CN103965398A - D-galactose/benzaldehyde nitrogen mustard/N-(2-hydroxypropyl) methacrylamide copolymer as well as preparation and application thereof - Google Patents
D-galactose/benzaldehyde nitrogen mustard/N-(2-hydroxypropyl) methacrylamide copolymer as well as preparation and application thereof Download PDFInfo
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Abstract
The invention provides a D-galactose/benzaldehyde nitrogen mustard/N-(2-hydroxypropyl) methacrylamide copolymer, and belongs to the field of high polymer chemistry and application. The D-galactose/benzaldehyde nitrogen mustard/N-(2-hydroxypropyl) methacrylamide copolymer is a macromolecular copolymer which has good biocompatibility and is formed in the way that D-galactose and benzaldehyde nitrogen mustard are linked to N-(2-hydroxypropyl) methacrylamide (HPMA) through covalent bonds. An experiment proves that the D-galactose/benzaldehyde nitrogen mustard/N-(2-hydroxypropyl) methacrylamide copolymer has the function of intelligently releasing drugs in a targeting manner, and has excellent inhibiting effect on a liver cancer HepG2 tumor cell; besides the macromolecular copolymer is an effective drug delivery system; therefore, the copolymer has excellent prospect as being used as an inhibitor for the HepG2 tumor cell and the like to be applied to the preparation of anti-tumor drugs.
Description
Technical field
The invention belongs to chemical field, relate to a kind of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer of the targeting anti-tumor activity with intelligence release medicine; The present invention also relates to the preparation method of this multipolymer simultaneously; The invention still further relates to this polymkeric substance as hepatoma Hep G 2 cells inhibitor in the application of preparing in cancer therapy drug.
Background technology
Nitrogen mustards compound belongs to alkylating agent series antineoplastic medicament, but because its toxic side effect is larger, has certain restriction in clinical application, is mainly used at present the treatment of malignant lymphoma.According to the difference of precursor structure, mustargen can be divided into benzoic acid nitrogen mustard, Chlorambucil, phenyl aldehyde mustargen and methyl mustargen.Wherein the structural formula of phenyl aldehyde mustargen is as follows:
Due to the poorly water-soluble of phenyl aldehyde mustargen, when application, need to select solubility promoter (dehydrated alcohol, DMSO etc.), can cause in vivo multiple toxic side effects.In addition, when administration, after water dilution, need a 0.25 μ m microporous membrane to filter, make administration inconvenience.
D-semi-lactosi is a kind of small-molecule substance that can have to surface of hepatocytes asialoglycoprotein receptor target identification and combination, and strong wetting ability can also increase the water-soluble of polymkeric substance.The structural formula of its D-semi-lactosi is as follows:
N-(2-hydroxypropyl) Methacrylamide (HPMA) is a kind of polymer drug carrier, there is good biocompatibility, not only can reduce the toxic side effect of medicine, reduce resistance, improve the stability in medicine body, can also increase the accumulation of medicine at tumor locus, make drug effect obtain the features such as better performance, be used as tumor-targeting drug carrier and be applied to clinical.Its structural formula is as follows:
Therefore, taking water-soluble good N-(2-hydroxypropyl) Methacrylamide as pharmaceutical carrier, by obtaining having the high-molecular copolymer of anti-tumor activity with double bond containing D-semi-lactosi, the copolymerization of phenyl aldehyde mustargen, be expected to provide more choices for anticancer field.
Summary of the invention
The object of the invention is to utilize the feature of phenyl aldehyde mustargen, D-semi-lactosi and N-(2-hydroxypropyl) Methacrylamide, a kind of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer is provided.
Another object of the present invention is to provide the preparation method of a kind of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer.
A further object of the invention, be just to provide D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as hepatoma Hep G 2 cells inhibitor in the application of preparing in antitumor drug.
