CN106083854B - A kind of hypoxemia activation AGT protein inhibitors and preparation method and application - Google Patents
A kind of hypoxemia activation AGT protein inhibitors and preparation method and application Download PDFInfo
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Abstract
The invention provides a kind of as led to AGT protein inhibitors that there is hypoxemia to activate characteristic shown in formula (I) and preparation method and application：R1=R2=H, or R1=H, R2=CH3, or R1=R2=CH3；R3=H, NO2, NH2, OCH3, CH2OH or CO2CH3.The compound has good hypoxemia activation characteristic and tumour cell targeting, can suppress the activity of AGT in solid tumor in targeting under low-oxygen environment, improve sensitiveness of the tumour cell to chemotherapeutics；The compounds of this invention has obvious inhibitory action with ACNU drug combinations to a variety of solid tumor cell systems；With alkylating agents anti-cancer agent in combination in use, sensitiveness of the tumour cell to cancer therapy drug can be significantly improved, the targeting combined chemotherapy available for malignant tumour.
Description
Technical field
The present invention relates to pharmaceutical field, and in particular to a kind of new targeting AGT protein inhibitors, its preparation method and
Using.
Background technology
Contain a variety of enzymes acted on alkanisation injury repair, wherein O in mammalian cell6- alkylguanine-DNA
Alkyl-transferase (AGT) is one of important DNA repair protein.AGT albumen can be by DNA guanines O6Methyl, chloroethene on position
Base, benzyl and benzyl etc. are covalently transferred on the 145th cysteine residues at activity itself center, so that the alkane of DNA plerosis
Change damage.For example, AGT enzymes can repair DNA alkylated products O caused by chloroethylnitrosoureas chemotherapeutics6- chloroethyl bird is fast
Purine and N1, O6- ethano- guanine, so as to block both alkylated products further to react to form dG-dC with cytimidine
It is crosslinked between stock, ultimately results in tumour cell and drug resistance is produced to chemotherapeutics.In order to block AGT to damage DNA of tumor cell alkanisation
The repair of wound to reduce drug resistance of the tumour cell to chemotherapeutics, design and develop effective AGT inhibitor and by its with
Alkylating agents Anticancer drug combination is in chemotherapy of tumors, it has also become enhancing tumour cell to the sensitiveness of chemotherapeutics, improve
The important channel of chemotherapy effect.
At present, coming into the AGT inhibitor of clinical experimental stage mainly includes O6- benzyl guanine and
Lomeguatrib (PaTrin-2), both compounds can consume the AGT enzymes in tumour cell, so as to reduce AGT work
Property.For example, O6- benzyl guanine can effectively reduce the AGT in human brain neuroglial cytoma SF767 and colon cancer cell HT29
Level, significantly improves sensitiveness of the tumour cell to Dichloroethyl nitroso ureas.However, existing AGT inhibitor does not have
Tumour cell targeting, during its being used in combination with chemotherapeutics, the AGT inhibitor of these non-target tropisms is suppressing swollen
Oncocyte AGT has been greatly reduced the activity of AGT in normal cell while active so that normal cell can not repair in time by
The DNA damage that chemotherapeutics is caused, so as to cause the bone marrow suppression toxicity of chemotherapeutics to significantly increase, ultimately results in chemotherapy mistake
Lose.
Hypoxemia is the key character in solid tumor evolution, and research shows, the partial pressure of oxygen in more than 70% solid tumor is bright
It is aobvious to be less than normal structure.There is the characteristic of hypoxemia microenvironment using tumor tissues, design and develop specific with hypoxemia activation
New A GT inhibitor precursor compounds, decompose release AGT with enabling the compound targeting under tumor tissues low-oxygen environment
Inhibitor, and AGT inhibitory action is not played under normal oxygen environment in the normal tissue, so that it is medication combined with alkylating agents
Use the targeted chemotherapy for realizing tumour.Therefore, the new A GT inhibitor that characteristic is activated with hypoxemia is designed and developed, can
Specifically act on tumour cell and play AGT inhibitory activity, for improving sensitivity of tumor cells, while protection is normal thin
Born of the same parents are not damaged by chemotherapeutics, so as to realize that the chemotherapeutic strategies of high-efficiency low-toxicity are significant.
The content of the invention
It is an object of the invention to provide a kind of new hypoxemia activation AGT inhibitor, its preparation method and application.
In order to realize the object of the invention, a kind of AGT inhibitor that characteristic is activated with hypoxemia that the present invention is provided, the suppression
Preparation is with as led to the compound shown in formula (I) structure：
R1=R2=H, or R1=H, R2=CH3, or R1=R2=CH3；
R3=H, NO2, NH2, OCH3, CH2OH, CO2CH3；
Preferably, R is worked as3=NO2When, the inhibitor is 4- nitrobenzyls-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-fast
Purine -2) carbamate (compound 1), 1- (4- nitrobenzene) ethyl-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amino
Formic acid esters (compound 2), 2- (4- nitrobenzene) propyl group-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate (are changed
Compound 3).
The present invention also provides the preparation method of the three of the above AGT protein inhibitors, and the reaction mechanism mechanism of reaction of methods described is：
The specific synthesis step that invention provides compound is as follows：
(1) 4- thienylmethanols are dissolved in DMF, sodium hydride and change is sequentially added into reaction solution
Compound a, stirring reaction, after reaction terminates, it is 1 to add ethyl acetate and water according to volume:The mixed liquor of 1 composition is extracted,
After organic phase is washed through saturated ammonium chloride solution, through anhydrous sodium sulfate drying, vacuum distillation, gained crude product is dissolved in methanol, mistake
Compound b is obtained after filtering out decontamination precipitation, recrystallization；
(2) ethanol solution containing caustic alcohol is added in compound b dissolves it, after stirring reaction 10-45min, vacuum
Solvent is drained, obtained solid is dissolved in organic solvent, chloromethyl pivalate is added into solution, stirred, after reaction terminates, plus
It is 1 to enter ethyl acetate and water according to volume:The mixed liquor of 1 composition is extracted, and organic phase is anhydrous after saturated common salt water washing
Sodium sulphate is dried, and vacuum distillation obtains crude on silica gel column chromatography and isolated and purified, obtains compound c；
(3) the compound c obtained by step (2) is dissolved in dichloromethane, adds triphosgene, stirred under inert gas shielding
Mix reaction, products therefrom compound d continue withReaction, vacuum distillation obtains crude on silica gel column chromatography
Isolate and purify, obtain compound e；
(4) the compound e obtained by step (3) is dissolved in the methanol solution containing ammoniacal liquor, stirring reaction, thin-layer chromatography prison
Reaction process is surveyed, after reaction terminates, solid is collected by filtration, compound f is obtained, i.e., of the invention has the AGT that hypoxemia activates characteristic
Protein inhibitor.
In the above method：
In the step (1)：
The mol ratio of the compound a, sodium hydride and 4- bromothiophene base methanol is 1:1-4:2-5, preferential 1:2-3:3-4；
Reaction time is controlled in 1-4h, preferably 2-3h；
Reaction temperature is controlled at 20-35 DEG C, preferably 25-35 DEG C；
Vacuum distillation temperature is controlled at 30-60 DEG C, preferably 30-40 DEG C.
