* 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.
With hydrogenchloride In 1,4-dioxane; water at 20℃; for 0.583333 h;
(S)-2-((2R,3R)-3-((S)- 1 -((6S ,9S, 12S, 1 3R)- 12-((S)-sec-butyl)-6,9-diisopropyl- 13- methoxy-2,2,5,11 -tetramethyl-4,7, 1 0-trioxo-3 -oxa-5 ,8,11 -triazapenta-decan- 15- oyl)pyrrolidin-2-yl)-3 -methoxy-2-methylpropanamido)-3 -phenylpropanoic acid (25 mg,0.030 mmol) in the mixture of HC1 (conc. 0.3 ml) and 1,4-dioxane (0.9 ml) was stirred at RT for 35 mm. The mixture was diluted with EtOH (1.0 ml) and toluene (1.0 ml), concentrated and re-evaporated with EtOH/toluene (2:1) to afford the title compound as a white solid (22 mg, -400percent yield) for the next step without further purification. LC-MS (ESI) mlz calcd. for C39H66N508 [M+Hj: 732.48, found: 732.60.
150 mg
With hydrogenchloride In 1,4-dioxane at 20℃; for 2 h;
Compound 12-5 was dissolved in 4N HClDioxane. The reactionmixture was stirred at room temperature for 2 hours and concentrated in vacuo and purified by HPLCto give 150mg of compound 12-6.
56 mg
With trifluoroacetic acid In dichloromethane at 20℃; for 5 h;
General procedure: LiOHH2O (5 mg, 0.12 mmol) was added to a solution of 18a (30 mg, 0.06 mmol) in MeOH (2 mL) and water (1 mL) at room temperature. After stirring at room temperature for 3 h, the reaction mixture was concentrated in vacuo and the obtained residue was redissolved in water (5 mL). The mixture was acidified to pH 4e5 by addition of 1 N HCl and a white precipitate was formed. The solid was collected and dried to afford the free acid species as a white solid. The obtained solid was dissolved in CH2Cl2 (2.5 mL), TFA (68 mg, 0.6 mmol) was added and the reaction mixture was stirred at room temperature for about 5 h and then concentrated under reduced pressure. The crude residue was purified by preparative HPLC (SB-C18 column, 5 mm, 9.4 250 mm, 40percent CH3CN/60percent H2O) to yield the desired product (24 mg, 93percent over 2 steps) as a white solid;
With sodium cyanoborohydride; acetic acid; In N,N-dimethyl-formamide; at 20℃; for 2h;
To a solution of compound 12-6 in DMF was added formylaldehyde (3eq) and 20 eq of acetic acid, followed by addition of 2 eq of sodium cyanoborohydride. The resulting mixture was stirred at ambient temperature for 2 hours. The reaction mixture was diluted with water and purified by HPLC to give compound 12-7.
With sodium cyanoborohydride; acetic acid; In N,N-dimethyl-formamide; for 2h;
To a solution of compound 12-6 in DMF was added formylaldehyde15 (3eq) and 20 eq of acetic acid, followed by addition of 2 eq of sodium cyanoborohydride. Theresulting mixture was stirred at ambient temperature for 2 hours. The reaction mixture was dilutedwith water and purified by HPLC to give compound 12-7.
With hydrogenchloride; In 1,4-dioxane; water; at 20℃; for 0.583333h;
(S)-2-((2R,3R)-3-((S)- 1 -((6S ,9S, 12S, 1 3R)- 12-((S)-sec-butyl)-6,9-diisopropyl- 13- methoxy-2,2,5,11 -tetramethyl-4,7, 1 0-trioxo-3 -oxa-5 ,8,11 -triazapenta-decan- 15- oyl)pyrrolidin-2-yl)-3 -methoxy-2-methylpropanamido)-3 -phenylpropanoic acid (25 mg,0.030 mmol) in the mixture of HC1 (conc. 0.3 ml) and 1,4-dioxane (0.9 ml) was stirred at RT for 35 mm. The mixture was diluted with EtOH (1.0 ml) and toluene (1.0 ml), concentrated and re-evaporated with EtOH/toluene (2:1) to afford the title compound as a white solid (22 mg, -400percent yield) for the next step without further purification. LC-MS (ESI) mlz calcd. for C39H66N508 [M+Hj: 732.48, found: 732.60.