(1) D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer
D-semi-lactosi/phenyl aldehyde mustargen/N-of the present invention (2-hydroxypropyl) methacrylamide copolymer is that D-semi-lactosi, phenyl aldehyde mustargen are connected to the upper high-molecular copolymer with good biocompatibility forming of N-(2-hydroxypropyl) Methacrylamide (HPMA) by covalent linkage.Its concrete preparation technology is as follows:
(1) compound
preparation: taking tetrahydrofuran (THF) as solvent, p-methyl benzenesulfonic acid is catalyzer, and molecular sieve, as water-retaining agent, under nitrogen protection, makes phenyl aldehyde mustargen react 12～15 h with 1.1.1-trimethylolethane in-5～5 DEG C; After phenyl aldehyde mustargen reacts completely, add ammonia neutralization p-methyl benzenesulfonic acid to pH=7～8, leach molecular sieve, concentrated filtrate, with methylene dichloride dilution, with the phosphoric acid buffer washing of pH=8, anhydrous sodium sulfate drying, silica gel column chromatography separates, and obtains compound
.Its structural formula is:
The mol ratio of phenyl aldehyde mustargen and 1.1.1-trimethylolethane is 1:2.9～1:3.3; The mol ratio of phenyl aldehyde mustargen and catalyzer p-methyl benzenesulfonic acid is 1:0.05～1:0.1; The add-on of molecular sieve is 15～20 times of phenyl aldehyde mustargen quality.
(2) compound
preparation: taking methylene dichloride as solvent, triethylamine, as acid binding agent, under nitrogen protection, makes compound
react 12～15 h with methacrylic chloride in-5～0 DEG C; After question response is complete, with saturated sodium bicarbonate washing, anhydrous sodium sulfate drying, silica gel column chromatography separates, and obtains compound
.Its structural formula is:
Compound
with the mol ratio of methacrylic chloride be 1:1～1:1.2; The mol ratio of methacrylic chloride and acid binding agent triethylamine is 1:1.1～1:1; Silica gel column chromatography eluent used is: ethyl acetate and sherwood oil are with the volume ratio of 1:10～1:30.
(3) compound
preparation: semi-lactosi is dissolved in acetone, adds the vitriol oil as dewatering agent, at room temperature stir spend the night, treat that solution becomes glassy yellow, add in solution of potassium carbonate and the vitriol oil, separate out white solid, suction filtration, steam acetone, with chloroform extraction, distilled water wash, anhydrous sodium sulfate drying, obtain water white transparency oily thing, be compound
.Its structural formula is:
The add-on of the vitriol oil is 2%～3% of semi-lactosi quality; The mass concentration 15%～20% of solution of potassium carbonate; Silica gel column chromatography eluent used is: ethyl acetate and sherwood oil are with the volume ratio of 1:10～1:30.
(4) compound
preparation: by compound
be dissolved in anhydrous diethyl ether, add triethylamine, be cooled to-5～5 DEG C, add methacrylic chloride, react 3～12 hours; Suction filtration is removed the precipitation of generation, and evaporate to dryness filtrate, and residuum n-hexane dissolution, removes insolubles, steams normal hexane, obtains colourless thick thing, is compound
.Its structural formula is:
Compound
with the mol ratio of methacrylic chloride be 1:1～1:1.2; Compound
with the mol ratio of triethylamine be 1:1.3～1:1.5; Methacrylic chloride add-on is compound
0.4～0.6 times of quality.
(5) compound
preparation: by compound
be dissolved in trifluoroacetic acid-water mixture, stirring at room temperature is fully hydrolyzed it, then separates out hydrolysate with ethanol, and suction filtration, obtains faint yellow solid, with ethyl alcohol recrystallization, obtains white solid and is compound
.Its structural formula is:
In trifluoroacetic acid-water mixture, the volume ratio of trifluoroacetic acid and water is 9:1～8:1.
(6) preparation of target compound: by N-(2-hydroxypropyl) Methacrylamide, compound
and compound
with DMSO and acetone solution; add initiator Diisopropyl azodicarboxylate; under nitrogen protection, react 20～24 h in 50～60 DEG C; by the mixed solution precipitation of acetone and ether; filter; with anhydrous methanol dissolution precipitation thing, the ultrafiltration and concentration centrifuge tube that is finally 3000 with molecular weight is centrifugal, removes small molecules and obtains target high-molecular copolymer.Its structure is as follows:
In formula, x=80～90 mol%, y=5～10 mol%, z=5～10 mol%; Number-average molecular weight Mn=2.1～2.8 × 10
4, Mw/Mn=1.33～1.81.
