CN104987504A - Pegylated lapatinib, injection and preparation method thereof - Google Patents
Pegylated lapatinib, injection and preparation method thereof Download PDFInfo
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- CN104987504A CN104987504A CN201510196919.7A CN201510196919A CN104987504A CN 104987504 A CN104987504 A CN 104987504A CN 201510196919 A CN201510196919 A CN 201510196919A CN 104987504 A CN104987504 A CN 104987504A
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- lapatinibditosylate
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Abstract
The present invention provides a pegylated lapatinib, an injection and a preparation method thereof, wherein the general formula of the pegylated lapatinib is defined in the specification, A is selected from a single-arm or multi-arm polyethylene glycol or poly ethylene glycol derivative, X is selected from the formula defined in the specification, Y and M are independently and respectively selected from double carboxylic acids having amino or corresponding acyl substituents, N is selected from an amino acid or peptide, LPT is lapatinib, a is 0 or 1, b is 0 or 1, c is 1 or 2, d is 1 or 2, and e is equal to the arm number of X. According to the present invention, the pegylated drug of lapatinib is synthesized, such that the lapatinib toxicity is reduced, the water solubility and the biological stability are improved, the drug resistance of cancer cells and cancer stem cells are avoided, the targeting property is enhanced, and the anticancer treatment effect is significantly improved.
Description
Technical field
The present invention relates to the conjugate field of lapatinibditosylate, in particular to Pegylation lapatinibditosylate and injection thereof and preparation method.
Background technology
Lapatinibditosylate (Lapatinib) is a kind of oral small molecule epidermal growth factor receptor tyrosine kinase inhibitor, be mainly used in associating capecitabine treatment ErbB-2 overexpression, and previously accepted the late period of anthracycline, taxol or Herceptin treatment or metastatic mammary cancer, come into the market in 2007 by U.S. FDA approval.Lapatinibditosylate can suppress cancer stem cell, as U87-MG cancer of the brain stem cell and SUM225 breast carcinoma stem cell.
The molecular weight of lapatinibditosylate is 581.06, belong to small molecule anti-cancer drug, there is water-soluble difference, biologically stable is poor, toxic side effect is large shortcoming, and micromolecular lapatinibditosylate is by diffusing into cancer cells or cancer stem cell can be gone out by p-glycoprotein membrane pump, and then causes resistance.And the polymer coupling anticancer stem cell small-molecule drug of long-acting slow-release greatly can reduce the toxicity of small-molecule drug, greatly promote its water-soluble and biologically stable, strengthen its passive targeting, relative to antibody coupling anticarcinogen (ADC), antibody anticarcinogen, the antibody of antibody anticancer stem cell drugs or immunotherapy is cheap, and cancer cells or the extremely strong resistance of cancer stem cell can be avoided, this is because polymer coupling anticarcinogen or polymer coupling anticancer stem cell drugs have larger molecular weight, cancer cells or cancer stem cell can only be absorbed it by endocytosis and pinosome.But for be anticarcinogen and the polymer coupling technology of the lapatinibditosylate of anticancer stem cell drugs report and product not yet find.
In view of this, special proposition the present invention.
Summary of the invention
The first object of the present invention is to provide Pegylation lapatinibditosylate, described Pegylation lapatinibditosylate is the macromolecule anticancer drug that lapatinibditosylate and polyoxyethylene glycol and derivative coupling thereof are formed, compared with lapatinibditosylate, its toxicity significantly reduces, water-soluble and biologically stable improves, and has significant passive targeting, much lower resistance, higher anticancer therapeutic.
The second object of the present invention is to provide a kind of preparation method of described Pegylation lapatinibditosylate, and the method has simple to operate, and reaction conditions is gentle, the fast and productive rate advantages of higher of speed of reaction.
The third object of the present invention is the injection providing a kind of described Pegylation lapatinibditosylate, and this injection has the advantages such as additive is few, solvability is high, drug effect is high, easy preparation.
In order to realize above-mentioned purpose of the present invention, spy by the following technical solutions:
Pegylation lapatinibditosylate, its general formula is:
A is selected from the polyoxyethylene glycol of single armed or multi-arm or the derivative of polyoxyethylene glycol; X is selected from
y and M is separately selected from the amino two carboxylic acid of band or corresponding acyl substituted thing; N is selected from amino acid or peptide; LPT is lapatinibditosylate;
A=0 or 1; B=0 or 1; C=1 or 2; D=1 or 2; E equals the arm number of X.
From above-mentioned general formula, Pegylation lapatinibditosylate take amino acid as connection chain, the macromolecular substance formed together with polyoxyethylene glycol is coupled at lapatinibditosylate, it not only remains lapatinibditosylate anti-cancer properties, and following advantage has been showed compared with single lapatinibditosylate: toxicity significantly reduces, water-soluble and biologically stable improves, and has significant passive targeting and much lower resistance.
In above-mentioned general formula, as c=2, represent and M is connected to two N-LPT.
Preferably, when a, b are 0, c, d are 1; Work as a=0, during b=1, d=1.The conjugate with above constructional feature is more easily prepared, and medicine cost is low.Below list the molecular structural formula of moiety thing:
Preferably, two carboxylic acids that described band is amino are L-glutamic acid or aspartic acid.
Preferably, A is selected from single armed, four arms or the polyoxyethylene glycol of eight arms or the derivative of polyoxyethylene glycol.
Preferably, the molecular weight of A is 12000,20000 or 40000 dalton.
Preferably, Y and M is separately selected from
These materials are all the substituents of the amino position of L-glutamic acid; following two objects can be realized: one is the more amidated LPT of high reactivity acyl group coupling utilizing L-glutamic acid or aspartic acid; two is increase side chain quantity, to improve the drug release rate of Pegylation lapatinibditosylate.In addition, the dimethylaminoethoxyethanol on side chain can reduce with polyoxyethylene glycol in conjunction with difficulty.
Preferably, N is glycine, and it is more easily combined with lapatinibditosylate, and therefore product more easily obtains.
Preferably, molecular formula is
experiment proves, the medicine over-all properties of this compound is better, more easily promotes clinically.
The preparation method of above-mentioned Pegylation lapatinibditosylate, comprises the following steps:
Steps A: make lapatinibditosylate and Amino Acid/Peptide generation amidate action, obtain the first intermediate product;
Step B: make described first intermediate product and M that amidate action occur, obtain the second intermediate product;
Step C: make described second intermediate product and Y that amidate action occur, obtain the 3rd intermediate product;
Step D: make the derivative of described 3rd intermediate product and polyoxyethylene glycol or polyoxyethylene glycol by amido linkage coupling, obtain product.
Above-mentioned preparation method take amino acid as cross structure, reduce the difficulty of polyoxyethylene glycol and the direct coupling of LPT, and three classes reactions involved in building-up process (LPT and amino acid react, reaction between amino acid, polyoxyethylene glycol and Y reaction) reaction conditions all very gentle, therefore low to the requirement of equipment and production environment, preparation cost is low.In addition, because the mechanism of reacting in building-up process is amidate action, the selectivity of amidate action is high, and speed of reaction is fast, and the by product of isomer class is few, and therefore the productive rate of this synthetic method is high, speed is fast.
Preferably, in described steps A, the condition of lapatinibditosylate and peptide or arbitrary amino acid generation amidate action is: with HBTU, HOBT, DMF and DIEA for auxiliary agent, react at-2 ~ 4 DEG C, and lapatinibditosylate, peptide or arbitrary amino acid, mol ratio between HBTU, HOBT and DIEA are: 1:0.8 ~ 1.2:1.3 ~ 1.7:1.3 ~ 1.7:4.2 ~ 4.8.
Above-mentioned reaction is as condensing agent using HBTU (O-benzotriazole-tetramethyl-urea phosphofluoric acid ester); HOBT (I-hydroxybenzotriazole) is as compensator; DIEA (N; N-diisopropylethylamine) as alkaloid; DMF (DMF) as solvent, and promotes the reaction of lapatinibditosylate and peptide/arbitrary amino acid with specific ratio; acyl group is reduced in conjunction with difficulty with amino, and reaction yield improves.
Preferably, also comprise before making lapatinibditosylate and peptide or amino acid generation amidate action: protect described peptide or described amino acid whose amino with tertbutyloxycarbonyl (Boc).
After above-mentioned sfgd. can avoid the amino of peptide/arbitrary amino acid to be substituted, cannot be combined with other amino acid.Concrete guard method can with reference to under type: be dissolved in Isosorbide-5-Nitrae-dioxane by amino acid, then add sodium hydroxide solution, more at room temperature add tert-Butyl dicarbonate (Boc
2o), at room temperature react, last separation and Extraction product; The mol ratio of amino acid, sodium hydroxide is 1:1.2-1.3.
Preferably, in described steps A, the method of lapatinibditosylate and peptide generation amidate action is: the free amine group acid-respons that the termination of lapatinibditosylate and described peptide relates to, the product obtained is again according to other free amine group acid-respons that sequence amino acid whose in described peptide relates to described peptide successively, and the total free aminoacids in above-mentioned reaction is before participation reaction: the carboxyl activating described total free aminoacids with N-hydroxy-succinamide.Improve speed of reaction by activation, improve productive rate, reduce coupling difficulty, aftertreatment is more prone to.
