WO2020151296A1 - 双核苷酸前体药物及其制备方法 - Google Patents

双核苷酸前体药物及其制备方法 Download PDF

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WO2020151296A1
WO2020151296A1 PCT/CN2019/115314 CN2019115314W WO2020151296A1 WO 2020151296 A1 WO2020151296 A1 WO 2020151296A1 CN 2019115314 W CN2019115314 W CN 2019115314W WO 2020151296 A1 WO2020151296 A1 WO 2020151296A1
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compound
alkyl
independently selected
aryl
group
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PCT/CN2019/115314
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English (en)
French (fr)
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袁建栋
孙占莉
林祥义
刘平
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博瑞生物医药（苏州）股份有限公司
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Publication of WO2020151296A1 publication Critical patent/WO2020151296A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7084—Compounds having two nucleosides or nucleotides, e.g. nicotinamide-adenine dinucleotide, flavine-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • 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
    • 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
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a dinucleotide prodrug with a novel structure and a preparation method thereof, and also relates to a dinucleotide prodrug with pure optical isomers and a preparation method thereof, and its preparation for the treatment of viral infections, especially type B Hepatitis virus (HBV) infection and application of HBV-related liver disease drugs.
  • viral infections especially type B Hepatitis virus (HBV) infection and application of HBV-related liver disease drugs.
  • HBV hepatitis B
  • HBV is considered to be an important pathogen that can cause many human cancers.
  • HBV infection also causes fulminant hepatitis, a fatal disease in which the liver is destroyed.
  • Chronic hepatitis infection leads to chronic persistent hepatitis, failure, cirrhosis, liver cancer, and death.
  • Hepatitis B virus is very harmful to patients.
  • Iyer et al. disclosed two or more compounds of deoxyribonucleoside and/or ribonucleoside monomers connected by a nucleoside linkage, and their excellent anti-HBV activity.
  • these nucleotides are negatively charged, are difficult to penetrate the intestinal mucosal barrier, and are easily decomposed in the gastrointestinal tract, so they are not suitable for oral administration.
  • prodrug strategies can be used to improve compound stability, increase targeting, overcome first-pass effects, and improve bioavailability.
  • concept of prodrugs is known and there are many strategies for preparing many compounds, including prodrugs of nucleosides and mononucleotides, those of ordinary skill in the art still cannot infer or clearly anticipate dinucleotide similarities.
  • the prodrug may have oral bioavailability and can therefore be developed for oral use. It is known that oral bioavailability is not only related to stability in the gastric mucosa. For example, even with improved stability, it is still unknown whether this relatively large molecular weight dinucleotide prodrug (molecular weight> 700 Daltons) can be transported across the mucosal barrier.
  • the present invention provides a dinucleotide prodrug with a completely new structure; on the other hand, the present invention also provides a method for preparing these dinucleotide prodrugs and a method for separating optical isomers of pure compounds, and their preparation The application of drugs for treating viral infections, especially HBV infection.
  • the present invention provides a compound represented by general formula (I) or a salt thereof:
  • n 1;
  • a and B are independently selected from -NR 6 -and -O-;
  • R 1 , R 2 , R 3 , R 4 , R 5 , and R 6 are independently selected from hydrogen, alkyl, cycloalkyl, aryl, and heterocyclyl; the alkyl, cycloalkyl, aryl, hetero
  • the cyclic group is optionally substituted by one or more substituents, and the substituents are independently selected from: the substituents are independently selected from: halogen, OH, NH 2 , CN, NO 2 , C 1-6 alkyl , C 1-6 alkoxy, and C 5-12 aryl.
  • R 1, R 2, R 3, R 4, R 5, R 6 are each independently selected from H, C 1 ⁇ 6 alkyl group, C 3 ⁇ 12 cycloalkyl, and C 5 ⁇ 12 aryl group
  • the C 1-6 alkyl group and C 5-12 aryl group are optionally substituted by one or more substituents, and the substituents are independently selected from halogen, -OH, C 1-6 alkoxy, -NO 2 , -CN, -NH 2 , C 1-6 alkylamino.
  • the C 1-6 alkyl group is preferably methyl, ethyl, propyl, isopropyl, tert-butyl, n-butyl;
  • the halogen is F, Cl, Br, I, preferably the halogen is F , Cl,
  • the C 5-12 aryl group is preferably phenyl, thienyl, pyrrolyl, furanyl.
  • G is a group having the following structure:
  • R 1 , R 2 , R 3 , R 4 , R 5 are independently selected from hydrogen, alkyl, cycloalkyl, aryl, heterocyclic group; the alkyl, cycloalkyl, aryl, heterocyclic group The group is optionally substituted with one or more substituents, and the substituents are independently selected from: halogen, C 1-6 alkyl, C 1-6 alkoxy, and C 5-12 aryl.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from hydrogen, C 1-6 alkyl substituted or unsubstituted by one or more substituents, C 6-12 cycloalkane Group, C 5-12 aryl, C 5-12 heterocyclic group; the substituents are independently selected from halogen, C 1-6 alkyl, C 1-6 alkoxy, C 5-12 aryl, OH , NH 2 , CN, NO 2 , and trifluoromethyl.
  • R 1 , R 2 , R 3 , R 4 , and R 5 are independently selected from hydrogen, C 1-6 alkyl, C 1-6 alkoxy, and halogen.
  • R 1 and R 2 are both hydrogen
  • R 3 , R 4 , and R 5 are independently selected from: hydrogen and C 1-6 alkyl
  • the C 1-6 alkyl includes methyl, ethyl, propyl Group, isopropyl, cyclopropyl, tert-butyl, n-butyl, n-propyl, cyclohexyl and n-hexyl.
  • G is a group having the following structure:
  • n 1;
  • R 1 , R 2 , and R 3 are independently selected from hydrogen, methyl, ethyl, propyl, isopropyl, t-butyl, n-butyl, cyclohexyl, cyclopropyl or trifluoromethyl. More preferably, R 1 , R 2 , and R 3 are independently selected from hydrogen, and R 3 is hydrogen or methyl.
  • the present invention also provides a compound represented by general formula Ib or a salt thereof:
  • R 3 , R 4 , and R 5 are independently selected from hydrogen, alkyl, cycloalkyl, aryl, and heterocyclyl; the alkyl, cycloalkyl, aryl, and heterocyclyl are optionally selected by one or more substituents, the substituents are independently selected from: halo, C 1-6 alkyl, C 1-6 alkoxy, and C 5- 12 aryl group.
  • R 3 , R 4 and R 5 are independently selected from hydrogen, C 1-6 alkyl, C 6-12 cycloalkyl, C 5-12 substituted or unsubstituted by one or more substituents Aryl, C 5-12 heterocyclic group; the substituents are independently selected from halogen, C 1-6 alkyl, C 1-6 alkoxy, C 5-12 aryl, OH, NH 2 , CN, NO 2 , and trifluoromethyl.
  • R 3 , R 4 , and R 5 are independently selected from hydrogen, C 1-6 alkyl, C 1-6 alkoxy, and halogen.
  • R 3 and R 5 are both hydrogen
  • R 4 is hydrogen, halogen or C 1-6 alkyl
  • the C 1-6 alkyl includes methyl, ethyl, propyl, isopropyl, cyclopropyl Group, tert-butyl, n-butyl, n-propyl, cyclohexyl and n-hexyl
  • the halogen is F, Cl, Br or I, preferably F or Cl. More preferably, R 3 and R 5 are both hydrogen, and R 4 is methyl.
