CN108350007A - A kind of substituted adenine compound and its pharmaceutical composition - Google Patents

Abstract

A kind of substituted adenine compound and its pharmaceutical composition, the substituted adenine compound is as being formula（I）Compound represented or its crystal form, pharmaceutically acceptable salt, prodrug, stereoisomer, hydrate or solvated compounds.The compound can inhibit nucleoside reverse transcriptase activity, while have more preferable pharmacodynamics/pharmacokinetics performance, and the applicability of compound is good, safe, can be used for preparing treatment virus and infects relevant disease, has the good prospect of marketing.

Description

A kind of substituted adenine compound and its pharmaceutical composition Technical field

The invention belongs to pharmaceutical technology field more particularly to a kind of substituted adenine compounds and its pharmaceutical composition, can be used for treating the related disease of virus infection.

Background technique

Efabirenz (NRTIs), it is the analog for synthesizing the DNA reverse transcriptase substrate deoxynucleotide of HIV, it is converted to active nucleoside triphosphate derivative in vivo, it is combined with natural deoxy-ribonucleoside triphosphate competitiveness and hiv reverse transcriptase (RT), the effect for inhibiting RT, hinders the synthesis of provirus.The structure of NRTIs is similar with nucleosides, is dideoxyribonucleoside derivative, reverse transcriptase can be competitively combined with intracellular nucleosides, to terminate reverse transcription reaction.

Ucleotides hiv reverse transcriptase inhibitor acts on active site of the reverse transcriptase in conjunction with its natural substrate nucleosides.Such drug is the class drug of natural nucleus glycoside, by multistep phosphorus acylation reaction, being metabolized as real bioactive molecule triphosphoric acid nucleosides (NRTI-ppp), they and endogenic dNTP competitively act on the substrate active position of enzyme after in vivo.Since the structure of NRTI-ppp is very much like in dNTP substrate, once this kind of drug can be mistakenly considered substrate and be embedded into just in extended DNA chain that these drugs enter DNA chain by enzyme, since 3 ' -5 ' the 3 '-hydroxyls that be connected can not be carried out in the structure of drug molecule with next dNTP.The extension for thus having blocked viral DNA chain, also may refrain from the duplication of HIV.

In recent ten years, resisting HIV (human im-munodeficiency virus, HIV) drug research achieves huge progress.Currently, United States Food and Drug Administration Guidelines has had been approved by least 27 kinds for treating the antiviral drugs of HIV infection.Inverase mainly includes 4 major class: efabirenz (NRTIs), non-nucleoside reverse transcriptase inhibitor (NNRTIs), protease inhibitors (PIs) and hiv integrase inhibitor, wherein NRTIs is one kind most using earliest, kind, it mainly include Zidovudine (AZT) that Lamivudine removes fixed carboxylic anhydride, Stamford, Abacavir and tenofovir.

AIDS is the serious disease as caused by HIV infection, and since Patient With Aids case in 1981 is reported, the whole world is accumulative to be had nearly 70,000,000 people and infected by AIDS virus, and more than 2,000 ten thousand people die of AIDS.In past 20 years, although effective drug therapy was once declined the death rate of AIDS, millions of people is still increased newly by HIV infection every year, the number of global AIDS patient is in rising trend always.

However, HIV generates drug resistance to the inverase of nearly all clinical use at present, the generation of drug resistance HIV is considered as the main reason for leading to inverase treatment failure.

In addition, chronic hepatitis is to threaten one of the serious infectious disease of global human health.There are about more than 20 hundred million people once hepatitis b virus infection (Hepatitis B Virus, HBV) in the whole world, every year due to HBV infection and dead number up to 1,000,000.HBV infection is not only to cause chronic hepatitis B and is the important biomolecule factor for causing primary carcinoma of liver.China, Southeast Asia and Africa are the districts occurred frequently of HBV infection, and the incidence of primary carcinoma of liver is significantly higher than the area Di Fa of the HBV infections such as in the middle part of America and south.The existing treatment of chronic hepatitis B mainly has interferon, nucleoside medicine, thymosin extrasin, but these long term combined uses will appear more serious toxic side effect or generate drug resistance, and expensive.Therefore, finding novel, effective Anti-HBV drugs is urgent problem.

Therefore, there is still a need for the compounds that exploitation has inhibitory activity or more preferable pharmacodynamics performance to nucleoside reverse transcriptase for this field.

Summary of the invention

Against the above technical problems, the invention discloses a kind of efabirenz, pharmaceutical composition and its applications, with better nucleoside reverse transcriptase inhibitory activity and/or have more preferable pharmacodynamics/pharmacokinetics performance.

In this regard, the technical solution adopted by the present invention are as follows:

A kind of efabirenz, the adenine compound or its crystal form, pharmaceutically acceptable salt, prodrug, stereoisomer, hydrate or solvated compounds replaced as shown in formula (I),

Wherein, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶It is each independently hydrogen, deuterium, halogen or trifluoromethyl；

Additional conditions are R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵And R²⁶In at least one be deuterated or deuterium.

As a further improvement of the present invention, R¹And R²It is each independently deuterium or hydrogen.

As a further improvement of the present invention, R³、R⁴、R⁵、R⁶、R⁷And R⁸It is each independently deuterium or hydrogen.

As a further improvement of the present invention, R⁹And R¹⁰It is each independently deuterium or hydrogen.

As a further improvement of the present invention, R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰And R²¹It is each independently deuterium or hydrogen.

As a further improvement of the present invention, R²³、R²⁴、R²⁵And R²⁶It is each independently deuterium or hydrogen.

As a further improvement of the present invention, the compound can be selected from following compounds or its pharmaceutically acceptable salt, but be not limited to following compounds:

It adopts this technical solution, shape and volume of the deuterium in drug molecule are substantially the same with hydrogen, if hydrogen is optionally replaced with deuterium in drug molecule, deuterated drug generally can also retain original bioactivity and selectivity.Inventor passes through it is experimentally confirmed that the combination of carbon deuterium key is more more stable than the combination of C-H bond simultaneously, the attributes such as absorption, distribution, metabolism and the excretion of some drugs can be directly affected, to improve the curative effect of drug, safety and tolerance.

Preferably, deuterium isotopic content of the deuterium in deuterated position is at least greater than natural deuterium isotopic content (0.015%), is preferably greater than 30%, even more preferably greater than 50%, even more preferably greater than 75%, even more preferably greater than 95%, even more preferably greater than 99%.

Specifically, R in the present invention¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵And R²⁶Deuterium isotopic content is at least 5% in each deuterated position, is preferably greater than 10%, even more preferably greater than 15%, even more preferably greater than 20%, even more preferably greater than 25%, even more preferably greater than 30%, even more preferably greater than 35%, even more preferably greater than 40%, even more preferably greater than 45%, even more preferably greater than 50%, even more preferably greater than 55%, even more preferably greater than 60%, even more preferably greater than 65%, even more preferably greater than 70%, even more preferably greater than 75%, even more preferably greater than 80%, even more preferably greater than 85%, more preferably Greater than 90%, even more preferably greater than 95%, even more preferably greater than 99%.

