CN106167504A - Acyclonucleosides phosphamide D amino acid ester derivative and the preparation of salt thereof and in the application of anti-virus aspect - Google Patents

Abstract

The invention belongs to medical chemistry antiviral field, relate to a kind of acyclonucleosides phosphamide D amino acid ester derivative and the preparation of salt thereof and the application at anti-virus aspect.The present invention also provides for the compound containing the present invention, its stereoisomer, pharmaceutically acceptable salt, hydrate, solvate or crystallization and pharmaceutical composition, and the compound of the present invention or compositions in treatment and/or prevent AIDS and the application of hepatitis B virus infection.The compound activity in vivo of the present invention is significantly better than the prodrug of acyclonucleosides phosphamide L amino-acid ester, has obvious clinical value.

Description

Acyclonucleosides phosphamide D-amino acid ester derivative and the preparation of salt thereof and disease-resistant Application in terms of Du

Technical field

The invention belongs to medical chemistry antiviral field, be specifically related to a class novel acyclonucleosides phosphamide D-amino-acid ester Derivant or its stereoisomer and containing acyclonucleosides phosphamide D-amino acid ester derivative or the group of its stereoisomer Compound is as the purposes of antiviral drugs, and particularly treatment and/or pre-preventing HIV infection is or/and the purposes of patient of HBV infection.

Background technology

HIV (human immunodeficiency virus) (HIV) has two types, HIV-1 type and HIV-2 type.The patient of severe infections HIV-1 Cause immunologic function to lack (ARC and AIDS), easily cause fatal infection.HIV is as the most serious disease of infectiousness in the world One of sick, there are 35,000,000 people's aids infections in the whole world in 2012, newly-increased 2,700,000 cases, there are 2,300,000 people to die from acquired immune deficiency syndrome (AIDS).? Recent studies on shows that in teenager, HIV number becomes to steeply rise trend, so need nonetheless remain for developing the medicine of new and effective low toxicity Thing and new drug regimen are used for treating and preventing acquired immune deficiency syndrome (AIDS).

If some are crucial in the drug main blocking-up of the HIV of exploitation treatment and prevention at present or regulation HIV life-cycle processes Step and key protein, as hiv reverse transcriptase, hiv protease and intergrase and the up-to-date HIV that can effectively block enter thin The medicine of born of the same parents.The HAART (HAART) of these medicines and drug combination can effectively control the duplication of HIV, reduces body Interior inhibition of HIV quantity, extends the life long of patient and improves the quality of living.Due to existing inverase or compound recipe all Can not inhibition of HIV in purged body, vaccine development seriously baffles, so HIV sufferers needs lifelong medication.For long-term and Life-long therapy HIV patient, the drug resistance of generation makes the pharmaceutically active treated reduce or lose activity；Additionally open in early days The medicine high toxicity sent out and various side effect are also serious problems for the patient of long-term prescription.It is thus desirable to exploitation is new Chemical entities, there is the structure of novelty and new model of action, and low toxicity and high activity, treated for new compositions Through producing the patient of drug resistance.

Hiv reverse transcriptase (RT) is a kind of important virus protease, plays a crucial role in the reproduction process of virus. The reverse transcriptase of HIV is heterodimer, comprises two subunits of P66 and P51.The reverse transcriptase inhibitors of FDA approval is divided into nucleoside Inhibitor (NRTIs) and non-nucleosidic inhibitors (NNRTIs).Wherein nucleoside medicine includes AZT, ddI, ddC, d₄T,3TC, Abacavir, emtricitabine and tenofovir etc..

(R)-9-(2-phosphate methoxy propyl group)-adenine (tenofovir, PMPA, TFV) has anti-AIDS and anti-second Liver activity, toxicity is far below adefovirdipivoxil.Due to highly acid and the polarity of TFV, it is difficult to, through cell membrane, cause bioavailability Difference.Additionally TFV is to be excreted by kidney proximal tubule, and this process has two transhipment enzymes to participate in: organic anion Transporter (OAT) and multidrug resistance-associated protein 4 (MRP4). the TFV of high concentration The infiltration burden of glomerule can be increased the weight of, especially for the patient of renal function defect.Serious renal tubules toxicity causes Fanconi Syndrome.The highly acid of TFV also has certain toxicity to sclerotin.

The improvement of TFV generally utilizes the highly acid of the principle shielding phosphate portion of prodrug, improves absorption and the reduction of cell TFV concentration in blood plasma.Prodrug be designed with two different directions: (1) is for the stability of pro-drugs and prodrug portion The hypotoxicity divided；(2) for the different metabolic mechanism of prodrug, metabolism in target cell is selected.

TDF (tenofovir disoproxil fumarate), as the fumarate of TFV dibasic acid esters prodrug, is lucky moral The prodrug of new generation being better than adefovir ester of scientific company exploitation, calendar year 2001 and 2008 by FDA (Food and Drug Adminstration) (USFDA) approval is for the infection of first-line drug treatment HIV and HBV.TDF greatly improves the penetrance of cell and oral suction Receive availability.TDF in-vitro screening shows that the highest AntiHIV1 RT activity and HBV are active, outstanding anti-drug resistance and good safety, Anti-AIDS aspect is widely used in various combined dosage form.Wherein the Troyes of one of compound recipe reaches (TRUVADA) is exactly TDF and grace song His shore immobilised compound, is widely used in the treatment of AntiHIV1 RT activity and HBV, and what additionally Disease Control and Prevention Center of the Ye Shi U.S. was recommended is used for preventing AIDS Sick medicine.The shortcoming of TDF is easily to be hydrolyzed to TFV in blood plasma.On the other hand, for the patient of renal function defect, TDF can The risk of nephrotoxicity can be caused.

In order to reduce the TDF TFV concentration in blood plasma metabolism, the medicine that Gilid Science Co. develops metabolism more stable replaces Nuo Fuweiaila phenol amine (Tenofovir alafenamide fumarate (TAF)), two compound recipes of TAF (Genvoya and Odefsey) HIV is treated by FDA (Food and Drug Adminstration) (USFDA) approval for first-line drug.Two three of TAF treatment HBV Phase clinic is successful, is waiting FDA approval listing.TAF is more stable than TDF in blood plasma, is difficult to be hydrolyzed enzyme hydrolysis, big portion Divide TAF to be absorbed into cell, rely on the metabolism in tissue and hepatocyte of Cathepsin A (CatA) and carbonic acid esterase 1 (CES1), And phosphoric acid turns to active component TFVDP.

TAF with TDF compares has significant difference at two aspects: (1) TAF TFV concentration of metabolism in blood plasma is lower than TDF 90%.(2) the TFV concentration of TAF metabolism is higher 4 times than TDF in immunocyte.So TAF can use the lowest clinically Dosage, significantly reduces nephrotoxicity and bone toxicity that TDF causes.

Although TAF uses the lowest dosage and the highest AntiHIV1 RT activity and HBV activity clinically, but TAF is at transport process In and in blood plasma, still have a small amount of hydrolysis, need nonetheless remain for clinically developing new compound and single-minded metabolic way, enter one Step improves medicine stability in blood plasma, improves at histiocytic drug level, thus effectively reduces the clinical agent of medicine The nephrotoxicity of amount and further reduction medicine and bone toxicity, preferably play treatment hepatitis B and the clinical effectiveness of acquired immune deficiency syndrome (AIDS).

Summary of the invention:

Creative work through the present inventor and the profound understanding to drug metabolism, this patent provides a kind of brand-new Acyclonucleosides phosphamide D-amino acid ester derivative, this kind of prodrug is extremely stable in blood plasma, and people's whole blood is educated 2 hours altogether, Blood plasma is not detected by metabolite TFV.Significantly increasing is had compared with TAF at histiocyte particularly hepatocyte drug concentration Add, so the acyclonucleosides phosphamide D-amino acid ester derivative of the present invention can significantly improve the treatment to hepatitis B and acquired immune deficiency syndrome (AIDS) Effect, and reduce nephrotoxicity and the bone toxicity that TDF or TAF causes greatly.Thus propose following invention.

A kind of acyclonucleosides phosphamide D-amino acid ester derivative or its stereoisomer, officinal salt, hydrate, solvent Compound or crystallization, have formula I, and the structure of formula I is,

Wherein:

(1)R₁It is independently selected from C_1-6Alkyl or aryl alkyl；R₄It is independently selected from methyl or hydrogen；

(2)R₂It is independently selected from alkyl, cycloalkyl-alkyl, cycloalkyl or benzyl；

(3) X is selected from oxygen, NH or sulfur；

(4)R₃Selected from aryl, heteroaryl, C_1-20Alkyl, cycloalkyl, alkoxyalkyl, alkanoyl oxyalkyl, aroyl oxygen Alkyl or alkoxy carbonyl group oxyalkyl.Aryl is selected from unsubstituted aryl or various substituted aryl；Heteroaryl is selected from unsubstituted Heteroaryl or various substituted heteroaryl；Substituent group on substituted aryl and substituted heteroaryl selected from deuterium, halogen, alkyl, Cycloalkyl, haloalkyl, halogenated cycloalkyl, cycloalkyl-alkyl, cycloalkyl haloalkyl, cyano group, substituted amino, thiazolinyl, benzene Base, alkyl silyl；Unsubstituting aromatic yl includes phenyl, xenyl and naphthyl.

