CN108350007B - Substituted adenine compound and pharmaceutical composition thereof - Google Patents

Abstract

A substituted adenine compound and a pharmaceutical composition thereof are disclosed, wherein the substituted adenine compound is a compound shown as a formula (I), or a crystal form, a pharmaceutically acceptable salt, a prodrug, a stereoisomer, a hydrate or a solvate thereof. The compound can inhibit the activity of nucleoside reverse transcriptase, has better pharmacodynamics/pharmacokinetic performance, good applicability and high safety, can be used for preparing and treating diseases related to virus infection, and has good market development prospect.

Description

Substituted adenine compound and pharmaceutical composition thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a substituted adenine compound and a pharmaceutical composition thereof, which can be used for treating related diseases of viral infection.

Background

Nucleoside Reverse Transcriptase Inhibitors (NRTIs), analogs of deoxynucleotides, the substrate of DNA reverse transcriptase for the synthesis of HIV, are converted in vivo into active nucleoside triphosphate derivatives which compete with the natural deoxynucleoside triphosphates for binding to HIV Reverse Transcriptase (RT), inhibiting the action of RT and preventing proviral synthesis. NRTIs are structurally similar to nucleosides and are dideoxynucleoside derivatives that compete with intracellular ribonucleosides for binding to reverse transcriptase, thereby terminating the reverse transcription reaction.

Nucleotide HIV reverse transcriptase inhibitors act at the active site where reverse transcriptase binds to its natural substrate nucleoside. The medicine is a natural nucleoside medicine, is metabolized into a real active molecule triphosphored nucleoside (NRTI-ppp) through multi-step phosphorylation reaction after entering a body, and acts on a substrate active site of enzyme competitively with endogenous dNTP. Since the structure of NRTI-ppp is very similar to that of the dNTP substrate, enzymes will mistake this class of drugs as substrates and insert them into the growing DNA strand once they have entered the DNA strand, since the structure of the drug molecule does not have a 3 ' -hydroxyl group that can be 3 ' -5 ' linked to the next dNTP. Thus blocking the extension of the viral DNA strand and thus inhibiting the replication of HIV.

In recent decades, great progress has been made in the research of anti-Human Immunodeficiency Virus (HIV) drugs. Currently, at least 27 antiviral drugs for the treatment of HIV infection have been approved by the united states food and drug administration. anti-HIV drugs mainly include 4 major classes: nucleoside Reverse Transcriptase Inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (Pls) and HIV integrase inhibitors, wherein NRTIs is the first most used and most diverse group, mainly comprising zidovudine (AZT), lamivudine, dehydroxyanhydride, stanfordine, abacavir and tenofovir.

AIDS, a serious disease caused by HIV infection, has been reported in the first AIDS case in 1981, nearly 7000 million people are infected by AIDS virus in the world, and more than 2000 million people die of AIDS. Although effective drug therapy has reduced the mortality rate of AIDS over the last 20 years, millions of people are infected with HIV every year and the number of AIDS patients worldwide has been on the rise.

However, HIV is currently resistant to almost all clinically used anti-HIV drugs, and the development of resistant HIV is considered to be the major cause of failure of anti-HIV drug therapy.

In addition, chronic hepatitis is one of the serious infectious diseases threatening the global human health. About 20 more million people worldwide have been infected with Hepatitis B Virus (HBV), and the number of deaths due to HBV infection is 100 ten thousand per year. HBV infection is an important biological factor causing not only chronic hepatitis b but also primary liver cancer. China, southeast Asia and Africa are high-incidence areas of HBV infection, and the incidence rate of primary liver cancer is obviously higher than that of low-incidence areas of HBV infection in the middle and south America and the like. The existing treatment of chronic hepatitis B mainly comprises interferon, nucleoside drugs and thymosin, but the drugs have serious toxic and side effects or generate drug resistance after being applied for a long time and are expensive. Therefore, the search for new and effective anti-HBV drugs is an urgent problem to be solved.

Therefore, there is still a need in the art to develop compounds having inhibitory activity or better pharmacodynamic properties against nucleoside reverse transcriptase.

Disclosure of Invention

In view of the above technical problems, the present invention discloses a nucleoside reverse transcriptase inhibitor, a pharmaceutical composition and an application thereof, which have better nucleoside reverse transcriptase inhibitory activity and/or better pharmacodynamic/pharmacokinetic properties.

In contrast, the technical scheme adopted by the invention is as follows:

a nucleoside reverse transcriptase inhibitor is a substituted adenine compound shown as formula (I), or its crystal form, pharmaceutically acceptable salt, prodrug, stereoisomer, hydrate or solvate,

wherein R is¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶Each independently is hydrogen, deuterium, halogen or trifluoromethyl;

with the proviso that 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 R²⁶At least one of which is deuterated or deuterium.

As a further improvement of the invention, R¹And R²Each independently is deuterium or hydrogen.

As a further improvement of the invention, R³、R⁴、R⁵、R⁶、R⁷And R⁸Each independently is deuterium or hydrogen.

As a further improvement of the invention, R⁹And R¹⁰Each independently is deuterium or hydrogen.

As a further improvement of the invention, R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰And R²¹Each independently is deuterium or hydrogen.

As a further improvement of the invention, R²³、R²⁴、R²⁵And R²⁶Each independently is deuterium or hydrogen.

As a further improvement of the present invention, the compound may be selected from the following compounds or pharmaceutically acceptable salts thereof, but is not limited to the following compounds:

by adopting the technical scheme, the shape and the volume of deuterium in a drug molecule are basically the same as those of hydrogen, and if the hydrogen in the drug molecule is selectively replaced by deuterium, the original biological activity and selectivity of the deuterium-substituted drug can be generally kept. Meanwhile, the inventor proves that the combination of carbon and deuterium bonds is more stable than the combination of carbon and hydrogen bonds, and the absorption, distribution, metabolism, excretion and other properties of some medicines can be directly influenced, so that the curative effect, safety and tolerance of the medicines are improved.

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

Specifically, in the present invention 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 R²⁶The deuterium isotope content in each deuterated position is at least 5%, preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20%, more preferably greater than 25%, more preferably greater than 30%, more preferably greater than 35%, more preferably greater than 40%, more preferably greater than 45%, more preferably greater than 50%, more preferably greater than 55%, more preferably greater than 60%, more preferably greater than 65%, more preferably greater than 70%, more preferably greater than 75%, more preferably greater than 80%, more preferably greater than 85%, more preferably greater than 90%, more preferably greater than 95%, more preferably greater than 99%.

