* 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.
Stage #1: at 70℃; Inert atmosphere Stage #2: With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In acetonitrile at 0 - 70℃;
EXAMPLE 1N-Maleimidopropionic acid NHS ester[0083] Maleic anhydride, β-alanine (1 mol eq.), and acetonitrile (ACN, 25 volumes) were charged to a vessel. The slurry was stirred under nitrogen and heated to 70 0C. The temperature was maintained at 70 0C for a further 5 to 8 hours. After cooling to 5 0C, NHS (1 mol eq.) was charged, followed by Λ/-(3-dimethylaminopropyl)-Λf'-ethylcarbodiimide hydrochloride (EDCI, 1 mol eq.). The temperature was maintained at 0 - 5 0C for 1 hour before adding a further charge of EDCI (1 mol eq.). The mixture was heated to 70 0C and maintained at this temperature for 7 hours. The reaction mixture was cooled to 200C and concentrated under vacuum at a maximum temperature of 45°C until the rate of solvent distillation was negligible. Dichloromethane (DCM, 40 volumes) was added to the residue, which was stirred until a solution was formed. This solution was washed with aqueous ammonium chloride (12 percent w/w, 25 wt eq.) then with aqueous sodium chloride (24 percent w/w, 25 wt eq.). The organic solution was dried by stirring with magnesium sulphate (1 wt eq.) at ambient temperature for 1 to 2 hours. The inorganics were filtered off under vacuum and the filter cake was washed with DCM (4 volumes). The filtrates were concentrated under vacuum, whilst gradually replacing DCM with isopropylacetate (IPAC). The residual slurry was cooled and stirred at ambient temperature for 1 hour before filtering under vacuum. The product filter cake was washed with IPAC (4 volumes) and then dried to constant mass under vacuum with slow rotation at 40 0C to yield the desired compound as an off white solid. The typical yield is 60 to 80 percent of the maximum theoretical yield.[0084] The product was characerised by NMR as follows: 1H NMR (400MHz, d6-DMSO): δ 2.80 (4H, br s), δ 3.05 (2H, t, J ~ 7Hz), δ 3.75 (2H, t J ~ 7Hz), δ 7.05 (2H, s).
60%
Stage #1: at 20℃; for 2.5 h; Stage #2: With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 0 - 20℃;
To a solution of maleic anhydride (2.00 g, 20.4 mmol) in 24 mL of DMF was added β-alanine (1.82 g, 20.4 mmol)and the mixture was stirred for 2.5 h at room temperature in which the solid was gradually dissolved to afford clear solution. The reaction was cooled in an ice bath and N-hydroxysuccinimide (HOSu) (2.88 g, 25 mmol) andN,N'-dicyclohexylcarbodiimide (8.24 g. 40.0 mmol) were successively added to the solution. After 30 min, the reaction was warmed to room temperature and stirred for 14 h. The reaction mixture was filtered through a cottonplug and the residue was washed with DCM and water. The filtrates were gathered and the organic layer was washed with satd NaHCO3 (three times) and brine, dried over Na2SO4 and concentrated to afford 1 (3.20 g, 60percent) as a white solid. 1H NMR (400 MHz, d6-DMSO): 2.78 (s, 4H), 3.03 (t, J = 6.9 Hz, 2H), 3.73 (t, J = 6.9 Hz, 2H),7.02 (s, 2H); 13C NMR (100 MHz, d6-DMSO): 25.4, 29.0, 32.7, 134.6, 166.7, 169.9, 170.5.
25%
With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 0 - 20℃; for 1 h;
On a solution of N-hydroxysuccinimide (12.6 mmol) and dicyclohexylcarbodiimide (20.3 mmol) at 0° C., corresponding acid (6 mmol) was added and allowed to react for 4 hours at room temperature. In the case of compound VIa, a solution of maleic anhydride (10 mmol) and β-alanine was added in N,N-dimethylformamide, which has been previously made react for 1 hour. After 4 hours, mixture was evaporated at reduced pressure and the crude was dissolved in dichloromethane and washed with water. Organic extracts were dried with anhydrous magnesium sulfate, filtered and evaporated to dryness. Resulting residue was recrystallized to give desired compound. [0112] Using this methodology, and corresponding acid, the following compounds were prepared: [0113] 3-succinimidyl maleimidopropionate (VIa, 25percent yield). 1H NMR (CDCl3) δ ppm: 2.82 (4H, s), 3.02 (2H, t, J=7.07 Hz), 3.94 (2H, t, J=7.07 Hz), 6.74 (2H, s); 13C NMR (CDCl3) δ ppm: 25.5 (2C, s), 29.7 (1C, s), 32.9 (1C, s), 134.3 (2C, s), 166.0 (1C, s), 168.7 (2C, s), 170.1 (2C, s).
Reference:
[1] Patent: WO2011/23680, 2011, A2, . Location in patent: Page/Page column 13-14
[2] Angewandte Chemie - International Edition, 2015, vol. 54, # 35, p. 10198 - 10201[3] Angew. Chem., 2015, vol. 127, p. 10336 - 10339
[4] Journal of the American Chemical Society, 2013, vol. 135, # 29, p. 10582 - 10585
[5] Journal of the American Chemical Society, 2004, vol. 126, # 3, p. 734 - 735
[6] Bioorganic and Medicinal Chemistry, 2011, vol. 19, # 5, p. 1721 - 1728
[7] Macromolecular Bioscience, 2012, vol. 12, # 9, p. 1209 - 1219
[8] Patent: US2013/273581, 2013, A1, . Location in patent: Paragraph 0111-0113
[9] Synthesis, 1991, # 10, p. 819 - 821
[10] Patent: WO2011/9047, 2011, A2, . Location in patent: Page/Page column 13
[11] Patent: US2013/157375, 2013, A1, . Location in patent: Paragraph 0103; 0104
[12] Chemical Communications, 2014, vol. 50, # 26, p. 3473 - 3475
[13] Patent: WO2014/100762, 2014, A1, . Location in patent: Paragraph 248
[14] Chemical Communications, 2015, vol. 51, # 53, p. 10758 - 10761
[15] Angewandte Chemie - International Edition, 2017, vol. 56, # 9, p. 2356 - 2360[16] Angew. Chem., 2017, vol. 129, # 9, p. 2396 - 2400,5
2
[ 6066-82-6 ]
[ 7423-55-4 ]
[ 55750-62-4 ]
Reference:
[1] Analytical Chemistry, 2016, vol. 88, # 22, p. 10837 - 10841
[2] Angewandte Chemie - International Edition, 2016, vol. 55, # 40, p. 12243 - 12247[3] Angew. Chem., 2016, vol. 128, # 40, p. 12431 - 12435,5
[4] International Journal of Molecular Sciences, 2017, vol. 18, # 9,
[5] Tetrahedron Letters, 1998, vol. 39, # 11, p. 1321 - 1324
[6] Organic Letters, 2004, vol. 6, # 20, p. 3561 - 3564
[7] Journal of Medicinal Chemistry, 2006, vol. 49, # 21, p. 6400 - 6407
[8] Chemical Communications, 2011, vol. 47, # 25, p. 7068 - 7070
[9] Chemistry - A European Journal, 2011, vol. 17, # 46, p. 13059 - 13067
[10] Patent: US2014/56810, 2014, A1, . Location in patent: Paragraph 0181
[11] Organic and Biomolecular Chemistry, 2014, vol. 12, # 34, p. 6624 - 6633
[12] Journal of Controlled Release, 2015, vol. 220, p. 660 - 670
[13] Patent: WO2015/196167, 2015, A1, . Location in patent: Paragraph 0269; 0277
3
[ 108-31-6 ]
[ 6066-82-6 ]
[ 56-41-7 ]
[ 55750-62-4 ]
Yield
Reaction Conditions
Operation in experiment
64%
Stage #1: at 60℃; for 3 h; Stage #2: With dicyclohexyl-carbodiimide In N,N-dimethyl-formamide at 0 - 22℃; for 18.5 h;
To a solution of maleic anhydride (8; 86.0 g, 877 mmol) in DMF(1.03 L) was added β-alanine (9; 78.14 g, 877 mmol). The resultingsuspension was heated to 60 °C and, after 1 h, a solution was obtained.After 2 h, the mixture was cooled to 0–5 °C and N-hydroxysuccinimide(29; 126.17 g, 1.09 mol) was added followed by DCC(361.9 g, 1.75 mol) over 30 min in several portions while the internaltemperature was held below 22 °C. The thick, white slurry wasthen stirred at 20 °C for 18 h. The slurry was filtered and the solid(dicyclohexyl urea) was washed with H2O (1 L) and CH2Cl2 (1 L)and discarded. To the filtrates was added CH2Cl2 (1 L) and the phaseswere separated. The aqueous layer was extracted with CH2Cl2(2 × 300 mL) and the combined organic extracts were washed withH2O [500 mL; a small amount of brine (50 mL) was added to facilitatephase separation], sat. aq NaHCO3 (2 × 300 mL), and dried(MgSO4). The solvent was removed to give an oily, light tan solidthat was slurried in EtOH (520 mL) for 2 h at 20 °C. The solid wasfiltered, washed with EtOH (2 × 75 mL) and dried under vacuum at30 °C for 72 h to give 186.35 g (64percent) of NHS ester 30 as a whitesolid;32 HPLC purity: 96.6percent (areapercent); mp 169–171 °C.IR (ATR cell): 3088, 2954, 1825, 1783, 1705, 1583, 1446, 1433,1382, 1325, 1298, 1250, 1211, 1149, 1070, 1049, 998, 956, 901,836, 813, 786, 767, 756, 696, 651, 634, 596, 561, 544, 528 cm–1.1H NMR (400 MHz, DMSO-d6): δ = 2.80 (s, 4 H), 3.05 (t, J = 6.90Hz, 2 H), 3.75 (t, J = 6.78 Hz, 2 H), 7.05 (s, 2 H).13C NMR (100 MHz, DMSO-d6): δ = 25.38, 29.00, 32.67, 134.63,166.72, 169.93, 170.52.HRMS (ESI): m/z [M + H]+ calcd for C11H11N2O6: 267.06116;found: 267.06147.