In another preferred example, in formula (I) compound R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵And R²⁶, at least one of which R contains deuterium, more preferably two R contain deuterium, more preferably three R contain deuterium, more preferably four R contain deuterium, more preferably five R contain deuterium, more preferably six R contain deuterium, more preferably seven R contain deuterium, more preferably eight R contain deuterium, more preferably nine R contain deuterium, more preferably ten R contain deuterium, more preferably 11 R contain deuterium, more preferably 12 R contain deuterium, more preferably 13 R contain deuterium, more preferably 14 R contain deuterium, more preferably 15 R contain deuterium, more preferably 16 R contain deuterium, more preferably 17 R contain deuterium, more preferably 18 R contain deuterium, more preferably 19 R contain deuterium, more preferably 20 R contain deuterium, more preferably 21 R contain deuterium, more preferably 22 R contain deuterium, more preferably 23 R contain deuterium, more preferably 24 R contain deuterium, more preferably 25 R contain deuterium, more preferably 26 R contain Deuterium.

In another preferred example, the compound does not include non-deuterated compound.

The invention also discloses a kind of pharmaceutical compositions, its pharmaceutical composition for containing pharmaceutically acceptable carrier with the efabirenz or its crystal form, pharmaceutically acceptable salt, hydrate or solvate as described above, stereoisomer, prodrug or isotopic variations.

As a further improvement of the present invention, the pharmaceutically acceptable carrier includes at least one of glidant, sweetener, diluent, preservative, dyestuff/colorant, flavoring reinforcing agent, surfactant, wetting agent, dispersing agent, disintegrating agent, suspending agent, stabilizer, isotonic agent, solvent or emulsifier.

As a further improvement of the present invention, described pharmaceutical composition is tablet, pill, capsule, pulvis, granule, paste, emulsion, suspending agent, solution, suppository, injection, inhalant, gelling agent, microballoon or aerosol.

The classical pathway for giving pharmaceutical composition of the present invention includes but is not limited to oral, rectum, saturating mucous membrane, through enteral administration, or part, percutaneous, sucking, parenteral, sublingual, intravaginal, it is intranasal, intraocularly, peritonaeum is interior, intramuscular, subcutaneous, intravenous administration.It is preferred that oral administration or drug administration by injection.

Pharmaceutical composition of the invention can be manufactured using method well known in the art, such as conventional mixing method, dissolution method, granulation, dragee method processed, levigate method, emulsion process, freeze-drying.

The present invention also provides a kind of methods for preparing pharmaceutical composition, comprising steps of by pharmaceutically acceptable carrier and efabirenz as described above, or its crystal form, pharmaceutically acceptable salt, hydrate or solvate are mixed, and pharmaceutical composition is formed.

It as a further improvement of the present invention, also include other reactive compounds, the reactive compound can be selected from: non-nucleoside reverse transcriptase inhibitor (NNRTIs), protease inhibitors.

Active constituent of the invention can also be used in combination with other active components.This combined selection based on treatment the case where, composition cross reactivity and united pharmaceutical properties.It is also possible to that any compound of the invention is made to combine one or more other active components, is concurrently or consecutively administered to a patient with single formulation.Combination therapy can concurrently or consecutively Dosage Regimens Dosage.When given continuously, application can be administered in joint two or more times.Combination therapy can provide " synergistic effect " or " synergistic effect ", in other words, when active constituent together It is greater than using the effect of acquisition and is used separately the sum of compound income effect.Work as active constituent: (1) by co-formulation and administration or in the form of combination preparation while delivering；(2) as independent preparation alternating delivery or parallel administration；Or (3) by some other dosage regimens when, can get synergistic effect.When delivering with alternating treatment, when compound sequential administration or release, such as with independent tablet, pill or capsule, or by the difference injection of independent syringe, synergistic effect can get.In general, during alternating treatment, every kind of active constituent effective dose is sequential, i.e., continuously give, and in combination therapy, the effective dose of two or more active constituents is given jointly.

The invention also discloses the purposes of adenosine class reverse transcriptase inhibitor that one kind replaces as described above, i.e. the compounds of this invention therapeutic agent that can be advantageously suitable as treating the symptom such as AIDS and hepatitis B.

It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and it can be combined with each other between each technical characteristic specifically described in below (e.g. embodiment), to form a new or preferred technical solution.Due to space limitations, I will not repeat them here.

Herein, unless otherwise instructed, " halogen " refers to F, Cl, Br and I.More preferably, halogen atom is selected from F, Cl and Br.

Herein, unless otherwise instructed, " deuterated " refers to one or more hydrogen in compound or group replaced deuterium；It is deuterated to can be a substitution, two replace, polysubstituted or full substitution.Term " one or more deuterated " is used interchangeably with " one or many deuterated ".

Herein, unless otherwise instructed, " non-deuterated compound " refers to that ratio containing D-atom is not higher than the compound of natural deuterium isotopic content (0.015%).

Composition of the invention optionally includes the salt of compound here, and especially pharmaceutically acceptable nontoxic salt contains, for example, Na⁺、Li⁺、K⁺、Ca⁺²And Mg⁺².These salt may include by suitable cation, such as alkali and alkaline-earth metal ions or ammonium and tetravalence amino ion and acid anion moiety, typically carboxylic acid in conjunction with and derivative salt.Such as want to obtain water soluble salt, then preferred monovalent salt.Metal salt reacts preparation with the compound of the present invention typically via making metal hydroxides.It is containing Li in the embodiment of the metal salt of this method preparation⁺、Na⁺And K⁺Salt.By adding suitable metai compounds, more insoluble metallic salt can be precipitated out from more soluble salt solutions.In addition, salt can be formed by adding certain organic acids and inorganic acid, for example, HCl, HBr, H₂SO₄、H₃PO₄Or organic sulfonic acid, basic center is arrived, typically amine, or formed to acidic-group.Finally, it will be understood that composition herein includes the water combination of the stoichiometric amount with the compounds of this invention existing for their unionized and zwitterionic forms, in He Yuru hydrate.It within the scope of the present invention also include the salt of parent compound and one or more amino acid.Any one above-mentioned amino acid is all suitable, especially as the amino acid of protein component discovery naturally occurred, although the amino acid is typically a kind of amino acid with the side chain with alkalinity or acidic-group, such as, lysine, arginine or glutamic acid, or the amino acid with the side chain with neutral group, for example, glycine, serine, threonine, alanine, isoleucine or leucine.

The compounds of this invention can have chiral centre, for example, chiral carbon or phosphorus atoms.Therefore the compounds of this invention includes the racemic mixture of all stereoisomers, including enantiomer, diastereomer and atropisomer.In addition, the compounds of this invention includes the enrichment on any or whole asymmetric chiral atoms or fractionation optical isomer.In other words, obvious chiral centre is provided as chiral isomer or racemic mixture from description.Racemic mixture and diastereomeric mixture, and be substantially free of they mapping or diastereomeric gametophyte, separation or synthesis independent optical isomer, fully fall in protection scope of the present invention.By known technology, for example, the auxiliary agent of separation optically active is for example, the diastereomeric salt that acid or alkali are formed, then goes back to as optically active material, racemic Mixture is separated into their single, substantial optically pure isomer.In most cases, conceivable optical isomer is synthesized since the suitable stereoisomer of desired raw material by stereospecific reaction.

In some cases, the compounds of this invention may also exist in the form of tautomer.Although only a kind of non-localized resonant structure may be described, but it is envisioned that all this kind of forms are all within the scope of the present invention.For example, for purine, pyrimidine, imidazoles, guanidine, amidine and tetrazolium system, there may be and their all possible tautomeric forms are all fallen in the scope of the present invention alkene-amine tautomer.