  • the compound I of the present invention preferably has the structure shown in the following formula Ic:
  • R 3 , R 4 , and R 5 are independently selected from hydrogen, alkyl, cycloalkyl, aryl, and heterocyclyl; the alkyl, cycloalkyl, aryl, and heterocyclyl are optionally selected by one or more substituents, the substituents are independently selected from: halo, C 1-6 alkyl, C 1-6 alkoxy, and C 5- 12 aryl group.
  • R 3 , R 4 and R 5 are independently selected from hydrogen, C 1-6 alkyl, C 6-12 cycloalkyl, C 5-12 substituted or unsubstituted by one or more substituents Aryl, C 5-12 heterocyclic group; the substituents are independently selected from halogen, C 1-6 alkyl, C 1-6 alkoxy, C 5-12 aryl, OH, NH 2 , CN, NO 2 , and trifluoromethyl.
  • R 3 , R 4 , and R 5 are independently selected from hydrogen, C 1-6 alkyl, C 1-6 alkoxy, and halogen.
  • R 3 and R 5 are both hydrogen
  • R 4 is hydrogen, halogen or C 1-6 alkyl
  • the C 1-6 alkyl includes methyl, ethyl, propyl, isopropyl, cyclopropyl Group, tert-butyl, n-butyl, n-propyl, cyclohexyl and n-hexyl
  • the halogen is F, Cl, Br or I, preferably F or Cl. More preferably, R 3 and R 5 are both hydrogen, and R 4 is methyl.
  • the preferred representative compound is represented by formula Ib (or its stereoisomer, tautomer, geometric isomer, racemate, or its pharmaceutically acceptable salt) wherein R 3 , R 4 , R 5 is shown in the following table:
  • aryl used in the present invention refers to a monocyclic or polycyclic carbocyclic ring system having one or two aromatic rings, preferably an aryl group containing 5-12 carbon atoms, the aromatic ring includes but is not limited to phenyl , Naphthyl, tetrahydronaphthyl, indanyl, indenyl, furanyl, pyrrolyl, thienyl, etc.
  • the aryl group of the present invention is optionally halogenated (such as F, Cl, or Br), nitro, C 1-6 alkoxy, amino or C 1-6 alkyl at any position on its aromatic ring such as benzene ring. replace.
  • aralkyl or "arylalkyl” refers to an alkyl group containing aryl substitution, such as benzyl, benzhydryl, trityl, phenethyl, and diphenylethyl.
  • the "-aralkyl-” refers to an aralkyl group containing two points of attachment, where the points of attachment can be both in the alkyl part, or both in the aryl part, or in both the alkyl and aryl parts.
  • a connection point for example
  • cycloalkyl refers to a monovalent group of a monocyclic or polycyclic saturated carbocyclic compound produced by removing a single hydrogen atom. Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.2.1]heptyl, and bicyclo[2.2.2]octyl.
  • heterocyclyl refers to a 5-membered, 6-membered or 7-membered non-aromatic ring or di- or tricyclic group fusion system, wherein (i) each ring contains one to three hetero Atoms, independently selected from oxygen, sulfur and nitrogen, (ii) each 5-membered ring has 0 to 1 double strands and each 6-membered ring has 0 to 2 double strands, (iii) nitrogen and thia Atoms can be optionally oxidized, (iv) nitrogen heteroatoms can be optionally quaternized, (iv) any of the above rings can be optionally fused with a benzene ring, and (v) other ring atoms Is a carbon atom that may be optionally substituted with oxygen.
  • Typical heterocycloalkyl groups include, but are not limited to, [1,3]dioxolane, pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinone, imidazolidinyl, piperidinyl, Piperazine, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolinyl, quinoxalinyl, pyridazinyl, and tetrahydrofuran.
  • Such heterocyclic groups may be further substituted.
  • the compound I of the invention When the compound I of the invention is in a free form, it can be converted into the target salt by a method known per se or a similar method. Conversely, when the obtained compound I is a salt, it can be converted into a free form or a different target salt by a method known per se or the like.
  • Compound I may be crystalline, and may be a single crystal or a mixed form of multiple crystals.
  • the crystal form can be prepared by crystallization according to a known crystallization method.
  • Compound I may be a solvate (such as a hydrate, etc.), and both solvate and unsolvate are included in the scope of the present invention.
  • the salt of compound I in the present invention includes any pharmaceutically acceptable salt, for example, compound I and inorganic bases such as sodium and potassium, alkaline earth metals such as calcium, magnesium, etc., organic bases such as organic amines such as triethyl Amine, trimethylamine, tert-butylamine, pyridine, etc., basic amino acids such as arginine, lysine, ornithine, etc., as well as salts of ammonia; the preferred salts shown are potassium or sodium salts.
  • inorganic bases such as sodium and potassium, alkaline earth metals such as calcium, magnesium, etc.
  • organic bases such as organic amines such as triethyl Amine, trimethylamine, tert-butylamine, pyridine, etc.
  • basic amino acids such as arginine, lysine, ornithine, etc.
  • salts shown are potassium or sodium salts.
  • Another aspect of the present invention provides the dinucleotide prodrugs, or pharmaceutically acceptable salts thereof, and their stereoisomers, tautomers, geometric isomers, racemates, etc. are used in preparation Application in medicines for the treatment of viral infections, the viral infections particularly referring to HBV infections.
  • the present invention also provides a therapeutically effective amount of the dinucleoside precursor compound of the present invention in combination with other drugs for the treatment of viral infections, such as hepatitis B virus (HBV), hepatitis C (HCV) or two
  • drugs for the treatment of viral infections such as hepatitis B virus (HBV), hepatitis C (HCV) or two
  • HBV hepatitis B virus
  • HCV hepatitis C
  • two The application of drugs for any disease of mixed virus infection.
  • the dinucleotide prodrugs according to the present invention are effective in preventing and treating HBV infection, HCV infection and other conditions caused by HBV and HCV, such as hepatitis and cirrhosis caused by HBV and HCV. , Acute hepatitis, fulminant hepatitis, chronic hepatitis, and other liver diseases.
  • the compounds and preparations of the present invention can also be used prophylactically to prevent the deterioration of the disease in patients infected with HBV.
  • the dinucleotide prodrugs of the present invention are prepared for the treatment of hepatitis B virus (HBV), hepatitis C virus (HCV) or any disease caused by mixed infection of the two viruses.
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • the application of the drug; the administration process can be administered alone or in combination with another or other anti-HBV, HCV agent, or with another or other anti-HBV, HCV agent sequentially administered.
  • any one of the diastereomers of the S or R configuration of the phosphorus atom is administered in combination with another antiviral agent, the activity may be increased than when used alone.
  • the method of use is simultaneous or sequential administration.
  • the "simultaneous administration" includes administration of the agents at the same time or at different times. Two or more agents can be administered simultaneously by a single formulation containing two or more active ingredients or by substantially simultaneously administering two or more dosage forms containing a single active ingredient.
  • a pharmaceutical composition comprising the dinucleotide prodrug of the present invention and at least one pharmaceutically acceptable carrier or excipient.
  • hepatitis B virus HBV
  • HCV hepatitis C
  • mixed infection of the two viruses any disease of hepatitis B virus (HBV), hepatitis C (HCV) or mixed infection of the two viruses.
  • the present invention also provides a preparation method of the dinucleotide prodrug.
  • Compound I can be prepared by the method shown below or an analogous method or the like.
