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Structure of 638-07-3 * Storage: {[proInfo.prStorage]}
* 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.
The synthesis of 2-(chloromethyl)-1H-benzo[d]imidazoles (3a–f) is the same as described for compound 3a. SnCl2 (0.1 mmol) was added to a stirred solution of ortho-phenylenediamine (1,1 mmol) and ethyl 4-chloro-3-oxobutanoate (2, 1 mmol) in EtOH (2mL) at rt.The mixture was stirred under reflux for 6 h. After completion of the reaction, monitored by thin-layer chromatography(TLC), the solvent was removed under reduced pressure. The crude product was subjected to column chromatography (hexane=AcOEt 84:16) to give the pure benzimidazoles.
Reference：
[1] Green Chemistry, 2017, vol. 19, # 24, p. 5818 - 5830
[2] Synthetic Communications, 2015, vol. 45, # 14, p. 1642 - 1651
4-chloroEthyl acetoacetate,100mmol thiourea, 50mL ethanol, reflux 4h, spin solvent was evaporated to yield an oilLiquid, was added 20mL of ethyl acetate was dissolved, was added dropwise under ice-cooling with stirring, concentrated hydrochloric acid, the precipitated white solid was suction filtered, ethyl acetateA white solid was washed ester 2-aminothiazol-4-acetate hydrochloride, the filter cake was dissolved in water, adjusted to pH 10 ammonia water, ethyl acetateExtracted, dried over anhydrous sodium sulfate, and rotation of the solvent afforded a white solid 2-aminothiazol-4-acetate, yield 78percent, m.p.94 ~ 95 .
Reference：
[1] Patent: CN105541752, 2016, A, . Location in patent: Paragraph 0035; 0036; 0037
[2] Journal of Heterocyclic Chemistry, 1980, vol. 17, p. 1255 - 1257
[3] Synlett, 1999, # 8, p. 1239 - 1240
4
[ 420-04-2 ]
[ 638-07-3 ]
[ 53266-94-7 ]
Reference：
[1] Journal of Heterocyclic Chemistry, 2017, vol. 54, # 5, p. 2703 - 2707
5
[ 638-07-3 ]
[ 108-46-3 ]
[ 69716-04-7 ]
Yield
Reaction Conditions
Operation in experiment
67%
Stage #1: at 20℃; for 2 h; Stage #2: With sodium hydroxide In water for 2 h; Reflux
Weigh 4-ethyl chloroacetoacetate (8. 2g, 50mmol) was dissolved in 20mL of concentrated sulfuric acid in an ice-water bath was cooled to 0 ° C, the reaction system was added in divided portions resorcinol (5. 5g, 50mmol) and naturally to room temperature, stirring was continued for 2 hours, The starting material until the reaction was complete. The reaction solution was poured into 200mL ice water mixture, large amount of solid precipitated, was filtered, and the resulting solid was repeatedly washed with water, dried, and the resulting crude product was used without further purification in the next reaction. The resulting crude product from the previous step was dissolved 400ml 1M in aqueous NaOH and heated at reflux for 2 hours. The reaction was cooled to room temperature, the aqueous solution was washed twice with ethyl acetate, the aqueous phase retained, concentrated sulfuric acid, 200 mL ethyl acetate three times, the combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give The product YZ-5 (6. 3g), 67percent yield over two steps.
General procedure: A mixture of phenol (1.0 mmol), β-ketoester (1.5 mmol) and MNESA (0.075 g) was stirred at 90 °C in a round-bottomed flask for the appreciated time. After completion of the reaction as confirmed by TLC, the reaction mixture was cooled down to room temperature and the catalyst was separated from the reaction mixture using an external magnetic Some field. water was then added to the reaction mixture and the product was extracted using EtOAc (2 9 10 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered and concentrated under vacuumto yield the crude product. For more purification, the crude product was purified by recrystallization in ethanol to obtain the desired purity.
91%
With Ag supported on the hydroxyapatite-core–shell magnetic γ-Fe2O3 nanoparticles In neat (no solvent) at 80℃; for 0.333333 h; Green chemistry
General procedure: In a round bottom flask, g-Fe2O3HAp-Ag NPs (10 mg) were added to the mixture of the phenolic compound (1 mmol) and ethyl acetoacetate (1 mmol) at 80 C and the reaction mixture stirred for the appropriate time (Table 3). The progress of the reaction was monitored by TLC (eluent, n-hexane:ethyl acetate, 4:1) analysis. Upon completion of the reaction, EtOH was added to the reaction mixture and the g-Fe2O3HAp-Ag NPs were separated with an external magnet. The solvent was then removed under reduced pressure and the resulting product was purified by recrystallization using ethanol. The coumarin derivatives were obtained in good to excellent isolated yields (83percent–96percent).
84%
at 20℃; for 2 h;
Reference Example 28; 4- (chloromethyl) -7-hydroxy-2H-chromen-2- one; <n="88"/>Under ice-cooling, ethyl 4-chloroacetoacetate (14.0 g, 85.0 mmol) was dissolved in concentrated sulfuric acid (30 mL) , resorcinol (8.81 g, 80.0 mmol) was added by small portions, and the mixture was stirred at room temperature for 2 hr. The reaction mixture was poured into ice water, and the precipitated solid was collected by filtration, washed with water, and air-dried to give the title compound (14.1 g, yield 84percent) as a beige powder. MS m/z 211 (M + H)+.
84%
at 20℃; Cooling with ice
Example 30: 7-(3-(4-(3-(6-fluoro-benzisoxazole)-1-piperidyl)-n-propoxy))-4-hydroxymethyl-2H-benzopyran-2-one (30) [0067] The target compound was synthesized according to Scheme 3. 1) 50 ml of concentrated sulfuric acid was stirred in an ice bath, to which was added 5.5 g of resorcinol and added 8 g of 4-chloro ethyl acetoacetate dropwsie. The solution turned yellowish and turbid slowly. The reaction was performed at room temperature overnight. The reaction liquid was poured into ice/water mixture, and a lot of white solid was precipitated, which was filtrated. The cake was washed with water. The cake was recrystallized with 40percent ethanol to give 7.5 g of white crystal. Melting point: 183-185°C, yield: 84percent.
84%
at 20℃;
1) 50 ml of concentrated sulfuric acid was stirred in an ice bath, to which was added 5.5 g of resorcinol and added 8 g of 4-chloro ethyl acetoacetate dropwise. The solution turned yellowish and turbid slowly. The reaction was performed at room temperature overnight. The reaction liquid was poured into ice/water mixture, and a lot of white solid was precipitated, which was filtrated. The cake was washed with water. The cake was recrystallized with 40percent ethanol to give 7.5 g of white crystal. Melting point: 183-185° C., yield: 84percent.
84%
With nanosilica molybdic acid 2 In neat (no solvent) at 80℃; for 0.333333 h; Green chemistry
General procedure: In a general experimental procedure, β-ketoester 3(1 mmol) was added to a mixture of substituted phenol4 (1 mmol) and SMA NPs 2 (5 molpercent) in a solvent-freetube. The reaction mixture was stirred in a preheated oilbath (80 °C). After the completion of the reaction, the precipitateobtained was extracted with ethyl acetate, washedwith water (3 × 10 ml) and dried to obtain the product.The remaining insoluble solid catalyst in aqueous phasewas separated by filtration, washed with ethyl acetate
75.6%
at 0 - 20℃; for 2 h;
Intermediate 88: 4-(Chloromethyl)-7-hydroxy-2tf-chromen-2-oneTo a 250 mL RB flask fitted with magnetic stirrer was charged with Ethyl 4- chloroacetoacetate (22.12 g, 134.9 mmol) was dissolved in concentrated sulfuric acid (48 mL) at 0 , and resorcinol (14.0 g, 127.3 mmol) was ad ded portionwise. The mixture was stirred at room temperature for 2 h. The reaction mixture was poured into ice-water, and the resulting solid was collected by filtration, washed with water, and dried to give 4- (chloromethyl)-7-hydroxy-2H-chromen-2-one (21.0 g, 75.6 percent) as a beige solid.
70%
at 65℃; for 5 h;
A resorcinol (5.5 g, 0.05 mol)Ethyl 4-chloroacetoacetate (9.9 g, 0.06 mol)Anhydrous bismuth chloride (0.8 g, 2.5 mmol)Mixed in a 100 mL round bottom flask,It was heated in the absence of a solvent The reaction was stirred 65 , 5h,After the reaction,Add 16 mL of 50percent aqueous ethanol solution,Continue to stir at 65 ° C for 10 min,Cooled to room temperature, suction filtration, solid parts washed to neutral,Dried to give 7.45 g of a white solid,Is 4-chloromethyl-7-hydroxycoumarin, the yield of 70percent.
67%
With sulfuric acid In acetic acid at 60℃; for 4 h; Cooling with ice
M-bisphenol (27.5 g, 250 mmol) was placed in acetic acid (60mL) heated to 50 ° C for use, ethyl 4-chloroacetoacetate(20.5 g, 125 mmol) was dissolved in acetic acid (20 mL), cooled in an ice-water bath, concentrated sulfuric acid (10 mL) was slowly added, then, the acetic acid solution of m-bisphenol was added to the ice-water bath, stirred at room temperature for 1 hour, and then reacted at 60 deg. C for 3 hours. After completion of the reaction, water (300 mL) was added and the mixture was stirred at room temperature for 1 hour. The resulting white solid was suction-washed three times with water (100 mL) and dried to give the product (17.6g, 67percent yield).
60%
With methanesulfonic acid In toluene at 80 - 110℃;
Resorcinol (2 g, 18.18 mmol) was dissolved in 50 mL of hottoluene (80 oC). Chloroacetoacetate (3.5 g, 18.18 mmol, 3 mL)was added and allowed to dissolve. The solution was heated to110 oC and methanesulfonic acid (1 mL) was added. After 1 h,the hot toluene containing the product was decanted. The product crystallized uponcooling, it was filtered and dried under vacuum. The resulting gum from thedecanting step was re-suspended in toluene, heated and decanted. The procedurewas repeated until most of product 5 was recovered as a light brown solid (2.3 g,60percent yield).
45.7%
at 0℃; for 2 h;
4-Chloromethyl-7-hydroxy-2H-chromen-2-oneResorcinol (7.0 g, 63.6 mmol), ethyl 4-chloroacetoacetate (9.5 ml, 69.9 mmol) and 104 ml of 96percent sulphuric acid were stirred for 2 hours at 0 0C. The reaction mixture was poured into ice water (200 ml) and extracted with ethyl acetate. The organic layers were collected, washed with NaHCO3 10percent aqueous solution, then with water, dried over sodium sulphate and evaporated under vacuum. The resulting oil was purified by column chromatography on silica gel (eluant CHCl3/AcOEt 7.5/2.5 v/v) yielding 5.22 g (45.7percent) of a white solid used without any further purification for the next step synthesis.1H-NMR (Acetone-d6) δ: 9.50 (s, IH, exchanges with D2O), 7.73 (d, IH5 J=8.8), 6.91(dd, IH, J=8.8, J=2.5), 6.80 (d, IH, J=2.5), 6.40 (s, IH), 4.92 (s, 2H).
23.4 g
at 40 - 50℃; for 2 h;
Step 1: 4-Chloromethyl-7-hydroxy-chromen-2-one [0239] 33.5 g (304 mmol) resorcine is dissolved in 105 ml acetic acid at 40° C. 25.0 g (152 mmol) 4-chloroacetic acid ethylester is added and the funnel is rinsed with 10 ml acetic acid. Then, 26.8 g (274 mmol) conc. H2SO4 (97percent) is added and the funnel is rinsed with 10 ml acetic acid. The mixture is heated to 50° C. for approx. 2 h. After full conversion, 330 ml water is added. The reaction mixture is cooled to r.t. and the suspension is stirred overnight. The product is filtered off, washed with water (2×50 ml) and dried. Yield: 23.4 g; Rf=0.42 (silica gel, PE/EtOAc=6/4); Mass spectrum (ESI): m/z=211 [M+H]+.
23.4 g
With sulfuric acid In acetic acid at 40 - 50℃; for 0.2 h;
Step 1: 4-chloromethyl-7-hydroxy-chromen-2-one 33.5 g (304 mmol) resorcine is dissolved in 105 ml acetic acid at 40 °C. 25.0 g (152 mmol) 4-chloroacetic acid ethylester is added and the funnel is rinsed with 10 ml acetic acid. Then, 26.8 g (274 mmol) cone. H2S04(97percent) is added and the funnel is rinsed with 10 ml acetic acid. The mixture is heated to 50 °C for approx. 2 h. After full conversion, 330 ml water is added. The reaction mixture is cooled to r.t. and the suspension is stirred overnight. The product is filtered off, washed with water (2 x 50 ml) and dried. Yield: 23.4 g; Rf= 0.42 (silica gel, PE/EtOAc = 6/4); Mass spectrum (ESI): m/z = 21 1 [M + H]+
5.6 g
at 0℃; for 2 h;
4-chloroacetoacetate (4.25 ml, 31.43 mmol) was dissolved in 20 ml of concentrated sulfuric acid at 0 ° C, The resulting pale yellow viscous solution was placed in an ice bath and cooled to about _5 ° C, Resorcinol (3.15 g, 28.57 mmol) was added in portions, Control the internal temperature below 0 ° C, plus complete, room temperature stirring 2 h, The reaction solution was poured into 50 ml of ice water and precipitated as a white solid. The filtrate was filtered, washed with water (5 ml x2) and dried to obtain 5.6 g of a white solid and 82percent of the crude product. [0085] Take the crude product (2 g, 9.50 mmol) in 200 ml single-necked flask and add IN NaOH solution (100 ml). The solution immediately became concentrated yellow. The solution was heated in an oil bath for 2 h. Finished, cooled to room temperature, with concentrated sulfuric acid regulation PH to 2-3, the resulting solution was extracted with ethyl acetate (30 ml X4). The organic phases were combined, washed with saturated brine (20 ml X2), dried over anhydrous sodium sulfate, filtered and the filtrate was evaporated under reduced pressure. Brown columnar crystals 1.3 g, crude yield 71.2percent.(1 g, 5.20 mmol) was suspended in 10 ml of methanol, 0.5 ml of concentrated sulfuric acid was added dropwise, and the reaction was refluxed for about 4 hours. After completion of the reaction, the methanol was distilled off under reduced pressure and the residual liquid was poured into 30 ml The mixture was extracted with ethyl acetate (20 ml x3) and the organic phases were combined, washed with saturated sodium bicarbonate solution (15 ml X), washed with saturated brine (15 ml X), dried over anhydrous sodium sulfate, filtered, The filtrate was evaporated under reduced pressure to give a yellowish brown oil which was purified by column chromatography (petroleum ether / ethyl acetate, 80: 20, v / v) to give 0.75 g of a pale yellow solid in 70percent yield.
0.188 g
at 0℃; Cooling with ice
Under the condition of the ice, will be 1.10 g resorcinol is dissolved in concentrated sulfuric acid in the amount of drying, stirring to completely dissolve, slowly dropping 1.62 ml of 4 - chloro acetyl ethyl acetate, its temperature is 0 °C, after dropping, is moved to the reaction at room temperature, stirring overnight, the reaction [...] poured into ice water, stirring, to be solid after fully depositing, filtering and drying, to obtain 0.188 g intermediate 1, name for 4 - chloro methyl -7 - hydroxy coumarin
Reference：
[1] Tetrahedron Letters, 2005, vol. 46, # 36, p. 6119 - 6121
[2] Journal of Organic Chemistry, 2008, vol. 73, # 1, p. 287 - 290
[3] Synthetic Communications, 2008, vol. 38, # 13, p. 2082 - 2088
[4] Journal of Chemical Research, 2008, # 4, p. 232 - 234
[5] Synthetic Communications, 2010, vol. 40, # 22, p. 3358 - 3364
[6] Tetrahedron Letters, 2002, vol. 43, # 50, p. 9195 - 9197
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[9] Catalysis Communications, 2011, vol. 14, # 1, p. 62 - 67
[10] Research on Chemical Intermediates, 2016, vol. 42, # 6, p. 6089 - 6103
[11] Chinese Chemical Letters, 2017, vol. 28, # 1, p. 75 - 82
[12] Heterocycles, 2007, vol. 71, # 3, p. 677 - 682
[13] RSC Advances, 2016, vol. 6, # 24, p. 20269 - 20275
[14] Journal of the Chinese Chemical Society, 2014, vol. 61, # 2, p. 213 - 216
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[16] Journal of Materials Chemistry B, 2018, vol. 6, # 17, p. 2547 - 2556
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[18] Patent: WO2008/1931, 2008, A2, . Location in patent: Page/Page column 86-87
[19] Journal of Medicinal Chemistry, 2012, vol. 55, # 4, p. 1538 - 1552
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9
[ 638-07-3 ]
[ 109-73-9 ]
[ 5349-24-6 ]
Yield
Reaction Conditions
Operation in experiment
99%
With 4-toluenesulfonyl azide In tetrahydrofuran at 20 - 25℃;
General procedure: To a solution of the corresponding β-keto ester 1 (1.0 mmol) and TsN3 (197 mg, 1.0 mmol) in THF (2.0 mL) under stirring at 25 °C was added the amine (1.1 mmol). Then the reaction mixture was stirred at room temperature until consumption of the starting material (monitored by TLC: 30 min to 29 h, see Table 1 and Scheme 4). Next, the mixture was diluted in 5 mL of CH2Cl2 and concentrated under reduced pressure. After complete removal of the solvent, the residue was triturated in ethyl ether and the resulting mixture was again concentrated under reduced pressure. The final solid residue was repeatedly triturated with hexane (for amides 3 and 4) or a 9:1 hexane/CH2Cl2 mixture (for amides 5 and 6) to separate out the insoluble TsNH2 by decantation. The resulting supernatants were filtered and concentrated under reduced pressure to give the known amides25 3-6 as oils with high degree of purity. Alternatively, further purification through column chromatography on silica gel using gradient mixtures of hexane/EtOAc as eluent was employed to furnish pure products in 80-99percent yield.
Stage #1: With n-butyllithium; diisopropylamine In tetrahydrofuran; hexane at 0℃; for 1 h; Inert atmosphere Stage #2: With methyl chlorosulfate In tetrahydrofuran; hexane at -78℃; for 0.5 h; Inert atmosphere
General procedure: To a solution of diisopropylamine (340 µL, 2.4 eq.) in dry THF (2 mL) in a flame dried round bottom flask under argon at 0 °C was added n-butyllithium (1.4 mL, 1.6 M in hexanes, 2.2 eq.), and the reaction mixture was stirred at this temperature for 15 minutes. At the same temperature a solution of dicarbonyl compound (3n) (1 mmol) in THF (2 mL) was slowly added. After 1 hour of stirring at 0 °C the solution was cooled to -78 °C and methyl chlorosulfate (110 µL, 1.2 eq.) was then added. After stirring at –78 °C for 30 minutes, the reaction was quenched with saturated ammonium chloride aqueous solution (5 mL). The mixture was then extracted with dichloromethane (3 x 5 mL), the combined organic phases were dried with anhydrous magnesium sulfate and the solvent evaporated affording the desired 4-chloro dicarbonyl compound 5n.
Reference：
[1] Russian Journal of Organic Chemistry, 2004, vol. 40, # 7, p. 924 - 927
13
[ 591-78-6 ]
[ 6082-74-2 ]
[ 638-07-3 ]
Reference：
[1] Russian Journal of Organic Chemistry, 2004, vol. 40, # 7, p. 924 - 927
14
[ 591-78-6 ]
[ 6082-74-2 ]
[ 638-07-3 ]
[ 141-97-9 ]
Reference：
[1] Russian Journal of Organic Chemistry, 2004, vol. 40, # 7, p. 924 - 927
15
[ 6082-74-2 ]
[ 100-52-7 ]
[ 638-07-3 ]
[ 1503-99-7 ]
Reference：
[1] Russian Journal of Organic Chemistry, 2004, vol. 40, # 7, p. 924 - 927
16
[ 67-56-1 ]
[ 638-07-3 ]
[ 66762-68-3 ]
Yield
Reaction Conditions
Operation in experiment
15.1%
With sodium hydride In N,N-dimethyl-formamide at 0 - 20℃; for 16 h;
NaH (7.71 g, 192.7 mmol) was suspended in DMF (110 mL). The solution of MeOH (2.64 g, 82.5 mmol) in DMF (55 mL) was added drop wise at 0° C. After that, the mixture was stirred at 0° C. for 30 min. The solution of compound 1 (9.54 g, 57.9 mmol) in DMF (55 mL) was added dropwise at 0° C., then the reaction mixture was allowed to warm slowly to RT and stirred for 16 h. The reaction mixture was diluted with 1N HCl (400 mL), extracted with MTBE (250 mL×3). The combined organic layer was washed with H2O (200 mL×5), dried over Na2SO4, filtered, concentrated, purified by silica gel column (PE:EA=10:1) afforded ethyl 4-methoxy-3-oxobutanoate (1.4 g, 15.1percent) as light yellow oil.
74 g
With sodium hydride In tetrahydrofuran; mineral oil at 15 - 25℃; for 9 h; Inert atmosphere
In 2L pre-reaction bottle by adding 250 ml tetrahydrofuran, under the protection of argon, are started to stir, the oven 25 °C, the inner temperature 15-25 °C added in batches under the condition of 55g (1.35mol) sodium hydride (containing 40percent mineral oil), continue adding tetrahydrofuran after adding 450 ml; the inner temperature 20 °C slowly dropping under the condition of 30g methanol and 100g (0.61mol) 4-chloro-acetyl-acetic acid ethyl ester of mixed solution, adjusting the stirring rate in 310 R/min the left and the right, about 4h finish; to temperature rise within 20-25 °C stirring 5h, TLC detection reaction finishes; cooling system, can be seen and the color is yellowish solution a large number of solid suspended, the inner temperature 11 °C add under the condition of 200 ml tetrahydrofuran, to be oven to -5 ° C slowly adding 100 ml molar concentration is 2mol/L hydrochloric acid solution, maintained in the oven 0 °C the following, about 20 min after adding, at this time the pH of the reaction system 11, in the process of dropping the solid significant increase in the reaction bottle, immediately after adding filtering, the filter cake is a white solid, cake obtained drying filters 92g solid; the resulting solid is added to the 770g in ethyl acetate, the temperature of the reaction system to 0 °C, slow instillment mole concentration is 6mol/L hydrochloric acid solution, maintaining the temperature of the reaction system is not variable, the dropping white solid gradually disappear in the process, the reaction system is adjusted to pH 3, reaction liquid contains a small amount of inorganic salt white solid, filtering the reaction solution, the filtrate layer, the organic phase is separated, the organic phase by adding 5g activated carbon, in the 30 °C under the condition of stirring 1h, filtered, filtrate in the 35 °C ethyl acetate under the condition of the vacuum distillation to the colorless liquid 74g, HPLC checking the purity of 99.6percent.
