WO2016192560A1 - 替诺福韦单苄酯磷酰胺前药、其制备方法及应用 - Google Patents

替诺福韦单苄酯磷酰胺前药、其制备方法及应用 Download PDF

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WO2016192560A1
WO2016192560A1 PCT/CN2016/083407 CN2016083407W WO2016192560A1 WO 2016192560 A1 WO2016192560 A1 WO 2016192560A1 CN 2016083407 W CN2016083407 W CN 2016083407W WO 2016192560 A1 WO2016192560 A1 WO 2016192560A1
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Prior art keywords
tenofovir
group
compound
tenofovir monobenzyl
compounds
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PCT/CN2016/083407
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English (en)
French (fr)
Inventor
王国成
吴会敏
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江苏天士力帝益药业有限公司
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Priority to US15/577,956 priority Critical patent/US10233202B2/en
Priority to ES16802483T priority patent/ES2806726T3/es
Priority to EP16802483.4A priority patent/EP3305795B1/en
Priority to AU2016270101A priority patent/AU2016270101B2/en
Priority to RU2017145357A priority patent/RU2719594C2/ru
Priority to SG11201709786WA priority patent/SG11201709786WA/en
Priority to KR1020177037627A priority patent/KR20180016437A/ko
Priority to CN201680031443.4A priority patent/CN107709340B/zh
Application filed by 江苏天士力帝益药业有限公司 filed Critical 江苏天士力帝益药业有限公司
Priority to MYPI2017704555A priority patent/MY191515A/en
Priority to CA2987473A priority patent/CA2987473A1/en
Priority to JP2017561871A priority patent/JP6679624B2/ja
Publication of WO2016192560A1 publication Critical patent/WO2016192560A1/zh
Priority to IL255892A priority patent/IL255892B/en
Priority to HK18106521.5A priority patent/HK1246799A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
    • C07F9/65616—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings containing the ring system having three or more than three double bonds between ring members or between ring members and non-ring members, e.g. purine or analogs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6561—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings

Definitions

  • the invention belongs to the field of medicinal chemistry, and particularly relates to a novel tenofovir monobenzyl ester phosphoramide compound or hydrate, solvate, pharmaceutically acceptable salt or single chiral isomer thereof, and preparation method thereof and medicament thereof Application on.
  • Tenofovir is a water-soluble oral anti-HIV and anti-hepatitis B virus drug that is stable in the stomach, enters the body with blood after intestinal absorption, and is evenly distributed in human tissues, less than 20%. Metabolism and activation into tenofovir parent drug by the action of enzyme ester and double-phosphorylation to form tenofovir diphosphate after the onset, the other about 80% of the original form of urine excreted.
  • a strategy of adding a masking group to the tenofovir phosphate group to form a fat-soluble prodrug is currently adopted, in which one masking group forms a phosphoramide structure with a phosphate group, and another group Compounds that form phospholipid structures with phosphate groups have been shown to have lymphatic and liver tissue targeting effects.
  • the ester-forming group includes various aromatic rings and aromatic heterocyclic rings, particularly substituted or unsubstituted phenyl groups (CN201310041647.4, WO02082841), and the patent (CN01813161) discloses a compound GS-7340 obtained by using such a prodrug strategy.
  • liver-targeting properties compared to tenofovir (TDF), while activity enhances toxicity.
  • TDF tenofovir
  • the metabolically active parent drug tenofovir can still be produced in the blood, thereby bringing about certain systemic toxicity, and the phenol itself produced by metabolism is also highly toxic.
  • a substituted benzylic tenofovir prodrug compound on a benzene ring has been shown to have liver-targeting activity.
  • Patent US20130210757, CN201380030061.6 discloses a masking group for amino acid esters and phosphoric acid groups to form phosphoramide, a masking A benzyl group having a group substituted with an electron-donating group such as a methyl group on the benzene ring or a p-phosphate group forms a substituted benzyl ester on the benzene ring.
  • an electron-donating group such as a methyl group on the benzene ring or a p-phosphate group
  • the masking group o-methylbenzyl group in the disclosed compound structure has high group leaving activity, low stability in blood enzyme ester metabolism, and the target group is relatively easy to fall off and cause activity in blood.
  • the relative increase of the parent drug and the active parent drug in the liver are relatively reduced, thereby affecting the activity and systemic toxicity.
  • the present invention provides a class of tenofovir monobenzyl phosphate phosphorus-amide compounds which are unsubstituted on a benzylbenzene ring, a preparation method thereof and a lymphoid target thereof Compared with GS-7340 and Compound 7, this prodrug is more stable to the enzyme ester than the GS-7340 and Compound 7, further enhancing the systemic stability and liver targeting resistance of tenofovir analogs. The role of the virus.
  • the inventors of the present invention invented a class of unsubstituted tenofovir monobenzyl phosphate phosphoramides on benzylbenzene rings, and unexpectedly found that the compounds of the invention can be metabolized into the active parent drug tenofovir in cellular experiments. (TFV), thus having antiviral activity.
  • TBV tenofovir
  • mice can be enriched in the liver and effectively metabolized into the active product tenofovir after gavage, and the compounds of the invention are more active against HBV than in the prior art, or in plasma. It is more stable and its metabolic fragments are safer, thus reducing systemic side effects caused by plasma metabolism.
  • the present invention provides a tenofovir monobenzyl phosphate phosphoramide compound having the general formula X, a hydrate, a solvate thereof, a pharmaceutically acceptable salt or a resolved single isomer thereof,
  • Z is selected from the group consisting of O, S, Se, NH- or -CH 2 -,
  • R 1 , R 2 , R 3 , R 4 , R 5 are each independently selected from H, a substituted or unsubstituted C 1 -C 10 linear hydrocarbon group, a C 3 -C 10 branched hydrocarbon group, a C 3 -C 10 ring a hydrocarbyl group, a C 6 -C 10 aromatic hydrocarbon group or a heteroaryl group, wherein the substitution is one to three heteroatoms independently selected from O, S, N, Se, or R 1 and R 2 , R 1 and R 3 R 2 and R 3 together with the moiety linking them form a substituted or unsubstituted 3-8 membered ring.
  • Z is selected from O or S
  • R 1 , R 2 , R 3 , R 4 , R 5 are each independently selected from H, a substituted or unsubstituted C 1 -C 6 linear hydrocarbon group, a C 3 -C 6 branched hydrocarbon group, a C 3 -C 6 ring Hydrocarbyl group, C 6 -C 10 aromatic hydrocarbon group or heteroaryl group.
  • Z is selected from O,
  • R 1 , R 2 , R 3 , R 4 , and R 5 are each independently selected from H, a substituted or unsubstituted C 1 -C 6 linear hydrocarbon group, a C 3 -C 6 branched hydrocarbon group, and a C 6 -C 10 aromatic group. Hydrocarbyl group.
  • the tenofovir monobenzyl phosphate phosphoramide compound of the present invention is selected from the compounds of Table 1.
  • the stereochemistry of prodrugs can affect their metabolic and antiviral activity in target tissues, and the chiral moiety is on the phosphorus atom and is also found in its masking group amino acids. For example, an amino acid having a natural configuration has a better metabolic activity, and an isomer having a P atom configuration of S in the compound 3 has a stronger activity. If the chiral sites are impure, chiral enrichment of these diastereomers or racemates is required, making the screening results more meaningful. Purification by chiral resolution affords the formation of a single isomer at the above chiral center such that each test compound is essentially a single chiral compound.
  • a substantially unitary compound or chiral enrichment means that the desired stereoisomer constitutes more than about 60% by weight of the compound, by more than 80%, preferably more than 95%.