Preferably, in described step B, the condition of described first intermediate product and M amidate action is: mol ratio >=2 of described first intermediate product and M.Controlling suitable mol ratio makes the M of a part can connect bimolecular first intermediate product, makes the number of connection of LPT double.
Preferably, described first intermediate product and M also comprise before there is amidate action: the carboxyl activating L-glutamic acid with N-hydroxy-succinamide; In like manner, improve speed of reaction by activation, reduce coupling difficulty.Similar, before amidate action occurs for described second intermediate product and Y, the carboxyl of Y also first can be activated with N-hydroxy-succinamide.
Preferably, in described step C, the condition of described second intermediate product and Y amidate action is: mol ratio >=2 of described second intermediate product and L-glutamic acid.That the ratio of control reactant is to increase the quantity of the LPT of combination equally.
Preferably, in described step D, also comprise before making described 3rd intermediate product and polyoxyethylene glycol generation amidate action: N, N'-succinimidyl carbonate, N, the derivative of N'-succinimide yl acetate or N, N'-succinimidyl glutarat activated polyethylene glycol or polyoxyethylene glycol.To reduce the coupling difficulty of polyoxyethylene glycol and LPT.
Preferably, the mol ratio of described 3rd intermediate product and polyoxyethylene glycol is greater than 1.5; Ensure the 3rd intermediate product and the complete coupling of polyoxyethylene glycol.
Preferably, with N, N'-succinimidyl carbonate, N, N'-succinimide yl acetate or N, the derivative of N'-succinimidyl glutarat activated polyethylene glycol or polyoxyethylene glycol, obtain the 4th intermediate product, described 3rd intermediate product and described 4th intermediate product take methylene dichloride as solvent, at 20-30 DEG C, amidate action occur: select gentle reaction conditions to reduce production difficulty.
The injection of Pegylation lapatinibditosylate, comprising: described Pegylation lapatinibditosylate and solvent, and described solvent is physiological saline and/or 95% ethanol.This injection has the advantages such as additive is few, solvability is high, drug effect is high, easy preparation.
Compared with prior art, beneficial effect of the present invention is:
(1) the polymer coupling drug of lapatinibditosylate has been synthesized, reduce the toxicity of the former medicine of lapatinibditosylate, improve water-soluble and biologically stable, avoid cancer cells and the very strong resistance of cancer stem cell, enhance targeting, significantly improve anticancer therapeutic.
(2) preparation method of Pegylation lapatinibditosylate is simple to operate, reaction conditions is gentle, speed of reaction is fast and productive rate is high, is applicable to suitability for industrialized production.
(3) dissimilar Pegylation lapatinibditosylate injection is provided, for the clinical application of Pegylation lapatinibditosylate provides foundation.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 to Fig. 5 is respectively 6-56,4-208,6-162,7-129, LPT curve to U87 inhibition of cancer cell rate;
Fig. 6 to Figure 10 is 6-56,4-208,6-162,7-129, LPT curve to N87 inhibition of cancer cell rate;
Figure 11 to Figure 13 is 6-56,6-162, LPT curve to CALU-3 inhibition of cancer cell rate;
Figure 14 to Figure 18 is 6-56,4-208,6-162,7-129, LPT curve to BT474 inhibition of cancer cell rate;
Figure 19 to Figure 23 is 6-56,4-208,6-162,7-129, LPT curve to SK-BR-3 inhibition of cancer cell rate;
The growth curve of tumour when Figure 24 is physiological saline, 7-129, LPT act on mouse;
Figure 25 to Figure 42 is respectively the Pegylation lapatinibditosylate that embodiment 1-18 provides
1h-NMR spectrogram;
The mass spectrum of the Pegylation lapatinibditosylate that Figure 43 to Figure 55 is respectively embodiment 1,3-5,9-17 provide.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only for illustration of the present invention, and should not be considered as limiting the scope of the invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, be and can buy by commercially available the conventional products obtained.
For meet concisely, clearly requirement, the part of compounds numeral number hereafter related to, such as 3-59,7-129, P1 ~ P43 etc.
Embodiment 1
Synthesis
method:
One, prepare
Glycine (19.1274g, 254.7943mmol) is dissolved in Isosorbide-5-Nitrae-dioxane (200mL), adds 2N NaOH solution (80uL, 160mmol), be down to after room temperature until solution temperature, add Boc
2o (77.8524g, 356.7120mmol), at room temperature react 12 hours, evaporation concentration, finally uses ethyl acetate (150mL × 2) to wash, with hydrochloric acid, pH value is adjusted to 4, use ethyl acetate (150mL × 5) to extract again, merge organic phase, with anhydrous sodium sulfate drying, concentrating filter liquor drying obtains product 44.6 grams, and productive rate is 100%.
Two, prepare
P1 (90.4mg is added in the flask of 50mL, 0.5163mmol), lapatinibditosylate (300g, 0.5163mmol), HBTU (290.6mg, 0.7745mmol) with HOBT (104.6mg, 107745mmol), dissolve with DMF (15mL), solution is cooled 20 minutes in 0 DEG C of cryogenic thermostat reactive bath technique, then DIEA (0.41mL, 2.3234mmol) is slowly dripped.Move to room temperature after 2 hours, stirring is spent the night.Pour reaction solution into saturated NaHCO
3in solution (200mL), be extracted with ethyl acetate (200mL × 2).Then use saturated sodium bicarbonate solution (150mL × 2) to clean, finally clean by saturated NaCl solution (100mL).Use anhydrous MgSO
4drying, filters, concentrating under reduced pressure evaporate to dryness.Silica gel column chromatography, uses 5%MeOH/ dichloromethane eluent.Collect product, evaporate to dryness obtains 380.0mg, productive rate 99.70%.
Three, prepare
In flask, add P2 (380.0mg, 0.5147mmol), dissolve with methylene dichloride (10mL), finally instill TFA (0.20mL, 2.5737mmol).Room temperature for overnight.Stopped reaction, evaporated under reduced pressure, dissolves with MeOH, adds sodium bicarbonate (211.2mg, 2.5137mmol), stir 10 minutes.Suction filtration, adds silica-gel powder (3 grams), is spin-dried for and makes solid solution, dry method loading in filtrate.Column chromatography, first uses 2%MeOH/ dichloromethane eluent, then uses 1%TEA, 5%MeOH/ dichloromethane eluent.Collect product, concentrating under reduced pressure evaporate to dryness obtains 209.9mg, productive rate 63.9%.
Four, 3-59 is prepared:
P3 (19.95mg is added in cylinder bottle, 0.025mmol) and molecular weight be the polyoxyethylene glycol M-SC-12K (200mg of the single armed of 12000,0.01667mmol), dissolve with methylene dichloride (5mL), stirring at room temperature one week, stopped reaction, is spin-dried for mixture, product water dissolution is put into dialysis tubing dialysis.Then concentrated by product water solution decompression, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, filter, vacuum drying obtains product 136.3mg, productive rate 65.3%.
Embodiment 2
Synthesis
method:
P3 (9.6mg is added in cylinder bottle, 0.015mmol) and molecular weight be the polyoxyethylene glycol M-SC-20K (200mg of the single armed of 20000,0.01mmol), dissolve with methylene dichloride (5mL), stirring at room temperature one week, stopped reaction, is spin-dried for mixture, product water dissolution is put into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, filter, vacuum drying obtains product 182.6mg, productive rate 89.0%.
Embodiment 3
Synthesis
method:
P3 (9.6mg is added in cylinder bottle, 0.015mmol), with the polyoxyethylene glycol M-SC-40K (200mg that molecular weight is the single armed of 40000,0.01mmol), dissolve with methylene dichloride (8mL), stirring at room temperature one week, stopped reaction, mixture is spin-dried for, product water dissolution is put into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, filter, vacuum drying obtains product 168.4mg, productive rate 83.1%.
Embodiment 4
Synthesis
P3 (19.1433mg is added in cylinder bottle, 0.03mmol) and molecular weight be the polyoxyethylene glycol 4ARM-SC-40K (200mg of four arms of 40000,0.01mmol), dissolve with methylene dichloride (8mL), stirring at room temperature two weeks, mixture is spin-dried for, product water dissolution is put into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, filter, obtain product, vacuum drying obtains product 120mg, productive rate 55.8%.
Embodiment 5
Synthesis
P3 (101.9mg is added in cylinder bottle, 0.16mmol), with the polyoxyethylene glycol 8ARM-SG-40K (400mg that molecular weight is eight arms of 40000,0.01mmol), dissolve with methylene dichloride (8mL), stirring at room temperature 2 weeks, is spin-dried for mixture, product water dissolution is put into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, filter, vacuum drying obtains product 236.9mg, productive rate 53.6%.
