CN105367573A - AGT protein inhibitor and preparation method and application thereof - Google Patents
AGT protein inhibitor and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a novel tumour cell targeting AGT protein inhibitor with high activity and a preparation method and application thereof. The inhibitor is a compound shown as the general formula (I). The compound can be specifically activated under low oxygen condition, a nitro group is reduced to an amino group, and a O6-benzylguanine derivative with AGT inhibitory activity is generated, so that the inhibitor can specifically act on the tumour cell and have an AGT inhibiting effect, and the purpose of improving sensibility of the tumour cell on an anti-cancer alkylating agent is realized. Besides, compared with existing AGT protein inhibitors, the compound has the advantages of higher activity and water solubility.
Description
Technical field
The present invention relates to pharmacy field, specifically, relate to a kind of novel AGT protein inhibitor, its preparation method and application.
Background technology
O
6-alkylguanine-DNA alkyl transferring enzyme (AGT) is a kind of DNA repair protein, and it can be repaired, by alkylguanine O the DNA molecular being subject to alkanisation damage
6the alkyl group that position combines is transferred to the cysteine residues in activity itself site from guanine, thus protection DNA is not by the damage of alkylating agent.Research shows, guanine O
6the groups such as the methyl on position, 2-chloroethyl, benzyl and pyridyl oxo butyl all can be removed by AGT.But in the clinical application of alkylating agent series antineoplastic medicament, the AGT in tumour cell makes tumour cell to the raw resistance of alkylating agent kind anti-cancer drugs produce by repairing the DNA damage that causes of alkylating agent, finally cause chemotherapy failure.Such as, for bifunctional alkylating agent Dichloroethyl nitrosourea (BCNU, carmustine), AGT can by repairing the O that BCNU causes
6-chloroethyl guanine intermediate and formation crosslinked between blocking dna stock, cause tumour cell to produce resistance to BCNU.Therefore, design and develop effective AGT inhibitor and itself and alkylating agent kind anti-cancer drugs thing be united and applied in chemotherapy of tumors, for strengthen tumour cell to the susceptibility of chemotherapeutics, reduce resistance, to improve chemotherapy effect significant.
O
6-benzyl guanine is current most widely used AGT inhibitor in clinical trial, the AGT in Human colorectal carcinoma HT29 cells can be suppressed completely active, improve tumour cell to the susceptibility of CCNU simultaneously in 15 minutes.But, due to O
6there is the problem of limited activity, poorly water-soluble in-benzyl guanine, does not especially have tumour cell targeting, therefore fails to be widely used clinically.Research shows, O
6-benzyl guanine also greatly reduces the content of AGT in normal cell while inhibition tumor cell AGT activity, thus causes the bone marrow depression toxicity of chemotherapeutics significantly to strengthen, and finally causes chemotherapy failure.Therefore; design and develop the new A GT inhibitor with tumour cell targeting; tumour cell can be acted on specifically and play AGT inhibit activities; be improve sensitivity of tumor cells, simultaneously protection normal cell not damage by chemotherapeutics, thus realize the important channel of the therapeutic strategy of high-efficiency low-toxicity.
Summary of the invention
The object of this invention is to provide and a kind ofly novel there is highly active tumour cell targeting AGT protein inhibitor, its preparation method and application.
Because the quick growth of tumour cell can cause inside tumor anoxic, therefore, hypoxemia is a kind of universal phenomenon existed in solid tumor.The present invention utilizes this feature of tumor hypoxia, and synthesized the AGT protein inhibitor that a class has hypoxemia activation, this compounds can not be activated in the normal tissue, and the AGT in normal cell thus can not be suppressed active; But can be activated under hypoxia in tumor cells condition specifically, produce the compound with AGT inhibit activities.Therefore, this compounds can the AGT of specific inhibition tumor cell active, targeting ground improves tumour cell to the susceptibility of chemotherapeutics, thus is united and applied in chemotherapy of tumors with alkylating agent series antineoplastic medicament.
In order to realize the object of the invention, a kind of AGT protein inhibitor with hypoxemia activation characteristic provided by the invention, described inhibitor is for having the compound as shown in general formula (I) structure:
Preferably, described inhibitor is O
6-(2-nitrobenzyl)-9 hydrogen-guanine (compound 1), O
6-(3-nitrobenzyl)-9 hydrogen-guanine (compound 2), O
6-(4-nitrobenzyl)-9 hydrogen-guanine (compound 3):
The present invention also provides the preparation method of described AGT protein inhibitor, comprises the following steps:
1) 2-amido-6-chloropurine (a) and 1-crassitude reacting generating compound (b), i.e. 1-(2-amino-9-hydrogen-purine-6-)-1-crassitude muriate;
2) formula (II) compound and compound (b) react and generate described inhibitor.