  • the invention is separated by reverse phase column separation or chiral column separation, and the mobile phase is an aqueous acetonitrile solution.
  • Another object of the present invention is to provide a process for preparing a tenofovir monobenzyl phosphate phosphoramide compound, which comprises the following steps:
  • tenofovir is reacted with benzyl halide or benzyl alcohol in the presence of a base to give a tenofovir monobenzyl ester intermediate;
  • the base may be various inorganic or organic bases, preferably an organic base; and the terminal NH group-containing compound in step B is preferably an amino acid ester compound or an amino acid.
  • An amide compound is preferably an amino acid ester compound or an amino acid.
  • DIPEA diisopropylethylamine
  • benzyl bromide or benzyl alcohol is sequentially added to the tenofovir acetonitrile suspension, and the mixture is heated to 50 ° C - 80 ° C, stirred under heating for 2-24 hours, and pyridine is added.
  • the invention further comprises a method for chiral separation of the compound, HPLC preparative column separation (preparation column: C18, mobile phase: 10%-50% aqueous acetonitrile (V/V)) or chiral column separation to collect the eluent for each retention time. Dry to obtain each chiral isomer.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the tenofovir monobenzyl ester phosphate amide compound, or a hydrate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof Or a single isomer of its resolution.
  • the pharmaceutically acceptable salt of the compound of the present invention can be obtained by acid-base neutralization using conventional techniques in the chemical field as needed. If the compound of the present invention is reacted with sulfuric acid, hydrochloric acid, hydrobromic acid, phosphoric acid, tartaric acid, fumaric acid, maleic acid, citric acid, acetic acid, formic acid, methanesulfonic acid, p-toluenesulfonic acid, oxalic acid or succinic acid, the corresponding reaction is obtained. Salt.
  • the compound of the present invention may be reacted with sodium hydroxide, potassium hydroxide, cesium hydroxide or the like, an alkali metal carbonate such as sodium carbonate, calcium carbonate or the like to obtain the corresponding salt.
  • the reaction can be carried out in a solvent such as water or an organic solvent such as ethanol, tetrahydrofuran, dioxane, ethylene glycol, acetic acid or the like, or a mixture of such an organic solvent and water.
  • the reaction can also be carried out without any solvent, if necessary.
  • the pharmaceutical composition of the present invention is preferably in the form of a unit dosage of a pharmaceutical preparation which can be formulated into any pharmaceutically acceptable dosage form, which is selected from the group consisting of: tablets, sugar-coated tablets, film-coated tablets, Enteric coated tablets, capsules, hard capsules, soft capsules, oral liquids, buccal preparations, granules, suspensions, solutions, injections, suppositories, ointments, plasters, creams, sprays, Patch.
  • Preferred are oral formulations, and tablets and capsules are most preferred.
  • composition of the present invention further comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical preparation can be prepared by a conventional technique of formulation, such as the tenofovir monobenzyl phosphate phosphoramide compound of the present invention, or a hydrate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable salt thereof
  • the single isomer is mixed with a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes, but is not limited to, mannitol, sorbitol, sorbic acid or potassium salt, sodium metabisulfite, sodium hydrogen sulfite, sodium thiosulfate, cysteine hydrochloride, thioglycolic acid, methionine, Vitamin A, Vitamin C, vitamin E, vitamin D, azone, EDTA disodium, EDTA calcium sodium, monovalent alkali metal carbonate, acetate, phosphate or its aqueous solution, hydrochloric acid, acetic acid, sulfuric acid, phosphoric acid, amino acid, rich Horse acid, sodium chloride, potassium chloride, sodium lactate, xylitol, maltose, glucose, fructose, dextran, glycine, starch, sucrose, lactose, mannitol, silicon derivatives, cellulose and its derivatives, Alginate, gelatin, polyvinylpyrrolidone, glycerin, propylene glyco
  • a unit dose of the medicament may contain 0.1 to 1000 mg of the pharmaceutically active substance of the present invention, and the balance is a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier may be from 0.1 to 99.9% by weight based on the total weight of the formulation.
  • the pharmaceutical composition of the present invention determines the usage amount according to the condition of the patient at the time of use.
  • the present invention finally provides the tenofovir monobenzyl phosphate phosphoramide compound, or a hydrate thereof, or a solvate thereof, or a pharmaceutically acceptable salt thereof, or a resolved single isomer thereof, in the preparation of a treatment
  • the use in a medicament for a viral infectious disease is preferably used in the preparation of a medicament for treating an HIV infection or a disease caused by hepatitis B or hepatitis B virus.
  • DIPEA (10 mmol) and benzyl bromide (5 mmol) were added to a suspension of tenofovir (5 mmol) in acetonitrile (20 mL). The mixture was heated to 80 ° C and stirred for 16 hr. Add pyridine (20 mL) to dissolve, and then add triethylamine (5 mL) and glycine isopropyl ester hydrochloride (10 mmol), and then stir to 50 ° C for 30 minutes, then add triphenylphosphine (15 mmol) and 2 at this temperature. 2'-Dithiodipyridine (15 mmol), and the mixture was stirred at 50 ° C for 3 hours, and then dried under reduced pressure. The residue was purified by silica gel chromatography eluting elut The yield was 48%.
  • DIPEA (10 mmol) and benzyl bromide (5 mmol) were added to a suspension of tenofovir (5 mmol) in acetonitrile (20 mL). The mixture was heated to 80 ° C and stirred for 16 hr. Add pyridine (20 mL) to dissolve, and then add triethylamine (5 mL) and glycine methyl ester hydrochloride (10 mmol), and then stir to 50 ° C for 30 minutes, then add triphenylphosphine (15 mmol) and 2 at this temperature. 2'-Dithiodipyridine (15 mmol) was stirred at 50 ° C for 3 hours and then dried under reduced pressure. The residue was purified by silica gel chromatography eluting elut The yield was 57%.
  • DIPEA (10 mmol) and benzyl bromide (5 mmol) were added to a suspension of tenofovir (5 mmol) in acetonitrile (20 mL). The mixture was heated to 80 ° C and stirred for 16 hr. Add pyridine (20 mL) to dissolve, and then add triethylamine (5 mL) and L-phenylalanine isopropyl ester hydrochloride (10 mmol), and then stir to 50 ° C for 30 minutes, then add triphenylphosphine at this temperature. (15 mmol) and 2,2'-dithiodipyridine (15 mmol) were stirred at 50 ° C for 3 hours and then dried under reduced pressure. The residue was purified by silica gel chromatography eluting elut The yield was 61%.
  • DIPEA (10 mmol) and benzyl bromide (5 mmol) were added to a suspension of tenofovir (5 mmol) in acetonitrile (20 mL). The mixture was heated to 80 ° C and stirred for 16 hr. Add pyridine (20 mL) to dissolve, and then add triethylamine (5 mL) and benzyl glycinate hydrochloride (10 mmol), and then stir to 50 ° C for 30 minutes, then add triphenylphosphine (15 mmol) and 2 at this temperature. 2'-Dithiodipyridine (15 mmol) was stirred at 50 ° C for 3 hours and then dried under reduced pressure. The residue was purified by silica gel chromatography eluting elut The yield was 58%.
  • HPLC reversed-phase column separation or HPLC chiral column separation Compound 2 (200 mg) of Example 2 was isolated by HPLC (preparation column: Diamonsil C18, 5 ⁇ m, 150 ⁇ 21.1 mm; mobile phase: 20% aqueous acetonitrile (V) /V)) Compound 2a (83 mg; retention time 14 min) and compound 2b (90 mg; retained) after isocratic elution Time 17min).
