* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Stage #1: With sodium hydroxide; water In tetrahydrofuran; methanol at 70℃; for 1 h; Stage #2: With hydrogenchloride In water
Methyl iodide (0.972 mL) was added to a mixture of 3-mercapto-benzoic acid (601 mg, 3.9 mmol) and potassium carbonate (2.7 g, 19.5 mmol) in DMF (8 mL) in an ice-bath. After the reaction was warmed to room temperature and stirred for 1 hour, the reaction mixture was diluted with ethyl acetate, washed with water (3X), dried over anhydrous sodium sulfate, filtered, and concentrated to afford 3-methylsulfanyl-benzoic acid methyl ester (684 mg, 96percent, yellow [OIL). 1H] NMR [(CDC13),] [S] (ppm): 7.90 (s, 1H), 7.80 (d, [1H),] 7.44 (d, 1H), 7.35 (t, 1H), 3.92 (s, 3H), 2.53 (s, 3H). 3-Methylsulfanyl-benzoic acid methyl ester (684mg, 3.8 mmol) and 1N [NAOH] (5.6 mL, 5.6 mmol) in methanol (8 [ML)] and THF (8 mL) were heated at [70°C] for 1 hour. The reaction mixture was concentrated and then the residue was diluted with water. After acidification with 1N [HC1] to [PH-2,] the aqueous layer was extracted with ethyl acetate and then washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford 3-methylsulfanyl-benzoic acid (616 mg, 97percent, white [SOLID). 1H] NMR (DMSO), [8] (ppm): 13.1 (bs, [1H),] 7.76 (s, 1H), 7.70 (d, 1H), 7.51 (d, 1H), 7.44 (t, 1H), 2.52 (s, 3H).
97%
Stage #1: With sodium hydroxide In tetrahydrofuran; methanol at 70℃; for 1 h; Stage #2: With hydrogenchloride In water
3-Methylsulfanyl-benzoic acid methyl ester (684 mg, 3.8 mmol) and [1N NAOH] (5.6 mL, 5.6 mmol) in methanol (8 [ML)] and THF (8 mL) were heated at [70°C] for 1 hour. The reaction mixture was concentrated and then the residue was diluted with water. After acidification with 1N [HC1] to [PH-2,] the aqueous layer was extracted with ethyl acetate and then washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford [3-METHYLSULFANYL-BENZOIC] acid (616 mg, 97percent, white [SOLID). LHNMR (DMSO), 6] (ppm): 13. 1 (bs, [1H),] 7.76 (s, 1H), 7.70 (d, 1H), 7.51 (d, 1H), 7.44 (t, 1H), 2.52 (s, 3H).
Stage #1: With hydrogenchloride; sodium nitrite In water Stage #2: With potassium ethyl xanthogenate; sodium carbonate In water at 70℃; Stage #3: With potassium hydroxide In water for 5 h; Heating / reflux
The aminobenzoic acid (54.8 g, 0.4 mol) was diazotized in the usual manner with sodium nitrite (27.6 g, 0.4 mol) and hydrochloric acid (40 mL) and the resulting diazonium salt solution poured into a hot (70° C.), freshly prepared solution of potassium ethyl xanthate (64.2 g, 0.4 mol) containing sodium carbonate (55.2 g, 0.4 mol) to neutralize acid in the diazonium salt solution. After the reaction was over, as indicated by the cessation of the evolution of gases, the mixture was cooled. It was then treated with potassium hydroxide (24.7 g, 0.44 mol) and dimethyl sulfate (50.4 g, 0.4 mol). The mixture was refluxed for 5 hours. On acidification with hydrochloric acid, the desired product was obtained (38.2 g, 57percent). MS: 167.8 (M+1+).