With hydrogenchloride; In 1,4-dioxane; at 20℃; for 2h;
Compound 12-5 was dissolved in 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo and purified by HPLC to give 150mg of compound 12-6.
150 mg
With hydrogenchloride; In 1,4-dioxane; at 20℃; for 2h;
Compound 12-5 was dissolved in 4N HClDioxane. The reactionmixture was stirred at room temperature for 2 hours and concentrated in vacuo and purified by HPLCto give 150mg of compound 12-6.
With hydrogenchloride; In 1,4-dioxane; water; at 20℃; for 0.583333h;
Compound 330 (25 mg, 0.030 mmol) in the mixture of conc. HCl (0.3 ml) and 1,4-dioxane (0.9 ml) was stirred at r.t. for 35 min. The mixture was diluted with EtOH (1.0 ml) and toluene (1.0 ml), concentrated and co-evaporated with EtOH/toluene (2:1) to afford the title compound 331 as a white solid (22 mg, ~100percent yield), which was used in the next step without further purification. LC-MS (ESI) m/z calcd.for [M+H]+: 732.48, found: 732.60.
56 mg
With trifluoroacetic acid; In dichloromethane; at 20℃; for 5h;
General procedure: LiOHH2O (5 mg, 0.12 mmol) was added to a solution of 18a (30 mg, 0.06 mmol) in MeOH (2 mL) and water (1 mL) at room temperature. After stirring at room temperature for 3 h, the reaction mixture was concentrated in vacuo and the obtained residue was redissolved in water (5 mL). The mixture was acidified to pH 4e5 by addition of 1 N HCl and a white precipitate was formed. The solid was collected and dried to afford the free acid species as a white solid. The obtained solid was dissolved in CH2Cl2 (2.5 mL), TFA (68 mg, 0.6 mmol) was added and the reaction mixture was stirred at room temperature for about 5 h and then concentrated under reduced pressure. The crude residue was purified by preparative HPLC (SB-C18 column, 5 mm, 9.4 250 mm, 40percent CH3CN/60percent H2O) to yield the desired product (24 mg, 93percent over 2 steps) as a white solid;
[0071] Example 6. Synthesis of 39-(2,5-dioxo-3,4-bis(2-pyridylsulfanyl)pyrrolyl)- -dodecaoxanonatriacontanoyl-MMAF [dPSPEG-MMAF] : [0072] 39-(2,5-Dioxo-3,4-bis(pyridin-2-ylthio)-2,5-dihydro-lH-pyrrol-l-yl)- 3,6, 9, 12,15, 18,21, 24,27, 30,33, 36-dodecaoxanonatriacontanoic acid was added to a clean, flame- dried 50 mL round bottom flask, and the carboxylic acid was activated with NHS in 3 mL of DMF in the presence of DCC. MMAF was predissolved in about 1 mL DMF and transferred to the NHS- activated acid via 22 gauge needle. DIPEA was added to the reaction mixture and stirred overnight. The crude reaction mixture was purified by reverse-phase HPLC on a 21.2 mm x 50 mm Agilent PREP-C18 column at a flow rate of 35 mL/min over 20 column volumes (about 30 minutes of gradient time). Enriched fractions were identified, pooled and lyophilized to give the dPSPEG- MMAF conjugate as a white semi-solid.
With sodium cyanoborohydride; In dimethyl sulfoxide; at 60℃; for 72h;
sodium cyanoborohydride (200fImol) and 6-O-propargyl-D-galactose (45flmol) were added to the solution of MMAF (2.7fImol) in dimethylsulphoxide (0.7m1) . The mixture was stirred at 60°C for three days.
