B. Latli et al.
radiochemical purity> 99%. 1H NMR (500 MHz, DMSO-d6): δ 12.47(s, 1H),
12.34(s, 1H), 8.56(s, 1H), 8.16(d, J= 9.1 Hz, 1H), 8.04(s, 1H), 7.46(s, 1H), 7.34(d,
J=9.1Hz, 1H), 7.00(d, J= 7.5 Hz, 1H), 5.72(m, 1H), 5.41(s, 1H), 5.20(m, 1H),
5.06(m, 1H), 4.61(m, 1H), 4.45(m, 1H), 4.37(m, 1H), 4.01(d, J = 11.6 Hz,
1H), 4.00(s, 3H), 3.95(dd, J = 8.3 Hz, 1H), 2.82(m, 1H), 2.56(m, 1H), 2.26
(m, 1H), 2.02(q, J = 8.6 Hz, 1H), 1.69(m, 1H), 1.60(m, 1H), 1.56(m, 1H),
1.26–1.81(m, 4H), 1.45(m, 1H), 1.31(m, 1H), 1.28(m, 1H), 1.16(d,
J = 7.5 Hz, 6H), 0.99(s, 9H).
stirred for 3 h. The pH was adjusted to ≈5 by addition of 5% HCl. The
mixture was worked up as usual giving an orange solid. The crude solid
was crystallized from n-propanol (3mL) to give 118 mg as light orange solid
after drying under high vacuum in 45% yield. A total of 8.07mCi at
55.8mCi/mmol was obtained. HPLC: tR = 15.51min, radiochemical purity
1
99%. H NMR (500 MHz, DMSO-d6): δ 12.45(s, 1H, COOH), 8.54(s, 1H, NH),
8.12(d, J= 9.12Hz, 1H), 7.71(d, J = 7.51Hz, 1H, NH), 7.51(s, 1H), 7.45(s, 1H)
7.33(d, J = 9.12Hz, 1H), 7.05(d, J = 8.58Hz, 1H, NH), 5.75(m, 1H), 5.43(s, 1H),
5.21(d, J = 16.45 Hz, 1H), 5.06(d, J = 11.76 Hz, 1H), 4.46(t, J = 8.34Hz, 1H),
4.32(d, J = 11.76 Hz, 1H), 4.15(d, J= 8.65 Hz 1H), 4.05(m, 2H), 4.01(S, 3H),
3.82(septet, J= 6.36 Hz, 1H), 3.74 (m, 2H), 2.52(m, 2H), 2.03(q, J = 8.64 Hz,
1H), 1.56(m, 2H), 1.41(m, 2H), 1.25(d, J = 6.36 Hz, 6H), 0.98(brs, 8H), 0.81(t,
J = 7.32Hz, 3H).
Synthesis of [14C]-4
[14C]-N-Carbamothioyl-3,3-dimethyl-butanamide, [14C]-7. 3,3-Dimethylbutanoyl
chloride (158 mg, 1.92 mmol) was added via a syringe to a solution of [14C]-
thiourea (100mCi, 1.92 mmol) in toluene (4.0mL) at room temperature in a
sealed tube. The mixture was heated at 110°C for 16 h before it was cooled
to room temperature. The mixture was worked up as usual to give 200 mg
of [14C]-N-tert-butylacetylthiourea in 60% yield as a yellow solid. TLC (50%
EtOAc : hexanes), Rf = 0.6, same as unlabeled authentic sample.23
Synthesis of deuterium-labeled inhibitors
(R)-1-[(1-(2S-Cyclopentyloxycarbonylamino-3,3-dimethyl-butyryl)-4-{2-[(14C)-
[2-(3,3-dimethyl-butyrylamino)-thiazol-4-yl]]-7-methoxy-quinolin-4R-yloxy}-
pyrrolidine-2S-carbonyl)-amino]-2-vinyl-cyclopropanecarboxylic acid,
Synthesis of [D7]-2
[D7]-N-Carbamothioyl-2-methyl-propanamide, [D7]-6. To a solution of [D7]-
2-methylpropionic acid (1.5 g, 15.78 mmol) in methylene chloride (10 mL)
was added oxalyl chloride (7 mL, 78.64 mmol) drop wise at 0 °C. The
[
of
14C]-4. α-bromoketone 11 (1.1g, 1.45 mmol) was added to a solution resulting colorless solution was warmed to room temperature and stirred