Wherein, the structural formula of compound (b) is as follows:
The general structure of formula (II) compound is as follows, and nitro is positioned at ortho position, a position or contraposition:
Above-mentioned reaction process is as follows:
Aforesaid method, step 1) be specially: 2-amido-6-chloropurine (a) is dissolved in organic solvent, add 1-crassitude, stirring reaction, reacting generating compound (b), reaction terminates to add acetone and continue to be stirred to compound (b) in backward solution to precipitate completely, filter and the solid that will collect, with washed with diethylether 2 times, vacuum-drying, obtain dry compound (b).
Step 1) in the mol ratio of 2-amido-6-chloropurine and 1-crassitude be 1:1-4, preferred 1:2-3; Described organic solvent is DMF or dimethyl sulfoxide (DMSO), preferred DMF, and consumption of organic solvent is by 2-amido-6-chloropurine 1mmol, with an organic solvent 10-15mL; Temperature of reaction controls at 20-40 DEG C, preferred 25-35 DEG C; Reaction times is 12-24 hour, preferred 16-20 hour.
Aforesaid method, step 2) be specially: formula (II) compound is dissolved in organic solvent, add step 1) compound (b) that obtains, sodium hydride and dimethyl aminopyridine, stirring reaction under protection of inert gas, the glacial acetic acid aqueous solution is added to neutralize excessive sodium hydride to pH value of solution=7 after reaction terminates, then saturated ammonium chloride is added and the ethyl acetate mixed solution that 1:1 forms by volume extracts, ethyl acetate layer after extraction uses deionized water wash 2 times again, vacuum-drying, obtain the crude product of formula (I) compound, by crude product purified by silica gel column chromatography purification, obtain described inhibitor.
Step 2) in the mol ratio of Dimethylamino pyridine, compound (b), formula (II) compound and sodium hydride be 1:6-12:10-50:30-60, preferred 1:8-10:20-30:40-50; Described organic solvent is DMF or dimethyl sulfoxide (DMSO), preferred DMF, and consumption of organic solvent is by compound (b) 1mmol, with an organic solvent 10-15mL; Temperature of reaction controls at 20-35 DEG C, preferred 25-30 DEG C; Reaction times is 3-6 hour, preferred 4-5 hour.
Preferably, step 2) in silica gel column chromatography purifying time eluent used be the mixed solution of methyl alcohol and methylene dichloride, adopt the method for gradient elution, in mixed solution, the volume ratio of methyl alcohol and methylene dichloride gradually becomes 1:10 by 1:50.
The AGT protein inhibitor that the present invention has hypoxemia activation characteristic has the following advantages:
(1) formula provided by the invention (I) compound can specificly activate under low oxygen conditions, and nitryl group is reduced to amino group, generates the O with AGT inhibit activities
6-benzyl guanine derivative, i.e. O
6-(aminobenzyl)-9 hydrogen-guanine, thus realize acting on tumour cell and playing AGT restraining effect of targeting, reach and improve tumour cell to the object of anticancer alkylating agent susceptibility.
(2) extracorporeal anti-tumor shaker test is carried out to formula (I) compound, result shows, under low oxygen conditions, formula (I) compound and BCNU drug combination, under low oxygen conditions, obvious restraining effect is all had to nasopharyngeal carcinoma cell KB, human cervical carcinoma cell HelaS3, human brain neuroglial cytoma SF763, Human Prostate Cancer Cells DU145, human colon cancer cell HT29 cell and lymphoma cell Raji; And under aerobic conditions, formula (I) compound and BCNU drug combination, not obvious to the restraining effect of above-mentioned tumour cell.Therefore, formula (I) compound has good targeting, can significantly improve the susceptibility of tumour cell to alkylating agent series antineoplastic medicament, can be used for the target combined chemotherapy of tumour.
(3) compared with existing AGT protein inhibitor, formula (I) compound also has active higher, water-soluble stronger advantage.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
The general structure of formula (II) compound related in following examples is as follows:
Wherein, nitro be positioned at ortho position, a position or contraposition time corresponding compound be respectively (c), (d), (e).
