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CAS No. : | 7423-55-4 | MDL No. : | MFCD00043030 |
Formula : | C7H7NO4 | Boiling Point : | - |
Linear Structure Formula : | - | InChI Key : | IUTPJBLLJJNPAJ-UHFFFAOYSA-N |
M.W : | 169.13 | Pubchem ID : | 573621 |
Synonyms : |
|
Num. heavy atoms : | 12 |
Num. arom. heavy atoms : | 0 |
Fraction Csp3 : | 0.29 |
Num. rotatable bonds : | 3 |
Num. H-bond acceptors : | 4.0 |
Num. H-bond donors : | 1.0 |
Molar Refractivity : | 42.16 |
TPSA : | 74.68 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) : | -7.3 cm/s |
Log Po/w (iLOGP) : | 0.68 |
Log Po/w (XLOGP3) : | 0.04 |
Log Po/w (WLOGP) : | -0.99 |
Log Po/w (MLOGP) : | -0.81 |
Log Po/w (SILICOS-IT) : | -0.34 |
Consensus Log Po/w : | -0.28 |
Lipinski : | 0.0 |
Ghose : | None |
Veber : | 0.0 |
Egan : | 0.0 |
Muegge : | 1.0 |
Bioavailability Score : | 0.56 |
Log S (ESOL) : | -0.72 |
Solubility : | 32.5 mg/ml ; 0.192 mol/l |
Class : | Very soluble |
Log S (Ali) : | -1.16 |
Solubility : | 11.7 mg/ml ; 0.069 mol/l |
Class : | Very soluble |
Log S (SILICOS-IT) : | 0.19 |
Solubility : | 261.0 mg/ml ; 1.54 mol/l |
Class : | Soluble |
PAINS : | 0.0 alert |
Brenk : | 1.0 alert |
Leadlikeness : | 1.0 |
Synthetic accessibility : | 2.14 |
Signal Word: | Warning | Class: | N/A |
Precautionary Statements: | P261-P305+P351+P338 | UN#: | N/A |
Hazard Statements: | 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 |
---|---|---|
1.2 g | 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). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
67% | at 170 - 180℃; for 1.5 h; | First, 3-maleimidopropionic acid was synthesized according todescribed literature methods with modification [26,27]. A suspensionof maleic anhydride (3.21 g, 32.78 mmol) and β-alanine (3 g,33.67 mmol) in glacial AcOH (30 mL) was heated to reflux (bath temperature:170–180 °C) for 90min. The solutionwas cooled to roomtemperatureand the solvent was evaporated in vacuo. Residual AcOH wasremoved by coevaporation with toluene under vacuum (2 × 50 mL).The residue was treated with water (100 mL) and EtOAc (200 mL).The organic layer was separated while the aqueous layer was extractedwith EtOAc (2 × 100 mL). The combined organic extracts were dried(Na2SO4) and evaporated in vacuo. The crude product was purified bysilica gel column chromatography, eluting with 50percent ethyl acetate inhexanes, to afford 3-maleimidopropionic acid (3.7 g, 21.88 mmol, 67percentyield), the spectroscopic data of which was consistent with reporteddata.(27) 1H NMR (CDCl3) δ 8.93 (br s, 1H), 6.71 (s, 2H), 3.80 (t, J =7.2 Hz, 2H), 3.62–3.66 (m, 24H), 2.67 (t, J = 7.2 Hz, 2H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
80% | With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 20℃; for 2h; | The product 2-11 (3.6 g, 21.4 mmol) obtained in the previous step and EDC-HCl (4.93 g, 25.7 mmol) were dissolved in 50 mL DCM, HOSu (2.96 g, 25.7 mol) was added. The reaction mixture was stirred for 2 hours at room temperature. 50 mL water was added, the mixture was extracted with EtOAc for 3 times (50 mL×3). The organic phases were combined, washed with saturated brine for 3 times (50 mL×3), dried over anhydrous sodium sulfate, and concentrated. The crude product was purified by silica gel column chromatography (PE/EtOAc=1:3) to give 4.55 g product as yellow oil, yield 80%. LCMS (ESI) m/z 267.1 (M+H)+. |
54% | With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 0 - 20℃; | N-(cyclopropylmethyl)-N-{(1S)-1-[1-(pyrimidin2-yl)-1H-1,2,4-triazol-5-yl]ethyl}-3-[(trifluoromethyl)sulfonyl]benzamide N-(cyclopropylmethyl)-N-{(1S)-1-[1-(pyrimidin2-yl)-1H-1,2,4-triazol-5-yl]ethyl}-3-[(trifluoromethyl)sulfonyl]benzamide |
With dicyclohexyl-carbodiimide; In N,N-dimethyl-formamide; at 20℃; | A solution of beta-alanine (1.8 g, 20 mmol) and maleic anhydride (2 g, 20 mmol) in DMF (30 mL) was stirred at room temperature for 3 h. After the solid was completely dissolved, N-hydroxysuccinimide (NHS; 2.3 g, 20 mmol) and dicyclohexylcarbodiimide (DCC; 4.6 g, 24 mmol) was added into the solution and the mixture was stirred at room temperature overnight. 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 3×50 ml 5% NaHCO3 and one time with brine. The organic layer was dried with Na2SO4 and dichloromethane removed under reduced pressure to obtain a white solid (55%). This solid does not need further purification and can be directly used for next coupling step. 1H NMR (CDCl3, 400 MHz): delta 2.82 (s, 4H, two succinimido-CH2), 3.02 (t, J=7.0 Hz, 2H, COCH2), 3.94 (t, J=7.0 Hz, 2H, NCH2), 6.74 (s, 2H, two CH). |
With dicyclohexyl-carbodiimide; In 1,2-dimethoxyethane; at 20℃; for 1h;Inert atmosphere; | 3-maleimidopropionic acid was esterified by adding Nhydroxysuccinimide(NHS) (23 mg, 0.20 mmol) and N,N?-dicyclohexylcarbodiimide (DCC) (50 mg, 0.24 mmol) into a stirredsolution of 3-maleimidopropionic acid (34 mg, 0.20 mmol) indimethoxyethane (5 mL) at room temperature under N2. After 1 h,the precipitate formed was removed by filtration. The filtrate wasconcentrated under vacuum to afford crude 3-maleimidopropionicNHS ester, which was used directly for the next step without furtherpurification. | |
With dicyclohexyl-carbodiimide; | [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. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
67% | With acetic acid; at 170 - 180℃; for 1.5h; | First, 3-maleimidopropionic acid was synthesized according todescribed literature methods with modification [26,27]. A suspensionof maleic anhydride (3.21 g, 32.78 mmol) and beta-alanine (3 g,33.67 mmol) in glacial AcOH (30 mL) was heated to reflux (bath temperature:170-180 C) for 90min. The solutionwas cooled to roomtemperatureand the solvent was evaporated in vacuo. Residual AcOH wasremoved by coevaporation with toluene under vacuum (2 × 50 mL).The residue was treated with water (100 mL) and EtOAc (200 mL).The organic layer was separated while the aqueous layer was extractedwith EtOAc (2 × 100 mL). The combined organic extracts were dried(Na2SO4) and evaporated in vacuo. The crude product was purified bysilica gel column chromatography, eluting with 50% ethyl acetate inhexanes, to afford 3-maleimidopropionic acid (3.7 g, 21.88 mmol, 67%yield), the spectroscopic data of which was consistent with reporteddata.(27) 1H NMR (CDCl3) delta 8.93 (br s, 1H), 6.71 (s, 2H), 3.80 (t, J =7.2 Hz, 2H), 3.62-3.66 (m, 24H), 2.67 (t, J = 7.2 Hz, 2H). |
44.4% | With acetic acid; for 6h;Reflux; | Maleic anhydride (20 g, 0.20 mol), beta-alanine (18.2 g, 0.20 mol) was dissolved in 140 mL of acetic acid, and heated to reflux for 6 h.After the reaction is completed, the acetic acid is rotated, and the obtained oily liquid is separated by column chromatography.White solid 15 g, yield 44.4%. |
42% | With acetic acid; for 16h;Reflux; Inert atmosphere; | Maleic anhydride (5 g, 51 mmol, 1 eq) and (-aminopropanoic acid (4.54 g, 51 mmol, 1 eq) were dissolved in 80 mL AcOH. The reaction mixture was heated to reflux and stirred overnight under nitrogen atmosphere. The reaction mixture was cooled to room temperature, AcOH was removed under reduced pressure. The residue was purified by silica gel column chromatography (DCM) to give 3.6 g product 2-11 as white solid, yield 42%. LCMS (ESI) m/z 170.1 (M+H)+. |
In N,N-dimethyl-formamide; at 20℃; for 3h; | A solution of beta-alanine (1.8 g, 20 mmol) and maleic anhydride (2 g, 20 mmol) in DMF (30 mL) was stirred at room temperature for 3 h. After the solid was completely dissolved, N-hydroxysuccinimide (NHS; 2.3 g, 20 mmol) and dicyclohexylcarbodiimide (DCC; 4.6 g, 24 mmol) was added into the solution and the mixture was stirred at room temperature overnight. 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 3×50 ml 5% NaHCO3 and one time with brine. The organic layer was dried with Na2SO4 and dichloromethane removed under reduced pressure to obtain a white solid (55%). This solid does not need further purification and can be directly used for next coupling step. | |
In N,N-dimethyl-formamide; | [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. | |
With acetic acid; at 20℃; for 20h;Reflux; | 2.1, maleic anhydride (5.5 g) and beta-alanine (5.0 g) were dissolved in 180 mL of glacial acetic acid,Stir at room temperature for 12 hours and then reflux for 8 hours. Acetic acid was removed in vacuo,The residue is recrystallized from ethyl acetate, filtered and dried to give the acylated product. | |
In N,N-dimethyl-formamide; at 20℃; | N-(cyclopropylmethyl)-N-{(1S)-1-[1-(pyrimidin2-yl)-1H-1,2,4-triazol-5-yl]ethyl}-3-[(trifluoromethyl)sulfonyl]benzamide |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With thionyl chloride; In dichloromethane; for 12h;Reflux; | 2.2, 2.1 The resulting material (1.69 g) was dissolved in 30 mL of methylene chloride,Add 6mL of thionyl chloride,Reflux for twelve hours.Then vacuum drying to remove thionyl chloride, the target acid chloride. | |
With oxalyl dichloride; N,N-dimethyl-formamide; In dichloromethane; at 20℃; for 0.5h;Sealed tube; | To a 10 L three-necked flask was added 3-maleimidopropionic acid (845.6 g, 5.0 mol)And dichloromethane, followed by the addition of oxalyl chloride (759.3 g, 6.0 mol) and a catalytic amount of DMF,The solution system oil seal is reacted at room temperature for half an hour and then returned to the oil seal without bubbles emerging.Cooling, concentrating, draining the oil pump, then diluting with dichloromethane to prepare a solution for use; | |
With oxalyl dichloride; N,N-dimethyl-formamide; In dichloromethane; at 20℃; for 2h; | 3-Maleido-propanoic acid (1.00 g, 5.91 mmol) in DCM (50 ml) was added oxalyl dichloride (2.70 g, 21.25 mmol) and DMF (50 muL) . The mixture was stirred at room temperature for 2 h, evaporated, and co-evaporated with DCM/toluene to obtain crude 3-maleido-propanoic acid chloride. To the compound di-tert-Butyl 3, 3'- (hydrazine-1, 2-diyl) dipropanoate (0.51 g, 1.76 mmol) in the mixture of DCM (35 ml) was added the crude 3-maleido-propanoic acid chloride. The mixture was stirred for overnight, evaporated, concentrated and purified on SiO 2 column eluted with EtOAc/DCM (1: 151: 8) to afford the title compound (738 mg, 71%yield) . ESI MS m/z+ C 28H 38N 4NaO 10 (M+Na) , cacld. 613.26, found 613.40. |
