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Discovery of Inhibitory Fragments That Selectively Target Spire2-FMN2 Interaction
Kitel, Radoslaw ; Surmiak, Ewa ; Borggrafe, Jan ; Kalinowska-Tluscik, Justyna ; Golik, Przemyslaw ; Czub, Miroslawa , et al.
Abstract: Here, we report the fragment-based drug discovery of potent and selective fragments that disrupt the Spire2-FMN2 but not the Spire1-FMN2 interaction. Hit fragments were identified in a differential scanning fluorimetry-based screen of an inhouse library of 755 compounds and subsequently validated in multiple orthogonal biophys. assays, including fluorescence polarization, microscale thermophoresis, and 1H-15N HSQC NMR. Extensive structure-activity relationships combined with mol. docking followed by chem. optimization led to the discovery of compound 13, which exhibits micromolar potency and high ligand efficiency (LE = 0.38). Therefore, this fragment represents a validated starting point for the future development of selective chem. probes targeting the Spire2-FMN2 interaction.
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Purchased from AmBeed: 63010-72-0 ; 391-82-2 ; 188111-79-7 ; 23432-40-8 ; 75090-52-7 ; 178984-56-0 ; 57260-73-8 ; 21617-12-9 ; 611-35-8 ; 68500-37-8 ; 346-55-4 ; 181950-57-2 ; 216854-23-8 ; 86-98-6 ; 309956-78-3 ; 5407-57-8 ; 625471-18-3 ; 63136-61-8 ; 82121-05-9 ; 259731-83-4 ; 82121-08-2 ; 1557337-03-7 ; 41460-18-8 ; 766544-91-6 ; 256923-25-8 ; 91066-18-1
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CAS No. : | 309956-78-3 |
Formula : | C10H20N2O2 |
M.W : | 200.28 |
SMILES Code : | O=C(OC(C)(C)C)N[C@H]1CNCCC1 |
MDL No. : | MFCD03093382 |
InChI Key : | WUOQXNWMYLFAHT-MRVPVSSYSA-N |
Pubchem ID : | 1514172 |
GHS Pictogram: |
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Signal Word: | Warning |
Hazard Statements: | H317-H319 |
Precautionary Statements: | P280-P305+P351+P338 |
* 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.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
95.3% | With potassium carbonate; In dimethyl sulfoxide; acetonitrile; at 84 - 86℃; for 20h; | a) At room temperature, add the compound 8-bromo-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione(100.00g, 220.6mmol), (R) -3-tert-butoxycarbonylaminopiperidine (57.50g, 287.0mmol),Potassium carbonate (122.00g, 883.0mmol),475ml of acetonitrile and 25ml of dimethyl sulfoxide, stirred and heated to 84-86 C, kept under stirring for about 20 hours. b) After the reaction is completed, add 750ml of purified water to the reaction bottle dropwise. After the dropwise addition, stir to dissolve, control the temperature to 65-67 , keep warm and stir. Hour, cool down to about 25 , stir for 1 hour,After filtration, after the filter cake was drained, rinse with 500ml of purified water and drain to obtain a white crystalline powder solid product D (120.40g, 210.2mmol) with a yield of 95.30% and a purity of 99.935%.The XRPD chart of the white crystalline powder is shown in FIG. 1, the DSC chart is shown in FIG. 2, and the TGA chart is shown in FIG. |
95.3% | With potassium carbonate; In dimethyl sulfoxide; acetonitrile; at 20 - 86℃; for 20h; | At room temperature, add 8-bromo-7- (2-butynyl) -3,7-dihydro-3-methyl-1-[(4-methyl-2-quinazoline) methyl to the reaction flask Group] -1H-purine-2,6-dione (100.00g, 220.6mmol), (R) -3-Boc-aminopiperidine (57.50g, 287mmol), potassium carbonate (122.00g, 883.0mol), acetonitrile 475ml, dimethyl sulfoxide (DMSO) 25ml, stir to warm up to 84-86 , keep stirring for about 20 hours, the reaction is over.b) Add 750ml of purified water dropwise to the reaction solution in step a). After the dropwise addition, stir to dissolve, control the temperature to 65 ~ 67 , keep warm