Term " solvate " refers to that the compounds of this invention and solvent molecule are coordinated the complex to form special ratios." hydrate " refers to that the compounds of this invention and water carry out the complex of coordination formation.

Compared with prior art, the invention has the benefit that the compound of the present invention has excellent inhibition to nucleoside reverse transcriptase；Change metabolism of the compound in organism by this technology of deuterate, makes compound that there is better pharmacokinetic parameter characteristic.In such a case, it is possible to change dosage and form durative action preparation, improve applicability；The drug concentration of compound in animal body is improved, curative effect of medication is improved due to its deuterium isotope effect with the hydrogen atom in deuterium substituted compound；With the hydrogen atom in deuterium substituted compound, certain metabolites can be inhibited, improve the safety of compound.

Specific embodiment

The preparation method of formula (I) structural compounds of the present invention is described more particularly below, but these specific methods do not form any restrictions to the present invention.Various synthetic methods describing in the present specification or known in the art can also optionally be combined and are easily made by the compounds of this invention, and such combination can be easy to carry out by those skilled in the art in the invention.

In general, in preparation flow, each reaction usually in atent solvent, room temperature to reflux temperature (such asIt is preferred that ) under carry out.Reaction time is usually -60 hours 0.1 hour, preferably 0.5-24 hours.

Embodiment 1 prepares 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } -2; 4; 6-d3- benzene oxygen phosphoryl) methoxyl group] propyl } adenine; i.e. compound T-1, molecular formula are as follows:

It is synthesized using following route:

The synthesis of step 1 (R) -9- (2- hydroxypropyl) adenine (compound 1).

Adenine (4.0g is added in reaction flask, 29.6mmol) and (R)-propene carbonate (3.45g, 33.8mmol), 4.5ml DMF dissolution is added, it is heated to 130 DEG C of reactions overnight, after contact plate detects fully reacting, it is cooled to 100 DEG C, 14ml toluene and 0.47g methanesulfonic acid (temperature is at 100-110 DEG C in keeping) is added, adds 11ml toluene and obtains a homogeneous suspension, be gradually cooled to room temperature, 0 DEG C is cooled to again to be kept for 1 hour, the white solid of filtering is dried in vacuo to obtain 5.77g product, yield 100%.LC-MS (APCI): m/z=194.3 (M+1)⁺。

The synthesis of step 2 diethyl [[(p-toluenesulfonyl) oxygroup] methyl] phosphate (compound 2).

Methylol diethyl phosphate (3.85g is added in reaction flask, 22.89mmol), the dissolution of 30ml anhydrous ether is added, triethylamine (3.38ml is added dropwise, 24.04mmol), it finishes, is cooled to -10 DEG C, paratoluensulfonyl chloride (4.58g is added dropwise, 10ml diethyl ether solution 24.04mmol), it finishes, is stirred to react at 0 DEG C 3 hours, then be warmed to room temperature reaction overnight.A small amount of ether dilution is added, is filtered to remove inorganic salts, filtrate concentration, silica gel column chromatography purifying is dried in vacuo to obtain product 5.4g, yield 73.2%.LC-MS (APCI): m/z=323.1 (M+1)⁺。

The synthesis of step 3 (R) -9- [2- (diethylphosphoryl methoxyl group) propyl] adenine (compound 3).

By 1 (1.0g of compound; 5.17mmol) dissolved with 40ml anhydrous DMF; it is cooled to 0 DEG C, is added under NaH (233.3mg) low temperature and reacts 40 minutes under nitrogen protection, 2 (1.75g of compound is added; 10ml anhydrous DMF dissolution 5.44mmol); it is warmed to room temperature reaction 18 hours, concentration removes solvent, silica gel column chromatography purifying after completion of the reaction for contact plate detection; product 0.88g, yield 50% are obtained after being evaporated.LC-MS (APCI): m/z=344.5 (M+1)⁺。

The synthesis of step 4 (R) -9- [2- (phosphatidyl methoxy) propyl] adenine (compound 4).

3 (2.276g of compound is added in dry reaction flask, 6.63mmol), the dissolution of 20ml anhydrous DMF is added, TMSBr (3.76g is added at room temperature, 24.57mmol), it is stirred to react 20 hours, contact plate detects after the reaction was completed, and concentration removes solvent, ammonium hydroxide tune PH to 8.0 is added, it is concentrated to give oily liquids, with dilute hydrochloric acid tune PH to 3.0, is evaporated again, isopropanol is added, yellow solid is precipitated, filtering, recrystallizes to obtain white solid 0.57g, yield 30.1% with isopropanol/water (31).LC-MS (APCI): m/z=286.7 (M-1)^-。

The synthesis of step 5 2,4,6-d3- phenol (compound 5).

Phenol (2.0g, 21.25mmol) and sodium hydroxide (0.425g, 10.63mmol) are added in microwave reaction bottle, the dissolution of 15ml heavy water is added, sealing, which is placed in microwave reactor, reacts 0.5 hour for 180 DEG C.It is down to room temperature, with dilute hydrochloric acid tune PH to acidity, ethyl acetate is extracted 3-4 times, merges organic phase, and saturated common salt water washing is concentrated, and silica gel column chromatography purifies to obtain compound 1.8g, yield: 90%.LC-MS (APCI): m/z=98.1 (M+1)⁺。

The synthesis of step 6 (R) -9- [2- (2,4,6-d3- phenoxy group phosphatidyl methoxy) propyl] adenine (compound 6).

4 (2.4g of compound is added in reaction flask, 8.36mmol), 5 (1.62g of compound, 16.72mmol) and 6.5ml NMP, 85 DEG C are heated to, is added triethylamine (1.04g, 10.3mmol), it is warming up to 100 DEG C, it is added dicyclohexylcarbodiimide (2.81g, 13.63mmol), then rises to 120 DEG C and be stirred to react 16 hours.Contact plate detection raw material is cooled to 45 DEG C after disappearing, and 4.8ml water is added, is down to room temperature, it is filtered to remove insoluble matter, with 2.5ml water washing filter cake, filtrate concentration, 4ml water is added, with NaOH tune PH to 11, chloroform is extracted 3-4 times, water phase concentrated hydrochloric acid tune PH to 3.1 is extracted 4-5 times with chloroform/isopropanol (3:1), merges organic phase, it is evaporated, the mashing purifying of a small amount of methanol is added, dry product 1.84g, yield 60.1% after filtering.LC-MS (APCI): m/z=367.3 (M+1)⁺。

Step 7 9- { (R) -2- [((R; S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } -2; 4,6-d3- benzene oxygen phosphoryl) methoxyl group] propyl adenine (compound 7) synthesis.

Compound 6 (0.866g, 2.364mmol) is added in reaction flask, is dissolved with 5ml acetonitrile, it is added thionyl chloride (635.3mg, 5.34mmol), is heated to 80 DEG C and reacts 2 hours, concentration removes solvent, and 4ml anhydrous methylene chloride is added, is cooled to -29 DEG C, the 3ml dichloromethane solution of alanine isopropyl ester (682.2mg, 5.2mmol) is added dropwise, finishes, triethylamine (717.6mg is added dropwise, 7.1mmol), reaction 1 hour, contact plate detection are warmed to room temperature.A small amount of water washing is added, saturated common salt water washing, silica gel column chromatography purifies after concentration, obtains 0.74g product, yield 65%.LC-MS (APCI): m/z=480.5 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 8.33 (d, J=7.1Hz, 1H), 7.98 (d, J=2.0Hz, 1H), 7.30 (s, 1H), 7.20 (s, 1H), 5.80 (s, 2H), 5.06-4.85 (m, 1H), 4.39 (m, 1H), 4.22-4.08 (m, 1H), 3.94 (m, 4H), 3.64 (m, 2H), 1.21 (m, 12H).

