* 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.
Reference：
[1] Patent: WO2012/97682, 2012, A1,
[2] Research on Chemical Intermediates, 2014, vol. 40, # 3, p. 1147 - 1155
14
[ 77-92-9 ]
[ 99-11-6 ]
Yield
Reaction Conditions
Operation in experiment
85.18%
With urea In water at 185℃; for 2 h; Sonication; High pressure
(1) Take 1.2188 g (0.0058 mol) of citric acid and 1.3932 g (0.0231 mol) of urea dissolved in 60 ml of deionized water, sonicate for 10 min, and mix well; (2) The mixture is charged into the reaction vessel, the oven temperature is set to 185 ° C, and the temperature is kept under hydrothermal conditions for 2 h; Cooling to room temperature to obtain a dark green solution; (3) The solution was placed in a constant temperature water bath at 60 ° C, magnetically stirred, and 98percent sulfuric acid was added dropwise to the solution until the pH of the solution was 2-3. A large amount of yellow solid precipitate was observed and centrifuged at 12000 r/min for 10 min. Remove the supernatant, wash it with deionized water multiple times, and drain it. The obtained yellow precipitate was dried in an oven at 60 ° C to obtain 85.18percent of the citrazinic acid product.
Reference：
[1] Patent: CN108164458, 2018, A, . Location in patent: Paragraph 0010; 0012; 0013
[2] Journal of the American Pharmaceutical Association (1912-1977), 1956, vol. 45, p. 478
Reference：
[1] Chemische Berichte, 1884, vol. 17, p. 2688
[2] Patent: DE957033, 1952, ,
[3] Journal of the Chemical Society, 1892, vol. 61, p. 1009
17
[ 725679-43-6 ]
[ 99-11-6 ]
Reference：
[1] Chemische Berichte, 1884, vol. 17, p. 2688
[2] Journal of the Chemical Society, 1892, vol. 61, p. 1009
18
[ 130839-31-5 ]
[ 99-11-6 ]
Reference：
[1] Chemische Berichte, 1884, vol. 17, p. 2688
[2] Journal of the Chemical Society, 1892, vol. 61, p. 1009
19
[ 5398-44-7 ]
[ 99-11-6 ]
Reference：
[1] Journal of the Chemical Society, 1897, vol. 71, p. 1082[2] Journal of the Chemical Society, 1898, vol. 73, p. 441
20
[ 1587-20-8 ]
[ 99-11-6 ]
Reference：
[1] Journal of the Chemical Society, 1892, vol. 61, p. 1009
[2] Chemische Berichte, 1884, vol. 17, p. 2688
[3] Cesko-Slovenska Farmacie, 1957, vol. 6, p. 369,370[4] Chem.Abstr., 1958, p. 10071
21
[ 57-13-6 ]
[ 77-92-9 ]
[ 99-11-6 ]
Reference：
[1] Patent: DE1011885, 1954, ,
22
[ 99-11-6 ]
[ 5398-44-7 ]
Yield
Reaction Conditions
Operation in experiment
89%
at 130 - 145℃; for 20 h;
Citrazinic acid (10.35 g, 66.7 mmol) and tetraethylammonium chloride (11.05 g, 66.7 mmol) were suspended in phosphoroxychloride (20 mL, excess) and heated at 130 °C for 18 h and then at 145 °C for 2 h. The reaction mixture was allowed to cool to RT and poured onto crushed ice (150 g). Extraction of ethyl acetate (3 x 100 mL), drying of the combined organic layers (MGS04) and evaporation in vacuo gave 11. 34 g (89 percent) of a white solid. IH NMR (400 MHz, DMSO-d6) 8 7.85 (s, 2H) 13.90 (br s, OH).
81%
Stage #1: at 140℃; for 24 h; Stage #2: at 0℃; for 2 h;
Citrazinic acid 1 (20.0 g, 129 mmol) and benzyltriethylammonium chloride (32.3 g, 142 mmol) in 40 ml of POCl3 were heated to 140 °C for 24 h under a CaCl2 drying tube. After being cooled to room temperature, the brown mixture was poured on ice (400 g) and stirred for 2 h. The resulting brown solid was filtered off, washed with water and dissolved in EtOAc (400 ml). The organic phase was then washed with saturated NH4Cl, dried over Na2SO4 and evaporated to dryness to afford 20 g (81percent) of the desired compound as a brown solid. 1H and 13C NMR were in accordance with those already reported.18
Reference：
[1] Chemical Communications, 2015, vol. 51, # 74, p. 14123 - 14126
[2] Patent: WO2004/63156, 2004, A1, . Location in patent: Page 35
[3] Tetrahedron Letters, 2011, vol. 52, # 5, p. 584 - 587
[4] Journal of Organic Chemistry, 1997, vol. 62, # 19, p. 6588 - 6597
[5] Chemical Communications, 1998, # 15, p. 1567 - 1568
[6] Tetrahedron, 2007, vol. 63, # 2, p. 291 - 298
[7] Tetrahedron, 2002, vol. 58, # 34, p. 6951 - 6963
[8] Organic Letters, 2000, vol. 2, # 22, p. 3421 - 3423
[9] Journal of the American Pharmaceutical Association (1912-1977), 1956, vol. 45, p. 478
[10] Yakugaku Zasshi, 1953, vol. 73, p. 969[11] Chem.Abstr., 1954, p. 11420
[12] Journal fuer Praktische Chemie (Leipzig), 1938, vol. <2> 151, p. 65,79
[13] Journal of Agricultural and Food Chemistry, 2005, vol. 53, # 25, p. 9775 - 9780
[14] Journal of the American Chemical Society, 2005, vol. 127, # 29, p. 10124 - 10125
[15] Organic Letters, 2003, vol. 5, # 7, p. 967 - 970
[16] Monatshefte fur Chemie, 2008, vol. 139, # 12, p. 1491 - 1498
[17] Monatshefte fur Chemie, 2008, vol. 139, # 11, p. 1409 - 1415
[18] Organic Letters, 2010, vol. 12, # 5, p. 940 - 943
[19] Bioorganic and Medicinal Chemistry Letters, 2011, vol. 21, # 5, p. 1429 - 1433
[20] Patent: WO2012/97682, 2012, A1, . Location in patent: Page/Page column 66
[21] Patent: WO2012/97479, 2012, A1, . Location in patent: Page/Page column 138
[22] Patent: WO2012/97683, 2012, A1, . Location in patent: Page/Page column 72-73
[23] ChemMedChem, 2013, vol. 8, # 6, p. 967 - 975
[24] Research on Chemical Intermediates, 2014, vol. 40, # 3, p. 1147 - 1155
[25] Patent: US2014/171429, 2014, A1, . Location in patent: Paragraph 0312
Reference：
[1] Journal of the American Chemical Society, 1958, vol. 80, p. 5481
25
[ 99-11-6 ]
[ 10025-87-3 ]
[ 5398-44-7 ]
Reference：
[1] Chemische Berichte, 1902, vol. 35, p. 2933
26
[ 99-11-6 ]
[ 2016-99-1 ]
Yield
Reaction Conditions
Operation in experiment
64%
Stage #1: at 180℃; for 3 h; Stage #2: at 0℃;
Example 15; Synthesis of 2, 6-Dibromoisonicotinic Acid; [0114] Citrazinic acid (2.33 g, 0.015 moles) and phosphorous oxybromide were combined and heated to 180 °C under a nitrogen atmosphere for three hours. The cooled reaction was carefully quenched with ice-water. The mixture was filtered and the aqueous portion extracted with dichloromethane (4 x 40 ml). The solids were extracted in a Soxhlet extractor with dichloromethane for twelve hours. The organic portion from the direct extraction was dried over sodium sulfate, filtered, and stripped to give 2.2 g of reddish solid. The organic portion from the Soxhlet extraction was dried over sodium sulfate, filtered, and stripped to give 0.5 g of reddish solid. The two portions of 2,6- dibromoisonicotinic acid (64 percent yield) were similar by NMR and were combined.'H NMR (CDC13) 8. 04 (2H, s).