The structural formula of the compound 1 related in following examples, compound 2 and compound 3 is as follows:
Embodiment 1O
6the synthesis of-(2-nitrobenzyl)-9 hydrogen-guanine (compound 1)
(1) synthesis of 1-(2-amino-9-hydrogen-purine-6-)-1-crassitude muriate (b)
356mg (2.1mmol) 2-amido-6-chloropurine (a) is dissolved in 30mLN, in dinethylformamide, add 0.33mL (6.2mmol) 1-crassitude, reactant all dissolves rear whole reaction system stirring reaction 17 hours under 25 DEG C of conditions, add 2mL acetone in the solution and continue to be stirred to after reaction terminates and precipitate completely, filter, collect solid through washed with diethylether 2 times, vacuum-drying, obtain compound (b) 300mg (1.18mmol), productive rate 56%.
The feature of compound (b) is as follows:
UVλ：289nm；
IR (KBr compressing tablet) v/cm
-1: 3445.4 (N-H), 3274.9 (NH
2), 2974.9 (C-H), 1719.3 (C=N), 1552.7 (C=C);
1H-NMR(DMSO-d
6)δ：1.73(m,2H,CH
2)，2.90(s,3H,CH
3)，3.24(m,2H,CH
2)，6.99(s,2H,NH
2)，8.57(s,1H,CH)，11.00(s,1H,NH)；
ESI-MS：255[M+H]
+。
(2) synthesis of compound 1
524mg (3.42mmol) compound (c) is dissolved in 15mLN; in dinethylformamide; to add in 147mg (6.11mmol) sodium hydride (NaH), 300mg (1.18mmol) step (1) obtained compound (b) and 17mg (0.14mmol) dimethyl aminopyridine solid successively, after all dissolving under argon shield 25 DEG C of environment stirring reactions 4 hours.The glacial acetic acid aqueous solution is added to neutralize excessive sodium hydride to pH value of solution=7 after reaction terminates, then saturated ammonium chloride is added and ethyl acetate is that the mixed solution that 1:1 forms extracts according to volume ratio, ethyl acetate layer after extraction uses deionized water wash 2 times again, vacuum-drying, obtains the crude product of compound 1.By crude product purified by silica gel column chromatography purification, eluent is methyl alcohol and methylene dichloride, adopts the method for gradient elution, gradually becomes 1:10 from ethanol/methylene (v/v) 1:50, obtain compound 1220mg (0.77mmol), productive rate 65%.
The feature of compound 1 is as follows:
UVλ：239nm；
IR (KBr compressing tablet) v/cm
-1: 3412.6 (N-H), 2924.4 (-CH
2-), 1592.5 (C=C), 1460.8 (C=N); 1397.5 (C-O-C);
1H-NMR(DMSO-d
6)δ：5.16(s,2H,CH
2)，6.99(s,2H,NH
2)，7.62-8.01(m,4H,C
6H
4)，8.57(s,1H,CH)，11.00(s,1H,NH)；
ESI-MS：287[M+H]
+。
Embodiment 2O
6the synthesis of-(2-nitrobenzyl)-9 hydrogen-guanine (compound 1)
(1) synthesis of 1-(2-amino-9-hydrogen-purine-6-)-1-crassitude muriate (b)
322mg (1.9mmol) 2-amido-6-chloropurine is dissolved in 27mLN, in dinethylformamide, add 0.30mL (5.6mmol) 1-crassitude, reactant all dissolves rear whole reaction system stirring reaction 16 hours under 27 DEG C of conditions, add 2mL acetone in the solution and continue to be stirred to after reaction terminates and precipitate completely, filter, collect solid through washed with diethylether 2 times, vacuum-drying, obtain compound (b) 278mg (1.09mmol), productive rate 57%.
The feature of compound (b) is as follows:
UVλ：289nm；
IR (KBr compressing tablet) v/cm
-1: 3446.5 (N-H), 3287.1 (NH
2), 2981.5 (C-H), 1723.9 (C=N), 1559.6 (C=C);
1H-NMR(DMSO-d
6)δ：1.79(m,2H,CH
2)，3.12(s,3H,CH
3)，3.37(m,2H,CH
2)，6.97(s,2H,NH
2)，8.62(s,1H,CH)，11.09(s,1H,NH)；
ESI-MS：255[M+H]
+。
(2) synthesis of compound 1
507mg (3.31mmol) compound (c) is dissolved in 15mLN; in dinethylformamide; to add in 142mg (5.92mmol) sodium hydride (NaH), 278mg (1.09mmol) step (1) obtained compound (b) and 16mg (0.13mmol) dimethyl aminopyridine solid successively, after all dissolving under argon shield 26 DEG C of environment stirring reactions 5 hours.The glacial acetic acid aqueous solution is added to neutralize excessive sodium hydride to pH value of solution=7 after reaction terminates, then saturated ammonium chloride is added and ethyl acetate is that the mixed solution that 1:1 forms extracts according to volume ratio, ethyl acetate layer after extraction uses deionized water wash 2 times again, vacuum-drying, obtains the crude product of compound 1.By crude product purified by silica gel column chromatography purification, eluent is methyl alcohol and methylene dichloride, adopts the method for gradient elution, gradually becomes 1:10 from ethanol/methylene (v/v) 1:50, obtain compound 1194mg (0.68mmol), productive rate 62%.
