Synthesis 20-1 -A; 6-Chloro-7H-purin-8(9H)-one; NXrNH2 A mixture of 4-chloropyrimidine-2,3-diamine (36 mg, 0.25 mmol) and di(/V-succinimidyl)carbonate (128 mg, 0.50 mmol) in acetonitrile (10 mL) was refluxed for 16 hours. The solids formed were collected, washed with acetonitrile (2 x 5 mL) and dried, to give the title compound as a light yellow powder (33 mg, 78percent).1H NMR (500 MHz, de-DMSO) δ 8.35 (1 H, s), 11.90 (2H, s, broad); LC-MS (2) R1 1.96 min; m/z (ESI) 171 [MH+].
Reference：
[1] Bioorganic and Medicinal Chemistry Letters, 2008, vol. 18, # 20, p. 5559 - 5562
5
[ 74124-79-1 ]
[ 1142-20-7 ]
[ 3401-36-3 ]
Reference：
[1] Bioorganic and Medicinal Chemistry Letters, 2008, vol. 18, # 20, p. 5559 - 5562
6
[ 74124-79-1 ]
[ 100-51-6 ]
[ 13139-17-8 ]
Reference：
[1] European Journal of Medicinal Chemistry, 1999, vol. 34, # 7-8, p. 625 - 638
[2] Patent: WO2008/64218, 2008, A2, . Location in patent: Page/Page column 72; 83-85; 86-87; 98-99; 125
7
[ 6066-82-6 ]
[ 28920-43-6 ]
[ 74124-79-1 ]
[ 102774-86-7 ]
Yield
Reaction Conditions
Operation in experiment
95 kg
With tributyl-amine In tetrahydrofuran at -2 - 5℃; Large scale
From the reactor solid feed port one - time investment HosuL43. 8Kg (l. 25KmOl),With stirring,So that the kettle liquid at -2 ° C ~ 0 ° C.The tributylamine300Kg (l. 62 kP)And tetrahydrofuran450Kg into the high slot,After mixing,In 10 ~ llhr slowly added to the reactor in the reaction,The amount of added per hour for the 50 ~ 57Kg;And keep the kettle temperature not to exceed 5 ° C,Because of the exothermic reaction,After adding tributylamine in tetrahydrofuran solution,The reaction vessel was allowed to warm to room temperature and the reaction was continued for 7.5 h.A large amount of white solid crystals were observed to be precipitated by DSC from the reaction vessel.(3) filtered DSC crude: the reactor material in lhr evenly introduced into a fully enclosed centrifuge in N2Under the protection of throwing dry, and then twice the introduction of about 0 ° C tetrahydrofuran solvent 100Kg in the centrifuge rinse, throw dry,The DSC wet goods 120Kg. The wet goods in a vacuum oven at 40 ~ 45 ° C drying 7 ~ 8hr to constant weight, get out of the white crystalline powder 95Kg; HPLC analysis of 99. 1percent. The resulting filtrate was centrifuged to proceed to the next step. (3) filtered to obtain DSC crude: the reactor material in 1hr evenly introduced into a fully enclosed centrifuge, in the N2Under the protection of throwing dry, and then twice the introduction of about 0 ° C tetrahydrofuran solvent 100Kg in the centrifuge rinse, throw dry, get DSC wet goods 120Kg.The wet product in a vacuum oven at 40 ~ 45 dry 7 ~ 8hr to constant weight, get out of the white crystalline powder 95Kg; HPLC analysis of 99.1percent.The resulting filtrate was centrifuged to proceed to the next step.(4) The centrifugal filtrate in step (3) is combined with two washing solvents and introduced into a 2000L distilling apparatus. The tetrahydrofuran solvent is removed at a vacuum control temperature of 0 to 5 DEG C, and the inside temperature of the kettle is kept at 40 to 42 DEG C .After the tetrahydrofuran was removed, 230 kg of dichloroethane was introduced, stirred at 40-50 ° C for 0.5 hr, and washed with 160 kg of water 3 times. The phases were separated and the dichloromethane was introduced into a 500 L autoclave.
With sodium hydrogencarbonate In N,N-dimethyl-formamide at 0℃; for 1 h; Inert atmosphere
6-Maleimidohexanoic acid N-hydroxysuccinimide ester 4. Sodium bicarbonate (0.0512 g, 0.483 mmol ) was added to a solution of 6-maleimidocaproic acid (0.211 g, 1.000 mmol) in DI water (10 mL). After the reagents were dissolved in DI water, the DI water was evaporated by blowing with air. The resulting solid was dissolved in anhydrous DMF (3 mL) and the solution was cooled to 0 °C. Disuccinimide carbonate (0.282 g, 1.10 mmol) was added into the solution and the reaction was stirred at 0 °C for 1 h. CH2C12 (25 mL) was added into reaction which was then washed with water (3 x 10 mL). The resulting organic layer was dried over Na2S04, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 25 g silica column [solvent A: MeOH; solvent B: CH2C12; gradient: 0percentA / 100percentB (1 CV), 0percentA / 100percentB→ 2percentA / 98percentB (10 CV), 2percentA / 98percentB (2 CV); flow rate: 25 mL/min; monitored at 210 and 280 nm] to afford ester 4 (0.215 g, 0.697 mmol, 70percent yield) as a light yellow liquid. [000194] NMR (500 MHz, CDC13) 66.69 (2H, s), 3.53 (2H, t, J= 7.1 Hz), 2.83 (4H, s), 2.60 (2H, t, .7=7.4 Hz), 1.78 (2H,p, .7=7.5 Hz), 1.63 (2H,p, .7=7.4 Hz), 1.41 (2H,p,J=7.6 Hz). [000195] 13CNMR(125 MHz, CDC13) δ 170.9, 169.2, 168.5, 134.2,37.5,30.8,28.1,25.9,25.7,24.1.