Reference：
[1] Journal of Medicinal Chemistry, 2013, vol. 56, # 12, p. 5079 - 5093
[2] Tetrahedron, 2007, vol. 63, # 2, p. 291 - 298
[3] Organic and Biomolecular Chemistry, 2005, vol. 3, # 8, p. 1460 - 1467
[4] Patent: WO2005/84439, 2005, A1, . Location in patent: Page/Page column 35
[5] Synthesis, 2000, # 8, p. 1138 - 1142
[6] Yakugaku Zasshi, 1953, vol. 73, p. 969[7] Chem.Abstr., 1954, p. 11420
[8] Tetrahedron Letters, 2001, vol. 42, # 35, p. 6113 - 6115
[9] Journal of Organic Chemistry, 2006, vol. 71, # 12, p. 4696 - 4699
[10] Chemical Communications, 2009, # 16, p. 2121 - 2123
Reference：
[1] Journal of Organic Chemistry, 1997, vol. 62, # 19, p. 6588 - 6597
29
[ 67-56-1 ]
[ 99-11-6 ]
[ 119308-57-5 ]
Yield
Reaction Conditions
Operation in experiment
79%
Stage #1: at 130℃; Stage #2: at 0℃;
A mixture of citrazinic acid (5.0 g, 32.2 mmol) and POBr3 (27.5 g, 96.8 mmol) is heated at 130 0C. Upon completion of the reaction, the thick slurry is cooled to 0 0C and the reaction is carefully quenched with MeOH (250 ml_). The reaction mixture is concentrated in vacuo and then extracted between dichloromethane and sat. aq. NaHCO3. Organic layer is dried over anhydrous Na2SO4 and concentrated in vacuo to give a tan solid that is clean enough by NMR/LCMS to use further (7.5 g, 79percent). (ESI) m/z 295.8 (M+1 ). 1H NMR (400 MHz, CD2CI2) δ ppm 8.10 (s, 2 H), 4.05 (s, 3 H).
50%
Stage #1: at 185℃; for 8 h; Inert atmosphere Stage #2: at 20℃; Reflux
The compound PBr3 (21.8 ml, 0.232 mol) was loaded into a 250 ml three -neck flask cooled in a room temperature water bath under a flow of nitrogen. Br 2 (11.9 ml, 0.231 mol) was then added dropwise with stirring to PBr 3 to yield a yellow solid (PBr5). 30 minutes was taken for addition of Br 2. P2O5 (12.0 g, 0.083 mol) was then <n="64"/>added and the solid mixture was mixed well with a spatula under a flow of nitrogen. A reflux condenser was added via a connection to a water -filled bubbler, to trap evolved HBr. The mixture was heated to 98 0C for 2 hours and then cooled to room temperature. Citrazinic acid (20.0 g, 0.128 mol) was then added and the solid was mixed thoroughly with a spatula under a flow of nitrogen. The mixture was heated at 1850C for 8 hours. The mixture was cooled back to room temperature. Methanol (150 ml) was slowly added (the reaction with methanol was exothermic, causing the solution to reflux). After the addition of methanol, the mixture was stirred until room temperature. Solid NaHCO3 was added slowly until bubbling stopped. After which, water (300 ml) was added and then additional NaHCO 3 was added until the bubbling stopped. A brown solid product was collected with filtration. The solid was washed with water and dried at room temperature under air. The product was purified by silica gel column using hexanes/ethyl acetate (9: 1) as a solvent. The final pure white product was obtained by recrystallization from methanol/water: 19.02 g (50.0percent).1H NMR (CDCl 3): δ 8.00 (s, 2H), 3.97 (s, 3H). 13C NMR (CDCl3): δ 162.74, 141.35, 141.25, 126.56, 53.38. MS (EI) m/z (percent): 294.8 (100percent) [M +].
Reference：
[1] Journal of Medicinal Chemistry, 2010, vol. 53, # 15, p. 5422 - 5438
[2] Patent: WO2009/150230, 2009, A1, . Location in patent: Page/Page column 65
[3] European Journal of Inorganic Chemistry, 2010, # 13, p. 1913 - 1928
[4] Journal of Organic Chemistry, 2006, vol. 71, # 12, p. 4696 - 4699
[5] Synthesis, 2008, # 17, p. 2764 - 2770
[6] Patent: WO2009/80799, 2009, A2, . Location in patent: Page/Page column 62-63
[7] Patent: US2010/261687, 2010, A1, . Location in patent: Page/Page column 61
[8] Dalton Transactions, 2010, vol. 39, # 41, p. 9872 - 9878
[9] Chemical Communications, 2012, vol. 48, # 21, p. 2704 - 2706
[10] Patent: WO2012/45803, 2012, A1, . Location in patent: Page/Page column 25
[11] Patent: US2012/264729, 2012, A1, . Location in patent: Page/Page column 9
[12] ACS Medicinal Chemistry Letters, 2013, vol. 4, # 6, p. 542 - 546
[13] Journal of Medicinal Chemistry, 2014, vol. 57, # 20, p. 8307 - 8318
[14] Journal of Medicinal Chemistry, 2016, vol. 59, # 23, p. 10719 - 10737
[15] Organic Process Research and Development, 2017, vol. 21, # 2, p. 231 - 240
30
[ 99-11-6 ]
[ 119308-57-5 ]
Reference：
[1] Patent: US2010/331509, 2010, A1,
31
[ 99-11-6 ]
[ 148332-36-9 ]
Reference：
[1] Synthesis, 2000, # 8, p. 1138 - 1142
Ethyl 2-ethoxy-6-hydroxyisonicotinate 154)
A suspension of 2,6-dihydroxyisonicotinic acid (40.00 g, 258 mmol) in EtOH (300 mL) was treated dropwise with H2S04 (40 mL, 18.4 M, 752 mmol). The solution was refluxed for 72 h then evaporated; the residue was treated with sat. aq. NaHC03 to pH 8 and then extracted with (0700) EtOAc (3 x 500 mL). The organic extracts were washed with sat. aq. NaHC03, brine, then dried and evaporated to give 154 (10.86 g, 20%). 1H MR (DMSO-d6) 5 11.15 (bs, 1H), 6.59 (d, J = (0701) 1.0 Hz, 1H), 6.57 (bs, 1H), 4.29 (q, J = 7.1 Hz, 2H), 4.25 (q, J = 7.1 Hz, 2H), 1.298 (t, J = 7.1 Hz, (0702) 3H), 1.296 (t, J = 7.1 Hz, 3H). Found: [M+H]=212.2
20%
With sulfuric acid for 72h; Reflux;
4.1.1.6. 6-Bromo-3-((2-ethoxy-6-propoxypyridin-4-yl)methyl)-2-methoxyquinoline(VI: Y = 4-aza, 3-OEt, 5-OPr). A suspension of2,6-dihydroxyisonicotinic acid (40.00 g, 258 mmol) in EtOH (300mL) was treated dropwise with H2SO4 (40 mL, 18.4 M, 752 mmol).The solution was refluxed for 72 h and then evaporated. The residuewas treated with sat. aq. NaHCO3 to pH 8 and extracted withEtOAc (3 200 mL). The organic extracts were washed with sat.aq. NaHCO3 and brine, then dried and evaporated to give ethyl 2-ethoxy-6-hydroxyisonicotinate (10.86 g, 20%) as a tan solid, mp102-104 C. 1H NMR (DMSO d6) d 11.45 (bs, 1H), 6.59 (d, J = 1.0Hz, 1H), 6.57 (s, 1H), 4.29 (q, J = 7.1 Hz, 2H), 4.25 (q, J = 7.0 Hz,2H), 1.298 (t, J = 7.1 Hz, 3H), 1.296 (t, J = 7.0 Hz, 3H).
20%
With sulfuric acid for 72h; Reflux;
4.1.1.25. 3-(Dimethylamino)-1-(2-ethoxy-6-isopropoxypyridin-4-yl)propan-1-one (Class X).
A suspension of 2,6-dihydroxyisonicotinic acid(40.00 g, 258 mmol) in EtOH (300 mL) was treated dropwise withH2SO4 (40 mL, 18.4 M, 752 mmol). The solution was refluxed for 72 hthen evaporated; the residue was treated with sat. aq. NaHCO3 to pH 8and then extracted with EtOAc (3×500 mL). The organic extracts werewashed with sat. aq. NaHCO3, brine, then dried and evaporated to giveethyl 2-ethoxy-6-hydroxyisonicotinate (10.86 g, 20%). 1H NMR(DMSO-d6) δ 11.15 (bs, 1H), 6.59 (d, J=1.0 Hz, 1H), 6.57 (bs, 1H),4.29 (q, J=7.1 Hz, 2H), 4.25 (q, J=7.1 Hz, 2H), 1.298 (t, J=7.1 Hz,3H), 1.296 (t, J=7.1 Hz, 3H). Found: [M+H]=212.2.
20%
With sulfuric acid for 72h; Reflux;
A suspension of 2,6-dihydroxyisonicotinic acid (40.00 g, 258 mmol) in EtOH (300 mL) was treated drop wise with H2SO4 (40 mL, 18.4 M,752 mmol). The solution was refluxed for 72 h then evaporated; the residue was treated with sat. aq. NaHCO3 to pH 8 and then extracted with EtOAc (3 × 500 mL). The organic extracts were washed with sat.aq. NaHCO3, brine, then dried and evaporated to give ethyl 2-ethoxy-6-hydroxyisonicotinate (S27) (10.86 g, 20%). 1H NMR (DMSO-d6) δ11.15 (bs, 1H), 6.59 (d, J = 1.0 Hz, 1H), 6.57 (bs, 1H), 4.29 (q,J = 7.1 Hz, 2H), 4.25 (q, J = 7.1 Hz, 2H), 1.298 (t, J = 7.1 Hz, 3H),1.296 (t, J = 7.1 Hz, 3H). Found: [M+H] = 212.2.
