Syntheses
Synthesis of [ 14 C]-SMX (5a), [14C]-SMM (7a)
Uniformly [phenyl-ring- l4 C]-labelled N-acetylaniline ( 2a )
To 14C-labelled aniline hydrochloride (1a, 3.70 × 108 Bq, 1.11 × 109 Bq/mmol, 99.0% purity) in deionized water (10 mL) were K2CO3 powder (360 mg) and acetic anhydride (187 µL) sequentially added with stirring at 25°C (Fig. 1). The mixture was stirred at 25°C for 1 h and then extracted five times with ethyl acetate (10 mL each). The extract was dried with anhydrous Na2SO4 and evaporated under vacuum to ~1 mL. The product in the extract was purified by flash chromatography (For details, see SI.1) with an elution gradient (SI, Table S1), giving 2a (3.33 × 108 Bq, 1.11 × 109 Bq/mmol) in 90.0% yield. TLC analysis using petroleum ether: ethyl acetate (1:4 / v: v), containing 0.2% CH3COOH as eluent (Rf value of 2a = 0.45) coupled to autoradiography (SI.2) showed a radiochemical purity of 99.0%.
Uniformly [phenyl-ring- l4 C]-labelled N-acetylsulfanilyl chloride ( 3a )
To 2a (2.59 × 108 Bq, 2.96 × 109 Bq/mmol, 99.0% purity) in CCl4 (1 mL) was ClSO3H (45 µL) added dropwise with stirring in an ice bath. The mixture was then stirred at 58°C for 2 h, NaCl (4 mg) was added (Fig. 1, Method I). The reaction mixture was stirred for another 2 h and then cooled down to the room temperature. The residual chlorosulfonic acid in the mixture was hydrolyzed with ice-cold water (10 mL), and the mixture was extracted twice with ethyl acetate (35 mL each). The extracts were dried with anhydrous Na2SO4 and evaporated to ~1 mL. The product in the extract was purified by flash chromatography (SI.1) with an elution gradient (SI, Table S1), giving 3a (1.39 × 108 Bq, 2.96 × 109 Bq/mmol) in 53.9% yield. TLC analysis using petroleum ether: ethyl acetate (1:4 / v: v), containing 0.2% CH3COOH as eluent (Rf value of 3a = 0.35) coupled to autoradiography (SI.2) showed a radiochemical purity of 96.0%.
Uniformly [phenyl-ring- l4 C]-labelled N-acetylsulfamethoxazole ( 4a ), Uniformly [phenyl-ring-l4C]-labelled N-acetylsulfamonomethoxine (6a)
Synthesis of 4a: To 3a (4.07 × 105 Bq, 7.40× 106 Bq/mmol) in 200 uL acetone were 3-amino-5-methylisoxazole (11, 13 mg), anhydrous pyridine (11 µL), and 5 pieces of molecular sieves (4 Å with a diameter of 1 mm) added with stirring in an ice bath, and then stirred for 5 h at 60°C. The resulting mixture were then diluted with methanol (200 µL) and separated by preparative TLC using petroleum ether: ethyl acetate (1:4 / v: v) containing 0.2% CH3COOH as eluent. The product band of 4a (Rf = 0.53) was scraped from the TLC plate and extracted six times with ethyl acetate (15 mL each). After concentration by evaporation, 4a was obtained (2.07 × 105 Bq, 7.40× 106 Bq/mmol, 51.0% yield) with 95.0% purity as determined by TLC coupled to autoradiography (SI.2).
Synthesis of 6a: To 3a (3.7 × 107 Bq, 7.55 × 108 Bq/mmol) in 200 uL acetone were 4-amino-6-methoxypyrimidine (12, 16.1 mg), anhydrous pyridine (10 µL) and 5 pieces of molecular sieves (4 Å with a diameter of 1 mm) sequentially added with stirring in an ice bath, and then stirred for 23 h at 60°C. The subsequent purification of 6a (Rf = 0.18) was the same as for 4a. 6a (5.77 × 106 Bq, 7.55 × 108 Bq/mmol, 15.6% yield) was obtained with 95.0% purity as determined by TLC coupled to autoradiography (SI.2).
Uniformly [phenyl-ring- l4 C]-SMX ( 5a ), Uniformly [phenyl-ring- l4 C]-SMM ( 7a )
4a (2.04 × 105 Bq, 7.40 × 106 Bq/mmol), 6a (4.81 × 106 Bq, 7.55 × 108 Bq/mmol) were heated in NaOH solution (10%, 1 mL) for 3 h at 100°C and neutralized with 6 M HCl to pH 6. The products were extracted with ethyl acetate (15 mL each) eight times. The extracts were dried with anhydrous Na2SO4, evaporated to around 0.5 mL, and purified by preparative TLC using petroleum ether: ethyl acetate (1:4 / v: v) containing 0.4% CH3COOH as eluent. The product bands of 5a and 7a (Rf = 0.6 and 0.51, respectively) were scraped from the plates and extracted with ethyl acetate (15 mL each) six times. The extracts were evaporated to around 0.1 mL, giving 5a (1.85 × 105 Bq) and 7a (3.18 × 106 Bq) in 90.9% and 66.2% yield, respectively, with purities of 98.1% and 98.3%, respectively, as determined by HPLC (SI.4). Chemical structure of 5a and 7a were characterized by 1H-NMR, 13C-NMR (SI.5) and LC-Q-TOF-MS/MS (SI.6) using the corresponding unlabelled compounds synthesized with the same procedures.