The feature of compound 1 is as follows:
UVλ：239nm；
IR (KBr compressing tablet) v/cm
-1: 3341.1 (N-H), 1671.1 (C=C), 1474.6 (C=N), 1262.7 (C-O-C);
1H-NMR(DMSO-d
6)δ：5.35(s,2H,CH
2)，6.31(s,2H,NH
2)，7.73-8.13(m,4H,C
6H
4)，8.87(s,1H,CH)，11.26(s,1H,NH)；
ESI-MS：287[M+H]
+。
Embodiment 3O
6the synthesis of-(3-nitrobenzyl)-9 hydrogen-guanine (compound 2)
(1) synthesis of 1-(2-amino-9-hydrogen-purine-6-)-1-crassitude muriate (b)
390mg (2.3mmol) 2-amido-6-chloropurine is dissolved in 30mLN, in dinethylformamide, add 0.36mL (6.8mmol) 1-crassitude, reactant all dissolves rear whole reaction system stirring reaction 19 hours under 27 DEG C of conditions, add 2mL acetone in the solution and continue to be stirred to after reaction terminates and precipitate completely, filter, collect solid through washed with diethylether 2 times, vacuum-drying, obtain compound (b) 310mg (1.22mmol), productive rate 53%.
The feature of compound (b) is as follows:
UVλ：289nm；
IR (KBr compressing tablet) v/cm
-1: 3455.9 (N-H), 3289.8 (NH
2), 2989.7 (C-H), 1744.7 (C=N), 1574.3 (C=C);
1H-NMR(DMSO-d
6)δ：1.81(m,2H,CH
2)，2.85(s,3H,CH
3)，3.10(m,2H,CH
2)，6.85(s,2H,NH
2)，8.69(s,1H,CH)，11.12(s,1H,NH)；
ESI-MS：255[M+H]
+。
(2) synthesis of compound 2
583mg (3.81mmol) compound (d) is dissolved in 15mLN; in dinethylformamide; to add in 163mg (6.79mmol) sodium hydride (NaH), 310mg (1.22mmol) step (1) obtained compound (b) and 18mg (0.15mmol) dimethyl aminopyridine solid successively, after all dissolving under argon shield 27 DEG C of environment stirring reactions 4 hours.The glacial acetic acid aqueous solution is added to neutralize excessive sodium hydride to pH value of solution=7 after reaction terminates, then saturated ammonium chloride is added and ethyl acetate is that the mixed solution that 1:1 forms extracts according to volume ratio, ethyl acetate layer after extraction uses deionized water wash 2 times again, vacuum-drying, obtains the crude product of compound 2.By crude product purified by silica gel column chromatography purification, eluent is methyl alcohol and methylene dichloride, adopts the method for gradient elution, gradually becomes 1:10 from ethanol/methylene (v/v) 1:50, obtain compound 2232mg (0.81mmol), productive rate 66%.
The feature of compound 2 is as follows:
UVλ：262nm；
IR (KBr compressing tablet) v/cm
-1: 3400.6 (N-H), 2924.9 (-CH
2-), 1529.5 (N=O), 1403.2 (C=N), 1141.8 (C-N);
1H-NMR(DMSO-d
6)δ：5.22(s,2H,CH
2)，6.87(s,2H,NH
2)，7.86-8.25(m,4H,C
6H
4)，8.32(s,1H,CH)，11.26(s,1H,NH)；
ESI-MS：287[M+H]
+。
Embodiment 4O
6the synthesis of-(3-nitrobenzyl)-9 hydrogen-guanine (compound 2)
(1) synthesis of 1-(2-amino-9-hydrogen-purine-6-)-1-crassitude muriate (b)
424mg (2.5mmol) 2-amido-6-chloropurine is dissolved in 30mLN, in dinethylformamide, add 0.39mL (7.4mmol) 1-crassitude, reactant all dissolves rear whole reaction system stirring reaction 20 hours under 28 DEG C of conditions, add 2mL acetone in the solution and continue to be stirred to after reaction terminates and precipitate completely, filter, collect solid through washed with diethylether 2 times, vacuum-drying, obtain compound (b) 328mg (1.29mmol), productive rate 52%.