With tetrabutyl-ammonium chloride; trichlorophosphate at 140℃;
89%
With tetraethylammonium chloride; trichlorophosphate at 130 - 145℃; for 20h;
16 Example 16-2,6-dichloroisonicotinic acid
Citrazinic acid (10.35 g, 66.7 mmol) and tetraethylammonium chloride (11.05 g, 66.7 mmol) were suspended in phosphoroxychloride (20 mL, excess) and heated at 130 °C for 18 h and then at 145 °C for 2 h. The reaction mixture was allowed to cool to RT and poured onto crushed ice (150 g). Extraction of ethyl acetate (3 x 100 mL), drying of the combined organic layers (MGS04) and evaporation in vacuo gave 11. 34 g (89 %) of a white solid. IH NMR (400 MHz, DMSO-d6) 8 7.85 (s, 2H) 13.90 (br s, OH).
85%
With trichlorophosphate at 40 - 105℃;
6 Example 6: Preparation of 2,6-Dichloroisonicotinic acid
Add 40 mL of phosphorus oxychloride to a clean and anhydrous 100 mL four-necked flask. Add 10 g of 2,6-dihydroxyisonicotinic acid in batches under stirring at 20-25°C, control the temperature to be less than 40°C, and slowly increase the temperature toReact at 100-105°C for 4-6 hours,After the reaction of the raw materials is complete, the phosphorus oxychloride is removed by distillation under reduced pressure, 50mL of water is added, and then 2N sodium hydroxide solution is added dropwise to control the pH of the system = 8-9. When the pH does not change, add 2N hydrochloric acid to pH = 6-7 The solid precipitated out, filtered and dried to obtain 10.5 g of the product with a yield of 85%.
81%
Stage #1: citrazinic acid With N-benzyl-N,N,N-triethylammonium chloride; trichlorophosphate at 140℃; for 24h;
Stage #2: With water at 0℃; for 2h;
Compound 2:
Citrazinic acid 1 (20.0 g, 129 mmol) and benzyltriethylammonium chloride (32.3 g, 142 mmol) in 40 ml of POCl3 were heated to 140 °C for 24 h under a CaCl2 drying tube. After being cooled to room temperature, the brown mixture was poured on ice (400 g) and stirred for 2 h. The resulting brown solid was filtered off, washed with water and dissolved in EtOAc (400 ml). The organic phase was then washed with saturated NH4Cl, dried over Na2SO4 and evaporated to dryness to afford 20 g (81%) of the desired compound as a brown solid. 1H and 13C NMR were in accordance with those already reported.18
78%
With POPCl3; tetramethlyammonium chloride at 130 - 142℃; for 2h;
78%
With trichlorophosphate at 160℃;
77%
With tetramethlyammonium chloride; trichlorophosphate
75%
With N-benzyl-N,N,N-triethylammonium chloride; trichlorophosphate In neat (no solvent) at 140℃; for 24h; Reflux;
72%
With trichlorophosphate at 150℃; for 24h;
71%
With tetramethlyammonium chloride; trichlorophosphate at 140 - 150℃; for 29h;
71%
With tetraethylammonium chloride; trichlorophosphate
With trichlorophosphate at 125℃;
With trichlorophosphate at 185 - 190℃;
With trichlorophosphate at 210 - 220℃;
With phosphorus pentachloride; trichlorophosphate at 180℃;
With trichlorophosphate at 125℃; for 24h;
Multi-step reaction with 2 steps
1: 76 percent / POCl3 / 18 h / 130 °C
2: 100 percent / aq. LiOH / tetrahydrofuran / 0.5 h / 23 °C
Multi-step reaction with 2 steps
1: 76 percent / POCl3 / 18 h / 130 °C / Heating
2: 100 percent / lithium hydroxide / tetrahydrofuran; H2O / 1.5 h / 20 °C
With trichlorophosphate In N,N-dimethyl-formamide
With triethylamine; trichlorophosphate
Stage #1: citrazinic acid With tetramethlyammonium chloride; trichlorophosphate Heating; Inert atmosphere;
Stage #2: With methanol Inert atmosphere;
Stage #3: With lithium hydroxide In tetrahydrofuran; water Inert atmosphere;
With tetramethlyammonium chloride; trichlorophosphate at 130℃; for 48h;
With trichlorophosphate at 160 - 165℃; for 6h; Sealed tube;
1.1A
EXAMPLE 1 7-(2,6-dichlorobenzyl)-5-{ f2-methoxy-4-(piperazin-l -yl)phenyl]amino}-2,3- dihydropyrido[4,3-d]pyrimidin-4( lH)-one EXAMPLE 1 A 2,6-dichloropyridine-4-carboxylic acid A mixture of 2,6-dihydroxypyridine-4-carboxylic acid ( 15. 1 g, 100 mmol) and phosphoryl trichloride (45 ml) was heated for 6 hours at 160-165°C in a 200 mL sealed tube. After cooling to ambient temperature, the mixture was poured into crushed ice (300 g) and stirred for 1 hours. The mixture was extracted with ethyl acetate (5 x 60 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the of crude product which was recrystallized from 40 mL of 2/1 ethyl acetate/petroleum ether to afford the title compound. NMR (DMSO-cfe) δ ppm 7.89 (s, 2H). MS; 192 (M+l).
With trichlorophosphate at 160 - 165℃; for 6h; Sealed tube;
5.5A
A mixture of 2,6-dihydroxypyridine-4-carboxylic acid (15.1 g, 100 mmol) and phosphoryl trichloride (45 ml) was heated for 6 hours at 160-165°C in a 200 mL sealed tube. After cooling to ambient temperature, the mixture was poured into crushed ice (300 g) and stirred for 1 hours. The mixture was extracted with ethyl acetate (5 x 60 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the of crude product which was recrystallized from 40 mL of 2/1 ethyl acetate/petroleum ether to afford the title compound. NMR (DMSCWe) δ ppm 7.89 (s, 2H). MS: 192 (M+l ).
With trichlorophosphate at 160 - 165℃; for 6h; Sealed tube;
5.5A
A mixture of 2,6-dihydroxypyridine-4-carboxylic acid (15.1 g, 100 mmol) and phosphoryl trichloride (45 ml) was heated for 6 hours at 160-165°C in a 200 mL sealed tube. After cooling to ambient temperature, the mixture was poured into crushed ice (300 g) and stirred for 1 hours. The mixture was extracted with ethyl acetate (5 x 60 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the of crude product which was recrystallized from 40 mL of 2/1 ethyl acetate/petroleum ether to afford the title compound. NMR (DMSCWe) δ ppm 7.89 (s, 2H). MS: 192 (M+l ).
Multi-step reaction with 2 steps
1: trichlorophosphate / 24 h / 130 °C
2: lithium hydroxide monohydrate / tetrahydrofuran; water / 0.33 h / 25 °C
With triethylamine; trichlorophosphate
With trichlorophosphate at 160 - 165℃; for 6h; Sealed tube;
5A 2,6-dichloropyridine-4-carboxylic acid
A mixture of 2,6-dihydroxypyridine-4-carboxylic acid (15.1 g, 100 mmol) and phosphoryl trichioride (45 ml)was heated for 6 hours at 160-165°C. in a 200 mL sealedtube. Afier cooling to ambient temperature, the mixture was poured into crushed ice (300 g) and stirred for 1 hours. The mixture was extracted with ethyl acetate (5x60 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo to give the of crude product which was recrystallized from 40 mL of 2/1 ethyl acetate/ petroleum ether to afford the title compound. ‘H NMR (DMSO-d5) ö ppm 7.89 (s, 2H). MS: 192 (M+1).
Example 15; Synthesis of 2, 6-Dibromoisonicotinic Acid; [0114] Citrazinic acid (2.33 g, 0.015 moles) and phosphorous oxybromide were combined and heated to 180 C under a nitrogen atmosphere for three hours. The cooled reaction was carefully quenched with ice-water. The mixture was filtered and the aqueous portion extracted with dichloromethane (4 x 40 ml). The solids were extracted in a Soxhlet extractor with dichloromethane for twelve hours. The organic portion from the direct extraction was dried over sodium sulfate, filtered, and stripped to give 2.2 g of reddish solid. The organic portion from the Soxhlet extraction was dried over sodium sulfate, filtered, and stripped to give 0.5 g of reddish solid. The two portions of 2,6- dibromoisonicotinic acid (64 % yield) were similar by NMR and were combined.'H NMR (CDC13) 8. 04 (2H, s).