and stir, wait for white solid to precipitate, continue to keep warm for about 2 hours, slowly Reduce the temperature to about 25 C, stir for 1 hour, filter, after the filter cake is drained, rinse with 500ml of purified water and drain to obtain a white crystalline powder solid product 1-[(4-methyl-quinazoline-2- Yl) methyl] -3-methyl-7- (2-butyn-1-yl) -8-[(R) -3- (tert-butoxycarbonylamino) -piperidin-1-yl] -2 , 6-Dione-2,3,6,7-tetrahydro-1H-purine (120.40g, 210.2mmol), the resulting white solid is in the form of Form A, with a yield of 95.30% and a purity of 99.935%. |
91% | With potassium carbonate; potassium iodide; In dimethyl sulfoxide; at 80 - 85℃; | 800 ml of DMSO, 53.2 g of (R) 3-Boc-Aminopiperidine(0.2654 moles), 100 g of 1- [(4-methyl-quinazolin-2yl)methyl]-3-methyl-7-(2-butyn-l -yl)-8-Bromo-xanthine (0.2212 moles prepared as per Example-3), 0.5g of potassium iodide and 91.5g of potassium carbonate (0.6620 moles) were charged into a 5 lit round bottom flask equipped with overhead stirrer and thermo pocket at 20-30C. The reaction mixture temperature was raised to 80-85 C and maintained for 4-5 hrs at same temperature. After completion of the reaction, reaction mixture was cooled to 30-35C, slowly added 1600 ml of chilled DM water and stirred for 60 min at 25-35C. The solid was filtered and washed with 200 ml of DM water. The wet material again washed with DM water. The wet material charged into RB flask and charged 700 ml of dichloromethane and stirred for 30 mins and layers were separated. The organic layer was washed with DM water and treated with activated charcoal followed by filtration through hyflo and washing with dichloromethane. The solvent was distilled out U/V at 35-40C till ~1.5V Dichloromethane remained inside. In another RB flask charged 800 ml of hexanes/cyclohexane and above dichloromethane solution was added slowly 35- 40C and stirred for 30-60 minutes at 30-35C. The solid was filtered and washed with 200 ml of hexanes/cyclohexane. The wet material was dried under vacuum at 40-45 C for 5-8 hours to get title compound (1 15 g, 91%, purity >97%). |
91% | With potassium carbonate; In N,N-dimethyl-formamide; at 75℃; for 16h; | In 100 mL of DMF, 4.53 g (10 mmol) of compound 5 and 2.41 g (12 mmol) of compound 6, 2.76 g (20 mmol) of K2CO3 were added, and the reaction was performed at 75 C for 16 hours.Monitor the progress of the reaction by TLC, cool to room temperature, filter, and add 500 mL of distilled water to the filtrate.A solid was precipitated, and the filtrate was cooled to 0 C, filtered, washed with water, and dried to obtain 8-[(3R) -3-tert-butoxycarbonylamino-1-piperidinyl] -7- (2-butynyl) -3, 7-dihydro-3-methyl-1-[(4-methyl-2-quinazolinyl) methyl] -1H-purine-2,6-dione 5.21 g (9.1 mmol) in a yield of 91%. |
89.6% | With potassium carbonate; In acetonitrile; at 80 - 85℃;Autoclave; | The intermediate (e) is reacted with (R) -3-Boc-aminopiperidine (f)To give intermediate (g).Steps:Was charged into a 10 L autoclave700 g (1.54 mol) of intermediate (e), 464.1 g (2.32 mol)(R) -3-Boc-aminopiperidine (f),854 g (6.18 mol) of potassium carbonate,Acetonitrile.Turn on agitation,Heated to reflux (micro-reflux),Reaction temperature 80 ~ 85 ,Reaction 28 ~ 35h after the end of the reaction.4.5 L of 70 C hot water was added slowly,Stirring slowly at room temperature,Precipitation of solid.filter.The resulting solid was filtered through 8 L65 ~ 75 hot water beating,Slowly drop to room temperature and filter.The filter cake was washed with water,Dried, light yellow solid product 792.7 g.The yield was 89.6%Purity 99.6%. |
89.6% | With potassium carbonate; In acetonitrile; at 80 - 85℃; | The intermediate (e) is reacted with (R)-3-Boc-aminopiperidine (f) to give the intermediate (g). Procedure: To a 10 L reaction vessel was charged with 700 g (1.54 mol) of intermediate (e), 464.1 g (2.32 mol) of (R)-3-Boc-aminopiperidine (f), 854 g (6.18 mol) of potassium carbonate, and 3.5 L of acetonitrile. Agitation was turned on, heated to reflux (micro-reflux), reaction temperature 80 ~ 85 deg. C, reaction 28 ~ 35h after the end of the reaction. 4.5 L of 70 C hot water was added slowly, stirring slowly at room temperature, precipitation of solid. Filtered. The resulting solid was filtered 8L 65 ~ 75 hot water, slowly dropped to room temperature and filtered. The filter cake was washed with water, dried, light yellow solid product 792.7 g. Yield 89.6%, purity 99.6%. |
88.5% | With potassium carbonate; potassium iodide; In N,N-dimethyl-formamide; at 90℃; | Into a 2L four-neck reaction flask, compound formula VI (29.2g, 64.5mmol), anhydrous DMF (450mL), (R) -3-Boc-aminopiperidine (16.5g, 82.3mmol), KI (200mg, 1.2 mmol) and K2CO3 (20.1g, 146mmol). After the addition was complete, the system was started to stir. The oil bath was slowly heated to 90C and stirred overnight. After the reaction, the system naturally cools downTo 10C, CH2CI2 (1L) and H2O (600 mL) were added to the reaction system, and the organic phase was separated after stirring for 30 minutes. The organic phase was washed 3 times with saturated brine (3 × 400 mL). The organic phase was desolvated under reduced pressure until about 100 ml of the system remained, and then petroleum ether (00 mL) was added to the system. The temperature of the system was raised to 50C, and after the system was fully stirred, the organic solvent was removed under reduced pressure until 200 mL of solvent remained in the system. The system was cooled to 0C and stirred for 3 hours, filtered through a Buchner funnel, and the filter cake was blown to dry at 40C overnight. Light yellow solid (compound formula VII) (32.6g, 88.5%) |
86% | With sodium carbonate; In 1-methyl-pyrrolidin-2-one; at 85 - 90℃; for 8h; | Quinazoline bromoxanthine (Formula III; 140 g), R-Boc-aminopiperidine (Formula IV; 68 g), and sodium carbonate (66 g) were added into a reaction vessel containing N-methyl-2-pyrrolidone (560 mL) at ambient temperature. The reaction mixture was heated to 85C to 90C and stirred for 8 hours. Progress of the reaction was monitored by HPLC. The reaction mixture was cooled to 26C and water (1 120 mL) was added at 26C to 40C. The reaction mixture was stirred at 30C to 35C for 30 minutes. The solid obtained was filtered and washed with water (700 mL) to obtain a wet solid (910 g). The wet solid (388g) was added into a reaction vessel containing water (87 mL) and acetonitrile (400 mL). The reaction mixture was heated to 70C to 75C for 30 minutes, and then cooled to 40C to 50C. The reaction mixture was stirred for 1 hour, further cooled to 25C to 30C, and stirred for 1 hour. The solid obtained was filtered, washed with a mixture of acetonitrile (50 mL) and water (50 mL), and then dried at 50C to 55C under reduced pressure to obtain the pure intermediate of Formula II. Yield: 86% HPLC Purity: 99.95% Impurity of Formula V: Not detected. |
86.2% | With potassium carbonate; methyl tributylammonium chloride; In toluene; | 1-[(4-methyl-quinazolin-2-yl)methyl]-3-methyl-7-(2-butyn-1-yl)-8-bromoxanthine (Compound VIII, 1.86 g, 0.0041 mol), potassium carbonate (base 3, 0.85 g, 0.0062 mol), (R)-3-tert-butoxycarbonyl-aminopiperidine (Compound IV, 0.82 g, 0.0041 mol), tributylmethylammonium chloride (PTC, 0.09 g, 0.0004 mol) and 50 mL of toluene were added to a reaction flask. The mixture was heated to reflux for 8 to 10 hours, cooled to room temperature, added with 50 mL of water, stirred and filtered. The filter cake was