at 20℃; for 2h; | To a solution of 3-maleimide-propionic acid (1.00 g, 5.91 mmol) in dichloromethane (50 mL) was added oxalyl chloride (2.70 g, 21.25 mmol) and DMF (50 muL).The mixture was stirred at room temperature for 2 hours, concentrated, and co-concentrated with dichloromethane / toluene to give crude 3-maleimide-propionyl chloride.To a mixture of 3,3 '-(hydrazine-1,2-diyl) di-t-butyl dipropionate (0.51 g, 1.76 mmol) in dichloromethane (35 mL) was added 3-maleimide-propane Crude acid chloride.The mixture was stirred overnight, concentrated and purified on a silica gel column,Elution with ethyl acetate / dichloromethane (1: 15-1: 8) gave the title compound (738 mg, 71% yield). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With 1-methyl-1H-imidazole; 1-(mesitylene-2-sulfonyl)-3-nitro-1H-1,2,4-triazole; N-ethyl-N,N-diisopropylamine; In dichloromethane; at 20℃; for 2h; | 300 mg of 4-hydroxy-3-methoxy-phenylacetic acid and 570 mul DIEA were dissolved in 5 ml DCM and added to 0.5 g 2-chlorotrityl-chloride resin (1.5 mmol/g). The suspension was agitated for 1 h at RT and resin was washed with DCM. Resin was resuspended in a solution of 190 mg 3-maleimidopropionic acid, 333 mg MSNT and 73 mul N-methyl imidazole in 3 ml DCM and agitated for 1h. After washing of the resin with DCM, cleavage was performed by agitation of the resin for 30 min in 10 ml 4/1 (v/v) DCM/TFA. Solvent was removed under nitrogen flow and compound 6 was purified by RP-HPLC. MS [MNa]+ (MW+Na calculated): 356 g/mol (356 g/mol) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Synthesis of compound 10 [] 10 was obtained according to the standard solid-phase synthesis protocol. The amino acids Fmoc-Dpr(Boc)-OH, Fmoc-Dpr(Fmoc)-OH, Fmoc-Dpr(Fmoc)-OH, Fmoc-Ado-OH, and Fmoc-Dpr(Fmoc)-OH were coupled to TentaGel Sieber amide resin. After final finoc removal the resin was agitated with 5 eq maleimidopropionic acid and 5 eq DIC in relation to amino groups in DMF for 30 min. 10 was cleaved from resin with TFA/TES/water 95/3/2 (v/v/v). After evaporation of solvent, product 10 was purified by RP-HPLC. MS: [M+H]+ = 2494.6 (MW calculated = 2495.4 g/mol) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
53% | With benzotriazol-1-ol; dicyclohexyl-carbodiimide; In tetrahydrofuran; at 20℃; | Example 5 Synthesis of tert-Butyldimethylsiloxy-9-methyl-2-(amino-3-maleimidopropionate) fluorene To a solution of 13.5 g 9-tert-Butyldimethylsiloxymethyl-2-aminofluorene (41.5 mmol) in dry THF (freshly distilled from sodium) 9.42 g N,N'-dicyclohexylcarbodiimide (75.7 mmol, 1.1 equiv) and 651 mg 1-hydroxybenzotriazole (4.8 mmol, 0.1 equiv) were added. 13.5 g (41.5 mmol, 1.1 equiv) 3-maleimidopropionic acid was dissolved in 50 ml dry THF and added dropwise. The reaction mixture was stirred at room temperature over night under argon atmosphere and the product formation was monitored by TLC [starting material Rf=0.6, title product Rf=0.18, PE-MTBE (1:2)]. As soon as the starting material could not be detected, dicyclohexylurea was filtered out and THF was eliminated by rotary evaporation. The residual solid was dissolved in 200 ml CH2Cl2, washed with 50 ml 5% NaHCO3 and 50 ml brine and dried over Na2SO4. The brown crystals were digerated in 20 ml MTBE. After filtration the residue was washed with small portions of MTBE until the washing solution maintained colorless. The yellow crystals (10.5 g, 53% yield) were analyzed by NMR. 1H NMR (200 MHz/DMSO) delta=10.06 (1H; s; NH); 7.92 (1H; s; H1); 7.82-7.70 (2H; m; H4 & H5); 7.62 (1H; d; J=7.20 Hz; H3); 7.49 (1H; d; J=8.08 Hz; H8); 7.41-7.18 (2H; m; H6 & H7); 7.03 (2H; s; 2*Mal-CH); 4.02 (1H; t; J=6.63 Hz; H9); 3.95-3.66 (4H; m; Prop-CH2-N & CH2-OTBDMS); 2.61 (2H; t; J=7.07 Hz; Prop-CH2-C=O); 0.84 (9H; s; 3*t-Bu CH3); 0.10-1-0.56 (6H; m; 2*CH3-Si) 13C NMR (50 MHz/DMSO) delta=170.81 (C=O Mal); 168.30 (C=O Amid); 145.12 (C9a); 144.62 (C8a); 140.68 (C4b); 138.05 (C2); 136.02 (C4a); 134.62 (2*CH Mal); 127.34 (C6); 126.02 (C7); 125.15 (C8); 119.99 (C4); 119.37 (C5); 118.63 (C3); 116.31 (C1); 65.54 (CH2OTBDMS); 49.85 (C9); 35.01 (Prop-CH2-N); 33.91 (Prop-CH2-CO); 25.84 (3*CH3 (t-Bu); 18.01 (Cqu t-Bu); -5.40 (CH3-Si); -5.44 (CH3-Si) |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
72% | With 4-methoxy-phenol; at 80 - 90℃; for 8h; | To a pre-heated (90C) three-neck round bottom flask was added 4- MEHQ (223mg, 1.8 mmol) and diethyleneglycol divinyl ether (22.1 g, 140 mmoi). MPA (50 g, 296 mmol) was added into the reaction mixture in ten portions over 1.5 hours. The reaction temperature was maintained at 80- 90C. After the reaction mixture was stirred for another 6.5 hours at 85C, the reaction mixture was cooled to ambient temperature. A very smal. amount of gel was found in the reaction mixture. Toluene (200 mL) was added to dissolve the product mixture after which Amberlyst A 21 resin (50 g) was added and the product mixture stirred for one hour. After filtration, the solvent was stripped under vacuum to give 50 g (72%) of a yellow viscous liquid with viscosity of 13,000 mPa.s (25 C). 1H-NMR (DMSO-Cf6, in ppm): delta <n="34"/>7.0 (S, 4H), 5.9 (q, 2H), 3.54-3.7 (m, 12H), 2.6 (t, 4H), 1.2 (m, 6H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Stage #1: C25H21ClN3O2Pol; Fmoc-L-His(Fmoc)-OH With N-ethyl-N,N-diisopropylamine; HATU for 0.666667h; Automated synthesizer; solid phase reaction; Stage #2: With piperidine for 0.216667h; Automated synthesizer; solid phase reaction; Stage #3: 3-maleimidepropionic acid; Fmoc-L-His(Fmoc)-OH Further stages; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With acetic acid; at 115℃; for 12h; | AMI was prepared following a reported procedure with some modifications (Scheme 1 ) (Mantovani et al., 2005). An acetic acid solution of maleic anhydride (5.00 g in 50 mL) was added dropwise to an acetic acid solution of beta-alanine (4.54 g in 50 mL). The mixture was stirred for 3 h at room temperature and a white suspension was obtained. After that period, 70 mL of AcOH were added, the temperature was raised until 115 C and the mixture was stirred overnight. After one hour of reaction a limpid colourless solution was observed. At the end of the whole process, an orange oil was obtained. The solvent excess was removed under reduced pressure and the product was washed with toluene (3 × 30 mL), which was again removed under reduced pressure. The product was then purified by flash chromatography using a silica column (DCM/ethylacetate 9:1). A white solid was obtained. Yield: 29.15%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
80% | With dmap; O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; triethylamine; In N,N-dimethyl-formamide; for 14h; | To a solution of 4 (30 mg, 0.18 mmol) in anhydrous DMF, were added triethylamine (50 mg, 0.49 mmol), HBTU (68 mg, 0.18 mmol), a catalytic amount of DMAP and 1 (70 mg, 0.16 mmol). The reaction mixture was stirred for 14 hours and then the solvent was removed under vacuum. The residue was dissolved in ethyl acetate and washed with water. The ethyl acetate fraction was then dried using Na2SO4 and evaporated under vacuum to obtain the residue, which was purified using column chromatography (5% MeOH/dichloromethane) to obtain the compound 5 (65 mg, 80%). |
80% | With O-(1H-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate; triethylamine;dmap; In N,N-dimethyl-formamide; for 14h; | Synthesis of Compound 21: N-(4-((4-amino-2-butyl-1H-imidazo[4,5-c]quinolin-1-yl)methyl)benzyl)-3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide To a solution of 20 (30 mg, 0.18 mmol) in anhydrous DMF, were added triethylamine (50 mg, 0.49 mmol), HBTU (68 mg, 0.18 mmol), a catalytic amount of DMAP and 7d (70 mg, 0.16 mmol). The reaction mixture was stirred for 14 hours and then the solvent was removed under vacuum. The residue was dissolved in ethyl acetate and washed with water. The ethyl acetate fraction was then dried using Na2SO4 and evaporated under vacuum to obtain the residue, which was purified using column chromatography (5% MeOH/dichloromethane) to obtain compound 21 (65 mg, 80%). 1H NMR (500 MHz, MeOD) delta 7.97 (dd, J=8.4, 0.7 Hz, 1H), 7.74-7.71 (m, 1H), 7.64 (ddd, J=8.4, 7.2, 1.2 Hz, 1H), 7.38 (ddd, J=8.4, 7.2, 1.2 Hz, 1H), 7.26 (d, J=8.3 Hz, 2H), 7.04 (d, J=8.3 Hz, 2H), 6.73 (s, 2H), 5.93 (s, 2H), 4.27 (s, 2H), 3.75 (t, J=7.0 Hz, 2H), 3.02-2.97 (m, 2H), 2.47 (t, J=7.0 Hz, 2H), 1.85 (dt, J=21.1, 7.6 Hz, 2H), 1.46 (dq, J=14.8, 7.4 Hz, 2H), 0.94 (t, J=7.4 Hz, 3H). 13C NMR (126 MHz, MeOD) delta 172.88, 172.10, 159.09, 150.46, 140.15, 137.66, 135.44, 135.36, 135.21, 130.99, 129.71, 126.83, 126.50, 125.89, 123.02, 119.59, 114.25, 49.85, 43.65, 35.59, 35.40, 30.35, 27.78, 23.33, 14.12. MS (ESI) calculated for C29H30N6O3, m/z 510.24. found 511.25 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
63% | With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 0 - 20℃; | A 500 ml three-neck flask equipped with a magnetic stirring bar and inside thermometer was charged with 300 ml DCM and 1.5 g (1.76 mmol) paclitaxel. 445 mg (2.64 mmol) of N-maleoyl-beta-alanine were added and the mixture was allowed to stir for 15 minutes. The flask was cooled in an ice-water bath to 0 C. 97 mg (0.79 mmol) DMAP and 408 mg (2.64 mmol) EDC were added to the reaction mixture and the mixture was allowed to warm up to room temperature. The course of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was washed twice with 150 ml of a 0.5% sodium bicarbonate solution and twice with 150 ml of 0.1 N hydrochloric acid. The organic phase was further washed with 300 ml of water followed by 300 ml of brine, dried over sodium sulfate and evaporated to dryness. The crude product was applied on silica and purified by column chromatography on silica (DCM/ethyl acetate 1:1). Yield was 1.12 g (1.11 mmol, 63%) of a colorless solid. (1190) 1H-NMR: (CDCl3, 200 MHz) delta=8.17-8.04 (m, 2H); 7.88-7.74 (m, 2H); 7.60-7.17 (m, 10H); 6.48-6.38 (m, 2H); 6.26-5.93 (m, 4H); 5.67-5.55 (m, 1H); 5.46-5.37 (m, 1H); 4.97-4.82 (m, 1H); 4.47-4.07 (m, 3H); 3.98-3.57 (m, 2H); 2.85-2.60 (m, 2H); 2.60-0.95 (m, 26H). (1191) TLC: (DCM/ethyl acetate 1:1) Rf=0.55. |
With dicyclohexyl-carbodiimide; In dichloromethane; at 20℃; for 12h; | Paclitaxel (8.6 mg, 10 mumol) was incubated with 3-Maleimidopropionic acid (1.0 mg, 6 mumol) and dicyclohexylcarbodiimide (4.1 mg, 20 mumol) in 500 muL anhydrous dichloromethane for 12 h at RT (refPreviewPlaceHolderScheme 1). Dicyclohexylurea was removed by filtration. The organic solvent was removed under vacuum and the solid was resuspended in 1 mL CH3OH:H2O (50:50 V/V) containing 0.1% trifluoroacetic acid. The reaction mixture was purified by reverse phase HPLC with eluents of H2O/0.1% TFA (eluent A) and acetonitrile/0.1% TFA (eluent B). The following elution profile (referred to as Method A) was utilized: 0-1 min, 70% A, 30% B; 1-71 min, eluent B was increased from 30% to 100% at a flow rate of 10 mL/min. Elution of the conjugate was monitored by UV absorbance at 220 nM. The purified conjugate was characterized by MALDI Mass Spectra and 1H NMR. Compound 2 (C54H56N2O17): MALDI MNa+ (monoisotopic mass calculated/found: 1027.36/1027.19). 1H NMR reported in ppm (CDCl3): 1.82 (s, 1H, 1-OH); 5.68 (d, 1H, H-2); 3.80 (d, 1H, H-3); 2.45 (s, 3H, 4-OAc); 4.97 (d, 1H, H-5); 2.54 (m, 1H, Ha-6); 1.98 (m, 1H, Hb-6); 4.43 (m, 1H, H-7); 2.40 (d, 1H, 7-OH); 6.28 (s, 1H, H-10); 2.23 (s, 3H, 10-OAc); 6.19 (t, 1H, H13); 2.20-2.30 (band, 2H, H14); 1.24 (s, 3H, Me-16); 1.14 (s, 3H, Me-17); 1.68 (s, 3H, Me-18); 1.93 (s, 3H, Me-19); 4.32 (d, 1H, Ha-20); 4.21 (d, 1H, Hb-20); 5.47 (d, 1H, H-2'); 6.06 (dd, 1H, H-3'); 6.98 (d, 1H, 3'-NH); 7.62-8.13 (m, 5H, C2-OBz); 7.32-7.52 (m, 5H, C3'-Ph); 7.49-7.77 (m, 5H, C3'-NBz) and 2.75 (t, 2H); 3.84 (t, 2H); 6.51 (s, 2H) contributed to 3-maleimidopropionic acid. | |