The feature of compound (b) is as follows:
UVλ：289nm；
IR (KBr compressing tablet) v/cm
-1: 3457.8 (N-H), 3290.3 (NH
2), 2977.6 (C-H), 1751.2 (C=N), 1579.2 (C=C);
1H-NMR(DMSO-d
6)δ：1.84(m,2H,CH
2)，2.87(s,3H,CH
3)，3.14(m,2H,CH
2)，6.79(s,2H,NH
2)，8.61(s,1H,CH)，11.07(s,1H,NH)；
ESI-MS：255[M+H]
+。
(2) synthesis of compound 2
625mg (4.08mmol) compound (d) is dissolved in 15mLN; in dinethylformamide; to add in 175mg (7.28mmol) sodium hydride (NaH), 328mg (1.29mmol) step (1) obtained compound (b) and 20mg (0.16mmol) dimethyl aminopyridine solid successively, after all dissolving under argon shield 25 DEG C of environment stirring reactions 5 hours.The glacial acetic acid aqueous solution is added to neutralize excessive sodium hydride to pH value of solution=7 after reaction terminates, then saturated ammonium chloride is added and ethyl acetate is that the mixed solution that 1:1 forms extracts according to volume ratio, ethyl acetate layer after extraction uses deionized water wash 2 times again, vacuum-drying, obtains the crude product of compound 2.By crude product purified by silica gel column chromatography purification, eluent is methyl alcohol and methylene dichloride, adopts the method for gradient elution, gradually becomes 1:10 from ethanol/methylene (v/v) 1:50, obtain compound 2277mg (0.97mmol), productive rate 75%.
The feature of compound 2 is as follows:
UVλ：262nm；
IR (KBr compressing tablet) v/cm
-1: 3406.1 (N-H), 2782.8 (-CH
2-), 1632.7 (C=C), 1273.9 (C-O-C), 1085.2 (C-N);
1H-NMR(DMSO-d
6)δ：5.33(s,2H,CH
2)，6.28(s,2H,NH
2)，7.51-8.03(m,4H,C
6H
4)，8.81(s,1H,CH)，11.42(s,1H,NH)；
ESI-MS：287[M+H]
+。
Embodiment 5O
6the synthesis of-(4-nitrobenzyl)-9 hydrogen-guanine (compound 3)
(1) synthesis of 1-(2-amino-9-hydrogen-purine-6-)-1-crassitude muriate (b)
339mg (2.0mmol) 2-amido-6-chloropurine is dissolved in 30mLN, in dinethylformamide, add 0.31mL (5.9mmol) 1-crassitude, reactant all dissolves rear whole reaction system stirring reaction 18 hours under 29 DEG C of conditions, add 2mL acetone in the solution and continue to be stirred to after reaction terminates and precipitate completely, filter, collect solid through washed with diethylether 2 times, vacuum-drying, obtain compound (b) 288mg (1.13mmol), productive rate 57%.
The feature of compound (b) is as follows:
UVλ：289nm；
IR (KBr compressing tablet) v/cm
-1: 3434.7 (N-H), 3225,3 (NH
2), 2957.4 (C-H), 1733.5 (C=N), 1523.5 (C=C);
1H-NMR(DMSO-d
6)δ：1.62(m,2H,CH
2)，3.14(s,3H,CH
3)，3.31(m,2H,CH
2)，7.01(s,2H,NH
2)，8.49(s,1H,CH)，10.97(s,1H,NH)；
ESI-MS：255[M+H]
+。
(2) synthesis of compound 3
545mg (3.56mmol) compound (e) is dissolved in 15mLN; in dinethylformamide; to add in 153mg (6.37mmol) sodium hydride (NaH), 288mg (1.13mmol) step (1) obtained compound (b) and 17mg (0.14mmol) dimethyl aminopyridine solid successively, after all dissolving under argon shield 26 DEG C of environment stirring reactions 4 hours.The glacial acetic acid aqueous solution is added to neutralize excessive sodium hydride to pH value of solution=7 after reaction terminates, then saturated ammonium chloride is added and ethyl acetate is that the mixed solution that 1:1 forms extracts according to volume ratio, ethyl acetate layer after extraction uses deionized water wash 2 times again, vacuum-drying, obtains the crude product of compound 3.By crude product purified by silica gel column chromatography purification, eluent is methyl alcohol and methylene dichloride, adopts the method for gradient elution, gradually becomes 1:10 from ethanol/methylene (v/v) 1:50, obtain compound 3183mg (0.64mmol), productive rate 57%.