Removing outside active hydrogen in formula I, remaining all of hydrogen atom can separately be replaced by deuterium.

The phosphorus atoms of the acyclonucleosides phosphamide D-amino acid ester derivative of described formula I has chirality, and its configuration is S- Configuration or R-configuration, or S-configuration and the mixture of R-configuration.

Described officinal salt becomes the medicine of salt with acyclonucleosides phosphamide D-amino acid ester derivative purine amino part On, acceptable acid is selected from phosphoric acid, hydrochloric acid, sulphuric acid, hydrobromic acid, citric acid, maleic acid, malonic acid, mandelic acid, succinic acid, richness Horse acid, acetic acid, lactic acid, nitric acid, sulfonic acid, p-methyl benzenesulfonic acid, malic acid or methanesulfonic acid；Acid and acyclonucleosides phosphamide D-aminoacid Ester derivant mol ratio is in the range of 0.5 to 1.

A kind of medical composition, containing the acyclonucleosides phosphamide D-amino acid ester derivative described in dose therapeutically effective or Its stereoisomer, officinal salt, hydrate, solvate or crystallization, described compositions is tablet, capsule, injection or receives Metric system agent.

Described medical composition, comprises other therapeutic agent and reinforcing agent.Wherein said other therapeutic agent is independent Group selected from consisting of: hiv protease inhibitor, hiv reverse transcriptase non-nucleosidic inhibitors, hiv reverse transcriptase nucleoside suppress Agent, hiv reverse transcriptase nucleotide inhibitor, hiv integrase inhibitor and CCR5 inhibitor, HBV capsid inhibitor (capsid Inhibitor), cccDNA forms inhibitor, cccDNA epigenetic modification agent or hbv rna i medicine；Reinforcing agent includes Li Tuo That Wei and than west he (Cobicistat).

Described acyclonucleosides phosphamide D-amino acid ester derivative, its stereoisomer, pharmaceutically acceptable salt, water Compound, solvate or crystallization or pharmaceutical composition are in preparation is used for the medicine for the treatment of and/or preventing viral infection Application；Wherein said viral infectious disease includes the infectious disease that HIV and/or HBV virus causes.

At least one purposes in the item of following (1)-(2):

(1) HIV (human immunodeficiency virus) (HIV) or hepatitis B and the side of hepatitis B virus are infected in a kind for the treatment of and/or prevention Method, including described acyclonucleosides phosphamide D-amino acid ester derivative or its stereoisomer, officinal salt, hydrate, molten Agent compound, crystallization or described pharmaceutical composition are used for treatment and/or the medicine of pre-preventing HIV infection or hepatitis B and second in preparation Application in the medicine that hepatovirus infects.

(2) a kind of method simultaneously treating and preventing HIV and HBV, including described acyclonucleosides phosphamide D-aminoacid Ester derivant or its stereoisomer, officinal salt, hydrate, solvate, crystallization or described pharmaceutical composition are in preparation Application in the medicine treating and/or preventing HIV and HBV infection.

Acyclonucleosides phosphamide D-amino acid ester derivative or its stereoisomer, pharmaceutically acceptable salt, hydrate, molten Agent compound or crystallization, described in there is the compound shown in formula I or pharmaceutically acceptable salt (officinal salt) includes following knot Structure:

Wherein:

(1)R₁It is independently selected from C_1-6Alkyl or aryl alkyl；R₄It is independently selected from methyl or hydrogen；

(2)R₂It is independently selected from alkyl, cycloalkyl-alkyl, cycloalkyl or benzyl；

(3) X is selected from oxygen, NH or sulfur；

(4)R₃Selected from aryl, heteroaryl, C_1-20Alkyl, cycloalkyl, alkoxyalkyl, alkanoyl oxyalkyl, aroyl oxygen Alkyl or alkoxy carbonyl group oxyalkyl；Aryl is selected from unsubstituted aryl or substituted aryl；Heteroaryl is selected from unsubstituted heteroaryl Base or substituted heteroaryl；Substituent group on substituted aryl and substituted heteroaryl is selected from deuterium, halogen, alkyl, cycloalkyl, halogen Substituted alkyl, halogenated cycloalkyl, cycloalkyl-alkyl, cycloalkyl haloalkyl, cyano group, substituted amino, thiazolinyl, phenyl, alkyl silicon Base；Unsubstituting aromatic yl includes phenyl, xenyl and naphthyl.

(5) acid is independently selected from phosphoric acid, sulphuric acid, hydrochloric acid, hydrobromic acid, citric acid, maleic acid, malonic acid, mandelic acid, succinum Acid, fumaric acid, acetic acid, lactic acid, nitric acid, sulfonic acid, p-methyl benzenesulfonic acid, malic acid, Loprazolam.

The described acyclonucleosides phosphamide D-amino acid ester derivative, its stereoisomer or the medicine that have shown in formula I On, acceptable salt includes following structure:

The present invention also provides for a kind of pharmaceutical composition, the acyclonucleosides phosphamide of the present invention containing dose therapeutically effective D-amino acid ester derivative or its isomer, officinal salt, hydrate, solvate or crystallization.

Said medicine can also be added one or more pharmaceutically acceptable carriers, such as pharmaceutically acceptable dilution Agent, excipient, filler, binding agent, disintegrating agent, absorption enhancer, surfactant, lubricant, flavouring agent, sweeting agent etc..

With the compounds of this invention for medicine prepared by active component can be tablet, powder, capsule, granule, oral liquid with And the various ways such as ejection preparation.The medicine of above-mentioned various dosage form all can be prepared by the conventional method of pharmaceutical field.

The pharmaceutical composition composition of the present invention can be made up of lower proportioning:

The compound of this patent design further increases tenofovir prodrug stability in blood plasma, the D-ammonia of synthesis Base acid esters can only be hydrolyzed by CES1, and corresponding l-amino acid ester prodrugs (TAF) can be by two kinds of enzyme hydrolysiss of CatA and CES1.This Plant compound based on mechanismic design and compare with TAF dramatically different in metabolic way.(1) stability aspect: owing to CES1 exists The concentration of intestinal wall cell is low, and the CatA of correspondence has the highest concentration and activity at intestinal wall cell and histiocyte, so this The compound of Patent design through intestinal wall cell and in blood plasma the most highly stable, and people's whole blood educate altogether 2 hours in blood plasma It is not detected by any metabolite.Corresponding TAF is passing through intestinal wall cell and is having considerable degree of metabolism to hydrolyze in blood plasma. (2) metabolic drug (TFV) distributes at blood plasma and histiocytic concentration: the compound of present invention synthesis is easily absorbed by cell, In liver, the highest concentration is particularly had, so the metabolic drug concentration (TFV) in liver cell is phase at histiocyte With 4-10 times of dosage TAF, the specificity of display medicine Liver targeting.In conjunction with the characteristic in terms of the two, this patent provides one Plant the hepatic targeting drug that potent low toxicity is brand-new.

Term defines

Unless otherwise defined, all technology used herein and scientific terminology have and common skill of the art The identical implication that art personnel are generally understood that.

Term " stereoisomer " refers to by isomer produced by molecule Atom spatially arrangement mode difference.Bag Include cis-trans-isomer, enantiomer and conformer.All stereoisomers belong to the scope of the present invention.The present invention's The independent stereoisomer of compound maybe can be mixed into such as racemic modification without other isomer, or with all other Stereoisomer mixes.

Term " salt " refers to the pharmaceutically acceptable salt that compound of the present invention is formed with acid, and described acid is optional From: phosphoric acid, sulphuric acid, hydrochloric acid, hydrobromic acid, citric acid, maleic acid, malonic acid, mandelic acid, succinic acid, fumaric acid, acetic acid, lactic acid, Nitric acid, sulfonic acid, p-methyl benzenesulfonic acid, malic acid, Loprazolam or its analog.

Term " solvate " refers to of the present inventionization of the coordination compound by forming solid-state or liquid with solvent molecule coordination The form of compound.Hydrate is the specific form of solvate, is wherein coordinated with water.Within the scope of the present invention, solvent closes Thing is preferably hydrate.

Term " crystallizes " the various solid forms referring to that compound of the present invention is formed, including crystal formation, amorphous.