  • Test Example The beneficial effects of the present invention are exemplified by the following tests
  • the prodrug compounds For prodrug compounds, the most critical is the stability of the prodrug in the system and the metabolic activity in the target organ part, the higher the stability in the system (gastrointestinal, blood, etc.), in the target organ (lymph, The higher the activity of metabolizing into a parent drug in the liver, the lower the toxicity of the compound and the higher the drug effect.
  • the prodrugs such as the compound of the present invention and the reference compound were all metabolized to the active parent drug tenofovir (TFV) to exert an antiviral action.
  • the currently similar prodrug compounds are compounds of the CN201380030061.6 claim (abbreviated as compound 7 and its single chiral isomers 7a, 7b) and Gilead recently applied to the FDA for the market treatment of hepatitis B drug TAF (GS-7340). It contains the same parent drug structure as the compound of the present invention, but the liver targeting fragment is different.
  • the compounds of the present invention are advantageous in that the activity is higher or the system is more stable and the system is less toxic. Further, the benzoic acid compound produced by the metabolism of the compound of the present invention is relatively safe relative to GS-7340, overcoming the GS-7340. The release of toxic phenol has the advantage of superior toxicity and lower toxicity. Further, in contrast to the compound of the CN201380030061.6 claim, since the compound target group benzyl group of the present invention is more stable than o-methylbenzyl group, the benzyl detachment activity is lower in blood enzyme ester metabolism, and thus is in the blood. The active parent drug is relatively reduced, and the active parent drug in the liver is relatively increased, thereby exhibiting better activity. The benzyl group of the present invention has less toxicity after detachment, and has superior system stability and lower toxicity. details as follows:
  • Test Example 1 Comparison of anti-HBV activity and cytotoxicity at the cellular level
  • HepG2.2.15 cells (4 x 10 4 cells/well) were seeded into 96-well plates and incubated overnight at 37 ° C, 5% CO 2 . The next day, fresh culture medium containing different concentrations of compounds was added to the culture wells. The compound arrangement is shown in Table 2. On the fifth day, the old culture solution in the culture well was aspirated and fresh culture medium containing different concentrations of the compound was added. On the eighth day, the supernatant in the culture well was collected for extracting HBV DNA from the supernatant. The qPCR assay was used to detect the HBV DNA content in the supernatant of HepG2.2.15.
  • %Inh. [(HBV quantity of DMSO control-HBV quantity of sample)/HBV quantity of DMSO control] ⁇ 100%
  • %cell viability (fluorescence of sample–fluorescence of medium control)/(fluorescence of DMSO control-fluorescence of medium control) ⁇ 100%
  • test compounds 3a, 3b showed better anti-hepatitis B virus activity, EC 50 value below 10 nM, 4 test compounds 1b, 2a 5a, 5b anti-hepatitis B virus activity is relatively weak, EC 50 value between 200nM-1000nM; the other two test compounds 1a, 2a anti-hepatitis B virus activity EC 50 value is higher than the maximum test concentration of 1000nM.
  • the compounds 1, 2, 4, 5, and 6 of the present invention are structurally similar to the compound 3, and thus have similar pharmacodynamic effects.
  • Test Example 2 Comparison of cell level anti-HBV activity and cytotoxicity
  • the compounds 3a and 3b of the present invention showed better anti-hepatitis B virus activity, and the effects were remarkably superior to those of the reference compounds 7a, 7b and GS-7340. Both had no significant effect on HepG2.2.15 cytotoxicity (CC 50 >100 ⁇ M)
  • Test Example 3 Cell level anti-HIV activity and cytotoxicity test
  • MT-4 cells were infected with 24 TCID50 HIV-1 IIIB/1x105 cells (2.4 TCID50/well) for 1 hour at 37 ° C and seeded in 96-well plates containing different concentrations of compounds (4 ⁇ 10). 4 cells/well), cultured at 37 ° C, 5% CO 2 for 5 days. Activity was calculated EC 50 value was determined by CellTiter Glo.
  • Compounds 3a and 3b were more active against HIV than 7b and GS-7340; while 3a and 3b were less toxic to MT-4 cells than GS-7340 and 7b.
  • CHO cells stably expressing hERG potassium channels were obtained from AViva Biosciences, and the cells were incubated at 37 ° C, 5% CO 2 , constant humidity.
  • the compound and the positive control compound amitriptyline (Amitriptyline, Sigma-Aldrich, BCBJ8594V) were diluted in 100% dimethyl sulfoxide (DMSO) and the final concentration of DMSO in the extracellular fluid was not higher than 0.30%. Stand by at -20 °C.
  • the compounds were tested on a Multiclamp patch-clamp amplifier at room temperature using whole-cell patch clamp technique.
  • the output signals were digitized using a DIgiDAta 1440A/D-D/A plate, and the Pclamp10 software was used for recording control.
  • the minimum sealing resistance was set to 500 MOhms and the minimum specific hERG current was 0.4 nA for quality control.
  • ICR mice Male, weight 30 ⁇ 5 g, purchased from Vitallihua Animal Center
  • 12 h 12 h before dosing, and given free water during fasting.
  • 30 mg of compound 3 was accurately weighed, dissolved in 100 ⁇ L of 75% ethanol, further added with physiological saline to 6 mL, vortexed and mixed, and ultrasonicated for use.
  • the tenofovir prodrug was administered at a dose of 50 mg/kg and the dose was 10 mL/kg.
  • Sample collection protocol 0.5 mL of blood was taken from the eyelids at 15 min, 30 min, 1 h and 3 h after intragastric administration. The liver tissue was washed and weighed. The liver was added to the physiological saline homogenate at a ratio of 1:1. Store in a 40 ° C refrigerator for testing.
  • internal standard 200 ng/ml theophylline
  • internal standard 200 ng/ml theophylline
  • Thermo TSQquantum LC/MS and column Thermo Hypersil GOLD (2.1 ⁇ 150mm), internal standard Theophylline, HPLC-MS injection, gradient elution analysis, recording internal standard, compound 1 and metabolite tenofovir
  • the retention time and peak area of (TFV) were analyzed by SRM quantitative detection method.