In another preferred embodiment, R of the compound of formula (I)¹、R²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵And R²⁶At least one of R, preferably two of R, more preferably three of R, more preferably four of R, more preferably five of R, more preferably six of R, more preferably seven of R, more preferably eight of R, more preferably nine of R, more preferably ten of R, more preferably eleven of R, more preferably twelve of R, more preferably thirteen of R, more preferably fourteen of R, more preferably fifteen of R, more preferably sixteen of R, more preferably seventeen of R, more preferably eighteen of R, more preferably nineteen of R, more preferably twenty two of R, more preferably twenty three of R, more preferably twenty four of R, more preferably twenty five of R, more preferably twenty three of R, more preferably twenty four of R, more preferably twenty five of R, more preferably four of R, more preferably six of R, more preferably seven of R, more preferably twelve of R, more preferably eight of R, more preferably twenty three of R, more.

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

The invention also discloses a pharmaceutical composition which contains a pharmaceutically acceptable carrier and the nucleoside reverse transcriptase inhibitor, or a pharmaceutical composition of a crystal form, a pharmaceutically acceptable salt, a hydrate or a solvate, a stereoisomer, a prodrug or an isotopic variant thereof.

As a further improvement of the invention, the pharmaceutically acceptable carrier comprises at least one of a glidant, a sweetener, a diluent, a preservative, a dye/colorant, a flavor enhancer, a surfactant, a wetting agent, a dispersing agent, a disintegrant, a suspending agent, a stabilizer, an isotonic agent, a solvent, or an emulsifier.

As a further improvement of the present invention, the pharmaceutical composition is a tablet, pill, capsule, powder, granule, paste, emulsion, suspension, solution, suppository, injection, inhalant, gel, microsphere or aerosol.

Typical routes of administration of the pharmaceutical compositions of the present invention include, but are not limited to, oral, rectal, transmucosal, enteral, or topical, transdermal, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration. Oral administration or injection administration is preferred.

The pharmaceutical compositions of the present invention may be manufactured by methods well known in the art, such as conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, lyophilizing, and the like.

The present invention also provides a method of preparing a pharmaceutical composition comprising the steps of: mixing a pharmaceutically acceptable carrier and the nucleoside reverse transcriptase inhibitor or the crystal form, the pharmaceutically acceptable salt, the hydrate or the solvate thereof to form the pharmaceutical composition.

As a further development of the invention, it also comprises further active compounds, which can be selected from: non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors.

The active ingredients of the present invention may also be used in combination with other active ingredients. The choice of such combination is based on the condition of the treatment, the cross-reactivity of the ingredients and the pharmaceutical properties of the combination. It is also possible to combine any of the compounds of the invention with one or more other active ingredients for simultaneous or sequential administration to a patient in a single dosage form. The combination therapy may be administered on a simultaneous or sequential dosing regimen. When administered sequentially, the combination may be administered in two or more administrations. Combination therapy may provide a "synergistic effect" or "synergy", in other words, the effect obtained when the active ingredients are used together is greater than the sum of the effects obtained when the compounds are used separately. When the active ingredients are: (1) are co-formulated and administered or delivered simultaneously in a combined formulation; (2) as separate formulations administered alternately or in parallel; or (3) by some other dosing regimen, a synergistic effect may be obtained. When delivered in alternating treatments, synergy can be obtained when the compounds are administered or released sequentially, e.g., in separate tablets, pills or capsules, or by different injections with separate syringes. Generally, during alternation therapy, the effective dose of each active ingredient is administered sequentially, i.e. consecutively, whereas in combination therapy, the effective doses of two or more active ingredients are co-administered.

The present invention also discloses the use of a substituted adenine nucleoside reverse transcriptase inhibitor as described above, i.e. the compounds of the invention are advantageously useful as therapeutic agents for the treatment of conditions such as aids and hepatitis b.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Herein, "halogen" means F, Cl, Br, and I, unless otherwise specified. More preferably, the halogen atom is selected from F, Cl and Br.

Herein, "deuterated", unless otherwise specified, means that one or more hydrogens of a compound or group are replaced with deuterium; deuterium can be mono-, di-, poly-, or fully substituted. The terms "deuterated one or more" and "deuterated one or more" are used interchangeably.

Herein, unless otherwise specified, "non-deuterated compound" means a compound containing deuterium at an atomic ratio of deuterium not higher than the natural deuterium isotope content (0.015%).

The compositions of the invention optionally comprise salts of the compounds herein, particularly pharmaceutically acceptable non-toxic salts, containing, for example, Na⁺、Li⁺、K⁺、Ca⁺²And Mg⁺². These salts may include those derived from a combination of suitable cations, such as alkali and alkaline earth metal ions or ammonium and quaternary amino ions and acid anion moieties, typically carboxylic acids. Monovalent salts are preferred if water soluble salts are desired. The metal salts are typically prepared by reacting a metal hydroxide with a compound of the invention. An example of a metal salt prepared in this way is a salt containing Li⁺、Na⁺And K⁺A salt. By adding a suitable metal compound, the more insoluble metal salt can be precipitated from the more soluble salt solution. In addition, salts may be formed by the addition of certain organic and inorganic acids, e.g., HCl, HBr, H₂SO₄、H₃PO₄Or organic sulfurAcids, to basic centers, typically amines, or to acidic groups. Finally, it will be understood that the compositions herein comprise the compounds of the present invention in their unionized, as well as zwitterionic forms, in combination with a stoichiometric amount of water, as in the hydrate. Also included within the scope of the invention are salts of the parent compound with one or more amino acids. Any of the above amino acids is suitable, particularly naturally occurring amino acids found as protein components, although typically the amino acid is one with a side chain having a basic or acidic group, e.g., lysine, arginine or glutamic acid, or a neutral group, e.g., glycine, serine, threonine, alanine, isoleucine or leucine.

The compounds of the invention may have chiral centers, for example, chiral carbon or phosphorus atoms. The compounds of the present invention thus include racemic mixtures of all stereoisomers, including enantiomers, diastereomers and atropisomers. In addition, the compounds of the present invention include enriched or resolved optical isomers at any or all of the asymmetric chiral atoms. In other words, the chiral centers apparent from the description are provided as chiral isomers or racemic mixtures. Racemic mixtures and diastereomeric mixtures, as well as individual optical isomers isolated or synthesized substantially free of their enantiomeric or diastereomeric partners, are all within the scope of the invention. Racemic mixtures are separated into their individual, substantially optically pure isomers by known techniques, e.g., separation of diastereomeric salts formed with optically active auxiliaries, e.g., acids or bases, followed by conversion back to the optically active substance. In most cases, the desired optical isomer is synthesized by stereospecific reactions starting from the appropriate stereoisomer of the desired starting material.

In certain instances, the compounds of the present invention may also exist as tautomers. Although only one delocalized resonance structure may be described, it is contemplated that all such forms fall within the scope of the invention. For example, ene-amine tautomers may exist for purine, pyrimidine, imidazole, guanidine, amidine, and tetrazole systems, and all their possible tautomeric forms fall within the scope of the invention.

The term "solvate" refers to a complex of a compound of the present invention coordinated to solvent molecules in a specific ratio. "hydrate" refers to a complex formed by the coordination of a compound of the present invention with water.