Step 8 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } -2; 4,6-d3- benzene oxygen phosphoryl) methoxyl group] propyl adenine (compound T-1) separation.

Raceme compound 7 (100mg) is carried out by isolated target product T-1 using chiral supercritical fluid chromatography column (SFC), weigh to obtain 45mg after dry, yield: 90%.LC-MS (APCI): m/z=480.5 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 8.33 (d, J=7.1Hz, 1H), 7.98 (d, J=2.0Hz, 1H), 7.30 (s, 1H), 7.20 (s, 1H), 5.80 (s, 2H), 5.06-4.85 (m, 1H), 4.39 (m, 1H), 4.22-4.08 (m, 1H), 3.94 (m, 4H), 3.64 (m, 2H), 1.21 (m, 12H).

Embodiment 2 prepares 9- { (R) -2- [((S)-{ [(S) -1- (d7- butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } adenine; that is compound T-2, molecular formula are as follows:

Using following synthetic route:

The synthesis of step 1 (R) -9- [2- (phenoxy group phosphatidyl methoxy) propyl] adenine (compound 8).

4 (2.4g of compound is added in reaction flask, 8.36mmol), phenol (1.62g, 16.72mmol) and 6.5ml NMP, 85 DEG C are heated to, is added triethylamine (1.04g, 10.3mmol), it is warming up to 100 DEG C, it is added dicyclohexylcarbodiimide (2.81g, 13.63mmol), then rises to 120 DEG C and be stirred to react 16 hours.Contact plate detection raw material is cooled to 45 DEG C after disappearing, and 4.8ml water is added, is down to room temperature, it is filtered to remove insoluble matter, with 2.5ml water washing filter cake, filtrate concentration, 4ml water is added, with NaOH tune PH to 11, chloroform is extracted 3-4 times, water phase concentrated hydrochloric acid tune PH to 3.1 is extracted 4-5 times with chloroform/isopropanol (3:1), merges organic phase, it is evaporated, the mashing purifying of a small amount of methanol is added, dry product 1.24g, yield 40.4% after filtering.LC-MS (APCI): m/z=364.3 (M+1)⁺。

The synthesis of step 2L- alanine-d7- isopropyl ester (compound 9).

L-Alanine (0.88g, 9.87mmol) and deuterated isopropanol (5.0g, 73.4mmol) are added in reaction flask, is added to flowing back, is added dropwise trim,ethylchlorosilane (1.79g, 16.5mmol), finishes, back flow reaction is overnight.Concentration removes solvent, and the 5ml tetrahydrofuran solution of triethylene diamine (1.11g, 9.87mmol) is added, white opacity liquid, diatomite drainage are obtained, filter cake is washed twice with methylene chloride, filtrate is concentrated to give crude product, and vacuum drying obtains 1.7g, yield 85%.LC-MS (APCI): m/z=139.1 (M+1)⁺。

The synthesis of step 3 9- { (R) -2- [((R, S)-{ [(S) -1- (d7- butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } adenine (compound 10).

Compound 8 (0.325g, 0.894mmol) is added in reaction flask, is dissolved with 3ml acetonitrile, it is added thionyl chloride (240.5mg, 2.02mmol), is heated to 80 DEG C and reacts 2 hours, concentration removes solvent, and 4ml anhydrous methylene chloride is added, is cooled to -29 DEG C, the 3ml dichloromethane solution of compound 9 (271.7mg, 1.97mmol) is added dropwise, finishes, triethylamine (271.4mg is added dropwise, 2.682mmol), reaction 1 hour, contact plate detection are warmed to room temperature.A small amount of water washing is added, saturated common salt water washing, silica gel column chromatography purifies after concentration, obtains 0.17g product, yield 39.35%.LC-MS (APCI): m/z=484.5 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 8.33 (d, J=7.0Hz, 1H), 7.98 (d, J=2.2Hz, 1H), 7.31 (d, J=8.0Hz, 1H), 7.22-7.03 (m, 3H), 6.98 (d, J=8.3Hz, 1H), 5.81 (s, 2H), 4.39 (m, 1H), 4.03 (m, 4H), 3.82-3.47 (m, 2H), 1.28-1.17 (m, 6H).

The separation of step 4 9- { (R) -2- [((S)-{ [(S) -1- (d7- butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } adenine (compound T-2).

Raceme compound 10 (170mg) is carried out by isolated target product T-2 using chiral supercritical fluid chromatography column (SFC), weigh to obtain 78.4mg after dry, yield: 46.1%.LC-MS (APCI): m/z=484.5 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 8.33 (d, J=7.0Hz, 1H), 7.98 (d, J=2.2Hz, 1H), 7.31 (d, J=8.0Hz, 1H), 7.22-7.03 (m, 3H), 6.98 (d, J=8.3Hz, 1H),5.81(s,2H),4.39(m,1H),4.03(m,4H),3.82-3.47(m,2H),1.28-1.17(m,6H)。

Embodiment 3 prepares 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino }-d5- benzene oxygen phosphoryl) methoxyl group] propyl } adenine; that is compound T-3, molecular formula are as follows:

Using synthetic route below:

The synthesis of step 1 2,3,4,5,6-d5- phenol (compound 11).

Phenol (2.0g is added in reaction flask, 21.25mmol), 5%Pt/C (0.4g, 20wt%) and 34ml heavy water, replacing hydrogen 3-4 times, it reacts 24 hours at room temperature, Filtration of catalyst, filter cake are washed with methylene chloride, merge organic phase, with saturated common salt water washing, anhydrous sodium sulfate through column chromatographic purifying obtains 1.6g compound 11, yield 80% after drying, filtering concentration.LC-MS (APCI): m/z=100.4 (M+1)⁺。

The synthesis of step 2 (R) -9- [2- (d5- phenoxy group phosphatidyl methoxy) propyl] adenine (compound 12).

4 (2.4g of compound is added in reaction flask, 8.36mmol), 11 (1.66g of compound, 16.72mmol) and 6.5ml NMP, 85 DEG C are heated to, is added triethylamine (1.04g, 10.3mmol), it is warming up to 100 DEG C, it is added dicyclohexylcarbodiimide (2.81g, 13.63mmol), then rises to 120 DEG C and be stirred to react 16 hours.Contact plate detection raw material is cooled to 45 DEG C after disappearing, and 4.8ml water is added, is down to room temperature, it is filtered to remove insoluble matter, with 2.5ml water washing filter cake, filtrate concentration, 4ml water is added, with NaOH tune PH to 11, chloroform is extracted 3-4 times, water phase concentrated hydrochloric acid tune PH to 3.1 is extracted 4-5 times with chloroform/isopropanol (3:1), merges organic phase, it is evaporated, the mashing purifying of a small amount of methanol is added, dry product 1.12g, yield 36.1% after filtering.LC-MS (APCI): m/z=369.3 (M+1)⁺。

The synthesis of step 3 9- { (R) -2- [((R, S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino }-d5- benzene oxygen phosphoryl) methoxyl group] propyl } adenine (compound 13).