[
14C]-7 (200 mg, 1.13 mmol) in isopropanol (25mL) at room overnight. The solution was then concentrated in vacuo and then diluted
temperature and heated at 70 °C for 1.5h. The mixture was cooled to with methylene chloride (10 mL ×2) and concentrated to remove excess
room temperature and concentrated under reduced pressure to a solid oxalyl chloride. The remaining residue of deuterium-labeled 2-
residue. The residue was worked up as usual giving 1.3g of a solid. methylpropanoyl chloride and thiourea (1.22 g, 15.86 mmol) were heated
The crude was purified by silica gel flash chromatography using 50% in toluene (20 mL) at 110 °C for 12 h. After cooling to room temperature,
ethyl acetate/hexane to give 460 mg of light yellow foam (HPLC the mixture was concentrated in vacuo to a yellow solid residue and
radiochemical purity 99%) and 337mg (HPLC radiochemical purity dissolved in ethyl acetate (20 mL) and worked up as usual. Crystallization
97%) as a yellow solid for a total of 797 mg in 66% yield. The total from (1:1) hexane: ethyl acetate gave 0.8 g of a pale yellow solid in 33%
activity of the pure product was found to be 30.6mCi, with a specific
yield. 1HNMR (400 MHz, CDCl3): δ 10.01(s, 1H), 9.57(s, 1H), 7.72(s, 1H).
activity of 55.55 mCi/mmol. The activity of the 97% pure product was 13CNMR (90 MHz, CDCl3): δ 182.44, 178.02, 39.2(m), 17.63(m). MS (ES+)
found to be 22.5mCi. A solution of LiOH (40mg, 1.74mmol) in H2O
(2.5mL) was added to a solution of the above 99% pure material
(167 mg, 11.1mCi, 0.2mmol) in THF (5mL) and MeOH (2.5mL) at room
temperature. The mixture was stirred at room temperature overnight.
The reaction mixture was concentrated and then diluted with ethyl
acetate (10 mL) and brine (10 mL). The pH was adjusted to ca. 5.0 by
addition of 1N aqueous HCl and worked up as usual giving 164 mg as
a light yellow solid. The total activity of the pure product was found
to be 11.02 mCi, with a specific activity of 55.1 mCi/mmol. HPLC, tR = 16.5
min (99% radiochemical purity). 1H NMR (500 MHz, DMSO-d6): δ 12.47
(s, 1H, OH), 12.32(s, 1H, NH), 8.53(s, 1H, NH), 8.14(d, J = 8.6 Hz, 1H), 8.03
(s, 1H), 7.51(s, 1H), 7.32(s, 1H), 7.02(d, J= 8.6Hz, 1H), 7.00(s, 1H, NH),
5.85(m, 1H), 5.44(s, 1H), 5.22(d, J = 16.5 Hz, 2H), 5.08(d, J= 11.8 Hz, 1H),
4.82(m, 1H), 4.47(t, J = 8.7Hz, 1H), 4.35(d, J = 11.8Hz, 1H), 4.21
(d, J= 8.7Hz, 1H), 3.95(s, 3H), 3.93(m, 1H), 2.65(m, 1H), 2.34(s, 2H), 2.21
(m, 1H), 2.02(m, 1H), 1.21–1.85(m, 9H), 1.04(s, 9H), 0.99(s, 9H).
calculated for C5H3N2OSD7 + H+ 154.25, found 154.20.