The feature of compound 3 is as follows:
UVλ：272nm；
IR (KBr compressing tablet) v/cm
-1: 3389.4 (N-H), 1524.3 (N=O), 1415.3 (C=N), 1347.6 (C-O-C), 1014.1 (C-N);
1H-NMR(DMSO-d
6)δ：5.29(s,2H,CH
2)，6.74(s,2H,NH
2)，7.62-8.19(m,4H,C
6H
4)，8.45(s,1H,CH)，11.17(s,1H,NH)；
ESI-MS：287[M+H]
+。
Embodiment 6O
6the synthesis of-(4-nitrobenzyl)-9 hydrogen-guanine (compound 3)
(1) synthesis of 1-(2-amino-9-hydrogen-purine-6-)-1-crassitude muriate (b).
305mg (1.8mmol) 2-amido-6-chloropurine is dissolved in 26mLN, in dinethylformamide, add 0.28mL (5.3mmol) 1-crassitude, reactant all dissolves rear whole reaction system stirring reaction 16 hours under 32 DEG C of conditions, add 2mL acetone in the solution and continue to be stirred to after reaction terminates and precipitate completely, filter, collect solid through washed with diethylether 2 times, vacuum-drying, obtain compound (b) 260mg (1.02mmol), productive rate 57%.
The feature of compound (b) is as follows:
UVλ：289nm；
IR (KBr compressing tablet) v/cm
-1: 3436.3 (N-H), 3237.5 (NH
2), 2956.2 (C-H), 1737.4 (C=N), 1531.7 (C=C);
1H-NMR(DMSO-d
6)δ：1.69(m,2H,CH
2)，2.97(s,3H,CH
3)，3.29(m,2H,CH
2)，6.94(s,2H,NH
2)，8.51(s,1H,CH)，10.92(s,1H,NH)；
ESI-MS：255[M+H]
+。
(2) synthesis of compound 3
472mg (3.08mmol) compound (e) is dissolved in 15mLN; in dinethylformamide; to add in 131mg (5.47mmol) sodium hydride (NaH), 260mg (1.02mmol) step (1) obtained compound (b) and 15mg (0.12mmol) dimethyl aminopyridine solid successively, after all dissolving under argon shield 27 DEG C of environment stirring reactions 5 hours.The glacial acetic acid aqueous solution is added to neutralize excessive sodium hydride to pH value of solution=7 after reaction terminates, then saturated ammonium chloride is added and ethyl acetate is that the mixed solution that 1:1 forms extracts according to volume ratio, ethyl acetate layer after extraction uses deionized water wash 2 times again, vacuum-drying, obtains the crude product of compound 3.By crude product purified by silica gel column chromatography purification, eluent is methyl alcohol and methylene dichloride, adopts the method for gradient elution, gradually becomes 1:10 from ethanol/methylene (v/v) 1:50, obtain compound 3168mg (0.59mmol), productive rate 58%.
The feature of compound 3 is as follows:
UVλ：272nm；
IR (KBr compressing tablet) v/cm
-1: 3339.1 (N-H), 1671.4 (C=C), 1474.5 (C=N), 1262.6 (C-O-C);
1H-NMR(DMSO-d
6)δ：5.27(s,2H,CH
2)，6.23(s,2H,NH
2)，7.56-8.11(m,4H,C
6H
4)，8.76(s,1H,CH)，11.47(s,1H,NH)；
ESI-MS：287[M+H]
+。
Embodiment 7 tumour cell AGT protein inhibitor activity rating
1, experiment material and instrument
Test compound: compound 1,2,3 obtained in above-described embodiment 1-6;
Clone: nasopharyngeal carcinoma cell KB, human cervical carcinoma cell HelaS3, human brain neuroglial cytoma SF763, Human Prostate Cancer Cells DU145, human colon cancer cell HT29 and lymphoma cell Raji.
