* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Reference：
[1] Journal of Organic Chemistry, 1992, vol. 57, # 9, p. 2755 - 2756
2
[ 1676-73-9 ]
[ 24424-99-5 ]
[ 13574-13-5 ]
Yield
Reaction Conditions
Operation in experiment
100%
at 0℃;
Example 28 - Formula 125 - Compound 28aIntermediate B: (S)-5-(Benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid To a solution of compound A (5.0 g, 21 .1 mmol) in dixoane and water (1 :1 , 40 mL) at 0 °C was added Boc20 (5.06 g, 23.1 mmol) and the mixture was stirred overnight. The solvent was removed under reduced pressure and the residue was diluted with water (30 mL), basified with Na2C03 (0.7 g) and washed with EtOAc (3 x 20 mL). The aqueous layer was adjusted to pH 2-3 with a 5 M aqueous HCI solution and extracted with EtOAc (4 x 50 mL). The combined organic extracts were washed with brine, dried over Na2S04 and the solvent was removed under reduced pressure to afford (S)-5-(benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid (7.1 g, 100percent) as a viscous colourless oil.LC-MS (Agilent): Rt 3.40 min; m/z calculated for C17H23NO6 [M+Na]+ 360.15, found 360.1 .
100%
at 0℃;
To a solution of compound A (5.0 g, 21.1 mmol) in dixoane and water (1:1, 40 mL) at 0° C. was added Boc2O (5.06 g, 23.1 mmol) and the mixture was stirred overnight. The solvent was removed under reduced pressure and the residue was diluted with water (30 mL), basified with Na2CO3 (0.7 g) and washed with EtOAc (3*20 mL). The aqueous layer was adjusted to pH 2-3 with a 5 M aqueous HCl solution and extracted with EtOAc (4*50 mL). The combined organic extracts were washed with brine, dried over Na2SO4 and the solvent was removed under reduced pressure to afford (S)-5-(benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid (7.1 g, 100percent) as a viscous colourless oil. LC-MS (Agilent): Rt 3.40 min; m/z calculated for C17H23NO6 [M+Na]+ 360.15. found 360.1.
Reference：
[1] Patent: WO2012/63085, 2012, A2, . Location in patent: Page/Page column 115
[2] Patent: US2013/225594, 2013, A1, . Location in patent: Paragraph 0425; 0426
[3] Synthetic Communications, 1990, vol. 20, # 15, p. 2235 - 2249
[4] Bioorganic and Medicinal Chemistry, 2005, vol. 13, # 2, p. 501 - 517
[5] Organic Syntheses, 1985, vol. 63, p. 160 - 160
[6] Acta chemica Scandinavica. Series B: Organic chemistry and biochemistry, 1986, vol. 40, # 4, p. 242 - 249
[7] Acta chemica Scandinavica. Series B: Organic chemistry and biochemistry, 1986, vol. 40, # 4, p. 250 - 256
[8] Molecular Crystals and Liquid Crystals (1969-1991), 1989, vol. 170, p. 195 - 214
[9] Doklady Chemistry, 2006, vol. 408, # 1, p. 57 - 60
[10] Organic and Biomolecular Chemistry, 2007, vol. 5, # 12, p. 1915 - 1923
[11] Bioorganic and Medicinal Chemistry Letters, 2009, vol. 19, # 12, p. 3220 - 3224
[12] Journal of Organic Chemistry, 2016, vol. 81, # 20, p. 9903 - 9911
[13] Chemical Communications, 2017, vol. 53, # 37, p. 5155 - 5158
3
[ 132090-12-1 ]
[ 13574-13-5 ]
Reference：
[1] Journal of Organic Chemistry, 2007, vol. 72, # 3, p. 782 - 786
Reference：
[1] Justus Liebigs Annalen der Chemie, 1968, vol. 716, p. 175 - 185
12
[ 1070-19-5 ]
[ 1676-73-9 ]
[ 13574-13-5 ]
Reference：
[1] Justus Liebigs Annalen der Chemie, 1964, vol. 673, p. 196 - 207
13
[ 1676-73-9 ]
[ 16965-08-5 ]
[ 13574-13-5 ]
Reference：
[1] Journal of the Chemical Society, Perkin Transactions 1: Organic and Bio-Organic Chemistry (1972-1999), 1972, p. 1523 - 1526
14
[ 13574-13-5 ]
[ 72086-72-7 ]
Reference：
[1] Organic and Biomolecular Chemistry, 2007, vol. 5, # 12, p. 1915 - 1923
[2] Organic and Biomolecular Chemistry, 2007, vol. 5, # 9, p. 1459 - 1465
[3] Chemical Communications, 2005, # 37, p. 4652 - 4654
[4] Tetrahedron Letters, 1998, vol. 39, # 15, p. 2099 - 2102
[5] Bioorganic and Medicinal Chemistry Letters, 1997, vol. 7, # 3, p. 331 - 336
[6] Chemical and Pharmaceutical Bulletin, 1996, vol. 44, # 8, p. 1498 - 1509
[7] Chemical and Pharmaceutical Bulletin, 1985, vol. 33, # 11, p. 4836 - 4846
[8] Journal of Organic Chemistry, 1987, vol. 52, # 24, p. 5331 - 5341
[9] Patent: WO2012/109164, 2012, A1,
[10] Tetrahedron Letters, 2014, vol. 55, # 14, p. 2274 - 2276
[11] Heterocycles, 2015, vol. 90, # 2, p. 1309 - 1316
15
[ 13574-13-5 ]
[ 24277-39-2 ]
Reference：
[1] Chemistry - An Asian Journal, 2012, vol. 7, # 9, p. 2052 - 2060
[2] Journal of Medicinal Chemistry, 2016, vol. 59, # 4, p. 1531 - 1544
16
[ 13574-13-5 ]
[ 74-88-4 ]
[ 59279-58-2 ]
Yield
Reaction Conditions
Operation in experiment
82%
With potassium carbonate In N,N-dimethyl-formamide at 20℃; for 3 h;
Step 1 Methyl iodide (1.01 mL, 16.3 mmol) was added dropwise to a solution of (2S)-5-(benzyloxy)-2-[(tert-butoxy)carbonyl]amino}-5-oxopentanoic acid XII (5.00 g, 14.82 mmol) and K2CO3 (2.25 g, 16.3 mmol) in DMF (25 mL) at r.t. The reaction mixture was stirred about 3 h at r.t. before adding additional methyl iodide (1.01 mL, 16.3 mmol). EtOAc was then added to the reaction and washed 3*10percent Na2S2O3 and dried over MgSO4. The solvent was removed under reduced pressure and the crude product was purified on a silica gel column (100:1 and then 50:1 CHCl3/MeOH) to give 5-benzyl 1-methyl (2S)-2-[(tert-butoxy)carbonyl]amino}pentanedioate XIII (4.20 g, 12.23 mmol, 82percent yield). ESIMS found for C18H25NO6 m/z 352 (M+H).
82%
With potassium carbonate In N,N-dimethyl-formamide at 20℃; for 3 h;
Step 1[00181] Methyl iodide (1.01 mL, 16.3 mmol) was added dropwise to a solution of (2S)-5-(benzyloxy)-2-[(tert-butoxy)carbonyl]amino}-5-oxopentanoic acid XII (5.00 g, 14.82 mmol) and K2CO3 (2.25 g, 16.3 mmol) in DMF (25 mL) at room temperature The reaction mixture was stirred about 3 h at room temperature before adding additional methyl iodide (1.01 mL, 16.3 mmol). EtOAc was then added to the reaction and washed 3x 10percent Na2S203 and dried over MgSC^. The solvent was removed under reduced pressure and the crude product was purified on a silica gel column (100: 1 and then 50: 1 CHCl3/MeOH) to give 5-benzyl 1-methyl (2S)-2-[(tert-butoxy)carbonyl]amino} pentanedioate XIII (4.20 g, 12.23 mmol, 82percent yield). ESIMS found for Ci8H25N06 mlz 352 (M+H).
Reference：
[1] Journal of Medicinal Chemistry, 2016, vol. 59, # 5, p. 1899 - 1913
[2] Patent: US2008/318957, 2008, A1, . Location in patent: Page/Page column 41-42
[3] Patent: WO2012/109164, 2012, A1, . Location in patent: Page/Page column 39
[4] Organic and Biomolecular Chemistry, 2007, vol. 5, # 12, p. 1915 - 1923
[5] Organic Letters, 2016, vol. 18, # 9, p. 1968 - 1971
17
[ 13574-13-5 ]
[ 77-78-1 ]
[ 59279-58-2 ]
Yield
Reaction Conditions
Operation in experiment
95%
Stage #1: With potassium carbonate In acetone for 0.166667 h; Inert atmosphere Stage #2: at 20℃; for 1 h; Inert atmosphere
Boc-L-Glu(OBn)OH 3 (5 g,14.82 mmol) was added to a 50 mL round bottom flask containing acetone (25 mL).Potassium carbonate (4097 mg, 29.64 mmol) was then added. The reaction mixture wasstirred for 10 minutes and dimethylsulphate (1.55 mL, 16.3 mmol) was addedand stirred for 1 hour at rt. Thereaction was monitored by TLC and showed complete disappearance of startingmaterial. After removal of acetone, water was added and extracted with ethylacetate. The resultant organic layer was dried over sodium sulphate andconcentrated to obtain compound 4 asa colorless liquid.(4.95 g , 95percent). [α]21D =23 (c 0.1, CHCl3). 1HNMR: (CDCl3, 400 MHz) 1.39(s, 9H), 1.88-2.03 (m, 1H), 2.13-2.20 (m, 1H), 2.35-2.49 (m, 2H), 3.66 (s, 3H),4.30-4.31 (m, 1H), 5.07 (s, 2H), 5.25-5.27 (d, 1H, J = 7.6 Hz), 7.24-7.33 (m, 5H). 13C NMR: (CDCl3,100 MHz) 27.6, 28.2, 30.2, 52.3, 52.8, 66.4, 79.9, 128.2, 128.5, 135.8, 155.4,172.4, 172.7. IR (KBr) 3372, 2978, 1716, 1513, 1455, 1392,1367, 1252, 1214, 1165, 1051, 1028, 873, 781, 751, 699, 579 cm-1.
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; isobutyl chloroformate In tetrahydrofuran at -10℃; for 1h; Inert atmosphere;
Stage #2: diazomethane In diethyl ether at 0 - 20℃; Inert atmosphere;
4-tert-Butoxycarbonylamino-6-diazo-5-oxo-hexanoic acid benzyl ester (5)
Boc-Glu(OBn)-OH (5.06 g, 15.00 mmol) was dissolved in anhydrous THF (80 mL) under an atmosphere of N2, and cooled to -10 °C. NMM (2.14 ml, 19.50 mmol) was added, followed by IBCF (2.95 ml, 22.50 mmol). The reaction was stirred at -10°C for 1 h. The white precipitate formed was eliminated by filtration. The filtrate was cooled to 0 °C and freshly prepared diazo methane in diethyl ether was added (2 eq, prepared from Diazald and KOH). The mixture was warmed up to room temperature and stirred overnight. The reaction was then quenched with the addition of acetic acid (1 ml), and the mixture was vigorously stirred (750 rpm) for two hours. An excess of acetic acid was then added (1 ml), and the mixture was then neutralised with a saturated aqueous NaHC03 solution (70 ml) and stirred until the effervescence disappeared. The organic phase was separated and washed with 1 N KHSO4 (aq) (60 ml), brine (60 ml), dried over MgSC>4, and the solvents were evaporated in vacuo. The crude obtained was purified by flash chromatography on silica gel (gradient cyclohexane-30% ethyl acetate), to afford the pure product as a yellow solid (3.94 g, 73 %). 1H NMR (CDC13, 300 MHz) δ: 7.46- 7.29 (m, 5H), 5.47 (s, 1H), 5.25 (m, 1H), 5.14 (s, 2H), 4.26 (m, 1H), 2.60-2.38 (m, 2H), 2.23- 2.10 (m, 1H), 1.93-1.77 (m, 1H), 1.45 (s, 9H).
With dicyclohexyl-carbodiimide In 1,2-dimethoxyethane at 5℃; for 18h;
66%
With dicyclohexyl-carbodiimide In ethyl acetate at 4℃;
With dicyclohexyl-carbodiimide In tetrahydrofuran Ambient temperature;
0.38 g
With dicyclohexyl-carbodiimide In ethyl acetate at 20℃; for 17h;
With dicyclohexyl-carbodiimide In 1,4-dioxane at -5℃;
With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at -10℃; for 12h;
1
1) BOC-Glu(OBzl)-OSu, N-oxysuccinimide ester of N-tert.butyloxycarbonyl-(γ-benzyl)glutamic acid (I). N-tert.butyloxycarbonyl-(γ-benzyl)glutamic acid BOC-Glu(OBzl)-OH (33,7 g, 0,1 mole) was dissolved in 50 ml of N,N'-dimethylformamide, cooled to -10°C; cooled (4-6°C) solutions of N,N'-dicyclohexylcarbodiimide (23,0 g, 0,11 mole) were added while stirring in 30 ml of N,N'-dimethylformamide and N-hydroxysuccinimide (13,0 g, 0,11 mole) in 20 ml N,N'-dimethylformamide. Reactive mixture was stirred for 12 hours, cooled with ice, and then for 24 hours at room temperature. The residue N,N'-dicyclohexylurea was filtered out and the obtained solution of activated ester was used without extracting during the next step.
Example 28 - Formula 125 - Compound 28aIntermediate B: (S)-5-(Benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid To a solution of compound A (5.0 g, 21 .1 mmol) in dixoane and water (1 :1 , 40 mL) at 0 C was added Boc20 (5.06 g, 23.1 mmol) and the mixture was stirred overnight. The solvent was removed under reduced pressure and the residue was diluted with water (30 mL), basified with Na2C03 (0.7 g) and washed with EtOAc (3 x 20 mL). The aqueous layer was adjusted to pH 2-3 with a 5 M aqueous HCI solution and extracted with EtOAc (4 x 50 mL). The combined organic extracts were washed with brine, dried over Na2S04 and the solvent was removed under reduced pressure to afford (S)-5-(benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid (7.1 g, 100%) as a viscous colourless oil.LC-MS (Agilent): Rt 3.40 min; m/z calculated for C17H23NO6 [M+Na]+ 360.15, found 360.1 .
100%
In 1,4-dioxane; water; at 0℃;
To a solution of compound A (5.0 g, 21.1 mmol) in dixoane and water (1:1, 40 mL) at 0 C. was added Boc2O (5.06 g, 23.1 mmol) and the mixture was stirred overnight. The solvent was removed under reduced pressure and the residue was diluted with water (30 mL), basified with Na2CO3 (0.7 g) and washed with EtOAc (3*20 mL). The aqueous layer was adjusted to pH 2-3 with a 5 M aqueous HCl solution and extracted with EtOAc (4*50 mL). The combined organic extracts were washed with brine, dried over Na2SO4 and the solvent was removed under reduced pressure to afford (S)-5-(benzyloxy)-2-(tert-butoxycarbonyl)-5-oxopentanoic acid (7.1 g, 100%) as a viscous colourless oil. LC-MS (Agilent): Rt 3.40 min; m/z calculated for C17H23NO6 [M+Na]+ 360.15. found 360.1.
83.55%
With sodium hydroxide; In 1,4-dioxane; water; at 0 - 20℃; for 16h;
To a stirred solution of Int- A (8 g, 33.75 mmol) in 1, 4 dioxane and hLO (80 mL, 1:1) were added NaOH (2.7 g, 67.51 mmol) and Boo20 (8.10 g, 37.13 mmol) at 0 C. The reaction mixture was stirred at RT for 16 h. After consumption of the starting material (by TLC), the reaction mixture was acidified with 2 N HC1 (pH~4) and extracted with EtOAc (3 x 100 mL). The combined organic extracts were dried over anhydrous Na2S04 and concentrated under reduced pressure to afford Int-B (9.5 g, 83.55%) as a yellow syrup. LCMS (ESI): (m/z) 238.05 [M-Boc]+.
72%
With sodium hydroxide; In tetrahydrofuran; water; at 0 - 20℃; for 12h;
General procedure: To a stirred suspension of amino acid (3 mmol) in THF/H2O (v : v = 10 mL : 10 mL), was added NaOH (12 mmol). The solution was cooled down to 0oC, Boc2O (3.3 mmol) was added (6.3 mmol for Tyrosine, Lysine and Histidine and 9.3 mmol for Arginine) and the resulting solution was stirred for 12h at room temperature. THF was removed under reduced pressure and the aqueous layer was extracted (diethyl ether, 2 x 5 mL). The aqueous layer was acidified with HCl (1M) to pH 2, and then extracted (dichloromethane, 3 x 10 mL). The organic phase was dried (Na2SO4) and the solvent was evaporated under reduced pressure to give the protected amino acid.
With N-(3-dimethylaminopropyl)-N-ethylcarbodiimide; triethylamine In dichloromethane
6.8 g
With dmap; benzotriazol-1-ol; dicyclohexyl-carbodiimide In dichloromethane
6
10158] 6.47 g (0.01 mol) N-tert-butyloxycarbonyl dipeptide of benzyl glutamate (compound 2) was dissolved in 15 mE dichloromethane. 6 mE trifluoroacetic acid was added and the system was reacted at room temperature for 2 h. The solvent was removed, and 100 mE dichloromethane was added and 5% sodium bicarbonate solution was used to adjust pH to 7-8. The solution was separated by extract, and the organic phase was washed by 5% sodium bicarbonate solution for two times and then dried through the anhydrous sodium sulfate. After filtration, the filter liquid was directly added to the reaction bottle, and 3.37 g (0.01 mol) tert-butyloxycarbonyl-E-glutamic acid-5-benzyl ester, 1.22 g (0.01 mol) DMAP and 1.35 g (0.01 mol) HOSt were added under the protection of the nitrogen gas. After they were dissolved completely, the dichloromethane solution containing 2.39 g (0.011 mol) DCC was added. Afier all the reagents were dropped, the system was sealed and reacted overnight. TEC was used to monitor that the end of the reaction. After filtration, the system was washed sequentially by 10% citric acid (30 mE*3), 5% sodium bicarbonate (30 mE*3) and 5% saturated sodium chloride aqueous solution (30 mE*3) and then dried by the anhydrous sodium sulfate. Afier the filtration, the solvent was removed and 25 mE ethyl acetate was added to the concentrated solution. The solid was removed by the filtration and 400 mE petroleum ether was added to the mother solution to precipitate it. The system was filtered to produce 6.8 g N-tert-butyloxycarbonyl tripeptide of benzyl glutamate (compound 14).
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; isobutyl chloroformate In tetrahydrofuran at -15℃; for 0.0833333h; Inert atmosphere;
Stage #2: H-Glu(OBzl)-Glu(OBzl)-OBzl In tetrahydrofuran at 20℃; Inert atmosphere;
2.2.1. Synthesis of Glu(4-6) peptides 4a-c
General procedure: The bone-targeting peptides were synthesized by a conventional liquid phase peptides synthetic method from Boc-Glu-Obzl and NH2-Glu-Obzl2 utilizing isobutyl chloroformate (IBCF) and N-methyl morpholine (NMM) to carry out the condensation. The -COOH part was dissolved in dry tetrahydrofuran and cooled to -15 °C, then N-methyl morpholine and isobutyl chloroformate were added, after stirred for 5 min, the -NH2 part in tetrahydrofuran was added to the reaction mixture. After the completion of coupling reaction at room temperature, the mixture was purified by silica column chromatography using CH2Cl2 and CH3OH as an eluent, then deprotected in trifluoroacetic acid (TFA) to give the peptides. Compounds 4a-c were obtained by repeating this operation.
benzyl (S)-4-((tert-butoxycarbonyl)amino)-5-(((S)-1-methoxy-4-methyl-1-oxopentan-2-yl)amino)-5-oxopentanoate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
94%
With triethylamine; dicyclohexyl-carbodiimide In dichloromethane at 0 - 20℃; for 4h;
5.2. General procedure for the synthesis of N-Boc-dipeptide methyl ester (1)
General procedure: To a solution of l-amino acid methyl ester hydrochloride in dichloromethane, triethylamine (1 equiv) and N-Boc-l-amino acid were added at -15 °C, and DCC was added as a dichloromethane solution at 0 °C. The reaction mixture was stirred at 0 °C for 1 h. Then the solution was stirred at room temperature for 3 h. The dicyclohexylurea was removed by filtration from the reaction mixture. The solution was washed with 5% NaHCO3, 1 M HCl, and brine. The organic layer was dried over anhydrous Na2SO4 and evaporated. The product was purified by silica gel column chromatography.
92.2%
With 4-methyl-morpholine; benzotriazol-1-ol; dicyclohexyl-carbodiimide In dichloromethane at 0 - 20℃; for 22h;
With dmap; benzotriazol-1-ol; dicyclohexyl-carbodiimide; In dichloromethane;Inert atmosphere; Sealed tube;
10136] 30 g (0.06 mol) of E-(+)-glutamic acid dibenzyl ester p-toluenesulfonate (compound 1) was dissolved in 500 mE dichloromethane. Then, 20.86 g (0.062 mol) tert-butyloxycarbonyl-E-glutamic acid-5-benzyl ester, 7.55 g (0.062 mol) DMAP and 8.35 g (0.07 mol) HOSt were added. The dichloromethane solution containing 14.3 g DCC was added under the protection of nitrogen gas. Afier all the reagents were dropped, the system was sealed and reacted overnight. TEC was used to monitor the completion of the reaction. The solvent was removed by filtration and 20 mE ethyl acetate was added to the concentrated solution. The solid was removed by filtration and 400 mE petroleum ether was added to the mother liquid to precipitate it. The product of 15.8 g N-tertbutyloxycarbonyl dipeptide of benzyl glutamate (compound 2) was produced afier the filtration.
With ammonium hydroxide; triethylamine; isobutyl chloroformate In N,N-dimethyl-formamide at 20℃; for 2h;
Multi-step reaction with 2 steps
1: N-methylmorpholine (NMM) / tetrahydrofuran / 0.03 h / -10 °C
2: 25percent aq. ammonia / tetrahydrofuran / 1.5 h / 10 - 20 °C
Multi-step reaction with 2 steps
1: triethylamine / tetrahydrofuran / 0.75 h / 25 °C
2: NH3 gas / tetrahydrofuran / 0.75 h
Multi-step reaction with 2 steps
1: N-methylmorpholine / 1,2-dimethoxy-ethane / 0.25 h / -13 °C
2: NH3 (g) / 1,2-dimethoxy-ethane / a.) -13 deg C, 5 min, b.) RT, 15 min
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; chloroformic acid ethyl ester In tetrahydrofuran at -10℃; for 0.333333h;
Stage #2: With ammonia In tetrahydrofuran; water at -10℃;
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; 1,3,5-trichloro-2,4,6-triazine In 1,2-dimethoxyethane at 20℃; for 3h;
Stage #2: With sodium tetrahydroborate In water at 0℃;
83.4%
Stage #1: Boc-Glu(OBzl)-OH With chloroformic acid ethyl ester; triethylamine In tetrahydrofuran at -5℃; for 0.5h; Inert atmosphere;
Stage #2: With sodium tetrahydroborate In tetrahydrofuran; water at -5 - 5℃; for 1h;
1.1
Boc-Glu (OBn) -OH (5.5 g) was dissolved in 80 mL of anhydrous THF, triethylamine (2.8 mL) was added,After cooling to -5 ° C under argon atmosphere, ethyl chloroformate (1.75 mL) was added dropwise and the mixture was stirred at -5 ° C for 30 min.20 [mu] L of water was added, then sodium borohydride (1.85 g, 48.9 mmol, 3.0 eq) was added portionwise,After the formation of the air bubble, the temperature was naturally raised to 5 ° C and stirring was continued for 1 hour until liquid-mass spectrometry (LC-MS)After addition of water, the reaction was quenched and concentrated under reduced pressure to remove most of the solvent. The concentrate was diluted with ethyl acetate,Washed with saturated sodium bicarbonate, 1 M KHSO4, saturated salt, dried over anhydrous sodium sulfate and concentrated under reduced pressureWas viscous material, dissolved in ether, ice bath while stirring slowly dropping n-hexane to a large number of white solid analysisThe filter cake was washed with petroleum ether-ethyl acetate (10: 1) and dried in vacuo to give 4.40 g of white powderThe yield was 83.4%
81%
Stage #1: Boc-Glu(OBzl)-OH With 1-hydroxy-pyrrolidine-2,5-dione; dicyclohexyl-carbodiimide In ethyl acetate at 20℃;
Stage #2: With sodium tetrahydroborate In tetrahydrofuran at 0℃; for 0.5h;
81%
Stage #1: Boc-Glu(OBzl)-OH With 1-hydroxy-pyrrolidine-2,5-dione; dicyclohexyl-carbodiimide In ethyl acetate at 20℃;
Stage #2: With sodium tetrahydroborate In tetrahydrofuran; ethanol at 0℃; for 0.5h;
60%
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; chloroformic acid ethyl ester In tetrahydrofuran at -10℃; for 0.416667h; Inert atmosphere;
Stage #2: With sodium tetrahydroborate In tetrahydrofuran; methanol at 0℃; for 1.16667h;
Stage #3: With hydrogenchloride In tetrahydrofuran; methanol; water
28.C
Intermediate C: (S)-Benzyl 4-(tert-butoxycarbonyl)-5-hydroxypentanoate To a solution of intermediate B (6.5 g, 20 mmol) in THF (20 mL) under nitrogen at -10 °C was added N-methylmorphline (2.0 g, 20 mmol) and ethyl chloroformate (2.3 g, 20 mmol) and the mixture was stirred at -10 °C for 25 min. Sodium borohydride (2.2 g, 60 mmol) was then added to the mixture followed by a slow addition of MeOH (60 mL) over a period of 1 h at 0 °C. The mixture was stirred at 0 °C for an additional 10 min and then quenched with a 1 M aqueous HCI solution (20 mL). The organic solvents were removed under reduced pressure and the aqueous mixture was extracted with EtOAc. The combined organic extracts were washed with a 1 M aqueous HCI solution, water and a 5% aqueous NaHC03 solution, dried over Na2S04 and the solvent was removed under reduced pressure. The residue was purified by column chromatography (Pet. ether/EtOAc, 5/1 , 2/1 , 1/1 , v/v) to give (S)-benzyl 4-(tert- butoxycarbonyl)-5-hydroxypentanoate (3.7 g, 60%) as a yellow oil.LC-MS (Waters): Rt 5.54 min; m/z calculated for C17H25NO5 [M+Na]+ 346.17, found 346.0.
60%
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; chloroformic acid ethyl ester In tetrahydrofuran at -10℃; for 0.416667h; Inert atmosphere;
Stage #2: With methanol; sodium tetrahydroborate In tetrahydrofuran at 0℃; for 1.16667h;
28 Intermediate C: (S)-Benzyl 4-(tert-butoxycarbonyl)-5-hydroxypentanoate
To a solution of intermediate B (6.5 g, 20 mmol) in THF (20 mL) under nitrogen at -10° C. was added N-methylmorphline (2.0 g, 20 mmol) and ethyl chloroformate (2.3 g, 20 mmol) and the mixture was stirred at -10° C. for 25 min. Sodium borohydride (2.2 g, 60 mmol) was then added to the mixture followed by a slow addition of MeOH (60 mL) over a period of 1 h at 0° C. The mixture was stirred at 0° C. for an additional 10 min and then quenched with a 1 M aqueous HCl solution (20 mL). The organic solvents were removed under reduced pressure and the aqueous mixture was extracted with EtOAc. The combined organic extracts were washed with a 1 M aqueous HCl solution, water and a 5% aqueous NaHCO3 solution, dried over Na2SO4 and the solvent was removed under reduced pressure. The residue was purified by column chromatography (Pet. ether/EtOAc, 5/1, 2/1, 1/1, v/v) to give (S)-benzyl 4-(tert-butoxycarbonyl)-5-hydroxypentanoate (3.7 g, 60%) as a yellow oil. LC-MS (Waters): Rt 5.54 min; m/z calculated for C17H25NO5 [M+Na]+ 346.17. found 346.0.
Multi-step reaction with 2 steps
1: 0.38 g / 1,3-dicyclohexylcarbodiimide / ethyl acetate / 17 h / 20 °C
2: 0.14 g / NaBH4; MeOH / tetrahydrofuran / 0.75 h / 0 °C
Multi-step reaction with 2 steps
1: 4-methyl-morpholine / tetrahydrofuran / 0.25 h / 0 °C
2: sodium tetrahydroborate / water; tetrahydrofuran / 0 - 20 °C
Multi-step reaction with 2 steps
1: 4-methyl-morpholine / 1,2-dimethoxyethane / Inert atmosphere
2: sodium tetrahydroborate / water / Inert atmosphere
4.71 g
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine In tetrahydrofuran for 0.166667h;
Stage #2: With chloroformic acid ethyl ester In tetrahydrofuran at -10℃; for 1h;
Stage #3: With sodium tetrahydroborate In water at 20℃; for 1h;
With TEA In dichloromethane at -10℃; for 0.333333h;
With triethylamine In toluene
With triethylamine In tetrahydrofuran at 0℃; for 0.0333333h;
With triethylamine In tetrahydrofuran at -25℃; for 0.25h;
With triethylamine In dichloromethane at 0℃; for 0.75h;
With 4-methyl-morpholine In tetrahydrofuran at -20 - -5℃;
Stage #1: Boc-Glu(OBzl)-OH With triethylamine In tetrahydrofuran at 20℃; for 0.0833333h;
Stage #2: chloroformic acid ethyl ester In tetrahydrofuran at -10℃; for 0.166667h;
With triethylamine In dichloromethane at 0℃; for 0.583333h; Inert atmosphere;
With triethylamine In tetrahydrofuran at -25℃; for 0.25h;
With tributyl-amine In tetrahydrofuran at 20℃; for 0.111667h; Flow reactor;
With 4-methyl-morpholine In tetrahydrofuran at -15℃; for 0.0666667h;
S2.4. Synthesis of benzyl 4-((tert-butoxycarbonyl)amino)-5-(isopropylamino)-5-oxopenta noate (Boc-Glu(OBn)-Ipr) (D)
To a cold (-15 °C) stirring solution of Boc-Glu(OBn)-OH (1 gm, 2.96 mmol) in dry tetrahydrofuran (THF) (15 ml) was added N-methyl morpholine (NMM) (483 μl, 4.4 mmol) and ethyl chloroformate (ECF) (297 μl, 3.1 mmol) and stirred until (4 min) TLC indicated that all the acid had been consumed to form corresponding mixed anhydride. To this mixture was added isopropylamine (515 μl, 5.93 mmol) followed by NMM (807 μl, 7.44 mmol). After 2 h the icesalt bath was removed and the mixture was stirred at ambient conditions for further 4 h. Removal of solvent resulted in a residue which was dissolved in ethyl acetate (EtOAc) (20 mL), washed with water (2 X 5 mL), 1N HCl (2 X 5 mL) and saturated NaHCO3 solution (2 X 5 mL). The organic layer was dried over anhydrous sodium sulphate (anhyd. Na2SO4) and concentrated to get a residue which was subjected to purification by silica gel flash column chromatography (EtOAc : Hexane - 1:5) to yield the desired product as a white solid (955 mg, 2.53 mmol, 85% yield ); m.p. - 100 °C; (TLC- EtOAc:Hexane (1:0) - Rf = 0.5); H NMR (400 MHz, CDCl3) δ ppm: 7.36 - 7.33 (m, 5H), 5.98 (bs, 1H), 5.24 (d, J = 5.8, 1H), 5.13 (s, 2H), 4.08 - 4.01 (m, 1H), 4.08 - 4.01 (m, 1H), 2.54 (quin, J = 7.3 Hz, 1H), 2.43 (quin, J = 7.0 Hz, 1H), 2.09 (m, 1H), 1.92 (m, 1H), 1.43 (s, 9H), 1.13 (d, J = 6.5 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ ppm: 173.2, 170.4, 155.7, 135.8, 128.6, 128.3, 128.26, 80.1, 66.6, 53.8, 41.5, 30.5, 28.3, 28.1, 22.65, 22.61; HRMS m/z Calcd for C20H30N2O5Na 401.2052, Found 401.2056.
(S)-4-tert-Butoxycarbonylamino-5-isobutoxycarbonyloxy-5-oxo-pentanoic acid benzyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With 4-methyl-morpholine In 1,2-dimethoxyethane at -13℃; for 0.25h;
With triethylamine In tetrahydrofuran at -15℃; for 0.25h;
With 4-methyl-morpholine In tetrahydrofuran at -23℃; for 0.25h;
With 4-methyl-morpholine In tetrahydrofuran at -10℃; for 0.0333333h;
With triethylamine In N,N-dimethyl-formamide at -10℃; for 0.25h;
With 4-methyl-morpholine In tetrahydrofuran at -20℃; for 0.05h;
With triethylamine
With triethylamine
With triethylamine In tetrahydrofuran at -23 - 0℃; for 1.58333h;
14.1; C
A stirred solution of (25)-5-(benzyloxy)-2-[(rer/-butoxycarbonyl)amino]-5- oxopentanoic acid (3.37 g, 10 mmol) and Et3N (1.7 mL, 12.2 mmol) in THF (8 mL) was cooled to -23°C. Then a solution of isobutyl chloroformate (1.4 mL, 10.8 mmol) in THF (2 mL) was slowly added over 5 min (following procedure in J Org. Chem., 1993, 55, 1586). The mixture was stirred at 0°C for 1.5 hour. The white precipitate of triethylammonium chloride was filtered off and washed with THF (8 mL), and the combined filtrates and the washings were slowly added over 10 min to a solution of sodium borohydride (764 mg, 20 mmol) in water (8 mL) at - 10°C. After the addition was complete, the reaction mixture was stirred at 0 °C for 1.5 h prior to acidification with 1N hydrochloric acid. The reaction mixture separated into two layers and the aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic extracts were washed with brine, dried over Na2SO4 and concentrated. The crude product was purified by column chromatography on silica gel eluting with a gradient of 40% EtOAc/hexanes -» 100% EtOAc/hexanes to give the title compound as a white solid
With 4-methyl-morpholine In N,N-dimethyl-formamide at -20℃; for 0.166667h;
With 4-methyl-morpholine In 1,2-dimethoxyethane Inert atmosphere;
With 4-methyl-morpholine In tetrahydrofuran at -10℃; for 0.333333h;
6 Example 6 [00264] Benzyl (S)-6-bromo-4-((tert-butoxycarbonyl)amino)-5-oxohexanoate (58-3):
Isobutyl chloroformate (5.84 mL, 45.0 mmol, 1.5 equiv.) was added dropwise to a solution of 0V)-5-(benzyloxy)-2-((tert-butoxycarbonyl)amino)-5-oxopentanoic acid (58-1) (10.12 g, 30.0 mmol, 1 equiv.) and A-methylmorpholine (5.28 mL, 48 mmol, 1.6 equiv.) in THF (100 mL) at - 10°C. After stirring at -10°C for 20 minutes, the reaction was filtered through celite and concentrated under reduced pressure to give the mixed anhydride as a colorless oil, which was used subsequently. [00265] Diazomethane preparation: A solution of A methyl-An-nitroso-p-toluenesulfon- amide (Diazald, 19.28 g, 90.0 mmol, 1 equiv.) in diethyl ether (66 mL) was added through an addition funnel to a mixture of potassium hydroxide (15.12 g, 270 mmol, 3 equiv.) in ethanol (30 mL) and water (26 mL) in an oil bath at 65 °C. The receiving flask to collect the ethereal solution of diazomethane was cooled in an ice-bath and the Diazald solution was added at such a rate as that allowed for a dropwise distillation into the receiving flask. When all of the Diazald solution had been added, additional diethyl ether (10 mL) was added through the addition funnel until the distillate was clear (no remaining diazomethane). After cooling to room temperature, the mixture in the distillation flask was quenched slowly with acetic acid until the yellow color disappeared. [00266] A solution of freshly prepared mixed anhydride above (12.70 g, 30.0 mmol, 1 equiv.) in diethyl ether (110 mL) was placed in a clear-seal joint flask and cooled to 0 °C in an ice bath. The freshly prepared diazomethane ethereal solution (-90.0 mmol, 3 equiv.) was added through an addition funnel dropwise while keeping it cold. The resulting mixture was stirred at 0 °C for 15 minutes, warmed to room temperature and stirred for 30 minutes. The reaction was cooled to 0 °C. Meanwhile a mixture of 48% aqueous HBr (24 mL, 210.0 mmol, 7 equiv.) and acetic acid (24.0 mL) was cooled to 0 °C and added to the above reaction mixture slowly at 0 °C. The mixture was stirred at 0 °C for 15 minutes, warmed to room temperature and stirred for 30 minutes. The mixture was diluted with diethyl ether (80 mL), washed with water (3 x 100 mL), dried over sodium sulfate, filtered and concentrated under reduced pressure. The residue was purified on an InterChim automated chromatography system (220 g SorbTech silica gel column), eluting with a gradient of 0 to 40% ethyl acetate in heptanes to give compound 58-3 (8.6 g, 69% yield) as a colorless oil.
With water In hexane at 40℃; for 24h; Yield given;
With 4-methyl-morpholine; (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate In methanol; acetonitrile
4.1
Manufacturing Example 4-1 (S)-2-tert-butoxycarbonylamino-pentanedioic acid 5-benzyl ester 1-ethyl ester To a mixture of (S)-2-tert-butoxycarbonylamino-pentanedioic acid 5-benzyl ester (300 mg, 0.89 mmol) and methanol (4 mL) were added N-methylmorpholine (98 μL, 0.89 mmol), benzotriazol-1-yloxytris-(dimethylamino)-phosphonium hexafluorophosphate (390 mg, 0.89 mmol), and acetonitrile (2 mL) at 0° C., which was stirred overnight at room temperature. A 1N sodium hydroxide aqueous solution was added to the reaction mixture, which was extracted with ethyl acetate. The organic layer was washed first with water and then with saturated brine, and dried over anhydrous magnesium sulfate. The solvent was evaporated under a reduced pressure, so as to obtain the titled compound (410 mg, purity of 63%) in a crude form. 1H-NMR Spectrum (CDCl3) δ (ppm): 1.27 (3H, t, J=7.1 Hz), 1.44 (9H, s), 1.91-2.01 (1H, m), 2.18-2.21 (1H, m), 2.39-2.52 (2H, m), 4.16-4.22 (2H, m), 4.32-4.33 (1H, m), 5.10-5.12 (3H, m), 7.31-7.39 (5H, m).
With benzotriazol-1-ol; dicyclohexyl-carbodiimide In dichloromethane at 0 - 20℃;
50.1 (Step 1) Synthesis ofBoc-Glu-OEt
1.01g (3.0 mmol) Boc-Glu (OBn)-OH, 1.01g (3.0 mmol), 620 mg (3.1 mmol) ofN,N-dicyclohexylamide (DCC) and 475 mg (3.1 mmol) of 1-hydroxybenzotriazole monohydrate (HOBt·H2O) were suspended in 12 ml of methylene chloride. The suspension was cooled to 0°C and 175 µl of ethyl alcohol was added thereto. After reverting to room temperature, stirring was continued overnight. The mixture was extracted with ethyl acetate/water. After the organic layer was washed with brine, sodium sulfate was added thereto and dried. The organic layer was concentrated under reduced pressure. The resulting residue was dissolved in 12 ml of methanol, and 100 mg of 10% Pd/C was added to the solution. The mixture was stirred in a hydrogen atmosphere overnight and the purification step A was applied to give the crude product of the title compound.ESI (m/z): 276 [M+H]+
8.3 g
With dmap; dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 4h;
1
The 7.8g type (6) the compound is dissolved in 100 ml dichloromethane solution, then adding 4.8g dicyclohexyl carbodiimide, 0.2g 4 - dimethylamino pyridine, 5 ml anhydrous ethanol, stirring at the room temperature reaction 4 hours, suction filtering to eliminate solvent to obtain 8.3g type (5) compound of formula.
With diazomethyl-trimethyl-silane; In hexane; dichloromethane; at 0 - 20℃; for 0.5h;
To a suspension of S1(400 mg, 1.19 mmol, 1.0 equiv.) in CH2Cl2 (15 mL) wasadded CH3OH (5 mL) and 2.04 M (trimethylsilyl)diazomethane in n-hexane (700 muL, 1.43 mmol, 1.2 equiv.)at 0 C. Afterstirring for room temperature for 30 min, the reaction mixture was concentratedin vacuo to obtain 473 mg (quant.) ofS2 as a yellow oil; 1HNMR (CDCl3, 500 MHz) 7.38-7.31(5H, m), 5.30 (2H, s), 5.11 (1H, s), 4.34 (1H, s), 3.73 (3H, s), 2.52-2.41 (2H, m), 2.21-2.19 (1H, m), 2.00-1.94 (1H, m), 1.43 (9H,s).
Manufacturing Example 2-1 (S)-2-tert-Butoxycarbonylamino-pentanedioic acid 5-benzyl ester 1-methyl ester To a mixture of (S)-2-tert-butoxycarbonylamino-pentanedioic acid 5-benzyl ester (300 mg, 0.89 mmol) and methanol (4 mL) were added benzotriazol-1-yloxytris-(dimethylamino)-phosphonium hexafluorophosphate (390 mg, 0.89 mmol), N-methylmorpholine (98 muL, 0.89 mmol), and acetonitrile (1 mL) at 0 C., which was stirred overnight at room temperature. A 1N sodium hydroxide aqueous solution was added to the reaction mixture, which was extracted with ethyl acetate. The organic layer was washed first with water and then with saturated brine, and dried over anhydrous magnesium sulfate. The solvent was evaporated under a reduced pressure, so as to obtain the titled compound (380 mg, purity of 66%) in a crude form. 1H-NMR Spectrum (CDCl3) delta (ppm): 1.43 (9H, s), 1.92-2.01 (1H, m), 2.18-2.23 (1H, m), 2.39-2.51 (2H, m), 3.73 (3H, s), 4.34-4.36 (1H, m), 5.10-5.12 (3H, m), 7.31-7.39 (5H, m).
Stage #1: Boc-Glu(OBzl)-OH With dicyclohexyl-carbodiimide In dichloromethane at 0℃; for 0.25h; Sonographic reaction;
Stage #2: aniline In dichloromethane at 0 - 20℃; for 0.833333h;
1
To a stirred solution of Boc-L-glutamic acid 5-benzyl ester [4a] (41 g, 122 mmol) in anhydrous CH2Cl2 (45 ml) at 0° C., was added during 15 minutes a solution of DCC (30.1 g, 146 mmol) in anhydrous CH2Cl2 (45 ml). The resulting white solid was sonicated. After that, anhydrous aniline was added dropwise to the reaction mixture over 10 minutes at 0° C. (11.1 ml, 122 mmol). The mixture was stirred at room temperature for 40 minutes and filtered through Celite to remove insoluble material. The resulting liquid was evaporated to dryness and chromatographically purified, yielding the desired product (47.2 g, 94%).1H-NMR (400 MHz, CDCl3): δ: 8.40 (br, 1H, CONHPh), 7.43 (d, 2H, J=7.7 Hz, 2Ha), 7.28 (d, 2H, J=7.7 Hz, Hb), 7.20 (m, 5H, 5×Hd), 7.02 (t, 1H, J=7.4 Hz, He), 5.35 (d, 1H, J=7.8 Hz, CHNHBoc), 5.04 (d, 2H, J=2.6 Hz, BnOCH2), 4.26 (sa, 1H, CH2CHNHBoc), 2.60-2.52 (mc, 1H, 1×OCOCH2CH2), 2.46-2.38 (mc, 1H, 1×OCOCH2CH2), 2.21-2.12 (mc, 1H, 1×OCOCH2CH2), 1.99-1.90 (mc, 1H, 1×OCOCH2CH2), 1.40 (s, 9H, NHCO2C(CH3)3) ppm.MS: Positive mode [M+Na]+=435.MS: Negative mode [M+2H2O-H]-=447.CAS nr: [126349-57-3]
94%
With dicyclohexyl-carbodiimide In dichloromethane at 0 - 20℃; for 0.833333h;
1
Benzyl 4-(tert-butoxycarbonylamino)-5-oxo-5-(phenylamino) pentanoate To a stirred solution of Boc-L-glutamic acid 5-benzyl ester [4a] (41 g, 122 mmol) in anhydrous CH2Cl2 (45 ml) at 0°C, was added during 15 minutes a solution of DCC (30.1 g, 146 mmol) in anhydrous CH2Cl2 (45 ml). The resulting white solid was sonicated. After that, anhydrous aniline was added dropwise to the reaction mixture over 10 minutes at 0°C (11.1 ml, 122 mmol). The mixture was stirred at room temperature for 40 minutes and filtered through Celite to remove insoluble material. The resulting liquid was evaporated to dryness and chromatographically purified, yielding the desired product (47.2 g, 94%). 1H-NMR (400 MHz, CDCl3): δ: 8.40 (br, 1H, CONHPh), 7.43 (d, 2H, J=7.7 Hz, 2Ha), 7.28 (d, 2H, J=7.7 Hz, Hb), 7.20 (m, 5H, 5xHd), 7.02 (t, 1H, J=7.4 Hz, Hc), 5.35 (d, 1H, J=7.8 Hz, CHNHBoc), 5.04 (d, 2H, J=2.6 Hz, BnOCH2), 4.26 (sa, 1H, CH2CHNHBoc), 2.60-2.52 (mc, 1H, 1xOCOCH2CH2), 2.46-2.38 (mc, 1H, 1x OCOCH2CH2), 2.21-2.12 (mc, 1H, 1xOCOCH2CH2), 1.99-1.90 (mc, 1H, 1x OCOCH2CH2), 1.40 (s, 9H, NHCO2C(CH3)3) ppm. MS: Positive mode [M + Na]+ = 435. MS: Negative mode [M + 2 H2O - H]- = 447. CAS nr: [126349-57-3]
Nα-t-butoxycarbonyl-N5-(N4-benzoyl-5'-deoxy-5'-cytidyl)-L-isoglutamine benzyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
90%
Stage #1: Boc-Glu(OBzl)-OH With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 0℃; for 0.00833333h;
Stage #2: 5'-amino-5'-deoxy-N4-benzoylcytidine In N,N-dimethyl-formamide at 20℃; for 1h;
With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide
Stage #1: Boc-Glu(OBzl)-OH With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 0℃; for 0.00833333h;
Stage #2: 5'-amino-5'-deoxyuridine In N,N-dimethyl-formamide at 20℃; for 1h;
With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide
Stage #1: Boc-Glu(OBzl)-OH With potassium carbonate In N,N-dimethyl-formamide at 5℃; for 0.166667h;
Stage #2: ethyl bromide In N,N-dimethyl-formamide at 5 - 20℃; for 4.5h;
1; 3 Synthesis of 2-(S)-tert-Butoxycarbonylamino-pentanedioic acid 5-benzyl ester 1 -ethyl ester (2)
K2CO3 (1.1 eq.) was added to a cooled solution of Boc-Glu(OBzl)-OH (1 eq.) in DMF (1.14 L) at 5 °C. The mixture was stirred for 10 min s before charging with ethyl bromide (1.5 eq.) and stirred for a further 30 mins at 5 °C. The reaction mixture was warmed to room temperature and stirred for a further 4 hrs. The mixture was then diluted with EtOAc (1.14 L), washed with water (2 x 1.14 L), brine (1.14 L), and concentrated under vacuum to give (2) (Yield ~ 97%) which was used without further purification.
91.2%
Stage #1: Boc-Glu(OBzl)-OH With caesium carbonate In methanol; water at 20℃; for 24h;
Stage #2: ethyl bromide In N,N-dimethyl-formamide at 20℃; for 24h;
1 General method for preparation of compounds 6a-c
General procedure: 2-(S)-tert-Butoxycarbonylamino-pentanedioic acid 5-benzylester 5 was dissolved in methanol with 10% water (10 mL). Thesolution was neutralized to pH 7 by drop-wise addition of aqueousCs2CO3 (20%). Following evaporation under high vacuum, theresulting syrup was dissolved in dry DMF (20 mL) and treated withthe appropriate alkyl bromide (2.88 molar equivalents). The reactionwas stirred at room temperature overnight. Ethyl acetate(20 mL) was added and the organic layer washed with water(3 20 mL), 10% K2CO3 (2 20 mL), and brine (20 mL), and thendried over magnesium sulfate. The resulting solution was concentratedunder reduced pressure to afford the product as a whitesolid.4.1.1. 2-(S)-tert-Butoxycarbonylaminopentanedioic acid 5-benzylester 1-propyl ester (6b)Following the procedure described in 4.1, the reaction of 2-tertbutoxycarbonylaminopentanedioicacid 5-benzyl ester 5 (1.00 g,2.96 mmol) with propyl bromide (1.05 mL, 8.52 mmol) gaveproduct 6b (1.01 g, 90%)
With sodium hydrogencarbonate In N,N-dimethyl-formamide at 20℃;
With potassium carbonate; In N,N-dimethyl-formamide; at 20℃; for 3h;
Step 1 Methyl iodide (1.01 mL, 16.3 mmol) was added dropwise to a solution of (2S)-5-(benzyloxy)-2-[(tert-butoxy)carbonyl]amino}-5-oxopentanoic acid XII (5.00 g, 14.82 mmol) and K2CO3 (2.25 g, 16.3 mmol) in DMF (25 mL) at r.t. The reaction mixture was stirred about 3 h at r.t. before adding additional methyl iodide (1.01 mL, 16.3 mmol). EtOAc was then added to the reaction and washed 3*10% Na2S2O3 and dried over MgSO4. The solvent was removed under reduced pressure and the crude product was purified on a silica gel column (100:1 and then 50:1 CHCl3/MeOH) to give 5-benzyl 1-methyl (2S)-2-[(tert-butoxy)carbonyl]amino}pentanedioate XIII (4.20 g, 12.23 mmol, 82% yield). ESIMS found for C18H25NO6 m/z 352 (M+H).
82%
With potassium carbonate; In N,N-dimethyl-formamide; at 20℃; for 3h;
Step 1[00181] Methyl iodide (1.01 mL, 16.3 mmol) was added dropwise to a solution of (2S)-5-(benzyloxy)-2-[(tert-butoxy)carbonyl]amino}-5-oxopentanoic acid XII (5.00 g, 14.82 mmol) and K2CO3 (2.25 g, 16.3 mmol) in DMF (25 mL) at room temperature The reaction mixture was stirred about 3 h at room temperature before adding additional methyl iodide (1.01 mL, 16.3 mmol). EtOAc was then added to the reaction and washed 3x 10% Na2S203 and dried over MgSC^. The solvent was removed under reduced pressure and the crude product was purified on a silica gel column (100: 1 and then 50: 1 CHCl3/MeOH) to give 5-benzyl 1-methyl (2S)-2-[(tert-butoxy)carbonyl]amino} pentanedioate XIII (4.20 g, 12.23 mmol, 82% yield). ESIMS found for Ci8H25N06 mlz 352 (M+H).
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane; N,N-dimethyl-formamide at 0 - 20℃;
1
To a stirred solution of (S)-2-tert-butoxycarbonylamino-pentanedioic acid 5-benzyl ester (5g, 14.8mmol) in a mixture of DCM (50ml) and DMF (3OmI) at O0C was added cyclopentanol (2.7ml, 29.6mmol), EDCI (4.25g, 22.2mmol) and DMAP (0.18g, 1.48mmol). Stirring was continued at r. t. overnight, after which time LCMS showed completion of reaction. DCM was removed under reduced pressure. The reaction mixture was diluted with EtOAc (200ml), washed with water (100ml), 1M aq HCl (50ml) followed by sat aq NaHCO3 (50ml). The EtOAc layer was dried (Na2SO4), filtered and concentrated in vacuo to give a viscous oil which solidified on standing overnight. Trituration with Et2O (2 x 10ml) afforded the title compound as a white solid (43.78g, 80%). LCMS purity 94%.
80%
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane; N,N-dimethyl-formamide at 0 - 20℃;
C
A mixture of 1-cyclopentyl 5-benzyl (2S)-2-[(tert-butoxycarbonyl)amino] pentanedioate (1.3g, 3.20mmol), and 10% Pd/ C (0.5g) in EtOH (150ml) was stirred under H2 (balloon) at RT for 4h, after which time LC showed completion of reaction. The reaction mixture was filtered through a pad of celite, washed with EtOH (20ml) and concentrated in vacuo to give a white solid. To remove residual EtOH the solid was dissolved in toluene/ THF mixture (5/1) (20ml) and concentrated in vacuo. Yield= 0.8g, 79%. 1H NMR (400 MHz, MeOD), δ: 1.35 (9H, s, t-Bu), 1.60-2.10 (1OH, m, 5xCH2), 4.05 (1 H, m, CH), 5.20 (1 H, m, CH).The benzyl ester used as starting material in the above process was prepared as followsTo a stirred solution of (2S)-5-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5- oxopentanoic acid (5g, 14.8mmol) in a mixture of DCM (50ml) and DMF (30ml) at 0 0C was added cyclopentanol (2.7ml, 29.6mmol), EDC (4.25g, 22.2mmol) and DMAP (0.18g, 1.48mmol). Stirring was continued at RT overnight, after which time LCMS showed completion of reaction. DCM was removed under reduced pressure. The reaction mixture was diluted with EtOAc (200ml), washed with water (100ml), 1 M aq HCI (50ml) followed by sat aq NaHCO3 (50ml). The EtOAc layer dried (Na2SO4), filtered and concentrated in vacuo to give a viscous oil which solidified on standing overnight. Trituration with Et2O (2 x 10ml) afforded the product as a white solid (43.78g, 80%). LC purity= 94%, m/z 406 [M+H]
71%
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane for 20h;
5.1; 7.1
To a solution of (2S)-5-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid (10 g, 30 mmol) in DCM (100 mL) was added cyclopentanol (30 mL, 33 mmol), EDC (6.25 g, 33 mmol) and DMAP (362 mg, 3 mmol). The reaction was allowed to stir for 20 hours for complete reaction. The reaction was diluted with DCM, washed with 1M HCl, sat NaHCO3, brine, dried (MgSO4) and concentrated in vacuo. The residue was purified by column chromatography (20% EtOAc/Heptane) to provide the title compound as a white solid (8.48 g, 71% yield). m/z 406 [M+H]+.
69%
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0 - 20℃; for 12h;
The (S)-2-tert-Butoxycarbonylamino-pentanedioic acid 5-benzyl ester 1 -cyclopentyl ester used as starting material in the above process was prepared as follows: To a solution of Boc-L-Glu(OBzl)-OH (1δg, 44.5mmol) in dichloromethane (220ml) in an ice-bath, was added cyclopentanol (4.8ml, 53.3mmol, 1.2eq), EDC (9.4g, 48.9mmol, 1.1eq) and DMAP (543mg, 4.4mmol, 0.1eq). The reaction mixture was allowed to warm to room temperature and stirred for 12 hours for complete reaction. The reaction mixture was diluted with DCM (200ml) and washed with 1M HCI, 1M Na2CO3 and brine. The organic layer was then dried over magnesium sulphate and evaporated under reduced pressure. The product was purified by column chromatography using ethyl acetate/heptane (1 :4) to give 12.4g, 69% yield of title compound as a white solid. 1H NMR (300 MHz, CDCI3), δ: 7.38 (5H, m), 5.70 (1H, m), 5.10 (2H, s), 5.05 (1H, m), 4.25 (1 H, m), 2.47 (2H, m), 2.15 (1H, m), 1.95-1.55 (9H, bm), 1.47 (9H, s).
69%
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0 - 20℃; for 12h;
3C.1
Stage 1; - 5-Benzyl 1 -cyclopentyl λ/-(teA?-butoxycarbonyl)-L-glutamate To a solution of (2S)-5-(benzyloxy)-2-[(teAt-butoxycarbonyl)amino]-5-oxopentanoic acid (15g, 44.5mmol) in DCM (22OmL) at O0C was added cyclopentanol (4.8mL, 53.3mmol), EDC (9.4g, 48.9mmol) and DMAP (543mg, 4.4mmol). The reaction mixture was allowed to warm to RT and stirred for a further 12 hours. The reaction mixture was diluted with DCM (20OmL) and washed with 1M HCI (5OmL), 1M Na2CO3 (3OmL) and brine (5OmL). The organic layer was dried (MgSO4) and concentrated under reduced pressure. The residue was purified by column chromatography (25% EtOAc/heptane) to give the product as a white solid (12.4g, 69% yield). ESMS: m/z 406 [M+H]+.
69%
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0 - 20℃; for 12h;
2.1
Cyclopentanol (4.8 ml_, 53.3 mmol), EDC (9.40 g, 48.9 mmol) and DMAP (543 mg, 4.4 mmol) were added to a cold (0 0C) solution of L-glutamic acid, λ/-[(1 ,1-dimethyl- ethoxy)carbonyl]-5-(phenylmethyl) ester (15 g, 44.5 mmol) in DCM (220 ml_). The reaction mixture was allowed to warm to room temperature, stirred for 12 hours, diluted with DCM (200 ml_), washed with 1 M HCI, 1 M Na2CO3 and brine, dried (MgSO4), filtered and concentrated under reduced pressure. Purification by column chromatography (20 % EtOAc in heptane) afforded the title compound as a white solid (12.4 g, 69 % yield). 1H NMR (300 MHz, CDCI3) 7.38 (5H1 m), 5.70 (1 H, m), 5.10 (2H, s), 5.05 (1 H, m), 4.25 (1 H, m), 2.47 (2H, m), 2.15 (1H, m), 1.95-1.55 (9H, m), 1.47 (9H, s).
69%
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0 - 20℃; for 12h;
1.1
Stage 1 : Cyclopentyl (S)-2-tert-Butoxycarbonylaminopentanedioic acid 5-benzyl ester 1 -cyclopentyl esterTo a solution of Boc-L-Glu(OBzl)-OH (15 g, 44.5 mmol) in dicHloromethane (220 ml) in an ice-bath, was added cyclopentanol (4.8 ml, 53.3 mmol, 1.2 eq), EDC (9.4 g, 48.9 mmol^l.l eq)-and DMAP (543 mg, 4.4 mmol, 0.1 eq). The reaction mixture-was-allowed - to warm to room temperature and stirred for 12 hours for complete reaction. The reaction mixture was diluted with DCM (200 ml) and washed with IM HCl, IM Na2CO3 and brine. The organic layer was then dried over magnesium sulphate and evaporated under reduced pressure. The product was purified by column chromatography using ethyl acetate/heptane (1 :4) to afford the title compound as a white solid (12.4 g, 69 %).1H NMR (300 MHz, CDCl3) δ: 7.38 (5H, m), 5.70 (IH, m), 5.10 (2H, s), 5.05 (IH, m), 4.25 (IH, m), 2.47 (2H, m), 2.15 (IH, m), 1.95-1.55 (9H, m), 1.47 (9H, s).
69%
With dmap; N-(3-dimethylaminopropyl)-N-ethylcarbodiimide In dichloromethane at 0 - 20℃; for 12h;
1.1
Intermediate 1Scheme 9Stage 1- 5-Benzyl 1 -cyclopentyl N-(tert-butoxycarbonyl)-L-glutamateTo a solution of (2S)-5-(benzyloxy)-2-[tert-butoxycarbonyl)amino]-5-oxopentanoic acid (15 g, 44.5 mmol) in DCM (220 mL) in an ice-bath, was added cyclopentanol (4.8 mL, 53.3 mmol, 1.2 eq), EDC (9.4 g, 48.9 mmol, 1.1 eq) and DMAP (543 mg, 4.4 mmol, 0.1 eq). The reaction mixture was allowed to warm to room temperature and stirred for 12 hours for complete reaction. The reaction mixture was diluted with DCM (200 mL) and washed with 1M HCI, 1 M Na2CO3 and brine. The organic layer was then dried (MgSO4) and evaporated under reduced pressure. The product was purified by column chromatography using ethyl acetate/heptane (1 :4) to afford the title compound as a white solid (12.4 g, 69% yield). 1H NMR (300 MHz, CDCI3) δ: 7.38 (5H1 m), 5.70 (1 H, m), 5.10 (2H, s), 5.05 (1H1 m), 4.25 (1H, m), 2.47 (2H, m), 2.15 (1 H1 m), 1.95-1.55 (9H, m), 1.47 (9H, s).
69%
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; Cooling with ice-bath;
Intermediate C-I - 5-Benzyl 1 -cyclopentyl N-(tert-butoxycarbonyl)-L-glutamate was prepared as follows:To a solution of (2S)-5-(benzyloxy)-2-[tert-butoxycarbonyl)amino]-5- oxopentanoic acid (15 g, 44.5 mmol) in DCM (220 mL) in an ice-bath, was added cyclopentanol (4.8 mL, 53.3 mmol), EDCI (9.4 g, 48.9 mmol) and DMAP (543 mg, 4.4 mmol). The reaction mixture was allowed to warm to RT and stirred for 12 hours for complete reaction. The reaction mixture was diluted with DCM (200 mL) and washed with IM HCl, IM Na2CO3 and brine. The organic layer was then dried (MgSO4) and evaporated under reduced pressure. The product was purified by column chromatography (EtOAc / heptane 1 :4) to afford the title compound as a white solid (12.4 g, 69 %). 1H NMR (300 MHz, CDCl3) δ: 7.38 (5H, m), 5.70 (IH, m), 5.10 (2H, s), 5.05 (IH, m), 4.25 (IH, m), 2.47 (2H, m), 2.15 (IH, m), 1.95-1.55 (9H, m), 1.47 (9H, s).
69%
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0 - 20℃; for 12h;
10.1
To a solution of Boc-L-Glu(OBzl)-OH (15g, 44.5mmol) in dichloromethane (220ml) in an ice- bath, was added cyclopentanol (4.8ml, 53.3mmol), EDC (9.4g, 48.9mmol) and DMAP (543mg, 4.4mmol). The reaction mixture was allowed to warm to room temperature and stirred for 12 hours for complete reaction. The reaction mixture was diluted with DCM (200ml) and washed with 1 M HCI, 1 M Na2CO3 and brine. The organic layer was then dried over magnesium sulphate and evaporated under reduced pressure. The product was purified by column chromatography using ethyl acetate/heptane (1 :4) to give 12.4g, 69% yield of title compound as a white solid. 1H NMR (300 MHz, CDCI3), δ: 7.38 (5H, m), 5.70 (1 H, m), 5.10 (2H, s), 5.05 (1 H, m), 4.25 (1 H, m), 2.47 (2H, m), 2.15 (1 H1 m), 1.95-1.55 (9H, bm), 1.47 (9H, s).
69%
With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0 - 20℃; for 12h;
29.1
To a solution of Boc-L-Glu(OBzl)-OH (15 g, 44.5 mmol) in dichloromethane (220 ml) in an ice-bath, was added cyclopentanol (4.8 ml, 53.3 mmol, 1.2 eq), EDC (9.4 g, 48.9 mmol, 1.1 eq) and DMAP (543 mg, 4.4 mmol, 0.1 eq). The reaction mixture was allowed to warm to room temperature and stirred for 12 hours for complete reaction. The reaction mixture was diluted with DCM (200 ml) and washed with 1M HCI, 1M Na2CO3 and brine. The organic layer was then dried over magnesium sulphate and evaporated under reduced pressure. The product was purified by column chromatography using ethyl acetate/heptane (1 :4) to afford the title compound as a white solid (12.4 g, 69 %).1H NMR (300 MHz, CDCI3) δ: 7.38 (5H, m), 5.70 (1 H, m), 5.10 (2H1 s), 5.05 (1 H, m), 4.25 (1 H1 m), 2.47 (2H, m), 2.15 (1H, m), 1.95-1.55 (9H, m), 1.47 (9H, s).
With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline In tetrahydrofuran at 0 - 20℃; for 16h;
9.a
24.7 g (100 mmol) of EEDQ (2-ethoxy-1,2-dihydroquinoline-1-carboxylic acid ethyl ester) is added at 0° C. to 16.87 g (50 mmol) of N-tert-butyloxycarbonyl-L-glutaminic acid-5-benyl ester (Bachem) and 23.86 g (50 mmol) of the title compound of Example 1a in 200 ml of THF, and it is stirred for 16 hours at room temperature. It is evaporated to the dry state in a vacuum, and the residue is chromatographed on silica gel (mobile solvent: dichloromethane/methanol 20:1). Yield: 35.1 g (88% of theory) of a colorless, viscous oil. Elementary Analysis: Cld.: C 42.22 H 3.67 N 3.52 F 40.55 Fnd.: C 42.39 H 3.65 N 3.55 F 40.38
With N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline In tetrahydrofuran at 20℃; for 16h;
16.a
9.88 g (40 mmol) of EEDQ (ethyl 2-ethoxy-1,2-dihydroquinoline-1-carboxylate) are added at 0° C. to 6.75 g (20 mmol) of N-tert-butyloxycarbonyl-L-glutamic acid 5-benzyl ester (Bachem) and 9.10 g (20 mmol) of the title compound from Example 3c in 200 ml of THF and the mixture is stirred at room temperature for 16 h. It is evaporated to dryness in vacuo and the residue is chromatographed on silica gel (eluent: dichloromethane/methanol 20:1). Yield: 13.2 g (85% of theory) of a colourless viscous oil.