With urea In water at 185℃; for 2h; Sonication; High pressure;
3 Example 3 Preparation of citrazinic acid
(1) Take 1.2188 g (0.0058 mol) of citric acid and 1.3932 g (0.0231 mol) of urea dissolved in 60 ml of deionized water, sonicate for 10 min, and mix well; (2) The mixture is charged into the reaction vessel, the oven temperature is set to 185 ° C, and the temperature is kept under hydrothermal conditions for 2 h; Cooling to room temperature to obtain a dark green solution; (3) The solution was placed in a constant temperature water bath at 60 ° C, magnetically stirred, and 98% sulfuric acid was added dropwise to the solution until the pH of the solution was 2-3. A large amount of yellow solid precipitate was observed and centrifuged at 12000 r/min for 10 min. Remove the supernatant, wash it with deionized water multiple times, and drain it. The obtained yellow precipitate was dried in an oven at 60 ° C to obtain 85.18% of the citrazinic acid product.
With methanol; toluene-4-sulfonic acid Erhitzen des Reaktionsgemisches mit wss. NH3 auf 130grad und anschliessenden Erwaermen mit NaOH;
With tetramethlyammonium chloride; trichlorophosphate at 130℃; for 18h;
Multi-step reaction with 3 steps
1: 76 percent / POCl3 / 18 h / 130 °C / Heating
2: 100 percent / lithium hydroxide / tetrahydrofuran; H2O / 1.5 h / 20 °C
3: oxalyl chloride / tetrahydrofuran; CH2Cl2 / 4 h
With tetramethlyammonium chloride; trichlorophosphate
Scheme 1
Synthesis of 4 begins with pyridine derivative citrazinic acid. Treatment with POC13 at elevated temperature afforded the corresponding 2-6, dichloroisonicotinoyl chloride. To facilitate purification, the reaction was quenched with methanol; methyl ester 1 was isolated in 76% yield after passage through a plug of silica gel to remove colored impurities. Saponification then provided acid 2 in quantitative yield without purification. Transformation to 3 was effected by conversion to the acyl azide, thermal Curtius rearrangement, and hydrolysis of the resulting trifluoroacetamide to provide 2, 6-DICHLORO-4-AMINOPYRIDINE (3), (Pfister, J. R. , Wymann, W. E. Synthesis 1983,38). While this method for converting 2 to 3 is nominally 3 steps, it requires only a single extractive workup and the steps can thus be carried out in rapid succession. 3 was converted directly to 4 by diazotization and reaction with potassium iodide (it is necessary to stir 3 in cold hydrochloric acid for several hours prior to diazotization in order to obtain an acceptable yield) providing 4 in reasonable yield and excellent purity after trituration with acetone. This short sequence of reactions allows preparation of 4 IN-35% overall yield, requires no chromatography beyond a single filtration through a plug of silica gel, and has allowed the routine preparation of 5-10 g quantities of this intermediate. Unlike many 4-halopyridines, 4 is stable for several months at room temperature if protected from light. 4 can be ready transformation to fluorophores of the present invention.
Multi-step reaction with 3 steps
1.1: trichlorophosphate / 18 h / 130 °C
2.1: lithium hydroxide / tetrahydrofuran; water / 0.33 h / 20 °C
2.2: 2 h / 0 °C
3.1: oxalyl dichloride / tetrahydrofuran / 2 h / Reflux
Multi-step reaction with 3 steps
1: trichlorophosphate / 24 h / 130 °C
2: lithium hydroxide monohydrate / tetrahydrofuran; water / 0.33 h / 25 °C
3: oxalyl dichloride / dichloromethane; tetrahydrofuran / 4 h / 25 °C
A mixture of citrazinic acid (5.0 g, 32.2 mmol) and POBr3 (27.5 g, 96.8 mmol) is heated at 130 0C. Upon completion of the reaction, the thick slurry is cooled to 0 0C and the reaction is carefully quenched with MeOH (250 ml_). The reaction mixture is concentrated in vacuo and then extracted between dichloromethane and sat. aq. NaHCO3. Organic layer is dried over anhydrous Na2SO4 and concentrated in vacuo to give a tan solid that is clean enough by NMR/LCMS to use further (7.5 g, 79%). (ESI) m/z 295.8 (M+1 ). 1H NMR (400 MHz, CD2CI2) delta ppm 8.10 (s, 2 H), 4.05 (s, 3 H).
50%
The compound PBr3 (21.8 ml, 0.232 mol) was loaded into a 250 ml three -neck flask cooled in a room temperature water bath under a flow of nitrogen. Br 2 (11.9 ml, 0.231 mol) was then added dropwise with stirring to PBr 3 to yield a yellow solid (PBr5). 30 minutes was taken for addition of Br 2. P2O5 (12.0 g, 0.083 mol) was then <n="64"/>added and the solid mixture was mixed well with a spatula under a flow of nitrogen. A reflux condenser was added via a connection to a water -filled bubbler, to trap evolved HBr. The mixture was heated to 98 0C for 2 hours and then cooled to room temperature. Citrazinic acid (20.0 g, 0.128 mol) was then added and the solid was mixed thoroughly with a spatula under a flow of nitrogen. The mixture was heated at 1850C for 8 hours. The mixture was cooled back to room temperature. Methanol (150 ml) was slowly added (the reaction with methanol was exothermic, causing the solution to reflux). After the addition of methanol, the mixture was stirred until room temperature. Solid NaHCO3 was added slowly until bubbling stopped. After which, water (300 ml) was added and then additional NaHCO 3 was added until the bubbling stopped. A brown solid product was collected with filtration. The solid was washed with water and dried at room temperature under air. The product was purified by silica gel column using hexanes/ethyl acetate (9: 1) as a solvent. The final pure white product was obtained by recrystallization from methanol/water: 19.02 g (50.0%).1H NMR (CDCl 3): delta 8.00 (s, 2H), 3.97 (s, 3H). 13C NMR (CDCl3): delta 162.74, 141.35, 141.25, 126.56, 53.38. MS (EI) m/z (%): 294.8 (100%) [M +].
In a three necked round bottom flask equipped with a mechanic stirrer and gas outlet solid cirtrazinic acid (15 g) and phosphorous oxybromide (45 g) were thoroughly mixed and heated to 140 C. for 12 to 14 h. The resulting mixture was cooled to 0 C. before MeOH (100 ml) was added carefully under vigorous stirring. The resulting mixture was then poured into a cooled (0 C.) aqueous sodium carbonate solution (1 M, 500 ml), before Chloroform (500 ml) was added. The biphasic mixture was filtered through a paper filter, before the organic layer was separated. After filtering through charcoal, the solution was concentrated in vacuum. The residue was purified by MPLC (DCM:MeOH=100:3 to 100:6) to yield methyl 2,6 dibromo-isonicotinate (13.7 g).HPLC (Rt)=1.62 (method D)
Step 2: To a stirred solution of citrazinic acid (15 g) is added phosphorous oxybromide (45 g) and the mixture heated to140C. After 14 h the mixture is cooled to 0C and MeOH (100 ml) added carefully under vigorous stirring. The mixture is then poured into a cooled (0C) aqueous sodium carbonate solution (1 M, 500ml), and chloroform (500 ml) is added. The biphasic mixture is filtered and the organic layer separated. After filtering through charcoal, the solution is concentrated in vacuum. The residue is purified by MPLC (dichloromethane:MeOH 100:3 to 100:6) to yield methyl 2,6-dibromoisonicotinate (13.7 g). HPLC (Rt) = 1 .62 (method D). To a stirred solution of I5 (2.6 g) in dioxane (30 ml) under argon is added methyl 2,6-dibromoisonicotinate (2.2g), palladium acetate (167mg), Xanthphos (432mg) and CS2CO3 (5.6g) and the mixture refluxed for 1 h. The mixture is allowed to cool to room temperature and then added to water and extracted with EtOAc. The organic extracts are washed with brine, dried under Na2SO4, filtered and the solvent evaporated under reduced pressure. The crude product is purified by HPLC (Method E) affording I6 (0.6 g).
