Purity | Size | Price | VIP Price | USA Stock *0-1 Day | Global Stock *5-7 Days | Quantity | |||||
{[ item.p_purity ]} | {[ item.pr_size ]} |
{[ getRatePrice(item.pr_usd, 1,1) ]} {[ getRatePrice(item.pr_usd,item.pr_rate,item.mem_rate) ]} |
{[ getRatePrice(item.pr_usd, 1,1) ]} | Inquiry {[ getRatePrice(item.pr_usd,item.pr_rate,item.mem_rate) ]} {[ getRatePrice(item.pr_usd,1,item.mem_rate) ]} | {[ item.pr_usastock ]} | Inquiry - | {[ item.pr_chinastock ]} | Inquiry - |
* Storage: {[proInfo.prStorage]}
CAS No. : | 55750-63-5 | MDL No. : | MFCD00043043 |
Formula : | C14H16N2O6 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | VLARLSIGSPVYHX-UHFFFAOYSA-N |
M.W : | 308.29 | Pubchem ID : | 5091655 |
Synonyms : |
EMCS
|
Num. heavy atoms : | 22 |
Num. arom. heavy atoms : | 0 |
Fraction Csp3 : | 0.5 |
Num. rotatable bonds : | 8 |
Num. H-bond acceptors : | 6.0 |
Num. H-bond donors : | 0.0 |
Molar Refractivity : | 80.42 |
TPSA : | 101.06 Ų |
GI absorption : | High |
BBB permeant : | No |
P-gp substrate : | No |
CYP1A2 inhibitor : | No |
CYP2C19 inhibitor : | No |
CYP2C9 inhibitor : | No |
CYP2D6 inhibitor : | No |
CYP3A4 inhibitor : | No |
Log Kp (skin permeation) : | -8.54 cm/s |
Log Po/w (iLOGP) : | 2.07 |
Log Po/w (XLOGP3) : | -0.51 |
Log Po/w (WLOGP) : | -0.68 |
Log Po/w (MLOGP) : | 0.31 |
Log Po/w (SILICOS-IT) : | 0.67 |
Consensus Log Po/w : | 0.37 |
Lipinski : | 0.0 |
Ghose : | None |
Veber : | 0.0 |
Egan : | 0.0 |
Muegge : | 0.0 |
Bioavailability Score : | 0.55 |
Log S (ESOL) : | -0.9 |
Solubility : | 38.6 mg/ml ; 0.125 mol/l |
Class : | Very soluble |
Log S (Ali) : | -1.14 |
Solubility : | 22.1 mg/ml ; 0.0717 mol/l |
Class : | Very soluble |
Log S (SILICOS-IT) : | -1.56 |
Solubility : | 8.55 mg/ml ; 0.0277 mol/l |
Class : | Soluble |
PAINS : | 0.0 alert |
Brenk : | 1.0 alert |
Leadlikeness : | 1.0 |
Synthetic accessibility : | 3.01 |
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 |
---|---|---|
95% | With dicyclohexyl-carbodiimide In acetonitrile at 0 - 20℃; Inert atmosphere | Under nitrogen protection, 4.7 g (22 mmol) of MC and 25 g (22 mmol) of HOSu were added to 50 ml of acetonitrile.Another 4.5 g (22 mmol) of DCC was dissolved in 25 ml of acetonitrile,Keeping the internal temperature around 0°C,It was slowly dropped into the reaction solution.The reaction solution was reacted at 0°C for 2 hours and then reacted overnight at room temperature.filter,The filter cake was washed with acetonitrile 10ml×3.The filtrate was concentrated to dryness under reduced pressure.The resulting oil was dried under reduced pressure at room temperature for 6 h to give 6.4 g of light brown solid.Yield 95percent. |
70% | With dicyclohexyl-carbodiimide In dichloromethane at 20℃; for 3 h; | Dicyclohexyl-carbodiimide (2.68 g, 13.02 mmol) and N-hydroxysuccinimide (1 .36 g, 1 1 .84 mmol) are added at room temperature to a stirrer solution of compound [4] (2.5 g, 1 1 .84 mmol) in anhydrous dichloromethane (15 mL) and the mixture stirred at room temperature for 3 hours. The white solid is filtered with dichloromethane to remove the dicyclohexylurea, the organic phase is washed with HCI 0.1 N and water, then dried over anhydrous sodium sulfate and the solvent removed by rotatory evaporation. The resulting residue is subjected to flash column chromatography with a medium pressure system Sepacore® Buchi (silica gel; gradient A: petroleum ether/B: ethyl acetate; Bpercent 0-80 in 15 minutes) to give the activated acid [5] as a white solid, 2.4 g (70percent). MS: m/z 309 [M+H]+.1H NMR (400 MHz, MeOD) δ 6.78 (s, 2H), 3.49 - 3.46 (m, 2H), 2.82 (s, 4H), 2.62 - 2.59 (m, 2H), 1 .78 - 1 .65 (m, 2H), 1 .64 - 1 .50 (m, 2H), 1 .46 - 1 .26 (m, 2H).13C NMR (100 MHz, MeOD) δ 169.8, 169.0, 167.3, 132.5, 35.4, 28.6, 26.1 , 23.8, 23.6, 22.3. |
48% | With dicyclohexyl-carbodiimide In ethyl acetate at 0 - 20℃; for 20 h; | To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (3.08 g, 14.6 mmol, 1.0 eq) and N-hydroxysuccinimide (1.76 g, 15.3 mmol, 1.05 eq) in EtOAc (30 mL) at 0 °C, was added dicyclohexylcarbodiimide (3.16 g, 15.3 mmol, 1.05 eq). The reaction was then allowed to warm to rt. After 20 h, the reaction was filtered and washed with EtOAc and the filtrate concentrated. The residue was purified by flash chromatography to yield the title compound (2.16 g, 48percent) as a clear oil that solidified slowly to a waxy white solid. NMR (400 MHz, Chloroform-i/) δ 6.71 (s, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.86 (s, 4H), 2.63 (t, J = 7.4 Hz, 2H), 1.80 (p, J= 7.4 Hz, 2H), 1.73 - 1.57 (m, 2H), 1.50 - 1.35 (m, 2H). m/z calcd. for C14H16N206 = 308.10. Found [M+H]+ = 309.13. Rf = 0.28 (50percent EtOAc/Hex). |
48% | With dicyclohexyl-carbodiimide In ethyl acetate at 0 - 20℃; for 20 h; | To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-l-yl)hexanoate (3.08 g, 14.6 mmol, 1.0 eq) and N-hydroxysuccinimide (1.76 g, 15.3 mmol, 1.05 eq) in EtOAc (30 mL) at 0 °C,was added dicyclohexylcarbodiimide (3.16 g, 15.3 mmol, 1.05 eq). The reaction was then allowed towarm to rt. After 20 h, the reaction was filtered and washed with EtOAc and the filtrate concentrated. The residue was purified by flash chromatography to yield the title compound (2.16 g, 48percent) as a clear oil that solidified slowly to a waxy white solid. ‘H NMR (400 MHz, Chloroform-d) ö 6.71 (s, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.86 (s, 4H), 2.63 (t, J = 7.4 Hz, 2H), 1.80 (p, J = 7.4 Hz, 2H), 1.73 — 1.57 (m,2H), 1.50 1.35 (m, 2H). m/z calcd. for C,4H,6N206 = 308.10. Found [M+H] = 309.13. Rf= 0.28 (50percent EtOAc/Hex) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
70% | With sodium hydrogencarbonate In N,N-dimethyl-formamide at 0℃; for 1 h; Inert atmosphere | 6-Maleimidohexanoic acid N-hydroxysuccinimide ester 4. Sodium bicarbonate (0.0512 g, 0.483 mmol ) was added to a solution of 6-maleimidocaproic acid (0.211 g, 1.000 mmol) in DI water (10 mL). After the reagents were dissolved in DI water, the DI water was evaporated by blowing with air. The resulting solid was dissolved in anhydrous DMF (3 mL) and the solution was cooled to 0 °C. Disuccinimide carbonate (0.282 g, 1.10 mmol) was added into the solution and the reaction was stirred at 0 °C for 1 h. CH2C12 (25 mL) was added into reaction which was then washed with water (3 x 10 mL). The resulting organic layer was dried over Na2S04, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 25 g silica column [solvent A: MeOH; solvent B: CH2C12; gradient: 0percentA / 100percentB (1 CV), 0percentA / 100percentB→ 2percentA / 98percentB (10 CV), 2percentA / 98percentB (2 CV); flow rate: 25 mL/min; monitored at 210 and 280 nm] to afford ester 4 (0.215 g, 0.697 mmol, 70percent yield) as a light yellow liquid. [000194] NMR (500 MHz, CDC13) 66.69 (2H, s), 3.53 (2H, t, J= 7.1 Hz), 2.83 (4H, s), 2.60 (2H, t, .7=7.4 Hz), 1.78 (2H,p, .7=7.5 Hz), 1.63 (2H,p, .7=7.4 Hz), 1.41 (2H,p,J=7.6 Hz). [000195] 13CNMR(125 MHz, CDC13) δ 170.9, 169.2, 168.5, 134.2,37.5,30.8,28.1,25.9,25.7,24.1. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
70% | With sodium hydrogencarbonate; In N,N-dimethyl-formamide; at 0℃; for 1h;Inert atmosphere; | 6-Maleimidohexanoic acid N-hydroxysuccinimide ester 4. Sodium bicarbonate (0.0512 g, 0.483 mmol ) was added to a solution of <strong>[55750-53-3]6-maleimidocaproic acid</strong> (0.211 g, 1.000 mmol) in DI water (10 mL). After the reagents were dissolved in DI water, the DI water was evaporated by blowing with air. The resulting solid was dissolved in anhydrous DMF (3 mL) and the solution was cooled to 0 °C. Disuccinimide carbonate (0.282 g, 1.10 mmol) was added into the solution and the reaction was stirred at 0 °C for 1 h. CH2C12 (25 mL) was added into reaction which was then washed with water (3 x 10 mL). The resulting organic layer was dried over Na2S04, concentrated under reduced pressure, and purified by flash chromatography using a pre-packed 25 g silica column [solvent A: MeOH; solvent B: CH2C12; gradient: 0percentA / 100percentB (1 CV), 0percentA / 100percentB? 2percentA / 98percentB (10 CV), 2percentA / 98percentB (2 CV); flow rate: 25 mL/min; monitored at 210 and 280 nm] to afford ester 4 (0.215 g, 0.697 mmol, 70percent yield) as a light yellow liquid. [000194] NMR (500 MHz, CDC13) 66.69 (2H, s), 3.53 (2H, t, J= 7.1 Hz), 2.83 (4H, s), 2.60 (2H, t, .7=7.4 Hz), 1.78 (2H,p, .7=7.5 Hz), 1.63 (2H,p, .7=7.4 Hz), 1.41 (2H,p,J=7.6 Hz). [000195] 13CNMR(125 MHz, CDC13) delta 170.9, 169.2, 168.5, 134.2,37.5,30.8,28.1,25.9,25.7,24.1. |
With triethylamine; In N,N-dimethyl-formamide; at 20℃; for 6h; | A solution of compound 8 (0.860 g, 1.43 mmol,1.0 equiv) in DMF (0.2 M) was treated with triethylamine (4.00 mL, 28.6 mmol, 20.0 equiv) and stirred at room temperature for 24 hours. DMF and excess triethylamine were removed under reduced pressure to obtain the crude deprotected intermediate. Separately, to a solution of 6-maleimidohexanoic acid (0.452 g, 2.14 mmol, 1.5 equiv) and N,N'-disuccinimidyl carbonate (0.439 g, 1.72 mmol, 1.2 equiv) in DMF (0.2 M), triethylamine (0.40 mL, 3.0 mmol, 2.0 equiv) was added followed by stirring at room temperature for 6 hours. To this reaction mixture, the previously generated amine dissolved in DMF (0.2 M) was added and stirred at room temperature for 20 hours. DMF was removed under reduced pressure and the resulting residue was purified by flash column chromatography using 3-12percent MeOH-CH2Cl2 as solvent. The product was obtained as white solid. Yield: 91percent. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
43% | With sodium hydrogencarbonate; In tetrahydrofuran; at 0 - 5℃; for 1h; | A solution of amine 8 (138.6 mg, 0.17 mmol) in 1 M aqueous solution [OF NAHC03] (1.5 mL) was added dropwise to a stirred solution of <strong>[55750-63-5]6-maleimidohexanoic acid N-hydroxylsuccinimide ester</strong> (272.5 mg, 0.88 mmol) in THF (3 mL), which was cooled with a bath of ice-water. The resulting mixture was stirred for 1 h at [0-5°C.] The mixture was then diluted with CHC13 (70 mL) and washed with brine [(3X10] mL). The organic phase was dried over Na2SO4, filtered, and concentrated. The oily residue was purified by flash chromatography [(CH2CL2/MEOH] 95: 5) to give maleimide 10 (103.6 mg, 43percent) ; [RU 0.] 62 [(CH2CL2/MEOH] 9: 1) [;APOS;H-NMR (CDC13/TMS)] : [A] 6.68 (s, 8 H, CH=CH), 6.26-6. 20 (m, 2 H, NH), 6.12 (t, [1] H, [J=6.] 8 Hz, NH), 6.04 (t, [1] H, [J=6.] 8 Hz, [NH),] 4.92 (d, [1] H, [J=3.] 2 Hz, [H-1),] 3.95-3. 86 (m, 2 H, H-4, and OCHHCH2CH2N), 3. [80-3.] 40 (m, 30 H, H-2, H-3, H-5, H-6, H-6', [OCH2CH2CH2S,] [SCH2CH2N,] NCH2(CH2) 4 and [OCH2CH3),] 2.75-2. 60 (m, 16 H, [OCH2CH2CH2SCH2CH2N),] 2.18 (t, 8 H, J=7.3 Hz, N (CH2) [4CH2] ), 1.96-1. 82 (m, 8 H, OCH2CH2CH2S), 1.74-1. 54 (m, 16 H, [NCH2CH2CH2CH2CH2),] 1.37-1. 24 (m, 8 H, N [(CH2)] 2CH2 (CH2) 2), 1.22 (t, 3 H, J=7.1 Hz, OCH2CH3) ; ES-MS: 1450.01 (M+H) [+,] 725.56 (M+2H) 2+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With N-ethyl-N,N-diisopropylamine In dichloromethane |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
60% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 0.666667h; | The mixture of Doxorubicin (hydrochloride salt) (38.4 mg, 0.065 mmol), (2)(30 mg, 0.08 mmol), diisopropylethylamine (28 muL, 0.16 mmol) and DMF (1 mL) was prepared and stirred at room temperature (Figure 4B), while progress of the reaction was monitored by TLC (chloroform : methanol : ammonia = 85 : 15 : 2). After 40 min, the reaction was completed. The reaction mixture was diluted with dichloromethane (2 mL) and precipitated with hexanes (50 mL). The obtained solid was separated from solvents by centrifugation. The product was separated by column chromatography (SilicaGel 60, Merck) using chloroform, chloroform : methanol 98 : 2, 95 : 5 as eluents. Fractions containing WP936 were pooled together, evaporated to dryness, dissolved in chloroform (1 mL) and precipitated with hexanes (25 mL). The obtained solid was dried under vacuum to give 24 mg of WP936 (yield 60percent). The 1H NMR spectrum was obtained for WP936 and was in agreement with the proposed structure.[0108] 1H NMR (CDCl3, delta) ppm: 14.0, 13.25 (2s, IH ea, 6,11-OH), 8.04 (dd, IH, J =7.6 Hz, J = 0.7 Hz, H-I), 7.72 (dd, IH, J = J = 8.4 Hz, H-2), 7.4 (d, IH, J = 8.1 Hz, H-3), 6.67 (s, 2H, CH maleimid), 5.82 (d, IH, J = 8.6 Hz, NH), 5.50 (d, IH, J = 3.5 Hz, H-I '), 5.38 (bs, IH, H-7), 4.76 (s, 2H, 14-CH2), 4.56 (s, IH, 9-OH), 4.16 (q, IH, J = 6.1 Hz, H-5'), 4.08 (s, 3H, OMe), 3.63 (bs, IH, H-4), 3.49 (t, 2H, J = 7.1 Hz, CH2-linker), 3.28 (dd, IH, J = 18.8 <n="37"/>Hz, J = 1.8 Hz, H-IO), 3.24 (d, IH, J = 18.8 Hz, H-IO), 3.02 (m, IH, H-3'), 2.34 (d, IH, J = 14.7 Hz, H-8), 2.17 (dd, IH, J = 14.7 Hz, J = 4.1 Hz, H-8), 2.12 (dd, 2H, J = 7.2 Hz, J = 2.4 Hz, CH2 from linker), 1.83 (dd, IH, J = 12.7 Hz, J = 5.5 Hz, H-2'e), 1.78 (ddd, IH, J = J = 12.7 Hz, J = 4.1 Hz, H-2'a), 1.63 - 1.55 (m, 4H, CH2 from linker), 1.29 (d, 3H, J = 6.1 Hz, H-6'), 1,30 - 1.25 (m, 2H, CH2 from linker).[0109] Anal. Elem. For C37H40N2Oi4 x 2H2O Calc.C, 57.51; H, 5.74; N, 3.63, Found:C, 57.90; H, 5.42; N, 3.60 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
47% | With sodium hydrogencarbonate; In tetrahydrofuran; water; at 0℃; for 1h; | A solution of compound 16 (55.4 mg, 73.8 [PMOL)] in 1 M aqueous solution [OF NAHC03] (2 [ML)] was added dropwise to a stirred solution of N-succimidyl 6-maleimidohexanoic acid ester (135.6 mg, 0.44 mmol) in THF (3 [ML)] cooled with a bath of ice-water. The resulting mixture was stirred for 1 h at 0 [°C,] and then diluted with ethyl acetate (60 [ML)] and washed with brine [(3X10] mL). The organic phase was dried over [NA2SO4,] filtered and concentrated. The oily residue was purified through flash chromatography to give compound 17 (47.0 mg, 47percent) [; LH-NMR (CDCL3/TMS)] : [No.] 6.89 (s, 8 H, CH=CH), 6.12 (t, 4 H, [J=5.] 4 Hz, [NH),] 3.51 (t, 8 H, J=7.2 Hz, [CCH20CH2CH2CH2S),] 3.48-3. 40 (m, 16 H, [SCH2CH2NH,] [NCH2] (CH2) 4), 3.34 (s, 8 H, [CCH2OCH2CH2CH2S),] 2.65 (t, 8 H, J=6.4 Hz, [SCH2CH2NH),] 2.58 (t, 8 H, J=7.4 Hz, [CCH2OCH2CH2CH2S),] 2.18 (t, 8 H, [J=7.] 4 Hz, [NHCOCH2CH2CH2CH2CH2N),] 1.81 (p, 8 H, J=6.8 Hz, [OCH2CH2CH2S),] 1.72-1. 54 (m, 16 H, [NCH2CH2CH2CH2CH2),] 1.38-1. 24 (m, 8 H, N (CH2) [2CH2] [(CH2)] 2); ESI-MS: 1399.98 (M+Na) [+,] 1377.97 (M+H) [+,] 689.75 (M+2H) 2+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