Fmoc-Asp-Cys(Acm)-His-Leu-Gln-Ala-Val-Val-Leu-His-Leu-Ala-Arg-Arg-Ser-Val-Cys(Acm)-Val-His-Pro-Gln-Asn-Arg-Ser-Leu-Ala-Arg-Trp-Leu-Glu-Arg-Gln-Gly-S-CH2CH2CO-Leu-NH2[ No CAS ]
12-(2'-(N-tBOC-glutamic-γ-benzylester)-α-amide)deoxoartemisinin[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
84%
Stage #1: Boc-Glu(OBzl)-OH With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; for 0.5h;
Stage #2: 12-(2'-aminoethyl)deoxoartemisinin In dichloromethane at 20℃; for 3h;
2; 8.1 Example 8: Synthesis of 12-[2'-(N-glutamic)-α,β-amide]deoxoartemisinin dimer (IVd).; (1) Synthesis of 12-[2'-(N-tBOC-glutamic-γ-benzylester)-α-amide]deoxoartemisinin (VIII).; Scheme 2
N-tBOC-L-glutamic acid-γ-benzylester (35mg, O.11mmol) was dissolved in dry CH2Cl2 (5mL) and HOBt (52mg, 0.342mmol) and EDC (63mg, 0. 342MMOL) was added. The reaction mixture was allowed to stir at room temperature for 30 minutes and 12- (2'-aminoethyl) deoxoartemisinin (VI) (42mg, 0. 135mmol) was added. The resulting reaction mixture was allowed to stir at room temperature for 3hours. The reaction mixture was extracted with ethyl acetate (20ML x 3) and washed with brine (10mL x 2). The extract was dried over MGS04, concentrated in vacuo, then purified by silica gel column (hexane/ethyl acetate = 1/2 as eluent) to afford 12-[2'-(N-tBOC-glutamic acid-γ-benzylester)-α-amide]deoxoartemisinin(VI) (71.5 mg, 84%) as a colorless oil.; [α] 25D= +73. 6 (c 0.47, CHC13).; 1H-NMR (CDC13, 250MHz) δ 7. 36 (s, 5H, aromatic H), 7.02 (br, 1H, NH), 5.33 (s, 1H, H-5), 5.11 (s, 2H, benzyl), 4.40-4. 39 (m, 1H, H-12), 4.29-4. 27 (m, 1H), 4.15-4. 13 (m, 1H), 3.58-3. 56 (m, 1H, H-2), 3.28-3. 24 (m, 1H, H-2), 2.61-2. 43 (m, 3H), 2.36-2. 14 (m, 3H), 2.12-1. 98 (m, 4H), 1.91-1. 65 (m, 4H), 1.44 (s, 9H, t-BOC), 1.42 (s, 3H, CH3-15), 1.35-1. 26 (m, 3H), 0.96 (d, 3H, J=5.6Hz, CH3-13), 0. 85 (d, 3H, J=7.6Hz, CH3-14), 0.82 (m, 1H).; 13C-NMR (CDC13, 63MHz) δ 173. 2,171. 5,155. 2,136. 1, 128. 8, 128. 8, 128. 5, 128. 5, 128. 4, 128. 4, 103. 8, 89.9, 81. 3,74. 4,66. 6,61. 2,53. 1,52. 3, 44. 0,39. 4, 37. 7,36. 7,34. 6, 30. 8, 28. 6, 28. 5, 28. 5,26. 2,25. 1,25. 0,20. 4, 12. 6.; IR (neat) umax 3364 (NH), 2932, 2876, 1736 (C=O), 1663 (C=O), 1538,1453, 1393,1249, 1163,1051, 880 (O-O), 702, 610 cm-1; HRMS (FAB) M/Z 631.3507 ([M+H]+, obsd), 630.3516(calcd for C34H50N2O9).
(2S)-pyrrolidine-2-carbonitrile hydrochloride[ No CAS ]
[ 1056640-52-8 ]
Yield
Reaction Conditions
Operation in experiment
80%
Stage #1: Boc-Glu(OBzl)-OH With 1-hydroxy-pyrrolidine-2,5-dione; dicyclohexyl-carbodiimide In 1,4-dioxane; dichloromethane at 0 - 20℃; for 1h;
Stage #2: (2S)-pyrrolidine-2-carbonitrile hydrochloride With triethylamine at 20℃; for 4h;
1.1
t-Butoxycarbonyl-L-glutamic acid 5-benzyl ester (A) (0.65 g, 2 mmol) and N- hydroxysuccinimide (HOSu, 0.23 g, 2 mmol) were dissolved in 6 ml of CH2CI2/4-dioxane (2/1). The solution was cooled in an ice-water bath and dicyclohexylcarbodimide (DCC, 0.45 g, 2.2 mmol) was added with stirring. The reaction mixture was stirred at room temperature for I hour and then pyrrolidine-2-carbonitrile hydrochloride (B) (0.27 g, 2 mmol) and triethylamine (Et3N, 0.22 g, 2.2 mmol) were added. After 4 hours at room temperature, DCC was removed by filtration and washed by CH2Cl2. The filtrate and washings were combined and washed with 10% aqueous citric acid and then saturated aqueous NaHC03, dried over MgS04, and concentrated in vacuo. Further purification by flash column chromatography (eluted with 5/4/1 hexane/CH2Cl2/EA) afforded compound (C) (80%) as a foam. (2) Preparation ofN-t-butoxycarbonyl-1- [2-amino-4- (3, 4-dihydro-1H-isoquinolin-2-yl)- 4-oxo-butyryl]-pyrrolidine-2-carbonitrile Compound (C) (0.40 g, 1 mmol) and 5% Pd/C (20 mg) in ethyl acetate (6 mL) and methanol (250 ; j. L) were stirred under H2 atmosphere for 7 hours. The reaction mixture was filtered and then concentrated in vacuo to give compound (D) as a white solid, which was used without further purification. A solution of compound (D) and HOSu (0.12 g, 1 mmol) in 3 ml of CH2CI2/1, 4-dioxane (2/1) was cooled in an ice-water bath. To this solution, DCC (0.23 g, 1.1 mmol) was added with stirring. After 1 hour, 1,2, 3,4-tetrahydro-isoquinoline (0.20 g, 1.5 mmol) was added. The reaction mixture was stirred for 4 hours at room temperature. Then, DCC was removed by filtration and washed by CH2CI2. The filtrate and washings were combined, washed with 10% aqueous citric acid and then saturated aqueous NaHC03, dried over MgS04, and concentrated in vacuo. Further purification by flash column chromatography (eluted with 3/6/1 hexane/CH2CI2/EA) afforded N-t-butoxycarbonyl-1- [2-amino-4- (3, 4-dihydro- lH-isoquinolin-2-yl)-4-oxo-butyryl]-pyrrolidine-2-carbonitrile (85%) as a foam. (3) Preparation of 1- [2-amino-4- (3, 4-dihydro-lH-isoquinolin-2-yl)-4-oxo-butyryl]- pyrrolidine-2-carbonitrile, trifluoroacetic acid N-t-butoxycarbonyl-l- [2-amino-4- (3, 4-dihydro-1 H-isoquinolin-2-yl)-4-oxo-butyryl]- pyrrolidine-2-carbonitrile (0.43 g, 1 mmol) was dissolved in cool TFA (2 mL). The resulting solution was stirred at room temperature for 10 minutes and then concentrated to provide Compound 1 as a pale yellow taffy. MS (ES+) m/z : 327.1 (M+H) +, 349.1 (M+Na) +.
With dicyclohexyl-carbodiimide In dichloromethane at 0 - 20℃; for 0.833333h;
1
To a stirred solution of Boc-Z-glutamic acid 5 -benzyl ester [4a] (41 g, 122 mmol) in anhydrous CH2Cl2 (45 ml) at O0C, was added during 15 minutes a solution of DCC (30.1 g, 146 mmol) in anhydrous CH2Cl2 (45 ml). The resulting white solid was sonicated. After that, anhydrous aniline was added dropwise to the reaction mixture over 10 minutes at 00C (11.1 ml, 122 mmol). The mixture was stirred at room temperature for 40 minutes and filtered through Celite to remove insoluble material. The resulting liquid was evaporated to dryness and chromatographically purified, yielding the desired product (47.2 g, 94%).1H-NMR (400 MHz, CDCl3): δ: 8.40 (br, IH, CONHPh), 7.43 (d, 2H, J=7.7 Hz, 2Ha), 7.28 (d, 2H, J=7.7 Hz, Hb), 7.20 (m, 5H, 5xHd), 7.02 (t, IH, J=7.4 Hz, Hc), 5.35 (d, IH, J=7.8 Hz, CHNHBoc), 5.04 (d, 2H, J=2.6 Hz, BnOCH2), 4.26 (sa, IH, CH2CHNHBoc), 2.60-2.52 (me, IH, IxOCOCH2CH2), 2.46-2.38 (me, IH, Ix OCOCH2CH2), 2.21-2.12 (me, IH, IxOCOCH2CH2), 1.99-1.90 (me, IH, Ix OCOCH2CH2), 1.40 (s, 9H, MS: Positive mode [M + Na]+ = 435.MS: Negative mode [M + 2 H2O - H]- = 447
94%
Stage #1: Boc-Glu(OBzl)-OH With dicyclohexyl-carbodiimide In dichloromethane at 0℃; for 0.25h;
Stage #2: aniline In dichloromethane at 0 - 20℃; for 0.833333h;
1
Example 1 Preparation of intermediate [6a]: Benzyl 4-(tert-butoxycarbonylamino)-5-oxo-5-(phenylamino) pentanoate To a stirred solution of Boc-L-glutamic acid 5-benzyl ester [4a] (41 g, 122 mmol) in anhydrous CH2Cl2 (45 ml) at 0°C, was added during 15 minutes a solution of DCC (30.1 g, 146 mmol) in anhydrous CH2Cl2 (45 ml). The resulting white solid was sonicated. After that, anhydrous aniline was added dropwise to the reaction mixture over 10 minutes at 0°C (11.1 ml, 122 mmol). The mixture was stirred at room temperature for 40 minutes and filtered through Celite to remove insoluble material. The resulting liquid was evaporated to dryness and chromatographically purified, yielding the desired product (47.2 g, 94%). 1H-NMR (400 MHz, CDCl3): δ: 8.40 (br, 1H, CONHPh), 7.43 (d, 2H, J=7.7 Hz, 2Ha), 7.28 (d, 2H, J=7.7 Hz, Hb), 7.20 (m, 5H, 5xHd), 7.02 (t, 1H, J=7.4 Hz, Hc), 5.35 (d, 1H, J=7.8 Hz, CHNHBoc), 5.04 (d, 2H, J=2.6 Hz, BnOCH2), 4.26 (sa, 1H, CH2CHNHBoc), 2.60-2.52 (mc, 1H, 1xOCOCH2CH2), 2.46-2.38 (mc, 1H, 1x OCOCH2CH2), 2.21-2.12 (mc, 1H, 1xOCOCH2CH2), 1.99-1.90 (mc, 1H, 1x OCOCH2CH2), 1.40 (s, 9H, NHCO2C(CH3)3) ppm. MS: Positive mode [M + Na]+ = 435. MS: Negative mode [M + 2 H2O - H]- = 447. CAS nr: [126349-57-3]
With diisopropyl-carbodiimide; In dichloromethane; at 0 - 20℃; for 4h;
General procedure: To an ice cooled stirred solution of 8-quinolinamine (1 or 3, 0.37 mmol) and orthogonally protected amino acid (0.41 mmol) in anhydrous CH2Cl2 (10 mL), DIC (0.41 mmol) was added. The reaction mixture was allowed to attain room temperature and stirring continued for another 4 h. The solvent was removed yielding the crude product. Purified by column chromatography on silica gel (100-200 mesh) using 0.7-1.2% CH3OH in CH2Cl2 to afford 45-49 as oil.
With diisopropyl-carbodiimide In dichloromethane at 0 - 20℃; for 4h;
General method for the synthesis of benzyl N2-(tert-butoxycarbonyl)-N-{4-[(6-methoxy-2-subs-tituted-quinolin-8-yl)amino]pentyl}-L-asparaginate/glutaminate (45-48) and tert-butyl N2-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-{4-[(6-methoxyquinolin-8-yl)amino]pentyl}-L-glutaminate (49) (Scheme 2)
General procedure: To an ice cooled stirred solution of 8-quinolinamine (1 or 3, 0.37 mmol) and orthogonally protected amino acid (0.41 mmol) in anhydrous CH2Cl2 (10 mL), DIC (0.41 mmol) was added. The reaction mixture was allowed to attain room temperature and stirring continued for another 4 h. The solvent was removed yielding the crude product. Purified by column chromatography on silica gel (100-200 mesh) using 0.7-1.2% CH3OH in CH2Cl2 to afford 45-49 as oil.
With 4-methyl-morpholine; benzotriazol-1-ol; dicyclohexyl-carbodiimide In tetrahydrofuran at 20℃; Cooling;
6.2.4. N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-caboline-3-carbonyl]-N'-(Boc-glycyl)-hydrazine (4a)
General procedure: At 0 °C to the solution of 394 mg (2.25 mmmol) of Boc-Gly, 304 mg (2.25 mmol) of HOBt, 507 mg (2.46 mmol) of DCC and 10 ml of anhydrous THF the solution of 500 mg (2.05 mmol) of N-[(1S,3S)-1-methyl-1,2,3,4-tetrahydro-β-carboline-3-carbonyl]hydrazine (3) and 10 ml of anhydrous THF was added. The reaction solution was adjusted to pH 9.5 with 0.6 ml of N-methylmorpholine, stirred at 0 °C for 1 h and at room temperature for 12 h and TLC (chloroform/methanol, 15:1) indicated the complete disappearance of 3. The reaction mixture was filtered to remove the resultant precipitates. The filtrate was evaporated under reduced pressure to remove the solvents. The residue was dissolved in 40 mL of ethyl acetate and washed successively with 5% sodium bicarbonate, 5% citric acid, and saturated sodium chloride, and the organic phase was separated and dried over anhydrous sodium sulfate. After filtration and evaporation under reduced pressure the residue was purified on a chromatographic column (CH2Cl2/CH3OH, 20:1) to provide 576 mg (70%) of the title compound as yellowing powders.
Stage #1: Boc-Glu(OBzl)-OH With benzotriazole-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 0.333333h;
Stage #2: 4-cyanobenzylamine hydrochloride In N,N-dimethyl-formamide at 20℃; for 1h;
7.15
Preparation 15Synthesis of(S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acid benzyl ester (Q). (Also: (S)-benzyl 4-(tert-butoxycarbonylamino)-5-(4-isocyanobenzylamino)-5- oxopentanoate)A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72g, 22.0 mmol) and DIEA (12.18 mL, 70.0 mmol) in DMF (50 mL) was maintained at room temperature for 20 min, followed by the addition of 4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). The reaction mixture was stirred at room temperature for an additional 1 h and diluted with EtOAc (500 mL). The obtained solution was extracted with water (100 mL), 5% aq. NaHC03 (100 mL) and water (2 x 100 mL). The organic layer was dried over MgSC^ evaporated and dried in vacuo to provide compound Q (9.65 g, yield exceeded quantitative) as yellowish oil. LC-MS [M+H] 452.0 (CisftgNsOs +H, calc: 452.4). Compound Q was used without further purification.
100%
Stage #1: Boc-Glu(OBzl)-OH With benzotriazole-1-yl-oxy-tris-(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 0.333333h;
Stage #2: 4-cyanobenzylamine hydrochloride In N,N-dimethyl-formamide at 20℃; for 1h;
29
Synthesis of (S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acid benzyl ester (Q).A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72g, 22.0 mmol) and DIEA (12.18 mL, 70.0 mmol) in DMF (50 mL) was maintained at room temperature for 20 min, followed by the addition of 4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). The reaction mixture was stirred at room temperature for an additional 1 h and diluted with EtOAc (500 mL). The obtained solution was extracted with water (100 mL), 5% aq. NaHC03 (100 mL) and water (2 x 100 mL), The organic layer was dried over MgS04> evaporated and dried in vacuo to provide compound Q (9.65 g, yield exceeded quantitative) as yellowish oil. LC-MS [M+H] 452.0 (C25H29N3O5 +H, calc: 452.4). Compound Q was used without further purification.
100%
With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 1.33333h;
7.15
Synthesis of (S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2- (phenylmethylsulfonamido)butanamido)pentanoic acid (Compound 108)108 Preparation 15: Synthesis of (S )-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)- butyric acid benzyl ester (Q).A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72g, 22.0 mmol) and DIEA (12.18 ml, 70.0 mmol) in DMF (50 ml) was maintained at room temperature for 20 min, followed by the addition of 4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). The reaction mixture was stirred at room temperature for an additional 1 h and diluted with EtOAc (500 ml). The obtained solution was extracted with water (100 ml), 5% aq. NaHC03 (100 ml) and water (2 x 100 ml). The organic layer was dried over MgS04; evaporated and dried in vacuo to provide compound Q (9.65 g, yield exceeded quantitative) as yellowish oil. LC-MS[M+H] 452.0 (C25H29N305 +H, calc: 452.4). Compound Q was used without further purification.