  • Compound I of general formula can be prepared by reacting compound SM3 or its salt with activated G or its derivative.
  • compound SM3 or its salt with activated G or its derivative.
  • the following scheme 1 describes the preparation method of compound Ia of general formula.
  • the compound NBIA (where L is a leaving group, such as Cl, Br, I, etc.) and SM3 in a solvent (preferably N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, 1,4-dioxane) in the reaction, preferably adding acid binding agent such as DIEA, and catalyst to the reaction system Sodium iodide.
  • a solvent preferably N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, 1,4-dioxane
  • NBIA The compound represented by the formula NBIA in the above scheme is synthesized according to the specific values of R1, R2, and R3.
  • R1, R2, and R3 are both H and R3 is methyl
  • NBIA can be prepared according to the following scheme 1a or scheme 1b:
  • R 3 is selected from H, C 1 ⁇ 6 alkyl group, C 3 ⁇ 12 cycloalkyl, and C 5 ⁇ 12 aryl group, preferably R3 is H, Me, Et, isopropyl, t-Bu, and the like, or Ph.
  • Reaction conditions (a) KO 2 CCH 2 CO 2 Et, MgCl 2 , Et 3 N/CH 3 CN; (b) PhCH 2 OH, DMAP (cat.), toluene; (c) 4-N-acetyl phenylsufonyllazide, Et 3 N/CH 3 CN; (d) Rh 2 (OAc) 4 (cat.), THF/H 2 O(2:1); (e) COCl 2 , iPr 2 Net; toluene or N,N'- Carbonyl diimidazole, iPr 2 NEt(cat.), CH 2 Cl 2 ; (f) H 2 , Pd(OH) 2 , EtOH; (g) i(COCl) 2 , DMF(cat.), CH 2 Cl 2 ; Ii Bu 4 NBH 4 , CH 2 Cl 2 ; (h) Ph 3 P, CBr 4 , CH 2 Cl 2 .
  • the dinucleotide prodrugs represented by the general formula Ib can be combined with the compound NBIB (where L is a leaving group, such as Cl, Br, I, etc.) and SM3 in nitrogen according to the method described in Scheme 2.
  • NBIB where L is a leaving group, such as Cl, Br, I, etc.
  • SM3 in nitrogen according to the method described in Scheme 2.
  • a solvent preferably N,N-dimethylformamide, N,N-dimethylacetamide, N-methylpyrrolidone, dimethyl sulfoxide, acetonitrile, tetrahydrofuran, 1,4-dioxane
  • an acid binding agent such as DIEA and a catalyst sodium iodide
  • the purified compound Ib was obtained by HPLC preparation and purification, and lyophilized, which was characterized by 1 H-NMR and 13 C-NMR.
  • the compound represented by the formula NBIB in the above scheme can be synthesized according to the following scheme according to the specific values of R3, R4, and R5.
  • the raw material SM1 is added with an initiator, and chlorine gas is introduced to obtain NBIB through a free radical reaction process.
  • the compound NBIB can be referred to according to the specific reaction situation.
  • the compounds BG001b to BG017b of the present invention can be prepared by referring to the method described in this scheme.
  • Compound Ic can be prepared by a method similar to that described in Scheme 2.
  • the above-mentioned method further includes: preparing and resolving the obtained compound mixture by high-performance liquid phase, or crystallization and other methods to obtain the optically pure compound diastereomers.
  • the present invention provides a dinucleotide prodrug with a novel structure and a preparation method thereof.
  • This type of compound exhibits excellent anti-HBV activity and good bioavailability in vivo, which is comparable to the existing dinucleotide prodrug.
  • the compound I provided by the present invention can significantly reduce liver HBV DNA, and these reductions are statistically significant; and experiments show that the dinucleoside precursor compound I provided by the present invention has low somatic cytotoxicity and good safety.
  • the present invention also provides a method for preparing compound I, which is easy to operate and has high yield and purity; in addition, the present invention also separates two diastereomeric pure compounds of compound I, and these separated isoforms
  • the structurally pure compounds provide a material basis and new ideas for research and development for those skilled in the art to further screen and study the physicochemical properties, pharmacodynamic activity and safety of such dinucleotide prodrugs, and also provide a better way for the development
  • the drugs that can meet clinical use provide new options.
  • BG002 Add 0.3g of BG002 to the reaction solution, dissolve it with 50mL of 50% CH 3 CN/H 2 O mixed solution, filter through a 0.45um organic filter membrane, and purify the filtrate by preparative HPLC, then preparative HPLC to remove salt, concentrate to remove most of the CH 3 CN, lyophilized to obtain white solid BG002R 47mg, BG002S 60mg, HPLC purity ⁇ 99%.
  • HBV transgenic positive C57 mice Male, 6-8 weeks old
  • mice Male, 6-8 weeks old
  • blood was taken from the mice, and the serum HBV DNA indicators were tested.
  • mice Using HBV mouse serum HBV DNA as the main indicator, 176 mice were selected for the experiment. 152 were divided into 19 groups of 8 animals. The specific groups and the administration of each group of mice are as follows:
  • Dosing frequency 1 Normal saline 0 Gavage every day 2 SB9200 50 Gavage every day 3 BG001b 50 Gavage every day 4 BG002b 50 Gavage every day 5 BG003b 50 Gavage every day 6 BG004b 50 Gavage every day 7 BG005b 50 Gavage every day 8 BG006b 50 Gavage every day 9 BG007b 50 Gavage every day 10 BG008b 50 Gavage every day 11 BG001c 50 Gavage every day 12 BG002c 50 Gavage every day 13 BG003c 50 Gavage every day 14 BG004c 50 Gavage every day 15 BG005c 50 Gavage every day 16 BG006c 50 Gavage every day 17 BG007c 50 Gavage every day 18 BG008c 50 Gavage every day 19 BG002 50 Gavage every day
  • mice in each group are weighed on D15/29/43/57, blood is taken from the orbit and the number of HBV DNA copies in the blood is detected. The animals were sacrificed after 8 weeks.
  • the anti-HBV effects of the 20 tested compounds were better than the control compound SB9200, and the antiviral effects of BG001b, BG001c, BG006b, BG006c and BG002 were all better.
  • BG002 has the most prominent effect. With the continuous increase in the number of days of administration, the viral DNA copy number decreases the most.
  • Each rat will collect about 0.15 mL of blood from the jugular vein at 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours after the administration, and place it in an EDTA-K2 anticoagulant tube, and store it on wet ice. And within 1h, centrifuge (1500 ⁇ 1600g) for 10min to separate the plasma, and store the obtained plasma sample at -40 ⁇ -20°C. After the experiment, the rats were euthanized by carbon dioxide asphyxiation method or cervical dislocation method.
  • the experimental results show that the cell proliferation IC 50 ( ⁇ M) of SB9200 and the test compounds BG001b ⁇ BG0017b, BG001c ⁇ BG0017c, and BG002 in the two cell lines are all>1000; it can be seen that the compound provided by the present invention has lower Somatic cell toxicity.