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Abstract

本发明涉及一种替诺福韦单苄酯磷酰胺前药、其制备方法及其在医药上的应用，具体而言，本发明涉及一种如通式(X)所示的化合物或其异构体、可药用盐、水合物或溶剂化物，它们的制备方法，以及它们在制备病毒感染性疾病，优选在制备艾滋病感染、乙型肝炎或乙肝病毒引起的疾病的药物中的应用。

Description

替诺福韦单苄酯磷酰胺前药、其制备方法及应用 技术领域
本发明属于药物化学领域，具体涉及一种新型替诺福韦单苄酯磷酰胺类化合物或其水合物、溶剂化物、可药用盐或单一手性异构体，及其制备方法与其在医药上的应用。
背景技术
替诺福韦酯(TDF)是一种水溶性口服抗HIV和抗乙肝病毒药物，在胃中稳定，于肠道吸收后随血液进入人体内，并均匀分布于人体组织内，不到20％在酶酯的作用下代谢活化成替诺福韦母药并双磷酸化生成替诺福韦二磷酸后起效，其他约80％以原形经尿液排出体外。为提高其生物利用度，目前多采取在替诺福韦磷酸基团上加入掩蔽基团从而形成脂溶性前药的策略，其中一个掩蔽基团与磷酸基团形成磷酰胺结构，另一个基团与磷酸基团形成磷酯类结构的化合物被证实有淋巴和肝组织靶向效应。成酯基团包括各种芳环和芳杂环，特别是取代或非取代苯基(CN201310041647.4，WO02082841)，专利(CN01813161)公开了一种采用此种前药策略得到的化合物GS-7340，它与替诺福韦酯(TDF)相比增加了肝靶向的特性，同时活性增强毒性降低。但是由于掩蔽基团苯酚基的稳定性不够强，在血液中仍可以发生代谢生成活性母药替诺福韦，从而带来一定的系统毒性，同时代谢生成的苯酚自身也具有较大毒性。苯环上有取代的苄基类替诺福韦前药化合物已被证实具有肝靶向活性，专利US20130210757，CN201380030061.6公开了一个掩蔽基团为氨基酸酯与磷酸基团形成磷酰胺，一个掩蔽基团为苯环上邻或对位有甲基等供电子基团取代的苄基与磷酸基团形成苯环上有取代的苄酯。而成酯基团为未取代苄基的替诺福韦前药化合物则未见合成与生物活性研究报道，可能是由于苯环上无取代的苄基在5-氟尿嘧啶核苷前药运用中无法代谢从而导致无活性的原因(WO02082841)。
CN201380030061.6公开的化合物结构中的掩蔽基团邻甲基苄基，基团离去活性高，在血液酶酯代谢中稳定性低，靶向基团相对更容易脱落而导致在血液中的活性母药相对增加，肝脏中的活性母药相对降低，从而影响活性和系统毒性。
Figure PCTCN2016083407-appb-000001
为增强替诺福韦的生物活性，升级其抗病毒活性，本发明提供了一类在苄基苯环上无取代的替诺福韦单苄酯磷-酰胺类化合物与其制备方法以及其淋巴靶向抗艾滋病感染、肝靶向抗乙肝治疗用途与GS-7340和化合物7相比这种前药对酶酯更稳定，进一步增强了替诺福韦类似物的系统稳定性和肝靶向性抗病毒作用。
发明内容
本发明的发明人发明了一类苄基苯环上无取代的替诺福韦单苄酯磷酰胺类化合物，并意外地发现本发明化合物在细胞实验中可以代谢成活性母药替诺福韦(TFV)，从而具有抗病毒活性。在动物体内实验中，小鼠灌胃后可以在肝部富集并有效代谢成活性产物替诺福韦，而且与现有技术相比，本发明化合物抗HBV病毒活性更强，或者在血浆中更稳定，其代谢片段更安全，从而降低了血浆代谢引起的系统毒副作用。
具体来说，本发明提供了一种具有通式X的替诺福韦单苄酯磷酰胺类化合物，其水合物、溶剂化物、药学上可接受的盐或其拆分的单一异构体，
Figure PCTCN2016083407-appb-000002
式中Z选自O，S，Se，NH-或-CH2-，
R1，R2，R3，R4，R5分别独立地选自H，取代或未取代的C1-C10直链烃基、C3-C10支链烃基、C3-C10环烃基、C6-C10芳香烃基或杂芳基，其中所述取代为一个到三个独立地选自O，S，N，Se的杂原子，或者R1与R2，R1与R3，R2与R3与连接它们的结构部分一起形成取代或未经取代的3-8元环。
优选的，
Z选自O或S，
R1，R2，R3，R4，R5分别独立地选自H，取代或未取代的C1-C6直链烃基、C3-C6支链烃基、C3-C6环烃基、C6-C10芳香烃基或杂芳基。
更优选的，
Z选自O，
R1，R2，R3，R4，R5分别独立地选自H，取代或未取代的C1-C6直链烃基、C3-C6支链烃基、C6-C10芳香烃基。
优选，本发明的替诺福韦单苄酯磷酰胺类化合物，选自表1的化合物
表1 化合物及结构式
Figure PCTCN2016083407-appb-000003
Figure PCTCN2016083407-appb-000004
我们发现，前药的立体化学能够影响其在靶组织中的代谢能力和抗病毒活性，手性部分在磷原子上，也发现在其掩蔽基团氨基酸上。例如天然构型的氨基酸具有更好的代谢活性，化合物3中的P原子构型为S的异构体具有较强的活性。如果各手性部位不纯，需要对这些非对映异构体或消旋体进行手性富集，从而使筛选的结果更有意义。通过手性拆分提纯得到在在上述手性中心上构型单一的异构体，使得每个实验化合物基本上是单一手性化合物。形成基本上单一的化合物或手性富集意味着所需的立体异构体构成超过化合物重量的约60％，通过超过80％，优选超过95％。本发明通过反向色谱柱分离或者手性色谱柱分离，流动相为乙腈水溶液。
本发明的另一个目的是提供替诺福韦单苄酯磷酰胺类化合物的制备方法，其特征在于，包括如下步骤：
A：替诺福韦在碱的存在下与卤化苄或苄醇反应得到替诺福韦单苄酯中间体；
B：替诺福韦单苄酯中间体与各种含末端NH基团的化合物反应生成本发明的替诺福韦单苄酯磷酰胺类化合物。
其中，步骤A中替诺福韦优选与苄溴或苄醇进行反应，碱可以是各种无机或有机碱，优选有机碱；步骤B中含末端NH基团的化合物优选氨基酸酯类化合物、氨基酸酰胺类化合物。
具体为：替诺福韦乙腈悬浮液中依次加入二异丙基乙基胺(DIPEA)、苄溴或苄醇，将此混合物加热到50℃-80℃，保温搅拌2-24小时，加入吡啶溶解，再依次加入三乙胺和甘氨酸苄酯盐酸盐、甘氨酸甲酯盐酸盐、L-丙氨酸异丙酯盐酸盐、L-苯丙氨酸异丙酯盐酸盐、甘氨 酸异丙酯盐酸盐、N-苯基甘氨酸异丙酯盐酸盐中任一种，加热到50℃-80℃-搅拌10-60分钟后在此温度下加入三苯基膦和2,2’-二硫二吡啶，保温在50℃-100℃搅拌3小时后减压旋干。残渣硅胶柱层析(甲醇/二氯甲烷洗脱)得到白色固体产物。
以下是合成路线：
Figure PCTCN2016083407-appb-000005
本发明进一步包括化合物手性分离的方法，HPLC制备柱分离(制备柱：C18，流动相:10％-50％乙腈水溶液(V/V))或者手性柱分离收集各保留时间的洗脱液，干燥得到各手性异构体。
本发明还提供了一种药物组合物，所述药物组合物含有所述的替诺福韦单苄酯磷酸酰胺类化合物、或其水合物、或其溶剂化物、或其药学上可接受的盐或其拆分的单一异构体。
根据需要，采用化学领域的常规技术，可用酸碱中和的方式得到本发明化合物的药用盐。如使本发明化合物和硫酸、盐酸、氢溴酸、磷酸、酒石酸、富马酸、马来酸、柠檬酸、乙酸、甲酸、甲磺酸、对甲苯磺酸、草酸或琥珀酸反应，得到相应的盐。或使本发明化合物和氢氧化钠、氢氧化钾、氢氧化钡等，碱金属碳酸盐，如碳酸钠、碳酸钙等反应得到相应的盐。反应可在溶剂中进行，例如水或有机溶剂，如乙醇、四氢呋喃、二噁烷、乙二醇、乙酸等，或这种有机溶剂与水的混合物。如需要，该反应还可在无任何溶剂中进行。