Embodiment 6
Synthesis
One, prepare
In flask, add Pidolidone (20.0620g, 13.36mmol), dissolve with water/DMF (100mL/200mL).Add triethylamine (38.00mL, 272.71mmol) while stirring under room temperature, then add Boc
2o (30.36g, 139.09mmol), finally add DMF (40mL), stirring is spent the night.Concentrated, dissolve by ethyl acetate (500mL), neutralize with 1M HCl (100mL).Isolate aqueous phase, in aqueous phase, add NaCl, to it no longer dissolves, be extracted with ethyl acetate aqueous phase, isolate organic phase.Above-mentioned organic phase merged, with water cleaning, with saturated aqueous common salt cleaning, with anhydrous magnesium sulfate drying, filter, evaporated under reduced pressure obtains product 19.75g, productive rate 58.58%.
Two, prepare
P4 (20g is added in the round-bottomed flask of 1L, 80.8898mmol) and N-hydroxy-succinamide (37.2384g, 323.5592mmol), dissolve with methylene dichloride (400mL), then add DMAP (1.9765g, 16.1780mmol), be placed in the cryogenic thermostat reactive bath technique of-5 DEG C, after 20 minutes, add DCC (66.7599g, 323.5592mmol) in batches, move to room temperature after 1 hour, stirring is spent the night.Filter, filtrate is concentrated.Solvent homogenate wet method dress post 16cm × 8.5cm is with sherwood oil.Wet method loading.Column chromatography, by 30% ethyl acetate/petroleum ether to 100% ethyl acetate gradient, TLC monitors, and potassium permanganate develops the color, and collects product, evaporate to dryness, and vacuum drying oven is dry obtains the finished product 41.6462g, productive rate 100%.
Three, prepare
In barrel reactor, add P5 (223.2mg, 0.5056mmol) and P3 (709.7mg, 1.1223mmol), dissolve with methylene dichloride, stirred overnight at room temperature.Direct wet method loading, silica gel column chromatography, with the NH of 2%
3h
2the NH of O, 4%MeOH/ methylene dichloride to 3%
3h
2o, 6%MeOH/ dichloromethane gradient, collect product point, concentrating under reduced pressure evaporate to dryness obtains product 620mg, productive rate 47.7%.
Four,
In flask, add P6 (620mg, 0.4168mmol), dissolve with methylene dichloride, add TFA (0.1597mL, 2.084mmol), stirred overnight at room temperature.Evaporated under reduced pressure, dissolves with MeOH, adds sodium bicarbonate (175.1mg, 2.084mmol) and stir 10 minutes.Filter, filtrate is made silica-gel powder solid solution, dry method loading.Column chromatography, with the NH of 2%
3h
2o, 4%MeOH/ dichloromethane eluent.Collect product, concentrating under reduced pressure evaporate to dryness obtains 413.3mg, productive rate 100%.
Five, prepare
P1 (5.0905g is added in flask, 29.0587mmol) and N-hydroxy-succinamide (3.6788g, 31.9646mmol), dissolve with methylene dichloride, then add DMAP (355.0mg, 2.9059mmol), be placed in the cryogenic thermostat reactive bath technique of 0 DEG C, after 20 minutes, add DCC (6.5953g, 31.9646mmol), move to room temperature after 1 hour, stirring is spent the night.Point TLC plate, launches in 20% ethyl acetate/petroleum ether, and phospho-molybdic acid colour developing display reacts completely.Filter, precipitate by washed with dichloromethane.In filtrate, add the silica-gel powder of 20g, be spin-dried for, dry method upper prop.With the eluent ethyl acetate of 30% ethyl acetate/petroleum ether to 50% ethyl acetate/petroleum ether to 100%, collect product point, concentrating under reduced pressure evaporate to dryness obtains product 4.8945g, productive rate 65.98%.
Six, prepare
In cylinder bottle, add P7 (220.2mg, 0.1587mmol) and P8 (43.213mg, 0.1587mmol), dissolve with methylene dichloride, stirred overnight at room temperature.Wet method loading column chromatography, uses 1.5%NH
3h
2o, 3%MeOH/ methylene dichloride is to 3%NH
3h
2o, 6%MeOH/ dichloromethane gradient, collect product point, concentrating under reduced pressure, vacuum drying obtains product 213.9mg, productive rate 89.8%.
Seven, prepare
In flask, add P9 (213mg, 0.1385mmol), dissolve with methylene dichloride, then add TFA (53uL, 0.6925mmol), stirred overnight at room temperature.Evaporated under reduced pressure, dissolves with MeOH, adds sodium bicarbonate (58.2mg, 0.6925mmol) and stir 10 minutes.Filter, filtrate is made silica-gel powder solid solution, dry method loading.Column chromatography, uses 3%NH
3h
2o, 6%MeOH/ dichloromethane eluent.Collect product, concentrating under reduced pressure evaporate to dryness obtains 166.8mg, productive rate 100%.
Eight, 3-137 is prepared
In cylinder bottle, add the polyoxyethylene glycol 4ARM-SCM-40K (300mg, 0.015mmol) that P10 (65.0mg, 0.045mmol) and molecular weight are four arms of 40000, dissolve with methylene dichloride (8mL), stirring at room temperature two weeks.Stopped reaction, is spin-dried for mixture, product water dissolution is put into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, filter, vacuum drying obtains product 289.6mg, productive rate 88.3%.
Embodiment 7
Synthesis
One, prepare
In flask, add P7 (4.8g, 3.3052mmol) and P5 (694.7187mg, 1.5739mmol.), dissolve with methylene dichloride, room temperature for overnight.Homogenate method dress silicagel column 25cm × 4.5cm.Wet method upper prop is separated, and uses 2%NH
3h
2o/4%MeOH/ methylene dichloride is to 2.5%NH
3h
2o/5%MeOH/ dichloromethane eluent, collect product point, concentrating under reduced pressure evaporate to dryness obtains product 3.3147g, productive rate 75.9%.
Two, prepare
In flask, add P11 (2.8356g, 0.9497mmol), dissolve with methylene dichloride (50mL), slowly add TFA (0.728mLL, 9.4967mmol) under stirring at room temperature, stirring is spent the night.Evaporated under reduced pressure, dissolves crude product anhydrous methanol, adds sodium bicarbonate (797.8177mg, 9.4967mmol) and stirs neutralization.Filter, filtrate is made silica-gel powder solid solution.Homogenate method dress silicagel column 20cm × 4.5cm.Dry method upper prop, uses 2.5%NH
3.H
2o/5%MeOH/ methylene dichloride is to 5%NH
3.H
2o/10%MeOH/ dichloromethane gradient, collect product, concentrating under reduced pressure evaporate to dryness obtains product 2.5983g, productive rate 94.8%.
Three, prepare
In cylinder bottle, add P12 (95mg, 0.0328mmol) and P8 (9.37mg, 0.0344mmol), dissolve with methylene dichloride (10mL), stirred overnight at room temperature.Methylene dichloride wet method homogenate dress post 20cm × 2cm.Wet method upper prop, column chromatography, uses 2%NH
3h
2o, 3%MeOH/ methylene dichloride is to 3%NH
3h
2o, 6%MeOH/ dichloromethane gradient, collect product point, concentrating under reduced pressure vacuum drying obtains product 99.8mg, productive rate 100%.
Four, prepare
In flask, add P13 (99.8mg, 0.0328mmol), dissolve with methylene dichloride, add TFA (12.6uL, 0.1640mmol), stirred overnight at room temperature.Evaporated under reduced pressure, dissolves with MeOH, adds sodium bicarbonate (0.3g), stir 10 minutes.Filter, filtrate is made silica-gel powder solid solution.Methylene dichloride wet method homogenate dress post 25cm × 2.5cm.Column chromatography, uses 4%NH
3h
2o, 8%MeOH/ methylene dichloride is to 5%NH
3h
2o, 10%MeOH/ dichloromethane gradient, collect product point, concentrating under reduced pressure vacuum drying obtains product 96.5mg, productive rate 100%.
Five, 3-153 is prepared
P14 (110.5mg is added in cylinder bottle, 0.03759mmol) and molecular weight be the polyoxyethylene glycol 4ARM-SCM-40K (250.6mg of four arms of 40000,0.006266mmol), dissolve with methylene dichloride (8mL), stirring at room temperature two weeks, mixture is spin-dried for, product water dissolution is put into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, suction filtration, vacuum drying obtains product 252.36mg, productive rate 77.2%.
Embodiment 8
Synthesis
One, prepare
H is added in the flask of 500mL
2nCH
2cH
2oCH
2cH
2oH (20g, 190.223mmol), uses CH
2cl
2(300mL) dissolve, then add triethylamine (53mL, 380.446mmol), under magnetic agitation, add Boc in batches
2o (49.8192 grams, 228.2670mmol), room temperature for overnight.TLC monitoring display consumption of starting material.Direct evaporate to dryness, with dissolve with methanol, adds in sodium bicarbonate (31.9575g, 380mmol) and triethylamine, filters, adds 60g silica-gel powder and be spin-dried in filtrate.With sherwood oil wet method homogenate dress post 10cm × 8.5cm.Dry method loading, with 20% ethyl acetate/petroleum ether to 50% ethyl acetate/petroleum ether and 2% methanol/ethyl acetate gradient elution.Collect product, concentrating under reduced pressure evaporate to dryness obtains 35.4485g, productive rate 90.79%.
