CN108558883B - A kind of nucleic acid base compound or its pharmaceutically acceptable salt and its preparation method and application - Google Patents

A kind of nucleic acid base compound or its pharmaceutically acceptable salt and its preparation method and application Download PDF

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CN108558883B
CN108558883B CN201810493926.7A CN201810493926A CN108558883B CN 108558883 B CN108558883 B CN 108558883B CN 201810493926 A CN201810493926 A CN 201810493926A CN 108558883 B CN108558883 B CN 108558883B
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hydroxyl
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CN108558883A (en
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王玉成
朱梅
王菊仙
白晓光
张国宁
董飚
彭宗根
岑山
王宇佳
王明华
赵跃
杜潇楠
张煊笛
邵端阳
李云鸽
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/40—Heterocyclic compounds containing purine ring systems with halogen atoms or perhalogeno-alkyl radicals directly attached in position 2 or 6
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C07D239/545—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/553—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms with halogen atoms or nitro radicals directly attached to ring carbon atoms, e.g. fluorouracil
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
    • C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
    • C07D405/04—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic System
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/645—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having two nitrogen atoms as the only ring hetero atoms
    • C07F9/6509—Six-membered rings
    • C07F9/6512—Six-membered rings having the nitrogen atoms in positions 1 and 3

Abstract

The present invention provides a kind of nucleic acid base compound or its pharmaceutically acceptable salt, the compound or its pharmaceutically acceptable salt have the apparent activity for inhibiting hiv protease and/or reverse transcriptase；Toxicity research shows it with good druggability, shows that such compound has a good application prospect as anti-AIDS drug.Experimental data according to the embodiment is it is found that the compound of the present invention has inhibitory activity to HIV-1 protease and HIV-1 reverse transcriptase, and all has lower cytotoxicity.Nucleic acid base compound of the invention or its pharmaceutically acceptable salt are expected to become while inhibiting double target spot inhibitor of hiv protease and reverse transcriptase.

Description

A kind of nucleic acid base compound or its pharmaceutically acceptable salt and preparation method thereof and Using
Technical field
The present invention relates to nucleic acid base technical field more particularly to a kind of nucleic acid base compound or its is pharmaceutically acceptable Salt and its preparation method and application.
Background technique
Acquired immunodeficiency syndrome (Acquired Immune Deficiency Syndrome, AIDS), also known as ends Disease is grown, is that the mankind lead to immune lack because of infection immunity defective virus (Human Immunodeficiency Virus, HIV) It falls into, and causes a series of syndrome of opportunistic infections and tumour.HIV is to be currently known differentiation, the highest virus of degree of variation, According to serological reaction, gene order difference and Characteristics of Geographical Distribution, two hypotypes: HIV-1 and HIV-2 can be divided into.HIV-1 is The pathogen for causing AIDS Global prevalence accounts for 95% absolute predominance on number of the infected；HIV-2 primary limitation is in Africa Portion and western some areas, number of the infected are relatively fewer.
It goes through formally to be used for clinic AIDS medicine from Zidovudine (zidovudine, AZT) conduct in 1987 is first Since object, existing more than 30 anti-AIDS drugs are applied to clinic at present, including a kind of entry inhibitors, 15 kinds of reverse transcriptions Enzyme (RT) inhibitor, 10 kinds of protease (PR) inhibitor, a kind of integrase inhibitor, a kind of fusion inhibitor and 5 kinds of complexing agents Deng.But prolonged application list target drug easily causes crossing drug resistant and serious toxic side effect.Chinese descendant in America's science in 1996 Family He great Yi is proposed " cocktail therapy " i.e. highly effective antiretroviral therapy (HAART), reduces what single drug was also easy to produce Drug resistance inhibits the duplication of virus to the maximum extent, greatly improves the quality of life of patient.However this therapy dosage Greatly, toxic side effect is strong, drug interaction is complicated, patient compliance is poor, and the severe situation faced above forces people constantly to visit Rope finds novel inverase.
Multiple target point drug designs (multitarget-directed ligands, MT-DLs) due to its uniform pharmacokinetics Property reduces drug interaction and improves the advantages such as therapeutic effect, it has also become the Disciplinary Frontiers of current medical design, for many The treatment of difficult diseases brings new hope.Reverse transcriptase inhibitor and protease inhibitors are the head in " cocktail therapy " Drug is selected, therefore the research of the bis- target spot inhibitor of HIV PR/RT has great importance.
HIV-1 reverse transcriptase (reverse transcriptase, RT) plays very in the reproduction process of inhibition of HIV Important role is catalyzed RNA reverse transcription synthetic dsdna, and double-stranded DNA enters in nucleus under the action of integrase, and turns Viral RNA is recorded, a part of host cell is become.Therefore, RT is the important target spot of inverase, while being also HIV earliest Study target spot.
HIV-1 protease is the species specificity aspartyl protease encoded by HIV gene, and active form is two The homodimer of identical peptide chain composition, every peptide chain are made of 99 amino acid residues, active site be located at two peptide chains it Between.HIV-1 protease inhibitors is made infected thin by preventing shearing of the protease to viral gag gag-pol gene Born of the same parents can only generate it is immature, do not have infective virus.Therefore, HIV-1PR is the important target spot for researching and developing inverase.
So far, there is not yet being able to suppress HIV-1 proteinase activity and reverse transcriptase activity and being applied to treatment Chinese mugwort Grow the relevant report of the derivative of disease.
Summary of the invention
The purpose of the present invention is to provide a kind of active nucleic acid bases for being able to suppress HIV-1 protease and reverse transcriptase Derivative or its pharmaceutically acceptable salt.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of nucleic acid base compound or its pharmaceutically acceptable salts, have structure shown in Formulas I:
In Formulas I, R1For amino, methoxyl group, nitro, aminomethyl or methylol；
X is-CH2,-O- ,-S- or-NH-；
Y is
Figure BDA0001668490030000021
When X is-CH2When, R Ra, Rb, Rc or Rd；
When X is-O- ,-S- or-NH-, R is Rc or Rd；
The Ra isThe Rb isThe Rc is
Figure BDA0001668490030000024
It is described Rd is
Wherein, R2For hydrogen, hydroxyl, amino or halogen；R3For hydrogen, hydroxyl, amino, halogen or methyl；R4For hydrogen or methyl； R5For hydrogen, hydroxyl, amino or phosphate group；
When Z is nitrogen, R6For hydrogen, hydroxyl, amino or phosphate group；When Z is oxygen or sulphur, R6It is not present.
Preferably, the nucleic acid base compound includes 2- (the chloro- 9H- purine -9- base of 6-)-N- ((2S, 3R) -3- hydroxyl - 4- (N- isobutyl group -4- methoxyphenyl sulphonyl amido) -1- phenyl butane -2- base)-acetamide, 2- (8- methyl -9H- purine - 9- yl)-N- [(2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphoxide imine base) -1- phenyl butyl -2- base] - Acetamide, 2- (2,4- dioxo -2H-3,4- dihydro-pyrimidin -1- base)-N- ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- first Phenyl sulfoamido) -1- phenyl butane -2- base)-acetamide, 2- (the fluoro- 2,4- dioxo -2H-3,4- dihydro-pyrimidin-of 5- 1- yl)-N- ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphonyl amido) -1- phenyl butane -2- base)-second Amide, ((2S, 5R) -5- (- 1 base of 1H-2- oxo -4- aminopyrimidine)-tetrahydrofuran -2- base) methyl N-((2S, 3R) -3- hydroxyl Base -4- (N- isobutyl group -4- aminocarbonyl phenyl sulfoamido) -1- phenyl butane -2- base))-carbamate or N- ((2R, 3S) - 3- (2- (the fluoro- 2,4- dioxo -2H-3,4- dihydro-pyrimidin -2H-1- base of 5-) acetamido) -2- hydroxy-4-phenyl butyl-N- Isobutyl group-P- (4- methoxyphenyl) phosphamic acid.
The present invention provides the preparation methods of the nucleic acid base compound described in above-mentioned technical proposal, comprising the following steps:
X is-CH in the compound of the structure shown in the Formulas I2, when R is Ra or Rb, by the change with structure shown in Formula II -1 It closes object and amine derivative carries out condensation reaction under the action of catalyst, obtain the compound with structure shown in Formulas I；
Figure BDA0001668490030000032
In Formula II -1, R is Ra or Rb, and the Ra is
Figure BDA0001668490030000041
The Rb is
Figure BDA0001668490030000042
Wherein, R2For hydrogen, hydroxyl, amino or halogen；R3For hydrogen, hydroxyl, amino, halogen or methyl；R4For hydrogen or methyl；
X is-CH in the compound of the structure shown in the Formulas I2,-O- ,-S- or-NH-, R be when being Rc or Rd, will have Formula II- Compound, carbonic acid trichloromethyl ester and the amine derivative of structure shown in 2 carry out condensation reaction under the action of catalyst, are had The compound of structure shown in Formulas I；
Formula II -2 R-XH,
In Formula II -2, X is-CH2,-O- ,-S- or-NH-；R is Rc or Rd, and the Rc is
Figure BDA0001668490030000043
The Rd is
Figure BDA0001668490030000044
Wherein, R2For hydrogen, hydroxyl, amino or halogen, R3For hydrogen, hydroxyl, amino, halogen or methyl；R4For hydrogen Or methyl；R5For hydrogen, hydroxyl, amino or phosphate group；When Z is nitrogen, R6For hydrogen, hydroxyl, amino or phosphate group；When Z is oxygen Or when sulphur, R6It is not any atom or group；
The amine derivative has structure shown in formula III:
Figure BDA0001668490030000045
In formula III, R1For amino, methoxyl group, nitro, aminomethyl or methylol；Y is
Figure BDA0001668490030000051
Figure BDA0001668490030000057
Preferably, the compound and amine derivative with structure shown in Formula II -1 is in carbodiimide hydrochloride, 1- Condensation reaction is carried out under the catalytic action of hydroxybenzotriazole and 4-dimethylaminopyridine；
The compound and carbonic acid trichloromethyl ester and amine derivative with structure shown in Formula II -2 is in N, N- diisopropyl Condensation reaction is carried out under the catalytic action of base ethamine.