本发明的药物组合物，优选的是单位剂量的药物制剂形式，在制成药物制剂时可以制成任何可药用的剂型，这些剂型选自：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、混悬剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、贴剂。优选的是口服制剂形式，最佳优选的是片剂，胶囊剂。
进一步的，本发明所述药物组合物还含有药学上可接受的载体。
可以采用制剂学常规技术制备该药物制剂，如将本发明的替诺福韦单苄酯磷酰胺类化合物、或其水合物、或其溶剂化物、或其药学上可接受的盐或其拆分的单一异构体与药学上可接受的载体混合。所述药学上可接受的的载体包括但不限于：甘露醇、山梨醇、山梨酸或钾盐、焦亚硫酸钠、亚硫酸氢钠、硫代硫酸钠、盐酸半胱氨酸、巯基乙酸、蛋氨酸、维生素A、 维生素C、维生素E、维生素D、氮酮、EDTA二钠、EDTA钙钠，一价碱金属的碳酸盐、醋酸盐、磷酸盐或其水溶液、盐酸、醋酸、硫酸、磷酸、氨基酸、富马酸、氯化钠、氯化钾、乳酸钠、木糖醇、麦芽糖、葡萄糖、果糖、右旋糖苷、甘氨酸、淀粉、蔗糖、乳糖、甘露糖醇、硅衍生物、纤维素及其衍生物、藻酸盐、明胶、聚乙烯吡咯烷酮、甘油、丙二醇、乙醇、土温60-80、司班-80、蜂蜡、羊毛脂、液体石蜡、十六醇、没食子酸酯类、琼脂、三乙醇胺、碱性氨基酸、尿素、尿囊素、碳酸钙、碳酸氢钙、表面活性剂、聚乙二醇、环糊精、β-环糊精、磷脂类材料、高岭土、滑石粉、硬脂酸钙、硬脂酸镁等。
本发明的药物组合物，在制成药剂时，单位剂量的药剂可含有本发明的药物活性物质0.1-1000mg，其余为药学上可接受的载体。药学上可接受的载体以重量计可以是制剂总重量的0.1-99.9％。
本发明的药物组合物在使用时根据病人的情况确定用法用量。
本发明最后还提供了所述替诺福韦单苄酯磷酰胺类化合物、或其水合物、或其溶剂化物、或其药学上可接受的盐或其拆分的单一异构体在制备治疗病毒感染性疾病的药物中的用途，优选在制备治疗艾滋病感染或乙型肝炎或者乙肝病毒引起的疾病的药物中的用途。
具体实施方式
以下结合具体实施例详细地解释本发明，使得本领域技术人员更全面地理解本专利。具体实施例仅用于说明本发明的技术方案，并不以任何方式限定本发明。
实施例1：化合物1的制备
Figure PCTCN2016083407-appb-000006
往替诺福韦(5mmol)的乙腈(20mL)悬浮液中依次加入DIPEA(10mmol)、苄溴(5mmol)，将此混合物加热到80℃，保温搅拌16小时后减压旋干。加入吡啶(20mL)溶解，再依次加入三乙胺(5mL)和甘氨酸异丙酯盐酸盐(10mmol)，加热到50℃搅拌30分钟后在此温度下加入三苯基膦(15mmol)和2,2’-二硫二吡啶(15mmol)，保温在50℃搅拌3小时后减压旋干。残渣硅胶柱层析(甲醇/二氯甲烷洗脱)得到白色固体产物。收率48％。
1H NMR(400MHz,CDCl3)δ8.30(s,1H),7.94,7.91(s,s,1H),7.37-7.28(m,5H),6.10,6.07(s,s,2H),5.07-4.89(m,3H),4.38-4.30(m,1H),4.14-4.05(m,1H),3.91-3.86(m,2H),3.71-3.48(m,4H),1.25-1.18(m,9H)；31P NMR(400MHz,CDCl3)δ25.76,25.66；MS(m/z)477.32(MH+),475.18(MH-)。
实施例2：化合物2的制备
Figure PCTCN2016083407-appb-000007
往替诺福韦(5mmol)的乙腈(20mL)悬浮液中依次加入DIPEA(10mmol)、苄溴(5mmol)，将此混合物加热到80℃，保温搅拌16小时后减压旋干。加入吡啶(20mL)溶解，再依次加入三乙胺(5mL)和甘氨酸甲酯盐酸盐(10mmol)，加热到50℃搅拌30分钟后在此温度下加入三苯基膦(15mmol)和2,2’-二硫二吡啶(15mmol)，保温在50℃搅拌3小时后减压旋干。残渣硅胶柱层析(甲醇/二氯甲烷洗脱)得到白色固体产物。收率57％。
1H NMR(400MHz,CDCl3)δ8.26(s,1H),7.93,7.92(s,s,1H),7.31-7.4(m,5H),6.37(s,2H),5.01-4.86(m,2H),4.33-4.25(m,1H),4.10-4.01(m,1H),3.93-3.80(m,2H),3.67-3.53(m,4H),1.40-1.14(m,6H)；31PNMR(400MHz,CDCl3)δ25.96,25.73；MS(m/z)449.30(MH+)。
实施例3：化合物3的制备
Figure PCTCN2016083407-appb-000008
往替诺福韦(5mmol)的乙腈(20mL)悬浮液中依次加入DIPEA(10mmol)、苄溴(5mmol)，将此混合物加热到在80℃，保温搅拌16小时后减压旋干。加入吡啶(20mL)溶解，再依次加入三乙胺(5mL)和L-丙氨酸异丙酯盐酸盐(10mmol)，加热到50℃搅拌30分 钟后在此温度下加入三苯基膦(15mmol)和2,2’-二硫二吡啶(15mmol)，保温在50℃搅拌3小时后减压旋干。残渣硅胶柱层析(甲醇/二氯甲烷洗脱)得到白色固体产物。收率54％。
1H NMR(400MHz,CDCl3)δ8.34,8.33(s,s,1H),7.93,7.92(s,s,1H),7.36-7.30(m,5H),6.00,5.99(s,s,2H),5.06-4.97(m,2H),4.94-4.89(m,1H),4.40-4.28(m,1H),4.14-4.06(m,1H),4.03-3.92(m,2H),3.89-3.78(m,2H),3.67-3.53(m,2H),1.33-1.18(m,12H)；31PNMR(400MHz,CDCl3)δ25.02,24.12；MS(m/z)491.32(MH+)。
实施例4：化合物4的制备
Figure PCTCN2016083407-appb-000009
往替诺福韦(5mmol)的乙腈(20mL)悬浮液中依次加入DIPEA(10mmol)、苄溴(5mmol)，将此混合物加热到80℃，保温搅拌16小时后减压旋干。加入吡啶(20mL)溶解，再依次加入三乙胺(5mL)和L-苯丙氨酸异丙酯盐酸盐(10mmol)，加热到50℃搅拌30分钟后在此温度下加入三苯基膦(15mmol)和2,2’-二硫二吡啶(15mmol)，保温在50℃搅拌3小时后减压旋干。残渣硅胶柱层析(甲醇/二氯甲烷洗脱)得到白色固体产物。收率61％。
1H NMR(400MHz,CDCl3)δ8.33(s,1H),7.90(s,1H),7.30-7.09(m,10H),6.23(s,2H),5.03-4.88(m,2H),4.33-4.29(m,1H),4.15-3.90(m,3H),3.81-3.71(m,1H),3.48-3.43(m,1H),3.21-3.02(m,3H),2.94-2.76(m,2H),1.47-1.42(m,3H),1.26-1.07(m,9H)；31PNMR(400MHz,CDCl3)δ20.78；MS(m/z)567.32(MH+)。
实施例5：化合物5的制备
Figure PCTCN2016083407-appb-000010
往替诺福韦(5mmol)的乙腈(20mL)悬浮液中依次加入DIPEA(10mmol)、苄溴(5mmol)，将此混合物加热到80℃，保温搅拌16小时后减压旋干。加入吡啶(20mL)溶解，再依次加入三乙胺(5mL)和甘氨酸苄酯盐酸盐(10mmol)，加热到50℃搅拌30分钟后在此温度下加入三苯基膦(15mmol)和2,2’-二硫二吡啶(15mmol)，保温在50℃搅拌3小时后减压旋干。残渣硅胶柱层析(甲醇/二氯甲烷洗脱)得到白色固体产物。收率58％。
