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Example 8
Characterization of Absorption, Distribution, Metabolism, and Excretion of Oral [14C]Vorasidenib with Concomitant Intravenous Microdose Administration of [13C315N3]Vorasidenib in Humans
Metabolite profiling and identification of vorasidenib (AG-881) was performed in plasma, urine, and fecal samples collected from five healthy subjects after a single 50-mg (100 μCi) oral dose of [14C]AG-881 and concomitant intravenous microdose of [13C3 15N3]AG-881.
Plasma samples collected at selected time points from 0 through 336 hour postdose were pooled across subjects to generate 0—to 72 and 96-336-hour area under the concentration-time curve (AUC)-representative samples. Urine and feces samples were pooled by subject to generate individual urine and fecal pools. Plasma, urine, and feces samples were extracted, as appropriate, the extracts were profiled using high performance liquid chromatography (HPLC), and metabolites were identified by liquid chromatography-mass spectrometry (LC-MS and/or LC-MS/MS) analysis and by comparison of retention time with reference standards, when available.
Due to low radioactivity in samples, plasma metabolite profiling was performed by using accelerator mass spectrometry (AMS). In plasma, AG-881 was accounted for 66.24 and 29.47% of the total radioactivity in the pooled AUC0-72 h and AUC96-336 h plasma, respectively. The most abundant radioactive peak (P7; M458) represented 0.10 and 43.92% of total radioactivity for pooled AUC0-72 and AUC96-336 h plasma, respectively. All other radioactive peaks accounted for less than 6% of the total plasma radioactivity and were not identified.
The majority of the radioactivity recovered in feces was associated with unchanged AG-881 (55.5% of the dose), while no AG-881 was detected in urine. In comparison, metabolites in excreta accounted for approximately 18% of dose in feces and for approximately 4% of dose in urine. M515, M460-1, M499, M516/M460-2, and M472/M476 were the most abundant metabolites in feces, and each accounted for approximately 2 to 5% of the radioactive dose, while M266 was the most abundant metabolite identified in urine and accounted for a mean of 2.54% of the dose. The remaining radioactive components in urine and feces each accounted for <1% of the dose.
Overall, the data presented indicate [14C]AG-881 underwent moderate metabolism after a single oral dose of 50-mg (100 μCi) and was eliminated in humans via a combination of metabolism and excretion of unchanged parent. AG-881 metabolism involved the oxidation and conjugation with glutathione (GSH) by displacement of the chlorine at the chloropyridine moiety. Subsequent biotransformation of GSH intermediates resulted in elimination of both glutamic acid and glycine to form the cysteinyl conjugates (M515 and M499). The cysteinyl conjugates were further converted by a series of biotransformation reactions such as oxidation, S-dealkylation, S-methylation, S-oxidation, S-acetylation and N-dealkylation resulting in the formation multiple metabolites.
A summary of the metabolites observed is included in Table 2
Example 2
N-(2-chloro-4-(trifluoromethyl)phenyl)-2-(5-ethyl-2-morpholino-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Intermediate B) (200 mg, 352 μmol) was suspended in DMF (5 mL). Perfluorophenyl 3-hydroxypicolinate (Intermediate CT) (215 mg, 703 μmol) and Et3N (97.0 μL, 703 μmol) were added and the RM was stirred at 70° C. for 3 hours. The RM was concentrated under reduced pressure. The crude product was first purified by column chromatography (Silica gel column: Silica 12 g, eluent DCM:MeOH 100:0 to 90:10). Then a second purification by reverse phase preparative HPLC (RP-HPLC acidic 9: 40 to 50% B in 2 min, 50 to 55% B in 10 min) afforded the title compound.
LC-MS: Rt=0.98 min; MS m/z [M+H]+ 690.6/692.6, m/z [M−H]− 688.4/690.3; UPLC-MS 1
LC-MS: Rt=4.84 min; MS m/z [M+H]+ 690.2/692.2 m/z [M−H]− 688.3/690.3; UPLC-MS 2
1H NMR (400 MHz, DMSO-d6) δ 10.37 (s, br, 1H), 10.34 (s, br, 1H), 8.05 (m, 2H), 7.96 (d, J=2.1 Hz, 1H), 7.72 (dd, J=2.1 Hz, 8.7 Hz, 1H), 7.28 (m, 2H), 5.21 (s, 2H), 4.53 (m, 1H), 3.66 (m, 4H), 3.46 (m, 3H), 3.38 (m, 4H), 3.20 (m, 1H), 2.92 (m, 3H), 2.76 (m, 1H), 2.58 (m, 1H), 1.16 (t, J=7.5 Hz, 3H)
Example 24
To the stirred solution of N-(2-chloro-6-(trifluoromethyl)pyridin-3-yl)-2-(5-ethyl-2-(4-methoxycyclohex-1-en-1-yl)-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Intermediate Y) (300 mg, 504 μmol), 4-chloro-3-hydroxypicolinic acid (140 mg, 807 μmol), HOBt (136 mg, 1.01 mmol) and EDC.HCl (193 mg, 1.01 mmol) in DCM (20 mL) was added pyridine (122 μL, 1.51 mmol) at 0° C. The RM was stirred at RT for 16 hours. The RM was quenched with NaHCO3 and extracted with DCM. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by column chromatography (Silica gel column: Silica 4 g, eluent DCM:MeOH 100:0 to 98:2). The residue was purified by preparative chiral HPLC (instrument: Agilent 1200 series, with single quad mass spectrometer; column: LUX CELLULOSE-4, 250 mm×21.1 mm, 5.0 μm; eluent: A=hexane, B=0.1% HCOOH in EtOH; flow rate: 15 mL/min; detection: 210 nm; injection volume: 0.9 mL; gradient: isocratic: 50(A):50(B)).