With N-ethyl-N,N-diisopropylamine In tetrahydrofuran; N,N-dimethyl-formamide at 20℃; for 2.5 h;
10152] 3-Maleimidopropionic acid (1.0 g, 5.9 mmol) was dissolved in tetrahydroffiran (20 ml). 2-Succinimido- 1,1,3,3- tetramethyluronium tetrafluoroborate (TSTU, 2.14 g, 7.1 mmol) and ethyldiisopropylamine (1.24 ml, 7.1 mmol) were added subsequently. N,N-Dimethylformamide (5 ml) was added. The reaction mixture was stirred at room temperature, while it was turning sluggish. The mixture was stirred for 2 mi N,N-Dimethylformamide (5 ml) was added. The mixture was stirred for 2.5 h at room temperature. It was diluted with dichloromethane (150 ml) and was washed subsequently with a 10percent aqueous solution of sodium hydrogensulphate (150 ml), a saturated aqueous solution of sodium hydrogencarbonate (150 ml) and water (150 ml). It was dried over magnesium sulphate. The solvent was removed in vacuo. The crude product was recrystallized from ethyl acetate to give 1.20 g of 3-(2,5-dioxo-2,5-dihydropyrrol-1-yl)propionic acid2,5-dioxopyrrolidiny-1 -yl ester. MS: mlz=289, required for [M+Na]: 28910154] ‘H-NMR (CDC13) ö 2.82 (m, 4H); 3.02 (t, 2H); 3.94 (t, 2H), 6.73 (s, 2H).
EXAMPLE 1N-Maleimidopropionic acid NHS ester[0083] Maleic anhydride, beta-alanine (1 mol eq.), and acetonitrile (ACN, 25 volumes) were charged to a vessel. The slurry was stirred under nitrogen and heated to 70 0C. The temperature was maintained at 70 0C for a further 5 to 8 hours. After cooling to 5 0C, NHS (1 mol eq.) was charged, followed by Lambda/-(3-dimethylaminopropyl)-Lambdaf'-ethylcarbodiimide hydrochloride (EDCI, 1 mol eq.). The temperature was maintained at 0 - 5 0C for 1 hour before adding a further charge of EDCI (1 mol eq.). The mixture was heated to 70 0C and maintained at this temperature for 7 hours. The reaction mixture was cooled to 200C and concentrated under vacuum at a maximum temperature of 45C until the rate of solvent distillation was negligible. Dichloromethane (DCM, 40 volumes) was added to the residue, which was stirred until a solution was formed. This solution was washed with aqueous ammonium chloride (12 % w/w, 25 wt eq.) then with aqueous sodium chloride (24 % w/w, 25 wt eq.). The organic solution was dried by stirring with magnesium sulphate (1 wt eq.) at ambient temperature for 1 to 2 hours. The inorganics were filtered off under vacuum and the filter cake was washed with DCM (4 volumes). The filtrates were concentrated under vacuum, whilst gradually replacing DCM with isopropylacetate (IPAC). The residual slurry was cooled and stirred at ambient temperature for 1 hour before filtering under vacuum. The product filter cake was washed with IPAC (4 volumes) and then dried to constant mass under vacuum with slow rotation at 40 0C to yield the desired compound as an off white solid. The typical yield is 60 to 80 % of the maximum theoretical yield.[0084] The product was characerised by NMR as follows: 1H NMR (400MHz, d6-DMSO): delta 2.80 (4H, br s), delta 3.05 (2H, t, J ~ 7Hz), delta 3.75 (2H, t J ~ 7Hz), delta 7.05 (2H, s).
60%
To a solution of maleic anhydride (2.00 g, 20.4 mmol) in 24 mL of DMF was added beta-alanine (1.82 g, 20.4 mmol)and the mixture was stirred for 2.5 h at room temperature in which the solid was gradually dissolved to afford clear solution. The reaction was cooled in an ice bath and N-hydroxysuccinimide (HOSu) (2.88 g, 25 mmol) andN,N'-dicyclohexylcarbodiimide (8.24 g. 40.0 mmol) were successively added to the solution. After 30 min, the reaction was warmed to room temperature and stirred for 14 h. The reaction mixture was filtered through a cottonplug and the residue was washed with DCM and water. The filtrates were gathered and the organic layer was washed with satd NaHCO3 (three times) and brine, dried over Na2SO4 and concentrated to afford 1 (3.20 g, 60%) as a white solid. 1H NMR (400 MHz, d6-DMSO): 2.78 (s, 4H), 3.03 (t, J = 6.9 Hz, 2H), 3.73 (t, J = 6.9 Hz, 2H),7.02 (s, 2H); 13C NMR (100 MHz, d6-DMSO): 25.4, 29.0, 32.7, 134.6, 166.7, 169.9, 170.5.
60%
General procedure: Thesynthesisofcompoundswascarriedoutaccordingtothemethoddescribedin[77,78].A portionof3mmolofanaminoacidisaddedtoasolutionofmaleicanhydride(0.3g,3mmol)in4mL ofDMF,stirredfor2hatroomtemperature.Aftercompletedissolution,atatemperatureof0C,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.
25%
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 0 - 20℃; for 1h;
On a solution of N-hydroxysuccinimide (12.6 mmol) and dicyclohexylcarbodiimide (20.3 mmol) at 0 C., corresponding acid (6 mmol) was added and allowed to react for 4 hours at room temperature. In the case of compound VIa, a solution of maleic anhydride (10 mmol) and beta-alanine was added in N,N-dimethylformamide, which has been previously made react for 1 hour. After 4 hours, mixture was evaporated at reduced pressure and the crude was dissolved in dichloromethane and washed with water. Organic extracts were dried with anhydrous magnesium sulfate, filtered and evaporated to dryness. Resulting residue was recrystallized to give desired compound. [0112] Using this methodology, and corresponding acid, the following compounds were prepared: [0113] 3-succinimidyl maleimidopropionate (VIa, 25% yield). 1H NMR (CDCl3) delta ppm: 2.82 (4H, s), 3.02 (2H, t, J=7.07 Hz), 3.94 (2H, t, J=7.07 Hz), 6.74 (2H, s); 13C NMR (CDCl3) delta ppm: 25.5 (2C, s), 29.7 (1C, s), 32.9 (1C, s), 134.3 (2C, s), 166.0 (1C, s), 168.7 (2C, s), 170.1 (2C, s).
beta-Alanine (1.00 eq.) was added to a solution of maleic anhydride (1.00 eq.) in dry dimethylformamide. The suspension was stirred for 1.0 h after the amino acid was dissolved. The resulting solution was cooled to O0C. N-hydroxysuccinimide (1.25 eq.) was added followed by dicyclohexylcarbodiimide (2.00 eq.). After 5.0 min, the ice bath was removed and the solution was stirred for additional 18 h. The reaction mixture was extracted with dichloromethane and washed with water. The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and recrystallized in ether.