Compared with the prior art, the invention has the beneficial effects that: the compound has excellent inhibition on nucleoside reverse transcriptase; the deuteration technology changes the metabolism of the compound in organisms, so that the compound has better pharmacokinetic parameter characteristics. In this case, the dosage can be varied and a depot formulation formed, improving the applicability; deuterium is used for replacing hydrogen atoms in the compound, and due to the deuterium isotope effect, the medicine concentration of the compound in an animal body is improved, and the medicine curative effect is improved; deuterium is used for replacing hydrogen atoms in the compound, so that certain metabolites can be inhibited, and the safety of the compound is improved.

Detailed Description

The following describes more specifically the processes for the preparation of the compounds of formula (I) according to the invention, but these particular processes do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining various synthetic methods described in the present specification or known in the art, and such combinations may be readily carried out by those skilled in the art to which the present invention pertains.

Generally, in the preparative schemes, each reaction is generally carried out in an inert solvent at room temperature to reflux temperature (e.g., at room temperature to reflux temperature)

Preference is given to

) The process is carried out as follows. The reaction time is usually 0.1 to 60 hours, preferably 0.5 to 24 hours.

Example 1 preparation of 9- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } -2, 4, 6-d 3-phenoxyphosphoryl) methoxy ] propyl } adenine, compound T-1, of the formula:

the synthesis was carried out using the following route:

step 1 Synthesis of (R) -9- (2-hydroxypropyl) adenine (Compound 1).

Adding adenine (4.0g, 29.6mmol) and (R) -propylene carbonate (3.45g, 33.8mmol) into a reaction bottle, adding 4.5ml of DMF to dissolve, heating to 130 ℃ to react overnight, cooling to 100 ℃ after the reaction is detected by a dot plate, adding 14ml of toluene and 0.47g of methanesulfonic acid (keeping the internal temperature at 100 ℃ and 110 ℃), adding 11ml of toluene to obtain a homogeneous suspension, gradually cooling to room temperature, cooling to 0 ℃ again for 1 hour, filtering to obtain a white solid, and drying in vacuum to obtain 5.77g of a product with the yield of 100%. LC-MS (APCI): 194.3(M +1)⁺。

Step 2 Synthesis of diethyl [ [ (p-toluenesulfonyl) oxy ] methyl ] phosphate (Compound 2).

Adding diethyl hydroxymethyl phosphate (3.85g, 22.89mmol) into a reaction bottle, adding 30ml of anhydrous ether for dissolving, dropwise adding triethylamine (3.38ml, 24.04mmol), cooling to-10 ℃ after adding, dropwise adding 10ml of ether solution of paratoluensulfonyl chloride (4.58g, 24.04mmol), stirring at 0 ℃ for reacting for 3 hours after adding, and raising the temperature to room temperature for reacting overnight. Adding a small amount of ether for dilution, filtering to remove inorganic salts, concentrating the filtrate, purifying by silica gel column chromatography, and drying in vacuum to obtain a product 5.4g with the yield of 73.2%. LC-MS (APCI): m/z 323.1(M +1)⁺。

Step 3 (R) -9- [2- (diethylphosphorylmethoxy) propyl ] adenine (Compound 3) synthesis.

Dissolving the compound 1(1.0g, 5.17mmol) with 40ml of anhydrous DMF, cooling to 0 ℃, adding NaH (233.3mg) under the protection of nitrogen, reacting for 40 minutes at low temperature, adding 10ml of anhydrous DMF of the compound 2(1.75g, 5.44mmol), dissolving, heating to room temperature, reacting for 18 hours, detecting by a dot plate, concentrating to remove the solvent after the reaction is finished, purifying by silica gel column chromatography, and evaporating to dryness to obtain 0.88g of product with the yield of 50%. LC-MS (APCI): 344.5(M +1) for M/z⁺。

Step 4 (R) -9- [2- (phosphorylmethoxy) propyl ] adenine (Compound 4) synthesis.

Adding compound 3(2.276g, 6.63mmol) into a dry reaction bottle, adding 20ml of anhydrous DMF for dissolving, adding TMSBr (3.76g, 24.57mmol) at room temperature, stirring for reacting for 20 hours, detecting the completion of the reaction by a dot plate, concentrating to remove the solvent, adding ammonia water to adjust the pH to 8.0, concentrating to obtain an oily liquid, adjusting the pH to 3.0 by using diluted hydrochloric acid, evaporating to dryness again, adding isopropanol to precipitate a yellow solid, filtering, recrystallizing with isopropanol/water (31) to obtain 0.57g of a white solid, wherein the yield is 30.1%. LC-MS (APCI): m/z 286.7(M-1)^-。

Step 52, synthesis of 4, 6-d 3-phenol (Compound 5).

Phenol (2.0g, 21.25mmol) and sodium hydroxide (0.425g, 10.63mmol) were added to a microwave reaction flask, dissolved in 15ml of heavy water, sealed and reacted in a microwave reactor at 180 ℃ for 0.5 hour. Cooling to room temperature, adjusting pH to acidity with dilute hydrochloric acid, extracting with ethyl acetate for 3-4 times, combining organic phases, washing with saturated saline solution, concentrating, and purifying with silica gel column chromatography to obtain compound 1.8g, yield: 90 percent. LC-MS (APCI): 98.1(M +1) M/z⁺。

Step 6 (R) -9- [2- (2, 4, 6-d 3-phenoxyphosphorylmethoxy) propyl ] adenine (Compound 6) synthesis.

Compound 4(2.4g, 8.36mmol), compound 5(1.62g, 16.72mmol) and 6.5ml of NMP were charged into a reaction flask, heated to 85 deg.C, triethylamine (1.04g, 10.3mmol) was added, the temperature was raised to 100 deg.C, dicyclohexylcarbodiimide (2.81g, 13.63mmol) was added, and the reaction was stirred to 120 deg.C for 16 hours. And (4) cooling to 45 ℃ after the raw materials disappear by point plate detection, and adding.8ml of water, cooling to room temperature, filtering to remove insoluble substances, washing a filter cake with 2.5ml of water, concentrating a filtrate, adding 4ml of water, adjusting the pH value to 11 by using NaOH, extracting for 3-4 times by using chloroform, adjusting the pH value of a water phase to 3.1 by using concentrated hydrochloric acid, extracting for 4-5 times by using chloroform/isopropanol (3: 1), combining organic phases, evaporating to dryness, adding a small amount of methanol, pulping and purifying, filtering, drying to obtain 1.84g of a product, wherein the yield is 60.1%. LC-MS (APCI): 367.3(M +1) M/z⁺。

Step 79 Synthesis of- { (R) -2- [ ((R, S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } -2, 4, 6-d 3-phenoxyphosphoryl) methoxy ] propyl } adenine (Compound 7).