Synthesis of [14C]-SDZ (10a)
Uniformly [phenyl-ring- l4 C]-labelled N-acetylsulfanilyl chloride ( 3a )
To 2a (2.48 × 108 Bq, 1.11 × 109 Bq/mmol, 99.0% purity) in CCl4 (0.5 mL) was ClSO3H (170 µL) added dropwise with stirring in an ice bath. The mixture was stirred at 58°C for 2 h, followed by addition of SOCl2 (25 µL) and another 2 h of heating at 58°C (Fig. 1, Method II). After the reaction, the mixture was cooled down to room temperature and extracted twice with ethyl acetate (35 mL each). The extracts were dried with anhydrous Na2SO4 and evaporated to ~1 mL. The purity of 3a (1.98 × 108 Bq) in the mixture was 93.0% as determined by TLC using petroleum ether: ethyl acetate (1:4 / v: v), containing 0.2% CH3COOH as eluent (Rf value of 3a = 0.35) coupled to autoradiography. The mixture without purification was directly used for subsequent synthesis of 8a. The yield of 3a according to its purity in the mixture was 74.3%.
Uniformly [phenyl-ring- l4 C]-labelled N-acetylsulfonamide ( 8a )
The mixture containing crude 3a (1.98 × 108 Bq, 6.29 × 108 Bq/mmol, 93.0% purity) was mixed with acetone (1 mL), after which ammonium hydroxide (0.5 mL, 28% NH3 in water) was added dropwise at 0°C. The mixture was vigorously stirred at room temperature for 1 h and the pH was adjusted to 6 with 6 M HCl. It was then extracted eight times with ethyl acetate (15 mL each), dried with anhydrous Na2SO4. The extract was evaporated and the product was purified by flash chromatography with an elution gradient (Table S1), resulting in 8a (1.81 × 108 Bq) in 98.3% yield with a purity of 99.0% as analyzed by TLC using petroleum ether: ethyl acetate (1:4 / v: v), containing 0.2% CH3COOH as eluent (Rf value of 8a = 0.26) coupled to autoradiography.
Uniformly [phenyl-ring- l4 C]-labelled N-acetylsulfadiazine ( 9a )
To 8a (1.74 × 108 Bq, 6.29 × 108 Bq/mmol, 99.0% radiochemical purity) in N, N-dimethylacetamide (800 µL) were 2-chloropyrimidine (13, 48.7 mg) and K2CO3 (58.6 mg) added sequentially with stirring at room temperature. The mixture was heated at 150°C for 4.5 h, and the solvent N, N-dimethylacetamide was then removed by evaporation. The crude product was dissolved in water and cooled in an ice bath. The mixture was adjusted to pH 6 with 6 M HCl and the precipitates were washed with ice-cold water, resulting in 9a (1.10 × 108 Bq) with 57% purity as analyzed by TLC (SI.2) coupled to autoradiography (Rf = 0.13) using petroleum ether: ethyl acetate (1:4 / v: v), containing 0.2% CH3COOH as eluent. The yield of 9a according to its purity was 36.0%.
Uniformly [phenyl-ring- l4 C]-labelled SDZ ( 10a )
The crude 9a (9.25 × 107 Bq, 6.29 × 108 Bq/mmol, 57.0% radiochemical purity) was reacted with NaOH solution (10%, 5 mL) for 3 h at 100°C and neutralized with 6 M HCl to pH 6. The products were extracted with ethyl acetate (15 mL each) eight times. The extracts were dried with anhydrous Na2SO4, evaporated to around 0.5 mL. The crude product was then recrystallized from boiling methanol. The precipitates were centrifuged and washed three times with methanol, resulting in 10a (3.11 × 107 Bq, 6.29 × 108 Bq/mmol). The purity of 10a was 98.3% as determined by HPLC (tR = 5.73 min. For details, see SI). The supernatant was further extracted five times with ethyl acetate (15 mL each), which was dried with anhydrous Na2SO4 and evaporated to dryness, giving solids containing 10a. The solid product was mixed with unlabelled SDZ (54 mg) and then recrystallized from boiling methanol. The precipitate was washed three times with methanol, resulting in another portion of 10a with a low specific activity (1.10 × 107 Bq, 7.40 × 107 Bq/mmol) with a radiochemical purity of 98.3%. The total amount of 10a was 4.21 × 107 Bq with a total yield of 79.9%. The chemical structure of 10a were characterized by 1H-NMR, 13C-NMR (SI.5) and LC-Q-TOF-MS/MS (SI.6) using the corresponding unlabelled compounds synthesized with the same procedures.