Compound 12 (1.0g, 2.71mmol) is added in reaction flask, is dissolved with 4ml acetonitrile, it is added thionyl chloride (730.5mg, 6.14mmol), is heated to 80 DEG C and reacts 2 hours, concentration removes solvent, and 4ml anhydrous methylene chloride is added, is cooled to -29 DEG C, the 3ml dichloromethane solution of alanine isopropyl ester (780.2mg, 5.96mmol) is added dropwise, finishes, triethylamine (822.7mg is added dropwise, 8.13mmol), reaction 1 hour, contact plate detection are warmed to room temperature.A small amount of water washing is added, saturated common salt water washing, silica gel column chromatography purifies after concentration, obtains 0.42g product, yield 32.3%.LC-MS (APCI): m/z=482.3 (M+1)⁺。¹H NMR(300MHz,CDCl₃)δ8.35(s,1H),7.98(s,1H),5.67(s,2H),5.01(m,1H),4.35(m,1H),4.17-4.00(m,2H),3.90(m,2H),3.65(m,2H),1.24(m,12H)。

The separation of step 4 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino }-d5- benzene oxygen phosphoryl) methoxyl group] propyl } adenine (compound T-3).

Raceme compound 13 (180mg) is carried out by isolated target product T-3 using chiral supercritical fluid chromatography column (SFC), weigh to obtain 78mg after dry, yield: 86.7%.LC-MS (APCI): m/z=482.3 (M+1)⁺。¹H NMR(300MHz,CDCl₃)δ8.35(s,1H),7.98(s,1H),5.67(s,2H),5.01(m,1H),4.35(m,1H),4.17-4.00(m,2H),3.90(m,2H),3.65(m,2H),1.24(m,12H)。

4 9- of embodiment { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl)-d4- ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } adenine; that is compound T-4, molecular formula are as follows:

It is synthesized using following route:

The synthesis of step 1L-d4- alanine isopropyl ester (compound 14).

L-d4- alanine (0.168g, 1.29mmol) and isopropanol (576mg, 9.6mmol) are added in reaction flask, is added to flowing back, is added dropwise trim,ethylchlorosilane (234mg, 2.15mmol), finishes, back flow reaction is overnight.Concentration removes solvent, and the 5ml tetrahydrofuran solution of triethylene diamine (145mg, 1.29mmol) is added, white opacity liquid, diatomite drainage are obtained, filter cake is washed twice with methylene chloride, filtrate is concentrated to give crude product, and vacuum drying obtains 148.2mg, yield 85%.LC-MS (APCI): m/z=136.2 (M+1)⁺。

The synthesis of step 2 9- { (R) -2- [((R, S)-{ [(S) -1- (butyloxycarbonyl)-d4- ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } adenine (compound 15).

Compound 8 (234.3g, 0.645mmol) is added in reaction flask, is dissolved with 2ml acetonitrile, it is added thionyl chloride (173.4mg, 1.46mmol), is heated to 80 DEG C and reacts 2 hours, concentration removes solvent, and 3ml anhydrous methylene chloride is added, is cooled to -29 DEG C, the 3ml dichloromethane solution of compound 14 (174.4mg, 1.29mmol) is added dropwise, finishes, triethylamine (195.8mg is added dropwise, 1.935mmol), reaction 1 hour, contact plate detection are warmed to room temperature.A small amount of water washing is added, saturated common salt water washing, silica gel column chromatography purifies after concentration, obtains 0.2g product, yield 64.5%.LC-MS (APCI): m/z=481.5 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 8.32 (d, J=7.1Hz, 1H), 8.00 (d, J=3.3Hz, 1H), 7.30 (d, J=7.8Hz, 1H), 7.23-7.05 (m, 3H), 6.98 (d, J=8.4Hz, 1H), 5.83 (s, 2H), 4.95 (m, 1H), 4.38 (m, 1H), 4.13 (m, 1H), 4.01-3.84 (m, 2H), 3.71-3.61 (m, 1H), 1.24-1.20 (m, 6H), 1.17 (dd, J=6.2,3.3Hz, 3H).

The separation of step 3 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl)-d4- ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } adenine (compound T-4).

Raceme compound 15 (150mg) is carried out by isolated target product T-4 using chiral supercritical fluid chromatography column (SFC), weigh to obtain 53mg after dry, yield: 70.7%.LC-MS (APCI): m/z=481.5 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 8.32 (d, J=7.1Hz, 1H), 8.00 (d, J=3.3Hz, 1H), 7.30 (d, J=7.8Hz, 1H), 7.23-7.05 (m, 3H), 6.98 (d, J=8.4Hz, 1H), 5.83 (s, 2H), 4.95 (m, 1H), 4.38 (m, 1H), 4.13 (m, 1H), 4.01-3.84 (m, 2H), 3.71-3.61 (m, 1H), 1.24-1.20 (m, 6H), 1.17 (dd, J=6.2,3.3Hz, 3H).

Embodiment 5 prepares 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } -2; 8-d2- adenine; that is compound T-5, molecular formula are as follows:

It is synthesized using following route:

The synthesis of step 1 2,8-d2- adenine (compound 16).

Adenine (1.0g, 7.4mmol) is added in microwave reaction bottle, heavy water (10ml) and Pd/C (100mg), replacing hydrogen, sealing are placed on 160 DEG C reaction 1.5-2 hours in microwave reactor, are down to room temperature, 0.65ml concentrated hydrochloric acid is added, being heated to 60 DEG C dissolves product, filters while hot, and filtrate is with ammonium hydroxide tune PH to 8.0, ice bath is cooling and is kept for 0.5 hour, white solid is filtered to obtain, 0.8g product, yield: 80% are dried in vacuo to obtain.LC-MS (APCI): m/z=138.3 (M+1)⁺。

The synthesis of step 2 (R) -9- (2- hydroxypropyl) -2,8-d2- adenine (compound 17).

16 (2.465g of compound is added in reaction flask, 17.97mmol) and (R)-propene carbonate (2.093g, 20.5mmol), 3ml DMF dissolution is added, it is heated to 130 DEG C of reactions overnight, after contact plate detects fully reacting, it is cooled to 100 DEG C, 8.5ml toluene and 0.3g methanesulfonic acid (temperature is at 100-110 DEG C in keeping) is added, adds 7ml toluene and obtains a homogeneous suspension, be gradually cooled to room temperature, 0 DEG C is cooled to again to be kept for 1 hour, the white solid of filtering is dried in vacuo to obtain 3.76g product, yield 100%.LC-MS (APCI): m/z=196.3 (M+1)⁺。

The synthesis of step 3 (R) -9- [2- (diethylphosphoryl methoxyl group) propyl] -2,8-d2- adenine (compound 18).

By 17 (1.0g of compound; 5.17mmol) dissolved with 40ml anhydrous DMF; it is cooled to 0 DEG C, is added under NaH (233.3mg) low temperature and reacts 40 minutes under nitrogen protection, 2 (1.75g of compound is added; 10ml anhydrous DMF dissolution 5.44mmol); it is warmed to room temperature reaction 18 hours, concentration removes solvent, silica gel column chromatography purifying after completion of the reaction for contact plate detection; product 0.89g, yield 51% are obtained after being evaporated.LC-MS (APCI): m/z=346.5 (M+1)⁺。

The synthesis of step 4 (R) -9- [2- (phosphatidyl methoxy) propyl] -2,8-d2- adenine (compound 19).