Two, prepare
Under nitrogen protection, in the there-necked flask of 1L, add P16 (35.4685g, 172.803mmol), dissolve with THF (200mL), mechanical stirring, is placed in the cryogenic thermostat reactive bath technique of 0 DEG C.After 20 minutes, add the NaH (27.6485g, 690.12mmol) of 60% in batches, release a large amount of bubble.After 20 minutes, add bromoacetic acid (36.0165g, 259.2045mmol), release a large amount of bubble and occur that white slurry precipitates.After adding, move to 60 DEG C of oil bath overnight.TLC detection display reacts completely.Drip water (30mL) and neutralize remaining NaH, then add water (300mL), water phase separated and organic phase, by sherwood oil/anhydrous diethyl ether (100mL × 2) wash water phase.Aqueous phase pH to 4 is regulated with 1M HCl.Then NaCl is added saturated to solution.Be extracted with ethyl acetate aqueous phase (100mL × 12).With anhydrous sodium sulfate drying, filter, concentrating under reduced pressure is dry obtains product 20.4653g, productive rate 44.99%.
Three, prepare
P16 (20.4653g is added in the flask of 500mL, 77.729mmol), N-hydroxy-succinamide (10.7311g, 93.275mmol) with DMAP (0.9488g, 7.7729mmol), dissolve with methylene dichloride (200mL), the cryogenic thermostat reactive bath technique being placed in 0 DEG C stirs, DCC (64.1513g, 310.916mmol) is added after 30 minutes.Move to room temperature after 3 hours, stirring is spent the night.TLC shows consumption of starting material.Filter, in filtrate, add 60g silica-gel powder be spin-dried for.With sherwood oil wet method homogenate dress post 20cm × 8.8cm.Dry method loading.With the ethyl acetate gradient of 20% ethyl acetate/petroleum ether to 100%, collect product point, concentrate drying obtains product 20.6374g, productive rate 73.88%.
Four, prepare
In 50mL flask, add P10 (644mg, 0.4458mmol) and P17 (1.6050g, 4.4583mmol), dissolve with methylene dichloride, stirred overnight at room temperature.Add 5g silica-gel powder after having reacted to be spin-dried for.With methylene dichloride wet method homogenate dress post 20cm × 3.5cm.Dry method loading, column chromatography.Use 1%NH
3h
2o, 2%MeOH/ methylene dichloride is to 5%NH
3h
2o, 10%MeOH/ dichloromethane gradient, collects product point, and concentrating under reduced pressure is dry obtains product 169.4mg, productive rate 22.27%.
Five, prepare
In 50mL flask, add P18 (320mg, 0.1894mmol) and TFA (0.145mL, 1.8939mmol), dissolve with methylene dichloride, stirred overnight at room temperature.TLC shows reaction to be completed, and direct evaporate to dryness, dissolves with MeOH, add sodium bicarbonate (2.3863g, 2.841mmol) and neutralize unnecessary TFA, add 5g silica-gel powder and be spin-dried for after having reacted.With methylene dichloride wet method homogenate dress post 15cm × 2.5cm.Dry method loading, column chromatography.Use 2%NH
3h
2o, 4%MeOH/ methylene dichloride is to 5%NH
3h
2o, 10%MeOH/ dichloromethane gradient, collects product point, and concentrating under reduced pressure is dry obtains product 228.7mg, productive rate 76.02%.
Six, 6-135-2 is prepared
P19 (160mg is added in 30mL cylinder bottle, 0.1006mmol, 10.0) and molecular weight be the polyoxyethylene glycol 4ARM-SCM-40K (400mg, 0.01mmol) of four arms of 40000, dissolve with methylene dichloride (3mL), stirring at room temperature two weeks.Mixture is spin-dried for, by product water dissolution, puts into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, filter, vacuum drying obtains product 432.1mg, productive rate 100%.
Embodiment 9
Synthesis
One, prepare
By 6-aminocaprolc acid (50g in the round-bottomed flask of 1L, 381.1847mmol) be dissolved in 1,4-dioxane (300mL), the 2N NaOH aqueous solution (18.2967g/230mL is added under 0 DEG C of condition stirs, 457.4217mmol), reaction is moved to room temperature, under agitation, adds Boc
2o, after 12 hours, reaction solution is concentrated into 30% of original system, then water (300mL) is added, by ethyl acetate cleaning (500mL × 2), with 1M HCl, aqueous phase PH is adjusted to about 4, is then extracted with ethyl acetate (500mL × 5), merge organic phase, use anhydrous Na
2sO
4dry 2 hours, filter, filtrate are spin-dried for, dewater three times, drain under vacuum pump with methylbenzene azeotropic, and then vacuum drying oven obtains product 103.79 grams, productive rate 100% in dry 5 hours.
Two, prepare
By P20 (20g, 86.4230mmol), N-hydroxy-succinamide (39.7857g, 345.6918mmol) and DMAP (2.1166g, 17.2846mmol) join in the round-bottomed flask of 2L, add methylene dichloride (500mL), stir 10 minutes under-5 DEG C of conditions, then add DCC (71g, 345.6918mmol) fast in batches, stir 1 hour under-5 DEG C of conditions, reaction is moved to stirred overnight at room temperature.Filter, by filtrate evaporate to dryness, with acetic acid ethyl dissolution, add about 60g silica-gel powder and be spin-dried for and make solid solution, dry method upper prop.Column chromatography, with the ethyl acetate/petroleum ether gradient elution of 10% ethyl acetate/petroleum ether to 50%.Collect product, concentrating under reduced pressure evaporate to dryness obtains product 17.8416 grams, productive rate 64%.
Three, prepare
P7 (2.5g, 1.7215mmol) and P21 (1.1292g, 3.4428mmol) is placed in 200mL round-bottomed flask, adds methylene dichloride (50mL), room temperature for overnight.P21 (649.599mg, 2.0658mmol) is added according to TLC monitoring in reaction.After reaction terminates, concentrated.Solvent homogenate wet method dress post is done with methylene dichloride.Wet method loading.Column chromatography, uses 1%NH
3h
2o/2% ethanol/methylene is to 2%NH
3h
2o/4% ethanol/methylene gradient elution, collect product, concentrating under reduced pressure evaporate to dryness obtains product 2.3933 grams, productive rate 83.4%.
Four, prepare
P22 (2.1g, 1.2611mmol) is placed in 250mL round-bottomed flask, adds methylene dichloride (20mL), slowly add TFA (12.6115mmol, 0.9664mL) under stirring at room temperature.After reaction terminates, reaction solution is spin-dried for, dissolves with anhydrous methanol, add sodium bicarbonate solid (12.6115mmol, 1.0595g) and neutralize unnecessary TFA, filter, in filtrate, add 6g silica-gel powder, do solid solution.Solvent homogenate dress post 10cm × 3.5cm is with methylene dichloride.Dry method loading.Column chromatography, first uses dichloromethane eluent, then uses 3%NH
3h
2o/6% ethanol/methylene is eluted to 5%NH
3h
2o, 10% ethanol/methylene gradient elution, collect product, concentrating under reduced pressure evaporate to dryness obtains product 1.5238 grams, productive rate 100%.
Five, 4-181 is prepared
By the polyoxyethylene glycol 4ARM-SCM-40K (2.5g of four arms, 0.0625mmol) be placed in 40mL straight tube bottle, then about methylene dichloride (30mL) is added, add P23 (782.57mg under stirring at room temperature, 0.5mmol), because solubleness is bad, add DMF (2mL), under room temperature, shading slowly stirs 2 weeks.Mixture is spin-dried for, product water dissolution is put into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, suction filtration, vacuum drying obtains product 2.4836g, productive rate 96.7%.
Embodiment 10
Synthesis
By single armed polyoxyethylene glycol M-SC-12K (1.0g, 0.08333mmol) be placed in 50mL straight tube bottle, add methylene dichloride (30mL), add P23 (260.9mg under stirring at room temperature, 0.1667mmol), because solubleness is bad, add DMF (10mL), under room temperature, shading slowly stirs 2 weeks.After reaction terminates, mixture is spin-dried for, product water dissolution is put into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, filter, vacuum drying obtains product 933.3mg, productive rate 93%.
Embodiment 11
Synthesis
One, prepare
P3 (2g is added in the flask of 500mL, 3.1343mmol), P1 (0.5491g, 3.1343mmol), HBTU (1.7644mg, 4.7015mmol) with HOBT (635.3mg, 4.7015mmol), dissolve with DMF (20mL), after solution is cooled 20 minutes in 0 DEG C of cryogenic thermostat reactive bath technique, drip DIEA (2.46mL14.1044mmol).Move to room temperature after 3 hours, stirring is spent the night.Pour reaction solution into saturated NaHCO
3solution (200mL), is extracted with ethyl acetate (200mL × 2).Organic phase saturated sodium bicarbonate solution (150mL) cleaning, cleans by saturated NaCl solution (100mL).Use anhydrous MgSO
4drying, filter, concentrating under reduced pressure, by solution evaporate to dryness, then dissolves with methylene dichloride.With methylene dichloride wet method homogenate dress post 10cm × 3.5cm.Wet method loading.Silica gel column chromatography.With 2%MeOH/ methylene dichloride to 4%MeOH/ dichloromethane gradient.Collect product evaporate to dryness and obtain 2.2062g, productive rate 88.5%.
