrile/water 0.1% TFA) with UV (254 nm)
and radiation detection.
2.2.2 | Fluorobenzyl-Candesartan (1-((2'-
(1H-tetrazol-5-yl)-[1,1'-biphenyl]-4-yl)
methyl)-2-ethoxy-7-(((4-(4-fluorophenyl)
but-3-yn-1-yl)oxy)methyl)-1H-benzo[d]
imidazole) (7)
The tetrazole-protected alkyne Candesartan (5) (20 mg,
0.03 mmol) was added under argon to a solution con-
taining Pd (PPh3)4 (0.70 mg, 0.6 μmol) and CuI (0.11 mg,
0.6 μmol) in 0.2-ml Et3N at 0ꢁC. After stirring for 10 min
under argon, FIB (6) (7.4 mg, 0.33 mmol) previously dis-
solved in Et3N (0.5 ml) was added dropwise and stirred at
0ꢁC to room temperature for 20 h. After TLC testing
(EtOAc/Hexanes 30:70, Rf = 0.4) for completion, the mix-
ture was quenched with saturated aqueous solution of
NH4Cl, extracted with ether (2 × 10 ml) and washed with
water (1 × 10 ml). A small aliquot was taken for
MS. ESI-MS: Calculated for C53H43FN6O2: 814.3432
Found (MH+): 815.3513. Subsequently, the product was
hydrolyzed with 0.250 ml of TFA/AcN (1:1.5) at 60ꢁC for
7 min. The final mixture was purified by semi-prep
HPLC, and the collected fractions were lyophilized
(FreeZone 12-84C, Labconco). The fluorobenzyl-
Candesartan (7) was obtained in 31% (white solid, 5 mg)
and a chemical purity of 98.9% (analytical HPLC using
Method 2). Using Chem3D® (Perkin Elmer), the logP
value was calculated to be 7.7. ESI-MS: Calculated for
C34H29FN6O2: 572.2336 Found (MH+): 573.2432. 1H
NMR (CDCl3) δ 8.04 (d, J = 6.6 Hz, 1H), 7.68-7.54 (m,
2H), 7.32–7.28 (m, 5H), 7.06–6.78 (m, 8H), 5.53 (s, 2H),
4.24 (s, 2H), 4.05 (s, 1H), 3.49 (t, J = 6.5 Hz, 2H), 2.58
(t, J = 6.4 Hz, 2H), 0.92 (t, J = 6.05 Hz, 3H). 13C NMR
(151 MHz, CD3CN): δ 162.98, 161.34, 157.35, 154.95,
141.29, 139.64, 138.79, 138.53, 138.19, 138.07, 133.47,
2.2 | Chemistry
Compounds (2), (3), and (4) were synthesized as reported
previously21 (see Data S1).
2.2.1 | Tetrazole-protected alkyne
Candesartan (7-((but-3-yn-1-yloxy)methyl)-
2-ethoxy-1-((2'-(1-trityl-1H-tetrazol-5-yl)-
[1,1'-biphenyl]-4-yl)methyl)-1H-benzo[d]
imidazole) (5)
But-3-yn-1-ol (12 mg, 0.18 mmol) was slowly added
dropwise to NaH (9.5 mg, 0.39 mmol) and Bu4N+I-
(13.2 mg, 0.04 mmol) in 1-ml anhydrous THF under
nitrogen and stirred for 1 h at 0ꢁC. Tetrazole-protected
bromide Candesartan (4) (0.158 g, 0.20 mmol) in 2-ml
anhydrous THF was added dropwise at 0ꢁC and stirring
continued overnight under N2. The reaction was then