CN110591076B - Low-oxygen targeting AGT inhibitor conjugate and preparation method and application thereof - Google Patents
Low-oxygen targeting AGT inhibitor conjugate and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a hypoxia targeting AGT inhibitor conjugate, a preparation method and application thereof. The amphiphilic low-oxygen targeting AGT inhibitor conjugate can form a nano carrier through self-assembly, is used for encapsulating a medicament and delivering the medicament to a tumor low-oxygen area in a targeting manner, thereby playing a targeting anti-tumor role and simultaneously having anti-drug resistance and good water solubility.
Description
Technical Field
The invention relates to the field of medicine preparation, and in particular relates to a hypoxia targeting AGT inhibitor conjugate, and a preparation method and application thereof.
Background
O6-alkylguanine-DNA alkyltransferase (O)6-alkylguanine-DNA alkyltransferase, AGT) is a class of DNA damage repair proteins capable of converting guanine to O6The alkyl group at position is transferred to its own cysteine residue at position 145, which restores the damaged guanine. However, the DNA damage repair of AGT to tumor cells can lead to tumor cells developing resistance to anticancer alkylating agents, thereby greatly reducing the effectiveness of chemotherapy. To improve AGT-mediated resistance, AGT inhibitors are often used clinically to deplete the over-expressed AGT from tumor cells, thereby increasing the sensitivity of the tumor cells to anticancer alkylating agents to increase alkaneTherapeutic effects of the agent. O is6-benzylguanine (O)6-BG) is a clinically common AGT inhibitor, O6-BG can transfer its own benzyl group to the active site of AGT, resulting in the formation of a structurally stable S-benzylcysteine leading to inactivation of AGT. Thus, O6the-BG can effectively inhibit the repair action of AGT on alkylated DNA and improve the sensitivity of tumor cells to anticancer alkylating agents.
Studies show that6the-BG is taken as an adjuvant and can obviously improve the inhibition effect of an alkylating agent on tumor cells after being combined with an anticancer alkylating agent Chloroethylnitrosourea (CENUs), temozolomide or cisplatin and the like. However, O6the-BG and the derivatives or the analogues thereof as AGT inhibitors have the defects of limited activity, poor water solubility, non-targeting property and the like, and particularly the compounds can inhibit the AGT of normal cells while inhibiting the AGT of tumor cells, so that the sensitivity of the normal cells to an alkylating agent is enhanced, and the toxic and side effects of the anticancer alkylating agent on the normal cells are increased. In addition, anticancer alkylating agents such as CENUs and the like have the defects of poor water solubility, poor stability, non-targeting property and the like and have enhanced toxic and side effects when being combined with AGT inhibitors. Therefore, CENUs is also limited in clinical application.
Although there are some studies on O6The conjugate of-BG or its derivative and CENUs is a molecule to avoid the compatibility problem of combined medicines, but the combined molecules still have the defects of non-tumor targeting, poor water solubility or poor stability and the like.
Disclosure of Invention
In view of the problems of the prior art, the first objective of the present invention is to provide a hypoxia targeting AGT inhibitor conjugate.
The invention provides a hypoxia targeting AGT inhibitor conjugate, which comprises an AGT inhibitor and a hydrophilic polymer material, wherein the AGT inhibitor and the hydrophilic polymer material are mutually coupled through a hypoxia response group.
The AGT inhibitor can block AGT mediated tumor drug resistance, and the AGT inhibitor is combined with a hypoxia response group and a hydrophilic polymer to generate an amphiphilic hypoxia targeting conjugate. The conjugate can form a nano-carrier through self-assembly, is used for encapsulating drugs and delivering the drugs to a tumor hypoxia area in a targeted manner, thereby playing a targeted anti-tumor role and simultaneously having anti-drug resistance and good water solubility.
Preferably, the hypoxia-responsive group is azobenzene, which is capable of undergoing azo bond cleavage in hypoxic regions of the tumor.
The AGT inhibitor is O6-benzylguanine or a derivative thereof.
The hydrophilic polymer material is methoxy polyethylene glycol amine, carboxymethyl dextran or polyoxyethylene, preferably methoxy polyethylene glycol amine, wherein polyethylene glycol has good biocompatibility and is inert, so that the circulation time of the medicament in vivo can be prolonged.
Preferably, the conjugate has a structure represented by general formula (I):
wherein R is H, NH2、CH3、CH2NH2、CH2One of the OH groups is selected from the group consisting of,
n1is an integer of 2 to 6, and,
n2is CH in methoxypolyethyleneglycolamine2CH2The number of O and the molecular weight of the methoxypolyethylene glycol amine are 1000-10000 Da, and specifically one of 1000, 2000, 5000 and 10000 Da.
In this case, the conjugate includes a hydrophobic O6-a BG group, a hypoxia-responsive group Azobenzene (AB) and hydrophilic polyethylene glycol (PEG) to form an amphiphilic hypoxia-targeted conjugate BG-AB-PEG.
Preferably, when R is H, the conjugate has better hypoxia targeting property and drug resistance.
Further preferably, when R is H, n1When the number is 2, the conjugate has better hypoxia targeting property and drug resistance.
Even more preferably, when R is H, n1Is 2 and the molecular weight of the methoxypolyethyleneglycol amine is 2000DaThe hypoxia targeting property and the drug resistance of the conjugate are further improved.
The second purpose of the invention is to provide a preparation method of the conjugate, which is prepared by amidation reaction of a low-oxygen response group, an AGT inhibitor and a hydrophilic polymer material, and has simple reaction steps and low cost.