1H NMR(400MHz,CDCl3)δ8.30,8.29(s,s,1H),7.93,7.92(s,s,1H),7.37-7.27(m,10H),6.14(s,2H),5.31(s,1H),5.15(s,1H),5.10(s,1H),5.04-4.87(m,2H),4.34-4.26(m,1H),4.09-4.00(m,1H),3.92-3.81(m,2H),3.76-3.54(m,1H),3.17-3.11(m,2H),1.18-1.16(m,3H)；31PNMR(400MHz,CDCl3)δ25.81,25.61；MS(m/z)525.19(MH+)。
实施例6：化合物6的制备
Figure PCTCN2016083407-appb-000011
往替诺福韦(5mmol)的乙腈(20mL)悬浮液中依次加入DIPEA(10mmol)、苄溴(5mmol)，将此混合物加热到80℃，保温搅拌16小时后减压旋干。加入吡啶(20mL)溶解，再依次加入三乙胺(5mL)和N-苯基甘氨酸异丙酯盐酸盐(10mmol)，加热到50℃搅拌30分钟后在此温度下加入三苯基膦(15mmol)和2,2’-二硫二吡啶(15mmol)，保温在50℃搅拌3小时后减压旋干。残渣硅胶柱层析(甲醇/二氯甲烷洗脱)得到白色固体产物。收率27％。
1HNMR(400MHz,CDCl3)δ8.29(s,1H),8.09(s,1H),7.50-7.14(m,10H),6.60(s,2H),5.07-4.90(m,3H),4.37-4.34(m,7H),3.17-3.12(m,3H),1.45-1.41(m,6H)；31PNMR(400MHz,CDCl3)δ24.43,24.15；MS(m/z)553.25(MH+)。
实施例7：化合物的手性分离制备
HPLC反向色谱柱分离或者HPLC手性色谱柱分离：取实施例2中化合物2(200mg)经HPLC制备分离(制备柱：Diamonsil C18，5μm，150x21.1mm；流动相：20％乙腈水溶液(V/V))等度洗脱后得到化合物2a(83mg；保留时间14min)和化合物2b(90mg；保留 时间17min)。
化合物2a：MS(m/z)449.26(MH+)；1H NMR(400MHz,CDCl3)δ8.28(s,1H),7.92(s,1H),7.32-7.24(m,5H),6.58(s,2H),5.02-4.88(m,2H),4.30-4.26(m,1H),4.16-4.02(m,1H),3.90-3.84(m,2H),3.69-3.65(m,5H),3.60-3.54(m,1H),1.16(s,3H)；31P NMR(400MHz，CDCl3)δ25.87；
化合物2b：MS(m/z)449.32(MH+)；1HNMR(400MHz,CDCl3)δ8.28(s,1H),7.92(s,1H),7.32-7.27(m,5H),6.64(s,2H),5.03-5.01(m,2H),4.34-4.30(m,1H),4.10-4.01(m,2H),3.93-3.84(m,2H),3.66-3.59(m,5H),1.14(s,3H)；31P NMR(400MHz，CDCl3)δ25.64。
化合物1,3,5采用与手性柱拆分化合物2类似的方法用手性柱进行拆分分别得到化合物1a,1b,3a,3b,5a,5b,。
化合物1a：1H NMR(400MHz,CDCl3)δ8.25(s,1H),7.93(s,1H),7.30-7.26(m,5H),6.17(s,2H),5.00-4.90(m,2H),4.34-4.29(m,1H),4.11-4.06(m,2H),3.92-3.81(m,2H),3.63-3.59(m,3H),1.18-1.23(m,9H)；31PNMR(400MHz，CDCl3)δ25.79；
化合物1b：1HNMR(400MHz,CDCl3)δ8.28(s,1H),7.92(s,1H),7.32-7.27(m,5H),6.64(s,2H),5.03-5.01(m,2H),4.34-4.30(m,1H),4.10-4.01(m,2H),3.93-3.84(m,2H),3.66-3.59(m,3H),1.16-1.14(m,9H)；31PNMR(400MHz，CDCl3)δ25.60。
化合物3a：1H NMR(400MHz,CDCl3)δ8.30(s,1H),7.90(s,1H),7.32-7.27(m,5H),6.19(s,2H),5.03-4.96(m,2H),4.92-4.87(m,1H),4.30-4.25(m,1H),4.09-4.03(m,1H),3.97-3.94(m,1H),3.90-3.76(m,2H),3.56-3.50(m,1H),1.30-1.15(m,12H)；31P NMR(400MHz，CDCl3)δ24.18；
化合物3b：1H NMR(400MHz,CDCl3)δ8.30(s,1H),7.91(s,1H),7.36-7.29(m,5H),6.09(s,2H),4.99-4.96(m,2H),4.94-4.87(m,1H),4.38-4.34(m,1H),4.12-4.06(m,1H),3.96-3.90(m,2H),3.87-3.81(m,1H),3.60-3.55(m,1H),3.45-3.40(m,1H),1.31-1.16(m,12H)；31P NMR(400MHz，CDCl3)δ25.04
化合物5a：1H NMR(400MHz,CDCl3)δ8.28(s,1H),7.95(s,1H),7.40-7.23(m,10H),6.33(s,2H),5.10-4.95(m,4H),4.32-4.28(m,1H),4.01-3.84(m,2H),3.82-3.55(m,4H),1.24(s,3H)；31P NMR(400MHz，CDCl3)δ25.88
化合物5b：1HNMR(400MHz,CDCl3)δ；8.27(s,1H),7.94(s,1H),7.34-7.27(m,10H),6.12(s,2H),4.96-4.84(m,4H),4.28-4.23(m,1H),3.83-3.51(m,6H),1.15(s,3H)；31P NMR(400MHz，CDCl3)δ25.59
表2 本发明手性化合物列表
Figure PCTCN2016083407-appb-000012
在上述化合物中a、b构型各占化合物比例50％。
实施例8
取实施例7表2任意一个手性化合物1.2kg，富马酸285g，和3L乙腈加入反应器中，加热回流混合物使固体溶解，趁热过滤，冷却后至5℃并保持16小时，过滤分离出产物，用乙腈冲洗，干燥得到白色粉末。
试验例：通过下述试验例证明本发明的有益效果
对前药化合物而言，最关键的是前药在系统中的稳定性和在靶器官部分的代谢活性，在系统(胃肠道，血液等)中稳定性越高，在靶器官(淋巴、肝脏)中代谢成母药的活性越高，则化合物毒性越低、药效越高。试验例中本发明化合物、参照化合物等前药均代谢成活性母药替诺福韦(TFV)后发挥抗病毒作用。
Figure PCTCN2016083407-appb-000013
目前结构相近的前药化合物是CN201380030061.6权利要求中的化合物(简称化合物7及其单一手性异构体7a，7b)和吉利德新近向FDA申请上市治疗乙肝的药物TAF(GS-7340)，其与本发明的化合物含有相同的母药结构，但肝靶向片段不同。