Term " alkyl " refers to straight chain, side chain or ring-type saturated hydrocarbyl, the alkyl below preferably 1-20 carbon atom.Alkane The embodiment of base include methyl, ethyl, n-pro-pyl, isopropyl, cyclopropyl, normal-butyl, isobutyl group, the tert-butyl group, cyclobutyl, positive penta Base, isopentyl, neopentyl, cyclohexyl, n-hexyl, isohesyl, 2,2 ,-methyl butyl and 2,3-dimethylbutyl, 16-alkyl, 18-alkyl.Term " C_1-20Alkyl " refer to the straight chain containing 1-20 carbon atom, side chain or ring-type saturated hydrocarbyl.Alkyl bag Include substituted and there is no substituted alkyl.When alkyl is replaced, substituent group can replace on any spendable junction point, Substituent group can be monosubstituted or polysubstituted.Substituent group independence selected from alkyl, thiazolinyl, alkynyl, alkoxyl, alkylthio group, alkyl Amino, deuterium, halogen, mercaptan, hydroxyl, nitro, carboxyl, ester group, cyano group, cycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycle alkane Epoxide, cycloalkylthio, oxo.

Term " cycloalkyl " refers to the saturated and/or unsaturated monocycle of part or multi-ring cyclic hydrocarbon radical.Monocycle can include 3-10 carbon Atom.The non-limiting example of monocyclic naphthenes base includes cyclopropyl, cyclobutyl, cyclopenta, cyclopentenyl, cyclohexyl, cyclohexene Base, cyclohexadienyl, suberyl etc..Polycyclic naphthene base includes the cycloalkyl of volution, condensed ring and bridged ring.Cycloalkyl includes unsubstituted Base and containing substituent group.Substituent group is selected from one or more substituted radicals, includes but are not limited to following group, and independent is selected from Alkyl, cycloalkyl, alkoxyl, halogen, carboxyl, ester group, amino, amide groups, hydroxyl, cyano group, nitro, aryl, heteroaryl.

Term " aryl " refers to the full carbon monocycle of 6-10 unit or multi-ring aromatic group, including phenyl, Nai Ji, xenyl etc..Virtue Base can be substituted and unsubstituted.Substituent group independence selected from alkyl, cycloalkyl (cyclopropane base, Tetramethylene. base and ring penta Alkyl etc.), thiazolinyl, alkynyl, nitrine, amino, deuterium, alkoxyl, alkylthio group, alkyl amino, halogen, mercaptan, hydroxyl, nitro, miscellaneous Cycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocyclylalkoxy groups, cycloalkylthio, heterocycle alkylthio group, alkyl silyl etc..

Term " heteroaryl " refers to comprise the group of 1-10 heteroatomic miscellaneous aroma system.Hetero atom includes oxygen, sulfur, nitrogen, Phosphorus etc..Wherein single heterocyclic radical includes but not limited to furan, thiophene, pyrroles, thiazole, imidazoles, 1,2,3-triazole, 1,2,4-tri-nitrogen Azoles, 1,2,3-thiadiazoles, azoles, 1,2,4-diazole, 1,3,4-diazole, pyridine, pyrimidine, pyridazine, pyrazine, oxolane, Nafoxidine, piperidines, piperazine, morpholine, isoxazolines etc..Condensed hetero ring base includes but not limited to quinoline, isoquinolin, indole, benzo Furan, benzothiophene, purine, acridine, carbazole, fluorenes, chromene ketone, Fluorenone, quinoxaline, 3,4-dihydro naphthalenone, dibenzofurans, hydrogen Change dibenzofurans, benzoxazolyl group etc..Heteroaryl can be substituted and unsubstituted.Substituent group independence selected from substituent group Independent selected from alkyl, cycloalkyl (cyclopropane base, Tetramethylene. base and Pentamethylene. base etc.), thiazolinyl, alkynyl, nitrine, amino, deuterium, Alkoxyl, alkylthio group, alkyl amino, halogen, mercaptan, hydroxyl, nitro, Heterocyclylalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycle Alkoxyl, cycloalkylthio, heterocycle alkylthio group, alkyl silyl etc..

Term " halogen " refers to fluorine, chlorine, bromine, iodine.

Term " haloalkyl " refers to the alkyl at least replaced by a halogen atom.

Term " heterocyclic radical " refers at least contain a heteroatomic cyclic group, and wherein hetero atom is nitrogen, oxygen, sulfur, sulfur Deng.Heterocyclic radical includes single heterocyclic radical and many heterocyclic radicals.

Detailed description of the invention

Following example can make those skilled in the art that the present invention is more fully understood, but limits this never in any form Invention.The equal warp of structure of all compounds¹H NMR or MS is determined.

In the embodiment of the present invention, the ratio of column chromatography purification agents useful for same is volume ratio.

Below in conjunction with specific embodiment, the present invention is described further:

Intermediate-1: phenyl hydrogen ((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) phosphonic acids

Commercially available tenofovir (TFV) (14.6g, 50.8mmol) and phenol (9.6g, 102.0mmol) are being vacuum dried After half an hour, it is dissolved in N-Methyl pyrrolidone (39g), under nitrogen protection, is warming up to 85 DEG C, after stirring 30 minutes, add three Ethamine (6.3g, 62.3mmol), reacts 10 minutes, is warming up to 100 DEG C, be slowly added dropwise dicyclohexylcarbodiimide (DCC) N-Methyl pyrrolidone (16g) solution of (17.1g, 82.9mmol), dropping in 6 hours is complete, continues reaction 20 hours.Reactant liquor Be cooled to room temperature, filter, filtrate concentrate, residue add methanol (30mL) stirring, the solid filtration drying of precipitation obtain intermediate- 1 (11.3g, yield: 61.4%).¹H NMR(400MHz,DMSO-d₆):δ0.99-1.11(d,3H,CH₃), 3.71-3.85 (m, 2H,OCH₂), 3.92-4.01 (m, 1H, OCH), 4.14-4.35 (m, 2H, NCH₂), 6.98-7.36 (m, 5H, phenol), 7.58 (s, 2H,NH₂), 8.14 (s, 2H, purine ring hydrogens), 8.15 (2H, s, purine ring hydrogens).ESI-MS:[M+H]⁺: 364.1；[M +Na]⁺: 386.1.

Reaction scheme 1

Embodiment 1:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base)-D-alanine isopropyl ester fumarate (18)

The synthesis of step 1:N-Boc-D-alanine isopropyl ester (16)

N-Boc-D-alanine 15 (5.0g, 26.4mmol) and isopropanol (1.92g, 31.9mmol) are dissolved in dichloromethane (50mL) in, under nitrogen protection, ice bath to 0 DEG C, add 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) (7.47g, 38.9mmol), adds DMAP (DMAP) (0.32g, 2.6mmol), slowly the most in three batches Be warmed to room temperature, react 12 hours, add dichloromethane (25mL) dilution, reactant liquor respectively with saturated sodium bicarbonate (20mL x2), Saturated aqueous common salt (20mL x2) washs, and anhydrous sodium sulfate is dried, and filters, and filtrate reduced in volume, by silica gel chromatographic column method with eluting Agent petroleum ether: ethyl acetate=10: it is different that 1 column chromatography purification (the concentrated sulphuric acid methanol solution of 3% develops the color) obtains N-Boc-D-alanine Propyl ester 16 (4.0g, productivity: 66%).

¹H NMR(400MHz,CDCl₃):δ1.20-1.30(m,6H,CH(CH₃)₂), 1.30-1.38 (d, 3H, CH₃), 1.44 (s,9H,(CH₃)₃), 4.20-4.30 (m, 1H, CHCH₃), 5.00-5.10 (m, 2H, CH and NH).

The synthesis of step 2:D-alanine isopropyl ester hydrochlorate (17)

N-Boc-D-alanine isopropyl ester 16 (5.0g, 21.6mmol) is joined the diethyl ether solution of the hydrogen chloride of 2.0M (21mL), after being stirred at room temperature 1 hour, concentration of reaction solution, residue over silica gel column chromatography purification (ethyl acetate: ethanol=10:1) Obtain D-alanine isopropyl ester hydrochlorate 17 (2.5g, yield: 71%).¹H NMR(400MHz,CDCl₃):δ1.28(s,6H,CH (CH₃)₂), 1.71 (s, 3H, CH₃), 4.21 (s, 1H, CH), 5.09 (s, 1H, CH), 8.64 (s, 3H ,-NH₂+HCl)。

Step 3:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base) synthesis of-D-alanine isopropyl ester (1)

By phenyl hydrogen ((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) phosphonic acids (intermediate- 1) (1g, 2.7mmol), D-alanine isopropyl ester hydrochlorate 17 (0.93g, 5.5mmol), two sulfur two pyridines (1.21g, 5.5mmol), triphenylphosphine (PPh₃) (1.44g, 5.5mmol) mixture be vacuum dried 30 minutes, add pyridine (12ml), Nitrogen is protected, and is warming up to 72 DEG C, adds triethylamine (3.84mL, 27.5mmol), continue reaction 12 hours after 30 minutes.Concentrate, Residue over silica gel column chromatography purification (ethyl acetate: ethanol=10:1) obtains tenofovir phosphamide derivant 1, and (0.64g receives Rate: 49%).¹H NMR(400MHz,CDCl₃):δ1.11-1.33(m,12H,4×CH₃), 3.55-3.79 (m, 2H, OCH₂P), 3.80-4.24(m,4H,NCH₂, NCH and OCH), 4.25-4.45 (m, 1H, NH), 4.90-5.10 (m, 1H, COOCH), 6.40(s,1H,NH₂), 6.80-7.38 (m, 5H, Ar-H), and 8.02 (s, 1H, the H on purine ring), 8.16-8.41 (d, 1H, fast H on purine ring).ESI-MS:[M+H]⁺: 477.2.

