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CAS No. : | 159857-81-5 | MDL No. : | MFCD22200431 |
Formula : | C35H43N7O11 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | HYSPJPGXSALJRR-DHIFEGFHSA-N |
M.W : | 737.76 | Pubchem ID : | 57587849 |
Synonyms : |
Maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate;MC-VC-PAB-PNP
|
Num. heavy atoms : | 53 |
Num. arom. heavy atoms : | 12 |
Fraction Csp3 : | 0.4 |
Num. rotatable bonds : | 25 |
Num. H-bond acceptors : | 11.0 |
Num. H-bond donors : | 4.0 |
Molar Refractivity : | 195.09 |
TPSA : | 266.35 Ų |
GI absorption : | Low |
BBB permeant : | No |
P-gp substrate : | Yes |
CYP1A2 inhibitor : | No |
CYP2C19 inhibitor : | No |
CYP2C9 inhibitor : | Yes |
CYP2D6 inhibitor : | No |
CYP3A4 inhibitor : | Yes |
Log Kp (skin permeation) : | -9.26 cm/s |
Log Po/w (iLOGP) : | 3.17 |
Log Po/w (XLOGP3) : | 2.17 |
Log Po/w (WLOGP) : | 2.04 |
Log Po/w (MLOGP) : | -0.08 |
Log Po/w (SILICOS-IT) : | 0.33 |
Consensus Log Po/w : | 1.53 |
Lipinski : | 2.0 |
Ghose : | None |
Veber : | 2.0 |
Egan : | 1.0 |
Muegge : | 4.0 |
Bioavailability Score : | 0.17 |
Log S (ESOL) : | -4.3 |
Solubility : | 0.0371 mg/ml ; 0.0000503 mol/l |
Class : | Moderately soluble |
Log S (Ali) : | -7.4 |
Solubility : | 0.0000296 mg/ml ; 0.0000000401 mol/l |
Class : | Poorly soluble |
Log S (SILICOS-IT) : | -6.06 |
Solubility : | 0.000646 mg/ml ; 0.000000875 mol/l |
Class : | Poorly soluble |
PAINS : | 0.0 alert |
Brenk : | 4.0 alert |
Leadlikeness : | 2.0 |
Synthetic accessibility : | 5.65 |
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P261-P305+P351+P338 | UN#: | N/A |
Hazard Statements: | H302-H315-H319-H335 | Packing Group: | N/A |
GHS Pictogram: |
* All experimental methods are cited from the reference, please refer to the original source for details. We do not guarantee the accuracy of the content in the reference.
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
57% | With N-ethyl-N,N-diisopropylamine In dichloromethane; N,N-dimethyl-formamide at 23℃; for 20 h; | To a solution of Compound 11 (500 mg, 0.87 mmol) and bis(4-nitrophenyl) carbonate (bis-PNP) (2.64 g, 8.72 mmol) in DCM:DMF (8:2, 25 mL) was added DIPEA (0.45 mL, 2.61 mmol). The reaction mixture was stirred for 20 h at 23 °C and poured onto a silica gel column (DCM:CH3OH, from 50:1 to 10:1 ) to afford pure target Compound 9 (364 mg, 57percent). Rf= 0.40 (CH2CI2:CH3OH, 9:1 ).1H NMR (400 MHz, CDCI3/CD3OD): δ 9.45 (s, 1 H), 8.23 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 6.65 (s, 2H), 5.20 (s, 2H), 4.56 (dt, J = 10.5, 5.4 Hz, 1 H), 4.15 (d, J = 7.2 Hz, 1 H), 3.46 (dd, J = 8.0, 6.4 Hz, 2H), 3.16-2.89 (m, 2H), 2.21 (dd, J = 8.3, 6.6 Hz, 2H), 2.06-1 .97 (m, 1 H), 1.90-1.83 (m, 1 H), 1.73-1.46 (m, 7H), 1.34- 1.20 (m, 2H), 0.91 (d, J = 6.7 Hz, 3H), 0.90 (d, = 6.7 Hz, 3H).13C NMR (125 MHz, CDCI3/CD3OD) δ 174.4, 172.4, 171.1 , 170.6, 160.5, 155.5, 152.5, 145.3, 138.7, 134.1 , 129.9, 129.5, 125.2, 121.8, 120.0, 70.6, 59.0, 53.2, 37.5, 35.8, 30.6, 29.6, 29.3, 28.1 , 26.2, 26.2, 25.1 , 19.1 , 18.1. ESI-MS m/z: Calcd. for CasH^N O^: 737.3. Found: 738.3 (M+H)+. |
57% | With N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide at 0 - 20℃; for 15 h; Inert atmosphere | Preparation Example 1-2: Preparation of Compound (II-3) (0054) (0055) Under an argon stream, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(5.0 g, 8.7 mmol, Dubowchik et al., Bioconjugate Chem., 2002, 13 (4), pp 855-869) was dissolved in 60 mL of anhydrous dimethylformamide, added with N, N-diisopropylethylamine (3.0 mL, 17.4 mmol), and then cooled to 0°C. To the mixture was added at once bis(4-nitrophenyl) carbonate (7.94 g, 26.1 mmol), followed by stirring at room temperature for 15 hrs. After completion of the reaction, the reaction mixture was concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain 4-((S)-2-((S)-2-(6-(2 ,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (3.65 g, 57 percent) as a pale yellow solid. (0056) 1H NMR (400 MHz, DMSO-d6) δ 0.83 (d, J = 6.8 Hz, 3 H), 0.86 (d, J = 6.8 Hz, 3 H), 1.09 (t, J = 7.2 Hz, 1 H), 1.19 (m, 2 H), 1.34 - 1.76 (m, 7 H), 1.96 (m, 1 H), 2.15 (m, 2 H), 2.99 (m, 2 H), 3.37 (m, 2 H), 4.19 (t, J= 7.8 Hz, 1 H), 4.39 (m, 1 H), 5.24 (s, 2 H), 5.41 (s, 2 H), 5.97 (brt, J= 5.6 Hz, 1 H), 7.00 (s, 2H), 7.41 (d, J= 8.4 Hz, 2 H), 7.57 (d, J= 7.2 Hz, 2 H), 7.65 (d, J= 8.4 Hz, 2 H), 7.80 (d, J= 8.4 Hz, 1 H), 8.09 (d, J = 7.2 Hz, 1 H), 8.31 (d, J = 7.2 Hz, 2 H), 10.05 (brs, 1 H) (0057) Under an argon stream, 4-((S)-2-((S)-2-(6-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (3.65 g, 4.95 mmol) was dissolved in 90 mL of anhydrous dimethylformamide, and stirred, together with t-butyl (2-aminoethyl)(methyl)carbamate (0.86 g, 4.95 mmol), at 20 - 25°C for 2 hrs. After completion of the reaction, the reaction mixture was completely concentrated in a high vaccum, and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-3) (3.8 g, 99percent). (0058) LC-MS m/z: 773.5 [M+H]+ (0059) To a solution of the amino-protected derivative of compound (II-3) (146 mg, 0.186 mmol) in 5 mL of dichloromethane was dropwise added 2 mL of trifluoroacetic acid, followed by stirring at 20 - 25°C for 2 hrs. After completion of the reaction, the reaction solvent was removed by vacuum concentration, and then trifluoroacetic acid was completely removed by adding 5 mL of toluene twice to obtain a concentrated TFA salt of the title compound. |
46% | Inert atmosphere | To stirred solution of 6-maleimidocaproyl-val-cit-PAB 38 in dry DMF under nitrogen, bis-(p-nitrophenyl)carbonate was added followed by DIPEA, resulting in a colour change from colourless to bright yellow. The solution was stirred at room temperature under nitrogen for 1 h after which the DMF was removed by high vacuum to give an oily residue. This was triturated with ethyl acetate for 15 min resulting in precipitation which was completed by the addition of ether. The solid was collected and washed well with ether and air dried to give an off-white solid. TLC [silica gel: 10 percent MeOH/DCM Rf0.46].This was purified by chromatography [silica gel: 5-10percent MeOH/DCM gradient elution] to give the activated linker 39 as a white solid 0.006 g, (46percent). MS (m/z) 738..3091 (M+H), HRMS (m/z) calculated for C35H43N7O11Na M+Na 760.2918 found 760.2922 |
40% | With pyridine In dichloromethane at 0 - 20℃; for 3.16667 h; Inert atmosphere | 168.6 mg (0.294 mmol) of mc-vc-PABOH was dissolved in 5 ml of anhydrous pyridine under nitrogen protection.Cool to about 0°C. Another 179 mg (3 eq) of PNP was dissolved in 5 ml of DCM, and it was slowly added to the reaction system. And inThe ice bath was removed after keeping at 0° C. for 10 min, and the reaction was stirred at room temperature for 3 h. After the reaction is completed, add 70ml EA and 100ml 15percentAqueous citric acid, dispense organic layer. The organic layer was washed successively with citric acid, water, saturated saline and dried over anhydrous sodium sulfate.Dry and filter. The filtrate was concentrated to dryness under reduced pressure to give a pale yellow oil. Methyl tert-butyl ether was added for crystallization to give an off-white solid.86mg, yield 40percent. |
40% | With pyridine In dichloromethane at 0 - 20℃; for 3.16667 h; Inert atmosphere | Under the protection of nitrogen, 168.6 mg (0.294 mmol) of mc-vc-PABOH was dissolved in 5 ml of anhydrous pyridine.The reaction system was cooled to about 0 °C. Further, PNP 179 mg (3 eq) was dissolved in 5 ml of DCM, and then slowly added to the reaction system.And after 10 minutes at 0 ° C, remove the ice bath.The reaction was further stirred at room temperature for 3 h. The reaction is completed,Add 70 ml EA and 100 ml 15percent aqueous citric acid solution.The organic layer was separated. The organic layer was washed successively with citric acid, water and saturated brine.Dry over anhydrous sodium sulfate and filter.The filtrate was concentrated to dryness under reduced pressure to give a pale yellow oil.Crystallization with methyl tert-butyl ether gave 86 mg of an off-white solid.The yield was 40percent. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
47% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 16h; | [0488] This material (67 mg, 0.118 mmole) and MC-vc-P AB-OCO-pNP (AB, 96 mg, 0.130 mmole) were then dissolved in DMF (2 ml) and treated with DIEA (41 muL, 0.236 mmole) and HOBT (7.7 mg, 0.056 mmole). After stirring for 16 hr at room temperature, the mixture was concentrated to near dryness, diluted with 2 ml of DMSO and purified by reverse phase preparative HPLC to give 64 mg (47%) of MC-vc-P AB-MeVaI- Val-Dil- ODMB as a glassy solid . LCMS (ESI) m/z: 1164.648 (MH)+, 1186.581 (M+Na)+, eluted at 13.49 min |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
95% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 25℃; | mc-Val-Cit-PABOH (1 eq.) was mixed with bis(4-nitrophenyl) carbonate (1.87 eq.) in N,N-dimethylformamide (8 vol.) at 20C. N,N-diisopropyl ethyl amine (1.75 eq.) was added at 25C. The reaction mixture was stirred at 25C for 2-6 hours until reaction was complete. Product was precipitated out of the reaction mixture by adding anhydrous ethyl acetate (12.5 vol.) at 25C and tert-Butyl methyl ether (12.5 vol.). The resulting slurry was stirred, then cooled to 0C and stirred for 10-30 minutes. The solids were isolated by filtration, washed and dried under vacuum before being re-slurried in ethyl acetate (12.5 vol.) at 20C, filtered and dried once more (95% yield). MS: m/e 738 (MH)+, 760 (M+Na)+. |
57% | With N-ethyl-N,N-diisopropylamine; In dichloromethane; N,N-dimethyl-formamide; at 23℃; for 20h; | To a solution of Compound 11 (500 mg, 0.87 mmol) and bis(4-nitrophenyl) carbonate (bis-PNP) (2.64 g, 8.72 mmol) in DCM:DMF (8:2, 25 mL) was added DIPEA (0.45 mL, 2.61 mmol). The reaction mixture was stirred for 20 h at 23 C and poured onto a silica gel column (DCM:CH3OH, from 50:1 to 10:1 ) to afford pure target Compound 9 (364 mg, 57%). Rf= 0.40 (CH2CI2:CH3OH, 9:1 ).1H NMR (400 MHz, CDCI3/CD3OD): delta 9.45 (s, 1 H), 8.23 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 6.65 (s, 2H), 5.20 (s, 2H), 4.56 (dt, J = 10.5, 5.4 Hz, 1 H), 4.15 (d, J = 7.2 Hz, 1 H), 3.46 (dd, J = 8.0, 6.4 Hz, 2H), 3.16-2.89 (m, 2H), 2.21 (dd, J = 8.3, 6.6 Hz, 2H), 2.06-1 .97 (m, 1 H), 1.90-1.83 (m, 1 H), 1.73-1.46 (m, 7H), 1.34- 1.20 (m, 2H), 0.91 (d, J = 6.7 Hz, 3H), 0.90 (d, = 6.7 Hz, 3H).13C NMR (125 MHz, CDCI3/CD3OD) delta 174.4, 172.4, 171.1 , 170.6, 160.5, 155.5, 152.5, 145.3, 138.7, 134.1 , 129.9, 129.5, 125.2, 121.8, 120.0, 70.6, 59.0, 53.2, 37.5, 35.8, 30.6, 29.6, 29.3, 28.1 , 26.2, 26.2, 25.1 , 19.1 , 18.1. ESI-MS m/z: Calcd. for CasH^N O^: 737.3. Found: 738.3 (M+H)+. |
57% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; for 15h;Inert atmosphere; | Preparation Example 1-2: Preparation of Compound (II-3) (0054) (0055) Under an argon stream, <strong>[159857-80-4]6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide</strong>(5.0 g, 8.7 mmol, Dubowchik et al., Bioconjugate Chem., 2002, 13 (4), pp 855-869) was dissolved in 60 mL of anhydrous dimethylformamide, added with N, N-diisopropylethylamine (3.0 mL, 17.4 mmol), and then cooled to 0C. To the mixture was added at once bis(4-nitrophenyl) carbonate (7.94 g, 26.1 mmol), followed by stirring at room temperature for 15 hrs. After completion of the reaction, the reaction mixture was concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain 4-((S)-2-((S)-2-(6-(2 ,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (3.65 g, 57 %) as a pale yellow solid. (0056) 1H NMR (400 MHz, DMSO-d6) delta 0.83 (d, J = 6.8 Hz, 3 H), 0.86 (d, J = 6.8 Hz, 3 H), 1.09 (t, J = 7.2 Hz, 1 H), 1.19 (m, 2 H), 1.34 - 1.76 (m, 7 H), 1.96 (m, 1 H), 2.15 (m, 2 H), 2.99 (m, 2 H), 3.37 (m, 2 H), 4.19 (t, J= 7.8 Hz, 1 H), 4.39 (m, 1 H), 5.24 (s, 2 H), 5.41 (s, 2 H), 5.97 (brt, J= 5.6 Hz, 1 H), 7.00 (s, 2H), 7.41 (d, J= 8.4 Hz, 2 H), 7.57 (d, J= 7.2 Hz, 2 H), 7.65 (d, J= 8.4 Hz, 2 H), 7.80 (d, J= 8.4 Hz, 1 H), 8.09 (d, J = 7.2 Hz, 1 H), 8.31 (d, J = 7.2 Hz, 2 H), 10.05 (brs, 1 H) (0057) Under an argon stream, 4-((S)-2-((S)-2-(6-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (3.65 g, 4.95 mmol) was dissolved in 90 mL of anhydrous dimethylformamide, and stirred, together with t-butyl (2-aminoethyl)(methyl)carbamate (0.86 g, 4.95 mmol), at 20 - 25C for 2 hrs. After completion of the reaction, the reaction mixture was completely concentrated in a high vaccum, and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-3) (3.8 g, 99%). (0058) LC-MS m/z: 773.5 [M+H]+ (0059) To a solution of the amino-protected derivative of compound (II-3) (146 mg, 0.186 mmol) in 5 mL of dichloromethane was dropwise added 2 mL of trifluoroacetic acid, followed by stirring at 20 - 25C for 2 hrs. After completion of the reaction, the reaction solvent was removed by vacuum concentration, and then trifluoroacetic acid was completely removed by adding 5 mL of toluene twice to obtain a concentrated TFA salt of the title compound. |
57% | With N-ethyl-N,N-diisopropylamine; In dichloromethane; N,N-dimethyl-formamide; at 23℃; for 20h; | To a solution of LIN 1 -2 (500 mg, 0.87 mmol) and bis(4-nitrophenyl) carbonate (bis-PNP) (2.64 g, 8.72 mmol) in DCM:DMF (8:2, 25 mL) was added DIPEA (0.45 mL, 2.61 mmol). The reaction mixture was stirred for 20 h at 23 SC and poured onto a silica gel column (DCM:CH30H, from 50:1 to 10:1 ) to afford pure target LIN 1 (364 mg, 57%). (1575) Rf= 0.40 (CH2Cl2:CH3OH, 9:1 ). (1576) 1 H NMR (400 MHz, CDCI3/CD3OD): d 9.45 (s, 1 H), 8.23 (d, J = 8.3 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.3 Hz, 2H), 7.34 (d, J = 8.5 Hz, 2H), 6.65 (s, 2H), 5.20 (s, 2H), 4.56 (dt, J = 10.5, 5.4 Hz, 1 H), 4.15 (d, J = 7.2 Hz, 1 H), 3.46 (dd, J = 8.0, 6.4 Hz, 2H), 3.16-2.89 (m, 2H), 2.21 (dd, J = 8.3, 6.6 Hz, 2H), 2.06-1 .97 (m, 1 H), 1 .90-1 .83 (m, 1 H), 1 .73-1 .46 (m, 7H), 1 .34-1 .20 (m, 2H), 0.91 (d, J = 6.7 Hz, 3H), 0.90 (d, J = 6.7 Hz, 3H). (1577) 13C NMR (125 MHz, CDCI3/CD3OD) d 174.4, 172.4, 171 .1 , 170.6, 160.5, 155.5, 152.5, 145.3, 138.7, 134.1 , 129.9, 129.5, 125.2, 121 .8, 120.0, 70.6, 59.0, 53.2, 37.5, 35.8, 30.6, 29.6, 29.3, (1578) 28.1 , 26.2, 26.2, 25.1 , 19.1 , 18.1 . (1579) ESI-MS m/z : Calcd. for C35H43N7O1 1 : 737.3. Found: 738.3 (M+H)+. |
46% | In N,N-dimethyl-formamide;Inert atmosphere; | To stirred solution of 6-maleimidocaproyl-val-cit-PAB 38 in dry DMF under nitrogen, bis-(p-nitrophenyl)carbonate was added followed by DIPEA, resulting in a colour change from colourless to bright yellow. The solution was stirred at room temperature under nitrogen for 1 h after which the DMF was removed by high vacuum to give an oily residue. This was triturated with ethyl acetate for 15 min resulting in precipitation which was completed by the addition of ether. The solid was collected and washed well with ether and air dried to give an off-white solid. TLC [silica gel: 10 % MeOH/DCM Rf0.46].This was purified by chromatography [silica gel: 5-10% MeOH/DCM gradient elution] to give the activated linker 39 as a white solid 0.006 g, (46%). MS (m/z) 738..3091 (M+H), HRMS (m/z) calculated for C35H43N7O11Na M+Na 760.2918 found 760.2922 |
40% | With pyridine; In dichloromethane; at 0 - 20℃; for 3.16667h;Inert atmosphere; | 168.6 mg (0.294 mmol) of mc-vc-PABOH was dissolved in 5 ml of anhydrous pyridine under nitrogen protection.Cool to about 0C. Another 179 mg (3 eq) of PNP was dissolved in 5 ml of DCM, and it was slowly added to the reaction system. And inThe ice bath was removed after keeping at 0 C. for 10 min, and the reaction was stirred at room temperature for 3 h. After the reaction is completed, add 70ml EA and 100ml 15%Aqueous citric acid, dispense organic layer. The organic layer was washed successively with citric acid, water, saturated saline and dried over anhydrous sodium sulfate.Dry and filter. The filtrate was concentrated to dryness under reduced pressure to give a pale yellow oil. Methyl tert-butyl ether was added for crystallization to give an off-white solid.86mg, yield 40%. |