Example 24a: The product containing fractions were concentrated at 40° C. and washed with n-pentane (5×10 mL), decanted and dried to give the title compound as an off-white solid—first eluting stereoisomer.
Chiral HPLC (C-HPLC 2): Rt=10.764 min
LC-MS: Rt=1.08 min; MS m/z [M+H]+ 750.5/752.5, m/z [M−H]− 748.4/750.4; UPLC-MS 1
LC-MS: Rt=5.29 min; MS m/z [M+H]+ 750.2/752.2, m/z [M−H]− 748.2/750.2; UPLC-MS 2
1H NMR (400 MHz, DMSO-d6) δ 10.68 (s, br, 2H), 8.56 (d, J=8.1 Hz, 1H), 7.98 (d, J=5.6 Hz, 1H), 7.94 (d, J=8.1 Hz, 1H), 7.50 (d, J=5.1 Hz, 1H), 6.72 (m, 1H), 5.34 (s, 2H), 4.53 (m, 1H), 3.52 (m, 4H), 3.28 (m, 4H), 2.98 (m, 3H), 2.80 (m, 1H), 2.63 (m, 1H), 2.55 (m, 1H), 2.46 (m, 1H), 2.16 (m, 2H), 1.95 (m, 1H), 1.68 (m, 1H), 1.17 (t, J=7.3 Hz, 3H)
Example 24b: The product containing fractions were concentrated at 40° C. and washed with n-pentane (5×10 mL), decanted and dried to give the title compound as an off-white solid—second eluting stereoisomer.
Chiral HPLC (C-HPLC 2): Rt=18.800 min
LC-MS: Rt=1.08 min; MS m/z [M+H]+ 750.1/752.1, m/z [M−H]− 748.2/750.2; UPLC-MS 1
LC-MS: Rt=5.30 min; MS m/z [M+H]+ 750.1/752.1, m/z [M−H]− 748.2/750.2; UPLC-MS 2
1H NMR (400 MHz, DMSO-d6) δ 10.83 (s, br, 1H), 10.55 (s, br, 1H), 8.56 (d, J=8.2 Hz, 1H), 8.06 (d, J=5.3 Hz, 1H), 7.92 (d, J=8.2 Hz, 1H), 7.55 (d, J=5.3 Hz, 1H), 6.72 (m, 1H), 5.35 (s, 2H), 4.54 (m, 1H), 3.54 (m, 4H), 3.28 (m, 3H), 3.25 (m, 1H), 2.99 (m, 3H), 2.81 (m, 1H), 2.62 (m, 1H), 2.41 (m, 2H), 2.16 (m, 2H), 1.96 (m, 1H), 1.66 (m, 1H), 1.18 (t, J=7.3 Hz, 3H)
Example 25
N-(2-chloro-6-(trifluoromethyl)pyridin-3-yl)-2-(5-ethyl-2-(4-methoxycyclohex-1-en-1-yl)-7-oxo-6-(piperazin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide.HCl (Intermediate Y) (120 mg, 190 μmol) and DIPEA (166 μL, 950 μmol) were dissolved in DCM (5 mL) and then 3-hydroxypicolinoyl chloride (Intermediate CV) (59.9 mg, 380 μmol) was added at 0° C. and stirred for 2 hours. 3-hydroxypicolinoyl chloride (Intermediate CV) (59.9 mg, 380 μmol) was added again and the reaction was continued under stirring for 12 hours. The RM was diluted with DCM and washed with water and aq NaHCO3 (2×20 mL), washed with water and brine, dried over Na2SO4, filtered and concentrated. The crude product was combined with another experiment and purified by column chromatography (Silica gel column: Silica 4 g, eluent DCM:MeOH 100:0 to 99:1) then further purified by reverse phase preparative HPLC (RP-HPLC acidic 10: 40 to 50% B in 2 min, 50 to 60% B in 8 min) to give the title compound as an off-white solid.
The racemate was purified by preparative chiral HPLC (instrument: Agilent 1200 series, with single quad mass spectrometer; column: CELLULOSE-4, 250 mm×21.2 mm; eluent: A=hexane, B=0.1% HCOOH in MeOH:EtOH 1:1; flow rate: 20 mL/min; detection: 210 nm; injection volume: 0.9 mL; gradient: isocratic 60(A):40(B)).
Example 25a: First eluting stereoisomer, off-white solid.