Reference：
[1] Journal of the American Chemical Society, 1994, vol. 116, # 14, p. 6101 - 6106
[2] Patent: WO2017/66668, 2017, A1, . Location in patent: Paragraph 000193-000195
With N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 40℃; for 1 h;
Lauric acid (1.2 g, 5.99 mmol, 1 eq) is dissolved in N, N-dimethylformamide (DMF, 70 ml). N, N-disuccinimidyl carbonate (DSC, 4.6 g, 17.97 mmol, 3 eq) and N, N-diisopropylethylamine Hunig base, 10.2 ml, 59.91 mmol, 10 eq), and the mixture was stirred at 40 ° C for 1 hour. After completion of the reaction, the compound was lyophilized to obtain Compound 2-1 (2.1 g, 100percent)
With triethylamine In acetonitrile at 20℃; for 3 h;
Compound 41; TEA (21.9 mL, 155.6 mmol, 3.0 eq.) was added to 2-trimethylsilanyl- ethanol (7.4 mL, 51.88 mmol, 1.0 eq.) in 260 mL MeCN, followed by di- succinimidyl carbonate (20 g, 1.5 eq.). The reaction mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated and extracted using EtOAc/ saturated NaHCO3. The organic layer was concentrated after dried over Na2SO4. Ether (100 mL) was added to the residue to form a precipitate. The precipitate was filtered and dried to give Compound 41 as a white solid (11.1 g, 84percent). 1H NMR (300 MHz, CDCl3): δ 4.42 (t, 3 H), 2.82 (s, 4 H), 1.16 (t, 2 H), 0.1 (s, 9 H).
82%
With triethylamine In acetonitrile at 25℃; for 16 h;
Example 5; methyl (l-l- ( {2- [ (2, 36)-3-amino-2-hydroxy-4-phenylbutyl]-2- benzylhydrazino} carbonyl)-2, 2-dimethylpropylcarbamate; Example 5A; 1- (f [2- (trimethylsilyl) ethoxy] carbonyl} oxy)-2, 5-pyrrolidinedione; Trimethylsilylethanol (7.4 mL, 52 mmol) was dissolved in acetonitrile (260 mL) and treated with disuccinimoyl carbonate (20 g, 1.5 equivalents) and triethylamine (33 mL, 3 equivalents) at 25°C for 16h. The solvents were evaporated, and the residue was partitioned between ethyl acetate and saturated sodium bicarbonate, the organic layer was separated and washed with brine, dried over sodium sulfate, filtered and concentrated. The crude residue was triturated with ether to form a solid which was filtered and dried to give 11.12 g (82percent) of the title compound.
82%
With triethylamine In acetonitrile
EXAMPLE 5A 1-([2-(trimethylsilyl)ethoxy]carbonyl}oxy)-2,5-pyrrolidinedione Trimethylsilylethanol (7.4 mL, 52 mmol) was dissolved in acetonitrile (260 mL) and treated with disuccinimoyl carbonate (20 g, 1.5 equivalents) and triethylamine (33 mL, 3 equivalents) at 25° C. for 16 h. The solvents were evaporated, and the residue was partitioned between ethyl acetate and saturated sodium bicarbonate, the organic layer was separated and washed with brine, dried over sodium sulfate, filtered and concentrated. The crude residue was triturated with ether to form a solid which was filtered and dried to give 11.12 g (82percent) of the title compound.
With pyridine In tert-butyl methyl ether at 25 - 40℃; for 62 h;
26.3 g of tert-butyl methyl ether,35.09 g (135 mmol) of di (N-succinimidyl) carbonate,And 20.00 g (the content of the compound (II-1): 73.0percent) of the compound (II-1) and the compound (III-2) obtained in the same manner as in Example 2 were mixed at room temperature And the temperature was raised to 40 ° C. 11.89 g (150 mmol) of pyridine was added dropwise to the obtained solution at 40 ° C., and the mixture was stirred at 40 ° C. for 22 hours. The whole amount of the solution was cooled to 20 ° C., 73.0 g of 2-propanol was added dropwise at 20 ° C. to precipitate compound (I-1), cooled to 0 to 5 ° C. and then cooled to 0 to 5 ° C. for 19 hours Followed by stirring.The precipitated compound (I-1) was filtered and washed to obtain 27.83 g of compound (I-1) (content: 97.8percent, yield 90percent, enantiomeric excess of compound (I-1) Rate> 99.9percent ee). 30.0 g of tert-butylmethyl ether, 11.87 g of the compound (II-0)(The diastereomer ratio of the compound (II-1) to the 3S, 3aS, 6aR-OH form: 91.1 / 8.9 containing 84.3percent diastereomer) and the enzyme (CHIRAZYME L-2c, -flyo, manufactured by Roche Diagnostics) was added to the mixture at a temperature of 25 ° C. 3.31 g (38.4 mmol) of vinyl acetate was added dropwise to the mixture,After stirring at 25 ° C. for 40 hours, insoluble matter was filtered off.The filtrate was concentrated to obtain 12.73 g of a mixture containing compound (II-1) and compound (III-2) (yield of compound (II-1): 90percent 3S, 3aS, 6aR-OH isomer: 100.0 / 0.0). (II-1) and the compound (III-2) obtained in the same manner as in Example 2, 26.3 g of tert-butyl methyl ether, 35.09 g (135 mmol) of di (N-succinimidyl carbonate) And 20.00 g of the compound (II-1) in an amount of 73.0percent) were mixed at room temperature, and the mixture was heated to 40 ° C. To the resulting solution, 11.89 g (150 mmol) of pyridine was added dropwise at 40 ° C. And the mixture was stirred for 22 hours at 40 ° C. The total amount of the solution was cooled to 20 ° C. and 73.0 g of 2-propanol was dropwise added at 20 ° C. to precipitate the compound (I-1) and cooled to 0 to 5 ° C. , And the mixture was stirred for 19 hours at 0 to 5 ° C. The precipitated compound (I-1) was filtered,(Yield: 97.8percent, yield: 90percent, enantiomer excess of compound (I-1)> 99.9percent ee) was obtained by filtration and washing with water to obtain 27.83 g of compound (I-1).