40% | With pyridine; In dichloromethane; at 0 - 20℃; for 3.16667h;Inert atmosphere; | Under the protection of nitrogen, 168.6 mg (0.294 mmol) of mc-vc-PABOH was dissolved in 5 ml of anhydrous pyridine.The reaction system was cooled to about 0 C. Further, PNP 179 mg (3 eq) was dissolved in 5 ml of DCM, and then slowly added to the reaction system.And after 10 minutes at 0 C, remove the ice bath.The reaction was further stirred at room temperature for 3 h. The reaction is completed,Add 70 ml EA and 100 ml 15% aqueous citric acid solution.The organic layer was separated. The organic layer was washed successively with citric acid, water and saturated brine.Dry over anhydrous sodium sulfate and filter.The filtrate was concentrated to dryness under reduced pressure to give a pale yellow oil.Crystallization with methyl tert-butyl ether gave 86 mg of an off-white solid.The yield was 40%. |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; for 1h; | [0439] The off-white solid intermediate (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12 M) and to the resulting solution was added bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and D3EA (3.66 mL, 21.0 mmol, 1.5 eq.). The reaction was complete in 1 h according to HPLC. The reaction mixture was concentrated to provide an oil that is precipitated with EtOAc, and then triturated with EtOAc (ca. 25 mL). The solute was further precipitated with ether (ca. 200 mL) and triturated for 15 min. The solid was filtered and dried under high vacuum to provide Compound AB which is 93% pure EPO <DP n="168"/>according to HPLC and used in the next step without further purification. Yield: 9.7 g (94%). | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; for 1h; | [0455] The off-white solid intermediate (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12 M) and to the resulting solution was added bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and DIEA (3.66 mL, 21.0 mmol, 1.5 eq.). The reaction was complete in 1 h according to HPLC. The reaction mixture was concentrated to provide an oil that was precipitated with EtOAc, and then triturated with EtOAc (ca. 25 mL). The solute was further precipitated with ether (ca. 200 mL) and triturated for 15 min. The solid was filtered and dried under high vacuum to provide Compound AB which was 93% pure according to HPLC and used in the next step without further purification. Yield: 9.7 g (94%). | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; for 1h; | The off-white solid intermediate (8.0 g, 14.0 mmol) was diluted with DMF (120 mL, 0.12 M) and to the resultingsolution was added bis(4-nitrophenyl)carbonate (8.5 g, 28.0 mmol, 2.0 eq.) and DIEA (3.66 mL, 21.0 mmol, 1.5 eq.).The reaction was complete in 1 h according to HPLC. The reaction mixture was concentrated to provide an oil that wasprecipitated with EtOAc, and then triturated with EtOAc (ca. 25 mL). The solute was further precipitated with ether (ca.200 mL) and triturated for 15 min. The solid was filtered and dried under high vacuum to provide Compound AB whichwas 93% pure according to HPLC and used in the next step without further purification. Yield: 9.7 g (94%). | |
0.42 g | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; | dissolving the reaction product of reaction step e in 12 mL of DMF,After dissolution, (PNP) 2CO (1.1 g, 3.6 mmol) and DIEA 0.5 mL were added.Stir at room temperature overnight. After the reaction is completed, the solvent is evaporated.Separation and purification on a silica gel column, the separation conditions are dichloromethane:Methanol = 20:1.The product is a yellowish solid0.42g. |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; | Compound 7 (1.3 g, 2.16 rnrnoi) and bis(4~nitrophenyl) carbonate (1.34 g, 4.34 rnrnol) were combined and dissolved in DMF (6 mb). To this solution was added DIPEA (0.75 mL, 4.35 mmol). The mixture was stirred overnight at room temperature. The reaction mixture was evaporated to dryness and the residue was washed with ether. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
38% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; for 1h; | Generalized procedure for the preparation of 47a-47f from 46a/46b: A solution of crude 46a or 46b (71 mumol) in DMF (1 mL) was cooled to 0 0C. p-nuitrophenyl carbonate-activated linker 57a, 57b, or 58 (0.142 mmol) and DIPEA (29 muL, 0.177 mmol) were added and the reaction temperature was subsequently increased to room temperature. After 1 h, the reaction mixture was concentrated to dryness. The residue was purified by column chromatography (CH2Cl2 / MeOH = 5:1) to afford the product as an off-white solid. Compounds 47e and 47f were prepared from their Aloe-protected analogs by dissolution in dichloromethane and addition of Pd(PPh3 )4 (0.1 equiv.) and morpholine (10 equiv.). After 1 h, the reaction mixture was concentrated, the residue was redissolved in dichloromethane, and the solution was acidified (pH 3) with trifluoroacetic acid. The solution was concentrated and the residue purified by preparative HPLC (acetonitrile / aqueous ammonium formate) to afford the product as a white solid. Compound 47a: 38% yield; 1H-NMR (400 MHz, CDCI3/CD3OD): delta = 0.94 (d, J = 6.4 Hz, 6 H, Hvai), 1.26 - 135 (m, 2 H, 2 x Hcapr), 1.49 - 1.74 (m, 10 H, 10-CH3, 7 x HCIt/Hcapr), 1.87 (m, 1 H, Hat), 2.06 (m, IH, Hvai), 2.26 (t, J = 7.6 Hz, 2 H, CH2C(O)), 2.49 (s, 6 H, N(CH3)2), 2.90 - 3.22 (m, 10 H, NCH2,clt, 2 x NCH3, OCH2CH2N), 3.46 - 3.83 (m, 6 eta, NCH2CH2N, CH2,capr), 4.09 (m, 1 H5 1-H), 4.15-4.21 (m, 3 H, OCH2CH2N, alpha-Hvai), 4.53 - 4.69 (m, 3 H, 2a-H, 10-H, alpha-Hclt), 4.86 (m, 1 H, 2b-H), 5.06-5.14 (m, 2 H, CH2OC(O)), 6.72 (s, 2 H, CH=CH), 7.00 - 7.06 (m, 2 H, 6'-H, 3'-H), 7.17 (m, IH, 4'-H), 7.23 - 7.61 & 7.79 - 7.93 (m, 9 H, 7'-H, 7-H, 8-H, 6-H, 9-H, 4 x HA^PABA), 8.22 (br. s, 1 H, 4-H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
38% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | Example 47Generalized procedure for the preparation of 47a-47f from 46a/46b: A solution of crude 46a or 46b (71 mumol) in DMF (1 mL) was cooled to 0 C. p-Nitrophenyl carbonate-activated linker 57a, 57b, or 58 (0.142 mmol) and DIPEA (29 muL, 0.177 mmol) were added and the reaction temperature was subsequently increased to room temperature. After 1 h, the reaction mixture was concentrated to dryness. The residue was purified by column chromatography (CH2Cl2/MeOH=5:1) to afford the product as an off-white solid. Compounds 47e and 47f were prepared from their Aloc-protected analogs by dissolution in dichloromethane and addition of Pd(PPh3)4 (0.1 equiv.) and morpholine (10 equiv.). After 1 h, the reaction mixture was concentrated, the residue was redissolved in dichloromethane, and the solution was acidified (H 3) with trifluoroacetic acid. The solution was concentrated and the residue purified by preparative HPLC (acetonitrile/aqueous ammonium formate) to afford the product as a white solid.Compound 47a: 38% yield; 1H-NMR (400 MHz, CDCl3/CD3OD): delta=0.94 (d, J=6.4 Hz, 6H, Hval), 1.26-135 (m, 2H, 2×Hcapr), 1.49-1.74 (m, 10H, 10-CH3, 7×Hcit/Hcapr), 1.87 (m, 1H, Hcit), 2.06 (m, 1H, Hval), 2.26 (t, J=7.6 Hz, 2H, CH2C(O)), 2.49 (s, 6H, N(CH3)2), 2.90-3.22 (m, 10H, NCH2,cit, 2×NCH3, OCH2CH2N), 3.46-3.83 (m, 6H, NCH2CH2N, CH2,capr), 4.09 (m, 1H, 1-H), 4.15-4.21 (m, 3H, OCH2CH2N, alpha-Hval), 4.53-4.69 (m, 3H, 2a-H, 10-H, alpha-Hcit), 4.86 (m, 1H, 2b-H), 5.06-5.14 (m, 2H, CH2OC(O)), 6.72 (s, 2H, CHCH), 7.00-7.06 (m, 2H, 6'-H, 3'-H), 7.17 (m, 1H, 4-H), 7.23-7.61 & 7.79-7.93 (m, 9H, 7'-H, 7-H, 8-H, 6-H, 9-H, 4×HAr,PABA), 8.22 (br. s, 1H, 4-H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
72% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 1.5h;Inert atmosphere; | A flame-dried flask was charged with benzylamine 7 (7.3 mg, 9.5 mumol, 1 eq) dissolved in anhydrous dimethylformamide (0.2 mL). Maleimidocaproyl-Valine-Citruiline-PAB-OCO-pNP (Dubowchik et al., Bioconjugate Chemistry, 2002, 13, 855-869) (7 mg, 9.5 pmol. 1 eq) was added, followed by diisopropylethylamine (16.5 pL, 95 muetaiotaomicronIota, 10 eq), the reaction was then stirred at room temperature under a nitrogen atmosphere. LC revealed conversion to product after 1.5 hours; the reaction was diluted with dichloromethane and loaded directly on to a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 80:20) to provide purified drug linker 9 (9.3 mg, 72%). TLC: Rf MS: tR 12.61 min, m/z (ES+) found 1366.8 (M+H)*. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
38% | With N-ethyl-N,N-diisopropylamine; In 1-methyl-pyrrolidin-2-one; at 23℃; for 9h; | DIPEA (10 muIota_, 0.06 mmol) was added to a solution of Compound 9 (13 mg, 0.02 mmol), prepared as shown in the Preparative Example above, and Compound 8 (20 mg, 0.02 mmol), prepared as described in Example 2 above, in NMP (6.5 mL) at 23 C. After 9 h the reaction mixture was diluted with H20 and extracted with EtOAc. The combined organic layers were dried over anhydrous Na2S04, filtered, and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (Si02, DCM:CH3OH, from 100:0 to 90:10) to afford pure target Compound 28 (9 mg, 38%).1H NMR (400 MHz, CDCI3/CD3OD): delta 9.40 (s, 1 H), 8.92 (d, J = 10.6 Hz, 1 H), 7.67 (d, J = 7.7 Hz, 1 H), 7.47 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 7.9 Hz, 2H), 7.16 (t, J = 1 1 .9 Hz, 1 H), 6.97 (t, J = 9.6 Hz, 2H), 6.82 (t, J = 1 1 .4 Hz, 1 H), 6.67-6.64 (m, 1 H), 6.64 (s, 2H), 6.08 (d, J = 1 1 .7 Hz, 1 H), 5.68 (d, J = 1 1 .4 Hz, 1 H), 5.62-5.58 (m, 1 H), 5.54-5.47 (m, 1 H), 5.35-5.29 (m, 1 H), 5.20 (d, J = 9.9 Hz, 1 H), 4.94 (s, 2H), 4.77 (q, J = 8.1 Hz, 1 H), 4.54-4.45 (m, 2H), 4.37 (d, J = 9.2 Hz, 1 H), 4.20-4.12 (m, 1 H), 4.08 (t, J = 7.8 Hz, 1 H), 3.58 (s, 3H), 3.42 (t, J = 7.2 Hz, 2H), 3.18- 3.00 (m, 7H), 2.81 -2.75 (m, 1 H), 2.35-2.30 (m, 3H), 2.29-2.25 (m, 3H), 2.17 (t, J = 7.2 Hz, 2H), 2.14-2.06 (m, 1 H), 2.04-1.92 (m, 1 H), 1 .86-1.74 (m, 1 H), 1 .76 (s, 3H), 1 .61 -1.42 (m, 10H), 1.54 (d, J = 6.3 Hz, 3H), 1 .30-1.14 (m, 4H), 1.08 (d, J = 6.4Hz, 3H), 0.94 (s, 9H), 0.86 (dd, J = 6.8, 4.3 Hz, 6H).13C NMR (100 MHz, CDCI3): delta 173.0, 172.1 , 171 .0, 170.2, 168.5, 167.1 , 162.0, 161.3, 157.2, 157.0, 145.0, 140.2, 137.7, 137.5, 137.1 , 134.0, 132.4, 128.8, 126.9, 124.9, 124.4, 124.3, 124.0, 120.5, 1 19.8, 108.6, 107.3. 81.9, 74.8, 66.1 , 60.4, 58.8, 55.4, 37.6, 37.2, 36.0, 34.8, 31.9, 31.6, 30.9, 30.7, 30.0, 29.7, 29.3, 28.1 , 26.5, 26.2, 26.1 , 25.1 , 19.2, 18.3, 17.1 , 16.5, 12.9. ESI-MS m/z: Calcd. for C63H90N10O15: 1226.7. Found: 1267.4 ( +H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
71% | With pyridine; benzotriazol-1-ol; In N,N-dimethyl-formamide; at 20℃; for 24h; | Example 29 Synthesis of N-alkyl maleimido-Val-Cit-PAB-MMAE mc-ValCit-PAB-OH PNP Carbonate "N-alkyl maleimide Val-Cit-PAB-MMAE" Maleimidocaproyl-valine-citrulline-/7-aminobenzylcarbonyl-mono- methyl-Auristatin-E (mc- ValCit-PABC-MMAE) was prepared according to the literature method with modifications.1 Thus, a mixture of mc-Val-Cit-p-aminobenzyl alcohol p-nitrophenylcarbonate (8) freshly prepared according to literature method2 (265 mg, 0.36 mmol, 1.5 eq.), MMAE (9) (169 mg, 0.24 mmol, 1 eq.) and N-hydroxybenzotriazole (HOBt) (6.48 mg, 0.048 mmol, 0.2 eq.) were stirred in DMF (5 mL) at r.t. for 2 min., followed by addition of pyridine (38 mg, 0.48 mmol, 2 eq.). After stirring for 24 h, volatile organics were removed under vacuum. The residue obtained was triturated with ethyl acetate and methanol (1L) to afford mc-Val-Cit-PAB- MMAE (10) as a white powder (220 mg, 0.17 mmol, 71 % yield) that was usually >95% pure by RP-HPLC analysis. If necessary, further purification can be carried out by C18 reversed- phase preparative HPLC or size exclusive column chromatography. Electrospray (ES)-MS m/z 1339 (M+Na, base peak), 1317 (M + 1). |
65% | With 2,6-dimethylpyridine; benzotriazol-1-ol; In N,N-dimethyl acetamide; at 40℃; | A compound of the following formula (1 eq.): and mc-Val-Cit-PABC-PNP (1.18 eq.) were solubilized in N,N-dimethylacetamide (7.87 vol.). l-Hydroxybenzotriazole (HOBt) hydrate (8.95 wt%) and 2,6-lutidine (2.315 vol.) were then added and the reaction mixture was stirred at 40C for 12-16 hours until reaction was complete. The reaction mixture was cooled to 20C and added into tert-Butyl methyl ether (168 vol.). The resultant slurry was stirred for 3-5 hours and filtered, washed and dried under vacuum. Crude product was purified by column purification and product-containing fractions were concentrated to dryness and slurried in Ethyl acetate (20 vol.) before being isolated by filtration, washed and dried (65% yield). MS: m/e 1317 (MH)+, 1339 (M+Na)+. |
Ca. 42% | 20 mg mc-vc-PAB-PNP (1.5 eq) and 3 mg HOBT were added to 2 ml DMF.After stirring for a while at room temperature,Add 13mg MMAE,0.5ml pyridine,25ulDIEA.The reaction was stirred at room temperature for 2 days.After the reaction is completed,The reaction solution is directly purified using a preparative column.Collect the required ingredients and lyophilize them after concentrationGet about 10mg product,The yield is about 42%. |
Ca. 42% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 48h; | 20mg of mc-vc-PAB-PNP (1.5 eq) and 3 mg of HOBT were added to 2 ml of DMF. After stirring for a while at room temperature,Add 13 mg MMAE, 0.5 ml pyridine,25ulDIEA. The reaction solution was stirred at room temperature for 2d.. After the reaction is completed, the reaction solution is directly purified by a preparative column.The desired components are collected and concentrated and lyophilized to obtain about 10 mg of the product.The yield is about 42%. |
With pyridine; benzotriazol-1-ol; In N,N-dimethyl-formamide; for 24h; | Preparation Method of Compound 20-10: MMAE (100.5 mg, 0.14 mmol, 1 eq. ) , Compound 20-9 (110.6 mg, 0.15 mmol, 1.1 eq. ) and HPBt (19 mg, 0.14 mmol, 1.0eq. ) were diluted with DMF (2 mL) . After 2 minutes, pyridine (0.5 mL) was added and the reaction was monitored by reverse-phase HPLC. The reaction was complete after about 24 hours. The mixture was concentrated and the resulting residue was purified using reverse phase preparative-HPLC (Varian Dynamax column 21.4 mm×25 cm, 5mu,through gradient elution of MeCN and Et3N-CO2 (pH 7) at 20 mL/minutes from 10 to 100 for 40 minutes. The relevant fractions were pooled and concentrated to give an off-white solid intermediate, i.e., Compound 20-10. MS [M] +: 1, 315.78. | |
10.7 g | MMAE (7.18g. lOmmol) was measured and added into a 250m1 three-neck flask, dissolved in anhydrous DMF, and stirred till clear at room temperature under N2 protection. Mc-Val-Cit-PAB-PNP (7.37g) and HOAt (72mg, 2mmol) were added into the solution and reacted for 5 mm, and then DIEA (3.5m1, 2Ommol) was added drop-wise, the reaction was continued for 30 mm at roomtemperature, and then the temperature was increased to 40?SOC and reacted for 20h, during which HPLC was used to monitor the reaction. DMF was removed by drying in vacuo, and the product Mc-Val-Cit-PAB-MMAE (10.7g. purity: 99.3%) was obtained by further HPLC purification. | |
With pyridine; benzotriazol-1-ol; In N,N-dimethyl-formamide; at 20℃; | MC-VC-PAB-PNP (93 mg, 0.125 mmol) and MMAE (60 mg, 0.083 mmol) were dissolved in 4 mL DMF.Add HoBt (10 mg, 0.074 mmol) and 0.5 mL of pyridine in sequence.The reaction was stirred at room temperature overnight.The reaction product is isolated and purified using a preparative liquid phase.The chromatographic conditions were as follows: phase A H2O + 0.08% TFA, phase B acetonitrile + 0.08% TFA. 0-35min B phase 65% isocratic elution,The pure product was collected and the reaction product was lyophilized. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
64% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 18h; | Synthesis of Maleimido-val-cit-PABQCQ-Tubulysin/Cytolysin-TAM461: TAM461 (Tubulysin/Cytolysin):DMF:3 mL30.0 mg (0.041 mmol)TAM465 (Linker):HOEt:DIPEA:35 mg (0.045 mmol)1.4 mg10 pLTAM461 and TAM465 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOEt and DIPEA. The reaction was stirred at RT for 18h. The reaction mixture was concentrated and the resulting oil was purified by column chromatography using 2-6% methanol: DCM to give 35 mg (64%) of TAM467 as a white solid. ESI-MS: m/z = 1371 [M+H] |
64% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 18h; | TAM461: 30.0 mg (0.041 mmol) DMF: 3 mL TAM465 : 35 mg (0.045 mmol) HOBt : 1.4 mg DIPEA: 10 pL TAM461 and TAM465 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18h. The reaction mixture was concentrated and the resulting oil was purified by column chromatography using 2-6% methanol: DCM to give 35 mg (64%) of TAM467 as a white solid. ESI-MS: m/z = 1371 [M+H] . |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
62% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 18h; | Synthesis of Maleimido-val-cit-PABQCQ-Tubulysin/Cytolysin-TAM47O: TAM47O (Tubulysin/Cytolysin):DMF: 5 mLTAM466 (Linker):HOEt:DIPEA:TAM47O and TAM466 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOEt and DIPEA. The reaction was stirred at RT for 18h and then analysed with TLC, indicating completion of reaction, The reaction mixture was concentrated and the resulting oil was purified with column chromatography using 4-12% methanol: DCM to give 56mg of TAM471 (yield: 62%) . ESI-MS: 1384.6 [P4+1]In vitro activity testing is performed. Functional activity is evaluated through microtubule inhibition assay, while cytotoxic activity is determined through crystal violet viability assay.0.07 mmol50 mg (0.065 mmol)2.4 mg18 pL |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
62% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 18h; | TAM470 and TAM466 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18h and then analysed with TLC, indicating completion of reaction, The reaction mixture was concentrated and the resulting oil was purified with column chromatography using 4-12% methanol: DCM to give 56mg of TA 471 (yield: 62%) . ESI-MS: 1384.6 [M+l] . |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
64% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 18h; | TAM461 and TAM465 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18h. The reaction mixture was concentrated and the resulting oil was purified by column chromatography using 2-6% methanol: DCM to give 35 mg (64%) of TAM467 as a white solid. ESI-MS: m/z = 1371 [M+H] . |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
62% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 18h; | TAM470 (Tubulysin/Cytolysin) : 0.07 mmol DMF: 5 mL TAM466 (Linker) : 50 mg (0.065 mmol) HOBt : 2.4 mg DIPEA: 18 pL TAM470 and TAM466 were dissolved in anhydrous DMF under dry conditions and the resulting solution was treated with HOBt and DIPEA. The reaction was stirred at RT for 18h and then analysed with TLC, indicating completion of reaction, The reaction mixture was concentrated and the resulting oil was purified with column chromatography using 4-12% methanol: DCM to give 56mg of TAM471 (yield: 62%) . ESI-MS: 1384.6 [M+l] . |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
99% | In N,N-dimethyl-formamide; at 20 - 25℃; for 2h;Inert atmosphere; | Preparation Example 1-2: Preparation of Compound (II-3) (0054) (0055) Under an argon stream, 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-((S)-1-(((S)-1-((4-(hydroxymethyl)phenyl)amino)-1-oxo-5-ureidopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(5.0 g, 8.7 mmol, Dubowchik et al., Bioconjugate Chem., 2002, 13 (4), pp 855-869) was dissolved in 60 mL of anhydrous dimethylformamide, added with N, N-diisopropylethylamine (3.0 mL, 17.4 mmol), and then cooled to 0C. To the mixture was added at once bis(4-nitrophenyl) carbonate (7.94 g, 26.1 mmol), followed by stirring at room temperature for 15 hrs. After completion of the reaction, the reaction mixture was concentrated in a high vacuum, and the concentrate was purified by silica gel column chromatography to obtain 4-((S)-2-((S)-2-(6-(2 ,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (3.65 g, 57 %) as a pale yellow solid. (0056) 1H NMR (400 MHz, DMSO-d6) delta 0.83 (d, J = 6.8 Hz, 3 H), 0.86 (d, J = 6.8 Hz, 3 H), 1.09 (t, J = 7.2 Hz, 1 H), 1.19 (m, 2 H), 1.34 - 1.76 (m, 7 H), 1.96 (m, 1 H), 2.15 (m, 2 H), 2.99 (m, 2 H), 3.37 (m, 2 H), 4.19 (t, J= 7.8 Hz, 1 H), 4.39 (m, 1 H), 5.24 (s, 2 H), 5.41 (s, 2 H), 5.97 (brt, J= 5.6 Hz, 1 H), 7.00 (s, 2H), 7.41 (d, J= 8.4 Hz, 2 H), 7.57 (d, J= 7.2 Hz, 2 H), 7.65 (d, J= 8.4 Hz, 2 H), 7.80 (d, J= 8.4 Hz, 1 H), 8.09 (d, J = 7.2 Hz, 1 H), 8.31 (d, J = 7.2 Hz, 2 H), 10.05 (brs, 1 H) (0057) Under an argon stream, 4-((S)-2-((S)-2-(6-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (3.65 g, 4.95 mmol) was dissolved in 90 mL of anhydrous dimethylformamide, and stirred, together with t-butyl (2-aminoethyl)(methyl)carbamate (0.86 g, 4.95 mmol), at 20 - 25C for 2 hrs. After completion of the reaction, the reaction mixture was completely concentrated in a high vaccum, and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-3) (3.8 g, 99%). (0058) LC-MS m/z: 773.5 [M+H]+ (0059) To a solution of the amino-protected derivative of compound (II-3) (146 mg, 0.186 mmol) in 5 mL of dichloromethane was dropwise added 2 mL of trifluoroacetic acid, followed by stirring at 20 - 25C for 2 hrs. After completion of the reaction, the reaction solvent was removed by vacuum concentration, and then trifluoroacetic acid was completely removed by adding 5 mL of toluene twice to obtain a concentrated TFA salt of the title compound. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
83% | With 2,6-dimethylpyridine; 1-hydroxy-7-aza-benzotriazole; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl acetamide; at 20℃; for 0.666667h; | To a stirring solution of 212 (750 mg, 1.02 mmol) and 213 tert-butyl methyl[2-(methylamino)ethyl]carbamate (192 mg, 1.02 mmol) in 6 mL of DMA, 2-6-Lutidine (0.236 mL, 2.03 mmol) was added followed by Hunig's base (0.354 mL, 2.03 mmol) and HOAT (69.1 mg, 0.5 mmol). Reaction was allowed to stir at room temperature for 40 minutes. Reaction was injected onto a 25 g C18 pre-column (which was previously equilibrated with acetonitrile and then water, with 0.02% TFA in each phase) and then purified by medium pressure reverse phase C18 chromatography (Gradient: 5% to 45% acetonitrile in water with 0.02% TFA in each phase) with the appropriate test tubes concentrated using a genevac producing 214 (663 mg, 83%) as a white solid. LC-MS (Protocol B): m/z 787.3 [M+H]+, retention time=1.45 minutes. |
71% | In N,N-dimethyl-formamide; at 20 - 25℃; for 19h;Inert atmosphere; | Preparation Example 1-3: Preparation of Compound (II-4)[0060][0061] Under an argon stream, <strong>[159857-81-5]4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate</strong> (6.8 g, 9.22 mmol) was dissolved in 150 mL of anhydrousdimethylformamide, and stirred, together with t-butyl methyl(2-(methylamino)ethyl)carbamate (1.82 g, 9.68 mmol) at 20- 25C for 19 hrs. After completion of the reaction, the reaction mixture was completely concentrated in a high vacuum,and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-4) (5.14 g, 71%).[0062]1H NMR (400 MHz, DMSO-d6) delta0.78-0.8 (d, 3 H), 0.8-0.83 (d, 3 H), 1.09 -1.99 (m, 10 H), 1.33 (s, 9 H), 1.92(m, 1 H), 2.07-2.19 (m, 2H), 2.66-2.73 (d, 3H), 2.8-2.81 (m, 3H), 2.9-3.0 (m, 2H), 3.37 (m, 2 H), 4.15 (t, 1 H), 4.34 (m,1 H), 4.94 (s, 2 H), 5.38 (s, 2 H), 5.94 (brt, 1 H), 6.96 (s, 2H), 7.23 (d, 2 H), 7.55 (d, 2 H), 7.77 (d, 1 H), 8.04 (d, 1 H),9.95 (brs, 1 H)[0063] LC-MS m/z: 787.5 [M+H]+[0064] With the exception of using the amino-protected derivative of compound (II-4), the same procedure as inPreparation Example 1-2 was repeated to obtain a concentrated TFA salt of the title compound. |
53% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 3h; | To a solution of MC-Val-Cit-PABC-PNP (93.0 mg, 0.126 mmol) in DMF (2 mL) was added N-Boc,N,N?-dimethylethylenediamine (23.7 mg, 0.126 mmol). Diisopropylethylamine (0.20 mL) was added, and the reaction stirred at room temperature for 3 h. The reaction mixture was acidified by adding acetic acid (0.50 mL), and purified by preparative HPLC (5% to 95% acetonitrile in water with 0.1% TFA) to give 19? (53.2 mg, 0.0676 mmol, 53% yield). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
59% | In N,N-dimethyl-formamide; at 20 - 25℃; for 15h;Inert atmosphere; | Preparation Example 1-4: Preparation of Compound (II-5)[0065][0066] Under an argon stream,4-((S)-2-((S)-2-(6-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (6.8 g, 9.22 mmol) was dissolved in 80 mL of anhydrousdimethylformamide, and stirred, together with t-butyl (2-aminoethyl)carbamate (1.63 g, 10.14 mmol), at 20 - 25C for 15hrs. After completion of the reaction, the reaction mixture was completely concentrated in a high vacuum, and theconcentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-5) (4.13 g, 59 %).[0067]1H NMR (400 MHz, DMSO-d6) delta0.78-0.8 (d, 3 H), 0.8-0.83 (d, 3 H), 1.09 -1.99 (m, 10 H), 1.33 (s, 9 H), 1.92(m, 1 H), 2.07-2.19 (m, 2H), 2.9-3.0 (m, 2H), 3.37 (m, 2 H), 4.15 (t, 1 H), 4.34 (m, 1 H), 4.94 (s, 2 H), 5.38 (s, 2 H), 5.94(brt, 1 H), 6.76 (m, 1 H), 6.96 (s, 2H), 7.15 (m, 1H), 7.23 (d, 2 H), 7.55 (d, 2 H), 7.77 (d, 1 H), 8.04 (d, 1 H), 9.95 (brs, 1H)[0068] LC-MS m/z: 759.5 [M+H]+[0069] With the exception of using the amino-protected derivative of compound (II-5), the same procedure as inPreparation Example 1-2 was repeated to obtain a concentrated TFA salt of the title compound |
53% | In N,N-dimethyl-formamide; at 0℃; for 2h; | 4-Nitrophenyl carbonate 14 (lOOmg, 0.136 mmol, 1 eq) was dissolved in 5 ml anhydrous DMF, cooled to 0 C and treated with tert-butyl (2-aminoethyl)carbamate (21.4 uL, 0.136 mmol, 1 eq). Reaction mixture was stirred for 2h, concentrated and purified by column chromatography (gradient 2-50% MeOH/DCM) to result in off-white solid (55mg, 53%). LCMS (analytical method A): Rt =1.73 min, [M+H]+ = 759.38. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
48% | In N,N-dimethyl-formamide; at 20 - 25℃; for 15h;Inert atmosphere; | Preparation Example 1-5: Preparation of Compound (II-6)[0070][0071] Under an argon stream, 4-((S)-2-((S)-2-(6-(2, 5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.74 g, 1 mmol) was dissolved in 30 mL of anhydrousdimethylformamide, and stirred, together with t-butyl (2-(methylamino)ethyl)carbamate (0.25 g, 1.4 mmol), at 20 - 25Cfor 15 hrs. After completion of the reaction, the reaction mixture was concentrated to the completion in a high vacuum,and the concentrate was purified by silica gel column chromatography to obtain an amino-protected derivative of compound (II-6) (0.36 g, 48 %).[0072]1H NMR (400 MHz, DMSO-d6) delta0.79 (d, 3 H), 0.82 (d, 3 H), 1.13-1.7 (m, 10 H), 1.32-1.39 (d, 9 H), 1.89-1.93(m, 1 H), 2.07-2.17 (m, 2 H), 2.79-2.82 (d, 2 H), 2.89-3.02(m, 5H), 3.14 (d, 1 H), 3.2 (m, 1H), 4.15 (t, 1 H), 4.34 (m, 1H), 4.9-4.94 (d, 2 H), 5.37 (s, 2 H), 5.941 (brt, 1 H), 6.81-6.85 (m, 1 H), 7.24 (d, 2H), 7.54 (d, 2 H), 7.76 (d, 1 H), 8.04(d, 1 H), 9.95 (brs, 1 H) [0073] LC-MS m/z: 773.5 [M+H]+[0074] With the exception of using the amino-protected derivative of compound (II-6), the same procedure as inPreparation Example 1-2 was repeated to obtain a concentrated TFA salt of the title compound. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
74% | With pyridine; benzotriazol-1-ol; In N,N-dimethyl-formamide; at 0 - 20℃; for 15h;Inert atmosphere; | Preparation Example 3-1: Preparation of Compound (IV-5) (0227) (0228) Under an argon stream, compound (II-1) (0.37 g, 0.50 mmol), prepared in Preparation Example 1-1, was added to a solution of compound (III-5) (0.36 g, 0.50 mmol), prepared in Preparation Example 2-1, in 14 mL of anhydrous dimethylformamide. The reaction mixture was cooled to 0C, and was sequentially added with HOBt (81 mg, 0.60 mmol) and 3.5 mL of anhydrous pyridine before being stirred at room temperature for 15 hrs. After completion of the reaction, vacuum concentration was carried out, and the residue was purified by silica gel column chromatography to afford the title compound as an ivory solid (0.49 g, 74%). (0229) MALDI-TOF MS m/z : 1345.6 [M+Na]+, 1361.6 [M+K]+ |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
73% | With pyridine; benzotriazol-1-ol; In N,N-dimethyl-formamide; at 0 - 20℃; for 17h;Inert atmosphere; | Preparation Example 3-7: Preparation of Compound (IV-11) (0245) (0246) Under an argon stream, compound (III-11)(89 mg, 0.116 mmol), obtained in Preparation Example 2-7, was dissolved in 8 mL of anhydrous dimethyl formamide, and added with compound (II-1)(78 mg, 0.106 mmol), obtained in Preparation Example 1-1. The reaction mixture was cooled to 0C, and was sequentially added with HOBt (17 mg, 0.128 mmol) and 2.0 mL of anhydrous pyridine before being stirred at room temperature for 17 hrs. After completion of the reaction, vacuum concentration was carried out, and the residue was purified by silica gel column chromatography to afford the title compound (106 mg, 73%). (0247) LC-MS m/z: 1377.8 [M+]+, 1400.8 [M+Na]+ |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
51% | With pyridine; benzotriazol-1-ol; In N,N-dimethyl-formamide; at 0 - 20℃; for 15h;Inert atmosphere; | Preparation Example 3-14: Preparation of Compound (IV-18) (0272) (0273) Under an argon stream, compound (III-18) (0.36 g, 0.48 mmol), obtained in Preparation Example 2-14, was dissolved in 10 mL of anhydrous dimethyl formamide, and added with compound (II-1)(0.32 g, 0.43 mmol), obtained in Preparation Example 1-1. The reaction mixture was cooled to 0C, and was sequentially added with HOBt (70 mg, 0.52 mmol) and 2.5 mL of anhydrous pyridine before being stirred at room temperature for 15 hrs. After completion of the reaction, vacuum concentration was carried out, and the residue was purified by silica gel column chromatography to afford the title compound as an ivory solid (0.29 g, 51%). (0274) LC-MS m/z : 1353 [M+]+, 1375 [M+Na]+ |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
82% | With pyridine; benzotriazol-1-ol; In N,N-dimethyl-formamide; at 0 - 20℃; for 15h;Inert atmosphere; | General procedure: Preparation Example 3-14: Preparation of Compound (IV-18) (0272) (0273) Under an argon stream, compound (III-18) (0.36 g, 0.48 mmol), obtained in Preparation Example 2-14, was dissolved in 10 mL of anhydrous dimethyl formamide, and added with compound (II-1)(0.32 g, 0.43 mmol), obtained in Preparation Example 1-1. The reaction mixture was cooled to 0C, and was sequentially added with HOBt (70 mg, 0.52 mmol) and 2.5 mL of anhydrous pyridine before being stirred at room temperature for 15 hrs. After completion of the reaction, vacuum concentration was carried out, and the residue was purified by silica gel column chromatography to afford the title compound as an ivory solid (0.29 g, 51%). (0274) LC-MS m/z : 1353 [M+]+, 1375 [M+Na]+ |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
71% | With pyridine; benzotriazol-1-ol; In N,N-dimethyl-formamide; at 20℃; for 24h; | Maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonylmonomethyl-Auristatin-E (mc-Val-Cit-PAB-MMAE) was preparedaccording to the literature method with modifications [28,29].A mixture of freshly prepared mc-Val-Cit-p-aminobenzyl alcoholp-nitrophenylcarbonate (265 mg, 0.36 mmol, 1.5 eq.), MMAE(169 mg, 0.24 mmol, 1 eq.) and N-hydroxybenzotriazole (HOBt)(6.48 mg, 0.048 mmol, 0.2 eq.) in DMF (5 mL) was stirred at r.t. for2 min., followed by addition of pyridine (38 mg, 0.48 mmol, 2 eq.).After stirring for 24 h, volatile organics were removed under vacuum.The residue obtained was successively triturated with ethyl acetateand methanol (1 L) to afford the target compound mc-Val-Cit-PABMMAE(4) (220 mg, 0.17 mmol, 71% yield) as white powder thatwas N95% pure by RP-HPLC analysis. MS (ESI+) m/z 1339 (M+Na,base peak), 1317 (M+1). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
44% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃;Sealed tube; | In a small vial, 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl 4-nitrophenyl carbonate 6 (5 mg, 0.006777 mmol, 1.0 equiv., 5 mg) and (3S)-3-[[(2S)-4-amino-2-[[(2S)-2-(decanoylamino)-3-(1H-indol-3-yl)propanoyl]amino]-4-oxo-butanoyl]amino]-4-[[(3S,6S,9S,15S,18R,21S,24R,30S,31S)-3-[2-(2-aminophenyl)-2-oxo-ethyl]-24-(3-aminopropyl)-15,21-bis(carboxymethyl)-9-(hydroxymethyl)-6-[(1S)-3-hydroxy-1-methyl-3-oxo-propyl]-18,31-dimethyl-2,5,8,11,14,17,20,23,26,29-decaoxo-1-oxa-4,7,10,13,16,19,22,25,28-nonazacyclohentriacont-30-yl]amino]-4-oxo-butanoic acid (<strong>[103060-53-3]daptomycin</strong>, Enzo Life Science, CatNo. BML-A201-0020, 1 equiv., 0.006777 mmol, 1.00 equiv., 10.98 mg) were taken up in DMF (0.2 mL, 3 mmol, 400 equiv., 200 mg). To this was added N,N-diisopropylethylamine (1.5 equiv., 0.01017 mmol, 1.500 equiv., 1.327 mg) followed by 1-hydroxybenzotriazole (HOBt, 0.3 equiv., 0.002033 mmol, 0.3000, 0.2775 mg). The mixture was stirred at RT sealed for 4 hours then stirred overnight. The mixture was diluted with DMF, purified via HPLC under acidic condition FAH2O/MeCN to give 6.6 mg of MC-vc-PAB-<strong>[103060-53-3]daptomycin</strong> 54 in 44% yield. M/Z=1622 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