(Z)-(1S,4R,6S,14S,18R-14-Cyclopentyloxycarbonylamino)-18-[2-(2-[D7]-
isobutyramidothiazol-4-yl)-7-methoxy-8-methylquinolin-4-yloxy)-2,15-dioxo-
3,16-diaza-tricyclo[14.3.0.04,6]nonadec-7-ene-4-carboxylic acid, [D7]-2. This
material was prepared as described for [14C]-2 from α-bromoketone 9
(0.4g, 0.487 mmol) and [D7]-6 (90mg, 0.59mmol) to give 390 mg of the
pure methyl ester product as a white solid in 96% yield. MS (ES+)
calculated for C43H47N6O9SD7 + H+ 839.05, found 839.27. The ester
(390 mg, 0.465 mmol) was hydrolyzed similarly and Purified by silica
gel flash chromatography using methylene chloride and 10 to 50%
EtOAc:CH2Cl2. The product was dissolved in 2 mL of methylene chloride
and precipitated by addition of hexanes to give 377 mg of a white solid
after drying in vacuo in 98% yield. HPLC, tR = 18.17 min (99% chemical
purity). MS (ES+): calculated for C42H45N6O9SD7 + H+ 825.02, found
825.6 1H NMR (400 MHz, DMSO-d6): δ 12.28 (s, 1H, NH), 12.21(s, 1H,
OH), 8.6(s, 1H, NH), 8.04(d, J = 9.1 Hz, 1H), 8.02(s, 1H), 7.43(s, 1H), 7.28
(s, 1H), 7.21(d, J = 9.1 Hz, 1H), 5.51(m, 2H), 5.45(s, 1H), 5.28
(d, J = 16.5 Hz, 1H), 4.64(m, 1H), 4.53(d, J = 8.3 Hz, 2H), 4.47(t, J = 8.3 Hz,
1H), 4.13(m, 1H), 3.93(s, 3H), 2.61(s, 3H), 2.53(m, 2H), 2.52(m, 2H), 2.2
(q, J = 8.6 Hz, 1H), 1.74(m, 2H), 1.71-1.58(m, 4H), 1.64-1.35(m, 4H), 1.46
(m, 2H), 1.38(m, 2H), 1.36(m, 2H), 1.33(m, 2H).
Synthesis of [14C]-5
(R)-1-{[(2S,4R)-4-[8-Bromo-2-(2-[2-14C]-2-isopropylamino-thiazol-4-yl)-7-methoxy-
quinolin-4-yloxy]-1-((S)-3,3-dimethyl-2-propoxycarbonylamino-butyryl)-pyrrolidine-
2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic acid, ([14C]-5. α-bromoketone
12 (1.43 g, 1.77 mmol) was added to a solution of [14C]-8 (172 mg,
1.46 mmol) in isopropanol (15 mL) at room temperature. The mixture was
heated at 70ºC for 1h. The reaction mixture was cooled to room
temperature and concentrated to a solid residue. This residue was diluted
with 5% Na2CO3 (10 mL), extracted with CH2Cl2 (10 mL ×3). The combined
extracts were dried over Na2SO4 and concentrated. The crude residue
was purified twice using CombiFlash Companion (40g, 40–60% EtOAc/
Synthesis of [D7]-BI 201335, [D7]-3
(1R,2S)-1-{[(2S,4R)-4-[8-Bromo-2-(2-[D7]-(isobutyrylamino-thiazol-4-yl)-7-
methoxy-quinolin-4-yloxy]-1-((S)-2-cyclopentyloxycarbonylamino-3,3-dimethyl-
butyryl)-pyrrolidine-2-carbonyl]-amino}-2-vinyl-cyclopropanecarboxylic
acid, [D7]-3. This material was prepared as previously described for [14C]-3
hexanes) to give 790 mg (48.1mCi) as light yellow solid in 65% yield. HPLC: from [D7]-6 (0.16 g, 1.04 mmol) and α-bromoketone 10 (0.41g, 0.46 mmol)
tR = 12.94 min (99% radiochemical purity), co-eluted with unlabeled to give after purification by flash chromatography using a 40 g RediSep
authentic material. A solution of LiOH (5.0mmol) in water (5.0mL) was disposable column and using CH2Cl2 then 10 to 50% EtOAc: CH2Cl2 as
added to a solution of the aforementioned material (263 mg, 0.32 mmol) eluent, 382 mg of methyl ester derivative as yellowish solid (98% chemical
in THF (10mL) and MeOH (5.0mL) at room temperature. The mixture was purity) in 94% yield. This ester (0.32 g, 0.36 mmol) was hydrolyzed as in
J. Label Compd. Radiopharm 2014, 57 350–357
Copyright © 2014 John Wiley & Sons, Ltd.