18 (2.276g of compound is added in dry reaction flask, 6.63mmol), the dissolution of 20ml anhydrous DMF is added, TMSBr (3.76g is added at room temperature, 24.57mmol), it is stirred to react 20 hours, contact plate detects after the reaction was completed, and concentration removes solvent, ammonium hydroxide tune PH to 8.0 is added, it is concentrated to give oily liquids, with dilute hydrochloric acid tune PH to 3.0, is evaporated again, isopropanol is added, yellow solid is precipitated, filtering, recrystallizes to obtain white solid 0.62g, yield 32.1% with isopropanol/water (3:1).LC-MS (APCI): m/z=288.6 (M-1)^-。

The synthesis of step 5 (R) -9- [2- (phenoxy group phosphatidyl methoxy) propyl] -2,8-d2- adenine (compound 20).

19 (296mg of compound is added in reaction flask, 1.023mmol), phenol (202.8mg, 2.05mmol) and 3ml NMP, 85 DEG C are heated to, is added triethylamine (127.3mg, 1.26mmol), it is warming up to 100 DEG C, it is added dicyclohexylcarbodiimide (344.6mg, 1.67mmol), then rises to 120 DEG C and be stirred to react 16 hours.Contact plate detection raw material is cooled to 45 DEG C after disappearing, and 2ml water is added, is down to room temperature, it is filtered to remove insoluble matter, with 2ml water washing filter cake, filtrate concentration, 3ml water is added, with NaOH tune PH to 11, chloroform is extracted 3-4 times, water phase concentrated hydrochloric acid tune PH to 3.1 is extracted 4-5 times with chloroform/isopropanol (3:1), merges organic phase, it is evaporated, the mashing purifying of a small amount of methanol is added, dry product 269mg, yield 72% after filtering.LC-MS (APCI): m/z=366.3 (M+1)⁺。

Step 6 9- { (R) -2- [((R; S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl -2,8-d2- adenine (compound 21) synthesis.

Compound 20 (312mg, 0.85mmol) is added in reaction flask, is dissolved with 3ml acetonitrile, it is added thionyl chloride (230mg, 1.93mmol), is heated to 80 DEG C and reacts 2 hours, concentration removes solvent, and 3ml anhydrous methylene chloride is added, is cooled to -29 DEG C, the 3ml dichloromethane solution of alanine isopropyl ester (253.8mg, 1.88mmol) is added dropwise, finishes, triethylamine (259mg is added dropwise, 2.56mmol), reaction 1 hour, contact plate detection are warmed to room temperature.A small amount of water washing is added, saturated common salt water washing, silica gel column chromatography purifies after concentration, obtains 0.15g product, yield 36.9%.LC-MS (APCI): m/z=479.1 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 7.34-7.27 (m, 1H), 7.23-7.07 (m, 3H), 6.98 (d, J=8.4Hz, 1H), 5.79 (s, 2H), 5.06-4.85 (m, 1H), 4.39 (m, 1H), 4.21-4.09 (m, 1H), 4.07-3.87 (m, 3H), 3.81-3.52 (m, 2H), 1.30-1.16 (m, 12H).

The separation of step 7 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } -2,8-d2- adenine (compound T-5).

Raceme compound 21 (135mg) is carried out by isolated target product T-5 using chiral supercritical fluid chromatography column (SFC), weigh to obtain 56mg after dry, yield: 83%.LC-MS (APCI): m/z=479.1 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 7.34-7.27 (m, 1H), 7.23-7.07 (m, 3H), 6.98 (d, J=8.4Hz, 1H), 5.79 (s, 2H), 5.06-4.85 (m, 1H), 4.39 (m, 1H), 4.21-4.09 (m, 1H), 4.07-3.87 (m, 3H), 3.81-3.52 (m, 2H), 1.30-1.16 (m, 12H).

Embodiment 6 prepares 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl)-d2- methoxyl group] propyl } adenine; that is compound T-6, molecular formula are as follows:

It is synthesized using route below:

The synthesis of step 1 diethyl [[(p-toluenesulfonyl) oxygroup]-d2- methyl] phosphate (compound 22).

Compound 2 (1.0g, 3.1mmol) is added in microwave reaction bottle, Anhydrous potassium carbonate (42.8mg, 10ml heavy water 0.31mmol) is added, sealing, which is placed in microwave reactor, is heated to 80 DEG C of reactions 1 hour, is cooled to room temperature, ethyl acetate extraction is added, closes And organic phase, it is successively washed twice, is concentrated with water and saturated salt solution, silica gel column chromatography purifying is dried in vacuo to obtain product 0.86g, yield 85%.LC-MS (APCI): m/z=325.1 (M+1)⁺。

The synthesis of step 2 (R) -9- [2- (diethylphosphoryl-d2- methoxyl group) propyl] adenine (compound 23).

By 1 (1.0g of compound; 5.17mmol) dissolved with 40ml anhydrous DMF; it is cooled to 0 DEG C, is added under NaH (233.3mg) low temperature and reacts 40 minutes under nitrogen protection, 22 (1.75g of compound is added; 10ml anhydrous DMF dissolution 5.44mmol); it is warmed to room temperature reaction 18 hours, concentration removes solvent, silica gel column chromatography purifying after completion of the reaction for contact plate detection; product 0.91g, yield 51.7% are obtained after being evaporated.LC-MS (APCI): m/z=346.5 (M+1)⁺。

The synthesis of step 3 (R) -9- [2- (phosphinylidyne-d2- methoxyl group) propyl] adenine (compound 24).

23 (2.276g of compound is added in dry reaction flask, 6.63mmol), the dissolution of 20ml anhydrous DMF is added, TMSBr (3.76g is added at room temperature, 24.57mmol), it is stirred to react 20 hours, contact plate detects after the reaction was completed, and concentration removes solvent, ammonium hydroxide tune PH to 8.0 is added, it is concentrated to give oily liquids, with dilute hydrochloric acid tune PH to 3.0, is evaporated again, isopropanol is added, yellow solid is precipitated, filtering, recrystallizes to obtain white solid 0.76g, yield 40.1% with isopropanol/water (3:1).LC-MS (APCI): m/z=288.2 (M-1)^-。

The synthesis of step 4 (R) -9- [2- (phenoxy group phosphinylidyne-d2- methoxyl group) propyl] adenine (compound 25).

24 (296mg of compound is added in reaction flask, 1.023mmol), phenol (202.8mg, 2.05mmol) and 3ml NMP, 85 DEG C are heated to, is added triethylamine (127.3mg, 1.26mmol), it is warming up to 100 DEG C, it is added dicyclohexylcarbodiimide (344.6mg, 1.67mmol), then rises to 120 DEG C and be stirred to react 16 hours.Contact plate detection raw material is cooled to 45 DEG C after disappearing, and 2ml water is added, is down to room temperature, it is filtered to remove insoluble matter, with 2ml water washing filter cake, filtrate concentration, 3ml water is added, with NaOH tune PH to 11, chloroform is extracted 3-4 times, water phase concentrated hydrochloric acid tune PH to 3.1 is extracted 4-5 times with chloroform/isopropanol (3:1), merges organic phase, it is evaporated, the mashing purifying of a small amount of methanol is added, dry product 295mg, yield 78.9% after filtering.LC-MS (APCI): m/z=366.3 (M+1)⁺。

The synthesis of step 5 9- { (R) -2- [((R, S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl)-d2- methoxyl group] propyl } adenine (compound 26).