Compound
, compound
, N-(2-hydroxypropyl) Methacrylamide mol ratio be 1:1:4～1:1:20; The consumption of described initiator Diisopropyl azodicarboxylate is compound
, compound
and N-(2-hydroxypropyl) Methacrylamide total mass 5%～10%; In the mixed solution of described acetone and ether, the volume ratio of acetone and ether is 7:3～7:1.
Fig. 1 is the proton nmr spectra with the intelligent targeting anti-tumor reactive polymer that discharges medicine prepared by aforesaid method.Can draw by hydrogen nuclear magnetic resonance spectrum analysis, the high molecular polymer that copolymerization obtains, chemical shift has in compound phenyl aldehyde mustargen the peak that goes out of hydrogen on aromatic ring between 6.5～7.5ppm, and chemical shift there will be characteristic peak-CH-(on HPMA to be connected with-OH at 3.70ppm and 2.81ppm) and-CH
2(being connected with-NH-), chemical shift has the peak of Compound D-semi-lactosi at 4.22ppm～5.12ppm, illustrate that the product obtaining with aforesaid method copolymerization is consistent with the structure designing above.
Two, anti-tumor activity experiment
1, extracorporeal suppression tumor cell growth experiment
Adopt tetrazolium reduction method (MTT) to test HepG2 cell strain: to get the liver cancer H22 cell in growth logarithmic phase, cell concn is adjusted to 2 × 10
4individual/ml adds 90 μ l/ holes in 96 well culture plates, and marginal pore is filled with aseptic PBS.At 5% CO
2, hatch for 37 DEG C, in incubator, place dosing again after adherent.For high molecular polymer of the present invention and phenyl aldehyde mustargen (4-bis--(2-chloroethyl) phenyl aldehyde), setting respectively concentration is 4 gradients of 1,5,25,125 μ g/ml.Experimental group and control group are all established 4 multiple holes, after dosing, cell continues to cultivate respectively after 24h in 37 DEG C of CO2gas incubator of temperature, take out first centrifugal, after discard the supernatant nutrient solution in 96 orifice plates, carefully rinse after 2～3 times with PBS, every hole adds people 20 μ l MTT (tetrazole, 5 mg/ml, i.e. 0.5% MTT) solution, be placed in continuation in 37 DEG C of CO2gas incubator and cultivate 4h.Stop cultivating, carefully suck nutrient solution in hole.Every hole adds the DMSO of 150 μ g l, puts low-speed oscillation 10 min on shaking table, and crystallisate is fully dissolved.Measure the extinction OD value in each hole at microplate reader 570 nm.The wherein IC of polymkeric substance
50=11.7 μ g/ml, the IC of phenyl aldehyde mustargen
50=25.9 μ g/mL.Inhibitory rate of cell growth is calculated as follows:
Inhibiting rate=[(the average OD value of 1-experimental group) the average OD value of/control group] × 100%
Test result is in table 1 and Fig. 2.
Can find out, with the high-molecular copolymer of target group, HepG2 cell is had to good restraining effect from the result of above-mentioned cell experiment.Pure phenyl aldehyde mustargen is because the solvability in the aqueous solution is very little on the one hand, substantially can not touch the surface of cell, on the other hand, pure phenyl aldehyde mustargen can only be impassive with target group by cellular uptake, so limited the restraining effect of phenyl aldehyde mustargen to HepG2 cell.And gather (HPMA-semi-lactosi-mustargen) so owing to being that water miscible macromolecular compound can be assembled and the specific recognition of acceptor asialoglycoprotein receptor by the target group semi-lactosi on polymkeric substance and the overexpression of HepG2 cell surface at HepG2 cell surface, under the mediation of this receptor by HepG2 cell endocytic, increase the intake of HepG2 cell to polymkeric substance, so significantly strengthened the restraining effect to HepG2 cell.Draw pure phenyl aldehyde mustargen and poly-(HPMA-semi-lactosi-mustargen) IC to HepG2 cell from interpretation of result
50value is respectively 25.9 μ g/ml and IC
50=11.7 μ g/ml, the IC of poly-(HPMA-semi-lactosi-mustargen)
50value is the half of pure phenyl aldehyde mustargen substantially.