Step 4:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base) synthesis of-D-alanine isopropyl ester fumarate (18)

Tenofovir phosphamide derivant 1 (1.0g, 2.1mmol) joins in acetonitrile (20mL), is heated to 80 DEG C to clear Clearly, adding fumaric acid (0.21g, 1.8mmol), after stirring 15 minutes, hot sucking filtration, filtrate is cooled to room temperature, continues to transfer at-12 DEG C Putting 12 hours and separate out crystallization, filter, filter cake is washed 1 time with cold acetonitrile, and drying at room temperature is positioned over the vacuum drying oven of 35 DEG C after 6 hours In be dried 12 hours fumarate 18 (1.0g, yield: 81%).¹H NMR(400MHz,DMSO-d₆):δ0.91-1.32(m, 12H,4×CH₃), 3.73-4.05 (m, 4H, OCH₂P, NCH and OCH), 4.10-4.35 (m, 4H, NCH₂), 4.71-4.94 (m, 1H, COOCH), 5.47-5.74 (m, 1H, NH), 6.63 (s, 2H, fumaric acid double bond hydrogen), 6.96-7.45 (m, 7H, NH₂and Ar-H), 8.06-8.13 (d, 1H, the H on purine ring), 8.13-8.20 (d, 1H, the H on purine ring), 13.1 (s, 2H, 2 × COOH).

Reaction scheme 2

Embodiment 2:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base)-D-alanine isobutyl ester fumarate (21)

The synthesis of step 1:N-Boc-D-alanine isobutyl ester (19)

N-Boc-D-alanine 15 (4.0g, 21.1mmol) and isobutanol (1.89g, 25.6mmol) are dissolved in dichloromethane (40mL) in, under nitrogen protection, ice bath to 0 DEG C, add EDC (5.9g, 30.7mmol), add DMAP the most in three batches (0.25g, 2.0mmol), is slowly increased to room temperature, reacts 12 hours, adds dichloromethane (18mL) dilution, and reactant liquor is respectively with full With sodium bicarbonate (16mL x2), saturated aqueous common salt (15mL x2) washing, anhydrous sodium sulfate is dried, and filters, filtrate reduced in volume, N-is obtained with silica gel column chromatography (eluant: petroleum ether: ethyl acetate=10: 1) purification (the concentrated sulphuric acid methanol solution of 3% develops the color) Boc-D-alanine isobutyl ester 19 (3.68g, productivity: 71%).

¹H NMR(400MHz,CDCl₃):δ0.85-1.02(d,6H,CH(CH₃)₂), 1.36-1.41 (d, 3H, CH₃), 1.44 (s,9H,(CH₃)₃), 1.85-2.08 (m, 1H, CH), 3.81-4.05 (m, 2H, OCH₂), 4.16-4.48 (m, 1H, CHCH₃), 5.06(s,1H,NH)。

The synthesis of step 2:D-alanine isobutyl ester hydrochlorate (20)

N-Boc-D-alanine isobutyl ester 19 (3.0g, 12.2mmol) is joined the diethyl ether solution of 2.0M hydrogen chloride (18mL), stirred overnight at room temperature, concentration of reaction solution, residue over silica gel chromatogram column technique purification (eluant: ethyl acetate: ethanol =10:1) obtain D-alanine isobutyl ester hydrochlorate 20 (1.73g, yield: 78%).

¹H NMR(400MHz,CDCl₃):δ0.82-1.05(d,6H,CH(CH₃)₂), 1.63-1.84 (d, 3H, CH₃), 1.86-2.08 (m, 1H, CH), 3.85-4.09 (m, 2H, CH₂), 4.17-4.40 (m, 1H, CH), 8.39 (s, 3H, NH₂+HCl)。

Step 3:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base) synthesis of-D-alanine isobutyl ester (3)

By phenyl hydrogen ((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) phosphonic acids (intermediate- 1) (1.5g, 4.1mmol), D-alanine isobutyl ester hydrochlorate 20 (1.5g, 8.2mmol), two sulfur two pyridines (1.81g, 8.2mmol), triphenylphosphine (PPh₃) (2.16g, 8.2mmol) mixture be vacuum dried 30 minutes, add pyridine (20ml), Nitrogen is protected, and is warming up to 72 DEG C, adds triethylamine (5.74mL, 41.2mmol), continue reaction 12 hours after 30 minutes.Reactant liquor Concentrating, residue over silica gel column chromatography purification (eluant: ethyl acetate: ethanol=10:1) obtains tenofovir phosphamide and derives Thing 3 (0.91g, yield: 45%).

¹H NMR(400MHz,CDCl₃):δ0.75-1.20(m,12H,4xCH₃), 1.73-1.88 (m, 1H, CH), 3.65- 4.07(m,6H,NCH₂,OCH₂P and 2 × CH), 4.10-4.35 (m, 2H, COOCH₂), 5.54-5.74 (m, 1H, NH), 6.96-7.41(m,7H,Ar-H and NH₂), 8.05-8.12 (d, 1H, the H on purine ring), 8.13 (s, 1H, on purine ring H)。ESI-MS:[M+H]⁺:491.2；[M+Na]⁺:513.2.

Step 4:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base) synthesis of-D-alanine isobutyl ester fumarate (21)

Tenofovir phosphamide derivant 3 (0.5g, 1.0mmol) joins in acetonitrile (10mL), is heated to 80 DEG C to clear Clearly, adding fumaric acid (0.107g, 0.92mmol), after stirring 15 minutes, hot sucking filtration, filtrate is cooled to room temperature, continues at-12 DEG C Placing 12 hours and separate out crystallization, filter, filter cake is washed 1 time with cold acetonitrile, and drying at room temperature is positioned over the vacuum drying of 35 DEG C after 6 hours Case is dried 12 hours to obtain fumarate 21 (0.48g, yield: 78%).

¹H NMR(400MHz,DMSO-d₆):δ0.71-1.23(m,12H,4xCH₃), 1.72-1.90 (m, 1H, CH), 3.67-4.04(m,6H,NCH₂,OCH₂P, 2xCH), 4.10-4.36 (m, 2H, COOCH₂), 5.52-5.764 (m, 1H, NH), 6.63 (s, 2H, fumaric acid double bond hydrogen), 6.97-7.41 (m, 7H, NH₂And Ar-H), (d, 1H, on purine ring for 8.00-8.11 H), 8.13 (s, 1H, the H on purine ring), 13.1 (s, 2H, 2xCOOH).

Reaction scheme 3

Embodiment 3:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base)-D-alanine cyclohexyl fumarate (24)

The synthesis of step 1:N-Boc-D-CHA (22)

N-Boc-D-alanine 15 (5g, 26.4mmol) and Hexalin (3.2g, 32mmol) are dissolved in dichloromethane (50mL) in, under nitrogen protection, ice bath to 0 DEG C, add EDC (7.47g, 38.9mmol), add DMAP the most in three batches (0.32g, 2.6mmol), is slowly increased to room temperature, reacts 12 hours, adds dichloromethane (30mL) dilution, and reactant liquor is respectively with full With sodium bicarbonate (25mL x2), saturated aqueous common salt (20mL x2) washing, anhydrous sodium sulfate is dried, and filters, filtrate reduced in volume, N-is obtained with silica gel column chromatography (eluant: petroleum ether: ethyl acetate=10:1) purification (the concentrated sulphuric acid methanol solution of 3% develops the color) Boc-D-CHA 22 (5.6g, productivity: 79%).

¹H NMR(400MHz,CDCl₃): δ 1.03-1.99 (m, 22H, 4xCH₃+5xCH₂),4.17-4.39(m,1H, OCH),4.70-4.92(m,1H,CHCH₃), 5.08 (s, 1H, NH).

The synthesis of step 2:D-CHA hydrochlorate (23)

N-Boc-D-CHA 22 (4.0g, 14.7mmol) is joined the diethyl ether solution of 2.0M hydrogen chloride (20mL), stirred overnight at room temperature, concentration of reaction solution, residue over silica gel column chromatography purification (eluant: ethyl acetate: ethanol= 10:1) obtain D-alanine cyclohexyl hydrochlorate 23 (2.44g, yield: 80%).