[0104] beta-Alanine (0.91 g, 10 mmol) was added to a solution of maleic anhydride (1.0 g, 10 mmol) in 12 ml DMF and stirred for 2 h at room temperature. When the solid dissolved, an ice bath was used to decrease the temperature to 0 C. N-Hydroxysuccinimide (NHS; 1.15 g, 10 mmol) was added into the solution followed by DCC (2.3 g, 12 mmol). After 10 min the ice bath was removed and the reaction was kept at room temperature overnight, which resulted in a white precipitate. The solution was filtered and the precipitate was washed with water (100 ml) and dichloromethane (100 ml). The filtrate was collected and the organic layer was washed with 3×20 ml 5% NaHCO3 and brine. The organic layer was dried with Na2SO4 and dichloromethane was removed under reduced pressure to obtain a white solid (55%) which can be used for next step reaction without further purification.
Referring to the scheme of synthesis of Compound O, beta-alanine was treated with maleic anhydride in DMF and the acid so obtained was reacted with N-hydroxysuccinimide (NHS) under DCC coupling to give NHS-ester.
2-(2-{2-[2-(2-acetylamino-6-amino-hexanoylamino)-acetylamino]-6-amino-hexanoylamino}-acetylamino)-6-amino-hexanoic acid [5-amino-1-(carbamoylmethyl-carbamoyl)-pentylcarbamoyl]-methyl}-amide[ No CAS ]
3-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]eth oxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoic acid[ No CAS ]
1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28,31,34,37,40-dodecaoxa-4-azatritetracontan-43-oic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
76%
With triethylamine; In dichloromethane; for 1h;
To Intermediate 1 (1 05mg, 0.16mmol) in DCM (6ml) were added Et3N (65mg, 0.64mmol) and maleimido propionic acid NHS ester (64mg, 0.24mmol). After 1 hour the solvent was removed, the residue dissolved in 0.1M HCI (25ml), washed with Et20 (4x25ml) and extracted into DCM (10x30ml). The combined DCM fractions were reduced to 30ml, extracted into water (10x50ml) and the water removed. The residue was dissolved in DCM, dried over MgS04 and the solvent removed to give the product 94mg, 76% as a colourless oil/gum. m/z (LCMS ES+, 70V) 769.0 (MH+). No.H (CDC13) 6.64 (2H, s), 6.59 (1H, br), 3.76 (2H, t, J7.lHz), 3.69 (2H, t, J6.3Hz), 3.57 (44H, br), 3.47 (2H, t, J4.9Hz), 3.34 (2H, br), 2.53 (2H, t, J 6.3Hz), 2.45 (2H, t, J7.lHz).
(S,S)-[(2-[2-(bis-carboxymethyl-amino)-propyl]-carboxymethyl-amino}-1-{4-[3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionylamino]-benzyl}-ethyl)-carboxymethyl-amino]-acetic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With N-ethyl-N,N-diisopropylamine; In DMF (N,N-dimethyl-formamide); at 0 - 20℃; for 4h;
800 mg (3 mmol) of 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (Aldrich) at 0 C. was added to a stirred solution of 1.02 g (2 mmol) of 3 and 774 mg (6 mmol) of diisopropylethylamine in 20 ml DMF. The reaction solution was stirred for 4 hours at room temperature. The solution was added drop by drop to 120 ml of vigorously stirred diethyl ether. The suspension was stirred for 30 minutes and filtered. The residue was dried at the oil pump. A portion of the residue was purified by semi-preparative RP-HPLC.
(S)-2-{2-[2-(2-tert-butoxycarbonylamino-acetylamino)-acetylamino]-acetylamino}-6-[3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionylamino]-hexanoic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
1 g of Pd/C (10%) was added to a solution of 4.18 g (6.5 mmol) of 16b in 40 ml of isopropanol. The atmosphere over the stirred solution was saturated with hydrogen. The reaction solution was stirred for 90 minutes, filtered and concentrated by evaporation. [0195] The residue was mixed at 0 C. with 20 ml of DMF, 1.5 g (12 mmol) of diisopropylethylamine and 2.4 mg (9 mmol) of 3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (Aldrich). The reaction solution was stirred for 4 hours at room temperature. The solution was added to HCl solution (pH 4) and extracted several times with ethyl acetate. The combined organic phases were washed with aqueous, saturated sodium chloride solution, dried with sodium sulfate and concentrated by evaporation. The residue was purified by column chromatography (SiO2 ethyl acetate?methanol:ethyl acetate 20:80). The desired product 16c was produced in a yield of 67% (2.5 g; 4.4 mmol).
With N-ethyl-N,N-diisopropylamine; In DMF (N,N-dimethyl-formamide); for 4h;
The purpose of this Example was to prepare a peptide-based hair conditioner by covalently linking the hair-binding, cysteine-attached D21 peptide, given as SEQ ID NO:64, with octylamine using the heterobifunctional cross-linking agent <strong>[55750-62-4]3-maleimidopropionic acid N-hydroxysuccinimide ester</strong>. Octylamine, obtained from Aldrich (Milwaukee, Wis.) was diluted by adding 11.6 mg to 0.3 mL of DMF. This diluted solution was added to a stirred solution containing 25 mg of <strong>[55750-62-4]3-maleimidopropionic acid N-hydroxysuccinimide ester</strong> (Aldrich) and 5 mg of diisopropylethylamine (Aldrich) in 0.2 mL of DMF in a 5 mL round bottom flask. The reaction mixture turned turbid immediately and then became clear several minutes later. The solution was stirred for another 4 h. The solution was then dried under high vacuum. The product, octylamine-attached maleimidopropionate, was purified by column chromatography using a Silica gel 60 (EMD Chemicals, formerly EM Science, Gibbstown, N.J.) column and DMF/ether as the eluent. Approximately 12 mg of the above product was placed into a 5 mL round bottom flask and 50 mg of cysteine-attached D21 peptide (obtained from SynPep, Dublin, Calif.), given as SEQ ID NO:64, and 0.5 mL of 0.1 M phosphate buffer at pH 7.2 were added. The cysteine-attached D21 peptide has 3 glycine residues and a cysteine attached to the end of the peptide binding sequence of the hair-binding D21 peptide (SEQ ID NO:46). This mixture was stirred at room temperature for 6 h. The final product, the C8-D21 peptide hair conditioner, was purified by extraction with water/ether.
[2-(2-[3-(2,5-dioxo-2,5-dihydro-pyrrol-1-yl)-propionylamino]-methyl}-benzo[1,3]dioxol-5-yl)-1-methyl-ethyl]-ethyl-carbamic acid tert-butyl ester[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
93%
With triethylamine; In DMF (N,N-dimethyl-formamide); at 20℃; for 18h;
A mixture containing 150 mg (0.44 mmol) of 2E and 130 mg (0.48 mmol) of succinimidyl 3-maleimido propionate in 2 mL of anhydrous DMF containing 100 muL (0.71 mmol) of triethylamine was allowed to stir at room temperature 18 hours and then concentrated under reduced pressure to dryness. The residue was purified by silica gel column chromatography using 15% methanol in chloroform to give 203 mg (0.41 mmol, 93%) of2N as white solid (M+Na, 510).