Reference：
[1] Patent: JP2015/214548, 2015, A, . Location in patent: Paragraph 0992; 0993
[2] Patent: CN105481694, 2016, A, . Location in patent: Paragraph 0015; 0016; 0017; 0018; 0019; 0020; 0021 - 0023
17
[ 638-07-3 ]
[ 66762-68-3 ]
Reference：
[1] Patent: US2006/189664, 2006, A1, . Location in patent: Page/Page column 8
[2] Patent: US5767131, 1998, A,
18
[ 638-07-3 ]
[ 100-51-6 ]
[ 67354-34-1 ]
Yield
Reaction Conditions
Operation in experiment
90%
Stage #1: With sodium tert-pentoxide In 1,3-dimethyl-2-imidazolidinone at 20 - 40℃; for 2 h; Inert atmosphere Stage #2: at 0 - 20℃; for 5 h; Cooling with ice
Example 2 First StepA solution of benzyl alcohol (0.66 g, 6.1 mmol) in DMI (3 ml) was added to a suspension of sodium tert-pentoxide (1.67 g, 15.2 mmol) in DMI (4 ml) at room temperature under a nitrogen atmosphere, and the mixture was stirred at 40° C. for 2 hours. This reaction solution was cooled in an ice bath, and a solution of Compound 2A (1.10 g, 6.68 mmol) in DMI (3 ml) was added dropwise at 0 to 10° C. The reaction solution was stirred at 0 to 5° C. for 2 hours, and at room temperature for 3 hours, and 2N hydrochloric acid (15 ml) was added, followed by extraction with ethyl acetate two times. The combined extracts were washed sequentially with water, saturated sodium bicarbonate water, water and saturated sodium chloride water, and then dried with anhydrous sodium sulfate. The solvent was distilled off, and the resulting oil product was purified by silica gel column chromatography (n-hexane-ethyl acetate 4:1, v/v) to obtain 1.29 g (yield 90percent) of Compound 2B as an oil product.1H-NMR (CDCl3) δ: 1.25 (3H, t, J=7.2 Hz), 3.54 (2H, s), 4.14 (2H, s), 4.17 (2H, q, J=7.2 Hz), 4.59 (2H, s), 7.28-7.40 (5H, m).
90%
Stage #1: at 40℃; for 2 h; Inert atmosphere Stage #2: at 0 - 20℃; for 5 h;
A DMI (3 ml) solution of benzyl alcohol (0.66 g, 6.1 mmol) was added to a DMI (4 ml) suspension of sodium tert-pentoxide (1.67 g, 15.2 mmol) at room temperature in a nitrogen atmosphere, and the mixture was stirred at 40°C for 2 hours. This reaction solution was cooled in an ice bath, and a DMI (3 ml) solution of compound 2A (1.10 g, 6.68 mmol) was added dropwise thereto at 0-10°C. The reaction solution was stirred at 0-5°C for 2 hours and at room temperature for 3 hours, and 2 N hydrochloric acid (15 ml) was then added thereto, followed by extraction two times with ethyl acetate. The combined extracts were washed with water, a saturated aqueous solution of sodium bicarbonate, water, and saturated saline in this order and then dried over anhydrous sodium sulfate. The solvent was distilled off, and the obtained oil was purified by silica gel column chromatography (n-hexane-ethyl acetate: 4:1, v/v) to obtain 1.29 g (yield: 90percent) of compound 2B as an oil. 1H-NMR (CDCl3) δ: 1.25 (3H, t, J = 7.2 Hz), 3.54 (2H, s), 4.14 (2H, s), 4.17 (2H, q, J = 7.2Hz), 4.59 (2H, s), 7.28-7.40 (5H, m).
85%
Stage #1: With sodium hydride In tetrahydrofuran; mineral oil at 10℃; for 1 h; Cooling with ice Stage #2: at 20℃; for 0.666667 h;
Benzyl alcohol (6.90 g, 63.8 mmol) was added dropwise to a stirred suspension of sodium hydride (60percent, 5.37 g, 134 mmol) in dry THE (50 mL) under ice bath to keep the temperature below 10°C. The mixture was stirred for an hour. Ethyl 4-chloro-3-oxobutanoate (10.0 g,60.8 mmcl) was added dropwise within 40 mm at rt, and the resulting mixture was stirred overnight. The reaction mixture was carefully added to HCI (5percent, 100 mL) at 5 °C and then extracted with EtOAc (20 mL x 3). The combined organic layers were washed with brine, dried over Na2504, filtered and concentrated. The crude was purified by column chromatography (PE/EtOAc 10/1) to give the title compound (12.1 g, yield 85percent) as ayellow oil.LCMS: [mobile phase: 5-95percent CH3CN in 2.5 minI, Rt 1.55 mm, MS Calcd: 236; MS Found:237 [M+H].1H NMR (300 MHz, CDCI3): 6 7.38-7.30 (m, 5H), 4.59 (s, 2H), 4.22-4.10 (m, 4H), 3.53 (s,2H), 1.25 Ct, J = 7.2 Hz, 3H).
78%
Stage #1: With sodium tert-pentoxide In tetrahydrofuran at 40 - 45℃; for 2 h; Stage #2: at 0 - 20℃; for 3 h;
Benzyl alcohol (95 mL, 1 eq) was added to a mixture of sodium tert- pentoxide (250 g, 2.5 eq) in tetrahydrofuran (750 mL, 5V) at 20-25°C. The reaction mass was heated to 40-45°C and stirred for 2 hrs. The reaction mass was then cooled to 0-5°C and ethyl-4-chloro acetoacetate (XII-C1,150 g, leq) in tetrahydrofuran (750 mL, 5V) was added to it. The reaction mass was then stirred for 3 hrs at room temperature. After reaction completion, the reaction mass was cooled to 0-5 °C and pH was adjusted to ~2 using 20percent hydrochloric acid. The reaction mass was extracted twice with ethyl acetate (2X5V). Organic layers were combined, washed with saturated bicarbonate solution (1X10V) followed by water (5V) and saturated brine solution (5V). Organic layer was dried over anhydrous sodium sulfate and distilled off the solvent completely to get the crude compound as brown oily thick liquid. The crude compound was purified by silica gel column chromatography (Eluent: EtOAc-hexane). The pure fractions were concentrated under vacuum to afford the title compound as pale yellow liquid (168 g, 78percent yield). MS (ES): m/z 237 (M+H)+.
69%
With sodium hydride In tetrahydrofuran at 40℃; for 2 h;
Ethyl 4-(benzyloxy)-3-oxobutanoate Sodium hydride (145 g, 6.06 mol) was suspended in tetrahydrofuran (3.0 L). Benzyl alcohol (328 g, 3.03 mol) and ethyl 4-chloro-3-oxobutanoate (500 g, 3.03 mol) were added to the suspension and the reaction mixture was stirred at 40° C. for 2 h. The reaction mixture was cooled to room temperature, quenched with ice water and extracted with ethyl acetate. The combined organic layers were dried over anhydrous sodium sulphate and concentrated under vacuum to afford 500 g (69percent) of Ethyl 4-(benzyloxy)-3-oxobutanoate. 1H NMR (400 MHz, CDCl3): δ1.29 (t, 3H), 3.53 (s, 2H), 4.22 (q, 2H), 4.22 (s, 2H), 4.60 (s, 2H), 7.38 (m, 5H).
20%
Stage #1: With sodium hydride In toluene at 20℃; for 1.66667 h; Stage #2: at 20℃; for 18 h;
A suspension of sodium hydride (60percent in oil) in tolene (200 mL) was mechanically stirred at room temperature under argon and treated dropwise with benzyl alcohol in toluene (40 mL) over 40 minutes. The mixture was stirred for 1 hour, treated with ethyl 4-chloroacetate, and then stirred over 18 hours at room temperature. [0744] At end of the reaction, citric acid; was added and layers were separated. The organic layers was washed water, dried over MgSO4 and concentrated to give the the title compound (7.2 g, 20percent). MS(ES) m/z 237 (M+H)+
Reference：
[1] Journal of Medicinal Chemistry, 2016, vol. 59, # 10, p. 5051 - 5062
[2] Journal of Organic Chemistry, 2011, vol. 76, # 22, p. 9444 - 9451
[3] Angewandte Chemie - International Edition, 2006, vol. 45, # 38, p. 6376 - 6380
[4] Patent: US2012/22251, 2012, A1, . Location in patent: Page/Page column 25
[5] Patent: EP2602260, 2013, A1, . Location in patent: Paragraph 0127
[6] Patent: WO2017/12576, 2017, A1, . Location in patent: Page/Page column 120
[7] Synlett, 2000, # 6, p. 844 - 846
[8] Organic and Biomolecular Chemistry, 2009, vol. 7, # 5, p. 962 - 975
[9] Patent: WO2015/110897, 2015, A2, . Location in patent: Paragraph 00138-00140
[10] Patent: WO2015/110897, 2015, A3, . Location in patent: Paragraph 00138-00140
[11] Patent: US2008/312255, 2008, A1, . Location in patent: Page/Page column 91
[12] New Journal of Chemistry, 2016, vol. 40, # 10, p. 8786 - 8808
[13] Tetrahedron Asymmetry, 2001, vol. 12, # 12, p. 1713 - 1718
[14] European Journal of Medicinal Chemistry, 2015, vol. 97, p. 42 - 54
[15] Patent: US2004/19190, 2004, A1, . Location in patent: Page 38
[16] Monatshefte fuer Chemie, 1989, vol. 120, p. 891 - 898
[17] Synthesis, 1995, # 8, p. 1014 - 1018
[18] Organic Letters, 2010, vol. 12, # 3, p. 416 - 419
[19] Patent: WO2011/76878, 2011, A1, . Location in patent: Page/Page column 52-53
[20] Patent: WO2015/164308, 2015, A1, . Location in patent: Page/Page column 90
[21] ChemMedChem, 2018, vol. 13, # 16, p. 1658 - 1663
19
[ 638-07-3 ]
[ 88150-42-9 ]
Reference：
[1] Journal of Medicinal Chemistry, 1986, vol. 29, # 9, p. 1696 - 1702
[2] Journal of Medicinal Chemistry, 1986, vol. 29, # 9, p. 1696 - 1702
20
[ 638-07-3 ]
[ 10488-69-4 ]
Yield
Reaction Conditions
Operation in experiment
98%
With hydrogen In ethanol at 95 - 105℃; for 0.5 - 8 h;
Example 1 (Comparative . A 600ml stainless steel Parr reactor was charged with ethanol (340ml) and ethyl-4-chloroacetoacetate (53g). The reactor agitator was started and the speed set to 600rpm. The reactor was pressurised using nitrogen to 7 bar and stirring continued for 5 minutes. After 5 minutes the reactor was slowly vented to ambient pressure, the pressurisation/depressurisation cycle was repeated for a total of five times to ensure complete removal of dissolved oxygen. At the end of the last cycle the reactor set-point temperature was adjusted to 95°C. (R)-[RuCI2 (BINAP)] ncatalyst was accurately weighed (23mg) into a catalyst transfer vessel and the vessel then purged using nitrogen for 5 minutes. The catalyst was flushed from the transfer vessel using deoxygenated solvent into a 100ml stainless steel injection bomb which was attached to the Parr reactor. When the Parr reactor temperature was between 95°C and 100°C the injection bomb was pressurised to 100bar using hydrogen. Appropriate valves were then opened to transfer the catalyst mixture and hydrogen into the reactor. The contents of the reactor were stirred at 600rpm for 30 minutes before being cooled to less than 30°C. The reactor was then slowly vented to ambient pressure. The reactor contents were transferred into a 1L rotary film evaporator flask and the mixture evaporated to constant weight by application of vacuum and by using a heated water bath. The residue was subjected to pot to pot distillation under vacuum to afford a clear colourless oily liquid product of ethyl (S)- (-)-4-chloro-3-hydroxybutyrate in >98percent yield, >98percent purity and 94percent enantiomeric excess.; Example 2 A feed tank was charged with 3.6L ethanol solvent. The solvent was deoxygenated by pumping it through a spray nozzle whilst pressurising to 7bar with nitrogen and then depressurising through a needle valve at a controlled rate. The pressurisation/depressurisation cycle was repeated three times and the entire process automated using a PLC-based control system. In a similar manner a second feed tank was charged with ethyl-4-chloroacetoacetate (3.6L) and deoxygenated using the same protocol to that described above. The catalyst, (R)- [RuCl2 (BINAP)] (149mg) was charged into a transfer vessel and the vessel purged using nitrogen before transferring the catalyst into the solvent feed tank. The catalyst solution had a concentration of 52.2mg/Kg. The two feed systems were connected to the continuous hydrogenation reactor system via two high-pressure pumps. The continuous hydrogenation reactor system was constructed of Hastalloy 276 and comprised a number of in-line static mixers to give a residence time of between 30 and 35 seconds. The static mixers also ensured good mixing of the process streams and rapid absorption of hydrogen. The reactor system was equipped with a recycle pump and an in-line valve which enabled operation as either a plug flow reactor (PFR, valve closed) or a continuous loop reactor (CLR, valve open). The system was equipped with a gas/liquid separator and the liquid level inside the separator controlled using a differential pressure sensor, which in turn operated an exit flow control valve. The reactor system was controlled using a PLC based control system. The hydrogenation reactor was pressurised using hydrogen and the pressure maintained between 90 and 100 bar by continually feeding hydrogen through a mass flow controller at a rate of 2.7g/h. The reaction liquors passed through a heat exchanger using a pump such that the process temperature was maintained between 102°C and 105°C. The system above was operated as a plug flow reactor. The flow rate of the ethyl-4-chloroacetoacetate was set to 2. 6ml/minute and the flow rate of the catalyst solution set to 8. 9ml/min. These flows gave a process concentration of 30percent w/w and a substrate to catalyst ratio of 20,000 : 1. Over a series of continuous runs, each varying between 4 and 8 hours, the reactor consistently converted >99percent ethyl-4-chloroacetoacetate to (S) -ethyl-4-chloro-3-hydroxybutyrate which was isolated after removing the solvents by evaporation to give a chemical yield of >98percent and an enantiomeric excess of 98- 99percent.; Example 3 The reactor was set up as Example 2, except it was operated as a continuous loop reactor. The flow rate of the ethyl-4-chloroacetoacetate was set to 2. 55ml/minute and the flow rate of the ethanol catalyst solution set to 6. 60ml/min at a catalyst concentration of 45. 8mg/kg. These flows gave a process concentration of 37percent w/w and a substrate to catalyst ratio of 65,000 : 1. Over a series of continuous runs, each varying between 4 and 8 hours, the reactor consistently converted >99percent ethyl-4-chloroacetoacetate to (S) -ethyl-4-chloro-3-hydroxybutyrate which was isolated after removing the solvents by evaporation to give a chemical yield of >98percent and an enantiomeric excess of 98- 99percent.
96%
With D-glucose In ethanol at 25℃; for 0.25 h; Microbiological reaction; aq. phosphate buffer
General procedure: Cells harvested by centrifugation were washed and resuspended (19 g cell dry weight/l) in a 10 mM potassium phosphate buffer, pH 6.8. Cell suspension (25 ml) was taken in a 150 ml Erlenmeyer flask capped with a cotton plug and pre-incubated in a water-bath shaker at 200 rpm and 25 °C for 15 min after adding glucose (50 g/l). A solution of 0.12 g of COBE in 0.5 ml of ethanol was then added to the reaction flask. Incubation was continued for 15 min [for (R)-CHBE, the reaction time was 1 h] after the addition of COBE after which the reaction mixture was extracted with ethyl acetate (2 .x. 100 ml). The organic layer was dried over anhydrous sodium sulfate and the pure product was obtained by silica gel chromatography using hexane/ethyl acetate (9:1) as a mobile phase. In order to determine the rate of formation of CHBE, a 0.5 ml sample was taken every 5 min and centrifuged (10,000 rpm, 10 min) at 4 °C to remove the cells. The supernatant was extracted with ethyl acetate (1 ml) of which 1 μl was injected into the GC to determine the concentrations of COBE and CHBE.
87.4%
With ketoreductase; NADP In acetic acid butyl ester; water for 7 - 18 h;
Example 9: Preparation of Ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE from Ethyl 4-chloro- acetoacetate. To a 100 mL vessel connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was charged a solution of glucose (7.5 g) in 100 mM TRIETHANOLAMINE pH 7 buffer (25 ML). To this solution was charged ketoreductase SEQ ID NO: 42 (100 mg); 50 mg GDH SEQ ID NO: 66 and NADP (6.25 mg). Butyl acetate (10 ml) was then charged. Then, ethyl 4-chloroacetoacetate (6 g) in butyl acetate (10 mL) was charged. The pH was maintained at 7 by the automatic titrater by the addition of 4M NaOH (7.5 mL) over 7 hrs. A sample of the reaction mixture was extracted with an equal volume of butyl acetate and the organic layer was analyzed by GC. The analysis showed 99percent conversion of the ethyl 4-CLLLOROACETOACETATE to ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE.Example 11 : Preparation of Ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE from Ethyl 4-chloro- acetoacetate. To a 100 mL vessel connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was charged a solution of glucose (12. g) in water (30 mL). To this solution was charged ketoreductase SEQ ID NO: 42 (100 mg); 50 mg GDH SEQ ID NO: 66 and NADP (6.25 mg). Butyl acetate (10 ML) WAS then charged. Ethyl 4-chloroacetoacetate (10 g) was then charged via syringe pump as follows : 1 mL was charged rapidly and the remainder was then charged at a rate of 1 mL/hr). The pH was maintained at 7 by the automatic titrater by the addition OF 4M NAOH over 18 hours hrs. The stirring was stopped and the phases allowed to separate. The organic layer included some emulsion. The organic layer, including some emulsion, was separated and washed with 10 mL of water. The combined aqueous layers were extracted twice WITH 20 mL of butyl acetate. The organic extracts were combined and rotary evaporated under vacuum to remove water. Additional butyl acetate was added during the evaporation to help remove the water. When the water was removed the butyl acetate solution was decanted from solids in the flask. Evaporation of the solvent under vacuum then gave 8. 85 g of ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE (87.4percent yield) of very good purity.
87.4%
With ketoreductase; NADP In acetic acid butyl ester; water for 7 - 18 h;
Example 9: Preparation of Ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE from Ethyl 4-chloro- acetoacetate. To a 100 mL vessel connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was charged a solution of glucose (7.5 g) in 100 mM TRIETHANOLAMINE pH 7 buffer (25 ML). To this solution was charged ketoreductase SEQ ID NO: 42 (100 mg); 50 mg GDH SEQ ID NO: 66 and NADP (6.25 mg). Butyl acetate (10 ml) was then charged. Then, ethyl 4-chloroacetoacetate (6 g) in butyl acetate (10 mL) was charged. The pH was maintained at 7 by the automatic titrater by the addition of 4M NaOH (7.5 mL) over 7 hrs. A sample of the reaction mixture was extracted with an equal volume of butyl acetate and the organic layer was analyzed by GC. The analysis showed 99percent conversion of the ethyl 4-CLLLOROACETOACETATE to ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE.Example 11 : Preparation of Ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE from Ethyl 4-chloro- acetoacetate. To a 100 mL vessel connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was charged a solution of glucose (12. g) in water (30 mL). To this solution was charged ketoreductase SEQ ID NO: 42 (100 mg); 50 mg GDH SEQ ID NO: 66 and NADP (6.25 mg). Butyl acetate (10 ML) WAS then charged. Ethyl 4-chloroacetoacetate (10 g) was then charged via syringe pump as follows : 1 mL was charged rapidly and the remainder was then charged at a rate of 1 mL/hr). The pH was maintained at 7 by the automatic titrater by the addition OF 4M NAOH over 18 hours hrs. The stirring was stopped and the phases allowed to separate. The organic layer included some emulsion. The organic layer, including some emulsion, was separated and washed with 10 mL of water. The combined aqueous layers were extracted twice WITH 20 mL of butyl acetate. The organic extracts were combined and rotary evaporated under vacuum to remove water. Additional butyl acetate was added during the evaporation to help remove the water. When the water was removed the butyl acetate solution was decanted from solids in the flask. Evaporation of the solvent under vacuum then gave 8. 85 g of ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE (87.4percent yield) of very good purity.
50%
at 37 - 40℃; for 60 h; Enzymatic reaction; Aqueous phosphate buffer
The General Methodology for the Reduction of Substituted β-Ketoesters; 100 mg of each of the compounds in Table 3, entry Nos. 1-10 and their related substituted β-ketoesters were added to a 100 ml flask containing 2 gm of crude extract of Daucus carota, and 50 ml of 0.1 M sodium phosphate buffer pH 6.0 to 7.5 were added. The reaction was incubated in a shaker for 50 to 70 hours for maximum product formation. The product formed was isolated, purified by column chromatography and the structure of the compound was confirmed by spectral data.
97.6 % ee
With hydrogen In ethanol at 80℃; for 2 h;
In a 150 mL autoclave under argon atmosphere RuCl3 (10.2 mg, 0.049 mmol), (-)-5,5'-bis(diphenylphosphanyl)-2,2,2',2'-tetrafluoro-4,4'-bi[benzo-1,3-dioxolyl] (i.e. (-)-Fluoxphos) (34.2 mg, 0.050 mmol) and ethyl 4-chloro-3-oxobutyrate (0.81 g, 4.9 mmol, approx. 98.5percent) is dissolved in degassed ethanol (30 mL). After flushing the autoclave with argon hydrogenation is carried out during 2 hours at 80 °C and at 4 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and enantiomeric excess (ee) (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100percent at an ee of 97.6percent.
89.5 % ee
With hydrogen In ethanol at 90℃; for 2.3 h;
In a 250 mL autoclave in an argon atmosphere [Ru2Cl4(cym)2] (6.7 mg, 0.011 mmol), (-)-5,5'-bis(diphenylphosphanyl)-4,4'-bi[benzo-1,3-dioxolyl] (14.3 mg, 0.023 mmol) and ethyl 4-chloro-3-oxobutyrate (9.10 g, 54.5 mmol, approx. 98.5percent) is dissolved in degassed ethanol (30 mL). After flushing the autoclave with argon hydrogenation is carried out during 2.3 hours at 90 °C and at 30 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100percent at an ee of 89.5percent.
97.4 % ee
With hydrogen In ethanol; dichloromethane at 100℃; for 2.83333 h;
In a 1 L autoclave under argon atmosphere [RuI((-)-Fluoxphos)(cym)]I (35 mg, 0.030 mmol, prepared from (-)-Fluoxphos and [Ru2I4(cym)2] as disclosed in WO 00/29370, and ethyl 4-chloro-3-oxobutyrate (33.4 g, 200 mmol, approx. 98.5percent) is dissolved in degassed ethanol (340 mL) and dichloromethane (80 mL). After flushing the autoclave with argon hydrogenation is carried out during 170 minutes at 100 °C and at 22 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100percent at an ee of 97.4percent.