Reference：
[1] Patent: JP2016/150901, 2016, A, . Location in patent: Paragraph 0016; 0017; 0023; 0024
[2] Journal of Organic Chemistry, 2004, vol. 69, # 23, p. 7822 - 7829
[3] European Journal of Organic Chemistry, 2016, vol. 2016, # 10, p. 1874 - 1880
[4] Journal of Medicinal Chemistry, 2005, vol. 48, # 6, p. 1813 - 1822
[5] Patent: WO2010/23322, 2010, A1, . Location in patent: Page/Page column 29
[6] Patent: US2008/85918, 2008, A1, . Location in patent: Page/Page column 18; Sheet 3
[7] Patent: WO2011/92687, 2011, A1, . Location in patent: Page/Page column 31
[8] Patent: WO2016/207907, 2016, A1, . Location in patent: Page/Page column 23
[9] Organic Process Research and Development, 2017, vol. 21, # 1, p. 98 - 106
[10] Patent: WO2007/126812, 2007, A2, . Location in patent: Page/Page column 41
[11] Patent: WO2008/133734, 2008, A2, . Location in patent: Page/Page column 28-29
A. N-hydroxysuccinimidyl-1-methoxypropane-3-carbonate A solution of 355 mg of <strong>[3513-81-3]2-methylene-1,3-propanediol</strong> in acetonitrile (30 mL) was added sequentially, at ambient temperature, 65 mg of sodium hydride and 0.25 mL iodomethane. The mixture was stirred for 12 h and concentrated in vacuo. The residue was then taken up in 15 mL of acetonitrile and treated sequentially, at ambient temperature under an atmosphere of nitrogen, with 1.3 g of N,N-disuccinimidyl carbonate and 1.6 mL of triethylamine. After stirring for 14 h, the reaction mixture was concentrated in vacuo and the residue was diluted CH2 Cl2, washed with saturated sodium bicarbonate solution and saturated brine, dried over magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography with EtOAc as eluant to give 95 mg of the title compound. (1 H)-NMR (CDCl3) consistent with structure.
With triethylamine; In acetonitrile;
A. N-hydroxysuccinimidyl-1-methoxypropane-3-carbonate A solution of 355 mg of <strong>[3513-81-3]2-methylene-1,3-propanediol</strong> in acetonitrile (30 mL) was added sequentially, at ambient temperature, 65 mg of sodium hydride and 0.25 mL iodomethane. The mixture was stirred for 12 h and concentrated in vacuo. The residue was then taken up in 15 mL of acetonitrile and treated sequentially, at ambient temperature under an atmosphere of nitrogen, with 1.3 g of N,N-disuccinimidyl carbonate and 1.6 mL of triethylamine. After stirring for 14 h, the reaction mixture was concentrated in vacuo and the residue was diluted CH2 Cl2, washed with saturated sodium bicarbonate solution and saturated brine, dried over magnesium sulfate, filtered and concentrated in vacuo. The residue was purified by silica gel chromatography with EtOAc as eluant to give 95 mg of the title compound. (1 H)-NMR (CDCl3) consistent with structure.
Into a three-necked flask equipped with a magnetic stirrer and placed under N2 is introduced tert-butyl (2S)-2-[(3,4-dichlorophenyl)(hydroxy)methyl]piperidine-1-carboxylate (1.9 g, 5.2 mmol) dissolved in acetonitrile (30 mL). N,N'-Disuccinimyl carbamate (2.05 g, 8 mmol) and triethylamine (2.19 mL, 15.6 mmol) are then added and the reaction medium is stirred for 4 hours at RT. After concentrating the reaction medium by evaporation under RP, the residue thus obtained is taken up in saturated aqueous sodium hydrogen carbonate solution and the aqueous phase is extracted with EtOAc (3.x.30 mL). The organic phase is washed with aqueous NaCl solution, dried over MgSO4 and concentrated by evaporation under RP. The residue obtained is diluted in DCM (15 mL). This solution is then added dropwise to solution, prepared beforehand and placed in a one-necked flask, of <strong>[129488-10-4]5-amino-N-tert-butoxycarbonyl-1H-indazole</strong> (1.45 g, 6.2 mmol), DCM (40 mL) and triethylamine (1.1 mL, 7.8 mmol). The reaction medium is stirred at RT overnight. 40 mL of DCM and 30 mL of saturated aqueous sodium hydrogen carbonate solution are then added. After separation of the phases by settling, the organic phase is washed with aqueous NaCl solution, dried over MgSO4, filtered and concentrated by evaporation under RP. The residue is purified by chromatography on silica gel eluted with a 3/1 cyclohexane/EtOAc mixture. tert-Butyl (2S)-2-[(S)-(3,4-dichlorophenyl)[(1-(tert-butoxycarbonyl)-1H-indazol-5-yl)carbamoyl]oxy}methyl]piperidine-1-carboxylate (0.215 g) is thus obtained the form of a colourless lacquer and tert-butyl (2S)-2-[(R)-(3,4-dichlorophenyl)[(1-(tert-butoxycarbonyl)-1H-indazol-5-yl)carbamoyl]oxy}methyl]piperidine-1-carboxylate (0.434 g) is obtained in the form of a white foam. (M-H)-=617.