2, in-vitro simulated release experiment
Gather in (HPMA-semi-lactosi-mustargen) because small-molecule drug mustargen phenyl aldehyde is by the acetal bonds of pH sensitivity is connected on polymer chain.In the body fluid of human normal (pH=7.4), this acetal bonds is stable, and in tumor locus (pH=4.5～6.9), this acetal bonds can be hydrolyzed, thereby discharges micromolecular medicine mustargen phenyl aldehyde.Poly-in order to test (HPMA-semi-lactosi-mustargen) small molecular medicine mustargen phenyl aldehyde under condition of different pH rate of release, the pH value that by concentration is respectively the aqueous solution of 0.2 mg/mL poly-(HPMA-semi-lactosi-mustargen) is transferred to 7.4 by phosphoric acid buffer, 5 and 4 and be positioned over the constant temperature of 37 DEG C water-soluble in, the mustargen phenyl aldehyde releasing with chloroform extraction in the different moment, be the absorbancy of its extraction liquid of 312nm place measurement at wavelength with ultraviolet-visual spectrometer, finally adding a concentrated hydrochloric acid is all hydrolyzed mustargen phenyl aldehyde, with the ratio of absorbancy in the same time not and total absorbancy as rate of release (see figure 3) in the same time not.Result shows that poly-(HPMA-semi-lactosi-mustargen) release of mustargen phenyl aldehyde in the phosphate buffered saline buffer of pH=7.4 is slow, in 100 hours, burst size approaches 20%, and under the condition of pH=4 and 5, discharge very fast, and in first 40 hours, there is an obvious burst type to discharge, then rate of release slows down relatively, and slow-release time extends.Presentation of results poly-(HPMA-semi-lactosi-mustargen) can stable existence in the damping fluid of pH=7.4, and under the condition of pH=4 and 5, can discharge micromolecular mustargen phenyl aldehyde, has reached the object that medicine slowly discharges under condition of different pH.
In sum, high-molecular copolymer of the present invention superposes the anti-tumor activity of D-semi-lactosi, phenyl aldehyde mustargen and N-(2-hydroxypropyl) Methacrylamide, further promote the restraining effect of polymkeric substance to tumour, greatly extended the residence time of cancer therapy drug in tumour simultaneously.Experimental results show that, D-semi-lactosi/phenyl aldehyde mustargen/N-of the present invention (2-hydroxypropyl) methacrylamide copolymer has the function of the intelligence release medicine of targeting, liver cancer HepG2 tumour cell is had to good restraining effect, and the poly-HPMA of polymer carrier has also reduced the toxicity of cancer therapy drug, embody good Bc simultaneously, thereby reduced the injury of normal tissue; This high molecular polymer is also a kind of effectively drug-loading system simultaneously.Therefore the preparation that, is applied to antitumor drug using this polymkeric substance as the agent of HepG2 inhibiting tumour cells has good prospect.
Brief description of the drawings
Fig. 1 is the proton nmr spectra that the present invention has the targeting anti-tumor reactive polymer of intelligence release medicine.
Fig. 2 is high molecular polymer thing of the present invention and the external 24h antitumour activity of phenyl aldehyde mustargen curve.
Fig. 3 is that the present invention has the targeting anti-tumor reactive polymer that intelligence discharges medicine discharge the amount of mustargen phenyl aldehyde and the relation of time in pH is respectively 7.4,5 and 4 phosphate buffered saline buffer.
Embodiment
Below by specific embodiment, the synthetic and structural characterization of high-molecular copolymer of the present invention is further described.
Embodiment 1
(1) compound
preparation: get phenyl aldehyde mustargen (1.0 g, 5.6 mmol, 1.0 equiv.), be dissolved in 75mL tetrahydrofuran (THF), add 15～20g molecular sieve, under nitrogen protection, add p-methyl benzenesulfonic acid (0.13 g, 0.75 mmol, 0.13 equiv.), 1,1,1-trimethylolethane (1.45g, 16mmol, 3.0equiv.), in ice-water bath reaction 12～15 h; Follow the tracks of and react with thin-layer chromatography, after phenyl aldehyde mustargen reacts completely, add ammonia neutralization p-methyl benzenesulfonic acid to pH=7～8, suction filtration goes out molecular sieve, concentrated filtrate, with methylene dichloride dilution, wash three times with the phosphoric acid buffer of pH=8, use anhydrous sodium sulfate drying 12h, separate (ethyl acetate/petroleum ether=1/101(v/v) through silica gel column chromatography), collect product component, boil off solvent, obtain compound
(1.08g).Productive rate (78%).