98.0 % ee
With hydrogen In ethanol; dichloromethane at 100℃; for 3 h;
In a 1 L autoclave under argon atmosphere [RuCl((-)-Fluoxphos)(cym)]BF4 (23 mg, 0.022 mmol, prepared from (-)-Fluoxphos and [Ru2Cl4(cym)2] and AgBF4 as disclosed in Mashima, K., J. Org. Chem. 1994, 59, 3064-3076, and ethyl 4-chloro-3-oxobutyrate (33.4 g, 200 mmol, approx. 98.5percent) is dissolved in degassed ethanol (340 mL) and dichloromethane (80 mL). After flushing the autoclave with argon hydrogenation is carried out during 180 minutes at 100 °C and at 22 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100percent at an ee of 98.0percent.
98.1 % ee
With hydrogen In ethanol; dichloromethane at 110℃; for 1.16667 h;
In a 1 L autoclave under argon atmosphere [Ru2Cl4(cym)2] (5.0 mg, 0.008 mmol, (-)-Fluoxphos (12.1 mg, 0.018 mmol) and ethyl 4-chloro-3-oxobutyrate (27.0 g, 162 mmol, approx. 98.5percent) is dissolved in degassed ethanol (340 mL) and dichloromethane (80 mL). After flushing the autoclave with argon hydrogenation is carried out during 70 minutes at 110 °C and at 22 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100percent at an ee of 98.1percent.
95.8 % ee
With hydrogen In ethanol at 90℃; for 2.3 h;
In a 250 mL autoclave in an argon atmosphere bis[(1-isopropyl-4-methylbenzene)dichloro ruthenium] (i.e. [Ru2Cl4(cym)2]) (6.7 mg, 0.011 mmol), (-)-Fluoxphos (16.0 mg, 0.023 mmol) and ethyl 4-chloro-3-oxobutyrate (9.11 g, 54.5 mmol, approx. 98.5percent) is dissolved in degassed ethanol (30 mL). After flushing the autoclave with argon hydrogenation is carried out during 2.3 hours at 90 °C and at 30 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100percent at an ee of 95.8percent.
80 % ee
With hydrogen In ethanol at 80℃; for 3 h;
Example 11: (5)-Ethyl4-chloro-3-hydrpxybutyrate; In a 150 mL autoclave under argon atmosphere bis(l-isopropyl-4-methylbenzene)dichloro-ruthenium (7.5 mg, 0.012 mmol), (+)-ligand Ic (14.4 mg, 0.025 mmol) and ethyl 4-chloro-3-oxobutyrate (0.83 g, 5.0 mmol) is dissolved in degassed ethanol (30 mL). After flushingthe autoclave with argon hydrogenation is carried out during 3 h at 80 °C and at 4 barhydrogen pressure. After cooling to room temperature the reaction solution is directlyanalyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column:Lipodex-E 25 m / 0.25 mm). Conversion is 100percent at an ee of 80percent.
80 % ee
With (+)-6-dicyclohexylphosphanyl-2'-diphenylphosphanyl-2-methoxy-1,1'-biphenyl; hydrogen In ethanol at 80℃; for 3 h;
In a 150 mL autoclave under argon atmosphere bis(1-isopropyl-4-methylbenzene)dichloro-ruthenium (7.5 mg, 0.012 mmol), (+)-ligand Ic (14.4 mg, 0.025 mmol) and ethyl 4-chloro-3-oxobutyrate (0.83 g, 5.0 mmol) is dissolved in degassed ethanol (30 mL). After flushing the autoclave with argon hydrogenation is carried out during 3 h at 80 °C and at 4 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100percent at an ee of 80percent.
80 % ee
With hydrogen In ethanol at 80℃; for 3 h;
Example 16: (5)-Ethyl 4-chloro-3-hydroxybutyrate; In a 150 mL autoclave under argon atmosphere bis(l-isopropyl-4-methylbenzene)dichloro-ruthenium (7.5 mg, 0.012 mmol), (+)-ligand Ic (14.4 mg, 0.025 mmol) and ethyl 4-chloro-3-oxobutyrate (0.83 g, 5.0 mmol) is dissolved in degassed ethanol (30 mL). After flushingthe autoclave with argon hydrogenation is carried out during 3 h at 80 °C and at 4 barhydrogen pressure. After cooling to room temperature the reaction solution is directlyanalyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column:Lipodex-E 25 m / 0.25 mm). Conversion is 100percent at an ee of 80percent.
98 % ee
With hydrogen In tetrahydrofuran; ethanol at 100℃; for 2 h;
Examples 3 to 17The reaction of example 1 was carried out with different catalysts and under varying conditions. The specific catalysts, conditions and results are summarized in table 1 in figure 2. Unless specified otherwise in figure 2, the reaction was carried out as described above for example 1. The solvent used in all reactions was EtOH THF. The ratio substrate/catalyst (S/C) was relatively high in most experiments. Thus only low amounts of catalyst are necessary in the inventive reaction. Further, the concentration of the substrate S was selected relatively high to 3 or 4 M. This is equivalent to a concentration of approximately 50percent (w/w). The temperature was 100°C. The pressures were adjusted to relatively low levels of between 15 and 40 bar.The results show, that for almost all catalysts a high absolute yield (conversion) and a high enantiomeric yield (ee) were obtained, although the total amounts of catalyst were very low. Further, the pressure was relatively low, which is advantageous for large scale industrial applications. High yields could be obtained even after relatively short reaction times of between 1.3 and 3 hours (examples 8 to 13). Overall, the examples show that the inventive reaction is efficient and can be carried out with low amounts of catalyst and solvent. The reaction is also energy-efficient, because the reaction time is low, the volume to be heated is small (due to low solvent levels) and the pressure is low.
> 99.9 % ee
at 30℃; for 8 h; Enzymatic reaction
General procedure: Asymmetric reductions of various carbonyl compounds by the purified enzymes were carried out at 30°C for 8h with mild shaking in a reaction mixture containing 0.1M potassium phosphate buffer (pH 6.5), 1gL−1 substrate, 10mM NADPH, and the purified enzyme of appropriate amount in a total volume of 2mL. In order to determine the absolute configuration of chiral alcohols, the reaction products were extracted with ethyl acetate or hexane and the organic layer was used for analysis. The optical purity of the reaction products were determined by chiral HPLC (HP 1100, Agilent, USA) equipped with Chiralcel OB-H column (4.6mm×250mm; Daicel Chemical Ind. Ltd., Japan) or chiral GC (7890A, Agilent, USA) equipped with FID detector and Chrompack Chirasil-Dex CB chiral capillary column (25m×0.25mm; Varian, USA) [21].
> 99 % ee
With D-glucose; nicotinamide adenine dinucleotide phosphate; recombinant Bacillus subtilis-derived glucose dehydrogenase; recombinant Gluconobacter oxydans carbonyl reductase 0525 In aq. phosphate buffer; ethanol at 30℃; for 12 h; Enzymatic reaction
General procedure: The enzyme activity toward the reduction ofketones was determined by spectrophotometricallymeasuring the oxidation of NAD(P)H at 340 nm(ε = 6.22mM−1 cm−1) in the presence of an excess amount of ketones. The change in absorbance of NAD(P)H was monitored at 340 nm using an ultraviolet-visiblespectrophotometer with a temperature-controlledcuvette holder (Shimadzu Co., Kyoto, Japan). One unit(U) activity was defined as the amount of enzymesrequired to catalyze the oxidation of 1 μmol NAD(P)Hper minute at 30 °C. The data were expressed as U/mgof protein. The standard reaction mixture contained100mM phosphate buffer (pH 7.0), 0.1mM NAD(P)H,5 mM substrate, and enzyme solvent in a total volumeof 1 mL. The reaction was initiated by adding 20 μLsolvent containing 10 μg–40 μg of enzymes. Blankswithout the enzyme were carried out for each substrate,and data were collected in triplicate. Protein concentrationswere determined with the bicinchoninic acid assayusing bovine serum albumin as a standard.NAD(P)H or NADH was assayed under standardreaction conditions for the study of coenzyme dependence.A range of substrates from 1 to 20 mM concentrationwas assayed under the standard reactionconditions for the study of kinetics. Apparent values ofMichaelis constant (Km) and kcat were calculated by fittingthe data into Michaelis–Menten equation using theSigmaPlot (Systat Software Inc., San Jose, CA, USA).All reactions followed Michaelis–Menten-type kinetics.Enantioselective reduction of ketones. The enantioselectivityof the enzymes was determined byexamining the reduction of aryl ketones, ethyl 4-chloroacetoacetate(COBE), and ethyl 2-oxo-4-phenylbutyrate(OPBE) using an NAD(P)H regeneration system consistingof BsGDH and glucose. The general procedurewas as follows: D-glucose (0.5percent), recombinant BsGDH(10 U), NAD(P)+ (0.1 mM), the recombinant cell(30 g L−1, wet weight), and ketone solvated in ethanol[1 g L−1, 10percent(v/v)] were mixed in a potassium phosphatebuffer (10 mL, 100 mM, pH 7.0). The mixturewas shaken at 30 °C for 12 h.22) Upon termination ofthe reaction, each sample was extracted twice withequivalent ethyl acetate. The organic layer wasremoved, dried, diluted in the mobile phase, and thensubjected to chiral high-performance liquid chromatography(HPLC) to determine the conversion and enantiomericexcess (e.e.). Chiral HPLC analysis wasperformed on an Agilent 1100 series HPLC system witha UV detector.23) Chiral CHBE was analyzed on a chiracelOB-H column (Daicel, Japan) at λ210 nm using hexane/2-propanol (90/10, v/v) as eluent at a flow rate of0.8 mL min−1 and a temperature of 25 °C. Chiral HPBEand 4-phenyl-2-butanol were analyzed on a chiracelOD-H column at λ210 nm and λ254 nm using hexane/2-propanol (98/2, v/v) as eluent at a flow rate of 1.0 mLmin−1 and a temperature of 30 °C. Authentic (relevant)standards were used for peak identification, and quantificationwas based on the peak area that was suitablycalibrated with standards of known concentration.
99 % ee
With D-glucose; sodium carbonate In aq. phosphate buffer; acetic acid butyl ester at 30℃; for 5 h; Microbiological reaction; Enzymatic reaction
Cells were harvested by centrifugation (10,000 g, 20 min, 4 °C) and washed with 100 mM potassium phosphate buffer (pH 6.2). The bioconversion of COBE to (S)-CHBE was performed in an aqueous–butyl acetate (4:1, v/v) biphase system containing 100 mM potassium phosphate buffer (pH 6.2), 1500 mM COBE, 1550 mM glucose, 1550 mM Na2CO3, Triton X-100 (1‰, v/v), 0.1 g of dry cell weight (DCW) of E. coli Rosseta (pET-22b-SOU1), and E. coli Rosseta (pET-22b-GDH), respectively, at a total volume of 25 mL. The reaction was performed at 30 °C and 220 rpm for 5 h. The organic layer was isolated to determine the product concentration and optical purity.
> 99 % ee
With aldo-keto reductase CaAKR; NADH In aq. phosphate buffer at 30℃; for 10 h; Enzymatic reaction
General procedure: The bioreduction was performed in 1.5 mL Eppendorf tubes containing 200 mmol/L potassium phosphate buffer (pH 7.0), 1 mmol/L of each substrate, 0.5 mmol/L NADH, and 1 mg/mL purified CaAKR in a total volume of 1.0 mL, shaking for 10 h at 30 °C. The reaction mixture was extracted twice with an equivalent volume of 300 μL ethyl acetate. The extracts were combined, dried with anhydrous sodium sulfate. The concentrations for each product were determined by GC or HPLC analysis.
0.91%
With alpha-D-glucopyranose In toluene at 50℃; for 11 h; Enzymatic reaction
Then 1.0 g COBE (6.1 mmol) and 1.2 g glucose was added. The second batch was completed within 70 min. After-wards, another 1.0 g COBE (6.1 mmol) and 1.2 g glucose was fedwhen COBE was converted thoroughly. At the 7th batch, at least3 h was required for a complete reaction. All 7.0 g COBE was fullyreduced into (S)-CHBE by merely 0.1 g RpCR-GDH dry cells within11 h without addition of external NADP+. After extraction, about6.29 g (S)-CHBE (>99percent ee) was recovered, with a molar yield ofabout 91percent. The substrate to catalyst ratio and the space-time yieldwere about 70 and 1480 g L−1d−1, which was attributed to the highcatalytic efciency of RpCR in the biphasic system with substrate-feeding.
99.95 % ee
With hydrogenchloride; dichloro-R-2,2′-bis(diphenylphosphino)-1,1′-binaphthyl ruthenium; hydrogen In methanol; water at 95 - 98℃;
In a 500 ml autoclave in a hydrogen atmosphere, was added Compound A: ethyl chloroacetoacetate (50 g, 0.30 mol), hydrochloric acid 5ml (AR, content of 36percent to 38percent), methanol 300 ml, the reaction solution was stirred, a chiral ruthenium metal catalyst (R) -RuCl2 (BINAP) (50 mg) was added, the reaction solution was replaced with hydrogen twice, the pressure rose to 8 to 10 atmospheres, insulation 95 ~ 98 deg. C reaction 1 ~ 2h. The reaction solution was cooled to room temperature, and the reaction solution was directly analyzed by GC to measure the conversion (column: HP-10125 m / 0.2 mm) and the enantiomeric excess (column: Lipodex-E25 m / 0.25 mm). The enantiomeric excess was 99.95percent and the conversion was 100percent. The filtrate was dried and filtered, and the solvent was distilled off under reduced pressure to obtain the compound B, which was directly used in a one-step reaction.
458 g
With sodium formate In ethanol at 50℃;
5 liters of reaction flask 500g (3.03 mol) of ethyl chloroacetoacetate and 2 liters of ethanol, after stirring, add a chiral catalyst (0.15 mol, 0.05 eq.) and 250g (3.68 mol, 1.2 eq.) of sodium formate, heating to 50 °C to react; the gas phase detection reaction was complete, the solvent was removed by distillation,And distilled under reduced pressure to give 485g of chiral ethyl 4-chloro-3-hydroxybutyrate;
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21
[ 638-07-3 ]
[ 10488-69-4 ]
[ 10488-69-4 ]
Yield
Reaction Conditions
Operation in experiment
82.30 % ee
at 30℃; for 8 h; Enzymatic reaction
General procedure: Asymmetric reductions of various carbonyl compounds by the purified enzymes were carried out at 30°C for 8h with mild shaking in a reaction mixture containing 0.1M potassium phosphate buffer (pH 6.5), 1gL−1 substrate, 10mM NADPH, and the purified enzyme of appropriate amount in a total volume of 2mL. In order to determine the absolute configuration of chiral alcohols, the reaction products were extracted with ethyl acetate or hexane and the organic layer was used for analysis. The optical purity of the reaction products were determined by chiral HPLC (HP 1100, Agilent, USA) equipped with Chiralcel OB-H column (4.6mm×250mm; Daicel Chemical Ind. Ltd., Japan) or chiral GC (7890A, Agilent, USA) equipped with FID detector and Chrompack Chirasil-Dex CB chiral capillary column (25m×0.25mm; Varian, USA) [21].
11.84 % ee
at 30℃; for 8 h; Enzymatic reaction
General procedure: Asymmetric reductions of various carbonyl compounds by the purified enzymes were carried out at 30°C for 8h with mild shaking in a reaction mixture containing 0.1M potassium phosphate buffer (pH 6.5), 1gL−1 substrate, 10mM NADPH, and the purified enzyme of appropriate amount in a total volume of 2mL. In order to determine the absolute configuration of chiral alcohols, the reaction products were extracted with ethyl acetate or hexane and the organic layer was used for analysis. The optical purity of the reaction products were determined by chiral HPLC (HP 1100, Agilent, USA) equipped with Chiralcel OB-H column (4.6mm×250mm; Daicel Chemical Ind. Ltd., Japan) or chiral GC (7890A, Agilent, USA) equipped with FID detector and Chrompack Chirasil-Dex CB chiral capillary column (25m×0.25mm; Varian, USA) [21].
70 % ee
With D-glucose; D-glucose dehyrodenase; Lodderomyces elongisporus aldo–keto reductase 48; NADPH In aq. phosphate buffer at 30℃; Enzymatic reaction
General procedure: The enantioselectivity of LEAKRs was determined by employingNADPH regeneration system composed of D-glucose dehydrogenase(GDH) and D-glucose. The reaction mixture included 20 mMethyl 4-chloroacetoacetate, 0.2 mM NADPH, 0.03 mg LEAKR(1.2 mg for LEAKR 49), 100 mM D-glucose, 0.1 mg GDH, and100 mM sodium phosphate buffer (pH 7.0), with the final volumeof 0.5 mL. After shaking constantly at 30 C, the reaction mixturewas extracted with 1 mL ethyl acetate. The organic layer collectedby centrifugation and dried with anhydrous sodium sulfate wasthen analyzed to determine the conversion of the substrate andenantiomeric excess (e. e.) of the products by employing GC andHPLC as efficient and sensitive tools. A GC station (Agilent6890N) with a capillary column (DB-5) was used to analyze theconversion of ethyl 4-chloroacetoacetate while an HPLC station(Agilent 1100) with a Chiralcel OB-H column was employed forthe optical assay of the products.
58 % ee
With D-glucose; D-glucose dehyrodenase; Lodderomyces elongisporus aldo–keto reductase 49; NADPH In aq. phosphate buffer at 30℃; Enzymatic reaction
The enantioselectivity of LEAKRs was determined by employingNADPH regeneration system composed of D-glucose dehydrogenase(GDH) and D-glucose. The reaction mixture included 20 mMethyl 4-chloroacetoacetate, 0.2 mM NADPH, 0.03 mg LEAKR(1.2 mg for LEAKR 49), 100 mM D-glucose, 0.1 mg GDH, and100 mM sodium phosphate buffer (pH 7.0), with the final volumeof 0.5 mL. After shaking constantly at 30 C, the reaction mixturewas extracted with 1 mL ethyl acetate. The organic layer collectedby centrifugation and dried with anhydrous sodium sulfate wasthen analyzed to determine the conversion of the substrate andenantiomeric excess (e. e.) of the products by employing GC andHPLC as efficient and sensitive tools. A GC station (Agilent6890N) with a capillary column (DB-5) was used to analyze theconversion of ethyl 4-chloroacetoacetate while an HPLC station(Agilent 1100) with a Chiralcel OB-H column was employed forthe optical assay of the products.
21.6 % ee
With D-glucose; nicotinamide adenine dinucleotide phosphate; recombinant Bacillus subtilis-derived glucose dehydrogenase; recombinant Gluconobacter oxydans carbonyl reductase 0644 In aq. phosphate buffer; ethanol at 30℃; for 12 h; Enzymatic reaction
General procedure: The enzyme activity toward the reduction ofketones was determined by spectrophotometricallymeasuring the oxidation of NAD(P)H at 340 nm(ε = 6.22mM−1 cm−1) in the presence of an excess amount of ketones. The change in absorbance of NAD(P)H was monitored at 340 nm using an ultraviolet-visiblespectrophotometer with a temperature-controlledcuvette holder (Shimadzu Co., Kyoto, Japan). One unit(U) activity was defined as the amount of enzymesrequired to catalyze the oxidation of 1 μmol NAD(P)Hper minute at 30 °C. The data were expressed as U/mgof protein. The standard reaction mixture contained100mM phosphate buffer (pH 7.0), 0.1mM NAD(P)H,5 mM substrate, and enzyme solvent in a total volumeof 1 mL. The reaction was initiated by adding 20 μLsolvent containing 10 μg–40 μg of enzymes. Blankswithout the enzyme were carried out for each substrate,and data were collected in triplicate. Protein concentrationswere determined with the bicinchoninic acid assayusing bovine serum albumin as a standard.NAD(P)H or NADH was assayed under standardreaction conditions for the study of coenzyme dependence.A range of substrates from 1 to 20 mM concentrationwas assayed under the standard reactionconditions for the study of kinetics. Apparent values ofMichaelis constant (Km) and kcat were calculated by fittingthe data into Michaelis–Menten equation using theSigmaPlot (Systat Software Inc., San Jose, CA, USA).All reactions followed Michaelis–Menten-type kinetics.Enantioselective reduction of ketones. The enantioselectivityof the enzymes was determined byexamining the reduction of aryl ketones, ethyl 4-chloroacetoacetate(COBE), and ethyl 2-oxo-4-phenylbutyrate(OPBE) using an NAD(P)H regeneration system consistingof BsGDH and glucose. The general procedurewas as follows: D-glucose (0.5percent), recombinant BsGDH(10 U), NAD(P)+ (0.1 mM), the recombinant cell(30 g L−1, wet weight), and ketone solvated in ethanol[1 g L−1, 10percent(v/v)] were mixed in a potassium phosphatebuffer (10 mL, 100 mM, pH 7.0). The mixturewas shaken at 30 °C for 12 h.22) Upon termination ofthe reaction, each sample was extracted twice withequivalent ethyl acetate. The organic layer wasremoved, dried, diluted in the mobile phase, and thensubjected to chiral high-performance liquid chromatography(HPLC) to determine the conversion and enantiomericexcess (e.e.). Chiral HPLC analysis wasperformed on an Agilent 1100 series HPLC system witha UV detector.23) Chiral CHBE was analyzed on a chiracelOB-H column (Daicel, Japan) at λ210 nm using hexane/2-propanol (90/10, v/v) as eluent at a flow rate of0.8 mL min−1 and a temperature of 25 °C. Chiral HPBEand 4-phenyl-2-butanol were analyzed on a chiracelOD-H column at λ210 nm and λ254 nm using hexane/2-propanol (98/2, v/v) as eluent at a flow rate of 1.0 mLmin−1 and a temperature of 30 °C. Authentic (relevant)standards were used for peak identification, and quantificationwas based on the peak area that was suitablycalibrated with standards of known concentration.
88.8 % ee
With Kluyveromyces polysporus alcohol dehydrogenase S237R mutant; isopropyl alcohol; NADPH In aq. phosphate buffer at 30℃; Enzymatic reaction
General procedure: Bioconversion was conducted with 20 mM 1a–10a,20 U·mL−1KpADH variants, 40 mM isopropanol in PBS buffer (pH 7.0,100 mM) in total volume of 2 mL at 30 °C and 180 rpm overnight. Then,1 mL of the reaction mixture was withdrawn and extracted with ethylacetate. The organic phase was isolated by centrifugation and driedover anhydrous MgSO4. The conversion rate and enantioselectivity ofthe products were analyzed as described in supporting information.