3-(3-methylaminopropyl)-4-methoxyphenyl-bis(4-methoxyphenyl)methanol trifluoroacetate[ No CAS ]
[ 55750-62-4 ]
3-(3-maleimidopropyl)-4-methoxyphenyl-bis(4-methoxyphenyl)methanol[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
71%
With N-ethyl-N,N-diisopropylamine; In tetrahydrofuran; at 20℃; for 5h;
3-(3-Maleimidopropyl)-4-methoxyphenyl-bis(4-methoxyphenyl)methanol. To a stirred solution of 3-(3-methylaminopropyl)-4-methoxyphenyl-bis(4-methoxyphenyl)methanol trifluoroacetate (267 mg, 0.50 mmol) in dry THF (5 mL) 3-maleimidopropionic acid N-oxysuccinimide ester (136 mg, 0.6 mmol) and DIEA (105 muL, 0.6 mmol) were added in one portion and the mixture was stirred at ambient temperature for 5 h, then evaporated, diluted with EtOAc (100 mL), washed with with 5% NaHCO3 solution (50 mL), water (100 mL), dried over Na2SO4, and evaporated. The residue was chromatographed on silica gel in 5?25% EtOAc in toluene in the presence of 0.5% pyridine. Yield 202 mg (71 %), yellowish foam
(1'R,2R,5S)-5-([2-({1-carboxy-5-[3-(2,5-dioxo-2,5-dihydropyrrol-1-yl)propionylamino]pentyl}carboxymethyl-amino)ethyl]carboxymethyl-amino}methyl)-1-carboxymethyl-pyrrolidine-2-carboxylic acid[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
With N-ethyl-N,N-diisopropylamine; In DMF (N,N-dimethyl-formamide); at 0 - 20℃; for 4h;
Zu einer geruehrten Loesung von 981 mg (2 MMOL) 5f und 774 mg (6 MMOL) Diisopropylethylamin in 20 ml DMF wurden bei 0 C 800 mg (3 MMOL) 3- (2, 5-Dioxo-2,5- DIHYDRO-PYRROL-1-YL)-PROPIONSaeURE 2, 5-dioxo-pyrrolidin-1-yl ester (Aldrich) hinzugefuegt. Die Reaktionsloesung wurde fuer 4 Stunden bei Raumtemperatur geruehrt. Die Loesung wurde tropfenweise auf 120 ml heftig geruehrten Diethylether gegeben. Die Suspension wurde fuer 30 Minuten geruehrt und filtriert. Der Rueckstand wurde an der Oelpumpe getrocknet. Ein Teil des Rueckstands wurde durch semipraeparative RP-HPLC gereinigt. MS-FAB : 642 (M+ +1, 11)
With N-ethyl-N,N-diisopropylamine; In tetrahydrofuran; at 20℃; for 2.16667h;
To a MDA (4) (21.0 mg, 0.097 mmol) in THF (4 mL) solution was added N, N-diisopropyl ethyl amine (85 IlL, 0.49 mmol). The mixture was stirred for half-hour under nitrogen before adding-maleimidopropionic acid-NHS-ester (54) (38.9 mg, 0.146 mmol). The reaction was run under nitrogen at room temperature for one hour and forty minutes. The progress of the reaction was monitored by TLC with ethyl acetate as the developing solvent. [0160] Most of organic solvent was removed by rotary evaporator. Trace amount of solvent was removed over high vacuum for one hour. Crude product (92.4 mg) was purified by flash column chromatography with MeOH/CH2CI2 (2/98) as the eluent to give a white solid (55) (21.5 mg, 66% yield). 1H-NMR (CDCl3, 400 MHz) 8 : 6.66 (m, 5H), 5.93 (s, 2H), 5.45 (d, J= 8 Hz, 1H), 4.16 (m, 1H), 3.81 (t, J=8,2H), 2.67 (m, 2H), 2.47 (t, J=8, 2H), 1.08 (d, J=4. 0 Hz, 3H).
0.5 g of Y-shape branched PEG amine ((MeO-PEG)2-N-CH2CH2NH2) was dissolved in acetonitrile. 20 mg of NHS-3-maleimidopropionate was added to the solution. The solution was stirred overnight at room temperature. The solvent was removed under vacuum. The residue was added to 30 ml isopropyl alcohol. The precipitated was collected and dried under vacuum. Yield: 0.42 g (84%). NMR (DMSO): 3.5 (br m, H in PEG), 3.24 (s, 6H), 3.05 (t, 2H), 2.56 (t, 2H), 6.71 (s, 2H in maleimide).
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide;
N-hydroxy-Succinimidyl-Maleimido-Propionate (SMP, 1 eq dissolved in DMF) is added to doxorubicin chlorhydrate (1 eq) solubilised in dimethylformamide (DMF) in the presence of Disopropylethylamine (DIEA, 2 eq).The thiol function carrier peptide (1.2 eq in DMF) is then added to the reactional mix, and spontaneously combines with doxorubicin maleimidopropionate during an additional 20 minutes incubation.The coupling product is then purified on HPLC in preparation and then freeze dried.Each of the steps, and the final product are checked by analytic HPLC and mass spectrometry.
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; for 1h;
The peptide sequences were synthesized, see FIG. 1, in an automated peptide synthesizer (Advanced ChemTech, 396 multiple well peptide synthesizer) using a standard coupling procedure. 800 mul of a 0.5 M amino acid solution in DMF, 800 mul of a 0.5 M 6-Chloro-1-Hydroxybenzotriazole (HOBt) and O-benzotriazole, N,N,N',N'-tetramethyl-uronium-hexa-fluoro-phosphate (HBTU) solution in DMF and 800 mul of a 1 M diisopropyethylamine (DIPEA) solution were added to a well. The reaction mixture in the reaction block was mixed for 45 minutes at 800 rpm, afterwards the wells were emptied and washed with DMF before a new coupling cycle was started. After the last coupling the resin was washed with dichloromethane (CH2Cl2), tetrahydrofurane (THF) and DMF.The Fmoc (9-fluorenylmethyloxycarbonyl) protection groups were cleaved with 50% piperidine in DMF and subsequently the ivDde ([1-(4,4-dimethyl-2,6-dioxo-cyclohexylidene)3-methyl-butyl]) protection group was cleaved using 2% hydrazine in DMF to leave the free amine groups that are conjugated to maleimide building blocks. 0.6 mmol <strong>[55750-62-4]N-succinimidyl 3-maleimidopropionate</strong> and 0.6 mmol diisopropyl ethyl amine in 2 ml DMF were added to 0.2 mmol of the resin. The reaction mixture was stirred for one hour, afterwards the solvent was removed and the resin washed extensively with DMF. A ninhydrin standard test was employed to indicate unreacted free amine groups. Subsequently, the peptides were cleaved from the resin. During the cleavage reaction in 85% TFA, 5% phenol, 5% water, 5% triisopropansilane the tertiary butyl protection groups were eliminated as well. The cleaved peptides still contain one protection group, S-tBu, which protects the SH-group in cysteine.The peptides P1-P6, see Table 1, were prepared using automated peptide synthesis on an Apex 396 multiple well peptide synthesizer (Advanced ChemTech, Louisville, Ky.). Ten wells of a 40-well block were utilized with 0.1 mmol of resin in each well.Standard Coupling Procedure:0.8 ml of a 0.5 M amino acid solution in DMF, 0.8 ml of a 0.5 M 6-Chloro-1-Hydroxybenzotriazole (HOBt) and O-Benzotriazole,N,N,N',N'-tetramethyl-uronium-hexafluoro-phosphate (HBTU) solution in DMF, and 0.8 ml of a 1 M diisopropyethylamine (DIPEA) solution were added to a well of the reaction block. The reaction block containing the reaction mixtures was mixed at 800 rpm for 45 minutes. Afterwards, the wells were emptied with nitrogen and washed with DMF. The coupling was repeated until the desired peptide sequence was finished and subsequently, the resin was washed with CH2Cl2, THF, and DMF.Standard Fmoc Deprotection:1.8 ml of DMF was transferred to the well followed by 1.2 ml 50% piperidine in DMF. The reaction block was mixed at 800 rpm for 5 minutes then emptied and the wells were washed with DMF. The deprotection was repeated, this time for 20 minutes. The resin was then washed with DMF, CH2Cl2, and DMF.Deprotection of Iv-Dde Groups:2% hydrazine in DMF was added to the reaction vial with the resin. After 10 minutes the hydrazine solution was removed. The deprotection reaction was repeated twice. Afterwards the resin was washed five times with DMF. A ninhydrin standard test was performed to proof the deprotection success.Addition of Maleimide-Group:The reaction solution containing 160 mg (0.6 mmol) N-Succinimidyl 3-Maleimidopropionate and 130 mg (0.6 mmol) diisopropyl ethyl amine in 2 ml DMF was added to the resin. After one hour the solvent was removed and the resin washed extensively with DMF and dichloromethane. A ninhydrin standard test was used to indicate unreacted free amine groups.Cleavage of the Resin:All cleavages carried out using a mixture of 85% trifluoroacetic acid, 5% phenol, 5% triisopropylsilane, and 5% water. After five hours, the resin was removed by filtration, and washed with a small quantity of CH2Cl2. The peptide was then precipitated using cold t-butyl methyl ether (minimum ratio of 1:5 of eluent to ether), and cooled in anice-bath for 30 minutes. Afterwards, it was centrifuged for 5-10 minutes at 3000 rpm. The supernatant was decanted and additional ether added. The product was again cooled and centrifuged and decanted. The peptide was then dried under a flow of argon, lyophilized, and analyzed by HPLC and mass spectra.Purification of the Products:Purification of the peptides was carried out using preparative HPLC-MS and collecting the product according to the mass. Therefore the lyophilized samples were dissolved in 3-4 ml of a 80% water+0.1% TFA/20% acetonitrile+0.1% TFA solution before they were loaded on the column (YMC-pack ODS-AQ 150×20 mm).The fractions containing the product were combined and lyophilized. A HPLC and a mass spectra of the final product indicated the purity.Peptides P6 and P7 were bought on the resin and therefore the handling of these two peptides started with the Fmoc-deprotection step.