Reference：
[1] Patent: US2003/220335, 2003, A1, . Location in patent: Page/Page column 3; 5
[2] Journal of the Chemical Society, 1963, p. 1947 - 1954
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[1] Journal of Chemical Research, Miniprint, 1988, # 1, p. 201 - 223
[2] Journal of Organic Chemistry, 1996, vol. 61, # 4, p. 1310 - 1314
[3] Journal of Chemical Research, Miniprint, 1999, # 8, p. 2052 - 2074
[4] International Journal of Chemical Kinetics, 2000, vol. 32, # 10, p. 615 - 622
[5] Journal of Physical Organic Chemistry, 2001, vol. 14, # 9, p. 650 - 656
[6] Indian Journal of Chemistry - Section B Organic and Medicinal Chemistry, 2002, vol. 41, # 4, p. 832 - 838
[7] Journal of Organic Chemistry, 2000, vol. 65, # 11, p. 3322 - 3325
[8] Journal of the Indian Chemical Society, 2007, vol. 84, # 6, p. 582 - 587
[9] Journal of the Indian Chemical Society, 2008, vol. 85, # 12, p. 1281 - 1288
[10] Journal of the Indian Chemical Society, 2009, vol. 86, # 9, p. 927 - 935
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[12] Journal of the Indian Chemical Society, 2012, vol. 89, # 8, p. 1045 - 1052
5
[ 140-89-6 ]
[ 99-05-8 ]
[ 825-99-0 ]
Reference：
[1] Journal of the American Chemical Society, 1940, vol. 62, p. 220
6
[ 4025-64-3 ]
[ 825-99-0 ]
Reference：
[1] Patent: US2453232, 1943, ,
[2] Journal of the Chemical Society, 1921, vol. 119, p. 1796
7
[ 1227-49-2 ]
[ 77-78-1 ]
[ 825-99-0 ]
Reference：
[1] Chemische Berichte, 1937, vol. 70, p. 296,307
[2] Journal of the Chemical Society, 1921, vol. 119, p. 1796
8
[ 65052-48-4 ]
[ 825-99-0 ]
Reference：
[1] Chemische Berichte, 1913, vol. 46, p. 783
9
[ 4869-59-4 ]
[ 825-99-0 ]
Reference：
[1] Journal of the Chemical Society, 1921, vol. 119, p. 1796
10
[ 65-85-0 ]
[ 825-99-0 ]
Reference：
[1] Journal of the Chemical Society, 1921, vol. 119, p. 1796
11
[ 1783-81-9 ]
[ 825-99-0 ]
Reference：
[1] Chemische Berichte, 1913, vol. 46, p. 783
12
[ 4869-59-4 ]
[ 77-78-1 ]
[ 825-99-0 ]
Reference：
[1] Patent: US2453232, 1943, ,
13
[ 1227-49-2 ]
[ 77-78-1 ]
[ 825-99-0 ]
Reference：
[1] Journal of the Chemical Society, 1921, vol. 119, p. 1796
14
[ 7647-01-0 ]
[ 15436-23-4 ]
[ 90345-62-3 ]
[ 70-55-3 ]
[ 825-99-0 ]
Reference：
[1] Journal of the Chemical Society, 1927, p. 193
15
[ 825-99-0 ]
[ 5345-27-7 ]
Reference：
[1] Journal of the American Chemical Society, 1952, vol. 74, p. 1943,1944
[2] Patent: US2453232, 1943, ,
[3] Journal of the Chemical Society, 1921, vol. 119, p. 1796
To a suspension of <strong>[825-99-0]3-methylsulfanyl-benzoic acid</strong> (38 g, 0.226 mol) in Toluene (500 mL) was added 200 mL of Red-AID dropwise at 50 C. over 30 minutes. After stirring at room temperature for 3 hours, NaOH 10% (250 mL) was added while cooling with an ice-bath and then stirred overnight. The organic layer was separated and the water layer extracted twice with toluene (2*100 mL). The combined organic layers were dried (K2CO3), concentrated and distilled under reduced pressure to give the desired product (23.8 g, 68%). MS: 152.7 (M+131).