With dmap; diisopropyl-carbodiimide; In dichloromethane; at 0 - 20℃; for 18h; | Preparation of 2'-maleimide-paclitaxel: Dissolving one gram of paclitaxel (1.2 mmoles) in 160 mL of DCM, adding 0.12 mmole of DMAP and cooling the reaction mixture to 0 C. To the cooled reaction mixture, was added 2.4 mmoles of 3- maleimidopropionic acid, and followed by 1.2 mmoles of DIC under stirring. The reaction mixture was then slowly warmed up to room temperature and the coupling reaction was allowed to proceed at room temperature for 18 h under continuous stirring. Crude product of 2'- maleimide-paclitaxel was purified to a purity >90% and used for conjugation to a cyclic CXCR4 antagonist peptide. [0076] Most of the drugs disclosed herein as a cancer therapy to be conjugated to the high affinity CXCR4 binding ligand peptide conjugate can be activated and incorporated in a similar way known to the person of the art. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
56.5% | Step 62-(2-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)ethoxy)ethyl-4-(/V-(2- (naphthalen-2-yl)imidazo[1 ,2-a]pyrazin-8-yl)sulfamoyl)phenyl carbamate (JS199)17 16 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanoic acid (62.7 mg, 0.371 mmol) and H BTU (210.9 mg, 0.556 mmol) were dissolved in anhydrous DMF (15 ml) and the mixture was purged with Argon. DIPEA (197.4 muIota, 1.1 1 mmol) was added and a colourless to orange colour change was observed after stirring at RT for 20 min. JS178 (367 mg, 0.556 mmol) in anhydrous DMF (5 ml) was added and the reaction was stirred at RT for 16 h. Removal of the solvent was followed by diluting with EtOAc and washing with H20 (4x) and brine, drying (MgS04), filtering and concentrating in vacuo. Flash chromatography (CH2CI2; 2% to 3% to 4% MeOH/CH2CI2) afforded the title compound as an off white solid (146.2 mg, 0.210 mmol, 56.5%). Mpt: >200 C; Rf = 0.43 (10% MeOH/CH2CI2); IR (vmax/cm"1, thin film): 3254 (aromatic C-H stretch), 2925 (C-H and N-H Stretches), 1706 (C=0 stretch), 1589, 1527, 1405, 1224, 1 140; 1 H NMR (600 MHz, CD3CN): deltaEta = 2.38 (t, J = 7.1 Hz, 2H, 36-H), 3.27 (q, J = 5.5 Hz, 2H, 33-H), 3.48 (t, J = 5.6 Hz, 2H, 32-H), 3.64-3.67 (m, 4H, 20,37-H), 4.24-4.26 (m, 2H, 29-H), 6.58 (bs, 1 H, 34-H), 6.72 (s, 2H, 40-H), 7.06 (bd, J = 5.4 Hz, 1 H, 6-H), 7.51 -7.56 (m, 2H, 15,16-H), 7.62-7.65 (m, 3H, 5,24-H), 7.91-7.93 (m, 3H, 14,23-H), 7.95-7.98 (m, 2H, 12,17-H), 8.03 (dd, J = 8.5, 1 .6 Hz, 1 H, 11 -H), 8.20 (s, 1 H, 3-H), 8.31 (bs, 1 H, 20-H), 8.49 (s, 1 H, 19-H); 13C NMR (150 MHz, CD3CN): 5C = 33.8 (C-37), 33.9 (C-36), 38.4 (C-33), 64.0 (C-29), 68.3 (C-30), 68.9 (C-32), 1 10.3 (C-5) 1 14.5 (C-3), 1 15.8 (C-6) 1 17.6 (C-24), 123.4 (C-11 ), 124.2 (C-19), 126.1 (C-15), 126.2 (C-16), 127.1 (C-23), 127.3 (C-14), 127.9 (C-17), 128.2 (C-12), 129.4 (C-10) 132.9 (C-13), 133.2 (C-18), 133.9 (C-40), 135.4 (C-9), 135.8 (C-22), 145.2 (C-2), 142.5 (C-25), 144.7 (C-8) 153.1 (C-27), 169.8 (C-35), 170.5 (C-39); LRMS m/z (ES+): 698 [M+H]+; HRMS m/z (ES+): Found 698.2010; C34H32N7O8S requires 698.2033. |
Yield | Reaction Conditions | Operation in experiment |
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31.9% | With 2,6-dimethylpyridine; In dichloromethane; at 35℃;Inert atmosphere; | In the glove box under an atmosphere of dry nitrogen, to a 250 mL, 4-necked RB flask fitted with an immersion well, egg-shaped magnetic stir bar, and PTFE stoppers was added MPS (35.0 g, 131 mmol) and dry (over sieves) dichloromethane (75 ml) to form a white slurry. Separately, in an Erlenmeyer flask, Lysine-dPEG4-TBE (30.0 g, 66.7 mmol) and 2,6-Lutidine (20 ml, 172 mmol) were dissolved in dry (over sieves) dichloromethane (25 ml) to form a yellow solution. The reaction vessel (RB flask) was transferred to the fume hood, placed in an ice-water bath atop a magnetic stir plate, and fitted with an addition funnel connected by a gas inlet adapter to a bleed of dry nitrogen. A thermocouple was inserted in the immersion well to monitor temperature. The Lysine-dPEG(R)4-TBE solution was poured under a nitrogen shower into the addition funnel and then added slowly by drop. The reaction mixture exothermed strongly. When about [2/3] of the lysine-dPEG(R)4-TBE solution had added, the reaction suddenly congealed into an off-white paste. Added dry (over sieves) Dichloromethane (100 ml), placed the reaction vessel in a heating mantle set at 35[deg.] C., broke up the paste into small chunks so that stirring could resume, and then continued adding the lysine-dPEG(R)4-TBE solution. By the time all of the amine had added, the temperature of the reaction mixture was at 42[deg.] C., and the dichloromethane was boiling. Reaction mixture was yellow solution, a bit darker than before the reaction began. Removed the heating mantle and continued stirring at ambient until the reaction mixture cooled to 35[deg.] C. Added back the heating mantle, set at 30[deg.] C. and continued the reaction overnight at 30[deg.] C. The reaction had lost [1/2] of solvent and was an amber gel the next day. The heat was turned off, and 200 mL dry dichloromethane were added with stirring. All of the product went into solution, then gelled again. The gel was transferred to an Erlenmeyer flask and diluted with stirring to 700 mL with dichloromethane (not dry). Checked reaction by HPLC and TLC. Reaction was not complete. Additional attempts to force the reaction to completion were unsuccessful. TLC using ninhydrin showed no primary amine present, but on extended heating the impurity slowly turned red, suggesting that the amine had reacted but not coupled with a maleimide. The solvent was partially removed by rotary evaporation at 25[deg.] C. under reduced pressure, then dichloromethane was added to 700 mL volume followed by 35 mL methanol. The reaction mixture was washed 1*100 mL with ambient temperature saturated aqueous sodium bicarbonate. Time in contact with this wash solution was kept to a minimum. The reaction mixture was then placed in a salted ice-water bath and chilled with stirring. The mixture was washed 4*100 mL with 600 mM aqueous HCl (ice cold) then washed 2*250 mL with distilled water. The solvent was removed by rotary evaporation, and 55 grams (110%) of crude, impure material was recovered. This material was purified by normal phase flash chromatography on a Teledyne-Isco Torrent flash chromatography system to give 16 grams (31.9%). TLC: 10% Methanol, 90% DCM. HPLC: ACID60FF, 4.747 minutes (ELSD), column is Supelco Discovery HS C18 column, 4.6 mm*25 cm. ACID3045FF, 21.247 minutes (ELSD), same column as above. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With dicyclohexyl-carbodiimide; 4-(dimethylamino)pyridinium tosylate; In acetonitrile; at 20℃; | A solution of 3-maleimidopropionic acid (J. Am. Chem. Soc. 2005, 127, 2966), together with 1 eq of 2-(hydroxyphenyl)diphenylphosphine (Catalysis Today 1998, 42, 413) in anhydrous acetonitrile was treated with a catalytic amount of DPTS, followed by 1.2 eq of DCC and the reaction was stirred at room temperature until completion. The reaction was filtered and the filtrate was concentrated to give a residue, which was purified by flash chromatography on silica gel with ethyl acetate in hexane to give the desired product. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
51% | With dmap; diisopropyl-carbodiimide; In dichloromethane; | CPT (50 mg, 0.144 mmol) was suspended in DCM (8 ml) and DMAP (11 mg, 0.093 mmol), 3-maleimido-propionic acid (70 mg, 0.288 mmol) and DIC (109 mul, 0.698 mmol) were added. The mixture was stirred until complete dissolution of CPT had occurred (overnight), with TLC (3% MeOH in CHCl3) showing complete consumption. The solution was then diluted with CHCl3 (30 ml), extracted with H2O (20 ml), sat. NaHCO3 (20 ml), brine (20 ml), dried over Na2SO4 and concentrated in vacuo. The residue was purified by flash chromatography using 3% MeOH in CHCl3. Product fractions were identified by TLC, combined and solvent removed in vacuo to give CPT-mal as an off-white solid (37 mg, 51%). NMR showed a maleimide impurity, but only a single CPT compound-this was used without further purification; 1H NMR (CDCl3, 300 MHz, 298K): deltaH (ppm) 8.41 (s, 1H), 8.24 (d, 3JHH=8.0, 1H), 7.96 (d, 1H, 3JHH=8.1, 1H), 7.85 (dd, 3JHH=8.4, 1.5, 1H), 7.68 (dd, 3JHH=8.2, 1.2, 1H), 7.19 (s, 1H), 6.67 (s, 2H), 5.66 (d, 2JHH=17.2, 1H), 5.40 (d, 2JHH=17.2, 1H), 5.30 (s, 2H), 3.95-3.75 (m, 2H), 3.00-2.85 (m, 2H), 2.35-2.08 (m, 2H), 0.96 (t, 3JHH=7.5, 3H); 13C NMR (CDCl3, 100 MHz, 298K): 170.6, 169.9, 167.6, 165.8, 162.8, 157.4, 152.6, 149.2, 146.6, 145.8, 131.6, 131.0, 129.9, 128.6, 128.4, 120.6, 96.3, 67.5, 50.3, 33.5, 32.6, 32.1, 22.8, 21.1, 7.9 (FIG. 12); MS (ESI): 499.9 [M+H]+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
99% | With N-ethyl-N,N-diisopropylamine; HATU; In dichloromethane; N,N-dimethyl-formamide; at 120℃; for 1h; | General procedure: General protocol 3 for linker attachment To a solution of the carboxylic acid linker (1 .8 equiv.), HATU (2.1 equiv.) in DCM (0.2 M) were added /'Pr2EtN (1 1 equiv.) followed by the Boc-payload (1 equiv.) in DMF (1 :9 ratio with DCM). The reaction mixture was stirred at RT for 1 h. Upon completion of the reaction by LC/MS (uplc), the reaction mixture was diluted in EtOAc, and partitioned between EtOAc and H20. The organic layers were combined, dried over Na2S04, filtered and solvent evaporated under reduce pressure. Purification of the crude product by chromatography on silica elutuing with 0 - 100% ethylacetate in heptane afforded the title compound. (3R,4R)-tert-butyl 3-(((S)-2-acetoxy-N-((R)-(1 -benzyl-4-(2,5-difluorophenyl)-1 H-imidazol-2- yl)(1 -(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanoyl)piperidin-4- yl)methyl)propanamido)methyl)-4-fluoropyrrolidine-1 -carboxylate 99% yield; UPLC-MS: Rt = 2.58 mins; MS m/z [M+H]+ 849.5; Method E. |
Yield | Reaction Conditions | Operation in experiment |