To a stirred solution of citrazinic acid (15 g) is added phosphorous oxybromide (45 g) and the mixture heated to 140 C. After 14 h the mixture is cooled to 0 C. and MeOH (100 ml) added carefully under vigorous stirring. The mixture is then poured into a cooled (0 C.) aqueous sodium carbonate solution (1M, 500 ml), and chloroform (500 ml) is added. The biphasic mixture is filtered and the organic layer separated. After filtering through charcoal, the solution is concentrated in vacuum. The residue is purified by MPLC (dichloromethane:MeOH 100:3 to 100:6) to yield methyl 2,6-dibromoisonicotinate (13.7 g). HPLC (Rt)=1.62 (method D). To a stirred solution of I5 (2.6 g) in dioxane (30 ml) under argon is added methyl 2,6-dibromoisonicotinate (2.2 g), palladium acetate (167 mg), Xanthphos (432 mg) and Cs2CO3 (5.6 g) and the mixture refluxed for 1 h. The mixture is allowed to cool to room temperature and then added to water and extracted with EtOAc. The organic extracts are washed with brine, dried under Na2SO4, filtered and the solvent evaporated under reduced pressure. The crude product is purified by HPLC (Method E) affording I6 (0.6 g).
Citrazinic acid (1.0 kg, 6.4 mol, 1 eq.) and sulfolane (6.3 kg, 5 vol.), melted beforehand (in an oven at 60 C.), are placed in a 20 L reactor. The reaction medium is stirred at a temperature of 90 C. and a solution of POBr3 (2.77 kg, 9.7 mol, 1.5 eq.) in sulfolane (3.53 kg, 2.8 vol.), prepared beforehand, is then introduced over 39 minutes. [Ti=93.3 C.?m.p.=89.0 C.]. The reaction medium is stirred at a temperature of 125 C. for 2 hours at this temperature. The reaction progress is monitored by HPLC: disappearance of the citrazinic acid. The reaction medium is cooled to a temperature of 40 C., and methanol (2.61 L, 10 eq.) is then introduced over 30 minutes (Ti=43.9 C.?m.p.=47.0 C.]. The reaction medium is stirred at 45 C. for 30 minutes and the methanol is distilled off under vacuum (V=0.64 L). The reaction medium (m=13.38 kg) is placed in a suitable container. (0214) Water (8.0 kg, 8 vol.) is placed in a 20 L reactor, and the preceding reaction medium is then introduced at 20 C. so as to precipitate the crude intermediate product. The reaction medium is cooled to a temperature of 0 C. and then stirred at this temperature for 1 hour. The solid is filtered off and rinsed with water (4×3 L). The product is loaded back into the reactor and MeTHF (4.30 kg, 5 vol.) is then added, along with 0.68 kg of active charcoal. The reaction medium is heated at 50 C. and left stirring at this temperature for 30 minutes. The reaction medium is filtered through Clarcel and then rinsed with dichloromethane (3×1 L) and then rinsed with MeTHF (4×1.7 kg). The organic phase is washed with saturated NaCl solution (1.0 L, 1 vol.). (0215) The organic phase is codistilled with MeTHF (8 L) until the water content reaches 0.03%. Production of 4.84 kg of a solution of methyl 2,6-dibromoisonicotinate in MeTHF (mass assay at 20.45% w/w by HPLC-external calibration-i.e. 0.99 kg of methyl 2,6-dibromoisonicotinate). Mass yield=52%, HPLC purity=94%.
2,6-bis-tert-butoxycarbonyloxy-pyridine-4-carboxylic acid ethyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
15%
Stage #1: ethanol; citrazinic acid With sulfuric acid for 3h; Heating;
Stage #2: di-<i>tert</i>-butyl dicarbonate With dmap; triethylamine In dichloromethane at 20℃;
(2-{6-[2-(<i>tert</i>-butoxycarbonylamino-methyl)-4-methoxy-phenyl]-4-formyl-pyridin-2-yl}-5-methoxy-benzyl)-carbamic acid <i>tert</i>-butyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
Multi-step reaction with 8 steps
1: 76 percent / POCl3 / 18 h / 130 °C
2: 100 percent / aq. LiOH / tetrahydrofuran / 0.5 h / 23 °C
3: 93 percent / BH3*SMe2 / tetrahydrofuran / 12 h / 80 °C
4: 91 percent / imidazole; 4-dimethylaminopyridine / CH2Cl2 / 2 h / 23 °C
5: 1.49 g / Cs2CO3; Pd2(dba)3; (t-Bu)3P / tetrahydrofuran / 12 h / 80 °C
6: 53 percent / NiCl2; NaBH4 / methanol / 12 h / 20 °C
7: 88 percent / TBAF / tetrahydrofuran / 0.25 h / 0 °C
8: 80 percent / DMSO; (COCl)2; Et3N / CH2Cl2 / -78 - 20 °C
(2-{6-[2-(<i>tert</i>-butoxycarbonylamino-methyl)-4-methoxy-phenyl]-4-hydroxymethyl-pyridin-2-yl}-5-methoxy-benzyl)-carbamic acid <i>tert</i>-butyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
Multi-step reaction with 7 steps
1: 76 percent / POCl3 / 18 h / 130 °C
2: 100 percent / aq. LiOH / tetrahydrofuran / 0.5 h / 23 °C
3: 93 percent / BH3*SMe2 / tetrahydrofuran / 12 h / 80 °C
4: 91 percent / imidazole; 4-dimethylaminopyridine / CH2Cl2 / 2 h / 23 °C
5: 1.49 g / Cs2CO3; Pd2(dba)3; (t-Bu)3P / tetrahydrofuran / 12 h / 80 °C
6: 53 percent / NiCl2; NaBH4 / methanol / 12 h / 20 °C
7: 88 percent / TBAF / tetrahydrofuran / 0.25 h / 0 °C
{2-[6-[2-(<i>tert</i>-butoxycarbonylamino-methyl)-4-methoxy-phenyl]-4-(<i>tert</i>-butyl-dimethyl-silanyloxymethyl)-pyridin-2-yl]-5-methoxy-benzyl}-carbamic acid <i>tert</i>-butyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
Multi-step reaction with 6 steps
1: 76 percent / POCl3 / 18 h / 130 °C
2: 100 percent / aq. LiOH / tetrahydrofuran / 0.5 h / 23 °C
3: 93 percent / BH3*SMe2 / tetrahydrofuran / 12 h / 80 °C
4: 91 percent / imidazole; 4-dimethylaminopyridine / CH2Cl2 / 2 h / 23 °C
5: 1.49 g / Cs2CO3; Pd2(dba)3; (t-Bu)3P / tetrahydrofuran / 12 h / 80 °C
6: 53 percent / NiCl2; NaBH4 / methanol / 12 h / 20 °C
[2-(6-[2-(<i>tert</i>-butoxycarbonylamino-methyl)-4-methoxy-phenyl]-4-{2-[2-(2-methoxy-ethoxy)-ethylcarbamoyl]-vinyl}-pyridin-2-yl)-5-methoxy-benzyl]-carbamic acid <i>tert</i>-butyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
Multi-step reaction with 11 steps
1: 76 percent / POCl3 / 18 h / 130 °C
2: 100 percent / aq. LiOH / tetrahydrofuran / 0.5 h / 23 °C
3: 93 percent / BH3*SMe2 / tetrahydrofuran / 12 h / 80 °C
4: 91 percent / imidazole; 4-dimethylaminopyridine / CH2Cl2 / 2 h / 23 °C
5: 1.49 g / Cs2CO3; Pd2(dba)3; (t-Bu)3P / tetrahydrofuran / 12 h / 80 °C
6: 53 percent / NiCl2; NaBH4 / methanol / 12 h / 20 °C
7: 88 percent / TBAF / tetrahydrofuran / 0.25 h / 0 °C
8: 80 percent / DMSO; (COCl)2; Et3N / CH2Cl2 / -78 - 20 °C
9: 100 percent / t-BuOK / tetrahydrofuran / 0.25 h / 0 °C
10: aq. LiOH / tetrahydrofuran / 4 h / 23 °C
11: 0.260 g / iPr2NEt; DEPBT / tetrahydrofuran / 12 h / 23 °C
[2-(6-{2-[(2-<i>tert</i>-butoxycarbonylamino-acetylamino)-methyl]-4-methoxy-phenyl}-4-{2-[2-(2-methoxy-ethoxy)-ethylcarbamoyl]-vinyl}-pyridin-2-yl)-5-methoxy-benzylcarbamoyl]-methyl}-carbamic acid <i>tert</i>-butyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
Multi-step reaction with 13 steps
1: 76 percent / POCl3 / 18 h / 130 °C
2: 100 percent / aq. LiOH / tetrahydrofuran / 0.5 h / 23 °C
3: 93 percent / BH3*SMe2 / tetrahydrofuran / 12 h / 80 °C
4: 91 percent / imidazole; 4-dimethylaminopyridine / CH2Cl2 / 2 h / 23 °C
5: 1.49 g / Cs2CO3; Pd2(dba)3; (t-Bu)3P / tetrahydrofuran / 12 h / 80 °C
6: 53 percent / NiCl2; NaBH4 / methanol / 12 h / 20 °C
7: 88 percent / TBAF / tetrahydrofuran / 0.25 h / 0 °C
8: 80 percent / DMSO; (COCl)2; Et3N / CH2Cl2 / -78 - 20 °C
9: 100 percent / t-BuOK / tetrahydrofuran / 0.25 h / 0 °C
10: aq. LiOH / tetrahydrofuran / 4 h / 23 °C
11: 0.260 g / iPr2NEt; DEPBT / tetrahydrofuran / 12 h / 23 °C
12: HCl; iPr3SiH / dioxane / 1 h / 23 °C
13: 0.033 g / DIEA; DEPBT / tetrahydrofuran / 12 h / 23 °C
Multi-step reaction with 2 steps
1: POCl3; Me4NCl / 18 h / 130 °C
2: NaN3 / acetone; H2O
Multi-step reaction with 4 steps
1: trichlorophosphate / 24 h / 130 °C
2: lithium hydroxide monohydrate / tetrahydrofuran; water / 0.33 h / 25 °C
3: oxalyl dichloride / dichloromethane; tetrahydrofuran / 4 h / 25 °C
4: sodium azide / water; acetone / 0.03 h / 25 °C
Multi-step reaction with 3 steps
1.1: trichlorophosphate / 40 - 105 °C