Two, prepare
In 50mL flask, add P24 (2.2062g, 2.7778mmol), dissolve with methylene dichloride (5mL), then add TFA (0.212mL, 27.778mmol), stirred overnight at room temperature.TLC display reaction does not complete, and adds 1.5mL.Next day has reacted, and is spin-dried for, and dissolves with MeOH, adds sodium bicarbonate (3g) neutralization, adds 15g silica-gel powder and be spin-dried for after having reacted.With methylene dichloride wet method homogenate dress post 30cm × 3.5cm.Dry method loading.Column chromatography, first uses 2%MeOH/ dichloromethane eluent, then uses 1%NH
3.H
2o, 2%MeOH/ methylene dichloride is to 4%NH
3.H
2o, 8%MeOH/ dichloromethane gradient, collect product point, concentrating under reduced pressure, vacuum drying obtains product 1.639g, productive rate 84.88%.
Three, 6-162 is prepared
In flask, add the polyoxyethylene glycol 4ARM-SCM-40K (10g, 0.25mmol) that P25 (1.3903g, 2.00mmol) and molecular weight are four arms of 40000, dissolve with methylene dichloride (10mL), stirred at ambient temperature.Some insolubles, dissolves after adding DMF (80mL) completely.Move in the flask of 2L by reaction after two weeks, magnetic agitation adds ether 1.5L, and product is separated out completely, continues magnetic agitation 10min, filter to obtain product, dissolve with methylene dichloride, add anhydrous diethyl ether while stirring and be precipitated out, filter, vacuum drying obtains product 8.9895g, productive rate 88%.
Embodiment 12
Synthesis
One, prepare
P25 (5.0161g, 7.2157mmol) and P5 (1.5167g, 3.4361mmol) is placed in 500mL round-bottomed flask, adds methylene dichloride (200mL), at room temperature stirring reaction.After reaction terminates, add 10g silica-gel powder and be spin-dried for and make solid solution.Dry method loading, column chromatography.First use 0.5%NH
3h
2o/1% ethanol/methylene is to 4%NH
3h
2o/8% ethanol/methylene gradient elution, collect product, concentrating under reduced pressure evaporate to dryness obtains product 4.006g, productive rate 34.8%.
Two, prepare
In 500mL round-bottomed flask, add P26 (5.4g, 3.3718mmol), dissolve with methylene dichloride (50mL), slowly drip TFA (2.58mL, 33.7176mmol) under stirring at room temperature condition, stirring is spent the night.Reaction terminates, and evaporated under reduced pressure, dissolves thick product anhydrous methanol, adds sodium bicarbonate solid (2.8g, 33.7176mmol) neutralization, filters, add 15g silica-gel powder and be spin-dried in filtrate.Solvent homogenate wet method dress post 15cm × 4.5cm is with methylene dichloride.Dry method loading.Column chromatography, uses 1%NH
3.H
2o/2%MeOH/ methylene dichloride is to 5%NH
3.H
2o/10%MeOH/ dichloromethane gradient, collect product, concentrating under reduced pressure, vacuum drying obtains straight product 4.6g, productive rate 92%.
Three, prepare
P27 (3.4114g, 2.2719mmol) and P21 (2.2356g, 6.8158mmol) is placed in 500mL round-bottomed flask, then adds methylene dichloride (200mL), stirred at ambient temperature reacts.P21 (863.4mmg, 2.6323mmol) is added according to TLC monitoring in reaction.Reaction terminates rear concentrated.Solvent homogenate wet method dress post is done with methylene dichloride.Wet method loading.Column chromatography, uses 2%NH
3h
2o/4% ethanol/methylene is to 6%NH
3h
2o/12% methyl alcohol/CH
2cl
2gradient elution, collect product, concentrating under reduced pressure evaporate to dryness obtains product 2.3933 grams, productive rate 83.4%.
Four, prepare
In 500mL round-bottomed flask, add P28 (2.5g, 1.4581mmol), dissolve with methylene dichloride (50mL), slowly drip TFA (1.117mL, 14.5812mmol) under stirring at room temperature, stirred overnight at room temperature.Be spin-dried for after reaction terminates, thick product anhydrous methanol dissolved, adds sodium bicarbonate (1.2249g, 14.58124mmol) and neutralize remaining TFA, filter, in filtrate, add 8g silica-gel powder make solid solution.Solvent homogenate wet method dress post is done with methylene dichloride.Dry method loading.Column chromatography, uses 2%NH
3h
2o/4% ethanol/methylene is to 5%NH
3h
2o/10% ethanol/methylene gradient elution, collect product, concentrating under reduced pressure evaporate to dryness obtains product 3.1 grams, productive rate 100%.
Five, 4-199 is prepared
Be the polyoxyethylene glycol 4ARM-SCM-40K (1.0g of four arms of 40000 by molecular weight, 0.02409mmol) be placed in 30mL straight tube bottle, then methylene dichloride (20mL) is added, add P29 (311.2mg under stirring at room temperature, 0.19275mmol), because solubleness is bad, add DMF (2mL).The slow stirring reaction of shading 2 weeks under room temperature.After reaction terminates, add diethyl ether sedimentation, and filter, thick product methylene dichloride dissolves, then the sedimentation that adds diethyl ether, and filters, and collects product drying and obtain 1.0322g, productive rate 100%.
Embodiment 13
Synthesis
By the polyoxyethylene glycol M-SC-12K (1.0g of single armed, 0.08333mmol) be placed in 50mL straight tube bottle, then methylene dichloride (30mL) is added, P29 (269.1mg is added under stirring at room temperature, 0.1667mmol), because solubleness is bad, add DMF (10mL), the slow stirring reaction of shading 2 weeks under room temperature.After reaction terminates, add diethyl ether sedimentation, and filter, thick product methylene dichloride dissolves, then uses ether sedimentation, collects product drying and obtain 847.6mg, productive rate 76%.
Embodiment 14
Synthesis
One, prepare
P27 (4.4g, 2.9303mmol) and P5 (616.0mg, 51.3954mmol) is placed in 500mL round-bottomed flask, then adds methylene dichloride (100mL), at room temperature stirring reaction.After terminating to reaction, stopped reaction, adds 20g silica-gel powder and does solid solution.Solvent homogenate wet method dress post 12cm × 8.5cm is with methylene dichloride.Dry method loading.Column chromatography, uses 3%NH
3h
2o/6%MeOH/ methylene dichloride is to 5%NH
3h
2o/10%MeOH/ dichloromethane gradient, collect product, concentrating under reduced pressure evaporate to dryness obtains product 1.5g, productive rate 17%.
Two, prepare
In 500mL round-bottomed flask, add P31 (1.5g, 0.4725mmol), dissolve with methylene dichloride (50mL), under stirring at room temperature condition, slowly drip TFA (0.3621mL, 4.72526mmol), be placed in stirred overnight at room temperature.Reaction terminates, and stopped reaction, is spin-dried for, and is dissolved by thick product anhydrous methanol, adds sodium bicarbonate neutralization, filters, add proper amount of silicon rubber powder and make solid solution in filtrate.Solvent homogenate wet method dress post 3.5cm × 7cm is with methylene dichloride.Dry method loading.Column chromatography, uses 3%NH
3h
2o/6%MeOH/ methylene dichloride is to 4%NH
3h
2o/8%MeOH/ dichloromethane gradient, collect product, concentrating under reduced pressure vacuum drying obtains 2.2 grams, productive rate 100%.
Three, prepare
In 250mL round-bottomed flask, add P32 (2.5g, 0.8663mmol) and P21 (852.4mg, 2.5989mmol), dissolve with methylene dichloride (50mL), room temperature for overnight.Wet method dress silicagel column 12cm × 4.5cm.Wet method loading after reacting completely.Use 2%NH
3h
2o/4%MeOH/ methylene dichloride is to 3%NH
3h
2o/6%MeOH/ dichloromethane gradient, collect product point, concentrating under reduced pressure evaporate to dryness obtains product 26212g, productive rate 976%.
Four, prepare
In 50mL round-bottomed flask, add P32 (200mg, 0.06454mmol), dissolve with methylene dichloride (5mL), slowly add TFA (0.04945mL, 0.6454mmol) under stirring at room temperature, stirring is spent the night.Decompression is spin-dried for, and by thick product dissolve with methanol, adds in sodium bicarbonate stirring and TFA.Filter, filtrate is made silica-gel powder solid solution.Homogenate method dress silicagel column 4.5cm × 7.4cm.Dry method loading.Column chromatography, first uses dichloromethane eluent, then uses 2%MeOH/CH
2cl
2to 5%MeOH/ dichloromethane eluent, then use 2%NH
3h
2o/5%MeOH/ methylene dichloride is to 4%NH
3h
2o/8%MeOH/ dichloromethane gradient.Collect product, concentrating under reduced pressure evaporate to dryness obtains product 137.4mg, productive rate 70.9%.