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64% | 600 mg (1.18 mmol) of 7u was dissolved in 30 ml dry DCM under argon and cooled to 0C in an ice bath. Thereafter, EDC (339 mg, 1.7 mmol, 1.5eq) and DIPEA (413.7 muEpsilon, 2.32 mmol, 2eq) were added and the reaction mixture was stirred for 30 min. 3-Maleimidopropionic acid (240.1 mg, 1.4 mmol, 1.2 eq) was added, and the solution was stirred overnight. Then 40 mL DCM was added and the solution was washed with water (10 mL) and brine (10 mL). The organic layer was dried with Na2S04, filtered, and concentrated at high vacuum. Finally, the crude product was purified by column chromatography (97:3 DCM/MeOH) to yield a yellow oil (501.6 mg, 64%). 1H NMR (CDCI3, 300 MHz): delta 1.44 (s, 27H); 2.44 (t, J=6Hz, 6H); 2.51 (t, J=6Hz, 2H); 3.63 (t, J=6Hz, 6H); 3.67 (s, 6H); 3.80 (t, J=6Hz, 6H); 6.69 (s,2H). ES-MS (m/z): Calcd: 656.35; Found: 657.44 (MH+), 679.31 (M+Na). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
38% | With dmap; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In dichloromethane; at 0 - 20℃; | A 250 ml three-neck flask equipped with a magnetic stirring bar and inside thermometer was charged with 175 ml DCM and 861 mg (1.06 mmol) docetaxel. 270 mg (1.59 mmol) of N-maleoyl-beta-alanine were added and the mixture was allowed to stir for 15 minutes. The flask was cooled in an ice-water bath to 0 C. 58 mg (0.48 mmol) DMAP [4-(dimethylaminopyridine)] and 247 mg (1.59 mmol) EDC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide) were added and the reaction mixture allowed to warm up to room temperature. The course of the reaction was monitored by TLC. After completion of the reaction, the reaction mixture was washed twice with 100 ml of a 0.5% sodium bicarbonate solution and twice with 100 ml of 0.1 N hydrochloric acid. The organic phase was further washed with 200 ml of water followed by 200 ml of brine, dried over sodium sulfate and evaporated to dryness. The crude product was applied on silica and purified by column chromatography on silica (DCM/ethyl acetate 1:1). The yield was 0.38 g (0.40 mmol, 38%) of a colorless solid. (1144) 1H-NMR: (CDCl3, 200 MHz) delta=8.09-8.00 (m, 2H); 7.59-7.18 (m, 8H); 6.62-6.57 (m, 2H); 6.22-6.03 (m, 1H); 5.73-5.56 (m, 2H); 5.46-5.32 (m, 1H); 5.32-5.18 (m, 1H); 5.18-5.09 (m, 1H); 4.94-4.81 (m, 1H); 4.32-4.08 (m, 3H); 3.92-3.57 (m, 3H); 2.72-2.60 (m, 2H); 2.60-1.00 (m, 31H). (1145) TLC: (DCM/ethyl acetate 1:1) Rf=0.40. (1146) MS: (ESI; MeOH) 1013.3 [M+Na++MeOH], 981.3 [M+Na+]; 371.6 [100]. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
44% | With dmap; diisopropyl-carbodiimide; In 1,2-dichloro-ethane; at 0 - 2℃; for 2.5h; | A 100 ml 3-neck flask was equipped with a magnetic stirring bar, a dropping funnel and an inside thermometer. The flask was loaded with 500 mg of sirolimus, 120 mg of 3-maleimidopropionic acid and 33 mg of DMAP. The substances were dissolved in 20 ml of dichloroethane and the mixture cooled to 0 C. 0.17 ml of diisopropylcarbodiimide (DIC) was diluted with 5 ml of DCE and then added to the reaction mixture under control of the temperature (0 C. to 2 C.). The reaction was followed by HPLC. After 2.5 h at 0 C., the reaction mixture was diluted with 100 ml of DCM and quenched with 100 ml of a 0.5% NaHCO3 solution. After the phases were separated, the organic phase was washed with 100 ml of 0.1 N HCl solution and 50 ml of brine. The organic phase was dried with sodium sulfate. The solvent was evaporated under reduced pressure and the crude product purified by column chromatography on silica (DCM:methanol//60:1) to give the title compound (220 mg, 206 mmol, 44%) as a colorless solid. (1203) TLC: (DCM:MeOH//10:1), Rf=0.55. (1204) MS (ESI): m/z=1087.53 [M+Na]+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
With N-ethyl-N,N-diisopropylamine; In tetrahydrofuran; at 20℃;Inert atmosphere; | Step 1: 3-(2,5-Dioxo-2,5-dihydropyrrol-1-yl)propionic acid 2,5-dioxopyrrolidin-1-yl ester (0238) Malimidopropionic acid (500 mg) was dissolved in dry THF (15 ml). TSTU (790 mg) and DIPEA (0.62 ml) was added. The mixture was stirred at room temperature under nitrogen over night. The yellow thick suspension was concentrated. The residue was dissolved in DCM and extracted with 0.1 N HCl (2×) and brine (1×). The organic layer was dried (Na2SO4) and concentrated to give a white solid. LC-MS, M/z: 267.26 (M+1). | |
With N-ethyl-N,N-diisopropylamine; In tetrahydrofuran; at 20℃;Inert atmosphere; | Step 4: 3-(2,5-Dioxo-2,5-dihydropyrrol-1-yl)propionic acid 2,5-dioxopyrrolidin-1-yl ester Malimidopropionic acid (500 mg) was dissolved in dry THF (15 ml). TSTU (790 mg) and DIPEA (0.62 ml) was added. The mixture was stirred at room temperature under nitrogen over night. The yellow thick suspension was concentrated. The residue was dissolved in DCM and extracted with 0.1 N HCl (2*) and brine (1*). The organic layer was dried (Na2SO4) and concentrated to give a white solid. LCMS3: Theoretical mass: 266.21 Found: 267.26 (M+1). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
80% | With 4-methyl-morpholine; benzotriazol-1-yloxyl-tris-(pyrrolidino)-phosphonium hexafluorophosphate; In N,N-dimethyl-formamide; at 0 - 20℃; | To an ice-cooled solution of N-beta-maleimidopropionic acid (108 mg, 0.64 mmol), cPN216-amine (200 mg, 0.58 mmol), and PyBoP (333 mg, 0.64 mmol) in anhydrous DMF at 0 C. was added 0.4 M of N-methylmorpholine in DMF (128 muL, 1.16 mmol). The ice bath was removed after 2 hrs, and the mixture was stirred at room temperature overnight before being subjected to HPLC purification. The product was obtained as a white powder (230 mg, 80% yield). 1H-NMR (400 MHz, DMSO-d6): delta 1.35 (m, 2H), 1.43 (s, 12H), 1.56 (m, 5H), 1.85 (s, 6H), 2.33 (dd, J1=8 Hz, J2=4 Hz, 2H), 2.78 (m, 4H), 3.04 (m, 2H), 3.61 (dd, J1=8 Hz, J2=4 Hz, 2H), 7.02 (s, 2H), 8.02 (s, 1H), 8.68 (s, 4H), 11.26 (s, 2H); m/z=495.2 for [M+H]+ (C24H43N6O5, Calculated MW=495.3). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
85% | 100 mg of 3-maleimidopropionic acid was weighed out and placed in a 100 mL single-necked flask, dissolved in an appropriate amount of acetonitrile,After cooling in ice bath for 15 minutes, 370 mg of benzotriazol-1-yl-oxytripyrrolidinophosphate hexafluorophosphate was added to a one-necked flask and 120 mg of the intermediate 2 obtained by the method of Example S1 was taken, N-diisopropylethylamine 95mg start reaction, ice bath stirring 2h, thin layer chromatography to monitor the progress of the reaction, after the completion of the reaction, the rotary evaporate to remove The solvent was purified by column chromatography on a silica gel column using dichloromethane / methanol as the mobile phase, yielding 124 mg of the final product in 85% yield. | |
113 g | 115.9 mg (0.68 mM) of 3-maleimidopropionic acid was weighed, Put in 100mL single-necked bottle, Add appropriate amount of acetonitrile dissolved, Take 96.6 mg (0.77 mM) N, N-diisopropylcarbodiimide was added to a single neck flask, Stir at room temperature for 1 h. Take 120 mg of intermediate 2, Dissolved in acetonitrile and slowly added to the single-necked flask, stirred at room temperature for 3 h, Thin layer chromatography to monitor the reaction process, After completion of the reaction, The solvent was removed by rotary evaporation, Using methylene chloride / methanol system as mobile phase silica gel column chromatography purification, To give 113 mg of the final product CyP, The yield was 78.51%. | |
113 mg | S2: Weigh 115.9 mg (0.68 mM) of 3-maleimidopropionic acid,Into a 100mL single-necked bottle, add the appropriate amount of acetonitrile to dissolve,96.6 mg (0.77 mM) of N,N-diisopropylcarbodiimide was added to a single-necked flask.Stir at room temperature for 1 h. Take 120mg of intermediate 2,Dissolve in acetonitrile, slowly add to a single-necked flask, stir at room temperature for 3 h.The progress of the reaction was monitored by thin layer chromatography, and after completion of the reaction, the solvent was removed by rotary evaporation.Purification by column chromatography on a mobile phase using a dichloromethane/methanol system.113 mg of the final product CyP was obtained in a yield of 78.51%.The mass spectrum was verified to be 712.1, which is consistent with the theoretical calculation. Nuclear magnetic resonance spectrum: |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
6.9% | With 2,4,6-tripropyl-1,3,5,2,4,6-trioxatriphosphinane-2,4,6-trioxide; triethylamine; In dichloromethane; ethyl acetate; at 20℃; for 16h; | To a stirring solution of 3-(benzo[<i]thiazol-2-yl)-6-isopropyl-4, 5,6,7- tetrahydrothieno[2,3-c]pyridin-2-amine (15 mg, 0.0455 mmol) in DCM (0.5 mL) was added T3P (50 wt% in EtOAc, 32 uL, 0.0501 mmol) and Et3N (19 uL, 0.137 mmol) and the reaction was stirred at room temperature for 16 h. Upon completion, the reaction mixture was quenched with aqueous sat. NaHC03 solution and extracted with DCM (3 x 5 mL). The combined organic layers were washed with brine, dried over Na2S04, filtered, and concentrated under reduced pressure to provide a crude residue, which was purified by silica gel column chromatography eluting with 0-100% ethyl acetate in n-hexane to afford the title compound as a yellow solid (1.5 mg, yield 6.9%). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
A round bottom flask was charged with 5-(2-methoxy-5-(piperazin-i -ylmethyl)benzyl)-N4- pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (lnt-i, 1 .0 equiv.), HBTU (1 .2 equiv.), Huenig?s base (3.0 equiv.), 3-(2,5-dioxo-2,5-dihydro-i H-pyrrol-i -yl)propanoic acid (1.2 equiv.) and DMSO (0.1 M). The reaction mixture was stirred at room temperature for 3 hours and then the crude reaction mixture was purified by RP-HPLC (0.035% TFA in ACN:0.05% TFA in H20, C18colu mn) to afford 1 -(3-(4-(4-((2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl)-3- methoxybenzyl)piperazin-i-yl)-3-oxopropyl)-i H-pyrrole-2,5-dione (C-i) as a solid as the TFA salt: 1H NMR (CDCI3): 3 7.35 (d, 1H), 7.12 (d, 1H), 6.86 (d, 1H), 6.72 (5, 2H), 6.69 (d, 1H), 6.40(5, 1H), 5.46 Ct, 1H), 5.33 Cs, 2H), 4.16 Cs, 2H), 3.95 Cs, 3H), 3.82 Cm, 6H), 3.40 Cm, 4H), 3.21 Cm,2H), 2.67 Cm, 4H), 1.39 Cm, 2H), 1.26 Cm, 2H), 1.14 Cm, 2H), 0.86 Ct, 3H). LRMS [M+H] = 589.3. | ||
A round bottom flask was charged with 5-(2-methoxy-5-(piperazin-1-ylmethyl)benzyl)-N4-pentyl-5H-pyrrolo[3,2-d]pyrimidine-2,4-diamine (lnt-1 , 1 .0 equiv.), HBTU (1 .2 equiv.), Huenig's base (3.0 equiv.), <strong>[7423-55-4]3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanoic acid</strong> (1 .2 equiv.) and DMSO (0.1 M). The reaction mixture was stirred at room temperature for 3 hours and then the crude reaction mixture was purified by RP-HPLC (0.035% TFA in ACN:0.05% TFA in H20, C18 column) to afford 1 -(3-(4-(4-((2-amino-4-(pentylamino)-5H-pyrrolo[3,2-d]pyrimidin-5-yl)methyl)-3- methoxybenzyl)piperazin-1 -yl)-3-oxopropyl)-1 H-pyrrole-2,5-dione (C-1) as a solid as the TFA salt:1H NMR (CDCI3): delta 7.35 (d, 1 H), 7.12 (d, 1 H), 6.86 (d, 1 H), 6.72 (s, 2H), 6.69 (d, 1 H), 6.40 (s, 1 H), 5.46 (t, 1 H), 5.33 (s, 2H), 4.16 (s, 2H), 3.95 (s, 3H), 3.82 (m, 6H), 3.40 (m, 4H), 3.21 (m, 2H), 2.67 (m, 4H), 1 .39 (m, 2H), 1 .26 (m, 2H), 1 .14 (m, 2H), 0.86 (t, 3H). LRMS [M+H] = 589.3. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