With triethylamine; In N,N-dimethyl-formamide; at 50℃; for 5h;
512 mg (1 mmol) of [3-(4-Amino-phenyl)-2-(bis-carboxymethyl-amino)-propyl]-[2-(bis-carboxymethyl-amino)-propyl]-amino}-acetic acid (Macrocyclics Inc. Dallas, TX, U.S.A.) and 707 mg (7 mmol) triethylamine were dissolved in 3 ml dry DMF. 400 mg (1,5 mmol) of 3-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (Aldrich) in 1 ml dry DMF were added drop-wisely. The solution was stirred for 5 h at 50 C. 30 ml of diethylether were added slowly. The reaction mixture was stirred for further 30 min. The precipitate was collected by filtering. The crude product was purified by RP-HPLC (acetonitrile- : water- : trifluoracetic acid / 3 : 96,9 : 0, 1 ? 99,9 : 0 : 0,1). Yield: 61% (405 mg, 0,61 mmol). MS-ESI: 664 = M+ +1.
With triethylamine; In tetrahydrofuran; N,N-dimethyl-formamide; at 20℃; for 18h;
A solution of 200 mg (0.47 mmol) of mycophenolic acid amine (8) in 8 mL of anhydrous DMF is prepared. To this solution 165 DL (1.18 mmol) of triethylamine is added . This solution is added dropwise to a solution of 139 mg (0.52 mmol) of 3-maleimido-propionic acid N- hydroxysuccinimide ester in 8 mL of freshly distilled THF. The reaction mixture is allowed to stir at room temperature 18 h under argon atmosphere and concentrated under reduced pressure. <n="12"/>The crude product is purified by silica gel column chromatography to give the desired maleimido linked mycophenolic acid (14).
Part A To a solution of 6-amino-N-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}hexanamide (28 mg, 0.66 mmol, prepared as generally described in Part A-B of IRM Compound 1) in dichloromethane (0.5 mL) at room temperature was added N-succinimidyl-3-maleimidopropionate (18 mg, 0.68 mmol). The mixture was shaken until the reagent dissolved, allowed to stand for 30 minutes, then concentrated under reduced pressure. The foam was purified by reverse phase HPLC using 0.05% formic acid/acetonitrile in 0.05% formic acid/water as the eluent to yield 11 mg of N-{2-[4-amino-2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}-6-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}hexanamide as the monoformate salt. Off white glassy solid, MS (ESI) m/z 578 (M+H)+. 1H NMR (300 MHz, DMSO-d6) delta 8.34 (d, J=7.5 Hz, 1H), 8.22 (s, 2H), 7.90 (t, J=5.3 Hz, 1H), 7.65 (s, 1H), 7.60 (dd, J=8.4, 1.2 Hz, 1H), 7.42 (m, 1H), 7.22 (m, 1H), 7.00 (s, 2H), 6.67 (br s, 2H), 5.00 (br s, 2H), 4.74 (br, 2H), 3.59 (t, J=7.3 Hz, 2H), 3.51 (q, J=7.0 Hz, 2H), 2.98 (q, J=6.3 Hz, 2H), 2.31 (t, J=7.2 Hz, 2H), 2.04 (t, J=7.4 Hz, 2H), 1.47 (m, 2H), 1.36 (m, 2H), 1.20 (m, 8H), 1.13 (t, J=7.0 Hz, 3H).
N-{2-[2-(Ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}-6-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}hexanamide To a solution of 6-amino-N-{2-[2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}hexanamide (which was prepared as described above in Parts A and B of Control Compound 1, 163 mg, 0.40 mmol) in dichloromethane (4 mL) at room temperature was added N-succinimidyl-3-maleimidopropionate (111 mg, 0.42 mmol). The mixture was shaken until the reagent dissolved and allowed to stand overnight. The mixture was diluted with dichloromethane (25 mL), washed with 2M aqueous ammonia (10 mL), dried over magnesium sulfate and concentrated under reduced pressure to provide 158 mg of N-{2-[2-(ethoxymethyl)-1H-imidazo[4,5-c]quinolin-1-yl]-1,1-dimethylethyl}-6-[3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoyl]amino}hexanamide. Light yellow foam, MS (ESI) m/z 563 (M+H)+. 1H NMR (300 MHz, CDCl3) delta 9.30 (s, 1H), 8.55 (d, J=7.0 Hz, 1H), 8.31 (m, 1H), 7.69 (m, 2H), 6.68 (s, 2H), 5.79 (m, 1H), 5.57 (br s, 1H), 5.20 (m, 2H), 4.92 (m, 2H), 3.83 (t, J=7.1 Hz, 2H), 3.63 (q, J=6.9 Hz, 2H), 3.22 (q, J=6.7 Hz, 2H), 2.52 (t, J=7.2 Hz, 2H), 2.05 (t, J=7.3 Hz, 2H), 1.55 (m, 12H), 1.25 (t, J=7.0 Hz, 3H).
WHP.3: To a solution of WHP.l (1.5 mg, 0.0015 mmol) in 700 muL of a2:1 mixture of MeCN and pH 8.5 buffer (0.1 M NaH2PO4/ Na2HPO4) was added 3- maleimidopropionic acid jV-hydroxysuccinimide ester (2.1 mg, 0.0077 mmol, 5.0 equiv). The solution was stirred for 4 h at room temperature, then acidified with 50 muL of 1.0 M aqueous HCl. The mixture was lyophilized to give a solid material that was purified by reverse phase HPLC (Altima C 18, 10 mum, 1O x 250 mm column, eluting with a gradient flow over 14 min of 20:80 MeCN/10 mM aqueous C3F7CO2H ? 27:73 MeCN/10 mM aqueous C3F7CO2H, 214 nm UV detection). At a flow rate of 6 mL/min, WHP.3 had a retention time of 7.1 min and was isolated following lyophylization as a white powder (0.38 mg, 28%). 1H NMR (D2O, 500 MHz) delta 6.81 (s, 2H), 4.60 (s, IH), 4.31 (dd, IH, J= 11.3, 9.5 Hz), 3.96 (dd, IH, J= 12.0, 5.0 Hz), 3.81-3.76 (m, 2H), 3.75 (t, 2H, J= 6.0 Hz), 3.58-3.54 (m, IH), 3.31-3.20 (m, 2H), 3.14 (t, 2H, J = 10.5Hz), 2.47 (t, 2H, J= 6.5 Hz), 2.41-2.29 (m, 2H) ppm; LRMS (ES+) calcd for C19H27N9O7 493.47 found 494.59 (MH+).