Five, 4-226 is prepared
The polyoxyethylene glycol M-SCM-40K (352.5mg, 0.008813mmol) getting single armed is placed in straight tube bottle, adds P33 (72.6mg, 0.02421mmol), dissolves with methylene dichloride (5mL), stirs 2 weeks under room temperature lucifuge.Concentrated, add ether sedimentation, filter to obtain thick product; Dissolved by thick product methylene dichloride, then add ether sedimentation, vacuum drying obtains 150mg, productive rate 43%.
Embodiment 15
Synthesis
One, prepare
Get P3 (7.1g, 11.1267mmol) be placed in 500mL flask, add Boc-Leu (2.5735g, 11.1267mmol), HBTU (6.2636g, 16.6900mmol), HOBT (2.2552g, 16.6900mmol) and DMF (300mL), stir 30 minutes under 0 DEG C of condition, drip DIEA (8.7218mL, 50.0702mmol), after 1 hour, move to room temperature for overnight.Be poured into by reaction solution in saturated sodium bicarbonate solution, with ethyl acetate (400mL) extraction, aqueous phase uses ethyl acetate (200mL × 3) to extract again.Merge organic phase, with saturated sodium bicarbonate solution cleaning (200mL × 2), then clean (200mL × 2) by saturated NaCl solution.The anhydrous MgSO of organic phase
4drying, filters, evaporate to dryness.Thick product wet method loading crosses silica column purification, and with 3% ethanol/methylene to 5% ethanol/methylene gradient elution, collect product, concentrate drying obtains 9.96g, productive rate 100%.
Two, prepare
Get P34 (9.8394g, 11.5568mmol) and be placed in 500mL flask, add methylene dichloride (70mL) and dissolve, add TFA (10mL), room temperature for overnight.Evaporate to dryness, thick product dissolve with methanol, adds TFA in sodium bicarbonate He unnecessary.Filter, add silica-gel powder (40g) in filtrate, revolve and steam into solid solution, dry method loading crosses silica column purification.First use 3% ethanol/methylene to 5% ethanol/methylene wash-out, then use 2%NH
3h
2o/5% ethanol/methylene is to 3%NH
3h
2o/7% ethanol/methylene gradient elution, collect product, concentrate drying obtains 6.75 grams, and 77.74%.
Three, prepare
Get P35 (6.657g, 8.8610mmol) be placed in 500mL flask, add Boc-Phe (2.3509g, 8.8610mmol), HBTU (4.9882g, 13.2915mmol), HOBT (1.7960g, 13.2915mmol) with DMF (400mL), stir 30 minutes in 0 DEG C of lower magnetic force, drip DIEA (6.9485mL, 39.8745mmol), move to room temperature after 1 hour, stirring is spent the night.Be poured into by reaction solution in saturated sodium bicarbonate solution, with ethyl acetate (400mL) extraction, aqueous phase uses ethyl acetate (200mL × 3) to extract again.Merge organic phase, with saturated sodium bicarbonate solution cleaning (200mL × 3), then clean (200mL × 3) by saturated NaCl solution.Organic phase is with anhydrous MgSO
4drying, filters, evaporate to dryness.Thick product methylene dichloride dissolves, and wet method loading crosses silica column purification.First use dichloromethane eluent, then use 2% ethanol/methylene to 7% ethanol/methylene gradient elution, collect product, concentrate drying obtains 8.9151g, productive rate 100%.
Four, prepare
Get P36 (8.8209g, 8.8335mmol) and be placed in 250mL flask, add methylene dichloride (70mL), then add TFA (6.7688mL, 88.3350mmol), room temperature for overnight.Evaporate to dryness, thick product dissolve with methanol, add sodium bicarbonate neutralization, filter, add silica-gel powder (60g) in filtrate, revolve and steam into solid solution, dry method loading crosses silica column purification.First use dichloromethane eluent, then use 5% ethanol/methylene wash-out, then use 2%NH
3h
2o/5% ethanol/methylene is to 4%NH
3h
2o/8% ethanol/methylene gradient elution, collect product, concentrate drying obtains 8.8842g, productive rate 100%.
Five, prepare
Get P37 (8.7882g, 9.7815mmol) be placed in 1L flask, add P1 (1.7135g, 9.7815mmol), HBTU (5.5063g, 14.6723mmol), HOBT (1.9825g, 14.6723mmol) with DMF (400mL), stir 30 minutes under 0 DEG C of condition, drip DIEA (7.6673mL, 44.0168mmol), under moving to room temperature after 2 hours, stirring is spent the night.Be poured into by reaction solution in saturated sodium bicarbonate solution, with ethyl acetate (500mL) extraction, aqueous phase uses ethyl acetate (200mL × 3) to extract again.Merge organic phase saturated sodium bicarbonate solution cleaning (200mL × 3), then clean (200mL × 3) by saturated NaCl solution.The anhydrous MgSO of organic phase
4drying, filter, evaporate to dryness, thick product methylene dichloride dissolves, and wet method loading crosses silica column purification.First use dichloromethane eluent, then use 2% ethanol/methylene to 7% ethanol/methylene gradient elution, collect product, concentrate drying obtains product 8.2468g, productive rate 79.87%.
Six, prepare
Get P38 (8.1332g, 10.1936mmol) and be placed in 250mL flask, add methylene dichloride (70mL) and dissolve, slowly add TFA (10mL), room temperature for overnight.Add TFA (2mL) next day, react rear stopped reaction, evaporate to dryness, thick product dissolve with methanol, adds sodium bicarbonate neutralization, filters, add silica-gel powder (60g) in filtrate, be spin-dried for and make solid solution, dry method loading crosses silica column purification.First use dichloromethane eluent, then use 2% ethanol/methylene wash-out, finally use 1%NH
3h
2o/3% ethanol/methylene is to 4%NH
3h
2o/8% ethanol/methylene gradient elution, collect product, concentrate drying obtains product 6.4309g, productive rate 90.42%.
Seven, 7-129 is prepared
Get the polyoxyethylene glycol 4ARM-SCM-40K (3.9847g of four arms, 0.09962mmol) be placed in 500mL flask, add P39 (0.7616g, 0.7970mmol), dissolve with methylene dichloride (80mL), stir 2 weeks under room temperature lucifuge.Concentrated, add ether sedimentation, filter to obtain thick product, dissolved by thick product methylene dichloride, then use ether sedimentation, filtration drying obtains product 4.3973g, productive rate 92.93%.
Embodiment 16
Synthesis
One, prepare
Get P39 (3.6671g, 3.8379mmol) and be placed in 250mL flask, add P5 (806.7mg, 1.8275mmol), then add methylene dichloride (50mL), stirred at ambient temperature 5 days.Add methyl alcohol to be dissolved by product, then add silica-gel powder (30g) and make solid solution, dry method loading crosses silica column purification.Use 0.5%NH
3h
2o/1% ethanol/methylene is to 4%NH
3h
2o/7% ethanol/methylene gradient elution, collect product, concentrate drying obtains product 3.55g, productive rate 83.62%.
Two, synthesize
Get P40 (3.5g, 1.6492mmol) and be placed in 500mL flask, add methylene dichloride (50mL) and dissolve, more slowly add TFA (2mL), in room temperature for overnight, after add again TFA (4mL), spend the night.Stopped reaction, is spin-dried for, thick product dissolve with methanol, and add sodium bicarbonate neutralization, filter, add silica-gel powder (40g) in filtrate, be spin-dried for and make solid solution, dry method loading crosses silica column purification.First use 0.5L dichloromethane eluent, then use 2%NH
3h
2o/5% ethanol/methylene is to 4%NH
3h
2o/8% ethanol/methylene gradient elution, collect product, concentrate drying obtains product 3.1333g, productive rate 93.96%.
Three, prepare
Get P41 (3.0423g, 1.5045mmol) and be placed in 100mL flask, add P21 (0.9870g, 3.0091mmol) and methylene dichloride (20mL), stirred at ambient temperature 4 days.Concentrated, then add normal hexane sedimentation, filtration drying obtains product 2.9822g, productive rate 8869%.
Four, prepare
Get P42 (2.8756g, 1.2866mmol) and be placed in 500mL flask, add methylene dichloride (20mL), more slowly drip TFA (2mL), room temperature for overnight.Be spin-dried for, thick product dissolve with methanol, adds TFA in sodium bicarbonate He unnecessary.Solids removed by filtration impurity, add silica-gel powder (30g) in filtrate, be spin-dried for and make solid solution, dry method loading crosses silica column purification.First use dichloromethane eluent, then use 2% ethanol/methylene to 5% ethanol/methylene wash-out, then use 2%NH
3h
2o/5% ethanol/methylene is to 5%NH
3h
2o/10% ethanol/methylene gradient elution, collect product, concentrate drying obtains product 1.9896g, productive rate 72.43%.
Five, 7-172 is prepared
Get the polyoxyethylene glycol 4ARM-SCM-40K (2.5g of four arms, 0.0625mmol) be placed in straight tube bottle, add methylene dichloride (20mL) and P43 (1.0724g, 0.5mmol), stir 2 weeks under room temperature lucifuge, be transferred in 200mL flask with DMF and react 7 days again.Concentrated by rotary evaporation, then adds anhydrous diethyl ether sedimentation, filters to obtain thick product; Then by thick product methylene dichloride and dissolve with methanol, add ether sedimentation, filtration drying obtains product 1.7192g.