23% | With N-ethyl-N,N-diisopropylamine In N,N-dimethyl-formamide for 3h; | 7 MC-vc-PAB-Sitafloxacin 57 In a small vial, 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl 4-nitro henyl carbonate 6 (35 mg, 0.04744 mmol, 1.000, 35 mg) and 7-[(7S)-7-amino-5-azaspiro[2.4]heptan-5-yl]-8-chloro-6-fluoro-1-[(1R,2S)-2-fluorocyclopropyl]-4-oxo-quinoline-3-carboxylic acid (sitafloxacin, Toronto Research Chemicals CatNo.S490920, 1 equiv., 0.04744 mmol, 1.000, 19.44 mg) were taken up in DMF (0.2 mL, 3 mmol, 50, 200 mg). To this was added N,N-diisopropylethylaminde (1.5 equiv., 0.07116 mmol, 1.500, 9.290 mg). The reaction stirred for 3 hours, diluted with DMF, and purified directly on the HPLC under acidic condition FAH2O/MeCN to give MC-vc-PAB-sitafloxacin 57 23% yield. M/Z: 1008.6 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With acetic acid; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 2h; | Example 1.1.14 (72 mg) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl(4-nitrophenyl)carbonate (91 mg) in N,Ndimethylformamide(3 mL) was cooled in a water-ice bath and N,N-diisopropylethylamine (0.12 mL) wasadded. The mixture was stirred at 0 C. for 2 hours and acetic acid (0.057 mL) was added. Afterconcentration of the solvents, the residue was purified via HPLC (20-80% acetonitrile in 0.1% TFA/water) toprovide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) delta ppm 9.98 (s, 1H), 8.40 (s, 1H),8.06 (d, 1H), 8.00 (d, 1H), 7.74-7.89 (m, 4H), 7.59 (d, 2H), 7.46 (s, 2H), 7.37 (t, 1H), 7.18-7.32 (m, 4H), 6.99(s, 2H), 6.01 (s, 1H), 4.98 (s, 3H), 4.38 (d, 2H), 3.47 (d, 2H), 3.36 (t, 2H), 3.28 (t, 2H), 2.91-3.10 (m, 2H),2.79-2.91 (m, 4H), 2.19-2.25 (m, 3H), 2.06-2.20 (m, 2H), 1.89-2.02 (m, 3H), 1.53-1.74 (m, 2H), 1.30-1.55(m, 8H), 1.06-1.29 (m, 10H), 0.91-1.06 (m, 2H), 0.76-0.89 (m, 12H). MS (ESI) m/e 1356.3 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 2h; | Example 1.1.14 (72 mg) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (91 mg) in N,N-dimethylformamide (3 mL) was cooled in a water-ice bath and N,N- diisopropylethylamine (0.12 mL) was added. The mixture was stirred at 0 C for 2 hours and acetic acid (0.057 mL) was added. After concentration of the solvents, the residue was purified via HPLC (20-80% acetonitrile in 0.1% TF A/water) to provide the title compound. ]H NMR (400 MHz, dimethyl sulfoxide-^) delta ppm 9.98 (s, IH), 8.40 (s, IH), 8.06 (d, IH), 8.00 (d, IH), 7.74-7.89 (m, 4H), 7.59 (d, 2H), 7.46 (s, 2H), 7.37 (t, IH), 7.18-7.32 (m, 4H), 6.99 (s, 2H), 6.01 (s, IH), 4.98 (s, 3H), 4.38 (d, 2H), 3.47 (d, 2H), 3.36 (t, 2H), 3.28 (t, 2H), 2.91-3.10 (m, 2H), 2.79-2.91 (m, 4H , 2.19-2.25 (m, 3H), 2.06-2.20 (m, 2H), 1.89-2.02 (m, 3H), 1.53-1.74 (m, 2H), 1.30-1.55 (m, 8H), 1.06-1.29 (m, 10H), 0.91-1.06 (m, 2H), 0.76-0.89 (m, 12H). MS (ESI) m e 1356.3 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 2h; | Example l.l.I4 (72 mg) and 4-( (S)-2-( (S)-2-(6-(2,5-dioxo-2,5-dihydro-IH-pyrrol-I-25 yl)hexanamido )-3-methylbutanamido )-5-ureidopentanamido )benzyl ( 4-nitrophenyl) carbonate (9Img) in N,N-dimethylformamide (3 mL) was cooled in a water-ice bath and N,Ndiisopropylethylamine(O.I2 mL) was added. The mixture was stirred at 0 oc for 2 hours and aceticacid (0.057 mL) was added. After concentration of the solvents, the residue was purified via HPLC(20-SO% acetonitrile in O.I% TFA/water) to provide the title compound. 1H NMR (400 MHz,30 dimethyl sulfoxide-d6) 8 ppm 9.9S (s, IH), S.40 (s, IH), S.06 (d, IH), S.OO (d, IH), 7.74-7.S9 (m, 4H),7.59 (d, 2H), 7.46 (s, 2H), 7.37 (t, IH), 7.IS-7.32 (m, 4H), 6.99 (s, 2H), 6.0I (s, IH), 4.9S (s, 3H),4.3S (d, 2H), 3.47 (d, 2H), 3.36 (t, 2H), 3.2S (t, 2H), 2.9I-3.10 (m, 2H), 2.79-2.9I (m, 4H), 2.I9-2.25(m, 3H), 2.06-2.20 (m, 2H), l.S9-2.02 (m, 3H), 1.53-1.74 (m, 2H), 1.30-1.55 (m, SH), 1.06-1.29 (m,10H), 0.9I-1.06 (m, 2H), 0.76-0.S9 (m, I2H). MS (ESI) m/e 1356.3 (M+Ht. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With pyridine; benzotriazol-1-ol; In N,N-dimethyl-formamide; for 24h; | The activated linker 39 (50 mg, 0.068 mmol), MMAE (32.6 mg, 0.045 mmol) and N-hydroxybenzotriazole (1.4 mg, 0.0091 mmol) are stirred in dry DMF (1 ml) for 2 min. after which a drop of pyridine is added and the reaction stirred for 24 h. The solvent is then removed by high vacuum and the residue purified by reverse-phase preparative HPLC togive the desired product 40 after lyopholisation as a white powder; MS (m/z) 1316.7 (M+H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 3h; | [000920] A solution of Example 1.26.2 (0.040 g), 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro- lH- pyrrol- 1 -yl)hexanamido)-3 -methylbutanamido)-5 -ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.030 g) and N,N-diisopropylethylamine (0.020 inL) in N,N-dimethylformamide (0.5 mL) was stirred at room temperature. After stirring for 3 hours, the reaction was diluted with N,N- dimethylformamide ( 1.25 mL) and water (0.5 mL). The mixture was purified by reverse phase HPLC using a Gilson system, eluting with 10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid. The desired fractions were combined and freeze-dried to provide the title compound. NMR (500 MHz, dimethyl sulfoxide -d6) delta ppm 9.98 (s, IH), 9.26 (s, IH), 8.06 (d, IH), 8.05-8.01 (m, IH), 7.79 (d, 2H), 7.62 (d, IH), 7.61-7.57 (m, 2H), 7.52-7.42 (m, 3H), 7.38 (d, IH), 7.35 (d, IH), 7.32-7.26 (m, 3H), 6.99 (s, 2H), 6.95 (d, IH), 6.01 (s, IH), 4.99 (s, 2H), 4.96 (s, 3H), 4.44-4.33 (m, 2H), 4.18 (dd, 2H), 3.88 (t, 2H), 3.83 (s, 2H), 3.71-3.61 (m, 2H), 3.53 (t, 2H), 3.36 (t, 2H), 3.07-2.66 (m, 8H), 2.28-2.06 (m, 6H), 2.05- 1.92 (m, 2H), 1.92- 1.80 (m, 2H), 1.78-0.95 (m, 32H), 0.92-0.77 (m, 14H). MS (ESI) m/e 1549.5 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 3h; | A solution of Example 1.26.2 (0.040 g), 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.030 g) and N,N-diisopropylethylamine (0.020 mL) in N,N-dimethylformamide (0.5 mL) was stirred at room temperature. After stirring for 3 hours, the reaction was diluted with N,N- dimethylformamide (1.25 mL) and water (0.5 mL). The mixture was purified by reverse phase HPLC using a Gilson system, eluting with 10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid. The desired fractions were combined and freeze-dried to provide the title compound. 1H NMR (500 MHz, dimethyl sulfoxide-d6) delta ppm 9.98 (s, 1H), 9.26 (s, 1H), 8.06 (d, 1H), 8.05-8.01 (m, 1H), 7.79 (d, 2H), 7.62 (d, 1H), 7.61-7.57 (m, 2H), 7.52-7.42 (m, 3H), 7.38 (d, 1H), 7.35 (d, 1H), 7.32-7.26 (m, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.99 (s, 2H), 4.96 (s, 3H), 4.44-4.33 (m, 2H), 4.18 (dd, 2H), 3.88 (t, 2H), 3.83 (s, 2H), 3.71-3.61 (m, 2H), 3.53 (t, 2H), 3.36 (t, 2H), 3.07-2.66 (m, 8H), 2.28-2.06 (m, 6H), 2.05-1.92 (m, 2H), 1.92-1.80 (m, 2H), 1.78-0.95 (m, 32H), 0.92-0.77 (m, 14H). MS (ESI) m/e 1549.5 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 3h; | A solution of Example 1.26.2 (0.040 g), <strong>[159857-81-5]4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate</strong> (0.030 g) and N,N-diisopropylethylamine (0.020 mL) in N,N-dimethylformamide (0.5 mL) was stirred at room temperature. After stirring for 3 hours, the reaction was diluted with N,N-dimethylformamide (1.25 mL) and water (0.5 mL). The mixture was purified by reverse phase HPLC using a Gilson system, eluting with 10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid. The desired fractions were combined and freeze-dried to provide the title compound. 1H NMR (500 MHz, dimethyl sulfoxide-d6) delta ppm 9.98 (s, 1H), 9.26 (s, 1H), 8.06 (d, 1H), 8.05-8.01 (m, 1H), 7.79 (d, 2H), 7.62 (d, 1H), 7.61-7.57 (m, 2H), 7.52-7.42 (m, 3H), 7.38 (d, 1H), 7.35 (d, 1H), 7.32-7.26 (m, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.99 (s, 2H), 4.96 (s, 3H), 4.44-4.33 (m, 2H), 4.18 (dd, 2H), 3.88 (t, 2H), 3.83 (s, 2H), 3.71-3.61 (m, 2H), 3.53 (t, 2H), 3.36 (t, 2H), 3.07-2.66 (m, 8H), 2.28-2.06 (m, 6H), 2.05-1.92 (m, 2H), 1.92-1.80 (m, 2H), 1.78-0.95 (m, 32H), 0.92-0.77 (m, 14H). MS (ESI) m/e 1549.5 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; | [000915] A solution of Example 1.28 (0.0449 g), 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH- pyrrol- 1 -yl)hexanamido)-3 -methylbutanamido)-5 -ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.049 g) and N,N-diisopropylethylamine (0.044 mL) were stirred together in N,N- dimethylformamide (0.5 mL) at room temperature. The reaction mixture was stirred overnight and diluted with N,N-dimethylformamide (1 mL) and water (0.5 mL). The mixture was purified by reverse phase HPLC using a Gilson system, eluting with 10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid. The desired fractions were combined and freeze-dried to provide the title compound. NMR (400 MHz, dimethyl sulfoxide- g) delta ppm 12.85 (s, IH), 9.99 (s, IH), 8.04 (t, 2H), 7.78 (t, 2H), 7.65-7.58 (m, 3H), 7.54-7.41 (m, 3H), 7.38 (d, IH), 7.34 (d, IH), 7.32-7.24 (m, 3H), 6.99 (s, 2H), 6.95 (d, IH), 5.97 (s, IH), 5.01 (s, 2H), 4.96 (s, 2H), 4.38 (q, IH), 4.23-4.14 (m, IH), 4.05 (s, 2H), 3.88 (t, 2H), 3.80 (s, 2H), 3.36 (t, 2H), 3.26-2.86 (m, 8H), 2.27-2.02 (m, 6H), 2.02- 1.86 (m, 2H), 1.86-1.75 (m, 2H), 1.75-1.54 (m, 2H), 1.54-0.90 (m, 24H), 0.89-0.72 (m, 14H). MS (ESI) m/e 1485.2 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; | A solution of Example 1.28 (0.0449 g), 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.049 g) and N,N-diisopropylethylamine (0.044 mL) were stirred together in N,N- dimethylformamide (0.5 mL) at room temperature. The reaction mixture was stirred overnight and diluted with N,N-dimethylformamide (1 mL) and water (0.5 mL). The mixture was purified by reverse phase HPLC using a Gilson system, eluting with 10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid. The desired fractions were combined and freeze-dried to provide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.04 (t, 2H), 7.78 (t, 2H), 7.65-7.58 (m, 3H), 7.54-7.41 (m, 3H), 7.38 (d, 1H), 7.34 (d, 1H), 7.32-7.24 (m, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 5.97 (s, 1H), 5.01 (s, 2H), 4.96 (s, 2H), 4.38 (q, 1H), 4.23-4.14 (m, 1H), 4.05 (s, 2H), 3.88 (t, 2H), 3.80 (s, 2H), 3.36 (t, 2H), 3.26-2.86 (m, 8H), 2.27-2.02 (m, 6H), 2.02- 1.86 (m, 2H), 1.86-1.75 (m, 2H), 1.75-1.54 (m, 2H), 1.54-0.90 (m, 24H), 0.89-0.72 (m, 14H). MS (ESI) m/e 1485.2 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; | A solution of Example 1.28 (0.0449 g), <strong>[159857-81-5]4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate</strong> (0.049 g) and N,N-diisopropylethylamine (0.044 mL) were stirred together in N,N-dimethylformamide (0.5 mL) at room temperature. The reaction mixture was stirred overnight and diluted with N,N-dimethylformamide (1 mL) and water (0.5 mL). The mixture was purified by reverse phase HPLC using a Gilson system, eluting with 10-90% acetonitrile in water containing 0.1% v/v trifluoroacetic acid. The desired fractions were combined and freeze-dried to provide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.04 (t, 2H), 7.78 (t, 2H), 7.65-7.58 (m, 3H), 7.54-7.41 (m, 3H), 7.38 (d, 1H), 7.34 (d, 1H), 7.32-7.24 (m, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 5.97 (s, 1H), 5.01 (s, 2H), 4.96 (s, 2H), 4.38 (q, 1H), 4.23-4.14 (m, 1H), 4.05 (s, 2H), 3.88 (t, 2H), 3.80 (s, 2H), 3.36 (t, 2H), 3.26-2.86 (m, 8H), 2.27-2.02 (m, 6H), 2.02-1.86 (m, 2H), 1.86-1.75 (m, 2H), 1.75-1.54 (m, 2H), 1.54-0.90 (m, 24H), 0.89-0.72 (m, 14H). MS (ESI) m/e 1485.2 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 3h; | [0001016] A solution of Example 1.60 (0.026 g), 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH- pyrrol- 1 -yl)hexanamido)-3 -methylbutanamido)-5 -ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.024 g) and N,N-diisopropylethylamine (0.022 mL) were stirred together in N,N- dimethylformamide (0.8 mL) at room temperature for 3 hours. The reaction was diluted with a 1 : 1 mixture of N,N-dimethylformamide:water (2 mL). The mixture was purified by reverse phase HPLC using a Gilson system, eluting with 10-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid. The desired fractions were combined and freeze-dried to provide the title compound. NMR (400 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, IH), 9.99 (s, IH), 8.06 (d, IH), 8.03 (d, IH), 7.79 (dd, 2H), 7.60 (dd, 3H), 7.55-7.41 (m, 3H), 7.36 (td, 2H), 7.29 (t, 3H), 6.99 (s, 2H), 6.95 (d, IH), 5.99 (s, IH), 5.04-4.92 (m, 4H), 4.37 (q, IH), 4.34-4.24 (m, IH), 4.24-4.10 (m, 4H), 3.88 (t, 2H), 3.82 (s, 2H), 3.40-3.29 (m, 4H), 3.01 (t, 2H), 2.99-2.91 (m, IH), 2.87 (t, 2H), 2.25-2.06 (m, 5H), 1.95 (dt, IH), 1.68 (s, IH), 1.60 (s, IH), 1.54-1.24 (m, 12H), 1.24-0.94 (m, 9H), 0.90-0.78 (m, 12H); MS (ESI) m/e 1507.4 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 3h; | A solution of Example 1.60 (0.026 g), 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.024 g) and N,N-diisopropylethylamine (0.022 mL) were stirred together in N,N- dimethylformamide (0.8 mL) at room temperature for 3 hours. The reaction was diluted with a 1:1 mixture of N,N-dimethylformamide:water (2 mL). The mixture was purified by reverse phase HPLC using a Gilson system, eluting with 10-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid. The desired fractions were combined and freeze-dried to provide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.06 (d, 1H), 8.03 (d, 1H), 7.79 (dd, 2H), 7.60 (dd, 3H), 7.55-7.41 (m, 3H), 7.36 (td, 2H), 7.29 (t, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 5.99 (s, 1H), 5.04-4.92 (m, 4H), 4.37 (q, 1H), 4.34-4.24 (m, 1H), 4.24-4.10 (m, 4H), 3.88 (t, 2H), 3.82 (s, 2H), 3.40-3.29 (m, 4H), 3.01 (t, 2H), 2.99-2.91 (m, 1H), 2.87 (t, 2H), 2.25-2.06 (m, 5H), 1.95 (dt, 1H), 1.68 (s, 1H), 1.60 (s, 1H), 1.54-1.24 (m, 12H), 1.24-0.94 (m, 9H), 0.90-0.78 (m, 12H); MS (ESI) m/e 1507.4 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 3h; | A solution of Example 1.60 (0.026 g), <strong>[159857-81-5]4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate</strong> (0.024 g) and N,N-diisopropylethylamine (0.022 mL) were stirred together in N,N-dimethylformamide (0.8 mL) at room temperature for 3 hours. The reaction was diluted with a 1:1 mixture of N,N-dimethylformamide: water (2 mL). The mixture was purified by reverse phase HPLC using a Gilson system, eluting with 10-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid. The desired fractions were combined and freeze-dried to provide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.06 (d, 1H), 8.03 (d, 1H), 7.79 (dd, 2H), 7.60 (dd, 3H), 7.55-7.41 (m, 3H), 7.36 (td, 2H), 7.29 (t, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 5.99 (s, 1H), 5.04-4.92 (m, 4H), 4.37 (q, 1H), 4.34-4.24 (m, 1H), 4.24-4.10 (m, 4H), 3.88 (t, 2H), 3.82 (s, 2H), 3.40-3.29 (m, 4H), 3.01 (t, 2H), 2.99-2.91 (m, 1H), 2.87 (t, 2H), 2.25-2.06 (m, 5H), 1.95 (dt, 1H), 1.68 (s, 1H), 1.60 (s, 1H), 1.54-1.24 (m, 12H), 1.24-0.94 (m, 9H), 0.90-0.78 (m, 12H); MS (ESI) m/e 1507.4 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 3h; | [0001018] To a mixture of Example 1.61.2 (12.8 mg) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro- lH-pyrrol-l-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (10.4 mg) in N,N-dimethylformamide (0.5 mL) at 0 C was added N,N- diisopropylethylamine (24.54 mu). The mixture was stirred for 3 hours and purified by reverse phase HPLC using a Gilson system and a C 18 column, eluting with 20-60% acetonitrile in water containing 0.1% trifluoroacetic acid, to provide the title compound. lH NMR (400 MHz, dimethyl sulfoxide-^) delta ppm 12.85 (s, 1H), 9.97 (s, 1H), 8.97 (s, 1H), 8.04 (t, 2H), 7.79 (dd, 2H), 7.65-7.40 (m, 7H), 7.36 (td, 3H), 7.28 (d, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 5.98 (s, 1H), 4.95 (d, 4H), 4.49-4.30 (m, 1H), 4.24- 4.11 (m, 1H), 3.88 (t, 2H), 3.82 (s, 2H), 3.36 (t, 3H), 3.18-2.84 (m, 9H), 2.25-1.88 (m, 5H), 1.85-0.90 (m, 14H), 0.91-0.75 (m, 13H). MS (ESI) m/e (M+H | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 3h; | To a mixture of Example 1.61.2 (12.8 mg) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro- 