EXAMPLE 2Succinimidyl-6- [beta-maleimidopropionamido] hexanoate (SMPH)[0085] N-Maleimidopropionic acid (NHS ester), 6-aminohexanoic acid (1.04 mol eq.) and ACN (20 volumes) were charged to a vessel. The slurry was stirred and heated under nitrogen to 70 0C. This temperature was maintained for 5 to 8 hours. After cooling to 20 to 25 0C, the mixture was filtered through GF/F paper to remove fine insoluble particles. The filtrates were charged back to the vessel and cooled to 0 to 5 0C. EDCI (1.1 mol eq.) was charged and the reaction mixture was then heated to 21 0C. This temperature was maintained for 10 to 16 hours.[0086] The reaction mixture was discharged and concentrated under vacuum at a maximum temperature of 30 0C until the rate of solvent distillation was negligible. DCM (15 volumes) was added to the residue, which was stirred until a solution is formed.[0087] This solution was washed with aqueous ammonium chloride (12 % w/w, 10 wt eq.) then with aqueous sodium chloride (24 % w/w, 10 wt eq.). It was then dried by stirring with magnesium sulphate (1 wt eq.) at ambient temperature for 1 to 2 hours. The inorganics were filtered off under vacuum and the filter cake was washed with DCM (2 volumes). The filtrates were concentrated under vacuum at a maximum temperature of 30 0C, whilst gradually replacing DCM with IPAC. The residual slurry was cooled and stirred at ambient temperature for 1 hour before filtering under vacuum. The product filter cake was washed with IPAC (2 volumes) and then dried to constant mass under vacuum with slow rotation at 30 0C to yield crude SMPH as a yellow solid. The typical yield is 60 to 80 % of the maximum theoretical yield.[0088] Crude SMPH was added to ACN (3 volumes) and the slurry heated to 60 0C to completely solubilise the solid. t-Butyl methyl ether (TBME, 9 volumes) was slowly added, whilst maintaining temperature. Insoluble material was filtered off and the filtrates cooled to5 0C to crystallise. SMPH was filtered off as an off white solid. Typical recovery was 60 to80 %.[0089] Recrystallised SMPH was stirred with ACN (20 volumes) and silica (2 wt eq.) at 20 to 25 0C for 2 to 4 hours before charging to a bed of silica (4 wt eq.). The bed was pulled dry and more ACN (20 volumes) was charged to the silica bed to wash SMPH through. The filtrates were concentrated under vacuum at a maximum of 30 0C whilst gradually replacing the ACN with TBME. The resulting precipitate was filtered off under vacuum. The white solid was dried to constant mass under vacuum with slow rotation at 30 0C to yield high purity SMPH. Typical recovery was 60 to 80 %.[0090] The end product was characerised by NMR as follows: IH NMR (400MHz, d6-DMSO): delta 1.30 (4H, m), delta 1.60 (2H, m), delta 2.30 (2H, t, J ~ 7Hz), delta 2.65 (2H, t, J ~ 7Hz), delta2.80 (4H, t, J ~ 7Hz), delta 3.0 (2H, m), delta 3.60 (2H, t, J ~ 7Hz), delta 7.0 (2H, s); LCMS: m/z (ES),380 (M + H)+
bis[εN'-cyclo(L-arginyl-glycyl-L-asparagyl-D-phenylalanyl-L-lysyl)]-αN-[3-(1-maleimido)propionyl]glutamide bistrifluoroacetate[ No CAS ]
Yield
Reaction Conditions
Operation in experiment
86%
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 6h;
To a solution of E[c(RGDfK)2]·3TFA (143 mg, 0.086 mmol) and 1 (26.6 mg, 0.10 mmol) in 5 mL of DMF was added DIPEA (0.418 mL, 2.40 mmol) and the solution was stirred at room temperature. After 6 h, the solvent was removed in vacuo. Purification of the crude product by preparative RP-HPLC afforded 4 (109 mg, 86%) as bistrifluoroacetate salt of colorless freeze-dried amorphous. HRMS (ESI) m/z: calcd for C66H93N20O19+ [M+H]+ 1469.6920, found 1469.6906.
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 20℃; for 24h;
To a solution of c(RGDfK)·2TFA (0.333 g, 0.40 mmol) and 1 (0.107 g, 0.40 mmol) in 5 mL of DMF was added N,N-diisopropylethylamine (DIPEA) (0.279 mL, 1.60 mmol) and the solution was stirred at room temperature. After 24 h, the solvent was removed in vacuo. Purification of the crude product by preparative RP-HPLC afforded 2 (0.293 g, 84%) as trifluoroacetate salt of colorless freeze-dried amorphous. HRMS (FAB) m/z: calcd for C34H47N10O10 [M+H]+ 755.3471, found 755.3485.
The crude compound 4 was dissolved in 15 mL DMF, 460 mg (1.7 mmol) of 3-(maleimido)propionic acid N-hydroxysuccinimide ester was added to the solution. The reaction was stirred for 2 days. Then the solvent was removed under vacuum to offer a white solid. The crude product was purified by silica gel chromatography eluting with 5:1 CHCl3:MeOH to afford 100 mg of a N-Boc protected bismaleimide (22% for 3 steps).
1-(4-amidemethyl-fluorescein)-4,7,10-tris(2-amino-ethylamidemethyl)-1,4,7,10-tetraazacyclododecane[ No CAS ]
1-(4-amidemethyl-fluorescein)-4,10-di(2-aminoethylamidemethyl)-7-(2-(3-maleimidopropionate)-2-aminoethylamidemethyl)-1,4,7,10-tetraazacyclododecane[ No CAS ]
With sodium hydrogencarbonate; N-ethyl-N,N-diisopropylamine; In tetrahydrofuran; water;
Step 1 3-(2,5-Dioxo-2,5-dihydropyrrol-1-yl)propionic acid 2,5-dioxopyrrolidiny-1-yl ester 3-Maleimidopropionic acid (1.0 g, 5.9 mmol) was dissolved in tetrahydrofuran (20 ml). 2-Succinimido-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU, 2.14 g, 7.1 mmol) and ethyldiisopropylamine (1.24 ml, 7.1 mmol) were added subsequently. N,N-Dimethylformamide (5 ml) was added. The reaction mixture was stirred at room temperature, while it was turning sluggish. The mixture was stirred for 2 min. N,N-Dimethylformamide (5 ml) was added. The mixture was stirred for 2.5 h at room temperature. It was diluted with dichloromethane (150 ml) and was washed subsequently with a 10% aqueous solution of sodium hydrogensulphate (150 ml), a saturated aqueous solution of sodium hydrogencarbonate (150 ml) and water (150 ml). It was dried over magnesium sulphate. The solvent was removed in vacuo. The crude product was recrystallized from ethyl acetate to give 1.20 g of 3-(2,5-dioxo-2,5-dihydropyrrol-1-yl)propionic acid 2,5-dioxopyrrolidin-1-yl ester. MS: m/z =289, required for [M+Na]+: 289 1H-NMR (CDCl3) delta 2.82 (m, 4H); 3.02 (t, 2H); 3.94 (t, 2H), 6.73 (s, 2H).
In acetonitrile; at 20℃; for 2.08333h;pH 7;Aqueous phosphate buffer; Cooling with ice;
To a solution of 4.04 mg (9 mumol) of 3-O-(5-aminopentyl)carbamoyl-1-phosphate-D-myo-inositol (compound 10) prepared according to Example 8 in 2 ml of 0.1 M phosphate buffer pH 7 cooled in an ice bath are added 5.54 mg (20 mumol) of N-hydroxysuccinimidyl 3-maleimidopropionate (Aldrich) dissolved in 400 mul of acetonitrile in 100 mul fractions every 5 min, allowing the mixture to return to 20 C., and then the mixture is left to react for two hours. The conversion of the starting material peak to a novel compound (tR=12.7 min) [Lichrospher Merck RP18 E (5 mum) 125×4 mm, 1 ml/min, A: water containing 0.05% of TFA, B: acetonitrile, gradient 0 to 2% of B over 10 min, 2% to 15% B over 5 min, 15% to 100% B over 8 min, detection at 190 nm] is observed by HPLC. The compound is purified by RP-HPLC using the same solvents, after evaporation and drying under vacuum, 1.15 mg (21%) of IP1 maleimide derivative (compound 33) are obtained. MS (ES+) [MH]+=420 (30%) [M+Na]+=562 (50%) (Calc: C19H30N3O13P). The product is aliquoted and stored at -20 C. up to the time of use.
Solution of 39 mg (0.1 mmol) of 7-amino-4-carboxamido (6-aminohexyl) methyl-2(1H) quinolone (compound VI) in 0.4 ml of DMSO was supplemented with 30 mg (0.11 mmol) of MIpr-OSu and the mixture kept at room temperature. After 15 min incubation TLC analysis in chloroform-ethanol (4:1) developing system revealed nearly complete conversion of original compound (Rf = 0) to reaction product VIII with Rf = 0.3). DTPA acylation step (producing probe 14) and subsequent preparation of the sample for HPLC purification was performed as described above for probe 13. The sample was applied to HPLC column and the product eluted using linear gradient (560 ml) of methanol in water (0-40%) at the elution rate 2 ml/min. The fractions corresponding to desired product eluted between 140 and 210 min were combined and methanol evaporated under reduced pressure. After titration by TbCl3 as described in previous section the resulting solution was freeze dried. Yield: 20 mumol. LC/MS samples were prepared with final product and cysteine, the reaction mixture was purified in 2.5:1 acetonitrile H2O with Rf = 0.2 of reactant and Rf = 0.3 of Probe 14. UV: lambdamax = 332 nm (? = 18,900 M-1 cm-1). MS: Tb3+DTPA-cs124-CH2C(O)-NH(CH2)6NHC(O)CH2CH2-3MI-cysteine (+H) 1120.2 (found) 1119.9 (calculated). DTPA-cs124-CH2C(O)-NH(CH2)6NHC(O)CH2CH2-3MI-cysteine (+4H) 964.3356 (found) 964.026 (calculated).
Solution of 39 mg (0.1 mmol) of 7-amino-4-carboxamido (6-aminohexyl) methyl-2(1H) quinolone (compound VI) in 0.4 ml of DMSO was supplemented with 30 mg (0.11 mmol) of MIpr-OSu and the mixture kept at room temperature. After 15 min incubation TLC analysis in chloroform-ethanol (4:1) developing system revealed nearly complete conversion of original compound (Rf = 0) to reaction product VIII with Rf = 0.3). DTPA acylation step (producing probe 14) and subsequent preparation of the sample for HPLC purification was performed as described above for probe 13. The sample was applied to HPLC column and the product eluted using linear gradient (560 ml) of methanol in water (0-40%) at the elution rate 2 ml/min. The fractions corresponding to desired product eluted between 140 and 210 min were combined and methanol evaporated under reduced pressure. After titration by TbCl3 as described in previous section the resulting solution was freeze dried. Yield: 20 mumol. LC/MS samples were prepared with final product and cysteine, the reaction mixture was purified in 2.5:1 acetonitrile H2O with Rf = 0.2 of reactant and Rf = 0.3 of Probe 14. UV: lambdamax = 332 nm (? = 18,900 M-1 cm-1). MS: Tb3+DTPA-cs124-CH2C(O)-NH(CH2)6NHC(O)CH2CH2-3MI-cysteine (+H) 1120.2 (found) 1119.9 (calculated). DTPA-cs124-CH2C(O)-NH(CH2)6NHC(O)CH2CH2-3MI-cysteine (+4H) 964.3356 (found) 964.026 (calculated).
In dimethyl sulfoxide; at 20 - 50℃; for 0.166667h;
5.2 mg (14 mumol) of compound IX was dissolved in 200 mul of DMSO at 50 C, cooled to room temperature and mixed with the solution of 23 mumol (1.5 equiv.) of 4-nitrophenylbromoacetate. After 10 min reaction at room temperature TLC analysis in 9:1 acetonitrile-water developing system revealed complete conversion of the starting fluorophore (Rf = 0.4) to the reaction product X (Rf = 0.7). The mixture was supplemented with the solution of 56 mumol (4-fold excess) of DTPA anhydride e in 200 mul of DMSO. After 20 min incubation the reaction mixture was supplemented with 100 mul of water and left for another 10 min at room temperature. Five milliliters of BuOH and 2 ml of water were added and after the extraction the organic layer was collected. Extraction was repeated one more time, organic layers combined and the amount of the DTPA acylation product in butanol solution was determined by UV absorption. The solution was supplemented with 1.5 equiv. of 0.1 M TbCl3. After rigorous agitation the organic layer was separated and evaporated in vacuo to 2-3 ml. The product was collected by centrifugation and purified by preparative TLC in acetonitrile-water (4:1) developing system. The product (Rf = 0.24) was recovered by elution with 50% aqueous MeOH. Yield: 1.35 mumol (10%). MS: Tb3+DTPA-cs124-CH2-BiPh-CH2NHC(O)CH2Br (+H+) 1022.1 (found) 1021.7 (calculated). DTPA-cs124-CH2-BiPh-CH2NHC(O)CH2Br (+H+) 867.2 (found) 866.7 (calculated).
7-amino-4-trifluoromethyl-4-carboxamido(6-aminohexyl)methyl-2-(1H)quinolone[ No CAS ]
[ 1431329-86-0 ]
Yield
Reaction Conditions
Operation in experiment
In dimethyl sulfoxide; at 20℃; for 0.25h;
Solution of 39 mg (?100 mumol) of compound III was dissolved in 400 mul of DMSO and supplemented with 31 mg (?100 mumol) of N-succinimidyl-3-maleimidioproprionate (MIpr-OSu). After 15 min incubation at room temperature TLC analysis in chloroform-ethanol (4:1) developing system revealed nearly complete conversion of original compound (Rf = 0) to reaction product with Rf = 0.53. The mixture was supplemented with ?300 mumol of DTPA dianhydride dissolved in 400 muL of DMSO at 70 C and cooled to room temperature before addition. After 40 min incubation at 40 C the mixture was poured drop-wise into 5 ml of ice-cold 0.1 M lithium citrate pH 4.0. Subsequently, pH was adjusted to 5.0 by 1 M LiOH and the mixture was extracted by 5 ml of ethyl acetate followed by centrifugation of the water layer at 6000 rpm for 5 min at 4 C. The product from the supernatant was purified by preparative HPLC and converted to Eu3+ complex as described above. Yield: 11 mumol (11%). Cysteine reaction product was obtained from the probe as described below. UV: lambdamax = 347 nm (? = 14,300 M-1 cm-1). MS: Eu3+DTPA-cs124-CF3-CH2C(O)-NH(CH2)6NHC(O)3MI-cysteine (-H+) 1177.411 (found) 1178.988 (calculated). DTPA-cs124-CF3-CH2C(O)-NH(CH2)6NHC(O)3MI-cysteine 1032.358 (found) 1032.028 (calculated). Eu3+ DTPA-cs124-CF3-CH2C(O)-NH(CH2)6NHC(O)3MI 1059.838 (found) 1060.3092 (calculated).
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; for 18.166h;
Compound 5 N-[B-Maleimidopropyloxy]succinimide ester (50 mg, 1.25 equiv) was dissolved in 1.0 mL of dry DMF. 3-Butyn-l -amine hydrochloride (16 mg, 1.0 equiv) was dissolved in 0.5 mL of dry DMF and 26 of DIEA. This amine solution was added dropwise to the succinimide ester while keeping the ester solution in an ice bath. The mixture was stirred at 0C for 10 min. The solution was warmed up to room temperature and stirred for 18 h. The solvents were evaporated in vacuo and the residue was dissolved in 5 mL CH2CI2. The organic solution was extracted with brine (3 x 5 mL) and dried over MgS04. The solvent was removed under reduced pressure and the crude material was purified using flash chromatography (silica, MeOH/CF^Cy. The product (5) was purified from grease by dissolving the sample in a minimum amount of CH2CI2 (ca. 2 mL), followed by three washes with hexanes. The product (5) precipitated as a fluffy white solid. Characterization of the product was achieved using 1H-NMR. Yield: 8.2 mg (25%). 1H-NMR (500 MHz, CDC13): delta 2.02 (s, 1H), 2.41 (t, J = 5Hz, 2H), 2.57 (t, J = 5 Hz, 2H), 3.42 (dt, J= 5 Hz, 2H), 3.88 (t, J= 5 Hz), 5.90 (bs, 1H), 6.73 (s, 2H).