Into a one-necked flask equipped with a magnetic stirrer is introduced tert-butyl (2S)-2-[3-(ethoxycarbonyl)phenyl](hydroxy)methyl}piperidine-1-carboxylate (4.7 g, 12.8 mmol) dissolved in acetonitrile (85 mL) with N,N'-disuccinimidyl carbonate (13.1 g, 51 mmol). Triethylamine (8.95 mL, 64 mmol) is then added and the reaction medium is stirred for 4 hours at a temperature in the region of 20° C. The reaction medium is concentrated to dryness and the evaporation residue is taken up in saturated aqueous sodium hydrogen carbonate solution (50 mL) and extracted with twice 40 mL of EtOAc. A persistent insoluble material is removed by filtration of the organic phases through a sinter funnel. The filtrate is dried over MgSO4, filtered and concentrated to dryness under RP to give the activated intermediate. Into a second one-necked flask equipped with a magnetic stirrer is introduced tert-butyl 5-amino-indazole-1-carboxylate (3 g, 12.8 mmol) with DCM (125 mL) and triethylamine (2.7 mL, 19.1 mmol). Into this solution is poured the activated intermediate dissolved in DCM (40 mL) over about 10 minutes. The reaction medium is stirred in the region of 20° C. for 16 hours. The medium is hydrolysed with saturated aqueous sodium hydrogen carbonate solution (80 mL), the phases are separated by settling and the aqueous phase is re-extracted with DCM (30 mL). The combined organic extracts are dried over MgSO4, filtered and concentrated to dryness under RP. The garnet-coloured oil isolated is chromatographed on 420 g of silica gel 60, particle size 15-40 mum, contained in a column 5 cm in diameter, eluting with a 7/3v/v cyclohexane/EtOAc mixture, under an excess pressure of 0.6 bar of argon. The evaporation of the fractions gives 1.53 g of tert-butyl 5-[([(2S)-1-(tert-butoxycarbonyl)piperid-2-yl][3-(ethoxycarbonyl)phenyl]methoxy}carbonyl)amino]-1H-indazole-1-carboxylate in the form of a white-coloured foam. (M-H)-=621. 1H NMR (DMSO, 400 MHz): 70percent-30percent mixture of isomers, delta (ppm) from 0.98 to 2.00 (m, 27H); 2.98 (m, 1H); 3.90 (broad m, 1H); 4.33 (q, J=7.5 Hz, 2H); 4.50 (broad m, 1H); 6.09 (d, J=10.0 Hz, 0.7H); 6.23 (broad d, J=9.0 Hz, 0.3H); 7.50 (t, J=7.5 Hz, 0.7H); 7.58 (m, 1.3H); 7.68 (broad d, J=7.5 Hz, 0.7H); 7.75 (broad d, J=7.5 Hz, 0.3H); from 7.85 to 8.00 (m, 3H); 8.04 (broad s, 0.7H); 8.08 (broad s, 0.3H); 8.32 (s, 0.7H); 8.34 (s, 0.3H); 9.82 (broad m, 0.3H); 10.1 (s, 0.7H).
N-(3-(1H-imidazol-1-yl)propyl)-2-((4-fluorobenzyl)amino)-1,3-thiazole-5-carboxamide[ No CAS ]
N1-(4-fluorobenzyl)-N1-(5-[3-(1H-imidazol-1-yl)propyl]carbamoyl}-1,3-thiazol-2-yl)piperidine-1,3-dicarboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
General procedure: Example 6B (0.018 g, 0.05 mmol) was dissolved in acetonitrile (0.5 ml), and treated with potassium carbonate (0.021 g, 0.15 mmol) and 1 -(2-chloroethyl)pyrrolidine (0.017 g, 0.1 mmol). The reaction mixture was heated via microwave at 180 °C for 30 minutes, concentrated in vacuo, and submitted to reverse-phase HPLC (as described in Example 6C) to provide the title compound.
6-(4-aminophenyl)-2-cyclopentyl-5-methyl-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-[4-(1-cyclopentyl-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
68%
To a solution of Intermediate 9, 60 mg (0.22 minol), and 4-dimethylaminopyridine, 32 mg (0.26 minol), in tetrahydrofuran, 25 mL, was added N,N-disuccinimidyl carbonate, 67.9 mg, (0.26 minol). The reaction was stirred for 1 hour then triethylamine, 0.092 mL (0.66 minol), and 2,3-dihydro-IH-pyrrolo[3,4-c]pyridine hydrochloride (6000-50-6), 41.3 mg (0.26 minol), was added and the reaction was left to stir at room temperature for 18 hours. Water was added and the mixture was extracted with dichloromethane. The combined organics were dried over solid sodium sulfate and concentrated under vacuum. Purification by flash chromatography on silica gel 60 (eluent: ethyl acetate-heptane 0:1, 1:1, 1:0 and methanol-ethyl acetate 1:9) gave thedesired product, 62.8 mg (68percent).1H NMR (300 MHz, CDCI3): 6 [ppm] = 1.16 (d, 3H), 1.57-2.00 (m, 8H), 2.46 (d, IH), 2.64 (dd, IH), 3.27 (m, IH), 4.90 (d, 4H), 5.21 (m, IH), 6.40 (s, IH), 7.30 (d, IH), 7.52 (d, 2H), 7.76 (d, 2H), 8.58 (d, IH), 8.63 (s, IH).UPLC-MS (Method 4): R 1.93 min., 100percent. MS (ESIpos): mz[M+H]418.
6-(4-aminophenyl)-5-methyl-2-(2,2,2-trifluoroethyl)-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-{4-[4-methyl-6-oxo-1-(2,2,2-trifluoroethyl)-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
20%
A solution of 100mg of Intermediate 11(0.35 minol, 1.00 eq) in DMF (10 mL) was treated with108 mg of N,N?-disuccinimidyl carbonate (0.42 minol, 1.20 eq) and 51.4 mg of 4-dimethylaminopyridine (0.42 minol, 1.20 eq) and was left over night at room temperature. Asuspension of 81 .2 mg of 2,3-dihydro-1 H-pyrrolo[3,4-c]pyridine dihydrochloride (0.42 minol, 1 .20eq) and 667 pL of triethylamine (4.78 minol, 3.60 eq) in DMF (5 mL) was added. The reactionmixture was left for 3 days at room temperature. The mixture was poured into water. The aqueous phase was three times extracted with ethyl acetate. The combined organic layers were washed with brine, dried over Na2504 and the solved was removed under reduced pressure. The residue was purified by preparative reverse phase HPLC to yield the desired productExample 11(30 mg, 20percent).1H-NMR (400MHz, DMSO-d6): oe [ppm] = 1.09 (d, 3H), 2.40-2.47 (m, IH), 2.79-2.87 (m, IH),3.42-3.54 (m, IH), 4.26-4.38 (m, IH), 4.37-4.37 (m, IH), 4.80-4.83 (m, 4H), 7.41 -7.47 (m, I H), 7.65 - 7.72 (m, 2H), 7.72 - 7.79 (m, 2H), 8.50 (d, I H), 8.61 (s, I H), 8.66 (s, I H).U PLC-MS (Method 2): R= 0.81 min; MS (ESipos): mz [M÷H] 432.