Adding compound 6(0.866g, 2.364mmol) into a reaction flask, dissolving with 5ml acetonitrile, adding thionyl chloride (635.3mg, 5.34mmol), heating to 80 ℃ for reaction for 2 hours, concentrating to remove the solvent, adding 4ml anhydrous dichloromethane, cooling to-29 ℃, dropwise adding a solution of isopropyl alaninate (682.2mg, 5.2mmol) in 3ml dichloromethane, after the addition is finished, dropwise adding triethylamine (717.6mg, 7.1mmol), heating to room temperature for reaction for 1 hour, and detecting by a dot-plate method. Washing with a small amount of water, washing with saturated brine, concentrating, and purifying with silica gel column chromatography to obtain 0.74g of product with yield of 65%. LC-MS (APCI): m/z 480.5(M +1)⁺。¹H NMR(300MHz，CDCl₃)δ8.33(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 89 isolation of- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } -2, 4, 6-d 3-phenoxyphosphoryl) methoxy ] propyl } adenine (Compound T-1).

Chiral Supercritical Fluid Chromatography (SFC) is adopted to separate racemic compound 7(100mg) to obtain target product T-1, and the target product T-1 is dried and weighed to obtain 45mg, and the yield is as follows: 90 percent. LC-MS (APCI): m/z 480.5(M +1)⁺。¹H NMR(300MHz，CDCl₃)δ8.33(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)。

Example 2 preparation of 9- { (R) -2- [ ((S) - { [ (S) -1- (d 7-isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } adenine, compound T-2, of the formula:

the following synthetic route was used:

step 1 (R) -9- [2- (phenoxyphosphorylmethoxy) propyl ] adenine (compound 8) synthesis.

Compound 4(2.4g, 8.36mmol), phenol (1.62g, 16.72mmol) and 6.5ml NMP were charged into a reaction flask, heated to 85 deg.C, triethylamine (1.04g, 10.3mmol) was added, the temperature was raised to 100 deg.C, dicyclohexylcarbodiimide (2.81g, 13.63mmol) was added, and the reaction was stirred to 120 deg.C for 16 hours. And (3) detecting the disappearance of the raw materials by a dot plate, cooling to 45 ℃, adding 4.8ml of water, cooling to room temperature, filtering to remove insoluble substances, washing a filter cake by 2.5ml of water, concentrating the filtrate, adding 4ml of water, adjusting the pH to 11 by NaOH, extracting for 3-4 times by chloroform, adjusting the pH of a water phase to 3.1 by concentrated hydrochloric acid, extracting for 4-5 times by chloroform/isopropanol (3: 1), combining organic phases, evaporating to dryness, adding a small amount of methanol, pulping and purifying, filtering, and drying to obtain 1.24g of a product, wherein the yield is 40.4%. LC-MS (APCI): m/z 364.3(M +1)⁺。

Step 2 Synthesis of L-alanine-d 7-isopropyl ester (Compound 9).

L-alanine (0.88g, 9.87mmol) and deuterated isopropanol (5.0g, 73.4mmol) were added to a reaction flask, the mixture was added to reflux, chlorotrimethylsilane (1.79g, 16.5mmol) was added dropwise, and after the addition, the reaction was refluxed overnight. Concentrating to remove solvent, adding triethylene diamine (1.11g, 9.87mmol) in 5ml tetrahydrofuran to obtain white turbid liquid, filtering with diatomaceous earth, washing filter cake with dichloromethane twice, concentrating the filtrateThe crude product was condensed and dried in vacuo to give 1.7g, 85% yield. LC-MS (APCI): m/z 139.1(M +1)⁺。

Step 39 Synthesis of- { (R) -2- [ ((R, S) - { [ (S) -1- (d 7-isopropyloxycarbonyl) ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } adenine (Compound 10).

Compound 8(0.325g, 0.894mmol) was added to a reaction flask, dissolved in 3ml of acetonitrile, added with thionyl chloride (240.5mg, 2.02mmol), heated to 80 ℃ for reaction for 2 hours, concentrated to remove the solvent, added with 4ml of anhydrous dichloromethane, cooled to-29 ℃, added dropwise with 3ml of dichloromethane solution of Compound 9(271.7mg, 1.97mmol), added after completion of addition, added dropwise with triethylamine (271.4mg, 2.682mmol), warmed to room temperature for reaction for 1 hour, and detected on a dot-plate. Washing with a small amount of water, washing with saturated brine, concentrating, and purifying with silica gel column chromatography to obtain 0.17g of product with yield of 39.35%. LC-MS (APCI): m/z 484.5(M +1)⁺。¹HNMR(300MHz，CDCl₃)δ8.33(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)。

Step 49 isolation of- { (R) -2- [ ((S) - { [ (S) -1- (d 7-isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } adenine (compound T-2).

Chiral Supercritical Fluid Chromatography (SFC) is adopted to separate the racemic compound 10(170mg) to obtain a target product T-2, and the target product T-2 is dried and weighed to obtain 78.4mg, and the yield is as follows: 46.1 percent. LC-MS (APCI): m/z 484.5(M +1)⁺。¹H NMR(300MHz，CDCl₃)δ8.33(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)。

Example 3 preparation of 9- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } -d 5-phenoxyphosphoryl) methoxy ] propyl } adenine, compound T-3, of the formula:

the following synthetic route was used:

step 12, synthesis of 3, 4, 5, 6-d 5-phenol (Compound 11).

Phenol (2.0g, 21.25mmol), 5% Pt/C (0.4g, 20 wt%) and 34ml of heavy water were added to a reaction flask, hydrogen gas was replaced 3 to 4 times, reaction was carried out at room temperature for 24 hours, the catalyst was removed by filtration, the filter cake was washed with dichloromethane, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography to obtain 1.6g of compound 11 with a yield of 80%. LC-MS (APCI): m/z 100.4(M +1)⁺。

Step 2 Synthesis of (R) -9- [2- (d 5-phenoxyphosphorylmethoxy) propyl ] adenine (Compound 12).

Compound 4(2.4g, 8.36mmol), compound 11(1.66g, 16.72mmol) and 6.5ml NMP were charged into a reaction flask, heated to 85 deg.C, triethylamine (1.04g, 10.3mmol) was added, the temperature was raised to 100 deg.C, dicyclohexylcarbodiimide (2.81g, 13.63mmol) was added, and the reaction was stirred to 120 deg.C for 16 hours. And (3) detecting the disappearance of the raw materials by a dot plate, cooling to 45 ℃, adding 4.8ml of water, cooling to room temperature, filtering to remove insoluble substances, washing a filter cake by 2.5ml of water, concentrating the filtrate, adding 4ml of water, adjusting the pH to 11 by NaOH, extracting for 3-4 times by chloroform, adjusting the pH of a water phase to 3.1 by concentrated hydrochloric acid, extracting for 4-5 times by chloroform/isopropanol (3: 1), combining organic phases, evaporating to dryness, adding a small amount of methanol, pulping and purifying, filtering, and drying to obtain 1.12g of a product, wherein the yield is 36.1%. LC-MS (APCI): m/z 369.3(M +1)⁺。

Step 39 Synthesis of- { (R) -2- [ ((R, S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } -d 5-phenoxyphosphoryl) methoxy ] propyl } adenine (Compound 13).