51% | With sodium hydrogencarbonate; In methanol; water; at 0 - 5℃; for 1h; | To the stirred solution of compound 20 (44.6 mg, 10.0 [(IMOL)] and N-succimidyl 6-maleimidohexanoic acid ester (129.5 mg, 0.42 mmol) in methanol (2 mL) was added 1 M aqueous solution of [NAHC03] (1.0 mL) at 0- 5°C. The resulting mixture was stirred for 1 h at [0-5 °C,] and then diluted with Chloroform (60 mL) and washed with water [(3X10] mL). The organic phase was dried over Na2SO4, filtered and concentrated. The oily residue was purified through flash chromatography to give compound 21 (38.6 mg, 51percent) [;APOS;H-NMR (DMSO-D6)] : [No.7. ]96-7.86 (m, 21 H, [SCH2CH2NH),] 6.97 (s, 42 H, CH=CH), [5.] 11-5.00 (m, 7 H, [H-1),] 4.00-3. 05 (m, 168 H, H-2, H-3, H-4, H-5, H-6, [H-6APOS;,] [OCH2CH2CH2SCH2CH2NH,] NH (O) [CCH2CH2CH2CH2CH2N),] 2.65-2. 38 (m, 84 H, [OCH2CH2CH2SCH2CH2NH),] 2.08-1. 97 (m, 42 H, NH (O) [CCH2CH2CH2CH2CH2N),] 1.88-1. 65 (m, 42 H, [OCH2CH2CH2S),] 1.60-1. 35 (m, 84 H, [NCH2CH2CH2CH2CH2),] 1.30-1. 10 (m, 42 H, N (CH2) [ZCHZ] [(CH2)] [2)-] |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
100% | In 1-methyl-pyrrolidin-2-one; for 24h;Heating; | vc-PABOH 205 mg (0.54 mmol) and MC-OSu 184 mg (1.1 eq) were added to 10 ml of NMP.The reaction was stirred for 24 h under warming. After completion of the reaction, the mixture was concentrated under reduced pressure at 40 C., and 20 ml of methyl tert-butyl ether was added to the resulting oil.Crystallization. It was filtered and the cake was washed with methyl tert-butyl ether to give 310 mg of product in 100% yield. |
100% | In 1-methyl-pyrrolidin-2-one; at 20℃; for 24h; | VP-PABOH 205 mg (0.54 mmol) and MC-OSu 184 mg (1.1 eq) were added to 10 ml of NMP, and the reaction was stirred at room temperature for 24 h.The reaction is completed,Should be completed, concentrated at 40 C under reduced pressure,The obtained oil was added to 20 ml of methyl t-butyl ether and stirred to crystallize. filter,The filter cake was washed with methyl tert-butyl ether.Get 310mg of product,The yield is 100%. |
97% | In N,N-dimethyl-formamide; at 20 - 30℃; | To mc-OSu (1.7 eq.) was added anhydrous N,N-Dimethylformamide (3 vol.) and the resulting mixture was stirred at 20C until a clear colorless solution formed. A solution of Val-Cit-PABOH (1 eq.) in anhydrous N,N-Dimethylformamide (7 vol.) was then added over 30 minutes while keeping temperature below 30C. The reaction mixture was stirred at 30C for 5-6 hours until reaction was complete. Ethyl acetate (30 vol.) was then added over 30 minutes at 30C. The resulting suspension was stirred at that temperature for 10-20 minutes before being cooled to 20C and stirred at 20C for 2-4 hours. Filtered solids were solubilized in N.N-Dimethylformamide (10 vol.) and the resulting mixture was stirred at 30C for 30-60 minutes. Ethyl acetate (30 vol.) was added over 30 minutes at 30C. The resulting suspension was stirred at that temperature for 10-20 minutes before being cooled to 20C and stirred at 20C for 2-4 hours. The resulting solids were collected by filtration, washed and dried under vacuum (97% yield). MS: m/e 573 (MH)+, 595 (M+Na)+. |
96% | In N,N-dimethyl-formamide; for 2h; | [0438] Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol, 1.0 eq.; see, e.g., U.S. Patent No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) and to this solution was added a diethylamine (60 mL). The reaction was monitored by HPLC and found to be complete in 2 h. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (ca. 100 mL) under sonication over for 10 min. Ether (200 mL) was added and the precipitate was further sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring and was then filtered and dried under high vacuum to provide Val-cit-P AB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC-OSu (Willner et at, 1993, Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). The reaction was complete according to HPLC after 2 h. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonicating for 15 min, ether (400 mL) was added and the mixture was sonicated further until all large particles were broken up. The solution was then filtered and the solid dried to provide an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M-H] |
96% | In N,N-dimethyl-formamide; for 2h; | [0454] Fmoc-val-cit-P AB-OH (14.61 g, 24.3 mmol, 1.0 eq., see, e.g., U.S. Patent No. 6,214,345 to Firestone et al.) was diluted with DMF (120 mL, 0.2 M) and to this solution was added a diethylamine (60 mL). The reaction was monitored by HPLC and found to be complete in 2 h. The reaction mixture was concentrated and the resulting residue was precipitated using ethyl acetate (ca. 100 mL) under sonication for 10 min. Ether (200 mL) was added and the precipitate was further sonicated for 5 min. The solution was allowed to stand for 30 min. without stirring and was then filtered and dried under high vacuum to provide Val-cit-P AB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%). Val-cit-P AB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC-OSu (Willner et al, 1993, EPO <DP n="138"/>Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). The reaction was complete according to HPLC after 2 h. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). After sonicating for 15 min, ether (400 niL) was added and the mixture was sonicated further until all large particles were broken up. The solution was then filtered and the solid dried to provide an off-white solid intermediate. Yield: 11.63 g (96%); ES-MS m/z 757.9 [M-H] |
96% | In N,N-dimethyl-formamide; for 2h; | Fmoc-val-cit-PAB-OH (14.61 g, 24.3 mmol), 1.0 eq., U.S. Patent No. 6214345 to Firestone et al.) was dilutedwith DMF (120 mL, 0.2 M) and to this solution was added a diethylamine (60 mL). The reaction was monitored by HPLCand found to be complete in 2 h. The reaction mixture was concentrated and the resulting residue was precipitated usingethyl acetate (ca. 100 mL) under sonication over for 10 min. Ether (200 mL) was added and the precipitate was furthersonicated for 5 min. The solution was allowed to stand for 30 min. without stirring and was then filtered and dried under high vacuum to provide Val-cit-PAB-OH, which was used in the next step without further purification. Yield: 8.84 g (96%).Val-cit-PAB-OH (8.0 g, 21 mmol) was diluted with DMF (110 mL) and the resulting solution was treated with MC-OSu(Willner et al., (1993) Bioconjugate Chem. 4:521; 6.5 g, 21 mmol, 1.0 eq.). Reaction was complete according to HPLCafter 2 h. The reaction mixture was concentrated and the resulting oil was precipitated using ethyl acetate (50 mL). Aftersonicating for 15 min, ether (400 mL) was added and the mixture was sonicated further until all large particles werebroken up. The solution was then filtered and the solid dried to provide an off-white solid intermediate. Yield: 11.63 g(96%); ES-MS m/z 757.9 [M-H] |
92% | In 1-methyl-pyrrolidin-2-one; at 20℃; for 16h;Inert atmosphere; | Mc-Val-Cit-PABOH 5. Compound 3 (0.19 g, 0.49 mmol) was added to a solution of ester 4 (0.18 g, 0.57 mmol) in NMP (7 mL), and the reaction was stirred for 16 h. The solvent was evaporated at reduced pressure, and then the residue was triturated with Et20 (30 mL). The crude product was collected by filtration and washed with Et20 (3 x 15 mL). The crude product was purified by flash chromatography using a pre-packed 25 g silica column [solvent A: MeOH; solvent B: CH2C12; gradient: 0%A / 100%B (1 CV), 0%A / 100%B? 30%A / 70%B (10 CV), 30%A / 70%B (2 CV); flow rate: 25 mL/min; monitored at 254 and 280 nm] to afford linker 5 (0.26 g, 0.45 mmol, 92% yield) as a brown solid. [000197] NMR (500 MHz, DMSO-d6) delta 9.82 (1H, s), 7.98 (1H, d,J= 8.5 Hz), 7.73 (1H, d,J= 8.5 Hz), 7.46 (2H, d,J= 7.8 Hz), 7.14 (2H, d, 7= 8.0 Hz), 6.92 (2H, s), 5.89 (1H, t,J= 6.0 Hz), 5.33 (2H, s), 5.01 (1H, t, J= 5.7 Hz), 4.34 (2H, d, J= 4.5 Hz), 4.29 (1H, q,J= 7.4 Hz), 4.14 - 4.05 (1H, m), 3.29 (2H, t, .7=6.3 Hz), 2.99-2.82 (2H, m), 2.17- 1.96 (1H, m), 1.94- 1.80 (2H, m), 1.67-1.03 (8H, m), 0.76 (3H, d, .7=6.4 Hz), 0.73 (3H, d, .7=6.1 Hz). [000198] 13C NMR (125 MHz, DMSO-d6) delta 173.2, 172.7, 171.5, 170.8, 159.3, 138.0, 137.8, 134.9, 127.3, 119.2, 63.0, 58.0, 53.5, 37.4, 35.4, 30.8, 29.8, 28.2, 26.2, 25.7, 25.4, 19.7, 18.6. |
0.69 g | In N,N-dimethyl-formamide; at 20℃; | The product of reaction step d (0.5 g, 1.3 mmol) was dissolved in 18 mL of DMF and added to MC-OSu (0.45 g,1.4 mmol), the reaction was stirred at room temperature overnight. After the reaction is completed, the reaction solvent is spin-dried, washed with diethyl ether three times, and filtered to obtain a yellow solid.The body was 0.69 g without further purification. |
With triethylamine; In N,N-dimethyl-formamide; at 20℃; for 20h; | : Procedure A: A solution of compound 8 (0.270 g, 0.450 mmol, 1.0 equiv) in DMF (0.2 M) was treated with triethylamine (1.25 mL, 8.90 mmol, 19.8 equiv) followed by stirring at room temperature for 24 hours. DMF and excess triethylamine were removed under reduced pressure. The resulting residue was dissolved in DMF (0.1 M) and to the solution Mc-OSu (0.150 g, 0.500 mmol, 1.1 equiv) was added. The reaction mixture was stirred at room temperature for 20 hours. DMF was removed under reduced pressure and the resulting residue was purified by flash column chromatography using 3-12% MeOH-CH2Cl2 as solvent. The product was obtained as white solid. Yield: 86%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
75% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 25℃; for 1h; | Synthesis of compound 6: A solution of 4 (16 mg, 0.015 mmol) and 5 (6.2 mg, 0.02 mmol) in DMF (2mL) was treated with DIPEA (10 uL). The reaction mixture was stirred for 1 hr at 250C. The final product was purified by Prep HPLC (SymmetrPrep C18, 7mum, 19 x 150 mm column), eluted at 10 ml/min (0.01percent TFA in water/acetonitrile) with a gradient: 10percent acetonitrile in 5 min, 10percent to 50percent acetonitrile in 15 min, maintaining 50percent acetonitrile in 5 min, 50percent to 100percent acetonitrile in 5 min, to obtain 6 (13 mg, 75percent). MS: calcd for C5IH55BrN10O9 (M+H) m/z 1031.33 found 1031.6. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
79% | With N-ethyl-N,N-diisopropylamine; In dichloromethane; N,N-dimethyl-formamide; at 20℃; for 2h; | Synthesis of compound 14: To a solution of 11 (18 mg, 0.021 mmol) in 20percent DMF in dichloromethane (1 mL) were added diisopropylethylamine (11 muL, 0.63 mmol) and N- succinimidyl-6-maleimidohexanoate (10 mg, 0.031 mmol) at room temperature. The mixture thus obtained was stirred for 2 hours. The solvent was evaporated and the residue was purified by semi-preparative HPLC to give compound 14 as a white solid (19 mg, 79percent). 1H NMR (CD3OD) delta 1.32 (m, 2H ), 1.42 (s, 9H), 1.45-1.80 (m, 8H), 1.78 (m, IH), 1.85 (m, IH), 2.28 (t, 2H), 2.96 (s, 3H), 3.05 (t, 3H), 3.35-3.70 (bs, 8H), 3.47 (t, 2H), 3.60 (m, IH), 3.90 (m, IH), 4.05 (m, IH), 4.44 (m, 2H), 4.55 (m, IH), 6.76 (s, 2H), 6.96 (s, IH), 7.31 (bs, 2H), 7.42 (m, IH), 7.46 (m, IH), 7.71 (m, 3H), 7.86 (m, 3H), 7.99 (s, IH), 8.25 (bs, IH); LC-MS (ES+) 1059 (M+H) +, 1082 (M+Na)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
97% | With triethylamine; In N,N-dimethyl-formamide; at 20℃; | 150 mg (0.797 mmol) of <strong>[1115-74-8]L-valyl-L-alanine</strong> and 246 mg (0.797 mmol) of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione were dissolved in 4.0 ml of dimethylformamide, and 0.220 ml (1.6 mmol) of triethylamine was added. The reaction mixture was stirred at RT overnight. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250*30; 10mu, flow rate; 50 ml/min, MeCN/water). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 302 mg (97% of theory) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl) hexanoyl]-<strong>[1115-74-8]L-valyl-L-alanine</strong>. LC-MS (Method 12): Rt=1.02 min; MS (ESIpos): m/z=382 (M+H)+. 1H-NMR (400 MHz, DMSO-d6): delta [ppm]=0.82 (dd, 6H), 1.17 (m, 2H), 1.27 (d, 3H), 1.48 (m, 4H), 1.94 (m, 1H), 2.13 (m, 2H), 3.38 (t, 2H), 4.17 (m, 2H), 7.00 (s, 2H), 7.75 (d, 1H), 8.19 (d, 1H). |
70% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃;Inert atmosphere; | Maeilimidocaproy. N-hydroxysuccinimide (1.619 g, 5.25 mmo., 105 eq.) and H-Val-Aia-OH (0,941 g, 5 mmol, 1 eq.) were placed in a 25 ml recovery fiask with a stir bar and the flask was flushed with nitrogen, DMF (4,7 mL) was added and the resulting white siurry was stirred. DIPEA (0.87 mL, 5 mmol, 1 eq) was added and the mixture was allowed to stir at room temperature overnight. The mixture was coo.ed in an ice/water bath and 2M HCI (3 mL, 6 mmol) was added dropwise. The viscous mixture was transferred to a separator/ funnel and the reaction vessel rinsed with sat. NaCl (7 mL), EtOAc (10 mL), sat NaCl {10 mL) and EtOAc (5 mL). After separation of the aqueous phase, it was extracted with additionai EtOAc {2 x 15 mL). The combined organic extracts were washed with sat NaCi {4 x 15 mL), until the washings were pH ~3,5. The organic extracts were dried over Na2S04, filtered and concentrated under reduced pressure to give crude 5 as a white solid (2,1 2 g, 114% crude yield). Crude 5 was suspended in warm CHjCIa {35 mL) and filtered to remov a fine white solid. The solids wer rinsed with additional CHjCi2 (3 mL). Toiuene {5mL) was added and the mixture was cooled in an ice/water bath, which resulted in a thick siurry. The solids were collected by filtration, washed with a cold mixture of CHjC (12 mL) and toluene (2 mL) and dried by puiilng air through the sample overnight to give 5 as a White solid (1 ,327 g, 70% yield). TLC: Rf = 0.26, 10% MeOH in CH2CI2. 1 H NMR (CDCI3)(ppm) 0.95 {d, J - 17 Hz, 3H), 0.98 (d, J - 17 Hz, 3H), 1.30 (m, 2H), 1.40 (d, J = 17 Hz, 3H), 1.61 (m, 4H), 2.06 (m, 1 H), 2.25 (dt, J = 4, 19 Hz, 2H). 3.35 (s, 1 H), 3.49 (t, J - 17 Hz,2H), 4.20 (d, J = 18 Hz, 1 H), 4.38 (m, 1 H), 6.80 {s, 2H). Analytical HPLC (0.1% formic acid): tR 9.05 min. LC-MS: tR 11.17 min, m/z (ES+) found 381.9 (M+H)+, m/z (ES-) found 379.9 (M-H)-. |