Preferably, the preparation method of the compound with structure shown in Formula II -1, comprising the following steps:
Substituted pyrimidines base or substituted purin base and bromoacetic acid or bromoacetate are subjected to substitution reaction, replaced Product；
The substitution product is hydrolyzed, the compound with structure shown in Formula II -1 is obtained；
The substituted pyrimidines base has structure shown in formula a:
Figure BDA0001668490030000053
The substituted purin base has structure shown in formula b:
Figure BDA0001668490030000054
Preferably, when in the amine derivative Y beWhen, the amine derivative has structure shown in formula III -1:
Figure BDA0001668490030000056
The preparation method of the amine derivative with structure shown in formula III -1, comprising the following steps:
[(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] is carried out nucleophilic with isobutyl amine to take Generation reaction, obtains (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate；
The R of (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate by described in and structure shown in formula d1 Substituted phenylsulfonyl chloride carries out nucleophilic substitution, obtains the intermediate of structure shown in formula c；
By the intermediate Deprotection of structure shown in the formula c, the amine derivative with structure shown in formula III -1 is obtained；
Figure BDA0001668490030000061
Preferably, when in the amine derivative Y beWhen, the amine derivative has structure shown in formula III -2:
Figure BDA0001668490030000063
The preparation method of the amine derivative with structure shown in formula III -2, comprising the following steps:
[(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] is carried out nucleophilic with isobutyl amine to take Generation reaction, obtains (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate；
The R of (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate by described in and structure shown in formula d1 Substituted phenylsulfonyl chloride carries out nucleophilic substitution, obtains the intermediate of structure shown in formula c；
The intermediate of structure shown in the formula c and Sodium azide are subjected to imidization, obtain the centre of structure shown in formula e Body；
By the intermediate Deprotection of structure shown in the formula e, the amine derivative with structure shown in formula III -2 is obtained.
Figure BDA0001668490030000064
Preferably, when in the amine derivative Y beWhen, the amine derivative has structure shown in formula III -3:
Figure BDA0001668490030000072
The preparation method of the amine derivative with structure shown in formula III -3, comprising the following steps:
[(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] is carried out nucleophilic with isobutyl amine to take Generation reaction, obtains (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate；
By 4- substituted-phenyl phosphonyl dichloride, benzyl alcohol and (1S, the 2R) -1- benzyl -2- hydroxyl with structure shown in formula f Base -3- (isobutyl amine) t-butyl carbamate carries out Arbuzov and reacts to obtain the intermediate of structure shown in formula g；
By the intermediate and H of structure shown in the formula g2Reduction reaction is carried out, the intermediate of structure shown in formula h is obtained；
By the intermediate Deprotection of structure shown in the formula h, the amine derivative with structure shown in formula III -3 is obtained.
Figure BDA0001668490030000073
The present invention provides nucleic acid base compounds described in above-mentioned technical proposal or its pharmaceutically acceptable salt to prepare Application in hiv inhibitor, the hiv inhibitor is using hiv protease and reverse transcriptase as target spot.
Preferably, the dosage of the nucleic acid base compound or its pharmaceutically acceptable salt in hiv inhibitor is 0.01~100nM.
The present invention provides a kind of nucleic acid base compound or its pharmaceutically acceptable salt, the compound or its pharmaceutically Acceptable salt has the apparent activity for inhibiting hiv protease and/or reverse transcriptase；Toxicity research shows it with good Druggability shows that such compound has a good application prospect as anti-AIDS drug.Experimental data according to the embodiment It is found that the compound of the present invention has inhibitory activity (table 1) to HIV-1 protease and HIV-1 reverse transcriptase, and all have Lower cytotoxicity (table 2).Nucleic acid base compound of the invention or its pharmaceutically acceptable salt are expected to become while pressing down Double target spot inhibitor of hiv protease and reverse transcriptase processed.
Detailed description of the invention
The compound of Fig. 1 Formulas I structure；
The synthetic route chart of Fig. 2 compound 1；
The synthetic route chart of Fig. 3 compound 2；
The synthetic route chart of Fig. 4 compound 3；
The synthetic route chart of Fig. 5 compound 4；
The synthetic route chart of Fig. 6 compound 5；
The synthetic route chart of Fig. 7 compound 6.
Specific embodiment
The present invention provides a kind of nucleic acid base compound or its pharmaceutically acceptable salts, have structure shown in Formulas I:
Figure BDA0001668490030000081
In Formulas I, R1For amino, methoxyl group, nitro, aminomethyl or methylol；
X is-CH2,-O- ,-S- or-NH-；
Y is
Figure BDA0001668490030000082
When X is-CH2When, R Ra, Rb, Rc or Rd；
When X is-O- ,-S- or-NH-, R is Rc or Rd；
The Ra is
Figure BDA0001668490030000083
The Rb is
Figure BDA0001668490030000084
The Rc is
Figure BDA0001668490030000085
It is described Rd is
Wherein, R2For hydrogen, hydroxyl, amino or halogen；R3For hydrogen, hydroxyl, amino, halogen or methyl；R4For hydrogen or methyl； R5For hydrogen, hydroxyl, amino or phosphate group；
When Z is nitrogen, R6For hydrogen, hydroxyl, amino or phosphate group；When Z is oxygen or sulphur, R6It is not present.
In the present invention, the nucleic acid base compound preferably include 2- (the chloro- 9H- purine -9- base of 6-)-N- ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphonyl amido) -1- phenyl butane -2- base)-acetamide, 2- (8- first Base -9H- purine -9- base)-N- [(2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphoxide imine base) -1- phenyl Butyl -2- base]-acetamide, 2- (2,4- dioxo -2H-3,4- dihydro-pyrimidin -1- base)-N- ((2S, 3R) -3- hydroxyl -4- (N- Isobutyl group -4- methoxyphenyl sulphonyl amido) -1- phenyl butane -2- base)-acetamide, 2- (the fluoro- 2,4- dioxo -2H-3 of 5-, 4- dihydro-pyrimidin -1- base)-N- ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphonyl amido) -1- phenyl fourth Alkane -2- base)-acetamide, ((2S, 5R) -5- (- 1 base of 1H-2- oxo -4- aminopyrimidine)-tetrahydrofuran -2- base) methyl N - ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- aminocarbonyl phenyl sulfoamido) -1- phenyl butane -2- base))-carbamate Or N- ((2R, 3S) -3- (2- (the fluoro- 2,4- dioxo -2H-3,4- dihydro-pyrimidin -2H-1- base of 5-) acetamido) -2- hydroxyl - 4- phenyl butyl-N- isobutyl group-P- (4- methoxyphenyl) phosphamic acid.
The present invention provides the preparation methods of the nucleic acid base compound described in above-mentioned technical proposal, comprising the following steps:
X is-CH in the compound of the structure shown in the Formulas I2, when R is Ra or Rb, by the change with structure shown in Formula II -1 It closes object and amine derivative carries out condensation reaction under the action of catalyst, obtain the compound with structure shown in Formulas I；
In Formula II -1, R is Ra or Rb, and the Ra is
Figure BDA0001668490030000101
The Rb is
Figure BDA0001668490030000102
Wherein, R2For hydrogen, hydroxyl, amino or halogen；R3For hydrogen, hydroxyl, amino, halogen or methyl；R4For hydrogen or methyl；
X is-CH in the compound of the structure shown in the Formulas I2,-O- ,-S- or-NH-, R be when being Rc or Rd, will have Formula II- Compound, carbonic acid trichloromethyl ester and the amine derivative of structure shown in 2 carry out condensation reaction under the action of catalyst, are had The compound of structure shown in Formulas I；
Formula II -2 R-XH,
In Formula II -2, X is-CH2,-O- ,-S- or-NH-；R is Rc or Rd, and the Rc is
Figure BDA0001668490030000103
The Rd is
Figure BDA0001668490030000104
Wherein, R2For hydrogen, hydroxyl, amino or halogen, R3For hydrogen, hydroxyl, amino, halogen or methyl；R4For Hydrogen or methyl；R5For hydrogen, hydroxyl, amino or phosphate group；When Z is nitrogen, R6For hydrogen, hydroxyl, amino or phosphate group；When Z is When oxygen or sulphur, R6It is not any atom or group；
The amine derivative has structure shown in formula III:
Figure BDA0001668490030000105
In formula III, R1For amino, methoxyl group, nitro, aminomethyl or methylol；Y is
Figure BDA0001668490030000111
In the present invention, the compound and amine derivative with structure shown in Formula II -1 is preferably in carbodiimides salt Condensation reaction is carried out under the catalytic action of hydrochlorate, I-hydroxybenzotriazole and 4-dimethylaminopyridine, is obtained with knot shown in Formulas I The compound of structure, reaction process are as follows:
Figure BDA0001668490030000113
In the present invention, the compound with structure shown in Formula II -1, amine derivative, carbodiimide hydrochloride, The molar ratio of I-hydroxybenzotriazole and 4-dimethylaminopyridine is preferably 1:(1.0~1.1): (1.4~1.6): (1.0~ 1.2): (0.19~0.21), more preferably 1:1.05:1.5:1.1:0.2.In the present invention, the carbodiimide hydrochloride Preferably 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride.In the present invention, the condensation reaction preferably exists It is carried out under the conditions of organic solvent is existing；The present invention does not have the type of the organic solvent special restriction, using ability The organic solvent that condensation reaction can be made to go on smoothly known to field technique personnel, it is specific such as dimethylformamide (DMF). In the present invention, the condensation reaction preferably includes following steps:
Compound, amine derivative and DMF with structure shown in Formula II -1 are mixed, mixed under condition of ice bath to gained Carbodiimide hydrochloride and I-hydroxybenzotriazole are added in solution, carries out first 0.5~1.5h of condensation reaction at room temperature； Then 4-dimethylaminopyridine is added in resulting material, carries out second 1.5~2.5h of condensation reaction at room temperature.