Compound 12(1.0g, 2.71mmol) was charged into a reaction flask, dissolved in 4ml of acetonitrile, added with thionyl chloride (730.5mg, 6.14mmol), heated to 80 ℃ for reaction for 2 hours, concentrated to remove the solvent, and added with 4ml of anhydrous dichloromethaneThe temperature of the alkane is reduced to-29 ℃, a solution of isopropyl alaninate (780.2mg, 5.96mmol) in 3ml dichloromethane is added dropwise, triethylamine (822.7mg, 8.13mmol) is added dropwise after the addition, the temperature is raised to room temperature, the reaction is carried out for 1 hour, and the detection is carried out by a dot plate. Washing with a small amount of water, washing with saturated brine, concentrating, and purifying with silica gel column chromatography to obtain 0.42g of product with yield of 32.3%. LC-MS (APCI): m/z 482.3(M +1)⁺。¹HNMR(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)。

Step 49 isolation of- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } -d 5-phenoxyphosphoryl) methoxy ] propyl } adenine (compound T-3).

Chiral Supercritical Fluid Chromatography (SFC) is adopted to separate the racemate 13(180mg) to obtain a target product T-3, and the target product T-3 is dried and weighed to obtain 78mg, and the yield is as follows: 86.7 percent. 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)。

Example 49- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) -d 4-ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } adenine, compound T-4, of formula:

the synthesis adopts the following route:

step 1 Synthesis of L-d 4-isopropyl alaninate (Compound 14).

L-d 4-alanine (0.168g, 1.29mmol) and isopropanol (576mg, 9.6mmol) were added to the reaction flask, the mixture was brought to reflux, chlorotrimethylsilane (234mg, 2.15mmol) was added dropwise, and after the addition, the reaction was refluxed overnight. Concentrating to remove solvent, adding triethyleneDiamine (145mg, 1.29mmol) in 5ml tetrahydrofuran gave a white turbid liquid, celite assisted filtration, the filter cake washed twice with dichloromethane, the filtrate concentrated to give crude product, which was dried in vacuo to give 148.2mg, yield 85%. LC-MS (APCI): m/z 136.2(M +1)⁺。

Step 29 Synthesis of- { (R) -2- [ ((R, S) - { [ (S) -1- (isopropoxycarbonyl) -d 4-ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } adenine (Compound 15).

Compound 8(234.3g, 0.645mmol) was added to a reaction flask, dissolved in 2ml of acetonitrile, added thionyl chloride (173.4mg, 1.46mmol), heated to 80 ℃ for reaction for 2 hours, concentrated to remove the solvent, added with 3ml of anhydrous dichloromethane, cooled to-29 ℃, added dropwise with 3ml of dichloromethane solution of compound 14(174.4mg, 1.29mmol), added after addition, added dropwise with triethylamine (195.8mg, 1.935mmol), warmed to room temperature for reaction for 1 hour, and detected on a dot plate. Washing with a small amount of water, washing with saturated brine, concentrating, and purifying with silica gel column chromatography to obtain 0.2g product with yield of 64.5%. LC-MS (APCI): 481.5(M +1) with M/z⁺。¹HNMR(300MHz，CDCl₃)δ8.32(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)。

Step 39 isolation of- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) -d 4-ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } adenine (Compound T-4).

Chiral Supercritical Fluid Chromatography (SFC) is adopted to separate racemic compound 15(150mg) to obtain target product T-4, 53mg is weighed after drying, and the yield is as follows: 70.7 percent. LC-MS (APCI): 481.5(M +1) with M/z⁺。¹H NMR(300MHz，CDCl₃)δ8.32(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)。

Example 5 preparation of 9- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } -2, 8-d 2-adenine, compound T-5, of formula:

the synthesis adopts the following route:

step 12, Synthesis of 8-d 2-adenine (Compound 16).

Adding adenine (1.0g, 7.4mmol), heavy water (10ml) and Pd/C (100mg) into a microwave reaction bottle to replace hydrogen, sealing, placing in a microwave reactor to react at 160 ℃ for 1.5-2 hours, cooling to room temperature, adding 0.65ml concentrated hydrochloric acid, heating to 60 ℃ to dissolve the product, filtering while hot, adjusting the pH of the filtrate to 8.0 by ammonia water, cooling in an ice bath and keeping for 0.5 hour, filtering to obtain a white solid, and drying in vacuum to obtain 0.8g of the product, wherein the yield is as follows: 80 percent. LC-MS (APCI): 138.3(M +1) ═ M/z⁺。

Step 2 Synthesis of (R) -9- (2-hydroxypropyl) -2, 8-d 2-adenine (Compound 17).

Adding a compound 16(2.465g, 17.97mmol) and (R) -propylene carbonate (2.093g, 20.5mmol) into a reaction bottle, adding 3ml of DMF for dissolving, heating to 130 ℃ for reacting overnight, cooling to 100 ℃ after the reaction is detected by a dot plate, adding 8.5ml of toluene and 0.3g of methanesulfonic acid (keeping the internal temperature at 100 ℃ and 110 ℃), adding 7ml of toluene to obtain a homogeneous suspension, gradually cooling to room temperature, cooling to 0 ℃ for 1 hour, filtering to obtain a white solid, and drying in vacuum to obtain 3.76g of a product with the yield of 100%. LC-MS (APCI): 196.3(M +1)⁺。

Step 3 Synthesis of (R) -9- [2- (diethylphosphonomethoxy) propyl ] -2, 8-d 2-adenine (Compound 18).

Dissolving compound 17(1.0g, 5.17mmol) with 40ml anhydrous DMF, cooling to 0 ℃, adding NaH (233.3mg) under nitrogen protection, reacting at low temperature for 40 minutes, adding 10ml anhydrous DMF of compound 2(1.75g, 5.44mmol), dissolving, heating to room temperature, reacting for 18 hours, detecting reaction by dot plate, concentrating to remove solvent, purifying by silica gel column chromatography, evaporating to dryness to obtain 0.89g product with 51% yield. LC-MS (APCI): m/z 346.5(M +1)⁺。

Step 4 Synthesis of (R) -9- [2- (phosphorylmethoxy) propyl ] -2, 8-d 2-adenine (Compound 19).