Compound 25 (312mg, 0.85mmol) is added in reaction flask, is dissolved with 3ml acetonitrile, it is added thionyl chloride (230mg, 1.93mmol), is heated to 80 DEG C and reacts 2 hours, concentration removes solvent, and 3ml anhydrous methylene chloride is added, is cooled to -29 DEG C, the 3ml dichloromethane solution of alanine isopropyl ester (253.8mg, 1.88mmol) is added dropwise, finishes, triethylamine (259mg is added dropwise, 2.56mmol), reaction 1 hour, contact plate detection are warmed to room temperature.A small amount of water washing is added, saturated common salt water washing, silica gel column chromatography purifies after concentration, obtains 0.17g product, yield 41.8%.LC-MS (APCI): m/z=479.1 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 8.32 (d, J=7.1Hz, 1H), 8.00 (d, J=3.3Hz, 1H), 7.34-7.27 (m, 1H), 7.23-7.07 (m, 3H), 6.98 (d, J=8.4Hz, 1H), 5.79 (s, 2H), 5.06-4.85 (m, 1H), 4.39 (m, 1H), 4.21-4.09 (m, 1H), 4.07-3.87 (m, 1H), 3.81-3.52 (m, 1H), 1.30-1.16 (m, 12H).

The separation of step 6 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl)-d2- methoxyl group] propyl } adenine (compound T-6).

Raceme compound 26 (150mg) is carried out by isolated target product T-6 using chiral supercritical fluid chromatography column (SFC), Weigh to obtain 48mg after drying, yield: 64%.LC-MS (APCI): m/z=479.1 (M+1)⁺。¹H NMR(300MHz,CDCl₃) δ 8.32 (d, J=7.1Hz, 1H), 8.00 (d, J=3.3Hz, 1H), 7.34-7.27 (m, 1H), 7.23-7.07 (m, 3H), 6.98 (d, J=8.4Hz, 1H), 5.79 (s, 2H), 5.06-4.85 (m, 1H), 4.39 (m, 1H), 4.21-4.09 (m, 1H), 4.07-3.87 (m, 1H), 3.81-3.52 (m, 1H), 1.30-1.16 (m, 12H).

Biological activity test

(1) the external HIV-resistant activity of detection compound

Compound processing: untested compound and reference compound will with DMSO doubling dilution it is good after in addition tissue culture plate.Untested compound and reference compound will test 8 concentration, two multiple holes.

Virus infection and cell processing: by HIV-1 and MT-4 cell in 37 DEG C, 5%CO₂1h is co-cultured in incubator.Then infection cell is inoculated in tissue culture plate with certain density.DMSO final concentration of 0.5% in cell culture medium.Cell is placed in 37 DEG C, 5%CO₂It is cultivated 5 days in incubator.The cell of cytotoxicity test experiment is the MT-4 cell being uninfected by, and other experiment conditions are consistent with antiviral activity experiment.

Cytoactive detection: cell activity is measured by cytoactive detection reagent C ellTiter-Glo (Promega).Initial data is calculated for compound Anti-HIV-1 Active and cytotoxicity.Compound dose-effect curve and its EC₅₀And CC₅₀Value obtains after being analyzed by GraphPad Prism software, wherein A indicates EC₅₀< 10nM, B indicate 10nM≤EC₅₀≤ 100nM, C indicate 100nM < EC₅₀≤ 500nM, D indicate EC₅₀>500Nm；F indicates CC₅₀> 10000nM (as shown in table 1 below).

(2) the external Anti-HBV effect of detection compound

Experimental method: luciferase assay compound antihepatitis C virus activity is detected with Bright-Glo (Promega).Using GraphPad Prism software analysis data, matched curve simultaneously calculates EC₅₀And CC₅₀Value.

Experimental procedure:

Anticellular activities experiment: the effect on hepatitics B virus in vitro activity of 20 compounds is detected in HepG2.2.15 cell, TDF is as positive reference compound.First day kind cell is to 96 orifice plates, and addition compound handles cell, the 5th day culture solution containing compound more renewed within second day.Collection supernatant extracts DNA within 8th day.With the content of quantitative PCR detection HBV DNA.Untested compound and equal 3 times of TDF are serially diluted, 8 concentration points, are measured in parallel 2 multiple holes.Final concentration of the 0.5% of DMSO in culture solution.Suppression percentage calculation formula is as follows:

% inhibiting rate=(copy number of HBV in the copy number of HBV/DMSO control group in 1- sample) × 100

EC₅₀It is analyzed by Graphpad Prism software (four parameter logistic equations), wherein I indicates EC₅₀< 5nM, II indicate 5nM≤EC₅₀≤ 20nM, III indicate 20nM < EC₅₀≤ 100nM, IV indicate EC₅₀> 100nM (as shown in table 1 below).

Cytotoxicity experiment: compound plate-laying, compound process flow are consistent with HIV-resistant activity detection.After compound is handled cell six days, cell activity is measured.Cell-titer Blue reagent is added in every hole, and 37 DEG C are incubated for 3 hours, reads fluorescent value (560Ex/590Em)；It analyzes data and calculates versus cell vigor:

Cell activity percentage: % cell viability=(fluorescent reading-culture solution control fluorescence reading is calculated using following formula Number)/(fluorescence reading of DMSO control-culture solution control fluorescence reading) × 100.

The CC of compound is finally calculated using GraphPad Prism software₅₀Value, V indicate CC₅₀> 200000nM (as shown in table 1 below).

1 embodiment compound HBV activity of table and HIV activity

The experimental results showed that, the compounds of this invention has very strong HIV-resistant activity and HBV active (reaching nanomolar range), compared with the inverase (GS7340) of the newest listing of Gilid Science Co., drugmaker, the U.S., the HIV-resistant activity and Anti-HBV effect of the present embodiment compound are suitable with it, wherein, the Anti-HBV effect of embodiment compound T-5 and T-6 shows the activity better than GS7340.In addition, the compounds of this invention does not show toxicity (optimal CC in the cell line surveyed₅₀>200000nM)。

(3) liver particle metabolism

Microsomal assay: people's hepatomicrosome: 0.5mg/mL, Xenotech；Rat liver microsomes: 0.5mg/mL, Xenotech；Coenzyme (NADPH/NADH): 1mM, Sigma Life Science；Magnesium chloride: 5mM, 100mM phosphate buffer (pH 7.4).

The preparation of stock solution: precision weighs a certain amount of COMPOUNDS EXAMPLE 1-6 powder, and is dissolved to 5mM respectively with DMSO.

Phosphate buffer (100mM, pH7.4 preparation): the 0.5M dipotassium hydrogen phosphate solution of the 0.5M potassium dihydrogen phosphate 150mL and 700mL that prepare in advance is taken to mix, mixed liquor pH value is adjusted to 7.4 with 0.5M dipotassium hydrogen phosphate solution again, 5 times are diluted with ultrapure water using preceding, magnesium chloride is added, phosphate buffer (100mM) is obtained, wherein potassium phosphate containing 100mM, 3.3mM magnesium chloride, pH 7.4.

Prepare NADPH regenerative system solution (containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-P D, 3.3mM magnesium chloride), using it is preposition in it is wet on ice.