62%
To a solution of lithium aluminium hydride (1 M in diethyl ether, 5.35ml, 5.35mmol) in dry diethyl ether (25ml) was added dropwise, under nitrogen, over 15 minutes, a solution of <strong>[825-99-0]3-(methylthio)benzoic acid</strong> (1.Og, 5.9mmol) in diethyl ether (30ml). The reaction mixture was heated at reflux for one hour, cooled down to -30C and quenched with water (25ml, added dropwise over five minutes). The organic phase was separated and the aqueous phase was extracted with diethyl ether (3x25ml). The organic phases were combined, washed with water (3x100ml), dried over magnesium sulphate and concentrated under reduced pressure. The crude product was purified by flash chromatography on silica gel eluting with diethyl ethe?pentane (0:1 to 1 :2, by volume) to provide the title compound (568mg, 62%) as a colourless oil. 1H-NMR (400MHz, CDCI3): delta = 2.43 (s, 3H), 3.39 (s, 1 H), 4.51 (s, 2H), 7.04 (d, 1 H), 7.14 (d, 1 H), 7.20 (m, 2H).
With dimethylsulfide borane complex; In dichloromethane; at 0 - 20℃;
Step A A solution of 1.50 g (8.92 mmol) <strong>[825-99-0]3-(methylthio)benzoic acid</strong> (Aldrich) in 20 mL of dry dichloromethane is cooled to 0 C. and 1.86 mL (19.6 mmol) borane-methylsulfide complex (Aldrich) is added. The solution is allowed to reach room temperature and stirred overnight, then hydrochloric acid (4 M, 50 mL) is added and the resulting mixture is stirred for additional 2 h. The reaction mixture is extracted with ethyl acetate (50 mL, 3 times) and the collected organic phases are washed with 10% NaHCO3 solution and brine and volatiles evaporated under vacuum to yield 3-methylsulfanyl-phenyl)-methanol (1.20 g, content ca. 75%), which is directly used in the next step.
The aminobenzoic acid (54.8 g, 0.4 mol) was diazotized in the usual manner with sodium nitrite (27.6 g, 0.4 mol) and hydrochloric acid (40 mL) and the resulting diazonium salt solution poured into a hot (70 C.), freshly prepared solution of potassium ethyl xanthate (64.2 g, 0.4 mol) containing sodium carbonate (55.2 g, 0.4 mol) to neutralize acid in the diazonium salt solution. After the reaction was over, as indicated by the cessation of the evolution of gases, the mixture was cooled. It was then treated with potassium hydroxide (24.7 g, 0.44 mol) and dimethyl sulfate (50.4 g, 0.4 mol). The mixture was refluxed for 5 hours. On acidification with hydrochloric acid, the desired product was obtained (38.2 g, 57%). MS: 167.8 (M+1+).
Methyl iodide (0.972 mL) was added to a mixture of 3-mercapto-benzoic acid (601 mg, 3.9 mmol) and potassium carbonate (2.7 g, 19.5 mmol) in DMF (8 mL) in an ice-bath. After the reaction was warmed to room temperature and stirred for 1 hour, the reaction mixture was diluted with ethyl acetate, washed with water (3X), dried over anhydrous sodium sulfate, filtered, and concentrated to afford 3-methylsulfanyl-benzoic acid methyl ester (684 mg, 96%, yellow [OIL). 1H] NMR [(CDC13),] [S] (ppm): 7.90 (s, 1H), 7.80 (d, [1H),] 7.44 (d, 1H), 7.35 (t, 1H), 3.92 (s, 3H), 2.53 (s, 3H). 3-Methylsulfanyl-benzoic acid methyl ester (684mg, 3.8 mmol) and 1N [NAOH] (5.6 mL, 5.6 mmol) in methanol (8 [ML)] and THF (8 mL) were heated at [70C] for 1 hour. The reaction mixture was concentrated and then the residue was diluted with water. After acidification with 1N [HC1] to [PH-2,] the aqueous layer was extracted with ethyl acetate and then washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford 3-methylsulfanyl-benzoic acid (616 mg, 97%, white [SOLID). 1H] NMR (DMSO), [8] (ppm): 13.1 (bs, [1H),] 7.76 (s, 1H), 7.70 (d, 1H), 7.51 (d, 1H), 7.44 (t, 1H), 2.52 (s, 3H).