6-(4-aminophenyl)-5-methyl-2-(tetrahydro-2H-pyran-4-yl)-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-{4-[4-methyl-6-oxo-1-(tetrahydro-2H-pyran-4-yl)-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
11%
A solution of 177 mg of Intermediate 14 (0.31 minol, 50percent purity 1.00 eq) in DMF (19 mL) wastreated with 94.7 mg of N,N?-disuccinimidyl carbonate (0.37 minol, 1.20 eq) and 45.2 mg, of 4-dimethylaminopyridine (0.37 minol, 1.20 eq). The mixture was left over night at room temperature. A suspension of 71.4 mg of 2,3-dihydro-IH-pyrrolo[3,4-c]pyridine dihydrochloride (0.37 minol, 1.20 eq) and 155 pL of triethylamine (1.11 minol, 3.60 eq) on DMF (3 mL) wasadded. The reaction mixture was stirred 3 days at room temperature. The mixture was poured into water and extracted three times with ethyl acetate. The combined organic layers were washed with brine and dried over Na2SO4. The aqueous and the organic phase were evaporated under reduced pressure. Both residues were purified by preparative HPLC to yield in total 15.0mg of the desired product Example 21(0.03 minol, 11percent).1H-NMR (400MHz, DMSO-d6): oe [ppm]= 1.05 (d, 3H), 1.49- 1.61 (m, 2H), 1.80- 1.93 (m, IH),2.02 - 2.16 (m, I H), 2.29 - 2.37 (m, I H), 2.68 - 2.76 (m, I H), 3.88 - 3.99 (m, 2H), 4.70 - 4.80 (m,IH), 4.91 (d, 4H), 7.64 - 7.71 (m, 3H), 7.74 - 7.79 (m, 2H), 8.64 (d, IH), 8.72 (s, IH), 8.76 (s,I H). Three protons are not visible.U PLC-MS (Method 2): R 0.71 min; MS (ESIpos): mz [M÷H] 434.
6-(4-aminophenyl)-5-methyl-2-phenyl-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-[4-(4-methyl-6-oxo-1-phenyl-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
21%
A solution of 100 mg of Intermediate 23(0.36 minol, 1.00 eq) in DMF (11 mL) was treated with110 mg of N,N?-disuccinimidyl carbonate (0.43 minol, 1.20 eq) and 53.4 mg of 4-dimethylaminopyrdine (0.44 minol, 1.20 eq). The mixture was stirred over night at roomtemperature. A solution of 82.9 mg of 2,3-dihydro-1 H-pyrrolo[3,4-c]pyridine dihydrochloride (0.43minol, 1 .20 eq) and 2.99 mL of triethylamine (21.47 minol, 30 eq) in DMF (2 mL) was added. The mixture was stirred over night. THE was removed from under reduced pressure. The remaining solution was poured into water. The solid was removed by filtration, the filtrate was taken to dryness and the residue was purified by preparative reverse phase HPLC to yield 35.0mg of the desired product (21percent).1H-NMR (400MHz, DMSO-d6): oe [ppm] = 1.07 (d, 3H), 2.17 (s, 6H), 2.22-2.36 (m, IH), 2.40-2.48 (m, 2H), 2.64 - 2.70 (m, I H), 3.37 - 3.44 (m, I H), 3.59 - 3.75 (m, I H), 3.91 - 4.08 (m, I H),4.83 (d, 4H), 7.44 (d, IH), 7.63 - 7.71 (m, 2H), 7.71 - 7.79 (m, 2H), 8.50 (d, IH), 8.65 (s, IH),8.61 (s, IH).UPLC-MS (Method 1): R0.83 min; MS (ESIpos): mz[M+H]426.