With sodium cyanoborohydride; In dimethyl sulfoxide; at 60℃; for 72h;
sodium cyanoborohydride (28mumol) and 2-deoxy-D-glucose (2lfImol) were added to thesolution of MMAF (1.4mmuol) in DMSQ (0.6m1) . The mixture was stirred at 60°C for three days.
to the solution of MMAF (2.7mmuol) in dry DMF (0.6ml) was added NaH (54mumol) and 4-bromo-1-butyne (27mumol) . The mixture was stirred at 60°C for 4 hours. Reaction was quenched by adding dry methanol (0.2ml)
With sodium hydride; In N,N-dimethyl-formamide; mineral oil; at 20℃; for 3h;
to the solution of MMAF (1.4mumol) in dry DMF (0.4ml) was added NaH (7mol) muand 5-iodo-1-pentyne (7mumol) . The mixture was stirred at room temperature for3 hours. Reaction was quenched by adding dry methanol (0.2m1)
sodium cyanoborohydride (25mumol) and 6-O-(3-D-galactopyranosyl)- D-galactose (5.3mumol) were added to the solution of MMAF(07.mumol) in DMSO (0.25ml) . The mixture was stirred at 60°C for five days.
2-acetamido-2-deoxy-4-O-(-D-galactopyranosyl)-D-glucose[ No CAS ]
C53H90N6O18[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With sodium cyanoborohydride; In dimethyl sulfoxide; at 60℃; for 120h;
sodium cyanoborohydride (SOfImol) and 2-acetamido-2-deoxy-4-O-(-D-galactopyranosyl)-D-glucose (llmumol)were added to the solution of MMAF (1.4mumol) in DMSO (0.4m1) The mixture was stirred at 60°C for five days.
4-O-[4-O-(α-D-galactopyranosyl)-β-D-galactopyranosyl]-D-glucose[ No CAS ]
C57H97N5O23[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With sodium cyanoborohydride; In dimethyl sulfoxide; at 60℃; for 120h;
N-{4-O- [4-0- (-D-galactopyranosyl) --Dgalactopyranosyl]-D-glucosyl}-MMAF (10) : sodium cyanoborohydride (SOfImol) and 4-0-[4-0-(-D-galactopyranosyl)--D-galactopyranosyl]-D-glucose (llfImol) were added to the solution of MMAF (1.4imol) in DMS0 (0.4m1) . The mixture was stirred at 60°C for five days.
4-O-[3-O-(α-N-acetyl-neuraminyl)-β-D-galactopyranosyl]-D-glucose[ No CAS ]
C60H101N5O23[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With sodium cyanoborohydride; In dimethyl sulfoxide; at 60℃; for 120h;
N-{4-O-[3-O-(-N-acetylneuraminyl) -Dgalactopyranosyl]-D-glucosyl}-MMAF (11) : sodium cyanoborohydride(50mumol) and 4-0-[3-0-(-N-acetyl-neuraminyl)-D- galactopyranosyl]-D-glucose (1mu1 mol) were added to the solution of MMAF (1.4mumol) in DMS0 (0.4muml) . The mixture was stirred at 60°C for five days.
With N-ethyl-N,N-diisopropylamine; In acetonitrile; at 20℃;
N- (N-hydroxysuccinimidylglutaryl) -MMAF (14) : disuccinimidyl glutarate (20flmol) and diisopropylethylamine (20flmol) were added to the solution of MMAF (1.4imol) in ACN (0.4m1) . The mixture was stirred at room temperature overnight.
With sodium cyanoborohydride; In dimethyl sulfoxide; at 60℃; for 72h;
sodium cyanoborohydride (l60mumol) and 6-azido-6-deoxy-D-galactose (9Smmuol)were added to the solution of MMAF (2.7mumol) in DMSO (0.6ml)The mixture was stirred at 60°C for three days.
4-nitrophenyl-(3‘-nitropyridin-2’-yl)disulfenylethyl carbonate[ No CAS ]
N-(2-((3-nitropyridin-2-yl)disulfanyl)ethanoxycarbonyl)monomethylauristatin F[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
87%
With benzotriazol-1-ol; triethylamine; In tetrahydrofuran; at 20℃; for 72h;Inert atmosphere;
General procedure: To a solution of MMAE (4.8 mg, 0.007mmol) in THF (2 mL) was added 9b (4.8 mg, 2.0 eq), HOBt (1 mg, 1.0 eq) and Et3N (1 muL, 1.0 eq). After stirring for 72 h at rt, the mixture was evaporated and the residue was purified by column chromatography on silica gel (MeOH/DCM 0percent?15percent) to afford 16b (4.2 mg, rho=62percent).