本发明的化合物优势在于活性更高或由于结构更稳定从而系统毒性更小。进一步地相对于GS-7340而言，本发明的化合物代谢生成的苯甲酸类化合物则相对安全，克服了GS-7340 释放出毒性苯酚的缺陷，在活性优越的同时具备更低毒性的优势。进一步相对于CN201380030061.6权利要求中的化合物来说，由于本发明的化合物肝靶基团苄基比邻甲基苄基更稳定，在血液酶酯代谢中苄基脱落活性较低，因此血液中的活性母药相对减少，肝脏中的活性母药相对增加，从而体现出更好的活性。本发明的化合物苄基脱落后毒性更小，具有更优的系统稳定性和更低的毒性。具体如下：
试验例1：细胞水平抗HBV活性与细胞毒性对比实验
通过实时荧光定量PCR(qPCR)方法检测HepG2.2.15细胞上清中的HBV DNA的含量测定化合物在HepG2.2.15细胞的抗乙肝病毒活性，通过Cell-titer Blue检测受试化合物对HepG2.2.15细胞活性影响。
8.1.化合物稀释：体外抗HBV活性实验所有化合物起始浓度为1μM，3倍稀释，8个浓度；细胞毒性实验所有化合物起始浓度100μM，3倍稀释，8个浓度；用DMSO对化合物母液进行稀释。参照化合物TDF的体外抗HBV活性实验和细胞毒性实验的起始浓度均设为0.2μM，3倍稀释，8个浓度。
8.2.体外抗HBV活性实验：种HepG2.2.15细胞(4×104细胞/孔)到96孔板，在37℃，5％CO2培养过夜。第二天，加入含不同浓度化合物的新鲜培养液到培养孔中。化合物排布见表2。第五天，吸除培养孔中旧的培养液，加入含不同浓度化合物的新鲜培养液。第八天，收集培养孔中的上清，用于提取上清中的HBV DNA。qPCR实验检测HepG2.2.15上清中的HBV DNA含量。
8.3.细胞活力实验细胞处理：种HepG2.2.15细胞到96孔板(4×104细胞/孔)，在37℃，5％CO2培养过夜。第二天，加入含不同浓度化合物的新鲜培养液到培养孔中，化合物排布见表3。第五天，吸除培养孔中旧的培养液，加入含不同浓度化合物的新鲜培养液。第八天，每孔加入Cell-titer Blue试剂，酶标仪检测各孔的荧光值。
8.4.分析数据和计算抑制百分比和相对细胞活力：
应用如下公式计算抑制百分比：
％Inh.＝【(HBV quantity of DMSO control-HBV quantity of sample)/HBV quantity of DMSO control】×100％
应用如下公式计算细胞活性百分比：
％cell viability＝(fluorescence of sample–fluorescence of medium control)/(fluorescence of DMSO control-fluorescence ofmedium control)×100％
用GraphPad Prism软件计算化合物的50％有效浓度(EC50)值和50％细胞毒性浓度CC50值。
8.5.实验结果与结论：
表3：化合物抗HBV病毒实验结果EC50与CC50值
Figure PCTCN2016083407-appb-000014
本次实验中共有8个受试化合物，实验结果总结如下：2个受试化合物3a、3b显示出较好的抗乙肝病毒活性，EC50值在10nM级以下，4个受试化合物1b、2a、5a、5b抗乙肝病毒活性相对较弱，EC50值在200nM-1000nM之间；另外2个受试化合物1a、2a的抗乙肝病毒活性EC50值高于最大测试浓度1000nM。
本发明的化合物1、2、4、5、6与化合物3的结构相似，因此具有相似的药效作用。
试验例2、细胞水平抗HBV活性与细胞毒性对比实验
9.1药品：化合物3、参照化合物(CN201380030061.6，权利要求36所示的化合物简称化合物7及其异构体)稀释及浓度同实施例1中。
Figure PCTCN2016083407-appb-000015
9.2实验方法：按照实施例1进行
9.3结果与分析：
表4：化合物抗HBV病毒实验结果EC50与CC50值
Figure PCTCN2016083407-appb-000016
Figure PCTCN2016083407-appb-000017
从表4可见，本发明化合物3a、3b显示出较好的抗乙肝病毒活性，效果显著优于参照化合物7a、7b、GS-7340。两者对HepG2.2.15细胞毒性均未有明显影响(CC50>100μM)
试验例3：细胞水平抗HIV活性与细胞毒性实验
9.1.化合物、参照化合物(CN01813161GS-7340、TDF)稀释及浓度同实施例1中。
9.2.体外抗HIV活性实验：MT-4细胞在37℃用24 TCID50 HIV-1 IIIB/1x105 cells(2.4TCID50/well)感染1小时后种到含有不同浓度化合物的96孔板中(4×104细胞/孔)，37℃，5％CO2培养5天。用CellTiter Glo测定活性计算EC50值。
9.3.细胞活力实验细胞处理：用9.2中相同的方法，仅将含有不同浓度化合物的96孔板中替换成空白96孔板进行平行实验，用CellTiter Glo测定细胞活力计算CC50值。
9.4.分析数据和计算抑制百分比：应用如下公式计算活性百分比：
Activity(％)＝(Raw datacpd-AverageVC)/(AverageCC-AverageVC)*100
Cell Viability(％)＝Raw datacpd/AverageCC*100
用GraphPad Prism软件计算化合物的50％有效浓度(EC50)值和50％细胞毒性浓度CC50值。
9.5.实验结果与结论：
表5：化合物抗HIV病毒实验结果EC50与CC50值
Figure PCTCN2016083407-appb-000018
化合物3a和3b抗HIV病毒活性比7b和GS-7340更高；同时3a、3b对MT-4细胞的毒性要小于GS-7340和7b的毒性。
总结：实施例2、3可见，药效初步研究中抗HBV和抗HIV活性数据显示，化合物3a、3b具有较好的抗乙肝病毒活性，同时具有较好的抗HIV病毒活性，与TAF的活性成分GS-7340活性相比，具有显著的优势，同时明显优于另外两个对照化合物7a和7b。细胞毒性研究的结果：对HepG2.2.15细胞毒性均未有明显影响(CC50>100μM)；而对MT-4细胞的毒性，数 据显示化合物3a和3b与GS-7340和7b相比具有更低的MT-4细胞毒性。
试验例4：稳定性研究结果
下述稳定性试验方法按照现有技术进行，稳定性实验中表中显示的数据为测试条件下被试化合物在孵育不同时间段后的残留百分比。
10.1模拟胃液稳定性(表6)：
Figure PCTCN2016083407-appb-000019
10.2模拟肠液稳定性(测试浓度：10μM)(表7)：
Figure PCTCN2016083407-appb-000020
10.3人血稳定性(测试浓度：2μM)(表8)：
Figure PCTCN2016083407-appb-000021
10.4人肝S9稳定性(测试浓度：1μM)(表9)：
Figure PCTCN2016083407-appb-000022
上述(7-Ethoxycumarin)7-乙氧基香豆素、(7-Hydroxycoumarin)7-羟基香豆素、(Eucatropine)尤卡托品、(Chlorambucil)氯氨布西、(Omeprazole)奥美拉唑等相关对照品的实验数据可以验证本系列实验的有效性。
10.5数据分析与结论
稳定性初步研究实验数据显示，化合物3a、3b与GS-7340、7b相比，在人肝S9中稳定性相当，及代谢成活性母药的速率相当，预示在肝细胞中相同浓度的化合物具有相当的活性。
在模拟胃液中，但3a、3b稳定性与GS-7340相当，高于7b；在模拟肠液中的稳定性3a、3b显著高于7b和GS-7340。在人血中3a和3b的稳定性也好于对比化合物7b和GS-7340。综合比较化合物3a、3b与GS-7340和7b相比具有更高胃肠道和血液系统代谢稳定性，从而非病灶部分药物浓度更低，病灶部位药物浓度更高，预示着化合物3a、3b与GS-7340和7b相比具有更好的肝靶向性和更低的系统毒性。