Adding compound 18(2.276g, 6.63mmol) into a dry reaction flask, adding 20ml of anhydrous DMF for dissolving, adding TMSBr (3.76g, 24.57mmol) at room temperature, stirring for reacting for 20 hours, detecting the completion of the reaction by a dot plate, concentrating to remove the solvent, adding ammonia water to adjust the pH to 8.0, concentrating to obtain an oily liquid, adjusting the pH to 3.0 by using diluted hydrochloric acid, evaporating to dryness again, adding isopropanol to precipitate a yellow solid, filtering, and recrystallizing with isopropanol/water (3: 1) to obtain 0.62g of a white solid with the yield of 32.1%. LC-MS (APCI): m/z 288.6(M-1)^-。

Step 5 Synthesis of (R) -9- [2- (phenoxyphosphorylmethoxy) propyl ] -2, 8-d 2-adenine (Compound 20).

Compound 19(296mg, 1.023mmol), phenol (202.8mg, 2.05mmol) and 3ml NMP were added to a reaction flask, heated to 85 deg.C, triethylamine (127.3mg, 1.26mmol) was added, the temperature was raised to 100 deg.C, dicyclohexylcarbodiimide (344.6mg, 1.67mmol) was added, and the reaction was stirred to 120 deg.C for 16 hours. And (3) detecting the disappearance of the raw materials by using a dot plate, cooling to 45 ℃, adding 2ml of water, cooling to room temperature, filtering to remove insoluble substances, washing a filter cake by using 2ml of water, concentrating the filtrate, adding 3ml of water, adjusting the pH to 11 by using NaOH, extracting for 3-4 times by using chloroform, adjusting the pH of a water phase to 3.1 by using concentrated hydrochloric acid, extracting for 4-5 times by using chloroform/isopropanol (3: 1), combining organic phases, evaporating to dryness, adding a small amount of methanol, pulping and purifying, filtering, and drying to obtain a product of 269mg, wherein the yield is 72%. LC-MS (APCI): m/z 366.3(M +1)⁺。

Step 69 Synthesis of- { (R) -2- [ ((R, S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } -2, 8-d 2-adenine (Compound 21).

Compound 20(312mg, 0.85mmol) was added to a reaction flask, dissolved in 3ml of acetonitrile, added with thionyl chloride (230mg, 1.93mmol), heated to 80 ℃ for reaction for 2 hours, concentrated to remove the solvent, added with 3ml of anhydrous dichloromethane, cooled to-29 ℃, added dropwise with a solution of isopropyl alaninate (253.8mg, 1.88mmol) in 3ml of dichloromethane, added dropwise with triethylamine (259mg, 2.56mmol), warmed to room temperature for reaction for 1 hour, and detected on a dot-plate. Washing with a small amount of water, washing with saturated brine, concentrating, and purifying with silica gel column chromatography to obtain 0.15g of product with yield of 36.9%. LC-MS (APCI): m/z 479.1(M +1)⁺。¹HNMR(300MHz，CDCl₃)δ7.34-7.27(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)。

Step 79 isolation of- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } -2, 8-d 2-adenine (Compound T-5).

Chiral Supercritical Fluid Chromatography (SFC) is adopted to separate racemate compound 21(135mg) to obtain a target product T-5, and the target product T-5 is dried and weighed to obtain 56mg, and the yield is as follows: 83 percent. LC-MS (APCI): m/z 479.1(M +1)⁺。¹H NMR(300MHz，CDCl₃)δ7.34-7.27(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)。

Example 6 preparation of 9- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) -d 2-methoxy ] propyl } adenine, compound T-6, of formula:

the synthesis adopts the following route:

step 1 Synthesis of diethyl [ [ (p-toluenesulfonyl) oxy ] -d 2-methyl ] phosphate (Compound 22).

Adding the compound 2(1.0g, 3.1mmol) and anhydrous potassium carbonate (42.8mg, 0.31mmol) into a microwave reaction bottle, adding 10ml of heavy water, sealing, placing in a microwave reactor, heating to 80 ℃ for reaction for 1 hour, cooling to room temperature, adding ethyl acetate for extraction, combining organic phases, washing twice with water and saturated saline water in sequence, concentrating, purifying by silica gel column chromatography, and drying in vacuum to obtain 0.86g of a product with the yield of 85%. LC-MS (APCI): 325.1(M +1)⁺。

Step 2 Synthesis of (R) -9- [2- (diethylphosphoryl-d 2-methoxy) propyl ] adenine (Compound 23).

Dissolving compound 1(1.0g, 5.17mmol) with 40ml anhydrous DMF, cooling to 0 ℃, adding NaH (233.3mg) under nitrogen protection, reacting at low temperature for 40 minutes, adding 10ml anhydrous DMF of compound 22(1.75g, 5.44mmol), dissolving, heating to room temperature, reacting for 18 hours, detecting reaction by dot plate, concentrating to remove solvent, purifying by silica gel column chromatography, evaporating to dryness to obtain 0.91g product, yield 51.7%. LC-MS (APCI): m/z 346.5(M +1)⁺。

Step 3 Synthesis of (R) -9- [2- (phosphoryl-d 2-methoxy) propyl ] adenine (Compound 24).

Adding compound 23(2.276g, 6.63mmol) into a dry reaction flask, adding 20ml of anhydrous DMF for dissolving, adding TMSBr (3.76g, 24.57mmol) at room temperature, stirring for reacting for 20 hours, detecting the completion of the reaction by a dot plate, concentrating to remove the solvent, adding ammonia water to adjust the pH to 8.0, concentrating to obtain an oily liquid, adjusting the pH to 3.0 by using diluted hydrochloric acid, evaporating to dryness again, adding isopropanol to precipitate a yellow solid, filtering, and recrystallizing with isopropanol/water (3: 1) to obtain 0.76g of a white solid with the yield of 40.1%. LC-MS (APCI): m/z 288.2(M-1)^-。

Step 4 Synthesis of (R) -9- [2- (phenoxyphosphoryl-d 2-methoxy) propyl ] adenine (Compound 25).

Compound 24(296mg, 1.023mmol), phenol (202.8mg, 2.05mmol) and 3ml NMP were added to a reaction flask, heated to 85 deg.C, added triethylamine (127.3mg, 1.26mmol), warmed to 100 deg.C,dicyclohexylcarbodiimide (344.6mg, 1.67mmol) was added and the reaction stirred for 16 h rising to 120 ℃. And (3) detecting the disappearance of the raw materials by using a dot plate, cooling to 45 ℃, adding 2ml of water, cooling to room temperature, filtering to remove insoluble substances, washing a filter cake by using 2ml of water, concentrating the filtrate, adding 3ml of water, adjusting the pH to 11 by using NaOH, extracting for 3-4 times by using chloroform, adjusting the pH of a water phase to 3.1 by using concentrated hydrochloric acid, extracting for 4-5 times by using chloroform/isopropanol (3: 1), combining organic phases, evaporating to dryness, adding a small amount of methanol, pulping and purifying, filtering, and drying to obtain 295mg of a product, wherein the yield is 78.9%. LC-MS (APCI): m/z 366.3(M +1)⁺。

Step 59 Synthesis of- { (R) -2- [ ((R, S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) -d 2-methoxy ] propyl } adenine (Compound 26).