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Abstract

一种双核苷酸前体化合物及其在制备治疗乙型肝炎病毒感染药物中的应用。

Description

双核苷酸前体药物及其制备方法 技术领域
本发明涉及一种结构新颖的双核苷酸前体药物及其制备方法，还涉及光学异构体纯的双核苷酸前体药物及其制备方法，以及其在制备治疗病毒感染，特别是乙型肝炎病毒(HBV)感染和与HBV有关的肝脏疾病药物的应用。
背景技术
据估计，全世界有3亿5千万慢性HBV携带者，乙肝也是我国社会负担最大的疾病之一。根据疾病控制中心，每年将近3到7百万人死于与感染有关的并发症，例如，肝硬化和肝细胞癌变。大量接受肝移植的病人还持续需要抗-HBV治疗。HBV被认为是一种重要的病原，能够导致很多人类癌症。HBV感染还导致暴发型肝炎，这是一种致命的疾病，在此疾病中肝脏被破坏。慢性肝炎感染导致慢性持续的肝炎、衰竭、肝硬化、肝癌和死亡。乙肝病毒对患者的危害很大。患者感染乙肝病毒后，短期内对身体健康并不会造成很大的损失，而发病时，往往已发展成慢性乙肝，治疗困难，且预后较差。
目前临床上有多种药物用于HBV感染的治疗，例如拉米夫定(lamivudine)、恩替卡韦(entacavir)、替诺福韦艾拉酚胺(tenofovir alafenamide)、阿德福韦双特戊酰氧基甲酯(adefovir dipivoxil)等。但是由于耐药性的出现及剂量相关的毒副作用，这些药物远远不能满足临床需求。
Iyer等人在专利CN02810843中公开了两种或两种以上通过核苷内连接健相连接的脱氧核糖核苷和/或核糖核苷单体的化合物，及其优良的抗HBV活性。但是，这些核苷酸带有负电荷，较难穿过肠粘膜屏障的渗透，且易在胃肠道中分解，因此不适合口服给药。
使用一些前体药物策略可用来改善化合物的稳定性、提高靶向性、克服首过效应、改进生物利用率等。尽管前体药物的概念是已知的且对于制备许多化合物，包括核苷和单核苷酸的前体药物存在许多策略，但是本领域普通技术人员仍不能推知或明显的预料到双核苷酸类似的前体药物可能具有的口服生物利用率并因此可以发展成为可口服使用。已知口服生物利用率不仅仅与在胃粘膜中的稳定性有关系。例如，即使具有提高的稳定性，也仍然不知道这种相对大分子量的双核苷酸前体药物(分子量＞700道尔顿)是否能够被运输穿过粘膜屏障。实际上，人们对这种可以通过主动输送机制促进这些新型化合物传输穿过粘膜的具体转运装置是否存在知之甚少。按照里宾斯基原则(Lipinski’s rule)(Lipinski,C.A.,Adv.Drug Del.Rev.23,3,1997)，通过被动扩散用于口服吸收的药物分子应该具有小于500道尔顿的分子量、不超过5个氢键供体(OH和NH基团)、不超过10个氢键受体(值得注意的是氮和氧)、分子量低于500、LogP值低于5。实际上，双核苷酸前体药物都是具有较高分子量的化合物，因此在很多方面不能满足用于口腔吸收的里宾斯基标准。
发明内容
本发明提供了一种结构全新的双核苷酸前体药物；本发明另一方面还提供了这些双核苷酸前体药物的制备方法以及光学异构体纯的化合物的分离方法，及其在制备治疗病毒感染，特别是HBV感染的药物中的应用。
首先，本发明提供了通式(Ⅰ)表示的化合物或其盐：
Figure PCTCN2019115314-appb-000001
其中，n为0或1；G是具有如下结构的基团：
Figure PCTCN2019115314-appb-000002
优选，当G为
Figure PCTCN2019115314-appb-000003
时，n为1；
当G为
Figure PCTCN2019115314-appb-000004
时，n为0；
A、B独立的选自-NR 6-和-O-；
R 1、R 2、R 3、R 4、R 5、R 6独立的选自氢，烷基、环烷基、芳基、杂环基；所述烷基、环烷基、芳基、杂环基任选的被一个或多个取代基取代，所述取代基独立的选自：所述取代基独立的选自：卤素，OH，NH 2，CN，NO 2，C 1-6烷基，C 1-6烷氧基，和C 5-12芳基。
进一步的优选，R 1、R 2、R 3、R 4、R 5、R 6各自独立的选自H、C 1～6烷基、C 3～12环烷基、和C 5～12芳基；所述C 1～6烷基和C 5～12芳基任选的被一种或多种取代基取代，所述取代基独立的选自卤素、-OH，C 1～6烷氧基、-NO 2、-CN、-NH 2、C 1～6烷基氨基。
更进一步，所述C 1～6烷基优选为甲基，乙基，丙基，异丙基，叔丁基，正丁基；所述卤素为F，Cl，Br，I，优选卤素为F，Cl，所述C 5～12芳基优选为苯基，噻吩基，吡咯基，呋喃基。
在本发明的另一个优选实施方式中，所述化合物I中，G是具有如下结构的基团：
Figure PCTCN2019115314-appb-000005
其中，R 1、R 2、R 3、R 4、R 5独立的选自氢，烷基、环烷基、芳基、杂环基；所述烷基、环烷基、芳基、杂环基任选的被一个或多个取代基取代，所述取代基独立的选自：卤素，C 1-6烷基，C 1-6烷氧基，和C 5-12芳基。进一步的优选，R 1、R 2、R 3、R 4、R 5独立的选自氢，被一个或多个取代基取代或未被取代的C 1-6烷基、C 6-12环烷基、C 5-12芳基、C 5-12杂环基；所述取代基独立的选自卤素，C 1-6烷基，C 1-6烷氧基，C 5-12芳基，OH，NH 2，CN，NO 2，和三氟甲基。
更进一步的，优选上述化合物I中，R 1、R 2、R 3、R 4、R 5独立的选自氢、C 1-6烷基、C 1-6烷氧基、和卤素。最优选，R 1、R 2均为氢，R 3、R 4、R 5独立的选自：氢和C 1-6烷基，所述C 1-6烷基包括甲基，乙基，丙基，异丙基，环丙基，叔丁基，正丁基，正丙基，环己基和正己基。
更进一步的优选，当G是具有如下结构的基团：
Figure PCTCN2019115314-appb-000006
时，n为1；
当G是具有如下结构的基团：
Figure PCTCN2019115314-appb-000007
时，n为0。
在本发明的另一实施方式中，提供了具有如下式Ia所示的化合物或其盐：
Figure PCTCN2019115314-appb-000008
其中，R 1、R 2、R 3独立的选自氢，甲基，乙基，丙基，异丙基，叔丁基，正丁基，环己基，环丙基或三氟甲基。更进一步的优选，R 1、R 2、R 3独立的选自氢，R 3为氢或甲基。
本发明还提供了通式Ib所表示的化合物或其盐：
Figure PCTCN2019115314-appb-000009
或其外消旋体、对映异构体、非对映异构体、立体异构体、互变异构体；
其中，R 3、R 4、R 5独立的选自氢，烷基、环烷基、芳基、杂环基；所述烷基、环烷基、芳基、杂环基任选的被一个或多个取代基取代，所述取代基独立的选自：卤素，C 1-6烷基，C 1-6烷氧基，和C 5- 12芳基。
进一步的优选，R 3、R 4、R 5独立的选自氢，被一个或多个取代基取代或未被取代的C 1-6烷基、C 6-12环烷基、C 5-12芳基、C 5-12杂环基；所述取代基独立的选自卤素，C 1-6烷基，C 1-6烷氧基，C 5-12芳基，OH，NH 2，CN，NO 2，和三氟甲基。