1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (10.4 mg) in N,N-dimethylformamide (0.5 mL) at 0 oC was added N,N- diisopropylethylamine (24.54 muL). The mixture was stirred for 3 hours and purified by reverse phase HPLC using a Gilson system and a C18 column, eluting with 20-60% acetonitrile in water containing 0.1% trifluoroacetic acid, to provide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 1H), 9.97 (s, 1H), 8.97 (s, 1H), 8.04 (t, 2H), 7.79 (dd, 2H), 7.65-7.40 (m, 7H), 7.36 (td, 3H), 7.28 (d, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 5.98 (s, 1H), 4.95 (d, 4H), 4.49-4.30 (m, 1H), 4.24- 4.11 (m, 1H), 3.88 (t, 2H), 3.82 (s, 2H), 3.36 (t, 3H), 3.18-2.84 (m, 9H), 2.25-1.88 (m, 5H), 1.85-0.90 (m, 14H), 0.91-0.75 (m, 13H). MS (ESI) m/e (M+H). | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 3h; | To a mixture of Example 1.61.2 (12.8 mg) and <strong>[159857-81-5]4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate</strong> (10.4 mg) in N,N-dimethylformamide (0.5 mL) at 0 C. was added N,N-diisopropylethylamine (24.54 muL). The mixture was stirred for 3 hours and purified by reverse phase HPLC using a Gilson system and a C18 column, eluting with 20-60% acetonitrile in water containing 0.1% trifluoroacetic acid, to provide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) 6 ppm 12.85 (s, 1H), 9.97 (s, 1H), 8.97 (s, 1H), 8.04 (t, 2H), 7.79 (dd, 2H), 7.65-7.40 (m, 7H), 7.36 (td, 3H), 7.28 (d, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 5.98 (s, 1H), 4.95 (d, 4H), 4.49-4.30 (m, 1H), 4.24-4.11 (m, 1H), 3.88 (t, 2H), 3.82 (s, 2H), 3.36 (t, 3H), 3.18-2.84 (m, 9H), 2.25-1.88 (m, 5H), 1.85-0.90 (m, 14H), 0.91-0.75 (m, 13H). MS (ESI) m/e (M+H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | [000883] Example 1.2.9 (100 mg) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanamido)-3 -methylbutanamido)-5 -ureidopentanamido)benzyl (4-nitrophenyl) carbonate (purchased from Synchem, 114 mg) in N,N-dimethylformamide (7 mL) was cooled in an water-ice bath, and N,N-diisopropylethylamine (0.15 mL) was added. The mixture was stirred at 0 C for 30 minutes and then at room temperature overnight. The reaction was purified by a reverse phase HPLC using a Gilson system, eluting with 20-60% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. lH NMR (400 MHz, dimethyl sulfoxide- 6) delta ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.04 (t, 2H), 7.75-7.82 (m, 2H), 7.40-7.63 (m, 6H), 7.32-7.39 (m, 2H), 7.24-7.29 (m, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.83-5.08 (m, 4H), 4.29-4.48 (m, 1H), 4.19 (t, 1H), 3.84- 3.94 (m, 2H), 3.80 (d, 2H), 3.14-3.29 (m, 2H), 2.87-3.06 (m, 4H), 2.57-2.69 (m, 2H), 2.03-2.24 (m, 5H), 1.89-2.02 (m, 1H), 1.53-1.78 (m, 2H), 1.26-1.53 (m, 8H), 0.89-1.27 (m, 12H), 0.75-0.88 (m, 12H). MS (ESI) m/e 1452.2 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | Example 1.44.2 (100 mg) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (purchased from Synchem, 114 mg) in N,N-dimethylformamide (7 mL) was cooled in an water-ice bath, and N,N-diisopropylethylamine (0.15 mL) was added. The mixture was stirred at 0 C for 30 minutes and then at room temperature overnight. The reaction was purified by a reverse phase HPLC using a Gilson system, eluting with 20-60% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. NMR (400 MHz, dimethylsulfoxide-<i6) delta ppm 12.85 (s, IH), 9.99 (s, IH), 8.04 (t, 2H), 7.75-7.82 (m, 2H), 7.40-7.63 (m, ThetaEta), 7.32-7.39 (m, 2H), 7.24-7.29 (m, 3H), 6.99 (s, 2H), 6.95 (d, IH), 6.01 (s, IH), 4.83-5.08 (m, 4H), 4.29-4.48 (m, IH), 4.19 (t, IH), 3.84- 3.94 (m, 2H), 3.80 (d, 2H), 3.14-3.29 (m, 2H), 2.87-3.06 (m, 4H), 2.57-2.69 (m, 2H), 2.03-2.24 (m, 5H), 1.89-2.02 (m, IH), 1.53-1.78 (m, 2H), 1.26-1.53 (m, 8H), 0.89-1.27 (m, 12H), 0.75-0.88 (m, 12H). MS (ESI) m/e 1452.2 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | Example 1.44.2 (100 mg) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-IH-pyrrol-1-yl)hexanamido )-3-methylbutanamido )-5-ureidopentanamido )benzyl ( 4-nitrophenyl) carbonate(purchased from Synchem, 114 mg) in N,N-dimethylformamide (7 mL) was cooled in an water-icebath, and N,N-diisopropylethylamine (0.15 mL) was added. The mixture was stirred at 0 oc for 3035 minutes and then at room temperature overnight. The reaction was purified by a reverse phase HPLCusing a Gilson system, eluting with 20-60% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. 1H NMR (400 MHz, dimethylsulfoxide-d6) 8 ppm 12.85 (s, IH),9.99 (s, IH), 8.04 (t, 2H), 7.75-7.82 (m, 2H), 7.40-7.63 (m, 6H), 7.32-7.39 (m, 2H), 7.24-7.29 (m,3H), 6.99 (s, 2H), 6.95 (d, IH), 6.01 (s, IH), 4.83-5.08 (m, 4H), 4.29-4.48 (m, IH), 4.19 (t, IH), 3.84-3.94 (m, 2H), 3.80 (d, 2H), 3.14-3.29 (m, 2H), 2.87-3.06 (m, 4H), 2.57-2.69 (m, 2H), 2.03-2.24 (m,5 5H), 1.89-2.02 (m, IH), 1.53-1.78 (m, 2H), 1.26-1.53 (m, 8H), 0.89-1.27 (m, 12H), 0.75-0.88 (m,12H). MS (ESI) m/e 1452.2 (M+Ht. |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | Example 1.2.9 (100 mg) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1- yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (purchased from Synchem, 114 mg) in N,N-dimethylformamide (7 mL) was cooled in an water-ice bath, and N,N-diisopropylethylamine (0.15 mL) was added. The mixture was stirred at 0 oC for 30 minutes and then at room temperature overnight. The reaction was purified by a reverse phase HPLC using a Gilson system, eluting with 20-60% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.04 (t, 2H), 7.75-7.82 (m, 2H), 7.40-7.63 (m, 6H), 7.32-7.39 (m, 2H), 7.24-7.29 (m, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.83-5.08 (m, 4H), 4.29-4.48 (m, 1H), 4.19 (t, 1H), 3.84- 3.94 (m, 2H), 3.80 (d, 2H), 3.14-3.29 (m, 2H), 2.87-3.06 (m, 4H), 2.57-2.69 (m, 2H), 2.03-2.24 (m, 5H), 1.89-2.02 (m, 1H), 1.53-1.78 (m, 2H), 1.26-1.53 (m, 8H), 0.89-1.27 (m, 12H), 0.75-0.88 (m, 12H). MS (ESI) m/e 1452.2 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | Example 1.2. 9 ( 100 mg) and 4-( (S)-2-( (S)-2-( 6-(2,5-dioxo-2,5-dihydro-1H -pyrrol-1-yl)hexanamido )-3-methylbutanamido )-5-ureidopentanamido )benzyl ( 4-nitrophenyl) carbonate(purchased from Synchem, 114 mg) in N,N-dimethylformamide (7 mL) was cooled in an water-icebath, and N,N-diisopropylethylamine (0.15 mL) was added. The mixture was stirred at 0 oc for 3015 minutes and then at room temperature overnight. The reaction was purified by a reverse phase HPLCusing a Gilson system, eluting with 20-60% acetonitrile in water containing 0.1% v/v trifluoroaceticacid, to provide the title compound. 1H NMR ( 400 MHz, dimethyl sulfoxide-d6) 8 ppm 12.85 (s, 1H),9.99 (s, 1H), 8.04 (t, 2H), 7.75-7.82 (m, 2H), 7.40-7.63 (m, 6H), 7.32-7.39 (m, 2H), 7.24-7.29 (m,3H), 6.99 (s, 2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.83-5.08 (m, 4H), 4.29-4.48 (m, 1H), 4.19 (t, 1H), 3.84-20 3.94 (m, 2H), 3.80 (d, 2H), 3.14-3.29 (m, 2H), 2.87-3.06 (m, 4H), 2.57-2.69 (m, 2H), 2.03-2.24 (m,5H), 1.89-2.02 (m, 1H), 1.53-1.78 (m, 2H), 1.26-1.53 (m, 8H), 0.89-1.27 (m, 12H), 0.75-0.88 (m,12H). MS (ESI) m/e 1452.2 (M+Ht. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; for 0.5h; | Example 1.9.2 (100 mg) and 4-((S)-2-((S)-2-(6-(2,5 -dioxo-2,5-dihydro-1 H-pyrrol- 1 -yl)hexanamido)20 3-methylbutanamido)-5 -ureidopentanamido)benzyl (4-nitrophenyl) carbonate (purchased fromSynchem, 114 mg) in N,N-dimethylformamide (7 mL) was cooled in an water-ice bath, and N,Ndiisopropylethylamine (0.15 mL) was added. The mixture was stirred at 0 C for 30 minutes and then at room temperature overnight. The reaction was purified by a reverse phase HPLC using a Gilson system, eluting with 20-60% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to providethe title compound. ?H NMR (400 MHz, dimethylsulfoxide-d6) 6 ppm 12.85 (s, 111), 9.99 (s, 111),8.04 (t, 211), 7.75-7.82 (m, 211), 7.40-7.63 (m, 6H), 7.32-7.39 (m, 2H), 7.24-7.29 (m, 311), 6.99 (s,211), 6.95 (d, 1H), 6.01 (s, 1H), 4.83-5.08 (m, 411), 4.29-4.48 (m, 111), 4.19 (t, 111), 3.84-3.94 (m, 2H),3.80 (d, 2H), 3.14-3.29 (m, 2H), 2.87-3.06 (m, 411), 2.57-2.69 (m, 211), 2.03-2.24 (m, 5H), 1.89-2.02(m, 1H), 1.53-1.78 (m, 2H), 1.26-1.53 (m, 8H), 0.89-1.27 (m, 12H), 0.75-0.88 (m, 12H). MS (ESI)mle 1452.2 (M+H). | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; for 0.5h; | Example 1.2.9 (100 mg) and <strong>[159857-81-5]4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate</strong> (purchased from Synchem, 114 mg) in N,N-dimethylformamide (7 mL) was cooled in an water-ice bath, and N,N-diisopropylethylamine (0.15 mL) was added. The mixture was stirred at 0 C. for 30 minutes and then at room temperature overnight. The reaction was purified by a reverse phase HPLC using a Gilson system, eluting with 20-60% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. 1H NMR (400 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.04 (t, 2H), 7.75-7.82 (m, 2H), 7.40-7.63 (m, 6H), 7.32-7.39 (m, 2H), 7.24-7.29 (m, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.83-5.08 (m, 4H), 4.29-4.48 (m, 1H), 4.19 (t, 1H), 3.84-3.94 (m, 2H), 3.80 (d, 2H), 3.14-3.29 (m, 2H), 2.87-3.06 (m, 4H), 2.57-2.69 (m, 2H), 2.03-2.24 (m, 5H), 1.89-2.02 (m, 1H), 1.53-1.78 (m, 2H), 1.26-1.53 (m, 8H), 0.89-1.27 (m, 12H), 0.75-0.88 (m, 12H). MS (ESI) m/e 1452.2 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | Example 1.9.2 (100 mg) and <strong>[159857-81-5]4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate</strong> (purchased from Synchem, 114 mg) in N,N-dimethylformamide (7 mL) was cooled in an water-ice bath, and N,N-diisopropylethylamine (0.15 mL) was added. The mixture was stirred at 0 C. for 30 minutes and then at room temperature overnight. The reaction was purified by a reverse phase HPLC using a Gilson system, eluting with 20-60% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. 1H NMR (400 MHz, dimethylsulfoxide-d6) delta ppm 12.85 (s, 1H), 9.99 (s, 1H), 8.04 (t, 2H), 7.75-7.82 (m, 2H), 7.40-7.63 (m, 6H), 7.32-7.39 (m, 2H), 7.24-7.29 (m, 3H), 6.99 (s, 2H), 6.95 (d, 1H), 6.01 (s, 1H), 4.83-5.08 (m, 4H), 4.29-4.48 (m, 1H), 4.19 (t, 1H), 3.84-3.94 (m, 2H), 3.80 (d, 2H), 3.14-3.29 (m, 2H), 2.87-3.06 (m, 4H), 2.57-2.69 (m, 2H), 2.03-2.24 (m, 5H), 1.89-2.02 (m, 1H), 1.53-1.78 (m, 2H), 1.26-1.53 (m, 8H), 0.89-1.27 (m, 12H), 0.75-0.88 (m, 12H). MS (ESI) m/e 1452.2 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline / dichloromethane; methanol / 36 h / 20 °C / Darkness; Inert atmosphere 2: diethylamine / 1-methyl-pyrrolidin-2-one / 16 h / 20 °C / Inert atmosphere 3: 1-methyl-pyrrolidin-2-one / 16 h / 20 °C / Inert atmosphere 4: pyridine / dichloromethane / 12 h / 20 °C / Inert atmosphere | ||
Multi-step reaction with 4 steps 1: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline / dichloromethane; methanol / 36 h / 20 °C / Darkness; Inert atmosphere 2: diethylamine / 1-methyl-pyrrolidin-2-one / 16 h / 20 °C / Inert atmosphere 3: 1-methyl-pyrrolidin-2-one / 16 h / 20 °C / Inert atmosphere 4: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 18 h / 20 °C / Inert atmosphere | ||
Multi-step reaction with 4 steps 1: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline / dichloromethane; methanol / 36 h / 20 °C 2: piperidine / N,N-dimethyl-formamide / 2 h / 20 °C 3: N,N-dimethyl-formamide / 6 h / 20 °C 4: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 5 h / 20 °C |
Multi-step reaction with 4 steps 1: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline / dichloromethane; methanol / 48 h / 20 °C / Darkness 2: diethylamine / 24 h / 20 °C 3: 1-methyl-pyrrolidin-2-one / 24 h / Heating 4: pyridine / dichloromethane / 3.17 h / 0 - 20 °C / Inert atmosphere | ||
Multi-step reaction with 4 steps 1: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline / dichloromethane; methanol / 48 h / 20 °C / Darkness 2: diethylamine / 1-methyl-pyrrolidin-2-one / 24 h / 20 °C 3: 1-methyl-pyrrolidin-2-one / 24 h / 20 °C 4: pyridine / dichloromethane / 3.17 h / 0 - 20 °C / Inert atmosphere | ||
Multi-step reaction with 4 steps 1: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline / dichloromethane; methanol / 20 °C / Darkness 2: diethylamine / N,N-dimethyl-formamide / 20 °C 3: N,N-dimethyl-formamide / 20 °C 4: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 20 °C | ||
Multi-step reaction with 4 steps 1: N-ethyl-N,N-diisopropylamine; N-[(dimethylamino)-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate / water; N,N-dimethyl-formamide; ethyl acetate / 0 - 5 °C 2: diethylamine / N,N-dimethyl-formamide / 20 °C 3: N,N-dimethyl-formamide / 20 - 30 °C 4: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 25 °C | ||
Multi-step reaction with 4 steps 1: 1-bromo-2,3,4-tri-O-acetyl-α-D-glucuronic acid methyl ester / dichloromethane; methanol / 48 h / 20 °C / Darkness 2: diethylamine / N,N-dimethyl-formamide / 24 h 3: N,N-dimethyl-formamide / 20 °C 4: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 20 °C | ||
Multi-step reaction with 4 steps 1: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline / dichloromethane; methanol / 14 h / 20 °C 2: diethylamine / N,N-dimethyl-formamide 3: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline / N,N-dimethyl-formamide / 20 °C 4: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 16 h / 20 °C | ||
Multi-step reaction with 4 steps 1: N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline / dichloromethane; methanol / 36 h / 20 °C / Darkness 2: piperidine / N,N-dimethyl-formamide / 2 h / 20 °C 3: N,N-dimethyl-formamide / 20 °C 4: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 5 h / 20 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 5 steps 1: piperidine / N,N-dimethyl-formamide / 4 h / 20 °C / Inert atmosphere 2: N,N-dimethyl-formamide / 16 h / 20 °C / Inert atmosphere 3: diethylamine / 1-methyl-pyrrolidin-2-one / 16 h / 20 °C / Inert atmosphere 4: 1-methyl-pyrrolidin-2-one / 16 h / 20 °C / Inert atmosphere 5: pyridine / dichloromethane / 12 h / 20 °C / Inert atmosphere | ||
Multi-step reaction with 5 steps 1: piperidine / N,N-dimethyl-formamide / 4 h / 20 °C / Inert atmosphere 2: N,N-dimethyl-formamide / 16 h / 20 °C / Inert atmosphere 3: diethylamine / 1-methyl-pyrrolidin-2-one / 16 h / 20 °C / Inert atmosphere 4: 1-methyl-pyrrolidin-2-one / 16 h / 20 °C / Inert atmosphere 5: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 18 h / 20 °C / Inert atmosphere | ||
Multi-step reaction with 5 steps 1: piperidine / N,N-dimethyl-formamide / 2 h / 30 °C 2: N,N-dimethyl-formamide / 2 h / 30 °C 3: piperidine / N,N-dimethyl-formamide / 2 h / 30 °C 4: 2 h / 30 °C 5: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 16 h / 30 °C / Inert atmosphere |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
60% | With N-ethyl-N,N-diisopropylamine; In dichloromethane; at 20℃; | To compound 71 (16.0 mg, 21 .7 muiotatauiotaomicronIota) were added a solution of example 25 (14.3 mg, 19.7 muiotatauiotaomicronIota) in DCM (2.5 mL) and DIEA (3.4 muIota_, 19.7 muiotatauiotaomicronIota). The reaction medium was stirred at RT overnight then was added DMF (1 mL) and stirring was carried on at RT for 1 d. H20 (8 mL) was added to the reaction medium, stirrring carried on for 15 min then the aqueous phase was extracted with DCM (3 x 10 mL). The combined organic phases were dried over MgS04, filtered, concentrated in vacuo, co-evaporated twice with toluene and purified by flash chromatography on 5 g on silica gel (gradient elution DCM/MeOH) to give 15.8 mg of example 29 as a white solid (60%). RMN 1H (400 MHz, delta in ppm, DMSO-o(6): 0.81 (d, J = 7.0 Hz, 3H); 0.85 (d, J = 7.0 Hz, 3H); 0.86 (s, 9H); 0.89 (d, J = 6.8 Hz, 3H); 0.99 (s, 3H); 1 .04 (d, J = 7.0 Hz, 3H); 1.18 (s, 3H); 1 .19 (m, 2H); 1.31 to 1.75 (m, 9H); 1.80 (m, 1 H); 1 ,96 (m, 2H) 2.10 (m, 1 H); 2.18 (m, 1 H); 2,27 (m, 1 H); 2.61 (m, 1 H); 2,69 (dd, J = 10.8 and 14.3 Hz, 1 H); 2,90 (dd, J = 2.1 and 7.6 Hz, 1 H); 2.96 (m, 2H); 3.01 (m, 1 H); 3.37 (t, J = 7.2 Hz, 2H); 3.42 (m, 1 H); 3.80 (s, 3H); 3.90 (d, J = 2.1 Hz, 1 H); 4.08 (m, 1 H); 4.1 1 (m, 1 H); 4.20 (m, 3H); 4.39 (m, 1 H); 4.97 (s, 2H); 5.04 (m, 1 H); 5.40 (s, 2H); 5.89 (dd, J = 1 .7 and 15.5 Hz, 1 H); 5.97 (t, J = 6.2 Hz, 1 H); 6.45 (ddd, J = 4.8, 10.7 and 15.5 Hz, 1 H); 6.99 (s, 2H); 7.01 (d, J = 8.7 Hz, 1 H); 7.20 (dd, J = 2.2 and 8.7 Hz, 1 H); 7.21 to 7.30 (m, 6H); 7.31 (d, J = 2.2 Hz, 1 H); 7.59 (d, J = 8.7 Hz, 2H); 7.78 (m, 2H); 7.85 (d, J = 8.5 Hz, 1 H); 7.90 (d, J = 6.9 Hz, 1 H); 8.07 (d, J = 7.8 Hz, 1 H); 8.39 (d, J = 7.3 Hz, 1 H); 9.97 (s, 1 H). LCMS (A5): ES m/z = 662 [M+2H]2+; m/z = 1322 [M-H]"; m/z = 1324 [M+H]+; m/z = 1368 [M-H+HC02H]"; tR = 1.3 min. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
63% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 12h; | [00352] Eribulin (ER-000086526) (10 mg, 0.012 mmol) was dissolved in DMF (1 mL), and mixed with MC-Val-Cit-PAB-P P (9.02 mg, 0.012 mmol) and Hunig's Base (4.44 mu., 0.025 mmol). The mixture was then stirred at room temperature for 12 hours until HPLC analysis indicated the completion of the reaction. The reaction mixture was concentrated and purified by flash chromatography to yield Mal-(CH2)5-Val-Cit-PAB- eribulin (ER-001235638) as a white solid (11.3 mg, 63% yield). 1HNMR (400 MHz, CD3OD) delta ppm 7.57 (d, J= 8.4 Hz, 2H), 7.31 (d, J= 8.4 Hz, 2H), 6.79 (s, 2H), 5.13 (s, 1H), 5.05 (d, J= 12.4 Hz, 1H), 5.02 (s, 1H), 5.00 (d, J= 12.4 Hz, 1H), 4.87 (s, 1H), 4.83 (s, 1H), 4.71 (t, J= 4.4 Hz, 1H), 4.61 (t, J= 4.4 Hz, 1H), 4.56-4.46 (m, 3H), 4.35-4.27 (m, 2H), 4.20-4.07 (m, 4H), 3.98 (t, J= 10.8 Hz, 1H), 3.87-3.83 (m, 3H), 3.73-3.70 (m, 2H), 3.48 (t, J= 7.6 Hz, 2H), 3.38 (s, 3H), 3.20-3.08 (m, 4H), 2.93 (dd, J = 1.6, 9.6 Hz, 1H), 2.89-2.85 (m, 1H), 2.69 (dt, J= 11.2, 16.8 Hz, 2H), 2.44-2.33 (m, 5H), 2.27-1.83 (m, 13H), 1.78-1.68 (m, 5H), 1.66-1.27 (m, 14H), 1.1 1 (d, J= 7.2 Hz, 3H), 1.07-0.98 (m, 1H), 0.98 (d, J= 7.2 Hz, 3H), 0.96 (d, J= 7.2 Hz, 3H). LCMS (M+H)=1328.9. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
65% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 2h; | To a solution of doxorubicin (250 mg, 0.460 mmol) in DMF (5 mL) was added MC-Val-Cit-PABC-PNP1 (340 mg, 0.460 mmol) and diisopropylethylamine (0.175 mL, 0.920 mmol). The reaction was stirred at room temperature for 2 h, the solvent removed in vacuo, and the remaining residue purified by silica gel chromatography (10:1 dichloromethane:methanol) to give 12? (340 mg, 0.298 mmol, 65% yield). LCMS M/Z = 1142.4 [M + 1]. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
41 mg | After dissolving 100 mg of purchased 10-O-Boc-SN-38 in 10 ml of dry dichloromethane,Add 25.6 mg (1 eq) of DMAP,A solution of triphosgene in dichloromethane (62 mg of triphosgene dissolved in 2 ml of dichloromethane) was added dropwise at 0C.Di Bi,Continue to react at 0C for 12hDichloromethane is removed under reduced pressureAfter dissolving with 10 ml of dry DMF,Add 144mg mc-vc-PABOH,Stir to room temperature and stir for 24h.After the preparation of liquid phase separation mc-vc-PAB-SN-38 41mg,The two-step yield is a total of 19.7%. | |
41 mg | After 100 mg of purchased 10-O-Boc-SN-38 was dissolved in 10 ml of dry dichloromethane, 25.6 mg (1 eq) of DMAP was added.A solution of triphosgene in dichloromethane (62 mg of triphosgene dissolved in 2 ml of dichloromethane) was added dropwise at 0 C.Continue to react at 0 C for 12 h,The dichloromethane was removed under reduced pressure.After dissolving in 10 ml of dry DMF,Add 144 mg mc-vc-PABOH,Stir to room temperature and stir for 24 hours.After preparative liquid phase separation, mc-vc-PAB-SN-38 41mg was obtained.The two-step yield is 19.7%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
22 mg | To a stirred solution of tert-butyl (8-((6-(4-((2-aminoethyl)carbamoyl)benzyl)-5,6,7,8-tetrahydro-1,6-naphthyridin-3-yl)carbamoyl)-4-(dipropylcarbamoyl)-3H-benzo[b]azepin-2-yl)carbamate (80 mg, 0.109 mmol) and Hunig's base (0.057 mL, 0.326 mmol) in DMF (3.4 mL) under nitrogen cooled in an ice-water bath was added dropwise a solution of <strong>[159857-81-5]4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate</strong> (80 mg, 0.109 mmol) in DMF (2 mL). The reaction was stirred overnight while cooling bath expired. The reaction mixture was then concentrated and the residue neutralized with saturated NaHCO3 and purified by reverse phase column (Gold C18 30 g; 5-60% CH3CN in water, no TFA). Fractions pooled, concentrated to afford 100 mg of an off-yellow solid which was directly dissolved in 50 mL of DCM and treated with 10 mL of TFA. The resulting solution was stirred for 1 h then concentrated under reduced pressure. The residue was dried in vacuo, neutralized with saturated NaHCO3, and purified by reverse phase column chromatography (ISCO Gold C18 30 g; 5-70% MeCN in water gradient, no TFA). Major fractions were combined and lyophilized to provide 22 mg of an off-white solid. 1H NMR (CD3OD) delta 8.67 (d, J=2.5 Hz, 1H), 7.91 (d, J=2.5 Hz, 1H), 7.80 (d, J=8.0 Hz, 1H), 7.69 (d, J=2.5 Hz, 1H), 7.58-7.50 (m, 5H), 7.45 (d, J=8.0 Hz, 1H), 7.26 (d, J=8.5 Hz, 2H), 6.89 (s, 1H), 6.77 (s, 2H), 5.04 (s, 2H), 4.90 (m, 1H), 4.14 (d, J=7.5 Hz, 1H), 3.81 (s, 2H), 3.69 (s, 2H), 3.51-3.40 (m, 8H), 3.34 (m, 2H), 3.22 (m, 1H), 3.11 (m, 2H), 2.97 (m, 2H), 2.90 (m, 3H), 2.25 (t, J=7.5 Hz, 2H), 2.06 (m, 1H), 1.88 (m, 1H), 1.75-1.52 (m, 12H), 1.28 (m, 2H), 0.95 (t, J=7.5 Hz, 6H), 0.89 (bs, 6H). LCMS (M+H)=1235.9. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
67% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 23℃; for 0.5h;Inert atmosphere; | [00530] To a solution of Mal-Cap-Val-Cit-PABO-pNP (9.61 mg, 0.013 mmol, 3 equiv) in anhydrous dimethylformamide (72 mu) was added N,N-diisopropylethylamine (7.6 mu, 0.043 mmol, 10 equiv). Alkyl amine 4 (2.3 mg, 4.3 muiotaetaomicron, 1 equiv) was then added dropwise as a solution in anhydrous dimethylformamide (72 mu^). The reaction mixture was stirred at 23 C under argon for 30 min. The reaction mixture was diluted with dimethylformamide (800 uL) and purified directly by preparatory HPLC (Waters Prep-C18 SunFirecolumn, 5 muiotaeta, 19 x 250 mm, UV detection at 399 nm, gradient elution with 30?90% acetonitrile in water containing 0.1% formic acid, flow rate: 15 mL/min) to provide 43 (R02-B5) as a pale yellow solid (3.4 mg, 67%). 1H NMR (600 MHz, CD3OD) delta 7.51 (s, 1H), 7.47 (d, = 8.2 Hz, 2H), 7.19 (d, = 8.1 Hz, 2H), 6.77 (s, 2H), 5.22 (d, = 4.1 Hz, 1H), 5.04 - 5.00 (m, 2H), 4.94 (d, = 4.1 Hz, 1H), 4.65 (s, 1H), 4.49 (dd, = 9.1, 5.1 Hz, 1H), 4.09 - 4.04 (m, 2H), 3.79 (s, 3H), 3.56 (s, 3H), 3.45 (t, = 7.1 Hz, 2H), 3.43 - 3.41 (m, 2H), 3.40 (s, 3H), 3.24 - 3.17 (m, 1H), 3.14 - 3.07 (m, 1H), 3.06 - 3.02 (m, 2H), 2.82 (d, = 5.8 Hz, 1H), 2.74 (d, = 5.8 Hz, 1H), 2.71 - 2.64 (m, 2H), 2.58 (s, 3H), 2.27 (t, = 7.4 Hz, 2H), 2.11 - 2.06 (m, 1H), 2.06 - 2.00 (m, 2H), 1.93 - 1.85 (m, 1H), 1.79 - 1.71 (m, 1H), 1.63 (p, J = 7.5 Hz, 2H), 1.59 - 1.52 (m, 4H), 1.34 - 1.26 (m, 2H), 0.97 (app t, / = 6.4 Hz, 6H); HRMS (ESI): Calc'd for(C56H69N7O18+ H)+: 1128.4777, found: 1128.4668. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
37% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 23℃; for 0.75h;Inert atmosphere; | [00532] To a solution of Mal-Cap-Val-Cit-PABO-pNP (42.3 mg, 0.057 mmol, 3 equiv) in anhydrous dimethylformamide (319 mu) was added N,N-diisopropylethylamine (33.4 mu, 0.191 mmol, 10 equiv). Alkyl amine 7 (R02-A2) (10.4 mg, 0.019 mmol, 1 equiv) was then added dropwise as a solution in anhydrous dimethylformamide (319 mu). The reaction mixture was stirred at 23 C under argon for 45 min. The reaction mixture was diluted with dimethylformamide to a total of 900 uL and purified directly by preparatory HPLC (Waters Prep-C18 SunFirecolumn, 5 muiotaeta, 19 x 250 mm, UV detection at 399 nm, gradient elution with 30?90% acetonitrile in water containing 0.1 % formic acid, flow rate: 15 mL/min) to provide 45 (R02-A4) as a yellow solid (8.1 mg, 37%). TLC (15% methanol-dichloromethane + 2% 30% aqueous ammonium hydroxide solution): Rf= 0.57 (UV);1H NMR (600 MHz, CD3OD) delta 8.22 (d, J = 7.6 Hz, 1H), 7.96 (d, J = 7.8 Hz, 1H), 7.50 - 7.47 (m, 3H), 7.19 (d, / =8.2 Hz, 2H), 6.77 (s, 2H), 5.21 (d, = 4.1 Hz, 1H), 5.00 (d, = 4.0 Hz, 1H), 4.99 - 4.92 (m, 2H), 4.64 (s, 1H), 4.49 (dd, J = 9.1, 5.1 Hz, 1H), 4.17 - 4.15 (m, 1H), 4.06 - 4.01 (m, 2H), 3.78 (s, 3H), 3.55 (s, 3H), 3.45 (t, J = 7.1 Hz, 2H), 3.39 (s, 3H), 3.38 - 3.32 (m, 2H), 3.23 - 3.17 (m, 1H), 3.13 - 3.07 (m, 1H), 3.05 - 2.99 (m, 2H), 2.78 (d, 7 = 5.8 Hz, 1H), 2.71 (d, = 5.8 Hz, 1H), 2.69 - 2.63 (m, 2H), 2.56 (s, 3H), 2.26 (t, = 7.4 Hz, 2H), 2.10 - 2.05 (m, 1H),2.03 - 1.99 (m, 2H), 1.90 - 1.85 (m, 3H), 1.78 - 1.70 (m, 1H), 1.62 (p, 7 = 7.5 Hz, 2H), 1.59 - 1.52 (m, 4H), 1.32 - 1.25 (m, 2H), 0.96 (app t, J = 6.3 Hz, 6H); HRMS (ESI): Calc'd for (C57H71N7O18 + Na)+: 1164.4748, found: 1164.4704. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
47% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 23℃; for 0.75h;Inert atmosphere; | [00534] To a solution of Mal-Cap-Val-Cit-PABO-pNP (50.8 mg, 0.069 mmol, 3 equiv) in anhydrous dimethylformamide (383 mu) was added N,N-diisopropylethylamine (40.1 mu, 0.230 mmol, 10 equiv). Alkyl amine 10 (R02-A1) (12.8 mg, 0.023 mmol, 1 equiv) was then added dropwise as a solution in anhydrous dimethylformamide (383 mu). The reaction mixture was stirred at 23 C under argon for 45 min. The reaction mixture was diluted with dimethylformamide to a total of 900 uL and purified directly by preparatory HPLC (Waters Prep-C18 SunFirecolumn, 5 muiotaeta, 19 x 250 mm, UV detection at 399 nm, gradient elution with 30?90% acetonitrile in water containing 0.1% formic acid, flow rate: 15 mL/min) to provide 47 (R02-A3) as a yellow solid (12.4 mg, 47%). TLC (15% methanol- dichloromethane + 2% 30% aqueous ammonium hydroxide solution): Rf= 0.59 (UV); 1H NMR (600 MHz, CD3OD) delta 8.23 (d, = 7.6 Hz, 1H), 7.96 (d, = 7.9 Hz, 1H), 7.54 (d, = 8.3 Hz, 2H), 7.46 (d, = 1.1 Hz, 1H), 7.26 (d, = 8.5 Hz, 2H), 6.77 (s, 2H), 5.20 (d, = 4.1 Hz, 1H), 5.00 - 4.97 (m, 3H), 4.65 (s, 1H), 4.52 - 4.47 (m, 1H), 4.19 - 4.14 (m, 1H), 4.02 - 3.93 (m, 2H), 3.78 (s, 3H), 3.56 (s, 3H), 3.45 (t, = 7.1 Hz, 2H), 3.39 (s, 3H), 3.25 - 3.18 (m, 3H), 3.10 (dt, = 13.4, 6.6 Hz, 1H), 3.06 - 3.01 (m, 2H), 2.78 (d, = 5.8 Hz, 1H), 2.73 - 2.68 (m, 3H), 2.56 (s, 3H), 2.26 (t, = 7.4 Hz, 2H), 2.10 - 2.04 (m, 3H), 1.93 - 1.86 (m, 1H), 1.77 - 1.71 (m, 3H), 1.70 - 1.65 (m, 2H), 1.65 - 1.59 (m, 2H), 1.60 - 1.50 (m, 2H), 1.39 - 1.35 (m, 2H), 1.33 - 1.25 (m, 2H), 0.96 (app t, = 6.5 Hz, 6H); HRMS (ESI): Calc'd for (C58H73N7Oi8+ Na)+: 1178.4904, found: 1178.4875. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
46% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 23℃; for 0.333333h;Inert atmosphere; | [00536] To a solution of Mal-Cap-Val-Cit-PABO-pNP (14.0 mg, 0.019 mmol, 3 equiv) in anhydrous dimethylformamide (105 mu) was added N,N-diisopropylethylamine (11.0 mu, 0.063 mmol, 10 equiv). Alkyl amine 13 (R02-A10) (3.6 mg, 6.30 muiotaetaomicron, 1 equiv) was then added dropwise as a solution in anhydrous dimethylformamide (105 mu). The reaction mixture was stirred at 23 C under argon for 20 min. The reaction mixture was diluted with dimethylformamide to a total of 900 uL and purified directly by preparatory HPLC (Waters Prep-C18 SunFirecolumn, 5 muiotaeta, 19 x 250 mm, UV detection at 399 nm, gradient elution with 30?90% acetonitrile in water containing 0.1 % formic acid, flow rate: 15 mL/min) to provide 49 as a yellow solid (3.4 mg, 46%). TLC (15% methanol-dichloromethane + 2% 30% aqueous ammonium hydroxide solution): Rf= 0.60 (UV); 1H NMR (600 MHz, CD3OD) delta 8.52 (s, 1H), 7.55 (d, = 8.3 Hz, 2H), 7.48 (s, 1H), 7.28 (d, = 8.2 Hz, 2H), 6.78 (s, 2H), 5.21 (d, = 4.2 Hz, 1H), 4.99 (s, 2H), 4.98 (d, = 4.1 Hz, 1H), 4.81 (d, = 5.6 Hz, 2H), 4.66 (s, 1H), 4.49 (dd, = 9.0, 5.1 Hz, 1H), 4.15 (d, = 7.4 Hz, 1H), 4.00 - 3.94 (m, 2H), 3.78 (s, 3H), 3.56 (s, 3H), 3.46 (t, = 7.1 Hz, 2H), 3.40 (s, 3H), 3.22 - 3.17 (m, 1H), 3.15 (t, = 6.7 Hz, 2H), 3.13 - 3.07 (m, 1H), 3.07 - 3.03 (m, 2H), 2.80 (d, = 5.8 Hz, 1H), 2.74 - 2.70 (m, 3H), 2.57 (s, 3H), 2.26 (t, = 7.4 Hz, 2H), 2.10 - 2.03 (m, 3H), 1.93 - 1.86 (m, 1H), 1.76 - 1.70 (m, 2H), 1.66 - 1.53 (m, 10H), 1.53 - 1.46 (m, 2H), 1.32 - 1.26 (m, 2H), 0.96 (app t, / = 6.5 Hz, 6H); HRMS (ESI): Calc'd for (C59H75N7O18+ Na)+: 1192.5061, found: 1192.5012. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
64% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 23℃; for 0.666667h;Inert atmosphere; | [00531] To a solution of Mal-Cap-Val-Cit-PABO-pNP (12.22 mg, 0.017 mmol, 3.0 equiv) in dry dimethylformamide (200 mu) was added N,N-diisopropylethylamine (9.64 mu, 0.055 mmol, 10 equiv) at 23 C. Amine 16 (3 mg, 5.52 muiotaetaomicron, 1.0 equiv) in dry dimethylformamide (200 mu) was added dropwise to the solution and stirred for 40 min. The reaction solution was purified directly by preparatory HPLC (Waters Prep-C18 SunFirecolumn, 5 muiotaeta, 19 x 250 mm, UV detection at 399 nm, gradient elution with 30?90% acetonitrile in water containing 0.1% formic acid over 30 min, flow rate: 15 mL/min) to provide 44 (R02-B4) as yellow solid (4 mg, 64%). 1H NMR (600 MHz, CD3OD) delta: 7.52-7.24 (m, 8H), 6.78 (s, 2H), 5.20 - 4.78 (m, 7H), 4.63 (s, 1H), 4.48 - 4.46 (m, 1H), 4.19 - 4.17 (m, 1H), 4.10 - 4.08 (m, 2H), 3.80 (s, 3H), 3.58 (s, 3H), 3.45 - 3.44 (m, 2H), 3.40 (s, 3H), 3.23 - 3.18 (m, 2H), 3.10 - 3.04 (m, 6H), 2.81 - 2.62 (m, 5H), 2.58 (s, 3H), 2.26 - 2.24 (m, 2H), 2.09 - 2.05 (m, 2H), 1.90 - 1.53 (m, 11H), 0.95 - 0.93 (m, 6H); HRMS (ESI): Calc'd for (C57H7iN70i8+Na)+1164.4748, found 1164.4851. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
35% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 23℃; for 0.666667h;Inert atmosphere; | [00533] To a solution of Mal-Cap-Val-Cit-PABO-pNP (39.7 mg, 0.054 mmol, 3.0 equiv) in dry dimethylformamide (200 mu) was added N,N-diisopropylethylamine (31.3 mu, 0.179 mmol, 10 equiv) at 23 C. Amine 19 (10 mg, 0.018 mmol, 1.0 equiv) in drydimethylformamide (200 mu) was added dropwise to the solution and stirred for 40 min. The reaction solution was purified directly by preparatory HPLC (Waters Prep-C18 SunFirecolumn, 5 muiotaeta, 19 x 250 mm, UV detection at 399 nm, gradient elution with 30?90% acetonitrile in water containing 0.1% formic acid over 30 min, flow rate: 15 mL/min) to provide 46 (R02-A6) as a yellow solid (7.2 mg, 35%). 1H NMR (600 MHz, CD3OD) delta: 7.59- 7.21 (m, 8H), 6.78 (s, 2H), 5.20 - 4.63 (m, 8H), 4.50 - 4.48 (m, 1H), 4.20 - 4.18 (m, 1H), 4.05 - 3.94 (m, 2H), 3.78 (s, 3H), 3.58 (s, 3H), 3.43 - 3.42 (m, 2H), 3.39 (s, 3H), 3.23 - 3.05 (m, 2H), 3.06 - 2.94 (m, 6H), 2.78 - 2.64 (m, 5H), 2.56 (s, 3H), 2.26 - 2.24 (m, 2H), 2.07 - 2.05 (m, 2H), 1.95 - 1.51 (m, 13H), 0.96 - 0.94 (m, 6H); HRMS ESI): Calc'd for(C58H73N7Oi8+Na)+1178.4904, found 1178.4992. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
57% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 23℃; for 0.666667h;Inert atmosphere; | [00535] To a solution of Mal-Cap-Val-Cit-PABO-pNP (11.6 mg, 0.016 mmol, 3.0 equiv) in dry dimethylformamide (200 mu) was added N,N-diisopropylethylamine (9.17 mu, 0.052 mmol, 10 equiv) at 23 C. Amine 22 (3 mg, 5.25 muiotaetaomicron, 1.0 equiv) in dry dimethylformamide (200 mu) was added dropwise to the solution and stirred for 40 min. The reaction solution was purified directly by preparatory HPLC (Waters Prep-C18 SunFirecolumn, 5 muiotaeta, 19 x 250 mm, UV detection at 399 nm, gradient elution with 30?90% acetonitrile in water containing 0.1% formic acid over 30 min, flow rate: 15 mL/min) to provide 48 (R02-A8) as a yellow solid (3.5 mg, 57%). 1H NMR (600 MHz, CD3OD) delta: 7.95 (br, 1H), 7.59 - 7.18 (m, 7H), 6.78 (s, 2H), 5.20 - 4.93 (m, 7H), 4.64 (s, 1H), 4.48 - 4.46 (m, 1H), 4.19 - 4.16 (m, 1H), 4.00-3.94 (m, 2H), 3.78 (s, 3H), 3.56 (s, 3H), 3.45 - 3.44 (m, 2H), 3.38 (s, 3H), 3.23 - 3.08 (m, 2H), 3.06 - 3.04 (m, 2H), 2.92 - 2.90 (m, 4H), 2.77 - 2.71 (m, 5H), 2.56 (s, 3H), 2.26 - 2.24 (m, 2H), 2.07 - 2.05 (m, 2H), 1.90 - 1.89 (m, 1H), 1.74 - 1.29 (m, 14H), 0.95 - 0.92 (m, 6H); HRMS (ESI): Calc'd for (C59H75N7Oi8+Na)+1192.5061, found 1192.5151. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
74% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 23℃; for 0.666667h;Inert atmosphere; | [00537] To a solution of Mal-Cap-Val-Cit-PABO-pNP (6.30 mg, 8.54 muetaiotaomicron, 5.0 equiv) in dry dimethylformamide (200 mu) was added N,N-diisopropylethylamine (2.98 mu, 0.017 mmol, 10 equiv) at 23 C. Amine 26 (1 mg, 1.708 muiotaetaomicron, 1.0 equiv) in drydimethylformamide (200 mu) was added dropwise to the solution and stirred for 40 min. The reaction solution was purified directly by preparatory HPLC (Waters Prep-C18 SunFirecolumn, 5 muiotaeta, 19 x 250 mm, UV detection at 399 nm, gradient elution with 30?90% acetonitrile in water containing 0.1% formic acid over 30 min, flow rate: 15 mL/min) to provide 50 as a yellow solid (1.5 mg, 74%). 1H NMR (600 MHz, CD3OD) delta: 8.53 (s, 1H), 7.56 - 7.18 (m, 7H), 6.77 (s, 2H), 5.22 - 4.85 (m, 7H), 4.65 (s, 1H), 4.48 - 4.47 (m, 1H), 4.15 (d, 1H, 7.5 Hz), 4.02 - 3.94 (m, 2H), 3.78 (s, 3H), 3.56 (s, 3H), 3.45 (t, 2H, = 7.1 Hz), 3.40 (s, 3H), 3.20 - 3.08 (m, 2H), 3.06 - 3.04 (m, 2H), 2.92 - 2.89 (m, 4H), 2.80 - 2.71 (m, 5H), 2.59 (s, 3H), 2.26 - 2.24 (m, 2H), 2.12 - 2.07 (m, 2H), 1.90 - 1.89 (m, 1H), 1.74 - 1.43 (m, 16H), 0.95 - 0.91 (m, 6H); HRMS (ESI): Calc'd for (C6oH77N7Oi8+Na)+1206.5217, found 1206.5284. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
62% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; for 16h; | 54 mg (0.07 mmol) of MC-Val-Cit-PAB-PNP (CAS No. 159857-81-5) was added to a solution containing 40 mg (0.07 mmol) of 2-amino-N8-(5-(aminomethyl)pyridin-3-yl)-N4,N4-dipropyl- 3H-benzo[b]azepine-4,8-dicarboxamide in 1.0 mL of DMF and 32 pL (0.18 mmol) of DIPEA.. The reaction mixture was stirred for 16 h then purified directly by reverse phase chromatography (no TFA). The clean fractions were lyophilized to afford 60 mg (71 %) of the desired product which was dissolved in 5 mL of DCM and treated with 1 mL of TFA at room temperature. The mixture was stirred for 45 minutes and then evaporated. The resulting residue was purified by reverse phase chromatography (no TFA) to afford 34 mg (62 %) of Compound-Linker 2.1 as a white solid. 1H NMR (CD3OD) d 8.81 (s, 1H), 8.25 (s, 1H), 8.21 (s, 1H), 7.72 (s, 1H), 7.58 (m, 2H), 7.45 (d, J=8.2 Hz, 2H), 7.33 (d, J=8.4Hz, 2H), 6.91 (s, 1H), 6.75 (s, 2H), 5.08 (s, 2H), 4.49 (m, 1H), 4.39 (m, 2H), 4.14 (d, J=6.5Hz, 1H), 3.47 (t, J=7. lHz, 2H), 3.42 (m, 4H), 3.15 (m, 1H), 3.10 (m, 1H), 2.27 (t, J=7.4Hz, 2H), 2.05 (m, 1H), 1.88 (m, 1H), 1.75-1.52 (m, 13H), 1.31 (m, 2H), 0.97 (t, J=6.5Hz, 6H). LCMS [M+H] = 1033. |
62% | 54 mg (0.07 mmol) of MC-Val-Cit-PAB-PNP (CAS No. 159857-81-5) was added to a solution containing 40 mg (0.07 mmol) of 2-amino-N8-(5-(aminomethyl)pyridin-3-yl)-N4,N4-dipropyl-3H-benzo[b]azepine-4,8-dicarboxamide in 1.0 mL of DMF and 32 muL (0.18 mmol) of DIPEA. The reaction mixture was stirred for 16 h then purified directly by reverse phase chromatography (no TFA). The clean fractions were lyophilized to afford 60 mg (71%) of the desired product which was dissolved in 5 mL of DCM and treated with 1 mL of TFA at room temperature. The mixture was stirred for 45 minutes and then evaporated. The resulting residue was purified by reverse phase chromatography (no TFA) to afford 34 mg (62%) of Compound-Linker 2.1 as a white solid. 1H NMR (CD3OD) delta 8.81 (s, 1H), 8.25 (s, 1H), 8.21 (s, 1H), 7.72 (s, 1H), 7.58 (m, 2H), 7.45 (d, J=8.2 Hz, 2H), 7.33 (d, J=8.4 Hz, 2H), 6.91 (s, 1H), 6.75 (s, 2H), 5.08 (s, 2H), 4.49 (m, 1H), 4.39 (m, 2H), 4.14 (d, J=6.5 Hz, 1H), 3.47 (t, J=7.1 Hz, 2H), 3.42 (m, 4H), 3.15 (m, 1H), 3.10 (m, 1H), 2.27 (t, J=7.4 Hz, 2H), 2.05 (m, 1H), 1.88 (m, 1H), 1.75-1.52 (m, 13H), 1.31 (m, 2H), 0.97 (t, J=6.5 Hz, 6H). LCMS [M+H]=1033. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
5.03% | General procedure: To a solution of (2S,3S,6S,7R,10R,E)-7-acetoxy-10-hydroxy-2-((R,2E,4E)-6- hydroxy-7-((2R,3R)-3-((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4- dien-2-yl)-3,7-dimethyl-12-oxooxacyclododec-4-en-6-yl piperazine-1 -carboxylate (30 mg, 0.045 mmol) in DCM (0.1 M) was added (9H-fluoren-9-yl)methyl methyl(2-oxoethyl)carbamate (5 equiv.) and sodium triacetoxy borohydride (10 equiv.) and stirred for 30 minutes. The mixture then diluted with ethyl acetate and washed with water and brine. The organic layer was dried over anhydrous Na2SC>4, concentrated and purified by column chromatography (0-10% MeOH/DCM gradient) to isolate the desired product. The resulting material was then diluted with DMF (0.1M), charged with diethylamine (20 equiv.) and stirred for 20 minutes. The mixture was then concentrated to dryness and diluted again in DMF and charged with 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H- pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (1.2 equiv.), then Hunig?s base (3.0 equiv.). The mixture was stirred for 30 minutes, concentrated in vacuo, and purified via preparative HPLC purification to afford target product. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
56.1% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | Linker-Payload (ADL1-D81: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (7.5 mg,10.166 pmol) in DMF (315 pL,4.066 mmol) was added Hunig's base (5.33 pL, 0.03 mmol). The reaction was cooled to 0C, and added (1604) (2S,3S,6S,7R,10R,E)-6-acetoxy-10-hydroxy-2-((R,2E,4E)-6-hydroxy-7-((2R,3R)-3- ((2R,3S)-3-hydroxypentan-2-yl)oxiran-2-yl)-6-methylhepta-2,4-dien-2-yl)-3, 7-dimethyl- 12-oxooxacyclododec-4-en-7-yl piperazine-1-carboxylate (7.50 mg, 0.011 mmol). The reaction mixture was stirred at RT until consumption of starting material. The reaction mixture was concentrated in vacuo. Flash chromatography afforded ADL1-D8 (7.2 mg,5.70 pmol,56.1 % yield). LC/MS (ESI, m/z),1263.8 [M+H]+. General procedure 1 (1.3.1) can also be employed for the preparation of ADL1-1 10987. (1605) [961] 1H-NMR (400 MHz, CHLOROFORM-d): d ppm 0.80 - 1.02 (m,16 H) 1.20 - 1.35 (m, 6 H) 1.38 - 1.48 (m, 2 H) 1.57 - 1.69 (m, 7 H) 1.74 - 1.81 (m,4 H) 1.86 - 1.96 (m, 2 H) 2.00 - 2.1 1 (m,4 H) 2.31 (br d, J= 6.02 Hz, 2 H) 2.45 - 2.55 (m, 2 H) 2.59 - 2.74 (m, 2 H) 2.85 - 2.96 (m,1 H) 3.05 -3.25 (m,5 H) 3.40 -3.59 (m, 9 H) 3.62 -3.88 (m,1 H) (1606) 4.15 (d, J= 7.53 Hz,1 H) 5.05 - 5.22 (m,5 H) 5.56 - 5.77 (m, 2 H) 5.84 - 5.98 (m,1 H) 6.1 1 - 6.22 (m,1 H) 6.47 - 6.61 (m,1 H) 7.31 - 7.40 (m, 2 H) 7.56 - 7.65 (m, 2 H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
47.7% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | To 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H- pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (17 mg, .023 mmol) in DMF (714 pL, 9.217 mmol) was added Hunig's base (12.07 pL, 0.069 mmol). The reaction mixture was cooled to 0C, and added 2-(4-(3-fluoro-5-((E)-2-((2S,3S,6R,7S,10R,E)-10-hydroxy-3,7-dimethyl-12-oxo-6- ((piperazine-1-carbonyl)oxy)oxacyclododec-4-en-2-yl)prop-1-en-1-yl)phenyl)piperazin-1-yl)acetic acid (16.71 mg, 0.026 mmol). The reaction mixture was stirred at RT until LC/MS showed the reaction complete. The reaction mixture was concentrated in vacuo. Flash chromatography on silica gel and further HPLC purification gave the titled compound (13.5 mg,10.98 pmol,47.7 % yield). LC/MS (ESI, m/z),1229.5 [M+H]+. (1671) [992] 1H-NMR (400 MHz, MeOH-d4): d ppm 0.92 - 0.99 (m,10 H) 1.01 (d, J= 6.78 Hz,3 H) 1.24 - 1.46 (m, 9 H) 1.52 - 1.70 (m,10 H) 1.70 - 1.82 (m,1 H) 1.87 (d, =1.00 Hz,4 H) 1.89 - 2.00 (m, 2 H) 2.01 - 2.23 (m, 2 H) 2.27 (t, =7.40 Hz, 2 H) 2.46 (dd, =14.31,5.27 Hz,1 H) 2.57 - 2.71 (m, 2 H) 2.86 (d, =0.63 Hz,1 H) 3.00 (s,1 H) 3.06 -3.27 (m, (1672) 4 H) 3.35 (s,5 H) 3.41 -3.53 (m,19 H) 3.66 (s, 2 H) 3.76 -3.90 (m,1 H) 4.07 - 4.21 (m, (1673) 1 H) 4.39 - 4.69 (m,37 H) 5.09 (s,3 H) 5.13 (d, =10.54 Hz,1 H) 5.52 (dd, =14.56, (1674) 9.03 Hz, 2 H) 6.55 (br s, 2 H) 6.69 (d, =1.38 Hz, 2 H) 7.33 (d, =8.66 Hz, 2 H) 7.59 (d, =8.53 Hz, 2 H) 7.88 - 8.04 (m,1 H) 8.16 - 8.33 (m,1 H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
68% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; | Linker-Payload (AD1-D131: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol- 1-yl)hexanamido)-3-methylbutanamido)-5ureidopentanamido)benzyl (4-nitrophenyl) carbonate (4.2 mg,5.693 pmol) in DMF (176 pL, 2.277 mmol) was added Hunig's base (2.98 pL, 0.017 mmol). The reaction mixture was cooled to 0C, and D13 was added (4.06 mg, 6.262 pmol). The reaction mixture was stirred at RT until LC/MS showed the reaction complete. The reaction mixture was concentrated in vacuo. Flash (1655) chromatography of the residue on silica gel with DCM/MeOH gave the title compound (4.8 mg, 68% yield). LC/MS (ESI, m/z),1248.0 [M+H]+. (1656) [986] 1H-NMR (400 MHz, CHC l3-c): d ppm 0.58 - 0.98 (m,10 H) 0.99 - 1.06 (m, 2 H) 1.1 1 - 1.31 (m, 6 H) 1.57 - 1.75 (m,3 H) 2.1 1 - 2.26 (m,1 H) 2.30 - 2.73 (m, 2 H) 3.28 -3.80 (m, 7 H) 4.14 - 4.33 (m,1 H) 4.44 - 4.55 (m,1 H) 4.63 - 4.81 (m,1 H) 4.92 - 5.06 (m,1 H) 5.09 (s,1 H) 5.49 - 5.74 (m,1 H) 5.88 - 6.09 (m,1 H) 6.14 - 6.36 (m,1 H) 6.99 - 7.08 (m,1 H) 7.21 - 7.38 (m, 2 H) 7.53 - 7.67 (m,1 H) 7.95 (s,1 H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
24% | With triethylamine; In N,N-dimethyl-formamide; at 0 - 21℃; for 2h; | Compound C was linked to a valine-citrulline linker according to the general methodology in Scheme 6 below: L1 (122 mg, 0.165 mmol, 1.1 equiv.) and TEA (52 mI, 0.375 mmol, 2.5 equiv.) was added to a solution of Compound C (58 mg, 0.150 mmol, 1.0 equiv.) in DMF (2 ml) at 0 C and the reaction mixture was stirred at approximately 21 C for 2 hours to afford crude ADC- 1. The crude ADC-1 was purified by preparative HPLC to afford purified ADC-1 as a white solid (34 mg, 24 % yield). |
24% | With triethylamine; In N,N-dimethyl-formamide; at 0 - 21℃; for 2h; | L1 (122 mg, 0.165 mmol, 1.1 equiv.) and TEA (52 mul, 0.375 mmol, 2.5 equiv.) was added to a solution of Compound C (58 mg, 0.150 mmol, 1.0 equiv.) in DMF (2 ml) at 0 C. and the reaction mixture was stirred at approximately 21 C. for 2 hours to afford crude ADC-1. The crude ADC-1 was purified by preparative HPLC to afford purified ADC-1 as a white solid (34 mg, 24% yield). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
42% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; | In a small vial, MC-VC-PAB-PNP6 (7.2 mg, 0.0098 mmol) and compound 5 (10 mg,0.0089 mmol) were dissolved in 50 uL DMF. To this mixture was added (iPr)2NEt (1.8 uL,0.01 mmol) and the reaction was stirred at room temperature overnight (whereupon LCMSindicated the reaction was complete). The homogeneous mixture was diluted with DMF, andwas purified directly by HPLC using a 28 min method eluting with a gradient of 40-80%Acetonitrile: 0.05% TFA in Water on a Luna 10u C18, 250 x 30 mm column. Isolatedcompound 6 (6.5 mg, 42%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
47% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 40℃; for 21h; | To a mixture of 2-amino-N-(2-((l-(4-amino-2-(ethoxymethyl)-lH-imidazo[4,5- c]quinolin-l-yl)-2-methylpropan-2-yl)oxy)ethyl)-2-methylpropanamide 1.2 (78.0 mg, 0.18 mmol) and 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanamido)-3- methylbutanamido)-5-ureidopentanamido)benzyl (4-nitrophenyl) carbonate (0.9 equiv., 117 mg, 0.16 mmol) in DMF (5.0 mL) was added diisopropylethylamine (4.0 equiv., 123 pL, 0.77 mmol). The mixture was stirred at 40 C for 2lh. The crude reaction was purified via preparative RP-HPLC (0- 100% AcN in H20, 0.1% TFA). Pure fractions were pooled, frozen and dried via lyophilization to give 86 mg (47% yield) of the desired product as an off-white solid. (1049) LCMS (M + H) = 1042.2. (1050) 1H NMR (DMSO, 400 MHz) d 13.50 (s, 1H), 9.97 (s, 1H), 8.54 (d, 1H, J= 8.4 Hz), 8.12 (d, 1H, J = 6.8 Hz), 8.07 (d, 1H, 7.6 Hz), 7.79 (apparent t, 2H, j = 7.60 Hz), 7.68 (t, 1H, j = 8.0 Hz), 7.57 (d, 2H, J = 8.8 Hz), 7.53 (t, 1H, J = 7.6 Hz), 7.32-7.17 (m, 4H), 6.98 (s, 2H), 6.98 (d, 1H, j = 6.8 Hz), 6.92 (d, 1H, j = 6.8 Hz), 6.00 (bs, 1H), 4.87 (s, 3H), 4.35 (quintet, 3H, j = 6.8 Hz), 4.18 (dd, 2H, J = 8.4, 6.8 Hz), 3.55 (t, 2H, J =12 Hz), 3.36 (t, 2H, J = 6.8 Hz), 3.20-3.08 (m, 2H), 3.60-2.90 (m, 2H), 2.78-2.89 (m, 2H), 2.17 (quintet, 1H, J = 6.4 Hz), 2.13 (quintet, 1H, J = 6.4 Hz), 1.94 (septet, 1H, 7 = 6.4 Hz), 1.74-1.63 (m, 1H), 1.63-1.54 (m, 1H), 1.53-1.39 (m, 6H), 1.38-1.27 (m, 2H), 1.21 (s, 6H), 1.22-1.10 (m, 4H), 1.14 (t, 6H, 7 = 6.8 Hz), 0.83 (dd, 6H, J = 12.4, 6.8 Hz). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
68.4% | To the stirring solution of compound CR55e1 (10.0 mg, 0.01 mmol) in 2 mL CH2Cl2 was added 0.5 mL TFA. After stirring for 1 h at room temperature, TLC showed that the reaction was completed. The solution was concentrated and dried out by diaphragm pump to obtain a pale yellow oil paste. After that, Mc- Val-Cit-PAB-PNP (17.7 mg, 0.02 mmol) and 1 mL dry DMF were added and stirred, and to this solution was added 12 mL DIPEA. The mixturewas stirred at room temperature for 18 h and monitored by TLC. After completion, the solution was concentrated to remove DMF by oil pump and purified by preparative TLC (CH2Cl2/MeOH = 20/1) to obtain the target compound L2-CR55 as a creamy white solid (10.8 mg) in a 68.4 yield with HPLC purity of 95.7%. 1H NMR (400 MHz, CD3OD) delta 7.84-7.67 (m, 1H), 7.65-7.52 (m, 2H),7.39-7.33 (m, 2H), 7.32-7.25 (m, 5H), 7.19-7.15 (m, 1H), 6.96 (d,J = 8.4 Hz, 1H), 6.77 (s, 2H), 6.73-6.61 (m, 1H), 5.92 (d, J = 15.0 Hz,1H), 5.45 (d, J = 9.5 Hz, 1H), 5.11-5.01 (m, 2H), 5.00 (s, 2H),4.55-4.46 (m, 2H), 4.18 (d, J = 7.4 Hz, 1H), 3.83 (s, 3H), 3.46 (t,J = 7.1 Hz, 3H), 3.40 (d, J = 5.6 Hz, 2H), 3.21-3.14 (m, 2H), 3.14-3.05(m, 2H), 2.79-2.70 (m, 1H), 2.66-2.60 m, 2H), 2.27 (t, J = 7.4 Hz,2H), 2.12-2.03 (m, 1H), 1.93-1.83 (m, 2H), 1.82-1.76 (m, 2H),1.64-1.54 (m, 5H),1.31-1.26 (m, 4H),1.22 (s, 3H),1.18 (s, 3H),1.12 (d,J = 6.3 Hz, 3H), 1.00-0.94 (m, 12H). 13C NMR (101 MHz, CD3OD)delta 177.5, 174.9, 172.5, 172.3, 171.2, 170.2, 168.8, 166.8, 160.9, 157.2,154.0,142.5,138.0,137.7,133.9,130.1,128.6,128.3,128.1,127.9,123.8,121.9, 119.7, 112.2, 74.9, 71.2, 67.8, 67.2, 60.4, 59.1, 55.2, 53.5, 42.7,39.6, 37.0, 35.2, 30.3, 27.9, 26.4, 26.0, 25.0, 24.9, 22.3, 22.0, 20.7,18.4,18.1, 17.5, 8.6. HRMS (ESI) calcd for C67H87Cl2N9O17 [M+Na]+1382.5489, found 1382.5498. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
23.4% | To the stirring solution of compound CR55-3 (18.0 mg, 0.02 mmol) in 3mL CH2Cl2 was added 1.5mL TFA. After strirring for 30 minitues at room temperature, TLC showed that the reaction was completed. The solution was concentrated and dried out by diaphragm pump to obtain a pale yellow oil paste. After that, Mc-Val-Cit-PAB-PNP (15.0 mg, 0.02 mmol) and 2 mL dry DMF was added, and to this solution was added 50 mL DIPEA. The solution was stirred at room temperature for 48 h and monitored by TLC. After completion, the mixture was concentrated to remove DMF by oil pump and purified by preparative TLC (CH2Cl2/MeOH 0 10/1) to get the compound L3-CR55 as an offwhite solid (6.6 mg) in a 23.4% yield with HPLC purify of 95.5%.1H NMR (400 MHz, MeOD) delta 7.58-7.53 (m, 2H), 7.49-7.41 (m, 2H), 7.40-7.22 (m, 6H), 7.19-7.12(m, 1H), 7.01-6.90 (m, 1H), 6.77 (s, 1H), 6.71-6.52 (m, 1H),5.99-5.83 (m, 1H), 5.41-5.28 (m, 1H), 5.26-5.14 (m, 1H), 5.13-5.01(m, 2H), 5.00-4.92 (m, 1H), 4.61-4.43 (m, 2H), 4.17 (d, J 0 7.4 Hz,1H), 3.83-3.81 (m, 3H), 3.57-3.34 (m, 5H), 3.24-3.14 (m, 2H),3.13-3.04 (m, 2H), 3.05-2.91 (m, 2H), 2.91-2.78 (m, 3H),2.77-2.68 (m, 2H), 2.67-2.47 (m, 2H), 2.43-2.15 (m, 5H), 2.12e2.01(m, 2H), 1.96-1.82 (m, 1H), 1.81-1.67 (m, 2H), 1.65-1.54 (m, 5H),1.38-1.31 (m, 4H), 1.28 (s, 6H), 1.21 (s, 3H), 1.16 (s, 3H), 0.95 (d,J = 6.1 Hz, 6H), 0.93-0.85 (m, 6H). 13C NMR (101 MHz, MeOD)delta 177.4, 172.6, 172.3, 171.2, 170.9, 170.4, 166.7, 163.6, 156.5, 153.9,138.0,135.0,133.9,132.7,130.8,130.1,128.5,128.2,127.9,124.1,121.9,119.8, 112.2, 71.1, 66.6, 63.7, 61.3, 59.1, 56.1, 55.3, 53.6, 50.3, 46.0,42.7, 39.4, 37.0, 35.2, 33.7, 31.7, 30.3, 29.4, 29.1, 27.9, 26.4, 26.0, 25.0,24.7, 22.3, 22.0, 18.5, 17.6, 13.0. HRMS (ESI) calcd forC70H94Cl2N10O17 [M+Na]+ 1439.6073, found 1439.6069. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
52% | According to the preparation method of Example 1, maleimide caproyl-MMAF was prepared.Dissolve 63.0mg maleimide caproyl-MMAF in 2.0mL N,N-dimethylformamide, add 13.3mg Cys-Cys,Add 4.8uL N,N-diisopropylethylamine and stir at room temperature for 3h.Add 86.6mg Mc-VC-PAB-PNP to the reaction system,Add 58uLN,N-diisopropylethylamine and stir at room temperature for 15h.The solvent was distilled off under reduced pressure and purified by liquid phase preparation647.4 mg of compound was obtained with a yield of 52%, |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