¹H NMR(400MHz,CDCl₃): δ 1.19-1.95 (m, 13H, CH₃+5xCH₂), 4.07-4.27 (m, 1H, OCH), 4.70-5.00 (m, 1H, NCH), 8.71 (s, 3H, NH₂+HCl)。

Step 3:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base) synthesis of-D-alanine cyclohexyl (5)

By phenyl hydrogen ((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) phosphonic acids (intermediate- 1) (2.0g, 5.5mmol), D-alanine cyclohexyl hydrochlorate 23 (2.28g, 11.0mmol), two sulfur two pyridines (2.42g, 11.0mmol), triphenylphosphine (PPh₃) (2.88g, 11.0mmol) mixture be vacuum dried 30 minutes, add pyridine (12ml), nitrogen is protected, and is warming up to 72 DEG C, reacts 30 minutes, and dropping triethylamine (7.68mL, 55.0mmol) continues reaction 12 Hour.Concentrating, residue over silica gel column chromatography purification (eluant: ethyl acetate: ethanol=10:1) obtains tenofovir phosphinylidyne Amine derivative 5 (1.65g, yield: 58%).

¹H NMR(400MHz,CDCl₃): δ 1.09-1.90 (m, 16H, 2xCH₃5xCH on and cyclohexyl₂),3.56- 3.82 (m, 2H, OCH₂P), 3.85-4.24 (m, 4H, NCH₂, NCH and CH), 4.28-4.44 (m, 1H, the CH on cyclohexyl), 4.66-4.84(m,1H,NH),5.90-6.41(d,2H,NH₂), 6.91-7.39 (m, 5H, Ar-H), 7.93-8.09 (d, 1H, fast H on purine ring), 8.23-8.42 (d, 1H, the H on purine ring).ESI-MS:[M+H]⁺: 517.2；[M+Na]⁺: 539.3.

Step 4:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base) synthesis of-D-alanine cyclohexyl fumarate (24)

Tenofovir phosphamide derivant 5 (1.0g, 1.9mmol) joins in acetonitrile (20mL), is heated to 80 DEG C to clear Clearly, adding fumaric acid (0.2g, 1.7mmol), after stirring 15 minutes, hot sucking filtration, filtrate is cooled to room temperature, continues to transfer at-12 DEG C Putting 12 hours and separate out crystallization, filter, filter cake is washed 1 time with cold acetonitrile, and drying at room temperature is positioned over the vacuum drying oven of 35 DEG C after 6 hours In be dried 12 hours fumarate 24 (1.0g, yield: 82%).

¹H NMR(400MHz,DMSO-d₆):δ0.99-1.79(m,16H,2xCH₃5xCH on and cyclohexyl₂), 3.69- 4.39(m,6H,OCH₂P,NCH₂, 2 × CH), 4.49-4.71 (m, 1H, the CH on cyclohexyl), 5.46-5.76 (m, 1H, NH), 6.63 (s, 2H, fumaric acid double bond hydrogen), 6.97-7.43 (m, 7H, NH₂And Ar-H), (d, 1H, on purine ring for 8.06-8.12 H), 8.12-8.24 (d, 1H, the H on purine ring), 13.1 (s, 2H, 2xCOOH).

Reaction scheme 4

Embodiment 4:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base)-D-alanine N-butyl fumarate (27)

The synthesis of the different N-butyl of step 1:N-Boc-D-alanine (25)

N-Boc-D-alanine 15 (4.5g, 23.7mmol) and n-butyl alcohol (3.52g, 47.5mmol) are dissolved in dichloromethane (50mL) in, under nitrogen protection, ice bath to 0 DEG C, add EDC (10.92g, 57.0mmol), add DMAP the most in three batches (0.75g, 6.2mmol), is slowly increased to room temperature, reacts 12 hours, adds dichloromethane (30mL) dilution, and reactant liquor is respectively with full With sodium bicarbonate (25mL x2), saturated aqueous common salt (25mL x2) washing, anhydrous sodium sulfate is dried, and filters, filtrate reduced in volume, With silica gel column chromatography (eluant: petroleum ether: ethyl acetate=10:1) purification obtain N-Boc-D-alanine n-butyl ester 25 (4.72g, Productivity: 81%).

¹H NMR(400MHz,CDCl₃):δ0.94(t,3H,CH₂CH₃), 1.39 (d, 3H, CHCH₃), 1.40 (m, 2H, (CH₂)₂CH₂CH₃), 1.45 (s, 9H ,-(CH₃)₃), 1.64 (m, 2H, OCH₂CH₂CH₂CH₃), 4.16 (m, 2H, OCH₂CH₂), 4.3 (m,1H,CHCH₃), 5.06 (s, 1H, NH).

The synthesis of step 2:D-alanine n-butyl ester hydrochlorate (26)

N-Boc-D-alanine n-butyl ester 25 (4.0g, 16.3mmol) is joined the diethyl ether solution of 2.0M hydrogen chloride (21mL), stirred overnight at room temperature, concentration of reaction solution, the ethanol solution washing of residue with diethyl ether, it is dried, is just obtaining D-alanine Butyl ester hydrochlorate 26 (2.13g, yield: 72%).

¹H NMR(400MHz,CDCl₃): δ 0.85 (t, 3H, CH₂CH₃), 0.89-0.91 (m, 3H, CHCH₃), 1.25-1.35 (m,2H,CH₂CH₃), 1.53-1.63 (m, 2H, CH₂CH₂CH₃), 4.00-4.05 (m, lH, CHCH₃), 4.06-4.15 (m, 2H, OCH₂), 8.68 (d, 3H, NH₂+HCl)。

Step 3:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base) synthesis of-D-alanine N-butyl (6)

By phenyl hydrogen ((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) phosphonic acids (intermediate- 1) (1.0g, 2.7mmol), D-alanine N-butyl hydrochlorate 26 (1.0g, 5.5mmol), two sulfur two pyridines (1.21g, 5.5mmol), triphenylphosphine (PPh₃) (1.44g, 5.5mmol) mixture be vacuum dried 30 minutes, add pyridine (12ml), Nitrogen is protected, and is warming up to 72 DEG C, reacts 30 minutes, dropping triethylamine (3.84mL, 27.5mmol), continues reaction 12 hours.Dense Contracting, residue over silica gel column chromatography purification (ethyl acetate: ethanol=10:1) obtains tenofovir phosphamide derivant 6 (0.55g, yield: 41%).

¹H NMR(400MHz,CDCl₃):δ0.84-0.97(m,3H,CH₃), 1.14-1.41 (m, 8H, 2xCH₃and CH₂), 1.51-1.65(m,2H,CH₂), 3.59-4.24 (m, 8H, NCH₂,OCH₂P,COOCH₂And 2xCH), 4.29-4.42 (m, 1H, NH), 5.87-6.38 (d, 2H, NH₂), 6.93-7.37 (m, 5H, Ar-H), 7.95-8.05 (d, 1H, the H on purine ring), 8.27-8.37 (d, 1H, the H on purine ring).ESI-MS:[M+H]⁺: 491.2；[M+Na]⁺: 513.2.

Step 4:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (phenoxy group) phosphinylidyne Base) synthesis of-D-alanine N-butyl fumarate (27)

Tenofovir phosphamide derivant 6 (0.3g, 0.6mmol) joins in acetonitrile (7mL), is heated to 80 DEG C to clear Clearly, adding fumaric acid (64.6mg, 0.5mmol), after stirring 15 minutes, hot sucking filtration, filtrate is cooled to room temperature, continues at-12 DEG C Placing 12 hours and separate out crystallization, filter, filter cake is washed 1 time with cold acetonitrile, and drying at room temperature is positioned over the vacuum drying of 35 DEG C after 6 hours Case is dried 12 hours to obtain fumarate 27 (0.29g, yield: 79%).

¹H NMR(400MHz,DMSO-d₆):δ0.80-0.89(m,3H,CH₃), 1.00-1.20 (m, 6H, 2xCH₃), 1.21-1.34(m,2H,CH₂), 1.41-1.55 (m, 2H, CH₂), 3.75-4.04 (m, 6H, NCH₂,OCH₂P, 2xCH), 4.11- 4.34(m,2H,COOCH₂), 5.49-5.75 (m, 1H, NH), 6.63 (s, 2H, fumaric acid double bond hydrogen), 6.99-7.37 (m, 7H, NH₂And Ar-H), 8.00-8.12 (d, 1H, the H on purine ring), 8.13 (s, 1H, the H on purine ring), 13.02 (s, 2H, 2 ×COOH)。

Reaction scheme 5

Embodiment 5 (([1,1'-biphenyl]-4-base epoxide) ((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) Epoxide) methyl) phosphoryl)-D-alanine isopropyl ester fumarate (28)

Step 1:(([1,1'-biphenyl]-4-base epoxide) ((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) oxygen Base) methyl) phosphoryl) synthesis of-D-alanine isopropyl ester (7)

TFV (0.82g, 2.8mmol) is dissolved in pyridine (12mL), is then added sequentially xenol (2.43g, 14.2mmol), D-alanine isopropyl ester hydrochlorate 17 (0.86g, 5.1mmol), triethylamine (4.8mL, 34.4mmol), mixed liquor is 60 DEG C of reactions 6 minutes, add two sulfur two pyridine (4.41g, 20.0mmol), PPh₃(5.25g, 20.0mmol) is dissolved in the molten of pyridine (18mL) Liquid, under nitrogen protection, 80 DEG C of reactions overnight.Reactant liquor concentrates, and the residue obtained is dissolved in dichloromethane.Silica gel column chromatography, First with petroleum ether: ethyl acetate (100:20) eluting then dichloromethane: methanol=100:5-7 affords compound 7 (0.78g, productivity: 50%).¹H NMR(400MHz,CDCl₃):δ1.10-1.33(m,12H,4×CH₃), 3.55-3.79 (m, 2H,OCH₂P), 3.81-4.24 (m, 4H, NCH₂, NCH and OCH), 4.25-4.43 (m, 1H, NH), 4.90-5.10 (m, 1H, COOCH), 6.40 (s, 1H, NH₂), 6.80-7.38 (m, 9H, Ar-H), 8.02 (s, 1H, the H on purine ring), 8.16-8.41 (d, 1H, the H on purine ring).MS-ESI:(M+H)⁺: 553.2.