更进一步的，优选上述化合物Ib中，R 3、R 4、R 5独立的选自氢、C 1-6烷基、C 1-6烷氧基、和卤素。最优选，R 3、R 5均为氢，R 4为氢，卤素或C 1-6烷基，所述C 1-6烷基包括甲基，乙基，丙基，异丙基，环丙基，叔丁基，正丁基，正丙基，环己基和正己基，所述卤素为F，Cl，Br或I，优选为F或Cl。更进一步的优选，R 3、R 5均为氢，R 4为甲基。
优选的代表化合物(或其立体异构体、互变异构体，几何异构体、外消旋物、或其药学上可接受的盐)，用式Ib表示其中，R 3、R 4、R 5如下表所示：
Figure PCTCN2019115314-appb-000010
Figure PCTCN2019115314-appb-000011
另一方面，本发明所述化合物I优选具有如下式Ic所示的结构：
Figure PCTCN2019115314-appb-000012
或其外消旋体、对映异构体、非对映异构体、立体异构体、互变异构体；
其中，R 3、R 4、R 5独立的选自氢，烷基、环烷基、芳基、杂环基；所述烷基、环烷基、芳基、杂环基任选的被一个或多个取代基取代，所述取代基独立的选自：卤素，C 1-6烷基，C 1-6烷氧基，和C 5- 12芳基。
进一步的优选，R 3、R 4、R 5独立的选自氢，被一个或多个取代基取代或未被取代的C 1-6烷基、C 6-12环烷基、C 5-12芳基、C 5-12杂环基；所述取代基独立的选自卤素，C 1-6烷基，C 1-6烷氧基，C 5-12芳基，OH，NH 2，CN，NO 2，和三氟甲基。
更进一步的，优选上述化合物Ib中，R 3、R 4、R 5独立的选自氢、C 1-6烷基、C 1-6烷氧基、和卤素。最优选，R 3、R 5均为氢，R 4为氢，卤素或C 1-6烷基，所述C 1-6烷基包括甲基，乙基，丙基，异丙基，环丙基，叔丁基，正丁基，正丙基，环己基和正己基，所述卤素为F，Cl，Br或I，优选为F或Cl。更进一步的优选，R 3、R 5均为氢，R 4为甲基。
优选的代表化合物，用式Ib表示(或其立体异构体、互变异构体，几何异构体、外消旋物、或其药学上可接受的盐)其中，R 3、R 4、R 5如下表所示：
Figure PCTCN2019115314-appb-000013
Figure PCTCN2019115314-appb-000014
在本发明另一实施方式中，提供了如下化合物：
Figure PCTCN2019115314-appb-000015
Figure PCTCN2019115314-appb-000016
本发明所使用的“芳基”是指具有一个或两个芳环的单环或多环碳环系统，优选含有5～12个碳原子的芳基，所述芳环包括但不限于苯基，萘基，四氢萘基，茚满基，茚基，呋喃基，吡咯基，噻吩基等。本发明的芳基在其芳香环例如苯环上任意位置，任选的被卤素(如F，Cl，或Br)，硝基，C 1～6烷氧基，氨基或C 1～6烷基取代。
术语“烷基”是指饱和的、直链或含有支链的烃基基团，分别包含一到十二个碳原子，优选一到六个碳原子。C 1-C 12烷基基团的例子包括，但不仅限于，甲基、乙基、丙基、异丙基、正丁基、叔丁基、新戊基和正己基、辛基、癸基、十二烷基等。
术语“芳烷基”或“芳基烷基”是指包含芳基取代的烷基，例如苯甲基、二苯甲基、三苯甲基、苯乙基、和二苯基乙基。所述的“-芳烷基-”是指含有两个连接点的芳烷基，其中连接点可以都在烷基部分，也可以都在芳基部分，或者在烷基和芳基部分各有一个连接点，例如
Figure PCTCN2019115314-appb-000017
术语“环烷基”表示一种通过除去单一氢原子产生的单环或多环饱和碳环化合物的单价基团。例子包括但不限于，环丙基、环丁基、环戊基、环己基、二环[2.2.1]庚基、和二环[2.2.2]辛基。
术语“杂环基”是指一种5-元、6-元或7-元的非芳香族环或二或三环基团融合的系统，其中(i)每个环包含一到三个杂原子，独立的选自氧、硫和氮，(ii)每个5-元环具有0到1个双链且每个6-元环具有0到2个双链，(iii)氮和硫杂原子可以任选地被氧化，(iv)氮杂原子可以任选地被季铵化，(iv)上述任何一种环可以任选地与一种苯环融合，和(v)其他的环原子是可以被任选氧取代的碳原子。典型的杂环烷基基团包括，但不仅限于，[1，3]二氧戊环、吡咯烷基、吡唑啉基、吡唑烷基、咪唑啉酮、咪唑烷基、哌啶基、哌嗪、噁唑烷基、异噁唑烷基、吗啉基、噻唑烷基、异噻唑啉基、喹喔啉基、哒嗪基、和四氢呋喃。这种杂环基团可以进一步被取代。
发明的化合物I是游离形式时，通过本身已知的方法或类似的方法可以将其转化成目标盐。反之，当获得的化合物I是盐时，通过本身已知的方法或类似方法可以将其转化为游离形式或不同的目标盐。
当本化合物I存在光学异构体时，这样的单个光学异构体及其混合物当然包括在本发明范围内。
化合物I可以是结晶，并可以是单晶或多种结晶的混合形式。根据已知的结晶方法通过结晶可以制备晶型。
化合物I可以是溶剂化物(如水合物等)，溶剂化物和非溶剂化物都包含在本发明范围内。
本发明所述的化合物I的盐，包括任何一种药学上可接受的盐，比如，化合物I与无机碱如钠、 钾等，碱土金属如钙，镁等、有机碱如有机胺如三乙胺，三甲胺，叔丁胺，吡啶，等，碱性氨基酸如精氨酸，赖氨酸，鸟氨酸等，以及氨等形成的盐；优选的所示盐为钾盐或钠盐。
本发明的另一方面提供了所述双核苷酸前体药物，或其药学可接受盐，其立体异构体、互变异构体，几何异构体、外消旋物等在制备用于治疗病毒感染的药物中的应用，所述病毒感染特别是指HBV感染。
本发明还提供了与其他药物相结合的治疗有效量的本发明所述的双核苷前体化合物在制备用于治疗病毒感染，如乙型肝炎病毒(HBV)、丙型肝炎(HCV)或者两种病毒混合感染的任何病症的药物中的应用。
根据本发明的双核苷酸前体药物在预防和治疗HBV感染、HCV感染及其他由HBV、HCV所引起情况时是有效的，这里所述的由HBV、HCV所引起的情况例如肝炎、肝硬化、急性肝炎、暴发型肝炎、慢性肝炎、及其他肝脏疾病。本发明的化合物和制剂还可以预防性的使用，用于防止HBV感染的患者的疾病恶化。
以及，本发明所述双核苷酸前体药物，单独或与其他试剂相结合，在制备用于治疗乙型肝炎病毒(HBV)、丙型肝炎病毒(HCV)或者两种病毒混合感染的任何病症的药物中的应用；给药过程可以是单独给药也可以与另一种或其他抗HBV、HCV试剂结合给药，或者与另一种或其他抗HBV、HCV试剂顺序给药。
当化合物I，其磷原子的S或R构型的非对映异构体中的任一种和另一抗病毒剂联合施用时，活性可能比单独使用增加。当治疗是联合疗法时，使用方法是同时施用或序贯施用。所述的“同时施用”包括在相同时间或在不同时间施用药剂。可通过含有两种或更多种活性成分的单一制剂或通过基本上同时施用两种或更多种含有单一活性成分的剂型来同时施用两种或更多种药剂。
在本发明的另外一方面，提供了一种药物组合物，包含本发明所述双核苷酸前体药物和至少一种药学上可接受的载体或赋形剂。
以及所述药物组合物在制备用于治疗乙型肝炎病毒(HBV)、丙型肝炎(HCV)或者两种病毒混合感染的任何病症的药物中的应用。
另一方面，本发明还提供了所述双核苷酸前体药物的制备方法。
制备方法：
可通过下面所示的方法或其类似方法等制备化合物I。
尽管根据下面方法制备得到的化合物I的收率可以取决于所用的反应条件而改变，但可通过常规的分离方法或纯化方法(如结晶，柱层析，HPLC制备柱分离等)获得高纯度的化合物I。
通式化合物I可以有化合物SM3或其盐与活化的G或其衍生物进行反应，制备得到。例如：