97%
3-Methylsulfanyl-benzoic acid methyl ester (684 mg, 3.8 mmol) and [1N NAOH] (5.6 mL, 5.6 mmol) in methanol (8 [ML)] and THF (8 mL) were heated at [70C] for 1 hour. The reaction mixture was concentrated and then the residue was diluted with water. After acidification with 1N [HC1] to [PH-2,] the aqueous layer was extracted with ethyl acetate and then washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to afford [3-METHYLSULFANYL-BENZOIC] acid (616 mg, 97%, white [SOLID). LHNMR (DMSO), 6] (ppm): 13. 1 (bs, [1H),] 7.76 (s, 1H), 7.70 (d, 1H), 7.51 (d, 1H), 7.44 (t, 1H), 2.52 (s, 3H).
1-Hydroxybenzotriazole (HOBt) (3.7 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (4.1 g) were added to a solution of <strong>[825-99-0]3-(methylsulfanyl)benzoic acid</strong> (22) (15.0 g) in DMF (150 mL) at 0 C. After stirring for 30 min at room temperature, N,O-dimethylhydroxylamine hydrochloride (8.7 g) was added at 0 C. The reaction mixture was stirred at room temperature for 13 h and poured into saturated aqueous NaHCO3 at 0 C. The mixture was extracted with AcOEt (twice). The combined organic layer was washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo to give 23 (18.3 g, 97%) as a yellow oil. 1H NMR (CDCl3) delta 2.50 (3H, s), 3.36 (3H, s), 3.56 (3H, s), 7.28-7.45 (3H, m), 7.54 (1H, s); FAB MS m/e (M+H)+ 212.
Production Example 1; Synthesis of N-{4-[2-(4-[amino(imino)methyl]aminojphenyl)ethyl]-5-[3-(methylsulfonyl)benzyl]-1,3-thiazol-2-yl}acetamide; Step 1; A mixture of <strong>[825-99-0]3-(methylthio)benzoic acid</strong> (15 g), N,0-dimethylhydroxylamine hydrochloride (8.7 g), 1-hydroxybenzotriazole (3.71 g) and l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (4.07 g) in N,N-dimethylformamide (DMF, 150 ml) was stirred at ambienttemperature for 13 hours. The reaction mixture was pouredinto saturated NaHC03, and extracted with ethyl acetate(AcOEt, 2 times). The combined organic layer was washed with water and brine, dried over anhydrous MgS04, and concentrated in vacua to give N-methoxy-N-methyl-3-(methylthio)benzamide(18.3 g) as a yellow oil.
N-(2-methyl-5-[3-(methylthio)benzoyl]amino}phenyl)quinoxaline-6-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
73%
With N-ethyl-N,N-diisopropylamine; HATU; In N,N-dimethyl-formamide; at 25℃;
Example 1; N-(2-Methyl-5-{|'3-('methylthio)benzovnamino>phenyl)quinoxaline-6-carboxamide; 7V-(5-Amino-2-methylphenyl)quinoxaline-6-carboxamide hydrochloride (Method 4; 100 mg, 0.317 mmol), 3-(me%lthio)benzoic acid (59 mg, 0.348 mmol), HATU (145 mg, 0.380 mmol), anhydrous DMF (2 ml) and DIEA (275 muL, 1.585 mmol) were added to a 20 ml scintillation vial. The reaction mixture was shaken overnight at 25 0C. Water (10 ml) was added slowly to precipitate the product. The resulting precipitate was washed with water (10 ml), isolated and dried overnight in a vacuum oven at 70 C to give the title compound 98.4 mg, (73%) as a solid. NMR: 2.25 (s, 3H), 2.54 (s, 3H), 7.27 (d, IH), 7.46 (d, 2H), 7.62 (d, IH), 7.71 (t, IH), 7.79 (s, IH), 7.89 (s, IH), 8.25 (d, IH)5 8.37 (d, IH), 8.77 (s, IH), 9.17 (d, 2H), 10.32 (d, 2H); m/z: 429.