After completing the condensation reaction, the present invention preferably post-processes gained condensation material, obtains with shown in Formulas I The compound of structure.In the present invention, the post-processing preferably includes following steps:
It carries out gained condensation material that removal DMF is concentrated under reduced pressure, residue be mixed with water, using ethyl acetate to gained Mixture is extracted, and gained organic phase is dry with anhydrous sodium sulfate, and the organic phase after drying is concentrated to give crude product, The crude product is prepared thin-layer chromatography through silica gel to isolate and purify, obtains the compound with structure shown in Formulas I.
In the present invention, the preparation method of the compound with structure shown in Formula II -1, preferably includes following steps:
Substituted pyrimidines base or substituted purin base and bromoacetic acid or bromoacetate are subjected to substitution reaction, replaced Product；
The substitution product is hydrolyzed, the compound with structure shown in Formula II -1 is obtained；
The substituted pyrimidines base has structure shown in formula a:
Figure BDA0001668490030000121
The substituted purin base has structure shown in formula b:
Substituted pyrimidines base or substituted purin base and bromoacetic acid or bromoacetate preferably replace anti-by the present invention It answers, obtains substitution product.In the present invention, the substituted pyrimidines base with structure shown in formula a and there is structure shown in formula b Substituted purin base be preferably commercial product.In the present invention, the substituted pyrimidines base or substituted purin base and bromine second The molar ratio of acid or bromoacetate preferably stands alone as 10:(10~12), more preferably 10:11.In the present invention, the substitution Reaction preferably existing for the catalyst and organic solvent under the conditions of carry out；Kind of the present invention for the catalyst and organic solvent Class does not have special restriction, using the catalyst well known to those skilled in the art that substitution reaction can be made to go on smoothly and organic Solvent, it is specific if catalyst is sodium hydride, organic solvent DMF.
In the present invention, the substitution reaction preferably includes following steps:
Substituted pyrimidines base or substituted purin base are mixed with DMF, under condition of ice bath, sodium hydride is added drop-wise to gained In mixture, displacement 0.5~1.5h of reaction is carried out at room temperature, bromoacetic acid or bromoacetate are added drop-wise to gained and replace reactant In material, 0.5~1.5h of substitution reaction is carried out at room temperature.
After completing the substitution reaction, gained is preferably replaced material to post-process by the present invention, obtains substitution product.? In the present invention, the post-processing preferably includes following steps:
Replace material to mix with water gained, extracted with ethyl acetate, gained organic phase is done with anhydrous sodium sulfate It is dry, the organic phase after drying is concentrated to give crude product, the crude product is prepared into thin-layer chromatography through silica gel and is isolated and purified, Obtain substitution product.
In the present invention, the structural formula of the substitution product is preferably
Figure BDA0001668490030000131
After obtaining substitution product, the present invention preferably hydrolyzes the substitution product, obtains having structure shown in Formula II -1 Compound.In the present invention, the hydrolysis preferably existing for the sodium hydroxide solution under the conditions of carry out；The sodium hydroxide solution Concentration be preferably 3mol/L；The molar ratio of the substitution product and sodium hydroxide is preferably 1:3.In the present invention, described The temperature of hydrolysis is preferably 25~35 DEG C, specifically can be room temperature；The time of the hydrolysis is preferably 0.5~ 1.5h, more preferably 1h.
After completing the hydrolysis, the present invention preferably post-processes gained hydrolysis material, obtains with Formula II -1 The compound of shown structure.In the present invention, the post-processing preferably includes following steps:
Using hydrochloric acid solution by gained hydrolysis material pH value be adjusted to 3.5~4.5, under condition of ice bath stir 25~ Solid is precipitated in 35min, filters, and filter cake is washed with water, dries, and obtains the compound with structure shown in Formula II -1.
In the present invention, when in the amine derivative Y be
Figure BDA0001668490030000132
When, the amine derivative has knot shown in formula III -1 Structure:
Figure BDA0001668490030000133
The preparation method of the amine derivative with structure shown in formula III -1, preferably includes following steps:
[(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] is carried out nucleophilic with isobutyl amine to take Generation reaction, obtains (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate；
The R of (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate by described in and structure shown in formula d1 Substituted phenylsulfonyl chloride carries out nucleophilic substitution, obtains the intermediate of structure shown in formula c；
By the intermediate Deprotection of structure shown in the formula c, the amine derivative with structure shown in formula III -1 is obtained；
Figure BDA0001668490030000141
The present invention is preferably by [(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] and isobutyl amine Nucleophilic substitution is carried out, (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate is obtained.In the present invention In, 3 [(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formates] are preferred with the molar ratio of isobutyl amine For (73~78): (187~192), more preferably (75~76): (189~190), most preferably 76:189.In the present invention, 3 [(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formates] are preferably commercial product, specifically may be used To derive from lark prestige Science and Technology Ltd..In the present invention, the nucleophilic displacement of fluorine preferably existing for the organic solvent under the conditions of It carries out, the organic solvent is preferably acetonitrile.In the present invention, the temperature of the nucleophilic substitution is preferably 75~85 DEG C, More preferably 80 DEG C；The time of reaction is preferably 4.5~5.5h, more preferably 5h.After completing the nucleophilic substitution, this hair It is bright that preferably gained nucleophilic displacement of fluorine material is successively cooled down, is concentrated and recrystallized, obtain (1S, 2R) -1- benzyl -2- hydroxyl - 3- (isobutyl amine) t-butyl carbamate.In the present invention, recrystallization reagent used by the recrystallization is preferably acetic acid second Ester and n-hexane；The volume ratio of ethyl acetate and n-hexane is preferably 1:(8~10 in the recrystallization reagent), more preferably 1: 9。
After obtaining (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate, the present invention preferably will be described The R of (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate and structure shown in formula d1Substituted phenylsulfonyl chloride into Row nucleophilic substitution obtains the intermediate of structure shown in formula c.In the present invention, described (1S, the 2R) -1- benzyl -2- hydroxyl - 3- (isobutyl amine) t-butyl carbamate and N, N- diisopropylethylamine, 4-dimethylaminopyridine and R1Substituted phenylsulfonyl chloride rubs That ratio preferably 14~15:16~17:1~2:16~17, more preferably 14.86mmol:16.34mmol:1.49mmol: 16.34mmol.In the present invention, the nucleophilic substitution preferably existing for the organic solvent and catalyst under the conditions of carry out, The organic solvent is preferably tetrahydrofuran and THF, and the catalyst is preferably n,N-diisopropylethylamine and 4- dimethylamino Pyridine.The present invention is preferably first mixed by (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate and tetrahydrofuran It closes, n,N-diisopropylethylamine, 4-dimethylaminopyridine, R is then added under condition of ice bath1Substituted phenylsulfonyl chloride and THF, into Row nucleophilic substitution.In the present invention, the temperature of the nucleophilic substitution is preferably 0 DEG C of ice bath, and the nucleophilic displacement of fluorine is anti- The time answered preferably 0.2~1h, more preferably 0.5h.The present invention preferably passes through TLC and detects the extent of reaction.The nucleophilic to be done After substitution reaction, gained nucleophilic displacement of fluorine material is preferably concentrated under reduced pressure and removes THF by the present invention, then adopts and is extracted with ethyl acetate, Extraction gained organic phase is concentrated, by concentration gained crude product through flash column purification, obtains the intermediate of structure shown in formula c. In the present invention, eluant, eluent used by the flash column purification is preferably ethyl acetate and n-hexane, second in the eluant, eluent The volume ratio of acetoacetic ester and n-hexane is preferably 1:5.
After obtaining the intermediate of structure shown in formula c, the intermediate of structure shown in the formula c is preferably deprotected by the present invention Base obtains the amine derivative with structure shown in formula III -1.In the present invention, the Deprotection preferably in organic solvent and It is carried out under the conditions of catalyst is existing, the organic solvent is preferably CH2Cl2, the catalyst is preferably trifluoroacetic acid.At this In invention, the amount and CH of the substance of the intermediate of structure shown in the formula c2Cl2Volume ratio with trifluoroacetic acid is preferably 8~ 10mmol:9~11mL:9~11mL, more preferably 9.87mmol:10mL:10mL.In the present invention, the deprotection reaction Temperature be preferably 25~35 DEG C, be specifically as follows room temperature；The time of the deprotection reaction is preferably 2~4h, more preferably For 3h.After completing the deprotection reaction, then it is super that saturated sodium bicarbonate solution is added in the preferably first concentration of reaction solution of the present invention Solid is precipitated in sound, stirring, filters, and will filter products therefrom and carries out column chromatography for separation, obtains having structure shown in formula III -1 Amine derivative.In the present invention, eluant, eluent used by the column chromatography for separation is preferably CH2Cl2And MeOH, the eluant, eluent Middle CH2Cl2Volume ratio with MeOH is preferably 10:1.
In the present invention, when in the amine derivative Y beWhen, the amine derivative has knot shown in formula III -2 Structure:
Figure BDA0001668490030000161
The preparation method of the amine derivative with structure shown in formula III -2, preferably includes following steps:
3 [(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formates] are carried out nucleophilic with isobutyl amine to take Generation reaction, obtains (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate；
The R of (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate by described in and structure shown in formula d1 Substituted phenylsulfonyl chloride carries out nucleophilic substitution, obtains the intermediate of structure shown in formula c；
The intermediate of structure shown in the formula c and Sodium azide are subjected to imidization, obtain the centre of structure shown in formula e Body；
By the intermediate Deprotection of structure shown in the formula e, the amine derivative with structure shown in formula III -2 is obtained；
Figure BDA0001668490030000162
In the present invention, the specific preparation method of the intermediate of structure shown in the formula c is same as mentioned above, herein not It repeats again.