下述方案1描述了通式化合物Ia的制备方法，将化合物NBIA(其中L为离去基团，如Cl，Br，I等)与SM3在溶剂(优选N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮、二甲亚砜、乙腈、四氢呋喃、1,4-二氧六环)中反应，优选反应体系中加入缚酸剂如DIEA，以及催化剂碘化钠。
在反应结束后，经高效液相制备纯化，冻干，得到纯化后化合物Ia，使用 1H-NMR和 13C-NMR对 其定性。
Figure PCTCN2019115314-appb-000018
上述方案中式NBIA所示化合物根据R1，R2，R3的具体取值情况进行合成。举例说明的，例如当R1，R2均为H，R3为甲基时，NBIA可以按照如下方案1a或方案1b制备：
方案1a：
Figure PCTCN2019115314-appb-000019
R 3选自H、C 1～6烷基、C 3～12环烷基、和C 5～12芳基，优选R3为H，Me，Et，异丙基，t-Bu，或Ph等。
方案1b：
Figure PCTCN2019115314-appb-000020
反应条件：(a)KO 2CCH 2CO 2Et，MgCl 2，Et 3N/CH 3CN；(b)PhCH 2OH,DMAP(cat.)，甲苯；(c)4-N-acetyl phenylsufonyllazide,Et 3N/CH 3CN；(d)Rh 2(OAc) 4(cat.),THF/H 2O(2:1)；(e)COCl 2，iPr 2Net；甲苯或者N,N'-羰基二咪唑,iPr 2NEt(cat.)，CH 2Cl 2；(f)H 2，Pd(OH) 2,EtOH；(g)i(COCl) 2，DMF(cat.)，CH 2Cl 2；ii Bu 4NBH 4,CH 2Cl 2；(h)Ph 3P，CBr 4，CH 2Cl 2。
具体操作方法可以参考文献：Tetrahedron Letters 43(2002)1161-1164。
进一步的，举例说明，通式Ib所示的双核苷酸前体药物可按照方案2描述的方法，将化合物NBIB(其中L为离去基团，如Cl，Br，I等)与SM3在氮气保护下，在溶剂(优选N,N-二甲基甲酰胺、N,N-二甲基乙酰胺、N-甲基吡咯烷酮、二甲亚砜、乙腈、四氢呋喃、1,4-二氧六环)中反应，优选反应体系中加入缚酸剂如DIEA，以及催化剂碘化钠。
在反应结束后，经高效液相制备纯化，冻干，得到纯化后的化合物Ib，使用 1H-NMR和 13C-NMR对其定性。
Figure PCTCN2019115314-appb-000021
上述方案中式NBIB所示化合物根据R3，R4，R5的具体取值情况可根据如下方案进行合成。原料SM1加入引发剂，通入氯气，通过自由基反应过程，得到NBIB。
Figure PCTCN2019115314-appb-000022
其中化合物NBIB可以根据具体反应情况参考当。本发明所述化合物BG001b～BG017b均可参考该方案所述的方法制备得到。
可以采用类似方案2所述的方法制备化合物Ic。
优选的，上述方法，进一步还包括，将得到化合物混旋体经高效液相制备拆分，或者结晶等方法得光学纯化合物非对映异构体。
本发明提供了一种结构新颖的双核苷酸前体药物及其制备方法，这类化合物在体内显示出优异的抗HBV活性和良好的生物利用度，与现有的双核苷酸前体药物相比，本发明提供的化合物I能使肝HBV DNA显著降低，并且这些降低具有统计学意义；并且，实验表明本发明提供的双核苷前体化合物I体细胞毒性小，安全性好。本发明还提供了一种化合物I的制备方法，操作简便，收率和纯度高；另外，本发明还分离出化合物I的两种非对映异构体纯的化合物，这些已经分离出来的异构体纯的化合物，为本领域技术人员进一步筛选研究这类双核苷酸前体药物的理化性质，药代药效活性以及安全性等提供了物质基础和研发新思路，也为开发出更好的能满足临床使用的药物提供了新的选择方案。
具体实施例
以下结合具体实施例对本发明所述化合物的制备方法以及其具备有的有益效果做进一步阐释说明。
实施例1化合物BG002的合成方法：
Figure PCTCN2019115314-appb-000023
氮气保护下，反应瓶中，依次加入SM3(4.5g，7.7mmol)、DMF(100ml)、二异丙基乙胺(1.1g，8.5mmol)、碘化钠(0.58g，5.8mmol)、4-氯甲基-5-甲基-1,3-二氧杂环戊烯-2-酮(1.2g，8.4mmol)，加毕，室温搅拌反应过夜，反应液经柱层析，纯化得BG002 2.0g，纯度98.08％，收率57.3％。
MS Calcd:699；MS Found:700[M+H] +。
1H-NMR(DMSO-d 6,400Hz):
δ2.0177-2.0411(d,3H)；2.3059-2.3700(m,1H)；2.8201-2.9104(m,1H)；3.33(s,3H)；3.5887(s,2H)；3.9426-3.9976(m,2H)；4.0326-4.1349(m,3H)；4.1831-4.2859(m,1H)；4.3294-4.4024(m,1H)；4.4879-4.5206(m,1H)；4.9859-5.0385(m,1H)；5.3439-5.3860(t,1H)；5.5369-5.5509(dd,1H)；5.7020-5.7325(m,1H)；5.8897-5.9135(m,1H)；6.3617-6.3993(m,1H)；7.2728(s,2H)；7.8637-7.8938(m,1H)；8.1423(s,1H)；8.2848-8.2965(d,1H)；11.4335(s,1H)。
13C-NMR(DMSO-d 6,400Hz):
δ9.13,9.18,23.34,38.66,38.84,58.35,58.42,60.74,60.85,67.97,70.87,70.92,75.21,80.88,
80.93,81.06,83.96,84.04,84.11,84.24,85.09,103.01,119.68,119.74,134.25,134.29,138.01,139.96,140.15,140.62,149.49,149.53,151.03,151.04,152.18,153.01,156.50,163.39。
分离光学纯化合物BG002R、BG002S：
Figure PCTCN2019115314-appb-000024
向反应液中加入0.3g BG002用50mL的50％CH 3CN/H 2O混合液溶解，经0.45um有机滤膜过滤，滤液经制备HPLC纯化，然后制备HPLC除盐，浓缩除去大部分的CH 3CN，冻干，得到白色固体BG002R 47mg，BG002S 60mg，HPLC纯度≥99％。
制备纯化方法
Figure PCTCN2019115314-appb-000025
Figure PCTCN2019115314-appb-000026
经测定分离得到的BG002的两种光学纯的非对映异构体的 1H-NMR和 13C-NMR数据如下：
(1)、 1H-NMR(DMSO-d 6,400Hz):δ2.02(d,3H)；2.31-2.37(m,1H)；2.82-2.91(m,1H)；3.33(s,3H)；3.59(s,2H)；3.94-4.00(m,2H)；4.03-4.13(m,3H)；4.18-4.29(m,1H)；4.33-4.40(m,1H)；4.49-4.52(m,1H)；4.99-5.04(m,1H)；5.34-5.3860(t,1H)；5.54-5.55(dd,1H)；5.70-5.73(m,1H)；5.89-5.91(m,1H)；6.36-6.40(m,1H)；7.27(s,2H)；7.86-7.89(m,1H)；8.15(s,1H)；8.31(s,1H)；11.43(s,1H)；