In the step (2)：
The compound b, caustic alcohol and chloromethyl pivalate mol ratio are 1:1-3:2-5, preferential 1:1-2:2-3；
Selected organic solvent is DMF or dimethyl sulfoxide (DMSO), preferably DMF；
The reaction time of compound b and caustic alcohol is controlled in 10-45min, preferably 20-30min；Reaction temperature control exists
20-35 DEG C, preferably 25-35 DEG C；Vacuum distillation temperature is controlled at 30-60 DEG C, preferably 30-40 DEG C；
The reaction time that compound b reacts obtained solid and chloromethyl pivalate with caustic alcohol controls in 1-3h, preferably
1-2h；Reaction temperature is controlled at 20-35 DEG C, preferably 25-35 DEG C；Vacuum distillation temperature is controlled at 30-60 DEG C, preferably 30-40 DEG C；
Eluant, eluent used in silica gel column chromatography is petroleum ether and ethyl acetate, using gradient elution, petrol ether/ethyl acetate
Volume ratio is 1:1-1:4.
In the step (3)：
The compound c, triphosgene,Mol ratio be 1:1-4:2-5, preferably 1:1-2:2-3；
Selected inert protective gas is nitrogen or argon gas, preferably argon gas；
The reaction temperature of compound c and triphosgene is controlled at 0-25 DEG C, and the reaction time is 3-8h, preferably 4-6h, is changed
Compound d；
Compound d withReaction temperature control at 25-40 DEG C, preferably 25-35 DEG C；
Reaction time is 3-8h, preferably 4-6h；
Vacuum distillation temperature is controlled at 25-40 DEG C, preferably 25-35 DEG C；
Eluant, eluent used in silica gel column chromatography is petroleum ether and ethyl acetate, using gradient elution, petrol ether/ethyl acetate
Volume ratio is 1:2-1:5.
In the step (4)：
The mol ratio of the compound e and ammoniacal liquor are 1:0.1-1(mmol:ML), preferably 1:0.1-0.5(mmol:mL)；
Reaction time is controlled in 20-40h, preferably 30-40h；
Reaction temperature is controlled at 20-35 DEG C, preferably 25-35 DEG C.
Present invention also offers a kind of pharmaceutical composition, including a) alkylating agents antitumoral compounds and b) of the present invention
Have hypoxemia activate characteristic targeting AGT protein inhibitors.
The alkylating agents antitumoral compounds are Nimustine.
Purposes of the hypoxemia activation AGT protein inhibitors of the present invention in antineoplastic is prepared belongs to the present invention's
Protection domain.
Purposes of the aforementioned pharmaceutical compositions of the present invention in antineoplastic is prepared falls within protection scope of the present invention.
The antitumor activity that hypoxemia activates AGT protein inhibitors, described tumour are embodied in an embodiment of the present invention
For leukaemia, lung cancer, melanoma, T lymthomas, glioma brain tumour, liver cancer.
The new hypoxemia activation AGT inhibitor that the present invention is provided has advantages below：
(1) formula (I) compound for providing of the present invention be capable of selectivity under low-oxygen environment by solid tumor cell also
Protoenzyme is activated, and 4- nitrobenzyls are reduced to 4- nitro aminos or 4- nitro azanols, discharges AGT counterfeit substrate O6- 4- bromothiophenes
Methyl guanine, thus targeting suppress the activity of AGT in solid tumor, improve sensitiveness of the tumour cell to chemotherapeutics.
(2) compared with existing AGT protein inhibitors, formula (I) compound that the present invention is provided has higher targeting
Property, AGT inhibitory activity and water solubility.
(3) the extracorporeal anti-tumor screening test that the present invention is carried out to the compound in formula (I) shows, under low oxygen conditions
Compound in formula (I) acts on human leukemia cell line HL-60, human lung cancer cell A549, human melanin with ACNU drug combinations
Oncocyte A375 cells, human T lymphoma cell HUT102, human brain neuroglial cytoma H4, human liver cancer cell BEL-7402 etc.
A variety of solid tumor cell systems show obvious inhibitory action；And under aerobic conditions, the compound in formula (I) is to above-mentioned swollen
The inhibitory action of oncocyte is not obvious.Therefore, there is the compound in formula (I) good hypoxemia to activate characteristic and tumour cell
Targeting, with alkylating agents anti-cancer agent in combination in use, sensitiveness of the tumour cell to cancer therapy drug can be significantly improved, can be used
In the targeting combined chemotherapy of malignant tumour.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.It is related in following examples
Medicine without specified otherwise, it is commercially available to obtain；The operation being related to, no specified otherwise is the conventional operation in this area.
The compound 1 that is related in following examples, compound 2, compound 3 structural formula it is as follows：
The 4- nitrobenzyls of embodiment 1-(6- (4- bromothiophenes methoxyl group)-9 hydrogen-purine-2) carbamate (compound 1)
Synthesis
1) synthesis of (6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine
Weigh 4- bromothiophene base methanol (1.38g, 7.2mmol) to be added in 100mL round-bottomed flasks, add 20mL N, N-
Dimethylformamide, sodium hydride (0.115g, 4.8mmol) and 2-aminopurine -6- leptodactyline chlorine are added into reaction solution
Compound (0.547g, 2.4mmol), 25 DEG C, stirring reaction 2h.After reaction terminates, it is 1 to add ethyl acetate and water according to volume:1
The mixed liquor of composition is extracted, after organic phase is washed through saturated ammonium chloride solution, through anhydrous sodium sulfate drying, and 30 DEG C of decompressions are steamed
Evaporate, gained crude product is dissolved in methanol, be filtered to remove after contamination precipitation, recrystallization and obtain (6- (4- bromothiophenes methoxyl group) -9 hydrogen-fast
Purine -2) amine (0.52g, 1.6mmol), yield 67%.
UVλ:246,283nm。
IR (KBr tablettings) v/cm-1:3380.1(N-H)；2956.8(C-H)；1695.7 (C=C)；1094.8(C-N)；
703.8(C-Br)；610.8(C-S).
1H NMR(400MHz,CDCl3)δ:5.16(s,2H,OCH2)；6.54(s,2H,NH2)；6.65-6.87(s,2H,
C4H2BrS)；8.25(s,1H,H8)；13.25(s,H,NH).
ESI-MS:m/z326(M+H)+。
2) synthesis of (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate
(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine (0.52g, 1.6mmol) is weighed in 100mL round-bottomed flasks
In, the ethanol solution that addition 3.2mL contains caustic alcohol (0.218g, 3.2mmol) dissolves it.25 DEG C, react after 20min, very
Sky drains solvent, and obtained solid is dissolved in the anhydrous DMFs of 10mL.Chloromethyl pivalate is added into solution
(0.692mL, 4.8mmol), 25 DEG C, stirring reaction 1h.After question response terminates, it is 1 to add ethyl acetate and water according to volume:1
The mixed liquor of composition is extracted, and organic phase is after saturated common salt water washing, anhydrous sodium sulfate drying, 30 DEG C, and vacuum distillation is obtained
Isolated and purified to crude on silica gel column chromatography, eluant, eluent is petroleum ether and ethyl acetate, using gradient elution, petroleum ether/
The volume ratio of ethyl acetate is from 1:1 progressively increases to 1:4, obtain (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) valeric acid
Methyl esters (0.505g, 1.15mmol), yield 72%.
UVλ:266,283nm。
IR (KBr tablettings) v/cm-1:3380.4(N-H)；2956.8(C-H)；1690.8 (C=C)；1215.9(C-O-C)；
1087.5(C-N)；706.3(C-Br)；614.6(C-S).
1H NMR(400MHz,CDCl3)δ：1.16(s,3H,CH3)；5.18(s,2H,OCH2)；5.37(s,2H,NCH2)；
6.56(s,2H,NH2)；6.67-6.88(s,2H,C4H2BrS)；8.21(s,1H,H8).