A round bottom flask was charged with (2R,3R,4R,55)-6-(4-(((4-(4-((2-amino-4- (pentylamino)-5H-pyrrolo[3,2-d]pyrimid in-5-yl)methyl)-3-methoxybenzyl)piperazine-1 - carbonyl)oxy)methyl)-2-(3-aminopropanamido)phenoxy)-3 ,4 ,5-trihydroxytetrahyd ro-2H-pyran-2- carboxylic acid (1.0 equiv.), 3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1-yl)propanoic acid (2.0 equiv.), Huenig?s base (6.0 equiv.), HBTU (1 .8 equiv.) and DMF (0.003 M). The reaction was keptstirring at room temperature for 15 minutes. The reaction mixture was stirred at room temperature for 2 hours. The crude reaction mixture was then purified by RP-HPLC (0.035% TFA in ACN:0.05% TFA in H20, C18 column) to afford (2R,3R,4R,5S)-6-(4-(((4-(4-((2-amino-4- (pentylamino)-5H-pyrrolo[3,2-d]pyrimid in-5-yl)methyl)-3-methoxybenzyl)piperazine-1 - carbonyl)oxy)methyl)-2-(3-(3-(2,5-dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanamido)propanamido)phenoxy)-3,4,5-trihydroxytetrahyd ro-2H-pyran-2-carboxylic acid (C-31) as a solid as the TFA salt: LCMS [M+H] = 1001.3. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
31.4% | A compound having a structure of formula (21)(0.2 g, 0.221 mmol) was dissolved in 3 mL of dichloromethane,3-maleimidopropionic acid (0.0374 g, 0.221 mmol) was added,After all the addition, N, N-dicyclohexylcarbodiimide (0.0456 g, 0.221 mmol) was added,At room temperature reaction stirring overnight, TCL detection is not finished, then add1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride(0.127 g, 0.663 mmol) and N, N-dicyclohexylcarbodiimide (0.075 g, 0.422 mmol), TCLTest reaction is completed, spin dry methylene chloride, column purification,To give 50 mg of a colorless oily liquid having the structure of formula (22), the yield of this step being 31.4%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
[00246] Commercially available Rink amide polystyrene resin (265mg, 1/16 mmol) was placed in the reaction vessel of a CS Biosystems (CA) automatic peptide synthesizer programmed to perform the following reaction cycle: (a) wash with dimethylformamide (DMF), (b) remove the Fmoc protecting group by mixing with 20% piperidine in DMF (20 min) ), and (c) couple (4 h) a protected amino acid (1.5 mmole) to the deprotected resin using diisopropylcarbodiimide (DIC) (1.5 mmole) and hydroxybenzotriazole (HOBt) (3.00 mmole) in N-methylpyrolidone, beginning with the C-terminal amino acid. The following protected amino acids were coupled: Fmoc-DSer(Trt), Fmoc-Lys(Mtt), Fmoc-DSer(Trt), Fmoc-Ser(Trt), Fmoc-Thr(Trt), Fmoc-Ser(Trt), Fmoc-DSer(Trt), Fmoc-Asp(tBu), Fmoc-DAsp(tBu), Fmoc-Asp(tBu), Fmoc-DAsp(tBu). At each stage the Fmoc group on the growing protected peptide chain was removed by mixing with 20% piperidine in DMF (20 min). The protected peptide resin containing a free N-terminal amino group was mixed with tacrolimus- 32-thiol-3-mercaptoproprionic acid disulfide (150 mg, 0.16 mmol), DIC (3 equiv), and HOBt (3 equiv), and allowed to react overnight and then washed with (0370) dichloromethane. (0371) [00247] The Mtt group on the epsilon amino group of the Lys residue was removed selectively by washing 10 times with a 1.9% solution of trifluoroacetic acid in dichloromethane followed by washing 5 times with N-methylpyrolidone. The resin was then reacted (3 h) with a 3-fold excess of N-maleoyl-b-alanine using an equivalent amount of diisopropylcarbodiimide / HOBT. After washing with dichloromethane, the tacrolimus maleimide peptide was cleaved from the resin and all protecting groups were removed from amino acid side chains by treatment (2 hours) with 10 ml of a mixture of DCM:trifluoroacetic acid:1,3- dimethoxybenzene:triisopropylsilane (6.75:2.5:0.5:0.25). The peptide conjugate was precipitated 3 times with ethyl ether and spun down on centrifuge (3000 rpm). The crude tacrolimus peptide maleimide was purified on silica gel using BAW 4:1:1 (butanol:acetic acid:water). Fractions containing pure compound were collected and lyophilized to yield a white powder. MALDI MS gave a MW of 2199 (see FIG.4C). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
50.0% | Compound 2 (1 g, 5.9 mmol) was dispersed in 12 mL of acetone, and under a nitrogen atmosphere, TEA (0.9 mL, 6.5 mmol) was added to the reaction mixture at -5 C, and the reaction liquid was clarified.5 mL of isopropyl chloroformate (0.68 mL, 6.5 mmol) in acetone was slowly added dropwise, and the solution was reacted for 30 min after the addition.Sodium azide (0.38 g, 5.9 mmol) was added thereto, and the reaction was continued for 2 h.After the reaction was completed, the reaction solution was poured into 30 mL of water and extracted twice with 40 mL of toluene.The organic phase is combined, washed with saturated sodium chloride, filtered through silica gel, and then partially evaporated.It was dried over anhydrous magnesium sulfate overnight.The filtrate (Compound 3) obtained after filtration was then heated under reflux for 1 h under nitrogen.After the reaction is complete, the solvent is removed.Vacuum drying gave 0.5 g of a yellow oily liquid as Compound 4 in a yield of 50.0%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
52% | With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; N-ethyl-N,N-diisopropylamine; In N,N-dimethyl-formamide; at 60℃; for 1h; | To a solution of N-(trans-4-((6-aminohexyl)carbamoyl)cyclohexyl)-4-(2-(ethyl(m- tolyl)amino)-2-oxoethyl)-4H-thieno[3,2-b]pyrrole-5-carboxamide (50 mg, 0.088 mmol) in DMF (4 mL), HOBT (27 mg, 0.18 mmol), EDC (34 mg, 0.18 mmol), diisopropyethylamine (34 mg, 0.26 mmol), and 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoic acid (22 mg, 0.13 mmol) was added. The solution was heated to 60C for 1 h. The mixture was diluted with ethyl acetate and washed with water. The organic layers were combined, dried with sodium sulfate, filtered, concentrated, and purified with silica gel chromatography to afford the desired product (33 mg, 52%) as a white solid. 1H MR (300 MHz, DMSO-i) delta 7.95 - 7.84 (m, 2H), 7.72 - 7.64 (m, 1H), 7.45 - 7.34 (m, 2H), 7.33 - 7.17 (m, 3H), 7.11 - 7.05 (m, 2H), 6.99 (s, 2H), 4.96 (s, 2H), 3.74 - 3.48 (m, 5H), 3.05 - 2.89 (m, 4H), 2.36 (s, 3H), 2.29 (t, J= 7.2, 2H), 2.12 - 1.94 (m, 1H), 1.89 - 1.68 (m, 4H), 1.52 - 1.15 (m, 12H), 1.08 - 0.95 (m, 3H); ESI MS m/z 717 [M + H]+; HPLC 98.5 % (AUC), TR5.63 min; UV (MeOH) lambda 289 nm, epsilon 25000. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
19.6% | With benzotriazol-1-ol; 1,2-dichloro-ethane; In N,N-dimethyl-formamide; at 20℃; for 2h;Inert atmosphere; | H2N-L-Ala-D-Ala-L-Ala-CH2-S-(CH2)5-CO-DM (8 mg, 7.82 jtmol), was dissolved in DMF (2 mL), treated with 3-maleimidopropanoic acid (1.32 mg, 7.82 jtmol), EDC (2.25 mg, 0.012 mmol) and HOBt (1.198 mg, 7.82 imol). The reaction was allowed to proceed at room temperature with magnetic stirring under an argron atmosphere for 2 h. The crude material was purified via semi-prep HPLC using a XDB-C18, 21.2x5mm, 5 im eluting with deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% over 30 mm at 20 milmin. Fractions containing desired product were immediately combined and frozen then lypholized to give 1.8 mg (19.60 % yield) of white solid. HRMS (M+H) calcd. 1173.4940, found 1173.493 1. ?H NMR (400 MHz, DMSO-d6) 0.71 (s, 3H), 1.02- 1.14 (m, 15H), 1.16 - 1.25 (m, 3H), 1.30- 1.44 (m, 5H), 1.52 (s, 3H), 1.92-2.03 (m, 1H), 2.03 -2.17 (m, 1H), 2.23-2.39 (m, 4H), 2.63 (s, 3H), 2.73 (d, J= 9.6 Hz, 1H), 3.02 (s, 3H), 3.18 (s, 4H), 3.33 -3.46 (m, 2H), 3.52 (t, J = 7.3 Hz, 2H), 3.86 (s, 3H), 3.95 -4.17 (m, 7H), 4.45 (dd, J =12.0, 2.9 Hz, 1H), 5.27 (q, J = 6.7 Hz, 1H), 5.44 - 5.56 (m, 1H), 5.85 (s, 1H), 6.39 - 6.64 (m, 3H), 6.81 (s, 1H), 6.86 (s, 1H), 6.92 (s, 2H), 7.11 (d, J= 1.7 Hz, 1H), 7.89 (d, J= 7.4 Hz, 1H),8.10 (d, I = 7.3 Hz, 1H), 8.17 (d, J = 6.7 Hz, 1H), 8.28 (t, J = 6.3 Hz, 1H), 8.43 (s, 1H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
tert-Butyl 2-(hydroxymethyl)piperazine-l-carboxylate (200 mg, 0.93 mmol) was dissolved in dichloromethane (10 mL) at 0 C. Diisopropylethylamine (359 mg, 2.78 mmol) and HATU (422 mg, 1.11 mmol) were added and the solution was stirred 15 min at 0 C. 3-maleimidopropionic acid (187 mg, 1.11 mmol) was added, and then the reaction was stirred 12 h at room temperature under argon. The reaction was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate and concentrated. Purification by flash chromatography (neutral alumina, 2% methanol : ethyl acetate provided the title material L-12a. LCMS (method 6): retention time = 2.87 min. | ||
tert-Butyl 2-(hydroxymethyl)piperazine-1-carboxylate (200 mg, 0.93 mmol) was dissolved in dichloromethane (10 mL) at 0 C. Diisopropylethylamine (359 mg, 2.78 mmol) and HATU (422 mg, 1.11 mmol) were added and the solution was stirred 15 min at 0 C. 3-maleimidopropionic acid (187 mg, 1.11 mmol) was added, and then the reaction was stirred 12 h at room temperature under argon. The reaction was diluted with water and extracted with dichloromethane. The organic layer was washed with brine, dried over sodium sulfate and concentrated. Purification by flash chromatography (neutral alumina, 2% methanol:ethyl acetate provided the title material L-12a. LCMS (method 6): retention time=2.87 min. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 4 steps 1.1: oxalyl dichloride; N,N-dimethyl-formamide / dichloromethane / 0.5 h / 20 °C / Sealed tube 2.1: N-ethyl-N,N-diisopropylamine / dichloromethane / 20 - 25 °C / Large scale 2.2: 20 °C / Large scale 3.1: oxalyl dichloride; N,N-dimethyl-formamide / dichloromethane / 0.5 h / 20 °C 4.1: triethylamine / dichloromethane / 20 - 25 °C 4.2: 20 °C | ||
Multi-step reaction with 3 steps 1: 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride / dichloromethane / 2 h / 20 °C 2: sodium hydrogencarbonate / water; 1,2-dimethoxyethane / 2 h / 20 °C 3: 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride / dichloromethane / 2 h / 20 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
76.8% | Compound 2 (161 mg, 0.95 mmol) was dispersed in 10 mL DCM, and HATU (433 mg, 1.14 mmol) was added thereto, DIEAAfter reacting (0.5 mL, 2.85 mmol) for 15 min, compound 24 (600 mg, 0.95 mmol) was added thereto and allowed to react at room temperature overnight.After the reaction is completed, the reaction solution is diluted with DCM, and the organic phase is washed with water and saturated sodium chloride and dried over anhydrous sodium sulfate.Chromatographic separationAfter the reaction, 500 mg of yellow solid was obtained, and the yield was 76.8%. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