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[58] Journal of Molecular Catalysis B: Enzymatic, 2016, vol. 123, p. 67 - 72
[59] European Journal of Inorganic Chemistry, 2017, vol. 2017, # 20, p. 2762 - 2773
[60] Organic Process Research and Development, 2017, vol. 21, # 9, p. 1349 - 1354
[61] Catalysis Communications, 2018, vol. 108, p. 1 - 6
22
[ 638-07-3 ]
[ 10488-69-4 ]
Reference：
[1] Journal of Molecular Catalysis B: Enzymatic, 2011, vol. 69, # 3-4, p. 89 - 94
23
[ 3891-07-4 ]
[ 638-07-3 ]
[ 88150-75-8 ]
Yield
Reaction Conditions
Operation in experiment
93%
With sodium hydride In tetrahydrofuran at -10 - 32℃;
a, 2400 ml of tetrahydrofuran was charged into the reaction flask A, and the mixture was cooled to -10 °C or less, 750 g of N-hydroxyethyl phthalimide and 345 g of NaH were added successively (while stirring at a low speed, N-hydroxyethyl phthalimide, NaH added to the beginning of the dropwise addition of ethyl 4-chloroacetoacetate time interval within 30min); the reaction flask temperature down to -10 ° C below, 732 g of ethyl 4-chloroacetoacetate was added slowly dropwise, and the temperature was controlled below 0 °C and the addition was completed in 1.5 to 2 hours. The temperature was raised to 28 to 32 °C. After 5 to 6 hours of reaction, 1500 ml of toluene was added, The temperature dropped below 5 °C to give a toluene dilution of the reaction solution; b, add 1700 ml of toluene, 2800 ml of water and 1178 g of acetic acid to the reaction flask B at a temperature below 5 °C; add the toluene dilution of the above reaction solution with 1 to 1.5 h at a temperature below 10 °C; After stirring for 10 min, the mixture was allowed to stand for 15 min, and the aqueous layer was removed. The toluene layer was washed 5 times with a mass fraction of 4percent brine (2500 ml each). After the washed toluene solution was added anhydrous magnesium sulfate, And the toluene solution was concentrated under reduced pressure to remove toluene (the amount of toluene removed was 2700 ml or more and the toluene content was not more than a predetermined value) to give ethyl-4- (2-phthalimidoethoxy) acetoacetate in a yield of 93percent
51%
Stage #1: With sodium hydride In tetrahydrofuran at -10℃; for 0.583333 h; Stage #2: at -10 - 20℃; for 18 h;
Preparation of Ethyl 4-[2-(PHTHALIMIDO) ETHOXY] ACEFOACETATE (X1 A 250 mL two-necked flask, equipped with a magnetic stirring bar, thermometer and a pressure equalized addition funnel was charged with 75 mL of tetrahydrofuran under nitrogen atmosphere. 7.53 g Sodium hydride (60percent dispersed in oil) was added and the resulting suspension was cooled to-10 °C and 20 g of N- (2-HYDROXYETHYL) PHTHALIMIDE was added slowly over 5 minutes. The resulting slurry was stirred at-10 °C for 30 minutes. To this mixture a solution of 16.35 g ethyl 4-chloroacetoacetate in 30 ML of TETRAHYDRAFURAN was added at-10 °C in 40 minutes. The reaction mixture was warmed to room temperature and then stirred at room temperature for 18 hours. The reaction mixture was placed in an ice bath and quenched by dropwise addition of 10 mL ethanol. The mixture was then poured into 150 mL of IN hydrochloric acid solution in crushed ice and 200 ML of ethyl acetate was added. The resulting mixture was transferred into a separatory funnel and the aqueous phase was separated. The organic phase was first washed with 70 mL of ethyl acetate was added. The resulting mixture was transferred into a separatory funnel and the aqueous phase was separated. The organic phase was first washed with 70 mL of 5 wt. percent OF NAHC03 solution, then with 150 ML of water, dried over 5 G of MgS04. MgS04 was filtered off and the filtrate was concentrated in vacuo to give a light brown oily product. The oil was washed with 15 mL of hexane to remove the mineral oil to give 17.2 g of ethyl 4- [2- (phthalimido) ethoxy] acetoacetate light brown product. (yield 51percent, rel. compound purity > 80percent). IR (KBR, CM~L) : 2995 and 1716. 1H-NMR (CDC13) 6 7.76 (dd, 2H), 7.65 (dd, 2H), 4.11 (S, 2H), 4.02 (q, 2H), 3.80 (t, 2H), 3.70 (t, 2H), 3.37 (s, 2H), 1. 16 (t, 3H). 13C-NMR (CDC13) 8 201.7, 168.4, 167.2, 134.3, 132.2, 123.5, 75.6, 68.7, 61.6, 46.0, 37.4, 14.3.
50%
With sodium hydride In DMF (N,N-dimethyl-formamide) at 0 - 20℃;
Example 7 : 2-({2-[2-(4-{(2S)-2-Hydroxy-3-[(methylethyl)amino]propoxy}phenoxy) acetylamino] ETHOXY} METHYL)-4-(2-CHLOROPHENYL)-5-CYANO-6-METLLYL-1, 4- dihydropyridine-3-carboxylate is synthesized according to Scheme VI. SCHEME VI 0 Ho- p OEt HO out C1 Ni C1 Me NC Me N N CHO CHO ci NC Me H 0 1. acetone, II. -- . HO /O 2. II wEthyl 4- [2-FL, 3-DIOXOISOMDOLM-2-YDETHOXY1-3-OXOBUTANOATE. A solution of 2- (2- hydroxyethyl) isoindoline-1, 3-dione (10 g; 52.31 mmol) in DMF (150 ml) is cooled to 0°C and treated with 1. 1 equivalents of a 60percent dispersion of sodium hydride in mineral oil. After gas evolution has ceased, ethyl 4-chloro-3-oxobutanoate (7.75 g; 47.08 mmol) in 20 ml of DMF is added drop-wise, and the resulting solution is stirred overnight at room temperature. The pH of the mixture is then adjusted to 6-7 by the addition of 1 N HC1, and the product is partitioned between H2O and EtOAc. The aqueous phase is extracted again with EtOAc, and the combined organic phases are washed with water and brine, dried over magnesium sulfate, and concentrated to a crude residue, which is purified on a silica gel column, eluting with 25percent EtOAc in hexane to obtain 7.52 g (23.54 mmol ; 50percent) of the product as an oil.
40.5%
Stage #1: With sodium hydride In tetrahydrofuran; N,N-dimethyl-formamide; mineral oil at 20℃; for 4 h; Stage #2: at 0 - 20℃;
To a mechanically stirred suspension of NaH (15g, 0. 375 mol, 60percent dispersion in mineral oil) in THF (250 mL) and DMF (25 mL) was added N- (2-hydroxyethyl)-phtalimide (43g, 0. 225 mol). The mixture was stirred at room temperature for about 4 h and then cooled to about 0°C in an ice bath. Ethyl-4- chloroacetate (21. 4 mL, 0. 15 mol) in THF (4 mL) was then added via an addition funnel over a period of about 0. 5 h and the resulting mixture allowed to stir overnight. The mixture was then poured into a 2 L sepratory funnel containing 500 mL ice water and 700 mL EtOAc and separated. The organic layer was washed 2x with 500 mL of water and dried over MGS04. The solution was then filtered through a 4-inch plug of silica and then CONCENTRATED IN VACUO. The resulting yellow oil was then dissolved in 250 mL CH3CN and washed 2x with 30 mL hexane. The CH3CN layer was the concentrated in vacuo to yield 19. 4g (40. 5percent) OF 4- [2- (1, 3-DIOXO-1, 3-DIHYDRO-ISOINDOL-2-YL)-ETHOXY]-3-OXO-BUTYRIC acid ethyl ester as a yellow oil. This crude compound was used for the next step
40.5%
Stage #1: With sodium hydride In tetrahydrofuran; N,N-dimethyl-formamide; mineral oil at 20℃; for 4 h; Stage #2: at 0 - 20℃;
To a mechanically stirred suspension of NaH (15g, 0. 375 mol, 60percent dispersion in mineral oil) in THF (250 mL) and DMF (25 mL) was added N- (2-hydroxyethyl)-phtalimide (43g, 0. 225 mol). The mixture was stirred at room temperature for about 4 h and then cooled to about 0°C in an ice bath. Ethyl-4- chloroacetate (21. 4 mL, 0. 15 mol) in THF (4 mL) was then added via an addition funnel over a period of about 0. 5 h and the resulting mixture allowed to stir overnight. The mixture was then poured into a 2 L sepratory funnel containing 500 mL ice water and 700 mL EtOAc and separated. The organic layer was washed 2x with 500 mL of water and dried over MGS04. The solution was then filtered through a 4-inch plug of silica and then CONCENTRATED IN VACUO. The resulting yellow oil was then dissolved in 250 mL CH3CN and washed 2x with 30 mL hexane. The CH3CN layer was the concentrated in vacuo to yield 19. 4g (40. 5percent) OF 4- [2- (1, 3-DIOXO-1, 3-DIHYDRO-ISOINDOL-2-YL)-ETHOXY]-3-OXO-BUTYRIC acid ethyl ester as a yellow oil. This crude compound was used for the next step
Reference：
[1] Patent: CN106749187, 2017, A, . Location in patent: Paragraph 0031; 0032; 0033; 0034
[2] Patent: WO2004/58711, 2004, A1, . Location in patent: Page 18, 19
[3] Patent: WO2005/16885, 2005, A2, . Location in patent: Page/Page column 47-49
[4] Patent: WO2005/25507, 2005, A2, . Location in patent: Page/Page column 166-167
[5] Patent: WO2005/25507, 2005, A2, . Location in patent: Page/Page column 166-167
[6] Journal of Medicinal Chemistry, 1986, vol. 29, # 9, p. 1696 - 1702
[7] Patent: US2004/44218, 2004, A1, . Location in patent: Page 3
[8] Patent: US4572908, 1986, A,
[9] Patent: US4572909, 1986, A,
[10] Patent: US2007/260065, 2007, A1, . Location in patent: Page/Page column 5-6
[11] Patent: CN108358833, 2018, A, . Location in patent: Paragraph 0013; 0015; 0016
[12] Patent: CN108456160, 2018, A, . Location in patent: Paragraph 0006; 0014; 0015; 0016; 0020
24
[ 504-29-0 ]
[ 638-07-3 ]
[ 101820-69-3 ]
Yield
Reaction Conditions
Operation in experiment
92.8%
With copper(I) triflate; triethylamine In ethanol at 55 - 60℃;
Dissolve 23.5 g (250 mmol) of 2-aminopyridine with 100 mL of absolute ethanol, add 35.4 g of triethylamine, 2.4 g of copper trifluoromethanesulfonate to the solution, and add dropwise 100 mL of absolute ethanol to the solution 47.3 while stirring. g ethyl 4-chloroacetoacetate, heated to 55-60° C., reacted for 4-5 hours, TLC (developer: chloroform:methanol=2:1) was used to monitor the reaction solution to 2-aminopyridine.The spots disappeared, and 1percent citric acid solution was added to the solution until the pH of the solution was 7.6. The insolubles were removed by filtration and the filtrate was extracted with ethyl acetate. The organic layer was collected, dried, filtered and concentrated to give a pale yellow viscous liquid. 47.4 g, yield 92.8percent (calculated as 2-aminopyridine), purity 97.57percent, impurity 2-(imidazo[1,2-a]pyridin-2-yl)acetic acid ethyl ester 0.43percent (HPLC normalization method) .
A mixture of 2-amino-6-methylpyridine (10.00 g, 92.47 mmol), ethyl 4-chloro- acetoacetate (16.24 mL, 120.2 mmol), and polyphosphoric acid (50.00 g) was stirred at 125 0C. After 5.5 h, the mixture was removed from the heat. To the cooled mixture was added ice-water (200 mL) and neutralized with 2 N NaOH (400 mL) to pH 6-7. The resulting precipitate was collected by filtration, washed with water (~ 400 mL), and dried to give 2-(chloromethyl)-6-methyl-4H-pyrido- [l,2-a]pyrimidin-4-one as a dark brown solid: 1H NMR (400 MHz, DMSO-d6) delta ppm 7.68 (1 H, dd, J=9.0, 7.0 Hz), 7.40 (1 H, dd, J=9.0, 0.8 Hz), 6.93 (1 H, d, J=6.7 Hz), 6.36 (1 H, s), 4.58 (2 H, s), 2.93 (3 H, s); Mass Spectrum (ESI) m/e = 208.9 (M + 1).
With sodium hydride; In tetrahydrofuran; at -10 - 32℃;
a, 2400 ml of tetrahydrofuran was charged into the reaction flask A, and the mixture was cooled to -10 C or less, 750 g of N-hydroxyethyl phthalimide and 345 g of NaH were added successively (while stirring at a low speed, N-hydroxyethyl phthalimide, NaH added to the beginning of the dropwise addition of ethyl 4-chloroacetoacetate time interval within 30min); the reaction flask temperature down to -10 C below, 732 g of ethyl 4-chloroacetoacetate was added slowly dropwise, and the temperature was controlled below 0 C and the addition was completed in 1.5 to 2 hours. The temperature was raised to 28 to 32 C. After 5 to 6 hours of reaction, 1500 ml of toluene was added, The temperature dropped below 5 C to give a toluene dilution of the reaction solution; b, add 1700 ml of toluene, 2800 ml of water and 1178 g of acetic acid to the reaction flask B at a temperature below 5 C; add the toluene dilution of the above reaction solution with 1 to 1.5 h at a temperature below 10 C; After stirring for 10 min, the mixture was allowed to stand for 15 min, and the aqueous layer was removed. The toluene layer was washed 5 times with a mass fraction of 4% brine (2500 ml each). After the washed toluene solution was added anhydrous magnesium sulfate, And the toluene solution was concentrated under reduced pressure to remove toluene (the amount of toluene removed was 2700 ml or more and the toluene content was not more than a predetermined value) to give ethyl-4- (2-phthalimidoethoxy) acetoacetate in a yield of 93%
51%
Preparation of Ethyl 4-[2-(PHTHALIMIDO) ETHOXY] ACEFOACETATE (X1 A 250 mL two-necked flask, equipped with a magnetic stirring bar, thermometer and a pressure equalized addition funnel was charged with 75 mL of tetrahydrofuran under nitrogen atmosphere. 7.53 g Sodium hydride (60% dispersed in oil) was added and the resulting suspension was cooled to-10 C and 20 g of N- (2-HYDROXYETHYL) PHTHALIMIDE was added slowly over 5 minutes. The resulting slurry was stirred at-10 C for 30 minutes. To this mixture a solution of 16.35 g ethyl 4-chloroacetoacetate in 30 ML of TETRAHYDRAFURAN was added at-10 C in 40 minutes. The reaction mixture was warmed to room temperature and then stirred at room temperature for 18 hours. The reaction mixture was placed in an ice bath and quenched by dropwise addition of 10 mL ethanol. The mixture was then poured into 150 mL of IN hydrochloric acid solution in crushed ice and 200 ML of ethyl acetate was added. The resulting mixture was transferred into a separatory funnel and the aqueous phase was separated. The organic phase was first washed with 70 mL of ethyl acetate was added. The resulting mixture was transferred into a separatory funnel and the aqueous phase was separated. The organic phase was first washed with 70 mL of 5 wt. % OF NAHC03 solution, then with 150 ML of water, dried over 5 G of MgS04. MgS04 was filtered off and the filtrate was concentrated in vacuo to give a light brown oily product. The oil was washed with 15 mL of hexane to remove the mineral oil to give 17.2 g of ethyl 4- [2- (phthalimido) ethoxy] acetoacetate light brown product. (yield 51%, rel. compound purity > 80%). IR (KBR, CM~L) : 2995 and 1716. 1H-NMR (CDC13) 6 7.76 (dd, 2H), 7.65 (dd, 2H), 4.11 (S, 2H), 4.02 (q, 2H), 3.80 (t, 2H), 3.70 (t, 2H), 3.37 (s, 2H), 1. 16 (t, 3H). 13C-NMR (CDC13) 8 201.7, 168.4, 167.2, 134.3, 132.2, 123.5, 75.6, 68.7, 61.6, 46.0, 37.4, 14.3.
50%
With sodium hydride; In DMF (N,N-dimethyl-formamide); at 0 - 20℃;
Example 7 : 2-({2-[2-(4-{(2S)-2-Hydroxy-3-[(methylethyl)amino]propoxy}phenoxy) acetylamino] ETHOXY} METHYL)-4-(2-CHLOROPHENYL)-5-CYANO-6-METLLYL-1, 4- dihydropyridine-3-carboxylate is synthesized according to Scheme VI. SCHEME VI 0 Ho- p OEt HO out C1 Ni C1 Me NC Me N N CHO CHO ci NC Me H 0 1. acetone, II. -- . HO /O 2. II wEthyl 4- [2-FL, 3-DIOXOISOMDOLM-2-YDETHOXY1-3-OXOBUTANOATE. A solution of 2- (2- hydroxyethyl) isoindoline-1, 3-dione (10 g; 52.31 mmol) in DMF (150 ml) is cooled to 0C and treated with 1. 1 equivalents of a 60% dispersion of sodium hydride in mineral oil. After gas evolution has ceased, ethyl 4-chloro-3-oxobutanoate (7.75 g; 47.08 mmol) in 20 ml of DMF is added drop-wise, and the resulting solution is stirred overnight at room temperature. The pH of the mixture is then adjusted to 6-7 by the addition of 1 N HC1, and the product is partitioned between H2O and EtOAc. The aqueous phase is extracted again with EtOAc, and the combined organic phases are washed with water and brine, dried over magnesium sulfate, and concentrated to a crude residue, which is purified on a silica gel column, eluting with 25% EtOAc in hexane to obtain 7.52 g (23.54 mmol ; 50%) of the product as an oil.
40.5%
To a mechanically stirred suspension of NaH (15g, 0. 375 mol, 60% dispersion in mineral oil) in THF (250 mL) and DMF (25 mL) was added N- (2-hydroxyethyl)-phtalimide (43g, 0. 225 mol). The mixture was stirred at room temperature for about 4 h and then cooled to about 0C in an ice bath. Ethyl-4- chloroacetate (21. 4 mL, 0. 15 mol) in THF (4 mL) was then added via an addition funnel over a period of about 0. 5 h and the resulting mixture allowed to stir overnight. The mixture was then poured into a 2 L sepratory funnel containing 500 mL ice water and 700 mL EtOAc and separated. The organic layer was washed 2x with 500 mL of water and dried over MGS04. The solution was then filtered through a 4-inch plug of silica and then CONCENTRATED IN VACUO. The resulting yellow oil was then dissolved in 250 mL CH3CN and washed 2x with 30 mL hexane. The CH3CN layer was the concentrated in vacuo to yield 19. 4g (40. 5%) OF 4- [2- (1, 3-DIOXO-1, 3-DIHYDRO-ISOINDOL-2-YL)-ETHOXY]-3-OXO-BUTYRIC acid ethyl ester as a yellow oil. This crude compound was used for the next step
With sodium hydride; In toluene; at -15 - 45℃; for 13h;
N-(2-hydroxyethyl) phthalimide (100 g, 0.52 M) was coupled with 4-chloroethyl acetoacetate (50 g, 0.30 M) in the presence of sodium hydride in toluene under nitrogen atmosphere at ?11 to ?15 C. and at 25-30 C. for 12 hours and at 45 C. for 1 hour. The reaction mass was quenched in acidic water and extracted with toluene. The toluene extract was subjected to distillation and residue was degassed below 60 C.
With hydrogenchloride; In tetrahydrofuran; ethyl acetate;
(i) Preparation of Ethyl-4-(2-phthalimidoethoxy)acetoacetate STR141 To a slurry of sodium hydride (57% dispersion in oil, 66.1 g; 1.57M), in tetrahydrofuran (500 ml) cooled to 0 under nitrogen was added B 2-phthalimidoethanol (150 g; 0.785M) followed by ethyl 4-chloroacetoacetate (129 g; 0.785M) in tetrahydrofuran (250 ml) over 1 hour. The mixture was stirred at room temperature overnight then poured into a mixture of 1M hydrochloric acid (800 ml) and ethyl acetate (750 ml). The organic phase was separated and the solvent was evaporated at reduced pressure. The residue separated into two layers and the upper layer of mineral oil was removed to leave the title compound (243 g) as a crude product which was used without further purification.
In tetrahydrofuran;
PREPARATION 7 Preparation of ethyl 4-[2-(phthalimido)ethoxy]acetoacetate STR52 Sodium hydride (57% [by weight] in oil, 66.1 g) was stirred in dry tetrahydrofuran (500 ml) under nitrogen at -10 while N-(2-hydroxyethyl)phthalimide (150 g) was added. To this slurry was added at -10 a solution of ethyl 4-chloroacetoacetate (129.3 g), in dry tetrahydrofuran, over 1 hour. The reaction mixture was then allowed to warm to room temperature and stirring was continued for 18 hours. This mixture was poured into 1N hydrochloric acid (800 ml) and ethyl acetate was added (750 ml). The aqueous layer was washed with ethyl acetate (300 ml) and the organic solutions were combined. After washing with water (300 ml), the ethyl acetate was evaporated to give the title compound as a crude oil (243 g), sufficiently pure for further use.
EXAMPLE 1 Preparation of Ethyl 4-(2-(Phthalimido)Ethoxy)Acetoacetate (Formula V) 300 liters of toluene was taken into a clean and dry reactor and 50 Kg of sodium hydride was added under a nitrogen atmosphere. 50 Kg of [N-{2-hydroxyethyl}] phthalimide was added to the reactor and the resultant reaction mass was subjected to agitation and heating to 57 C. The reaction mass was maintained at 56-57 C. for 30 minutes. Another 50 Kg of [N-{2-hydroxyethyl}] phthalimide was added to the above reaction mass in 18 lots of 2.7 kg each and a further lot of 1.4 kg, at 60 C. Liberation of hydrogen was observed. The reaction mass was maintained at 57 C. until hydrogen gas liberation ceased. A solution of 94 Kg of ethyl 4-chloroacetoacetate in 287 liters of toluene was added to the above reaction mass at 57 C. and maintained at the same temperature for 20 minutes. The reaction mass was then cooled to 30 C. and then further cooled to 17 C. 200 liters of glacial acetic acid was added to the above obtained reaction mass in three equal lots at 17 C. and stirred for 30 minutes. 1000 liters of water was added to the reaction mass and stirred for 20 minutes. The organic layer was separated and the aqueous layer was extracted into 220 liters of toluene. The combined toluene layer was progressed to the next stage.