Synthesis of [ 13 C]-SMX (5b), [13C]-SMM (7b), and [13C]-SDZ (10b)
Uniformly [phenyl-ring- l3 C]-labelled N-acetylaniline ( 2b )
To 13C-labelled aniline hydrochloride (1b, 3.00 g, 99% of 13C atom) in a 200-mL flask were K2CO3 solution (0.32 g/mL, 30 mL) and acetic anhydride (4.70 g) added sequentially with stirring at 25°C. The mixture was further stirred at 25°C for 1 h and then extracted five times with ethyl acetate (15 mL each). The extract was washed with 20 mL of H2O and then evaporated, resulting in 2b (3.01 g, 99% of 13C atom, 99.0% purity (For detail, see SI.4) in 95.7% yield.
Uniformly [phenyl-ring- l3 C]-labelled N-acetylsulfanilyl chloride ( 3b )
To [13C]-2 (3.00 g, 99% of 13C atom) in CCl4 (5 mL) was ClSO3H (19.8 g) added dropwise with stirring in an ice bath. The mixture was stirred at 58°C for 2 h and SOCl2 (2.67 g) was then added (Fig. 1, Method II). The mixture was heated at 58°C for another 2 h and cooled down to room temperature. White crystals were formed after dropwise addition of ice-cold water (10 mL) to the mixture and were washed twice with ice-cold water (each 10 mL) by filtration, resulting in 3b (4.29 g, 99% of 13C atom, 96.0% purity (SI.4)) in 82.8% yield.
Uniformly [phenyl-ring- l3 C]-labelled N-acetylsulfamethoxazole ( 4b ), N-acetylsulfamonomethoxine ( 6b ), and N-acetylsulfadiazine ( 9b )
Synthesis of 4b: To 3b (500 mg, 99% of 13C atom) in acetone (2 mL) were 3-amino-5-methylisoxazole (11, 412 mg), anhydrous pyridine (339 µL) and 10 pieces of molecular sieves (diameter 1 mm, 4 Å) were added sequentially with stirring in an ice bath. The mixture was then stirred for 7 h at 60°C. The molecular sieves were removed and acetone was evaporated. The crude product 4b (470 mg, 99% of 13C atom, 95.0% purity (SI.4)) was obtained in 73.8% yield after crystallization in ice-cold water.
Synthesis of 6b: To 3b (500 mg, 99% of 13C atom) in acetone (2 mL) were 4-amino-6-methoxypyrimidine (12, 526 mg), anhydrous pyridine (339 µL, 4.2 mmol), and 10 pieces of molecular sieves (diameter 1 mm, 4 Å) added with stirring in an ice bath. The reaction conditions and workup were similar to the synthesis of 4b as describe above. The crude product 6b (286 mg, 99% of 13C atom, 93.0% purity (SI.4)) was obtained in 42.3%.
Synthesis of 8b: To 3b (1.0 g, 99% of 13C atom) in acetone (10 mL) were ammonium hydroxide (5 mL, 28% NH3 in water) added with stirring in an ice bath. The mixture was vigorously stirred at 25°C for 1 h. Then acetone was removed by evaporation. After addition of ice-cold water and adjusting with 6 M HCl to pH about 6, 8b (672 mg, 99% of 13C atom, 98.0% purity (SI.4)) was obtained in 73.0% by filtration and washing with ice-cold water.
Synthesis of 9b: To 8b (450 mg, 99% of 13C atom) in N, N-dimethylacetamide (3.5 mL) were 2-chloropyrimidine (13, 361 mg), K2CO3 (439 mg) added with stirring in room temperature. Then the mixture was stirred for 5 h at 150°C. The subsequent procedures were similar to the synthesis of 9a as describe above, to obtain 9b (494 mg, 99% of 13C atom, 93.0% purity (SI.4)) in 74.8% yield.
Uniformly [phenyl-ring- l3 C]-labelled SMX ( 5b ), SMM ( 7b ), and SDZ ( 10b )
4b (300 mg, 99% of 13C atom), 6b (280 mg, 99% of 13C atom), and 9b (350 mg, 99% of 13C atom) were individually hydrolyzed in NaOH solution (10%, 3 mL) for 3 h at 100°C. The reaction mixtures were neutralized to pH 6 with 6 M HCl and cooled down in an ice bath. The precipitates were washed with ice-cold water six times (1 mL each) and dissolved in boiling methanol (SAs: methanol = 1: 1 / w: v). The methanol solutions were cooled in an ice bath to recrystallize the products, which were then separated by centrifugation (10 min, 2810 g) and washed twice with ice-cold methanol, giving 5b (238 mg, 99.0% purity (SI.4)), 7b (204 mg, 98.0% purity (SI.4)), and 10b (276 mg, 98.0% purity (SI.4)) in 92.5%, 83.7%, and 91.7% yield, respectively.