100%
Stage #1: Boc-Glu(OBzl)-OH With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 0.333333h;
Stage #2: 4-cyanobenzylamine hydrochloride In N,N-dimethyl-formamide at 20℃; for 1h;
7.15
Preparation 15Synthesis of(S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acid benzyl ester (Q).A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72g, 22.0 mmol) andDIEA (12.18 ml, 70.0 mmol) in DMF (50 ml) was maintained at room temperature for 20 min, followed by the addition of 4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). The reaction mixture was stirred at room temperature for an additional 1 h and diluted with EtOAc (500 ml). The obtained solution was extracted with water (100 ml), 5% aq. NaHC03 (100 ml) and water (2 x 100 ml). The organic layer was dried over MgS04; evaporated and dried in vacuo to provide compound Q (9.65 g, yield exceeded quantitative) as yellowish oil. LC-MS [M+H] 452.0 (C25H29N305 +H, calc: 452.4). Compound Q was used without further purification.Preparation 16
96.2%
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; isobutyl chloroformate In N,N-dimethyl-formamide at -20 - -15℃; for 0.166667h;
Stage #2: 4-cyanobenzylamine hydrochloride With 4-methyl-morpholine at -20 - 20℃;
Stage #1: Boc-Glu(OBzl)-OH With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 0.333333h;
Stage #2: 4-cyanobenzylamine hydrochloride In N,N-dimethyl-formamide at 20℃; for 1h;
7.15
Preparation 15 Synthesis of (S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acid benzyl ester (Q) A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72 g, 22.0 mmol) and DIEA (12.18 ml, 70.0 mmol) in DMF (50 ml) was maintained at room temperature for 20 min, followed by the addition of 4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). The reaction mixture was stirred at room temperature for an additional 1 h and diluted with EtOAc (500 ml). The obtained solution was extracted with water (100 ml), 5% aq. NaHCO3 (100 ml) and water (2*100 ml). The organic layer was dried over MgSO4, evaporated and dried in vacuo to provide compound Q (9.65 g, yield exceeded quantitative) as yellowish oil. LC-MS [M+H] 452.0 (C25H29N3O5+H, calc: 452.4).
9.65 g
Stage #1: Boc-Glu(OBzl)-OH With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 0.333333h;
Stage #2: 4-cyanobenzylamine hydrochloride at 20℃; for 1h;
19 Synthesis of (S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acid benzyl ester (HH)
A solution of Boc-Glu(OBz1)-OH (7.08 g, 21.0 mmol), BOP (9.72 g, 22.0 mmol) and DIEA (12.18 ml, 70.0 mmol) in DMF (50 ml) was maintained at room temperature for 20 min, followed by the addition of 4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). The reaction mixture was stirred at room temperature for an additional 1 h and diluted with EtOAc (500 ml). The obtained solution was extracted with water (100 ml), 5% aq. NaHCO3 (100 ml) and water (2×100 ml). The organic layer was dried over MgSO4, evaporated and dried in vacuo to provide compound HH (9.65 g, yield exceeded quantitative) as yellowish oil. LC-MS [M+H] 452.0 (C25H29N3O5+H, calc: 452.4). Compound HH was used without further purification.
9.65 g
Stage #1: Boc-Glu(OBzl)-OH With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 0.333333h;
Stage #2: 4-cyanobenzylamine hydrochloride In N,N-dimethyl-formamide at 20℃; for 1h;
7.15 Synthesis of (S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acid benzyl ester (Q)
A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72g, 22.0 mmol) and DIEA (12.18 mL, 70.0 mmol) in DMF (50 mL) was maintained at room temperature for 20 min, followed by the addition of 4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). The reaction mixture was stirred at room temperature for an additional 1 h and diluted with EtOAc (500 mL). The obtained solution was extracted with water (100 mL), 5% aq. NaHCO3 (100 mL) and water (2 x 100 mL). The organic layer was dried over MgSO4, evaporated and dried in vacuo to provide compound Q (9.65 g, yield exceeded quantitative) as yellowish oil. LC-MS [M+H] 452.0 (C25H29N3O5 +H, calc: 452.4). Compound Q was used without further purification.
4-(aminomethyl)benzonitrile hydrochloride[ No CAS ]
[ 13574-13-5 ]
[ 1221593-80-1 ]
Yield
Reaction Conditions
Operation in experiment
100%
Stage #1: Boc-Glu(OBzl)-OH With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 0.333333h;
Stage #2: 4-(aminomethyl)benzonitrile hydrochloride In N,N-dimethyl-formamide at 20℃; for 1h;
29
Example 29Synthesis of (S)-5-(4-carbamimidoylbenzylamino)-5-oxo-4-((R)-4-phenyl-2- (phenylmethylsulfonamido)butanamido)pentanoic acid (Compound 108)108 Atty Dkt: PFOR-0801WOPreparation 30Synthesis of (S)-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)-butyric acid benzyl ester (Q).A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72g, 22.0 mmol) and DIEA (12.18 mL, 70.0 mmol) in DMF (50 mL) was maintained at room temperature for 20 min, followed by the addition of 4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). The reaction mixture was stirred at room temperature for an additional 1 h and diluted with EtOAc (500 mL). The obtained solution was extracted with water (100 mL), 5% aq. NaHCC>3 (100 mL) and water (2 x 100 mL). The organic layer was dried over MgSC^, evaporated and dried in vacuo to provide compound Q (9.65 g, yield exceeded quantitative) as yellowish oil. LC-MS [M+H] 452.0 (C25H29N3O5 +H, calc: 452.4). Compound Q was used without further purification.
4-(aminomethyl)benzonitrile hydrochloride[ No CAS ]
[ 13574-13-5 ]
[ 1221593-80-1 ]
Yield
Reaction Conditions
Operation in experiment
100%
Stage #1: Boc-Glu(OBzl)-OH With (benzotriazo-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 0.333333h;
Stage #2: 4-(aminomethyl)benzonitrile hydrochloride In ethyl acetate; N,N-dimethyl-formamide at 20℃; for 1h;
7.15
Preparation 15: Synthesis of (S )-4-tert-butoxycarbonylamino-4-(4-cyano-benzylcarbamoyl)- butyric acid benzyl ester (Q).A solution of Boc-Glu(OBzl)-OH (7.08 g, 21.0 mmol), BOP (9.72g, 22.0 mmol) and DIEA (12.18 mL, 70.0 mmol) in DMF (50 mL) was maintained at room temperature for 20 min, followed by the addition of 4-(aminomethyl)benzonitrile hydrochloride (3.38 g, 20.0 mmol). The reaction mixture was stirred at room temperature for an additional 1 h and diluted with EtOAc (500 mL). The obtained solution was extracted with water (100 mL), 5% aq. NaHC03 (100 mL) and water (2 x 100 mL). The organic layer was dried over MgS04; evaporated and dried in vacuo to provide compound Q (9.65 g, yield exceeded quantitative) as yellowish oil. LC-MS [M+H] 452.0 (C25H29N305 +H, calc: 452.4). Compound Q was used without further purification.
1-methyl-β-carboline-3-carboxylic acid hydrazide[ No CAS ]
[ 1346115-07-8 ]
Yield
Reaction Conditions
Operation in experiment
30%
With 4-methyl-morpholine; benzotriazol-1-ol; dicyclohexyl-carbodiimide In tetrahydrofuran at 0 - 20℃; for 13h;
4.2.5. N-(1-methyl-β-caboline-3-carbonyl)-N'-(Boc-glycyl)-hydrazine (5a)
General procedure: At 0 °C to the solution of 400 mg (2.29 mmmol) of Boc-Gly, 304 mg (2.25 mmol) of HOBt, 507 mg (2.46 mmol) of DCC and 10 ml of anhydrous THF the solution of 500 mg (2.08 mmol) of 1-methyl-β-carboline-3-carbonylhydrazine (4) and 10 ml of anhydrous THF was added. The reaction solution was adjusted to pH 9.5 with 0.6 ml of N-methylmorpholine, stirred at 0 °C for 1 h and at room temperature for 12 h and TLC (CHCl3/CH3OH,15:1) indicated the complete disappearance of 4. The reaction mixture was filtered to remove the resultant precipitates. The filtrate was evaporated under reduced pressure to remove the solvents. The residue was dissolved in 40 ml of ethyl acetate and washed successively with 5% sodium bicarbonate, 5% citric acid, and saturated sodium chloride, and the organic phase was separated and dried over anhydrous sodium sulfate. After filtration and evaporation under reduced pressure the residue was purified on a chromatographic column (CH2Cl2/CH3OH, 20:1) to provide 419 mg (50%) of the title compound as yellowing powder.
With dmap; dicyclohexyl-carbodiimide In benzene at 20℃; for 12h; Inert atmosphere;
2.2.8. 20-Oxo-5β-[9,12,12-2H3]pregnan-3α-yl (2S)-5-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5-oxo-pentanoate (9)
General procedure: Compound 8 (245 mg, 0.8 mmol) and Boc-Glu(OBzl)-OH (257 mg, 0.8 mmol) were dissolved in dry benzene (8.5 mL). Then, 4-dimethylaminopyridine (10 mg, 0.1 mmol) and dicyclohexylcarbodiimide (1 M in benzene, 0.84 mL, 0.84 mmol) were added under inert atmosphere and the reaction mixture was stirred at room temperature. After 12 h, the reaction mixture was poured into saturated solution of sodium bicarbonate (50 mL) and the product was extracted with ethyl acetate (3 × 15 mL). Combined organic extracts were washed with water (2 × 15 mL). Precipitated N,N'-dicyclohexylurea was filtered off, filtrate was dried over anhydrous sodium sulfate, and the solvent was removed in vacuo. Chromatography on silica gel (15 g, 10% ether in petroleum ether) gave oily conjugate 9 (456 mg, 93%):
Stage #1: Boc-Glu(OBzl)-OH With 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane-2,4,6-trioxide; 1,8-diazabicyclo[5.4.0]undec-7-ene In ethyl acetate; acetonitrile at 0℃; for 0.166667h;
Stage #2: N,O-dimethylhydroxylamine*hydrochloride With triethylamine In ethyl acetate; acetonitrile at 0℃; for 0.5h;
General procedure for the synthesis of N-Fmoc/Boc/Cbz-α-amino/peptidyl Weinreb amides 2 and 4
General procedure: To a solution of N-protected amino/peptide acid (1.0 mmol) in CH3CN (2.0 mL), DBU (1.1 mmol, 0.16 mL), and T3P (50% in EtOAc, 1.02 mL, 3.2 mmol) were added at 0 °C and the solution was stirred for about 10 min. Then, N,O-dimethylhydroxylamine (1.1 mmol, 0.11 g, hydrochloride salt was neutralized by the addition of 1.1 mmol, 0.12 mL TEA) was added to the reaction mixture and the reaction was allowed to stir till the completion of the reaction as indicated by TLC. After the evaporation of solvent, the crude product was extracted into EtOAc and the organic phase was washed with 5% citric acid (10 mL × 2), 5% Na2CO3 (10 mL × 2), water and brine solution and dried over anhydrous Na2SO4. The solvent was evaporated and pure product was isolated after followed by triturating with ether.
Stage #1: Boc-Glu(OBzl)-OH With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 20℃; for 0.166667h; Inert atmosphere;
Stage #2: N,O-dimethylhydroxylamine*hydrochloride With N-ethyl-N,N-diisopropylamine In dichloromethane at 20℃; Inert atmosphere;
With 4-methyl-morpholine; isobutyl chloroformate In dichloromethane
Stage #1: Boc-Glu(OBzl)-OH With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0℃; for 0.0833333h;
Stage #2: N,O-dimethylhydroxylamine*hydrochloride With 4-methyl-morpholine In dichloromethane at 0 - 20℃;
4.1.7 tert-Butyl N-{(S)-5-hydroxy-1-[methoxy(methyl)amino]-1-oxopentan-2-yl}carbamate (21)
To a solution of Boc-Glu(OBn)-OH (50.0g, 148mmol) in CH2Cl2 (296mL) were added HOBt·H2O (25.0g, 163mmol) and EDC·HCl (34.1g, 178mmol) at 0°C, and the mixture was stirred for 5min. To the mixture were added MeNHOMe·HCl (16.6g, 170mmol) and NMM (19.6mL, 178mmol) at 0°C, followed by stirring overnight at room temperature. The mixture was then poured into EtOAc and 1M HCl. After concentration under reduced pressure, the residue was extracted with EtOAc. The organic layer was washed with 1M HCl, saturated aqueous NaHCO3 and brine and dried over Na2SO4.Concentration under reduced pressure obtained the crude Weinreb amide. The crude product was used in the next step without purification. The crude Weinreb amide was treated with 10% Pd/C (5.64g) in EtOAc (494mL) under a H2 atmosphere at room temperature overnight. After filtration, the filtrate was concentrated to afford crude deprotected product. The crude product was used in the next step without purification. To a solution of the crude deprotected product and triethylamine (TEA; 22.7mL, 163mmol) in tetrahydrofuran (THF; 494mL) was slowly added isobutyl chloroformate (IBCF; 23.1mL, 178mmol) at -40°C. The reaction mixture was stirred for 40min at the same temperature, and then NaBH4 (16.8g, 445mmol) and H2O (74.0mL) were slowly added, followed by stirring for 1h at 0°C. The reaction was quenched by adding saturated aqueous NH4Cl at 0°C. After concentration under reduced pressure, the whole was extracted with EtOAc. The organic layer was washed with brine and dried over Na2SO4. Concentration under reduced pressure followed by flash chromatography over silica gel using n-hexane-EtOAc (1:3) yielded compound 21 (34.1g, 83%, over 3 steps) as a colorless oil: [α]21D [α]D21 -0.279 (c 1.05, CHCl3);1H NMR (500MHz, CDCl3) δ 5.35 (d, J=8.0Hz, 1H), 4.74 (br s, 1H), 3.78 (s, 3H), 3.70-3.64 (m, 2H), 3.21 (s, 3H), 2.05 (s, 1H), 1.85-1.80 (m, 1H), 1.69-1.60 (m, 3H), 1.44 (s, 9H); 13C NMR (75MHz, CDCl3) δ 173.1, 155.7, 79.7, 62.3, 61.7, 50.1, 32.1, 29.7, 28.4 (4C); HRMS (FAB) m/z calcd for C12H25N2O5 ([M+H]+) 277.1763, found 277.1751.
(S)-benzyl 5-(benzyl(2-ethoxy-2-oxoethyl)amino)-4-((tert-butoxycarbonyl)amino)-5-oxopentanoate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
75%
With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3h;
To a solution of BOC-GLU(OBZL)-OH (22.56 g, 66.80 mmol) in 250 ml of DCM was added HOBt (1 1.28 g, 73.6 mmol) followed by 1 equivalent of EDC (12.8 g, 67.2 mmol) and the resulting mixture was stirred for 2 minutes at room temperature. Then, ethyl 2- (benzylamino) acetate (14.20 g, 73.6 mmol) was added dropwise over 5 minutes, and the resulting mixture stirred for 5 minutes. An addition equivalent of EDC (12.8 g, 67.2 mmol) was then added and the resulting mixture was sirred at rt for 3 hrs. The mixture was diluted with 200 ml of DCM and washed by sat'd aq. soln of NaHC( (200 ml). The aqueous layer was extracted with DCM (2 x 100 ml) and the organic layers were combined, washed with NaHC( and brine, dried over sodium sulfate, filtered, and concentrated in vacuo to afford 37.6 g of crude product.. The material was purified by ISCO column (2 x 220g silica gel) eluting with a gradient of 0-60 % EtOAc in hexane (3000 ml). The tubes containing the product were collected and the solvent removed under reduced pressure to afford the title compound (25.6 g, 75%). MS: m/z = 513 (M+H+).
75%
With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 3.16667h;
Step 1. (5)-benzyl 5-(benzyl(2-ethoxy-2-oxoethyl)amino)-4-((ter?-butoxycarbonyl)amino)-5- oxopentanoate To a solution of BOC-GLU(OBZL)-OH (22.56 g, 66.80 mmol) in 250 ml of DCM was added HOBt (1 1.28 g, 73.6 mmol) followed by 1 equivalent of EDC (12.8 g, 67.2 mmol) and the resulting mixture was stirred for 2 minutes at room temperature. Then, ethyl 2- (benzylamino) acetate (14.20 g, 73.6 mmol) was added dropwise over 5 minutes, and the resulting mixture stirred for 5 minutes. An addition equivalent of EDC (12.8 g, 67.2 mmol) was then added and the resulting mixture was sirred at rt for 3 hrs. The mixture was diluted with 200 ml of DCM and washed by sat'd aq. soln of NaHC( (200 ml). The aqueous layer was extracted with DCM (2 x 100 ml) and the organic layers were combined, washed with NaHCC and brine, dried over sodium sulfate, filtered, and concentrated in vacuo to afford 37.6 g of crude product.. The material was purified by ISCO column (2 x 220g silica gel) eluting with a gradient of 0-60 % EtOAc in hexane (3000 ml). The tubes containing the product were collected and the solvent removed under reduced pressure to afford the title compound (25.6 g, 75%). MS: m/z = 513 (M+H+).