Step 2:(([1,1'-biphenyl]-4-base epoxide) ((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) oxygen Base) methyl) phosphoryl) synthesis of-D-alanine isopropyl ester fumarate (28)

Tenofovir phosphamide derivant 7 (0.6g, 1.0mmol) joins in acetonitrile (10mL), is heated to 80 DEG C to clear Clearly, adding fumaric acid (0.107g, 0.92mmol), after stirring 15 minutes, hot sucking filtration, filtrate is cooled to room temperature, continues at-12 DEG C Placing 12 hours and separate out crystallization, filter, filter cake is washed 1 time with cold acetonitrile, and drying at room temperature is positioned over the vacuum drying of 35 DEG C after 6 hours Case is dried 12 hours to obtain fumarate 28 (0.54g, yield: 74.7%).¹H NMR(400MHz,DMSO-d₆):δ0.92- 1.32(m,12H,4×CH₃), 3.73-4.05 (m, 4H, OCH₂P, NCH and OCH), 4.12-4.35 (m, 4H, NCH₂), 4.71-4.94 (m, 1H, COOCH), 5.47-5.74 (m, 1H, NH), 6.63 (s, 2H, fumaric acid double bond hydrogen), 6.96-7.45 (m, 11H,NH₂And Ar-H), 8.06-8.13 (d, 1H, the H on purine ring), 8.13-8.20 (d, 1H, the H on purine ring), 13.1 (s,2H,2×COOH)。

Reaction scheme 6

Embodiment 6:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (4-cyclopropyl-phenyl Epoxide) phosphoryl)-D-alanine isopropyl ester fumarate (29)

Step 1:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (4-cyclopropyl-phenyl oxygen Base) phosphoryl) synthesis of-D-alanine isopropyl ester (8)

TFV (1.09g, 3.8mmol) is dissolved in pyridine (16mL), be added sequentially 4-cyclopropylphenol (2.56g, 19.08mmol), D-alanine isopropyl ester hydrochlorate 17 (1.15g, 6.8mmol) and triethylamine (6.4mL, 45.76mmol), instead Answer liquid to react 6 minutes at 60 DEG C, add two sulfur two pyridine (5.88g, 26.6mmol), triphenylphosphine (7.01g, 26.7mmol) molten Solution is in the solution of pyridine (24mL), N₂Under protection, 80 DEG C of reactions overnight.Concentrating, the residue obtained is dissolved in dichloromethane.Silicon Plastic column chromatography, first with petroleum ether: ethyl acetate (100:20) eluting then dichloromethane: ethanol=affording of 100:5-7 Compound 8 (1.08g, 55%).¹H NMR(400MHz,CDCl₃):δ0.49-0.60(m,2H,CH₂CH₂), 0.83-0.92 (m, 2H, CH₂CH₂), 1.10-1.33 (m, 12H, 4 × CH₃), 1.12-1.86 (m, 1H, CH), 3.55-3.79 (m, 2H, OCH₂P), 3.81- 4.24(m,4H,NCH₂, NCH and OCH), 4.25-4.43 (m, 1H, NH), 4.90-5.10 (m, 1H, COOCH), 6.40 (s, 1H,NH₂), 6.80-7.38 (m, 4H, Ar-H), 8.02 (s, 1H, the H on purine ring), (d, 1H, on purine ring for 8.16-8.41 H)。MS-ESI:(M+H)⁺: 517.2.

Step 2:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (4-cyclopropyl-phenyl oxygen Base) phosphoryl) synthesis of-D-alanine isopropyl ester fumarate (29)

Tenofovir phosphamide derivant 8 (0.8g, 1.5mmol) joins in acetonitrile (18mL), is heated to 80 DEG C to clear Clearly, adding fumaric acid (163mg, 1.4mmol), after stirring 15 minutes, hot sucking filtration, filtrate is cooled to room temperature, continues to transfer at-12 DEG C Putting 12 hours and separate out crystallization, filter, filter cake is washed 1 time with cold acetonitrile, and drying at room temperature is positioned over the vacuum drying oven of 35 DEG C after 6 hours In be dried 12 hours fumarate 29 (0.73g, yield: 75%).¹H NMR(400MHz,DMSO-d₆):δ0.51-0.63(m, 2H,CH₂CH₂), 0.85-0.94 (m, 2H, CH₂CH₂), 0.92-1.32 (m, 12H, 4 × CH₃), 1.15-1.88 (m, 1H, CH), 3.73-4.05(m,4H,OCH₂P, NCH and OCH), 4.12-4.35 (m, 4H, NCH₂), 4.71-4.94 (m, 1H, COOCH), 5.47-5.74 (m, 1H, NH), 6.63 (s, 2H, fumaric acid double bond hydrogen), 6.96-7.45 (m, 6H, NH₂And Ar-H), 8.06- 8.13 (d, 1H, the H on purine ring), 8.13-8.20 (d, 1H, the H on purine ring), 13.0 (s, 2H, 2 × COOH).

Embodiment 7:(R)-9-[2-[[hexadecane oxygen propyl group [(R)-1-(butyloxycarbonyl)] ethyl] phosphoramidic acid methoxy] Propyl group] adenine (31)

The preparation of compound 31 is with reference to the preparation method of compound 12 in patent CN102786549.TFV(3.6g, 12.5mmol) it is dried overnight in an oven, cooling, adds acetonitrile (100mL), under nitrogen protection, be warming up to 50 DEG C, drip two Chlorine sulfoxide (0.9mL, 12.5mmol), is warming up to 80 DEG C after dropping, reactant liquor stir 2 hours, be evaporated off under decompression acetonitrile and Thionyl chloride, adds dichloromethane (100mL), add at-30 DEG C solid D-alanine isopropyl ester hydrochlorate 17 (2.08g, 12.5mmol), it is slowly added dropwise the dichloromethane solution (50mL) of triethylamine (8.3mL, 60mmol), is warming up to-10 DEG C, react 1 Hour, add the sodium dihydrogen phosphate washing organic facies of 10%, organic facies is dried, and concentrates, column chromatography, obtain compound 30 (2g, 39.8%).Compound 30 (2g, 5.0mmol) is dissolved in DMF (25mL), is subsequently added into 1-(3-bromine propoxyl group) Hexadecane (1.8g, 5.0mmol) and triethylamine (0.85mL, 6.0mmol), reactant liquor stirs 6 hours at 80 DEG C, removes N under reduced pressure, Dinethylformamide, addition ethyl acetate and the mixture (100mL) that ethanol is volume ratio 1:1, be sufficiently stirred for, and filters solid Body, filtrate concentrates, and column chromatography obtains compound 31 (2.1g, productivity: 61%).

¹H NMR(400MHz,CDCl₃):δ0.89(t,3H,CH₃), 1.21-1.39 (m, 38H, 13xCH₂and 4xCH₃), 1.48-1.61(m,2H,CH₂), 1.91-1.96 (m, 2H, CH₂),3.26-3.57(m,6H,3xOCH₂), 3.81-4.11 (m, 6H, OCH₂P,NCH₂And 2xOCH), 4.05-4.24 (m, 1H, NCH), 4.94-5.03 (m, 1H, NH), 6.04 (s, 2H, NH₂), 8.00 (s, 1H, the H on purine ring), 8.34 (s, 1H, the H on purine ring).MS-ESI:(M+H)⁺: 683.4；(M+Na)⁺: 705.4。

Embodiment 8:(((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (((isopropyl) oxygen Base) methoxyl group) phosphoryl)-D-alanine N-butyl (33)

Commercially available tenofovir disoproxil (Tenofovir disoproxil) (3g, 5.7mmol) is joined acetonitrile (15mL) With in water (25mL), it is warming up between 40-45 DEG C, under stirring, drips ammonia, maintain solution alkalescence, be stirred overnight, under low temperature (25-30 DEG C) concentrating under reduced pressure, add dichloromethane (15mL), be stirred at room temperature, cross filter solid obtain ((((((R)-1-(6-amino- 9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (hydroxyl) phosphoryl) epoxide) (1.5g produces isopropyl methyl carbonic ester 32 Rate: 64%).¹H NMR(400H_Z, CDCl₃): δ 0.96 (d, 3H, CH₃), 1.17-1.19 (m, 6H, 2xCH₃), 3.35-3.49 (m, 2H ,-CH₂P), 3.81-3.88 (m, 1H ,-CHO), 4.11-4.25 (m, 2H ,-CH₂-N), 4.70-4.76 (m, 1H, CHO), 5.31-5.4 (m, 2H ,-CH₂P), 8.12 (s, 1H, the H-8 on purine ring), 8.22 (s, 1H, the H-2 on purine ring).MS- ESI:(M-H)⁺: 402.1.