Compound 25(312mg, 0.85mmol) was added to a reaction flask, dissolved in 3ml of acetonitrile, added with thionyl chloride (230mg, 1.93mmol), heated to 80 ℃ for reaction for 2 hours, concentrated to remove the solvent, added with 3ml of anhydrous dichloromethane, cooled to-29 ℃, added dropwise with a solution of isopropyl alaninate (253.8mg, 1.88mmol) in 3ml of dichloromethane, added dropwise with triethylamine (259mg, 2.56mmol), warmed to room temperature for reaction for 1 hour, and detected on a dot-plate. Washing with a small amount of water, washing with saturated brine, concentrating, and purifying with silica gel column chromatography to obtain 0.17g of product with yield of 41.8%. LC-MS (APCI): m/z 479.1(M +1)⁺。¹HNMR(300MHz，CDCl₃)δ8.32(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)。

Step 69 isolation of- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) -d 2-methoxy ] propyl } adenine (Compound T-6).

Chiral Supercritical Fluid Chromatography (SFC) is adopted to separate racemic compound 26(150mg) to obtain target product T-6, and the target product T-6 is dried and weighed to obtain 48mg, and the yield is as follows: and 64 percent. LC-MS (APCI): m/z 479.1(M +1)⁺。¹H NMR(300MHz，CDCl₃)δ8.32(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 assay

(1) Testing compounds for in vitro anti-HIV activity

Compound treatment: test and reference compounds will be diluted in DMSO at double ratios and added to the cell culture plate. Test and reference compounds will be tested at 8 concentrations, two replicate wells.

Viral infection and cell treatment: HIV-1 and MT-4 cells were incubated at 37 ℃ with 5% CO₂Co-culturing for 1h in an incubator. Infected cells are then seeded at a density in cell culture plates. The final concentration of DMSO in the cell culture medium was 0.5%. The cells were incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 5 days. The cells tested in the cytotoxicity test are uninfected MT-4 cells, and other test conditions are consistent with the antiviral activity test.

And (3) detecting the activity of the cells: cell activity was measured by using cell activity assay reagent CellTiter-glo (Promega). The raw data were used for compound anti-HIV-1 activity and cytotoxicity calculations. Dose response curves of compounds and their EC₅₀And CC₅₀Values will be obtained by analysis with GraphPad Prism software, where a represents EC₅₀Less than 10nM, B means EC < 10nM₅₀Less than or equal to 100nM, C means 100nM < EC₅₀Less than or equal to 500nM, D represents EC₅₀More than 500 Nm; f represents CC₅₀> 10000nM (as shown in Table 1 below).

(2) Detection of in vitro anti-HBV Activity of Compounds

The experimental method comprises the following steps: luciferase assay Compounds were assayed for anti-hepatitis C virus activity using Bright-glo (Promega). Data were analyzed using GraphPad Prism software, curves were fitted and EC calculated₅₀And CC₅₀The value is obtained.

The experimental steps are as follows:

anti-cell activity assay: in vitro anti-hepatitis b virus activity of 20 compounds was tested in hepg2.2.15 cells with TDF as a positive control compound. Cells were seeded into 96-well plates on the first day, compound-treated cells were added the next day, and the culture broth containing the compound was replaced on the fifth day. And collecting supernatant on the eighth day to extract DNA. The content of HBV DNA was detected by quantitative PCR. Both test compound and TDF were serially diluted 3-fold, 8 concentration points, and 2 replicate wells were assayed in parallel. The final concentration of DMSO in the culture broth was 0.5%. The percent inhibition calculation formula is as follows:

% inhibition ═ 100 (1-copy number of HBV in sample/copy number of HBV in DMSO control group) × 100

EC₅₀Analysis by Graphpad Prism software (four parameter logistic equines), where I denotes EC₅₀Less than 5nM, II denotes EC < 5nM₅₀Less than or equal to 20nM, III denotes 20nM < EC₅₀Less than or equal to 100nM, IV denotes EC₅₀(> 100nM (as shown in Table 1 below).

Cytotoxicity experiments: the compound plate arrangement and the compound treatment process are consistent with the detection of anti-HIV activity. Six days after compound treatment of the cells, cell activity was measured. Cell-titer Blue reagent was added to each well, incubated at 37 ℃ for 3 hours, and fluorescence values were read (560Ex/590 Em); analysis of data and calculation of relative cell viability:

the percentage of cell activity was calculated using the following formula: percent cell viability ═ 100 (sample fluorescence reading-culture control fluorescence reading)/(DMSO control fluorescence reading-culture control fluorescence reading).

Finally CC of the compound was calculated using GraphPad Prism software₅₀Value, V denotes CC₅₀> 200000nM (as shown in Table 1 below).

Table 1 compounds of the examples HBV activity and HIV activity

The experimental result shows that the compound of the invention has strong anti-HIV activity and HBV activity (both reaching nanomolar level), and compared with the anti-HIV drug (GS7340) which is newly marketed by Gilidde science of the pharmaceutical company of America, the anti-HIV activity and anti-HBV activity of the compound of the embodiment are both equivalent, wherein the anti-HBV activity of the compounds T-5 and T-6 of the embodiment shows better activity than that of GS 7340. In addition, as measuredThe compounds of the invention showed no toxicity in cell lines (optimal CC)₅₀＞200000nM)。

(3) Liver microparticle metabolism test

Microsome experiment: human liver microsomes: 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).

Preparing a stock solution: an amount of the compound of example 1-6 was weighed out precisely and dissolved in DMSO to 5mM each.

Preparation of phosphate buffer (100mM, pH 7.4): 150mL of 0.5M potassium dihydrogenphosphate and 700mL of a 0.5M dipotassium hydrogenphosphate solution prepared in advance were mixed, the pH of the mixture was adjusted to 7.4 with the 0.5M dipotassium hydrogenphosphate solution, the mixture was diluted 5-fold with ultrapure water before use, and magnesium chloride was added to obtain a phosphate buffer (100mM) containing 100mM potassium phosphate and 3.3mM magnesium chloride at a pH of 7.4.

NADPH regenerating system solution (containing 6.5mM NADP, 16.5mM G-6-P, 3U/mL G-6-P D, 3.3mM magnesium chloride) was prepared and placed on wet ice before use.

Preparing a stop solution: acetonitrile solution containing 50ng/mL propranolol hydrochloride and 200ng/mL tolbutamide (internal standard). 25057.5 mu L of phosphate buffer solution (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of human liver microsome is respectively added and mixed evenly, and liver microsome dilution liquid with the protein concentration of 0.625mg/mL is obtained. 25057.5 mu L of phosphate buffer (pH7.4) is taken to a 50mL centrifuge tube, 812.5 mu L of SD rat liver microsome is respectively added, and the mixture is mixed evenly to obtain liver microsome dilution with the protein concentration of 0.625 mg/mL.