4-((((4-nitrophenoxy)carbonyl)oxy)methyl)phenyl (2-((2-(2-azidoethoxy)ethyl)disulfanyl)ethyl)(methyl)carbamate[ No CAS ]
C55H85N9O13S2[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
60%
With triethylamine; In N,N-dimethyl-formamide; at 20℃;Inert atmosphere;
4.2.8 4-((Carbamoyloxy)methyl)phenyl(2-((2-(2-azidoethoxy)ethyl)disulfanyl)ethyl)(methyl)<strong>[745017-94-1]monomethylauristatin F</strong> (23) Triethylamine (6.5 muL, 0.048 mmol) was added to 15 (8.8 mg, 0.016 mmol), MMAF (12 mg, 0.016 mmol) in anhydrous DMF (800 muL). The solution was stirred at room temperature overnight. After evaporation the crude product was purified over silica gel (MeOH/DCM, 0:100-8:92) to obtain compound 23 (11 mg, 60percent). Rf = 0.23(MeOH/DCM, 10:90). MS (MALDI-TOF+ for C55H85N9O13S2): m/z 1166 [M+Na+], calcd 1133.
2,5-dioxopyrrolidin-1-yl-3 (2-(2-azidoethoxy)ethoxy)propanoate[ No CAS ]
(2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S)-1-azido-17-((R)-sec-butyl)-11,14-diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
22.5 g
In aq. phosphate buffer; N,N-dimethyl-formamide;pH 7.5;
Example 41. (2S)-2-((2R,3R)-3-((2S)- 1 -((11S, 14S, 17S)- 1 -azido- 17-((R)-sec-butyl)-11, 14-diisopropyl- 1 8-methoxy- 10,1 6-dimethyl-9, 12,1 5-trioxo-3 ,6-dioxa- 10,13,1 6-triazai- cosan-20-oyl)pyrrolidin-2-yl)-3 -methoxy-2-methylpropanamido)-3 -phenylpropanoic acid To the crude compound 101 (22 mg, 0.030 mmol) in the mixture of DMA (0.8 ml) and NaH2PO4 buffer (0.7 ml, 1.0 M, pH 7.5) was added compound 88 (18.0 mg, 0.060 mmol) in four portions in 2 h. The mixture was stirred overnight, concentrated and purified on Si02 column eluted with CH3OH/CH2C12/HOAc (1:8:0.01) to afford the titlecompound (22.5 mg, 82percent yield). LC-MS (ESI) mlz calcd. for C46H77N8011 [M+Hj:917.56, found: 917.60.
2,5-dioxopyrrolidin-1-yl-3 (2-(2-azidoethoxy)ethoxy)propanoate[ No CAS ]
(2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S)-1-amino-17-((R)-sec-butyl)-11,14-diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoicacid[ No CAS ]
6-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid[ No CAS ]
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5R)-4-((S)-2-((S)-2-(6-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
58%
Procedure: DIPC (34 mg, 0.271 mmol) and DIPEA (35 mg, 0.271 mmol) were added to a solution of 6-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)hexanoic acid (3) (250 mg, 0.677 mmol) in DCM (3 mL) and the resulting solution was stirred for 1 h at room temperature. (S)-2-((2R,3R)-3-((S)-1 -((3R4S,5S)-4-((S)- N,3-Dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy- 5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3- phenylpropanoic acid hydrochloride (MMAF.HCI) (208 mg, 0.271 mmol) was added in 50 mg portions over a 4 hr period and the resulting solution was stirred for a further 16 h. The DCM was removed under vacuum and the residue was purified by preparative HPLC. Lyophilization of the appropriate fractions gave (S)-2-((2R,3R)-3- ((S)-1 -((3R,4S,5S)-4-((S)-2-((S)-2-(6-(3,4-dibromo-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)-N-methylhexanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3- methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3- phenylpropanoic acid (19) (170 mg, 0.156 mmol, 58percent). 1 H NMR (500 MHz, CDCI3) 7.15-7.26 (m, 5H), 4.60-4.92 (m, 4H), 3.70-4.20 (m, 4H), 3.59-3.63 (m, 2H), 3.39- 3.42 (m, 1 H), 3.26-3.35 (m, 6H), 2.93-3.09 (m, 6H), 2.20-2.60 (m, 6H), 1 .70-2.15 (m, 4H), 1 .61 -1 .69 (m, 8H), 1 .25-1 .37 (m, 3H), 1 .15 (dd, J = 18.5, 7.5 Hz, 2H), 0.81 -1 .05 (m, 20H). LC/MS 4.297 min (5-95percent acetonitrile in water over 5 min), m/z 1083.3 [M+H].
6-(3-bromo-2-bromomethylpropionylamino)hexanoic acid[ No CAS ]
6-(3-bromo-2-bromomethylpropanamido)caproamido-monomethylauristatin F[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
44%
Compound 13 (0.040 g, 0.11 mmol) and HATU (0.033 g, 0.087 mmol) were weighed into a dry round-bottom flask, dissolved in DMF (2.0 mL), and treated with DIEA (0.030 mL, 0.17 mmol) via syringe. The flask was purged with argon, sealed with a rubber septum, and stirred at ambient temperature for 0.5 h. A solution of Monomethyl Auristatin F 27 (0.034 g, 0.046 mmol) in DMF (1.0 mL) was then added, the flask purged again with argon and resealed, and the reaction stirred another 18 h. The reaction was diluted with water (3 mL) and purified on a lOOg CI 8 Aq RediSep Gold column via ISCO CombiFlash system (20 - 80percent MeCN in water, 0.05percent HO Ac both, over 17 mins, 50 mL/min). The product-containing fractions by LCMS were combined, frozen in a dry ice/acetone bath, and lyophilized giving the title compound as a white solid (0.024 g, 44percent). MS (ESI, pos.): calc'd for C49H8oBr2N6010, 1072.4 (most abundant isotope); found 1073.5 (M+H).
2,5-dioxopyrrolidin-1-yl-3 (2-(2-azidoethoxy)ethoxy)propanoate[ No CAS ]
(2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S)-1-azido-17-((R)-sec-butyl)-11,14-diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
22.5 mg
With sodium dihydrogenphosphate; In N,N-dimethyl acetamide; water;pH 7.5;
To the crude compound 331 (22 mg, 0.030 mmol) in a mixture of DMA (0.8 ml) and NaH2PO4 buffer solution (pH 7.5, 1.0 M, 0.7 ml) was added compound 166 (18.0 mg, 0.060 mmol) in four portions in 2 h. The mixture was stirred overnight, concentrated and purified on SiO2 column chromatography (CH3OH/CH2Cl2/HOAc 1:8:0.01) to afford the title compound (22.5 mg, 82percent yield). LC-MS (ESI) m/z calcd.for C46H77N8O11 [M+H]+: 917.56, found: 917.60.
2,5-dioxopyrrolidin-1-yl-3 (2-(2-azidoethoxy)ethoxy)propanoate[ No CAS ]
(2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S)-1-amino-17-((R)-sec-butyl)-11,14-diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid[ No CAS ]
6-(3-(4-cyanophenoxy)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoic acid[ No CAS ]
(S)-2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(6-(3-(4-cyanophenoxy)-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-methylhexanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
63%
With N-ethyl-N,N-diisopropylamine; HATU; In N,N-dimethyl-formamide; at 20℃; for 0.25h;
Procedure: 6-(3-(4-cyanophenoxy)-2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 - yl)hexanoic acid (6) (10.2 mg) and HATU (1 1 .8 mg) were dissolved in 0.25 mL dimethylformamide. DIPEA (12 mg) was added and after stirring solution for 1 min (S)-2-((2R3R)-3-((S)-1 -((3R4S,5S)-4-((S)-N,3-D^ (methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidi yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid hydrochloride (MMAF.HCI) (26.2 mg) was added. After 15 min of stirring at room temperature the product was purified by preparative HPLC. Lyophilization of the appropriate fractions gave ((S)-2-((2R3R)-3-((S)-1 -((3R4S,5S)-4-((S)-2-((S)-2-(6-(3-(4-cyanophenoxy)- 2, 5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)-N-methylhexanamido)-3-methylbutanamido)- N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2- methylpropanamido)-3-phenylpropanoic acid (20 mg, 63percent) (20). 1H NMR (400 MHz, CD3OD) 7.8 (m, 2H), 7.5 (d, 2H) 7.15-7.26 (m, 5H), 5.6 (s, 1 H), 4.50-4.92 (m, 4H), 3.60-4.20 (m, 4H), 3.45-3.55 (m, 2H), 3.39-3.42 (m, 1 H), 3.26-3.35 (m, 6H), 2.85- 3.09 (m, 6H), 2.20-2.50 (m, 6H), 1 .70-2.15 (m, 4H), 1 .50-1 .69 (m, 8H), 1 .25-1 .37 (m, 3H), 1 .15 (dd, 2H), 0.81 -1 .05 (m, 20H). LC/MS 1 .88 min (5-95percent acetonitrile/water + 0.1 percent formic acid over 2 min, hold at 95percent for 0.5 min, then 95-5percent over 0.1 min, and hold at 5percent for 0.4 min. Column used was Waters BEH C18 1 .7 muiotatauiota, 2.1 x 50 mm, flowrate was 0.8 mL/min.), m/z 1042.65 [M+H]+.