52% | In N,N-dimethyl-formamide; at 20℃; for 48h; | (a) (S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3- methylbutanamido)propanoic acid (23)A suspension of dipeptide (22) (0.1 g, 0.54 mmol, 1 eq.) and 6-maleimidohexanoic acid succinimide ester (0.165 g, 0.54 mmol, 1 eq.) in anhydrous DMF (5 mL) was stirred at room temperature for 24 hours at which time LCMS indicated 50% conversion to a new product. The reaction mixture was diluted with anhydrous DMF (5 mL) and the reaction was allowed to continue for a further 24 hours. The solvent was evaporated under reduced pressure to give a colourless residue. Diethyl ether (60 mL) was added and the mixture was sonicated for 5 min, the ether was decanted and the process was repeated (x 2). The final ethereal portion was filtered to isolate the product (23) as a white powder which was dried under vacuum (0.105 g, 52%). Analytical Data: RT 2.28 min; MS (ES+) m/z (relative intensity) 382 ([M + H]+ , 90), MS (ES") m/z (relative intensity) 380 ([M - H])-, 100). |
With triethylamine; | 150 mg (0.797 mmol) of <strong>[1115-74-8]L-valyl-L-alanine</strong> and 246 mg (0.797 mmol) of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione were dissolved in 4.0 ml of dimethylformamide, and 0.220 ml (1.6 mmol) of triethylamine were added. The reaction mixture was stirred at RT overnight. The reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250*30; 10mu, flow rate: 50 ml/min, MeCN/water). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 302 mg (97% of theory) of N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-<strong>[1115-74-8]L-valyl-L-alanine</strong>. LC-MS (Method 12): Rt=1.02 min; MS (ESIpos): m/z=382 (M+H)+. 1H-NMR (400 MHz, DMSO-d6): delta [ppm]=0.82 (dd, 6H), 1.17 (m, 2H), 1.27 (d, 3H), 1.48 (m, 4H), 1.94 (m, 1H), 2.13 (m, 2H), 3.38 (t, 2H), 4.17 (m, 2H), 7.00 (s, 2H), 7.75 (d, 1H), 8.19 (d, 1H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
85% | Stage #1: maleic anhydride; 6-aminohexanoic acid In N,N-dimethyl-formamide at 20℃; for 3h; Inert atmosphere; Stage #2: 1-hydroxy-pyrrolidine-2,5-dione With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In N,N-dimethyl-formamide at 20℃; Inert atmosphere; Cooling with ice; | |
63% | Stage #1: maleic anhydride; 6-aminohexanoic acid In N,N-dimethyl-formamide for 2h; Inert atmosphere; Stage #2: 1-hydroxy-pyrrolidine-2,5-dione With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In N,N-dimethyl-formamide at 0 - 35℃; for 12h; Inert atmosphere; | |
60% | Stage #1: maleic anhydride; 6-aminohexanoic acid In N,N-dimethyl-formamide at 20℃; for 2h; Stage #2: 1-hydroxy-pyrrolidine-2,5-dione With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 0 - 20℃; | 3.2.2.Synthesisof(N-maleimidoalkyl)succinimideEsters(4-6) General procedure: Thesynthesisofcompoundswascarriedoutaccordingtothemethoddescribedin[77,78].A portionof3mmolofanaminoacidisaddedtoasolutionofmaleicanhydride(0.3g,3mmol)in4mL ofDMF,stirredfor2hatroomtemperature.Aftercompletedissolution,atatemperatureof0°C,0.43 g(3.75mmol)ofN-hydroxysuccinimide(NHS)and1.24g(6mmol)ofdicyclohexylcarbodiimide (DCC)areaddedtothesolution.Thereactionmassisstirredinanicebath5-10minandthen incubatedatroomtemperatureforaday.Theformationofawhiteprecipitateofdicyclohexylureais observed.Theprecipitateiswashedwith20mLofwaterand20mLofmethylenechloride.The organiclayeristreatedwith10mLofwater,extractedwithmethylenechloride,then10mLof5% NaHCO3areaddedandextractedwithmethylenechloride(theprocedureiscarriedoutthreetimes). TheorganiclayerwasdriedoverNa2SO4,thesolutionwasfilteredandthesolventwasevaporatedat the reduced pressure. The residue was purified by column chromatography on SiO2 with CHCl3/MeOH(from1:0to100:1)asaneluenttogivecompounds4-6. |
27% | Stage #1: maleic anhydride; 6-aminohexanoic acid In N,N-dimethyl-formamide at 25℃; for 2.5h; Inert atmosphere; Stage #2: 1-hydroxy-pyrrolidine-2,5-dione With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 0 - 25℃; for 18h; Inert atmosphere; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
40% | With triethylamine; In dichloromethane; N,N-dimethyl-formamide; at 20℃; for 18h;Inert atmosphere; | Under nitrogen H2N-Val-Ala-PABC-DOXO 21 (0.44 g, 0.51 mmol, 1 eq) was dissolved in a mixture of dichloromethane (abs.) and dry N,N-dimethylformamide (5:1, 42 mL). After addition of 234 mg 6-maleimimidocaproic acid N-hydroxysuccinimide ester (EMC-OSu) (0.76 mmol, 1.5 eq), the solution was treated with 71 muL triethylamine (0.51 mmol, 1 eq) and stirred for 18 h at room temperature. The crude product was precipitated with a mixture of 600 mL hexane and 100 mL diethyl ether. The red solid was washed twice with ether, dried in vacuo and purified by flash chromatography (CHCl3/MeOH 31:1) on silica gel to give the product 22 as a red solid (213 mg, 40percent). HPLC analysis was carried out with a Waters Symmetry 300 C18 5 mum [4.6×250 mm] with pre-column [3.9×20 mm]; chromatographic conditions: flow: 1.0 mL/min, mobile phase A: 15percent CH3CN, 85percent 20 mM sodium phosphate buffer (pH 7.0); mobile phase B: 30percent CH3CN, 70percent 20 mM sodium phosphate buffer (pH 7.0); injection volume: 50 muL; gradient: 0-1.5 min 100percent mobile phase A; 1.5-20 min increase to 30percent CH3CN, 70percent 20 mM sodium phosphate buffer; 20-45 min 70percent CH3CN, 30percent 20 mM sodium phosphate buffer; 45-55 min decrease to initial mobile phase; 55-58 min 100percent mobile phase A. C53H61N5O18, HRMS ESI-TOF: calculated [M+Na]+ 1078.39. found 1078.2. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
61% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 2h;Inert atmosphere; | A flame-dried flask was charged with glucuronide linker 13 (2 mg, 1.7 mumol, 1 eq), maleimidocaproic acid N-hydroxysuccinimide ester (0.8 mg, 2.6 pmol, 1.5 eq), and anhydrous dimethylformamide (85 mu). Diisopropylethylamine (1.5 uL, 8.5 pmol, 5 eq) was added, the reaction was then stirred at room temperature under nitrogen. After 2 hours HPLC revealed conversion to product. The reaction was diluted in dimethylsulfoxide and purified by preparative HPLC to provide PBD glucuronide linker 14 (1.4 mg, 61percent). LC- MS: tR 12.30 min, m/z (ES+) found 1373.7 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
89% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 3h;Inert atmosphere; | Alloc-protected di-mine PBD 5 (19 mg, 19 mumomicronL) was added to a flame-dried flask and dissolved in anhydrous dichloromethane (1.9 mL). Triphenylphosphine (0.25 mg, 1 mumomicronL), pyrollidine (3.1 pL, 38 muL), and tetrakis(triphenylphosphine)palladium(0) (0.5 mg, 0.5 mumol) were added, the reaction was then stirred under nitrogen for 30 minutes at room temperature, at which time LC-MS revealed complete alloc-deprotection. The reaction was loaded directly onto a 1 mm chromatotron plate eluted with CH2Cl2; MeOH mixtures (100:0 to 80:20 CH2Cl2/MeOH) to provide the free amine (14 mg, 79percent). Analytical HPLC: tR 9.32 min. LC-MS: tR 11.61 min, m/z (ES+) found 938.5 (M+H)+. The free amine was then dissolved in anhydrous DMF (0.37 mL) and maleimidocaproyl NHS ester was added (6.9 mg, 22 pmol), followed by diisopropylethylamine (13 muL, 75 muL). The reaction was stirred at room temperature for three hours, at which time LC-MS revealed complete consumption of starting material. The reaction mixture was diluted with dichloromethane and purified by radial chromatography on a 1 mm chromatotron plate eluted with CH2Cl2/MeOH mixtures (100:0 to 90:10 CH2Cl2/MeOH) to provide 4 (15 mg, 89percent). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
34 mg | With triethylamine; In dichloromethane; at 20℃; | Step 7 A -[6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)hexanoyl]-L-valyl-/V-[4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9- dioxa-4,7-diazaundec-1-yl]phenyl}-/V5-carbamoyl-L-omithinamide To a solution of the crude product A -[6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)hexanoyl]-L-valyl-A -{4-[11-(biphenyl-4- yl) ,6,6,8,11-pentamethyl-3,9-dioxo-2,10-dioxa ,8-diazado ( 0.0629 mmol) in anhydrous dimethylformamide (1.5 ml), 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1 H-pyrrole-2,5- dione (0.189 mmol, 58.3 mg) and triethylamine ( 0.252 mmol, 25.5 mg) were added. The reaction mixture was stirred at room temperature until no starting material was detectable. The solvent was evaporated under vacuum and the crude was purified by flash column chromatography (eluantEtOHiCh C = 1 :9) on silica gel (230-400 mesh), affording the desidered product (34 mg, white wax) MS (ESI): 925 (MH+) 1H NMR (500 MHz, DMSO-d6) delta ppm 0.83 (dd, J=15.79, 6.79 Hz, 6 H) 1.31 - 1.40 (m, 1 H) 1.60 (br. s., 1 H) 1.69 (s, 6 H) 1.90 - 2.02 (m, 1 H) 2.05 - 2.23 (m, 2 H) 2.89 - 2.96 (m, 3 H) 3.01 (br. s., 1 H) 3.34 - 3.38 (m, 2 H) 3.46 (m, J=8.24 Hz, 2 H) 4.19 (t, J=7.40 Hz, 1 H) 4.38 (br. s., 1 H) 4.95 - 5.05 (m, 2 H) 5.40 (s, 2 H) 5.97 (d, J=12.20 Hz, 1 H) 6.98 - 7.02 (m, 2 H) 7.30 (d, J=8.85 Hz, 2 H) 7.37 (dd, J=15.40, 7.93 Hz, 3 H) 7.45 (m, J=7.17 Hz, 2 H) 7.52 - 7.67 (m, 6 H) 7.80 (d, J=8.24 Hz, 1 H) 8.08 (d, J=6.71 Hz, 1 H) 9.76 - 10.18 (m, 1 H) |
34 mg | With triethylamine; | Step 7 N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl]phenyl}-N5-carbamoyl-L-ornithinamide To a solution of the crude product N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]L-valyl-N-{4-[11-(biphenyl-4-yl)-4,6,6,8,11-pentamethyl-3,9-dioxo-2,10-dioxa-4,8-diazadodec-1-yl]phenyl}-N5-carbamoyl-L-ornithinamide (0.0629 mmol) in anhydrous dimethylformamide (1.5 ml), 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (0.189 mmol, 58.3 mg) and triethylamine (0.252 mmol, 25.5 mg) were added. The reaction mixture was stirred at room temperature until no starting material was detectable. The solvent was evaporated under vacuum and the crude was purified by flash column chromatography (eluant:EtOH:CH2Cl2=1:9) on silica gel (230-400 mesh), affording the desired product (34 mg, white wax) MS (ESI): 925 (MH+) 1H NMR (500 MHz, DMSO-d6) delta ppm 0.83 (dd, J=15.79, 6.79 Hz, 6H) 1.31-1.40 (m, 1H) 1.60 (br. s., 1H) 1.69 (s, 6H) 1.90-2.02 (m, 1H) 2.05-2.23 (m, 2H) 2.89-2.96 (m, 3H) 3.01 (br. s., 1H) 3.34-3.38 (m, 2H) 3.46 (m, J=8.24 Hz, 2H) 4.19 (t, J=7.40 Hz, 1H) 4.38 (br. s., 1H) 4.95-5.05 (m, 2H) 5.40 (s, 2H) 5.97 (d, J=12.20 Hz, 1H) 6.98-7.02 (m, 2H) 7.30 (d, J=8.85 Hz, 2H) 7.37 (dd, J=15.40, 7.93 Hz, 3H) 7.45 (m, J=7.17 Hz, 2H) 7.52-7.67 (m, 6H) 7.80 (d, J=8.24 Hz, 1H) 8.08 (d, J=6.71 Hz, 1H) 9.76-10.18 (m, 1H) |
With triethylamine; In dichloromethane; at 20℃; | To a solution of N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl]phenyl}-N5-carbamoyl-L-ornithinamide (200 mg, 0.21 mmol) in dry DMF (6 mL) was added piperidine (0.105 mL, 1 mmol). The resulting solution was stirred at room temperature for 2 hours, concentrated to dryness to afford L-valyl-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9-dioxa-4,7-diazaundec-1-yl]phenyl}-N5-carbamoyl-L-ornithinamide which was used without further purification. The crude intermediate was dissolved in dry DCM (6 mL) and 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (130 mg, 0.42 mmol) and triethylamine (0.088 mL, 0.63 mmol) were added. The resulting solution was stirred at room temperature overnight, solvents were evaporated and the residue was purified by column chromatography (DCM / EtOH = 10 / 1) affording N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-valyl-N-{4-[10-(biphenyl-4-yl)-4,7,10-trimethyl-3,8-dioxo-2,9dioxa-4,7-diazaundec-1-yl]phenyl}-N5-carbamoyl-L-ornithinamide (150 mg, 77percent yield).ESI MS: m/z 925 (MH+)1H NMR (500 MHz, DMSO-de) delta 0.81 (d, J = 6.7 Hz, 3 H), 0.84 (d, J = 6.7 Hz, 3 H), 1.18 (m, 2 H), 1.36 (m, 1 H), 1.40-1.52 8m, 6H), 1.60 (m, 1 H), 1.69 (s, 6 H), 1.97 (m, 1 H), 2.08 - 2.23 (m, 2 H), 2.63 - 2.96 (m, 8 H), 2.90 (m, 1 H), 3.01 (m, 1 H), 3.23- 3.34 (m, 2H), 3.36 (m, 2 H), 3.45 (m, 2 H), 4.19 (t, J = 7.4 Hz, 1 H), 4.38 (m, 1 H), 4.95 - 5.05 (m, 2 H), 5.40 (s, 2 H), 5.97 (d, J = 5.6 Hz, 1 H), 6.99 (s, 2 H), 7.25-7.41 (m, 5 H), 7.45 (m, 2 H), 7.53 - 7.66 (m, 6 H), 7.80 (d, J =9 Hz, 1 H), 8.08 (d, J = 7.0 Hz, 1 H), 9.99 (m, 1 H), 10.76 (br.s., 1 H) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
41% | With triethylamine; In dichloromethane; at 20℃; for 3h; | General procedure: Step g N-[6-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1^ methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1 H-i thieno[3,2-e]indol -yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)ph [(IV)] (Compd 52) To a solution of L-valyl-W5-carbamoyl-W-[4-({^ yl)ethoxy]-1 H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4- yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinam (4.6 mg, 0.0039 mmol) in 1 mL of DCM (10percent DMF), were added 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1 H-pyrrole-2,5-dione (3.6 mg, 0.0117 mmol) and triethylamine (2.2 mL, 0.0156 mmol). The mixture was stirred at room temperature for 3 hours, solvents were evaporated and the title compound (2.2 mg, 41percent yield) was isolated by column chromatography purification (DCM/MeOH 8:2). ESI MS: m/z 1380 (MH+) 1H NMR (500 MHz, methanol-*) delta ppm 1.98 (br. s., 4 H) 2.60 - 2.63 (m, 3 H) 2.89 - 3.11 (m, 6 H) 3.39 - 3.72 (m,10H) 4.07 - 4.75 (m,7H) 4.98 - 5.17 (m,2H) 6.74 (br. s., 2 H) 7.00 (m, 2 H) 7.07 - 7.45 (m, 8 H) 7.52 (br. s., 3 H) 8.07 - 8.11 (m, 1 H) |