dissolved in 50 mL of dichloromethane, and washed with 5% diluted HCl, water and saturated sodium chloride solution, respectively. The above dichloromethane solution was concentrated to give the xanthine precursor, i.e., 1-[(4-methyl-quinazolin-2-yl)methyl]-3-methyl-7-(2-butyn-1-yl)-8-(3-(R)-tert-butoxycarbonylaminopiperidin-1-yl)xanthine (Compound V). (0086) Yield: 2.02 g (86.2% of theoretical value) (0087) MS: [M+H]+=573.4 (0088) 1H-NMR (400 MHz, DMSO): delta 1.39 (s, 9H), 1.63-1.70 (m, 1H), 1.76 (s, 3H), 1.76-1.85 (m, 2H), 2.84-2.88 (broad, s, CH3, CH, 4H), 3.00 (m, 1H), 3.34 (s, 1H), 3.39 (s, 3H), 3.56-3.59 (m, 2H), 3.65-3.68 (m, 1H), 4.87 (d, J=1.6 Hz, 2H), 5.32 (s, 2H), 7.02 (d, J=8.0 Hz, 1H), 7.66 (t, J=7.6 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.90 (dd, J=7.2, 1.2 Hz, 1H), 8.23 (d, J=8.0 Hz, 1H). |
76.3% | With potassium carbonate; In N,N-dimethyl-formamide; at 75℃; for 15h;Large scale; | Adding dipole aprotic organic solvent N, N-dimethylformamide 50 kg,Anhydrous potassium carbonate 3.4 kg,(R) -3-tert-butoxycarbonylaminopiperidine (2.75 kg)Intermediate I5.5 kg,Stir,The temperature was raised to 75 C and stirred for 15 hours.Detection of intermediate I residues remaining no more than 2.0%Cooling to below 40 ,Stirring under the uniform addition of 70kg of purified water,Continue to cool down and cool to 15 C for 2 hours,Centrifugal,The solid was rinsed with 15 kg of purified water,Wet goods with dichloromethane 66kg stirring dissolved,Plus 30kg of purified water,Layered abandoned water phase,Add anhydrous sodium sulfate 3kg room temperature stirring drying 1hr,Filtered under reduced pressure to dry,Concentrate with methanol 32kg temperature reflux to dissolve,Plus isopropyl alcohol 32kg,Slowly cooled to 15 insulation stirring crystallization hr, centrifugal, solid with 15kg of isopropyl alcohol leaching, get yellowish wet goods, set double cone dryer 50 under reduced pressure drying until the weight loss does not exceed 2.5%Dew yellow dry intermediate II5.3kg, purity 98.9%The yield range was 76.3% |
With potassium carbonate; In N,N-dimethyl-formamide; at 75℃; for 6h; | A mixture of 4b (88 mg, 0.2 mmol), (S)-3-(N-Boc-amino)piperidine (44 mg, 0.22 mmol) and K2CO3 (55 mg, 0.4 mmol) in DMF (6 mL) was stirred at 75 C for 6 h. After cooling to r.t., the mixture was poured into water (12 mL) and extracted with DCM (3 * 10 mL). The combined organic layer was washed with saturated brine, dried over anhydrous Na2SO4, and concentrated. The crude product was purified by flash chromatography (petroleum ether/ethyl acetate, 1:1) to give the Boc precursor of 1i as a colorless syrup (80 mg, 72%), which was dissolved in DCM (2 mL), and TFA (390 muL) was added. The solution was stirred at room temperature for 3 h and then poured into ice-cold water (4 mL). The organic phase was separated, and the aqueous phase was basified with K2CO3 and extracted with DCM (2 * 10 mL). The organic layers were combined and washed with saturated brine, dried over anhydrous Na2SO4, and concentrated. The crude product was purified by flash chromatography (DCM/MeOH/TEA, 100:0.5:0.5) to give pure 1i as a white solid (51 mg, 85%). | |
35 g | With potassium carbonate; In N,N-dimethyl acetamide; at 80 - 85℃; for 12h; | To the stirring mixture of Boc-Amino piperidine (14 g) in dimethylacetamide (175ml) were added potassium carbonate powder (31 g) and bromopurine (18 g) at 25-30 C. The reaction mixture was heated to 80-85C for 12 hr. To the reaction mixture, water was added (525 ml). After addition of water, reaction mass cooled to 35-40C and maintained for 30 min. Filtered reaction mixture to get Boc-Linagliptin (35 g). HPLC Purity: 98.03% |