1H NMR (400MHz, CDCl
3): δ7.61 (d,
J= 7.2 Hz, 2H, –CHC
6 H 2 H
2N(CH
2CH
2Cl)
2), 6.79 (d,
J= 7.2 Hz, 2H, –CHC
6H
2 H 2 N(CH
2CH
2Cl)
2), 5.18 (s, 1H, –C
HC
6H
4N(CH
2CH
2Cl)
2), 4.45 (s, 2H, –OC
H 2 C(CH
3)(CH
2O–)
2), 3.48-3.86 (m, 12H, –OCH
2C(CH
3)(C
H 2 O–)
2and –O(–O)CHC
6H
4N(C
H 2 C
H 2 Cl)
2)), 0.83 (s, 3H, –OCH
2C(C
H 3 )(CH
2O–)
2)。
(2) compound
preparation: get compound
(1.56 g, 4.5 mmol) and triethylamine (0.68 g, 6.7 mmol) are dissolved in the methylene dichloride of 50mL, and ice-water bath is cooled to 0 DEG C of left and right; Get methacrylic chloride (0.56 g, 5.4 mmol) and be dissolved in the methylene dichloride of 10mL, be slowly added drop-wise in above-mentioned solution, reaction 12～15h.Follow the tracks of and react with thin-layer chromatography, until compound
react, saturated sodium bicarbonate solution washing three times for reaction solution, with anhydrous sodium sulfate drying 10～12h, suction filtration, after filtrate is concentrated, thick product separates (ethyl acetate/petroleum ether=1/20(v/v) with silicagel column), collect product component, boil off solvent, obtain white solid 1.05g, productive rate 56%.
1H NMR (400 MHz, CDCl
3, δ, ppm): 7.61 (d,
J= 7.2 Hz, 2H, –CHC
6 H 2 H
2N(CH
2CH
2Cl)
2), 6.79 (d,
J= 7.2 Hz, 2H, –CHC
6H
2 H 2 N(CH
2CH
2Cl)
2), 6.13 (s, 1H, –OC(CH
3)C=CH
H), 5.57 (s, 1H, –OC(CH
3)C=C
HH), 5.18 (s, 1H, –C
HC
6H
4N(CH
2CH
2Cl)
2), 4.45 (s, 2H, –OC
H 2 C(CH
3)(CH
2O–)
2), 3.48-3.86 (m, 12H, –OCH
2C(CH
3)(C
H 2 O–)
2and –O(–O)CHC
6H
4N(C
H 2 C
H 2 Cl)
2)), 1.97 (s, 3H, –OC(C
H 3 )C=CH
2), 0.83 (s, 3H, –OCH
2C(C
H 3 )(CH
2O–)
2)。
(3) compound
preparation: take 1.0g(5.6mmol) D-semi-lactosi be dissolved in the acetone of 50mL, add the vitriol oil of 0.5～1.2mL, solution at room temperature stirs and spends the night, and solution becomes glassy yellow, adds 20% solution of potassium carbonate 10mL, separate out a large amount of white solids, suction filtration, steams the acetone in filtrate, and remaining filtrate is with the chloroform extraction of 5 × 15ml, the distilled water wash chloroform of using again 2 × 5ml, adds anhydrous Na
2sO
4after dry 5h, steam chloroform, take out 20 minutes with oil pump, obtain water white oily matter 1.2g, productive rate 83%.
1H NMR (400 MHz, CDCl
3, δ, ppm): 1.32–1.50 (m, 12H), 4.07 (m, 1H), 4.23–4.37 (m, 4 H), 4.62 (dd, 1H), 5.53 (d, 1H)。
(4) compound
preparation: get 1.2g(4.7mmol) compound
be dissolved in the anhydrous diethyl ether of 60mL, add 0.474g(4.7mmol) triethylamine, in ice-water bath, cool to 0 DEG C of left and right, by 0.489g(4.7mmol) methacrylic chloride be dissolved in 30mL ether, slowly be added drop-wise in above-mentioned solution with separating funnel, after dripping off, react again 3h.The precipitation that suction filtration falls to produce, filtrate evaporate to dryness, adds 50mL normal hexane, has a small amount of insolubles, after removing, steams except normal hexane, obtains colourless thick thing 1.03g, productive rate 68%.
1H NMR (400 MHz, CDCl
3, δ, ppm): 1.32–1.50 (m, 12H), 1.94 (s, 3H), 4.07 (m, 1H), 4.23–4.37 (m, 4H), 4.62 (dd, 1H), 5.53 (d, 1H). 5.56 (s, 1H), 6.13 (s, 1H)。
(5) compound
preparation: get 1.03g(3.1mmol) compound
in the round-bottomed flask of 50mL, adding 5mL volume ratio is the mixture of trifluoroacetic acid-water of 9:1, at room temperature stirs 15min, adds ethanol, and suction filtration, except desolventizing, obtains faint yellow solid, with ethyl alcohol recrystallization, obtains white solid 0.68g, productive rate 87%.
1H NMR (400 MHz, D
2O, δ, ppm): 1.95 (s, 3H, –OC(C
H 3 )C=CH
2), 4.08, 4.19 4.42, 4.63, and 5.04 (7H, sugar moiety), 6.13 (s, 1H, –OC(CH
3)C=CH
H), 5.57 (s, 1H, –OC(CH
3)C=C
HH).
(6) preparation of multipolymer: take HPMA (0.218g, 1.52 mmol, 69 mol%) and join in Shleck bottle, by the DMSO heating for dissolving of 0.5 mL, then add the acetone of 0.5 mL; Take compound
(0.052 g, 0.12 mmol, 6.5 mol%) and compound
(0.054g, 0.22 mmol, 12 mol%) join in Shleck bottle, stir until dissolve, to be cooledly add 0.016 g (5%, wt) azo and isopropyl cyanide (AIBN) during to room temperature, vacuum nitrogen filling gas circulation 3～5 times, after sealing, keep temperature greatly about 55 DEG C of left and right, react 24 hours.By mixed solution (volume ratio the is 7:3) precipitation of acetone and ether, after filtering, with the anhydrous methanol dissolution precipitation of 1mL, the ultrafiltration and concentration centrifuge tube that is 3000 with molecular weight carries out centrifugal, removes small molecules, obtains polymkeric substance 207mg, productive rate 64%.
M
n=2.7×10
4，M
w/M
n=1.28.
1H-NMR (400 MHz, DMSO, δ, ppm): δ 7.26 (–CHC
6 H 2 H
2N(CH
2CH
2Cl)
2), 6.79 (–CHC
6H
2 H 2 N(CH
2CH
2Cl)
2), 5.05, 4.14 (sugar moiety), 3.69-4.02 ((–OCH
2C(CH
3)(C
H 2 O–)
2and –O(–O)CHC
6H
4N(C
H 2 C
H 2 Cl)
2)), 3.68 (CH
3C
H(OH)CH
2NH– of HPMA), 3.06 (CH
3CH(OH)C
H 2 NH– of HPMA ), 1.33–1.80 (–C
H 2 – of polymer backbone), 0.59–0.98 (–C
H 3 )。
Embodiment 2
Compound
,
,
,
,
preparation with embodiment 1.
The preparation of multipolymer: take HPMA (0.159 g, 1.11 mmol, 82 mol%) and join in Shleck bottle, by the DMSO heating for dissolving of 0.5 mL, then add the acetone of 0.5 mL; Take compound
(0.038 g, 0.09 mmol, 7 mol%) and compound
(0.023 g, 0.09 mmol, 7 mol%) join in Shleck bottle, stir until dissolve, to be cooledly add 0.018 g (8%, wt) azo and isopropyl cyanide (AIBN) during to room temperature, vacuum nitrogen filling gas circulation 3～5 times, after sealing, keep temperature greatly about 55 DEG C of left and right, react 24 hours.By mixed solution (volume ratio the is 7:3) precipitation of acetone and ether, after filtering, with the anhydrous methanol dissolution precipitation of 1ml, the ultrafiltration and concentration centrifuge tube that is 3000 with molecular weight carries out centrifugal, removes small molecules, obtains polymkeric substance 0.153g, productive rate 70%.
M
n=2.4×10
4, M
w/M
n=1.16.
1H-NMR (400 MHz, DMSO, δ, ppm): δ 7.26 (–CHC
6 H 2 H
2N(CH
2CH
2Cl)
2), 6.79 (–CHC
6H
2 H 2 N(CH
2CH
2Cl)
2), 5.05, 4.14 (sugar moiety), 3.69-4.02 ((–OCH
2C(CH
3)(C
H 2 O–)
2and –O(–O)CHC
6H
4N(C
H 2 C
H 2 Cl)
2)), 3.68 (CH
3C
H(OH)CH
2NH– of HPMA), 3.06 (CH
3CH(OH)C
H 2 NH– of HPMA ), 1.33–1.80 (–C
H 2 – of polymer backbone), 0.59–0.98 (–C
H 3 )。
Embodiment 3
Compound
,
,
,
,
preparation with embodiment 1.
The preparation of multipolymer: take HPMA (0.159 g, 1.11 mmol, 80 mol%) and join in Shleck bottle, by the DMSO heating for dissolving of 0.5 mL, then add the acetone of 0.5 mL; Take compound
(0.057g, 0.14 mmol, 10 mol%) and compound 5 (0.034 g, 0.14 mmmol, 10 mol%) join in Shleck bottle, stir until dissolve, to be cooledly add 0.025 g (10%, wt) azo and isopropyl cyanide (AIBN) during to room temperature, vacuum nitrogen filling gas circulation 3～5 times, after sealing, keep temperature greatly about 55 DEG C of left and right, react 24 hours.By mixed solution (volume ratio the is 7:3) precipitation of acetone and ether, after filtering, with the anhydrous methanol dissolution precipitation of 1ml, the ultrafiltration and concentration centrifuge tube that is 3000 with molecular weight carries out centrifugal, removes small molecules, obtains polymkeric substance 0.16 g, productive rate 64%.
M
n=2.6×10
4, M
w/M
n=1.23.
1H-NMR (400 MHz, DMSO, δ, ppm): δ 7.26 (–CHC
6 H 2 H
2N(CH
2CH
2Cl)
2), 6.79 (–CHC
6H
2 H 2 N(CH
2CH
2Cl)
2), 5.05, 4.14 (sugar moiety), 3.69-4.02 ((–OCH
2C(CH
3)(C
H 2 O–)
2and –O(–O)CHC
6H
4N(C
H 2 C
H 2 Cl)
2)), 3.68 (CH
3C
H(OH)CH
2NH– of HPMA), 3.06 (CH
3CH(OH)C
H 2 NH– of HPMA ), 1.33–1.80 (–C
H 2 – of polymer backbone), 0.59–0.98 (–C
H 3 )。
Claims (10)
1.D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer, its structure is as follows:
In formula, x=80～90 mol%, y=5～10 mol%, z=5～10 mol%.
2. the preparation method of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as claimed in claim 1, comprises following processing step:
(1) compound
preparation: taking tetrahydrofuran (THF) as solvent, p-methyl benzenesulfonic acid is catalyzer, and molecular sieve is as water-retaining agent, under nitrogen protection, makes phenyl aldehyde mustargen and 1,1,1-trimethylolethane in-5～5 DEG C of reaction 12～15 h; After phenyl aldehyde mustargen reacts completely, add ammonia neutralization p-methyl benzenesulfonic acid to pH=7～8, leach molecular sieve, concentrated filtrate, with methylene dichloride dilution, with the phosphoric acid buffer washing of pH=8, anhydrous sodium sulfate drying, silica gel column chromatography separates, and obtains compound
;
(2) compound
preparation: taking methylene dichloride as solvent, triethylamine, as acid binding agent, under nitrogen protection, makes compound
react 12～15 h with methacrylic chloride in-5～0 DEG C; After question response is complete, with saturated sodium bicarbonate washing, anhydrous sodium sulfate drying, silica gel column chromatography separates, and obtains compound
;
(3) compound
preparation: semi-lactosi is dissolved in acetone, adds the vitriol oil as dewatering agent, at room temperature stir spend the night, treat that solution becomes glassy yellow, add in solution of potassium carbonate and the vitriol oil, separate out white solid, suction filtration, steam acetone, with chloroform extraction, distilled water wash, anhydrous sodium sulfate drying, obtain water white transparency oily thing, be compound
;
(4) compound
preparation: by compound
be dissolved in anhydrous diethyl ether, add triethylamine, be cooled to-5～5 DEG C, add methacrylic chloride, react 3～12 hours, by compound
be dissolved in anhydrous diethyl ether, add triethylamine, be cooled to-5～5 DEG C, add methacrylic chloride, react 3～12 hours; Suction filtration is removed the precipitation of generation, and evaporate to dryness filtrate, and residuum n-hexane dissolution, removes insolubles, steams normal hexane, obtains colourless thick thing, is compound
;
(5) compound
preparation: by compound
be dissolved in trifluoroacetic acid-water mixture, stirring at room temperature is fully hydrolyzed it, separates out hydrolysate with ethanol, and suction filtration, obtains faint yellow solid, with ethyl alcohol recrystallization, obtains white solid and is compound
;
(6) preparation of target compound: by N-(2-hydroxypropyl) Methacrylamide, compound
and compound
with DMSO and acetone solution; add initiator Diisopropyl azodicarboxylate; under nitrogen protection, react 20～24 h in 50～60 DEG C; by the mixed solution precipitation of acetone and ether; filter; with anhydrous methanol dissolution precipitation thing, the ultrafiltration and concentration centrifuge tube that is finally 3000 with molecular weight is centrifugal, removes small molecules and obtains target high-molecular copolymer.
3. the preparation method of described D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as claimed in claim 2, it is characterized in that: in step (1), the mol ratio of phenyl aldehyde mustargen and 1.1.1-trimethylolethane is 1:2.9～1:3.3; The mol ratio of phenyl aldehyde mustargen and catalyzer p-methyl benzenesulfonic acid is 1:0.05～1:0.1; The add-on of molecular sieve is 15～20 times of phenyl aldehyde mustargen quality.
4. the preparation method of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as claimed in claim 2, is characterized in that: in step (2), the mol ratio of methacrylic chloride and acid binding agent triethylamine is 1:1.1～1:1; Compound
with the mol ratio of methacrylic chloride be 1:1～1:1.2.
5. the preparation method of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as claimed in claim 2, it is characterized in that: in step (1) and step (2), described silica gel column chromatography eluent used is: ethyl acetate and sherwood oil are with the volume ratio of 1:10～1:30.
6. the preparation method of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as claimed in claim 2, is characterized in that: in step (3), the add-on of the vitriol oil is 2%～3% of semi-lactosi quality; The mass concentration 15%～20% of solution of potassium carbonate.
7. the preparation method of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as claimed in claim 2, is characterized in that: in step (4), and compound
with the mol ratio of methacrylic chloride be 1:1～1:1.2; Compound
with the mol ratio of triethylamine be 1:1.3～1:1.5; Methacrylic chloride add-on is compound
0.4～0.6 times of quality.
8. the preparation method of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as claimed in claim 2, it is characterized in that: in step (5), in trifluoroacetic acid-water mixture, the volume ratio of trifluoroacetic acid and water is 9:1～8:1.
9. the preparation method of D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as claimed in claim 2, is characterized in that: in step (6), and compound
, compound
, N-(2-hydroxypropyl) Methacrylamide mol ratio be 1:1:4～1:1:20; The consumption of described initiator Diisopropyl azodicarboxylate is compound
, compound
and N-(2-hydroxypropyl) Methacrylamide total mass 5%～10%; In the mixed solution of described acetone and ether, the volume ratio of acetone and ether is 7:3～7:1.
As claimed in claim 1 D-semi-lactosi/phenyl aldehyde mustargen/N-(2-hydroxypropyl) methacrylamide copolymer as hepatoma Hep G 2 cells inhibitor in the application of preparing in cancer therapy drug.
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| CN106822909A (en) * | 2017-02-14 | 2017-06-13 | 辽宁大学 | A kind of gambogicacid galactolipin HPMA high-molecular copolymers and its preparation method and application |
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