13C-NMR(DMSO-d 6,400Hz):δ9.13,23.37,38.65,,58.35,58.42,60.73,68.00,70.85,75.28,80.94,84.04,84.11,84.32,,103.02,119.73,134.21,134.25,138.01,140.20,140.61,149.48,151.04,152.18,152.91,156.44,163.39。
(2)、 1H-NMR(DMSO-d 6,400Hz):δ2.04(d,3H)；2.31-2.37(m,1H)；2.82-2.91(m,1H)；3.33(s,3H)；3.59(s,2H)；3.94-4.00(m,2H)；4.03-4.13(m,3H)；4.18-4.29(m,1H)；4.33-4.40(m,1H)；4.49-4.52(m,1H)；4.99-5.04(m,1H)；5.34-5.3860(t,1H)；5.54-5.55(dd,1H)；5.70-5.73(m,1H)；5.89-5.91(m,1H)；6.36-6.40(m,1H)；7.27(s,2H)；7.88-7.91(d,1H)；8.18(s,1H)；8.32(s,1H)；11.43(s,1H)；
13C-NMR(DMSO-d 6,400Hz):δ9.18,23.36,38.89,58.35,60.84,67.94,68.00,70.86,75.30,81.10,84.08,85.05,103.01,119.62,134.25,134.29,138.01,140.23,140.65,149.41,151.06,152.18,152.29,155.92,163.41.
实施例2化合物BG004b的合成方法：
Figure PCTCN2019115314-appb-000027
氯代碳酸乙烯酯合成：
取94g碳酸乙烯酯，加入引发剂过氧化二苯甲酰(BPO),搅拌溶解澄清。紫外线光照，通入氯气(流量120ml/min)，控温80-90℃，搅拌反应4h。经蒸馏纯化得无色油状物78g，收率59.5％，气相纯度98.5％。
氮气保护下，反应瓶中，依次加入SM3(4.5g，7.7mmol)、DMF(100ml)、二异丙基乙胺(1.1g，8.5mmol)、碘化钠(0.58g，5.8mmol)、氯代碳酸乙烯酯(1.0g，8.4mmol)，加毕，室温搅拌反应过夜，反应液经柱层析，纯化得BG004b 0.85g，纯度98.58％，收率16.4％。MS Calcd:673；MS Found:674[M+H] +。
实施例3化合物BG006b的合成方法
Figure PCTCN2019115314-appb-000028
氮气保护下，反应瓶中，依次加入SM3(4.5g，7.7mmol)、DMF(100ml)、二异丙基乙胺(2.2g，17mmol)、碘化钠(0.58g，5.8mmol)、4-氯-5,5-二甲基碳酸乙烯酯(2.3g，15.0mmol)，加毕，室温搅拌反应过夜，反应液经柱层析，纯化得BG006b 1.56g，纯度99.1％，收率28.9％。MS Calcd:701；MS Found:702[M+H]+。
参考实施例2和3或其类似方法合成化合物BG002b～BG005b以及BG007b～BG016b
化合物编号 MS Calcd MS Found：[M+H] + 化合物编号 MS Calcd MS Found：[M+H] +
BG001b 687 688 BG010b 861 862
BG002b 687 688 BG011b 715 716
BG003b 701 702 BG012b 771 772
BG005b 715 716 BG013b 779 780
BG007b 715 716 BG014b 741 742
BG008b 763 764 BG015b 739 740
BG009b 851 852 BG016b 755 756
实施例4化合物BG004c的合成方法
Figure PCTCN2019115314-appb-000029
氮气保护下，反应瓶中，依次加入SM3(4.5g，7.7mmol)、DMF(100ml)、二异丙基乙胺(1.1g，8.5mmol)、碘化钠(0.58g，5.8mmol)、4-氯丁内酯(1.0g，8.4mmol)，加毕，室温搅拌反应过夜，反应液经柱层析，纯化得BG004c 2.8g，纯度98.7％，收率54.2％。MS Calcd:671；MS Found:672[M+H] +。
实施例5化合物BG006c的合成方法
Figure PCTCN2019115314-appb-000030
氮气保护下，反应瓶中，依次加入SM3(4.5g，7.7mmol)、DMF(100ml)、二异丙基乙胺(1.1g，8.5mmol)、碘化钠(0.58g，5.8mmol)、4-氯-3,3-二甲基丁酸内酯(1.25g，8.4mmol)，加毕，室温搅拌反应过夜，反应液经柱层析，纯化得BG006c 1.21g，纯度97.6％，收率22.5％。MS Calcd:699；MS Found:700[M+H] +。
参考实施例4和5或其类似方法合成化合物BG002c～BG005c以及BG007c～BG016c
化合物编号 MS Calcd MS Found：[M+H] + 化合物编号 MS Calcd MS Found：[M+H] +
BG001c 685 686 BG010c 759 760
BG002c 685 686 BG011c 713 714
BG003c 699 700 BG012c 769 770
BG005c 713 714 BG013c 777 778
BG007c 713 714 BG014c 739 740
BG008c 761 762 BG015c 737 738
BG009c 749 750 BG016c 753 754
实施例6：受试化合物抗HBV活性研究
选取约170只HBV转基因阳性C57小鼠(雄性，6-8周龄)，称重和大体观察，对不适合实验的动物提前剔除掉。初筛后的小鼠取血，检测血清HBV DNA指标。
以HBV小鼠血清HBV DNA为主要指标，选取176只小鼠入组进行实验。152分成19每组8只。具体分组及每组小鼠给药情况如下：
序号 组别 剂量(mg/kg) 给药方式 给药频率
1 生理盐水 0 灌胃 每天
2 SB9200 50 灌胃 每天
3 BG001b 50 灌胃 每天
4 BG002b 50 灌胃 每天
5 BG003b 50 灌胃 每天
6 BG004b 50 灌胃 每天
7 BG005b 50 灌胃 每天
8 BG006b 50 灌胃 每天
9 BG007b 50 灌胃 每天
10 BG008b 50 灌胃 每天
11 BG001c 50 灌胃 每天
12 BG002c 50 灌胃 每天
13 BG003c 50 灌胃 每天
14 BG004c 50 灌胃 每天
15 BG005c 50 灌胃 每天
16 BG006c 50 灌胃 每天
17 BG007c 50 灌胃 每天
18 BG008c 50 灌胃 每天
19 BG002 50 灌胃 每天
给药当天为D1，随后的8周每天给药，各组小鼠在D15/29/43/57称重，眼眶取血并检测血液中HBV DNA拷贝数。8周后处死动物。
实验结果：