6-(4-aminophenyl)-2-[2-(dimethylamino)ethyl]-5-methyl-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-(4-{1-[2-(dimethylamino)ethyl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3- dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
13%
A solution of 100mg of Intermediate 24 (0.36 minol, 1.00 eq) in DMF (11 mL) was treated with112 mg of N,N?-disuccinimidyl carbonate (0.44 minol, 1.20 eq) and 53.4 mg of 4-dimethylaminopyrdine (0.44 minol, 1.20 eq). The mixture was stirred over night at roomtemperature. A solution of 84.4 mg of 2,3-dihydro-1 H-pyrrolo[3,4-c]pyridine dihydrochloride (0.44minol, 1 .20 eq) and 3.05 mL of triethylamine (21.87 minol, 30 eq) in DMF (2 mL) was added. The mixture was stirred over night. THE was removed from under reduced pressure. The remaining solution was poured into water. The solid was removed by filtration, the filtrate was taken to dryness and the residue was purified by preparative reverse phase HPLC to yield 20.0mg of the desired product (13percent).1H-NMR (400MHz, DMSO-d6): oe [ppm] = 1.07 (d, 3H), 2.17 (s, 6H), 2.22-2.36 (m, IH), 2.40-2.48 (m, 2H), 2.64 - 2.70 (m, I H), 3.37 - 3.44 (m, I H), 3.59 - 3.75 (m, I H), 3.91 - 4.08 (m, I H),4.83 (d, 4H), 7.44 (d, IH), 7.63 - 7.71 (m, 2H), 7.71 - 7.79 (m, 2H), 8.50 (d, IH), 8.65 (s, IH),8.61 (s, IH).UPLC-MS (Method 1): R0.47 min; MS (ESIpos): mz[M+2H]421
6-(4-aminophenyl)-2-[4-(difluoromethoxy)benzyl]-5-methyl-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-(4-{1-[4-(difluoromethoxy)benzyl]-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
12%
A solution of 200 mg of Intermediate 25 (0.56 minol, 1.00 eq) in DMF (8 mL) was treated with171 mg of N,N?-disuccinimidyl carbonate (0.67 minol, 1.20 eq) and 82.0 mg of 4- dimethylaminopyrdine (0.67 minol, 1.20 eq). The mixture was stirred for 3 days at room temperature. A suspension of 129 mg of 2,3-dihydro-IH-pyrrolo[3,4-c]pyridine dihydrochloride (0.67 minol, 1.20 eq) and 279 pL of triethylamine (2.00 minol, 3.6 eq) in DMF (2 mL) was added. The mixture was stirred over night. The resulting suspension was filtered, the solid was removedand the filtrate was taken to dryness. The residue was purified by reverse phase preparative HPLC to yield 34.0mg of the desired product (12percent).1H-NMR (400MHz, DMSO-d6): oe [ppm]= 1.04 (d, 3H), 2.30 -2.40 (m, IH), 2.75 -2.86 (m, IH),3.38-3.48 (m, IH), 4.78 -4.87 (m, 5H), 4.99 (s, IH), 7.00- 7.22 (m, 3H), 7.34 -7.40 (m, 2H),7.44 (d, I H), 7.62 - 7.68 (m, 2H), 7.69 - 7.75 (m, 2H), 8.50 (d, I H), 8.63 (d, 2H).UPLC-MS (Method 2): Rt0.92 min; MS (ESIpos): mz [M÷H] 506.
N-[4-(4-oxo-3,4-dihydrophthalazin-1-yl)phenyl]-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1.5%
A solution of 100.0 mg of 4-(4-aminophenyl)-2-methylphthalazin-1(2H)-one (by S. Demirayak etal. in Eur. J. Med. Chem. 2004, 39, 1089?1095, 0.42 minol, 1.00 eq) in DMF (12 mL) wastreated with 129.6mg of N,N?-disuccinimidyl carbonate (0.51 minol, 1.20 eq) and 61.8mg of 4-dimethylaminopyridine (0.51 minol, 1.20 eq). The mixture was left of night at room temperature.A suspension of 97.7 mg of 2,3-dihydro-1 H-pyrrolo[3,4-c]pyridine dihydrochloride (0.51 minol,1.20 eq) and 1.76 mL of triethylamine (12.79 minol, 30 eq) in DMF (2 mL) was added and themixture was again stirred over night. The suspension was filtered, the filtrate was taken to dryness and the residue was purified by preparative reverse phase HPLC to obtain 2.5 mg of the desired material (1.5percent).1H-NMR (400MHz, DMSO-d6): oe [ppm] = 4.86 (d, 4H), 7.39 - 7.60 (m, 3H), 7.76 (d, 3H), 7.83 -8.00 (m, 2H), 8.24-8.40 (m, IH), 8.52 (d, IH), 8.67 (s, IH), 8.63 (s, IH), 12.80 (s, IH).UPLC-MS (Method 2): R0.80 min; MS (ESIpos): mz [M÷H] 384.
6-(4-aminophenyl)-5-methyl-2-[2-(morpholin-4-yl)ethyl]-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-(4-{4-methyl-1-[2-(morpholin-4-yl)ethyl]-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl}phenyl)-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
11%
A solution of 150 mg of Intermediate 34 (0.47 minol, 1.00 eq) in DMF (5 mL) was treated with145 mg of N,N?-disuccinimidyl carbonate (0.57 minol, 1.20 eq) and 181 mg of 4-dimethylaminopyrdine (0.57 minol, 1.20 eq). The mixture was stirred over night at roomtemperature. A suspension of 109 mg of 2,3-dihhydro-IH-pyrrolo[3,4-c]pyridine dihydrochloride(0.57 minol, 1.20 eq) and 2.00 mL of triethylamine in DMF (2 mL) was added. The mixture was stirred for 3 days. The solid was removed by filtration, the filtrate was taken to dryness and the residue was purified by preparative reverse phase HPLC to yield 24.2 mg of the desired product(11percent).1H-NMR(400MHz, DMSO-d6): oe [ppm]= 1.09-1.13 (m, 3H), 2.30 (dd, IH), 2.35 -2.45 (m, 4H),2.65-2.75 (m, IH), 3.36-3.43 (m, 4H), 3.50 (t, 4H), 3.59-3.68 (m, IH), 4.12 (dt, IH), 4.82 (d,4H), 7.44 (d, I H), 7.64 - 7.69 (m, 2H), 7.71 - 7.77 (m, I H), 8.50 (d, I H), 8.63 (d, 2H).UPLC-MS (Method 2): R = 0.49 min; MS (ESIpos): mz [M÷H] 463.