1.2: 20 - 60 °C
2.1: hydrazine hydrate / ethanol / 50 - 55 °C
3.1: hydrogenchloride; sodium nitrite / toluene; water / 0 - 10 °C
Multi-step reaction with 3 steps
1.1: trichlorophosphate / 40 - 105 °C
2.1: thionyl chloride / dichloromethane / 0 - 45 °C
2.2: 0 - 10 °C
3.1: hydrogenchloride; sodium nitrite / toluene; water / 0 - 10 °C
With sodium hydroxide; In water; at 27 - 30℃; for 0.5 - 25h;pH 7.0;Medium; fermentation;Product distribution / selectivity;
Example 2: Activity of preparing 2-hydroxyisonicotinic acid of isolated bacteria strains Ten mL of medium C was charged into a test tube (25 mL volume), and was sterilized by an autoclave at 121C, for 20 minutes. A piece of the bacteria of the Delftia species YGK-A649 (FERM BP-10389), Delftia species YGK-C217 (FERM BP-10388), or Acidovorax species YGK-A854 (FERM BP-10387), which was picked up by a platinum loop from a nutrient agar wherein it was maintained, was inoculated to the medium in the test tube respectively and the whole was incubated at 27C, for 25 hours while shaking respectively. The medium C for the Delftia species YGK-A649 was adjusted to pH 7.0 after sterilization, and those for other species was adjusted to pH8.5. After the culture was completed, the desired 2-hydroxyisonicotinic acid and 2,6-dihydroxyisonicotinic acid, which is a by-product, which are contained in the supernatant obtained by centrifugation from broth, were measured, and the molar rate (%) of 2-hydroxyisonicotinic acid converted from isonicotinic acid contained in the broth was calculated. The purity [P] of the 2-hydroxyisonicotinic acid was calculated from the following equation: [Show Image] wherein "a" is a molar amount of the 2-hydroxyisonicotinic acid, "b" is a molar amount of the 2,6-dihydroxyisonicotinic acid. In addition, the bacterial cells in 1 mL of the culture were collected by centrifugation, and the resulting bacterial cells were suspended in 0.5 mL of a 0.1 M potassium phosphate buffer (adjusted to pH 7.0) containing isonicotinic acid (0.5 w/v%). The suspension was incubated at 30C, for 30 minute, while shaking. The amount of resulting 2-hydroxyisonicotinic acid and the amount of resulting 2,6-dihydroxyisonicotinic acid were determined by HPLC analysis. An accumulating reaction activity of the 2-hydroxyisonicotinic acid with respect to 1 L of broth (U/L broth) [hereinafter referred to as an activity of resting bacterial cell (U/L broth)]was determined under the condition that an amount (1 mumol/min) of 2-hydroxyisonicotinic acid accumulated was 1 unit (U). Then, according to the above equation, the purity of 2-hydroxyisonicotinic acid in a reaction of resting bacterial cell was calculated. The results are shown in Table 1. The title "2HINA" in the Table 1 denotes 2-hydroxyisonicotinic acid.
With sodium hydroxide; In water; at 27 - 30℃; for 0.5 - 25h;pH 7.0 - 8.5;Medium; fermentation;Product distribution / selectivity;
Example 2: Activity of preparing 2-hydroxyisonicotinic acid of isolated bacteria strains Ten mL of medium C was charged into a test tube (25 mL volume), and was sterilized by an autoclave at 121C, for 20 minutes. A piece of the bacteria of the Delftia species YGK-A649 (FERM BP-10389), Delftia species YGK-C217 (FERM BP-10388), or Acidovorax species YGK-A854 (FERM BP-10387), which was picked up by a platinum loop from a nutrient agar wherein it was maintained, was inoculated to the medium in the test tube respectively and the whole was incubated at 27C, for 25 hours while shaking respectively. The medium C for the Delftia species YGK-A649 was adjusted to pH 7.0 after sterilization, and those for other species was adjusted to pH8.5. After the culture was completed, the desired 2-hydroxyisonicotinic acid and 2,6-dihydroxyisonicotinic acid, which is a by-product, which are contained in the supernatant obtained by centrifugation from broth, were measured, and the molar rate (%) of 2-hydroxyisonicotinic acid converted from isonicotinic acid contained in the broth was calculated. The purity [P] of the 2-hydroxyisonicotinic acid was calculated from the following equation: [Show Image] wherein "a" is a molar amount of the 2-hydroxyisonicotinic acid, "b" is a molar amount of the 2,6-dihydroxyisonicotinic acid. In addition, the bacterial cells in 1 mL of the culture were collected by centrifugation, and the resulting bacterial cells were suspended in 0.5 mL of a 0.1 M potassium phosphate buffer (adjusted to pH 7.0) containing isonicotinic acid (0.5 w/v%). The suspension was incubated at 30C, for 30 minute, while shaking. The amount of resulting 2-hydroxyisonicotinic acid and the amount of resulting 2,6-dihydroxyisonicotinic acid were determined by HPLC analysis. An accumulating reaction activity of the 2-hydroxyisonicotinic acid with respect to 1 L of broth (U/L broth) [hereinafter referred to as an activity of resting bacterial cell (U/L broth)]was determined under the condition that an amount (1 mumol/min) of 2-hydroxyisonicotinic acid accumulated was 1 unit (U). Then, according to the above equation, the purity of 2-hydroxyisonicotinic acid in a reaction of resting bacterial cell was calculated. The results are shown in Table 1. The title "2HINA" in the Table 1 denotes 2-hydroxyisonicotinic acid.
With sodium hydroxide; In water; at 27 - 30℃; for 0.5 - 25h;pH 7.0 - 8.5;Medium; fermentation;Product distribution / selectivity;
Example 2: Activity of preparing 2-hydroxyisonicotinic acid of isolated bacteria strains Ten mL of medium C was charged into a test tube (25 mL volume), and was sterilized by an autoclave at 121C, for 20 minutes. A piece of the bacteria of the Delftia species YGK-A649 (FERM BP-10389), Delftia species YGK-C217 (FERM BP-10388), or Acidovorax species YGK-A854 (FERM BP-10387), which was picked up by a platinum loop from a nutrient agar wherein it was maintained, was inoculated to the medium in the test tube respectively and the whole was incubated at 27C, for 25 hours while shaking respectively. The medium C for the Delftia species YGK-A649 was adjusted to pH 7.0 after sterilization, and those for other species was adjusted to pH8.5. After the culture was completed, the desired 2-hydroxyisonicotinic acid and 2,6-dihydroxyisonicotinic acid, which is a by-product, which are contained in the supernatant obtained by centrifugation from broth, were measured, and the molar rate (%) of 2-hydroxyisonicotinic acid converted from isonicotinic acid contained in the broth was calculated. The purity [P] of the 2-hydroxyisonicotinic acid was calculated from the following equation: [Show Image] wherein "a" is a molar amount of the 2-hydroxyisonicotinic acid, "b" is a molar amount of the 2,6-dihydroxyisonicotinic acid. In addition, the bacterial cells in 1 mL of the culture were collected by centrifugation, and the resulting bacterial cells were suspended in 0.5 mL of a 0.1 M potassium phosphate buffer (adjusted to pH 7.0) containing isonicotinic acid (0.5 w/v%). The suspension was incubated at 30C, for 30 minute, while shaking. The amount of resulting 2-hydroxyisonicotinic acid and the amount of resulting 2,6-dihydroxyisonicotinic acid were determined by HPLC analysis. An accumulating reaction activity of the 2-hydroxyisonicotinic acid with respect to 1 L of broth (U/L broth) [hereinafter referred to as an activity of resting bacterial cell (U/L broth)]was determined under the condition that an amount (1 mumol/min) of 2-hydroxyisonicotinic acid accumulated was 1 unit (U). Then, according to the above equation, the purity of 2-hydroxyisonicotinic acid in a reaction of resting bacterial cell was calculated. The results are shown in Table 1. The title "2HINA" in the Table 1 denotes 2-hydroxyisonicotinic acid.