Preferably, when the conjugate has a structure represented by the general formula (I) and R is H, n1When the number is 2, the preparation method comprises the following steps:
(1) mixing O with6-benzylguanine and dihaloalkane are subjected to substitution reaction to generate N9-halogenated ethyl-O6-benzylguanine;
(2) adopting a drape Rayle reaction, reacting the product obtained in the step (1) with phthalimide potassium, and then performing a hydrazinolysis reaction with hydrazine hydrate to generate N9- (2-amido) ethyl-O6-benzylguanine;
(3) reacting azobenzene-4, 4 '-dicarboxylic acid with methoxy polyethylene glycol amine to generate a methoxy polyethylene glycol-4, 4' -dicarboxamide azobenzene conjugate;
(4) and (4) reacting the products obtained in the step (2) and the step (3) to obtain the catalyst.
Further, the preparation method specifically comprises the following steps:
(1) N9-Haloethyl-O6Synthesis of-benzylguanine
Mixing O with6dissolving-BG in acetone, adding inorganic salt as catalyst, dropwise adding dihaloalkane, stirring to react, filtering, collecting filtrate, rotary steaming, separating and purifying by silica gel column chromatography, and vacuum drying to obtain N9-haloethyl-O6-benzylguanine;
(2) n9- (2-amino) ethyl-O6Synthesis of-benzylguanine
Dissolving the product obtained in the step (1) in an anhydrous organic solvent, adding a potassium phthalimide solid, stirring, refluxing, reacting, extracting, washing, drying and rotary steaming, dissolving the obtained product in the anhydrous organic solvent, adding hydrazine hydrate, stirring, refluxing, reacting to allow hydrazinolysis, extracting, washing, drying and rotary steaming to obtain N9- (2-amino) N9) ethyl-O6-benzylguanine;
(3) synthesis of methoxy polyethylene glycol-4, 4' -diformylamidoazo benzene conjugate
Dissolving azobenzene-4, 4 '-dimethyl acid in anhydrous pyridine, adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), reacting for a certain time, adding 4-Dimethoxypyridine (DMAP) and methoxypolyethyleneglycoamine to react, removing pyridine by rotary evaporation, dialyzing, and freeze-drying to obtain methoxypolyethyleneglycone-4, 4' -dimethylaminoazobenzene conjugate;
(4) methoxy polyethylene glycol-4, 4' -dicarboxamidazobenzene-N9-ethyl-O6Synthesis of-benzylguanine conjugates
And (3) dissolving the product obtained in the step (3) in anhydrous pyridine, adding EDC and NHS, reacting for a certain time, adding the product obtained in the step (2) and DMAP, stirring for reaction, dialyzing, and freeze-drying to obtain the target conjugate.
Preferably, in step (1), O6The molar ratio of the benzylguanine to the inorganic base to the dihaloalkane is 1:1-7:1-6, and the reaction temperature is 40-60 ℃. The dihaloalkane is preferably dibromo alkane or dichloroalkane, and is further preferably dibromo alkane, and the dibromoalkane has high reaction activity, so that smooth reaction can be ensured, and the reaction yield can be increased. The inorganic salt is preferably anhydrous potassium carbonate. The rotary evaporation temperature is preferably 35-45 ℃. And (3) separating and purifying the sample by adopting a column chromatography method, wherein a preferred column chromatography stationary phase is silica gel, and a preferred mobile phase is petroleum ether and ethyl acetate, and further preferred petroleum ether/ethyl acetate (v/v) ═ 1: 2-6.
Preferably, in step (2), N9-haloethyl-O6The molar ratio of benzylguanine to potassium phthalimide is 1:1-4, the reaction temperature is 40-75 ℃, and N9- (2- (phthalimido) ethyl) -O6The molar ratio of the benzylguanine to the hydrazine hydrate is 2-6:1, and the reaction temperature is 50-70 ℃. The organic solvent solution is preferably acetonitrile or N, N-Dimethylformamide (DMF).
Preferably, in the step (3), the molar ratio of the methoxypolyethyleneglycol amine to the azobenzene-4, 4' -dicarboxylic acid is 1:1-100, the reaction temperature is preferably 25-35 ℃, and the rotary evaporation temperature is preferably 50-70 ℃. The dialysate is preferably distilled water, and the dialysis time is preferably 24-48 h.
Preferably, in the step (4), the molar ratio of the product obtained in the step (3) to the product obtained in the step (2) is 1:1-100, the reaction temperature is preferably 25-35 ℃, and the dialysate is preferably distilled water.
When R in the structure shown in the general formula (I) is not H, n1If not 2, then O is added accordingly6-benzyl guanine by its derivative, and in step (1) the number of carbon atoms of dihaloalkane and n1Correspondingly, the preparation can still be carried out by adopting the method.
The third purpose of the invention is to provide the application of the conjugate in preparing anti-tumor drugs.
The tumor is one or more of brain tumor, myeloma, melanoma, lung cancer, breast cancer, leukemia, colon cancer and lymph cancer, preferably one or more of brain tumor, leukemia, colon cancer and lymph cancer.
The fourth purpose of the invention is to provide an anti-tumor nano preparation, which is prepared by self-assembling the conjugate to form a nano carrier to encapsulate an anti-tumor alkylating agent.
The nano preparation can deliver the anti-tumor alkylating agent to a tumor area in a targeted mode to play a targeted anti-tumor role, and simultaneously release the AGT inhibitor to play a role in inhibiting tumor drug resistance. The anti-tumor alkylating agent and the AGT inhibitor are targeted on tumor hypoxia parts, so that damage to normal cells can be avoided, and toxic and side effects of combined medication are reduced. Meanwhile, the nanometer material is used as a drug carrier, so that the problems of poor water solubility and poor stability of the anti-tumor alkylating agent are solved, and the circulation time of the drug in blood is prolonged. Namely, the nano preparation has the advantages of high anti-tumor activity, good stability, low toxic and side effects and the like.
Preferably, the antineoplastic alkylating agent is an alkylating agent capable of causing DNA alkylation damage, further preferably one or more of Chloroethylnitrosourea (CENUs), temozolomide and dacarbazine; more preferably carmustine (BCNU), semustine (Me-CCNU) or lomustine (CCNU) which is hydrophobic in CENUs.