2, experimental technique
Six kinds of tumour cells inoculate 96 orifice plates with 1000/ hole respectively, at 37 DEG C, and 5%CO
2cultivate after 24 hours, change with the carmustine (positive controls) of series concentration (1 μM, 5 μMs, 10 μMs, 50 μMs, 100 μMs, 200 μMs, 400 μMs and 1000 μMs), carmustine (BCNU)+compound 1, BCNU+ compound 2, BCNU+ compound 3, often organize 5 multiple holes, and blank group is set.Under normal oxygen and hypoxia condition, 48 hours (under normal oxygen condition, oxygen content is about 21%, and under hypoxia condition, oxygen content is about 1%) is acted on respectively by above-mentioned each group.Then, in every hole, add 10 μ LCCK-8 solution, act on 4 hours.Finally, be determined at the absorbance at 450nm place, calculate cytoactive as follows, and calculate half inhibiting rate IC by regression analysis
50.
Tumor cell survival (%)=(A
dosing group– A
blank group)/(A
control group– A
blank group) × 100
A
dosing groupfor having the absorbancy in the hole of tumour cell, CCK-8 solution and drug solution (BCNU+ compound 1/2/3);
A
blank groupfor there is substratum and CCK-8 solution and there is no the absorbancy in the hole of tumour cell;
A
control groupfor having tumour cell, CCK-8 solution and do not have the absorbancy in the hole of drug solution.
3, experimental result: in table 1.
Half inhibiting rate (the IC of table 1 tumour cell
50, μM)
Table 1 result shows, under normal oxygen environment, compared with positive controls (BCNU group), and the IC of BCNU+ compound 1,2 or 3 to six kinds of tumour cells
50be worth basically identical, show that compound 1,2 and 3 is very weak to the restraining effect of AGT activity in six kinds of tumour cells under normal oxygen environment.
Under low-oxygen environment, compared with positive controls (BCNU group), the IC of BCNU+ compound 1,2 or 3 to six kinds of tumour cells
50value obviously reduces, and shows that compound 1,2 and 3 can be generated O by specific reduction under low-oxygen environment
6-benzyl guanine analogue to suppress as AGT, thus effectively inhibits tumour cell AGT active, enhances the susceptibility of tumour cell to chemotherapeutics.
Contrast the IC of BCNU+ compound 1,2 or 3 under normal oxygen and low-oxygen environment
50value, can find out that low-oxygen environment is increased significantly than the inhibiting tumour cells activity of medicine under normal oxygen environment, show that compound 1,2 and 3 can be activated selectively in low-oxygen environment, thus plays AGT restraining effect.Therefore, compound 1,2 and 3 can specific suppression to be in the AGT of the solid tumor cell under low-oxygen environment active; The AGT simultaneously do not affected in the normal cell be under normal oxygen environment is active, makes normal cell from the damage of chemotherapeutics, thus reaches chemotherapeutics targeting in the object of tumour cell.
Above result shows, compound provided by the present invention under normal oxygen condition without obvious AGT restraining effect.But, under low-oxygen environment can AGT significantly in inhibition tumor cell active, and than existing AGT inhibitor O
6-benzyl guanine has the tumour cell targeting of stronger AGT inhibit activities, more highly water-soluble and hypoxemia activation.Therefore, compound provided by the present invention can be used for the adjuvant of allcylating treatment medicine, to realize, while raising tumour cell is to chemotherapy drug susceptibility, acts on tumour cell with making chemotherapeutics targeting, thus improving the chemotherapy effect of medicine.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (9)
1.AGT protein inhibitor, is characterized in that, described inhibitor is for having the compound as shown in general formula (I) structure:
2. inhibitor according to claim 1, is characterized in that, described inhibitor is:
3. the preparation method of inhibitor described in claim 1 or 2, is characterized in that, comprises the following steps:
1) 2-amido-6-chloropurine and 1-crassitude reacting generating compound (b);
2) formula (II) compound and compound (b) react and generate described inhibitor;
Wherein, the structural formula of compound (b) is as follows:
The general structure of formula (II) compound is as follows, and nitro is positioned at ortho position, a position or contraposition:
4. method according to claim 3, it is characterized in that, step 1) be specially: 2-amido-6-chloropurine is dissolved in organic solvent, adds 1-crassitude, stirring reaction, reacting generating compound (b), reaction terminates to add acetone and continue to be stirred to compound (b) in backward solution to precipitate completely, filters and the solid that will collect, with washed with diethylether 2 times, vacuum-drying, obtains dry compound (b).