2.2 mg | With triethylamine; In dichloromethane; N,N-dimethyl-formamide; at 20℃; for 3h; | Step g N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-valyl-N5-carbamoyl-N-[4-([{2-[([(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide [(IV)] (Compd 52) To a solution of L-valyl-N5-carbamoyl-N-[4-([{2-[([(8S)-8-(chloromethyl)-1-methyl-6-({5-[({5-[2-(pyrrolidin-1-yl)ethoxy]-1H-indol-2-yl}carbonyl)amino]-1H-indol-2-yl}carbonyl)-7,8-dihydro-6H-thieno[3,2-e]indol-4-yl]oxy}carbonyl)(methyl)amino]ethyl}(methyl)carbamoyl]oxy}methyl)phenyl]-L-ornithinamide (4.6 mg, 0.0039 mmol) in 1 mL of DCM (10percent DMF), were added 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione (3.6 mg, 0.0117 mmol) and triethylamine (2.2 mL, 0.0156 mmol). The mixture was stirred at room temperature for 3 hours, solvents were evaporated and the title compound (2.2 mg, 41percent yield) was isolated by column chromatography purification (DCM/MeOH 8:2). ESI MS: m/z 1380 (MH+) 1H NMR (500 MHz, methanol-d4) delta ppm 1.98 (br. s., 4H) 2.60-2.63 (m, 3H) 2.89-3.11 (m, 6H) 3.39-3.72 (m, 10H) 4.07-4.75 (m, 7H) 4.98-5.17 (m, 2H) 6.74 (br. s., 2H) 7.00 (m, 2H) 7.07-7.45 (m, 8H) 7.52 (br. s., 3H) 8.07-8.11 (m, 1H) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
93% | With triethylamine; In N,N-dimethyl-formamide; at 20℃; for 4h; | To a solution of amine 62 (10,4 mg, 0.00823 mmol) and 6-maleimidohexanoic acid NHS ester (Leonard and Brunckova 2010) (3.3 mg, 0.011 mmol) in DMF (80 uL) is added Et3N (0.9 mg, 0.009 mmol) and the mixture is stirred at rt. After 4 h, the mixture is concentrated under high vacuum, and the crude residue is purified by column chromatography on silica gel (MeOH/CH2CI2= 8:92 to 14:86) to afford the title compound CN211 as a white solid (1 1.2 mg, 93%).1H NMR (500 MHz, 2:1 CDCI3/CD3OD) delta 0.87-0.90 (m, 6 H), 0.94-0.97 (m, 6 H), 1.23- 1.36 (m, 70 H), 1.49-1.77 (m, 11 H), 1.87-1.94 (m, 1 H), 2.03-2.10 (m, 1 H), 2.24-2.30 (m, 2 H), 2.31-2.41 (m, 2 H), 3.09-3.14 (m, 1 H), 3.20-3.26 (m, 1 H), 3.51 (t, J = 7.2 Hz, 2 H), 3.66- 3.81 (m, 8 H), 3.85-3.87 (m, 2 H), 4.17 (d, J = 7.4 Hz, 1 H), 4.53 (dd, J = 5.1 , 8.6 Hz, 1 H), 4.85 (d, J = 3.8 Hz, 1 H), 4.92-4.98 (m, 2 H), 5.10-5.15 (m, 1 H), 6.74 (s, 2 H), 7.31 (d, J = 8.1 Hz, 2 H), 7.56 (d, J = 8.1 Hz, 2 H);13C NMR (126 MHz, 2:1 CDCI3/CD3OD) delta 14.2, 18.5, 19.4, 23.0, 25.4, 25.5, 25.6, 26.6, 28.5, 29.2, 29.5, 29.58, 29.63, 29.65, 29.73, 29.84, 29.86, 29.90, 29.93, 29.97, 30.01 , 31.0, 32.2, 34.9, 36.2, 37.9, 39.3, 52.5, 53.7, 59.4, 62.3, 66.7, 68.4, 69.4, 70.2, 70.6, 70.8, 72.3, 75.0, 100.3, 120.4, 129.0, 132.9, 134.5, 138.2, 157.0, 161.0, 171.0, 171.5, 172.8, 174.9; HRMS-ESI m/z calcd for C79H137N7Na017[M+Naf: 1478.9969, found 1478.9971. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
95% | With dicyclohexyl-carbodiimide; In acetonitrile; at 0 - 20℃;Inert atmosphere; | Under nitrogen protection, 4.7 g (22 mmol) of MC and 25 g (22 mmol) of HOSu were added to 50 ml of acetonitrile.Another 4.5 g (22 mmol) of DCC was dissolved in 25 ml of acetonitrile,Keeping the internal temperature around 0C,It was slowly dropped into the reaction solution.The reaction solution was reacted at 0C for 2 hours and then reacted overnight at room temperature.filter,The filter cake was washed with acetonitrile 10ml×3.The filtrate was concentrated to dryness under reduced pressure.The resulting oil was dried under reduced pressure at room temperature for 6 h to give 6.4 g of light brown solid.Yield 95%. |
95% | With dicyclohexyl-carbodiimide; In acetonitrile; at 0 - 20℃;Inert atmosphere; | 4.7 g (22 mmol) of MC and 25 g (22 mmol) of HOSu were added to 50 ml of acetonitrile under nitrogen. Another 4.5g (22mmol) DCC was dissolved in 25ml acetonitrile.The internal temperature was kept at about 0 C, and it was slowly dropped into the reaction liquid. The reaction solution was reacted at 0 C for 2 hours and then allowed to react at room temperature overnight. filter,The filter cake was washed with acetonitrile 10 ml × 3The filtrate was concentrated to dryness under reduced pressure. The obtained oil was dried under reduced pressure at room temperature for 6 h to give 6.4 g of pale brown solid. The yield was 95%. |
91.9% | With dicyclohexyl-carbodiimide; In dichloromethane; at 20℃; for 4h; | The method for preparing biotin maleimide according to the present example includes the following steps: <strong>[55750-53-3]6-maleimidocaproic acid</strong> compound 1 (1.28 g, 6 mmol, 1 eq), and N-hydroxysuccinimide (0.7 g, 6 mmol, 1 eq) were sequentially added to a 50 ml single-necked flask and 10 ml of dichloromethane, dissolved and clarified, and added dicyclohexylcarbodiimide DCC (1.25 g, 6 mmol, 1 eq) at room temperature, and stirred at room temperature for 4 hours.A large amount of dicyclohexylurea DCU was produced, and TLC monitored the reaction of the starting material completely.After cooling to room temperature, filtration, the filtrate was added with silica gel, and the column was chromatographed, and the product was eluted with PE: EtOAc = 1:1.1.7 g of an oily liquid was obtained as 6-maleimidohexanoic acid N-hydroxysuccinimide ester compound 2, MS+1: 309.10, yield 91.9percent. |
70% | With dicyclohexyl-carbodiimide; In dichloromethane; at 20℃; for 3h; | Dicyclohexyl-carbodiimide (2.68 g, 13.02 mmol) and N-hydroxysuccinimide (1 .36 g, 1 1 .84 mmol) are added at room temperature to a stirrer solution of compound [4] (2.5 g, 1 1 .84 mmol) in anhydrous dichloromethane (15 mL) and the mixture stirred at room temperature for 3 hours. The white solid is filtered with dichloromethane to remove the dicyclohexylurea, the organic phase is washed with HCI 0.1 N and water, then dried over anhydrous sodium sulfate and the solvent removed by rotatory evaporation. The resulting residue is subjected to flash column chromatography with a medium pressure system Sepacore Buchi (silica gel; gradient A: petroleum ether/B: ethyl acetate; B% 0-80 in 15 minutes) to give the activated acid [5] as a white solid, 2.4 g (70%). MS: m/z 309 [M+H]+.1H NMR (400 MHz, MeOD) delta 6.78 (s, 2H), 3.49 - 3.46 (m, 2H), 2.82 (s, 4H), 2.62 - 2.59 (m, 2H), 1 .78 - 1 .65 (m, 2H), 1 .64 - 1 .50 (m, 2H), 1 .46 - 1 .26 (m, 2H).13C NMR (100 MHz, MeOD) delta 169.8, 169.0, 167.3, 132.5, 35.4, 28.6, 26.1 , 23.8, 23.6, 22.3. |
48% | With dicyclohexyl-carbodiimide; In ethyl acetate; at 0 - 20℃; for 20h; | To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (3.08 g, 14.6 mmol, 1.0 eq) and N-hydroxysuccinimide (1.76 g, 15.3 mmol, 1.05 eq) in EtOAc (30 mL) at 0 C, was added dicyclohexylcarbodiimide (3.16 g, 15.3 mmol, 1.05 eq). The reaction was then allowed to warm to rt. After 20 h, the reaction was filtered and washed with EtOAc and the filtrate concentrated. The residue was purified by flash chromatography to yield the title compound (2.16 g, 48%) as a clear oil that solidified slowly to a waxy white solid. NMR (400 MHz, Chloroform-i/) delta 6.71 (s, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.86 (s, 4H), 2.63 (t, J = 7.4 Hz, 2H), 1.80 (p, J= 7.4 Hz, 2H), 1.73 - 1.57 (m, 2H), 1.50 - 1.35 (m, 2H). m/z calcd. for C14H16N206 = 308.10. Found [M+H]+ = 309.13. Rf = 0.28 (50% EtOAc/Hex). |
48% | With dicyclohexyl-carbodiimide; In ethyl acetate; at 0 - 20℃; for 20h; | To a stirred solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-l-yl)hexanoate (3.08 g, 14.6 mmol, 1.0 eq) and N-hydroxysuccinimide (1.76 g, 15.3 mmol, 1.05 eq) in EtOAc (30 mL) at 0 C,was added dicyclohexylcarbodiimide (3.16 g, 15.3 mmol, 1.05 eq). The reaction was then allowed towarm to rt. After 20 h, the reaction was filtered and washed with EtOAc and the filtrate concentrated. The residue was purified by flash chromatography to yield the title compound (2.16 g, 48%) as a clear oil that solidified slowly to a waxy white solid. ?H NMR (400 MHz, Chloroform-d) oe 6.71 (s, 2H), 3.56 (t, J = 7.2 Hz, 2H), 2.86 (s, 4H), 2.63 (t, J = 7.4 Hz, 2H), 1.80 (p, J = 7.4 Hz, 2H), 1.73 - 1.57 (m,2H), 1.50 1.35 (m, 2H). m/z calcd. for C,4H,6N206 = 308.10. Found [M+H] = 309.13. Rf= 0.28 (50% EtOAc/Hex) |
With dicyclohexyl-carbodiimide; In acetonitrile; at 0℃; for 2h;Inert atmosphere; | 4.7 g (22 mmol) of MC and 25 g (22 mmol) of HOSu were added to 50 ml of acetonitrile under nitrogen.Another 4.5g (22mmol) DCC was dissolved in 25ml acetonitrile.The internal temperature was kept at about 0 C, and it was slowly dropped into the reaction liquid.The reaction solution was reacted at 0 C for 2 hours and then allowed to react at room temperature overnight. filter,The filter cake was washed with acetonitrile (10 ml × 3) and the filtrate was concentrated to dryness.The obtained oil was dried under reduced pressure at room temperature for 6 h.Obtained 6.4 g of light brown solid.The yield was 95%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
64% | With N-ethyl-N,N-diisopropylamine; In dichloromethane; at 23℃; | To a solution of Compound 24 (40 mg, 0.064 mmol) ), prepared as described in step (b) above, in CH2CI2 (2 ml.) was added 6-maleimidohexanoic acid N- hydroxysuccinimide ester (21 .6 mg, 0.07 mmol). The reaction mixture was stirred at 23 °C overnight and concentrated under vacuum. The residue obtained was purified in a system for flash chromatography (Si02, Hex:EtOAc mixtures) to afford pure Compound 25 (33.5 mg, 64percent).1H N R (400 MHz, CDCI3): delta 8.67 (d, J = 10.7 Hz, 1 H), 7.29-7.23 (m, 1 H), 6.90 (t, J = 11.5 Hz, 1 H), 6.80 (t, J = 9.6 Hz, 1 H), 6.68 (s, 2H), 6.46 (d, J = 9.4 Hz, 1 H), 6.42 (bs, 1 H), 6.16 (d, J = 11.6 Hz, 1 H), 5.73-5.67 (m, 2H), 5.64 (dd, J = 6.2, 3.1 Hz, 1 H), 5.30 (d, = 9.6 Hz, 1 H), 4.86 (q, J = 8.4 Hz, 1 H), 4.63-4.54 (m, Eta), 4.44 (d, J = 9.4 Hz, 1 H), 4.30-4.18 (m, 1 H), 3.66 (s, 3H),3.50 (t, J = 7.2 Hz, 2H), 3.34-3.10 (m, 3H), 2.85 (dt, J = 9.9, 6.9 Hz, 1 H), 2.54-2.37 (m, 4H), 2.36-2.28 (m, 1 H), 2.16 (t, J = 7.6 Hz, 2H), 1.84 (d, J = 1.3 Hz, 3H), 1.83-1 .81 (m, 3H), 1.70-1.51 (m, 8H), 1.34-1.23 (m, 2H), 1.16 (d, J - 6.6 Hz, 3H), 1.05 (s, 9H).13C NMR (100 MHz, CDCI3): delta 173.4, 170.8, 168.1 , 166.4, 161.6, 157.0, 145.2, 140.3, 137.5, 134.3, 134.1 , 133.9, 124.2, 120.7, 108.2, 106.2, 81.8, 78.4, 74.3, 73.5, 60.7, 55.4, 37.7, 37.6, 37.3, 36.4, 35.9, 34.6, 32.8, 30.3, 29.9, 28.3, 26.7, 26.4, 26.2, 25.4, 25.2, 24.4, 17.2, 16.6, 3.6.ESI-MS m/r. Calcd. for C44H61CIN5O10: 819.44. Found: 820.4 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
46% | In N,N-dimethyl-formamide at 0 - 20℃; Inert atmosphere; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
49% | In N,N-dimethyl-formamide at 20℃; for 3h; Inert atmosphere; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
95% | With N-ethyl-N,N-diisopropylamine; In dimethyl sulfoxide; at 20℃; | A mixture of 59a (688 mg, 0.99 mmol), 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)hexanoate (SuOMC, 320 mg, 1.04 mmol), and DIPEA (190 1.09 mmol) in DMSO (10 mL) was stirred at room temperature overnight. All the volatile components were pumped off. The resultant residue was triturated with ethyl acetate to give (S)-tert-butyl l-(chloromethyl)-5-(4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanamido)-3-methylbutanamido)-5-ureidopentanamido)benzyloxy)-lH-benzo[e]indole- 3(2H)-carboxylate 59b as an off-white solid (832 mg, 95percent). 1H NMR (DMSO) delta 10.03 (s, 1H), 8.13-8.08 (m, 2H), 7.83-7.79 (m, 2H), 7.66 (d, J= 8.4 Hz, 2H), 7.55-7.48 (m, 3H), 7.35 (t, J= 7.5 Hz, 1H), 6.99 (s, 2H), 5.97 (t, J= 5.4 Hz, 1H), 5.41 (s, 2H), 5.21 (s, 2H), 4.43-4.38 (m, 1H), 4.22-3.98 (m, 4H), 3.84-3.80 (m, 1H), 3.38-3.33 (m, 3H), 3.08-2.90 (m, 2H), 2.24- 2.06 (m, 2H), 2.01-1.91 (m, 1H), 1.74-1.14 (m, 10H), 1.55 (s, 9H), 0.86 (d, J= 6.8 Hz, 3H), 0.83 (d, J= 6.7 Hz, 3H) ppm. HRMS (ESI) found m/z 910.3897 (M + Na). C46H58ClN7Na09 requires 910.3877. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
67% | With N-ethyl-N,N-diisopropylamine; In dimethyl sulfoxide; at 20℃; | A mixture of 66j (40 mg, 0.036 mmol), 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)hexanoate (SuOMC, 12 mg, 0.037 mmol), and DIPEA (6.8 muEpsilon, 0.039 mmol) in DMSO (1 mL) was stirred at room temperature overnight before all the volatile components were pumped off. The resultant residue was triturated with ether to give bis (di- tert-butylphosphate) N-(3-(2,5-bis((E)-3-((S)-l-(chloromethyl)-5-hydroxy-lH-benzo[e]indol- 3(2H)-yl)-3-oxoprop-l-enyl)phenylamino)-3-oxopropyl)-6-(2,5-dioxo-2,5-dihydro-lH- pyrrol-l-yl)hexanamide 66k as a yellow solid (32 mg, 67percent). 1H NMR (DMSO) delta 10.02 (s, 1H), 8.67 (s, 2H), 8.11-8.05 (m, 3H), 7.98-7.92 (m, 3H), 7.85-7.75 (m, 2H), 7.70 (d, J= 15.3 Hz, 1H), 7.63-7.58 (m, 2H), 7.51 (t, J= 7.9 Hz, 2H), 7.26 (dd, J= 4.4, 15.4 Hz, 2H), 6.96 (s, 2H), 4.66-4.52 (m, 4H), 4.44-4.35 (m, 2H), 4.07-3.95 (m, 4H), 3.45-3.30 (m, 4H), 2.52-2.50 (m, 2H), 2.08 (t, J= 7.2 Hz, 2H), 1.50-1.40 (m, 40H), 1.33-1.25 (m, 2H) ppm. 31P NMR (DMSO) delta -15.42, 15.45 ppm. HRMS (ESI) found m/z 1334.4515 (M + Na). C67H8iCl2N5NaOi4P2 requires 1334.4525. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
68% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; | A mixture of 67a (1.00 g, 2.40 mmol), 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)hexanoate (SuOMC, 740 mg, 2.40 mmol), and DIPEA (460 muEpsilon, 2.64 mmol) in DMF (10 mL) was stirred at room temperature overnight. All the volatile components were pumped off. The resultant residue was purified by column chromatography using a mixture of ethyl acetate and petroleum ether (v/v 2: 1) as eluent, followed by ethyl acetate alone, to give (2E,2'E)-tert-butyl 3,3'-(2-(3-(6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanamido)propanamido)-l,4-phenylene)diacrylate 67b as a white solid (1.00 g, 68percent). mp 69-72 °C. 1H NMR (CDC13) delta 7.95 (s, 1H), 7.73 (s, 1H), 7.65 (d, J= 15.9 Hz, 1H), 7.61 (d, J= 8.2 Hz, 1H), 7.53 (d, J= 16.0 Hz, 1H), 7.37 (d, J= 8.2 Hz, 1H), 6.69 (br s, 1H), 6.64 (s, 2H), 6.39 (d, J= 16.0 Hz, 1H), 6.38 (d, J= 15.8 Hz, 1H), 3.68-3.63 (m, 2H), 3.43 (d, J = 7.0 Hz, 2H), 2.69 (d, J= 5.5 Hz, 2H), 2.21 (t, J= 7.3 Hz, 2H), 1.67-1.50 (m, 4H), 1.53 (s, 9H), 1.52 (s, 9H), 1.31-1.24 (m, 2H) ppm. HRMS (ESI) found m/z 62,2292, 1 (M + Na). C33H43N3Na08 requires 632.2942. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