66.7% | Compound 2 (93 mg, 0.55 mmol) was dispersed in 10 mL DCM, and HATU (251 mg, 0.66 mmol), DIEA (212.8 mg, 1.65 mmol) was added thereto and reacted for 15 min.Compound 29 (300 mg, 0.55 mmol) was added thereto, and the mixture was reacted at room temperature overnight.After the reaction is completed, the reaction solution is diluted with DCM, and the organic phase is washed with water and saturated sodium chloride.Drying with anhydrous sodium sulfate, separation and purification by column chromatography to obtain 220 mgYellow solid, yield 66.7%, |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
61.0% | With N-ethyl-N,N-diisopropylamine; N-[(dimethylamino)-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate; In dichloromethane; at 20℃; for 12h; | Compound 2 (72 mg, 0.43 mol) was dispersed in 10 mL DCM.After adding HATU (176 mg, 0.46 mmol), DIEA (0.19 mL, 1.07 mmol), compound 33 (200 mg, 0.36 mmol),Reaction at room temperature for 12 h,After the reaction is completed, the reaction solution is diluted with water, extracted with DCM, and the organic phase is saturated aqueous ammonium chloride,After washing with saturated brine, it is dried with anhydrous Na2SO4 , filtered and dried, and then purified by column chromatography.Obtained 135mg of pale yellow solid, yield 61.0%, |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
1.2 g | With N-ethyl-N,N-diisopropylamine; In tetrahydrofuran; N,N-dimethyl-formamide; at 20℃; for 2.5h; | 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 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 acid2,5-dioxopyrrolidiny-1 -yl ester. MS: mlz=289, required for [M+Na]: 28910154] ?H-NMR (CDC13) oe 2.82 (m, 4H); 3.02 (t, 2H); 3.94 (t, 2H), 6.73 (s, 2H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
19.6% | With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20℃; for 2h;Inert atmosphere; | H2N-L-Ala-D-Ala-L-Ala-CH2-S-(CH2)5-CO-DM (8 mg, 7.82 miho), was dissolved in DMF (2 mL), treated with 3-maleimidopropanoic acid (1.32 mg, 7.82 pmol), EDC (2.25 mg, 0.012 mmol) and HOBt (1.198 mg, 7.82 pmol). The reaction was allowed to proceed at room temperature with magnetic stirring under an argron atmosphere for 2 h. The crude material was purified via semi-prep HPLC using a XDB-C18, 2l.2x5mm, 5 pm eluting with deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% over 30 min at 20 ml/min. Fractions containing desired product were immediately combined and frozen then lypholized to give 1.8 mg (19.60 % yield) of white solid. HRMS (M+H)+ calcd. 1173.4940, found 1173.4931. 1H NMR (400 MHz, DMSO-d6) d 0.71 (s, (1132) 3H), 1.02 - 1.14 (m, 15H), 1.16 - 1.25 (m, 3H), 1.30 - 1.44 (m, 5H), 1.52 (s, 3H), 1.92 - 2.03 (m, 1H), 2.03 - 2.17 (m, 1H), 2.23 - 2.39 (m, 4H), 2.63 (s, 3H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 (s, 3H), 3.18 (s, 4H), 3.33 - 3.46 (m, 2H), 3.52 (t, 7 = 7.3 Hz, 2H), 3.86 (s, 3H), 3.95 - 4.17 (m, 7H), 4.45 (dd, 7 = 12.0, 2.9 Hz, 1H), 5.27 (q, 7 = 6.7 Hz, 1H), 5.44 - 5.56 (m, 1H), 5.85 (s, 1H), 6.39 - 6.64 (m, 3H), 6.81 (s, 1H), 6.86 (s, 1H), 6.92 (s, 2H), 7.11 (d, 7 = 1.7 Hz, 1H), 7.89 (d, 7 = 7.4 Hz, 1H), 8.10 (d, J = 7.3 Hz, 1H), 8.17 (d, 7 = 6.7 Hz, 1H), 8.28 (t, 7 = 6.3 Hz, 1H), 8.43 (s, 1H). |
19.6% | With benzotriazol-1-ol; 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride; In N,N-dimethyl-formamide; at 20℃; for 2h;Inert atmosphere; | H2N-L-Ala-D-Ala-L-Ala-CH2-S-(CH2)5-CO-DM (8 mg, 7.82 miho), was dissolved in DMF (2 mL), treated with 3-maleimidopropanoic acid (1.32 mg, 7.82 pmol), EDC (2.25 mg, 0.012 mmol) and HOBt (1.198 mg, 7.82 pmol). The reaction was allowed to proceed at room temperature with magnetic stirring under an argron atmosphere for 2 h. The crude material was purified via semi-prep HPLC using a XDB-C18, 2l.2x5mm, 5 pm eluting with deionized water containing 0.1% formic acid and a linear gradient of acetonitrile from 5% to 95% over 30 min at 20 ml/min. Fractions containing desired product were immediately combined and frozen then lypholized to give 1.8 mg (19.60 % yield) of white solid. HRMS (M+H)+ calcd. 1173.4940, found 1173.4931. 1H NMR (400 MHz, DMSO-d6) d 0.71 (s, (1132) 3H), 1.02 - 1.14 (m, 15H), 1.16 - 1.25 (m, 3H), 1.30 - 1.44 (m, 5H), 1.52 (s, 3H), 1.92 - 2.03 (m, 1H), 2.03 - 2.17 (m, 1H), 2.23 - 2.39 (m, 4H), 2.63 (s, 3H), 2.73 (d, J = 9.6 Hz, 1H), 3.02 (s, 3H), 3.18 (s, 4H), 3.33 - 3.46 (m, 2H), 3.52 (t, 7 = 7.3 Hz, 2H), 3.86 (s, 3H), 3.95 - 4.17 (m, 7H), 4.45 (dd, 7 = 12.0, 2.9 Hz, 1H), 5.27 (q, 7 = 6.7 Hz, 1H), 5.44 - 5.56 (m, 1H), 5.85 (s, 1H), 6.39 - 6.64 (m, 3H), 6.81 (s, 1H), 6.86 (s, 1H), 6.92 (s, 2H), 7.11 (d, 7 = 1.7 Hz, 1H), 7.89 (d, 7 = 7.4 Hz, 1H), 8.10 (d, J = 7.3 Hz, 1H), 8.17 (d, 7 = 6.7 Hz, 1H), 8.28 (t, 7 = 6.3 Hz, 1H), 8.43 (s, 1H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
60% | With N-ethyl-N,N-diisopropylamine; N-[(dimethylamino)-3-oxo-1H-1,2,3-triazolo[4,5-b]pyridin-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate; In N,N-dimethyl-formamide; at 20℃; for 1.5h; | Compound 1 (105.7 mg, 0.388 mmol) was combined with maleimide (238.69 mg, 1.55 mmol) and dissolved in three mL dimethylformamide. hexafluorophosphate azabenzotriazole tetramethyl uranium (HATU) (393.8 mg, 1.861 mmol) was added to the mixture. See, FIG. 12. The reaction was stirred for 15 minutes at room temperature before adding N,N-diisopropylethylamine (DIPEA) (256.6 pL, 2.713 mmol). The reaction could proceed at room temperature for seventy-five minutes and monitored through TLC (20% ethyl acetate/80% hexanes). TLC Silica Gel 60 on glass plates is commercially available from MilliporeSigma (Billerica, MA, USA The crude reaction was transferred to a separatory funnel and partitioned between dichloromethane and 0.1 M HCI to separate the layers. The dichloromethane was rinsed again with HCI and then rinsed twice with water. All experiments used ultrapure deionized water (18.2 MQ*cm, commercially available from MilliporeSigma, Billerica, MA, USA). The combined organic layers were washed with brine and dried over sodium sulfate. The dichloromethane was then evaporated at reduced pressure. The conjugate was stored at -20C. Compound 3 was dissolved in dimethyl sulfoxide at a concentration of approximately fifty mg/ml and diluted with water. The suspension was stored at 4C overnight. Afterward, the suspension was centrifuged for twenty minutes at 4C and 5000 rpm. The supernatant was removed at the rinsing process was repeated. After the final rinse, the product was frozen, lyophilized, and stored at -20C (60% yield). Six mg of Compound 3 dissolved in deuterated-dimethyl sulfoxide were tested using 1H-NMR on a Bruker 400 MHz instrument to confirm conjugation. Deuterated dimethyl sulfoxide (DMSO-d6) and deuterium oxide are commercially available from Cambridge Isotope Laboratories (Andover, MA, USA). [00120] 1H-NMR indicated the disappearance of the 3-maleimidopropionic acid carboxylic group proton at 12.36 ppm, along with the appearance of the newly formed amide?s proton split at 8.98 and 9.06 ppm. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
72% | To a solution of 3-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)propanoic acid (500 mg, 2.96 mmol) in 20 ml of dry DCM was added 1.0 M DCC in DCM (3.55 ml, 3.55 mmol) and N-hydroxysuccinimide (408 mg, 3.55 mmol) and the solution stirred at r.t. until the reaction was complete by HPLC (approximately 1 hr). The urea byproduct was removed by filtration and to the solution was added 2-((3- ((3-(2-(2-(3-aminopropoxy)ethoxy)ethoxy)propyl)carbamoyl) -2-oxo-2H-chromen- 7-yl)oxy)acetic acid 2,2,2-trifluoroacetic acid salt( 1 : 1) (1.20 g, 2.00 mmol) and triethyl amine (700 pL, 5.00 mmol) and the reaction allowed to stir o.n. at room temperature. The crude reaction was filtered through a 0.2 micron filter and purified by preparative HPLC. The pure fractions were combined and lyophilized to give 889 mg (72% yield) of 2-((3-((l7-(2,5-dioxo-2,5-dihydro-lH-pyrrol-l-yl)-l5-oxo- 4,7,10-trioxa- l4-azaheptadecyl)carbamoyl)-2-oxo-2H-chromen-7-yl)oxy)acetic acid (compound 40). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
87% | Cl-T rtCI-resin (5 g, 1 .49 mmol/g) was placed in a filter plate. To the resin was added CH2CI2 (25 mL) and the mixture was stirred for 1 h at 23 SC. The solvent was eliminated by filtration over vacuum. A solution of Fmoc-Cit-OH (2.95 g, 7.44 mmol) and DIPEA (4.29 mL, 24.61 mmol) in CH2CI2 (20 mL) was added and the mixture was stirred for 10 min at 23 SC. DIPEA (8.70 mL, 49.99 mmol) was additionally added and the mixture was stirred for 1 h at 23 SC. The reaction was stopped by addition of MeOH (10 mL) and stirred 15 min at 23 SC. The Fmoc-Cit-O-TrtCI-resin was subjected to the following washing/treatments: CPI2CI2 (5 x 15 mL x 0.5 min), DMF (5 x 15 mL x 0.5 min), piperidine:DMF (1 :4, 15 mL, 1 x 1 min, 2 x 10 min), DMF (5 x 15 mL x 0.5 min), CH2CI2 (5 x 15 mL x 0.5 min). The loading was calculated: 1 .17 mmol/g. (1603) The NF -Cit-O-TrtCI-resin was washed with DMF (5 x 15 mL x 0.5 min) and a solution of Fmoc-Val-OH (7.80 g, 22.99 mmol) and HOBt (2.80 g, 24.5 mmol) in DMF (25 mL) was added to the NFb-Cit-O-TrtCI-resin followed by addition of DIPCDI (3.56 mL, 24.5 mmol) at 23 aC. The reaction mixture was stirred for 1 .5 h at 23 SC. The reaction was stopped by washing with DMF (5 x 15 mL x 0.5 min). The Fmoc-Val-Cit-O-TrtCI-resin was treated with piperidine:DMF (1 :4, 15 mL, 1 x 1 min, 2 x 10 min) and washed with DMF (5 x 15 mL x 0.5 min). (1604) A solution of 15-(9-Fluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoic acid (Fmoc-NH-PEG4-OH) (4.27 g, 8.75 mmol) and HOBt (1 .18 g, 8.72 mmol) in DMF (30 mL) was added to the NH2-Val-Cit-0-TrtCI-resin followed by addition of DIPCDI (1 .35 mL, 8.72 mmol) at 23 SC. The reaction mixture was stirred for 24 h at 23 SC. The reaction was stopped by washing with DMF (5 x 15 mL x 0.5 min). The Fmoc-NH-PEG4-Val-Cit-0-TrtCI-resin was treated with piperidine:DMF (1 :4, 15 mL, 1 x 1 min, 2 x 10 min) and washed with DMF (5 x 15 mL x 0.5 min). (1605) A solution of 3-(Maleimido)propionic acid (MC2-OH) (3.95 g, 23.35 mmol) and HOBt (3.16 g, 23.37 mmol) in DMF (30 mL) was added to the NH2-PEG4-Val-Cit-0-TrtCI-resin followed by addition of DIPCDI (3.62 mL, 23.37 mmol) at 23 SC. The reaction mixture was stirred for 2 h at 23 SC. The reaction was stopped by washing with DMF (5 x 15 mL x 0.5 min) and CH2CI2 (5 x 15 mL x 0.5 min). (1606) The peptide was cleaved from the resin by treatments with TFA:CH2Cl2 (1 :99, 5 x 50 mL). The resin was washed with CH2CI2 (7 x 50 mL x 0.5 min). The combined filtrates were evaporated to dryness under reduced pressure, the solid obtained was triturated with Et20 and filtrated to obtain LIN 2-1 (4.59 g, 87% yield) as a white solid. (1607) 1H NMR (300 MHz, CDCI3): d 7.67-7.57 (m, 1H), 7.44 (d, J= 8.3 Hz, 1H), 7.11 (t, J= 5.4 Hz, 1 H), 6.73 (s, 2H), 4.49 (d, J = 7.2 Hz, 1H), 4.35 (t, J = 7.7 Hz, 1H), 3.82 (t, J = 7.0 Hz, 2H), 3.74 (t, J= 6.2 Hz, 2H), 3.68-3.56 (m, 13H), 3.56-3.45 (m, 2H), 3.39 (q, J= 5.4 Hz, 2H), 3.17 (s, 2H), 2.55 (q, J= 7.0, 6.0 Hz, 4H), 2.16-1.99 (m, 1H), 1.91 (s, 1H), 1.75 (s, 1H), 1.43 (s, 2H), 0.94 (d, = 9.7 Hz, 3H), 0.93 (d, = 9.7 Hz, 3H). (1608) ESI-MS m/z: 673.3 (M+H)+. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