5. method according to claim 4, is characterized in that, step 1) in the mol ratio of 2-amido-6-chloropurine and 1-crassitude be 1:1-4, preferred 1:2-3; Described organic solvent is DMF or dimethyl sulfoxide (DMSO), preferred DMF, and consumption of organic solvent is by 2-amido-6-chloropurine 1mmol, with an organic solvent 10-15mL; Temperature of reaction controls at 20-40 DEG C, preferred 25-35 DEG C; Reaction times is 12-24 hour, preferred 16-20 hour.
6. method according to claim 3, it is characterized in that, step 2) be specially: formula (II) compound is dissolved in organic solvent, add step 1) compound (b) that obtains, sodium hydride and dimethyl aminopyridine, stirring reaction under protection of inert gas, the glacial acetic acid aqueous solution is added to neutralize excessive sodium hydride to pH value of solution=7 after reaction terminates, then saturated ammonium chloride is added and the ethyl acetate mixed solution that 1:1 forms by volume extracts, ethyl acetate layer after extraction uses deionized water wash 2 times again, vacuum-drying, obtain the crude product of formula (I) compound, by crude product purified by silica gel column chromatography purification, obtain described inhibitor.
7. method according to claim 6, it is characterized in that, step 2) in the mol ratio of Dimethylamino pyridine, compound (b), formula (II) compound and sodium hydride be 1:6-12:10-50:30-60, preferred 1:8-10:20-30:40-50; Described organic solvent is DMF or dimethyl sulfoxide (DMSO), preferred DMF, and consumption of organic solvent is by compound (b) 1mmol, with an organic solvent 10-15mL; Temperature of reaction controls at 20-35 DEG C, preferred 25-30 DEG C; Reaction times is 3-6 hour, preferred 4-5 hour.
8. method according to claim 6, it is characterized in that, step 2) in silica gel column chromatography purifying time eluent used be the mixed solution of methyl alcohol and methylene dichloride, adopt the method for gradient elution, in mixed solution, the volume ratio of methyl alcohol and methylene dichloride gradually becomes 1:10 by 1:50.
9. the application in antitumor drug prepared by inhibitor described in claim 1 or 2.
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| CN107235981A (en) * | 2017-05-25 | 2017-10-10 | 北京工业大学 | The nitro purine derivative of hypoxemia activation O6 benzyls 2 and preparation method and application |
| CN111467500A (en) * | 2020-03-23 | 2020-07-31 | 北京工业大学 | Low-oxygen dual-targeting AGT inhibitor conjugate and preparation method and application thereof |
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|---|---|---|---|---|
| CN1720332A (en) * | 2002-10-03 | 2006-01-11 | 洛桑生态综合技术联合公司 | Substrates for O6-alkylguanine-DNA alkyltransferase |
| US20070213279A1 (en) * | 2004-09-08 | 2007-09-13 | Government Of The United States Of America, Represented By The | O6-alkylguanine-dna alkyltransferase inactivators and beta-glucuronidase cleavable prodrugs |
| CN104844710A (en) * | 2015-04-13 | 2015-08-19 | 西北大学 | Preparation method and applications of oriented immobilized PEGA composite resin |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1720332A (en) * | 2002-10-03 | 2006-01-11 | 洛桑生态综合技术联合公司 | Substrates for O6-alkylguanine-DNA alkyltransferase |
| US20070213279A1 (en) * | 2004-09-08 | 2007-09-13 | Government Of The United States Of America, Represented By The | O6-alkylguanine-dna alkyltransferase inactivators and beta-glucuronidase cleavable prodrugs |
| CN104844710A (en) * | 2015-04-13 | 2015-08-19 | 西北大学 | Preparation method and applications of oriented immobilized PEGA composite resin |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107235981A (en) * | 2017-05-25 | 2017-10-10 | 北京工业大学 | The nitro purine derivative of hypoxemia activation O6 benzyls 2 and preparation method and application |
| CN107235981B (en) * | 2017-05-25 | 2019-10-25 | 北京工业大学 | Hypoxemia activates O6- benzyl -2- nitro purine derivative and preparation method and application |
| CN111467500A (en) * | 2020-03-23 | 2020-07-31 | 北京工业大学 | Low-oxygen dual-targeting AGT inhibitor conjugate and preparation method and application thereof |
| CN111467500B (en) * | 2020-03-23 | 2022-08-09 | 北京工业大学 | Low-oxygen dual-targeting AGT inhibitor conjugate and preparation method and application thereof |
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