ESI-MS:m/z440(M+H)+。
3) (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzyls) oxygen) carbonyl) hydrogen of amino-9-purine-9) valeric acid first
The synthesis of ester
Weigh triphosgene (0.341g, 1.15mmol) to be added in two mouthfuls of flask bottles, add the dissolving of 7mL dichloromethane, will
Gained solid (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.505g, 1.15mmol), adds 12mL bis-
Chloromethanes dissolves, and adds 0.5mL anhydrous pyridines, by (2- amino -6- (4- bromothiophene first under conditions of ice bath, argon gas protection
Epoxide) -9 hydrogen -9) methyl valerate solution is added drop-wise in triphosgene solution dropwise, and after completion of dropping, temperature is slowly raised into 25 DEG C,
Stirring reaction 4h；The dichloromethane solution that 7mL contains p nitrobenzyl alcohol (0.352g, 2.3mmol), 25 are added into reaction solution
DEG C, stirring reaction 4h, thin-layered chromatography monitoring reaction process.After question response is without raw material point, 30 DEG C of vacuum distillations remove solvent, use
Silica gel column chromatography is isolated and purified, and eluant, eluent is petroleum ether and ethyl acetate, using gradient elution, petrol ether/ethyl acetate
Volume ratio is from 1:2 progressively increase to 1:5,30 DEG C of vacuum drying obtain white solid (6- (4- bromothiophenes methoxyl group) -2- ((((4-
Nitrobenzyl) oxygen) carbonyl) hydrogen of amino-9-purine-9) methyl valerate (0.445g, 0.72mmol), yield 63%.
UVλ:264,285nm。
IR (KBr tablettings) v/cm-1:3425.6(N-H)；2956.8(C-H)；1791.6 (C=O)；1617.8 (C=C)；
1427.2(N-O)；1224.5(C-O-C)；1094.5(C-N)；713.6(C-Br)；618.1(C-S).
1H NMR(400MHz,CDCl3)δ：1.19(s,3H,CH3)；5.17(s,2H,OCH2)；5.34(s,2H,OCH2Ar)；
5.39(s,2H,NCH2)；6.68-6.89(s,2H,C4H2BrS)；7.76-8.42(m,4H,Ar)；8.06(s,1H,H8)；10.20
(s,H,NH)；.
ESI-MS：m/z619(M+H)+。
4) synthesis of 4- nitrobenzyls-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate
By (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzyls) oxygen) carbonyl) hydrogen of amino-9-purine-9) valeric acid first
Ester (0.445g, 0.72mmol) is dissolved in the 40mL methanol solutions containing 0.072mol/L ammonia (0.11mL ammoniacal liquor), 25 DEG C, stirring
40h is reacted, thin-layer chromatography monitoring reaction process, question response is collected by filtration solid, obtains solid 4- nitrobenzyls to after without raw material point
Base-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate (0.305g, 0.605mmol), yield is 84%.
UVλ:264,281nm。
IR (KBr tablettings) v/cm-1:3418.6(N-H)；2963.5(C-H)；1787.8 (C=O)；1625.3 (C=C)；
1431.7(N-O)；1232.6(C-O-C)；1134.1(C-N)；724.5(C-Br)；615.8(C-S).
1H NMR(400MHz,CDCl3)δ:5.18(s,2H,OCH2)；5.34(s,2H,OCH2Ar)；6.63-6.85(s,
2H,C4H2BrS)；7.74-8.27(m,4H,Ar)；8.06(s,1H,H8)；10.51(s,1H,NHCO)；13.27(s,H,H9).
ESI-MS:m/z505(M+H)+。
The 4- nitrobenzyls of embodiment 2-(6- (4- bromothiophenes methoxyl group)-9 hydrogen-purine-2) carbamate (compound 1)
Synthesis
1) synthesis of (6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine
Weigh 4- bromothiophene base methanol (1.32g, 6.9mmol) to be added in 100mL round-bottomed flasks, add 18mL N, N-
Dimethylformamide, sodium hydride (0.110g, 4.6mmol) and 2-aminopurine -6- leptodactyline chlorine are added into reaction solution
Compound (0.525g, 2.3mmol), 30 DEG C, stirring reaction 2h.After reaction terminates, it is 1 to add ethyl acetate and water according to volume:1
The mixed liquor of composition is extracted, after organic phase is washed through saturated ammonium chloride solution, through anhydrous sodium sulfate drying, and 30 DEG C of decompressions are steamed
Evaporate, gained crude product is dissolved in methanol, be filtered to remove after contamination precipitation, recrystallization and obtain (6- (4- bromothiophenes methoxyl group) -9 hydrogen-fast
Purine -2) amine (0.517g, 1.59mmol), yield 69%.
UVλ:246,283nm。
IR (KBr tablettings) v/cm-1:3385.3(N-H)；2963.5(C-H)；1689.3 (C=C)；1085.3(C-N)；
706.2(C-Br)；614.7(C-S).
1H NMR(400MHz,CDCl3)δ:5.18(s,2H,OCH2)；6.59(s,2H,NH2)；6.62-6.89(s,2H,
C4H2BrS)；8.31(s,1H,H8)；13.21(s,H,NH).
ESI-MS:m/z326(M+H)+。
2) synthesis of (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate
(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine (0.517g, 1.59mmol) is weighed in 100mL round-bottomed flasks
In, the ethanol solution that addition 3.18mL contains caustic alcohol (0.216g, 3.18mmol) dissolves it.30 DEG C, react after 20min,
Vacuum drains solvent, and obtained solid is dissolved in the anhydrous DMFs of 10mL.Chloromethyl pivalate is added into solution
(0.687mL, 4.77mmol), 30 DEG C, stirring reaction 1h.After reaction terminates, it is 1 to add ethyl acetate and water according to volume:1 group
Into mixed liquor extracted, organic phase is after saturated common salt water washing, anhydrous sodium sulfate drying, 30 DEG C, and vacuum distillation is obtained
Crude on silica gel column chromatography is isolated and purified, using gradient elution, and the volume ratio of petrol ether/ethyl acetate is from 1:1 gradually increases
It is added to 1:4, obtain (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.509g, 1.16mmol), yield
73%.
UVλ:266,283nm。
IR (KBr tablettings) v/cm-1:3385.1(N-H)；2963.6(C-H)；1696.1 (C=C)；1219.3(C-O-C)；
1085.8(C-N)；709.5(C-Br)；617.2(C-S).
1H NMR(400MHz,CDCl3)δ:1.15(s,3H,CH3)；5.19(s,2H,OCH2)；5.39(s,2H,NCH2)；
6.61(s,2H,NH2)；6.64-6.89(s,2H,C4H2BrS)；8.25(s,1H,H8).
ESI-MS:m/z440(M+H)+。
3) (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzyls) oxygen) carbonyl) hydrogen of amino-9-purine-9) valeric acid first
The synthesis of ester
Weigh triphosgene (0.344g, 1.16mmol) to be added in two mouthfuls of flask bottles, add the dissolving of 6mL dichloromethane, will
Gained solid (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.509g, 1.16mmol), adds 11mL bis-
Chloromethanes dissolves, and adds 0.45mL anhydrous pyridines, by (2- amino -6- (4- bromothiophenes under conditions of ice bath, argon gas protection
Methoxyl group) -9 hydrogen -9) methyl valerate solution is added drop-wise in triphosgene solution dropwise, and after completion of dropping, temperature is slowly raised into 25
DEG C, stirring reaction 4h；Into reaction solution add 6mL contain p nitrobenzyl alcohol (0.355g, 2.32mmol) dichloromethane it is molten
Liquid, 30 DEG C, stirring reaction 4h, thin-layered chromatography monitoring reaction process.After question response is without raw material point, 30 DEG C of vacuum distillations remove molten
Agent, is isolated and purified with silica gel column chromatography, and eluant, eluent is petroleum ether and ethyl acetate, using gradient elution, petroleum ether/acetic acid second
The volume ratio of ester is from 1:2 progressively increase to 1:5,30 DEG C of vacuum drying obtain white solid (6- (4- bromothiophenes methoxyl group) -2-
(((4- nitrobenzyls) oxygen) carbonyl) hydrogen of amino-9-purine-9) methyl valerate (0.464g, 0.75mmol), yield 65%.