Into the reaction tank, put 350 kg of toluene, stirring temperature, distillation of toluene, after the liquid in the water separator is filled, keep the reflux water for 60 minutes, and take a sample to measure the water, when the water content of the reflux liquid is ?0.2%, the reflux is changed to distillation, distilled 45kg of toluene (worked ethyl 4-chloroacetoacetate as a dilution solvent), stirring and lowering the temperature, when the temperature in the tank drops to 45 C, the reaction tank is filled with nitrogen, continue to cool down at-5 C, under stirring add 40 kg of N-(2-hydroxyethyl)-phthalimide, and the add 20 kg of sodium hydride, after the addition, cover the can lid, stirring at -5 C, into the metering tank draw 38.4 kg of ethyl 4-chloroacetoacetate, 45 kg of toluene and mix well, at -5 C condition added the drops of ethyl 4-chloroacetoacetate toluene solution, the dropping time is controlled at 4 hours, after the addition, the reaction is stirred and the temperature is slowly increased, the temperature of the reaction solution is slowly raised at 32 C within 5 hours, the reaction time is more than 12 hours, and the temperature is raised at 45 C to continue the heat preservation, until the reaction is complete, cool down, control the internal temperature of 25 C, add 61 kg of glacial acetic acid, after the addition, continue stirring to make the material in the reaction solution stir evenly, and the internal temperature is controlled at 25 C, add 180kg of water, add, continue to stir for 30 minutes, stop nitrogen filling, stop stirring, let stand, divide the water layer into the barrel and transfer the toluene layer to the washing tank, transfer the water layer into the reaction tank, into the reaction tank add 75kg of toluene, stir and let stand, the water layer is separated into barrels, and the toluene layer is transferred to a washing tank, the water layer is transferred into the reaction tank, 75 kg of toluene is added into the reaction tank, stirred, and allowed to stand, and the water layer was separated, combine the toluene layer into a water wash tank, add 100kg of 25% salt water to the washing tank, stir for 15 minutes, let stand for 30 minutes, and separate the water layer, obtained 4-[(2-phthalimido) ethyoxyl] ethyl acetoacetate toluene solution
With sodium hydride; In toluene; at 5 - 51℃;Inert atmosphere; Large scale;
a synthetic process of amlodipine besylate,The difference from Embodiment 1 is thatStep 2 specifically includes:In the reaction tank,Put 350kg of toluene,Stirring,Distilling toluene,After the liquid in the water separator is full,Keep the reflux water for 60 minutes,Sampling to determine water separation,When the water content of the reflux liquid is ?0.2%,Change the reflux to distillation,Distilling off toluene,Stir and cool down,When the temperature inside the tank drops to 45 C,Filling the reaction tank with nitrogen,Continue to cool down to 5 C,40 kg of N-(2-hydroxyethyl)-phthalimide was added with stirring.Then add 20kg of sodium hydride,Plus,Close the lid,Stir at 5 C,38.4 kg of ethyl 4-chloroacetoacetate was taken in the measuring tank.45kg of toluene and mix well,Drop into the reaction tank at 5 CAdd a solution of ethyl 4-chloroacetoacetate in toluene,The dripping time is controlled at 4 hours.Plus,Continue to stir the reaction for 2 hours.Slowly warming up,The temperature of the reaction solution was slowly raised to 38 C within 5 hours.The reaction time is more than 12 hours.Warm up to 51 C and keep warm for 1 hour.Until the reaction is complete,Cooling down,Control the internal temperature of 35 C,Add 61kg glacial acetic acid,Plus,Stirring was continued for 60 minutes to stir the material in the reaction solution evenly.The internal temperature is controlled at 35 C.Add 180kg of water,Plus,Continue to stir for 30 minutes.Stop filling with nitrogen,Stop stirring,Stand still and separate the water layer into the bucket.Transfer the toluene layer to the washing tank,Transfer the water layer to the reaction tank,Add 75 kg of toluene to the reaction tank.Stir,Stand still and separate the water layer into the bucket.Then transfer the toluene layer to the washing tank.Transfer the water layer to the reaction tank,Add 75 kg of toluene to the reaction tank.Stir,Rest,Separate the water layer,Combine the toluene layer into a water wash tank,Add 100kg of 25% salt water to the washing tank.Stir for 15 minutes,Let stand for 30 minutes,Divided into the water layer,Toluene solution of 4-[(2-phthalimido)ethoxy]acetic acid ethyl acetate;The yield of amlodipine besylate was 91.1%, and the purity was 99.3%.
With hydrogen;(R)-[RuCl2(BINAP)]n; In ethanol; at 95 - 105℃; under 67506.8 - 75007.5 Torr; for 0.5 - 8h;Conversion of starting material;
Example 1 (Comparative . A 600ml stainless steel Parr reactor was charged with ethanol (340ml) and ethyl-4-chloroacetoacetate (53g). The reactor agitator was started and the speed set to 600rpm. The reactor was pressurised using nitrogen to 7 bar and stirring continued for 5 minutes. After 5 minutes the reactor was slowly vented to ambient pressure, the pressurisation/depressurisation cycle was repeated for a total of five times to ensure complete removal of dissolved oxygen. At the end of the last cycle the reactor set-point temperature was adjusted to 95C. (R)-[RuCI2 (BINAP)] ncatalyst was accurately weighed (23mg) into a catalyst transfer vessel and the vessel then purged using nitrogen for 5 minutes. The catalyst was flushed from the transfer vessel using deoxygenated solvent into a 100ml stainless steel injection bomb which was attached to the Parr reactor. When the Parr reactor temperature was between 95C and 100C the injection bomb was pressurised to 100bar using hydrogen. Appropriate valves were then opened to transfer the catalyst mixture and hydrogen into the reactor. The contents of the reactor were stirred at 600rpm for 30 minutes before being cooled to less than 30C. The reactor was then slowly vented to ambient pressure. The reactor contents were transferred into a 1L rotary film evaporator flask and the mixture evaporated to constant weight by application of vacuum and by using a heated water bath. The residue was subjected to pot to pot distillation under vacuum to afford a clear colourless oily liquid product of ethyl (S)- (-)-4-chloro-3-hydroxybutyrate in >98% yield, >98% purity and 94% enantiomeric excess.; Example 2 A feed tank was charged with 3.6L ethanol solvent. The solvent was deoxygenated by pumping it through a spray nozzle whilst pressurising to 7bar with nitrogen and then depressurising through a needle valve at a controlled rate. The pressurisation/depressurisation cycle was repeated three times and the entire process automated using a PLC-based control system. In a similar manner a second feed tank was charged with ethyl-4-chloroacetoacetate (3.6L) and deoxygenated using the same protocol to that described above. The catalyst, (R)- [RuCl2 (BINAP)] (149mg) was charged into a transfer vessel and the vessel purged using nitrogen before transferring the catalyst into the solvent feed tank. The catalyst solution had a concentration of 52.2mg/Kg. The two feed systems were connected to the continuous hydrogenation reactor system via two high-pressure pumps. The continuous hydrogenation reactor system was constructed of Hastalloy 276 and comprised a number of in-line static mixers to give a residence time of between 30 and 35 seconds. The static mixers also ensured good mixing of the process streams and rapid absorption of hydrogen. The reactor system was equipped with a recycle pump and an in-line valve which enabled operation as either a plug flow reactor (PFR, valve closed) or a continuous loop reactor (CLR, valve open). The system was equipped with a gas/liquid separator and the liquid level inside the separator controlled using a differential pressure sensor, which in turn operated an exit flow control valve. The reactor system was controlled using a PLC based control system. The hydrogenation reactor was pressurised using hydrogen and the pressure maintained between 90 and 100 bar by continually feeding hydrogen through a mass flow controller at a rate of 2.7g/h. The reaction liquors passed through a heat exchanger using a pump such that the process temperature was maintained between 102C and 105C. The system above was operated as a plug flow reactor. The flow rate of the ethyl-4-chloroacetoacetate was set to 2. 6ml/minute and the flow rate of the catalyst solution set to 8. 9ml/min. These flows gave a process concentration of 30% w/w and a substrate to catalyst ratio of 20,000 : 1. Over a series of continuous runs, each varying between 4 and 8 hours, the reactor consistently converted >99% ethyl-4-chloroacetoacetate to (S) -ethyl-4-chloro-3-hydroxybutyrate which was isolated after removing the solvents by evaporation to give a chemical yield of >98% and an enantiomeric excess of 98- 99%.; Example 3 The reactor was set up as Example 2, except it was operated as a continuous loop reactor. The flow rate of the ethyl-4-chloroacetoacetate was set to 2. 55ml/minute and the flow rate of the ethanol catalyst solution set to 6. 60ml/min at a catalyst concentration of 45. 8mg/kg. These flows gave a process concentration of 37% w/w and a substrate to catalyst ratio of 65,000 : 1. Over a series of continuous runs, each varying between 4 and 8 hours, the reactor consistently converted >99% ethyl-4-chloroacetoacetate to (S) -ethyl-4-chloro-3-hydroxybutyrate which was isolated after removing the solvents by evaporation to give a chemical yield of >98% and an enantiomeric excess of 98- 99%.
96%
With D-glucose; In ethanol; at 25℃; for 0.25h;pH 6.8;Microbiological reaction; aq. phosphate buffer;Kinetics;
General procedure: Cells harvested by centrifugation were washed and resuspended (19 g cell dry weight/l) in a 10 mM potassium phosphate buffer, pH 6.8. Cell suspension (25 ml) was taken in a 150 ml Erlenmeyer flask capped with a cotton plug and pre-incubated in a water-bath shaker at 200 rpm and 25 C for 15 min after adding glucose (50 g/l). A solution of 0.12 g of COBE in 0.5 ml of ethanol was then added to the reaction flask. Incubation was continued for 15 min [for (R)-CHBE, the reaction time was 1 h] after the addition of COBE after which the reaction mixture was extracted with ethyl acetate (2 × 100 ml). The organic layer was dried over anhydrous sodium sulfate and the pure product was obtained by silica gel chromatography using hexane/ethyl acetate (9:1) as a mobile phase. In order to determine the rate of formation of CHBE, a 0.5 ml sample was taken every 5 min and centrifuged (10,000 rpm, 10 min) at 4 C to remove the cells. The supernatant was extracted with ethyl acetate (1 ml) of which 1 mul was injected into the GC to determine the concentrations of COBE and CHBE.
87.4%
With ketoreductase; NADP; In acetic acid butyl ester; water; for 7 - 18h;pH 7;
Example 9: Preparation of Ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE from Ethyl 4-chloro- acetoacetate. To a 100 mL vessel connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was charged a solution of glucose (7.5 g) in 100 mM TRIETHANOLAMINE pH 7 buffer (25 ML). To this solution was charged ketoreductase SEQ ID NO: 42 (100 mg); 50 mg GDH SEQ ID NO: 66 and NADP (6.25 mg). Butyl acetate (10 ml) was then charged. Then, ethyl 4-chloroacetoacetate (6 g) in butyl acetate (10 mL) was charged. The pH was maintained at 7 by the automatic titrater by the addition of 4M NaOH (7.5 mL) over 7 hrs. A sample of the reaction mixture was extracted with an equal volume of butyl acetate and the organic layer was analyzed by GC. The analysis showed 99% conversion of the ethyl 4-CLLLOROACETOACETATE to ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE.Example 11 : Preparation of Ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE from Ethyl 4-chloro- acetoacetate. To a 100 mL vessel connected to an automatic titrater by a pH electrode and a feeding tube for addition of base was charged a solution of glucose (12. g) in water (30 mL). To this solution was charged ketoreductase SEQ ID NO: 42 (100 mg); 50 mg GDH SEQ ID NO: 66 and NADP (6.25 mg). Butyl acetate (10 ML) WAS then charged. Ethyl 4-chloroacetoacetate (10 g) was then charged via syringe pump as follows : 1 mL was charged rapidly and the remainder was then charged at a rate of 1 mL/hr). The pH was maintained at 7 by the automatic titrater by the addition OF 4M NAOH over 18 hours hrs. The stirring was stopped and the phases allowed to separate. The organic layer included some emulsion. The organic layer, including some emulsion, was separated and washed with 10 mL of water. The combined aqueous layers were extracted twice WITH 20 mL of butyl acetate. The organic extracts were combined and rotary evaporated under vacuum to remove water. Additional butyl acetate was added during the evaporation to help remove the water. When the water was removed the butyl acetate solution was decanted from solids in the flask. Evaporation of the solvent under vacuum then gave 8. 85 g of ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE (87.4% yield) of very good purity.
50%
Daucas carota root; extract of; In water; at 37 - 40℃; for 60h;pH 7.0;Enzymatic reaction; Aqueous phosphate buffer;Conversion of starting material;
The General Methodology for the Reduction of Substituted beta-Ketoesters; 100 mg of each of the compounds in Table 3, entry Nos. 1-10 and their related substituted beta-ketoesters were added to a 100 ml flask containing 2 gm of crude extract of Daucus carota, and 50 ml of 0.1 M sodium phosphate buffer pH 6.0 to 7.5 were added. The reaction was incubated in a shaker for 50 to 70 hours for maximum product formation. The product formed was isolated, purified by column chromatography and the structure of the compound was confirmed by spectral data.
With hydrogen;ruthenium trichloride; (-)-5,5'-bis(diphenylphosphanyl)-2,2,2',2'-tetrafluoro-4,4'-bi[benzo-1,3-dioxolyl]; In ethanol; at 80℃; under 3000.3 Torr; for 2h;
In a 150 mL autoclave under argon atmosphere RuCl3 (10.2 mg, 0.049 mmol), (-)-5,5'-bis(diphenylphosphanyl)-2,2,2',2'-tetrafluoro-4,4'-bi[benzo-1,3-dioxolyl] (i.e. (-)-Fluoxphos) (34.2 mg, 0.050 mmol) and ethyl 4-chloro-3-oxobutyrate (0.81 g, 4.9 mmol, approx. 98.5%) is dissolved in degassed ethanol (30 mL). After flushing the autoclave with argon hydrogenation is carried out during 2 hours at 80 C and at 4 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and enantiomeric excess (ee) (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100% at an ee of 97.6%.
With hydrogen;5,5?-bis(diphenylphosphino)-4,4?-bi-1,3-benzodioxole; bis[(1-isopropyl-4-methylbenzene)dichloro ruthenium]; In ethanol; at 90℃; under 22502.3 Torr; for 2.3h;
In a 250 mL autoclave in an argon atmosphere [Ru2Cl4(cym)2] (6.7 mg, 0.011 mmol), (-)-5,5'-bis(diphenylphosphanyl)-4,4'-bi[benzo-1,3-dioxolyl] (14.3 mg, 0.023 mmol) and ethyl 4-chloro-3-oxobutyrate (9.10 g, 54.5 mmol, approx. 98.5%) is dissolved in degassed ethanol (30 mL). After flushing the autoclave with argon hydrogenation is carried out during 2.3 hours at 90 C and at 30 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100% at an ee of 89.5%.
With hydrogen;C56H54I2P2Ru; In ethanol; dichloromethane; at 100℃; under 16501.7 Torr; for 2.83333h;
In a 1 L autoclave under argon atmosphere [RuI((-)-Fluoxphos)(cym)]I (35 mg, 0.030 mmol, prepared from (-)-Fluoxphos and [Ru2I4(cym)2] as disclosed in WO 00/29370, and ethyl 4-chloro-3-oxobutyrate (33.4 g, 200 mmol, approx. 98.5%) is dissolved in degassed ethanol (340 mL) and dichloromethane (80 mL). After flushing the autoclave with argon hydrogenation is carried out during 170 minutes at 100 C and at 22 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100% at an ee of 97.4%.
With hydrogen;C56H56BClF4P2Ru; In ethanol; dichloromethane; at 100℃; under 16501.7 Torr; for 3h;
In a 1 L autoclave under argon atmosphere [RuCl((-)-Fluoxphos)(cym)]BF4 (23 mg, 0.022 mmol, prepared from (-)-Fluoxphos and [Ru2Cl4(cym)2] and AgBF4 as disclosed in Mashima, K., J. Org. Chem. 1994, 59, 3064-3076, and ethyl 4-chloro-3-oxobutyrate (33.4 g, 200 mmol, approx. 98.5%) is dissolved in degassed ethanol (340 mL) and dichloromethane (80 mL). After flushing the autoclave with argon hydrogenation is carried out during 180 minutes at 100 C and at 22 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100% at an ee of 98.0%.
With hydrogen;bis[(1-isopropyl-4-methylbenzene)dichloro ruthenium]; (-)-5,5'-bis(diphenylphosphanyl)-2,2,2',2'-tetrafluoro-4,4'-bi[benzo-1,3-dioxolyl]; In ethanol; dichloromethane; at 110℃; under 16501.7 Torr; for 1.16667h;
In a 1 L autoclave under argon atmosphere [Ru2Cl4(cym)2] (5.0 mg, 0.008 mmol, (-)-Fluoxphos (12.1 mg, 0.018 mmol) and ethyl 4-chloro-3-oxobutyrate (27.0 g, 162 mmol, approx. 98.5%) is dissolved in degassed ethanol (340 mL) and dichloromethane (80 mL). After flushing the autoclave with argon hydrogenation is carried out during 70 minutes at 110 C and at 22 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100% at an ee of 98.1%.
With hydrogen;bis[(1-isopropyl-4-methylbenzene)dichloro ruthenium]; (-)-5,5'-bis(diphenylphosphanyl)-2,2,2',2'-tetrafluoro-4,4'-bi[benzo-1,3-dioxolyl]; In ethanol; at 90℃; under 22502.3 Torr; for 2.3h;
In a 250 mL autoclave in an argon atmosphere bis[(1-isopropyl-4-methylbenzene)dichloro ruthenium] (i.e. [Ru2Cl4(cym)2]) (6.7 mg, 0.011 mmol), (-)-Fluoxphos (16.0 mg, 0.023 mmol) and ethyl 4-chloro-3-oxobutyrate (9.11 g, 54.5 mmol, approx. 98.5%) is dissolved in degassed ethanol (30 mL). After flushing the autoclave with argon hydrogenation is carried out during 2.3 hours at 90 C and at 30 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100% at an ee of 95.8%.
With hydrogen;(+)-6-dicyclohexylphosphanyl-2'-diphenylphosphanyl-2-methoxy-1,1'-biphenyl; bis(1-isopropyl-4-methylbenzene)dichloro-ruthenium; In ethanol; at 80℃; under 3000.3 Torr; for 3h;Product distribution / selectivity;
Example 11: (5)-Ethyl4-chloro-3-hydrpxybutyrate; In a 150 mL autoclave under argon atmosphere bis(l-isopropyl-4-methylbenzene)dichloro-ruthenium (7.5 mg, 0.012 mmol), (+)-ligand Ic (14.4 mg, 0.025 mmol) and ethyl 4-chloro-3-oxobutyrate (0.83 g, 5.0 mmol) is dissolved in degassed ethanol (30 mL). After flushingthe autoclave with argon hydrogenation is carried out during 3 h at 80 C and at 4 barhydrogen pressure. After cooling to room temperature the reaction solution is directlyanalyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column:Lipodex-E 25 m / 0.25 mm). Conversion is 100% at an ee of 80%.
Comparative Example 1 Asymmetric hydrogenation of ethyl 4-chloroacetoacetate The same operation as in Example 7 was conducted except that 13.5 mg (0.008 mmol) of Ru2 Cl4 ((R)-BINAP)2 NEt3 was put in a stainless steel autoclave, and at a conversion ratio of 100%, selectivity of 95.9% and optical purity of 94.4% ee, ethyl 4-chloro-(S)-3-hydroxybutyrate was obtained.
With D-glucose; In water; at 28℃; for 24 - 48h;Microbiological reaction;
Mucor circinelloides MTCC 5187 was maintained on Malt Extract Agar (MEA) plates prepared in sterilized MEA (5%) in distilled water.For growth in liquid medium a loopful of microbial cells was aseptically transferred from an agar plate to a 250 mL baffled conical flask containing 50 ml of medium having the following composition (per liter) - K2 HPO4 (1.9 g), NaH2 PO4.2H2 O (2.02 g) (NH4)2 SO4 (1.8 g), MgSO4 7H2 O (0.2 g), FeCb (0.97 mg) and trace elements solution (1 ml). Trace elements EPO <DP n="9"/>solution consisted of (per liter) CuSO45H2 O (0.02 g), MnSO44H2 O (0.1 g), ZnSO4JH2 O (0.1 g) and CaCO3 (1.8 g). The above minimal medium was supplemented with 0.2% (w/v) yeast extract and 2.25% (w/v) glucose.The microorganism was grown at 280C on an orbital shaker at 220 rpm for 24-48 hours. Microbial cells were harvested by centrifuging at 7000 rpm for 20 minutes at 10 C. The cell pellet was resuspended in 10 ml of 50 mM sodium phosphate buffer, pH 5.4 and the cells were washed by centrifuging as above. The cell pellet was finally resuspended in 10 ml of the above buffer. To the 10 ml of cell suspension in a 50 ml baffled flask glucose (10 g per liter) was added and the compound of formula VI (2 g per liter). The cells were incubated at 28 degree C on a rotary shaker at 150 rpm for 24-48 hours.The entire reaction broth was extracted with ethyl acetate. The separated ethyl acetate extracts were pooled together and dried over anhydrous sodium sulphate and the solvent removed by vacuum distillation to afford a golden oil. The extent of conversion of the compound of formula VI to the compound of formula V and the enantiomeric / diastereomeric composition of the compound of formula VI was determined by HPLC (high pressure liquid chromatography).
With (+)-6-dicyclohexylphosphanyl-2'-diphenylphosphanyl-2-methoxy-1,1'-biphenyl; hydrogen;bis(1-isopropyl-4-methylbenzene)dichloro-ruthenium; In ethanol; at 80℃; under 3000.3 Torr; for 3h;
In a 150 mL autoclave under argon atmosphere bis(1-isopropyl-4-methylbenzene)dichloro-ruthenium (7.5 mg, 0.012 mmol), (+)-ligand Ic (14.4 mg, 0.025 mmol) and ethyl 4-chloro-3-oxobutyrate (0.83 g, 5.0 mmol) is dissolved in degassed ethanol (30 mL). After flushing the autoclave with argon hydrogenation is carried out during 3 h at 80 C and at 4 bar hydrogen pressure. After cooling to room temperature the reaction solution is directly analyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column: Lipodex-E 25 m / 0.25 mm). Conversion is 100% at an ee of 80%.
With hydrogen;(+)-6-dicyclohexylphosphanyl-2'-diphenylphosphanyl-2-methoxy-1,1'-biphenyl; [RuCl2(p-cymene)2]; In ethanol; at 80℃; under 3000.3 Torr; for 3h;Product distribution / selectivity;
Example 16: (5)-Ethyl 4-chloro-3-hydroxybutyrate; In a 150 mL autoclave under argon atmosphere bis(l-isopropyl-4-methylbenzene)dichloro-ruthenium (7.5 mg, 0.012 mmol), (+)-ligand Ic (14.4 mg, 0.025 mmol) and ethyl 4-chloro-3-oxobutyrate (0.83 g, 5.0 mmol) is dissolved in degassed ethanol (30 mL). After flushingthe autoclave with argon hydrogenation is carried out during 3 h at 80 C and at 4 barhydrogen pressure. After cooling to room temperature the reaction solution is directlyanalyzed by GC for conversion (column: HP-101 25 m / 0.2 mm) and ee (column:Lipodex-E 25 m / 0.25 mm). Conversion is 100% at an ee of 80%.
With hydrogen;[(R)P-Phos-Ru(benzene)Cl]Cl; In tetrahydrofuran; ethanol; at 100℃; under 18751.9 Torr; for 2h;Product distribution / selectivity;
Examples 3 to 17The reaction of example 1 was carried out with different catalysts and under varying conditions. The specific catalysts, conditions and results are summarized in table 1 in figure 2. Unless specified otherwise in figure 2, the reaction was carried out as described above for example 1. The solvent used in all reactions was EtOH THF. The ratio substrate/catalyst (S/C) was relatively high in most experiments. Thus only low amounts of catalyst are necessary in the inventive reaction. Further, the concentration of the substrate S was selected relatively high to 3 or 4 M. This is equivalent to a concentration of approximately 50% (w/w). The temperature was 100C. The pressures were adjusted to relatively low levels of between 15 and 40 bar.The results show, that for almost all catalysts a high absolute yield (conversion) and a high enantiomeric yield (ee) were obtained, although the total amounts of catalyst were very low. Further, the pressure was relatively low, which is advantageous for large scale industrial applications. High yields could be obtained even after relatively short reaction times of between 1.3 and 3 hours (examples 8 to 13). Overall, the examples show that the inventive reaction is efficient and can be carried out with low amounts of catalyst and solvent. The reaction is also energy-efficient, because the reaction time is low, the volume to be heated is small (due to low solvent levels) and the pressure is low.