Chiral HPLC (C-HPLC 1): Rt=10.070 min
LC-MS: Rt=0.98 min; MS m/z [M+H]+ 716.5/718.6, m/z [M−H]− 714.3/716.3; UPLC-MS 1
LC-MS: Rt=4.76 min; MS m/z [M+H]+ 716.2/718.2, m/z [M−H]− 714.2/716.2; UPLC-MS 2
1H NMR (400 MHz, DMSO-d6) δ 10.46 (s, br, 2H), 8.56 (d, J=8.5 Hz, 1H), 8.05 (m, 1H), 7.90 (d, J=8.4 Hz, 1H), 7.28 (m, 2H), 6.72 (m, 1H), 5.30 (s, 2H), 4.54 (m, 1H), 3.47 (m, 4H), 3.27 (s, 3H), 3.21 (m, 1H), 2.96 (m, 3H), 2.79 (m, 1H), 2.59 (m, 3H), 2.43 (m, 1H), 2.14 (m, 1H), 1.95 (m, 1H), 1.67 (m, 1H), 1.17 (t, J=7.2 Hz, 3H)
Example 25b: Second eluting stereoisomer, off-white solid.
Chiral HPLC (C-HPLC 1): Rt=16.023 min
LC-MS: Rt=0.96 min; MS m/z [M+H]+ 716.3/718.3, m/z [M−H]− 714.3/716.3; UPLC-MS 1
LC-MS: Rt=4.77 min; MS m/z [M+H]+ 716.2/718.2, m/z [M−H]− 714.2/716.2; UPLC-MS 2
1H NMR (400 MHz, DMSO-d6) δ 10.39 (s, br, 2H), 8.56 (d, J=8.0 Hz, 1H), 8.06 (m, 1H), 7.93 (d, J=8.1 Hz, 1H), 7.28 (m, 2H), 6.72 (m, 1H), 5.32 (s, 2H), 4.54 (m, 1H), 3.46 (m, 4H), 3.27 (s, 3H), 3.20 (m, 1H), 2.96 (m, 3H), 2.79 (m, 1H), 2.59 (m, 3H), 2.41 (m, 1H), 2.14 (m, 1H), 1.95 (m, 1H), 1.68 (m, 1H), 1.17 (t, J=7.1 Hz, 3H)
Example 18
To the stirred solution of 3-hydroxypicolinic acid (166 mg, 1.19 mmol), EDC.HCl (228 mg, 1.19 mmol), HOBt (161 mg, 1.19 mmol) in DMF (3 mL) was added N-(2-chloro-6-(trifluoromethyl)pyridin-3-yl)-2-(5-ethyl-7-oxo-6-(piperazin-1-yl)-2-(pyrrolidin-1-yl)-[1,2,4]triazolo[1,5-a]pyrimidin-4(7H)-yl)acetamide (Intermediate Q) (330 mg, 596 μmol) and DIPEA (624 μL, 3.57 mmol) and the RM was at RT for 16 hours. The RM was concentrated under reduced pressure and water was added. The resultant brown solid was filtered off and dried under vacuum. The crude product was purified by reverse phase preparative HPLC (RP-HPLC acidic 4: 35 to 40% B in 2 min, 40 to 45% B in 10 min) to give the title compound.
LC-MS: Rt=0.94 min; MS m/z [M+H]+ 675.3/677.3, m/z [M−H]− 673.3/675.3; UPLC-MS 1
LC-MS: Rt=4.68 min; MS m/z [M+H]+ 675.2/677.2, m/z [M−H]− 673.2/675.2; UPLC-MS 2
1H NMR (400 MHz, DMSO-d6) δ 10.54 (s, br, 1H), 10.38 (s, br, 1H), 8.55 (d, J=8.4 Hz, 1H), 8.06 (m, 1H), 7.95 (d, J=8.4 Hz, 1H), 7.28 (m, 2H), 5.25 (s, 2H), 4.53 (m, 1H), 3.46 (m, 3H), 3.35 (m, 4H), 3.19 (m, 1H), 2.91 (m, 3H), 2.75 (m, 1H), 2.57 (m, 1H), 1.89 (m, 4H), 1.15 (t, 3H)
Example 8
The SFME sample processing can be done in fused silica tubing of smaller diameter which are commonly used as liquid line in liquid chromatography system (e.g., tubing having an inner diameter of 500 μm or less). The extraction can be induced by applying a push and pull force on one side of the tubing. The extract can be either directly analyzed by nanoESI or stored for further operations.
Example 10
The objective of this study was to develop an acute model of homocystinuria in nonhuman primates. Male cynomolgus monkeys of approximately 2-5 years of age (average weight of 3.4 kg) were fasted overnight and orally administered a methionine load at 100 or 300 mg/kg, and plasma was collected at 0-, 0.5-, 1-, 2-, 4-, 6-, and 24-hours post-dose for methionine and total homocysteine measurements by LC-MS/MS.
Oral administration of methionine (100 or 300 mg/kg) resulted in a dose-dependent increase in plasma methionine levels, with peak concentration recorded at 30 minutes and 1 hour post dose for 100 mg/kg and 300 mg/kg, respectively (
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