Stage #1: citrazinic acid With tetramethlyammonium chloride; trichlorophosphate at 130℃; for 18h; Inert atmosphere;
Stage #2: diisopropylamine In diethyl ether at 0 - 20℃; for 16h; Inert atmosphere;
Stage #1: citrazinic acid With phosphorus(V) oxybromide In methoxybenzene at 120℃; for 5h; Inert atmosphere;
Stage #2: diisopropylamine In methoxybenzene at 25℃; for 1h; Inert atmosphere;
Stage #1: citrazinic acid; isopropyl alcohol at 10℃; Reflux;
Stage #2: With sodium hydrogencarbonate In water Alkaline aqueous solution;
2.1
To a 2, -dihydroxy-isonicotinicacid (1Og, 64.5 mmole) in isopropanol (60 mL) was added concentrated sulphuric acid (10 mL) slowly at 10 0C. After complete addition, reaction mixture was re fluxed for 72 hrs. Isopropanol was removed using reduced pressure and basified the reaction mixture with saturated sodium bicarbonate solution (pH = 8) and extracted with ethyl acetate (200 ml x 3). The organic layer was washed with saturated sodium bicarbonate solution (2 x 100 mL), brine (2 x 100 mL), dried over sodium sulfate and concentrated using high vacuum. Crude product was purified by column chromatography over silica gel (60-120 mesh), with 5% ethyl acetate in hexane to give 2-hydroxy-6-isopropoxy-isonicotinicacid isopropyl ester as a white solid (1.5 g, 9.74 %); MS m/z 240 (M+H+); 1H NMR δ (300 MHz, CDCl3): 1.4 (d, 12H), 3.2 (m, 2H), 6.6 (d, 2H), 11.1 (bs, IH).
With sulfuric acid at 0 - 100℃;
16.a
Example 164-{4-[4-(2-aminoethyl)piperidine-1-carbonyl]-6-isopropoxypyridine-2-yloxy}-benzamidinea) 2-Hydroxy-6-isopropoxy-isonicotinic Acid isopropyl Ester2,6-Dihydroxyisonicatonic acid (2.0 g, 12.9 mmol) was dissolved in 20 ml of propane-2-ol at 0° C. 2 ml of concentrated sulfuric acid was added to the stirred solution of dihydroxy acid at 0° C. over a period of 10 min and then the contents of the flask were refluxed overnight at 100° C. The solvent was removed under reduced pressure and the crude residual mixture was dissolved in 250 ml of ethylacetate and washed with water. The organic phase was dried over anhydrous sodium sulphate, concentrated under reduced pressure and subjected to column chromatography, using chloroform:ethylacetate (8:2) as eluant to afford 0.5 g of required product. 1H NMR (CDCl3): δ 1.2 (6H, d), 1.35 (6H, d), 4.8 (1H, m), 5.25 (1H, m), 6.85 (1H, s), 6.95 (1H, s), 10.52 (1H, brs).
Combine 2,6-dihydroxyisonicotinic acid Int-J14a (10g, 64.47mmol) andConcentrated sulfuric acid (18.00g, 183.67mmol, 10mL)The mixture in methanol (200 mL) was heated to reflux and stirred for 72 hours.The reaction solution was concentrated, the resulting residue was diluted with ethyl acetate, washed with saturated sodium chloride solution and dried, concentrated, and the resulting residue was purified by silica gel column chromatography (ethyl acetate rinse),The title compound Int-J14b (8.1 g, 44.22 mmol, yield: 68.59%) was obtained.
39.4%
Stage #1: methanol; citrazinic acid at 10℃; Reflux;
Stage #2: With sodium hydrogencarbonate In water Alkaline aqueous solution;
1.1
To a solution of 2, 6-dihydroxy-isonicotinicacid (1Og, 64.5 mmole) in methanol (60 mL) was added concentrated sulfuric acid (10 mL) slowly at 10 0C. After complete addition, reaction mixture was refluxed for 72 hrs. Methanol was removed at reduced pressure and the resultant was basified with saturated sodium bicarbonate solution (pH = 8) and extracted with ethyl acetate (200 ml x 3). The organic layer was washed with saturated sodium bicarbonate solution (2 x 100 mL), brine (2 x 100 mL), dried over sodium sulfate and concentrated at reduced pressure. Crude product was purified by column chromatography over silica gel (60-120 mesh), with 5% ethyl acetate in hexane to give 2- hydroxy-6-methoxy-isonicotinic acid methyl ester as a white solid (4.3 g, 39.4%); MS m/z 184 (M+H+); 1H NMR δ (300 MHz, CDCl3): 3.8 (s, 3H), 3.9 (s, 3H), 6.6 (s, 2H), 11.2 (bs, IH).
30%
With sulfuric acid for 72h; Reflux;
Methyl 2-hydroxy-6-methoxyisonicotinate (139)
A suspension of 2,6-dihydroxyisonicotinic acid (10.00 g, 64.5 mmol) in MeOH (60 mL) was treated dropwise with H2S04 (10 mL, 18.4 M, 184 mmol). The solution was refluxed for 72 h and then evaporated. The residue was treated with sat. aq. NaHC03 to pH 8 and extracted with EtOAc (3 x 200 mL). The organic extracts were washed with sat. aq. NaHC03 and brine, then dried and evaporated to give 139 (3.55 g, 30%). 1H MR (DMSO-d6) δ 11.2 (bs, 1H), 6.61 (bs, 2H), 3.84 (s, 3H), 3.83 (s, 3H). Found: [M+H]=184.2.
30%
With sulfuric acid for 72h; Reflux;
4.1.1.3. 6-Bromo-3-((2-isopropoxy-6-methoxypyridin-4-yl)methyl)-2-methoxyquinoline (VI: Y = 4-aza, 3-OMe, 5-OiPr). A suspension of2,6-dihydroxyisonicotinic acid (10.00 g, 64.5 mmol) in MeOH (60mL) was treated dropwise with H2SO4 (10 mL, 18.4 M, 184 mmol).The solution was refluxed for 72 h and then evaporated. The residuewas treated with sat. aq. NaHCO3 to pH 8 and extracted withEtOAc (3 200 mL). The organic extracts were washed with sat.aq. NaHCO3 and brine, then dried and evaporated to give methyl2-hydroxy-6-methoxyisonicotinate (3.55 g, 30%) as a tan solid,mp 183-185 C. 1H NMR (DMSO d6) d 11.2 (bs, 1H), 6.61 (bs, 2H),3.84 (s, 3H), 3.83 (s, 3H). Found: [M+H] = 184.2.
30%
With sulfuric acid for 72h; Reflux;
A suspension of 2,6-dihydroxyisonicotinic acid (10.00 g,64.5 mmol) in MeOH (60 mL) was treated dropwise with H2SO4(10 mL, 18.4 M, 184 mmol). The solution was refluxed for 72 h andthen evaporated. The residue was treated with sat. aq. NaHCO3 to pH 8and extracted with EtOAc (3 × 200 mL). The organic extracts were washed with sat. aq. NaHCO3 and brine, then dried and evaporated to give methyl 2-hydroxy-6-methoxyisonicotinate (S4) (3.55 g, 30%). 1HNMR (DMSO-d6) δ 11.2 (bs, 1H), 6.61 (bs, 2H), 3.84 (s, 3H), 3.83 (s,3H). Found: [M+H] = 184.2.
Stage #1: citrazinic acid With tetramethlyammonium chloride; trichlorophosphate at 130℃; for 16h;
Stage #2: isopropyl alcohol In dichloromethane
Stage #3: With sodium acetate; sodium hydroxide In dichloromethane; water
a
a) A suspension of citrazinic acid (40 g, 0.258 mol) and tetramethylammonium chloride (29.4 g, 0.268 mol) in POCl3 (71 mL, 0.774 mol) is heated up to 130° C. for 16 h. The mixture is then cooled down to rt and diluted with DCM (250 mL). The dark solution is added dropwise to isopropanol (1 L). After addition, the solvents are removed by distillation and the crude product is dissolved in DCM (200 mL), and added dropwise to a well-stirred 200 mL 10% NaOAc solution. The pH is kept around 7-8 by addition of 4N aq. NaOH. The layers are separated and the org. phase is washed with water (500 mL) followed with brine (150 mL) and evaporated to dryness. The dark solid is purified by distillation under HV (90° C.) to yield 2,6-dichloro-isonicotinic acid isopropyl ester as a white crystalline solid (60 g); LC-MS: tR=1.03 min, [M+H]+=233.97. 1H NMR (CDCl3): δ 1.41 (d, J=6.0 Hz, 6H), 5.29 (m, 1H), 7.81 (s, 2H).