With Saccharomyces cerevisiae reductase R264H/S260E mutant; NADPH;Enzymatic reaction;Kinetics;
Using the purified wild-type and six mutants (S260D, R264H, R264Y, R264H/R27Y, R264H/S259E, and R264H/S260E), kinetic studies were conducted. In order to elucidate the Km and kcat values of wild-type and mutants toward ECOB, enzyme assays were conducted in the reaction mixtures containing 1-15 mM ECOB, 0.5 mM NAD(P)H, and 0.19-14 muM purified enzyme. The Km and kcat values of each enzyme toward ECOB were calculated from the Lineweaver-Burk plots. Then, in order to determine the Km values of the wild-type and mutants toward NAD(P)H cofactor, enzyme assays were conducted in the reaction mixtures containing excess ECOB (10-fold concentration of Km ECOB) and 0.01-0.5 mM NAD(P)H cofactor, and 0.19-14 muM purified enzyme. The Km values of each enzyme were calculated from the Lineweaver-Burk plots.
With potassium phosphate; Candida parapsilosis aldo-keto reductase CPAR2; NADPH; at 30℃; for 8h;pH 6.5;Enzymatic reaction;
General procedure: Asymmetric reductions of various carbonyl compounds by the purified enzymes were carried out at 30C for 8h with mild shaking in a reaction mixture containing 0.1M potassium phosphate buffer (pH 6.5), 1gL-1 substrate, 10mM NADPH, and the purified enzyme of appropriate amount in a total volume of 2mL. In order to determine the absolute configuration of chiral alcohols, the reaction products were extracted with ethyl acetate or hexane and the organic layer was used for analysis. The optical purity of the reaction products were determined by chiral HPLC (HP 1100, Agilent, USA) equipped with Chiralcel OB-H column (4.6mm×250mm; Daicel Chemical Ind. Ltd., Japan) or chiral GC (7890A, Agilent, USA) equipped with FID detector and Chrompack Chirasil-Dex CB chiral capillary column (25m×0.25mm; Varian, USA) [21].
With D-glucose; nicotinamide adenine dinucleotide phosphate; recombinant Bacillus subtilis-derived glucose dehydrogenase; recombinant Gluconobacter oxydans carbonyl reductase 0525; In aq. phosphate buffer; ethanol; at 30℃; for 12h;pH 7.0;Enzymatic reaction;Kinetics;
General procedure: The enzyme activity toward the reduction ofketones was determined by spectrophotometricallymeasuring the oxidation of NAD(P)H at 340 nm(epsilon = 6.22mM-1 cm-1) in the presence of an excess amount of ketones. The change in absorbance of NAD(P)H was monitored at 340 nm using an ultraviolet-visiblespectrophotometer with a temperature-controlledcuvette holder (Shimadzu Co., Kyoto, Japan). One unit(U) activity was defined as the amount of enzymesrequired to catalyze the oxidation of 1 mumol NAD(P)Hper minute at 30 C. The data were expressed as U/mgof protein. The standard reaction mixture contained100mM phosphate buffer (pH 7.0), 0.1mM NAD(P)H,5 mM substrate, and enzyme solvent in a total volumeof 1 mL. The reaction was initiated by adding 20 muLsolvent containing 10 mug-40 mug of enzymes. Blankswithout the enzyme were carried out for each substrate,and data were collected in triplicate. Protein concentrationswere determined with the bicinchoninic acid assayusing bovine serum albumin as a standard.NAD(P)H or NADH was assayed under standardreaction conditions for the study of coenzyme dependence.A range of substrates from 1 to 20 mM concentrationwas assayed under the standard reactionconditions for the study of kinetics. Apparent values ofMichaelis constant (Km) and kcat were calculated by fittingthe data into Michaelis-Menten equation using theSigmaPlot (Systat Software Inc., San Jose, CA, USA).All reactions followed Michaelis-Menten-type kinetics.Enantioselective reduction of ketones. The enantioselectivityof the enzymes was determined byexamining the reduction of aryl ketones, ethyl 4-chloroacetoacetate(COBE), and ethyl 2-oxo-4-phenylbutyrate(OPBE) using an NAD(P)H regeneration system consistingof BsGDH and glucose. The general procedurewas as follows: D-glucose (0.5%), recombinant BsGDH(10 U), NAD(P)+ (0.1 mM), the recombinant cell(30 g L-1, wet weight), and ketone solvated in ethanol[1 g L-1, 10%(v/v)] were mixed in a potassium phosphatebuffer (10 mL, 100 mM, pH 7.0). The mixturewas shaken at 30 C for 12 h.22) Upon termination ofthe reaction, each sample was extracted twice withequivalent ethyl acetate. The organic layer wasremoved, dried, diluted in the mobile phase, and thensubjected to chiral high-performance liquid chromatography(HPLC) to determine the conversion and enantiomericexcess (e.e.). Chiral HPLC analysis wasperformed on an Agilent 1100 series HPLC system witha UV detector.23) Chiral CHBE was analyzed on a chiracelOB-H column (Daicel, Japan) at lambda210 nm using hexane/2-propanol (90/10, v/v) as eluent at a flow rate of0.8 mL min-1 and a temperature of 25 C. Chiral HPBEand 4-phenyl-2-butanol were analyzed on a chiracelOD-H column at lambda210 nm and lambda254 nm using hexane/2-propanol (98/2, v/v) as eluent at a flow rate of 1.0 mLmin-1 and a temperature of 30 C. Authentic (relevant)standards were used for peak identification, and quantificationwas based on the peak area that was suitablycalibrated with standards of known concentration.
With D-glucose; sodium carbonate; In aq. phosphate buffer; acetic acid butyl ester; at 30℃; for 5h;pH 6.2;Microbiological reaction; Enzymatic reaction;
Cells were harvested by centrifugation (10,000 g, 20 min, 4 C) and washed with 100 mM potassium phosphate buffer (pH 6.2). The bioconversion of COBE to (S)-CHBE was performed in an aqueous-butyl acetate (4:1, v/v) biphase system containing 100 mM potassium phosphate buffer (pH 6.2), 1500 mM COBE, 1550 mM glucose, 1550 mM Na2CO3, Triton X-100 (1?, v/v), 0.1 g of dry cell weight (DCW) of E. coli Rosseta (pET-22b-SOU1), and E. coli Rosseta (pET-22b-GDH), respectively, at a total volume of 25 mL. The reaction was performed at 30 C and 220 rpm for 5 h. The organic layer was isolated to determine the product concentration and optical purity.
With aldo-keto reductase CaAKR; NADH; In aq. phosphate buffer; at 30℃; for 10h;Enzymatic reaction;
General procedure: The bioreduction was performed in 1.5 mL Eppendorf tubes containing 200 mmol/L potassium phosphate buffer (pH 7.0), 1 mmol/L of each substrate, 0.5 mmol/L NADH, and 1 mg/mL purified CaAKR in a total volume of 1.0 mL, shaking for 10 h at 30 C. The reaction mixture was extracted twice with an equivalent volume of 300 muL ethyl acetate. The extracts were combined, dried with anhydrous sodium sulfate. The concentrations for each product were determined by GC or HPLC analysis.
Ca.91%
With alpha-D-glucopyranose; In toluene; at 50℃; for 11h;pH 5;Enzymatic reaction;Kinetics;
Then 1.0 g COBE (6.1 mmol) and 1.2 g glucose was added. The second batch was completed within 70 min. After-wards, another 1.0 g COBE (6.1 mmol) and 1.2 g glucose was fedwhen COBE was converted thoroughly. At the 7th batch, at least3 h was required for a complete reaction. All 7.0 g COBE was fullyreduced into (S)-CHBE by merely 0.1 g RpCR-GDH dry cells within11 h without addition of external NADP+. After extraction, about6.29 g (S)-CHBE (>99% ee) was recovered, with a molar yield ofabout 91%. The substrate to catalyst ratio and the space-time yieldwere about 70 and 1480 g L-1d-1, which was attributed to the highcatalytic efciency of RpCR in the biphasic system with substrate-feeding.
With hydrogenchloride; dichloro-R-2,2?-bis(diphenylphosphino)-1,1?-binaphthyl ruthenium; hydrogen; In methanol; water; at 95 - 98℃; under 6080.41 - 7600.51 Torr;
In a 500 ml autoclave in a hydrogen atmosphere, was added Compound A: ethyl chloroacetoacetate (50 g, 0.30 mol), hydrochloric acid 5ml (AR, content of 36% to 38%), methanol 300 ml, the reaction solution was stirred, a chiral ruthenium metal catalyst (R) -RuCl2 (BINAP) (50 mg) was added, the reaction solution was replaced with hydrogen twice, the pressure rose to 8 to 10 atmospheres, insulation 95 ~ 98 deg. C reaction 1 ~ 2h. The reaction solution was cooled to room temperature, and the reaction solution was directly analyzed by GC to measure the conversion (column: HP-10125 m / 0.2 mm) and the enantiomeric excess (column: Lipodex-E25 m / 0.25 mm). The enantiomeric excess was 99.95% and the conversion was 100%. The filtrate was dried and filtered, and the solvent was distilled off under reduced pressure to obtain the compound B, which was directly used in a one-step reaction.
458 g
With sodium formate; In ethanol; at 50℃;
5 liters of reaction flask 500g (3.03 mol) of ethyl chloroacetoacetate and 2 liters of ethanol, after stirring, add a chiral catalyst (0.15 mol, 0.05 eq.) and 250g (3.68 mol, 1.2 eq.) of sodium formate, heating to 50 C to react; the gas phase detection reaction was complete, the solvent was removed by distillation,And distilled under reduced pressure to give 485g of chiral ethyl 4-chloro-3-hydroxybutyrate;
With NADP; In acetic acid butyl ester; water; for 18h;pH 6.8 - 7;Aqueous phosphate buffer;Product distribution / selectivity;
Example 5: Preparation of ethyl (R)-4-CYANO-3-LIVDROXYBUTYRATE from ethyl 4-CHLOROACETOACETATE (via ethyl (S ?-4-CHLORO-3-HYDROXYBUTYRATE .. To a well-stirred solution of 100 mM potassium phosphate buffer, 500 MM NACL, pH 7 (1 L) at room temperature was added glucose (160g, 830 mmoles, 1.1 equiv). To this was added ketoreductase SEQ ID NO: 2 (0.9g), glucose dehydrogenase S06 SEQ ID NO: 10 (0.5 g) and NADP (0. 5G) as lyophilized powders. Once dissolved, butyl acetate (500 mL) was added to form an emulsion. To this emulsion was added a solution of ethyl 4-chloroacetoacetate (100g, 608 mmoles) in butyl acetate (500 mL), dropwise over 3 hours. The pH was maintained between 6. 8 and 7 by an automatic titrater that dispensed NA2CO3 (2M in water, about 160 mL total). After 40 hours the automated addition of the base had ceased and there was no residual starting material by gas chromatography. The layers were separated, and the aqueous phase was washed with ethyl acetate (500 ML). The combined organics were dried over anhydrous sodium sulfate, filtered and evaporated on a rotary evaporator, to give essentially pure (-97%) ethyl (S)-4-chloro-3-hydroxybutyrate.Example 7: Preparation of Ethyl (S)-4-CHLORO-3-HVDROXYBUTYRATE FROM ETHYL 4-chloro- acetoacetate. To a 3-necked jacketed 3L flask equipped with a mechanical stiner and connected to an automatic titrater by a pH electrode and a feeding tube for addition of base, was charged triethanolamine (6.6 mL) and H2O (492 ML) to make 100 mM TRIETHANOLAMINE solution. The pH was adjusted to 7 with 37% HCI. Then, D-Glucose (125 g) was added. The water circulating to the flask jacket was set to 30 C. After 10 minutes, ketoreductase SEQ ID NO : 2 (5.7g) and glucose dehydrogenase S06 SEQ ID NO: 10 (3.1 g) powder were added. After 10 minutes, ss-NAD (125 mg) was added and the resulting mixture was allowed to stir for 5 minutes. Then, butyl acetate (250 mL) was charged. Using an addition funnel, 2.4 M ethyl 4-chloroacetoacetate (250 mL, 100 g in 167 mL of butyl acetate) was slowly added over 3 hrs. The pH was maintained at 7 by the automatic titrater by the addition OF 2 M NA2CO3 (152 mL) over 15 hrs. Subsequently, gas chromatography of a reaction sample showed complete conversion to PRODUCT. CELITE (16 g) was added and the reaction mixture was allowed to stir for 10 minutes. The solution was filtered through a celite pad and the organic layer was separated. The aqueous layer was extracted with butyl acetate (2x 200 mL). The organic layers were combined and the solvent removed under vacuum by rotary evaporation to obtain 87 g of the ethyl (S) 4-chloro-3-hydroxybutyrate. The ENANTIOMERIC excess was >99%, as determined after its conversion to ethyl (R)-4-CYANO-3-HYDROXYBUTYRATE in Example 8.
With ketoreductase; In acetic acid butyl ester; water; for 11h;pH 7;Product distribution / selectivity;
EXAMPLE 10 : Preparation of Ethyl (S)-4-CHLORO-3-HYDROXVBUTVRATE FROM Ethyl 4-chloro- acetoacetate. The procedure was identical to Example 9 with the exceptions that 400 mg of the ketoreductase SEQ ID NO: 42 was used and NAD+ (12.5 mg) was added in place of the NADP. The addition of the NAOH solution by the automatic titrater was complete in 11 hours and the GC analysis showed 99% conversion of the ethyl 4-chloroacetoacetate to ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE.
With glucose dehydrogenase; ketoreductase; In acetic acid butyl ester; water; at 13℃; for 13h;pH 7;Product distribution / selectivity;
Example 23: Preparation of ethyl (R)-4-CYANO-3-HYDROXYBUTERRATE from ethyl 4-chloroacetoacetate (via ethyl (USD .-4-CHLORO-3-HYDROXYBUTRATE .. To a 100 mL vessel connected to an automatic titrater by a pH electrode and a feeding tube for addition of base (4M NAOH) was charged a solution (25 mL) of glucose (7.5 g) in 100 mM triethanolamine buffer, pH 7. To this solution was charged ketoreductase SEQ ID NO: 50 (50 mg), glucose dehydrogenase SEQ ID NO: 62 (20 mg) and NADP (1.5 mg). Butyl acetate (10 ml) and ethyl 4-chloroacetoacetate (6 g) in additional butyl acetate (10 mL) were then charged. The pH was maintained at 7 by the automatic titrater by the addition OF 4M NAOH to the stirring mixture over 13 hrs. The phases were then allowed to separate for 30 minutes and the organic layer (25 mL), containing the ethyl (S)-4-CHLORO-3-HYDROXYBUTYRATE intermediate, was removed.
With D-glucose; In ethanol; at 25℃;pH 6.8;Microbiological reaction; aq. phosphate buffer;Kinetics;
General procedure: Cells harvested by centrifugation were washed and resuspended (19 g cell dry weight/l) in a 10 mM potassium phosphate buffer, pH 6.8. Cell suspension (25 ml) was taken in a 150 ml Erlenmeyer flask capped with a cotton plug and pre-incubated in a water-bath shaker at 200 rpm and 25 C for 15 min after adding glucose (50 g/l). A solution of 0.12 g of COBE in 0.5 ml of ethanol was then added to the reaction flask. Incubation was continued for 15 min [for (R)-CHBE, the reaction time was 1 h] after the addition of COBE after which the reaction mixture was extracted with ethyl acetate (2 × 100 ml). The organic layer was dried over anhydrous sodium sulfate and the pure product was obtained by silica gel chromatography using hexane/ethyl acetate (9:1) as a mobile phase. In order to determine the rate of formation of CHBE, a 0.5 ml sample was taken every 5 min and centrifuged (10,000 rpm, 10 min) at 4 C to remove the cells. The supernatant was extracted with ethyl acetate (1 ml) of which 1 mul was injected into the GC to determine the concentrations of COBE and CHBE.
(2,2'-bis(diphenylphosphino)-1,1'-binaphathalene)chloro(p-cymene)ruthenium chloride; In ethanol; at 100℃; under 30003 Torr; for 4h;Product distribution / selectivity;
Examples 18 to 22 The reaction was carried out with (S)-(-) BINAP CI(cymene)RuCI in the presence of different solvents. The solvents, conditions and results are summarized in table 2 in figure 3. Examples 18, 21 and 22 are comparative. Unless specified otherwise in table 2, the reaction was carried out as described above for example 1. The results show, that the highest enantiomeric yields are obtained with EtOH THF and EtOH/acetone, although the reaction times are significantly shorter.
With potassium phosphate; Candida parapsilosis aldo-keto reductase CPAR5; NADPH; at 30℃; for 8h;pH 6.5;Enzymatic reaction;
General procedure: Asymmetric reductions of various carbonyl compounds by the purified enzymes were carried out at 30C for 8h with mild shaking in a reaction mixture containing 0.1M potassium phosphate buffer (pH 6.5), 1gL-1 substrate, 10mM NADPH, and the purified enzyme of appropriate amount in a total volume of 2mL. In order to determine the absolute configuration of chiral alcohols, the reaction products were extracted with ethyl acetate or hexane and the organic layer was used for analysis. The optical purity of the reaction products were determined by chiral HPLC (HP 1100, Agilent, USA) equipped with Chiralcel OB-H column (4.6mm×250mm; Daicel Chemical Ind. Ltd., Japan) or chiral GC (7890A, Agilent, USA) equipped with FID detector and Chrompack Chirasil-Dex CB chiral capillary column (25m×0.25mm; Varian, USA) [21].
With potassium phosphate; Candida parapsilosis aldo-keto reductase CPAR6; NADPH; at 30℃; for 8h;pH 6.5;Enzymatic reaction;
General procedure: Asymmetric reductions of various carbonyl compounds by the purified enzymes were carried out at 30C for 8h with mild shaking in a reaction mixture containing 0.1M potassium phosphate buffer (pH 6.5), 1gL-1 substrate, 10mM NADPH, and the purified enzyme of appropriate amount in a total volume of 2mL. In order to determine the absolute configuration of chiral alcohols, the reaction products were extracted with ethyl acetate or hexane and the organic layer was used for analysis. The optical purity of the reaction products were determined by chiral HPLC (HP 1100, Agilent, USA) equipped with Chiralcel OB-H column (4.6mm×250mm; Daicel Chemical Ind. Ltd., Japan) or chiral GC (7890A, Agilent, USA) equipped with FID detector and Chrompack Chirasil-Dex CB chiral capillary column (25m×0.25mm; Varian, USA) [21].
With reductase from Rhodococcus sp. ECU1014; NADH; In aq. phosphate buffer;pH 6;Enzymatic reaction;
General procedure: The reductase activity was analyzed spectrophotometrically at 30C by monitoring the decrease in the absorbance of NADH at 340nm. The standard analysis mixture (1mL) was composed of 100mM Na2HPO4-NaH2PO4 buffer (pH 6.0), 2.0mM ketoesters, 0.1mM NADH and an appropriate amount of enzyme. One unit was defined as the amount of enzyme leading to the consumption of 1mumol of NADH per min.
With D-glucose; D-glucose dehyrodenase; Lodderomyces elongisporus aldo?keto reductase 48; NADPH; In aq. phosphate buffer; at 30℃;pH 7;Enzymatic reaction;
General procedure: The enantioselectivity of LEAKRs was determined by employingNADPH regeneration system composed of D-glucose dehydrogenase(GDH) and D-glucose. The reaction mixture included 20 mMethyl 4-chloroacetoacetate, 0.2 mM NADPH, 0.03 mg LEAKR(1.2 mg for LEAKR 49), 100 mM D-glucose, 0.1 mg GDH, and100 mM sodium phosphate buffer (pH 7.0), with the final volumeof 0.5 mL. After shaking constantly at 30 C, the reaction mixturewas extracted with 1 mL ethyl acetate. The organic layer collectedby centrifugation and dried with anhydrous sodium sulfate wasthen analyzed to determine the conversion of the substrate andenantiomeric excess (e. e.) of the products by employing GC andHPLC as efficient and sensitive tools. A GC station (Agilent6890N) with a capillary column (DB-5) was used to analyze theconversion of ethyl 4-chloroacetoacetate while an HPLC station(Agilent 1100) with a Chiralcel OB-H column was employed forthe optical assay of the products.
With D-glucose; D-glucose dehyrodenase; Lodderomyces elongisporus aldo?keto reductase 49; NADPH; In aq. phosphate buffer; at 30℃;pH 7;Enzymatic reaction;
The enantioselectivity of LEAKRs was determined by employingNADPH regeneration system composed of D-glucose dehydrogenase(GDH) and D-glucose. The reaction mixture included 20 mMethyl 4-chloroacetoacetate, 0.2 mM NADPH, 0.03 mg LEAKR(1.2 mg for LEAKR 49), 100 mM D-glucose, 0.1 mg GDH, and100 mM sodium phosphate buffer (pH 7.0), with the final volumeof 0.5 mL. After shaking constantly at 30 C, the reaction mixturewas extracted with 1 mL ethyl acetate. The organic layer collectedby centrifugation and dried with anhydrous sodium sulfate wasthen analyzed to determine the conversion of the substrate andenantiomeric excess (e. e.) of the products by employing GC andHPLC as efficient and sensitive tools. A GC station (Agilent6890N) with a capillary column (DB-5) was used to analyze theconversion of ethyl 4-chloroacetoacetate while an HPLC station(Agilent 1100) with a Chiralcel OB-H column was employed forthe optical assay of the products.