87% | Cl-T rtCI-resin (5 g, 1 .49 mmol/g) was placed in a filter plate. To the resin was added CH2CI2 (25 mL) and the mixture was stirred for 1 h at 23 SC. The solvent was eliminated by filtration over vacuum. A solution of Fmoc-Ala-OH (2.31 g, 7.41 mmol) and DIPEA (4.28 mL, 24.61 mmol) in CH2CI2 (20 mL) was added and the mixture was stirred for 10 min at 23 SC. DIPEA (8.60 mL, 49.37 mmol) was additionally added and the reaction mixture was stirred for 1 h at 23 SC. The reaction was stopped by addition of MeOH (10 mL) and stirred 15 min at 23 SC. The Fmoc-Ala-O-TrtCI-resin was subjected to the following washing/treatments: CH2CI2 (5 x 15 mL x 0.5 min), DMF (5 x 15 mL x 0.5 min), piperidine:DMF (1 :4, 15 mL, 1 x 1 min, 2 x 10 min), DMF (5 x 15 mL x 0.5 min), CH2CI2 (5 x 15 mL x 0.5 min). The loading was calculated: 1 .34 mmol/g. (1635) The NFL-Ala-O-TrtCI-resin was washed with DMF (5 x 15 mL x 0.5 min) and a solution of Fmoc-Val-OH (9.09 g, 26.79 mmol) and HOBt (3.62 g, 26.79 mmol) in DMF (25 mL) was added to the NFL-Ala-O-TrtCI-resin followed by addition DIPCDI (4.14 mL, 26.79 mmol) at 23 aC. The mixture was stirred for 1 .5 h at 23 SC. The reaction was stopped by washing with DMF (5 x 15 mL x 0.5 min). The Fmoc-Val-Ala-O-TrtCI-resin was treated with piperidine:DMF (1 :4, 15 mL, 1 x 1 min, 2 x 10 min) and washed with DMF (5 x 15 mL x 0.5 min). (1636) A solution of 15-(9-Fluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoic acid (Fmoc-NH-PEG4-OH) (4.90 g, 8.75 mmol) and HOBt (1 .35 g, 9.98 mmol) in DMF (30 mL) was added to the NH2-Val-Ala-0-TrtCI-resin followed by addition DIPCDI (1 .55 mL, 10.0 mmol) at 23 SC. The reaction mixture was stirred for 22 h at 23 SC. The reaction was stopped by washing with DMF (5 x 15 mL x 0.5 min). The Fmoc-NH-PEG4-Val-Ala-0-TrtCI-resin was treated with piperidine:DMF (1 :4, 15 mL, 1 x 1 min, 2 x 10 min) and washed with DMF (5 x 15 mL x 0.5 min). (1637) A solution of 3-(Maleimido)propionic acid (MC2-OH) (4.53 g, 26.78 mmol) and HOBt (3.62 g, 26.77 mmol) in DMF (30 mL) was added to the NH2-PEG4-Val-Ala-0-TrtCI-resin followed by addition of DIPCDI (4.15 mL, 26.80 mmol) at 23 SC. The reaction mixture was stirred for 2 h at 23 SC. The reaction was stopped by washing with DMF (5 x 15 mL x 0.5 min) and CH2CI2 (5 x 15 mL x 0.5 min). (1638) The peptide was cleaved from the resin by treatments with TFA:CH2Cl2 (1 :99, 5 x 50 mL). The resin was washed with CH2CI2 (7 x 50 mL x 0.5 min). The combined filtrates were evaporated to dryness under reduced pressure, the solid obtained was triturated with Et20 and filtrated to obtain L 3-1 (4.73 g, 87% yield) as a white solid. 1H NMR (500 MHz, CDCIs): d 7.67 (bs, 1H), 7.31 (d, J= 8.9 Hz, 1H), 7.17 (d, J= 7.0 Hz, 1H), 6.85 (t, J = 5.6 Hz, 1 H), 6.72 (s, 2H), 4.51 (q, J = 7.1 Hz, 1 H), 4.38 (dd, J = 8.9, 6.9 Hz, 1 H), 3.84 (t, J= 7.1 Hz, 2H), 3.75 (t, J= 5.9 Hz, 2H), 3.69-3.59 (m, 12H), 3.55 (t, J= 5.1 Hz, 2H), 3.41 (qd, J= 5.0, 1.7 Hz, 2H), 2.62-2.49 (m, 4H), 2.19-2.01 (m, 1H), 1.44 (d, J= 7.2 Hz, 3H), 0.95 (d, J = 11.9 Hz, 1 H), 0.94 (d, J = 11.9 Hz, 1 H). |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
88% | TEA (6.7 g, 0.066 mmol) and HATU (5.0 mg, 0.013 mmol) was added to a solution of 3-(2,5- dioxo-2,5-dihydro-1 H-pyrrol-1 -yl)propanoic acid (2.2 mg, 0.013 mmol)) in DMF (1 L) and the mixture was stirred for 5 mins CDN intermediate (CDNi-3) (15 mg, 0.013 mmol) in DMF (1 mi) was then added and the mixture was stirred for 18 hrs at room temperature and then concentrated. The residue was dissolved in DMSO (2 ml) and then purified by mass triggered reverse phase HPLC, using C18 column, eluted with 5-25% acetonitrile-H20 containing 0.05% TFA. Fractions containing desired product were lyophilized to obtain Compound (C17) (14.3 mg, 88 % yield) as TFA salt. LCMS M+1 =943.1 tr= 0.561 min. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
59% | TEA (1 .3 mg, 0.013 mmol) and HATU (5 mg, 0.013 mmol) were added to a solution of 3-(2,5- dioxo-2,5-dlhydro-1 H-pyrrol-1 -yl)propanoic acid (2.2 mg, 0.013 mmol) in DMF (1 mL) and the mixture was stirred for 5 mins. A solution of CDN intermediate (CDiSSS-1 ) TFA salt (15 mg, 0.013 mmol) in DMF (1 ml) was then added and the mixture was stirred for 18 hrs at room temperature and then concentrated. The residue was dissoived in DMSO (2 ml) and then purified by mass triggered reverse phase HPLC using C18 column, eluted with 5-25% acetoniirile-H20 containing 0.05% TFA. Fractions containing desired product were iyophilized to obtain Compound (C2) (8.7 rng, 59 % yieid) as TFA salt. LCMS M+1 =947.1 , tr= 0.646 min. |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
84% | Stage #1: 3-maleimidepropionic acid With 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride In dichloromethane at 0℃; for 0.25h; Stage #2: 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethanamine In dichloromethane at 0 - 20℃; for 12h; |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
Multi-step reaction with 2 steps 1.1: 1-ethyl-(3-(3-dimethylamino)propyl)-carbodiimide hydrochloride / dichloromethane / 0.25 h / 0 °C 1.2: 12 h / 0 - 20 °C 2.1: tris[(1-benzyl-1H-1,2,3-triazol-4yl)methyl]amine; copper(ll) sulfate pentahydrate; sodium L-ascorbate / dichloromethane; water / 18 h / 20 °C 2.2: 12 h / 20 °C |
Yield | Reaction Conditions | Operation in experiment |
---|---|---|
87% | Stage #1: N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine With N-ethyl-N,N-diisopropylamine In dichloromethane at 23℃; for 2.16667h; Stage #2: With piperidine In dichloromethane; N,N-dimethyl-formamide Stage #3: 3-maleimidepropionic acid; Fmoc-Val-OH; 15–(9–fluorenylmethyloxycarbonyl)amino–4,7,10,13–tetraoxa–pentadecanoic acid Further stages; | 2.a (a) Preparation of LIN 3-1: MC2-PEG4-Val-Ala-OH Cl-TrtCl-resin (5 g, 1.49 mmol/g) was placed in a filter plate. To the resin was added CH2CI2 (25 mL) and the mixture was stirred for 1 h at 23 °C. The solvent was eliminated by filtration over vacuum. A solution of Fmoc-Ala-OH (2.31 g, 7.41 mmol) and DIPEA (4.28 mL, 24.61 mmol) in CH2CI2 (20 mL) was added and the mixture was stirred for 10 min at 23 °C. DIPEA (8.60 mL, 49.37 mmol) was additionally added and the reaction mixture was stirred for 1 h at 23 °C. The reaction was stopped by addition of MeOH (10 mL) and stirred 15 min at 23 °C. The Lmoc-Ala-O-TrtCl-resin was subjected to the following washing/treatments: CH2CI2 (5 x 15 mL x 0.5 min), DML (5 x 15 mL x 0.5 min), piperidine :DML (1:4, 15 mL, l x l min, 2 x 10 min), DML (5 x 15 mL x 0.5 min), CH2CI2 (5 x 15 mL x 0.5 min). The loading was calculated: 1.34 mmol/g.The NPh-Ala-O-TrtCl-resin was washed with DML (5 x 15 mL x 0.5 min) and a solution of Lmoc-Val-OH (9.09 g, 26.79 mmol) and HOBt (3.62 g, 26.79 mmol) in DML (25 mL) was added to the NPL-Ala-O-TrtCl-resin followed by addition DIPCDI (4.14 mL, 26.79 mmol) at 23 °C. The mixture was stirred for 1.5 h at 23 °C. The reaction was stopped by washing with DML (5 x 15 mL x 0.5 min). The Lmoc-Val-Ala-O-TrtCl-resin was treated with piperidine: DML (1:4, 15 mL, l x l min, 2 x 10 min) and washed with DML (5 x 15 mL x 0.5 min).A solution of 15-(9-Lluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoic acid (Lmoc-NH-PEG4-OH) (4.90 g, 8.75 mmol ) and HOBt (1.35 g, 9.98 mmol ) in DML (30 mL) was added to the NH2-Val-Ala-O-TrtCl-resin followed by addition DIPCDI (1.55 mL, 10.0 mmol) at 23 °C. The reaction mixture was stirred for 22 h at 23 °C. The reaction was stopped by washing with DML (5 x 15 mL x 0.5 min). The Lmoc-NH-PEG4-Val-Ala-O- TrtCl-resin was treated with piperidine: DML (1:4, 15 mL, l x l min, 2 x 10 min) and washed with DML (5 x 15 mL x 0.5 min).A solution of 3-(Maleimido)propionic acid (MC2-OH) (4.53 g, 26.78 mmol) and HOBt (3.62 g, 26.77 mmol) in DML (30 mL) was added to the NH2-PEG4-Val-Ala-O-TrtCl-resin followed by addition of DIPCDI (4.15 mL, 26.80 mmol) at 23 °C. The reaction mixture was stirred for 2 h at 23 °C. The reaction was stopped by washing with DML (5 x 15 mL x 0.5 min) and CH2CI2 (5 x 15 mL x 0.5 min).The peptide was cleaved from the resin by treatments with TLA:CH2Cl2 (1:99, 5 x 50 mL). The resin was washed with CH2CI2 (7 x 50 mL x 0.5 min). The combined filtrates were evaporated to dryness under reduced pressure, the solid obtained was triturated with Et20 and filtrated to obtain L 3-1 (4.73 g, 87% yield) as a white solid. NMR (500 MHz, CDC13): d 7.67 (bs, 1H), 7.31 (d, J = 8.9 Hz, 1H), 7.17 (d, J = 7.0 Hz, 1H), 6.85 (t, J = 5.6 Hz, 1H), 6.72 (s, 2H), 4.51 (q, /= 7.1 Hz, 1H), 4.38 (dd, J = 8.9, 6.9 Hz, 1H), 3.84 (t, J = 7.1 Hz, 2H), 3.75 (t, J = 5.9 Hz, 2H), 3.69-3.59 (m, 12H), 3.55 (t, J = 5.1 Hz, 2H), 3.41 (qd, /= 5.0, 1.7 Hz, 2H), 2.62-2.49 (m, 4H), 2.19-2.01 (m, 1H), 1.44 (d, /= 7.2 Hz, 3H), 0.95 (d, J = 11.9 Hz, 1H), 0.94 (d, J = 11.9 Hz, 1H). |
87% | Stage #1: N-[(9H-fluoren-9-ylmethoxy)carbonyl]-L-alanine With N-ethyl-N,N-diisopropylamine In dichloromethane at 23℃; for 2.16667h; Stage #2: With piperidine In dichloromethane; N,N-dimethyl-formamide Stage #3: 3-maleimidepropionic acid; Fmoc-Val-OH; 15–(9–fluorenylmethyloxycarbonyl)amino–4,7,10,13–tetraoxa–pentadecanoic acid Further stages; | 2.a (a) Preparation of LIN 3-1: MC2-PEG4-Val-Ala-OH Cl-TrtCl-resin (5 g, 1.49 mmol/g) was placed in a filter plate. To the resin was added CH2CI2 (25 mL) and the mixture was stirred for 1 h at 23 °C. The solvent was eliminated by filtration over vacuum. A solution of Fmoc-Ala-OH (2.31 g, 7.41 mmol) and DIPEA (4.28 mL, 24.61 mmol) in CH2CI2 (20 mL) was added and the mixture was stirred for 10 min at 23 °C. DIPEA (8.60 mL, 49.37 mmol) was additionally added and the reaction mixture was stirred for 1 h at 23 °C. The reaction was stopped by addition of MeOH (10 mL) and stirred 15 min at 23 °C. The Lmoc-Ala-O-TrtCl-resin was subjected to the following washing/treatments: CH2CI2 (5 x 15 mL x 0.5 min), DML (5 x 15 mL x 0.5 min), piperidine :DML (1:4, 15 mL, l x l min, 2 x 10 min), DML (5 x 15 mL x 0.5 min), CH2CI2 (5 x 15 mL x 0.5 min). The loading was calculated: 1.34 mmol/g.The NPh-Ala-O-TrtCl-resin was washed with DML (5 x 15 mL x 0.5 min) and a solution of Lmoc-Val-OH (9.09 g, 26.79 mmol) and HOBt (3.62 g, 26.79 mmol) in DML (25 mL) was added to the NPL-Ala-O-TrtCl-resin followed by addition DIPCDI (4.14 mL, 26.79 mmol) at 23 °C. The mixture was stirred for 1.5 h at 23 °C. The reaction was stopped by washing with DML (5 x 15 mL x 0.5 min). The Lmoc-Val-Ala-O-TrtCl-resin