Stage #1: citrazinic acid With tetramethlyammonium chloride; trichlorophosphate at 130℃; for 16h;
Stage #2: isopropyl alcohol In dichloromethane
Stage #3: With sodium acetate; sodium hydroxide In dichloromethane; water
a
A suspension of citrazinic acid (40 g, 0.258 mol) and tetramethylammonium chloride (29.4 g, 0.268 mol) in POCI3 (71 mL, 0.774 mol) is heated up to 1300C for 16 h. The mixture is then cooled down to rt and diluted with DCM (250 mL). The dark solution is added dropwise to isopropanol (1 L). After addition, the solvents are removed by distillation and the crude product is dissolved in DCM (200 mL), and added dropwise to a well-stirred 200 mL 10% NaOAc solution. The pH is kept around 7-8 by addition of 4N aq. NaOH. The layers are separated and the org. phase is washed with water (500 mL) followed with brine (150 mL) and evaporated to dryness. The dark solid is purified by distillation under HV (900C) to yield 2,6-dichloro-isonicotinic acid isopropyl ester as a white crystalline solid (60 g); LC-MS: tR = 1.03 min, [M+H]+ = 233.97. 1H NMR (CDCI3): £ 1.41 (d, J = 6.0 Hz, 6 H), 5.29 (m, 1 H), 7.81 (s, 2 H).
Stage #1: citrazinic acid With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In N,N-dimethyl-formamide at 20℃; for 0.0833333h;
Stage #2: N-(3-(aminomethyl)benzyl)-3,5-dichloro-N-(4-fluorobenzyl)-2-hydroxybenzenesulfonamide With dmap In N,N-dimethyl-formamide
2
To a solution of citrazinic acid (60 mg, 0.39 mmol) in DMF (4 mL) at rt was added l-(3-(dimethylamino)propyl)-3-ethyl-carbodiimde hydrochloride(EDCI.HC1, 90 mg, 0.46 mmol) followed by HOBt-H2O (52 mg) at rt. After 5 min. the free base of sulfonamide 5 (180 mg, 0.38 mmol) and DMAP (50 mg) were added to the mixture, and the reaction mixture was stirred overnight. The next morning it was mostly concentrated in vacuo, and the residue was purified by preparative HPLC to obtain the product 8 as a light brown solid (60 mg, 25 %).[00169] 1H NMR (CD3OD): δ 7.57 (s, 1H), 7.52 (s, 1H), 7.10-6.99 (m, 6H), 6.81 (t, J= 8.8 Hz, 2H), 4.35 (s, 2H), 4.34 (s, 2H), 4.32 (s, 2H); MS (ESI): (M+H)+ « 606.38
Stage #1: citrazinic acid With phosphorus(V) oxybromide at 130℃; for 3h;
Stage #2: With ammonium hydroxide at 0 - 20℃;
60.A
A mixture of citrazinic acid (10.0 g, 64.4 mmol) and POBr3 (64.0 g, 225.4 mmol) is placed in a flask with HBr receiver (a saturated KOH reservoir is attached with condenser through a funnel) and heated at 130 0C for 3 h. The mixture is allowed to cool to 0 0C and the reaction is carefully quenched with NH4OH (about 350 ml_). The mixture is stirred at room temperature over night. The solid is collected through filtration to give 1st batch of title compound (5.6 g). [(ESI) m/z 280.8 (M+1 ), 1H NMR (400 MHz, CDCI3) δ ppm 7.80 (s, 2 H), 5.65 - 6.23 (m, 2 H). The filtrate is extracted with CH2CI2 (X2). Combined organic layer is washed with brine, dried (Na2SO4), filtered and concentrated to 2nd batch of title compound (2.76 g). [(ESI) m/z 278.8, 280.8, 282.8 (M+1 )]. The total 8.4 g of title compound 2,6- dibromo-isonicotinamide is obtained in 47% yield.
With P2O5; bromine; phosphorus tribromide; sodium hydrogencarbonate; In methanol; water; ethyl acetate;
Synthesis of Methyl 2,6-dibromoisonicotinate (YZ-I-131) The compound PBr3 (21.8 ml, 0.232 mol) was loaded into a 250 ml three-neck flask cooled in a room temperature water bath under a flow of nitrogen. Br2 (11.9 ml, 0.231 mol) was then added dropwise with stirring to PBr3 to yield a yellow solid (PBr5). 30 minutes was taken for addition of Br2. P2O5 (12.0 g, 0.083 mol) was then added and the solid mixture was mixed well with a spatula under a flow of nitrogen. A reflux condenser was added via a connection to a water-filled bubbler, to trap evolved HBr. The mixture was heated to 98 C. for 2 hours and then cooled to room temperature. Citrazinic acid (20.0 g, 0.128 mol) was then added and the solid was mixed thoroughly with a spatula under a flow of nitrogen. The mixture was heated at 185 C. for 8 hours. The mixture was cooled back to room temperature. Methanol (150 ml) was slowly added (the reaction with methanol was exothermic, causing the solution to reflux). After the addition of methanol, the mixture was stirred until room temperature. Solid NaHCO3 was added slowly until bubbling stopped. After which, water (300 ml) was added and then additional NaHCO3 was added until the bubbling stopped. A brown solid product was collected with filtration. The solid was washed with water and dried at room temperature under air. The product was purified by silica gel column using hexanes/ethyl acetate (9:1) as a solvent. The final pure white product was obtained by recrystallization from methanol/water: 19.02 g (50.0%). 1H NMR (CDCl3): delta 8.00 (s, 2H), 3.97 (s, 3H). 13C NMR (CDCl3): delta 162.74, 141.35, 141.25, 126.56, 53.38. MS (EI) m/z (%): 294.8 (100%) [M+].
(9Z,12Z)-octadeca-9,12-dien-1-yl 2,6-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)isonicotinate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
5.2 g
With potassium carbonate In N,N-dimethyl-formamide at 80℃;
24.24a Intermediate 24a: (9Z, 12Z)-octadeca-9, 12-dien-1-yl 2,6-bis((9Z, 12Z)-octadeca-9,12-dien-1- yloxy)isonicotinate
Intermediate 24a: (9Z, 12Z)-octadeca-9, 12-dien-1-yl 2,6-bis((9Z, 12Z)-octadeca-9,12-dien-1- yloxy)isonicotinate A solution of citrazinic acid (1.8 g, 1 1.6 mmol) in DMF (60 mL) was stirred at room temperature and linoleyl mesylate (16.0 g, 46.4 mmol) and potassium carbonate (8.02 g, 58.0 mmol) were added. The mixture was heated to 80 deg C overnight and then cooled to room temperature and water (50 mL) and EtOAc (100 mL) were added. The organic phase was collected and dried over sodium sulfate and then the volatiles removed under reduced pressure. The resulting crude material was purified on silica using heptanes/EtOAc as eluent, providing 5.2 g of the desired product. 3C NMR (100 MHz, CDCI3) δ 165.3, 163.5, 142.9, 130.2, 130.1 , 130.1 , 120.8, 127.9, 101.2, 66.6, 65.7, 31.6, 29.7, 29.7, 29.5, 29.4, 29.4, 29.3, 29.3, 29.1 , 28.6, 27.2, 26.1 , 26.0, 25.6, 22.6, 14.1 ppm.
2,6-bis(methylsulfanyl)pyridine-4-carboxylic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
90%
With N,N-dimethyl-formamide at 0 - 150℃; for 18h;
To a solution of 2,6-dichloroisonicotinic acid (4.00 g, 20.8 mmol) in DMF (40 mL) at 0 °C was added sodium thiomethoxide (4.38 g,65.5 mmol). The reaction mixture was stirred at 150 °C for 18 h. Water(40 mL) was added to the resultant solution and the pH was adjusted to ~ 3 using 2 M HCl solution. The aqueous solution was extracted with EtOAc (3× 40 mL), dried with MgSO4, filtered and concentrated underreduced pressure to give 2,6-bis(methylthio)isonicotinic acid (S35) asan orange solid, which was recrystallized from methanol (4.01 g, 90%).1H NMR (CDCl3) δ 7.44 (s, 2H), 2.61 (s, 6H). Found: [M+H] = 216.5.
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