By ((((((R)-1-(6-amino-9H-purine-9-base) acrylate-2-yl) epoxide) methyl) (hydroxyl) phosphoryl) oxygen Base) isopropyl methyl carbonic ester 32 (1.44g, 3.5mmol), D-alanine N-butyl hydrochlorate 26 (1.29g, 7.1mmol), two Sulfur two pyridine (1.57g, 7.1mmol), triphenylphosphine (1.87g, 7.1mmol) are dissolved in 32ml pyridine, N₂Under protection, 60 DEG C stirring 6 minutes after, add triethylamine (2.86ml, 20.8mmol), 80 DEG C reaction overnight.Reactant liquor concentrates, and obtain is residual Staying thing to be dissolved in dichloromethane, silica gel column chromatography, first with petroleum ether: ethyl acetate (100:20) eluting then dichloromethane: methanol =100:5-8 affords compound 33 (0.67g, productivity: 35%).

¹H NMR(400MHz,CDCl₃): δ 0.97 (t, 3H, CH₃), 1.15-1.41 (m, 14H, 4xCH₃and CH₂), 1.53-1.67(m,2H,CH₂), 3.58-3.94 (m, 3H, OCH, COOCH, and NCH), 3.96-4.07 (m, 2H, COOCH₂), 4.08-4.41(m,4H,OCH₂P and NCH₂), 4.83-4.95 (m, 1H, NH), 5.52-5.72 (m, 2H ,-OCH₂O-), 6.24 (s,2H,NH₂), 7.94-8.02 (d, 1H, the H on purine ring), 8.31 (s, 1H, the H on purine ring).ESI-MS:[M+H]⁺: 531.2；[M+Na]⁺: 553.2.

Embodiment 9: the compound antiviral activity detection of the present invention

1.HIV active testing

By 293T cell by every hole 6 × 10⁴Individual density is added on 24 orifice plates, dissolves testing compound with DMSO, and joins Making different concentration, add in cell culture fluid in infecting first 15 minutes, blank made by DMSO solvent, adds 0.5mL sick Virus stock solution used (is diluted to 0.1 0.5ng p24/mL according to p24 concentration) by venom.Infect latter 48 hours, remove supernatant, often Hole adds 50 μ L cell pyrolysis liquid (Promega) cell lysis, then 20 μ L product of cell lysis are added to 30 μ L fluoresceins In zymolyte (Promega), with the relative activity of FB15 fluorescence detector (Sirius) Instrument measuring cell fluorescence element enzyme, with DMSO compares, and calculates the half-inhibition concentration that compounds against wild type HIV-1 replicates.

The 293T cell of exponential phase is seeded in 96 orifice plates by the cell density in 8000-10000/hole, every hole 100 μ L, 37 DEG C, 5%CO₂After incubator is cultivated 24 hours, add testing compound, and (the denseest with DMSO as blank Degree is 0.1%), 37 DEG C, 5%CO₂Incubator continues cultivate 44 hours.Add in every hole that 20 μ L MTS/PMS now join is mixed Conjunction liquid, 37 DEG C, 5%CO₂Develop the color after incubator continuing cultivate 4 hours.On enzyme connection detector, at wavelength 490nm and 650nm Detect the absorbance value (OD) in each hole, at Victor³Glimmering on detection plate in V1420 multiple labeling register (Perkin Elmer) Light, CC obtained by application Microsoft Excel and XLfit4.1 software₅₀Value.

Table 1: the HIV (human immunodeficiency virus)-resistant activity evaluation of synthesis compound:

Experiment conclusion: the compound of synthesis has potent HIV (human immunodeficiency virus)-resistant activity.Contrast with the HIV (human immunodeficiency virus)-resistant activity of TAF, synthesize chemical combination The activity of thing is less than more than 4 times.The reason that HIV (human immunodeficiency virus)-resistant activity reduces be CES1 level expressed in test cell system of institute very Low, being not enough to metabolism in cell provides the TFVDP of suitable concentration.If the cell line measuring HIV can transfect CES1 gene, close The activity becoming compound just can show really.In the cell line surveyed, do not show toxicity.

2. the Anti-HBV effect evaluation of synthesis compound:

Owing to CES1 expresses the lowest at HepG2 2.2.15 cell, so before test synthesis compound, containing complete HBV The HepG2 2.2.15 cell of genome is by using transfection process based on lipid/histone, with mankind CES1 (GenBank Accession number.NM_001025194) transient transfection HepG2 2.2.15 cell.After transfecting 24 hours and 48 hours, use anti-CESI Tag antibody, confirmed the transfection whether success of CESI by Western blotting.

Confirm that the HepG2 2.2.15 cell of transfection CESI has complete HBV gene group, and stably express viral RNA, CccDNA and virus protein.The method being quantified virion DNA by qPCR can measure viral duplication.Testing compound It is dissolved as the storage liquid of 30mM with DMSO and is saved in-20 DEG C.10,000, every hole HepG2 is added in 96 porocyte culture plates 2.2.15 cell, every hole 200 μ L cell culture medium, at 37 DEG C, 5%CO₂Cell culture incubator is cultivated and within 3 days, covers with to cell.Abandon Fall old culture medium and add 200 detection culture medium (5%FBS) fresh for μ L.The compound 1 μ L of addition 100%DMSO dilution: It is diluted to the different test concentrations specified, at CO₂Hatching in incubator 10 days, every other day (the 2nd, 4,6,8,10 day) changes one Not good liquor (5%FBS), and add the compound of Fresh concentration.At the 11st day, every hole took 150 μ L of supernatant and extracts viral DNA.Carefully Cellular toxicity detection plate is also carried out similar process: maximum concentration is 150 μMs.The extraction test kit of virus genom DNA is QIAamp 96DNA Blood Kit.Through conventional centrifugal and QPCR process.With plasmid (viral copy number: the 2* comprising HBV gene group 10E6,2*10E5,2*10E4,2*10E3) do standard curve, and calculate viral copy number with standard curve.The meter of suppression ratio Calculation formula is as follows: the suppression ratio=100-(detected value-HPE meansigma methods) of antiviral/(ZPE meansigma methods-HPE meansigma methods) * 100 (ZPE: least concentration compound well meansigma methods, HPE: maximum concentration compound well meansigma methods).Suppression ratio data are passed through Graphpad Prism 5 software processes also draws curve, EC₅₀And EC₉₀Calculated by four parameter nonlinear regression models.Cell Toxicity %=100-(detected value/DMSO control wells meansigma methods * 100).Cytotoxicity % data pass through Graphpad Prism 5 Software processes also draws curve, CC₅₀Calculated by four parameter nonlinear regression models.

Table 2: the Anti-HBV effect evaluation of synthesis compound:

Experiment conclusion: the compound of nearly all synthesis has the strongest Anti-HBV effect, is better than the work of document report TDF Property.Compound and the TAF of synthesis compare, and except compound 31, other compounds all show higher antiviral activity.Institute In the cell line surveyed, do not show toxicity.

Owing to acyclonucleosides phosphamide D-amino acid ester derivative is compared with TAF, there is significant metabolism specificity, be only subject to CES1 metabolism hydrolyzes.CES1 is relatively low in the concentration of intestinal epithelial cell, can significantly alleviate synthesis compound in absorption process generation Thank, it addition, D-amino-acid ester is also more difficult to hydrolysis than l-amino acid in blood plasma, and be easier to be absorbed by cell, so synthesis Compound can effectively reduce TFV concentration in blood plasma, the concentration at hepatocyte TFV significantly improves, and reduces further and faces The nephrotoxicity of bed and raising bioavailability.CES1 dense and activity in hepatocyte is the strongest, the D-amino of synthesis Ester compound can be in liver effectively and rapid metabolization narrow spectrum Liver targeting prodrug, therefore at the HepG2 of transfection CES1 2.2.15, in cell, majority of compounds shows more excellent anti-hepatitis B activity.Owing to the screening system of this HBV can not be effective Expression phospho-esterase c, so compound 31 activity is the lowest.Owing to the compound of this Patent design decomposes less in blood plasma, Being prone to be absorbed by histiocyte and hepatocyte and metabolism efficiently and effectively, toxicity is less, has fabulous clinical value.

Embodiment 10: choose the compound 18,21 and 31 stability study in people's whole blood and compound and metabolism is produced Thing is in blood plasma and the distribution of peripheral blood lymphocytes (PBMCs)

1. by the synthesis compound of close concentration and TAF at the common incubation of human whole blood 1 hour and 2 hours of 37 DEG C After, isolate blood plasma and PBMCs cell (Ficolli density－gradient centrifuga－tion method), discard erythrocyte.