With D-glucose; nicotinamide adenine dinucleotide phosphate; recombinant Bacillus subtilis-derived glucose dehydrogenase; recombinant Gluconobacter oxydans carbonyl reductase 0644; In aq. phosphate buffer; ethanol; at 30℃; for 12h;pH 7.0;Enzymatic reaction;Kinetics;
General procedure: The enzyme activity toward the reduction ofketones was determined by spectrophotometricallymeasuring the oxidation of NAD(P)H at 340 nm(epsilon = 6.22mM-1 cm-1) in the presence of an excess amount of ketones. The change in absorbance of NAD(P)H was monitored at 340 nm using an ultraviolet-visiblespectrophotometer with a temperature-controlledcuvette holder (Shimadzu Co., Kyoto, Japan). One unit(U) activity was defined as the amount of enzymesrequired to catalyze the oxidation of 1 mumol NAD(P)Hper minute at 30 C. The data were expressed as U/mgof protein. The standard reaction mixture contained100mM phosphate buffer (pH 7.0), 0.1mM NAD(P)H,5 mM substrate, and enzyme solvent in a total volumeof 1 mL. The reaction was initiated by adding 20 muLsolvent containing 10 mug-40 mug of enzymes. Blankswithout the enzyme were carried out for each substrate,and data were collected in triplicate. Protein concentrationswere determined with the bicinchoninic acid assayusing bovine serum albumin as a standard.NAD(P)H or NADH was assayed under standardreaction conditions for the study of coenzyme dependence.A range of substrates from 1 to 20 mM concentrationwas assayed under the standard reactionconditions for the study of kinetics. Apparent values ofMichaelis constant (Km) and kcat were calculated by fittingthe data into Michaelis-Menten equation using theSigmaPlot (Systat Software Inc., San Jose, CA, USA).All reactions followed Michaelis-Menten-type kinetics.Enantioselective reduction of ketones. The enantioselectivityof the enzymes was determined byexamining the reduction of aryl ketones, ethyl 4-chloroacetoacetate(COBE), and ethyl 2-oxo-4-phenylbutyrate(OPBE) using an NAD(P)H regeneration system consistingof BsGDH and glucose. The general procedurewas as follows: D-glucose (0.5%), recombinant BsGDH(10 U), NAD(P)+ (0.1 mM), the recombinant cell(30 g L-1, wet weight), and ketone solvated in ethanol[1 g L-1, 10%(v/v)] were mixed in a potassium phosphatebuffer (10 mL, 100 mM, pH 7.0). The mixturewas shaken at 30 C for 12 h.22) Upon termination ofthe reaction, each sample was extracted twice withequivalent ethyl acetate. The organic layer wasremoved, dried, diluted in the mobile phase, and thensubjected to chiral high-performance liquid chromatography(HPLC) to determine the conversion and enantiomericexcess (e.e.). Chiral HPLC analysis wasperformed on an Agilent 1100 series HPLC system witha UV detector.23) Chiral CHBE was analyzed on a chiracelOB-H column (Daicel, Japan) at lambda210 nm using hexane/2-propanol (90/10, v/v) as eluent at a flow rate of0.8 mL min-1 and a temperature of 25 C. Chiral HPBEand 4-phenyl-2-butanol were analyzed on a chiracelOD-H column at lambda210 nm and lambda254 nm using hexane/2-propanol (98/2, v/v) as eluent at a flow rate of 1.0 mLmin-1 and a temperature of 30 C. Authentic (relevant)standards were used for peak identification, and quantificationwas based on the peak area that was suitablycalibrated with standards of known concentration.
With Kluyveromyces polysporus alcohol dehydrogenase S237R mutant; isopropyl alcohol; NADPH; In aq. phosphate buffer; at 30℃;pH 7;Enzymatic reaction;
General procedure: Bioconversion was conducted with 20 mM 1a-10a,20 U·mL-1KpADH variants, 40 mM isopropanol in PBS buffer (pH 7.0,100 mM) in total volume of 2 mL at 30 C and 180 rpm overnight. Then,1 mL of the reaction mixture was withdrawn and extracted with ethylacetate. The organic phase was isolated by centrifugation and driedover anhydrous MgSO4. The conversion rate and enantioselectivity ofthe products were analyzed as described in supporting information.
With bis(1,5-cyclooctadiene)diiridium(I) dichloride; C26H25N2P; potassium tert-butylate; hydrogen; In isopropyl alcohol; at 20 - 80℃; under 38002.6 Torr; for 24h;Inert atmosphere; Autoclave;
In this embodiment, under the protection of nitrogen,The substance [Ir(COD)CL] 2 (0.00125 mmol, 0.0005 mol%),Chiral phosphine - pyridine ligand (0.00275 mmol, 0.0011 mol%)Potassium tert-butoxide (0.0125 mmol, 0.005 mol%) was dissolved in isopropanol (10 mL) and stirred at room temperature for 10 minutes.A solution of the substrate 4-chloroacetoacetate (0.25 mol) in isopropanol (10 ml) was added, which was placed in a high pressure autoclave and replaced with hydrogen three times.Then, hydrogen gas was introduced to 50 atm. and reacted at 80 C for 24 hours.Slowly release hydrogen,After removing the solvent, it was separated with a silica gel column to give ethyl 3-hydroxy-4-chlorobutanoate.
4-chloroEthyl acetoacetate,100mmol thiourea, 50mL ethanol, reflux 4h, spin solvent was evaporated to yield an oilLiquid, was added 20mL of ethyl acetate was dissolved, was added dropwise under ice-cooling with stirring, concentrated hydrochloric acid, the precipitated white solid was suction filtered, ethyl acetateA white solid was washed ester 2-aminothiazol-4-acetate hydrochloride, the filter cake was dissolved in water, adjusted to pH 10 ammonia water, ethyl acetateExtracted, dried over anhydrous sodium sulfate, and rotation of the solvent afforded a white solid 2-aminothiazol-4-acetate, yield 78percent, m.p.94 ~ 95 .
A mixture of ethyl 4-chloro-3-oxobutanoate (100 gm), polyphosphoric acid (400 gm) and urea (109.4 gm) was heated to 90-95C and stirred the reaction mixture for 3 hr at the same temperature. Further heated the reaction mixture to i20-i25C and stirred for iO hr at the same temperature. Reduced the temperature of the reaction mixture to 90-95C and chilled water was slowly added to it at the same temperature. Cooled the reaction mixture to 0-5C and stirred for 3 hr at the same temperature. Filtered the solid and washed with chilled water. Methanol (500 ml) and water (500 ml) were added to the obtained compound at 25-30C. Heated the reaction mixture to 70-75C and stirred for 40 mm at the same temperature. Charcoal (8 gm) was added to the reaction mixture at 70-75C and stirred for 40 mm at the same temperature. Filtered the reaction mixture through hyflow bed and washed the hyflow bed with methanol. Cooled the filtrate to 0-5C and stirred for 3 hr at the same temperature. Filtered the precipitated solid, washed with chilled water and dried the material to get the title compound.Yield: 23.0 gm; M.R.: 253-255C.
Ethyl 4-chloroacetoacetate (6.4 g, 39 mmol) was added to a stirred suspension of methyl-2-thiopseudourea sulphate (10 g, 36 mmol) and Ca (OH) 2 (3 g, 40 mmol) in water (60 ml) and ethanol (180 ml). The mixture was stirred for 36 h and acidified with 2N HCl and extracted with EtOAc. After drying (MGS04) and evaporation the crude product was suspended in DCM and filtered. Yield 3.1 g (45%). 'H NMR (400 MHz, CDC13) 8 2.58 (s, 3 H) 4.34 (s, 2 H) 6.40 (s, 1 H) 12.82 (br. s. , 1 H). MS (70eV) m/z (%) 189 (M-, 100)
With sodium carbonate; In water; at 17 - 25℃;
6-(Chloromethviy2-methylsulfanyl-pyrimidin-4-ol S-Methyl-2-thiopseudourea sulphate (20 g, 71.85 mmol), ethyl 4-chloroacetoacetate(10.755 ml, 79.04 mmol) and sodium carbonate (13.925 g, 107.78 mmol) were dissolved in water (100 ml) and stirred at room temperature overnight. The reaction was monitored by TLC, and once complete, the reaction precipitate was collected and the supernatant was neutralised with 6N hydrochloric acid to yield more reaction precipitate which was also collected. The accumulated precipitate was then washed with water (x3) and an off-white solid was obtained. This was dried in vacuo at 60 C for 48 hours to yield the desired compound as a pale yellow/white solid, 43.2g.NMR Spectrum: 1U NMR (300.132 MHz, CDCl3) 52.59 (s, 3H), 4.35 (s, 2H), 6.41 (s, IH), 12.70 (s, IH) ppm Mass Spectrum: M+ 190
With sodium carbonate; In water; at 17 - 25℃;
6-(Chloromethyl)-2-methylsulfanyl-pyrimidin-4-ol S-Methyl-2-thiopseudourea sulphate (20 g, 71.85 mmol), ethyl 4-chloroacetoacetate (10.76 ml, 79.04 mmol) and sodium carbonate (13.93 g, 107.78 mmol) were dissolved in water (100 mL) and stirred at RT overnight. The reaction was monitored by TLC, and once complete, the <n="98"/>reaction precipitate was collected and the supernatant was neutralised with 6N hydrochloric acid to yield more reaction precipitate which was also collected. The accumulated precipitate was then washed with water and an off-white solid was obtained. This was dried in vacuo at 600C for 48 hours to yield the desired compound as a pale yellow/white solid (43.2 g). NMR Spectrum: 1H NMR (300.132 MHz, CDCl3) delta 2.59 (s, 3H), 4.35 (s, 2H), 6.41 (s, IH), 12.70 (s, IH) Mass Spectrum: M+ 190
A mixture of 2-carbamoylnaphthalene (2.64 g, 15.4 mmol) and ethyl 4-chloroacetoacetate (2.06 g, 12.5 mmol) was stirred at 160°C for 1 hr. and then diluted with ethyl acetate (200 ml) at room temperature. The solution was washed with saturated sodium bicarbonate aqueous solution and brine in turn, and then dried. The solvent was distilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (n-hexane/ethyl acetate = 8 - 4) to give ethyl ester of [2-(2-naphthyl)oxazol-4-yl]acetic acid(459 mg, 13 percent) as yellow crystals. m.p.: 61.5 - 64 °C; IR (Nujol): 1737, 1591 cm-1; 1H-NMR (CDCl3): delta 1.31 (3H, t, J = 7.1 Hz), 3.73 (2H, d, J = 1.1 Hz), 4.24 (2H, q, J = 7.1 Hz), 7.49-7.57 (2H, m), 7.76 (1H, t, J = 1.1 Hz), 7.82-7.95 (3H, m), 8.11 (1H, dd, J = 1.7, 8.7 Hz), 8.58 (1H, br d, J = 1.1 Hz); APCI-MS m/z: 282 [M+H]+.
13%
at 160℃; for 1h;
2) A mixture of 2-carbamoylnaphthalene (2.64 g, 15.4 mmol) and ethyl 4-chloroacetoacetate (2.06 g, 12.5 mmol)was stirred at 160° C. for 1 hr. and then diluted with ethyl acetate (200 ml) at room temperature. The solution waswashed with saturated sodium bicarbonate aqueous solution and brine in turn, and then dried. The solvent wasdistilled off under reduced pressure, and the residue was purified by column chromatography on silica gel (nhexane/ethyl acetate=8-4) to give ethyl ester of [2- (2-naphthyl) oxazol-4-yl] acetic acid (459 mg, 13percent) as yellow crystals.m.p.: 61.5-64° C.; IR (Nujol): 1737, 1591 cm-1; 1H-NMR(CDCl3): delta1.31 (3H, t, J=7.1 Hz), 3.73 (2H, d, J=1.1 Hz),4.24 (2H, q, J=7.1 Hz), 7.49-7.57 (2H, m), 7.76 (1H, t, J=1.1 Hz), 7.82-7.95 (3H, m), 8.11 (1H, J=1.7, 8.7 Hz), 8.58(1H, br d, J=1.1 Hz); APCI-MS m/z: 282 [M+H]+
ethyl 2-isopropyl-4-methyl-1,3-thiazole-5-carboxylate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
93%
In ethanol; dichloromethane; ethyl acetate;
B. 2-(1-Methylethyl)-4-methylthiazole-5-carboxylic Acid Ethyl Ester A solution of <strong>[13515-65-6]2-methylpropanethioamide</strong> (3.1 g, 30 mmol) and ethyl 2-chloroacetoacetate (3.29 g, 20 mmol) in ethanol (40 mL) was heated at reflux for 3 h. The reaction mixture was evaporated to dryness and the residue was taken up in dichloromethane (50 mL) and the solution was washed with saturated sodium bicarbonate solution (2*25 mL). The aqueous layers were back-washed with dichloromethane (25 mL), then the combined dichloromethane layers were dried (MgSO4), filtered and concentrated under vacuum. The residue was purified using a Waters Prep 500 apparatus (silica gel dual column; 10% ethyl acetate/hexanes). Evaporation of the appropriate fractions afforded 2-(1-methylethyl)-4-methylthiazole-5-carboxylic acid ethyl ester (4.0 g, 93% yield).
PREPARATION 5 A mixture of ethyl 4-chloro-3-oxobutanoate (12.1 g), benzo-[c]-furazan-4-aldehyde (10 g), acetic acid (ml 0.2) and benzylamine (ml 0.37) is stirred for 24 hours at room temperature and is then diluted with acetonitrile (ml 95). After addition of 8.5 g of methyl 3-aminocrotonate, the solution is heated for three hours to 60 C., cooled to 35 C. and then treated with g 4.8 of p-toluenesulphonic acid. After further 30 minutes, the stirred solution is treated with 2 ml of a 28% solution of ammonium hydrate, concentrated under vacuum to 1/3 of its volume and finally diluted with water (150 ml). After extraction with ethyl acetate (3*40 Ml) and usual workup, by evaporation of the solvent and crystallization, 18 g of 2-chloromethyl-6-methyl-3-carboethoxy-5-carbomethoxy-4 -(benzo-[c]-furazan-4-yl)-1,4-dihydropyridine are obtained m.p. 121-122 C. (from MeOH); m.p. 92-94 C. (from Et2 O).
4-(1-benzylpiperid-4-ylmethoxy)acetoacetate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
4-(1-benzylpiperid-4-ylmethoxy)acetoacetate Treatment of ethyl 4-chloroacetoacetate with 1-benzylpiperidine-4-methanol according to the method of Example 22(i) gave the title compound as an oil which was used directly in the next step.
6-(Chloromethyl)-4-hydroxy-2-(methoxymethyl)-pyrimidine[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With sodium methylate; In methanol; water;
EXAMPLE 5 6-(Chloromethyl)-4-hydroxy-2-(methoxymethyl)-pyrimidine Sodium methylate (11.90 g, 0.22 mole) was added to a solution of <strong>[1903-91-9]methoxyacetamidine hydrochloride</strong> (12.45 g, 0.1 mole) and ethyl 4-chloroacetoacetate (16.50 g, 0.1 mole) in methanol (50 ml) at 5 to 10 C. with stirring. The resulting orange reaction mixture was stirred at 5 to 10 C. for 2 hours and then at 20 C. for 3 hours, and neutralized with conc. hydrochloric acid. The precipitate was filtered, and the filtrate was concentrated under reduced pressure to obtain a residue which was then dissolved in water (300 ml). The aqueous solution was extracted with chloroform, and the chloroform extract was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crystalline residue. The residue was suspended in ether and then filtered to obtain 17.8 g of 6-(chloromethyl)-4-hydroxy-2-(methoxymethyl)pyrimidine as pale brown needle-like crystals (m.p. 124.0-125.5 C.).
With hydrogenchloride; sodium methylate; In methanol; water;
EXAMPLE 3 2,6-Bis(chloromethyl)-4-hydroxypyrimidine Sodium methylate (5.4 g, 0.01 mole) was added to a solution of <strong>[10300-69-3]chloroacetamidine hydrochloride</strong> (6.45 g, 0.05 mole) and ethyl 4-chloroacetoacetate (8.3 g, 0.05 mole) in methanol (30 ml) at room temperature with stirring. The mixture was stirred at room temperature for 6 hours and concentrated under reduced pressure. The residue obtained was dissolved in water (50 ml). The pH of this aqueous solution was adjusted to 4 to 5 with conc. hydrochloric acid, followed by extraction with chloroform. The chloroform extract was washed with water, dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 6.8 g of a yellow residue. The residue was recrystallized from ether to obtain 6.0 g of 2,6-bis(chloromethyl)-4-hydroxypyrimidine as colorless needle-like crystals (m.p. 136-138 C.)
7-chloromethyl-2-methylthio-5H-1,3,4-thiadiazolo[3,2-a]pyrimidine-5-one[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With sodium hydroxide; In PPA;
EXAMPLE 3 <strong>[5319-77-7]2-amino-5-methylthio-1,3,4-thiadiazole</strong> (9.4 g) was reacted with ethyl 4-chloro-acetoacetate (15.8 g) in polyphosphoric acid (50 g) under stirring at 100 C. for 1 hour. After cooling, dilution with ice water and neutralization with 35% NaOH, the precipitate was filtered and washed with water until neutral to give 7-chloromethyl-2-methylthio-5H-1,3,4-thiadiazolo[3,2-a]pyrimidine-5-one, m.p. 168-169 C. (12.4 g), which was reacted with triphenylphosphine (14.4 g) in acetonitrile (250 ml) under stirring at reflux temperature for 24 hours.
4-[N-(4-chlorophenyl)aminocarbonylamino]-2(5H)-furanone[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
In 1,4-dioxane; N-methyl-acetamide; methanol; diethyl ether; acetone;
EXAMPLE 2 4-[N-(4-chlorophenyl)aminocarbonylamino]-2(5H)-furanone To a magnetically stirred suspension of <strong>[140-38-5]4-chlorophenylurea</strong> (2 g) in dioxane (4 ml), ethyl 4-chloroacetoacetate (1.63 ml) are added and the suspension is heated to reflux for two hours, then kept at toom temperature overnight. The yellowish gray precipitate is recovered by filtration and washed with the mother liquor, with methanol (2*1 ml) and finally with diethyl ether (2 ml). After drying under vacuum at 40 C. overnight, 1.34 g of crude product were obtained. The crude product is then suspended under reflux in 20 ml of dioxane for 45 minutes, then the suspension is allowed to return to room temperature and the solid is recovered by filtration and washed with dioxane (1 ml) and then with diethyl ether (2 ml). After drying overnight under vacuum at 40 C., 1.1 g of the product are obtained, which still contains some impurities. It is therefore dissolved in hot dimethylformamide (2.2 ml) and when a complete solution is obtained the heating bath is removed and the product reprecipitate in a short time. When the suspension reaches room temperature 11.2 ml of acetone are added and after stirring for 2 hours the solid is recovered by filtration and washed first with the mother liquor and then with acetone (2*3 ml). After drying under vacuum at 40 C. overnight, 0.73 g of off-white product are obtained, m.p.>254-256 C. TLC (silica gel, eluant methylene chloride/methanol 9:1; UV detection at 254 nm): single spot at Rf=0.39. Elem. Anal. (% calcd/found): C 52.29/52.05; H 3.59/3.60; N 11.09/10.99; Cl 14.03/14.05.
With glucose dehydrogenase; ketoreductase; In acetic acid butyl ester; water; at 30 - 40℃; for 16h;Aqueous phosphate buffer;Product distribution / selectivity;
Example 24: Preparation of ethyl (R)-4-CVANO-3-HYDROXYRATE from ethyl 4-CHLOROACETOACETATE (via ethyl (S)-4-chloro-3-hydroxybutyrate). To a 20 mL screw cap vial was added NACN (125 MG, 2. 55 MMOL), NAH2PO4 (415 mg, 3.46 mmol) and glucose (750 mg, 3. 8 mmol). Water (5 mL) was added followed by NADP (2 mg), ketoreductase SEQ ID NO: 56 (50 mg), glucose dehydrogenase SEQ ID NO: 62 (50 mg), and halohydrin dehalogenase SEQ ID NO: 32 (100 mg). Then ethyl 4-chloro- acetoacete (24 mg, 0.15 mmol) in 0.5 ML BUTYL acetate was added. The vial was capped and heated in an oil bath at 30 C ; After 1 HR, GC analysis of a butyl acetate extract of a reaction sample showed 100% conversion of the ethyl 4-CHLORO-ACETOACETE to ethyl (S)-4-chloro- 3-hydroxybutyrate, at 96% selectivity, and ethyl (R)-4-CYANO-3-HYDROXYBUTYRATE at 4% selectivity. Then, the reaction vial was heated to 40 C for 15 hrs. GC analysis of a butyl acetate extract then showed 2% of the ethyl (S) -4-chloro-3-hydroxy-butyrate remaining, with overall 98% yield of ethyl (R) -4-cyano-3-hydroxybutyrate based on the starting ethyl 4-chloroacetate. This example shows the process of the invention wherein a 4-CYANO-3-HYDROXYBUTYRIC acid ester (ethyl 4-CYANO-3- (R)-HYDROXYBUTYRATE) is produced, via an intermediate 4-halo-3- hydroxybutyric acid ester (ethyl 4-CHLORO-3- (S)-HYDROXYBUTYRATE), by contacting a 4-halo-3- ketobutyric acid ester (ethyl 4-chloroacetoacetate) with a ketoreductase, a cofactor (NADPH, provided as NADP) a cofactor regeneration system (glucose and glucose dehydrogenase), a halohydrin dehalogenase, and cyanide (provided by an cyanide salt, NaCN) with all the reactants simultaneously present in the reaction mixture.
To a solution of 2,4-diamino-6-hydroxypyrimidine, 11, (45 g, 0.36 mol) and sodium acetate (29.5 g, 0.36 mol) in water (1 L) at reflux was added, dropwise, ethyl-4-chloroacetoacetae, 12 (60.9 g, 0.37 mol). Within an hour, a thick white precipitate appeared. The mixture was refluxed for 18 h. The suspension was cooled to room temperature, filtered, washed with water (2 * 150 mL), acetone (2 * 100 mL) and dried to afford 13 as an off-white powder (45.6 g, 54%); mp: 275-276 C (lit. mp > 250 C); 1H NMR (400 MHz, DMSO-d6): delta = 1.18-1.22 (t, 3H, -COOCH2CH3), 3.58 (s, 2H, -CH2COO CH2CH3), 4.07-4.19 (q, 2H, -COOCH2CH3), 6.01 (s, 1H, 5-H), 6.04 (s, 2H, 2-NH2), 10.20 (s, 1H, 3-H), 10.90 (s, 1H, 7-H); ESI-MS (m/z): 237.1 [M+H]+.
To a stirred suspension of 58,8 g 60 percent (1.47 mol) sodium hydride in 600 ml anhydrous tetrahydrofuran, a solution of 94 g (0.7 mol) 2,2-diethoxy-ethanol in 160 ml tetrahydrofuran was added dropwise, so that the temperature was kept below 40 'C. After completion of the addition, the reaction mixture was stirred for further 30 minutes. Then 115 g (0.7 mol) ethyl 4-chloroacetoacetate in 500 ml anhydrous tetrahydrofuran was added dropwise within 3 hours, so that the temperature was kept between 10 °C and 40 °C, preferentially at about 20 'C. The mixture was stirred overnight at room temperature. Then 90 ml ethanol was added dropwise, and the mixture was poured into 900 g of ice after which pH was adjusted to 6 with hydrochloric acid. The organic phase was separated and dried over MgSO4. The tetrahydrofuran was evaporated off and the product was separated from the oily layer in a separation funnel. Then the product was dissolved in toluene and purified by filtration through a short column of silica. The toluene was evaporated off, leaving the product as a light yellow oil. The product was purified by distillation in vacuo. yield: 130.9 g = 71.4 percent bp. = 112-114 'C at 0.2 mm Hg[] Elemental analysis: CalculatedC 54.9percentH 8.5percentFoundC 54.48percentH 8.7percentIR: 2986 cm-1; 1726 cm-1; 1748 cm-1; 1119 cm-1; 1067 cm-1 (Between KBr plates) NMR: 250 MHz 1H-NMR (CDCl3) (delta ppm): 4.646 (t, H, CH); 4.286 (s, 2H, CH2); 4.224 (s, 2H, CH2); 3,708 (q, 2H, CH2); 3.568 (q, 2H, CH2); 3.556 (d, 2H, CH2) ; 1.30 (t, 3H, CH3); 1.21 (m, 6H, CH3).