92% | In N,N-dimethyl-formamide; at 20℃; for 2h; | Amine 10(4.37 g, 10.8 mmol, 1.0 eq) and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-l -yl) hexanoate 11(3.67 g, 11.9 mmol, 1.1 eq) were dissolved in anhydrous DMF (100 mL) with stirring. The reaction mixture was stirred at room temperature for 2 hours. The solvent wasevaporated under reduced pressure to give the product 12 as a viscous yellow oil (6.0 g, 92percent). Analytical Data: RT 1.20 mm; MS (E5) m/z (relative intensity) 597 ([M+ H]b,100). |
54% | In N,N-dimethyl-formamide; at 16 - 20℃; for 2h; | Compound 13 (390 mg, 0.965 mmol) and compound 14 (327 mg, 1.06 mmol) were dissolved in DMF (10 mL) at 16°C. The mixture was stirred at r.t. for 2 h. The mixture was concentrated in vacuum and purified by column chromatography (PE/EtOAc=3/1) to give desire product 15 (400 mg, yield: 54percent)LCMS: (5-95, AB, 1.5 mm), 0.726 mi MS=597.1 [M+1];1H NMR (400 MHz, DMSO-d6) 10.52 (s, 1 H), 8.09 (d, J= 9.2 Hz, 1 H), 8.03 (s, 1 H), 7.53 (d, J= 8.4 Hz, 2 H), 7.25 (d, J 8.4 Hz, 2 H), 7.00 (s, 2 H), 6.03 - 6.00 (t, J= 5.6 Hz, 1 H), 5.45 (s, 1H), 5.42 (s, 2 H), 5.14 -5.11 (t, J= 5.8 Hz, 1 H)), 4.91 -4.87 (m, 1 H), 4.43 (d, J 5.2 Hz, 2 H),3.38 (s, 2 H), 3.03 -2.98 (m, 2 H), 2.14 -2.05 (m, 4 H), 1.50 - 1.46 (m, 4 H), 1.27 - 1.18 (m, 4 H),0.82 - 0.77 (m, 6 H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
76% | With triethylamine; In N,N-dimethyl-formamide; at 20℃; for 1h; | To an N,N-dimethylfomiamide (1.2 mL) solution of the compound (337 mg, 0.353 mmol) obtained in above Process 4, triethylamine (44.3 mL, 0.3 18 mmol) and Nsuccinimidyl 6-maleimidehexanoate (119.7 mg, 0.388 mmol) were added and stirred at room temperature for 1 hour. The solvent was removed under reduced pressure and the obtained residues were purified by silica gel column chromatography [chloroform - chloroform : methanol = 5: 1 (v/v)j to yield the titled compound as a pale yellow solid (278.0 mg, 76percent).1HNMR (400 MHz, DMSO-d6) delta: 0.87 (3H, t, J=7.3 Hz), 1.12-1.22 (2H, m),1.40-1.51 (4H, m), 1.66-1.76 (2H, m), 1.80-1.91 (2H, m), 2.05-2.21 (6H, m), 2.39 (3H, s), 2.79 (1H, dd, J=14.0, 9.8 Hz), 2.98-3.21 (5H, m), 3.55-3.77 (8H, m), 4.41-4.48 (1H, m), 5.15 (1H, d, J=18.9 Hz), 5.24 (1H, d, J=18.9 Hz), 5.40 (1H, d, J=17.1 Hz), 5.44 (1H, d, J=17.1 Hz), 5.54-5.60 (1H, m), 6.53 (1H, s), 6.99 (2H, s), 7.20-7.27 (5H, m), 7.30 (1H, s), 7.70 (1H, t, J=5.5 Hz), 7.80 (1H, d, J=11.0 Hz), 8.03 (1H, t, J=5.8 Hz), 8.08 (1H, t, J=5.5 Hz), 8.14 (1H, d, J=7.9 Hz), 8.25 (1H, t, J=6.1 Hz), 8.46 (1H, d, J=8.5 Hz).MS (APCI) mlz: 1032 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
70% | With N-ethyl-N,N-diisopropylamine; In acetonitrile; at 20℃; for 6h; | Compound E-12: Compound E-12-3 (5.6 mg, 4 jimol, 1.0 eq.) was dissolved in acetonitrile (0.5 mL), and DIEA (5 jiL, 7 eq.) was added, followed by 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (2.5 mg, 8 jimol, 2 eq.). The mixture was stirred for 6 hours at room temperature. After controlling the reaction by LC-MS, 200 jiL of water was added, and the resulting solution purified by preparative HPLC (Waters 600E, SunFire Prep C18 OBD column, 5 jim, 19 x 100 mm; Eluting phase:water / MeCN buffered with 0.1 percent TFA; Gradient of 5 percent to 100 percent MeCN in 15minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combinedand lyophilised to furnish compound E-12 as a white solid (4.6 mg, 70 percent). mlz (Q-TOF MS ESI+) 739.4389 (100 percent, (MH2)2, C78H118N12016 requires 739.4389). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
70% | With N-ethyl-N,N-diisopropylamine; In acetonitrile; at 20℃; for 6h; | Compound E-12: Compound E-12-3 (5.6 mg, 4 mumol, 1.0 eq.) was dissolved in acetonitrile (0.5 mL), and DIEA (5 mu, 7 eq.) was added, followed by 2,5-dioxopyrrolidin-l-yl 6-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (2.5 mg, 8 muiotaetaomicron, 2 eq.). The mixture was stirred for 6 hours at room temperature. After controlling the reaction by LC-MS, 200 mu, of water was added, and the resulting solution purified by preparative HPLC (Waters 600E, SunFire Prep C 18 OBD column, 5 muiotaeta, 19 x 100 mm; Eluting phase: water / MeCN buffered with 0.1 percent TFA; Gradient of 5 percent to 100 percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combined and lyophilised to furnish compound E-12 as a white solid (4.6 mg, 70 percent). m/z (Q-TOF MS ESI+) 739.4389 (100 percent, (MH2)2+, C78Hi i8Ni20i6 requires 739.4389). |
70% | With N-ethyl-N,N-diisopropylamine; In acetonitrile; at 20℃; for 6h; | General procedure: Compound E-12-3 (5.6 mg, 4 mumol, 1.0 eq.) was dissolved in acetonitrile (0.5 mL), and DIEA (5 mu, 7 eq.) was added, followed by 2,5-dioxopyrrolidin-l-yl 6-(2,5- dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (2.5 mg, 8 muiotaetaomicron, 2 eq.). The mixture was stirred for 6 hours at room temperature. After controlling the reaction by LC-MS, 200 mu, of water was added, and the resulting solution purified by preparative HPLC (Waters 600E, SunFire Prep C18 OBD column, 5 muiotaeta, 19 x 100 mm; Eluting phase: water / MeCN buffered with 0.1 percent TFA; Gradient of 5 percent to 100 percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combined and lyophilised to furnish compound E-12 as a white solid (4.6 mg, 70 percent). m/z (Q-TOF MS ESI+) 739.4389 (100 percent, (MH2)2+, C78Hi i8Ni20i6 requires 739.4389). |
70% | With N-ethyl-N,N-diisopropylamine; In acetonitrile; at 20℃; for 6h; | Compound E-12-3 (5.6 mg, 4 mumol, 1.0 eq.) was dissolved in acetonitrile (0.5 mL), and DIEA (5 muL.EL, 7 eq.) was added, followed by 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (2.5 mg, 8 mumol, 2 eq.). The mixture was stirred for 6 hours at room temperature. After controlling the reaction by LC-MS, 200 muL of water was added, and the resulting solution purified by preparative HPLC (Waters 600E, SunFire Prep C18 OBD column, 5 mum, 19×100 mm; Eluting phase: water/MeCN buffered with 0.1percent TFA; Gradient of 5percent to 100percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combined and lyophilised to furnish compound E-12 as a white solid (4.6 mg, 70percent). m/z (Q-TOF MS ESI+) 739.4389 (100percent, (MH2)2+, C78H118N12O16 requires 739.4389). |
70% | With N-ethyl-N,N-diisopropylamine; In acetonitrile; at 20℃; for 6h; | Compound E-12-3 (5.6 mg, 4 jimol, 1.0 eq.) was dissolved in acetonitrile (0.5 mL), and DIEA (5 jiL, 7 eq.) was added, followed by 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (2.5 mg, 8 jimol, 2 eq.). The mixture was stirred for 6 hours at room temperature. After controlling the reaction by LC-MS, 200 jiL of water was added, and the resulting solution purified by preparative HPLC (Waters 600E, SunFire Prep C 18 OBD column, 5 jim, 19 x 100 mm; Eluting phase: water / MeCN buffered with 0.1 percent TFA; Gradient of 5 percent to 100 percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm).The selected fractions were combined and lyophilised to furnish compound E-12 as a white solid (4.6 mg, 70 percent).mlz (Q-TOF MS ESI+) 739.4389 (100 percent, (MH2)2, C78H118N12016 requires 739.4389). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
35% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; acetonitrile; at 20℃; for 3h; | Compound E-13: ((2^,3^)-3-((5)-1-((3^,4^,55)-4-((5)-2-((5)-2-((4-((((4-((5)-2- ((iS)-2-(6-(2,5-dioxo-2,5-dihydro- IH-pyrrol- 1 -yl)hexanamido)-3-methylbutanamido)-5- ureidopentanamido)benzyl)oxy)carbonyl)(methyl)amino)phenethyl)(methyl)amino)-3- methylbutanamido)-N,3-dimethylbutanamido)-3-methoxy-5-methylheptanoyl) pyrrolidin-2-yl)-3-methoxy-2-methylpropanoyl)-Z-phenylalanine, 2,2,2-trifluoroacetate Compound E-13-2 (80 mg, 0.058 mmol, 1.0 eq.) was dissolved in a mixture of acetonitrile (1.5 mL) and DMF (0.4 mL). DIEA (50 mu,, 0.289 mmol, 5 eq.) was added, followed by 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoate (36 mg, 0.1 16 mmol, 2 eq.). The mixture was stirred for 3 hours at room temperature. After controlling the reaction by LC-MS, the solvent was evaporated under reduced pressure and the residue purified by preparative HPLC (Waters 600E, SunFire Prep CI 8 OBD column, 5 muiotaeta, 19 x 100 mm; Eluting phase: water / MeCN buffered with 0.1 percent TFA; Gradient of 5 percent to 100 percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combined and lyophilised to furnish compound E-13 as a white solid (32 mg, 35 percent). m/z (Q-TOF MS ESI-) 1461.8336 (100 percent, (M-H)~, C77H113N12O16 requires 1461.8403). m/z (Q-TOF MS ESI+) 1463.8565 (2 percent, MH+, CvvHi isNizOig requires 1463.8549), 732.4317 (100 percent, (MH2)2+, CvvHi igNizOig requires 732.431 1). |
35% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; acetonitrile; at 20℃; for 3h; | Compound E-13-2 (80 mg, 0.058 mmol, 1.0 eq.) was dissolved in a mixture of acetonitrile (1.5 mL) and DMF (0.4 mL). DIEA (50 mu,, 0.289 mmol, 5 eq.) was added, followed by 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoate (36 mg, 0.1 16 mmol, 2 eq.). The mixture was stirred for 3 hours at room temperature. After controlling the reaction by LC-MS, the solvent was evaporated under reduced pressure and the residue purified by preparative HPLC (Waters 600E, SunFire Prep CI 8 OBD column, 5 muiotaeta, 19 x 100 mm; Eluting phase: water / MeCN buffered with 0.1 percent TFA; Gradient of 5 percent to 100 percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combined and lyophilised to furnish compound E-13 as a white solid (32 mg, 35 percent). m/z (Q-TOF MS ESI-) 1461.8336 (100 percent, (M-H)~, C77H113N12O16 requires 1461.8403). m/z (Q-TOF MS ESI+) 1463.8565 (2 percent, MH+, CvvHi isNizOig requires 1463.8549), 732.4317 (100 percent, (MH2)2+, CvvHi igNizOig requires 732.431 1). |
35% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; acetonitrile; at 20℃; for 3h; | Compound E-13-2 (80 mg, 0.058 mmol, 1.0 eq.) was dissolved in a mixture of acetonitrile (1.5 mL) and DMF (0.4 mL). DIEA (50 muL, 0.289 mmol, 5 eq.) was added, followed by 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (36 mg, 0.116 mmol, 2 eq.). The mixture was stirred for 3 hours at room temperature. After controlling the reaction by LC-MS, the solvent was evaporated under reduced pressure and the residue purified by preparative HPLC (Waters 600E, SunFire Prep C18 OBD column, 5 mum, 19×100 mm; Eluting phase: water/MeCN buffered with 0.1percent TFA; Gradient of 5percent to 100percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combined and lyophilised to furnish compound E-13 as a white solid (32 mg, 35percent). (0474) m/z (Q-TOF MS ESI-) 1461.8336 (100percent, (M-H)?, C77H113N12O16 requires 1461.8403). (0475) m/z (Q-TOF MS ESI+) 1463.8565 (2percent, MH+, C77H115N12O16 requires 1463.8549), 732.4317 (100percent, (MH2)2+, C77H116N12O16 requires 732.4311). |
35% | With N-ethyl-N,N-diisopropylamine; In acetonitrile; N,N-dimethyl-d6-formamide; at 20℃; for 3h; | Compound E-13-2 (80 mg, 0.058 mmol, 1.0 eq.) was dissolved in a mixture of acetonitrile (1.5 mL) and DMF (0.4 mL). DIEA (50 jiL, 0.289 mmol, 5 eq.) was added, followed by 2,5 -dioxopyrrolidin- 1 -yl 6-(2,5 -dioxo -2 ,5 -dihydro- 1 H-pyrrol- 1 -yl)hexanoate (36mg, 0.116 mmol, 2 eq.). The mixture was stirred for 3 hours at room temperature. After controlling the reaction by LC-MS, the solvent was evaporated under reduced pressure and the residue purified by preparative HPLC (Waters 600E, SunFire Prep C18 OBD column, 5 jim, 19 x 100 nm; Eluting phase: water / MeCN buffered with 0.1 percent TFA; Gradient of 5 percent to 100 percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractionswere combined and lyophilised to furnish compound E-13 as a white solid (32 mg, 35 percent).mlz (Q-TOF MS ESI-) 1461.8336 (100 percent, (M-H), C77H113N12016 requires 1461.8403). m/z (Q-TOF MS ESI+) 1463.8565 (2 percent, MH, C77H115N12016 requires 1463.8549), 732.4317 (100 percent, (MH2)2, C77H1 16N12016 requires 732.4311). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
70% | With N-ethyl-N,N-diisopropylamine; In acetonitrile; for 6h; | Compound F-61-5 (28.3 mg, 0.020 mmol, 1 eq.) was dissolved in acetonitrile (0.5 mL), followed by 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoate (9 mg, 0.029 muiotaetaomicron, 1.4 eq.) and DIEA (25 mu, 0.143 mmol, 7 eq.). The mixture was stirred for 4.5 hours, after which time HPLC analysis showed the presence of starting material but complete consumption of the succinimide. Supplementary 2,5- dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate was therefore added (3 mg, 0.01 muiotaetaomicron, 0.5 eq.) and the reaction stirred for 1.5 hours. HPLC analysis showed complete consumption of the starting material. The solvent was evaporated to dryness and the residue triturated twice with a mixture of EtOAc/Et20 (80/20) to yield compound F-61 as an off-white solid (19.4 mg, 70 percent). m/z (Q-TOF MS ESI+) 1361.7725 (2 percent, MNa+, C70Hio6Ni2NaOi2S requires 1361.7666), 670.3961 (100 percent, (MH2)2+, C7oHi08Ni2Oi2S requires 670.3960). |