2. take 100 μ L blood plasma and be separately added into 20 μ L internal standard liquid (400ng/mL SN-38 solution), 5 μ L methanol/water (v/v:1: 1) and 200 μ L acetonitriles, eddy current mixes, centrifugal 5 minutes, extracts 20 μ L of supernatant liquid and 180 μ L water, and eddy current mixes, and takes 10 μ L mixing Liquid carries out LC/MS/MS analysis.

3. count PBMCs cell with cell counter, and be that 200fL (ascending to heaven) calculates by each PBMCs cell, take 100 μ L PBMCs cell, adds 20 μ L internal standard liquid (400ng/mL SN-38 solution), 5 μ L methanol/water (v/v:1:1) and 200 μ L second Nitrile, eddy current mixes, centrifugal 5 minutes, extracts 20 μ L of supernatant liquid and 180 μ L water, and eddy current mixes, and takes 10 μ L mixed liquors and carries out LC/MS/ MS analyzes.

Table 3: synthesis compound stability in people's whole blood and synthesis compound and metabolite TFV at blood plasma and Distribution in PBMCs cell

Experiment conclusion: TAF has metabolism slowly, in animal experiment and clinical trial, the speed of TAF metabolism in people's whole blood The degree TAF to be far longer than speed in static blood plasma.In comparison, compound 18,21 and 31 is the most steady in people's whole blood Fixed.Compound 18,21 and 31 is easily absorbed by cell, and metabolism is rapid, so compound 18,21 and 31 is at PBMCs cell The concentration of middle TFV is significantly superior to TAF.

More than test result indicate that, the compound of the present invention has efficient AntiHIV1 RT activity and the ability of HBV virus in vivo, with Positive control drug TDF with TAF compares, and these compounds will have more excellent antiviral activity in vivo, and nephrotoxicity is little, safety Property good, have treatment HIV and HBV virus infect good clinical landscapes.

Although the detailed description of the invention of the present invention has been described in detail, but according to it will be appreciated by those skilled in the art that On the premise of without departing from the spirit and scope of the present invention, the present invention can be carried out various modifications and changes all the present invention's Within protection domain.The interest field of the present invention is not limited to the detailed description made above, and should belong to the power of the present invention Profit claim.

Claims (9)

1. an acyclonucleosides phosphamide D-amino acid ester derivative or its stereoisomer, officinal salt, hydrate, solvent conjunction Thing or crystallization, it is characterised in that: having formula I, the structure of formula I is,

Wherein:

(1)R₁It is independently selected from C_1-6Alkyl or aryl alkyl；R₄It is independently selected from methyl or hydrogen；

(2)R₂It is independently selected from alkyl, cycloalkyl-alkyl, cycloalkyl or benzyl；

(3) X is selected from oxygen, NH or sulfur；

(4)R₃Selected from aryl, heteroaryl, C_1-20Alkyl, cycloalkyl, alkoxyalkyl, alkanoyl oxyalkyl, aroyl oxyalkyl Or alkoxy carbonyl group oxyalkyl；Aryl is selected from unsubstituted aryl or substituted aryl；Heteroaryl selected from unsubstituted heteroaryl or Substituted heteroaryl；Substituent group on substituted aryl and substituted heteroaryl is selected from deuterium, halogen, alkyl, cycloalkyl, alkyl halide Base, halogenated cycloalkyl, cycloalkyl-alkyl, cycloalkyl haloalkyl, cyano group, substituted amino, thiazolinyl, phenyl, alkyl silyl；Not Substituted aryl is selected from phenyl, xenyl or naphthyl.

Acyclonucleosides phosphamide D-amino acid ester derivative the most according to claim 1 or its stereoisomer, pharmaceutically acceptable Salt, hydrate, solvate or crystallization, it is characterised in that: removing outside active hydrogen in the compound of formula I, remaining is all of Hydrogen atom can separately be replaced by deuterium.

Acyclonucleosides phosphamide D-amino acid ester derivative the most according to claim 1 or its stereoisomer, pharmaceutically acceptable Salt, hydrate, solvate or crystallization, it is characterised in that: the compound phosphorus atoms in formula I has chirality, and its configuration is S- Configuration or R-configuration, or S-configuration and the mixture of R-configuration.

Acyclonucleosides phosphamide D-amino acid ester derivative the most according to claim 1 or its stereoisomer, pharmaceutically acceptable Salt, hydrate, solvate or crystallization, it is characterised in that: with acyclonucleosides phosphamide D-aminoacid in described officinal salt Ester derivant purine amino part becomes the pharmaceutically acceptable acid of salt selected from phosphoric acid, hydrochloric acid, sulphuric acid, hydrobromic acid, citric acid, horse Come sour, malonic acid, mandelic acid, succinic acid, fumaric acid, acetic acid, lactic acid, nitric acid, sulfonic acid, p-methyl benzenesulfonic acid, malic acid or first sulphur Acid；Acid and acyclonucleosides phosphamide D-amino acid ester derivative mol ratio are in the range of 0.5 to 1.

5. a medical composition, it is characterised in that: non-according to any one of the Claims 1-4 containing dose therapeutically effective Ring nucleoside phosphoramidite D-amino acid ester derivative or its stereoisomer, officinal salt, hydrate, solvate or crystallization, institute The compositions stated is tablet, capsule, injection or nanometer formulation.

Medical composition the most according to claim 5, it is characterised in that: comprise other therapeutic agent and reinforcing agent；Wherein Described other therapeutic agent is independently selected from the group of consisting of: the non-nucleoside of hiv protease inhibitor, hiv reverse transcriptase suppresses Agent, hiv reverse transcriptase nucleosidic inhibitors, hiv reverse transcriptase nucleotide inhibitor, hiv integrase inhibitor and CCR5 inhibitor, HBV capsid inhibitor (capsid inhibitor), cccDNA form inhibitor, cccDNA epigenetic modification agent or hepatitis B RNAi medicine；Reinforcing agent include ritonavir and than west he (Cobicistat).

7. at least one purposes in following (1)-(2) item:

(1) HIV (human immunodeficiency virus) (HIV) or hepatitis B and the method for hepatitis B virus are infected in a kind for the treatment of and/or prevention, its It is characterised by: include the acyclonucleosides phosphamide D-amino acid ester derivative according to any one of Claims 1-4 or its solid Isomer, officinal salt, hydrate, solvate, crystallization or the pharmaceutical composition described in claim 5 or 6 are used in preparation Application in the medicine for the treatment of and/or the medicine of pre-preventing HIV infection or hepatitis B and hepatitis B virus infection；

(2) a kind of method simultaneously treating and preventing HIV and HBV, it is characterised in that: include institute any one of Claims 1-4 The acyclonucleosides phosphamide D-amino acid ester derivative stated or its stereoisomer, officinal salt, hydrate, solvate, knot Pharmaceutical composition described in crystalline substance or claim 5 or 6 is in preparing the medicine for treating and/or prevent HIV and HBV infection Application.

Acyclonucleosides phosphamide D-amino acid ester derivative the most according to claim 1 or its stereoisomer, pharmaceutically acceptable Salt, hydrate, solvate or crystallization, it is characterised in that: there is the acyclonucleosides phosphamide D-amino-acid ester shown in formula I and spread out Biological officinal salt includes following structure:

Wherein:

(1)R₁It is independently selected from C_1-6Alkyl or aryl alkyl；R₄It is independently selected from methyl or hydrogen；

(2)R₂It is independently selected from alkyl, cycloalkyl-alkyl, cycloalkyl or benzyl；

(3) X is selected from oxygen, NH or sulfur；

(4)R₃Selected from aryl, heteroaryl, C_1-20Alkyl, cycloalkyl, alkoxyalkyl, alkanoyl oxyalkyl, aroyl oxyalkyl Or alkoxy carbonyl group oxyalkyl；Aryl is selected from unsubstituted aryl or substituted aryl；Heteroaryl selected from unsubstituted heteroaryl or Substituted heteroaryl；Substituent group on substituted aryl and substituted heteroaryl is selected from deuterium, halogen, alkyl, cycloalkyl, alkyl halide Base, halogenated cycloalkyl, cycloalkyl-alkyl, cycloalkyl haloalkyl, cyano group, substituted amino, thiazolinyl, phenyl, alkyl silyl；Not Substituted aryl includes phenyl, xenyl and naphthyl；

(5) acid be independently selected from phosphoric acid, sulphuric acid, hydrochloric acid, hydrobromic acid, citric acid, maleic acid, malonic acid, mandelic acid, succinic acid, Fumaric acid, acetic acid, lactic acid, nitric acid, sulfonic acid, p-methyl benzenesulfonic acid, malic acid, Loprazolam.

Acyclonucleosides phosphamide D-amino acid ester derivative the most according to claim 1 or its stereoisomer, officinal salt, water Compound, solvate or crystallization, it is characterised in that: the described acyclonucleosides phosphamide D-amino-acid ester having shown in formula I Derivant, its stereoisomer or officinal salt include following structure: