Journal of Medicinal Chemistry
Article
[α]20D −22.6°
1.32° (c 0.10, DMSO). 1H NMR (400 MHz,
reaction and without any workup, the reaction mixture was
concentrated and the residue was adsorbed on LiChroprep RP18
packing material (Merck), charged on a RP-18 PuriFlash 15PT C18T
column (Interchim) and eluted consecutively with the following
solvents:
DMSO-d6) δ, ppm: 10.77−10.86 (m, 1H), 10.53 (s, 1H), 8.15 (d, J =
8.59 Hz, 1H), 8.05 (dd, J = 5.56, 8.84 Hz, 2H), 7.81−7.87 (m, 2H),
7.58−7.63 (m, 2H), 7.40 (t, J = 8.84 Hz, 2H), 7.30 (t, J = 8.34 Hz,
2H), 7.08 (d, J = 2.27 Hz, 1H), 7.02 (t, J = 8.08 Hz, 1H), 6.90−6.96
(m, 1H), 3.88−3.95 (m, 1H), 3.05 (dd, J = 6.82, 14.40 Hz, 1H), 2.86
(dd, J = 7.58, 14.40 Hz, 1H). 13C NMR (101 MHz, DMSO-d6) δ,
ppm: 172.5, 165.5, 164.8, 163.0, 158.0, 142.4, 136.0, 135.2, 131.0,
130.6, 130.5, 127.3, 126.9, 123.9, 120.8, 119.4, 118.3, 117.9, 115.5,
115.3, 111.4, 108.9, 56.6, 28.3. 19F NMR (376 MHz, DMSO-d6) δ,
ppm: −111.9 (m). HRMS (TOF MS ES+): calcd for C24H21FN3O5S
([M + H]+), 482.1186; found 482.1183.
(1) an aqueous 0.1% solution H-X (X = tosylate, bromide, triflate,
or perchlorate, according to the desired salt form) (500 mL/1 g
crude product);
(2) water (500 mL/1 g crude product);
(3) a gradient of acetonitrile in water, from 20% to 90% over 20
min.
The product was collected by monitoring UV signal (215−360
nm, PDA detector) and lyophilized.
Synthesis of Labeling Precursors. The reaction of oxidative
formation of iodonium compound was adapted from the published
procedure.15
Tosylate (23b). The analytical data correspond to those described
above; integrals of the tosylate signals indicate the presence of 0.9−1.0
equiv tosylate with respect to the cation.
(4-Carboxyphenyl)(4-methoxyphenyl)iodonium 4-methylbenze-
nesulfonate (21). 4-Iodobenzoic acid methyl ester (10 g, 38.2
mmol), 3-chloroperoxybenzoic acid (17.8 g, 72 mmol), and anisole
(6.2 g, 57.2 mmol) were dissolved in dichloromethane/trifluoroetha-
nol (1:1, 200 mL). 4-Toluenesulfonic acid (10.89 g, 57.2 mmol) was
added, and the reaction mixture was stirred at room temperature for 3
days. To the obtained suspension ether (1500 mL) was added and the
precipitate was filtered and washed with ether. The solvent was
evaporated to yield 22.8 g of the crude intermediate methyl ester. MS
(ES+): m/z 368.61 ([M]+) (iodonium cation).
The methyl ester was dissolved in TFA/water (1:1, 600 mL) and
heated at 120 °C (oil bath) for 48 h. After cooling to room
temperature, the solvents were removed and the oily residue was
triturated with ether (500 mL), filtered, and dried to give an off-white
Triflate (23c). The triflate could be qualitatively confirmed by 19F
NMR δ −77.7 and 13C NMR δ 120.7 (q, J = 322 Hz). Because tosylate
could not be detected, a complete exchange to triflate was presumed.
Bromide (23d). The presence of bromide was not directly
confirmed. Because the tosylate, which was present in the crude
product, could not be detected, a complete exchange to bromide was
presumed.
Perchlorate (23e). The presence of perchlorate was not directly
confirmed. Because the tosylate, which was present in the crude
product, could not be detected, a complete exchange to perchlorate
was presumed.
Enantiomeric Stability under Basic Hydrolysis Conditions. Methyl
ester of compound 7 was hydrolyzed under conditions similar to the
hydrolysis reaction during radiosynthesis. For this, 20 mg of the
methyl ester, 26 mg of Cs2CO3, 5 mL pf DMF, and 100 μL of water
were combined in a reaction vial. Then, 1 mL of 4N NaOH was added
and the mixture was heated on an oil bath at 100 °C for 10 or 20 min.
The reactions were removed from the oil bath and neutralized with 4
mL of 1N HCl to pH = 7.5. Analysis on chiral HPLC confirmed that
the reactions were chemically clean and full conversion occurred at
both time points. The product eluted at 4.0 min, which corresponds to
R-enantiomer. The expected retention time for the S-enantiomer was
6.25 min.
Inhibitory Potency Determination (Fluorescent Assay). Recombi-
nant human full length MMP2 (902-MP, R&D Systems) or MMP9
(911-MP, R&D Systems) was chemically activated using p-amino-
phenylmercuric acetate (APMA) according to the manufacturer’s
protocol.21 To the activated enzyme (final concentration 0.1 nM, 24
μL) in reaction buffer (50 mM Tris/HCl pH 7.5, 10 mM CaCl2, 150
mM NaCl, 0.05% Brij-35), compound of interest (in DMSO, suitable
concentrations, e.g., 1 nM to 30 μM, 1 μL) was added in a 384-MTP
white plate. Reaction was started by addition of internally quenched
substrate Mca-Pro-Leu-Gly-Leu-Dpa(Dnp)-Ala-Arg-NH2 (final con-
centration 10 μM; Mca = (7-methoxycoumarin-4-yl)acetyl; Dpa(Dnp)
= N-3(2,4-dinitrophenyl)-L-2,3-diaminopropionyl, R&D Systems) to
yield a total volume of 50 μL. Progress of the MMP reaction was
monitored by fluorescence intensity measurement (excitation, 320 nm;
emission, 410 nm) over 120 min at 32 °C. IC50 values were
determined by plotting the compound concentration versus the
percentage of MMP activity by interpolation.