To a solution of <strong>[825-99-0]3-(methylthio)benzoic acid</strong> (200 mg, 1.2 mmol) in DMF (4.5 mL) was added in succession N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydroiodide (345 mg, 1.75 mmol) 1-hydroxybenzotriazole hydrate (186 mg, 1.2 mmol) and diisopropylethylamine (0.630 mL, 3.6 mmol). After stirring at room temperature for 10 min, 2,2-dimethyl-1-(5-(piperidin-3-ylamino)-1H-indazol-1-yl)propan-1-one (300 mg, 1.0 mmol) was added in one portion. The solution was stirred under a nitrogen atmosphere overnight. The solution was poured into 1.0 M HCl (20 mL), extracted twice with EtOAc, and the organic phases were washed with 10% NaOH, dried over Na2SO4 and evaporated. Chromatography of the residue on silica gel, eluting with EtOAc/heptane, gave the title compound as a pale yellow foam (240 mg, 53%).
Acetic acid (3S,4S,4aR,6S,6aS,12R,12aS,12bS)-4-acetoxymethyl-6,12-dihydroxy-4,6a,12b-trimethyl-11-oxo-9-pyridin-3-yl-1,3,4,4a,5,6,6a,12,12a,12b-decahydro-2H,11H-7,10-dioxa-benzo[a]anthracen-3-yl ester[ No CAS ]
Example 13Preparation of Ethyl 5-(methylthio)benzo[b]thiophene-2-carboxylateTwo hundred and fifty grams (1.23 moles) of <strong>[825-99-0]5-(methylthio)benzoic acid</strong> was added portionwise to 600 ml of thionyl chloride over a period of 30 minutes and the resulting reaction mixture was heated under reflux for about 4-5 h. Excess thionyl chloride was removed by distillation and the residue was redistilled under high vacuum to afford 203 g (75 %) of the acid chloride as a liquid which crystallized on standing, mp 28-30C; 1H-NMR (400 MHz, CDC13): delta 2.53 (s 3), 7.34-7.40 (m 2), 7.88 (d 1); IR (DCM) 1772 cm-1 (C=0). The crude acid chloride was used as such in the next step.
N-(3-methylthiobenzoyl)-3-hydroxyanthranilic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With pDRf1-(4-coumaroyl:CoA ligase 5 from Arabidopsis thaliana)-(hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase 1 from Dianthus caryophyllus) recombinant yeast; In dimethyl sulfoxide; at 30℃; for 24h;
General procedure: An overnight culture from a single colony of the pDRf1-4CL5-HCBT1 recombinant yeast grown on 2X YNB medium without amino acids, supplemented with 6% glucose and 2X CSM-Ura, was used to inoculated 4 mL of fresh minimal medium at an OD600 = 0.15 and shaken at 200 rpm at 30C. All precursors were prepared in DMSO and added 5 hours post inoculation at the concentrations indicated in S1, S2 and S3 Tables. The anthranilate acceptors were added to the medium at a final concentration of 300 muM (for anthranilate, 3-hydroxyanthranilate, 3-methylanthranilate, and 5-nitroanthranilate) or 50 muM (for 3-chloroanthranilate, 5-methylanthranilate, 3-methoxyanthranilate, 5-fluoroanthranilate, 5-iodoanthranilate, and 5-chloroanthranilate). These concentrations were selected to limit toxicity and growth inhibition due to either the supplied precursors or the metabolites produced. The cultures were shaken at 200 rpm at 30C for 24 h in the presence of the precursors for the production of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates. Yeast colonies harboring the pDRf1-HCBT1 or pDRf1-4CL5 control vectors were grown under similar conditions. For the detection of metabolites, an aliquot of the culture medium was collected and cleared by centrifugation (21,000xg for 5 min at 4C), mixed with an equal volume of cold methanol:water (1:1, v/v), and filtered using Amicon Ultra centrifugal filters (3,000 Da MW cutoff regenerated cellulose membrane; Millipore, Billerica, MA) prior to LC-TOF MS analysis. The separation and identification of the metabolites were performed using high-performance liquid chromatography (HPLC), electrospray ionization (ESI), and time-of-flight (TOF) mass spectrometry (MS) as previously described [35]. For each compound, the measured masses agreed with the expected theoretical masses within less than 5 ppm mass error. Standard solutions of DHavnD and dianthramide B were prepared in methanol:water (1:1, v/v). Values obtained for the production of DHavnD and dianthramide B are the average of four replicates (n = 4). ESI-MS spectra of other cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates were obtained from single feeding experiments for each combination of precursors.