25%
With N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 0 - 20℃; for 7.36667h;
N-[beta-Maleimidopropyloxy]succinimide ester (50 mg, 1.25 equiv) was dissolved in 1.0 mL of dry DMF. 3-Butyn-1-amine hydrochloride (16 mg, 1.0 equiv) was dissolved in 0.5 mL of dry DMF and 26 muL of DIEA. This amine solution was added dropwise to the succinimide ester while keeping the ester solution in an ice bath. The mixture was stirred at 0 C. for 10 min. The solution was warmed up to room temperature and stirred for 18 h. The solvents were evaporated in vacuo and the residue was dissolved in 5 mL CH2Cl2. The organic solution was extracted with brine (3×5 mL) and dried over MgSO4. The solvent was removed under reduced pressure and the crude material was purified using flash chromatography (silica, MeOH/CH2Cl2). The product (5) was purified from grease by dissolving the sample in a minimum amount of CH2Cl2 (ca. 2 mL), followed by three washes with hexanes. The product (5) precipitated as a fluffy white solid. Characterization of the product was achieved using 1H-NMR. Yield: 8.2 mg (25%). 1H-NMR (500 MHz, CDCl3): delta 2.02 (s, 1H), 2.41 (t, J=5 Hz, 2H), 2.57 (t, J=5 Hz, 2H), 3.42 (dt, J=5 Hz, 2H), 3.88 (t, J=5 Hz), 5.90 (bs, 1H), 6.73 (s, 2H).
The solution containing B was filtered and the filtrate was added to amine 3 (1.72 g, 4 mmol). The reaction mixture was stirred at room temperature overnight, followed by the addition of succinimido 3-maleimidopropanoate (A) prepared above (1.33 g, 5 mmol). The mixture was stirred for 2 days at room temperature. After evaporation to remove DMF, the residue was purified on column chromatography first eluting with MeOH:DCM=1:9 to remove all by-products then increasing the polarity of the eluent to afford compound 4 (1.2 g, 35% yield based on the amine 3). 1H-NMR (400 MHz, DMSO-d6) delta: 2.15 and 2.20 (two s, CH3, 3H), 2.59-3.94 (m, CH2, 16H), 4.99 and 5.02 (two s, CH2, 2H), 6.06 (d, J=7.4 Hz, pyridinone 5-CH, 1H), 6.70 (s, maleimide CH, 2H), 7.27-7.40 (m, BnH and pyridinone 6-CH, 6H), 6.20-8.44 (m, fluorescein CH, 9H). ESI-MS: Calculated for C47H43N5O11 853.3 (monoisotopic molecular weight M). found 854.2 (M+H)+.
Synthesis of Compound 22 To a solution of N-BOC-l,3-diaminopropane (Compound 20, 115 mg, 0.660 mmol) in 1 : 1 CH2CI2 /CH3CN (1 mL) at 0 C was added a solution of <strong>[55750-62-4]3-maleimidopropionic acid N-hydroxysuccinimide ester</strong> (Compound 21, 185 mg, 0.695 mmol) dissolved in acetonitrile (4 mL) and CH2CI2 (1 mL). The mixture was stirred for 1 h and concentrated in vacuo. The residue was purified by silica gel chromatography (0 - 5% MeOH/ CH2CI2 to give product Compound 22. Calculated mass: [M+H] : C15H24N3O5, 326.2; observed: 326.3.
In dichloromethane; acetonitrile; at 0℃; for 1h;
Synthesis of Compound 22 To a solution of N-BOC-l,3-DIAMINOPROPANE (Compound 20, 115 mg, 0.660 mmol) in 1 : 1 CH2C12 /CH3CN (1 mL) at 0 C was added a solution of 3- maleimidopropionic acid N-hydroxysuccinimide ester (Compound 21, 185 mg, 0.695 mmol) dissolved in acetonitrile (4 mL) and CH2C12 (1 mL). The mixture was stirred for 1 h and concentrated in vacuo. The residue was purified by silica gel chromatography (0 - 5% MeOH/ CH2C12 to give product Compound 22. Calculated mass: [M+H]+: C15H24N3O5, 326.2; observed: 326.3.
4.03 g
With N-ethyl-N,N-diisopropylamine; In chloroform; at 25℃; for 18h;
In 250 ml round bottomed flask were taken 3.0 g of1-(tert-Butoxycarbonyl) amino 3-aminopropane) and 100mL of anhydrous chloroform. The reaction mixture wasstirred at 25 C. until a clear solution was formed. To thissolution was added <strong>[55750-62-4]N-succinimidyl 3-maleimidopropionate</strong>(5.05 g) with stirring until dissolved followed 3.42 mL ofdiisoproylethylamine. The resulting reaction mixture wasstirred at 25 C. for 18 h. The solution was washed with 1Mhydrochloric acid (50mL), 10% brine (50 mL), saturatedsodium bicarbonate (50 mL), semi-saturated brine (50 mL).The organic phase was isolated and was dried over anhydroussodium sulfate. After removing the sodium lo sulfateby filtration, the solution was concentrated under reducedpressure and the residue was purified by silica gel colurm1chromatography using ethyl acetate: hexanes gradient as themobile phase. Removal of the solvent under reduced pressurefollowed by vacuum drying offered the desired productas an off white solid ( 4.03 g).
4.03 g
With N-ethyl-N,N-diisopropylamine; In chloroform; at 25℃; for 18h;
Boct In 250 ml round bottomed flask were taken 3.0 g of l-(tert-Butoxycarbonyl)amino 3-aminopropane) and 100 ml_ of anhydrous chloroform. The reaction mixture was stirred at 25C until a clear solution was formed. To this solution was added /V-succinimidyl 3- maleimidopropionate (5.05g) with stirring until dissolved followed 3.42ml_ ofdiisoproylethylamine. The resulting reaction mixture was stirred at 25C for 18h. The solution was washed with 1 M hydrochloric acid (50ml_), 10% brine (50ml_), saturated sodium bicarbonate (50ml_), semi-saturated brine (50ml_). The organic phase was isolated and was dried over anhydrous sodium sulfate. After removing the sodium sulfate by filtration, the solution was concentrated under reduced pressure and the residue was purified by silica gel column chromatography using ethyl acetate: hexanes gradient as the mobile phase. Removal of the solvent under reduced pressure followed by vacuum drying offered the desired product as an off white solid ( 4.03 g).
With triethylamine; In N,N-dimethyl-formamide; for 1h;Inert atmosphere;
Di-macrocycle 12 (10.12 mg, 6.45 muiotaetaomicron), was dissolved in dimethylformamide (100 mu) and triethylamine (10 mu), and added to 3- maleimidoproprionic acid, N-hydroxysuccinimide ester (3.4 mg, 13 muiotaetaomicron). The resulting solution was mixed at 800 rpm under inert atmosphere for 1 hour. Ether (ca. 1.5 mL) was added, and the resulting suspension placed at 4 C for 60 minutes. The tube was centrifuged at 12,000 rpm for 3 minutes, decanted, the pellet was washed with ether (ca. 1.5 mL) and allowed to air dry. The pellet was dissolved in methanol (300 mu) then precipitated and washed with ether as described above. The pellet was dried in vacuo to provide di-macrocycle, 3-maleimidopropyl derivative 99 (7.42 mg, 74.8%). FTMS pESI: calculated for C75H105 14O21 [M+H]+, 1537.7573, found, 1537.7561.
With N-ethyl-N,N-diisopropylamine; In dichloromethane; at 20℃;
Referring to the scheme of synthesis of Compound O, beta-alanine was treated with maleic anhydride in DMF and the acid so obtained was reacted with N-hydroxysuccinimide (NHS) under DCC coupling to give NHS-ester. The BOC protective group in commercially available t-blc-N-amido-dPEG4-acid was removed by treatment with TFA to give the TFA salt of the amine, which was reacted with previously synthesized NHS ester. The carboxylic acid so obtained was isolated and was coupled with N-hydroxysuccinimide using EDCI to furnish NHS ester Compound O.
0.227g
With N-ethyl-N,N-diisopropylamine; In dichloromethane; at 20℃;
[0277] Referring to the scheme of synthesis of Compound O, beta-alanine was treated with maleic anhydride in DMF and the acid so obtained was reacted with N-hydroxysuccinimide (NHS) under DCC coupling to give NHS-ester. The BOC protective group in commercially available t-boc-N-amido-dPEG4-acid was removed by treatment with TFA to give the TFA salt of the amine, which was reacted with previously synthesized NHS ester. The carboxylic acid so obtained was isolated and was coupled with N-hydroxysuccinimide using EDCI to furnish NHS ester Compound O.