1
solid (18.5 g, 35.1 mmol, 92%). H NMR (400 MHz, DMSO-d6) δ,
ppm: 13.50 (br. s., 1 H), 8.34−8.25 (m, 2 H), 8.24−8.15 (m, 2 H),
8.04−7.94 (m, 2 H), 7.47 (d, J = 8.1 Hz, 2 H), 7.15−7.02 (m, 4 H),
3.80 (s, 3 H), 2.28 (s, 3 H). 13C NMR (101 MHz, DMSO-d6) δ, ppm:
166.1, 162.1, 145.8, 137.5, 137.4, 134.9, 133.6, 131.9, 128.0, 125.5,
121.3, 117.5, 105.4, 55.7, 20.8. MS (ES+): m/z 355.11 ([M]+)
(iodonium cation). MS (ES−): m/z 171.07 ([M]−) (tosylate anion).
Methyl N-{[4-({4-[(4-Methoxyphenyl)iodonio]benzoyl}amino)-
phenyl]sulfonyl}-D-tryptophanate Salts (23b−e). Aniline I (200
mg, 0.54 mmol), (4-carboxyphenyl)(4-methoxyphenyl)iodonium 4-
methylbenzenesulfonate 21 (423 mg, 0.8 mmol), and 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (1.03 g,
5.4 mmol) were suspended in acetonitrile/pyridine (2:1, 30 mL),
and the reaction was stirred at room temperature for 4 days. The
resulted clear yellow solution was acidified to pH 1 using 2N aqueous
HCl. The solution was extracted with dichloromethane (3 × 200 mL),
and the organic phases were combined, dried (MgSO4), and
concentrated. The residue was dissolved in water/acetonitrile (1:1,
10 mL). Toluenesulfonic acid monohydrate (101 mg, 0.53 mmol) was
added, and the mixture was lyophilized (1H NMR indicated an excess
of tosylate in this material). The product (12 mg) was stirred in
dichloromethane, filtered, and dried to obtain the title compound as
approximately 35−40% tosylate salt based on 1H NMR. The
remaining 60−65% were assumed to be the corresponding chloride
salt. The yield, when calculated for both counterions together, was in a
1
range of 70−80%. H NMR (400 MHz, DMSO-d6) δ, ppm: 10.94−
10.81 (m, 1 H), 10.70 (s, 1 H), 8.41 (d, J = 8.6 Hz, 1 H), 8.30 (d, J =
8.3 Hz, 2 H), 8.19−8.09 (m, 2 H), 8.00−7.91 (m, J = 8.6 Hz, 2 H),
7.87−7.79 (m, J = 8.8 Hz, 2 H), 7.65−7.57 (m, J = 8.8 Hz, 2 H), 7.48
(d, J = 8.1 Hz, 0.8 H, tosylate), 7.26 (d, J = 7.8 Hz, 1 H), 7.29 (d, J =
8.1 Hz, 1 H), 7.13−6.99 (m, 5 H), 6.96−6.89 (m, 1 H), 3.96 (q, J =
7.6 Hz, 1 H), 3.78 (s, 3 H), 3.33 (s, 3 H), 3.04 (dd, J = 7.5, 14.3 Hz, 1
H), 2.88 (dd, J = 7.1, 14.4 Hz, 1 H), 2.28 (s, 1.1 H, tosylate). 13C
NMR (101 MHz, DMSO-d6) δ, ppm: 171.4, 164.9, 161.6, 145.7,
142.2, 137.6, 137.0, 137.0, 136.0, 135.1, 134.6, 130.3, 128.0, 127.3,
126.7, 125.5, 123.9, 123.1, 120.9, 119.6, 118.4, 117.6, 117.2, 111.5,
108.6, 108.3, 56.7, 55.6, 51.7, 28.2, 20.8. MS (ES+): m/z 710.22
([M]+) (iodonium cation). MS (ES−): m/z 171.03 ([M]−) (tosylate
anion).
18F-Fluoride Production. No-carrier-added (nca) 18F-fluoride was
produced via the 18O(p,n)18F nuclear reaction in a fixed-energy
Cyclone 18/9 cyclotron (IBA, Belgium). For this, >98% isotopically
enriched 18O-water (Nukem GmbH, Germany) was irradiated by 18
MeV proton beam. Produced 18F-fluoride/18O-water solution was
transferred using a helium stream from the target to a shielded hot cell
equipped with a manipulator where radiosynthesis was performed.
Typical production of 18F-fluoride at end of bombardment (EOB) of
the 2.5 mL target for 20 mAh (∼45 min) was 47−60 GBq.
Radiochemistry. 18F-fluoride (∼ 80−100 GBq) was trapped on an
anion exchange Sep-Pak Light Accell Plus QMA cartridge (Waters)
preconditioned with 5 mL of 0.5 M potassium carbonate solution, 10
mL of water, and flushed with 10 mL of air. It was eluted with 1 mL
Introduction of Counterions. The synthesis was conducted as
described above (scale: 2 g of aniline I). After completion of the
G
dx.doi.org/10.1021/jm400156p | J. Med. Chem. XXXX, XXX, XXX−XXX