1-N-{2-[1,3-di-(2,3,4,6-tetra-O-acetyl-β-D-galacto-pyranosyl-(1→4)-2,3,6-tri-O-acetyl-β-D-glucopyranosyloxy)propyl]amino}-N-[(t-butoxycarbonyl)amino]-5-oxo-glutamic benzyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
79%
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; benzotriazol-1-ol; dicyclohexyl-carbodiimide In tetrahydrofuran at 0℃; for 0.5h;
Stage #2: C55H77NO36 In tetrahydrofuran at 0 - 20℃; for 38h;
General procedure: N-Boc-L-glutamic acid-5-benzylester (0.51g, 1.52mmol) was dissolved in dry THF (1mL) that was cooled to 0°Cfollowed by the addition of EDC•HCl (0.35g, 1.82mmol), HOBt (0.25g, 1.82mmol)and NMM (50μL). The mixture was stirred at 0°Cfor 0.5h. A solution of compound 2a (1.16g,1.52mmol) in dry THF (2 mL) was added dropwise and the mixture was stirred at 0°C for2 h, followed by stirring at room temperature for 36 h. The mixture wasconcentrated and the residue was diluted with CH2Cl2. Thesolution was filtered and the filtrate was washed with saturated aqueous sodiumbicarbonate, aqueous citric acid and water, dried (Na2SO4)and evaporated under diminished pressure. The crude product was purified byflash chromatography with PE/EtOAc = 1:1 as eluent to give compound 3a (1.24g) as a colorless syrup in 76% yield
1-N-{2-[1,3-di-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyloxy)propyl]amino}-N-[(t-butoxycarbonyl)amino]-5-oxo-glutamicacid benzyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
76%
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In tetrahydrofuran at 0℃; for 0.5h;
Stage #2: C31H45NO20 In tetrahydrofuran at 0 - 20℃; for 38h;
1-N-{2-[1,3-di-(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyloxy)propyl]amino}-N-[(t-butoxycarbonyl)amino]-5-oxo-glutamicacid benzyl ester (3a)
N-Boc-L-glutamic acid-5-benzyl ester (0.51g, 1.52mmol) was dissolved in dry THF (1mL) that was cooled to 0°C followed by the addition of EDC•HCl (0.35g, 1.82mmol), HOBt (0.25g, 1.82mmol) and NMM (50μL). The mixture was stirred at 0°C for 0.5h. A solution of compound 2a (1.16g,1.52mmol) in dry THF (2 mL) was added dropwise and the mixture was stirred at 0°C for 2 h, followed by stirring at room temperature for 36 h. The mixture was concentrated and the residue was diluted with CH2Cl2. The solution was filtered and the filtrate was washed with saturated aqueous sodium bicarbonate, aqueous citric acid and water, dried (Na2SO4) and evaporated under diminished pressure. The crude product was purified by flash chromatography with PE/EtOAc = 1:1 as eluent to give compound 3a (1.24g) as a colorless syrup in 76%yield; [a]25D = +22.6 (c = 0.53 in CHCl3); 1HNMR (400 MHz, CDCl3) δ7.38 - 7.32 (m, 5H, -COCH2C6H5-), 5.49 (d, J = 7.5 Hz, 1H), 5.37 - 5.21 (m,6H), 5.14 (s, 2H, -COCH2-),4.86 (s, 2H, H-1, H-1’), 4.39 - 3.98 (m, 8H), 3.85 (m, 2H), 3.63 (dd, 1H), 3.53(dd, 1H), 2.65 - 2.45 (m, 4H, -CH2CH2CO-), 2.18 , 2.11, 2.03,2.11-1.98 (8s, 24H, CH3CO-),1.42 (s, 9H, -C(CH3)3)ppm; 13C NMR (100 MHz, CDCl3) δ 173.6, 171.9, 170.8, 170.7, 170.1, 170.0, 169.9, 169.8 (CO), 135.8,128.6, 128.3 (Ph), 97.9, 97.8, 80.0, 69.3, 69.1, 68.9, 66.6, 66.3, 66.0, 65.9,62.4, 53.3, 47.8, 30.5, 29.7, 28.3, 20.9-20.7 (CH3) ppm; HRESI-TOFMS: m/z calcd for C48H67N2O25[M+H]+ 1071.4027,found 1071.4014; C48H66N2NaO25[M+Na]+ 1093.3847,found 1093.3817
2-(S)-tert-butoxycarbonylaminopentanedioic acid 5-benzyl ester 1-butyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
85.4%
Stage #1: Boc-Glu(OBzl)-OH With caesium carbonate In methanol; water at 20℃; for 24h;
Stage #2: 1-bromo-butane In N,N-dimethyl-formamide at 20℃; for 24h;
1 General method for preparation of compounds 6a-c
General procedure: 2-(S)-tert-Butoxycarbonylamino-pentanedioic acid 5-benzylester 5 was dissolved in methanol with 10% water (10 mL). Thesolution was neutralized to pH 7 by drop-wise addition of aqueousCs2CO3 (20%). Following evaporation under high vacuum, theresulting syrup was dissolved in dry DMF (20 mL) and treated withthe appropriate alkyl bromide (2.88 molar equivalents). The reactionwas stirred at room temperature overnight. Ethyl acetate(20 mL) was added and the organic layer washed with water(3 20 mL), 10% K2CO3 (2 20 mL), and brine (20 mL), and thendried over magnesium sulfate. The resulting solution was concentratedunder reduced pressure to afford the product as a whitesolid.4.1.1. 2-(S)-tert-Butoxycarbonylaminopentanedioic acid 5-benzylester 1-propyl ester (6b)Following the procedure described in 4.1, the reaction of 2-tertbutoxycarbonylaminopentanedioicacid 5-benzyl ester 5 (1.00 g,2.96 mmol) with propyl bromide (1.05 mL, 8.52 mmol) gaveproduct 6b (1.01 g, 90%)
2-(S)-tert-butoxycarbonylaminopentanedioic acid 5-benzyl ester 1-propyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
90.1%
Stage #1: Boc-Glu(OBzl)-OH With caesium carbonate In methanol; water at 20℃; for 24h;
Stage #2: propyl bromide In N,N-dimethyl-formamide at 20℃; for 24h;
1 General method for preparation of compounds 6a-c
General procedure: 2-(S)-tert-Butoxycarbonylamino-pentanedioic acid 5-benzylester 5 was dissolved in methanol with 10% water (10 mL). Thesolution was neutralized to pH 7 by drop-wise addition of aqueousCs2CO3 (20%). Following evaporation under high vacuum, theresulting syrup was dissolved in dry DMF (20 mL) and treated withthe appropriate alkyl bromide (2.88 molar equivalents). The reactionwas stirred at room temperature overnight. Ethyl acetate(20 mL) was added and the organic layer washed with water(3 20 mL), 10% K2CO3 (2 20 mL), and brine (20 mL), and thendried over magnesium sulfate. The resulting solution was concentratedunder reduced pressure to afford the product as a whitesolid.4.1.1. 2-(S)-tert-Butoxycarbonylaminopentanedioic acid 5-benzylester 1-propyl ester (6b)Following the procedure described in 4.1, the reaction of 2-tertbutoxycarbonylaminopentanedioicacid 5-benzyl ester 5 (1.00 g,2.96 mmol) with propyl bromide (1.05 mL, 8.52 mmol) gaveproduct 6b (1.01 g, 90%): Mp 51e52 C; 1H NMR (300 MHz, CDCl3)dH 0.95 (t, J 7.1 Hz, 3H, 30-CH3), 1.43 (s, 9H, t-Boc CH3), 1.68 (sextet,J 7.1 Hz, 2H, 20-CH2), 1.96 (m, 1H, 3-CHa), 2.21 (m, 1H, 3-CHb), 2.41(dd, J 16.5 and 6.8 Hz, 1H, 4-CHa), 2.50 (dd, J 16.5 and 6.8 Hz, 1H,4-CHb), 4.09 (t, J 7.1 Hz, 2H, 10-CH2), 4.33 (m, 1H, 2-CH), 5.08 (br d,1H, NH), 5.12 (s, 2H, CH2Ph), 7.35 (m, 5H, CH2Ph); 13C NMR (75 MHz,CDCl3) dC 10.3 (30-C), 21.9 (20-C), 28.0 (3-C), 28.3 (t-Boc CH3), 30.4(4-C), 53.0 (2-C), 62.1 (10-C), 66.5 (CH2Ph), 80.0 (quat., t-Boc C),128.3 (Ph CH),128.3 (Ph CH),128.6 (Ph CH),135.9 (quat., Ph C),155.4(quat., NCO2), 172.2 (quat., 5-C), 172.5 (quat., 1-C); IR cm1 3361(NeH), 1760 (C]O, ester), 1720 (C]O, ester), 1686 (C]O, carbamate),1365 (CeN), 1169 (CeO), 750, 698 (Ar CeH); Anal. calculatedfor C20H29NO6 (Mr 379.45) C 63.31, H 7.70, N 3.69%; Found C 63.51, H7.68, N 3.73%; ESI MS 401.1 [MNa].
(3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol[ No CAS ]
C37H55NO6[ No CAS ]
(4S)-4-amino-5-oxo-5-(((3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)pentanoic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
91%
Stage #1: Boc-Glu(OBzl)-OH; (3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol With dmap; dicyclohexyl-carbodiimide In benzene at 20℃; Inert atmosphere;
Stage #2: L-glutamic acid 5-methyl ester; C37H55NO6 With palladium 10% on activated carbon In methanol at 20℃; for 2h;
73
To a stirred mixture of alcohol 77 (500 mg, 1.72 mmol) and Boc-Glu(OBzl)-OH (640 mg, 1.90 mmol) in freshly dried benzene (35 ml) were added 4-(N,N-dimethylamino)pyridine (21 mg, 0.172 mmol) and dicyclohexylcarbodiimide (IM solution in benzene, 2.5 ml) under inert atmosphere at room temperature. After 18 h, the reaction mixture was poured into saturated sodium bicarbonate solution (40 ml), the product was extracted into ethyl acetate (3 x 30 ml), and the combined organic phases were washed with water (2 x 10 ml). The precipitated Ν,Ν'-dicyclohexyIurea was filtered off, the filtrate was dried over anhydrous sodium sulfate and the solvents evaporated under reduced pressure. Further portions of Ν,Ν'-dicyclohexylurea was crystallised from ether and filterred off. The filtrate containing the desired product was evaporated under reduced pressure. Chromatography of the residue on silica gel (10% ethyl acetate in petroleum ether) gave solid white foam of protected glutamate (960 mg, 91%). The product was characterized by NMR and used crude for the next step. NMR (400 MHz, CDCi3): δ 0.53 (3H, s, H-18), 0.83 (3H, d, J = 6.8, H-20), 0.94 (3H, s, H-19), 1.43 (9H, s, t-Bu), 2.26-2.43 (2H, m, H-4'), 4.32-4.41 (1H, m, CH- 2'), 4.72 (1H, tt, J, = 1 1.3, J2 = 4.7, H-3), 5.17 (2H, d, J = 5.1, CH2-benzyI), 7.34-7.38 (5H, m, phenyl). 13C NMR (101 MHz, CDC13): δ 172.41 (C-Γ), 172.37 (C-5'), 155.50 (OCONH), 135.48 (C-r, phenyl), 128.74 (C-3 ', C-5 '} phenyl), 128.54 (C-4r, phenyl), 128.35 (C-2', C-6', phenyl), 80.15 (t-Bu), 74.93 (C-3), 67.32, 55.99, 53.30, 45.32, 42.34, 42.19, 41.01 , 37.86, 36.21, 35.30, 34.90, 32.38, 30.91, 30.41, 28.46 (3 x C, t-Bu), 27.87, 27.20, 26.78, 26.69, 24.88, 23.53, 20.76, 13.98, 12.17. To a solution of the protected ester of glutamic acid (950 mg, 1.56 mmol) in absolute methanol (55 ml) was added palladium catalyst on charcoal (10%, 50 mg). The reaction mixture was stirred at room temperature under vigorous stirring and a slight positive pressure of hydrogen for 2 h. The catalyst was filtered off and the solvents evaporated under reduced pressure. The residue was dissolved in dichloromethane (8 ml) and stirring was dropwise added trifluoracetic acid (5 ml). The reaction mixture was stirred at room temperature for 30 min and then evaporated under reduced pressure to dryness. To the residue was added a mixture of methanol (10 ml) and pyridine (1 ml) and the solution was dropwise added into water (60 ml) with crushed ice. The whole mixture was kept overnight at 5 °C. The solids were collected by filtration, washed with water and dried to afford the monoester 81 (541 mg, 67%, two steps): mp 169-171°C (metanol), [a]D20 +52.5 (c 0.099, CHCl3/MeOH, 1.7:0.8). H NMR (400 MHz, CDC13/CD30D, 6: 1): δ 0.48 (3H, s, H-18), 0.78 (3H, d, J= 6.7, H-20), 0.90 (3H, s, H-19), 2.42-2.55 (2H, m, H-4'), 3.52-3.61 (1H, m, CH-2'), 4.604.72 (1H, m, H-3). I3C NMR (101 MHz, CDCI3/CD3OD, 6: 1): δ 173.36 (COOH, COO), 75.22 (C-3), 55.86, 55.83, 45.23, 42.25, 42.17, 40.95, 37.76, 36.14, 35.25, 34.83, 32,31, 31.16, 30.32, 29.76, 27.19, 26.65, 26.57, 24.81, 23.47, 20.70, 13.86, 12.05. IR spectrum (KBr): 2717, 1540, 1491 (NH3+); 1745, 1718 (C=0, glutamyl ester); 1732 (CO); 1635 (COO-); 1022 (COC). MS (ESI) m/z: 442.3 (100 %, M + Na), 420.4 (45 %, M + H). HR-MS (ESI) m/z: For C2;H4204N (M+H) calcd: 420.3108; found: 420.3108.