40% | With N-ethyl-N,N-diisopropylamine; In 1-methyl-pyrrolidin-2-one; at 23℃; for 72h; | To a solution of 11-R (100 mg, 0.12 mmol) and LIN 1 (465 mg, 0.63 mmol) in /V-Methyl-2- pyrrolidone (NMP) (15 mL) was added A/,A/-diisopropylethylamine (DIPEA) (111 m, 0.63 mmol) at 23 SC. The reaction mixture was stirred for 3 days at 23 SC, diluted with EtOAc (50 mL) and washed with H2O (4 x 30 mL) and a saturated aqueous solution of NaCI (30 mL). The combined organic layers were dried over anhydrous Na2SC>4, filtered, and concentrated under vacuum. The residue was purified by flash chromatography on silica gel (CH2Cl2:CH30H, from 99:1 to 90:10) to obtain DL 1 which was purified by HPLC preparative to yield pure DL 1 (69 mg, 40% yield). 1H NMR (400 MHz, CD3OD/CDCI3): d 7.85 (d, J = 7.9 Hz, 1H), 7.59 (d, J= 8.1 Hz, 2H), 7.36 (d, J= 8.2 Hz, 2H), 7.30 (d, J= 7.9 Hz, 1H), 7.22 (d, J= 8.2 Hz, 1H), 7.01 (t, J= 7.6 Hz, 1H), 6.91 (t, J = 7.5 Hz, 1 H), 6.72 (s, 2H), 6.58 (s, 1H), 6.26 (s, 1H), 6.07 (s, 1H), 5.25-5.14 (m, 2H), 5.14-5.01 (m, 2H), 4.67 (bs, 1H), 4.51 (d, J= 6.7 Hz, 2H), 4.28 (dd, J= 16.2, 7.1 Hz, 4H), 4.21-4.05 (m, 3H), 3.71 (s, 3H), 3.51-3.40 (m, 2H), 3.36-3.32 (m, 2H), 3.23-2.99 (m, 2H), (1655) 2.99-2.72 (m, 2H), 2.65 (d, J= 14.9 Hz, 2H), 2.28 (s,-3H), 2.25 (s,-3H), 2.10 (s,-3H), 2.04 (1656) (S,-3H), 1.96-1.83 (m, 1H), 1.80-1.68 (m, 2H), 1.65-1.50 (m, 10H), 1.35-1.23 (m, 2H), 0.95 (d, J= 6.8Hz, 3H), 0.94 (d, J= 6.8Hz, 3H). 13C NMR (75 MHz, CD3OH/CDCI3): d 174.9, 172.5, 171.5, 171.1, 170.7, 169.5, 160.8, 157.4, 148.6, 146.1, 143.6, 141.1, 140.9, 138.0, 136.8, 133.9, 132.6, 130.5, 129.8, 129.6, 128.6, (1658) 126.0, 121.6, 120.4, 119.8, 119.0, 118.6, 118.0, 117.8, 116.7, 113.5, 112.8, 110.9, 109.1, (1659) 102.3, 66.0, 63.1, 62.9, 61.6, 60.2, 59.9, 59.2, 59.1, 58.9, 54.6, 54.6, 53.5, 50.7, 45.3, 42.1, 40.5, 37.1, 35.3, 30.3, 29.1, 27.9, 26.3, 26.0, 25.0, 24.5, 23.6, 19.4, 18.6, 17.7, 15.1, 8.6. ESI-MS m/z: 1391.4 (M+H)+. (1660) Rf= 0.40 (CH2Cl2:CH3OH, 9:1 ). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
58% | With benzotriazol-1-ol; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 23℃; for 18h; | To a solution of 11-S (30 mg, 0.037 mmol) and LIN 1 (56 mg, 0.075 mmol) in Dimethylformamide (DMF) (2 mL, 0.018 M) was added A/,A/-Diisopropylethylamine (DIPEA) (26 m, 0.15 mmol) and 1-Hydroxybenzotriazole (HOBt, 10 mg, 0.075 mmol) at 23 SC. After 18 hours the reaction mixture was purified by HPLC preparative to yield pure DL 8 (30 mg, 58% yield). 1H NMR (400 MHz, CDsOD): d 7.59 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 7.9 Hz, 1 H), 7.23 (d, J = 8.2 Hz, 1 H), 7.00 (t, J = 7.6 Hz, 1 H), 6.91 (t, J = 7.5 Hz, 1 H), 6.74 (s, 2H), 6.50 (s, 1 H), 6.27 (s, 1H), 6.09 (s, 1H), 5.20-5.03 (m, 2H), 4.65 (bs, 1H), 4.54-4.46 (m, 1 H), 4.43-4.37 (m, 1H), 4.34-4.30 (m, 1H), 4.17-4.12 (m, 1H), 3.75 (s, 3H), 3.45 (t, J= 7.0 Hz, 4H), 3.32-3.23 (m, 2H), 3.38 (d, J = 7.6 Hz, 2H), 3.23-2.99 (m, 2H), 3.21-2.97 (m, 3H), 2.94- (1713) 2.83 (m, 3H), 2.61-2.53 (m, 2H), 2.48-2.34 (m, 2H), 2.28 (s,-3H), 2.27-2.22 (m, 1H), 2.21 (1714) (S,-3H), 2.11 (S,-3H), 2.08-2.02 (m, 1H), 1.99 (s,-3H), 1.91-1.82 (m, 1H), 1.77-1.68 (m, 1H), 1.65-1.50 (m, 6H), 1.31-1.24 (m, 2H), 0.94 (d, J= 6.8Hz, 3H), 0.93 (d, J= 6.8Hz, 3H). (1715) 13C NMR (100 MHz, CDsOD): d 174.9, 174.8, 172.5, 172.0, 171.2, 170.8, 169.4, 160.8, 157.5, 148.6, 146.0, 143.6, 140.9, 140.8, 138.0, 136.6, 136.5, 134.0, 132.7, 130.2, 129.9, 129.7, (1716) 128.7, 128.4, 126.3, 121.6, 120.5, 120.0, 119.8, 119.3, 118.7, 118.0, 117.7, 113.5, 112.9, (1717) 111.0, 107.8, 102.4, 65.9, 63.5, 61.4, 60.6, 59.7, 59.3, 59.2, 59.1, 58.8, 54.6, 54.6, 53.4, 44.8, 42.3, 40.6, 38.3, 37.1, 35.3 (x2), 30.3, 29.1, 28.0, 26.3, 26.0, 25.1, 24.0, 23.7, 19.5, 18.6, (1718) 17.7, 15.2, 8.6. (1719) ESI-MS m/z: 1391.4 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
39.2% | Stage #1: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-([(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide; Y-27632 With triethylamine In dimethyl sulfoxide at 25℃; for 0.75h; Stage #2: trifluoroacetic acid In lithium hydroxide monohydrate; acetonitrile | 8.1.4 8.1.4. Synthesis and analytical characterization of Mal-Val-Cit-PAB-Y27632 (1f) Y27632 x 2 HCI (20 mg, 62 mmol, 1.0 eq.), Mal-Val-Cit-PAB-PNP (48 mg, 66 mmol, 1.05 eq.) and triethylamine (22.2 mL, 125 mmol, 2.0 eq.) were dissolved in dry DMSO (1 mL) and the reaction mixture was stirred for 45 min at 25 °C. The reaction mixture was then diluted with MilliQ/MeOH (1:1, 3 mL) after which the suspension was filtered through a 0.2 mm syringe filter. Purification was performed by preparative reverse-phase HPLC (Grace Alltima C18 5 mm column, 22 x 250 mm; gradient: 20% to 40% B, whereas eluent A: 95/5 water/MeCN (+0.1% TFA) and eluent B: 5/95 water/MeCN (+0.1% TFA) in 80 min.). Product containing fractions were collected and lyophilized and the product If was obtained as a yellow solid (23.5 mg, 39.2% yield). (0200) HRMS (ESI+): C43H60N9O9 [M+H]+ 846.4509, found 846.4472. (0201) HPLC (Grace Alltima C18 5 mm column, 25 x 4.6 mm) indicated that the product was 95.6% pure (retention time 10.5 min.; gradient: 20% to 100% MeCN/0.1% TFA in water/0.1% TFA in 20 min. (0202) measured at a wavelength of 273 nm). |
39.2% | Stage #1: N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-([(4-nitrophenoxy)carbonyl]oxy}methyl)phenyl]-L-ornithinamide; Y-27632 With triethylamine In dimethyl sulfoxide at 25℃; for 0.75h; Stage #2: trifluoroacetic acid | 9.1.4 9.1.4. Synthesis and analytical characterization of Mal-Val-Cit-PAB-Y27632 (1f) Y27632 X 2 HCI (20 mg, 62 pmol, 1.0 eq.), Mal-Val-Cit-PAB-PNP (48 mg, 66 pmol, 1.05 eq.) and triethylamine (22.2 pL, 125 pmol, 2.0 eq.) were dissolved in dry DMSO (1 mL) and the reaction mixture was stirred for 45 min at 25°C. The reaction mixture was then diluted with MilliQ/MeOH (1 :1, 3 mL) after which the suspension was filtered through a 0.2 pm syringe filter. Purification was performed by preparative reverse-phase HPLC (Grace Alltima C18 5 pm column, 22 x 250 mm; gradient: 20% to 40% B, whereas eluent A: 95/5 water/MeCN (+0.1 % TFA) and eluent B: 5/95 water/MeCN (+0.1 % TFA) in 80 min.). Product containing fractions were collected and lyophilized and the product 1f was obtained as a yellow solid (23.5 mg, 39.2% yield). HRMS (ESF): C43H60N9O9 [M+H]+ 846.4509, found 846.4472. HPLC (Grace Alltima C18 5 pm column, 25 x 4.6 mm) indicated that the product was 95.6% pure (retention time 10.5 min.; gradient: 20% to 100% MeCN/0.1 % TFA in water/0.1 % TFA in 20 min. measured at a wavelength of 273 nm). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
200 mg | With 2,6-dimethylpyridine; 2-hydroxy-pyridine N-oxide; In N,N-dimethyl-formamide; for 48h;Inert atmosphere; Sealed tube; | To a scintillation vial were added N-Methyl-L-valine (6.2 equiv.) and mc-Val- Cit-PABC-PNP (500 mg, 1.0 equiv.). The vial was purged with nitrogen (x 3), and the solids were suspended in 2,6-lutidine (4.0 vol.) and DMF (4.0 vol.). Solid HOPO (1.2 equiv.) was added in one portion, the vessel was sealed, and the reaction was stirred vigorously for 48 h. The reaction was poured into MTBE (200 vol.), and the resulting mixture was vacuum filtered (washing with MTBE) to deliver a grey solid. The solid was solubilized in minimal AcOH (4.0 vol.), and the resulting solution was chromatographically purified on silica gel (5% MeOH in CH2Cl2 to 20%) to provide mc-Val-Cit-PAB-<strong>[2480-23-1]N-Me-Val-OH</strong> as a pale-yellow residue (200 mg). |
Tags: 159857-81-5 synthesis path| 159857-81-5 SDS| 159857-81-5 COA| 159857-81-5 purity| 159857-81-5 application| 159857-81-5 NMR| 159857-81-5 COA| 159857-81-5 structure
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P309 | IF exposed or if you feel unwell: |
P310 | Immediately call a POISON CENTER or doctor/physician. |
P311 | Call a POISON CENTER or doctor/physician. |
P312 | Call a POISON CENTER or doctor/physician if you feel unwell. |
P313 | Get medical advice/attention. |
P314 | Get medical advice/attention if you feel unwell. |
P315 | Get immediate medical advice/attention. |
P320 | |
P302 + P352 | IF ON SKIN: wash with plenty of soap and water. |
P321 | |
P322 | |
P330 | Rinse mouth. |
P331 | Do NOT induce vomiting. |
P332 | IF SKIN irritation occurs: |
P333 | If skin irritation or rash occurs: |
P334 | Immerse in cool water/wrap n wet bandages. |
P335 | Brush off loose particles from skin. |
P336 | Thaw frosted parts with lukewarm water. Do not rub affected area. |
P337 | If eye irritation persists: |
P338 | Remove contact lenses, if present and easy to do. Continue rinsing. |
P340 | Remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P341 | If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P342 | If experiencing respiratory symptoms: |
P350 | Gently wash with plenty of soap and water. |
P351 | Rinse cautiously with water for several minutes. |
P352 | Wash with plenty of soap and water. |
P353 | Rinse skin with water/shower. |
P360 | Rinse immediately contaminated clothing and skin with plenty of water before removing clothes. |
P361 | Remove/Take off immediately all contaminated clothing. |
P362 | Take off contaminated clothing and wash before reuse. |
P363 | Wash contaminated clothing before reuse. |
P370 | In case of fire: |
P371 | In case of major fire and large quantities: |
P372 | Explosion risk in case of fire. |
P373 | DO NOT fight fire when fire reaches explosives. |
P374 | Fight fire with normal precautions from a reasonable distance. |
P376 | Stop leak if safe to do so. Oxidising gases (section 2.4) 1 |
P377 | Leaking gas fire: Do not extinguish, unless leak can be stopped safely. |
P378 | |
P380 | Evacuate area. |
P381 | Eliminate all ignition sources if safe to do so. |
P390 | Absorb spillage to prevent material damage. |
P391 | Collect spillage. Hazardous to the aquatic environment |
P301 + P310 | IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician. |
P301 + P312 | IF SWALLOWED: call a POISON CENTER or doctor/physician IF you feel unwell. |
P301 + P330 + P331 | IF SWALLOWED: Rinse mouth. Do NOT induce vomiting. |
P302 + P334 | IF ON SKIN: Immerse in cool water/wrap in wet bandages. |
P302 + P350 | IF ON SKIN: Gently wash with plenty of soap and water. |
P303 + P361 + P353 | IF ON SKIN (or hair): Remove/Take off Immediately all contaminated clothing. Rinse SKIN with water/shower. |
P304 + P312 | IF INHALED: Call a POISON CENTER or doctor/physician if you feel unwell. |
P304 + P340 | IF INHALED: Remove victim to fresh air and Keep at rest in a position comfortable for breathing. |
P304 + P341 | IF INHALED: If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P305 + P351 + P338 | IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. |
P306 + P360 | IF ON CLOTHING: Rinse Immediately contaminated CLOTHING and SKIN with plenty of water before removing clothes. |
P307 + P311 | IF exposed: call a POISON CENTER or doctor/physician. |
P308 + P313 | IF exposed or concerned: Get medical advice/attention. |
P309 + P311 | IF exposed or if you feel unwell: call a POISON CENTER or doctor/physician. |
P332 + P313 | IF SKIN irritation occurs: Get medical advice/attention. |
P333 + P313 | IF SKIN irritation or rash occurs: Get medical advice/attention. |
P335 + P334 | Brush off loose particles from skin. Immerse in cool water/wrap in wet bandages. |
P337 + P313 | IF eye irritation persists: Get medical advice/attention. |
P342 + P311 | IF experiencing respiratory symptoms: call a POISON CENTER or doctor/physician. |
P370 + P376 | In case of fire: Stop leak if safe to Do so. |
P370 + P378 | In case of fire: |
P370 + P380 | In case of fire: Evacuate area. |
P370 + P380 + P375 | In case of fire: Evacuate area. Fight fire remotely due to the risk of explosion. |
P371 + P380 + P375 | In case of major fire and large quantities: Evacuate area. Fight fire remotely due to the risk of explosion. |
Storage | |
Code | Phrase |
P401 | |
P402 | Store in a dry place. |
P403 | Store in a well-ventilated place. |
P404 | Store in a closed container. |
P405 | Store locked up. |
P406 | Store in corrosive resistant/ container with a resistant inner liner. |
P407 | Maintain air gap between stacks/pallets. |
P410 | Protect from sunlight. |
P411 | |
P412 | Do not expose to temperatures exceeding 50 oC/ 122 oF. |
P413 | |
P420 | Store away from other materials. |
P422 | |
P402 + P404 | Store in a dry place. Store in a closed container. |
P403 + P233 | Store in a well-ventilated place. Keep container tightly closed. |
P403 + P235 | Store in a well-ventilated place. Keep cool. |
P410 + P403 | Protect from sunlight. Store in a well-ventilated place. |
P410 + P412 | Protect from sunlight. Do not expose to temperatures exceeding 50 oC/122oF. |
P411 + P235 | Keep cool. |
Disposal | |
Code | Phrase |
P501 | Dispose of contents/container to ... |
P502 | Refer to manufacturer/supplier for information on recovery/recycling |
Physical hazards | |
Code | Phrase |
H200 | Unstable explosive |
H201 | Explosive; mass explosion hazard |
H202 | Explosive; severe projection hazard |
H203 | Explosive; fire, blast or projection hazard |
H204 | Fire or projection hazard |
H205 | May mass explode in fire |
H220 | Extremely flammable gas |
H221 | Flammable gas |
H222 | Extremely flammable aerosol |
H223 | Flammable aerosol |
H224 | Extremely flammable liquid and vapour |
H225 | Highly flammable liquid and vapour |
H226 | Flammable liquid and vapour |
H227 | Combustible liquid |
H228 | Flammable solid |
H229 | Pressurized container: may burst if heated |
H230 | May react explosively even in the absence of air |
H231 | May react explosively even in the absence of air at elevated pressure and/or temperature |
H240 | Heating may cause an explosion |
H241 | Heating may cause a fire or explosion |
H242 | Heating may cause a fire |
H250 | Catches fire spontaneously if exposed to air |
H251 | Self-heating; may catch fire |
H252 | Self-heating in large quantities; may catch fire |
H260 | In contact with water releases flammable gases which may ignite spontaneously |
H261 | In contact with water releases flammable gas |
H270 | May cause or intensify fire; oxidizer |
H271 | May cause fire or explosion; strong oxidizer |
H272 | May intensify fire; oxidizer |
H280 | Contains gas under pressure; may explode if heated |
H281 | Contains refrigerated gas; may cause cryogenic burns or injury |
H290 | May be corrosive to metals |
Health hazards | |
Code | Phrase |
H300 | Fatal if swallowed |
H301 | Toxic if swallowed |
H302 | Harmful if swallowed |
H303 | May be harmful if swallowed |
H304 | May be fatal if swallowed and enters airways |
H305 | May be harmful if swallowed and enters airways |
H310 | Fatal in contact with skin |
H311 | Toxic in contact with skin |
H312 | Harmful in contact with skin |
H313 | May be harmful in contact with skin |
H314 | Causes severe skin burns and eye damage |
H315 | Causes skin irritation |
H316 | Causes mild skin irritation |
H317 | May cause an allergic skin reaction |
H318 | Causes serious eye damage |
H319 | Causes serious eye irritation |
H320 | Causes eye irritation |
H330 | Fatal if inhaled |
H331 | Toxic if inhaled |
H332 | Harmful if inhaled |
H333 | May be harmful if inhaled |
H334 | May cause allergy or asthma symptoms or breathing difficulties if inhaled |
H335 | May cause respiratory irritation |
H336 | May cause drowsiness or dizziness |
H340 | May cause genetic defects |
H341 | Suspected of causing genetic defects |
H350 | May cause cancer |
H351 | Suspected of causing cancer |
H360 | May damage fertility or the unborn child |
H361 | Suspected of damaging fertility or the unborn child |
H361d | Suspected of damaging the unborn child |
H362 | May cause harm to breast-fed children |
H370 | Causes damage to organs |
H371 | May cause damage to organs |
H372 | Causes damage to organs through prolonged or repeated exposure |
H373 | May cause damage to organs through prolonged or repeated exposure |
Environmental hazards | |
Code | Phrase |
H400 | Very toxic to aquatic life |
H401 | Toxic to aquatic life |
H402 | Harmful to aquatic life |
H410 | Very toxic to aquatic life with long-lasting effects |
H411 | Toxic to aquatic life with long-lasting effects |
H412 | Harmful to aquatic life with long-lasting effects |
H413 | May cause long-lasting harmful effects to aquatic life |
H420 | Harms public health and the environment by destroying ozone in the upper atmosphere |
Sorry,this product has been discontinued.
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