6-((tert-butoxycarbonyl)amino)hexanoic acid (46.5 mg,0.201 mmol) was dissolved in DMSO (1 mE) and Huenig?s Base (0.08 mE, 0.458 mmol) and HATU (76 mg, 0.201 mmol) were added. The reaction stirred at RT for 30 minutes before adding the reaction mixture to MMAF (50 mg, 0.067 mmol). The reaction then stirred at RT for 4 hours. The reaction mixture was loaded directly onto 35 g c-i 8 colunm for purification by column chromatography (5-75percent MeCN/Water 0.1percent formic acid). The solvent was removed on rotovap and placed on high vac overnight.The resulting product was treated with TFA (2 mE) at 00 C. and brought to room temp to stir for 5 mm. The reaction was loaded directly onto a 35 g C-18 colunm for column purification. The solvent was removed under reduced pressure toyield 30 mg (52percent yield).
(S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((tert-butoxycarbonyl)-(methyl)amino)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate[ No CAS ]
4,4-difluoro-4-bora-3α,4α-diaza-5,7-dimethyl-s-indacen-3-ylpentanoic acid[ No CAS ]
{N-(5-{2-[(3,5-dimethyl-1H-pyrrol-2-yl-kappaN)methylidene]-2H-pyrrol-5-yl-kappaN}pentanoyl)-N-methyl-L-valyl-N-[(3R,4S,5S)-1-{(2S)-2-[(1R,2R)-3-[(1S)-1-carboxy-2-phenylethyl]amino}-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl}-3-methoxy-5-methyl-1-oxoheptan-4-yl]-N-methyl-L-valinamidato}(difluoro)boron[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
63%
With N-ethyl-N,N-diisopropylamine; HATU; In N,N-dimethyl-formamide; at 20℃;
To a vial containing 4,4-DIFLUORO-5,7-DIMETHYL-4-BORA-3A,4A-DIAZA-S-INDACENE-3- PENTANOIC ACID (93, 3.2 mg , 0.01 mmol) in DCM (0.4 mL) and DMF (0.1 mL) was added DIPEA (1 drop) and HATU (3.9 mg, 0.01 mmol). The mixture was stirred for 0.5 h and transferred to a vial containing Monomethyl Auristatin-F (CAS 74501 7-94-1 ) (7.3 mg, 0.01 mmol). The reaction was stirred at rt overnight, concentrated in vacuo, and the residue purified by reverse phase HPLC (Method B) to give the title compound B81 (5.9 mg , 63percent) as a white solid . LC-MS (Protocol D): m/z 1034.6; Retention time = 7.80 and 8.01 min
4-(3-(((5-(azidomethyl)pyrimidin-2-yl)thio)methyl)-1,1,3,3-tetramethyldisiloxanyl)butanoic acid[ No CAS ]
((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(4-(3-(((5-(azidomethyl)pyrimidin-2-yl)thio)methyl)-1,1,3,3-tetramethyldisiloxaneyl)-N-methylbutanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
Step 3. Synthesis of ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-2-((S)-2-(4-(3-(((5- (azidomethyl)pyrimidin-2-yl)thio)methyl)-1,1,3,3-tetramethyldisiloxaneyl)-N- methylbutanamido)-3-methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5- methylheptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-L-phenylalanine (0216) [00125] A solution of 4-(3-(((5-(azidomethyl)pyrimidin-2-yl)thio)methyl)-1,1,3,3- tetramethyldisiloxanyl)butanoic acid (16.38 mg, 0.041 mmol) and HATU (15.58 mg, 0.041 mmol) in DMF (1997 mul) was stirred for 15 min then charged with a solution of (S)-2- ((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2- (methylamino)butanamido)butanamido)-3-methoxy-5-methylheptanoyl)pyrrolidin-2-yl)-3- methoxy-2-methylpropanamido)-3-phenylpropanoic acid (20 mg, 0.027 mmol) and TEA (6.47 muL, 0.046 mmol) in DMF (1331 muL). After stirring at rt for 7 hrs, the reaction was diluted with DCM (20 mL) and washed with water (2x10 mL). The filtrate was washed with brine, dried over MgSO4, filtered, and concentrated. This material was carried on to the hydrolysis step. MS (ES+): m/z = 1,113.74 [M+H]+; LCMS: tR = 2.77 min.