was treated with piperidine: DML (1:4, 15 mL, l x l min, 2 x 10 min) and washed with DML (5 x 15 mL x 0.5 min).A solution of 15-(9-Lluorenylmethyloxycarbonyl)amino-4,7,10,13-tetraoxa-pentadecanoic acid (Lmoc-NH-PEG4-OH) (4.90 g, 8.75 mmol ) and HOBt (1.35 g, 9.98 mmol ) in DML (30 mL) was added to the NH2-Val-Ala-O-TrtCl-resin followed by addition DIPCDI (1.55 mL, 10.0 mmol) at 23 °C. The reaction mixture was stirred for 22 h at 23 °C. The reaction was stopped by washing with DML (5 x 15 mL x 0.5 min). The Lmoc-NH-PEG4-Val-Ala-O- TrtCl-resin was treated with piperidine: DML (1:4, 15 mL, l x l min, 2 x 10 min) and washed with DML (5 x 15 mL x 0.5 min).A solution of 3-(Maleimido)propionic acid (MC2-OH) (4.53 g, 26.78 mmol) and HOBt (3.62 g, 26.77 mmol) in DML (30 mL) was added to the NH2-PEG4-Val-Ala-O-TrtCl-resin followed by addition of DIPCDI (4.15 mL, 26.80 mmol) at 23 °C. The reaction mixture was stirred for 2 h at 23 °C. The reaction was stopped by washing with DML (5 x 15 mL x 0.5 min) and CH2CI2 (5 x 15 mL x 0.5 min).The peptide was cleaved from the resin by treatments with TLA:CH2Cl2 (1:99, 5 x 50 mL). The resin was washed with CH2CI2 (7 x 50 mL x 0.5 min). The combined filtrates were evaporated to dryness under reduced pressure, the solid obtained was triturated with Et20 and filtrated to obtain L 3-1 (4.73 g, 87% yield) as a white solid. NMR (500 MHz, CDC13): d 7.67 (bs, 1H), 7.31 (d, J = 8.9 Hz, 1H), 7.17 (d, J = 7.0 Hz, 1H), 6.85 (t, J = 5.6 Hz, 1H), 6.72 (s, 2H), 4.51 (q, /= 7.1 Hz, 1H), 4.38 (dd, J = 8.9, 6.9 Hz, 1H), 3.84 (t, J = 7.1 Hz, 2H), 3.75 (t, J = 5.9 Hz, 2H), 3.69-3.59 (m, 12H), 3.55 (t, J = 5.1 Hz, 2H), 3.41 (qd, /= 5.0, 1.7 Hz, 2H), 2.62-2.49 (m, 4H), 2.19-2.01 (m, 1H), 1.44 (d, /= 7.2 Hz, 3H), 0.95 (d, J = 11.9 Hz, 1H), 0.94 (d, J = 11.9 Hz, 1H). |
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Precautionary Statements-General | |
Code | Phrase |
P101 | If medical advice is needed,have product container or label at hand. |
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P103 | Read label before use |
Prevention | |
Code | Phrase |
P201 | Obtain special instructions before use. |
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P221 | Take any precaution to avoid mixing with combustibles |
P222 | Do not allow contact with air. |
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P231 | Handle under inert gas. |
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P233 | Keep container tightly closed. |
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P334 | Immerse in cool water/wrap n wet bandages. |
P335 | Brush off loose particles from skin. |
P336 | Thaw frosted parts with lukewarm water. Do not rub affected area. |
P337 | If eye irritation persists: |
P338 | Remove contact lenses, if present and easy to do. Continue rinsing. |
P340 | Remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P341 | If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P342 | If experiencing respiratory symptoms: |
P350 | Gently wash with plenty of soap and water. |
P351 | Rinse cautiously with water for several minutes. |
P352 | Wash with plenty of soap and water. |
P353 | Rinse skin with water/shower. |
P360 | Rinse immediately contaminated clothing and skin with plenty of water before removing clothes. |
P361 | Remove/Take off immediately all contaminated clothing. |
P362 | Take off contaminated clothing and wash before reuse. |
P363 | Wash contaminated clothing before reuse. |
P370 | In case of fire: |
P371 | In case of major fire and large quantities: |
P372 | Explosion risk in case of fire. |
P373 | DO NOT fight fire when fire reaches explosives. |
P374 | Fight fire with normal precautions from a reasonable distance. |
P376 | Stop leak if safe to do so. Oxidising gases (section 2.4) 1 |
P377 | Leaking gas fire: Do not extinguish, unless leak can be stopped safely. |
P378 | |
P380 | Evacuate area. |
P381 | Eliminate all ignition sources if safe to do so. |
P390 | Absorb spillage to prevent material damage. |
P391 | Collect spillage. Hazardous to the aquatic environment |
P301 + P310 | IF SWALLOWED: Immediately call a POISON CENTER or doctor/physician. |
P301 + P312 | IF SWALLOWED: call a POISON CENTER or doctor/physician IF you feel unwell. |
P301 + P330 + P331 | IF SWALLOWED: Rinse mouth. Do NOT induce vomiting. |
P302 + P334 | IF ON SKIN: Immerse in cool water/wrap in wet bandages. |
P302 + P350 | IF ON SKIN: Gently wash with plenty of soap and water. |
P303 + P361 + P353 | IF ON SKIN (or hair): Remove/Take off Immediately all contaminated clothing. Rinse SKIN with water/shower. |
P304 + P312 | IF INHALED: Call a POISON CENTER or doctor/physician if you feel unwell. |
P304 + P340 | IF INHALED: Remove victim to fresh air and Keep at rest in a position comfortable for breathing. |
P304 + P341 | IF INHALED: If breathing is difficult, remove victim to fresh air and keep at rest in a position comfortable for breathing. |
P305 + P351 + P338 | IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. |
P306 + P360 | IF ON CLOTHING: Rinse Immediately contaminated CLOTHING and SKIN with plenty of water before removing clothes. |
P307 + P311 | IF exposed: call a POISON CENTER or doctor/physician. |
P308 + P313 | IF exposed or concerned: Get medical advice/attention. |
P309 + P311 | IF exposed or if you feel unwell: call a POISON CENTER or doctor/physician. |
P332 + P313 | IF SKIN irritation occurs: Get medical advice/attention. |
P333 + P313 | IF SKIN irritation or rash occurs: Get medical advice/attention. |
P335 + P334 | Brush off loose particles from skin. Immerse in cool water/wrap in wet bandages. |
P337 + P313 | IF eye irritation persists: Get medical advice/attention. |
P342 + P311 | IF experiencing respiratory symptoms: call a POISON CENTER or doctor/physician. |
P370 + P376 | In case of fire: Stop leak if safe to Do so. |
P370 + P378 | In case of fire: |
P370 + P380 | In case of fire: Evacuate area. |
P370 + P380 + P375 | In case of fire: Evacuate area. Fight fire remotely due to the risk of explosion. |
P371 + P380 + P375 | In case of major fire and large quantities: Evacuate area. Fight fire remotely due to the risk of explosion. |
Storage | |
Code | Phrase |
P401 | |
P402 | Store in a dry place. |
P403 | Store in a well-ventilated place. |
P404 | Store in a closed container. |
P405 | Store locked up. |
P406 | Store in corrosive resistant/ container with a resistant inner liner. |
P407 | Maintain air gap between stacks/pallets. |
P410 | Protect from sunlight. |
P411 | |
P412 | Do not expose to temperatures exceeding 50 oC/ 122 oF. |
P413 | |
P420 | Store away from other materials. |
P422 | |
P402 + P404 | Store in a dry place. Store in a closed container. |
P403 + P233 | Store in a well-ventilated place. Keep container tightly closed. |
P403 + P235 | Store in a well-ventilated place. Keep cool. |
P410 + P403 | Protect from sunlight. Store in a well-ventilated place. |
P410 + P412 | Protect from sunlight. Do not expose to temperatures exceeding 50 oC/122oF. |
P411 + P235 | Keep cool. |
Disposal | |
Code | Phrase |
P501 | Dispose of contents/container to ... |
P502 | Refer to manufacturer/supplier for information on recovery/recycling |
Physical hazards | |
Code | Phrase |
H200 | Unstable explosive |
H201 | Explosive; mass explosion hazard |
H202 | Explosive; severe projection hazard |
H203 | Explosive; fire, blast or projection hazard |
H204 | Fire or projection hazard |
H205 | May mass explode in fire |
H220 | Extremely flammable gas |
H221 | Flammable gas |
H222 | Extremely flammable aerosol |
H223 | Flammable aerosol |
H224 | Extremely flammable liquid and vapour |
H225 | Highly flammable liquid and vapour |
H226 | Flammable liquid and vapour |
H227 | Combustible liquid |
H228 | Flammable solid |
H229 | Pressurized container: may burst if heated |
H230 | May react explosively even in the absence of air |
H231 | May react explosively even in the absence of air at elevated pressure and/or temperature |
H240 | Heating may cause an explosion |
H241 | Heating may cause a fire or explosion |
H242 | Heating may cause a fire |
H250 | Catches fire spontaneously if exposed to air |
H251 | Self-heating; may catch fire |
H252 | Self-heating in large quantities; may catch fire |
H260 | In contact with water releases flammable gases which may ignite spontaneously |
H261 | In contact with water releases flammable gas |
H270 | May cause or intensify fire; oxidizer |
H271 | May cause fire or explosion; strong oxidizer |
H272 | May intensify fire; oxidizer |
H280 | Contains gas under pressure; may explode if heated |
H281 | Contains refrigerated gas; may cause cryogenic burns or injury |
H290 | May be corrosive to metals |
Health hazards | |
Code | Phrase |
H300 | Fatal if swallowed |
H301 | Toxic if swallowed |
H302 | Harmful if swallowed |
H303 | May be harmful if swallowed |
H304 | May be fatal if swallowed and enters airways |
H305 | May be harmful if swallowed and enters airways |
H310 | Fatal in contact with skin |
H311 | Toxic in contact with skin |
H312 | Harmful in contact with skin |
H313 | May be harmful in contact with skin |
H314 | Causes severe skin burns and eye damage |
H315 | Causes skin irritation |
H316 | Causes mild skin irritation |
H317 | May cause an allergic skin reaction |
H318 | Causes serious eye damage |
H319 | Causes serious eye irritation |
H320 | Causes eye irritation |
H330 | Fatal if inhaled |
H331 | Toxic if inhaled |
H332 | Harmful if inhaled |
H333 | May be harmful if inhaled |
H334 | May cause allergy or asthma symptoms or breathing difficulties if inhaled |
H335 | May cause respiratory irritation |
H336 | May cause drowsiness or dizziness |
H340 | May cause genetic defects |
H341 | Suspected of causing genetic defects |
H350 | May cause cancer |
H351 | Suspected of causing cancer |
H360 | May damage fertility or the unborn child |
H361 | Suspected of damaging fertility or the unborn child |
H361d | Suspected of damaging the unborn child |
H362 | May cause harm to breast-fed children |
H370 | Causes damage to organs |
H371 | May cause damage to organs |
H372 | Causes damage to organs through prolonged or repeated exposure |
H373 | May cause damage to organs through prolonged or repeated exposure |
Environmental hazards | |
Code | Phrase |
H400 | Very toxic to aquatic life |
H401 | Toxic to aquatic life |
H402 | Harmful to aquatic life |
H410 | Very toxic to aquatic life with long-lasting effects |
H411 | Toxic to aquatic life with long-lasting effects |
H412 | Harmful to aquatic life with long-lasting effects |
H413 | May cause long-lasting harmful effects to aquatic life |
H420 | Harms public health and the environment by destroying ozone in the upper atmosphere |
Sorry,this product has been discontinued.
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