Example 1; Part A; To the solution of 3-phenyl-1H-1 ,2,4-triazol-5-amine (0.8 g, 5 mmol) in acetic acid (6 mL) was added ethyl 4-chloro-3-oxobutanoate (0.75 mL, 5.5 mmol). The reaction mixture was stirred for 24 hours at 80 0C, and then cooled to room temperature. The reaction mixture was filtered. The precipitates were washed with ACN and dried to give compound 1-1 in a white powder (872 mg, 67percent yield). 1H NMR (400 MHz, CD3OD) delta 8.24-8.18 (m, 2H), 7.54-7.46 (m, 3H), 6.21 (s, 1H), 4.65 (s, 2H).; Example 6; Part A; To the solution of 3-phenyl-1 H-1 ,2,4-triazol-5-amine (0.8 g, 5 mmol) in acetic acid (6 ml_) was added ethyl 4-chloro-3-oxobutanoate (0.75 ml_, 5.5 mmol).The reaction mixture was stirred for 24 hours at 80 0C, and then cooled to room temperature. The reaction mixture was filtered. The precipitates were washed with ACN and dried to give compound 1-1 in a white powder (872 mg,67percent yield). 1H NMR (400 MHz, CD3OD) delta 8.24-8.18 (m, 2H), 7.54-7.46 (m,3H), 6.21 (s, 1 H), 4.65 (s, 2H).; Example 12; Part A; To the solution of 3-phenyl-1 H-1 ,2,4-triazol-5-amine (0.8 g, 5 mmol) in acetic acid (6 mL) was added ethyl 4-chloro-3-oxobutanoate (0.75 mL, 5.5 mmol). The reaction mixture was stirred for 24 hours at 80 0C, and then cooled to room temperature. The reaction mixture was filtered. The precipitates were washed with ACN and dried to give compound 1-1 in a white powder (872 mg, 67percent yield). 1H NMR (400 MHz, CD3OD) delta 8.24-8.18 (m, 2H), 7.54-7.46 (m, 3H), 6.21 (s, 1 H), 4.65 (s, 2H).
65.1%
With acetic acid; at 80℃;
<strong>[4922-98-9]3-phenyl-1H-1,2,4-triazole-5-amine</strong> (0.95 g, 5.93 mmol) obtained in Step 3-2 was dissolved in 15 mL of acetic acid and ethyl 4-chloroacetoacetate (1.07 g , 6.52 mmol), and the mixture was stirred at 80 ° C. overnight. After completion of the reaction, the precipitate was suction filtered to obtain 5-methylchloro-2-phenyl-[1,2,4]triazolo[1,5-a]pyrimidin-7(4H)-one (1.0 g, 86 mmol; yield 65.1percent)
EXAMPLES; Example 1; To a 30 gallon glass-lined reactor was charged water (26 L) and p-anisidine (8.5 kg; 68.1 moles). The reactor was inerted, and 20.1 kg 37percent HCl (3 eq.) were charged to the reactor followed by a line flush of 2 L purified water. The batch was heated to 40° C. and checked to ensure dissolution of p-anisidine (total time at 40° C. was 25 minutes). The batch was cooled to -2° C. and 40percent aqueous sodium nitrite (12.0 kg; 69.5 moles) was charged to the reactor while keeping the temperature at -2+/-3° C. The addition time for the aqueous sodium nitrite was 35 minutes. The sodium nitrite line was flushed with 2 L of purified water. The reaction mixture was sampled and analyzed for reaction completion. To the batch was added 3.0 kg of 11percent aqueous sulfamic acid via nitrogen pressure, keeping the reactor contents at -2+/-3° C. In a separate 100 gallon glass-lined reactor, 28 L of purified water was charged followed by 11.2 kg of solid sodium acetate. The reactor was inerted, and the batch was heated to 35° C. until the solid dissolved. The batch was cooled to 15° C. and 18 kg acetone was added to the reactor. To the reactor was added 12.4 kg ethyl-2-chloroacetoacetate by deadhead vacuum, followed by 2.1 kg of acetone to flush the addition line. The batch was cooled to -2+/-3° C. The contents of the above 30 gallon reactor were then transferred to the 100 gallon reactor keeping the batch in the 100 gallon reactor at -2+/-3° C. The transfer time was 45 minutes. The transfer line was flushed with 5.0 kg of acetone, and the batch was allowed to mix for one hour. To the reactor was charged 32.0 kg of acetone, and the batch was mixed for 15 minutes. The batch was allowed to settle for 30 minutes. To the reactor was charged 10.2 kg of acetone and the batch was mixed for 5 minutes. Agitation was stopped and the batch was allowed to settle for 30 minutes. The aqueous phase was discharged to a drum (140.2 kg; waste). To the reactor was charged 13.4 kg of methanol, controlling the reactor temperature to 0+/-3° C. The batch was held at 0+/-3° C. and sampled every hour until the reaction was complete. To the reactor was charged 75 L of purified water. The batch was heated to 5+/-5° C. and allowed to mix for 15 minutes. The batch was isolated using a portable centrifuge with a 5-7 micron polypropylene bag. The isolation lasted 10 minutes (109 kg of centrate). To the reactor was charged 26 L of purified water and 20.2 kg of methanol. The mixture was cooled to 5+/-3° C. and then discharged to the centrifuge to wash the cake. The wet cake (14.5 kg) was transferred to drying trays covered with FEP liners. The cake was dried at 40° C. for 80 hours to give 13.8 kg of the desired product.
PPA (10 mL) was added to the mixture of compound 3 (1 g, 6 mmol) and compound 4 (1 g, 6 mmol). The mixture was heated to 1000C for 0.5 hours. Then it was poured into water and extracted with EA, washed with brine and dried over Na2SO4. Concentration and chromatography gave a yellow solid (135 mg). Yield: 8.5%.1HNMR (400 MHz, CDCl3): delta (ppm): 8.91 (IH, d, Ar-H), 7.32-7.47 (2H, m, Ar-H), 6.59 (IH, s, Ar-H), 4.47 (2H, s, -CH2-), 2.52 (3H, s, CH3-).
To ethyl 4-chloro-3-oxobutanoate (4.91 mL) was added sulfuric acid (12.91 mL) at 0C, then was added 2- chlorobenzene-1, 3-diol (5.0 g) in several portions at 0C. The mixture was warmed to room temperature and stirred at the same temperature for 2 h. The reaction mixture was poured into water under stirring at 0C. The precipitate was collected by filtration and washed with hexane to give the title compound (2.78 g) . MS (ESI-) : [M-H]" 243.1.
With aluminum (III) chloride; In nitrobenzene; at 90 - 130℃; for 3h;Inert atmosphere;
A mixture of <strong>[41175-50-2]8-hydroxyjulolidine</strong> (300 mg, 1.59 mmol) and ethyl 4-chloroacetoacetate (260 ul, 1.90 mmol) was stirred at ?90° C. under argon atmosphere. To this mixture was added a solution of aluminum chloride (420 mg, 3.2 mmol) in nitrobenzene (5 ml) and the whole reaction mixture was stirred at ?130° C. for 3 hours. After the reaction mixture was cooled to room temperature, it was purified by column chromatography over silica gel eluting first with 5percent ethyl acetate in hexane and then with chloroform to obtain the desired product as a yellow-brown solid (200 mg, 43percent yield). TLC: Rf=0.56 (silica gel, 1:1 hexane/ethyl acetate). 1NMR (DMSO-d6): chemical shift (ppm) 7.21 (s, 1H, ArH), 6.15 (s, 1H, ArH), 4.86 (s, 2H, CH2), 3.28-3.25 (m, 4H, CH2), 2.76-2.69 (m, 4H, CH2), 1.90-1.87 (m, 4H, CH2). absorption maximum: 405 nm in methanol, emission maximum: 493 nm in methanol. MS: m/e 290.20.
With sulfuric acid; In water; at 20℃;
General procedure: To a solution of 3-methoxy-2-naphthol (0.104 g, 5.97 x 10-4 mol) in 70percent aqueous sulphuric acid (5 mL), ethyl 4-chloro-3-oxobutanoate (0.089 mL, 6.57 x 10-4 mol) was added. The reaction was followed by TLC (ethyl acetate/n-hexane, 1:4), and stirred at room temperature for 96 h. The mixture was poured into ice water and stirred for 2 h to give a fine pale precipitate. The solid was collected by filtration, washed with cold water, dried and purified by column chromatography, using ethyl acetate/light petroleum as eluent, with mixtures of increasing polarity. Compound 3 was obtained as pale brown solid (0.012 g, 7percent).
With piperidine; hydrogenchloride; iodine; acetic acid; In ethanol; at 80℃; for 12h;Inert atmosphere; Green chemistry;
General procedure: Ethyl 4-chloroacetoacetate (2 mmol) and ethanal (1mmol) were placed in a flask under an atmosphere of nitrogen. Piperidine (1 mmol), glacial acetic acid (1 mmol), iodine (1 mmol) concentrated hydrochloric acid (0.01 ml) and ethanol (10 ml) was added. The resulting mixture was heated at 80 °C for 12 h. After cooling to room temperature, the solvent was removed by evaporation. The residue was poured into 50 ml of water and extracted with ether. The organic layer was separated and was washed with saturated sodium chloride solution. After dried over sodium sulfate, the solvent was removed and the residue was purified by column chromatography on silica gel to give the product 1a (0.15 g, white powder) in 63 percent yield.
10-(chloromethyl)-7-thia-1,9-diazatricyclo[6.4.0.02,6]dodeca-2(6),8,10-trien-12-one[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
4%
With polyphosphoric acid; at 110℃; for 1h;
A mixture of 4H,5H,6H-cyclopenta[d][l,3]thiazol-2-amine (2.00 g, 14.3 mmol) and ethyl 4- chloro-3-oxobutanoate (3.50 g, 21.3 mmol) in polyphosphoric acid (15 mL) was stirred for 1 h at 110 C. The reaction mixture cooled to room temperature, diluted with water (30 mL) and stirred for 1 h at 80 C. After cooling to room temperature, the reaction was quenched by water (200 mL), and the pH value ofthe solution was adjusted to pH 8-9 with potassium carbonate, extracted with dichloromethane (100 mL x 3), washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by chromatogtaphy with 20% ethyl acetate in petroleum ether to afford 10-(chloromethyl)-7-thia-l,9- diazatricyclo[6.4.0.0' [2,6]]dodeca-2(6),8,10-trien-12-one as a brown solid (150 mg, 4.0%). LCMS (ESI): M+H+ =241.1.
3-(methoxymethyl)-1-(3-nitrophenyl)-1H-pyrazol-5(4H)-one[ No CAS ]
5-ethoxy-3-(methoxymethyl)-1-(3-nitrophenyl)-1H-pyrazole[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
58%; 10%
With bismuth(lll) trifluoromethanesulfonate; for 14h;Reflux;
The syntheses of 3-(chloromethyl)-1-phenyl-1H-pyrazol-5(4H)-ones (7a-o and 8a-j) are the same as described for those of compound 7a. Bi(OTf)3 (0.1 mmol) was added to a stirred solution of phenylhydrazine hydrochloride (1, 1 mmol) and ethyl 4-chloro-3-oxobutanoate (2, 1 mmol) in alcohols such as MeOH and EtOH (3mL). The mixture was stirred under reflux for 16 h. After completion of the reaction(TLC), the solvent was removed under reduced pressure. The crude product was subjected to column chromatography (hexane/AcOEt 8:2) to give pure pyrazolones and pyrazoles.
3-(ethoxymethyl)-1-(3-nitrophenyl)-1H-pyrazol-5(4H)-one[ No CAS ]
5-ethoxy-3-(ethoxymethyl)-1-(3-nitrophenyl)-1H-pyrazole[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
51%; 11%
With bismuth(lll) trifluoromethanesulfonate; for 20h;Reflux;
General procedure: The syntheses of 3-(chloromethyl)-1-phenyl-1H-pyrazol-5(4H)-ones (7a-o and 8a-j) are the same as described for those of compound 7a. Bi(OTf)3 (0.1 mmol) was added to a stirred solution of phenylhydrazine hydrochloride (1, 1 mmol) and ethyl 4-chloro-3-oxobutanoate (2, 1 mmol) in alcohols such as MeOH and EtOH (3mL). The mixture was stirred under reflux for 16 h. After completion of the reaction(TLC), the solvent was removed under reduced pressure. The crude product was subjected to column chromatography (hexane/AcOEt 8:2) to give pure pyrazolones and pyrazoles.
3-(chloromethyl)-1-(3-nitrophenyl)-1H-pyrazol-5(4H)-one[ No CAS ]
3-(chloromethyl)-5-ethoxy-1-(3-nitrophenyl)-1H-pyrazole[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
52%; 10%
With bismuth(lll) trifluoromethanesulfonate; In tetrahydrofuran; for 18h;Reflux;
General procedure: The syntheses of 3-(chloromethyl)-1-phenyl-1H-pyrazol-5(4H)-ones (5a,b and 6a-e) are the same as described for those of compound 6a. Bi(OTf)3 (0.1 mmol) was added to a stirred solution of phenylhydrazine hydrochloride (1, 1 mmol) and ethyl 4-chloro-3-oxobutanoate (2, 1 mmol) in THF (3mL). The mixture was stirred under reflux for 12 h. After completion of the reaction (TLC), the solvent was removed under reduced pressure. The crude product was subjected to column chromatography (hexane=AcOEt 8:2) to give pure pyrazolones and pyrazoles.
(6-bromo-7-fluoroimidazo[1,2-a]pyridin-2-yl)acetic acid ethyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
83%
In ethanol; at 80℃; for 14h;Inert atmosphere;
To a solution of Intermediate 13 (2.014 g, 10.54 mmol, 1.0 eq.) in EtOH (25 mL) heated at 80C was added 4-chloro-3-oxo-butyric acid ethyl ester (1.91 g, 11.6 mmol, 1.1 eq.) drop wise. The reaction mixture was heated for 14 h, then concentrated to dryness to give a beige solid, taken up in 2 x 20 mL EtOAc and filtered. The solid was again taken up in 50 mL EtOAc, washed with 50 mL of an aq. sat. sol. of NaHC03. The aqueous phase was extracted with 3 x 50 mL EtOAc. The organic phase was washed with brine, dried over MgS04and concentrated to yield the title compound as a black solid (2.65 g, 83 %). 1H NMR (400 MHz, CDC13) delta 8.26 (d, J 6.4 Hz, 1 H), 7.55 (s, 1 H), 7.27 (m, 1 H), 4.21 (m, 2 H), 3.83 (s, 2 H), 1.28 (m, 3 H). LCMS (ES+) RT 4.3 min, 301.0/303.0 (M+H)+.
ethyl (2E/Z)-(7-bromo-2H-1,4-benzoxazin-3(4H)-ylidene)acetate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
56%
With triethylamine; In tetrahydrofuran; at 50℃; for 16.5h;
Intermediate 25 (2E or 2Z)-(7-bromo-2H-1,4-benzoxazin-3(4H)-ylidene)acetate Ethyl 4-chloro-3-oxobutanoate (45 mL, 333 mmol) was added to a solution of <strong>[38191-34-3]2-amino-5-bromophenol</strong> (55.7 g, 296 mmol) in THF (400 mL) at rt. The mixture was heated to 50 C., and TEA (57 mL, 326 mmol) was added into slowly over 30 min. The resulting mixture was stirred at this temperature for 16 h. The mixture was diluted with water (500 mL) and extracted with EtOAc (500 mL*3). The combined organic phase was washed with brine (500 mL*2), dried over anhydrous Na2SO4, filtered and concentrated by evaporation. The residue was purified by a silica chromatography using a solvent gradient from 1.25% to 2% of EtOAc in PE. The pure fraction was evaporated to dryness to afford the title compound (47 g, 56%) as a light yellow solid. 1H NMR (300 MHz, CDCl3) delta 1.29 (3H, t), 4.18 (2H, q), 4.56 (2H, s), 4.70 (1H, s), 6.70 (1H, d), 7.02-7.06 (2H, m), 10.14 (1H, brs). MS m/z 298 (M+H)+.
7-chloro-2-(chloromethyl)-9-hydroxy-4H-pyrido[1,2-a]pyrimidin-4-one[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
100%
With phosphoric acid; at 110.0℃; for 2.5h;
<strong>[40966-87-8]5-chloro-2-amino-pyridinol</strong> 12-1(4.3 g, 29.7 mmol), 4-chloroacetoacetate (8.5 mL) were heated together in polyphosphoric acid (20 mL) at 110°C for 2.5 h. The reaction mixturewas cooled, crushed ice (30 g) was added and the pH of the mixture was adjusted to 5, by the addition of 2N NaOH. A brown precipitate formed, that was collected by filtration, washing with H20 until the washings were colourless. The product was dried to afford the chioromethyl derivative as a brown powder (7.27 g, 100percent). 1H NMR (500 mHz, d6- DMSO) 4.67 (s, 2H), 6.59 (s, 1 H), 7.27 (d, J=2.OHz, 1 H), 8.46 (d, J=2.OHz, 1 H).
Weighing 4-ethyl chloroacetoacetate (1.64g, 10mmol) dissolved in 10 ml of concentrated sulfuric acid, ice water bath cooling to 0 C, for assigning the batch reaction system by adding <strong>[75996-29-1]5-fluoro resorcinol</strong> (1.28g, 10mmol), then naturally to room temperature, and then continuously stirred for 2 hours, until the raw material the reaction is complete. The reaction solution is poured into 200 ml ice water mixed solution, a large amount of solid precipitated, filtering, the plurality of times to get the solid washing with water, drying, the resulting product 2a.
Weigh 4-ethyl chloroacetoacetate (8. 2g, 50mmol) was dissolved in 20mL of concentrated sulfuric acid in an ice-water bath was cooled to 0 C, the reaction system was added in divided portions resorcinol (5. 5g, 50mmol) and naturally to room temperature, stirring was continued for 2 hours, The starting material until the reaction was complete. The reaction solution was poured into 200mL ice water mixture, large amount of solid precipitated, was filtered, and the resulting solid was repeatedly washed with water, dried, and the resulting crude product was used without further purification in the next reaction. The resulting crude product from the previous step was dissolved 400ml 1M in aqueous NaOH and heated at reflux for 2 hours. The reaction was cooled to room temperature, the aqueous solution was washed twice with ethyl acetate, the aqueous phase retained, concentrated sulfuric acid, 200 mL ethyl acetate three times, the combined organic phase was washed with water and saturated brine, dried over anhydrous sodium sulfate, filtered, and concentrated to give The product YZ-5 (6. 3g), 67% yield over two steps.
ethyl 2-(6-bromoimidazo[1,2-b]pyridazin-2-yl)acetate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
65%
In ethanol; at 120℃; for 2h;Microwave irradiation;
A solution of ethyl 4-chloro-3-oxobutanoate (1.99 g, 811.49 mmol) and 6-bromopyridazin-3 -amine (1.0 g, 6.90 mmol, PharmaBlock) in EtOH (15 mL) was heated in a microwave at 120 C for 2 h. The reaction was concentrated under reduced pressure. The residue was dissolved in EtOAc (about 250 mL) and washed with water, sat. sodium bicarbonate, and then brine. The organics were collected, dried over MgS04, filtered and concentrated under reduced pressure. The residue was purified by column chromatography on silica (EtOAc /heptane 0-50 %) to give the title compound (1.06 g, 65%) LC/MS (Table 1, Method i) = 0.73 min; MS m/z: 284, 286 (M+H)+ .
With 4-toluenesulfonyl azide; In tetrahydrofuran; at 20 - 25℃;
General procedure: To a solution of the corresponding beta-keto ester 1 (1.0 mmol) and TsN3 (197 mg, 1.0 mmol) in THF (2.0 mL) under stirring at 25 C was added the amine (1.1 mmol). Then the reaction mixture was stirred at room temperature until consumption of the starting material (monitored by TLC: 30 min to 29 h, see Table 1 and Scheme 4). Next, the mixture was diluted in 5 mL of CH2Cl2 and concentrated under reduced pressure. After complete removal of the solvent, the residue was triturated in ethyl ether and the resulting mixture was again concentrated under reduced pressure. The final solid residue was repeatedly triturated with hexane (for amides 3 and 4) or a 9:1 hexane/CH2Cl2 mixture (for amides 5 and 6) to separate out the insoluble TsNH2 by decantation. The resulting supernatants were filtered and concentrated under reduced pressure to give the known amides25 3-6 as oils with high degree of purity. Alternatively, further purification through column chromatography on silica gel using gradient mixtures of hexane/EtOAc as eluent was employed to furnish pure products in 80-99% yield.
ethyl 2-(8-amino-5-bromoimidazo[1,2-a]pyridin-2-yl)acetate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
263 mg
In ethanol;Microwave irradiation;
A mixture of ethyl 4-chloroacetoacetate (205 mu, 1.51 mmol) and 6-bromo-pyridine-2,3- diamine (237 mg, 1.26 mmol) in EtOH (20 mL) was refluxed overnight. After the solvent was removed on a rotary evaporator, the residue was dissolved in EtOAc (30 ml) and washed with saturated Na2C03and brine. The organic layer was dried over Na2S04and concentrated in vacuo. The residue was purified by flash chromatography on silica gel to give ethyl 2-(8- bromoH-imidazo[l,2-a]pyridin-2-yl)acetate (263 mg, 0.88 mmol). 1H MR (CDC13) delta 7.71 (s, 1H), 6.79 (d, J= 7.8 Hz, 1H), 6.28 (d, J= 7.8 Hz, 1H), 4.53 (br, 2H), 4.21(q, J= 7.1 Hz, 2H), 3.86 (s, 2H), 1.29 (t, J = 9.1 Hz, 3H).
(2E)-2-(4-benzylpiperazin-2-ylidene)acetic acid ethyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
35.3%
In methanol; at 0 - 30℃; for 1h;Inert atmosphere;
4-Chloro-3-carbonyl-butyric acid ethyl ester (3.38 mL, 25 mmol) at 0 CAdd to a solution of N'-benzylethyl-1,2-diamino 2a (5.7 mL, 38 mmol) in MeOH (25 mL)The reaction was carried out for 1 hour at room temperature.The solvent was removed by EtOAc (EtOAc)EtOAc.Wash with water (50mL × 2) and saturated sodium chloride solution (50mL × 2), dry anhydrous sodium sulfateThe residue was purified by silica gel column chromatography eluting2b (2.3 g, yield 35.3%) as a yellow solid.
Chemistry
Pharmaceutical Intermediates
Inhibitors/Agonists
Material Science
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110K+ Compounds
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