After obtaining the intermediate of structure shown in formula c, the present invention is preferably by the intermediate and Sodium azide of structure shown in the formula c Imidization is carried out, the intermediate of structure shown in formula e is obtained.In the present invention, the intermediate of structure shown in the formula c and folded The molar ratio of nitrogen sodium is preferably 6~7:6~7, more preferably 6.30:6.90.In the present invention, the imidization preferably exists It is carried out under the conditions of organic solvent and catalyst are existing, the organic solvent is preferably anhydrous chloroform, and the catalyst is excellent It is selected as the concentrated sulfuric acid.In the imidization, the present invention is preferably first by the intermediate of structure shown in formula c and anhydrous chloroform Mixing, Sodium azide is then added under protection of argon gas, the concentrated sulfuric acid then is added dropwise under the conditions of 0 DEG C of ice bath.The present invention preferably exists The process that the concentrated sulfuric acid is added dropwise is completed in 5min.In the present invention, the temperature of the imidization is preferably 40~50 DEG C, more excellent It is selected as 45 DEG C；The time of the imidization is preferably 10~12h.Present invention preferably employs TIC to monitor reaction process.To complete After the imidization, present invention preferably uses chloroforms to extract gained reactant material, then uses saturated common salt water washing Organic phase is concentrated in organic phase after drying, is purified by silica gel column chromatography to enriched product, obtains the intermediate of structure shown in formula e. In the present invention, eluant, eluent used by the silica gel column chromatography purifies is preferably ethyl acetate and methanol, in the eluant, eluent The volume ratio of ethyl acetate and methanol is preferably 30:1.
After obtaining the intermediate of structure shown in formula e, the present invention obtains the intermediate Deprotection of structure shown in the formula e To the amine derivative with structure shown in formula III -2.The intermediate of structure shown in formula e is carried out the specific of Deprotection by the present invention Method is identical as the above-mentioned method of intermediate Deprotection by structure shown in formula c, and details are not described herein.
In the present invention, when in the amine derivative Y be
Figure BDA0001668490030000171
When, the amine derivative has shown in formula III -3 Structure:
Figure BDA0001668490030000172
The preparation method of the amine derivative with structure shown in formula III -3, preferably includes following steps:
[(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] is carried out nucleophilic with isobutyl amine to take Generation reaction, obtains (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate；
By 4- substituted-phenyl phosphonyl dichloride, benzyl alcohol and (1S, the 2R) -1- benzyl -2- hydroxyl with structure shown in formula f Base -3- (isobutyl amine) t-butyl carbamate carries out Arbuzov reaction, obtains the intermediate of structure shown in formula g；
By the intermediate and H of structure shown in the formula g2Reduction reaction is carried out, the intermediate of structure shown in formula h is obtained；
By the intermediate Deprotection of structure shown in the formula h, the amine derivative with structure shown in formula III -3 is obtained；
Figure BDA0001668490030000181
The present invention carries out [(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] and isobutyl amine Nucleophilic substitution, obtain the specific method of (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate with it is upper It is identical to state content, details are not described herein.
After obtaining (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate, the present invention will preferably have 4- substituted-phenyl phosphonyl dichloride, benzyl alcohol and (1S, the 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) of structure shown in formula f T-butyl carbamate carries out Arbuzov reaction, obtains the intermediate of structure shown in formula g.In the present invention, described that there is formula f The 4- substituted-phenyl phosphonyl dichloride and (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate of shown structure The amount of substance and the volume ratio of benzyl alcohol be preferably 4~5mol:3~4mL:3~4mol, more preferably 4.16:3.78: 3.78.In the present invention, Arbuzov reaction preferably existing for the organic solvent and catalyst under the conditions of carry out；It is described to have Solvent is preferably benzene, and the catalyst is selected as n,N-diisopropylethylamine (DIEA) and 1-H- tetrazole.In the present invention, institute It states Arbuzov reaction and preferably includes following steps:
4- substituted-phenyl phosphonyl dichloride, 1-H- tetrazole with structure shown in formula f are dissolved in benzene, under condition of ice bath to Successively be added dropwise benzyl alcohol, n,N-diisopropylethylamine (DIEA) in gained mixed solution, carry out the first ice bath reaction 25~ 35min；After completing the reaction of the first ice bath, first 2~3h of room temperature reaction is carried out at room temperature；After completing the first room temperature reaction, (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) carbamic acid uncle is sequentially added under condition of ice bath into gained reactant material Butyl ester and DIEA carry out the second ice bath and react 25~35min；After completing the reaction of the second ice bath, the second room temperature is carried out at room temperature React 3~4h.
After completing Arbuzov reaction, present invention preferably employs reversed-phase column chromatography to gained Arbuzov reaction mass into Row isolates and purifies, and obtains the intermediate of structure shown in formula g.In the present invention, the mobile phase of the reversed-phase column chromatography is preferably second The mixed liquor of alcohol and water, the volume ratio of ethyl alcohol and water is preferably 5:1 in the mobile phase.
After obtaining the intermediate of structure shown in formula g, the present invention is preferably by the intermediate and H of structure shown in the formula g2It carries out Reduction reaction obtains the intermediate of structure shown in formula h.In the present invention, the reduction reaction is preferably in organic solvent and catalysis It is carried out under the conditions of agent is existing, the organic solvent is preferably tetrahydrofuran, and the catalyst is preferably Pd/C.In the reduction In reaction, the present invention is preferably under protection of argon gas by the intermediate of structure shown in formula g and tetrahydrofuran, Pd/C, K2CO3It is mixed with water It closes, then replaces the argon gas using hydrogen, carry out the reduction reaction at room temperature.In the present invention, the reduction reaction Time be preferably 3~5h, more preferably 4h, the temperature of the reduction reaction is preferably 25~35 DEG C, specifically can be room Temperature.After completing the reduction reaction, present invention preferably uses miillpore filters to be filtered reaction solution obtained by reduction reaction, in ice Under the conditions of bath, the pH value of gained filtrate is adjusted to 5.0 using hydrochloric acid, is then extracted using ethyl acetate, with anhydrous sulphur The dry organic phase of sour sodium, is concentrated organic phase, obtains the intermediate of structure shown in formula h.
After obtaining the intermediate of structure shown in formula h, the intermediate of structure shown in the formula h is preferably deprotected by the present invention Base obtains the amine derivative with structure shown in formula III -3.The intermediate of structure shown in formula h is carried out Deprotection by the present invention Specific method it is identical as the above-mentioned method of intermediate Deprotection by structure shown in formula c, details are not described herein.
In the present invention, the compound and carbonic acid trichloromethyl ester and amine derivative with structure shown in Formula II -2 are excellent It is selected under the catalytic action of n,N-diisopropylethylamine and carries out condensation reaction, obtain the compound with structure shown in Formulas I, react Process is as follows:
Figure BDA0001668490030000191
In the present invention, compound, carbonic acid trichloromethyl ester, amine derivative and the N with structure shown in Formula II -2, The molar ratio of N- diisopropylethylamine is preferably 0.5:(0.45~0.55): (0.5~0.6): (0.5~0.6), more preferably 0.5:0.5:0.55:0.55.In the present invention, the condensation reaction preferably existing for the organic solvent under the conditions of carry out；It is described Organic solvent is preferably anhydrous organic solvent.The present invention does not have the type of the organic solvent special restriction, using this The organic solvent that condensation reaction can be made to go on smoothly known to the technical staff of field, it is specific such as methylene chloride and/or four Hydrogen furans.In the present invention, the condensation reaction preferably includes following steps:
Carbonic acid trichloromethyl ester is mixed with methylene chloride under the conditions of ice bath, nitrogen protection, obtains the first mixed solution； Compound with structure shown in Formula II -2, n,N-diisopropylethylamine are mixed with tetrahydrofuran, the second mixed solution is obtained； Second mixed solution is added drop-wise in first mixed solution under condition of ice bath, it is anti-to carry out the first condensation at room temperature 25~35min is answered, the first condensation material is obtained；
Amine derivative is mixed with methylene chloride, n,N-diisopropylethylamine is added drop-wise in gained mixed solution, in room Temperature is lower to carry out second 4~6min of condensation reaction, obtains the second condensation material；
First condensation material is mixed with the second condensation material, carries out 0.5~1.5h of condensation reaction at room temperature.
After completing the condensation reaction, the present invention preferably post-processes gained condensation material, obtains with shown in Formulas I The compound of structure.In the present invention, the post-processing preferably includes following steps:
Gained condensation material is mixed with methylene chloride, is washed with saturated ammonium chloride solution, gained organic phase is used Anhydrous sodium sulfate is dry, and the organic phase after drying is concentrated to give crude product, the crude product is prepared thin-layer chromatography through silica gel It is isolated and purified, obtains the compound with structure shown in Formulas I.In the present invention, the method for the thin-layer chromatography with it is above-mentioned Scheme is identical, and details are not described herein.
In the present invention, the compound with structure shown in Formula II -2 is preferably commercially available substituted nucleosides or deoxidation core Glycosides.
In the present invention, the preparation method of the amine derivative is identical as above scheme, and details are not described herein.
The present invention provides nucleic acid base compounds described in above-mentioned technical proposal or its pharmaceutically acceptable salt to prepare Application in hiv inhibitor, the hiv inhibitor is using hiv protease and reverse transcriptase as target spot.