70% | With N-ethyl-N,N-diisopropylamine; In acetonitrile; for 6h; | Compound F-61-5 (28.3 mg, 0.020 mmol, 1 eq.) was dissolved in acetonitrile (0.5 mL), followed by 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (9 mg, 0.029 mumol, 1.4 eq.) and DIEA (25 muL, 0.143 mmol, 7 eq.). The mixture was stirred for 4.5 hours, after which time HPLC analysis showed the presence of starting material but complete consumption of the succinimide. Supplementary 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate was therefore added (3 mg, 0.01 mumol, 0.5 eq.) and the reaction stirred for 1.5 hours. HPLC analysis showed complete consumption of the starting material. The solvent was evaporated to dryness and the residue triturated twice with a mixture of EtOAc/Et2O (80/20) to yield compound F-61 as an off-white solid (19.4 mg, 70percent). (0513) m/z (Q-TOF MS ESI+) 1361.7725 (2percent, MNa+, C70H106N12NaO12S requires 1361.7666), 670.3961 (100percent, (MH2)2+, C70H108N12O12S requires 670.3960). |
19.4 mg | With N-ethyl-N,N-diisopropylamine; In acetonitrile; for 4.5h; | Compound F-61: Compound F-61-5 (28.3 mg, 0.020 mmol, 1 eq.) was dissolved in acetonitrile (0.5 mL), followed by 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l- yl)hexanoate (9 mg, 0.029 muiotaetaomicron, 1.4 eq.) and DIEA (25 mu, 0.143 mmol, 7 eq.). The mixture was stirred for 4.5 hours, after which time HPLC analysis showed the presence of starting material but complete consumption of the succinimide. Supplementary 2,5- dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate was therefore added (3 mg, 0.01 muiotaetaomicron, 0.5 eq.) and the reaction stirred for 1.5 hours. HPLC analysis showed complete consumption of the starting material. The solvent was evaporated to dryness and the residue triturated twice with a mixture of EtOAc/Et20 (80/20) to yield compound F-61 as an off-white solid (19.4 mg, 70 percent). m/z (Q-TOF MS ESI+) 1361.7725 (2 percent, MNa+, C70Hio6Ni2NaOi2S requires 1361.7666), 670.3961 (100 percent, (MH2)2+, C7oHi08Ni2Oi2S requires 670.3960). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
36% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; acetonitrile; at 20℃; for 4.5h; | Compound F-63: Amine F-63-2 (100 mg, 0.082 mmol, 1 eq.) was dissolved in a mixture acetonitrile (2 mL) and DMF (0.5 mL), and 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)hexanoate (45 mg, 0.147 mmol, 1.8 eq.) and DIEA (71 mu,, 0.409 mmol, 5 eq.) were added. After stirring at room temperature for 4.5 hours, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC (Waters 600E, SunFire Prep C 18 OBD column, 5 muiotaeta, 19 x 100 mm; Eluting phase: water / MeCN buffered with 0.1 percent TFA; Gradient of 5 percent to 100 percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combined and lyophilised to furnish compound F-63 as a white solid after (42 mg, 36 percent). |
36% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; acetonitrile; at 20℃; for 4.5h; | Amine F-63-2 (100 mg, 0.082 mmol, 1 eq.) was dissolved in a mixture of acetonitrile (2 mL) and DMF (0.5 mL), and 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (45 mg, 0.147 mmol, 1.8 eq.) and DIEA (71 muL, 0.409 mmol, 5 eq.) were added. After stirring at room temperature for 4.5 hours, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC (Waters 600E, SunFire Prep C18 OBD column, 5 mum, 19×100 mm; Eluting phase: water/MeCN buffered with 0.1percent TFA; Gradient of 5percent to 100percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combined and lyophilised to furnish compound F-63 as a white solid after (42 mg, 36percent). (0529) m/z (Q-TOF MS ESI+) 1300.7901 (2percent, MH+, C68H106N11O14 requires 1300.7915), 650.8990 (100percent, (MH2)2+, C68H107N11O14 requires 650.8994). |
36 mg | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; acetonitrile; | Amine F-63-2 (100 mg, 0.082 mmol, 1 eq.) was dissolved in a mixture of acetonitrile (2 mL) and DMF (0.5 mL), and 2,5-dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5- dihydro-lH-pyrrol-l-yl)hexanoate (45 mg, 0.147 mmol, 1.8 eq.) and DIEA (71 mu,, 0.409 mmol, 5 eq.) were added. After stirring at room temperature for 4.5 hours, the solvent was evaporated under reduced pressure. The crude product was purified by preparative HPLC (Waters 600E, SunFire Prep C 18 OBD column, 5 muiotaeta, 19 x 100 mm; Eluting phase: water / MeCN buffered with 0.1 percent TFA; Gradient of 5 percent to 100 percent MeCN in 15 minutes; Waters 2487 UV Detector at 220 nm). The selected fractions were combined and lyophilised to furnish compound F-63 as a white solid after (42 mg, 36 percent). m/z (Q-TOF MS ESI+) 1300.7901 (2 percent, MH+, CesHioe nOn requires 1300.7915), 650.8990 (100 percent, (MH2)2+, CesHiov nOn requires 650.8994). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 0.25h; | To Example 2.35.8 (200 mg) in N,N-dimethylformamide (5 mL) at 0 C was added 2,5- dioxopyrrolidin-l -yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (105 mg) and N,N- diisopropylethylamine (0.12 mL). The mixture was stirred at 0 C for 15 minutes, warmed to room temperature and purified by reverse-phase HPLC on a Gilson system using a lOOg CI 8 column, eluting with 30-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. NMR (500 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 2H) 9.07 (s, 1H) 8.18 (s, 1H) 8.03 (d, 1H) 7.87 (t, 1H) 7.79 (d, 1H) 7.61 (d, 1H) 7.41-7.53 (m, 3H) 7.36 (q, 2H) 7.28 (s, 1H) 7.03- 7.09 (m, 1H) 6.96-7.03 (m, 3H) 6.94 (d, 1H) 4.95 (s, 4H) 4.82 (t, 1H) 3.88 (t, 3H) 3.80 (d, 2H) 3.01 (t, 2H) 2.86 (d, 3H) 2.54 (t, 2H) 2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d, 2H) 0.90-1.28 (m, 12H) 0.82 (d, 6H). MS (ESI) m/e 1365.3 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 0.25h; | To Example 2.32.25 (200 mg) in N,N-dimethylformamide (5 mL) at 0 C. was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (105 mg) and N,N-diisopropylethylamine (0.12 mL).The mixture was stirred at 0 C. for 15 minutes, warmed to room temperature and purified by reverse-phaseHPLC on a Gilson system using a 100 g C18 column, eluting with 30-80% acetonitrile in water containing0.1% v/v trifluoroacetic acid, to provide the title compound. 1H NMR (500 MHz, dimethyl sulfoxide-d6) deltappm 12.85 (s, 2H) 9.07 (s, 1H) 8.18 (s, 1H) 8.03 (d, 1H) 7.87 (t, 1H) 7.79 (d, 1H) 7.61 (d, 1H) 7.41-7.53 (m,3H) 7.36 (q, 2H) 7.28 (s, 1H) 7.03-7.09 (m, 1H) 6.96-7.03 (m, 3H) 6.94 (d, 1H) 4.95 (s, 4H) 4.82 (t, 1H) 3.88(t, 3H) 3.80 (d, 2H) 3.01 (t, 2H) 2.86 (d, 3H) 2.54 (t, 2H) 2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d,2H) 0.90-1.28 (m, 12H) 0.82 (d, 6H). MS (ESI) m/e 1365.3 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 0.25h; | [0001053] To Example 2.99.8 (200 mg) in N,N-dimethylformamide (5 mL) at 0 C was added 2,5- dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (105 mg) and N,N- diisopropylethylamine (0.12 mL). The mixture was stirred at 0 C for 15 minutes, warmed to room temperature and purified by reverse-phase HPLC on a Gilson system using a lOOg CI 8 column, eluting with 30-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. NMR (500 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 2H) 9.07 (s, 1H) 8.18 (s, 1H) 8.03 (d, 1H) 7.87 (t, 1H) 7.79 (d, 1H) 7.61 (d, 1H) 7.41-7.53 (m, 3H) 7.36 (q, 2H) 7.28 (s, 1H) 7.03- 7.09 (m, 1H) 6.96-7.03 (m, 3H) 6.94 (d, 1H) 4.95 (s, 4H) 4.82 (t, 1H) 3.88 (t, 3H) 3.80 (d, 2H) 3.01 (t, 2H) 2.86 (d, 3H) 2.54 (t, 2H) 2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d, 2H) 0.90-1.28 (m, 12H) 0.82 (d, 6H). MS (ESI) m/e 1365.3 (M+H)+. |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 0.25h; | To Example 2.32.25 (200 mg) in N,N-dimethylformamide (5 mL) at 0 C was added 2,5- dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (105 mg) and N,N- diisopropylethylamine (0.12 mL). The mixture was stirred at 0 C for 15 minutes, warmed to room temperature and purified by reverse-phase HPLC on a Gilson system using a lOOg CI 8 column, eluting with 30-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. NMR (500 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 2H) 9.07 (s, IH) 8.18 (s, IH) 8.03 (d, IH) 7.87 (t, IH) 7.79 (d, IH) 7.61 (d, IH) 7.41-7.53 (m, 3H) 7.36 (q, 2H) 7.28 (s, IH) 7.03- 7.09 (m, IH) 6.96-7.03 (m, 3H) 6.94 (d, IH) 4.95 (s, 4H) 4.82 (t, IH) 3.88 (t, 3H) 3.80 (d, 2H) 3.01 (t, 2H) 2.86 (d, 3H) 2.54 (t, 2H) 2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d, 2H) 0.90-1.28 (m, 12H) 0.82 (d, 6H). MS (ESI) m/e 1365.3 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 0.25h; | To Example 2.32.25 (200 mg) in N,N-dimethylformamide (5 mL) at 0 oc was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-IH-pyrrol-1-yl)hexanoate (105 mg) and N,Ndiisopropylethylamine(0.12 mL). The mixture was stirred at 0 oc for 15 minutes, warmed to roomtemperature and purified by reverse-phase HPLC on a Gilson system using a lOOg CIS column,20 eluting with 30-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the titlecompound. 1H NMR (500 MHz, dimethyl sulfoxide-d6) 8 ppm 12.85 (s, 2H) 9.07 (s, IH) 8.18 (s, IH)8.03 (d, IH) 7.87 (t, IH) 7.79 (d, IH) 7.61 (d, IH) 7.41-7.53 (m, 3H) 7.36 (q, 2H) 7.28 (s, IH) 7.03-7.09 (m, IH) 6.96-7.03 (m, 3H) 6.94 (d, IH) 4.95 (s, 4H) 4.82 (t, IH) 3.88 (t, 3H) 3.80 (d, 2H) 3.01(t, 2H) 2.86 (d, 3H) 2.54 (t, 2H) 2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d, 2H) 0.90-1.2825 (m, 12H) 0.82 (d, 6H). MS (ESI) m/e 1365.3 (M+Ht. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 0.25h; | To Example 2.99.8 (200 mg) in N,N-dimethylformamide (5 mL) at 0 C was added 2,5- dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (105 mg) and N,N- diisopropylethylamine (0.12 mL). The mixture was stirred at 0 C for 15 minutes, warmed to room temperature and purified by reverse-phase HPLC on a Gilson system using a 100g C18 column, eluting with 30-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. 1H NMR (500 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 2H) 9.07 (s, 1H) 8.18 (s, 1H) 8.03 (d, 1H) 7.87 (t, 1H) 7.79 (d, 1H) 7.61 (d, 1H) 7.41-7.53 (m, 3H) 7.36 (q, 2H) 7.28 (s, 1H) 7.03- 7.09 (m, 1H) 6.96-7.03 (m, 3H) 6.94 (d, 1H) 4.95 (s, 4H) 4.82 (t, 1H) 3.88 (t, 3H) 3.80 (d, 2H) 3.01 (t, 2H) 2.86 (d, 3H) 2.54 (t, 2H) 2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d, 2H) 0.90-1.28 (m, 12H) 0.82 (d, 6H). MS (ESI) m/e 1365.3 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 0.25h; | To Example 2.35.8 (200 mg) in N,N-dimethylformaniide (5 niL) at 0 C was added 2,5- dioxopyrrolidill- l-yl 6-(2,5-dioxo-2,5 -dihydro- 1 H-pyrrol- 1 -yl)hexanoate (105 mg) and N,N5 diisopropylethylarnine (0.12 mL). The mixture was stirred at 0 C for 15 minutes, warmed to roomtemperature and purified by reverse-phase HPLC on a Gilsoll system using a lOOg C18 column, eluting with 30-80% acetollitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. 1H NMR (500 MHz, dimethyl sulfoxide-d6) oe ppm 12.85 (s, 2H) 9.07 (s, 1H) 8.18 (s, 1H) 8.03 (d, 1H) 7.87 (t, 1H) 7.79 (d, 1H) 7.61 (d, 1H) 7.41-7.53 (m, 3H) 7.36 (q, 2H) 7.28 (s, 1H) 7.03-7.09 (m, 1H) 6.96-7.03 (m, 3H) 6.94 (d, 1H) 4.95 (s, 4H) 4.82 (t, 1H) 3.88 (t, 3H) 3.80 (d, 2H) 3.01 (t,2H) 2.86 (d, 3H) 2.54 (t, 2H) 2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d, 2H) 0.90-1.28 (m,12H) 0.82 (d, 6H). MS (ESI) nile 1365.3 (M+H). | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 0.25h; | To Example 2.99.8 (200 mg) in N,N-dimethylformamide (5 mL) at 0 C. was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (105 mg) and N,N-diisopropylethylamine (0.12 mL). The mixture was stirred at 0 C. for 15 minutes, warmed to room temperature and purified by reverse-phase HPLC on a Gilson system using a 100 g C18 column, eluting with 30-80% acetonitrile in water containing 0.1% v/v trifluoroacetic acid, to provide the title compound. 1H NMR (500 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 2H) 9.07 (s, 1H) 8.18 (s, 1H) 8.03 (d, 1H) 7.87 (t, 1H) 7.79 (d, 1H) 7.61 (d, 1H) 7.41-7.53 (m, 3H) 7.36 (q, 2H) 7.28 (s, 1H) 7.03-7.09 (m, 1H) 6.96-7.03 (m, 3H) 6.94 (d, 1H) 4.95 (s, 4H) 4.82 (t, 1H) 3.88 (t, 3H) 3.80 (d, 2H) 3.01 (t, 2H) 2.86 (d, 3H) 2.54 (t, 2H) 2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d, 2H) 0.90-1.28 (mn, 12H) 0.82 (d, 6H). MS (ESI) m/e 1365.3 (M+H)+. | |
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0℃; for 0.25h; | To Example 2.35.8 (200 mg) in N,N-dimethylformamide (5 mL) at 0° C. was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (105 mg) and N,N-diisopropylethylamine (0.12 mL). The mixture was stirred at 0° C. for 15 minutes, warmed to room temperature and purified by reverse-phase HPLC on a Gilson system using a 100 g C18 column, eluting with 30-80percent acetonitrile in water containing 0.1percent v/v trifluoroacetic acid, to provide the title compound. 1H NMR (500 MHz, dimethyl sulfoxide-d6) delta ppm 12.85 (s, 2H) 9.07 (s, 1H) 8.18 (s, 1H) 8.03 (d, 1H) 7.87 (t, 1H) 7.79 (d, 1H) 7.61 (d, 1H) 7.41-7.53 (m, 3H) 7.36 (q, 2H) 7.28 (s, 1H) 7.03-7.09 (m, 1H) 6.96-7.03 (m, 3H) 6.94 (d, 1H) 4.95 (s, 4H) 4.82 (t, 1H) 3.88 (t, 3H) 3.80 (d, 2H) 3.01 (t, 2H) 2.86 (d, 3H) 2.54 (t, 2H) 2.08 (s, 3H) 2.03 (t, 2H) 1.40-1.53 (m, 4H) 1.34 (d, 2H) 0.90-1.28 (m, 12H) 0.82 (d, 6H). MS (ESI) m/e 1365.3 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
73.6% | With triethylamine; In N,N-dimethyl-formamide; at 20℃; for 2h;Inert atmosphere; | (0102) Ac-Lys-Gly-Asp-Glu-Val-Asp-PABC-doxorubicin (SEQ ID NO: 32) (20 mg, 0.013 mmol) and N-(epsilon-maleimidocaproyloxy)succinimide ester (EMCS; 8.26 mg, 26.8 mol, 2 eq; Pierce, Rockford, Ill.) are dissolved in anhydrous DMF (1.5 ml; Sigma-Aldrich, St. Louis, Mo.) under inert gas. Then Et3N (4.64 mul, 2.5 eq; Sigma-Aldrich) is added to the reaction mixture and stirred at room temperature for 2 hours. The final product is purified with semi-preparative HPLC (Shimadzu, Kyoto, Japan) using an ODS-A 5 mum reverse phase semi-preparative column (150 mm×20 mm) in a gradient system (Water and CH3CN with 0.05percent TFA as an additive, CH3CN 20-50percent over 50 min, 8 ml/min) to obtain Ac-Lys(EMC)-Gly-Asp-Glu-Val-Asp-PABC-doxorubicin (SEQ ID NO: 39) (red amorphous solid, 16.7 mg, 73.6percent). The peaks are monitored at 280 nm. The purity of the final products is confirmed by analytical HPLC (Agilent 1300 series, Agilent Technologies, Santa Clara, Calif.) using ODS-A 5 mum analytical column (150 mm×3 mm; YMC) in a gradient system (Water and CH3CN with 0.1percent TFA as an additive, CH3CN 5-95percent/5-30 min, 1 ml/min). The peaks are monitored under a UV detector (214 nm) and fluorescent detector (excitation 470 nm, emission 580 nm). The purity of the final compound is determined to be >95percent. ESI-MS (m/z): 1593.3 [M+Na]+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