20种受试化合物的抗HBV效果均优于对照化合物SB9200，其中BG001b、BG001c、BG006b、BG006c和BG002的抗病毒效果都较好。在所有受试化合物中，BG002的效果最突出，随着给药天数的持续增加，病毒DNA拷贝数下降幅度最大。
化合物各次检测的HBV DNA拷贝数
Figure PCTCN2019115314-appb-000031
上述结果表明，与对照组相比，结构修饰的前体化合物BG001b～BG008b以及化合物BG001c～BG008c，特别是BG002能使肝HBV DNA显著降低，并且这些降低具有统计学意义。
实施例7：本发明化合物在SD大鼠体内的药代动力学研究
选取6-8周龄雄性SD大鼠63只(维通利华实验动物技术有限公司)，动物饲养在SPF动物房内。动物房装备空调系统，通风良好，室内温度维持在20～26℃范围，湿度维持在40％～70％范围内。动物房内采用人工照明，明暗各12小时(因实验操作、清洁需开启工作照明等情况除外)，实验动物自由采食和饮水。经兽医检验，体征状况良好的大鼠入选本实验，每只大鼠均用尾标号标记。所有动物给药前禁食，禁食时间不少于12h，于给药后4h恢复给食，保持所有动物在实验过程中均可自由饮水。SB9200和受试化合物均以0.5％CMC-Na配制灌胃制剂，每组小鼠单次给药，给药途径均为灌胃。
动物分组及给药信息详见下表。
Figure PCTCN2019115314-appb-000032
每只大鼠于给药前、给药结束后0.25、0.5、1、2、4、8、12和24h由颈静脉采血约0.15mL，置于EDTA-K2抗凝管中，存放于湿冰上，并于1h之内，离心(1500～1600g)10min，分离血浆，将所得血浆样品保存于-40～-20℃环境中。实验结束后，采用二氧化碳窒息法或者颈椎脱臼法对大鼠实施安乐死。
建立测定SD大鼠血浆中活性代谢产物SB9000浓度测定的LC-MS/MS分析方法，用于本实验获得的生物样品的浓度测定。采用Pharsight Phoenix 8.0中的非房室模型计算相应的药代动力学参数。
实验结果如下所示：
Figure PCTCN2019115314-appb-000033
Figure PCTCN2019115314-appb-000034
从上述实验结果中可以看出，SD大鼠灌胃给予本发明所述化合物后，其活性代谢产物SB9000的药代动力学性质均优于对照化合物SB9200，其中BG001b、BG001c、BG006b、BG006c和BG002的药代动力学性质较为突出。在所有受试化合物中，BG002的药代性质最突出，Cmax和AUC 0-t均为对照化合物SB9200的2.5倍左右。
实施例8：体外细胞毒性：
用CTG方法测试化合物BG001b～BG0017b，以及BG001c～BG0017c，BG002和SB9200在人正常肺成纤维细胞(MRC-9)和人脐静脉内皮细胞(HUVEC)的细胞增殖50％抑制浓度(IC 50)。
收集处于指数生长期的细胞并用Vi-Cell XR细胞计数仪进行活细胞计数。用各细胞相应培养基调整细胞悬液浓度。每孔加90μl细胞悬液于96-孔细胞培养板，最终细胞浓度为3000细胞/孔。以DMSO溶解各供试化合物为10mM储存液。用储存液和DMSO制备3.16X系列梯度稀释液。然后分别用培养基稀释100倍。最后每株细胞每孔分别加入10μl相应的10倍溶液，每个药物浓度各3个复孔，置于37℃,5％CO 2孵箱中培养72小时。药物处理72小时后，按照CTG操作说明，每孔加入50μl(1/2培养体积)预先融化并平衡到室温的CTG溶液，用微孔板震荡器混匀2分钟，于室温放置10分钟后用Envision2104读板仪测定萤光信号值。应用GraphPad Prism 5.0软件,使用非线性回归模型绘制S型剂量-存活率曲线并计算IC 50值。实验结果显示SB9200以及供试化合物BG001b～BG0017b，以及BG001c～BG0017c，BG002在2个细胞系的细胞增殖IC 50(μM)均＞1000；由此可以看出，本发明提供的化合物具有较低的体细胞毒性。

Claims (11)

  1. 由式(Ⅰ)表示的化合物或其盐：
    Figure PCTCN2019115314-appb-100001
    其中，n为0或1；G是具有如下结构的基团：
    Figure PCTCN2019115314-appb-100002
    A、B独立的选自-NR 6-和-O-；
    R 1、R 2、R 3、R 4、R 5、R 6独立的选自氢，烷基、环烷基、芳基、杂环基；所述烷基、环烷基、芳基、杂环基任选的被一个或多个取代基取代，所述取代基独立的选自：卤素，OH，NH 2，CN，NO 2，C 1-6烷基，C 1-6烷氧基，和C 5-12芳基。
  2. 根据权利要求1所述化合物，其特征在于，
    当G为
    Figure PCTCN2019115314-appb-100003
    时，n为1；
    当G为
    Figure PCTCN2019115314-appb-100004
    时，n为0；
    其中，R 1、R 2、R 3、R 4、R 5独立的选自氢，烷基、环烷基、芳基、杂环基；所述烷基、环烷基、芳基、杂环基任选的被一个或多个取代基取代，所述取代基独立的选自：卤素，C 1-6烷基，C 1-6烷氧基，和C 5- 12芳基。
  3. 根据权利要求2所述化合物，其特征在于，R 1、R 2、R 3、R 4、R 5独立的选自氢，被一个或多个取代基取代或未被取代的C 1-6烷基、C 6-12环烷基、C 5-12芳基、C 5-12杂环基；所述取代基独立的选自卤素，C 1- 6烷基，C 1-6烷氧基，C 5-12芳基，和三氟甲基。
  4. 根据权利要求2所述化合物，其特征在于，R 1、R 2、R 3、R 4、R 5独立的选自氢、C 1-6烷基、C 1-6烷氧 基、和卤素。
  5. 根据权利要求2所述化合物，其特征在于，R 1、R 2均为氢，R 3、R 4、R 5独立的选自：氢和C 1-6烷基。
  6. 根据权利要求1～5任一项所述化合物，其特征在于，具有如下结构：
    Figure PCTCN2019115314-appb-100005
  7. 根据权利要求6所述化合物，其特征在于，R 1、R 2、R 3、R 4、R 5独立的选自氢，甲基，乙基，丙基，异丙基，叔丁基，正丁基，环己基，环丙基或三氟甲基；优选，R 1、R 2、R 3、R 4、R 5独立的选自氢和甲基。
  8. 化合物，其特征在于，具有如下结构：
    Figure PCTCN2019115314-appb-100006
    Figure PCTCN2019115314-appb-100007
  9. 根据权利要求1～5任一项所述化合物，其特征在于，所述化合物中手性磷原子P为S构型，所述化合物是磷原子为S构型的非对映异构体化合物，或者所述化合物中手性磷原子P为R构型，所述化合物是磷原子为R构型的非对映异构体化合物，或者所述化合物是磷原子为S构型和R构型的非对映异构体混合物。
  10. 权利要求1～3或7～8任一项所述化合物在制备用于治疗病毒感染的药物中的应用。
  11. 根据权利要求10所述化合物或其盐的用途，其特征在于，所述病毒感染是指乙肝病毒(HBV)，丙肝病毒(HCV)感染或HBV与HCV混合感染。
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