N-[4-(1,4-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
83%
A solution of 108 mg of Intermediate 4 (0.50 minol, 1.00 eq) in THE (18 mL) was treated with154 mg N,N?-disuccinimidyl carbonate (0.60 minol, 1.20 eq) and 73.3 mg of 4-dimethylaminopyrindine. The reaction mixture was stirred at room temperature over night. Asuspension of 116mg 2,3-dihydro-IH-pyrrolo[3,4-c]pyridine dihydrochloride (0.6 minol, 1.20 eq)and 251 pL of triethylamine (1.80 minol, 3.60 eq) was added. The reaction mixture was stirred at room temperature and additionally 250 pL triethylamine were added. After stirring for 3 days the precipitate was filtered off and discarded. The filtrate was taken to dryness. The remaining residue was purified by preparative reverse phase HPLC to provide 150 mg of the desiredproduct Example 4 (83percent).1H-NMR (400MHz, DMSO-d6): oe [ppm] = 1.07 (d, 3H), 2.30 (dd, IH), 2.65-2.75 (m, IH), 3.32(s, 3H), 4.83 (d, 4H), 7.41 -7.46 (m, IH), 7.64-7.69 (m, 2H), 7.71 -7.76 (m, 2H), 8.14 (s, IH),8.50 (d, I H), 8.62 (d, 2H). One proton under the water protons.LC-MS (Method 2): R = 0.62 min; MS (ESIpos): mz = 364 [M÷H]
6-(6-aminopyridin-3-yl)-2,5-dimethyl-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-[5-(1,4-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)pyridin-2-yl]-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
21%
To a solution of Intermediate 83 45 mg (0.21 minol) in acetonitrile, 6mL, was added N,Ndisuccinimidyl carbonate, 63.38 mg (0.25 minol). The reaction was stirred at room temperature for 18 hours in a sealed tube. To a suspension of 2,3-dihydro-IH-pyrrolo[3,4-c]pyridinehydrochloride, 32 mg (0.21 minol) in acetonitrile, I mL, was added triethylamine, 0.057 mL (0.41 minol), the slurry was transferred to the tube and the reaction was left to stir at room temperature for 18 hours. The reaction mixture was diluted with water and extracted with ethyl acetate. The combined organics were dried over solid sodium sulfate and concentrated under vacuum.Purification by MDAP (Eluent: Acetonitrile: 0.1percent NH4OH 5:90, 40:60) gave 102.4, 15.9 mg (21percent) as a white solid.1H NMR (300 MHz, CDCI3): 6 [ppm] = 1.23 (d, 3H), 2.50 (d, IH), 2.70 (dd, IH), 3.27 (m, IH),3.47 (s, 3H), 4.92 (d, 4H), 7.32 (m, IH), 8.14 (m, IH), 8.22 (m, IH), 8.58-8.64 (m, 3H).UPLC-MS (Method 4): R= 1.31min., 99percent ES (ESIpos) [M÷H] 365.
(5S)-6-(4-aminophenyl)-2,5-dimethyl-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-{4-[(4S)-1,4-dimethyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl]phenyl}-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
83%
A solution of 500 mg of Intermediate 6 (2.30 minol, 1.00 eq) in THE (60 mL) was treated with707 mg of N,N?-disuccinimidyl carbonate (2.76 minol, 1.20 eq) and 338 mg of 4-dimethylaminopyridine (2.76 minol, 1.20 eq). The mixture was stirred for 3 days at roomtemperature. A suspension of 533 mg 2,3-dihydro-IH-pyrrolo[3,4-c] pyrimidine dihydrochloride(2.76 minol, 1.20 eq) and 1.16 mL of triethylamine (8.29 minol, 3.60 eq) in DMF (3 mL) wasadded. Additionally 5 mL of DMF were added to the mixture and it was stirred again over night at room temperature. The precipitate was filtered off and discarded. The filtrate was poured into water. The resulting suspension was stirred over night, the precipitate collected by filtration, washed with water and dried to provide the desired product Example 5 (732 mg, 83percent).1H-NMR (400MHz, DMSO-d6): oe [ppm] = 1.07 (d, 3H), 2.30 (dd, IH), 2.70 (dd, IH), 3.35-3.45 (m, I H), 4.82 (d, 4H), 7.44 (d, I H), 7.63 - 7.70 (m, 2H), 7.71 - 7.76 (m, 2H), 8.50 (d, I H), 8.62 (d,I H). The methyl group is under the water protons.LC-MS (Method 2): R = 0.60 min; MS (ESIpos): mz = 364 [M÷H].Optical rotation (Method 5): [a] = + 35750 (c = 1.00, DMSO).
6-(4-aminophenyl)-5-isopropyl-2-methyl-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-[4-(4-isopropyl-1-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
42%
To a solution of 6-(4-aminophenyl)-5-isopropyl-2-methyl-4, 5-dihydropyridazin-3(2H )-one Intermediate 91, 50 mg (0.20 minol) and 4-dimethylaminopyridine, 30 mg (0.25 minol) in tetrahydrofuran, 5 mL, N,N-disuccinimidyl carbonate, 63 mg (0.25 minol) was added. After 2 hours at room temperature 2,3-dihydro-1 H-pyrollo[3,4-c]pyridine dihydrochloride, 59 mg (6000- 50-6, 0.31 minol) and triethylamine, 0.29 mL, (2.04 minol) dissolved in N,N dimethylformide (3mL) were added and the mixture was stirred for a further 16 hours. The reaction mixture was diluted with a saturated solution of aminonium chloride and extracted with ethyl acetate. The combined organic layers were dried over solid sodium sulfate, filtered and concentrated undervacuum. The crude compound was purified by reverse phase chromatography (BIOTAGE SP4, 30 g Biotage cartridge) using acetonitrile and water containing 10min aminonium bicarbonate pH 10 buffer (3:97 to 100:0) to give Example 78, 31 mg (42percent) as a colourless solid.1H NMR (400 MHz, CDCI3): 6 [ppm] = 0.89 (m, 6H), 1.96 (m, IH), 2.56 (dd, IH), 2.70 (d, IH),3.04 (m, IH), 3.41 (s, 3H), 4.89 (d, 4H), 6.44 (s, IH), 7.33 (m, IH), 7.51 (d, 2H), 7.73 (d, 2H),8.59 (m, IH), 8.65 (m, IH).UPLC (Method 3): R = 0.61 min., 95percent. MS (ESIpos): mz = [M÷H] 392.