Embodiment 17
Synthesis
Get the polyoxyethylene glycol M-SC-12K (1g of single armed, 0.0833mmol) be placed in straight tube bottle, add methylene dichloride (5mL) and P43 (357.4796mg, 0.1667mmol), stir 2 weeks under room temperature lucifuge, be transferred in 100mL flask with DMF and continue reaction 9 days.Concentrated by rotary evaporation, adds anhydrous diethyl ether sedimentation, filters to obtain thick product; Then by thick product methylene dichloride and dissolve with methanol, add ether sedimentation, filtration drying obtains product 0.9927g, productive rate 84.93%.
Embodiment 18
Synthesis
One, prepare
In flask, add L-Aspartic acid (15g, 112.70mmol) H2O/DMF (150mL/120mL) dissolve.Add triethylamine (31.41mL, 225.39mmol) while stirring under room temperature condition, then add Boc2O (25.09g, 114.95mmol), finally add DMF (60mL) and stir, spend the night.With the direct evaporate to dryness of Rotary Evaporators, dissolve by ethyl acetate (500mL), neutralize with 1M HCl (90mL).Isolate aqueous phase, in aqueous phase, add NaCl, to it no longer dissolves, extract by ethyl acetate (300mL × 3).Merge organic phase washed with water (200ml) and saturated aqueous common salt (200ml) cleaning.With anhydrous sodium sulfate drying, filter, concentrating under reduced pressure, vacuum-drying obtains product 302g, productive rate 100%.
Two, prepare
5-70 (1.8462g is entered in the three-necked flask of 500mL, 7.9180mmol), 5-64 (10.6125g, 16.6278mmol), HBTU (8.9146g, 23.7539mmol) and HOBT (3.2096g, 23.7539mmol), dissolve with DMF (300mL), solution is cooled 20 minutes in-5 DEG C of cryogenic thermostat reactive bath techniques, then DIEA (12.63mL, 71.2617mmol) is dripped.Move to stirring at room temperature after 30 minutes, spend the night.Reaction stops, and pours reaction solution into saturated NaHCO
3in solution (200mL), be extracted with ethyl acetate (400mL), then use saturated NaHCO
3solution (200mL × 3) cleaning organic phase, then clean by saturated NaCl solution (300mL × 2).The anhydrous MgSO of final organic phase
4drying, filter, then concentrating under reduced pressure is by solution evaporate to dryness.Crude product methylene dichloride is dissolved, adds 30g silica-gel powder and make solid solution, column chromatography, do solvent homogenate wet method dress post with methylene dichloride, use 1%NH
3.H
2o/2%MeOH/CH
2cl
2to 2%NH
3.H
2o/4%MeOH/CH
2cl
2gradient elution.Collect product, evaporate to dryness obtains 6.2g, productive rate 53.2%.
Three, prepare
In flask, add 5-75 (6g, 4.0715mmol), use CH
2cl
2(50ml) dissolve, slowly drip TFA (3.1199mL, 40.7152mmol) under stirring at room temperature, room temperature for overnight.Reaction terminates, and evaporated under reduced pressure, dissolves crude product anhydrous methanol, add NaHCO
3solid neutralizes.Filter, filtrate is made silica-gel powder solid solution.Solvent homogenate wet method dress post is done with methylene dichloride.Dry method loading.Column chromatography, uses 2%NH
3.H
2o/2%MeOH/CH
2cl
2to 2.5%NH
3.H
2o/5%MeOH/CH
2cl
2gradient elution.Collect product, concentrating under reduced pressure evaporate to dryness obtains 3.3g productive rate 60%.
Four, prepare
5-78 (1.0g, 0.7280mmol) and P21 (478.1mg, 1.4561mmol) is placed in 200mL round-bottomed flask, adds methylene dichloride (50mL), room temperature for overnight.After reaction terminates, concentrated.Solvent homogenate wet method dress post is done with methylene dichloride.Wet method loading.Column chromatography, uses 1%NH
3h
2o/2% ethanol/methylene is to 2%NH
3h
2o/4% ethanol/methylene gradient elution, collect product, concentrating under reduced pressure evaporate to dryness obtains product 5-85:808.7mg, productive rate 70%.
Five, prepare
5-85 (2.0g, 1.2602mmol) is placed in 250mL round-bottomed flask, adds methylene dichloride (20mL), under stirring at room temperature, slowly add TFA (12.6022mmol, 0.9657mL).After reaction terminates, reaction solution is spin-dried for, with dissolve with methanol, adds TFA in sodium bicarbonate solid He unnecessary, filter, in filtrate, add silica-gel powder make solid solution, dry method loading.Column chromatography 10cm × 3.5cm, does solvent homogenate dress post with methylene dichloride.First use dichloromethane eluent, then use 3%NH
3h
2o/6% ethanol/methylene is eluted to 5%NH
3h
2o/10% ethanol/methylene gradient gradient elution, collect product, concentrating under reduced pressure evaporate to dryness obtains product 5-99:1.78 gram, productive rate 95.0%.
Six, 5-123 is prepared
By the polyoxyethylene glycol 4ARM-SCM-40K (1.0g of four arms, 0.02409mmol) be placed in 50mL straight tube bottle, add methylene dichloride (20mL), 5-99 (286.6mg is added under stirring at room temperature, 0.01928mmol), because solubleness is bad, add DMF (15mL), slowly stir 2 weeks under room temperature shading.After reaction terminates, mixture is spin-dried for, product water dissolution is put into dialysis tubing dialysis.Then by the aqueous solution concentrating under reduced pressure of product, dissolve with methylene dichloride after vacuum-drying, add anhydrous diethyl ether while stirring and be precipitated out, suction filtration, vacuum drying obtains product 5-123:1.132g, productive rate 100%.
Test above-described embodiment
1h-NMR spectrogram and mass spectrum, as shown in Figure 25-55.
The Performance experiment of Pegylation lapatinibditosylate
Experiment 1, cytotoxicity test
Utilize MTS detection method, measure the restraining effect that compound is bred people's Malignant glioma cells strain (U87), human stomach cancer cell line (N87), human lung carcinoma cell line (CALU-3), Breast cancer lines (BT474) and Breast cancer lines (SK-BR-3).
Testing compound totally 13 groups.First and second group is respectively the DMSO solution of lapatinibditosylate and BAY 43-9006, and concentration is 0.1M.Three to eight group is respectively the normal saline solution of 6-56,6-162,7-172,4-181,4-208,7-129.Nine to ten three components Wei 95% ethanolic soln of 6-56,6-162,7-172,4-181,7-129.
One, for U87, CALU-3, N87 and BT474 tetra-strain cell (compound to be determined adopts stroke-physiological saline solution to dissolve)
U87 and CALU-3 EMDM+10%FBS+1%P/S cultivates, N87 and BT474 1640+10%FBS+1%P/S cultivates, and in 37 DEG C, cultivates in the incubator of 5%CO2.Within 2 ~ 3 days, go down to posterity once.When cell to grow at the bottom of culturing bottle about 80%, trypsin digestion cell, accurate counting, accesses in 96 orifice plates with the amount in 3000/100 μ L/ holes, and blank group directly adds not celliferous nutrient solution, uses after 24 hours after cell attachment.Suck the cell culture fluid in orifice plate, correspondence adds testing compound gradient dilution application liquid.Cell is placed in 37 DEG C, 5%CO
2incubator in cultivate, suck nutrient solution after 72 hours, continue to add testing compound gradient dilution application liquid, cultivate 48 hours.After arriving action time, every hole adds 10 μ L MTS, hatches 3 hours for 37 DEG C, detects OD 492nM in Sunrise microplate reader.
Two, for the mensuration (compound employing anhydrous alcohol solution to be determined) of SK-BR-3 cell
SK-BR-3 adopts 1640+10%FBS+1%P/S to cultivate.Other condition, the step of cell cultures are identical with above-mentioned four strain cells.The mode of action of testing compound is also identical with above-mentioned four strain cells.
Experimental result
Testing compound, can draw to draw a conclusion to the curve of inhibition of cancer cell rate as shown in Fig. 1-2 3 from figure:
1, compared with other testing compound, the solubleness of compound 4-208 in physiological saline is better.The solvability of all testing compounds in 95% ethanol is greater than the solvability in physiological saline.
2, all testing compounds all show inhibit activities to a certain degree in physiological saline, and the activity of solubleness good compound 4-208 is the strongest.Pharmaceutical activity in all testing compound 95% ethanol is greater than the pharmaceutical activity in physiological saline.The IC 6-56 and 6-162 two compounds can be inferred
50value is near 20 μMs.
3, the former medicine lapatinibditosylate (LPT) in this experiment shows good activity, the IC of the positive drug Sorafenib of this experiment
50for a μM rank, this and literature value are consistent, illustrate that native system is reliable.
The animal tolerance test of experiment 2, polyethylene glycol conjugation lapatinibditosylate
One, the tolerance of animal during 6-56 and 6-162 single tail intravenously administrable is investigated
On Balb/c female mice, give tested material 6-56 and the 6-162 single tail intravenously administrable of various dose, be specially: mouse is divided into 7 groups at random, often organize 3, administration one day, administration same day is D1, to observation post administration Mouse Weight and clinical symptom one week, D7 puts to death.