Stage #1: Boc-Glu(OBzl)-OH With 4-methyl-morpholine; isobutyl chloroformate In tetrahydrofuran at -10℃; for 1h; Inert atmosphere;
Stage #2: N-methyl-N-nitrosotoluene-p-sulfonamide With potassium hydroxide In tetrahydrofuran; diethyl ether at 0 - 20℃;
4-tert-Butoxycarbonylamino-6-diazo-5-oxo-hexanoic acid benzyl ester (5)
4-tert-Butoxycarbonylamino-6-diazo-5-oxo-hexanoic acid benzyl ester (5) Boc-Glu(OBn)-OH (5.06 g, 15.00 mmol) was dissolved in anhydrous THF (80 mL) under an atmosphere of N2, and cooled to -10 °C. NMM (2.14 ml, 19.50 mmol) was added, followed by IBCF (2.95 ml, 22.50 mmol). The reaction was stirred at -10°C for 1 h. The white precipitate formed was eliminated by filtration. The filtrate was cooled to 0 °C and freshly prepared diazo methane in diethyl ether was added (2 eq, prepared from Diazald and KOH). The mixture was warmed up to room temperature and stirred overnight. The reaction was then quenched with the addition of acetic acid (1 ml), and the mixture was vigorously stirred (750 rpm) for two hours. An excess of acetic acid was then added (1 ml), and the mixture was then neutralised with a saturated aqueous NaHC03 solution (70 ml) and stirred until the effervescence disappeared. The organic phase was separated and washed with 1 N KHSO4 (aq) (60 ml), brine (60 ml), dried over MgSC>4, and the solvents were evaporated in vacuo. The crude obtained was purified by flash chromatography on silica gel (gradient cyclohexane-30% ethyl acetate), to afford the pure product as a yellow solid (3.94 g, 73 %). FontWeight="Bold" FontSize="10" H NMR (CDC13, 300 MHz) δ: 7.46- 7.29 (m, 5H), 5.47 (s, 1H), 5.25 (m, 1H), 5.14 (s, 2H), 4.26 (m, 1H), 2.60-2.38 (m, 2H), 2.23- 2.10 (m, 1H), 1.93-1.77 (m, 1H), 1.45 (s, 9H).
benzyl (S)-4-((tert-butoxycarbonyl)amino)-5-oxo-5-(3-oxo-2-azaspiro[4.5]decan-2-yl)pentanoate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
60%
With N-ethyl-N,N-diisopropylamine; N-[(dimethylamino)-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate In N,N-dimethyl-formamide at 0 - 20℃; for 5h;
AE Synthesis of benzyl (S)-4-((tert-butoxycarbonyl)amino)-5-oxo-5-(3-oxo-2-azaspiro[4.5]decan-2-yl)pentanoate (1):
To a stirred solution of SM (4.0 g, 26.1 mmol) in DMF (30 mL), (S)-5-(benzyloxy)-2- ((/ - h u to x y c arbo n y 1) a m i n o) - 5 - o x ope n ta no i c acid (Int-B) (8.80 g, 26.1 mmol), DIPEA (15.53 mL, 91.3 mmol), were added at 0 °C and reaction mixture stirred for 10 min. HATU (34.7 g, 91.3 mmol) was added to the reaction mixture and stirred at room temperature for 5 h. After consumption of the starting material (by TLC), reaction mixture was quenched with water (30 mL) and extracted with EtOAc (3 x 30 mL). The combined organic layer was washed with brine (30 mL), dried over NanSQ* and concentrated under reduced pressure. The crude product was purified by column chromatography eluting with EtOAc/n-hexane to afford compound 1 (7.50 g, 60%) as a colourless oil. LCMS (ESI): m/z 373.2 [M++l-Boc]
2-(4'-bromo-2'-chlorophenoxy)-N-(4'-sulfamoylphenyl)acetamide[ No CAS ]
N-(4’-(((5’’-benzoxy-2’’-(S)-((tert-butoxycarbonyl)amino)-5’’-oxo) pentanoicylamino)sulfonyl)phenyl)-2-(4'-bromo-2'-chlorophenoxy) acetamide[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
84.8%
With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0 - 20℃;
5.1.4. General procedure for preparation of 6-8, 10, 13, 15, 17-18
General procedure: To a mixture of compound 5 (1.0 eq.) and corresponding carboxylicacid (1.2e2.0 eq.) in DCM EDCI (5.0 eq.) and DMAP (3.0 eq.)were added at 0 C, then the mixturewas warmed to r.t. and stirredovernight. The mixturewas washed with 0.5MHCl (30 mL 3) andbrine (30 mL), dried over anhydrous Na2SO4 and concentrated togive the residue which was purified by appropriate method to givethe product.
(3S)-2-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester[ No CAS ]
(3S)-N-(6-tert-butoxycarbonylbenzyloxyglutamylamino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
31%
Stage #1: Boc-Glu(OBzl)-OH With benzotriazol-1-ol; dicyclohexyl-carbodiimide In tetrahydrofuran for 0.666667h; Cooling with ice;
Stage #2: (3S)-2-(6-amino-n-hexanoyl)-2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester With 4-methyl-morpholine In tetrahydrofuran at 20℃; for 12h; Cooling with ice;
5 Example 5 Preparation of (3S) -N- (6-Boc-Glu (OBzl) -amino-n-hexanoyl) -2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester (5a)
Dissolve 2.44 g (7.2 mmol) of Boc-Glu (OBzl) and 0.97 g (7.2 mmol) of N-hydroxybenzotriazole (HOBt) with 30 mL of anhydrous tetrahydrofuran,Add 1.48 g (7.2 mmol) of N, N-dicyclohexylcarbodiimide (DCC) under ice bath, and stir for 40 minutes. Thereafter, 3.00 g (6.6 mmol) of (3S) -N- (6-amino-n-hexanoyl) -2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester was added to the obtained reaction solution. With 30 mL of a solution of anhydrous tetrahydrofuran, adjust the pH to 8 with N-methylmorpholine under an ice bath, and stir at room temperature for 12 hours. TLC showed that (3S) -N- (6-amino-n-hexanoyl) -2,3,4,9-tetrahydro-β-carboline-3-carboxylic acid benzyl ester disappeared, the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to leave Add 100 mL of ethyl acetate to dissolve. The solution was washed with saturated NaHCO3 aqueous solution (40mL × 3), saturated NaCl aqueous solution (40mL × 3), saturated KHSO4 aqueous solution (40mL × 3), saturated NaCl aqueous solution (40mL × 3), and saturated NaHCO3 aqueous solution (40mL × 3), washed with a saturated NaCl aqueous solution (40 mL × 3). The ethyl acetate layer was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane: methanol = 60: 1) to obtain 1.52 g (31%) of the target compound as a pale yellow solid.
(25R)-spirost-5-en-3β-yl N-(t-butoxycarbonyl)-5-benzyl-L-glutamate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
94%
Stage #1: Boc-Glu(OBzl)-OH With dmap; dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 0.166667h;
Stage #2: diosgenin In dichloromethane at 20℃; for 24h;
2.3.1. General procedure for the synthesis of compounds 2-16
General procedure: 2.3.1.1. Synthesis of analogue 2 - (25R)-spirost-5-en-3β-yl N-(t-butoxycarbonyl)-O-benzyl- L-serinate. DMAP (40 mg, 0.327 mmol) andDCC (280 mg, 1.357 mmol) were added to the solution of Boc-L-Ser(Bzl)-OH (400 mg, 1.354 mmol) in anhydrous CH2Cl2 (10 mL) and themixture was stirred for 10 min at room temperature. Then diosgenin(406 mg, 0.979 mmol) was added and the reaction mixture was stirredat room temperature for 24 h (TLC control). After the reaction had beencompleted, the DCU precipitate was filtered off. The filtrate was washedsuccessively with the NaHCO3 aq. solution and NaCl aq. solution. Theextract was dried over anhydrous MgSO4, filtered and evaporated todryness. The crude product was purified through a silica gel columneluted with ethyl acetate-hexane. Yield 93%, white solid, m.p. 161.6°C;[M+H]+ calcd for C42H62NO7: 692.4526, found: 692.4531,[M+Na]+ calcd for C42H61NO7Na: 714.4346, found: 714.4352.
With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 20℃; for 16h;
1.1-3 Compound 7
5-benzyl N-(tert-butoxycarbonyl)-L-glutamate (0.10 g, 0.29 mmol), 1-hydroxybenzotriazole (HOBt, 80 mg, 2.0 eq.) and DIEA (0.18 mL, 3.5 eq.) were dissolved in 1 mL of DMF, and EDCI (0.11 g, 2.0 eq.) and compound 1 (0.13 g, 1.2 eq.) were added to the solution. After stirring at room temperature for 16 hours, the solution was diluted with ethyl acetate (EtOAc), and then washed with a 0.5 M aqueous solution of citric acid, a saturated aqueous solution of sodium hydrogen carbonate (NaHCO3) and a saturated aqueous solution of sodium chloride (NaCl). The organic layer was separated, dried with sodium sulfate (Na2SO4), and then filtered, and the filtrate was distilled under reduced pressure to remove the solvent. The remaining mixture was purified by SiO2 column chromatography to obtain compound 4 as a white solid (0.16 g, 76%). 1HNMR (400 MHz, CDCl3): δ (ppm)=9.49 (brs, 1H), 7.687.83 (m, 15H), 7.277.35 (m, 5H), 5.87 (d, J=9.2 Hz, 1H), 5.08 (s, 2H), 4.184.23 (m, 1H), 3.613.87 (m, 4H), 2.432.47 (m, 2H), 2.122.22 (m, 1H), 1.942.01 (m,1H), 1.43 (s, 9H); 13CNMR (100 MHz, CDCl3): δ (ppm)=172.8, 172.7, 155.2, 135.8, 135.3 (d, 4JCP=3.0 Hz), 133.4 (d, 3JCP=10.4 Hz), 130.5 (d, 2JCP=12.7 Hz), 128.4, 128.1, 128.0, 117.4(d, 1JCP=85.9 Hz), 79.2, 66.1, 53.9, 33.3, 30.4, 28.4, 28.3, 22.2 (d, 1JCP=49.7 Hz); 31P NMR (121 MHz, CDCl3): δ (ppm)=22.1; HRMS (m/z): [M]+625.2826.
Stage #1: Boc-Glu(OBzl)-OH With dmap; 2-(6-chloro-1H-benzo[d][1,2,3]triazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (V) In dichloromethane; N,N-dimethyl-formamide for 1h; Inert atmosphere;
Stage #2: (S)-2,2'-diamino-1,1'-binaphthalene In dichloromethane; N,N-dimethyl-formamide at 20℃; for 16h; Inert atmosphere;
4.1 Synthesis of BINAM-glutamic acid 13
L-Boc-glutamic acid benzyl ester (7.11 g,21.1 mmol, 3 equiv), HCTU (8.71 g, 21.1 mmol, 3equiv) and DMAP (4.30 g, 35.1 mmol, 5 equiv) weredissolved in DCM (100 mL) and DMF (1 mL) andstirred for one hour. Subsequently (S)-BINAM (2.00g, 7.02 mmol, 1 equiv) was added and the reactionmixture was stirred for 16 hours at room temperature.The solvent was evaporated in vacuo and the residue was redissolved in EtOAc. The organic layer waswashed with water (3 × 50 mL) and brine (3 × 50 mL) and dried over MgSO4. The solvent wasevaporated and the crude product purified by flash chromatography ( = 6 cm, h = 50 cm, CyHex:EtOAc= 5:1) to obtain the product 13 (4.35 g, 4.71 mmol, 67%) as a white foam.Rf: 0.52 (CyHex:EtOAc = 5:1). 1H-NMR: (300 MHz, DMSO-d6) δ [ppm] = 1.27 - 1.37 (m, 22 H, 2 xC-(CH3)3 + 2 x CH2), 1.85 - 1.92 (m, 4 H, 2 x CH2), 3.76 - 3.84 (m, 2 H, 2 x CH), 5.01 (s, 4 H, 2 x CH2),6.75 (d, 3J = 7.82 Hz, 2 H, 2 x BINOL-H), 6.85 (d, 3J = 8.44 Hz, 2 H, 2 x BINOL-H), 7.20 (d, 3J = 7.50Hz, 2 H, 2 x BINOL-H), 7.32 - 7.38 (m, 12 H, 2 x BINOL-H + 2 x C6H5), 7.89 (d, 7.82 Hz, 2 H, 2 xBINOL-H), 7.92 (br s, 2 H, 2 x NH), 7.99 (d, 3J = 9.07 Hz, 2 H, 2 x BINOL-H), 8.90 (br s, 2 H, 2 xNH). 13C-NMR: (75 MHz, DMSO-d6) δ [ppm] = 17.8, 30.3, 34.3, 54.8, 64.1, 81.5, 120.5, 122.5, 125.0,125.8, 127.2, 128.9, 129.2, 132.3, 133.5, 136.9, 138.9, 140.7, 142.5, 149.3, 160.7, 176.0, 180.9. HRMS:(ESI, MeOH): m/z = 923.4187 ([M+H]+), calcd. 923.4226 (for [C54H59N4O10]+). FT-IR: (ATR): [cm-1] = 2979, 1675, 1596, 1496, 1452, 1365, 1247, 1160, 1052, 865, 811, 746, 696. Melting point: 67°C.
With dmap; dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 3h; Cooling with ice;
18 Preparation of Intermediate 18.1
Add to the 100mL reaction flask1.3 (3.00g, 7.19mmol, 1.0eq),L-HO-Glu-Boc-5-OBn (2.91g, 8.63mmol, 1.2eq),DMAP (0.10g, 0.72mmol, 0.1eq)And dichloromethane 20mL,Stir until homogeneous under ice bath;DCC (1.78g, 8.63mmol, 1.2eq)Dissolved in 10mL of dichloromethane,Then added to the reaction solution,Then stir at room temperature for 3 hours,The end of the reaction was detected by TLC.Filter out the white solid DCU,The filtrate was washed with saturated NaHCO3.The organic phase is dried with anhydrous sodium sulfate,Concentrate under reduced pressure.The crude product was subjected to column chromatography (petroleum ether (60-90)/ethyl acetate = 10:1) to obtain a white solid (4.7 g, yield 88.85%).
benzyl N2-(tert-butoxycarbonyl)-N-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-alpha-glutaminate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
45%
With N-ethyl-N,N-diisopropylamine; N-[(dimethylamino)-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate In N,N-dimethyl-formamide at 20℃;
benzyl N2-(tert-butoxycarbonyl)-N-2,5,8,11,14,17,20,23-octaoxapentacosan-25-yl-L-alpha- glutaminate
(2S)-5-(benzyloxy)-2-[(tert-butoxycarbonyl)amino]-5-oxopentanoic acid (691 mg, 2.05 mmol) was dissolved in DMF (25 ml, 330 mmol). HATU (934 mg, 2.46 mmol), N,N- diisopropylethylamine (1.1 ml, 6.1 mmol) and 2,5,8,11,14,17,20,23-octaoxapentacosan-25- amine (890 l, 2.5 mmol) were added and the mixture was stirred at room temperature overnight. Water was added and the mixture was extracted with DCM (3x). The combined organic layers were dried and evaporated under reduced pressure. Purification by preparative HPLC (ACN/water + 0.1% TFA, gradient) provided the title compound (645 mg, 45% yield). Instrument: Gilson Abimed 305 pump, KNAUER UV detector K-2501, Varian fraction collector model 701A, Prepcon 5 software. Column: Chromatorex C18, 10m, 125x40 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 10-70% B; rate 50 ml/min; temperature 25°C. Preparative HPLC: Column Chromatorex C18, 10 m, 125x40 mm. Eluent A: water + 0.1 Vol-% TFA; Eluent B: acetonitrile; gradient: 0-22 min 10-70% B. Rate 50 ml/min; temperature 25°C. LC-MS (METHOD 6): Rt = 1.73 min; MS (ESIpos): m/z = 703 [M+H]+ H-NMR (400 MHz, DMSO-d6) d [ppm]: -0.008 (0.67), 1.370 (4.36), 3.235 (5.50), 3.395 (0.76), 3.409 (0.75), 3.411 (0.76), 3.420 (0.88), 3.427 (0.67), 3.434 (1.07), 3.484 (5.31), 3.491 (5.17), 3.501 (15.77), 3.503 (16.00), 3.507 (3.51), 3.515 (1.03), 3.517 (1.01), 5.078 (1.94), 7.357 (2.65).
With N-(3-dimethylaminopropyl)-N-ethylcarbodiimide In dichloromethane at 20℃; for 264h; Inert atmosphere;
1.3.4 1.3.4. Synthesis of Boc-Glu (OBzl) -OH-DL-G1-U2
DL-G1-U2 (109.5 mg, 0.1273 mmol) synthesized in 1.2 was dissolved in 15 mL of dichloromethane. This solution was added dropwise to 25 mL of dichloromethane with Boc-Glu (OBzl) -OH (375.9 mg, 1.114 mmol) and the condensing agent WSC (137.6 mg, 0.713 mmol). After enclosing Ar gas, the mixture was stirred at room temperature for 11 days, the reaction solution was transferred to a separating funnel, and the mixture was washed 3 times with saturated brine. Then, anhydrous sodium sulfate was added to the separated organic layer, and the mixture was dried overnight. The obtained crude product was separated and purified by column chromatography (developing solvent; chloroform / methanol = 1/1, filler: silica gel). The solvent was distilled off by a rotary evaporator and then vacuum dried. The obtained purified product was dissolved in methanol and further separated and purified by an LH-20 column. A yellowish oily substance was obtained with a yield of 155.5 mg and a yield of 81.5%.
81.5%
With N-(3-dimethylaminopropyl)-N-ethylcarbodiimide In dichloromethane at 20℃; for 264h; Inert atmosphere;
1.3.4 1.3.4. Synthesis of Boc-Glu (OBzl) -OH-DL-G1-U2
DL-G1-U2 (109.5 mg, 0.1273 mmol) synthesized in 1.2 was dissolved in 15 mL of dichloromethane. This solution was added dropwise to 25 mL of dichloromethane with Boc-Glu (OBzl) -OH (375.9 mg, 1.114 mmol) and the condensing agent WSC (137.6 mg, 0.713 mmol). After enclosing Ar gas, the mixture was stirred at room temperature for 11 days, the reaction solution was transferred to a separating funnel, and the mixture was washed 3 times with saturated brine. Then, anhydrous sodium sulfate was added to the separated organic layer, and the mixture was dried overnight. The obtained crude product was separated and purified by column chromatography (developing solvent; chloroform / methanol = 1/1, filler: silica gel). The solvent was distilled off by a rotary evaporator and then vacuum dried. The obtained purified product was dissolved in methanol and further separated and purified by an LH-20 column. A yellowish oily substance was obtained with a yield of 155.5 mg and a yield of 81.5%.
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