600 mg | Stage #1: C42H55N5O8 With piperidine In N,N-dimethyl-formamide at 20℃; for 0.5h; Stage #2: Boc-Val-ONSu With triethylamine In N,N-dimethyl-formamide for 1h; Stage #3: 6-maleimidohexanoic acid N-hydroxylsuccinimide ester Further stages; | 9 Compound 68a. The mixture of compound 67 from the above reaction was dissolved in 10 mL of DMF (with 5% piperidine) at RT. After 30 min, the mixture was evaporated and dried with a high vacuum pump overnight. The residue was mixed with compound 48 (1.5 g, 3.45 mmol) in 5 mL DMF. Et3N was used to adjust the pH of the solution to 12. After 1 h the reaction mixture was taken up in EtOAc, which was washed with 10% citric acid, sat. aq. NaHCO3 solution and brine. The organic phase was dried, filtered and evaporated to dryness. The residue was deprotected with 5% piperidine in DMF in the same way as the deprotection of compound 67. The amine obtained in this step and compound 21a (1 g, 3.27 mmol) were mixed in 10 mL of DMF. Et3N was used to adjust the pH of the solution to 12 at RT. After allowing the reaction to proceed overnight, the mixture was taken up in EtOAc, which was washed with 10% citric acid, sat. aq. NaHCO3 and brine. The organic phase was dried, filtered and evaporated to give a residue. The residue was passed through a regular silica column to give a mixture of compounds 68a and 68b (1 g, 35% yield from compound 66, [M+1]+, calculated 828.5. found 828.5). After separation on a preparative HPLC column, 600 mg of the major epimer was obtained (structure tentatively assigned as compound 68a, with compound 68b assigned as the minor one). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
84% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 5h; | To 10.0 mg (8.83 pmol) 6?-O-(-6-aminohexyl)-a-amanitin (HDP 30.0134, synthesized as disclosed in EP 2621536), dissolved in 400 p1 dry DMF were added subsequently 663 p1 of 20 mM 6-(maleimido)hexanoic acid N-hydroxysuccinimide ester (EMCS) in DMF, and 17.7 p1 of 1M DIPEA in DMF. After 5 h at room temperature 100 p1 water was added to the reaction mixture and the volatiles were evaporated. The crude product purified by RP18 HPLC with a water-methanol gradient and the pure fractions were lyophilized from t-butanol/water: 9.02mg (84percent) HDP 30.1948 as colorless powder.MS (ESl) found: 1210.99; found: 1233.32;calc.: 1211.54 [MH] (C55H79N12017S)calc.: 1233.52 [M+Na] (C55H78N12NaO17S) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1: N,N-dimethyl-formamide / 6 h / 20 °C 2: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 5 h / 20 °C | ||
Multi-step reaction with 2 steps 1: N,N-dimethyl-formamide / 20 °C 2: N-ethyl-N,N-diisopropylamine / N,N-dimethyl-formamide / 5 h / 20 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
47% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 18h;Inert atmosphere; | Step B: 6-Maleimidyl-caproamidyl-L-valine-L-citrulline (4) (0752) To a 25 mL round bottom flask equipped with a magnetic stirrer and 132 nitrogen inlet was charged 137 L-valine-L-citrulline (2, 0.4 g; 1.3 mmol), 139 6-maleimido-caproic acid succinate ester (3, 0.6 g; 2.0 mmol) in 140 DMF (3 mL) followed by 141 DIEA (0.4 mL; 2.0 mmol). The solution was stirred at ambient temperature for 18 hours, diluted with water (2 mL) and loaded onto a C18 column eluting with 5-95percent MeCN in 135 water (containing 0.05percent 142 HOAc). The fractions containing product were combined, frozen and lyophilized to afford the 22 title compound (0.3 g, 47percent yield) as a white solid. MS (ESI, pos. & neg.): calc'd for C21H33N5O7, 467.2; found 468.4 (M+H), 466.4 (M?H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
63% | With sodium hydrogencarbonate; In water; acetonitrile; at 20℃; for 20h;Sealed tube; Inert atmosphere; | Step E: Maytan-3-O-carbamoyl-N-phenyl-2-chloro-4-(amino-Cit-Val-Cap-Mal) (19) (0769) The product of the preceding step (18, 14 mg, 0.013 mmol) and 191 6-maleimidyl-caproic acid succinate ester (3, 20 mg, 0.065 mmol) were dissolved in 146 MeCN (2.0 mL) and 135 water (0.5 mL), treated with one drop of saturated aqueous NaHCO3, the flask sealed with a rubber septum, purged with argon, and the reaction stirred at ambient temperature. After 20 h the reaction was acidified with a few drops of 10percent 152 aqueous acetic acid, dissolved in MeCN (ca. 2 mL), and purified on a C18 Aq RediSep Gold column via ISCO (gradient elution: 30-90percent MeCN in water, 0.05percent acetic acid in both). The fractions containing product were combined, partially concentrated in vacuo, frozen on dry ice, and lyophilized for 4 d giving the 192 title compound as a white solid (10 mg, 63percent). MS (ESI, pos.): calc'd for C56H73Cl2N9O15, 1181.5; found 1183.1 (M+H), 1204.1 (M+Na). 1H NMR (500 MHz, DMSO-d6) delta: 10.16 (s, 1H), 8.14 (d, J=7.3 Hz, 1H), 7.93 (dd, J=15.2, 2.3 Hz, 1H), 7.81 (d, J=9.1 Hz, 2H), 7.71-7.59 (m, 1H), 7.46 (dd, J=8.8, 2.4 Hz, 1H), 7.21 (d, J=1.7 Hz, 1H), 7.07 (s, 1H), 6.99 (d, J=11.8 Hz, 2H), 6.90 (d, J=8.2 Hz, 1H), 6.57 (dd, J=15.1, 11.2 Hz, 1H), 6.45 (d, J=10.6 Hz, 1H), 5.98 (d, J=6.2 Hz, 1H), 5.91-5.84 (m, 1H), 5.47-5.34 (m, 3H), 4.45 (dd, J=12.0, 2.6 Hz, 1H), 4.40-4.29 (m, 1H), 4.19 (dd, J=8.6, 6.8 Hz, 1H), 4.13 (t, J=11.1 Hz, 1H), 3.94 (s, 3H), 3.57 (d, J=12.4 Hz, 1H), 3.48 (d, J=9.1 Hz, 1H), 3.21 (d, J=3.7 Hz, 4H), 3.07 (s, 3H), 3.05-2.98 (m, 1H), 2.98-2.88 (m, 1H), 2.78 (d, J=9.8 Hz, 1H), 2.43 (t, J=13.1 Hz, 1H), 2.24-2.05 (m, 2H), 2.01 (d, J=13.8 Hz, 1H), 1.98-1.91 (m, 1H), 1.69 (d, J=7.9 Hz, 1H), 1.65 (s, 3H), 1.61 (dd, J=9.2, 4.9 Hz, 1H), 1.54-1.40 (m, 8H), 1.40-1.30 (m, 1H), 1.19 (td, J=15.8, 14.8, 8.2 Hz, 3H), 1.11 (d, J=6.4 Hz, 3H), 0.88 (d, J=6.7 Hz, 1H), 0.84 (dd, J=17.0, 6.8 Hz, 6H), 0.81 (s, 2H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
63% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 40h;Inert atmosphere; | 2, 5-dioxopyrrolidin- 1 -yl 6-(2, 5-dioxo-2, 5-dihydro- 1H-pyrrol- 1 -yl)hexanoate (62.1 mg. 0.201mmol) was dissolved in DMF (1 mL) and tert-butyl (2-(2-(2aminoethoxy)ethoxy)ethyl)carbamate (50 mg, 0,201 mmol) in DMF (1 mL) was addeddropwise under argon, followed by the addition of DIPEA (0,053 ml, 0,302 mmol). The reaction was stirred for 40 h at room temperature and subsequently concentrated on silica. Purification by flash chromatography (1-4percent MeOH in DCM) afforded the product (10) (56 mg, 63 percent) as a colorless oil.?H-NIVIR (500 IVIHz, CDC13) 6.66 (s, 2 H), 6.05 (br s, 1 H), 5.02 (br s, 1 H), 3.70-3.36 (m, 12 H), 3.36-3.20 (m, 2 H), 2.15 (t, J= 7.4 Hz, 2 H), 1.66-1.54 (m, 4 H), 1.42 (s, 9 H), 1.3 1-1.25(m,2H).?3C-NIVIR (500 IVIFIz, CDC13) 172.7, 170.8, 155.9, 134.0, 79.3, 70.2, 70.1, 70.1, 69.9, 40.2, 39.1, 37.6, 36.3, 28.3, 28.2, 26.3, 25.0.HRMS (ESIj C21H35N307 [M+Na] calc 464.2367, found 464.2363 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
77% | With 4-methyl-morpholine; In N,N-dimethyl-formamide; at 20℃; | 64.8 mg (0.357 mmol) of tert-butyl beta-alaninate hydrochloride (1:1) and 100 mg (0.324 mmol) of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione were dissolved in 4 ml of dimethylformamide, and 65.6 mg (0.649 mmol) of N-methylmorpholine were added. The reaction mixture was stirred at RT overnight. 0.048 ml (0.838 mmol) of acetic acid was added and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250*30; 10mu, flow rate: 50 ml/min, MeCN/water/0.1percent TFA). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 84.5 mg (77percent, purity 100percent) of tert-butyl N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alaninate. LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=339 (M+H)+. |
77%, purity 100% | With 4-methyl-morpholine; acetic acid; trifluoroacetic acid; | 64.8 mg (0.357 mmol) of tert-butyl beta-alaninate hydrochloride (1:1) and 100 mg (0.324 mmol) of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione were dissolved in 4 ml of dimethylformamide, and 65.6 mg (0.649 mmol) of N-methylmorpholine were added. The reaction mixture was stirred at RT overnight. 0.048 ml (0.838 mmol) of acetic acid were added and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 250*30; 10mu, flow rate: 50 ml/min, MeCN/water/0.1percent TFA). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 84.5 mg (77percent, purity 100percent) of tert-butyl N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-beta-alaninate. LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=339 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
73% | With 4-methyl-morpholine; In N,N-dimethyl-formamide; at 20℃; | 200 mg (0.594 mmol) of <strong>[811442-84-9]tert-butyl (14-amino-3,6,9,12-tetraoxatetradec-1-yl)carbamate</strong> and 202 mg (0.654 mmol) of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione were dissolved in 4.0 ml of dimethylformamide, and 0.130 ml (1.2 mmol) of N-methylmorpholine were added. The reaction mixture was stirred at RT overnight. 0.085 ml (1.5 mmol) of acetic acid was added and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 125×30; 10mu, flow rate: 50 ml/min, MeCN/water/0.1% TFA). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 275 mg (73% of theory) of tert-butyl [21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-16-oxo-3,6,9,12-tetraoxa-15-azahenicos-1-yl]carbamate. LC-MS (Method 1): Rt=0.81 min; MS (ESIpos): m/z=530 (M+H)+. |
With 4-methyl-morpholine; acetic acid; trifluoroacetic acid; | 200 mg (0.594 mmol) of <strong>[811442-84-9]tert-butyl (14-amino-3,6,9,12-tetraoxatetradec-1-yl)carbamate</strong> and 202 mg (0.654 mmol) of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione were dissolved in 4.0 ml of dimethylformamide, and 0.130 ml (1.2 mmol) of N-methylmorpholine were added. The reaction mixture was stirred at RT overnight. 0.085 ml (1.5 mmol) of acetic acid were added and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 125*30; 10mu, flow rate: 50 ml/min, MeCN/water/0.1% TFA). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 275 mg (73% of theory) of tert-butyl [21-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-16-oxo-3,6,9,12-tetraoxa-15-azahenicos-1-yl]carbamate. LC-MS (Method 1): Rt=0.81 min; MS (ESIpos): m/z=530 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
70% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 3h; | S-{11-[(R)-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl](cyclohexyl) methyl]-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl}-N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-cysteine 34.8 mg (0.27 mmol) of N,N-diisopropylethylamine were added to a mixture of 100 mg (0.13 mmol) of S-{11-[(R)-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl](cyclohexyl)methyl]-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl}-L-cysteine (1:1) (Intermediate C84) and 41.5 mg (0.13 mmol) of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione in 4.0 ml of DMF, and the reaction mixture was stirred at RT for 3 h. Without workup, the mixture was purified by preparative HPLC. This gave 88 mg (70percent of theory) of the title compound. LC-MS (Method 12): Rt=2.71 min; MS (ESIpos): m/z=936 (M+H)+. |
With N-ethyl-N,N-diisopropylamine; | S-{11-[(R)-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl](cyclohexyl)methyl]-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl}-N-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]-L-cysteine 34.8 mg (0.27 mmol) of N,N-diisopropylethylamine were added to a mixture of 100 mg (0.13 mmol) of S-{11-[(R)-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl](cyclohexyl)methyl]-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl}-L-cysteine (1:1) (Intermediate C84) and 41.5 mg (0.13 mmol) of 1-{6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl}-1H-pyrrole-2,5-dione in 4.0 ml of DMF, and the reaction mixture was stirred at RT for 3 h. Without work-up, the mixture was purified by preparative HPLC. This gave 88 mg (70percent of theory) of the title compound. LC-MS (Method 12): Rt=2.71 min; MS (ESIpos): m/z=936 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