UVλ:264,284nm。
IR (KBr tablettings) v/cm-1:3429.3(N-H)；2964.2(C-H)；1782.8 (C=O)；1625.3 (C=C)；
1435.1(N-O)；1236.8(C-O-C)；1081.3(C-N)；718.5(C-Br)；613.7(C-S).
1H NMR(400MHz,CDCl3)δ:1.18(s,3H,CH3)；5.19(s,2H,OCH2)；5.37(s,2H,OCH2Ar)；
5.42(s,2H,NCH2)；6.65-6.87(s,2H,C4H2BrS)；7.74-8.45(m,4H,Ar)；8.09(s,1H,H8)；10.34
(s,H,NH)；.
ESI-MS:m/z619(M+H)+。
4) synthesis of 4- nitrobenzyls-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate
By (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzyls) oxygen) carbonyl) hydrogen of amino-9-purine-9) valeric acid first
Ester (0.464g, 0.75mmol) is dissolved in the 40mL methanol solutions containing 0.075mol/L ammonia (0.12mL ammoniacal liquor), 30 DEG C, stirring
38h is reacted, thin-layer chromatography monitoring reaction process, question response is collected by filtration solid, obtains solid 4- nitrobenzyls to after without raw material point
Base-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate (0.322g, 0.638mmol), yield is 85%.
UVλ：264,282nm.
IR (KBr tablettings) v/cm-1:3426.2(N-H)；2972.1(C-H)；1779.3 (C=O)；1631.6 (C=C)；
1437.9(N-O)；1235.6(C-O-C)；1128.5(C-N)；721.7(C-Br)；613.6(C-S).
1H NMR(400MHz,CDCl3)δ:5.19(s,2H,OCH2)；5.37(s,2H,OCH2Ar)；6.61-6.87(s,
2H,C4H2BrS)；7.72-8.26(m,4H,Ar)；8.09(s,1H,H8)；10.52(s,1H,NHCO)；13.25(s,H,H9).
ESI-MS:m/z505(M+H)+。
The 1- of embodiment 3 (4- nitrobenzene) ethyl-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate (is changed
Compound 2)
1) synthesis of (6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine
Weigh 4- bromothiophene base methanol (1.50g, 7.8mmol) to be added in 100mL round-bottomed flasks, add 20mLN, N- bis-
NMF, sodium hydride (0.125g, 5.2mmol) and 2-aminopurine -6- leptodactyline chlorinations are added into reaction solution
Thing (0.593g, 2.6mmol), 25 DEG C, stirring reaction 2h.After reaction terminates, it is 1 to add ethyl acetate and water according to volume:1 group
Into mixed liquor extracted, after organic phase is washed through saturated ammonium chloride solution, through anhydrous sodium sulfate drying, 30 DEG C of decompressions are steamed
Evaporate, gained crude product is dissolved in methanol, be filtered to remove after contamination precipitation, recrystallization and obtain (6- (4- bromothiophenes methoxyl group) -9 hydrogen-fast
Purine -2) amine (0.585g, 1.8mmol), yield 69%.
UVλ:246,283nm。
IR (KBr tablettings) v/cm-1:3375.4(N-H)；2967.3(C-H)；1683.4 (C=C)；1086.4(C-N)；
705.2(C-Br)；615.3(C-S).
1H NMR(400MHz,CDCl3)δ:5.18(s,2H,OCH2)；6.57(s,2H,NH2)；6.61-6.87(s,2H,
C4H2BrS)；8.23(s,1H,H8)；13.24(s,H,NH).
ESI-MS:m/z326(M+H)+。
2) synthesis of (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate
(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine (0.585g, 1.8mmol) is weighed in 100mL round-bottomed flasks
In, the ethanol solution that addition 3.6mL contains caustic alcohol (0.245g, 3.6mmol) dissolves it, 25 DEG C, stirring reaction 20min
Afterwards, vacuum drains solvent, and obtained solid is dissolved in the anhydrous DMFs of 12mL.Pivalic acid chloromethane is added into solution
Ester (0.778mL, 5.4mmol), 25 DEG C, stirring reaction 1h.After reaction terminates, it is 1 to add ethyl acetate and water according to volume:1
The mixed liquor of composition is extracted, and organic phase is after saturated common salt water washing, anhydrous sodium sulfate drying, 30 DEG C, and vacuum distillation is obtained
Isolated and purified to crude on silica gel column chromatography, using gradient elution, the volume ratio of petrol ether/ethyl acetate is from 1:1 gradually
Increase to 1:4, obtain (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.584g, 1.33mmol), yield
74%.
UVλ:267,283nm。
IR (KBr tablettings) v/cm-1:3349.5(N-H)；2972.3(C-H)；1685.9 (C=C)；1226.5(C-O-C)；
1093.4(C-N)；709.2(C-Br)；626.8(C-S).
1H NMR(400MHz,CDCl3)δ:1.15(s,3H,CH3)；5.19(s,2H,OCH2)；5.36(s,2H,NCH2)；
6.58(s,2H,NH2)；6.69-6.87(s,2H,C4H2BrS)；8.25(s,1H,H8).
ESI-MS:m/z440(M+H)+。
3) (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzene) ethyoxyl) carbonyl) hydrogen of amino-9-purine-9) valeric acid
The synthesis of methyl esters
Weigh triphosgene (0.395g, 1.33mmol) to be added in two mouthfuls of flask bottles, add the dissolving of 9mL dichloromethane, will
Gained solid (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.584g, 1.33mmol), adds 14mL bis-
Chloromethanes dissolves, and adds 0.6mL anhydrous pyridines, by (2- amino -6- (4- bromothiophene first under conditions of ice bath, argon gas protection
Epoxide) -9 hydrogen -9) methyl valerate solution is added drop-wise in triphosgene solution dropwise, and after completion of dropping, temperature is slowly raised into 25 DEG C,
Stirring reaction 4h；Into reaction solution, addition 9mL contains the dichloromethane to 1- (4- nitrobenzophenones) ethanol (0.445g, 2.66mmol)
Alkane solution, 25 DEG C, stirring reaction 4h, thin-layered chromatography monitoring reaction process.After question response is without raw material point, 30 DEG C of vacuum distillations are removed
Solvent is removed, is isolated and purified with silica gel column chromatography, eluant, eluent is petroleum ether and ethyl acetate, using gradient elution, petroleum ether/second
The volume ratio of acetoacetic ester is from 1:2 progressively increase to 1:5,30 DEG C vacuum drying obtain white solid (6- (4- bromothiophenes methoxyl group)-
2- (((4- nitrobenzene) ethyoxyl) carbonyl) hydrogen of amino-9-purine-9) methyl valerate (0.512g, 0.81mmol), yield 61%.
UVλ:267,285nm。
IR (KBr tablettings) v/cm-1:3468.3(N-H)；2937.9(C-H)；1764.2 (C=O)；1636.4 (C=C)；
1435.6(N-O)；1237.5(C-O-C)；1125.7(C-N)；721.3(C-Br)；624.5(C-S).
1H NMR(400MHz,CDCl3)δ:1.15(s,3H,C(CH3)3)；1.65(d,3H,CH3)；5.16(s,2H,
OCH2)；5.45(s,2H,NCH2)；6.06(s,H,CH3 CH)；6.61-6.84(s,2H,C4H2BrS)；7.56-8.26(m,4H,
Ar)；8.04(s,1H,H8)；10.34(s,H,NH).
ESI-MS:m/z633(M+H)+。
4) synthesis of 1- (4- nitrobenzene) ethyl-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate
By (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzene) ethyoxyl) carbonyl) hydrogen of amino-9-purine-9) valeric acid
Methyl esters (0.512g, 0.81mmol) is dissolved in the 45mL methanol solutions containing 0.081mol/L ammonia (0.14mL ammoniacal liquor), 25 DEG C, is stirred
Reaction 40h is mixed, thin-layer chromatography monitoring reaction process, question response is collected by filtration solid, obtains solid 1- (4- to after without raw material point
Nitrobenzene) ethyl-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate (0.357g, 0.69mmol), yield is
85%.
UVλ:267,280nm。
IR (KBr tablettings) v/cm-1:3425.3(N-H)；2974.8(C-H)；1769.6 (C=O)；1639.1 (C=C)；
1435.3(N-O)；1237.4(C-O-C)；1136.7(C-N)；728.2(C-Br)；621.5(C-S).
1H NMR(400MHz,CDCl3)δ:1.84(d,3H,CH3)；5.19(s,2H,OCH2)；5.96(q,H,CH3 CH)；
6.64-6.88(s,2H,C4H2BrS)；7.76-8.31(m,4H,Ar)；8.09(s,1H,H8)；10.34(s,H,NHCO)；13.22
(s,H,H9)。
ESI-MS:m/z519(M+H)+。
The 1- of embodiment 4 (4- nitrobenzene) ethyl-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate (is changed
Compound 2)
1) synthesis of (6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine
Weigh 4- bromothiophene base methanol (1.56g, 8.1mmol) to be added in 100mL round-bottomed flasks, add 25mLN, N- bis-
NMF, sodium hydride (0.130g, 5.4mmol) and 2-aminopurine -6- leptodactyline chlorinations are added into reaction solution
Thing (0.616g, 2.7mmol), 30 DEG C, stirring reaction 2h.After reaction terminates, it is 1 to add ethyl acetate and water according to volume:1 group
Into mixed liquor extracted, after organic phase is washed through saturated ammonium chloride solution, through anhydrous sodium sulfate drying, 30 DEG C of decompressions are steamed
Evaporate, gained crude product is dissolved in methanol, be filtered to remove after contamination precipitation, recrystallization and obtain (6- (4- bromothiophenes methoxyl group) -9 hydrogen-fast
Purine -2) amine (0.605g, 1.86mmol), yield 69%.
UVλ:246,283nm。
IR (KBr tablettings) v/cm-1:3352.6(N-H)；2968.1(C-H)；1685.2 (C=C)；1087.3(C-N)；
708.1(C-Br)；618.1(C-S).
1H NMR(400MHz,CDCl3)δ:5.19(s,2H,OCH2)；6.58(s,2H,NH2)；6.62-6.84(s,2H,
C4H2BrS)；8.24(s,1H,H8)；13.25(s,H,NH).
ESI-MS:m/z326(M+H)+。
2) synthesis of (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate
(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine (0.605g, 1.86mmol) is weighed in 100mL round-bottomed flasks
In, the ethanol solution that addition 3.72mL contains caustic alcohol (0.253g, 3.72mmol) dissolves it, 30 DEG C, stirring reaction 20min
Afterwards, vacuum drains solvent, and obtained solid is dissolved in the anhydrous DMFs of 15mL.Pivalic acid chloromethane is added into solution
Ester (0.804mL, 5.58mmol), 30 DEG C, stirring reaction 1h.After reaction terminates, it is 1 to add ethyl acetate and water according to volume:1
The mixed liquor of composition is extracted, and organic phase is after saturated common salt water washing, anhydrous sodium sulfate drying, 30 DEG C, and vacuum distillation is obtained
Isolated and purified to crude on silica gel column chromatography, using gradient elution, the volume ratio of petrol ether/ethyl acetate is from 1:1 gradually
Increase to 1:4, obtain (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.615g, 1.4mmol), yield
75%.
UVλ:266,283nm。
IR (KBr tablettings) v/cm-1:3342.4(N-H)；2975.3(C-H)；1686.3 (C=C)；1231.7(C-O-C)；
1087.5(C-N)；712.5(C-Br)；624.3(C-S).
1H NMR(400MHz,CDCl3)δ:1.16(s,3H,CH3)；5.24(s,2H,OCH2)；5.34(s,2H,NCH2)；
6.59(s,2H,NH2)；6.64-6.89(s,2H,C4H2BrS)；8.27(s,1H,H8).
ESI-MS:m/z440(M+H)+。
3) (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzene) ethyoxyl) carbonyl) hydrogen of amino-9-purine-9) valeric acid
The synthesis of methyl esters
Weigh triphosgene (0.415g, 1.4mmol) to be added in two mouthfuls of flask bottles, add the dissolving of 10mL dichloromethane, will
Gained solid (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.615g, 1.4mmol), adds 15mL bis-
Chloromethanes dissolves, and adds 0.7mL anhydrous pyridines, by (2- amino -6- (4- bromothiophene first under conditions of ice bath, argon gas protection
Epoxide) -9 hydrogen -9) methyl valerate solution is added drop-wise in triphosgene solution dropwise, and after completion of dropping, temperature is slowly raised into 25 DEG C,
Stirring reaction 4h；Into reaction solution, addition 10mL contains the dichloromethane to 1- (4- nitrobenzophenones) ethanol (0.468g, 2.8mmol)
Alkane solution, 30 DEG C, stirring reaction 4h, thin-layered chromatography monitoring reaction process.After question response is without raw material point, 30 DEG C of vacuum distillations are removed
Solvent is removed, is isolated and purified with silica gel column chromatography, eluant, eluent is petroleum ether and ethyl acetate, using gradient elution, petroleum ether/second
The volume ratio of acetoacetic ester is from 1:2 progressively increase to 1:5,30 DEG C vacuum drying obtain white solid (6- (4- bromothiophenes methoxyl group)-
2- (((4- nitrobenzene) ethyoxyl) carbonyl) hydrogen of amino-9-purine-9) methyl valerate (0.556g, 0.88mmol), yield 63%.
UVλ:267,285nm。
IR (KBr tablettings) v/cm-1:3471.2(N-H)；2935.8(C-H)；1758.5 (C=O)；1627.5 (C=C)；
1438.4(N-O)；1239.2(C-O-C)；1128.5(C-N)；725.1(C-Br)；625.6(C-S).
1H NMR(400MHz,CDCl3)δ:1.18(s,3H,C(CH3)3)；1.66(d,3H,CH3)；5.18(s,2H,
OCH2)；5.47(s,2H,NCH2)；6.08(s,H,CH3 CH)；6.63-6.85(s,2H,C4H2BrS)；7.54-8.28(m,4H,
Ar)；8.05(s,1H,H8)；10.35(s,H,NH).
ESI-MS:m/z633(M+H)+。
4) synthesis of 1- (4- nitrobenzene) ethyl-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate
By (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzene) ethyoxyl) carbonyl) hydrogen of amino-9-purine-9) valeric acid
Methyl esters (0.556g, 0.88mmol) is dissolved in the 48mL methanol solutions containing 0.088mol/L ammonia (0.16mL ammoniacal liquor), 30 DEG C, is stirred
Reaction 38h is mixed, thin-layer chromatography monitoring reaction process, question response is collected by filtration solid, obtains solid 1- (4- to after without raw material point
Nitrobenzene) ethyl-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate (0.394g, 0.76mmol), yield is
86%.
UVλ:267,281nm。
IR (KBr tablettings) v/cm-1:3429.4(N-H)；29741.5(C-H)；1765.8 (C=O)；1635.4 (C=C)；
1438.2(N-O)；1235.6(C-O-C)；1084.7(C-N)；723.6(C-Br)；625.2(C-S).
1H NMR(400MHz,CDCl3)δ:1.86(d,3H,CH3)；5.17(s,2H,OCH2)；5.94(q,H,CH3 CH)；
6.61-6.87(s,2H,C4H2BrS)；7.73-8.31(m,4H,Ar)；8.07(s,1H,H8)；10.32(s,H,NHCO)；13.21
(s,H,H9)。
ESI-MS:m/z519(M+H)+。
The 2- of embodiment 5 (4- nitrobenzyls) propyl group-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate
The synthesis of (compound 3)
1) synthesis of (6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine
Weigh 4- bromothiophene base methanol (1.44g, 7.5mmol) to be added in 100mL round-bottomed flasks, add 20mLN, N- bis-
NMF, sodium hydride (0.12g, 5.0mmol) and 2-aminopurine -6- leptodactyline chlorides are added into reaction solution
(0.570g, 2.5mmol), stirring, reacts 2h by 25 DEG C.After reaction terminates, it is 1 to add ethyl acetate and water according to volume:1 group
Into mixed liquor extracted, after organic phase is washed through saturated ammonium chloride solution, through anhydrous sodium sulfate drying, 30 DEG C of decompressions are steamed
Evaporate, gained crude product is dissolved in methanol, be filtered to remove after contamination precipitation, recrystallization and obtain (6- (4- bromothiophenes methoxyl group) -9 hydrogen-fast
Purine -2) amine (0.553g, 1.7mmol), yield 68%.
UVλ:246,283nm。
IR (KBr tablettings) v/cm-1:3432.6(N-H)；2947.2(C-H)；1670.1 (C=C)；1098.5(C-N)；
707.3(C-Br)；615.4(C-S).
1H NMR(400MHz,CDCl3)δ:5.18(s,2H,OCH2)；6.53(s,2H,NH2)；6.67-6.83(s,2H,
C4H2BrS)；8.25(s,1H,H8)；13.21(s,H,NH).
ESI-MS:m/z326(M+H)+。
2) synthesis of (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate
(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine (0.553g, 1.7mmol) is weighed in 100mL round-bottomed flasks
In, the ethanol solution that addition 3.4mL contains caustic alcohol (0.231g, 3.4mmol) dissolves it, 25 DEG C, stirring reaction 20min
Afterwards, vacuum drains solvent, and obtained solid is dissolved in the anhydrous DMFs of 12mL.Pivalic acid chlorine is added into reaction solution
Methyl esters (0.735mL, 5.1mmol), 25 DEG C, stirring reaction 1h, after reaction terminates, it is 1 to add ethyl acetate and water according to volume:
The mixed liquor of 1 composition is extracted, and organic phase is after saturated common salt water washing, anhydrous sodium sulfate drying, 30 DEG C of vacuum distillations, obtains
Isolated and purified to crude on silica gel column chromatography, using gradient elution, the volume ratio of petrol ether/ethyl acetate is from 1:1 gradually
Increase to 1:4, obtain (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.544g, 1.24mmol), yield
73%.
UVλ:266,283nm。
IR (KBr tablettings) v/cm-1:3438.3(N-H)；2942.4(C-H)；1685.7 (C=C)；1236.3(C-O-C)；
1094.6(C-N)；712.5(C-Br)；609.7(C-S).
1H NMR(400MHz,CDCl3)δ:1.15(s,3H,CH3)；5.19(s,2H,OCH2)；5.35(s,2H,NCH2)；
6.58(s,2H,NH2)；6.69-6.85(s,2H,C4H2BrS)；8.25(s,1H,H8).
ESI-MS:m/z440(M+H)+。
3) (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzene) propoxyl group) carbonyl) hydrogen of amino-9-purine-9) valeric acid
The synthesis of methyl esters
Weigh triphosgene (0.368g, 1.24mmol) to be added in two mouthfuls of flask bottles, add the dissolving of 8mL dichloromethane, will
Gained solid (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.544g, 1.24mmol), adds 14mL bis-
Chloromethanes dissolves, and adds 0.55mL anhydrous pyridines, by (2- amino -6- (4- bromothiophenes under conditions of ice bath, argon gas protection
Methoxyl group) -9 hydrogen -9) methyl valerate solution is added drop-wise in triphosgene solution dropwise, and after completion of dropping, temperature is slowly raised into 25
DEG C, stirring reaction 4h；8mL is added into reaction solution to contain to 2- (4- nitrobenzophenones) -2- propyl alcohol (0.449g, 2.48mmol)
Dichloromethane solution, 25 DEG C, stirring reaction 4h, thin-layered chromatography monitoring reaction process.After question response is without raw material point, 30 DEG C of decompressions
Solvent is distilled off, is isolated and purified with silica gel column chromatography, eluant, eluent is petroleum ether and ethyl acetate, using gradient elution, stone
The volume ratio of oily ether/ethyl acetate is from 1:2 progressively increase to 1:5,30 DEG C of vacuum drying obtain white solid (6- (4- bromothiophenes
Methoxyl group)-2- (((4- nitrobenzene) ethyoxyl) carbonyl) hydrogen of amino-9-purine-9) methyl valerate (0.504g, 0.78mmol),
Yield 63%.
UVλ:268,285nm。
IR (KBr tablettings) v/cm-1:3445.3(N-H)；2948.5(C-H)；1784.7 (C=O)；1627.4 (C=C)；
1431.7(N-O)；1219.8(C-O-C)；1083.6(C-N)；715.7(C-Br)；614.3(C-S).
1H NMR(400MHz,CDCl3)δ:1.13(s,3H,CH3)；1.87(s,3H,CH3)；5.18(s,2H,OCH2)；
5.58(s,2H,NCH2)；6.63-6.87(s,2H,C4H2BrS)；7.29-8.19(m,4H,Ar)；7.97(s,1H,H8)；10.36
(s,H,NH)。
ESI-MS:m/z647(M+H)+。
4) synthesis of 2- (4- nitrobenzene) propyl group-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate
By (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzene) propoxyl group) carbonyl) hydrogen of amino-9-purine-9) valeric acid
Methyl esters (0.504g, 0.78mmol) is dissolved in the 45mL methanol solutions containing 0.078mol/L ammonia (0.135mL ammoniacal liquor), 25 DEG C,
Stirring reaction 40h, thin-layer chromatography monitoring reaction process, question response is collected by filtration solid, obtains solid 2- to after without raw material point
(4- nitrobenzene) propyl group-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate (0.353g, 0.663mmol), production
Rate is 85%.
UVλ:268,283nm。
IR (KBr tablettings) v/cm-1:3439.2(N-H)；2958.9(C-H)；1792.5 (C=O)；1637.2 (C=C)；
1426.8(N-O)；1235.9(C-O-C)；1095.6(C-N)；715.8(C-Br)；627.4(C-S).
1H NMR(400MHz,CDCl3)δ:1.84(s,3H,CH3)；5.18(s,2H,OCH2)；6.67-6.85(s,2H,
C4H2BrS)；7.46-8.34(m,4H,Ar)；8.19(s,1H,H8)；10.36(s,1H,NHCO)；13.21(s,H,H9).
ESI-MS:m/z533(M+H)+。
The 2- of embodiment 6 (4- nitrobenzyls) propyl group-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate
The synthesis of (compound 3)
1) synthesis of (6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine
Weigh 4- bromothiophene base methanol (1.27g, 6.6mmol) to be added in 100mL round-bottomed flasks, add 18mLN, N- bis-
NMF, sodium hydride (0.106g, 4.4mmol) and 2-aminopurine -6- leptodactyline chlorinations are added into reaction solution
Thing (0.502g, 2.2mmol), stirring, reacts 2h by 30 DEG C.After reaction terminates, it is 1 to add ethyl acetate and water according to volume:1
The mixed liquor of composition is extracted, after organic phase is washed through saturated ammonium chloride solution, through anhydrous sodium sulfate drying, and 30 DEG C of decompressions are steamed
Evaporate, gained crude product is dissolved in methanol, be filtered to remove after contamination precipitation, recrystallization and obtain (6- (4- bromothiophenes methoxyl group) -9 hydrogen-fast
Purine -2) amine (0.494g, 1.52mmol), yield 69%.
UVλ:246,283nm。
IR (KBr tablettings) v/cm-1:3429.2(N-H)；2937.5(C-H)；1675.3 (C=C)；1085.9(C-N)；
709.5(C-Br)；618.1(C-S).
1H NMR(400MHz,CDCl3)δ:5.19(s,2H,OCH2)；6.56(s,2H,NH2)；6.64-6.85(s,2H,
C4H2BrS)；8.25(s,1H,H8)；13.25(s,H,NH).
ESI-MS:m/z326(M+H)+。
2) synthesis of (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate
(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) amine (0.494g, 1.52mmol) is weighed in 100mL round-bottomed flasks
In, the ethanol solution that addition 3.04mL contains caustic alcohol (0.207g, 3.04mmol) dissolves it, 30 DEG C, stirring reaction 20min
Afterwards, vacuum drains solvent, and obtained solid is dissolved in the anhydrous DMFs of 10mL.Pivalic acid chloromethane is added into solution
Ester (0.657mL, 4.56mmol), 30 DEG C, stirring reaction 1h, after reaction terminates, it is 1 to add ethyl acetate and water according to volume:1
The mixed liquor of composition is extracted, and organic phase is after saturated common salt water washing, anhydrous sodium sulfate drying, 30 DEG C of vacuum distillations, is obtained
Isolated and purified to crude on silica gel column chromatography, using gradient elution, the volume ratio of petrol ether/ethyl acetate is from 1:1 gradually
Increase to 1:4, obtain (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.492g, 1.12mmol), yield
74%.
UVλ:266,283nm。
IR (KBr tablettings) v/cm-1:3426.1(N-H)；2937.5(C-H)；1674.2 (C=C)；1247.5(C-O-C)；
1105.7(C-N)；713.6(C-Br)；613.2(C-S).
1H NMR(400MHz,CDCl3)δ:1.16(s,3H,CH3)；5.21(s,2H,OCH2)；5.37(s,2H,NCH2)；
6.56(s,2H,NH2)；6.65-6.84(s,2H,C4H2BrS)；8.26(s,1H,H8).
ESI-MS:m/z440(M+H)+。
3) (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzene) propoxyl group) carbonyl) hydrogen of amino-9-purine-9) valeric acid
The synthesis of methyl esters
Weigh triphosgene (0.332g, 1.12mmol) to be added in two mouthfuls of flask bottles, add the dissolving of 5mL dichloromethane, will
Gained solid (2- amino -6- (4- bromothiophenes methoxyl group) -9 hydrogen -9) methyl valerate (0.492g, 1.12mmol), adds 10mL bis-
Chloromethanes dissolves, and adds 0.4mL anhydrous pyridines, by (2- amino -6- (4- bromothiophene first under conditions of ice bath, argon gas protection
Epoxide) -9 hydrogen -9) methyl valerate solution is added drop-wise in triphosgene solution dropwise, and after completion of dropping, temperature is slowly raised into 25 DEG C,
Stirring reaction 4h；Into reaction solution, addition 5mL contains two to 2- (4- nitrobenzophenones) -2- propyl alcohol (0.406g, 2.24mmol)
Chloromethanes solution, 30 DEG C, stirring reaction 4h, thin-layered chromatography monitoring reaction process.After question response is without raw material point, 30 DEG C of decompressions are steamed
Solvent is removed in distillation, is isolated and purified with silica gel column chromatography, and eluant, eluent is petroleum ether and ethyl acetate, using gradient elution, oil
The volume ratio of ether/ethyl acetate is from 1:2 progressively increase to 1:5,30 DEG C of vacuum drying obtain white solid (6- (4- bromothiophene first
Epoxide)-2- (((4- nitrobenzene) ethyoxyl) carbonyl) hydrogen of amino-9-purine-9) methyl valerate (0.470g, 0.728mmol), production
Rate 65%.
UVλ:268,285nm。
IR (KBr tablettings) v/cm-1:3425.4(N-H)；2935.2(C-H)；1785.9 (C=O)；1632.6 (C=C)；
1435.6(N-O)；1215.6(C-O-C)；1085.1(C-N)；716.8(C-Br)；617.4(C-S).
1H NMR(400MHz,CDCl3)δ:1.14(s,3H,CH3)；1.89(s,3H,CH3)；5.19(s,2H,OCH2)；
5.57(s,2H,NCH2)；6.62-6.87(s,2H,C4H2BrS)；7.28-8.17(m,4H,Ar)；8.03(s,1H,H8)；10.35
(s,H,NHCO)；.
ESI-MS:m/z 647(M+H)+。
4) synthesis of 2- (4- nitrobenzene) propyl group-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate
By (6- (4- bromothiophenes methoxyl group)-2- (((4- nitrobenzene) propoxyl group) carbonyl) hydrogen of amino-9-purine-9) valeric acid
Methyl esters (0.470g, 0.728mmol) is dissolved in the 40mL methanol solutions containing 0.0728mol/L ammonia (0.112mL ammoniacal liquor), and 30
DEG C, stirring reaction 40h, thin-layer chromatography monitoring reaction process, question response is collected by filtration solid, obtains solid to after without raw material point
2- (4- nitrobenzene) propyl group-(6- (4- bromothiophenes methoxyl group) -9 hydrogen-purine -2) carbamate (0.333g, 0.626mmol),
Yield is 86%.
UVλ:268,283nm。
IR (KBr tablettings) v/cm-1:3418.7(N-H)；2943.5(C-H)；1783.2 (C=O)；1629.7 (C=C)；
1435.3(N-O)；1238.1(C-O-C)；1127.6(C-N)；718.2(C-Br)；614.9(C-S).
1H NMR(400MHz,CDCl3)δ:1.85(s,3H,CH3)；5.17(s,2H,OCH2)；6.65-6.87(s,2H,
C4H2BrS)；7.45-8.36(m,4H,Ar)；8.21(s,1H,H8)；10.35(s,1H,NHCO)；13.23(s,H,H9).
ESI-MS:m/z533(M+H)+。
The AGT protein inhibitors of embodiment 7 are to cellular biology of tumor activity rating
1st, experiment material and instrument
Test compound：Obtained compound 1,2 and 3 in above-mentioned preparation embodiment；
Cell line：Human leukemia cell line HL-60, human lung cancer cell A549, human melanoma cell A375 cells, people T drench
Bar oncocyte HUT102, human brain neuroglial cytoma H4, human liver cancer cell BEL-7402.
2nd, experimental method sets Nimustine (ACNU) group (experimental group 1), ACNU+ respectively under normal oxygen and hypoxia condition
The drug combination group (experimental group 2) of compound 1, the drug combination group (experimental group 3) of ACNU+ compounds 2, the joint of ACNU+ compounds 3 are used
Medicine group (experimental group 4), while setting blank group and control group.
Six kinds of tumour cells are inoculated with 96 orifice plates with 2000/ hole respectively, in 37 DEG C, 5%CO2After culture 24 hours, experimental group 1
Cell is handled with the ACNU of series concentration (0.05mM, 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM, 1.2mM)
24h；Experimental group 2,
3rd, 4 handle cell 3h through compound 1,2,3 respectively after, change and cell 24h (normal oxygen bars handled with the ACNU of series concentration
Oxygen content is about 21% under part, and 1%) oxygen content is about under hypoxia condition.Then, CCK-8 is contained to every μ L of Kong Zhongjia 10
Culture medium, act on 4 hours.Finally, the absorbance at 450nm is determined, cytoactive is calculated as follows, and pass through
Regression analysis calculates half inhibiting rate IC50。
Cell survival rate (%)=(AExperimental group–ABlank group)/(AControl group–ABlank group)×100
AExperimental groupFor the absorbance in the hole with tumour cell, CCK-8 and medicine (ACNU+ compounds 1/2/3)；
ABlank groupThere was only culture medium and CCK-8, the absorbance in the hole without tumour cell and medicine；
AControl groupFor with tumour cell and CCK-8, the absorbance in the hole without medicine.
3rd, experimental result：It is shown in Table 1
Half inhibiting rate (the IC of the tumour cell of table 150,μM)
The result of table 1 is shown, under normal oxygen environment, compared with ACNU groups, and compound 1,2 or 3 is with ACNU drug combinations to six
Plant the IC of tumour cell50Value is close, shows under the conditions of normal oxygen, AGT activity in compound 1,2 and 3 pairs of six kinds of tumour cells
Inhibitory action is very weak.
But under low-oxygen environment, compared with ACNU groups, compound 1,2 or 3 and ACNU drug combinations are thin to six kinds of tumours
The IC of born of the same parents50Value is significantly reduced, and shows that compound 1,2 and 3 can specifically be reduced under low-oxygen environment and discharge O6- benzyl
Base guanine analog reduces the activity of AGT in tumour cell as AGT inhibitor, so as to effectively enhance tumour cell
To ACNU sensitiveness, inhibitory activity of the medicine to tumour cell is improved.
Contrast the IC of compound 1,2 and 3 under normal oxygen and hypoxia condition50Value, it can be seen that under the conditions of hypoxia condition is than normal oxygen
Three kinds of compounds are significantly improved to the AGT inhibitory activity of six kinds of tumour cells, show that compound 1,2 and 3 can specifically exist
It is activated under hypoxia condition.Therefore, compound 1,2 and 3 can targeting in the tumour cell under hypoxia, it is to avoid
AGT activity in influence normal cell, so as to reach chemotherapeutics targeting in the purpose of tumour cell.
Test result indicates that, the compound that the present invention is provided has stronger AGT protein inhibiting activities under low oxygen conditions,
Without obvious inhibiting effect under the conditions of normal oxygen.With existing AGT protein inhibitors O6- benzyl guanine is compared, with significant
Hypoxemia targeting, and preferably water-soluble and AGT inhibitory activity.Therefore, the compound that the present invention is provided is used as alkane
The adjuvant of agent based chemotherapy medicine, acts on tumour cell to targeting, so as to improve tumour cell to the quick of chemotherapeutics
Perception, enhances the chemotherapy effect of alkylating agents medicine.
Although above making with a general description of the specific embodiments to the present invention
Description, but on the basis of the present invention, it can be made some modifications or improvements, this is to people in the art in detail
It is obvious for member.Therefore, without departing from spirit of the invention
On the basis of these modifications or improvements, belong to the scope of protection of present invention.
Claims (10)
1. a kind of hypoxemia activates AGT protein inhibitors, the AGT protein inhibitors have as led to the chemical combination shown in formula (I) structure
Thing：
Wherein, R1=R2=H, or R1=H, R2=CH3, or R1=R2=CH3；
R3=H, NO2, NH2, OCH3, CH2OH or CO2CH3。
2. AGT protein inhibitors as claimed in claim 1, it is characterised in that the AGT protein inhibitors are：
3. the hypoxemia described in claim 1 or 2 activates the preparation method of AGT protein inhibitors, it is characterised in that methods described
The reaction mechanism mechanism of reaction is：
Including following reactions steps：
(1) 4- bromothiophene -2- base methanol is dissolved in DMF, sodium hydride and change is sequentially added into reaction solution
Compound a, stirring reaction, after question response terminates, it is 1 to add ethyl acetate and water according to volume:The mixed liquor of 1 composition is extracted
Take, after organic phase is washed through saturated ammonium chloride solution, through anhydrous sodium sulfate drying, vacuum distillation is recrystallized to give compound b；
(2) ethanol solution containing caustic alcohol is added in compound b dissolves it, and after stirring reaction 10-45min, vacuum is drained
Solvent, obtained solid is dissolved in organic solvent, and chloromethyl pivalate is added into solution, stirring, after reaction terminates, and is added
Ethyl acetate and water are 1 according to volume:The mixed liquor of 1 composition is extracted, and organic phase is after saturated common salt water washing, anhydrous sulphur
Sour sodium is dried, and vacuum distillation obtains crude on silica gel column chromatography and isolated and purified, obtains compound c；
(3) the compound c obtained by step (2) is dissolved in dichloromethane, adds triphosgene, stir anti-under inert gas shielding
Should, products therefrom compound d withReaction, vacuum distillation obtains the separation of crude on silica gel column chromatography pure
Change, obtain compound e；
(4) the compound e obtained by step (3) is dissolved in the methanol solution containing ammoniacal liquor, stirring reaction, thin-layer chromatography monitoring is anti-
Process is answered, reacts after terminating, solid is collected by filtration, the i.e. compound f of hypoxemia activation AGT protein inhibitors is obtained.
4. the preparation method described in claim 3, it is characterised in that in the step (1), compound a, sodium hydride and 4- bromine thiophenes
The mol ratio of fen base methanol is 1:1-4:2-5；The temperature control of stirring reaction is at 20-35 DEG C, and vacuum distillation temperature is 30-60
℃。
5. the preparation method described in claim 3, it is characterised in that in the step (2), the compound b, caustic alcohol and spy
The mol ratio of valeric acid chloromethyl ester is 1:1-3:2-5, organic solvent is DMF or dimethyl sulfoxide (DMSO), two stirrings
Reaction temperature is controlled at 20-35 DEG C, and vacuum distillation temperature is 30-60 DEG C；Eluant, eluent used in the silica gel column chromatography
For petroleum ether and ethyl acetate, using gradient elution, petroleum ether is 1 with ethyl acetate volume ratio:1-1:4.
6. the preparation method described in claim 3, it is characterised in that step (3) the compound c, triphosgene,
Mol ratio be 1:1-4:2-5；
The reaction temperature of compound c and triphosgene is controlled at 0-25 DEG C, and the reaction time is 3-8h, obtains compound d；
Compound d withReacted under the conditions of 25-40 DEG C, the reaction time is 3-8h；
Eluant, eluent used in silica gel column chromatography is petroleum ether and ethyl acetate, using gradient elution, petrol ether/ethyl acetate body
Product is than being 1:2-1:5.
7. the preparation method described in claim 3, it is characterised in that the molal volume of compound e and ammoniacal liquor described in step (4)
Than for 1mmol:0.1-1mL；Reaction time is 20-40h；Reaction temperature is controlled at 20-35 DEG C.
8. a kind of pharmaceutical composition, it is characterised in that including a) alkylating agents antitumoral compounds；B) claim 1-2 is any
The targeting AGT inhibitor with hypoxemia activation characteristic described in one.
9. the pharmaceutical composition described in claim 8, it is characterised in that the alkylating agents antitumoral compounds are Ni Mosi
Spit of fland.
10. the pharmaceutical composition described in hypoxemia activation AGT protein inhibitors or claim 8 or 9 described in claim 1 or 2
Purposes in antineoplastic is prepared.
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