N-(3-methylthiobenzoyl)-3-methylanthranilic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With pDRf1-(4-coumaroyl:CoA ligase 5 from Arabidopsis thaliana)-(hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase 1 from Dianthus caryophyllus) recombinant yeast; In dimethyl sulfoxide; at 30℃; for 24h;
General procedure: An overnight culture from a single colony of the pDRf1-4CL5-HCBT1 recombinant yeast grown on 2X YNB medium without amino acids, supplemented with 6% glucose and 2X CSM-Ura, was used to inoculated 4 mL of fresh minimal medium at an OD600 = 0.15 and shaken at 200 rpm at 30C. All precursors were prepared in DMSO and added 5 hours post inoculation at the concentrations indicated in S1, S2 and S3 Tables. The anthranilate acceptors were added to the medium at a final concentration of 300 muM (for anthranilate, 3-hydroxyanthranilate, 3-methylanthranilate, and 5-nitroanthranilate) or 50 muM (for 3-chloroanthranilate, 5-methylanthranilate, 3-methoxyanthranilate, 5-fluoroanthranilate, 5-iodoanthranilate, and 5-chloroanthranilate). These concentrations were selected to limit toxicity and growth inhibition due to either the supplied precursors or the metabolites produced. The cultures were shaken at 200 rpm at 30C for 24 h in the presence of the precursors for the production of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates. Yeast colonies harboring the pDRf1-HCBT1 or pDRf1-4CL5 control vectors were grown under similar conditions. For the detection of metabolites, an aliquot of the culture medium was collected and cleared by centrifugation (21,000xg for 5 min at 4C), mixed with an equal volume of cold methanol:water (1:1, v/v), and filtered using Amicon Ultra centrifugal filters (3,000 Da MW cutoff regenerated cellulose membrane; Millipore, Billerica, MA) prior to LC-TOF MS analysis. The separation and identification of the metabolites were performed using high-performance liquid chromatography (HPLC), electrospray ionization (ESI), and time-of-flight (TOF) mass spectrometry (MS) as previously described [35]. For each compound, the measured masses agreed with the expected theoretical masses within less than 5 ppm mass error. Standard solutions of DHavnD and dianthramide B were prepared in methanol:water (1:1, v/v). Values obtained for the production of DHavnD and dianthramide B are the average of four replicates (n = 4). ESI-MS spectra of other cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates were obtained from single feeding experiments for each combination of precursors.