6-(4-aminophenyl)-2-isopropyl-5-methyl-4,5-dihydropyridazin-3(2H)-one[ No CAS ]
[ 6000-50-6 ]
N-[4-(1-isopropyl-4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin-3-yl)phenyl]-1,3-dihydro-2H-pyrrolo[3,4-c]pyridine-2-carboxamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
25%
A solution of 100mg of Intermediate 22 (0.41 minol, 1.00 eq) in DMF (12 mL)was treated with125 mg of N,N?-disuccinimidyl carbonate (0.49 minol, 1.20 eq) and 59.7 mg of 4-dimethylaminopyrdine (0.49 minol, 1.20 eq). The mixture was stirred over night at roomtemperature. A solution of 94.4 mg of 2,3-dihydro-I H-pyrrolo[3,4-c]pyridine dihydrochloride (0.49minol, 1 .20 eq) and 1.70 mL of triethylamine (12.37 minol, 30 eq) in DMF (2 mL) was added.The mixture was stirred over night. THE was removed from under reduced pressure. The remaining solution was poured into water. The solid was removed by filtration, the filtrate was taken to dryness and the residue was purified by preparative reverse phase HPLC to yield 40.3 mg of the desired product (25percent).H-NMR (400MHz, DMSO-d6): oe [ppm] = 1.04 (d, 3H), 1.15 (d, 3H), 1.24 (d, 3H), 2.29 (dd, IH),2.68 (dd, IH), 3.21 - 3.42 (m, IH), 4.83 (d, 4H), 4.90 (quin, IH), 7.44 (d, IH), 7.60 - 7.72 (m,2H), 7.72 - 7.83 (m, 2H), 8.50 (d, I H), 8.62 (d, 2H).UPLC-MS (Method 1): RO.78 min; MS (ESIpos): mz [M÷2H] 393.
5-carboxy-2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)benzoate[ No CAS ]
C108H153N23O19S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
25 pmol of NH2-Fx-r-Fx-r-Fx-r-K(Mtt) on resin was reacted with S-trityl-2- mercaptoproprionic acid (4 eq), PyBOP (4 eq), and DIPEA (8 eq) in 1 mL DMF. The peptide was washed (2 x DMF/MeOH/DCM) and deprotected with trifluoroacetic (0146) acid:triisopropylsilane:DCM (5:3:92, 2 x 15 minutes). The peptide washed and equilibrated in acetonitrile:water (5: 1 ). Cysteamine (20 eq) was dissolved in 1 mL acetonitrile:water (5: 1 ) and added to the reaction mixture followed by iodine (10 eq). The reaction was stirred for 30 minutes. The peptide was washed (2 x DMF:MeOH:DCM) and reacted with 5- Carboxytetramethylrhodamine (2 eq), HBTU (2 eq), and DIPEA (4 eq) in 0.5 mL DMF for 2 hours. The peptide was washed, cleaved from resin using trifluoroacetic (0147) acid:triisopropylsilane:water (95:2.5:2.5) and precipitated in ether at -20°C for 1 hour. The precipitate was purified by HPLC and lyophilized. 5-(2,4-Dihydroxy-5-isopropylphenyl)-N-ethyl- 4-(4-(morpholinomethyl)phenyl)isoxazole-3-carboxamide (Luminespib, 3 eq, Adooq Bioscience, Irvine CA) was reacted with Nu,Nu'-Disuccinimidyl carbonate (3 eq, Sigma-Aldrich) and 4- (Dimethylamino)pyridine (12 eq, Sigma-Aldrich) in 0.4 mL DMF for 1 hour. The peptide was dissolved in 0.1 mL DMF and added to the reaction mixture and the solution was left stirring overnight. The peptide was precipitated in ether and purified by HPLC. The earlier eluting isomer was purified and tested due to its higher relative abundance. The solution was frozen in dry ice as the compound eluted from the column and lyophilized. The peptide was identified by ESI mass spectrometry, expected m/z = 2140.12, found m/z = 2140.12. The peptide was quantified using the 5-Carboxytetramethylrhodamine absorbance at 547 nm with an extinction coefficient of 92000 M"1cm"1.
25 muetaetaomicronIota of NH2-Fx-r-Fx-r-Fx-r on resin was reacted with S-trityl-2- mercaptoproprionic acid (4 eq), PyBOP (4 eq), and DIPEA (8 eq) in 1 mL DMF. The peptide was washed (2 x DMF/MeOH/DCM), cleaved from resin using trifluoroacetic (0139) acid:triisopropylsilane:water (95:2.5:2.5) and precipitated in ether at -20°C for 1 hour. The precipitate was purified by RP-HPLC, dried under vacuum and dissolved in 0.5 mL (0140) acetonitrile:water (5:1). 2-mercpatoethanol (20 eq, Sigma-Aldrich) was added to the reaction mixture followed by iodine (10 eq) and the reaction was stirred for 30 minutes. The peptide was purified by HPLC and lyophilized. 5-(2,4-Dihydroxy-5-isopropylphenyl)-N-ethyl-4-(4- (morpholinomethyl)phenyl)isoxazole-3-carboxamide (Luminespib, 3 eq, Adooq Bioscience, Irvine CA) was reacted with Nu,Nu'-Disuccinimidyl carbonate (3 eq, Sigma-Aldrich) and 4- (Dimethylamino)pyridine (12 eq, Sigma-Aldrich) in 0.4 mL DMF for 1 hour. The peptide was dissolved in 0.1 mL DMF and added to the reaction mixture and the solution was left stirring overnight. The peptide was precipitated in ether and purified by HPLC. Two isomers were identified during HPLC purification, likely due to attachment to either of the two resorcinol hydroxyls. The earlier eluting isomer was purified and tested due to its higher relative abundance. The solution was frozen in dry ice as the compound eluted from the column and lyophilized. The peptide was identified by ESI mass spectrometry, expected m/z = 1599.88, found m/z = 1599.88. The peptide was quantified via absorbance spectrophotometry using a SpectraMax M5 spectrophotometer. The absorbance profile of Compound 5 was found to be shifted as compared to Luminespib itself, therefore the peptide was quantified by cleavage in 25 mM TCEP in PBS pH 7.4 for 10 minutes, then measuring free Luminespib absorbance at 305 nm with an extinction coefficient of 8520 M"1cm"1. TCEP was not found to affect the extinction coefficient of Luminespib.
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