Preferably, the dosage of the nucleic acid base compound or its pharmaceutically acceptable salt in hiv inhibitor is 0.01-100nM。
Nucleic acid base compound provided by the invention or its pharmaceutically acceptable salt are carried out below with reference to embodiment detailed Thin explanation, but they cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
6-chloropurine (0.62g, 4mmol) and Anhydrous potassium carbonate (1.66g, 12mmol) are added in 10mL eggplant-shape bottle, Anhydrous solvent n,N-Dimethylformamide (DMF) 2mL is added, room temperature is vigorously stirred reaction 1h under nitrogen protection；By bromoacetic acid (0.64g, 4.6mmol) is dissolved in anhydrous solvent n,N-Dimethylformamide (DMF) 1mL, is slowly dropped in reaction solution, is added It finishes, continues to be stirred at room temperature reaction overnight.5mL distilled water is added into reaction solution, with diatomite filtering solution, washing is obtained Yellow clarified solution body adjusts pH to 3.0 with 4M HCl, stirs 10min under ice bath, stand, filter under ice bath stirring state, Filter cake is washed with a small amount, dry, obtains target product yellow greenish powder solid i.e. intermediate 1 (0.37g, 43.4%).Intermediate 1 LC-MS (ESI, M+H+)m/z 213.4。
By (S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate (20.0g, 75.94mmol), second Nitrile 80mL and isobutyl amine (19.02mL, 189.46mmol) are added in 200mL eggplant-shape bottle, and 80 DEG C of mixture are stirred 5 hours.Reaction After reaction solution be cooled to room temperature, be concentrated under reduced pressure remove solvent.Crude product obtains after being recrystallized with ethyl acetate/n-hexane (1:9) White object product (21.2g, 83%), the white object product are intermediate b1, LC-MS (ESI, M+H+)m/z 337.2。
Intermediate b1 (5.0g, 14.86mmol), tetrahydrofuran (THF) (40mL) are added in 250mL eggplant-shape bottle, ice bath Under be slowly added to n,N-diisopropylethylamine (DIEA) (3.68mL, 16.34mmol) and 4-dimethylaminopyridine (DMAP) (0.18g, 1.49mmol), is then added 4- Methoxybenzenesulfonyl chloride (3.38g, 16.34mmol) and the mixing of THF (10mL) is molten Liquid.It is stirred 0.5 hour under ice bath and moves to room temperature.TLC detection is concentrated under reduced pressure after completion of the reaction removes THF, ethyl acetate extraction (30 × 3mL), organic phase concentration.Crude product through flash column purification ethyl acetate-hexane (1:5) target product white solid be in LC-MS (ESI, M+H+) m/z 507.0 of mesosome b2 (6.14g, 82%), intermediate b2.
Intermediate b2 (5.0g, 9.87mmol) is added in 100mL eggplant-shape bottle, CH is added at room temperature2Cl2(10mL) and three Fluoroacetic acid (10mL).It finishes, reacts at room temperature 3h.Concentration of reaction solution after completion of the reaction, and 200mL saturated sodium bicarbonate solution is added Ultrasound, stirring, there is solid precipitation, filter to obtain crude product, and column chromatographs CH2Cl2- MeOH (10:1) target product white solid be in Mesosome B1a (2.71g, 68%).LC-MS (ESI, M+H+) m/z 407.3 of intermediate B 1a.
Intermediate 1 (0.21g, 1mmol), intermediate B 1a (0.43g, 1.05mmol) are added in 10mL eggplant-shape bottle, are added 2mL anhydrous DMF, is placed in ice bath, is slowly added to 1- ethyl-(3- dimethylaminopropyl) carbodiimide hydrochloride hydrochloride (EDCHCl) (0.29g, 1.5mmol), I-hydroxybenzotriazole (HOBt) (0.15g, 1.1mmol), finishes and is transferred to room temperature It is stirred to react 1h, is added DMAP (0.024g, 0.2mmol), continues to be stirred to react 2h.DMF is concentrated under reduced pressure after completion of the reaction, remains 6mL water is added in object, is extracted with ethyl acetate (6 × 3mL), and organic phase is dry with anhydrous sodium sulfate, is concentrated to give crude product.Crude product is through silicon Glue prepare thin-layer chromatography isolated and purified (solvent be ethyl acetate and methanol, by volume, ethyl acetate: methanol=8: 1) white powder solid (0.54g, 89.3%) to get to compound 1:2- (the chloro- 9H- purine -9- base of 6-)-N- ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphonyl amido) -1- phenyl butane -2- base)-acetamide.
The synthetic route of compound 1 is shown in Fig. 2, and the analysis result of compound 1 is as follows: LC-MS (ESI, M+H+)m/z 601.7。1H NMR(400MHz,CDCl3)δ7.81(s,1H),7.62(s,1H),7.18-7.12(m,7H),7.01(brs, 2H),4.50(m,2H),4.25-4.23(m,1H),4.06(m.1H),3.90(s,3H),3.37(m,2H),3.04-2.88(m, 4H),1.92(m,1H),0.82(s,6H).13CNMR(101MHz,CDCl3)δ174.2,165.3,162.8,153.6,147.4, 138.1,137.0,130.3,129.5,128.3,127.0,114.3,71.6,57.5,55.7,54.6,47.6,34.5,26.8, 20.0。
Embodiment 2
8- methyl purine (1.34g, 10mmol) is added in 50mL eggplant-shape bottle, 10mL anhydrous DMF is added, is placed in ice bath In, sodium hydride (0.44g, 11mmol) is slowly added portionwise, finishes and is transferred to room temperature and carries out reaction 1h, bromoacetic acid second is slowly added dropwise Ester (1.84g, 11mmol), the reaction was continued 1h.Reaction is terminated, 10mL water is added, is extracted with ethyl acetate (15 × 3mL), it is organic It is mutually dry with anhydrous sodium sulfate, it is concentrated to give yellow oily crude product.Through flash column purification, (eluant, eluent used is ethyl acetate to crude product And hexane, by volume, ethyl acetate: hexane=1:2) obtain colorless needle crystals, that is, intermediate 2 (1.53g, 69.6%).It is intermediate LC-MS (ESI, the M+H of body 2+)m/z 221.4。
NaOH (0.65g, 16.2mmol) is dissolved in 5mL water, is added to containing intermediate 2 (1.19g, 5.4mmol) In 100mL eggplant-shape bottle, be stirred at room temperature reaction 1h, eggplant-shape bottle is placed in ice bath, under stirring with 4M HCl adjust pH to 4.0,30min is stirred under ice bath, a large amount of solids are precipitated, is stood, and is filtered, filter cake is washed with water, and it is dry, obtain target product white powder Last solid, that is, intermediate 3 (0.58g, 56.3%).LC-MS (ESI, the M-H of intermediate 3-)m/z 191.4。
Intermediate b2 (3.19g, 6.30mmol) is added in the dry there-necked flask of 50mL, the anhydrous chloroform of 8mL is added, It stirs evenly, is slowly added to Sodium azide (0.45g, 6.90mmol) under protection of argon gas, reaction flask is placed in ice bath, at 0 DEG C It is lower to be slowly added dropwise with dropping funel into the 1.53mL concentrated sulfuric acid, it is added dropwise in 5min.Then reaction flask is transferred to 45 DEG C of conditions Under be stirred to react overnight.TIC monitors reaction process, and reaction is quenched with water, and chloroform extracts (25 × 3mL), merges organic phase, uses 20mL saturated common salt water washing, dry, organic phase concentration.Crude product is purified by silica gel column chromatography acetate-methanol (30:1) and obtains Target product white solid, that is, intermediate b3 (2.16g, 68%), LC-MS (ESI, M+H+) m/z 506.4 of intermediate b3.
Referring to the synthesis of the intermediate B 1a of embodiment 1, difference, which is only that, replaces intermediate b2 for the synthesis of intermediate B 2a For equimolar intermediate b3, intermediate B 2a, yield 54.3%, LC-MS (ESI, M+H+) m/z of intermediate B 2a are obtained 406.3。
The synthesis of synthesis reference 1 compound 1 of embodiment of compound 2, the difference with the synthesis of compound 1 are only that by Mesosome 1 replaces with equimolar intermediate 3, and B1a is replaced with equimolar B2a, and obtaining compound 2:2-, (8- methyl -9H- is fast Purine -9- base)-N- [(2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphoxide imine base) -1- phenyl butyl -2- Base]-acetamide, yield 68.7%.
The synthetic route of compound 2 is shown in Fig. 3, and the analysis result of compound 2 is as follows: LC-MS (ESI, M+H+)m/z 583.7。1H NMR (600MHz, Acetone) δ 7.84 (s, 1H), 7.73 (d, J=8.8Hz, 2H), 7.63 (s, 1H), 7.26- 7.24 (m, 2H), 7.21-7.18 (m, 3H), 7.03 (d, J=8.8Hz, 2H), 4.04 (td, J=9.2,4.8Hz, 1H), 3.84 (s, 3H), 3.79 (m, 1H), 3.24 (dd, J=15.0,3.3Hz, 1H), 3.01 (ddd, J=23.4,14.4,6.0Hz, 2H), 2.91-2.86 (m, 2H), 2.67 (dd, J=13.8,10.6Hz, 1H), 1.92-1.88 (m, 1H), 0.86 (d, J=6.6Hz, 6H), 0.84 (d, J=6.6Hz, 6H) .13C NMR (151MHz, Acetone) δ 166.8,163.7,157.3,149.5, 145.9,141.3,139.4,132.0,130.2,130.0129.5,129.0,126.9,115.0,72.7,58.5,56.2, 55.1,53.5,46.6,35.4,27.5,20.1,20.0。
Embodiment 3
The synthesis of synthesis reference 2 intermediate 2 of embodiment of intermediate 4, the difference with the synthesis of intermediate 2 are only that 8- Methyl purine replaces with equimolar uracil, obtains 4 white powder solid of intermediate, yield 49.1%, structural formula such as Fig. 3 It is shown.LC-MS (ESI, the M+H of intermediate 4+)m/z 199.3。
The synthesis of synthesis reference 2 intermediate 3 of embodiment of intermediate 5, the difference with the synthesis of intermediate 3 are only that by Mesosome 2 replaces with equimolar intermediate 4, obtains 5 white powder solid of intermediate, yield 61.2%.The LC-MS of intermediate 5 (ESI,M-H?)m/z 169.4。
The synthesis of synthesis reference 1 compound 1 of embodiment of compound 3, the difference with the synthesis of compound 1 are only that by Mesosome 1 replaces with equimolar intermediate 5, obtains compound 3:2- (2,4- dioxo -2H-3,4- dihydro-pyrimidin -1- base)-N- ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphonyl amido) -1- phenyl butane -2- base)-acetamide is received Rate 97.5%.
The synthetic route of compound 3 is shown in Fig. 4, and the analysis result of compound 3 is as follows: LC-MS (ESI, M+H+)m/z 559.71H NMR(500MHz,CDCl3) δ 8.89 (s, 1H), 7.75 (d, J=8.6Hz, 2H), 7.29-7.27 (m, 3H), 7.23-7.22 (m, 2H), 7.01 (d, J=8.6Hz, 2H), 6.70 (d, J=7.8Hz, 1H), 5.68 (d, J=7.8Hz, 1H), 4.26 (d, J=15.4Hz, 2H), 4.17 (d, J=15.7Hz, 1H), 3.99-3.98 (m, 1H), 3.90 (s, 3H), 3.13 (d, J =5.7Hz, 2H), 2.94-2.86 (m, 4H), 1.93-1.84 (m, 1H), 0.90 (d, J=6.0Hz, 6H) .13C NMR (101MHz,CDCl3)δ166.6,163.9,163.1,151.3,144.9,137.9,130.0,129.5,129.4,128.5, 126.5,114.4,102.6,72.3,58.3,55.7,54.1,53.0,50.4,34.7,27.0,20.1,20.0。
Embodiment 4
Anhydrous solvent n,N-Dimethylformamide (DMF) 5mL is added in 25mL eggplant-shape bottle, under stirring slowly 5 FU 5 fluorouracil (1.05g, 8.0mmol, Beijing are coupled Science and Technology Ltd.) is added, stirs evenly and is placed in ice bath, slowly Sodium hydride (0.23g, 9.6mmol) is added portionwise, finishes to be transferred to and reacts 1h at room temperature, be slowly added dropwise into bromoacetate (1.60g, 9.6mmol), the reaction was continued 2h.Filtering, is washed three times, merging filtrate with water (5 × 3mL), and 0.96g hydroxide is added Sodium (24.0mmol) is stirred at room temperature reaction 1h, is placed in ice bath, adjusts pH to 3.0 with 4M HCl, stirs 30min under ice bath, quiet It sets, filters, filter cake is washed with water, and it is dry, obtain target product white powder solid i.e. intermediate 6 (0.58g, 38.7%).It is intermediate LC-MS (ESI, the M-H of body 6-)m/z 187.4。
The synthesis of synthesis reference 1 compound 1 of embodiment of compound 4, the difference with the synthesis of compound 1 are only that by Mesosome 1 replaces with equimolar intermediate 6, obtains compound 4:2- (fluoro- 2,4- dioxo -2H-3, the 4- dihydro-pyrimidin -1- of 5- Base)-N- ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphonyl amido) -1- phenyl butane -2- base)-acetyl Amine, yield 96.0%.
The synthetic route of compound 4 is shown in Fig. 5, and the analysis result of compound 4 is as follows: LC-MS (ESI, M+H+)m/z 577.7。1H NMR(500MHz,CDCl3) δ 10.44 (s, 1H), 8.07 (s, 1H), 7.79 (d, J=7.4Hz, 2H), 7.44- 7.38 (m, 1H), 7.33-7.24 (m, 1H), 7.15-7.14 (m, 2H), 7.06 (d, J=7.4Hz, 1H), 6.98 (d, J= 7.4Hz, 2H), 4.33 (td, J=9.4,4.8Hz, 1H), 4.29-4.20 (m, 2H), 4.16-4.13 (m, 1H), 4.01 (m, 1H),3.85(s,3H),3.29-3.26(m,1H),3.20-3.10(m,2H),2.97-2.96(m,1H),2.93-2.90(m, 2H),1.99-1.92(m,1H),0.90(brs,6H).13C NMR(101MHz,CDCl3)δ166.5,162.9,157.9, 150.0,139.1,138.0,130.0,129.5,129.4,128.4,126.4,114.4,72.3,58.1,55.7,54.1, 52.9,50.1,36.6,26.9,20.1,20.0。
Embodiment 5
Triphosgene (0.15g, 0.50mmol) is weighed in 50mL dry eggplant-shape bottle, is placed in ice bath, in nitrogen protection Under be slowly added to 4mL anhydrous methylene chloride solvent, stirred under ice bath.By Zalcitabine (0.106g, 0.50mmol) It is dissolved in 4mL anhydrous tetrahydro furan solvent with n,N-diisopropylethylamine (0.065g, 0.50mmol).Under condition of ice bath slowly It is added dropwise in the dichloromethane solution for filling triphosgene, finishes, be transferred to room temperature reaction 30min.
The synthesis of intermediate b4 is similar with the synthesis of intermediate b2, and difference, which is only that, replaces with 4- Methoxybenzenesulfonyl chloride Equimolar 4- nitrobenzene sulfonyl chloride；
The synthesis of intermediate B 1b is similar with the synthesis of B1a, and difference, which is only that, replaces with equimolar centre for intermediate b2 Body b4；
Intermediate B 1b (0.23g, 0.55mmol) is dissolved in 4mL anhydrous methylene chloride solvent, N, N- diisopropyl are added dropwise to Base ethamine (0.071g, 0.55mmol) is stirred at room temperature reaction 5min, is then slowly added dropwise under condition of ice bath into three chloromethane of carbonic acid Base ester, Zalcitabine reaction solution in, finish, be transferred to room temperature reaction 1h.Reaction is terminated, is added into reaction solution 10mL methylene chloride is washed (20 × 3mL) with saturated ammonium chloride solution, and organic phase is dry with anhydrous sodium sulfate, is concentrated to give crude product. Crude product through silica gel prepare thin-layer chromatography isolated and purified (solvent be ethyl acetate, hexane and ammonium hydroxide, by volume, acetic acid Ethyl ester: hexane: NH3·H2O=2:3:0.1%) white powder solid, that is, intermediate 7 (0.078g, 23.7%) is obtained.Intermediate 9 LC-MS(ESI,M+H+)m/z 659.6。
Intermediate 7 (0.040g, 0.06mmol) is dissolved in the in the mixed solvent of 1mL methanol and 1mL ethyl acetate, first is added Sour ammonium (0.031g, 0.49mmol), 10%Pd/C (0.040g, humidity 36.68%), heating reflux reaction one hour, mistake while hot Filter reaction solution, methanol washing, used after concentration prepare thin-layer chromatography isolated and purified (solvent for ethyl acetate and methanol, By volume, ethyl acetate: methanol=4:1), it obtains white powder solid (0.035g, 91.4%), i.e. compound 5:((2S, 5R) -5- (- 1 base of 1H-2- oxo -4- aminopyrimidine)-tetrahydrofuran -2- base) ((N- is different by (2S, 3R) -3- hydroxyl -4- for methyl N - Butyl -4- aminocarbonyl phenyl sulfoamido) -1- phenyl butane -2- base))-carbamate.
The synthetic route of compound 5 is shown in Fig. 6, and the analysis result of compound 5 is as follows: LC-MS (ESI, M+H+)m/z 629.7。1HNMR(500MHz,CDCl3) δ 9.02 (s, 1H), 7.65 (d, J=8.6Hz, 2H), 7.29-7.27 (m, 3H), 7.23- 7.22 (m, 2H), 6.61 (d, J=8.6Hz, 2H), 5.48 (d, J=7.8Hz, 1H), 5.23-5.19 (m, 1H), 4.80-4.75 (m, 1H), 4.52-4.47 (m, 2H), 4.17 (d, J=15.7Hz, 1H), 3.99-3.98 (m, 1H), 3.89 (s, 3H), 3.13 (d, J=5.7Hz, 2H), 2.99-2.84 (m, 6H), 1.93-1.84 (m, 2H), 1.71-1.68 (m, 1H), 0.93 (d, J= 6.6Hz, 3H), 0.91 (d, J=6.6Hz, 3H) .13C NMR (101MHz, MeOD) δ 169.6,168.2,164.5,158.9, 147.8,140.0,132.1,130.7,130.5,129.4,129.1,111.6,97.3,95.7,81.2,73.5,70.5, 56.2,54.0,52.4,36.3,30.2,28.1,20.6,20.5.
Embodiment 6
The synthesis of synthesis reference 1 compound 1 of embodiment of compound 6, the difference with the synthesis of compound 1 are only that by Mesosome 1 replaces with equimolar intermediate 6, and intermediate B 1a is replaced with equimolar intermediate B 3a, obtains compound 6:N- ((2R, 3S) -3- (2- (the fluoro- 2,4- dioxo -2H-3,4- dihydro-pyrimidin -2H-1- base of 5-) acetamido) -2- hydroxyl -4- benzene Base butyl-N- isobutyl group-P- (4- methoxyphenyl) phosphamic acid, yield 70.2%.
The synthesis process of intermediate B 3a are as follows:
4- methoxyphenyl phosphonyl dichloride (0.85g, 4.16mmol), 1-H- tetrazole (0.027g, 0.038mmol) is molten In 8mL dry benzene, reaction flask is placed in ice bath, be successively slowly added dropwise under protection of argon gas into benzyl alcohol (0.39mL, 3.78mmol), DIEA (0.72mL, 4.16mmol).Finish, 30min be stirred to react in ice bath, be then transferred at room temperature after Continuous reaction 2.5 hours.Reaction flask is placed in ice bath, be successively slowly added to 4 (1.27g, 3.78mmol), DIEA (0.72mL, 4.16mmol), be stirred to react 30min in ice bath, after be transferred to that the reaction was continued at room temperature 3.5 hours.Filtering is adopted after filtrate concentration (MeOH/H is isolated and purified with reversed-phase column chromatography2O:5/1), white powder i.e. intermediate b5 (0.95g, 42.3%) is obtained, in LC-MS (ESI, M+H+) m/z 597.7 of mesosome b5；
By intermediate b5 (0.10g, 0.17mmol), 1mL tetrahydrofuran, 0.010g 10%Pd/C, K2CO3(0.038g, 0.27mmol) and 1mL water is added in dry hydrogenation bottle, is vigorously stirred under protection of argon gas uniformly, then with hydrogen displacement 3 It is secondary, room temperature catalytic hydrogenation 4 hours under 40psi pressure.Reaction solution filtering with microporous membrane, filtrate are placed in ice bath, use 2M HCl adjusts pH to 5.0, stirs 30min, is extracted with ethyl acetate (5 × 3mL), and organic phase is dry with anhydrous sodium sulfate, dense Contracting, obtains white powder solid i.e. intermediate b6 (0.072g, 83.9%), LC-MS (ESI, the M+H of intermediate b6+)m/z 507.7；
Referring to the synthesis of the intermediate B 1a of embodiment 1, difference, which is only that, replaces intermediate b2 for the synthesis of intermediate B 3a For equimolar intermediate b6, intermediate B 3a, yield 54.3%, LC-MS (ESI, M+H+) m/ of intermediate B 3a are obtained z406.7。
The synthetic route of compound 6 is shown in Fig. 7, and the analysis result of compound 6 is as follows: LC-MS (ESI, M+H+)m/z 577.7。1H NMR(500MHz,CDCl3) δ 10.21 (s, 1H), 8.04 (s, 1H), 7.75 (d, J=7.4Hz, 2H), 7.44- 7.38 (m, 1H), 7.33-7.24 (m, 2H), 7.03-6.99 (m, 2H), 6.98 (d, J=7.4Hz, 2H), 4.28 (td, J= 9.4,5.0Hz,1H),4.28-4.21(m,2H),4.19-4.15(m,1H),4.03(m,1H),3.85(s,3H),3.29-3.26 (m,1H),2.97-2.96(m,1H),2.93-2.90(m,2H),2.63-2.52(m,2H),1.95-1.87(m,1H),0.84 (d, J=6.6Hz, 3H), 0.79 (d, J=6.6Hz, 3H) .13C NMR (101MHz, CDCl3)δ168.2,163.3,157.7, 150.0,139.0,138.2,133.5,133.2,131.1,129.5,128.4,126.4,72.3,58.1,56.1,55.7, 52.9,48.5,36.2,27.1,20.1,20.0。
Embodiment 7
Compound 1~6 prepared by Examples 1 to 6 is dissolved with DMSO, and carries out gradient dilution with distilled water and obtains not Solution with concentration measures above compound to HIV-1 protease inhibiting activity and cell toxicant as sample by the following method Property.
According to document (foundation [J] China of Dong Biao, Zhang Tian, Tao Peizhen high throughput fluorescence substrate HIV-1 Protease models AIDS venereal disease, 2006 (05): 402-405.) method to compound 1~6 carry out HIV-1 protease inhibiting activity test:
Substrate is (Arg-Glu (EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys (DABCYL)- Arg) (AnaSpec), substrate point of contact two sides mark Edans and Dabcyl chromophore respectively.The fluorescence hair color spectrum of Edans with The absorption spectrum of Dabcyl is overlapped, and generates fluorescent quenching by fluorescence resonance energy transfer in distance close enough, is made complete Substrate almost without fluorescence.When fluorogenic substrate is after hiv protease is cut, Edans chromophore is far from Dabcyl group, fluorescence Quenched conditions disappear, and at this moment Edans just generates fluorescence under the exciting light of 340nm at 490nm, after untested compound is added, Then substrate product is reduced when compound is strong to enzyme inhibition activity, and fluorescence intensity reduces, otherwise fluorescence intensity increases.
According to document (foundation [J] China of Dong Biao, Zhang Tian, Tao Peizhen high throughput fluorescence substrate HIV-1 Protease models AIDS venereal disease, 2006 (05): 402-405) method the measurement of HIV-1PR inhibitory activity is carried out to sample with 96 orifice plates, often Substrate (5 μM) and 185 μ L of buffer is added in hole, and 5 μ L sample solution are added, and measurement blank absorbs, 10 μ L HIV-1PR are added, incubate The absorbance for measuring 490nm wavelength after 5min is educated, the inhibiting rate of sample under each concentration is calculated, is calculated with Graphpad software Obtain IC50Value is the positive with DRV (Darunavir) (being purchased from U.S. ARP (American Research Product) company) Control.
Wherein, HIV-1PR is according to (expression, purifying and its inhibitor in-vitro screening side of the .HIV-1 protease such as Wang Yunhua Method establishes CHINA virus March phase .2006 of volume 21 2) in method in expression in escherichia coli and purify, HIV-1PR Use PD-10 column desalination.
With HIV-1 protease inhibitors DRV (Darunavir) be positive control, according to the method described above measure embodiment 1~ 1~6 pair of protease (PR) inhibitory activity of compound and cytotoxicity prepared by 6.The result shows that compound 2, compound 5 and change It closes object 6 and positive control medicine HIV-1 protease inhibitors DRV is better than to the inhibitory activity of HIV-1 protease；Compound 1, chemical combination Object 3 and compound 4 are living to the inhibitory activity of HIV-1 protease and the inhibition of positive control medicine HIV-1 protease inhibitors DRV Property it is close or quite (table 1).
The inhibitory activity of 1 1~6 pair of HIV-1 protease of compound of table
Compound PRIC50(nM)
Compound 1 2.11±0.37
Compound 2 0.94±0.32
Compound 3 2.53±0.42
Compound 4 1.95±0.32
Compound 5 0.46±0.22
Compound 6 0.032±0.019
DRV 1.12±0.59
The test method of cytotoxicity:
Compound Cytotoxicity is measured using kit Cell Counting Kit-8 (CCK-8 kit).With 96 orifice plates Cytotoxicity test is carried out to sample, every hole is added 20,000,293T cell, and 1 μ L sample is added in incubation afterwards for 24 hours, continues to be incubated for For 24 hours, be added 10 μ L CCK-8,2h after with measure absorbance under 450nm, calculate the percentage of each concentration survivaling cell, use CC is calculated in Graphpad software50Value, using DMSO as blank control, with DRV (Darunavir) for positive control.
The cytotoxicity of 2 compound 1~6 of table
Figure BDA0001668490030000281
The result shows that above compound 1~6 all has lower cytotoxicity (table 2).
Embodiment 8
Compound 1~6 is dissolved with DMSO, and use distilled water carry out gradient dilution obtain the solution of various concentration as Sample measures above compound to HIV-1 albumen enzyme inhibition rate by the following method.
Using 293T cell as virus host, measurement sample inhibits pseudotype virus reporter gene uciferase activity.It adopts With envelope glycoprotein pHCMV-G (VSV-G) the cotransfection 293T of pNL-Luc-E- Strain and expression vesicular stomatitis virus Generate VSV-G-HIV.
According to document, [Wang Ping, Chen Huan, Luo Ronghua wait .VSVG/HIV-1NL4-3Luc pseudovirus screens anti-HIV-1 medicines Condition optimizing and apply [J] Chinese Pharmacological Bulletin, 2016,32 (3): 433-438.] method carry out the suppression of HIV-1 protease The test of rate processed:
Envelope glycoprotein pHCMV-G (VSV-G) plasmid of plasmid pNL-Luc-E- and expression vesicular stomatitis virus is total It transfects 293T cell and prepares VSV-G-HIV pseudovirus, a certain concentration protease inhibitor sample is added after transfecting 5h, sets 5% CO2, 37C culture 48 hours.Every hole takes 10 μ l inoculation 293T cell of supernatant, 96 well culture plate later, after culture 48 hours, Uciferase activity in infection cell is measured, the inhibiting rate of each sample is calculated.
With HIV-1 protease inhibitors DRV (Darunavir) for positive control, according to the method described above under 10nM concentration Determine the inhibiting rate of 1~6 pair of hiv protease (PR) of above compound.The result shows that above-mentioned 6 compounds all have it is higher Inhibiting rate, wherein compound 5 and 6 inhibiting rate of compound (table 3) are higher than positive control.As shown in table 3.
The inhibiting rate of 3 1~6 pair of HIV-1 protease of compound of table
Figure BDA0001668490030000282
Figure BDA0001668490030000291
Embodiment 9
Compound 1~6 is dissolved with DMSO, and use distilled water carry out gradient dilution obtain the solution of various concentration as Sample measures above compound to HIV-1 reverse transcriptase inhibiting rate by the following method.
The test method of HIV-1 reverse transcriptase inhibitory activity is as follows:
Primer is 5 '-CAG CAG TACAAATGG CAG TATTC-3 ', is marked in the position T19 with Cyanine 5 (Cy5) Note, with 3 '-TGT CGT CAT GTT TAC CGT CATAAG TAGGTG TTACTAGTC CGATTT CCC CTAGTC CGACCCATG-5 ' is template, is marked in the position T2 with carboxytetramethylrhodamine (TMR), TMR excitation wavelength For 540nm, launch wavelength 580nm, using FRET as donor.The measurement of HIV-1RT inhibitory activity is carried out to sample with 96 orifice plates, 100nM HIV-1RT is mixed with the bis- index object/template composites of 100nM, and kinetics of polymerization reaction is opened by 100 μM of dNTPs are added It is dynamic.HIV-1RT and double index object/templates is added, the absorbance of 580nm wavelength is measured after incubation 5min, calculates each concentration IC is calculated with Graphpad software in the inhibiting rate of lower sample50Value, with EFV (Efavirenz) for positive control.If without spy Different explanation, all tests are carried out in buffer at 20 DEG C.Buffer 4M hydrochloric acid (pH7.5), 10mM KCl and 6mM MgCl configuration.
With HIV-1 reverse transcriptase inhibitor EFV (Efavirenz) for positive control, determined under 10nM concentration above-mentioned The inhibiting rate of 1~6 pair of hiv reverse transcriptase (RT) of compound.The result shows that above-mentioned 6 compounds all have higher inhibiting rate, Wherein compound 5 and 6 inhibiting rate of compound (table 4) are suitable with positive control inhibiting rate.As shown in table 4.
The inhibiting rate of 4 1~6 pair of HIV-1 reverse transcriptase of compound of table
Figure BDA0001668490030000292
As seen from the above embodiment, the present invention provides a kind of nucleic acid base compound or its pharmaceutically acceptable salt, The compound or its pharmaceutically acceptable salt have the apparent activity for inhibiting hiv protease and/or reverse transcriptase；Toxicity is ground Study carefully and shows that it, with good druggability, shows that such compound has a good application prospect as anti-AIDS drug.Root According to the experimental data of embodiment it is found that the compound of the present invention has inhibition to live in HIV-1 protease and HIV-1 reverse transcriptase Property (table 1), and all have lower cytotoxicity (table 2).Nucleic acid base compound of the invention or its is pharmaceutically acceptable Salt be expected to become while inhibiting double target spot inhibitor of hiv protease and reverse transcriptase.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.

Claims (10)

1. a kind of nucleic acid base compound or its pharmaceutically acceptable salt have structure shown in Formulas I:
Figure FDA0002118423450000011
In Formulas I, R1For amino, methoxyl group, nitro, aminomethyl or methylol；
X is-CH2,-O- ,-S- or-NH-；
Y is
Figure FDA0002118423450000012
When X is-CH2When, R Ra, Rb or Rc；
When X is-O- ,-S- or-NH-, R Rc；
The Ra is
Figure FDA0002118423450000013
The Rb isThe Rc is
Figure FDA0002118423450000015
Wherein, R2For hydrogen, hydroxyl, amino or halogen；R3For hydrogen, hydroxyl, amino, halogen or methyl；R4For hydrogen or methyl；R5For Hydrogen, hydroxyl, amino or phosphate group；
When Z is nitrogen, R6For hydrogen, hydroxyl, amino or phosphate group；When Z is oxygen or sulphur, R6It is not present.
2. nucleic acid base compound according to claim 1 or its pharmaceutically acceptable salt, which is characterized in that the core Soda acid based compound includes 2- (the chloro- 9H- purine -9- base of 6-)-N- ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyl group PhenylSulphon amido) -1- phenyl butane -2- base)-acetamide, 2- (8- methyl -9H- purine -9- base)-N- [(2S, 3R) -3- hydroxyl Base -4- (N- isobutyl group -4- methoxyphenyl sulphoxide imine base) -1- phenyl butyl -2- base]-acetamide, 2- (2,4- dioxo - 2H-3,4- dihydro-pyrimidin -1- base)-N- ((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphonyl amido) -1- Phenyl butane -2- base)-acetamide, 2- (the fluoro- 2,4- dioxo -2H-3,4- dihydro-pyrimidin -1- base of 5-)-N- ((2S, 3R) -3- Hydroxyl -4- (N- isobutyl group -4- methoxyphenyl sulphonyl amido) -1- phenyl butane -2- base)-acetamide, ((2S, 5R) -5- (- 1 base of 1H-2- oxo -4- aminopyrimidine)-tetrahydrofuran -2- base) methyl N-((2S, 3R) -3- hydroxyl -4- (N- isobutyl group -4- Aminocarbonyl phenyl sulfoamido) -1- phenyl butane -2- base))-carbamate or N- ((2R, 3S) -3- (2- (fluoro- 2,4- bis- of 5- Oxo -2H-3,4- dihydro-pyrimidin -2H-1- base) acetamido) -2- hydroxy-4-phenyl butyl-N- isobutyl group-P- (4- methoxyl group Phenyl) phosphamic acid.
3. the preparation method of nucleic acid base compound of any of claims 1 or 2, comprising the following steps:
X is-CH in the compound of the structure shown in the Formulas I2, R be Ra or Rb when, by with structure shown in Formula II -1 compound with Amine derivative carries out condensation reaction under the action of catalyst, obtains the compound with structure shown in Formulas I；
Figure FDA0002118423450000021
In Formula II -1, R is Ra or Rb, and the Ra is
Figure FDA0002118423450000022
The Rb is
Figure FDA0002118423450000023
Wherein, R2For Hydrogen, hydroxyl, amino or halogen；R3For hydrogen, hydroxyl, amino, halogen or methyl；R4For hydrogen or methyl；
X is-CH in the compound of the structure shown in the Formulas I2,-O-,-S- or-NH-, R be when being Rc, will have structure shown in Formula II -2 Compound, carbonic acid trichloromethyl ester and amine derivative carry out condensation reaction under the action of catalyst, obtain with shown in Formulas I tie The compound of structure；
Formula II -2 R-XH,
In Formula II -2, X is-CH2,-O- ,-S- or-NH-；R is Rc, and the Rc is
Figure FDA0002118423450000031
Wherein, R2For hydrogen, hydroxyl, Amino or halogen, R3For hydrogen, hydroxyl, amino, halogen or methyl；R4For hydrogen or methyl；R5For hydrogen, hydroxyl, amino or phosphate Group；When Z is nitrogen, R6For hydrogen, hydroxyl, amino or phosphate group；When Z is oxygen or sulphur, R6It is not any atom or group；
The amine derivative has structure shown in formula III:
Figure FDA0002118423450000032
In formula III, R1For amino, methoxyl group, nitro, aminomethyl or methylol；Y is
Figure FDA0002118423450000034
4. preparation method according to claim 3, which is characterized in that the compound with structure shown in Formula II -1 with Amine derivative is condensed under the catalytic action of carbodiimide hydrochloride, I-hydroxybenzotriazole and 4-dimethylaminopyridine Reaction；
The compound and carbonic acid trichloromethyl ester and amine derivative with structure shown in Formula II -2 is in N, N- diisopropyl second Condensation reaction is carried out under the catalytic action of amine.
5. preparation method according to claim 3, which is characterized in that the compound with structure shown in Formula II -1 Preparation method, comprising the following steps:
Substituted pyrimidines base or substituted purin base and bromoacetic acid or bromoacetate are subjected to substitution reaction, obtain replacing production Object；
The substitution product is hydrolyzed, the compound with structure shown in Formula II -1 is obtained；
The substituted pyrimidines base has structure shown in formula a:
Figure FDA0002118423450000035
The substituted purin base has structure shown in formula b:
Figure FDA0002118423450000041
6. preparation method according to claim 3, which is characterized in that when Y is in the amine derivative
Figure FDA0002118423450000042
When, it is described Amine derivative has structure shown in formula III -1:
Figure FDA0002118423450000043
The preparation method of the amine derivative with structure shown in formula III -1, comprising the following steps:
[(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] and isobutyl amine progress nucleophilic displacement of fluorine is anti- It answers, obtains (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate；
The R of (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate by described in and structure shown in formula d1Replace Benzene sulfonyl chloride carries out nucleophilic substitution, obtains the intermediate of structure shown in formula c；
By the intermediate Deprotection of structure shown in the formula c, the amine derivative with structure shown in formula III -1 is obtained；
Figure FDA0002118423450000044
7. preparation method according to claim 3, which is characterized in that when Y is in the amine derivative
Figure FDA0002118423450000045
When, institute Amine derivative is stated with structure shown in formula III -2:
The preparation method of the amine derivative with structure shown in formula III -2, comprising the following steps:
[(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] and isobutyl amine progress nucleophilic displacement of fluorine is anti- It answers, obtains (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate；
The R of (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate by described in and structure shown in formula d1Replace Benzene sulfonyl chloride carries out nucleophilic substitution, obtains the intermediate of structure shown in formula c；
The intermediate of structure shown in the formula c and Sodium azide are subjected to imidization, obtain the intermediate of structure shown in formula e；
By the intermediate Deprotection of structure shown in the formula e, the amine derivative with structure shown in formula III -2 is obtained.
Figure FDA0002118423450000052
8. preparation method according to claim 3, which is characterized in that when Y is in the amine derivative
Figure FDA0002118423450000053
When, institute Amine derivative is stated with structure shown in formula III -3:
Figure FDA0002118423450000061
The preparation method of the amine derivative with structure shown in formula III -3, comprising the following steps:
[(S) -1- ((S)-ethylene oxide -2- base) -2- benzene ethylamino t-butyl formate] and isobutyl amine progress nucleophilic displacement of fluorine is anti- It answers, obtains (1S, 2R) -1- benzyl -2- hydroxyl -3- (isobutyl amine) t-butyl carbamate；
By 4- substituted-phenyl phosphonyl dichloride, benzyl alcohol and (1S, the 2R) -1- benzyl -2- hydroxyl-with structure shown in formula f 3- (isobutyl amine) t-butyl carbamate carries out Arbuzov and reacts to obtain the intermediate of structure shown in formula g；
By the intermediate and H of structure shown in the formula g2Reduction reaction is carried out, the intermediate of structure shown in formula h is obtained；
By the intermediate Deprotection of structure shown in the formula h, the amine derivative with structure shown in formula III -3 is obtained.
Figure FDA0002118423450000062
9. nucleic acid base compound as claimed in claim 1 or 2 or its pharmaceutically acceptable salt are preparing answering in hiv inhibitor With the hiv inhibitor is using hiv protease and reverse transcriptase as target spot.
10. application according to claim 9, which is characterized in that the nucleic acid base compound or its is pharmaceutically acceptable Dosage of the salt in hiv inhibitor be 0.01~100nM.
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