Result is as shown in table 1.6-56 administration is after 3-4 days, weight loss 2.8%-10.1%, administration after 7 days body weight substantially recover; And body weight is always in rising trend after Ctrl group and tested material 2 administration; And all groups all do not observe animal dead during administration.Illustrate that the tolerance of 6-56 is lower, and 6-162 has no side effect substantially.
The body weight change of animal when table 1 6-56 and 6-162 single tail intravenously administrable
Two, the tolerance of animal during 6-56 and 6-162 many tail intravenously administrables is investigated
On Balb/c female mice, give tested material 6-56 and 6-162 many tail intravenously administrables of various dose, be specially: mouse is divided into 3 groups at random, often organize 3, respectively 1,6,11, administration in 16 days, administration gives (6 hours interval times), administration 8 times altogether when natural gift 2 times.Administration same day is D1, to observation post administration Mouse Weight and clinical symptom, puts to death after the administration of D16 last in one week.
Result is as shown in table 2, and 6-56 (20mg/kg) administration group mouse was administration about 6 days, and Normal-weight rises; During administration 6-16 days, body weight change is less, substantially remains unchanged.And body weight is always in rising trend after solvent control group and the administration of 6-162 (60mg/kg) administration group mouse.All groups, under selected dosage, do not observe animal dead.
The body weight change of animal during table 2 6-56 and 6-162 many tail intravenously administrables
Three, 7-129 drug resistance experiment
BALB/c mouse, adopt anhydrous alcohol solution, ultrasonic wave added, reaches medicine dissolution effect after adding physiological saline again after complete drug dissolution.Medicine is divided into saline control group and 7-129 drug model group.Medicine dissolution mode be 7.75mg medicine ultrasound suspending add water after 15ul dehydrated alcohol 45ul dissolve, 37 DEG C of lasting water-baths.Experiment grouping: control group single, model group single (injecting 20mg/kg altogether), control group repeatedly, model group repeatedly (morning and afternoon each tail vein injection once, per injection amount is 10mg/kg).
Experimental result
1, drug resistance experiment single group: through the observation of 1 week, compare with physiological saline, hair indifference; Body weight change no significant difference; Without nausea,vomiting,diarrhea, appetite, maldigestion, xerosis cutis, fash, red swelling of the skin, scratch where it itches, pain, backache, expiratory dyspnea, the situation such as tired and insomnia occur.Intracorporeal organ naked eyes viewing indifference.Mice sleep and enliven situation indifference; Mouse anus and defecation situation indifference, do not have diarrhoea situation.
2, drug resistance tests repeatedly group: through the observation of 1 week, compare with physiological saline, hair has some setbacks, brightless smooth.Body weight change no significant difference; Without nausea,vomiting,diarrhea, appetite, maldigestion, xerosis cutis, fash, red swelling of the skin, scratch where it itches, pain, backache, expiratory dyspnea, the situation such as tired and insomnia occur.Intracorporeal organ naked eyes viewing indifference.Mice sleep and enliven situation indifference; Mouse anus and defecation situation indifference, do not have diarrhoea situation.
The mouse live body curative effect of experiment 3, polyethylene glycol conjugation lapatinibditosylate
Mouse becomes knurl: SK-BR-3 cell cultures is to after several bottles, and trysinization, uses serum-free 1640 substratum resuspended after PBS washing counting, be that solution density reaches 1*10E7/mL, trash ice placed stand-by.Hair is shaved at NOD mouse inoculation position, and use disposable sterilized injector inoculation SK-BR-3 cell, 0.2mL/ props up, and uses cotton ball soaked in alcohol wiping to kill the cell of spilling, put back in cage and normally raise after inoculation.
Live body is treated:
Former medicine LPT: physiological saline disperses, and 1g is scattered in 200mL physiological saline, dispersed.Oral administration gavage 20mg/kg at noon.7-129: be divided into and respectively inject a 10mg/kg (conversion dosage) in the morning and afternoon, interval time, 6-7 hour, amounted to 20mg/kg.
Experiment grouping: former medicine multiple dosing group; 7-129 multiple dosing group; Physiological saline group.
Experimental result
As shown in figure 24, compared with former medicine, 7-129 shows higher inhibition to tumour to tumor growth curve, and illustrate that lapatinibditosylate is after Pegylation, drug effect significantly improves.
Although illustrate and describe the present invention with specific embodiment, however it will be appreciated that can to make when not deviating from the spirit and scope of the present invention many other change and amendment.Therefore, this means to comprise all such changes and modifications belonged in the scope of the invention in the following claims.
Claims (10)
1. Pegylation lapatinibditosylate, its general formula is:
A is selected from the polyoxyethylene glycol of single armed or multi-arm or the derivative of polyoxyethylene glycol; X is selected from
y and M is separately selected from the amino two carboxylic acid of band or its corresponding acyl substituted thing; N is selected from amino acid or peptide; LPT is lapatinibditosylate;
A=0 or 1; B=0 or 1; C=1 or 2; D=1 or 2; E equals the arm number of A.
2. Pegylation lapatinibditosylate according to claim 1, is characterized in that, when a, b are 0, c, d are 1; Work as a=0, during b=1, d=1; Preferably, two carboxylic acids that described band is amino are L-glutamic acid or aspartic acid.
3. Pegylation lapatinibditosylate according to claim 1, is characterized in that, A is selected from single armed, four arms or the polyoxyethylene glycol of eight arms or the derivative of polyoxyethylene glycol; Preferably, the molecular weight of A is 12000,20000 or 40000 dalton; Preferably, Y and M is separately selected from
preferably, N is glycine.
4. Pegylation lapatinibditosylate according to claim 1, is characterized in that, molecular formula is
5. the preparation method of Pegylation lapatinibditosylate according to claim 1, is characterized in that, comprise the following steps:
Steps A: make lapatinibditosylate and Amino Acid/Peptide generation amidate action, obtain the first intermediate product;
Step B: make described first intermediate product and M that amidate action occur, obtain the second intermediate product;
Step C: make described second intermediate product and Y that amidate action occur, obtain the 3rd intermediate product;
Step D: make the derivative of described 3rd intermediate product and polyoxyethylene glycol or polyoxyethylene glycol by amido linkage coupling, obtain product.
6. the preparation method of Pegylation lapatinibditosylate according to claim 5, it is characterized in that, in described steps A, the condition of lapatinibditosylate and peptide/amino acid generation amidate action is: with HBTU, HOBT, DMF and DIEA for auxiliary agent, react at-2 ~ 4 DEG C, and lapatinibditosylate, peptide/arbitrary amino acid, mol ratio between HBTU, HOBT and DIEA are: 1:0.8 ~ 1.2:1.3 ~ 1.7:1.3 ~ 1.7:4.2 ~ 4.8; Preferably, also comprise before making lapatinibditosylate and peptide or amino acid generation amidate action: protect described peptide or amino acid whose amino with tertbutyloxycarbonyl;
Preferably, in described steps A, the method of lapatinibditosylate and peptide generation amidate action is: the free amine group acid-respons that the termination of lapatinibditosylate and described peptide relates to, the product obtained is again according to other free amine group acid-respons that sequence amino acid whose in described peptide relates to described peptide successively, and the total free aminoacids in above-mentioned reaction is before participation reaction: the carboxyl activating described total free aminoacids with N-hydroxy-succinamide.
7. the preparation method of Pegylation lapatinibditosylate according to claim 5, is characterized in that, in described step B, the condition of described first intermediate product and M amidate action is: mol ratio >=2 of described first intermediate product and M; Preferably, if M is L-glutamic acid, described first intermediate product and M also comprise before there is amidate action: the carboxyl activating M with N-hydroxy-succinamide; Preferably, in described step C, the condition of described second intermediate product and Y amidate action is: mol ratio >=2 of described second intermediate product and Y; Preferably, if M is L-glutamic acid, described second intermediate product and Y also comprise before there is amidate action: the carboxyl activating Y with N-hydroxy-succinamide.
8. the preparation method of Pegylation lapatinibditosylate according to claim 5, it is characterized in that, in described step D, also comprise before making described 3rd intermediate product and polyoxyethylene glycol or derivatives thereof generation amidate action: with N, N'-succinimidyl carbonate, N, the derivative of N'-succinimide yl acetate or N, N'-succinimidyl glutarat activated polyethylene glycol or polyoxyethylene glycol; Preferably, the mol ratio of described 3rd intermediate product and polyoxyethylene glycol is greater than 1.5.
9. the preparation method of Pegylation lapatinibditosylate according to claim 8, it is characterized in that, with N, N'-succinimidyl carbonate, N, the derivative of N'-succinimide yl acetate or N, N'-succinimidyl glutarat activated polyethylene glycol or polyoxyethylene glycol, obtains the 4th intermediate product, described 3rd intermediate product and described 4th intermediate product take methylene dichloride as solvent, at 20-30 DEG C, amidate action occur.
10. the injection of Pegylation lapatinibditosylate according to claim 1, is characterized in that, comprising: described Pegylation lapatinibditosylate and solvent, and described solvent is physiological saline and/or 95% ethanol.
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