试验例5：心脏毒性研究
11.1.实验细胞与化合物配制
实验采用从AVivaBiosciences公司获得的能稳定表达hERG钾离子通道的CHO细胞，细胞在37℃，5％CO2，恒定湿度环境中孵育。
化合物和阳性对照化合物阿米替林(Amitriptyline，Sigma-Aldrich,BCBJ8594V)溶解于100％二甲基亚砜(DMSO)后等度稀释，细胞外液中DMSO的最终浓度不高于0.30％，保存于-20℃备用。
11.2.手动膜片钳记录
化合物于室温下在Multiclamp patch-clamp amplifier上采用全细胞膜片钳技术进行测试，输出信号采用DIgiDAta 1440A/D-D/A板进行数字化，Pclamp10软件进行记录控制。设置最小密封电阻为500MOhms，最小特异hERG电流为0.4nA进行质量控制。
11.3.数据分析
采用Clampfit(V10.2,Molecular Devices)，Excel 2003和GraphPad Prism 5.0进行数据分析。电流计算公式：
I/Icontrol＝Bottom+(Top-Bottom)/(1+10^((LogIC50-Log C)*Hillslope
11.4.实验结果与结论(表10)：
Figure PCTCN2016083407-appb-000023
Figure PCTCN2016083407-appb-000024
结论：hERG实验中化合物3a、3b和GS-7340、7b对IC50相当，均在10μM以上，在心脏毒性方面是安全的，符合新药开发中对化合物成药性研究中hERG数据的一般性要求。
试验例6：小鼠体内代谢与组织分布实验
12.1.实验动物、药物配制方法与给药方案
12只ICR小鼠(雄性，体重30±5g，购自维通利华动物中心)随机分为4组，每组3只，给药前先禁食12h，禁食期间自由给水。分析天平上精密称取30mg化合物3，加入100μL 75％乙醇溶解，进一步加入生理盐水至6mL，涡旋混匀，超声待用。替诺福韦前体药物给药剂量为50mg/kg，给药量为10mL/kg。
12.2.样品采集方案与处理方法
样品采集方案：小鼠灌胃给药后于15min，30min，1h和3h眼眶取血0.5mL，处死，取肝组织洗净，称重，肝按1：1比例加入生理盐水匀浆，于-40℃冰箱保存待测。
血浆样品处理方法：取小鼠血浆100μL置于1.5ml塑料EP管中，加入100μL内标(200ng/ml茶碱)溶液，加入600μL乙腈，涡旋振荡2min，离心3min(12500rpm)，取上清液，氮气吹干，用100μL流动相(水：甲醇＝95:5)复溶，进样10μL。
组织样品处理方法：取小鼠组织样品200μL置于1.5mL塑料EP管中，加入100μL内标(200ng/ml茶碱)溶液，加入600μL乙腈，涡旋振荡2min，离心3min(12500rpm)，取上清液，氮气吹干，用100μL流动相(水：甲醇＝95:5)复溶，进样20μL。
12.3.样品分析方法
采用Thermo TSQquantum液质联用仪和色谱柱：Thermo Hypersil GOLD(2.1×150mm)，内标Theophylline，HPLC-MS进样后进行梯度洗脱分析，记录内标、化合物1和代谢产物替诺福韦(TFV)的保留时间和峰面积，通过SRM定量检测方法进行分析。
12.4.样品分析结果与结论(表11)
Figure PCTCN2016083407-appb-000025
Figure PCTCN2016083407-appb-000026
C(化合物3+TFV)肝组织/C(化合物3+TFV)血浆＝377
CTFV血浆/C化合物3血浆/＝0.72
CTFV肝组织/C化合物3肝组织＝166
结果显示3h后，化合物3和其代谢产物替诺福韦TFV在肝脏中的浓度均高于在血液中的浓度，肝脏中两者总浓度为在血液中的377倍，说明化合物3可以在肝脏有效富集；同时血液中TFV的浓度仅为母药化合物3的浓度的0.72倍，而在肝脏中母药TFV的浓度为前药化合物3的浓度的166倍，说明化合物3在小鼠血液中比较稳定，在肝脏有效代谢成活性母药替诺福韦。因而化合物3在动物体内实验中具有血液稳定性和肝靶向性抗HBV活性。

Claims (10)

  1. 一种具有通式X的替诺福韦单苄酯磷酰胺类化合物，其水合物、溶剂化物、药学上可接受的盐或其拆分的单一异构体，
    Figure PCTCN2016083407-appb-100001
    式中Z选自O，S，Se，NH-或-CH2-，
    R1，R2，R3，R4，R5分别独立地选自H，取代或未取代的C1-C10直链烃基、C3-C10支链烃基、C3-C10环烃基、C6-C10芳香烃基或杂芳基，其中所述取代为一个到三个独立地选自O，S，N，Se的杂原子，或者R1与R2，R1与R3，R2与R3与连接它们的结构部分一起形成取代或未经取代的3-8元环。
  2. 根据权利要求1所述的替诺福韦单苄酯磷酰胺类化合物，其中，
    Z选自O或S，
    R1，R2，R3，R4，R5分别独立地选自H，取代或未取代的C1-C6直链烃基、C3-C6支链烃基、C3-C6环烃基、C6-C10芳香烃基或杂芳基。
  3. 根据权利要求2所述的替诺福韦单苄酯磷酰胺类化合物，其中，
    Z选自O，
    R1，R2，R3，R4，R5分别独立地选自H，取代或未取代的C1-C6直链烃基、C3-C6支链烃基、C6-C10芳香烃基。
  4. 根据权利要求3所述的替诺福韦单苄酯磷酰胺类化合物，其中，化合物选自：
    Figure PCTCN2016083407-appb-100002
    Figure PCTCN2016083407-appb-100003
  5. 根据要求4所述的替诺福韦单苄酯磷酸酰胺类化合物，其中化合物1、2、3、5的异构体分别为1a和1b、2a和2b、3a和3b、5a和5b，结构：
    Figure PCTCN2016083407-appb-100004
    Figure PCTCN2016083407-appb-100005
  6. 根据权利要求1～5任一所述的替诺福韦单苄酯磷酰胺类化合物的制备方法，其特征在于，包括如下步骤：
    A：替诺福韦在碱的存在下与卤化苄或苄醇反应得到替诺福韦单苄酯中间体；
    B：替诺福韦单苄酯中间体与与各种含末端NH基团的化合物，优选氨基酸酯类化合物、氨基酸酰胺类化合物反应生成本发明的替诺福韦单苄酯磷酰胺类化合物。
  7. 根据权利要求6任一所述的替诺福韦单苄酯磷酰胺类化合物的制备方法，其中，步骤A中替诺福韦优选与苄溴或苄醇进行反应，碱可以是各种无机或有机碱，优选有机碱。
  8. 一种药物组合物，其特征在于，所述药物组合物含有权利要求1-4任一项所述的替诺福韦单苄酯磷酰胺类化合物、或其水合物、或其溶剂化物、或其药学上可接受的盐或其拆分的单一异构体；其中，所述药物组合物还含有药学上可接受的载体。
  9. 根据权利要求1～5任一所述替诺福韦单苄酯磷酰胺类化合物、或其水合物、或其溶剂化物、或其药学上可接受的盐或其拆分的单一异构体在制备治疗病毒感染性疾病的药物中的用途。
  10. 根据权利要求9所述的替诺福韦单苄酯磷酸胺类化合物、或其水合物、或其溶剂化物、或其药学上可接受的盐或其拆分的单一异构体在制备治疗艾滋病感染或乙型肝炎或者乙肝病毒引起的疾病的药物中的用途。
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