With potassium carbonate; potassium iodide; In dimethyl sulfoxide; at 82℃; | To a 3000 mL glass vessel equipped with a stirrer, condenser and a thermometer probewere added Formula III (100.0 g, 0.22 mol) Formula IV (50.81 g, 0.25 mol), potassiumiodide (3.66 g, 0.02 mol), potassium carbonate (36.65 g, 0.26 mol) and DMSO (400 mL). The mass was heated to 82±2 C. The reaction mass was maintained at 82±2 C under stirring for 6 - 9h. The reaction mass was cooled to 25±5 C, MDC (400 mL) & water (600 mL) was added to the reaction mass under constant stirring for 1 to 2h. Layers wereseparated. Re-extracted the aqueous layer with MDC (2x200 mL). Combined the MDC layers and washed with water (200 mL). Separated the layers and partially concentrated the MDC layer to obtain the Formula V in MDC solution. Purification of crude Formula V:To the compound of Formula V in MDC solution was added acetonitrile and concentrated. Added another lot of acetonitrile and heated the reaction mass to 78±3 C for 2 h. Charge water at temperature 70±5C. Maintain at 75±5C for 2 hours. Reaction mass was slowlycooled to 25±5C. Stir the mass for 1 hour at 25±5C. The resulting product was filtered off, washed with acetonitrile followed by water, suck dried and dried at 70±5 C under vacuum for 16-18h to obtain compound of Formula V as a pale yellow solid.The novel crystalline Linagliptin intermediate of Formula V which is prepared as per Example-2 is characterized by XPRD as represented in Figure-i.The novel crystalline Linagliptin intermediate of Formula V which is prepared as per Example-2 is characterized by DSC as represented in Figure-2.The novel crystalline Linagliptin intermediate of Formula V which is prepared as per Example-2 is characterized by FTIR as represented in Figure-3. | |
1.2 g | With potassium carbonate; potassium iodide; In dimethyl sulfoxide; at 80 - 85℃;Inert atmosphere; | To a suspension of quinazolinyl xanthine [Formula 4] (1.0 g, 2.21 mmol, 1 eq) in DMSO (10 mL) BOC-aminopiperidine [Formula 9] (0.53 g, 2.65 mmol, 1.2 eq), potassium carbonate (0.46 g, 3.32 mmol, 1.5 eq) and potassium iodide (50 mg, 10 w/w%) were added under nitrogen atmosphere. The reaction mixture was heated to 80-85 C and stirred for 4-5 hrs. The reaction was followed by TLC (silica; DCM:MeOH = 95:5). The reaction mixture was cooled down to room temperature, water (40 mL) was added dropwise and the reaction mixture was stirred at 20-25 C for half hour. The precipitate was filtered, washed with water and dried under vacuum at 50 C to get title compound [Formula 2] as a pale yellow powder (1.2 g, HPLC: 96.7%). |
With potassium carbonate; In N,N-dimethyl-formamide; at 60℃; for 5h;Large scale; | S3. Take another reaction kettle, add 9kg of N, N-dimethylformamide to it, and start stirring,The stirring speed was 450 rpm, and 3.8 kg of the intermediate I obtained in step S2 was added thereto while stirring.R-3-tert-butoxycarbonylaminopiperidine 1.67kg, anhydrous potassium carbonate 1.52kg,After heating up to 60 C and keeping the reaction under stirring for 5h, the reaction was monitored by HPLC,Turn on stirring, add 40kg of purified water, stir for 1.0h, collect the cake by spinning and drying,4.7 kg of intermediate II was obtained; the detection spectrum of intermediate II is shown in Figure 2; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
72% | With formaldehyd; sodium cyanoborohydride; In methanol; water; at 20.0℃; | 163I. (R)-1-methylpiperidin-3-amine Sodium cyanoborohydride (4.51 g, 0.075 mol) was added in portion to a mixture of (R)-tert-butyl piperidin-3-ylcarbamate (10 g, 0.05 mol), 30% water solution of formaldehyde (7.5 mL), and methanol (75 mL) at 0 C. The reaction mixture was stirred at room temperature overnight and concentrated in vacuo. The residue was dissolved in ethyl acetate and water. After extraction, the organic layers were washed with water, brine, and dried over Na2SO4. Concentration in vacuo, gave the N-methyl compound as an oil which was used directly without further purification. To a solution of the crude material previously obtained in methanol (60 mL) was added 4N HCl dioxane (10 mL). The reaction mixture was stirred at room temperature for 6 h. After concentration in vacuo, the residue was triturated with ether. The resulting precipitate was filtered and washed with ice-cold methanol to give the title compound as a solid (4.01 g, 72%). 1H NMR (CD3OD, 400 MHz) delta 3.54 (1H, m), 2.81 (1H, m), 2.62 (1H, m), 2.23 (3H, s), 1.97 (1H, m), 1.67-1.87 (3H, m), 1.56-1.61 (1H, m), 1.41 (9H, s), 1.15-1.42 (1H, m). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
General procedure: A mixture of 14 (5.00 g; 20.4 mmol) and 13a (4.37 g; 20.4 mmol) in CH3CN (60 ml) was stirred at 50 C for 1 h. After cooling to room temperature, 1,8-diazabicyclo[5,4,0]undeca-7-ene (DBU) (6.20 g; 40 mmol) and 2-bromo-5-fluoro-benzylamine (4.99 g; 24.4 mmol) were added to the mixture, and the resulting mixture was stirred at 80 C for 10 h. After removal of the solvent, the residue was diluted with EtOAc, and washed with H2O, 10%KHSO4 aq, 10%NaOH aq and brine. The EtOAc layer was dried over Na2SO4 and the filtrate was concentrated. The residue was dissolved in DMF (60 mL), and ethyl bromoacetate (2.80 mL; 28 mmol) and K2CO3 (8.5 g) were added. The resulting slurry was then stirred at 50 C for 2 h and cooled to room temperature. The slurry was diluted with EtOAc, filtered through Celite, and washed with satd NH4Cl aq and brine. The organic layer was dried over Na2SO4, and the filtrate was concentrated. To tert-butyl alcohol (30 mL) was added lithium amide (1.16 g; 48 mmol). After stirring at 80 C for 1 h, the resulting mixture was cooled to 30 C, and CH3CN (40 mL) was added. A solution of the alkylated product from the third step in toluene (10 mL) was next added dropwise, and the reaction mixture was stirred for 2 h at 30 C. The solvent was removed, and to the residue was added EtOAc. The resulting mixture was finally washed with satd NH4Cl aq and brine, the organic layer was dried over Na2SO4, and the filtrate was concentrated. The residue was purified by SiO2 column chromatography (Hexane/EtOAc = 6/1 to 3/1) to give 5.80 g (44% from 14) of 15a as amorphous solid. In addition to the target product 15a, the ester exchange product (R2 = tert-butyl) was also obtained as an amorphous (415 mg). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
78.6% | With triethylamine; In ethanol; at 75℃; for 8h; | To a solution of Intermediate 1 (5.00 g, 31.5 mmol, 1 eq), Intermediate 2 (12.6 g, 63.0 mmol, 2 eq) in EtOH (30.0 mL) was added TEA (6.38 g, 63.0 mmol, 8.78 mL, 2 eq). The mixture was stirred at 75C for 8 h. TLC (Petroleum ether/Ethyl acetate = 3/1, Rf = 0.22) showed the reaction was completed. The reaction mixture was concentrated under reduced pressure to remove EtOH. The residue was diluted with 0 (30.0 mL) and extracted with EtOAc (30.0 mL x 3). The combined organic layers were washed with brine (50.0 mL), dried over Na2S04, filtered and concentrated under reduced pressure to give a residue. The residue was purified by column chromatography (S1O2, Petroleum ether/Ethyl acetate = 50/1 to 0/1). Give Intermediate 3 (8.00 g, 24.8 mmol, 78.6% yield) as a yellow solid. NMR : CDCI3 400 MHz 8.20 (d, J= 5.9 Hz, 1H), 7.58 (d, J= 2.4 Hz, 1H), 6.94 (br d, J = 3.5 Hz, 1H), 4.57 (br s, 1H), 3.86 (br d, J= 12.3 Hz, 1H), 3.57 - 3.74 (m, 2H), 3.14 - 3.33 (m, 2H), 2.02 - 1.98 (m, 1H), 1.77 - 1.90 (m, 1H), 1.64 - 1.71 (m, 1H), 1.52 - 1.61(m, 1H), 1.44 (br s, 9H) |