Compound 68 (45.0 mg, 0.14 mmol), HOSt (19.0 mg, 0.14 mmol), DIC (20.5 IL, 0.14 mmol) and DIPEA (23.2 IL, 0.14 mmol,) were added to DMF (10 mE) and stirred at 0° C. in an ice bath for 1 hout Compound 63 (76.9 mg, 0.11 mmol) was added to the solution. The mixture was allowed to warm to room temperature and lefi to react for 36 hours. The reaction mixture was purified by prep-HPEC to yield compound 69 (64.3 mg, 44.3percent) as a white solid. EC-MS mlz (ESj, 1039.49 (M+H).
The compound 62 (57.0 mg, 0.2 mmol), HOSt (27.2 mg, 0.2 mmol), DIC (29.3 IL, 0.2 mmol) and DIPEA (0.2 mmol, 33.1 pL) were added to DMF (6 mE). Afier 30 mm, the compound 63 (109.8 mg, 0.15 mmol) and was added at 0° C. The mixture was allowed to stir at room temperature for 24 hours. The solution was concentrated and purified by prep-HPEC to give compound 64 (94.8 mg, 47.6percent) as a white solid. EC-MS mlz (ES), 1000.53 (M+H).
2,5-dioxopyrrolidin-1-yl-3 (2-(2-azidoethoxy)ethoxy)propanoate[ No CAS ]
(2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S)-1-azido-17-((R)-sec-butyl)-11,14-diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid[ No CAS ]
(S)-methyl 2-((2R,3R)-3-((S)-1-((3R,4S,5S)-4-((S)-N,3-dimethyl-2-((S)-3-methyl-2-(methylamino)butanamido)butanamido)-3-methoxy-5-methyl-heptanoyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoate[ No CAS ]
(2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S)-1-amino-17-((R)-sec-butyl)-11,14-diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-3,6-dioxa-10,13,16-triazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid[ No CAS ]
(2S)-2-((2R,3R)-3-((2S)-1-((11S,14S,17S,18R)-17-((S)-sec-butyl)-11,14-diisopropyl-18-methoxy-10,16-dimethyl-9,12,15-trioxo-1-((bis(2-propioloylhydrazinyl)phosphoryl)amino)-3,6-dioxa-10,13,16-triazaicosan-20-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid[ No CAS ]
(S)-2,5-dioxopyrrolidin-1-yl 2-((S)-2-(2,2-dipropiolamido-acetamido)propanamido)propanoate[ No CAS ]
(S)-2-((2R,3R)-3-((S)-1-((8S,11S,14S,17S,20S,21R)-20-((S)-sec-butyl)-14,17-diisopropyl-21-methoxy-8,11,13,19-tetramethyl-3,6,9,12,15,18-hexaoxo-5-propiolamido-4,7,10,13,16,19-hexaazatricos-1-yn-23-oyl)pyrrolidin-2-yl)-3-methoxy-2-methylpropanamido)-3-phenylpropanoic acid[ No CAS ]