Incubation of the samples: the stock solutions of the corresponding compounds were diluted to 0.25mM each with an aqueous solution containing 70% acetonitrile, and used as working solutions. 398. mu.L of human liver microsome or rat liver microsome dilutions were added to a 96-well plate (N2), 2. mu.L of 0.25mM working solution was added, and mixed well.

Determination of metabolic stability: 300. mu.L of pre-cooled stop solution was added to each well of a 96-well deep-well plate and placed on ice as a stop plate. The 96-well incubation plate and the NADPH regeneration system are placed in a 37 ℃ water bath box, shaken at 100 rpm and pre-incubated for 5 min. 80. mu.L of the incubation solution was taken out of each well of the incubation plate, added to the stop plate, mixed well, and supplemented with 20. mu.L of NADPH regenerating system solution as a 0min sample. Then 80. mu.L of NADPH regenerating system solution was added to each well of the incubation plate, the reaction was started, and the timer was started. The reaction concentration of the corresponding compound was 1. mu.M, and the protein concentration was 0.5 mg/mL. When the reaction was carried out for 10min, 30min and 90min, 100. mu.L of each reaction solution was added to the stop plate and vortexed for 3min to terminate the reaction. The stop plates were centrifuged at 5000 Xg for 10min at 4 ℃. And (3) taking 100 mu L of supernatant to a 96-well plate in which 100 mu L of distilled water is added in advance, mixing uniformly, and performing sample analysis by adopting LC-MS/MS.

And (3) data analysis: and detecting peak areas of the corresponding compound and the internal standard through an LC-MS/MS system, and calculating the peak area ratio of the compound to the internal standard. The slope is determined by plotting the natural logarithm of the percentage of compound remaining against time and calculating t according to the following formula_1/2And CL_intWhere V/M is equal to 1/protein concentration.

Table 2 evaluation of liver microparticle metabolism of the compounds of the examples

The experimental results are shown in table 2 above, compared with GS7340, the compound of the present invention has longer half-life and smaller clearance, shows better metabolic stability in both human liver microsome and rat liver microsome experiments, and is more suitable for being used as anti-HIV or anti-HBV drugs.

(4) Pharmacokinetic experiment of rat

Purpose of the experiment: study the pharmacokinetic behaviour of the compounds of the invention was investigated after administration of 9- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) -methoxy ] propyl } adenine, the compounds of examples 1-6, to rats.

Experimental animals:

species and strain: SD rat grade: SPF stage

Sex and amount: male, 6

Body weight range: 180 to 220g (actual weight range 187 to 197g)

The source is as follows: shanghai Xipulbikai laboratory animals Co., Ltd

Experimental and animal certification numbers: SCXK (Shanghai) 2013-0016

The experimental process comprises the following steps:

before blood sample collection, 20L of 2M sodium fluoride solution (esterase inhibitor) was added to an EDTA-K2 anticoagulation tube, dried in an 80 ℃ oven, and stored in a 4 ℃ refrigerator.

Rats, males, weighing 187-197 g, were randomized into 2 groups, fasted overnight but with free access to water starting the afternoon of the day before the experiment, and given food 4h after administration. Group A was administered with 3mg/kg of 9- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } adenine, and group B was administered with 3mg/kg of the compound of examples 1-6, and 100L of blood was collected from orbital veins of rats at 15min, 30min, 1, 2, 3, 5, 8, and 10h after administration, and placed in 0.5mL Eppendorf tubes anticoagulated with EDTA-K2, immediately mixed, after anticoagulation, the tubes were gently mixed 5-6 times, the blood was collected and placed in an ice box, and the blood sample was centrifuged at 4000rpm for 10min and 4 ℃ for 30min to separate plasma, and the whole plasma was collected and immediately stored at-20 ℃. Plasma concentrations were determined in plasma at each time point after sample collection at all time points.

From the mean plasma concentration-time data after administration obtained above, pharmacokinetic-related parameters of i.g administration of 9- { (R) -2- [ ((S) - { [ (S) -1- (isopropyloxycarbonyl) ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } adenine (3mg/kg) and the compound of example 1-6 (3mg/kg) to male SD rats, respectively, were calculated by non-atrial-moment theory using Winnonin software.

Experiments show that compared with 9- { (R) -2- [ ((S) - { [ (S) -1- (isopropoxycarbonyl) ethyl ] amino } phenoxyphosphoryl) methoxy ] propyl } adenine, the compound has better activity and excellent pharmacokinetic property, so the compound is more suitable to be used as a compound for inhibiting nucleoside reverse transcriptase and is further suitable to be used for preparing a medicine for treating antiviral infection.

It is to be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention, and that experimental procedures not specifically identified in the examples will generally be performed under conventional conditions, or under conditions recommended by the manufacturer. Parts and percentages are parts and percentages by weight unless otherwise indicated.

The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the invention pertains, several simple deductions or substitutions may be made without departing from the spirit of the invention, which should be construed as belonging to the scope of the invention.

Claims (9)

1. A nucleoside reverse transcriptase inhibitor, comprising: an adenine compound represented by the formula (1), or a pharmaceutically acceptable salt thereof,

wherein R is¹、R²、R⁹、R¹⁰、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵、R²⁶Each independently is hydrogen, deuterium, halogen or trifluoromethyl;

R³、R⁴、R⁵、R⁶、R⁷、R⁸、R¹¹、R¹²、R¹³and R¹⁴Is hydrogen;

with the proviso that R¹、R²、R⁹、R¹⁰、R¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰、R²¹、R²²、R²³、R²⁴、R²⁵And R²⁶Is deuterium, and does not include the following compounds:

2. the nucleoside reverse transcriptase inhibitor of claim 1, wherein: r¹And R²Is deuterium.

3. The nucleoside reverse transcriptase inhibitor of claim 1, wherein: r⁹And R¹⁰Is deuterium.

4. The nucleoside reverse transcriptase inhibitor of claim 1, wherein: r¹⁵、R¹⁶、R¹⁷、R¹⁸、R¹⁹、R²⁰And R²¹Is deuterium.

5. The nucleoside reverse transcriptase inhibitor of claim 1, wherein: r²²、R²⁴And R²⁶Is deuterium.

6. The nucleoside reverse transcriptase inhibitor of claim 1, wherein: the compound may be selected from the following compounds or pharmaceutically acceptable salts thereof:

7. a pharmaceutical composition characterized by: a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the nucleoside reverse transcriptase inhibitor of any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof.

8. Use of a nucleoside reverse transcriptase inhibitor according to any one of claims 1 to 6, wherein: can be used for preparing medicine for treating viral infection diseases.

9. The use according to claim 8, wherein said viral infection is selected from the group consisting of AIDS and hepatitis B.