Prepare terminate liquid: the acetonitrile solution containing 50ng/mL Propranolol Hydrochloride and 200ng/mL orinase (internal standard).It takes 25057.5 μ L phosphate buffers (pH7.4) into 50mL centrifuge tube, is separately added into 812.5 μ L people's hepatomicrosomes, mix, obtain the hepatomicrosome dilution that protein concentration is 0.625mg/mL.It takes 25057.5 μ L phosphate buffers (pH7.4) into 50mL centrifuge tube, is separately added into 812.5 μ L SD rat liver microsomes, mix, obtain the hepatomicrosome dilution that protein concentration is 0.625mg/mL.

The incubation of sample: being diluted to 0.25mM for the stock solution of respective compound with containing the aqueous solution of 70% acetonitrile respectively, as working solution, It is spare.It takes people's hepatomicrosome of 398 μ L or rat liver microsomes dilution that 96 holes are added respectively to be incubated in plate (N=2), is separately added into the working solution of 2 μ L 0.25mM, mixes.

The measurement of metabolic stability: the terminate liquid of 300 μ L pre-cooling is added in every hole of 96 hole deep-well plates, is placed on ice, as termination plate.96 holes are incubated for plate and NADPH regenerative system is placed in 37 DEG C of water baths, 5min is incubated in 100 revs/min of concussions in advance.80 μ L Incubating Solutions addition termination plate is taken out from the every hole of plate is incubated for, mixes, 20 μ L NADPH regenerative system solution is supplemented, as 0min sample.Again to the NADPH regenerative system solution for being incubated for 80 μ L of the every hole addition of plate, starting reaction starts timing.The reaction density of respective compound is 1 μM, protein concentration 0.5mg/mL.When reacting 10,30,90min, 100 μ L reaction solutions are respectively taken, are added in termination plate, vortex 3min terminates reaction.Termination plate is centrifuged 10min under the conditions of 5000 × g, 4 DEG C.It takes 100 μ L supernatants to being previously added in 96 orifice plates of 100 μ L distilled water, mixes, sample analysis is carried out using LC-MS/MS.

Data analysis: by LC-MS/MS system detection respective compound and interior target peak area, compound and internal standard peak area ratio are calculated.Slope is measured by the natural logrithm of the percentage of compound surplus and time mapping, and calculates t according to the following formula_1/2And CL_int, wherein V/M is equal to 1/ protein concentration.

The liver particle metabolic evaluation of 2 embodiment compound of table

Shown in experimental result table 2 as above, compared with GS7340, the half-life period of the compounds of this invention is longer, and clearance rate is smaller, all shows preferably metabolic stability in people's hepatomicrosome and rat liver microsomes experiment, is more suitable for AntiHIV1 RT activity or the drug of HBV.

(4) pharmacokinetics in rats is tested

Experiment purpose: after research rat gives 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl)-methoxyl group] propyl } adenine, embodiment 1-6 compound, the pharmacokinetics behavior of the compounds of this invention is investigated.

Experimental animal:

Type and strain: SD rat grade: SPF grades

Gender and quantity: male, 6

Weight range: 180~220g (actual weight range is 187~197g)

Source: the western Poole Bi Kai experimental animal Co., Ltd in Shanghai

Experiment and animal certificate number: SCXK (Shanghai) 2013-0016

Experimentation:

Before blood specimen collection, the 2M Fluorinse (esterase inhibitor) of 20L is added in EDTA-K2 anticoagulant tube in advance, after 80 degree of drying in oven, is placed in 4 degree of refrigerator storages.

Rat, male, 187~197g of weight is randomly divided into 2 groups, be fasted in experiment noon before that day overnight but can free water, 4h is to food after administration.A group gives 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } adenine 3mg/kg, B group gives embodiment 1-6 compound 3mg/kg, distinguish 15min after administration, 30min, 1, 2, 3, 5, 8, 10h takes blood 100-200L or so from rat orbital vein, it is placed in the Eppendorf pipe through EDTA-K2 anticoagulant 0.5mL, it mixes immediately, after anticoagulant, after test tube is gently mixed by inversion 5-6 times as early as possible, blood is placed in ice chest after taking, blood sample in 4000rpm in 30min, 10min, centrifugal separation plasma under the conditions of 4 DEG C, it is saved immediately in -20 DEG C after collecting whole blood plasma.The blood concentration in the blood plasma of each time point is measured after all time point sample acquisitions.

According to mean blood plasma concentration-time data after above-mentioned resulting administration; using Winnonin software, seeking calculation male SD rat by non-chamber statistical moment theory, i.g gives the pharmacokinetics relevant parameter after 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } adenine (3mg/kg), embodiment 1-6 compound (3mg/kg) respectively.

Experiment shows; compared with 9- { (R) -2- [((S)-{ [(S) -1- (butyloxycarbonyl) ethyl] amino } benzene oxygen phosphoryl) methoxyl group] propyl } adenine; the compounds of this invention has more preferably active; and there is excellent pharmacokinetic property; therefore it is more suitable for inhibiting the compound of nucleoside reverse transcriptase, and then is suitble to the drug of preparation treatment viral infection resisting.

It should be understood that these examples are only for illustrating the present invention and are not intended to limit the scope of the present invention, test method without specific conditions in embodiment, usually according to normal condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise parts and percentages are parts by weight and weight percent.

The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, without departing from the inventive concept of the premise, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to protection scope of the present invention.

Claims (10)

1. A kind of efabirenz, it is characterised in that: the adenine compound as shown in formula (I) or its crystal form, pharmaceutically acceptable salt, prodrug, stereoisomer, hydrate or solvated compounds,

   Wherein, R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶It is each independently hydrogen, deuterium, halogen or trifluoromethyl；

   Additional conditions are R¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵And R²⁶In at least one be deuterated or deuterium.
2. Efabirenz according to claim 1, it is characterised in that: R¹And R²It is each independently deuterium or hydrogen.
3. Efabirenz according to claim 1, it is characterised in that: R³、R⁴、R⁵、R⁶、R⁷And R⁸It is each independently deuterium or hydrogen.
4. Efabirenz according to claim 1, it is characterised in that: R⁹And R¹⁰It is each independently deuterium or hydrogen.
5. Efabirenz according to claim 1, it is characterised in that: R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰And R²¹It is each independently deuterium or hydrogen.
6. Efabirenz according to claim 1, it is characterised in that: R²³、R²⁴、R²⁵And R²⁶It is each independently deuterium or hydrogen.
7. Efabirenz according to claim 1, it is characterised in that: the compound can be selected from following compounds or its pharmaceutically acceptable salt:
8. A kind of pharmaceutical composition, it is characterized by: its pharmaceutical composition for containing pharmaceutically acceptable carrier and efabirenz as described in claim 1~7 any one or its crystal form, pharmaceutically acceptable salt, hydrate or solvate, stereoisomer, prodrug or isotopic variations.
9. A kind of purposes of the efabirenz as described in claim 1~7 any one, it is characterised in that: the drug of the disease for the treatment of virus infection is used to prepare, such as AIDS, hepatitis B.
10. A method of disease relevant to virus is treated and/or prevented in subject, the method includes to pharmaceutical composition described in formula (I) compound of the snibject as described in claim 1~7 any one or its polymorphic, pharmaceutically acceptable salt, prodrug, stereoisomer, isotopic variations, hydrate or solvated compounds or claim 8.