The invention also provides a preparation method of the anti-tumor nano preparation.
Preferably, when the antitumor alkylating agent is BCNU with strong lipid solubility, the method specifically comprises the following steps: dissolving the conjugate and BCNU in an organic solvent, hydrating with deionized water under ultrasonic treatment, dialyzing, and freeze-drying to obtain the anti-tumor nano preparation.
Preferably, the BCNU and the conjugate are dissolved in an organic solvent in a mass ratio of 1:2 to 1: 20; the organic solvent is preferably one or more of tetrahydrofuran, methanol, chloroform and dimethyl sulfoxide, and is further preferably tetrahydrofuran.
The amphiphilic low-oxygen targeting AGT inhibitor conjugate can form a nano carrier through self-assembly, is used for encapsulating a medicament and delivering the medicament to a tumor low-oxygen area in a targeting manner, thereby playing a targeting anti-tumor role and simultaneously having anti-drug resistance and good water solubility.
Drawings
FIG. 1 is a particle size distribution and electron micrograph of BG-AB-PEG/BCNU1 in example 4 of the present invention;
FIG. 2 is a graph showing the particle size distribution and electron microscopy of BG-AB-PEG/BCNU 2 in example 5 of the present invention;
FIG. 3 is a graph showing the particle size distribution and electron microscopy of BG-AB-PEG/BCNU 3 in example 6 of the present invention;
FIG. 4 shows the results of stability experiments of BG-AB-PEG/BCNU in Phosphate Buffered Saline (PBS) and high-sugar medium containing 10% serum (FBS) in Experimental example 1;
FIG. 5 shows the results of the hypoxia-reduction experiment of BG-AB-PEG/BCNU in Experimental example 2;
FIG. 6 shows the results of in vitro BG-AB-PEG/BCNU release experiments under hypoxic conditions in Experimental example 3;
FIG. 7 shows the results of the in vitro BG-AB-PEG/BCNU release test in Experimental example 3 under normoxic conditions;
FIG. 8 shows the results of the BG-AB-PEG/BCNU cell clone formation experiment in Experimental example 4;
FIG. 9 shows the results of the cell invasion assay of BG-AB-PEG/BCNU in Experimental example 5.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The structures of conjugate 1, conjugate 2 and conjugate 3 referred to in the following examples are as follows:
example 1: preparation of hypoxia targeting AGT inhibitor conjugate 1(BG-AB-PEG 1)
1) N9-bromoethyl-O6Synthesis of-benzylguanine
Weighing O6-benzylguanine (960mg,4mmol) is put in a 100mL round-bottom flask, then 50mL acetone is added to dissolve the benzylguanine, anhydrous potassium carbonate (1660mg,12mmol) is added, the mixture is slowly heated to 50 ℃,1, 2-dibromoethane (1.4mL,16mmol) is dripped into the mixture, the mixture is stirred and reacts for 72 hours after dripping, the reaction solution is filtered, the filtrate is collected, the solvent is dried by spinning under reduced pressure at 40 ℃, the separation and purification are carried out by silica gel column chromatography, the mixed solution of petroleum ether and ethyl acetate is used as eluent, gradient elution is adopted (the mixed solution of petroleum ether and ethyl acetate V/V is 1:2-1: 6), and the white solid N9-bromoethyl-O is obtained by vacuum drying at 40 DEG6-benzylguanine (970mg,2.8mmol), yield 69%.
UVλ:248,283nm；
IR (KBr pellet) v/cm-1:3467.1(-(CH2)2-N-H)；2957.3(-CH2-)；1633.6(C＝C)；1249.9(C-O-C)；1065.7(C-N)；687.8(C-Br)；
1H NMR(400MHz,DMSO)δ:3.78(t,2H,CH2)；4.23(t,2H,CH2)；5.46(s,2H,CH2)；6.99(s,2H,NH2)；7.38-7.47(m,5H,C6H5)；8.05(s,1H,CH)；
ESI-MS M/z 348.0457 and 350.0436(M + H)+。
2)N9-(2-amino) ethyl-O6Synthesis of-benzylguanine
The solid N9-bromoethyl-O obtained is weighed6-benzylguanine (970mg,2.8mmol) in a 50mL round bottom flask, dissolved in 20mL anhydrous N 'N-Dimethylformamide (DMF), added phthalimide potassium solid (1680mg,9.1mmol), stirred at 65 ℃ under reflux for 4h, extracted with ethyl acetate and deionized water, the organic phase washed with saturated aqueous sodium chloride, dried over night over anhydrous sodium sulfate, dried at 35 ℃ under vacuum to give a white solid, this product (996mg,2.4mmol) was weighed out dissolved in 10mL anhydrous N' N-Dimethylformamide (DMF), adding 0.8mL of hydrazine hydrate, controlling the temperature at 32 ℃, stirring, refluxing and reacting for 4h to allow hydrazinolysis to occur, extracting with dichloromethane and deionized water, washing an organic phase with a saturated sodium chloride aqueous solution, drying over night anhydrous sodium sulfate, and drying in vacuum at 35 ℃ to obtain N9- (2-amino) ethyl-O.6Benzylguanine (505mg,1.8mmol), 66% yield.
UVλ:250,283nm；
IR (KBr pellet) v/cm-1:3467.7(N-(CH2)2-)；3320.4(N-H)；2954.5(-CH2-)；1613.7(C＝C)；1251.5(C-O-C)；1073.7(C-N)；
1H NMR(400MHz,DMSO)δ:2.90(t,2H,NH2)；3.99(t,2H,CH2)；5.50(s,2H,CH2)；6.50(s,2H,NH2)；7.33-7.52(m,5H,C6H5)；7.88(s,1H,CH)；
ESI-MS:m/z 284.1458(M+H)+。
3) Preparation of methoxy polyethylene glycol-4, 4' -dicarboxamidazobenzene conjugate
Azobenzene-4, 4 ' -dicarboxylic acid (150mg,0.6mmol) was weighed into a 100mL round-bottomed flask, dissolved in 60mL anhydrous pyridine solution, EDC (130mg,0.8mmol) and NHS (92mg,0.8mmol) were added, after 2h reaction DMAP (7mg,0.06mmol) and methoxypolyethyleneglycoamine 2000(1110mg,0.6mmol) were added, reaction was carried out for 12h, pyridine was removed by rotary evaporation at 50 ℃, dissolved in deionized water and centrifuged to remove free azobenzene-4, 4 ' -dicarboxylic acid to give methoxypolyethyleneglycone-4, 4 ' -dicarboxamidazobenzene conjugate (340mg,0.15mmol) in 25% yield.
UVλ：445nm；
1H NMR(400MHz,DMSO)δ:2.93(s,3H,CH3),3.24(t,2H,CH2)，3.50(s,176H,CH2),3.87.(t,2H,CH2)；8.03(s,1H,NH),8.10-8.33(m,8H,C6H4)。
4) Preparation of methoxypolyethylene glycol-4, 4' -dicarboxamidazobenzene-N9-ethyl-O6-benzyl guanine conjugate (BG-AB-PEG 1)
Dissolving methoxypolyethylene glycol-4, 4' -dicarboxamidazobenzene conjugate (1020mg,0.6mmol) in 60mL of anhydrous pyridine, adding EDC (130mg,0.8mmol) and NHS (92mg,0.8mmol), reacting for 2h, and reacting with N9- (2-amino) ethyl-O6-benzylguanine (170mg,0.6mmol) was put into deionized water and stirred at room temperature for reaction for 24h, the resulting solution was dialyzed against deionized water for 2 days, and lyophilized to give BG-AB-PEG 1(433mg, 0.17mmol) with 28% yield.
UVλ：445nm；
1H NMR(400MHz,DMSO)δ:3.11(s,3H,CH3)；3.23(m,2H,CH2)；3.65(m,176H,CH2)；3.74(t,2H,CH2)；3.92(t,2H,CH2)；3.97(t,2H,CH2)；5.54(s,2H,CH2)；6.57(s,2H,NH2)；7.27-7.52(m,5H,C6H5)；7.67(t,1H,NH)；7.74(t,1H,NH)；7.67-8.23(d,8H,C6H4)；8.52(s,1H,CH)。
Example 2: preparation of hypoxia targeting AGT inhibitor conjugate 2(BG-AB-PEG 2)
1) N9-Bromopropyl-O6Synthesis of-benzylguanine
Weighing O6-benzylguanine (480mg,2mmol) in 50mL round-bottom flask, then adding acetone 25mL to dissolve it, adding anhydrous potassium carbonate (830mg,6mmol), slowly heating the mixture to 50 ℃, dropwise adding 1, 3-dibromopropane (576. mu.L, 8mmol), stirring to react for 72h after dripping, filtering the reaction solution, collecting the filtrate, spinning off the solvent under reduced pressure at 40 ℃, separating and purifying by silica gel column chromatography, using the mixed solution of petroleum ether and ethyl acetate as eluent, and adopting gradient elution (petroleum ether/ethyl acetate)V/V is 1:2-1: 6), and vacuum drying is carried out at 40 ℃ to obtain white solid N9-bromopropyl-O6Benzylguanine (447mg,1.5mmol), yield 75%.
UVλ:248,283nm；
IR (KBr pellet) v/cm-1:3464.4(-(CH2)2-N-H)；2955.7(-CH2-)；1636.2(C＝C)；1247.3(C-O-C)；1067.3(C-N)；684.8(C-Br)；
1H NMR(400MHz,DMSO)δ:3.25(m,2H,CH2)；3.74(t,2H,CH2)；4.26(t,2H,CH2)；5.44(s,2H,CH2)；6.93(s,2H,NH2)；7.38-7.45(m,5H,C6H5)；8.01(s,1H,CH)；
ESI-MS M/z 362.0614 and 364.0595(M + H)+。
2) N9- (2-amino) propyl-O6Synthesis of-benzylguanine
The solid N9-bromopropyl-O was weighed6-benzylguanine (417mg,1.4mmol) in a 25mL round bottom flask in 10mL anhydrous DMF solution, adding potassium phthalimide solid (0.6g,3.3mmol), stirring at 65 ℃ for reflux reaction for 4h, extracting with ethyl acetate and deionized water, washing the organic phase with saturated aqueous sodium chloride solution, drying overnight over anhydrous sodium sulfate, vacuum drying at 35 ℃ to obtain white solid, weighing the product (513mg,1.2mmol) and dissolving in 10mL anhydrous N' N-Dimethylformamide (DMF) solution, adding 0.4mL hydrazine hydrate, stirring at 32 ℃ for reflux reaction for 4h to allow hydrazinolysis, extracting with dichloromethane and deionized water, washing the organic phase with saturated aqueous sodium chloride solution, drying overnight over anhydrous sodium sulfate, vacuum drying at 35 ℃ to obtain N9- (2-amino) propyl-O6Benzylguanine (268mg, 0.9mmol), yield 65%.
UVλ:250,283nm；
IR (KBr pellet) v/cm-1:3466.6(N-(CH2)2-)；3323.4(N-H)；2953.6(-CH2-)；1616.2(C＝C)；1256.4(C-O-C)；1072.8(C-N)。
1H NMR(400MHz,DMSO)δ:2.96(t,2H,NH2)；3.21(m,2H,CH2)；3.78(t,2H,CH2)；4.32(t,2H,CH2)；5.58(s,2H,CH2)；6.56(s,2H,NH2)；7.37-7.52(m,5H,C6H5)；7.83(s,1H,CH)；
ESI-MS:m/z 299.1619(M+H)+。
3) Preparation of methoxy polyethylene glycol-4, 4' -dicarboxamidazobenzene conjugate
Azobenzene-4, 4 ' -dicarboxylic acid (75mg,0.3mmol) was weighed into a 50mL round-bottomed flask, dissolved in 30mL anhydrous pyridine solution, EDC (65mg,0.4mmol) and NHS (46mg,0.4mmol) were added, after 2h reaction DMAP (4mg,0.03mmol) and methoxypolyethyleneglycoamine 2000(555mg,0.3mmol) were added, reaction was carried out for 12h, pyridine was removed by rotary evaporation at 50 ℃, dissolved in deionized water and centrifuged to remove free azobenzene-4, 4 ' -dicarboxylic acid to give methoxypolyethyleneglycone-4, 4 ' -dicarboxamidazobenzene conjugate (250mg,0.11mmol) in 37% yield.
UVλ：445nm；
1H NMR(400MHz,CDCl3)δ3.09(s,3H,CH3)；3.48(t,2H,CH2)；3.67(t,176H,CH2)；4.06(t,2H,CH2)；8.11(s,1H,NH)；8.46-8.89(m,8H,C6H4)。
4) Preparation of methoxypolyethylene glycol-4, 4' -dicarboxamidazobenzene-N9-propyl-O6-benzyl guanine conjugate (BG-AB-PEG 2)
EDC (65mg,0.4mmol) and NHS (46mg,0.4mmol) were added to methoxypolyethylene glycol-4, 4' -dicarboxamidazobenzene conjugate (681mg,0.3mmol), reacted for 2h, and reacted with N9- (2-amino) propyl-O6-benzylguanine (90mg,0.3mmol) was put into deionized water and stirred for reaction at room temperature for 24h, and the resulting solution was dialyzed against deionized water for 2 days and lyophilized to give BG-AB-PEG 2(191mg,0.075mmol) with 25% yield.
UVλ：445nm；
1H NMR(400MHz,DMSO)δ:3.20(m,2H,CH2)；3.08(s,3H,CH3)；3.67(m,176H,CH2)；3.88(t,2H,CH2)；3.93(t,2H,CH2)；3.45(m,2H,CH2)；3.67(t,2H,CH2)；6.97(s,2H,NH2)；5.29(s,2H,CH2)；7.12-7.65(m,5H,C6H5)；7.77(t,1H,NH)；7.91(t,1H,NH)；8.36-8.68(d,8H,C6H4)；8.79(s,1H,CH)。
Example 3: preparation of hypoxia targeting AGT inhibitor conjugate 3(BG-AB-PEG 3)
1) N9-bromoethyl-O6Synthesis of-benzylguanine
Weighing O6-benzylguanine (1920mg,8mmol) is placed in a 250mL round-bottom flask, then 100mL acetone is added to dissolve the benzylguanine, anhydrous potassium carbonate (3320mg,24mmol) is added, the mixture is slowly heated to 50 ℃,1, 2-dibromoethane (2.8mL,32mmol) is dropwise added to the mixture, the mixture is stirred to react for 72 hours after the dropwise addition, reaction liquid is filtered, filtrate is collected, the solvent is dried in a spinning mode under reduced pressure at 40 ℃, the separation and purification are carried out through silica gel column chromatography, the mixed liquid of petroleum ether and ethyl acetate is used as eluent, gradient elution is adopted (the mixed liquid of petroleum ether and ethyl acetate V/V is 1:2-1: 6), and white solid N9-bromoethyl-O is obtained after vacuum drying at 40 DEG6-benzylguanine (1645mg,4.7mmol), yield 59%.
UVλ:248,283nm；
IR (KBr pellet) v/cm-1:3463.1(-(CH2)2-N-H)；2954.7(-CH2-)；1635.2(C＝C)；1252.7(C-O-C)；1068.1(C-N)；687.3(C-Br)；
1H NMR(400MHz,DMSO)δ:3.71(t,2H,CH2)；4.19(t,2H,CH2)；5.08(s,2H,CH2)；6.91(s,2H,NH2)；7.29-7.38(m,5H,C6H5)；8.21(s,1H,CH)；
ESI-MS M/z 348.0455 and 350.0436(M + H)+。
2) N9- (2-amino) ethyl-O6Synthesis of-benzylguanine
The solid N9-bromoethyl-O obtained is weighed6-benzylguanine (1948mg,5.6mmol) in a 100mL round bottom flask, dissolved in 50mL anhydrous DMF solution, added solid potassium phthalimide (3360mg,18.2mmol), stirred at 65 ℃ under reflux for 4h, extracted with ethyl acetate and deionized water, the organic phase washed with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate overnight, dried at 35 ℃ under vacuum to give a white solidWeighing the product (1992mg,4.8mmol), dissolving in 20mL of anhydrous DMF solution, adding 1.6mL of hydrazine hydrate, stirring and refluxing at 32 ℃ for 4h to allow hydrazinolysis, extracting with dichloromethane and deionized water, washing the organic phase with saturated aqueous sodium chloride solution, drying over night anhydrous sodium sulfate, and vacuum drying at 35 ℃ to obtain N9- (2-amino) ethyl-O6Benzylguanine (855mg,3mmol), yield 53%.
UVλ:250,283nm；
IR (KBr pellet) v/cm-1:3463.8(N-(CH2)2-)；3319.3(N-H)；2958.1(-CH2-)；1617.3(C＝C)；1253.1(C-O-C)；1072.6(C-N)；
1H NMR(400MHz,DMSO)δ:2.94(t,2H,NH2)；3.91(t,2H,CH2)；5.64(s,2H,CH2)；6.538(s,2H,NH2)；7.27-7.63(m,5H,C6H5)；7.95(s,1H,CH)；
ESI-MS:m/z 285.1453(M+H)+。
3) Preparation of methoxy polyethylene glycol-4, 4' -dicarboxamidazobenzene conjugate
Azobenzene-4, 4 ' -dicarboxylic acid (300mg,1.2mmol) was weighed into a 120mL round bottom flask, dissolved in 120mL anhydrous pyridine solution, EDC (260mg,1.6mmol) and NHS (184mg,1.6mmol) were added, after 2h reaction DMAP (14mg,0.12mmol) and methoxypolyethyleneglycoamine 1000(1110mg,1.2mmol) were added, reaction 12h, pyridine was removed by rotary evaporation at 50 ℃ and dissolved in deionized water and centrifuged to remove free azobenzene-4, 4 ' -dicarboxylic acid to give methoxypolyethyleneglycone-4, 4 ' -dicarboxamidazobenzene conjugate (411mg,0.32mmol) in 27% yield.
UVλ：445nm；
1H NMR(400MHz,DMSO)δ:3.28(t,3H,CH3)；3.43(m,2H,CH2)；3.54(t,88H,CH2)；3.78(t,2H,CH2)；7.45(s,1H,NH)；7.63-8.35(m,8H,C6H4)。
4) Preparation of methoxypolyethylene glycol-4, 4' -dicarboxamidazobenzene-N9-ethyl-O6-benzylguanine conjugate (BG-AB-PEG 3).
Weighing methoxyl groupPolyethylene glycol-4, 4' -dicarboxamidazobenzene conjugate (1524mg,1.2mmol) was dissolved in 120mL of anhydrous pyridine in a 250mL round-bottomed flask, EDC (260mg,1.6mmol) and NHS (184mg,1.4mmol) were added, and after reacting for 2h, the mixture was reacted with N9- (2-amino) ethyl-O6-benzylguanine (342mg,1.2mmol) was put into deionized water and stirred at room temperature for reaction for 24h, the resulting solution was dialyzed against deionized water for 2 days, and lyophilized to give BG-AB-PEG 3(558mg,0.36mmol) with a yield of 30%.
UVλ：445nm；
1H NMR(400MHz,DMSO)δ:3.17(s,3H,CH3)；3.43(m,2H,CH2)；3.68(m,88H,CH2)；3.84(m,2H,CH2)；3.98(t,2H,CH2)；4.02(t,2H,CH2)；5.34(s,2H,CH2)；6.93(s,2H,NH2)；7.31-7.76(m,5H,C6H5)；7.87(t,1H,NH)；7.87(t,1H,NH)；8.06-8.43(d,8H,C6H4)；8.76(s,1H,CH2)。
Example 4: preparation of anti-tumor nano preparation 1(BG-AB-PEG/BCNU 1)
40mg of BG-AB-PEG 1 and 5mg of BCNU were weighed out and dissolved in tetrahydrofuran, hydrated under ultrasound with 10mL of deionized water, and the solution was dialyzed against deionized water for 4h to remove unencapsulated BCNU. The product is lyophilized to obtain BG-AB-PEG/BCNU1, and the particle size distribution and electron microscopy are shown in figure 1.
Example 5: preparation of anti-tumor nano preparation 2(BG-AB-PEG/BCNU 2)
40mg of BG-AB-PEG 2 and 5mg of BCNU were weighed out and dissolved in tetrahydrofuran, hydrated under ultrasound with 10mL of deionized water, and the solution was dialyzed against deionized water for 4h to remove unencapsulated BCNU. The product was lyophilized to obtain BG-AB-PEG/BCNU 2, the particle size distribution and electron microscopy are shown in FIG. 2.
Example 6: preparation of anti-tumor nano preparation 3(BG-AB-PEG/BCNU 3)
25mg BG-AB-PEG 3 and 5mg BCNU were weighed into tetrahydrofuran, hydrated with 10mL deionized water under sonication, and the solution dialyzed against deionized water for 4h to remove unencapsulated BCNU. The product was lyophilized to obtain BG-AB-PEG/BCNU 3, the particle size distribution and electron microscopy are shown in FIG. 3.
Experimental example 1: stability testing of BG-AB-PEG/BCNU
BG-AB-PEG/BCNU prepared in examples 4-6 were dissolved in PBS or DMEM medium containing 10% FBS, and the particle size change of the three nano-formulations was measured at 0, 6, 12, 24, 36, and 48h after filtration through a filter, respectively, as shown in FIG. 4. As can be seen from the figure, the particle sizes of the three nano preparations in the two solvents are not obviously changed within 48h, which indicates that BG-AB-PEG/BCNU has good stability.
Experimental example 2: BG-AB-PEG/BCNU hypoxia reduction sensitivity test
BG-AB-PEG/BCNU prepared in examples 4-6 was dissolved in an appropriate amount of PBS, rat liver microsomes and Nicotinamide Adenine Dinucleotide Phosphate (NADPH) were added, the mixture was incubated at 37 ℃ under normoxic and hypoxic conditions, and the particle size of each NanoPrep was measured after the reaction was completed, as shown in FIG. 5. As can be seen from FIG. 5, under the hypoxic condition, the particle size of the nano-preparation is significantly increased, which indicates that the nano-drug carrier is cracked due to the reduction of azo bonds by rat liver microsomes and NADPH under the hypoxic condition, thereby releasing the entrapped drug; under the condition of normal oxygen, the particle size of the nano preparation has no obvious change, which shows that azo bonds can not be reduced under the condition of normal oxygen, and nano drug carriers can not be cracked, so that the drug can not be released. The result shows that BG-AB-PEG/BCNU is sensitive to hypoxia, and can specifically generate nanoshell fragmentation and release of the drug through azo bond reduction under the hypoxia condition.
Experimental example 3: BCNU release in BG-AB-PEG/BCNU
BG-AB-PEG/BCNU and free BCNU prepared in examples 4-6 were placed in dialysis bags (1000Da), respectively, and the dialysis bags were placed in PBS containing rat liver microsomes and NADPH, and dialyzed under normoxic and hypoxic conditions, respectively. Taking 1mL PBS out of the dialysis bag at 5 min, 10 min, 20 min, 40 min, 60min, 90 min, 120 min, 150 min, 180 min, 240 min, 300 min and measuring the content of BCNU in the PBS by using high performance liquid chromatography, thereby obtaining the in vitro release condition of the 3 nano-drugs. As shown in FIG. 6, under low oxygen condition, BCNU can be effectively released (60-70% release) because azo bond in BG-AB-PEG carrier is reduced, resulting in the cracking of nano carrier. Under the normal oxygen condition (as shown in figure 7), the azo bond in BG-AB-PEG carrier can not be reduced, so that the nano carrier can not be cracked, and therefore, BCNU loaded in the nano preparation can hardly be released (the release amount is less than 15%). The result shows that the BG-AB-PEG/BCNU nano preparation prepared by the invention not only has good stability, but also is beneficial to improving the circulation time of BCNU in vivo; and has ideal hypoxia responsiveness, and can ensure that BCNU is delivered to the hypoxic part of the tumor in a targeted way.
Experimental example 4: cell clone formation experiment of BG-AB-PEG/BCNU
A human brain glioma SF763 cell and an SF126 cell are selected to carry out clone formation experiments, wherein the AGT of the SF763 cell is high in expression and easy to generate drug resistance, and the AGT of the SF126 cell is low in expression and low in drug resistance. SF763 and SF126 cells with good growth state are respectively digested by pancreatin to prepare single cell suspension, the cell suspension is diluted by culture solution respectively containing PBS, BCNU, BG-AB-PEG/BCNU1, BG-AB-PEG/BCNU 2 and BG-AB-PEG/ BCNU 3, 500 cells in each hole are inoculated in a six-hole plate, and three cells in each group are arranged in parallel. After 9 days of culture, staining counts were performed and the results are shown in FIG. 8. Because the AGT-mediated DNA repair effect can cause the tumor cells to generate drug resistance to BCNU, the clone formation rate of SF763 cells with high AGT expression is higher than that of SF126 cells with low AGT expression after BCNU treatment. In each group of cells treated by the nano-drug BG-AB-PEG/BCNU, the normoxic group and the hypoxic group have obvious difference: under the condition of normal oxygen, because the nano-drug carrier can not be disintegrated, the encapsulated BCNU can not be effectively released, the cloning formation rate of the two cells is close to that of a blank control group, and the three nano-preparations can not play the role of inhibiting the tumor cells under the condition of normal oxygen; under the condition of low oxygen, azobenzene groups in the three nano preparations can be reduced, so that the carrier is cracked, and not only the entrapped BCNU is released, but also the AGT inhibitor O with the drug resistance effect is released6the-BG derivative obviously reduces the clone formation rate of two cells, and has more obvious inhibition effect on SF763 cells with drug resistance. The above resultsThe three nano preparations show good inhibition effect on two cells under the condition of hypoxia. Compared with the clinical common CENUs antineoplastic agents, the nanometer preparation BG-AB-PEG/BCNU has good tumor hypoxia targeting property, drug resistance and higher antineoplastic activity.
Experimental example 5: cell invasion assay of BG-AB-PEG/BCNU
Treating SF763 and SF126 cells with PBS, BCNU, BG-AB-PEG/BCNU1, BG-AB-PEG/BCNU 2 and BG-AB-PEG/BCNU 3 for 24h, respectively, and treating at 1 × 104One cell/well was seeded in a Matrigel-plated chamber and cultured for 48h, and the number of invading cells was recorded by photography. As can be seen in fig. 9, after BCNU alone treatment, SF763 cells that highly expressed AGT protein invaded more efficiently than SF126 cells that underexpressed AGT protein; after BG-AB-PEG/BCNU treatment, the cell invasion rate of the normoxic group is obviously higher than that of the hypoxic group. The result shows that the nano preparation synthesized by the invention has no obvious inhibition effect on the invasion capacity of tumor cells under the normoxic condition; however, under the hypoxia condition, the invasion number of SF763 cells treated by BG-AB-PEG/BCNU is lower than that of each group under the normoxic condition, which shows that azo bonds in BG-AB-PEG/BCNU are reduced and broken under the hypoxia condition, so that the nano-carrier is cracked, and entrapped BCNU and AGT inhibitor O are released6-BG derivatives, thus inhibiting the resistance of tumor cells while BCNU exerts anti-tumor activity. The results show that the nanometer preparation BG-AB-PEG/BCNU provided by the invention can specifically inhibit the invasion capacity of tumor cells under the hypoxia condition, and the nanometer preparation has good tumor hypoxia targeting property and anti-tumor activity.
Experimental example 6: experiment on antitumor Activity of BG-AB-PEG/BCNU
1. Experimental Material
Test compounds: BG-AB-PEG/BCNU1, BG-AB-PEG/BCNU 2 and BG-AB-PEG/BCNU 3 prepared in examples 4-6, and BCNU, which is a commonly used clinical antitumor agent.
Cell line: human brain glioma cell SF763, human brain glioma cell SF126, human breast cancer cell MCF-7, human lung cancer cell A549, human melanoma cell A375, and human colon cancer cell HT 29.
2. Experimental methods
The 6 cells were seeded in 96-well plates at 1000/well with 5% CO at 37 deg.C2After 24h of incubation, 6 replicates of each group were treated with anti-tumor nanopreparations BG-AB-PEG/BCNU1, 2, 3 and unencapsulated BCNU at concentrations of 10 μ M, 20 μ M, 50 μ M, 100 μ M, 200 μ M, 400 μ M, 600 μ M and 800 μ M, and a control group was set. The normoxic group and the hypoxic group are set, namely, the groups are respectively incubated under normoxic (21% oxygen) and hypoxic (1% oxygen) for 24 hours. Then 10. mu.L of CCK-8 solution was added to each well and allowed to act for 4 hours. Finally, the absorbance value is detected at a wavelength of 450nm, the cell survival rate is calculated according to the following formula, and the half inhibition rate IC of the drug is calculated50。
Tumor cell survival rate (%) ═ aMedicine adding device-ABlank group)/(AControl group-ABlank group)×100％
AMedicine adding deviceAbsorbance values for wells with media, tumor cells, drug solution and CCK-8 solution;
Ablank groupAbsorbance values for wells with media and CCK-8 solution, but no tumor cells and drug;
Acontrol groupAbsorbance values for wells with media, tumor cells, CCK-8 solution, but no drug solution.
3. The experimental results are as follows: see Table 1
TABLE 1 half inhibition rate (IC) of tumor cells50，μM)
As shown in Table 1, the IC of BG-AB-PEG/BCNU1, BG-AB-PEG/BCNU 2 and BG-AB-PEG/BCNU 3 formed by entrapping BCNU with nano-drug under the atmospheric condition50The values were significantly higher than free BCNU. This is because the nano carrier BG-AB-PEG effectively encapsulates BCNU and can not be cracked under the normal oxygen condition, so that the medicine can not be released under the normal oxygen condition, thereforeIt cannot exert tumor cell inhibiting effect. This prevents BCNU from being released and causing DNA damage before the formulation reaches the hypoxic region of the tumor in vivo, thereby effectively reducing the toxic side effects of BCNU on normal tissues.
And IC of BG-AB-PEG/BCNU1, BG-AB-PEG/BCNU 2 and BG-AB-PEG/BCNU 3 in hypoxic environment50The values were significantly lower than the normoxic and free BCNU groups. The result shows that the synthesized nano preparation has good low-oxygen targeting property, and azo bonds in the nano preparation can be effectively reduced under the low-oxygen condition, so that the nano shell is cracked, and the BCNU and the AGT inhibitor O encapsulated in the nano shell are released6-BG or a derivative thereof. O is6the-BG derivative can effectively inhibit the activity of AGT, block the repair of DNA alkylation damage caused by the AGT to BCNU, and increase the sensitivity of tumor cells to the BCNU, so that the BCNU can sufficiently exert the anti-tumor activity in a tumor hypoxia area in a targeted manner.
The experimental results show that compared with the traditional CENUs antineoplastic drugs, the nano preparation synthesized by the invention has good low-oxygen targeting property and drug resistance, and can specifically reduce azobenzene groups under the low-oxygen condition to crack the nano shell, so that CENUs and O in the nano particles are released6the-BG or the derivative thereof also inhibits the AGT mediated drug resistance in the tumor in a targeted way while delivering the anti-tumor drug, thereby realizing the three-in-one effect of anti-cancer, anti-drug resistance and targeted delivery. In addition, the water solubility and stability of CENUs are also obviously improved through the entrapment of the nano-carrier BG-AB-PEG, and the improvement of the anticancer activity is also facilitated.
Finally, the examples are only preferred embodiments and are not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (12)
1. A hypoxia-targeted AGT inhibitor conjugate having a structure represented by general formula (I):
wherein R is H, NH2、CH3、CH2NH2、CH2One of the OH groups is selected from the group consisting of,
n1is an integer of 2 to 6, and,
n2is CH in methoxypolyethyleneglycolamine2CH2The number of O and the molecular weight of the methoxypolyethylene glycol amine are 1000-10000 Da.
2. The conjugate of claim 1, wherein R is H, and n1Is 2.
3. The conjugate of claim 2, wherein R is H and n1Is 2 and the molecular weight of the methoxypolyethyleneglycol amine is 2000 Da.
4. A method for preparing the conjugate of claim 1, comprising the steps of:
(1) mixing O with6-benzylguanine and dihaloalkane are subjected to substitution reaction to generate N9-halogenated ethyl-O6-benzylguanine;
(2) adopting a drape Rayle reaction, reacting the product obtained in the step (1) with phthalimide potassium, and then performing a hydrazinolysis reaction with hydrazine hydrate to generate N9- (2-amido) ethyl-O6-benzylguanine;
(3) reacting azobenzene-4, 4 '-dicarboxylic acid with methoxy polyethylene glycol amine to generate a methoxy polyethylene glycol-4, 4' -dicarboxamide azobenzene conjugate;
(4) and (4) reacting the products obtained in the step (2) and the step (3) to obtain the catalyst.
5. Use of the conjugate of any one of claims 1 to 3 for the preparation of an anti-tumor medicament.
6. The use of claim 5, wherein the tumor is one or more of a brain tumor, myeloma, melanoma, lung cancer, breast cancer, leukemia, colon cancer, and lymphoma.
7. The use of claim 6, wherein the tumor is one or more of a brain tumor, leukemia, colon cancer, and lymphoma.
8. An anti-tumor nano preparation, which is prepared by self-assembling the conjugate of any one of claims 1 to 3 to form a nano carrier to encapsulate an anti-tumor alkylating agent.
9. The anti-tumor nanoformulation according to claim 8, wherein the anti-tumor alkylating agent is an alkylating agent that causes DNA alkylation damage.
10. The anti-tumor nano-formulation according to claim 9, wherein the anti-tumor alkylating agent is one or more of chloroethylnitrosourea, temozolomide and dacarbazine.
11. The anti-tumor nano-formulation according to claim 10, wherein the anti-tumor alkylating agent is carmustine, semustine or lomustine in chloroethyl nitrosourea.
12. The method for preparing an anti-tumor nano-formulation according to any one of claims 8 to 11, comprising the steps of: dissolving the conjugate and the anti-tumor alkylating agent in an organic solvent, and hydrating, dialyzing and freeze-drying the conjugate by using deionized water under ultrasonic treatment to obtain the anti-tumor drug.
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