With pDRf1-(4-coumaroyl:CoA ligase 5 from Arabidopsis thaliana)-(hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase 1 from Dianthus caryophyllus) recombinant yeast; In dimethyl sulfoxide; at 30℃; for 24h;
General procedure: An overnight culture from a single colony of the pDRf1-4CL5-HCBT1 recombinant yeast grown on 2X YNB medium without amino acids, supplemented with 6% glucose and 2X CSM-Ura, was used to inoculated 4 mL of fresh minimal medium at an OD600 = 0.15 and shaken at 200 rpm at 30C. All precursors were prepared in DMSO and added 5 hours post inoculation at the concentrations indicated in S1, S2 and S3 Tables. The anthranilate acceptors were added to the medium at a final concentration of 300 muM (for anthranilate, 3-hydroxyanthranilate, 3-methylanthranilate, and 5-nitroanthranilate) or 50 muM (for 3-chloroanthranilate, 5-methylanthranilate, 3-methoxyanthranilate, 5-fluoroanthranilate, 5-iodoanthranilate, and 5-chloroanthranilate). These concentrations were selected to limit toxicity and growth inhibition due to either the supplied precursors or the metabolites produced. The cultures were shaken at 200 rpm at 30C for 24 h in the presence of the precursors for the production of cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates. Yeast colonies harboring the pDRf1-HCBT1 or pDRf1-4CL5 control vectors were grown under similar conditions. For the detection of metabolites, an aliquot of the culture medium was collected and cleared by centrifugation (21,000xg for 5 min at 4C), mixed with an equal volume of cold methanol:water (1:1, v/v), and filtered using Amicon Ultra centrifugal filters (3,000 Da MW cutoff regenerated cellulose membrane; Millipore, Billerica, MA) prior to LC-TOF MS analysis. The separation and identification of the metabolites were performed using high-performance liquid chromatography (HPLC), electrospray ionization (ESI), and time-of-flight (TOF) mass spectrometry (MS) as previously described [35]. For each compound, the measured masses agreed with the expected theoretical masses within less than 5 ppm mass error. Standard solutions of DHavnD and dianthramide B were prepared in methanol:water (1:1, v/v). Values obtained for the production of DHavnD and dianthramide B are the average of four replicates (n = 4). ESI-MS spectra of other cinnamoyl, dihydrocinnamoyl, and benzoyl anthranilates were obtained from single feeding experiments for each combination of precursors.
(1) Under an argon atmosphere, an aqueous solution (6.77 mL) of sodium periodate (421 mg) was added dropwise to a solution of <strong>[825-99-0]3-methylsulfanylbenzoic acid</strong> (569 mg) in methanol (6.77 mL) under ice cooling. This mixture was stirred at the same temperature for 2 hours, and then at room temperature for 2 hours, and the precipitate was filtered off and washed with a small amount of methanol. The filtrate and washing solution were combined and concentrated under reduced pressure, and brine (10 mL) was added to the residue and the mixture was extracted with a mixed solution (chloroform/methanol = 9:1) three times. The obtained organic layer was collected, dehydrated by a phase separator, and concentrated under reduced pressure. 1,1'-Carbonyldiimidazole (594 mg) was added to a suspension of the obtained residue in tetrahydrofuran (13.3 mL) and the mixture was stirred at room temperature for 30 minutes. Methanol (1.1 mL) was added to this mixture, the container was heated with a drier to reflux for a short time and left standing to cool, and the mixture was then stirred at room temperature for 30 minutes. An aqueous solution of saturated sodium hydrogen carbonate was added to the mixture and the mixture was extracted with ethyl acetate three times. The obtained organic layer was collected, washed sequentially with brine, 1 mol/L hydrochloric acid, and then brine, separated by a phase separator, and concentrated under reduced pressure. The residue was purified by column chromatography (n-hexane/ethyl acetate = 4:1 to ethyl acetate only) to give methyl 3-methylsulfinylbenzoate (465 mg) as a light yellow oily substance.
With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; triethylamine; In N,N-dimethyl-formamide; at 0 - 20℃; for 12h;
To a suspension of <strong>[825-99-0]3-(methylthio)benzoic acid</strong> (2 g, 11.88 mmol) in DMF (20 mL) at 0 C was added Nu,Omicron-dimethylhydroxylamine (1.45 g, 23.77 mmol), HOBT (2.18 g, 14.26 mmol), EDC.HC1 (2.73 g, 14.26 mmol) and triethylamine (2.40 g, 23.77 mmol). The reaction mixture was then stirred at ambient temperature. After 12 h, the reaction mixture was diluted with 10 % aqueous sodium bicarbonate solution (50 mL) and extracted with dichloromethane (3 x 50 mL). The combined organic extract were dried over anhydrous sodium sulphate and evaporated to dryness to afford N- methoxy-N-methyl-3-(methylthio)benzamide (2.1 g) as an off- white solid. NMR (400 MHz, CDC13) delta 7.54 (s, 1H), 7.43 (t, 1H, J = 1.6 Hz), 7.35-7.27 (m, 2H), 3.56 (s, 3H), 3.36 (s, 3H), 2.50 (s, 3H); MS: m/z 212.2 (M+l).
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