72% | Compound 1 was prepared according to the methods described in International Publication No. WO 2007/011968, which is incorporated herein by reference for such disclosure and compounds 15 was prepared as previously described by Burke, P.J. et al. "Design, synthesis, and biological evaluation of antibody-drug conjugates comprised of potent camptothecin analogues" Bioconj. Chem. (2009) 20: 1242-1250 which is incorporated herein by reference for such disclosure. Stepwise deprotection of compound 15 by a method of the present invention, which reduces the amount of impurities from beta- elimination of the C-4 acetate within the glucuronic acid residue as compared to global deprotection by aqueous base, is achieved in the manner as described for Example 1. Deprotection step (1) of Scheme 3 converts the lactone of compound 15 in variable amount to the methyl ester of its ring opened form during the transesterification for removing the acetate protecting groups. However, Li OH hydrolysis in deprotection step (2) will convert that ester and hydrolyze any remaining lactone to the carboxylate ring opened form, which provides the Drug linker intermediate compound 16 as the predominate reaction product. On recovery of the product, closure to reform the lactone ring occurs. Further elaboration of compound 17 is by condensing that fully deprotected compound with 2,5- dioxopyrrolidin-l-yl 6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (mc-OSu, 8) in DMF in the presence of di-isopropyldiethylamine (DIPEA) as follows: To a solution of 17 (23.0 mg, 27.6 muiotaetaomicron) dissolved in 1.5 mL anhydrous DMF was added maleimidocaproyl NHS ester (8, 12.8 mg, 41.4 muiotaetaomicron), followed by DIPEA (14.4\L, 82.8 muiotaetaomicron). The reaction was stirred at room temperature under nitrogen for 4 h, at which time LC-MS revealed conversion to product. The crude reaction was diluted in DMSO and purified by preparative HPLC (Method A) to give 10 (20.5 mg, 72percent). NMR (DMF-dv) delta (ppm) 0.99 (m, 6H), 1.26 (p, J = 8.4 Hz, 2H), 1.57 (m, 6H), 1.81 (p, J = 7.2 Hz, 2H), 2.01 (m, 2H), 2.16 (t, J = 7.2 Hz, 2H), 2.68 (t, J = 6.8 Hz, 2H), 3.21 (t, J = 8.0 Hz, 2H), 3.57 (m, 10H), 4.10 (d, J = 9.6 Hz, 2H), 5.03 (m, 1H), 5.23 (s, 2H), 5.36 (s, 2H), 5.52 (d, J = 2.4 Hz, 2H), 6.52 (bs, 1H), 7.01 (s, 2H), 7.19 (dd, J = 8.4, 2.0 Hz, 1H), 7.27 (d, J = 8.4 Hz, 1H), 7.43 (s, 1H), 7.94 (t, J = 5.6 Hz, 1H), 8.09 (dd, J = 9.2, 2.0 Hz, 1H), 8.18 (d, J = 9.2 Hz, 1H), 8.48 (s, 1H), 8.57 (s, 1H), 9.36 (s, 1H), 10.37 (s, 1H). UV max 267 nm and 385 nm. Analytical HPLC: gradient A, tR = 11.93 min; gradient B, tR = 5.76 min. LC-MS: m/z (ES+) found 1025.40 (M+H)+, m/z (ES ) found 1023.29 (M-H)\ |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
60% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 1.5h; | 2,5 -Dioxopyrrolidin- 1 -yl 6-(2, 5-dioxo-2, 5-dihydro- 1H-pyrrol- 1 -yl)hexanoate (1.6 mg, 5.2 iimol) was dissolved in DMF (100 pL). The resulting solution was added to compound 2 (3.6 mg, 4.7 iimol) followed by addition of DIPEA (2.5 1iL, 14iimol). The reaction was mixed vigorously, then incubated at room temperature for90 minutes. The reaction was diluted with DMSO and purified by preparative HPLC to provide the title compound (2.7 mg, 2.8 iimol, 60percent) referred to as mc-GlucQFK866. LCMS: tR = 0.96 mm; mlz = 953.5 [Mr. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
81% | 88.6 mg (0.357 mmol) of N-Boc-2,2?-(ethylenedioxy)diethylamine and 100 mg (0.324 mmol) of N-succinimidyl 6-maleimidohexanoate were dissolved in 4.0 ml of dimethylformamide, and 0.071 ml (0.650 mmol) of N-methylmorpholine were added. The reaction mixture was stirred at RT overnight. 0.048 ml (0.838 mmol) of acetic acid was added and the reaction mixture was purified directly by preparative RP-HPLC (column: Reprosil 125×30; 10mu, flow rate: 75 ml/min, MeCN/water/0.1percent TFA). The solvents were evaporated under reduced pressure and the residue was dried under high vacuum. This gave 127 mg (81percent of theory) of tert-butyl {2-[2-(2-[6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl]amino}ethoxy)ethoxy]ethyl}carbamate. LC-MS (Method 1): Rt=0.78 min; MS (ESIpos): m/z=442 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
86% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 2h;Inert atmosphere; | HOP 30.1702 (23.49 mg, 17.9 pmol) was dissolved in dry DME (400 p1). 6-(maleimido)hexanoic acid N-hydroxysuccinimide ester (ECMS) (11 .07 mg, 35.9 pmol) dissolved in DME (562 p1), and DIPEA (12.21 p1, 7.16 pmol) were added. After 2 h at room temperature under argon, mixture is dripped into 40 ml of cold MTBE and centrifuged at 000. The precipitate was collected and washed with 40 ml of MTBE and centrifuged again. The crude product was dried and purified by RP18-HPLC [A= 305 nm; gradient: 0-5 mm 5percent B; 20-25 mm 100percent B; 27-35 mm 5percent B; A= water with0.05percent TEA; B= methanol with 0.05percent TEA]. The pure fractions were lyophilized to yield 21 .03 mg (86percent) of title product 6?-[(6-Maleidohexanamido)-Val-Ala-PAB]-a- amanitin as white powder.[00266] MS (ESI-?-): mlz found: 1145.7 calc.: 1144.99 [M+Na]. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
0.78 g | With hydrazine hydrate In tetrahydrofuran at 20℃; for 4h; | 1; 2 6-maleimidohexanoic acid N-hydroxysuccinimide ester compound 2 (1.7 g) was dissolved in THF, and an excess of hydrazine hydrate solution (1.70 g, 28.86 mmol, content 85%) was added dropwise, magnetic force The reaction was carried out for 4 h at room temperature with stirring, and the reaction of the starting material was monitored by TLC. It was extracted three times with ethyl acetate and concentrated to give 6-maleimidohexanoylhydrazide compound 3 (0.78 g). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
72% | With N-ethyl-N,N-diisopropylamine In dimethyl sulfoxide at 20℃; for 4h; | 14 Step 14. Synthesis of Compound 142 To a slurry of L-valine (Compound 141, 0.50 g, 4.27 mmol, 1.00 equiv) in DMSO (10 mL) was added 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxopyrrol-1-yl)hexanoate (1.32 g, 4.28 mmol, 1.00 equiv) and DIEA (1103 mg, 8.54 mmol, 2.00 equiv).The resulting mixture was stirred at room temperature for 4 h. LCMS indicated the reaction was completed. The reaction mixture was purified by reverse flash chromatography with the following conditions: column, C18 silica gel; mobile phase, ACN in water (0.1%FA), 0% to 60% gradient in 30 min; detector, UV 220 nm to give (2S)-2-[6-(2,5-dioxopyrrol-1-yl)hexanamido]-3-methylbutanoic acid (Compound 142, 1.2 g, 72%) as a brown solid. LCMS (ES, m/z): 311 [M+H]+ |
46% | With triethylamine In N,N-dimethyl-formamide at 20℃; for 8h; | 6; 1.2q (S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanoic acid (5i) To a solution of L-valine (0.12 g, 1.0 mmol) in anhydrous DMF (2 mL) were added compound 5a (0.31 g, 1.0 mmol) and triethylamine (0.51 g, 5.0 mmol) at RT. The reaction mixture was stirred at RT for 8 hours until compound 5a was totally consumed, which was monitored by LCMS. The reaction mixture was filtered through filtering membrane and the filtrate was directly purified by prep-HPLC (method A) to give the title compound 5i(0.14 g, yield 46%) as colorless oil. ESI m/z: 311 (M + H)+. |
46% | With triethylamine In N,N-dimethyl-formamide at 20℃; for 8h; | 2q (S)-2-(6-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanoic acid (5i) To a solution of L-valine (0.12 g, 1.0 mmol) in anhydrous DMF (2 mL) were added compound 5a (0.31 g, 1.0 mmol) and triethylamine (0.51 g, 5.0 mmol) at RT. The reaction mixture was stirred at RT for 8 hours until compound 5a was totally consumed, which was monitored by LCMS. The reaction mixture was filtered through filtering membrane and the filtrate was directly purified by prep-HPLC (method A) to give the title compound 5i(0.14 g, yield 46%) as colorless oil. ESI m/z: 311 (M + H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
68% | Chemical Formula: C76H1 01N7O35Exact Mass: 1671 ,63 To the solution of 32-azido-3,6,9,l2,l5,l8,2l,24,27,30-decaoxadotriacontan-l-amine (1.5 eq., 11.82 mg, 22.45 pmol) in dry DMF (0.2 mL) was added a solution of 2,5-dioxopyrrolidin- 1 -yl6-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)hexanoate (1.5 eq., 6.92 mg, 22.45 pmol) in dry DMF (0.2 mL). The mixture was stirred at 2lC for 1 hour and then S8b (1 eq., 12 mg, 14.97 pmol) in DMSO (0.2 mL) was added. A THPTA-Cu(II) complex was prepared by mixing tris(3- hydroxypropyltriazolylmethyl)amine (1 eq., 0.1 M in DMF, 150 pL, 14.97 pmol) and copper sulfate pentahydrate (1 eq., 0.1 M in H20, 150 pL, 14.97 pmol) and incubating at 2lC for 1 minute. The THPTA-Cu(II) complex (1 eq., 0.05 M in DMF/FLO 50/50, 300 pL, 14.97 pmol) was added to the reaction mixture followed by sodium ascorbate (2 eq., 0.5 M in H20, 60 pL, 29.94 miho). The resulting mixture was incubated at 2lC for 15 min and purified by preparative HPLC (method 1) to yield S8 (15.5 mg, 10.18 pmol, 68 %) as a red solid.HPLC (Method 2) RT: 3.65 minMS (Method 3) m/z : 836.9 [M+2H]2+/2 |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
67% | With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 0.0833333h; | To the crude H-PEG8-Val-Lys(Boc)-Gly-7-MAD-MDCPT (from procedure above for the preparation of compound Ex_l0-la) 0.0367M in DMF (0.50 mL, 0.018 mmol) was added MCOSu (17.0 mg, 0.0550 mmol, TCI America: S0428, CAS: 55750-63-5), followed by DIPEA (9.6 pL, 0.055 mmol). The reaction was stirred at room temperature for 5 minutes at which point complete conversion was observed. The reaction was quenched AcOH (0.02 mL), and purified by prep-HPLC 10 x 250 mm Max-RP 5-60-95% MeCN in H20 0.1% Formic Acid. Fractions containing the desired product were concentrated in vacuo to afford MC-PEG8-Val-Lys(Boc)- Gly-7-MAD-MDCPT as a yellow solid (17.4 mg, 18.3 pmol, 67%). Rt = 1.63 min General Method UPLC. MS (m/z) [M + H]+ calc for C69HIOON9023 1422.69, found 1422.27. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
44% | With sodium hydrogencarbonate; In water; acetonitrile; at 4 - 20℃; for 16h;pH 8.0; | A solution of R18b (700 mg, 1.47 mmol, 1.0 eq) in water (8 mL) was diluted with 2 mL of aqueous NaHCO3 solution at 4 C and the mixture (pH = 8.0) was treated with commercially available 2,5-dioxopyrrolidin-1-yl 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoate (408 mg, 0.9 eq) in 10 mL of acetonitrile. The suspension was stirred at room temperature for 16 h until the reaction was complete. The crude product was concentrated under reduced pressure and diluted with DMSO (5 mL). The crude product was purified by an ISCO 150g C18 column (eluents: 10 - 95% MeCN in water, 0.05 % in AcOH). Pure fractions were combined and lyophilized to afford 368 mg (44%) of compound R18 572.30; found 573.6 (M+H), (2M+H), 1145.9.1H NMR (500 MHz; DMSO-d6): ' 9.89 (s, 1H), 8.05 (d, J = 7.33 Hz, 1H), 7.81 (d, J = 8.79 Hz, 1H), 7.52 - 7.57 (m, J = 8.79 Hz, 2H), 7.21 - 7.26 (m, J = 8.79 Hz, 2H), 6.99 - 7.02 (m, 2H), 5.98 (br. s., 1H), 5.40 (s, 2H), 5.10 (t, J = 5.62 Hz, 1H), 4.35 - 4.45 (m, 3H), 4.18 (dd, J = 6.84, 8.30 Hz, 1H), 3.26 - 3.33 (m, 2H), 2.91 - 3.06 (m, 2H), 2.08 - 2.22 (m, 2H), 1.93 - 2.01 (m, 1H), 1.66 - 1.74 (m, 1H), 1.59 (dd, J = 4.40, 9.28 Hz, 1H), 1.43 - 1.55 (m, 5H), 1.32 - 1.43 (m, 1H), 1.19 (quin, J = 7.57 Hz, 2H), 0.84 (d, J = 8.30 Hz, 3H), 0.80 - 0.89 (m, 3H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
81% | With potassium carbonate In water; N,N-dimethyl-formamide at 25℃; for 1h; | 2 Specific example 2 The specific synthesis process and synthesis conditions are as follows: add T3 (1g, 1eq) to DMF (15ml), dissolve potassium carbonate (0.31g, 1.5eq) in water (15ml), and then mix and dissolve with T3 DMF suspension. Then dissolve 6-(maleimido)hexanoic acid succinimidyl ester (0.46g, 1eq) in DMF (5ml), add dropwise to the weakly alkaline solution of T3, and react at 25°C for 1 hour.Adjust the pH to acidic with dilute hydrochloric acid, extract 3 times with EA, wash 2 times with saturated aqueous NaCl,Purification by column, the eluent is DCM: methanol=50:1, and purification by column, obtain 1.09g of light yellow solid product, the yield is 81%, and the HPLC purity is 98.12%. |
Tags: 55750-63-5 synthesis path| 55750-63-5 SDS| 55750-63-5 COA| 55750-63-5 purity| 55750-63-5 application| 55750-63-5 NMR| 55750-63-5 COA| 55750-63-5 structure
Precautionary Statements-General | |
Code | Phrase |
P101 | If medical advice is needed,have product container or label at hand. |
P102 | Keep out of reach of children. |
P103 | Read label before use |
Prevention | |
Code | Phrase |
P201 | Obtain special instructions before use. |
P202 | Do not handle until all safety precautions have been read and understood. |
P210 | Keep away from heat/sparks/open flames/hot surfaces. - No smoking. |
P211 | Do not spray on an open flame or other ignition source. |
P220 | Keep/Store away from clothing/combustible materials. |
P221 | Take any precaution to avoid mixing with combustibles |
P222 | Do not allow contact with air. |
P223 | Keep away from any possible contact with water, because of violent reaction and possible flash fire. |
P230 | Keep wetted |
P231 | Handle under inert gas. |
P232 | Protect from moisture. |
P233 | Keep container tightly closed. |
P234 | Keep only in original container. |
P235 | Keep cool |
P240 | Ground/bond container and receiving equipment. |
P241 | Use explosion-proof electrical/ventilating/lighting/equipment. |
P242 | Use only non-sparking tools. |
P243 | Take precautionary measures against static discharge. |
P244 | Keep reduction valves free from grease and oil. |
P250 | Do not subject to grinding/shock/friction. |
P251 | Pressurized container: Do not pierce or burn, even after use. |
P260 | Do not breathe dust/fume/gas/mist/vapours/spray. |
P261 | Avoid breathing dust/fume/gas/mist/vapours/spray. |
P262 | Do not get in eyes, on skin, or on clothing. |
P263 | Avoid contact during pregnancy/while nursing. |
P264 | Wash hands thoroughly after handling. |
P265 | Wash skin thouroughly after handling. |
P270 | Do not eat, drink or smoke when using this product. |
P271 | Use only outdoors or in a well-ventilated area. |
P272 | Contaminated work clothing should not be allowed out of the workplace. |
P273 | Avoid release to the environment. |
P280 | Wear protective gloves/protective clothing/eye protection/face protection. |
P281 | Use personal protective equipment as required. |
P282 | Wear cold insulating gloves/face shield/eye protection. |
P283 | Wear fire/flame resistant/retardant clothing. |
P284 | Wear respiratory protection. |
P285 | In case of inadequate ventilation wear respiratory protection. |
P231 + P232 | Handle under inert gas. Protect from moisture. |
P235 + P410 | Keep cool. Protect from sunlight. |
Response | |
Code | Phrase |
P301 | IF SWALLOWED: |
P304 | IF INHALED: |
P305 | IF IN EYES: |
P306 | IF ON CLOTHING: |
P307 | IF exposed: |
P308 | IF exposed or concerned: |
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.
Home
* Country/Region
* Quantity Required :
* Cat. No.:
* CAS No :
* Product Name :
* Additional Information :